Targeted chimeric proteins and uses thereof

Abstract

To provide chimeric proteins and the like useful for treating diseases, etc. The present invention relates in part to chimeric proteins comprising at least one targeting moiety that recognizes and binds to SIRP1α and their use as diagnostic and therapeutic agents. The present invention further relates to pharmaceutical compositions comprising the chimeric proteins and their use in the treatment of various diseases.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 454,993, filed February 6, 2017, the entire contents of which are incorporated herein by reference.

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

[0003] Sequence Listing This application contains a Sequence Listing that has been submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy of which, created on January 31, 2018, is named ORN-027PC-Sequence_Listing_ST25.txt and is 282,624 bytes in size. [Background technology]

[0004] Cancer is a global health problem that causes nearly 7 million deaths worldwide each year and has proven to date to be largely untreatable despite major medical advances. Frustratingly, cancer appears to have developed ways to evade detection and eradication by the immune system, thereby evading the body's defenses against disease. For example, one mechanism by which cancer cells avoid phagocytosis by macrophages is through upregulation of CD47, which engages an inhibitory receptor on macrophages, namely signal regulatory protein alpha-1 (SIRP1α). Specifically, the interaction of CD47 with SIRP1α on cancer cells results in a "don't eat me" signal that inhibits phagocytosis of cancer cells. The Myc oncogene induces expression of CD47 in cancer cells, an immunosuppressive mechanism that has been implicated in the potent in vivo tumor growth promoting activity of Myc.

[0005] Activation of cellular oncogenes of the Myc family is one of the most common events involved in oncogenesis in human cancers. The Myc protein family encodes three closely related cellular nuclear phosphoproteins (c-Myc, N-Myc, and L-Myc) that are believed to function as sequence-specific transcription factors. Myc proteins activate a variety of genes important for biological processes including growth, proliferation, apoptosis, metabolism, differentiation, autorenewal, and angiogenesis. Despite efforts to inhibit highly active Myc proteins in cancer cells, this oncogene remains highly resistant to therapeutic targeting. Furthermore, evidence suggests that active Myc proteins promote tumor resistance to a variety of anticancer drugs.

[0006] Thus, there is a need for novel therapeutic agents that can effectively target cancers, including Myc-driven cancers. Summary of the Invention

[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 chimeric proteins of the present disclosure have applications, for example, in recruiting macrophage cells directly or indirectly to a site of interest. In various embodiments, the chimeric proteins further comprise signaling agents, such as, but not limited to, interferons, interleukins, and tumor necrosis factors, which may be modified to attenuate their activity. In various embodiments, the chimeric proteins comprise additional targeting moieties that bind to other targets of interest (e.g., antigens, receptors). In certain embodiments, the other targets of interest (e.g., antigens, receptors) are present on tumor cells. In other embodiments, the other targets of interest (e.g., antigens, receptors) are present on immune cells. In some embodiments, the chimeric proteins of the present disclosure may directly or indirectly recruit immune cells (e.g., macrophages) to a site of action (such as, for non-limiting example, the tumor microenvironment). In some embodiments, the chimeric proteins of the present disclosure promote phagocytosis of target cells (e.g., tumor cells) by macrophages.

[0008] In various embodiments, the chimeric proteins of the present disclosure are used to treat a variety of diseases or disorders, such as cancer, infectious diseases, immune disorders, and other diseases and disorders, and the present invention encompasses a variety of methods of treatment. [Brief description of the drawings]

[0009] [Figure 1A] Figure 1: Binding assay using anti-mouse Sirp1α VHH. Serial dilutions of anti-mouse Sirp1α VHH were tested against mouse Sirp1α expressing cells in a FACS-based mSIRPA binding assay. The geometric mean of the fluorescence intensity was plotted. [Figure 1B] Figure 1: Binding assay using anti-mouse Sirp1α VHH. Serial dilutions of anti-mouse SIRP1α VHH were tested in the mouse CD47-mouse SIRPα binding assay. Mean values ​​± standard deviations of triplicate determinations are plotted. [Figure 2A]Figure 1 shows a B16 in vivo study using anti-mouse Sirp1α VHH / human IFN Q124R chimera. Tumor growth is compared to a PBS control. Anti-mouse Sirp1α VHH / human IFN Q124R chimera is the lower curve, PBS is the upper curve. [Figure 2B] Figure 2 shows B16 in vivo study using anti-mouse Sirp1α VHH / human IFN Q124R chimera. Various safety parameters were evaluated in mice from the tumor study in Figure 2A, namely white blood cell count ("wbc"), lymphocyte count ("ly"), neutrophil count ("ne"), monocyte count ("mo"), red blood cell count ("rbc"), hemoglobin ("hb"); hemocrit ("hct"), platelets ("plt"), and mean platelet volume ("mpv"). In each set, the left bar is PBS and the right bar is anti-mouse Sirp1α VHH / human IFN Q124R. [Diagram 3] Figure 1 shows B16 cells stimulated (or not) with 100ng / ml of the chimera and stained for phosphorylated STAT1. Data are plotted as mean fluorescence intensity. Anti-mouse Sirp1α VHH / anti-mouse PD-L1 VHH / human IFN Q124R and a monospecific anti-BcII10 VHH fusion to modified human IFNα Q124R (non-targeting IFN Q124R control) were analyzed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention is based in part on the discovery of targeting chimeric proteins having a targeting moiety that specifically recognizes and binds to signal regulatory protein alpha-1 (SIRP1α). In some embodiments, the chimeric proteins are multispecific and include one or more targeting moieties. In some embodiments, the chimeric proteins further include a modified signaling agent (e.g., interferon) that has reduced affinity for one or more receptors. In various embodiments, the chimeric proteins can bind to immune cells, such as macrophages, and directly or indirectly recruit those immune cells to the site (e.g., tumor or tumor microenvironment) where a therapeutic action is required. In some embodiments, the chimeric proteins induce and / or promote phagocytosis of tumor cells by macrophages. The chimeric proteins of the present disclosure exhibit beneficial therapeutic properties and reduced side effects.

[0011] Targeting Chimeric Proteins In various embodiments, the present invention relates to a targeting chimeric protein comprising a targeting moiety that specifically recognizes and binds to signal regulatory protein alpha-1 (SIRP1α). SIRP1α (also known as SIRPα) belongs to a family of cellular immune receptors that includes inhibitory members (SIRPα), activating members (SIRPβ), non-signaling members (SIRPγ), and soluble members (SIRPδ). SIRP1α is expressed primarily on myeloid cells, including macrophages, granulocytes, myeloid dendritic cells (DCs), mast cells, and their precursors, including hematopoietic stem cells. SIRP1α acts as an inhibitory receptor that interacts with the ubiquitously expressed transmembrane glycoprotein CD47 to regulate phagocytosis. In particular, binding of CD47 expressed on target cells to SIRP1α on macrophages results in an inhibitory signal that negatively regulates phagocytosis of the target cells.

[0012] In various embodiments, the present invention relates to chimeric targeting proteins that include a targeting moiety that specifically recognizes and binds to SIRP1α on macrophages.

[0013] In various embodiments, the present invention relates to chimeric targeting proteins that include a targeting moiety that specifically recognizes and binds to SIRP1α on monocytes.

[0014] In various embodiments, the present invention relates to a targeting chimeric protein comprising a targeting moiety that specifically recognizes and binds to SIRP1α on TAMs (tumor associated macrophages).

[0015] In various embodiments, the present invention relates to chimeric targeting proteins comprising a targeting moiety that specifically recognizes and binds to SIRP1α on dendritic cells, including but not limited to cDC2s and pDCs.

[0016] In various embodiments, the chimeric protein of the present invention comprises a targeting moiety having a recognition domain that recognizes SIRP1α. In some embodiments, the recognition domain recognizes one or more linear epitopes present on SIRP1α. As used herein, a linear epitope refers to any continuous sequence of amino acids present on 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 sections of amino acids (which may be discontinuous) that form a three-dimensional surface that has a characteristic and / or shape and / or tertiary structure that can be recognized by the antigen recognition domain.

[0017] In some embodiments, the chimeric protein comprises a targeting moiety that may bind to full length and / or mature and / or isoforms and / or splice variants and / or fragments and / or any other natural or synthetic analogs, variants, or mutants of SIRP1α. In an embodiment, SIRP1α is human SIRP1α. In various embodiments, the chimeric protein comprises a targeting moiety that may bind to any form of human SIRP1α, including monomers, dimers, heterodimers, multimers, and aggregates. In an embodiment, the targeting moiety binds to the monomeric form of SIRP1α. In another embodiment, the targeting moiety binds to the dimeric form of SIRP1α.

[0018] In an embodiment, the chimeric protein of the present disclosure comprises a targeting moiety having a recognition domain that recognizes one or more epitopes present on human SIRP1α. In an embodiment, the targeting moiety comprises a recognition domain that recognizes human SIRP1α that includes a signal peptide sequence. An exemplary human SIRP1α polypeptide that includes a signal peptide sequence (underlined) is shown below. [ka]

[0019] In certain embodiments, the targeting moiety comprises a recognition domain that recognizes human SIRP1α without the signal peptide sequence. An exemplary human SIRP1α polypeptide without the signal peptide sequence is shown below. [ka]

[0020] In certain embodiments, the targeting moiety comprises a recognition domain that recognizes a polypeptide encoding human SIRP1α isoform 2. [ka]

[0021] In certain embodiments, the targeting moiety comprises a recognition domain that recognizes a polypeptide encoding human SIRP1α isoform 4. [ka]

[0022] In various embodiments, the targeting moiety of the present invention can be any proteinaceous substance capable of specific binding, such as an antibody or a derivative thereof. In certain embodiments, 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 comprises one variable region (e.g., V H ) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain comprises one variable region (V L ) and one constant region (C L ). The variable regions determine the specificity of the antibody. Each variable region contains three hypervariable regions, also known as complementarity determining regions (CDRs), flanked by four relatively conserved framework regions (FRs). The three CDRs, termed 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.

[0023] In some embodiments, the targeting moiety comprises an antibody derivative or antibody format. In some embodiments, the targeting moiety of the chimeric protein of the present disclosure can be any of the antibodies described in U.S. Patent No. 7,417,130, U.S. Patent Application Publication No. 2004 / 132094, U.S. Patent No. 5,831,012, U.S. Patent Application Publication No. 2004 / 023334, U.S. Patent No. 7,250,297, U.S. Patent No. 6,818,418, U.S. Patent Application Publication No. 2004 / 209243, U.S. Patent No. 7,838,629, U.S. Patent No. 7,186,524, U.S. Patent No. 7,186,524, U.S. Patent No. 7,250,297, U.S. Patent No. 6,818,418, U.S. Patent Application Publication No. 2004 / 209243, U.S. Patent No. 7,838,629 ...

[0023] A single molecule, such as those described in U.S. patents or U.S. patent applications, such as U.S. Pat. No. 6,004,746, U.S. Pat. No. 5,475,096, U.S. Patent Application Publication No. 2004 / 146938, U.S. Patent Application Publication No. 2004 / 157209, U.S. Pat. No. 6,994,982, U.S. Pat. No. 6,794,144, U.S. Patent Application Publication No. 2010 / 239633, U.S. Pat. No. 7,803,907, U.S. Patent Application Publication No. 2010 / 119446, and / or U.S. Pat. No. 7,166,697. Domain antibodies, recombinant heavy chain antibodies (VHH), single chain antibodies (scFv), shark heavy chain antibodies (VNAR), microproteins (cysteine ​​knot proteins, knottins), DARPin, tetranectin, affibody, transbody, anticalin, adnectin, affilin, microbody, peptide aptamer, alterase, plastic antibody, phylomer, stradobody, maxibody, shrimp body, fy nomer, armadillo repeat protein, Kunitz domain, avimer, atrimer, probody, immunobody, triomab, troibody, pepbody, vaccibody, unibody, affimer, duobody, Fv, Fab, Fab', F(ab')2, peptidomimetic molecule, or synthetic molecule. See also Storz, MAbs., May-June 2011, 3(3):pp.310-317.

[0024] In one embodiment, the targeting moiety comprises a single domain antibody, such as a VHH derived from an organism that produces VHH antibodies, e.g., camelid VHH, shark VHH, or designed VHH. VHHs are antibody-derived therapeutic proteins that contain the structural and functional properties unique to natural heavy chain antibodies. VHH technology is based on fully functional antibodies derived from 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.

[0025] In certain embodiments, the targeting moiety comprises a VHH. In some embodiments, the VHH is a humanized VHH or a camelized VHH.

[0026] In some embodiments, the VHH is a fully human V H In some embodiments, the fully human V H A domain, e.g., HUMABODY, is monovalent, bivalent, or trivalent. In some embodiments, a fully human V H Domains, e.g., HUMABODYs, can be monospecific or multispecific, e.g., monospecific, bispecific, or trispecific. Exemplary fully human V H Domains such as HUMABODY are described, for example, in WO 2016 / 113555 and WO 2016 / 113557, the disclosures of which are incorporated by reference in their entireties.

[0027] For example, in some embodiments, the chimeric protein of the invention comprises one or more antibodies, antibody derivatives or antibody formats, peptides or polypeptides, VHHs, 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 to SIRP1α. In some embodiments, the chimeric protein comprises a targeting moiety that is a camelid heavy chain antibody (VHH) that specifically binds to SIRP1α.

[0028] In some embodiments, the chimeric protein comprises a targeting moiety that is a VHH comprising a single amino acid chain having four "framework regions" (FR) and three "complementarity determining regions" (CDRs). As used herein, "framework region" or "FR" refers to a region in a variable domain located between the CDRs. As used herein, "complementarity determining region" or "CDR" refers to a variable region in a VHH that comprises an amino acid sequence capable of specifically binding to an antigenic target. In various embodiments, the chimeric protein of the present disclosure comprises a VHH having a variable domain comprising at least one of a CDR1 sequence, a CDR2 sequence, and / or a CDR3 sequence.

[0029] In various embodiments, the targeting moieties of the invention may comprise any combination of heavy chain sequences, light chain sequences, heavy chain variable region sequences, light chain variable region sequences, complementarity determining region (CDR) sequences, and framework region sequences that are known to recognize and bind to SIRP1α.

[0030] In various embodiments, the technology contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs (collectively referred to herein as "analogs") of the SIRP1α targeting moieties described herein. In various embodiments, the amino acid sequence of the SIRP1α targeting moiety further comprises amino acid analogs, amino acid derivatives, or other non-classical amino acids.

[0031] In various embodiments, the chimeric protein comprises a targeting moiety comprising a sequence that is at least 60% identical to any one of the sequences disclosed herein. For example, the chimeric protein comprises a targeting moiety that is 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 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%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95 ...5%, at least about 95%, at least about 95%, at least about 95%, at least about 95%, at least about 95%, at least about 95%, at least about %, 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 (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about In some embodiments, the targeting moiety may comprise a sequence that is about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identical).

[0032] In various embodiments, the chimeric proteins of the present disclosure include a targeting moiety that includes an amino acid sequence that has one or more amino acid mutations with respect to any targeting moiety sequence known to recognize and bind to SIRP1α. In various embodiments, the chimeric proteins of the present disclosure include a targeting moiety that includes an amino acid sequence that has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, or 50 amino acid mutations with respect to any targeting moiety sequence known to recognize and bind to SIRP1α. In some embodiments, the one or more amino acid mutations can be independently selected from substitutions, insertions, deletions, and truncations.

[0033] In some embodiments, the amino acid mutations are amino acid substitutions, which may include conservative and / or non-conservative substitutions.

[0034] "Conservative substitutions" may be made, for example, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the amino acid residues involved. The 20 naturally occurring amino acids can be grouped into six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

[0035] As used herein, "conservative substitution" is defined as the replacement of an amino acid with another amino acid described in the same group of the six standard amino acid groups above. For example, replacement of Asp with Glu maintains one negative charge in the modified polypeptide. Furthermore, glycine and proline can be substituted for each other based on their ability to disrupt alpha helices.

[0036] As used herein, a "non-conservative substitution" is defined as the replacement of an amino acid with another amino acid that is described in a different group among the six standard amino acid groups (1) to (6) above.

[0037] In various embodiments, substitutions can also include non-typical amino acids. Exemplary non-typical amino acids generally include, but are not limited to, selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA, and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of 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, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methylamino acids, C α-methylamino acids, N α-methylamino acids, and amino acid analogs.

[0038] In various embodiments, the amino acid mutations may be in the CDRs of the targeting moiety (e.g., the CDR1, CDR2, or CDR3 regions). In another embodiment, the amino acid changes may be in the framework regions (FRs) of the targeting moiety (e.g., the FR1, FR2, FR3, or FR4 regions).

[0039] Amino acid sequence modifications can be made using any method known in the art, such as site-directed mutagenesis or PCR-based mutagenesis, as described, for example, in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Plainview, NY, 1989, and Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1989.

[0040] In various embodiments, the mutations do not substantially reduce the ability of the chimeric protein of the present disclosure to specifically recognize and bind to SIRP1α. In various embodiments, the mutations do not substantially reduce the ability of the chimeric protein of the present disclosure to specifically bind to SIRP1α and not modulate (e.g., partially or fully neutralize) the function of SIRP1α.

[0041] In various embodiments, the binding affinity of the chimeric proteins of the present disclosure to the full-length and / or mature form and / or isoforms and / or splice variants and / or fragments and / or monomeric and / or dimeric forms of SIRP1α and / or any other natural or synthetic analogs, variants, or mutants is determined by the equilibrium dissociation constant (K DIn various embodiments, the chimeric proteins of the present disclosure have a K of less than about 1 uM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 10 nM, or less than about 5 nM, or less than about 1 nM to full-length and / or mature forms and / or isoforms and / or splice variants and / or fragments and / or any other natural or synthetic analogs, variants, or mutants of SIRP1α, including monomeric and / or dimeric forms. D The targeting moiety binds at

[0042] In various embodiments, the chimeric protein of the present disclosure includes a targeting moiety that binds to an antigen of interest, i.e., SIRP1α, but does not modulate its function. For example, in various embodiments, the targeting moiety of the chimeric protein merely targets the antigen and does not substantially functionally modulate (e.g., does not substantially inhibit, reduce, or neutralize) the biological effect of the antigen. In various embodiments, the targeting moiety of the chimeric protein of the present disclosure binds to an epitope that is physically separated from the site of the antigen that is important for biological activity (e.g., the active site of the antigen).

[0043] In other embodiments, the chimeric proteins of the present disclosure include a targeting moiety that binds to and modulates the function of an antigen of interest, i.e., SIRP1α. For example, in various embodiments, the targeting moiety of the chimeric protein targets an antigen, i.e., SIRP1α, and functionally modulates (e.g., inhibits, reduces, or neutralizes) the biological effect of the antigen. Such binding and associated functional modulation may be utilized in various embodiments of the present invention, including methods of using the chimeric proteins of the present disclosure to directly or indirectly recruit active immune cells to a site of need via effector antigens.

[0044] For example, in various embodiments, the chimeric proteins of the present disclosure can be used to directly or indirectly recruit macrophages to tumor cells via SIRP1α in a method to reduce or eliminate tumors (e.g., the chimeric proteins of the present disclosure can include a targeting moiety having an anti-SIRP1α antigen recognition domain and a targeting moiety having a recognition domain (e.g., an antigen recognition domain) directed to a tumor antigen or receptor). There is evidence that tumor cells often upregulate CD47, which engages with SIRP1α to avoid phagocytosis. Thus, in various embodiments, it may be desirable to directly or indirectly recruit macrophages to tumor cells and cause phagocytosis of tumor cells by macrophages by inhibiting, reducing, or neutralizing the inhibitory activity of SIRP1α. In various embodiments, the chimeric proteins of the present disclosure promote phagocytosis of tumor cells or any other undesirable cells by macrophages.

[0045] Therapeutic Agents Comprising the Chimeric Proteins of the Disclosure Chimeras and fusions with signal transduction molecules In various embodiments, the chimeric proteins of the invention are part of a chimera or fusion with one or more signaling agents. Thus, for example, the invention provides chimeric or fusion proteins that include a targeting moiety against SIRP1α and one or more signaling agents.

[0046] In various embodiments, the signaling agent is modified to reduce affinity or activity for one or more of its receptors, thereby attenuating activity (including agonism or antagonism) and / or preventing non-specific signaling or undesired sequestration of the chimeric or fusion protein. In various embodiments, the signaling agent is antagonistic in wild type and has one or more mutations that attenuate its antagonist activity. In various embodiments, the signaling agent is antagonistic due to one or more mutations, e.g., an agonistic signaling agent is converted to an antagonistic signaling agent, and such a converted signaling agent may also optionally have one or more mutations that attenuate its antagonist activity (e.g., as described in WO2015 / 007520, the entire contents of which are incorporated herein by reference).

[0047] Thus, in various embodiments, the signaling agent is a modified (e.g., mutant) form of a signaling agent having one or more mutations. In various embodiments, the modifications (e.g., mutations) result in the modified signaling agent having attenuated one or more activities, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity, compared to the unmodified or unmutated, i.e., wild-type, signaling agent (e.g., comparing the wild-type and modified or mutant forms of the same signaling agent). In some embodiments, mutations that attenuate or reduce binding or affinity include mutations that substantially reduce or eliminate binding or affinity. In some embodiments, mutations that attenuate or reduce binding or affinity are different from mutations that substantially reduce or eliminate binding or affinity. As a result, in various embodiments, the mutations result in the signaling agent having improved safety, e.g., reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to the unmutated, i.e., wild-type, signaling agent (e.g., comparing the wild-type and modified (e.g., mutant) forms of the same signaling agent).

[0048] As described herein, an agent may have one or more modifications, e.g., mutations, to improve its safety. In various embodiments, improved safety means that the chimeric proteins of the present disclosure provide reduced toxicity (e.g., systemic toxicity and / or tissue / organ-related toxicity), and / or reduced or substantially eliminated side effects, and / or increased tolerability, reduced or substantially eliminated adverse events, and / or reduced or substantially eliminated off-target effects, and / or an increased therapeutic window.

[0049] In various embodiments, the signaling agent is modified to have one or more mutations that reduce its binding affinity or activity to one or more of its receptors. In some embodiments, the signaling agent is modified to have one or more mutations that substantially reduce or eliminate its binding affinity or activity to the receptor. In some embodiments, the activity of the wild-type signaling agent is agonism at the receptor (e.g., activation of a cellular effect at the treatment site). For example, the wild-type signaling agent may activate the receptor. In said embodiments, the mutations result in the modified signaling agent having reduced or eliminated activation activity at the receptor. For example, the mutations may result in the modified signaling agent delivering a reduced activation signal to the target cell or may eliminate the activation signal. In some embodiments, the activity of the wild-type signaling agent is antagonism at the receptor (e.g., inhibition or attenuation of a cellular effect at the treatment site). For example, the wild-type signaling agent may attenuate or inhibit the receptor. In these embodiments, the mutations result in the modified signaling agent having reduced or eliminated antagonizing activity at the receptor. For example, the mutation may result in the altered signal transduction agent delivering a reduced inhibitory signal to the target cell, or the inhibitory signal may be eliminated. In various embodiments, the signal transduction agent is antagonistic due to one or more mutations, e.g., an agonistic signal transduction agent is converted to an antagonistic signal transduction agent (e.g., as described in WO2015 / 007520, the entire contents of which are incorporated herein by reference). Optionally, such a converted signal transduction agent may have one or more mutations that reduce its binding affinity or activity to one or more of its receptors, or substantially reduce or eliminate its binding affinity or activity to one or more of its receptors.

[0050] In some embodiments, the loss of affinity or activity at the receptor is reversible by binding to one or more of the targeting moieties as described herein (e.g., a targeting moiety to SIRP1α, or any other targeting moiety described herein), in other embodiments, the loss of affinity or activity at the receptor is not substantially reversible by the activity of one or more of the targeting moieties.

[0051] In various embodiments, the chimeric proteins of the invention have reduced off-target effects because their signaling agents have mutations that weaken or eliminate binding affinity or activity at the receptor. In various embodiments, such reduced side effects are observed, for example, compared to wild-type signaling agents. In various embodiments, the signaling agents are active on the target cells because the targeting moiety compensates for the lost / insufficient binding (and / or binding activity, including but not limited to) required for substantial activation. In various embodiments, the modified signaling agents are substantially inactive on the way to the therapeutic active site and act substantially on the specifically targeted cell type, greatly reducing unwanted side effects.

[0052] In some embodiments, a signaling agent may include one or more mutations that attenuate or reduce binding or affinity to one receptor (i.e., a therapeutic receptor) and one or more mutations that substantially reduce or eliminate binding or affinity at a second receptor. In said embodiments, these mutations may be at the same or different positions (i.e., the same mutation or mutations). In some embodiments, the mutation that reduces binding and / or activity at one receptor is different from the mutation that substantially reduces or eliminates binding and / or activity at another receptor. In some embodiments, the mutation that reduces binding and / or activity at one receptor is the same as the mutation that substantially reduces or eliminates binding and / or activity at another receptor. In some embodiments, the chimeric proteins of the present disclosure have modified signaling agents that have both mutations that attenuate binding and / or activity at a therapeutic receptor, thereby allowing for a more controlled on-target therapeutic effect (e.g., compared to a wild-type signaling agent), and mutations that substantially reduce or eliminate binding and / or activity at another receptor, thereby reducing side effects (e.g., compared to a wild-type signaling agent).

[0053] In some embodiments, the substantial reduction or elimination of binding or activity is not substantially restored by a targeting moiety (e.g., a targeting moiety for SIRP1α or any other targeting moiety described herein). In some embodiments, the substantial reduction or elimination of binding or activity can be restored by a targeting moiety. In various embodiments, the substantial reduction or elimination of binding or activity at the second receptor can also prevent adverse effects mediated by the other receptor. Alternatively, or in addition, the substantial reduction or elimination of binding or activity at the other receptor can improve therapeutic efficacy by reducing or eliminating sequestering of the therapeutic chimeric protein away from the therapeutic action site. For example, in some embodiments, this eliminates the need for high doses of the chimeric protein of the present disclosure to compensate for loss at the other receptor. Such ability to reduce doses further reduces the possibility of side effects.

[0054] In various embodiments, the altered signaling agent reduces, substantially reduces, or eliminates, e.g., binding (e.g., K D ) and / or activation (e.g., the modified signal transduction substance is, e.g., K A and / or EC 50 agonism of its receptor, measurable as a K I and / or IC 50 The modified signaling agent comprises one or more mutations that render the signaling agent measurable as an antagonist of the receptor (when the signaling agent is an antagonist of the receptor measurable as an antagonist of the receptor). In various embodiments, activity (including agonism or antagonism) is attenuated by a decrease in affinity of the signaling agent at the receptor. In such embodiments, the modified signaling agent has an affinity for the receptor that 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%-20%, about 20%-40%, about 50%, about 40%-60%, about 60%-80%, or about 80%-100% of that of 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-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 20-fold lower, at least about 25-fold lower, at least about 30-fold lower, at least about 35-fold lower, at least about 40-fold lower, at least about 45-fold lower, at least about 50-fold lower, at least about 100-fold lower, at least about 150-fold lower, or about 10-50-fold lower, about 50-100-fold lower, about 100-150-fold lower, about 150-200-fold lower, or more than 200-fold lower, as compared to a wild-type signaling agent.

[0055] In embodiments where the chimeric protein comprises an altered signaling agent having a mutation that reduces binding at one receptor and a mutation that substantially reduces or eliminates binding at a second receptor, the binding affinity of the altered signaling agent for one receptor is reduced or decreased by less than substantially reduces or eliminates affinity for the other receptor. In some embodiments, the binding affinity of the altered signaling agent for one receptor is reduced or decreased by about 1%, or by about 3%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% less than the substantially reduced or eliminated affinity for the other receptor. In various embodiments, substantially reduced or eliminated refers to a greater reduction in binding affinity and / or activity than reduced or eliminated.

[0056] In various embodiments, the modified signaling agent comprises one or more mutations that reduce the intrinsic activity of the signaling agent by about 75%, or about 70%, or about 60%, or about 50%, or about 40%, or about 30%, or about 25%, or about 20%, or about 10%, or about 5%, or about 3%, or about 1%, for example, as compared to a wild-type signaling agent.

[0057] In some embodiments, the modified signaling agent comprises one or more mutations that result in a reduced affinity of the signaling agent to the receptor, the binding affinity being lower than the binding affinity of the targeting moiety to the receptor. In some embodiments, the difference in binding affinity is the difference between the binding affinity of the signaling agent / receptor and the binding affinity of the targeting moiety / receptor in the same cell. In some embodiments, the difference in binding affinity allows the signaling agent, e.g., the mutant signaling agent, to localize the on-target effect and minimize the off-target effect that underlies the side effects seen with the wild-type signaling agent. In some embodiments, the binding affinity is at least about 2-fold lower, at least about 5-fold lower, at least about 10-fold lower, at least about 15-fold lower, at least about 25-fold lower, at least about 50-fold lower, at least about 100-fold lower, or at least about 150-fold lower.

[0058] Receptor binding activity can be measured using methods known in the art. For example, affinity and / or avidity can be assessed by Scatchard plot analysis and computer fitting of binding data (e.g., Scatchard, 1949), or by reflectance interferometry under flow-through conditions as described by Brecht et al. (1993), the entire contents of which are incorporated herein by reference.

[0059] In various embodiments, the signaling agent is an immunomodulator, such as one or more of an interleukin, an interferon, and a tumor necrosis factor.

[0060] In some embodiments, the signaling agent is an interleukin or modified interleukin, such as 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 fragments, variants, analogs, or family members thereof. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes, and macrophages. Known functions include stimulating the proliferation of immune cells (e.g., 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, i.e., those in Matthews et al., Lymphokines and Interferons: A Practical Approach, edited by Clemens et al., IRL Press, Washington, DC, 1987, 221-225, and Orencole and Dinarello, (1989) Cytokine, 1, pp. 14-20.

[0061] In some embodiments, the signaling agent is an interferon or modified forms of interferon, such as interferon types I, II, and III. Examples of interferons include, for example, interferon-α-1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, and 21, interferon-β and interferon-γ, interferon-κ, interferon-ε, interferon-τ, and interferon-ω.

[0062] In some embodiments, the signal transduction agent is tumor necrosis factor (TNF) or modified 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.

[0063] The amino acid sequences of the wild-type signaling agents described herein are well known in the art. Thus, in various embodiments, the modified signaling agents are 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 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%, or at least about 100%, or at least about 102%, or at least about 104%, or at least about 105%, or at least about 106%, or at least about 107%, or at least about 108%, or at least about 109%, or at least about 110%, or at least about 111%, or at least about 112%, or at least about 113%, or at least about 114%, or at least about 115%, or at least about 116%, or at least about 117%, or at least about 118%, or at least about 1 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 (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65% , about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence.

[0064] In various embodiments, the modified signaling agent has 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 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%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 1 %, 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%, or at least about 99% sequence identity (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69% , about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence.

[0065] In various embodiments, the modified signal transduction agent comprises an amino acid sequence having one or more amino acid mutations. In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, which may include conservative and / or non-conservative substitutions as described herein.

[0066] In various embodiments, substitutions can also include non-typical amino acids as described anywhere herein.

[0067] As described herein, modified signaling agents have mutations that affect affinity and / or activity at one or more receptors. In various embodiments, the affinity and / or activity is reduced at a therapeutic receptor, e.g., a receptor that mediates a desired therapeutic effect (e.g., agonism or antagonism). In various embodiments, modified signaling agents have mutations that substantially reduce or eliminate affinity and / or activity at a receptor, e.g., a receptor that does not mediate a desired therapeutic effect (e.g., as a result of promiscuous binding). Receptors for any signaling agent as described herein are known in the art.

[0068] Examples of mutations that result in reduced affinity and / or activity (e.g., agonist activity) in receptors can be found in WO 2013 / 107791 and PCT / EP2017 / 061544 (e.g., for interferons), WO 2015 / 007542 (e.g., for interleukins), and WO 2015 / 007903 (e.g., for TNF), the contents of each of which are incorporated herein by reference in their entirety. Examples of mutations that result in reduced affinity and / or activity (e.g., antagonist activity) in therapeutic receptors can be found in WO 2015 / 007520, the contents of which are incorporated herein by reference in their entirety.

[0069] In some embodiments, the altered signal transduction agent comprises one or more mutations that reduce the affinity and / or activity of the signal transduction agent for a type I cytokine receptor, a type II cytokine receptor, a chemokine receptor, a receptor in the tumor necrosis factor receptor (TNFR) superfamily, a TGF-β receptor, a receptor in the immunoglobulin (Ig) superfamily, and / or a receptor in the tyrosine kinase superfamily.

[0070] In various embodiments, the receptor of the signal transduction 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 (β-subunit), IL3, IL4, IL5, IL6, IL7, IL9, IL11, IL12, GM-CSF, G-CSF, LIF, and CNTF, as well as receptors for thrombopoietin (TPO), prolactin, and growth hormone. Examples of 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 receptor.

[0071] In various embodiments, the receptor of the signal transduction substance is a type II cytokine receptor. Type II cytokine receptors are multimeric receptors composed of heterogeneous subunits, and are mainly receptors for interferons. Receptors of this family include, but are not limited to, receptors for interferon-α, interferon-β, and interferon-γ, IL10, IL22, and tissue factor. Examples of type II cytokine receptors include, but are not limited to, IFN-α receptors (e.g., IFNAR1 and IFNAR2), IFN-β receptors, IFN-γ receptors (e.g., IFNGR1 and IFNGR2), and type II IL receptors.

[0072] In various embodiments, the receptor of the signal transduction substance is a G protein-coupled receptor. Chemokine receptors are G protein-coupled receptors with seven transmembrane structures, and are coupled to G proteins 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). Exemplary chemokine receptors include, but are not limited to, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CXCR1, CXCR2, CXCR3, CXCR3B, CXCR4, CXCR5, CSCR6, CXCR7, XCR1, and CX3CR1.

[0073] In various embodiments, the signal transduction receptor is a TNFR family member. Tumor necrosis factor receptor (TNFR) family members share a cysteine-rich domain (CRD) formed from three disulfide bonds around a CXXCXXC core motif that forms an extended molecule. Exemplary tumor necrosis factor receptor family members include CD120a (TNFRSF1A), CD120b (TNFRSF1B), lymphotoxin beta receptor (LTBR, ​​TNFRSF3), CD134 (TNFRSF4), CD40 (CD40, TNFRSF5), FAS (FAS, TNFRSF6), TNFRSF6B (TNFRSF6B), CD27 (CD27, TNFRSF7), CD30 (TNFRSF8), CD137 (TNFRSF9), TNFRSF10A (TNFRSF10A), TNFRSF10B, (TNFRSF10B), TNFRSF10C (TNFRSF10C) , TNFRSF10D (TNFRSF10D), RANK (TNFRSF11A), osteoprotegerin (TNFRSF11B), TNFRSF12A (TNFRSF12A), TNFRSF13B (TNFRSF13B), TNFRSF13C (TNFRSF13C), TNFRSF14 (TNFRSF14), nerve growth factor receptor (NGFR, TNFRSF16), TNFRSF17 (TNFRSF17), TNFRSF18 (TNFRSF18), TNFRSF19 (TNFRSF19), 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 (TNFRSF1B) or TNF-R2.

[0074] In various embodiments, the receptor of the signal transduction agent is a TGF-β receptor. TGF-β receptors are single-pass serine / threonine kinase receptors. TGF-β receptors include, but are not limited to, TGFBR1, TGFBR2, and TGFBR3.

[0075] In various embodiments, the signal transduction receptor is an Ig superfamily receptor. Immunoglobulin (Ig) superfamily receptors share structural homology with immunoglobulins. Ig superfamily receptors include, but are not limited to, interleukin-1 receptor, CSF-1R, PDGFR (e.g., PDGFRA and PDGFRB), and SCFR.

[0076] In various embodiments, the receptor of the signal transduction substance is a tyrosine kinase superfamily receptor. The receptor of the tyrosine kinase superfamily is well known in the art. There are about 58 known receptor tyrosine kinases (RTKs), which are grouped into 20 subfamilies. The receptor of the tyrosine kinase superfamily includes, but is not limited to, FGF receptors and their various isoforms, such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR5.

[0077] In some embodiments the modified signal transduction agent is interferon alpha. In said embodiments the modified IFN-alpha agent has reduced affinity and / or activity for the IFN-alpha / beta receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments the modified IFN-alpha agent has substantially reduced or eliminated affinity and / or activity for the IFN-alpha / beta receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0078] Mutant forms of interferon alpha are known to those of skill in the art. In an exemplary embodiment, the modified signal transduction agent is an IFN-alpha 2a allelic form having the amino acid sequence of SEQ ID NO:46.

[0079] In an exemplary embodiment, the modified signal transduction agent is an IFN-α2b allelic form having the amino acid sequence of SEQ ID NO:47 (which differs from IFN-α2a at amino acid position 23).

[0080] In some embodiments, the IFN-α2 mutant (IFN-α2a or IFN-α2b) comprises one or more amino acid mutations at amino acid positions 144-154, e.g., 148, 149, and / or 153. In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from L153A, R149A, and M148A. Such mutants are described, for example, in WO 2013 / 107791 and Piehler, et al., (2000) J. Biol. Chem, 275:40425-33, the entire contents of which are incorporated herein by reference.

[0081] In some embodiments, the IFN-α2 mutant has 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 WO 2010 / 030671, the contents of which are incorporated herein by reference in their entirety.

[0082] In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from K133A, R144A, R149A, and L153A as described in WO 2008 / 124086, the entire contents of which are incorporated herein by reference.

[0083] In some embodiments, the IFN-α2 mutant comprises one or more mutations selected from R120E and R120E / K121E as described in WO 2015 / 007520 and WO 2010 / 030671, the entire contents of which are incorporated herein by reference. In said embodiments, the IFN-α2 mutant antagonizes wild-type IFN-α2 activity. In said embodiments, the mutant IFN-α2 has reduced affinity and / or activity for IFNAR1 while maintaining affinity and / or activity for IFNR2.

[0084] In some embodiments, the human IFN-α2 mutant comprises one or more mutations selected from (1) R120E and R120E / K121E, which, without wishing to be bound by theory, result in an antagonistic effect, and (2) one or more mutations selected from, for example, K133A, R144A, R149A, and L153A, which, without wishing to be bound by theory, attenuate the effect at IFNAR2. In certain embodiments, the human IFN-α2 mutant comprises R120E and L153A.

[0085] In some embodiments, the human IFN-α2 variant 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 disclosed in WO 2013 / 059885, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the human IFN-α2 variant comprises the H57Y, E58N, Q61S, and / or L30A mutations as disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 variant comprises the H57Y, E58N, Q61S, and / or R33A mutations as disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 variant comprises the H57Y, E58N, Q61S, and / or M148A mutations as disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 variant comprises the H57Y, E58N, Q61S, and / or L153A mutations as disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 mutant comprises the mutations N65A, L80A, Y85A, and / or Y89A, as disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 mutant comprises the mutations N65A, L80A, Y85A, Y89A, and / or D114A, as disclosed in WO 2013 / 059885. In some embodiments, the human IFN-α2 mutant comprises one or more mutations selected from R144X1, A145X2, and R33A, where X1 is selected from A, S, T, Y, L, and I, and X2 is selected from G, H, Y, K, and D.

[0086] In some embodiments the modified signal transduction agent is interferon beta. In said embodiments the modified interferon beta agent has reduced affinity and / or activity for the IFN-α / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments the modified interferon beta agent has substantially reduced or eliminated affinity and / or activity for the IFN-α / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0087] In certain embodiments, the modified signal transduction agent is interferon beta. In said embodiments, the modified interferon beta agent has reduced affinity and / or activity for the IFN-α / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains. In some embodiments, the modified interferon beta agent has substantially reduced or eliminated affinity and / or activity for the IFN-α / β receptor (IFNAR), i.e., the IFNAR1 and / or IFNAR2 chains.

[0088] In an exemplary embodiment, the modified signal transduction 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 from any species. In one embodiment, the chimeric protein includes a modified mouse IFN-β. In another embodiment, the chimeric protein includes a modified human IFN-β. Human IFN-β is a polypeptide containing 166 amino acid residues and has a molecular weight of approximately 22 kDa. The amino acid sequence of human IFN-β is SEQ ID NO:48.

[0089] In some embodiments, the human IFN-β is IFN-β-1a, a glycosylated form of human IFN-β. In some embodiments, the human IFN-β is IFN-β-1b, a non-glycosylated form of human IFN-β with a deletion of Met-1 and a mutation of Cys-17 to Ser.

[0090] In various embodiments, the modified IFN-β has one or more mutations that reduce binding or affinity to the IFNAR1 subunit of IFNAR. In some embodiments, the modified IFN-β has reduced affinity and / or activity at IFNAR1. In various embodiments, the modified IFN-β is human IFN-β and has one or more mutations at positions F67, R71, L88, Y92, I95, N96, K123, and R124. In some embodiments, the one or more mutations are substitutions selected from F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, I95A, N96G, K123G, and R124G. In some embodiments, the modified IFN-β comprises a mutation at F67G. In some embodiments, the modified IFN-β comprises a mutation at K123G. In some embodiments, the modified IFN-β comprises F67G and R71A mutations. In some embodiments, the modified IFN-β comprises L88G and Y92G mutations. In some embodiments, the modified IFN-β comprises Y92G, I95A, and N96G mutations. In some embodiments, the modified IFN-β comprises K123G and R124G mutations. In some embodiments, the modified IFN-β comprises F67G, L88G, and Y92G mutations. In some embodiments, the modified IFN-β comprises F67S, L88S, and Y92S mutations.

[0091] In some embodiments, the modified IFN-β has one or more mutations that reduce binding or affinity to the IFNAR2 subunit of IFNAR. In some embodiments, the modified IFN-β has reduced affinity and / or binding to 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, the one or more mutations are substitutions selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, and Y155G. In some embodiments, the modified IFN-β comprises a W22G mutation. In some embodiments, the modified IFN-β comprises a L32A mutation. In some embodiments, the modified IFN-β comprises a L32G mutation. In some embodiments, the modified IFN-β comprises a R35A mutation. In some embodiments, the modified IFN-β comprises a R35G mutation. In some embodiments, the modified IFN-β comprises a V148G mutation. In some embodiments, the modified IFN-β comprises a R152A mutation. In some embodiments, the modified IFN-β comprises a R152G mutation. In some embodiments, the modified IFN-β comprises a Y155G mutation. In some embodiments, the modified IFN-β comprises a W22G and R27G mutation. In some embodiments, the modified IFN-β comprises a L32A and R35A mutation. In some embodiments, the modified IFN-β comprises a L151G and R152A mutation. In some embodiments, the modified IFN-β comprises a V148G and R152A mutation.

[0092] 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 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.

[0093] In some embodiments, the modified IFN-β has one or more of the following mutations in combination with any of the other IFN-β mutations described herein: R35A, R35T, E42K, M62I, G78S, A141Y, A142T, E149K, and R152H.

[0094] The crystal structure of human IFN-β is known and described in Karpusas et al., (1998) PNAS, 94(22):11813-11818. Specifically, the structure of human IFN-β has been shown to include five α-helices (i.e., A, B, C, D, and E) and four loop regions (i.e., AB, BC, CD, and DE loops) connecting these helices. In various embodiments, the modified IFN-β has one or more mutations in the A helix, B helix, C helix, D helix, E helix, and / or AB, BC, CD, and DE loops that reduce binding affinity or activity at a therapeutic receptor, such as IFNAR. Exemplary mutations are described in WO 2000 / 023114 and U.S. Patent Application Publication No. 2015 / 0011732, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the modified IFN-β is a 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 a human IFN-β comprising alanine substitutions at amino acid positions 28-30, 32, and 33. In an exemplary embodiment, the modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 36, 37, 39, and 42. In an exemplary embodiment, the modified IFN-β is a 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 modified IFN-β is a human IFN-β comprising alanine substitutions at amino acid positions 71-73. In an exemplary embodiment, the modified IFN-β is a human IFN-β that comprises alanine substitutions at amino acid positions 92, 96, 99, and 100. In an exemplary embodiment, the modified IFN-β is a human IFN-β that comprises alanine substitutions at amino acid positions 128, 130, 131, and 134.In exemplary embodiments, the modified IFN-β is a human IFN-β that comprises alanine substitutions at amino acid positions 149, 153, 156, and 159. In some embodiments, the mutated IFN-β comprises SEQ ID NO:48 and comprises a mutation at W22, wherein the mutation is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0095] In some embodiments, the mutated IFNβ comprises SEQ ID NO: 48 and comprises a mutation at R27, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0096] In some embodiments, the mutated IFNβ comprises SEQ ID NO:48 and comprises a mutation at W22, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and a mutation at R27, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0097] In some embodiments, the mutated IFNβ comprises SEQ ID NO: 48 and comprises a mutation at L32, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0098] In some embodiments, the mutated IFNβ comprises SEQ ID NO: 48 and comprises a mutation at R35, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0099] In some embodiments, the mutated IFNβ comprises SEQ ID NO:48 and comprises a mutation at L32, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and a mutation at R35, which 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 mutated IFNβ comprises SEQ ID NO: 48 and comprises a mutation at F67, which 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 mutated IFNβ comprises SEQ ID NO: 48 and comprises a mutation at R71, which 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 comprises a mutation at F67, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and a mutation at R71, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0103] In some embodiments, the mutated IFNβ comprises SEQ ID NO: 48 and comprises a mutation at L88, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0104] In some embodiments, the mutated IFNβ comprises SEQ ID NO: 48 and comprises a mutation at Y92, which 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 mutated IFNβ comprises SEQ ID NO:48 and comprises a mutation at F67, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); a mutation at L88, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V); and a mutation at Y92, which 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 mutated IFNβ comprises SEQ ID NO:48 and comprises a mutation at L88, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and a mutation at Y92, which 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 mutated IFNβ comprises SEQ ID NO:48 and comprises a mutation at I95, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V), and a mutation at Y92, which 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 mutated IFNβ comprises SEQ ID NO:48 and comprises a mutation at N96, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and a mutation at Y92, which 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 mutated IFNβ comprises SEQ ID NO:48 and comprises a mutation at Y92, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V); a mutation at I95, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), methionine (M), and valine (V); and a mutation at N96, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0110] In some embodiments, the mutated IFNβ comprises SEQ ID NO: 48 and comprises a mutation at K123, which 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 mutated IFNβ comprises SEQ ID NO: 48 and comprises a mutation at R124, which 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 mutated IFNβ comprises SEQ ID NO:48 and comprises a mutation at K123, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and a mutation at R124, which 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 mutated IFNβ comprises SEQ ID NO: 48 and comprises a mutation at L151, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V).

[0114] In some embodiments, the mutated IFNβ comprises SEQ ID NO: 48 and comprises a mutation at R152, which 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 comprises a mutation at L151, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), isoleucine (I), methionine (M), and valine (V), and a mutation at R152, which 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 comprises a mutation at V148, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), and methionine (M).

[0117] In some embodiments, the mutated IFNβ comprises SEQ ID NO:48 and comprises a mutation at V148, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and a mutation at R152, which is an aliphatic hydrophobic residue selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V).

[0118] In some embodiments, the mutated IFNβ comprises SEQ ID NO: 48 and comprises a mutation at Y155, which 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 invention relates to a chimeric protein comprising (a) a modified IFN-β having an amino acid sequence of SEQ ID NO: 48 and a mutation at position W22, wherein the mutation is an aliphatic hydrophobic residue, and (b) one or more targeting moieties comprising a recognition domain that specifically binds to an antigen or receptor of interest (e.g., Clec9A), wherein the modified IFN-β and the one or more targeting moieties are optionally linked by one or more linkers. 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.

[0120] Additional exemplary IFNβ mutants are set forth in International Application No. PCT / EP2017 / 061544, the disclosure of which is incorporated herein by reference in its entirety.

[0121] In some embodiments, the modified signal transduction agent is interferon gamma. In said embodiments, the modified interferon gamma agent has reduced affinity and / or activity for the interferon-gamma receptor (IFNGR), i.e., the IFNGR1 and IFNGR2 chains. In some embodiments, the modified interferon gamma agent has substantially reduced or eliminated affinity and / or activity for the interferon-gamma receptor (IFNGR), i.e., the IFNGR1 and IFNGR2 chains.

[0122] In some embodiments, the modified signal transduction agent is a consensus interferon. The consensus interferon is generated by scanning the sequences of several human non-allelic IFN-α subtypes and selecting the most frequently observed amino acids at the corresponding positions. The consensus interferon differs from IFN-α2b in 20 of 166 amino acids (88% homology), and shows identity at more than 30% of the amino acid positions when compared to IFN-β. In various embodiments, the consensus interferon comprises the amino acid sequence of SEQ ID NO: 49 below.

[0123] 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 does not include the initial methionine residue of SEQ ID NO:49.

[0124] In various embodiments, the consensus interferon comprises a modified consensus interferon, i.e., a consensus interferon variant, as a signaling agent, which in various embodiments includes a functional derivative, analog, precursor, isoform, splice variant, or fragment of consensus interferon.

[0125] In some embodiments, the consensus interferon variant is selected from consensus interferon variants as disclosed in U.S. 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 comprise the amino acid sequence of IFN-CON2 or IFN-CON3 as disclosed in U.S. Patent Nos. 4,695,623, 4,897,471, and 5,541,293. In some embodiments, the consensus interferon variant comprises the amino acid sequence of IFN-CON2 (SEQ ID NO:51).

[0126] In one embodiment, the consensus interferon variant comprises the amino acid sequence of IFN-CON3 (SEQ ID NO:52).

[0127] In some embodiments, the consensus interferon variant comprises the amino acid sequence of any one of the variants disclosed in U.S. Patent No. 8,496,921. For example, the consensus variant may comprise the amino acid sequence of SEQ ID NO:53.

[0128] In another embodiment, the consensus interferon variant may comprise the amino acid sequence of SEQ ID NO:54.

[0129] In some embodiments, the consensus interferon variant may be PEGylated, i.e., comprises a PEG moiety. In some embodiments, the consensus interferon variant may comprise a PEG moiety attached to position S156C of SEQ ID NO:54.

[0130] In some embodiments, the modified interferon is a variant of human IFN-α2a having an insertion of Asp at position 41 in the sequence Glu-Glu-Phe-Gly-Asn-Gln (SEQ ID NO:275) to obtain the sequence Glu-Glu-Phe-Asp-Gly-Asn-Gln (SEQ ID NO:276) (thus renumbering the sequence relative to the IFN-α2a sequence) and the following mutations: Arg23Lys, Leu26Pro, Glu53Gln, Thr54Ala, Pro56Ser, Asp86Glu, Ile104Thr, Gly106Glu, Thr110Glu, Lys117Asn, Arg125Lys, and Lys136Thr. All embodiments herein describing consensus interferon apply to this modified interferon as well.

[0131] In various embodiments, the consensus interferon variant comprises an amino acid sequence having one or more amino acid mutations, which in some embodiments may be independently selected from a substitution, an insertion, a deletion, and a truncation.

[0132] In some embodiments, the amino acid mutations are amino acid substitutions, which can include conservative and / or non-conservative substitutions.

[0133] In various embodiments, substitutions may also include non-classical amino acids (e.g., generally, selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA, and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of 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, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids such as β-methylamino acids, C α-methylamino acids, N α-methylamino acids, and amino acid analogs).

[0134] In various embodiments, the consensus interferon is modified to have one or more mutations. In some embodiments, the mutations cause the consensus interferon variant to have one or more reduced activities, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity, compared to the unmutated, e.g., wild-type, consensus interferon (e.g., consensus interferon having the amino acid sequence of SEQ ID NO: 49 or 50). For example, the reduced activity, such as reduced binding affinity, reduced intrinsic activity, and reduced specific biological activity, compared to the unmutated, e.g., wild-type, consensus interferon, may occur at a therapeutic receptor, such as IFNAR. As a result, in various embodiments, the mutations cause the consensus interferon variant to have reduced systemic toxicity, reduced side effects, and reduced off-target effects, compared to the unmutated, e.g., wild-type, consensus interferon.

[0135] In various embodiments, the consensus interferon is modified to have a mutation that reduces binding affinity or activity at a therapeutic receptor, such as IFNAR. In some embodiments, the activity provided by the consensus interferon is agonism at a therapeutic receptor (e.g., activation of a cellular effect at a therapeutic site). For example, the consensus interferon may activate a therapeutic receptor. In such embodiments, the mutation results in the consensus interferon variant having reduced activation activity at the therapeutic receptor.

[0136] In some embodiments, the loss of affinity or activity at the therapeutic receptor can be restored by binding to a targeting moiety (e.g., SIRPα). In other embodiments, the loss of affinity or activity at the therapeutic receptor is not substantially restored by binding to a targeting moiety. In various embodiments, the therapeutic chimeric proteins of the invention have reduced off-target effects because the consensus interferon variant has a mutation that weakens its binding affinity or activity at the therapeutic receptor. In various embodiments, this reduces the side effects seen, for example, with wild-type consensus interferon. In various embodiments, the consensus interferon variant is substantially inactive on the way to the site of therapeutic activity and acts substantially against the specifically targeted cell type, greatly reducing unwanted side effects.

[0137] In various embodiments, the consensus interferon variants are characterized by their affinity, e.g., binding (e.g., K D ) and / or activation (e.g., K A and / or EC 50 In various embodiments, the therapeutic receptor has one or more mutations that attenuate or reduce activity and / or signaling from the therapeutic receptor due to reduced affinity at the therapeutic receptor.

[0138] In various embodiments, the consensus interferon variant has one or more mutations that reduce binding or affinity to the IFNAR1 subunit of IFNAR. In certain embodiments, 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 binding or affinity to the IFNAR2 subunit of IFNAR. In some embodiments, the consensus interferon variant has one or more mutations that reduce binding or affinity to both the IFNAR1 and IFNAR2 subunits.

[0139] In some embodiments, a consensus interferon variant has one or more mutations that reduce binding or affinity to IFNAR1 and one or more mutations that substantially reduce or eliminate binding or affinity to IFNAR2. In some embodiments, chimeric proteins with such consensus interferon variants can provide target-selective IFNAR1 activity (e.g., IFNAR1 activity can be restored by targeting via a targeting moiety, such as SIRPα).

[0140] In some embodiments, a consensus interferon variant has one or more mutations that reduce binding or affinity to IFNAR2 and one or more mutations that substantially reduce or eliminate binding or affinity to IFNAR1. In some embodiments, chimeric proteins comprising such consensus interferon variants may provide target-selective IFNAR2 activity (e.g., IFNAR2 activity is restoreable by targeting via a targeting moiety, such as SIRPα).

[0141] In some embodiments, a consensus interferon variant has one or more mutations that reduce binding or affinity to IFNAR1 and one or more mutations that reduce binding or affinity to IFNAR2. In some embodiments, chimeric proteins comprising such consensus interferon variants may provide target-selective IFNAR1 and / or IFNAR2 activity (e.g., IFNAR1 and / or IFNAR2 activity is restoreable by targeting via a targeting moiety, such as SIRPα).

[0142] In some embodiments, the consensus interferon is modified to have a mutation at one or more of amino acid positions 145-155 in SEQ ID NO:50, such as amino acid positions 149, 150, and / or 154. In some embodiments, the consensus interferon is modified to have a mutation at one or more of amino acid positions 145-155 in SEQ ID NO:50, such as amino acid positions 149, 150, and / or 154, where the substitutions are optionally hydrophobic and selected from alanine, valine, leucine, and isoleucine. In some embodiments, the consensus interferon variant comprises one or more mutations selected from M149A, R150A, and L154A in SEQ ID NO:50.

[0143] In one embodiment, the consensus interferon is modified to have a mutation at amino acid position 121 (i.e., K121) in SEQ ID NO: 50. In one embodiment, the consensus interferon comprises a K121E mutation in SEQ ID NO: 50.

[0144] In various embodiments, the modified signal transduction substances are selected from modified 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, N-methionyl human 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), hepatocyte growth factor, fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-α and tumor necrosis factor-β, Müllerian inhibitory substance, mouse gonadotropin-related peptide, inhibin, activin, vascular endothelial growth factor, integrins, thrombopoietin (TPO), nerve growth factors such as NGF-α, platelet-derived growth factor, and transforming augmentation factors such as TGF-α and TGF-β. interferons, such as interferon-α, interferon-β, and interferon-γ (as well as interferons types I, II, and III); colony stimulating factors (CSFs), such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF); interleukins (ILs), such as IL-1, IL-1α, 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; tumor necrosis factors, such as TNF-α or TNF-β, and other polypeptide factors, including, for example, LIF and kit ligand (KL). As used herein, cytokines, growth factors, and hormones include proteins of natural origin or produced from recombinant bacterial, eukaryotic, or mammalian cell culture systems, and biologically active equivalents of the native sequence cytokines.

[0145] In some embodiments, the modified signal transduction agent is a modified form of a growth factor selected from, but not limited to, 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 factors such as insulin-like growth factor-I and insulin-like growth factor-II, fibroblast growth factor (FGF), heregulin, platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF).

[0146] In some embodiments, the growth factor is a modified fibroblast growth factor (FGF). Examples of FGF 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, FGF21, FGF22, and FGF23.

[0147] In some embodiments, the modified signal transduction agent is vascular endothelial growth factor (VEGF). VEGF plays a major role in physiological as well as pathological angiogenesis, and is a potent growth factor that can control vascular permeability and act as a growth factor for VEGF receptor expressing cells. Among other functions, other functions include stimulating cell migration in macrophage lineage cells and endothelial cells. There are several members of the VEGF family of growth factors and at least three receptors (VEGFR-1, VEGFR-2, and VEGFR-3). Members of the VEGF family can bind to and activate more than one VEGFR. For example, VEGF-A binds to VEGFR-1 and VEGFR-2, while VEGF-C can bind to VEGFR-2 and VEGFR-3. Activation of VEGFR-1 and VEGFR-2 controls angiogenesis, while activation of VEGFR-3 is associated with lymphangiogenesis. The main pro-angiogenic signal comes from activation of VEGFR-2. It has been reported that activation of VEGFR-1 may be associated with a negative role in angiogenesis. Transduction of VEGFR-1 signaling is important for in vivo tumor growth via bone marrow-derived VEGFR-1 positive cells (which contribute to the formation of the pre-metastatic niche in bone). Several therapies based on VEGF-A directed / neutralizing therapeutic antibodies have been developed for use in the treatment of various human tumors that are primarily dependent on angiogenesis. However, these therapies are not without side effects. This may not be surprising considering that these antibodies act as general non-cell / tissue specific VEGF / VEGFR interaction inhibitors. It would therefore be preferable to restrict VEGF (e.g. VEGF-A) / VEGFR-2 inhibition to specific target cells (e.g. tumor vascular endothelial cells).

[0148] In some embodiments, the VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D, or VEGF-E, as well as VEGF 121 , VEGF 121 b, VEGF 145 , VEGF 165 , VEGF 165b, VEGF 189 , and VEGF 206 and isoforms thereof, including various isoforms of VEGF-A such as VEGFR-1 (Flt-1) and / or VEGFR-2 (KDR / Flk-1). 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 some embodiments, 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). The embodiments find use, for example, in wound healing methods (without wishing to be bound by theory, via the effects of VEGFR-2 on endothelial cell function and angiogenesis) or in treating ischemia-related diseases. In various embodiments, binding to VEGFR-1 (Flt-1) is avoided, which leads to cancer and pro-inflammatory activity. In various embodiments, VEGFR-1 (Flt-1) acts as a decoy receptor, thus substantially reducing or eliminating affinity at this receptor, and avoiding sequestration of the therapeutic agent. In some embodiments, 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 said 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.

[0149] Angiogenic therapies are also important in various diseases (e.g., ischemic heart disease, hemorrhage, etc.), and these include VEGF-based therapies. Activation of VEGFR-2 is proangiogenic (acting on endothelial cells). Activation of VEGFR-1 can lead to stimulation of migration of inflammatory cells (including macrophages), leading to inflammation-associated hyperpermeability of blood vessels. Activation of VEGFR-1 can also promote bone marrow-associated tumor niche formation. Therefore, in this case, a VEGF-based therapy that is selective for VEGFR-2 activation would be desirable. Furthermore, cell-specific targeting, e.g., to endothelial cells, would be desirable.

[0150] In some embodiments, the modified signal transduction agent has reduced affinity and / or activity for VEGFR-2 (e.g., antagonist activity) and / or substantially reduced or eliminated affinity and / or activity for VEGFR-1. When targeted to tumor vascular endothelial cells via a targeting moiety that binds to tumor endothelial cell markers (e.g., PSMA and others), such constructs inhibit VEGFR-2 activation, particularly on cells positive for such markers, while not activating VEGFR-1 on the way to and (in the absence of activity) on the target cells, thus eliminating induction of inflammatory responses, etc. This results in a more selective and safer antiangiogenic therapy for many types of tumors compared to VEGF-A neutralizing therapy.

[0151] In some embodiments, the modified signaling agent has reduced affinity and / or activity for VEGFR-2 (e.g., agonist activity) and / or substantially reduced or eliminated affinity and / or activity for VEGFR-1. Through targeting to the vascular endothelium, in some embodiments, such constructs promote angiogenesis without inducing a VEGFR-1-associated inflammatory response. Thus, such constructs will have a targeted pro-angiogenic effect with substantially reduced risk of side effects caused by systemic activation of VEGFR-2 as well as VEGR-1.

[0152] In an exemplary embodiment, the modified signal transduction agent is VEGF 165 and has the amino acid sequence of SEQ ID NO:55.

[0153] In another exemplary embodiment, the altered signal transduction agent is VEGF 165b and has the amino acid sequence of SEQ ID NO:56.

[0154] In these embodiments, it is believed that the modified signal transduction agent may have a mutation at amino acid I83 (e.g., a substitution mutation at position I83, e.g., I83K, I83R, which may result in reduced receptor binding affinity; see, e.g., U.S. Pat. No. 9,078,860, the entire contents of which are incorporated herein by reference).

[0155] In some embodiments, the altered signal transduction substance may be, but is not limited to, human chorionic gonadotropin, gonadotropin releasing hormone, androgen, 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, thyrotropin releasing hormone ... and modified forms of hormones selected from: insulin, parathyroid hormone, glucocorticoids, mineralocorticoids, adrenaline, noradrenaline, 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.

[0156] In some embodiments, the modified signal transduction agent is TNF-α. TNF is a pleiotropic cytokine with a wide variety of functions, including cell growth control, differentiation, apoptosis, tumorigenesis, viral replication, autoimmunity, immune cell function and trafficking, inflammation, and septic shock, and binds to two distinct membrane receptors on target cells, namely TNFR1 (p55) and TNFR2 (p75). While TNFR1 shows a very broad expression pattern, TNFR2 is preferentially expressed in certain populations of lymphocytes, Tregs, endothelial cells, certain neuronal cells, microglia, cardiomyocytes, and mesenchymal stem cells. Although there is some overlap, very different biological pathways are activated in response to receptor activation. In principle, without wishing to be bound by theory, TNFR1 signaling is associated with the induction of apoptosis (cell death), and TNFR2 signaling is associated with the activation of cell survival signals (e.g., activation of the NFkB pathway). Administration of TNF is systemically toxic, which is mainly due to the involvement of TNFR1. However, it should be noted that activation of TNFR2 is also associated with a wide range of activities and, as is the case for TNFR1, targeting TNF and controlling TNF activity is important with a view to developing TNF-based therapies.

[0157] In some embodiments, the modified signaling agent 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, whereas TNFR2 is involved in cell survival signaling. Thus, in embodiments relating to methods for 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 protein can be targeted to cells where apoptosis is desired, such as tumor cells or tumor vascular endothelial cells. In embodiments relating to methods for promoting cell survival in neurodegeneration, for example for treating neurodegenerative disorders, the modified signaling agent has reduced affinity and / or activity for TNFR2 and / or substantially reduced or eliminated affinity and / or activity for TNFR1. In other words, the chimeric proteins of the present disclosure, in some embodiments, comprise modified TNF-α agents that allow for a preference for either a death signal or a survival signal.

[0158] In some embodiments, the chimeric protein comprises a modified TNF with reduced affinity and / or activity for TNFR1 and / or substantially reduced or eliminated affinity and / or activity for TNFR2. Such chimeras are in some embodiments more potent inducers of apoptosis compared to wild-type TNF and / or chimeras with only mutations that result in reduced affinity and / or activity for TNFR1. Such chimeras are in some embodiments used to induce tumor cell death or tumor vascular endothelial cell death (e.g., in the treatment of cancer). In some embodiments, these chimeras are also used to inhibit TNF activation, e.g., via TNFR2. reg Preventing or inhibiting cellular activation further supports TNFR1-mediated anti-tumor activity in vivo.

[0159] In some embodiments, the chimeric protein has a modified TNF with reduced affinity and / or activity for TNFR2 and / or substantially reduced or eliminated affinity and / or activity for TNFR1. In some embodiments, such chimeras are more potent activators of cell survival of certain cell types, which may be of particular therapeutic interest in various disease settings, including, but not limited to, stimulating neurogenesis. Furthermore, such TNFR2-preferring chimeras are also useful for treating autoimmune diseases (e.g., Crohn's disease, diabetes, MS, colitis, etc., and many others described herein). In some embodiments, the chimeras target autoreactive T cells. In some embodiments, the chimeras target T reg Promotes cell activation and indirect suppression of cytotoxic T cells.

[0160] In some embodiments, the chimeras (e.g., modified TNF with reduced affinity and / or activity for TNFR2 and / or substantially reduced or eliminated affinity and / or activity for TNFR1) cause cell death of autoreactive T cells, e.g., by activation of TNFR2 and / or by evasion of TNFR1. Without wishing to be bound by theory, these autoreactive T cells alter their cellular apoptosis / cell survival signaling, e.g., by altered activity / signaling of the NFkB pathway. In some embodiments, the chimeras cause cell death of autoreactive T cells with impairment or alteration in the NFkB pathway, which underpins an imbalance, and optionally altered sensitivity, of the cell death (apoptosis) / survival signaling profile of the autoreactive T cells to certain cell death-inducing signals (e.g., activation of TNFR2).

[0161] In some embodiments, TNFR-2 based chimeras have other therapeutic uses, particularly in diseases including autoimmune diseases, various cardiovascular diseases, demyelinating neurodegenerative disorders, and infectious diseases.

[0162] In one embodiment, the wild-type TNF-α has the amino acid sequence of SEQ ID NO:57.

[0163] In such embodiments, the modified TNF-α agent has mutations at one or more of amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147 to produce modified TNF-α with reduced receptor binding affinity. See, e.g., U.S. Patent No. 7,993,636, the entire contents of which are incorporated herein by reference.

[0164] In some embodiments, the modified human TNF-α portion has mutations at one or more of the following amino acid positions: R32, N34, Q67, H73, L75, T77, S86, Y87, V91, I97, T105, P106, A109, P113, Y115, E127, N137, D143, A145, and E146 (numbering based on the human TNF sequence in Genbank Accession No. BAG70306, Update No. BAG70306.1, GI:197692685), e.g., as described in WO 2015 / 007903, the entire contents of which are incorporated herein by reference. In some embodiments, the modified human TNF-α portion has a substitution mutation selected from L29S, R32G, R32W, N34G, Q67G, H73G, L75G, L75A, L75S, T77A, S86G, S86T, Y87Q, Y87L, Y87A, Y87F, Y87H, V91G, V91A, I97A, I97Q, I97S, T105G, P106G, A109Y, P113G, Y115G, Y115A, E127G, N137G, D143N, A145G, A145R, A145T, E146D, E146K, and S147D. In some embodiments, the human TNF-α portion has a mutation selected from Y87Q, Y87L, Y87A, Y87F, and Y87H. In other embodiments, the human TNF-α portion has a mutation selected from I97A, I97Q, and I97S. In other embodiments, the human TNF-α portion has a mutation selected from Y115A and Y115G. In some embodiments, the human TNF-α portion has an E146K mutation. In some embodiments, the human TNF-α portion has a Y87H mutation and an E146K mutation. In some embodiments, the human TNF-α portion has a Y87H mutation and an A145R mutation. In some embodiments, the human TNF-α portion has an R32W mutation and an S86T mutation. In some embodiments, the human TNF-α portion has an R32W mutation and an E146K mutation. In some embodiments, the human TNF-α portion has an L29S mutation and an R32W mutation. In some embodiments, the human TNF-α portion has a D143N mutation and an A145R mutation.In some embodiments, the human TNF-α portion has a D143N mutation and an A145R mutation.In some embodiments, the human TNF-α portion has an A145T mutation, an E146D mutation, and an S147D mutation. In some embodiments, the human TNF-α portion has an A145T mutation and an S147D mutation.

[0165] In some embodiments, the modified TNF-α substance has one or more mutations selected from N39Y, S147Y, and Y87H, as described in WO 2008 / 124086, the entire contents of which are incorporated herein by reference.

[0166] In some embodiments, the modified human TNF-α portion has mutations that result in receptor selectivity as described in International Application No. PCT / IB2016 / 001668, the entire contents of which are incorporated herein by reference. In some embodiments, the mutations to TNF are TNF-R1 selective. In some embodiments, the mutations to TNF that are TNF-R1 selective are at one or more of positions R32, S86, and E146. In some embodiments, the mutations to TNF that are TNF-R1 selective are at one or more of positions R32W, S86T, and E146K. In some embodiments, the mutations to TNF that are TNF-R1 selective are at one or more of positions R32W, R32W / S86T, R32W / E146K, and E146K. In some embodiments, the mutations to TNF are TNF-R2 selective. In some embodiments, the mutations to TNF that are TNF-R2 selective are at one or more of positions A145, E146, and S147. In some embodiments, the mutations to TNF that are TNF-R2 selective are one or more of A145T, A145R, E146D, and S147D. In some embodiments, the mutations to TNF that are TNF-R2 selective are one or more of A145R, A145T / S147D, and A145T / E146D / S147D.

[0167] In certain embodiments, the altered signal transduction agent is TNF-β. TNF-β can form homodimers or heterodimers with LT-β (LT-α1β2). In some embodiments, the altered signal transduction agent has substantially reduced or eliminated affinity and / or activity for TNFR1 and / or TNFR2 and / or herpes virus entry mediator (HEVM) and / or LT-βR.

[0168] In one embodiment, the wild-type TNF-β has the amino acid sequence of SEQ ID NO:58.

[0169] In said embodiments, the modified TNF-β agent may contain a mutation at one or more amino acids at positions 106-113 that produces a modified TNF-β with reduced receptor binding affinity for TNFR2. In certain embodiments, the modified signaling agent has one or more substitution mutations at amino acid positions 106-113. In exemplary embodiments, the 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.

[0170] In some embodiments, the modified agent is a TNF family member (e.g., TNF-α, TNF-β), which may be in a single-chain trimer form as described in International Publication No. WO 2015 / 007903 and International Application No. PCT / IB2016 / 001668, the entire contents of which are incorporated herein by reference.

[0171] In some embodiments, the modified agent is a TNF family member (e.g., TNF-α, TNF-β) that has reduced affinity and / or activity, i.e., antagonist activity, at TNFR1 (e.g., naturally occurring antagonist activity or antagonist activity that is the result of one or more mutations, see, e.g., WO 2015 / 007520, the contents of which are incorporated herein by reference in their entirety). In these embodiments, the modified agent is a TNF family member (e.g., TNF-α, TNF-β) that has substantially reduced or optionally eliminated affinity and / or activity for TNFR2. In some embodiments, the modified agent is a TNF family member (e.g., TNF-α, TNF-β) with reduced affinity and / or activity, i.e., antagonist activity, at TNFR2 (e.g., naturally occurring antagonist activity or antagonist activity that is the result of one or more mutations, see, e.g., WO 2015 / 007520, the contents of which are incorporated herein by reference in their entirety). In these embodiments, the modified agent is a TNF family member (e.g., TNF-α, TNF-β) that may have substantially reduced or eliminated affinity and / or activity at TNFR1. The constructs of the above embodiments are used, for example, in methods of attenuating TNF responses in a cell-specific manner. In some embodiments, the antagonistic TNF family member (e.g., TNF-α, TNF-β) is a single-chain trimer as described in WO 2015 / 007903.

[0172] In certain embodiments, the modified signal transduction agent 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 substantially reduced or eliminated affinity and / or activity for DR4 (TRAIL-RI), and / or DR5 (TRAIL-RII), and / or DcR1, and / or DcR2.

[0173] In one embodiment, wild-type TRAIL has the amino acid sequence of SEQ ID NO:59.

[0174] In said embodiment, the modified TRAIL agent may have mutations at amino acid positions T127 to R132, E144 to R149, E155 to H161, Y189 to Y209, T214 to 1220, K224 to A226, W231, E236 to L239, E249 to K251, T261 to H264, and H270 to E271 (numbering based on the human sequence of Genbank Accession No. NP_003801, Update No. 10NP_003801.1, GI:4507593; see above).

[0175] 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 may specifically bind to TRAIL-R2. Exemplary mutations include mutations at one or more of the following amino acid positions: Y189, R191, Q193, H264, I266, and D267. For example, the mutations may be one or more of Y189Q, R191K, Q193R, H264R, I266L, and D267Q. In certain embodiments, the modified TRAIL agent includes the following mutations: Y189Q, R191K, Q193R, H264R, I266L, and D267Q.

[0176] 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 may specifically bind to TRAIL-R1. Exemplary mutations include mutations at one or more of the following amino acid positions: G131, R149, S159, N199, K201, and S215. For example, the mutations may be one or more of G131R, R149I, S159R, N199R, K201H, and S215D. In certain embodiments, the modified TRAIL agent includes the following mutations: G131R, R149I, S159R, N199R, K201H, and S215D. Other TRAIL mutations are described, for example, in Trebing, et al. (2014) Cell Death and Disease, 5:e1035, the disclosure of which is incorporated herein by reference in its entirety.

[0177] In certain embodiments, the modified signal transduction 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).

[0178] In some embodiments, the modified signaling agent is TGFβ. In said 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 may have reduced, substantially reduced or eliminated affinity and / or activity for TGFBR3, which, without wishing to be bound by theory, may act as a reservoir of ligands for the TGF-β receptor. In some embodiments, TGFβ may prefer TGFBR1 over TGFBR2, and may prefer TGFBR2 over TGFBR1. Similarly, without wishing to be bound by theory, LAP may act as a reservoir of ligands for the TGF-β receptor. In some embodiments, the altered signal transduction agent has reduced affinity and / or activity for TGFBR1 and / or TGFBR2 and / or has substantially reduced or eliminated affinity and / or activity for latency associated peptide (LAP). In some embodiments, such chimeras are used in Kamurati-Engelman disease or other disorders associated with inappropriate TGFβ signaling.

[0179] In some embodiments, the modified agent is a TGF family member (e.g., TGFα, TGFβ) that has reduced affinity and / or activity, i.e., antagonist activity (e.g., naturally occurring antagonist activity or antagonist activity that is the result of one or more mutations, see, e.g., WO 2015 / 007520, the contents of which are incorporated herein by reference in their entirety) at one or more of TGFBR1, TGFBR2, TGFBR3. In these embodiments, the modified agent is a TGF family member (e.g., TGFα, TGFβ) that optionally has substantially reduced or eliminated affinity and / or activity at one or more of TGFBR1, TGFBR2, TGFBR3.

[0180] In some embodiments, the modified agent is a TGF family member (e.g., TGFα, TGFβ) that has reduced affinity and / or activity, i.e., antagonist activity, at TGFBR1 and / or TGFBR2 (e.g., naturally occurring antagonist activity or antagonist activity that is the result of one or more mutations, see, e.g., WO 2015 / 007520, the contents of which are incorporated herein by reference in their entirety). In these embodiments, the modified agent is a TGF family member (e.g., TGFα, TGFβ) that optionally has substantially reduced or eliminated affinity and / or activity at TGFBR3.

[0181] In some embodiments, the modified signaling agent is an interleukin. In some embodiments, the modified signaling agent is IL-1. In some embodiments, the modified signaling agent is IL-1α or IL-1β. 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 modified IL-1 agents of the present disclosure avoid interactions at IL-1R2, thereby substantially reducing their function as a decoy and / or sink for therapeutic agents.

[0182] In one embodiment, the wild-type IL-1β has the amino acid sequence of SEQ ID NO:60.

[0183] IL1 is a pro-inflammatory cytokine and an important regulator of the immune system. IL1 is a potent activator of CD4 T cell responses, increasing the proportion of Th17 cells and enhancing the proliferation of IFNγ- and IL-4-producing cells. IL-1 inhibits CD8 + It is also a potent regulator of T cells and antigen-specific CD8 + It enhances T cell proliferation, differentiation, peripheral migration, and memory. IL-1 receptors include IL-1R1 and IL-1R2. Binding to and signaling from IL-1R1 constitutes the mechanism by which IL-1 mediates many of its biological (and pathological) activities. IL1-R2 can function as a decoy receptor, thereby reducing the availability of IL-1 for interaction with and signaling from IL-1R1.

[0184] In some embodiments, the modified IL-1 has reduced affinity and / or activity for IL-1R1 (e.g., agonist activity). In some embodiments, the modified IL-1 has substantially reduced or eliminated affinity and / or activity for IL-1R2. In said embodiments, there is restorable IL-1 / IL-1R1 signaling and prevention of loss of the therapeutic chimera at IL-R2, and a resulting reduction in the required IL-1 dose (e.g., compared to wild-type or chimeras having only attenuating mutations for IL-R1). Such constructs are used, for example, in methods of treating cancer, including stimulating the immune system to mount an anti-cancer response.

[0185] In some embodiments, the modified IL-1 has reduced affinity and / or activity for IL-1R1 (e.g., antagonist activity, e.g., naturally occurring antagonist activity or antagonist 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). In some embodiments, the modified IL-1 has substantially reduced or eliminated affinity and / or activity for IL-1R2. In said embodiments, there is no recovery of IL-1 / IL-1R1 signaling and no loss of the therapeutic chimera at IL-R2, resulting in a reduction in the required IL-1 dose (e.g., compared to wild type or chimeras having only attenuating mutations for IL-R1). Such constructs are used, for example, in methods of treating autoimmune diseases, including suppressing the immune system.

[0186] In said embodiment, the modified signal transduction agent has a deletion of amino acids 52-54 that produces a modified human IL-1β with reduced binding affinity to type I IL-1R and reduced biological activity, see, e.g., WO 1994 / 000491, the entire contents of which are incorporated herein by reference. In some embodiments, the modified human IL-1β is selected from the group consisting of A117G / P118G, R120X, L122A, T125G / L126G, R127G, Q130X, Q131G, K132A, S137G / Q138Y, L145G, H146X, L145A / L147A, Q148X, Q148G / Q150G, Q150G / D151A, M152G, F16G, F17G, F20G, F21G, F30G, F40G, F41G, F50G, F60G, F70G, F80G, F90G, F100G, F110G, F120G, F130G, F210G, F310G, F410G, F50G, F60G, F70G, F80G, F90G, F100G, F110G, F120G, F130G, F140G, F150G, F150G, F16 ... 2A, F162A / Q164E, F166A, Q164E / E167K, N169G / D170G, I172A, V174A, K208E, K209X, K209A / K210A, K219X, E221X, E221S / N224A, N224S / K225S, E244K, N245Q (wherein X can be any amino acid change, e.g. a non-conservative change) (numbering based on the human IL-1β sequence of Genbank Accession No. NP_000567, Update No. 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 some embodiments, the modified human IL-1β comprises Q131G and Q148G mutations. In some embodiments, the modified human IL-1β comprises Q148G and K208E mutations. In some embodiments, the modified human IL-1β comprises R120G and Q131G mutations. In some embodiments, the modified human IL-1β comprises R120G and H146A mutations. In one embodiment, the modified human IL-1β comprises the mutations R120G and H146N.In some embodiments, the modified human IL-1β comprises R120G and H146R mutations. In some embodiments, the modified human IL-1β comprises R120G and H146E mutations. In some embodiments, the modified human IL-1β comprises R120G and H146G mutations. In some embodiments, the modified human IL-1β comprises R120G and K208E mutations. In some embodiments, the modified human IL-1β comprises R120G, F162A, and Q164E mutations.

[0187] In certain embodiments, the modified signaling agent is IL-2. In said embodiments, 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. The embodiments may be suitable for the treatment of cancer, for example, where the modified IL-2 is agonistic at IL-2Rβ and / or IL-2Rγ. For example, constructs of the present disclosure may be used to target CD8 cells bearing IL2 receptors β and γ (which may provide anti-tumor activity). + Advantageous for attenuating the activity of T cells and containing IL2 receptors α, β, and γ (which may provide an immunosuppressive tumor-promoting effect) reg Furthermore, in some embodiments, the preference for IL-2Rβ and / or IL-2Rγ over IL-2Rα avoids IL-2 side effects such as pulmonary edema. IL-2-based chimeras are also useful for treating diseases (e.g., autoimmune diseases) where the modified IL-2 is antagonistic at IL-2Rβ and / or IL-2Rγ (e.g., naturally occurring antagonist activity or antagonist 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 example, constructs of the present disclosure can be used to treat CD8 +It is favorable for attenuating the activity of T cells (thus attenuating the immune response) and is favorable for T cells that have IL2 receptors α, β, and γ. reg Alternatively, in some embodiments, the chimera with IL-2 is reg Activation of CD8 + For example, these constructs are used to treat diseases or conditions that may benefit from immunosuppression, such as autoimmune diseases.

[0188] In some embodiments, the chimeric protein is CD8 + In some embodiments, these constructs include a targeting moiety as described herein that is directed to T cells, and a modified IL-2 agent that has reduced affinity and / or activity for IL-2Rβ and / or IL-2Rγ, and / or has substantially reduced or eliminated affinity and / or activity for IL-2Rα. + Provides T cell activity and reg IL-2 is generally inactive (or has substantially reduced activity) against T cells. In some embodiments, such constructs have enhanced immunostimulatory effects compared to wild-type IL-2 (e.g., by not stimulating Tregs, without wishing to be bound by theory), while eliminating or reducing systemic toxicity associated with IL-2.

[0189] In one embodiment, the wild-type IL-2 has the amino acid sequence of SEQ ID NO:61.

[0190] In said embodiment, the modified IL-2 agents comprise one or more mutations at amino acid L72 (L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, or L72K), amino acid F42 (F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, or F42K), and amino acid Y45 (Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, or Y45K). Without wishing to be bound by theory, it is believed that compared to wild-type IL-2, these modified IL-2 agents have reduced affinity for the high affinity IL-2 receptor and maintain affinity for the intermediate affinity IL-2 receptor. See, for example, U.S. Patent Application Publication No. 2012 / 0244112, the entire contents of which are incorporated herein by reference.

[0191] 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 at C125. For example, the mutation may be C125S. In such embodiments, the modified IL-2 agent may have a substantially reduced affinity and / or activity for IL-2Rα, e.g., as described in Carmenate, et al., (2013) The Journal of Immunology, 190:6230-6238, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the modified IL-2 agent having mutations at R38, F42, Y45, and / or E62 is capable of inducing proliferation of effector cells, including CD8+ T cells and NK cells, but is unable to induce proliferation of Treg cells. In some embodiments, modified IL-2 agents having mutations at R38, F42, Y45, and / or E62 are less toxic than wild-type IL-2 agents. Chimeric proteins comprising modified IL-2 agents with substantially reduced affinity and / or activity for IL-2Rα may find applications in oncology, etc.

[0192] In other embodiments, the modified IL-2 agent may have substantially reduced affinity and / or activity for IL-2Rβ, e.g., as described in WO 2016 / 025385, the disclosure of which is incorporated herein by reference in its entirety. In said embodiments, the modified IL-2 agent is capable of inducing proliferation of Treg cells, but is unable to induce proliferation of effector cells, such as CD8+ T cells and NK cells. Chimeric proteins comprising modified IL-2 agents having substantially reduced affinity and / or activity for IL-2Rβ may be applied in the treatment of autoimmune diseases, etc. In some embodiments, the modified IL-2 agent may include one or more mutations at amino acids N88, D20, and / or A126. For example, the modified IL-2 agent may include one or more of N88R, N88I, N88G, D20H, Q126L, and Q126F.

[0193] In various embodiments, the modified IL-2 agent may include a mutation at D109 or C125. For example, the mutation may be D109C or C125S. In some embodiments, a modified IL-2 having a mutation at D109 or C125 may be utilized for conjugation to a PEG moiety.

[0194] In certain embodiments, the altered signaling agent is IL-3. In some embodiments, the altered signaling agent has reduced affinity and / or activity for the IL-3 receptor, a heterodimer that contains a unique alpha chain paired with a common beta (beta or CD131) subunit. In some embodiments, the altered signaling agent has substantially reduced or eliminated affinity and / or activity for the IL-3 receptor, a heterodimer that contains a unique alpha chain paired with a common beta (beta or CD131) subunit.

[0195] In certain embodiments, the altered signaling agent is IL-4. In said embodiments, the altered signaling agent has reduced affinity and / or activity for type 1 and / or type 2 IL-4 receptors. In said embodiments, the altered signaling agent has substantially reduced or eliminated affinity and / or activity for type 1 and / or type 2 IL-4 receptors. Type 1 IL-4 receptor is composed of an IL-4Rα subunit and a common γ chain, which specifically binds IL-4. Type 2 IL-4 receptor comprises an IL-4Rα subunit bound to a different subunit known as IL-13Rα1. In some embodiments, the altered signaling agent has substantially reduced or eliminated affinity and / or activity for type 2 IL-4 receptors.

[0196] In one embodiment, the wild-type IL-4 has the amino acid sequence of SEQ ID NO:62.

[0197] In said embodiment, the modified IL-4 agents have one or more mutations at amino acid R121 (R121A, R121D, R121E, R121F, R121H, R121I, R121K, R121N, R121P, R121T, R121W), amino acid E122 (E122F), Y124 (Y124A, Y124Q, Y124R, Y124S, Y124T), and amino acid S125 (S125A). Without wishing to be bound by theory, it is believed that these modified IL-4 agents maintain type I receptor mediated activity but significantly reduce other receptor mediated biological activities. See, e.g., U.S. Patent No. 6,433,157, the entire contents of which are incorporated herein by reference.

[0198] In some embodiments, the modified signaling agent is IL-6. IL-6 signals through a cell surface type I cytokine receptor complex that includes the ligand-binding IL-6R chain (CD126) and the signaling element gp130. IL-6 may also bind to the extracellular portion of IL-6R, soluble IL-6R (sIL-6R). The sIL-6R / IL-6 complex may be involved in neurite outgrowth and neuronal survival and thus may be important for neuronal regeneration via remyelination. Thus, 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.

[0199] In one embodiment, the wild-type IL-6 has the amino acid sequence of SEQ ID NO:63.

[0200] In such embodiments, the modified signal transduction agent has one or more mutations at amino acid positions 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 to IL-6Rα and reduced biological activity. See, e.g., WO 97 / 10338, the entire contents of which are incorporated herein by reference.

[0201] In one embodiment, the altered signaling agent is IL-10. In said embodiment, the altered signaling agent has reduced affinity and / or activity for IL-10 receptor-1 and IL-10 receptor-2. In some embodiments, the altered signaling agent has substantially reduced or eliminated affinity and / or activity for IL-10 receptor-1 and IL-10 receptor-2.

[0202] In one embodiment, the altered signaling agent is IL-11. In said embodiment, the altered signaling agent has reduced affinity and / or activity for IL-11Rα and / or IL-11Rβ and / or gp130. In said embodiment, the altered signaling agent has substantially reduced or eliminated affinity and / or activity for IL-11Rα and / or IL-11Rβ and / or gp130.

[0203] In one embodiment, the altered signaling agent is IL-12. In said embodiment, the altered signaling agent has reduced affinity and / or activity for IL-12Rβ1 and / or IL-12Rβ2. In said embodiment, the altered signaling agent has substantially reduced or eliminated affinity and / or activity for IL-12Rβ1 and / or IL-12Rβ2.

[0204] In one embodiment, the altered signaling agent is IL-13. In said embodiment, the altered signaling agent has reduced affinity and / or activity for IL-4 receptor (IL-4Rα) and IL-13Rα1. In some embodiments, the altered signaling agent has substantially reduced or eliminated affinity and / or activity for IL-4 receptor (IL-4Rα) or IL-13Rα1.

[0205] In one embodiment, the wild-type IL-13 has the amino acid sequence of SEQ ID NO:64.

[0206] In such embodiments, the modified IL-13 agents have 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-13 agents have reduced biological activity. See, e.g., WO 2002 / 018422, the contents of which are incorporated herein by reference in their entirety.

[0207] In certain embodiments, 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-18Rβ. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for type II IL-18Rα, an isoform of IL-18Rα that lacks the TIR domain required for signal transduction.

[0208] In one embodiment, wild-type IL-18 has the amino acid sequence of SEQ ID NO:65.

[0209] In said embodiments, the modified IL-18 agent may comprise one or more mutations at amino acids or amino acid regions selected from Y37 to K44, R49 to Q54, D59 to R63, E67 to C74, R80, M87 to A97, N127 to K129, Q139 to M149, K165 to K171, R183, and Q190 to N191 (numbering based on the human IL-18 sequence of Genbank Accession No. AAV38697, Update No. AAV38697.1, GI:54696650), as described in WO 2015 / 007542, the contents of which are incorporated herein by reference in their entirety.

[0210] In one embodiment, the altered signaling agent is IL-33. In said embodiment, the altered signaling agent has reduced affinity and / or activity for the ST-2 receptor and IL-1RAcP. In some embodiments, the altered signaling agent has substantially reduced or eliminated affinity and / or activity for the ST-2 receptor and IL-1RAcP.

[0211] In one embodiment, the wild-type IL-33 has the amino acid sequence of SEQ ID NO:66.

[0212] In said embodiments, the modified IL-33 agent may include one or more mutations at amino acids or amino acid regions selected from I113 to Y122, S127 to E139, E144 to D157, Y163 to M183, E200, Q215, L220 to C227, and T260 to E269, as described in WO 2015 / 007542, the contents of which are incorporated herein by reference in their entirety (numbering based on the human sequence of Genbank Accession No. NP_254274, Update No. NP_254274.1, GI:15559209).

[0213] In one embodiment, the modified signal transduction agent is epidermal growth factor (EGF). EGF is a member of a family of potent growth factors. Members include EGF, HB-EGF, and others, such as TGFα, amphiregulin, neuregulin, epiregulin, and betacellulin. EGF family receptors include EGFR (ErbB1), ErbB2, ErbB3, and ErbB4. These receptors may function as homodimeric and / or heterodimeric receptor subtypes. Different EGF family members show different selectivity for various receptor subtypes. For example, EGF binds to ErbB1 / ErbB1, ErbB1 / ErbB2, ErbB4 / ErbB2, and several other heterodimeric subtypes. HB-EGF has a similar pattern, but also binds to ErbB4 / 4. Modulation of EGF (EGF-like) growth factor signaling is of high therapeutic interest, both positive and negative. For example, inhibition of EGFR signaling is of interest in the treatment of various cancers in which EGFR signaling constitutes a major growth-promoting signal, whereas stimulation of EGFR signaling is of therapeutic interest, for example, in promoting wound healing (acute and chronic) and in oral mucositis (a major side effect of various cancer therapies, including but not limited to radiation therapy).

[0214] In some embodiments, the modified signaling agent has reduced affinity and / or activity for ErbB1, ErbB2, ErbB3, and / or ErbB4. The embodiments are used, for example, in methods for treating wounds. In some embodiments, the modified signaling agent binds to one or more of ErbB1, ErbB2, ErbB3, and ErbB4 and antagonizes the activity of the receptor. In the embodiments, the modified signaling agent has reduced affinity and / or activity for ErbB1, ErbB2, ErbB3, and / or ErbB4, thereby antagonizing the activity of the receptor such that it is attenuated. The embodiments are used, for example, in the treatment of cancer. In some embodiments, the modified signaling agent has reduced affinity and / or activity for ErbB1. ErbB1 is the therapeutic target of kinase inhibitors (e.g., gefitinib, erlotinib, afatinib, brigatinib, and icotinib), most of which have side effects due to their limited selectivity. In some embodiments, the attenuation of ErbB1 signaling by the antagonist is more targeted and has fewer side effects than other agents that target the EGF receptor.

[0215] In some embodiments, the altered signal transduction agent has reduced affinity and / or activity for ErbB1 (e.g., antagonist activity, e.g., naturally occurring antagonist activity or antagonist 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), and / or has substantially reduced or eliminated affinity and / or activity for ErbB4 or other subtypes with which it may interact. Specific targeting via the targeting moiety will achieve cell-selective inhibition (antagonism, e.g., naturally occurring antagonist activity or antagonist 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), of ErbB1 / ErbB1 receptor activation, but not other receptor subtypes that may be associated with inhibition-related side effects. Thus, in contrast to EGFR kinase inhibitors, which inhibit EGFR activity in all cell types in the body, such constructs would provide selective anti-EGFR (ErbB1) drug action in cells (e.g., tumor cells in which EGFR signaling is activated due to receptor amplification, overexpression, etc.) with reduced side effects.

[0216] In some embodiments, the altered signaling agent has reduced affinity and / or activity (e.g., agonist activity) for ErbB4 and / or other subtypes with which it may interact. Selective activation of ErbB1 signaling (e.g., epithelial cells) is achieved by targeting to specific target cells via a targeting moiety. In some embodiments, such constructs are used in the treatment of wounds with reduced side effects (promoting wound healing), particularly in the treatment of chronic conditions, and in applications other than local application of therapeutic agents (e.g., systemic wound healing).

[0217] In certain embodiments, the modified signal transduction agent is insulin or an insulin analog. 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 substantially reduced or eliminated affinity and / or activity for the insulin receptor and / or the IGF1 or IGF2 receptor. Attenuated response at the insulin receptor allows for management of diabetes, obesity, metabolic disorders, etc., while away from the IGF1 or IGF2 receptor avoids cancer-promoting effects.

[0218] In some embodiments, the modified signaling agent is insulin-like growth factor-I or insulin-like growth factor-II (IGF-1 or IGF-2). In some embodiments, the modified signaling agent is IGF-1. In said embodiments, the modified signaling agent has reduced affinity and / or activity for insulin receptor and / or IGF1 receptor. In some embodiments, the modified signaling agent can bind to IGF1 receptor and antagonize the activity of that receptor. In said embodiments, the modified signaling agent has reduced affinity and / or activity for IGF1 receptor, thereby antagonizing the activity of that receptor attenuated. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for insulin receptor and / or IGF1 receptor. In some embodiments, the modified signaling agent has reduced affinity and / or activity for IGF2 receptor, thereby antagonizing the activity of that receptor attenuated. In some embodiments, the modified signaling agent has substantially reduced or eliminated affinity and / or activity for insulin receptor, thereby antagonizing the activity of that receptor. In various embodiments, this applies to the treatment of cancer. In various embodiments, the agents of the present disclosure prevent IR isoform A from becoming resistant to cancer treatment.

[0219] In some embodiments, the modified signaling agent is EPO. In various embodiments, the modified EPO agonist has reduced affinity and / or activity for the EPO receptor (EPOR) receptor and / or ephrin receptor (EphR) compared to wild-type EPO or other EPO-based agonists described herein. In some embodiments, the modified EPO agonist has substantially reduced or eliminated affinity and / or activity for the EPO receptor (EPOR) receptor and / or Eph receptor (EphR). Examples of EPO receptors include, but are not limited to, the EPOR homodimer or the EPOR / CD131 heterodimer. The beta common receptor (βcR) is also included as an EPO receptor. Examples of 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 one or more EPO receptors or receptors, including Eph receptors (e.g., heterodimers, heterotrimers, including, but not limited to, EPOR-EPHB4, EPOR-βcR-EPOR). Receptors are also provided in EP2492355, the entire contents of which are incorporated herein by reference, including, but not limited to, NEPOR.

[0220] In some embodiments, the human EPO has the amino acid sequence of SEQ ID NO:67 (the first 27 amino acids are the signal peptide).

[0221] In some embodiments, the human EPO protein is a mature form of EPO (with the signal peptide truncated), a 166 amino acid residue glycoprotein having the sequence of SEQ ID NO:68.

[0222] The structure of the human EPO protein is predicted to comprise a four-helix bundle including helix A, helix B, helix C, and helix D. In various embodiments, the modified EPO protein comprises one or more mutations located within four regions of the EPO protein that are important for biological activity, namely, amino acid residues 10-20, 44-51, 96-108, and 142-156. In some embodiments, the one or more mutations are located at residues 11-15, 44-51, 100-108, and 147-151. These residues are located in helix A (Val11, Arg14, and Tyr15), helix C (Ser100, Arg103, Ser104, and Leu108), helix D (Asn147, Arg150, Gly151, and Leu155), and the A / B connecting loop (mp residues 42-51). In some embodiments, the modified EPO protein contains mutations at residues between amino acids 41 and 52, and at amino acids 147, 150, 151, and 155. Without wishing to be bound by theory, mutations at these residues are believed to have a significant effect on both receptor binding and in vitro biological activity. In some embodiments, the modified EPO protein contains mutations at residues 11, 14, 15, 100, 103, 104, and 108. Without wishing to be bound by theory, it is believed that mutations of these residues have a moderate effect on receptor binding activity and a greater effect on in vitro biological activity. Exemplary substitutions include, but are not limited to, one or more of Val11Ser, Arg14Ala, Arg14Gln, Tyr15Ile, Pro42Asn, Thr44Ile, Lys45Asp, Val46Ala, Tyr51Phe, Ser100Glu, Ser100Thr, Arg103Ala, Ser104Ile, Ser104Ala, Leu108Lys, Asn147Lys, Arg150Ala, Gly151Ala, and Leu155Ala.

[0223] In some embodiments, the modified EPO protein contains mutations that confer biological activity but not binding, such as those described in Eliot, et al., Mapping of the Active Site of Recombinant Human Erythropoietin, Jan. 15, 1997, Blood, 89(2), the entire contents of which are incorporated herein by reference.

[0224] In some embodiments, the modified EPO protein comprises one or more mutations that involve surface residues of the EPO protein that are involved in contact with the receptor. Without wishing to be bound by theory, it is believed that mutations of these surface residues are unlikely to affect protein folding, and therefore some biological activity may be retained. Examples of surface residues that can be mutated include, but are not limited to, residues 147 and 150. In an exemplary embodiment, the mutation is a substitution, and includes one or more of N147A, N147K, R150A, and R150E.

[0225] In some embodiments, the modified EPO protein contains one or more mutations at residues N59, E62, L67, and L70, and one or more mutations that affect 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 buried positions, thus indirectly affecting biological activity.

[0226] In one embodiment, the modified EPO protein contains a K20E substitution, which significantly reduces binding to the receptor. See Elliott, et al., (1997) Blood, 89:493-502, the entire contents of which are incorporated herein by reference.

[0227] Other EPO mutations that can be incorporated into the chimeric EPO proteins of the present invention are disclosed, for example, in Elliott, et al., (1997) Blood, 89:493-502, the entire contents of which are incorporated herein by reference, and Taylor, et al., (2010) PEDS, 23(4):251-260, the entire contents of which are incorporated herein by reference.

[0228] In an embodiment, a chimeric protein of the present disclosure has (i) a targeting moiety to SIRP1α and (ii) a targeting moiety that targets tumor cells with any of the modified or mutant signaling agents described herein. In an embodiment, a chimeric protein of the present disclosure has a targeting moiety that targets SIRP1α on macrophages and a second targeting moiety that targets PD-L1 or PD-L2 on tumor cells.

[0229] In various embodiments, the signaling agent is a toxin or toxic enzyme. In some embodiments, the toxin or toxic enzyme is derived from plants and bacteria. Examples of toxins or toxic enzymes include, but are not limited to, diphtheria toxin, Pseudomonas aeruginosa toxin, anthrax toxin, ribosome-inactivating proteins (RIPs) such as ricin and saponin, modeccin, abrin, gelonin, and pokeweed antiviral protein. Other toxins include those disclosed in Mathew, et al., (2009) Cancer Sci, 100(8):1359-65, the disclosure of which is incorporated herein by reference in its entirety. In said embodiments, the chimeric proteins of the invention can be utilized to induce cell death in a cell type specific manner. In said embodiments, the toxin can be modified, e.g., mutated, to reduce the affinity and / or activity of the toxin to attenuate the effect, as described for other signaling agents herein.

[0230] Multispecific Chimeras and Fusions with Signal Transduction Agents In various embodiments, the chimeric proteins of the invention comprise one or more signal transduction agents described herein and / or one or more additional targeting moieties (i.e., in addition to a targeting moiety for SIRP1α).Thus, the invention provides chimeric or fusion proteins that comprise one or more signal transduction agents, a targeting moiety for SIRP1α, and / or one or more additional targeting moieties.

[0231] In various embodiments, the chimeric proteins of the invention have targeting moieties that target two different cells (e.g., to create a synapse) or target the same cell (e.g., to obtain a more focused signal transducer action).

[0232] In various embodiments, the chimeric proteins of the invention are multispecific, i.e., the chimeric proteins comprise two or more targeting moieties having recognition domains (e.g., antigen recognition domains) that recognize and bind to two or more targets (e.g., antigens, or receptors, or epitopes). In such embodiments, the chimeric proteins of the invention may comprise two or more targeting moieties having recognition domains that recognize and bind to two or more epitopes on the same antigen, on 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 certain embodiments, the chimeric proteins of the invention comprise two targeting moieties and are bispecific, i.e., they recognize and bind to two epitopes on the same antigen, on different antigens, or on different receptors.

[0233] In various embodiments, the multispecific chimeric protein of the invention comprises two or more targeting moieties, each of which is an antibody or antibody derivative as described herein. In an exemplary embodiment, the multispecific chimeric protein of the invention comprises at least one antibody or antibody derivative (e.g., VHH) that comprises an antigen recognition domain against SIRP1α, and one antibody or antibody derivative that comprises a recognition domain against a tumor antigen.

[0234] In various embodiments, a multispecific chimeric protein of the present disclosure has two or more targeting moieties that target different antigens or receptors, and one targeting moiety may be attenuated for that antigen or receptor, e.g., the targeting moiety binds to the antigen or receptor with low affinity or avidity (e.g., includes an affinity or avidity that is lower than the affinity or avidity that the other targeting moiety has for that antigen or receptor, e.g., the difference between the binding affinities can be about 10-fold, 25-fold, 50-fold, 100-fold, 300-fold, 500-fold, 1000-fold, or 5000-fold. For example, a targeting moiety with low affinity or avidity may have a K in the mid-to-high nM range or in the low-to-mid μM range. D A targeting moiety may bind to its antigen or receptor with a K in the mid-to-high pM range or a K in the low-to-mid nM range, whereas a targeting moiety with high affinity or avidity may bind to its antigen or receptor with a K in the mid-to-high pM range or a K in the low-to-mid nM range. D For example, in some embodiments, the multispecific chimeric proteins of the present disclosure include attenuated targeting moieties directed to promiscuous antigens or receptors, which may improve targeting to cells of interest (e.g., via other targeting moieties) and prevent action across multiple cell types, including cells not targeted by the therapy (e.g., by binding to the promiscuous antigen or receptor with higher affinity than provided in these embodiments).

[0235] The multispecific chimeric proteins of the present invention can be constructed using methods known in the art. See, for example, U.S. Pat. No. 9,067,991, U.S. Patent Application Publication No. 2011 / 0262348, and WO 2004 / 041862, the contents of which are incorporated herein by reference in their entirety. In an exemplary embodiment, the multispecific chimeric proteins of the present invention comprising two or more targeting moieties can be constructed by chemical cross-linking by reacting amino acid residues with organic derivatizing agents, as described, for example, in Blattler, et al., Biochemistry, 24, 1517-1524 and European Patent No. 294703, the contents of which are incorporated herein by reference in their entirety. In another exemplary embodiment, the multispecific chimeric proteins comprising two or more targeting moieties are constructed by genetic fusion, i.e., by constructing a single polypeptide comprising the polypeptides of their individual targeting moieties. For example, a single polypeptide construct can be formed that encodes a first antibody or antibody derivative (e.g., VHH) having an antigen recognition domain for SIRP1α and a second antibody or antibody derivative having a recognition domain for a tumor antigen. Methods for making bivalent or multivalent VHH polypeptide constructs are disclosed in PCT Patent Application WO 96 / 34103, the entire contents of which are incorporated herein by reference. In other exemplary embodiments, the multispecific chimeric proteins of the present invention can be constructed by using a linker. For example, the carboxy terminus of a first antibody or antibody derivative (e.g., VHH) having an antigen recognition domain for SIRP1α can be linked to the amino terminus of a second antibody or antibody derivative having a recognition domain for a tumor antigen (or vice versa). Examples of linkers that can be used are described herein. In some embodiments, the components of the multispecific chimeric proteins of the present invention are directly linked to each other without the use of a linker.

[0236] In various embodiments, the multispecific chimeric proteins of the present invention recognize and bind to SIRP1α and one or more antigens found on one or more immune cells. Immune cells may include, but are not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer cells, T lymphocytes (e.g., cytotoxic T lymphocytes, helper T cells, natural killer T cells), B lymphocytes, plasma cells, dendritic cells, or a subset thereof. In some embodiments, the chimeric proteins specifically bind to an antigen of interest and effectively recruit one or more immune cells directly or indirectly.

[0237] In various embodiments, the multispecific chimeric proteins of the present disclosure recognize and bind to SIRP1α and one or more antigens found on tumor cells. In these embodiments, the chimeric proteins of the present disclosure can directly or indirectly recruit immune cells (e.g., macrophages) to tumor cells or the tumor microenvironment. In said embodiments, the chimeric proteins of the present disclosure promote phagocytosis of tumor cells by macrophages.

[0238] In some embodiments, the chimeric proteins of the present disclosure can alter the balance of immune cells in favor of immune attack of tumors, or can be used in methods involving such alteration. For example, the chimeric proteins of the present disclosure can alter the ratio of immune cells at clinically important sites in favor of cells that can kill and / or suppress tumors (e.g., anti-tumor macrophages (e.g., M1 macrophages), T cells, cytotoxic T lymphocytes, helper T cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, and dendritic cells) and against cells that protect tumors (e.g., 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 chimeric proteins of the present disclosure can increase the ratio of effector T cells to regulatory T cells.

[0239] In some embodiments, the multispecific chimeric proteins of the invention comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with a tumor cell. In some embodiments, the targeting moiety directly or indirectly recruits tumor cells. For example, in some embodiments, recruitment of tumor cells is to one or more effector cells (e.g., macrophages) that can phagocytose, kill, and / or suppress the tumor cells.

[0240] Tumor cells or cancer cells are associated with uncontrolled proliferation of cells or tissues and / or abnormal increase in cell survival and / or inhibition of apoptosis that interferes with the normal functioning of the body's organs and systems. For example, tumor cells include benign and malignant cancers, polyps, hyperplasias, and dormant tumors or micrometastases. Examples of tumor cells include basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, digestive cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma, hepatocellular carcinoma, liver cancer, intraepithelial neoplasia, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, leukemia, and leukemia). Lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), melanoma, myeloma, neuroblastoma, oral cancer (lips, tongue, mouth, and pharynx), ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, respiratory cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, stomach cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary tract cancer, vulvar cancer, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. Lymphomas, including Hodgkin's lymphoma and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, intermediate-grade diffuse NHL, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-dividing cell NHL, bulky mass disease NHL, mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phacomatosis, edema (e.g., edema associated with a brain tumor), and cells of Meigs' syndrome.

[0241] Tumor or cancer cells include, but are not limited to, carcinomas (e.g., various subtypes including adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcomas (including, e.g., osteosarcoma and soft tissue sarcoma), leukemias (including, e.g., acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and hairy cell leukemia), lymphomas and myelomas (including, e.g., Hodgkin's and non-Hodgkin's lymphoma, light chain myeloma, non-secretory myeloma, MGUS, and plasmacytoma), and central nervous system cancers (e.g., brain cancers (e.g., gliomas (e.g., astrocytoma, oligodendroglioma, and ependymoma), meningiomas, pituitary adenomas, and neuromas), and spinal tumors (e.g., meningiomas and neurofibromas)).

[0242] Examples of tumor antigens include MART-1 / MelanA, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colon cancer associated antigen (CRC)-0017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate specific membrane antigen (PSMA), T cell receptor / CD3 ζ chain, MAGE family 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-A13, MAGE-A14, MAGE-A15, MAGE-A16, MAGE-A17, MAGE-A18, MAGE-A19, MAGE-A20, MAGE-A21, MAGE-A22, MAGE-A23, MAGE-A24, MAGE-A25, MAGE-A26, MAGE-A27, MAGE-A28, MAGE-A29, MAGE-A30, MAGE-A31, MAGE-A32, MAGE-A33, MAGE-A34, MAGE-A35, MAGE-A36, MAGE-A37, MAGE-A38, MAGE-A39 ... E-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), GAGE ​​family tumor antigens (e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GA 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, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis coli protein (APC), fodrin, connexin 37, Ig idiotype, p15, gp75, GM2 ganglioside and GD2 ganglioside, viral products such as human papillomavirus proteins, Smad family tumor antigens, lmp-1, NA, EBV-encoded nuclear antigen (EBNA)-1, brain glycogen phosphorylase, S These include, but are not limited to, SX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1CT-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.

[0243] In some embodiments, the multispecific chimeric proteins of the present disclosure recognize and bind to SIRP1α as well as antigens on tumor cells, hi some embodiments, the multispecific chimeric proteins directly or indirectly recruit macrophages to tumor cells or the tumor microenvironment.

[0244] In some embodiments, the multispecific chimeric proteins of the invention comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with a T cell. In some embodiments, the targeting moiety directly or indirectly recruits a T cell. In certain embodiments, the antigen recognition domain specifically binds to an effector T cell. In some embodiments, the antigen recognition domain directly or indirectly recruits an effector T cell, for example, in some embodiments, to a treatment site (e.g., a location that contains one or more diseased cells or cells where a therapeutic effect is to be modulated). Examples of effector T cells include cytotoxic T cells (e.g., αβTCR, CD3 + , CD8 + , CD45RO + ), CD4 +Effector T cells (e.g., αβTCR, CD3 + , CD4 + , CCR7 + , CD62Lhi, IL - 7R / CD127 + );CD8 + Effector T cells (e.g., αβTCR, CD3 + , CD8 + , CCR7 + , CD62Lhi, IL - 7R / CD127 + ); effector memory T cells (e.g., CD62Llow, CD44 + , TCR, CD3 + , I.L. - 7R / CD127 + , IL-15R + , CCR7low); central memory T cells (e.g., CCR7 + , CD62L + , CD27 + , or CCR7hi, CD44 + , CD62Lhi, TCR, CD3 + , IL-7R / CD127 + , IL-15R + );CD62L + Effector T cells; early effector memory T cells (CD27 + CD62L - ) and late effector memory T cells (CD27 - CD62L - ) (TemE and TemL, respectively) + Effector memory T cells (TEM), CD127 ( + )CD25(low / -) effector T cells;CD127( - )CD25( - ) Effector T cells; CD8 + Stem cell memory effector cells (TSCM) (e.g., CD44(low)CD62L(high)CD122(high)sca( + )); TH1 effector T cells (e.g., CXCR3 + , CXCR6 + and CCR5 +, or αβTCR, CD3 + , CD4 + , IL-12R + , IFNγR + , CXCR3 + ); TH2 effector T cells (e.g., CCR3 + , CCR4 + and CCR8 + , or αβTCR, CD3 + , CD4 + , IL-4R + , IL-33R + , CCR4 + , IL-17RB + , CRTH2 + ); TH9 effector T cells (e.g., αβTCR, CD3 + , CD4 + );TH17 effector T cells (e.g. αβTCR, CD3 + , CD4 + , 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 that secrete IL-2, IL-4 and / or IFN-γ.

[0245] Examples of T cell antigens of interest (and, where applicable, the extracellular domains) include, for example, 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, CXCR1 / IL-SRA, CCR5, CCR6, IL-10Rα, CCR7, IL-10Rβ, CCRS, IL-12Rβ1, CCR9, IL-12Rβ2, CD2, I L-13Rα1, IL-13, CD3, CD4, ILT2 / CDS5j, ILT3 / CDS5k, ILT4 / CDS5d, ILT5 / CDS5a, integrin α4 / CD49d, CDS, integrin αE / CD103, CD6, integrin αM / CD11b, CDS, integrin αX / CD11c, integrin β2 / CDlS, KIR / CD15S, CD27 / TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30 / TNFRSFS, KIR2DL4 / CD15Sd, CD31 / PE CAM-1, KIR2DS4, CD40 ligand / TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS3, leukotriene B4-R1, CDS4 / SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229 / SLAMF3, NKG2D, CD2F-10 / SLAMF9, NT-4, CD69, NTB-A / SLAMF6, common gamma chain / IL-2Rgamma, osteopontin, CRACC / SLAMF7, PD-1, CRTAM, PSGL-1, CTLA -4, RANK / TNFRSF11A, CX3CR1, CX3CL1, L-selectin, CXCR3, SIRPβ1, CXCR4, SLAM, CXCR6, TCCR / WSX-1, DNAM-1, thymopoietin, EMMPRIN / CD147, TIM-1, EphB6, TIM-2, Fas / TNFRSF6, TIM-3, Fas ligand / TNFSF6, TIM-4, FcγRIII / CD16, TIM-6, TNFR1 / TNFRSF1A, granulysin, TNFRIII / TNFRSF1B, TRAIL R1 / TNFRSF10A, ICAM-1 / CD54, TRAILR2 / TNFRSF10B, ICAM-2 / CD102, TRAILR3 / TNFRSF10C, IFN-γR1, TRAILR4 / TNFRSF10D, IFN-γR2, TSLP, IL-1R1, and TSLP R. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these examples of T cell antigens.

[0246] As non-limiting examples, in various embodiments, the chimeric proteins of the present disclosure have a targeting moiety directed to one or more of checkpoint markers expressed on T cells, such as PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2aR.

[0247] In some embodiments, the multispecific chimeric proteins of the invention comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with B cells. In some embodiments, the targeting moiety directly or indirectly recruits B cells, for example, in some embodiments, to a therapeutic site (e.g., a location that contains one or more diseased cells or cells where a therapeutic effect is to be modulated). Examples of 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 examples of B cell antigens.

[0248] In some embodiments, a 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 receptor) associated with a natural killer cell. In some embodiments, the targeting moiety directly or indirectly recruits natural killer cells, e.g., in some embodiments, to a therapeutic site (e.g., a location that contains one or more diseased cells or cells where a therapeutic effect is to be modulated). Examples of 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, Kir1α, DNAM-1, LMIR5 / CD300LB, Fc-εRII, LMIR6 / CD300LE, Fc-γR1 / CD64, MICA, Fc-γRIIB / CD32b, MICB, Fc-γRIIC / CD32c, MULT-1, Fc-γRIIA / CD32a, nectin-2 / CD112, Fc-γRIII / CD16, NKG2A, FcRH1 / IRTA5, NKG 2C, FcRH2 / IRTA4, NKG2D, FcRH4 / IRTA1, NKp30, FcRH5 / IRTA2, NKp44, Fc receptor-like molecule 3 / CD16-2, NKp46 / NCR1, NKp80 / KLRF1, NTB-A / SLAMF6, Rae-1, Rae-1α, Rae-1β, Rae-1δ, H60, Rae-1ε, 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 examples of NK cell antigens.

[0249] In some embodiments, a 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 receptor) associated with macrophages / monocytes. In some embodiments, the targeting moiety directly or indirectly recruits macrophages / monocytes, e.g., in some embodiments, to a therapeutic site (e.g., a location that contains one or more diseased cells or cells where a therapeutic effect is to be modulated). Examples of macrophage / monocyte antigens of interest include, for example, SIRP1a, B7-1 / CD80, ILT4 / CD85d, B7-H1, ILT5 / CD85a, common beta chain, integrin α4 / CD49d, BLAME / SLAMF8, integrin αX / CD11c, CCL6 / C10, integrin β2 / CD18, CD155 / PVR, integrin β3 / CD61, CD31 / PECAM-1, latexin, CD36 / SR-B3, leukotriene B4R1, CD40 / TNFRSF5, LIMPIIISR-B2, CD43, LMIR1 / CD300A, CD45, LMIR2 / CD300c, CD68, LMIR 3 / 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-γRI / CD64, osteopontin, Fc-γRIIB / CD32b, PD-L2, Fc-γRIIC / CD32c, Siglec-3 / CD33, Fc-γRIIA / CD32a, SIGNR1 / CD209, Fc-γRIII / CD16, SLAM, GM-CSFRα, TCCR / WSX-1, ICAM-2 / CD102, TLR3, IFN-γR1, TLR4, IFN-γR2, TREM-1, IL-1RII, TREM-2, ILT2 / CD85j, TREM-3, ILT3 / CD85k, TREML1 / TLT-1, 2B4 / SLAMF4, IL-10Rα, ALCAM, IL-10Rβ, aminopeptidase N / ANPEP, ILT2 / CD85j, common β chain, ILT3 / CD85k, C1qR1 / CD93, ILT4 / CD85d, CCR1, ILT5 / CD85a, CCR2, CD206, integrin α4 / CD49d, CCR5, integrin αM / C D11b, CCR8, integrin αX / CD11c, CD155 / PVR, integrin β2 / 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-gammaRI / CD64, PSGL-1, Fc-gammaRIIIICD16, RP105, G-CSF R, L-selectin, GM-CSF Ra, Siglec-3 / CD33, HVEM / TNFRSF14, SLAM, ICAM-1 / CD54, TCCR / WSX-1, ICAM-2 / CD102, TREM-1, IL-6R, TREM-2, CXCR1 / IL-8RA, TREM-3, and TREML1 / TLT-1. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these examples of macrophage / monocyte antigens.

[0250] In some embodiments, the multispecific chimeric proteins of the invention comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with a dendritic cell. In some embodiments, the targeting moiety directly or indirectly recruits dendritic cells, for example, in some embodiments, to a treatment site (e.g., a location that contains one or more diseased cells or cells where a therapeutic effect is to be modulated). Examples of 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-P1 / COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand / TNFSF9, IL-12 / IL-23, and the like. p40, 4-amino-1, 8-naphthalimide, ILT2 / CD85j, CCL21 / 6Ckine, ILT3 / CD85k, 8-oxo-dG, ILT4 / CD85d, 8D6A, ILT5 / CD85a, A2B5, integrin α4 / CD49d, Aag, integrin β2 / CD18, AMICA, Langerin, B7-2 / CD86, leukotriene B4R1, B7-H3, LMIR1 / CD300A, BLAME / SLAMF8, LMIR2 / CD300c, C1qR1 / CD93, LMIR3 / CD300LF, CCR6, LMIR5 / CD300LBCCR7, LMIR6 / CD300LE, CD40 / TNFRSF5, MAG / Siglec-4-a, CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1 / CLEC5A, CD84 / SLAMF5, MMR, CD97, NCAML1, CD2F-10 / SLAMF9, osteoactivin, GPNMB, Chern23, 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, Sig These include REC-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-γR1 / CD64, TLR3, Fc-γRIIB / CD32b, TREM-1, Fc-γRIIC / CD32c, TREM-2, Fc-γRIIA / CD32a, TREM-3, Fc-γRIII / 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 examples of DC antigens.

[0251] In some embodiments, a 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 receptor) associated with an immune cell selected from, but not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or a subset thereof. In some embodiments, the antigen recognition domain directly or indirectly recruits megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or a subset thereof, e.g., in some embodiments, to a treatment site (e.g., a location that contains one or more diseased cells or cells where a therapeutic effect is to be modulated).

[0252] In some embodiments, the multispecific chimeric proteins of the invention comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with megakaryocytes and / or platelets. Examples of megakaryocyte and / or platelet antigens of interest include, for example, GPIIb / IIIa, GPIb, vWF, PF4, and TSP. In various embodiments, the chimeric proteins comprise a targeting moiety that binds to one or more of these examples of megakaryocyte and / or platelet antigens.

[0253] In some embodiments, the multispecific chimeric proteins of the invention comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with red blood cells. Examples of red blood cell antigens of interest include, for example, CD34, CD36, CD38, CD41a (platelet glycoprotein IIb / IIIa), CD41b (GPIIb), CD71 (transferrin receptor), CD105, glycoprotein A, glycoprotein C, c-kit, HLA-DR, H2 (MHC-II), and Rh antigen. In various embodiments, the chimeric proteins comprise a targeting moiety that binds to one or more of these examples of red blood cell antigens.

[0254] In some embodiments, the multispecific chimeric proteins of the invention comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with a mast cell. Examples of mast cell antigens of interest include, for example, SCFR / CD117, Fc ε These include RI, CD2, CD25, CD35, CD88, CD203c, C5R1, CMA1, FCER1A, FCER2, TPSAB1. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these mast cell antigens.

[0255] In some embodiments, a 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 receptor) associated with a basophil. Examples of basophil antigens of interest include, for example, Fc εThese include RI, CD203c, CD123, CD13, CD107a, CD107b, and CD 164. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these basophil antigens.

[0256] In some embodiments, the multispecific chimeric proteins of the invention comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with neutrophils. Examples of neutrophil antigens of interest include, for example, 7D5, CD10 / CALLA, CD13, CD16 (FcRIII), CD18 protein (LFA-1, CR3, and p150, 95), CD45, CD67, and CD177. In various embodiments, the chimeric proteins comprise a targeting moiety that binds to one or more of these neutrophil antigens.

[0257] In some embodiments, the multispecific chimeric proteins of the invention comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., an antigen or receptor) associated with eosinophils. Examples of 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.

[0258] In various embodiments, the multispecific chimeric proteins of the present invention comprise a targeting moiety having a recognition domain that specifically binds to a suitable antigen or cell surface marker known to one of skill in the art. In some embodiments, the antigen or cell surface marker is a tissue-specific marker. Examples of tissue-specific markers include endothelial cell surface markers such as ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAM1, PROCR, SELE, SELP, TEK, THBD, VCAM1, VWF; smooth muscle cell surface markers such as ACTA2, MYH10, MYH11, MYH9, MYOCD; fibroblast surface markers such as ALCAM, CD34, COL1A1, COL1A2, COL3A1, FAP, PH-4, etc. These include, but are not limited to, blast (stromal) cell surface markers; epithelial cell surface markers such as CD1D, K6IRS2, KRT10, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUC1, TACSTD1; angiogenesis 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 comprises a targeting moiety that binds to one or more of these antigens. In various embodiments, the targeting moiety of the chimeric protein binds to one or more of the cells that bear these antigens.

[0259] In various embodiments, the multispecific chimeric proteins of the invention comprise one or more targeting moieties directed to one or more of checkpoint markers, such as 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.

[0260] As non-limiting examples, in various embodiments, the chimeric proteins of the present disclosure (i) have a targeting moiety directed to a checkpoint marker expressed on T cells, e.g., one or more of PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, Cd27, CD40L, TIM3, and A2aR, and (ii) have a targeting moiety directed to tumor cells in conjunction with any of the modified (e.g., mutant) signal transducers described herein.

[0261] In various embodiments, the multispecific chimeric proteins of the present disclosure have one or more targeting moieties directed to PD-1. In some embodiments, the chimeric proteins have one or more targeting moieties that selectively bind to a PD-1 polypeptide. In some embodiments, the chimeric proteins comprise one or more antibodies, antibody derivatives or antibody formats, peptides or polypeptides, or fusion proteins that selectively bind to a PD-1 polypeptide.

[0262] In some embodiments, the targeting moiety comprises the anti-PD-1 antibody pembrolizumab (also known as MK-3475, Keytruda) or a fragment thereof. Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed in Hamid, et al., (2013) New England Journal of Medicine, 369(2):pp.134-44, U.S. Patent No. 8,354,509, and WO 2009 / 114335, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, pembrolizumab, or an antigen-binding fragment for use in the methods described 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.

[0263] In certain embodiments, the targeting moiety comprises the anti-PD-1 antibody nivolumab (also known as BMS-936558, MDX-1106, ONO-4538, OPDIVO) or a fragment thereof. Nivolumab (clone 5C4), and other human monoclonal antibodies that specifically bind to PD-1, are disclosed in U.S. Pat. No. 8,008,449 and WO 2006 / 121168, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 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.

[0264] In one embodiment, the targeting moiety comprises the anti-PD-1 antibody pidilizumab (also known as CT-011, hBAT or hBAT-1) or a fragment thereof. Pidilizumab and other humanized anti-PD-I monoclonal antibodies are disclosed in U.S. Patent Application Publication No. 2008 / 0025980 and WO 2009 / 101611, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, the anti-PD-1 antibody, or antigen-binding fragment thereof for use in the methods described herein, comprises a light chain comprising an amino acid sequence selected from SEQ ID NOs:15-18 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:15 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:73), SEQ ID NO:16 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:74), SEQ ID NO:17 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:75), and SEQ ID NO:18 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:76)). The heavy chain comprises a variable region and / or an amino acid sequence selected from SEQ ID NOs:20-24 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:20 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:77), SEQ ID NO:21 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:78), SEQ ID NO:22 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:79), SEQ ID NO:23 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:80), and SEQ ID NO:24 of U.S. Patent Application Publication No. 2008 / 0025980 (SEQ ID NO:81)).

[0265] In one embodiment, the targeting moiety comprises a light chain (SEQ ID NO: 76) comprising SEQ ID NO: 18 of U.S. Patent Application Publication No. 2008 / 0025980 and a heavy chain (SEQ ID NO: 79) comprising SEQ ID NO: 22 of U.S. Patent Application Publication No. 2008 / 0025980.

[0266] In certain embodiments, the targeting moiety comprises AMP-514 (also known as MEDI-0680).

[0267] In one embodiment, the targeting moiety comprises AMP-224, a PD-L2-Fc fusion protein, as disclosed in WO 2010 / 027827 and WO 2011 / 066342, the disclosures of which are incorporated herein by reference in their entireties. In such embodiments, the targeting moiety may comprise a targeting domain comprising SEQ ID NO:4 of WO 2010 / 027827 (SEQ ID NO:82), and / or a B7-DC fusion protein comprising SEQ ID NO:83 of WO 2010 / 027827 (SEQ ID NO:83).

[0268] In some embodiments, the targeting moiety comprises an AUNP12 peptide or any of the other peptides disclosed in U.S. Patent Application Publication No. 2011 / 0318373 or U.S. Patent No. 8,907,053. For example, the targeting moiety may comprise AUNP12 having the following sequence (SEQ ID NO:84) (i.e., compound 8 or SEQ ID NO:49 of U.S. Patent Application Publication No. 2011 / 0318373): [ka]

[0269] In one embodiment, the targeting moiety comprises 1E3, an anti-PD-1 antibody, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the disclosure of which is incorporated herein by reference in its entirety. In an exemplary embodiment, 1E3 or an antigen-binding fragment thereof for use in the methods described 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.

[0270] In one embodiment, the targeting moiety comprises 1E8, an anti-PD-1 antibody, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the disclosure of which is incorporated herein by reference in its entirety. In an exemplary embodiment, 1E8, or an antigen-binding fragment thereof, for use in the methods described 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.

[0271] In one embodiment, the targeting moiety comprises 1H3, an anti-PD-1 antibody, or a fragment thereof, as disclosed in U.S. Patent Application Publication No. 2014 / 0044738, the disclosure of which is incorporated herein by reference in its entirety. In an exemplary embodiment, 1H3, or an antigen-binding fragment thereof, for use in the methods described herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:89, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:90.

[0272] In certain embodiments, the targeting moiety comprises a VHH directed against PD-1, e.g., as disclosed in U.S. Patent No. 8,907,065 and WO 2008 / 071447, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, the VHH against PD-1 comprises SEQ ID NOs:347-351 of U.S. Patent No. 8,907,065 (SEQ ID NO:347 of U.S. Patent No. 8,907,065 (SEQ ID NO:91), SEQ ID NO:348 of U.S. Patent No. 8,907,065 (SEQ ID NO:92), SEQ ID NO:349 of U.S. Patent No. 8,907,065 (SEQ ID NO:93), SEQ ID NO:350 of U.S. Patent No. 8,907,065 (SEQ ID NO:94), and SEQ ID NO:351 of U.S. Patent No. 8,907,065 (SEQ ID NO:95)).

[0273] In certain embodiments, the targeting moiety comprises any one of the anti-PD-1 antibodies or fragments thereof as disclosed in U.S. Patent Application Publication No. 2011 / 0271358 and WO 2010 / 036959, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibodies or antigen-binding fragments thereof for use in the methods described herein are selected from the group consisting of SEQ ID NOs:25-29 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO:25 (SEQ ID NO:96) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO:26 (SEQ ID NO:97) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO:27 (SEQ ID NO:98) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO:28 (SEQ ID NO:99) of U.S. Patent Application Publication No. 2011 / 0271358, and SEQ ID NO:30 (SEQ ID NO:31) of U.S. Patent Application Publication No. 2011 / 0271358. No. 2011 / 0271358), and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 30 to 33 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 30 (SEQ ID NO: 101) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 31 (SEQ ID NO: 102) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 32 (SEQ ID NO: 103) of U.S. Patent Application Publication No. 2011 / 0271358, and SEQ ID NO: 33 (SEQ ID NO: 104) of U.S. Patent Application Publication No. 2011 / 0271358).

[0274] In various embodiments, the multispecific chimeric proteins of the present disclosure comprise one or more anti-PD-1 antibodies or antibody fragments thereof selected from TSR-042 (Tesaro), REGN2810 (Regeneron Pharmaceuticals), PDR001 (Novartis Pharmaceuticals), and BGB-A317 (BeiGene).

[0275] In various embodiments, the multispecific chimeric proteins of the present disclosure comprise one or more targeting moieties directed to PD-L1. In some embodiments, the chimeric proteins comprise one or more targeting moieties that selectively bind to a PD-L1 polypeptide. In some embodiments, the chimeric proteins comprise one or more antibodies, antibody derivatives or antibody formats, peptides or polypeptides, or fusion proteins that selectively bind to a PD-L1 polypeptide.

[0276] In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody MEDI4736 (also known as durvalumab) or a fragment thereof. MEDI4736 is selective for PD-L1 and inhibits binding of PD-L1 to the PD-1 receptor and the CD80 receptor. MEDI4736 and antigen-binding fragments thereof for use in the methods described herein comprise a heavy chain and a light chain, or a heavy chain variable region and a light chain variable region. The sequence of MEDI4736 is disclosed in WO 2016 / 06272, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, MEDI4736 or antigen-binding fragments thereof for use in the methods described herein comprise 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.

[0277] In an exemplary embodiment, MEDI4736, or an antigen-binding fragment thereof, for use in the methods described 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).

[0278] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody atezolizumab (also known as MPDL3280A, RG7446), or a fragment thereof. In an exemplary embodiment, atezolizumab or an antigen-binding fragment thereof for use in the methods described 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.

[0279] In some embodiments, the targeting moiety comprises the anti-PD-L1 antibody avelumab (also known as MSB0010718C), or a fragment thereof. In an exemplary embodiment, avelumab, or an antigen-binding fragment thereof, for use in the methods described 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.

[0280] In one embodiment, the targeting moiety comprises BMS-936559 (also known as 12A4, MDX-1105), an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Publication No. 2013 / 0309250 and WO 2007 / 005874, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, BMS-936559 or an antigen-binding fragment thereof for use in the methods described 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.

[0281] In one embodiment, the targeting moiety comprises 3G10, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 3G10, or an antigen-binding fragment thereof, for use in the methods described 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.

[0282] In one embodiment, the targeting moiety comprises 10A5, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the disclosures of which are incorporated by reference in their entireties. In an exemplary embodiment, 10A5, or an antigen-binding fragment thereof, for use in the methods described 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.

[0283] In one embodiment, the targeting moiety comprises 5F8, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 5F8, or an antigen-binding fragment thereof, for use in the methods described 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.

[0284] In one embodiment, the targeting moiety comprises 10H10, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 10H10, or an antigen-binding fragment thereof, for use in the methods described 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.

[0285] In one embodiment, the targeting moiety comprises 1B12, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 1B12, or an antigen-binding fragment thereof, for use in the methods described 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.

[0286] In one embodiment, the targeting moiety comprises 7H1, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 7H1, or an antigen-binding fragment thereof, for use in the methods described 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.

[0287] In one embodiment, the targeting moiety comprises 11E6, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Publication No. 2013 / 0309250 and WO 2007 / 005874, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 11E6, or an antigen-binding fragment thereof, for use in the methods described 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.

[0288] In one embodiment, the targeting moiety comprises 12B7, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Application Publication No. 2013 / 0309250 and WO 2007 / 005874, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 12B7, or an antigen-binding fragment thereof, for use in the methods described 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.

[0289] In one embodiment, the targeting moiety comprises 13G4, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Publication No. 2013 / 0309250 and WO 2007 / 005874, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 13G4, or an antigen-binding fragment thereof, for use in the methods described 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.

[0290] In one embodiment, the targeting moiety comprises 1E12, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Application Publication No. 2014 / 0044738, the disclosure of which is incorporated herein by reference in its entirety. In an exemplary embodiment, 1E12, or an antigen-binding fragment thereof, for use in the methods described 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.

[0291] In one embodiment, the targeting moiety comprises 1F4, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Application Publication No. 2014 / 0044738, the disclosure of which is incorporated herein by reference in its entirety. In an exemplary embodiment, 1F4, or an antigen-binding fragment thereof, for use in the methods described 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.

[0292] In one embodiment, the targeting moiety comprises 2G11, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Application Publication No. 2014 / 0044738, the disclosure of which is incorporated herein by reference in its entirety. In an exemplary embodiment, 2G11, or an antigen-binding fragment thereof, for use in the methods described 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.

[0293] In one embodiment, the targeting moiety comprises 3B6, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Application Publication No. 2014 / 0044738, the disclosure of which is incorporated herein by reference in its entirety. In an exemplary embodiment, 3B6, or an antigen-binding fragment thereof, for use in the methods described 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.

[0294] In one embodiment, the targeting moiety comprises 3D10, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in US Patent Application Publication No. 2014 / 0044738 and WO 2012 / 145493, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 3D10, or an antigen-binding fragment thereof, for use in the methods described 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.

[0295] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies as disclosed in US Patent Application Publication No. 2011 / 0271358 and WO 2010 / 036959, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibodies or antigen-binding fragments thereof for use in the methods described herein are selected from the group consisting of SEQ ID NOs: 34-38 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 143 of US Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 35 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 144 of US Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 36 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 145 of US Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 37 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 146 of US Patent Application Publication No. 2011 / 0271358, and SEQ ID NO: 147 of US Patent Application Publication No. 2011 / 0271358). No. 58), and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 39 to 42 of U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 39 (SEQ ID NO: 148) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 40 (SEQ ID NO: 149) of U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 41 (SEQ ID NO: 150) of U.S. Patent Application Publication No. 2011 / 0271358, and SEQ ID NO: 42 (SEQ ID NO: 151) of U.S. Patent Application Publication No. 2011 / 0271358).

[0296] In some embodiments, the targeting moiety comprises 2.7A4, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 2.7A4, or an antigen-binding fragment thereof, for use in the methods described 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).

[0297] In one embodiment, the targeting moiety comprises 2.9D10, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 2.9D10, or an antigen-binding fragment thereof, for use in the methods described 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).

[0298] In some embodiments, the targeting moiety comprises 2.14H9, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 2.14H9, or an antigen-binding fragment thereof, for use in the methods described 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).

[0299] In some embodiments, the targeting moiety comprises 2.20A8, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 2.20A8, or an antigen-binding fragment thereof, for use in the methods described 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).

[0300] In some embodiments, the targeting moiety comprises 3.15G8, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 3.15G8, or an antigen-binding fragment thereof, for use in the methods described 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).

[0301] In some embodiments, the targeting moiety comprises 3.18G1, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 3.18G1, or an antigen-binding fragment thereof, for use in the methods described 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).

[0302] In some embodiments, the targeting moiety comprises 2.7A4OPT, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 2.7A4OPT or an antigen-binding fragment thereof for use in the methods described 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).

[0303] In some embodiments, the targeting moiety comprises 2.14H9OPT, an anti-PD-L1 antibody, or a fragment thereof, as disclosed in WO 2011 / 066389, U.S. Patent No. 8,779,108, and U.S. Patent Application Publication No. 2014 / 0356353, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, 2.14H9OPT or an antigen-binding fragment thereof for use in the methods described 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).

[0304] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies as disclosed in WO 2016 / 061142, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibodies or antigen-binding fragments thereof for use in the methods described herein are selected from the group consisting of SEQ ID NOs: 18, 30, 38, 46, 50, 54, 62, 70, and 78 of WO 2016 / 061142 (SEQ ID NO: 18 of WO 2016 / 061142 (SEQ ID NO: 168), SEQ ID NO: 30 of WO 2016 / 061142 (SEQ ID NO: 169), SEQ ID NO: 38 of WO 2016 / 061142 (SEQ ID NO: 170), SEQ ID NO: 40 of WO 2016 / 061142 (SEQ ID NO: 41 of WO 2016 / 061142 (SEQ ID NO: 42 of WO 2016 / 061142 (SEQ ID NO: 43 of WO 2016 / 061142 (SEQ ID NO: 44 of WO 2016 / 061142 (SEQ ID NO: 45 of WO 2016 / 061142 (SEQ ID NO: 46 of WO 2016 / 061142 (SEQ ID NO: 47 of WO 2016 / 061142 (SEQ ID NO: 48 of WO 2016 / 061142 (SEQ ID NO: 49 of WO 2016 / 061142 (SEQ ID NO: 50 of WO 2016 / 061142 (SEQ ID NO: 51 of SEQ ID NO: 46 (SEQ ID NO: 171) of Publication No. WO 2016 / 061142, SEQ ID NO: 50 (SEQ ID NO: 172) of Publication No. WO 2016 / 061142, SEQ ID NO: 54 (SEQ ID NO: 173) of Publication No. WO 2016 / 061142, SEQ ID NO: 62 (SEQ ID NO: 174) of Publication No. WO 2016 / 061142, SEQ ID NO: 70 (SEQ ID NO: 175) of Publication No. WO 2016 / 061142, and SEQ ID NO: 78 (SEQ ID NO: 176) of Publication No. WO 2016 / 061142 22, 26, 34, 42, 58, 66, 74, 82, and 86 of WO 2016 / 061142 (SEQ ID NO: 22 of WO 2016 / 061142 (SEQ ID NO: 177), SEQ ID NO: 26 of WO 2016 / 061142 (SEQ ID NO: 178), SEQ ID NO: 34 of WO 2016 / 061142 (SEQ ID NO: 179), SEQ ID NO: 42 of WO 2016 / 061142 ( and SEQ ID NO:185 of WO 2016 / 061142 (SEQ ID NO:186), SEQ ID NO:58 of WO 2016 / 061142 (SEQ ID NO:181), SEQ ID NO:66 of WO 2016 / 061142 (SEQ ID NO:182), SEQ ID NO:74 of WO 2016 / 061142 (SEQ ID NO:183), SEQ ID NO:82 of WO 2016 / 061142 (SEQ ID NO:184), and SEQ ID NO:86 of WO 2016 / 061142 (SEQ ID NO:185).

[0305] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies as disclosed in WO 2016 / 022630, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibodies or antigen-binding fragments thereof for use in the methods described herein are selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, and 46 of WO 2016 / 022630 (SEQ ID NO: 2 of WO 2016 / 022630 (SEQ ID NO: 186), SEQ ID NO: 6 of WO 2016 / 022630 (SEQ ID NO: 187), SEQ ID NO: 10 of WO 2016 / 022630 (SEQ ID NO: 188), SEQ ID NO: 10 of WO 2016 / 022630 (SEQ ID NO: 189), SEQ ID NO: 11 of WO 2016 / 022630 (SEQ ID NO: 190), SEQ ID NO: 12 of WO 2016 / 022630 (SEQ ID NO: 191), SEQ ID NO: 13 of WO 2016 / 022630 (SEQ ID NO: 192), SEQ ID NO: 14 of WO 2016 / 022630 (SEQ ID NO: 193), SEQ ID NO: 15 of WO 2016 / 022630 (SEQ ID NO: 194), SEQ ID NO: 16 of WO 2016 / 022630 (SEQ ID NO: 195), SEQ ID NO: 17 of WO 2016 / 022630 (SEQ ID SEQ ID NO:14 (SEQ ID NO:189), SEQ ID NO:18 of WO 2016 / 022630 (SEQ ID NO:190), SEQ ID NO:22 of WO 2016 / 022630 (SEQ ID NO:191), SEQ ID NO:26 of WO 2016 / 022630 (SEQ ID NO:192), SEQ ID NO:30 of WO 2016 / 022630 (SEQ ID NO:193), SEQ ID NO:34 of WO 2016 / 022630 (SEQ ID NO:194), SEQ ID NO:38 of WO 2016 / 022630 (SEQ ID NO:195), and / or a heavy chain comprising an amino acid sequence selected from SEQ ID NO: 42 (SEQ ID NO: 196) of WO 2016 / 022630, and SEQ ID NO: 46 (SEQ ID NO: 197) of WO 2016 / 022630), and / or a heavy 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 WO 2016 / 022630 (SEQ ID NO: 4 (SEQ ID NO: 198) of WO 2016 / 022630, SEQ ID NO: 8 (SEQ ID NO: 199) of WO 2016 / 022630, SEQ ID NO: 9 (SEQ ID NO: 191) of WO 2016 / 022630). SEQ ID NO: 12 of International Publication No. 630 (SEQ ID NO: 200), SEQ ID NO: 16 of International Publication No. 2016 / 022630 (SEQ ID NO: 201), SEQ ID NO: 20 of International Publication No. 2016 / 022630 (SEQ ID NO: 202), SEQ ID NO: 24 of International Publication No. 2016 / 022630 (SEQ ID NO: 203), SEQ ID NO: 28 of International Publication No. 2016 / 022630 (SEQ ID NO: 204), SEQ ID NO: 32 of International Publication No. 2016 / 022630 (SEQ ID NO: 205), SEQ ID NO: 36 of International Publication No. 2016 / 022630 (SEQ ID NO: 206),The light chain comprises an amino acid sequence selected from SEQ ID NO: 40 of WO 2016 / 022630 (SEQ ID NO: 207), SEQ ID NO: 44 of WO 2016 / 022630 (SEQ ID NO: 208), and SEQ ID NO: 48 of WO 2016 / 022630 (SEQ ID NO: 209).

[0306] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies as disclosed in WO 2015 / 112900, the contents of which are incorporated herein by reference in their entirety. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods described 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 heavy chain comprising an amino acid sequence selected from SEQ ID NOs: 42, 46, 54, 58, 62, 66, 70, 74, and 78 of WO 2015 / 112900 (SEQ ID NO: 214). and SEQ ID NO: 78 of WO 2015 / 112900 (SEQ ID NO: 222).

[0307] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies as disclosed in WO 2010 / 077634 and US Patent No. 8,217,149, the disclosures of which are incorporated herein by reference in their entireties. In exemplary embodiments, the anti-PD-L1 antibodies, or antigen-binding fragments thereof, for use in the methods described herein comprise 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).

[0308] In some embodiments, the targeting moiety comprises any one of the anti-PD-L1 antibodies obtainable from the hybridomas available under CNCM Accession Nos. CNCM I-4122, CNCM I-4080, and CNCM I-4081, as disclosed in U.S. Patent Application Publication No. 2012 / 0039906, the disclosure of which is incorporated herein by reference in its entirety.

[0309] In certain embodiments, the targeting moiety comprises a VHH against PD-L1, e.g., as described in U.S. Patent No. 8,907,065 and WO 2008 / 071447, the disclosures of which are incorporated herein by reference in their entireties. In exemplary embodiments, the VHH against PD-L1 includes SEQ ID NOs: 394-399 of U.S. Patent No. 8,907,065 (SEQ ID NO: 394 of U.S. Patent No. 8,907,065 (SEQ ID NO: 225), SEQ ID NO: 395 of U.S. Patent No. 8,907,065 (SEQ ID NO: 226), SEQ ID NO: 396 of U.S. Patent No. 8,907,065 (SEQ ID NO: 227), SEQ ID NO: 397 of U.S. Patent No. 8,907,065 (SEQ ID NO: 228), SEQ ID NO: 398 of U.S. Patent No. 8,907,065 (SEQ ID NO: 229), and SEQ ID NO: 399 of U.S. Patent No. 8,907,065 (SEQ ID NO: 230)).

[0310] In various embodiments, the multispecific chimeric proteins of the present disclosure have one or more targeting moieties directed to 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 derivatives or antibody formats, peptides or polypeptides, or fusion proteins that selectively bind to a PD-L2 polypeptide.

[0311] In one embodiment, the targeting moiety comprises a VHH directed against PD-L2, e.g., as described in U.S. Pat. No. 8,907,065 and WO 2008 / 071447, the disclosures of which are incorporated herein by reference in their entireties. In an exemplary embodiment, the VHH against PD-1 comprises SEQ ID NOs:449-455 of U.S. Patent No. 8,907,065 (SEQ ID NO:449 (SEQ ID NO:231) of U.S. Patent No. 8,907,065, SEQ ID NO:450 (SEQ ID NO:232) of U.S. Patent No. 8,907,065, SEQ ID NO:451 (SEQ ID NO:233) of U.S. Patent No. 8,907,065, SEQ ID NO:452 (SEQ ID NO:234) of U.S. Patent No. 8,907,065, SEQ ID NO:453 (SEQ ID NO:235) of U.S. Patent No. 8,907,065, SEQ ID NO:454 (SEQ ID NO:236) of U.S. Patent No. 8,907,065, and SEQ ID NO:455 (SEQ ID NO:237) of U.S. Patent No. 8,907,065).

[0312] In some embodiments, the targeting moiety comprises any one of the anti-PD-L2 antibodies disclosed in US Patent Application Publication No. 2011 / 0271358 and WO 2010 / 036959, the contents of which are incorporated herein by reference in their entireties. In exemplary embodiments, the antibodies or antigen-binding fragments thereof for use in the methods described herein are selected from the group consisting of SEQ ID NOs: 43-47 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 43 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 238), SEQ ID NO: 44 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 239), SEQ ID NO: 45 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 240), SEQ ID NO: 46 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 241), and SEQ ID NO: 47 of US Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 242). No. 58 (SEQ ID NO: 47 (SEQ ID NO: 242)), and / or a light chain comprising an amino acid sequence selected from SEQ ID NOs: 48 to 51 in U.S. Patent Application Publication No. 2011 / 0271358 (SEQ ID NO: 48 (SEQ ID NO: 243) in U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 49 (SEQ ID NO: 244) in U.S. Patent Application Publication No. 2011 / 0271358, SEQ ID NO: 50 (SEQ ID NO: 245) in U.S. Patent Application Publication No. 2011 / 0271358, and SEQ ID NO: 51 (SEQ ID NO: 246) in U.S. Patent Application Publication No. 2011 / 0271358).

[0313] In various embodiments, the targeting moieties of the invention have a targeting affinity of 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 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 (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical to any of the sequences disclosed herein (e.g., about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 7 ...5%, about 96%, about 97%, about 98%, about 99%, or 100% identical 3%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% sequence identity) of sequences targeting PD-1, PD-L1, and / or PD-L2.

[0314] In various embodiments, targeting moieties of the invention may comprise any combination of heavy chain sequences, light chain sequences, heavy chain variable region sequences, light chain variable region sequences, complementarity determining region (CDR) sequences, and framework region sequences that target PD-1, PD-L1, and / or PD-L2 as disclosed herein.

[0315] Other antibodies, antibody derivatives or formats, peptides or polypeptides, or fusion proteins that selectively bind to or target PD-1, PD-L1, and / or PD-L2 are described in WO 2011 / 066389, U.S. Patent Application Publication No. 2008 / 0025980, U.S. Patent Application Publication No. 2013 / 0034559, U.S. Patent No. 8,779,108, U.S. Patent Application Publication No. 201 4 / 0356353, U.S. Patent No. 8,609,089, U.S. Patent Application Publication No. 2010 / 028330, U.S. Patent Application Publication No. 2012 / 0114649, WO 2010 / 027827, WO 2011 / 066342, U.S. Patent No. 8,907,065, WO 2016 / 062722, WO 2009 / 101611, WO 2010 / 027827, WO 2012 / 0114649, No. 011 / 066342, WO 2007 / 005874, WO 2001 / 014556, U.S. Patent Application Publication No. 2011 / 0271358, WO 2010 / 036959, WO 2010 / 077634, U.S. Patent No. 8217149, U.S. Patent Application Publication No. 2012 / 0039906, WO 2012 / 145493, U.S. Patent Application Publication No. 2011 / 0318373 No. 8,779,108, U.S. Patent Application Publication No. 2014 / 0044738, WO 2009 / 089149, WO 2007 / 00587, WO 2016 / 061142, WO 2016 / 02263, WO 2010 / 077634, and WO 2015 / 112900, the disclosures of which are incorporated herein by reference in their entireties.

[0316] In some embodiments, the targeting moiety is a natural ligand, such as a chemokine. Exemplary chemokines that may be included in the chimeric proteins of the invention include, but are not limited to, CCL1, CCL2, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CLL25, CCL26, CCL27, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, HCC-4, and LDGF-PBP. In an exemplary embodiment, the targeting moiety may be XCL1, a chemokine that recognizes and binds to the dendritic cell receptor XCR1. In another exemplary embodiment, the targeting moiety is CCL1, a chemokine that recognizes and binds to CCR8. In another exemplary embodiment, the targeting moiety is CCL2, a chemokine that recognizes and binds to CCR2 or CCR9. In another exemplary embodiment, the targeting moiety is CCL3, a chemokine that recognizes and binds to CCR1, CCR5, or CCR9. In another exemplary embodiment, the targeting moiety is CCL4, a chemokine that recognizes and binds to CCR1, CCR5, or CCR9. In another exemplary embodiment, the targeting moiety is CCL5, a chemokine that recognizes and binds to CCR1, CCR3, CCR,4, or CCR5. In another exemplary embodiment, the targeting moiety is CCL6, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL7, a chemokine that recognizes and binds to CCR2 or CCR9. In another exemplary embodiment, the targeting moiety is CCL8, a chemokine that recognizes and binds to CCR1, CCR2, CCR2B, CCR5, or CCR9. In another exemplary embodiment, the targeting moiety is CCL9, a chemokine that recognizes and binds to CCR1. In another exemplary embodiment, the targeting moiety is CCL10, a chemokine that recognizes and binds to CCR1.In another exemplary embodiment, the targeting moiety is CCL11, a chemokine that recognizes and binds CCR2, CCR3, CCR5, or CCR9. In another exemplary embodiment, the targeting moiety is CCL13, a chemokine that recognizes and binds CCR2, CCR3, CCR5, or CCR9. In another exemplary embodiment, the targeting moiety is CCL14, a chemokine that recognizes and binds CCR1 or CCR9. In another exemplary embodiment, the targeting moiety is CCL15, a chemokine that recognizes and binds CCR1 or CCR3. In another exemplary embodiment, the targeting moiety is CCL16, a chemokine that recognizes and binds CCR1, CCR2, CCR5, or CCR8. In another exemplary embodiment, the targeting moiety is CCL17, a chemokine that recognizes and binds CCR4. In another exemplary embodiment, the targeting moiety is CCL19, a chemokine that recognizes and binds CCR7. In another exemplary embodiment, the targeting moiety is CCL20, a chemokine that recognizes and binds CCR6. In another exemplary embodiment, the targeting moiety is CCL21, a chemokine that recognizes and binds CCR7. In another exemplary embodiment, the targeting moiety is CCL22, a chemokine that recognizes and binds CCR4. In another exemplary embodiment, the targeting moiety is CCL23, a chemokine that recognizes and binds CCR1. In another exemplary embodiment, the targeting moiety is CCL24, a chemokine that recognizes and binds CCR3. In another exemplary embodiment, the targeting moiety is CL25, a chemokine that recognizes and binds C, CCR9. In another exemplary embodiment, the targeting moiety is CCL26, a chemokine that recognizes and binds CCR3. In another exemplary embodiment, the targeting moiety is CCL27, a chemokine that recognizes and binds CCR10. In another exemplary embodiment, the targeting moiety is CCL28, a chemokine that recognizes and binds CCR3 or CCR10. In another exemplary embodiment, the targeting moiety is CXCL1, a chemokine that recognizes and binds to CXCR1 or CXCR2. In another exemplary embodiment, the targeting moiety is CXCL2, a chemokine that recognizes and binds to CXCR2.In another exemplary embodiment, the targeting moiety is CXCL3, a chemokine that recognizes and binds CXCR2. In another exemplary embodiment, the targeting moiety is CXCL4, a chemokine that recognizes and binds CXCR3B. In another exemplary embodiment, the targeting moiety is CXCL5, a chemokine that recognizes and binds CXCR2. In another exemplary embodiment, the targeting moiety is CXCL6, a chemokine that recognizes and binds CXCR1 or CXCR2. In another exemplary embodiment, the targeting moiety is CXCL8, a chemokine that recognizes and binds CXCR1 or CXCR2. In another exemplary embodiment, the targeting moiety is CXCL9, a chemokine that recognizes and binds CXCR3. In another exemplary embodiment, the targeting moiety is CXCL10, a chemokine that recognizes and binds CXCR3. In another exemplary embodiment, the targeting moiety is CXCL11, a chemokine that recognizes and binds CXCR3 or CXCR7. In another exemplary embodiment, the targeting moiety is CXCL12, a chemokine that recognizes and binds CXCR4 or CXCR7. In another exemplary embodiment, the targeting moiety is CXCL13, a chemokine that recognizes and binds to CXCR5. In another exemplary embodiment, the targeting moiety is CXCL16, a chemokine that recognizes and binds to CXCR6. In another exemplary embodiment, the targeting moiety is LDGF-PBP, a chemokine that recognizes and binds to CXCR2. In another exemplary embodiment, the targeting moiety is XCL2, a chemokine that recognizes and binds to XCR1. In another exemplary embodiment, the targeting moiety is CX3CL1, a chemokine that recognizes and binds to CX3CR1.

[0317] In various embodiments, the chimeric protein of the present disclosure comprises various combinations of targeting moieties. In an exemplary embodiment, the chimeric protein of the present disclosure may comprise two targeting moieties, both of which are antibodies or antibody derivatives. In another exemplary embodiment, the chimeric protein of the present disclosure may comprise two targeting moieties, both of which are natural ligands of a cell receptor. In other exemplary embodiments, the chimeric protein of the present disclosure may comprise two targeting moieties, one of which is an antibody or antibody derivative, and the other of which is natural ligands of a cell receptor.

[0318] In various embodiments, the recognition domain of the chimeric protein of the present disclosure modulates (by way of non-limiting example, partially or completely neutralizes) the function of a target of interest (e.g., antigen, receptor), e.g., substantially inhibits, reduces, or neutralizes the biological effect of the antigen. For example, various recognition domains may be directed to one or more tumor antigens that actively suppress or have the ability to suppress the immune system, such as in a patient with a tumor. For example, in some embodiments, the chimeric protein of the present disclosure modulates the function of one or more of immune suppressive signals (e.g., checkpoint inhibitors), such as 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 chimeric proteins of the present disclosure are modified to disrupt, block, inhibit, and / or inhibit the transmission of immunosuppressive signals through, by way of non-limiting examples, the binding of PD-1 to PD-L1 or PD-L2, and / or the binding of CTLA-4 to one or more of AP2M1, CD80, CD86, SHP-2, and PPP2R5A.

[0319] In various embodiments, the recognition domain of the chimeric protein of the present disclosure binds to a target of interest (e.g., an antigen, a receptor) but does not modulate its function, e.g., the recognition domain is or resembles a binding antibody. For example, in various embodiments, the recognition domain simply targets an antigen or receptor but does not substantially inhibit, suppress, or modulate the biological action of the antigen or receptor. For example, some of the smaller antibody formats described above (e.g., compared to full-length antibodies, etc.) have the ability to target epitopes that are less accessible and provide specific binding at a wider range of locations. In various embodiments, the recognition domain binds to an epitope that is physically distant from the site of the antigen or receptor that is important for biological activity (e.g., the active site of the antigen).

[0320] Such non-neutralizing binding is used in various embodiments of the present invention, including methods of using the chimeric proteins of the present disclosure to directly or indirectly recruit active immune cells to a site of need via an effector antigen, such as any of the effector antigens described herein. For example, in various embodiments, the chimeric proteins of the present disclosure can be used to directly or indirectly recruit cytotoxic T cells to tumor cells via CD8 in methods of reducing or eliminating tumors (e.g., the chimeric proteins of the present disclosure can include an anti-CD8 recognition domain and an anti-tumor antigen recognition domain). In such embodiments, it is desirable to directly or indirectly recruit CD8-expressing cytotoxic T cells but not functionally modulate CD8 activity. Conversely, in these embodiments, CD8 signaling is important for tumor reduction or tumor elimination. As another example, the chimeric proteins of the present disclosure can be used to directly or indirectly recruit dendritic cells (DCs) via CLEC9A in various methods of reducing or eliminating tumors (e.g., the chimeric proteins of the present disclosure can include an anti-CLEC9A recognition domain and an anti-tumor antigen recognition domain). In said embodiments, it is desirable to directly or indirectly recruit CLEC9A-expressing DCs but not functionally modulate CLEC9A activity - in contrast, in these embodiments, CLEC9A signaling is important for tumor reduction or elimination.

[0321] In various embodiments, the recognition domain of the chimeric protein of the present disclosure binds to XCR1 on a dendritic cell, for example, the recognition domain comprises, in some embodiments, all or a portion of XCL1, or a non-neutralizing anti-XCR1 factor.

[0322] In various embodiments, the recognition domain of the chimeric protein of the present disclosure binds to an immunomodulatory (e.g., immunostimulatory or immunosuppressive) antigen. In various embodiments, the immunomodulatory antigen is one or more of 4-1BB, 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, such immunostimulatory antigens are expressed on tumor cells. In various embodiments, the recognition domain of the chimeric protein of the present disclosure binds to such immunostimulatory antigens but does not modulate their function, thus allowing the recruitment of cells expressing the antigen without reducing or eliminating their potential tumor reduction or tumor elimination capabilities.

[0323] In various embodiments, the recognition domains of the chimeric proteins of the present disclosure may be in the form of a chimeric protein that includes two recognition domains with neutralizing activity, two recognition domains with non-neutralizing (e.g., binding) activity, or one recognition domain with neutralizing activity and one recognition domain with non-neutralizing (e.g., binding) activity.

[0324] In various embodiments, the multispecific chimeric protein has a targeting moiety that has a recognition domain that specifically binds to a target (e.g., antigen, receptor) that is a non-cellular structural moiety. In some embodiments, the antigen or receptor is not an integral component of an intact cell or cellular structure. In some embodiments, the antigen or receptor is an extracellular antigen or receptor. In some embodiments, the target is a non-proteinaceous non-cellular marker, including, but not limited to, nucleic acid, including DNA or RNA, e.g., DNA released from necrotic tumor cells, or extracellular deposits such as cholesterol.

[0325] In some embodiments, the target of interest (e.g., antigen, receptor) is part of the non-cellular components of the stroma or extracellular matrix (ECM) or their associated markers. As used herein, stroma refers to the connective supporting framework of a tissue or organ. The stroma may include a collection of cells, such as fibroblasts / myofibroblasts, glial cells, epithelial cells, adipocytes, immune cells, vascular cells, smooth muscle cells, and immune cells, with the extracellular matrix (ECM) and extracellular molecules. In various embodiments, the target of interest (e.g., antigen, receptor) is part of the non-cellular components of the stroma, such as the extracellular matrix and extracellular molecules. As used herein, ECM refers to the non-cellular components present in all tissues and organs. The ECM is composed of many biochemically distinct components, including but not limited to proteins, glycoproteins, proteoglycans, and polysaccharides. These components of the ECM are usually produced by adjacent cells and secreted into the ECM by exocytosis. When secreted, the ECM components often aggregate to form complex network structures of macromolecules. In various embodiments, the chimeric proteins of the invention include a targeting moiety that recognizes a target (e.g., an antigen or receptor or a nonproteinaceous molecule) located on any component of the ECM, including, but not limited to, proteoglycans, non-proteoglycan polysaccharides, fibers, and other ECM proteins or ECM non-proteins, such as polysaccharides and / or lipids, or ECM associated molecules (e.g., proteins or non-proteins, such as polysaccharides, nucleic acids, and / or lipids).

[0326] In some embodiments, the targeting moiety recognizes targets (e.g., antigens, receptors) on ECM proteoglycans. Proteoglycans are glycosylated proteins. The basic proteoglycan unit comprises one or more covalently attached glycosaminoglycan (GAG) chains with a core protein. Proteoglycans have a net negative charge that attracts positively charged sodium ions (Na+), thereby attracting water molecules via osmosis, keeping the ECM and resident cells hydrated. Proteoglycans may also help capture and store growth factors within the ECM. Examples of proteoglycans that may be targeted by the chimeric proteins of the present invention include, but are not limited to, heparan sulfate, chondroitin sulfate, and keratan sulfate. In certain embodiments, the targeting moiety recognizes targets (e.g., antigens, receptors) on non-proteoglycan polysaccharides such as hyaluronic acid.

[0327] 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 collagen or collagen fibers. Collagen is the most abundant protein in the ECM. Collagen exists in the ECM as a fibrous protein and provides structural support to resident cells. In one or more embodiments, the targeting moiety recognizes and binds to various types of collagen 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 (types VIII, X), basement membrane collagens (type IV), and / or collagens VI, VII, or XIII. Elastin fibers provide elasticity to tissues, allowing them to return to their original state after stretching when needed. In some embodiments, the targeting moiety recognizes one or more epitopes on elastin or elastin fibers.

[0328] In some embodiments, the targeting moiety recognizes one or more ECM proteins, including, but not limited to, tenascin, fibronectin, fibulin, laminin, or nidogen / entactin.

[0329] In certain embodiments, the targeting moiety recognizes and binds to tenascin. The tenascin (TN) family of glycoproteins includes at least four members: tenascin-C, tenascin-R, tenascin-X, and tenascin-W. The primary structure of tenascin proteins contains several common motifs arranged in the same consecutive sequence: an amino-terminal heptad repeat, an epidermal growth factor (EGF)-like repeat, a fibronectin type III domain repeat, and a carboxyl-terminal fibrinogen globular domain. Each protein member is associated with a typical diversity in the number and nature of the EGF-like and fibronectin type III repeats. There are also isoform variants, particularly for tenascin-C. More than 27 splice variants and / or isoforms of tenascin-C are known. In certain embodiments, the targeting moiety recognizes and binds to tenascin-CA1. Similarly, tenascin-R has various splice variants and isoforms. Tenascin-R usually exists as a dimer or trimer. Tenascin-X is the largest member of the tenascin family and is known to exist as a trimer. Tenascin-W exists as a trimer. In some embodiments, the targeting moiety recognizes one or more epitopes on the tenascin protein. In some embodiments, the targeting moiety recognizes monomeric and / or dimeric and / or trimeric and / or hexameric forms of the tenascin protein.

[0330] In some embodiments, the targeting moiety recognizes and binds fibronectin. Fibronectin is a glycoprotein that connects cells to collagen fibers in the ECM, allowing cells to migrate through the ECM. Upon binding to integrins, fibronectin unfolds 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 exemplary embodiments, the targeting moiety recognizes fibronectin extracellular domain A (EDA) or fibronectin extracellular domain B (EDB). Elevated levels of EDA are associated with 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 utilized 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 utilized to target the chimeric protein to tumor cells, including tumor neovasculature.

[0331] In some embodiments, the targeting moiety recognizes and binds to fibrin. Fibrin is another protein substance often found in the matrix network structure of ECM. Fibrin is formed by the action of thrombin protease on fibrinogen, which causes the polymerization of fibrin. In some embodiments, the targeting moiety recognizes one or more epitopes on fibrin. In some embodiments, the targeting moiety recognizes monomeric and polymeric forms of fibrin.

[0332] In certain embodiments, the targeting moiety recognizes and binds to laminin. Laminin is a major component of basement membranes, the protein network infrastructure for cells and organs. Laminin is a heterotrimeric protein that includes 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.

[0333] In certain embodiments, the targeting moiety recognizes and binds nidogen or entactin. Nidogen / entactin are a family of highly conserved sulfated glycoproteins. These glycoproteins constitute the main structural components of basement membranes and function to link 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.

[0334] 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 an embodiment, the antigen recognition domain recognizes one or more linear epitopes present on a protein. As used herein, a linear epitope refers to any continuous sequence of amino acids present on a protein. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on a protein. As used herein, a conformational epitope refers to one or more sections of amino acids (which may be discontinuous) that form a three-dimensional surface having a characteristic and / or shape and / or tertiary structure that can be recognized by the antigen recognition domain.

[0335] In various embodiments, the targeting moiety may bind to full length and / or mature forms and / or isoforms and / or splice variants and / or fragments and / or any other natural or synthetic analogs, variants or mutants of any of the targets (e.g., ECM proteins) described herein. In various embodiments, the targeting moiety may bind to any form of protein described herein, including monomers, dimers, trimers, tetramers, heterodimers, multimers and aggregates. In various embodiments, the targeting moiety may bind to post-translationally modified forms of proteins described herein, such as glycosylated and / or phosphorylated forms.

[0336] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an extracellular molecule, such as DNA. In some embodiments, the targeting moiety comprises an antigen recognition domain that recognizes DNA. In some embodiments, the DNA is released into the extracellular space from necrotic or apoptotic tumor cells or other diseased cells.

[0337] In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures associated with atherosclerotic plaque. Two types of atherosclerotic plaque are known: Fibro-lipidic (fibro-fatty) plaques are characterized by the accumulation of fat-containing cells beneath the intima of the artery. Beneath the endothelium is a fibrous cap that covers the atheromatous core of the plaque. The core contains adipocytes (macrophages and smooth muscle cells) with high tissue cholesterol and cholesterol ester content, fibrin, proteoglycans, collagen, elastin, and cellular debris. In advanced plaques, the central core of the plaque usually contains extracellular cholesterol deposits (released from dead cells), thereby forming an area containing cholesterol crystals with empty needle-like clefts. At the periphery of the plaque are relatively young foam cells and capillaries. Fibrous plaques are localized within the arterial wall, even beneath the intima, resulting in thickening and enlargement of the wall, and sometimes localized patchy plaques narrowing the lumen and causing some atrophy of the muscle layer. Fibrous plaques contain collagen fibers (eosinophilic), calcium deposits (hematoxylinophilic), and fat-containing 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 nucleic acid, such as DNA or RNA, released from dead cells.

[0338] 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 localized in amyloid plaques found in the brains of patients with Alzheimer's disease. For example, the targeting moiety may recognize and bind to amyloid beta peptide, a major component of amyloid plaques. In some embodiments, the targeting moiety recognizes and binds to one or more non-cellular structures localized 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 diseases, such as Lewy body dementia and inclusion body myositis.

[0339] Linkers and Functional Groups In various embodiments, the chimeric proteins of the present disclosure may include one or more functional groups, residues, or moieties. In various embodiments, one or more functional groups, residues, or moieties are attached to or genetically fused to any of the signal transduction or targeting moieties (e.g., SIRP1α) described herein. In some embodiments, such functional groups, residues, or moieties impart one or more desired properties or functions to the chimeric proteins of the present disclosure. Examples of such functional groups and methods for introducing them into the chimeric proteins of the present disclosure are known in the art, see, for example, Remington's Pharmaceutical Sciences, 16th Edition, Mack Publishing, Easton, PA (1980).

[0340] In various embodiments, the chimeric proteins of the present disclosure may be conjugated and / or fused to another agent to extend half-life or improve pharmacodynamic and pharmacokinetic properties. In some embodiments, the chimeric proteins may be fused or conjugated to one or more of PEG, XTEN (e.g., rPEG, etc.), polysialic acid (POLYXEN), albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, etc. In some embodiments, the chimeric proteins of the present disclosure may be fused or conjugated to an antibody or an antibody fragment, such as an Fc fragment. For example, the chimeric proteins may be fused to either the N-terminus or 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.

[0341] In some embodiments, the functional group, residue, or moiety comprises a suitable pharma- ceutically acceptable polymer, such as poly(ethylene glycol) (PEG) or a derivative thereof (such as methoxypoly(ethylene glycol), i.e., mPEG). In some embodiments, the attachment of a 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 PEGylation used in the art of antibodies and antibody fragments (including, but not limited to, single domain antibodies such as VHHs). See, for example, Chapman, Nat. Biotechnol., 54, pp. 531-545 (2002); Veronese and Harris, Adv. Drug Deliv. Rev., 54, pp. 453-456 (2003); Harris and Chess, Nat. Rev. Drug. Discov., 2 (2003), and WO 04 / 060965, the entire contents of which are incorporated herein by reference. Various reagents for PEGylation of proteins are also commercially available, for example from Nektar Therapeutics, USA. In some embodiments, site-specific PEGylation, particularly via cysteine ​​residues, is used (see, for example, Yang, et al., Protein Engineering, 16, 10, pp. 761-770 (2003), the entire contents of which are incorporated herein by reference). For example, for this purpose, PEG may be attached to a cysteine ​​residue that is naturally present in the chimeric protein of the present invention. In some embodiments, the chimeric protein of the present invention may be modified to appropriately introduce one or more cysteine ​​residues for PEG attachment using methods known in the art, or an amino acid sequence containing one or more cysteine ​​residues for PEG attachment may be fused to the amino and / or carboxy terminus of the chimeric protein of the present disclosure.

[0342] In some embodiments, the functional group, residue, or moiety comprises an N-linked or O-linked glycosylation. In some embodiments, the N-linked or O-linked glycosylation is introduced as part of a co-translational and / or post-translational modification.

[0343] In some embodiments, the functional group, residue, or moiety comprises one or more detectable labels or other signal-generating groups or moieties. Suitable labels and methods for attaching, using, and detecting such labels are known in the art and include fluorescent labels (such as fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, 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 salt, oxalate ester, dioxetane, or GFP and its analogs), radioisotopes, metals, metal chelates, or metal cations, or in vivo, in vitro, or in vivo detection. Other metals or metal cations that are particularly suitable for use in situ diagnosis and imaging include, but are not limited to, chromophores and enzymes such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triosephosphate isomerase, biotin avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase, and acetylcholinesterase. Other suitable labels include moieties detectable using nuclear magnetic resonance or electron spin resonance spectroscopy. Such labelled VHHs and polypeptides of the invention may be used, for example, for in vitro assays (including immunoassays known per se, such as ELISA, RIA, EIA and other "sandwich assays"), in vivo assays or in situ assays, as well as for in vivo diagnostic and imaging purposes depending on the choice of the particular label.

[0344] In some embodiments, the functional group, residue, or moiety comprises a tag attached or genetically fused to the chimeric protein of the present disclosure. In some embodiments, the chimeric protein of the present disclosure may comprise a single tag or may comprise multiple tags. The tag is, for example, a peptide, sugar, or DNA molecule that does not inhibit or interfere with the binding of the chimeric protein of the present disclosure to SIRP1α or any other antigen of interest, such as a tumor antigen. In various embodiments, the tag is at least about 3-5 amino acids in length, 5-8 amino acids in length, 8-12 amino acids in length, 12-15 amino acids in length, or 15-20 amino acids in length. Examples of tags are described, for example, in U.S. Patent Application Publication No. 2013 / 0058962. In some embodiments, the tag is an affinity tag, such as a glutathione-S-transferase (GST) tag and a histidine (His) tag. In certain embodiments, the chimeric protein of the present disclosure comprises a His tag.

[0345] In some embodiments, the functional group, residue, or moiety comprises a chelating group, for example, for chelating one of a metal or metal cation. For example, suitable chelating groups include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0346] In some embodiments, the functional group, residue, or moiety comprises a functional group that is part of a specific binding pair, such as a biotin-(strept)avidin binding pair. Such functional groups can be used to link the chimeric protein of the present invention to another protein, polypeptide, chemical compound that is bound to the other member of the binding pair, i.e., through the formation of a binding pair. For example, the chimeric protein of the present invention may be conjugated with biotin and linked to another protein, polypeptide, compound, or carrier that is bound to avidin or streptavidin. For example, such a complexed chimeric protein of the present disclosure may be used as a reporter, such as in a diagnostic system in which a detectable signal generating substance is bound to avidin or streptavidin. Such binding pairs may also be used for binding of the chimeric protein of the present disclosure to a carrier, including carriers suitable for pharmaceutical purposes. One non-limiting example is the liposomal formulation described in Cao and Suresh, Journal of Drug Targeting, 8, 4, pp. 257 (2000). Such binding pairs may also be used to link therapeutically active substances to the chimeric protein of the present disclosure.

[0347] In some embodiments, the chimeric proteins of the present disclosure optionally include one or more linkers. In some embodiments, the chimeric proteins of the present disclosure include a linker that connects the targeting moiety and the signaling agent. In some embodiments, the chimeric proteins of the present disclosure include a linker in the signaling agent (e.g., in the case of single-chain TNF, which includes two linkers to obtain a trimer).

[0348] In some embodiments, vectors are provided that encode the chimeric proteins of the present disclosure linked by any of the linkers described herein as a single nucleotide sequence and can be used to prepare such chimeric proteins.

[0349] In some embodiments, the length of the linker allows the targeting moiety and the signal transduction agent to efficiently bind to their receptors.For example, in some embodiments, the length of the linker allows the efficient binding of one of the targeting moieties and the signal transduction agent to a receptor on the same cell, and the efficient binding of the other binding moiety to another cell.Examples of cell combinations are described elsewhere herein.

[0350] In some embodiments, the length of the linker is at least equal to the smallest distance between the binding site of one of the targeting moieties to a receptor on the same cell and the binding site of the signal transduction agent. In some embodiments, the length of the linker is at least 2 times, or 3 times, or 4 times, or 5 times, or 10 times, or 20 times, or 25 times, or 50 times, or 100 times, or more than the smallest distance between the binding site of one of the targeting moieties to a receptor on the same cell and the binding site of the signal transduction agent.

[0351] As described herein, the length of the linker allows for efficient binding of one of the targeting moieties and the signaling agent to a receptor on the same cell, and the binding occurs sequentially, e.g., binding of the targeting moiety to the receptor precedes binding of the signaling agent to the receptor.

[0352] In some embodiments, two linkers are present in a single chimera, each linker connecting a signaling agent to a targeting moiety, In various embodiments, the linkers are of a length that allows for the formation of sites that are free of steric hindrance that would interfere with the regulation of either disease or effector cells.

[0353] The present invention contemplates the use of a variety of linker sequences. In various embodiments, the linker may be derived from a natural multidomain protein or may be an empirical linker, such as those 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 may 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 Eng., 13(5):309-312, the entire contents of which are incorporated herein by reference. In various embodiments, the linker may be functional. For example, without limitation, a linker may function to improve folding and / or stability, improve expression, improve pharmacokinetics, and / or improve biological activity of a chimeric protein of the present disclosure.

[0354] In some embodiments, the linker is a polypeptide. In some embodiments, the length of the linker is less than about 100 amino acids. For example, the length of the linker may be less than about 100 amino acids, less than about 95 amino acids, less than about 90 amino acids, less than about 85 amino acids, less than about 80 amino acids, less than about 75 amino acids, less than about 70 amino acids, less than about 65 amino acids, less than about 60 amino acids, less than about 55 amino acids, less than about 50 amino acids, less than about 45 amino acids, less than about 40 amino acids, less than about 35 amino acids, less than about 30 amino acids, less than about 25 amino acids, less than about 20 amino acids, less than about 19 amino acids, less than about 18 amino acids, less than about 17 amino acids, less than about 16 amino acids, less than about 15 amino acids, less than about 14 amino acids, less than about 13 amino acids, less than about 12 amino acids, less than about 11 amino acids, less than about 10 amino acids, less than about 9 amino acids, less than about 8 amino acids, less than about 7 amino acids, less than about 6 amino acids, less than about 5 amino acids, less than about 4 amino acids, less than about 3 amino acids, or less than about 2 amino acids. In some embodiments, the linker is a polypeptide. In some embodiments, the length of the linker is greater than about 100 amino acids. For example, the length of the linker may be greater than about 100 amino acids, greater than about 95 amino acids, greater than about 90 amino acids, greater than about 85 amino acids, greater than about 80 amino acids, greater than about 75 amino acids, greater than about 70 amino acids, greater than about 65 amino acids, greater than about 60 amino acids, greater than about 55 amino acids, greater than about 50 amino acids, greater than about 45 amino acids, greater than about 40 amino acids, greater than about 35 amino acids, greater than about 30 amino acids, greater than about 25 amino acids, greater than about 20 amino acids, greater than about 19 amino acids, greater than about 18 amino acids, greater than about 17 amino acids, greater than about 16 amino acids, greater than about 15 amino acids, greater than about 14 amino acids, greater than about 13 amino acids, greater than about 12 amino acids, greater than about 11 amino acids, greater than about 10 amino acids, greater than about 9 amino acids, greater than about 8 amino acids, greater than about 7 amino acids, greater than about 6 amino acids, greater than about 5 amino acids, greater than about 4 amino acids, greater than about 3 amino acids, or greater than about 2 amino acids. In some embodiments, the linker is flexible. In other embodiments, the linker lacks flexibility.

[0355] In some embodiments, a linker interconnects two targeting moieties, the linker being short in length, and a linker connects a targeting moiety and a signaling agent, the linker being longer than the linker connecting the two targeting moieties. For example, the difference in amino acid length between a linker connecting two targeting moieties and a linker connecting a targeting moiety and a signaling agent can be about 100 amino acids, about 95 amino acids, about 90 amino acids, about 85 amino acids, about 80 amino acids, about 75 amino acids, about 70 amino acids, about 65 amino acids, about 60 amino acids, about 55 amino acids, about 50 amino acids, about 45 amino acids, about 40 amino acids, about 35 amino acids, about 30 amino acids, about 25 amino acids, about 20 amino acids, about 19 amino acids, about 18 amino acids, about 17 amino acids, about 16 amino acids, about 15 amino acids, about 14 amino acids, about 13 amino acids, about 12 amino acids, about 11 amino acids, about 10 amino acids, about 9 amino acids, about 8 amino acids, about 7 amino acids, about 6 amino acids, about 5 amino acids, about 4 amino acids, about 3 amino acids, or about 2 amino acids.

[0356] In various embodiments, the linker is substantially composed of glycine and serine residues (e.g., about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 97% glycine and serine). For example, in some embodiments, the linker is (Gly4Ser) n where n is from about 1 to about 8, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NO: 247 to SEQ ID NO: 254). In one embodiment, the linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 255). Other exemplary linkers include LE, GGGGS (SEQ ID NO: 247), (GGGGS) n (n = 1 to 4) (SEQ ID NO: 247 to SEQ ID NO: 250), (Gly)8 (SEQ ID NO: 256), (Gly)6 (SEQ ID NO: 257), (EAAAK) n (n = 1 to 3) (SEQ ID NO: 258 to SEQ ID NO: 260), A(EAAAK) nA (n = 2 to 5) (SEQ ID NO: 261 to SEQ ID NO: 264), AEAAAKEAAAKA (SEQ ID NO: 261), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO: 265), PAPAP (SEQ ID NO: 266), KESGSVSSEQLAQFRSLD (SEQ ID NO: 267), EGKSSGSGSESKST (SEQ ID NO: 268), GSAGSAAGSGEF (SEQ ID NO: 269), and X represents any amino acid, for example, Ala, Lys, or Glu (XP) n In various embodiments, the linker is GGS.

[0357] 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), GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 273), and a linker consisting of G, S, and E randomly spaced every 4 amino acid interval.

[0358] In some embodiments, the linker is a hinge region of an antibody (e.g., of the IgG, IgA, IgD, and IgE subclasses, e.g., IgG1, IgG2, IgG3, and IgG4, as well as IgA1 and IgA2). In various embodiments, the linker is a hinge region of an antibody (e.g., of the IgG, IgA, IgD, and IgE subclasses, e.g., IgG1, IgG2, IgG3, and IgG4, as well as 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 portions to move freely in space. In contrast to constant regions, hinge domains are structurally diverse and differ in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region differs among IgG subclasses. The hinge region of IgG1 encompasses amino acids 216-231 and is flexible enough that the Fab fragment can rotate around its axis of symmetry and move within a sphere centered on the first of the two inter-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1, containing 12 amino acid residues and 4 disulfide bridges. The hinge region of IgG2 lacks glycine residues, is relatively short, and contains a rigid polyproline double helix stabilized by an additional inter-heavy chain disulfide bond. These properties limit the mobility of the IgG2 molecule. IgG3 differs from the other subclasses by a unique long hinge region (approximately four times longer than the IgG1 hinge) that contains 62 amino acids (including 21 prolines and 11 cysteines) and forms a non-mobile polyproline double helix. In IgG3, the Fab fragment is relatively far from the Fc fragment, which allows the molecule to be more mobile. The long hinge of IgG3 also accounts for its large molecular weight compared to other subclasses. The hinge region of IgG4 is shorter than that of IgG1, and its flexibility is intermediate between that of IgG1 and IgG2. The flexibility of the hinge region is reported to decrease in the order IgG3>IgG1>IgG4>IgG2.

[0359] Crystallographic studies have shown that the immunoglobulin hinge region can be functionally further subdivided into three regions: the upper hinge region, the core region, and the lower hinge region. See Shin, et al., 1992, Immunological Reviews, 130:87. The upper hinge region is H1 It includes the amino acids from the carboxy terminus of C to the first residue in the hinge that restricts movement, typically the first cysteine ​​residue that forms the interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the interchain disulfide bridges, while the lower hinge region contains the C H2 linked to the amino terminus of the domain and H2 The core hinge region of wild-type human IgG1 comprises the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 274), which upon dimerization by disulfide bond formation results in a cyclic octapeptide that is believed to act as a pivot point, thereby providing flexibility. In various embodiments, the linkers of the present disclosure comprise one, two, or three of the upper hinge region, core region, and lower hinge region of any antibody (e.g., of IgG, IgA, IgD, and IgE, including subclasses such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2). The hinge region may also contain one or more glycosylation sites, which include a number of structurally distinct types of sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within a 17 amino acid segment of the hinge region that confer resistance of the hinge region polypeptide to intestinal proteases, a property believed to be advantageous for secretory immunoglobulins. In various embodiments, the linker of the invention contains one or more glycosylation sites. In various embodiments, the linker is the hinge-CH2-CH3 domain of a human IgG4 antibody.

[0360] Optionally, the chimeric protein of the present disclosure is H 2 domain and C HIt can be linked to an antibody Fc region comprising one or both of the three domains, and can be linked to a hinge region, if desired. For example, such a polypeptide can be prepared using a vector encoding the chimeric protein of the present disclosure linked to an Fc region as a single nucleotide sequence.

[0361] In some embodiments, the linker is a synthetic linker, such as PEG.

[0362] In various embodiments, the linker may be functional. For example, but not limited to, the linker may function to improve the folding and / or stability, improve expression, improve pharmacokinetics, and / or improve biological activity of the chimeric protein of the present disclosure. In another example, the linker may function to target the chimeric protein to a particular cell type or a particular location.

[0363] Engineering and creation of chimeric proteins In various embodiments, the chimeric protein of the present disclosure comprises a targeting moiety that is a VHH (e.g., SIRP1α). In various embodiments, the VHH is not limited to a particular biological origin or to a particular method of preparation. For example, a VHH is typically: (1) a VHH of a natural heavy chain antibody; H (2) by isolating the natural V H (3) by expressing a nucleotide sequence encoding the H domain of a naturally occurring V H "Humanizing" the H domain, or such a humanized V H(4) by "camelizing" a naturally occurring VH domain from any animal species, such as a mammalian species, e.g. from human, or by expressing a nucleic acid encoding such a camelized VH domain; (5) by "camelizing" a "domain antibody" or "Dab" as described in the art, or by expressing a nucleic acid encoding such a camelized VH domain; (6) by using synthetic or semi-synthetic methods for preparing proteins, polypeptides, or other amino acid sequences known in the art; (7) by preparing a VHH-encoding nucleic acid using nucleic acid synthesis methods known in the art and then expressing the resulting nucleic acid; and / or (8) by any combination of one or more of the foregoing.

[0364] In one embodiment, the chimeric protein comprises the V and VD of a natural heavy chain antibody directed against human SIRP1α. H In some embodiments, such a VHH comprises a VHH corresponding to the H domain. H The H sequence is typically determined by suitably immunizing an animal of the camelid species with a SIRP1α molecule (i.e. immunizing so as to generate an immune response and / or heavy chain antibodies directed against SIRP1α), obtaining a suitable biological sample from the camelid (e.g. a blood sample or any B cell sample), and isolating from the sample the V H sequence against SIRP1α using any suitable known method. H In some embodiments, the naturally occurring VH sequence for SIRP1α can be generated or obtained by generating H The H domain is a member of the camelid V HA naive library of H sequences can be obtained, for example, by screening such libraries with SIRP1α, or at least a portion, fragment, antigenic determinant, or epitope thereof, using one or more screening methods known in the art. Such libraries and methods are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020, and WO 03 / 035694, the contents of which are incorporated herein by reference in their entirety. In some embodiments, a naive V sequence can be obtained by screening such libraries with SIRP1α, or at least a portion, fragment, antigenic determinant, or epitope thereof, using one or more screening methods known in the art. Such libraries and methods are described, for example, in WO 99 / 37681, WO 01 / 90190, WO 03 / 025020, and WO 03 / 035694, the contents of which are incorporated herein by reference in their entirety. H Improved synthetic or semi-synthetic libraries derived from the H library can be used, e.g., by methods such as random mutagenesis and / or CDR shuffling, as described in WO 00 / 43507, the entire contents of which are incorporated herein by reference. H V obtained from the H library H In some embodiments, a VH library directed against SIRP1α is available. H Another method for obtaining the H sequence involves appropriately immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., immunizing to generate an immune response and / or heavy chain antibodies directed against SIRP1α), obtaining a suitable biological sample from the transgenic mammal (e.g., a blood sample or any B cell sample), and isolating from the sample the V sequence against SIRP1α using any suitable known method. H For example, for this purpose, the heavy chain antibody expressing mice and other methods and techniques described in WO 02 / 085945 and WO 04 / 049794 (the entire contents of which are incorporated herein by reference) can be used.

[0365] In one embodiment, the chimeric protein is a VHH that is "humanized", i.e. HThe present invention also includes VHHs that have been humanized by replacing one or more amino acid residues in the amino acid sequence of the H sequence (particularly in the framework sequences) with one or more of the amino acid residues present at the corresponding positions in the VH domain from a conventional four-chain antibody of human origin. This can be done using humanization methods known in the art. In some embodiments, possible humanizing substitutions or combinations of humanizing substitutions can be determined by methods known in the art, for example, by comparison between the sequence of the VHH and the sequence of a naturally occurring human VH domain. In some embodiments, the humanizing substitutions are selected such that the resulting humanized VHH still retains advantageous functional properties. Typically, the VHHs of the present invention are humanized to a degree similar to that of the corresponding naturally occurring VH domain as a result of humanization. H In various embodiments, the humanized VHH of the present invention can be obtained by any suitable method known in the art, and thus can be obtained by using a natural VHH as a starting material. H It is not strictly limited to polypeptides obtained using a polypeptide containing an H domain.

[0366] In one embodiment, the chimeric protein is a VHH that has been "camelized", i.e., one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional four-chain antibody have been replaced with those of a VH domain from a camelid heavy chain antibody. HThe VHHs include those that have been camelized by replacing one or more of the amino acid residues present at the corresponding positions in the H domain. In some embodiments, such "camelization" substitutions are inserted at amino acid positions forming and / or present at the VH / VL interface and / or at the so-called camelid hallmark residues (see, for example, WO 94 / 04678, the entire contents of which are incorporated herein by reference). In some embodiments, the VH sequence used as starting material or starting point for generating or designing a camelized VHH is an animal-derived VH sequence, e.g. a human VH sequence, such as a VH3 sequence. In various embodiments, the camelized VHHs can be obtained by any suitable method known in the art (i.e., as indicated in (1) to (8) above), and are not strictly limited to polypeptides obtained using a polypeptide comprising a natural VH domain as starting material.

[0367] In various embodiments, "humanized" and "camelized" refer to naturally occurring V H This can be done by providing a nucleotide sequence encoding the H or VH domain and then exchanging one or more codons in said nucleotide sequence, by methods known in the art, such that the new nucleotide sequence encodes a "humanized" VHH or a "camelized" VHH, respectively. This nucleic acid can then be expressed by methods known in the art to provide the desired VHH of the invention. Alternatively, the naturally occurring VHH can be expressed by methods known in the art to provide the desired VHH of the invention. H Based on the amino acid sequence of the H domain or the natural VH domain, respectively, the amino acid sequence of the desired humanized VHH or camelized VHH of the present invention can be designed and then synthesized de novo using peptide synthesis methods known in the art. HBased on the amino acid or nucleotide sequence of the H domain or the native VH domain, respectively, a nucleotide sequence encoding the desired humanized or camelized VHH can then be designed and then synthesized de novo using nucleic acid synthesis methods known in the art, and the resulting nucleic acid can then be expressed by methods known in the art to provide the desired VHH of the invention. H Other suitable methods and techniques for obtaining the VHHs of the invention and / or nucleic acids encoding them starting from a VH sequence are known in the art and can be used to, for example, obtain one or more parts of one or more naturally occurring VH sequences (such as one or more FR sequences and / or CDR sequences), one or more naturally occurring VH sequences, H It may involve appropriately combining one or more parts of the H sequence (such as one or more FR sequences or CDR sequences) and / or one or more synthetic or semi-synthetic sequences to provide a VHH of the present invention or a nucleotide sequence or nucleic acid encoding same.

[0368] Methods for producing the chimeric proteins of the present invention are described herein. For example, DNA sequences encoding the chimeric proteins of the present invention (e.g., DNA sequences encoding modified signal transduction agents, targeting moieties, and linkers) can be chemically synthesized using methods known in the art. The synthetic DNA sequences can be ligated to other appropriate nucleotide sequences, including expression control sequences, to generate gene expression constructs encoding the desired chimeric proteins. Thus, in various embodiments, the present invention provides isolated nucleic acids comprising nucleotide sequences encoding the chimeric proteins of the present invention.

[0369] The nucleic acid encoding the chimeric protein of the invention can be incorporated (ligated) into an expression vector, which can be introduced into an expression host cell by transfection, transformation, or transduction. For example, the nucleic acid encoding the chimeric protein of the invention can be introduced into a host cell by retroviral transduction. Examples of host cells include E. coli cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and myeloma cells. The transformed host cells can be cultured under conditions that allow the host cells to express the gene encoding the chimeric protein of the invention. Thus, in various embodiments, the invention provides an expression vector comprising a nucleic acid encoding the chimeric protein of the invention. In various embodiments, the invention further provides a host cell comprising such an expression vector.

[0370] Specific expression and purification conditions will vary depending on the expression system used.For example, if the gene is expressed in E. coli, the gene is first cloned into an expression vector by placing the modified gene downstream of a suitable bacterial promoter such as Trp or Tac and a prokaryotic signal sequence.In another example, if the modified gene is expressed in a eukaryotic host cell such as a CHO cell, the modified gene is first inserted into an expression vector that includes, for example, a suitable eukaryotic promoter, secretion signal, enhancer, and various introns.The gene construct can be introduced into the host cell using transfection, transformation, or transduction methods.

[0371] The chimeric proteins of the present invention can be produced by culturing a host cell transfected with an expression vector encoding the protein under conditions that allow expression of the chimeric protein. After expression, the protein can be recovered and purified by methods well known in the art, for example, using affinity tags such as glutathione-S-transferase (GST) tags and histidine tags, or by chromatography.

[0372] Thus, in various embodiments, the invention provides a nucleic acid encoding a chimeric protein of the invention.In various embodiments, the invention provides a host cell comprising a nucleic acid encoding a chimeric protein of the invention.

[0373] In various embodiments, the SIRP1α targeting moiety or chimeric protein comprising a SIRP1α targeting moiety of the present disclosure may be expressed in vivo, for example, in a patient. For example, in various embodiments, the SIRP1α targeting moiety or chimeric protein comprising a SIRP1α targeting moiety of the present disclosure may be administered in the form of a nucleic acid encoding the SIRP1α targeting moiety or chimeric protein comprising a SIRP1α targeting moiety of the present disclosure. In various embodiments, the nucleic acid is DNA or RNA. In some embodiments, the SIRP1α targeting moiety or chimeric protein comprising a SIRP1α targeting moiety of the present disclosure is encoded by a modified mRNA, i.e., an mRNA comprising one or more modified nucleotides. In some embodiments, the modified mRNA comprises one or more of the modifications found in U.S. 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, s2U, Ψ, and 2'-O-methyl-U. In some embodiments, the invention relates to the administration of modified mRNA encoding one or more of the chimeric proteins of the present disclosure. In some embodiments, the invention relates to a gene therapy vector comprising the modified mRNA. In some embodiments, the invention relates to a gene therapy method comprising the administration of the modified mRNA. In various embodiments, the nucleic acid is in the form of an oncolytic virus, such as an adenovirus, a reovirus, a measles virus, a herpes simplex virus, a Newcastle disease virus, or a vaccinia virus.

[0374] Pharmaceutically acceptable salts and excipients The chimeric proteins described herein may have sufficiently basic functional groups capable of reacting with inorganic or organic acids, or carboxyl groups capable of reacting with inorganic or organic bases, to form pharma- ceutically acceptable salts. As is well known in the art, pharma- ceutically acceptable acid addition salts are formed from pharma- ceutically acceptable acids. Such salts include, for example, the pharma- ceutically acceptable salts described in Journal of Pharmaceutical Science, 66, pp. 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use., edited by P. H. Stahl and C. G. Wermuth, Verlag, Zurich (Switzerland) 2002, the entire contents of which are incorporated herein by reference.

[0375] Pharmaceutically acceptable salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, superphosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, 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, xylenesulfonate, and tartrate salts.

[0376] The term "pharmaceutically acceptable salt" also refers to base salts of the compositions of the present invention having an acidic functional group, such as a carboxylic acid functional group. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia, and organic amines such as unsubstituted or hydroxy-substituted mono-, di-, or tri-alkylamines, 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-(hydroxyl-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.

[0377] In some embodiments, the compositions described herein are in the form of a pharma- ceutically acceptable salt.

[0378] Pharmaceutical Compositions and Formulations In various embodiments, the present invention relates to pharmaceutical compositions comprising the chimeric protein described herein and a pharma- ceutically acceptable carrier or excipient. Any of the pharmaceutical compositions described herein can be administered to a subject as a component of a composition that includes a pharma- ceutically acceptable carrier or vehicle. Such compositions may include a suitable amount of a pharma- ceutically acceptable excipient to make the composition in a form for proper administration.

[0379] In various embodiments, the pharmaceutical excipients may be liquids such as water and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. These pharmaceutical excipients may be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary agents, stabilizers, thickening agents, lubricants, and coloring agents may be used. In some embodiments, the pharma- ceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any of the agents described herein are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid excipients, particularly for injectables. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. Any of the agents described herein may contain small amounts of wetting or emulsifying agents, or pH buffering agents. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences, pp.1447-1676 (edited by Alfonso R. Gennaro, 19th Edition, 1995), which is incorporated herein by reference.

[0380] The present invention includes the pharmaceutical compositions (and / or additional therapeutic agents) described herein in a variety of dosage forms. Any of the pharmaceutical compositions (and / or additional therapeutic agents) of the present invention described herein may be in the form of a liquid, suspension, emulsion, drops, tablets, pills, pellets, capsules, liquid-containing capsules, gelatin capsules, powders, sustained release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powders, frozen suspensions, dry powders, 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 the form of a tablet. In yet another embodiment, the pharmaceutical composition is formulated in the form of a soft gel capsule. In other embodiments, the pharmaceutical composition is formulated in the form of a gelatin capsule. In yet another embodiment, the pharmaceutical composition is formulated as a liquid.

[0381] The pharmaceutical compositions (and / or other agents) of the present invention may also contain solubilizing agents, if necessary. Additionally, the agents may be delivered by suitable vehicles or delivery devices known in the art. The combination therapies outlined herein may be co-delivered in a single delivery vehicle or delivery device.

[0382] The formulations of the present invention, including the pharmaceutical composition (and / or other agents) of the present invention, may conveniently be provided 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 into association the therapeutic agent with the carrier, which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with liquid carriers, finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired formulation (e.g., by wet or dry granulation, powder blending, or the like, followed by tableting using conventional methods known in the art).

[0383] In various embodiments, any of the pharmaceutical compositions (and / or other agents) described herein are routinely formulated into compositions compatible with the methods of administration described herein.

[0384] Routes of administration include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, by inhalation, or topical. Administration may be local or systemic. In some embodiments, administration is accomplished orally. In other embodiments, administration is by parenteral injection. The method of administration may be at the discretion of the physician and will depend in part on the site of the disease. In most cases, any of the agents described herein will be released into the bloodstream when administered.

[0385] In some embodiments, the chimeric proteins described herein are routinely formulated as compositions adapted for oral administration. Compositions for oral delivery may be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs. Orally administered compositions may contain one or more agents to provide a pharma- ceutically palatable preparation, such as sweeteners, such as fructose, aspartame, or saccharin; flavorings, such as peppermint, wintergreen oil, or cherry; coloring agents, and preservatives. Additionally, 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 an extended period of time. Selectively permeable membranes surrounding the osmotically active portion of any of the chimeric proteins described herein are also suitable for orally administered compositions. In these latter platforms, the fluid in the environment surrounding the capsule is absorbed by the driving compound, which swells and pushes the agent or agent composition through a hole. These delivery platforms can produce an essentially zero order delivery profile, as opposed to the spiked profiles of immediate release formulations. Time-delay materials such as glycerol monostearate or glycerol stearate can also be useful. Oral compositions can include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. In some embodiments, the excipients are of pharmaceutical grade. Suspensions can contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, and the like, and mixtures thereof, in addition to the active compound.

[0386] Suitable dosage forms for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous, and intraarticular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. These dosage forms may be prepared in the form of a sterile solid composition (e.g., a lyophilized composition) that can be dissolved or suspended in a sterile injectable medium immediately before use. The sterile solid composition may, for example, contain a suspending agent or dispersing agent known in the art. Suitable formulation components for parenteral administration include a sterile diluent such as water for injection, physiological saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates, or phosphates; and osmolality regulators such as sodium chloride or glucose.

[0387] Suitable carriers for intravenous administration include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The carrier must be stable under the conditions of manufacture and storage and must be preserved against microorganisms. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0388] The compositions provided herein, alone or in combination with other suitable components, can be formulated into aerosol formulations (i.e., "nebulized") to be administered via inhalation. The aerosol formulations can be mixed into pressurized propellants, such as dichlorodifluoromethane, propane, and nitrogen.

[0389] Any of the pharmaceutical compositions (and / or other agents) of the present invention described herein can be administered by controlled or sustained release means or delivery devices well known to those skilled in the art, including, but not limited to, those described in U.S. 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 for achieving controlled or sustained release of one or more active ingredients, for example, using hydropropyl cellulose, hydropropyl methyl cellulose, polyvinylpyrrolidone, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof in various ratios to achieve desired release profiles.Suitable controlled or sustained release formulations known to those skilled in the art, including those described herein, can be easily selected for use with the active ingredients of the agents described herein.Thus, the present invention provides single unit dosage forms suitable for oral administration, for example, but not limited to, tablets, capsules, gel caps, and caplets adapted for controlled or sustained release.

[0390] Controlled or sustained release of an active ingredient can be stimulated by various conditions including, but not limited to, changes in pH, temperature changes, stimulation with light of appropriate wavelengths, enzyme concentration or availability, water concentration or availability, or other physiological conditions or compounds.

[0391] In another embodiment, a controlled release system can be placed in proximity to the target area to be treated, thus requiring only a fraction of the systemic dose. (See, e.g., Goodson, in Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138 (1984)). Other controlled release systems discussed in the review by Langer, 1990, Science, 249:1527-1533 may also be used.

[0392] Pharmaceutical preparations 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 either prior to or after lyophilization and reconstitution.

[0393] Administration and Dosage It is understood that the actual dose of the chimeric protein of the present disclosure administered according to the present invention will vary depending on the specific dosage form and method of administration. Those skilled in the art can take into account numerous factors that may modify the action of the chimeric protein of the present disclosure (e.g., body weight, sex, diet, administration time, administration route, excretion rate, condition of the subject, drug combinations, genetic disposition, and reaction sensitivity). Administration can be performed by continuous administration or one or more divided administrations within the maximum tolerated dose. Those skilled in the art can use conventional dose administration tests to ascertain the optimal administration rate for a given condition.

[0394] In some embodiments, a suitable dosage of a chimeric protein of the present disclosure is within the range of about 0.01 mg to about 10 g per kg of subject body weight, about 0.01 mg to about 1 g per kg of subject body weight, about 0.01 mg to about 100 mg per kg of subject body weight, about 0.01 mg to about 10 mg per kg of subject body weight, e.g., 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 10 ... , 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 1.1 mg, about 1.2 mg, about 1.3 mg, about 1.4 mg, about 1.5 mg, about 1.6 mg, about 1.7 mg, about 1.8 mg, 1.9 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 100 mg, about 1 g, and about 10 g, including all values ​​and ranges between these upper and lower values.

[0395] Individual doses of the chimeric protein are, for example, about 0.01 mg to about 100 g, about 0.01 mg to about 75 g, about 0.01 mg to about 50 g, about 0.01 mg to about 25 g, about 0.01 mg to about 10 g, about 0.01 mg to about 7.5 g, about 0.01 mg to about 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 The active ingredient can be administered as a unit dosage form (e.g., a tablet or capsule) containing about 0.1 mg to about 70 mg, about 0.1 mg to about 60 mg, about 0.1 mg to about 50 mg, or about 0.1 mg to about 40 mg of active ingredient per unit dosage form, about 0.1 mg to about 30 mg, about 0.1 mg to about 20 mg, about 0.1 mg to about 10 mg, about 0.1 mg to about 5 mg, about 0.1 mg to about 3 mg, about 0.1 mg to about 1 mg, or about 5 mg to about 80 mg per unit dosage form. For example, the 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, The amount may be 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 5 g, about 10 g, about 25 g, about 50 g, about 75 g, about 100 g, including all values ​​and ranges between these upper and lower values.

[0396] In certain embodiments, the chimeric protein is administered in a daily dose of about 0.01 mg to about 100 g, a daily dose of about 0.01 mg to about 75 g, a daily dose of about 0.01 mg to about 50 g, a daily dose of about 0.01 mg to about 25 g, an amount of about 0.01 mg to about 10 g, a daily dose of about 0.01 mg to about 7.5 g, an amount of about 0.01 mg to about 5 g, or an amount of about 0.01 mg daily dose of about 0.01 mg to about 2.5 g, daily dose of about 0.01 mg to about 1 g, daily dose of about 0.01 mg to about 100 mg, daily dose of about 0.1 mg to about 100 mg, daily dose of about 0.1 mg to about 95 mg, daily dose of about 0.1 mg to about 90 mg, daily dose of about 0.1 mg to about 85 mg, daily dose of about 0.1 mg to about 80 mg, daily dose of about 0.1 mg to about 75 mg daily dose, about 0.1 mg to about 70 mg daily dose, about 0.1 mg to about 65 mg daily dose, about 0.1 mg to about 60 mg daily dose, about 0.1 mg to about 55 mg daily dose, about 0.1 mg to about 50 mg daily dose, about 0.1 mg to about 45 mg daily dose, about 0.1 mg to about 40 mg daily dose, about 0.1 mg to about 35 mg daily dose, about 0.1 mg to about 3 The drug is administered in a daily dose of about 0.0 mg, a daily dose of about 0.1 mg to about 25 mg, a daily dose of about 0.1 mg to about 20 mg, a daily dose of about 0.1 mg to about 15 mg, a daily dose of about 0.1 mg to about 10 mg, a daily dose of about 0.1 mg to about 5 mg, a daily dose of about 0.1 mg to about 3 mg, a daily dose of about 0.1 mg to about 1 mg, or a daily dose of about 5 mg to about 80 mg.In various embodiments, the chimeric protein is present at 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 The compound is administered in a daily dose of 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 5 g, about 7.5 g, about 10 g, about 25 g, about 50 g, about 75 g, about 100 g, including all values ​​and ranges between these upper and lower values.

[0397] According to certain embodiments of the present invention, a pharmaceutical composition comprising a chimeric protein of the present disclosure may be administered, for example, more than once a day (e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 times a day), about once a day, about once every 2 days, about once every 3 days, about once a week, about once every 2 weeks, about once a month, about once every 2 months, about once every 3 months, about once every 6 months, or about once a year.

[0398] Combination and add-on therapies In various embodiments, the pharmaceutical compositions of the invention are co-administered with an additional therapeutic agent. The co-administration may be simultaneous or sequential.

[0399] In some embodiments, the additional therapeutic agent and the chimeric protein of the present disclosure are administered to the subject simultaneously. The term "simultaneous" as used herein means that the additional therapeutic agent and the chimeric protein of the present disclosure are administered within about 60 minutes, e.g., within about 30 minutes, within about 20 minutes, within about 10 minutes, within about 5 minutes, or within about 1 minute. The administration of the additional therapeutic agent and the chimeric protein of the present disclosure may be by simultaneous administration of a single formulation (e.g., a formulation containing the additional therapeutic agent and the chimeric protein of the present disclosure) or by simultaneous administration of separate formulations (e.g., a first formulation containing the additional therapeutic agent and a second formulation containing the chimeric protein of the present disclosure).

[0400] Co-administration does not require that the therapeutic agents be administered simultaneously, provided that the timing of administration of the therapeutic agents is such that the pharmacological activity of the additional therapeutic agent and the chimeric protein of the present disclosure overlap in time, thereby exerting a combined therapeutic effect. For example, the additional therapeutic agent and the chimeric protein of the present disclosure can be administered sequentially. The term "sequentially," as used herein, means that the additional therapeutic agent and the chimeric protein of the present disclosure are administered about 60 minutes or more apart. For example, the time between the sequential administration of the additional therapeutic agent and the chimeric protein of the present disclosure can be about 60 minutes, about 2 hours, about 5 hours, about 10 hours, about 1 day, about 2 days, about 3 days, about 1 week, about 2 weeks, or about 1 month or more. The optimal administration time depends on the metabolic rate, excretion rate, and / or pharmacodynamic activity of the additional therapeutic agent and the chimeric protein of the present disclosure administered. Either the additional therapeutic agent or the chimeric protein cells of the present disclosure can be administered first.

[0401] Co-administration also does not require that the therapeutic agents be administered to a subject by the same route of administration. Rather, each therapeutic agent can be administered by any suitable route, e.g., parenterally or non-parenterally.

[0402] In some embodiments, the chimeric proteins described herein act synergistically when co-administered with another therapeutic agent, in which the chimeric proteins of the present disclosure and the additional therapeutic agent may be administered at lower doses than those used when those agents are used in conjunction with monotherapy.

[0403] In some embodiments, the present invention relates to a chemotherapeutic agent as an additional therapeutic agent, for example and without limitation, such a combination of a chimeric protein of the present disclosure and a chemotherapeutic agent is used to treat cancer, as described anywhere herein. Exemplary chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN (cyclophosphamide); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (e.g., , cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin A; spongistatins; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novoembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gamma II and calicheamicin omega II (e.g., Agnew.Chem Intl. Ed. Engl., 33:183-186 (1994); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycin, anramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN (doxorubicin) (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, and deoxydoxorubicin). mitomycins such as mitomycin C; mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); denopterin, metoprolol, methadone ... Folic acid analogues such as thotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folinic acid Which folic acid supplements;aceglatone;aldophosphamide glycosides;aminolevulinic acid;eniluracil;amsacrine;bestrabulil;bisantrene;edatrexate;demecolcine;diaziquone;elformithine;elliptinium acetate;epothilone;etoglucide;gallium nitrate;hydroxyurea;lentinan;lonidamine;maytansinoids such as maytansine and ansamitocins;mitoguazone;mitoxantrone;mopidanmol;nitraerine;Pentostatin; Phenamet; Pirarubicin; Rosoxantrone; Podophyllic acid; 2-Ethylhydrazide; Procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, OR); Razoxane; Rhizoxin; Sizofiran; Spirogermanium; Tenuazonic acid; Triazicon; 2,2',2"-Trichlorotriethylamine; Trichothecenes (e.g., T-2 toxin, verracurin A, roridin A, and anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., Taxol-paclitaxel (Bristol-Myers Squibb oncology, Princeton, NJ), cremophor-free ABRAXANE (an albumin-loaded nanoparticle formulation of paclitaxel) (American Pharmaceutical Partners, Schaumburg, IL), and taxotere docetaxel (Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopropane Thelin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11) (including treatment regimens consisting of irinotecan with 5-FU and leucovorin); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); Tykerb (lapatinib);These include, but are not limited to, PKC-α inhibitors, Raf inhibitors, H-Ras inhibitors, EGFR inhibitors (e.g., Terceva (erlotinib)), and VEGF-A inhibitors that reduce cell proliferation, as well as pharma- ceutically acceptable salts, acids, or derivatives of any of the above. In addition, the method of treatment may fur...

Claims

1. (a) a targeting moiety comprising a recognition domain comprising a recombinant heavy chain antibody (VHH) that recognizes and binds to signal regulatory protein alpha-1 (SIRP1α); and (b) an altered signal transduction agent comprising human interferon alpha 2 (IFNα2) comprising an amino acid sequence having at least 98% identity to a sequence selected from SEQ ID NO: 46 or SEQ ID NO: 47, the altered signal transduction agent comprising human interferon alpha 2 (IFNα2) having an R149A mutation that results in reduced affinity or activity for the IFN-alpha receptor compared to wild-type IFNα2; Including, A chimeric protein, wherein the targeting moiety and the modified interferon alpha 2 signaling agent are linked by one or more linkers.

2. The chimeric protein of claim 1 further comprising one or more additional targeting moieties.

3. The chimeric protein of claim 2 , wherein the one or more additional targeting moieties comprise a recognition domain that recognizes and binds to an antigen or receptor on a tumor cell.

4. The chimeric protein of claim 2 , wherein the one or more additional targeting moieties comprise a recognition domain that recognizes and binds to an antigen or receptor on an immune cell.

5. The chimeric protein of claim 4 , wherein the immune cell is selected from a macrophage, a monocyte, and a dendritic cell.

6. 3. The chimeric protein of claim 2, wherein the one or more additional targeting moieties recognize one or more of PD-L1, PD-L2, PD-1, and Clec9A.

7. The chimeric protein of claim 1 , wherein the recognition domain functionally regulates an antigen or receptor of interest.

8. The chimeric protein of claim 1 , wherein the recognition domain recognizes and binds to an antigen or receptor of interest but does not functionally modulate them.

9. A recombinant nucleic acid encoding the chimeric protein according to any one of claims 1 to 8.

10. A host cell comprising the recombinant nucleic acid of claim 9.

11. A medicament for use in treating cancer, comprising the chimeric protein according to any one of claims 1 to 8.

12. The cancer is selected from the group consisting of 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 cancer, connective tissue cancer, digestive cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, stomach cancer, gastrointestinal cancer, glioblastoma, hepatocellular carcinoma, intraepithelial neoplasia, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, and buccal cancer.

12. The pharmaceutical of claim 11, selected from one or more of: pulmonary cancer, salivary gland cancer, sarcoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, urinary tract cancer, vulvar cancer, lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, and other carcinomas and sarcomas, and post-transplant lymphoproliferative disorders (PTLDs), and abnormal blood vessel proliferation associated with phacomatosis and Meigs syndrome.

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