TMIGD2 AND ITS DERIVATIVES AS BLOCKERS OR BINDERS OF CANCER-EXPRESSED HHLA2 FOR IMMUNOTHERAPIES

Abstract

Provided are methods of treating an HHLA2-bearing tumor in a subject with a fusion protein comprising an IgV-like domain of a TMIGD2 sufficient to treat the HHLA2-bearing tumor. A fusion protein comprising an IgV-like domain of a TMIGD2 and related compositions and encoding nucleic acids are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional application of U.S. patent application Ser. No. 16/351,574, filed Mar. 13, 2019, which is a divisional of U.S. patent application Ser. No. 15/300,294, filed Sep. 29, 2016, now U.S. Pat. No. 10,280,208, issued May 7, 2019, which is a 371 of International Application No. PCT/US2015/027429, filed Apr. 24, 2019, which claims the benefit of U.S. Provisional Patent Application No. 61/986,238, filed on Apr. 30, 2014, the contents of which are herein incorporated by reference in their entirety into the present application.

BACKGROUND OF THE INVENTION

[0004] Throughout this application various publications are referred to in parentheses. Full citations for these references may be found at the end of the specification. The disclosures of these publications, and all patents, patent application publications and books referred to herein, are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.

[0005] The B7 and CD 28 families are part of the immune system. The B7 ligand family binds to the CD28 receptor family on T cells and other immune cells, which critically regulates functions of immune cells. The currently known members of the B7 family include B7-1 (CD80), B7-2 (CD86), B7h (CD275), PD-L1 (B7-H1, CD274), PD-L2 (B7-DC, CD273), B7-H3 (CD276), B7x (B7-H4/B7s1) and HHLA2, whereas the CD28 family contains CD28, CTLA-4 (CD152), ICOS (CD278) and PD-1 (CD279)(1-3). The B7/CD28 pathways are very attractive therapeutic targets (3-9). The FDA has approved five new drugs developed from the B7/CD28 families.

[0006] HHLA2 has recently been characterized as a member of the B7 family. In 2013, by homology search of various databases using amino acid sequences of human B7x and B7-H3, HERV-H LTR-associating 2 (HHLA2) was identified as a new member of the B7 family (1). The human HHLA2 gene is located in the q13.13 region of the chromosome 3 and is near the B7-1 and B7-2 genes (q13.3-q21). HHLA2 shares varying levels of amino acid similarity with human B7-1 (23%), B7-2 (29%), B7h (30%), PD-L1 (26%), PD-L2 (27%), B7-H3 long form (32%) and short form (33%), and B7x (30%) (1), which are comparable to the homologies exhibited by other members of the family; for example, B7-1, the founding member of the B7 family, shares 22-37% amino acid similarity with other human B7 molecules. HHLA2 protein is expressed on the cell surface and inhibits both human CD4 and CD8 T cell functions (1).

[0007] HHLA2 is widely expressed in many human cancers. It was recently shown that HHLA2 protein can be detected in the epithelium of the placenta, gut, kidney, gallbladder and breast, but not in most other organs (10). In contrast, HHLA2 protein was widely expressed in human cancers from the breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney, esophagus and hematological malignancies of leukemia and lymphoma (10). A more detailed analysis was performed on a cohort of 50 patients with triple-negative breast cancer; 56% of patients had aberrant expression of HHLA2 on their tumors. This expression significantly correlated with advanced breast cancer stage and metastatic spread to lymph nodes (10). These results demonstrate that the endogenous HHLA2 protein is absent in most normal tissues, but is widely over-expressed in human cancers. Therefore the HHLA2 pathway represents a novel immunosuppressive mechanism within the tumor microenvironment and is an attractive target for human cancer therapy.

[0008] The present invention addresses the need for improved therapies and therapeutics based on the HERV-H LTR Associating Protein 2 (HHLA2) pathway based on identifying a binding partner protein for HHLA2.

SUMMARY OF THE INVENTION

[0009] A method of treating an HHLA2-bearing tumor in a subject is provided comprising administering to the subject an amount of a fusion protein comprising an IgV-like domain (or >80% identity) of a TMIGD2 sufficient to treat the HHLA2-bearing tumor.

[0010] Also provided is a method of treating an HHLA2-bearing tumor in a subject comprising administering to the subject an amount of an agent comprising an IgV-like domain (or >80% identity) of a TMIGD2 conjugated to a cytotoxic agent, and/or administered in combination with radiation therapy, sufficient to treat the HHLA2-bearing tumor.

[0011] Also provided is a method of inhibiting immunosuppression in a subject by an HHLA2-bearing tumor comprising administering an amount of (i) a fusion protein, comprising an IgV-like domain (or >80% identity) of a TMIGD2 and an Fc portion of an immunoglobulin G, or (ii) an amount of a purified protein comprising an IgV-like domain (or >80% identity) of a TMIGD2 but not comprising SEQ ID NO:4, sufficient to inhibit immunosuppression in a subject by an HHLA2-bearing tumor.

[0012] Also provided is an isolated fusion protein comprising an IgV-like domain (or >80% identity) of a TMIGD2 and an Fc portion of an immunoglobulin G. Also provided is a composition comprising such a fusion protein. Also provided is an isolated recombinant nucleic acid encoding such a fusion protein. Also provided is a composition comprising such an isolated recombinant nucleic acid encoding such a fusion protein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1A. Transmembrane and Immunoglobulin Domain Containing 2 (TMIGD2) is an immunoglobulin family member that binds to HHLA2. TMIGD2 is composed of a signal peptide, a single extracellular immunoglobulin domain, transmembrane domain, and a cytoplasmic tail. There are 3 predicted sites of N-linked glycosylation found within the Ig domain (arrows) and four sites of predicted phosphorylation (asterisks) and one potential SH3-binding domain (dot) within the cytoplasmic tail. The whole sequence set forth in FIG. 1A is SEQ ID NO:4.

[0014] FIG. 1B. HHLA2-Ig binds to TMIGD2-expressing 3T3 cells but not to CTLA-4-expressing 3T3 cells. TMIGD2-Ig binds to HHLA2-expressing 3T3 cells but not to CTLA-4-expressing 3T3 cells. Cells were stained with HHLA2-Ig or TMIGD2-Ig (open histograms) or control Ig (shaded histograms) followed by anti-human IgG-APC.

[0015] FIG. 1C. Phylogenetic tree of the human B7 and CD28 families. The phylogenetic comparison of human B7 molecules was generated by PAUP and was divided into three groups. Receptors for human B7 molecules are also shown. TMIGD2 and HHLA2 are in the group 3.

DETAILED DESCRIPTION OF THE INVENTION

[0016] A method of treating an HHLA2-bearing tumor in a subject is provided comprising administering to the subject an amount of a fusion protein comprising an IgV-like domain (or >80% identity of an IgV-like domain) of a TMIGD2 sufficient to treat the HHLA2-bearing tumor.

[0017] In an embodiment, the fusion protein comprising an IgV-like domain of a TMIGD2 comprises an Fc portion of an immunoglobulin G. In an embodiment, the C-terminal residue of the IgV-like domain of a TMIGD2 of the fusion protein is fused via a peptide bond to an N-terminal residue of an Fc portion of an immunoglobulin G. In an embodiment, the immunoglobulin G has the sequence of a human immunoglobulin G. In an embodiment, the immunoglobulin G is an immunoglobulin G1.

[0018] Also provided is a method of treating an HHLA2-bearing tumor in a subject comprising administering to the subject an amount of an agent comprising an IgV-like domain (or >80% identity) of a TMIGD2 conjugated to a cytotoxic agent, and/or administered in combination with radiation therapy, sufficient to treat the HHLA2-bearing tumor.

[0019] As used herein, "treating" a tumor means that one or more symptoms of the disease, such as the tumor itself, metastasis thereof, vascularization of the tumor, or other parameters by which the disease is characterized, are reduced, ameliorated, prevented, placed in a state of remission, or maintained in a state of remission. "Treating" a tumor also means that one or more hallmarks of the tumor may be eliminated, reduced or prevented by the treatment. Non-limiting examples of such hallmarks include uncontrolled degradation of the basement membrane and proximal extracellular matrix, migration, division, and organization of the endothelial cells into new functioning capillaries, and the persistence of such functioning capillaries. Preferably, the treatment is effective to reduce tumor growth and/or size.

[0020] In an embodiment of the methods, the IgV-like domain of a TMIGD2 comprises consecutive amino acid residues having the sequence set forth in SEQ ID NO:3.

[0021] Also provided is a method of inhibiting immunosuppression in a subject by an HHLA2-bearing tumor comprising administering an amount of (i) a fusion protein, comprising an IgV-like domain of a TMIGD2 and an Fc portion of an immunoglobulin G, or (ii) an amount of a purified protein comprising an IgV-like domain of a TMIGD2 but not comprising SEQ ID NO:4, sufficient to inhibit immunosuppression in a subject by an HHLA2-bearing tumor.

[0022] In an embodiment of the methods, the HHLA2 of the HHLA2-bearing tumor is a human HHLA2.

[0023] In an embodiment of the methods, the HHLA2-bearing tumor is a tumor of a breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney, esophagus, or is a hematological tumor. In an embodiment of the methods, the HHLA2-bearing tumor is a hematological tumor and is a leukemia or a lymphoma. In an embodiment of the methods, the HHLA2-bearing tumor is a tumor of the breast and is a triple negative breast cancer.

[0024] In an embodiment of the inventions described herein, the fusion protein comprises consecutive amino acid residues having the sequence set forth in SEQ ID NO:2. In an embodiment, the fusion protein comprises consecutive amino acid residues having the sequence set forth in SEQ ID NO:3.

[0025] Also provided is an isolated fusion protein comprising an IgV-like domain of a TMIGD2 and an Fc portion of an immunoglobulin G. In an embodiment, the isolated fusion protein comprises SEQ ID NO:2. In an embodiment, the isolated fusion protein comprises SEQ ID NO:3.

[0026] An isolated chimeric nucleic acid encoding an isolated fusion protein as described herein is provided.

[0027] A composition comprising the isolated fusion protein as described herein and a carrier is provided.

[0028] In an embodiment, the composition is a pharmaceutical composition, and the carrier is a pharmaceutical carrier.

[0029] The term "TMIGD2-Ig" fusion protein as used herein means a fusion protein constructed of a portion of an immunoglobulin and an active portion of a TMIGD2, or proteins having >80% identical sequence thereto. In a preferred embodiment, the active portion of a TMIGD2 is an extracellular domain of a TMIGD2. In an embodiment, the TMIGD2 has the sequence of a human TMIGD2. In an embodiment, the active portion of a TMIGD2 is the IgV-like domain of an active portion of a TMIGD2. In an embodiment, the portion of an immunoglobulin is a portion of an IgG or an IgM. In an embodiment, it as a portion of an IgG. The IgG portion of the fusion protein can be, e.g., any of an IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgG4 or a portion thereof. In an embodiment, the portion is an Fc region. In an embodiment the fusion protein comprises a sequence identical to an Fc portion of a human IgG1, human IgG2, human IgG2a, human IgG2b, human IgG3 or human IgG4. In an embodiment the fusion protein comprises a sequence identical to an Fc portion of a human IgG1. The term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine of the Fc region may be removed, for example, by recombinantly engineering the nucleic acid encoding the fusion protein.

[0030] In an embodiment, the Fc portion of the Ig is used in the fusion protein. The presence of the Fc domain markedly increases the plasma half-life of the attached protein (e.g. the IgV-like domain of the TMIGD2), which prolongs therapeutic activity. In addition, the Fc domain also enables the fusion protein to interact with Fc-receptors. In an embodiment, the TMIGD2-Ig comprises a TMIGD2 portion linked to an Fc domain. In an embodiment, the TMIGD2 portion is bound directly by a peptide bond to the Fc domain. In an embodiment, the TMIGD2 portion is linked to the Fc domain through a linker. In an embodiment, it is linked via a peptide linker which permits flexibility. In an embodiment, the linker is rigid. In an embodiment the linker is cleavable. Non-limiting examples of flexible linkers within the scope of the invention are Gn, and GGGGS (SEQ ID NO:7), and (GGGGS).sub.n (SEQ ID NO:7), where n=2, 3, 4 or 5. Non-limiting examples of rigid linkers within the scope of the invention are EAAAK (SEQ ID NO:8), (EAAAK).sub.n (SEQ ID NO:8) and (XP).sub.n, where X can be any amino acid. Non-limiting examples of cleavable linkers within the scope of the invention include disulfide links and protease cleavable linkers. In a preferred embodiment, the linker is a peptide linker.

[0031] In an embodiment, the Fc domain has the same sequence or 95% or greater sequence similarity with a human IgG1 Fc domain. In an embodiment, the Fc domain has the same sequence or 95% or greater sequence similarity with a human IgG2 Fc domain. In an embodiment, the Fc domain has the same sequence or 95% or greater sequence similarity with a human IgG3 Fc domain. In an embodiment, the Fc domain has the same sequence or 95% or greater sequence similarity with a human IgG4 Fc domain. In an embodiment, the Fc domain is not mutated. In an embodiment, the Fc domain is mutated at the CH2-CH3 domain interface to increase the affinity of IgG for FcRn at acidic but not neutral pH (11, 12).

[0032] In an embodiment, the fusion protein described herein is recombinantly produced. In an embodiment, the fusion protein is produced in a eukaryotic expression system. In an embodiment, the fusion protein produced in the eukaryotic expression system comprises glycosylation at a residue on the Fc portion corresponding to Asn297.

[0033] In an embodiment, the fusion protein is a homodimer. In an embodiment, the fusion protein is monomeric. In an embodiment, the fusion protein is polymeric.

[0034] In an embodiment, of all aspects of the invention described herein reciting a subject, the subject is a human. In an embodiment, of all aspects of the invention described herein reciting HHLA2, the HHLA2 is a human HHLA2.

[0035] In an embodiment, the HHLA2 is a human HHLA2 comprising the following sequence: (SEQ ID NO:1)

TABLE-US-00001 MKAQTALSFFLILITSLSGSQGIFPLAFFIYVPMNEQIVIGRLDEDIILP SSFERGSEVVIHWKYQDSYKVHSYYKGSDHLESQDPRYANRTSLFYNEIQ NGNASLFFRRVSLLDEGIYTCYVGTAIQVITNKVVLKVGVFLTPVMKYEK RNTNSFLICSVLSVYPRPIITWKMDNTPISENNMEETGSLDSFSINSPLN ITGSNSSYECTIENSLLKQTWTGRWTMKDGLHKMQSEHVSLSCQPVNDYF SPNQDFKVTWSRMKSGTFSVLAYYLSSSQNTIINESRFSWNKELINQSDF SMNLMDLNLSDSGEYLCNISSDEYTLLTIHTVHVEPSQETASHNKGLWIL VPSAILAAFLLIWSVKCCRAQLEARRSRHPADGAQQERCCVPPGERCPSA PDNGEENVPLSGKV.

[0036] In an embodiment, the TMIGD2-Ig fusion protein comprises the following sequence (SEQ ID NO:2):

TABLE-US-00002 PNLLQVRQGSQATLVCQVDQATAWERLRVKWTKDGAILCQPYITNGSLSL GVCGPQGRLSWQAPSHLTLQLDPVSLNHSGAYVCWAAVEIPELEEAEGNI TRLFVDPDDPTQNRNRIASFGGRSKTSGSEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK.

[0037] In a further embodiment, the TMIGD2-Ig fusion protein comprises the following sequence (SEQ ID NO:3):

TABLE-US-00003 LSVQQGPNLLQVRQGSQATLVCQVDQATAWERLRVKWTKDGAILCQPYIT NGSLSLGVCGPQGRLSWQAPSHLTLQLDPVSLNHSGAYVCWAAVEIPELE EAEGNITRLFVDPDDPTQNRNRIASFGGRSKTSGSEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK.

[0038] In an embodiment, the IgV-like domain of TMIGD2 comprises the following sequence (SEQ ID NO:5):

TABLE-US-00004 PNLLQVRQGSQATLVCQVDQATAWERLRVKWTKDGAILCQPYITNGSLSL GVCGPQGRLSWQAPSHLTLQLDPVSLNHSGAYVCWAAVEIPELEEAEGNI TRLFVD.

[0039] In an embodiment, the fusion protein comprises a portion comprising the following sequence including an IgV-like domain of TMIGD2 (SEQ ID NO:6):

TABLE-US-00005 PNLLQVRQGSQATLVCQVDQATAWERLRVKWTKDGAILCQPYITNGSLSL GVCGPQGRLSWQAPSHLTLQLDPVSLNHSGAYVCWAAVEIPELEEAEGNI TRLFVDPDDPTQNRNRIASF.

[0040] In an embodiment, the methods can be performed, mutatis mutandis, wherein a nucleic acid encoding a TMIGD2-Ig is administered in a fashion such that it can express inside the subject, in place of the TMIGD2-Ig fusion protein.

[0041] Cancers, including tumors, treatable by the invention include e.g. tumors of the nasopharynx, pharynx, lung, bone, brain, salivary gland, stomach, esophagus, testes, ovary, uterus, endometrium, liver, small intestine, appendix, colon, rectum, gall bladder, pancreas, kidney, urinary bladder, breast, cervix, vagina, vulva, prostate, thyroid, and skin, or a glioma. In an embodiment, the cancer treated is a metastatic melanoma. In an embodiment, the cancer treated comprises a tumor. In an embodiment, the cancer treated comprises an HHLA2-bearing tumor.

[0042] This invention also provides a composition comprising a fusion protein as described herein. In an embodiment, the composition is a pharmaceutical composition. In an embodiment the composition or pharmaceutical composition comprising one or more of the fusion proteins described herein is substantially pure with regard to the fusion protein. A composition or pharmaceutical composition comprising one or more of the fusion proteins described herein is "substantially pure" with regard to the antibody or fragment when at least about 60 to 75% of a sample of the composition or pharmaceutical composition exhibits a single species of the fusion protein. A substantially pure composition or pharmaceutical composition comprising one or more of the fusion proteins described herein can comprise, in the portion thereof which is the fusion protein, 60%, 70%, 80% or 90% of the fusion protein of the single species, more usually about 95%, and preferably over 99%. Fusion protein purity or homogeneity may be tested by a number of means well known in the art, such as polyacrylamide gel electrophoresis or HPLC.

[0043] The invention also encompasses compositions comprising the described fusion proteins and a carrier. The carrier may comprise one or more pharmaceutically-acceptable carrier components. Such pharmaceutically-acceptable carrier components are widely known in the art. Examples of acceptable pharmaceutical carriers include, but are not limited to, additive solution-3 (AS-3), saline, phosphate buffered saline, Ringer's solution, lactated Ringer's solution, Locke-Ringer's solution, Krebs Ringer's solution, Hartmann's balanced saline solution, and heparinized sodium citrate acid dextrose solution. The pharmaceutically acceptable carrier used can depend on the route of administration. The pharmaceutical composition can be formulated for administration by any method known in the art, including but not limited to, oral administration, parenteral administration, intravenous administration, transdermal administration, intramuscular administration, intranasal administration, direct injection into a tumor site, and administration through an osmotic mini-pump.

[0044] Cytotoxic agents, as referred to herein, include chemotherapuetic agents. In an embodiment, the cytotoxic agent is doxorubicin. In an embodiment, the cytotoxic agent is a maytansinoid. In an embodiment, the cytotoxic agent an alkylating agent, an anti-metabolite, a plant alkaloid or terpenoid, or a cytotoxic antibiotic. In embodiments, the cytotoxic agent is cyclophosphamide, bleomycin, etoposide, platinum agent (cisplatin), fluorouracil, vincristine, methotrexate, taxol, epirubicin, leucovorin (folinic acid), or irinotecan. Radiation therapy can be used in place of or in combination with one or more cytotoxic agents.

[0045] In an embodiment, the fusion protein of the invention is administered systemically in the methods described herein. In an embodiment, the fusion protein of the invention is administered locally in the methods described herein. In an embodiment, the fusion protein of the invention is administered directly to the tumor in the methods described herein, for example by injection or cannulation.

[0046] In an embodiment, the agent comprising an IgV-like domain of a TMIGD2 conjugated to a cytotoxic agent of the invention is administered systemically in the methods described herein. In an embodiment, the agent comprising an IgV-like domain of a TMIGD2 conjugated to a cytotoxic agent of the invention is administered locally in the methods described herein. In an embodiment, the agent comprising an IgV-like domain of a TMIGD2 conjugated to a cytotoxic agent of the invention is administered directly to the tumor in the methods described herein, for example by injection or cannulation.

[0047] In an embodiment, "determining" as used herein means experimentally determining.

[0048] All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0049] This invention will be better understood from the Experimental Details, which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.

EXPERIMENTAL DETAILS

Example 1

[0050] Identification of immunoglobulin domain-containing protein 2 (TMIGD2) as one of the receptors for HHLA2: Receptors for HHLA2 are widely expressed on both naive and activated T cells as well as dendritic cells, monocytes, and B cells (1). As HHLA2 is a member of the immunoglobulin superfamily and has orthologs in humans and monkeys but not in mice or rats, it was hypothesized the receptors for HHLA2 may belong to the immunoglobulin superfamily and have the same expression pattern due to the co-evolution. From more than 500 members of the immunoglobulin superfamily, a list was compiled of the immunoglobulin family members expressed in humans and monkeys but not in mice or rats. This list was further refined by only including members with predicted transmembrane domains. Their ability to bind to HHLA2 was tested. It was also found that a fusion protein of Transmembrane and Immunoglobulin Domain Containing (2) (TMIGD2, FIG. 1A), the extracellular domain of TMIGD2 linked to the Fc region of human IgG1 (TMIGD2-Ig), bound strongly to 3T3 cells overexpressing HHLA2 (FIG. 1B) but not other human B7 molecules in flow cytometry. TMIGD2 contains an N-terminal signal peptide, an extracellular IgV-like domain, three potential sites for N-linked glycosylation, a transmembrane region, and a cytoplasmic tail with two potential sites for phosphorylation (FIG. 1A). Thus, TMIGD2 was identified as a receptor for HHLA2.

[0051] The human TMIGD2-Ig fusion protein (SEQ ID NO:3) was generated with the extracellular domain of TMIGD2 linked to the Fc region of human IgG1. TMIGD2-Ig protein was generated in Drosophila S2 cells and purified using Protein G columns.

Prophetic Example

[0052] TMIGD2-Ig is administered to humanized NOD-scid/IL2Rgnull (NSG) mice bearing HHLA2 positive human tumors. It will be seen that the tumors are reduced in size in the animals treated with TMIGD2-Ig compared to controls. In addition, a TMIGD2-drug conjugate is administered to humanized NOD-scid/IL2Rgnull (NSG) mice bearing HHLA2 positive human tumors. It will be seen that the tumors are reduced in size in the animals treated with TMIGD2-drug conjugate compared to controls.

Discussion

[0053] TMIDG2 and its derivatives as blockers or binders of HHLA2 for immunotherapies: Given the new finding that TMIGD2 very strongly binds HHLA2 (FIG. 1), together with the previous findings that HHLA2 is an inhibitor of the human immune system (1) and that many human cancers over-express HHLA2 as a novel immunosuppressive mechanism (10), TMIGD2 and its derivatives can be used as blockers or binders of HHLA2 for immunotherapies of cancers and other diseases. There are at least two possible mechanisms by which the TMIGD2 protein and its derivatives can treat cancers: (1) TMIGD2 protein and its derivatives can bind cancer-expressed HHLA2 and therefore enhance anti-tumor immunity by blocking HHLA2-mediated immunosuppression; and (2) TMIGD2-drug conjugates and its derivatives-drug conjugates can bind HHLA2 positive human cancer cells and directly kill them. Accordingly, the following are within the scope of the invention:

[0054] i) TMIGD2-Ig fusion proteins can be used to treat human cancers by blocking immunosuppression of tumor-expressed HHLA2, including cancers of the breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney, esophagus, leukemia and lymphoma;

[0055] ii) derivatives of TMIGD2 protein (such as high-affinity and low-affinity mutants, IgV-like domain of TMIDG2) can be used to treat human cancers by blocking immunosuppression of tumor-expressed HHLA2;

[0056] iii) TMIGD2-drug conjugates can be used as new drugs to treat human cancers by directly killing HHLA2 positive cancer cells; and

[0057] iv) TMIGD2-derivative-drug conjugates can be used as new drugs to treat human cancers by directly killing HHLA2 positive cancer cells.

Materials and Methods

[0058] Bioinformatic Analysis: The NCBI database was queried for proteins of the immunoglobulin family with homologues in humans and monkeys, but not in mice or rats. The sequences of the resulting list of proteins were analyzed by various domain-prediction programs to determine if these proteins contained Ig, IgC, IgC-like, IgV, or IgV-like domains. The list was further refined by excluding proteins that did not contain a transmembrane domain. MacVector 10.6. was used for sequence alignment and homology comparison. The phylogenetic tree was generated by PAUP (4.0b10) after removal of significant inserts and trimming C- and N-terminal extensions from sequence alignments. Motifs and domains were analyzed with EMBL-EBI tools, SMART, and CBS Prediction. For gene copy number variations, the cBioPortal for Cancer Genomics database and the Cancer Genome Atlas were analyzed.

[0059] Fusion Protein Production and Purification: TMIGD2-Ig fusion protein was prepared by PCR-amplifying the extracellular domain of the protein without the signal peptide. The amplified product was inserted into a human IgG1 Fc tag of plasmid pMT/BiP. Drosophila Schneider 2 cells were co-transfected with this construct and a plasmid inducing hygromycin resistance. The fusion protein was expressed in Express Five serum-free medium (Life Technologies) and purified using Protein G Plus Agarose columns (Pierce). The purity of the fusion protein was confirmed by SDS-PAGE.

[0060] Identification of immunoglobulin domain--containing protein 2 (TMIGD2) as one of the receptors for HHLA2: Receptors for HHLA2 are widely expressed on both naive and activated T cells as well as dendritic cells, monocytes, and B cells. As HHLA2 is a member of the immunoglobulin superfamily and has orthologs in humans and monkeys but not in mice or rats, it was hypothesized that the receptors for HHLA2 may belong to the immunoglobulin superfamily and have the same phylogenetic pattern due to co-evolution.

[0061] From more than 500 members of the immunoglobulin superfamily, a list was compiled of the immunoglobulin family members expressed in humans and monkeys but not in mice or rats. This list was further refined by only including members with predicted transmembrane domains. Their ability to bind to HHLA2 was tested. A fusion protein of Transmembrane and Immunoglobulin Domain Containing 2 (TMIGD2), the extracellular domain of TMIGD2 linked to the Fc region of human IgG1 (TMIGD2-Ig), bound strongly to 3T3 cells expressing HHLA2 but not other human B7 molecules in flow cytometry. TMIGD2 contains an N-terminal signal peptide, an extracellular IgV-like domain, three potential sites for N-linked glycosylation, a transmembrane region, and a cytoplasmic tail with two potential sites for phosphorylation. By sequence analysis, it was found that TMIGD2, the immunoglobulin-containing and proline-rich receptor-1 (IGPR-1), and CD28 homologue (CD28H) are the same molecule. IGPR-1 was originally identified as a adhesion molecule involved in angiogenesis, while CD28H was recently reported as a receptor by a high-throughput screen of transmembrane proteins. Thus TMIGD2/IGPR-1/CD28H is one of the receptors for HHLA2.

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[0073] 12. Yeung Y A, Leabman M K, Marvin J S, Qiu J, Adams C W, Lien S, Starovasnik M A, Lowman H B. Engineering human IgG1 affinity to human neonatal Fc receptor: impact of affinity improvement on pharmacokinetics in primates. J Immunol. 182(12), 7663-71 (2009).

Sequence CWU 1

1

81414PRTArtificial Sequencefusion protein based on human sequence 1Met Lys Ala Gln Thr Ala Leu Ser Phe Phe Leu Ile Leu Ile Thr Ser1 5 10 15Leu Ser Gly Ser Gln Gly Ile Phe Pro Leu Ala Phe Phe Ile Tyr Val 20 25 30Pro Met Asn Glu Gln Ile Val Ile Gly Arg Leu Asp Glu Asp Ile Ile 35 40 45Leu Pro Ser Ser Phe Glu Arg Gly Ser Glu Val Val Ile His Trp Lys 50 55 60Tyr Gln Asp Ser Tyr Lys Val His Ser Tyr Tyr Lys Gly Ser Asp His65 70 75 80Leu Glu Ser Gln Asp Pro Arg Tyr Ala Asn Arg Thr Ser Leu Phe Tyr 85 90 95Asn Glu Ile Gln Asn Gly Asn Ala Ser Leu Phe Phe Arg Arg Val Ser 100 105 110Leu Leu Asp Glu Gly Ile Tyr Thr Cys Tyr Val Gly Thr Ala Ile Gln 115 120 125Val Ile Thr Asn Lys Val Val Leu Lys Val Gly Val Phe Leu Thr Pro 130 135 140Val Met Lys Tyr Glu Lys Arg Asn Thr Asn Ser Phe Leu Ile Cys Ser145 150 155 160Val Leu Ser Val Tyr Pro Arg Pro Ile Ile Thr Trp Lys Met Asp Asn 165 170 175Thr Pro Ile Ser Glu Asn Asn Met Glu Glu Thr Gly Ser Leu Asp Ser 180 185 190Phe Ser Ile Asn Ser Pro Leu Asn Ile Thr Gly Ser Asn Ser Ser Tyr 195 200 205Glu Cys Thr Ile Glu Asn Ser Leu Leu Lys Gln Thr Trp Thr Gly Arg 210 215 220Trp Thr Met Lys Asp Gly Leu His Lys Met Gln Ser Glu His Val Ser225 230 235 240Leu Ser Cys Gln Pro Val Asn Asp Tyr Phe Ser Pro Asn Gln Asp Phe 245 250 255Lys Val Thr Trp Ser Arg Met Lys Ser Gly Thr Phe Ser Val Leu Ala 260 265 270Tyr Tyr Leu Ser Ser Ser Gln Asn Thr Ile Ile Asn Glu Ser Arg Phe 275 280 285Ser Trp Asn Lys Glu Leu Ile Asn Gln Ser Asp Phe Ser Met Asn Leu 290 295 300Met Asp Leu Asn Leu Ser Asp Ser Gly Glu Tyr Leu Cys Asn Ile Ser305 310 315 320Ser Asp Glu Tyr Thr Leu Leu Thr Ile His Thr Val His Val Glu Pro 325 330 335Ser Gln Glu Thr Ala Ser His Asn Lys Gly Leu Trp Ile Leu Val Pro 340 345 350Ser Ala Ile Leu Ala Ala Phe Leu Leu Ile Trp Ser Val Lys Cys Cys 355 360 365Arg Ala Gln Leu Glu Ala Arg Arg Ser Arg His Pro Ala Asp Gly Ala 370 375 380Gln Gln Glu Arg Cys Cys Val Pro Pro Gly Glu Arg Cys Pro Ser Ala385 390 395 400Pro Asp Asn Gly Glu Glu Asn Val Pro Leu Ser Gly Lys Val 405 4102361PRTArtificial SequenceFusion protein based on human sequence 2Pro Asn Leu Leu Gln Val Arg Gln Gly Ser Gln Ala Thr Leu Val Cys1 5 10 15Gln Val Asp Gln Ala Thr Ala Trp Glu Arg Leu Arg Val Lys Trp Thr 20 25 30Lys Asp Gly Ala Ile Leu Cys Gln Pro Tyr Ile Thr Asn Gly Ser Leu 35 40 45Ser Leu Gly Val Cys Gly Pro Gln Gly Arg Leu Ser Trp Gln Ala Pro 50 55 60Ser His Leu Thr Leu Gln Leu Asp Pro Val Ser Leu Asn His Ser Gly65 70 75 80Ala Tyr Val Cys Trp Ala Ala Val Glu Ile Pro Glu Leu Glu Glu Ala 85 90 95Glu Gly Asn Ile Thr Arg Leu Phe Val Asp Pro Asp Asp Pro Thr Gln 100 105 110Asn Arg Asn Arg Ile Ala Ser Phe Gly Gly Arg Ser Lys Thr Ser Gly 115 120 125Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 130 135 140Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys145 150 155 160Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 165 170 175Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 180 185 190Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 195 200 205Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 210 215 220Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys225 230 235 240Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 245 250 255Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 260 265 270Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 275 280 285Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 290 295 300Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu305 310 315 320Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 325 330 335Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 340 345 350Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 3603367PRTHomo sapiens 3Leu Ser Val Gln Gln Gly Pro Asn Leu Leu Gln Val Arg Gln Gly Ser1 5 10 15Gln Ala Thr Leu Val Cys Gln Val Asp Gln Ala Thr Ala Trp Glu Arg 20 25 30Leu Arg Val Lys Trp Thr Lys Asp Gly Ala Ile Leu Cys Gln Pro Tyr 35 40 45Ile Thr Asn Gly Ser Leu Ser Leu Gly Val Cys Gly Pro Gln Gly Arg 50 55 60Leu Ser Trp Gln Ala Pro Ser His Leu Thr Leu Gln Leu Asp Pro Val65 70 75 80Ser Leu Asn His Ser Gly Ala Tyr Val Cys Trp Ala Ala Val Glu Ile 85 90 95Pro Glu Leu Glu Glu Ala Glu Gly Asn Ile Thr Arg Leu Phe Val Asp 100 105 110Pro Asp Asp Pro Thr Gln Asn Arg Asn Arg Ile Ala Ser Phe Gly Gly 115 120 125Arg Ser Lys Thr Ser Gly Ser Glu Pro Lys Ser Cys Asp Lys Thr His 130 135 140Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val145 150 155 160Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 165 170 175Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 180 185 190Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 195 200 205Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 210 215 220Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys225 230 235 240Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 245 250 255Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 260 265 270Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 275 280 285Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 290 295 300Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser305 310 315 320Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 325 330 335Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 340 345 350His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 355 360 3654282PRTHomo sapiens 4Met Gly Ser Pro Gly Met Val Leu Gly Leu Leu Val Gln Ile Trp Ala1 5 10 15Leu Gln Glu Ala Ser Ser Leu Ser Val Gln Gln Gly Pro Asn Leu Leu 20 25 30Gln Val Arg Gln Gly Ser Gln Ala Thr Leu Val Cys Gln Val Asp Gln 35 40 45Ala Thr Ala Trp Glu Arg Leu Arg Val Lys Trp Thr Lys Asp Gly Ala 50 55 60Ile Leu Cys Gln Pro Tyr Ile Thr Asn Gly Ser Leu Ser Leu Gly Val65 70 75 80Cys Gly Pro Gln Gly Arg Leu Ser Trp Gln Ala Pro Ser His Leu Thr 85 90 95Leu Gln Leu Asp Pro Val Ser Leu Asn His Ser Gly Ala Tyr Val Cys 100 105 110Trp Ala Ala Val Glu Ile Pro Glu Leu Glu Glu Ala Glu Gly Asn Ile 115 120 125Thr Arg Leu Phe Val Asp Pro Asp Asp Pro Thr Gln Asn Arg Asn Arg 130 135 140Ile Ala Ser Phe Pro Gly Phe Leu Phe Val Leu Leu Gly Val Gly Ser145 150 155 160Met Gly Val Ala Ala Ile Val Trp Gly Ala Trp Phe Trp Gly Arg Arg 165 170 175Ser Cys Gln Gln Arg Asp Ser Gly Asn Ser Pro Gly Asn Ala Phe Tyr 180 185 190Ser Asn Val Leu Tyr Arg Pro Arg Gly Ala Pro Lys Lys Ser Glu Asp 195 200 205Cys Ser Gly Glu Gly Lys Asp Gln Arg Gly Gln Ser Ile Tyr Ser Thr 210 215 220Ser Phe Pro Gln Pro Ala Pro Arg Gln Pro His Leu Ala Ser Arg Pro225 230 235 240Cys Pro Ser Pro Arg Pro Cys Pro Ser Pro Arg Pro Gly His Pro Val 245 250 255Ser Met Val Arg Val Ser Pro Arg Pro Ser Pro Thr Gln Gln Pro Arg 260 265 270Pro Lys Gly Phe Pro Lys Val Gly Glu Glu 275 2805106PRTHomo sapiens 5Pro Asn Leu Leu Gln Val Arg Gln Gly Ser Gln Ala Thr Leu Val Cys1 5 10 15Gln Val Asp Gln Ala Thr Ala Trp Glu Arg Leu Arg Val Lys Trp Thr 20 25 30Lys Asp Gly Ala Ile Leu Cys Gln Pro Tyr Ile Thr Asn Gly Ser Leu 35 40 45Ser Leu Gly Val Cys Gly Pro Gln Gly Arg Leu Ser Trp Gln Ala Pro 50 55 60Ser His Leu Thr Leu Gln Leu Asp Pro Val Ser Leu Asn His Ser Gly65 70 75 80Ala Tyr Val Cys Trp Ala Ala Val Glu Ile Pro Glu Leu Glu Glu Ala 85 90 95Glu Gly Asn Ile Thr Arg Leu Phe Val Asp 100 1056120PRTHomo sapiens 6Pro Asn Leu Leu Gln Val Arg Gln Gly Ser Gln Ala Thr Leu Val Cys1 5 10 15Gln Val Asp Gln Ala Thr Ala Trp Glu Arg Leu Arg Val Lys Trp Thr 20 25 30Lys Asp Gly Ala Ile Leu Cys Gln Pro Tyr Ile Thr Asn Gly Ser Leu 35 40 45Ser Leu Gly Val Cys Gly Pro Gln Gly Arg Leu Ser Trp Gln Ala Pro 50 55 60Ser His Leu Thr Leu Gln Leu Asp Pro Val Ser Leu Asn His Ser Gly65 70 75 80Ala Tyr Val Cys Trp Ala Ala Val Glu Ile Pro Glu Leu Glu Glu Ala 85 90 95Glu Gly Asn Ile Thr Arg Leu Phe Val Asp Pro Asp Asp Pro Thr Gln 100 105 110Asn Arg Asn Arg Ile Ala Ser Phe 115 12075PRTArtificial Sequencesynthesized linker sequence 7Gly Gly Gly Gly Ser1 585PRTArtificial Sequencesynthesized linker sequence 8Glu Ala Ala Ala Lys1 5

Claims

1. A method of treating an HHLA2-bearing tumor in a subject comprising administering to the subject an amount of a fusion protein comprising an IgV-like domain of a TMIGD2 sufficient to treat the HHLA2-bearing tumor.

2. The method of claim 1, wherein the fusion protein comprising an IgV-like domain of a TMIGD2 comprises an Fe portion of an immunoglobulin G.

3. The method of claim 1, wherein the C-terminal residue of the IgV-like domain of a TMIGD2 of the fusion protein is fused via a peptide bond to an N-terminal residue of an Fe portion of an immunoglobulin G.

4. The method of claim 2, wherein the immunoglobulin G has the sequence of a human immunoglobulin G.

5. The method of claim 2, wherein the immunoglobulin G 1s an immunoglobulin G 1.

6. A method of treating an HHLA2-bearing tumor in a subject comprising administering to the subject an amount of an agent comprising an IgV-like domain of a TMIGD2 conjugated to a cytotoxic agent, and/or administered in combination with radiation therapy, sufficient to treat the HHLA2-bearing tumor.

7. The method of claim 1, wherein the IgV-like domain of a TMIGD2 comprises consecutive amino acid residues having the sequence set forth in SEQ ID NO:3.

8. A method of inhibiting immunosuppression in a subject by an HHLA2-bearing tumor comprising administering an amount of (i) a fusion protein, comprising an IgV-like domain of a TMIGD2 and an Fe portion of an immunoglobulin G, or (ii) an amount of a purified protein comprising an IgV-like domain of a TMIGD2 but not comprising SEQ ID N0:4, sufficient to inhibit immunosuppression in a subject by an HHLA2-bearing tumor.

9. The method of claim 1, wherein the HHLA2 of the HHLA2-bearing tumor is a human HHLA2.

10. The method of claim 1, wherein the HHLA2-bearing tumor is a tumor of a breast, lung, thyroid, melanoma, pancreas, ovary, liver, bladder, colon, prostate, kidney, or esophagus, or is a hematological tumor.

11. The method of claim 10, wherein the HHLA2-bearing tumor is a hematological tumor and is a leukemia or a lymphoma.

12. The method of claim 10, wherein the HHLA2-bearing tumor is a tumor of the breast and is a triple negative breast cancer.

13. The method of claim 1, wherein the fusion protein comprises consecutive amino acid residues having the sequence set forth in SEQ ID N0:2.

14. The method of claim 1, wherein the fusion protein comprises consecutive amino acid residues having the sequence set forth in SEQ ID N0:3.

15. An isolated fusion protein comprising an IgV-like domain of a TMIGD2 and an Fc portion of an immunoglobulin G, wherein the isolated fusion protein comprises SEQ ID NO:2 or SEQ ID NO:3.

16. The isolated fusion protein of claim 15, comprising SEQ ID NO:2.

17. The isolated fusion protein of claim 15, comprising SEQ ID NO:3.

18. An isolated chimeric nucleic acid encoding the isolated fusion protein of claim 15.

19. A composition comprising the isolated fusion protein of claim 15 and a carrier.

20. The composition of claim 19, which is a pharmaceutical composition, and wherein the carrier is a pharmaceutical carrier.