阅读说明：本技术 靶向嵌合蛋白及其用途 (Targeted chimeric proteins and uses thereof ) 是由 N·克雷 J·塔威尼尔 于 2018-02-05 设计创作，主要内容包括：本发明部分涉及包含至少一个识别和结合SIRP1α的靶向部分的嵌合蛋白及其作为诊断和治疗剂的用途。本发明还涉及包含所述嵌合蛋白的药物组合物及其在治疗多种疾病中的用途。(The present invention relates in part to chimeric proteins comprising at least one targeting moiety that recognizes and binds SIRP1a and their use as diagnostic and therapeutic agents. The invention also relates to pharmaceutical compositions comprising said chimeric proteins and their use in the treatment of various diseases.)

1. A chimeric protein comprising:

(a) A targeting moiety comprising a recognition domain that recognizes and binds SIRP1 a; and

(b) A modified signaling agent having one or more mutations that confer improved safety relative to a wild-type signaling agent as compared to the wild-type signaling agent, and

Wherein the targeting moiety and modified signaling agent are optionally linked to 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 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 an antigen or receptor on an immune cell.

5. The chimeric protein according to claim 4, wherein the immune cell is selected from the group consisting of macrophages, monocytes and dendritic cells.

6. The chimeric protein of any one of claims 2-5, wherein the one or more additional targeting moieties recognize one or more of PD-L1, PD-L2, PD-1, and Clec 9A.

7. The chimeric protein according to any one of the preceding claims, wherein the recognition domain comprises a full length antibody, a single domain antibody, a recombinant heavy chain only antibody (VHH), a single chain antibody (scFv), a shark heavy chain only antibody (VNAR), a micro-protein (e.g. cystine knot protein, knottin), a darpin, an anticalin, an adnectin, an aptamer, an Fv, a Fab ', a F (ab')2, a peptidomimetic molecule, a natural ligand of a receptor, or a synthetic molecule.

8. The chimeric protein of any of the above claims, wherein the recognition domain functionally modulates an antigen or receptor of interest.

9. The chimeric protein according to any of the above claims, wherein the recognition domain recognizes and binds to, but does not functionally modulate, an antigen or receptor of interest.

10. The chimeric protein of any one of the above claims, wherein the modified signaling agent comprises one or more mutations that confer a reduced affinity or activity at a receptor of the signaling agent relative to a wild-type signaling agent.

11. The chimeric protein of any one of the above claims, wherein the modified signaling agent comprises one or more mutations that confer upon the receptor a substantially reduced or eliminated affinity or activity relative to the wild-type signaling agent.

12. The chimeric protein of any one of the above claims, wherein the modified signaling agent comprises both: (a) one or more mutations that confer a significantly reduced or eliminated affinity to the receptor relative to the wild-type signaling agent, and (b) one or more mutations that confer a reduced affinity or activity to the receptor relative to the wild-type signaling agent; and wherein the receptors are different.

13. The chimeric protein of claim 10, wherein the one or more mutations allow activity to be attenuated.

14. The chimeric protein of claim 13, wherein agonistic or antagonistic activity is reduced.

15. The chimeric protein of claim 13 or 14, wherein the modified signaling agent comprises one or more mutations that switch its activity from agonistic to antagonistic.

16. The chimeric protein of claim 10, wherein a mutation confers reduced affinity or activity that can be restored by attachment to one or more targeting moieties.

17. The chimeric protein of claim 11, wherein a mutation confers a significantly reduced or eliminated affinity or activity that is not substantially recoverable by attachment to one or more targeting moieties.

18. The chimeric protein according to any of the above claims, wherein the modified signaling agent is selected from one or more of an interferon, an interleukin, and a tumor necrosis factor.

19. The chimeric protein according to any one of the preceding claims, wherein the signaling agent is an interferon.

20. The chimeric protein of any one of the above claims, wherein the chimeric protein is suitable for use in a patient having one or more of: cancer, infection, immune disorder, autoimmune disease, cardiovascular disease, trauma, ischemia-related disease, neurodegenerative disease and/or metabolic disease.

21. A chimeric protein comprising:

(a) A first targeting moiety comprising a recognition domain that recognizes and binds SIRP1 a;

(b) A second targeting moiety comprising a recognition domain that recognizes and binds PD-1; and

(b) Human interferon alpha 2 having one or more mutations conferring reduced affinity or activity at a receptor of a signaling agent compared to a wild type signaling agent, said mutations optionally being selected from L153A, R149A, M148A, R144X1, a145X2, R33A, wherein

X1 is selected from the group consisting of A, S, T, Y, L and I,

X2 is selected from G, H, Y, K and D, and

Wherein the targeting moiety and modified signaling agent are optionally linked to one or more linkers.

22. A recombinant nucleic acid composition encoding one or more chimeric proteins according to any one of the preceding claims.

23. A host cell comprising the nucleic acid of claim 22.

24. A method of treating cancer, comprising administering to a patient in need thereof an effective amount of a chimeric protein according to any of the above claims.

25. The method of claim 24, wherein the cancer is selected from one or more of: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancers of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); a glioblastoma; hepatocellular carcinoma; liver cancer; an intraepithelial neoplasm; kidney or renal cancer; laryngeal cancer; leukemia; cancer of the liver; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; a myeloma cell; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; a sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas including Hodgkin's and non-Hodgkin's lymphomas, as well as B-cell lymphomas (including low grade/follicular non-Hodgkin's lymphomas (NHLs), Small Lymphocytic (SL) NHLs, intermediate grade/follicular NHLs, intermediate grade diffuse NHLs, high grade immunoblastic NHLs, high grade lymphoblastic NHLs, high grade small non-nucleated NHLs, large tumors NHLs, mantle cell lymphomas, AIDS-related lymphomas, and Wallace's macroglobulinemia, Chronic Lymphocytic Leukemia (CLLs), Acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, as well as other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), and abnormal vascular hyperplasia associated with macular-hamartoma, edema (e.g., edema associated with brain tumors), and Megges syndrome.

26. The method of any one of claims 24 or 25, wherein the cancer overexpresses Myc protein.

27. The method of any one of claims 24-26, wherein the method induces and/or enhances phagocytosis of cancer cells by macrophages.

28. The chimeric protein according to any of the preceding claims for use as a medicament.

29. The chimeric protein of any one of the above claims, for use in the treatment of cancer.

30. The chimeric protein according to any of the preceding claims, for use in the treatment of hepatitis.

31. Use of a chimeric protein according to any of the preceding claims in the manufacture of a medicament.

Technical Field

The present invention relates in part to chimeric proteins comprising at least one targeting moiety that recognizes and binds SIRP1a and their use as diagnostic and therapeutic agents. The invention also relates to pharmaceutical compositions comprising said chimeric proteins and their use in the treatment of various diseases, including cancer.

Sequence listing

This application contains a sequence listing, which has been filed in ASCII format via EFS-Web, and is incorporated herein by reference in its entirety. The ASCII copy created on 31 st 1/2018 was named ORN-027PC-Sequence _ Listing _ st25.txt and was 282,624 bytes in size.

Background

Cancer is a global health challenge causing nearly 7 million deaths worldwide each year, and to date, despite major advances in medicine, has proven largely untreatable. Frustrating is that cancer appears to develop strategies to evade immunodetection and destruction to circumvent the body's primary protective role against the disease. For example, one mechanism by which cancer cells evade macrophage phagocytosis is through the upregulation of CD47, CD47 binding to an inhibitory receptor on macrophages, the signal-regulatory protein α -1(SIRP1 α). Specifically, the interaction between CD47 and SIRP1 α on cancer cells generates a "eat me" signal to inhibit phagocytosis of cancer cells. The Myc oncogene induces expression of CD47 in cancer cells-an immunosuppressive mechanism that involves the potent in vivo tumor growth promoting activity of Myc.

Activation of the Myc family of cellular oncogenes is one of the most common oncogenic events in human cancer. The Myc protein family encodes three highly related nuclear phosphoproteins (c-Myc, N-Myc and L-Myc) which are thought to function as sequence-specific transcription factors. Myc protein activates a variety of genes important to biological processes including growth, proliferation, apoptosis, metabolism, differentiation, self-renewal, and angiogenesis. Despite efforts to inhibit the highly active Myc protein in cancer cells, the oncogene remains significantly resistant to therapeutic targeting. In addition, there is evidence that active Myc protein promotes tumor resistance to a variety of cancer drugs.

Thus, there remains a need for new therapeutic agents that can effectively target cancers, including Myc-driven cancers.

Disclosure of Invention

In various aspects, the invention relates to chimeric proteins having at least one targeting moiety that specifically binds SIRP1 a. In various embodiments, the chimeric proteins of the invention can be used, for example, to recruit macrophages, either directly or indirectly, to a target site. In various embodiments, the chimeric protein further comprises a signaling agent, such as, but not limited to, interferons, interleukins, and tumor necrosis factors, which may be modified to attenuate activity. In various embodiments, the chimeric protein comprises additional targeting moieties that bind to other targets of interest (e.g., antigens, receptors). In one embodiment, the other target of interest (e.g., antigen, receptor) is present on a tumor cell. In another embodiment, the other target of interest (e.g., antigen, receptor) is present on an immune cell. In some embodiments, the chimeric proteins of the invention can directly or indirectly recruit immune cells (e.g., macrophages) to a site of action (e.g., as a non-limiting example, a tumor microenvironment). In some embodiments, the chimeric proteins of the invention promote phagocytosis of target cells (e.g., tumor cells) by macrophages.

In various embodiments, the chimeric proteins of the invention can be used to treat a variety of diseases or disorders, such as cancer, infections, immune disorders, and other diseases and disorders, and the invention includes a variety of methods of treatment.

Drawings

FIGS. 1A-B show binding assays with anti-mouse Sirp1 α VHH. In FIG. 1A, serial dilutions of anti-murine SIRP1 α VHH were tested on cells expressing murine SIRP1 α in a FACS-based mSRPA binding assay. Geometric mean values of fluorescence intensity were plotted. In FIG. 1B, serial dilutions of anti-murine SIRP 1. alpha. VHH were tested in a murine CD 47-murine SIRP. alpha. binding assay. Mean-/+ standard deviation of triplicate measurements are plotted.

FIGS. 2A-B show B16 in vivo studies using an anti-mouse Sirp1 α VHH/human IFN Q124R chimera. In fig. 2A, tumor growth was compared to PBS control. The anti-mouse Sirp1 α VHH/human IFN Q124R chimera is the bottom curve and PBS is the top curve. In fig. 2B, various safety parameters of the tumor study in the mice of fig. 2A were evaluated: white blood cell count ("wbc"), lymphocyte count ("ly"), neutrophil count ("ne"), monocyte count ("mo"), red blood cell count ("rbc"), hemoglobin ("hb"); hematocrit ("hct"), platelet ("plt"), and mean platelet volume ("mpv"). In each group, the left bar is PBS and the right bar is anti-mouse Sirp 1. alpha. VHH/human IFN Q124R.

FIG. 3 shows B16 cells stimulated (or unstimulated) with 100ng/ml chimera and stained for phosphoSTAT 1. Data are plotted as mean fluorescence intensity. Monospecific fusions of anti-murine Sirp1 α VHH/anti-murine PD-L1 VHH/human IFN Q124R and anti-Bcll 10 VHH with modified human IFN α Q124R (untargeted IFNQ124R control) were analyzed.

Detailed Description

The present invention is based, in part, on the discovery of targeted chimeric proteins having a targeting moiety that specifically recognizes and binds signal-regulating protein alpha-1 (SIRP1 alpha). In some embodiments, the chimeric protein is multispecific and includes one or more targeting moieties. In some embodiments, the chimeric protein further comprises a modified signaling agent (e.g., an interferon) having reduced affinity for one or more receptors. In various embodiments, the chimeric protein can bind to and recruit, directly or indirectly, immune cells, such as macrophages, to a site in need of therapeutic action (e.g., a tumor or tumor microenvironment). In some embodiments, the chimeric protein induces and/or enhances phagocytosis of tumor cells by macrophages. The chimeric proteins of the invention exhibit beneficial therapeutic properties and reduced side effects.

Targeted chimeric proteins

In various embodiments, the invention relates to targeted chimeric proteins comprising a targeting moiety that specifically recognizes and binds to signal-regulatory protein alpha-1 (SIRP1 alpha). SIRP 1a (also known as sirpa) belongs to a family of cellular immune receptors that includes inhibitory members (sirpa), activating members (sirpa), non-signaling members (SIRP γ), and soluble members (SIRP δ). SIRP1 α is expressed primarily on bone marrow cells including macrophages, granulocytes, bone marrow Dendritic Cells (DCs), mast cells and their precursors, including hematopoietic stem cells. SIRP 1a acts as an inhibitory receptor that interacts with the widely expressed transmembrane glycoprotein CD47 to regulate phagocytosis. In particular, SIRP 1a produces an inhibitory signal that negatively regulates phagocytosis of target cells by binding of CD47 expressed on target cells to macrophages.

In various embodiments, the present invention relates to targeted chimeric proteins comprising a targeting moiety that specifically recognizes and binds SIRP1 α on macrophages.

In various embodiments, the present invention relates to targeted chimeric proteins comprising a targeting moiety that specifically recognizes and binds SIRP1 α on monocytes.

In various embodiments, the present invention relates to targeting chimeric proteins comprising a targeting moiety that specifically recognizes and binds SIRP1 α on TAMs (tumor associated macrophages).

In various embodiments, the present invention relates to targeted chimeric proteins comprising a targeting moiety that specifically recognizes and binds SIRP1 α on dendritic cells, including but not limited to cD2 and pDC.

In various embodiments, the chimeric proteins of the invention comprise a targeting moiety having a recognition domain that recognizes SIRP1 a. In one embodiment, the recognition domain recognizes one or more linear epitopes present on SIRP1 a. As used herein, a linear epitope refers to any contiguous amino acid sequence present on SIRP1 a. In another embodiment, the recognition domain recognizes one or more conformational epitopes present on SIRP1 a. As used herein, a conformational epitope refers to one or more portions of amino acids (which may be discontinuous) that form a three-dimensional surface having features and/or shape and/or tertiary structure capable of being recognized by an antigen recognition domain.

In some embodiments, the chimeric protein comprises a targeting moiety that can bind to full-length and/or mature forms and/or isoforms and/or splice variants and/or fragments and/or any other naturally occurring or synthetic analogs, variants or mutants of SIRP1 a. In one embodiment, SIRP 1a is human SIRP1 a. In various embodiments, the chimeric protein comprises a targeting moiety that can bind any form of human SIRP 1a, including monomers, dimers, heterodimers, multimers, and related forms. In one embodiment, the targeting moiety binds a monomeric form of SIRP1 a. In another embodiment, the targeting moiety binds to a dimeric form of SIRP1 α.

In one embodiment, the chimeric protein of the invention comprises a targeting moiety with a recognition domain that recognizes one or more epitopes present on human SIRP1 a. In one embodiment, the targeting moiety comprises a recognition domain that recognizes human SIRP 1a having a signal peptide sequence. An exemplary human SIRP 1a polypeptide having a signal peptide sequence (underlined) is provided below:

MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQW FRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSV RAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTRE DVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRT ETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNI YIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPN NHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK(SEQ ID NO:1)

In one embodiment, the targeting moiety comprises a recognition domain that recognizes human SIRP1a without a signal peptide sequence. An exemplary human SIRP1a polypeptide without a signal peptide sequence is provided below: EEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK (SEQ ID NO:2)

In one embodiment, the targeting moiety comprises a recognition domain that recognizes a polypeptide encoding human SIRP1a subtype 2:

MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQW FRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSV RAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTRE DVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRT ETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNI YIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQVQSLDTNDITYADLNLPKGKKPAPQA AEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK(SEQ ID NO:3)

In one embodiment, the targeting moiety comprises a recognition domain that recognizes a polypeptide encoding human SIRP 1a subtype 4:

MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQW FRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDVEFKSGAGTELSVR AKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTRED VHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTE TASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIY IVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNN HTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK(SEQ ID NO:4)

In various embodiments, the targeting moiety of the present invention may be any protein-based agent capable of specific binding, such as an antibody or derivative thereof. In one embodiment, the targeting moiety comprises an antibody. In various embodiments, the antibody is a full-length multimeric protein comprising two heavy chains and two light chains. Each heavy chain comprises a variable region (e.g., VH) and at least three constant regions (e.g., CH1, CH2, and CH3), and each light chain comprises a variable region (VL) and a constant region (CL). The variable region determines the specificity of the antibody. Each variable region comprises three hypervariable regions, also known as Complementarity Determining Regions (CDRs), flanked by four relatively conserved Framework Regions (FRs). Three CDRs, designated CDR1, CDR2, and CDR3, contribute to the antibody binding specificity. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody.

In some embodiments, the targeting moiety comprises an antibody derivative or form. In some embodiments, the targeting moiety of the chimeric proteins of the invention is a single domain antibody, a recombinant heavy chain-only antibody (VHH), a single chain antibody (scFv), a shark heavy chain-only antibody (VNAR), a micro-protein (cysteine knot protein, knottin), a DARPin; tetranectin (Tetranectin); affibody (Affibody); transbody (Tansbody); anticalin; AdNectin; affilin; microbodies (microbodies); a peptide aptamer; alterase; a plastic antibody; ferulomer (phylomer); stradobody (Stradobody); the macrocode (maxibody); the evibody (evibody); feinuobody (fynomer), armadillo repeat protein, Kunitz domain, avimer, atrimer, prokaryote (probody), immunomer (immunobody), triomab, troybody; body of perps (pepbody); vaccine body (vaccibody), monospecific body (UniBody); affinity multimers (affimers), bispecific bodies (duobodies), Fv, Fab ', F (ab')2, peptidomimetic molecules, or synthetic molecules, as described in U.S. patent No. or patent publication nos. US 7,417,130, US 2004/132094, US 5,831,012, US 2004/023334, US 7,250,297, US 6,818,418, US 2004/209243, US 7,838,629, US 7,186,524, US 6,004,746, US 5,475,096, US 2004/146938, US 2004/157209, US 6,994,982, US 6,794,144, US 2010/239633, US 7,803,907, US 2010/119446, and/or US 7,166,697, the contents of which are incorporated herein by reference in their entirety. See also Storz mabs.2011, months 5-6; 3(3):310-317.

In one embodiment, the targeting moiety comprises a single domain antibody, such as a VHH or a designed VHH from, for example, a VHH antibody producing organism (e.g. camel, shark). VHH are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally occurring heavy chain antibodies. VHH technology is based on fully functional antibodies from camelids lacking light chains. These heavy chain antibodies contain a single variable region (VHH) and two constant regions (CH2 and CH 3). VHH is commercially available under the trademark NANOBODY or NANOBODIES.

In one embodiment, the targeting moiety comprises a VHH. In some embodiments, the VHH is a humanized VHH or a camelized VHH.

In some embodiments, the VHH comprises a fully human VH domain, such as HUMABODY (credence Biologics, Cambridge, UK). In some embodiments, a fully human VH domain, e.g., HUMABODY, is monovalent, divalent, or trivalent. In some embodiments, a fully human VH domain, e.g., HUMABODY, is monospecific or multispecific, e.g., monospecific, bispecific, or trispecific. Exemplary fully human VH domains, such as HUMABODIES, are described, for example, in WO 2016/113555 and WO2016/113557, the entire disclosures of which are incorporated herein by reference.

For example, in some embodiments, a chimeric protein of the invention comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, VHHs, or fusion proteins that selectively bind SIRP1 α. In some embodiments, the chimeric protein comprises a targeting moiety that is an antibody or derivative thereof that specifically binds SIRP1 a. In some embodiments, the chimeric protein comprises a targeting moiety that is a camelidae heavy chain antibody (VHH) that specifically binds SIRP1 a.

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

In various embodiments, the targeting moiety of the present invention may comprise any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, Complementarity Determining Regions (CDRs), and framework region sequences known to recognize and bind SIRP1 α.

In various embodiments, the present technology contemplates the use of any natural or synthetic analogs, mutants, variants, alleles, homologs, and orthologs (collectively referred to herein as "analogs") of the SIRP 1a targeting moieties described herein. In various embodiments, the amino acid sequence of the SIRP 1a targeting moiety further includes amino acid analogs, amino acid derivatives, or other non-canonical amino acids.

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

In various embodiments, the chimeric proteins of the invention comprise a targeting moiety comprising an amino acid sequence having one or more amino acid mutations relative to any targeting moiety sequence known to recognize and bind SIRP1 α. In various embodiments, the chimeric proteins of the invention comprise a targeting moiety comprising an amino acid sequence having one, or two, or three, or four, or five, or six, or visible, or eight, or nine, or ten, or fifteen, twenty, thirty, forty, or fifty amino acid mutations relative to any targeting moiety sequence known to recognize and bind SIRP1 a. 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 mutation is an amino acid substitution, and may include conservative and/or non-conservative substitutions.

"conservative substitutions" can be made, for example, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids can be divided into the following 6 standard amino acid groups: (1) hydrophobicity: met, Ala, Val, Leu, Ile; (2) neutral hydrophilicity: cys, Ser, Thr, Asn, Gln; (3) acidity: asp and Glu; (4) alkalinity: his, Lys, Arg; (5) residues that influence chain orientation: gly, Pro; and (6) aromatic: trp, Tyr, Phe.

As used herein, "conservative substitutions" are defined as the replacement of one amino acid by another amino acid listed in the same group of the six standard amino acid groups indicated above. For example, exchange of Asp by Glu retains a negative charge in the polypeptide so modified. In addition, glycine and proline may be substituted for each other based on their ability to disrupt alpha-helices.

As used herein, "non-conservative substitution" is defined as the replacement of one amino acid by another amino acid listed in a different one of the six standard amino acid groups (1) to (6) shown above.

In various embodiments, substitutions may also include non-classical amino acids. Exemplary non-classical amino acids include, but are not limited to, selenocysteine, pyrrolysine, N-formylmethionine beta-alanine, GABA and delta-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2, 4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminocaproic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoroamino acids, design amino acids such as beta-methyl amino acids, C α -methyl amino acids, N α -methyl amino acids and amino acid analogs in general.

In various embodiments, the amino acid mutation can be in a CDR (e.g., CDR1, CDR2, or CDR3 region) of the targeting moiety. In another embodiment, the amino acid change may be in a Framework Region (FR) of the targeting moiety (e.g., FR1, FR2, FR3, or FR4 region).

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

In various embodiments, the mutation does not substantially reduce the ability of the chimeric proteins of the invention to specifically recognize and bind SIRP1 a. In various embodiments, the mutation does not substantially reduce the ability of a chimeric protein of the invention to specifically bind SIRP1a and does not functionally modulate (e.g., partially or fully neutralize) SIRP1 a.

In various embodiments, the binding affinity of a chimeric protein of the invention to the full-length and/or mature form and/or subtype and/or splice variant and/or fragment and/or monomeric and/or dimeric form of SIRP1a and/or any other naturally occurring or synthetic analog, variant or mutant can be described by the equilibrium dissociation constant (KD). In various embodiments, a chimeric protein of the invention comprises a targeting moiety that binds to a full-length and/or mature form and/or subtype and/or splice variant and/or fragment of SIRP1a and/or any other naturally occurring or synthetic analog, variant, or mutant (including monomeric and/or dimeric forms) with a KD of less than about 1 μ M, about 900nM, about 800nM, about 700nM, about 600nM, about 500nM, about 400nM, about 300nM, about 200nM, about 100nM, about 90nM, about 80nM, about 70nM, about 60nM, about 50nM, about 40nM, about 30nM, about 20nM, about 10nM, or about 5nM or about 1 nM.

In various embodiments, the chimeric proteins of the invention comprise a targeting moiety that binds to, but does not functionally modulate, an antigen of interest, SIRP1 a. For example, in various embodiments, the targeting moiety of the chimeric protein simply targets the antigen, but is substantially incapable of functionally modulating (e.g., significantly inhibiting, reducing, or neutralizing) the biological effect possessed by the antigen. In various embodiments, the targeting moiety of the chimeric proteins of the invention binds to an epitope physically separated from the antigenic site important to their biological activity (e.g., the active site of the antigen).

In other embodiments, the chimeric proteins of the invention comprise a targeting moiety that binds to but functionally modulates a target antigen, SIRP1 a. For example, in various embodiments, the targeting portion of the chimeric protein targets the antigen, i.e., SIRP1a, and functionally modulates (e.g., inhibits, reduces, or neutralizes) the biological effect that the antigen has. Such binding and functional modulation may be used in various embodiments of the invention, including methods in which the chimeric proteins of the invention are used to recruit active immune cells to a desired site, either directly or indirectly, via an effector antigen.

For example, in various embodiments, the chimeric proteins of the invention may be used to recruit macrophages directly or indirectly to tumor cells via SIRP 1a in a method of reducing or eliminating tumors (e.g., the chimeric proteins of the invention may comprise a targeting moiety having an anti-SIRP 1a antigen recognition domain and a targeting moiety having a recognition domain (e.g., antigen recognition domain) for a tumor antigen or receptor). Evidence suggests that tumor cells frequently upregulate CD47, while CD47 binds SIRP1 α to evade phagocytosis. Thus, in various embodiments, it may be desirable to recruit macrophages directly or indirectly to tumor cells and functionally inhibit, reduce, or neutralize the inhibitory activity of SIRP1 α, thereby allowing the macrophages to phagocytose the tumor cells. In various embodiments, the chimeric proteins of the present invention enhance phagocytosis of tumor cells or any other undesirable cells by macrophages.

Therapeutic agents comprising the chimeric proteins of the invention

Chimeras and fusions with signaling agents

In various embodiments, the chimeric proteins of the invention are part of a chimera or fusion with one or more signaling agents. Accordingly, the present invention provides a chimeric or fusion protein comprising a targeting moiety, e.g., to SIRP 1a, and one or more signaling agents.

In various embodiments, the signaling agent is modified to have reduced affinity or activity for one or more of its receptors, which may attenuate the activity of the chimeric or fusion protein (including agonism or antagonism) and/or prevent non-specific signaling or unwanted sequestration (sequestration). In various embodiments, the signaling agent is antagonistic in its wild-type form and has one or more mutations that diminish its antagonistic activity. In various embodiments, the signaling agent has an antagonistic effect due to one or more mutations, e.g., an agonistic signaling agent is converted to an antagonistic signaling agent, and such converted signaling agent optionally further has one or more mutations that diminish its antagonistic activity (e.g., as described in WO2015/007520, the entire contents of which are incorporated herein by reference).

Thus, in various embodiments, the signaling agent is a modified (e.g., mutated) form of the signaling agent having one or more mutations. In various embodiments, the modification (e.g., mutation) is such that the modified signaling agent has one or more reduced activities, e.g., one or more of reduced binding affinity, reduced endogenous activity, and reduced specific biological activity, relative to the unmodified or unmutated signaling agent, i.e., the wild-type signaling agent (e.g., comparing the wild-type form to the same signaling agent in the modified or mutated form). In some embodiments, mutations that reduce or decrease binding or affinity include those that significantly reduce or eliminate binding or activity. In some embodiments, the mutations that reduce or decrease binding or affinity are different from those that significantly reduce or eliminate binding or activity. Thus, in various embodiments, the mutation results in a signaling agent having improved safety, e.g., reduced systemic toxicity, reduced side effects, and reduced off-target effects, relative to an unmutated, i.e., wild-type, signaling agent (e.g., comparing a wild-type form to a modified (e.g., mutated) form of the same signaling agent).

As described herein, the agents may have improved safety due to one or more modifications, such as mutations. In various embodiments, improved safety means that the chimeric proteins of the invention result in less toxicity (e.g., systemic toxicity and/or tissue/organ related toxicity); and/or reduced or substantially eliminated side effects; and/or increased tolerance, reduced or substantially eliminated adverse events; and/or a reduction or substantial elimination of off-target effects; and/or increased therapeutic window.

In various embodiments, the signaling agent is modified to have one or more mutations that reduce its binding affinity or activity for one or more of its receptors. In some embodiments, the signaling agent is modified to have one or more mutations that substantially reduce or eliminate binding affinity or activity for the receptor. In some embodiments, the activity produced by the wild-type signaling agent is agonistic at the receptor (e.g., activating a cellular effect at the treatment site). For example, a wild-type signaling agent may activate its receptor. In such embodiments, the mutation results in the modified signaling agent having reduced or eliminated activation activity at the receptor. For example, the mutation may result in a reduction in the activation signal delivered by the modified signaling agent to the target cell, or may eliminate the activation signal. In some embodiments, the activity provided by the wild-type signaling agent is antagonistic at the receptor (e.g., blocks or inhibits a cellular effect at the treatment site). For example, a wild-type signaling agent may antagonize or inhibit a receptor. In these embodiments, the mutation results in the modified signaling agent having reduced or eliminated antagonistic activity at the receptor. For example, the mutation may result in a reduction in inhibitory signal delivered by the modified signaling agent to the target cell, or may eliminate the inhibitory signal. In various embodiments, a signaling agent has an antagonistic effect due to one or more mutations, e.g., an agonist signaling agent is converted to an antagonistic signaling agent (e.g., as described in WO2015/007520, the entire contents of which are incorporated herein by reference), and such converted signaling agent optionally further has one or more mutations that reduce its binding affinity or activity to one or more of its receptors, or that significantly reduce or eliminate its binding affinity or activity to one or more of its receptors.

In some embodiments, the reduced affinity or activity at the receptor is recoverable by linkage to one or more targeting moieties described herein (e.g., a targeting moiety for SIRP 1a or any other targeting moiety described herein). In other embodiments, the reduced affinity or activity at the receptor is not substantially restored by the activity of the one or more targeting moieties.

In various embodiments, the chimeric proteins of the invention reduce off-target effects because their signaling agents have mutations that reduce or eliminate binding affinity or activity at the receptor. In various embodiments, a reduction in such side effects relative to, for example, a wild-type signaling agent is observed. In various embodiments, the signaling agent is active on the target cell because the targeting moiety compensates for the lack/insufficient binding (e.g., without limitation and/or avidity) required for substantial activation. In various embodiments, the modified signaling agents are substantially inactive in the pathway to the therapeutically active site and have a significant effect on the specifically targeted cell type, which greatly reduces undesirable side effects.

In some embodiments, a signaling agent may include one or more mutations that reduce or decrease binding or affinity to one receptor (i.e., a therapeutic receptor), and one or more mutations that substantially reduce or eliminate binding or activity at a second receptor. In such embodiments, the 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 a mutation that significantly reduces or eliminates at another receptor. In some embodiments, the mutation that reduces binding and/or activity at one receptor is the same as the mutation that significantly reduces or eliminates at another receptor. In some embodiments, the chimeric proteins of the invention have a modified signaling agent that has both a mutation that attenuates binding and/or activity at a therapeutic receptor and thus allows for a more controlled on-target therapeutic effect (e.g., relative to the wild-type signaling agent) and a mutation that significantly reduces or eliminates binding and/or activity at another receptor and thus reduces side effects (e.g., relative to the wild-type signaling agent).

In some embodiments, a significant reduction or elimination of binding or activity is substantially not recoverable with a targeting moiety (e.g., a targeting moiety directed to SIRP1a or any other targeting moiety described herein). In some embodiments, a significant reduction or elimination of binding or activity can be restored with the targeting moiety. In various embodiments, a significant reduction or elimination of binding or activity at a second receptor may also prevent deleterious effects mediated by another receptor. Alternatively or additionally, a significant reduction or elimination of binding or activity at another receptor results in an increase in therapeutic efficacy, as the sequestration of the therapeutic chimeric protein away from the site of therapeutic action is reduced or eliminated. For example, in some embodiments, this avoids the need for high doses of the chimeric proteins of the invention to compensate for losses at other receptors. This ability to reduce the dose further provides a lower potential for side effects.

In various embodiments, the modified signaling agent comprises one or more mutations that result in the signaling agent having reduced, significantly reduced, or eliminated affinity for one or more of its receptors, e.g., binding (e.g., KD) and/or activation (e.g., measurable as, e.g., KA and/or EC50 when the modified signaling agent is an agonist of its receptor) and/or inhibition (e.g., measurable as, e.g., KI and/or IC50 when the modified signaling agent is an antagonist of its receptor). In various embodiments, the decrease in affinity at the signaling agent receptor results in a decrease in activity (including agonism or antagonism). In such embodiments, the affinity of the modified signaling agent for the receptor 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%, about 80% -100% 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 150-fold lower, or more than 200-fold lower relative to the wild-type signaling agent.

In embodiments where the chimeric protein comprises a modified signaling agent having a mutation that reduces binding at one receptor and substantially reduces or eliminates binding at a second receptor, the reduction or reduction in binding affinity of the modified signaling agent for one receptor is less than the substantial reduction or elimination of affinity for the other receptor. In some embodiments, the reduction or decrease in binding affinity of the modified signaling agent for one receptor is less than 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%, or about 95% less than the significant reduction or elimination of the affinity for another receptor. In various embodiments, a significant reduction or elimination refers to a reduction in binding affinity and/or activity that is greater than the reduction or reduction.

In various embodiments, the modified signaling agent comprises one or more mutations that reduce the endogenous activity of the signaling agent, e.g., to 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% relative to the wild-type signaling agent.

In some embodiments, the modified signaling agent comprises one or more mutations that result in a signaling agent with reduced affinity for its receptor and lower binding affinity than the targeting moiety(s) for its receptor. In some embodiments, such a binding affinity difference is a difference between a signaling agent/receptor and a targeting moiety/receptor on the same cell. In some embodiments, this difference in binding affinity allows for a signaling agent (e.g., a mutated signaling agent) to have a localized on-target effect and minimizes off-target effects exhibited by side effects observed with wild-type signaling agents. In some embodiments, the binding affinity is at least about 2-fold, or at least about 5-fold, or at least about 10-fold, or at least about 15-fold, or at least about 25-fold, or at least about 50-fold, or at least about 100-fold, or at least about 150-fold lower.

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

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

In some embodiments, the signaling agent is an interleukin or a modified interleukin, including, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-35, IL-36, or fragments, variants, analogs, or family members thereof. Interleukins are a group of multifunctional cytokines synthesized by lymphocytes, monocytes and macrophages. Known functions include stimulation of immune cell (e.g., T helper cell, B cell, eosinophil, and lymphocyte) proliferation, neutrophil and T lymphocyte chemotaxis, and/or interferon inhibition. Interleukin activity can be determined using assays known in the art: matthews et al, in Lymphokines and interferences A Practical Approach, Clemens et al, eds, IRL Press, Washington, D.C.1987, pp.221-225; and Orencole & Dinarello (1989) Cytokine 1, 14-20.

In some embodiments, the signaling agent is an interferon or a modified form of an interferon, such as I, II and type III interferon. Exemplary interferons include, for example, interferons 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 τ

In some embodiments, the signaling agent is Tumor Necrosis Factor (TNF) or a modified form of Tumor Necrosis Factor (TNF) or a protein in the TNF family, including but not limited to TNF- α, TNF- β, LT- β, CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, and TRAIL.

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

In various embodiments, the modified signaling agent comprises an amino acid sequence that has 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% sequence identity (e.g., about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71%, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or both, Or about 98%, or about 99% sequence identity).

In various embodiments, the modified signaling 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 mutation is an amino acid substitution, and may include conservative and/or non-conservative substitutions as described elsewhere herein.

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

As described herein, the modified signaling agent carries a mutation that affects affinity and/or activity at one or more receptors. In various embodiments, there is reduced affinity and/or activity at a therapeutic receptor, for example at a receptor through which a desired therapeutic effect (e.g., agonism or antagonism) is mediated. In various embodiments, the modified signaling agent bears a mutation that significantly reduces or eliminates affinity and/or activity at a receptor, e.g., a receptor that does not mediate a desired therapeutic effect (e.g., due to a confounding of binding). As described herein, receptors for any signaling agent are known in the art.

Exemplary mutations that provide reduced affinity and/or activity (e.g., agonism) at the receptor are found in WO2013/107791 and PCT/EP2017/061544 (e.g., for interferons), WO2015/007542 (e.g., for interleukins), and WO2015/007903 (e.g., for TNF), the entire contents of each of which are incorporated herein by reference. Exemplary mutations that provide reduced affinity and/or activity (e.g., antagonism) at a therapeutic receptor are found in WO2015/007520, which is incorporated by reference in its entirety.

In some embodiments, the modified signaling agent comprises one or more mutations that result in the signaling agent having reduced affinity and/or activity 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.

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

In various embodiments, the receptor for the signaling agent is a type II cytokine receptor. Type II cytokine receptors are multimeric receptors composed of heterologous subunits, and are primarily receptors for interferons. This family of receptors includes, but is not limited to, receptors for interferon- α, interferon- β and interferon- γ, IL10, IL22, and tissue factor. Exemplary type II cytokine receptors include, but are not limited to, IFN-alpha receptors (e.g., IFNAR1 and IFNAR2), IFN-beta receptors, IFN-gamma receptors (e.g., IFNGR1 and IFNGR2), and type II IL receptors.

In various embodiments, the receptor of the signaling agent is a G protein-coupled receptor. Chemokine receptors are G protein-coupled receptors with 7 transmembrane structures and coupled to G proteins for signal transduction. Chemokine receptors include, but are not limited to, the CC chemokine receptor, the CXC chemokine receptor, the CX3C chemokine receptor, and the XC chemokine receptor (XCR 1). 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 CX3CR 1.

In various embodiments, the receptor for the signaling agent is a TNFR family member. Tumor Necrosis Factor Receptor (TNFR) family members share a cysteine-rich domain (CRD) formed by three disulfide bonds that encircle the core motif of CXXCXXC, creating an elongated molecule. Exemplary tumor necrosis factor receptor family members include: CDl20a (TNFRSFlA), CD120 b (tnfrsffb), lymphotoxin beta receptor (LTBR, TNFRSF3), CD 134(TNFRSF4), CD40(CD40, TNFRSF5), FAS (FAS, TNFRSF6), TNFRSF6B (TNFRSF6B), CD27(CD27, TNFRSF7), CD 7(TNFRSF 7), CD137(TNFRSF 7), TNFRSF10 7(TNFRSF 10 7), TNFRSF10 7, (TNFRSF10 7), TNFRSF10 7(TNFRSF 10 7), TNFRSF17), TNfrsf11 7, TNFRSF12 7(TNFRSF 12 7), TNFRSF 3613 (TNFRSF 7), TNFRSF 7(TNFRSF 7), tnfr3672 (TNFRSF 7), tnfrs 7(tnfrs 7), tnfrs 7(tnfrs 7), tn. . In one embodiment, the TNFR family member is CD120a (TNFRSF1A) or TNF-R1. In another embodiment, the TNFR family member is CD120 b (TNFRSF1B) or TNF-R2.

In various embodiments, the receptor for the signaling agent is a TGF- β receptor. TGF-beta receptors are single-spanning serine/threonine kinase receptors. TGF β receptors include, but are not limited to, TGFBR1, TGFBR2, and TGFBR 3.

In various embodiments, the receptor for the signaling agent is an Ig superfamily receptor. Receptors in the immunoglobulin (Ig) superfamily share structural homology with immunoglobulins. Receptors in the Ig superfamily include, but are not limited to, interleukin-1 receptor, CSF-1R, PDGFR (e.g., PDGFRA and PDGFRB), and SCFR.

In various embodiments, the receptor for the signaling agent is a receptor of the tyrosine kinase superfamily. Receptors in the tyrosine kinase superfamily are well known in the art. There are about 58 known Receptor Tyrosine Kinases (RTKs), which are divided into 20 subfamilies. Receptors in the tyrosine kinase superfamily include, but are not limited to, FGF receptors and their various subtypes, such as FGFR1, FGFR2, FGFR3, FGFR4, and FGFR 5.

In some embodiments, the modified signaling agent is interferon alpha. In such embodiments, the modified IFN- α agent has reduced affinity and/or activity for the IFN- α/β receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chain. In some embodiments, the modified IFN- α agents have significantly reduced or eliminated affinity and/or activity for the IFN- α/β receptor (IFNAR), i.e., the IFNAR1 and/or IFNAR2 chains.

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

In an exemplary embodiment, the modified signaling agent is an allelic form of IFN- α 2b having the amino acid sequence of SEQ ID NO: 47 (which differs from IFN-. alpha.2a at amino acid position 23).

In some embodiments, the IFN-. alpha.2 mutant (IFN-. alpha.2a or IFN-. alpha.2b) is mutated at one or more of amino acids 144-154, e.g., amino acids 148, 149, and/or 153. In some embodiments, the IFN- α 2 mutant comprises one or more mutations selected from the group consisting of L153A, R149A, and M148A. These mutants are described, for example, in WO2013/107791 and Piehler et al, (2000) J.biol.chem,275:40425-33, the entire contents of which are incorporated herein by reference.

In some embodiments, the IFN- α 2 mutant has reduced affinity and/or activity for IFNAR 1. In some embodiments, the IFN- α 2 mutant comprises one or more mutations selected from F64A, N65A, T69A, L80A, Y85A, and Y89A, as described in WO2010/030671, the entire contents of which are incorporated herein by reference.

In some embodiments, the IFN- α 2 mutant comprises one or more mutations selected from K133A, R144A, R149A, and L153A, as described in WO2008/124086, the entire contents of which are incorporated herein by reference.

In some embodiments, the IFN- α 2 mutant comprises one or more mutations selected from R120E and R120E/K121E, as described in WO2015/007520 and WO2010/030671, the entire contents of which are incorporated herein by reference. In such embodiments, the IFN- α 2 mutant antagonizes wild-type IFN- α 2 activity. In such embodiments, the mutant IFN- α 2 has reduced affinity and/or activity for IFNAR1, while the affinity and/or activity of IFNR2 is retained.

In some embodiments, the human IFN- α 2 mutant comprises (1) one or more mutations selected from R120E and R120E/K121E, without wishing to be bound by theory, that produce an antagonistic effect, and (2) one or more mutations selected from K133A, R144A, R149A, and L153A, without wishing to be bound by theory, that allow for a reduced effect at, for example, IFNAR 2. In one embodiment, the human IFN- α 2 mutant comprises R120E and L153A.

In some embodiments, the human IFN- α 2 mutant comprises one or more mutations selected from L15A, a19W, R22A, R23A, L26A, F27A, L30A, L30V, K31A, D32A, R33K, R33A, R33Q, H34A, D35A, Q40A, D114R, L117A, R120A, R125A, K134A, R144A, a145G, a145M, M148A, R149A, S152A, L153A, and N156A disclosed in WO2013/059885, the entire disclosure of which is incorporated herein by reference. In some embodiments, the human IFN- α 2 mutant comprises mutations H57Y, E58N, Q61S, and/or L30A as disclosed in WO 2013/059885. In some embodiments, the human IFN- α 2 mutant comprises mutations H57Y, E58N, Q61S, and/or R33A as disclosed in WO 2013/059885. In some embodiments, the human IFN- α 2 mutant comprises mutations H57Y, E58N, Q61S, and/or M148A as disclosed in WO 2013/059885. In some embodiments, the human IFN- α 2 mutant comprises mutations H57Y, E58N, Q61S, and/or L153A as disclosed in WO 2013/059885. In some embodiments, the human IFN- α 2 mutant comprises the mutations N65A, L80A, Y85A and/or Y89A disclosed in WO 2013/059885. In some embodiments, the human IFN- α 2 mutant comprises the mutations N65A, L80A, Y85A, Y89A and/or D114A disclosed in WO 2013/059885. In some embodiments, the human IFN- α 2 mutant comprises one or more mutations selected from R144X1, a145X2, and R33A, wherein X1 is selected from A, S, T, Y, L and I, and wherein X2 is selected from G, H, Y, K and D.

In some embodiments, the modified signaling agent is interferon-beta. In such embodiments, the modified interferon beta agent has reduced affinity and/or activity for an IFN- α/β receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chain. In some embodiments, the modified interferon beta agent has significantly reduced or eliminated affinity and/or activity for the IFN-alpha/beta receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chains.

In one embodiment, the modified signaling agent is interferon beta. In such embodiments, the modified interferon beta agent has reduced affinity and/or activity for an IFN- α/β receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chain. In some embodiments, the modified interferon beta agent has significantly reduced or eliminated affinity and/or activity for the IFN-alpha/beta receptor (IFNAR), i.e., IFNAR1 and/or IFNAR2 chains.

In an exemplary embodiment, the modified signaling agent is IFN- β. In various embodiments, IFN- β comprises a functional derivative, analog, precursor, subtype, splice variant or fragment of IFN- β. In various embodiments, IFN- β comprises IFN- β derived from any species. In one embodiment, the chimeric protein comprises a modified form of mouse IFN- β. In another embodiment, the chimeric protein comprises a modified form of human IFN- β. Human IFN- β is a polypeptide of about 22kDa in molecular weight and comprises 166 amino acid residues. The amino acid sequence of human IFN-beta is SEQ ID NO: 48.

In some embodiments, the human IFN- β is IFN- β -1a, which is a glycosylated form of human IFN- β. In some embodiments, the human IFN- β is IFN- β -1b, which is a non-glycosylated form of human IFN- β, having a Met-1 deletion and a mutation from Cys-17 to Ser.

In various embodiments, the modified IFN- β has one or more mutations that reduce its binding to the IFNAR1 subunit of the IFNAR or its affinity for the IFNAR1 subunit. In one embodiment, the modified IFN- β has reduced affinity and/or activity at IFNAR 1. 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 is a substitution selected from the group consisting of F67G, F67S, R71A, L88G, L88S, Y92G, Y92S, I95A, N96G, K123G, and R124G. In one embodiment, the modified IFN- β comprises the F67G mutation. In one embodiment, the modified IFN- β comprises a K123G mutation. In one embodiment, the modified IFN- β comprises F67G and R71A mutations. In one embodiment, the modified IFN- β comprises L88G and Y92G mutations. In one embodiment, the modified IFN- β comprises Y92G, I95A, and N96G mutations. In one embodiment, the modified IFN- β comprises K123G and R124G mutations. In one embodiment, the modified IFN- β comprises F67G, L88G, and Y92G mutations. In one embodiment, the modified IFN- β comprises F67S, L88S, and Y92S mutations.

In some embodiments, the modified IFN- β has one or more mutations that reduce its binding to the IFNAR2 subunit of IFNAR or its affinity for the IFNAR2 subunit. In one embodiment, the modified IFN- β has reduced affinity and/or activity at IFNAR 2. In various embodiments, the modified IFN- β is a 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 is a substitution selected from W22G, R27G, L32A, L32G, R35A, R35G, V148G, L151G, R152A, R152G, and Y155G. In one embodiment, the modified IFN- β comprises a W22G mutation. In one embodiment, the modified IFN- β comprises the L32A mutation. In one embodiment, the modified IFN- β comprises the L32G mutation. In one embodiment, the modified IFN- β comprises the R35A mutation. In one embodiment, the modified IFN- β comprises the R35G mutation. In one embodiment, the modified IFN- β comprises the V148G mutation. In one embodiment, the modified IFN- β comprises the R152A mutation. In one embodiment, the modified IFN- β comprises the R152G mutation. In one embodiment, the modified IFN- β comprises a Y155G mutation. In one embodiment, the modified IFN- β comprises W22G and R27G mutations. In one embodiment, the modified IFN- β comprises L32A and R35A mutations. In one embodiment, the modified IFN- β comprises L151G and R152A mutations. In one embodiment, the modified IFN- β comprises the V148G and R152A mutations.

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: combinations of R35A, R35T, E42K, M62I, G78S, a141Y, a142T, E149K and R152H with C17S or C17A.

In some embodiments, the modified IFN- β has one or more of the following mutations: R35A, R35T, E42K, M62I, G78S, a141Y, a142T, E149K, and R152H in combination with any other IFN- β mutations described herein.

The crystal structure of human IFN- β is known and described in Karpusas et al, (1998) PNAS, 94 (22): 11813-11818. Specifically, it has been shown that the structure of human IFN- β comprises five α -helices (i.e., A, B, C, D and E) and four loop regions connecting these helices (i.e., AB, BC, CD and DE loops). In various embodiments, the modified IFN- β has one or more mutations in the A, B, C, D, E helix and/or the AB, BC, CD and DE loops that reduce its binding affinity or activity at a therapeutic receptor, such as an IFNAR. Exemplary mutations are described in WO2000/023114 and US20150011732, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the modified IFN- β is a human IFN- β comprising an alanine substitution 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 one 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 an alanine substitution at amino acid positions 71-73. In an exemplary embodiment, the modified IFN- β is a human IFN- β comprising alanine substitutions at amino acid positions 92, 96, 99 and 100. In an exemplary embodiment, the modified IFN- β is a human IFN- β comprising alanine substitutions at amino acid positions 128, 130, 131 and 134. In an exemplary embodiment, the modified IFN- β is a human IFN- β comprising alanine substitutions at amino acid positions 149, 153, 156, and 159. In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at W22 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at R27 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at W22, said mutation being an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V), and a mutation at R27, said mutation being an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at L32 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at R35 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at L32 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), isoleucine (I), methionine (M) and valine (V), and a mutation at R35 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at F67, said mutation being an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at R71 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at F67 which is an aliphatic hydrophobic residue selected from the group consisting of 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 the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at L88 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at Y92 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at F67 of an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V), and a mutation at L88 of an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), isoleucine (I), methionine (M) and valine (V), and a mutation at Y92 of an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at L88 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), isoleucine (I), methionine (M) and valine (V), and a mutation at Y92 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and 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).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at N96, said mutation being an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V), and a mutation at Y92, said mutation being an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

in some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at Y92 of an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V), and a mutation at I95 of an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), methionine (M) and valine (V), and a mutation at N96 of an aliphatic hydrophobic residue selected from glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at K123 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M) and valine (V). .

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at R124 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and 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).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at L151 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (A), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at R152 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and 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).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at V148 which is an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I) and methionine (M).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at V148, which is an aliphatic hydrophobic residue selected from the group consisting of 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 the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the mutant IFN β comprises SEQ ID NO: 48 and a mutation at Y155, said mutation being an aliphatic hydrophobic residue selected from the group consisting of glycine (G), alanine (a), leucine (L), isoleucine (I), methionine (M) and valine (V).

In some embodiments, the present invention relates to a chimeric protein comprising: (a) a modified IFN- β having the amino acid sequence of SEQ ID NO: 48 and a mutation at position W22, wherein the mutation is 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), said modified IFN- β and said one or more targeting moieties optionally being linked with 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.

Additional exemplary IFN beta mutants are provided in PCT/EP2017/061544, the entire disclosure of which is incorporated herein by reference.

In some embodiments, the modified signaling agent is interferon gamma. In such embodiments, the modified interferon gamma agent has reduced affinity and/or activity for the interferon-gamma receptor (IFNGR), i.e., the IFNGR1 and IFNGR2 chains. In some embodiments, the modified interferon gamma agent has a substantially reduced or eliminated affinity and/or activity for the interferon gamma receptor (IFNGR), i.e., the IFNGR1 and/or IFNGR2 chain.

In some embodiments, the modified signaling agent is a consensus interferon. Consensus interferon was generated by scanning the sequences of several human non-allelic IFN- α subtypes and assigning the most frequently observed amino acids at each corresponding position. Consensus interferon differs from IFN-. alpha.2b by 20 amino acids out of 166 (88% homology), and comparison with IFN-. beta.shows identity at more than 30% of the amino acid positions. In various embodiments, the consensus interferon comprises SEQ ID NO: 49.

In some embodiments, the consensus interferon comprises SEQ ID NO: 50, which is identical to SEQ ID NO: 49, i.e. SEQ ID NO: 50 lacks SEQ ID NO: 49 of the initiator methionine residue.

In various embodiments, the consensus interferon comprises a modified form of consensus interferon, i.e., a consensus interferon variant, as a signaling agent. In various embodiments, the consensus interferon variant comprises a functional derivative, analog, precursor, isoform, splice variant, or fragment of consensus interferon.

In one embodiment, the consensus interferon is selected from the group consisting of consensus interferon variants disclosed in U.S. patent nos. 4,695,623, 4,897,471, 5,541,293, and 8,496,921, all of which are incorporated herein by reference in their entirety. For example, consensus interferon variants may comprise the amino acid sequence of IFN-CON2 or IFN-CON3, as disclosed in U.S. Pat. Nos. 4,695,623, 4,897,471, and 5,541,293. In one embodiment, the consensus interferon variant comprises the amino acid sequence of IFN-CON 2(SEQ ID NO: 51).

In one embodiment, the consensus interferon variant comprises the amino acid sequence of IFN-CON 3(SEQ ID NO: 52).

In one embodiment, the consensus interferon variant comprises the amino acid sequence of any of the variants disclosed in U.S. patent No.8,496,921. For example, a composite variant may comprise SEQ ID NO: 53, or a pharmaceutically acceptable salt thereof.

In another embodiment, the consensus interferon may comprise the amino acid sequence of SEQ ID NO: 54, or a pharmaceutically acceptable salt thereof.

In some embodiments, the consensus interferon variant may be pegylated, i.e., comprise a PEG moiety. In one embodiment, the consensus interferon variant may comprise a consensus interferon that binds to the amino acid sequence of SEQ ID NO: 54 at position S156C.

In some embodiments, the engineered interferon is a variant of human IFN- α 2a having an Asp inserted at about position 41 in the sequence Glu-Glu-Phe-Gly-Asn-Gln (SEQ ID NO: 275) to produce Glu-Glu-Phe-Asp-Gly-Asn-Gln (SEQ ID NO: 276) (which results in renumbering the sequence relative to the IFN- α 2a sequence) and the following mutations: arg23Lys, Leu26Pro, Glu53Gln, Thr54Ala, Pro56Ser, Asp86Glu, Ile104Thr, Gly106Glu, Thr110Glu, Lys117Asn, Arg125Lys, and Lys136 Thr. All embodiments of the consensus interferon described herein are equally applicable to the engineered interferon.

In various embodiments, the consensus interferon variant 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 mutation is an amino acid substitution, and may include conservative and/or non-conservative substitutions.

In various embodiments, substitutions may also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine beta-alanine, GABA and delta-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2, 4-diaminobutyric acid, alpha-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, gamma-Abu, epsilon-Ahx, 6-aminocaproic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoroamino acids, etc, Designer amino acids such as beta-methyl amino acids, C alpha-methyl amino acids, N alpha-methyl amino acids, and amino acid analogs in general).

In various embodiments, the consensus interferon is modified to have one or more mutations. In some embodiments, the mutation results in the consensus interferon variant having one or more reduced activities, such as one or more of reduced binding affinity, reduced endogenous activity, and reduced specific biological activity, relative to the unmutated, e.g., wild-type, form of consensus interferon (e.g., consensus interferon having the amino acid sequence of SEQ ID NO: 49 or 50). For example, one or more attenuated activities, e.g., reduced binding affinity, reduced endogenous activity, and reduced specific biological activity, relative to an unmutated, e.g., wild-type, form of consensus interferon may be at a therapeutic receptor, e.g., an IFNAR. Thus, in various embodiments, the mutations result in the consensus interferon variant having reduced systemic toxicity, reduced side effects, and reduced off-target effects relative to the unmutated, e.g., wild-type, form of consensus interferon.

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

In some embodiments, the reduced affinity or activity at the therapeutic receptor is recoverable by attachment to a targeting moiety (e.g., sirpa). In other embodiments, the reduced affinity or activity at the therapeutic receptor is substantially non-recoverable by attachment to a targeting moiety. In various embodiments, the therapeutic chimeric proteins of the invention reduce off-target effects because the consensus interferon variant has a mutation that reduces binding affinity or activity at the therapeutic receptor. In various embodiments, this reduces side effects such as those observed with wild-type consensus interferon. In various embodiments, the consensus interferon variant is substantially inactive in the pathway of the therapeutically active site and exerts its effect substantially on the cell type specifically targeted, which greatly reduces undesirable side effects.

In various embodiments, the consensus interferon has one or more mutations that result in a consensus interferon variant having reduced or decreased affinity for one or more therapeutic receptors, e.g., binding (e.g., KD) and/or activation (e.g., as measurable by KA and/or EC 50). In various embodiments, the reduced affinity at the therapeutic receptor results in a reduction in activity and/or signaling from the therapeutic receptor.

In various embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity to the IFNAR1 subunit of IFNAR. In one embodiment, the consensus interferon variant has reduced affinity and/or activity at IFNAR 1. In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity to the IFNAR2 subunit of IFNAR. In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity to both IFNAR1 and IFNAR2 subunits.

In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity to IFNAR1, and one or more mutations that substantially reduce or eliminate its binding or affinity to IFNAR 2. In some embodiments, a chimeric protein having such a consensus interferon variant may provide target-selective IFNAR1 activity (e.g., IFNAR1 activity may be restored by targeting with a targeting moiety (e.g., sirpa).

In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity to IFNAR2, and one or more mutations that substantially reduce or eliminate its binding or affinity to IFNAR 1. In some embodiments, a chimeric protein having such a consensus interferon variant may provide target-selective IFNAR2 activity (e.g., IFNAR2 activity may be restored by targeting with a targeting moiety (e.g., sirpa).

In some embodiments, the consensus interferon variant has one or more mutations that reduce its binding or affinity to IFNAR1 and one or more mutations that reduce its binding or affinity to IFNAR 2. In some embodiments, chimeric proteins having such consensus interferon variants can provide target-selective IFNAR1 and/or IFNAR2 activity (e.g., IFNAR1 and/or IFNAR2 activity is restored by targeting with a targeting moiety (e.g., sirpa)).

In some embodiments, the consensus interferon is modified with reference to SEQ ID NO: 50 has a mutation at one or more of amino acids 145-155, for example at amino acids 149, 150 and/or 154. In some embodiments, the consensus interferon is modified with reference to SEQ ID NO: 50 has a mutation at one or more of amino acids 145-155, for example at amino acids 149, 150 and/or 154, said substitution optionally being hydrophobic and selected from the group consisting of alanine, valine, leucine and isoleucine. In some embodiments, the consensus interferon is given with reference to SEQ ID NO: 50 comprises one or more mutations selected from M149A, R150A, and L154A.

In one embodiment, the consensus interferon is modified to have the amino acid sequence shown in SEQ ID NO: 50 has a mutation at amino acid position 121 (i.e., K121). In one embodiment, the consensus interferon is given with reference to SEQ ID NO: 50 contained the K121E mutation.

In various embodiments, the modified signaling agent is selected from modified forms of cytokines, growth factors, and hormones. Illustrative examples of such cytokines, growth factors and hormones include, but are not limited to, lymphokines, monokines, traditional polypeptide hormones such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; pro-relaxin; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), and Luteinizing Hormone (LH); a liver growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and tumor necrosis factor-beta; a Muller inhibitor; mouse gonadotropin-related peptides; a statin; an activin; vascular endothelial growth factor; an integrin; thrombopoietin (TPO); nerve growth factors such as NGF-alpha; platelet growth factor; transforming Growth Factors (TGF) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and insulin-like growth factor-II; an osteoinductive factor; interferons such as interferon- α, interferon- β and interferon- γ (and I, II and type III interferons), Colony Stimulating Factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-1 α, 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-alpha or TNF-beta; and other polypeptide factors including, for example, LIF and Kit Ligand (KL). As used herein, cytokines, growth factors and hormones include biologically active equivalents of the proteins and native sequence cytokines obtained from natural sources or produced from recombinant bacterial, eukaryotic or mammalian cell culture systems.

in some embodiments, the modified signaling agent is a modified form of a growth factor selected from, but not limited to, Transforming Growth Factors (TGF) such as TGF-alpha and TGF-beta (and subtypes thereof, including various subtypes of TGF-beta, including TGF-beta 1, TGF-beta 2, and TGF-beta 3), Epidermal Growth Factor (EGF), insulin-like growth factors such as insulin-like growth factors-I and-II, Fibroblast Growth Factor (FGF), heregulin, Platelet Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF).

In one embodiment, the growth factor is a modified form of a Fibroblast Growth Factor (FGF). Exemplary FGFs include, but are not limited to, FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, murine FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF22, and FGF 23.

In some embodiments, the modified signaling agent is Vascular Endothelial Growth Factor (VEGF). VEGF is a potent growth factor that plays a major role in physiological and pathological angiogenesis, regulates vascular permeability, and acts as a growth factor on cells expressing VEGF receptors. Additional functions include stimulating cell migration in macrophage lineages and endothelial cells. There are several members of the VEGF family of growth factors, as well as at least three receptors (VEGFR-1, VEGFR-2, and VEGFR-3). Members of the VEGF family can bind and activate more than one VEGFR type. For example, VEGF-A binds VEGFR-1 and-2, while VEGF-C can bind VEGFR-2 and-3. VEGFR-1 and-2 activation regulates angiogenesis, while VEGFR-3 activation is associated with lymphangiogenesis. The main pro-angiogenic signal is produced by the activation of VEGFR-2. VEGFR-1 activation has been reported to be possibly associated with a negative effect in angiogenesis. VEGFR-1 signaling has also been reported to be important for the progression of tumors in vivo through bone marrow derived VEGFR-1 positive cells (contributing to the formation of pre-metastatic niches in bone). Several therapies based on VEGF-a-directed/neutralizing therapeutic antibodies have been developed, primarily for the treatment of a variety of human tumors that are dependent on angiogenesis. These are not without side effects. This may not be surprising given that these act as general, non-cell/tissue specific VEGF/VEGFR interaction inhibitors. Thus, it is desirable to limit VEGF (e.g., VEGF-A)/VEGFR-2 inhibition to specific target cells (e.g., tumor vascular endothelial cells).

In some embodiments, the VEGF is VEGF-A, VEGF-B, VEGF-C, VEGF-D or VEGF-E and subtypes thereof, including multiple subtypes of VEGF-A, such as VEGF121, VEGF121b, VEGF145, VEGF165b, VEGF189, and VEGF 206. 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 significantly reduced or eliminated affinity and/or activity for VEGFR-1(Flt-1) and/or VEGFR-2 (KDR/Flk-1). In one embodiment, the modified signaling agent has reduced affinity and/or activity for VEGFR-2(KDR/Flk-1) and/or significantly reduced or eliminated affinity and/or activity for VEGFR-1 (Flt-1). Such embodiments may be useful, for example, in methods of wound healing or treating ischemia-related diseases (not wishing to be bound by theory, mediated by the effects of VEGFR-2 on endothelial cell function and angiogenesis). In various embodiments, binding to VEGFR-1(Flt-1), which is associated with cancer and pro-inflammatory activity, is avoided. In various embodiments, VEGFR-1(Flt-1) acts as a decoy receptor, thereby significantly reducing or eliminating affinity at the receptor, avoiding sequestration of the therapeutic agent. In one embodiment, the affinity and/or activity of the modified signaling agent for VEGFR-1(Flt-1) is substantially reduced or eliminated, and/or the affinity and/or activity for VEGFR-2(KDR/Flk-1) is substantially reduced or eliminated. In some embodiments, the VEGF is VEGF-C or VEGF-D. In such embodiments, the modified signaling agent has reduced affinity and/or activity for VEGFR-3. Alternatively, the affinity and/or activity of the modified signaling agent for VEGFR-3 is substantially reduced or eliminated.

Pro-angiogenic therapies are also important in a variety of diseases (e.g., ischemic heart disease, hemorrhage, etc.) and include VEGF-based therapies. VEGFR-2 activation is pro-angiogenic (acting on endothelial cells). Activation of VEFGR-1 can cause stimulation of inflammatory cell (including, for example, macrophages) migration and result in vascular hyperpermeability associated with inflammation. Activation of VEFGR-1 may also promote bone marrow-related tumor niche formation. Thus, in this case, a VEGF-based therapy selected for VEGFR-2 activation is desired. Furthermore, cell-specific targeting, e.g., targeting of endothelial cells, is desirable.

In some embodiments, the modified signaling agent has reduced affinity and/or activity (e.g., antagonistic) for VEGFR-2 and/or substantially reduced or eliminated affinity and/or activity for VEGFR-1. When tumor vascular endothelial cells are targeted by targeting moieties that bind to tumor endothelial cell markers (e.g., PSMA and others), the construct specifically inhibits VEGFR-2 activation on the marker-positive cells, but does not activate VEGFR-1 (if activity is abolished) in the pathway and on the target cells, thereby abrogating, for example, the induction of an inflammatory response. This would provide a more selective and safer anti-angiogenic therapy for many tumor types than VEGF-a neutralizing therapy.

In some embodiments, the modified signaling agent has reduced affinity and/or activity (e.g., agonist) for VEGFR-2 and/or substantially reduced or eliminated affinity and/or activity for VEGFR-1. In some embodiments, such constructs promote angiogenesis without inducing the VEGFR-1-associated inflammatory response by targeting vascular endothelial cells. Thus, such a construct would have a targeted pro-angiogenic effect, significantly reducing the risk of side effects caused by systemic activation of VEGFR-2 as well as VEGR-1.

In an exemplary embodiment, the modified signaling agent is VEGF165, having the amino acid sequence of SEQ ID NO: 55) the amino acid sequence of (a).

In another exemplary embodiment, the modified signaling agent is VEGF165b, having the amino acid sequence of SEQ ID NO: 56.

In these embodiments, the modified signaling agent has a mutation at amino acid I83 (e.g., has a substitution mutation at I83, e.g., I83K, I83R, or I83H). Without wishing to be bound by theory, it is believed that such mutations may result in a decrease in receptor binding affinity. See, for example, U.S. patent No. 9,078,860, which is incorporated herein by reference in its entirety.

In some embodiments, the modified signaling agent is a modified form of a hormone selected from, but 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, thyroid stimulating hormone releasing hormone, growth hormone releasing hormone, adrenocorticotropic hormone releasing hormone, somatostatin, dopamine, melatonin, thyroxine, calcitonin, parathyroid hormone, glucocorticoid, mineralocorticoid, epinephrine, norepinephrine, progesterone, insulin, glucagon, amyloid toxin, calcitriol, calciferol, atrial natriuretic peptide, gastrin, secretin, cholecystokinin, neuropeptide Y, ghrelin (ghrelin), PYY3-36, a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable salt thereof, Insulin-like growth factor (IGF), leptin, thrombopoietin, Erythropoietin (EPO), and angiotensinogen.

In some embodiments, the modified signaling agent is TNF- α. TNF is a pleiotropic cytokine with many diverse functions including regulation of cell growth, differentiation, apoptosis, tumorigenesis, viral replication, autoimmunity, immune cell function and trafficking, inflammation, and septic shock. It binds to two different membrane receptors on the target cell: TNFR1(p55) and TNFR2(p 75). TNFR1 exhibits a very broad expression pattern, whereas TNFR2 is preferably expressed on certain populations of lymphocytes, tregs, endothelial cells, certain neurons, microglia, cardiomyocytes, and mesenchymal stem cells. Very different biological pathways are activated in response to receptor activation, although there is also some overlap. As a general rule, without wishing to be bound by theory, TNFR1 signaling is associated with induction of apoptosis (cell death), while TNFR2 signaling is associated with activation of cell survival signals (e.g., activation of the NFkB pathway). Administration of TNF is systemically toxic, primarily due to the involvement of TNFR 1. However, it should be noted that activation of TNFR2 is also associated with a broad range of activities, and as with TNFR1, control of TNF targeting and activity is important in the development of TNF-based therapeutics.

In some embodiments, the modified signaling agent has reduced affinity and/or activity for TNFR1 and/or TNFR 2. In some embodiments, the affinity and/or activity of the modified signaling agent for TNFR1 and/or TNFR2 is substantially reduced or eliminated. TNFR1 is expressed in most tissues and is involved in cell death signaling, in contrast to TNFR2, which is involved in cell survival signaling. Thus, in embodiments directed to methods of treating cancer, the affinity and/or activity of the modified signaling agent for TNFR1 is reduced and/or the affinity and/or activity for TNFR2 is significantly reduced or eliminated. In these embodiments, the chimeric protein may target cells in which apoptosis is desired, such as tumor cells or endothelial cells of tumor vasculature. In embodiments directed to methods of promoting cell survival, for example, in neurogenesis for treating a neurodegenerative disease, the modified signaling agent has reduced affinity and/or activity for TNFR2 and/or significantly reduced or eliminated affinity and/or activity for TNFR 1. In other words, in some embodiments, the chimeric proteins of the invention comprise a modified TNF-alpha agent that allows for promotion of death or survival signaling.

In some embodiments, the chimeric protein has a modified TNF with reduced affinity and/or activity for TNFR1 and/or significantly reduced or eliminated affinity and/or activity for TNFR 2. In some embodiments, such chimeras are more potent inducers of apoptosis than wild-type TNF and/or chimeras with only mutations that result in reduced affinity and/or activity to TNFR 1. In some embodiments, such chimeras find use in inducing tumor cell death or tumor vasculature endothelial cell death (e.g., in the treatment of cancer). Moreover, in some embodiments, these chimeras avoid or reduce activation of Treg cells by TNFR2, e.g., thus further supporting TNFR 1-mediated anti-tumor activity in vivo.

In some embodiments, the chimeric protein has a modified TNF with reduced affinity and/or activity for TNFR2 and/or significantly reduced or eliminated affinity and/or activity for TNFR 1. In some embodiments, such chimeras are more effective activators of cell survival of some cell types, which may be a specific therapeutic target in a variety of disease settings, including but not limited to stimulation of neurogenesis. In addition, such TNFR 2-preferred chimeras can also be used to treat autoimmune diseases (e.g., crohn's disease, diabetes, MS, colitis, and the like, and many other diseases described herein). In some embodiments, the chimeras target autoreactive T cells. In some embodiments, the chimeras promote Treg cell activation and indirect suppression of cytotoxic T cells.

In some embodiments, the chimeras cause death of autoreactive T cells, for example, by activating TNFR2 and/or avoiding TNFR1 (e.g., a modified TNF having reduced affinity and/or activity for TNFR2 and/or significantly reduced or eliminated affinity and/or activity for TNFR 1). Without wishing to be bound by theory, the apoptosis/survival signals of these autoreactive T cells are altered, for example, by altered NFkB pathway activity/signaling. In some embodiments, the chimeras cause death of autoreactive T cells with lesions or modifications in the nfkb pathway, demonstrating an imbalance in their cell death (apoptosis)/survival signaling properties and optionally altered sensitivity to certain death-inducing signals (e.g., TNFR2 activation).

In some embodiments, TNFR-2 based chimeras have additional therapeutic applications in diseases including autoimmune diseases, various heart diseases, demyelinating and neurodegenerative disorders, and infectious diseases, among others.

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

In such embodiments, the modified TNF- α agent has a mutation at one or more of amino acid positions 29, 31, 32, 84, 85, 86, 87, 88, 89, 145, 146, and 147 that results in a modified TNF- α with reduced receptor binding affinity. See, for example, U.S. patent No. 7,993,636, which is incorporated herein by reference in its entirety.

In some embodiments, the modified human TNF- α portion has a mutation at one or more of amino acid positions R32, N34, Q67, H73, L75, T77, S86, Y87, V91, I97, T105, P106, a109, P113, Y115, E127, N137, D143, a145, and E146, as described in WO/2015/007903, the entire contents of which are incorporated herein by reference (according to human TNF sequence numbering, Genbank accession number BAG70306, version BAG70306.1 GI: 197692685). In some embodiments, the modified human TNF- α moiety has a substitution mutation selected from the group consisting of 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, E127G, N137G, D143G, a145G, E146G, and S147G. In some embodiments, the human TNF- α moiety has a mutation selected from the group consisting of Y87Q, Y87L, Y87A, Y87F, and Y87H. In another embodiment, the human TNF- α moiety has a mutation selected from the group consisting of I97A, I97Q, and I97S. In another embodiment, the human TNF- α moiety has a mutation selected from the group consisting of Y115A and Y115G. In some embodiments, the human TNF- α portion has the E146K mutation. In some embodiments, the human TNF- α portion has the Y87H and E146K mutations. In some embodiments, the human TNF- α portion has the Y87H and a145R mutations. In some embodiments, the human TNF- α moiety has the R32W and S86T mutations. In some embodiments, the human TNF- α moiety has the R32W and E146K mutations. In some embodiments, the human TNF- α portion has the L29S and R32W mutations. In some embodiments, the human TNF- α portion has the D143N and a145R mutations. In some embodiments, the human TNF- α portion has the D143N and a145R mutations. In some embodiments, the human TNF- α moiety has the a145T, E146D, and S147D mutations. In some embodiments, the human TNF- α moiety has the a145T and S147D mutations.

In some embodiments, the modified TNF-a agent has one or more mutations selected from N39Y, S147Y, and Y87H, as described in WO2008/124086, the entire contents of which are incorporated herein by reference.

In some embodiments, the modified human TNF- α moiety has a mutation that provides receptor selectivity as described in PCT/IB2016/001668, which is incorporated herein by reference in its entirety. In some embodiments, the mutation of TNF is TNF-R1 selective. In some embodiments, TNF-R1-selective TNF mutations are at one or more of positions R32, S86, and E146. In some embodiments, the TNF-R1-selective TNF mutation is one or more of R32W, S86T, and E146K. In some embodiments, the TNF-selective mutation of TNF-R1 is one or more of R32W, R32W/S86T, R32W/E146K, and E146K. In some embodiments, the mutation of TNF is TNF-R2 selective. In some embodiments, TNF-R2-selective TNF mutations are at one or more of positions A145, E146, and S147. In some embodiments, the TNF-R2-selective TNF mutation is one or more of A145T, A145R, E146D, and S147D. In some embodiments, the TNF-R2-selective TNF mutation is one or more of A145R, A145T/S147D, and A145T/E146D/S147D.

In one embodiment, the modified signaling agent is TNF- β. TNF-beta can form homotrimers or heterotrimers with LT-beta (LT-alpha 1 beta 2). In some embodiments, the affinity and/or activity of the modified signaling agent for TNFR1 and/or TNFR2 and/or a herpes virus entry mediator (HEWM) and/or LT- β R is substantially reduced or eliminated.

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

In such embodiments, the modified TNF- β agent may comprise a mutation at one or more amino acids at position 106-113 which results in a modified TNF- β having reduced receptor binding affinity for TNFR 2. In one embodiment, the modified signaling agent has one or more substitution mutations at amino acid positions 106-113. In exemplary embodiments, the substitution mutation is 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 position 106-113.

In some embodiments, the modified agent is a TNF family member (e.g., TNF- α, TNF- β), which may be in the form of a single chain trimer as described in WO2015/007903 and PCT/IB2016/001668, the entire contents of which are incorporated herein by reference.

In some embodiments, the modified agent is a TNF family member (e.g., TNF- α, TNF- β) having reduced affinity and/or activity at TNFR1, i.e., antagonistic activity (e.g., natural antagonistic activity or antagonistic activity due to one or more mutations, see, e.g., WO2015/007520, the entire contents of which are incorporated herein by reference). In these embodiments, the modified agent is a TNF family member (e.g., TNF- α, TNF- β), which also optionally has significantly reduced or eliminated affinity and/or activity for TNFR 2. In some embodiments, the modified agent is a TNF family member (e.g., TNF- α, TNF- β) having reduced affinity and/or activity at TNFR2, i.e., antagonistic activity (e.g., natural antagonistic activity or antagonistic activity due to one or more mutations, see, e.g., WO2015/007520, the entire contents of which are incorporated herein by reference). In these embodiments, the modified agent is a TNF family member (e.g., TNF- α, TNF- β), which also optionally has significantly reduced or eliminated affinity and/or activity for TNFR 1. The constructs of these embodiments are useful, for example, in methods of inhibiting a TNF response in a cell-specific manner. In some embodiments, the antagonistic TNF family member (e.g., TNF- α, TNF- β) is in the form of a single chain trimer as described in WO 2015/007903.

In one embodiment, the modified signaling 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 DcR 2. In some embodiments, the modified TRAIL agent has significantly reduced or eliminated affinity and/or activity for DR4(TRAIL-RI) and/or DR5(TRAIL-RII) and/or DcR1 and/or DcR 2.

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

In these embodiments, the modified TRAIL agent may comprise mutations at amino acid positions T127-R132, E144-R149, E155-H161, Y189-Y209, T214-1220, K224-A226, W231, E236-L239, E249-K251, T261-H264 and H270-E271 (according to human sequence numbering, Genbank accession NP-003801, version 10 NP-003801.1, GI: 4507593; see above).

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

In some embodiments, the modified TRAIL agent may comprise one or more mutations that significantly reduce its affinity and/or activity for TRAIL-R2. In such embodiments, the modified TRAIL agent can specifically bind TRIL-R1. Exemplary mutations include mutations at one or more of amino acid positions G131, R149, S159, N199, K201, and S215. For example, the mutation may be one or more of G131R, R149I, S159R, N199R, K201H, and S215D. In one embodiment, the modified TRAIL agent comprises the mutations G131R, R149I, S159R, N199R, K201H and S215D. Other TRAIL mutations are described, for example, in trebin et al, (2014) Cell Death and Disease, 5: e1035, the entire disclosure of which is incorporated herein by reference.

In one embodiment, the modified signaling agent is TGF α. In such embodiments, the modified TGF α agent has reduced affinity and/or activity for Epidermal Growth Factor Receptor (EGFR). In some embodiments, the modified TGF α agents have a substantially reduced or eliminated affinity and/or activity for Epidermal Growth Factor Receptor (EGFR).

In one embodiment, the modified signaling agent is TGF β. In such embodiments, the modified signaling agent has reduced affinity and/or activity for TGFBR1 and/or TGFBR 2. In some embodiments, the modified signaling agent has significantly reduced or eliminated affinity and/or activity for TGFBR1 and/or TGFBR 2. In some embodiments, the modified signaling agent optionally has reduced or significantly reduced or eliminated affinity and/or activity for TGFBR3, and without wishing to be bound by theory, TGFBR3 may act as a reservoir for a ligand for the TGF β receptor. In some embodiments, TGF β may be more biased towards TGFBR1 than TGFBR2, or more biased towards TGFBR2 than TGFBR 1. Similarly, without wishing to be bound by theory, LAP may act as a reservoir for ligands of the TGF β receptor. In some embodiments, the modified signaling agent has reduced affinity and/or activity for TGFBR1 and/or TGFBR2 and/or significantly reduced or eliminated affinity and/or activity for Latency Associated Peptide (LAP). In some embodiments, such chimeras are useful for Camurati-Engelmann disease or other diseases associated with inappropriate TGF β signaling.

In some embodiments, the modified agent is a TGF family member (e.g., TGF α, TGF β) that has reduced affinity and/or activity, i.e., antagonistic activity (e.g., natural antagonistic activity or antagonistic activity as a result of one or more mutations, see, e.g., WO2015/007520, the entire contents of which are incorporated herein by reference) at one or more of TGFBR1, TGFBR2, TGFBR 3. In these embodiments, the modified agent is a TGF family member (e.g., TGF α, TGF β), which also optionally has significantly reduced or eliminated affinity and/or activity at one or more of TGFBR1, TGFBR2, TGFBR 3.

In some embodiments, the modified agent is a TGF family member (e.g., TGF α, TGF β) that has reduced affinity and/or activity, i.e., antagonistic activity, at TGFBR1 and/or TGFBR2 (e.g., natural antagonistic activity or antagonistic activity as a result of one or more mutations, see, e.g., WO2015/007520, the entire contents of which are incorporated herein by reference). In these embodiments, the modified agent is a TGF family member (e.g., TGF α, TGF β), which also optionally has significantly reduced or eliminated affinity and/or activity at TGFBR 3.

In one embodiment, the modified signaling agent is an interleukin. In one embodiment, the modified signaling agent is IL-1. In one embodiment, the modified signaling agent is IL-1 α or IL-1 β. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-1R1 and/or IL-1 RAcP. In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and/or activity for IL-1R1 and/or IL-1 RAcP. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-1R 2. In some embodiments, the affinity and/or activity of the modified signaling agent for IL-1R2 is substantially reduced or eliminated. For example, in some embodiments, the modified IL-1 agents of the invention avoid interactions at IL-1R2 and thus significantly reduce their function as decoys and/or reservoirs (sinks) for therapeutic agents.

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

IL1 is a pro-inflammatory cytokine and an important regulator of the immune system. It is a potent activator of CD 4T cell responses, increasing the proportion of Th17 cells and the expansion of IFN γ and IL-4 producing cells. IL-1 is also a potent regulator of CD8+ T cells, enhancing the expansion, differentiation, migration to the periphery and memory of antigen-specific CD8+ T cells. IL-1 receptors include IL-1R1 and IL-1R 2. Binding to IL-1R1 and signaling through IL-1R1 constitute the mechanism by which IL-1 mediates many of its biological (and pathological) activities. IL1-R2 may function as decoy receptors, thereby reducing the availability of IL-1 through IL-1R1 interactions and signaling.

In some embodiments, the modified IL-1 has reduced affinity and/or activity (e.g., agonist activity) for IL-1R 1. In some embodiments, the modified IL-1 has a significantly reduced or eliminated affinity and/or activity for IL-1R 2. In such embodiments, there is recoverable IL-1/IL-1R1 signaling and prevention of loss of the therapeutic chimera at IL-R2, and thus a reduction in the dose of IL-1 required (e.g., relative to wild-type or chimeras carrying only attenuating mutations for IL-R1). Such constructs find use in, for example, methods of treating cancer, including, for example, stimulating the immune system to mount an anti-cancer response.

In some embodiments, the modified IL-1 has reduced affinity and/or activity (e.g., antagonistic activity, e.g., natural antagonistic activity or antagonistic activity as a result of one or more mutations, see, e.g., WO2015/007520, the entire contents of which are incorporated herein by reference) for IL-1R 1. In some embodiments, the modified IL-1 has a significantly reduced or eliminated affinity and/or activity for IL-1R 2. In such embodiments, there is unrecoverable IL-1/IL-1R1 signaling and loss of the therapeutic chimera at IL-R2 is prevented, and thus the dose of IL-1 required is reduced (e.g., relative to wild-type or chimeras carrying only attenuating mutations for IL-R1). Such constructs find use, for example, in methods of treating autoimmune diseases, including, for example, suppression of the immune system.

In such embodiments, the modified signaling agent has a deletion of amino acids 52-54, which results in a modified human IL-1 β with reduced binding affinity for type I IL-1R and reduced bioactivity. See, for example, WO1994/000491, the entire contents of which are incorporated herein by reference. In some embodiments, the modified human IL-1 β has one or more substitution mutations selected from a117G/P118G, R120X, L122A, T125G/L126G, R127G, Q130X, Q131X, K132X, S137X/Q138X, L145X, H146X, L145X/L147X, Q148X/Q150X, Q150X/D151X, M152X, F162X/Q164, F166X, Q164X/E167X, N169X/D170X, I172X, V174X, K208, K209X, K209/K X, K36219, E221/N221, E221/N X, NP 36245 NP X, NP-NP X, NP 36244, GI: 10835145). In some embodiments, the modified human IL-1 β may have one or more mutations selected from R120A, R120G, Q130A, Q130W, H146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209A, K209D, K219S, K219Q, E221S, and E221K. In one embodiment, the modified human IL-1 β comprises the mutations Q131G and Q148G. In one embodiment, the modified human IL-1 β comprises the mutations Q148G and K208E. In one embodiment, the modified human IL-1 β comprises the mutations R120G and Q131G. In one embodiment, the modified human IL-1 β comprises the mutations R120G and H146A. In one embodiment, the modified human IL-1 β comprises the mutations R120G and H146N. In one embodiment, the modified human IL-1 β comprises the mutations R120G and H146R. In one embodiment, the modified human IL-1 β comprises the mutations R120G and H146E. In one embodiment, the modified human IL-1 β comprises the mutations R120G and H146G. In one embodiment, the modified human IL-1 β comprises the mutations R120G and K208E. In one embodiment, the modified human IL-1 β comprises the mutations R120G, F162A, and Q164E.

In one embodiment, the modified signaling agent is IL-2. In such embodiments, the modified signaling agent has reduced affinity and/or activity for IL-2R α and/or IL-2R β and/or IL-2R γ. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-2R β and/or IL-2R γ. In some embodiments, the affinity and/or activity of the modified signaling agent for IL-2 ra is substantially reduced or eliminated. Such embodiments may be relevant to the treatment of cancer, for example, when the modified IL-2 is agonistic at IL-2R β and/or IL-2R γ. For example, the constructs of the invention may favour attenuated activation of CD8+ T cells with IL2 receptors β and γ (which may provide an anti-tumour effect) and against tregs with IL2 receptors α, β and γ (which may provide an immunosuppressive pro-tumour effect). Furthermore, in some embodiments, the preference for IL-2R β and/or IL-2R γ over IL-2R α avoids IL-2 side effects such as pulmonary edema. Furthermore, IL-2 based chimeras may be useful in the treatment of diseases (e.g., autoimmune diseases), for example when the modified IL-2 is antagonistic at IL-2R β and/or IL-2R γ (e.g., natural antagonistic activity or antagonistic activity caused by one or more mutations, see e.g., WO2015/007520, the entire contents of which are incorporated herein by reference). For example, the constructs of the invention may favour the attenuated suppression of CD8+ T cells bearing IL2 receptors β and γ (and hence the suppression of the immune response) over tregs bearing IL2 receptors α, β and γ. Alternatively, in some embodiments, chimeras with IL-2 favor activation of tregs and thus immunosuppression, as well as disfavor activation of CD8+ T cells. For example, these constructs may be used to treat diseases or disorders that would benefit from immunosuppression, such as autoimmune disorders.

In some embodiments, the chimeric protein has a targeting moiety described herein for CD8+ T cells, and a modified IL-2 agent with reduced affinity and/or activity for IL-2R β and/or IL-2R γ and/or significantly reduced or eliminated affinity and/or activity for IL-2R α. In some embodiments, these constructs provide targeted CD8+ T cell activity, and are generally inactive (or significantly reduced in activity) against Treg cells. In some embodiments, such constructs have enhanced immunostimulatory effects (e.g., without wishing to be bound by theory, by not stimulating tregs) compared to wild-type IL-2, while eliminating or reducing systemic toxicity associated with IL-2.

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

In such embodiments, the modified IL-2 agent has one or more mutations at amino acids L72(L72G, L72A, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, or L72K), F42(F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, or F42K) and Y45(Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, or Y45K). Without wishing to be bound by theory, it is believed that these modified IL-2 agents have reduced affinity for the high affinity IL-2 receptor and retained affinity for the intermediate affinity IL-2 receptor compared to wild-type IL-2. See, for example, U.S. patent publication No. 2012/0244112, which is incorporated herein by reference in its entirety.

In some embodiments, the modified IL-2 agent has one or more mutations at amino acids R38, F42, Y45, and E62. For example, a modified IL-2 agent may comprise one or more of R38A, F42A, Y45A, and E62A. In some embodiments, the modified IL-2 agent may comprise a mutation at C125. For example, the mutation may be C125S. In such embodiments, The modified IL-2 agent may have a significantly reduced affinity and/or activity for IL-2R α, as described, for example, in Carmenate et al (2013) The Journal of Immunology,190: 6230-. In some embodiments, a modified IL-2 agent having a mutation at R38, F42, Y45, and/or E62 is capable of inducing expansion of effector cells, including CD8+ T cells and NK cells but not Treg cells. In some embodiments, a modified IL-2 agent having a mutation at R38, F42, Y45, and/or E62 is less toxic than a wild-type IL-2 agent. Chimeric proteins comprising modified IL-2 agents with significantly reduced affinity and/or activity for IL-2 ra may find application, for example, in oncology.

In other embodiments, the modified IL-2 agent may have a significantly reduced affinity and/or activity for IL-2R β, as described, for example, in WO2016/025385, the entire disclosure of which is incorporated herein by reference. In such embodiments, the modified IL-2 agent may induce expansion of Treg cells, but not effector cells such as CD8+ T cells and NK cells. Chimeric proteins comprising modified IL-2 agents with significantly reduced affinity and/or activity for IL-2R β may find application, for example, in the treatment of autoimmune diseases. In some embodiments, the modified IL-2 agent may comprise one or more mutations at amino acids N88, D20, and/or a 126. For example, a modified IL-2 agent may comprise one or more of N88R, N88I, N88G, D20H, Q126L, and Q126F.

In various embodiments, the modified IL-2 agent may comprise a mutation at D109 or C125. For example, the mutation may be D109C or C125S. In some embodiments, modified IL-2 with mutations at D109 or C125 can be used to bind PEG moieties.

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

In one embodiment, the modified signaling agent is IL-4. In such embodiments, the modified signaling agent has reduced affinity and/or activity for a type 1 and/or type 2 IL-4 receptor. In such embodiments, the modified signaling agent has significantly reduced or eliminated affinity and/or activity for type 1 and/or type 2 IL-4 receptors. The type 1 IL-4 receptor consists of IL-4R α subunits having a common γ chain and specifically binds IL-4. The type 2 IL-4 receptor includes an IL-4R α subunit that binds to a different subunit called IL-13R α 1. In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and/or activity for the type 2 IL-4 receptor.

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

In such embodiments, the modified IL-4 agent has one or more mutations at amino acids R121(R121A, R121D, R121E, R121F, R121H, R121I, R121K, R121N, R121P, R121T, R121W), E122(E122F), Y124(Y124A, Y124Q, Y124R, Y124S, Y124T), and S125 (S125A). Without wishing to be bound by theory, it is believed that these modified IL-4 agents retain activity mediated by type I receptors, but significantly reduce biological activity mediated by other receptors. See, for example, U.S. patent No. 6,433,157, which is incorporated herein by reference in its entirety.

In one embodiment, the modified signaling agent is IL-6. IL-6 signals through a cell surface type I cytokine receptor complex that includes a ligand-binding IL-6R chain (CD126) and a signal transduction component gp 130. IL-6 can also bind to a soluble form of IL-6R (sIL-6R), which is the extracellular portion of IL-6R. The sIL-6R/IL-6 complex may be involved in axonal growth and survival of neurons and, therefore, may be important in nerve regeneration through 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 significantly reduced or eliminated affinity and/or activity for IL-6R/gp130 and/or sIL-6R.

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

In such embodiments, the modified signaling agent has one or more mutations at amino acids 58, 160, 163, 171, or 177. Without wishing to be bound by theory, it is believed that these modified IL-6 agents exhibit reduced binding affinity and reduced bioactivity for IL-6 ra. See, for example, WO97/10338, the entire contents of which are incorporated herein by reference.

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

In one embodiment, the modified signaling agent is IL-11. In such embodiments, the modified signaling agent has reduced affinity and/or activity for IL-11R α and/or IL-11R β and/or gp 130. In such embodiments, the modified signaling agent has significantly reduced or eliminated affinity and/or activity for IL-11R α and/or IL-11R β and/or gp 130.

In one embodiment, the modified signaling agent is IL-12. In such embodiments, the modified signaling agent has reduced affinity and/or activity for IL-12R β 1 and/or IL-12R β 2. In such embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and/or activity for IL-12R β 1 and/or IL-12R β 2.

In one embodiment, the modified signaling agent is IL-13. In such embodiments, the modified signaling agent has reduced affinity and/or activity for IL-4 receptor (IL-4R α) and IL-13R α 1. In some embodiments, the modified signaling agent has significantly reduced affinity and/or activity for IL-4 receptor (IL-4R α) or IL-13R α 1.

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

In such embodiments, the modified IL-13 agent has one or more mutations at amino acids 13, 16, 17, 66, 69, 99, 102, 104, 105, 106, 107, 108, 109, 112, 113, and 114. Without wishing to be bound by theory, it is believed that these modified IL-13 agents exhibit reduced biological activity. See, for example, WO2002/018422, the entire contents of which are incorporated herein by reference.

In one embodiment, the modified signaling agent is IL-18. In some embodiments, the modified signaling agent has reduced affinity and/or activity for IL-18R α and/or IL-18R β. In some embodiments, the modified signaling agent has a substantially reduced or eliminated affinity and/or activity for IL-18R α and/or IL-18R β. In some embodiments, the modified signaling agent has significantly reduced or eliminated affinity and/or activity for type II IL-18 Ra, which is a subtype of IL-18 Ra that lacks a TIR domain required for signaling.

In one embodiment, wild-type IL-18 has the amino acid sequence of SEQ ID NO: 65, or a pharmaceutically acceptable salt thereof.

In such embodiments, the modified IL-18 agent may comprise one or more mutations in an amino acid or amino acid region selected from the group consisting of Y37-K44, R49-Q54, D59-R63, E67-C74, R80, M87-A97, N127-K129, Q139-M149, K165-K171, R183, and Q190-N191, as described in WO/2015/007542, the entire contents of which are incorporated herein by reference (based on human IL-18 sequence numbering, Genbank accession No. AAV38697, AAV38697.1 version, GI: 54696650).

In one embodiment, the modified signaling agent is IL-33. In such embodiments, the modified signaling agent has reduced affinity and/or activity for the ST-2 receptor and IL-1 RAPcP. In some embodiments, the modified signaling agent has significantly reduced or eliminated affinity and/or activity for ST-2 receptor and IL-1 RAPcP.

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

In such embodiments, the modified IL-33 agent may comprise one or more mutations in an amino acid or amino acid region selected from the group consisting of I113-Y122, S127-E139, E144-D157, Y163-M183, E200, Q215, L220-C227, and T260-E269, as described in WO/2015/007542, the entire contents of which are incorporated herein by reference (based on human sequence numbering, Genbank accession No. NP-254274, version NP-254274.1, GI: 15559209).

In one embodiment, the modified signaling agent is Epidermal Growth Factor (EGF). EGF is a member of the efficient growth factor family. Members include EGF, HB-EGF, and others such as TGF alpha, amphiregulin, neuregulin, epidermal regulatory protein, betacellulin. EGF family receptors include EGFR (ErbB1), ErbB2, ErbB3, and ErbB 4. These may function as homodimeric and/or heterodimeric receptor subtypes. Different EGF family members exhibit different selectivity for multiple receptor subtypes. For example, EGF is associated with ErbB1/ErbB1, ErbB1/ErbB2, ErbB4/ErbB2, and some other heterodimer isoforms. HB-EGF has a similar pattern, although it is also associated with ErbB 4/4. There is considerable therapeutic interest in the positive or negative regulation of EGF (EGF-like) growth factor signaling. For example, inhibition of EGFR signaling is of interest in the treatment of various cancers in which EGFR signaling constitutes the primary growth promoting signal. Alternatively, stimulation of EGFR signaling is of therapeutic interest in, for example, promoting wound healing (acute and chronic), oral mucositis (a major side effect of various cancer treatments, including but not limited to radiation therapy).

In some embodiments, the modified signaling agent has reduced affinity and/or activity for ErbB1, ErbB2, ErbB3, and/or ErbB 4. These embodiments are useful, for example, in methods of 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 such embodiments, the modified signaling agent has reduced affinity and/or activity for ErbB1, ErbB2, ErbB3, and/or ErbB4, which results in the activity of the receptor being antagonized in a reduced manner. These embodiments are useful, for example, in the treatment of cancer. In one embodiment, the modified signaling agent has reduced affinity and/or activity for ErbB 1. ErbB1 are therapeutic targets for kinase inhibitors, most of which have side effects because they are not very selective (e.g., gefitinib, erlotinib, afatinib, brigatinib, and ercetinib). In some embodiments, the attenuated antagonistic ErbB1 signaling is more targeted than other agents that target the EGF receptor and has fewer side effects.

In some embodiments, the affinity and/or activity (e.g., antagonistic, e.g., natural antagonistic activity or antagonistic activity as a result of one or more mutations, see, e.g., WO2015/007520, the entire contents of which are incorporated herein by reference) of the modified signaling agent for ErbB1 is reduced and/or the affinity and/or activity for ErbB4 or other subtypes with which it may interact is substantially reduced or eliminated. By specific targeting of the targeting moiety, cell-selective inhibition of ErbB1/ErbB1 receptor activation (antagonism, e.g., natural antagonism or antagonism resulting from one or more mutations, see, e.g., WO2015/007520, the entire contents of which are incorporated herein by reference) can be achieved without binding to 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 vivo, such constructs will provide anti-EGFR (ErbB1) drug effects with reduced cell selectivity (e.g., tumor cells with activated EGFR signaling due to receptor amplification, overexpression, etc.), side effects.

In some embodiments, the modified signaling agent has reduced affinity and/or activity (e.g., agonist) for ErbB4 and/or other isoforms with which it may interact. Selective activation of ErbB1 signaling (e.g., epithelial cells) is achieved by targeting a specific target cell with a targeting moiety. In some embodiments, such constructs are useful for treating wounds (promoting wound healing) with reduced side effects, particularly for treating chronic conditions and for administration other than topical administration of therapeutic agents (e.g., systemic wound healing).

in one embodiment, the modified signaling 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 IGF1 or IGF2 receptor. In some embodiments, the modified insulin or insulin analog has significantly reduced or eliminated affinity and/or activity for the insulin receptor and/or IGF1 or IGF2 receptor. Impaired response at the insulin receptor may control diabetes, obesity, metabolic disorders, etc., while being remote from IGF1 or IGF2 receptors, avoiding pro-cancer (pro-cancer) effects.

In one embodiment, the modified signaling agent is insulin-like growth factor-I or insulin-like growth factor-II (IGF-1 or IGF-2). In one embodiment, the modified signaling agent is IGF-1. In such embodiments, the modified signaling agent has reduced affinity and/or activity for the insulin receptor and/or IGF1 receptor. In one embodiment, the modified signaling agent may bind to the IGF1 receptor and antagonize the activity of the receptor. In such embodiments, the modified signaling agent has reduced affinity and/or activity for the IGF1 receptor, which allows the activity of the receptor to be antagonized in a reduced manner. In some embodiments, the modified signaling agent has significantly reduced or eliminated affinity and/or activity for the insulin receptor and/or IGF1 receptor. In some embodiments, the modified signaling agent has reduced affinity and/or activity for the IGF2 receptor, which allows the activity of the receptor to be antagonized in a reduced manner. In one embodiment, the modified signaling agent has a substantially reduced or eliminated affinity and/or activity for the insulin receptor and therefore does not interfere with insulin signaling. In various embodiments, this is applicable to cancer therapy. In various embodiments, the agents of the invention can prevent IR subtype a from causing resistance to cancer therapy.

in some embodiments, the modified signaling agent is EPO. In various embodiments, the modified EPO agents have reduced affinity and/or activity for EPO receptor (EPOR) and/or ephrin receptor (EphR) relative to wild-type EPO or other EPO-based agents described herein. In some embodiments, the modified EPO agent has a substantially reduced or abolished affinity and/or activity for EPO receptor (EPOR) and/or Eph receptor (EphR). Exemplary EPO receptors include, but are not limited to, EPOR homodimers or EPOR/CD131 heterodimers. Also included as EPO receptors are the beta-co-receptors (β cR). Exemplary Eph receptors include, but are not limited to, EPHA1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHA9, EPHA10, EPHB1, EPHB2, EPHB3, EPHB4, EPHB5, and EPHB 6. In some embodiments, the modified EPO protein comprises one or more mutations that result in a EPO protein having a reduced affinity for a receptor comprising one or more different EPO receptors or Eph receptors (e.g., heterodimers, heterotrimers, and the like, including but not limited to EPOR-EPHB4, EPOR- β cR-EPOR). Receptors of EP patent publication No. 2492355 (the entire contents of which are incorporated herein by reference) are also provided, including but not limited to NEPOR.

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

In some embodiments, the human EPO protein is the mature form of EPO (with the signal peptide cleaved off), which is a polypeptide having the sequence of SEQ ID NO: 68, of 166 amino acid residues.

The structure of the human EPO protein is expected to include four helical bundles, including helix A, B, C and D. In various embodiments, the modified EPO protein comprises one or more mutations in four regions of the EPO protein 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 at residues 11-15, 44-51, 100-108 and 147-151. These residues are located on 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 (residues 42-51). In some embodiments, the modified EPO protein comprises residues between amino acids 41-52 and mutations in amino acids 147, 150, 151, and 155. Without wishing to be bound by theory, it is believed that mutation of these residues has a significant effect on both receptor binding and in vitro biological activity. In some embodiments, the modified EPO protein comprises mutations at residues 11, 14, 15, 100, 103, 104, and 108. Without wishing to be bound by theory, it is believed that mutations of these residues have a modest effect on receptor binding activity and have a much greater effect on in vitro biological activity. Exemplary substitutions include, but are not limited to, one or more of Val11Ser, Arg14Ala, Arg14Gln, Tyr15lle, Pro42Asn, Thr44lle, Lys45Asp, Val46Ala, Tyr51Phe, Ser100Glu, Ser100Thr, Arg103Ala, Ser104lle, Ser104Ala, Leu108Lys, Asn147Lys, Arg150Ala, Gly151Ala, and Leu155 Ala.

In some embodiments, the modified EPO protein comprises a mutation that affects biological activity without affecting binding, e.g., Eliot et al Mapping of the Active Site of a Recombinant Human erythropoetin 1997 1, 15; blood:89(2), the entire contents of which are incorporated herein by reference.

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

in some embodiments, the modified EPO protein comprises 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 expected to be in cryptic positions and thus indirectly affect biological activity.

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

Other EPO mutations that may 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.

In one embodiment, the chimeric protein of the invention has (i) a targeting moiety that is SIRP1a and (ii) a targeting moiety that is directed against a tumor cell, as well as any modified or mutated signaling agent described herein. In one embodiment, the chimeric protein of the invention has a targeting moiety to SIRP1a on macrophages and a second targeting moiety to PD-L1 or PD-L2 on tumor cells.

In various embodiments, the signaling agent is a toxin or a toxic enzyme. In some embodiments, the toxin or toxic enzyme is derived from plants and bacteria. Exemplary toxins or toxic enzymes include, but are not limited to, diphtheria toxin, pseudomonas toxin, anthrax toxin, Ribosome Inactivating Proteins (RIP) such as ricin and saporin, moducin, abrin, gelonin, and pokeweed antiviral protein. Other toxins include Mathew et al, (2009) Cancer Sci 100(8):1359-65, the entire disclosure of which is incorporated herein by reference. In these embodiments, the chimeric proteins of the invention can be used to induce cell death in a cell-type specific manner. In such embodiments, the toxin may be modified, e.g., mutated, to reduce the affinity and/or activity of the toxin to achieve an attenuated effect, as described herein for other signaling agents.

Multispecific chimeras and fusions with signaling agents

In various embodiments, the chimeric proteins of the invention comprise one or more signaling agents described herein and/or one or more additional targeting moieties (i.e., in addition to a targeting moiety to SIRP1 a). Accordingly, the present invention provides chimeric or fusion proteins comprising one or more signaling agents, a targeting moiety for SIRP 1a, and/or one or more additional targeting moieties.

In various embodiments, the chimeric proteins of the invention have targeting moieties that target two different cells (e.g., produce synapses) or the same cell (e.g., achieve a more focused signaling agent effect).

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 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 or different antigens or different receptors. In various embodiments, such multispecific chimeric proteins exhibit advantageous properties, such as increased avidity and/or improved selectivity. In one embodiment, the chimeric protein of the invention comprises two targeting moieties and is bispecific, i.e. binds and recognizes two epitopes on the same antigen or on different antigens or on different receptors.

In various embodiments, the multispecific chimeric proteins of the present invention comprise two or more targeting moieties, each targeting moiety being 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) comprising an antigen recognition domain against SIRP1 α and one antibody or antibody derivative comprising a recognition domain against a tumor antigen.

In various embodiments, the multispecific chimeric proteins of the present invention have two or more targeting moieties that target different antigens or receptors, and one targeting moiety may be attenuated for its antigen or receptor, e.g., the targeting moiety binds its antigen or receptor with low affinity or avidity (including, e.g., less than the affinity or avidity of the other targeting moiety for its antigen or receptor, e.g., the difference between binding affinities may be about 10-fold, or 25-fold, or 50-fold, or 100-fold, or 300-fold, or 500-fold, or 1000-fold, or 5000-fold; e.g., a lower affinity or avidity targeting moiety may bind its antigen or receptor with a KD in the mid to high nM or low to mid μ M range, while a higher affinity or avidity targeting moiety may bind its antigen or receptor with a KD in the mid to high pM or low to mid nM range). For example, in some embodiments, multispecific chimeric proteins of the invention comprise attenuated targeting moieties directed against promiscuous antigens or receptors, which can improve targeting to target cells (e.g., by other targeting moieties) and prevent effects across multiple types of cells, including those that are not targeted for therapy (e.g., by binding to a promiscuous antigen or receptor with higher affinity than provided in these embodiments).

Multispecific chimeric proteins of the invention can be constructed using methods known in the art, see, e.g., U.S. patent No. 9,067,991, U.S. patent publication No. 20110262348, and WO2004/041862, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, multispecific chimeric proteins of the invention comprising two or more targeting moieties may be constructed by chemical crosslinking, e.g., by reaction of amino acid residues with an organic derivatizing agent, as described by Blattler et al, Biochemistry 24,1517-1524 and EP294703, the entire contents of which are incorporated herein by reference. In another exemplary embodiment, a multispecific chimeric protein comprising two or more targeting moieties is constructed by genetic fusion, i.e., the construction of a single polypeptide comprising polypeptides of separate targeting moieties. For example, a single polypeptide construct may be formed that encodes a first antibody or antibody derivative (e.g., VHH) having an antigen recognition domain against SIRP1 α and a second antibody or antibody derivative having a recognition domain against a tumor antigen. PCT patent application WO96/34103, the entire content of which is incorporated herein by reference, discloses a method for producing bivalent or multivalent VHH polypeptide constructs. In another exemplary embodiment, the multispecific chimeric protein of the present invention may 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 against SIRP1 α may be linked to the amino terminus of a second antibody or antibody derivative having a recognition domain against a tumor antigen (or vice versa). Exemplary linkers that may 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.

In various embodiments, the multispecific chimeric proteins of the present invention recognize and bind SIRP1a and one or more antigens found on one or more immune cells, which may include, but are not limited to, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer cells, T lymphocytes (e.g., cytotoxic T lymphocytes, T helper cells, natural killer T cells), B lymphocytes, plasma cells, dendritic cells, or subpopulations thereof. In some embodiments, the chimeric protein specifically binds to an antigen of interest and is effective to recruit one or more immune cells, either directly or indirectly.

In various embodiments, the multispecific chimeric proteins of the present invention recognize and bind SIRP 1a and one or more antigens found on tumor cells. In these embodiments, the chimeric proteins of the invention can recruit immune cells (e.g., macrophages) directly or indirectly to tumor cells or the tumor microenvironment. In such embodiments, the chimeric proteins of the invention enhance phagocytosis of tumor cells by macrophages.

In some embodiments, the chimeric proteins of the invention can be used or found useful in methods involving altering the balance of immune cells to facilitate immune attack of a tumor. For example, the chimeric proteins of the invention can alter the proportion of immune cells at a clinically important site in favor of cells that can kill and/or inhibit tumors (e.g., anti-tumor macrophages (e.g., M1 macrophages), T cells, cytotoxic T lymphocytes, T helper cells, Natural Killer (NK) cells, natural killer T (nkt) cells, B cells, and dendritic cells) and against cells that protect tumors (e.g., bone marrow-derived suppressor cells (MDSCs), regulatory T cells (tregs), tumor-associated neutrophils (TAN), M2 macrophages, tumor-associated macrophages (TAMs), or subpopulations thereof). In some embodiments, the chimeric proteins of the invention are capable of increasing the ratio of effector T cells to regulatory T cells.

In some embodiments, the multispecific chimeric proteins of the present invention comprise a targeting moiety with a recognition domain that specifically binds to a target (e.g., antigen or receptor) associated with a tumor cell. In some embodiments, the targeting moiety recruits tumor cells directly or indirectly. For example, in some embodiments, recruitment of tumor cells is to one or more effector cells (e.g., macrophages) capable of phagocytosing, killing, and/or inhibiting the tumor cells.

Tumor cells or cancer cells refer to the uncontrolled growth and/or abnormal increase in cell survival and/or inhibition of apoptosis of cells or tissues that interfere with the normal function of body organs and systems. For example, tumor cells include benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases. Exemplary tumor cells include, but are not limited to, the following: basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancers of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); a glioblastoma; hepatocellular carcinoma (hepatic carcinoma); liver cancer (hepatoma); intraepithelial neoplasms; renal cancer (kidney cancer) or renal cancer (renal cancer); laryngeal cancer; leukemia; liver cancer (liver cancer); lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; a myeloma cell; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; a sarcoma; skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas include Hodgkin's and non-Hodgkin's lymphomas, as well as B-cell lymphomas (including low grade/follicular non-Hodgkin's lymphomas (NHLs), Small Lymphocytic (SL) NHLs, intermediate grade/follicular NHLs, intermediate grade diffuse NHLs, high grade immunoblastic NHLs, high grade lymphoblastic NHLs, high grade small non-nucleated NHLs, large masses (bulk diseases) NHLs, mantle cell lymphomas, AIDS related lymphomas, and Wallace macroglobulinemia, Chronic Lymphocytic Leukemia (CLLs), Acute Lymphoblastic Leukemia (ALL), hairy cell leukemia, chronic myeloblastic leukemia, as well as other carcinomas and sarcomas, and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular hyperplasia associated with nevus hamartoma, edema (e.g., edema associated with brain tumors), and Meger's syndrome.

Tumor cells or cancer cells also include, but are not limited to, epithelial cell carcinomas (carcinoma), such as various subtypes including, for example, adenocarcinomas, basal cell carcinomas, squamous cell carcinomas, and transitional cell carcinomas), sarcomas (sarcomas) (including, for example, bone and soft tissue), leukemias (including, for example, acute bone marrow, acute lymphoblasts, chronic bone marrow, chronic lymphocytes and hair cells), lymphomas and myelomas (including, for example, hodgkin's and non-hodgkin's lymphomas, light chain non-secretory MGUS, and plasmacytomas), and central nervous system carcinomas (including, for example, brain (such as gliomas (e.g., astrocytomas, oligodendrogliomas, and ependymomas), meningiomas, pituitary adenomas, and neuroma (e.g., meningiomas and neurofibromas).

Exemplary tumor antigens include, but are not limited to, MART-1/Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin b, colorectal-associated antigen (CRC) -0017-1A/GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor/CD 3-zeta chain, tumor antigen MAGE family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2(MAGE-B2), MAGE-Xp3(MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), tumor antigen GAGE families (e.g. GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, E-1, NAG, GnT-V, MUM-1, CDK 38, tyrosinase, p53, MUC family, HER2/neu, p21ras, RCAS1, alpha-fetoprotein, E-calnexin, alpha-catenin, beta-catenin and gamma-catenin, NYth-catenin, Pyjn 120, Pcyl protein, Pcel-APC-100, Pmel protein, Pcel-2, Pmel-protein, Pcel-2, Pcel-protein, Pcel-Pcel, Pcel-3, Pcl protein, Pcl 3, and its derivatives, The cell-lining proteins, connexin 37, Ig-idiotypes, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papilloma virus proteins, tumor antigen Smad family, lmp-1, NA, EBV-encoded nuclear antigen (EBNA) -1, brain glycogen phosphorylase, SSX-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 TNFRSF 17. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these tumor antigens.

In some embodiments, the multispecific chimeric proteins of the present invention recognize and bind SIRP 1a as well as antigens on tumor cells. In some embodiments, the multispecific chimeric protein recruits macrophages, directly or indirectly, to a tumor cell or tumor microenvironment.

In some embodiments, the multispecific chimeric proteins of the present invention comprise a targeting moiety with a recognition domain that specifically binds to a target (e.g., antigen or receptor) associated with a T cell. In some embodiments, the targeting moiety recruits T cells directly or indirectly. In one embodiment, the antigen recognition domain specifically binds to effector T cells. In some embodiments, the antigen recognition domain recruits effector T cells directly or indirectly, e.g., in some embodiments, recruits effector T cells to a treatment site (e.g., with the location of one or more disease cells or cells to be modulated to obtain a therapeutic effect). Exemplary effector T cells include cytotoxic T cells (e.g., α β TCR, CD3+, CD8+, CD45RO +); CD4+ effector T cells (e.g.,. alpha. beta.TCR, CD3+, CD4+, CCR7+, CD62Lhi, IL-7R/CD127 +); CD8+ effector T cells (e.g.,. alpha. beta.TCR, CD3+, CD8+, CCR7+, CD62Lhi, IL-7R/CD127 +); effector memory T cells (e.g., CD62L low, CD44+, TCR, CD3+, IL-7R/CD127+, IL-15R +, CCR7 low); central memory T cells (e.g., CCR7+, CD62L +, CD27 +; or CCR7hi, CD44+, CD62Lhi, TCR, CD3+, IL-7R/CD127+, IL-15R +); CD62L + effector T cells; CD8+ effector memory T cells (TEM), including early effector memory T cells (CD27+ CD62L-) and late effector memory T cells (CD27-CD62L-) (TemE and TemL, respectively); CD127(+) CD25 (low /) effector T cells; CD127(-) CD25(-) effector T cells; CD8+ stem cell memory effector cells (TSCMs) (e.g., CD44 (low) CD62L (high) CD122 (high) sca (+); TH1 effector T cells (e.g., CXCR3+, CXCR6+, and CCR5 +; or α β TCR, CD3+, CD4+, IL-12R +, IFN γ R +, CXCR3+), TH2 effector T cells (e.g., CCR3+, CCR4+, and CCR8 +; or α β TCR, CD3+, CD4+, IL-4R +, IL-33R +, CCR4+, IL-17RB +, CRTH2 +); TH9 effector T cells (e.g., α β TCR, CD3+, CD4 +); TH17 effector T cells (e.g.. alpha. beta.TCR, CD3+, CD4+, IL-23R +, CCR6+, IL-1R +); CD4+ CD45RO + CCR7+ effector T cells, ICOS + effector T cells; CD4+ CD45RO + CCR7(-) effector T cells; and IL-2, IL-4 and/or IFN-gamma secreting effector T cells.

Exemplary T cell antigens of interest include, for example (and including the extracellular domain, if applicable): CD8, CD3, SLAMF4, IL-2 Ra, 4-1BB/TNFRSF9, IL-2 Rbeta, ALCAM, B7-1, IL-4R, B7-H3, BLAME/SLAMF, CEACAM1, IL-6R, CCR3, IL-7 Ra, CCR4, CXCRl/IL-S RA, CCR5, CCR6, IL-10 Ra, CCR7, IL-l 0 Rbeta, CCRS, IL-12 Rbeta 1, CCR7, IL-12 Rbeta 2, CD7, IL-13 Ralpha 1, IL-13, CD7, ILT 7/CDS 5/CDS 72, ILT 7/CDS 57, ILT 7/CDS 5, luteolin (telugnin) alpha 4/CD 7, CDS 72/CDS 72, integrin alpha/7, CD 7/CDS 11/CD 7, integrin/7, CD 7/CDS 11/CD 7, integrin alpha/11, CDS, CD27/TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30/TNFRSF, KIR2DL4/CD15Sd, CD31/PECAM-1, KIR2DS4, CD40 ligand/TNFRSF 5, LAG-3, CD43, LAIR1, CD45, LAIR2, CDS 2, leukotriene B2-R2, CDS 2/SLAMF 2, NCAM-L2, CD2, NKG 22, CD2, NKNKG 22, CD229/SLAMF 2, NKG 22, CD2 2-10/SLAMF 2, NT-4, CD2, NTB-A/SLAMF 2, common gamma chain/IL-2R gamma, osteopontin, FasCCC/2, CRACAM 1-CX-1, CTCR 3, CTCXCR 1/TCCR 3, CTCR 11, CTMCR-TCCR 3, CTFR-2, CTLA-2, CTLA-11, CTLA-2, CTCR 3-2, CTLA-2, CTCR 1-11, CTLA-2, CTCR 3-2, CTLA-11, CTLA-2, CTMCR gamma-2, CTMCR-2, CTLA-2, CTMCR gamma-36, TIM-4, Fc gamma RIII/CD16, TIM-6, TNFR1/TNFRSF1A, granulysin, TNF RIII/TNFRSF1B, TRAIL Rl/TNFRSF10A, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102, TRAIL R3/TNFRSF10C, IFN-gamma R1, TRAIL R4/TNFRSF10D, IFN-gamma R2, TSLP, IL-1R1 and TSLP R. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these exemplary T cell antigens.

As a non-limiting example, in various embodiments, the chimeric proteins of the invention have targeting moieties directed against checkpoint markers expressed on T cells, such as one or more of PD-1, CD28, CTLA4, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, and A2 aR.

In some embodiments, the multispecific chimeric proteins of the present invention comprise a targeting moiety with a recognition domain that specifically binds to a target (e.g., antigen or receptor) associated with a B cell. In some embodiments, the targeting moiety recruits B cells directly or indirectly, e.g., in some embodiments, to a treatment site (e.g., a site with one or more disease cells or cells to be modulated for a therapeutic effect). Exemplary B cell antigens of interest include, for example, CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CD 75, CDw76, CD77, CD78, CD79a/B, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDw130, CD138, and CDw 150. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these exemplary B cell antigens.

In some embodiments, the multispecific chimeric proteins of the present invention comprise a targeting moiety with a recognition domain that specifically binds to a target (e.g., antigen or receptor) associated with a natural killer cell. In some embodiments, the targeting moiety recruits natural killer cells directly or indirectly, e.g., in some embodiments, recruits natural killer cells to the treatment site (e.g., with one or more disease cells or location of cells to be modulated to obtain a therapeutic effect). Exemplary 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, Kir1 α, DNAM-1, LMIR5/CD300LB, Fc- εRII, LMIR LB/CD 300LB, Fc- γ RI/CD LB, MICA, Fc- γ RII/CD 32 LB, MICB, Fc- γ RIIC/CD32 LB, FcRIIC/CD 32 LB, FcRIIA/CD 32 LB, FcRIII/CD LB, linker-2/CD 36112, Fc- γ RIII/CD LB, RaG 2 LB, NKH LB/IRTA LB, NKH 2/NKTA 2, NKH 2/LB, NKTA-IRTA-LB, NKIRTA- α IRP LB, NKIRCR LB, NKIRF-LB, NKIRCR-LB, NKIRC-LB, NKIRF-LB, NKIRC-LB-like, NKIRF/LB, NKIRF-LB, NKIR, Rae-1 beta, Rae-1 delta, H60, Rae-1 epsilon, ILT2/CD85j, Rae-1 gamma, 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 proteins comprise a targeting moiety that binds to one or more of these exemplary NK cell antigens.

In some embodiments, the multispecific chimeric proteins of the present invention comprise a targeting moiety with a recognition domain that specifically binds to a target (e.g., antigen or receptor) associated with a macrophage/monocyte. In some embodiments, the targeting moiety recruits macrophages/monocytes directly or indirectly, e.g., in some embodiments, to a treatment site (e.g., with one or more disease cells or a location of cells to be modulated to obtain a therapeutic effect). Exemplary macrophage/monocyte antigens of interest include, e.g., SIRP1a, B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, consensus beta chain, integrin alpha 4/CD49d, BLAME/SLAMF8, integrin alpha X/CDllc, CCL 6/C6, integrin beta 2/CD 6, CD155/PVR, integrin beta 3/CD6, CD 6/PECAM-1, lactolin, CD 6/SR-B6, leukotriene B6R 6, CD 6/TNFRSF 6, 6-B6, CD6, LMIR6/CD300, CD 6/6, CD 6/CD 6, LMIR6/CD 6, CD 6/CD 6, EMAMF SLIR 6, CRA 3/CD6, CD 6/CD 6, EMAMF 6, CD 6/CD 6, CD 6/CD 36, MGL2, endoglin/CD 105, osteoactivin/GPNMB, Fc- γ RI/CD64, osteopontin, Fc- γ RIIB/CD32B, PD-L2, Fc- γ RIIC/CD32c, Siglec-3/CD33, Fc- γ RIIA/CD32a, SIGNR1/CD209, Fc- γ RIII/CD16, SLAM, GM-CSF Ra, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN- γ RI, TLR4, IFN- γ R2, TREM-L, IL-L RII, TREM-2, ILT2/CD85j, TREM-3, ILT3/CD85k, TRML 1/TLT-1, TREM 4/SLIF 4, IL-10 Ra, IL-10, ALC-10. beta.7, CLT-R28/NLR 8653, CLT-8485, CLT-43/ILT-2, CLILT-2/CD 93, CLT-2/CD 8653, CLILT-2, CLT-2, CLILR-2, CLT-R-3, ILT4/CD85d, CCR1, ILT5/CD85a, CCR2, CD206, integrin alpha 4/CD49d, CCR5, integrin alpha M/CDll B, CCR8, integrin alpha X/CDllc, CD155/PVR, integrin beta 2/CD18, CD14, integrin beta 3/CD61, CD36/SR-B3, IR1, CD43, LAIR 43, CD43, leukotriene B43-R43, CD43, 43-B43, CD 43/SLAMF 43, LMIR 43/CD 300, CD43, LMIR 43/CD 300 43, CD163, LMIR 43/CD 43, blood coagulation factor III/tissue factor, LMIR 72/CD 36300, CSF 3CR 43, LRP 3/CX 300, CX/CD 5872, CD 1/CX/CDLR 1-CD 43, CD 43-CD 43, CD 43/CD 43, CD 43-CD 43, CD 43-CX/CX-1-CD 43, CD 3624, CD-CD 43, CD 3624, PSGL-1, Fc-gamma RIIIICD16, RP105, G-CSF R, L-selectin, GM-CSF R alpha, Siglec-3/CD33, HVEM/TNFRSF14, SLAM, ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-L, IL-6R, TREM-2, CXCRl/IL-8RA, TREM-3, and TREL/TLT-1. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these exemplary macrophage/monocyte antigens.

In some embodiments, the multispecific chimeric proteins of the present invention comprise a targeting moiety with a recognition domain that specifically binds to a target (e.g., antigen or receptor) associated with a dendritic cell. In some embodiments, the targeting moiety recruits dendritic cells directly or indirectly, e.g., in some embodiments, recruits dendritic cells to a treatment site (e.g., with one or more disease cells or a location of a cell to be modulated to obtain a therapeutic effect). Exemplary dendritic cell antigens of interest include, for example, CLEC9A, XCR1, RANK, CD36/SRB3, LOX-1/SR-E1, CD68, MARCO, CD163, SR-A1/MSR, CD5L, SREC-1, CL-PI/COLEC12, SREC-II, LIMPIIISRB2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-IBB ligand/TNFSF 9, IL-12/IL-23p40, 4-amino-1, 8-naphthalimide, ILT2/CD85j, CCL21/6Ckine, ILT 21/CD 21/85, 8-oxo-dG, ILT 21/CD 85, CD 368D 6, 21/CD 21, CD 21/CD 3685, CD21, LARG 21/GRR 21, LARG 21/CD 21, LARG, BLAME/SLAMF, LMIR/CD 300, Clq R/CD, LMIR/CD 300, CCR, LMIR/CD 300, CD/TNFRSF, MAG/SIGlec-4-a, CD, MCAM, CD, MD-1, CD, MD-2, CD, MDL-1/CLEC5, CD/SLAMF, MMR, CD, NCAMLl, CD 2-10/SLAMF, osteoactivin GPNMB, Chern, PD-L, CLEC-1, RP105, CLEC-2, CLEC-8, Siglec-2/CD, CRACC/SLAMF, Siglec-3/CD, DC-SIGN, CLEE 205, Siglec-5, DC-SIGNR/CD299, Sig-6, SigDCDCC, SigDCC-7, DCDCDCC/CLEC 4, Siglec-9, DEC-205, DEC-10, CLEC-1/CD-1, CD-6, SIGC-1/CD-1, CD-5, CD-CD 299, SIGC-8, SIGC-2/CD, CRAC-3, SIGC-C, SIGC-3/CD, SIGC-C-, SIGNR1/CD209, DEP-1/CD148, SIGNR4, DLEC, SLAM, EMMPRIN/CD147, TCCR/WSX-1, Fc- γ RI/CD64, TLR3, Fc- γ RIIB/CD32b, TREM-1, Fc- γ RIIC/CD32c, TREM-2, Fc- γ RIIA/CD32a, TREM-3, Fc- γ RIII/CD16, TREML1/TLT-1, ICAM-2/CD102, and capsaicin R1. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these exemplary DC antigens.

In some embodiments, the multispecific chimeric protein of the present invention comprises a targeting moiety with 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, a megakaryocyte, a platelet, an erythrocyte, a mast cell, a basophil, a neutrophil, an eosinophil, or a subpopulation thereof. In some embodiments, the antigen recognition domain recruits, directly or indirectly, megakaryocytes, platelets, erythrocytes, mast cells, basophils, neutrophils, eosinophils, or a subset thereof, e.g., in some embodiments, to the treatment site (e.g., with the location of one or more disease cells or cells to be modulated, to achieve a therapeutic effect).

In some embodiments, the multispecific chimeric proteins of the present invention comprise a targeting moiety with a recognition domain that specifically binds to a target (e.g., antigen or receptor) associated with a megakaryocyte and/or platelet. Exemplary megakaryocyte and/or platelet antigens of interest include, for example, GP IIb/IIIa, GPIb, vWF, PF4, and TSP. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these exemplary megakaryocyte and/or platelet antigens.

In some embodiments, the multispecific chimeric proteins of the present invention comprise a targeting moiety with a recognition domain that specifically binds to a target (e.g., antigen or receptor) associated with a red blood cell. Exemplary red blood cell antigens of interest include, for example, CD34, CD36, CD38, CD41a (platelet glycoprotein IIb/IIIa), CD41b (GPIIb), CD71 (transferrin receptor), CD105, glycophorin A, glycophorin C, C-kit, HLA-DR, H2(MHC-II), and rhesus antigens. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these exemplary red blood cell antigens.

In some embodiments, the multispecific chimeric proteins of the present invention comprise a targeting moiety with a recognition domain that specifically binds to a target (e.g., antigen or receptor) associated with a mast cell. Exemplary mast cell antigens of interest include, for example, SCFR/CD117, fcsri, CD2, CD25, CD35, CD88, CD203C, C5R1, CMAI, FCERIA, FCER2, TPSABI. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these mast cell antigens.

In some embodiments, the multispecific chimeric proteins of the present invention comprise a targeting moiety with a recognition domain that specifically binds to a target (e.g., antigen or receptor) associated with a basophil. Exemplary target basophil antigens include, for example, fceri, 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.

In some embodiments, the multispecific chimeric proteins of the present invention comprise a targeting moiety having a recognition domain that specifically binds to a target (e.g., antigen or receptor) associated with a neutrophil. Exemplary neutrophil antigens of interest include, for example, 7D5, CD 10/cala, CD13, CD16(FcRIII), CD18 protein (LFA-1, CR3 and p150, 95), CD45, CD67 and CD 177. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these neutrophil antigens.

In some embodiments, the multispecific chimeric protein of the present invention comprises a targeting moiety with a recognition domain that specifically binds to an eosinophil-associated target (e.g., an antigen or receptor). Exemplary eosinophil antigens of interest include, for example, CD35, CD44, and CD 69. In various embodiments, the chimeric protein comprises a targeting moiety that binds to one or more of these eosinophil antigens.

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 those of skill in the art. In some embodiments, the antigen or cell surface marker is a tissue-specific marker. Exemplary tissue-specific markers include, but are not limited to, endothelial cell surface markers such as ACE, CD14, CD34, CDH5, ENG, ICAM2, MCAM, NOS3, PECAMI, PROCR, SELE, SELP, TEK, THBD, VCAMI, VWF; smooth muscle cell surface markers ACTA2, MYHIO, MYHI 1, MYH9, MYOCD; fibroblast (stromal) cell surface markers such as ALCAM, CD34, COL1Al, COL1A2, COL3A1, FAP, pH-4; epithelial cell surface markers such as CDID, K6IRS2, KRTIO, KRT13, KRT17, KRT18, KRT19, KRT4, KRT5, KRT8, MUCI, tactdi; neovasculature markers such as CD13, TFNA, alpha-V beta-3(α V β 3), 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 cells bearing these antigens.

In various embodiments, the multispecific chimeric proteins of the invention have one or more targeting moieties to checkpoint markers, for example one or more of PD-1/PD-L1 or PD-L2, CD28/CD80 or CD86, CTLA4/CD80 or CD86, ICOS/ICOSL or B7RP1, BTLA/HVEM, KIR, LAG3, CD137/CD137L, OX40/OX40L, CD27, CD40L, TIM3/Gal9, and A2 aR.

By way of non-limiting example, in various embodiments, the chimeric proteins of the present invention have targeting moieties directed to: (i) checkpoint markers 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) targeting moieties directed against tumor cells, as well as any modified (e.g., mutated) signaling agents described herein.

In various embodiments, the multispecific chimeric proteins of the present invention have one or more targeting moieties for PD-1. In some embodiments, the chimeric protein has one or more targeting moieties that selectively bind to the PD-1 polypeptide. In some embodiments, the chimeric protein comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a PD-1 polypeptide.

In one embodiment, the targeting moiety comprises an anti-PD-1 antibody, palboclizumab (pembrolizumab, aka MK-3475, KEYTRUDA), or a fragment thereof. Pabollizumab and other humanized anti-PD-1 antibodies are disclosed in Hamid, et al (2013) New England Journal of Medicine 369(2):134-44, US8,354,509 and WO 2009/114335, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, the palivizumab or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 69; and/or comprises a light chain comprising SEQ ID NO: 70.

In one embodiment, the targeting moiety comprises an anti-PD-1 antibody, nivolumab (aka 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 US8,008,449 and WO 2006/121168, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, the nivolumab, or antigen-binding fragment thereof, comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 71; and/or a light chain comprising SEQ ID NO: 72.

In one embodiment, the targeting moiety comprises an anti-PD-1 antibody pidilizumab (aka CT-011, hBAT, or hBAT-1), or a fragment thereof. Pilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in US2008/0025980 and WO 2009/101611, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, the anti-PD-1 antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a light chain variable region comprising an amino acid sequence selected from SEQ ID NO:15-18 of US2008/0025980 (SEQ ID NO:15(SEQ ID NO:73) of US 2008/002598), SEQ ID NO:16 of US2008/0025980 (SEQ ID NO:74), SEQ ID NO:17 of US2008/0025980 (SEQ ID NO:75), and SEQ ID NO:18 of US2008/0025980 (SEQ ID NO: 76)); and/or a heavy chain comprising an amino acid sequence selected from SEQ ID NO:20-24 of US2008/0025980 (SEQ ID NO:20 of US2008/0025980 (SEQ ID NO: 77); SEQ ID NO:21 of US2008/0025980 (SEQ ID NO: 78); SEQ ID NO:22 of US2008/0025980 (SEQ ID NO: 79); SEQ ID NO:23 of US2008/0025980 (SEQ ID NO: 80); and SEQ ID NO:24 of US2008/0025980 (SEQ ID NO: 81)).

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

In one embodiment, the targeting moiety comprises AMP-514(aka MEDI-0680).

In one embodiment, the targeting moiety comprises PD-12-Fc fusion protein AMP-224, which is disclosed in WO2010/027827 and WO 2011/066342, the entire disclosures of which are incorporated herein by reference. In such embodiments, the targeting moiety may comprise a peptide comprising SEQ ID NO: 4(SEQ ID NO:82) and/or a targeting domain comprising SEQ ID NO of WO 2010/027827: 83(SEQ ID NO: 83) of B7-DC fusion protein.

In one embodiment, the targeting moiety comprises the peptide AUNP12 or any other peptide disclosed in US2011/0318373 or 8,907,053. For example, the targeting moiety may comprise AUNP12 (i.e., compound 8 or SEQ ID NO: 49 of US 2011/0318373) having a sequence (SEQ ID NO: 84).

In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1E3 or fragment thereof as disclosed in US2014/0044738, the entire disclosure of which is incorporated herein by reference. In exemplary embodiments, 1E3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 85; and/or a light chain variable region comprising SEQ ID NO: 86.

In one embodiment, the targeting moiety comprises an anti-PD-1 antibody 1E8 or fragment thereof as disclosed in US2014/0044738, the entire disclosure of which is incorporated herein by reference. In exemplary embodiments, 1E8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 87; and/or a light chain variable region of SEQ ID NO: 88.

In one embodiment, the targeting moiety comprises the anti-PD-1 antibody 1H3 or fragment thereof as disclosed in US2014/0044738, the entire disclosure of which is incorporated herein by reference. In exemplary embodiments, 1H3 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 89; and/or a light chain variable region of SEQ ID NO: 90.

In one embodiment, the targeting moiety comprises a VHH directed against PD-1, as disclosed in, for example, US8,907,065 and WO 2008/071447, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, the VHH directed against PD-1 comprises SEQ ID NO: 347-351 (SEQ ID NO: 347(SEQ ID NO: 91) of US8,907,065; SEQ ID NO: 348(SEQ ID NO: 92) of US8,907,065; SEQ ID NO: 349(SEQ ID NO: 93) of US8,907,065; SEQ ID NO: 350(SEQ ID NO: 94) of US8,907,065; and SEQ ID NO: 351(SEQ ID NO: 95) of US8,907,065).

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

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

In various embodiments, the multispecific chimeric proteins of the present invention have one or more targeting moieties to PD-L1. In some embodiments, the chimeric protein has one or more targeting moieties that selectively bind to the PD-L1 polypeptide. In some embodiments, the chimeric protein comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a PD-L1 polypeptide.

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody MEDI4736(aka deluzumab) or a fragment thereof. MEDI4736 is selective for PD-L1 and blocks the binding of PD-L1 to the PD-1 and CD80 receptors. MEDI4736 and antigen-binding fragments thereof for use in the methods provided herein comprise heavy and light chains or heavy and light chain variable regions. The sequence of MEDI4736 is disclosed in WO/2016/06272, the entire contents of which are incorporated herein by reference. In exemplary embodiments, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 105; and/or a light chain comprising SEQ ID NO: 106.

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

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody atelizumab (atezolizumab, aka MPDL32880A, RG7446) or a fragment thereof. In exemplary embodiments, the atuzumab, or antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 109; and/or a light chain comprising SEQ ID NO: 110.

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody avilumab (avelumab, aka MSB0010718C) or a fragment thereof. In exemplary embodiments, avizumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 111; and/or a light chain comprising SEQ ID NO: 112.

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody BMS-936559(aka12a4, MDX-1105) or a fragment thereof, as disclosed in US2013/0309250 and WO2007/005874, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, BMS-936559 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 113; and/or a light chain variable region comprising SEQ ID NO: 114.

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3G10 or a fragment thereof, as disclosed in US2013/0309250 and WO2007/005874, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 3G10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 115, or an amino acid sequence of seq id no; and/or a light chain variable region of SEQ ID NO: 116.

In one embodiment, the targeting moiety comprises anti-PD-L1 antibody 10a5 or a fragment thereof, as disclosed in US2013/0309250 and WO2007/005874, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 10a5 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 117; and/or a light chain variable region of SEQ ID NO: 118.

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 5F8 or a fragment thereof, as disclosed in US2013/0309250 and WO2007/005874, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 5F8 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 119; and/or a light chain variable region of SEQ ID NO: 120.

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 10H10 or fragment thereof, as disclosed in US2013/0309250 and WO2007/005874, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 10H10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 121; and/or a light chain variable region of SEQ ID NO: 122.

In one embodiment, the targeting moiety comprises anti-PD-L1 antibody 1B12 or a fragment thereof, as disclosed in US2013/0309250 and WO2007/005874, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 1B12 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 123; and/or a light chain variable region of SEQ ID NO: 124.

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 7H1 or fragment thereof, as disclosed in US2013/0309250 and WO2007/005874, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 7H1 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 125; and/or a light chain variable region of SEQ ID NO: 126, or a pharmaceutically acceptable salt thereof.

In one embodiment, the targeting moiety comprises anti-PD-L1 antibody 11E6 or a fragment thereof, as disclosed in US2013/0309250 and WO2007/005874, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 11E6 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 127; and/or a light chain variable region of SEQ ID NO: 128.

In one embodiment, the targeting moiety comprises anti-PD-L1 antibody 12B7 or a fragment thereof, as disclosed in US2013/0309250 and WO2007/005874, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 12B7 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 129; and/or a light chain variable region of SEQ ID NO: 130.

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 13G4 or a fragment thereof, as disclosed in US2013/0309250 and WO2007/005874, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 13G4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 131; and/or a light chain variable region of SEQ ID NO: 132.

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1E12 or fragment thereof as disclosed in US2014/0044738, the entire disclosure of which is incorporated herein by reference. In exemplary embodiments, 1E12 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 133; and/or a light chain variable region of SEQ ID NO: 134, or a pharmaceutically acceptable salt thereof.

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 1F4 or fragment thereof, as disclosed in US2014/0044738, the entire disclosure of which is incorporated herein by reference. In exemplary embodiments, 1F4 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 135; and/or a light chain variable region of SEQ ID NO: 136, or a pharmaceutically acceptable salt thereof.

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2G11 or a fragment thereof, as disclosed in US2014/0044738, the entire disclosure of which is incorporated herein by reference. In exemplary embodiments, 2G11 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 137; and/or a light chain variable region of SEQ ID NO: 138, or a pharmaceutically acceptable salt thereof.

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3B6 or fragment thereof, as disclosed in US2014/0044738, the entire disclosure of which is incorporated herein by reference. In exemplary embodiments, 3B6 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 139; and/or a light chain variable region comprising SEQ ID NO: 140.

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3D10 or fragment thereof, as disclosed in US2014/0044738 and WO2012/145493, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 3D10 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 141; and/or a light chain variable region comprising SEQ ID NO: 142.

In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in US2011/0271358 and WO2010/036959, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 34-38 (SEQ ID NO: 34(SEQ ID NO: 143) of US 2011/0271358; SEQ ID NO: 35(SEQ ID NO: 144) of US 2011/0271358; SEQ ID NO: 36(SEQ ID NO: 145) of US 2011/0271358; SEQ ID NO: 37(SEQ ID NO: 146) of US 2011/0271358; and SEQ ID NO: 38(SEQ ID NO: 147) of US 2011/0271358); and/or comprises a light chain comprising SEQ ID NO: 39-42 (SEQ ID NO: 39 of US2011/0271358 (SEQ ID NO: 148); SEQ ID NO: 40 of US2011/0271358 (SEQ ID NO: 149); SEQ ID NO: 41 of US2011/0271358 (SEQ ID NO: 150); and SEQ ID NO: 42 of US2011/0271358 (SEQ ID NO: 151)).

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7a4 or fragment thereof, as disclosed in WO2011/066389, US8,779,108, and US2014/0356353, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 2.7a4 or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2(SEQ ID NO: 152); and/or a light chain variable region comprising SEQ ID NO: 7(SEQ ID NO: 153).

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.9D10 or fragment thereof, as disclosed in WO2011/066389, US8,779,108, and US2014/0356353, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 2.9D10, or antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12(SEQ ID NO: 154); and/or comprising SEQ ID NO of WO 2011/066389: 17(SEQ ID NO: 155).

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9 or fragment thereof, as disclosed in WO2011/066389, US8,779,108, and US2014/0356353, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 2.14H9, or antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:22(SEQ ID NO: 156); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 27(SEQ ID NO: 157).

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.20a8 or fragment thereof, as disclosed in WO2011/066389, US8,779,108, and US2014/0356353, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 2.20A8, or antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 32(SEQ ID NO: 158); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 37(SEQ ID NO: 159).

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3.15G8 or a fragment thereof, as disclosed in WO2011/066389, US8,779,108, and US2014/0356353, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 3.15G8, or antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 42(SEQ ID NO: 160); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 47(SEQ ID NO: 161).

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 3.18G1 or a fragment thereof, as disclosed in WO2011/066389, US8,779,108, and US2014/0356353, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, 3.18G1, or antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 52(SEQ ID NO: 162); and/or a light chain variable region comprising SEQ ID NO: 57(SEQ ID NO: 163).

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.7A4OPT or a fragment thereof, as disclosed in WO2011/066389, US8,779,108 and US2014/0356353 and US2014/0356353, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, the 2.7A4OPT, or antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 62(SEQ ID NO: 164); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 67(SEQ ID NO: 165).

In one embodiment, the targeting moiety comprises the anti-PD-L1 antibody 2.14H9OPT or a fragment thereof, as disclosed in WO2011/066389, US8,779,108, and US2014/0356353, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, the 2.14H9OPT, or antigen-binding fragment thereof, for use in the methods provided herein comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 72(SEQ ID NO: 166); and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:77 (SEQ ID NO: 167).

In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in WO2016/061142, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 18. 30, 38, 46, 50, 54, 62, 70 and 78(SEQ ID NO: 18 of WO2016/061142 (SEQ ID NO: 168); SEQ ID NO: 30 of WO2016/061142 (SEQ ID NO: 169); SEQ ID NO: 38 of WO2016/061142 (SEQ ID NO: 170); SEQ ID NO: 46 of WO2016/061142 (SEQ ID NO: 171); SEQ ID NO: 50 of WO2016/061142 (SEQ ID NO: 172); SEQ ID NO: 54 of WO2016/061142 (SEQ ID NO: 173); SEQ ID NO: 62 of WO2016/061142 (SEQ ID NO: 174); SEQ ID NO: 70 of WO2016/061142 (SEQ ID NO: 175); and SEQ ID NO: 78 of WO2016/061142 (SEQ ID NO: 176)); and/or a light chain comprising the amino acid sequence of SEQ ID NO: 22. 26, 34, 42, 58, 66, 74, 82 and 86(SEQ ID NO: 22(SEQ ID NO: 177) of WO 2016/061142), SEQ ID NO: 26(SEQ ID NO: 178) of WO2016/061142, SEQ ID NO: 34(SEQ ID NO: 179) of WO2016/061142, SEQ ID NO: 42(SEQ ID NO: 180) of WO2016/061142, SEQ ID NO: 58(SEQ ID NO: 181) of WO2016/061142, SEQ ID NO: 66(SEQ ID NO: 182) of WO2016/061142, SEQ ID NO: 74(SEQ ID NO: 183) of WO2016/061142, SEQ ID NO: 82(SEQ ID NO: 184) of WO2016/061142, and SEQ ID NO: 86(SEQ ID NO: 185) of WO 2016/061142).

In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in WO2016/02263, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 2. 6, 10, 14, 18, 22, 26, 30, 34, 38, 42 and 46(SEQ ID NO:2 of WO2016/022630 (SEQ ID NO: 186); SEQ ID NO: 6 of WO2016/022630 (SEQ ID NO: 187); SEQ ID NO: 10 of WO2016/022630 (SEQ ID NO: 188); SEQ ID NO: 14 of WO2016/022630 (SEQ ID NO: 189); SEQ ID NO:18 of WO2016/022630 (SEQ ID NO: 190); SEQ ID NO:22 of WO2016/022630 (SEQ ID NO: 191); SEQ ID NO:26 of WO2016/022630 (SEQ ID NO: 192); SEQ ID NO: 30 of WO2016/022630 (SEQ ID NO: 193); SEQ ID NO: 34 of WO2016/022630 (SEQ ID NO: 194); SEQ ID NO: 38 of WO 2016/022630); SEQ ID NO: 2016; SEQ ID NO: 195); SEQ ID NO: 42 of WO2016/022630 (SEQ ID NO: 2016/02242); and SEQ ID NO: 187/2016/022630); SEQ ID NO: 187/2016 SEQ ID NO: 46(SEQ ID NO: 197)); and/or a light chain comprising SEQ ID NO: 4. the amino acid sequences of 8, 12, 16, 20, 24, 28, 32, 36, 40, 44 and 48(SEQ ID NO: 4 of WO2016/022630 (SEQ ID NO: 198); SEQ ID NO:8 of WO2016/022630 (SEQ ID NO: 199); SEQ ID NO: 12 of WO2016/022630 (SEQ ID NO: 200); SEQ ID NO:16 of WO2016/022630 (SEQ ID NO: 201); SEQ ID NO:20 of WO2016/022630 (SEQ ID NO: 202); SEQ ID NO:24 of WO2016/022630 (SEQ ID NO: 203); SEQ ID NO: 28 of WO2016/022630 (SEQ ID NO: 204); SEQ ID NO: 32 of WO2016/022630 (SEQ ID NO: 205); SEQ ID NO: 36 of WO2016/022630 (SEQ ID NO: 206); SEQ ID NO: 40 of WO2016/022630 (SEQ ID NO: 207); SEQ ID NO: 208 of WO 2016/022630); SEQ ID NO: 2016/022630 (SEQ ID NO: 208/2016/199); and 2016/022630 (SEQ ID NO: 199/199) SEQ ID NO: 48(SEQ ID NO: 209)).

In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in WO2015/112900, the entire contents of which are incorporated herein by reference. In an exemplary embodiment, the antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 38. amino acid sequences of 50, 82 and 86(SEQ ID NO: 38 of WO2015/112900 (SEQ ID NO: 210); SEQ ID NO: 50 of WO2015/112900 (SEQ ID NO: 211); SEQ ID NO: 82 of WO2015/112900 (SEQ ID NO: 212); and SEQ ID NO: 86 of WO2015/112900 (SEQ ID NO: 213)); and/or a light chain comprising the amino acid sequence of SEQ ID NO: 42. 46, 54, 58, 62, 66, 70, 74 and 78(SEQ ID NO: 42 of WO2015/112900 (SEQ ID NO: 214); SEQ ID NO: 46 of WO2015/112900 (SEQ ID NO: 215); SEQ ID NO: 54 of WO2015/112900 (SEQ ID NO: 216); SEQ ID NO: 58 of WO2015/112900 (SEQ ID NO: 217); SEQ ID NO: 62 of WO2015/112900 (SEQ ID NO: 218); SEQ ID NO: 66 of WO2015/112900 (SEQ ID NO: 219); SEQ ID NO: 70 of WO2015/112900 (SEQ ID NO: 220); SEQ ID NO: 74 of WO2015/112900 (SEQ ID NO: 221); and SEQ ID NO: 78 of WO2015/112900 (SEQ ID NO: 222)).

In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies disclosed in WO2010/077634 and US8,217,149, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, the anti-PD-L1 antibodies or antigen-binding fragments thereof for use in the methods provided herein comprise a heavy chain region comprising the amino acid sequence of SEQ ID NO:20(SEQ ID NO: 223); and/or comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO:21(SEQ ID NO: 224).

In one embodiment, the targeting moiety comprises any of the anti-PD-L1 antibodies obtainable from a hybridoma obtained from CNCM accession numbers CNCM I-4122, CNCM I-4080 and CNCM I-4081 as disclosed in US 20120039906, the entire disclosures of which are incorporated herein by reference.

In one embodiment, the targeting moiety comprises a VHH directed to PD-L1, as disclosed in, for example, US8,907,065 and WO 2008/071447, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, the VHH directed against PD-L1 comprises the amino acid sequence of SEQ ID NO: 394-399 (SEQ ID NO: 394 of US8,907,065 (SEQ ID NO: 225); SEQ ID NO: 395 of US8,907,065 (SEQ ID NO: 226); SEQ ID NO: 396 of US8,907,065 (SEQ ID NO: 227); SEQ ID NO: 397 of US8,907,065 (SEQ ID NO: 228); SEQ ID NO: 398 of US8,907,065 (SEQ ID NO: 229); SEQ ID NO: 399 of US8,907,065 (SEQ ID NO: 230)).

In various embodiments, the multispecific chimeric proteins of the present invention have one or more targeting moieties to PD-L2. In some embodiments, the chimeric protein has one or more targeting moieties that selectively bind to the PD-L2 polypeptide. In some embodiments, the chimeric protein comprises one or more antibodies, antibody derivatives or forms, peptides or polypeptides, or fusion proteins that selectively bind to a PD-L2 polypeptide.

In one embodiment, the targeting moiety comprises a VHH directed to PD-L2, as disclosed in, for example, US8,907,065 and WO 2008/071447, the entire disclosures of which are incorporated herein by reference. In exemplary embodiments, the VHH directed against PD-1 comprises SEQ ID NO: 449-455 (SEQ ID NO: 449 of US8,907,065 (SEQ ID NO: 231), SEQ ID NO: 450 of US8,907,065 (SEQ ID NO: 232), SEQ ID NO: 451 of US8,907,065 (SEQ ID NO: 233), SEQ ID NO: 452 of US8,907,065 (SEQ ID NO: 234), SEQ ID NO: 453 of US8,907,065 (SEQ ID NO: 235), SEQ ID NO: 454 of US8,907,065 (SEQ ID NO: 236), and SEQ ID NO: 455 of US8,907,065 (SEQ ID NO: 237)).

In one embodiment, the targeting moiety comprises any of the anti-PD-L2 antibodies disclosed in US2011/0271358 and WO2010/036959, the entire contents of which are incorporated herein by reference. In exemplary embodiments, the antibody or antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 43-47 (SEQ ID NO: 43 of US2011/0271358 (SEQ ID NO: 238); SEQ ID NO: 44 of US2011/0271358 (SEQ ID NO: 239); SEQ ID NO: 45 of US2011/0271358 (SEQ ID NO: 240); SEQ ID NO: 46 of US2011/0271358 (SEQ ID NO: 241); and SEQ ID NO: 47 of US2011/0271358 (SEQ ID NO: 242)); and/or a light chain comprising SEQ ID NO: 48-51 (SEQ ID NO: 48(SEQ ID NO: 243) of US 2011/0271358); SEQ ID NO of US 2011/0271358: 49(SEQ ID NO: 244); SEQ ID NO of US 2011/0271358: 50(SEQ ID NO: 245); and SEQ ID NO of US 2011/0271358: 51(SEQ ID NO: 246)).

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

In various embodiments, the targeting moieties of the present invention can include any combination of heavy chain, light chain, heavy chain variable region, light chain variable region, Complementarity Determining Regions (CDRs), and framework region sequences that target PD-1, PD-L1, and/or PD-L2 as disclosed herein.

Other antibodies, antibody derivatives or forms, peptides or polypeptides or fusion proteins that selectively bind or target PD-1, PD-L1 and/or PD-L2 are disclosed in WO2011/066389, US 2008/0025980, US 2013/0034559, US8,779,108, US2014/0356353, US8,609,089, US 2010/028330, US 2012/0114649, WO 2010/027827, WO 2011/066342, US8,907,065, WO 2016/062722, WO 2009/101611, WO 2010/027827, WO 2011/066342, WO2007/005874, WO 2001/014556, US2011/0271358, WO2010/036959, WO 2010/077634, US8,217,149, US 2012/0039906, WO 2012/145493, US2011/0318373, US patent No.8,779,108, US 20140044738, WO 2009/089149, WO2007/00587, WO2, WO 2016061142, WO2016,02263, WO 2010/077634 and WO2015/112900, the disclosures of which are incorporated herein by reference.

In some embodiments, the targeting moiety is a natural ligand, such as a chemokine. Exemplary chemokines that can be included in a chimeric protein of the invention include, but are not limited to, CCL1, CCL2, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CXCL9, XCL9, xccl 3, PBP-PBP. In an exemplary embodiment, the targeting moiety can be XCL1, which is a chemokine that recognizes and binds to dendritic cell receptor XCR 1. In another exemplary embodiment, the targeting moiety is CCL1, a chemokine that recognizes and binds CCR 8. In another exemplary embodiment, the targeting moiety is CCL2, a chemokine that recognizes and binds CCR2 or CCR 9. In another exemplary embodiment, the targeting moiety is CCL3, which is a chemokine that recognizes and binds CCR1, CCR5, or CCR 9. In another exemplary embodiment, the targeting moiety is CCL4, which is a chemokine that recognizes and binds CCR1 or CCR5 or CCR 9. In another exemplary embodiment, the targeting moiety is CCL5, which is a chemokine that recognizes and binds CCR1 or CCR3 or CCR4 or CCR 5. In another exemplary embodiment, the targeting moiety is CCL6, a chemokine that recognizes and binds CCR 1. In another exemplary embodiment, the targeting moiety is CCL7, a chemokine that recognizes and binds CCR2 or CCR 9. In another exemplary embodiment, the targeting moiety is CCL8, which is a chemokine that recognizes and binds CCR1 or CCR2 or CCR2B or CCR5 or CCR 9. In another exemplary embodiment, the targeting moiety is CCL9, a chemokine that recognizes and binds CCR 1. In another exemplary embodiment, the targeting moiety is CCL10, a chemokine that recognizes and binds CCR 1. In another exemplary embodiment, the targeting moiety is CCL11, which is a chemokine that recognizes and binds CCR2 or CCR3 or CCR5 or CCR 9. In another exemplary embodiment, the targeting moiety is CCL13, which is a chemokine that recognizes and binds CCR2 or CCR3 or CCR5 or CCR 9. In another exemplary embodiment, the targeting moiety is CCL14, a chemokine that recognizes and binds CCR1 or CCR 9. In another exemplary embodiment, the targeting moiety is CCL15, a chemokine that recognizes and binds CCR1 or CCR 3. In another exemplary embodiment, the targeting moiety is CCL16, which is a chemokine that recognizes and binds CCR1, CCR2, CCR5, or CCR 8. In another exemplary embodiment, the targeting moiety is CCL17, a chemokine that recognizes and binds CCR 4. In another exemplary embodiment, the targeting moiety is CCL19, a chemokine that recognizes and binds CCR 7. In another exemplary embodiment, the targeting moiety is CCL20, a chemokine that recognizes and binds CCR 6. In another exemplary embodiment, the targeting moiety is CCL21, a chemokine that recognizes and binds CCR 7. In another exemplary embodiment, the targeting moiety is CCL22, a chemokine that recognizes and binds CCR 4. In another exemplary embodiment, the targeting moiety is CCL23, a chemokine that recognizes and binds CCR 1. In another exemplary embodiment, the targeting moiety is CCL24, a chemokine that recognizes and binds CCR 3. In another exemplary embodiment, the targeting moiety is CCL25, a chemokine that recognizes and binds CCR 9. In another exemplary embodiment, the targeting moiety is CCL26, a chemokine that recognizes and binds CCR 3. In another exemplary embodiment, the targeting moiety is CCL27, a chemokine that recognizes and binds CCR 10. In another exemplary embodiment, the targeting moiety is CCL28, a chemokine that recognizes and binds CCR3 or CCR 10. In another exemplary embodiment, the targeting moiety is CXCL1, which is a chemokine that recognizes and binds to CXCR1 or CXCR 2. In another exemplary embodiment, the targeting moiety is CXCL2, which is a chemokine that recognizes and binds CXCR 2. In another exemplary embodiment, the targeting moiety is CXCL3, which is a chemokine that recognizes and binds CXCR 2. In another exemplary embodiment, the targeting moiety is CXCL4, which is a chemokine that recognizes and binds CXCR 3B. In another exemplary embodiment, the targeting moiety is CXCL5, which is a chemokine that recognizes and binds CXCR 2. In another exemplary embodiment, the targeting moiety is CXCL6, which is a chemokine that recognizes and binds to CXCR1 or CXCR 2. In another exemplary embodiment, the targeting moiety is CXCL8, which is a chemokine that recognizes and binds to CXCR1 or CXCR 2. In another exemplary embodiment, the targeting moiety is CXCL9, which is a chemokine that recognizes and binds CXCR 3. In another exemplary embodiment, the targeting moiety is CXCL10, which is a chemokine that recognizes and binds CXCR 3. In another exemplary embodiment, the targeting moiety is CXCL11, which is a chemokine that recognizes and binds to CXCR3 or CXCR 7. In another exemplary embodiment, the targeting moiety is CXCL12, which is a chemokine that recognizes and binds to CXCR4 or CXCR 7. In another exemplary embodiment, the targeting moiety is CXCL13, which is a chemokine that recognizes and binds CXCR 5. In another exemplary embodiment, the targeting moiety is CXCL16, which is a chemokine that recognizes and binds CXCR 6. In another exemplary embodiment, the targeting moiety is LDGF-PBP, which is a chemokine that recognizes and binds to CXCR 2. In another exemplary embodiment, the targeting moiety is XCL2, which is a chemokine that recognizes and binds XCR 1. In another exemplary embodiment, the targeting moiety is CX3CL1, which is a chemokine that recognizes and binds CX3CR 1.

In various embodiments, the chimeric proteins of the present invention comprise multiple combinations of targeting moieties. In an exemplary embodiment, the chimeric protein of the invention may comprise two targeting moieties, wherein both targeting moieties are antibodies or derivatives thereof. In another exemplary embodiment, the chimeric protein of the invention may comprise two targeting moieties, wherein both targeting moieties are natural ligands of a cellular receptor. In another exemplary embodiment, the chimeric protein of the invention may comprise two targeting moieties, wherein one targeting moiety is an antibody or derivative thereof and the other targeting moiety is a natural ligand for a cellular receptor.

In various embodiments, the recognition domain of the chimeric proteins of the invention functionally modulates (without limitation, partially or fully neutralizes) a target of interest (e.g., antigen, receptor), e.g., significantly inhibits, reduces or neutralizes a biological effect that the antigen has. For example, the plurality of recognition domains may be directed against one or more tumor antigens that actively suppress, or have the ability to suppress, the immune system of, for example, a patient having a tumor. For example, in some embodiments, the chimeric proteins of the invention functionally modulate an immunosuppressive signal (e.g., a checkpoint inhibitor), e.g., one or more of TIM-3, BTLA, PD-1, CTLA-4, B7-H4, GITR, galectin-9, HVEM, PD-L1, PD-L2, B7-H3, CD244, CD160, TIGIT, sirpa, ICOS, CD172a, and TMIGD 2. For example, in some embodiments, the chimeric proteins of the invention are engineered to disrupt, block, reduce and/or inhibit the transmission of immunosuppressive signals, by way of non-limiting example, binding of PD-1 to PD-L1 or PD-L2 and/or binding of CTLA-4 to one or more of AP2M1, CD80, CD86, SHP-2, and PPP2R 5A.

In various embodiments, the recognition domain of the chimeric protein of the invention binds to but does not functionally modulate a target of interest (e.g., antigen, receptor), e.g., the recognition domain is or resembles a binding antibody. For example, in various embodiments, the recognition domain targets only the antigen or receptor, but does not significantly inhibit, reduce, or functionally modulate a biological effect that the antigen or receptor has. For example, some of the smaller antibody formats described above (e.g., as compared to, e.g., a full antibody) have the ability to target difficult to access epitopes and provide a broader spectrum of specific binding sites. In various embodiments, the recognition domain binds to an epitope that is physically separated from the antigen or receptor site important for its biological activity (e.g., the active site of the antigen).

Such non-neutralizing binding may be useful in various embodiments of the invention, including methods in which the chimeric proteins of the invention are used to recruit active immune cells to a desired site, either directly or indirectly, via an effector antigen (e.g., any of those described herein). For example, in various embodiments, the chimeric proteins of the invention can be used to recruit cytotoxic T cells directly or indirectly via CD8 in a method of reducing or eliminating a tumor (e.g., the chimeric protein can comprise an anti-CD 8 recognition domain and a recognition domain for a tumor antigen). In these embodiments, it is desirable to recruit directly or indirectly cytotoxic T cells expressing CD8, but not to functionally modulate CD8 activity. Rather, in these embodiments, CD8 signaling is an important part of the tumor reducing or eliminating effect. As a further example, in various methods of reducing or eliminating tumors, the chimeric proteins of the invention are used to recruit Dendritic Cells (DCs) directly or indirectly through CLEC9A (e.g., the chimeric proteins may comprise an anti-CLEC 9A recognition domain and a recognition domain for a tumor antigen). In such embodiments, it is desirable to recruit directly or indirectly DC expressing CLEC9A, but not to functionally modulate CLEC9A activity. Rather, in these embodiments, CLEC9A signaling is an important part of the tumor reducing or eliminating effect.

In various embodiments, the recognition domain of the chimeric proteins of the invention binds XCR1, e.g., XCR1 on dendritic cells. For example, in some embodiments, the recognition domain comprises all or part of XCL1 or a non-neutralizing anti-XCR 1 agent.

In various embodiments, the recognition domain of the chimeric proteins of the invention binds to an immunomodulatory antigen (e.g., immunostimulatory or immunosuppressive). 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 ligand, CD137 ligand, and TL 1A. In various embodiments, such immunostimulatory antigens are expressed on tumor cells. In various embodiments, the recognition domain of the chimeric proteins of the invention binds to, but does not functionally modulate, these immunostimulatory antigens, thus allowing recruitment of cells expressing these antigens without reducing or losing their potential tumor-reducing or eliminating capacity.

In various embodiments, the recognition domain of the chimeric protein of the invention may be in the case of a chimeric protein comprising two recognition domains with neutralizing activity, or in the case of a chimeric protein comprising two recognition domains with non-neutralizing (e.g., binding) activity, or in the case of a chimeric protein comprising one recognition domain with neutralizing activity and one recognition domain with non-neutralizing (e.g., binding) activity.

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

In some embodiments, the target of interest (e.g., antigen, receptor) is part of a non-cellular component of the matrix or extracellular matrix (ECM) or a marker associated therewith. As used herein, stroma refers to the connective and supportive framework of a tissue or organ. The matrix may include a compilation of cells (compilations) such as fibroblasts/myofibroblasts, glial cells, epithelial cells, adipocytes, immune cells, vascular cells, smooth muscle cells and immune cells, as well as extracellular matrix (ECM) and extracellular molecules. In various embodiments, the target of interest (e.g., antigen, receptor) is part of a non-cellular component of the matrix, such as an extracellular matrix and extracellular molecules. As used herein, ECM refers to the acellular component present in all tissues and organs. The ECM is composed of a large collection of biochemically distinct components, including but not limited to proteins, glycoproteins, proteoglycans, and polysaccharides. These components of the ECM are typically produced by neighboring cells and secreted into the ECM by exocytosis. Once secreted, ECM components typically aggregate to form complex macromolecular networks. In various embodiments, the chimeric proteins of the invention comprise a targeting moiety that recognizes a target (e.g., an antigen or receptor or a non-proteinaceous molecule) located on any component of the ECM. Exemplary components of the ECM include, but are not limited to, proteoglycans, non-proteoglycan polysaccharides, fibers and other ECM proteins or ECM non-proteins such as polysaccharides and/or lipids, or ECM-related molecules (e.g., proteins or non-proteins such as polysaccharides, nucleic acids and/or lipids).

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

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

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

In one embodiment, the targeting moiety recognizes and binds tenascin. The Tenascin (TN) family of glycoproteins includes at least four members: tenascin-C, tenascin-R, tenascin-X and tenascin W. The primary structure of tenascin comprises several common motifs arranged in the same contiguous sequence: an amino-terminal heptad repeat, an Epidermal Growth Factor (EGF) -like repeat, a fibronectin type III domain repeat, and a carboxy-terminal fibrin-like globular domain. Each protein member is associated with typical variations in the number and nature of EGF-like and fibronectin type III repeats. Subtype variants also exist in particular for tenascin-C. tenascin-C is known to have more than 27 splice variants and/or subtypes. In particular embodiments, the targeting moiety recognizes and binds tenascin-CA 1. Similarly, tenascin-R also has a number of splice variants and subtypes. tenascin-R is usually present 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 is present as a trimer. In some embodiments, the targeting moiety recognizes one or more epitopes on tenascin. In some embodiments, the targeting moiety recognizes monomeric and/or dimeric and/or trimeric and/or hexameric forms of tenascin.

In one embodiment, the targeting moiety recognizes and binds fibronectin. Fibronectin is a glycoprotein that links cells to collagen fibers in the ECM, allowing the cells to move through the ECM. By binding to integrins, fibronectin unfolds to form functional dimers. In some embodiments, the targeting moiety recognizes the monomeric and/or dimeric form 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 EDA levels are associated with a variety of diseases and disorders, including psoriasis, rheumatoid arthritis, diabetes, and cancer. In some embodiments, the targeting moiety recognizes fibronectin containing EDA subtypes and can be used to target the chimeric protein to diseased cells, including cancer cells. In some embodiments, the targeting moiety recognizes fibronectin containing EDB subtypes. In some embodiments, such targeting moieties may be used to target the chimeric protein to tumor cells, including tumor neovasculature.

In one embodiment, the targeting moiety recognizes and binds fibrin. Fibrin is another proteinaceous substance often found in the matrix network of the ECM. Fibrin is formed by the action of the protease thrombin on fibrinogen, which results in fibrin polymerization. In some embodiments, the targeting moiety recognizes one or more epitopes on the fibrin. In some embodiments, the targeting moiety recognizes fibrin in monomeric as well as in polymerized form.

In one embodiment, the targeting moiety recognizes and binds laminin. Laminin is the major component of the basal lamina, which underlies the protein network of cells and organs. Laminins are heterotrimeric proteins containing an alpha chain, a beta chain, and a gamma chain. In some embodiments, the targeting moiety recognizes one or more epitopes on a laminin. In some embodiments, the targeting moiety recognizes monomeric, dimeric, and trimeric forms of laminin.

In one embodiment, the targeting moiety recognizes and binds nestin or endoglin. Nestin/lactolin is a family of highly conserved sulfated glycoproteins. They constitute the main structural component of the basement membrane and function to link the laminin and collagen IV networks in the basement membrane. Members of this family include nestin-1 and nestin-2. In various embodiments, the targeting moiety recognizes an epitope on nestin-1 and/or nestin-2.

In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes an epitope present on any target described herein (e.g., an ECM protein). In one embodiment, the antigen recognition domain recognizes one or more linear epitopes present on the protein. As used herein, a linear epitope refers to any contiguous amino acid sequence present on a protein. In another embodiment, the antigen recognition domain recognizes one or more conformational epitopes present on the protein. As used herein, a conformational epitope refers to one or more portions of amino acids (which may be discontinuous) that form a three-dimensional surface having features and/or shape and/or tertiary structure capable of being recognized by an antigen recognition domain.

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 naturally occurring or synthetic analogs, variants or mutants of any of the targets (e.g., ECM proteins) described herein. In various embodiments, the targeting moiety can bind to any form of the proteins described herein, including monomers, dimers, trimers, tetramers, heterodimers, multimers, and related forms. In various embodiments, the targeting moiety can bind to any post-translationally modified form of the protein described herein, e.g., glycosylated and/or phosphorylated forms.

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 one embodiment, DNA is shed from necrotic or apoptotic tumor cells or other diseased cells into the extracellular space.

In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more non-cellular structures associated with atherosclerotic plaques. Two types of atherosclerotic plaques are known. Fibrolipid (fibro-fatty) plaques are characterized by the accumulation of lipid-filled cells under the arterial subintium. Beneath the endothelium is a fibrous cap that covers the atheromatous core of the plaque. The core comprises lipid-filled cells (macrophages and smooth muscle cells), fibrin, proteoglycans, collagen, elastin, and cell debris with elevated tissue cholesterol and cholesterol ester content. In late-stage plaques, the central core of the plaque usually contains extracellular cholesterol deposits (released from dead cells) that form areas of cholesterol crystals with empty needle-like fissures. At the periphery of the plaque are younger foam cells and capillaries. Fibrous plaque is also located under the intima within the arterial wall, resulting in thickening and dilation of the wall and sometimes local punctiform narrowing of the lumen with some atrophy of the muscle layer. Fibrous plaques contain collagen fibers (eosinophils), calcium precipitates (hematoxylin affinity) and lipid-filled cells. In some embodiments, the targeting moiety recognizes and binds one or more 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 cell debris is nucleic acid, such as DNA or RNA, released from dead cells.

In various embodiments, the targeting moiety comprises an antigen recognition domain that recognizes one or more acellular structures found in brain plaques associated with neurodegenerative diseases. In some embodiments, the targeting moiety recognizes and binds to one or more acellular structures located in amyloid plaques found in the brain of alzheimer's disease patients. For example, the targeting moiety can recognize and bind the peptide amyloid β, which is a major component of amyloid plaques. In some embodiments, the targeting moiety recognizes and binds to one or more acellular structures located in brain plaques found in huntington's disease patients. In various embodiments, the targeting moiety recognizes and binds to one or more acellular structures found in plaques associated with other neurodegenerative or musculoskeletal diseases such as dementia with lewy bodies and inclusion body myositis.

Linker and functional group

In various embodiments, the chimeric proteins of the present invention may include one or more functional groups, residues, or moieties. In various embodiments, the one or more functional groups, residues, or moieties are linked or genetically fused to any of the signaling agents or targeting moieties described herein (e.g., SIRP1 a). In some embodiments, these functional groups, residues, or moieties impart one or more desired properties or functions to the chimeric proteins of the invention. Examples of such functional groups and techniques for introducing them into the chimeric proteins of the invention are known in the art, see, for example, Remington's Pharmaceutical Sciences, 16 th edition, Mack Publishing co., Easton, Pa. (1980).

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

In some embodiments, the functional group, residue, or moiety comprises a suitable pharmaceutically acceptable polymer, such as poly (ethylene glycol) (PEG) or a derivative thereof (e.g., methoxypoly (ethylene glycol) or mPEG). In some embodiments, the conjugation of the PEG moiety increases the half-life of the SIRP 1a binding protein and/or reduces immunogenicity. In general, any suitable form of pegylation may be used, such as pegylation used in the art for antibodies and antibody fragments (including but not limited to single domain antibodies, such as VHH); see, e.g., Chapman, nat. biotechnol, 54,531-545 (2002); veronese and Harris, adv.drug deliv.rev.54,453-456(2003), Harris and Chess, nat.rev.drug discov.,2, (2003) and WO04060965, the entire contents of which are incorporated herein by reference. Various reagents for protein pegylation are also commercially available, for example, from Nektar Therapeutics, USA. In some embodiments, site-directed pegylation is used, particularly via cysteine residues (see, e.g., Yang et al, Protein Engineering,16,10,761-770(2003), the entire contents of which are incorporated herein by reference). For example, PEG may be attached to cysteine residues naturally occurring in the chimeric proteins of the invention for this purpose. In some embodiments, the chimeric proteins of the invention are modified to suitably introduce one or more cysteine residues for PEG attachment, or an amino acid sequence comprising one or more cysteine residues for PEG attachment may be fused to the amino terminus and/or the carboxy terminus of the chimeric proteins of the invention, using techniques known in the art.

In some embodiments, the functional group, residue or moiety comprises N-linked or O-linked glycosylation. In some embodiments, N-linked or O-linked glycosylation is introduced as part of co-translational and/or post-translational modifications.

In some embodiments, the functional group, residue or moiety comprises one or more detectable labels or other signal generating groups or moieties. Suitable labels (labels) and techniques for attaching, using and detecting them are known in the art and include, but are not limited to, fluorescent labels (e.g., fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescein and fluorescent metals such as Eu or other metals from the lanthanide series), phosphorescent labels, chemiluminescent labels or bioluminescent labels (e.g., luminol, isoluminol, thermoaromatic acridinium ester, imidazole, acridinium salt, oxalate ester, dioxyethane or GFP and analogs thereof), radioisotopes, metals, metal chelates or metal cations or other metals or metal cations particularly suitable for in vivo, in vitro or in situ diagnostics and imaging, as well as chromophores and enzymes (e.g., malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, beta-gamma-glucosidase, gamma-glucosidase, and the like, Yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate 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 that can be detected using NMR or ESR spectroscopy. Such labelled VHH and polypeptides of the invention may for example be used for in vitro, in vivo or in situ assays (including per se known immunoassays such as ELISA, RIA, EIA and other "sandwich assays" etc.) as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label.

In some embodiments, the functional group, residue, or moiety comprises a tag (tag) linked or genetically fused to the chimeric protein. In some embodiments, the chimeric protein may comprise a single tag or multiple tags. For example, the tag is a peptide, sugar or DNA molecule that does not inhibit or prevent binding of the chimeric protein to its target 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. Exemplary labels are described, for example, in U.S. patent publication No. US 2013/00558962. In some embodiments, the tag is an affinity tag, such as a glutathione-S-transferase (GST) and histidine (His) tag. In one embodiment, the chimeric protein comprises a His-tag.

In some embodiments, the functional group, residue, or moiety comprises a chelating group, for example to chelate a metal or one of the metal cations. Suitable chelating groups include, for example, but are not limited to, diethyltriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

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 a functional group can be used to link the chimeric protein of the invention to another protein, polypeptide or compound that is bound to the other half of the binding pair, i.e., by forming the binding pair. For example, the chimeric protein of the invention may be conjugated to biotin and linked to another avidin or streptavidin conjugated protein, polypeptide, compound or carrier. For example, such conjugated chimeric proteins can be used as reporters, e.g., in diagnostic systems in which a detectable signal generating agent is conjugated to avidin or streptavidin. Such binding pairs may also be used, for example, to bind the chimeric protein to a carrier, including a carrier suitable for pharmaceutical purposes. One non-limiting example is the liposome formulation described by Cao and Suresh, Journal of Drug Targeting,8,4,257 (2000). Such binding pairs may also be used to link a therapeutically active agent to the chimeric protein of the invention.

In some embodiments, the chimeric proteins of the invention optionally comprise one or more linkers. In some embodiments, the chimeric proteins of the invention comprise a linker connecting the targeting moiety and the signaling agent. In some embodiments, the chimeric proteins of the invention comprise a linker within the signaling agent (e.g., in the case of single-chain TNF, it may comprise two linkers to produce a trimer).

In some embodiments, vectors encoding the chimeric proteins of the invention linked as a single nucleotide sequence to any linker described herein are provided and can be used to make such chimeric proteins.

In some embodiments, the linker length may allow for efficient binding of the targeting moiety and signaling agent to their receptors. For example, in some embodiments, the linker length allows one of the targeting moieties and the signaling agent to effectively bind to a receptor on the same cell, and the other targeting moiety to effectively bind to another cell. Exemplary cell pairs are provided elsewhere herein.

In some embodiments, the linker length is at least equal to the minimum distance between one of the targeting moieties and the binding site of the signaling agent to a receptor on the same cell. In some embodiments, the linker length is at least two, or three, or four, or five, or ten, or twenty, or 25, or 50, or one hundred, or more times the minimum distance between one of the targeting moieties and the binding site of the signaling agent to a receptor on the same cell.

As described herein, the linker length allows for efficient binding of one of the targeting moieties and the signaling agent to a receptor on the same cell, which binding is sequential, e.g., targeting moiety/receptor binding precedes signaling agent/receptor binding.

In some embodiments, there are two linkers in a single chimera, each linker linking the signaling agent to the targeting moiety. In various embodiments, the length of the linker allows for the formation of a site with disease cells and effector cells without steric hindrance that would prevent either cell from regulating.

The present invention contemplates the use of multiple linker sequences. In various embodiments, the linker may be derived from a naturally occurring multidomain Protein, or may be a desired empirical linker, such as described in Chichil et al, (2013), Protein Sci.22(2): 153-. In some embodiments, linkers can be designed using a linker design database and computer programs, such as those described in Chen et al, (2013), Adv Drug Deliv Rev.65(10): 1357-. In various embodiments, the linker may be functional. For example, without limitation, a linker may act to improve folding and/or stability, improve expression, improve pharmacokinetics, and/or improve biological activity of the chimeric proteins of the invention.

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

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

In various embodiments, the linker consists essentially of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or 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, such as1, 2, 3, 4,5, 6,7, or 8(SEQ ID NO: 247-SEQ ID NO: 254, respectively). In one embodiment, the linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 255). Other exemplary connectors include, but are not limited to, connectors having the following sequence: LE, GGGGS (SEQ ID NO:247), (GGGGS) n (n ═ 1-4) (SEQ ID NO: 247-SEQ ID NO:250), (Gly)8(SEQ ID NO:256), (Gly)6(SEQ ID NO:257), (EAAAK) n (n ═ 1-3) (SEQ ID NO:258-SEQ ID NO:260), a (EAAAK) nA (n ═ 2-5) (SEQ ID NO:261-SEQ ID NO:264), AEAAAKEAAAKA (SEQ ID NO:261), a (EAAAK)4alea (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 (XP) n, wherein X represents any amino acid, such as Ala, Lys or Glu. In various embodiments, the linker is a GGS.

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 linkers of G, S and E randomly placed every 4 amino acid intervals.

In some embodiments, the linker is a hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE, including subclasses such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA 2). In various embodiments, the linker is a hinge region of an antibody (e.g., IgG, IgA, IgD, and IgE, including subclasses such as IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA 2). The hinge region found in IgG, IgA, IgD and IgE class antibodies acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant regions, hinge regions are structurally diverse, differing in both sequence and length between immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies between IgG subclasses. The hinge region of IgG1 comprises amino acids 216 and 231 and because it is freely flexible, the Fab fragment can rotate about its axis of symmetry and move within a sphere centered on the first of the two inter-heavy chain disulfide bonds. IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bonds. The hinge region of IgG2 lacks glycine residues, is relatively short, and contains a rigid polyproline double helix stabilized by additional inter-heavy chain disulfide bonds. These properties limit the flexibility of the IgG2 molecule. IgG3 differs from the other subclasses by its unique extended hinge region (approximately four times as long as the IgG1 hinge region), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible polyproline double helix. In IgG3, the Fab fragment is relatively distant from the Fc fragment, giving the molecule greater flexibility. The extended hinge in IgG3 also results in a higher 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 IgG 2. It is reported that the flexibility of the hinge region decreases in the order of IgG 3> IgG1 > IgG4 > IgG 2.

According to crystallographic studies, the immunoglobulin hinge region can be further functionally subdivided into three regions: an upper hinge region, a core region, and a lower hinge region. See Shin et al, 1992Immunological Reviews 130: 87. The upper hinge region includes amino acids from the carboxy terminus of CH1 to the first residue in the hinge that restricts motion (typically the first cysteine residue that forms an interchain disulfide bond between the two heavy chains). The length of the upper hinge region is related to the fragment flexibility of the antibody. The core hinge region contains the inter-heavy chain disulfide bonds, while the lower hinge region connects the amino-termini of the CH2 domain and includes residues in CH 2. As above. The core hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 274), which when dimerized by formation of disulfide bonds, produces a cyclic octapeptide thought to act as a pivot, thereby imparting flexibility. In various embodiments, the linkers of the invention comprise one or two or three of the upper, core and lower hinge regions of any antibody (e.g., IgG, IgA, IgD and IgE, including subclasses such as IgG1, IgG2, IgG3 and IgG4, and IgA1 and IgA 2). The hinge region may also contain one or more glycosylation sites, which include a number of structurally different types of sites for sugar attachment. For example, IgA1 contains 5 glycosylation sites within a 17 amino acid segment of the hinge region, conferring resistance to enteroproteases to hinge region polypeptides, which is considered an advantageous property of secretory immunoglobulins. In various embodiments, linkers of the present invention comprise one or more glycosylation sites. In various embodiments, the linker is the hinge-CH 2-CH3 domain of human IgG4 antibody.

If desired, the chimeric proteins of the invention may be linked to an antibody Fc region comprising one or both of the CH2 and CH3 domains, and optionally a hinge region. For example, vectors encoding the chimeric proteins of the invention linked to an Fc region as a single nucleotide sequence may be used to prepare such polypeptides.

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

In various embodiments, the linker may be functional. For example, without limitation, the linker may act to improve folding and/or stability, improve expression, improve pharmacokinetics, and/or improve biological activity of the chimeric proteins of the invention. In another example, a linker may serve to target the chimeric protein to a particular cell type or location.

Modification and production of chimeric proteins

In various embodiments, the chimeric proteins of the invention comprise a targeting moiety that is a VHH (e.g., SIRP1 a). In various embodiments, VHH is not limited to a particular biological source or a particular method of preparation. For example, VHH can be obtained generally as follows: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) by expressing a nucleotide sequence encoding a naturally occurring VHH domain; (3) by "humanization" of naturally occurring VHH domains or by expression of nucleic acids encoding such humanized VHH domains; (4) by "camelising" a naturally occurring VH domain from any animal species, for example from a mammalian species, for example from a human, or by expressing a nucleic acid encoding such a camelised VH domain; (5) by "camelization" of a "domain antibody" or "DAb" as described in the art, or by expression of a nucleic acid encoding such a camelized VH domain; (6) preparing proteins, polypeptides or other amino acid sequences known in the art by using synthetic or semi-synthetic techniques; (7) preparing a nucleic acid encoding a VHH by using nucleic acid synthesis techniques known in the art, and then expressing the nucleic acid thus obtained; and/or (8) by any combination of one or more of the foregoing.

In one embodiment, the chimeric protein comprises a VHH that corresponds to the VHH domain of a naturally occurring heavy chain antibody directed to human SIRP1 a. In some embodiments, such VHH sequences may typically be generated or obtained by suitably immunizing a camelid species with a SIRP1a molecule (i.e. so as to generate an immune response and/or heavy chain antibodies against SIRP 1a), by obtaining a suitable biological sample (e.g. a blood sample, or any B cell sample) from a camelid, and by generating VHH sequences against SIRP1a starting from the sample using any suitable known technique. In some embodiments, a naturally occurring VHH domain directed to SIRP1a may be obtained from a naive library of camelid VHH sequences, e.g., by screening such library using SIRP1a or at least a portion, fragment, epitope or epitope thereof using one or more screening techniques known in the art. Such libraries and techniques are described, for example, in WO9937681, WO0190190, WO03025020 and WO03035694, the entire contents of which are incorporated herein by reference. In some embodiments, improved synthetic or semi-synthetic libraries derived from the naive VHH library may be used, for example a VHH library obtained from the naive VHH library by e.g. random mutagenesis and/or CDR shuffling techniques, as described in WO0043507, the entire content of which is incorporated herein by reference. In some embodiments, another technique to obtain a VHH sequence directed to SIRP1a involves suitably immunizing a transgenic mammal capable of expressing heavy chain antibodies (i.e., so as to elicit an immune response and/or heavy chain antibodies directed to SIRP 1a), obtaining a suitable biological sample (e.g., a blood sample, or any B cell sample) from the transgenic mammal, and then generating a VHH sequence directed to SIRP1a from the sample using any suitable known technique. For example, mice expressing heavy chain antibodies and other methods and techniques described in WO02085945 and WO04049794 (the entire contents of which are incorporated herein by reference) can be used for this purpose.

in one embodiment, the chimeric protein comprises a VHH which has been "humanized", i.e. by replacement of one or more amino acid residues in the amino acid sequence of a naturally occurring VHH sequence (and in particular in the framework sequence) with one or more amino acid residues present at corresponding positions in the VH domain of a conventional 4 chain antibody from human. This can be done using humanization techniques known in the art. In some embodiments, possible humanized substitutions or combinations of humanized substitutions may be determined by methods known in the art, for example by comparison between the VHH sequence and the naturally occurring human VH domain sequence. In some embodiments, the humanized substitutions are selected such that the resulting humanized VHH still retains favorable functional properties. In general, as a result of humanization, the VHH of the invention may become more "human-like" while still retaining advantageous properties, such as reduced immunogenicity, compared to the corresponding naturally occurring VHH domain. In various embodiments, the humanized VHH of the invention may be obtained in any suitable manner known in the art and is therefore not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring VHH domains as starting material.

In one embodiment the chimeric protein comprises a VHH which has been "camelised", i.e. by substituting one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain of a conventional 4 chain antibody with one or more amino acid residues present at corresponding positions in a VHH domain of a camelid heavy chain antibody. In some embodiments, such "camelized" substitutions are inserted at amino acid positions formed and/or present at the VH-VL interface and/or at marker residues of the so-called Camelidae (Camelidae) (see, e.g., WO9404678, the entire contents of which are incorporated herein by reference). In some embodiments, the VH sequences used as starting materials or starting points for the generation or design of camelised VHHs are VH sequences from mammals, for example human VH sequences such as VH3 sequences. In various embodiments, camelized VHHs may be obtained in any suitable manner known in the art (i.e. as shown below points (1) - (8) above) and are therefore not strictly limited to polypeptides obtained using polypeptides comprising naturally occurring VH domains as starting material.

In various embodiments, "humanization" and "camelization" may both be performed by providing a nucleotide sequence encoding a naturally occurring VHH domain or VH domain, respectively, and then altering one or more codons in the nucleotide sequence in a manner known in the art such that the new nucleotide sequence encodes a "humanized" or "camelized" VHH, respectively. The nucleic acid may then be expressed in a manner known in the art to provide the desired VHH of the invention. Alternatively, the amino acid sequence of a humanized or camelized VHH required for the present invention may be designed based on the amino acid sequence of a naturally occurring VHH domain or VH domain, respectively, and then synthesised de novo using peptide synthesis techniques known in the art. Furthermore, based on the amino acid sequence or nucleotide sequence of a naturally occurring VHH domain or VH domain, respectively, a nucleotide sequence encoding a desired humanized or camelized VHH may be designed and then synthesised de novo using nucleic acid synthesis techniques known in the art, after which the nucleic acid thus obtained may be expressed in a manner known in the art to provide the desired VHH of the invention. Other suitable methods and techniques for obtaining a VHH of the invention and/or nucleic acids encoding the same, starting from naturally occurring VH sequences or VHH sequences, are known in the art and may, for example, comprise combining one or more portions of one or more naturally occurring VH sequences (e.g., one or more FR sequences and/or CDR sequences), one or more portions of one or more naturally occurring VHH sequences (e.g., one or more FR sequences or CDR sequences), and/or one or more synthetic or semi-synthetic sequences in a suitable manner to provide a VHH of the invention or nucleotide sequences or nucleic acids encoding the same.

Methods of producing the chimeric proteins of the invention are described herein. For example, DNA sequences encoding the chimeric proteins of the invention (e.g., DNA sequences encoding the modified signaling agents and targeting moieties and linkers) can be chemically synthesized using methods known in the art. The synthetic DNA sequence may be linked to other suitable nucleotide sequences, including, for example, expression control sequences, to produce a gene expression construct encoding the desired chimeric protein. Thus, in various embodiments, the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding a chimeric protein of the present invention.

The nucleic acid encoding the chimeric protein of the invention may be introduced (ligated) into an expression vector, which may be introduced into a host cell by transfection, transformation or transduction techniques. For example, a nucleic acid encoding a chimeric protein of the invention can be introduced into a host cell by retroviral transduction. Exemplary host cells are 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 cell may be grown under conditions that allow the host cell to express the gene encoding the chimeric protein of the invention. Thus, in various embodiments, the present invention provides expression vectors comprising a nucleic acid encoding a chimeric protein of the present invention. In various embodiments, the present invention also provides host cells comprising such expression vectors.

The specific expression and purification conditions will vary depending on the expression system used. For example, if a gene is to be expressed in E.coli, the engineered gene is first cloned into an expression vector by locating it downstream of an appropriate bacterial promoter (e.g., Trp or Tac) and prokaryotic signal sequences. In another example, if the engineered gene is to be expressed in a eukaryotic host cell, such as a CHO cell, it is first inserted into an expression vector containing, for example, a suitable eukaryotic promoter, secretion signal, enhancer, and various introns. The genetic construct may be introduced into the host cell using transfection, transformation or transduction techniques.

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

Thus, in various embodiments, the invention provides nucleic acids encoding the chimeric proteins of the invention. In various embodiments, the invention provides host cells comprising a nucleic acid encoding a chimeric protein of the invention.

In various embodiments, the SIRP 1a targeting moiety of the invention or a chimeric protein comprising the same may be expressed in vivo, e.g., in a patient. For example, in various embodiments, the SIRP 1a targeting moiety of the invention or a chimeric protein comprising the same may be administered in the form of a nucleic acid encoding the SIRP 1a targeting moiety of the invention or a chimeric protein comprising the same. In various embodiments, the nucleic acid is DNA or RNA. In some embodiments, the SIRP 1a targeting moiety of the invention or a chimeric protein comprising the same 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 modifications found in U.S. patent No.8,278,036, which is incorporated herein by reference in its entirety. 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 administering a modified mRNA encoding one or more chimeric proteins of the invention. In some embodiments, the invention relates to a vector for gene therapy comprising the above-described vector. In some embodiments, the invention relates to methods of gene therapy comprising the above. In various embodiments, the nucleic acid is in the form of an oncolytic virus, such as an adenovirus, reovirus, measles virus, herpes simplex virus, newcastle disease virus, or vaccinia virus.

Pharmaceutically acceptable salts and excipients

The chimeric proteins described herein can have functional groups that are sufficiently basic to be reactive with inorganic or organic acids, or have carboxyl groups that can be reactive with inorganic or organic bases to form pharmaceutically acceptable salts. As is well known in the art, pharmaceutically acceptable acid addition salts are formed from pharmaceutically acceptable acids. These salts include, for example, the pharmaceutically acceptable salts listed below: journal of Pharmaceutical Science, 66, 2-19(1977) and The Handbook of Pharmaceutical Salts; properties, Selection, and use, p.h.stahl and c.g.wermuth (eds.), Verlag, Zurich (switzerland) 2002, the entire contents of which are incorporated herein by reference.

As non-limiting examples, pharmaceutically acceptable salts include sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, salts of benzoic acid, salts of benzoic, Naphthalene-2-benzoate, isobutyrate, phenylbutyrate, α -hydroxybutyrate, butyne-1, 4-dicarboxylate, hexyne-1, 4-dicarboxylate, caprate, octanoate, cinnamate, glycolate, heptanoate, hippurate, malate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, phthalate, terephthalate, propionate, phenylpropionate, sebacate, suberate, p-bromobenzenesulfonate, chlorobenzenesulfonate, ethylsulfonate, 2-isethionate, methylsulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, naphthalene-1, 5-sulfonate, xylenesulfonate and tartrate.

the term "pharmaceutically acceptable salt" also refers to salts of the compositions of the present invention that have an acidic functionality, such as a carboxylic acid functionality, and a base. 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-methylamine, N-ethylamine; diethylamine; triethylamine; mono-, bis-or tris- (2-OH-lower alkyl amines), such as mono-, bis-or tris- (2-hydroxyethyl) amine, 2-hydroxy-tert-butylamine or tris- (hydroxymethyl) methylamine, N-di-lower alkyl-N- (hydroxy-lower alkyl) -amines, such as N, N-dimethyl-N- (2-hydroxyethyl) amine or tris- (2-hydroxyethyl) amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.

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

Pharmaceutical compositions and formulations

In various embodiments, the present invention relates to pharmaceutical compositions comprising a chimeric protein described herein and a pharmaceutically acceptable carrier or excipient. Any of the pharmaceutical compositions described herein can be administered to a subject as a component of a composition comprising a pharmaceutically acceptable carrier or vehicle. Such compositions may optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide a form for proper administration.

In various embodiments, the pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The pharmaceutical excipient may be, for example, saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, or the like. In addition, auxiliaries, stabilizers, thickeners, lubricants and colorants may be used. In one embodiment, the pharmaceutically acceptable excipient is 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 may also be employed as liquid excipients, particularly for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk (chalk), silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. Any of the reagents described herein may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. Other examples of suitable Pharmaceutical excipients are described in Remington's Pharmaceutical Sciences 1447-.

The invention encompasses such pharmaceutical compositions (and/or additional therapeutic agents) in a variety of formulations. Any of the pharmaceutical compositions of the invention described herein (and/or the additional therapeutic agent) may take the form of a solution, suspension, emulsion, drop, tablet, pill, capsule, liquid-containing capsule, gelatin capsule, powder, sustained release formulation, suppository, emulsion, aerosol, spray, suspension, lyophilized powder, frozen suspension, dried powder, or any other suitable form 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 another embodiment, the pharmaceutical composition is formulated in the form of a soft gel capsule. In another embodiment, the pharmaceutical composition is formulated in the form of a gelatin capsule. In another embodiment, the pharmaceutical composition is formulated as a liquid.

The pharmaceutical compositions (and/or additional agents) of the present invention may also include a solubilizing agent, if desired. Likewise, the agents may be delivered using 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.

Formulations comprising the pharmaceutical compositions (and/or other agents) of the present invention may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. These methods generally include the step of bringing the therapeutic agent into association with a carrier which constitutes one or more accessory ingredients. Generally, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired dosage form for formulation (e.g., wet or dry granulation, powder mixing, and the like, followed by tableting using conventional methods known in the art).

In various embodiments, any of the pharmaceutical compositions (and/or other agents) described herein are formulated according to conventional methods into compositions suitable for the modes of administration described herein.

Routes of administration include, for example: oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, inhalation, or topical administration. Administration may be local or systemic. In some embodiments, the administration is oral. In another embodiment, administration is by parenteral injection. The mode of administration may be at the discretion of the physician and will depend in part on the location of the medical condition. In most cases, administration results in the release of any of the agents described herein into the bloodstream.

In one embodiment, the chimeric proteins described herein are formulated in accordance with conventional methods into compositions suitable 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. Compositions for oral administration may comprise one or more agents, for example sweetening agents, such as fructose, aspartame or saccharin; flavoring agents, such as peppermint, oil of wintergreen, or cherry; a colorant; and a preservative to provide a pharmaceutically pleasing formulation. In addition, when in tablet or pill form, the compositions may be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained action over an extended period of time. Selectively permeable membranes surrounding an osmotic active driving any of the chimeric proteins described herein are also suitable for use in compositions for oral administration. In these latter platforms, fluid from the capsule environment is absorbed by the driving compound, which expands to displace the agent or agent composition through the pores. These delivery platforms can provide a substantially zero order delivery profile, as opposed to the spike profile of immediate release formulations. A time delay material such as glyceryl monostearate or glyceryl stearate may also be used. Oral compositions may include standard excipients such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. In one embodiment, the excipient is pharmaceutical grade. Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth, and the like, and mixtures thereof.

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

For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ), or Phosphate Buffered Saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be antimicrobial. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

The compositions provided herein (alone or in combination with other suitable components) can be formulated as an aerosol (i.e., "spray") for administration by inhalation. The aerosol may be placed in a pressurized acceptable propellant, such as dichlorodifluoromethane, propane, nitrogen, and the like.

Any of the pharmaceutical compositions of the present invention (and/or other agents) described herein can be administered by controlled or sustained release means known to those of ordinary skill in the art or by a delivery device. Examples include, but are not limited to, those disclosed in U.S. Pat. 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 used to provide controlled or sustained release of one or more active ingredients, for example using hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone, other polymeric matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof, to provide the desired release profile in varying proportions. Suitable controlled or sustained release formulations known to those skilled in the art, including those described herein, can be readily 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, such as, but not limited to, tablets, capsules, gel capsules and caplets suitable for controlled or sustained release.

Controlled or sustained release of the active ingredient may be stimulated by a variety of conditions, including but not limited to a change in pH, a change in temperature, stimulation by light of an appropriate wavelength, concentration or availability of an enzyme, concentration or availability of water, or other physiological conditions or compounds.

In another embodiment, a Controlled Release system may be placed near 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 reviews by Langer, 1990, Science 249: 1527-.

The pharmaceutical formulation is preferably sterile. Sterilization can be accomplished, for example, by filtration through sterile filtration membranes. When the composition is lyophilized, it may be filter sterilized before or after lyophilization and reconstitution.

Administration and dosage

It will be appreciated that the actual dosage of the chimeric protein administered according to the invention will vary depending upon the particular dosage form and mode of administration. One skilled in the art can consider many factors that can alter the action of the chimeric protein (e.g., body weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the subject, drug combination, genetic disposition, and response sensitivity). Administration may be continuous within the maximum tolerated dose, or in one or more discrete doses. For a given set of conditions, the optimal rate of administration can be determined by one skilled in the art using conventional administration dosage tests.

In some embodiments, suitable dosages of the chimeric protein are from about 0.01mg/kg to about 10g/kg of subject weight, from about 0.01mg/kg to about 1g/kg of subject weight, from about 0.01mg/kg to about 100mg/kg of patient weight, from about 0.01mg/kg to about 10mg/kg of patient weight, e.g., about 0.01mg/kg, about 0.02mg/kg, about 0.03mg/kg, about 0.04mg/kg, about 0.05mg/kg, about 0.06mg/kg, about 0.07mg/kg, about 0.08mg/kg, about 0.09mg/kg, about 0.1mg/kg, about 0.2mg/kg, about 0.3mg/kg, about 0.4mg/kg, about 0.5mg/kg, about 0.6mg/kg, about 0.7mg/kg, about 0.8mg/kg, about 0.9mg/kg, about 1mg/kg, about 1.1mg/kg, About 1.2mg/kg, about 1.3mg/kg, about 1.4mg/kg, about 1.5mg/kg, about 1.6mg/kg, about 1.7mg/kg, about 1.8mg/kg, 1.9mg/kg, about 2mg/kg, about 3mg/kg, about 4mg/kg, about 5mg/kg, about 6mg/kg, about 7mg/kg, about 8mg/kg, about 9mg/kg, about 10mg/kg body weight, about 100mg/kg body weight, about 1g/kg body weight, about 10g/kg body weight, including all values and ranges therebetween.

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

In one embodiment, the chimeric protein is administered in the following amounts: from about 0.01mg to about 100g per day, from about 0.01mg to about 75g per day, from about 0.01mg to about 50g per day, from about 0.01mg to about 25g per day, from about 0.01mg to about 10g per day, from about 0.01mg to about 7.5g per day, from about 0.01mg to about 5g per day, from about 0.01mg to about 2.5g per day, from about 0.01mg to about 1g per day, from about 0.01mg to about 100mg per day, from about 0.1mg to about 95mg per day, from about 0.1mg to about 90mg per day, from about 0.1mg to about 85mg per day, from about 0.1mg to about 80mg per day, from about 0.1mg to about 75mg per day, from about 0.1mg to about 70mg per day, from about 0.1mg to about 65mg per day, from about 0.1mg to about 60mg per day, from about 0.1mg to about 0.55 mg per day, from about 0.1mg to about 0.1mg per day, from about 0.1mg to about 30mg per day, about 0.1mg to about 20mg per day, about 0.1mg to about 15mg per day, about 0.1mg to about 10mg per day, about 0.1mg to about 5mg per day, about 0.1mg to about 3mg per day, about 0.1mg to about 1mg per day, or about 5mg to about 80mg per day. In various embodiments, the chimeric protein is administered at the following daily doses: about 0.01mg, about 0.02mg, about 0.03mg, about 0.04mg, about 0.05mg, about 0.06mg, about 0.07mg, about 0.08mg, about 0.09mg, about 0.1mg, about 0.2mg, about 0.3mg, about 0.4mg, about 0.5mg, about 0.6mg, about 0.7mg, about 0.8mg, about 0.9mg, about 1mg, about 2mg, about 3mg, about 4mg, about 5mg, about 6mg, about 7mg, about 8mg, about 9mg, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 55mg, about 60mg, about 65mg, about 70mg, about 75mg, about 80mg, about 85mg, about 95mg, about 100mg, about 200mg, about 500mg, about 1mg, about 5g, about 5.5 g, about 5g, and all values therebetween.

According to certain embodiments of the invention, a pharmaceutical composition comprising a chimeric protein may be administered, for example, more than once per day (e.g., about two, about three, about four, about five, about six, about seven, about eight, about nine, or about ten times per day), about once per day, about once per two days, about once per three days, about once per week, about once per two weeks, about once per month, about once per two months, about once per three months, about once per six months, or about once per year.

Combination therapy and additional therapeutic agents

In various embodiments, the pharmaceutical compositions of the present invention are administered in combination with an additional therapeutic agent. The combined administration may be simultaneous or sequential.

In one embodiment, the additional therapeutic agent and the chimeric protein of the invention are administered to the subject simultaneously. The term "simultaneously" as used herein refers to the administration of the additional therapeutic agent and the chimeric protein at intervals of no more than about 60 minutes, such as no more than about 30 minutes, no more than about 20 minutes, no more than about 10 minutes, no more than about 5 minutes, or no more than about 1 minute. Administration of the additional therapeutic agent and the chimeric protein can be performed by administering a single formulation (e.g., a formulation comprising the additional therapeutic agent and the chimeric protein) simultaneously or administering separate formulations (e.g., a first formulation comprising the additional therapeutic agent and a second formulation comprising the chimeric protein).

The co-administration does not require simultaneous administration of the therapeutic agents if the administration times are such that the pharmacological activities of the additional therapeutic agent and the chimeric protein overlap in time to exert a combined therapeutic effect. For example, the additional therapeutic agent and the chimeric protein may be administered sequentially. The term "sequential" as used herein refers to the administration of additional therapeutic agents and chimeric proteins at intervals greater than about 60 minutes. For example, the time between successive administrations of the additional therapeutic agent and the chimeric protein can be spaced more than about 60 minutes, more than about 2 hours, more than about 5 hours, more than about 10 hours, more than about 1 day, more than about 2 days, more than about 3 days, more than about 1 week, more than about 2 weeks, or more than about 1 month apart. The optimal time of administration will depend on the additional therapeutic agent administered and the metabolic rate, rate of excretion, and/or pharmacokinetic activity of the chimeric protein. Additional therapeutic agents or chimeric protein cells may be administered first.

The combined administration also does not require that the therapeutic agents be administered to the subject by the same route of administration. Rather, each therapeutic agent may be administered by any suitable route, e.g., parenterally or non-parenterally.

In some embodiments, the chimeric proteins described herein act synergistically when co-administered with another therapeutic agent. In such embodiments, the chimeric protein and the additional therapeutic agent may be administered at lower doses than would be used if the agents were used in a monotherapy setting.

In some embodiments, the invention relates to chemotherapeutic agents as additional therapeutic agents. For example, but not limited to, such a combination of a chimeric protein of the invention and a chemotherapeutic agent may be used to treat cancer, as described elsewhere herein. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa (thiotepa) and CYTOXAN cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzotepa, carboquone, metoclopramide and uretepa; vinyl imines and methyl melamines, including hexamethylmelamine (altretamine), tritylamine (triethyleneamine), triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine (trimethlomelamine); polyacetyls (acetogenin) (e.g., bullatacin and bullatacin); camptothecin (camptothecin) (including the synthetic analogue topotecan); bryostatin; sponge statin (cally statin); CC-1065 (including its adozelesin (adozelesin), carvelesin (carzelesin), and bizelesin (bizelesin) synthetic analogs); cryptophycins (e.g., cryptophycins 1 and cryptophycins 8); dolastatin (dolastatin); duocarmycins (duocarmycins) (including the synthetic analogs KW-2189 and CB1-TM 1); eislobin (eleutherobin); coprinus atrata base (pancratistatin); sarcandra glabra alcohol (sarcodictyin); spongistatin (spongistatin); nitrogen mustards such as chlorambucil (chlorambucil), chlorambucil (chloranaphazine), cholorophosphamide (cholorophosphamide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine oxide hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), novembechin (novembichin), benzene mustard cholesterol (phenyleneterester), prednimustine (prednimustine), trofosfamide (trofosfamide), uramustine (uracil mustard); nitrosoureas such as carmustine (carmustine), chlorouretocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine) and ranimustine (ranirnustine); antibiotics such as enediynes antibiotics (e.g., calicheamicin, especially calicheamicin γ II and calicheamicin ω II (see, e.g., Agnew, chem. Intl. Ed. Engl.,33:183-186(1994)), daptomycin (dynemicin), including daptomycin A; bisphosphonates, such as clodronate (clodronate), esperamicin (esperamicin), and neocarcinostatin (neocarzinostatin chromophoropterin) and related chromoprotein enediynes antibiotics chromophores), aclacinomycin (acalomycin), actinomycin (actinomycin), antrocin (oxytocin), aureomycin (aureomycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (calcimycin), carbamycin (carbamycin), actinomycin (oxytocin), oncomycin (oxytocin), paramycin (oxytocin), adromycin (oxytetracycline), adriamycin (monochromycin), monochromycin (monochromycin), monochromycin (monochromycin D-5-D), monochromycin D-6-D (monochromycin D), monochromycin D-D (monochromycin D), and a, ADRIAMYCIN doxorubicin (doxorubicin) (including morpholino-doxorubicin), cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin and doxorubicin), epirubicin (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), mariomycin (marcellomycin), mitomycins (mitomycins) such as mitomycin C, mycophenolic acid (mycophenolic acid), nogomycin (nogalamycin), olivomycin (oleamycin), pelomycin (peplomycin), profomycin (potfiromycin), puromycin (puromycin), quinamycin (quelabubycin), rodobicin (rodorubicin), streptonigromycin (streptamycin), streptozocin (streptozocin), urotucin (puromycin), medryucin (merin), mebendacin (zorubicin), tubercidin (zorubicin); antimetabolites such as methotrexate (methotrexate) and 5-fluorouracil (5-FU); folic acid analogs such as dinotexine (denopterin), methotrexate, pteropterin (pteropterin), trimetrexate (trimetrexate); purine analogs, such as fludarabine (fludarabine), 6-mercaptopurine, thiamiazine (thiamirhine), thioguanine; pyrimidine analogs, such as cyclocytidine (ancitabine), azacitidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), deoxyfluorouridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine); androgens such as caridotestosterone (calusterone), dromostanolone propionate (dromostanolone propionate), epitioandrostanol (epitiostanol), mepiquitane (mepiquitane), testolactone (testolactone); anti-adrenal agents such as aminoglutethimide (minoglutethimide), mitotane (mitotane), trostane (trilostane); folic acid supplements, such as folinic acid (frilic acid); acetoglucurolactone (acegultone); (ii) an aldophosphamide glycoside; aminolevulinic acid (aminolevulinic acid); eniluracil (eniluracil); amsacrine (amsacrine); bessburyl (beslabucil); bisantrene; edatrexate (edatraxate); colchicine (demecolcine); diazaquinone (diaziqutone); iloxanel (elformithine); ammonium etitanium acetate; epothilone (epothilone); etoglut (etoglucid); gallium nitrate; a hydroxyurea; mushroom polysaccharides (lentinan); lonidamine (lonidainine); maytansinoids such as maytansinoids and ansamitocins; mitoguazone (mitoguzone); mitoxantrone (mitoxantrone); mopidanol (mopidanmol); diamine nitracridine (nitrarine); pentostatin (pentostatin); methionine mustard (phenamett); pirarubicin (pirarubicin); losoxantrone (losoxantrone); podophyllinic acid (podophyllic acid); 2-ethyl hydrazide; procarbazine (procarbazine); PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane (rizoxane); rhizomycin (rhizoxin); azofurans (sizofurans); germanium spiroamines (spirogyranium); tenuizonic acid (tenuazonic acid); triimine quinone (triaziquone); 2,2' -trichlorotriethylamine; trichothecenes (trichothecenes) such as T-2 toxin, myxomycin a (veracurin a), bacillus a (roridin a), and serpentin (anguidine)); urethane (urethan); vindesine (vindesine); dacarbazine (dacarbazine); mannomustine (manomostine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromane (pipobroman); gatifloxacin (gacytosine); cytarabine (arabine) ("Ara-C"); cyclophosphamide; thiotepa; taxanes (taxoids), such as TAXOL paclitaxel (paclitaxel) (Bristol-Myers Squibb Oncology, Princeton, n.j.), albumin engineered nanoparticle formulations of ABRAXANE paclitaxel (Cremophor) free of polyoxyethylated castor oil (Cremophor) (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE docetaxel (doxetaxel) (Rhone-Poulenc ror, Antony, France); chlorambucil; GEMZAR gemcitabine (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin (cissplatin), oxaliplatin (oxaliplatin) and carboplatin (carboplatin); vinblastine (vinblastine); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (vincristine); navelbine vinorelbine (vinorelbine); norfloxacin (novantrone); teniposide (teniposide); edatrexae; daunorubicin (daunomycin); aminopterin (aminopterin); (xiloda); ibandronate (ibandronate); irinotecan (irinotecan) (Camptosar, CPT-11) (including irinotecan with 5-FU regimens) and leucovorin (leucovorin)); topoisomerase inhibitor RFS 2000; difluoromethyl ornithine (DMFO); retinoids, such as retinoic acid (retinic acid); capecitabine (capecitabine); combretastatin (combretastatin); folinic acid (LV); oxaliplatin (oxaliplatin), including oxaliplatin treatment regimen (FOLFOX); lapatinib (typerb); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)), and VEGF-A that reduce cell proliferation; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Additionally, the method of treatment may further comprise the use of radiation. In addition, the method of treatment may further comprise administering photodynamic therapy.

In one embodiment, the invention relates to any agent that targets the spliceosome, including any component of the spliceosome, as an additional therapeutic agent in the treatment of cancer.

In one embodiment, the invention relates to any agent that targets Myc (i.e., an anti-Myc therapeutic agent) as an additional therapeutic agent in the treatment of cancer.

In some embodiments, including but not limited to infectious disease applications, the present invention relates to anti-infective agents as additional therapeutic agents. In some embodiments, the anti-infective agent is an antiviral agent, including, but not limited to, Abacavir (Abacavir), Acyclovir (Acyclovir), Adefovir (Adefovir), Amprenavir (Amprenavir), Atazanavir (Atazanavir), Cidofovir (Cidofovir), Darunavir (daunarvir), Delavirdine (Delavirdine), Didanosine (Didanosine), Docosanol (Docosanol), Efavirenz (Efavirenz), elvitevir (Elvitegravir), Emtricitabine (Emtricitabine), enz (Enfuvirtide), Etravirine (Etravirine), Famciclovir (Famciclovir) and Foscarnet (Foscarnet). In some embodiments, the anti-infective agent is an antibacterial agent, including, but not limited to, cephalosporins (cefotaxin, cefuroxime, cefazamide, cefazolin, cefalothin, cefaclor, cefamandole, cefoxitin, cefprozil, cefditoren, cefuroxime, cefaclor, cefuroxime axetil, cefuroxime axetil, cefuroxime, cef; fluoroquinolone antibiotics (ciprofloxacin), levofloxacin (Levaquin), ofloxacin (floxin), gatifloxacin (tequin), moxifloxacin (avelox), and norfloxacin (norflox)); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and deoxycycline); penicillin antibiotics (amoxicillin), ampicillin (ampicillin), penicillin V, dicloxacillin (dicloxacillin), carbenicillin (carbenicillin), vancomycin (vancomycin), and methicillin (methicillin)); a monoamide ring antibiotic (aztreonam); and carbapenem antibiotics (ertapenem), doripenem (doripenem), imipenem (imipenem)/cilastatin (cilastatin), and meropenem (meropenem)). In some embodiments, the anti-infective agent includes antimalarials (e.g., chloroquine (chloroquine), quinine (quinine), mefloquine (mefloquine), primaquine (primaquine), doxycycline (doxycycline), artemether/lumefantrine (lumefantrine), atovaquone (atovaquone)/proguanil (proguanil), and sulfadoxine (sulfadoxine)/pyrimethamine (pyrimethanmine)), metronidazole (metronidazole), tinidazole (tinidazole), ivermectin (ivermectin), pyrantel pamoate (pyrantel pamoate), and albendazole (albendazole).

In some embodiments, including but not limited to autoimmune applications, the additional therapeutic agent is an immunosuppressive agent. In some embodiments, the immunosuppressive agent is an anti-inflammatory agent, such as a steroidal anti-inflammatory agent or a non-steroidal anti-inflammatory agent (NSAID). Steroids, particularly adrenal corticosteroids and their synthetic analogs, are well known in the art. Examples of corticosteroids that may be used in the present invention include, but are not limited to, hydroxytryptazinone (hydroxytryptaminolone), alpha-methyl dexamethasone (alpha-methyl dexamethasone), beta-methyl dexamethasone, beclomethasone dipropionate (beclomethasone dipropionate), betamethasone benzoate (betamethasone benzoate), betamethasone dipropionate, betamethasone valerate, clobetasol valerate (clobetasol valerate), desonide (desonide), desoximetasone (desoxyymethasone), dexamethasone (desoxymethasone), diflorasone diacetate (diflorasone diacetate), diflucortolone (diflucortolone valerate), fludrolone hydrofluoride (fludroxydol), fluocinonide (fluidolone acetonide), fluocinonide (fluflurandrenolide), fluocinonide (flunisolide), fluoxynisolone (flunisolide), fluoxyflunisolide (flunisolide), fluoxynil (flunisolide), fluocinonide/fluocinonide), fluocinonide (fluocinonide/fluocinonide), fluocinonide (fluocinolone/fluocinonide), fluocinolone/fluocinonide (fluocinolone/fluocinonide), fluocinolone, fluocinonide, fluocinolone/fluocinolone (fluocinonide), fluocinonide (fluocinolone/fluocinonide (fluocinonide), fluocinolone, Hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cotolone, fluocinolone acetonide, fludrocortisone, diflorasone diacetate, fludrolone acetonide, medrysone, amcinafelon, amcinamide, betamethasone and its corresponding esters, prednisolone chloride, clocortolone (clocotrione), cinolone (clesinolone), dichloropine (dichlorrisone), difluprednate (difluprednate), fluocinolone (fluclone ide), flunisolide (flutolide), fluorometholone (fluorometholone), fluoropolylone (fluperolone), flupredone (fluprednilone), hydrocortisone (hydrocortisone), methylprednisolone (meprednisone), paramethasone (paramethasone), prednisolone (prednisone), prednisone (prednisone), betamethasone dipropionate. Examples of (NSAIDS) that may be used in the present invention include, but are not limited to, salicylic acid, acetylsalicylic acid, methyl salicylate, glycol salicylate, salicylamide, benzyl-2, 5-diacetoxybenzoic acid, ibuprofen (ibuprofen), sulindac (fulindac), naproxen (naproxen), ketoprofen (ketoprofen), etofenamate (etofenamate), phenylbutazone (phenylbutazone), and indomethacin (indomethacin). In some embodiments, the immunosuppressive agent can be a cytostatic agent, such as alkylating agents, antimetabolites (e.g., azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g., basiliximab, daclizumab (daclizumab), and muromab), anti-immunophilins (anti-immunophilins) (e.g., cyclosporine, tacrolimus, sirolimus), interferons, opioids (opioids), TNF binding proteins, mycophenolates (mycophenolate), and small biological agents (e.g., fingolimod, myriocin). Other anti-inflammatory agents are described, for example, in U.S. patent No.4,537,776, the entire contents of which are incorporated herein by reference.

In some embodiments, the present invention relates to agents useful as additional therapeutic agents for the treatment of obesity. Exemplary agents for treating obesity include, but are not limited to orlistat (orlistat) (e.g., ALL1, XENICAL), lorcaserin (loraricin) (e.g., BELVIQ), phentermine-topiramate (e.g., QSYMIA), sibutramine (e.g., reducil or merdia), rimonabant (rimonabant) (acompla), exenatide (e.g., BYETTA), pramlintide (e.g., SYMLIN) phentermine, benzphetamine (benzphentermine), diethylbenzophenone (diethylpropion), phendimetrazine (pherylmutame), bupropion (buproppion), and metformin (metmin). Among the additional agents are agents that interfere with the body's ability to absorb specific nutrients in food, such as orlistat (e.g., ALU, XENICAL), glucomannan, and guar gum. Among such additional agents are agents that suppress appetite, for example catecholamines and derivatives thereof (such as phentermine (phenteimine) and other amphetamine-based drugs), various antidepressants and mood stabilizers (such as bupropion and topiramate), anorectic agents (such as dexedrine, digoxin). Agents that increase body metabolism are also among the additional agents.

In some embodiments, the additional therapeutic agent may be selected from appetite suppressants, neurotransmitter re-uptake inhibitors, dopaminergic agonists, serotonergic agonists, gabaergic signaling modulators, anticonvulsants, antidepressants, monoamine oxidase inhibitors, substance P (NK1) receptor antagonists, melanocortin receptor agonists and antagonists, lipase inhibitors, fat absorption inhibitors, modulators of energy uptake or metabolism, camel receptor modulators, agents for treating addiction, agents for treating metabolic syndrome, peroxisome proliferator-activated receptor (PPAR) modulators; dipeptidyl peptidase 4(DPP-4) antagonists, agents for treating cardiovascular disease, agents for treating elevated triglyceride levels, agents for treating low HDL, agents for treating hypercholesterolemia, and agents for treating hypertension. Some agents for cardiovascular disease include statins (e.g., lovastatin, atorvastatin, fluvastatin, rosuvastatin, simvastatin, and pravastatin) and omega-3 agents (e.g., LOVAZA, EPANQVA, VASCEPA, esterified omega-3, typically fish oil, krill oil, algae oil). In some embodiments, the additional agent may be selected from amphetamines, benzodiazepines, sulfonylureas, meglitinides (meglitinides), thiazolidinediones, biguanides, beta-blockers, XCE inhibitors, diuretics, nitrates, calcium channel blockers, phentermine, sibutramine, lorcaserin, cetilistat (cetilistat), rimonabant, tylenab (taranabant), topiramate, gabapentin (gabapentin), valproic acid (valproate), Vigatran (vigabatrin), bupropion, tiagabine (tiagabine), sertraline (sertraline), felinine (fluxetine), trazodone (trazodone), zonisamide (zonisamide), methylphenidate (methaphenidate), varenicline (varenicline), naltrexone (naltrexone), diethylpropion, phendimetrazine, rapalogen (rpaglinide), nateglinide (nateglinide), glimepiride (glimepiride), metformin, pioglitazone (pioglitazone), rosiglitazone (rolisigazolone), and sitagliptin (sitagliptin).

In some embodiments, the invention relates to agents useful as additional therapeutic agents for the treatment of diabetes. Exemplary antidiabetic agents include, but are not limited to, sulfonylureas (e.g., DYMELOR (acesulfame-cylurea), DIABINESE (chlorpropamide), ORINASE (tolbutamide) and TOLINASE (tolazamide), GLUCOTROL (glipizide), GLUCOTROL XL (extended release), DIABETA (glyburide), MICRONASE (glyburide), GLYNASE PRESTAB (glyburide), and AMARYL (glyburide)); biguanides (e.g., metformin (GLUCOPHAGE, GLUCOPHAGE XR, RIOMET, FORTAMET, and glucetza)); thiazolidinediones (e.g. ACTOS (pioglitazone) and AVANDIA (rosiglitazone); alpha-glucosidase inhibitors (e.g. PRECOSE (acarbose) and GLYSET (miglitol); meglitinide (e.g. PRANDIN (repaglinide) and STARLIX (nateglinide)); dipeptidyl peptidase IV (DPP-IV) inhibitors (e.g. JANUVIA (sitagliptin), NESIA (alogliptin), ONGLYZA (saxagliptin) and TRADJENTA (linagliptin)); sodium-glucose co-transporter 2(SGLT2) inhibitors (e.g. INVOKANA (canagliflozin)), and combination pills (e.g. GLOVANCE combining glibenclamide (sulfonylurea) and metformin, METAGLIP combining metin (sulfonylurea) and metformin (AVANDIA) AVANDAMET, KAZANO, aloglipin), aloglipin one pill, metformin (Acoglitazone (Aclinaglitazone) and alogliptin (Aclinaglitazone), Oral preparation of Methoformin, ACTOS, Byetta, Januvia, Welchol, Janunum, glipizide, glimepiride, Glucophage, Lantus, Glibenclamide, ONGLYZA, Amaryl, Lantus SOLOASTAR, Bydoreon, Levemir FLEXPEN, ACTOPLUS MET, GLUMETZA, TRADJENTA, bromocriptine, KOIGLYZE XR, INKANA, PRANDIN, LEVELURMIR, PARRTLODEL, Primopiopiopionone, VONOLOG, EXNOLOG, GLYCOMLOG FLEXLOPEN, VICTOXA 2-PAZON, Byeta, GLYETTA, GLYCOMETTA, GLYCOMETUS FLEXUR, GLYCOMETUROXE, GLYCOMETHYPER-OVE, GLYCOMETUROXA-E, GLYCOMETHYPERMETHA, GLYCOMETHYPERMETHYPER-E, GLYCOMETHYPER-ALPHA-E, GLYCOMETHYPERMETHYLON, GLYCOME, GLYCOMETHYLON-MAG-E, GLYCOME-C-E, GLYCOMOROCLON-E, GLYCOME-C-O-E, GLYCOMORC-E-C-E, GLYCOMLOG-E, GLYCOMLOG-, DUETACT oral preparation, sitagliptin oral preparation, SYMLINPEN 120 subcutaneous preparation, HUMALOGKWIKPEN subcutaneous preparation, JANUET XR oral preparation, GLIPIZIDE-METFORMIN oral preparation, CYCLOSET oral preparation, HUMALOG MIX 75-25 subcutaneous preparation, nateglinide oral preparation, HUMALOG MIX 75-25KWIKPEN subcutaneous preparation, eucrine 70/30 subcutaneous preparation, PRECOSE oral preparation, APIDRA subcutaneous preparation, eucrine R injection, Jentadeutero oral preparation, Victorza 3-Pak subcutaneous preparation, Novolin 70/30 subcutaneous preparation, NOVOLIN subcutaneous preparation, insulin subcutaneous preparation, micronized glibenclamide oral preparation, GLYNASE oral preparation, eucrine N subcutaneous preparation, glargine subcutaneous insulin preparation, RIOMET oral preparation, pioglitazone-METFORMIN oral preparation, APRA, SOLIDLOLIN 70/30 subcutaneous insulin preparation, PeVIOLITIN subcutaneous preparation, PeVIOLITON subcutaneous preparation, PeVIOLITANIN oral preparation, GLYNEIDIN subcutaneous preparation, and PeVITAL-DIVITAL, DIABETA oral agent, conventional human insulin injection, Yorelin N Pen subcutaneous agent, Exenatide subcutaneous agent, HUMALOG Mix 50-50 KWIKEN subcutaneous agent, liraglutide subcutaneous agent, KAZANO oral agent, repaglinide oral agent, chlorpropamide oral agent, insulin aspart subcutaneous agent, NOVOLOG Mix 70-30 subcutaneous agent, HUMALOG Mix 50-50 subcutaneous agent, saxagliptin oral agent, ACTOPLUS MetXR oral agent, miglitol oral agent, NPH recombinant subcutaneous agent, NPH insulin and conventional human insulin subcutaneous agent, azotemide oral agent, mifepristone oral agent, insulin aspart insulin subcutaneous agent, repaglinide-metformin oral agent, saxagliptin-metformin oral agent, linagliptin-metformin oral agent, SINNEA oral agent, OSI oral agent, gliptin, glibenclamide oral agent, gliptin, Insulin lispro protamine subcutaneous, pramlintide subcutaneous, insulin glulisine subcutaneous, pioglitazone-glimepiride oral, PRANDIMET oral, NOVOLOG penphil subcutaneous, linagliptin oral, exenatide microsphere subcutaneous, KORLYM oral, alogliptin-pioglitazone oral, alogliptin-metformin oral, canagliflozin oral, insulin lispro (hulog); insulin aspart (NOVOLOG); insulin glulisine (APIDRA); regular (NOVOLIN R or eurelin R); NPH (NOVOLIN N or kyrine N); insulin glargine (LANTUS); insulin detemir (LEVEMIR); youngin or NOVOLIN 70/30; and NOVOLOG Mix 70/30HUMALOG Mix 75/25 or 50/50.

In some embodiments, the invention relates to combination therapy with blood transfusion. For example, the composition of the invention may be used to supplement blood transfusion. In some embodiments, the present invention relates to combination therapy with iron supplements.

In some embodiments, the invention relates to combination therapy with one or more EPO-based agents. For example, the compositions of the present invention may be used as adjuvants for other EPO-based agents. In some embodiments, the compositions of the invention are used as maintenance therapies for other EPO-based agents. Other EPO-based agents include the following: epoetin α, including, but not limited to, darbepoetin (aransep), epocept (lupin pharma), nanogine (nanogen pharmaceutical), epofit (intas pharma), epogen (amgen), epogen, EPREX, (JANSSEN-cilg), BINOCRIT7 (santoz), crirt; epothilones beta, including but not limited to neorcormon (HOFFMANN-LA ROCHE), reormon, methoxypolyethylene glycol-epothilones beta (mirconea, ROCHE); epoetin δ, including but not limited to dynope (erythropoietin, SHIRE PLC); epoetin ω, including but not limited to EPOMAX; epoetin ζ, including but not limited to silapo (stada) and regenerative (hospira) and other EPO's, including but not limited to epocept (lupin PHARMACEUTICALs), epotrue (panacea BIOTEC LTD), erpylo SAFE (BIOCON LTD), regenerative (sodium INSTITUTE OF INDIA LIMITED), vintor (organic PHARMACEUTICALs), fit (inter phaermaceae), regenerative (inter phaeric), and regenerative (organic chemical), and regenerative (CADILA HEALTHCARE LTD), regenerative (catalytic) and regenerative (catalytic) EPO.

In some embodiments, the invention relates to combination therapy with one or more immune modulators, such as, but not limited to, agents that modulate immune checkpoints. In various embodiments, the immunomodulator targets one or more of PD-1, PD-L1, and PD-L2. In various embodiments, the immunomodulator is a PD-1 inhibitor. In various embodiments, the immunomodulator is an antibody specific for one or more of PD-1, PD-L1, and PD-L2. For example, in some embodiments, the immunomodulator is an antibody, such as, but not limited to, nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), Pabrizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK-3475(MERCK), BMS 936559(BRISTOL MYERS SQUIBB), MPDL328OA (ROCHE). In some embodiments, the immunomodulator targets one or more of CD137 or CD 137L. In various embodiments, the immunomodulator is an antibody specific for one or more of CD137 or CD 137L. For example, in some embodiments, the immunomodulator is an antibody, such as, but not limited to, Urelumab (also known as BMS-663513 and anti-4-1 BB antibodies). In some embodiments, the chimeric proteins of the invention are used in combination with udersumab (optionally with one or more of nivolumab, liriluzumab and udersumab) for the treatment of solid tumors and/or B-cell non-hodgkin's lymphoma and/or head and neck cancer and/or multiple myeloma. In some embodiments, the immunomodulatory agent is an agent that targets one or more of CTLA-4, AP2M1, CD80, CD86, SHP-2, and PPP2R 5A. In various embodiments, the immunomodulator is an antibody specific for one or more of CTLA-4, AP2M1, CD80, CD86, SHP-2 and PPP2R 5A. For example, in some embodiments, the immunomodulator is an antibody, such as, but not limited to, ipilimumab (MDX-010, MDX-101, Yervoy, BMS) and/or tremelimumab (tremelimumab) (Pfizer). In some embodiments, the chimeric proteins of the invention are used in combination with ipilimumab (optionally with bazedoxifene) to treat one or more of melanoma, prostate cancer, and lung cancer. In various embodiments, the immunomodulator is targeted to CD 20. In various embodiments, the immunomodulator is an antibody specific for CD 20. For example, in some embodiments, the immunomodulator is an antibody, such as, but not limited to, ofatumumab (genemab), obituzumab (gainyva), AME-133v (applied MOLECULAR evotion), Ocrelizumab (Ocrelizumab) (GENENTECH), TRU-015 (trubiton/EMERGENT), vetuzumab (IMMU-106).

In some embodiments, the chimeric proteins of the invention exert a synergistic effect when used in combination with Chimeric Antigen Receptor (CAR) T cell therapy. In an exemplary embodiment, the chimeric proteins exert a synergistic effect when used in combination with CAR T cell therapy to treat a tumor or cancer. In one embodiment, the chimeric protein agent exerts a synergistic effect when used in combination with CAR T cell therapy to treat a blood-based tumor. In one embodiment, the chimeric proteins exert a synergistic effect when used in combination with CAR T cell therapy to treat a solid tumor. For example, the use of chimeric proteins and CAR T cells can act synergistically to reduce or eliminate, or slow the growth and/or progression and/or metastasis of, a tumor or cancer. In various embodiments, the chimeric proteins of the invention induce CAR T cell division. In various embodiments, the chimeric proteins of the invention induce CAR T cell proliferation. In various embodiments, the chimeric proteins of the invention prevent anergy of CAR T cells.

In various embodiments, the CAR T cell therapy includes CAR T cells that target antigens (e.g., tumor antigens) such as, but not limited to, carbonic anhydrase ix (caix), 5T4, CD19, CD20, CD22, CD30, CD33, CD38, CD47, CS1, CD138, Lewis-Y, L1-CAM, MUC16, ROR-1, IL13R α 2, gp100, Prostate Stem Cell Antigen (PSCA), Prostate Specific Membrane Antigen (PSMA), B Cell Maturation Antigen (BCMA), human papilloma virus 16E6 type (HPV-16E6), CD171, folate receptor α (FR- α), GD2, human epidermal growth factor receptor 2(HER2), mesothelin, EGFRvIII, Fibroblast Activation Protein (FAP), carcinoembryonic antigen (CEA), and vascular growth factor receptor 2(VEGF-R2), as well as other tumor antigens well known in the art. Other exemplary tumor antigens include, but are not limited to, MART-1/Melan-A, gp, dipeptidyl peptidase IV (DPPIV), adenosine deaminase binding protein (ADAbp), cyclophilin B, colorectal-associated antigen (CRC) -0017-1A/GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2 and PSA-3, T cell receptor/CD 3-zeta chain, tumor antigens of the MAGE-family (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-A2 (B-2B), MAGE-Xp3(MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), tumor antigens of the GAGE-family (e.g. GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, GnT-4, tyrosinase, p53, GP-family, HER2/neu, p21ras, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-and gamma-catenin, p120ctn, Pmel 100, PRAME 117, MURCO 27, adenoma, sarcoidosis, colon carcinoma-related protein, colon cancer cell receptor, colon carcinoma, ig-idiotypes, p15, gp75, GM2 and GD2 gangliosides, viral products such as human papillomavirus proteins, Smad family tumor antigens, lmp-1, NA, EBV encoded nuclear antigen (EBNA) -1, brain glycogen phosphorylase, SSX-1, SSX-2(HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1CT-7, c-erbB-2, CD19, CD37, CD56, CD70, CD74, CD138, AGS16, MUC1, GPNMB, Ep-CAM, PD-L1 and PD-L2.

Examples of CAR T cell therapy include, but are not limited to, JCAR014(Juno Therapeutics), JCAR015(Juno Therapeutics), JCAR017(Juno Therapeutics), JCAR018(Juno Therapeutics), JCAR020(Juno Therapeutics), JCAR023(Juno Therapeutics), JCAR024(Juno Therapeutics), CTL019(Novartis), KTE-C19(Kite Pharma), BPX-401 (Bellim Pharmaceuticals), BPX-601 (Bellium Pharmaceuticals), BPX 2121(Blubird Bio), CD-19 sleep cells (Ziopcol ART), CAR 56 (cell 56), cells 123 (Biotic chromatography), UC-123 (Biotic cells), and developed Biotic cells for Ovatia 6335 (Biotic cells).

In some embodiments, the chimeric proteins of the invention are used in methods of treating Multiple Sclerosis (MS) in combination with one or more MS therapeutic agents, including, but not limited to, 3-interferon, glatiramer acetate, T-interferon, IFN- β -2 (U.S. patent publication No. 2002/0025304), spirogermanium (spirogemaniums) (e.g., N- (3-dimethylaminopropyl) -2-aza-8, 8-dimethyl-8-germylspiro [4:5] decane, N- (3-dimethylaminopropyl) -2-aza-8, 8-diethyl-8-germylspiro [4:5] decane, N- (3-dimethylaminopropyl) -2-aza-8, 8-dipropyl-8-germylspiro [4:5] decane, and N- (3-dimethyiaminopropyl) -2-aza-8, 8-dipropyl-8-germylspiro [4:5] decane Dimethylaminopropyl) -2-aza-8, 8-dibutyl-8-germylspirane [4:5] decane), vitamin D analogs (e.g., 1,25(OH)2D3 (see, e.g., U.S. Pat. No. 5,716,946)), prostaglandins (e.g., latanoprost, brimonidine, PGE1, PGE2, and PGE3, see, e.g., U.S. Pat. No. 2002/0004525), tetracyclines and derivatives (e.g., minocycline and doxycycline, see, e.g., U.S. Pat. No. 20020022608), VLA-4 binding antibodies (see, e.g., U.S. Pat. No. 2009/0202527), adrenocorticotropin, prednisone, methylprednisolone, 2-chlorodeoxyadenosine, mitoxantrone, sulfasalane, methotrexate, azathioprine, cyclophosphamide, cyclosporine, fumarate, cyclosporin, fumarate, fludarabiraterone, fludarabine, anti-CD 20 antibodies (e.g., rituximab) and tizanidine hydrochloride.

In some embodiments, the chimeric protein is used in combination with one or more therapeutic agents that treat one or more symptoms or side effects of MS. Such agents include, but are not limited to, amantadine (amantadine), baclofen (baclofen), papaverine (papaverine), meclizine (meclizine), hydroxyzine (hydroxyzine), sulfamethoxazole (sulfmethozole), ciprofloxacin (ciprofloxacin), docusate (docusate), pemoline (pemoline), dantrolene (dantrolene), desmopressin (desmopressin), dexamethasone (dexamethone), tolterodine (tolterodine), phenytoin (phenyloin), oxybutynin (oxybutynin), bisacodyl (bisacodyl), venlafaxine (venlafaxine), amitriptyline (amitriptyline), urotropine (penoxepine), clonidine (clozapine), clonazepam (clonazepam), isoniazide (isoniazide), chlorpheniramine (dihydrocarnitine), procarbazine (dihydropterine), procarbazine (theophylline), procarbazine (dihydropterosin (dihydropteridine), procarbazine (procarbazine), procarbazine (procarbazine), and procarbazine (procarbazine), procarbazine (procarba, Fluoxetine (fluoxetine), phenazopyridine (), methylprednisolone (methylprednisone), carbamazepine (carbamazepine), imipramine (imipramine), diazepam (diazepam), sildenafil (sildenafil), bupropion (bupapion) and sertraline (sertraline).

In some embodiments, the chimeric proteins are used in a method of treating multiple sclerosis in combination with one or more Disease Modifying Therapies (DMTs) described herein (e.g., agents of table a). In some embodiments, the present invention provides improved therapeutic efficacy compared to the use of one or more DMTs described herein (e.g., the agents listed in table a below) without one or more of the disclosed binding agents. In one embodiment, the combination of the chimeric protein and one or more DMTs produces a synergistic therapeutic effect.

Exemplary disease modifying therapies

MS disease progression may be the strongest and most devastating at the earliest stages of disease progression. Thus, in contrast to many reimbursement policies and physician practices (e.g., in view of cost and side effect mitigation), it may be most beneficial for a patient's long-term disease state to begin treatment with the most intense DMT (e.g., so-called second-line therapy). In some embodiments, the patient is treated with a combination of a regimen of chimeric proteins and second line therapy. Such combination is useful for reducing the side effects of one or more second line therapies. In some embodiments, the combination is used to reduce the frequency or dose of administration of one or more second line treatments. For example, in the case of combinations, the dosages of the agents listed in the tables above may be reduced by about 50%, or about 40%, or about 30%, or about 25%, and/or the frequency of administration may be reduced to typically half, or typically one third, or may be reduced from, for example, daily to every other day or weekly, every other day to weekly or biweekly, weekly to biweekly or monthly, etc. Thus, in some embodiments, the chimeric proteins increase patient compliance by allowing for more convenient treatment regimens. In addition, some DMTs have recommended lifetime dose limits, such as mitoxantrone, and lifetime cumulative doses should be severely limited to 140mg/m2, or 2-3 years of treatment. In some embodiments, the supplemental chimeric protein protects the patient's access to mitoxantrone by allowing less or less frequent dosing with the DMT.

In some embodiments, the patient is a naive (naive) patient that has not received one or more DMT treatments, and the chimeric protein is used to buffer side effects of second-line therapy. Thus, the primary patient can benefit from the long-term benefits of second-line therapy at the onset of the disease. In some embodiments, the chimeric protein is used as an entry therapy prior to the use of second line therapy. For example, the chimeric protein may be administered for an initial treatment period of about 3 months to stabilize the disease, and then the patient may be switched to maintenance therapy with a second-line drug.

It is generally believed that naive patients are more likely to respond to treatment than patients who received and may have failed one or more DMTs. In some embodiments, the chimeric protein is used in patients who have received and may have failed one or more DMTs. For example, in some embodiments, the chimeric protein increases the therapeutic effect in patients who have received and may have failed one or more DMTs, and may allow these patients to respond as if they were naive.

In some embodiments, the patient has received or is receiving treatment with one or more DMTs and is not responding well. For example, a patient may not respond or respond poorly to one or more DMTs. In some embodiments, the patient does not respond or responds poorly to one or more of the following: teriflunomide (aubagio (genzyme)); interferon beta-1 a (avonex (biogen idec)), interferon beta-1 b (betaseroon (BAYER helthchcae PHARMACEUTICALS, INC.), glatiramer acetate (copaxone (teva neurosience)), interferon beta-1 b (EXTAVIA (NOVARTIS PHARMACEUTICALS CORP.), fingolimod (gleynia (NOVARTIS pharmacranthes CORP.), alemtramumab (lenszyme), mitoxantrone (novatropine seresen), pegylated interferon beta-1 a (megriddy (polyglidge), interferon beta-1 a (red (EMD ono, INC., dimethyl fumarate (BG-12), and brityle TECFIDERA(BIOGEN IDEC) (brit, INC.), one or more of the disclosed binding agents results in the therapeutic benefit of one or more DMTs in a patient, this allows for higher doses or frequencies of DMT(s) to be reserved for patient treatment, for example.

In patients with more aggressive disease, one approach is to induce a model of treatment in which a therapy with strong efficacy but strong safety concerns is given first, followed by maintenance therapy. Examples of such models may include initial treatment with alemtuzumab followed by IFN- β, GA, or BG-12. In some embodiments, one or more of the disclosed binding agents are used to prevent the need to switch therapy for maintenance. In some embodiments, one or more of the disclosed binding agents is used in maintenance therapy, including second line therapy, of one or more DMTs. In some embodiments, one or more of the disclosed binding agents are used as a first therapy in induction, followed by another DMT as a maintenance therapy, e.g., first line therapy.

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

In various embodiments, one or more of the disclosed binding agents are used to reduce one or more side effects of DMT, including but not limited to any of the agents disclosed herein. For example, one or more of the disclosed binding agents may be used in a regimen that allows for a dose savings of one or more DMTs and thus results in fewer side effects. For example, in some embodiments, one or more of the disclosed binding agents can reduce one or more side effects of AUBAGIO or related agents, which can include hair thinning, diarrhea, influenza, nausea, abnormal liver testing, and abnormal numbness or tingling of the hands or feet (paresthesia), white blood cell levels, which can increase the risk of infection; an increase in blood pressure; and severe liver damage. In some embodiments, one or more of the disclosed binding agents may reduce one or more side effects of AVONEX or related agents, including flu-like symptoms, depression, mild anemia, liver abnormalities, allergies, and cardiac problems after injection. In some embodiments, one or more of the disclosed binding agents may reduce one or more side effects of BETASERON or related agents, including flu-like symptoms after injection, injection site reactions, allergic reactions, depression, liver abnormalities, and low white blood cell counts. In some embodiments, the disclosed one or more binding agents may reduce one or more side effects of COPAXONE or related agents, including injection site reactions, vasodilation (vasodilation); chest pain; reactions occurring immediately after injection include anxiety, chest pain, palpitations, shortness of breath and flushing. In some embodiments, one or more of the disclosed binding agents can reduce one or more side effects of EXTAVIA or related agents, including flu-like symptoms after injection, injection site reactions, allergic reactions, depression, liver abnormalities, and low white blood cell counts. In some embodiments, one or more of the disclosed binding agents can reduce one or more side effects of gileya or a related agent, including headache, influenza, diarrhea, back pain, elevated liver enzymes, cough, decreased heart rate after first administration, infection, and swelling of the eyes. In some embodiments, one or more of the disclosed binding agents can reduce one or more side effects of lemrada or a related agent, including rash, headache, fever, nasal congestion, nausea, urinary tract infection, fatigue, insomnia, upper respiratory tract infection, urticaria, pruritus, thyroid disorder, fungal infection, joint and limb and back pain, diarrhea, vomiting, flushing, and infusion reactions (including nausea, urticaria, pruritus, insomnia, chills, flushing, fatigue, shortness of breath, altered taste, dyspepsia, dizziness, pain). In some embodiments, one or more of the disclosed binding agents may reduce one or more side effects of NOVANTRONE or related agents, which includes blue-green urine 24 hours after administration; infection, bone marrow depression (fatigue, bruising, low blood counts), nausea, thinning of hair, bladder infection, canker sores and severe liver and heart damage. In some embodiments, one or more of the disclosed binding agents can reduce one or more side effects of PLEGRIDY or related agents, including flu-like symptoms after injection, injection site reactions, depression, mild anemia, liver abnormalities, allergies, and cardiac problems. In some embodiments, one or more of the disclosed binding agents can reduce one or more side effects of REBIF or related agents, including flu-like symptoms after injection, injection site reactions, liver abnormalities, depression, allergic reactions, and low red or white blood cell counts. In some embodiments, one or more of the disclosed binding agents can reduce TECFIDERA or related agents for one or more side effects including flushing (hot or itchy feeling on the skin and flushing), gastrointestinal problems (nausea, diarrhea, abdominal pain), rash, protein in the urine, elevated liver enzymes; and a decrease in blood lymphocyte (leukocyte) count. In some embodiments, one or more of the disclosed binding agents can reduce one or more side effects of TYSABRI or a related agent, including headache, fatigue, urinary tract infections, depression, respiratory tract infections, joint pain, stomach discomfort, abdominal discomfort, diarrhea, vaginitis, arm or leg pain, rash, hypersensitivity or hypersensitivity reactions within 2 hours of infusion (dizziness, fever, rash, itching, nausea, flushing, hypotension, dyspnea, chest pain).

In some embodiments, the present invention relates to combination therapy with one or more of the chimeric agents described in WO2013/10779, WO 2015/00736, WO2015/007520, WO2015/007542, and WO2015/007903, the entire contents of which are incorporated herein by reference in their entirety.

In some embodiments, the chimeric proteins described herein include modified derivatives, i.e., covalently linked to a component (composition) through any type of molecule, such that the covalent linkage does not hinder the activity of the component. For example, but not by way of limitation, derivatives include compositions modified by the following methods: glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a number of chemical modifications may be made by known techniques, including but not limited to specific chemical cleavage of tunicamycin, acetylation, formylation, metabolic synthesis, and the like.

In other embodiments, the chimeric proteins described herein further comprise a cytotoxic agent, which in exemplary embodiments comprises a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death. These agents may be conjugated to the compositions described herein.

Thus, the chimeric proteins described herein can be post-translationally modified to add effector moieties, such as chemical linkers, detectable moieties, such as fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties, such as streptavidin, avidin, biotin, cytotoxins, cytotoxic agents, and radioactive materials.

Exemplary cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine; alkylating agents such as nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, streptozocin (streptozocin), mitomycin C, cis-dichlorodiammineplatinum (II) (DDP), cisplatin, and carboplatin (balastatin); anthracyclines include zorubicin (formerly daunomycin), doxorubicin (adriamycin), ditorbin (detoribicin), carminomycin (carminomycin), idarubicin, epirubicin, mitoxantrone, and bisantrene; antibiotics include actinomycin D (dactinomycin/actinomycin D), bleomycin, calicheamicin, mithramycin (mithramycin) and Antrocin (AMC); and antimitotic agents such as vinca alkaloids (vinca alkloids), vincristine (vincristine) and vinblastine (vinblastine). Other cytotoxic agents include paclitaxel (paclitaxel), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emidine (emetine), etoposide, teniposide, colchicin (colchicin), dihydroxyanthracenedione, 1-dehydrotestosterone, glucocorticoids, procaine (procaine), tetracaine (tetracaine), lidocaine (lidocaine), propranolol (propranolol), puromycin (puromycin), procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (O, P' - (DDD)), interferons, and mixtures of these cytotoxic agents.

Other cytotoxic agents include, but are not limited to, chemotherapeutic agents, such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGF antagonists, EGFR antagonists, platins (platins), taxanes, irinotecan, 5-fluorouracil, gemcitabine, leucovorin, steroids, cyclophosphamide, melphalan, vinblastines (e.g., vinblastine, vincristine, vindesine, and vinorelbine), mustine (mustine), tyrosine kinase inhibitors, radiation therapy, sex hormone antagonists, selective androgen receptor modulators, selective estrogen receptor modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists, interleukins (e.g., IL-12 or IL-2), An IL-12R antagonist, a toxin-conjugated monoclonal antibody, a tumor antigen-specific monoclonal antibody, Erbitux (Erbitux), Avastin (Avastin), Pertuzumab (Pertuzumab), an anti-CD 20 antibody, Rituxan (Rituxan), ocrelizumab, ofatumumab, DXL625, or any combination thereof. Toxic enzymes from plants and bacteria, such as ricin, diphtheria toxin and pseudomonas toxin, may be conjugated to these therapeutic agents (e.g., antibodies) to produce cell type specific killing agents (Youle et al, proc. nat ' l acad. sci. usa 77:5483 (1980); Gilliland et al, proc. nat ' l acad. sci. usa 77:4539 (1980); Krolick et al, proc. nat ' l acad. sci. usa 77:5419 (1980)).

Other cytotoxic agents include cytotoxic ribonucleases as described by golden in U.S. patent No. 6,653,104. Embodiments of the invention also relate to radioimmunoconjugates in which alpha-or beta-particle emitting radionuclides are stably coupled to a chimeric protein, with or without the use of a complex forming agent. Such radionuclides include beta-emitters such as phosphorus-32, scandium-47, copper-67, gallium-67, yttrium-88, yttrium-90, iodine-125, iodine-131, samarium-153, lutetium-177, rhenium-186, or rhenium-188; and alpha-emitters, such as astatine-211, lead-212, bismuth-213, or actinium-225.

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

Method of treatment

The methods and compositions described herein may be applied to the treatment of a variety of diseases and disorders, including, but not limited to, cancer, infections, immune disorders, anemia, autoimmune diseases, cardiovascular diseases, wound healing, ischemia-related diseases, neurodegenerative diseases, metabolic diseases, and numerous other diseases and disorders.

In addition, any of the agents of the present invention may be used to treat or manufacture a medicament for treating a variety of diseases and disorders, including but not limited to cancer, infections, immune disorders, inflammatory diseases or conditions, and autoimmune diseases.

In some embodiments, the invention relates to the treatment of one or more of the following diseases or patients suffering from one or more of the following diseases: chronic granulomatous disease, osteopetrosis, idiopathic pulmonary fibrosis, friedreich's ataxia, atopic dermatitis, chagas ' disease, cancer, heart failure, autoimmune disease, sickle cell disease, thalassemia, blood loss, transfusion reactions, diabetes, vitamin B12 deficiency, collagen vascular disease, schwarckmann-syndrome, thrombocytopenic purpura, celiac disease, endocrine deficiency states such as hypothyroidism or addison's disease, autoimmune diseases such as crohn's disease, systemic lupus erythematosus, rheumatoid arthritis or juvenile rheumatoid arthritis, ulcerative colitis immune disorders such as eosinophilic fasciitis, hypoimmunoglobulinemia or thymoma/carcinoma, graft-versus-host disease, pre-leukemia, non-hematologic syndromes (e.g., down's syndrome, Dubowwitz, Seckel), Felty syndrome (Felty syndrome), Hemolytic uremic syndrome, myelodysplastic syndrome, nocturnal paroxysmal hemoglobinuria, myelofibromas, pancytopenia, pure red blood cell aplasia, Schoenlein-Henoch purpura, malaria, protein deficiency, menorrhagia, systemic sclerosis, cirrhosis, hypometabolic state, and congestive heart failure.

In some embodiments, the invention relates to the treatment of one or more of the following diseases or patients suffering from one or more of the following diseases: chronic granulomatosis, osteopetrosis, idiopathic pulmonary fibrosis, friedreich's ataxia, atopic dermatitis, chagas disease, mycobacterial infection, cancer, scleroderma, hepatitis c, septic shock and rheumatoid arthritis.

In some embodiments, the invention relates to the treatment of patients suffering from cancer or patients suffering from cancer. As used herein, cancer refers to any uncontrolled cell growth that may interfere with the normal function of body organs and systems, and includes both primary and metastatic tumors. A primary tumor or a cancer that migrates from its original location and colonizes a vital organ may ultimately lead to death of the subject by a decline in function of the affected organ. Metastasis is a cancer cell or group of cancer cells that appear at a location remote from the primary tumor due to dissemination of the cancer cells from the primary tumor to other parts of the body. The metastasis may eventually lead to death of the subject. For example, cancer may include benign and malignant cancers, polyps, hyperplasia, and dormant tumors or micrometastases.

Exemplary cancers that may be treated include, but are not limited to, epithelial cell carcinoma (carcinoma) (e.g., various subtypes including, for example, adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, and transitional cell carcinoma), sarcomas including, for example, osteosarcoma and soft tissue sarcoma, leukemias including, for example, acute myelogenous, acute lymphoblastic, chronic myelogenous, chronic lymphocytic, and hairy cell leukemia, lymphomas and myelomas including, for example, hodgkin's and non-hodgkin's lymphomas, light chain, non-secretory, MGUS, and plasmacytomas, and central nervous system carcinomas including, for example, brain cancers (e.g., gliomas (e.g., astrocytomas, oligodendrogliomas, and ependymomas)), meningiomas, pituitary adenomas, and neuroma (e.g., meningiomas and fibroneuromas).

Exemplary cancers that may be treated include, but are not limited to, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancers; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; cancers of the digestive system; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); a glioblastoma; liver cancer; hepatocellular carcinoma; an intraepithelial neoplasm; kidney or renal cancer; laryngeal cancer; leukemia; cancer of the liver; lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); melanoma; a myeloma cell; neuroblastoma; oral cancer (lip, tongue, mouth and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland cancer; sarcomas (e.g., kaposi's sarcoma); skin cancer; squamous cell carcinoma; gastric cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulvar cancer; lymphomas including hodgkin's and non-hodgkin's lymphomas, and B cell lymphomas (including low grade/follicular non-hodgkin's lymphomas (NHL); small Lymphocyte (SL) NHL; medium/follicular NHL; intermediate diffuse NHL; higher-order immunoblastic NHL; higher lymphoblasts NHL; high-grade small non-nucleated cell NHL; giant tumor mass NHL; mantle cell lymphoma; AIDS-related lymphomas; 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), and abnormal vascular proliferation associated with nevus destructor, edema (e.g., edema associated with brain tumors), and megs syndrome.

In various embodiments, the invention relates to the treatment of Myc-driven cancers, i.e., cancer cells that overexpress Myc. In some embodiments, the cancer cell overexpresses any of c-Myc, N-Myc, and/or L-Myc. In some embodiments, the methods of the invention sensitize cancer cells to treatment with any one of the anti-cancer therapeutic agents described herein. In some embodiments, the methods of the invention reduce the transcriptional activity of a cancer cell.

In some embodiments, the invention relates to the treatment of microbial and/or chronic infections, or patients suffering from microbial and/or chronic infections. Exemplary infections include, but are not limited to, Chagas' disease, HIV/AIDS, tuberculosis, osteomyelitis, hepatitis B, hepatitis C, Epstein-Barr virus (Epstein-Barr virus) or parvovirus, T-cell leukemia virus, bacterial overgrowth syndrome, fungal or parasitic infections.

In various embodiments, the compositions of the invention are used to treat or prevent one or more inflammatory diseases or conditions, such as inflammation, acute inflammation, chronic inflammation, respiratory disease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopic dermatitis, septic shock, rheumatoid arthritis, inflammatory bowel disease, inflammatory pelvic disease, pain, ocular inflammatory disease, celiac disease, Leigh Syndrome, glycerol kinase deficiency, Familial Eosinophilia (FE), autosomal spasmodic ataxia, laryngeal inflammatory disease; tuberculosis, chronic cholecystitis, bronchiectasis, silicosis and other pneumoconiosis.

In various embodiments, the compositions of the invention are used to treat or prevent one or more autoimmune diseases or conditions, such as multiple sclerosis, diabetes, lupus, celiac disease, crohn's disease, ulcerative colitis, Guillain-Barre syndrome (Guillain-Barre syndrome), scleroderma, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, rasmassen's encephalitis, primary biliary sclerosis, sclerosing cholangitis, autoimmune hepatitis, edison's disease, Hashimoto's thyroiditis, fibromyalgia, meniere's syndrome; transplant rejection (e.g., prevention of allograft rejection), pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease, and other autoimmune diseases.

In various embodiments, the compositions of the present invention are used to treat, control or prevent cardiovascular disease, such as diseases or conditions affecting the heart and blood vessels, including, but not limited to, Coronary Heart Disease (CHD), cerebrovascular disease (CVD), aortic stenosis, peripheral vascular disease, atherosclerosis, arteriosclerosis, myocardial infarction (heart attack), cerebrovascular disease (stroke), Transient Ischemic Attack (TIA), angina (stable and unstable), atrial fibrillation, arrhythmia, vascular disease, and/or congestive heart failure.

In various embodiments, the compositions of the present invention are used to treat or prevent one or more metabolic-related disorders. In various embodiments, the present invention may be used to treat, control or prevent diabetes, including type 1 diabetes and type 2 diabetes, as well as diabetes associated with obesity. The compositions and methods of the invention are useful for treating or preventing diabetes-related disorders, including, but not limited to, diabetic nephropathy, hyperglycemia, impaired glucose tolerance, insulin resistance, obesity, lipid disorders, dyslipidemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, low HDL levels, high LDL levels, atherosclerosis and its sequelae, vascular restenosis, irritable bowel syndrome, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), other inflammatory conditions, pancreatitis, abdominal obesity, neurodegenerative diseases, retinopathy, neoplastic conditions, adipose cell tumors, adipose cell cancers such as liposarcoma, prostate cancer and other cancers (including gastric, breast, bladder and colon cancers), angiogenesis, Alzheimer's disease, psoriasis, hypertension, metabolic syndrome (e.g., a human presenting three or more of abdominal obesity, psoriasis, hypertension, and metabolic syndrome), Hypertriglyceridemia, low HDL cholesterol, hypertension and high fasting plasma glucose), ovarian hyperandrogenism (polycystic ovary syndrome), and other conditions in which insulin resistance is a component, such as sleep apnea. The compositions and methods of the present invention are useful for treating, controlling or preventing obesity (including genetic or environmental) and obesity-related disorders. The obesity-related disorders herein are related to, caused by, or the result of obesity. Examples of obesity-related disorders include obesity, diabetes, excessive eating, binge eating and bulimia, hypertension, elevated plasma insulin concentrations and insulin resistance, dyslipidemia, hyperlipidemia, endometrial cancer, breast cancer, prostate cancer, kidney and colon cancers, osteoarthritis, obstructive sleep apnea, gallstones, heart disease, arrhythmias and arrhythmia, myocardial infarction, congestive heart failure, coronary heart disease, sudden death, stroke, polycystic ovarian disease, craniopharyngioma, Prader-Willi Syndrome, fleshing's Syndrome, GH-deficient subjects, normal short stature of variability, Turner's Syndrome, and other conditions exhibiting reduced metabolic activity or reduced resting energy expenditure (percentage of total non-fat containing material), such as children with acute lymphoblastic leukemia. Other examples of obesity-related disorders are metabolic syndrome, insulin resistance syndrome, reproductive hormone abnormalities, sexual and reproductive dysfunction such as impaired fertility, infertility, male hypogonadism and female hirsutism, fetal defects associated with maternal obesity, gastrointestinal motility disorders such as obesity-related gastroesophageal reflux, respiratory disorders such as obese hypoventilation syndrome (pickwick syndrome), shortness of breath, cardiovascular disorders, inflammation such as systemic vascular inflammation, arteriosclerosis, hypercholesterolemia, lower back pain, gallbladder disease, hyperuricemia, gout, and kidney cancer, and an increased risk of numbness. The compositions and methods of the invention are also useful for treating alzheimer's disease.

In various embodiments, the compositions of the present invention are used to treat or prevent one or more respiratory diseases, such as Idiopathic Pulmonary Fibrosis (IPF), asthma, Chronic Obstructive Pulmonary Disease (COPD), bronchiectasis, allergic rhinitis, sinusitis, pulmonary vasoconstriction, inflammation, allergy, respiratory obstruction, respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, Hantavirus Pulmonary Syndrome (HPS), luffier's syndrome, Goodpasture's syndrome, pleuritis, pneumonia, pulmonary edema, pulmonary fibrosis, sarcoidosis, complications associated with respiratory syncytial virus infection, and other respiratory diseases.

In some embodiments, the invention is used to treat or prevent one or more neurodegenerative diseases. Exemplary neurodegenerative diseases include, but are not limited to, Friedreich's Ataxia, multiple sclerosis (including, but not limited to, benign multiple sclerosis, relapsing-remitting multiple sclerosis (RRMS), Secondary Progressive Multiple Sclerosis (SPMS), Progressive Relapsing Multiple Sclerosis (PRMS), and Primary Progressive Multiple Sclerosis (PPMS)), alzheimer's disease (including, but not limited to, early-onset alzheimer's disease, late-onset alzheimer's disease, Familial Alzheimer's Disease (FAD)), parkinson's disease and parkinsonism (including, but not limited to, idiopathic parkinson's disease, vascular parkinson's syndrome, drug-induced parkinson's syndrome, lewy body dementia, hereditary parkinson's disease, juvenile parkinson's disease), Huntington's disease (Huntington's disease), amyotrophic lateral sclerosis (ALS, including but not limited to sporadic ALS, familial ALS, western pacific ALS, juvenile ALS, mountain sickness (Hiramaya Disease)).

In various embodiments, the chimeric proteins of the invention are used to treat wounds, such as non-healing wounds, ulcers, burns or frostbites, chronic or acute wounds, open or closed wounds, internal wounds, or trauma (exemplary trauma are penetrating and non-penetrating wounds). In various embodiments, the chimeric proteins of the invention are used to treat ischemia, by way of non-limiting example, ischemia associated with acute coronary syndrome, Acute Lung Injury (ALI), Acute Myocardial Infarction (AMI), Acute Respiratory Distress Syndrome (ARDS), arterial occlusive disease, arteriosclerosis, articular cartilage defects, sterile systemic inflammation, atherosclerotic cardiovascular disease, autoimmune disease, bone fracture, cerebral edema, cerebral hypoperfusion, Buerger's disease, burns, cancer, cardiovascular disease, cartilage injury, cerebral infarction, cerebral ischemia, stroke, cerebrovascular disease, chemotherapy-induced neuropathy, chronic infection, chronic mesenteric ischemia, claudication, congestive heart failure, connective tissue injury, contusion, Coronary Artery Disease (CAD), Critical Limb Ischemia (CLI), Crohn's disease, deep vein thrombosis, deep wound, delayed ulcer healing, delayed wound healing, diabetes (type I and type II), diabetic neuropathy, diabetes-induced ischemia, Disseminated Intravascular Coagulation (DIC), embolic cerebral ischemia, cold injury, graft-versus-host disease, hereditary hemorrhagic telangiectasia ischemic vascular disease, hyperoxic injury, hypoxia, inflammation, inflammatory bowel disease, inflammatory disease, tendon injury, intermittent claudication, intestinal ischemia, ischemic encephalopathy, ischemic heart disease, ischemic peripheral vascular disease, placental ischemia, ischemic nephropathy, ischemic vascular disease, ischemic reperfusion injury, laceration, left main coronary artery disease, limb ischemia, lower limb ischemia, myocardial infarction, myocardial ischemia, organ ischemia, osteoarthritis, osteoporosis, osteosarcoma, Parkinson's disease, Peripheral Arterial Disease (PAD), peripheral arterial disease, peripheral ischemia, peripheral neuropathy, peripheral vascular disease, pre-cancer, pulmonary edema, pulmonary embolism, remodeling disorders, renal ischemia, retinal ischemia, retinopathy, sepsis, skin ulcers, solid organ transplantation, spinal cord injury, stroke, subchondral bone cyst, thrombosis, thrombotic cerebral ischemia, tissue ischemia, Transient Ischemic Attack (TIA), traumatic brain injury, ulcerative colitis, renal vascular disease, vasculitic conditions, von Hippel-lindau syndrome (von Hippel-lindau syndrome), or tissue or organ trauma.

In various embodiments, the invention relates to the treatment of one or more anemias, including anemias caused by chronic kidney disease (e.g., by dialysis) and/or anti-cancer agents (e.g., chemotherapy and/or HIV treatment (e.g., Zidovudine (INN) or Zidovudine (AZT)), inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), anemia associated with inflammatory conditions (e.g., arthritis, lupus, IBD), anemia associated with diabetes, schizophrenia, cerebral malaria, aplastic anemia and myelodysplasia resulting from cancer therapy (e.g., chemotherapy and/or radiation) and various myelodysplastic syndromes (e.g., sickle cell anemia, hemoglobinopathy, alpha and beta thalassemia, post-preterm neonatal anemia, and comparable conditions).

In some embodiments, the invention relates to the treatment of patients with anemia, i.e. a condition in which the number of red blood cells and/or the amount of hemoglobin found in the red blood cells is below normal. In various embodiments, the anemia can be acute or chronic. For example, anemias of the invention include, but are not limited to, iron-deficiency anemia, renal anemia, chronic diseases/inflammatory anemia, pernicious anemia such as macrogastric juice-deficiency anemia, juvenile pernicious anemia and congenital pernicious anemia, cancer-related anemia, anticancer-related anemia (e.g., chemotherapy-related anemia, radiotherapy-related anemia), aplastic anemia, refractory anemia with excess blasts, aplastic anemia, X-linked siderocytic anemia (siderobic anemia), hemolytic anemia, sickle cell anemia, anemia resulting from impaired ESA production, myelodysplastic syndrome, hypochromatic anemia, microcytic anemia, siderobytic anemia, autoimmune hemolytic anemia, couley's anemia (Cooley's anemia), thalassemia, wearable-buybi anemia (Diamond blackman anemia), Fanconi's anemia (Fanconi's anemia) and drug-induced immune hemolytic anemia. Anemia can lead to serious symptoms including hypoxia, chronic fatigue, inattention, pale skin, hypotension, dizziness and heart failure.

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

In some embodiments, the invention relates to the treatment of anemia in chronic kidney disease patients who are not undergoing dialysis. For example, the invention relates to patients at stage 1 CKD or stage 2 CKD or stage 3 CKD or stage 4 CKD or stage 5 CKD. In some embodiments, the patient of the invention is stage 4 CKD or stage 5 CKD. In some embodiments, the patient of the invention has undergone a kidney transplant. In some embodiments, the invention relates to treating anemia in a patient suffering from Acute Kidney Injury (AKI).

In some embodiments, the anemia is chemotherapy-induced. For example, the chemotherapy may be any myelosuppressive chemotherapy. In some embodiments, the chemotherapy is one or more of lenalidomide, thalidomide, dexamethasone, doxorubicin (Adriamycin), and Doxil. In some embodiments, the chemotherapy is one or more platinum-based drugs, including cisplatin (e.g., placanol) and carboplatin (e.g., parapelatin). In some embodiments, the chemotherapy is any of the chemotherapeutic agents described herein. In some embodiments, the chemotherapy is any agent described in Groopman et al, J Natl Cancer Inst (1999)91(19):1616-1634, the contents of which are incorporated herein by reference. In some embodiments, the compositions and methods of the invention are used to treat chemotherapy-associated anemia in a patient with advanced stage cancer (e.g., stage IV or stage III or stage II cancer). In some embodiments, the compositions and methods of the invention are used to treat chemotherapy-associated anemia in cancer patients who have received dose-intensive chemotherapy or other invasive chemotherapy regimens.

In some embodiments, the invention relates to treating anemia in patients with one or more blood-based cancers, such as leukemia, lymphoma, and multiple myeloma. Such cancers may directly affect bone marrow. In addition, the present invention relates to metastatic cancer that has spread to bone or bone marrow. In some embodiments, the invention relates to treating anemia in a patient undergoing radiation therapy. Such radiation therapy may damage the bone marrow, thereby reducing its ability to produce red blood cells. In other embodiments, the invention relates to treating anemia in a patient having a reduction or deficiency in one or more of iron, vitamin B12, and folic acid. In other embodiments, the invention relates to treating anemia in patients with excessive blood loss (including but not limited to post-surgery or due to tumors causing internal bleeding). In other embodiments, the invention relates to the treatment of anemia in patients with chronic anemia.

In some embodiments, the methods and compositions of the invention stimulate red blood cell production. In some embodiments, the methods and compositions of the present invention stimulate the division and differentiation of committed erythroid progenitors in the bone marrow.

Certain embodiments of the invention are particularly useful for treating chemotherapy-induced anemia in cancer patients. In some embodiments, the methods and compositions of the invention allow for continued administration of the chimeric protein after the cancer patient has concluded chemotherapy. In some embodiments, the methods and compositions of the invention allow for the treatment of cancer patients at doses that are not reduced relative to non-cancer patients. In some embodiments, the methods and compositions of the invention allow for the treatment of cancer patients who are receiving chemotherapy and are considered to be curable. In various embodiments, the cancer patient has one or more of a history of thrombosis, recent surgery, prolonged bed rest or limited mobility, and treatment with a chemotherapeutic agent.

Reagent kit

The invention also provides kits for administering any of the agents described herein (e.g., chimeric proteins with or without administration of a variety of additional therapeutic agents). The kit is a combination of materials or components including at least one inventive pharmaceutical composition described herein. Thus, in some embodiments, the kit contains at least one pharmaceutical composition described herein.

The exact nature of the components configured in the kit will depend on their intended purpose. In one embodiment, the kit is configured for the purpose of treating a human subject.

Instructions for use may be included in the kit. Instructions for use typically include tangible representations that describe the techniques to be employed to achieve the desired result, e.g., in the treatment of cancer, using the components of the kit. Optionally, the kit also contains other useful components as would be readily understood by one skilled in the art, such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, dressings or other useful accessories.

The materials and components assembled in the kit can be provided to the practitioner for storage in any convenient and suitable manner that maintains their operability and utility. For example, these components may be provided at room temperature, refrigeration temperature, or freezing temperature. These components are typically contained in a suitable packaging material. In various embodiments, the packaging material is constructed by well-known methods, preferably to provide a sterile, non-contaminating environment. The packaging material may have an external label indicating the contents and/or target of the kit and/or its components.

Definition of

As used herein, "a/an" or "the" may mean one or more than one.

The term "or" as used herein is understood to include and encompass "or" and "both" unless otherwise indicated herein or clearly contradicted by context.

Furthermore, the term "about" when used in conjunction with a recitation of a numerical indication means that the recited numerical indication is plus or minus at most 10% of the recited numerical indication, such as (plus or minus) 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the recited value. For example, the language "about 50" covers the range of 45 to 55.

An "effective amount" when used in conjunction with medical use is an amount effective to provide measurable treatment, prevention, or reduction of the incidence of a disease of interest.

As used herein, a property is "reduced" if a readout of activity and/or effect is reduced by a significant amount, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or more, up to and including at least about 100%, in the presence of an agent or stimulus relative to the absence of such modulation. As will be appreciated by one of ordinary skill in the art, in some embodiments, the activity is reduced and some downstream readouts will be reduced but others may be increased.

Conversely, an activity is "increased" if a readout of activity and/or effect is increased by a significant amount, e.g., at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or more, up to and including at least about 100% or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold in the presence of an agent or stimulus relative to the absence of such agent or stimulus.

As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word "comprise" and variations thereof is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the term "may" and variations thereof is intended to be non-limiting, such that a listing that an embodiment may contain certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

Although the open-ended term "comprising" is used herein as a term such as comprising, containing, or having synonyms for the same, to describe and claim the present invention, or embodiments thereof, may alternatively be described using alternative terms such as "consisting of … …" or "consisting essentially of … …".

As used herein, the words "preferred" and "preferably" refer to embodiments of the technology that provide certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the technology.

The amount of the compositions described herein required to achieve a therapeutic effect can be determined empirically for a particular purpose according to routine procedures. Generally, for administration of a therapeutic agent for therapeutic purposes, the therapeutic agent is administered in a pharmaceutically effective dose. "pharmaceutically effective amount," "pharmaceutically effective dose," "therapeutically effective amount," or "effective amount" refers to an amount sufficient to produce a desired physiological effect or to achieve a desired result, particularly for treating a condition or disease. As used herein, an effective amount will include an amount sufficient to, for example, delay the development of, alter the course of (e.g., slow the progression of), reduce or eliminate one or more symptoms or manifestations of a disorder or disease, and reverse the symptoms of a disorder or disease. Therapeutic benefit also includes interrupting or slowing the progression of the underlying disease or condition, regardless of whether an improvement is achieved.

Effective amount, toxicity and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., determining LD50 (the dose lethal to about 50% of the population) and ED50 (the dose therapeutically effective in about 50% of the population). The dosage may vary depending on the dosage form used and the route of administration used. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED 50. In some embodiments, compositions and methods that exhibit a greater therapeutic index are preferred. The therapeutically effective dose can be initially estimated by in vitro tests including, for example, cell culture assays. In addition, the dose may be formulated in animal models to achieve a circulating plasma concentration range that includes IC50 as determined in cell culture or in an appropriate animal model. The content of the described composition in plasma can be measured, for example, by high performance liquid chromatography. The effect of any particular dose can be monitored by a suitable bioassay. The dosage can be determined by a physician and adjusted as necessary to accommodate the observed therapeutic effect.

In certain embodiments, the effect will result in a quantifiable change of at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 70%, or at least about 90%. In some embodiments, the effect will result in a quantifiable change of about 10%, about 20%, about 30%, about 50%, about 70%, or even about 90% or more. Therapeutic benefit also includes interrupting or slowing the progression of the underlying disease or condition, whether or not improvement is achieved.

As used herein, "method of treatment" is equally applicable to the use of a composition for the treatment of a disease or condition described herein and/or to one or more uses of a composition for the manufacture of a medicament for the treatment of a disease or condition described herein. The invention is further illustrated by the following non-limiting examples.

Examples

The term "AcTaferon" is occasionally used herein to refer to interferon-based chimeras.

In the following examples, unless otherwise indicated, the mutation of IFN is relative to human IFN-alpha 2.

The Q124R mutation represents attenuated human IFN alpha 2 mutant, which can be in a mouse model in vivo test. Specifically, Q124R is a human IFN mutation applicable to mice (i.e., it is a human mutant IFN that plays a role in mice). See nat.comm.2014; 5:3016.doi:10.1038/ncomms4016, the entire contents of which are incorporated herein by reference.