TARGETED CHIMERIC PROTEINS AND THEIR USES - US20220281992A1 (2023)

CROSS REFERENCE TO RELATED APPS

[0001]This application claims the benefit of and priority to US Provisional Patent Application No. 62/454,993, filed Feb. 6, 2017, the contents of which are hereby incorporated by reference in their entirety.

CAMPO

[0002]The present invention relates in part to chimeric proteins comprising at least one targeting moiety that recognizes and binds SIRP1α and their use as diagnostic and therapeutic agents. The present invention further relates to pharmaceutical compositions comprising chimeric proteins and their use in the treatment of various diseases including cancer.

SEQUENCE LIST

[0003]The immediate request contains a sequence listing transmitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. This ASCII copy created on January 31, 2018 is called ORN-027PC-Sequence_Listing_ST25.txt and is 282,624 bytes in size.

FONDS

[0004]Cancer is a global health challenge that causes nearly 7 million deaths worldwide each year and, despite major advances in medicine, has proven largely intractable to date. Frustratingly, cancers seem to adopt strategies to evade immunological recognition and destruction, thereby bypassing the body's main defenses against disease. For example, one mechanism by which cancer cells evade phagocytosis by macrophages is the upregulation of CD47, which couples to an inhibitory receptor on macrophages, i. H. the regulatory signaling protein α-1 (SIRP1α). In particular, the interaction between CD47 on cancer cells and SIRP1α provides a "don't eat me" signal that inhibits phagocytosis of cancer cells. The Myc oncogene induces CD47 expression on cancer cells, an immunosuppressive mechanism that has been implicated in Myc's potent tumor growth-promoting activity in vivo.

[0005]Activation of the Myc family of cellular oncogenes is one of the most common oncogenic events in human cancers. The Myc family of proteins encodes three highly related nuclear phosphoproteins (c-Myc, N-Myc, and L-Myc) that are thought to function as sequence-specific transcription factors. The Myc protein activates several genes important for biological processes including growth, proliferation, apoptosis, metabolism, differentiation, self-renewal and angiogenesis. Despite efforts to inhibit overactive Myc proteins in cancer cells, this oncogene remains remarkably resistant to therapeutic targeting. In addition, evidence suggests that active Myc proteins promote tumor resistance to various anticancer drugs.

[0006]Accordingly, there remains a need for new therapeutic agents that can effectively target cancers, including cancers caused by Myc.

SUMMARY

[0007]In various aspects, the present invention relates to chimeric proteins having at least one targeting moiety that specifically binds to SIRP1α. In various embodiments, the present chimeric proteins find use in, for example, direct or indirect recruitment of a macrophage cell to a site of interest. In various embodiments, the chimeric proteins further comprise a signaling agent, such as, without limitation, an interferon, an interleukin, and a tumor necrosis factor that can be modified to attenuate activity. In various embodiments, the chimeric protein includes additional targeting moieties that bind to other targets of interest (e.g., antigens, receptors). In one embodiment, the other targets of interest (e.g., antigens, receptors) are present on the tumor cells. In another embodiment, the other targets of interest (e.g., antigens, receptors) are present on the immune cells. In some embodiments, the present chimeric protein can directly or indirectly recruit an immune cell (e.g., a macrophage) to a site of action (such as, for example, as a non-limiting example, the tumor microenvironment). In some embodiments, the present chimeric protein facilitates phagocytosis of a target cell (e.g., a tumor cell) by macrophages.

[0008]In various embodiments, the present chimeric proteins find use in the treatment of various diseases or disorders, such as cancer, infections, immune disorders, and other diseases and disorders, and various methods of treatment are encompassed by the present invention.

DETAILED DESCRIPTION

[0012]The present invention is based in part on the discovery of chimeric targeting proteins that have a targeting moiety that specifically recognizes and binds the signaling regulatory protein α-1 (SIRP1α). In some embodiments, the chimeric protein is multispecific and includes one or more targeting residues. In some embodiments, the chimeric protein further comprises a modified signaling agent (e.g., an interferon) with reduced affinity for one or more receptors. In various embodiments, the chimeric protein can bind directly or indirectly to immune cells such as macrophages and recruit them to sites in need of a therapeutic effect (e.g., a tumor or the tumor microenvironment). In some embodiments, the chimeric protein induces and/or enhances phagocytosis of tumor cells by macrophages. The present chimeric protein exhibits beneficial therapeutic properties and reduced side effects.

[0013]chimeric Zielproteine

[0014]In various embodiments, the present invention relates to chimeric targeting proteins comprising a targeting moiety that specifically recognizes and binds signaling regulatory protein α-1 (SIRP1α). SIRP1α (also known as SIRPα) belongs to a family of cellular immune receptors that includes inhibitory (SIRPα), activator (SIRPβ), non-signalling (SIRPγ), and soluble (SIRPδ) members. SIRP1α is mainly expressed on myeloid cells including macrophages, granulocytes, myeloid dendritic cells (DC), mast cells and their progenitors including hematopoietic stem cells. SIRP1α functions as an inhibitory receptor that interacts with a widely distributed CD47 transmembrane glycoprotein to regulate phagocytosis. Specifically, binding of SIRP1α to macrophages by CD47 expressed on target cells generates an inhibitory signal that negatively regulates target cell phagocytosis.

[0015]In various embodiments, the present invention relates to chimeric targeting proteins comprising a targeting moiety that specifically recognizes and binds SIRP1α on macrophages.

[0016]In various embodiments, the present invention relates to chimeric targeting proteins comprising a targeting moiety that specifically recognizes and binds SIRP1α on monocytes.

[0017]In various embodiments, the present invention relates to chimeric targeting proteins comprising a targeting moiety that specifically recognizes and binds SIRP1α on TAM (tumor-associated macrophages).

[0018]In various embodiments, the present invention relates to chimeric targeting proteins comprising a targeting moiety that specifically recognizes and binds SIRP1α on dendritic cells, including but not limited to cDC2 and pDC.

[0019]In various embodiments, the chimeric protein of the invention comprises a targeting moiety having a recognition domain that recognizes SIRP1α. In one embodiment, the recognition domain recognizes one or more linear epitopes present on SIRP1α. As used herein, a linear epitope refers to any continuous amino acid sequence present in SIRP1α. In another embodiment, the recognition domain recognizes one or more conformational epitopes present on SIRP1α. As used herein, a conformational epitope refers to one or more stretches of amino acids (which may be discontinuous) that form a three-dimensional surface with features and/or shapes and/or tertiary structures that can be recognized by an epitope recognition domain. Antigen.

[0020]In some embodiments, the chimeric protein comprises a targeting moiety capable of binding to full-length and/or mature forms and/or isoforms and/or variants and/or splice fragments and/or any other analogues, variants, natural or synthetic. or SIRP1α mutants. In one embodiment, the SIRP1α is human SIRP1α. In various embodiments, the chimeric protein comprises a targeting moiety capable of binding to any form of human SIRP1α, including monomeric, dimeric, heterodimeric, multimeric, and associated forms. In one embodiment, the targeting moiety binds to the monomeric form of SIRP1α. In another embodiment, the targeting moiety binds to a dimeric form of SIRP1α.

[0021]In one embodiment, the present chimeric protein comprises a targeting moiety having a recognition domain that recognizes one or more epitopes present on human SIRP1α. In one embodiment, the targeting moiety comprises a recognition domain that recognizes human SIRP1α having a signal peptide sequence. An exemplary human SIRP1α polypeptide having a signal peptide sequence (underlined) is provided below:

[0022]In one embodiment, the targeting moiety comprises a recognition domain that recognizes human SIRP1α without a signal peptide sequence. An exemplary human SIRP1α polypeptide without a signal peptide sequence is provided below:

[0023]In one embodiment, the targeting moiety comprises a recognition domain that recognizes a polypeptide encoding human SIRP1α isoform 2:

[0024]In one embodiment, the targeting moiety comprises a recognition domain that recognizes a polypeptide encoding human SIRP1α isoform 4:

[0025]In various embodiments, the targeting moieties of the present invention can be any protein-based agent capable of specific binding, such as an antibody or derivatives thereof. In one embodiment, the targeting moiety comprises an antibody. In various embodiments, the antibody is a full-length, multimeric protein comprising two heavy chains and two light chains. Each heavy chain contains a variable region (e.g. V_H) and at least three constant regions (e.g. CH₁, CH₂and CH₃) and each light chain includes a variable region (V_L) and a constant region (C_L). The variable regions determine the specificity of the antibody. Each variable region includes three hypervariable regions, also known as complementarity determining regions (CDRs), flanked by four relatively conserved framework regions (FRs). The three CDRs, designated CDR1, CDR2 and CDR3, contribute to the binding specificity of the antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

[0026]In some embodiments, the targeting moiety comprises antibody derivatives or formats. In some embodiments, the targeting moiety of the present chimeric protein is a single domain antibody, a recombinant heavy chain (VHH) antibody, a single chain antibody (scFv), a shark heavy chain only (VNAR) antibody, a Microprotein (cysteine). knot protein, knottin), a DARPin; a tetranectin; an affibody; a transbody; an anticalina; an adnectin; an affilin; a microbody; a peptide aptamer; an alterase; plastic antibodies; a phyllomer; a strado body; a maxi body; an evibody; a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunebody, a triomab, a troybody; an animated body; a vaccibody, a unibody; Affimer, DuoBody, Fv, Fab, Fab′, F(ab′)₂B. a peptide mimetic molecule or a synthetic molecule as described in US patent numbers or patent publication numbers. No. 7,417,130, US 2004/132094, US Patent No. 5,831,012, US 2004/02334, US Patent No. 146938, US 2004/157209, US Patent No. 6,994,982, 6,794,144, US 2010/239633, US Patent 7.8 7.8 No. 7.8 US 2010/119446 and/or US Patent No. 7,166,697, the contents of which are incorporated herein by reference in their entirety. See also Storz MAbs. 2011 May-June; 3(3): 310-317.

[0027]In one embodiment, the targeting moiety comprises a single domain antibody such as VHH, for example from an organism that produces VHH antibodies such as camelids, sharks or genetically engineered VHH. VHHs are antibody-derived therapeutic proteins that share the unique structural and functional properties of naturally occurring heavy-chain antibodies. VHH technology is based on fully functional camelid antibodies that lack light chains. These heavy chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). VHHs are commercially available under the trademark NANOBODY or NANOBODIES.

[0028]In one embodiment, the addressing unit includes a VHH. In some embodiments, the VHH is a humanized VHH or a camel VHH.

[0029]In some embodiments, the VHH comprises a fully human V_Hdomain, e.g. to HUMABODY (Crescendo Biologics, Cambridge, UK). In some embodiments, V is fully human_Hdomain, e.g. A HUMAN BODY is monovalent, bivalent or trivalent. In some embodiments, the fully human V_HDomain, S. HUMABODY is monospecific or multispecific, as monospecific, bispecific or trispecific. Totally Human Illustrative V_Hdomains, e.g. a HUMABODIES are described, for example, in WO 2016/113555 and WO 2016/113557, the full description of which is incorporated by reference.

[0030]For example, in some embodiments, the chimeric protein of the invention comprises one or more antibodies, antibody derivatives or formats, peptides or polypeptides, VHH, or fusion proteins that selectively bind to SIRP1α. In some embodiments, the chimeric protein comprises a targeting moiety that is an antibody or derivative thereof that specifically binds SIRP1α. In some embodiments, the chimeric protein comprises a targeting moiety that is a camelid heavy chain (VHH) antibody that specifically binds to SIRP1α.

[0031]In some embodiments, the chimeric protein comprises a targeting moiety that is a VHH comprising a single amino acid chain with four "framework regions" or FRs and three "complementary determining regions" or CDRs. As used herein, "framework region" or "FR" refers to a region in the variable domain that lies between the CDRs. As used herein, "complementary determining region" or "CDR" refers to variable regions in VHH that contain amino acid sequences capable of specifically binding to antigenic targets. In various embodiments, the present chimeric protein comprises a VHH with a variable domain comprising at least one CDR1, CDR2 and/or CDR3 sequence.

[0032]In various embodiments, the targeting moieties of the invention may comprise any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR) and framework region sequences, of which known to recognize and bind to SIRP1α.

[0033]In various embodiments, the present technology contemplates the use of any natural or synthetic analog, mutant, variant, allele, homolog, and ortholog (collectively referred to herein as "analogs") of the SIRP1α targeting moiety described herein. In various embodiments, the amino acid sequence of the SIRP1α targeting residue further includes an amino acid analog, an amino acid derivative, or other non-classical amino acids.

[0034]In various embodiments, the chimeric protein comprises a targeting moiety comprising a sequence that is at least 60% identical to any sequence described herein. For example, the chimeric protein may comprise a targeting moiety comprising a sequence comprising at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 65% 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any of the sequences described herein (eg. B. about 60%, about 61%, about 62%, about 63%, about 64%, about 65% or about 66%, or about 67% or about 68% or about 69% or about 70% or about 71% or about 72% or about 73% or about 74% or about 75% or about 76%, or about 77% or about 78% or about 79% or about 80% or about 81% or about 82% or about 83% or about 84% or about 85% or about 86% or about 87% or about 88% or about 89% or about 90% or about 91% or about 92% or about 93% or about 94% or about 95% or about 96 % or about 97% or about 98%, about 99% or about 100% sequence identity with any of the sequences described herein).

[0035]In various embodiments, the present chimeric proteins comprise a targeting moiety comprising an amino acid sequence having one or more amino acid mutations relative to any targeting moiety sequence known to recognize and bind SIRP1α. In various embodiments, the present chimeric protein comprises a targeting moiety comprising an amino acid sequence having one, two, three, four, five, six, seven, eight, nine, ten, or fifteen, twenty, thirty, forty, or fifty amino acid mutations relative to any targeting residue sequence known to recognize and bind SIRP1α. In some embodiments, the one or more amino acid mutations can be selected independently of substitutions, insertions, deletions, and truncations.

[0036]In some embodiments, the amino acid mutations are amino acid substitutions and may include conservative and/or non-conservative substitutions.

[0037]"Conservative substitutions" can be made, for example, based on similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) Acid: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0038]As used herein, "conservative substitutions" are defined as replacing an amino acid with another amino acid listed within the same set of six standard amino acid groups shown above. For example, replacing Asp with Glu retains a negative charge on the polypeptide so modified. In addition, glycine and proline can be interchanged due to their ability to break α-helices.

[0039]As used herein, "non-conservative substitutions" are defined as replacing an amino acid with another amino acid listed in a group other than the six standard amino acid groups (1) through (6) shown above.

[0040]In various embodiments, the substitutions can also include non-classical amino acids. Examples of non-classical amino acids include selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-acid -diaminobutyric acid, α- Aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosm, citrulline, homocitrulline, cysteic acid , t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methyl amino acids, C α-methyl amino acids, Nα-methyl amino acids and amino acid analogues in general.

[0041]In various embodiments, the amino acid mutation can be present in the CDRs of the targeting moiety (e.g., the CDR1, CDR2, or CDR3 regions). Alternatively, the amino acid change may be in the framework (FR) regions of the targeting moiety (e.g., the FR1, FR2, FR3, or FR4 regions).

[0042]Modification of amino acid sequences can be performed using any technique known in the art, for example site-directed mutagenesis or PCR-based mutagenesis. Such techniques are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., 1989 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York. . . , New York, 1989.

[0043]In various embodiments, the mutations do not substantially reduce the ability of the present chimeric protein to specifically recognize and bind SIRP1α. In various embodiments, the mutations do not substantially reduce the ability of the present chimeric protein to specifically bind SIRP1α without functionally modulating (e.g., partially or completely neutralizing) SIRP1α.

[0044]In various embodiments, the binding affinity of the present chimeric protein is for the full-length and/or mature forms and/or isoforms and/or splice variants and/or fragments and/or monomeric and/or dimeric forms and/or any other naturally occurring forms that may exist or synthetic analogues, variants or mutants of SIRP1α by the equilibrium dissociation constant (K_D). In various embodiments, the present chimeric protein comprises a targeting moiety that binds to the full-length and/or mature forms and/or isoforms and/or splice variants and/or fragments and/or any other analogues, variants, natural or synthetic. or mutants (including monomeric and/or dimeric forms) of SIRP1α with a K_Dless than about 1 µM, about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 200 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM or about 5 nM or about 1 nM.

[0045]In various embodiments, the present chimeric protein comprises a targeting moiety that binds to, but does not functionally modulate, the antigen of interest, ie, SIRP1α. For example, in various embodiments, the targeting moiety of the chimeric protein simply targets the antigen, but does not substantially functionally modulate (e.g., substantially inhibit, reduce, or neutralize) a biological effect that the antigen has. In various embodiments, the targeting moiety of the present chimeric protein binds to an epitope that is physically distinct from an antigenic site important to its biological activity (e.g., the active site of an antigen).

[0046]In other embodiments, the present chimeric protein comprises a targeting moiety that binds to but functionally modulates the antigen of interest, ie, SIRP1α. For example, in various embodiments, the targeting moiety of the chimeric protein targets the antigen, ie, SIRP1α, and functionally modulates (e.g., inhibits, reduces, or neutralizes) a biological effect that the antigen has. Such binding along with functional modulation may find use in various embodiments of the present invention, including methods in which the present chimeric protein is used to recruit active immune cells to a needed site directly or indirectly via an effector antigen.

[0047]For example, in various embodiments, the present chimeric protein can be used to recruit macrophages directly or indirectly via SIRP1α to a tumor cell in a method of shrinking or eliminating a tumor (p. SIRP1α antigen recognition domain and a targeting moiety having a recognition domain) . (e.g. antigen recognition domain) directed against an antigen or a tumor receptor). There is evidence that tumor cells often upregulate CD47 binding to SIRP1α to avoid phagocytosis. Accordingly, in various embodiments, it may be desirable to recruit macrophages directly or indirectly to tumor cells and functionally inhibit, reduce, or neutralize the inhibitory activity of SIRP1α, resulting in phagocytosis of tumor cells by macrophages. In various embodiments, the present chimeric protein enhances phagocytosis of tumor cells or any other undesired cell by macrophages.

[0048]Therapeutic agents comprising the present chimeric protein

[0049]Chimeras and fusions with signaling substances

[0050]In various embodiments, the chimeric protein of the invention is part of a chimera or fusion with one or more signaling agents. Accordingly, the present invention provides chimeric or fusion proteins comprising, for example, an anti-SIRP1α moiety and one or more signaling molecules.

[0051]In various embodiments, the signaling agent is modified to have reduced affinity or activity for one or more of its receptors, allowing for attenuation of activity (including agonism or antagonism) and/or preventing non-specific signaling or undesired sequestration of the chimera receptor . or fusion protein. In various embodiments, the signaling agent is antagonistic in its natural form and has one or more mutations that decrease its antagonistic activity. In various embodiments, the signaling molecule is antagonistic due to one or more mutations, e.g. an agonistic signaling substance is converted into an antagonistic signaling substance, and the converted signaling substance optionally also has one or more mutations that reduce its antagonistic activity (for example as described in WO 2015/007520, the entire content of which is hereby incorporated by reference).

[0052]Accordingly, in various embodiments, the signaling molecule is a modified (e.g., mutant) form of the signaling agent having one or more mutations. In various embodiments, the modifications (e.g., mutations) enable the modified signaling molecule to exhibit one or more attenuated activities, such as one or more reduced binding affinity, reduced endogenous activity, and reduced specific bioactivity compared to unmodified or unmutated, i. H. the wild-type form of the signaling agent (e.g., comparing the same signaling agent in a wild-type versus a modified or mutant form). In some embodiments, mutations that weaken or reduce binding or affinity include mutations that substantially reduce or eliminate binding or activity. In some embodiments, mutations that weaken or reduce binding or affinity differ from those mutations that substantially reduce or abolish binding or activity. Accordingly, in various embodiments, the mutations allow the signaller to have increased security, e.g. reduced systemic toxicity, reduced side effects and reduced off-target effects compared to the unmutated, i. H. Wild-type signaling substance (e.g. comparison of the same signaling substance in a wild-type with a modified form (e.g. mutant)). As described in this document, due to one or more modifications, e.g. Mutations In various embodiments, improved safety means that the subject chimeric protein provides less toxicity (e.g., systemic toxicity and/or associated tissue/organ toxicity); and/or reduces or substantially eliminates side effects; and/or increased tolerability, reduced or substantially eliminated side effects; and/or reduces or substantially eliminates side effects; and/or an enlarged therapeutic window.

[0053]In various embodiments, the signaling agent is modified to have one or more mutations that reduce its binding affinity or activity for one or more of its receptors. In some embodiments, the signaling agent is modified to have one or more mutations that substantially reduce or abolish binding affinity or activity for receptors. In some embodiments, the activity provided by the wild-type signaling agent is agonism at the receptor (e.g., activation of a cellular effect at a therapeutic site). For example, the wild-type signaling substance can activate its receptor. In such embodiments, the mutations result in the modified signaling agent having reduced or eliminated activation activity in the receptor. For example, the mutations may result in the modified signaling agent delivering a reduced activation signal to a target cell, or the activation signal could be eliminated. In some embodiments, the activity provided by the wild-type signaling agent is receptor antagonism (e.g., blocking or attenuating a cellular effect at a therapeutic site). For example, the wild-type signaling agent can antagonize or inhibit the receptor. In these embodiments, the mutations result in the modified signaling agent having reduced or eliminated antagonizing activity in the recipient. For example, the mutations may result in the modified signaling agent delivering a reduced inhibitory signal to a target cell, or the inhibitory signal may be eliminated. In various embodiments, the signaling molecule is antagonistic due to one or more mutations, e.g. an agonistic signaling molecule is converted into an antagonistic signaling molecule (e.g. as described in WO 2015/007520, the entire contents of which are incorporated herein by reference), and optionally the converted signaling molecule also has one or more reducing mutations on its binding affinity or activity for one or more of its receptors or which substantially reduce or abolish its binding affinity or activity for one or more of its receptors.

[0054]In some embodiments, reduced affinity or activity at the receptor can be restored by ligation with one or more of the targeting moieties described herein (e.g., anti-SIRP1α targeting moiety or any other targeting moiety described herein). In other embodiments, the reduced affinity or activity at the receptor is substantially unrecoverable by the activity of one or more of the targeting moieties.

[0055]In various embodiments, the chimeric proteins of the present invention reduce off-target effects because their signaling molecules have mutations that weaken or abolish binding affinity or activity at a receptor. For example, in various embodiments, this reduction in side effects is observed relative to wild-type signaling agents. In various embodiments, the signaling agent is active in target cells because the targeting moiety(s) compensate for the missing/inadequate binding (e.g., unrestrained and/or greedy) required for substantial activation. In various embodiments, the modified signaling agent is essentially inactive en route to the site of therapeutic activity and acts essentially on specifically targeted cell types, greatly reducing undesirable side effects.

[0056]In some embodiments, the signaling agent may comprise one or more mutations that decrease or decrease binding or affinity for a receptor (i.e., a therapeutic receptor), and one or more mutations that significantly decrease or eliminate binding or activity at a second recipient. In such embodiments, these mutations may be at the same position or at different positions (ie, same mutation or multiple mutations). In some embodiments, the mutation(s) that reduce binding and/or activity at a receptor differ from the mutation(s) that reduce or eliminate in a substantially different recipient. In some embodiments, the mutation(s) that reduce(s) binding and/or activity at one receptor is/are the same as the mutation(s) that substantially reduce(s) in another recipient. In some embodiments, the present chimeric proteins feature a modified signaling molecule containing mutations that attenuate binding and/or activity at a therapeutic receptor, thus allowing for a more controlled therapeutic effect on the target (e.g., a relative wild-type signal substance). and mutations that substantially reduce or eliminate binding and/or activity at another receptor and thus reduce side effects (e.g., compared to wild-type signaling agent).

[0057]In some embodiments, a substantial reduction or ablation of binding or activity with a targeting moiety (e.g., a SIRP1α targeting moiety or any other targeting moiety described herein) cannot be substantially restored. In some embodiments, a substantial reduction or ablation of binding or activity can be restored with a targeting moiety. In various embodiments, substantially reducing or abolishing binding or activity at a second receptor may also prevent deleterious effects mediated by the other receptor. Alternatively or additionally, a substantial reduction or elimination of binding or activity at the other receptor enhances the therapeutic effect because there is reduced or eliminated sequestration of the therapeutic chimeric proteins away from the site of therapeutic effect. For example, in some embodiments, this avoids the need for high doses of the present chimeric proteins that compensate for the loss of the other receptor. Such an opportunity to further reduce the dose provides a lower likelihood of side effects.

[0058]In various embodiments, the modified signaling molecule comprises one or more mutations that cause the signaling molecule to have a reduced, substantially reduced, or eliminated affinity, e.g. bond (e.g. K_D) and/or activation (e.g. when the modified signaling substance is an agonist of its receptor, measurable as e.g. K_Aj/o CE₅₀) and/or inhibition (e.g. when the modified signaling substance is an antagonist of its receptor, measurable as e.g. K₁j/o CI₅₀), for one or more of its receptors. In various embodiments, reduced affinity at the signaling receptor allows for attenuation of activity (including agonism or antagonism). In such embodiments, the modified signaling substance is about 1% or about 3%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45% , about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 10% to 20%, about 20% -40% , about 50%, about 40%-60%, about 60%-80%, about 80%-100% affinity for the receptor relative to the wild-type signaling agent. In some embodiments, the binding affinity is at least about 2-fold lower, about 3-fold lower, about 4-fold lower, about 5-fold lower, about 6-fold lower, about 7-fold lower, about 8-fold lower, about 9 times lower, at least about 10 times lower, at least about 15 times lower, at least about 20 times lower, at least about 25 times lower, at least about 30 times lower, at least about 35 times lower, at least about 40 times less, at least about 45 times less, at least about 50 times less, at least about 100 times less, at least about 150 times less, or about 10-50 times less, about 50-100 times less, about 100-150 times less, about 150-200-fold less or more than 200-fold less relative to the wild-type signaling agent.

[0059]In embodiments where the chimeric protein comprises a modified signaling molecule having mutations that reduce binding to one receptor and substantially reduce or eliminate binding to a second receptor, the attenuation or reduction in binding affinity of the modified signaling molecule for a receptor is less as the substantial reduction or abolition of affinity for the other receptor. In some embodiments, the attenuation or reduction in binding affinity of the modified signaling agent for one receptor by about 1%, or about 3%, is about 5% less than the substantial reduction or ablation in affinity for the other receptor. about 10%. , about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95%. In various embodiments, a substantial reduction or ablation refers to a greater reduction in binding activity and/or affinity than an attenuation or reduction.

[0060]In various embodiments, the modified signaling molecule comprises one or more mutations that reduce the endogenous activity of the signaling molecule to about 75%, about 70%, about 60%, about 50%, about 40%, or about 30%. B. % or about 25% or about 20% or about 10% or about 5% or about 3% or about 1% based on the signal substance wild type.

[0061]In some embodiments, the modified signaling molecule comprises one or more mutations that cause the signaling molecule to have a reduced affinity for its receptor that is less than the binding affinity of the targeting moiety(s) for its recipient(s). In some embodiments, this binding affinity differential exists between the signaling agent/receptor and the targeting moiety/receptor in the same cell. In some embodiments, this binding affinity differential allows the signaling agent, e.g. mutated signaling agent to have localized on-target effects and minimize off-target effects underlying the side effects observed with the wild-type signaling agent. In some embodiments, this binding affinity is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold less, or at least about 25-fold or equal to at least about 50-fold less, or at least 100-fold or at least 150 times smaller.

[0062]Receptor binding activity can be measured using methods known in the art. For example, binding affinity and/or activity can be determined by Scatchard plot analysis and computational fitting of the binding data (e.g. Scatchard, 1949) or by reflectometric interference spectroscopy under continuous flow conditions as described by Brecht et al. (1993), the entire contents of which are incorporated herein by reference.

[0063]In various embodiments, the signaling agent is an immunomodulatory agent, e.g. one or more of interleukin, interferon and tumor necrosis factor.

[0064]In some embodiments, the signaling agent is an interleukin or a modified interleukin including, for example, IL-1; IL-2; IL-3; IL-4; IL-5; IL-6; IL-7; IL-8; IL-9; IL-10; IL-11; IL-12; IL-13; IL-14; IL-15; IL-16; IL-17; IL-18; IL-19; IL-20; IL-21; IL-22; IL-23; IL-24; IL-25; IL-26; IL-27; IL-28; IL-29; IL-30; IL-31; IL-32; IL-33; IL-35; IL-36 or a fragment, variant, analogue or relative thereof. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes and macrophages. Known functions include stimulating proliferation of immune cells (eg, helper T cells, B cells, eosinophils, and lymphocytes), chemotaxis of neutrophils and T lymphocytes, and/or inhibition of interferons. Interleukin activity can be determined using assays known in the art: Matthews et al., inLymphokines and interferons: a practical approach, Clemens et al., eds., IRL Press, Washington, DC. 1987, pp. 221-225; and Orencole & Dinarello (1989) Cytokine 1 , 14-20.

[0066]In some embodiments, the signaling agent is tumor necrosis factor (TNF) or a modified version of tumor necrosis factor (TNF) or a protein of the TNF family including but not limited to TNF-α, TNF-β, LT-β, ​​CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L and TRAIL.

[0067]The amino acid sequences of the wild-type signaling agents described herein are well known in the art. Accordingly, in various embodiments, the modified signaling substance comprises an amino acid sequence that is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67% or at least about 68% or at least about 69% or at least about at least about 70% or at least about 71% or at least about 72% or at least about 73% or at least about 74% or at least about 75% or at least about 76% or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86% , or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95% or at least about 96% or at least about 97% or at least about 98% or at least about 99% sequence identity to known wild-type sequences of amino acids of the signaling substances described herein (e.g. about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69% or about 70%, about 71%, about 72% %, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79% or about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89% or about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97 %, about 98%, about 99% sequence identity).

[0068]In various embodiments, the modified signaling agent comprises an amino acid sequence that is at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 63%, about 64%, or at least at least 65%, or at least 66% or at least 67% or at least 68% or at least 69% or at least 70% or at least 71% or at least about 72% or at least about 73% or at least about 74% or at least about 75% or at least about 76% or at least about 77% or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87% , or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% sequence identity with any amino acid sequence of the signaling substances described herein (eg. about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69% or about 70%, about 71%, about 72% %, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79% or about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89% or about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97 %, about 98%, about 99% sequence identity).

[0069]In various embodiments, the modified signaling agent comprises an amino acid sequence that has one or more amino acid mutations. In some embodiments, the one or more amino acid mutations can be selected independently of substitutions, insertions, deletions, and truncations. In some embodiments, amino acid mutations are amino acid substitutions and may include conservative and/or non-conservative substitutions as described elsewhere herein.

[0070]In various embodiments, substitutions may also include non-classical amino acids, as described elsewhere herein.

[0071]As described herein, modified signaling agents carry mutations that affect affinity and/or activity at one or more receptors. In various embodiments, there is reduced affinity and/or activity at a therapeutic receptor, e.g. a receptor through which a desired therapeutic effect is mediated (e.g., agonism or antagonism). In various embodiments, the modified signaling substances carry mutations that affect affinity and/or activity at a receptor, e.g. a receptor through which a desired therapeutic effect is not mediated (e.g., as a result of junctional promiscuity). Receptors for any signaling substance as described herein are known in the art.

[0072]Illustrative mutations providing affinity and/or activity can be found in WO 2013/107791 and PCT/EP2017/061544 (relating to interferons for example), WO 2015/007542 (relating to interleukins for example). agonist) at a receptor. and WO 2015/007903 (e.g. relating to TNF), the entire content of which is incorporated herein by reference. Illustrative mutations providing affinity and/or reduced activity (e.g., antagonist) at a therapeutic receptor can be found in WO 2015/007520, the entire content of which is incorporated herein by reference.

[0073]In some embodiments, the modified signaling agent comprises one or more mutations that cause the signaling agent to have reduced affinity and/or activity for a type I cytokine receptor, a type II cytokine receptor, a chemokine receptor, a tumor necrosis factor receptor (TNFR) superfamily, TGF-beta receptors, a receptor in the immunoglobulin (Ig) superfamily, and/or a receptor in the tyrosine kinase superfamily.

[0074]In various embodiments, the receptor for the signaling agent is a type I cytokine receptor. Type I cytokine receptors are known in the art and include, but are not limited to, receptors for IL2 (beta subunit), IL3, IL4, IL5, IL6 , IL7, IL9, IL11, IL12, GM-CSF, G-CSF, LIF, CNTF, as well as thrombopoietin (TPO), prolactin and growth hormone receptors. Illustrative Type I cytokine receptors include, but are not limited to, GM-CSF receptor, G-CSF receptor, LIF receptor, CNTF receptor, TPO receptor, and Type I IL receptors.

[0075]In various embodiments, the receptor for the signaling agent is a type II cytokine receptor. Type II cytokine receptors are multimeric receptors composed of heterologous subunits and are primarily receptors for interferons.

[0076]This family of receptors includes, inter alia, receptors for interferon-.alpha., interferon-.beta. and interferon-γ, IL10, IL22 and tissue factor. Illustrative Type II cytokine receptors include, but are not limited to, IFN-α receptor (e.g., IFNAR1 and IFNAR2), IFN-β receptor, IFN-γ receptor (e.g., IFNGR1 and IFNGR2), and IL type II receptors.

[0077]In various embodiments, the receptor for the signaling agent is a G protein-coupled receptor. Chemokine receptors are G protein-coupled receptors with seven transmembrane structures and are G protein-coupled for signal transduction. Chemokine receptors include, but are not limited to, CC chemokine receptors, CXC chemokine receptors, CX3C chemokine receptors, and XC chemokine receptors (XCR1). Examples of chemokine receptors include CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CSCR6, CXCR7, XCR1, and CX3CR1.

[0078]In various embodiments, the signaling receptor is a member of the TNFR family. Members of the tumor necrosis factor receptor (TNFR) family share a cysteine-rich domain (CRD) composed of three disulfide bonds surrounding a central CXXCXXC motif, creating an elongated molecule. Exemplary members of the tumor necrosis factor receptor family include: CDI 20a (TNFRSFIA), CD 120b (TNFRSFIB), lymphotoxin receptor beta (LTBR, ​​TNFRSF3), CD 134 (TNFRSF4), CD40 (CD40, TNFRSF5), FAS (FAS, TNFRSF6), TNFRSF6B (TNFRSF6B), CD27 (CD27, TNFRSF7), CD30 (TNFRSF8), CD137 (TNFRSF9), TNFRSFIOA (TNFRSFIOA), TNFRSFIOB, (TNFRSFIOB), TNFRSFIOC (TNFRSFIOC), TNFRSFIOD (TNFRSFIOD), RANK (TNFRSFI IA), osteoprotegerin (TNFRSF12A), TNFRSF12A (TNFRSF12A), TNFRSF13B (TNFRSF13B), TNFRSF13C (TNFRSF13C), TNFRSF14 (TNFRSF14), TNFRSF21 (TNFRSF21) and TNFRSF25 (TNFRSF25). In one embodiment, the TNFR family member is CD120a (TNFRSF1A) or TNF-R1. In another embodiment, the TNFR family member is CD120b (TNFRSFIB) or TNF-R2.

[0079]In various embodiments, the signaling receptor is a TGF-beta receptor. TGF-beta receptors are single-stage serine/threonine kinase receptors. TGF-beta receptors include, but are not limited to, TGFBR1, TGFBR2, and TGFBR3.

[0080]In various embodiments, the receptor for the signaling agent is a receptor of the Ig superfamily. Immunoglobulin (Ig) superfamily receptors share structural homology with immunoglobulins. Ig superfamily receptors include, but are not limited to, receptors for interleukin-1, CSF-1R, PDGFR (eg, PDGFRA and PDGFRB), and SCFR.

[0081]In various embodiments, the receptor for the signaling agent is a tyrosine kinase superfamily receptor. The receptor tyrosine kinase superfamily is well known in the art. There are approximately 58 known receptor tyrosine kinases (RTKs), classified into 20 subfamilies. Tyrosine kinase superfamily receptors include, but are not limited to, FGF receptors and their various isoforms, such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5.

[0082]In some embodiments, the modified signaling agent is alpha interferon. In such embodiments, the modified IFN-α agent has reduced affinity and/or activity for the IFN-α/β receptor (IFNAR), ie, IFNAR1 and/or IFNAR2 chains. In some embodiments, the modified IFN-α agent has substantially reduced or suppressed affinity and/or activity for the IFN-α/13 receptor (IFNAR), ie, IFNAR1 and/or IFNAR2 chains.

[0083]Mutant forms of α-interferon are known to those skilled in the art. In an illustrative embodiment, the modified signaling agent is the IFN-α2a allelic form having the amino acid sequence of SEQ ID NO: 46.

[0084]In an illustrative embodiment, the modified signaling agent is the allelic form IFN-α2b having the amino acid sequence of SEQ ID NO: 47 (differing from IFN-α2a at amino acid position 23).

[0085]In some embodiments, the IFN-α2 mutant (IFN-α2a or IFN-α2b) is mutated at one or more amino acids at positions 144-154, such as amino acid positions 148, 149, and/or 153. In some embodiments, the IFN-α2 α2 mutant comprises one or more mutations selected from L153A, R149A and M148A. Such mutants are described, for example, in WO2013/107791 and Piehler et al., (2000) J. Biol. Chem, 275:40425-33, the entire content of which is incorporated herein by reference.

[0086]In some embodiments, IFN-α2 mutants have reduced affinity and/or activity for IFNAR1. In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A and Y89A as described in WO2010/030671, the entire content of which is incorporated herein by reference.

[0087]In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from K133A, R144A, R149A and L153A as described in WO2008/124086, the entire content of which is incorporated herein by reference.

[0088]In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from R120E and R120E/K121E as described in WO2015/007520 and WO2010/030671, the entire content of which is incorporated herein by reference. In these embodiments, the mutant IFN-α2 antagonizes the activity of wild-type IFN-α2. In such embodiments, the mutant IFN-α2 has reduced affinity and/or activity for IFNAR1 while retaining IFNR2 affinity and/or activity.

[0089]In some embodiments, the human IFN-α2 mutant comprises (1) one or more selected R120E and R120E/K121E mutations that, without wishing to be bound by theory, produce an antagonistic effect, and (2) one or more selected K133A mutations B. R144A, R149A and L153A which, without wishing to be bound by theory, allow for an attenuated effect in IFNAR2, for example. In one embodiment, the human IFN-α2 mutant comprises R120E and L153A.

[0090]In some embodiments, the human IFN-α2 mutant comprises one or more mutations selected from L15A, A19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, D114R , L117A, R120A, R125A, K134A, R144A, A145G, A145M, M148A, R149A, S152A, L153A and N156A as described in WO 2013/059885, the full descriptions of which are incorporated herein by reference. In some embodiments, the human IFN-α2 mutant comprises the H57Y, E58N, Q61S and/or L30A mutations as described in WO 2013/059885. In some embodiments, the human IFN-α2 mutant comprises the H57Y, E58N, Q61S and/or R33A mutations as described in WO 2013/059885. In some embodiments, the human IFN-α2 mutant comprises the H57Y, E58N, Q61S and/or M148A mutations as described in WO 2013/059885. In some embodiments, the human IFN-α2 mutant comprises the H57Y, E58N, Q61S and/or L153A mutations as described in WO 2013/059885. In some embodiments, the human IFN-α2 mutant comprises the N65A, L80A, Y85A and/or Y89A mutations as described in WO 2013/059885. In some embodiments, the human IFN-α2 mutant comprises the N65A, L80A, Y85A, Y89A and/or D114A mutations as described in WO 2013/059885. In some embodiments, the human IFN-α2 mutant comprises one or more selected R144X mutations.₁, A145X₂, and R33A, where X is selected from A, S, T, Y, L and I, and where X₂is selected from G, H, Y, K and D.

[0091]In some embodiments, the modified signaling agent is β-interferon. In such embodiments, the modified interferon-β agent has reduced affinity and/or activity for the IFN-α/β receptor (IFNAR), ie, the IFNAR1 and/or IFNAR2 chains. In some embodiments, the modified interferon-β agent has substantially reduced or eliminated affinity and/or activity for the IFN-α/β receptor (IFNAR), ie, the IFNAR1 and/or IFNAR2 chains.

[0092]In one embodiment, the modified signaling agent is β-interferon. In such embodiments, the modified interferon-β agent has reduced affinity and/or activity for the IFN-α/β receptor (IFNAR), ie, the IFNAR1 and/or IFNAR2 chains. In some embodiments, the modified interferon-1 agent has substantially reduced or suppressed affinity and/or activity for the IFN-α/β receptor (IFNAR), ie, the IFNAR1 and/or IFNAR2 chains.

[0093]In an illustrative embodiment, the modified signaling agent is IFN-β. In various embodiments, IFN-β includes functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of IFN-β. In various embodiments, IFN-β includes IFN-β derived from any species. In one embodiment, the chimeric protein comprises a modified version of the mouse IFN-R. In another embodiment, the chimeric protein comprises a modified version of human IFN-β. Human IFN-β is a polypeptide with a molecular weight of approximately 22 kDa, comprising 166 amino acid residues. The amino acid sequence of human IFN-β is SEQ ID NO: 48.

[0094]In some embodiments, the human IFN-? IFN-?-1a, which is a glycosylated form of human IFN-? is. In some embodiments, the human IFN-? IFN-?-1b, which is a non-glycosylated form of human IFN-? with a deletion of Met-1 and a mutation of Cys-17 to Ser.

[0095]In various embodiments, the modified IFN-β has one or more mutations that reduce its binding or affinity for the IFNAR1 subunit of IFNAR. In one embodiment, the modified IFN-β has reduced affinity and/or activity towards IFNAR1. In various embodiments, the modified IFN-β is human IFN-β and has one or more mutations at positions F67, R71, L88, Y92, 195, N96, K123 and R124. In some embodiments, one or more mutations are selected substitutions of F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, 195A, N96G, K123G, and R124G. In one embodiment, the modified IFN-β comprises the F67G mutation. In one embodiment, the modified IFN-β comprises the K123G mutation. In one embodiment, the modified IFN-P comprises the F67G and R71A mutations. In one embodiment, the modified IFN-β comprises the L88G and Y92G mutations. In one embodiment, the modified IFN-β comprises the Y92G, 195A and N96G mutations. In one embodiment, the modified IFN-β includes the K123G and R124G mutations. In one embodiment, the modified IFN-β includes the F67G, L88G, and Y92G mutations. In one embodiment, the modified IFN-β includes the F67S, L88S, and Y92S mutations.

[0096]In some embodiments, the modified IFN-β has one or more mutations that reduce its binding or affinity for the IFNAR2 subunit of IFNAR. In one embodiment, the modified IFN-β has reduced affinity and/or activity towards IFNAR2. In various embodiments, the modified IFN-β is human IFN-β and has one or more mutations at positions W22, R27, L32, R35, V148, L151, R152 and Y155. In some embodiments, one or more mutations are selected substitutions of W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, and Y155G. In one embodiment, the modified IFN-β comprises the W22G mutation. In one embodiment, the modified IFN-β comprises the L32A mutation. In one embodiment, the modified IFN-β comprises the L32G mutation. In one embodiment, the modified IFN-β comprises the R35A mutation. In one embodiment, the modified IFN-β comprises the R35G mutation. In one embodiment, the modified IFN-β comprises the V148G mutation. In one embodiment, the modified IFN-β comprises the R152A mutation. In one embodiment, the modified IFN-β comprises the R152G mutation. In one embodiment, the modified IFN-β comprises the Y155G mutation. In one embodiment, the modified IFN-R includes the W22G and R27G mutations. In one embodiment, the modified IFN-β comprises the L32A and R35A mutation. In one embodiment, the modified IFN-β comprises the L151G and R152A mutations. In one embodiment, the modified IFN-β comprises the V148G and R152A mutations.

[0097]In some embodiments, the modified IFN-β has one or more of the following mutations: R35A. R35T, E42K, M21, G78S, A141Y, A142T, E149K and R152H. In some embodiments, the modified IFN-β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H in combination with C17S or C17A.

[0098]In some embodiments, the modified IFN-β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H in combination with any of the other IFN-β mutations described Document.

[0099]The crystal structure of human IFN-β is known and is described in Karpusas et al., (1998) PNAS, 94(22):11813-11818. In particular, the structure of human IFN-β has been shown to include five α-helices (ie, A, B, C, D, and E) and four loop regions connecting these helices (ie, AB, BC, CD, and loops). FROM). In various embodiments, the modified IFN-β has one or more mutations in the A, B, C, D, E helices and/or AB, BC, CD and DE loops that affect its binding affinity or decrease activity at a therapeutic receptor. . as IFNAR. Exemplary mutations are described in WO2000/023114 and US20150011732, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the modified IFN-β is human IFN-β comprising alanine substitutions at amino acid positions 15, 16, 18, 19, 22 and/or 23. In an exemplary embodiment, the modified IFN-β is human IFN-β comprising alanine substitutions at amino acid positions 28-30, 32, and 33. In an exemplary embodiment, the modified IFN-β is human IFN-β comprising alanine substitutions at amino acid positions 36, 37, 39, and 42. An exemplary embodiment is the modified IFN-β is human IFN-β comprising alanine substitutions at amino acid positions 64 and 67 and a serine substitution at position 68. In an exemplary embodiment, the IFN-β-modified β is human IFN-β comprising alanine substitutions at amino acid positions 71-73. In an exemplary embodiment, the modified IFN-β is human IFN-β comprising alanine substitutions at amino acid positions 92, 96, 99 and 100. In an exemplary embodiment, the modified IFN-β is human IFN-β comprising alanine substitutions at amino acid positions 128, 130, 131 and 134. In an exemplary embodiment, the modified IFN-β is human IFN-β comprising alanine substitutions at amino acid positions 149, 153, 156 and 159. In some embodiments, the mutant IFNβ comprises SEQ ID NO:48 and a mutation in W22, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V). .

[0100]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in R27, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0101]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in W22, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V) and a mutation in R27, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V ). ).

[0102]In some embodiments, the mutated IFNβ comprises SEQ ID NO: 48 and a mutation in L32, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine . (V).

[0103]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in R35, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0104]In some embodiments, the mutated IFNβ comprises SEQ ID NO: 48 and a mutation in L32, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine . (V) and a mutation in R35, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0105]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in F67, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0106]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in R71, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0107]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in F67, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V) and a mutation in R71, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V ). ).

[0108]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in L88, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine . (V). In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in Y92, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0109]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in F67, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V) and a mutation in L88, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M) and valine (V) and a mutation in Y92, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0110]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in L88, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine . (V) and a mutation in Y92, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0111]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation at 195, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine . (V) and a mutation in Y92, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0112]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in N96, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V) and a mutation in Y92, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V ). ).

[0113]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in Y92, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V) and a mutation in 195, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M) and valine (V) and a mutation in N96, the mutation being an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0114]In some embodiments, the mutated IFNβ comprises SEQ ID NO: 48 and a mutation in K123, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0115]In some embodiments, the mutated IFNβ comprises SEQ ID NO: 48 and a mutation in R124, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0116]In some embodiments, the mutated IFNβ comprises SEQ ID NO: 48 and a mutation in K123, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V) and a mutation in R124, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V ).

[0117]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in L151, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M) and valine . (V).

[0118]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in R152, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0119]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in L151, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M) and valine . (V) and a mutation in R152, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0120]In some embodiments, the mutated IFNβ comprises SEQ ID NO: 48 and a mutation in V148, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), and methionine ( METER).

[0121]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in V148, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V) and a mutation in R152, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V ). ).

[0122]In some embodiments, the mutant IFNβ comprises SEQ ID NO: 48 and a mutation in Y155, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

[0123]In some embodiments, the present invention relates to a chimeric protein comprising: (a) a modified IFN-β having the amino acid sequence of SEQ ID NO: 48 and a mutation at position W22, wherein the mutation is a hydrophobic aliphatic residue; and (b) one or more targeting moieties, wherein the targeting moieties comprise recognition domains that specifically bind to antigens or receptors of interest (e.g., Clec9A), the modified IFN-β, and one or more targeting moieties optionally with a or more linkers are connected. In various embodiments, the mutation at position W22 is an aliphatic hydrophobic residue selected from G, A, L, I, M and V. In various embodiments, the mutation at position W22 is G.

[0124]Additional illustrative IFNβ mutants are provided in PCT/EP2017/061544, the full description of which is incorporated herein by reference.

[0125]In some embodiments, the modified signaling agent is interferon-gamma. In such embodiments, the modified interferon-gamma agent has reduced affinity and/or activity for the interferon-gamma receptor (IFNGR), ie, IFNGR1 and IFNGR2 chains. In some embodiments, the modified interferon-γ agent has a substantially reduced or suppressed affinity and/or activity for the interferon-γ receptor (IFNGR), ie, IFNGR1 and/or IFNGR2 chains.

[0126]In some embodiments, the modified signaling agent is a consensus interferon. Consensus interferon is generated by scanning the sequences of various non-allelic human IFN-α subtypes and assigning the most frequently observed amino acid at each corresponding position. Consensus interferon differs from IFN-α2b in 20 of 166 amino acids (88% homology), and comparison with IFN-β shows identity at more than 30% amino acid positions. In various embodiments, the consensus interferon comprises the following amino acid sequence of SEQ ID NO: 49.

[0127]In some embodiments, the consensus interferon comprises the amino acid sequence of SEQ ID NO: 50, which differs from the amino acid sequence of SEQ ID NO: 49 by one amino acid, ie SEQ ID NO: 50 lacks the methionine residue SEQ ID NO: 49.

[0128]In various embodiments, the consensus interferon comprises a modified version of the consensus interferon, ie a consensus interferon variant, as a signaling substance. In various embodiments, the consensus interferon variant includes functional derivatives, analogs, precursors, isoforms, splice variants, or fragments of the consensus interferon.

[0129]In one embodiment, the consensus interferon variants are selected from the consensus interferon variants described in US Patent Nos. 4,695,623, 4,897,471, 5,541,293 and 8,496,921, the entire contents of which are incorporated herein by reference. For example, the consensus interferon variant may include the amino acid sequence of IFN-CON₂o IFN-CON₃as described in U.S. Patent Nos. 4,695,623, 4,897,471 and 5,541,293. In one embodiment, the consensus interferon variant comprises the amino acid sequence of IFN-CON₂(SEQ ID NO: 51).

[0130]In one embodiment, the consensus interferon variant comprises the amino acid sequence of IFN-CON₃(SEQ ID NO: 52).

[0131]In one embodiment, the consensus interferon variant comprises the amino acid sequence of any of the variants described in US Patent No. 8,496,921. For example, the consensus variant may include the amino acid sequence of SEQ ID NO:53.

[0132]In another embodiment, the consensus interferon variant may comprise the amino acid sequence of SEQ ID NO:54.

[0133]In some embodiments, the consensus interferon variant may be PEGylated, ie it comprises a PEG moiety. In one embodiment, the consensus interferon variant may comprise a PEG moiety linked to position S156C of SEQ ID NO:54.

[0134]In some embodiments, the engineered interferon is a variant human IFN-α2a with an Asp insertion at approximately position 41 in the sequence Glu-Glu-Phe-Gly-Asn-Gln (SEQ ID NO: 275) to convert to Glu-Glu produce -Phe-Asp-Gly-Asn-Gln (SEQ ID NO:276) (resulting in a sequence renumbering relative to the IFN-α2a sequence) and the following Arg23Lys, Leu26Pro, Glu53Gln, Thr54Ala mutations, Pro56Ser, Asp86Glu, Ile104Thr, Gly106Glu B. Thr110Glu, Lys117Asn, Arg125Lys and Lys136Thr. All embodiments describing consensus interferons herein apply equally to this designed interferon.

[0135]In various embodiments, the consensus interferon variant comprises an amino acid sequence that has one or more amino acid mutations. In some embodiments, the one or more amino acid mutations can be selected independently of substitutions, insertions, deletions, and truncations.

[0136]In some embodiments, the amino acid mutations are amino acid substitutions and may include conservative and/or non-conservative substitutions.

[0137]In various embodiments, substitutions may also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine-β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of the common amino acids, 2,4 -Diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosmus, citrulline , homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methyl amino acids, C α-methyl amino acids, Nα-methyl amino acids and amino acid analogues in general).

[0138]In various embodiments, the consensus interferon is modified to have one or more mutations. In some embodiments, the mutations allow the consensus interferon variant to have one or more attenuated activities, such as one or more reduced binding affinity, reduced endogenous activity, and reduced specific bioactivity compared to the unmutated form, e.g. B. Wild-type consensus interferon (e.g., the consensus interferon having an amino acid sequence of SEQ ID NO: 49 or 50). For example, one or more of attenuated activity, such as reduced binding affinity, reduced endogenous activity, and reduced specific bioactivity relative to non-mutated, e.g. the wild-type form of the consensus interferon can be present in a therapeutic receptor such as IFNAR. Accordingly, in various embodiments, the mutations allow the consensus interferon variant to have reduced systemic toxicity, reduced side effects, and reduced off-target effects relative to the unmutated, e.g. the wild-type form of consensus interferon.

[0139]In various embodiments, the consensus interferon is modified to have a mutation that reduces its binding affinity or activity at a therapeutic receptor, such as IFNAR. In some embodiments, the activity provided by the consensus interferon is agonism at the therapeutic receptor (e.g., activation of a cellular effect at a therapeutic site). For example, consensus interferon can activate the therapeutic receptor. In such embodiments, the mutation results in the consensus interferon variant having reduced activation activity at the therapeutic receptor.

[0140]In some embodiments, reduced affinity or activity at the therapeutic receptor can be restored by ligation with a targeting moiety (e.g., SIRPα). In other embodiments, reduced affinity or activity at the therapeutic receptor cannot be substantially restored by binding to the targeting moiety. In various embodiments, the therapeutic chimeric proteins of the present invention reduce off-target effects because the consensus interferon variant has mutations that weaken binding affinity or activity at a therapeutic receptor. In various embodiments, this reduces the side effects seen with consensus wild-type interferon, for example. In various embodiments, the consensus interferon variant is essentially inactive en route to the site of therapeutic activity and acts essentially on specifically targeted cell types, thereby greatly reducing undesirable side effects.

[0141]In various embodiments, the consensus interferon variant has one or more mutations that cause the consensus interferon variant to have an attenuated or reduced affinity, e.g. bond (e.g. K_D) and/or activation (measurable as e.g. K_Aj/o CE₅₀) for one or more therapeutic receptors. In various embodiments, decreased affinity at the therapeutic receptor allows for a decrease in therapeutic receptor activity and/or signaling.

[0142]In various embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity for the IFNAR1 subunit of IFNAR. In one embodiment, the consensus interferon variant has reduced affinity and/or activity at IFNAR1. In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity for the IFNAR2 subunit of IFNAR. In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity for the IFNAR1 and IFNAR2 subunits.

[0143]In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity for IFNAR1 and one or more mutations that substantially reduce or abolish its binding or affinity for IFNAR2. In some embodiments, chimeric proteins with such a consensus interferon variant can provide target-selective IFNAR1 activity (e.g., IFNAR1 activity can be restored by targeting by the target moiety, e.g., SIRPα).

[0144]In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity for IFNAR2 and one or more mutations that substantially reduce or eliminate its binding or affinity for IFNAR1. In some embodiments, chimeric proteins with the consensus interferon variant can provide target-selective IFNAR2 activity (e.g., IFNAR2 activity can be restored by targeting by the target moiety, e.g., SIRPα).

[0145]In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity for IFNAR1 and one or more mutations that reduce its binding or affinity for IFNAR2. In some embodiments, chimeric proteins with such a consensus interferon variant can provide target-selective IFNAR1 and/or IFNAR2 activity (e.g., IFNAR1 and/IFNAR2 activity can be mediated by targeting by the target moiety, e.g. SIRPα ) to be restored.

[0146]In some embodiments, the consensus interferon is modified to have a mutation in one or more amino acids at positions 145-155, such as amino acid positions 149, 150 and/or 154 relative to SEQ ID NO: 50. In some In embodiments, the consensus interferon is modified to have a mutation in one or more amino acids at positions 145-155, such as amino acid positions 149, 150 and/or 154, relative to SEQ ID NO: 50, with substitutions being optional hydrophobic and selected from alanine, valine, leucine and isoleucine. In some embodiments, the consensus interferon mutant comprises one or more mutations selected from M149A, R150A and L154A and with reference to SEQ ID NO: 50.

[0147]In one embodiment, the consensus interferon is modified to have a mutation at amino acid position 121 (ie, K121), referring to SEQ ID NO:50. In one embodiment, the consensus interferon comprises a K121E mutation referenced to SEQ ID NO: 50.

[0148]In various embodiments, the modified signaling agent is selected from modified versions of cytokines, growth factors and hormones. Illustrative examples of such cytokines, growth factors and hormones include lymphokines, monokines, conventional polypeptide hormones such as human growth hormone, human N-methionyl growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; Insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH); liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-α and tumor necrosis factor-β; Müllerian inhibitor; mouse gonadotropin-associated peptide; inhibin; activine; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-α; platelet growth factor; transforming growth factors (TGFs) such as TGF-α and TGF-β; insulin-like growth factor-I and -II; osteoinductive factors; interferons such as interferon-α, interferon-β and interferon-γ (and interferon types I, II and III), colony stimulating factors (CSF) such as macrophage CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte CSF (G-CSF); Interleukins (IL) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL -11, IL-12, IL-13 and IL-18; a tumor necrosis factor such as TNF-α or TNF-3; and other polypeptide factors including, for example, LIF and Kit Ligand (KL). As used herein, cytokines, growth factors and hormones include proteins obtained from natural sources or produced from recombinant bacterial, eukaryotic or mammalian cell culture systems, and biologically active equivalents of the sequence cytokines.

[0149]In some embodiments, the modified signaling agent is a modified version of a growth factor selected from, among other things, transforming growth factors (TGFs) such as TGF-α and TGF-β (and subtypes thereof, including the various subtypes of TGF-β, including TGFβ1, TGFβ2 and TGFβ3 ), epidermal growth factor (EGF), insulin-like growth factor, such as insulin-like growth factor I and II, fibroblast growth factor (FGF), heregulin, platelet growth factor (PDGF) B. vascular endothelial growth factor (VEGF).

[0150]In one embodiment, the growth factor is a modified version of a fibroblast growth factor (FGF). Illustrative FGFs include, but are not limited to, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, mouse FGF15, FGF16, FGF17, FGF18, FGF19, FGF20 . B. FGF21, FGF22 and FGF23.

[0151]In some embodiments, the modified signaling agent is vascular endothelial growth factor (VEGF). VEGF is a potent growth factor that plays an important role in both physiological and pathological angiogenesis, regulates vascular permeability, and can act as a growth factor in cells expressing VEGF receptors. Additional functions include, but are not limited to, stimulating cell migration in the macrophage lineage and endothelial cells. There are several members of the VEGF family of growth factors, as well as at least three receptors (VEGFR-1, VEGFR-2, and VEGFR-3). VEGF family members can bind to and activate more than one type of VEGFR. For example, VEGF-A binds VEGFR-1 and -2, while VEGF-C can bind VEGFR-2 and -3. Activation of VEGFR-1 and -2 regulates angiogenesis, while activation of VEGFR-3 is associated with lymphangiogenesis. The main proangiogenic signal is generated by activation of VEGFR-2. It has been reported that VEGFR-1 activation may be associated with a negative role in angiogenesis. It has also been reported that VEGFR-1 signaling is important for tumor progression in vivo via bone marrow-derived VEGFR-1 positive cells (contributing to the formation of a pre-metastatic niche in bone). Various therapies based on neutralization/targeting of therapeutic antibodies against VEGF-A have been developed, mainly for use in the treatment of various angiogenesis-dependent human tumors. However, these are not without side effects. This may not be surprising given that these act as general non-cell/tissue specific inhibitors of the VEGF/VEGFR interaction. Therefore, it would be desirable to restrict VEGF (e.g., VEGF-A)/VEGFR-2 inhibition to specific target cells (e.g., endothelial cells of tumor vasculature).

[0152]In some embodiments, the VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E and its isoforms, including the various VEGF-A isoforms such as VEGF.₁₂₁, VEGF₁₂₁b, VEGF₁₄₅, VEGF₁₆₅, VEGF₁₆₅b, VEGF₁₈₉and VEGF₂₀₆. In some embodiments, the modified signaling agent has reduced affinity and/or activity for VEGFR-1 (Flt-1) and/or VEGFR-2 (KDR/Flk-1). In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for VEGFR-1 (Flt-1) and/or VEGFR-2 (KDR/Flk-1). In one embodiment, the modified signaling agent has reduced affinity and/or activity for VEGFR-2 (KDR/Flk-1) and/or substantially reduced or eliminated affinity and/or activity for VEGFR-1 (Flt-1). Such an embodiment finds use, for example, in methods of wound healing or treatment of ischemia-related diseases (without wishing to be bound by theory, VEGFR-2-mediated effects on endothelial cell function and angiogenesis). In various embodiments, binding to VEGFR-1 (Flt-1), which is associated with cancer and pro-inflammatory activities, is prevented. In various embodiments, VEGFR-1 (Flt-1) acts as a decoy receptor, thereby substantially reducing or eliminating affinity at this receptor and preventing sequestration of the therapeutic agent. In one embodiment, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for VEGFR-1 (Flt-1) and/or substantially reduced or eliminated affinity and/or activity for VEGFR-2 (KDR/Flk-1) . . In some embodiments, the VEGF is VEGF-C or VEGF-D. In such embodiments, the modified signaling agent has reduced affinity and/or activity for VEGFR-3. Alternatively, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for VEGFR-3.

[0153]Proangiogenetic therapies are also important in various diseases (e.g. ischemic heart disease, bleeding, etc.) and include VEGF-based therapies. VEGFR-2 activation is proangiogenic (acts on endothelial cells). Activation of VEGF-1 can cause stimulation of inflammatory cell migration (including, for example, macrophages) and result in inflammation-associated hypervascular permeability. Activation of VEFGR-1 may also promote formation of bone marrow-associated tumor niches. Therefore, in this case, a selective VEGF-based therapeutic for VEGFR-2 activation would be desirable. In addition, the specific targeting of cells, e.g. to endothelial cells, would be desirable.

[0154]In some embodiments, the modified signaling agent has reduced affinity and/or activity (e.g., antagonist) for VEGFR-2 and/or has substantially reduced or eliminated affinity and/or activity for VEGFR-1. When such a construct is directed to endothelial cells of the tumor vasculature via a targeting moiety that binds to a tumor endothelial cell marker (e.g., PSMA and others), such a construct inhibits VEGFR-2 activation specifically in those cells encoding the marker are positive, while VEGFR is not activated -1 in the signaling pathway and in target cells (if the activity is removed), eliminating, for example, the induction of inflammatory responses. This would provide a more selective and safer anti-angiogenic therapy for many tumor types compared to VEGF-A neutralizing therapies.

[0155]In some embodiments, the modified signaling agent has reduced affinity and/or activity (e.g., agonist) for VEGFR-2 and/or has substantially reduced or eliminated affinity and/or activity for VEGFR-1. In some embodiments, by targeting vascular endothelial cells, such a construct promotes angiogenesis without causing the induction of VEGFR-1-associated inflammatory responses. Therefore, such a construct would have targeted proangiogenic effects with a substantially reduced risk of side effects caused by systemic activation of VEGFR-2 as well as VEGR-1.

[0156]In an illustrative embodiment, the modified signaling agent is VEGF.₁₆₅having the amino acid sequence of SEQ ID NO: 55).

[0157]In another illustrative embodiment, the modified signaling agent is VEGF._165bhaving the amino acid sequence of SEQ ID NO:56.

[0158]In these embodiments, the modified signaling agent has a mutation at amino acid 183 (e.g., a substitution mutation at 183, e.g., 183K, 183R, or 183H). Without wishing to be bound by theory, it is believed that such mutations may result in reduced receptor binding affinity. See, for example, US Patent No. 9,078,860, the entire contents of which are incorporated herein by reference.

[0159]In some embodiments, the modified signaling agent is a modified version of a hormone selected from, but not limited to, human chorionic gonadotropin, gonadotropin releasing hormone, an androgen, an estrogen, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, prolactin , growth hormone, adrenocorticotropic hormone, antidiuretic hormone, oxytocin, thyrotropin-releasing hormone, growth hormone-releasing hormone, corticotropin-releasing hormone, somatostatin, dopamine, melatonin, thyroxine, calcitonin, parathyroid hormone, glucocorticoids, mineralocorticoids, adrenaline, norepinephrine , progesterone , Insulin, Glucagon, Amylin, Calcitriol, Calciferol, Atrial Natriuretic Peptide, Gastrin, Secretin, Cholecystokinin, Neuropeptide Y, Ghrelin, PYY3-36, Insulin-Like Growth Factor (IGF), Leptin, Thrombopoietin, Erythropoietin (EPO), and Angiotensinogen

[0160]In some embodiments, the modified signaling agent is TNF-α. TNF is a pleiotropic cytokine with many diverse functions including regulation of cell growth, differentiation, apoptosis, tumorigenesis, viral replication, autoimmunity, immune cell function and transport, inflammation, and septic shock. It binds to two different membrane receptors on target cells: TNFR1 (p55) and TNFR2 (p75). TNFR1 shows a very broad expression pattern, while TNFR2 is preferentially expressed in certain populations of lymphocytes, Tregs, endothelial cells, certain neurons, microglia, cardiomyocytes and mesenchymal stem cells. Very different biological pathways are activated in response to receptor activation, although there is also some overlap. As a general rule, without wishing to be bound by theory, TNFR1 signaling is associated with the induction of apoptosis (cell death) and TNFR2 signaling with the activation of cell survival signals (e.g. cell survival activation) via NFKB. TNF administration is systemically toxic, mainly due to the involvement of TNFR1. However, it should be noted that TNFR2 activation is also associated with a wide range of activities and, as with TNFR1, in the context of developing TNF-based therapies, control over the targeting and activity of TNF is important.

[0161]In some embodiments, the modified signaling molecule has reduced affinity and/or activity for TNFR1 and/or TNFR2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for TNFR1 and/or TNFR2. TNFR1 is expressed in most tissues and is involved in cell death signaling, while TNFR2, in contrast, is involved in cell survival signaling. Accordingly, in embodiments directed to methods of treating cancer, the modified signaling agent has reduced affinity and/or activity for TNFR1 and/or substantially reduced or eliminated affinity and/or activity for TNFR2. In these embodiments, the chimeric proteins can be targeted to a cell for which apoptosis is desired, e.g. a tumor cell or an endothelial cell of the tumor vasculature. In embodiments directed to methods of promoting cell survival, for example in neurogenesis to treat neurodegenerative diseases, the modified signaling agent has reduced affinity and/or activity for TNFR2 and/or substantially reduced or eliminated affinity and/or activity for TNFR1. Stated another way, in some embodiments, the present chimeric proteins comprise a modified TNF-α agent that allows for an enhancement of death or survival signals.

[0162]In some embodiments, the chimeric protein comprises a modified TNF that has reduced affinity and/or activity for TNFR1 and/or substantially reduced or eliminated affinity and/or activity for TNFR2. Such a chimera is, in some embodiments, a more potent inducer of apoptosis compared to a wild-type TNF and/or a chimera carrying only mutations that cause reduced affinity and/or activity for TNFR1. Such a chimera finds use in some embodiments for inducing tumor cell death or endothelial cell death of tumor vessels (e.g., in the treatment of cancer). Additionally, in some embodiments, these chimeras prevent or reduce the activation of T_recordCells, for example, via TNFR2, further supporting TNFR1-mediated antitumor activity in vivo.

[0163]In some embodiments, the chimeric protein comprises a modified TNF that has reduced affinity and/or activity for TNFR2 and/or substantially reduced or eliminated affinity and/or activity for TNFR1. This chimera, in some embodiments, is a potent activator of cell survival in some cell types, which may be a specific therapeutic target in various disease settings, including but not limited to stimulating neurogenesis. In addition, such TNFR2-promoting chimeras are also useful in the treatment of autoimmune diseases (e.g., Crohn's disease, diabetes, MS, colitis, etc. and many others described herein). In some embodiments, the chimera targets autoreactive T cells. In some embodiments, the chimera promotes T_recordCell activation and indirect suppression of cytotoxic T cells.

[0164]In some embodiments, the chimera causes autoreactive T cell death, e.g. by TNFR2 activation and/or TNFR1 avoidance (eg, a modified TNF with reduced affinity and/or activity for TNFR2 and/or substantially reduced or eliminated affinity and/or activity for TNFR1). Without wishing to be bound by theory, these autoreactive T cells have impaired apoptosis/survival signals, e.g. by NFkB pathway activity/signaling changes. In some embodiments, the chimera kills autoreactive T cells that have lesions or modifications of the NFκB pathway resulting in an imbalance in their cell death/survival signaling properties (apoptosis) and optionally an altered susceptibility to certain death-triggering signals (e.g., TNFR2 activation) underlie. .

[0165]In some embodiments, a TNFR-2-based chimera has additional therapeutic uses in diseases including, but not limited to, autoimmune diseases, various cardiac diseases, demyelinating and neurodegenerative diseases, and infectious diseases.

[0166]In one embodiment, wild-type TNF-α has the amino acid sequence of SEQ ID NO: 57.

[0167]In such embodiments, the modified TNF-α agent has mutations at one or more amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146 and 147 that produce a modified TNF-α with reduced receptor binding affinity. See, for example, US Patent No. 7,993,636, the entire contents of which are incorporated herein by reference.

[0168]In some embodiments, the modified human TNF-α unit has mutations at one or more amino acid positions R32, N34, Q67, H73, L75, T77, S86, Y87, V91, 197, T105, P106, A109, P113, Y115. B. E127, N137, D143, A145 and E146 as described for example in WO/2015/007903, the entire content of which is hereby incorporated by reference (human TNF sequence numbering, Genbank accession number BAG70306, version BAG70306. 1 GI: 197692685). ). In some embodiments, the modified human TNF-α unit has substitution mutations selected from L29S, R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F, Y87H , V91G, V91A, 197A, 197Q, 197S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, A145R, A145T, E146D, E146K and S147D. In some embodiments, the human TNF-α moiety has a mutation selected from Y87Q, Y87L, Y87A, Y87F and Y87H. In another embodiment, the human TNF-α unit has a mutation selected from 197A, 197Q and 197S. In another embodiment, the human TNF-α moiety has a mutation selected between Y115A and Y115G. In some embodiments, the human TNF-α unit has an E146K mutation. In some embodiments, the human TNF-α unit has a Y87H and E146K mutation. In some embodiments, the human TNF-α unit has a Y87H and A145R mutation. In some embodiments, the remainder of human TNF-α has an R32W and S86T mutation. In some embodiments, the human TNF-α unit has an R32W and E146K mutation. In some embodiments, the remainder of human TNF-α has an L29S and R32W mutation. In some embodiments, the human TNF-α unit has a D143N and A145R mutation. In some embodiments, the human TNF-α unit has a D143N and A145R mutation. In some embodiments, the human TNF-α unit has an A145T, E146D, and S147D mutation. In some embodiments, the human TNF-α unit has an A145T and S147D mutation.

[0169]In some embodiments, the modified TNF-α agent has one or more mutations selected from N39Y, S147Y and Y87H as described in WO2008/124086, the entire content of which is incorporated herein by reference.

[0170]In some embodiments, the modified human TNF-α moiety has mutations that confer receptor selectivity as described in PCT/IB2016/001668, the entire contents of which are incorporated herein by reference. In some embodiments, the TNF mutations are selective for TNF-R1. In some embodiments, the TNF mutations selective for TNF-R1 are located at one or more of positions R32, S86 and E146. In some embodiments, the TNF mutations that are selective for TNF-R1 are one or more of R32W, S86T, and E146K. In some embodiments, the TNF mutations that are selective for TNF-R1 are one or more of R32W, R32W/S86T, R32W/E146K, and E146K. In some embodiments, the TNF mutations are selective for TNF-R2. In some embodiments, TNF mutations selective for TNF-R2 are located at one or more of positions A145, E146, and S147. In some embodiments, the TNF mutations that are selective for TNF-R2 are one or more of A145T, A145R, E146D, and S147D. In some embodiments, the TNF mutations that are selective for TNF-R2 are one or more of A145R, A145T/S147D, and A145T/E146D/S147D.

[0171]In one embodiment, the modified signaling agent is TNF-β. TNF-β can form a homotrimer or a heterotrimer with LT-β (LT-α1β2). In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for TNFR1 and/or TNFR2 and/or herpesvirus entry mediator (HEVM) and/or LT-βR.

[0172]In one embodiment, wild-type TNF-β has the amino acid sequence of SEQ ID NO: 58.

[0173]In such embodiments, the modified TNF-β agent may comprise mutations in one or more amino acids at positions 106-113 that produce a modified TNF-β with reduced receptor binding affinity for TNFR2. In one embodiment, the modified signaling agent has one or more substitution mutations at amino acid positions 106-113. In illustrative embodiments, substitution mutations are selected from Q107E, Q107D, S106E, S106D, Q107R, Q107N, Q107E/S106E, Q107E/S106D, Q107D/S106E and Q107D/S106D. In another embodiment, the modified signaling agent has an insertion of about 1 to about 3 amino acids at positions 106-113.

[0174]In some embodiments, the modified agent is a member of the TNF family (e.g., TNF-alpha, TNF-beta), which may be a single-chain trimeric version, as described in WO 2015/007903 and PCT/IB2016/001668, their full contents are incorporated by reference.

[0175]In some embodiments, the modified agent is a member of the TNF family (e.g., TNF-alpha, TNF-beta) that has reduced affinity and/or activity, i. H. antagonistic activity (e.g. antagonistic activity), natural or antagonistic activity that is the result of one or more mutations, see for example WO 2015/007520, the entire content of which is incorporated herein by reference) in TNFR1. In these embodiments, the modified agent is a member of the TNF family (e.g., TNF-alpha, TNF-beta) that optionally also has substantially reduced or eliminated affinity and/or activity for TNFR2. In some embodiments, the modified agent is a member of the TNF family (e.g., TNF-alpha, TNF-beta) that has reduced affinity and/or activity, i. H. antagonistic activity (e.g. antagonistic activity), natural or antagonistic activity that is the result of one or more mutations, see for example WO 2015/007520, the entire content of which is incorporated herein by reference) in TNFR2. In these embodiments, the modified agent is a member of the TNF family (e.g., TNF-alpha, TNF-beta) that optionally also has substantially reduced or eliminated affinity and/or activity for TNFR1. The constructs of such embodiments find use, for example, in methods for attenuating the TNF response in a cell-specific manner. In some embodiments, the antagonistic member of the TNF family (e.g., TNF-alpha, TNF-beta) is a single-chain trimeric version, as described in WO 2015/007903.

[0176]In one embodiment, the modified signaling means is TRAIL. In some embodiments, the modified TRAIL agent has reduced affinity and/or activity for DR4 (TRAIL-RI) and/or DR5 (TRAIL-RII) and/or DcR1 and/or DcR2. In some embodiments, the modified TRAIL agent has a substantially reduced or suppressed affinity and/or activity for DR4 (TRAIL-RI) and/or DR5 (TRAIL-RII) and/or DcR1 and/or DcR2.

[0177]In one embodiment, the wild-type TRAIL has the amino acid sequence of SEQ ID NO: 59.

[0178]In such embodiments, the modified TRAIL agent may comprise a mutation at amino acid positions T127-R132, E144-R149, E155-H161, Y189-Y209, T214-1220, K224-A226, W231, E236-L239, E249-K251. T261-H264 and H270-E271 (numbering based on human sequence, Genbank accession number NP_003801, version 10 NP_003801.1, GI: 4507593; see above).

[0179]In some embodiments, the modified TRAIL agent may include one or more mutations that substantially reduce its affinity and/or activity for TRAIL-R1. In such embodiments, the modified TRAIL agent can specifically bind to TRIL-R2. Examples of mutations include mutations at one or more amino acid positions Y189, R191, Q193, H264, I266 and D267. For example, the mutations can be one or more of Y189Q, R191K, Q193R, H264R, I266L and D267Q. In one embodiment, the modified TRAIL agent comprises the Y189Q, R191K, Q193R, H264R, I266L and D267Q mutations.

[0180]In some embodiments, the modified TRAIL agent may include one or more mutations that substantially reduce its affinity and/or activity for TRAIL-R2. In such embodiments, the modified TRAIL agent can specifically bind to TRIL-R1. Examples of mutations include mutations at one or more amino acid positions G131, R149, S159, N199, K201 and S215. For example, the mutations can be one or more of G131R, R1491, S159R, N199R, K201H and S215D. In one embodiment, the modified TRAIL agent comprises the G131R, R1491, S159R, N199R, K201H, and S215D mutations. Additional TRAIL mutations are described, for example, in Trebing et al., (2014) Cell Death and Disease, 5:e1035, the full description of which is incorporated herein by reference.

[0181]In one embodiment, the modified signaling agent is TGFα. In such embodiments, the modified TGFα agent has reduced affinity and/or activity for the epidermal growth factor receptor (EGFR). In some embodiments, the modified TGFα agent has substantially reduced or eliminated affinity and/or activity for the epidermal growth factor receptor (EGFR).

[0182]In one embodiment, the modified signaling agent is TGFβ. In such embodiments, the modified signaling agent has reduced affinity and/or activity for TGFBR1 and/or TGFBR2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for TGFBR1 and/or TGFBR2. In some embodiments, the modified signaling agent optionally has reduced or substantially reduced or abolished affinity and/or activity for TGFBR3, which, without wishing to be bound by theory, can act as a ligand reservoir for TGF-beta receptors. In some embodiments, TGFβ may prefer TGFBR1 over TGFBR2 or TGFBR2 over TGFBR1. Similarly, without wishing to be bound by theory, LAP may act as a ligand reservoir for TGF-beta receptors. In some embodiments, the modified signaling agent has reduced affinity and/or activity for TGFBR1 and/or TGFBR2 and/or substantially reduced or eliminated affinity and/or activity for latency-associated peptide (LAP). In some embodiments, such chimeras find use in Camurati-Engelmann disease or other diseases associated with inappropriate TGFβ signaling.

[0183]In some embodiments, the modified agent is a member of the TGF family (e.g., TGFα, TGFβ) that has reduced affinity and/or activity, e.g. H. antagonistic activity (e.g. natural antagonistic activity or antagonistic activity resulting from one or more mutations, see for example WO 2015/007520, the entire content of which is incorporated herein by reference) in one or more of TGFBR1, TGFBR2, TGFBR3. In these embodiments, the modified agent is a member of the TGF family (e.g., TGFα, TGFβ) that optionally also has substantially reduced or eliminated affinity and/or activity for one or more of TGFBR1, TGFBR2, TGFBR3.

[0184]In some embodiments, the modified agent is a member of the TGF family (e.g., TGFα, TGF) that has reduced affinity and/or activity, e.g. H. antagonistic activity (eg, natural or antagonistic activity resulting from one or more mutations). , see for example WO 2015/007520, the entire content of which is incorporated herein by reference) in TGFBR1 and/or TGFBR2. In these embodiments, the modified agent is a member of the TGF family (e.g., TGFα, TGFβ) that optionally also has substantially reduced or eliminated affinity and/or activity for TGFBR3.

[0185]In one embodiment, the modified signaling agent is an interleukin. In one embodiment, the modified signaling agent is IL-1. In one embodiment, the modified signaling agent is IL-1a or IL-1p. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-1R1 and/or IL-1RAcP. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-1R1 and/or IL-1RAcP. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-1R2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-1R2. For example, in some embodiments, the present modified IL-1 agents prevent IL-1R2 from interacting and therefore substantially reduce its function as a decoy and/or sink for therapeutic agents.

[0186]In one embodiment, the wild-type IL-1β has the amino acid sequence of SEQ ID NO: 60.

[0187]IL1 is a pro-inflammatory cytokine and an important regulator of the immune system. It is a potent activator of CD4 T cell responses, increases Th17 cell percentage and expansion of IFNγ and IL-4 producing cells. IL-1 is also a potent regulator of CD8+ T cells, enhancing antigen-specific CD8+ T cell expansion, differentiation, peripheral migration and memory. IL-1 receptors include IL-1R1 and IL-1R2. Binding and signaling by IL-1R1 constitute the mechanism by which IL-1 mediates many of its biological (and pathological) activities. IL1-R2 can act as a decoy receptor, thus reducing the availability of IL-1 for interaction and signaling via IL-1R1.

[0188]In some embodiments, the modified IL-1 has reduced affinity and/or activity (e.g., agonist activity) for IL-1R1. In some embodiments, the modified IL-1 has substantially reduced or eliminated affinity and/or activity for IL-1R2. In such embodiments, there is recoverable IL-1/IL-1R1 signaling and prevention of loss of therapeutic IL-R2 chimeras, and therefore a reduction in IL-1 dose is required (eg, compared to wild-type or a chimera containing only one). a knockout mutation for IL-R1). Such constructs find use, for example, in methods of treating cancer, including, for example, stimulating the immune system to elicit an anti-cancer response.

[0189]In some embodiments, the modified IL-1 has reduced affinity and/or activity (e.g., antagonistic activity, e.g., natural antagonistic activity, or antagonistic activity that is the result of one or more mutations, see e.g. WO 2015/007520). the entire contents of which are incorporated herein by reference) for IL-1R1. In some embodiments, the modified IL-1 has substantially reduced or eliminated affinity and/or activity for IL-1R2. In such embodiments, IL-1/IL-1R1 signaling is unrecoverable and prevents loss of therapeutic chimeras in IL-R2 and therefore requires a reduction in IL-1 dose (e.g., relative to wild-type or a chimera with only a knockout). mutation for IL-R1). Such constructs find use, for example, in methods of treating autoimmune diseases, including, for example, suppression of the immune system.

[0190]In such embodiments, the modified signaling agent has a deletion at amino acids 52-54 that produces a modified human IL-1β with reduced binding affinity for Type I IL-1R and reduced biological activity. See, for example, WO 1994/000491, the entire content of which is incorporated herein by reference. In some embodiments, the modified human IL-1β has one or more substitution mutations selected from A117G/P118G, R120X, L122A, T125G/L126G, R127G, Q130X, Q131G, K132A, S137G/Q138Y, L145G, H146X, L145A/L147A, Q148X, Q148G/Q150G, Q150G/D151A, M152G, F162A, F162A/Q164E, F166A, Q164E/E167K, N169G/D170G, I172A, V174A, K208E, K209X, K209A EX221/K2 /N224A, N224S/K225S, E244K, N245Q (where X can be any amino acid change, e.g. a non-conservative change) showing reduced binding to IL-1R, such as e.g. B. described in WO2015/007542 and WO/2015/007536, the entire contents of which are incorporated by reference (base numbering in human IL-1β sequence, Genbank accession number NP_000567, version NP-000567.1, GI: 10835145). In some embodiments, the modified human IL-1β may have one or more mutations selected from R120A, R120G, Q130A, Q130W, H146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209A, K209D, K219S, K219Q, E221S and E221K. In one embodiment, the modified human IL-1β comprises the Q131G and Q148G mutations. In one embodiment, the modified human IL-1β comprises the Q148G and K208E mutations. In one embodiment, the modified human IL-1β comprises the R120G and Q131G mutations. In one embodiment, the modified human IL-1β comprises the R120G and H146A mutations. In one embodiment, the modified human IL-1β comprises the R120G and H146N mutations. In one embodiment, the modified human IL-1β comprises the R120G and H146R mutations. In one embodiment, the modified human IL-1β comprises the R120G and H146E mutations. In one embodiment, the modified human IL-1β comprises the R120G and H146G mutations. In one embodiment, the modified human IL-1β comprises the R120G and K208E mutations. In one embodiment, the modified human IL-1β comprises the R120G, F162A and Q164E mutations.

[0191]In one embodiment, the modified signaling agent is IL-2. In such an embodiment, the modified signaling agent has reduced affinity and/or activity for IL-2Rα and/or IL-2Rβ and/or IL-2Rγ. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-2Rβ and/or IL-2Rγ. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-2Rα. Such embodiments may be relevant, for example, to the treatment of cancer when the modified IL-2 is an agonist at IL-2Rβ and/or IL-2Rγ. For example, the present constructs can promote attenuated activation of CD8⁺T cells (which can have antitumor effects), which have IL2-β and γ receptors and T_Rules(which may have an immunosuppressive, tumor-promoting effect) that have IL2-α, -β and -γ receptors. Additionally, in some embodiments, preferences for IL-2Rβ and/or IL-2Rγ over IL-2Rα prevent IL-2 side effects such as pulmonary edema. In addition, IL-2-based chimeras are useful for the treatment of diseases (e.g. autoimmune diseases), for example when the modified IL-2 is antagonistic (e.g. natural antagonistic activity or antagonistic activity resulting from a or further mutations, see for example WO 2015/007520, the entire content of which is incorporated herein by reference) in IL-2Rβ and/or IL-2Rγ. For example, the present constructs can promote attenuated suppression of CD8⁺T cells (thereby suppressing the immune response), which have IL2-β and γ receptors and T_Rulesthat have IL2 α, β and γ receptors. Alternatively, in some embodiments, IL-2-bearing chimeras promote activation of T_Rulesand thus immunosuppression and disadvantaged activation of CD8⁺T cells For example, these constructs find use in the treatment of diseases or conditions that would benefit from immunosuppression, e.g. B. Autoimmune diseases.

[0192]In some embodiments, the chimeric protein has targeting moieties as described herein that target CD8+ T cells and a modified IL-2 agent that has reduced affinity and/or activity for IL-2Rβ and/or IL -2Rγ and/or IL-2Rβ has substantially reduced affinity and/or activity when ablated by IL-2Rα. In some embodiments, these constructs provide targeted CD8⁺T cell activity and are generally inactive (or have significantly reduced activity) towards T_recordcells. In some embodiments, such constructs have enhanced immunostimulatory activity compared to wild-type IL-2 (e.g., not wishing to be bound by theory by not stimulating Tregs), while minimizing the systemic toxicity associated with IL-2 eliminate or reduce.

[0193]In one embodiment, the wild-type IL-2 has the amino acid sequence of SEQ ID NO: 61.

[0194]In such embodiments, the modified IL-2 agent has one or more mutations in amino acids L72 (L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R or L72K), F42 (F42A, F42G, F42S, F42T , F42Q, F42E, F42N, F42D, F42R or F42K) and Y45 (Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R or Y45K). Without wishing to be bound by theory, it is believed that these modified IL-2 agents have reduced affinity for the high affinity IL-2 receptor and affinity for the IL-2 relative to the IL-2 receptor -Retain medium affinity receptor type receptor. IL-2. See, for example, US Patent Publication No. 2012/0244112, the entire contents of which are incorporated herein by reference.

[0195]In some embodiments, the modified IL-2 agent has one or more mutations at amino acids R38, F42, Y45, and E62. For example, the modified IL-2 agent may include one or more of R38A, F42A, Y45A, and E62A. In some embodiments, the modified IL-2 agent may include a mutation in C125. For example, the mutation can be C125S. In such embodiments, the modified IL-2 agent may have substantially reduced affinity and/or activity for IL-2Rα, such as described in Carmenate et al. (2013) The Journal of Immunology, 190:6230-6238, the entire disclosure of which is incorporated herein by reference. In some embodiments, the IL-2 agent modified with mutations in R38, F42, Y45, and/or E62 can induce expansion of effector cells, which include CD8+ T cells and NK cells, but not Treg cells. In some embodiments, the IL-2 agent modified with mutations in R38, F42, Y45, and/or E62 is less toxic than wild-type IL-2 agents. For example, a chimeric protein comprising the modified IL-2 agent with substantially reduced affinity and/or activity for IL-2Rα may find application in oncology.

[0196]In other embodiments, the modified IL-2 agent may have substantially reduced affinity and/or activity for IL-2Rβ, for example as described in WO2016/025385, the full disclosure of which is incorporated herein by reference. In such embodiments, the modified IL-2 agent can induce expansion of Treg cells but not effector cells such as CD8+ T cells and NK cells. For example, a chimeric protein comprising the modified IL-2 agent having substantially reduced affinity and/or activity for IL-2Rβ may find use in the treatment of autoimmune diseases. In some embodiments, the modified IL-2 agent may include one or more mutations in amino acids N88, D20, and/or A126. For example, the modified IL-2 agent can include one or more of N88R, N881, N88G, D20H, Q126L, and Q126F.

[0197]In various embodiments, the modified IL-2 agent may include a mutation at D109 or C125. For example, the mutation can be D109C or C125S. In some embodiments, IL-2 modified with a mutation at D109 or C125 can be used to bind to a PEG moiety.

[0198]In one embodiment, the modified signaling agent is IL-3. In some embodiments, the modified signaling agent has reduced affinity and/or activity for the IL-3 receptor, which is a heterodimer with a single alpha chain paired with the common beta subunit (beta c or CD131). In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for the IL-3 receptor, which is a heterodimer with a single alpha chain paired with the common beta subunit (beta c or CD131). is.

[0199]In one embodiment, the modified signaling agent is IL-4. In such an embodiment, the modified signaling molecule has a reduced affinity and/or activity for type 1 and/or type 2 IL-4 receptors. In such an embodiment, the modified signaling molecule has a substantially reduced affinity and/or activity, reduced or eliminated by type 1 and/or type 2 IL-4 receptors. Type 1 IL-4 receptors consist of the IL-4Rα subunit with a common γ chain and specifically bind IL-4. Type 2 IL-4 receptors comprise an IL-4Rα subunit linked to another subunit known as IL-13Rα1. In some embodiments, the modified signaling agent has substantially reduced or suppressed affinity and/or activity for IL-4 type 2 receptors.

[0200]In one embodiment, wild-type IL-4 has the amino acid sequence of SEQ ID NO: 62.

[0201]In such embodiments, the modified IL-4 agent has one or more mutations in amino acids R121 (R121A, R121D, R121E, R121F, R121H, R1211, R121K, R121N, R121P, R121T, R121W), E122 (E122F), Y124 ( Y124A, Y124Q, Y124R, Y124S, Y124T) and S125 (S125A). Without wishing to be limited by theory, it is believed that these modified IL-4 agents retain Type I receptor-mediated activity but significantly decrease biological activity mediated by the other receptors. See, for example, US Patent No. 6,433,157, the entire contents of which are incorporated herein by reference.

[0202]In one embodiment, the modified signaling agent is IL-6. IL-6 signals via a cell surface type I cytokine receptor complex comprising the ligand binding chain IL-6R (CD126) and the signal transduction component gp130. IL-6 can also bind to a soluble form of IL-6R (sIL-6R), which is the extracellular portion of IL-6R. The sIL-6R/IL-6 complex may be involved in neurite outgrowth and neuronal survival and may therefore be important in nerve regeneration through remyelination. Accordingly, in some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-6R/gp130 and/or sIL-6R. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-6R/gp130 and/or sIL-6R.

[0203]In one embodiment, wild-type IL-6 has the amino acid sequence of SEQ ID NO: 63.

[0204]In such embodiments, the modified signaling agent has one or more mutations at amino acids 58, 160, 163, 171 or 177. Without wishing to be bound by theory, it is believed that these modified IL-6 agents exhibit reduced binding affinity IL. -6R alpha and reduced biological activity. See, for example, WO 97/10338, the entire contents of which are incorporated herein by reference.

[0205]In one embodiment, the modified signaling agent is IL-10. In such an embodiment, the modified signaling agent has reduced affinity and/or activity for IL-10 receptor 1 and IL-10 receptor 2. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL- 10 receptor 1 and IL-10 receptor 2.

[0206]In one embodiment, the modified signaling agent is IL-11. In such an embodiment, the modified signaling agent has reduced affinity and/or activity for IL-11Rα and/or IL-11Rβ and/or gp130. In such an embodiment, the modified signaling agent has a substantially reduced or suppressed affinity and/or activity for IL-11Rα and/or IL-11Rβ and/or gp130.

[0207]In one embodiment, the modified signaling agent is IL-12. In such an embodiment, the modified signaling agent has reduced affinity and/or activity for IL-12Rβ1 and/or IL-12Rβ2. In such an embodiment, the modified signaling agent has a substantially reduced or suppressed affinity and/or activity for IL-12Rβ1 and/or IL-12Rβ2.

[0208]In one embodiment, the modified signaling agent is IL-13. In such an embodiment, the modified signaling agent has reduced affinity and/or activity for the IL-4 receptor (IL-4Ra) and IL-13Rα1. In some embodiments, the modified signaling agent has substantially reduced or suppressed affinity and/or activity for the IL-4 receptor (IL-4Ra) or IL-13Rα1.

[0209]In one embodiment, the wild-type IL-1β has the amino acid sequence of SEQ ID NO: 64.

[0210]In such embodiments, the modified IL-1β agent has one or more mutations at amino acids 13, 16, 17, 66, 69, 99, 102, 104, 105, 106, 107, 108, 109, 112, 113 and 114 Without wishing to be bound by theory, it is believed that these modified IL-1β agents have reduced biological activity. See, for example, WO 2002/018422, the entire content of which is incorporated herein by reference.

[0211]In one embodiment, the modified signaling agent is IL-18. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-18Rα and/or IL-18Rβ. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-18Rα and/or IL-18RR. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for IL-18Rα type II, which is an IL-18Rα isoform that lacks the TIR domain required for signaling.

[0212]In one embodiment, wild-type IL-18 has the amino acid sequence of SEQ ID NO: 65.

[0213]In such embodiments, the modified IL-18 agent may comprise one or more mutations in amino acids or amino acid regions selected from Y37-K44, R49-Q54, D59-R63, E67-C74, R80, M87-A97, N127-K129, Q139 -M149, K165-K171, R183 and Q190-N191 as described in WO/2015/007542, the entire contents of which are hereby incorporated by reference (numbering based on human IL-18 sequence, number Genbank Registry AAV38697, version AAV38697. 1, GI: 54696650).

[0214]In one embodiment, the modified signaling agent is IL-33. In such an embodiment, the modified signaling agent has reduced affinity and/or activity for the ST-2 receptor and IL-1RAcP. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for the ST-2 receptor and IL-1RAcP.

[0215]In one embodiment, wild-type IL-33 has the amino acid sequence of SEQ ID NO: 66.

[0216]In such embodiments, the modified IL-33 agent may comprise one or more mutations in amino acids or amino acid regions selected from 1113-Y122, S127-E139, E144-D157, Y163-M183, E200, Q215, L220-C227 and T260. -E269 as described in WO/2015/007542, the entire contents of which are incorporated herein by reference (numbering based on human sequence, Genbank accession number NP_254274, version NP_254274.1, GI:15559209).

[0217]In one embodiment, the modified signaling agent is epidermal growth factor (EGF). EGF is a member of a family of potent growth factors. Members include EGF, HB-EGF and others like TGFalpha, Amphiregulin, Neureguline, Epiregulin, Betacellulin. The EGF family of receptors includes EGFR (ErbB1), ErbB2, ErbB3 and ErbB4. These can function as homodimeric and/or heterodimeric receptor subtypes. The different members of the EGF family show different selectivity for the different receptor subtypes. For example, EGF associates with ErbB1/ErbB1, ErbB1/ErbB2, ErbB4/ErbB2, and some other heterodimeric subtypes. HB-EGF has a similar pattern, although it is also associated with ErbB4/4. The positive or negative modulation of EGF growth factor (EGF-like) signaling is of considerable therapeutic interest. For example, inhibition of EGFR signaling is of interest in the treatment of various cancers where EGFR signaling is an important growth-promoting signal. Alternatively, stimulation of EGFR signaling is of therapeutic interest, for example to promote wound healing (both acute and chronic) or oral mucositis (a major side effect of various cancer therapies, including but not limited to radiotherapy).

[0218]In some embodiments, the modified signaling agent has reduced affinity and/or activity for ErbB1, ErbB2, ErbB3, and/or ErbB4. Such embodiments find use, for example, in methods of treating wounds. In some embodiments, the modified signaling agent binds to one or more ErbB1, ErbB2, ErbB3, and ErbB4 and antagonizes receptor activity. In such embodiments, the modified signaling agent has reduced affinity and/or activity for ErbB1, ErbB2, ErbB3 and/or ErbB4, thereby allowing receptor activity to be antagonized in a reduced manner. Such embodiments find application in cancer treatments, for example. In one embodiment, the modified signaling agent has reduced affinity and/or activity for ErbB1. ErbB1 is the therapeutic target of kinase inhibitors; most have side effects because they are not highly selective (eg, gefitinib, erlotinib, afatinib, brigatinib, and icotinib). In some embodiments, the attenuated ErbB1 antagonist signaling is more targeted and has fewer side effects than other agents that target EGF receptors.

[0219]In some embodiments, the modified signaling agent has reduced affinity and/or activity (e.g. antagonist, e.g. natural antagonistic activity or antagonistic activity that is the result of one or more mutations, see e.g. WO 2015/ 007520, the entirety of which is incorporated herein by reference) for ErbB1 and/or a substantially reduced or eliminated affinity and/or activity for ErbB4 or other subtypes with which it can interact. By specific targeting via the targeting moiety, selective repression of cells (antagonism, e.g. natural antagonistic activity or antagonistic activity that is the result of one or more mutations, see e.g. WO 2015/007520, the full content of which is herein incorporated by reference) activation of the ErbB1/ErbB1 receptor without involving other receptor subtypes potentially associated with the side effects associated with inhibition. Thus, in contrast to EGFR kinase inhibitors, which inhibit EGFR activity in all cell types in the body, such a construct would target a selective cell (e.g., tumor cells with activated EGFR signaling due to receptor amplification, overexpression, etc.) anti -EGFR (ErbB1) drug effect with reduced side effects.

[0220]In some embodiments, the modified signaling agent has reduced affinity and/or activity (e.g., agonist) for ErbB4 and/or other subtypes with which it can interact. By targeting specific target cells via the targeting moiety, selective activation of ErbB1 signaling (e.g. epithelial cells) is achieved. Such a construction finds use in some embodiments in the treatment of wounds (promotion of healing) with reduced side effects, particularly for the treatment of chronic conditions and uses other than topical application of a therapeutic agent (e.g., systemic wound healing). . . ).

[0221]In one embodiment, the modified signaling agent is insulin or insulin analogs. In some embodiments, the modified insulin or insulin analog has reduced affinity and/or activity for the insulin receptor and/or the IGF1 or IGF2 receptor. In some embodiments, the modified insulin or insulin analog has a substantially reduced or suppressed affinity and/or activity for the insulin receptor and/or the IGF1 or IGF2 receptor. The attenuated response at the insulin receptor enables control of diabetes, obesity, metabolic disorders and the like, while removal of the IGF1 or IGF2 receptor prevents cancer-promoting effects.

[0222]In one embodiment, the modified signaling agent is insulin-like growth factor I or insulin-like growth factor II (IGF-1 or IGF-2). In one embodiment, the modified signaling agent is IGF-1. In such an embodiment, the modified signaling agent has reduced affinity and/or activity for the insulin receptor and/or IGF1 receptor. In one embodiment, the modified signaling agent can bind to the IGF1 receptor and antagonize receptor activity. In such an embodiment, the modified signaling agent has reduced affinity and/or activity for the IGF1 receptor, thereby allowing the receptor activity to be antagonized in a reduced manner. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and/or activity for the insulin receptor and/or the IGF1 receptor. In some embodiments, the modified signaling agent has reduced affinity and/or activity for the IGF2 receptor, thereby allowing receptor activity to be antagonized in a reduced manner. In one embodiment, the modified signaling agent has substantially reduced or abolished affinity and/or activity for the insulin receptor and thus does not interfere with insulin signaling. In various embodiments, this applies to the treatment of cancer. In various embodiments, the present agents can prevent IR isoform A from causing resistance to cancer treatments.

[0223]In some embodiments, the modified signaling agent is EPO. In various embodiments, the modified EPO agent has reduced affinity and/or activity for the EPO receptor (EPOR) receptor and/or the ephrin receptor (EphR) compared to wild-type EPO or other EPO-based agents in EPO , which are described herein document. In some embodiments, the modified EPO agent has substantially reduced or suppressed affinity and/or activity for the EPO receptor (EPOR) and/or the Eph receptor (EphR). Illustrative EPO receptors include, but are not limited to, an EPOR homodimer or an EPOR/CD131 heterodimer. Also included as an EPO receptor is the common beta receptor (βcR). Illustrative Eph receptors include, but are not limited to, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB6. In some embodiments, the modified EPO protein comprises one or more mutations that cause the EPO protein to have reduced affinity for receptors comprising one or more different EPO receptors or Eph receptors (e.g. restriction: EPOR -EPHB4, EPOR-βcR-EPOR). Also provided are the recipients of EP Patent Publication No. 2492355, the entire contents of which are incorporated herein by reference, including but not limited to NEPORs.

[0224]In some embodiments, human EPO has the amino acid sequence of SEQ ID NO: 67 (the first 27 amino acids are the signal peptide).

[0225]In some embodiments, the human EPO protein is the mature form of EPO (where the signal peptide is cleaved) which is a 166 amino acid residue glycoprotein having the sequence of SEQ ID NO:68.

[0226]The structure of human EPO protein is predicted to comprise bundles of four helices, including helices A, B, C, and D. In various embodiments, the modified EPO protein comprises one or more mutations located in four major regions of the EPO proteins are located for bioactivity, ie amino acid residues 10-20, 44-51, 96-108 and 142-156. In some embodiments, one or more mutations are located at residues 11-15, 44-51, 100-108, and 147-151. These residues are found in the A-helix (Val11, Arg14, and Tyr15), the C-helix (Ser100, Arg103, Ser104, and Leu108), the D-helix (Asn147, Arg150, Gly151, and Leu155), and the A/B- connecting loop (residues 42-51). In some embodiments, the modified EPO protein comprises mutations at residues between amino acids 41-52 and amino acids 147, 150, 151 and 155. Without wishing to be bound by theory, it is believed that mutations at these residues have substantial effects on both have receptor binding as biological activity in vitro. In some embodiments, the modified EPO protein comprises mutations at residues 11, 14, 15, 100, 103, 104, and 108. Without wishing to be bound by theory, it is believed that mutations at these residues have modest effects on EPO Binding activity have recipients. and much larger effects on biological activity in vitro. Illustrative substitutions include, but are not limited to, one or more of val11ser, arg14ala, arg14 gln, tyr15ile, pro42asn, thr44ile, LYS45asp, Val46ala, Tyr51phe, ser100glu, ser100thr, arg103ala, ser104ile, ser10, and Leu155Ala.

[0227]In some embodiments, the modified EPO protein includes mutations that affect bioactivity and do not bind, e.g. that of Eliot et al. Mapping of the active site of recombinant human erythropoietin, January 15, 1997; Sangre:89(2), the entire contents of which are incorporated herein by reference.

[0228]In some embodiments, the modified EPO protein comprises one or more mutations affecting surface residues of the EPO protein involved in receptor contact. Without wishing to be bound by theory, it is believed that mutations in these surface residues are less likely to affect the folding of the protein, thereby retaining some biological activity. Illustrative surface residues that can be mutated include, but are not limited to, residues 147 and 150. In illustrative embodiments, the mutations are substitutions involving one or more of N147A, N147K, R150A, and R150E.

[0229]In some embodiments, the modified EPO protein comprises one or more mutations at residues N59, E62, L67 and L70 and one or more mutations affecting disulfide bond formation. Without wishing to be bound by theory, it is believed that these mutations affect folding and/or are predicted to be in hidden positions and thus indirectly affect biological activity.

[0230]In one embodiment, the modified EPO protein includes a K20E substitution that significantly decreases receptor binding. See Elliott, et al., (1997)Sangre,89:493-502, the entire contents of which are incorporated herein by reference.

[0231]Additional EPO mutations that can be engineered into the chimeric EPO protein of the invention are described, for example, in Elliott et al., (1997).Sangre,89:493-502, the entire contents of which are incorporated herein by reference, and Taylor et al., (2010)pediatrics,23(4): 251-260, the entire contents of which are incorporated herein by reference.

[0232]In one embodiment, the present chimeric protein has (i) an anti-SIRP1α-directed residue and (ii) an anti-tumor cell-directed residue, together with any of the modified or mutated signaling agents described herein. In one embodiment, the present chimeric protein has one residue directed against SIRP1α in macrophages and a second residue directed against PD-L1 or PD-L2 in tumor cells.

[0233]In various embodiments, the signaling agent is a toxin or a toxic enzyme. In some embodiments, the toxin or toxic enzyme is derived from plants and bacteria. Illustrative toxic enzymes or toxins include diphtheria toxin,PseudomonasAnthrax toxin, ribosome-inactivating proteins (RIPs) such as ricin and saporin, modecin, abrin, gelonin, and pokeweed antiviral protein. Additional toxins include those described in Mathew et al., (2009) Cancer Sci 100(8):1359-65, the full descriptions of which are incorporated herein by reference. In such embodiments, the chimeric proteins of the invention can be used to induce cell death in a cell-type specific manner. In such embodiments, the toxin can be modified, e.g. mutated to reduce toxin affinity and/or activity for attenuated effect, as described with other signaling agents herein.

[0234]Chimaera and multispecies fusions with signaling agents

[0235]In various embodiments, the chimeric protein of the invention comprises one or more signaling substances as described herein and/or one or more additional targeting moieties (ie in addition to the anti-SIRP1α targeting moiety). Accordingly, the present invention provides chimeric or fusion proteins comprising one or more signaling molecules, a SIRP1α targeting moiety and/or one or more additional targeting moieties.

[0236]In various embodiments, the chimeric proteins of the present invention have targeting moieties that target two different cells (e.g., to create a synapse) or the same cell (e.g., to obtain a targeting agent effect). aim (more focused marking).

[0237]In various embodiments, the chimeric protein of the invention is multispecific, i. H. the chimeric protein comprises two or more targeting moieties with recognition domains (e.g., antigen recognition domains) that address two or more targets (e.g., antigens, receptors, or epitopes). ). In such embodiments, the chimeric protein of the invention may comprise two further targeting moieties having recognition domains that recognize and bind to two or more epitopes on the same or different antigens or on different receptors. In various embodiments, such multispecific chimeric proteins exhibit advantageous properties such as increased avidity and/or improved selectivity. In one embodiment, the chimeric protein of the invention comprises two targeting moieties and is bispecific, ie it binds and recognizes two epitopes on the same antigen or on different antigens or different receptors.

[0238]In various embodiments, the multispecific chimeric protein of the invention comprises two or more targeting moieties, each targeting moiety being an antibody or an antibody derivative as described herein. In an exemplary embodiment, the multispecific chimeric protein of the invention comprises at least one antibody or antibody derivative (for example a VHH) comprising an antigen recognition domain against SIRP1α and an antibody or antibody derivative comprising a SIRP1α antigen recognition domain tumor antigen.

[0239]In various embodiments, the present multispecific chimeric proteins have two or more targeting moieties targeting different antigens or receptors, and one targeting moiety may be attenuated for its antigen or receptor, e.g. the targeting moiety binds its antigen or receptor with low affinity or avidity (including, for example, an affinity or avidity that is less than the affinity or avidity that the other targeting moiety has for its antigen or receptor, e.g. the Difference between binding affinities can be approximately 10-fold, 25-fold, 50-fold, 100-fold, 300-fold, 500-fold, 1000-fold or 5000-fold, for example the lower fraction can be directed affinity or avidity bind to its antigen or receptor in a K_Din the mid to high nM or low to mid μM range, while the targeting moiety can bind to its antigen or receptor on a K with higher affinity or avidity_Din the mid to high pM or low to mid nM range). For example, in some embodiments, the present multispecific chimeric protein comprises an attenuated targeting moiety directed against a promiscuous antigen or receptor, which improves targeting to a cell of interest (e.g. via the other targeting moiety) and Can prevent effects on several types. of cells, including those that are not the targets of therapy (e.g., by binding a promiscuous antigen or receptor with a higher affinity than contemplated in these embodiments).

[0240]The multispecific chimeric protein of the invention can be constructed using methods known in the art, see for example US Patent No. 9,067,991, US Patent Publication No. 20110262348 and WO 2004/041862, the entire contents of which are incorporated herein by reference. In an illustrative embodiment, the multispecific chimeric protein of the invention comprising two or more targeting moieties can be constructed by chemical cross-linking, for example by reacting amino acid residues with an organic derivatizing agent, as described by Blattler et al., Biochemistry 24, 1517 - 1524 and EP294703, the entire contents of which are incorporated herein by reference. In another illustrative embodiment, the multispecific chimeric protein comprising two or more targeting moieties is constructed by genetic fusion, ie, a single polypeptide is constructed comprising the polypeptides of the individual targeting moieties. For example, a single polypeptide construct can be generated encoding a first antibody or antibody derivative (e.g., a VHH) having an antigen recognition domain against SIRP1α and a second antibody or antibody derivative having a recognition domain against a tumor antigen. A method for preparing divalent or polyvalent VHH polypeptide constructs is described in PCT patent application WO 96/34103, the entire contents of which are incorporated herein by reference. In another illustrative embodiment, the multispecific chimeric protein of the invention can be constructed using linkers. For example, the carboxy terminus of a first antibody or antibody derivative (e.g., a VHH) with a SIRP1α antigen recognition domain can be linked to the amino terminus of a second antibody or antibody derivative with a SIRP1α antigen recognition domain antigen . (or the other way around). Illustrative linkers that can be used are described in this document. In some embodiments, the components of the multispecific chimeric protein of the invention are linked directly to one another without the use of linkers.

[0241]In various embodiments, the multispecific chimeric protein of the invention recognizes and binds SIRP1α and one or more antigens found on one or more immune cells, which may include, but are not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils. neutrophils. . eosinophils, monocytes, macrophages, natural killer cells, T cells (e.g. cytotoxic T cells, helper T cells, natural killer T cells), B cells, plasma cells, dendritic cells or subsets thereof. In some embodiments, the chimeric protein specifically binds to an antigen of interest and directly or indirectly recruits one or more immune cells.

[0242]In various embodiments, the multispecific chimeric protein of the invention recognizes and binds SIRP1α and one or more antigens found on tumor cells. In these embodiments, the present chimeric protein can directly or indirectly recruit an immune cell (e.g., a macrophage) to a tumor cell or tumor microenvironment. In such embodiments, the present chimeric protein enhances phagocytosis of tumor cells by macrophages.

[0243]In some embodiments, the present chimeric proteins are capable of shifting the balance of immune cells in favor of an immune attack by a tumor, or have use in methods involved. For example, the present chimeric protein may alter the proportion of immune cells at a clinically important site in favor of cells capable of killing and/or suppressing tumor (e.g., antitumor macrophages (e.g., M1 macrophages), T cells , cytotoxic T cells, lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells and dendritic cells) and in contrast to tumor-protective cells (e.g. tumor-protective cells) derived from myeloid suppressor cells (MDSCs), regulatory T cells (Tregs), tumor-associated neutrophils (TANs), M2 macrophages, tumor-associated macrophages (TAMs), or subsets thereof). In some embodiments, the present chimeric protein is capable of increasing the ratio of effector T cells to regulatory T cells.

[0244]In some embodiments, the multispecific chimeric protein of the invention comprises a targeting moiety having a recognition domain that specifically binds to a target (eg, antigen or receptor) associated with tumor cells. In some embodiments, the targeting entity directly or indirectly recruits tumor cells. For example, in some embodiments, the tumor cell is recruited by one or more effector cells (e.g., a macrophage) that can engulf, kill, and/or suppress the tumor cell.

[0245]Tumor cells or cancer cells refer to uncontrolled cell or tissue growth and/or abnormal increase in cell survival and/or inhibition of apoptosis that disrupts the normal functioning of body organs and systems. For example, tumor cells include benign and malignant cancers, polyps, hyperplasia, and latent tumors or micrometastases. Illustrative tumor cells include, but are not limited to, cells from: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; cancer of the brain and central nervous system; breast cancer; peritoneal cancer; Cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; endometrial carcinoma; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver carcinoma; hepatoma; intraepithelial neoplasia; kidney or renal cancer; throat cancer; Leukemia; liver cancer; lung cancer (eg, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth and throat); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; colon cancer; cancer of the respiratory tract; salivary gland carcinoma; Sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; Lymphoma, including Hodgkin and non-Hodgkin lymphoma, and B-cell lymphoma (including low grade follicular non-Hodgkin lymphoma (NHL); small lymphocyte (SL) NHL; follicular/moderate NHL; moderate diffuse NHL; high grade NHL immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small cell NHL; NHL with severe disease; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia, chronic myeloblastic leukemia, as well as others Carcinomas and sarcomas and post-transplant lymphoproliferative disorder (PTLD) and abnormal vascular proliferation associated with phakomatosis, edema (e.g. associated with brain tumors) and Meigs syndrome.

[0246]Tumor cells or cancer cells also include carcinomas, e.g. various subtypes including, for example, adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcomas (including, for example, bone and soft tissue), leukemias (including, for example, acute myeloid, lymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cells), lymphomas, and myelomas (including, for example, Hodgkin and non-Hodgkin lymphomas, light chain, non-secretory, MGUS and plasmacytomas) and cancers of the central nervous system (including, for example, brain (e.g. gliomas (e.g. astrocytoma, oligodendroglioma and ependymoma), meningiomas , pituitary adenomas and neuromas, and spinal cord tumors (eg, meningiomas and neurofibromas).

[0247]Representative tumor antigens include, but are not limited to, MART-1/Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-0017-1A/GA733, carcinoembryonic Antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, amI1, Prostate Specific Antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA-3, Prostate Specific Antigen (PSMA) , T cell receptor/CD3 zeta chain, MAGE family of tumor antigens (e.g. MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE -A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), the GAGE ​​family of tumor antigens (eg, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE- 6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1 , α-fetoprotein, E-cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli (APC) protein, fodrin, connexin 37, Ig idiotype, gangliosides p15, gp75, GM2 and GD2, viral products such as human papillomavirus proteins, Smad family of tumor antigens, Imp-1, NA, EBV-encoded core antigen (EBNA)-1, brain glycogen phosphorylase, SSX -1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 CT-7, c-erbB-2, CD19, CD20, CD22, CD30, CD33, CD37 , CD56, CD70, CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1, PD-L2, PMSA and BCMA (TNFRSF17). In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these tumor antigens.

[0248]In some embodiments, the present multispecific chimeric protein SIRP1α recognizes and binds to an antigen on a tumor cell. In some embodiments, the multispecific chimeric protein recruits macrophages directly or indirectly into the tumor cell or tumor microenvironment.

[0249]In some embodiments, the multispecific chimeric protein of the invention comprises a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or a receptor) associated with T cells. In some embodiments, the targeting entity directly or indirectly recruits T In one embodiment, the antigen recognition domains specifically bind effector T cells. In some embodiments, the antigen recognition domain directly or indirectly recruits effector T cells, e.g. e.g., in some embodiments, to a therapeutic site (e.g., a site containing one or more disease cells or cells to be modulated) for a therapeutic effect). Illustrative effector T cells include cytotoxic T cells (eg, αβ TCR, CD3⁺, CD8⁺, CD45RO⁺); CD4⁺Effector T cells (e.g. αβ TCR, CD3⁺, CD4⁺, CCR7⁺B. CD62Lhi, IL-7R/CD127⁺); CD8⁺Effector T cells (e.g. αβ TCR, CD3⁺, CD8⁺, CCR7⁺B. CD62Lhi, IL-7R/CD127⁺); Effector memory T cells (e.g. CD62LIow, CD44⁺, TCR, CD3⁺B. IL-7R/CD127⁺B. THE 15R⁺, CCR7low); Central memory T cells (such as CCR7⁺, CD62L⁺, CD27⁺; from CCR7 hours, CD44⁺, CD62Lhi, TCR, CD3⁺B. IL-7R/CD127⁺B. THE 15R⁺); CD62L⁺effector T cells; CD8⁺Effector memory T cells (TEM), including early effector memory T cells (CD27⁺CD62L⁻) and late effector memory T cells (CD27⁻CD62L⁻) (TemE or TemL); CD127 (⁺) CD25 effector T lymphocytes (low/-); CD127 (⁻(CD25)⁻) effector T cells; CD8⁺Memory effector stem cells (TSCM) (eg, CD44 (low) CD62L (high) CD122 (high) sca (⁺)); TH1 effector T cells (e.g. CXCR3⁺, CXCR6⁺and CCR5⁺; o αβ TCR, CD3⁺, CD4⁺B. IL-12R⁺, IFNγR⁺, CXCR3⁺), TH2 effector T cells (eg, CCR3⁺, CCR4⁺and CCR8⁺; o αβ TCR, CD3⁺, CD4⁺, IL-4R⁺B. IL-33R⁺, CCR4⁺B. THE 17RB⁺, CRTH2⁺); TH9 effector T cells (e.g. αβ TCR, CD3⁺, CD4⁺); TH17 effector T cells (e.g. αβ TCR, CD3⁺, CD4⁺B. Il-23R⁺, CCR6⁺, IL-1R⁺); CD4⁺CD45RO⁺CCR7⁺Effector T cells, ICOS⁺effector T cells; CD4⁺CD45RO⁺CCR7 (⁻) effector T cells; and effector T cells secreting IL-2, IL-4 and/or IFN-γ.

[0250]Los Antigenos de Células T Ilustrativos de Interés incluyen, por ejemplo (e inclusive Los Dominions Extracellulares, Cuando Corresponda): CD8, CD3, SLAMF4, IL-2Rα, 4-1BB/TNFRSF9, IL-2Rβ, ALCAM, B7-1, IL - 4R, B7-H3, BLAME/SLAMFS, CEACAM1, IL-6R, CCR3, IL-7Rα, CCR4, CXCRI/IL-S RA, CCR5, CCR6, IL-10Rα, CCR 7, IL-10Rβ, CCRS, IL - E/CD103, CD6, Integrin α M/CD11b, CDS, Integrin α X/CD11c, Integrin β 2/CDIS, KIR/CD15S, CD27/TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30/TNFRSFS, KIR2DL4/CD15Sd, CD31 /PECAM-1, KIR2DS4, Ligand CD40/TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, Leucotrieno B4-R1, CDS4/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3 , NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Cadena γ común/IL-2Rγ, Osteopontina, CRACC/SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK /TNFRSF11A, CX3CR1, CX3CL1, L-Selectina, CXCR3, SIRPβ1, CXCR4, SLAM, CXCR6, TCCR/WSX-1, DNAM-1, Timopoyetina, EMMPRIN/CD147, TIM-1, EphB6, TIM-2, Fas/TNFRSF6 , TIM-3, Ligando Fas/TNFSF6, TIM-4, Fcγ RIII/CD16, TIM-6, TNFR1/TNFRSF1A, Granulisina, TNF RIII/TNFRSF1B, TRAIL RI/TNFRSFIOA, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2 /CD102, TRAILR3/TNFRSF10C, IFN-γR1, TRAILR4/TNFRSF10D, IFN-γ R2, TSLP, IL-1R1 und TSLP R. En diverses realizaciones, la proteína quimérica comprende un resto de direccionamiento que se une a uno o más de estos ejemplos illustrivos de células T. antigenos

[0251]As a non-limiting example, in various embodiments, the present chimeric protein has a targeting moiety directed against a checkpoint marker expressed on a T cell, e.g. one or more of PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3 and A2aR.

[0252]In some embodiments, the multispecific chimeric protein of the invention comprises a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or a receptor) associated with B cells. In some embodiments, the targeting entity directly or indirectly recruits B cells, e.g. e.g., in some embodiments, to a therapeutic site (e.g., a site containing one or more disease cells or cells to be modulated for a therapeutic effect). Illustrative B cell antigens of interest include, for example, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b , CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138 and CDw150. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these exemplary B cell antigens.

[0253]In some embodiments, the multispecific chimeric protein of the invention comprises a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or a receptor) associated with natural killer cells. In some embodiments, the targeting moiety directly or indirectly recruits natural killer cells, e.g. e.g., in some embodiments, to a therapeutic site (e.g., a site containing one or more disease cells or cells to be modulated for a therapeutic effect). Illustrative natural killer cell antigens of interest include, for example, TIGIT, 2B4/SLAMF4, KIR2DS4, CD155/PVR, KIR3DL1, CD94, LMIR1/CD300A, CD69, LMIR2/CD300c, CRACC/SLAMF7, LMIR3/CD300LF, Kir1alpha, DNAM-1, LMIR5/CD300LB, Fc-epsilon RII, LMIR6/CD300LE, Fc-γ RI/CD64, MICA, Fc-γ RIIB/CD32b, MICB, Fc-γ RIIC/CD32c, MULT-1, Fc-γ RIIA /CD32a , Nectin -2/CD112, Fc-γRIII/CD16, NKG2A, FcRH1/IRTA5, NKG2C, FcRH2/IRTA4, NKG2D, FcRH4/IRTA1, NKp30, FcRH5/IRTA2, NKp44, Fc receptor-like 3/CD16 -2, NKp46/ NCR1, NKp80/KLRF1, NTB-A/SLAMF6, Rae-1, Rae-1α, Rae-1β, Rae-1 Delta, H60, Rae-1 Epsilon, ILT2/CD85j, Rae-1γ, ILT3 /CD85k, TREM- 1, ILT4/CD85d, TREM-2, ILT5/CD85a, TREM-3, KIR/CD158, TREML1/TLT-1, KIR2DL1, ULBP-1, KIR2DL3, ULBP-2, KIR2DL4/CD158d and ULBP-3. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these exemplary NK cell antigens.

[0254]In some embodiments, the multispecific chimeric protein of the invention comprises a targeting moiety having a recognition domain that specifically binds a target (e.g., an antigen or a receptor) associated with macrophages/monocytes. In some embodiments, the targeting moiety directly or indirectly recruits macrophages/monocytes, e.g. e.g., in some embodiments, to a therapeutic site (e.g., a site containing one or more disease cells or cells to be killed) to modulate them for therapeutic effect). Illustrative macrophage/monocyte antigens of interest include, for example, SIRP1α, B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, common β chain, α4/CD49d integrin, BLAME/SLAMF8, αX integrin/CDIIc , CCL6/C10, Integrin β 2/CD18, CD155/PVR, Integrin β 3/CD61, CD31/PECAM-1, Latex, CD36/SR-B3, Leukotriene B4R1, CD40/TNFRSF5, LIMPIIISR-B2, CD43, LMIR1/ CD300A, CD45, LMIR2/CD300c, CD68, LMIR3/CD300LF, CD84/SLAMF5, LMIR5/CD300LB, CD97, LMIR6/CD300LE, CD163, LRP-1, CD2F-10/SLAMF9, MARCO, CRACC/SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN/CD147, MGL2, Endoglin/CD105, Osteoactivin/GPNMB, Fc-γ RI/CD64, Osteopontin, Fc-γ RIIB/CD32b, PD-L2, Fc-γ RIIC/CD32c, Siglec -3/CD33, Fc-γ RIIA/CD32a, SIGNR1/CD209, Fc-γ RIII/CD16, SLAM, GM-CSF Rα, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN-γ RI, TLR4 , IFN-gannna R2, TREM-I, IL-I RII, TREM-2, ILT2/CD85j, TREM-3, ILT3/CD85k, TREML1/TLT-1, 2B4/SLAMF 4, IL-10Rα, ALCAM, IL- 10Rβ, aminopeptidase N/ANPEP, ILT2/CD85j, common β chain, ILT3/CD85k, Clq R1/CD93, ILT4/CD85d, CCR1, ILT5/CD85a, CCR2, CD206, integrin α4/CD49d, CCR5, integrin α M/ CDII b, CCR8, integrin α X/CDIIc, CD155/PVR, integrin β 2/CD18, CD14, integrin β3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45, leukotriene B4-R1, CD68 , LIMPIIISR - B2, CD84/SLAMF5, LMIR1/CD300A, CD97, LMIR2/CD300c, CD163, LMIR3/CD300LF, Coagulation Factor III/Tissue Factor, LMIR5/CD300LB, CX3CR1, CX3CL1, LMIR6/CD300LE, CXCR4, LRP-1, CXCR6, M - CSF R, DEP-1/CD148, MD-1, DNAM-1, MD-2, EMMPRIN/CD147, MMR, Endoglin/CD105, NCAM-L1, Fc-γRI/CD64, PSGL-1, Fc-γ RIIIICD16, RP105, G-CSF R, L-selectin, GM-CSF R a, Siglec-3/CD33, HVEM/TNFRSF14, SLAM, ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM -I , IL-6R, TREM-2, CXCRI/IL-8 RA, TREM-3 and TREMLI/TLT-1.

[0255]In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these exemplary macrophage/monocyte antigens.

[0256]In some embodiments, the multispecific chimeric protein of the invention comprises a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or a receptor) associated with dendritic cells. In some embodiments, the targeting moiety directly or indirectly recruits dendritic cells, e.g. e.g., in some embodiments, to a therapeutic site (e.g., a site containing one or more disease cells or cells to be modulated for a specific effect). Illustrative dendritic cell antigens of interest include, for example, CLEC9A, XCR1, RANK, CD36/SRB3, LOX-1/SR-E1, CD68, MARCO, CD163, SR-A1/MSR, CD5L, SREC-1, CL-PI/COLEC12, SREC -II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, Ligand 4-IBB/TNFSF9, IL-12/IL-23 p40, 4-amino-1,8-naphthalimide , ILT2/CD85j , CCL21/6Ckine, ILT3/CD85k, 8-oxo-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, A2B5, Integrin α 4/CD49d, Aag, Integrin β 2/CD18, AMICA, Langerin, B7 -2/CD86 , Leukotriene B4 RI, B7-H3, LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300c, Clq R1/CD93, LMIR3/CD300LF, CCR6, LMIR5/CD300LB CCR7, LMIR6/CD300LE, CD40/TNFRSF5, MAG /Siglec-4 -a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1/CLEC5A, CD84/SLAMF5, MMR, CD97, NCAMLI, CD2F-10/SLAMF9, Osteoactivin GPNMB, Chern 23, PD -L2, CLEC-1, RP105, CLEC-2, CLEC-8, Siglec-2/CD22, CRACC/SLAMF7, Siglec-3/CD33, DC-SIGN, DCE205, Siglec-5, DC-SIGNR /CD299, Siglec -6, DCAR, Siglec-7, DCIR/CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1/CLEC7A, Siglec-F, Dectin-2/CLEC6A, SIGNR1/CD209 , DEP -1/CD148 , SIGNR4, DLEC, SLAM, EMMPRIN/CD147, TCCR/WSX-1, Fc-γ R1/CD64, TLR3, Fc-γ RIIB/CD32b, TREM-1, Fc-γ RIIC/CD32c, TREM- 2, Fc- γ RIIA/CD32a, TREM-3, Fc-γ RIII/CD16, TREML1/TLT-1, ICAM-2/CD102 and vanilloid R1. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these exemplary DC antigens.

[0257]In some embodiments, the multispecific chimeric protein of the invention comprises a targeting moiety having a recognition domain that specifically binds to a target (e.g. an antigen or a receptor) associated with immune cells selected from megakaryocytes, platelets, erythrocytes, but not limited to B. mast cells, basophils, neutrophils, eosinophils, or subsets thereof. In some embodiments, the antigen recognition domains directly or indirectly recruit megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or subsets thereof, e.g. e.g., in some embodiments, to a therapeutic site (e.g., a locus containing one or more disease cells or cells to be modulated for a therapeutic effect).

[0258]In some embodiments, the multispecific chimeric protein of the invention comprises a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or a receptor) associated with megakaryocytes and/or platelets. Illustrative megakaryocyte and/or platelet antigens of interest include, for example, GP Ib/IIIa, GPIb, vWF, PF4 and TSP. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these exemplary megakaryocyte and/or platelet antigens.

[0259]In some embodiments, the multispecific chimeric protein of the invention comprises a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or a receptor) associated with erythrocytes. Illustrative erythrocyte antigens of interest include, for example, CD34, CD36, CD38, CD41a (platelet glycoprotein Ib/IIIa), CD41b (GPIIb), CD71 (transferrin receptor), CD105, glycophorin A, glycophorin C, c-kit, HLA -DR, H2 (MHC-II) and Rhesus antigens. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these exemplary red cell antigens.

[0260]In some embodiments, the multispecific chimeric protein of the invention comprises a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or a receptor) associated with mast cells. Illustrative mast cell antigens of interest include, for example, SCFR/CD117, Fc_miIR, CD2, CD25, CD35, CD88, CD203c, C5R1, CMAI, FCERIA, FCER2, TPSABI. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these mast cell antigens.

[0261]In some embodiments, the multispecific chimeric protein of the invention comprises a targeting moiety having a recognition domain that specifically binds a target (e.g., an antigen or a receptor) associated with basophils. Illustrative basophil antigens of interest include, for example, Fc_miIR, CD203c, CD123, CD13, CD107a, CD107b and CD164. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these basophil antigens.

[0262]In some embodiments, the multispecific chimeric protein of the invention comprises a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or a receptor) associated with neutrophils. Illustrative neutrophil antigens of interest include, for example, 7D5, CD10/CALLA, CD13, CD16 (FcRIII), CD18 proteins (LFA-1, CR3, and p150,95), CD45, CD67, and CD177. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these neutrophil antigens.

[0263]In some embodiments, the multispecific chimeric protein of the invention comprises a targeting moiety having a recognition domain that specifically binds a target (e.g., an antigen or a receptor) associated with eosinophils. Illustrative eosinophil antigens of interest include, for example, CD35, CD44 and CD69. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these eosinophil antigens.

[0264]In various embodiments, the multispecific chimeric protein of the invention comprises a targeting moiety having a recognition domain that specifically binds to an appropriate cell surface marker or antigen known to those skilled in the art. In some embodiments, the cell surface marker or antigen is a tissue specific marker. Illustrative tissue-specific markers include, but are not limited to, endothelial cell surface markers such as ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAMI, PROCR, SELE, SELP, TEK, THBD, VCAMI, VWF; smooth muscle cell surface markers such as ACTA2, MYHIO, MYHI 1, MYH9, MYOCD; fibroblast cell surface (stromal) markers such as ALCAM, CD34, COLIAI, COL1A2, COL3A1, FAP, PH-4; epithelial cell surface markers such as CDID, K6IRS2, KRTIO, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUCI, TACSTDI; neovascular markers such as CD13, TFNA, alpha-v-beta-3 (αVβ3), E-selectin; and adipocyte surface markers such as ADIPOQ, FABP4 and RETN. In various embodiments, the chimeric protein includes a targeting moiety that binds to one or more of these antigens. In various embodiments, a targeting moiety of the chimeric protein binds to one or more of the cells bearing those antigens.

[0265]In various embodiments, the multispecific chimeric protein of the invention has one or more targeting residues directed against a checkpoint marker, e.g. one or more of PD-1/PD-L1 or PD-L2, CD28/CD80 or CD86, CTLA4/CD80 or CD86, ICOS/ICOSL or B7RP1, BTLA/HVEM, KIR, LAG3, CD137/CD137L, OX40/OX40L, CD27, CD40L, TIM3/Gal9 and A2aR.

[0266]As a non-limiting example, in various embodiments, the present chimeric protein has a targeting moiety directed against (i) a checkpoint marker expressed on a T cell, e.g. one or more of PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, Cd27, CD40L, TIM3 and A2aR and (ii) a targeting moiety is directed against a tumor cell, together with any of those herein modified signaling agents (e.g., mutants) described.

[0267]In various embodiments, the present multispecific chimeric protein has one or more targeting residues directed against PD-1. In some embodiments, the chimeric protein has one or more targeting moieties that selectively bind to a PD-1 polypeptide. In some embodiments, the chimeric protein comprises one or more antibodies, antibody formats or derivatives, peptides or polypeptides, or fusion proteins that selectively bind to a PD-1 polypeptide.

[0268]In one embodiment, the targeting entity comprises the anti-PD-1 antibody pembrolizumab (also known as MK-3475, KEYTRUDA) or fragments thereof. Pembrolizumab and other humanized anti-PD-1 antibodies are described in Hamid et al. (2013) New England Journal of Medicine 369(2):134-44, US Patent No. 8,354,509 and WO 2009/114335, the entire disclosures of which are incorporated herein by reference. In illustrative embodiments, pembrolizumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 69; and/or a light chain comprising the amino acid sequence of SEQ ID NO: 70).

[0269]In one embodiment, the targeting entity comprises the anti-PD-1 antibody, nivolumab (also known as BMS-936558, MDX-1106, ONO-4538, OPDIVO), or fragments thereof. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are described in US Patent No. 8,008,449 and WO 2006/121168, the entire disclosures of which are incorporated herein by reference. In illustrative embodiments, nivolumab or an antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 71; and/or a light chain comprising the amino acid sequence of SEQ ID NO:72.

[0270]In one embodiment, the targeting entity comprises the anti-PD-1 antibody pidilizumab (also known as CT-011, hBAT or hBAT-1) or fragments thereof. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are described in US 2008/0025980 and WO 2009/101611, the full disclosures of which are incorporated herein by reference. In illustrative embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises light chain variable regions comprising an amino acid sequence selected from SEQ ID NOS: 15-18 US 2008/0025980 ( SEQ ID NO: 15 US 2008/0025980 (SEQ ID NO: 73); SEQ ID NO: 16 US 2008/0025980 (SEQ ID NO: 74); SEQ ID NO: 17 US 2008/0025980 (SEQ ID NO: 75) and SEQ ID NO: 18 of US 2008/0025980 (SEQ ID NO: 76)); and/or a heavy chain comprising an amino acid sequence selected from SEQ ID NOS: 20-24 of US 2008/0025980 (SEQ ID NO: 20 of US 2008/0025980 (SEQ ID NO: 77); SEQ ID NO: 21 of US 2008/0025980 (SEQ ID NO: 78), SEQ ID NO: 22 of US 2008/0025980 (SEQ ID NO: 79), SEQ ID NO: 23 of US 2008/0025980 (SEQ ID NO: 80) and SEQ ID NO .: 24 of US 2008/0025980 (SEQ ID NO: 81)).

[0271]In one embodiment, the targeting moiety comprises a light chain comprising SEQ ID NO: 18 of US 2008/0025980 (SEQ ID NO: 76) and a heavy chain comprising SEQ ID NO: 22 of US 2008/0025980 (SEQ ID NO: 79) includes ).

[0272]In one embodiment, the addressing unit comprises AMP-514 (also known as MEDI-0680).

[0273]In one embodiment, the targeting moiety comprises the PD-L2-Fc-AMP-224 fusion protein described in WO 2010/027827 and WO 2011/066342, the full descriptions of which are incorporated herein by reference. In such an embodiment, the targeting moiety may be a targeting domain comprising SEQ ID NO: 4 of WO2010/027827 (SEQ ID NO: 82) and/or the B7-DC fusion protein comprising SEQ ID NO: 83 of WO2010/ 027827 (SEQ ID NO:83).

[0274]In one embodiment, the targeting moiety comprises the AUNP 12 peptide or one of the other peptides described in US 2011/0318373 or 8,907,053. For example, the targeting moiety may comprise AUNP 12 (ie compound 8 or SEQ ID NO: 49 of US 2011/0318373) having the sequence (SEQ ID NO: 84).

[0000]

[00001]

[0275]In one embodiment, the targeting entity comprises the anti-PD-1 antibody 1E3 or fragments thereof as described in US 2014/0044738, the full disclosure of which is incorporated herein by reference. In illustrative embodiments, 1E3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 85; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:86.

[0276]In one embodiment, the targeting entity comprises the 1E8 anti-PD-1 antibody or fragments thereof as described in US 2014/0044738, the entire disclosure of which is incorporated herein by reference. In illustrative embodiments, 1E8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:88.

[0277]In one embodiment, the targeting entity comprises the anti-PD-1 antibody 1H3 or fragments thereof as described in US 2014/0044738, the full disclosure of which is incorporated herein by reference. In illustrative embodiments, 1H3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 89; and/or the light chain variable region comprising the amino acid sequence of SEQ ID NO:90.

[0278]In one embodiment, the targeting moiety comprises a VHH directed against PD-1, such as described in US Patent No. 8,907,065 and WO 2008/071447, the full disclosures of which are incorporated herein by reference. In illustrative embodiments, VHHs against PD-1 include SEQ ID NO: 347-351 of US Patent No. 8,907,065 (SEQ ID NO: 347 of US Patent No. 8,907,065 (SEQ ID NO: 91); SEQ ID NO: 348 of US Patent No. 8,907,065 (SEQ ID NO: 92), SEQ ID No: 349 of US Patent No. 8,907,065 (SEQ ID NO: 93), SEQ ID No: 350 of US Patent No. 8,907,065 (SEQ ID NO: 94) and SEQ ID NO: 351 of US Patent No. 8,907,065 (SEQ ID NO: 95)).

[0279]In one embodiment, the targeting moiety comprises one of the anti-PD-1 antibodies or fragments thereof as described in US2011/0271358 and WO2010/036959, the entire contents of which are incorporated herein by reference. In illustrative embodiments, the antibody or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOS: 25-29 of US2011/0271358: (SEQ ID NO: 25 of US2011 /0271358 (SEQ ID NO: 96) SEQ ID NO: 26 of US2011/0271358 (SEQ ID NO: 97) SEQ ID NO: 27 of US2011/0271358 (SEQ ID NO: 98) SEQ ID NO: 28 of US2011/0271358 (SEQ ID NO: 99) and SEQ ID NO: 29 of US2011/0271358 (SEQ ID NO: 100)); and/or a light chain comprising an amino acid sequence selected from SEQ ID NOS: 30-33 of US2011/0271358 (SEQ ID NO: 30 of US2011/0271358 (SEQ ID NO: 101); SEQ ID NO: 31 of US2011/0271358 (SEQ ID NO: 102), SEQ ID NO: 32 of US2011/0271358 (SEQ ID NO: 103) and SEQ ID NO: 33 of US2011/0271358 (SEQ ID NO: 104)).

[0280]In various embodiments, the present multispecific chimeric protein comprises one or more antibodies directed against PD-1, or antibody fragments thereof, selected from TSR-042 (Tesaro, Inc.), REGN2810 (Regeneron Pharmaceuticals, Inc.), PDR001 (Novartis Pharmaceuticals ) and BGB-A317 (BeiGene Ltd.)

[0281]In various embodiments, the present multispecific chimeric protein has one or more targeting residues directed against PD-L1. In some embodiments, the chimeric protein has one or more targeting residues that selectively bind to a PD-L1 polypeptide. In some embodiments, the chimeric protein comprises one or more antibodies, antibody formats or derivatives, peptides or polypeptides, or fusion proteins that selectively bind to a PD-L1 polypeptide.

[0282]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody MEDI4736 (also known as durvalumab) or fragments thereof. MEDI4736 is selective for PD-L1 and blocks PD-L1 binding to PD-1 and CD80 receptors. MEDI4736 and antigen-binding fragments thereof for use in the methods provided herein comprise a heavy chain and a light chain or a heavy chain variable region and a light chain variable region. The MEDI4736 sequence is described in WO/2016/06272, the entire content of which is incorporated herein by reference. In illustrative embodiments, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 105; and/or a light chain comprising the amino acid sequence of SEQ ID NO:106.

[0283]In illustrative embodiments, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 of WO/2016/06272 (SEQ ID NO: 107); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 3 of WO/2016/06272 (SEQ ID NO: 108).

[0284]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody atezolizumab (also known as MPDL3280A, RG7446) or fragments thereof. In illustrative embodiments, atezolizumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 109; and/or a light chain comprising the amino acid sequence of SEQ ID NO:110.

[0285]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody avelumab (also known as MSB0010718C) or fragments thereof. In illustrative embodiments, avelumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 111; and/or a light chain comprising the amino acid sequence of SEQ ID NO:112.

[0286]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody BMS-936559 (also known as 12A4, MDX-1105) or fragments thereof as described in US 2013/0309250 and WO 2007/005874, the full descriptions of which are contained herein. incorporated by reference. In illustrative embodiments, BMS-936559 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:113; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:114.

[0287]In one embodiment, the targeting moiety comprises the anti-PD-L1 3 G10 antibody or fragments thereof as described in US 2013/0309250 and WO 2007/005874, the full disclosures of which are incorporated herein by reference. In illustrative embodiments, 3G10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 115; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:116.

[0288]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 10A5 or fragments thereof as described in US 2013/0309250 and WO 2007/005874, the full disclosures of which are incorporated herein by reference. In illustrative embodiments, 10A5 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 117; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:118.

[0289]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 5F8 or fragments thereof as described in US 2013/0309250 and WO 2007/005874, the full disclosures of which are incorporated herein by reference. In illustrative embodiments, 5F8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 119; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:120.

[0290]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 10H10 or fragments thereof as described in US 2013/0309250 and WO 2007/005874, the full disclosures of which are incorporated herein by reference. In illustrative embodiments, 10H10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 121; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:122.

[0291]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 1B12 or fragments thereof as described in US 2013/0309250 and WO 2007/005874, the full disclosure of which is incorporated herein by reference. In illustrative embodiments, 1812 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 123; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:124.

[0292]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 7H1 or fragments thereof as described in US 2013/0309250 and WO 2007/005874, the full disclosures of which are incorporated herein by reference. In illustrative embodiments, 7H1 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 125; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:126.

[0293]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 11E6 or fragments thereof as described in US 2013/0309250 and WO 2007/005874, the full disclosures of which are incorporated herein by reference. In illustrative embodiments, 11E6 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 127; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:128.

[0294]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 12B7 or fragments thereof as described in US 2013/0309250 and WO 2007/005874, the full disclosures of which are incorporated herein by reference. In illustrative embodiments, 12B7 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 129; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:130.

[0295]In one embodiment, the targeting entity comprises the 13 G4 anti-PD-L1 antibody or fragments thereof as described in US 2013/0309250 and WO 2007/005874, the full disclosures of which are incorporated herein by reference. In illustrative embodiments, 13G4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 131; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:132.

[0296]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 1E12 or fragments thereof as described in US 2014/0044738, the entire disclosure of which is incorporated herein by reference. In illustrative embodiments, 1E12 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:134.

[0297]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 1F4 or fragments thereof as described in US 2014/0044738, the full disclosure of which is incorporated herein by reference. In illustrative embodiments, 1F4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:136.

[0298]In one embodiment, the targeting moiety comprises the anti-PD-L1 2 G11 antibody or fragments thereof as described in US 2014/0044738, the full disclosure of which is incorporated herein by reference. In illustrative embodiments, 2G11 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 137; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:138.

[0299]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 3B6 or fragments thereof as described in US 2014/0044738, the full disclosure of which is incorporated herein by reference. In illustrative embodiments, 3B6 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 139; and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:140.

[0300]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 3D10 or fragments thereof as described in US 2014/0044738 and WO 2012/145493, the full disclosures of which are incorporated herein by reference. In illustrative embodiments, 3D10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of (SEQ ID NO: 141; and/or a light chain variable region comprising the amino acid Sequence of SEQ ID NO: 142.

[0301]In one embodiment, the targeting moiety comprises one of the anti-PD-L1 antibodies described in US2011/0271358 and WO2010/036959, the entire contents of which are incorporated herein by reference. In illustrative embodiments, the antibody or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 34-38 of US2011/0271358 (SEQ US2011 ID NO: 34/0271358 (SEQ ID NO: 143), US2011 SEQ ID NO: 35/0271358 (SEQ ID NO: 144), SEQ ID NO: 37 of US2011/0271358 (SEQ ID NO: 146) and SEQ ID NO: 38 of US2011/0271358 (SEQ ID NO: 147)); and/or a light chain comprising an amino acid sequence selected from SEQ ID NO: 39-42 of US2011/0271358 (SEQ ID NO: 39 of US2011/0271358 (SEQ ID NO: 148); SEQ ID NO: 40 of US2011/0271358 (SEQ ID NO: 149), SEQ ID NO: 41 of US2011/0271358 (SEQ ID NO: 150) and SEQ ID NO: 42 of US2011/0271358 (SEQ ID NO: 151)).

[0302]In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4 or fragments thereof as described in WO 2011/066389, US Patent No. 8,779,108 and US2014/0356353, the full descriptions of which are incorporated herein by reference. In illustrative embodiments, 2.7A4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 of WO 2011/066389 (SEQ ID NO: 152); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 7 of WO 2011/066389 (SEQ ID NO: 153).

[0303]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 2.9D10 or fragments thereof as described in WO 2011/066389, US Patent No. 8,779,108 and US2014/0356353, the full descriptions of which are incorporated herein by reference. In illustrative embodiments, 2.9D10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12 of WO 2011/066389 (SEQ ID NO: 154); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 17 of WO 2011/066389 (SEQ ID NO: 155).

[0304]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 2.14H9 or fragments thereof as described in WO 2011/066389, US Patent No. 8,779,108 and US2014/0356353, the full descriptions of which are incorporated herein by reference. In illustrative embodiments, 2.14H9 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 22 of WO 2011/066389 (SEQ ID NO: 156); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 27 of WO 2011/066389 (SEQ ID NO: 157).

[0305]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 2.20A8 or fragments thereof as described in WO 2011/066389, US Patent No. 8,779,108 and US2014/0356353, the full descriptions of which are incorporated herein by reference. In illustrative embodiments, 2.20A8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 32 of WO 2011/066389 (SEQ ID NO: 158); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37 of WO 2011/066389 (SEQ ID NO: 159).

[0306]In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3.15 G8 or fragments thereof as described in WO 2011/066389, US Patent No. 8,779,108 and US2014/0356353, the full descriptions of which are incorporated herein by reference. In illustrative embodiments, 3.15 G8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42 of WO 2011/066389 (SEQ ID NO: 160); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 47 of WO 2011/066389 (SEQ ID NO: 161).

[0307]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 3.18 G1 or fragments thereof as described in WO 2011/066389, US Patent No. 8,779,108 and US2014/0356353, the full descriptions of which are incorporated herein by reference. In illustrative embodiments, 3.18 G1 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 52 of WO 2011/066389 (SEQ ID NO: 162); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 57 of WO 2011/066389 (SEQ ID NO: 163).

[0308]In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A40PT or fragments thereof as described in WO 2011/066389, US Patent No. 8,779,108 and US2014/0356353 and US2014/0356353, the full descriptions of which are here incorporated herein by reference. In illustrative embodiments, 2.7A40PT or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 62 of WO 2011/066389 (SEQ ID NO: 164); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 67 of WO 2011/066389 (SEQ ID NO: 165).

[0309]In one embodiment, the targeting entity comprises the anti-PD-L1 antibody 2.14H90PT or fragments thereof as described in WO 2011/066389, US Patent No. 8,779,108 and US2014/0356353, the full descriptions of which are incorporated herein by reference. In illustrative embodiments, 2.14H90PT or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72 of WO 2011/066389 (SEQ ID NO: 166); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 77 of WO 2011/066389 (SEQ ID NO: 167).

[0310]In one embodiment, the targeting moiety comprises one of the anti-PD-L1 antibodies described in WO2016/061142, the entire content of which is incorporated herein by reference. In illustrative embodiments, the antibody or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 18, 30, 38, 46, 50, 54, 62, 70 and 78 from WO2016/061142 (SEQ ID NO: 18 from WO2016/061142 (SEQ ID NO: 168); SEQ ID NO: 30 from WO2016/061142 (SEQ ID NO: 169); SEQ ID WO2016/061142 SEQ ID NO: 46 (SEQ ID No.: 171); WO2016/061142 SEQ ID No.: 50 (SEQ ID No.: 172); WO2016/061142 SEQ ID No.: 54/061142 (SEQ ID No.: 173), WO2016/ 061142 SEQ ID NO: 62 (SEQ ID NO: 174), WO2016/061142 SEQ ID NO: 70 (SEQ ID NO: 175) and SEQ ID NO: 78 of WO2016/061142 (SEQ ID NO: 176)); and/or a light chain comprising an amino acid sequence selected from SEQ ID NO: 22, 26, 34, 42, 58, 66, 74, 82 and 86 of WO2016/061142; SEQ ID NO: 22 of WO2016/061142 (SEQ ID NO: 177); SEQ ID NO: 26 of WO2016/061142 (SEQ ID NO: 178); SEQ ID NO: 34 of WO2016/061142 (SEQ ID NO: 179); SEQ ID NO: 42 of WO2016/061142 (SEQ ID NO: 180); SEQ ID NO: 58 of WO2016/061142 (SEQ ID NO: 181); SEQ ID NO: 66 of WO2016/061142 (SEQ ID NO: 182); SEQ ID NO: 74 of WO2016/061142 (SEQ ID NO: 183); SEQ ID NO: 82 of WO2016/061142 (SEQ ID NO: 184); and SEQ ID NO: 86 of WO2016/061142 (SEQ ID NO: 185)).

[0311]In one embodiment, the targeting moiety comprises one of the anti-PD-L1 antibodies described in WO2016/022630, the entire content of which is incorporated herein by reference. In illustrative embodiments, the antibody or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 2, 6, 10, 14, 18, 22, 26, 30 , 34, 38, 42 and 46 of WO2016/022630 (SEQ ID NO: 2 of WO2016/022630 (SEQ ID NO: 186); SEQ ID NO: 6 of WO2016/022630 (SEQ ID NO: 187); SEQ ID NO : 10 of WO2016/022630 (SEQ ID No.: 188);SEQ ID No.: 14 of WO2016/022630 (SEQ ID No.:189);SEQ ID No.: 18 of WO2016/022630 (SEQ ID No.: 190); SEQ ID NO: 18 of WO2016/022630 (SEQ ID NO: 190); NO: 30 of WO2016/022630 (SEQ ID NO: 193); SEQ ID NO: 34 of WO2016/022630 ( SEQ ID NO: 194); SEQ ID NO: 38 of WO2016/022630 (SEQ ID SEQ ID NO: 42 of WO2016/022630 (SEQ ID NO: 196) and SEQ ID NO: 46 of WO2016/022630 (SEQ ID NO: 197)); and/or a light chain comprising an amino acid sequence selected from SEQ ID NO: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48 of WO2016/022630 (SEQ ID NO: WO2016 /022630 4 (SEQ ID NO: 198); WO2016/022630 SEQ ID NO: 8 (SEQ ID NO: 199); WO2016/022630 SEQ ID NO: 12 (SEQ ID NO: 200); SEQ ID NO: WO2016/022630 16 (SEQ ID NO: 201); SEQ ID NO: WO2016/022630 20 (SEQ ID NO: 202); WO2016/022630 SEQ ID NO: 24 (SEQ ID NO: 203); SEQ ID WO2016/022630 SEQ ID NO: 32 (SEQ ID NO: 205); WO2016/022630 SEQ ID NO: 36 (SEQ ID NO: 206); WO2016/022630 ID NO: 36 (SEQ ID NO: 206), WO2016/022630 ID NO: 40 (SEQ ID NO: 206). NO: 207), WO2016/022630 SEQ ID NO: 44 (SEQ ID NO: 208) and SEQ ID NO: 48 of WO2016/022630 (SEQ ID NO: 209)).

[0312]In one embodiment, the targeting moiety comprises one of the anti-PD-L1 antibodies described in WO2015/112900, the entire content of which is incorporated herein by reference. In illustrative embodiments, the antibody or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 38, 50, 82 and 86 of WO 2015/112900 (SEQ ID No.: 38 of WO 2015/112900 (SEQ ID No.: 210); SEQ ID No.: 50 of WO 2015/112900 (SEQ ID No.: 211); SEQ ID No.: 82 of WO 2015/112900 ( SEQ ID NO: 212) and SEQ ID NO: 86 of WO 2015/112900 (SEQ ID NO: 213)); and/or a light chain comprising an amino acid sequence selected from SEQ ID NO: 42, 46, 54, 58, 62, 66, 70, 74 and 78 of WO 2015/112900 (SEQ ID NO: 42 of WO 2015/112900 ( SEQ ID NO: 214); SEQ ID NO: 46 of WO 2015/112900 (SEQ ID NO: 215); SEQ ID NO: 54 of WO 2015/112900 (SEQ ID NO: 216); SEQ ID NO: 58 of WO 2015/112900 (SEQ ID NO: 217) SEQ ID NO: 62 of WO 2015/112900 (SEQ ID NO: 218) SEQ ID NO: 66 of WO 2015/112900 (SEQ ID NO: 219) SEQ ID WO 2015/112900 SEQ ID NO: 70 (SEQ ID NO: 220), WO 2015/112900 SEQ ID NO: 74 (SEQ ID NO: 221) and WO 2015/112900 SEQ ID NO: 78 (SEQ ID NO: 222)) .

[0313]In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies described in WO 2010/077634 and US Patent No. 8,217,149, the entire disclosures of which are incorporated herein by reference. In illustrative embodiments, the anti-PD-L1 antibody or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain region comprising the amino acid sequence of SEQ ID NO: 20 of WO 2010/077634 (SEQ ID NO:223 ); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 21 of WO 2010/077634 (SEQ ID NO: 224).

[0314]In one embodiment, the targeting moiety comprises one of the anti-PD-L1 antibodies obtainable from the hybridoma accessible under CNCM accession numbers CNCM I-4122, CNCM I-4080 and CNCM I-4081, as described in US 20120039906, the disclosures described in their entirety, which are incorporated herein by reference.

[0315]In one embodiment, the targeting moiety comprises a VHH directed against PD-L1, such as described in US Patent No. 8,907,065 and WO 2008/071447, the entire disclosures of which are incorporated herein by reference. In illustrative embodiments, VHHs against PD-L1 include SEQ ID NO: 394-399 of US Patent No. 8,907,065 (SEQ ID NO: 394 of US Patent No. 8,907,065 (SEQ ID NO: 225); SEQ ID NO: 395 of US Patent No. 8,907,065 (SEQ ID NO: 226), SEQ ID No: 396 of US Patent No. 8,907,065 (SEQ ID NO: 227), SEQ ID No: 397 of US Patent No. 8,907,065 (SEQ ID NO: 228), SEQ ID NO: 398 of US Patent No. 8,907,065 (SEQ ID NO: 229) and SEQ ID NO: 399 of US Patent No. 8,907,065 (SEQ ID NO: 230)).

[0316]In various embodiments, the present multispecific chimeric protein has one or more targeting residues directed against PD-L2. In some embodiments, the chimeric protein has one or more targeting moieties that selectively bind to a PD-L2 polypeptide. In some embodiments, the chimeric protein comprises one or more antibodies, antibody formats or derivatives, peptides or polypeptides, or fusion proteins that selectively bind to a PD-L2 polypeptide.

[0317]In one embodiment, the targeting moiety comprises a VHH directed against PD-L2, such as described in US Patent No. 8,907,065 and WO 2008/071447, the full disclosures of which are incorporated herein by reference. In illustrative embodiments, VHHs against PD-1 include SEQ ID NO: 449-455 of US Patent No. 8,907,065 (SEQ ID NO: 449 of US Patent No. 8,907,065 (SEQ ID NO: 231) SEQ ID NO: 450 of the US -Patents No. 8,907,065 (SEQ ID No: 232) SEQ ID No: 451 of US Patent No. 8,907,065 (SEQ ID No: 233), SEQ ID No: 452 of US Patent No. 8,907,065 (SEQ ID No: 234 ), SEQ ID No.: 453 of US Patent No. 8,907,065 (SEQ ID No.: 235), US Patent No.: 454 No. 8,907,065 (SEQ ID No.: 236) and US Patent No. 8,907,065, SEQ ID NO: 455 (SEQ ID NO: 237). )).

[0318]In one embodiment, the targeting moiety comprises one of the anti-PD-L2 antibodies described in US2011/0271358 and WO2010/036959, the entire contents of which are incorporated herein by reference. In illustrative embodiments, the antibody or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 43-47 of US2011/0271358 (SEQ US2011 ID NO: 43/0271358 (SEQ ID NO: 238), US2011 SEQ ID NO: 44/0271358 (SEQ ID NO: 239), SEQ ID NO: 46 of US2011/0271358 (SEQ ID NO: 241) and SEQ ID NO: 47 of US2011/0271358 (SEQ ID NO: 242)); and/or a light chain comprising an amino acid sequence selected from SEQ ID NO: 48-51 of US2011/0271358 (SEQ ID NO: 48 of US2011/0271358 (SEQ ID NO: 243); SEQ ID NO: 49 of US2011/0271358 (SEQ ID NO: 244), SEQ ID NO: 50 of US2011/0271358 (SEQ ID NO: 245) and SEQ ID NO: 51 of US2011/0271358 (SEQ ID NO: 246)).

[0319]In various embodiments, the targeting moieties of the invention may comprise a sequence that targets PD-1, PD-L1, and/or PD-L2 that is at least about 60%, at least about 61%, at least about 61%, 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any of the sequences described herein (eg. about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69% or about 70%, about 71%, about 72% %, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79% or about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97% % or about 98%, about 99% or about 100% sequence identity with any of the sequences described herein).

[0320]In various embodiments, the targeting moieties of the invention may comprise any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, complementarity determining region (CDR) and flanking region sequences on PD-1, PD-L1 and/or PD-L2 as described herein.

[0321]Additional antibodies, derivatives or antibody formats, peptides or polypeptides or fusion proteins that selectively bind or attack PD-1, PD-L1 and/or PD-L2. , US patent no. 8,779,108, US 2014/0356353, US patent 8,609,089, US 2010/028330, US 2012/0114649, WO 2010/027827, WO 2011/066342, patent US no. 8,907,065 WO 2016/06 101611 WO 2010/027827 WO 2011/066342 WO 2007/005874 WO 2001/014556 US2011/0271358 WO 2011/0271358 2016.02263, WO 2010/077634 and WO 2015/112900, the full descriptions of which are incorporated herein by reference.

[0322]In some embodiments, the targeting moiety is a natural ligand, such as a chemokine. Exemplary chemokines that can be included in the chimeric protein of the invention include, but are not limited to, CCL1, CCL2, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16 restricted , CL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CLL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL143, , CXCL16, CXCL17, XCL1, XCL2, CX3CL1, HCC-4 and LDGF-PBP. In an illustrative embodiment, the targeting moiety may be XCL1, which is a chemokine that recognizes and binds to the XCR1 dendritic cell receptor. In another illustrative embodiment, the targeting moiety is CCL1, which is a chemokine that recognizes and binds CCR8. In another illustrative embodiment, the targeting moiety is CCL2, which is a chemokine that recognizes and binds to CCR2 or CCR9. In another illustrative embodiment, the targeting moiety is CCL3, which is a chemokine that recognizes and binds CCR1, CCR5, or CCR9. In another illustrative embodiment, the targeting moiety is CCL4, which is a chemokine that recognizes and binds CCR1 or CCR5 or CCR9. In another illustrative embodiment, the targeting moiety is CCL5, which is a chemokine that recognizes and binds to CCR1 or CCR3 or CCR4 or CCR5. In another illustrative embodiment, the targeting moiety is CCL6, which is a chemokine that recognizes and binds CCR1. In another illustrative embodiment, the targeting moiety is CCL7, which is a chemokine that recognizes and binds to CCR2 or CCR9. In another illustrative embodiment, the targeting moiety is CCL8, which is a chemokine that recognizes and binds CCR1 or CCR2 or CCR2B or CCR5 or CCR9. In another illustrative embodiment, the targeting moiety is CCL9, which is a chemokine that recognizes and binds CCR1. In another illustrative embodiment, the targeting moiety is CCL10, which is a chemokine that recognizes and binds CCR1. In another illustrative embodiment, the targeting moiety is CCL11, which is a chemokine that recognizes and binds CCR2 or CCR3 or CCR5 or CCR9. In another illustrative embodiment, the targeting moiety is CCL13, which is a chemokine that recognizes and binds CCR2 or CCR3 or CCR5 or CCR9. In another illustrative embodiment, the targeting moiety is CCL14, which is a chemokine that recognizes and binds to CCR1 or CCR9. In another illustrative embodiment, the targeting moiety is CCL15, which is a chemokine that recognizes and binds CCR1 or CCR3. In another illustrative embodiment, the targeting moiety is CCL16, which is a chemokine that recognizes and binds to CCR1, CCR2, CCR5, or CCR8. In another illustrative embodiment, the targeting moiety is CCL17, which is a chemokine that recognizes and binds CCR4. In another illustrative embodiment, the targeting moiety is CCL19, which is a chemokine that recognizes and binds CCR7. In another illustrative embodiment, the targeting moiety is CCL20, which is a chemokine that recognizes and binds CCR6. In another illustrative embodiment, the targeting moiety is CCL21, which is a chemokine that recognizes and binds CCR7. In another illustrative embodiment, the targeting moiety is CCL22, which is a chemokine that recognizes and binds CCR4. In another illustrative embodiment, the targeting moiety is CCL23, which is a chemokine that recognizes and binds CCR1. In another illustrative embodiment, the targeting moiety is CCL24, which is a chemokine that recognizes and binds CCR3. In another illustrative embodiment, the targeting moiety is CCL25, which is a chemokine that recognizes and binds CCR9. In another illustrative embodiment, the targeting moiety is CCL26, which is a chemokine that recognizes and binds CCR3. In another illustrative embodiment, the targeting moiety is CCL27, which is a chemokine that recognizes and binds CCR10. In another illustrative embodiment, the targeting moiety is CCL28, which is a chemokine that recognizes and binds to CCR3 or CCR10. In another illustrative embodiment, the targeting moiety is CXCL1, which is a chemokine that recognizes and binds to CXCR1 or CXCR2. In another illustrative embodiment, the targeting moiety is CXCL2, which is a chemokine that recognizes and binds CXCR2. In another illustrative embodiment, the targeting moiety is CXCL3, which is a chemokine that recognizes and binds CXCR2. In another illustrative embodiment, the targeting moiety is CXCL4, which is a chemokine that recognizes and binds CXCR3B. In another illustrative embodiment, the targeting moiety is CXCL5, which is a chemokine that recognizes and binds CXCR2. In another illustrative embodiment, the targeting moiety is CXCL6, which is a chemokine that recognizes and binds CXCR1 or CXCR2. In another illustrative embodiment, the targeting moiety is CXCL8, which is a chemokine that recognizes and binds CXCR1 or CXCR2. In another illustrative embodiment, the targeting moiety is CXCL9, which is a chemokine that recognizes and binds CXCR3. In another illustrative embodiment, the targeting moiety is CXCL10, which is a chemokine that recognizes and binds CXCR3. In another illustrative embodiment, the targeting moiety is CXCL11, which is a chemokine that recognizes and binds to CXCR3 or CXCR7. In another illustrative embodiment, the targeting moiety is CXCL12, which is a chemokine that recognizes and binds CXCR4 or CXCR7. In another illustrative embodiment, the targeting moiety is CXCL13, which is a chemokine that recognizes and binds CXCR5. In another illustrative embodiment, the targeting moiety is CXCL16, which is a chemokine that recognizes and binds CXCR6. In another illustrative embodiment, the targeting moiety is LDGF-PBP, which is a chemokine that recognizes and binds CXCR2. In another illustrative embodiment, the targeting moiety is XCL2, which is a chemokine that recognizes and binds to XCR1. In another illustrative embodiment, the targeting moiety is CX3CL1, which is a chemokine that recognizes and binds CX3CR1.

[0323]In various embodiments, the present chimeric protein comprises targeting moieties in various combinations. In an illustrative embodiment, the present chimeric protein may comprise two targeting moieties, both targeting moieties being antibodies or derivatives thereof. In another illustrative embodiment, the present chimeric protein may comprise two targeting moieties, where both targeting moieties are natural ligands for cellular receptors. In another illustrative embodiment, the present chimeric protein may comprise two targeting moieties, one of the targeting moieties being an antibody or derivative thereof and the other targeting moiety being a natural ligand for a cellular receptor.

[0324]In various embodiments, the recognition domain of the present chimeric protein functionally modulates (without limitation partially or completely neutralizes) the target (e.g., antigen, receptor) of interest, e.g. B. by inhibiting, reducing or substantially neutralizing a biological effect that the antigen has. For example, different recognition domains may be directed against one or more tumor antigens that actively suppress or have the ability to suppress the immune system of, for example, a patient with a tumor. For example, in some embodiments, the present chimeric protein operatively modulates immune-inhibitory signals (e.g., checkpoint inhibitors), e.g. B. one or more of TIM-3, BTLA, PD-1, CTLA-4, B7-H4, GITR, Galectin -9, HVEM, PD-L1, PD-L2, B7-H3, CD244, CD160, TIGIT, SIRPα, ICOS, CD172a and TMIGD2. For example, in some embodiments, the present chimeric protein is designed to disrupt, block, reduce and/or inhibit the transmission of an immune inhibitory signal, such as binding of PD-1 to PD-L1 or PD-L2 and/or CTLA-4 binding to one or more of AP2M1, CD80, CD86, SHP-2 and PPP2R5A.

[0325]In various embodiments, the recognition domain of the present chimeric protein binds the target (e.g. antigen, receptor) of interest but does not functionally modulate it, e.g. e.g., the recognition domain is or is similar to a binding antibody. For example, in various embodiments, the recognition domain simply targets the antigen or receptor, but does not substantially inhibit, reduce, or modulate a biological effect that the antigen or receptor has. For example, some of the smaller antibody formats described above (e.g. compared to e.g. whole antibodies) have the ability to target difficult to access epitopes and provide a broader range of specific binding sites. In various embodiments, the recognition domain binds to an epitope that is physically distinct from an antigen or a receptor site important to its biological activity (e.g., the antigen's active site).

[0326]Such non-neutralizing binding finds use in various embodiments of the present invention, including methods in which the present chimeric protein is used to target active immune cells directly or indirectly via an effector antigen, such as this document. . For example, in various embodiments, the present chimeric protein can be used to directly or indirectly recruit cytotoxic T cells via CD8 to a tumor cell in a method of shrinking or eliminating a tumor (e.g., the chimeric protein can have an anti-CD8 detection include mechanism). domain and a recognition domain directed against a tumor antigen). In such embodiments, it is desirable to directly or indirectly recruit cytotoxic T cells that express CD8 but do not functionally modulate CD8 activity. Rather, in these embodiments, CD8 signaling is an important part of the tumor reduction or elimination effect. As another example, the present chimeric protein is used in various methods of reducing or eliminating tumors to directly or indirectly recruit dendritic cells (DC) via CLEC9A (e.g., the chimeric protein can contain a recognition domain, an anti-CLEC9A domain and a recognition domain directed against a tumor antigen). In such embodiments, it is desirable to directly or indirectly recruit DCs that express CLEC9A but do not functionally modulate CLEC9A activity. Rather, in these embodiments, CLEC9A signaling is an important part of the tumor reduction or elimination effect.

[0327]In various embodiments, the recognition domain of the present chimeric protein binds to XCR1, e.g. B. on dendritic cells. For example, in some embodiments, the recognition domain comprises all or part of XCL1 or a non-neutralizing anti-XCR1 agent.

[0328]In various embodiments, the recognition domain of the present chimeric protein binds to an immunomodulatory (e.g., immunostimulatory or immunoinhibitory) antigen. In various embodiments, the immunomodulatory antigen is one or more of 4-1 BB, OX-40, HVEM, GITR, CD27, CD28, CD30, CD40, ICOS Ligand; OX-40 Ligand, LIGHT (CD258), GITR Ligand, CD70, B7-1, B7-2, CD30 Ligand, CD40 Ligand, ICOS, ICOS Ligand, CD137 Ligand, and TL1A. In various embodiments, the immunostimulatory antigens are expressed on a tumor cell. In various embodiments, the recognition domain of the present chimeric protein binds, but does not functionally modulate, the immunostimulatory antigens, thus allowing recruitment of cells expressing these antigens without the reduction or loss of their potential tumor reduction or elimination capacity.

[0329]In various embodiments, the recognition domain of the present chimeric protein may be associated with a chimeric protein comprising two recognition domains that exhibit neutralizing activity or two recognition domains that exhibit non-neutralizing (e.g., binding) activity. or comprises a recognition domain with neutralizing activity and a recognition domain with non-neutralizing (e.g. binding) activity.

[0330]In various embodiments, the multispecific chimeric protein has targeting moieties that have recognition domains that specifically bind to a target (e.g., antigen, receptor) that is part of a non-cellular structure. In some embodiments, the antigen or receptor is not an integral part of an intact cell or cell structure. In some embodiments, the antigen or receptor is an extracellular antigen or receptor. In some embodiments, the target is a non-cellular, non-proteinaceous marker, including but not limited to, nucleic acids, including DNA or RNA, such as DNA released from necrotic tumor cells or extracellular deposits such as cholesterol.

[0331]In some embodiments, the target (e.g., antigen, receptor) of interest is part of the non-cellular component of the stroma or extracellular matrix (ECM) or marker associated therewith. As used herein, stroma refers to the supporting and connecting framework of a tissue or organ. The stroma may comprise an assemblage of cells such as fibroblasts/myofibroblasts, glial, epithelial, adipose, immune, vascular, smooth muscle and immune cells along with extracellular matrix (ECM) and extracellular molecules. In various embodiments, the target (e.g., antigen, receptor) of interest is part of the non-cellular component of the stroma, such as. B. the extracellular matrix and extracellular molecules. As used herein, ECM refers to the non-cellular components present in all tissues and organs. The ECM consists of a large collection of biochemically diverse components including proteins, glycoproteins, proteoglycans and polysaccharides, among others. These ECM components are normally produced by neighboring cells and secreted into the ECM by exocytosis. Once secreted, the ECM components often aggregate to form a complex network of macromolecules. In various embodiments, the chimeric protein of the invention comprises a targeting moiety that recognizes a target (e.g. an antigen or a receptor or a non-proteinaceous molecule) located on any component of the ECM. Illustrative components of the ECM include, without limitation, proteoglycans, non-proteoglycan polysaccharides, fibers, and other ECM proteins or non-ECM proteins, e.g. Polysaccharides and/or lipids or ECM-associated molecules (e.g. proteins or non-proteins, e.g. polysaccharides, nucleic acids and/or lipids).

[0332]In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on ECM proteoglycans. Proteoglycans are glycosylated proteins. The basic proteoglycan unit comprises a core protein with one or more covalently linked glycosaminoglycan (GAG) chains. Proteoglycans have a net negative charge that attracts positively charged sodium ions (Na+), which attract water molecules through osmosis, keeping the ECM and resident cells hydrated. Proteoglycans can also help capture and store growth factors within the ECM. Illustrative proteoglycans that can be targeted by the chimeric proteins of the invention include, but are not limited to, heparan sulfate, chondroitin sulfate, and keratan sulfate. In one embodiment, the targeting moiety recognizes a target (eg, antigen, receptor) on non-proteoglycan polysaccharides such as hyaluronic acid.

[0333]In some embodiments, the targeting moiety recognizes a target (e.g., antigen, receptor) on ECM fibers. ECM fibers include collagen fibers and elastin fibers. In some embodiments, the targeting moiety recognizes one or more epitopes on collagens or collagen fibers. Collagens are the most abundant proteins in the ECM. Collagens are present in the ECM as fibrillar proteins and provide structural support for the resident cells. In one or more embodiments, the targeting moiety recognizes and binds to various types of collagens present in the ECM, including but not limited to fibrillar collagens (types I, II, III, V, XI), facit collagens ( types IX, XII, XIV), short-chain collagens (type VIII, X), basement membrane collagens (type IV) and/or type VI, VII or XIII collagens. Elastin fibers give tissues elasticity, allowing them to stretch when needed and then return to their original state. In some embodiments, the target moiety recognizes one or more epitopes on elastins or elastin fibers.

[0334]In some embodiments, the targeting moiety recognizes one or more ECM proteins including, but not limited to, tenascin, fibronectin, fibrin, laminin, or nidogen/entactin.

[0335]In one embodiment, the targeting moiety recognizes and binds tenascin. The tenascin (TN) family of glycoproteins includes at least four members, tenascin-C, tenascin-R, tenascin-X, and tenascin W. The primary structures of tenascin proteins share several common motifs, arranged in the same sequence: amino-terminal heptad- repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III domain repeats, and a carboxyl-terminal fibrinogen-like globular domain. Each protein member is associated with typical variations in the number and nature of fibronectin type III and EGF type repeats. Deviating isoforms also exist, particularly with regard to tenascin-C. More than 27 splice variants and/or isoforms of tenascin-C are known. In a particular embodiment, the targeting moiety recognizes and binds tenascin-CA1. Similarly, tenascin-R also has multiple variants and splicing isoforms. Tenascin-R generally exists as dimers or trimers. Tenascin-X is the largest member of the tenascin family and is known to exist as trimers. Tenascin-W exists as trimers. In some embodiments, the targeting moiety recognizes one or more epitopes on a tenascin protein. In some embodiments, the targeting moiety recognizes monomeric and/or dimeric and/or trimeric and/or hexameric forms of a tenascin protein.

[0336]In one embodiment, the targeting moieties recognize and bind to fibronectin. Fibronectins are glycoproteins that connect cells to collagen fibers in the ECM and allow cells to move across the ECM. Upon binding to integrins, fibronectins unfold to form functional dimers. In some embodiments, the targeting moiety recognizes monomeric and/or dimeric forms of fibronectin. In some embodiments, the targeting moiety recognizes one or more epitopes on fibronectin. In illustrative embodiments, the targeting moiety recognizes extracellular fibronectin domain A (EDA) or extracellular fibronectin domain B (EDB). Elevated EDA levels have been linked to various diseases and disorders, including psoriasis, rheumatoid arthritis, diabetes and cancer. In some embodiments, the targeting moiety recognizes fibronectin containing the EDA isoform and can be used to target the chimeric protein to diseased cells, including cancer cells. In some embodiments, the targeting moiety recognizes fibronectin containing the EDB isoform. In various embodiments, such targeting moieties can be used to target the chimeric protein to tumor cells, including tumor neovasculature.

[0337]In one embodiment, the targeting moiety recognizes and binds to fibrin. Fibrin is another protein substance commonly found in the ECM matrix network. Fibrin is formed by the action of thrombin protease on fibrinogen, causing fibrin to polymerize. In some embodiments, the targeting moiety recognizes one or more epitopes on fibrin. In some embodiments, the targeting moiety recognizes monomeric and polymerized forms of fibrin.

[0338]In one embodiment, the targeting moiety recognizes and binds to laminin. Laminin is a major component of the basal lamina, which is a protein network foundation for cells and organs. Laminins are heterotrimeric proteins containing an α chain, a β chain, and a γ chain. In some embodiments, the targeting moiety recognizes one or more epitopes on laminin. In some embodiments, the targeting moiety recognizes monomeric, dimeric, and trimeric forms of laminin.

[0339]In one embodiment, the targeting moiety recognizes and binds a nidogen or entactin. The nidogenes/entactins are a family of highly conserved sulfated glycoproteins. They form the main structural component of basement membranes and bind the laminin and collagen IV networks in basement membranes. Members of this family include nidogen-1 and nidogen-2. In various embodiments, the targeting moiety recognizes an epitope on nidogen-1 and/or nidogen-2.

[0340]In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an epitope present on any of the targets (e.g., ECM proteins) described herein. In one embodiment, the antigen recognition domain recognizes one or more linear epitopes present on the protein. As used herein, a linear epitope refers to any continuous sequence of amino acids present in the protein. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on the protein. As used herein, a conformational epitope refers to one or more stretches of amino acids (which may be discontinuous) that form a three-dimensional surface with features and/or shapes and/or tertiary structures that can be recognized by an epitope recognition domain. Antigen.

[0341]In various embodiments, the targeting moiety may be provided with full-length and/or mature forms and/or isoforms and/or variants and/or splice fragments and/or any other natural or synthetic analogue, variant or mutant of any target (e.g. ECM- proteins) described herein. In various embodiments, the targeting moiety can bind to any form of the proteins described herein, including monomeric, dimeric, trimeric, tetrameric, heterodimeric, multimeric, and associated forms. In various embodiments, the targeting moiety can bind to any post-translationally modified form of the proteins described herein, such as e.g. B. glycosylated and / or phosphorylated forms.

[0342]In various embodiments, the targeting moiety includes an antigen recognition domain that recognizes extracellular molecules such as DNA. In some embodiments, the targeting moiety includes an antigen recognition domain that recognizes DNA. In one embodiment, DNA is shed into the extracellular space from necrotic or apoptotic tumor cells or other diseased cells.

[0343]In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures associated with atherosclerotic plaques. Two types of atherosclerotic plaque are known. Fibrolipid plaque (fibrofat) is characterized by an accumulation of lipid-laden cells beneath the intima of arteries. Underneath the endothelium is a fibrous cap covering the atheromatous core of the plaque. The nucleus includes lipid-laden cells (macrophages and smooth muscle cells) with elevated tissue cholesterol and cholesterol esters, fibrin, proteoglycans, collagen, elastin, and cellular debris. In advanced plaques, the central core of the plaque often contains extracellular cholesterol deposits (released from dead cells) that form arrays of cholesterol crystals with empty, needle-like slits. At the periphery of the plate are capillaries and younger foam cells. Fibrous plaque is also located below the intima within the wall of the artery, causing thickening and distension of the wall, and sometimes localized irregular narrowing of the lumen with some atrophy of the muscular layer. Fibrous plaque contains collagen fibers (eosinophil), calcium precipitates (hematoxylinophil), and lipid-laden cells. In some embodiments, the targeting moiety recognizes and binds to one or more of the non-cellular components of these plaques, such as fibrin, proteoglycans, collagen, elastin, cellular debris, and calcium or other mineral deposits or precipitates. In some embodiments, the cellular debris is a nucleic acid, e.g. DNA or RNA released from dead cells.

[0344]In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures found in brain plaques associated with neurodegenerative diseases. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures found in amyloid plaques found in the brains of patients with Alzheimer's disease. For example, the targeting moiety can recognize and bind to amyloid beta peptide, which is a major component of amyloid plaques. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures found in brain plaques found in patients with Huntington's disease. In various embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures found in plaques associated with other neurodegenerative or musculoskeletal disorders such as Lewy body dementia and inclusion body myositis.

[0345]Linkers and functional groups

[0346]In various embodiments, the present chimeric protein may comprise one or more functional groups, residues or moieties. In various embodiments, one or more functional groups, residues, or moieties are genetically linked or fused to any of the signaling agents or targeting moieties (e.g., SIRP1α) described herein. In some embodiments, the functional groups, residues, or moieties impart one or more desired properties or functionality to the subject chimeric protein of the invention. Examples of such functional groups and techniques for their introduction into the present chimeric protein are known in the art, see e.gRemington Pharmaceutical Sciences,16. Ausgabe, Mack Publishing Co., Easton, Pennsylvania (1980).

[0347]In various embodiments, the present chimeric protein may be conjugated and/or fused to another agent to increase half-life or otherwise improve pharmacodynamic and pharmacokinetic properties. In some embodiments, the present chimeric protein may be conjugated with one or more of PEG, XTEN (e.g. as rPEG), polysialic acid (POLYXEN), albumin (e.g. human serum albumin or HAS), a protein like elastin (ELP) , PAS, PAH, GLK, CTP, transferrin and the like. In some embodiments, the present chimeric protein can be fused or conjugated to an antibody or an antibody fragment such as an Fc fragment. For example, the chimeric protein can be fused to either the N-terminus or the C-terminus of the Fc domain of human immunoglobulin (Ig)G. In various embodiments, each of the individual chimeric proteins is fused to one or more of the agents described in BioDrugs (2015) 29:215-239, the entire contents of which are incorporated herein by reference.

[0348]In some embodiments, the functional groups, moieties, or moieties comprise a suitable pharmacologically acceptable polymer, such as polyethylene glycol (PEG) or derivatives thereof (such as methoxypoly(ethylene glycol) or mPEG). In some embodiments, binding of the PEG moiety increases the half-life and/or reduces the immunogenicity of the SIRP1α-binding protein. In general, any suitable form of pegylation can be used, such as e.g. B. Pegylation, used in the art for antibodies and antibody fragments (including but not limited to single domain antibodies such as VHH); see for example Chapman,nat. Biotech.,54 , 531-545 (2002); by Veronese and Harris,Extended drug delivery Rev.54, 453-456 (2003), by Harris and Chess,nat. Rev. Drug. Discovery,2, (2003) and in WO04060965, the entire content of which is incorporated herein by reference. Various reagents for pegylation of proteins are also commercially available, for example from Nektar Therapeutics, USA. In some embodiments, site-directed pegylation is used, particularly through a cysteine ​​residue (see, for example, Yang et al., Protein Engineering, 16, 10, 761-770 ( 2003), the entire contents of which are incorporated herein by reference). For example, PEG can be linked to a cysteine ​​residue naturally found in the present chimeric protein of the invention for this purpose. In some embodiments, the present chimeric protein of the invention is modified to appropriately introduce one or more cysteine ​​residues for PEG binding, or an amino acid sequence comprising one or more cysteine ​​residues for PEG binding can be fused to the amino acid. and/or the carboxy terminus of the present chimeric protein using techniques known in the art.

[0349]In some embodiments, the functional groups, moieties, or moieties include N-linked or O-linked glycosylation. In some embodiments, N-linked or O-linked glycosylation is introduced as part of a co-translational and/or post-translational modification.

[0350]In some embodiments, the functional groups, moieties, or moieties include one or more detectable labels or other signal-generating groups or moieties. Labels and suitable techniques for attaching, using and detecting the same are known in the art and include, but are not limited to, fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine and fluorescent Metals such as Eu or other metals of the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (such as luminal, isoluminol, theromatic acridinium ester, imidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogues), radioisotopes, metals, chelates of metals or metal cations or other metals or metal cations particularly suitable for use in diagnostics and imaging in vivo, in vitro or in situ, as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta isomerase - V-steroid, yeast alcohol Dehydrogenase, Alpha-Glycerophosphate Dehydrogenase, Triose Phosphate Isomerase, Biotinavidin Peroxidase, Horseradish Peroxidase, Alkaline Phosphatase, Asparaginase, Glucose Oxidase, Beta-Galactosidase, Ribonuclease, Urease, Catalase, Glucose VI Phosphate Dehydrogenase, Glucoamylase , and acetylcholinesterase). Other suitable labels include fractions that can be detected by NMR or ESR spectroscopy. For example, the labeled polypeptides and VHHs of the invention can be used for in vitro, in vivo or in situ assays (including immunoassays known per se as ELISA, RIA, EIA and other "sandwich assays" etc.) as well are used for research, diagnostics and in vivo imaging, depending on the choice of specific label.

[0351]In some embodiments, the functional groups, residues, or moieties comprise a tag that is genetically linked or fused to the chimeric protein. In some embodiments, the present chimeric protein may contain a single tag or multiple tags. The tag is, for example, a peptide, sugar or DNA molecule that does not inhibit or prevent binding of the subject chimeric protein to SIRP1α or any other antigen of interest, such as tumor antigens. In various embodiments, the tag is at least about: three to five amino acids in length, five to eight amino acids in length, eight to twelve amino acids in length, twelve to fifteen amino acids in length, or fifteen to twenty amino acids in length. Illustrative labels are described, for example, in US Patent Publication No. US2013/0058962. In some embodiments, the tag is an affinity tag, such as B. Glutathione S-transferase (GST) and histidine (His) tag. In one embodiment, the present chimeric protein comprises a His tag.

[0352]In some embodiments, the functional groups, moieties or moieties include a chelating group, e.g. B. to chelate one of the metals or metal cations. Suitable chelating groups include, for example, without limitation, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0353]In some embodiments, the functional group, moiety, or moiety comprises a functional group that is part of a specific binding pair, such as the biotin-(strepto)avidin binding pair. Such a functional group can be used to link the present chimeric protein of the invention to another protein, polypeptide or chemical which binds to the other half of the binding pair, ie through binding pair formation. For example, a subject chimeric protein of the invention may be biotin-conjugated and linked to another avidin- or streptavidin-conjugated protein, polypeptide, compound or carrier. For example, such a conjugated subject chimeric protein can be used as a reporter, e.g. B. in a diagnostic system in which a detectable signal generating agent is conjugated to avidin or streptavidin. Such binding pairs can also be used, for example, to link the present chimeric protein to a carrier, including carriers suitable for pharmaceutical use. A non-limiting example is the liposomal formulations described by Cao and Suresh.Drug-Targeting-Magazine,8, 4, 257 (2000). Such binding pairs can also be used to attach a therapeutically active agent to the chimeric protein of the invention.

[0354]In some embodiments, the subject chimeric protein optionally comprises one or more linkers. In some embodiments, the subject chimeric protein comprises a linker connecting the targeting moiety and the signaling agent. In some embodiments, the present chimeric protein comprises a linker within the signaling agent (eg, in the case of single-chain TNF, which may comprise two linkers to produce a trimer).

[0355]In some embodiments, vectors are provided that encode the present chimeric proteins linked as a single nucleotide sequence with any of the linkers described herein and can be used to produce such chimeric proteins.

[0356]In some embodiments, the length of the linker allows for efficient binding of a targeting moiety and a signaling agent to their receptors. For example, in some embodiments, the length of the linker allows for efficient binding of one of the targeting moieties and the signaling agent to receptors in the same cell and efficient binding of the other targeting moiety to a different cell. Illustrative cell pairs are provided elsewhere herein.

[0357]In some embodiments, the length of the linker is at least equal to the minimum distance between the binding sites of one of the targeting moieties and the signaling agent at receptors on the same cell. In some embodiments, the length of the linker is at least 2, 3, 4, 5, 10, 20, 25, 50, 100 or more times the minimum distance between the binding sites of one of the targeting moieties and the signaling molecule receptors on the same cell.

[0358]As described herein, the length of the linker allows for efficient binding of one of the targeting moieties and the signaling agent to receptors on the same cell, with binding occurring sequentially, e.g. Target moiety/receptor binding preceding receptor/signalling agent binding.

[0359]In some embodiments there are two linkers in a single chimera, each connecting the signaling means to an addressing unit. In various embodiments, the linkers have lengths that allow formation of a site containing a diseased cell and an effector cell without steric hindrance that would prevent modulation of either cell.

[0360]The invention contemplates the use of a variety of linker sequences. In various embodiments, the linker can be derived from natural multidomain proteins or empirical linkers, such as described in Chichili et al., (2013) Protein Sci. 22(2):153-167, Chen et al., (2013) Adv Drug Deliv Rev. 65(10):1357-1369, the entire contents of which are incorporated herein by reference. In some embodiments, the linker can be designed using linker design databases and computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10):1357-1369 and Crasto et al., (2000) , Protein Ing. 13(5): 309-312, the entire contents of which are incorporated herein by reference. In various embodiments, the linker can be functional. For example, without limitation, the linker can serve to enhance folding and/or stability, enhance expression, enhance pharmacokinetics, and/or enhance bioactivity of the present chimeric protein.

[0361]In some embodiments, the linker is a polypeptide. In some embodiments, the linker is less than about 100 amino acids in length. For example, the linker can be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length. In some embodiments, the linker is a polypeptide. In some embodiments, the linker is longer than about 100 amino acids. For example, the linker can be greater than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids in length. In some embodiments, the linker is flexible. In another embodiment, the linker is rigid.

[0362]In some embodiments, a linker connects the two targeting entities together and this linker is short in length and a linker connects a targeting entity and a signaling agent. This linker is longer than the linker connecting the two orientation units. For example, the difference in amino acid length between the linker connecting the two targeting moieties and the linker connecting a targeting moiety and a signaling agent can be about 100, about 95, about 90, about 85, about 80, about 75 , about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3 or about 2 amino acids.

[0363]In various embodiments, the linker consists essentially of glycine and serine residues (e.g., about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%, or about 95%). % or about 97% glycines and serines). For example, in some embodiments (Gly₄Be)_Norte, where n is from about 1 to about 8, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NO: 247 - SEQ ID NO: 254). In one embodiment, the linker sequence is GGGGGSGGGGGSGGGGS (SEQ ID NO: 255). Additional illustrative linkers include, but are not limited to, linkers having the sequence LE, GGGGS (SEQ ID NO: 247), (GGGGS)._Norte(n = 1–4) (SEQ ID NO: 247 – SEQ ID NO: 250), (Gly)₈(SEQ ID NO: 256), (Gly)₆(SEQ ID NO: 257), (EAAAK)_Norte(n = 1–3) (SEQ ID NO: 258 – SEQ ID NO: 260), A (EAAAK)_NorteA (n=2-5) (SEC-ID-Nr.: 261-SEC-ID-Nr.: 264), AEAAAAKEAAAKA (SEC-ID-Nr.: 261), A (EAAAK)₄ALEA (EAAAAK)₄A (SEC ID No: 265), PAPAP (SEC ID No: 266), KESGSVSSEQLAQFRSLD (SEC ID No: 267), EGKSSGSGSESKST (SEC ID No: 268), GSAGSAAGGEF ( SEC-ID-Nr.: 269) y (XP)_Norte, where X denotes any amino acid, e.g. B. Ala, Lys or Glu. In various embodiments, the linker is GGS.

[0364]In some embodiments, the linker is one or more of GGGSE (SEQ ID NO: 270), GSESG (SEQ ID NO: 271), GSEGS (SEQ ID NO: 272), GEGGGSGEGSSGEGSSSSEGGGGSEGGGSEGGGSEGGS (SEQ ID NO: 273), and a linker from G , S and E randomly placed every 4 amino acid intervals.

[0365]In some embodiments, the linker is a hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). In various embodiments, the linker is a hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region, found in antibodies of the IgG, IgA, IgD, and IgE classes, acts as a flexible spacer, allowing the Fab portion to move freely in space. Unlike constant regions, hinge domains are structurally diverse, varying in both sequence and length between immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies between IgG subclasses. The IgG1 hinge region spans amino acids 216–231, and because it is freely flexible, Fab fragments can rotate about their axes of symmetry and move within a sphere centered on the first of the two disulfide bridges between the heavy chains. IgG2 has a shorter hinge than IgG1 with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacks a glycine residue, is relatively short, and contains a rigid polyproline double helix stabilized by additional heavy chain disulfide bonds. These properties limit the flexibility of the IgG2 molecule. IgG3 is distinguished from the other subclasses by its unique extended hinge region (approximately four times longer than the IgG1 hinge) containing 62 amino acids (including 21 prolines and 11 cysteines) forming an inflexible polyproline double helix. In IgG3, the Fab fragments are relatively distant from the Fc fragment, giving the molecule greater flexibility. The elongated hinge in IgG3 is also responsible for its higher molecular weight compared to the other subclasses. The hinge region of IgG4 is shorter than that of IgG1 and its flexibility is between that of IgG1 and IgG2. It is reported that the flexibility of the hinge regions decreases in the order of IgG3 > IgG1 > IgG4 > IgG2.

[0366]Based on crystallographic studies, the immunoglobulin hinge region can be functionally divided into three regions: the upper hinge region, the central region, and the lower hinge region. See Shin et al., 1992.Immunological reviews130:87. The upper hinge region includes amino acids from the carboxyl terminus of C_H1to the first residue in the hinge that restricts movement, usually the first cysteine ​​residue that forms a disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The central hinge region contains the disulfide bonds between heavy chains, and the lower hinge region binds to the N-terminus of C_H2Domain y contains residues in C_H2. ID. The core-hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 274) which, when dimerized via disulfide bond formation, results in a cyclic octapeptide from which it is assumed to act as a fulcrum. giving flexibility. In various embodiments, the present linker comprises one, two or three of the superior hinge region, the central region and the inferior hinge region of any antibody (e.g. IgG, IgA, IgD and IgE, including subclasses (e.g. . , IgG1, IgG2, IgG3 and IgG4 and IgA1 and IgA2)). The hinge region may also contain one or more glycosylation sites, including several structurally distinct types of carbohydrate binding sites. For example, IgA1 contains five glycosylation sites within a 17-amino acid segment of the hinge region, conferring resistance to intestinal proteases on the hinge region polypeptide, which is considered an advantageous property for a secretory immunoglobulin. In various embodiments, the linker of the present invention comprises one or more glycosylation sites. In various embodiments, the linker is a hinge CH2-CH3 domain of a human IgG4 antibody.

[0367]If desired, the present chimeric protein can be linked to an antibody Fc region encoding one or both of C_H2 years c_H3 domains and optionally a hinge region. For example, vectors encoding the present chimeric proteins linked as a single nucleotide sequence to an Fc region can be used to produce such polypeptides.

[0368]In some embodiments, the linker is a synthetic linker such as PEG.

[0369]In various embodiments, the linker can be functional. For example, without limitation, the linker can serve to enhance folding and/or stability, enhance expression, enhance pharmacokinetics, and/or enhance bioactivity of the present chimeric protein. In another example, the linker can serve to target the chimeric protein to a particular cell type or location.

[0370]Modifications and production of chimeric proteins

[0371]In various embodiments, the present chimeric protein comprises a targeting moiety (e.g., SIRP1α) that is a VHH. In various embodiments, the VHH is not limited to a specific biological source or manufacturing process. For example, the VHH can usually be obtained by: (1) isolating the V_HH domain of a naturally occurring heavy chain antibody; (2) by expression of a nucleotide sequence encoding a wild-type V_Hdomain H; (3) by "humanizing" a natural V_HH domain or by expression of a nucleic acid encoding the humanized V domain_Hdomain H; (4) by "camelizing" a naturally occurring VH domain from any animal species, such as a mammalian species, such as a human, or by expressing a nucleic acid encoding the camelated VH domain; (5) by "camelization" of a "domain antibody" or "Dab" as described in the prior art, or by expression of a nucleic acid encoding such a camelized VH domain; (6) use of synthetic or semi-synthetic techniques to produce proteins, polypeptides or other amino acid sequences known in the art; (7) preparing a nucleic acid encoding a VHH using techniques for nucleic acid synthesis known in the art, followed by expression of the resulting nucleic acid; and/or (8) any combination of one or more of the foregoing.

[0372]In one embodiment, the chimeric protein comprises a VHH corresponding to V_HH domains of naturally occurring heavy chain antibodies directed against human SIRP1α. In some embodiments, like V_HH sequences can generally be generated or obtained by appropriately immunizing a camelid species with a SIRP1α molecule (i.e., to generate an immune response and/or heavy chain antibodies directed against SIRP1α) by obtaining a sample of the correct biology of the camelid becomes. (e.g. a blood sample or any B cell sample) and the generation of V_HH sequences directed against SIRP1α from the sample using any known suitable technique. In some embodiments, naturally occurring V_HH domains against SIRP1α can be obtained from naïve camelid V libraries_HB. by screening such a library using SIRP1α or at least a part, fragment, antigenic determinant or epitope thereof using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO 9937681, WO 0190190, WO 03025020 and WO 03035694, the entire contents of which are incorporated herein by reference. In some embodiments, enhanced synthetic or semi-synthetic libraries derived from naïve V_HH libraries can be used, like V_HLibraries H obtained from naïve V_HH libraries by techniques such as random mutagenesis and/or CDR shuffling, such as those described in WO0043507, the entire contents of which are incorporated herein by reference. In some embodiments, another technique for obtaining V_HH sequences directed against SIRP1α include appropriately immunizing a transgenic mammal capable of expressing anti-heavy chain antibodies (i.e., to generate an immune response and/or anti-SIRP1α heavy chain antibodies), obtaining an appropriate biological sample from the mammal. like a blood sample or a B cell sample) and then generate V_HH sequences directed against SIRP1α from the sample using any known suitable technique. For example, mice expressing heavy chain antibodies and additional methods and techniques described in WO02085945 and WO04049794 (the entire contents of which are incorporated herein by reference) can be used for this purpose.

[0373]In one embodiment, the chimeric protein comprises a VHH that has been "humanized," ie, by replacing one or more amino acid residues in the naturally occurring V amino acid sequence._HH sequence (and particularly flanking sequences) by one or more of the amino acid residues found at the corresponding position(s) in a VH domain of a conventional 4-chain antibody of an individual human subject. This can be done using humanization techniques known in the art. In some embodiments, potential humanizing substitutions or combinations of humanizing substitutions can be determined by methods known in the art, for example, by comparing the sequence of a VHH and the sequence of a natural human VH domain. In some embodiments, the humanizing substitutions are chosen such that the resulting humanized VHHs still retain advantageous functional properties. In general, the VHHs of the invention may become more "human-like" as a result of humanization, while retaining favorable characteristics, such as e.g. B. a reduced immunogenicity maintained._HH domains. In various embodiments, the humanized VHHs of the invention may be obtained in any suitable manner known in the art and are therefore not strictly limited to polypeptides obtained using a polypeptide comprising a natural V._HH domain as starting material.

[0374]In one embodiment, the chimeric protein comprises a VHH that has been "camelized," i. H. by replacing one or more amino acid residues in the amino acid sequence of a native VH domain of a conventional 4-chain antibody with one or more of the following: amino acid residues found at the corresponding position(s) in a V_HH domain of a camelid heavy chain antibody. In some embodiments, such "camelization" substitutions are introduced at amino acid positions that form and/or are present at the VH-VL interface and/or at so-called Camelidae signature residues (see, for example, WO9404678, the entirety of which is incorporated herein by reference). reference is included). In some embodiments, the VH sequence used as starting material or starting point for generating or constructing the camel VHH is a mammalian VH sequence, for example the human VH sequence, such as a VH3 sequence. In various embodiments, camelized VHHs can be obtained in any suitable manner known in the art (i.e., as specified in (1)-(8) above) and are therefore not strictly limited to polypeptides obtained using a polypeptide comprising a Naturally occurring VH domain as starting material.

[0375]In various embodiments, both "humanization" and "camelization" can be performed by providing a nucleotide sequence encoding a wild-type V._HH domain or VH domain and then changing one or more codons in the nucleotide sequence in a manner known in the art such that the new nucleotide sequence encodes a "humanized" or "camelized" VHH. This nucleic acid can then be expressed in a manner known in the art to provide the desired VHH of the invention. Alternatively, based on the amino acid sequence of a natural V_HIn the H domain or VH domain, the desired humanized or camelized VHH amino acid sequence of the invention can be designed and then synthesized de novo using techniques known in the art for peptide synthesis. Furthermore, depending on the amino acid sequence or nucleotide sequence of a natural V_HH domain or VH domain, a nucleotide sequence encoding the desired humanized or camelized VHH, respectively, can be designed and then synthesized de novo using art-known nucleic acid synthesis techniques, after which it can thus obtain the nucleic acid to be expressed. in a manner known in the art to provide the desired VHH of the invention. Other suitable methods and techniques for obtaining the VHHs of the invention and/or the nucleic acids encoding them from natural VH or V sequences_HH sequences are known in the art and can, for example, be combining one or more parts of one or more natural VH sequences (such as one or more FR sequences and/or CDR sequences) with one or more parts of one or more more natural v_HH sequences (such as one or more FR sequences or CDR sequences) and/or one or more synthetic or semi-synthetic sequences, suitable to provide a VHH of the invention or a nucleotide sequence or nucleic acid encoding the same.

[0376]Methods for producing the chimeric proteins of the invention are described herein. For example, the DNA sequences encoding the chimeric proteins of the invention (e.g., the DNA sequences encoding the modified signaling agent and the targeting moiety and linker) can be isolated using those known in the art Methods are chemically synthesized. The synthetic DNA sequences can be ligated to other suitable nucleotide sequences, including, for example, expression control sequences, to produce gene expression constructs encoding the desired chimeric proteins. Accordingly, in various embodiments, the present invention provides isolated nucleic acids comprising a nucleotide sequence encoding the chimeric protein of the invention.

[0377]Nucleic acids encoding the chimeric protein of the invention can be incorporated (ligated) into expression vectors, which can be introduced into host cells by transfection, transformation, or transduction techniques. For example, nucleic acids encoding the chimeric protein of the invention can be introduced into host cells by retroviral transduction. Illustrative host cells areE coliCells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney (COS) cells, human hepatocellular carcinoma cells (e.g. Hep G2) and myeloma cells. The transformed host cells can be cultured under conditions which allow the host cells to express the genes encoding the chimeric protein of the invention. Accordingly, in various embodiments, the present invention provides expression vectors comprising nucleic acids encoding the chimeric protein of the invention. In various embodiments, the present invention further provides host cells comprising such expression vectors.

[0378]Specific expression and purification conditions will vary depending on the expression system used. For example, when a gene is to be expressedE coliit is first cloned into an expression vector by placing the modified gene downstream of an appropriate bacterial promoter, eg Trp or Tac, and a prokaryotic signal sequence. In another example, if the designed gene is expressed in eukaryotic host cells, e.g. B. CHO cells, is to be expressed, it is first inserted into an expression vector z. B. contains a suitable eukaryotic promoter, a secretion signal, enhancers and various introns. The gene construct can be introduced into host cells using transfection, transformation, or transduction techniques.

[0379]The chimeric protein of the invention can be produced by culturing a host cell transfected with an expression vector encoding the chimeric protein under conditions allowing expression of the protein. After expression, the protein can be harvested and purified using techniques well known in the art, for example affinity tags such as glutathione-S-transferase (GST) and histidine tags, or by chromatography.

[0380]Accordingly, in various embodiments, the present invention provides a nucleic acid encoding a chimeric protein of the present invention. In various embodiments, the present invention provides a host cell comprising a nucleic acid encoding a chimeric protein of the present invention.

[0381]In various embodiments, the present SIRP1α targeting moiety or chimeric protein comprising same may be expressed in vivo, e.g. B. in a patient. For example, in various embodiments, the subject SIRP1α targeting entity, or chimeric proteins comprising the same, may be administered in the form of a nucleic acid encoding the subject SIRP1α targeting entity, or chimeric proteins comprising the same. In various embodiments, the nucleic acid is DNA or RNA. In some embodiments, the present SIRP1α targeting moiety or chimeric protein comprising it is encoded by a modified mRNA, ie an mRNA comprising one or more modified nucleotides. In some embodiments, the modified mRNA comprises one or more modifications found in US Patent No. 8,278,036, the entire contents of which are incorporated herein by reference. In some embodiments, the modified mRNA comprises one or more of m5C, m5U, m6A, ε2U, Ψ, and 2'-O-methyl-U. In some embodiments, the present invention relates to the administration of a modified mRNA encoding one or more of the present chimeric proteins. In some embodiments, the present invention relates to gene therapy vectors comprising the same. In some embodiments, the present invention relates to gene therapy methods comprising the same. In various embodiments, the nucleic acid is in the form of an oncolytic virus, e.g. an adenovirus, reovirus, measles, herpes simplex, Newcastle disease virus or vaccinia.

[0382]Pharmaceutically acceptable salts and excipients

[0383]The chimeric proteins described herein can possess a sufficiently basic functional group that can react with an inorganic or organic acid, or a carboxyl group that can react with an inorganic or organic base to form a pharmaceutically acceptable salt. A pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid as is well known in the art. Such salts include the pharmaceutically acceptable salts listed, for example, inJournal of Pharmaceutical Sciences,66, 2-19 (1977) andThe Handbook of Pharmaceutical Salts; Properties, selection and use. P.H. Stahl and C.G. Wermuth (eds.), Verlag, Zurich, Switzerland 2002, which are incorporated herein by reference in their entirety.

[0384]Pharmaceutically acceptable salts include, as a non-limiting example, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, hydrogen phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate , Succinate, Maleate, Gentisinate, Fumarate, Gluconate, Glucoronate, Saccharate, Formate, Benzoate, Glutamate, Methanesulfonate, Ethanesulfonate, Benzenesulfonate, p-Toluenesulfonate, Camphorsulfonate, Pamoate, Phenylacetate, Trifluoroacetate, Acrylate, Chlorobenzoate, Dinitrobenzoate, Hydroxybenzoate, Methoxybenzoate, Methylbenzoate , o-acetoxybenzoate, naphthalene-2-benzoate, isobutyrate, phenylbutyrate, α-hydroxybutyrate, butyne-1,4-dicarboxylate, hexyne-1,4-dicarboxylate, caprate, caprylate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate , Malonate, Mandelate, Mesylate, Nicotinate, Phthalate, Terephthalate, Propiolate, Propionate, Phenylpropionate, Sebacate, Suberate, p-Bromobenzenesulfonate, Chlorobenzenesulfonate, Ethylsulfonate, 2-Hydroxyethylsulfonate, Methylsulfonate, Naphthalene-1-sulfonate, Naphthalene-2-sulfonate, Naphthalene -1,5-sulfonate, xylene sulfonate and tartrate salts.

[0385]The term "pharmaceutically acceptable salt" also refers to a salt of the compositions of the present invention with an acid functional group, such as a carboxylic acid functional group, and a base. Suitable bases include alkali metal hydroxides such as sodium, potassium and lithium; alkaline earth metal hydroxides such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; Ammonia and organic amines such as mono-, di- or trialkylamines, unsubstituted or hydroxy-substituted dicyclohexylamine; tributylamine; pyridine; N-methyl, N-ethylamine; diethylamine; triethylamine; mono-, bis- or tris-(2-OH-lower alkylamines) such as mono-; Bis- or tris-(2-hydroxyethyl)amine, 2-hydroxy-tert-butylamine or tris-(hydroxymethyl)methylamine, N,N-di-lower-alkyl-N-(hydroxy-lower-alkyl)-amines, such as N ,N-dimethyl-N-(2-hydroxyethyl)amine or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine and the like.

[0386]In some embodiments, the compositions described herein are in the form of a pharmaceutically acceptable salt.

[0387]Compositions and pharmaceutical formulations

[0388]In various embodiments, the present invention relates to pharmaceutical compositions comprising the chimeric proteins described herein and a pharmaceutically acceptable carrier or excipient. Any pharmaceutical composition described herein can be administered to a patient as part of a composition that includes a pharmaceutically acceptable carrier or vehicle. Such compositions may optionally comprise a suitable amount of a pharmaceutically acceptable excipient to provide a form for convenient administration.

[0389]In various embodiments, the pharmaceutical excipients can be liquid, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Pharmaceutical excipients can be, for example, saline, acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliaries, stabilizers, thickeners, lubricants and colorants can be used. In one embodiment, the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful adjuvant when any agent described herein is administered intravenously. Saline and aqueous dextrose and glycerol solutions may also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol. and the same. Any composition described herein may also comprise, if desired, minor amounts of wetting or emulsifying agents or pH buffering agents. Further examples of suitable pharmaceutical excipients are described inRemington Pharmaceutical Sciences1447-1676 (Alfonso R. Gennaro ed., 19th ed. 1995), incorporated herein by reference.

[0390]The present invention encompasses the described pharmaceutical compositions (and/or additional therapeutic agents) in various formulations. All of the pharmaceutical compositions of the invention (and/or additional therapeutic agents) described herein may be in the form of solutions, suspensions, emulsions, drops, tablets, pills, granules, capsules, liquid capsules, gelatine capsules, powders, sustained release formulations, suppositories, emulsions, Aerosol, spray, suspension, lyophilized powder, frozen suspension, dried powder, or any other form suitable for use. In one embodiment, the composition is in the form of a capsule. In another embodiment, the composition is in tablet form. In yet another embodiment, the pharmaceutical composition is formulated in the form of a soft gelatin capsule. In another embodiment, the pharmaceutical composition is formulated in the form of a gelatin capsule. In yet another embodiment, the pharmaceutical composition is formulated as a liquid.

[0391]If required, the pharmaceutical compositions (and/or additional agents) according to the invention may also contain a solubilizing agent. In addition, the agents can be administered with any suitable delivery vehicle or device such as are known in the art. The combination therapies described herein can be administered simultaneously in a single delivery vehicle or delivery device.

[0392]Formulations comprising the inventive pharmaceutical compositions (and/or additional agents) of the present invention are conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier that is one or more accessory ingredients. Formulations are generally prepared by uniformly and intimately bringing into association the therapeutic agent with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., dry granulation, powder blends). etc., followed by tabletting using conventional methods known in the art).

[0393]In various embodiments, each pharmaceutical composition (and/or additional agent) described herein is formulated according to routine methods as a composition adapted for a mode of administration described herein.

[0394]Routes of administration include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, inhalation, or topical. Administration can be local or systemic. In some embodiments, administration is by the oral route. In another embodiment, administration is by parenteral injection. The mode of administration can be left to the discretion of the physician and will depend in part on the site of the medical condition. In most cases, administration will result in the release of any agent described herein into the bloodstream.

[0395]In one embodiment, the chimeric protein described herein is formulated according to routine methods as a composition adapted for oral administration. Compositions for oral administration may, for example, be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs. Orally administered compositions may comprise one or more active ingredients, for example sweeteners such as fructose, aspartame or saccharin; flavoring agents such as peppermint, wintergreen or cherry oil; dyes; and preservatives to provide a pharmaceutically palatable preparation. In addition, when in tablet or pill form, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a prolonged period of time. Selectively permeable membranes surrounding an osmotically active protein driving any chimeric protein described herein are also suitable for orally administered compositions. In these latter platforms, liquid from the environment surrounding the capsule is absorbed by the propellant compound, which swells to force the agent or agent composition through an orifice. These delivery platforms can provide an essentially zero-order delivery profile compared to the spiked profiles of immediate-release formulations. A time delay material such as glycerol monostearate or glycerol stearate may also be useful. Oral compositions may contain standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose and magnesium carbonate. In one embodiment, the excipients are pharmaceutical grade. The suspensions may contain, in addition to the active ingredients, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, etc., and mixtures. of that.

[0396]Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be prepared in the form of sterile solid compositions (e.g. lyophilized compositions) which can be dissolved or suspended in a sterile injectable medium immediately before use. For example, they may contain suspending or dispersing agents known in the art. Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and tonicity adjusters such as sodium chloride or dextrose.

[0397]For intravenous administration, suitable vehicles include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). The vehicle must be stable under the conditions of manufacture and storage and preserved against microorganisms. The carrier may be a solution or dispersion medium containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol) and suitable mixtures thereof.

[0398]The compositions provided herein, alone or in combination with other suitable components, may be converted into aerosol (ie, "nebulized") formulations for administration by inhalation. Aerosol formulations can be placed in pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

[0399]All of the pharmaceutical compositions of the invention (and/or additional agents) described herein may be administered by controlled release or sustained release means or by delivery devices well known to those skilled in the art. Examples include, but are not limited to, those disclosed in US Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms may be useful in providing controlled or sustained release of one or more active ingredients using, for example, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, other polymeric matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled or sustained release formulations known to those skilled in the art, including those described herein, can readily be selected for use with the active ingredients of the compositions described herein. Thus the invention provides unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps and capsules adapted for controlled or sustained release.

[0400]The controlled or sustained release of an active ingredient can be stimulated by a variety of conditions, including but not limited to pH changes, temperature changes, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water. or other physiological conditions or connections.

[0401]Alternatively, a controlled release system can be placed in close proximity to the target area to be treated, requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra, Vol. 2, pages 115 -138 (1984)). Other controlled release systems discussed in the review by Langer, 1990, Science 249:1527-1533 can be used.

[0402]Pharmaceutical formulations are preferably sterile. Sterilization can be achieved, for example, by filtration through sterile filtration membranes. If the composition is lyophilized, filter sterilization can be performed before or after lyophilization and reconstitution.

[0403]administration and dosage

[0404]It is understood that the actual dose of chimeric protein to be administered according to the present invention will vary depending on the particular dosage form and mode of administration. Many factors can be considered that can modify the effect of the chimeric protein (eg, body weight, gender, diet, time of administration, route of administration, rate of clearance, condition of the subject, drug combinations, genetic disposition, and responsiveness). ). considered by experts in this field. Administration can be continuous or in one or more discrete doses within the maximum tolerated dose. Those skilled in the art can determine optimal delivery rates for a given set of conditions using standard dose delivery tests.

[0405]In some embodiments, a suitable dosage of the chimeric protein is in a range of about 0.01 mg / kg to about 10 g / kg of subject body weight, about 0.01 mg / kg to about 1 g / kg of subject body weight, about 0 .01 mg/kg to about 100 mg/kg subject body weight, about 0.01 mg/kg to about 10 mg/kg subject body weight, e.g. B. about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0 .4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/ kg, about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg /kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg body weight, about 100 mg/kg body weight, about 1 g/kg body weight, about 10 g/kg body weight, including all values ​​and ranges in between.

[0406]Single doses of the chimeric protein can be administered in unit dosage forms (e.g., tablets or capsules) containing, for example, from about 0.01 mg to about 100 g, from about 0.01 mg to about 75 g, from about 0.01 mg to about 50 g, from about 0.01 mg to about 25 g, from about 0.01 mg to about 10 g, from about 0.01 mg to about 7.5 g, from about 0.01 mg to about 0.01 mg 5 g, about 0.01 mg to about 2.5 g, about 0.01 mg to about 1 g, about 0.01 mg to about 100 mg, about 0.1 mg to about 100 mg, about 0.1 mg to about 90 mg, about 0.1 mg to about 80 mg, about 0.1 mg to about 70 mg, about 0.1 mg to about 60 mg mg, from about 0.1 mg to about 50 mg, from about 0 1 mg to about 40 mg active ingredient, from about 0.1 mg to about 30 mg, from about 0.1 mg to about 20 mg, from about 0.1 mg to about 10 mg B. from about 0.1 mg to from about 5 mg, from about 0.1 mg to about 3 mg, from about 0.1 mg to about 1 mg per unit dosage form, or from about 5 mg to about 80 mg per unit dosage form. For example, a unit dosage form may be about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg , about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75mg, about 80mg, about 85mg, about 90mg, about 95mg, about 100mg, about 200mg, about 500mg, about 1g, about 2.5g, about 5g, about 10g, about 25g, about 50g, about 75g, about 100g, including all values ​​and varies between.

[0407]In one embodiment, the chimeric protein is administered in an amount from about 0.01 mg to about 100 g per day, from about 0.01 mg to about 75 g per day, from about 0.01 mg to about 50 g per day, from administered from about 0.01 mg to about 50 g per day, from about 0.01 mg to about 10 g daily, from about 0.01 mg to about 7.5 g daily, from about 0.01 mg to about 25 g daily mg to about 5 g daily, from about 0.01 mg to about 2.5 g daily, from about 0.01 mg to about 1 g daily, from about 0.01 mg to about 100 mg daily, from about 0.1 mg to about 100 mg daily, from about 0.1 mg to about 95 mg daily, from about 0.1 mg to about 90 mg daily, from about 0.1 mg to about 85 mg daily, from about 0.1 mg to about 80 mg daily, from about 0.1 mg to about 75 mg daily, about 0.1 mg to about 70 mg daily, about 0.1 mg to about 65 mg daily, about 0.1 mg to about 60 mg mg daily , about 0.1 mg to about 55 mg daily, about 0.1 mg to about 50 mg daily, about 0.1 mg to about 45 mg daily, about 0.1 mg to about 40 mg daily, about 0.1 mg to about 35 mg mg daily, about 0.1 mg to about 30 mg daily, about 0.1 mg to about 25 mg daily, about 0.1 mg to about 20 mg daily, about 0.1 mg to about 15 mg daily , from about 0.1 mg to about 10 mg daily, from about 0.1 mg to about 5 mg daily, from about 0.1 mg to about 3 mg daily, from about 0.1 mg to about 1 mg daily, or from about 5 mg to about 80 mg daily. In various embodiments, the chimeric protein is provided at a daily dose of about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0. 07mg, about 0.08mg, about 0.09mg administered, about 0.1mg, about 0.2mg, about 0.3mg, about 0.4mg, about 0.5mg, about 0.6mg mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 85 mg, about 90 mg, about 95 mg, about 100 mg, about 200 mg, about 500 mg, about 1 g, about 2.5 g, about 5g, about 7.5g, about 10g, about 25g, about 50g, about 75g, about 100g, including all values ​​and ranges in between.

[0408]According to certain embodiments of the invention, the pharmaceutical composition comprising the chimeric protein can be administered, for example, more than once per day (for example about twice, about three times, about four times, about five times, about six times, about seven times, about eight times, about nine or about ten times a day), about once a day, about every other day, about every third day, about once a week, about once every two weeks, about once a month, about every two months, about every three months, about every six months or about once a year.

[0409]Combination therapy and additional therapeutics

[0410]In various embodiments, the pharmaceutical composition of the present invention is administered in conjunction with one or more additional therapeutic agents. The co-administration can be simultaneous or sequential.

[0411]In one embodiment, the additional therapeutic agent and the chimeric protein of the present invention are administered to a subject concurrently. The term "simultaneously" as used herein means that the additional therapeutic agent and the chimeric protein are separated in time by no more than 60 minutes, such as no more than 30 minutes, no more than 20 minutes, no more than be administered 20 minutes. more than about 10 minutes, no more than about 5 minutes, or no more than about 1 minute. The administration of the additional therapeutic agent and the chimeric protein can be carried out by simultaneous administration of a single formulation (e.g. a formulation comprising the additional therapeutic agent and the chimeric protein) or separate formulations (e.g. a single formulation comprising the additional therapeutic agents and the chimeric protein) first formulation comprising the additional therapeutic agent and the chimeric protein). a second formulation containing the chimeric protein).

[0412]Co-administration does not require the therapeutic agents to be administered simultaneously if the timing of their administration is such that the pharmacological activities of the additional therapeutic agent and the chimeric protein overlap in time, thereby exerting a combined therapeutic effect. For example, the additional therapeutic agent and the chimeric protein can be administered sequentially. As used herein, the term "sequential" means that the additional therapeutic agent and the chimeric protein are administered more than about 60 minutes apart. For example, the time between sequential administration of the additional therapeutic agent and the chimeric protein can be greater than 60 minutes, greater than 2 hours, greater than 5 hours, greater than 10 hours, greater than 1 day, greater than about 2 days. more than about 3 days apart, more than about 1 week apart, more than about 2 weeks apart, or more than about a month apart. Optimal administration times will depend on the metabolic rates, clearance, and/or pharmacodynamic activity of the additional therapeutic agent and chimeric protein being administered. The additional therapeutic agent or chimeric protein cell can be administered first.

[0413]Also, co-administration does not require that the therapeutic agents be administered to the subject by the same route of administration. Rather, any therapeutic agent can be administered by any suitable route, e.g. B. parenterally or non-parenterally.

[0414]In some embodiments, the chimeric protein described herein acts synergistically when co-administered with another therapeutic agent. In such embodiments, the chimeric protein and the additional therapeutic agent can be administered at doses lower than the doses used when the agents are used in conjunction with monotherapy.

[0415]In some embodiments, the present invention relates to chemotherapeutic agents as additional therapeutic agents. For example, without limitation, such a combination of the present chimeric proteins and chemotherapeutic agent finds utility in the treatment of cancer as described elsewhere herein. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquon, meturedopa and uredopa; ethyleneimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g. bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callous statin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (e.g. cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, colophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitroureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; Antibiotics such as the enediyne antibiotics (e.g. calicheamicin, particularly calicheamicin gammall and calicheamicin omegall (see e.g. Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemycin, including dynemicin A; bisphosphonates B. clodronate; an esperamycin; as well as neocarzinostatin chromophore and related chromoprotein antibiotics chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophyllin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6 -diazo- 5-oxo-L-norleucine, ADRIAMYCIN doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, Olivomycins B. Peplomycin, Potfiromycin, Puromycin, Chelamycin, Rhodorubicin, Streptongrin, Streptozocin, Tubercidin, Ubenimex, Zinostatin, Zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as callusterone, dromostanolone propionate, epitiostanol, mepitiostan, testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as frolic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestabucil; bisanthrene; edatraxate; demecolcin; diaziquon; Elformitine; elliptinium acetate; an epothilone; ethoglycide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; Mitoguazón; mitoxantrone; mopidanmol; nitraerin; pentostatin; freak pyrarubicin; losoxantrone; podophyllic acid; 2-ethyl hydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oregon); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triazicuon; 2,2',2''-trichlorotriethylamine; trichothecenes (eg, T-2 toxin, verracurin A, roridin A, and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronite; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; Taxoids, for example TAXOL paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE Cremophor, albumin-modified nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111) and TAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France ); chloranbucil; gemcitabine GEMZAR; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; Platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVEL. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the regimen of irinotecan with 5-FU and leucovorin); RFS 2000 topoisomerase inhibitor; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin regimen (FOLFOX); lapatinib (Tykerb); PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)), and VEGF-A inhibitors that reduce cell proliferation, and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Additionally, the treatment methods may further include the use of radiation. In addition, methods of treatment may further include the use of photodynamic therapy.

[0416]In one embodiment, the present invention relates to any agent that targets the spliceosome, including all components of the spliceosome, as adjunct therapeutic agents in the treatment of cancer.

[0417]In one embodiment, the present invention relates to any Myc-targeting agent (ie, anti-Myc therapeutic agent) as an adjunct therapeutic agent in the treatment of cancer.

[0418]In some embodiments, including but not limited to infectious disease applications, the present invention relates to anti-infectives as additional therapeutic agents. In some embodiments, the anti-infective is an anti-viral agent including, but not limited to, abacavir, aciclovir, adefovir, amprenavir, atazanavir, cidofovir, darunavir, delavirdine, didanosine, docosanol, efavirenz, elvitegravir, emtricitabine, enfuvirtide, etravirine, famciclovir, and Foscarnet. In some embodiments, the anti-infective is an antibacterial agent, including but not limited to cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (Cipro, Levaquin, Floxin, Tequin, Avelox, and Norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactam antibiotics (aztreonam); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem). In some embodiments, anti-infective agents include anti-malarials (eg, chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil, and sulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate, and albendazole. .

[0419]In some embodiments, including but not limited to autoimmune applications, the additional therapeutic agent is an immunosuppressive agent. In some embodiments, the immunosuppressive agent is an anti-inflammatory agent, such as a steroidal anti-inflammatory agent or a non-steroidal anti-inflammatory agent (NSAID). Steroids, particularly adrenal corticosteroids and their synthetic analogs, are well known in the art. Examples of corticosteroids useful in the present invention include, without limitation, hydroxyl triamcinolone, alpha-methyl dexamethasone, beta-methyl betamethasone, beclomethasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, clobetasolvalerate, desonide, deoxymethasone, dexamethasone, diflorason diacetate, diflucortolone acetone, fluocortolon acetonide, fluadrenolone acetonide Flumethasone pivalate, fluocinolone acetonide, fluocinonide, fluocortin butyl ester, fluocortolone, flupredniden (fluorenylidene) acetate, flurandrenolide, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, fluradoxonide acetonide, medrisone, amcinafel, amcinafel, and the rest of its beta, chlorredop, betamethasone, chlorredop, Clocortelone, clescinolone, dichlorosone, difluprednate, fluchloronide, flunisolide, fluorometholone, fluperolone, fluprednisolone, hydrocortisone, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate) that can be used in the present invention include, but are not limited to, salicylic acid, acetylsalicylic acid , methyl salicylate, glycol salicylate, salicylmide, benzyl-2,5-diacetoxybenzoic acid, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone and indomethacin. In some embodiments, the immunosuppressive agent can be cytostatic, such as alkylating agents, antimetabolites (e.g., azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g., basiliximab, daclizumab, and muromonab), anti-immunophyllines (e.g., cyclosporine, tacrolimus, sirolimus). ), interferons, opioids, TNF-binding proteins, mycophenolates, and small biologics (eg, fingolimod, myriocin). Additional anti-inflammatory agents are described, for example, in US Patent No. 4,537,776, the entire contents of which are incorporated herein by reference.

[0420]In some embodiments, the present invention relates to various agents used to treat obesity as additional therapeutic agents. Illustrative agents used to treat obesity include orlistat (eg, ALL1, XENICAL), loracaserin (eg, BELVIQ), phentermine topiramate (eg, QSYMIA), sibutramine (eg, REDUCTIL or MERJDIA), rimonabant (ACOMPLLA), exenatide (eg, BYETTA), pramlintide (eg, SYMLIN), phentermine, benzphetamine, diethylpropion, phendimetrazma, bupropion, and metformin. Substances that affect the body's ability to absorb certain nutrients from food include additional substances such as: Orlistat (e.g. ALU, XENICAL), glucomannan and guar gum. Additional drugs also include appetite suppressants, e.g. catecholamines and their derivatives (such as phenetamine and other amphetamine-based drugs), various antidepressants and mood stabilizers (e.g. bupropion and topiramate), anorexigenics (e.g. dexedrine, digoxin). Additional active ingredients also include active ingredients that increase the body's metabolism.

[0421]In some embodiments, additional therapeutic agents may be selected from appetite suppressants, neurotransmitter reuptake inhibitors, dopamine agonists, serotonergic agonists, GABAergic signaling modulators, anticonvulsants, antidepressants, monoamine oxidase inhibitors, substance P (NK1) receptor antagonists, melanocortin receptor agonists and antagonists, lipase inhibitors, Fat absorption inhibitors, energy intake or metabolism regulators, cannabinoid receptor modulators, addiction treatment agents, drugs for the treatment of metabolic syndrome, peroxisome proliferator-activated receptor (PPAR) modulators; Dipeptidylpeptidase 4 (DPP-4) antagonists, agents for treating cardiovascular diseases, agents for treating elevated triglyceride levels, agents for treating low HDL, agents for treating hypercholesterolemia, and agents for treating hypertension. Some cardiovascular disease drugs include statins (eg, lovastatin, atorvastatin, fluvastatin, rosuvastatin, simvastatin, and pravastatin) and omega-3 drugs (eg, LOVAZA, EPANQVA, VASCEPA, omega-3 esterified oils). in general, fish, krill oils, algae oils). In some embodiments, additional agents may be selected from amphetamines, benzodiazepines, sulfonylureas, meglitinides, thiazolidinediones, biguanides, beta blockers, XCE inhibitors, diuretics, nitrates, calcium channel blockers, phentermine, sibutramine, lorcaserin, cetilistat, rimonabant, taranabant. , topiramate, gabapentin, valproate, vigabatrin, bupropion, tiagabine, sertraline, fluoxetine, trazodone, zonisamide, methylphenidate, varenicline, naltrexone, diethylpropion, phendimetrazine, repaglinide, nateglinide, glimepiride, metformin, pioglitazone, rosiglitazone and sitagliptin.

[0422]In some embodiments, the present invention relates to an agent used to treat diabetes as an adjunct therapeutic agent. Illustrative antidiabetic drugs include sulfonylurea (eg, DYMELOR (acetohexamide), DIABINESE (chlorpropamide), ORINASE (tolbutamide), and TOLINASE (tolazamide), GLUCOTROL (glipizide), GLUCOTROL XL (extended release), DIABETA (glyburide), MICRONASE (glyburide) , GLYNASE PRESTAB (glyburide) and AMARYL (glimepiride)); a biguanide (e.g., metformin (GLUCOPHAGE, GLUCOPHAGE XR, RIOMET, FORTAMET, and GLUMETZA)); a thiazolidinedione (eg, ACTOS (pioglitazone) and AVANDIA (rosiglitazone); an alpha-glucosidase inhibitor (eg, PRECOSE (acarbose) and GLYSET (miglitol); a meglitinide (eg, PRANDIN (repaglinide) and STARLIX (nateglinide)); dipeptidyl peptidase IV (DPP-IV) inhibitor (e.g. JANUVIA (sitagliptin), NESIN (alogliptin), ONGLYZA (saxagliptin) and TRADJENTA (linagliptin)), inhibitor sodium glucose cotransporter 2 (SGLT2 ) (e.g. INVOKANA (canagliflozin) ) and a combination pill (e.g. GLUCOVANCE, which combines glyburide (a sulphonylurea) and metformin, MTAGLIP, which combines glipizide (a sulphonylurea) and metformin, and AVANDAMET, which combines metformin and rosiglitazone (AVANDIA) used in a single pill, KAZANO (alogliptin and metformin), OSENI (alogliptin plus pioglitazone), oral METFORMIN, oral ACTOS, subcutaneous BYETTA, oral JANUVIA, oral WELCHOL, JANUMET oral, glipizide oral, glimepiride oral, GLUCOPHAGE oral, LANTUS subcutaneous, Glyburid oral, ONGLYZA oral, AMARYI oral, LANTUS SOLOSTAR subcutaneous, BYDUREON subcutaneous, LEVEMIR FLEXPEN subcutaneous, ACTOPLUS MET oral, GLUMETZA oral, TRADJENTA oral, bromocriptine oral, KOMBIGLYZE oral XR, oral INVOKANA, oral PRANDIN, subcutaneous LEVEMIR, oral PARLODEL, oral pioglitazone, subcutaneous NOVOLOG, subcutaneous NOVOLOG FLEXPEN, subcutaneous VICTOZA 2-PAK, subcutaneous HUMALOG, oral STARLIX, oral FORTAMET, oral GLUCOVANCE, oral GLUCOPHAGE XR, oral NOVOLOG Mix 70-30 FLEXPEN subcutaneous, GLYBURIDE-METFORMIN oral, acarbose oral , SYMLINPEN 60 subcutaneous, GLUCOTROI XL oral, NOVOLIN R inj, GLUCOTROL oral, DUETACT oral, sitagliptin oral, SYMLINPEN 120 subcutaneous, HUMALOG KWIKPEN subcutaneous, JANUMET XR oral, GLIPFORMIN-METFORMIN oral, CYCLOSET oral, HUMALOG MIX 75-25 subcutaneous, nateglinide oral, HUMALOG Mix 75-25 KWIKPEN subcutaneous, HUMULIN 70/30 subcutaneous, PRECOSE oral, APIDRA subcutaneous, Humulin R inj, Jentadueto oral, Victoza 3-Pak subcutaneous, Novolin 70/30 subcutaneous, NOVOLIN N subcutaneous, insulin detemir subcutaneous, micronized Glyburide oral, GLYNASE oral, HUMULIN N subcutaneous, insulin glargine subcutaneous, RIOMET oral, pioglitazone-metformin oral, APIDRA SOLOSTAR subcutaneous, insulin lispro subcutaneous, GLYSET oral, HUMULIN 70/30 pen subcutaneous, colesevelam oral, sitagliptin-metformin oral, DIABETA oral , regular human insulin for injection, HUMULIN N Pen subcutaneous, exenatide subcutaneous, HUMALOG Mix 50-50 KWIKPEN subcutaneous, liraglutide subcutaneous, KAZANO oral, repaglinide oral, chlorpropamide oral, insulin aspart subcutaneous, NOVOLOG Mix 70-30 subcutaneous, HUMALOG Mix 50-50 subcutaneous, oral saxagliptin, oral ACTOPLUS Met XR, oral miglitol, recombinant human NPH insulin subcutaneous, NPH and regular human insulin subcutaneous, oral tolazamide, oral mifepristone, insulin aspart protam insulin aspart subcutaneous, oral repaglinide-metformin, oral saxagliptin- Metformin, Oral Linagliptin-Metformin, Oral NESIN, Oral OSENI, Oral Tolbutamide, Subcutaneous Insulin Lispro, Protamine and Lispro, Subcutaneous Pramlintide, Subcutaneous Insulin Glulisine, Oral Pioglitazone Glimepiride, Oral PRANDIMET, Subcutaneous NOVOLOG PenFill, Oral Linagliptin, Subcutaneous Exenatide Microspheres, oral KORLYM, oral alogliptin, oral alogliptin-pioglitazone, oral alogliptin-metformin, oral canagliflozin, lispro (HUMALOG); aspart (NOVOLOG); glulisine (APIDRA); Regular (NOVOLIN R or HUMULIN R); NPH (NOVOLINA N or HUMULIN N); glargine (LANTUS); Detemir(LEVEMIR); HUMULIN or NOVOLIN 70/30; and NOVOLOG Mix 70/30 HUMALOG Mix 75/25 or 50/50.

[0423]In some embodiments, the present invention relates to combination therapy with blood transfusion. For example, the present compositions can supplement a blood transfusion. In some embodiments, the present invention relates to combination therapy with iron supplements.

[0424]In some embodiments, the present invention relates to combination therapy with one or more EPO-based agents. For example, the present compositions can be used as an adjunct to other EPO-based agents. In some embodiments, the present compositions are used as maintenance therapy for other EPO-based agents. Other EPO-based agents include the following: Epoetin alfa, including but not limited to, DARBEPOETIN (ARANESP), EPOCEPT (LUPIN PHARMA), NANOKINE (NANOGEN PHARMACEUTICAL), EPOFIT (INTAS PHARMA), EPOGEN (AMGEN), EPOGIN, EPREX , (JANSSEN-CILAG), BINOCRIT (SANDOZ), PROCRIT; epoetin beta, including but not limited to, NEORECORMON (HOFFMANN-LA ROCHE), RECORMON, methoxypolyethylene glycol epoetin beta (MIRCERA, ROCHE); epoetin delta, including without limitation DYNEPO (erythropoiesis-stimulating protein, SHIRE PLC); Epoetin Omega, including but not limited to EPOMAX; Epoetin zeta including but not limited to SILAPO (STADA) and RETACRIT (HOSPIRA) and other EPOs including but not limited to EPOCEPT (LUPIN PHARMACEUTICALS), EPOTRUST (PANACEA BIOTEC LTD), ERYPRO SAFE (BIOCON LTD.), REPOITIN (SERUM INSTITUTE OF INDIA LIMITED), VINTOR (EMCURE PHARMACEUTICALS), EPOFIT (INTAS PHARMA), ERYKINE (INTAS BIOPHARMACEUTICA), WEPOX (WOCKHARDT BIOTECH), ESPOGEN (LG LIFE SCIENCES), RELIPOIETIN (RELIANCE LIFE SCIENCE), SHANPOIETIN (SHANTHA BIOTECHNICS LTD), ZYROP (CADILA HEALTHCARE LTD.), EPIAO (RHUEPO) (SHENYANG SUNSHINE PHARMACEUTICAL CO. LTD), CINNAPOIETIN (CINNAGEN).

[0425]In some embodiments, the present invention relates to combination therapy with one or more immunomodulatory agents, e.g. B. without limitation, immune checkpoint modulating agents. In various embodiments, the immunomodulatory agent targets one or more of PD-1, PD-L1, and PD-L2. In various embodiments, the immunomodulatory agent is a PD-1 inhibitor. In various embodiments, the immunomodulatory agent is an antibody specific for one or more of PD-1, PD-L1 and PD-L2. For example, in some embodiments, the immunomodulatory agent is an antibody such as nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), MPDL3280A (ROCHE). In some embodiments, the immunomodulatory agent targets one or more of CD137 or CD137L. In various embodiments, the immunomodulatory agent is an antibody specific for one or more of CD137 or CD137L. For example, in some embodiments, the immunomodulatory agent is, but is not limited to, an antibody such as urelumab (also known as BMS-663513 and anti-4-1BB antibody). In some embodiments, the present chimeric protein is combined with urelumab (optionally with one or more of nivolumab, lirilumab and urelumab) for the treatment of solid tumors and/or B-cell non-Hodgkin's lymphoma and/or head and neck cancer / or multiple myeloma. In some embodiments, the immunomodulatory agent is an agent that targets one or more of CTLA-4, AP2M1, CD80, CD86, SHP-2, and PPP2R5A. In various embodiments, the immunomodulatory agent is an antibody specific for one or more of CTLA-4, AP2M1, CD80, CD86, SHP-2, and PPP2R5A. For example, in some embodiments, the immunomodulatory agent is an antibody such as, but not limited to, ipilimumab (MDX-010, MDX-101, Yervoy, BMS) and/or tremelimumab (Pfizer). In some embodiments, the present chimeric protein is combined with ipilimumab (optionally with bavituximab) for the treatment of one or more of melanoma, prostate cancer and lung cancer. In various embodiments, the immunomodulatory agent targets CD20. In various embodiments, the immunomodulatory agent is a CD20-specific antibody. For example, in some embodiments, the immunomodulatory agent is an antibody such as, but not limited to, ofatumumab (GENMAB), obinutuzumab (GAZYVA), AME-133v (APLIED MOLECULAR EVOLUTION), ocrelizumab (GENENTECH), TRU-015 (TRUBION/EMERGENT) , veltuzumab (IMMU-106).

[0426]In some embodiments, the present chimeric protein acts synergistically when used in combination with chimeric antigen receptor (CAR) T cell therapy. In an exemplary embodiment, the chimeric protein acts synergistically when used in combination with CAR-T cell therapy in the treatment of tumors or cancer. In one embodiment, the chimeric protein acts synergistically when used in combination with CAR-T cell therapy in the treatment of blood tumors. In one embodiment, the chimeric protein acts synergistically when used in combination with CAR-T cell therapy in the treatment of solid tumors. For example, use of the chimeric protein and CAR-T cells can act synergistically to shrink or eliminate the tumor or cancer, or slow the growth and/or progression and/or metastasis of the tumor or cancer. In various embodiments, the chimeric protein of the invention induces division of CAR-T cells. In various embodiments, the chimeric protein of the invention induces proliferation of CAR-T cells. In various embodiments, the chimeric protein of the invention prevents CAR T cell anergy.

[0427]In various embodiments, CAR T cell therapy includes CAR T cells that target antigens (e.g., tumor antigens) such as, but not limited to, carbonic anhydrase IX (CAIX), 5T4, CD19, CD20, CD22, CD30 , CD33, CD38, CD47, CS1, CD138, Lewis-Y, L1-CAM, MUC16, ROR-1, IL13Rα2, gp100, Prostate Stem Cell Antigen (PSCA), Prostate Specific Membrane Antigen (PSMA), Cell Maturation Antigen (BCMA), human papillomavirus type 16 E6 (HPV-16 E6), CD171, folate receptor alpha (FR-α), GD2, human epidermal growth factor receptor 2 (HER2), mesothelin, EGFRvIII, fibroblast activating protein (FAP), carcinoembryonic antigen (CEA) , and vascular endothelial growth factor receptor 2 (VEGF-R2), as well as other tumor antigens well known in the art. Additional representative tumor antigens include, but are not limited to, MART-1/Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal associated antigen (CRC)-0017-1A /GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, amI1, prostate specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA-3, receptor T cells / CD3 zeta chain, MAGE family of tumor antigens (eg, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE- A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE -C3 , MAGE-C4, MAGE-C5), the GAGE ​​family of tumor antigens (e.g. GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7 , GAGE ​​​​-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, α- Fetoprotein, E -cadherin, α-catenin, β-catenin and γ-catenin, p120ctn, gp100 Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli (APC) protein, fodrin, connexin 37, Ig idiotype , p15, gp75 , GM2 and GD2 gangliosides, viral products, such as -MEL-40), SSX-1, SSX-4, SSX-5, SCP-1 CT-7, c-erbB-2, CD19, CD37, CD56, CD70, CD74, CD138, AGS16, MUC1, GPNMB, Ep -CAM, PD-L1 and PD-L2.

[0428]Examples of CAR T cell therapy include JCAR014 (Juno Therapeutics), JCAR015 (Juno Therapeutics), JCAR017 (Juno Therapeutics), JCAR018 (Juno Therapeutics), JCAR020 (Juno Therapeutics), JCAR023 (Juno Therapeutics), JCAR024 (Juno Therapeutics), CTL019 (Novartis), KTE-C19 (Kite Pharma), BPX-401 (Bellicum Pharmaceuticals), BPX-501 (Bellicum Pharmaceuticals), BPX-601 (Bellicum Pharmaceuticals), bb2121 (Bluebird Bio), CD-19 Sleeping Beauty cells (Ziopharm Oncology), UCART19 (Cellectis), UCART123 (Cellectis), UCART38 (Cellectis), UCARTCS1 (Cellectis), OXB-302 (Oxford BioMedica, MB-101 (Mustang Bio) and CAR-T cells developed by Innovative cell therapeutics.

[0429]In some embodiments, the chimeric protein is used in a method of treating multiple sclerosis (MS) in combination with one or more MS therapies including, but not limited to, 3-interferons, glatiramer acetate, T-interferon, IFN-β 2 (US Patent Publication No. 2002/0025304), spirogermanium (e.g. N-(3-dimethylaminopropyl)-2-aza-8,8-dimethyl-8-germanspiro[4:5]decane, N-(3 -dimethylaminopropyl) -2-aza-8,8-diethyl-8-germaspiro[4:5]decane, N-(3-dimethylaminopropyl)-2-aza-8,8-dipropyl-8-germaspiro[4:5] decane and N-(3-dimethylaminopropyl)-2-aza-8,8-dibutyl-8-germaspiro[4:5]decane), vitamin D analogues (e.g. 1,25(OH)2D3, ( see e.g. US Pat. No. 5,716,946), prostaglandins (e.g. latanoprost, brimonidine, PGE1, PGE2 and PGE3, see e.g. US Patent Publication No. 2002/0004525), tetracycline and derivatives (e.g B. minocycline and doxycycline, see B. US Patent Publication No. 20020022608 ), a VLA-4 binding antibody (see for example US Patent Publication No. 2009/0202527 ), adrenocorticotropic hormone, corticosteroid, prednisone, methylprednisone, 2-chlorodeoxyadenosine , mitoxantrone, sulfasalazine, methotrexate B. azathioprine, cyclophosphamide, cyclosporine, fumarate, anti-CD20 antibodies (eg. rituximab) and tizanidine hydrochloride.

[0430]In some embodiments, the chimeric protein is used in combination with one or more therapeutic agents that treat one or more symptoms or side effects of MS. Such agents include, but are not limited to, amantadine, baclofen, papaverine, meclizine, hydroxyzine, sulfamethoxazole, ciprofloxacin, docusate, pemoline, dantrolene, desmopressin, dexamethasone, tolterodine, phenyloine, oxybutynin, bisacodyl, venlafaxine, amitriptyline, methenamine, clonazepam, isoniazid, vardenafil, nitrofurantoin,Psiliohydrophilic muciloid, alprostadil, gabapentin, nortriptyline, paroxetine, propantheline bromide, modafinil, fluoxetine, phenazopyridine, methylprednisolone, carbamazepine, imipramine, diazepam, sildenafil, bupropion, and sertraline.

[0431]In some embodiments, the chimeric protein is used in a method of treating multiple sclerosis in combination with one or more disease modifying therapies (DMTs) described herein (e.g., the agents in Table A). In some embodiments, the present invention provides an improved therapeutic effect compared to using one or more of the DMTs described herein (e.g., the agents listed in the table below) without the described combination of one or more agents. In one embodiment, the combination of the chimeric protein and one or more DMTs produces synergistic therapeutic effects.

[0432]MS disease progression can be more intense and damaging in the earliest stages of disease progression. Consequently, in contrast to many medical practices and reimbursement policies, starting treatment with the more intensive DMTs may be more beneficial to a patient's long-term medical condition in terms of, for example, cost and side effect reduction. so-called second-line therapies. In some embodiments, a patient is treated with a regimen of the chimeric protein in combination with second-line therapy. Such a combination is used to reduce the side effect profile of one or more second-line therapies. In some embodiments, the combination is used to reduce the dose or frequency of administration of one or more second-line therapies. For example, the dosages of the agents listed in the table provided above may be reduced by about 50%, about 40%, about 30%, or about 25% in conjunction with the combination and/or frequency of dosing to which they may be reduced in half or one One-third the frequency or it can be reduced, for example, from daily to every other day or weekly, from every other day to weekly or biweekly, from weekly to biweekly or monthly, etc. Accordingly, in some embodiments, the chimeric protein increases patient adherence by more convenient treatment regimens. Additionally, some DMTs have a recommended lifetime dose limit, e.g. for mitoxantrone the cumulative lifetime dose should be strictly limited to 140 mg/m², or 2 to 3 years of therapy. In some embodiments, supplementation with the chimeric protein preserves the patient's access to mitoxantrone by allowing for lower or less frequent dosing with that DMT.

[0433]In some embodiments, the patient is a treatment-naïve patient who has not received treatment with one or more DMTs, and the chimeric protein is used to buffer the side effects of second-line therapy. Consequently, early in the disease, the naïve patient can reap the long-term benefits of second-line therapy. In some embodiments, the chimeric protein is used as an initial therapy that precedes the use of second-line therapy. For example, the chimeric protein can be administered for an initial treatment period of about 3 months to stabilize the disease, and then the patient can be switched to maintenance therapy with a second-line agent.

[0434]In general, it is believed that naïve patients are more likely to respond to therapy than patients who have received one or more DMTs and may have failed. In some embodiments, the chimeric protein finds use in patients who have received one or more DMTs and may have failed. For example, in some embodiments, the chimeric protein increases the therapeutic effect in patients who have received one or more DMTs and may have failed, and may allow these patients to respond as naïve patients.

[0435]In some embodiments, the patient is on or receiving treatment with one or more DMTs and does not respond well. For example, the patient may be refractory or respond poorly to one or more DMTs. In some embodiments, the patient is refractory or poorly responsive to one or more of teriflunomide (AUBAGIO (GENZYME)); Interferon beta-1a (AVONEX (BIOGEN IDEC); Interferon beta-1b (BETASERON (BAYER HEALTHCARE PHARMACEUTICALS, INC.); Glatiramer acetate (COPAXONE (TEVA NEUROSCIENCE); Interferon beta-1b (EXTAVIA (NOVARTIS PHARMACEUTICALS CORP.)); Fingolimod (GILENYA (NOVARTIS PHARMACEUTICALS CORP.); alemtuzumab (LEMTRADA (GENZYME); mitoxantrone (NOVANTRONE (EMD SERONO); pegylated interferon beta-1a (PLEGRIDY (BIOGEN IDEC); interferon beta-1a (REBIF (EMD SERONO, INC.); dimethyl fumarate (BG-12) (TECFIDERA (BIOGEN IDEC)); and natalizumab (TYSABRI (BIOGEN IDEC)) reduce or eliminate non-responsiveness to DMT, which may, for example, save the patient from therapy with one or more DMTs at higher doses or frequency.

[0436]For patients with more aggressive disease, an induction treatment model is an approach in which therapy with high efficacy but high safety concerns is given first, followed by maintenance therapy. An example of such a model might include initial treatment with alemtuzumab followed by IFN-β, GA, or BG-12. In some embodiments, the one or more excipients described are used to avoid the need to change maintenance therapies. In some embodiments, the one or more excipients described are used as maintenance therapy for one or more DMTs, including second-line therapies. In some embodiments, one or more of the disclosed binding agents are used as the first therapy in an induction, followed by another DMT as maintenance therapy, such as. B. a first-line therapy.

[0437]In some embodiments, one or more of the described binding agents can be administered for an initial treatment period of about 3 months to stabilize the disease, and then the patient can be switched to maintenance therapy with a first-line agent.

[0438]In various embodiments, one or more excipients described are used to reduce one or more side effects of a DMT, including but not limited to any agent described herein. For example, the one or more excipients described can be used in a regimen that allows for dose savings for one or more DMTs and therefore results in fewer side effects. For example, in some embodiments, one or more of the excipients described can reduce one or more side effects of AUBAGIO or related agents, which can include hair loss, diarrhea, flu, nausea, abnormal liver function tests, and unusual numbness or tingling in the hands. . or feet (paraesthesia), levels of white blood cells that may increase the risk of infection; increased blood pressure; and severe liver damage. In some embodiments, one or more excipients described may reduce one or more side effects of AVONEX or related agents, including post-injection flu-like symptoms, depression, mild anemia, liver abnormalities, allergic reactions, and heart problems. In some embodiments, one or more excipients described may reduce one or more side effects of BETASERON or related agents, including post-injection flu-like symptoms, injection site reactions, allergic reactions, depression, abnormalities, liver problems, and low white blood cell counts. . In some embodiments, one or more excipients described can reduce one or more side effects of COPAXONE or related agents, including injection site reactions, vasodilation (blood vessel dilatation); chest pain; a reaction immediately after the injection, including anxiety, chest pain, palpitations, shortness of breath and flushing. In some embodiments, one or more excipients described may reduce one or more side effects of EXTAVIA or related agents, including post-injection flu-like symptoms, injection site reactions, allergic reactions, depression, abnormalities, liver problems, and low white blood cell counts. . In some embodiments, one or more excipients described can reduce one or more side effects of GILENYA or related agents, including headache, flu, diarrhea, back pain, elevated liver enzymes, cough, slow heart rate after the first dose, infection, and swelling in the eye. In some embodiments, one or more excipients described may reduce one or more side effects of LEMTRADA or related agents, including rash, headache, fever, nasal congestion, nausea, urinary tract infection, fatigue, insomnia, respiratory infection, hives. , itching, thyroid disorders, fungal infection, joint, body and back pain, diarrhea, vomiting, flushing and infusion reactions (including nausea, hives, itching, insomnia, chills, flushing, tiredness, shortness of breath, changes in taste, indigestion, dizziness, Pains). In some embodiments, one or more excipients described can reduce one or more side effects of NOVANTRONE or related agents, including blue-green urine 24 hours after administration; Infections, bone marrow suppression (tiredness, bruising, low blood cell count), nausea, thinning hair, bladder infections, mouth sores, and severe liver and heart damage. In some embodiments, one or more excipients described may reduce one or more side effects of PLEGRIDY or related agents, including post-injection flu-like symptoms, injection site reactions, depression, mild anemia, abnormalities, liver problems, allergic reactions, and heart problems. . In some embodiments, one or more excipients described may reduce one or more side effects of REBIF or related agents, including post-injection flu-like symptoms, injection site reactions, liver abnormalities, depression, reactions, allergies, and low red or white blood cell levels. cell counts. In some embodiments, one or more of the excipients described may reduce one or more side effects of TECFIDERA or related agents, including flushing (hot or stinging sensation and redness of the skin), gastrointestinal issues (nausea, diarrhea, abdominal pain), rash, protein in the urine, increased liver enzymes; and reduction in the number of lymphocytes (white blood cells) in the blood. In some embodiments, one or more excipients described can reduce one or more side effects of TYSABRI or related agents, including headache, fatigue, urinary tract infection, depression, respiratory tract infection, joint pain, upset stomach, abdominal discomfort, diarrhea, vaginitis, pain in arms or legs, rash , allergic or hypersensitivity reactions within two hours of the infusion (dizziness, fever, rash, itching, nausea, flushing, low blood pressure, shortness of breath, chest pain).

[0439]In some embodiments, the present invention relates to combination therapy with one or more chimeric agents described in WO 2013/10779, WO 2015/007536, WO 2015/007520, WO 2015/007542 and WO 2015/007903, the entire contents of which are incorporated herein is by reference in its entirety.

[0440]In some embodiments, the chimeric protein described herein includes derivatives that are modified, ie, by covalently attaching any type of molecule to the composition, such that the covalent attachment does not impede the activity of the composition. By way of example, but not limitation, derivatives include compositions modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be performed by known techniques including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc.

[0441]In still other embodiments, the chimeric protein described herein further comprises a cytotoxic agent, which in illustrative embodiments comprises a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death. Such agents can be conjugated with a composition described herein.

[0442]The chimeric protein described herein can be post-translationally modified to include effector moieties such as chemical linkers, detectable moieties such as fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials and chemiluminescent moieties, or functional moieties such as e.g. B. add streptavidin. Avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.

[0443]Illustrative cytotoxic agents include methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine; Alkylating agents such as mechlorethamine, thioepa, chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamineplatinum(II) (DDP) cisplatin and carboplatin (paraplatin); Anthracyclines include daunorubicin (formerly daunomycin), doxorubicin (adriamycin), detorubicin, carminomycin, idarubicin, epirubicin, mitoxantrone, and bisanthrene; Antibiotics include dactinomycin (actinomycin D), bleomycin, calicheamicin, mithramycin, and anthramycin (AMC); and antimitotic agents such asVincaalkaloids, vincristine and vinblastine. Other cytotoxic agents include paclitaxel (Taxol), ricin,PseudomonasExotoxin, Gemcitabine, Cytochalasin B, Gramicidin D, Ethidium Bromide, Emetine, Etoposide, Tenoposide, Colchicine, Dihydroxyanthracinedione, 1-Dehydrotestosterone, Glucocorticoids, Procaine, Tetracaine, Lidocaine, Propranolol, Puromycin, Procarbazine, Hydroxyurea, Asparaginase, Corticosteroids, Mitotane (O, P'-(DDD)), interferons and mixtures of these cytotoxic agents.

[0444]Other cytotoxic agents include but are not limited to chemotherapeutics such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGF antagonists, EGFR antagonists, platinum , Taxols, Irinotecan, 5-Fluorouracil, Gemcitabine, Leucovorin, Steroids, Cyclophosphamide, Melphalan,VincaAlkaloids (e.g. vinblastine, vincristine, vindesine and vinorelbine), mustines, tyrosine kinase inhibitors, radiotherapy, sex hormone antagonists, selective androgen receptor modulators, selective estrogen receptor modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists, interleukins (e.g. IL-12 or IL-2), IL-12R antagonists, toxin-conjugated monoclonal antibodies, tumor antigen-specific monoclonal antibodies, Erbitux, Avastin, Pertuzumab, anti-CD20 antibodies, Rituxan, Ocrelizumab, Ofatumumab, DXL625, HERCEPTIN® , or any combination thereof. Toxic enzymes from plants and bacteria such as ricin, diphtheria toxin andPseudomonasthe toxin can be conjugated to therapeutic agents (eg, antibodies) to generate cell-type specific killing reagents (Youle, et al., Proc. Nat'l Acad. Sci. USA 77:5483 (1980); Gilliland, et al. e.g., Proc Nat'l Acad Sci USA 77:4539 (1980), Krolick et al., Proc Nat'l Acad Sci USA 77:5419 (1980)).

[0445]Other cytotoxic agents include cytotoxic ribonucleases as described by Goldenberg in US Patent No. 6,653,104. Embodiments of the invention also relate to radioimmunoconjugates in which an alpha or beta particle emitting radionuclide is stably coupled to the chimeric protein, with or without the use of a chelating agent. Such radionuclides include beta emitters such as phosphorus-32, scandium-47, copper-67, gallium-67, yttrium-88, yttrium-90, iodine-125, iodine-131, samarium-153, lutetium-177, rhenium-186 or rhenium -188 and alpha emitters such as Astatine-211, Lead-212, Bismuth-212, Bismuth-213 or Actinium-225.

[0446]Illustrative detectable entities also include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta-galactosidase, and luciferase. Other exemplary fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin, and dansyl chloride. Other exemplary chemiluminescent moieties include, but are not limited to, luminol. Other exemplary bioluminescent materials include, but are not limited to, luciferin and aequorin. Other exemplary radioactive materials include, but are not limited to, iodine-125, carbon-14, sulfur-35, tritium, and phosphorus-32.

[0447]treatment methods

[0448]The methods and compositions described herein have utility in treating various diseases and disorders, including but not limited to cancer, infections, immune disorders, anemia, autoimmune diseases, cardiovascular diseases, wound healing, ischemia-related diseases, neurodegenerative diseases, metabolic diseases, and many other diseases and disruptions.

[0449]In addition, any of the present agents can be used in the treatment or manufacture of a medicament for treating a variety of diseases and disorders including, but not limited to, cancer, infections, immunological disorders, inflammatory diseases or conditions, and autoimmune diseases. .

[0450]In some embodiments, the present invention relates to the treatment of chronic granulomatous disease, osteopetrosis, idiopathic pulmonary fibrosis, Friedreich's ataxia, atopic dermatitis, Chagas disease, cancer, heart failure, autoimmune disease, sickle cell disease, or a patient having one or more of these diseases. Thalassemia, blood loss, transfusion reaction, diabetes, vitamin B12 deficiency, collagenosis, Shwachman syndrome, thrombocytopenic purpura, celiac disease, endocrine deficiency such as hypothyroidism or Addison's disease, autoimmune disease such as Crohn's disease, systemic lupus erythematosus, rheumatoid arthritis or juvenile rheumatoid arthritis, colitis colitis, immune disorders such as eosinophilic fasciitis, hyperimmunoglobulinemia or thymoma/thymic carcinoma, graft-versus-host disease, preleukemia, non-hematological syndrome (e.g. Down's syndrome, Dubowwitz, Seckel), Felty's syndrome, hemolytic-uremic syndrome, myelodysplastic syndrome , paroxysmal nocturnal hemoglobinuria, osteomyelofibrosis, pancytopenia, erythrocyte aplasia, Henoch-Schoenlein purpura, malaria, protein deficiency, menorrhagia, systemic sclerosis, liver cirrhosis, hypometabolic states and congestive heart failure.

[0451]In some embodiments, the present invention relates to the treatment of one or more chronic granulomatous diseases, osteopetrosis, idiopathic pulmonary fibrosis, Friedreich's ataxia, atopic dermatitis, Chagas disease, mycobacterial infections, cancer, scleroderma, hepatitis, hepatitis or a patient with these C, septic shock and rheumatoid arthritis.

[0452]In some embodiments, the present invention relates to treating a patient with cancer. As used herein, cancer refers to any uncontrolled growth of cells that can disrupt the normal functioning of body organs and systems and includes both primary and metastatic tumors. Primary tumors or cancers that migrate from their original site and colonize vital organs can eventually lead to patient death due to impaired function of the affected organs. A metastasis is a cancer cell or group of cancer cells different from the site of the primary tumor, resulting from the spread of cancer cells from the primary tumor to other parts of the body. Metastases can eventually lead to the death of a subject. For example, cancers can include benign and malignant cancers, polyps, hyperplasia, and latent tumors or micrometastases.

[0453]Illustrative cancers that can be treated include carcinomas, e.g. various subtypes including, for example, adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcomas (including, for example, bone and soft tissue), leukemias (including, for example, acute myeloid, lymphoblastic, chronic myeloid, chronic lymphocytic, and hairy cells), lymphomas, and myelomas (including, for example, Hodgkin and non-Hodgkin lymphomas, light chain, non-secretory, MGUS and plasmacytomas) and cancers of the central nervous system (including, for example, brain (e.g. gliomas (e.g. astrocytoma, oligodendroglioma and ependymoma), meningiomas , pituitary adenomas and neuromas, and spinal cord tumors (eg, meningiomas and neurofibromas).

[0454]Illustrative cancers that can be treated include basal cell carcinoma, bile duct cancer; bladder cancer; bone cancer; cancer of the brain and central nervous system; breast cancer; peritoneal cancer; Cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancer of the digestive system; endometrial carcinoma; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma; liver carcinoma; hepatoma; intraepithelial neoplasia; kidney or renal cancer; throat cancer; Leukemia; liver cancer; lung cancer (eg, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth and throat); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; colon cancer; cancer of the respiratory tract; salivary gland carcinoma; Sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; Lymphoma, including Hodgkin and non-Hodgkin lymphoma, and B-cell lymphoma (including low grade follicular non-Hodgkin lymphoma (NHL); small lymphocyte (SL) NHL; follicular/moderate NHL; moderate diffuse NHL; high grade NHL immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small cell NHL; NHL with severe disease; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia, chronic myeloblastic leukemia, as well as others Carcinomas and sarcomas and post-transplant lymphoproliferative disorder (PTLD) and abnormal vascular proliferation associated with phakomatosis, edema (e.g. associated with brain tumors) and Meigs syndrome.

[0455]In various embodiments, the present invention relates to the treatment of cancers caused by Myc, ie cancer cells that overexpress Myc. In some embodiments, cancer cells overexpress one of c-Myc, N-Myc, and/or L-Myc. In some embodiments, the methods of the invention render cancer cells amenable to treatment with any of the cancer therapeutics described herein. In some embodiments, the methods of the invention reduce transcriptional activities of cancer cells.

[0456]In some embodiments, the present invention relates to treating a patient suffering from a microbial infection and/or a chronic infection. Illustrative infections include, but are not limited to, Chagas' disease, HIV/AIDS, tuberculosis, osteomyelitis, hepatitis B, hepatitis C, Epstein-Barr virus or parvovirus, T-cell leukemia virus, bacterial overgrowth syndrome, fungal or parasitic infections.

[0457]In various embodiments, the present compositions are used to treat or prevent one or more inflammatory diseases or conditions, such as inflammation, acute inflammation, chronic inflammation, respiratory disease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopic dermatitis, septic shock, rheumatism . Arthritis, inflammatory bowel disease, pelvic inflammatory disease, pain, ocular inflammatory disease, celiac disease, Leigh syndrome, glycerol kinase deficiency, familial eosinophilia (FE), autosomal recessive spastic ataxia, inflammatory laryngeal disease; Tuberculosis, chronic cholecystitis, bronchiectasis, silicosis and other pneumoconiosis.

[0458]In various embodiments, the present compositions are used to treat or prevent one or more autoimmune diseases or conditions, such as multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barré syndrome, scleroderma, Goodpasture's Syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, fibromyalgia, Meniere's syndrome; Transplant rejection (e.g. prevention of allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Graves' disease and other autoimmune diseases.

[0459]In various embodiments, the present compositions are used to treat, control, or prevent cardiovascular diseases such as: B. a disease or condition affecting the heart and vasculature, including but not limited to coronary artery disease (CHD), cerebrovascular disease (CVD), coronary artery disease (CAD), aortic stenosis, peripheral vascular disease, atherosclerosis, arteriosclerosis, Myocardial infarction (heart attack), cerebrovascular disease (stroke), transient ischemic attack (TIA), angina (stable and unstable), atrial fibrillation, arrhythmia, valvular heart disease and/or congestive heart disease.

[0460]In various embodiments, the present compositions are used to treat or prevent one or more metabolic disorders. In various embodiments, the present invention is useful for the treatment, control, or prevention of diabetes, including type 1 and type 2 diabetes and diabetes associated with obesity. The compositions and methods of the present invention are useful for the treatment or prevention of diabetes-related disorders, including but not limited to diabetic nephropathy, hyperglycemia, glucose intolerance, insulin resistance, obesity, lipid disorders, dyslipidemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, hypoglycemia. HDL levels, high LDL levels, atherosclerosis and its consequences, vascular restenosis, irritable bowel syndrome, inflammatory bowel diseases including Crohn's disease and ulcerative colitis, other inflammatory diseases, pancreatitis, abdominal obesity, neurodegenerative diseases, retinopathy, neoplastic conditions, adipocyte tumors, adipocyte carcinomas such as liposarcoma , prostate cancer and other cancers, including gastric, breast, bladder and colon cancer, angiogenesis, Alzheimer's disease, psoriasis, hypertension, metabolic syndrome (for example, a person has three or more of the following conditions: abdominal obesity, hypertriglyceridemia, low HDL cholesterol, high blood pressure and high fasting plasma glucose), ovarian hyperandrogenism (polycystic ovarian syndrome) and other disorders in which insulin resistance is a component such as sleep apnea. The compositions and methods of the present invention are useful for treating, controlling, or preventing obesity, including genetic or environmental obesity-related disorders. The obesity-related disorders herein are associated with, are caused by, or result from obesity. Examples of obesity-related disorders are obesity, diabetes, overeating, binge eating and bulimia, hypertension, elevated plasma insulin levels and insulin resistance, dyslipidemia, hyperlipidemia, endometrial, breast, prostate, kidney and colon cancer, osteoarthritis, obstructive sleep apnea , gallstones , heart disease, abnormal heart rhythms and arrhythmias, myocardial infarction, congestive heart failure, coronary artery disease, sudden death, stroke, polycystic ovarian disease, craniopharyngioma, Prader-Willi syndrome, de Frohlich syndrome, individuals with GH deficiency, normal short stature variant , Turner syndrome and other pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total lean mass, e.g. B. Children with acute lymphoblastic leukemia. Other examples of obesity-related disorders include metabolic syndrome, insulin resistance syndrome, reproductive hormone abnormalities, sexual and reproductive dysfunction such as fertility problems, infertility, male hypogonadism and female hirsutism, birth defects in fetuses associated with maternal obesity, gastrointestinal motility disorders, B. obesity- related gastroesophageal reflux disease, respiratory diseases such. B. Obesity hypoventilation syndrome (Pickwick syndrome), shortness of breath, cardiovascular disease, inflammation, such as z , gout and kidney cancer and increased risk of anesthesia. The compositions and methods of the present invention are also useful for treating Alzheimer's disease.

[0461]In various embodiments, the present compositions are used to treat or prevent one or more respiratory diseases such as idiopathic pulmonary fibrosis (IPF), asthma, chronic obstructive pulmonary disease (COPD), bronchiectasis, allergic rhinitis, sinusitis, pulmonary vasoconstriction, allergies, shortness of breath, Respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, hantavirus pulmonary syndrome (HPS), Loeffler syndrome, Goodpasture syndrome, pleurisy, pneumonitis, pulmonary edema, pulmonary fibrosis, sarcoidosis, complications associated with respiratory diseases syncytial virus infection and others respiratory diseases.

[0462]In some embodiments, the present invention is used to treat or prevent one or more neurodegenerative diseases. Illustrative neurodegenerative diseases include, but are not limited to, Friedreich's ataxia, multiple sclerosis (including but not limited to benign multiple sclerosis; relapsing remitting multiple sclerosis (RRMS); secondary progressive multiple sclerosis (SPMS); relapsing progressive multiple sclerosis (PRMS) and primary progressive multiple sclerosis (PPMS)), Alzheimer's. (including but not limited to early onset Alzheimer's disease, late onset Alzheimer's disease and familial Alzheimer's disease (FAD), Parkinson's disease and parkinsonism (including but not limited to idiopathic Parkinson's disease, vascular parkinsonism, drug-induced Parkinsonism, Lewy foreign body dementia, hereditary Parkinson's, juvenile Parkinson's), Huntington's disease, amyotrophic lateral sclerosis (ALS, including but not limited to sporadic ALS, familial ALS, West Pacific ALS, juvenile ALS, Hiramaya disease).

[0463]In various embodiments, the present chimeric proteins find use in the treatment of wounds, for example a non-healing wound, an ulcer, a burn or frostbite, a chronic or acute wound, an open or closed wound, an internal or external wound ( Wounds Exemplary external wounds are penetrating and blunt wounds In various embodiments, the present chimeric proteins find use in the treatment of ischemia, for example, ischemia associated with acute coronary syndrome, acute lung injury (ALI), acute myocardial infarction (AMI), acute respiratory distress syndrome ( ARDS), arterial occlusive disease, arteriosclerosis, articular cartilage defect, aseptic systemic inflammation, atherosclerotic cardiovascular disease, autoimmune disease, bone fracture, bone fracture, cerebral edema, cerebral hypoperfusion, Morbus Bürger, burns, cancer, cardiovascular disease, cartilage damage, cerebral infarction, cerebral Ischemia, Stroke, Cerebrovascular Disease, Chemotherapy-Induced Neuropathy, Chronic Infection, Chronic Mesenteric Ischemia, Claudication, Congestive Heart Failure, Connective Tissue Damage, Contusion, Coronary Artery Disease (CAD) Disease, Critical Limb Ischemia (CLI), Crohn's Disease, Deep Vein Thrombosis, Deep Wound, delayed ulcer healing, delayed wound healing, diabetes (type I and type II), diabetic neuropathy, diabetes-induced ischemia, disseminated intravascular coagulation (DIC), embolic cerebral ischemia, frostbite, graft-versus-host disease, hereditary hemorrhagic vascular disease, ischemic telengiectasia, hyperoxic Injury, hypoxia, inflammation, inflammatory bowel disease, inflammatory disease, injured tendons, intermittent claudication, intestinal ischemia, ischemia, ischemic cerebral disease, ischemic heart disease, ischemic peripheral vascular disease, ischemic placenta, ischemic kidney disease, ischemic vascular disease, ischemic reperfusion injury, laceration, disease of the Left main coronary artery, limb ischemia, ischemic limb disease, myocardial infarction, myocardial ischemia, organic ischemia, osteoarthritis, osteoporosis, osteosarcoma, Parkinson's disease, peripheral arterial disease (PAD), peripheral arterial disease, peripheral ischemia, peripheral neuropathy, peripheral vascular disease, precancerous lesions, pulmonary edema, pulmonary embolism, remodeling disorder , renal ischemia, retinal ischemia, retinopathy, sepsis, skin ulcers, organ transplantation, spinal cord injury, stroke, subchondral bone cyst, thrombosis, thrombotic brain ischemia, tissue ischemia, transient ischemic attack (TIA), traumatic brain injury, ulcerative colitis, renal vascular disease, inflammatory vascular conditions, von Hippel - Lindau syndrome or dialysis tissue or organ injury) and/or an anticancer drug (e.g. B. chemotherapy and/or HIV treatment [S. zidovudine (INN) or azidothymidine (AZT)], inflammatory bowel disease (e.g. Crohn's disease and ulcerative colitis), anemia associated with inflammatory conditions (e.g. arthritis, lupus, IBD), anemia associated with diabetes , schizophrenia, cerebral malaria such as aplastic anemia and myelodysplasia caused by cancer treatment (e.g., chemotherapy and/or radiation) and various disorders of myelodysplastic syndrome (e.g., sickle cell disease, hemoglobin SC disease, hemoglobin C disease, alpha and beta thalassemias, neonatal anemia after prematurity and comparable conditions).

[0464]In some embodiments, the present invention relates to the treatment of a patient suffering from anemia, that is, a condition in which the red blood cell count and/or the amount of hemoglobin found in the red blood cells is below normal. In various embodiments, the anemia can be acute or chronic. For example, the present anemias include iron deficiency anemia, renal anemia, anemia of chronic disease/inflammation, pernicious anemia such as e.g. B. large cell achyl anemia, juvenile pernicious anemia and congenital pernicious anemia, cancer-related anemia, anti-cancer anemia - chemotherapy-related anemia (e.g. chemotherapy-related anemia, radiotherapy-related anemia), pure erythrocyte aplasia, refractory anemia with excessive Blasts, aplastic anemia, X-line sideroblastic anemia, hemolytic anemia, sickle cell anemia, anemia caused by defective ESA production, myelodysplastic syndromes, hypochromic anemia, microcytic anemia, sideroblastic anemia, autoimmune hemolytic anemia, Cooley's anemia, Mediterranean anemia, Diamond-Blackfan Anemia, Fanconi anemia, and drug-induced immune-induced hemolytic anemia. Anemia can cause serious symptoms, including hypoxia, chronic fatigue, poor concentration, pale skin, low blood pressure, dizziness, and heart failure.

[0465]In some embodiments, the present invention relates to the treatment of anemia resulting from chronic renal failure. In some embodiments, the present invention relates to the treatment of anemia resulting from the use of one or more renal replacement therapies, including dialysis, hemodialysis, peritoneal dialysis, hemofiltration, hemodiafiltration, and kidney transplantation.

[0466]In some embodiments, the present invention relates to the treatment of anemia in patients with chronic kidney disease who are not on dialysis. For example, the present invention relates to patients with stage 1 CKD or stage 2 CKD or stage 3 CKD or stage 4 CKD or stage 5 CKD. In some embodiments, the affected patient has stage 4 CKD or stage 5 CKD in some embodiments, the affected patient has undergone a kidney transplant. In some embodiments, the present invention relates to treating anemia in a patient with Acute Kidney Injury (AKI).

[0467]In some embodiments, the anemia is induced by chemotherapy. For example, the chemotherapy can be any myelosuppressive chemotherapy. In some embodiments, the chemotherapy is one or more of Revlimid, Thalomid, dexamethasone, Adriamycin, and Doxil. In some embodiments, the chemotherapy consists of one or more platinum-based drugs including cisplatin (e.g., PLATINOL) and carboplatin (e.g., PARAPLATIN). In some embodiments, chemotherapy is one of the chemotherapeutic agents described herein. In some embodiments, chemotherapy is any agent described in Groopman et al. J. Natl. Cancer Inst. (1999) 91(19):1616-1634, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the present compositions and methods are used in the treatment of chemotherapy-related anemia in patients with advanced cancer (e.g., stage IV, stage III, or stage II cancer). In some embodiments, the present compositions and methods are used in the treatment of chemotherapy-related anemia in cancer patients receiving dose-dense chemotherapy or other aggressive chemotherapy regimens.

[0468]In some embodiments, the present invention relates to treating anemia in a patient with one or more blood cancers such as leukemia, lymphoma and multiple myeloma. Such cancers can directly affect the bone marrow. Furthermore, the present invention relates to metastatic cancer that has spread to bone or bone marrow. In some embodiments, the present invention relates to treating anemia in a patient undergoing radiation therapy. Such radiation therapy can damage the bone marrow and reduce its ability to make red blood cells. In further embodiments, the present invention relates to the treatment of anemia in a patient who is depleted or deficient in one or more of iron, vitamin B12 and folic acid. In further embodiments, the present invention relates to the treatment of anemia in a patient with excessive bleeding, including but not limited to, following surgery or a tumor causing internal bleeding. In further embodiments, the present invention relates to the treatment of anemia in a patient with anemia from a chronic disease.

[0469]In some embodiments, the present methods and compositions stimulate red blood cell production. In some embodiments, the present methods and compositions stimulate the division and differentiation of established erythroid progenitors in the bone marrow.

[0470]Certain embodiments of the present invention are particularly useful for treating chemotherapy-induced anemia in cancer patients. In some embodiments, the present methods and compositions allow for continued administration of the chimeric protein after a cancer patient's chemotherapy is complete. In some embodiments, the present methods and compositions allow for the treatment of a cancer patient without reducing the dose relative to a non-cancer patient. In some embodiments, the present methods and compositions enable treatment of a cancer patient who is receiving chemotherapy and is believed to be curable. In various embodiments, the cancer patient has one or more histories of blood clots, recent surgery, prolonged periods of bed rest or restricted activity, and treatment with a chemotherapeutic agent.

[0471]kits

[0472]The invention also provides kits for the administration of any agent described herein (e.g., the chimeric protein with or without various additional therapeutic agents). The kit is a kit of materials or components containing at least one of the pharmaceutical compositions of the invention described in this document. Thus, in some embodiments, the kit contains at least one of the pharmaceutical compositions described herein.

[0473]The exact nature of the components configured in the kit depends on their intended use. In one embodiment, the kit is configured for the purpose of treating human subjects.

[0474]Instructions for use may be included with the kit. Instructions for use often include a tangible phrase that describes the technique to be employed when using kit components to achieve a desired result, such as: B. a cancer treatment. Optionally, the kit also contains other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, dressings, or other useful paraphernalia, as will be readily known to those skilled in the art. .

[0475]The materials and components assembled in the kit may be provided to the physician, stored in any convenient and appropriate manner that preserves their functionality and usefulness. For example, the components can be provided at room temperature, refrigerated, or frozen. The components are usually contained in suitable packaging materials. In various embodiments, the packaging material is manufactured by well-known methods, preferably to provide a contamination-free and sterile environment. The packaging material may have an external label stating the contents and/or purpose of the kit and/or its components.

The definition

[0476]As used in this document, "a", "an" or "the" can mean one or more than one.

[0477]Unless expressly stated or apparent from the context, as used in this document, the term "or" should be understood broadly and includes both "or" and "and".

[0478]In addition, the term "above" when used in connection with a referenced numeric specification means the referenced numeric specification plus or minus up to 10% of that referenced numeric specification, e.g. within (plus or minus) 10%, 9% , 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the displayed value. For example, the language “approx. 50” covers the range from 45 to 55.

[0479]An "effective amount," when used in connection with medicinal uses, is an amount effective in providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disease of interest.

[0480]As used herein, something is "reduced" when a measure of activity and/or effect is reduced by a significant amount, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50 %, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or more, up to and including at least about 100%, in the presence of an agent or stimulus relative to the lack of modulation. As will be appreciated by those skilled in the art, in some embodiments activity will decrease and some downstream readings will decrease, but others may increase.

[0481]Conversely, when an activity and/or effect measure increases by a significant amount, e.g. B. at least 10%, at least 20%, at least 30%, at least 40%. %, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100% or more, at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times, at least about 6 times, at least about 7 times, at least about 8 times, at least about 9 times, at least about 10 times, at least about 50 times, at least about 100 times, in the presence of an agent or stimulus relative to the absence of such agent or stimulus.

[0482]As mentioned herein, all composition percentages are by weight of the total composition unless otherwise indicated. As used in this document, the word "include" and its variants are not intended to be limiting, such that recitation of elements in a list does not exclude other similar elements that may also be useful in the compositions and methods of this technology. Likewise, the terms "may" and "can" and their variants are not intended to be limiting, such that the listing that one embodiment may include particular elements or features does not exclude other embodiments of the present technology that do not include those elements or features.

[0483]Although the open-ended term "comprising" is used interchangeably with terms such as comprising, containing or having to describe and claim the invention, the present invention or its embodiments may alternatively be described using alternative terms such as "consisting of" or "consisting essentially of".

[0484]As used in this document, the words "preferred" and "preferred" refer to embodiments of technology that offer certain advantages in certain circumstances. However, other embodiments may be preferred under the same or different circumstances. Furthermore, the listing of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the technology.

[0485]The amount of the compositions herein required to produce a therapeutic effect can be determined empirically using conventional methods for the particular purpose. To administer therapeutic agents for therapeutic purposes, the therapeutic agents will generally be administered at a pharmacologically effective dose. A "pharmacologically effective amount", "pharmacologically effective dose", "therapeutically effective amount" or "effective amount" refers to an amount sufficient to produce the desired physiological effect, or an amount capable of to achieve the desired result, particularly for the treatment of the disorder or illness. An effective amount as used herein would include an amount sufficient to, for example, delay the development of a symptom of the disorder or disease, alter the course of a symptom of the disorder or disease (e.g., delay progression of a symptom). the disorder or illness). disease), reduce or eliminate one or more symptoms or manifestations of the disorder or disease and reverse a symptom of a disorder or disease. Therapeutic benefit also includes stopping or slowing the progression of the underlying disease or disorder, whether or not improvement is achieved.

[0486]Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical techniques in cell cultures or experimental animals, for example, around the LD50 (the lethal dose for approximately 50% of the population) and ED50 (the lethal dose for approximately 50% of the population) therapeutically effective at about 50% of the population). The dosage may vary depending on the dosage form and route of administration used. The dose ratio between toxic and therapeutic effect is the therapeutic index and can be expressed as LD50/ED50 ratio. In some embodiments, compositions and methods that exhibit high therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from in vitro assays including, for example, cell culture assays. In addition, a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 determined in cell culture or in an appropriate animal model. Plasma concentrations of the disclosed compositions can be measured, for example, by high performance liquid chromatography. The effects of a given dosage can be monitored by an appropriate bioassay. The dosage can be determined by a physician and, if necessary, adjusted to take account of the observed effects of the treatment.

[0487]In certain embodiments, the effect results in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect results in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefit also includes stopping or slowing the progression of the underlying disease or disorder, whether or not improvement is achieved.

[0488]As used herein, "methods of treatment" are equally applicable to use of a composition for treating the diseases or disorders described herein and/or compositions for use and/or uses in the manufacture of medicaments for treating the diseases or disorders described herein.

EXAMPLES

[0489]The term "AcTaferon" is sometimes used herein to denote an interferon-based chimera.

[0490]In the following examples, unless otherwise noted, IFN mutations refer to human IFN-α2.

[0491]The Q124R mutant is representative of an attenuated human IFN-alpha-2 mutant that can be tested in vivo in a mouse model. In particular, Q124R is a human IFN mutation suitable for use in the mouse (ie, it is a human IFN mutant that functions in the mouse).See Nat. How.2014; 5:3016. doi: 10.1038/ncomms4016, the entire contents of which are incorporated herein by reference.

Example 1. VHH directed against murine SIRP1α binds to SIRP1α and neutralizes the SIRP1α/CD47 interaction

[0492]HEK293T cells were transiently transfected with a murine SIRP1α expression plasmid and maintained in DMEM medium supplemented with 10% FBS at 37°C for 48 h. Cells were detached, washed with PBS and treated for 1 hour with the indicated concentration of purified His-tagged VHH in PBS supplemented with 1% FBS. Samples were washed with PBS and incubated with Alexa488-coupled anti-His antibody (RnD Systems) in PBS supplemented with 1% FBS for 1 hour. The samples were measured on a FACSCalibur analyzer (BD Biosciences).

[0493]As shown inFEIGE. 1Aa serial dilution of murine anti-SIRP1α VHH was tested in a FACS-based mSIRPA binding assay in cells expressing murine SIRP1α. The geometric mean of the fluorescence intensity was shown. Murine anti-SIRP1α VHH specifically binds to murine SIRP1α (COWARDLY. 1B).

[0494]It was also examined whether murine anti-SIRP1α VHH neutralizes the interaction with CD47 (COWARDLY. 1B).

[0495]HEK293T cells were transiently transfected with a murine SIRP1α expression plasmid and seeded in 96-well plates in DMEM medium supplemented with 1% anti-murine SIRP1α-HHV at the indicated concentration for 1 hour at 37°C. The cells were then treated with conditioned medium containing a fusion protein derived from murine CD47 (N-terminal 158 amino acids) coupled to SEAP (secreted alkaline phosphatase) for 2 hours at 37°C. Cells were washed 4 times with PBS + 0.05% Tween -20 and phosphatase activity was measured using the PhosphaLight assay according to the manufacturer's instructions (ThermoFisher Scientific).

[0496]As shown inCOWARDLY. 1Ba serial dilution of murine anti-SIRP1α VHH was tested in a murine CD47-murine SIRPα binding assay. The mean -/+ standard deviation of the triplicate measurements was plottedCOWARDLY. 1B. . . . Murine anti-SIRP1α VHH inhibits CD47 binding in a dose-dependent manner. The data showed that murine anti-SIRP1α VHH neutralizes the SIRP1α-CD47 interaction.

Example 2. In vivo antitumor effects of bispecific SIRP1α chimeras

[0497]An in vivo mouse study was performed using an anti-mouse Sirp1α VHH/human IFN chimera Q124R in model B16.

[0498]A SIRP1α targeting chimera (hIFNα2Q124R coupled via a 20xGGS linker to an N-terminal neutralizing VHH specific for mouse SIRP1α) was constructed in a pHen6 vector and large-scale productions of His-tagged SIRP1α were performed .E coli. Bacteria were grown to stationary phase (OD600 of 0.7-0.8) after which IPTG (BioScientific) was added to activate the LacZ promoter. Cell supernatant was collected after overnight cultivation. Proteins from the periplasmic fraction were released by osmotic shock using sucrose solution and purified by immobilized metal ion chromatography (IMAC) on a cobalt ion-loaded HiTrap sepharose resin (Clontech, Takara Biotechnology). After protein binding, the columns were washed with 0.5% EMPIGEN (Calbiochem, Millipore), 0.5% CHAPS (Sigma-Aldrich) and PBS. Imidazole (Merck) was used for elution and removed using PD-10 gel filtration columns (GE Healthcare). Protein concentration was determined using absorbance at 280 nm and purity was assessed by SDS-PAGE. LPS levels were quantified using Limulus amebocyte lysate (LAL) QCL-1000 (Lonza). If still present, LPS was removed using endotoxin removal resin (Thermo Scientific). The biological activities of all products were determined by a functional assay using the mouse luciferase reporter cell line LL171 against the WHO international mouse IFNa standard Ga02-901-511 as previously described (born how.2014; 5:3016. doi: 10.1038/ncomms4016).

[0499]Mice were housed under pathogen-free conditions in a temperature-controlled environment with 12/12 hour light/dark cycles and received food and water ad libitum. Female C57BL/6J mice (Charles River Laboratories, Saint-Germain sur I'Arbresle, France) were inoculated at 5 x 10 6⁶Clone B16-mCD20 cells (B16B16 cells stably transfected with a plasmid containing the expression cassette for mCD20) at 8 weeks of age using a 30 G insulin syringe, in 50 µL suspension, onto the shaved flank from briefly sedated mice (using 4% isoflurane).

[0500]Tumor treatments were performed perilesional (p.l.), which s.c. at the edge of the tumor, starting from day 7 after tumor inoculation. Mice (n=5) received SIRP1α chimeric treatments on days 7, 8, 9, 10, 11, 14, 15 and 16. As a control, mice (n=4) were treated with 100 µl PBS. The chimeras were administered at a rate of 5500 IU per treatment, which corresponds to 35 µg protein (1.6 mg/kg). One day after the last tumor treatment, blood was collected from the tail vein into EDTA-coated Microvette tubes (Sarstedt) and analyzed in a Hemavet 950FS whole blood counter (Drew Scientific, Waterbury, USA). WBC, lymphocytes, neutrophils and monocytes are expressed in K/μl, rbc in M/μl, hemoglobin in g/dl and hematocrit in %; Platelets in K/μl and mean platelet volume in fl.

[0501]As shown inHIGO. 2A-B, significant inhibition of tumor growth was achieved with the anti-mouse Sirp1α VHH/human IFN chimera Q124R (lower curve) compared to the PBS control (upper curve). In addition, the Sirp1α VHH/human IFN chimera Q124R has been shown to be safe as measured by several blood cell-based parameters (white blood cell count ("wbc"), lymphocyte count ("ly"), neutrophil count (" ne"), monocyte count ("mo"), erythrocyte count ("rbc"), hemoglobin ("hb"), hemocrit ("warm"), platelets ("plt"), and mean platelet volume ("mpv")). Importantly, the anti-mouse Sirp1α VHH/human IFN chimera Q124R and PBS show almost no difference in these parameters. It is known that wild-type interferon is poorly tolerated in vivo.

Example 3. Bispecific SIRP1α chimeras

[0502]An anti-mouse Sirp1α-VHH/anti-mouse PD-L1 VHH/human Q124R-IFN bispecific chimera was examined. In particular, FACS analysis was performed to quantify STAT1 phosphorylation in the mouse PD-L1 positive cell line B16.

[0503]B16 cells were stimulated with the bispecific Sirp1α chimera for 15 minutes at 37°C in DMEM medium supplemented with 10% FBS. After stimulation, the cells were fixed by adding 1 volume of fixation buffer I (BD Biosciences) for 10 minutes at 37° C. and permeabilized by resuspending in 2 volumes of Perm III buffer I (BD Biosciences) on ice for 30 minutes. The samples were stained with an anti-STAT1 antibody pY701 (BD Biosciences) for 20 min at 4°C and analyzed with a FACSCalibur (BD Biosciences) and the CellQuest Pro Version 4.0.2 software (BD Biosciences).

[0504]As shown inFEIGE. 3B16 cells were stimulated with 100 ng/ml bispecific anti-mouse Sirp1α VHH/anti-mouse PD-L1 VHH/human IFN-Q124R chimera and Bcl10-HHV-human IFN-Q124R chimera or for Leave unstimulated for 15 minutes at 37°C. °C. After fixation and permeabilization, cells were stained for Phospho-STAT1 and analyzed on FACS. The data clearly illustrate that targeting PD-L1 significantly increased STAT1 phosphorylation by the bispecific chimeras compared to the untargeted chimera (Bcll10 VHH).

Example 4: Bispecific SIRP1α chimeras

[0505]In this example, chimeric proteins are constructed and characterized comprising a mutant human IFNalpha2 (IFNα2) and a recombinant heavy chain-only (VHH) antibody targeting human SIRP1α.

[0506]The above chimeric proteins are assayed for FACS by quantifying STAT1 phosphorylation in CD20-positive and CD20-negative peripheral blood mononuclear cells (PBMC).

[0507]Generation, production and purification of chimeric proteins

[0508]To generate mutant IFNα2-based chimeric proteins, a nucleic acid sequence encoding wild-type human IFNα2 is fused via a flexible 20x Gly-Gly-Ser flexible linker to the sequence of a human SIRP1α-targeting VHH. The wild-type human IFNα2 in the resulting nucleic acid construct is mutated with one of the following mutations: R33A, R144A, R144S, R144T, R144Y, R144L, R1441, A145G, A145H, A145Y, A145K, A145D, M148A, R149A and L153A. Nucleic acid constructs containing only wild-type IFNα2 or wild-type IFNα2 fused to a VHH targeting human SIRP1α are used as controls.

[0509]The nucleic acid constructs described above are inserted into the pHEN6C vector for bacterial expression. Protein expression is induced overnight with 1mM IPTG, cells are pelleted and periplasmic extracts are prepared using TES buffer (0.2M Tris pH 8.0, 0.5mM EDTA, 0.5M sucrose) and TES /4 manufactured. Proteins are purified from extracts using TALON metal affinity resin according to manufacturer's guidelines and imidazole is removed from samples using PD10 columns (GE Healthcare).

[0510]STAT1-Phosphorylierung in PBMC

[0511]Buffy coat PBMCs from healthy donors are isolated by density gradient centrifugation with Lymphoprep (StemCell Technologies). The cells are washed twice with FACS buffer (2% FBS, 1 mM EDTA in PBS) and stained with FITC anti-human CD20 (SinoBiologicals) for 20 min at 4°C. After two washes, cells are stimulated with a serial dilution of wild-type IFNα2-like chimeric IFNα2/SIRP1α proteins or one of the IFNα2/SIRP1α mutant chimeric proteins described above for 15 minutes at 37°C. After fixation (10 min, 37 °C, Fix Buffer I; BD Biosciences), permeabilization (30 min, on ice, Perm III Buffer I; BD Biosciences) and washing, the cells are treated with anti-STAT1 pY701 Ab (BD Biosciences). Samples are collected with a FACSCalibur (BD Biosciences) and analyzed with FlowJo software version 10.2 (LLC).

[0512]Results

[0513]It is predicted that CD20 positive and CD20 negative cells are comparably sensitive to wild-type human IFNα2 alone. Fusion of wild-type human IFNα2 to a VHH targeting SIRP1α is also predicted to result in a significant increase in STAT1 phosphorylation in CD20-positive cells compared to CD20-negative cells, ie the ImmunoKine effect.

[0514]Mutant IFNα2 chimeric proteins are predicted to have little, if any, activity in CD20 negative cells. It is further predicted that the loss of activity of mutant IFNα2 chimeric proteins is largely restored when the CD20 antigen is expressed on PBMCs.

Example 5. In vivo antitumor effects of bispecific SIRP1α chimeras

[0515]An in vivo mouse study is performed with anti-mouse VHH SIRP1α-VHH/human IFNα2 chimeric mutant proteins of these examples in the B16 model.

[0516]Nucleic acid constructs encoding a mutant human SIRP1α-VHH/IFNα2 chimeric protein comprising a mutant human IFNα2 fused via a 20xGGS connector to a mouse SIRP1α-specific N-terminal neutralizing VHH are expressed in a pHen6 vector constructed. The mutation in human IFNα2 is selected from R33A, R144A, R144S, R144T, R144Y, R144L, R1441, A145G, A145H, A145Y, A145K, A145D, M148A, R149A and L153A. Large-scale productions of mutant His-tagged human SIRP1α-VHH/IFNα2 chimeric proteins are underwayE coli. Bacteria are grown to stationary phase (OD600 of 0.7-0.8) after which IPTG (BioScientific) is added to activate the LacZ promoter. Cell supernatant is collected after overnight cultivation. Proteins from the periplasmic fraction are released by osmotic shock using sucrose solution and purified by immobilized metal ion chromatography (IMAC) on a cobalt ion-loaded HiTrap sepharose resin (Clontech, Takara Biotechnology). After protein binding, the columns are washed with 0.5% EMPIGEN (Calbiochem, Millipore), 0.5% CHAPS (Sigma-Aldrich) and PBS. Imidazole (Merck) is used for elution and removed using PD-10 gel filtration columns (GE Healthcare). Protein concentration is determined using absorbance at 280 nm and purity is assessed by SDS-PAGE. LPS levels are quantitated using Limulus amebocyte lysate (LAL) QCL-1000 (Lonza). If still present, the LPS is removed with endotoxin removal resin (Thermo Scientific). The biological activities of all products are determined by a functional assay using the mouse luciferase reporter cell line LL171 against the WHO international mouse IFNα standard Ga02-901-511 as previously described (born how.2014; 5:3016. doi: 10.1038/ncomms4016).

[0517]Mice are housed under pathogen-free conditions in a temperature-controlled environment with 12/12 hour light/dark cycles and receive food and water ad libitum. Female C57BL/6J mice (Charles River Laboratories, Saint-Germain sur I'Arbresle, France) are inoculated with 5 x 10 6⁶Clone B16-mCD20 cells (B16B16 cells stably transfected with a plasmid containing the expression cassette for mCD20) at 8 weeks of age using a 30 G insulin syringe in 50 µl suspension onto the shaved flank of sedated mice briefly (using 4% isoflurane).

[0518]Tumor treatments are administered perilesionally (p.l.), which means s.c. at the edge of the tumor, starting from day 7 after tumor inoculation. Mice (n=5) receive SIRP1α chimeric protein treatments on days 7, 8, 9, 10, 11, 14, 15 and 16. As a control, mice are treated with 100 µl PBS (n=4). Mice treated with chimeric protein receive 5500 IU per treatment, corresponding to 35 µg protein (1.6 mg/kg). One day after the last tumor treatment, blood is collected from the tail vein into EDTA-coated Microvette tubes (Sarstedt) and analyzed in a Hemavet 950FS whole blood counter (Drew Scientific, Waterbury, USA). WBC, lymphocytes, neutrophils and monocytes are expressed in K/μl, rbc in M/μl, hemoglobin in g/dl and hematocrit in %; Platelets in K/μl and mean platelet volume in fl.

[0519]A significant inhibition of tumor growth is predicted to be observed in mice treated with the human IFNα2/anti-mouse SIRP1αVHH chimeric mutant proteins compared to the PBS control. Mutant human SIRP1αVHH/IFNα chimeric proteins are also expected to be shown to be safe, as assessed by several blood cell-based parameters (white blood cell count ("wbc"), lymphocyte count ("ly"), neutrophil count ("ne" )), monocyte count (“mo”), erythrocyte count (“rbc”), hemoglobin (“hb”), hemocrit (“hct”), platelets (“plt”), and mean platelet volume (“mpv”)). It is further predicted that the SIRP1α-VHH/IFNα2 human chimeric anti-mouse mutant proteins and PBS will show almost no difference in these parameters. It is known that wild-type interferon is poorly tolerated in vivo.

EQUIVALENTS

[0520]While the invention has been described in connection with specific embodiments thereof, it should be understood that it is susceptible to further modification and this application is intended to cover any variation, use or adaptation of the invention which generally follows the principles of the invention and includes departures from the present invention Description falling within known or usual practice in the field to which the invention pertains and which may be applicable to the essential features set forth above and as follows within the scope of the appended claims.

[0521]Those skilled in the art will recognize, or be able to determine, using no more than routine experimentation, numerous equivalents to the specific embodiments described herein. Such equivalents are intended to be included within the scope of the following claims.

RECORDING BY REFERENCE

[0522]All patents and publications referred to in this document are incorporated herein by reference in their entirety.

[0523]The publications discussed in this document are provided for disclosure prior to the filing date of the present application only. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of any prior invention.

[0524]As used in this document, all headings are organizational only and are not intended to limit disclosure in any way. The content of each individual section may apply equally to all sections.