IL256261A - Hide1 compositions and methods - Google Patents

Description

HIDE1 COMPOSITIONS AND METHODS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 to USSN 62/191,775, filed July 13, 2015, and USSN 62/191,804, filed July 13, 2015, both of which are expressl y incorporated herei nby reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] Naive T cell smust receive two independent signals from antigen-presenting cell s (APC) in order to become productivel actiy vated. The firs t,Signal 1, is antigen-specific and occurs when T cell antigen receptor encounters the appropriate antigen-MHC complex on the APC. The fate of the immune response is determined by a second, antigen-independent signal (Signal 2) which is delivered through a T cel lcostimulatory molecul thate engages its APC- expressed ligand. This second signal could be either stimulatory (positive costimulati on)or inhibitory (negative costimulation or coinhibition). In the absence of a costimulatory signal , or in the presence of a coinhibitory signal, T-cel lactivation is impaire dor aborted whic, h may lead to a state of antigen-specific unresponsiveness (known as T-cel lanergy), or may resul int T-cel lapoptotic death.

[0003] Costimulatory molecul paire susually consis oft ligands expressed on APCs and their cognate receptor express sed on T cells. The prototy lipegand/recept pairor sof costimulatory molecule ares B7/CD28 and CD40/CD40L. The B7 family consists of structurall relaty ed, cell-surfac proteie ligan nds, which may provide stimulatory or inhibito ryinput to an immune response. Members of the B7 family are structurall relaty ed, with the extracellula domainr containing at leas tone variabl eor constant immunoglobulin domain.

[0004] Both positive and negative costimulatory signals play critic alrole ins the regulati onof cell-mediate immd une responses, and molecule thats mediate thes esignal shave proven to be effective targets for immunomodulation. Based on thi sknowledge, several therapeuti c approaches that involve targeting of costimulatory molecules have been developed, and were shown to be useful for prevention and treatment of cancer by turning on, or preventing the turning off, of immune responses in cancer patients and for prevention and treatment of autoimmune diseases and inflammatory diseases, as well as rejection of allogenic transplantat eachion, by turning off uncontroll immedune responses or, by induction of “off 1WO 2017/009712 PCT/IB2016/001079 signal” by negative costimulation (or coinhibition) in subject swith thes pathe ologica l conditions.

[0005] Manipulation of the signals delivered by B7 ligands has shown potential in the treatment of autoimmunit y,inflammatory diseases, and transplant rejection. Therapeuti c strategi includees blocking of costimulati usingon monoclonal antibodie tos the ligand or to the receptor of a costimulatory pair, or using solubl fusione proteins composed of the costimulatory receptor that may bind and block its appropriat ligand.e Another approac his induction of co-inhibition using solubl fusione protei ofn an inhibitor ligand.y These approaches rely, at least partiall ony, the eventual deletion of auto -or allo-react Tive cells (which are responsible for the pathogeni processesc in autoimmune diseases or transplantat respecion, tivel y),presumably because in the absence of costimulati (whichon induces cell survival genes) T cells become highl ysusceptible to induction of apoptosis.

Thus, novel agents that are capable of modulating costimulatory signals, without compromising the immune system’s abilit yto defend against pathogens, are highly advantageous for treatment and prevention of such pathological conditions.

[0006] Costimulatory pathways play an important role in tumor development Inter. estingly, tumor haves been shown to evade immune destructi byon impeding T cell activation through inhibition of co-stimulatory factors in the B7-CD28 and TNF families, as well as by affecting regulatory T cells, whic hinhibit anti-tumor T cel lresponses (see Wang (2006), “Immune Suppression by Tumor Specific CD4+ Regulatory T cells” in Cancer. Semin. Cancer. Biol. 16:73-79; Greenwal d,et al. (2005), “The B7 Family Revisited”, Ann. Rev. Immunol. 23:515- 48; Watt s(2005) “TNF/TNFR Family Members in Co-stimulation of T Cell Responses”, Ann. Rev. Immunol. 23:23-68; Sadum, et al. (2007), “Immune Signatures of Murine and Human Cancers Reveal Unique Mechanisms of Tumor Escape and New Target sfor Cancer Immunotherapy”, Clin. Cane. Res. 13(13): 4016-4025). Such tumo rexpressed co-stimulatory molecule haves become attracti cancerve biomarkers and may serve as tumor-associa ted antigens (TAAs). Furthermor costie, mulatory pathways have been identified as immunologic checkpoin tsthat attenuat Te cell dependent immune responses, both at the level of initiation and effector function within tumor metastases. As engineered cancer vaccines continue to improve, it is becoming clear that such immunologic checkpoint ares a major barrie tor the 2WO 2017/009712 PCT/IB2016/001079 vaccines’ abilit yto induce therapeutic anti-tumor responses In. that regard, costimulatory molecule cans serve as adjuvant sfor active (vaccination) and passive (antibody-mediated ) cancer immunotherapy, providing strategie tos thwart immune tolerance and stimula tethe immune system.

[0007] In addition, such agents could be of use in other types of cancer immunotherapy, such as adoptive immunotherapy, in whic htumor-specific T cell populations are expanded and directed to attack and kill tumor cells. Agents capable of augmenting such anti-tumor response have great therapeutic potentia andl may be of value in the attempt to overcome the obstacles to tumor immunotherapy. Recently, novel agents that modulate several costimulatory pathways were indeed introduced to the clinic as cancer immunotherapy.

[0008] Regulating costimulati usingon agonists and/or antagonis tsto various costimulat ory proteins has been extensively studied as a strategy for treating autoimmune diseases, graft rejection, allergy and cancer. This field has been clinical lypioneered by CTLA4-Ig (Abatacept, Orencia®) which is approved for treatment of RA, mutated CTLA4-Ig (Belatacept Nuloji, x@) for prevention of acute kidney transplant rejection and by the anti - CTLA4 antibody (Ipilimumab, Yervoy®), recently approved for the treatment of melanoma .

Other costimulation regulators are currently in advanced stages of clinical developme nt including anti-PD-1 antibody (BMS-936558) which is in development for treatment of Non- Smal lCell Lung cancer and other cancers. Furthermor suche, agents are also in clinical development for viral infections, for example the anti PD-1 Ab, MDX-1106, which is being tested for treatme ntof hepatiti C,s and the anti-CTLA-4 Ab CP-675,206 (tremelimumab) which is in a clinical trial in hepatitis C virus-infected patients wit hhepatocellul carcinomar a.

[0009] In addition recently researcher haves developed compounds whic htarget regulatory T cells (iTregs) for use in immunotherapy. With respec theretot it is known that inducible regulatory T cells (iTregs) are commonly seen in many tumors, and form the major subset of immune suppressor cells in the tumor tissue and moreover represe nta major tumo resistr ance mechanism from immune surveillance. Accordingly, iTregs are therefore viewed as important cellula targer ts for cancer therapy. 3WO 2017/009712 PCT/IB2016/001079

[0010] Multiple immune-checkpoi ntreceptors, such as CTLA4, PD-1, TIM3 and LAG3, are expressed at high level son the surfac eof iTregs and direct lypromot Trege cell-mediated suppression of effector immune responses. Therefore, some immune-checkpoi ntantibodies may, in addition to increasin gCTL immunity, furth erblock the immunosuppressive activit y of iTregs and thereby enhance anti-tumor immunity .For example, CTLA4 blockade by ipilimuma bboth enhances effector T cel lactivity, and inhibits Treg immunosuppressive activity.

[0011] B cells play a critic alrole in recognition of foreign antigens and they produce the antibodie necess sary to provide protecti againston various types of infectious agents .T cell help to B cells is a pivotal proces ofs adaptive immune responses Foll. icul helpear Tr (Tfh) cells are a subset of CD4+ T cells specialize din B cel lhelp (reviewed by Crotty, Annu. Rev.

Immunol. 29: 621-663, 2011). Tfh cells express the B cel lhoming chemokine receptor, CXCR5, which drives Tfh cel lmigration into B cel lfollicl wites hin lymph nodes in a CXCL13-dependent manner. The requirement of Tfh cells for B cel lhelp and T cell - dependent antibody responses indicate sthat thi scel ltype is of great importance for protecti ve immunity against various types of infectious agents ,as well as for rational vaccine design. In addition, regulatory B cell s(Bregs) have a role in impairing effective clearance of tumors.

The mechanism sfor Bregs effects in cancer are not well understood, however one proposed mechanism is through inhibition of cytotoxic CD8+T cells.

[0012] NK cell sare effector lymphocyte ofs the innate immune system that are known to be involved in killing of pathologica orl diseased cell ssuch as cancer and infected cell sand pathogens. Natural killer cells have the capacity to kill cellula targr ets and produce cytokines without prior specific sensitization. NK cells are unique, as they have the abilit yto recognize stressed cell sin the absence of antibodie ands MHC, allowin forg a much faster immune reaction. This role is especially important because harmful cells that are missing MHC 1 markers cannot be detected and destroyed by othe immr une cell s,such as T cells.

[0013] Induction of immune tolerance has long been considered the “holy grail” for autoimmune disease therapy The. immune system has the reciproc taskal tos protect the host against invading pathogens, but simultaneously to preven tdamage resulting from unwanted reactions to self-antigens. The latte partr is known as immune tolerance and performe byd a 4WO 2017/009712 PCT/IB2016/001079 complex set of interactive and complementary pathways whic, h regulat imme une responses.

T cells have the abilit yto react to a variety of antigens, both self and nonself. Therefore, there are many mechanism sthat exist naturall toy eliminate potential self-rely active responses - thi sis known as natural tolerance The. main mechanism for eliminating potential auto- reactive T cell soccurs in the thymus and is known as central tolerance Some. potentially autoreactive T cell sescape central tolerance and, therefore, peripheral tolerance mechanisms also exist. Despite thes mechae nisms some, self-reactive T cell smay ' escape' and be present in the repertoire it is; believed that their activation may lead to autoimmune disease.

[0014] Studies on therapeutic tolerance have attempt edto induce and amplify potent physiologica mechanisl ms of tolerance in order to eliminat ore neutralize self-reactive T cell s and preven tor trea autt oimmune diseases. One way to induce tolerance is by manipulation of the interaction between costimulatory ligand sand receptor ons antigen presenting cells (APCs) and lymphocytes.

[0015] CTLA-4 is the most extensively studied costimulatory molecul whice hdown- regulates immune responses The. attributes of immunosuppressive qualities and capacity to induce tolerance have made its recognition as a potential immuno-therapeutic agent for autoimmune mediated inflammatory disorders. Abatacept (commercia name:l Orencia) is a fusion protei compn osed of the ECD (extracellular domain) of CTLA-4 fused to the Fc fragment of hlgGl Abat. acept is believed to induce costimulation blockade whic, h has been approved for treating patients wit hrheumatoi arthrid tibys, effectively interfering with the inflammatory cascade.

[0016] Induction of disease control wit hthe current therapies foll, owed by progress ive withdrawal in parallel with re-establishing immune tolerance, may be an attracti approachve in the futur eof autoimmune therapies Furthe. rmor duee, to thei imr mune specificity, in the absence of global immunosuppressi on,such therapies should be safe for chronic use.

[0017] Certain mechanism sare known to be widespread in various autoimmune diseases T. helper type 1 (Thl) cell sare induced by IL-12 and produce IFN-y; while T helpe typer 2 (Th2) cells secret IL-4,e IL-5 and IL-13. Thl cells can mediate proinflammatory or cell - mediated immune responses, wherea Th2s cells mainly promote certain types of humor al 5WO 2017/009712 PCT/IB2016/001079 immunity. Some immune relate diseased s, such as autoimmune reactions, inflammation, chronic infection and sepsis are, characterize byd a dysregulat ionof the pro- versus anti- inflammatory tendencies of the immune system as, well as an imbalance in the Thl versus Th2 cytokine balance. During inflammation, induction of a shift in the balance from Thl to Th2 protects the organism from systemi c'overshooti witng' hThl/pro-inflammator y cytokines, by reducing the inflammatory tendencies of the immune system.

Immunomodulatory therapi esthat are associated with a Thl to Th2 immune shift have protective effects in Thl-mediat edautoimmune diseases, such as multiple sclerosi ands rheumatoid arthriti Fors. example, Laquinimod, which has demonstrate efficacyd in animal models of several autoimmune diseases including MS, shows immunomodulat oryeffects through Thl/Th2 shift, and does not lead to immunosuppression. Glatirame acetater (Copaxone® )also induces Thl/Th2 shift with decreased secretion of proinflammatory cytokines, and increased secretion of antiinflammatory cytokines Furt. hermore, glatiram er acetate -specific Th2 cells are able to migrate across the blood-brain barrie andr cause in sit u bystander suppression of autoaggressive Thl T cells.

[0018] Certain immune cell sand immune cell signal transducti pathwayson are promising targets for new agents for treating immune disorders. For example Thl, Thl7, Th2 and regulatory T cell s(Tregs )play important role ins modulati ngautoimmunit andy inflammatio n.Mounting evidence from numerous studie showss the importance of these immune cell sin disorders such as rheumatoid arthriti infls, ammator bowely disease, multipl e sclerosis, psoriasi lupuss, erythematosus, type 1 diabetes and uveitis. Most existing therapi es target only one pathway at a time.

[0019] Accordingly, there is a need for additional agonist ands antagonist ofs immune checkpoint pathways.

BRIEF SUMMARY OF THE INVENTION

[0020] According to at leas tsome embodiment s,the present invention provides for a method of activating T cells of a patient comprising administering an anti-HIDEl antibody to said patient, wherein a subset of the T cells of said patient are activated. 6WO 2017/009712 PCT/IB2016/001079

[0021] According to at leas tsome embodiment s,the present invention provides for a method of activating cytotoxic T cells (CTLs) of a patient comprising administering an anti-HIDE 1 antibody to said patient, wherei an subset of the CTLs of said patient are activated.

[0022] According to at least some embodiment s,the present inventio nalso provides for a method of activating NK cell sof a patient comprising administering an anti-HIDE 1 antibody to said patient, wherei an subset of the NK cell sof said patient are activated.

[0023] According to at least some embodiment s,the present inventio nalso provides for a method of activating y5 T cell sof a patient comprising administering an anti-HIDEl antibody to said patient, wherei an subset of the y5 T cells of said patient are activated.

[0024] According to at least some embodiment s,the present inventio nalso provides for a method of activating Thl cells of a patient comprising administering an anti-HIDEl antibody to said patient, wherei an subset of the Thl cells of said patient are activated.

[0025] According to at least some embodiment s,the present inventio nalso provides for a method of decreasing or eliminating cell number and/or activity of at least one of regulatory T cell s(Tregs) in a patient comprising administering an anti-HIDEl antibody to said patient.

[0026] According to at least some embodiment s,the present inventio nalso provides for a method of increasing interferon-y production and/or pro-inflammatory cytokine secretion in a patient comprising administering an anti-HIDEl antibody to said patient.

[0027] According to at least some embodiment s,the present inventio nalso provides for a method of modulating myeloid cel lpolarizatio in na patient comprising administering an anti - HIDE1 antibody to said patient.

[0028] According to at least some embodiment s,the present inventio nalso provides for a method of modulating myeloid cel lshifting toward a pro-inflammatory response in a patient comprising administering an anti-HIDEl antibody to said patient.

[0029] According to at least some embodiments, the present invention also provide fors a method of shifting myeloid from M2 toward Ml phenotype in a patient comprising administering an anti-HIDEl antibody to said patient. 7WO 2017/009712 PCT/IB2016/001079

[0030] According to at least some embodiment s,the present inventio nalso provides for a method of modulating myeloid cel lin the TME to support anti-cancer immune respons ine a patient comprising administering an anti-HIDEl antibody to said patient.

[0031] According to at least some embodiment s,the present inventio nalso provides for a method of restricti theng pro-tumorigeni effectsc of the myeloid cell sin the tumor microenvironment in a patient comprising administering an anti-HIDEl antibody to said patient.

[0032] According to at least some embodiment s,the present inventio nalso provides for a method to elicit one or more of the followi effectsng on immunity in a patient by administering an anti-HIDEl antibody to said patient, wherei saidn effect is selected from the group consisting of: i) increases immune response, (ii) increases T cell activity, (iii) increases activation of a,|3 and/or y5 T cells, (iv) increases cytotoxic T cel lactivity, (v) increases NK and/or NKT cel lactivit y,(vi) alleviates a,|3 and/or y5 T-cel lsuppressio (viin, ) increases pro- inflammatory cytokine secretio (viiin, ) increases IL-2 secretio (ix)n; increases interferon-Y production, (x) increases Thl response, (xi) decrease Th2 response, (xii) decreases or eliminates cel lnumber and/or activity of at least one of regulatory T cell s(Tregs), myeloi d derived suppressor cell s(MDSCs), iMCs, mesenchyma lstromal cell s,TIE2-expressing monocyte s,(xiii) reduce sregulatory cell activity, and/or the activit yof one or more of myeloid derived suppressor cell s(MDSCs), iMCs, mesenchymal stromal cell s,TIE2- expressing monocytes, (xiv) decrease ors eliminate M2s macrophages, (xv) reduces M2 macrophage pro-tumorigenic activit y,(xvi) decreases or eliminates N2 neutrophils, (xvii) reduce sN2 neutrophil pro-tumoriges activitnic y,(xviii) reduce sinhibition of T cell activation (xix), reduces inhibition of CTL activation (xx), reduce sinhibition of NK and/or NKT cel lactivation (xxi), reverses a,|3 and/or y5 T cel lexhaustion, (xxii) increases a,[3 and/o r y5 T cel lresponse, (xxiii) increases activit yof cytotoxic cell s,(xxiv) stimulates antigen- specific memory responses, (xxv) elicits apoptos oris lysi sof cancer cells, (xxvi) stimulates cytotoxic or cytostat effectic on cancer cell s,(xxvii) induces direc killt ing of cancer cells , (xxviii) increases Thl 7 activity and (xxix) modulati ngmyeloid cell polarizati on,(xxx) modulating myeloid cel lshifting toward a pro-inflammatory response, (xxxi) shifting myeloid from M2 toward Ml phenotype, (xxxii) modulating myeloid cel lin the TME to 8WO 2017/009712 PCT/IB2016/001079 support anti-cancer immune response, (xxxiii) restricti theng pro-tumorigenic effects of the myeloid cell sin the TME, (xxxiv) enhancing myeloid and lymphoi infild trati intoon the tumor cite thereby shifting the tumor into more immunogenic, (xxxv) induces complement dependent cytotoxicit and/ory antibody dependent cell-mediated cytotoxicity.

[0033] According to at least some embodiment s,the present inventio nalso provides for a method of depleting myeloid cell sor othe circr ulati ngtumor cell sexpressing HIDE1 from a patient or patient sample ,said method comprising: i) contacting said patient or said patient sample with an anti-HIDEl antibody, wherein said anti-HIDEl antibody binds to HIDE1 expressing cell s,ii) identifying cells to which said anti-HIDEl antibody has bound, and iii) removing said cells in step ii) from said patient or said patient sample.

[0034] According to at least some embodiment s,the present inventio nalso provides for a method of treating cancer in a patient, comprising administering an anti-HIDEl antibody to said patient, wherei saidn cancer is treated. In some embodiments said patient has cancer. In some embodiments, the cancer is selected from the group consisting of Acute Myeloid Leukemia, Acute Myeloid Leukemia Induction Failure, Acute Lymphoblast ic Leukemia, Diffuse Large B—cell Lymphoma, Malignant Lymphoma, Non-Hodgkin Lymphoma, Diffuse Large B-Cel lLymphoma, Glioblastoma multiform Mesote, heliom a, Thymoma ,Testicula Germr Cel lTumors, Kidney renal clear cell carcinoma Sarcoma,, Brain Lower Grade Glioma, Chronic Lymphocytic Leukemia, Non-Hodgkin Lymphoma - Follicul Lymphomar a, Uterine Carcinosarcoma, Pediatri Brainc Tumors, Lung adenocarcinom Cervica, al squamous cel lcarcinoma, endocervical adenocarcinoma , Pancreatic adenocarcinoma Skin, Cutaneous Melanoma, Kidney renal papillary cell carcinoma, Liver hepatocellular carcinoma Bladder; Urotheli Carcial noma, Colo n adenocarcinom Heada, and Neck squamous cel lcarcinoma, Lung squamous cell carcinoma, Rectum adenocarcinoma, and Stomach adenocarcinoma In. some embodiment s,the cancer is a cancer having high immune infiltra ofte myeloid cells expressing HIDE1.

[0035] In some embodiments, the anti-HIDEl antibody is selected from the group consisting of CPA. 12.001 human IgG4, CPA. 12.002 human IgG4, CPA. 12.003 human IgG4, CPA. 12.004 human IgG4, CPA. 12.005 human IgG4, CPA. 12.006 human IgG4, CPA. 12.007 9WO 2017/009712 PCT/IB2016/001079 human IgG4, CPA. 12.008 human IgG4, CPA. 12.009 human IgG4, CPA. 12.011 human IgG4, CPA. 12.012 human IgG4, CPA. 12.013 human IgG4, CPA. 12.014 human IgG4, and CPA. 12.015 human IgG4. In some embodiment s,the anti-HIDEl antibody is selected from the group consisting of CPA.12.006-H4, CPA.12.007-H4, and CPA.12.0012-H4.

[0036] In some embodiments, the anti-HIDEl antibody is selected from the group consisting of CPA.12.001, CPA.12.002, CPA.12.003, CPA.12.004, CPA.12.005, CPA.12.006, CPA. 12.007, CPA. 12.008, CPA. 12.009, CPA. 12.011, CPA. 12.012, CPA. 12.013, CPA. 12.014, and CPA. 12.015. In some embodiments, the anti-HIDEl antibody is selected from the group consisting of CPA.12.006, CPA. 12.007, and CPA. 12.0012.

[0037] According to at least some embodiment s,the present inventio nalso provides for a method of diagnosing cancer comprising: a) contacting a tissue from a patient with an anti - HIDE1 antibody; and b) determining the presence of over-expression of HIDE1 in said tissue as an indication of the presenc eof cancer. In some embodiments, the tissue is a blood sample.

In some embodiments, the tissue is a biopsy of a soli tumor.d

[0038] In some embodiments of the diagnosti metc hod, the anti-HIDEl antibody is labeled.

In some embodiments, a second labeled antibody that binds to said anti-HIDEl antibody is contacted wit hsaid sample.

[0039] According to at least some embodiment s,the present inventio nalso provides for an anti-HIDEl antigen-binding domain comprising: a) a heavy chain variabl edomain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti-HIDEl antibod y;and b) a light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from said anti-HIDEl antibody; wherei saidn anti-HIDEl antibody is selected from the group consisting of CPA.12.001 human IgG4, CPA.12.002 human IgG4, CPA.12.003 human IgG4, CPA.12.004 human IgG4, CPA.12.005 human IgG4, CPA.12.006 human IgG4, CPA. 12.007 human IgG4, CPA. 12.008 human IgG4, CPA. 12.009 human IgG4, CPA. 12.011 human IgG4, CPA. 12.012 human IgG4, CPA.12.013 human IgG4, CPA.12.014 human IgG4, and CPA.12.015 human IgG4. 10WO 2017/009712 PCT/IB2016/001079

[0040] According to at least some embodiment s,the present inventio nalso provides for an anti-HIDEl antigen-binding domain comprising: a) a heavy chain variabl edomain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti-HIDEl antibod y;and b) a light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from said anti-HIDEl antibody; wherei saidn anti-HIDEl antibody is selected from the group consisting of CPA. 12.001, CPA. 12.002, CPA. 12.003, CPA. 12.004, CPA. 12.005, CPA. 12.006, CPA. 12.007, CPA. 12.008, CPA. 12.009, CPA. 12.011, CPA. 12.012, CPA. 12.013, CPA. 12.014, and CPA. 12.015.

[0041] In some embodiments, the anti-HIDEl antigen binding domain of the antibody is a singl echain Fv (scFv), wherei saidn heavy chain variable domain and said light chain variabl edomain are covalentl atty ached via a scFv linker.

[0042] According to at least some embodiment s,the present inventio nalso provides for an anti-HIDEl antibody comprising: a) a heavy chain variabl edomain comprising a vhCDRl , vhCDR2, and vhCDR3 from an anti-HIDEl antibod y;and b) a light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from said anti-HIDEl antibody; wherein said anti-HIDEl antibody is selected from the group consisting of CPA. 12.001 human IgG4, CPA. 12.002 human IgG4, CPA. 12.003 human IgG4, CPA. 12.004 human IgG4, CPA. 12.005 human IgG4, CPA. 12.006 human IgG4, CPA. 12.007 human IgG4, CPA. 12.008 human IgG4, CPA. 12.009 human IgG4, CPA. 12.011 human IgG4, CPA. 12.012 human IgG4, CPA. 12.013 human IgG4, CPA. 12.014 human IgG4, and CPA. 12.015 human IgG4.

[0043] According to at least some embodiment s,the present inventio nalso provides for an anti-HIDEl antibody comprising: a) a heavy chain variabl edomain comprising a vhCDRl , vhCDR2, and vhCDR3 from an anti-HIDEl antibod y;and b) a light chain variable domain comprising a vlCDRl, vlCDR2, and vlCDR3 from said anti-HIDEl antibody; wherein said anti-HIDEl antibody is selected from the group consisting of CPA. 12.001, CPA. 12.002, CPA. 12.003, CPA. 12.004, CPA. 12.005, CPA. 12.006, CPA. 12.007, CPA. 12.008, CPA. 12.009, CPA. 12.011, CPA. 12.012, CPA. 12.013, CPA. 12.014, and CPA. 12.015. 11WO 2017/009712 PCT/IB2016/001079

[0044] According to at least some embodiment s,the present inventio nalso provides for an anti-HIDEl antibody that competes for binding with an antibody comprising: a) a heavy chain variabl edomain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti-HIDEl antibody; and b) a light chain variable domain comprising a vlCDRl, a vlCDR2 and vlCDR3, from said anti-HIDEl antibody; wherei saidn anti-HIDEl antibody is selected from the group consisting of CPA. 12.001 human IgG4, CPA. 12.002 human IgG4, CPA. 12.003 human IgG4, CPA. 12.004 human IgG4, CPA. 12.005 human IgG4, CPA. 12.006 human IgG4, CPA. 12.007 human IgG4, CPA. 12.008 human IgG4, CPA. 12.009 human IgG4, CPA. 12.011 human IgG4, CPA. 12.012 human IgG4, CPA. 12.013 human IgG4, CPA. 12.014 human IgG4, and CPA. 12.015 human IgG4.

[0045] According to at least some embodiment s,the present inventio nalso provides for an anti-HIDEl antibody that competes for binding with an antibody comprising: a) a heavy chain variabl edomain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti-HIDEl antibody; and b) a light chain variable domain comprising a vlCDRl, a vlCDR2 and vlCDR3, from said anti-HIDEl antibody; wherei saidn anti-HIDEl antibody is selected from the group consisting of CPA. 12.001, CPA. 12.002, CPA. 12.003, CPA. 12.004, CPA. 12.005, CPA. 12.006, CPA. 12.007, CPA. 12.008, CPA. 12.009, CPA. 12.011, CPA. 12.012, CPA.12.013, CPA.12.014, and CPA.12.015.

[0046] According to at least some embodiment s,the present inventio nalso provides for an anti-HIDEl antibody that competes for binding with an antibody comprising: a) a heavy chain variabl edomain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti-HIDEl antibody; and b) a light chain variable domain comprising a vlCDRl, a vlCDR2 and vlCDR3, from said anti-HIDEl antibody; wherei saidn anti-HIDEl antibody is selected from the group consisting of AB-506, AB-507, AB-508, AB-509, and AB-510.

[0047] According to at leas tsome embodiment s,the present inventio nalso provides for an anti-HIDEl antibody that competes for binding with an antibody comprising: a) a heavy chain variabl edomain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti-HIDEl antibody; and b) a light chain variable domain comprising a vlCDRl, a vlCDR2 and vlCDR3, 12WO 2017/009712 PCT/IB2016/001079 from said anti-HIDE1 antibody; wherei saidn anti-HIDEl antibody is selected from the group consisting of 33B4, 36C1, and 39A7.

[0048] According to at least some embodiment s,the present inventio nalso provides for an anti-HIDEl antibody that competes for functional activity with an antibody comprising: a) a heavy chain variable domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti - HIDE1 antibod y;and b) a light chain variabl edomain comprising a vlCDRl, a vlCDR2 and vlCDR3, from said anti-HIDEl antibody; wherei sain d anti-HIDEl antibody is selected from the group consisting of CPA. 12.001 human IgG4, CPA. 12.002 human IgG4, CPA. 12.003 human IgG4, CPA. 12.004 human IgG4, CPA. 12.005 human IgG4, CPA. 12.006 human IgG4, CPA. 12.007 human IgG4, CPA. 12.008 human IgG4, CPA. 12.009 human IgG4, CPA. 12.011 human IgG4, CPA. 12.012 human IgG4, CPA. 12.013 human IgG4, CPA. 12.014 human IgG4, and CPA. 12.015 human IgG4.

[0049] According to at leas tsome embodiment s,the present inventio nalso provides for an anti-HIDEl antibody that competes for functional activity with an antibody comprising: a) a heavy chain variable domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti - HIDE1 antibod y;and b) a light chain variabl edomain comprising a vlCDRl, a vlCDR2 and vlCDR3, from said anti-HIDEl antibody; wherei sain d anti-HIDEl antibody is selected from the group consisting of CPA.12.001, CPA.12.002, CPA.12.003, CPA.12.004, CPA.12.005, CPA. 12.006, CPA. 12.007, CPA. 12.008, CPA. 12.009, CPA. 12.011, CPA. 12.012, CPA.12.013, CPA.12.014, and CPA.12.015.

[0050] According to at least some embodiment s,the present inventio nalso provides for an anti-HIDEl antibody that competes for functional activity with an antibody comprising: a) a heavy chain variable domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti - HIDE1 antibod y;and b) a light chain variabl edomain comprising a vlCDRl, a vlCDR2 and vlCDR3, from said anti-HIDEl antibody; wherei sain d anti-HIDEl antibody is selected from the group consisting of AB-506, AB-507, AB-508, AB-509, and AB-510.

[0051] According to at least some embodiment s,the present inventio nalso provides for an anti-HIDEl antibody that competes for functional activity with an antibody comprising: a) a 13WO 2017/009712 PCT/IB2016/001079 heavy chain variable domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti - HIDE1 antibod y;and b) a light chain variabl edomain comprising a vlCDRl, a vlCDR2 and vlCDR3, from said anti-HIDEl antibody; wherei sain d anti-HIDEl antibody is selected from the group consisting of 33B4, 36C1, and 39A7.

[0052] In some embodiments, in the composition comprising an anti-HIDEl antibody comprise an antibody selected from the group consisting of CPA. 12.001 human IgG4, CPA. 12.002 human IgG4, CPA. 12.003 human IgG4, CPA. 12.004 human IgG4, CPA. 12.005 human IgG4, CPA. 12.006 human IgG4, CPA. 12.007 human IgG4, CPA. 12.008 human IgG4, CPA. 12.009 human IgG4, CPA. 12.011 human IgG4, CPA. 12.012 human IgG4, CPA. 12.013 human IgG4, CPA. 12.014 human IgG4, and CPA. 12.015 human IgG4. In some embodiment s,in the composition comprising an anti-HIDEl antibody, the antibody is selected from the group consisting CPA.12.006-H4, CPA.12.007-H4, and CPA.12.0012-H4.

[0053] In some embodiments, in the composition comprising an anti-HIDEl antibody comprise an antibody selected from the group consisting of CPA. 12.001, CPA. 12.002, CPA. 12.003, CPA. 12.004, CPA. 12.005, CPA. 12.006, CPA. 12.007, CPA. 12.008, CPA. 12.009, CPA. 12.011, CPA. 12.012, CPA. 12.013, CPA. 12.014, and CPA. 12.015. In some embodiments, in the compositi oncomprising an anti-HIDEl antibody, the antibody is selected from the group consisting CPA.12.006-H4, CPA.12.007-H4, and CPA.12.0012-H4.

[0054] According to at least some embodiment s,the present inventio nalso provides for a nucleic acid compositi oncomprising: a) a firs nucleit c acid encoding a heavy chain variabl e domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti-HIDEl antibody; and b) a second nucleic acid encoding a light chain variabl domaine comprising a vlCDRl, vlCDR2 , and vlCDR3 from said anti-HIDEl antibody; wherein said anti-HIDEl antibody is selected from the group consisting of CPA. 12.001 human IgG4, CPA. 12.002 human IgG4, CPA. 12.003 human IgG4, CPA. 12.004 human IgG4, CPA. 12.005 human IgG4, CPA. 12.006 human IgG4, CPA. 12.007 human IgG4, CPA. 12.008 human IgG4, CPA. 12.009 human IgG4, CPA. 12.011 human IgG4, CPA. 12.012 human IgG4, CPA. 12.013 human IgG4, CPA. 12.014 human IgG4, and CPA. 12.015 human IgG4. 14WO 2017/009712 PCT/IB2016/001079

[0055] According to at least some embodiment s,the present inventio nalso provides for a nucleic acid compositi oncomprising: a) a firs nucleit c acid encoding a heavy chain variabl e domain comprising a vhCDRl, vhCDR2, and vhCDR3 from an anti-HIDEl antibody; and b) a second nucleic acid encoding a light chain variabl domaine comprising a vlCDRl, vlCDR2 , and vlCDR3 from said anti-HIDEl antibody; wherein said anti-HIDEl antibody is selected from the group consisting of CPA.12.001, CPA.12.002, CPA.12.003, CPA.12.004, CPA. 12.005, CPA. 12.006, CPA. 12.007, CPA. 12.008, CPA. 12.009, CPA. 12.011, CPA.12.012, CPA.12.013, CPA.12.014, and CPA.12.015.

[0056] According to at least some embodiment s,the present inventio nalso provides for an expression vector compositi oncomprising: a) a firs expresst ion vector comprising a first nucleic acid as describe dabove and herein and; b) a second expression vector comprising said second nucleic as described above and herein. In some embodiment s,the expression vector compositi oncompris esan expression vector comprising the firs nuclet ic acid of as described above and herei nand the second nucleic acid of as described above and herein.

[0057] In some embodiments, the inventio nprovides a host cel lcomprising the expression vector compositi onas described above and herein.

[0058] According to at least some embodiment s,the present inventio nalso provides for a method of making an anti-HIDEl antibody comprising: a) culturin theg host cell as describe d above and herein under conditions wherein said antibody is expressed; and b) recovering said antibody.

[0059] According to at least some embodiment s,the present inventio nalso provides for a method of activating cytotoxi Tc cells (CTLs) of a patient comprising administering an anti - HIDE1 antibody to said patient, wherein a subset of the CTLs of said patient are activated. In some embodiments, the antibody is optional anly antibody as described above and herein.

[0060] According to at least some embodiment s,the present inventio nalso provides for a method of activating NK cell sof a patient comprising administering an anti-HIDEl antibody to said patient, wherei an subset of the NK cell sof said patient are activated. In some embodiment s,the antibody is optionall any antibody as described above and herein. 15WO 2017/009712 PCT/IB2016/001079

[0061] According to at least some embodiment s,the present inventio nalso provides for a method of activating y5 T cell sof a patient comprising administering an anti-HIDEl antibody to said patient, wherei an subset of the y5 T cells of said patient are activated. In some embodiment s,the antibody is optionall any antibody as described above and herein.

[0062] According to at least some embodiment s,the present inventio nalso provides for a method of activating Thl cells of a patient comprising administering an anti-HIDEl antibody to said patient, wherei an subset of the Thl cell sof said patient are activated. In some embodiment s,the antibody is optionall any antibody as described above and herein.

[0063] According to at least some embodiment s,the present inventio nalso provides for a method of decreasing or eliminating cell number and/or activity of at least one of regulatory T cell s(Tregs) in a patient comprising administering an anti-HIDEl antibody to said patient.

In some embodiments, the antibody is optionall any antibody as described above and herein.

[0064] According to at least some embodiment s,the present inventio nalso provides for a method of increasing interferon-y production and/or pro-inflammatory cytokine secretion in a patient comprising administering an anti-HIDEl antibody to said patient In. some embodiment s,the antibody is optionall any antibody as described above and herein.

[0065] According to at least some embodiment s,the present inventio nalso provides for a method of activating monocytes of a patient comprising administeri ngan anti-HIDEl antibody to said patient, wherei an subset of the monocyte cell sof said patient are activated.

In some embodiments, the antibody is optionall any antibody as described above and herein.

[0066] According to at least some embodiment s,the present inventio nalso provides for a method of treating cancer in a patient comprising administering an anti-HIDEl antibody to said patient In. some embodiments, the antibody is optionall any antibody as described above and herein.

[0067] In some embodiments, the treatment is an increase in immune response. In some embodiment s,the treatment is an increase in activation of a,|3 and/or y5 T cells. In some embodiment s,the treatment is an increase in cytotoxic T cel lactivity. In some embodiment s, 16WO 2017/009712 PCT/IB2016/001079 the treatment is an increase in natural kill er(NK) and/or NKT cel lactivit y.In some embodiment s,the treatment is an increase in aP and/or y5 T-cel lactivity. In some embodiment s,the treatment is an increase in pro-inflammatory cytokine secretion In. some embodiment s,the treatment is increase in IL-2 secretion In. some embodiments, the treatme nt is an increase in interferon-Y production. In some embodiment s,the treatment is an increase in Thl response. In some embodiments, the treatment is a decrease in the cell number and/or activity of regulatory T cells. In some embodiments, the treatment decreases cel lnumber and/or activit yof at leas tone or more cells selected from the group consisting of regulatory T cells (Tregs), myeloi dderived suppressor cell s(MDSCs), iMCs, mesenchyma lstromal cells , and TIE2-expressing monocytes. In some embodiment s,the treatment is decreases the cell activit y,and/or the activit yof one or more cells selected from the group consisting of myeloid derived suppressor cell s(MDSCs), iMCs, mesenchymal stromal cell s,and TIE2- expressing monocytes. In some embodiments, the said treatment is a decrease in M2 macrophages. In some embodiment s,the treatment is a decrease in M2 macrophage activit y.

In some embodiments, the treatme ntis a decrease in N2 neutrophil Ins. some embodiment s, the treatment is a decrease in N2 neutrophil actsivity In. some embodiments, the treatment is a decrease in inhibition of T cel lactivation. In some embodiments, the treatment is a decrease in inhibition of CTL activation. In some embodiments, the treatment is a decrease in inhibition of NK cel lactivation. In some embodiment s,the treatment is a decrease in «P and/or y5 T cel lexhaustion. In some embodiment s,the treatment is an increase in «P and/o r y5 T cel lresponse. In some embodiment s,the treatment is an increase in activit yof cytotoxic cells. In some embodiments, the treatment is an inductio ofn antigen-specifi cmemory responses In. some embodiments, the treatment induces apoptosis or lysi sof cells. In some embodiment s,the treatment is an increase in cytotoxic or cytostatic effect on cells. In some embodiment s,the treatment induces direc killt ing of cells. In some embodiments, the treatment is an increase in Thl7 activity. In some embodiments, the treatment induces complement dependent cytotoxicit and/y or antibody dependent cell-mediate cytotd oxicity.

[0068] According to at least some embodiment s,the present inventio nalso provides for a method of treating an immune disorde comprisr, ing administering to a patient a compositi on comprising an enhancer of HIDE1 associated immune suppressio ton, effect treatment. In 17WO 2017/009712 PCT/IB2016/001079 some embodiments, the treatment is a decrease in immune response In. some embodiment s, the treatment is a decrease in activation of aP and/or y5 T cells In. some embodiments, the treatment is a decrease in cytotoxic T cel lactivity In. some embodiments, the treatment is a decrease in NK and/or NKT cell activit y.In some embodiment s,the treatment is a decrease of aP and/or y5 T-cel lactivit y.In some embodiment s,the treatment is a decrease in pro- inflammatory cytokine secretion In. some embodiments, the treatment is a decrease in IL-2 secretion. In some embodiments, the treatment is a decrease in interferon-y production. In some embodiments, the treatment is a decrease in Thl response. In some embodiment s,the treatment is a decrease in Th2 response In. some embodiment s,the treatment is an increase in inhibition of T cel lactivit y.In some embodiments, the treatment is an increase in inhibition of CTL activity. In some embodiments, the treatment is an increase in inhibition of NK cell activity In. some embodiment s,the treatment is an increase in «P and/or y5 T cel lexhaustion.

In some embodiments, the treatme ntis a decrease in «P and/or y5 T cel lresponse. In some embodiment s,the treatment is a decrease in activit yof cytotoxic cells. In some embodiment s, the treatment is a reduction in antigen-specific memory responses In. some embodiments, the treatment is an inhibition of apoptosis or lysi sof cells. In some embodiment s,the treatment is a decrease in cytotoxic or cytostat effeic ct on cells. In some embodiments, the treatment is a reduction in direc killt ing of cells. In some embodiments, the treatment is a decrease in Thl 7 activity In. some embodiment s,the treatment is a reduction of complement dependent cytotoxicit and/ory antibody dependent cell-mediated cytotoxicity.

[0069] According to at least some embodiment s,the present inventio nalso provides a method to elicit one or more of the followi effectsng on immunity in a patient by administering a HIDE1 peptide to said patient, wherei sain d effect is selected from the group consisting of: i) decrease ims mune response, (ii) decreases «P and/or y5 T cel lactivation, (iii) decreases T cel lactivity, (iv) decrease cytotoxics T cel lactivity, (v) decrease naturs al killer (NK) and/or NKT cel lactivity, (vi) decreases «P and/or y5 T-cel lactivit y,(vii) decreases pro- inflammatory cytokine secretio (viiin, ) decreases IL-2 secretion; (ix) decrease interferon-ys production, (x) decreases Thl response, (xi) decrease Th2s response, (xii) increases cell number and/or activity of regulatory T cell s,(xiii) increases regulatory cel lactivit yand/or one or more of myeloid derived suppressor cell s(MDSCs), iMCs, mesenchyma lstromal 18WO 2017/009712 PCT/IB2016/001079 cell s,TIE2-expressing monocyte s,(xiv) increases regulatory cel lactivit yand/or the activit y of one or more of myeloid derive dsuppressor cell s(MDSCs), iMCs, mesenchymal stromal cell s,TIE2-expressing monocyte s,(xv) increases M2 macrophages, (xvi) increases M2 macrophage activit y,(xvii) increases N2 neutrophil (xviiis, ) increases N2 neutrophil s activit y,(xix) increases inhibition of T cel lactivation (xx), increases inhibition of CTL activation (xxi), increases inhibition of NK cel lactivation, (xxii) increases and/or y5 T cell exhaustion, (xxiii) decrease saP and/or y5 T cel lresponse, (xxiv) decreases activity of cytotoxic cell s,(xxv) reduce santigen-specific memory responses, (xxvi) inhibits apoptosis or lysi sof cell s,(xxvii) decrease cytots oxic or cytostat effectic on cell s,(xxviii) reduce sdirec t killing of cells (xxix), decrease Thl7s activit y,(xxx) modulat esmyeloid cel lpolarizati on, and/or modulates myeloid cel lshifting toward an anti-inflammatory response, (xxxi) reduces complement dependent cytotoxicit and/y or antibody dependent cell-mediate cytotd oxicity.

[0070] According to at least some embodiment s,the present inventio nalso provides for a method of treating an immune disorde comprisr, ing administering to a patient a compositi on comprising an enhancer of HIDE 1 associated immune suppression to effect treatment In . some embodiments, the enhancer is aHIDEl peptide. In some embodiments, the HIDE1 peptide is a HIDE1 ECD. In some embodiments, the HIDE1 peptide is a HIDE1 polypeptide consisting of aHIDEl polypeptide ECD domain having at least 95% identity to the ECD domain of an amino acid sequence selected from the group consisting of the sequences depicted in Figure 66.

[0071] According to at least some embodiment s,the present inventio nalso provides for a method of activating cytotoxi Tc cells (CTLs) of a patient comprising administering the HIDE1 peptide as describe aboved and herein to said patient, wherein a subset of the CTLs of said patient are inhibited.

[0072] According to at least some embodiment s,the present inventio nalso provides for a method of activating NK cell sof a patient comprising administering the HIDE1 peptide as described above and herei nto said patient, wherein a subset of the NK cell sof said patient are inhibited. 19WO 2017/009712 PCT/IB2016/001079

[0073] According to at least some embodiment s,the present inventio nalso provides for a method of activating y5 T cell sof a patient comprising administering the HIDE1 peptide as described above and herei nto said patient, wherei an subset of the y5 T cells of said patient are inhibited.

[0074] According to at least some embodiment s,the present inventio nalso provides for a method of activating Thl cells of a patient comprising administering the HIDE1 peptide as described above and herei nto said patient, wherein a subset of the Thl cell sof said patient are inhibited.

[0075] According to at leas tsome embodiments of the present inventio nalso provides for a method of increasing cel lnumber and/or activit yof at least one of regulatory T cells (Tregs ) in a patient comprising administering the HIDE1 peptide as describe dabove and herein to said patient.

[0076] According to at least some embodiment s,the present inventio nalso provides for a method of decreasing interferon-y production and/or pro-inflammatory cytokine secretion in a patient comprising administering the HIDE1 peptide as described above and herein to said patient.

[0077] According to at least some embodiment s,the present inventio nalso provides for a method of treating an autoimmune disease in a patient comprising administering the HIDE1 peptide as described above and herei nto said patient. In some embodiments, the patient has an immune disord er.In some embodiments, the immune disord iser selected from the group consisting of an autoimmune disease, organ transplant rejectio andn inflammation. . In some embodiment s,the autoimmune disease is selected from the group consisting of rheumatoi d arthri tilupus,s, Inflammator bowey ldisease, psoriasi mults, iple sclerosi ands diabetes type I.

[0078] In some embodiments, the enhancer of HIDE 1 is selected from the group consisting of a prote inand a nucleic acid. In some embodiments, the prote incompris esan extracellul ar domain (ECD) of HIDE1. In some embodiment s,the prote inis a fusion protei comprin sing said ECD and a fusion partner. . In some embodiments, the fusion partner is selected from the group consisting of a human IgG Fc domain and a human serum albumin (HSA). 20WO 2017/009712 PCT/IB2016/001079

[0079] According to at least some embodiment s,the present inventio nalso provides for a compositi oncomprising an isolated HIDE1 polypepti deconsisting of aHIDEl polypeptide ECD domain having at least 95% identity to the ECD domain of an amino acid sequence selected from the group consisting of the sequences depicted in Figure 66. In some embodiments of the composition, the isolated HIDE1 polypeptide has at least 99% identit toy an amino acid sequence selected from the group consisting of the sequences depicted in Figure 66. In some embodiments of the composition the, isolated HIDE1 polypeptide is selected from the group consisting of the sequences depicted in Figure 66.

[0080] According to at least some embodiment s,the present inventio nalso provides for a compositi oncomprising aHIDEl fusion polypeptide comprising: a) an ECD from aHIDEl polypeptide; and b) a covalentl atty ached fusion partner moiety. In some embodiments of the composition, the fusion partner moiety is selected from the group consisting of a human IgG Fc domain, a human serum albumin (HSA) and a polyethylene glycol (PEG). In some embodiments of the composition, the ECD has an amino acid sequence selected from the group consisting of the sequences depicted in Figure 66. In some embodiments of the composition the, HIDE1 polypeptide and said fusion partner moiety are direct lycovalentl y attached. In some embodiments of the composition, the fusion partne moir ety is a polyethylene glycol (PEG) moiety. In some embodiments of the composition the, HIDE1 polypeptide and said fusion partner moiety are covalent lyattached using an exogenous linker.

In some embodiments of the composition, the exogenous linke isr selected from the group consisting of those depicted in Figure 66. In some embodiments of the composition the, exogenous linke hasr the formul (GGGSa )n, wherei nn is from 1 to 5. In some embodiments of the composition, the fusion partner moiety is a human serum albumin (HSA). In some embodiments of the composition the, fusion partner moiety is an Fc domain. In some embodiments of the composition, the Fc domain is a human IgG Fc domain. In some embodiments of the composition, the human IgG Fc domain is selected from the group consisting of the Fc domain of human IgGl ,the Fc domain of human IgG2, the Fc domain of human IgG3, and the Fc domain of human IgG4. In some embodiments of the composition, the Fc domain is a variant human Fc domain from IgGl or IgG2. 21WO 2017/009712 PCT/IB2016/001079

[0081] In some embodiments of the composition, the compositi oncomprises a pharmaceutical acceptly able carrier.

[0082] According to at least some embodiment s,the present inventio nalso provides for a method of suppressing T cel lactivation of a patient comprising administeri nga composition as described above and herei nto said patient such that said patien’ts immune response is suppressed as a resul oft treatment. In some embodiments, the patient has an immune disord er.In some embodiment s,the immune disorder is selected from the group consisting of an autoimmune disease, and organ transplant rejection. In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthri tilupus,s, Inflammatory bowel disease, psoriasis mult, iple sclerosis, and Diabetes type I.

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1: Schematic presentati ofon elevation of endogenous expression of the immune checkpoi ntligand (PDL-1) by induction of anti-tumor immunity.

[0084] Figure 2: Shows inductio ofn HIDE 1 expression in DSS model of IBD.

[0085] Figure 3: Correlati ofon HIDE1 to CSF1R in human colorecta cancerl (TCGA data).

[0086] Figure 4: Shows inductio ofn HIDE1 expression in DSS + AOM model of IBD.

[0087] Figure 5: Correlati ofon HIDE1 to CD86 and CD68 in human colorecta cancerl (TCGA data).

[0088] Figure 6: Presents correlati ofHIon DEl and CDllb in multipl autoime mune patien’ts derived samples.

[0089] Figure 7: Expression ofHIDEl in both monocytic and granulocytic MDSCs derived from mouse tumor model Mean. expression values for each group are shown as gray horizonta linesl .

[0090] Figure 8: Presents cancers wit ha strong myeloid infiltration, based on the CSF1R expression profile.

[0091] 22WO 2017/009712 PCT/IB2016/001079

[0092] Figure 9: HIDE1 expression in normal tissues (GTEx data).

[0093] Figure 10: HIDE1 expression pattern in BioGPS.

[0094] Figure 11: HIDE1 expression in cancer.

[0095] Figure 12: HIDE1 is upregulated in several tumor types. Examples from ovary cancer, melanoma and kidne ycancers are shown.

[0096] Figure 13: FACS analysis of ectopical lyexpressed HEK293 cells expressing human HIDE1 Flag pMSCV vector HEK293 cells expressing the human HIDE1 Flag were analyzed by FACS using rabbit anti Human HIDE1 (GenScript light, blue line). Rabbit IgG (Jackson, pink line) was used as isotype control Det. ection was carrie dout using donkey anti-rabbit PE- conjugated secondary antibody and analysi sby FACS.

[0097] Figure 14: FACS analysis of ectopical lyexpressed HEK293 cells expressing human HIDE1 Flag pMSCV vector using anti human HIDE1 mAbs. HEK293 cells expressing the human HIDE1 Flag or HEK293 pMSCV empty vector were analyzed by FACS using mouse anti Human HIDE1 mAbs (Biotem A-C,, green line or orang eline respectively) or using mouse IgG isotype control (light blue or pink line respectivel y).Detection was carried out using goat anti mouse-PE-conjugated secondary Ab.

[0098] Figure 15: FACS analysis of ectopical lyexpressed SK-MEL-5 cell sexpressing human HIDE1 Flag pMSCV vector SK-MEL-5 cell sexpressing the human HIDE1 Flag were analyzed by FACS using Rabbit anti Human HIDE1 (GenScript light, blue line). Rabbit IgG (Jackson, pink line) was used as isotype control Dete. ction was carried out using donkey anti - rabbit PE-conjugated secondary antibody and analysis by FACS .

[0099] Figure 16: Top 10 most enriched interactions, pathway ands diseases for genes highly correla tedwith HIDE1 in a variet yof cancers .Gradient black to gray scale reflect -logls O(p- values). Rows are ranked order by the sum of each row.

[00100] Figure 17: FACS analysi sof ectopically expressed HEK293 cells expressing mouse HIDE1 Flag pMSCV vector. HEK293 cell sover expressing the mouse HIDE1 Flag or HEK293 cells transduced wit han empty vector were analyzed by FACS using Rabbit polyclonal anti mouse HIDE1 (GenScript #488536 13). Rabbit IgG (Jackson) was used as an 23WO 2017/009712 PCT/IB2016/001079 isotype control Dete. ction was carried out using Donkey Anti-Rabbi tPE-conjugated secondary Ab.

[00101] Figure 18: FACS analysi sof EL4 cells ectopicall expressy ing mouse HIDE1 Flag pMSCV vector. EL4 cell sover expressing the mouse HIDE1 Flag or EL4 cells transduce withd an empty vector were analyzed by FACS using Rabbit (Rb) polyclonal anti mouse HIDE1 (GenScript #488536 13). Rabbit IgG (Jackson) was used as an isotype control Detect. ion was carrie dout using Donkey Anti-Rabbi tPE-conjugated secondary Ab.

[00102] Figure 19: WB analysis using anti Human or mouse HIDE1 pAbs on HEK293 cells expressing human or mouse HIDE1 protein. Whole cell sextract ofs HEK293 cell s expressing the human HIDE1 flag (lane 2), HEK293 cell sexpressing the mouse HIDE1 flag (lane 3) or HEK293 transfected with an empty vector (lanes 1) were analyzed using anti Flag antibody (A), a commercial antibody anti human HIDE1 (Sigma, D), pAb anti human-HIDEl (GenScript B), or with pAb anti mouse-HIDEl (GenScript C)., Detection was carried out using goat anti rabbit-HRP (except anti flag which is already conjugated to HRP).

[00103] Figure 20: present WBs analysis of ectopical lyexpressed human HIDE1 Flag pCDNA3.1 vector. Whole cell extract 0fHEK293s cel lpools, transfect edwit hexpression construct encoding human HIDEl-flag (lane 2) or wit hempty vector (lane 1) were analyzed by WB using an anti-flag antibody (left panel) or anti-HIDEl antibodie (rights panel).

[00104] Figure 21: FACS analysi susing anti Human HIDE1 pAbs on HEK293 or SKMEL5 cells expressing Human HIDE1 protein. HEK293 cells expressing the human HIDE1 Flag (A) or SKMEL-5 expressing the human HIDE1 Flag (B) were analyzed by FACS using Rabbit anti Human HIDE1 (GenScript, light blue line). Rabbit IgG (Jackson, pink line) was used as isotype control Det. ection was carrie dout using donkey anti-rabbit PE- conjugated secondary antibody and analysi sby FACS.

[00105] Figure 22: WB analysi susing anti Human HIDE1 mAbs on HEK293 cells expressing human or mouse HIDE1 protein. Whole cells extract ofs HEK293 cell sexpressing the human HIDE1 flag (lanes 2), HEK293 cell sexpressing the mouse HIDE1 flag (lanes 3) or HEK293 transfect edwit han empty vector (lanes 1) were analyzed by WB using mAbs anti 24WO 2017/009712 PCT/IB2016/001079 human HIDE1 (Biotem): 33B4-2F7(A), 36C1-2F6 (B) or 39A7-3A10-3G8 (C). Detection was carried out using Goat Anti Mouse-HRP (Anti flag is conjugated to HRP).

[00106] Figure 23A & 23B: FACS analysi susing anti human HIDE1 Fab’s on HEK293 cells over-expressing human HIDE1 Flag protein. (A)HEK293 cells over- expressing the human HIDE1 Flag (blue line) or HEK293 cell sover-expressing the mouse HIDE1 Flag (orange line) or HEK293 transduce witd hempty vector (red line) were analyzed by FACS using Serotec anti-Human HIDE1 Fab’s (1-16). Detection was carried out using Goat Ant iHuman IgG F(ab')2-PE secondary Ab. (B) Summary table of Serotec anti-human HIDE1 Fab’s 1-16 FACS analysis, the Geo mean rati oof HEK293 cell sover-expressi theng human HIDE1 Flag/ HEK293 transduce withd empty vector. And the cross reactive validation, the Geo-mean ratio of HEK293 cell sover-expressing the mouse HIDE1 Flag/ HEK293 transduce withd empty vector.

[00107] Figure 24A & 24B: FACS analysi susing anti-mous HIDe E1 Fab’s on HEK293 cells over-expressing mouse HIDE1 Flag protein. (A)HEK293 cells over-expressi ng the mouse HIDE1 Flag (orange line) or HEK293 cell sover-expressing the human HIDE1 Flag (blue line) or HEK293 transduce witd hempty vector (red line) were analyzed by FACS using Serotec anti-mouse HIDE1 Fab’s (1-13). Detection was carried out using Goat Ant i Human IgG F(ab')2-PE secondary Ab. (B) Summary table of Serotec anti-mouse HIDE1 Fab’s 1-13 FACS analysis, the Geo mean ratio of HEK293 cells over-expressin theg mouse HIDE1 Flag/ HEK293 transduce withd empty vector. And the cross reactive validation, the Geo mean rati oof HEK293 cell sover-expressing the human HIDE1 Flag/ HEK293 transduce withd empty vector.

[00108] Figure 25: Schematic representation of the exon structure of Human (A) and Mouse (B) HIDE1 in the mRNA transcript (Introns are not represent ined a real proportion).

Primers that were used for the qRT-PCR are represented as a red arrows. TaqMan probes are indicated by different colors.

[00109] Figure 26: Transcript expression of human HIDE1 in various Human cancer cel llines. Verification of the human transcript in several cell lines was performe byd qRT- PCR using TaqMan probes. Colum ndiagram represents data observed using TaqMan probe 25WO 2017/009712 PCT/IB2016/001079 Hs01128131_ml. Ct values are detailed in the table. Analysis indicating high transcript in HL-60 KG-1 and U937 And lower levels in THP1, A704 and NCI-H28 cell lines.

[00110] Figure 27: Membrane expression of human HIDE1 prote inin various human cancer cel llines. Human cel llines were staine dwith monoclonal anti-HIDEl Abs BIOTEM 33B4-2F7 (Orange, upper panel) or 36C1-2F6 (Orange, lower panel) or with IgGl isotype control antibody (light blue). Followi celng lwashing, PE-Goat anti-mouse secondar y conjugated Ab was added. Cell thats were stained wit hthe secondary antibody only are represent byed a red color. Binding was evaluated by FACS. Histograms represent living cells gated using viability stai n450 (BD Bioscience).

[00111] Figure 28: Membrane expression of human HIDE1 prote inin U937 human cancer cel lline. U937 cel lline was stained with various anti-human HIDE1 F(ab’)2, Serotec.

Only 3 F(ab’)2 showed specific membrane staining of U937 cell line as shown by the black arrow ins the Figure: Ab3295 (A. Purple line,), AbD333 (B. Olive line) and with Ab332 (B.

Green line). Followi celng lwashing, PE-Goat anti-F(ab’)2 secondary conjugated Ab was added. Cells that were stained wit hthe secondary antibody only are represent byed a red color. Binding was evaluated by FACS. Histogram represes ntliving cells gated using viability stain 450 (BD Bioscience).

[00112] Figure 29: qRT-PCR analysi sof human HIDE1 transcr iptin HL-60 and U937 cel llines transfect edwit hHIDE1 siRNA. HL-60 (A) or U973 (B) human cancer cell lines, untreated, transfect edwith human HIDE1 siRNA or wit hscramble siRNd A were analyzed by qRT-PCR using human HIDE1 TaqMan probe # HsOl 128131ml. Ct values are detailed in the table. Standard deviation of technical triplicates of the PCR reaction are indicated.

[00113] Figure 30: Membrane expression of human HIDE1 prote inin HL-60 and U937 human cel lline stransfected with human HIDE1 siRNA. HL-60 or U937 Cell s transfect edwit hHuman HIDE1 siRNA were stained wit hmonoclonal anti-HIDEl Abs BIOTEM, 33B4-2F7 (green) or wit hIgGl isotype control antibody (blue). Cell stransfect ed with Scrambled siRNA were stained with the same anti-HIDEl (orange) or isotype control (red). Followi celng lwashing, PE-Goat anti-mous secondarye conjugated Ab was added. 26WO 2017/009712 PCT/IB2016/001079

[00114] Figure 31: Membrane expression of human HIDE1 prote inin THP1 human cel lline transfected with human HIDE1 siRNA. THP1 cell stransfect edwit hHuman HIDE1 siRNA were stained wit hmonoclonal anti-HIDE 1 Abs BIOTEM (conjugated to AF647), Ab- 263 (green) or wit hIgGl isotype control-AF647 antibody (blue). Cell stransfect edwit h Scrambled siRNA were stained wit hthe same anti-HIDEl (orange) or isotype control (red).

[00115] Figure 32: Membrane expression of human HIDE1 prote inin U937 human cel lline transfected with human HIDE1 siRNA. U937 cells transfect edwith Human HIDE1 siRNA were stained with 3 different anti-HIDEl F(ab’)2, Serotec (A. Ab329, B. Ab332 and C. Ab333- green line), or wit hnon-relevant F(ab’)2, Ab 307 (blue). Cells transfect edwit h Scrambled siRNA were stained wit hthe same anti-HIDEl (orange) or isotype control (red).

[00116] Figure 33: Transcript expression of mouse HIDE1 in various mouse cel llines.

Verification of the mouse transcriinpt several cell line swas performe byd qRT-PCR using specific primers (A) or TaqMan probe (B). Ct values are indicated in the tables Analys. is indicating relatively high transcript level in J774A.1 EL4, YAC-1, A20 as wel las in RAW264.7, and P388D1 cel llines

[00117] Figure 34: Membrane expression of mouse HIDE1 prote inin various mouse cel llines. Mouse cel llines were staine dwith rabbit anti-HIDEl pAb (Genescript, ID 488536 13) (Orange) or wit hrabbit IgG isotype control (light blue). Followi celng lwashing, PE-donke yanti-rabbit secondary conjugated Ab was added. Cell sthat were stained wit hthe secondary antibody only are represent byed a red color. Binding was evaluated by FACS.

Histogram represes ntliving cells gated using viability stain 450 (BD Bioscience).

[00118] Figure 35: Membrane expression of mouse HIDE1 prote inin EL4 and J774A.2 mouse cancer cel llines. EL4 cel lline was staine dwith various anti-mouse HIDE1 F(ab’)2, Serotec. Only 3 F(ab’)2 showed specific membrane staining of EL4 cell line (indicate dwith the black arrow ins the figure): Ab345 (A. Purple line), Ab360 and Ab359 (B. yellow and blue lines respectivel y),whereas only 2 F(ab’)2 showed specific staining of J774A.2 cel lline (indicated by the black arrow ins the figure): Ab360 and Ab359 (C. yellow and blue lines respectivel only.y) Followi celng lwashing, PE-Goat anti-F(ab’)2 secondary conjugated Ab was added. Cell thats were stained wit hthe secondary antibody only are 27WO 2017/009712 PCT/IB2016/001079 represent byed a red color. Binding was evaluated by FACS. Histograms represent living cells gated using viability stai n450 (BD Bioscience).

[00119] Figure 36: qRT-PCR analysis of mouse HIDE1 transcript in EL4 mouse cell line EL4 cells: untreated (lef tcolumn), transfect edwith mouse HIDE1 siRNA (right column) or with scramble siRNd A (middl column)e were analyzed by qRT-PCR using mouse HIDE1 TaqMan custom probe # HOJEX12_CCAAZKY. Ct values are indicated in the table.

Standard deviation of technical triplicate ofs the PCR reaction are indicate dare indicated.

[00120] Figure 37: Membrane expression of mouse HIDE1 prote inin EL4 mouse cell line transfect edwith mouse HIDE1 siRNA. EL4 mouse cel lline transfect edwith mouse HIDE1 siRNA was stained (48 hours post transfection) with rabbit anti-HIDEl pAbs (Genscript, ID. 488536 13, green) or with Rabbit IgG isotype control antibody (blue). Cells transfect edwit hScrambled siRNA were stained wit hthe same anti-HIDEl (orange) or isotype control (red). Follow ingcell washing, PE-Donkey anti-Rabbit secondary conjugated Ab was added.

[00121] Figure 38: HIDE1 Expression profil one fresh PBMCs. Freshly thawe d PBMCs from 2 donors were thawed and stained with viability dye, washed and pre-blocked with Fc blocking soluti onfollowed by surfac estaining with surfac emarkers in the presence of 2 anti-hHIDEl antibodie ors isotype control as describe ind materia lsand methods. HIDE1 surfac eexpression level swere analyzed by flow cytometry. (A) Gating strategy for flow cytometry analysis gating on CD14+ and CD3+ cell sis shown (B) Histogram plotss represent the staining of anti-hHIDEl Abs gating on CD 14+ cells from 2 donor s.(C) Histogram plotss represe ntthe staining of anti-hHIDEl Abs gating on CD3+ cells. Values represent geometri meac n fluorescent intensity (gMFI).

[00122] Figure 39: HIDE1 expression profil one fresh PBMCs. PBMCs from 4 donors were pre-blocked with human Ig Fc fragments (200|Jg/ml) in addition to Fc-blocking soluti on (Biolegend) to avoid non-specific binding via Fc receptors Cells. were then stained with anti - hHIDEl antibodie ors isotype control as described in materials and analyzed by flow cytometry. Histogram plotss represent the staining of anti-hHIDEl Abs gating on Monocytes 28WO 2017/009712 PCT/IB2016/001079 (FSC-A, SSC-A). Values represe ntgeometr icmean fluorescent intensity ratio (MFIr) compar eto isotype control.

[00123] Figure 40: Transcript expression of mouse HIDE1 in subpopulati oncells (CD1 lb+ ,CD1 lb-) and in whol tume or cells. Verification of the mouse transcr iptwas performe byd qRT-PCR using TaqMan probe. Colum ndiagram represents data observe d using TaqMan probe HOJEX12-CCAAZKY. Ct values are detaile ind the table. Analysis indicating high transcript in CD1 lb+ subpopulation, and lower level swhole tumor cells and in CD 11b- subpopulation.

[00124] Figure 41: Transcript expression of mouse HIDE1 in subpopulati oncells (A.

CD1 lb+, CD1 lb-) and in whol tumore cells. Verification of the mouse transcript was performe byd qRT-PCR using TaqMan probe. Colum ndiagram represents data observe d using TaqMan probe HOJEX12-CCAAZKY. Ct values are detaile ind the table. Analysis indicating high transcript in CD1 lb+ subpopulation, and lower level swhole tumor cells and in CD 11b- subpopulation.

[00125] Figure 42: Transcript expression of mouse HIDE1 in splee ncells.

Verification of the mouse transcriwaspt performe byd qRT-PCR using TaqMan probe.

Colum ndiagram represents data observed using TaqMan probe HOJEX12-CCAAZKY. Ct values are detailed in the table. Analysis indicating high transcript in spleen cells.

[00126] Figure 43: Transcript expression of mouse HIDE1 in splee ncells.

Verification of the mouse transcriwaspt performe byd qRT-PCR using TaqMan probe.

Colum ndiagram represent datas observed using TaqMan probe HOJEX12-CCAAZKY. Ct values are detailed in the table. Analysis indicating high transcript in colon lamima propia and tota colonl cell scompared to colon epitheli cela ls.

[00127] Figure 44: Transcript expression of mouse HIDE1 in intestine cell populations. Verification of the mouse transcript was performe byd qRT-PCR using TaqMan probe. Column diagram represent datas observed using TaqMan probe HOJEX12- CCAAZKY. Ct values are detaile ind the table. Analysis indicating high transcr iptin lymph nodes, spleen and peyer patches population, and lower level sin colon and small intestine cells population. 29WO 2017/009712 PCT/IB2016/001079

[00128] Figure 45: Effect of various HIDEl-ECD-Ig (SEQ ID NO: 18) on mouse CD4 T cell activation. Plates were coated with anti-CD3 mAh (2pg/mL) in the presenc eof 10ug/ml HIDEl-Ig (batch #195) ybar is the mean of duplicat cultures,e the error bars indicating the standard deviation. (Figures 45B-45C) Culture supernatants were collect edat 48 h post-stimulat andion mouse IL-2 and IFNy levels were analyzed by ELISA. Results are shown as Mean ± Standard errors of duplicat sample es. One experiment out of two performed is presented.

[00129] Figure 46: Demonstrate inhibis tion of human T cel lproliferation induced by anti-CD3 and anti-CD28 in the presence of irradiate autold ogous PBMCs by human HIDE1 ECD-Ig (SEQ ID NO: 17). Figure 46A shows averages of three donors tested. Figures 46B- 46D show the individual data of each donor. The control Ig is Synagis.

[00130] Figure 47: Schemati cillustra tionof the experiment alsystem.

[00131] Figure 48: FACS analysi son HIDE1 transduce PBLsd using an anti-FLAG antibody wit hintra-cellula starining. The percent of cells staining positive (relati veto empty vector transduced) for the protei isn provided

[00132] Figure 49: FACS analysi sperformed on TCR transduce stid mulated PBLs using a specific monoclonal antibody that recognizes the extra-cellula domainr of the beta- chain from the transduce specifid c TCR. The percentage of cell sstaining positive is provided

[00133] Figure 50: Expression of HIDE1 on the F4 expressing PBLs causes a reduction of IFNy secretion upon co-culture wit hSK-MEL23, MEL-624 and MEL-624.38 in comparison to expression of an empty vector

[00134] Figure 51: Expression of HIDE1 on F4 expressing PBLs causes a reduction of secretion upon co-culture with SK-MEL23, MEL-624 and MEL-624.38.

[00135] Figure 52: Expression of HIDE 1 on F4 expressing PBLs causes a reduction in the expression of CD137 (4-1BB) upon co-culture wit hSK-MEL23, MEL-624 and MEL- 624.38.

[00136] Figure 53: HIDE1 expression and H2Db expression on EL4 cell s.(A) Mock cells and HIDE1 transuded EL4 cells (0.5xl05 per sample) were staine dwith rabbit anti- 30WO 2017/009712 PCT/IB2016/001079 HIDE1 (Genscript, 10|Jg/ml )followed by PE donkey anti-rabbit IgG and analyzed by flow cytometry. (B) Mock and PDL1 transduce EL4d clone cell swere stained wit hAPC-anti- PDL1 and PE-anti-H2Db antibodies.

[00137] Figure 54: Schematic illustra tionof pmel-1 experimental system Prima. ry stimulation: Spleen cell swere culture ind the presence of lug/ml of gplOO-peptide and 25ng/ml of IL-2 for 4 days. CD8+ cells were then isolated and cultured in the presence of 25ng/ml of IL-2 for overnight rest Secondary. stimulation: pmel-1 cultures were washed and co-cultured overnight wit hgp-100 pulse dEL4 cell sas described in materia andl methods.

Surface CD137 and CD25 expression level son gated pmel-1 CD8+ T cell swere examined by flow cytometry. Cytokines secretion level swas tested by Thl/Th2/Thl cytoki7 ne CBA kit.

[00138] Figure 55: The effect of HIDE1 and PDL1 on pmel-1 CD8+ T cells activation upon co-culture wit hEL4 cell spulse dwith gp-100. Pre-activated pmel-1 splenocytes were culture aloned (5xl04/well) or together wit hgp-100 (0.3 or Ing/ml) pulse dEL4 cells (25xl04/well) at 2:1 E:T ratio. (A) Gating strategy for flow cytometry analysis of resting pmel-1 CD8+ cell sfollowin 4dg activation of pmel-1 splenocyt eswit hgp-100 peptide and IL2 as described in material ands methods. (B) Values of histogram represes ntCD137 expression (gMFI) levels of activated CD8+ pmel-1 cells co-cultured wit hPDL1- transduced EL4 as positive control treat edwith or without blocking anti-PDL1. (C) Values of histogram s represe ntCD137 expression (gMFI) (left or) IFNy (right level) sof activated CD8+ pmel-1 cells co-cultured with HIDE1- transduced EL4 cells. Each bar is the mean ± SEM of triplicate samples. One representat experiive ment out of three independent experiment performes isd shown.

[00139] Figure 56: Therapeutic effect of HIDEl-ECD-Ig (SEQ ID: 18) in the PLP139-151-induced R-EAE model in SIL mice. HIDEl-ECD-Ig (SEQ ID: 18) was administered in a therapeutic mode from the onset of disease remission, at 100 microg/mouse i.p. 3 times per week for two weeks. Therapeut iceffects on clinical symptom ares demonstrat ased reduction in Mean Clinical Score.

[00140] Figure 57: Presents HIDE1 tetrame bindingr to H9, Jurkat and U937 human cel llines. Cells were stained wit hviability dye, then incubated wit hHIDE1 tetramer (orange) 31WO 2017/009712 PCT/IB2016/001079 and control EGFR and B7H4 tetramers (dark green and light green, respectively) at 3pg/well (60pg/ml )and evaluated by flow cytometry. The binding of HIDE 1 is shown compared to control EGFR gated on live cell sfollowi sinng gle gating.t Values of histogram represes ntthe geometri meac n fluorescent intensity (Geo Mean) of gated cells. Shown is one representati ve experiment out of three independent experiment performed.s

[00141] Figure 58A, 58B, & 58C: Shows detection of CD137 and PD-1 surface expression. CD8+ T cell s,CD4+ T cells and TILs were activated and monitored over time at 4 time-points as described in M&M. Resting or activated cells were first gated for lymphocyt (FSC-Aes vs. SSC-A), followed by live cell sgating, further gated for singlets (FSC-H vs. FSC-A), CD4/CD8 positive cell sand furth ergated for CD137 and PD1. Shown is surfac eexpression of PD-1 (left) and CD 137 (right) on (A) CD8+ T cell s(B) CD4+ T cell s and (C) TILs at different time-points normalized to isotype control over the time course of activation.

[00142] Figure 59A, 59B, & 59C: Shows HIDE1 binding to resting and activated CD4+ T and CD8+ T cells. CD4+ and CD8+ T cells were activated and monitored over time at 4 time-points as described in M&M. Cell swere staine dwit hviability dye, then incubated with HIDE1 tetrame andr controls (3pg/well) and, evaluated by flow cytometry. (A) Binding of HIDE1 to CD4+ T cells. Binding of HIDE1 to live resting (time 0) and activated CD4+ cells follow ingsingle gatingt for 24, 48, 72h and 144h compared to EGFR control (B).

Binding of HIDE1 to CD8+ T cells. Binding of HIDE1 to live resting (time 0) and activated CD8+ cell sfollowi sinng gle gatit ng for 24, 48, 72h and 144h compared to EGFR control.

Shown are the Geometric Mean of the fluorescent intensity values obtained. (C) Fold change of HIDE1 tetramer binding normalized to EGFR tetramer control over the time course of activation.

[00143] Figure 60A, 60B, & 60C: Shows HIDE1 binding to resting and activated TILs. TIL Marti and 209 were activated and monitored over time at 4 time-points as described in M&M. Cell swere stained with viability dye, then incubated with HIDE1 tetramer and controls (3pg/well), and evaluated by flow cytometry. (A) Binding of HIDE1 to TIL Mart i.Binding of HIDE1 to live resting (time 0) and activated TIL follow ingsingle t 32WO 2017/009712 PCT/IB2016/001079 gating for 24, 48, 72h and 144h compared to EGFR control (B). Binding of HIDE1 to TIL 209. Binding of HIDE1 to live resting (time 0) and activated TIL followi sinng gle gatingt for 24, 48, 72h and 144h compared to EGFR control Shown. are the Geometric Mean of the fluorescent intensity values obtained. (C) Fold change of HIDE 1 tetramer binding normalized to EGFR tetramer control over the time course of activation.

[00144] Figure 61: Surface expression of HIDE1 by healthy PBLs was evaluated using whol bloode taken from 3 different healthy donors using anti Hidel mAbs 33B4- 2F7 as compared to staining with isotype control Isotype. Red: line. Anti HIDE1 mAh: Blue line

[00145] Figure 62: Surface expression of HIDE1 by whole blood taken from 3 different AML patients using anti Hidel mAbs 33B4-2F7 as compared to staining with isotype control Isot. ype: Red line .Ant iHIDE1 mAh: Blue line.

[00146] Figure 63: Shows levels of HIDE1 RNA expression in patients treate withd PD-1 inhibitor

[00147] Figure 64A-N: Anti-HIDE1 antibody sequences CPA. 12.001 human IgG4, CPA. 12.002 human IgG4, CPA. 12.003 human IgG4, CPA. 12.004 human IgG4, CPA. 12.005 human IgG4, CPA. 12.006 human IgG4, CPA. 12.007 human IgG4, CPA. 12.008 human IgG4, CPA. 12.009 human IgG4, CPA. 12.011 human IgG4, CPA. 12.012 human IgG4, CPA. 12.013 human IgG4, CPA. 12.014 human IgG4, and CPA. 12.015 human IgG4

[00148] Figure 65: IgGl, IgG2, IgG3, and IgG4 sequences.

[00149] Figure 66: HIDE1 ECD and peptide seqeunces.

[00150] Figure 67: Enhanced proliferati andon cytokine secretion observed for HIDE1 siRNA knockdown (A) schemati representatc ionof THP1-KD cell sco-cultured with CD3 T cells polyclonal activated at lug/m limmobilized anti-CD3. (B) Histogram depicting level sof HIDE1, PDL1, CD86, HLA-I & HLA-II expression compared to isotype control HIDE1. and PDL1 knockdown level scalculate wered 71% and 61% respectively, as noted in histograms.

Fold change of CD86, HLAI & HLA-II noted in histogram calculateds showed no variations between HIDE1, PDL1 and SCR KD cells. (C) Levels of CD4 and CD8 T cell proliferation 33WO 2017/009712 PCT/IB2016/001079 was determined by dilution of CFSE and quantified for triplicates D. Levels of IFNY and TNFa was determined from the supernata ntusing TH-1/2/17 CBA kit (BD biosciences) and quantified for triplicates.

[00151] Figure 68: Effect of anti-HIDE 1 antibodie ons IFNy and TNFa secretion in THP-1 Treated T cell poly activation assay. Graph depict snormalizat ionof IFNy & TNFa levels, where each bar represent thes effect of a specific aHIDE-1 hlgGl or hIgG4 Ab on IFNy or TNFa secretion from A. Donor 18 B. Donor 19.

[00152] Figure 69: Effect of anti-HIDE1 antibodie ons IFNy secretion inHIDEl over- expression CHOS-OKT3 assay Graph depict snormalizat ionof IFNy levels, where each bar represent thes effect of a specific aHIDE-1 hlgGl Ab on IFNy secretion from Donor 19.

[00153] Figure 70: Effect of anti-HIDE 1 antibodie ons T cell proliferati inon immature DC-MLR. Graph depict snormalizati ofon proliferation, where each bar represent s the effect of a specific aHIDE-1 hlgGl Ab on CD4 or CDS T cel lproliferation from Donor 16 (A) and Donor 17 (B).

[00154] Figure 71: hHIDEl/ hPD-Ll and HLA-A2 surface expression on Mel-526 and Mel-624 cells. Mock and hPDLl/HIDEl transduce Meld -526 and Mel-624 cells (0.5xl05 per sample) were stained with APC-anti-PDLl (5pg/ml )or AF647 anti HIDE1 (lug/mL) and PE-anti-HLA-A2 (5pg/ml) antibodies gMFI. levels of PE, APC and AF647 were compared between over expressing cell sto mock transduce celd ls. HLA-A2 level scomparison is indicated by percentage HID. E1 or PD-L1 expression is indicate dby fold of expression.

[00155] Figure 72: Effect of hPDLl and hHIDEl on TIL activation upon co-culture with Mel-526 and Mel-624 cells. (A-B) gplOO or MART-1 reactive TILs were co-cultured with hPDLl (A,C) or hHIDEl (B,D) overexpressing Mel-526 and Mel-624 cell sat 1:1 effector to target rati oas describe ind materia andl methods. Values of histogram represes nt CD137 expression (gMFI) or IFNy secretion from TIL-209, TIL-154 and TIL-MART1. Each bar is the mean ± SEM of triplicate samples.

[00156] Figure 73: HIDE1 expression and HLA-A2 expression on Mel-526 and Mel- 624 cells (A). Mock and hPDLl transduce Meld -526 and Mel-624 cell s(0.5xl05 per sample) were stained with APC-anti-PDLl (5pg/ml) and PE-anti-HLA-A2 (5pg/ml )antibodies (B). 34WO 2017/009712 PCT/IB2016/001079 Mock cell sand hHIDEl transuded Mel-526 and Mel-624 cell swere stained wit hAF647 anti- HIDE1 (In-house, Ipg/ml) and analyzed by flow cytometry.

[00157] Figure 74: Effect of hPDLl and hHIDEl on TIL activation upon co-culture with Mel-526 and Mel-624 cells. (A-B) gplOO or MART-1 reactive TILs were co-culture d with hPDLl (A) or hHIDEl (B) overexpressi Melng -526 and Mel-624 cell sat 1:1 effector to target rati oas describe ind materia andl methods. Values of histogram represents CD137 expression (gMFI) or IFNy secretion from TIL-209, TIL-154 and TIL-MART-1. Each bar is the mean ± SEM of triplicat same ples.

[00158] Figure 75: Experimenta systl em and B7H4-Ig effect on T cel lactivit y.(A) Schemati cillustra tionof the experiment alsystem Plat. es were coated wit hanti-CD3 mAh (2ug/mL) in the presence of tested compound, as described in materia andl methods. The effect of the tested prote inon T cell activation manifes, ted by activation markers and cytokine secretion, was analyzed. (B) Culture supernatants were collect ed48 h post- stimulation and mouse IL-2 and IFNy level swere analyzed by ELISA. Results are shown as Mean ± Standard errors of duplicat same ples.

[00159] Figure 76: Effect of HIDEl-Fc on mouse CD4 T cell activation.

Plates were coated wit hanti-CD3 mAh (2pg/mL) in the presence of 10ug/ml HIDEl-Fc or control mIgG2a as described in materia lsand methods. Wells were plated with IxlO5 CD4+CD25- mouse T cells per well in the presence of 2ug/ml of soluble anti-CD28. (A) The expression of CD69 was analyzed by flow cytometry at 48h post-stimulati (B-C)on. Culture supernatants were collected at 48 h post-stimulat andion mouse IFNy or IL-2 levels were analyzed by ELISA. Results are shown as Mean ± Standard errors of duplicate samples.

[00160] Figure 77: Anti-HIDE1 Fabs injected over HIDE1- HH-1 captured to a GLC chip (black lines). A 1:1 kinetic binding model (red line) provided rough estimates of the binding parameters. Panels where no fitting lines are shown indicate complex kinetics.

[00161] Figure 78: Anti-HIDE 1 Fabs injected over CD 155 control fusion protein captured to a GLC chip (black lines). There were either no binding respons esor the binding respons eswere minimal compared to the same HIDE1 Fabs injected over HIDE1- fusion protei (Figun re 77). 35WO 2017/009712 PCT/IB2016/001079

[00162] Figure 79: Transcript expression of human HIDE1 in cell sderived from three different areas (TILs, Invasive Front and Strom a)from MSI and MSS colorec talcancer patients. Expression of human transcript of HIDE1 was analyzed by qRT-PCR using 2 specific TaqMan probes HsOl 128131ml (B) and HsOl 128129 ml (A). Analysis indicating higher transcript expression in TIL, Invasive front and Stroma areas in MSI patients (3/3) compared to MSS patients and higher expression in strom anda IF areas compared to TIL area.

[00163] Figure 80: Serotec antibody sequences. Complementarit determiningy regions (CDRs) are underlined. CDR definition is according to standard definitions (Krebs, B, et al., J Immunol Methods 2001, 254:67-84). Constant domains CHI and CL sequence are in italics, dimerization domain sequence (AP) is in green (ref. 4), linke sequencer s are in bold, FLAG® tag (ref. 5) is in pink and His6 tag (ref. 6) is in blue.

[00164] Figure 81: Anti-HIDE1 antibody seqeunces 33B4, 36C1, and 39A7.

[00165] Figure 82: Schemati crepresentation of 33B4-Vlk and 33B4-Clk.

[00166] Figure 83: Schemati crepresentation of 33B4-VH and 33B4-CH.

[00167] Figure 84: Schemati crepresentation of 36C1-Vlk and 36C1-Clk.

[00168] Figure 85: Schemati crepresentation of 36C1-VH and 36C1-CH.

[00169] Figure 86: Schemati crepresentation of 39A7-Vlk and 39A7-Clk.

[00170] Figure 87: Schemati crepresentation of 39A7-VH and 39A7-CH.

[00171] Figure 88: HIDE1 expression in blood cells and tissues with enriched blood cells (GTEx data).

[00172] Figure 89: HIDE1 expression pattern in BioGPS.

[00173] Figure 90 A-B: FACS analysi susing anti-human HIDE1 and anti-mouse HIDE1 Fab’s on HEK293 cell sover-expressing cyno HIDE1 Flag protein. (A)HEK293 cells over-expressing the cyno HIDE1 (lower panel) or HEK293 transduce withd empty vector (upper panel) were analyzed by FACS using Serotec anti-human HIDE1 Fab’s (1-16) and anti-mouse HIDE1 Fab’s (4,5). Detection was carried out using Goat Ant iHuman IgG 36WO 2017/009712 PCT/IB2016/001079 F(ab')2-PE secondary Ab. (B) Summary tabl eof Serotec anti-human HIDE1 Fab’s (1-16) and anti-mouse HIDE1 Fab’s (4,5) FACS analysis, the Geo mean ratio of HEK293 cell sover- expressing the cyno HIDE1 Flag/ HEK293 transduced with empty vector.

[00174] Figure 91: Serotec summary for the epitope binning data for anti human HIDE1 antibodies group, 1.

[00175] Figure 92: Serotec summary for the epitope binning data for anti human HIDE1 antibodies group, 2.

[00176] Figure 93: Serotec summary for the epitope binning data for anti human HIDE1 antibodies group, 3.

[00177] Figure 94: Serotec summar yfor the epitope binning data for anti mouse HIDE1 antibodies

[00178] Figure 95: FACS analysis using anti human HIDE1 Reformatted Fab’s on HEK293 cells over-expressing human/mouse/cyno HIDE1 Flag protein. (A)HEK293 cell s over-expressing the human HIDE1 Flag (orange line) or HEK293 cells over-expressing the mouse HIDE1 Flag (light green line) or HEK293 cell sover-expressin theg cyno HIDE1 (dark green line) or HEK293 transduced/transf ectwithed empty vector (blue and red line respectivel werey) analyzed by FACS using Serotec anti-Human HIDE1 reformatte Fabd’s (1-5). Detection was carrie dout using Goat Ant iHuman IgG-PE secondary Ab.

[00179] Figure 96: Affinity measurement usings FACS application for the anti-human HIDE1 reformatted Fab’s on CHO-S cell sover-expressin humang HIDE1 Flag protein.

(A)CHO-S cells over-expressi theng human HIDE1 Flag (circl edots) or HEK293 transduced with empty vector (square dots) were analyzed by FACS using Serotec anti-Human HIDE1 reformatte antibodied (1-5)s in 7 concentrations seri- es dilution 1:3, 10-0.0lug/ml . Detection was carried out using Goat Anti Human-PE secondary Ab. (B) Summary tabl eof Serot ec anti-human HIDE1 reformatte antibodied 1-5s FACS analysis, the Geo mean ratio of HEK293 cells over-expressing the human HIDE1 Flag/ HEK293 transduced with empty vector and the cross reactive validation, the Geo-mean ratio of HEK293 cells over-expressi ng the mouse/cyno HIDE1 Flag/ HEK293 transduced/transf ectewitd hempty vector. And Kd (nM) for each reformatte Fab.d 37WO 2017/009712 PCT/IB2016/001079

[00180] Figure 97: FACS analysi sof CHO-S OKT3 cells ectopically expressing human HIDE1 Flag pcDNA3.1 vector. CHO-S OKT3 cells over expressing human HIDE1 Flag or CHO-S OKT3 cells transfect edwith an empty vector were analyzed by FACS using mouse monoclonal anti human HIDE1 (BIOTEM, 33B4-2F7- Alexa 647). Mouse IgG (Biotem, F1150528d-2695- Alexa 647) was used as an isotype control

[00181] Figure 98: FACS analysi sof hek293 cell sectopical lyexpressing cyno HIDE1 pcDNA3.1 vector. Hek293 cells over expressing cyno HIDE1 or HEK293 cell stransfect ed with an empty vector were analyzed by FACS using mouse monoclonal anti human HIDE1 (BIOTEM, 33B4-2F7- Alexa 647). Mouse IgG (Biotem F1150528d-2695-, Alexa 647) was used as an isotype control.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

[00182] Cancer can be considered as an inability of the patient to recognize and eliminate cancerous cells. In many instances, thes transforme (e.g.ed cancerous) cell s counteract immunosurveillance There. are natural control mechanism sthat limi tT-cel l activation in the body to preven tunrestrained T-cel lactivit y,which can be exploited by cancerous cells to evade or suppress the immune response. Restoring the capacity of immune effector cells—especially T cell—s to recognize and eliminat cancee r is the goal of immunotherapy. The field of immuno-oncology, sometimes referre tod as “immunotherapy” is rapidly evolving with, several recent approvals of T cell checkpoint inhibitor anty ibodies such as Yervoy, Keytruda and Opdivo. These antibodie ares generall referredy to as “checkpoint inhibitors” because they block normally negative regulators of T cel limmunity.

It is generally understood that a variety of immunomodulat orysignals, both costimulatory and coinhibitory, can be used to orchestrate an optima antil gen-specific immune response.

Generally, thes antibodiee binds to checkpoi ntinhibitor proteins such as CTLA-4 and PD-1, which under normal circumstances prevent or suppress activation of cytotoxic T cell s (CTLs). By inhibiting the checkpoi ntprotein, for example throu ghthe use of antibodie thats bind thes proteinse an, increased T cell response against tumors can be achieved. That is, thes ecancer checkpoint proteins suppress the immune response; when the protei nsare 38WO 2017/009712 PCT/IB2016/001079 blocked, for example using antibodie tos the checkpoi ntprotein, the immune system is activated, leading to immune stimulation, resulting in treatment of conditions such as cancer and infectious disease.

[00183] According to at leas tsome embodiments of the present inventio nis directed to the use of antibodies to HIDE1. HIDE1 is expressed on the cel lsurfac eof myeloid cells including but not limite tod monocytes dendri, tic cell s,macrophages, M1/M2 tumor associated macrophages, neutrophil Myelois, d-derive suppressor cells (MDSC), and share s several similariti toes other known immune checkpoints.

[00184] Functiona effectl s of HIDE1 blocking antibodie ons myeloid cell sincluding, for example, but not limited to monocytes, dendritic cell s,macrophages, M1/M2 tumor associated macrophages, neutrophil Myelois, d-deri suppresve sor cells (MDSC), and/or on NK and T-cells can be assessed in vitro (and in some cases in vivo, as described more full y below) by measuring changes in the followi parang meters: proliferati cytokion, ne release and cell-surfac makers.e For NK cell s,increases in cel lproliferat ion,cytotoxici (abilty it yto kill targe cellt sas measured by increases in CD107a, granzyme ,and perforin expression, or by direct lymeasuring target cell skilling), cytokine production (e.g. IFN-y and TNF), and cell surfac ereceptor expression (e.g, CD25) is indicative of immune modulation, e.g. enhanced killing of cancer cells. For T-cells incre, ases in proliferat ion,increase sin expression of cell surfac emarkers of activation (e.g.,CD25, CD69, CD137, and PD1), cytotoxici (abity lity to kill targe cellt s), and cytokine production (e.g, IL-2, IL-4, IL-6, IFNy, TNF-a, IL-10, IL- 17A) are indicative of immune modulation, e.g. enhanced killi ngof cancer cells. For myeloid cells: effect on myeloid cells polarizati on,such as M2 to Ml shift, improvement of antigen presentation by myeloid cell sincluding more efficient cross-presnta tibyon professional as wel las non-professional antigen-present ingcell s,enhanced antigen uptake and processing by antigen-presenting cells. Additional effects can include relief of T cell suppression (i.e. indirect effect on T cel lactivation), effect on cel lrecruitmen (i.et. influx of immune cell sand shift to more “inflamed tumors”).

[00185] In some embodiments, the anti-HIDE 1 antibody is a depleting HIDE1 antibody. In some embodiment s,a depleting anti-HIDEl antibody binds to cel lsurface HIDE1. In some embodiments, the anti HIDE1 depleting antibody preferably is able to 39WO 2017/009712 PCT/IB2016/001079 deplet HIDE1e expressing cells including but not limite tod monocytes, dendritic cells, macrophages, M1/M2 tumor associated macrophages, neutrophils, Myeloid-derive suppressor cells (MDSC) and as a resul reducet the number of HIDE1 expressing cell sin a patient treate withd the anti HIDE1 depleting antibody. Such depletion may be achieved via various mechanism ssuch as antibody-dependent cell mediated cytotoxicit (ADCy C) and/or complement dependent cytotoxicit (CDy C), inhibition of HIDE 1 expressing cells proliferation and/or inductio ofn HIDE 1+ cel ldeat h(e.g. via apoptosis).

[00186] HIDE1 expressing cel ldepleting anti HIDE1 antibody might optionall bey conjugated wit hor fused to a cytotoxic agent.

[00187] “Complement dependent cytotoxici” tyor “CDC” refers to the lysi sof a target cel lin the presenc eof complement. Activation of the classical complement pathwa isy initiated by the binding of the firs componet nt of the complement system to antibodie whics h are bound to thei cognar te antigen. To asses scomplement activation, a CDC assay, e.g. as described in Gazzano- Santoro et al. (1997), or any other CDC assay known in the art, can be performe ord employed with the anti-HIDEl antibodies of the invention.

[00188] “Antibody-depende cell-mnt ediated cytotoxici” tyor “ADCC” refers to a form of cytotoxici inty whic hsecreted antibodie bounds onto Fc receptor (FcRs)s present on certai ncytotoxic cell s(e.g., Natural Killer (NK) cell s,neutrophil monocytess, and macrophages) allow the cytotoxi effectc or cells to bind specifically to an antigen-bearing target cell and subsequentl killy the targe cellt .

[00189] Accordingly, in at least some embodiments of the present inventio nprovides antibodies, includin gantigen binding domains, that bind to human HIDE1 and methods of activating T cells and/or NK cell sand/or methods of activating myeloid cell swhich induce activatory, migratory, secretory effect enhancing T/NK cel lfunction and migration to treat diseases such as cancer and infectious diseases, and other conditions where increased immune activit yresult ins treatment. According to at least some embodiments of the present invention also provide antibs odies includin, gantigen binding domains,that bind to human HIDE1 for use in methods of depleting myeloid cell s,or other circulating tumor cell s,in order to trea diset ases such as cancer. 40WO 2017/009712 PCT/IB2016/001079

[00190] Furthermore wi,thout wishing to be limited by a singl ehypothesis, HIDE1 shows potentiati effectsng on the followi immng une functions: induction or differentiati on and proliferation of inducible T regulatory or suppressor cells (iTregs). These cells are known to be involved in eliciting tolerance to self-antigens and to suppress anti-tumor immunity.Again without wishing to be limited by a singl ehypothesis, HIDE1 contribut toes a non-functional phenotype of CDS T cell from the tumor environment, also known as T cell exhaustion.

[00191] The flip side of immuno-oncology is the suppression of T cell activation in conditions where the immune system is too active, or is launching an immune response to an auto-antigen, etc. Thus, by providing HIDE1 proteins (for example as fusion proteins, as discussed below), treatment of immune conditions such as auto-immune disease, inflammatio andn allergi disec ases can be treated. That is, as HIDE1 has an inhibitor effecty on specific immune cells such as CD4+ T cell s,CD8+ T cell sor CTLs, and NK cells, which cells are known to be involved in the pathology of certain immune conditions such as autoimmune and inflammatory disorders, as well as eliciting a potentiat ingeffect on Tregs or immuno-supressi vemyeloid cells, HIDE1 polypeptides which potentiate or agonize the effects of HIDE 1 on immunity may optionall bey used for treating conditions wherei then suppression of T cel lor NK mediate dimmunity and/or the induction of immune tolerance or prolonged suppression of antigen-specific immunity is therapeuticall desiry able, e.g, the treatment of autoimmune, inflammator ory allergi condic tion and/ors, the suppression of undesire dimmune respons essuch as to cell or gene therapy, adverse immune responses during pregnancy, and adverse immune respons esto transplant heteroled ogous, allogenei orc xenogeneic cells, organs and tissues and for inhibiting or preventing the onset of graft versus host disease (GVHD) after transplant.

[00192] Therefore in, one embodiment the present inventio nbroadly relat esto the development of novel “immunomodulat oryprotei”ns wherei thin sincludes HIDE1 polypeptides that antagonize or block the effects of HIDE 1 on immunity and particularl they effects of HIDE1, on specific types of immune cell sand cytokine production (z. e., immunostimulatory HIDE1 polypeptides or fusion proteins and/or immunostimulatory HIDE1 antibodies). 41WO 2017/009712 PCT/IB2016/001079

[00193] Additional thely, inventio nrelat esto HIDE1 polypeptides that agonize or mimic the effects of HIDE 1 on immunity and particularl they effects of HIDE 1 on specific types of immune cells and cytokine production (i.e., "immunoinhibito HIDE1ry polypeptide s or fusion protei”ns or HIDE1 polypeptides that increase and/or enhance in immune suppression), as well as immune suppressing polypeptides which mimic HIDE1 immune suppressi veactivit ythrough binding to the HIDE1 binding/signalling partner and can be referre tod as enhancers of HIDE1 associated immune suppression.

II. HIDE1 Mechanism of Action

[00194] Accordingly, as discussed herein, HIDE1 is an immune checkpoint protein, sometimes referred to as “an immuno-oncology protei”.n As has been shown for PD-1 and CTLA-4, among others im, mune checkpoint proteins can be exploited in several ways, to either immunopotentiate the immune system to increase immune activity, such as through the activation of T cells for treatments of diseases such as cancer and infectious disease, or through immunoinhibition, where immunosuppressio is ndesired, for example in allergic reactions, autoimmune diseases and inflammatio n.HIDE1 as shown herein exhibit snegative signaling on the immune system, by suppressing T cell activation and other pathways as outlined herein. Thus, by reducing the activity of HIDE 1, for example by inhibiting it s binding ability to its ligand Q.e., inhibiting binding to its binding partner or signaling partner), the suppression is decreased and the immune system can be activated or stimulated to trea t cancer, for example. Conversely, by increasing the activity of HIDE 1 (“stimulating” the activit ywit ha “stimulat”or and/or wit han enhancer of HIDE 1 associated immune suppression), for example by adding recombinan HIDt E1 ECD that mimics HIDE1 immune suppressi veactivit iythrough binding to HIDE1 counterpart (a “stimulat ofor HIDE1” or an enhancer of HIDE1 associated immune suppressio i.e.,n; HIDE1 binding and/or signaling partner) to a host in the form of a solubl ECDe (and optionally a fusion protein), the suppression is increased and the immune system is suppresse d,allowing for treatment of diseases associated with increased immune function such as autoimmune diseases and others outlined herein. As shown in the Example section, HIDE1 tetramers have been shown to bind to activated T cell sand TILs, activation status of T cell shas been manifeste dby 42WO 2017/009712 PCT/IB2016/001079 increased PD-1 and CD137 expression Addi. tional introductily, ofon HIDE1 (for example, as an ECD Fc-fusion protei n)was shown to inhibit the activation of T cells, as shown in the Examples. Accordingly, anti-HIDEl antibodie cans be used to trea conditt ions for which T cel lor NK cel lactivation is desired such as cancer.

[00195] Accordingly, in some embodiment ofs the present inventio nis directed to compounds that either suppres thes signaling pathwa triy ggered by the binding interaction of HIDE1 and its binding and/or signaling partner (leading to increased T cell and NK cell activation, among other things leading, to treatment of diseases such as cancer and pathogen infection), or activate the signaling pathway triggered by the binding interaction of HIDE 1 and its binding and/or signaling partne (lear ding to decreased T cel land NK cell activation , among othe thir ngs), leading to treatment of diseases such as autoimmune diseases and inflammation.

[00196] Thus, specific mechanisms of action are provided for the immunostimulatory actions of, for example, anti-HIDEl antibodies that, are useful for increasin gimmune function, for example for the treatment of cancer. These include but, are not limited to, (i) increases immune response, (ii) increase sT cel lactivit y,(iii) increases activation of a,|3 and/or y5 T cell s,(iv) increases cytotoxic T cel lactivity, (v) increases NK and/or NKT cell activit y,(vi) alleviat esa,|3 and/or y5 T-cel lsuppressio (vii)n, increases pro-inflammatory cytokine secretion, (viii) increases IL-2 secretion; (ix) increases interferon-Y production, (x) increases Thl respons e,(xi) decrease Th2 response, (xii) decrease ors eliminates cell number and/or activit yof at leas tone of regulatory T cell s(Tregs), myeloid derived suppressor cell s (MDSCs), iMCs, mesenchyma lstroma celll s,TIE2-expressing monocytes (xiii, ) reduces regulatory cel lactivit y,and/or the activit yof one or more of myeloid derived suppressor cell s (MDSCs), iMCs, mesenchyma lstroma celll s,TIE2-expressing monocytes, (xiv) decrease ors eliminates M2 macrophages, (xv) reduce sM2 macrophage pro-tumorigeni activitc y,(xvi) decreases or eliminates N2 neutrophil (xvii)s, reduce sN2 neutrophil pro-tumoris genic activit y,(xviii) reduce sinhibition of T cel lactivation (xix), reduce sinhibition of CTL activation (xx), reduce sinhibition ofNK and/or NKT cel lactivation (xxi), reverses a,[3 and/or y5 T cel lexhaustion, (xxii) increases a,[3 and/or y5 T cel lresponse, (xxiii) increases activit yof cytotoxic cell s,(xxiv) stimulates antigen-specific memory responses, (xxv) elicits apoptosis 43WO 2017/009712 PCT/IB2016/001079 or lysi sof cancer cells, (xxvi) stimulates cytotoxic or cytostati effecc t on cancer cell s,(xxvii) induces direc killt ing of cancer cell s,(xxviii) increases Thl7 activity and/or (xxix) modulating myeloid cel lpolarizati on,(xxx) modulating myeloid cel lshifting toward a pro- inflammatory respons e,(xxxi) shifting myeloid from M2 toward Ml phenotype, (xxxii) modulating myeloid cel lin the TME to support anti-cancer immune response, (xxxiii) restricti theng pro-tumorigenic effects of the myeloid cell sin the TME, (xxxiv) enhancing myeloid and lymphoi infild trati intoon the tumor cite thereby shifting the tumor into more immunogenic, (xxxv) induces complement dependent cytotoxici and/orty antibody dependent cell-mediated cytotoxicity.

[00197] Functiona effectl s of HIDE1 blocking antibodie ons myeloid cell sincludin g but not limited to monocytes, dendritic cell s,macrophages, M1/M2 tumor associated macrophages, neutrophil Myelois, d-deri suppresve sor cells (MDSC), and/or on NK and T- cells can be assessed in vitro (and in some cases in vivo, as described more fully below) by measuring changes in the follow ingparameters: proliferat ion,cytokine release and cell - surfac emakers .For NK cells, increases in cel lproliferati cytotoxicion, (abilty it yto kill target cells as measured by increases in CD107a, granzyme ,and perforin expression, or by direct lymeasuring targe cellt skilling), cytokine production (e.g. IFN-y and TNF), and cell surfac ereceptor expression (e.g. CD25) is indicative of immune modulation, e.g. enhanced killing of cancer cells. For T-cells incre, ases in proliferat ion,increase sin expression of cell surfac emarkers of activation (e.g. CD25, CD69, CD 137, and PD1), cytotoxici (abity lity to kill targe celt ls), and cytokine production (e.g. IL-2, IL-4, IL-6, IFNy, TNF-a, IL-10, IL-17A) are indicative of immune modulation, e.g. enhanced killing of cancer cells. For monocytes , increases in monocyte differentiati on,Ml-M2 skuwing and vise versa detected by specific differentiat ingmarkers (Ml -iNOS, IL-12p35, TNFa, IL-lb; M2 - IL-10, OH-1, CCL17, CCL22, Msr2 ,MRC1); Monocyte activation detected by cell surfac emarkers of activation (e.g MHC, CD80/86, PDL1) and cytokine and chemokine secretion (CCL19/21). The afforimentioned effects could be examined upon differentiat ingprimary myeloid cell sas well as cellslines towards macrophage-like cell susing M1/M2 polarazing conditions. Indirect suppressi veeffect of HYDE1-expressing myeloid cells on effector immune cells, includin g but not limited to CD4+ T cell s,CD8+ T cell s,NK cell s,NKT cell s,could be reverse witd h 44WO 2017/009712 PCT/IB2016/001079 HIDE1-blocking Ab upon poly-clona orl antigen-specific activation of immune effector cells in the presence of HIDE1- expressing myeloid cells.

[00198] Accordingly, in some embodiment thes present inventio nprovide antis bodies, including antigen binding domains, that bind to human HIDE1 and methods of activating myeloid cell sincludin gbut not limited to monocyte s,dendritic cell s,macrophages, M1/M2 tumor associated macrophages, neutrophil Myelois, d-derive suppressor cells (MDSC), and/or on, T cell sand/or NK cell sto trea diset ases such as cancer and infectious diseases, and othe r conditions where increased immune activit yresult ins treatment. According to at least some embodiments of the present inventio nalso provides antibodies including, antigen binding domains that, bind to human HIDE1 for use in methods of depleting myeloid cells, or othe r circulati ngtumo rcell s,in order to trea diseat ses such as cancer.

[00199] According to at least some embodiments, the present invention provides immunoinhibitory HIDE1 therapeutic agents (e.g., a compound, including but not limite tod a HIDE1 peptide that, mimics HIDE1 immune suppressi veactivit iythrough binding to HIDE1 counterpart i.e.,, the HIDE1 binding and/or signalling partner; such compounds can also be referre tod as enhancers of HIDE 1 associated immune suppression), wherei saidn agents are used for treatment of immune related diseases and/or for reducing the undesirab leimmune activation that follows gene therapy, and wherei saidn agents mediate at leas tone of the followi imngmune effects: (i) decreases immune response, (ii) decrease a,[3s and/or y5 T cell activation (iii, ) decrease Ts cel lactivit y,(iv) decreases cytotoxic T cel lactivit y,(v) decreases natural killer (NK) and/or NKT cel lactivit y,(vi) decreases a,[3 and/or y5 T-cel lactivit y,(vii) decreases pro-inflammatory cytokine secretio (viiin, ) decreases IL-2 secretion; (ix) decreases interferon-y production, (x) decrease Thls respons e,(xi) decreases Th2 response, (xii) increases cel lnumber and/or activity of regulatory T cells, (xiii) increases regulatory cell activity and/or one or more of myeloid derived suppressor cell s(MDSCs), iMCs, mesenchyma lstroma celll s,TIE2-expressing monocytes, (xiv) increases regulatory cell activit yand/or the activity of one or more of myeloid derive dsuppressor cells (MDSCs), iMCs, mesenchyma lstroma celll s,TIE2-expressing monocytes, (xv) increases M2 macrophages, (xvi) increases M2 macrophage activit y,(xvii) increases N2 neutrophil (xviis, i) increases N2 neutrophi activitls y,(xix) increases inhibition of T cel lactivation (xx), increases 45WO 2017/009712 PCT/IB2016/001079 inhibition of CTL activation, (xxi) increases inhibition of NK cel lactivation (xxii), increases aP and/or y5 T cel lexhaustion, (xxiii) decreases aP and/or y5 T cel lrespons e,(xxiv) decreases activity of cytotoxi cellc s,(xxv) reduces antigen-specific memory responses (xxvi), inhibits apoptosis or lysi sof cell s,(xxvii) decrease cytots oxic or cytostati effecc t on cells , (xxviii) reduce sdirec killit ngof cell s,(xxix) decreases Thl7 activity, and/or (xxx) modulates myeloid cel lpolarizati on,and/or modulates myeloid cell shifting toward an anti- inflammatory respons e,(xxxi) reduce scomplement dependent cytotoxici and/orty antibody dependent cell-mediated cytotoxici ty.Again without wishing to be limited by a single hypothesis, HIDE1 shows potentiat ingeffects on the follow ingimmune functions indu: ction or differentiat ionand proliferati ofon inducible T regulatory or suppressor cells (iTregs).

These cells are known to be involved in eliciting tolerance to self-antige nsand to suppres s anti-tumor immunity.

[00200] Again without wishing to be limite byd a singl ehypothesi HIDE1s, contribut toes a non-functional phenotype of CDS T cel lfrom the tumor environment, also known as T cel lexhaustion.

[00201] The flip side of immuno-oncology is the suppression of T cell activation in conditions where the immune system is too active, or is launching an immune response to an auto-antigen, etc. Thus, by providing HIDE1 proteins (for example as fusion proteins, as discussed below), treatment of immune conditions such as auto-immune disease, inflammatio andn allergi disec ases can be treated. That is, as HIDE1 has an inhibitor effecty on specific immune cells such as CD4+ T cell s,CD8+ T cell sor CTLs, and NK cells, which cells are known to be involved in the pathology of certain immune conditions such as autoimmune and inflammatory disorders, as wel las eliciting a potentiat ingeffect on Tregs or immuno-supressi vemyeloid cells, HIDE1 polypeptides which potentiate or agonize the effects of HIDE1 on immunity may optionall bey used for treating conditions wherei then suppression of T cel lor NK mediate dimmunity and/or the induction of immune tolerance or prolonged suppression of antigen-specific immunity is therapeuticall desiry able, e.g, the treatment of autoimmune, inflammator ory allergi condic tion and/ors, the suppression of undesired immune respons essuch as to cell or gene therapy, adverse immune responses during pregnancy, and adverse immune respons esto transplant heteroled ogous, allogenei orc 46WO 2017/009712 PCT/IB2016/001079 xenogeneic cells, organs and tissues and for inhibiting or preventing the onset of graft versus host disease (GVHD) after transplant.

[00202] Furthermore, in some embodiments a HIDE1 ECD (for example, in the form of an Fc fusion, for example) binds to the HIDE1 binding and/or signaling and interrupt ones or more inhibitory signals (via HIDE1) and thus acts in an immunoinhibitory manner (including increasing and/or enhancing immune suppression). These include, but are not limite to,d i) decreases immune response, (ii) decrease a(3s and/or y5 T cel lactivation, (iii) decreases T cel lactivity, (iv) decrease cytotoxics T cel lactivity, (v) decrease naturs al killer (NK) and/or NKT cel lactivity, (vi) decreases a,|3 and/or y5 T-cel lactivit y,(vii) decreases pro- inflammatory cytokine secretio (viiin, ) decreases IL-2 secretion; (ix) decrease interferon-ys production, (x) decreases Thl response, (xi) decrease Th2s response, (xii) increases cell number and/or activity of regulatory T cell s,(xiii) increases regulatory cel lactivit yand/or one or more of myeloid derived suppressor cell s(MDSCs), iMCs, mesenchyma lstromal cell s,TIE2-expressing monocyte s,(xiv) increases regulatory cel lactivit yand/or the activity of one or more of myeloid derive dsuppressor cell s(MDSCs), iMCs, mesenchymal stromal cell s,TIE2-expressing monocyte s,(xv) increases M2 macrophages, (xvi) increases M2 macrophage activit y,(xvii) increases N2 neutrophil (xviiis, ) increases N2 neutrophil s activit y,(xix) increases inhibition of T cel lactivation (xx), increases inhibition of CTL activation (xxi), increases inhibition of NK cel lactivation, (xxii) increases a,|3 and/or y5 T cell exhaustion, (xxiii) decrease a(3s and/or y5 T cel lresponse, (xxiv) decreases activity of cytotoxic cell s,(xxv) reduce santigen-specific memory responses, (xxvi) inhibits apoptosis or lysi sof cell s,(xxvii) decrease cytots oxic or cytostat effectic on cell s,(xxviii) reduce sdirec t killing of cells (xxix), decrease Thls 7 activit y,(xxx) modulat esmyeloid cell polarizati on, and/or modulates myeloid cel lshifting toward an anti-inflammatory response, and/or (xxxi) reduce scomplement dependent cytotoxicit and/ory antibody dependent cell-mediated cytotoxicity.

[00203] Therefore in, one embodiment the present inventio nbroadly relat esto the development of novel immunomodulat oryproteins wherein thi sincludes HIDE1 polypeptides that antagonize or block the effects of HIDE 1 on immunity and particularl they effects of HIDE1, on specific types of immune cell sand cytokine production (z. e., 47WO 2017/009712 PCT/IB2016/001079 immunostimulatory HIDE1 polypeptides or fusion proteins and/or immunostimulatory HIDE1 antibodies).

[00204] Additional thely, inventio nrelat esto HIDE 1 polypeptides that agonize or mimic the effects of HIDE 1 on immunity and particularl they effects of HIDE 1 on specific types of immune cells and cytokine production (i.e., immunoinhibitory HIDE1 polypeptide s or fusion proteins or HIDE1 polypeptides that increase and/or enhance in immune suppression), as well as immune enhancing polypeptides whic hmimi HIDE1 immune suppressi veactivit ythrough binding to the HIDE1 binding/signalling partner and can be referre tod as enhancers of HIDE1 associated immune suppression.

III. Methods of Screening

[00205] Accordingly, in some embodiments the present inventio nprovide methodss of screening for modulators of the interaction of HIDE 1 and the binding and/or signaling partner for HIDE1, which either leads to immunostimulati oron immunoinhibition, as outlined herein.

For example, compounds that inhibi tthe interaction of HIDE1 and the binding and/or signaling partner for HIDE1, whic hnormall leay ds to the suppression of myeloid cells including but not limite tod monocytes dendri, tic cells, macrophages, tumor associate d macrophages, neutrophil Myelois, d-deri suppresve sor cells (MDSC), and/or T cel land/or NK cel lactivation and migration and thus increase the immune respons toe allow for the ultimate administration to patients for the treatment of cancer and pathogen infections for, example. Conversely, compounds that increase the signaling due to the interaction of HIDE1 and the binding and/or signaling partner for HIDE1, lead to increased suppression of myeloi d cells includin gbut not limite tod monocyte s,dendritic cells, macrophages, tumor associate d macrophages, neutrophil Myelois, d-deri suppresve sor cells (MDSC), and/or T cel land/or NK cel lactivation and migration, thus resulting in decreased immune responses, to allo wfor the ultimate administration to patients for the treatment of diseases associated wit hincreased immune function such as autoimmune diseases and inflammatio n.In thi slatter case, the increase of signaling is termed “stimulation of binding”, whic hcan be effected, for example, by adding a compound (a “stimulat”or) such as the ECD of HIDE1, resulting in stimulated binding of the ECD to the endogenous binding and/or signaling partne forr HIDE1 and triggering the signaling pathway. 48WO 2017/009712 PCT/IB2016/001079 A. Cell Based Assays

[00206] Accordingly, in one embodiment, the inventio nprovides assays to screen for inhibitors of the binding association of HIDE1 and the binding and/or signaling partner for HIDE1.

[00207] In one embodiment, the methods of screening provide cells that comprise an exogenous recombinant nucleic acid encoding a human HIDE1 protein, generally the full length protei includinn gthe transmembrane domain, such that the HIDE1 protei isn expressed in the corre ctorientation, resulting in the extracellul domainar (ECD) being on the surfac eof the cell .By “exogenous” in thi scontext herei nis meant that the gene (and any required expression vector sequences) is not endogenous (natural lyoccurring in the genome) to the cell .In the case of non-human cell line sto be used in the assays herein, thi smeans that the non-human cel lline has a human gene transfect edinto the cell .In the case where human cel llines are used (preferabl ine most instances), and thus contai nan endogenous HIDE1 gene, the cell scontain at least an addition al,recombinan humant gene, if not additional copies as well.

[00208] In thi sembodiment, cell sexpressing exogenous HIDE1 are contacted wit h candidate agent(s )as is more fully outlined below, and a labeled binding and/or signaling partner for HIDE1 (generall they ECD domain). By comparing the result tos a reference standard not includin gthe candidate agent, where binding is known to occur, the lack of bound label means the candidate agent binds to the HIDE1 in such a way as to prevent binding to the binding and/or signaling partner for HIDE1.

[00209] In one embodiment, the methods are reverse d,and thus use cells that comprise an exogenous recombinan nuclet ic acid encoding a binding and/or signaling partne forr HIDE1, generally the full length protei includingn the transmembra nedomain, such that the binding and/or signaling partner for HIDE1 protei isn expressed in the correct orientation, resulting in the extracellul domainar (ECD) being on the surface of the cell.

[00210] In thi sembodiment, cell sexpressing exogenous binding and/or signaling partner for HIDE1 are contacted with candidate agent(s) as is more fully outlined below, and a labeled binding and/or signaling partner for HIDE1 (generall they ECD domain). By 49WO 2017/009712 PCT/IB2016/001079 comparing the resul tsto a reference standard not includin gthe candidate agent, where binding is known to occur the, lack of bound label means the candidate agent binds to the binding and/or signaling partner for HIDE1 in such a way as to preven tbinding to binding and/or signaling partner for HIDE1.

[00211] As wil bel appreciate byd those in the art, these assays can be done on surface s such as in microtit plater es.

B. Support Based Assays

[00212] In one embodiment, the screening assay is a soli dsupport assay, where one or the other of HIDE 1 and/or the binding and/or signaling partner for HIDE1 is attache d,for example to a microtit plater e.Candidate agents and labeled proteins e.g., the “other” of the HIDE1 and/or the binding and/or signaling partner for HIDE1 is added. If the candidat e agent blocks binding, thi scan be determined using the read out. For example, in one embodiment, HIDE1 is attached to the soli dsuppor t,generally at discre telocations. A candidate agent and the binding and/or signaling partner for HIDE1 is added, for example that is either direct lylabeled (for example with a fluorophore as outlined below), or indirect ly labeled (for example using a labeled anti-bindin gand/or signaling partne forr HIDE1 antibody). After allowing a sufficient period of time and after washing, if the candidate agent blocks the interaction of HIDE1 and the binding and/or signaling partner for HIDE1, no signal will be seen. If the agent does not, the signal will be generated and bound to the support. Similarl thiy, scan be done using attachment of the binding and/or signaling partner for HIDE1 to the soli supportd and adding labeled HIDE1 and candidate agents.

C. FRET Based Assays

[00213] In some embodiments, thi sbinding assay can be done using fluorescent resonance energy transfe (FRET)r assays ,as is well known in the art, where one of the receptor-ligand pair of HIDE 1 and the binding and/or signaling partner for HIDE1 has a FRET donor and the other has a FRET acceptor. Upon binding of the two, FRET occurs. If the candidate agent prevents binding, the FRET signal wil bel lost This. is also useful in competiti onassays, to determine whether the binding of the agent to HIDE1 is stronger than the binding of the binding and/or signaling partner for HIDE1. 50WO 2017/009712 PCT/IB2016/001079 D. Functional Assays

[00214] These identified candidate agents that bind and block the interaction of HIDE1 and/or the binding and/or signaling partner for HIDE1 can then be further teste tod see their effect on the signaling pathwa y.That is, the binding/blocking agents can be run in assays that measure immuno-supressive function of myeloid cells including but not limite tod monocyte s,dendritic cells, macrophages, M1/M2 tumo rassociated macrophages, neutrophil s, Myeloid-derive suppressor cells (MDSC), and/or T cel lor NK cel lactivation and or migration, for example, to determine whether the blocking agents are immunostimulatory (increasing immune function such that diseases such as cancer can be treated) or immunoinhibitory (decreasing immune function to trea autoit mmune diseases and inflammatio orn increasing or ehancing immune suppression).

[00215] According to at leas tsome embodiment ofs the present invention also provides antibodies, includin gantigen binding domains, that bind to human HIDE1 for use in methods of depleting myeloid cell s,or other circulati ngtumor cell s,in order to trea diset ases such as cancer.

[00216] In addition, the assays below can also be used to assess treatment efficacy, as is more further outlined below.

[00217] In some embodiments, the functional assay uses CTLs. The CTLs express a T cel lreceptor (TCR) recognizing a specific antigen (Ag) presented on an MHC molecule .

Upon TCR Ag engagement, CTLs undergo activation as manifested by cell proliferat ion,up- regulati onof activation markers (e.g. CD25, CD137 etc.), and cytokine secretion (e.g. interferon gamma, IL2, TNFa etc.) and cytotoxic activity. Upon contact with the binding and/or signaling partner for HIDE1 expressed on cancer cell sor antigen presentin celg ls, HIDE1 mediate sa negative signal to CTLs thereby causing down-regulat ofion CTL activation as manifested by the above readouts. Thus, contacting candidate agents that have shown binding and/or inhibition of receptor-ligand binding wit hCTLs wil tol interrupt the HIDE1 and/or the binding and/or signaling partner for HIDE1 interaction and thereby release the negative signal mediated by HIDE1 and enhances antigen specific CTL activation as 51WO 2017/009712 PCT/IB2016/001079 manifeste dby cel lproliferati up-regulon, ation of activation markers (e.g. CD25, CD137 etc.), and cytokine secretion (e.g. interferon gamma, IL2, TNF alfa etc.).

[00218] Similarly, in some embodiment s,the functional assay uses NK cells. The NK cells express various activating and inhibito ryreceptors The. execution of NK cytotoxic activit yis determined by the balance between the activatory and inhibitory signals derived from thes recee ptors Upon. engagement of NK cells with certain target cells, NK cells underg oactivation as manifested by cel lproliferat ion,cytokine secretion (e.g. interferon gamma, IL2, TNF alfa etc.) and cytotoxic activit y.Upon contact with the HIDE1 and/or the binding and/or signaling partner expresse don cancer target cells, HIDE1 mediates a negative signal to NK cell sthereby causing down-regulation of NK cel lactivation as manifested by the above readouts Contact. ing of candidate agents with NK cells will interrupt the HIDE1 and/or the binding and/or signaling partner for HIDE1 interaction and thereby releas thee negative signal mediated by HIDE1 and enhances NK cel lactivation as manifested by cell proliferat ion,cytokine secretion (e.g. interferon gamma, IL2, TNF alfa etc.) and cytotoxic activity.

[00219] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in immune response as measured for an example by phosphorylati or onde- phosphorylat ofion different factors, or by measuring other post translational modifications.

An increase in activity indicate simmunostimulator actiyvity and a decrease indicate s immunoinhibitory activit y(or an increase and/or enhancement in immune suppression) .

Appropriat incree ases or decreases in activity are outlined below.

[00220] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in activation of a,|3 and/or y5 T cell sas measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in activit yindicate simmunostimulatory activit yand a decrease indicate simmunoinhibitory activit y(or an increase and/or enhancement in immune suppression). Appropriat incree ases or decreases in activit yare outlined below. 52WO 2017/009712 PCT/IB2016/001079

[00221] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in cytotoxi Tc cel lactivity as measured for an example by direc kilt lin ofg target cells like for an example cancer cells or by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc .

An increase in activity indicate simmunostimulator actiyvity and a decrease indicate s immunoinhibitory activit y(or an increase and/or enhancement in immune suppression) .

Appropriat incree ases or decreases in activity are outlined below.

[00222] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in NK and/or NKT cell activity as measured for an example by direc kilt ling of target cells like for an example cancer cell sor by cytokine secretion or by changes in expression of activation markers like for an example CD107a, etc. An increase in activity indicate simmunostimulator actiyvit yand a decrease indicates immunoinhibitory activity(or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.I onen embodiment, the signaling pathwa assy ay measure s increases or decreases in 0,(3 and/or y5 T-cel lsuppression, as measured for an example by cytokine secretion or by proliferati oron by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc .An increase in activit yindicates immunostimulatory activit yand a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriat incree ases or decreases in activity are outlined below.

[00223] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in pro-inflammatory cytokine secretion as measured for example by ELISA or by Luminex or by Multiple beadx based methods or by intracellula stainingr and FACS analysis or by Alispot etc. An increase in activit yindicates immunostimulatory activit yand a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriat incree ases or decrease ins activity are outlined below.

[00224] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in IL-2 secretion as measured for example by ELISA or by Luminex or by Multiple beadx based methods or by intracellul staiarning and FACS analysi sor by Alispot 53WO 2017/009712 PCT/IB2016/001079 etc. An increase in activity indicate simmunostimulatory activity and a decrease indicates immunoinhibitory activit y(or an increase and/or enhancement in immune suppression).

[00225] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in interferon-Y production as measured for example by ELISA or by Luminex or by Multiple beadx based methods or by intracellul staiarning and FACS analysi sor by Alispot etc. An increase in activit yindicates immunostimulatory activity and a decrease indicate simmunoinhibitory activit y(or an increase and/or enhancement in immune suppression). Appropriat incree ases or decrease ins activity are outlined below.

[00226] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in Thl response as measured for an example by cytokine secretion or by changes in expression of activation markers An. increase in response indicate simmunostimulatory activit yand a decrease indicate simmunoinhibitory activit y(or an increase and/or enhancement in immune suppression). Appropriat incree ases or decreases in activit yare outlined below.

[00227] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in Th2 response as measured for an example by cytokine secretion or by changes in expression of activation markers An. increase in response indicate simmunostimulatory activit yand a decrease indicate simmunoinhibitory activit y(or an increase and/or enhancement in immune suppression). Appropriat incree ases or decreases in activity are outlined below.

[00228] In one embodiment, the signaling pathwa yassay measure sincreases or decreases cel lnumber and/or activit yof at least one of regulatory T cells (Tregs), as measured for example by flow cytometry or by IHC. A decrease in response indicates immunostimulatory activit yand an increase indicate simmunoinhibitory activit y(or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.

[00229] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in M2 macrophages cel lnumbers ,as measured for example by flow cytomet ryor by IHC. A decrease in response indicates immunostimulator actyivit yand an increase 54WO 2017/009712 PCT/IB2016/001079 indicate simmunoinhibitory activit y(or an increase and/or enhancement in immune suppression). Appropriat incree ases or decreases in activit yare outlined below.

[00230] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in M2 macrophage pro-tumorigenic activit y,as measured for an example by cytokine secretion or by changes in expression of activation markers. A decrease in response indicate simmunostimulator actiyvit yand an increase indicate simmunoinhibitory activit y(or an increase and/or enhancement in immune suppression).

[00231] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in N2 neutrophil increase,s as measured for example by flow cytometry or by IHC.

A decrease in response indicate simmunostimulatory activity and an increase indicates immunoinhibitory activit y(or an increase and/or enhancement in immune suppression) .

Appropriat incree ases or decreases in activity are outlined below.

[00232] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in N2 neutrophil pro-tumoriges activinic ty, as measured for an example by cytokine secretion or by changes in expression of activation markers. A decrease in response indicate simmunostimulator actiyvit yand an increase indicate simmunoinhibitory activit y(or an increase and/or enhancement in immune suppression). Appropriat incree ases or decreases in activity are outlined below.

[00233] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in inhibition of T cel lactivation, as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in response indicate simmunostimulatory activit yand a decrease indicate simmunoinhibitor actiy vity (or an increase and/or enhancement in immune suppression). Appropriat incree ases or decrease ins activit yare outlined below.

[00234] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in inhibition of CTL activation as measured for an example by direc killit ngof target cells like for an example cancer cell sor by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc .

An increase in response indicate simmunostimulatory activit yand a decrease indicates 55WO 2017/009712 PCT/IB2016/001079 immunoinhibitory activit y(or an increase and/or enhancement in immune suppression) .

Appropriat incree ases or decreases in activity are outlined below.

[00235] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in a(3 and/or y5 T cell exhaustion as measured for an example by changes in expression of activation markers A. decrease in response indicate simmunostimulatory activity and an increase indicate simmunoinhibitory activit y(or an increase and/or enhancement in immune suppression). Appropriat incree ases or decreases in activit yare outlined below.

[00236] In one embodiment, the signaling pathwa yassay measure sincreases or decreases a,[3 and/or y5 T cel lresponse as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD 13 7, CD 107a, PD1, etc .An increase in activity indicates immunostimulatory activit yand a decrease indicate simmunoinhibitory activit y(or an increase and/or enhancement in immune suppression) Appropriat. incree ases or decrease ins activit yare outlined below.

[00237] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in stimulation of antigen-specific memory responses as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD45RA, CCR7 etc. An increase in activity indicate s immunostimulatory activit yand a decrease indicate simmunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriat incree ases or decreases in activity are outlined below.

[00238] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in apoptos oris lysi sof cancer cells as measured for an example by cytotoxici ty assays such as for an example MTT, Cr releas e,Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc . An increase in activity indicate simmunostimulatory activity and a decrease indicates immunoinhibitory activit y(or an increase and/or enhancement in immune suppression). Appropriat incree ases or decrease ins activit yare outlined below. 56WO 2017/009712 PCT/IB2016/001079

[00239] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in stimulation of cytotoxic or cytostat effecic t on cancer cells, as measured for an example by cytotoxicit assaysy such as for an example MTT, Cr releas e,Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc. An increase in activit yindicates immunostimulator actiyvit yand a decrease indicates immunoinhibitory activit y(or an increase and/or enhancement in immune suppression) .

Appropriat incree ases or decreases in activity are outlined below.

[00240] In one embodiment, the signaling pathwa yassay measure sincreases or decreases direc killingt of cancer cell sas measured for an example by cytotoxicit assaysy such as for an example MTT, Cr release, Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc . An increase in activity indicate simmunostimulator actiyvit yand a decrease indicates immunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriate increases or decreases in activity are outlined below.

[00241] In one embodiment, the signaling pathwa yassay measure sincreases or decreases Thl7 activit yas measured for an example by cytokine secretion or by proliferati on or by changes in expression of activation markers. An increase in activit yindicates immunoinhibitory activity and a decrease indicate simmunostimulatory activity (or an increase and/or enhancement in immune suppression). Appropriat incree ases or decreases in activity are outlined below.

[00242] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in induction of complement dependent cytotoxici and/orty antibody dependent cell - mediated cytotoxicity, as measured for an example by cytotoxici assty ays such as for an example MTT, Cr releas e,Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc. An increase in activit yindicates immunostimulatory activit yand a decrease indicate simmunoinhibitory activity (or an increase and/or enhancement in immune suppression). Appropriat incree ases or decreases in activity are outlined below. 57WO 2017/009712 PCT/IB2016/001079

[00243] In one embodiment the, assay measure sincreases or decrease ins cell proliferation as a function of activation or inhibition, using well known methodolo giessuch as thymidine incorporat ionand CFSE dilution.

[00244] Appropriat incree ases in activit yor response (or decreases, as appropriat ase outlined above), are increases of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98 to 99% percent over the signal in either a reference sample or in control samples, for example test samples that do not contain an anti-HIDEl antibody of the invention. Similarly, increases of at leas tone-, two-, three-, four- or five-fold as compared to reference or control samples show efficacy.

E. Candidate Agents

[00245] The assays are run by contacting candidate agents with the HIDE1 and the binding and/or signaling partner for HIDE1. By “candidate agent”, “candidate bioactive agent” or “candidate drugs” or grammatical equivalent hereis nis meant any molecule, e.g. proteins (which herei nincludes proteins poly, peptide ands, peptides), smal lorganic or inorganic molecules, polysaccharides, polynucleotides, etc. whic hare to be tested for binding to HIDE1, inhibition of the HIDE1 and the binding and/or signaling partner for HIDE1 interaction, or activation of the binding and/or signaling partner for HIDE1. Candidate agents encompass numerous chemical classes. In a preferre embodimed nt, the candidate agents are organic molecules, particularly small organic molecules, comprising functional groups necessary for structural interaction wit hproteins, particularly hydrogen bonding, and typicall incly ude at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups The. candidate agents often comprise cyclical carbon or heterocycli strc uctures and/or aromat icor polyaroma ticstructures substitut wited hone or more chemical functional groups.

[00246] Candidate agents are obtained from a wide variet yof sources as, will be appreciate byd thos ine the art, including libraries of synthet oric natural compounds As. will be appreciate byd thos ine the art, in some embodiments the present invention provides a rapid and easy method for screening any library of candidate agents ,includin gthe wide variet yof known combinator ialchemistry-typ librarie es. 58WO 2017/009712 PCT/IB2016/001079

[00247] In a preferred embodiment, candidate agents are synthet comic pounds Any. number of techniques are available for the random and directed synthesis of a wide variet yof organic compounds and biomolecul es,including expression of randomized oligonucleoti des.

Alternative aly, preferred embodiment utilizes libraries of natural compounds in the form of bacterial fungal,, plant and animal extract thats are available or readily produced.

[00248] Additional naturly, al or syntheticall producey librad ries and compounds are readil modiy fied through conventional chemical, physical and biochemica lmeans. Known pharmacologic agenal ts may be subjected to directed or random chemical modificatio ns, including enzymatic modificatio ns,to produce structural analogs.

[00249] In a preferred embodiment, candidate bioactive agents include proteins, nucleic acids ,and chemical moieties.

[00250] In a preferred embodiment, the candidate bioactive agents are proteins. By “protei” nherei nis meant at least two covalentl atty ached amino acids, which includes proteins polypepti, des, oligopeptides and peptides. The protei mayn be made up of naturall y occurring amino acids and peptide bonds, or synthet peptidic omimetic structures. Thus “amino acid”, or “peptide residue”, as used herein means both natural lyoccurring and syntheti aminoc acids. For example, homo-phenylalani citrulne, line and noreleucine are considered amino acids for the purposes of the invention. “Amino acid” also includes imino acid residues such as proli neand hydroxyproline. The side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L- configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to preven tor retard in vivo degradations.

[00251] In a preferred embodiment, the candidate bioactive agents are naturall y occurring proteins or fragments of naturall occurriy ng proteins Thus,. for example, cellular extract contais ning proteins, or rando mor directed digests of proteinaceous cellula extracr ts, may be attached to beads as is more fully describe belod w. In thi sway libraries of procaryotic and eucaryoti proteic nsmay be made for screening against any number of targets.

Particularly preferred in thi sembodiment are libraries of bacterial, fungal, viral, and 59WO 2017/009712 PCT/IB2016/001079 mammalian proteins with, the latt erbeing preferred, and human proteins being especially preferred.

[00252] In many embodiment s,the candidate agents are antibodies to HIDE1, generated as is known in the art and outlined herein.

[00253] In many embodiment s,the candidate agents are ECDs of HIDE1, includin g fusion proteins and variants as, is known in the art and more fully outlined herein.

[00254] In a preferred embodiment, the candidate bioactive agents are peptides of from about 2 to about 50 amino acids, with from about 5 to about 30 amino acids being preferred, and from about 8 to about 20 being particularl preferred.y The peptides may be digests of natural lyoccuring proteins as is outlined above, rando mpeptides or, “biased” rando m peptide s.By “randomized” or grammatical equivalents herei nis meant that each nucleic acid and peptide consists of essentiall randomy nucleotides and amino acids, respectivel Sincey. generall thesy randome peptides (or nucleic acids ,discussed below) are chemically synthesized, they may incorporate any nucleoti deor amino acid at any position. The synthet ic proces cans be designed to generat erandomized proteins or nucleic acids ,to allow the formation of all or most of the possibl combinae tions over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.

[00255] The librar shouldy provide a sufficiently structural diversely population of randomized agents to effect a probabilistic alsufficly ient range of diversity to allow binding to a particular target Accordi. ngly, an interaction library must be large enough so that at least one of its members wil havel a structure that gives it affinity for the target Alt. hough it is difficult to gauge the required absolute size of an interaction librar naturey, provides a hint with the immune response a: diversi tyof 107 -108 different antibodie providess at least one combination wit hsufficient affinity to interact wit hmost potential antigens faced by an organism. Publishe ind vitro selection techniques have also shown that a library size of 107 to 108 is sufficient to find structures wit haffinity for the target A. library of all combinations of a peptide 7 to 20 amino acids in length, such as generally proposed herein, has the potential to code for 207 (109) to 2020. Thus, with libraries of 107 to 108 different molecule thes present methods allo wa “working” subset of a theoretica compllly et interacte ion library for 7 amino 60WO 2017/009712 PCT/IB2016/001079 acids, and a subset of shapes for the 202° library. Thus, in a preferre embodiment,d at least .sup.6, preferably at least 107, more preferabl aty least 108 and most preferably at least 109 different sequences are simultaneously analyzed in the subject methods. Preferred methods maximize librar sizy e and diversity.

[00256] In one embodiment, the library is fully randomized, wit hno sequence preferences or constant ats any position. In a preferred embodiment, the librar isy biased .

That is, some positions within the sequence are either held constan t,or are selected from a limited number of possibiliti Fores. example, in a preferred embodiment, the nucleotide ors amino acid residues are randomized within a defined class, for example, of hydrophob ic amino acids ,hydrophil resiic dues ster, icall biasedy (either smal lor large) residues, towar ds the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylat sitiones, etc., or to purines, etc.

[00257] In some embodiments, the present inventio nprovides a method of screening for inhibitors of the binding association of HIDE 1 polypepti dewit ha HIDE1 binding and/or signaling partner polypeptide, said method comprising: a) providing a surfac ecomprising a firs ligandt prote incomprising HIDE1 polypeptide or HIDE1 binding and/or signaling partner polypeptide; b) contacting said surfac ewith a candidate agent under physiologi cal conditions, wherei ifn said candidate agent binds to said first ligand protei itn forms a first binding complex c), contacting said surfac ewit ha second ligand prote incomprising the othe r of HIDE1 polypeptide or HIDE1 binding and/or signaling partne polypeptr ide; and d) determining whether said HIDE1 polypepti deand said HIDE1 binding and/or signaling partner polypeptide are bound as an indication of whether said candidate agent inhibits said binding association.

[00258] In some embodiments, the present inventio nprovides a method of screening for inhibitors of the binding association of HIDE 1 polypepti dewit hHIDE1 binding and/o r signaling partner polypeptide, said method comprising: a) providing a cel lcomprising an exogeneous recombinan nuclet ic acid encoding a human HIDE1 polypeptide, wherei saidn cel lexpresses said human HIDE1 polypeptide; b) contacting said cel lwith a candidate agent and a labeled HIDE1 binding and/or signaling partner polypeptide; c) determining whether said HIDE1 polypeptide binds to HIDE1 binding and/or signaling partner polypeptide as an 61WO 2017/009712 PCT/IB2016/001079 indication of whether said candidate agent inhibit thes binding of HIDE 1 polypeptide wit h HIDE1 binding and/or signaling partner polypeptide.

[00259] In some embodiments, the present inventio nprovides a method of screening for inhibitors of the binding association of HIDE 1 polypepti dewit hHIDE1 binding and/o r signaling partner polypeptide, said method comprising: a) providing a cel lcomprising an exogeneous recombinan nucleict acid encoding a human HIDE1 binding and/or signaling partner polypeptide, wherein said cel lexpresses said human HIDE1 binding and/or signaling partner polypeptide; b) contacting said cel lwit ha candidate agent and a labeled HIDE1 polypeptide; c) determining whether said HIDE1 binding and/or signaling partne r polypeptide binds to HIDE1 as an indication of whether said candidate agent inhibits the binding of HIDE 1 polypeptide wit hHIDE1 binding and/or signaling partner polypeptide.

[00260] In some embodiments, the present inventio nprovides a method of screening for inhibitors of the binding association of HIDE 1 polypepti dewit hHIDE1 binding and/o r signaling partner polypeptide, said method comprising:a providing) a test soluti on comprising: i) a HIDE1 polypeptide comprising a first FRET label; ii) a HIDE1 binding and/or signaling partner polypeptide comprising a second FRET label; c) providing a candidate agent; d) detecting a FRET signal between said firs andt second label, wherei an difference in said FRET signal in the presenc eor absence of said candidate agent indicate sthat the candidate agent inhibits said binding association. In some embodiments of the method, a plural ityof candidate agents are tested In. some embodiments of the method, thecandidate agent is a protein. In some embodiments of the method, the prote inis an anti- HIDE1 antibody. In some embodiments of the method, the prote incomprises an extracellular domain (ECD) of HIDE 1. In some embodiments of the method, the prote inis a fusion prote in comprising said ECD and a fusion partner.

[00261] In some embodiments, the fusion partner is selected from the group consisting of a human IgG Fc domain and a human serum albumin (HSA).

[00262] In some embodiments of the invention, said the method furth ercomprise s:a) contacting said candidate agent with a population of cytotoxic T cells (CTLs) under 62WO 2017/009712 PCT/IB2016/001079 conditions wherei saidn CTLs would normall bey activated; and b) determining the effect of said agent on said activation.

[00263] In some embodiments of the invention, the method further comprises: a) contacting said candidate agent with a population of cytotoxic T cells (CTLs); an b) determining the effect of said agent on IFNy production.

[00264] In some embodiments of the invention, themethod further comprises: a) contacting said candidate agent with a population of y5 T cells under conditions wherein said y5 T cells would normally be activated; and b) determining the effect of said agent on said activation.

[00265] In some embodiments of the invention, the method further comprises:a) contacting said candidate agent with a population of Thl cells under conditions wherei saidn Thl cell swould normall bey activated; and b) determining the effect of said agent on said activation.

[00266] In some embodiments of the invention, the method further comprises: a) contacting said candidate agent with a population of regulatory T cells (Tregs )under conditions and determining the effect of said agent on Treg cel lnumber or activity.

[00267] In some embodiments of the invention, the determinati ison done by measuring the presence or absence of increased expression of a protei seln ected from the group consisting of IFNg, TNFa, GM-CSF, CD25, CD 13 7, CD69, PD1, CD 107 A, HLA-DR, IL-2, IL-6, IL-4, IL-5, IL-10 and IL-13, wherei increasn ed expression is an indication of activation.

IV. HIDE1 Proteins

[00268] According to at leas tsome embodiments of the present inventio nis directed to the use of human HIDEl ,as outlined below. As used herein, the term "HIDEl" or "HIDEl protei” nor “HIDEl polypepti”de may optionall incly ude any such protein, or variant s, conjugates, or fragments thereof, includin gbut not limite tod known or wil dtype HIDEl ,as described herein, includin gbut not limite tod SEQ ID NO: 1, and also other HIDEl variants, including but not limite tod SEQ ID NOs: 7-11, as well as any solubl HIDEle protein, including but not limite tod any of SEQ ID NOs: 2,3,6,19-47, and/or variants thereof 63WO 2017/009712 PCT/IB2016/001079 possessin atg least 80% sequence identity, more preferably at least 90% sequence identit y therewit andh even more preferably at least 95, 96, 97, 98 or 99% sequence identit y therewith, and/or fusions and or conjugates thereof and/, or polynucleoti desencoding same.

As used herein, the term "HIDE1" or “HIDE1 protei” nor “HIDE1 polypepti”de may optionally include any such protein, or variants, conjugates, or fragments thereof, includin g but not limited to non-human HIDE1 orthologs such, as for example, mouse HIDE1 prote in as set forth in SEQ ID NO: 12, and/or its correspond ingexracellular domain, as set forth in any of SEQ IDNOs: 13-16.

[00269] The HIDE 1 sequence, as describe hereid nunder SEQ ID NO: 1, is copied below.

MPWTILLFAAGSLAIPAPSIRLVPPYPSSQEDPIHIACMAPGNFPGANFTLYRGGQVVQLLQAPTDQRGVTFNLS GGSSKAPGGPFHCQYGVLGELNQSQLSDLSEPVNVSFPVPTWILVLSLSLAGALFLLAGLVAVALVVRKVKLRNL QKKRDRESCWAQINFDSTDMSFDNSLFTVSAKTMPEEDPATLDDHSGTTATPSNSRTRKRPTSTSSSPETPEFST FRACQ

[00270] The HIDE1 ECD sequence, as describe hereid nunder SEQ ID NO:2, copied below.

IPAPSIRLVPPYPSSQEDPIHIACMAPGNFPGANFTLYRGGQVVQLLQAPTDQRGVTFNLSGGSSKAPGGPFHCQ YGVLGELNQSQLSDLSEPVNVSFPVPTWIL

[00271] The HIDE1 ECD sequence, as describe hereid nunder SEQ ID NO:3, copied below.

IPAPSIRLVPPYPSSQEDPIHIACMAPGNFPGANFTLYRGGQVVQLLQAPTDQRGVTFNLSGGSSKAPGGPFHCQ YGVLGELNQSQLSDLSEPVNVSFPVPTW

[00272] The term “soluble” form of HIDE 1 is also used interchangeably wit hthe term s “solubl ectodoe mai (ECD)n ” or “ectodomain” or “fragments of HIDE 1 polypeptides” or “extracellular domain” , and whic hmay refer broadly to one or more of the follow ing optional polypeptides:

[00273] Optionally, the HIDE1 ECD proteins and fragments thereof refe rto any one of the polypepti desequences liste ind any of SEQ ID NOs: 2-6, 21-47, and/or variants thereof possessin atg least 80% sequence identity, more preferably at least 90% sequence identit y 64WO 2017/009712 PCT/IB2016/001079 therewit andh even more preferably at least 95, 96, 97, 98 or 99% sequence identit y therewith, and/or mouse ortholog thereof listed in any one of SEQ ID NOs: 13-16, and/or fusions and or conjugate sthereof, and/or polynucleoti desencoding same.

[00274] Optionall they, fragment is of at least about 95 and so forth amino acids of the extracellul domainar of HIDE1 protein, set forth in SEQ ID NO: 2, up to 115 amino acids of the HIDE1 prote inextracellul domaar in, optionall includingy any integral value between 95 and 115 amino acids in length. Preferably, the fragment is of at leas tabout 101 and up to 109 amino acids of the HIDE1 protei extran cellula domair n, optional inclly uding any integral value between 101 and 109 amino acids in length. Also preferabl they fragment is of at least about 103 up to 105 amino acids of the HIDE1 prote inextracellula domar in, optional ly including any integral value between 103 and 105 amino acids in length. More preferably, the fragment is about 105 amino acids. The HIDE1 fragment protei accordin ng to at least some embodiments of the present invention may or may not include a signal peptide sequence, and may or may not include 1, 2, 3, 4, or 5 contiguous amino acids from the HIDE1 transmembrane domain.

[00275] In particular, the fragments of the extracellula domainr of HIDE1 can include any sequence correspond toing any portion of or comprising the Ig domain of the extracellul domainar of HIDE1, having any sequence corresponding to residues of HIDE1 (SEQ ID NO:1) starting from any position between 17 and 21 and ending at any position between 109 and 113.

[00276] Without wishing to be limite byd a singl ehypothesi thes, HIDE1 protei ns contain an immunoglobulin domain within the extracellula domair n. The Ig domain may optionally be responsibl fore receptor binding, by analogy to the other B7 family members. The Ig domain of the extracellula domainr includes one disulfide bond formed between intra domain cysteine residues, as is typica lfor thi sfold and may optionall bey important for structure-function In SEQ. ID NO: 2 these cysteines are located at residues 19 and 69.

[00277] In one embodiment, there is provided a solubl fragmee nt of HIDE1; as described in greater deta ilbelow with regard to the section on fusion proteins, such a soluble 65WO 2017/009712 PCT/IB2016/001079 fragment may optionally be described as a first fusion partner. Useful fragments are those that alone or when comprised in fusion proteins or multimerized retain the ability to bind to their natural molecular partner or partners, e.g, expressed on antigen presenting T, and NK cell s,and/or which modulate T cel land/or NK cell activation. A HIDE1 polypeptide that is a fragment of full-leng HIDE1th typicall hasy at least 20 percent, 30 percent, 40 percent, 50 percent, 60 percent, 70 percent, 80 percent, 90 percent, 95 percent, 98 percent, 99 percent, 100 percent, or even more than 100 percent of the abilit yto bind its natural molecula r partner(s) and/or of the modulati on(preferably enhancing and/or agonizing of one or more of the functional effects of HIDE1 on immunity and on specific immune cells as compared to full-length HIDE1; optionall suchy a percentage may be any integral value between 20 and 100 percent.

[00278] Soluble HIDE1 polypeptide fragments are fragments of HIDE1 polypeptides that may optionally be shed, secreted or otherwi extrase cte fromd the producing cells. In othe embodimer nts, the solubl fragmene ts of HIDE1 polypeptides include fragments of the HIDE1 extracellul domainar that retain HIDE1 biologica activil ty, such as fragments that retain the abilit yto bind to thei naturalr receptor or receptor and/ors whic hmodulate T or NK cel lactivation. The extracellular domain can include 1, 2, 3, 4, or 5 contiguou amis no acids from the transmembrane domain, and/or 1, 2, 3, 4, or 5 contiguous amino acids from the signal sequence. Alternative thely, extracellul domainar can have 1, 2, 3, 4, 5 or more amino acids removed from the C-terminus, N-terminus or, both.

[00279] In some embodiments the HIDE1 extracellul domainar polypeptide comprises the amino acid sequence of the Ig domain as set forth in any one of SEQ ID NO: 6, 24-47, or fragments or variants thereof. In othe embodimr ents the HIDE1 extracellula domair n polypeptide consists essentiall ofy the amino acid sequence of the Ig domain as set forth in any one of SEQ ID NOs: 6, 24-47.

[00280] Generall y,the SEQ ID NOs:6, 24-47polypeptide fragments are expressed from nucleic acids that include sequences that encode a signal sequence. The signal sequence is generally cleaved from the immature polypepti deto produce the mature polypeptide lacking the signal sequence. The signal sequence of HIDE 1 can be replaced by the signal sequence of another polypeptide using standard molecul biologye technique sto affect the 66WO 2017/009712 PCT/IB2016/001079 expression levels, secretio solubilin, ty,or other property of the polypeptide The. signal peptide sequence that is used to replace the HIDE1 signal peptide sequence can be any known in the art.

[00281] In one embodiment such “solubl ecte odomain (ECD)” or “ectodomain” or “soluble” form of HIDE1 will modulate (preferabl enhancey and/or agonize) one or more of HIDEl’s effects on immunity and specific types of immune cells such as T helpe r,cytotoxi c or effector T cell s,Tregs NK cell sand antigen presentin celg ls.

[00282] Optionally, the HIDE1 ECD fragments refer also to any one of the polypeptide sequences listed in any of SEQ ID NOs: 19-20, whic hare reasonabl expecty ed to comprise functional regions of the HIDE1 protein. This expectation is based on a systematic analysis of a set of prote incomplexes with solved 3D structures whic, hcontaine dcomplexes of Ig proteins (for example PDB ID li85 which describe the complex of CTLA4 and CD86). The intermolecular contact residues from each co-structure were collect edand projected on the sequence of HIDE1. Several regions with clusters of interacti ngresidues support edby several contact maps were identified and are reasonabl expectedy to mimic the structure of the intact full length prote inand thereby modulate one or more of the effects of HIDE1 on immunity and on specific immune cel ltypes.The HIDE1 extracellular domain polypeptides according to at least some embodiments of the present inventio nare expected to be useful for treatment of autoimmune diseases whic hhave proved resistant to othe drugr treatment s, because, without wishing to be limited by a singl ehypothesis, the mode of action of thes e HIDE1 extracellula domainr polypeptides is expected to enable them to overcome such resistance. Methods of treatment are further described herein, below under the methods of using section.

[00283] According to at least some embodiments there is provided an isolated or recombinan HIDE1t new variant polypepti deor a fragment thereof, consisting essentially of an amino acid sequence as set forth in any of SEQ ID NOs: 7-11, or variant thereof that possesses at least 90% or 95% , 96%, 97%, 98%, or 99% sequence identity therewith.

[00284] According to at least some embodiments there is provided an isolated or recombinan HIDEt 1 polypeptide comprising aHIDEl ECD, consisting essential lyof an 67WO 2017/009712 PCT/IB2016/001079 amino acid sequence as set forth in any of SEQ ID NOs: 2-5, 21-23, or a fragment thereof or variant there ofthat possess esat least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewi th,or mouse orthol correspondog toing amino acid sequence as set forth in any one of SEQ ID NOs: 13-16.

[00285] According to at least some embodiments there is provided an isolated or recombinan HIDE1t ECD fragment consis, ting essentiall ofy an amino acid sequence as set forth in any of SEQ ID NOs: 6, 19-20, 24-47 or variant thereof that possesses at leas t90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewith.

[00286] According to at least some embodiments there is provided an isolated or recombinan HIDE1t polypeptide comprising a multimer or a fusion polypeptide comprising a HIDE1 ECD, consisting essential lyof an amino acid sequence as set forth in any of SEQ ID NOs: 2-5, 21-23 or variant there ofthat possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewi th,or mouse ortholog correspondin to gamino acid sequence as set forth in any one of SEQ ID NOs: 13-16, or HIDE1 ECD fragment thereof, whic h fragment consists essentiall ofy an amino acid sequence as set forth in any of SEQ ID NOs: 6, 19-20, 24-47 or variant thereof that possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewi th,wherein such multimer or fusion polypeptide may optional ly comprise one or more of such HIDE1 ECD polypeptides or fragments thereof e.g,, 1-10 fragments direc, tly linked or attached to one another or fused via a linke orr multimerizati on domain.

[00287] According to at leas tsome embodiments there is provided a fusion prote in comprising the polypepti decomprising aHIDEl ECD, consisting essentiall ofy an amino acid sequence as set forth in any of SEQ ID NOs:2-5, 21-23, or variant there ofthat possesses at leas t90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewi th,or mouse ortholog correspond toing amino acid sequence as set forth in any one of SEQ ID NOs: 13-16, or HIDE1 ECD fragment thereof, whic hfragment consists essential lyof an amino acid sequence as set forth in any of SEQ ID NOs: 6, 19-20, 24-47 or variant there ofthat possesse s at leas t90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewith, joined to a second fusion partner composed of a heterologous sequence (z.e., anon- HIDE1 polypeptide), fused together direct lyor indirectl viay a peptide linke sequencer or a chemical linker. Optionally, 68WO 2017/009712 PCT/IB2016/001079 the heterologous sequence compris esat least a portion of an immunoglobulin molecul e.

Optionally and preferably, the immunoglobul molin ecul porte ion is an immunoglobulin heavy chain constant region Fc fragment Optional. lyand more preferably, the immunoglobul heavyin chain constant regio nis derive dfrom an immunoglobulin isotype selected from the group consisting of an IgGl ,IgG2, IgG3, IgG4, IgM, IgE, IgA and IgD.

Optionally, the fusion prote inhas the amino acid sequence set forth in any one of SEQ ID NOs: 17, 18, and also optionally modulates immune cel lrespons ine vitro or in vivo.

[00288] According to at least some embodiments, the subject inventio nprovides isolated nucleic acid sequences encoding any one of the foregoing HIDE1 polypeptides comprising a HIDE1 ECD, consisting essentiall ofy an amino acid sequence as set forth in any of SEQ ID NOs: 2-5, 21-23, or variant there ofthat possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewi th,or mouse ortho logcorrespondin to gamino acid sequence as set forth in any one of SEQ ID NOs: 13-16, or HIDE1 ECD fragment thereof, which fragment consists essentiall ofy an amino acid sequence as set forth in any of SEQ ID NOs: 6, 19-20, 24-47 or variant thereof that possesses at least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewit orh, multimers or fusion proteins thereof.

[00289] According to at least some embodiments, there is provided an expression vector or a virus, containing at least one isolated nucleic acid sequence as described herein.

According to at least some embodiment s,there is provided a recombinan celt lcomprising an expression vector or a virus containing an isolated nucleic acid sequence as describe hereid n, wherei then cell constitutivel or yinducibly expresses the polypeptide encoded by the DNA segment. According to at least some embodiments, there is provided a method of producing any one of the foregoing HIDE1 polypeptides comprising aHIDEl ECD, consisting essential lyof an amino acid sequence as set forth in any of SEQ ID NOs: 2-5, 21-23, or variant there ofthat possess esat least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewi th,or mouse orthol correspondog toing amino acid sequence as set forth in any one of SEQ ID NOs: 13-16, or HIDE1 ECD fragment thereof, which fragment consist s essential lyof an amino acid sequence as set forth in any of SEQ ID NOs: 6, 19-20, 24-47, or variant there ofthat possess esat least 90% or 95%, 96%, 97%, 98%, or 99% sequence identity therewi th,or fusion proteins thereof, comprising culturin theg recombinant cel las 69WO 2017/009712 PCT/IB2016/001079 described herein, under conditions whereby the cel lexpresses the polypepti deencoded by the DNA segment or nucleic acid and recovering said polypeptide.

V. HIDE1 Fusion Proteins

[00290] In some embodiments, the invention uses HIDE1 polypeptides in the form of fusion proteins, wherei then HIDE1 polypeptide (generall any ECD) is fused, recombinantly in frame to a fusion partner.

A. Fusion Partners

[00291] In many embodiment s,the HIDE1 polypeptide is fused to a “fusion partner” (also referred to herei nas a “fusion partner moiety”), either direct lyor indirect lythrough the use of a linke asr is more fully describe belowd .As will be appreciate byd those in the art, the fusion partner can be any moiety that is fused to the HIDE1 polypeptide for any number of biochemical and/or biological reasons. In some embodiment s,the fusion partner moiety increases the half life of the HIDE1 fusion protei asn is described below. In some embodiment s,the fusion partner moiety adds an additional biologic or biochemical function to the HIDE1 polypeptide.

[00292] In some embodiments, the fusion partner is generally linked at either the N- terminus or the C-terminus of the HIDE1 polypeptide, optionall usingy a linke asr described herein, such that the fusion protei hasn a formul sela ected from the group consisting of NH2- HIDE1 polypeptide-fus ionpartner-COO H,HIDE1 polypeptide, NH2-fusion partner-L- HIDE1 polypeptide-COOH, and NH2-fusion partner-HIDE1-polypeptide-COOH. 1. HSA

[00293] In some embodiments, the HIDE1 fusion partne isr a human serum albumin (HSA), as is known in the art. In particular, fusions to HSA are known to increase serum half life of the fusion protein, as compared to the protei itn self .These can include standard flexibl linkerse such as described herei nand shown in Figure 66. 70WO 2017/009712 PCT/IB2016/001079 2. Fc Domains

[00294] In some embodiments, the HIDE1 polypeptide is fused to a fusion partner that is an Fc domain. By “Fc domain” herei nis meant the CH2-CH3 domains of an antibody, as is known in the art, optional includinly gsome or all of the hinge region residues. The Fc domain is generall derivey dfrom a human IgG protein, generally IgGl ,IgG2, IgG3 or IgG4, the sequences of whic hare shown in (Figure 65 depict sthe sequences of human IgGl ,IgG2, IgG3 and IgG4). The choice of Fc domain may depend on whether effector function is desired (e.g, such that IgGl is chosen), or not (IgG2 and IgG4, for example).

[00295] In addition, there are a number of Fc domain variants that can be optional ly and independent lyincluded as amino acid substitutions. By “amino acid substituti” onor “substitut”ion herein is meant the replacement of an amino acid at a particular position in a parent polypepti desequence wit ha different amino acid . In particular, in some embodiment s,the substitution is to an amino acid that is not naturall occurringy at the particular position, either not natural lyoccurring within the organism or in any organism .

For example, the substitution E272Y refer sto a variant polypeptide, in thi scase an Fc variant in, which the glutamic acid at position 272 is replaced with tyrosin e.For clarit y,a protei whicn h has been engineered to change the nucleic acid coding sequence but not change the starti ngamino acid (for example exchanging CGG (encoding arginine) to CGA (stil l encoding arginine) to increase host organism expression levels) is not an “amino acid substituti”on; that is, despit thee creation of a new gene encoding the same protein, if the protei hasn the same amino acid at the particular position that it started with, it is not an amino acid substitution.

[00296] In some embodiments, amino acid substituti onscan be made in the Fc region, in genera lfor altering binding to FcyR receptors. By “Fc gamma receptor”, "FcyR" or “FcgammaR” as used herein is meant any member of the family of protei nsthat bind the IgG antibody Fc region and is encoded by an FcyR gene. In humans thi sfamily includes but is not limite tod FcyRI (CD64), includin gisoform FcyRIas , FcyRIb, and FcyRIc; FcyRII (CD32), including isoform FcyRIIas (including allotypes H131 and R131), FcyRIIb (including FcyRIIb-1 and FcyRIIb-2), and FcyRIIc; and FcyRIII (CD16), includin gisoforms FcyRIIIa (including allotypes V158 and F158) and FcyRIIIb (including allotypes FcyRIIIb-NAl and 71WO 2017/009712 PCT/IB2016/001079 FcyRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirely incorporat byed reference), as well as any undiscovered human FcyRs or FcyR isoform ors allotypes An.

FcyR may be from any organism, includin gbut not limited to humans, mice, rats rabbit, s,and monkeys. Mouse FcyRs include but are not limite tod FcyRI (CD64), FcyRII (CD32), FcyRIII-1 (CD16), and FcyRIII-2 (CD16-2), as well as any undiscovered mouse FcyRs or FcyR isoform ors allotypes.

[00297] There are a number of useful Fc substitutions that can be made to alter binding to one or more of the FcyR receptors. Substitutions that resul int increased binding as well as decrease dbinding can be useful. For example, it is known that increase dbinding to FcyRIIIa generally result ins increased ADCC (antibody dependent cell-mediated cytotoxici ty;the cell - mediated reaction wherei nonspecifin ccytotoxic cell sthat express FcyRs recognize bound antibody on a targe cellt and subsequentl causey lysi sof the targe celt l. Similarl decreasedy, binding to FcyRIIb (an inhibitory receptor) can be beneficial as wel inl some circumstances.

Amino acid substitutions that find use in the present inventio ninclude thos liste ed in U.S.

Ser. Nos. 11/124,620 (particularl FIG.y 41) and U.S. Patent No. 6,737,056, both of which are expressl incorporatedy herein by reference in their entirety and specifically for the variant s disclosed therei n.Particul varianar ts that find use include, but are not limited to, 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y, 239D, 332E/330L, 299T and 297N.

[00298] In addition, the antibodie ofs the invention are modified to increase its biologic alhalf-life. Various approaches are possible. For example, one or more of the followi mutang tions can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternative toly, increase the biologic alhalf-lif e,the antibody can be alter edwithin the CHI or CL region to contain a salvage receptor binding epitope take nfrom two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. ,869,046 and 6,121,022 by Prest eta al. Additional mutations to increase serum half life are disclosed in U.S. Patent Nos. 8,883,973, 6,737,056 and 7,371,826, and include 428L, 434A, 434S, and 428L/434S.

[00299] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue wit ha different amino acid residue to alter the effector functions of the 72WO 2017/009712 PCT/IB2016/001079 antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approac his described in furth erdetai inl U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

[00300] In anoth erexample, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicit (CDCy ). This approac his described in furth erdeta ilin U.S. Pat. Nos. 6,194,551 by Idusogie et al.

[00301] In anoth erexample, one or more amino acid residues within amino acid positions 231 and 239 are alter edto thereby alter the ability of the antibody to fix complement. This approac his described furth erin PCT Publication WO 94/29351 by Bodmer et al.

[00302] In yet another example, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellula cytotoxir cit (ADCy C) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids at the followi positing ons: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approac his described furth erin PCT Publication WO 00/42072 by Presta. Moreover, the binding site son human IgGl for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al. (2001) J. Biol.

Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 are shown to improve binding to FcyRIII. Additional thely, follow ingcombination mutants are shown to improve FcyRIII binding :T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A. Furthermore, mutations such as M252Y/S254T/T256E or 73WO 2017/009712 PCT/IB2016/001079 M428L/N434S improve binding to FcRn and increase antibody circulati onhalf-life (see Chan CA and Carte PJr (2010) Nature Rev Immunol 10:301-316).

[00303] In still another embodiment, the glycosylati ofon an Fc domain is modified.

For example, an aglycosylate Fcd domain can be made Q.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen or reduce effector function such as ADCC. Such carbohydrat e modifications can be accomplished by, for example, altering one or more site sof glycosylati witon hin the antibody sequence, for example N297. For example, one or more amino acid substituti onscan be made that resul int elimination of one or more variable region framework glycosylat ionsites to thereby eliminate glycosylat ionat that site.

[00304] Additional orly alternatively, an Fc domain can be made that has an altere d type of glycosylation, such as a hypofucosylated antibody having reduce damounts of fucosy l residues or an Fc domain having increased bisecting GlcNac structures Such. altered glycosylati patteon rns have been demonstrated to increase the ADCC abilit yof antibodies.

Such carbohydrat modie fications can be accomplished by, for example, expressing the antibody in a host cell wit haltered glycosylat ionmachinery. Cell swit haltered glycosylati on machinery have been described in the art and can be used as host cells in whic hto expres s recombinan antibodiet accords ing to at least some embodiments of the inventio nto thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransfera gene,se FUT8 (a (1,6) fucosyltransferase such), that antibodie express sed in the Ms704, Ms705, and Ms709 cel llines lack fucose on their carbohydrates The. Ms704, Ms705, and Ms709 FUT8 cell line sare created by the target ed disrupti onof the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S.

Patent Publication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 by Hanai et al. describes a cel lline wit ha functionally disrupt edFUT8 gene, which encodes a fucosyl transferase, such that antibodie express sed in such a cell line exhibit hypofucosylation by reducing or eliminating the a 1,6 bond-relat enzymeed . Hanai et al. also describe cel llines which have a low enzyme activit yfor adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activit y,for example the rat myeloma cel lline 74WO 2017/009712 PCT/IB2016/001079 YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 by Prest desca ribe as variant CHO cel lline, Led 3 cells, with reduce dability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylat ionof antibodie express sed in that host cell (see also Shields R., L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describe cels lline sengineered to express glycoprotein-modifying glycosyl transferases (e.g., P(l,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodie express sed in the engineered cell lines exhibi tincreased bisecting GlcNac structures whic hresul tsin increased ADCC activity of the antibodie (sees also Umana et al. (1999) Nat. Biotech. 17:176-180). Alternative thely, fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase a-L-fucosidase removes fucosyl residues from antibodies (Tarentino, A. L. et al. (1975) Biochem. 14:5516-23). 3. Polyethylene Glycol Fusion Partners

[00305] In some embodiments, the fusion partner moiety is one or more polyethylene glycol (PEG) moieties. As is well known in the art, the modification of therapeutic prote in drugs, such as erythropoie GM-Ctin, SF, interferon alpha and beta and human growt h hormone, is frequently done to increase to alter a number of pharmacologi calproperti es, including, but not limite to,d increased solubility, extended serum half-life, decreased dosage frequency, increased stabilit decreasedy, immunogenicit yand enhanced protecti fromon proteases.

[00306] As is known in the art, generall ay number of PEG molecules are “loade”d onto each protein, depending on a number of factors and, the PEG molecul esmay be of varying length.

[00307] In general, the PEG moieties are covalently attached directly to the amino acid side chains of the HIDE1 polypeptide, using activated PEG derivatives as is well known in the art. That is, generally no additional linkers are used, e.g. there are no extra atoms between the PEG and the amino acid side chain. In othe emr bodiment s,linkers such as those outlined below are used. 75WO 2017/009712 PCT/IB2016/001079 4. Additional Heterologous Fusion Partners

[00308] In addition to half life extension fusion partner moieties, HIDE1 polypeptides can be fused (generall buty optionall usingy linkers as outlined herein), with heterologous polypeptide that give additional biochemical functionalitie tos the HIDE1 polypeptides.

These heterologous fusion partner moieties including, but are not limite to,d receptors, hormones cytokines, ant, igens, B-cell target NKs, cel ltarget Ts, cel ltargets, TNF receptor superfamil memy bers, Hedgehog family members, a receptor tyrosi nekinases, a proteoglycan-relate molecd ules a ,TGF-P superfamil memy bers, Wnt-relate molecd ules, receptor ligands, dendrit cellic target myels, oid cel ltarget monocyte/s, macrophage cell targets or angiogenesi stargets.

[00309] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise a T cel ltarge selt ected from the group consisting of 2B4/SLAMF4, IL-2 Ra, 4-1BB/TNFRSF9, IL-2RP, ALCAM, B7-1/CD80, IL-4R, B7-H3, BLAME/SLAMF8, BTLA, IL-6R, CCR3, IL-7 Ra, CCR4, CXCR1/IL-8 RA, CCR5, CCR6, IL-10 R a, CCR7, IL-10 Rp, CCR8, IL-12 Rpi, CCR9, IL-12 Rp 2, CD2, IL-13Ral ,IL-13, CD3, CD4, ILT2/CD85j, ILT3/CD85k, ILT4/CD85d, ILT5/CD85a, Integrin a 4/CD49d, CD5, IntegrinaE/CD103, CD6, Integrin aM/CDllb CD8,, Integrin aX/CD1lc ,Integri n P2/CD18, KIR/CD158, CD27/TNFRSF7, KIR2DL1, CD28, KIR2DL3, CD30/TNFRSF8, KIR2DL4/CD158d, CD31/PECAM-1, KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43, LAIR1, CD45, LAIR2, CD83, Leukotriene B4 RI, CD84/SLAMF5, NCAM-L1, CD94, NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69, NTB-A/SLAMF6, Common y Chain/IL-2 Ry, Osteopont in,CRACC/SLAMF7, PD-1, CRTAM, PSGL-1, CTLA-4, RANK/TNFRSF11A, CX3CR1, CX3CL1, L-Selectin, CXCR3, SIRP pi, CXCR4, SLAM, CXCR6, TCCR/WSX-1, DNAM-1, Thymopoiet in, EMMPRIN/CD147, TIM-1, EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, Fey RIII/CD16, TIM-6, GITR/TNFRSF18, TNF R1/TNFRSF1A, Granulysi n,TNF R11/TNFRSFIB, HVEM/TNFRSF14, TRAIL R1/TNFRSF10A, ICAM-1/CD54, TRAIL R2/TNFRSF1OB, ICAM-2/CD102, TRAIL R3/TNFRSF10C, IFN-yRl, TRAIL R4/TNFRSF10D, IFN-yR2, TSLP, IL-1 RI and TSLP R. 76WO 2017/009712 PCT/IB2016/001079

[00310] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise at least one monocyte/macrophag cele ltarget selected from the group consisting of B7-1/CD80, ILT4/CD85d, B7-H1, ILT5/CD85a, Common (3 Chain, Integri na 4/CD49d, BLAME/SLAMF8, IntegrinaX/CD11c, CCL6/C10, Integri n(3 2/CD18, CD155/PVR, Integri n(3 3/CD61, CD31/PECAM-1, Latexin, CD36/SR-B3, Leukotrie neB4 RI, CD40/TNFRSF5, LIMPII/SR-B2, CD43, LMIR1/CD300A, CD45, LMIR2/CD300c, CD68, LMIR3/CD300LF, CD84/SLAMF5, LMIR5/CD300LB, CD97, LMIR6/CD300LE, CD163, LRP-1, CD2F-10/SLAMF9, MARCO, CRACC/SLAMF7, MD-1, ECF-L, MD-2, EMMPRIN/CD147, MGL2, Endoglin/CD105, Osteoactivin/GPNMB FcyRl/C, D64, Osteopontin, Fey RIIB/CD32b, PD-L2, FcyRIIC/CD32c, Siglec-3/CD33, Fey RIIA/CD32a, SIGNR1/CD209, Fey RIII/CD16, SLAM, GM-CSF R a, TCCR/WSX-1, ICAM-2/CD102, TLR3, IFN-y RI, TLR4, IFN-y R2, TREM-1, IL-1 RII, TREM-2, ILT2/CD85j, TREM-3, ILT3/CD85k, TREML1/TLT-1, 2B4/SLAMF4, IL-10 R a, ALCAM, IL-10 R (3, Aminopeptidas N/Ae NPEP, ILT2/CD85j, Common (3 Chain, ILT3/CD85k, Clq R1/CD93, ILT4/CD85d, CCR1, ILT5/CD85a, CCR2, Integrin a 4/CD49d, CCR5, Integrin a M/CDllb, CCR8, Integrin a X/CD lie, CD155/PVR, Integrin (3 2/CD18, CD 14, Integri n(3 3/CD61, CD36/SR-B3, LAIR1, CD43, LAIR2, CD45, Leukotrie neB4 RI, CD68, LIMPII/SR-B2, CD84/SLAMF5, LMIR1/CD300A, CD97, LMIR2/CD300c, CD163, LMIR3/CD300LF, Coagulation Factor III/Tissue Factor, LMIR5/CD300LB, CX3CR1, CX3CL1, LMIR6/CD300LE, CXCR4, LRP-1, CXCR6, M-CSF R, DEP-1/CD148, MD-1, DNAM-1, MD-2, EMMPRIN/CD147, MMR, Endoglin/CD105, NCAM-L1, FcyRl/CD64, PSGL-1, FcyRIII/CD16, RP105, G-CSF R, L-Selectin, GM-CSF R a, Siglec-3/CD33, HVEM/TNFRSF14, SLAM, ICAM-1/CD54, TCCR/WSX-1, ICAM-2/CD102, TREM-1, IL- 6 R, TREM-2, CXCR1/IL-8 RA, TREM-3 and TREML1/TLT-1.

[00311] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise at least one Dendrit iccell targe ist selected from the group consisting of CD36/SR-B3, LOX-1/SR-E1, CD68, MARCO, CD163, SR-AI/MSR, CD5L, SREC-I, CL-P1/COLEC12, SREC-II, LIMPII/SR-B2, RP105, TLR4, TLR1, TLR5, TLR2, TLR6, TLR3, TLR9, 4-1BB Ligand/TNFSF9, IL-12/IL-23 p40, 4-Amino-l,8-naphthalim ide, ILT2/CD85j, CCL21/6Ckine, ILT3/CD85k, 8-oxo-dG, ILT4/CD85d, 8D6A, ILT5/CD85a, 77WO 2017/009712 PCT/IB2016/001079 A2B5, Integri na 4/CD49d, Aag, Integri np 2/CD18, AMIGA, Langerin, B7-2/CD86, Leukotrie neB4 RI, B7-H3, LMIR1/CD300A, BLAME/SLAMF8, LMIR2/CD300c, Clq R1/CD93, LMIR3/CD300LF, CCR6, LMIR5/CD300LB, CCR7, LMIR6/CD300LE, CD40/TNFRSF5, MAG/Siglec-4a ,CD43, MCAM, CD45, MD-1, CD68, MD-2, CD83, MDL-1/CLEC5A, CD84/SLAMF5, MMR, CD97, NCAM-L1, CD2F-10/SLAMF9, Osteoactivin/GPNMB, Chern 23, PD-L2, CLEC-1, RP105, CLEG-2, Siglec-2/CD22, CRACC/SLAMF7, Siglec-3/CD33, DC-SIGN, Siglec-5, DC-SIGNR/CD299, Siglec-6, DCAR, Siglec-7, DCIR/CLEC4A, Siglec-9, DEC-205, Siglec-10, Dectin-1/CLEC7 A, Siglec - F, Dectin-2/CLEC6A ,SIGNR1/CD209, DEP-1/CD148, SIGNR4, DLEC, SLAM, EMMPRIN/CD147, TCCR/WSX-1, FcyRl/CD64, TLR3, FcyRIIB/CD32b, TREM-1, FcyRIIC/CD32c, TREM-2, FcyRIIA/CD32a, TREM-3, FcyRIII/CD16, TREML1/TLT-1, ICAM-2/CD102 and Vanilloid RI.

[00312] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise at least one TNF receptor superfamil memy ber is selected from the group consisting of 4-1BB/TNFRSF9, NGF R/TNFRSF16, BAFF R/TNFRSF13C, Osteoprotegerin/TNFRSFllB BCMA, /TNFRSF17, OX40/TNFRSF4, CD27/TNFRSF7, RANK/TNFRSF11A, CD30/TNFRSF8, RELT/TNFRSF19L, CD40/TNFRSF5, TACI/TNFRSF13B, DcR3/TNFRSF6B, TNF RI/TNFRSF1A, DcTRAIL R1/TNFRSF23, TNF RII/TNFRSF1IB, DcTRAIL R2/TNFRSF22, TRAIL R1/TNFRSF10A, DR3/TNFRSF25, TRAIL R2/TNFRSF10B, DR6/TNFRSF21, TRAIL R3/TNFRSF10C, ED AR, TRAIL R4/TNFRSF10D, Fas/TNFRSF6, TROY/TNFRSF19, GITR/TNFRSF18, TWEAK R/TNFRSF12, HVEM/TNFRSF14, XEDAR, Lymphotoxin P R/TNFRSF3, 4-IBB Ligand/TNFSF9, Lymphotoxin, APRIL/TNFSF13, Lymphotoxin B/TNFSF3, BAFF/TNFSF13C, OX40 Ligand/TNFSF4, CD27 Ligand/TNFSF7, TL1A/TNFSF15, CD30 Ligand/TNFSF8, TNF-a/TNFSFlA, CD40 Ligand/TNFSF5, TNF-p/TNFSFIB, EDA-A2, TRAIL/TNFSF10, Fas Ligand/TNFSF6, TRANCE/TNFSF11, GITR Ligand/TNFSF18, TWEAK/TNFSF12 and LIGHT/TNFSF14.

[00313] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise at least one Hedgehog family member selected from the group consisting of Patched and Smoothened. 78WO 2017/009712 PCT/IB2016/001079

[00314] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise at least one receptor tyrosi nekinase selected from the group consisting of Axl, FGF R4, Clq R1/CD93, FGF R5, DDR1, Flt-3, DDR2, HGF R, Dtk, IGF- I R, EGF R, IGF-II R, Eph, INSRR, EphAl, Insuli nR/CD220, EphA2, M-CSF R, EphA3, Mer, EphA4, MSP R/Ron, EphA5, MuSK, EphA6, PDGF R a, EphA7, PDGF R (3, EphA8, Ret ,EphBl, ROR1, EphB2, ROR2, EphB3, SCF R/c-kit, EphB4, Tie-1, EphB6, Tie-2, ErbB2, TrkA, ErbB3, TrkB, ErbB4, TrkC, FGF RI, VEGF Rl/Flt-1, FGF R2, VEGF R2/Flk- 1, FGF R3 and VEGF R3/Flt-4.

[00315] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise at least one Transforming Growth Factor (TGF)-[3 superfamil memy ber selected from the group consisting of Activin RIA/ALK-2, GFR a-1, Activin RIB/ALK-4, GFR 02, Activin RHA, GFR 0-3, Activin RUB, GFR 0-4, ALK-1, MIS RII, ALK-7, Ret, BMPR-IA/ALK-3, TGF-bet|3a Rl/ALK-5, BMPR-IB/ALK-6, TGF-0 RII, BMPR-II, TGF-P Rllb, Endoglin/C D105 and TGF-P RIII.

[00316] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise at least one Wnt-relate mold ecul sele ected from the group consisting of Frizzled-1, Frizzled-8, Frizzled-2, Frizzled-9, Frizzled-3, sFRP-1, Frizzled-4, sFRP-2, Frizzled-5, sFRP-3, Frizzled-6, sFRP-4, Frizzled-7, MFRP, LRP 5, LRP 6, Wnt-1, Wnt-8a, Wnt-3a, Wnt-lOb, Wnt-4, Wnt-11, Wnt-5a ,Wnt-9a and Wnt-7a.

[00317] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise at least one receptor ligand selected from the group consisting of 4-1BB Ligand/TNFSF9, Lymphotoxin, APRIL/TNFSF13, Lymphotoxin P/TNFSF3, BAFF/TNFSF13C, 0X40 Ligand/TNFSF4, CD27 Ligand/TNFSF7, TL1A/TNFSF15, CD30 Ligand/TNFSF8, TNF-a/TNFSFlA, CD40 Ligand/TNFSF5, TNF- P/TNFSF1B, EDA-A2, TRAIL/TNFSF10, Fas Ligand/TNFSF6, TRANCE/TNFSF11, GITR Ligand/TNFSF18, TWEAK/TNFSF12, LIGHT/TNFSF14, Amphiregulin, NRG1 isofor m GGF2, Betacelluli NRGn, 1 Isoform SMDF, EGF, NRG1-a/HRG1-a, Epigen, NRG1-P 1/HRG1-P 1, Epiregulin, TGF-a, HB-EGF, TMEFFl/Tomoregulin-1, Neuregulin-3, TMEFF2, IGF-I, IGF-II, Insulin, Activin A, Activin B, Activin AB, Activin C, BMP-2, BMP-7, BMP-3, BMP-8, BMP-3b/GDF-10, BMP-9, BMP-4, BMP-15, BMP-5, 79WO 2017/009712 PCT/IB2016/001079 Decapentaplegi c,BMP-6, GDF-1, GDF-8, GDF-3, GDF-9, GDF-5, GDF-11, GDF-6, GDF- , GDF-7, Artemin, Neurturin, GDNF, Persephin, TGF-0, TGF-0 2, TGF-P 1, TGF-P 3, LAP (TGF-P 1), TGF-P 5, Latent TGF-P 1, Latent TGF-P bpl, TGF-P 1.2, Lefty, Nodal, MIS/AMH, FGF acidic, FGF-12, FGF basic, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, Neuropilin-1, Pl GF, Neuropilin-2, P1GF-2, PDGF, PDGF-A, VEGF, PDGF-B, VEGF-B, PDGF-C, VEGF-C, PDGF-D, VEGF-D and PDGF-AB.

[00318] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise at least one tumor antigen selected from the group consisting of Squamous Cell Carcinoma Antigen 1 (SCCA-1), (PROTEIN T4-A), Squamous Cell Carcinoma Antigen 2 (SCCA-2), Ovarian carcinoma antigen CA125 (1A1-3B; KIAA0049).

MUCIN 1 (TUMOR-ASSOCIATED MUCIN; Carcinoma-Associate Mucin;d Polymorphi c Epitheli Mucial n; PEM; PEMT; EPISIALIN; Tumor-Associated Epitheli Membraal ne Antigen; EMA; H23AG; Peanut-Reactive Urinary Mucin; PUM; and Breast Carcinoma- Associate Antid gen DF3), CTCL tumor antigen sel-1, CTCL tumor antigen sel4-3, CTCL tumor antigen se20-4, CTCL tumor antigen se20-9, CTCL tumor antigen se33-l, CTCL tumor antigen se37-2, CTCL tumor antigen se57-l, CTCL tumor antigen se89-l, Prosta te- specific membrane antigen ,5T4 oncofeta trophobll glycoast protei Orf73n, Kaposi's sarcoma- associated herpesvirus MAG, E-CI (cancer/test antiis gen CT7), MAGE-B1 ANTIGEN (MAGE-XP Antigen; DAM 10), MAGE-B2 Antigen (DAM6), MAGE-2 ANTIGEN, MAGE- 4a antigen ,MAGE-4b antigen ,Colon cancer antigen NY-CO-45, Lung cancer antigen NY- LU-12 variant A, Cancer associate surfacd eantigen, Adenocarcinom antia gen ART1, Paraneoplas ticassociated brain-testis-canc antier gen (onconeuronal antigen MA2; paraneoplast neuronalic antigen), Neuro-oncologic vental ral antigen 2 (NOVA2), Hepatocellular carcinoma antigen gene 520, Tumor-Associated Antigen CO-029, Tumor- associated antigen MAGE-X2, Synovial sarcoma X, breakpoi 2,nt Squamous cel lcarcinoma antigen recognized by T cell, Serologica llydefined colon cancer antigen 1, Serological ly defined breast cancer antigen NY-BR-15, Serologica llydefined breast cancer antigen NY- BR-16, Chromograni A,n parathyroi secred tory protei 1,n DUPAN-2, CA 19-9, CA 72-4, CA 195 and L6. 80WO 2017/009712 PCT/IB2016/001079

[00319] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise at least one B cel ltarge selet cted from the group consisting of CD10, CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD39, CD40, CD72, CD73, CD74, CDw75, CDw76, CD77, CD78, CD79a/b, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD89, CD98, CD126, CD127, CDwl30, CD138 and CDwl50.

[00320] In some embodiment HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise at least one angiogenesis target is selected from the group consisting of Angiopoietin-1, Angiopoietin-like 2, Angiopoietin-2, Angiopoietin-like 3, Angiopoietin-3, Angiopoietin-l ike7/CDT6, Angiopoietin-4, Tie-1, Angiopoietin-like 1, Tie- 2, Angiogenin, iNOS, Coagulation Factor III/Tissue Factor, nNOS, CTGF/CCN2, NOV/CCN3, DANCE, OSM, EDG-1, Plfr EG-VEGF/, PK1, Proliferin, Endostatin, ROBO4, Erythropoiet Thrombospondin-in, 1, Kininostat in,Thrombospondi n-2,MFG-E8, Thrombospondi n-4,Nitric Oxide ,VG5Q, eNOS, EphAl, EphA5, EphA2, EphA6, EphA3, EphA7, EphA4, EphA8, EphBl, EphB4, EphB2, EphB6, EphB3, Ephrin-A l,Ephrin-A4, Ephrin-A2, Ephrin-A5, Ephrin-A3, Ephrin-Bl, Ephrin-B3 Ephrin, -B2, FGF acidic, FGF-12, FGF basic, FGF-13, FGF-3, FGF-16, FGF-4, FGF-17, FGF-5, FGF-19, FGF-6, FGF-20, FGF-8, FGF-21, FGF-9, FGF-23, FGF-10, KGF/FGF-7, FGF-11, FGF RI, FGF R4, FGF R2, FGF R5, FGF R3, Neuropilin-1, Neuropilin-2, Semaphorin 3 A, Semaphori 6B,n Semaphorin 3C, Semaphori 6C,n Semaphori 3E,n Semaphori 6D,n Semaphori 6A,n Semaphorin 7A, MMP, MMP-11, MMP-1, MMP-12, MMP-2, MMP-13, MMP-3, MMP-14, MMP-7, MMP- , MMP-8, MMP-16/MT3-MMP, MMP-9, MMP-24/MT5-MMP, MMP-10, MMP-25/MT6- MMP, TIMP-1, TIMP-3, TIMP-2, TIMP-4, ACE, IL-13 Rai, IL-13, Clq R1/CD93, Integri na 4/CD49d, VE-Cadherin, Integrin (3 2/CD18, CD31/PECAM-1, KLF4, CD36/SR- B3, LYVE-1, CD151, MCAM, CL-P1/COLEC12, Nectin-2/CD112, Coagulation Factor III/Tissue Factor, E-Selectin, D6, P-Selectin, DC-SIGNR/CD299, SLAM, EMMPRIN/CD147, Tie-2, Endoglin/CD105, TNF RI/TNFRSF1A, EPCR, TNF RII/TNFRSF1IB, Erythropoie R,tin TRAIL R1/TNFRSF10A, ESAM, TRAIL R2/TNFRSF1OB, FABP5, VCAM-1, ICAM-1/CD54, VEGF R2/Flk-1, ICAM-2/CD102, VEGF R3/Flt-4, IL-1 RI and VG5Q. 81WO 2017/009712 PCT/IB2016/001079 B. Linkers

[00321] In many embodiments of fusion proteins comprising a HIDE1 polypeptide and a fusion partner moiety, optiona flexil ble linkers are used to join the sequences in frame . A “flexibl linkere ” herein refers to a peptide or polypepti decontaining two or more amino acid residues joined by peptide bond(s) that provides increased rotational freedom for two polypeptides linked thereby than the two linked polypeptides would have in the absence of the flexibl line ker. Such rotational freedom allow eachs component of the fusion prote into interact wit hits intended targe witt hout hindrance. Generally thes linkerse are mixtures of glycine and serine, such as -(GGGS)n-, where n is from 1, 2, 3, 4, or 5 (SEQ ID NO:1).

[00322] Other suitable peptide/polypepti linkede domainsr optionall includey naturall y occurring or non-naturall occurringy peptides or polypeptide Peptides. linke sequenr ces are at least 2 amino acids in length. Optionally the peptide or polypeptide domains are flexibl e peptides or polypeptides Exempl. ary flexibl peptides/e polypeptides include but, are not limited to, the amino acid sequences Gly-Ser (SEQ ID NO:55), Gly-Ser-Gly-Ser (SEQ ID NO:56), Ala-Ser (SEQ ID NO:57), Gly-Gly-Gly-Ser (SEQ ID NO:58), Gly4-Ser (SEQ ID NO:59), (Gly4-Ser2 )(SEQ ID NO:60), (Gly4-Ser)3 (SEQ ID NO:61), (Gly4-Ser)4 (SEQ ID NO: 62), [Gly4-Ser]2 Gly-Ala-Gly-Ser-Gl4-Sery (SEQ ID NO: 72), Gly-(Gl4y-Ser)2 (SEQ ID NO:73), Gly4-Ser-Gl y(SEQ ID NO:74), Gly-Ser-Gly2 (SEQ ID NO:75) and Gly-Ser-Gly2- Ser (SEQ ID NO:76). Additional flexible peptide/polypeptide sequences are well known in the art. Other suitable peptide linker domains optionall includey the TEV linke ENLYFr QG, a linear epitope recognized by the Tobacco Etch Virus proteas Exemple. ary peptides/polypeptides include, but are not limited to, GSENLYFQGSG (SEQ ID NO:68).

Other suitable peptide linker domains include helix forming linkers such as Ala-(Glu-Al a- Ala-Ala-Lys)n-A la(n= 1-5). Additional helix forming peptide/polypepti sequende ces are well known in the art. Non-limiti ngexamples of such linkers are depicted in SEQ ID NOs:63-67, 69-76.

[00323] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise a HIDE1 polypeptide, and at least one heterologous polypeptide and/or binding moiety or HIDE1 polypeptides are linked to one another by an amino acid spacer. 82WO 2017/009712 PCT/IB2016/001079

[00324] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nand at least one heterologous polypeptide and/or or binding moiety or HIDE1 polypeptides are linked to one anoth erby an amino acid spacer of sufficient length of amino acid residues so that the different moieties can successfully bind to thei indivir dual targets.

[00325] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise 2-10 of any of the HIDE1 ECD polypeptide fragments disclosed herein.

[00326] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise one or more of HIDE1 polypeptide(s) and at least one heterologous polypeptide optional intervenly ed by a heterologous linke whicr hoptional ly comprises a polypeptide that is not a fragment of a HIDE1 polypeptide.

[00327] In some embodiments HIDE1 polypeptides or fusion proteins according to the present invention will comprise a linke whicr his a peptide comprising 5-50 amino acid residues more, preferabl 5-25y amino acid residues.

[00328] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise a linke whicr h comprises, consists essentiall of,y glycine, serine, and/or alanine residues.

[00329] In some embodiments HIDE1 polypeptides or fusion proteins according to the present inventio nwill comprise a linke whicr h comprises 5-50, 5-25, 5-15, 4-14, 4-12, or more amino acid residues, e.g., which may optional includely or consis oft glycine, serine, and/or alanine residues.

[00330] In some optionally embodiments HIDE1 fragment s,e.g., ECD fragments, are linked to each other (multimer and/ors) one or more HIDE1 fragment s,e.g., ECD fragments, are linked to a heterologous polypeptide such as an immunoglobulin or fragment thereof, especiall any immunoglobul heavyin chain or fragment there ofby a peptide linker, preferably a “flexibl elinker” sequence. The linker sequence should allow effective positioning of the HIDE1 fragments and the heterologous polypeptide such as an immunoglobul polypeptidein or domains thereof to allow functional activit yof both moieties and the domains thereof. 83WO 2017/009712 PCT/IB2016/001079 Successful presentation of the polypeptide fusion can modulate the activit yof a cel leither to induce or to inhibi tT-cel lproliferat ion,or to initiat ore inhibi tan immune response to a particular site. This can be determined in appropriate assays such as disclosed herei nbelow, including the in vitro assays that includes sequential steps of culturin Tg cell sto proliferate same, and contacting the T cell swith a fusion polypepti deaccording to the present invention or a cel lexpressing same and then evaluating whether the fusion polypepti depromot ores inhibits T cel lproliferation.

[00331] As used herein, the phrase “effective positioning of the heterologous polypeptide and the HIDE1 polypepti”de, or other similar phrase, is intended to mean that the domains of thes moie eties are positione sod that HIDE1 domains and heterologo us polypeptide domains are capable of interacting with immune or other targe cellt s,e.g., HIDE1 expressing cell sto initiat ore inhibi tan immune reaction, or to inhibi tor stimula tecell development.

[00332] With respec tot HIDEl-Ig fusion proteins the linke sequencer also preferably permits effective positioning of the Fc domain and HIDE1 domains to allo wfunctional activity of each domain. In certain embodiments, the Fc domains are effectively positioned to allow proper fusion protei compln ex formation and/or interaction withs Fc receptor ons immune cell sor protei nsof the complement system to stimulate Fc-mediated effects including opsonization cel, llysis, degranulation of mast cells, basophil ands, eosinophil ands, othe Fcr receptor-dependent processes; activation of the complement pathway; and enhanced in vivo half-life of the fusion protei compln ex.

[00333] Linker sequences are discussed supra in connection with fusion proteins according to some embodiments of the present invention. Linker sequences can optionall bey used to link two or more HIDE1 polypeptides of the biologically active polypeptide to generat ea single-chai molen cule with the desired functional activity. In some preferred embodiments the linke sequenr ce comprises from about 5 to 20 amino acids, more preferably from about 7 or 8 to about 16 amino acids. The linke sequenr ce is preferably flexibl eso as not hold the HIDE1 polypeptide and moiety linked thereto, e.g., an effector molecul ine a singl eundesired conformation. The linke sequencer can be used, e.g., to space the recognition sit efrom the fused molecule Specif. ically, the peptide linke sequer nce can be positione d 84WO 2017/009712 PCT/IB2016/001079 between the biologicall acty ive HIDE1 polypeptide and the effector molecule, e.g, to chemically cross-link same and to provide molecular flexibility. The linker in some embodiments will predominantl compriy se amino acids wit hsmall side chains, such as glycine, alanine and serine, to provide for flexibilit Preferablyy. about 80 or 90 percent or greater of the linke sequer nce comprise glycine ,alanine or serine residues, particula rly glycine and serine residues. Other suitable linke sequencer s include flexible linke desir gns that have been used successfull toy join antibody variable regions together, see Whitlow, M. et al., (1991) Methods: A Companion to Methods in Enzymology 2:97-105. In some examples for, covalentl linkiy ng an effector molecule to aHIDEl molecul e,the amino sequence of the linke shouldr be capable of spanning a suitable distance from the C-terminal residue of the HIDE1 polypeptide to the N-terminal residue of the effector molecul e.Suitabl e linke sequencesr can be readily identified empirically. Additional suitaly, ble size and sequences of linke sequencesr also can be determined by known computer modeling techniques based on the predicted size and shape of the fusion polypeptide Ot. her linker sequences are discussed supra in connection with fusion proteins according to some embodiments of the present invention.

[00334] Optionall a ypolypeptide as describe hereind comprises 2-20 of any of the HIDE1 ECD polypepti defragments linked together, such that the polypepti dehas less than 95% homology to any of the HIDE1 sequence as described herein.

[00335] Optionall they fragments are intervened by a heterologous linke whicr h optionally comprises a polypeptide that is not a fragment of a HIDE1 polypeptide.

[00336] Optionall they linke isr a peptide comprising 5-50 amino acid residues, more preferably 5-25 amino acid residues.

[00337] Optionall they linke comprisr es, consists essentiall of,y or consists of 4-12 glycine, serine, and/or alanine residues. 1. Dimerization, multimerization and targeting domains

[00338] The fusion proteins disclosed herei noptionall contay in a dimerization or multimerizat ionor oligomerization domain that functions to dimerize, oligomeri or ze multimerize two or more fusion proteins, whic hmay optionally be the same or different 85WO 2017/009712 PCT/IB2016/001079 (heteromultimers or homomultimers). For example aHIDEl fusion protei mayn optionally be attached to another HIDE1 fusion prote inor another moiety, e.g. anoth ercostimulatory fusion protein. The domain that functions to dimerize or multimeriz thee fusion proteins can either be a separat domae in, or alternativel cany be contained within one of the other domains (HIDE1 polypeptide, second polypeptide, or peptide/polypepti linkede domar in) of the fusion protein.

[00339] Dimerization or multimerizat ioncan occur between or among two or more fusion proteins through dimerizati onor multimerizati domaon ins. Alternative dimly, erizati on or multimerizati ofon fusion proteins can occur by chemical crosslinking. The dimers or multimers that are formed can be homodimeric/homomult imor ericheterodimeric / heteromultim ericThe .second polypepti de“partner” in the HIDE1 fusion polypeptides may optionally be comprised of one or more other proteins protei, fragmenn ts or peptides as described herein, includin gbut not limited to any immunoglobul (Ig)in prote inor portion thereof, preferably the Fc region, or a portion of a biologicall ory chemically active protein such as the papillomavirus E7 gene product, melanoma-associated antigen p97), and HIV env protei (gp!20).n The “partner” is optionall sely ected to provide a solubl dimer/me ultim er and/or for one or more other biologica actil vities as described herein.

[00340] A “dimerizati ondomain” is formed by the association of at least two amino acid residues or of at least tw opeptides or polypeptides (which may have the same, or different, amino acid sequences). The peptides or polypeptides may optionally interact wit h each other through covalent and/or non- covalent associations). Optiona diml erizati on domains contain at least one cysteine that is capable of forming an intermolecular disulfide bond with a cysteine on the partner fusion protein. The dimerization domain can contain one or more cysteine residues such that disulfide bond(s) can form between the partner fusion proteins. In one embodiment, dimerization domains contain one, two or three to about ten cysteine residues. In a further embodiment, the dimerizati ondomain is the hinge region of an immunoglobulin.

[00341] Additional exemplary dimerizati ondomains can be any known in the art and include, but not limited to, coiled coils, acid patches, zinc fingers, calcium hands, a CHI-CL pair, an “interface” with an engineered “knob” and/or “protuberance” as described in U. S. 86WO 2017/009712 PCT/IB2016/001079 Patent No. 5,821,333, leucine zippers (e.g., fromjun and/or fos) (U. S. Patent No. 5,932,448), and/or the yeast transcriptional activator GCN4, SH2 (src homology 2), SH3 (src Homology 3) (Vidal, et al, Biochemistry, 43, 7336- 44 ((2004)), phosphotyrosine binding (PTB) (Zhou, et al, Nature, 378:584- 592 (1995)), WW (Sudol, Prog. Biochys. Mol Biol., 65:113-132 (1996)), PDZ (Kim, et al, Nature, 378: 85-88 (1995); Komau, et al, Science, 269. 1737-1740 (1995)) 14-3-3, WD40 (Hu5 et al, J Biol Chem., 273: 33489- 33494 (1998)) EH, Lim, “An isoleucine zipper, a receptor dimer pair (e.g, interleukin-8 receptor (IL-8R); and integrin heterodime suchrs as LFA-I and GPIIIb/IIIa), or the dimerization region(s) thereof, dimeric ligand polypeptides (e.g, nerve growth factor (NGF), neurotrophin-3 (NT-3), interleuki n-8 (IL-8), vascular endothel growthial factor (VEGF), VEGF-C, VEGF-D, PDGF members, and brain-derived neurotrop facthic or (BDNF) (Arakawa, et al., J Biol. Chem., 269(45): 27833- 27839 (1994) and Radziejewski, et al., Biochem., 32(48): 1350 (1993)) and can also be variants of thes domainse in which the affinity is altered. The polypeptide pair scan be identified by methods known in the art, includin gyeast two hybri dscreens Yea. st two hybrid screens are describe ind U. S. Pat. Nos. 5,283,173 and 6,562,576. Affinities between a pair of interacting domains can be determined using methods known in the art, including as described in Katahir a,et al., J. Biol Chem, 277, 9242-9246 (2002)). Alternatively a libra, ry of peptide sequences can be screened for heterodimerizati foron, example, using the methods described in WO 01/00814. Useful methods for protein-prot inteineractions are also describe d inU. S. Patent No. 6,790,624.

[00342] A "multimerizat iondomain” or “oligomerizati domaon in” referre tod herein is a domain that causes three or more peptides or polypeptides to intera ctwith each othe r through covalent and/or non-covalent association(s). Suitable multimerizat ionor oligomerization domains include, but are not limite to,d coiled-coil domains. A coiled-coi isl a peptide sequence wit ha contiguous pattern of mainly hydropho residuesbic spaced 3 and 4 residues apart, usually in a sequence of seven amino acids (heptad repeat or) eleven amino acids (undecad repeat), whic hassembles (folds) to form a multimeric bundle of helices.

Coiled-coil withs sequences including some irregular distributi ofon the 3 and 4 residues spacing are also contemplate Hydrophobicd. residues are in particular the hydropho bicamino 87WO 2017/009712 PCT/IB2016/001079 acids Vai, He, Leu, Met ,Tyr, Phe and Trp. “Mainly hydrophobic” means that at least 50% of the residues must be selected from the mentioned hydrophobi amic no acids.

[00343] The coiled coil domain may optionall bey derived from laminin. In the extracellula spacr e, the heterotrimeric coiled coil protei lamin nin plays an important role in the formation of basement membranes .Apparently, the multifunctional oligomeri structc ure is required for laminin function. Coiled coil domains may optionall alsoy be derive dfrom the thrombospondins in which three (TSP-I and TSP-2) or five (TSP-3, TSP-4 and TSP-5) chains are connected or, from COMP (COMPcc) (Guo, et at, EMBO J, 1998, 17: 5265-5272) whic h folds into a parallel five-strand edcoiled coil (Malashkevich, et al., Science, 274: 761-765 (1996)). Additional non-limiti ngexamples of coiled-coil domains derived from othe r proteins and, othe domainsr that mediate polypepti demultimerizati areon known in the art such as the vasodilator-stimulat phosphoped rotei (VASP)n domain, matrilin-1 (CMP), viral fusion peptides solubl, NSFe (N-ethylmaleimide-sens itfactoive r) Attachment Protein receptor (SNARE) complexes, leucine-ric repeah ts, certain tRNA synthetases, are suitable for use in the disclosed fusion proteins.

[00344] In anoth erembodiment, HIDE1 polypeptides, fusion proteins, or fragment s thereof can be induced to form multimers by binding to a second multivalent polypeptide, such as an antibody. Antibodies suitable for use to multimeriz HIDe E1 polypeptides, fusion proteins or, fragments thereof include, but are not limited to, IgM antibodie ands cross - linked, multivalent IgG, IgA, IgD, or IgE complexes.

[00345] Dimerization or multimerizat ioncan occur between or among two or more fusion proteins through dimerizati onor multimerizati domains,on including those described above. Alternatively dimeri, zation or multimerizati ofon fusion proteins can occur by chemical crosslinking. Fusion protei dimen rs can be homodimers or heterodimer Fusios. n protei muln timers can be homomultimers or heteromultimer Fusios. n prote indimers as disclosed herei nare of formul II:a N-R1-R2-R3-C N-R4-R5-R6-C or, alternative arely, of formula III: N-R1-R2-R3-C C-R4-R5-R6-N wherein the fusion proteins of the dimer provided by formul IIa are defined 88WO 2017/009712 PCT/IB2016/001079 as being in a parallel orientati andon the fusion proteins of the dimer provided by formul IIIa are defined as being in an antiparall orientel ati on.Parall eland antiparalle dimersl are also referre tod as cis and trans dimers respe, ctively. “N” and “C” represent the N- and C-termini of the fusion protein, respectively. The fusion protei constin tuents “RI”, “R2” and "R3" are as defined above wit hrespect to formul I.a With respect to both formul IIa and formul III,a "R4" is aHIDEl polypeptide or a second polypeptide, “R5” is an optional peptide/polypepti linkede domar in, and "R6" is aHIDEl polypeptide or a second polypeptide, wherein "R6" is a HIDE1 polypeptide when “R4” is a second polypeptide, and "R6"" is a second polypeptide when “R4” is a HIDE1 polypeptide In. one embodiment “,RI” is a HIDE1 polypeptide, “R4” is also a HIDE1 polypeptide, and “R3” and “R6” are both second polypeptides.

[00346] Fusion prote indimers of formula II are defined as homodimers when “RI” = “R4”, “R2” = “R5” and “R3” = “R6” Similarly, fusion prote indimers of formul IIIa are defined as homodimers when “RI” = “R6”, “R2” = “R5” and “R3” = “R4” Fusion protei dimersn are defined as heterodim erswhen thes econditions are not met for any reason.

For example, heterodime mayrs optionally contai ndomain orientations that meet these conditions (i. e., for a dimer according to formula II, “RI” and "R4" are both HIDE1 polypeptide "R2"s, and “R5” are both peptide/polypepti linkede domainsr and "R3" and "R6" are both second polypeptides), however the species of one or more of thes domainse is not identical. For example, although "R3" and "R6" may optionall bothy be HIDE1 polypeptides, one polypeptide may optionall contay in a wild-typ HIDE1e amino acid sequence whil thee othe polypr epti demay optionally be a variant HIDE1 polypeptide An. exemplary variant HIDE1 polypeptide is HIDE1, polypeptide that has been modified to have increased or decrease dbinding to a targe cellt increas, ed activity on immune cell s,increased or decreased half-lif ore stability. Dimers of fusion proteins that contain either a Chi or Cl region of an immunoglobul asin part of the polypeptide linker domain preferably form heterodimers wherei onen fusion prote inof the dimer contains a Chi region and the other fusion protei ofn the dimer contains a Cl region.

[00347] Fusion proteins can also be used to form multimers. As with dimers, multimers may optionall bey parallel multimer ins, whic hall fusion proteins of the multimer 89WO 2017/009712 PCT/IB2016/001079 are aligned in the same orientation with respect to their N- and C- termini. Multime rsmay optionally be antiparalle multl imers in, which the fusion proteins of the multimer are alternatively aligned in opposite orientations with respec tot thei N-r and C-termini.

Multimer (paralls orel antiparallel can) be either homomultimers or heteromultimers.

The fusion protei isn optionall producey ind dimeric form; more preferabl they, fusion is performe atd the genetic level as described below, by joining polynucleoti seqdeuences correspond toing the tw o(or more) proteins portions, of proteins and/or peptide s,such that a joined or fused prote inis produced by a cel laccording to the joined polynucleotide sequence.

A description of preparati onfor such fusion proteins is describe withd regard to US Patent No. 5,851,795 to Linsley et al, which is hereby incorporated by reference as if fully set fort h herei nas a non-limiti ngexample only. 2. Targeting Domains

[00348] The HIDE1 polypeptides and fusion proteins can contai na targeting domain to target the molecul toe specific site sin the body. Optional targeting domains target the molecul toe areas of inflammatio n.Exemplary targeting domains are antibodies, or antigen binding fragments thereof that are specific for inflamed tissue or to a proinflammatory cytokine includin gbut not limite tod IL 17, IL-4, IL-6, IL-12, IL-21, IL-22, IL-23, MIF, TNF- a, and TNF-(3 and combinations thereof. In the case of neurological disorders such as Multiple Sclerosi thes, targeting domain may optionall targey thet molecul toe the CNS or may optionall bindy to VCAM-I on the vascula repithelium. Additional targeting domains can be peptide aptamers specific for a proinflammatory molecul e.In other embodiments, the HIDE1 fusion prote incan include a binding partne specr ific for a polypeptide displayed on the surfac eof an immune cell for, example a T cell. In sti llother embodiment s,the targeting domain specifically targets activated immune cells Optiona. iml mune cells that are target ed include ThO, Thl, Th 17, Th2 and Th22 T cells, other cell sthat secret e,or cause other cell sto secret infle ammatory molecules including, but not limited to, IL-1(3, TNF-a, TGF-(3, IFN-y, IL-17, IL-6, IL-23, IL-22, IL-21, and MMPs, and Tregs. For example, a targeting domain for Tregs may optionall bindy specifically to CD25.

[00349] Other targeting moieties or heterologo polypeus ptides that optional mayly optionally be attached or containe dwithin HIDE1 polypeptides or fusion proteins according 90WO 2017/009712 PCT/IB2016/001079 to some embodiments of the present inventio nare discussed supra in connection wit hthe synthese isof exemplary fusion proteins according to some embodiments of the present invention.

[00350] The above changes are intended as illustrations only of optiona changesl and are not meant to be limiting in any way. Furthermor thee, above explanation is provided for descripti vepurposes only, without wishing to be bound by a singl ehypothesis.

C. ADDITION OF GROUPS

[00351] If a prote inaccording to some embodiments of the present inventio nis a linear molecule, it is possible to place various functional groups at various point son the linear molecul whice h are susceptible to or suitable for chemical modification. Functional groups can be added to the termini of linear forms of the protei accordingn to at leas tsome embodiments of the present invention. In some embodiment s,the functional groups improve the activity of the prote inwit hregar dto one or more characterist includingics, but not limited to, improvement in stabilit penety, ration (through cellula memr branes and/or tissue barriers), tissue localization, efficacy, decreased clearance, decreased toxicit y,improved selectivity, improved resistanc toe expulsion by cellula pumps,r and the like. For convenience sake and without wishing to be limiting, the free N-terminus of one of the sequences contained in the compositions according to at least some embodiments of the present invention will be termed as the N-terminus of the composition, and the free C-terminal of the sequence wil bel considered as the C-terminus of the composition. Either the C-terminus or the N-terminus of the sequences ,or both, can be linked to a carboxylic acid functional groups or an amine functional group, respectively.

[00352] Non-limiti ngexamples of suitable functional groups are described in Green and Wuts ,“Protectin Groupsg in Organic Synthesi”s, John Wiley and Sons, Chapters 5 and 7, (1991), the teachings of which are incorporat hereined by reference. Preferr edprotecting groups are those that facilitate transport of the active ingredient attached thereto into a cell, for example, by reducing the hydrophilicity and increasin gthe lipophilicit of ythe active ingredient thes, beinge an example for “a moiety for transport across cellula membrar nes”. 91WO 2017/009712 PCT/IB2016/001079

[00353] These moieties can optionally and preferably be cleaved in vivo, either by hydrolysis or enzymaticall y,inside the cell. (Bitter et al., J. Pharm Sci. 57:783 (1968); Bitter et al., J. Pharm. Sci. 57:828 (1968); Bitter et al., J. Pharm. Sci. 58:557 (1969); King et al., Biochemistry 26:2294 (1987); Lindberg et al., Drug Metabolism and Disposition 17:311 (1989); and Tunek et al., Biochem. Pharm. 37:3867 (1988), Anderson et aV,Arch. Biochem.

Biophys. 239:538 (1985) and Singhal et al., FASEBJ. 1:220 (1987)). Hydroxyl protecti ng groups include esters, carbonat esand carbamate protecting groups Ami. ne protecti groupsng include alkoxy and aryloxy carbonyl groups, as describe dabove for N-terminal protecti ng groups Carboxylic. acid protecti groupsng include aliphati benzylic, c and aryl esters, as described above for C-terminal protecti groupsng In. one embodiment, the carboxyl icacid group in the side chain of one or more glutamic acid or asparti acic d residue in a composition accorind to some embodiments of the present invention is protected, preferably wit ha methyl, ethyl benzyl, or substitut benzyled ester more, preferably as a benzyl ester.

[00354] Non-limiting ill, ustrat exampive les of N-terminal protecti groupsng include acyl groups (-CO-R1) and alkoxy carbonyl or aryloxy carbonyl groups (-CO-O-R1), wherein RI is an aliphati substic, tut aliped hati benzyl,c, substituted benzyl, aromati orc a substitut ed aromat icgroup. Specific examples of acyl groups include but are not limited to acetyl, (ethyl)-CO- n-propyl-CO-,, iso-propyl-CO n-butyl-CO-, -,sec-butyl-CO- t-butyl, -CO- hexyl, lauroyl, palmitoyl, myristoyl, stearyl oleoyl, phenyl-CO-, substitut phenyl-COed -, benzyl-CO- and (substituted benzyl)-CO-. Examples of alkoxy carbonyl and aryloxy carbonyl groups include CH3-O-CO-, (ethyl)-O-CO-, n-propyl-O-CO iso-prop-, yl-O-CO-, n-butyl-O-CO -,sec-butyl-O-CO- t-butyl-O-CO-, phenyl-O- CO-, substitut ed phenyl-O-CO -and benzyl-O-CO-, (substituted benzyl)- O-CO-, Adamantan, naphtale n, myristoleyl, toluen, biphenyl, cinnamoyl, nitrobenzoy, toluoyl furoyl,, benzoyl, cyclohexane, norboman e,or Z-caproic. In order to facilitate the N-acylation, one to four glycine residues can be present in the N-terminus of the molecule.

[00355] The carboxyl group at the C-terminus of the compound can be protect ed,for example, by a group including but not limite tod an amide (i. e., the hydroxyl group at the C-terminus is replaced wit h-NH 2, -NHR2 and -NR2R3) or ester (i. e., the hydrox ylgroup at the C-terminus is replaced wit h-OR2). R2 and R3 are optionall independently any aliphatic, 92WO 2017/009712 PCT/IB2016/001079 substitut aliedphati benzyl,c, substitut benzyl,ed aryl or a substitut aryled group. In addition, taken together with the nitrogen atom, R2 and R3 can optionally form a C4 to C8 heterocycli ringc with from about 0-2 additional heteroatoms such as nitrogen, oxygen or sulfur. Non-limiti ngsuitable examples of suitable heterocycli ringsc include piperidinyl, pyrrolidi nyl,morpholino, thiomorpholino or piperazinyl Exampl. es of C-terminal protecti ng groups include but are not limited to -NH2, -NHCH3, -N(CH3)2 -NH(ethyl), -N(ethyl)2 , -N(methyl) (ethyl), -NH(benzyl), -N(C1-C4 alky !)(benzyl) ,-NH(phenyl), -N(C1-C4 alkyl) (phenyl), -OCH3 .

O-(ethyl ),-O-(n-propyl) -O-(n-bu, tyl), -O-(iso-propyl -O-(s), ec- butyl), -O-(t-butyl), -O-benz yl and -O-phenyl.

D. SUBSTITUTION BY PEPTIDOMIMETIC MOIETIES

[00356] A "peptidomimet organicic moiet”y can optional bely substitut fored amino acid residues in the compositi onof thi sinventio nboth as conservative and as non- conservative substitutions. These moieties are also termed "non-natur alamino acids” and may optionall replay ce amino acid residues, amino acids or act as spacer groups within the peptides in lieu of deleted amino acids. The peptidomimeti organicc moieties optionall andy preferably have steric, electronic or configurational properti simes ilar to the replaced amino acid and such peptidomimetics are used to replace amino acids in the essential positions, and are considered conservative substitutions. However such similarit iesare not necessarily required. According to some embodiments of the present invention, one or more peptidomimetics are selected such that the compositi onat least substantial retaily ns its physiologica actil vity as compared to the native protei accordin ng to some embodiments of the present invention.

[00357] Peptidomimeti maycs optionall bey used to inhibit degradation of the peptides by enzymatic or othe degradatr ive processes. The peptidomimetics can optionall andy preferably be produced by organic syntheti techc niques. Non-limiti ngexamples of suitable peptidomimetics include D amino acids of the corresponding L amino acids, tetrazol (Zabrocki et al., J. Am. Chem. Soc. 110:5875-5880 (1988)); isosteres of amide bonds (Jones et al., Tetrahedron Lett. YL 3853-3856 (1988)); LL-3-amino-2-propenidone-6-carboxylic 93WO 2017/009712 PCT/IB2016/001079 acid (LL-Acp) (Kemp et al., J. Org. Chem. 50:5834-5838 (1985)). Similar analogs are shown in Kemp et al, Tetrahedron Lett. 29:5081-5082 (1988) as wel las Kemp et al, Tetrahedron Lett. 29:5057-5060 (1988), Kemp et al., Tetrahedron Lett. 29:4935-4938 (1988) and Kemp et al, J. Org. Chem. 54:109-115 (1987). Other suitable but exemplary peptidomimetics are shown in Nagai and Sato, Tetrahedron Lett. 26:647-650 (1985); Di Maio et al., J. Chem. Soc.

Perkin Trans., 1687 (1985); Kahn et al., Tetrahedron Lett. 30:2317 (1989); Olso net al., J.

Am. Chem. Soc. 112:323-333 (1990); and Garvey et al., J. Org. Chem. 56:436 (1990). Further suitable exemplary peptidomimeti includecs hydroxy- 1,2,3,4-tetrahydroisoquinoli 3-carboxylane- te(Miyake et al., J. Takeda Res. Labs 43:53-76 (1989)); 1, 2,3,4-tetrahydro- isoquinoline-3-carboxylat (Kazme ierski et al., J. Am.

Chem. Soc. 133:2275-2283 (1991)); histidine isoquinolon carboxylie acic d (HIC) (Zechel et al.,/«t. J. Pep. Protein Res. 43 (1991)); (2S, S)-methyl-phenylalani (2S,ne, 3R)-methyl-phenylalanine (2R,, 3S)-methyl- phenylalanin ande (2R, 3R)-methyl-phenylalanine (Kazmierski and Hruby ,Tetrahedron Lett. (1991)).

[00358] Exemplary, illustrat butive non-limiti ngnon-natural amino acids include (3- amino acids (|33 and (32), homo-amino acids ,cyclic amino acids ,aromat icamino acids ,Pro and Pyr derivatives, 3-substitut Alanied ne derivatives, Glycine derivatives, ring-substituted Phe and Tyr Derivatives, linea rcore amino acids or diamino acids .They are availabl efrom a variet yof suppliers, such as Sigma-Aldrich (USA).

E. PROTEIN CHEMICAL MODIFICATIONS

[00359] In the present invention, according to at least some embodiment s,any part of a protei accordn ing to at leas tsome embodiments of the present invention may optionall bey chemically modified, i. e. changed by addition of functional groups. For example the side amino acid residues appearing in the native sequence may optionally be modified, although as described below alternativel othery part sof the prote inmay optional bely modified, in addition to or in place of the side amino acid residues. The modification may optionally be performe durind gsynthes isof the molecul ife a chemical synthet processic is follow ed,for example by adding a chemicall modiy fied amino acid. However, chemical modification of an amino acid when it is already present in the molecul (e“in situ” modification) is also possible. 94WO 2017/009712 PCT/IB2016/001079

[00360] The amino acid of any of the sequence regions of the molecul cane optional ly be modified according to any one of the follow ingexemplary types of modification (in the peptide conceptuall viewey d as “chemicall modifiy ed”). Non-limiti ngexemplary types of modification include carboxymethylat ion,acylation, phosphorylat glycoion, sylat ionor fatt y acylation. Ethe rbonds can optionall bey used to join the serine or threonine hydrox ylto the hydrox ylof a sugar. Amide bonds can optional bely used to join the glutamate or aspartat e carboxyl groups to an amino group on a sugar (Garg and Jeanloz, Advances in Carbohydrate Chemistry and Biochemistry, Vol .43, Academic Press (1985); Kunz, Ang. Chern. Int. Ed.

English 26:294-308 (1987)). Acetal and ketal bonds can also optionally be formed between amino acids and carbohydrates. Fatt yacid acyl derivatives can optionally be made, for example, by acylation of a free amino group (e.g, lysine) (Toth et al., Peptides: Chemistry, Structure and Biology, Rivier and Marshal, eds., ESCOM Publ., Leiden, 1078-1079 (1990)).

[00361] As used herein the term “chemical modification”, when referrin tog a protein or peptide according to some embodiments of the present invention, refers to a protei or n peptide where at least one of its amino acid residues is modified either by natural processes , such as processi ngor other post-translational modificatio ns,or by chemical modification techniques which are well known in the art. Examples of the numerous known modifications typicall includey but, are not limite to:d acetylation, acylation, amidation, ADP-ribosylati on, glycosylati on,GPI anchor formation, covalent attachment of a lipi dor lipi dderivative, methylati on,myristoylati pegylaon, tion, prenylation, phosphoryla tiubiqon,uitination, or any similar process.

[00362] Other types of modifications optionally include the addition of a cycloalkane moiety to a biological molecul e,such as a protein, as described in PCT Application No. WO 2006/050262, hereby incorporat byed reference as if fully set forth herein. These moieties are designed for use with biomolecul esand may optional bely used to impart various propertie s to proteins.

[00363] Furthermore, optionall anyy point on a prote inmay be modified. For example, pegylation of a glycosylati moieon ty on a prote inmay optionall bey performed, as described in PCT Application No. WO 2006/050247, hereby incorporat byed reference as if fully set forth herein. One or more polyethylene glycol (PEG) groups may optionall bey added to O- 95WO 2017/009712 PCT/IB2016/001079 linked and/or N-linked glycosylati on.The PEG group may optionall bey branched or linear.

Optionally any type of water-soluble polymer may be attached to a glycosylation sit eon a protei throughn a glycosyl linker.

F. ALTERED GLYCOSYLATION

[00364] Proteins according to at least some embodiments of the present invention may optionally be modified to have an altered glycosylati patternon (i. e., altered from the original or native glycosylati patton ern). As used herein, “altered” means having one or more carbohydrate moieties deleted, and/or having at least one glycosylati siton eadded to the original protein.

[00365] Glycosylati ofon proteins is typicall eity her N-linked or O-linked. N-linked refer sto the attachment of the carbohydr atemoiety to the side chain of an asparagine residue.

The tripepti sequede nces ,asparagine-X-serine and asparagine-X-threoni wherene, X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylati sitone. O-linked glycosylati referson to the attachment of one of the sugars N-acetylgalactosam ine,galactos e, or xylos toe a hydroxyamino acid, most commonly serine or threonine, although 5- hydroxyprol orine 5-hydroxylysine may optionally also be used.

[00366] Addition of glycosylati siteon sto proteins according to at least some embodiments of the present inventio nis convenientl accomply ishe byd altering the amino acid sequence of the protei suchn that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylati siteon s). The alterat ionmay optionally also be made by the addition of, or substitution by, one or more serine or threonine residues in the sequence of the original prote in(for O-linked glycosylat ionsites). The protein's amino acid sequence may optionall alsoy be altered by introducing changes at the DNA level.

[00367] Another means of increasing the number of carbohydrate moieties on proteins is by chemical or enzymatic coupling of glycosides to the amino acid residues of the protein.

Depending on the coupling mode used, the sugars may optionall bey attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, 96WO 2017/009712 PCT/IB2016/001079 (d) free hydroxyl groups such as thos ofe serine, threonine, or hydroxy proline, (e) aromat ic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87/05330, and in Aplin and Wriston, CRC Crit. Rev. Biochem., 22: 259-306 (1981).

[00368] Removal of any carbohydr atemoieties present on protei nsaccording to at least some embodiments of the present invention may optionally be accomplished chemically or enzymatically Chem. ica ldeglycosylati requireson exposure of the protei ton trifluoromethanesu lfoniacid, orc an equivalent compound. This treatment result ins the cleavage of most or all sugars except the linking sugar (N-acetylglucosami orne N- acetylgalactosami ne),leaving the amino acid sequence intact.

[00369] Chemical deglycosylatio is descrin bed by Hakimuddin et al., Arch. Biochem.

Biophys., 259: 52 (1987); and Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on proteins can be achieved by the use of a variet yof endo- and exo-glycosidases as describe byd Thotakura et al., Meth. Enzymol., 138: 350 (1987).

VI. Particular Constructs

[00370] The disclose HIDd E1 fusion proteins optionally contain a peptide or polypeptide linke domainr that separat esthe HIDE1 polypeptide from the second polypeptide (see, Figure 66). Various non-limiti ngexamples of such linke domainsr are described herein.

In one embodiment the, linke domainr contains the hinge region of an immunoglobulin. In a furth erembodiment, the hinge region is derived from a human immunoglobulin. Suitabl e human immunoglobulins that the hinge can be derived from include IgG, IgD and IgA. In a furth erembodiment, the hinge region is derive dfrom human IgG. Amino acid sequences of immunoglobul hingein regions and other domains are well known in the art. In one embodiment, HIDE1 fusion polypeptides contain the hinge, Ch2 and Ch3 regions of a human immunoglobul Cylin chain, optionall withy the Cys at position 220 (according to full lengt h human IgGl, position 5 in SEQ ID NO:48) replaced with a Ser having at least 85%, 90%, 95%, 99% or 100% sequence homology to amino acid sequence set forth in SEQ ID NO:49: SEQ ID NO:48 97WO 2017/009712 PCT/IB2016/001079 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQ ID NO:49 EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK SEQ ID NO:50 EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

[00371] The hinge can be further shortened to remove amino acids 1, 2, 3, 4, 5, or combinations thereof of any one of SEQ ID NOs: 48-50. In one embodiment, amino acids 1-5 of any one of SEQ ID NOs:48-50 are deleted. Exemplary HIDE1 fusion polypeptides comprised of the hinge, CH2 and CH3 regions of a human immunoglobul Cylin chain wit h the Cys at position 220 replaced with a Ser are set forth in SEQ ID NO: 17.

SEQ ID NO: 17 IPAPSIRLVPPYPSSQEDPIHIACMAPGNFPGANFTLYRGGQVVQLLQAPTDQRGVTFNLSGGSSKAPGGPFHCQ YGVLGELNQSQLSDLSEPVNVSFPVPTWEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTOKSLSLSPGK

[00372] In anoth erembodiment, the HIDE1 fusion polypeptides contain the CH2 and CH3 regions of human immunoglobulin Cyl chain having N297A mutation (SEQ ID NO:50) or the human Fc carrying the C220S, C226 and C229S mutations (SEQ ID NO:86).

[00373] In anoth erembodiment, HIDE1 fusion polypeptides contain the Ch2 and Ch3 regions of a human immunoglobul Cylin chain having at leas t85%, 90%, 95%, 99% or 100% sequence homology to amino acid sequence set forth in SEQ ID NO:51: SEQ ID NO:51 APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV 98WO 2017/009712 PCT/IB2016/001079 LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTOKSLSLSPGK

[00374] In another embodiment the, HIDE1 fusion polypeptides contain the hinge, CH2 and CH3 regions of a murine immunoglobulin Cy2a chain at least 85%, 90%, 95%, 99% or 100% sequence homology to amino acid sequence set forth in SEQ ID NO: 52: SEQ ID NO:52 EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQT QTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVT LTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTK SFSRTPGK

[00375] In anoth erembodiment, the HIDE1 fusion polypeptides contain the CH2 and CH3 regions of a murine immunoglobul Cy2ain chain having N297A mutation (SEQ ID NO:53) or the murine Fc without the Hinge (SEQ ID NO:54).

SEQ ID NO53 EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQT QTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVT LTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTK SFSRTPGK SEQ ID NO:54 APNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSA LPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVE WTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[00376] In another embodiment, the linker domain optionally contains a hinge region of an immunoglobulin as described above, and furth erincludes one or more additional immunoglobul domain ins.

A. Peptide or polypeptide linker domain

[00377] The disclosed HIDE1 fusion proteins optionall conty ain a peptide or polypeptide linke domainr that separat esthe HIDE1 polypeptide from the second polypeptide Vari. ous non-limiti ngexamples of such linke domainsr are described herein. In 99WO 2017/009712 PCT/IB2016/001079 one embodiment, the linke domainr contains the hinge region of an immunoglobulin. In a furth erembodiment, the hinge region is derived from a human immunoglobulin. Suitabl e human immunoglobulins that the hinge can be derived from include IgG, IgD and IgA. In a furth erembodiment, the hinge region is derive dfrom human IgG. Amino acid sequences of immunoglobul hingein regions and other domains are well known in the art. In one embodiment, HIDE1 fusion polypeptides contain the hinge, CH2 and CH3 regions of a human immunoglobul Cylin chain, optionall withy the Cys at position 220 (according to full length human IgGl ,position 5 in SEQ ID NO:48) replaced with a Ser having at least 85%, 90%, 95%, 99% or 100% sequence homology to amino acid sequence set forth in SEQ ID NO:49: EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

[00378] The hinge can be further shortened to remove amino acids 1, 2, 3, 4, 5, or combinations thereof of any one of SEQ ID NOs: 48-50. In one embodiment, amino acids 1-5 of any one of SEQ ID NOs: 48-50 are deleted. Exemplary HIDE1 fusion polypeptides comprised of the hinge, CH2 and CH3 regions of a human immunoglobul Cylin chain wit h the Cys at position 220 replaced with a Ser are set forth in SEQ ID NO: 17.

SEQ ID NO: 17 IPAPSIRLVPPYPSSQEDPIHIACMAPGNFPGANFTLYRGGQVVQLLQAPTDQRGVTFNLSGGSSKAPGGPFHCQ YGVLGELNQSQLSDLSEPVNVSFPVPTWEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTOKSLSLSPGK

[00379] In anoth erembodiment, the HIDE1 fusion polypeptides contain the CH2 and CH3 regions of human immunoglobulin Cyl chain having N297A mutation (SEQ ID NO:50) or the human Fc carrying the C220S, C226 and C229S mutations (SEQ ID NO:86).

SEQ ID NO:86 EPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSL 100WO 2017/009712 PCT/IB2016/001079 TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGK

[00380] In anoth erembodiment, HIDE1 fusion polypeptides contain the CH2 and CH3 regions of a human immunoglobul Cylin chain having at leas t85%, 90%, 95%, 99% or 100% sequence homology to amino acid sequence set forth in SEQ ID NO:51: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTOKSLSLSPGK

[00381] In another embodiment the, HIDE1 fusion polypeptides contain the hinge, CH2 and CH3 regions of a murine immunoglobulin Cy2a chain at least 85%, 90%, 95%, 99% or 100% sequence homology to amino acid sequence set forth in SEQ ID NO: 52: EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQT QTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVT LTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTK SFSRTPGK

[00382] In anoth erembodiment, the HIDE1 fusion polypeptides contain the CH2 and CH3 regions of a murine immunoglobul Cy2ain chain having N297A mutation (SEQ ID NO:53) or the murine Fc without the Hinge (SEQ ID NO:54).

SEQ ID NO 53 EPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQT QTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVT LTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTK SFSRTPGK SEQ ID NO:54 APNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSA LPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVE WTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK 101WO 2017/009712 PCT/IB2016/001079

[00383] In another embodiment, the linker domain optionally contains a hinge region of an immunoglobulin as described above, and furth erincludes one or more additional immunoglobul domain ins.

VII. Nucleic Acid Compositions

[00384] Nucleic acid compositions encoding the HIDE1 polypeptides of the invention are also provide d,as well as expression vectors containing the nucleic acids and host cell s transforme withd the nucleic acid and/or expression vector compositions.

[00385] The nucleic acid compositions that encode the HIDE1 polypeptides are generally put into a singl eexpression vectors is known in the art, transforme intod host cells, where they are expressed to form the HIDE1 proteins (or fusion proteins) of the invention.

The nucleic acids can be put into expression vectors that contain the appropriate transcriptional and translational control sequences ,including, but not limited to, signal and secretion sequences, regulatory sequences, promoters origi, ns of replication, selection genes, etc.

[00386] For example, to express the prote inDNA, DNAs can be obtained by standard molecular biology technique s(e.g., PCR amplification or gene synthesis) and the DNAs can be inserted into expression vectors such that the genes are operativel linkedy to transcriptional and translational control sequences. In thi scontext, the term “operativel y linked” is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve thei intendedr function of regulati ngthe transcript andion translat ionof the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cel lused. The protei genesn are inserted into the expression vector by standard methods (e.g, ligation of complementary restricti siontes on the gene fragment and vector, or blunt end ligation if no restricti siteon sare present). Additional orly alternatively, the recombinan t expression vector can encode a signal peptide that facilitate secrets ion of the protei n (including fusion proteins) from a host cell. The gene can be cloned into the vector such that the signal peptide is linke din-frame to the amino terminus of the gene. The signal peptide can be an immunoglobul signin al peptide or a heterologous signal peptide (z.e., a signal peptide from a non-immunoglobuli protein n). 102WO 2017/009712 PCT/IB2016/001079

[00387] In addition to the protei genen s, the recombinan expresst ion vectors according to at least some embodiments of the invention carr yregulatory sequences that control the expression of the genes in a host cell. The term “regulatory sequence” is intended to include promoters enhancers, and other expression control element s(e.g., polyadenylatio signaln s) that control the transcript orion translat ionof the genes. Such regulatory sequences are described, for example, in Goeddel (“Gene Expression Technology”, Methods in Enzymology 185, Academic Press San, Diego, Cali f(1990)). It will be appreciate byd thos skie ll edin the art that the design of the expression vector, including the selection of regulatory sequences, may depend on such factors as the choice of the host cel lto be transformed, the level of expression of protei desirn ed, etc .Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protei expressn ion in mammalian cell s,such as promoters and/or enhancers derive dfrom cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP) and polyoma. Alternative nonviraly, regulal tory sequences may be used, such as the ubiquitin promoter or B-globin promoter Stil. further,l regulatory element scomposed of sequences from different sources such, as the SR a. promoter system, whic hcontains sequences from the SV40 earl ypromoter and the long terminal repeat of human T cel lleukemi avirus type 1 (Takebe, Y. et al. (1988)Mol. Cell. Biol. 8:466-472).

[00388] In addition to the protei genesn and regulatory sequences ,the recombinant expression vectors according to at least some embodiments of the invention may carry additional sequences, such as sequences that regulat replicate ion of the vector in host cell s (e.g., origins of replicatio andn) selectable marker genes. The selectable marker gene facilitate selects ion of host cells into which the vector has been introduced (see, e.g, U.S. Pat.

Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example, typically the selectab lemarker gene confers resistanc toe drugs, such as G418, hygromycin or methotrexate, on a host cel linto whic hthe vector has been introduced. Preferred selectabl e marker genes include the dihydrofolate reducta se(DHFR) gene (for use in dhfr- host cell s with methotrexate selection/amplificatio andn) the neo gene (for G418 selection).

[00389] For expression of the proteins of the invention, an expression vector encoding the protei isn transfect edinto a host cell by standard techniques. The various forms of the 103WO 2017/009712 PCT/IB2016/001079 term “transfection” are intended to encompass a wide variet yof techniques commonly used for the introducti ofon exogenous DNA into a prokaryo orti ceukaryoti hostc cell, e.g., electroporat calciumion, -phosphat precie pitati on,DEAE-dextran transfecti onand the like.

Although it is theoreticall possibley to expres sthe proteins according to at least some embodiments of the invention in either prokaryotic or eukaryoti hostc cell s,expression of antibodie ins eukaryoti cellc s,and most preferably mammalian host cells, is the most preferred.

[00390] Preferred mammalian host cells for expressing the recombinan proteit ns according to at least some embodiments of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cell s,described in Urlaub and Chasin, (1980) Proc. Natl.

Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R.

J. Kaufman and P. A. Shar p(1982)Mol. Biol. 159:601-621), NSO myeloma cell s,COS cell s and SP2 cells. In particular, for use wit hNSO myeloma cells, another preferre expressid on system is the GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338,841. When recombinant expression vectors encoding protei genesn are introduced into mammalian host cell s,the proteins are produce byd culturing the host cell sfor a period of time sufficient to allow for expression of the protei inn the host cell sor, more preferably, secretion of the prote ininto the culture medium in whic hthe host cells are grown. Antibodies can be recovered from the culture medium using standard protei purifn ication methods.

[00391] HIDE1 protei codingn sequences of interest include those encoded by native sequences ,as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to the disclose nucled ic acids ,and variants thereof. Variant polypeptides can include amino acid substitutions as discussed herein. Techniques for in vitro mutagenesi sof clone dgenes are known. Also included in the subject inventio nare polypeptides that have been modified using ordinary molecular biologic altechniques so as to improve their resistanc toe proteolyti degradationc or to optimize solubilit property iesor to render them more suitable as a therapeutic agent.

[00392] The inventio nfurther provides nucleic acids which encode a HIDE1 protein according to the invention, or a fragment or conjugat ethereof. The nucleic acids may be present in whol celle s,in a cell lysate, or in a partially purified or substantial purely form .A 104WO 2017/009712 PCT/IB2016/001079 nucleic acid is “isolat”ed or “rendered substantial purely ” when purified away from othe r cellula componentsr or other contaminant s,e.g., other cellula nucleicr acids or proteins by, standard techniques, including alkaline/SDS treatment, CsCl banding, colum n chromatography, agarose gel electrophoresi and sothers well known in the art. See, F.

Ausubel ,et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. A nucleic acid according to at least some embodiments of the invention can be, for example, DNA or RNA and may or may not contain intronic sequences.

[00393] Nucleic acid compositions encoding the anti-HIDE antibodie ofs the invention are also provide d,as well as expression vectors containing the nucleic acids and host cell s transforme withd the nucleic acid and/or expression vector compositions. As wil bel appreciate byd thos ine the art, the prote insequences depicted herei ncan be encoded by any number of possible nucleic acid sequences ,due to the degenerac yof the genetic code.

[00394] The nucleic acid compositions that encode the HIDE1 antibodies will depend on the format of the antibody. For traditional, tetrameric antibodie contais ning tw oheavy chains and two light chains are encoded by two different nucleic acids, one encoding the heavy chain and one encoding the light chain. These can be put into a singl eexpression vector or two expression vectors, as is known in the art, transforme intod host cell s,where they are expressed to form the antibodi esof the invention. In some embodiments, for example when scFv constructs are used, a singl enucleic acid encoding the variabl heavye chain-linker-variabl lighte chain is generally used, which can be inserted into an expression vector for transformat intoion host cells. The nucleic acids can be put into expression vectors that contain the appropriate transcriptional and translational control sequences ,including, but not limited to, signal and secretion sequences, regulatory sequences, promoters, origins of replication, selection genes, etc.

[00395] Preferred mammalian host cells for expressing the recombinan antibodiet s according to at least some embodiments of the invention include Chinese Hamster Ovary (CHO cells), PER.C6, HEK293 and others as is known in the art. The nucleic acids may be present in whol celle s,in a cell lysate, or in a partially purified or substantial purely form .A nucleic acid is “isolat”ed or “rendered substantial purely ” when purified away from othe r cellula componentsr or other contaminant e.g.,s, other cellula nucleicr acids or proteins by, 105WO 2017/009712 PCT/IB2016/001079 standard techniques, including alkaline/SD treatmS ent, CsCl banding, colum n chromatography, agarose gel electrophoresi and sothers well known in the art.

[00396] To create a scFv gene, the Vh- and VL-encoding DNA fragments are operatively linke dto anoth erfragment encoding a flexible linker, e.g, encoding the amino acid sequence (Gly4-Ser3 )(SEQ ID NO: 61), such that the Vh and Vl sequences can be expressed as a contiguou singls e-chai proten in, wit hthe Vl and Vh regions joined by the flexibl linkee (seer e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc.

Natl. Acad. Set. USA 85:5879-5883; McCaffert yet al., (1990) Nature 348:552-554).

VIII. Formulations

[00397] The therapeutic compositions used in the practice of the foregoing methods can be formulat intoed pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carrier includes any materia thatl when combined with the therapeutic compositi onretains the anti-tumor function of the therapeutic compositi onand is generall non-ry eactive wit hthe patient' ims mune system Exampl. es include, but are not limite to,d any of a number of standard pharmaceutic alcarrier suchs as steri lephosphat e buffered saline solutions, bacteriostat watic er, and the like (see, generall y,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980). Acceptabl ecarriers, excipients or, stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate aceta, te, and other organic acids ;antioxidants includin g ascorbi acic d and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol resorci; nol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide proteis; ns, such as serum albumin, gelatin, or immunoglobulins hydrophilic; polymers such as polyvinylpyrroli done;amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose ,or dextrins chel; ating agents such as EDTA; sugars such as sucrose mannitol, tre, halose or sorbitol sweet; eners and other flavoring agents ;fille rssuch as microcrystal cellline ulose, lactose com, and other starches; binding agents ;additives; coloring agents ;salt-form ingcounter-ion suchs as sodium met; al complexes (e.g. Zn-protein 106WO 2017/009712 PCT/IB2016/001079 complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[00398] In a preferred embodiment, the pharmaceutical compositi onthat comprises the antibodie ofs the invention may be in a water-soluble form, such as being present as pharmaceutical acceptly able salts, which is meant to include both acid and base addition salts “.Pharmaceutically acceptable acid addition salt” refers to thos sale ts that retain the biologic aleffectivenes sof the free bases and that are not biologicall ory otherwi se undesirabl formede, wit hinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitri acid,c phosphoric acid and the like, and organic acids such as acetic acid, propioni acid,c glycol icacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfoni acid,c p-toluenesulfoni acid,c salicyli acic d and the like.

“Pharmaceutically acceptable base addition salt” sinclude thos derivede from inorganic bases such as sodium, potassium, lithium, ammonium ,calcium, magnesium, iron, zinc, copper, manganese, aluminum salt ands the like. Particularly preferred are the ammonium ,potassium, sodium calci, um, and magnesium salt s.Salt sderive dfrom pharmaceutically acceptable organic non-toxi basec s include salt ofs primary, secondary, and tertiary amines, substitut ed amines including naturall occurringy substitut amied nes, cyclic amines and basic ion exchange resins such, as isopropylami trimne, ethylamin diete, hylamine, triethylami ne, tripropylamine, and ethanolamine. The formulations to be used for in vivo administrati areon preferrabl stey rile. This is readily accomplished by filtrati throughon steril file trati on membranes or othe metr hods.

[00399] In some embodiments of, administration of the pharmaceutic alcomposition comprising antibodie ofs the present invention, preferably in the form of a steri leaqueous solution, may be done in a variety of ways, including, but not limited to subcutaneously and intravenous ly.Subcutaneous administration may be preferabl ine some circumstances because the patient may self-administer the pharmaceutic alcomposition. Many prote in therapeuti arecs not sufficientl potenty to allow for formulation of a therapeuticall effecty ive dose in the maximum acceptable volume for subcutaneous administration. This problem may be addresse ind part by the use of protei formulan tions comprising arginine-HCl hist, idine, 107WO 2017/009712 PCT/IB2016/001079 and polysorbate (see WO 04091658). Fc polypeptides of some embodiments of the present invention may be more amenable to subcutaneous administration due to, for example, increased potency, improved serum half-life, or enhanced solubility.

[00400] As is known in the art, protei therapeutin arecs often delivered by IV infusion or bolus. In some embodiments, the antibodie ofs the present inventio nmay also be delivered using such methods. For example, administration may venious be by intravenous infusion with 0.9% sodium chloride as an infusion vehicle.

[00401] In addition, any of a number of delivery systems are known in the art and may be used to administer the Fc variants of the present inventio nin various embodiments.

Examples include, but are not limited to, encapsulation in liposomes, microparticle s, microspheres (eg. PLA/PGA microspheres), and the like Alte. rnative anly, implant of a porous, non-porous, or gelatinous material, includin gmembranes or fibers, may be used.

Sustained releas syste ems may comprise a polymeri matc erial or matri xsuch as polyeste rs, hydrogels poly, (vinylalcohol), polylactides, copolyme rsof L-glutamic acid and ethyl-L- gutamate, ethylene-vinyl acetate, lacti cacid-glycolic acid copolymers such as the LUPRON DEPOTr™, and poly-D-(-)-3-hydroxyburyric acid. The antibodies disclose hereind may also be formulated as immunoliposome As. liposome is a smal lvesicl ecomprising various types of lipids phospho, lipids and/or surfactant that is useful for delivery of a therapeutic agent to a mammal .Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., 1985, Proc Nat lAcad Sci USA, 82:3688; Hwang et al., 1980, Proc Nat lAcad Sci USA, 77:4030; U.S. Pat. No. 4,485,045; U.S. Pat. No. 4,544,545; and PCT WO 97/38731. Liposomes wit henhanced circulati ontime are disclosed in U.S. Pat. No. ,013,556. The components of the liposome are commonly arranged in a bilayer formation , similar to the lipi darrangement of biologica membral nes. Particularly useful liposomes can be generated by the revers phasee evaporation method wit ha lipid composition comprising phosphatidylchol choline, este roland PEG-derivatized phosphatidylethanola (PEG-PE).mine Liposomes are extruded through filte rsof defined pore size to yield liposomes with the desired diamete r.A chemotherapeutic agent or other therapeutically active agent is optional ly contained within the liposom (Gabie zon et al., 1989, J National Cancer Inst 81:1484). 108WO 2017/009712 PCT/IB2016/001079

[00402] The antibodie mays also be entrapped in microcapsul preparedes by methods including but not limite tod coacervation techniques, interfacial polymerizat ion(for example using hydroxymethylcellulose or gelatin-microcapsules or poly-(met, hylmethacyl ate) microcapsules), colloidal drug delivery systems (for example, liposome albums, in microspheres mic, roemulsion nano-particless, and nanocapsules), and macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980. Sustained-rele asepreparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrice ofs soli hydrophobicd polymer, whic hmatrice s are in the form of shaped articles, e.g. films, or microcapsul es.Examples of sustained-releas e matrice incls ude polyesters, hydrogel (fors example poly(2-hydroxyethyl-methacryla or te), poly(vinylalcohol polylac)), tides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-glutamat non-de, egradable ethylene-vinyl acetate, degradabl lacte ic acid- glycol icacid copolymers such as the LUPRON DEPOT.RTM. (which are injectable microspher composedes of lactic acid-glycolic acid copolymer and leuproli acetde ate ),poly- D-(-)-3-hydroxybutyric acid, and ProLeasRe™ (commercial lyavailable from Alkermes ), which is a microsphere-based delivery system composed of the desire bioacd tive molecule incorporated into a matri xof poly-DL-lactide-co-glycolide (PEG).

[00403] The dosing amounts and frequencies of administration are, in a preferred embodiment, selected to be therapeutical orly prophylactical effeclytive. As is known in the art, adjustments for protei degradatin on, systemi versuc slocalized delivery, and rate of new protea sesynthesi s,as well as the age, body weight, general healt h,sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable wit hroutine experimentation by thos skile led in the art.

[00404] The concentrati onof the antibody in the formulation may vary from about 0.1 to 100 weight %. In a some embodiment s,the concentration of the Fc variant is in the range of 0.003 to 1.0 molar In. order to trea at patient, a therapeuticall effecty ive dose of the Fc variant of the present invention may be administered. By “therapeutically effective dose” herei nis meant a dose that produc esthe effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skill edin the art using known techniques. Dosage smay range from 0.0001 to 100 mg/kg of body weight or 109WO 2017/009712 PCT/IB2016/001079 greater, for example 0.1, 1, 10, or 50 mg/kg of body weight wit, h1 to 10 mg/kg being preferred.

[00405] In some embodiments, the present inventio nprovides a composition e.g.,, a pharmaceutic alcomposition, containing one or a combination of the HIDE1 therapeutic agent, according to at least some embodiments of the present invention. Thus, the present invention features a pharmaceutical compositi oncomprising a therapeutically effective amount of a therapeutic agent according to at least some embodiments of the present invention.

[00406] The pharmaceutical compositi onaccording to at least some embodiments of the present inventio nis furth erpreferably used for the treatme ntof immune related disorder.

[00407] A compositi onis said to be a “pharmaceutical acceptly able carrier” if its administration can be tolerated by a recipient patient As. used herein, “pharmaceutical ly acceptable carrier” includes any and all solvents dispersi, onmedia ,coatings, antibacterial and antifungal agents, isotonic and absorpti ondelaying agents, and the like that are physiologica llcompatiy ble. Preferably, the carrier is suitable for intravenous, intramuscula r, subcutaneous parente, ral, spina lor epidermal administration (e.g., by injection or infusion).

[00408] Such compositions include steri lewater buffe, red saline (e.g, Tris-HC l, acetate, phosphate), pH and ionic strength and optional additivesly such as detergent ands solubilizi ngagents (e.g, Polysorbate 20®, Polysorbate 80®), antioxidant (e.s g, ascorbic acid, sodium metabisulfit e),preservatives (e. g, Thimersol@ benzyl, alcoho andl) bulking substances (e.g, lactose man, nitol). Non-aqueous solven tsor vehicles may optionally also be used as detailed below.

[00409] Examples of suitable aqueous and nonaqueous carriers that may optionally be employed in the pharmaceutic alcompositions according to at least some embodiments of the present inventio ninclude water ethanol,, polyol (suchs as glycerol propylene, glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectabl eorganic esters, such as ethyl oleate Proper. fluidity can be maintained, for example, by the use of coating material suchs, as lecithin, by the maintenance of the required particl sizee in the case of dispersions, and by the use of surfactants. Depending on the route 110WO 2017/009712 PCT/IB2016/001079 of administration, the active compound, i. e., soluble polypeptide, fusion prote inor conjugate containing the ectodomai ofn the HIDE1 antigen, may optionall bey coated in a materia tol protect the compound from the action of acids and othe naturalr conditions that may optionally inactivate the compound. The pharmaceutic alcompounds according to at least some embodiments of the present inventio nmay optionall incly ude one or more pharmaceutical acceptly able salts A. “pharmaceutical acceptablely salt” refers to a salt that retains the desired biologic alactivit yof the parent compound and does not impart any undesired toxicological effects (see e.g., Berge, S. M., et al. J. Pharm. Sci. 66: 1-19 . (1977)).

Examples of such salt includes acid addition salt ands base addition salt s.Acid addition salt s include thos derivede from nontoxic inorganic acids ,such as hydrochloric nitri, c,phosphoric, sulfuri c,hydrobrom hydriodicic, phosphorous, and the like, as wel las from nontoxic organic acids such as aliphat icmono- and dicarboxylic acids ,phenyl-substitut alkanoiced acids, hydroxy alkanoic acids, aromat icacids ,aliphat icand aromat icsulfon icacids and the like.

Base addition salts include thos derivede from alkaline earth metal s,such as sodium , potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N,N'-dibenzylethylenediam N-metine, hylglucamine chloroprocai, choline,ne, diethanolamine, ethylenediamin procainee, and the like.

[00410] A pharmaceutic alcompositi onaccording to at least some embodiments of the present inventio nalso may optionall incly ude a pharmaceutical acceptly able anti-oxidan t.

Examples of pharmaceutical acclyeptable antioxidant incls ude: (1) water solubl e antioxidant suchs, as ascorbi acid,c cysteine hydrochloride, sodium bisulfat e,sodium metabisulfite sodium, sulfite and the like; (2) oil-solub antioxile dants, such as ascorbyl palmitate, butylate hydroxyad nisole (BHA), butylate hydroxytold uene(BHT), lecithi n, propyl gallate, a-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraaceti acicd (EDTA), sorbitol tar, tar aciicd, phosphoric acid, and the like.

[00411] These compositions may optional alsoly contain adjuvants such as preservatives, wetting agents ,emulsifying agents and dispersing agents. Prevention of presence of microorganisms may optional bely ensured both by sterilization procedures , supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, pheno lsorbic acid, and the like. It may optionall alsoy be desirable to 111WO 2017/009712 PCT/IB2016/001079 include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.

In addition, prolonged absorpti onof the injectabl pharmae ceutic alform may optionall bey brought about by the inclusion of agents which dela yabsorption such as aluminum monostearat ande gelatin.

[00412] Pharmaceutically acceptable carriers include steri leaqueous solutions or dispersions and steri lepowders for the extemporaneous preparation of steril inje ectable solutions or dispersion. The use of such media and agents for pharmaceuticall acty ive substances is known in the art. Except insofar as any conventional media or agent is incompatible wit hthe active compound, use there ofin the pharmaceutic alcompositions according to at leas tsome embodiments of the present inventio nis contemplat ed.

Supplementary active compounds can also be incorporat intoed the compositions.

[00413] Therapeut iccompositions typically must be steril ande stabl undere the conditions of manufacture and storage. The composition can be formulat ased a solution, microemulsion lipo, some, or other ordered structure suitable to high drug concentrati on.The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propyle neglycol, and liquid polyethyl eneglycol, and the like ),and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particl sizee in the case of dispersi onand by the use of surfactants. In many cases ,it will be preferabl toe include isotonic agents, for example, sugars, polyalcohol suchs as mannitol sorbit, ol,or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by includin gin the compositi onan agent that delays absorptio forn, example, monostearat salte ands gelatin. Steril injee ctabl solutie ons can be prepared by incorporati ng the active compound in the require damount in an appropriate solvent with one or a combination of ingredient enumers ated above, as required, followed by sterilizat ion microfiltrat Generalion. ly, dispersions are prepared by incorporat theing active compound into a steri levehicle that contains a basic dispersion medium and the require dothe r ingredients from those enumerate dabove. In the case of steri lepowders for the preparation of steri leinjectable solutions, the preferre methodsd of preparation are vacuum drying and 112WO 2017/009712 PCT/IB2016/001079 freeze-drying (lyophilizati thaton) yield a powder of the active ingredient plus any additional desired ingredient from a previousl steriy le-filter soluted ion thereof.

[00414] Steril inje ectabl solutie ons can be prepared by incorporat theing active compound in the required amount in an appropriat solvente wit hone or a combination of ingredients enumerate dabove, as required, followed by sterilization microfiltration.

Generall y,dispersions are prepared by incorporat theing active compound into a steri le vehicle that contains a basic dispersion medium and the required other ingredient froms those enumerated above. In the case of steri lepowders for the preparation of steri leinjectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilizati thaton) yield a powder of the active ingredient plus any additional desired ingredient from a previous lysterile-filtered solution thereof.

[00415] The amount of active ingredient which can be combined wit ha carrier material to produce a singl edosage form will vary depending upon the subject being treated, and the particular mode of administrati on.The amount of active ingredient whic hcan be combined wit ha carrier material to produce a singl edosage form will generally be that amount of the compositi onwhic hproduc esa therapeutic effect .Generall y,out of one hundred per cent, thi samount will range from about 0. 01 per cent to about ninety-nine percent of active ingredient prefer, ably from about 0. 1 per cent to about 70 per cent, most preferably from about I per cent to about 30 per cent of active ingredient in combination wit h a pharmaceutical acclyeptable carrier.

[00416] Dosage regimens are adjusted to provide the optimum desire responsed (e.g., a therapeutic response). For example, a singl ebolus may optionally be administere severad, l divided doses may optionall bey administered over time or the dose may optionall bey proportional reducedly or increased as indicated by the exigencies of the therapeut ic situation. It is especiall advantageousy to formulat parente eral compositions in dosage unit form for ease of administration and uniformit ofy dosage .Dosage unit form as used herein refers to physical lydiscret unitse suite das unitar ydosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association wit hthe require dpharmaceutic alcarrier. The specification for the dosage unit forms according to at leas tsome embodiments of the present inventio nare 113WO 2017/009712 PCT/IB2016/001079 dictated by and direct lydependent on (a) the unique characteris ticof thes active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivi tyin individuals.

[00417] For fusion proteins as described herein, optionall a simy ilar dosage regimen is followed; alternative thely, fusion proteins may optionally be administered in an amount between 0. 0001 to 100 mg/kg weight of the patient/day, preferably between 0. 001 to 10. 0 mg/kg/day, according to any suitable timing regimen. A therapeutic compositi onaccording to at leas tsome embodiments of the present inventio ncan be administered, for example, three times a day, twice a day, once a day, three times weekl y,twice weekl yor once weekly, once every two weeks or 3, 4, 5, 6, 7 or 8 weeks. Moreover, the compositi oncan be administered over a short or long period of time (e.g, 1 week, 1 month, 1 year, 5 years).

[00418] Alternatively, therapeutic agent can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-lif ofe the therapeutic agent in the patient The. half-lif fore fusion proteins may vary widely. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic In. prophylactic applications, a relative lylow dosage is administered at relatively infrequent interval overs a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeuti c applications, a relatively high dosage at relatively short interval iss sometimes require duntil progression of the disease is reduced or terminate andd, preferably unti lthe patient shows partial or complet amelie oration of symptom ofs disease. Thereafte ther, patient can be administere ad prophylact regime.ic

[00419] In some embodiments, the actual dosage levels of the active ingredient ins the pharmaceutic alcompositions of the present inventio nmay be varied so as to obtain an amount of the active ingredient whic his effective to achieve the desired therapeutic response for a particular patient, composition and, mode of administration, without being toxic to the patient In. some embodiment s,he selected dosage level will depend upon a variety of pharmacokineti factorsc including the activit yof the particular compositions of the present invention employed, or the ester salt, or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the 114WO 2017/009712 PCT/IB2016/001079 duration of the treatment, othe drugs,r compounds and/or material useds in combination wit h the particular compositions employed, the age, sex, weight, condition, general health and prior medical histor ofy the patient being treated, and like factors well known in the medical arts.

[00420] In some embodiments, a composition of the present inventio ncan be administered via one or more routes of administration using one or more of a variet yof methods known in the art. As wil bel appreciate byd the skill edartisa n,the route and/or mode of administration will vary depending upon the desired result Routess. of administration for therapeutic agents according to at least some embodiments of the present invention include intravascula delr ivery (e.g, injection or infusion), intravenous, intramuscula intrr, adermal , intraperitone subcutaneous,al, spinal, oral, enteral rectal, pulmonary, (e.g, inhalation), nasal, topical (including transderm buccalal, and sublingual), intravesica intl, ravitrea l, intraperitone vaginalal, ,brain delivery (e.g. intra-cerebroventri cular,intra-cerebral and , convection enhanced diffusion), CNS delivery (e.g., intrathecal peris, pinal, and intra-spina l) or parenteral (including subcutaneous int, ramuscula intrr, avenous and intraderma l), transmucosa (e.g.,l sublingual administration), administration or administration via an implant, or other parenteral routes of administration, for example by injection or infusion, or othe delivr ery rout esand/or forms of administration known in the art. The phrase “parenteral administrat”ion as used herein means modes of administration othe thanr enteral and topical administration, usually by injection, and includes wit, hout limitation, intravenous, intramuscular int, raarterial intr, athecal intr, acapsular int, raorbit intal,racardi ac,intradermal , intraperitone traal,nstrachea subcutaneous,l, subcuticular, intraarticul subcaar, psula r, subarachnoid, intraspinal, epidura andl intrasternal injection and infusion. In a specific embodiment, a protein, a therapeutic agent or a pharmaceutical compositi onaccording to at least some embodiments of the present invention can be administered intraperitoneal or ly intravenous ly.Alternatively a HIDE1, therapeutic agent can be administere viad a non- parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectall sublingualy, lyor topically.

[00421] The active compounds can be prepared with carrier thats will protect the compound against rapid release, such as a controll released forme ulation, including implants, 115WO 2017/009712 PCT/IB2016/001079 transdermal patches, and microencapsulated delivery systems Biodeg. radabl biocome, patibl e polymers can be used, such as ethylene vinyl acetate, polyanhydrid polyglycolices, acid, collagen, poly orthoesters and polyl, acti acid.c Many methods for the preparation of such formulations are patented or generally known to thos skie ll edin the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marce lDekker, Inc., New York, 1978.

[00422] Therapeutic compositions can be administered with medical devices known in the art. For example, in a preferre embodiment,d a therapeutic composition according to at least some embodiments of the present invention can be administere withd a needles hypoderm injic ection device, such as the devices disclosed in U. S. Pat. Nos. 5,399,163; ,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; or 4,596,556. Examples of well - known implants and modules useful in some embodiments of the present inventio ninclude: U. S. Pat. No. 4,487,603, which discloses an implantabl micre o-infusion pump for dispensing medication at a controll rateed U.; S. Pat. No. 4,486,194, which discloses a therapeutic device for administeri ngmedicaments through the skin; U. S. Pat. No. 4,447,233, whic hdiscloses a medication infusion pump for delivering medication at a precise infusion rate U.; S. Pat. No. 4,447,224, which discloses a variabl eflow implantable infusion apparatus for continuous drug delivery; U. S. Pat. No. 4,439,196, whic hdiscloses an osmot icdrug delivery system having multi-chamber compartments; and U. S. Pat. No. 4,475,196, which disclose ans osmot icdrug delivery system These. patent sare incorporat hereied nby reference. Many othe suchr implants, delivery system s,and modules are known to those skill edin the art.

[00423] In certain embodiments, HIDE1 soluble proteins, ectodomains and/or, fusion proteins can, be formulat toed ensure proper distributi inon vivo. For example, the blood- brain barrier (BBB) excludes many highly hydrophilic compounds To. ensure that the therapeutic compounds according to at leas tsome embodiments of the present invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U. S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. The liposomes may optionall compriy se one or more moieties which are selectively transported into specific cell sor organs, thus enhance targeted drug delivery (see, e.g, V. V. Ranade J.

Clin. Pharmacol. 29:685 (1989)). Exemplary targeting moieties include folat ore biotin (see, 116WO 2017/009712 PCT/IB2016/001079 e.g, U. S. Pat. No. 5,416,016 to Low et al.); mannosides (Umezawa et al, Biochem. Biophys.

Res. Commun. 153:1038 (1988)); antibodie (P.s G. Bloeman et al. FEBS Lett. 357:140 (1995); M. Owais et al. Antimicrob. Agents Chemother. 39:180 (1995)); surfactant prote inA receptor (Briscoe et al. ץ Am. J Physiol. 1233:134 (1995)); pl20 (Schreier et al. J. Biol. Chern. 269:9090) (1994); see also K. Keinanen; M. L. Laukkanen FEBS Lett 346:123. (1994); and Killion and Fidler Immunomethods 4:273 (1994).

A. Formulations for parental administration

[00424] In a furth erembodiment, compositions disclosed herein, including those containing peptides and polypeptides, are administered in an aqueous solution, by parenteral injection. The formulation may optional alsoly be in the form of a suspension or emulsion. In general, pharmaceutic alcompositions are provided includin geffective amounts of a peptide or polypeptide, and optionall includey pharmaceutical acclyeptable diluents, preservatives, solubilizer emuls, sifiers, adjuvant sand/or carriers. Such compositions optional incluly de one or more for the following: diluents steri, lewater, buffered saline of various buffer content (e.g., Tris-HCl aceta, te, phosphate), pH and ionic strengt andh; additives such as detergent s and solubilizing agents (e.g, TWEEN 20® (polysorbate-20), TWEEN 80® (polysorbat e- 80)), anti-oxidants (e.g, water soluble antioxidant suchs as ascorbi acid,c sodium metabisulfite cystei, ne hydrochloride, sodium bisulfate, sodium metabisulfite sodium, sulfite; oil-solub antioxidantle suchs, as ascorbyl palmitate, butylate hydroxyanisoled (BHA), butylated hydroxy toluene (BHT), lecithin, propyl gallate, a-tocopherol and; metal chelating agents ,such as citric acid, ethylenediamine tetraaceti acidc (EDTA), sorbitol tart, aric acid, phosphoric acid), and preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactos mannie, tol ).Examples of non-aqueous solven tsor vehicles are ethanol, propylene glycol, polyethyle glycolne veget, able oils, such as olive oil and com oil, gelatin, and injectable organic esters such as ethyl oleate The. formulations may optionall bey freeze dried (lyophilize ord) vacuum dried and redissolved/resuspende immedid atel beforey use.

The formulation may optionall bey sterilized by, for example, filtrat ionthrough a bacteria retaining filter by, incorporat ingsterilizi agentsng into the compositions by, irradiating the compositions or ,by heating the compositions. 117WO 2017/009712 PCT/IB2016/001079 B. Formulations for topical administration

[00425] HIDE1 polypeptides, fragments, fusion polypeptide nucles, ic acids, and vectors disclosed herein can be applied topically. Topical administration does not work well for most peptide formulations, although it can be effective especially if applied to the lungs, nasal ,oral (sublingual, buccal), vaginal, or recta mucosa.l

[00426] Compositio canns be delivered to the lungs while inhaling and traverse acros s the lung epithelial lining to the blood strea mwhen delivered either as an aerosol or spray dried particl eshaving an aerodynami diamc ete rof less than about 5 microns. A wide range of mechanical devices designed for pulmonary delivery of therapeutic products can be used, including but not limite tod nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices are the Ultravent nebulizer (Mallinckrodt Inc., St. Louis, Mo.); the Acom II nebulizer (Marquest Medical Products, Englewood, Colo.); the Ventolin metered dose inhaler (Glaxo Inc., Research Triangl Park,e N. C.); and the Spinhaler powder inhaler (Fisons Corp., Bedford, Mass. ). Nektar Alke, rmes and Mannkind all have inhalable insulin powder preparations approve dor in clinical trials where the technology could be applied to the formulations described herein.

[00427] Formulations for administration to the mucosa will typically be spra ydrie d drug particles, whic hmay optionally be incorporat intoed a tablet, gel ,capsule, suspension or emulsion. Standard pharmaceutic alexcipient sare availabl frome any formulator. Oral formulations may optionally be in the form of chewing gum, gel strips, tablets or lozenges.

[00428] Transdermal formulations may optionall alsoy be prepared. These will typicall bey ointments, lotions, sprays, or patches, all of which can be prepared using standard technology. Transdermal formulations will require the inclusion of penetration enhancers.

C. Controlled delivery polymeric matrices

[00429] HIDE1 polypeptides, fragments, fusion polypeptide nucles, ic acids, and vectors disclosed herein may optionall alsoy be administered in control ledrelease formulations. Controll released polymerie devicec s can be made for long term release 118WO 2017/009712 PCT/IB2016/001079 systemical lyfollow ingimplantation of a polymeri devicec (rod, cylinder, film ,disk) or injection (microparticles) The. matrix can be in the form of microparticles such as microspheres where, peptides are dispers edwithin a soli dpolymeri matric xor microcapsul es, where the core is of a different materia thanl the polymeri shellc and, the peptide is disperse d or suspended in the core, which may optionally be liquid or soli din nature Unless. specifically defined herein, microparticles mic,rospheres and, microcapsul arees used interchangeably. Alternative thely, polymer may be cast as a thin slab or film ,ranging from nanometers to four centimeters a ,powder produce byd grinding or othe stanr dard techniques , or even a gel such as a hydrogel.

[00430] Either non-biodegradable or biodegradabl mate rice cans be used for delivery of polypeptides or nucleic acids encoding the polypeptides, although biodegradable matrice s are preferred. These may be natural or synthet polymersic although, syntheti polymec rs are preferred due to the better characterizati ofon degradation and release profiles The. polyme r is selected based on the period over whic hreleas ise desired. In some cases linea rrelease may be most useful, although in others a pulse release or “bulk releas”e may provide more effective result Thes. polymer may be in the form of a hydroge (typicall lyin absorbing up to about 90% by weight of water), and can optionally be crosslinke withd multivalent ions or polymers.

[00431] The matrice cans be formed by solvent evaporation, spra ydrying, solvent extraction and other methods known to those skill edin the art. Bioerodibl miecrospher canes be prepared using any of the methods developed for making microspheres for drug delivery, for example, as described by Mathiowi andtz Langer, J. Controlled Release, 5:13-22 (1987); Mathiowit etz, al., Reactive Polymers, 6:275-283 (1987); and Mathiowit etz, al., J. Appl Polymer Set, 35:755-774 (1988).

[00432] The devices can be formulat fored local releas toe trea thet area of implantation or injection - which wil typicl all delivery a dosage that is much less than the dosage for treatment of an entir bodye - or systemi cdelivery. These can be implanted or injected subcutaneousl intoy, the muscle, fat, or swallowed. 119WO 2017/009712 PCT/IB2016/001079 IX. Antibodies

[00433] Accordingly, the invention provides anti-HIDEl antibodies. The antibodie ofs the invention are specific for the HIDE1 extracellul domainar as more fully outlined herein.

[00434] As is discussed below the, term “antibody” is used generally. Antibodi esthat find use in some embodiments of the present inventio ncan take on a number of formats as described herein, includin gtraditional antibodie ass well as antibody derivatives, fragments and mimetics, described below In. general, the term “antibody” includes any polypeptide that includes at least one antigen binding domain, as more fully described below .Antibodies may be polyclonal, monoclonal, xenogeneic, allogeneic syngene, ic, or modified forms thereof, as describe hereid n, wit hmonoclonal antibodie findings particular use in many embodiments. In some embodiment s,antibodie ofs the invention bind specifically or substantial specifily cally to HIDE1 molecules. The term s“monoclonal antibodie” sand “monoclonal antibody composition”, as used herein, refer to a population of antibody molecules that contain only one species of an antigen-binding sit ecapable of immunoreacting with a particular epitope of an antigen ,whereas the term “polyclonal antibodies” and “polyclonal antibody composition” refer to a population of antibody molecules that contain multiple species of antigen-binding site scapable of interacting with a particular antigen. A monoclonal antibody composition typic, all disply ays a singl ebinding affinity for a particular antigen with which it immunoreacts.

[00435] Traditional full length antibody structural units typically comprise a tetramer.

Each tetramer is typically composed of two identical pair sof polypeptide chains, each pair having one “light” (typically having a molecular weight of about 25 kDa) and one “heavy” chain (typical lyhaving a molecular weight of about 50-70 kDa). Human light chains are classifie das kappa and lambda light chains .According to at leas tsome embodiments of the present inventio nis directed to the IgG class, which has several subclasses incl, uding, but not limited to IgGl ,IgG2, IgG3, and IgG4. Thus, “isotype” as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristi ofcs their constant regions. While the exemplary antibodie hereis ndesignated below and in Figure 64 are based on IgG4 and and in Figure 80 and 81 are based on IgGl heavy constant regions, as shown in the figures, the anti-HIDEl antibodies of the invention include thos usinge IgGl, 120WO 2017/009712 PCT/IB2016/001079 IgG2, IgG3 and IgG4 sequences, or combinations thereof. In some embodiments, the anti - HIDE1 antibodies of the inventio ninclude IgG4 sequences. For example, as is known in the art, different IgG isotypes have different effector functions whic hmay or may not be desirable. Accordingly, the anti-HIDEl antibodie ofs the inventio ncan also swap out the IgG4 constant domains for IgGl ,IgG2 or IgG3 constant domains or can swap out the IgGl for IgG2, IgG3, or IgG4, wit hIgG2 and IgG4 finding particular use in a number of situations, for example for ease of manufacture or when reduced effector function is desired, the latt er being desire ind some situations.

Table 1: CPA. 12.001 human IgG4 Domain Sequence SEQ ID NO: Variable OVOLVESGGGLVOPGRSLRLSCAASGFTFSSYGMHWVROAP 87 heavy (vh) GKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLQ domain MNSLRAEDTAVYYCASEGVDFWSGLDYWGOGTLVTVSS vhCDRl GFTFSSYG 88 vhCDR2 ISYDGSNK 89 vhCDR3 ASEGVDFWSGLDY 90 Full length OVOLVESGGGLVOPGRSLRLSCAASGFTFSSYGMHWVROAP 91 HC GI MNSLRAEDTAVYYCASEGVDFWSGLDYWGOGTLVTVSSAST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP SNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSPGK OSALTOPRSASGSAGOSVTISCTGTSSDVGGHNYVSWYOOFP 92 Variable GKAPKLLIYEVSHRPSGVPDRFSGSKSGNTASLTVSGLQAEDE light (vl) domain ADYYCSSYADLNNLMFGGGTKLTVLG vlCDRl SSDVGGHNY 93 121WO 2017/009712 PCT/IB2016/001079 vlCDR2 94 EVS V1CDR3 SSYADLNNLM 95 Full length QSALTQPRSASGSAGQSVTISCTGTSSDVGGHNYVSWYQQFP 96 light chain GKAPKLLIYEVSHRPSGVPDRFSGSKSGNTASLTVSGLQAEDE ADYYCSSYADLNNLMFGGGTKLTVLGOPKAAPSVTLFPPSSE ELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPS KQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVA PTECS Table 2: CPA.12.002 human IgG4 Domain Sequence SEQ ID NO: Variable EVQLVESGGGLVOPGRSLRLSCAASGFTFSSYAMHWVROVPG 97 heavy (vh) KGLEWVAVISYDGSNI domain NSLRAEDTAVYYCAKPMYSSGWYPLGYWGOGTLVTVSS vhCDRl GFTFSSYA 98 vhCDR2 ISYDGSNK 99 vhCDR3 AKPMYSSGWYPLGY 100 Full length EVQLVESGGGLVOPGRSLRLSCAASGFTFSSYAMHWVROVPG 101 HC I NSLRAEDTAVYYCAKPMYSSGWYPLGYWGOGTLVTVSSAST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP SNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSPGK Variable DIVMTOTPLSLPVTPGOPASISCRSSQSPLDTDGNTYLDWYLO 102 light (vl) RPGQSPQLLIYTLSNRASGVPDRFSGSGSGTDFTLKISRVEAED domain VGIYYCMQRIQYPLTFGPGTRLEIKR vlCDRl QSPLDTDGNTY 103 122WO 2017/009712 PCT/IB2016/001079 V1CDR2 104 TLS V1CDR3 MQRIQYPLT 105 Full length DIVMTOTPLSLPVTPGOPASISCRSSQSPLDTDGNTYLDWYLO 106 light chain RPGQSPQLLIYTLSNRASGVPDRFSGSGSGTDFTLKISRVEAED VGIYYCMQRIQYPLTFGPGTRLEIKRTVAAPSVFIFPPSDEQLK SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC Table 3: CPA.12.003 human IgG4 Domain Sequence SEQ ID NO: Variable OVOLOESGPGLVTPSETLSLTCTVSGGSISSVSYYWGWVROPP 107 heavy (vh) GKGLEWIGSIYYSGTTAHNPSLKSRVTMAVDTSKNOFSLSLSS domain VTAADTAVYFCARGWRYYEDYYFDHWGOGTLVTVSS vhCDRl GGSISSVSYY 108 vhCDR2 IYYSGTT 109 vhCDR3 ARGWRYYEDYYFDH 110 Full length OVOLOESGPGLVTPSETLSLTCTVSGGSISSVSYYWGWVROPP 111 HC GKGLEWIGSIYYSGTTAHNPSLKSRVTMAVDTSKNOFSLSLSS VTAADTAVYFCARGWRYYEDYYFDHWGOGTLVTVSSASTK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSPGK Variable DIOMTOSPSTLSASVGDRVTITCRASQSINRWLAWYOOKPGK 112 light (vl) APKILIYTASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYY domain COOYNSYPITFGOGTRLEIKR vlCDRl QSINRW 113 123WO 2017/009712 PCT/IB2016/001079 V1CDR2 114 TAS V1CDR3 QQYNSYPH 115 Full length DIQMTQSPSTLSASVGDRVTITCRASOSINRWLAWYQQKPGK 116 light chain APKILIYTASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYY COOYNSYPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Table 4: CPA.12.004 human IgG4 Domain SEQ ID Sequence NO: Variable EVQLVETGGGLIOPGRSLRLSCVASDFRFSNHAMHWVROAPG 117 heavy (vh) KELEWVAVISSDGSNRQYAYSVKGRFTISRDNSKNTLYLEMNI domain LG P E DTAVYFCVRSHLGPEWYYGMDVWGOGTTVTV S S vhCDRl DFRFSNHA 118 vhCDR2 ISSDGSNR 119 vhCDR3 120 VRSHLGPEWYYGMDV Full length EVOLVETGGGLIOPGRSLRLSCVASDFRFSNHAMHWVROAPG 121 HC KELEWVAVISSDGSNRQYAYSVKGRFTISRDNSKNTLYLEMNI LGP E DTAVYFCVRSHLGPEWYYGMDVWGOGTTVTV S S A STK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSPGK DIQMTQSPSSLSTSVGDSVTITCRSSOSISTFLSWFQHKPGKAP 122 Variable light (vl) NLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC domain QQSDYLPFTFGPGTKV EIK R vlCDRl QSISTF 123 124WO 2017/009712 PCT/IB2016/001079 V1CDR2 124 DAS V1CDR3 QQSDYLPFT 125 Full length DIQMTQSPSSLSTSVGDSVTITCRSSOSISTELSWFQHKPGKAP 126 light chain NLLIYDASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC OOSDYLPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Table 5: CPA.12.005 human IgG4 Domain SEQ ID Sequence NO: Variable OVOLVESGGGVVOPGRSLRVSCAVSGFTFSSYGMHWVROAP 127 heavy (vh) GGGLEWVAVMSYEGSFRHYADSVKGRFTISRDNSKNTLFLO domain MDSLRAEDTAVYYCARDRPAGYTSGWGILDYWGOGTLVTVS S vhCDRl GFTFSSYG 128 vhCDR2 MSYEGSFR 129 vhCDR3 ARDRPAGYTSGWGILDY 130 Full length OVOLVESGGGVVOPGRSLRVSCAVSGFTFSSYGMHWVROAP 131 HC GGGLEWVAVMSYEGSFRHYADSVKGRFTISRDNSKNTLFLO MDSLRAEDTAVYYCARDRPAGYTSGWGILDYWGOGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNV DHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPK DTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEALHNHYTQKSLSLSPGK Variable EIVMTOSPATLSLSPGERATLSCRASOSVSSYLAWYOQKPGOA 132 light (vl) PRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC domain OQRSNWPLTFGGGTKV DIK R vlCDRl QSVSSY 133 125WO 2017/009712 PCT/IB2016/001079 V1CDR2 134 DAS V1CDR3 QQRSNWPLT 135 Full length EIVMTOSPATLSLSPGERATLSCRASQSVSSYLAWYOOKPGOA 136 light chain PRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYC OQRSNWPLTFGGGTKVDIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Table 6: CPA.12.006 human IgG4 Domain SEQ ID Sequence NO: Variable EVQLVESGGGVVOPGRSLRLSCAASGFTFSSFAMHWVROAPG 137 KGPEWLGIILYEGGHVVYADSVRGRLTISRDNSKNTLYLQMD heavy (vh) SLRDEDTAVYYCAKGFYHAFDVWGOGTMVTVSS domain vhCDRl GFTFSSFA 138 vhCDR2 139 ILYEGGHV vhCDR3 140 AKGFYHAFDV Full length EVOLVESGGGVVOPGRSLRLSCAASGFTFSSFAMHWVROAPG 141 HC KGPEWLGIILYEGGHVVYADSVRGRLTISRDNSKNTLYLQMD SLRDEDTAVYYCAKGFYHAFDVWGOGTMVTVSSASTKGPSV FPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKV DKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNST YRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKG QPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSPGK QAVLTQPASLSASPGASASLTCTLRSGINVGTYRIYWYQQKPG 142 Variable light (vl) SPPOYLLRYKSDSDK domain QQGSGVPSRFSGSKDASANAGILLISGLQSEDEADYYCMIWHS SAWVFGGGTOLTVLG vlCDRl SGINVGTYR 143 126WO 2017/009712 PCT/IB2016/001079 V1CDR2 YKSDSDK 144 V1CDR3 MIWHSSAWV 145 Full length QAVLTQPASLSASPGASASLTCTLRSGINVGTYRIYWYQQKPG 146 light chain SPPOYLLRYKSDSDK QQGSGVPSRFSGSKDASANAGILLISGLQSEDEADYYCMIWHS SAWVFGGGTOLTVLGOPKAAPSVTLFPPSSEELOANKATLVC LISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASS YLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Table 7: CPA.12.007 human IgG4 Domain Sequence SEQ ID NO: Variable EVQLVESGGGVVOPGESLRLSCAASGFTFSSYGFHWVROSPG 147 heavy (vh) EGLEWLAVISYDGTSKSYADSVKGRFTVSRDNSKETLYLOLSS domain LRREDTALYYCARDTWGYYYGMDVWGOGTTVTVSS vhCDRl GFTFSSYG 148 vhCDR2 ISYDGTSK 149 vhCDR3 ARDTWGYYYGMDV 150 Full length EVQLVESGGGVVOPGESLRLSCAASGFTFSSYGFHWVROSPG 151 HC EGLEWLAVISYDGTSKSYADSVKGRFTVSRDNSKETLYLOLSS LRREDTALYYCARDTWGYYYGMDVWGOGTTVTVSSASTKG PSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSN TKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQF NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSPGK Variable DIQMTQSPSFLSGSVGDRVSISCRASQDIRSHLAWYQRKPGEA 152 light (vl) PKLLIYTASSLQGGVPLRFSGSGSGTEFTLTISSLQPEDSATYYC domain QHLHLYPLTFGGGTKLEIKR vlCDRl QDIRSH 153 127WO 2017/009712 PCT/IB2016/001079 V1CDR2 154 TAS V1CDR3 QHLHLYPLT 155 Full length DIQMTQSPSFLSGSVGDRVSISCRASODIRSHLAWYQRKPGEA 156 light chain PKLLIYTASSLQGGVPLRFSGSGSGTEFTLTISSLQPEDSATYYC OHLHLYPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Table 8: CPA.12.008 human IgG4 Domain SEQ ID Sequence NO: Variable OVOLOESGPGLVTPSETLSLTCTVSGGSISSVSYYWGWVROPP 157 heavy (vh) GKGLEWIGSIYYSGTTAHNPSLKSRVTMAVDTSKNOFSLSLSS domain VTAADTAVYFCARGWRYYEDYYFDHWGOGTLVTVSS vhCDRl GGSISSVSYY 158 vhCDR2 IYYSGTT 159 vhCDR3 ARGWRYYEDYYFDH 160 Full length OVOLOESGPGLVTPSETLSLTCTVSGGSISSVSYYWGWVROPP 161 HC GKGLEWIGSIYYSGTTAHNPSLKSRVTMAVDTSKNOFSLSLSS VTAADTAVYFCARGWRYYEDYYFDHWGOGTLVTVSSASTK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSPGK AIRMTOSPSSLSASVGDRVTITCOASODISNYLNWYOOKPGKA 162 Variable light (vl) PKSLIYAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYC domain QQYYSYPLTFGGGTKVEIKR vlCDRl QDISNY 163 128WO 2017/009712 PCT/IB2016/001079 V1CDR2 164 AAS V1CDR3 QQYYSYPLT 165 Full length AIRMTQSPSSLSASVGDRVTITCQASODISNYLNWYQQKPGKA 166 light chain PKSLIYAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYC O0YYSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Table 9: CPA.12.009 human IgG4 Domain SEQ ID Sequence NO: Variable OMOLVOSGAEVKKPGASVKVSCKASGYTFTTHAFSWVROAP 167 heavy (vh) GLGLEWMGWISTYNGNTKYEQKFQGRVTLTTDSSTNTAYME domain LRSLRSDDTAVYYCVRDSRAFDVWGOGTMVTVSS vhCDRl GYTFTTHA 168 vhCDR2 ISTYNGNT 169 vhCDR3 170 VRDSRAFDV Full length OMOLVOSGAEVKKPGASVKVSCKASGYTFTTHAFSWVROAP 171 HC GLGLEWMGWISTYNGNTKYEOKFOGRVTLTTDSSTNTAYME LRSLRSDDTAVYYCVRDSRAFDVWGOGTMVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVMHEALHNHYTQKSLSLSPGK AIRMTOSPSSLSASVGDRVTITCOASODISNYLNWYOOKPGKA 172 Variable light (vl) PKSLIYAASSLQSGVPSKFSGSGSGTDFTLTISSLQPEDFATYYC domain QQYYSYPLTFGGGTKVEIKR DIOLTOSPSFLSASVGDRVTITCRASOGINSYLAWYOOKPGAA PKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYC 129WO 2017/009712 PCT/IB2016/001079 QQLNTYPLIFGOGTRLEIKR vlCDRl QG1NSY 173 vlCDR2 AAS 174 V1CDR3 QQLNTYPLI 175 Full length 176 DIOLTOSPSFLSASVGDRVTITCRASOGINSYLAWYOOKPGAA light chain PKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYC QQLNTYPLIFGOGTRLEIKRTVAAPSVFIFPPSDEOLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Table 10: CPA.12.011 human IgG4 Domain Sequence SEQ ID NO: 177 Variable EVOLVESGGGLVQPGGTLRLSCVVSGFTFSSYAMSWVROAPG KGLEWVSGISGSGGSTYISSGRTYRSTYYADSVKGRFTIARDN heavy (vh) domain SENTLFLOLNSLRAEDTAIYYCAKVNSGEYAHTFDYWGOGTL VTVSS vhCDRl GFTFSSYA 178 vhCDR2 ISGSGGSTYISSGRTYRST 179 vhCDR3 AKVNSGEYAHTFDY 180 Full length 181 EVOLVESGGGLVQPGGTLRLSCVVSGFTFSSYAMSWVROAPG KGLEWVSGISGSGGSTYISSGRTYRSTYYADSVKGRFTIARDN HC SENTLFLQLNSLRAEDTAIYYCAKVNSGEYAHTFDYWGOGTL VTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYT CNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKG LPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK Variable DIQMTQSPSSLSASVGDRVTITCRASQGISNYLVWYQQKPGKA 182 light (vl) PKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC domain OQYNYYPITFGOGTRLEIKR 130WO 2017/009712 PCT/IB2016/001079 vlCDRl QGISNY 183 vlCDR2 AAS 184 V1CDR3 QQYNYYPIT 185 Full length DIQMTQSPSSLSASVGDRVTITCRASOGISNYLVWYQQKPGKA 186 light chain PKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQYNYYPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASV VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Table 11: CPA.12.012 human IgG4 Domain Sequence SEQ ID NO: Variable OVOLVESGGGVVOPGRSLRLSCAASGFTFSSYAMHWVROAP 187 GKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLYLO heavy (vh) MNSLRAEDTAVYYCARSSGSSWSNIAYWGOGTLVTVSS domain vhCDRl GFTFSSYA 188 vhCDR2 ISYDGSNK 189 vhCDR3 ARSSGSSWSNIAY 190 Full length OVOLVESGGGVVOPGRSLRLSCAASGFTFSSYAMHWVROAP 191 HC GI MNSLRAEDTAVYYCARSSGSSWSNIAYWGOGTLVTVSSAST KGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP SNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREE QFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGN VFSCSVMHEALHNHYTQKSLSLSPGK Variable OSVLTOPPSVSGAPGORVTISCIGSSSNIGAGYDVHWYROLPG 192 light (vl) TAPKLLIYDNTNRPSGVPDRFSASKSGISASLAITGLQAEDEAD domain YYCOSYDSNLSGVFGGGTOLTV LG 131WO 2017/009712 PCT/IB2016/001079 vlCDRl SSNIGAGYD 193 V1CDR2 DNT 194 V1CDR3 QSYDSNLSGV 195 Full length OSVLTOPPSVSGAPGORVTISCIGSSSNIGAGYDVHWYROLPG 196 light chain TAPKLLIYDNTNRPSGVPDRFSASKSGISASLAITGLQAEDEAD YYCQSYDSNLSGVFGGGTOLTVLGOPKAAPSVTLFPPSSEELQ ANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQS NNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTE CS Table 12: CPA.12.013 human IgG4 Domain Sequence SEQ ID NO: Variable EVQLVETGGGLIQPGRSLRLSCVASDFRFSNHAMHWVRQAPG 197 heavy (vh) 1 domain LGP E DTAVYFCVRSHLGPEWYYGMDVWGOGTTVTV S S vhCDRl DFRFSNHA 198 vhCDR2 ISSDGSNR 199 vhCDR3 VRSHLGPEWYYGMDV 200 Full length EVOLVETGGGLIOPGRSLRLSCVASDFRFSNHAMHWVROAPG 201 HC KELEWVAVISSDGSNRQYAYSVKGRFTISRDNSKNTLYLEMNI LGP E DTAVYFCVRSHLGPEWYYGMDVWGOGTTVTV S S A STK GPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPS NTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMIS RTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQ FNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNV FSCSVMHEALHNHYTQKSLSLSPGK Variable DIOMTOSPSSLSASVGDRVTITCOASHDIRKFLNWYOOHPGKA 202 light (vl) PKLLIYDAAYSEIGVPSRFSGGGSGTDFTFTINNLQPDDFATYY domain CQQYESLPFTFGPGTKV DIK R 132WO 2017/009712 PCT/IB2016/001079 vlCDRl HDIRKF 203 V1CDR2 DAA 204 V1CDR3 QQYESLPFT 205 Full length DIOMTOSPSSLSASVGDRVTITCOASHDIRKFLNWYOQHPGKA 206 light chain PKLLIYDAAYSEIGVPSRFSGGGSGTDFTFTINNLQPDDFATYY COQYESLPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Table 13: CPA.12.014 human IgG4 Domain Sequence SEQ ID NO: Variable OVTLKESGPALVTPTONLTLTCTVSGGSISSSSYYWGWIROPP 207 GI heavy (vh) TAADTAVYYCARRRNSSGWFYFDYWGOGTLVTVSS domain vhCDRl GGSISSSSYY 208 vhCDR2 IYYSGST 209 vhCDR3 ARRRNSSGWFYFDY 210 Full length OVTLKESGPALVTPTONLTLTCTVSGGSISSSSYYWGWIROPP 211 HC G I TAADTAVYYCARRRNSSGWFYFDYWGOGTLVTVSSASTKGP SVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNT KVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRT PEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFN STYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKA KGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF SCSVMHEALHNHYTQKSLSLSPGK Variable OSVLTOPPSVSGAPRORVTISCSGSRSNIGDNAVNWYOHPPGK 212 light (vl) APKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADY domain YCATWDDSLNGHVFGTGTKLTVLG 133WO 2017/009712 PCT/IB2016/001079 vlCDRl RSNIGDNA 213 V1CDR2 YDD 214 V1CDR3 ATWDDSLNGHV 215 Full length QSVLTQPPSVSGAPRQRVTISCSGSRSNIGDNAVNWYQHPPGK 216 light chain APKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADY YCATWDDSLNGHVFGTGTKLTVLGOPKAAPSVTLFPPSSEEL QANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQ SNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPT ECS Table 14: CPA.12.015 human IgG4 Domain Sequence SEQ ID NO: QVOLOESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIROPPGKGLEWIGSIYYS Variable 217 GSTHYNPSLKSRVTISVDTSKNOFSLKLSSVTAADTAVYYCARVYYGSGTGGAFDI heavy (vh) WGOGTMVTVSS domain GGsisissYY vhCDRl 218 IYYSGST vhCDR2 219 ARVYYGSGTGGAFDI vhCDR3 220 Full length 221 GsiOiisiiGiOEsOtsiiQBsitOBsyi^iBBBYORWWsriTGGAFBT HC WGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGA LT SGVHTFPAVLQS SGLYS LSSWTVPS S SLGTKTYTCNVDHKPSNTKVDKRVES K YGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNW YVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE KTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSP GK gsyLTQPPSySiGAPGQRyTTGqTGSiSSgTGA/،yH»YQ@LPGTAPKiiTiYGgTTR Variable 222 PSGBpSRBs light (vl) G domain SSNIGAAYD vlCDRl 223 GDT vICDR2 224 134WO 2017/009712 PCT/IB2016/001079 V1CDR3 225 QSVLTOPPSVSGAPGORVTISCTGSSSNIGAAYDVHWYQOLPGTAPKLLIYGDTIR Full length 226 BG:GWBBRFSiSlSGYSAgEARYGEOEBEABYYGlSYSSSEGG،7FGGGY@E؛I؛WE light chain GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETT TPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS Table 15: 33B4 Antibody Domain Sequence SEQ ID NO: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTY 227 Variable ADDFKGRFAFSLETgASTAYLQINNLKNEDMATYFC VREHFYALDY WGQGTSVTVSS heavy (vh) domain GYTFTNYG vhCDRl 228 INTYTGEP vhCDR2 229 vhCDR3 230 @E@EB@S:GBEEKKgGEYWESGOOE7:iENYG،،OBGKGEf،^ Full length 231 ADDFKGREA HC FSiEiiASiAYrigYiKiKNEb$B7'YFGyREHFYAi0YSrigG$SyTySSAK$Y®®S®YRiA PGSAAQTNS MWEiCiyKGYFlERBBTWiSiSiSriGyHiEROESSBEYYiSrisVlVW CNVAHPASS |FS»FyBS® EyhTi|S|BiEE@FMS^YFRsysEEg^tiH@SriEMGKEiKGRy11S/BFgAgYEKTESETKGR BKAB|$Y$:E ggRiE|MAKBOSEYGMYYBFFgEBYY®EriQWKGggAENYKNY@RYB0,BBSSYFyYSKEN KIVLTOSPASLAVSLRORATISCRASESVDSYGNSFMHWYOOKPGOPPKLLIYRAS Variable 232 NLESGVPARFSGSGSRTDFTLTIDPVEADDAATYYCOOSNEDPRTFGGGTKLEIK light (vl) domain ESVDSYGNSF vlCDRl 233 RAS vICDR2 234 Q9SNEDPRT vlCDR3 235 KIVLTOSPASLAVSLRORATISCRASESVDSYGNSEMHWYQQKPGQPPKLLIYRASNLE Full length 236 iGiglRisiiGiRii،iiigt،0BB،s،iiiO،i،lO، 135WO 2017/009712 PCT/IB2016/001079 VSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERONGVLNSWTDODSKDSTY light chain Table 16: 36C1 Antibody Domain Sequence SEQ ID NO: QIQLVQSGPELKKPGETVKISCKASGYTFTNYVMNWVKQAPGKGLKWMGWINIYTG 237 Variable EPTYADDFKGRFAFSLETSASTSYLQINNLKNEDMATYFC ARWGDGYPWFAY heavy (vh) WGQGTLVTVSA domain GYTFTNYV vhCDRl 238 INIYTGEP vhCDR2 239 ARWGDGYPWFAY vhCDR3 240 QIQLVQSGPELKKPGETVKISCKASGYTFTNYVMNWVKQAPGKGLKWMGWINIYTG Full lengt h 241 EPTYADDFKGRFAFSLETSASTSYLQINNLKNEDMATYFCARWGDGYPWFAYWGQG HC TLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSG VHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCK PCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEV HTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTK GRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQP IMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK NIVMTQSPKSMSMSVGERVTLSCKASENVDTYVSWYQOKPEQSPKLLIYGASNRYT Variable 242 GVPDRFTGSGSATDFTLIISSVQAEDLADYHC GQSYSYPLT FGAGTKLELK light (vl) domain ENVDTY vlCDRl 243 GAS vlCDR2 244 GQSYSYPLT vlCDR3 245 NIVMTQSPKSMSMSVGERVTLSCKASENVDTYVSWYQOKPEQSPKLLIYGASNRYT Full length 246 GVPDRFTGSGSATDFTLIISSVQAEDLADYHCGQSYSYPLTFGAGTKLELKRADAA light chain PTVSIFPPSSEQLTSGGASVVCFLNNFYPRDINVKWKIDGSERQNGVLNSWTDQDS KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Table 17: 39A7Antibody Domain Sequence SEQ ID NO: 136WO 2017/009712 PCT/IB2016/001079 QVQLQQSGAELVRPGASVKLSCKAL GYTFTDYE 247 Variable MHWVKQTPVHGLEWIGD IYPGSGGT heavy (vh) domain AYNQKFKGKATLTADKSSSTAYMELSSLTSEDSAVYYC TRKGRSFAY WGQGTLVTVSA vhCDRl GYTFTDYE 248 vhCDR2 IYPGSGGT 249 vhCDR3 TRKGRSFAY 250 Full length 251 QVQLQQSGAELVRPGASVKLSCKALGYTFTDYEMHWVKQTP VHGLEWIGDIYPGSGGTAYNQKFKGKAT HC LTADKSSSTAYMELSSLTSEDSAVYYCTRKGRSFAYWGQGTL VTVSAAKTTPPSVYPLAPGSAAQTNSM VTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLS SSVTVPSSTWPSETVTCNVAHPASST KVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKV TCVVVDISKDDPEVQFSWFVDDVE VHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNS AAFPAPIEKTISKTKGRPKAPQVYTIP PPKEQMAKDKVSLTCMITDFFPEDITVEWQWNAQPAENYKN TQPIMDTDGSYFVYSKLNVQKSNWEAGN TFTCSVLHEGLHNHHTEKSLSHSPGK NIVMTQSPKSMSMSVGERVTLSCKAS ENVDTY 252 Variable VSWYQQKPEQSPKLLIY GAS light (vl) NRYTGVPDRFTGSGSATDFTLIISSVQAEDLADYHC domain GQSYRYPLT FGAGTKLELK vlCDRl ENVDTY 253 vlCDR2 GAS 254 vlCDR3 GQSYRYPLT 255 Full length NIVMTQSPKSMSMSVGERVTLSCKASENVDTYVSWYQQKPE 256 QSPKLLIYGASNRYTGVP light chain DRFTGSGSATDFTLIISSVQAEDLADYHCGQSYRYPLTFGAGT KLELKRADAAPTVSIF PPSSEQLTSGGASVVCFLNNFYPRDINVKWKIDGSERQNGVLN SWTDQDSKDSTYSMSS TLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

[00436] The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition, generally 137WO 2017/009712 PCT/IB2016/001079 referre tod in the art and herein as the “Fv domain” or “Fv region”. In the variabl region,e three loops are gathered for each of the V domains of the heavy chain and light chain to form an antigen-binding site. Each of the loops is referred to as a complementarity-determi ning region (hereinafter referred to as a "CDR"), in which the variation in the amino acid sequence is most significant “.Variable” refers to the fact that certai nsegments of the variable region differ extensively in sequence among antibodies Vari. abilit withiny the variabl eregion is not evenly distributed. Instead, the V regions consis oft relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extrem e variabilit caly led “hypervariable regions”.

[00437] Each Vh and Vl is composed of three hypervariable regions (“complementa ry determining regions,” “CDRs”) and four FRs, arranged from amino-termin usto carboxy- terminus in the follow ingorder: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[00438] The hypervariable region generall encompy asses amino acid residues from about amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56 (LCDR2) and 89- 97 (LCDR3) in the light chain variabl eregion and around about 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain variabl e region, although sometimes the numbering is shifted slightly as wil bel appreciate byd those in the art; Rabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, th Ed. Public Health Service, National Institutes of Health, Bethesda Md., (1991) and/or those residues forming a hypervariable loop (e.g. residues 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variabl eregion and 26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain variable region; Chothia and Lesk (1987) J. Mol.

Biol. 196:901-917. Specific CDRs of the inventio nand/or for use in the methods of the invention are describe dabove and shown in Figures 64, and 80-81. In some emboidments, specific CDRs of the invention include those in Figure 64, as well as Tables 1-14. In some embodiment s,specific CDRs of the invention include thos ine Figure 81, as wel las Tables -17.

[00439] The carboxy-termin portal ion of each chain defines a constant regio nprimarily responsibl fore effector function. Rabat et al. collect numeroused primary sequences of the variabl eregions of heavy chains and light chains .Based on the degree of conservation of the 138WO 2017/009712 PCT/IB2016/001079 sequences, they classified individual primary sequences into the CDR and the framework and made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5 th edition, NIH publication, No. 91-3242, E. A. Rabat et al., entirely incorporated by reference).

[00440] In the IgG subclas sof immunoglobulins there, are several immunoglobulin domains in the heavy chain. By “immunoglobulin (Ig) domain” herei nis meant a region of an immunoglobul havingin a distinct tertiary structure. Of interest in some embodiments of the present invention are the heavy chain domains includi, ng, the constant heavy (Ch) domains and the hinge domains. In the context of IgG antibodies the, IgG isotypes each have three CH regions. Accordingl “y,Ch” domains in the context of IgG are as follows “C:hi” refers to positions 118-220 according to the EU index as in Rabat. “Ch2” refers to positions 237-340 according to the EU index as in Rabat, and “Ch3” refer sto positions 341-447 according to the EU index as in Rabat.

[00441] Accordingly, the invention provides variabl heavye domains, variable light domains heavy, constant domains, light constant domains and Fc domains to be used as outlined herein. By “variabl regione ” as used herei nis meant the region of an immunoglobul thatin comprises one or more Ig domains substantial encodedly by any of the Vk or VX, and/or Vh genes that make up the kappa ,lambda ,and heavy chain immunoglobulin genetic loci respectively. Accordingly, the variabl heavye domain comprises vhFRl-vhCDRl-vhFR2-vhCDR2-vhFR3-vhCDR3-vhFR4 and the, variabl elight domain comprises vlFRl-vlCDRl-vlFR2-vlCDR2-vlFR3-vlCDR3-vlFR By “4.heavy constant region” herei nis meant the Cm-hinge-Cm-Cm portion of an antibody. By “Fc” or “Fc region” or “Fc domain” as used herei nis meant the polypeptide comprising the constant region of an antibody excluding the firs constt ant region immunoglobul domainin and in some cases, part of the hinge. Thus Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobul domaiin ns of IgE and IgM, and the flexible hinge N-terminal to thes edomains. For IgA and IgM, Fc may include the J chain. For IgG, the Fc domain comprises immunoglobulin domains Cy2 and Cy3 (Cy2 and Cy3) and the lower hinge region between Cyl (Cyl) and Cy2 (Cy2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usuall y defined to include residues C226 or P230 to its carboxyl-terminus, wherei then numbering is 139WO 2017/009712 PCT/IB2016/001079 according to the EU index as in Kabat. In some embodiment s,as is more fully described below, amino acid modifications are made to the Fc region, for example to alter binding to one or more FcyR receptor ors to the FcRn receptor.

[00442] Thus, “Fc variant” or “variant Fc” as used herei nis meant a prote in comprising an amino acid modification in an Fc domain. In some embodiments, the Fc variants of the present inventio nare defined according to the amino acid modifications that compose them. Thus, for example, N434S or 434S is an Fc variant with the substituti on serine at position 434 relati veto the parent Fc polypeptide, wherei then numbering is according to the EU index. Likewise M428L/N43, 4S defines an Fc variant with the substitutions M428L and N434S relati veto the parent Fc polypeptide The. identity of the WT amino acid may be unspecified ,in whic hcase the aforementioned variant is referred to as 428L/434S. It is noted that the order in which substitutions are provided is arbitrary, that is to say that, for example, 428L/434S is the same Fc variant as M428L/N434S, and so on. For all positions discussed in the present inventio nthat relat toe antibodies, unless otherwise noted, amino acid position numbering is according to the EU index.

[00443] By “Fab” or “Fab region” as used herein is meant the polypepti dethat comprises the Vh, Chi, Vl, and Cl immunoglobul domain ins. Fab may refer to thi sregion in isolation, or thi sregion in the context of a full length antibody, antibody fragment or Fab fusion protein. By “Fv” or “Fv fragment” or “Fv region” as used herei nis meant a polypeptide that comprises the Vl and Vh domains of a singl eantibody. As will be appreciate byd those in the art, thes generae lly are made up of two chains.

[00444] Throughout the present specification, either the IMTG numbering system or the Kabat numbering system is generally used when referri ngto a residue in the variabl e domain (approximatel residuesy, 1-107 of the light chain variable region and residues 1-113 of the heavy chain variabl eregion) (e.g, Kabat et al., supra (1991)). EU numbering as in Kabat is generally used for constant domains and/or the Fc domains.

[00445] The CDRs contribut toe the formation of the antigen-binding, or more specificall y,epitope binding sit eof antibodies “.Epitope” refer sto a determinant that interact s with a specific antigen binding sit ein the variabl regie on of an antibody molecul knowne as a 140WO 2017/009712 PCT/IB2016/001079 paratope. Epitopes are groupings of molecules such as amino acids or sugar side chains and usually have specific structural characterist asics, well as specific charge characteristi A cs. singl eantigen may have more than one epitope.

[00446] The epitope may comprise amino acid residues direct lyinvolved in the binding (also called immunodominant component of the epitope) and other amino acid residues whic, h are not direct lyinvolved in the binding, such as amino acid residues which are effectively blocked by the specificall antiy gen binding peptide; in other words, the amino acid residue is within the footpri ofnt the specifically antigen binding peptide.

[00447] Epitopes may be either conformational or linear. A conformational epitope is produce byd spatiall juxtay posed amino acids from different segments of the linear polypeptide chain. A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain. Conformational and nonconformational epitopes may be distinguished in that the binding to the former but not the latte isr lost in the presence of denaturing solvents.

[00448] An epitope typicall incly udes at least 3, and more usually, at leas t5 or 8-10 amino acids in a unique spatial conformation. Antibodies that recognize the same epitope can be verified in a simple immunoassa yshowing the ability of one antibody to block the binding of another antibody to a target antigen, for example “binning”. Specific bins are described below.

[00449] Included within the definition of “antibody” is an “antigen-binding portion” of an antibody (also used interchangeably wit h“antigen-binding fragment”, “antibody fragment” and “antibody derivative”). That is, for the purposes of the invention, an antibody of the invention has a minimum functional requirement that it bind to a HIDE1 antigen. As will be appreciate byd those in the art, there are a large number of antigen fragments and derivatives that retain the abilit yto bind an antigen and yet have alternat ivestructures, including, but not limite to,d (i) the Fab fragment consisting of Vl, Vh, Cl and CHI domains, (ii) the Fd fragment consisting of the Vh and Chi domains (ii, i) F(ab')2 fragments a, bivalent fragment comprising two linked Fab fragments (vii) singl echain Fv molecules (scFv), wherei an Vh domain and a Vl domain are linked by a peptide linke whicr h allows the two domains to associate to form an antigen binding sit e(Bird et al., 1988, Science 242:423-426, 141WO 2017/009712 PCT/IB2016/001079 Huston et al., 1988, Proc. Natl. Acad. Set. USA 85:5879-5883, entirely incorporat byed reference), (iv) "diabodies" or "triabodies" multi, valent or multispecif fragmentsic construct byed gene fusion (Tomlinson et. al., 2000, Methods Enzymol. 326:461-479; WO94/13804; Holliger et al., 1993, Proc. Natl. Acad. Set. USA 90:6444-6448, all entirely incorporated by reference), (v) “domain antibodies” or "dAb" (sometimes referred to as an “immunoglobul singlin evariabl edomain”, including singl eantibody variable domains from othe specir es such as rodent (for example, as disclosed in WO 00/29004), nurse shark and Camelid V-HH dAbs, (vi) SMIPs (small molecul imme unopharmaceuticals cam), elbodies, nanobodies and IgNAR.

[00450] Stil further,l an antibody or antigen-binding portion there of(antigen-bindin g fragment anti, body fragment anti, body portion) may be part of a large immunoadhesr ion molecules (sometimes also referred to as “fusion proteins”), formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptide s.Examples of immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecul ande use of a cysteine residue, a marker peptide and a C-terminal polyhistidi tagne to make bivalent and biotinylat scFved molecules.

Antibody portions, such as Fab and F(ab')2 fragment s,can be prepared from whol antibodiese using conventional techniques, such as papain or pepsin digestion, respectivel ofy, whole antibodies Moreov. er, antibodies anti, body portions and immunoadhesion molecul escan be obtained using standard recombinant DNA techniques, as described herein.

[00451] In general, the anti-HIDE 1 antibodie ofs the invention are recombinant .

“Recombinant” as used herein, refers broadly with reference to a product, e.g, to a cell or, nucleic acid, protein, or vector, indicate sthat the cell, nucleic acid, protei orn vector, has been modified by the introduct ionof a heterologous nucleic acid or protei orn the alterat ion of a native nucleic acid or protein, or that the cel lis derived from a cel lso modified. Thus, for example, recombinan cellst express genes that are not found within the native (non- recombinant form) of the cell or express native genes that are otherw iseabnormally expressed, under expressed or not expressed at all.

[00452] The term “recombinan antibodyt ”, as used herein, includes all antibodies that are prepared, expressed creat, ed or isolated by recombinant means, such as (a) antibodies 142WO 2017/009712 PCT/IB2016/001079 isolated from an animal (e.g, a mouse )that is transgeni orc transchromosom foral human immunoglobul genesin or a hybridoma prepare therd efrom (described further below), (b) antibodie isolas ted from a host cel ltransform toed express the human antibody, e.g, from a transfectom (c)a, antibodie isolas ted from a recombinant combi, nator ialhuman antibody librar y,and (d) antibodie prepared,s expressed, created or isolated by any other means that involve splicing of human immunoglobul genein sequences to other DNA sequences. Such recombinan humant antibodie haves variabl eregions in which the framework and CDR regions are derive dfrom human germline immunoglobul sequein nces . In certain embodiment s,however, such recombinan humant antibodie cans be subjected to in vitro mutagenesi s(or, when an animal transgenic for human Ig sequences is used, in vivo somati c mutagenesis) and thus the amino acid sequences of the Vh and Vl regions of the recombinan t antibodie ares sequences that, while derived from and related to human germline Vh and Vl sequences ,may not natural lyexist within the human antibody germline repertoire in vivo.

A. Optional Antibody Engineering

[00453] The antibodie ofs the inventio ncan be modified, or engineered, to alter the amino acid sequences by amino acid substitutions.

[00454] By “amino acid substituti” onor “substituti” onherei nis meant the replacement of an amino acid at a particular position in a parent polypeptide sequence with a different amino acid. In particular, in some embodiments, the substitution is to an amino acid that is not naturall occurringy at the particular position, either not naturall occuy rring within the organism or in any organism .For example, the substitution E272Y refer sto a variant polypeptide, in thi scase an Fc variant, in which the glutamic acid at position 272 is replaced with tyrosine For. clarit y,a protei whicn h has been engineered to change the nucleic acid coding sequence but not change the starting amino acid (for example exchanging CGG (encoding arginine) to CGA (still encoding arginine) to increase host organism expression levels is) not an “amino acid substituti”on; that is, despit thee creation of a new gene encoding the same protein, if the prote inhas the same amino acid at the particular position that it started with, it is not an amino acid substitution.

[00455] As discussed herein, amino acid substituti onscan be made to alter the affinity of the CDRs for the HIDE1 protei (incln uding both increasing and decreasing binding, as is 143WO 2017/009712 PCT/IB2016/001079 more fully outlined below), as well as to alter additional functional propertie ofs the antibodies For. example, the antibodie mays be engineered to include modifications within the Fc region, typicall toy alte oner or more functional properti ofes the antibody, such as serum half-lif e,complement fixation, Fc receptor binding, and/or antigen-dependen cellt ular cytotoxici ty.Furthermor ane, antibody according to at least some embodiments of the invention may be chemically modified (e.g., one or more chemical moieties can be attache d to the antibody) or be modified to alte itsr glycosylation, again to alter one or more functional propertie ofs the antibody. Such embodiments are describe furtherd below. The numbering of residues in the Fc region is that of the EU index of Kabat.

[00456] In one embodiment, the hinge region of Chi is modifie dsuch that the number of cysteine residues in the hinge region is altered, e.g, increased or decreased. This approach is described further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of CHI is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[00457] In anoth erembodiment, the Fc hinge region of an antibody is mutated to decrease the biologic alhalf-lif ofe the antibody. More specificall y,one or more amino acid mutations are introduced into the CH2-CH3 domain interfac eregion of the Fc-hinge fragment such that the antibody has impaired Staphylococcyl protei An (SpA) binding relati veto native Fc-hinge domain SpA binding. This approac his described in further deta ilin U.S. Pat. No. 6,165,745 by Ward et al.

[00458] In some embodiments, amino acid substituti onscan be made in the Fc region, in genera lfor altering binding to FcyR receptors. By “Fc gamma receptor”, "FcyR" or "FcgammaR" as used herein is meant any member of the family of protei nsthat bind the IgG antibody Fc region and is encoded by an FcyR gene. In humans thi sfamily includes but is not limite tod FcyRI (CD64), includin gisoform FcyRIas , FcyRIb, and FcyRIc; FcyRII (CD32), including isoform FcyRIIas (including allotypes H131 and R131), FcyRIIb (including FcyRIIb-1 and FcyRIIb-2), and FcyRIIc; and FcyRIII (CD16), includin gisoforms FcyRIIIa (including allotypes V158 and F158) and FcyRIIIb (including allotypes FcyRIIIb-NAl and FcyRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirely incorporat byed reference), as well as any undiscovered human FcyRs or FcyR isoform ors allotypes An. 144WO 2017/009712 PCT/IB2016/001079 FcyR may be from any organism, includin gbut not limited to humans, mice, rats rabbit, s,and monkeys. Mouse FcyRs include but are not limite tod FcyRI (CD64), FcyRII (CD32), FcyRIII-1 (CD16), and FcyRIII-2 (CD16-2), as well as any undiscovered mouse FcyRs or FcyR isoform ors allotypes.

[00459] There are a number of useful Fc substitutions that can be made to alter binding to one or more of the FcyR receptors. Substitutions that resul int increased binding as well as decrease dbinding can be useful. For example, it is known that increase dbinding to FcyRIIIa generally result ins increased ADCC (antibody dependent cell-mediated cytotoxici ty;the cell - mediated reaction wherei nonspecifin ccytotoxic cell sthat express FcyRs recognize bound antibody on a targe celt land subsequentl causey lysi sof the targe celt l. Similarl decreasedy, binding to FcyRIIb (an inhibitory receptor) can be beneficial as wel inl some circumstances.

Amino acid substituti onsthat find use in some embodiments of the present inventio ninclude those liste ind U.S. Ser. Nos. 11/124,620 (particularly FIG. 41) and U.S. Patent No. 6,737,056, both of which are expressl incorporaty hereined by reference in their entirety and specifically for the variants disclosed therei n.Particula variar nts that find use include, but are not limited to, 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y, 239D, 332E/330L, 299T and 297N.

[00460] In addition, the antibodie ofs the invention are modified to increase its biologic alhalf-life. Various approaches are possible. For example, one or more of the followi mutang tions can be introduced: T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternative toly, increase the biologic alhalf-lif e,the antibody can be alter edwithin the Chi or Cl region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. ,869,046 and 6,121,022 by Prest eta al. Additional mutations to increase serum half life are disclosed in U.S. Patent Nos. 8,883,973, 6,737,056 and 7,371,826, and include 428L, 434A, 434S, and 428L/434S.

[00461] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue wit ha different amino acid residue to alter the effector functions of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that 145WO 2017/009712 PCT/IB2016/001079 the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approac his described in furth erdetai inl U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

[00462] In anoth erexample, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished complement dependent cytotoxicit (CDCy ). This approac his described in furth erdeta ilin U.S. Pat. Nos. 6,194,551 by Idusogie et al.

[00463] In anoth erexample, one or more amino acid residues within amino acid positions 231 and 239 are alter edto thereby alter the ability of the antibody to fix complement. This approac his described furth erin PCT Publication WO 94/29351 by Bodmer et al.

[00464] In yet another example, the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellula cytotr oxici (ADCCty ) and/or to increase the affinity of the antibody for an Fey receptor by modifying one or more amino acids at the followi positing ons: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This approac his described furth erin PCT Publication WO 00/42072 by Presta. Moreover, the binding site son human IgGl for FcyRI, FcyRII, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al. (2001) J. Biol.

Chern. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 are shown to improve binding to FcyRIII. Additional thely, follow ingcombination mutants are shown to improve FcyRIII binding :T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A. Furthermore, mutations such as M252Y/S254T/T256E or M428L/N434S improve binding to FcRn and increase antibody circulati onhalf-life (see Chan CA and Carte PJr (2010) Nature Rev Immunol 10:301-316). 146WO 2017/009712 PCT/IB2016/001079

[00465] In still another embodiment, the antibody can be modified to abrogat ine vivo Fab arm exchange. Specifically, thi sprocess involves the exchange of IgG4 half-molecules (one heavy chain plus one light chain) between other IgG4 antibodi esthat effectively result s in bispecific antibodie whics h are functionall monovalenty Mut. ations to the hinge regio n and constant domains of the heavy chain can abrogat thie sexchange (see Aalberse, RC, Schuurma nJ., 2002, Immunology 105:9-19).

[00466] In still another embodiment, the glycosylati ofon an antibody is modified. For example, an aglycosylate antid body can be made (i.e., the antibody lacks glycosylation).

Glycosylati canon be altered to, for example, increase the affinity of the antibody for antigen or reduce effector function such as ADCC. Such carbohydr atemodifications can be accomplished by, for example, altering one or more site sof glycosylati withinon the antibody sequence, for example N297. For example, one or more amino acid substitutions can be made that resul int elimination of one or more variabl eregion framework glycosylati siteon sto thereby eliminate glycosylati aton that site.

[00467] Additional orly alternatively, an antibody can be made that has an altered type of glycosylati on,such as a hypofucosylat antibodyed having reduced amount sof fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altere d glycosylati patteon rns have been demonstrated to increase the ADCC abilit yof antibodies.

Such carbohydrat modie fications can be accomplished by, for example, expressing the antibody in a host cell wit haltered glycosylat ionmachinery. Cell swit haltered glycosylati on machinery have been described in the art and can be used as host cells in whic hto expres s recombinan antibodiet accords ing to at least some embodiments of the inventio nto thereby produce an antibody with altered glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransfera gene,se FUT8 (a (1,6) fucosyltransferase such), that antibodie express sed in the Ms704, Ms705, and Ms709 cel llines lack fucose on their carbohydrates The. Ms704, Ms705, and Ms709 FUT8 cell line sare created by the target ed disrupti onof the FUT8 gene in CHO/DG44 cells using two replacement vectors (see U.S.

Patent Publication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 by Hanai et al. describe as cel lline wit ha functionally disrupt edFUT8 gene, which encodes a fucosyl transferase, such 147WO 2017/009712 PCT/IB2016/001079 that antibodie express sed in such a cell line exhibit hypofucosylation by reducing or eliminating the a 1,6 bond-relat enzymeed . Hanai et al. also describe cel llines which have a low enzyme activit yfor adding fucose to the N-acetylglucosamine that binds to the Fc region of the antibody or does not have the enzyme activit y,for example the rat myeloma cel lline YB2/0 (ATCC CRL 1662). PCT Publication WO 03/035835 by Prest desca ribe as variant CHO cel lline, Led 3 cells, with reduce dability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylat ofion antibodie express sed in that host cell (see also Shields R., L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99/54342 by Umana et al. describe cels lline sengineered to express glycoprotein-modifying glycosyl transferases (e.g, P(l,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that antibodie express sed in the engineered cell lines exhibit increased bisecting GlcNac structures whic hresul tsin increased ADCC activity of the antibodie (sees also Umana et al. (1999) Ato. Biotech. 17:176-180). Alternative thely, fucose residues of the antibody may be cleaved off using a fucosidase enzyme. For example, the fucosidase a-L-fucosidase removes fucosyl residues from antibodies (Tarentino, A. L. et al. (1975) Biochem. 14:5516-23).

[00468] Another modification of the antibodie hereis nthat is contemplat byed the invention is pegylation or the addition of othe watr er solubl moie eties, typicall polymersy , e.g, in order to enhance half-life. An antibody can be pegylate dto, for example, increase the biologic al(e.g, serum) half-life of the antibody. To pegylat ean antibody, the antibody, or fragment thereof, typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivati veof PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylati reacton ion wit ha reactive PEG molecul (ore an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompas sany of the forms of PEG that have been used to derivati zeother proteins, such as mono (C1-C10) alkoxy- or aryloxy-pol ethyly ene glycol or polyethylene glycol-mal eimide.

In certain embodiments, the antibody to be pegylated is an aglycosylate antibody.d Methods for pegylating proteins are known in the art and can be applied to the antibodie accords ing to at leas tsome embodiments of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikaw eta al. 148WO 2017/009712 PCT/IB2016/001079

[00469] In addition to substituti onsmade to alter binding affinity to FcyRs and/or FcRn and/or increase in vivo serum half life ,additional antibody modifications can be made, as described in further deta ilbelow.

[00470] In some cases, affinity maturatio isn done. Amino acid modifications in the CDRs are sometime referres tod as “affinity maturati”on. An “affinity matured” antibody is one having one or more alteration(s) in one or more CDRs which result ins an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not posses thoses alteration(s). In some cases, although rare, it may be desirable to decrease the affinity of an antibody to its antigen ,but thi sis generall noty preferred.

[00471] In some embodiments, one or more amino acid modifications are made in one or more of the CDRs of the VISG1 antibodie ofs the invention. In general, only 1 or 2 or 3- amino acids are substitut ined any singl eCDR, and generall noy more than from 1, 2, 3. 4, 5, 6, 7, 8 9 or 10 changes are made within a set of CDRs. However, it should be appreciate thatd any combination of no substitutions, 1, 2 or 3 substitutions in any CDR can be independentl y and optionally combined with any othe substir tution.

[00472] Affinity maturati oncan be done to increase the binding affinity of the antibody for the HIDE1 antigen by at least about 10% to 50-100-150% or more, or from 1 to fold as compared to the “parent” antibody. Preferred affinity mature dantibodi eswil havel nanomol aror even picomol araffinities for the HIDE1 antigen. Affinity matured antibodies are produced by known procedures. See, for example, Marks et al., 1992, Biotechnology :779-783 that describes affinity maturatio byn variabl heavye chain (VH) and variable light chain (VL) domain shuffling. Random mutagenesi sof CDR and/or framework residues is described in: Barbas, et al. 1994, Proc. Nat. Acad. Sci, USA 91:3809-3813; Shier et al., 1995, Gene 169:147-155; Yelton et al., 1995, J. Immunol .155:1994-2004; Jackson et al., 1995, J.

Immunol. 154(7):3310-9; and Hawkins et al, 1992, J. Mol. Biol. 226:889-896, for example.

[00473] Alternatively, amino acid modifications can be made in one or more of the CDRs of the antibodie ofs the invention that are “silent”, e.g. that do not significantl altery the affinity of the antibody for the antigen. These can be made for a number of reasons , 149WO 2017/009712 PCT/IB2016/001079 including optimizing expression (as can be done for the nucleic acids encoding the antibodi es of the invention).

[00474] Thus, included within the definition of the CDRs and antibodie ofs the invention are variant CDRs and antibodies that; is, the antibodie ofs the inventio ncan include amino acid modifications in one or more of the CDRs of the enumerate dantibodi esof the invention. In addition, as outlined below, amino acid modifications can also independentl y and optionally be made in any region outside the CDRs, includin gframework and constant regions.

B. HIDE1 Antibodies

[00475] According to at leas tsome embodiments of the present inventio nprovides anti-HIDEl antibodies (For. convenience, "anti-HIDE1 antibodie” sand "HIDEl antibodie” s are used interchangeably). The anti-HIDEl antibodies of the inventio nspecificall bindy to human HIDEl ,and preferably the ECD of human HIDEl ,as depicted in Figure 66.

[00476] Specific binding for HIDEl or a HIDEl epitope can be exhibited, for example, by an antibody having a KD of at least about 104־ M, at least about 105־ M, at least about 106־ M, at leas tabout 107־ M, at leas tabout 108־ M, at least about 109־ M, alternativel at yleast about 1010־ M, at least about 1011־ M, at least about 1012־ M, or greater, where KD refers to a dissociati onrate of a particular antibody-antigen interaction. Typicall y,an antibody that specifically binds an antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-, ,000- or more times greater for a control molecule relati veto the HIDEl antigen or epitope

[00477] However, as shown in the Examples, for optimal binding to HIDEl expressed on the surface of myeloid cell s,the antibodie preferabls havey a KD less 50 nM and most preferably less than 1 nM, with less than 0.1 nM and less than 1 pM and 0.1 pM finding use in the methods of the invention.

[00478] Also, specific binding for a particular antigen or an epitope can be exhibited, for example, by an antibody having a KA or Ka for a HIDEl antigen or epitope of at least -, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater for the epitope relati veto a 150WO 2017/009712 PCT/IB2016/001079 control, wher KAe or Ka refer sto an association rate of a particular antibody-antige n interaction.

[00479] In some embodiments, the anti-HIDEl antibodie ofs the invention bind to human HIDE1 wit ha Kd of 100 nM or less 50, nM or less, 10 nM or less, or 1 nM or les s (tha is,t higher binding affinity), or IpM or less wherei, Kn D is determined by known methods, e.g. surface plasmon resonanc (SPR,e e.g. Biacore assays), ELISA, KinExA, and most typically SPR at 25° or 37° C.

C. Specific anti-HIDEl antibodies

[00480] The inventio nprovide antis gen binding domains, includin gfull length antibodies, which contain a number of specific, enumerate dset sof 6 CDRs, including for use in the methods of the invention. See, Figures 64 and 80-81. In some emboidments, the CDRs are those in Figure 64, as wel las Tables 1-14. In some embodiment s,the CDRs are those in Figure 81, as well as Tables 15-17.

[00481] As above, thes sete sof CDRs may also be amino acid variants as describe d above.

[00482] In addition, the framework regions of the variable heavy and variabl elight chains can be humanized as is known in the art (with occasional variants generated in the CDRs as needed) ,and thus humanized variants of the Vh and Vl chains of Figures 64 and 80-81 can be generated (see, also, Tables 1-17). Furthermore, the humanized variable heavy and light domains can then be fused with human constant regions, such as the constant regions from IgGl ,IgG2, IgG3 and IgG4.

[00483] In particular, as is known in the art, murine Vh and Vl chains can be humanized as is known in the art, for example, using the IgBLAST program of the NCBI website, as outlined in Ye et al. Nucleic Acids Res. 41:W34-W40 (2013), herei nincorporat ed by reference in its entirety for the humanization methods. IgBLAST takes a murine VH and/or VL sequence and compares it to a library of known human germline sequences. As shown herein, for the humanized sequences ,the databases that can be used are IMGT human Vh genes (F+ORF, 273 germline sequences) and IMGT human Vl kappa genes (F+ORF, 74 151WO 2017/009712 PCT/IB2016/001079 germline sequences). CDRs were and will be defined according to the AbM definition (see, the World Wide Web at bioinfo.org.uk/).abs

[00484] Specific humanized antibodie ofs CPA antibodie includes those shown in Figure 64, as well as Tables 1-14 above. As will be appreciated by those in the art, each humanized variable heavy (Humanized Heavy; HH) and variable light (Humanized Light, HL) sequence can be combined with the constant regions of human IgGl ,IgG2, IgG3 and IgG4 in order to generat efirs t,second , etc., humanized sequences.

[00485] In some embodiments, the anti-HIDEl antibodies of the present inventio n include anti-HIDEl antibodies wherei then Vh and Vl sequences of different anti-HIDEl antibodie cans be “mixed and matched” to create other anti-HIDEl antibodies HIDE1. binding of such “mixed and matched” antibodie cans be tested using the binding assays described above, e.g., ELISAs). In some embodiments, when Vh and Vl chains are mixed and matched, a Vh sequence from a particular VH/VL pairing is replaced with a structural ly similar Vh sequence. Likewise in, some embodiments, a Vl sequence from a particular VH/VL pairing is replaced with a structural simlyilar Vl sequence. For example, the Vh and Vl sequences of homologous antibodie ares particularl amyenable for mixing and matching.

[00486] Accordingl they, antibodie ofs the invention comprise CDR amino acid sequences selected from the group consisting of (a) sequences as listed herein; (b) sequences that differ from those CDR amino acid sequences specified in (a) by 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions; (c) amino acid sequences having 90% or greater, 95% or greater, 98% or greater, or 99% or greater sequence identity to the sequences specified in (a) or (b); (d) a polypepti dehaving an amino acid sequence encoded by a polynucleotide having a nucleic acid sequence encoding the amino acids as liste herein.d

[00487] Additional includedly in the definition of HIDE 1 antibodie ares antibodie thats share identit toy the HIDE1 antibodie enumes rate dherein. That is, in certain embodiment s, an anti-HIDEl antibody according to the invention comprises heavy and light chain variabl e regions comprising amino acid sequences that are homologous to isolated anti-HIDEl amino acid sequences of preferred anti-HIDEl immune molecules, respectively, wherei then antibodie rets ain the desired functional properti ofes the parent anti-HIDEl antibodies. The 152WO 2017/009712 PCT/IB2016/001079 percent identity between the tw osequences is a function of the number of identical positions share dby the sequences (z.e., % homology=# of identical positions/total # of positions X 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematica algoril thm, as described in the non-limiti ngexamples below.

[00488] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporat intoed the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalt yof 12 and a gap penalty of 4. In addition, the percen t identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available commerciall y),using either a Blossum 62 matri xor a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[00489] Additiona llyor alternatively, in some embodiments the protei sequencesn of the present inventio ncan further be used as a “query sequence” to perfor ma search against public database to,s for example, identify relat edsequences .Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10.

BLAST protei searchn es can be performe witd hthe XBLAST program score=50,, wordlength=3 to obtai namino acid sequences homologous to the antibody molecule s according to at leas tsome embodiments of the invention. To obtai ngapped alignment fors comparison purposes Gappe, d BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST program thes, default parameters of the respective programs (e.g, XBLAST and NBLAST) can be used.

[00490] In general, the percentage identity for comparison between HIDE1 antibodies is at least 75%, at least 80%, at least 90%, wit hat least about 95, 96, 97, 98 or 99% percen t identity being preferred. The percentage identity may be along the whol aminoe acid sequence, for example the entir heavye or light chain or along a portion of the chains . For 153WO 2017/009712 PCT/IB2016/001079 example, included within the definition of the anti-HIDE 1 antibodie ofs the invention are those that share identity along the entir variabe le region (for example, where the identity is 95 or 98% identical along the variable regions), or along the entir econstant region, or along just the Fc domain.

[00491] In addition, also included are sequences that may have the identical CDRs but changes in the variabl domaine (or entire heavy or light chain). For example, HIDE1 antibodie ofs the invention and/or for use in the invention, include those wit hCDRs identical to thos showne in Figure 64 and 80-81, as well as Tables 1-17, but whose identity along the variabl eregion can be lower, for example 95 or 98% percent identical.

[00492] According to at least some embodiments of the present invention provides not only the enumerate dantibodie buts additional antibodies that compete with the enumerated antibodie (thes Figure 64 and 80-81 numbers enumerate dherei nthat specificall bindy to HIDE1) to specificall bindy to the HIDE1 molecul e.As is shown above and in Figures 64 and 80-81, as well as Tables 1-17. 1. Generation of Additional Antibodies

[00493] Additional antibodie tos human HIDE1 can be done as is well known in the art, using wel knownl methods such as those outlined in the examples .Thus, additional anti - HIDE1 antibodie cans be generated by traditional methods such as immunizing mice (sometime usings DNA immunization, for example, such as is used by Aldevron), follow ed by screening against human HIDE1 prote inand hybridoma generation, wit hantibody purification and recovery.

D. Nucleic Acid Compositions

[00494] Nucleic acid compositions encoding the anti-HIDEl antibodi esof the invention are also provided, as wel las expression vectors containing the nucleic acids and host cells transforme witd hthe nucleic acid and/or expression vector composition Ass. will be appreciate byd those in the art, the protei sequencesn depicte dherein can be encoded by any number of possibl nuclee ic acid sequences ,due to the degenerac yof the genetic code.

[00495] The nucleic acid compositions that encode the HIDE1 antibodie wills depend on the format of the antibody. For traditional, tetrameric antibodie contais ning tw oheavy 154WO 2017/009712 PCT/IB2016/001079 chains and two light chains are encoded by two different nucleic acids, one encoding the heavy chain and one encoding the light chain. These can be put into a singl eexpression vector or two expression vectors, as is known in the art, transforme intod host cells, where they are expressed to form the antibodi esof the invention. In some embodiments, for example when scFv constructs are used, a singl enucleic acid encoding the variabl heavye chain-linker-variabl lighte chain is generally used, which can be inserted into an expression vector for transformat intoion host cells. The nucleic acids can be put into expression vectors that contain the appropriate transcriptional and translational control sequences ,including, but not limited to, signal and secretion sequences, regulatory sequences, promoters, origins of replication, selection genes, etc.

[00496] Preferred mammalian host cells for expressing the recombinan antibodiet s according to at least some embodiments of the invention include Chinese Hamster Ovary (CHO cells), PER.C6, HEK293 and others as is known in the art.

[00497] The nucleic acids may be present in whol celle s,in a cel llysate or, in a partially purified or substantial purely form. A nucleic acid is “isolat”ed or “rendered substantial purely ” when purified away from other cellula componentsr or othe r contaminant e.g.,s, other cellula nucleir c acids or proteins by, standard techniques, includin g alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophores is and othe rswell known in the art.

[00498] To create a scFv gene, the Vh- and VL-encoding DNA fragments are operatively linke dto anoth erfragment encoding a flexible linker, e.g, encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO:61), such that the Vh and Vl sequences can be expressed as a contiguou singls e-chai proten in, wit hthe Vl and Vh regions joined by the flexibl linkee (seer e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc.

Natl. Acad. Set. USA 85:5879-5883; McCaffert yet al., (1990) Nature 348:552-554).

E. Formulations of Anti-HIDE1 Antibodies

[00499] The therapeutic compositions used in the practice of the foregoing methods can be formulat intoed pharmaceutical compositions comprising a carrier suitable for the desired delivery method. Suitable carrier includes any materia thatl when combined with the 155WO 2017/009712 PCT/IB2016/001079 therapeutic compositi onretains the anti-tumor function of the therapeutic compositi onand is generall non-ry eactive wit hthe patient' ims mune system Exampl. es include, but are not limite to,d any of a number of standard pharmaceutic alcarrier suchs as steri lephosphat e buffered saline solutions, bacteriostat watic er, and the like (see, generall y,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980). Acceptabl ecarriers, excipients or, stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate aceta, te, and other organic acids ;antioxidants includin g ascorbi acic d and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol resorci; nol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptide proteis; ns, such as serum albumin, gelatin, or immunoglobulins; hydrophi lic polymers such as polyvinylpyrroli done;amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose ,or dextrins chel; ating agents such as EDTA; sugars such as sucrose mannitol, tre, halose or sorbitol sweet; eners and other flavoring agents ;fille rssuch as microcrystal cellline ulose, lactose com, and other starches; binding agents ;additives; coloring agents ;salt-form ingcounter-ion suchs as sodium met; al complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[00500] In some embodiments, the pharmaceutical compositi onthat comprises the antibodie ofs the invention may be in a water-soluble form, such as being present as pharmaceutical acceptly able salts, which is meant to include both acid and base addition salts “.Pharmaceutically acceptable acid addition salt” refers to thos sale ts that retain the biologic aleffectivenes sof the free bases and that are not biologicall ory otherwi se undesirabl formede, wit hinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitri acid,c phosphoric acid and the like, and organic acids such as acetic acid, propioni acid,c glycol icacid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfoni acid,c p-toluenesulfoni acid,c salicyli acic d and the like. 156WO 2017/009712 PCT/IB2016/001079 “Pharmaceutically acceptable base addition salt” sinclude thos derivede from inorganic bases such as sodium, potassium, lithium, ammonium ,calcium, magnesium, iron, zinc, copper, manganese, aluminum salt ands the like. Particularly preferred are the ammonium, potassium, sodium calci, um, and magnesium salt s.Salt sderive dfrom pharmaceutically acceptable organic non-toxi basec s include salt ofs primary, secondary, and tertiary amines, substitut ed amines including naturall occurringy substitut amied nes, cyclic amines and basic ion exchange resins such, as isopropylami trimne, ethylamin diete, hylamine, triethylami ne, tripropylamine, and ethanolamine. The formulations to be used for in vivo administrati areon preferrabl stey ril Thise. is readily accomplished by filtrati throughon steri lefiltrati on membranes or othe metr hods.

[00501] In some embodiments, administration of the pharmaceutic alcomposition comprising antibodie ofs the present invention, preferably in the form of a steri leaqueous solution, may be done in a variety of ways, including, but not limited to subcutaneously and intravenous ly.Subcutaneous administration may be preferabl ine some circumstances because the patient may self-administer the pharmaceutic alcomposition. Many prote in therapeuti arecs not sufficientl potenty to allow for formulation of a therapeuticall effecty ive dose in the maximum acceptable volume for subcutaneous administration. This problem may be addresse ind part by the use of protei formulan tions comprising arginine-HCl hist, idine, and polysorbate (see WO 04091658). In some embodiments, Fc polypeptides of the present invention may be more amenable to subcutaneous administration due to, for example, increased potency, improved serum half-life, or enhanced solubility.

[00502] As is known in the art, protei therapeutin arecs often delivered by IV infusion or bolus. In some embodiments, the antibodie ofs the present inventio nmay also be delivered using such methods. For example, administration may venious be by intravenous infusion with 0.9% sodium chloride as an infusion vehicle.

[00503] In addition, any of a number of delivery systems are known in the art and may be used to administer the Fc variants of the various embodiments of the present invention.

Examples include, but are not limited to, encapsulation in liposomes, microparticle s, microspheres (eg. PLA/PGA microspheres), and the like Alte. rnative anly, implant of a porous, non-porous, or gelatinous material, includin gmembranes or fibers, may be used. 157WO 2017/009712 PCT/IB2016/001079 Sustained releas syste ems may comprise a polymeri matc erial or matri xsuch as polyeste rs, hydrogels poly, (vinylalcohol), polylactides, copolyme rsof L-glutamic acid and ethyl-L- gutamate, ethylene-vinyl acetate, lactic acid-glycolic acid copolymers such as the LUPRON DEPOT.RTM., and poly-D-(-)-3-hydroxyburyric acid. The antibodie discloseds herei nmay also be formulat edas immunoliposome As. liposome is a smal lvesicl ecomprising various types of lipids phospholi, pids and/or surfactant that is useful for delivery of a therapeutic agent to a mammal .Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., 1985, Proc Natl Acad Sci USA, 82:3688; Hwang et al., 1980, Proc Natl Acad Sci USA, 77:4030; U.S. Pat. No. 4,485,045; U.S. Pat. No. 4,544,545; and PCT WO 97/38731. Liposome withs enhanced circulati ontime are disclose d in U.S. Pat. No. 5,013,556. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipi darrangement of biologic almembranes. Particularl y useful liposom escan be generated by the revers phasee evaporation method wit ha lipid compositi oncomprising phosphatidylcholine, cholester andol PEG-derivatized phosphatidylethanolam (PEGine-PE). Liposomes are extruded through filte rsof defined pore size to yield liposomes wit hthe desired diamete r.A chemotherapeutic agent or othe r therapeutically active agent is optional contaly ined within the liposom (Gabie zon et al., 1989, !National Cancer Inst 81:1484).

[00504] The antibodie mays also be entrapped in microcapsul preparedes by methods including but not limite tod coacervation techniques, interfacial polymerizat ion(for example using hydroxymethylcellulose or gelatin-microcapsules or poly-(met, hylmethacyl ate) microcapsules), colloidal drug delivery systems (for example, liposome albums, in microspheres mic, roemulsion nano-particless, and nanocapsules), and macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed., 1980. Sustained-rele asepreparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrice ofs soli hydrophobicd polymer, whic hmatrice s are in the form of shaped articles, e.g. films, or microcapsul es.Examples of sustained-releas e matrice incls ude polyesters, hydrogel (fors example poly(2-hydroxyethyl-methacryla or te), poly(vinylalcohol polylac)), tides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma ethyl-L-glutamat non-de, egradable ethylene-vinyl acetate, degradabl lacte ic acid- 158WO 2017/009712 PCT/IB2016/001079 glycol icacid copolymers such as the LUPRON DEPOT.RTM. (which are injectable microspher compes osed of lactic acid-glycolic acid copolymer and leuproli acetde ate ),poly- D-(-)-3-hydroxy butyric acid, and ProLease RTM.. (commercial lyavailable from Alkermes ), which is a microsphere-based delivery system composed of the desire bioacd tive molecule incorporated into a matri xof poly-DL-lactide-co-glycolide (PEG).

[00505] The dosing amounts and frequencies of administration are, in a preferred embodiment, selected to be therapeutical orly prophylactical effeclytive. As is known in the art, adjustments for protei degradatin on, systemi versuc slocalized delivery, and rate of new protea sesynthesi s,as well as the age, body weight, general healt h,sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable wit hroutine experimentation by thos skile led in the art.

[00506] The concentrati onof the antibody in the formulation may vary from about 0.1 to 100 weight %. In a preferred embodiment the, concentrati onof the Fc variant is in the range of 0.003 to 1.0 molar In. order to trea at patient, in some embodiments a therapeutical ly effective dose of the Fc variant of the present inventio nmay be administered. By “therapeutical effecly tive dose” herei nis meant a dose that produc esthe effects for which it is administered. The exact dose wil dependl on the purpose of the treatment, and wil bel ascertainable by one skilled in the art using known techniques Dosa. ges may range from 0.0001 to 100 mg/kg of body weight or greater, for example 0.1, 1, 10, or 50 mg/kg of body weight with, 1 to 10 mg/kg being preferred.

F. Methods of Using Anti-HIDEl Antibodies Compositions 1. Therapeutic Uses

[00507] A “therapeuticall effecty ive dosage” of HIDE 1 solubl proteie orn HIDE1 ectodomai orn fusion protei contain ning same, according to at least some embodiments of the present invention preferably result ins a decrease in severity of disease symptom s,an increase in frequency and duration of disease symptom-free periods, an increase in lifespan, disease remission, or a prevention or reduction of impairment or disability due to the disease affliction. Alternatively thi, sproperty of a compositi oncan be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled 159WO 2017/009712 PCT/IB2016/001079 practitioner. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorat syme ptom ins a subject.

[00508] One of ordinary skill in the art would be able to determine a therapeutical ly effective amount based on such factors as the subject's size, the severity of the subject's symptoms and, the particular compositi onor route of administration selected.

[00509] The anti-HIDE 1 antibodie ofs the invention find use in treating patients, such as human subjects, generall wity ha condition associated with HIDE1. The term “treatment” as used herein, refer sto both therapeutic treatme ntand prophylact oric preventative measures whic, hin thi sexample relat esto treatment of cancer; however, also as describe d below, uses of antibodie ands pharmaceutic alcompositions are also provided for treatment of infectious disease, sepsis, and/or autoimmune condition and/s, or for inhibiting an undesirabl e immune activation that follow genes therapy Those. in need of treatment include those already with cancer as well as those in which the cancer is to be prevented. Hence, the mammal to be treat edherei nmay have been diagnosed as having the cancer or may be predispos ored susceptible to the cancer. As used herein the term “treating” refers to preventing, delaying the onset of, curing, reversing, attenuating, alleviating, minimizing, suppressin g,halting the deleterious effects or stabilizing of discernibl syme ptom ofs the above-described cancerous diseases, disorder ors conditions. It also includes managing the cancer as described above. By “manage” it is meant reducing the severity of the disease, reducing the frequency of episodes of the disease, reducing the duration of such episodes, reducing the severity of such episodes, slowing/reducing cancer cel lgrowt orh proliferation, slowing progressio ofn at least one symptom amel, iorat ionof at leas tone measurabl e physical parameter and the like For. example, immunostimulator antiy -HIDEl immune molecules should promot myele oid cell T, cel lor NK or cytokine immunity against target cell s,e.g., cancer, infected or pathogen cell sand thereby trea cancert or infectious diseases by depleting the cell sinvolved in the disease condition. Conversely, immunoinhibitory anti - HIDE1 immune molecules should reduce myeloid cell T, cel lor NK activit yand/or or the secretion of proinflammatory cytokine whics h are involved in the disease pathology of some immune disease such as autoimmune, inflammatory or allergi conditic ons and thereby trea ort 160WO 2017/009712 PCT/IB2016/001079 ameliorate the disease pathology and tissue destructi thaton may be associate witd hsuch conditions (e.g, joint destruct ionassociate witd hrheumatoid arthri conditis tions).

[00510] The HIDE1 antibodie ofs the invention are provided in therapeutical ly effective dosages. A “therapeutically effective dosage” of an anti-HIDE1 immune molecul e according to at leas tsome embodiment ofs the present invention preferably resul tsin a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, an increase in lifespan, disease remission, or a prevention or reduction of impairment or disabilit duey to the disease affliction. For example, for the treatment of HIDE1 positive tumors (including, for example tumor thats express HIDE1 on the cellular membrane as well as tumor thats express HIDE 1 in the tumor microenvironmen alsot, referre tod as the TME), a “therapeutically effective dosage” preferably inhibits cel lgrowt h or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and sti llmore preferably by at least about 80% relati veto untreated subjects The. ability of a compound to inhibit tumor growth can be evaluated in an animal model system predicti veof efficacy in human tumors. Alternative thily, sproperty of a composition can be evaluated by examining the abilit yof the compound to inhibit, such inhibition in vitro by assays known to the skill edpractitioner. A therapeutical effectly ive amount of a therapeutic compound can decrease tumor size, or otherwi amelise orate symptom ins a subject.

[00511] One of ordinary skill in the art would be able to determine a therapeutical ly effective amount based on such factors as the subject's size, the severity of the subject's symptoms and, the particular compositi onor route of administration selected. a. Cancer Treatment

[00512] The HIDE1 antibodie ofs the invention find particular use in the treatment of cancer. In general, the antibodie ofs the inventio nare immunomodulatory, in that rathe thanr direct lyattack cancerous cells, the anti-HIDE 1 antibodie ofs the invention stimula tethe immune system gene, rall byy inhibiting the action of HIDE1. Thus, unlike tumor-targeted therapies whic, hare aimed at inhibiting molecular pathways that are crucial for tumor growt h and developmen t,and/or depleting tumor cell s,cancer immunotherapy is aimed to stimula te the patien’ts own immune system to eliminate cancer cell s,providing long-lived tumor 161WO 2017/009712 PCT/IB2016/001079 destruction. Various approaches can be used in cancer immunotherapy, among them are therapeutic cancer vaccines to induce tumor-specifi Tc cell responses, and immunostimulatory antibodie Q.e.,s antagonis tsof inhibitory receptor = simmune checkpoints) to remove immunosuppressive pathways.

[00513] Clinical responses wit htarget edtherapy or conventional anti-cancer therapies tend to be transient as cancer cell sdevelop resistance, and tumor recurrence takes place.

However, the clinical use of cancer immunotherapy in the past few years has shown that thi s type of therapy can have durable clinical responses showi, ng dramat icimpact on long term survival. However, although responses are long term, only a small number of patients respond (as opposed to conventional or target edtherapy, where a large number of patients respond, but respons esare transient).

[00514] By the time a tumor is detected clinically, it has already evaded the immune- defense system by acquiring immunoresista andnt immunosuppressive properti andes creating an immunosuppressive tumor microenvironment through various mechanism sand a variet yof immune cells.

[00515] Accordingly, the anti-HIDEl antibodie ofs the inventio nare useful in treating cancer. Due to the nature of an immuno-oncology mechanism of action, HIDE1 does not necessarily need to be overexpresse ond or correlat withed a particular cancer type; that is, the goal is to have the anti-HIDEl antibodies de-suppre ssmyeloid, T cell and NK cell activation such, that the immune system will go after the cancers.

[00516] “Cancer”, as used herein, refer sbroadly to any neoplasti diseasec (whether invasive or metastati charac) cterize byd abnormal and uncontroll celed ldivision causing malignant growth or tumor (e.g, unregulated cell growth.) The term “cancer” or “cancerous” as used herein should be understood to encompass any neoplastic disease (whether invasive, non-invasive or metastati whicc) his characterized by abnormal and uncontroll celed ldivision causing malignant growth or tumor, non-limiti ngexamples of which are described herein.

This includes any physiologica condil tion in mammals that is typicall charay cterize byd unregulated cel lgrowt Examplh. es of cancer are exemplified in the working examples and also are described within the specification. 162WO 2017/009712 PCT/IB2016/001079

[00517] Non-limiting examples of cancer that can be treat edusing anti-HIDEl antibodie include,s but are not limite to,d carcinoma, lymphoma, blastoma, sarcoma and, leukemia. More particular examples of such cancers include squamous cel lcancer, lung cancer (including small-cel lungl cancer, non-small cel llung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum hepatocel, lular cancer, gastric or stomach cancer (including gastrointest cancer),inal pancreat iccancer, glioblastoma , cervical cancer, ovaria ncancer, liver cancer, bladder cancer, hepatoma breast, cancer, colon cancer, colorecta cancl er, endometrial or uterine carcinoma, salivary gland carcinoma kidney, or renal cancer, liver cancer, prostat cance er, vulval cancer, thyro cancer,id hepatic carcinoma and various types of head and neck cancer, as wel las B-cel llymphoma (including low grade/follicul non-Hodgkin'sar lymphoma (NHL); small lymphocytic (SL) NHL; intermedia grade/te follicul NHLar; intermedia gradete diffus eNHL; high grade immunoblasti NHLc ; high grade lymphoblast NHLic ; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-relat edlymphoma; and Waldenstrom’s Macroglobulinemi chronica); lymphocyt leukemiic a(CLL); acute lymphoblast leukemiic a(ALL); Hairy cel lleukemia ;chronic myeloblasti leukemc ia; multipl e myeloma and post-transpl lymphoprant olifer ativedisorder (PTLD).

[00518] In some embodiments, othe cancersr amenable for treatment by the present invention include, but are not limite to,d carcinoma, lymphoma, blastoma, sarcoma and, leukemi aor lymphoi mald ignancies. More particular examples of such cancers include colorectal, bladde r,ovarian, melanoma, squamous cel lcancer, lung cancer (including small- cel llung cancer, non-small cel llung cancer, adenocarcinoma of the lung ,and squamous carcinoma of the lung), cancer of the peritoneum hepat, ocellul cancarer, gastr icor stomach cancer (including gastrointesti cancer),nal pancreatic cancer, glioblastom cervicala, cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorecta l cancer, endometri oral uterine carcinoma sali, vary gland carcinoma, kidney or renal cancer, liver cancer, prostate cancer, vulval cancer, thyroi cancer,d hepatic carcinoma and various types of head and neck cancer, as well as B-cel llymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocyt (SL)ic NHL; intermedia te grade/follicul NHLar; intermedia gradete diffuse NHL; high grade immunoblasti NHLc ; high 163WO 2017/009712 PCT/IB2016/001079 grade lymphoblast NHicL; high grade smal lnon-cleaved cel lNHL; bulky disease NHL; mantle cell lymphoma; AIDS-relat edlymphoma and; Waldenstr’oms Macroglobulinem ia); chronic lymphocytic leukemi a(CLL); acute lymphoblast leukemiic a(ALL); Hairy cell leukemia; chronic myeloblasti leukemc ia; and post-transpl lymphoprant olifer ativedisorder (PTLD), as well as abnormal vascular proliferati associon ated wit hphakomatoses, edema (such as that associated wit hbrain tumors), and Meigs' syndrom e.Preferably, the cancer is selected from the group consisting of colorecta cancer,l breast cancer, recta cancl er, non- small cel llung cancer, non-Hodgkin’s lymphom (NHLa ), renal cell cancer, prostat cancer,e liver cancer, pancreati cancc er, soft-tissue sarcoma Kaposi, ’s sarcoma carci, noid carcinoma, head and neck cancer, melanoma, ovarian cancer, mesothelioma, and multiple myeloma. In an exemplary embodiment the cancer is an earl yor advanced (including metastat ic)bladder , ovarian or melanoma. In another embodiment the cancer is colorecta cancer.l In some embodiment s,the cancerous conditions amenable for treatment of the present invention include cancers that express or do not express HIDE1 and furth erinclude non-metastati orc non-invasive as well as invasive or metastat cancersic wherei HIDE1n expression by immune, strom oral disease dcell ssuppress antitumor responses and anti-invasive immune responses The. method of the present inventio nis particularl suitay ble for the treatment of vascularized tumors.

[00519] As shown in the Examples, HIDE1 is over expressed and/or correlates wit h tumor leukocyte infiltrati (ason demonstrated by correlati toon CSFR1,CD68, CD86, CDllb, IL-4, IL-10, IL-13 and IFN-y expression) in a number of different tumor ofs various origins, and thus is useful in treating any cancer, including but not limite to,d prostat cance er, liver cancer (HCC), colorecta cancl er, ovaria ncancer, endometri cancer,al breast cancer, pancreati cancc er, stomach cancer, cervical cancer, head and neck cancer, thyro cancer,id test iscancer, urotheli cancal er, lung cancer, melanoma, non melanoma skin cancer (squamou sand basal cell carcinoma), glioma renal, cancer (RCC), lymphom (non-a Hodgkins’ lymphoma (NHL) and Hodgkin’s lymphom (HDa )), Acute myeloid leukemia (AML), T cell Acute Lymphoblastic Leukemia (T-ALL), Diffuse Large B cel llymphoma, testicula germr cel ltumors, mesotheliom anda esophageal cancer. In some embodiment s,the tumor ares myeloid or circulati ngtumor types .In some embodiment s,the cancer includes 164WO 2017/009712 PCT/IB2016/001079 but is not limited to Acute Myeloid Leukemia, Acute Myeloid Leukemia Induction Failure, Acute Lymphoblast Leukemiic a, Diffuse Large B-cel lLymphoma, Malignant Lymphoma , Non-Hodgkin Lymphoma ,Diffuse Large B-Cel lLymphoma, Glioblastom multa iform e, Mesothelioma, Thymoma ,Testicul arGerm Cell Tumors, Kidney renal clear cel lcarcinoma, Sarcoma, Brain Lower Grad eGlioma, Chroni Lymc phocytic Leukemia, Non-Hodgkin Lymphoma - Follicul Lymphoma,ar Uterine Carcinosarcoma, Pediatri Brainc Tumors, Lung adenocarcinom Cervica, al squamous cel lcarcinoma, endocervic aladenocarcinoma , Pancreatic adenocarcinoma Skin, Cutaneous Melanoma, Kidney renal papillary cell carcinoma, Liver hepatocellular carcinoma Bladder; Urotheli Carcial noma, Colo n adenocarcinom Heada, and Neck squamous cel lcarcinoma, Lung squamous cel lcarcinoma, Rectum adenocarcinoma, and Stomach adenocarcinoma.

[00520] “Cancer therapy” herein refers to any method which prevent ors treat cancers or ameliorates one or more of the symptoms of cancer. Typically such therapies will comprises administration of immunostimulatory anti-HIDEl antibodie (incls uding antigen- binding fragments eit) her alone or in combination with chemothera pyor radiotherapy or othe biologir esand for enhancing the activit ythereof, i.e., in individual whereis expresn sion of HIDE1 suppresse antits umor respons esand the efficacy of chemotherapy or radiotherapy or biologi efficac cy.

[00521] In some embodiments, the cancer for treatment is a cancer having high immune infiltrate of myeloid cell sexpressing HIDE1 wherei sain d cancer could be treat edby administering HIDE1 antibodies In. some embodiments, said cancer is a myeloid cancer. In some embodiments, the myeloid cells include but are not limite tod monocytes dendri, tic cell s,macrophages, M1/M2 tumor associated macrophages, neutrophil ands, myeloid-deri ve suppressor cells (MDSC). In some embodiments, the anti-HIDEl antibody for use in cancer treatment is a depleting HIDE1 antibody. In some embodiment s,a depleting anti-HIDEl antibody binds to cel lsurfac eHIDE1. In some embodiments, the anti HIDE1 depleting antibody preferably is able to deplet HIDE1e expressing cell sincludin gbut not limite tod monocyte s,dendritic cells, macrophages, M1/M2 tumo rassociated macrophages, neutrophil s, Myeloid-derive suppressor cell s(MDSC) and as a resul reducet the number of HIDE1 expressing cells in a patient treate withd the anti HIDE1 depleting antibody. Such depletion 165WO 2017/009712 PCT/IB2016/001079 may be achieved via various mechanisms such as antibody-dependent cell mediated cytotoxicit (ADCC)y and/or complement dependent cytotoxici (CDC),ty inhibition of HIDE1 expressing cell sproliferati and/oron inductio ofn HIDE1+ cel ldeat h(e.g., via apoptosis). b. Assessment of Treatment

[00522] Generall they anti-HIDE 1 antibodie ofs the invention are administered to patients wit hcancer, and efficacy is assessed, in a number of ways as described herein. Thus, while standar assaysd of efficacy can be run, such as cancer load, size of tumor, evaluation of presence or extent of metastasi etc.,s, immuno-oncology treatments can be assessed on the basis of immune status evaluations as wel l.This can be done in a number of ways, including both in vitro and in vivo assays. For example, evaluation of changes in immune status (e.g. presence of ICOS+ CD4+ T cells follow ingipi treatment) along with “old fashioned” measurements such as tumor burden, size, invasiveness, LN involvement met, astasi etc.s, can be done. Thus, any or all of the follow ingcan be evaluated: the inhibitor effectsy of HIDE 1 on CD4+ T cel lactivation or proliferat ion,CD8+ T (CTL) cel lactivation or proliferati on, CD8+ T cell-mediated cytotoxic activit yand/or CTL mediated cel ldepletion, NK cel lactivit y and NK mediated cel ldepletion, the potentiati effectsng of HIDE1 on Treg cell differentiat ionand proliferati andon Treg- or myeloid derived suppressor cel l(MDSC)- mediated immunosuppression or immune tolerance and/or, the effects of HIDE1 on proinflammatory cytokine production by immune cell s,e.g., IL-2, IFN-y or TNF-a production by T or other immune cells.

[00523] In some embodiments, assessment of treatment is done by evaluating immune cel lproliferat ion,using for example, CFSE dilution method, Ki67 intracellul stainingar of immune effector cell s,and 3H-Thymidine incorporati method,on

[00524] In some embodiments, assessment of treatment is done by evaluating the increase in gene expression or increased prote inlevels of activation-associated markers , including one or more of: CD25, CD69, CD137, ICOS, PD1, GITR, OX40, and cell degranulati onmeasured by surface expression of CD107A.

[00525] In general, gene expression assays are done as is known in the art. See for example Goodkind et al., Computers andChem. Eng. 29(3):589 (2005), Han et al., 166WO 2017/009712 PCT/IB2016/001079 Bioinform. Biol. Insight 11/15/15s 9(Suppl. !):29-46, Campo etal.,Nod. Pathol. 2013 Jan; 26 suppl. 1 :S97-S110, the gene expression measuremen techt nique sof which are expressl y incorporated by reference herein.

[00526] In general, protei expressn ion measurement ares also similar lydone as is known in the art, see for example, Wang etal., Recent Advances in Capillary Electrophoresis- BasedProteom Techniic ques for Biomarker Discovery, Methods. Mol. Biol. 2013:984:1-12; Taylor et al., BioMed Res. Volum e2014, Article ID 361590, 8 pages, Becerk et al., Mutat. Res 2011 June 17:722(2): 171-182, the measurement techniques of which are expressl incorporatedy herein by reference.

[00527] In some embodiments, assessment of treatment is done by assessing cytotoxic activit ymeasured by targe celt lviability detection via estimating numerous cell parameters such as enzyme activit y(including protease activity ),cel lmembrane permeabilit celly, adherence, ATP production, co-enzyme production, and nucleotide uptake activit y.Specific examples of thes assae ys include, but are not limite to,d Trypan Blue or PI staining, 5Cr or 35S releas method,e LDH activity, MTT and/or WST assays, Calcein-AM assay, Luminescent based assay, and others.

[00528] In some embodiments, assessment of treatment is done by assessing T cell activity measured by cytokine production, measure either intracellularl in culty ure supernatant using cytokine incls uding, but not limited to, IFNy, TNFa, GM-CSF, IL2, IL6, IL4, IL5, IL 10, IL 13 using well known techniques.

[00529] Accordingly, assessment of treatment can be done using assays that evaluat e one or more of the following: (i) increases immune response, (ii) increases T cel lactivity, (iii) increase sactivation of a,[3 and/or y5 T cell s,(iv) increases cytotoxi Tc cel lactivity, (v) increases NK and/or NKT cel lactivit y,(vi) alleviat esa[3 and/or y5 T-cel lsuppression, (vii) increases pro-inflammatory cytokine secretion, (viii) increases IL-2 secretion; (vix) increases interferon-y production, (x) increase sThl response, (xi) decrease Th2 respons e,(xii) decreases or eliminates cel lnumber and/or activit yof at least one of regulatory T cells (Tregs), myeloid derive dsuppressor cells (MDSCs), iMCs, mesenchyma lstromal cells, TIE2-expressing monocyte s,(xiii) reduces regulatory cel lactivit y,and/or the activit yof one 167WO 2017/009712 PCT/IB2016/001079 or more of myeloid derived suppressor cell s(MDSCs), iMCs, mesenchyma lstroma cell ls, TIE2-expressing monocyte s,(xiv) decreases or eliminates M2 macrophages, (xv) reduces M2 macrophage pro-tumorigenic activit y,(xvi) decreases or eliminates N2 neutrophils, (xvii) reduce sN2 neutrophil pro-tumoriges activitnic y,(xviii) reduce sinhibition of T cell activation (xix), reduces inhibition of CTL activation (xx), reduce sinhibition of NK and/or NKT cel lactivation (xxi), reverses and/or y5 T cel lexhaustion, (xxii) increases aP and/o r y5 T cel lresponse, (xxiii) increases activit yof cytotoxic cell s,(xxiv) stimulates antigen- specific memory responses, (xxv) elicits apoptos oris lysi sof cancer cells, (xxvi) stimulates cytotoxic or cytostat effectic on cancer cell s,(xxvii) induces direc killingt of cancer cells , (xxviii) increases Thl7 activity and/or (xxix) modulating myeloid cel lpolarizati on,(xxx) modulating myeloid cel lshifting toward a pro-inflammatory response, (xxxi) shifting myeloid from M2 toward Ml phenotype, (xxxii) modulating myeloid cel lin the TME to support anti-cancer immune response, (xxxiii) restricti theng pro-tumorigenic effects of the myeloid cell sin the TME, (xxxiv) enhancing myeloid and lymphoi infild trati intoon the tumor cite thereby shifting the tumor into more immunogenic, (xxxv) induces complement dependent cytotoxicit and/ory antibody dependent cell-mediated cytotoxicity.

[00530] Accordingly, assessment of treatment can be done using assays that evaluate one or more of the following: (i) decrease ims mune response, (ii) decrease «Ps and/or y5 T cel lactivation (iii, ) decreases T cel lactivit y,(iv) decrease cytotoxics T cell activity, (v) decreases natural killer (NK) and/or NKT cell activity, (vi) decreases «P and/or y5 T-cel l activit y,(vii) decrease pro-inflas mmatory cytokine secretion, (viii) decreases IL-2 secretion; (ix) decreases interferon-Y production, (x) decreases Thl response, (xi) decrease Th2s response, (xii) increases cel lnumber and/or activit yof regulatory T cell s,(xiii) increases regulatory cel lactivit yand/or one or more of myeloid derived suppressor cell s(MDSCs), iMCs, mesenchyma lstroma celll s,TIE2-expressing monocytes (xiv), increases regulatory cel lactivit yand/or the activit yof one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchyma lstroma celll s,TIE2-expressing monocytes, (xv) increases M2 macrophages, (xvi) increases M2 macrophage activit y,(xvii) increases N2 neutrophil (xviis, i) increases N2 neutrophi activitls y,(xix) increases inhibition of T cel lactivation (xx), increases inhibition of CTL activation, (xxi) increases inhibition of NK cel lactivation, (xxii) increases 168WO 2017/009712 PCT/IB2016/001079 aP and/or y5 T cel lexhaustion, (xxiii) decreases aP and/or y5 T cel lrespons e,(xxiv) decreases activity of cytotoxi cellc s,(xxv) reduces antigen-specific memory responses (xxvi), inhibits apoptosis or lysi sof cell s,(xxvii) decrease cytots oxic or cytostati effecc t on cells , (xxviii) reduce sdirec killit ngof cell s,(xxix) decreases Thl7 activity, and/or (xxx) modulates myeloid cel lpolarizati on,and/or modulates myeloid cell shifting toward an anti- inflammatory respons e,(xxxi) reduce scomplement dependent cytotoxici and/orty antibody dependent cell-mediated cytotoxici ty.Again without wishing to be limited by a single hypothesis, HIDE1 shows potentiat ingeffects on the follow ingimmune functions indu: ction or differentiat ionand proliferati ofon inducible T regulatory or suppressor cells (iTregs).

These cells are known to be involved in eliciting tolerance to self-antige nsand to suppres s anti-tumor immunity. c. Assays to measure efficacy

[00531] In some embodiments, T cel lactivation is assessed using a Mixed Lymphocyte Reaction (MLR) assay as is described in Example 2. An increase in activity indicate simmunostimulator activity y.Appropriat incree ases in activit yare outlined below.

[00532] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in immune response as measured for an example by phosphorylati or onde- phosphorylat ofion different factors, or by measuring other post translational modifications.

An increase in activit yindicate simmunostimulatory activit y.Appropriat incree ases in activity are outlined below.

[00533] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in activation of a,|3 and/or y5 T cell sas measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in activit yindicate simmunostimulatory activity .Appropriate increase sin activit yare outlined below.

[00534] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in cytotoxi Tc cel lactivity as measured for an example by direc killingt of target cells like for an example cancer cells or by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. 169WO 2017/009712 PCT/IB2016/001079 An increase in activit yindicate simmunostimulatory activit y.Appropriat incree ases in activity are outlined below.

[00535] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in NK and/or NKT cell activity as measured for an example by direc kilt ling of target cells like for an example cancer cell sor by cytokine secretion or by changes in expression of activation markers like for an example CD107a, etc. An increase in activity indicate simmunostimulator activity y.Appropriat incree ases in activit yare outlined below.

[00536] In one embodiment, the signaling pathway assay measures increases or decreases in myeloid cell-mediated suppression of T or NK cel lactivit yas measured for an example by direc killit ngof target cells like for an example cancer cell sor by cytokine secretion or by changes in expression of activation markers like for an example CD107a, etc.

An increase in activit yindicate simmunostimulatory activit y.Appropriat incree ases in activity are outlined below

[00537] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in myeloid cell-mediated suppression of T or NK cel lmigration as measured for an example by T or NK cel lmigration in two-chambe assar y system An. increase in migration indicate simmunostimulator actiyvity.

[00538] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in pro-inflammatory cytokine secretion as measured for example by ELISA or by Luminex or by Multiple beadx based methods or by intracellula stainingr and FACS analysi s or by Alispot etc. An increase in activity indicates immunostimulatory activity .Appropriate increases in activit yare outlined below.

[00539] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in IL-2 secretion as measured for example by ELISA or by Luminex or by Multiple beadx based methods or by intracellul staiarning and FACS analysi sor by Alispot etc. An increase in activity indicate simmunostimulatory activity .Appropriat incree ases in activity are outlined below.

[00540] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in interferon-Y production as measured for example by ELISA or by Luminex or 170WO 2017/009712 PCT/IB2016/001079 by Multiple beadx based methods or by intracellul staiarning and FACS analysi sor by Alispot etc. An increase in activit yindicates immunostimulatory activit y.Appropriate increases in activit yare outlined below.

[00541] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in Thl response as measured for an example by cytokine secretion or by changes in expression of activation markers An. increase in activity indicate simmunostimulatory activity .Appropriate increase sin activit yare outlined below.

[00542] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in Th2 response as measured for an example by cytokine secretion or by changes in expression of activation markers An. increase in activity indicate simmunostimulatory activity .Appropriate increase sin activit yare outlined below.

[00543] In one embodiment, the signaling pathwa yassay measure sincreases or decreases cel lnumber and/or activit yof at least one of regulatory T cells (Tregs), as measured for example by flow cytometry or by IHC. A decrease in response indicates immunostimulatory activit y.Appropriate decreases are the same as for increases, outlined below.

[00544] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in M2 macrophages cel lnumbers ,as measured for example by flow cytomet ryor by IHC. A decrease in response indicate simmunostimulator actiyvity. Appropriat decree ase s are the same as for increases, outlined below.

[00545] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in M2 macrophage pro-tumorigenic activit y,as measured for an example by cytokine secretion or by changes in expression of activation markers. A decrease in response indicate simmunostimulator activity y.Appropriat decree ase ares the same as for increases, outlined below.

[00546] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in N2 neutrophil increase,s as measured for example by flow cytometry or by IHC.

A decrease in response indicates immunostimulatory activit y.Appropriate decrease ares the same as for increases, outlined below. 171WO 2017/009712 PCT/IB2016/001079

[00547] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in N2 neutrophil pro-tums origeni activic ty, as measured for an example by cytokine secretion or by changes in expression of activation markers. A decrease in response indicate simmunostimulator activity y.Appropriat decree ase ares the same as for increases, outlined below.

[00548] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in inhibition of T cel lactivation, as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in activit yindicates immunostimulatory activit y.

Appropriat incree ases in activity are outlined below.

[00549] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in inhibition of CTL activation as measured for an example by direc killit ngof target cells like for an example cancer cell sor by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc .

An increase in activit yindicate simmunostimulatory activit y.Appropriat incree ases in activity are outlined below.

[00550] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in 0,(3 and/or y5 T cell exhaustion as measured for an example by changes in expression of activation markers A. decrease in response indicate simmunostimulatory activity Appropriat. decree ase ares the same as for increases, outlined below.

[00551] In one embodiment, the signaling pathwa yassay measure sincreases or decreases a,[3 and/or y5 T cel lresponse as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD137, CD107a, PD1, etc. An increase in activit yindicates immunostimulatory activit y.

Appropriat incree ases in activity are outlined below.

[00552] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in stimulation of antigen-specific memory responses as measured for an example by cytokine secretion or by proliferation or by changes in expression of activation markers 172WO 2017/009712 PCT/IB2016/001079 like for an example CD45RA, CCR7 etc. An increase in activity indicate s immunostimulatory activit y.Appropriat incree ases in activit yare outlined below ..

[00553] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in apoptos oris lysi sof cancer cells as measured for an example by cytotoxici ty assays such as for an example MTT, Cr releas e,Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc. An increase in activity indicate simmunostimulatory activity .Appropriat incree ases in activity are outlined below.

[00554] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in stimulation of cytotoxic or cytostat effecic t on cancer cells, as measured for an example by cytotoxicit assaysy such as for an example MTT, Cr releas e,Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc. An increase in activit yindicates immunostimulatory activit y.Appropriat incree ases in activity are outlined below.

[00555] In one embodiment, the signaling pathwa yassay measure sincreases or decreases direc killingt of cancer cell sas measured for an example by cytotoxicit assaysy such as for an example MTT, Cr release, Calcine AM, or by flow cytomet rybased assays like for an example CFSE dilution or propidium iodide staining etc . An increase in activit y indicate simmunostimulator activity y.Appropriate increases in activit yare outlined below.

[00556] In one embodiment, the signaling pathwa yassay measure sincreases or decreases Thl7 activit yas measured for an example by cytokine secretion or by proliferati on or by changes in expression of activation markers. An increase in activit yindicates immunostimulatory activit y.Appropriat incree ases in activit yare outlined below.

[00557] In one embodiment, the signaling pathwa yassay measure sincreases or decreases in induction of complement dependent cytotoxicit and/ory antibody dependent cell - mediated cytotoxicity, as measured for an example by cytotoxici assty ays such as for an example MTT, Cr releas e,Calcine AM, or by flow cytometry based assays like for an example CFSE dilution or propidium iodide staining etc. An increase in activit yindicates immunostimulatory activit y.Appropriat incree ases in activit yare outlined below. 173WO 2017/009712 PCT/IB2016/001079

[00558] In one embodiment, T cel lactivation is measured for an example by direc t killing of target cells like for an example cancer cells or by cytokine secretion or by proliferation or by changes in expression of activation markers like for an example CD 137, CD107a, PD1, etc .For T-cell s,increases in proliferat ion,cel lsurface markers of activation (e.g. CD25, CD69, CD137, PD1), cytotoxicit (abily it yto kill target cells), and cytokine production (e.g. IL-2, IL-4, IL-6, IFNy, TNF-a, IL-10, IL-17A) would be indicative of immune modulation that would be consistent wit henhanced killing of cancer cells.

[00559] In one embodiment, NK cell activation is measured for example by direct killing of target cells like for an example cancer cells or by cytokine secretion or by changes in expression of activation markers like for an example CD 107a, etc. For NK cells, increases in proliferat ion,cytotoxici (abity lity to kill target cells and increases CD 107 a, granzyme, and perfor inexpression), cytokine production (e.g. IFNy and TNF ), and cell surfac ereceptor expression (e.g. CD25) would be indicative of immune modulation that would be consiste ntwit henhanced killing of cancer cells.

[00560] In one embodiment, y5 T cel lactivation is measured for example by cytokine secretion or by proliferation or by changes in expression of activation markers.

[00561] In one embodiment, Thl cel lactivation is measured for example by cytokine secretion or by changes in expression of activation markers.

[00562] Appropriat incree ases in activit yor response (or decreases, as appropriat ase outlined above), are increases of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 98 to 99% percent over the signal in either a reference sample or in control samples, for example test samples that do not contain an anti-HIDEl antibody of the invention. Similarly, increases of at leas tone-, two-, three-, four- or five-fold as compared to reference or control samples show efficacy. 2. Combinations

[00563] As is known in the art, combination therapies comprising a therapeutic antibody targeting an immunotherapy target and an additional therapeutic agent, specific for the disease condition, are showing great promise. For example, in the area of immunotherapy, there are a number of promising combination therapies using a 174WO 2017/009712 PCT/IB2016/001079 chemotherapeutic agent (either a smal lmolecul druge or an anti-tumor antibody) with immuno-oncology antibodies like anti-PD-1, and as such, the anti-HIDE 1 antibodie outlis ned herei ncan be substituted in the same way. Any chemotherapeutic agent exhibiting anticancer activit ycan be used according to various embodiments of the present invention; various non- limitin examg ples are describe ind the specification.

[00564] The underlyin scientg ific rational fore the dramat icincreased efficacy of combination therapy claims that immune checkpoint blockade as a monothera wilpy inducel tumor regressions only when there is pre-existing strong anti-tumor immune respons toe be “unleashed” when the pathway is blocked. However, in most patients and tumor types the endogenous anti-tumor immune respons esare weak, and thus the inductio ofn anti-tumor immunity is required for the immune checkpoint blockade to be effective, as shown in the Figure 1. According to at leas tsome embodiment ofs the presen invent tion, HIDE !-specific antibodies, antibody fragment s,conjugate sand compositions comprising same, are used for treatment of all types of cancer in cancer immunotherapy in combination therapy.

[00565] The term s“in combination wit”h and "co-administrati on"are not limite tod the administration of said prophylact oric therapeutic agents at exactl ythe same time. Instead, it is meant that the anti-HIDE 1 antibody and the other agent or agents are administered in a sequence and within a time interval such that they may act together to provide a benefit that is increased versus treatment wit honly either anti-HIDE 1 antibody of the various embodiments of the present inventio nor the other agent or agents .It is preferred that the anti - HIDE1 antibody and the other agent or agents act additively, and especially preferred that they act synergisticall Suchy. molecules are suitably present in combination in amounts that are effective for the purpose intended. The skill edmedical practitioner can determine empirically, or by considering the pharmacokineti andcs modes of action of the agents, the appropriat dosee or dose sof each therapeutic agent, as wel las the appropriat time ings and methods of administration.

[00566] Accordingly, in some embodiments, the antibodie ofs the present inventio n may be administered concomitantly with one or more other therapeutic regimens or agents .

The additional therapeutic regimes or agents may be used to improve the efficacy or safet yof the anti-HIDEl antibody. Also, the additional therapeutic regimes or agents may be used to 175WO 2017/009712 PCT/IB2016/001079 trea thet same disease or a comorbid ityrathe thanr to alter the action of the HIDE1 antibody.

For example, a HIDE 1 antibody of the various embodiments of the present invention may be administere tod the patient along wit hchemotherapy, radiation therapy, or both chemothera pyand radiation therapy.

[00567] The HIDE1 antibodies of the various embodiments of the present inventio n may be administered in combination with one or more other prophylacti or ctherapeuti c agents ,includin gbut not limite tod cytotoxic agents, chemotherapeuti agents,c cytokine s, growth inhibitory agents, anti-hormonal agents ,kinase inhibitors, anti-angiogenic agents, cardioprotecta imnts,munostimulatory agents ,immunosuppressive agents, agents that promote proliferati ofon hematologic cellal s,angiogenesi sinhibitors, prote intyrosi nekinase (PTK) inhibitors or ,other therapeutic agents.

[00568] According to at least some embodiments, the anti-HIDEl immune molecul es could be used in combination with any of the known in the art standard of care cancer treatment (as can be found, for example, on the World Wide Web at cancer.gov/cancertopi). cs

[00569] In some embodiment s,conventional/class icalanti-cancer agents suitable for use with the present inventio ninclude but are not limited to platinum based compounds, antibiotics wit hanti-cance ractivit y,Anthracyclines, Anthracenediones, alkylating agents, antimetabolites Antim. itoti agentsc ,Taxanes, Taxoids, microtubule inhibitors, Folat e antagonis tsand/or foli cacid analogs, Topoisomeras inhibite ors, Aromatas inhibite ors, GnRh analogs, inhibitors of 5a-reductase bisp, hosphonate pyrimidines; analogs, purine analogs and related inhibitors, vinca alkaloid epips, odophyllotoxins antibi, otics L-Aspar, aginase, topoisomerase inhibitor, interferons, platinum coordination complexes, anthracenedione substitut urea,ed methyl hydrazine derivatives, adrenocorti suppressant,cal adrenocorticosteroid progests, ins, estrogens, antiestrogen, androgens, antiandrogen, and gonadotropin-releasi hormoneng analog.

[00570] Specific but non-limiti ngexamples of these categori esof drugs are as follows: platinum based compounds such as oxaliplati cisplan, tin, carboplati Antin; bioti cswith anti- cancer activity, such as dactinomycin, bleomycin, mitomycin-C, mithramycin and 176WO 2017/009712 PCT/IB2016/001079 Anthracy clines, such as doxorubicin, daunorubicin, epirubicin, idarubicin; Anthracenedione s, such as mitoxantrone; Alkylating agents, such as dacarbazine, melphalan, cyclophosphami de, temozolomi de,chlorambuci busulphan,l, nitrogen mustard, nitrosoureas; Antimetabolites , such as fluorouraci raltl, itrexed, gemcitabine, cytosine arabinoside, hydroxyurea and Folate antagonist suchs, as methotrexate, trimethoprim pyrimet, hamine, pemetrexed; Antimitoti c agents such as polokinase inhibitors and Microtubule inhibitors, such as Taxanes and Taxoids, such as paclitaxel docetaxel;, Vinca alkaloids such as vincristine, vinblastine, vindesine ,vinorelbine; Topoisomeras inhibie tors such, as etoposide, teniposide, amsacrine, topotecan, irinotecan, camptotheci Cytosn; tat agentsic including Antiestrogens such as tamoxifen, fulvestra nt,toremifene, raloxifene droloxifene,, iodoxyfene, Antiandrogens such as bicalutamide, flutamide, nilutamide and cyproterone acetate, Progestogens such as megestrol acetate, Aromatase inhibitors such as anastrozole let, rozol vorozole,e, exemestane; GnRH analogs, such as leuprorel goserin, elin buserel, in, degarelix; inhibitors of 5a-reductase such as finasteride.

[00571] More preferabl they, chemotherapeuti agentc is selected from the group consisting of 5-fluoroura (5-FU),cil leucovorin (LV), irenotecan, oxaliplat in,capecitabine, paclitaxel and doxetaxel. Two or more chemotherapeutic agents can be used in a cockta ilto be administered in combination with administrati ofon the anti-VEGF antibody. One preferred combination chemotherapy is fluorouracil-based, comprising 5-FU and one or more othe chemr otherapeuti agentc (s). Suitable dosing regimens of combination chemotherapi es are known in the art and describe in,d for example, Saltz et al. (1999) ProcASCO 18:233a and Douilla rdet al. (2000) Lancet 355:1041-7. The biologic may optional bely another immune potentiat orssuch as antibodie tos PD-L1, PD-L2, CTLA-4, or VISTA as well as PD- LI, PD-L2, CTLA-4 or VISTA fusion proteins as well as cytokines growth, factor antagonis tsand agonist s,hormones and anti-cytokine antibodies.

[00572] According to at least some embodiments of the present invention, target ed therapies used as agents for combination with anti HIDE1 antibodie fors treatme ntof cancer are selected from the group consisting of but not limite to:d histone deacetylas (HDe AC) inhibitors, such as vorinost romiat, depsin, panobinostat beli, nostat moceti, nostat abexinost, at, entinost at,resminostat givinost, at, quisinostat sodium, butyrate; Proteasome inhibitors, such 177WO 2017/009712 PCT/IB2016/001079 as bortezom ib,carfilzomib, disulfiram mTOR; pathwa inhibity ors, such as temsirolim us, rapamycin, everolimus; PI3K inhibitors, such as perifosine, CAL101, PX-866, IPI-145, BAY 80-6946; B-raf inhibitors such as vemurafenib, sorafenib; JAK2 inhibitors such, as lestaurti nib,pacritinib; tyrosi nekinase inhibito rs(TKIs), such as erlotinib, imatinib, sunitinib , lapatini b,gefitinib ,sorafeni b,nilotinib, tocerani b,bosutini b,neratini b,vatalanib, regorafenib, cabozantinib; other protei kinan se inhibitors, such as crizotinib; inhibitors of serine/threoni ne kinase sfor example Ras/Raf signalling inhibitors such as famesyl transferase inhibitors; inhibitors of serine proteas esfor example matriptas hepsin,e, urokinas e;inhibitors of intracellula signalr ing such as tipifamib, perifosine; Inhibitors of cel lsignalling through MEK and/or AKT kinases; aurora kinase inhibitors such as AZDI 152, PH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528, AX39459; cyclin dependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors; inhibitors of survival signaling proteins includin g Bcl-2, Bcl-XL, such as ABT-737; HSP90 inhibitors; therapeutic monoclonal antibodies such, as anti-EGFR mAbs cetuximab, panitumumab ,nimotuzumab, anti-ERBB2 mAbs trastuzumab, pertuzumab, anti-CD20 mAbs such as rituxima b,ofatumumab ,veltuzumab and mAbs targeting othe tumorr antigens such as alemtuzumab, labetuzumab, adecatumumab, oregovomab, onartuzumab; TRAIL pathway agonist s,such as dulanermin (soluble rhTRAIL) ,apomab, mapatumumab, lexatumumab, conatumumab, tigatuzumab; antibody fragments bi-specifi, c antibodie ands bi-specific T-cel lengagers (BiTEs), such as catumaxomab ,blinatumomab; antibody drug conjugate s(ADC) and othe r immunoconjugate s,such as ibritumom abtriuxetan, tositumom ab,brentuximab vedoti n, gemtuzumab ozogamicin, clivatuzumab tetraxet an,pemtumomab, trastuzumab emtansine; anti-angiogenic therapy such as bevacizumab, etaracizumab, volociximab, ramucirumab, aflibercept, sorafenib, sunitinib, regorafenib axiti, nib, nintedanib, motesani b,pazopanib, cediranib; metalloproteinas inhiebitors such as marimastat; inhibito rsof urokinase plasminogen activator receptor function; inhibitors of cathepsin activity.

[00573] Other therapeutic antibodie whics h may optional bely used in combination with an immunostimulatory antibody according to at least some embodiments of the present invention include but are not limited to cetuximab, panitumumab, nimotuzumab, trastuzumab, pertuzumab, rituximab, ofatumumab ,veltuzumab, alemtuzumab, labetuzumab, 178WO 2017/009712 PCT/IB2016/001079 adecatumumab, oregovomab, onartuzuma b;apomab, mapatumumab, lexatumumab, conatumumab, tigatuzumab, catumaxomab, blinatumoma b,ibritumom abtriuxetan, tositumomab, brentuximab vedoti n,gemtuzumab ozogamicin, clivatuzumab tetraxetan, pemtumomab, trastuzumab emtansine ,bevacizumab, etaracizumab, volociximab, ramucirumab, aflibercept.

[00574] Therapeutic agents targeting immunosuppressive cell ssuch as Tregs and/or MDSCs whic hmay optionall bey used in combination with an immunostimulatory antibody according to at leas tsome embodiments of the present invention include but are not limite tod antimitoti drugs,c cyclophosphamide gemcit, abine, mitoxantrone, fludarabine, thalidomi de, thalidomi derivativede COX-2s, inhibitors, depleting or killi ngantibodies that directl targety Tregs through recogniti onof Treg cel lsurfac ereceptors, anti-CD25 daclizumab, basiliximab, ligand-directed toxins denil, eukin diftit ox(Ontak), a fusion prote inof human IL-2 and diphtheri toxin,a or LMB-2, a fusion between an scFv against CD25 and the pseudomonas exotoxin antibodie, targets ing Treg cell surfac ereceptors, TLR modulators, agents that interfere with the adenosinergic pathway, ectonucleotidase inhibitors or ,inhibitors of the A2A adenosine receptor, TGF-(3 inhibitors, chemokine receptor inhibitors, retinoic acid, all- trans retinoic acid (ATRA), Vitamin D3, phosphodiesteras 5 inhie bitors, sildenafi ROSl, inhibitors and nitroaspirin.

[00575] Other immunostimulator antibodiey whics hmay according to at least some embodiments optionally be used in combination wit han immunostimulator antiybody according to at leas tsome embodiments of the present invention include but are not limite tod agonisti orc antagonisti antibodiec targes ting one or more of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, VISTA, and/or agonist icor antagonisti antibodiec targes ting one or more of CD40, CD137, OX40, GITR, CD27, CD28 or ICOS, or fusion protei ns containing any of the foregoing or fragments there ofwhich function as immune agonist ors antagonists.

[00576] As described infra, without wishing to be limited by a singl ehypothesis, the HIDE1 prote inapparentl intey ract withs a receptor expressed by NK cells. Accordingly, the subject immunostimulatory antibody or immunostimulatory antigen-binding fragments may optionally be used on combination or coupled to an antibody or antigen-binding fragment 179WO 2017/009712 PCT/IB2016/001079 thereof, or othe moier ty which specifically binds to an NK cell receptor. Such moieties whic h specifically bind to an NK cel lreceptor may optionall agonizey or antagonize the effect of said NK cel lreceptor. Various non-limiti ngexamples are given herein. Such NK receptors include thos ofe unknown function, as well as those known to inhibit NK cell activit ysuch as KIR2DL1, KIR2DL2/3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, NKG2E and LILRB5 and those known to promot ore activate NK cel lactivity such as NKp30, NKp44, NKp46, NKp46, NKG2D, KIR2DS4 CD2, CD16, CD69, DNAX accessory molecule-1 (DNAM-1), 2B4, NK1.1; a killer immunoglobul (Ig)-likein activating receptor (KAs R); ILTs/LIRs; NKRP-1, CD69; CD94/NKG2C and CD94/NKG2E heterodimers, NKG2D homodim erKIR2DS and KIR3DS.

[00577] Therapeutic cancer vaccines may also optionall bey used in combination wit h an immunostimulatory antibody or fragment thereof according to at least some embodiments of the present invention, includin gbut not limite tod exogenous cancer and infectious agent vaccines includin gproteins or peptides used to mount an immunogenic response to a tumor antigen or an infectious agent, recombinan virust and bacteri vectorsa encoding tumor antigens, DNA-based vaccines encoding tumor antigens, proteins targeted to dendritic cells, dendritic cell-based vaccines, whole tumor cell vaccines, gene modified tumor cells expressing GM-CSF, ICOS and/or Flt3-ligand, oncolyt virusic vaccines.

[00578] Cytokines which according to at least some embodiments may be used in combination wit han immunostimulator antiybody according to at least some embodiments of the present inventio ninclude but are not limite tod one or more cytokine suchs as interferons, interleukin colonys, stimulating factors, and tumor necrosis factors such as IL-2, IL-7, IL-12, IL-15, IL-17, IL-18, IL-21, IL-23, IL-27, GM-CSF, IFNa (interferon a), IFNa-2b, IFNP, IFNy, TNF-a, TNF- and combinations thereof.

[00579] Adoptive cell transfe therar py according to at least some embodiments that may optionall bey used in combination with an immunostimulator antiy body according to at least some embodiments of the present invention include but are not limite tod an ex vivo treatment selected from expansion of the patient autologous natural lyoccurring tumor specific T cells or genetic modification of T cells to confer specificit yfor tumor antigens. 180WO 2017/009712 PCT/IB2016/001079 a. Treatment of Pathogen Infections

[00580] According to at least some embodiments, anti-HIDE 1 antibodies may optionally be used for treating infectious disease, for the same reasons that cancer can be treated: chronic infections are often characterized by varying degrees of functional impairment of virus-specific T-cel lresponses, and thi sdefect is a principa lreason for the inability of the host to eliminat thee persisti ngpathogen. Although functional effector T cells are initiall generatedy during the early stages of infection, they gradually lose function during the course of the chronic infection as a resul oft persistent exposure to foreign antigen, giving rise to T cell exhaustion. Exhausted T cells express high level sof multiple co-inhibitory receptor suchs as CTLA-4, PD-1, and LAG3 (Crawford et al., Curr Opin Immunol. 2009;21:179-186; Kaufmann et al., J Immunol 2009;182:5891-5897, Sharpe et al., Nat Immunol 2007;8:239-245). PD-1 overexpressi byon exhausted T cells was observed clinicall iny patients suffering from chronic viral infections including HIV, HCV and HBV (Crawford et al., Curr Opin Immunol 2009;21:179-186; Kaufmann et al., J Immunol 2009;182:5891-5897, Sharpe et al., Nat Immunol 2007;8:239-245). There has been some investigation into thi spathway in additional pathogens, includin gothe virur ses bacte, ria, and parasites (Hofmeyer et al., J Biomed Biotechnol. Vol 2011, Art ID. 451694, Bhadra et al., Proc Natl. AcadSci. 2011;108(22):9196-201). For example, the PD-1 pathway was shown to be involved in controlling bacteria infecl tion using a sepsis model induced by the standard cecal ligation and puncture method. The absence of PD-1 in knockout mice protect fromed sepsis-induce deatd hin thi smodel (Huang et al., PNAS 2009: 106; 6303-6308).

[00581] T cell exhaustion can be reverse byd blocking co-inhibitory pathways such as PD-1 or CTLA-4 (Rivas et al., J Immunol. 2009 ;183:4284-91; Golden-Mason et al., J Virol. 2009;83:9122-30; Hofmeyer et al., J Biomed Biotechnol. Vol 2011, Art. ID 451694), thus allowin restog rati ofon anti-viral immune function. The therapeutic potential of co-inhibition blockade for treating vira linfection was extensively studied by blocking the PD-1/PD-L1 pathway, which was shown to be efficacious in several animal models of infection includin g acute and chronic simian immunodeficienc yvirus (SIV) infection in rhesu macas ques (Valu et al., Nature 2009;458:206-210) and in mouse model ofs chronic viral infection, such as lymphocyt choriic omeningi tiviruss (LCMV) (Barber et al., Nature. 2006;439:682-7), and 181WO 2017/009712 PCT/IB2016/001079 Theiler’s murine encephalomyeli tiviruss (TMEV) model in SJL/J mice (Duncan and Miller PLoS One. 201 l;6:e l8548). In thes modele PD-1/s PD-L1 blockade improved anti-viral respons esand promot edclearance of the persisti ngviruses. In addition, PD-1/PD-L1 blockade increased the humoral immunity manifested as elevated production of specific anti - virus antibodie ins the plasma, whic hin combination wit hthe improved cellula responsesr leads to decrease in plasma viral loads and increased survival.

[00582] As used herein the term “infectious disorder and/or disease” and/or “infection” used interchangeably, includes any disorder, disease and/or condition caused by presence and/or growth of pathogeni biolc ogica agentl in an individual host organism As. used herein the term “infection” comprises the disorder disease, and/or condition as above, exhibiting clinicall evidy ent illness Q.e., characteristi medic cal signs and/or symptom ofs disease) and/or which is asymtomat foric much or all of it course. As used herei nthe term “infection” also comprises disorder, disease and/or condition caused by persistenc ofe foreign antigen that lead to exhaustion T cell phenotype characterize byd impaired functionali tywhic his manifeste das reduced proliferation and cytokine production. As used herein the term “infectious disorder and/or disease” and/or “infection”, furth erincludes any of the below listed infectious disorders, diseases and/or conditions, caused by a bacteria infectil on, viral infection, fungal infection and /or parasit infecte ion.

[00583] Anti-HIDE 1 antibodie cans be administered alone or in combination wit hone or more additional therapeutic agents used for treatment of bacteria infectl ions, viral infection, fungal infections, optionall asy described herein.

[00584] That is, an infected subject is administered an anti-HIDEl antibodie thats antagonize sat least one HIDE1 mediated effect on immunity, e.g., its inhibitory effect on myeloid cell function, cytotoxic T cells or NK activit yand/or its inhibitor effey ct on the production of proinflammatory cytokine s,or inhibits the stimulatory effect of HIDE 1 on TRegs thereby prompting the depletion or killi ngof the infected cells or the pathogen, and potential resully ting in disease remission based on enhanced killi ngof the pathogen or infected cells by the subject’s immune cells. 182WO 2017/009712 PCT/IB2016/001079

[00585] In at least some embodiments, the present inventio nprovides at leas ton of the HIDE1-specific immunostimulator antiboy dy, antigen-binding fragment or conjugat eor compositi oncontaining according to at leas tsome embodiments of the present invention, pharmaceutic alcompositions and/or, uses thereof for treatment and/or diagnos isof infectious disease, wherei saidn infectious disease is e.g, a disease caused by bacterium virus, ,fungus or yeast, my coplasm or a parasite or sepsis associate thed rewith.

[00586] As used herein the term “vira linfection” comprises any infection caused by a virus ,optional includingly but not limited to Retroviridae (e.g, human immunodeficiency viruses, such as HIV-1 or HIV-2, acquired immune deficiency (AIDS) also referre tod as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolat es,such as HIV-LP; Picomaviridae (e.g, poli oviruses hepatiti, As virus ;enteroviruses human, coxsacki eviruses, rhinoviruses, echoviruses Calci); viridae (e.g., strain thats cause gastroenteri tis);Togaviridae (e.g., equine encephalitis viruses, rubell virusa es); Flaviridae (e.g., dengue viruses , encephalit viruses,is yellow fever viruses); Coronaviridae (e.g., coronaviruses) ; Rhabdoviridae (e.g., vesicula rstomati tiviruses,s rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses mum, ps virus ,measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses );Bungaviridae (e.g., Hantaan viruses, bunga viruses phle, boviruses and Nairo viruses); Arena viridae (hemorrha gicfever virus); Reoviridae (e.g., reoviruses orbivir, uses and rotaviruses) ; Birnaviridae׳, Hepadnaviridae (Hepatiti Bs virus); Parvoviridae (parvoviruses ); Papovaviridae (papilloma viruses polyom, virusesa );Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoste virusr ,cytomegalovirus (CMV), herpes viruses); Poxviridae (variol virusesa vaccinia, viruses pox, viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathi thees, agent of delt hepatia tides (thought to be a defective satell itofe hepatiti Bs virus), the agents of non-A, non-B hepatitis (class 1— internally transmitted; class 2—parenteral transmly it ted(i.e., Hepatitis C); Norwalk and related viruses and, astroviruses) as well as Severe acute respiratory syndrome virus and respiratory syncytial virus (RSV). 183WO 2017/009712 PCT/IB2016/001079

[00587] As used herein the term “fungal infection” comprises any infection caused by a fungus, optionally includin gbut not limited to Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.

[00588] As used herein the term “parasite infection” compris esany infection caused by a parasit e,optional includingly but not limite tod protozoa such, as Amebae, Flagellates, Plasmodium falciparum, Toxoplasma gondii, Ciliates, Coccidia, Microsporidia, Sporozoa; helminthes. Nematode (Roundworms s),Cestode (Tapews orms) Trema, todes (Flukes) , Arthropods, and aberrant proteins known as prions.

[00589] An infectious disorder and/or disease caused by bacteri maya optional ly comprise one or more of Sepsis, septi cshock, sinusiti s,skin infections, pneumonia, bronchiti meningis, tis Bacte, rial vaginosis, Urinary trac infect tion (UCI), Bacteria l gastroenterit Impis,etigo and erysipela s,Erysipelas, Cellulit antis, hrax whooping, cough, lyme disease, Brucellosis enteriti, acutes, enteriti Tetas, nus, diphtheria Pseudo, membranous coliti Gass, gangrene, Acute food poisoning, Anaerobic cellulit Nosocomis, ial infections, Diarrhea, Meningitis in infants, Traveller' diarrs hea, Hemorrhagi colic ti Hems, olytic-uremic syndrome, Tularemia Peptic, ulcer, Gastric and Duodena lulcers, Legionnaire's Disease, Pontiac fever, Leptospirosis List, eriosi Leprosys, (Hansen's disease) Tubercul, osis , Gonorrhea, Ophthalmia neonatorum Septi, carthriti Meningococcs, al disease includin g meningitis, Waterhouse-Friderichsen syndrome, pseudomonas infection, Rocky Mountain spott edfever, Typhoid fever type salmonellosis, Salmonellos withis gastroenterit and is enterocoli Bacitis, llary dysentery/Shigell osiCoagulass, e-positive staphylococcal infections: Localized skin infections includin gDiffuse skin infection (Impetigo ),Deep localized infections, Acute infective endocarditi Septis, cemia, Necrotizi ngpneumonia, Toxinoses such as Toxic shock syndrome and Staphylococcal food poisoning, Cystitis, Endometrit Otitis, is media ,Streptococcal pharyngitis Scarl, et fever, Rheumatic fever, Puerperal fever, Necrotizi ngfasciitis, Cholera Plagu, e (including Bubonic plague and Pneumonic plague), as well as any infection caused by a bacteri sela ected from but not limite tod Helicobacter pyloris, Boreliai burgdorferi, Legionella pneumophila, Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. Intracellulare, M. kansaii, M gordonae), Staphylococcus aureus, 184WO 2017/009712 PCT/IB2016/001079 Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococc us), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, corynebacterium diphtheriae, corynebacterium sp., Erysipelothrixrhusiopathiae, Clostridiumperfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, and Actinomyces israelii.

[00590] Non limiting examples of infectious disorder and/or disease caused by virus is selected from the group consisting of but not limited to acquired immune deficiency (AIDS), West Nile encephalit is,coronavir usinfection, rhinovirus infection, influenza, dengue, hemorrha gicfever; an otologica infectil on; severe acute respiratory syndrome (SAKS), acute febrile pharyngit is,pharyngoconjuncti valfever, epidemic keratoconjunctivit infantis, il e gastroenterit infecis,tious mononucleosi Burkitts, lymphoma, acute hepatit is,chronic hepatit is,hepatic cirrhosis hepat, ocellular carcinoma, primary HSV-1 infection, (gingivostomat itin ischildren, tonsilli &tis pharyngiti ins adult s,keratoconjunctiviti lates),nt HSV-1 infection (herpes labialis cold, sores ),aseptic meningitis, Cytomegalovirus infection, Cytomegali incluc sion disease, Kaposi sarcoma Castlem, an disease, primary effusion lymphoma, influenza, measles, encephaliti posts, infectious encephalomyelit mumps,is, hyperplast epitic heli lesial ons (common, flat, plantar and anogenital wart s,laryngea l papillomas, epidermodysplasia verruciform is),croup, pneumonia, bronchioliti s, Poliomyelit Rabieis, s, bronchioli tipneumonia,s, German measles, congenita rubelll a, Hemorrhagi Fever,c Chickenpox, Dengue, Ebola infection, Echovirus infection, EBV infection, Fifth Disease, Filovirus, Flavivirus, Hand, foot and mout hdisease, Herpes Zoster Virus (Shingles), Human Papilloma Virus Associate Epidd ermal Lesions, Lassa fever, Lymphocytic choriomeningitis, Parainfluenza Virus Infection, Paramyxovirus, Parvovirus Bl9 Infection, Picomavirus, Poxviruses infection, Rotavirus diarrhea, Rubella, Rubeola, Varicella, Variola infection. 185WO 2017/009712 PCT/IB2016/001079

[00591] An infectious disorder and/or disease caused by fungi optionally includes but is not limited to allergi bronchoc pulmonary aspergillos Aspeis, rgillom Aspea, rgillosi s, Basidiobolomycos Blais,stomycosi Candidias, sis, Chroni pulmc onary aspergillosis , Chytridiomycosis, Coccidioidomycosis Conid, iobolomycosis, covered smut (barley), Cryptococcosi Dermas, tophy te,Dermatophyt Dermid, atophytosis Endothrix,, Entomopathogenic fungus, Epizootic lymphangiti Epizos, otic ulcerati vesyndrom e, Esophageal candidiasi s,Exothrix, Fungemia, Histoplasmosis, Lobomycosi s,Massospora cicadina, Mycosis, Mycosphaerell fragariaea Myrin, gomy cosis Paracoc, cidioidomycosi s, Pathogenic fungi, Penicilliosi Thousands, cankers disease, Tinea, Zeaspora Zygomy, cosis .

Non limiting examples of infectious disorder and/or disease caused by parasit esis selected from the group consisting of but not limited to Acanthamoeba, Amoebiasis, Ascariasis, Ancylostomias Aniis, sakiasi s,Babesiosi s,Balantidias is,Baylisascariasi Blasts, ocystosi s, Candiru, Chagas disease, Clonorchia sisCochli, omyia, Coccidia, Chinese Liver Fluke Cryptosporidiosi Dientams, oebiasis, Diphyllobothrias Dioctis, ophyme renalis infection, Dracunculiasis, Echinococcosi Eles, phantiasis Enterobia, sis Fasc, iolias is,Fasciolopsiasi s, Filarias is,Giardiasis Gnat, hostomias Hymenolis, epiasis Halzoun, Syndrome, Isosporiasis , Katayama fever, Leishmaniasi s,lymphati filc ariasis, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Primary amoebic meningoencephalit Parasis, itic pneumonia, Paragonimiasis, Scabies, Schistosomiasi Sleeps, ing sickness, Strongyloidias Sparis, ganosis, Rhinosporidiosis, River blindness Taenia, sis (cause of Cysticercosis), Toxocariasis, Toxoplasmos Trichiis, nosi Trics, homoniasi Trichurs, iasi Trypanosos, miasis, and Tapeworm infection.

[00592] Some optiona butl particular examples of infectious disease include a disease caused by any of hepatit B,is hepatitis C, infectious mononucleosi EBVs, , cytomegalovir us, AIDS, HIV-1, HIV-2, tuberculosi mals, aria and schistosomiasis.

[00593] The therapeutic agents and/or a pharmaceutical compositi oncomprising same, as recited herein, can be administered in combination wit hone or more additional therapeutic agents used for treatment of bacteria infecl tions, including, but not limited to, antibiotics including Aminoglycosides Carbapenems, ,Cephalospori Macrolns, ides Lincos, amides, Nitrofurans peni, cillins, Polypeptides, Quinolones, Sulfonamides, Tetracyclines, drugs 186WO 2017/009712 PCT/IB2016/001079 against mycobacteri includinga but not limite tod Clofazimine, Cycloserine, Cycloserine, Rifabutin, Rifapentine ,Streptomyci andn other antibacteria drugsl such as Chloramphenicol , Fosfomycin, Metronidazole Mupiroci, n, and Tinidazole.

[00594] The therapeutic agents and/or a pharmaceutical compositi oncomprising same, as recited herein, can be administered in combination wit hone or more additional therapeutic agents used for treatment of viral infections, including, but not limited to, antivira drugsl such as oseltamivi (brandr name Tamiflu®) and zanamivir (brand name Relenza® )Arbidol® - adamantane derivatives (Amantadine® Rimantadi, ne®) - neuraminidase inhibito rs (Oseltamivir®, Laninamivir@, Peramivir@, Zanamivir®) nucleotide analog reverse transcripta inhise bitor includin gPurine analogue guanine (Aciclovir®/Valacyclovi r®, Ganciclovir®/Valganciclovir® Penci, clovir®/Famciclovi r®)and adenine (Vidarabine®) , Pyrimidine analogue, uridine (Idoxuridine@, Trifluridine@, Edoxudine® ),thymine (Brivudine®), cytosine (Cytarabine®) Fosca; met; nucleoside analogues/NARTIs: Entecavir, Lamivudine@, Telbivudine®, Clevudine®; nucleotide analogues/NtRTIs: Adefovir®, Tenofovir; nucleic acid inhibitors such as Cidofovir®; Interferoninterferon alfa-2b, Peginterfer ona-2a; Ribavirin®/Taribavirin®; antiretrovira drugsl includin gzidovudine, lamivudine, abacavir, lopinavir ritonavi, tenofovir/r, emtricitabine efavi,renz each of them alone or a various combinations, gp41 (Enfuvirtide), Raltegravir® protease, inhibitors such as Fosamprenavir®, Lopinavir® and Atazanavir®, Methisazone® Docosa, nol® , Fomivirsen®,and Tromantadine@.

[00595] The therapeutic agents and/or a pharmaceutical compositi oncomprising same, as recited herein, can be administered in combination wit hone or more additional therapeutic agents used for treatment of fungal infections, including, but not limite to,d antifungal drugs of the Polyene antifungals Imi. dazole, triazol ande, thiazole antifungals All. ylamines, Echinocandins or othe antir -funga ldrugs. b. Treatment of Sepsis

[00596] According to at least some embodiments, anti-HIDEl antibodies be used for treating sepsis. As used herein, the term “sepsi”s or “sepsis related condition” encompasses Sepsis, Severe sepsis ,Septi cshock, Systemic inflammator responsey syndrome (SIRS), Bacteremia, Septicemia, Toxemia, Septi csyndrome. 187WO 2017/009712 PCT/IB2016/001079

[00597] Upregulation of inhibitor proteinsy has lately emerged as one of the critica l mechanism sunderlying the immunosuppression in sepsis. The PD-l/PDL-1 pathway, for example, appears to be a determining factor of the outcome of sepsis, regulati ngthe delicate balance between effectivenes sand damage by the antimicrobia imlmune response Duri. ng sepsis in an experimental model perit, oneal macrophages and blood monocytes markedly increased PD-1 levels, which was associated with the development of cellula dysfuncr tion (Huang et al 2009 PNAS 106:6303-6308). Similarly, in patients with septic shock the expression of PD-1 on peripheral T cell sand of PDL-1 on monocytes was dramatically upregulated (Zhang et al 2011 Crit. Care 15:R70). Recent animal studie haves shown that blockade of the PD-l/PDL-1 pathway by anti-PD1 or anti-PDL1 antibodies improved survival in sepsis (Brahmamdam et al 2010 J. Leukoc. Biol. 88:233-240; Zhang et al 2010 Critical Care 14:R220; Chang et al 2013 Critica Carel 17:R85). Similarly, blockade of CTLA-4 with anti-CTLA4 antibodie improvs ed survival in sepsis (Inoue et al 2011 Shock 36:38-44; Chang et al 2013 Critical Care 17:R85). Taken together, thes findingse suggest that blockade of inhibitory proteins includin, gnegative costimulatory molecules is, a potenti theraal peutic approac hto preven tthe detriment effectsal of sepsis (Goyert and Silver, J Leuk. Biol., 88(2): 225-226, 2010).

[00598] According to some embodiments, the inventio nprovides treatment of sepsis using anti-HIDEl antibodies eit, her alone or in combination wit hknown therapeutic agent effective for treating sepsis such, as thos therapie esthat block the cytokine storm in the initial hyperinflammatory phase of sepsis and/, or wit htherapi esthat have immunostimulatory effect in order to overcome the sepsis-induced immunosuppressio phase.n

[00599] Combination with standard of care treatment fors sepsis, as recommended by the “Internationa Guidell ines for Management of Severe Sepsis and Septi cShock” (Dellinger et al 2013 Intensive Care Med 39:165-228), some of which are describe belowd .

[00600] Optionall they sepsis is selected from sepsis seve, re sepsis septic, shock, systemic inflammator responsey syndrome (SIRS), bacteremi a,septicemia toxem, ia, and septi csyndrome. 188WO 2017/009712 PCT/IB2016/001079

[00601] Optionall they treatment is combined with another moiety useful for treating sepsis.

[00602] According to at least some embodiments there is provided a diagnosti metc hod for determining whether to perfor mthe use or to administ eran antibody compositi onas described herein, comprising performing the diagnosti metc hod as described herein.

[00603] According to at least some embodiments of the present invention, there is provided use of a combination of the therapeutic agents and/or a pharmaceutical composition comprising same, as recited herein, can be combined with standard of care or novel treatments for sepsis with, therapies that block the cytokine storm in the initial hyperinflammatory phase of sepsis and/, or wit htherapi esthat have immunostimulatory effect in order to overcome the sepsis-induced immunosuppressio phase.n

[00604] Combination with standard of care treatment fors sepsis, as recommended by the “Internationa Guidell ines for Management of Severe Sepsis and Septi cShock” (Dellinger et al 2013 Intensive Care Med 39:165-228), some of which are describe belowd .

[00605] Broad spectrum antibiotics having activity against all likely pathogens (bacteria and/orl fungal - treatment starts when sepsis is diagnosed, but specific pathogen is not identified) - example Cefotaxime (Claforan@), Ticarcill andin clavulanate (Timentin®), Piperacillin and tazobacta m(Zosyn®), Imipenem and cilastat (Priin maxin®), Meropenem (Merrem®), Clindamycin (Cleocin), Metronidazole (Flagyl®), Ceftriaxone (Rocephin®), Ciprofloxacin (Cipro®), Cefepime (Maxipime®), Levofloxacin (Levaquin@), Vancomycin or any combination of the listed drugs.

[00606] Vasopressors: example Norepinephrine, Dopamine, Epinephrine, vasopressin

[00607] Steroids: example: Hydrocortis one,Dexamethasone or, Fludrocortisone, intravenous or otherwise

[00608] Inotropic therapy example: Dobutamine for sepsis patients wit hmyocardi al dysfunction

[00609] Recombinant human activated prote inC (rhAPC), such as drotrecogi alfan (activated) (DrotAA). 189WO 2017/009712 PCT/IB2016/001079

[00610] -blockers additionally reduce local and systemi inflac mmation.

[00611] Metabol icinterventions such as pyruvate, succinat eor high dose insulin substitutions.

[00612] Combination with novel potential therapi esfor sepsis:

[00613] Selective inhibitors of sPLA2-IIA (such as LY315920NA/S-5920). Rational e: The Group IIA secretory phospholipase A2 (sPLA2-IIA), released during inflammation, is increased in severe sepsis, and plasma level sare inversely related to survival.

[00614] Phospholipid emulsion (such as GR270773). Rationale: Preclinical and ex vivo studies show that lipoprote bindins and neutraliz endotoxin,e and experimental animal studie demonsts rate protecti fromon septi cdeat hwhen lipoprotei arens administered.

Endotoxin neutralizati correlon ates wit hthe amount of phospholipi in dthe lipoprote in particles.

[00615] anti-TNF-a antibod y:Rationale: Tumor necrosi factor-as (TNF-a) induces many of the pathophysiological signs and symptom observeds in sepsis

[00616] anti-CD14 antibody (such as IC14). Rationale: Upstrea mrecognition molecules like, CD 14, play key role ins the pathogenesi Bactes. rial cel lwall components bind to CD14 and co-receptors on myeloid cells, resulti ngin cellula actr ivation and production of proinflammatory mediators. An anti-CD14 monoclonal antibody (IC 14) has been shown to decrease lipopolysaccharide-ind responsesuced in animal and human model ofs endotoxemia..

[00617] Inhibitors of Toll-like receptor (TLRs)s and their downstream signaling pathways. Rationale: Infecting microbes displa highlyy conserved macromolecules (e.g., lipopolysaccharide peptidoglycans)s, on their surface .When thes macrome olecules are recognized by pattern-recognit receptorion (calls ed Toll-like receptor [TLRs])s on the surface of immune cell s,the host’s immune response is initiated. This may optionally contribut toe the excess systemic inflammator responsey that characterizes sepsis. Inhibition of several TLRs is being evaluated as a potential therapy for sepsis, in particular TLR4, the receptor for Gram-negative bacteri outera membrane lipopolysacchar oride endotoxi n.Various drugs 190WO 2017/009712 PCT/IB2016/001079 targeting TLR4 expression and pathway have a therapeuti potentc ial in sepsis (Wittebole et al 2010 Mediators of Inflammation Vol 10 Article ID 568396). Among thes aree antibodies targeting TLR4, solubl TLR4,e Statins (such as Rosuvastatin®, Simvastatin® Ket), amine, nicotini canalogues, eritoran (E5564), resatorvid (TAK242). In addition, antagonis tsof othe r TLRs such as chloroqui ne,inhibition of TLR-2 wit ha neutralizi ngantibody (anti-TLR-2).

[00618] Lansoprazole through its action on SOCS1 (suppress orof cytokine secretion)

[00619] Talactofer rinor Recombinant Human Lactoferrin. Rationale: Lactoferrin is a glycoprotein with anti-infective and anti-inflammatory propertie founds in secretions and immune cells. Talactoferrin alfa, a recombinan formt of human lactoferr hasin, similar propertie ands plays an important role in maintaining the gastrointest mucoinal sal barrie r integrit y.Talactoferrin showed efficacy in animal model ofs sepsis, and in clinical trials in patients wit hsevere sepsis (Guntupal liet al Crit Care Med. 2013;41(3):706-716).

[00620] Milk fat globule EGF factor VIII (MFG-E8) - a bridging molecul betwe een apoptot cellic sand phagocytes, which promotes phagocytosi ofs apoptoti celc ls.

[00621] Agonist ofs the ‘cholinergic anti-inflammatory pathway’, such as nicotine and analogues. Rationale: Stimulating the vagus nerve reduces the production of cytokine s,or immune system mediators, and blocks inflammation. This nerve “circuitry”, called the “inflammator refly ex”, is carrie dout through the specific action of acetylcholi releasedne, from the nerve endings, on the a7 subunit of the nicotini cacetylcholi receptne or (a7n AChR) expressed on macrophages, a mechanism termed ‘the cholinergic anti-inflammatory pathwa’.y Activation of thi spathway via vagus nerve stimulation or pharmacolog a7ic agonists prevents tissue injury in multiple model ofs systemi inflc ammation, shock, and sepsis (Matsuda et al 2012 J Nippon Med Sch,19A-l%; Huston 2012 Surg. Infect. 13:187- 193).

[00622] Therapeutic agents targeting the inflammasome pathways. Rationale: The inflammasome pathways greatl conty ribut toe the inflammatory response in sepsis and, critical element sare responsibl fore driving the transition from localized inflammatio ton deleterious hyperinflammator hosty response (Cinel and Opal 2009 Crit. Care Med. 37:291- 304; Matsuda et al 2012 J Nippon Med Sc/2.79:4-18). 191WO 2017/009712 PCT/IB2016/001079

[00623] Stem cel ltherap y.Rationale: Mesenchymal stem cell s(MSCs) exhibi t multiple beneficial propertie throughs their capacity to home to injured tissue, activate resident stem cells, secrete paracrine signals to limi tsystemi cand local inflammatory response, beneficially modulate immune cell s,promote tissue healing by decreasing apoptosis in threatened tissues and stimulating neoangiogenesis, and exhibit direc t antimicrobial activity. These effects are associated wit hreduced organ dysfunction and improved survival in sepsi sanimal models, whic hhave provided evidence that MSCs may optionally be useful therapeutic adjunct s(Wannemuehler et al 2012 J. Surg. Res. 173:113- 26).

[00624] Combination of anti-HIDEl antibody with other immunomodulat oryagents , such as immunostimulatory antibodies cytokine, therapy, immunomodulatory drugs. Such agents bring about increased immune responsiveness, especially in situations in whic h immune defenses (whethe innatr e and/or adaptive) have been degraded, such as in sepsis- induced hypoinflammatory and immunosuppressive condition. Reversal of sepsis-induced immunoparalysis by therapeutic agents that augments host immunity may optionally reduce the incidence of secondary infections and improve outcom ine patients who have documente d immune suppression (Hotchkiss et al 2013 Lancet Infect. Dis. 13:260-268; Payen et al 2013 Crit Care. 17:118).

[00625] Immunostimulat oryantibodie promots imme une responses by directly modulating immune functions i., e. blocking other inhibitory proteins or by enhancing costimulatory proteins. Experiment almodels of sepsis have shown that immunostimulat ion by antibody blockade of inhibitor proteinsy such, as PD-1, PDL-1 or CTLA-4 improved survival in sepsis (Brahmamdam et al 2010 J. Leukoc. Biol. 88:233-240; Zhang et al 2010 Critical Care 14:R220; Chang et al 2013 Critical Care 17:R85; Inoue et al 2011 Shock 36:38-44), pointing to such immunostimulatory agents as potenti therapiesal for preventing the detriment effectal s of sepsis-induced immunosuppression (Goyert and Silver J Leuk. Biol. 88(2):225-226, 2010). Immunostimulat oryantibodie incls ude: 1) Antagonistic antibodies targeting inhibitory immune checkpoints include anti-CTLA4 mAbs (such as ipilimumab, tremelimumab), Anti-PD-1 (such as nivolumab BMS-936558/ MDX-1106/ONO-4538, AMP224, CT-011, lambrozilum MK-3475),ab Anti-PDL-1 antagonist (suchs as BMS- 192WO 2017/009712 PCT/IB2016/001079 936559/ MDX-1105, MEDI4736, RG-7446/MPDL3280A); Anti-LAG-3 such as IMP-321), Anti-TIM-3, Anti-BTLA, Anti-B7-H4, Anti-B7-H3, anti-VISTA. 2) Agonisti antibodiesc enhancing immunostimulatory proteins include Anti-CD40 mAbs (such as CP-870,893, lucatumumab, dacetuzumab), Anti-CD137 mAbs (such as BMS-663513 urelumab, PF- 05082566), Anti-OX40 mAbs (such as Anti-OX40) ,Anti-GITR mAbs (such as TRX518), Anti-CD27 mAbs (such as CDX-1127), and Anti-ICOS mAbs.

[00626] Cytokin eswhic hdirectly stimula teimmune effector cells and enhance immune responses can be used in combination wit hanti-GEN antibody for sepsis therapy: IL-2, IL-7, IL-12, IL-15, IL-17, IL-18 and IL-21, IL-23, IL-27, GM-CSF, IFNa (interferon a), IFNp, IFNy. Rationale: Cytokine-based therapi esembody a direc attemptt to stimula tethe patien’ts own immune system Experime. ntal model ofs sepsis have shown administration of cytokines, such as IL-7 and IL-15, promote T cell viability and resul int improved survival in sepsis (Unsinger et al 2010 J. Immunol. 184:3768-3779; Inoue et al 2010 J. Immunol. 184:1401-1409). Interferon-Y (IFNy) reverses sepsis-induced immunoparalysis of monocytes in vitro. An in vivo study showed that IFNy partial reversesly immunoparalysis in vivo in humans. IFNy and granulocyte-macrophag colone y-stimulatin factgor (GM-CSF) restore immune competence of ex vivo stimulated leukocyte ofs patients with sepsi s(Mouktaroudi et al Crit Care. 2010; 14: P17; Leentjens et al Am J Respir Grit Care MedN 01 186, pp 838-845, 2012).

[00627] Immunomodulatory drugs such as thymosin al. Rationale: Thymosin a 1 (Tai) is a naturall occurringy thymi cpeptide whic hacts as an endogenous regulat orof both the innate and adaptive immune systems It. is used worldwi forde treating diseases associate d with immune dysfunction including viral infections such as hepatiti Bs and C, certai n cancers, and for vaccine enhancement. Notabl y,recent development in immunomodulat ory research has indicate dthe beneficial effect of Tai treatme ntin septi cpatients (Wu et al.

Critical Care 2013, 17:R8).

[00628] In the above-described sepsis therapies preferably a subject with sepsis or at risk of developing sepsis because of a virulent infection, e.g., one resistant to antibiotics or othe drugs,r wil bel administered an immunostimulatory anti-HIDE 1 antibody or antigen- binding fragment according to at least some embodiments of the present invention, which 193WO 2017/009712 PCT/IB2016/001079 antibody antagonizes at leas tone HIDE1 mediated effect on immunity, e.g., its inhibito ry effect on myeloid cel lfunction, cytotoxic T cell sor NK activity and/or its inhibitory effect on the production of proinflammatory cytokines, or inhibits the stimulatory effect of HIDE1 on TRegs thereby promoting the depletion or killi ngof the infected cells or the pathogen, and potential resully ting in disease remission based on enhanced killi ngof the pathogen or infected cells by the subject’s endogenous immune cells. Because sepsis may optional ly rapidly resul int organ failure, in thi sembodiment it may optionall bey beneficial to administe antir -HIDE 1 antibody fragments such as Fabs rathe thanr intact antibodie ass they may optionall reachy the sit eof sepsis and infection quicker than intact antibodies. In such treatment regimens antibody half-lif maye optionall bey of lesse concernr due to the sometimes rapid morbidity of thi sdisease. c. Treatment of Autoimmune Diseases

[00629] According to at least some embodiments, HIDE1 therapeutic agents and/or a pharmaceutic alcompositi oncomprising same, as described herein, which function as HIDE1 agonizing therapeutic agents ,may optionally be used for treating an immune system related disease In. some instances the immune system related condition comprises an immune relat ed condition, including but not limite tod autoimmune, inflammatory or allergic diseases such as recited herein, transplant rejection and graft versus host disease.

[00630] In some instances the immune condition is selected from autoimmune disease, inflammatory disease, allergi disease,c transplant rejection, undesired gene or cel ltherapy immune responses, or graft versus host disease.

[00631] In some embodiments the treatme ntis combined with another moiety useful for treating immune related condition. Non limiting examples thereof include immunosuppressa ntssuch as corticosteroi cyclds,ospori cyclophosphamin, prednisone,de, azathioprine, methotrexate rapam, ycin, tacrolim us,leflunomide or an analog thereof; mizoribine; mycophenol acid;ic my cophenolat mofetie l;15-deoxyspergual ineor an analog thereof; biologic alagents such as TNF-a blockers or antagonist ors, any other biological agent targeting any inflammatory cytokine, nonsteroidal antiinflammatory drugs/Cox-2 inhibitors, hydroxychloroquine, sulphasalazopryine, gold salts, etanercept, infliximab, my cophenola mofetite l,basiliximab, atacicept, rituximab, cytoxan, interferon [3-1 a, interferon 194WO 2017/009712 PCT/IB2016/001079 P־lb, glatiram aceer tate, mitoxantro hydrochloride,ne anakinra and/or othe biologir esand/or intravenous immunoglobul (IVIGin ), interferons such as IFN־p1a (REBIF®. AVONEX® and CINNOVEX ®) and IFN-pib (BETASERON®); EXTAVIA®, BETAFERON, ZIFERON®); glatiram aceer tat e(COPAXONE®), a polypeptide; natalizumab (TYSABRI®), mitoxantrone (NOVANTRONEG). a cytotoxic agent, a calcineurin inhibitor, e.g. Cyclospori An or FK506; an immunosuppressive macrolide, e.g. Rapamycin or a derivati vethereof; e.g. 40-O-(2-hydroxy)ethyl-rapamyc a lympin, hocyte homing agent, e.g.

FTY720 or an analog thereof, corticosteroi cyclods; phospham ide;azathioprene ; methotrexate; leflunomi deor an analog thereof; mizoribine; mycophenol aciic d; my cophenolat mofetie l;15-deoxy sperguali neor an analog thereof; immunosuppressive monoclonal antibodies e.g.,, monoclonal antibodie tos leukocyte receptors, e.g, MHC, CD2, CD3, CD4, CDlla/CD18, CD7, CD25, CD27, B7, CD40, CD45, CD58, CD137, ICOS, CD150 (SLAM), 0X40, 4-1BB or their ligands; or other immunomodulat orycompounds , e.g. CTLA4-Ig (abatacept, ORENCIA®, belatacept) CD28-Ig, , B7-H4-Ig, or othe r costimulatory agents, or adhesion molecul inhie bitors, e.g. mAbs or low molecular weight inhibitors includin gLFA-1 antagonist Selects, in antagonis tsand VLA-4 antagonist ors, another immunomodulat oryagent.

[00632] In particular, treatment of multiple sclerosi usings HIDE1 immunoinhibitory proteins according to some embodiments of the present inventio nmay optional e.g,ly be combined with, any therapeutic agent or method suitable for treating multiple sclerosis Non-. limitin examg ples of such known therapeutic agent or method for treating multiple scleros is include interferon class, IFN־P־la (REBIF®. AVONEX® and CINNOVEX ®) and IFN־p־lb (BETASERON®, EXTAVIA®, BETAFERON®, ZIFERON®); glatiramer acetate (COPAXONE®), a polypeptide; natalizuma b(TYSABRI®); and mitoxantrone (NOVANTRONE®), a cytotoxic agent, Fampridine (AMPYRA®). Other drugs include corticosteroi metds,hotrexate, cyclophosphamide aza,thioprine, and intravenous immunoglobul (IVIin G), inosine, Ocrelizuma b(RI 594), Mylinax (Caldribine® ), alemtuzuma b(Campath®), daclizuma b(Zenapax®), Panaclar/ dimethy fumal rate (BG-12), Teriflunomi de(HMR1726), fmgolimod (FTY720), laquinimod (ABR216062), as well as Hematopoietic stem cell transplantat NeuroVion, ax®, Rituximab (Rituxan@) BCG vaccine, 195WO 2017/009712 PCT/IB2016/001079 low dose naltrexone, helminthic therapy, angioplasty, venous stent s,and alternat ivetherap y, such as vitamin D, polyunsaturate fatsd ,medical marijuana.

[00633] Similarl treatmy, ent of rheumatoid arthri tiusings, HIDE1 immunoinhibitory proteins according to some embodiments of the present inventio nmay optionally be combined with, for example, any therapeutic agent or method suitable for treating rheumatoid arthri tis.Non-limiti ngexamples of such known therapeutic agents or methods for treati ng rheumatoid arthr itiincludes glucocortico ids,nonsteroidal anti-inflammatory drug (NSAID) such as salicylates or, cyclooxygenase-2 inhibitors, ibuprofen and naproxen, diclofenac , indomethacin, etodolac Disease-modifying antirheumat drugsic (DMARDs)- Ora lDMARDs: Auranofin (Ridaura@), Azathiopri (Imurne an®), Cyclospor ine(Sandimmune®, Gengraf, Neoral, generic), D-Penicillamin (Cuprime ine), Hydroxychloroqu (Plaqueine nil® ),IM gold Gold sodium thiomal ate(Myochrysine@) Aurothiogluc (Solganaose l@), Leflunomide (Arava®), Methotrexate (Rheumatrex®), Minocycline (Minocin®), Staphylococcal prote inA immunoadsorption (Prosor column),ba Sulfasalazine (Azulfidine@ ).Biologic DMARDs: TNF-a blockers including Adalimumab (Humira®) Etanercept (Enbrel® ),Infliximab (Remicade®) ,golimumab (Simponi®), certolizuma pegolb (Cimzia@), and other biological DMARDs, such as Anakinra (Kineret®), Rituximab (Rituxan@), Tocilizumab (Actemra@ ), CD28 inhibitor includin gAbatacept (Orencia®) and Belatacept.

[00634] Thus, treatment of IBD, using the agents according to at leas tsome embodiments of the present inventio nmay optionally be combined with, for example, any known therapeutic agent or method for treating IBD. Non-limiti ngexamples of such known therapeutic agents or methods for treating IBD include immunosuppressio to ncontrol the symptom, such as prednisone, Mesalazine (including Asacol® Pentasa®, ,Lialda®, Aspiro®, azathioprine (Imuran®), methotrexate, or 6-mercaptopuri ne,steroids, Ondansetron®, TNF-a blockers (including infliximab, adalimumab golimumab, certolizuma pegol),b Orencia® (abatacept), ustekinumab (Stelara®), Briakinuma b(ABT-874), Certolizuma pegolb (Cimzia®), ITF2357 (Givinostat® Nat), alizumab (Tysabri®), Firategrast® (SB-683699), Remicade® (infliximab), vedolizumab (MLN0002), othe drugsr including GSK1605786 CCX282-B (Traficet-EN®), AJM300, Stelara® (ustekinumab), Semapimod® (CNI-1493) tasocitinib (CP-690550), LMW Heparin MMX, Budesonide MMX, Simponi® (golimumab), 196WO 2017/009712 PCT/IB2016/001079 MultiStem ®,Gardasil® HPV vaccine, Epaxal® (virosomal hepatitis A vaccine), surgery, such as bowe lresection, stricturepla orsty a temporary or permanent colostomy or ileostomy; antifungal drugs such as nystat in(a broad spectrum gut antifungal and) either itraconazole (Sporanox) or fluconazole (Diflucan); alternat ivemedicine, prebiotic ands probiotics, cannabis, Helminthic therapy or ova of the Trichuris suis helminth.

[00635] Thus, treatment of psoriasi usings, the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating psoriasi Non-ls. imiti ngexamples of such known therapeuti forcs treating psorias inclis ude topical agents ,typically used for mild disease, phototherap fory moderat disease,e and systemi cagents for severe disease Non-. limitin examg ples of topica agentsl :bath solutions and moisturizers, mineral oil, and petroleum jelly; ointment and creams containing coal tar, dithranol (anthralin), corticoster oids like desoximetasone (Topicort), Betamethasone, fluocinonide, vitamin D3 analogues (for example, calcipotri ol),and retinoids. Non-limiti ngexamples of photothera sunlight;py: wavelength ofs 311-313 nm, psoral enand ultraviolet A phototherap (PUVAy ). Non-limiting examples of systemic agents :biologies, such as interleuki antagonistn TNF-as, blockers including antibodies such as infliximab (Remicade®), adalimumab (Humira®), golimumab, certolizumab pegol and, recombinan TNF-at decoy receptor, etanercept (Enbrel®); drugs that target T cells, such as efalizumab (Xannelim®/Raptiva®), alefacept (Ameviv®), dendritic cells such Efalizumab ;monoclonal antibodie (MAbss ) targeting cytokine s,including anti -IL- 12/IL-23 (ustekinumab (Stelara®)) and anti-Interleukin-17; Briakinumab® (ABT-874); small molecules, includin gbut not limite tod ISA247; immunosuppressants, such as methotrexat e, cyclosporine; vitamin A and retinoid (synthes tic forms of vitamin A); and alternat ivetherapy, such as changes in diet and lifestyle fasti, ng periods, low energy diet sand vegetarian diets , diet ssupplemented wit hfish oil rich in vitamin A and vitamin D (such as cod liver oil ),fish oil srich in the two omega-3 fatty acids eicosapentaenoi acic d (EPA) and docosahexaenoi c acid (DHA) and contain vitamin E, ichthyotherapy, hypnotherapy, and cannabis.

[00636] Thus, treatment of type 1 diabetes using, the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating type !diabetes. Non-limiti ngexamples of 197WO 2017/009712 PCT/IB2016/001079 such known therapeuti forcs treating type 1 diabetes include insulin, insulin analogs isl, et transplantat ion,stem cel ltherapy including PROCHYMAL®, non-insuli therapiesn such as il-1p inhibitors including Anakinra (Kineret ),Abatacept (Orencia®), Diamyd, alefacept (Ameviv®), Otelixizumab, DiaPep277 (Hsp60 derive dpeptide), a 1-Antitrypsi Prednin, sone, azathioprine, and Cyclospori El-INn, T (an injectabl islete neogenesi stherapy comprising an epidermal growt facth or analog and a gastrin analog), statins includin gZocor®, Simlup@, Simcard®, Simvacor®, and Sitagliptin® (dipeptidyl peptidase (DPP-4) inhibitor), anti-CD3 mAh (e.g., Teplizumab®); CTLA4-Ig (abatacept), anti-IL-1p (Canakinumab), Anti-CD20 mAh (e. g, rituximab) and combinations thereof.

[00637] Thus, treatment of uveitis, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating uveitis. Non-limiti ngexamples of such known therapeuti forcs treating uveitis include corticosteroids topic, al cycloplegics such, as atropine or homatropine, or injection of PSTTA (posteri subtenonor triamcinolone acetate ), antimetaboli medite cations, such as methotrexat TNFe, -a blockers (including infliximab, adalimumab, etanercept, golimumab, and certolizumab pegol).

[00638] Thus, treatment for Sjogren’s syndrome, using the agents according to at least some embodiments of the present inventio nmay optionall bey combined with, for example, any known therapeutic agent or method for treating for Sjogren’s syndrome. Non-limiting examples of such known therapeuti forcs treating for Sjogren’s syndrome include Cyclosporine, pilocarpine (Salagen@) and cevimeline (Evoxac®), Hydroxychloroquine (Plaquenil), cortisone (prednisone and others) and/or azathioprine (Imuran®) or cyclophosphami (Cytoxade n®), Dexamethasone, Thalidomide, Dehydroepiandrosterone, NGX267, Rebamipide@, FID 114657, Etanercept® Rapti, va®, Belimumab, MabThera® (rituximab); Anakinra® int, ravenous immune globulin (IVIG), Allogenei c Mesenchymal Stem Cell s(A110MSC®), and Automat icneuro-electrostimulat by "Saionliwel l Crow”n.

[00639] Thus, treatment for systemi clupus erythematosus, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating for systemic lupus 198WO 2017/009712 PCT/IB2016/001079 erythematosus. Non-limiti ngexamples of such known therapeuti forcs treating for systemic lupus erythematosus include corticosteroid and Dises ase-modifying antirheumat drugsic (DMARDs), commonly anti-malarial drugs such as plaqueni land immunosuppressa nts(e.g. methotrexate and azathioprine Hydroxychloroquine,) cytotoxic drugs (e.g, cyclophosphami andde my cophenolate) Hydroxychl, oroquine (HCQ), Benlysta ® (belimumab), nonsteroidal anti-inflammatory drugs, Prednisone, Cellcept®, Prograf® , Atacicept®, Lupuzor®, Intravenous Immunoglobuli ns(IVIGs), CellCept® (mycophenolate mofetil Orencia), ®, CTLA4-IgG4m (RG2077), rituxima b,Ocrelizumab, Epratuzumab, CNTO 136, Sifalimumab (MEDI-545), A-623 (formerly AMG 623), AMG 557, Rontalizuma b,paquinimod (ABR-215757), LY2127399, CEP-33457, Dehydroepiandrosterone, Levothyroxine, abetimus sodium (LIP 394), Memantine®, Opiates , Rapamycin®, renal transplantat steion,m cel ltransplanta tionand combinations of any of the foregoing.

[00640] The immunoinhibitory HIDE1 therapeutic agents and/or a pharmaceutical compositi oncomprising same, as recited herein, according to at least some embodiments of the present invention, may optionall bey administere asd the sole active ingredient or together with othe drugsr in immunomodulating regimens or other anti-inflammatory agents e.g. for the treatment or prevention of allo- or xenograft acute or chronic rejection or inflammatory or autoimmune disorders, or to induce tolerance. d. Use of the HIDE1 agents and/or pharmaceutical compositions for adoptive immunity:

[00641] One of the cardinal features of some model ofs tolerance is that once the tolerance state has been established, it can be perpetuated to naive recipients by the adoptive transfer of donor-speci ficregulatory cells. Such adoptive transfer studie haves also addressed the capacity of T-cel lsubpopulations and non-T cell sto transfer tolerance Such. tolerance can be induced by blocking costimulati oron upon engagement of a co-inhibitory B7 with its counter receptor. This approach, that has been successfully applied in animals and is evaluated in clinical tria lsin humans, (Scalapino KJ and Daikh DI. PLoS One, 4(6):e6031 (2009); Riley et al., Immunity 30(5): 656-665 (2009)) provides a promising treatment optio n for autoimmune disorders and transplantat Accordingion. to at least some embodiments of the 199WO 2017/009712 PCT/IB2016/001079 present invention, HIDE1 secreted or solubl forme or ECD and/or variants and/or, orthologs , and/or fusions and/or conjugates thereof, are used for adoptive immunotherapy. Thus, according to at least some embodiment s,the present inventio nprovides methods for in vivo or ex vivo tolerance induction, comprising administering effective amount of HIDE1 secreted or solubl forme or ECD and/or variants, and/or orthologs and/, or fusions and/or conjugates thereof, to a patient or to leukocyt esisolated from the patient, in order to induce differentiat ionof tolerogenic regulatory cells; followed by ex-vivo enrichment and expansion of said cells and reinfusion of the tolerogenic regulatory cell sto said patient.

[00642] In anoth erembodiment, a method of inhibiting immune responses involves isolating immune cells from a patient, transfecting them wit ha nucleic acid molecule encoding a form of HIDE1, such that the cells express all or a portion of the HIDE1 polypeptide according to various embodiments of the present inventio non thei surface,r and reintroducing the transfect edcell sinto the patient. The transfect edcells have the capacity toinhibitimmune respons esin the patient. 3. Diagnostic Uses

[00643] The anti-HIDEl antibodi esprovided also find use in the in vitro or in vivo diagnosis, including imaging, of tumors that over-expre ssHIDE1. It should be noted, howeve r,that as discussed herein, HIDE1, as an immuno-oncology targe protein,t is not necessarily overexpresse ond cancer cells rathe witr hin the immune infiltrat ines the cancer.

In some instances it is; rather, the mechanism of action, activation of immune cell ssuch as T cells and NK cells, that resul tsin cancer diagnosis. Accordingly, anti-HIDEl antibodies can be used to diagnose cancer.

[00644] In particular, immune cell sinfiltrati theng tumors that over express HIDE1, and thus can be diagnosed by anti-HIDEl antibodies include,, but are not limited to, squamous cel lcancer, lung cancer (including small-ce lungll cancer, non-smal cell llung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, hepatocellula cancer,r gastric or stomach cancer (including gastrointest inal cancer), pancreatic cancer, glioblastom cervia, cal cancer, ovaria ncancer, liver cancer, bladd ercancer, hepatom a,breast cancer, colon cancer, colorecta cancer,l endometri oral uterine carcinoma, salivary gland carcinoma, kidne yor renal cancer, liver cancer, prosta te 200WO 2017/009712 PCT/IB2016/001079 cancer, vulval cancer, thyroi cancd er, hepatic carcinoma and various types of head and neck cancer, as wel las B-cel llymphom (incla uding low grade/follicul non-Hodgkar in's lymphom a (NHL); small lymphocyt (SL)ic NHL; intermedia grade/te follicul NHLar; intermedia gradete diffus eNHL; high grade immunoblasti NHLc ; high grade lymphoblast NHLic ; high grade small non-cleaved cel lNHL; bulky disease NHL; mantle cel llymphoma; AIDS-related lymphoma; and Waldenstr’oms Macroglobulinemi chronica); lymphocytic leukemi a(CLL); acute lymphoblast leukemiic a(ALL); Hairy cell leukemia; chronic myeloblasti leukemic a; multiple myeloma and post-transpl lymantphoproliferati disorve der (PTLD).

[00645] Generall y,diagnos iscan be done in several ways. In one embodiment, a tissue from a patient, such as a biopsy sample, is contacted wit ha HIDE1 antibody, generall y labeled, such that the antibody binds to the endogenous HIDE1. The level of signal is compared to that of norma lnon-cancerous tissue either from the same patient or a reference sample, to determine the presence or absence of cancer. The biopsy sample can be from a soli tumd or, a blood sampl e(for lymphomas and leukemias such as ALL, T cel llymphoma, etc).

[00646] In general, in thi sembodiment, the anti-HIDE 1 is labeled, for example with a fluorophore or othe opticalr label, that is detected using a fluorome teror other optical detection system as is well known in the art. In an alternat embodiment,e a secondary labeled antibody is contacted with the sample ,for example using an anti-human IgG antibody from a different mammal (mouse ,rat, rabbit, goat, etc.) to form a sandwich assay as is known in the art. Alternative thely, anti-HIDE 1 mAh could be direct lylabeled (i.e. biotin) and detection can be done by a secondary Ab directed to the labeling agent in the art.

[00647] Once over-expression of HIDE 1 is seen, treatment can proceed with the administration of an anti-HIDE 1 antibody according to the invention as outlined herein.

[00648] In other embodiments, in vivo diagnosis is done. Generall y,in thi s embodiment, the anti-HIDEl antibody (including antibody fragments) is injected into the patient and imaging is done. In thi sembodiment, for example, the antibody is generally labeled wit han optical label or an MRI label, such as a gadolinium chelat e,radioactive labeling of mAb (including fragments). 201WO 2017/009712 PCT/IB2016/001079

[00649] In some embodiments, the antibodie descris bed herei nare used for both diagnosis and treatment or, for diagnosis alone. When anti-HIDEl antibodie ares used for both diagnosis and treatment some, embodiments rel yon two different anti-HIDEl antibodies to two different epitope s,such that the diagnosti antic body does not compete for binding wit h the therapeuti antic body, although in some cases the same antibody can be used for both. For example, thi scan be done using antibodie thats are in different bins, e.g, that bind to different epitopes on HIDE1, such as outlined herein. Thus included in the invention are compositions comprising a diagnosti antic body and a therapeutic antibody, and in some embodiment s,the diagnosti antic body is labeled as described herein. In addition the, compositi onof therapeuti andc diagnosti antibodiec cans also be co-administered wit hothe r drugs as outlined herein.

[00650] Particularly useful antibodie fors use in diagnosis include, but are not limited to thes eenumerate dantibodies or, antibodie thats utilize the CDRs with variant sequences

[00651] In many embodiment s,a diagnosti antic body is labeled. By “labeled” herein is meant that the antibodie discloseds herei nhave one or more elements, isotopes or, chemical compounds attache tod enable the detection in a screen or diagnosti procedure.c In general, label sfall into several classes: a) immune labels, whic hmay be an epitope incorporated as a fusion partner that is recognized by an antibody, b) isotopi labelsc whic, h may be radioactive or heavy isotopes c), smal lmolecul labee ls, whic hmay include fluorescent and colorimetri c dyes, or molecules such as biotin that enable other labeling methods, and d) label ssuch as particles (including bubbles for ultrasound labeling) or paramagnet iclabel sthat allow body imagining. Labels may be incorporated into the antibodie ats any position and may be incorporated in vitro or in vivo durin gprote inexpression, as is known in the art.

[00652] Diagnosi scan be done either in vivo, by administrati ofon a diagnosti c antibody that allows whol bodye imaging as described below, or in vitro, on samples removed from a patient “.Sample” in thi scontext includes any number of things incl, uding, but not limited to, bodily fluids (including, but not limited to, blood, urine, serum ,lymph, saliva ,anal and vaginal secretions, perspiration and semen), as well as tissue samples such as resul fromt biopsies of relevant tissues. 202WO 2017/009712 PCT/IB2016/001079

[00653] In some embodiment s,in vivo imaging is done, includin gbut not limite tod ultrasound CT, scans, X-rays, MRI and PET scans, as well as optical techniques, such as those using optical label sfor tumors near the surfac eof the body.

[00654] In vivo imaging of diseases associated with HIDE1 may be performe byd any suitable technique For. example, ״Tc-labeling or labeling wit hanother .beta.-ray emitting isotope may be used to label anti-HIDEl antibodies Variat. ions on thi stechniqu emay include the use of magnetic resonanc imae ging (MRI) to improve imaging over gamma camera techniques.

[00655] In one embodiment, the present inventio nprovides an in vivo imaging method wherei ann anti-HIDEl antibody is conjugated to a detection-promoting agent, the conjugated antibody is administered to a host, such as by injection into the bloodstrea andm, the presence and location of the labeled antibody in the host is assayed. Through thi stechnique and any othe diagnostir metc hod provided herein, in some embodiments the present invention provides a method for screening for the presence of disease-related cells in a human patient or a biologic alsampl etaken from a human patient.

[00656] For diagnosti imac ging, radioisotopes may be bound to an anti-HIDEl antibody either directl ory, indirectl byy using an intermediary functional group. Useful intermedia ryfunctional groups include chelators, such as ethylenediaminetetraaceti acid and c diethylenetriaminepentaac acieticd (see for instance U.S. Pat. No. 5,057,313), in such diagnost icassays involving radioisotope-conjuga antited-HIDEl antibodies the, dosage of conjugated anti-HIDEl antibody delivered to the patient typicall isy maintained at as low a level as possible through the choice of isotope for the best combination of minimum half-life , minimum retention in the body, and minimum quantity of isotope, which will permit detection and accurate measurement.

[00657] In addition to radioisotopes and radio-opaque agents ,diagnosti methodsc may be perform edusing anti-HIDEl antibodie thats are conjugated to dyes (such as wit hthe biotin-streptavidi complex)n ,contrast agents, fluorescent compounds or molecules and enhancing agents (e.g. paramagnet icions) for magnetic resonanc ime aging (MRI) (see, e.g, U.S. Pat. No. 6,331,175, which describes MRI technique sand the preparati onof antibodies 203WO 2017/009712 PCT/IB2016/001079 conjugated to a MRI enhancing agent). Such diagnostic/detect!on agents may be selected from agents for use in magnetic resonanc eimaging, and fluorescent compounds.

[00658] In order to load an anti-HIDEl antibody wit hradioactive metals or paramagneti ions,c it may be necessary to react it wit ha reagent having a long tai lto which are attached a multiplici ofty chelating groups for binding the ions. Such a tail may be a polymer such as a polylysine, polysaccharide, or othe derir vatize ord derivatizable chain having pendant groups to whic hcan be bound chelating groups such as, e.g., porphyri ns, polyamines, crown ethers, bisthiosemicarbazones poly, oximes, and like groups known to be useful for thi spurpose.

[00659] Chelates may be coupled to anti-HIDEl antibodie usings standard chemistries .

A chelat ise normal lylinked to an anti-HIDEl antibody by a group that enables formation of a bond to the molecul withe minimal los ofs immunoreactivit andy minimal aggregation and/or internal cross-linking.

[00660] Examples of potentially useful metal-chel atcombinae tions include 2-benzyl- DTP A and its monomethyl and cyclohex ylanalogs, used wit hdiagnosti isotopec ins the general energy range of 60 to 4,000 keV, such as 125I, 123I, 124I, 62Cu, 6Cu, 1SF, 111In, 67Ga, 99Tc, PTe, 1c, "N, 5O, and 76Br, for radio-imaging.

[00661] Labels include a radionucli de,a radiological contrast agent, a paramagneti c ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photoact iveagent. Such diagnosti agentsc are well known and any such known diagnosti c agent may be used. Non-limiti ngexamples of diagnost agenic ts may include a radionucli de such as 110In, 111In, 177Lu, ISF, S2Fe. 62Cu, 64Cu, 67Cu, 67Ga, 68Ga, *y, 90Y, 89Zr, 94mTc, 94Tc, "mTc, 120I, 123I, 124I, 125I, 131I, 154158־Gd, 32P, I’c, IN, 15o, 186Re, 188Re, ‘Mn, 52mMn, 55Co, 72As, 75Br, 76Br, 82mRb, 83Sr, or other .gamma.-, .beta.-, or positron- emitters.

[00662] Paramagneti ionsc of use may include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (III), copper (III), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) 204WO 2017/009712 PCT/IB2016/001079 or erbium (III), Metal contrast agents may include lanthanum (III), gold (III), lead (II) or bismuth (III).

[00663] Ultrasound contrast agents may comprise liposome suchs, as gas filled liposomes. Radiopaque diagnosti agentsc may be selected from compounds, barium compounds, gallium compounds, and thalli umcompounds.

[00664] These and similar chelate s,when complexed with non-radioact ivemetals, such as manganese, iron, and gadolinium may be useful for MRI diagnost icmethods in connection wit hanti-HIDEl antibodies Macrocycl. icchelates such as NOTA, DOTA, and TETA are of use with a variet yof metals and radiometal mosts, particularl wity h radionuclide ofs gallium, yttrium and, copper, respectivel Suchy. metal-chel atecomplexes may be made very stabl bye tailoring the ring size to the metal of interest. Other ring-type chelat essuch as macrocyclic poly ethers, which are of interest for stably binding nuclides, such as 223Ra may also be suitable in diagnosti metc hods.

[00665] Thus, in some embodiments the present invention provides diagnost icanti - HIDE1 antibody conjugates, wherei then anti-HIDEl antibody conjugat eis conjugated to a contrast agent (such as for magnetic resonance imaging, computed tomography, or ultrasound contrast-enhanc agent)ing or a radionuclide that may be, for example, a .gamma.-, .beta.-, .alpha.-, Auger electron-, or positron-emit tingisotope.

[00666] Anti-HIDEl antibodie mays also be useful in, for example, detecting expression of an antigen of interest in specific cell s,tissues, or serum .For diagnosti c applications, the antibody typicall willy be labeled wit ha detectable moiety for in vitro assays. As will be appreciate byd those in the art, there are a wide variety of suitable labels for use in in vitro testing. Suitable dyes for use in thi saspect of the invention include but, are not limited to, fluorescen lanthat nide complexes, including thos ofe Europium and Terbium , fluorescein, rhodamine, tetramethylrhodami eosinne, ,erythrosi coumn, arin, methyl- coumarins quantum, dots (also referre tod as “nanocrystals”; see U.S. Ser. No. 09/315,584, hereby incorporated by reference), pyrene, Malacit egreen, stilbene, Lucifer Yellow, Cascade Blue.TM., Texas Red, Cy dyes (Cy3, Cy5, etc.), alexa dyes (including Alexa, phycoerythi n, 205WO 2017/009712 PCT/IB2016/001079 bodipy, and othe rsdescribed in the 6th Edition of the Molecul arProbes Handbook by Richard P. Haugland ,hereby expressl incorporatedy by reference.

[00667] Stained tissues may then be assessed for radioactivi countty ing as an indicator of the amount of HIDE !-associat edpeptides in the tumor. The images obtained by the use of such techniques may be used to assess biodistribut ofion HIDE! in a patient, mammal, or tissue, for example in the context of using HIDE! as a biomarker for the presence of invasive cancer cells. 4. Diagnostic Uses of HIDE1 polypeptides

[00668] Soluble HIDE! polypeptides according to at least some embodiments of the present inventio nmay optionally also be modified wit ha label capable of providing a detectable signal, either direct lyor indirectly, including, but not limited to, radioisotopes and fluorescent compounds. Such labeled polypeptides can be used for various uses, includin gbut not limited to, diagnosis prognosis,, prediction, screening, earl ydiagnosis determi, nation of progressi on,therapy selection and treatment monitori ngof disease, disorder and/or an indicative condition, as detailed herein.

[00669] As used herein the term “diagnosis” refers to the proces ofs identifying or aiding in the identificati onof a medical condition or disease by its signs, symptoms, and in particular from the result ofs various diagnost proceduresic including, e.g., using labeled HIDE! polypeptides according to at leas tsome embodiments of the present invention, as described herein. Furthermor ase, used herei nthe term “diagnosis” encompasses screening for a disease, detecting a presence or a severity of a disease, providing prognosis of a disease, monitori ngdisease progression or relapse, as well as assessment of treatment efficacy and/o r relapse of a disease, disorder or condition, as well as selecting a therapy and/or a treatment for a disease, optimization of a given therapy for a disease, monitori ngthe treatment of a disease, and/or predicti ngthe suitability of a therapy for specific patients or subpopulations or determining the appropriate dosing of a therapeutic produ ctin patients or subpopulations.

The diagnost procedureic can be performe ind vivo or in vitro.

[00670] According to at least some embodiments, the present invention provides a method for imaging an organ or tissue, the method comprising: (a) administering to a subject 206WO 2017/009712 PCT/IB2016/001079 in need of such imaging, a labeled polypeptide; and (b) detecting the labeled polypeptide to determine wher thee labeled polypeptide is concentrated in the subject. When used in imaging applications, the labeled polypeptides according to at least some embodiments of the present inventio ntypicall havey an imaging agent covalently or monovalentl atty ache d theret Suito. able imaging agents include but, are not limited to, radionuclides, detectable tags , fluorophores fluorescent, proteins enzym, atic proteins, and the like .One of skill in the art will be familiar with other methods for attaching imaging agents to polypeptides For. example, the imaging agent can be attached via site-specifi conjugation,c e.g, covalent attachment of the imaging agent to a peptide linke suchr as a polyargini nemoiety having five to seven arginines present at the carboxy 1-terminus of and Fc fusion molecule The. imaging agent can also be direct lyattache viad non-site specific conjugation, e.g., covalent attachment of the imaging agent to primary amine groups present in the polypeptide One. of skil inl the art will appreciat thate an imaging agent can also be bound to a protei vian noncovalent interactions (e.g., ionic bonds, hydrophobic interactions, hydrogen bonds, Van der Waals forces, dipole- dipole bonds, etc)..

[00671] In certain instances, the polypeptide is radiolabeled with a radionucli deby direct lyattaching the radionuclide to the polypeptide In. certai nother instances, the radionuclide is bound to a chelating agent or chelating agent-linker attached to the polypeptide Suita. ble radionuclide fors direc conjt ugation include, without limitation, 18 F, 124I, 125I, 131I, and mixture thereof.s Suitable radionuclides for use wit ha chelating agent include wi, thout limitation 47Sc, 6Cu, 67Cu, 89Sr, 86Y, 87Y, PY, 105Rh, I‘‘Ag, 111In, 149P, II’mSn. 149Pm, 1S‘sm 166Ho, 177Lu, 186Re, 188Re, 211At, 212Bi and mixtures thereof.

[00672] Preferably, the radionuclide bound to a chelating agent is 64Cu, 90Y, 111In, or mixtures thereof. Suitable chelating agents include, but are not limited to, DOTA, BAD, TETA, DTP A, EDTA, NT A, HDTA, their phosphonate analogs, and mixtures thereof. One of skil inl the art will be familiar wit hmethods for attaching radionuclides, chelating agents, and chelating agent-linkers to polypeptides of various embodiments of the present invention.

In particular, attachment can be conveniently accomplished using, for example, commercially available bifunctiona linkil ng groups (generally heterobifunctional linking groups) that can be 207WO 2017/009712 PCT/IB2016/001079 attached to a functional group present in a non-interferi posing tion on the polypepti deand then further linked to a radionuclide, chelating agent, or chelating agent-linker.

[00673] Non-limiti ngexamples of fluorophore or fluoress cent dyes suitable for use as imaging agents include Alexa Fluor® dyes (Invitrogen Corp.; Carlsbad, Calif .), fluorescein, fluorescei isothiocyanatn (FITC),e Oregon Green™; rhodamine, Texas red, tetrarhodam ine isothiocynat (TRITC),e CyDye™ fluors (e.g, Cy2, Cy3, Cy5), and the like.

[00674] Examples of fluorescent proteins suitable for use as imaging agents include , but are not limite to,d green fluorescent protein, red fluorescent protei (e.g.,n DsRed), yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, and variants thereof (see, e.g, U. S. Pat. Nos. 6,403,374, 6,800,733, and 7,157,566). Specific examples of GFP variants include, but are not limite to,d enhanced GFP (EGFP), destabilized EGFP, the GFP variants described in Doan et al., Mol. Microbiol., 55:1767-1781 (2005), the GFP variant describe ind Crameri et al., Nat. Biotechnol., 14:315-319 (1996), cerulean fluorescent proteins described in Rizzo et al., Nat. Biotechnol, 22:445 (2004) and Tsien, Annu. Rev.

Biochem., 67:509 (1998), and the yellow fluorescent protei descrin bed in Nagai et al., Nat.

Biotechnol., 20:87-90 (2002). DsRed variants are describe in,d e.g., Shaner et al., Nat.

Biotechnol., 22:1567-1572 (2004), and include mStrawberry, mCherry, mOrange, mBanana, mHoneydew and, mTangerine .Additional DsRed variants are described in, e.g., Wang et al., Proc. Natl. Acad. Sci. U. S. A., 101:16745-16749 (2004) and include mRaspberr andy mPlum. Furthe exampr les of DsRed variants include mRFPmars described in Fischer et al., FEBSLett., 577:227-232 (2004) and mRFPruby described in Fischer et al., FEBS Lett., 580:2495-2502 (2006).

[00675] In other embodiments, the imaging agent that is bound to a polypeptide according to at least some embodiments of the present inventio ncomprises a detectabl tage such as, for example, biotin avidi, n, streptavidin, or neutravidin. In furth erembodiment s,the imaging agent comprises an enzymatic prote inincluding, but not limited to, luciferase , chloramphenicol acetyltransferase B-gala, ctosidase, B-glucuronidase, horseradis peroxidaseh , xylanase ,alkaline phosphatas ande, the like. 208WO 2017/009712 PCT/IB2016/001079

[00676] Any device or method known in the art for detecting the radioacti emive ssions of radionuclides in a subject is suitable for use in the various embodiments of the present invention. For example, methods such as Single Photon Emission Computerized Tomography (SPECT), whic hdetect thes radiation from a singl ephoton y-emitting radionuclide using a rotating y camera, and radionuclide scintigraphy, which obtains an image or series of sequential images of the distributi ofon a radionuclide in tissues, organs, or body system s using a scintillatio gammn a camera, may optionall bey used for detecting the radiation emitted from a radiolabel polypeptideed of the various embodiments of the present invention.

Positr onemission tomograph (PET)y is another suitable technique for detecting radiation in a subject. Miniature and flexibl radiate ion detectors intended for medical use are produced by Intra-Medical LLC (Santa Monica, Calif). Magnetic Resonance Imaging (MRI) or any othe r imaging techniqu eknown to one of skill in the art is also suitable for detecting the radioacti ve emissions of radionuclide Regards. less of the method or device used, such detection is aimed at determining where the labeled polypepti deis concentrated in a subject ,with such concentration being an indicator of diseas eactivity.

[00677] Non-invasive fluorescence imaging of animals and humans can also provide in vivo diagnost inforic mation and be used in a wide variety of clinical specialtie s.For instance , techniques have been developed over the years for simple ocular observations follow ingUV excitation to sophisticat spected roscopic imaging using advanced equipment (see, e.g, Andersson-Enge etls al., Phys. Med. Biol., 42:815-824 (1997)). Specific devices or methods known in the art for the in vivo detection of fluorescence e.g.,, from fluorophore or s fluorescent proteins include,, but are not limited to, in vivo near-infrared fluorescence (see, e.g., Frangioni, Curr. Opin. Chem. Biol., 7:626-634 (2003)), the Maestro™ in vivo fluorescence imaging system (Cambridge Research & Instrumentation, Inc.; Wobum, Mass. ), in vivo fluorescence imaging using a flying-spot scanner (see, e.g., Ramanujam et al., IEEE Transactions on Biomedical Engineering, 48:1034-1041 (2001), and the like.

[00678] Other methods or devices for detecting an optical respons include,e without limitation, visual inspection, CCD cameras, video cameras ,photographic film ,laser-scanning devices, fluoromet ers,photodiodes, quantum counters, epifluorescenc microe scopes , 209WO 2017/009712 PCT/IB2016/001079 scanning microscopes flow, cytometers fluores, cence microplate readers, or signal amplification using photomultipli tubeers.

. Theranostics

[00679] The term theranostics describes the use of diagnosti tesc ting to diagnose the disease, choose the correct treatme ntregime according to the result ofs diagnosti testc ing and/or monitor the patient response to therapy according to the result ofs diagnost tesic ting.

Theranosti testc cans be used to select patients for treatments that are particularly likel toy benefit them and unlikely to produce side-effect s.They can also provide an early and objective indication of treatment efficacy in individual patients so, that (if necessary) the treatment can be altered wit ha minimum of delay. For example: DAKO and Genentech together created HercepTest® and Herceptin® (trastuzuma forb) the treatment of breast cancer, the firs theranostt testic approved simultaneously wit ha new therapeutic drug. In addition to HercepTest (which is an immunohistochem icaltest ),other theranosti testc ares in development whic huse traditional clinical chemistry, immunoassay, cell-based technologi es and nucleic acid test s.PPGx's recently launche dTPMT (thiopurine S-methyltransferas teste) , which is enabling docto tors identify patients at ris kfor potentially fatal advers reactie ons to 6-mercaptopurine an, agent used in the treatme ntof leukemia. Also, Nova Molecular pioneered SNP genotypin gof the apolipoprotein E gene to predict Alzheimer's disease patients responses' to cholinomime tictherapies and it is now widel yused in clinical trial ofs new drugs for thi sindication. Thus, the field of theranosti represecs nts the intersecti ofon diagnost tesic ting informati onthat predicts the response of a patient to a treatment wit hthe selection of the appropriat treate ment for that particular patient.

[00680] The term theranostics describes the use of diagnosti tesc ting to diagnose the disease, choose the correct treatme ntregime according to the result ofs diagnosti testc ing and/or monitor the patient response to therapy according to the result ofs diagnost tesic ting.

Theranosti testc cans be used to select patients for treatments that are particularly likel toy benefit them and unlikely to produce side-effect s.They can also provide an early and objective indication of treatment efficacy in individual patients, so that (if necessary) the treatment can be altered wit ha minimum of delay. For example: DAKO and Genentech together created HercepTest® and Herceptin® (trastuzuma forb) the treatment of breast 210WO 2017/009712 PCT/IB2016/001079 cancer, the firs thet ranost testic approved simultaneously wit ha new therapeutic drug. In addition to HercepTest® (which is an immunohistochem icaltest ),othe theranostr testic ares in development which use traditional clinical chemistry, immunoassay, cell-based technologie ands nucleic acid test s.PPGx's recently launched TP MT (thiopurine S- methyltransferas test,e) which is enabling docto tors identify patients at risk for potentially fatal adverse reactions to 6-mercaptopurine an, agent used in the treatment of leukemia. Also, Nova Molecula pioneeredr SNP genotyping of the apolipoprote E ingene to predict Alzheimer's disease patients responses' to cholinomime tictherapies and it is now widely used in clinical trials of new drugs for thi sindication. Thus, the field of theranosti representcs thes intersection of diagnosti testc ing informati onthat predic tsthe response of a patient to a treatment wit hthe selection of the appropriate treatment for that particular patient.

[00681] As described herein, the term “theranostic” may optional referly to firs testt ing the subject ,such as the patient, for a certai nminimum level of HIDE1, for example optionally in the cancerous tissue and/or in the immune infiltrate as ,described herein as a sufficient level of HIDE1 expression Test. ing may optional bely performed ex vivo, in whic h the sample is removed from the subject ,or in vivo.

[00682] If the cancerous tissue and/or the immune infiltra havete been shown to have the minimum level of HIDE1, then an anti-HIDEl antibody, alone or optionall wity hothe r treatment modalit iesas described herein, may optionally be administered to the subject. 6. Surrogate Markers - HIDE1 antibodies

[00683] A surrogat markere is a marker, that is detectable in a laboratory and/or according to a physical sign or symptom on the patient, and that is used in therapeutic trials as a substitute for a clinicall meaningfuly endpoin t.The surroga markerte is a direc meat sure of how a patient feels, functions, or survives whic his expected to predict the effect of the therapy The. need for surroga marte kers mainly arises when such markers can be measured earlier more, conveniently, or more frequently than the endpoints of interest in terms of the effect of a treatment on a patient, whic hare referre tod as the clinical endpoint s.Ideally, a surrogat markere should be biologicall plauy sibl e,predictive of disease progression and measurable by standardize assd ays (including but not limite tod traditional clinical chemistry, 211WO 2017/009712 PCT/IB2016/001079 immunoassay, cell-based technologie recepts, or occupancy assay nucleic acid test ands imaging modalities).

[00684] The therapeutic compositions (e.g, human antibodies multi, specif andic bispecific molecules and immunoconjugates) according to at least some embodiments of the present inventio nwhic hhave complement binding sites, such as portions from IgGl ,IgG2, or IgG3 or IgM which bind complement can, also be used in the presence of complement In. one embodiment, ex vivo treatment of a population of cells comprising target cells wit ha binding agent according to at least some embodiments of the present invention and appropriate effector cells can be supplemented by the addition of complement or serum containing complement. Phagocytosis of target cell scoated wit ha binding agent according to at least some embodiments of the present inventio ncan be improved by binding of complement proteins. In another embodiment target cells coated wit hthe compositions (e.g, human antibodies, multispecif andic bispecific molecules accord) ing to at least some embodiments of the present inventio ncan also be lysed by complement In. yet anoth erembodiment, the compositions according to at least some embodiments of the present invention do not activat e complement.

[00685] The therapeutic compositions (e.g, human antibodies multi, specif andic bispecific molecules and immunoconjugates) according to at least some embodiments of the present inventio ncan also be administered together wit hcomplement Thus,. according to at least some embodiments of the present invention there are compositions, comprising human antibodies, multispecif oric bispecific molecules and serum or complement These. compositions are advantageous in that the complement is located in close proximity to the human antibodies, multispecific or bispecific molecules. Alternatively, the human antibodies , multispecif oric bispecific molecules according to at least some embodiments of the present invention and the complement or serum can be administered separately.

[00686] In some embodiments, the present inventio nprovides a method for determining whether an anti-HIDE 1 antibody has produced a desired immunomodulat ory effect in a human (e.g, a cancer patient). The method includes detecting an increase or decrease of at least one immunomodulat orybiomarke (somr etimes referred to herei nas an "anti-HIDE lantibody-associate imdmunomodulat orybiomarke”)r described herein in a 212WO 2017/009712 PCT/IB2016/001079 blood sampl eobtained from a patient who has been administered an anti-HIDE 1 antibody to thereby determine whether the anti-HIDE 1 antibody has produce and immunostimulatory effect .The immunostimulator effecy t can be characterized by a change (e.g, an increase or a decrease) in at least one biomarker, e.g, an anti-HIDEl antibody-associat ed immunomodulat orybiomarker described herein, the change selected from the group consisting of: (i) a reduce dconcentration of regulatory T cells, relati veto the concentration of regulatory T cell sof the same histologi typecal in the human prior to the firs admit nistrati on of the antibody; (ii) an increased concentration of CTL cell s,relative to the concentrati onof CTL cell sof the same histologi typecal in the human prior to the firs administ tration of the antibody; (iii) an increased concentrati onof activated T cell s,relati veto the concentration of activated T cell sof the same histologic typeal in the human prior to the first administrati ofon the antibody; (iv) an increased concentrati onof NK cells, relati veto the concentration of NK cells of the same histologi typecal in the human prior to the firs admit nistrati ofon the antibody; (v) a ratio of percent activated T cells to percent regulatory T cell s(T regs) of at least 2:1 (e.g, at least 3:1, at least 4:1, at least 5:1, at least 6:1, or at least 7:1), relati veto the rati oof activated T cell sto T regs in the human prior to the first administration of the antibody; (vii) a changed level of HIDE1 expression by a plural ityof leukocyt esin a biologic alsample obtained from a patient prior to administration to the patient of an anti- HIDE1 antibody, relati veto the level of HIDE 1 expression by a plural ityof leukocyte ofs the same histologic typeal in a biological sampl efrom the patient prior to administration of the antibody; viii) increasing T and/or NK cel linfiltaration of tumors, (vix) alleviati ngmyeloid- cel lsuppressio (vx)n, eliminating (depleting) immuno-supressive myeloid cell s,such as MDSCs (Myeloid-derived suppressor cells), TAMs (Tumor-associated macrophages).

[00687] It is understood that in some embodiments, a change in expression can be a change in prote inexpression or a change in mRNA expression That. is, for example, the methods can interrogate a population of leukocyte froms a patient to determine if a reduction in the level of HIDE 1 mRNA and/or HIDE1 prote inexpression has occurred relati, veto a control level of mRNA and/or prote inexpression. Methods for measuring protei andn mRNA expression are wel lknown in the art and described herein. 213WO 2017/009712 PCT/IB2016/001079

[00688] In some embodiments, any of the methods described herein (e.g, the methods for determining whether an anti-HIDE 1 has produce ad desire imd munostimulatory effect in a human) can include measuring the concentration of the specified cel ltype (e.g. CD4+ T cell s,CTLs, NK cells etc.), or quantifying the level of expression of a specified expression marker on a specified cel ltype (e.g. Foxp3, CD25, CD69, etc.), in a biologic alsampl e obtained from the human prior to administration of the antibody. 7. Surrogate Markers - HIDE1 peptides

[00689] The HIDE1 protei composn itions according to at least some embodiments of the present inventio ncan be used as a surroga marker.te A surrogat markere is a marker, that is detectable in a laborator and/ory according to a physica lsign or symptom on the patient, and that is used in therapeutic trial ass a substitute for a clinical lymeaningful endpoint. The surrogat markere is a direc meat sure of how a patient feels, functions, or survives whic his expected to predict the effect of the therapy. The need for surrogat markerse mainly arises when such markers can be measured earlier, more conveniently, or more frequently than the endpoints of interest in terms of the effect of a treatme nton a patient, whic hare referre tod as the clinical endpoints. Ideally, a surrogat markere should be biologicall plausiy ble, predicti ve of disease progression and measurable by standardiz assed ays (including but not limite tod traditional clinical chemistry, immunoassay, cell-based technologies, nucleic acid test ands imaging modalities).

[00690] HIDE1 prote incompositions according to at leas tsome embodiments of the present inventio nwhic hhave complement binding sites, such as portions from IgGl ,IGg2, or IgG3 or IgM which bind complement can, also be used in the presence of complement In. one embodiment, ex vivo treatment of a population of cell scomprising targe cellt swith a binding agent according to at least some embodiments of the present invention and appropriate effector cells can be supplemented by the addition of complement or serum containing complement. Phagocytosis of target cell scoated wit ha binding agent according to at least some embodiments of the present inventio ncan be improved by binding of complement proteins. In another embodiment target cells coated wit hthe compositions according to at least some embodiments of the present invention can also be lysed by complement. In yet 214WO 2017/009712 PCT/IB2016/001079 another embodiment, the compositions according to at least some embodiments of the present invention do not activate complement.

[00691] The HIDE1 protei composn itions according to at least some embodiments of the present inventio ncan also be administered together wit hcomplement Thus,. according to at leas tsome embodiments of the present inventio nthere is any of the HIDE1 protein composition, comprising human HIDE1 solubl proteie andn serum or complement. These compositions are advantageous in that the complement is located in close proximity to the human HIDE1 solubl molecule es. Alternative thely, human HIDE1 solubl molee cule s according to at leas tsome embodiments of the present invention and the complement or serum can be administere separatd ely.

[00692] In some embodiments the present invention provides the use of an immunostimulatory antibody, antigen-binding fragment or conjugat ethereof according to at least some embodiments of the present invention or a pharmaceutic alcompositi oncontaining the same, to perfor mone or more of the followin ing a subject in need thereof: (a) upregulating pro-inflammatory cytokine s;(b) increasin gT-cel lproliferati and/oron expansion; (c) increasing interferon-Y or TNF-a production by T-cells (d); increasing IL-2 secretion; (e) stimulating antibody responses; (f) inhibiting cancer cel lgrowth; (g) promoti ng antigenic specific T cel limmunity; (h) promoting CD4+ and/or CD8+ T cel lactivation; (i) alleviati ngT-cel lsuppression (j); promoting NK cel lactivit y;(k) promoting apoptosis or lysi sof cancer cells; (1) cytotoxic or cytostat effecic t on cancer cell s,(m) increasin gT and/or NK cel linfiltaration of tumors, (n) alleviati ngmyeloid-cell suppressio (0)n, eliminating (depleting) immuno-supressiv myele oid cell s,such as MDSCs (Myeloid-derived suppressor cells), TAMs (Tumor-associated macrophages).

[00693] In some embodiments the present invention provides the use of an immunostimulatory antibody, antigen-binding fragment or conjugat eaccording to at least some embodiments of the present inventio nfor diagnosing a disease in a subject ,or for aiding in the diagnosis of a disease, wherei then disease is selected from the group consisting of cancer, wherei then diagnost metic hod is performe exd vivo, by contacting a tissue or othe r sample from the subject wit hthe immune molecule or antibody as described herei nex vivo and detecting specific binding thereto. 215WO 2017/009712 PCT/IB2016/001079

[00694] In other embodiments the present inventio nprovides the use of an immunostimulatory antibody, antigen-binding fragment or conjugat eaccording to at least some embodiments of the present inventio nin diagnosti methodsc for diagnosing or aiding in the diagnosis of a disease in a subject ,wherei then disease is selected from the group consisting of cancer, and/or an infectious disease wherei then diagnost metic hod is performed in vivo, comprising administering the immune molecul ore antibody as described herein to the subject ,preferabl labeledy with a detectable agent such as a radionucli de,or fluorophore and detecting specific binding of the immunostimulatory antibody, antigen-binding fragment or conjugate as described herei nto a tissue of the subject. Alternative thely method may optionally be performe ind vitro in a sampl etaken from the subject.

[00695] Optionall suchy diagnosti metc hod may be performe befored concurrent or after administering an immunostimulator antiy body, antigen-binding fragment or conjugat e or compositi oncontaining according to at leas tsome embodiment ofs the present invention.

[00696] Optionall they diagnost useic or method further comprises determining the level of HIDE1 in a tissue of the subject before administering the immunostimulatory antibody, antigen-binding fragment or conjugate or compositi oncontaining according to at least some embodiments of the present inventio nto the subject. In some embodiments the immunostimulatory antibody, antigen-binding fragment or conjugat eor composition containing according to at least some embodiments of the present invention is only administered to the subject if said HIDE1 levelis at a threshol leveld deemed to be “sufficient” for the HIDE1 antibody to elicit a significant therapeutic benefit ,e.g, it is expressed at higher than norma llevels or it is expresse dat detectable level sby the treated disease cell s,e.g, specific types of cancer or immune or strom cellsal at the sit eof the disease, or is expressed at a level that based on in vitro or in vivo studie indis cate sthat the antibody is likel toy elicit a significan ttherapeutic benefit.

[00697] In some embodiments the expression level of HIDE1 is detecte upond initial diagnosis prior to the initiation of cancer therapy, or alternativel aftyer the start of cancer therapy, such as a combination therapy including use of an immunostimulator antyibod y, antigen-binding fragment or conjugat eaccording to at least some embodiments of the present 216WO 2017/009712 PCT/IB2016/001079 invention and another active such as a chemotherapeuti therac, peutic enzyme, radionuclide or radiation or another biologic.

[00698] In some embodiments the use or method further comprises determining said level of HIDE1 according to the expression level of said HIDE1.

[00699] In some embodiments the expression level of the HIDE1 is determined by use of an IHC (immunohistochemistr assy)ay or a gene expression assay in a subject’s tissue sample.

[00700] In some embodiments said IHC assay may optionall compriy se determining if the level of HIDE 1 expression is at least 1 on a scale of 0 to 3, e.g, in a tissue sampl e comprising cancer cell sand/or immune infiltrate and/or on immune and/or on stroma cell ls.

[00701] In some embodiments the level of HIDE 1 may optionall bey determined in a tissue by contacting the tissue with an immunostimulatory antibody, antigen-binding fragment or conjugat eor compositi oncontaining according to at least some embodiments of the present inventio nand detecting specific binding thereto.

[00702] In some embodiments the present invention provides assays for diagnosing or aiding in the diagnosis of a disease in a tissue sample take nfrom a subject ,comprising use of an immunostimulatory antibody, antigen-binding fragment or conjugat eas described herei n and at leas tone reagent for diagnosing a disease selected from the group consisting of cancer, infectious disease, and/or sepsis.

[00703] In some embodiments the present invention provides the use of anti-HIDEl antibody, antigen-binding fragment or conjugat eor compositi oncontaining same according to at least some embodiments of the present invention for screening for a disease or aiding in the diagnosis of a disease (particularl oney involving immunosuppression), detecting a presence or a severity of a disease, providing prognosis of a disease, monitori ngdisease progression or relapse, as well as assessment of treatment efficacy and/or relapse of a disease, disorder or condition, as well as selecting a therapy and/or a treatment for a disease, optimization of a given therapy for a disease, monitori ngthe treatment of a disease, and/or predicti ngthe suitability of a therapy for specific patients or subpopulations or determining the appropriat dosinge of a therapeutic product in patients or subpopulations. 217WO 2017/009712 PCT/IB2016/001079

[00704] In some embodiments, the present inventio nprovides anti-HIDE 1 immunostimulatory immune molecul ore compositi oncontaining same according to at least some embodiments of the present invention, and/or uses thereof for treatment and/or diagnosis of cancer, wherei then cancer, and/or immune cells infiltrati theng cancer, and/or stroma cellsl of the subject express HIDE1, e.g. prior to, or follow ingcancer therapy, and wherei saidn cancer is e.g., selected from the group consisting of but not limite tod breast cancer, cervical cancer, ovary cancer, endometri cancer,al melanoma, uveal melanoma, bladd ercancer, lung cancer, pancreati cancc er, colorecta cancl er, prostate cancer, leukemia, acute lymphocyt leukic emia, chronic lymphocyt leukic emia, B-cel llymphoma, Burkitt's lymphoma, multiple myeloma, Non-Hodgkin’s lymphoma, myeloid leukemia, acute myelogenous leukemia (AML), chronic myelogenous leukemia, thyroi cancd er, thyroid follicul cancar er, myelodysplastic syndrome (MDS), fibrosarcom andas rhabdomyosarcom as, teratocarcinoma, neuroblastom gliomaa, gli, oblastom benigna, tumo rof the skin, keratoacanthomas, renal cancer, anaplast iclarge-cel lymphoma,l esophageal cancer, follicul ar dendritic cel lcarcinoma, seminal vesicl etumor, epidermal carcinoma, spleen cancer, bladder cancer, head and neck cancer, stomach cancer, liver cancer, bone cancer, brain cancer, cancer of the retina, biliary cancer, smal lbowe lcancer, salivary gland cancer, cancer of uterus, cancer of testicle cances, r of connective tissue, myelodysplasi Waldensta, rom’s macroglobinaem ia,nasopharyngeal, neuroendocrine cancer, mesothelioma angiosarcoma,, Kaposi’s sarcoma carcino, id, fallopian tube cancer, peritoneal cancer, papillary serous mulleria cancn er, malignant ascites, gastrointesti stromnal tumal or (GIST), Li-Fraumeni syndrome, Von Hippel-Lindau syndrome (VHL), and othe cancersr as described herein, and cancer of unknown origi neither primary or metastat ic,wherein such cancers may be non- metastati invasic, ve, or metastatic.

[00705] In some embodiments the present invention provides the use of immunostimulatory anti-HIDEl immune molecul ore composition containing according to at least some embodiments of the present inventio nin treati ngand/or detecting or aiding in the diagnosis of cancers that express HIDE1 at level highers than othe cancr ers. 218WO 2017/009712 PCT/IB2016/001079 8. Methods of Using HIDE1 Peptide Compositions

[00706] As discussed herein, HIDE1 is involved in the immuno-oncology pathway, which means that manipulating certain signaling pathway cans have two different effects . On one hand, the HIDE1 prote insuppresses T cel lactivation and one or more of a number of othe pathwar ys, through binding to binding and/or signaling partner. Thus, by inhibiting the interaction of HIDE 1 and its binding and/or signaling partner, for example using antibodie tos HIDE1, the suppression is alleviated, thereby increasing an immune respons toe allow treatment of conditions for which a stronger immune response is desired, such as cancer and pathogen infection. This is referre tod as a "immuno-stimulatory" response. On the othe r hand, by increasing the amount of HIDE 1 in a patient, such as by adding HIDE1 ECD polypeptide thes, suppression is increased, thereby decreasing the immune respons toe allow treatment of conditions for which a decreased immune respons ise desired, such as autoimmune diseases and inflammation.

[00707] Accordingly, once made, the HIDE1 proteins of the inventio nfind use in a variet yof applications, includin gusing them in screening assays for additional immunomodulat oryagents, as well as treatment of patients as is more fully outlined below.

[00708] With regard to the immuno-stimulatory treatment usings anti-HIDEl antibodie ands HIDE1 proteins, reference is made to USSN 62/191,775, filed July 13, 2015, entitl ed“ANTI-HIDE 1 IMMUNE MOLECULES AND THE USE THEREOF IN THERAPY AND DIAGNOSIS” and USSN 62/191,804, filed July 13, 2015, entitl ed“HIDE1 POLYPEPTIDES AND USES THEREOF IN THERAPY”, all of which is expressl y incorporated by reference in its entirety herein.

G. Treatment

[00709] According to at leas tsome embodiments of the present inventio nprovides methods of treating a number of diseases and/or conditions associated wit han immune condition. An “immune condition” includes patients who would benefit from immunostimulatory action, such as cancer or pathogen infection, as well as patients who would benefit from immunoinhibitory action, such as autoimmune diseases and inflammation. 219WO 2017/009712 PCT/IB2016/001079

[00710] The term “autoimmune diseas”e as used herei nshould be understood to encompass any autoimmune disease and further includes chronic inflammatory conditions. In some embodiments, the HIDE1 polypeptides of the invention are used to trea autoit mmune diseases. Suitable autoimmune diseases include but are not limited to multiple sclerosi s, including relapsing-remitt multing iple sclerosis, primary progress ivemultipl sclee rosi and s, secondary progressi multve ipl sclee rosis, progress iverelapsing multipl sclee rosis, chroni c progressi multve iple sclerosis, transitional/progre multssiveiple sclerosis, rapidly worsening multiple sclerosi clinicas, lly-definit multe iple sclerosi mals, ignant multiple sclerosi alsos, known as Marburg's Variant, acute multiple sclerosis, conditions relating to multipl e sclerosis, psoriasi rheums, atoid arthri tipsoriats, arthriic tigouts, and pseudo-gout juven, ile idiopathic arthri tiStis,ll's disease, rheumatoid vasculiti s,conditions relating to rheumatoid arthri tidiscois, dlupus erythematosus, lupus arthri tilupuss, pneumonitis, lupus nephriti s, conditions relating to systemi clupus erythematosus include osteoarticular tuberculosi s, antiphospholipi antidbody syndrome, systemic lupus erythematosus (SLE); discoi dlupus erythematosus, inflammator bowely disease, ulcerative coliti s,Crohn 'sdisease, benign lymphocytic angiitis throm, bocytopenic purpura, idiopathi thrombocc ytopeni idia,opathic autoimmune hemolyti anemic a, pure red cel laplasia Sjogren, ’s syndrom e,rheumati diseac se, connective tissue disease, inflammatory rheumatism, degenerative rheumatis m,extra- articular rheumatis m,juvenile rheumatoi arthrid tisarthri, tisuratic a,muscular rheumatis m, chronic polyarthrit polymyalis, gia rheumatica, mixed connective tissue disease, systemic juvenile idiopathi arthric tireactis, ve arthri ticryoglobulinemis, vasculic tis ANCA-, associate d vasculitis, antiphospholipi syndromd e,myasthenia gravis, autoimmune hemolyt anaemiic a, Guillain-Barre syndrome, autoimmune thyroidi tiHass,himoto' thyroidits priis,mary myxedema, sympatheti ophthac lmia aut, oimmune uveitis anteri, or uveitis (or iridocyclitis) , intermedia uveitiste (pars planitis), posteri uveitisor (or chorioretini panuveitictis), form, hepatit is,chronic action hepatiti colls, agen diseases, ankylosing spondylitis peria, rthr itis humeroscapula ris,panarterit nodosa,is chondrocalcinosis Wegener, ’s granulomatosi s, microscopic polyangiiti chronics, urticari bulloa, us skin disorders, pemphigoid, bullous pemphigoid, cicatrici alpemphigoid, vitiligo, atopic eczema, eczema, chronic urticaria , autoimmune urticari normoca, omplementemic urticarial vasculiti s,hypocomplementemi c 220WO 2017/009712 PCT/IB2016/001079 urticari vasculal itis, autoimmune lymphoprolifer ativesyndrome, Devic's disease, sarcoidos is, pernicious anemia, childhood autoimmune hemolyti anemic a, idiopathi autc oimmune hemolyt anemiic a, Refracto ryor chronic Autoimmune Cytopenias, Prevention of development of Autoimmune Anti-Factor VIII Antibodi esin Acquired Hemophilia A, Col d Agglutinin Disease, Neuromyelit Optis ica ,Stiff Perso nSyndrome, gingivitis periodont, iti s, pancreatit is,myocarditi vasculs, itis, gastriti gout,s, gouty arthriti ands, inflammator skin y disorders, selected from the group consisting of psoriasi atopics, dermatitisrosac urticea, aria , acne, inflammation of various part ofs the heart such, as pericardit myocaris, dit is,and endocarditi lungs, and pleura inflammation, pleurit is,pleur aleffusion, chronic diffuse interstit lungial disease, pulmonary hypertension, pulmonary embol i,pulmonary hemorrha ge, and shrinking lung syndrome, lupus headache, idiopathic pericardit myosis, iti s, demyelinating syndrome, mononeuropath mononeuriy, tis multiple myelopatx, hy, cranial neuropathy, polyneuropathy, collagenous coliti lymphocytics, coliti iscs, haemi acoliti s, diversion coliti indets, erminate colitis idiopathi, autc oimmune hemolyt anemiic a, anti- synthetase syndrome, scleritis, macrophage activation syndrome, Behcet's Syndrome, PAPA Syndrome, Blau’s Syndrome, gout ,gouty arthri tiinfls, ammatory skin disorders selected from the group consisting of psoriasis, Non pustular Psoriasis including Psoriasis vulgaris and Psoriati erythrc oderm (erythrodermia psoriasc is), Pustul arpsorias includingis Generalized pustula psorir asis (pustul arpsorias ofis von Zumbusch), Pustulosi palms aris et plantar is (persistent palmoplantar pustulosis pustul, arpsoriasis of the Barbe rtype, pustul arpsoriasis of the extremitie s),Annular pustul arpsoriasi Acroderms, atitis continua .Impetigo herpetiformi s, drug-induced psoriasis, Inverse psoriasis, Napkin psoriasis, Seborrheic-li psoriasike s,Guttate psoriasis, Nail psoriasi adults, and juvenile Stil’sl disease, cryropyrinopat chronichy, immune polyneuropath idiopathiy, diabetc esjuvenile type !diabetes, maturit onsety diabetes of the young, latent autoimmune diabetes in adults, gestational diabetes, conditions relati ngto type 1 diabetes, membranous glomerulonephropat authy,oimmune gastritis Muckl, e-Wel ls syndrome, familial cold-induce autod -inflammatory syndrom e,neonata lonset multisystem ic inflammatory disease, familial Mediterrane fever,an chronic infantil neurole ogic cuta, neous and articular syndrome, Reiter’s syndrome, rheumati fever,c relapsing polychondri tis, Raynaud's phenomenon, vasculitis cryogl, obulinemic vasculiti tems, poral arteriti giants, cell 221WO 2017/009712 PCT/IB2016/001079 arterit Takayasis, u arteriti Behcet'ss, disease, chronic inflammatory demyelinating polyneuropath insuliny, dependent diabetes mellitus, type I diabetes Addison, ’s disease, membranous glomerulonephropat polyglandulahy, autoir mmune syndromes Goodpast, ure’s disease, autoimmune gastriti autoimmunes, atrophi gastrc iti pernics, ious anaemia, pemphigus, pemphigus vulgari s,cirrhosis prima, ry biliary cirrhosis idiopathic, pulmonary fibrosis, myositis, dermatomyosi juveniltis e dermatomyosi polymyositis, tis , fibromyositi myoges, losi cels, iac disease, celiac sprue dermatit immis, unoglobul A in nephropathy, Henoch-Schonl einpurpura, Evans syndrome, dermatit atopicis, dermatit is, psoriasis, psoriasis vulgari s,psorias arthropatis hica, Graves' disease, Graves' ophthalmopat sclerhy, oderma, systemic scleroderma, progressi systve emi cscleroderm a, diffuse scleroderma, localized scleroderma, Crest syndrome, asthma, allergi astc hma, allergy, primary biliary cirrhosis fibr, omyalgi a,chronic fatigue and immune dysfunction syndrome (CFIDS), autoimmune inner ear disease, Hyper IgD syndrome, Schnitzle’sr syndrom e, autoimmune retinopathy, age-relat edmacula rdegeneratio atherosn, cleros chronicis, prostatit alopeis, cia alopec, ia areata, alopeci univea rsali s,alopecia totali autois, mmune thrombocytopenic purpura, idiopathic thrombocytopenic purpura, pure red cel laplasia and , TNF receptor-associate period dic syndrom (TRAPS)e , .

[00711] In some preferre embodimed nts, the autoimmune disease includes but is not limite tod any of the types and subtypes of any of multiple sclerosis, rheumatoid arthrit is, type I diabetes psoriasi, systs, emi lupusc erythematosus, inflammator bowey ldisease, uveitis, or Sjogren’s syndrome or related conditions thereof.

[00712] Of particular interest in some embodiments is the treatment of rheumatoid arthri tilupus,s, inflammator bowely disease, psoriasi mults, ipl sclere osis and diabetes type I.

[00713] As described herein, HIDE1 polypeptides whic hmodulate (preferably inhibit ) immunity may optionall bey used to trea ort detect “immune related diseases (or disorders or conditions”. )These phrase ors terms are used interchangeably and encompas sany disease, disorder or condition selected from the group including but not limited to autoimmune diseases, inflammatory disorders, allergi disorc ders, e.g, chronic allergi disordersc such as asthma, and immune disorders associated with graft transplanta tionrejection, such as acute and chronic rejection of organ or tissue transplantation, allogeni stemc cel ltransplantati on, 222WO 2017/009712 PCT/IB2016/001079 autologous stem cel ltransplantat boneion, marro wtransplantation, and graft versus host disease.

[00714] As furth ernoted, the present HIDE1 polypeptides which modulate (preferably inhibit imm) unity may optionall bey used to trea “tinflammator disordersy ” and/or “inflammation”. These phrase ors term sare used interchangeably herein and include e.g, inflammatory abnormaliti characterizedes by dysregulat immed une response to harmful stimuli, such as pathogens, damaged cell s,or irritant Inflams. matory disorders underli avaste variet yof human diseases. Diseases wit hetiologica originsl in inflammatory processes include but are not limited to atherosclerosis, inflammator desty ruct ionof neurons and axons and ischemic heart disease .Examples of disorders associate withd inflammatio includen but are not limite to:d Glomerulonephri Hypersentis, sitiviti Peles,vic inflammatory disease, Reperfusion injury, Sarcoidosis, Interstiti cystial ti s,normocomplementemic urticari al vasculitis, pericardit myosiis, ti s,anti-synthetas syndrome,e sclerit is,macrophage activation syndrome, Behcet's Syndrome, PAPA Syndrome, Blau’s Syndrome, gout, adult and juvenile Still’s disease, cryropyrinopat Mucklhy, e-Wel lssyndrom e,familial cold-induced auto- inflammatory syndrome, neonatal onset multisystem inflic ammator diseay se, familial Mediterrane fever,an chronic infantil neurole ogic, cutaneous and articular syndrome, systemi juvenilc e idiopathic arthri tisHyper, IgD syndrome, Schnitzl’ers syndrome, TNF receptor-associate periodid syndromec (TRAPSP), gingivitis, periodontiti hepatits, is, cirrhosis panc, reatit is,myocardit is,inflammatory skin disorders, selected from the group consisting of psoriasis, atopic dermatit eczemis, a, rosacea, urticari anda, acne.

[00715] In some embodiments the treatme ntmay be optionall combiy ned wit hanother moiety useful for treating immune related condition, e.g, a moiety useful for treating immune related condition is selected from immunosuppressa ntssuch as corticosteroi cyclosds, porin, cyclophospham ide,prednisone, azathioprine, methotrexate, rapamycin, tacrolimus , leflunomide or an analog thereof mizori; bine; mycophenol acid;ic my cophenolat mofetie l; -deoxyspergual ineor an analog thereof; biologic alagents such as TNF-a blockers or antagonist ors, any other biologic alagent targeting any inflammatory cytokine, nonsteroid al antiinflammatory drugs/Cox-2 inhibitors, hydroxychloroquine, sulphasalazopryine, gold salts , etanercept, infliximab, my cophenolat mofetie l,basiliximab, atacicept, rituxima b,cytoxan, 223WO 2017/009712 PCT/IB2016/001079 interferon P־la, interferon P־lb, glatiramer acetate, mitoxantrone hydrochloride, anakinra and/or other biologi esand/or intravenous immunoglobulin (IVIG), interferons such as IFN-- la (REBIF®, AVONEX® and CINNOVEX ®) and IFN־p־lb (BETASERON®); EXTAVIA®, BETAFERON®, ZIFERON®); glatiram aceer tat e(COPAXONE®), a polypeptide; natalizumab (TYSABRI®), mitoxantrone (NOVANTRONE®), a cytotoxi c agent, a calcineurin inhibitor, e.g, cyclosporin A or FK506; an immunosuppressive macrolide, e.g. rapamycin or a derivative thereof; e.g. 40-O-(2-hydroxy) ethyl-rapamycin, a lymphocyte homing agent, e.g. FTY720 or an analog thereof, corticosteroids ; cyclophospham ide;azathioprene; methotrexate; leflunomide or an analog thereof; mizoribine; mycophenol acid;ic my cophenolat mofetie l;15-deoxyspergual ineor an analog thereof; immunosuppressive monoclonal antibodies, e.g., monoclonal antibodie tos leukocyt e receptors, e.g, MHC, CD2, CD3, CD4, CDlla/CD18, CD7, CD25, CD27, B7, CD40, CD45, CD58, CD137, ICOS, CD150 (SLAM), 0X40, 4-1BB or their ligands; or othe r immunomodulat orycompounds, e.g. CTLA4-Ig (abatacept, ORENCIA®, belatacept) CD28-, Ig, B7-H4-Ig, or othe costir mulatory agents ,or adhesion molecul inhibie tors e.g., mAbs or low molecular weight inhibitors includin gLFA-1 antagonist Selecs, tin antagonists and VLA- 4 antagonist ors, anoth erimmunomodulat oryagent.

[00716] According to at leas tsome embodiment ofs the present invention, the method of immunotherapy in a patient optionall compy, rises one of the following: • in vivo or ex vivo tolerance induction, comprising administering an effective amount of any of an isolated or recombinan HIDE1t polypeptide as describe hered in, or a multimer or fusion prote inas describe hereind a; nucleotide sequence as described herein; an expression vector as describe hereind a; host cel las described herein, or a pharmaceutical composition to ,a patient or to leukocyte isolas ted from the patient, in order to induce differentiat ionof tolerogenic regulatory cells; • ex vivo enrichment and expansion of said cells; and/o r • reinfusion of the tolerogenic regulatory cells to said patient.

[00717] According to at least some embodiments, any one of the foregoing therapeuti c agents according to the present inventio ncan be used for adoptive immunotherapy. Immune tolerance or immunological tolerance or prolonge immunosd uppression is the process by 224WO 2017/009712 PCT/IB2016/001079 which the immune system does not attack an antigen. It can be either 'natural or' 'self- toleranc wheree', the body does not mount an immune response to self-antigens, or 'induced toleranc wheree', tolerance to external antigens can be created by manipulating the immune system. It occurs in three forms: central tolerance, peripheral tolerance and acquired tolerance Wit. hout wishing to be bound by a singl etheory, tolerance employs regulatory immune cell s- including Tregs - or potentially other immunosuppressive cells such as MDSCs, iMSCs, monocyte s,neutrophil macrs, ophages, that direct lysuppress autoreact ive cell s,as well as several other immune cel lsubsets with immunoregulatory propertie - s including CD8+ T cells and other types of CD4+ T cells (Tri, Th3), Thl7 cells, in addition to natural killer (NK), NKT cells, dendritic cells (DC) and B cells.

[00718] Tolerance or prolonged immunosuppression can be induced by blocking costimulati oron upon engagement of a co-inhibitory B7 with its counter receptor. Transfer of tolerance involves isolation of the cells that have been induced for tolerance either in vivo (i. e., prior to cel lisolation) or ex-vivo, enrichment and expansion of these cell sex vivo, followed by reinfusion of the expanded cell sto the patient This. method can be used for treatment of autoimmune diseases as recited herein, immune related diseases as recited herein, transplanta tionand graft rejection. Thus, according to at least some embodiments, the present inventio noptional providesly methods for tolerance induction, comprising in vivo or ex vivo treatme ntadministration of effective amount of any one of isolated solubl HIDE1e polypeptide, or a polypepti decomprising the extracellular domain of HIDE1, or fragment thereof, or a fusion there ofto a heterologous sequence, to a patient or to leukocyte isolas ted from the patient, in order to induce differentiat ionof tolerogenic regulatory cell s,followed by ex-vivo enrichment and expansion of said cell sand reinfusion of the tolerogenic regulatory cells to said patient. 1. Treating by inhibiting the Interaction of HIDE1 and the HIDE1 binding and/or signaling partner

[00719] In some embodiments, the invention provides methods of treating subject sby inhibiting the interaction of HIDE 1 and the HIDE1 binding and/or signaling partner. As both HIDE1 and the HIDE1 binding and/or signaling partner contain transmembrane domain, the inhibition can be done by preventing the binding of the two, such as by using anti-HIDE 1 225WO 2017/009712 PCT/IB2016/001079 antibodies Alt. ernate ly,by administering a solubl HIDe E1 polypeptide, that will interact with the HIDE1 binding and/or signaling partner, preventing it from binding to the membrane bound endogenous HIDE1, thus preventing signaling (either by the los ofs signaling due to the lack of HIDE1 signaling, or by the prevention of free HIDE1 binding and/or signaling partner binding to another of its signaling partners).

[00720] In some embodiments, the invention provides methods of treating patients by modulating the interaction of HIDE1 and the HIDE1 binding and/or signaling partner by administering a HIDE 1 polypeptide as outlined herein.

[00721] In some embodiments, the invention provides methods of treating patients by inhibiting the Interacti onof HIDE1 and the HIDE1 binding and/or signaling partner by inhibiting the binding of HIDE1 and the HIDE1 binding and/or signaling partner by administering an anti-HIDEl antibody.

[00722] According to at least some embodiments, HIDE1 therapeutic agents and/or a pharmaceutic alcompositi oncomprising same, as described herein, which function as HIDE1 agonizing therapeutic agents ,may optionally be used for treating an immune system related disease In. some instances the immune system related condition comprises an immune relat ed condition, including but not limite tod autoimmune, inflammatory or allergic diseases such as recited herein, transplant rejection and graft versus host disease.

[00723] In some instances the immune condition is selected from autoimmune disease, inflammatory disease, allergi disease,c transplant rejection, undesired gene or cel ltherapy immune responses, or graft versus host disease.

[00724] According to at least some embodiments, HIDE1 therapeutic agents and/or a pharmaceutic alcompositi oncomprising same, as described herein, which function as HIDE1 agonizing therapeutic agents ,may optionally be used for treating an immune system related disease In. some instances the immune system related condition comprises an immune relat ed condition, including but not limite tod autoimmune, inflammatory or allergic diseases such as recited herein, transplant rejection and graft versus host disease. 226WO 2017/009712 PCT/IB2016/001079

[00725] In some instances the immune condition is selected from autoimmune disease, inflammatory disease, allergi disease,c transplant rejection, undesired gene or cel ltherapy immune responses, or graft versus host disease.

[00726] In some embodiments the treatme ntis combined with another moiety useful for treating immune related condition. Non limiting examples thereof include immunosuppressa ntssuch as corticosteroi cyclds,ospori cyclophosphamin, prednisone,de, azathioprine, methotrexate rapam, ycin, tacrolim us,leflunomide or an analog thereof; mizoribine; mycophenol acid;ic my cophenolat mofetie l;15-deoxyspergual ineor an analog thereof; biologic alagents such as TNF-a blockers or antagonist ors, any other biological agent targeting any inflammatory cytokine, nonsteroidal antiinflammatory drugs/Cox-2 inhibitors, hydroxychloroquine, sulphasalazopryine, gold salts, etanercept, infliximab, my cophenola mofetite l,basiliximab, atacicept, rituximab, cytoxan, interferon P־la, interferon P־lb, glatiram aceer tate, mitoxantrone hydrochloride, anakinra and/or othe biologir esand/or intravenous immunoglobul (IVIGin ), interferons such as IFN־p1a (REBIF®. AVONEX® and CINNOVEX ®) and IFN-pib (BETASERON®); EXTAVIA®, BETAFERON®, ZIFERON®); glatiram aceer tat e(COPAXONE®), a polypeptide; natalizumab (TYSABRI®), mitoxantrone (NOVANTRONEG). a cytotoxic agent, a calcineurin inhibitor, e.g. Cyclosporin A or FK506; an immunosuppressive macrolide, e.g. Rapamycin or a derivati vethereof; e.g. 40-O-(2-hydroxy)ethyl-rapamyc a lympin, hocyte homing agent, e.g.

FTY720 or an analog thereof, corticosteroi cyclods; phospham ide;azathioprene ; methotrexate; leflunomi deor an analog thereof; mizoribine; mycophenolic acid; my cophenolat mofetie l;15-deoxyspergual ineor an analog thereof; immunosuppressive monoclonal antibodies e.g.,, monoclonal antibodie tos leukocyte receptors, e.g, MHC, CD2, CD3, CD4, CDlla/CD18, CD7, CD25, CD27, B7, CD40, CD45, CD58, CD137, ICOS, CD150 (SLAM), OX40, 4-1BB or their ligands; or other immunomodulat orycompounds , e.g. CTLA4-Ig (abatacept, ORENCIA®, belatacept) CD28-Ig, , B7-H4-Ig, or othe r costimulatory agents, or adhesion molecul inhie bitors, e.g. mAbs or low molecular weight inhibitors includin gLFA-1 antagonist Selects, in antagonis tsand VLA-4 antagonist ors, another immunomodulat oryagent. 227WO 2017/009712 PCT/IB2016/001079

[00727] In particular, treatment of multiple sclerosi usings HIDE1 immunoinhibitory proteins according to the various embodiments of the present invention may optionally e.g, be combined with, any therapeutic agent or method suitable for treating multiple sclerosi s.

Non-limiti ngexamples of such known therapeutic agent or method for treating multipl e sclerosi incls ude interferon class, IFN־P־la (REBIF®. AVONEX® and CINNOVEX ®) and IFN-P-lb (BETASERON®, EXTAVIA®, BETAFERON®, ZIFERON®); glatiram aceter ate (COPAXONE®), a polypeptide; natalizuma b(TYSABRI®); and mitoxantrone (NOVANTRONE®), a cytotoxic agent, Fampr dinei (AMPYRA®). Other drugs include corticosteroi metds,hotrexate, cyclophosphamide azathi, oprine, and intravenous immunoglobul (IVIin G), inosine, Ocrelizuma b(RI 594), Mylinax (Caldribine® ), alemtuzuma b(Campath®), daclizuma b(Zenapax®), Panaclar/ dimethy fumal rate (BG-12), Teriflunomi de(HMR1726), fmgolimod (FTY720), laquinimod (ABR216062), as well as Hematopoietic stem cel ltransplantat NeuroVaion, x@, Rituximab (Rituxan@) BCG vaccine, low dose naltrexone, helminthic therapy, angioplasty, venous stent s,and alternat ivetherap y, such as vitamin D, polyunsaturate fatsd ,medical marijuana.

[00728] Similarl treatmy, ent of rheumatoid arthri tiusings, HIDE1 immunoinhibitory proteins according to the various embodiments of the present invention may optionally be combined with, for example, any therapeutic agent or method suitable for treating rheumatoid arthri tis.Non-limiti ngexamples of such known therapeutic agents or methods for treati ng rheumatoid arthr itiincludes glucocortico ids,nonsteroidal anti-inflammatory drug (NSAID) such as salicylates or, cyclooxygenase-2 inhibitors, ibuprofen and naproxen, diclofenac , indomethacin, etodolac Disease-modifying antirheumat drugsic (DMARDs)- Ora lDMARDs: Auranofin (Ridaura@), Azathiopri (Imurne an®), Cyclospor ine(Sandimmune®, Gengraf, Neoral, generic), D-Penicillamin (Cuprime ine), Hydroxychloroqu (Plaqueine nil® ),IM gold Gold sodium thiomal ate(Myochrysine@) Aurothiogluc (Solganaose l@), Leflunomide (Arava®), Methotrexate (Rheumatrex®), Minocycline (Minocin®), Staphylococcal prote inA immunoadsorption (Prosor column),ba Sulfasalazine (Azulfidine@ ).Biologic DMARDs: TNF-a blockers including Adalimumab (Humira®) Etanercept (Enbrel® ),Infliximab (Remicade®) ,golimumab (Simponi®), certolizuma pegolb (Cimzia@), and other biological 228WO 2017/009712 PCT/IB2016/001079 DMARDs, such as Anakinra (Kineret), Rituximab (Rituxan@), Tocilizumab (Actemra@ ), CD28 inhibitor includin gAbatacept (Orencia®) and Belatacept.

[00729] Thus, treatment of IBD, using the agents according to at leas tsome embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating IBD. Non-limiti ngexamples of such known therapeutic agents or methods for treating IBD include immunosuppressio to ncontrol the symptom, such as prednisone, Mesalazine (including Asacol® Pent, asa® ,Lialda®, Aspiro®, azathioprine (Imuran®), methotrexate, or 6-mercaptopuri ne,steroids, Ondansetron®, TNF-a blockers (including infliximab, adalimumab golimumab, certolizumab pegol), Orencia® (abatacept), ustekinumab (Stelara®), Briakinuma b(ABT-874), Certolizuma pegolb (Cimzia@), ITF2357 (Givinostat® Nat), alizumab (Tysabri®), Firategrast® (SB-683699), Remicade® (infliximab), vedolizumab (MLN0002), othe drugsr includin gGSK1605786 CCX282-B (Traficet-EN®), AJM300, Stelara® (ustekinumab), Semapimod® (CNI-1493) tasocitinib (CP-690550), LMW Heparin MMX, Budesonide MMX, Simponi® (golimumab), MultiStem ®,Gardasil@ HPV vaccine, Epaxal® (virosomal hepatiti As vaccine), surgery, such as bowe lresection, stricturepla orsty a temporary or permanent colostomy or ileostomy; antifungal drugs such as nystat in(a broad spectrum gut antifungal and) either itraconazole (Sporanox) or fluconazole (Diflucan); alternat ivemedicine, prebioti csand probiotics, cannabis, Helminthic therapy or ova of the Trichuris suis helminth.

[00730] Thus, treatment of psoriasi usings, the agents according to at least some embodiments of the present inventio nmay optional bely combined with, for example, any known therapeutic agent or method for treating psoriasi Non-ls. imiti ngexamples of such known therapeutic fors treating psorias inclis ude topical agents ,typically used for mild disease, phototherap fory moderat disease,e and systemi cagents for severe disease Non-. limitin examg ples of topica agentsl :bath solutions and moisturizers, mineral oil, and petroleum jelly; ointment and creams containing coal tar, dithranol (anthralin), corticoster oids like desoximetasone (Topicort), Betamethasone, fluocinonide, vitamin D3 analogues (for example, calcipotri ol),and retinoids. Non-limiti ngexamples of photothera sunlight;py: wavelength ofs 311-313 nm, psoral enand ultraviolet A phototherap (PUVAy ). Non-limiting examples of systemic agents :biologies, such as interleuki antagonistn TNF-s, a blockers 229WO 2017/009712 PCT/IB2016/001079 including antibodies such as infliximab (Remicade®), adalimumab (Humira®) ,golimumab, certolizumab pegol and, recombinan tTNF-a decoy receptor, etanercept (Enbrel®); drugs that target T cells, such as efalizumab (Xannelim®/Raptiva®), alefacept (Ameviv®), dendritic cells such Efalizumab ;monoclonal antibodie (MAbss ) targeting cytokine s,including anti -IL- 12/IL-23 (ustekinumab (Stelara®)) and anti-Interleukin-17; Briakinumab® (ABT-874); small molecules, includin gbut not limite tod ISA247; immunosuppressants, such as methotrexate, cyclosporine; vitamin A and retinoid (synthes tic forms of vitamin A); and alternat ivetherapy, such as changes in diet and lifestyle fasti, ng periods, low energy diet sand vegetarian diets , diet ssupplemented wit hfish oil rich in vitamin A and vitamin D (such as cod liver oil ),fish oil srich in the two omega-3 fatty acids eicosapentaenoi acic d (EPA) and docosahexaenoi c acid (DHA) and contain vitamin E, ichthyotherapy, hypnotherapy, and cannabis.

[00731] Thus, treatment of type 1 diabetes using, the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating type !diabetes. Non-limiti ngexamples of such known therapeuti forcs treating type 1 diabetes include insulin, insulin analogs isl, et transplantat ion,stem cel ltherapy including PROCHYMAL®, non-insuli therapiesn such as il-1p inhibitors including Anakinra (Kineret®), Abatacept (Orencia®), Diamyd, alefacept (Ameviv®), Otelixizumab, DiaPep277 (Hsp60 derive dpeptide), a 1-Antitrypsi Prednin, sone, azathioprine, and Cyclospori El-INn, T (an injectabl islete neogenesi stherapy comprising an epidermal growt facth or analog and a gastrin analog), statins includin gZocor®, Simlup@, Simcard®, Simvacor®, and Sitagliptin® (dipeptidyl peptidase (DPP-4) inhibitor), anti-CD3 mAb (e.g., Teplizumab®); CTLA4-Ig (abatacept), anti-IL-1p (Canakinumab), Anti-CD20 mAb (e.g, rituximab) and combinations thereof.

[00732] Thus, treatment of uveitis, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating uveitis. Non-limiti ngexamples of such known therapeuti forcs treating uveitis include corticosteroids topic, al cycloplegics such, as atropine or homatropine, or injection of PSTTA (posteri subtenonor triamcinolone acetate ), antimetaboli medite cations, such as methotrexat TNF-ae, blockers (including infliximab, adalimumab, etanercept, golimumab, and certolizum pegolab ). 230WO 2017/009712 PCT/IB2016/001079

[00733] Thus, treatment for Sjogren’s syndrome, using the agents according to at least some embodiments of the present inventio nmay optionall bey combined with, for example, any known therapeutic agent or method for treating for Sjogren’s syndrome. Non-limiting examples of such known therapeuti forcs treating for Sjogren’s syndrome include Cyclosporine, pilocarpine (Salagen@) and cevimeline (Evoxac®), Hydroxychloroquine (Plaquenil), cortisone (prednisone and others) and/or azathioprine (Imuran®) or cyclophosphami (Cytoxade n®), Dexamethasone, Thalidomide, Dehydroepiandrosterone, NGX267, Rebamipide@, FID 114657, Etanercept® Rapti, va®, Belimumab, MabThera® (rituximab); Anakinra@, intravenous immune globulin (IVIG), Allogenei c Mesenchymal Stem Cell s(A110MSC®), and Automat icneuro-electrostimulat by "Saionliwel l Crow”n.

[00734] Thus, treatment for systemi clupus erythematosus, using the agents according to at least some embodiments of the present invention may optionally be combined with, for example, any known therapeutic agent or method for treating for systemic lupus erythematosus. Non-limiti ngexamples of such known therapeuti forcs treating for systemic lupus erythematosus include corticosteroid and Dises ase-modifying antirheumat drugsic (DMARDs), commonly anti-malarial drugs such as plaqueni land immunosuppressa nts(e.g. methotrexate and azathioprine Hydroxychloroquine,) cytotoxic drugs (e.g, cyclophosphami andde my cophenolate) Hydroxychl, oroquine (HCQ), Benlysta ® (belimumab), nonsteroidal anti-inflammatory drugs, Prednisone, Cellcept®, Prograf® , Atacicept®, Lupuzor®, Intravenous Immunoglobuli ns(IVIGs), CellCept® (mycophenolate mofetil Orencia), ®, CTLA4-IgG4m (RG2077), rituximab, Ocrelizumab, Epratuzumab, CNTO 136, Sifalimumab (MEDI-545), A-623 (formerly AMG 623), AMG 557, Rontalizuma b,paquinimod (ABR-215757), LY2127399, CEP-33457, Dehydroepiandrosterone, Levothyroxine, abetimus sodium (LIP 394), Memantine®, Opiates , Rapamycin®, renal transplantat steion,m cel ltransplanta tionand combinations of any of the foregoing.

[00735] The immunoinhibitory HIDE1 therapeutic agents and/or a pharmaceutical compositi oncomprising same, as recited herein, according to at least some embodiments of the present invention, may optionall bey administered as the sole active ingredient or together 231WO 2017/009712 PCT/IB2016/001079 with othe drugsr in immunomodulating regimens or other anti-inflammatory agents e.g. for the treatment or prevention of allo- or xenograft acute or chronic rejection or inflammatory or autoimmune disorders, or to induce tolerance.

EXAMPLES

[00736] Specifically incorporat byed reference herein is USSN 62/191,775, filed July 13, 2015, and USSN 62/191,804, filed July 13, 2015, in thei entiretr andy, in particular for the Examples therein, and for the associated Figures and Legends.

EXAMPLE 1: RATIONAL FOR EVALUATING THE EFFECT OF ANTI-HIDE 1 ABS

[00737] The presented dry examples 2-6 below suggest a set of functional assays to evaluat ethe effect of anti-HIDEl on T cell function. In particular, the suggeste dassays will be used to evaluate the iinm uno-modulatory effect of anti-HIDEl antibodie (agnosts icor antagonist ic)that can be used to target monocyte s,Tumor associated, macrophages (TAMs) or other myeloid cells and screen for various functional activities includin, gmodulating the interaction between HIDE-1 and its putative receptor(s), modulati onof HIDE1 level sor direc signalt ing and attenuati onof negative signaling and/or depletion of HIDE! positive cells. Such recombinant antibodie mays be used as modulatory molecules to decrease or preven tHIDE1 from interacting with inhibitory receptor(s) on T cells or other cell sin the tumor microenvironmen theret, by releasing T cells or other functional cells from HIDE! check point (“break”)/suppressi vesignaling EXAMPLE 2: MIXED LYMPHOCYTE REACTION (MLR)

[00738] A mixed lymphocyt reactie on will be employed to demonstrat thee effect of blocking the HIDE1 pathway to lymphocyte effector cells. T cells in the assay will be tested for proliferation and cytokine secretion in the presence or absence of an anti-HIDEl human monoclonal antibodies Huma. n CD4+ or CD8+ T-cells will be purified from PBMC using a CD4+ or CD8+. Alloreactivi dendriticte cell swill be derived from purified monocytes culture withd 1000 U/ml of IL-4 and 500 U/ml of GM-CSF (R&D Biosystems) for seven days. Monocytes wil bel prepared using a monocyte negative selection kit (Mitenyi Biotech). 232WO 2017/009712 PCT/IB2016/001079 Each culture will contain 105 purified T-cells and 104 allogenei dendric tic cells in a total volume of 200 pl. Anti-HIDEl blocking or agonist icmAb at varying concentrations will be added to each culture at different antibody concentrations. Either no antibody or an isotype control antibody will be used as a negative control Afte. rday 5, the effect of anti-HIDEl antibodie ons T cel lproliferati (CFSEon dilution) and cytokine secretion (ELISA or TH1/2/17 CBA kits in) culture supernatants wil bel assessed.

EXAMPLE 3: THP-1-JURKAT OR PRIMARY T CELL CO-CULTURE

[00739] 96-well flat-bottom plates will be coated wit hmouse anti-human CD3 antibody (1 ug/ml in PBS; Clone HIT3a; BD Pharmingen Cat 555336) overnight at 4' C. The next day, Jurkat cell s(50,000) or CFSE-labeled primary human CD4+ or CD8+ T cells will be plated in the pre-coate platd es. Mytomicin C treated (50pg/ml, Ihr) THP-1 cell s,which express HIDE1, (50,000) will be added to the culture in the presenc eof HIDE1 blocking or agonisti mAbc at varying concentrations. After l-5d at 37'C and 5.0% CO2, the effect of anti- HIDE1 antibodie ons T cel lproliferati (CFSEon dilution and) cytokine secretion (ELISA or TH1/2/17 CBA kits in) culture supernatants wil bel assessed.

EXAMPLE 4: RECALL RESPONSE OF TETANUS TOXIN OR CMV SPECIFIC T CELLS

[00740] Purified human T cells will be labeled wit hCFSE and cultured wit h autologous monocyte-derived DCs in the presence of tetanus toxoid (TT) or CMV antigens.

The effect of the inclusion of HIDE 1 blocking or agonisti mAbc on TT or CMV specific T cel lresponse wil bel evaluated. Afte r5-10d at 37'C and 5.0% CO2, the effect of anti-HIDEl antibodie ons T cel lproliferati (CFSEon dilution) and cytokine secretion (ELISA or TH1/2/17 CBA kits in) culture supernatants wil bel assessed.

EXAMPLE 5: PLATE BOUND ANTI-CD3 AND HIDE1-FC ASSAY

[00741] Purified human T cells will be CFSE labeled and stimulate withd anti-CD3 Ab (OKT3, lug/ml) togethe withr plate-coated HIDEl-Fc or control prote in(FLAG-Fc). Control or HIDE1 mAb was added durin gcel lculture. Cells were gated on CD8+ T cells, and their division was analyzed based on the dilution of CFSE. Inclusion of a HIDE1 neutralizi ngmAb 233WO 2017/009712 PCT/IB2016/001079 is expected to enhance the T cel lfunction which will indicate that HIDE1 interact wits h putative receptor(s) expressed on T cells to inhibi tT cel lproliferation.

EXAMPLE 6: CHOS-OKT3 CO-CULTURE ASSAY

[00742] CFSE-labeled T cells will be stimulate withd stimulat cellsor (CHO cell s expressing expressing membrane-bound anti-CD3 mAb fragments). CHOS-stimulator cell s expressing human HIDE1 and control stimulator cells (empty vector) treate withd mitomyci n C before co-cultured with CFSE-labeled human T cell sat the rati oof 1:5. After 5d at 37°C and 5.0% CO2, the effect of anti-HIDE1 antibodie ons T cel lproliferation (CFSE dilution) and cytokine secretion (ELISA or TH1/2/17 CBA kits in) culture supernatants will be assessed.

EXAMPLE 7: EXPRESSION ANALYSIS OF HIDE1 PROTEINS

[00743] The purpose of thi sexample was to demonstrate that HIDE 1 is correlate to d CSF1R expression in cancer sample sof both human and mouse. Solid tumors contain a significant population of tumor-infiltrati myelng oid cell s(TIM). TIMs are now recognized as important mediators of not only tumor progression and metastasi buts, also therapeutic resistance, through promoting angiogenesis and suppressing antitumor immune responses.

The pro-tumorige rolenic of “alternativel” actiy vated macrophages, which are part of the TIM population, has been well established. Recently, another specific subtype of TIMs, namely myeloid-derived suppressor cells (MDSC; (Myeloid-Derived Suppressor Cells - an additional population of myeloid cells that is tumo promr otin Journag lfor ImmunoTherapy of Cancer 2013, 1:10 )), is receiving great attenti onin cancer researc h.MDSCs comprise a heterogeneous population of immature myeloid cells that originate in the bone marrow and are recruited to the tumor by a diverse arra yof cytokine and chemokine signals. Similar to tumor-associate macrophagesd (TAM), MDSCs have been shown to generat ean environment favorable for tumor bys heightening immunosuppressi on,angiogenesis and, invasion.

Various cel lsurface markers are used to identify TIM subsets TAM: s can be identified by CDllb and F4/80, and MDSCs by CDllb and Gr-1 coexpression in murine model s.

Macrophage colony-stimulating factor (M-CSF or CSF1) is a potent growt facth or that promot thees differentiation, proliferat ion,and migration of monocytes/macrophages via 234WO 2017/009712 PCT/IB2016/001079 signaling through its receptor tyrosi nekinase CSF1R (cFMS). It was recently showed that TAMs and MDSCs form a spectrum of bone marrow-derived myeloid cell sdependent on CSF1/CSF1R signaling for recruitmen intot the tumor and that they play critic alrole ins tumor growth In. addition, DeNard oand colleagues highlighte thed importance of CSF1/CSF1R signaling in the recruitment of TAMs in various cancers and further showed that CSF1R blockade can inhibi tTAMs and improve treatment outcom (Cancee r Res. 2013 May l;73(9):2782-94).

[00744] As can be seen in Table 18, HIDE1 was found to be highl correly ate to d CSF1R in various mouse tumor model s.This finding was also validate ind human tumor bys correlati HIDng E1 and CSF1R using The Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov/l. _dataFigure 3 demonstrate thes high correlati ofon HIDE1 to CSF1R in human colon cancer, as an example. Across all tumors, in TCGA, HIDE1 shows strong correlati toon myeloid/TAM markers such as CD86 and CD68 (Figure 5). HIDE1 is also found direct lyon MDSCs derived from mouse tumor model Figures 7. Specifically, as shown in Figure 7, in MDSC HIDE1 was found to be induced in later stages of tumor progressi on.Without wishing to be limited by a singl etheory, these data indicate that HIDE1 is expressed and inducible on tumor associated macrophages and thus can serve as a target for immunotherapy in cancer, particularl thosy cancerse wit ha strong myeloid infiltration, (such as Bladder cancer, Lower grade glioma, colon adenocarcinoma, glioblastoma multiform heade, and neck squamous cel lcancer, kidney renal clear cell cancer, kidney renal papillar cancy er, liver cancer, lung adenocarcinom lunga, squamous cel lcarcinoma, pancreatic adenocarcinom rectaa, adenocarcil noma, melanoma, stomach adenocarcinoma , testicula germr cel lcarcionoma and thyro carciona;id Figure 8 - correlati ofon CSF1R and HIDE1 in TCGA). In addition, HIDE1 showed higher expression in melanoma patients that did not respond to anti PD-1 therapy (Figure 63. Legend for figure: HIDE1 expression in patients treated wit hPD-1 inhibitor (ref:GSE78220, Hugo W, Zaretsky JM, Sun L, Song C et al. Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastat ic Melanoma. Cell 2016 Mar 24;165(l):35-44)). These data suggest anti HIDE1 therapy could be beneficial to patients resistant to anti PD1 therap y.Expression analysis of the Genotype Tissue Expression (GTEx) data (http://www.nature.com/ng/joumal/v45/n6/full/ng.2653.htm; l 235WO 2017/009712 PCT/IB2016/001079 http://www.gtexportal.org/) showshome/ high expression of HIDE1 in blood cells and tissues with enriched blood cell ssuch as the spleen (Figure 88). Analysis of HIDE1 expression in the BioGPS database indicate salso blood specific expression with a prominent myeloid expression (http://biogps.org/#goto=genereport&i;d=255809 Figure 89).

[00745] Figure 11 present integrs ate HIDE1d gene expression analysis in cancer tumors, based on the data analysi sfrom 5 major initiatives, namely, The Cancer Genome Characterizati Initiaton ive(http://cgap.ncinih.gov/cgc. i.htm), TARGl ET: Therapeutical ly Applicable Research To Generate Effective Treatment s (https://ocg.cancer.gov/programs/), targTCGAet: The Cancer Genome Atlas (https://tcga - data.nci.nih.gov/tcga/), ICGC: International cancer genome consortium (http://icgc.org), Multiple Myeloma Genomics Porta (https:l //www.broadinstitute.org/m).mgp/home This data shows that in addition to blood neoplasms like Acute myeloid leukemia and Diffuse large B cel llymphoma, HIDE1 is also expressed in a variet yof soli tumorsd like Glioblastoma, kidney lung and skin cancers (Figure 11). Furthermore analysi sof differential expression between norma land cancer using both TCGA and the GTEx data showed that HIDE1 is upregulated in breast, skin, kidney and ovarian cancers (Figure 12). Meta-analysi ofs gene enrichment demonstrated a strong signature of immune response, regulation by interferon gamma, leukocyte chemotaxi ands lymphocyt activatie on (Figure 16).

[00746] The analysis was performe usingd both human and mouse tumor expression data set sfrom Gene Expression Omnibus (GEO) (www.ncbi.nlm.nih.gov/GEO, the platform used were GPL570, GPL6244 for human, and GPL1261 and GPL6246 for mouse). The methodology of analysi sis described in the “methodolo sectigy on”. HIDE1 was found to be highl correly ate to dCSF1R and is a type I membrane protein (http://www.uniprot.org/uniprot/) A8MVin muriS5ne tumors Table. 18 shows the correlati on of AB124611 (the murine orthologue of human HIDE1) in several caner models in mice.

Methodology:

[00747] Raw data is downloaded from the GEO sit ein SOFT format In. case that the raw data is in MAS5 format data is take nwithout manipulation. If the data is Log MAS5 then 236WO 2017/009712 PCT/IB2016/001079 the Log is powered to linear data. If the data is in RMA format CEL file sare downloaded and re analyzed using MAS 5. If such data is not availabl thee RMA format is used.

[00748] Data is then normalized by multiplicat accordive ing to the 95th percentil fore Affy data.

[00749] Dataset analyzed:s GSE49910 , GSE47855, GSE39397, GSE36765, GSE27928, GDS4343, GDS4617, GDS4371, GDS3953, GSE35398, GSE21927.

Table 18: Correlation of HIDE1 to CSF1R in various tumor models. study correlati ofon tumor type tumo rmodel accession AB124611 toCSFIR number hepato cellula carcinomar Pdgf-c Tg GSE31431 0.790 breast cancer MMTV/c-MYC GDS3953 0.664 MYCN overexpressi on neuroblastoma and los ofs GDS4617 0.930 caspase-8 expression pancreati cancerc KrasG12D GDS4343 0.798

[00750] The purpose of thi sexample is to present the involvement of HIDE1 in autoimmune disorders.

[00751] The role of myeloid cells in autoimmune disease has been elucidated in recent years. Of the myeloid cell population Myeloid-Derived Suppressor Cells (MDSCs) seem to have a major role in autoimmunit y.Understandi ofng the origination and functions of MDSCs has come mainly from studies in tumo rmodel ands from cancer patients. MDSCs are 237WO 2017/009712 PCT/IB2016/001079 involved in a number of different autoimmune disorders, including multipl scle erosi (MS),s type 1 diabetes rheum, atoid arthri (RA),tis inflammator bowely diseas e(IBD) and autoimmune hepatiti Ins. steady state conditions, MDSCs resid primarie lyin the bone marrow. Under pathologica condil tions MDSC, populations expand and can be detected in the spleen, lymph nodes, cancerous tumors, and bloodstr eam(World J Immunol 2014 March 27; 4(1): 26-33). In the chronic inflammator condiy tion of IBD, there are complex interactions between several immune cells infiltrat inginto the intestinal mucosa, wit h epithelial cell seven ignoring the effects of microbiota. Among them, myeloid cell s,includin g neutrophils, macrophages, and MDSCs, have been a focus of study due to their divergent role in inflammation. In particular, the immunosuppressive function of MDSCs was suggeste din several mouse models of IBD. It was reported that CD1 lb+Gr-l+ MDSCs were accumulated in a murine colitis model, and they expressed nitric oxide synthase 2 and arginase ,which are known to be critical functional mediato rsof MDSCs. As wel las in a model of IBD, CD14+HLA-DR10W MDSCs wit hsuppressi vefunctions were reported to be increased in the peripheral blood of IBD patients (Intest Res. 2015 Apr; 13(2): 105-111.). The expression of murine HIDE1 is induced in colon tissues derived from 2 different mouse DSS model ofs IBD (Figure 2 and Figure 4). In Figure 2 the inductio isn higher as disease progre ss(highest expression on day 6). In Figure 4 the inductio ofn HIDE 1 occurs both in the proximal and the dist alcolon The. expression in autoimmune diseases is also derive dfrom the myeloid component as can be seen in Figure 6, in which the correlati betweenon HIDE1 and CD1 lb (marke rfor myeloid and MDSCs) is plotted in multiple autoimmune samples as well as allergy derive dhuman samples (sourc immunolande Omicsoft), the correlati whicon h is near 0.9 indicates that the expression of HIDE1 in thes sample es is derived from the myeloid component These. resul tsindicat ethat the expression of HIDE 1 is inducible in IBD specifically and in autoimmune in genera ltherefore its modulation can resul ist beneficial therapeutic effect in autoimmune conditions.

[00752] Expression analysis of the Genotype Tissue Expression (GTEx) data (http://www.nature.com/ng/joumal/v45/n6/full/ng.2653.htm; l http://www.gtexportal.org/) showedhome/ high expression of HIDE1 in blood cells and tissues wit henriched blood cells such as the splee n(Figure 9). Analysis of HIDE1 238WO 2017/009712 PCT/IB2016/001079 expression in the BioGPS database indicated also blood specific expression wit ha prominent myeloid expression (http://biogps.org/#goto=genereport&id=255809; Figure 10).

EXAMPLE 8: GENERATION AND CHARACTERIZATION OF HIDE1- EXPRESSING STABLE TRANSFECTANT CELL POOLS

[00753] Recombinant stabl poolse of cel lline soverexpressing HIDE1 human proteins were generated, for use in determining the effects of HIDE1 on immunity, for HIDE1 characterizati anti-Hon, IDEl antibody discovery, and to obtain cross-spec iesanti-HIDE1 immune molecules.

Materials & Methods Human HIDE1 Reagents DNA expression constructs: Human HIDE1 flag pUC57 (Genscript) Human HIDE1 flag pMSCV (in house) Recombinant cells: HEK293 Human HIDE1 flag pMSCV (in house) SK-MEL-5 Human HIDE1 flag pMSCV (in house) HIDE1 Specific Antibody: Anti cl90rf38 (HIDE1), sigma cat. HPA012150 Rabbit anti human HIDE1 pAb (GenScript, # 488536 4) Mouse anti human HIDE1 mAh (Biotem #33B4-2F7) Mouse anti human HIDE1 mAh (Biotem #36C1-2F6) Mouse anti human HIDE1 mAh (Biotem #39A7-3A10-3G8) Expression constructs

[00754] Full lengt hcloning of human HIDE1 was performe atd Genscript by gene synthesis using codon optimized sequence in pUC57 vector and subcloned into a retrovir al expression vector.

Construct encoding Human HIDE1-flag

[00755] Full lengt hcloning of human HIDE1 flag was performe byd gene synthesis in pUC57 vector and subcloned into a retrovir expressal ion vector, pMSCV, to create the expression plasmid. 239WO 2017/009712 PCT/IB2016/001079 Establishment of stable cells pools ectopically expressing HIDE1 proteins

[00756] The resulting expression constructs were transduce tod cell sand stabl poole cel llines were generated as detailed below The. prote insequences encoded by the expression constructs are detail edherein.

Establishment of stable pool HEK293 Flag human HIDE1 expressing cells.

[00757] GP2-293 packaging cell line (Clontech cat#631458) was transfect edwith pMSCV-human HIDE1 Flag or with pMSCV- empty vector using Lipofectamin 2000 reagen t (Invitrogen, cat No: 11668-019). 48 hours post transfection supernatants containing virions were collecte andd, direct lyused for infection of PT67 stabl viruse producing cells (Clontech #631510). 48 hour post infection Puromycin antibiot wasic added at concentration of 2pg/ml, and resistant colonies were selected for stabl poole generation. Afte rselection of PT67, stably human HIDE1 virions producing cells, supernatant containing virions were collect edand used for infection of HEK293 cel lline .48 hour post infection, Puromycin antibiot wasic added at concentration of lug/ml and, resistant colonies were selected for stabl poole generation.

Establishment of Stable pool SK-MEL-5 Flag human HIDE1 expressing cells.

[00758] GP2-293 packaging cell line (Clontech cat#631458) was transfect edwith pMSCV-human HIDE1 Flag or with pMSCV- empty vector using Lipofectamin 2000 reagen t (Invitrogen, cat No: 11668-019). 48 hours post transfection supernatants containing virions were collecte andd, direct lyused for infection of PT67 stabl viruse producing cells (Clontech #631510). 48 hour post infection Puromycin antibiot wasic added at concentration of 2pg/ml, and resistant colonies were selected for stabl poole generation. Afte rselection of PT67, stably human HIDE1 virions producing cell s,supernatant containing virions were collect edand used for infection of SK-Mel-5 cel lline .48 hour post infection, Puromycin antibiot wasic added at concentration of 0.5pg/ml, and resistant colonies were selected for stabl poole generation. 240WO 2017/009712 PCT/IB2016/001079 Expression validation Expression validation by Flow cytometry (FACS)

[00759] In order to validate the cel lsurfac eexpression of the human HIDE1 protei inn the recombinant stabl pools,e 2x105 cells were staine dwit hFixable viability stain 450 (BD, cat #562247) diluted 1:1000 in PBS, for 15 min at R.T. followed by cells washing with PBS.

Antibodi eswere used as following: • pAbs anti-human HIDE1 (GenScript #488536_, 4, #48853613) or Rabbit IgG whol mole ecul (Jae ckson, cat #011-000-003) were added to cell s(5pg/ml diluted in 0.5%BSA in PBS) followed by staining with Donky anti Rabbit-PE diluted 1:100 in 0.5%BSA in PBS. • mAbs anti-human HIDE1 (Biotem, #33B4-2F7, #36C1-2F6, #39A7-3A10- 3G8) or Mouse IgGl (Life technologies, Cat #MG100) were added to cell s(10ug/ml diluted in 0.5%BSA in PBS) followed by staining with Goat anti-mouse-PE (Jackson, cat #115-116-146), diluted 1:75 in 0.5%BSAin PBS.

Results

[00760] In order to veril ycell surface expression of human HIDE1 Flag protein, the cell s,describe dabove were analyzed by FACS using Rabbit anti human-HIDEl pAb (GenScript) as describe ind Material and Methods. As shown in Figure 13, the binding of anti-human HIDE1 pAbs to the HEK293 cells expressing human HIDE1 Flag prote in(light blue line) is higher than the binding of Rabbit IgG isotype control (pink line).

[00761] Figure 13 presennts FACS analysis of ectopically expressed HEK293 cells expressing human HIDE1 Flag pMSCV vector HEK293 cell sexpressing the human HIDE1 Flag were analyzed by FACS using Rabbit anti Human HIDE1 (GenScript light, blue line).

Rabbit IgG (Jackson, pink line) was used as isotype control. Detection was carried out using donkey anti-rabbit PE-conjugated secondary antibody and analysis by FACS.

[00762] The surfac eexpression of human HIDE1 Flag protei onn HEK293 cells transduce withd human HIDE1 Flag pMSCV vector or HEK293 cell stransduce withd the empty vector were further analyzed using mouse anti-human HIDE1 mAb (Biotem) as described in Material and Methods above. As shown in Figure 14, the binding of all the three 241WO 2017/009712 PCT/IB2016/001079 anti-human HIDE1 mAbs (A-C) to the HEK293 cells expressing human HIDE1 Flag prote in (green line) is higher than the binding of thes antibodiee tos the empty vector cells (orange line) or higher than the binding of the IgGl isotype control to HEK293 cell sexpressing human HIDE1 Flag prote in(light blue line) or to the empty vector cells (red line).

[00763] Figure 14 present FACSs analysi sof ectopicall expresy sed HEK293 cells expressing human HIDE1 Flag pMSCV vector using anti human HIDE1 mAbs. HEK293 cells expressing the human HIDE1 Flag or HEK293 pMSCV empty vector were analyzed by FACS using mouse anti Human HIDE1 mAbs (Biotem A-C,, green line or orang eline respectivel ory) using Mouse IgG isotype control (light blue or pink line respectively).

Detection was carried out using goat anti Mouse-PE-conjugat edsecondary Ab.

A. SK-MEL-5 cells overexpressing human HIDE1- flag pMSCV vector

[00764] SK-MEL-5 cells transduce witd hhuman HIDE1 Flag pMSCV vector or wit h the empty vector were analyzed by FACS for thei celr lsurfac eexpression using Rabbit anti human-HIDEl pAb (GenScript) as described in Materia andl Methods. As shown in Figure , the binding of anti-human HIDE1 pAbs to the SK-MEL-5 cell sexpressing human HIDE1 Flag prote in(light blue line) is higher than the binding of Rabbit IgG isotype control (pink line) to the same cells.

[00765] Figure 15 present FACSs analysi sof ectopically expressed SK-MEL-5 cell s expressing human HIDE1 Flag pMSCV vector SK-MEL-5 cells expressing the human HIDE1 Flag were analyzed by FACS using Rabbit anti Human HIDE1 (GenScript light, blue line). Rabbit IgG (Jackson, pink line) was used as isotype control Detect. ion was carried out using donkey anti-rabbit PE-conjugated secondary antibody Ab and analysi sby FACS.

B. Generation and Characterization of Stable Cell Pools Over-expressing HIDE1 Protein Materials & Methods Reagents DNA expression constructs: Human HIDE1 flag pUC57 (Genscript) Human HIDE1 flagpCDNA3.1 (Genscript) 242WO 2017/009712 PCT/IB2016/001079 Mouse HIDE1 flag pUC57 Mouse HIDE1 flag pCDNA3.1 Mouse HIDE1 flag pMSCV Recombinant cells: HEK293 Human HIDE1 flag pCDNA3.1 (GenScript) HEK293 Mouse HIDE1 flag pMSCV EL4 Mouse HIDE1 flag pMSCV HIDE1 Specific Antibody: Commercial pAb, rabbit anti cl90rf38 (HIDE1), sigma cat. HPA012150 Custom pAb, rabbit anti mouse HIDE1 (GenScript #, 488536 13) Expression constructs

[00766] Full length cloning of human and mouse HIDE1 was performe atd Genscript by gene synthes isusing codon optimized sequence in pUC57 vector and subcloned into a mammalian or retrovi ralexpression vector.

Construct encoding human HIDE1-flag

[00767] Full length cloning of human HIDE1 flag was performed by gene synthes isin pUC57 vector and subcloned into a mammalian expression vector, pcDNA3.1, to creat ethe expression plasmid (done by GenScript) Construct encoding mouse HIDE1-flag

[00768] The cloning of mouse HIDE1 -Flag retrovi ralexpressing construc ist described in example 8 section A.

Construct encoding cyno HIDEl-untagged

[00769] Full lengt hcloning of cyno HIDE1 untagged was performe byd gene synthesis in pUC57 vector and subcloned into a mammalian expression vector, pcDNA3.1, to create the expression plasmid (done by GenScript) Establishment of stable cells pools ectopically expressing HIDE1 proteins

[00770] The resulting expression constructs were transfect edor transduce tod cells and stabl poole cel lline swere generated as detailed below. The protei sequencesn encoded by the expression constructs are detaile below.d 243WO 2017/009712 PCT/IB2016/001079 Nucleic acid Sequence of human HIDE1-flag (SEQ ID NO:289) ATGCCCTGGA CTATTCTCCT CTTTGCCGCC GGTTCTCTCG CCATTCCCGC CCCTTCTATC AGACTCGTGC CACCCTACCC CTCTTCCCAG GAGGACCCTA TCCACATTGC TTGCATGGCA CCTGGCAACT TCCCAGGAGC AAATTTTACC CTGTACCGAG GAGGACAGGT GGTCCAGCTG CTCCAGGCCC CAACCGATCA GAGGGGCGTG ACATTCAACC TGTCTGGAGG TAGCTCCAAG GCACCAGGAG GACCATTTCA TTGTCAGTAT GGGGTGCTGG GCGAGCTCAA CCAGTCACAG CTGAGCGACC TCTCCGAACC CGTGAATGTC AGTTTCCCCG TGCCTACATG GATCCTGGTC CTCAGCCTGT CCCTCGCAGG AGCTCTGTTT CTGCTCGCTG GTCTGGTGGC AGTCGCCCTC GTGGTCAGGA AGGTGAAACT GAGAAACCTC CAGAAGAAAA GGGATAGAGA AAGCTGCTGG GCACAGATTA ACTTCGACTC TACAGATATG AGTTTCGACA ATTCACTGTT TACAGTGAGC GCCAAGACTA TGCCCGAGGA AGATCCTGCT ACTCTGGACG ATCACTCCGG AACCACAGCT ACTCCTTCTA ATAGTCGGAC CCGCAAACGA CCAACTAGCA CCTCTAGTTC ACCCGAGACA CCCGAGTTTA GCACATTCAG GGCTTGCCAG GACTACAAAG ACGATGATGA CAAATAA Amino acid Sequence of human HIDE1-flag (SEQ ID NO:77) MPWTILLFAAGSLAIPAPSIRLVPPYPSSQEDPIHIACMAPGNFPGANFTLYRGGQVVQLLQAPTDQRGVTFNLS GGSSKAPGGPFHCQYGVLGELNQSQLSDLSEPVNVSFPVPTWILVLSLSLAGALFLLAGLVAVALVVRKVKLRNL QKKRDRESCWAQINFDSTDMSFDNSLFTVSAKTMPEEDPATLDDHSGTTATPSNSRTRKRPTSTSSSPETPEFST FRACQDYKDDDDK Establishment of stable pool HEK293 human HIDE1 flag expressing cells

[00771] HEK293 cell sover expressing human HIDE1 flag were generated by GenScript Establishment of stable pool HEK293 and EL4 mouse HIDE1 Flag expressing cells

[00772] Supernatants from 48 hours culture of PT67 mouse HIDE1 virus producing cells or PT67 pMSCV empty vector were collect edand used for infection of HEK293 and EL4 cell lines. Four days post infection, puromycin antibioti wasc added at a concentrati onof l|Jg/ml for HEK293 cells or 4pg/ml for EL4 cells, and resista coloniesnt were selected for stabl poole generation.

Establishment of stable pool CHO-S-OKT3 human HIDE1 flag expressing cells

[00773] Stabl epool of CHO-S transduce withd OKT3, were electroporat by ed AMAXA device with 8ug of pcDNA3.1_human HIDE1 Flag construc generatedt at GS. 24hr post electroporat G-418ion, antibiot wasic added at a concentration of 300pg/ml in order 244WO 2017/009712 PCT/IB2016/001079 to generat establ poole cells. 2 weeks after selection process was finished, sorting for cells with highl yexpressed human HIDE1 prote inwas performed.

Establishment of stable pool HEK293 cyno HIDE1 expressing cells

[00774] The resulting expression constructs were transfect edto cells and stabl poole cel llines were generated as detailed below The. prote insequences encoded by the expression constructs are detail edbelow.

Nucleic acid Sequence of cyno HIDE1 (SEQ ID NO:290) ATGCCCTGGACCATCTTGCTTCTTGCAGCTGGCTCCTTGGCGATCCCGCGACCGTCCATC CGGCTGGTGCCCCCGCACCCAAGCAACCAAGAGGACCCCATCCACATCGCATGCATGGCC CCTGGGAACTTCCTGGGGGCGAATTTCACACTGTATCGAGGGGGGCAGGTGGTCCAGATC CTGCAGGCCCACGGAGACCAGCGCGGGGTGACATTTAACCTGAATGGCAGCAGCAGCGAG GCTTCAGGGGAACCCTTCCACTGCCAGTATGGAGTGTTAGGCGAGCTCAGCCAGCCCCAG CTGTCAGACCTCAGCGAGCCCGTGAACGTCTCCTTTCCAGTGCCCACTTGGATCTTGGTG CTCTCCCTGAGCCTGGCTGGTGCCGTCTTCCTCCTCGCTGGGCTGGTGGCTGTTGTCCTG GTGGTCAGAAGAGTTAAACTCAAAAATTTACAGAAGAAAAGAGATCGAGAATCCTGCTGG GCCCAGATTAACTTCAACAGCCCAGACATGTCCTTCGATAACTCCCTGTTTACCGTCTCT GGGAAAACGATGCCAGAAGAAGACCCGGCCACCTTGGATGATCACTCAGGCACCACTGCC ACCCCCAGCAACTCCAGGACCCGGAAGAGACCCACTTCTACGTCCTCCTTGCCTGAGATC CCGGAATTCAGCACTTTCCGGGCCTGCCAGTGA Amino acid Sequence of cyno HIDE1 (SEQ ID NO:291) MPWTILLLAAGSLAIPRPSIRLVPPHPSNQEDPIHIACMAPGNFLGANFTLYRGGQVVQI LQAHGDQRGVTFNLNGSSSEASGEPFHCQYGVLGELSQPQLSDLSEPVNVSFPVPTWILV LSLSLAGAVFLLAGLVAVVLVVRRVKLKNLQKKRDRESCWAQINFNSPDMSFDNSLFTVS GKTMPEEDPATLDDHSGTTATPSNSRTRKRPTSTSSLPEIPEFSTFRACQ

[00775] HEK-293 (ATCC, CRL-1573) cells were transfect edwith each of the constructs described above or with the empty vector (pcDNA3.1) as negative control, using polyplus JetPrime transf, ecti onreagent (polyplus transfection, catalog number 114-15).

Geneticin, G418 (Gibco, catalog number: 11811-031) resistant colonies were selected for stabl poole generation. 245WO 2017/009712 PCT/IB2016/001079 Expression validation Expression validation by Western blot (WB)

[00776] Whole cell extract ofs cells transfected or transduce withd human or mouse HIDE1 prote in(35 |Jg of total protei n)were analyzed for HIDE1 prote inexpression by WB.

As a negative control, whol cele lextracts of cells transfect edor transduce witd han empty vector were used.

[00777] Antibodi eswere used as following: • mAb mouse anti-Flag HRP, Sigma, cat.A8592 ,diluted 1:1000 in TTBS/5% BSA • Commercial antibody polyclonal Rabbit anti-human HIDE1, Sigma, cat #HPA012150, diluted 1:100 in TTBS/5% BSA) • Custom polyclonal Rabbit anti-mouse HIDE1 (GenScript, #488536 13) was diluted to a concentration of 1 |Jg/ml in TTBS/5% BSA

[00778] The polyclonal antibodie descs ribed above were follow byed a secondary antibody goat anti-Rabbit - Peroxidas (Jackson,e cat no. 111-035-003) diluted 1:20,000 in %milk/TTB Ssolution.

Expression validation by Flow cytometry (FACS)

[00779] In order to validate the cel lsurfac eexpression of the human and mouse HIDE1 prote inin the recombinan stablt pools,e 2x105 cell swere stained wit hFixable viability stai n450 (BD, cat #562247) dilut ed1:1000 in PBS, for 10 min at R.T. followed by cells washing wit hPBS. Antibodi eswere used as following: • For mouse HIDE1 validation: Custom pAbs anti- mouse HIDE1 (GenScript, #488536_13) or Rabbit IgG whol mole ecul e(Jackson, cat #011-000-003) were added to cells (5pg/ml diluted in 0.5%BSA in PBS) followed by staining wit h Donky anti Rabbit-PE diluted 1:100 in PBS/0.5%BSA.

• For human HIDE1 validation: Custom mouse monoclonal anti human HIDE1 Ab (Biotem , 33B4-2F7- Alexa 647) or mlgGl Isotype control (Biotem , lot.

F1150528d-2695- Alexa 647) were added to the cells in concentration of 5pg/ml (dilut edin 0.5%BSA/PBS). 246WO 2017/009712 PCT/IB2016/001079 Results Expression validation of HEK293 cells stable pool over expressing the human HIDE1 protein HEK293 cells over expressing human HIDE1- flag pCDNA3.1 vector (GenScript):

[00780] To verify expression of the HIDE1 protei inn the stably transfect edHEK293 pools (generated by GenScript ),whole cel lextract weres analyzed by WB using anti-flag antibody and commercial anti HIDE1 antibody (Sigma, cat #HPA012150), as described in Material and Methods. As shown in Figure 20, using anti Flag antibody (left panel) a band at -30 kDa is observed in the extracts of HEK293 cel lpools over expressing human HIDE1 vector, but not in the cell stransfect edwit hthe empty vector. Using the specific anti HIDE1 antibody (Sigma cat #HPA012150, right panel), two bands are observed, one is at ~30kDa as observed with the anti- flag Ab, and an additional higher band at- 40kDa. The Calculated Mw of the prote inis 25kDa.

[00781] In order to verify cell surface expression of the HIDE1 Flag protein, the same cells described above were analyzed by FACS using Rabbit anti-HIDEl pAb (GenScript, #488536_13) as described in Materia andl Methods. No cel lsurface expression was observed in thes ecell s(data is not shown).

Stable pool of HEK293 cells over expressing the mouse HIDE1 protein HEK293 cells overexpressing mouse HIDE1- flag pMSCV vector

[00782] To verify the expression of the mouse HIDE1 protei inn the stably transduced HEK293 cel lpools, whol cele lextracts of HEK293 stabl poolse transduced with pMSCV mouse HIDE1 flag vector or of HEK293 stable pools transduce witd hpMSCV empty vector, were analyzed by WB using anti-mouse HIDE1 pAb (GenScript #488536 13) as described in Material and Methods. The results, shown in Figure 19C, demonstrat a bande at ~30kDa in the extract ofs HEK293 cel lpools expressing mouse HIDE1, but not in the cells transfected with the empty vector or with the human HIDE1. Additional Band at higher Mw but lower intensities was observed.

[00783] These cell swere further analyzed by FACS for cel lsurface expression using Rabbit anti mouse-HIDEl pAb (GenScrip t#488536_13) as describe ind Material and 247WO 2017/009712 PCT/IB2016/001079 Methods. As shown in Figure 17. the binding of anti-mouse HIDE1 pAbs to the HEK293 cells over expressing mouse HIDE1 Flag prote in(green line) is higher than the binding of thi santibody to the empty vector cell s(orange line) and higher than the binding of the IgG isotype control to HEK293 cells expressing mouse HIDE1 Flag prote in(light blue line), or to the empty vector cells (red line).

Stable pool of EL4 cells over expressing the mouse HIDE1 protein EL4 cells overexpressing mouse HIDE1- flag pMSCV vector

[00784] To veril ymembrane expression, the cells were analyzed by FACS using Rabbit anti mouse-HIDEl pAb (GenScrip t#488536_13) as describe ind Material and Methods. As shown in Figure 18, the binding of anti-mouse HIDE1 pAbs to the EL4 cell s over expressing mouse HIDE1 Flag protei (greenn line) is higher than the binding of thi s antibody to the empty vector cells (orange line) and higher than the binding of the IgG isotype control to HEK293 cells expressing mouse HIDE1 Flag prote in(light blue line), or to the empty vector cells (red line).

Stable pool of CHO-S OKT3 cells over expressing human HIDE1 protein CHO-S OKT3 cells overexpressing human HIDE1- flag pcDNA3.1 vector

[00785] In order to veril ycell surface expression of human HIDE1 Flag protein, CHO- S OKT3 cells transfect edwith human HIDE !construc (describedt in Material & Methods) were analyzed by FACS using Mouse anti-human HIDE !monoclonal Ab (Biotem, 33B4- 2F7- Alexa 647) as describe ind Materia andl Methods. As shown in Figure 97. binding of anti-human HIDE! Ab was observed to CHO-S OKT3 over expressing human HIDE!protein, compared to isotype control (orange histogram) and empty vector cells (blue histogram).

Expression validation of HEK293 cells stable pool over expressing the cyno HIDElprotein HEK293 cells over expressing cyno HIDE1- flag pCDNA3.1 vector

[00786] To verify the expression of the cyno HIDE! prote inin the stably transfect ed HEK293 cel lpools, the cells were analyzed by FACS for cell surfac eexpression using mouse 248WO 2017/009712 PCT/IB2016/001079 anti human HIDE1 monoclonal Ab (Biotem 33B4-2F7- Alexa 647) as described in Material and Methods. As shown in Figure 98. the binding of mouse anti human HIDE1 monoclonal Ab to the HEK293 cell sover expressing cyno HIDE1 prote in(green line) is higher than the binding of thi santibody to the empty vector cells (orange line) and higher than the binding of the IgG isotype control to HEK293 cells expressing mouse HIDE1 Flag prote in(light blue line), or to the empty vector cell s(red line).

EXAMPLE 9: HIDE1-ECD Ig fusion protein production

[00787] HIDE1 mECD-mlg fusion prote in(SEQ ID NO: 18) batch #195, was generated in CHO-DG44 cells by culturing stabl cele lpools for 10 days, followed by Protein A mouse HIDE1 fused to the Fc of mouse IgG2a, was produce atd ProBioGen (Germany purification of cel lharvest and preparative SEC purification for aggregate removal. The final produ ctwas formulat ined lOrnM Na/K phosphate + 140mM NaCl pH 6.0 + 0.01% Tween.

SEQ ID NO: 18 HIDE1 MM-1 (Mouse_ECD + mouse_IgG2 Fc)_-_without_SP IPAPSISLVPPYPSSHEDPIYISCTAPGDILGANFTLFRGGEVVQLLQAPSDRPDVTFNVTGGGSGGGGEAAGGN FCCQYGVMGEHSQPQLSDFSQQVQVSFPVPTEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPI VTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIE RTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYS KLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK

[00788] HIDE1 mECD-mIg_fusion prote in(SEQ ID NO: 18, batch #233), composed of the ECD of mouse HIDE1 fused to the Fc of mouse IgG2a, was produced at ProBioGen (Germany) in CHO-DG44 cells by culturin stablg cele lpools for 12 days, followed by Protein A purification of cell harvest and preparative SEC purification for aggregate removal.

The final produ ctwas formulat ined 5mM Na citrate 5mM, Na/K phosphate, 140mM NaCl , 0.01% Tween pH5.5.

[00789] Expression vector used was ProBioGen’s PBG-GPEX6. HIDE1 gene was drive nby CMV/EF1 hybrid promoter followed by poly adenylation signal pA-1. The vector contained puromycin N-acetyl-transfera genese that allows selection of transfect edcells using puromycin, as well as dehydrofo latreductae segene that allow seles ction of transfected cells using methotrexate (MTX). 249WO 2017/009712 PCT/IB2016/001079

[00790] HIDE1 mECD-mIg_fusion prote in(SEQ ID NO: 18 batch #72), composed of the ECD of mouse HIDE1 fused to the Fc of mouse IgG2a, was produced at ExcellGene (Switzeland) by transient transfection in CHO-DG44 cells using Excellgene’s propriet ary vector system. Cell swere cultured for 10 days, followed by Prote inA purification of cell harvest The. final product was formulated in 10mM Na/K phosphate + 140mM NaCl pH 6.0 + 0.02% Tween.

[00791] HIDE1 hECD-hlg fusion prote in(SEQ ID NO: 17, batch #48), composed of the ECD of human HIDE1 fused to the Fc of human IgGl bearing C220 to S mutation at the hinge, was produce atd ExcellGene (Switzeland) by transient transfection in CHO-DG44 cells using Excellgene’s propriet aryvector system Cell. swere cultur edfor 10 days, followed by Protein A purification of cel lharvest The. final produ ctwas formulated in 0.1 M Glycine pH 6.

SEQ ID NO: 17 HIDE1 HH-1 _(Human_ECD_+_human_IgGl_Fc_mutated_C220S_at_hinge)_-_without_SP IPAPSIRLVPPYPSSQEDPIHIACMAPGNFPGANFTLYRGGQVVQLLQAPTDQRGVTFNLSGGSSKAPGGPFHCQ YGVLGELNQSQLSDLSEPVNVSFPVPTWEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTOKSLSLSPGK

[00792] HIDE1 hECD-hlg fusion prote in(SEQ ID NO: 17 batch #259), composed of the ECD of human HIDE !fused to the Fc of human IgGl bearing C220 to S mutation at the hinge, was produce atd GenScript (China) by transient transfection in CHO-3E7 cell susing GenScript’s vector system. Cell swere cultured for 6 days, followed by Protein A purification of cell harvest The. final product was formulat ined PBS pH 7.2.

[00793] HIDE1 hECD-hlg fusion prote in(SEQ ID NO: 17 batch #280), composed of the ECD of human HIDE1 fused to the Fc of human IgGl bearing C220 to S mutation at the hinge, was produce atd GenScript (China) by transient transfection in CHO-3E7 cell susing GenScript’s vector system. Cell swere cultured for 6 days, followed by Protein A purification of cell harvest The. final product was formulat ined 10 mM NaPhosphate, 150mM NaCl pH 7.0 +0.01% tween 20. 250WO 2017/009712 PCT/IB2016/001079 EXAMPLE 10: Generation and Characterization of custom Abs against HIDE1 protein

[00794] The aim of thi sexample was to raise polyclonal and monoclonal antibodies specific to HIDE1 protein.

Generation of rabbit polyclonal antibodies against human and mouse HIDE1 protein

[00795] Rabbit polyclonal antibodie weres raise dat GeneScript using the GenScript Poly Express™ custom polyclonal antibody production service.

Study Design

[00796] Generation of rabbit polyclonal antibodie wass performe atd GenScript (USA).

Antibodi esagainst the human HIDE1 protei weren raise dby using recombinan FCt fused protei ofn human HIDE1 ECD fused to FC domain of human IgGl (batch #280). Antibodies against the mouse HIDE1 protei weren raise dby using the recombinan Fct fusion protei ofn mouse HIDE1 ECD fused to FC domain of mouse IgG2a (batch #195).

Materials & Methods Reagents used in this study: 1. Stable pool of HEK293 cell sover expressing human HIDE1 flag protein. 2. Stable pool of HEK293 cell stransfect edwit hthe empty pMSCV vector. 3. Stable pool of SK-MEL-5 cells over expressing human HIDE1 flag protein. The generation of the cel llines 1-3 is described herein. 4. HIDE1 ECD (H:H) recombinant Fc fusion protei -Humn an ECD of HIDE 1 fused to the Fc domain of Human IgGl (Batch #280).

. HIDE1 ECD (M:M) recombinant Fc fusion protei -mousen ECD of HIDE1 fused to the Fc domain of mouse IgG2a (Batch #195). 6. Rabbit anti human HIDE1 (GenScript #, 488536 4). 7. Rabbit anti mouse HIDE1 (GenScript #, 488536 13). 8. Rabbit anti-human HIDE1, C19ORF38 (Sigma, cat #HPA012150). 9. Mouse anti Flag HRP, Sigma, cat.A8592.

. Goat Ant iRabbit-HRP (Jackson, Cat # 111-035-003). 11. Fixable viability stain 450 (BD Horizon cat #562247). 12. FACS buffer: 0.5% BSA in PBS. 251WO 2017/009712 PCT/IB2016/001079 13. Rabbit IgG whol molee cule (Jackson, cat #011-000-003). 14. R-Phycoerythrin-AffiniPure F (ab')2 Fragment Donkey Anti-Rabbit IgG, (Jackson, cat#711-116-152) Methods: Anti human or mouse HIDE1 pAbs production

[00797] Producti onof rabbit polyclonal antibody against Fc fusion prote inof mouse or human HIDE1 included immunizations of two rabbits production, bleeds and antibody purification, using GenScript 'spropriet aryfast immunization protocol. The immunoreactivit y of the test bleeds was analyzed follow ingthe third immunization by ELISA.

Analysis of the pAbs performance in WB application

[00798] Whole cell extract ofs cells (35ug of tota proteil n)expressing human or mouse HIDE1 prote inwere used to analyzed the pAbs anti-human or anti-mouse HIDE1 performance in WB. As negative control, whol cele lextracts of cell stransfect edwit hthe empty vector were used.

[00799] Antibodi eswere used as following: • Mouse anti Flag antibody (diluted 1:1000 in TTBS/5% BSA).

• Commercial antibody Rabbit anti-human HIDE1 (Sigma, diluted 1:100 in TTBS/5% BSA).

• Rabbit anti-human or anti-mouse HIDE1 antibodie (GenScrs ipt) were diluted to a concentrati onof 1 ug/ml in TTBS/5% BSA.

• The secondary antibody, goat anti rabbit HRP (Jackson) was diluted 1:20,000 in % millk/TTBS Analysis of the pAbs performance in FACS application

[00800] The performan ceof the rabbit pAbs, anti-human or anti-mouse HIDE1 in FACS application was tested using stabl celle sexpressing the mouse or the human HIDE1.

Cell s(2x105) were staine dwith Fixable viability stain diluted 1:1000 in PBS, for 10 min at R.T followed by cell swashing wit hPBS. The pAbs were then added to cells (5pg/ml were staine dwith Fixable viability stain diluted 1:1000 in PBS, for 10 min at R.T followed by cells washi 252WO 2017/009712 PCT/IB2016/001079 Results

[00801] The sera of the immunized rabbits were analyzed by ELISA against the respective Fc fusion proteins (human or mouse HIDE1). The negative control was pre immune serum. ELISA resul tsindicate dbinding of serum to the fusion proteins (data not shown).

Analysis of the purified rabbit polyclonal anti human or anti mouse HIDE1 by WB

[00802] The performan ceof the rabbit pAbs, anti-human or anti-mouse HIDE1 for WB application was tested using whol cele lextracts of HEK293 cell sexpressing human or mouse HIDE1 protein. As negative control, whole cel lextract ofs cells transfect edwith the empty vector were used.

[00803] Figure 19 present thes result ofs WB analysis using anti Human or mouse HIDE1 pAbs on HEK293 cell sexpressing human or mouse HIDE1 protein. Whole cells extract ofs HEK293 cell sexpressing the human HIDE1 flag (lanes 2), HEK293 cell s expressing the mouse HIDE1 flag (lanes 3) or HEK293 transfect edwit han empty vector (lanes 1) were analyzed using anti Flag antibody (A), a commercial antibody anti human HIDE1 (Sigma, D), pAb anti human-HIDEl (GenScript B), or with pAb anti mouse-HIDEl (GenScript C)., Detection was carried out using Goat Anti Rabbit-HRP (except anti flag which is already conjugated to HRP).

[00804] As shown in Figure 19, the human HIDE1 prote inexpressed on HEK293 cell s could be detected using the two positive controls antibodies anti, -flag antibody (A lane 2) and the commercial anti HIDE1 (D lane 2). The tested antibody, rabbit pAb anti human HIDE1 detected a band (B lane 2) in size of 35 kDa (higher than the calculate 25d kDa band), a similar band as detected by the positive antibodies This. prote incould not be detected using the anti-mouse HIDE1 tested antibody (C lane 2).

[00805] The mouse HIDE1 protei expresn sed on HEK293 cells could be detected by the tested antibody, rabbit anti mouse HIDE1 pAb (C lane 3) but not by the othe anti-humr an HIDE1 antibodie (coms mercia l,D lane 3 or the custom B lane 3) or by the anti-Flag antibody (A lane 3). 253WO 2017/009712 PCT/IB2016/001079 (Analysis of the purified rabbit polyclonal anti human HIDE1 by FACS

[00806] The performan ceof the purified polyclonal antibody against human HIDE1 Fc fusion protein, anti-human HIDE1 pAb (GenScript, ID 488536 4) in FACS application was tested using HEK293 cel lexpressing human HIDE1 Flag or SKMEL-5 cell sexpressing human HIDE1 Flag. Rabbit IgG (Jackson) was used an IgG control.

[00807] As shown in Figure 21, the binding of anti-human HIDE1 pAbs to the HEK293 cells expressing human HIDE1 Flag prote in(A, light blue line) or to SKMEL5 expressing human HIDE1 Flag (B, light blue line) is higher than the binding of Rabbit IgG isotype control to the same cells (A and B pink line).

[00808] Figure 21 present resuls tsof FACS analysi susing anti Human HIDE1 pAbs on HEK293 or SKMEL5 cells expressing Human HIDE1 protein. HEK293 cell sexpressing the human HIDE1 Flag (A) or SKMEL-5 expressing the human HIDE1 Flag (B) were analyzed by FACS using Rabbit anti Human HIDE1 (GenScript light, blue line). Rabbit IgG (Jackson, pink line) was used as isotype control Dete. ction was carried out using donkey anti - rabbit PE-conjugated secondary antibody and analysis by FACS.

(Analysis of the purified rabbit polyclonal anti mouse HIDE1 by FACS

[00809] The performan ceof the purified polyclonal antibody against mouse HIDE1 Fc fusion protein, anti-mouse HIDE1 pAb (GenScript ID, 488536 13) for FACS application was tested using HEK293 cel lexpressing mouse HIDE1 Flag. HEK293 cells transfect edwith an empty vector were used as a negative control.

[00810] As shown in Figure 17, the binding of anti-mouse HIDE1 pAbs to the HEK293 cells expressing mouse HIDE1 Flag prote in(green line) is higher than the binding of thi santibody to the empty vector cells (orange line) or higher than the binding of the IgG isotype control to HEK293 cells expressing mouse HIDE1 Flag prote in(light blue line) or to the empty vector cells (red line).

Generation of Mouse monoclonal antibodies against human HIDE1 protein

[00811] Mouse monoclonal antibodie weres raise dat BIOTEM (France) using BIOTEM R.A.D® (Rapid Antibody Development) protoc whicol h allows the breaking of 254WO 2017/009712 PCT/IB2016/001079 tolerance towards self-antigen ands the generation of more hybridomas than classical methods in a short time.

Study Design

[00812] Generation of murine monoclonal antibodie agais nst the extra-cellular domain of human HIDE1 were perform edat BIOTEM. The Antibodies were raise dusing the human HIDE1 Fc fusion protein, ECD of human HIDE1 fused to human IgGl (batch #295) for immunizations.

Materials & Methods Reagents used in this study: 1. Stabl epool of HEK293 cell sover expressing human HIDE1 flag protein 2. Stabl epool of HEK293 cell stransfect edwit hthe empty pMSCV vector. 3. HIDE1 ECD (H:H) recombinan Fct fusion protei -Humn an ECD of HIDE1 fused to the Fc domain of Human IgGl (Batch #259) 4. FACS buffer: 0.5% BSA in PBS.

. Fixable viability stai n450 (BD Horizo catn #562247) 6. Mouse mAb anti HIDE1, (Biotem #33B4-2F7) 7. Mouse mAb anti HIDE1, (Biotem #36C1-2F6) 8. Mouse mAbanti HIDE1, (Biotem #39A7-3A10-3G8) 9. Mouse IgGl (Life technologi es,Cat #MG100) . Goat Anti Mouse-PE (Jackson, cat #115-116-146) Methods mAbs anti human HIDE1 production

[00813] Producti onof mouse monoclonal antibodie includess the follow ingsteps.

[00814] The first phase of the project to raise anti-HIDEl mAbs included immunization of 2 BALB/c mice with human HIDE1 ECD fused to human IgGl.

[00815] The second phase of the protoc includedol fusion of the lymphocyt fromes the immunized mice with Sp2/O-Agl4 myeloma cell sand platin outg on 10 microtit 96-weller plates. 255WO 2017/009712 PCT/IB2016/001079

[00816] Mature clones were screened by ELISA using the HIDE1 H:H fusion proteins.

FACS analysis was subsequentl carriy ed out with the culture supernatant of ELISA-positive hybridoma clones, using anti mouse-PE (Jackson, cat .115-116-146) as secondary Ab for detection.

[00817] The thir phased included hybridoma cloning by limiting dilution and stabilization.

[00818] The fourth phase included further processing for production and purification.

Antibodi eswere produced by in vitro production and purification using protei An affinity chromatograph purifiy cation.

Analysis of the mAbs performance in WB application

[00819] Whole cell extract ofs cells (35ug of tota proteil n)expressing human or mouse HIDE1 prote inwere used to analyzed the mAbs anti-human HIDE1 performan cefor WB. As negative control, whole cel lextract ofs cells transfect edwith the empty vector were used.

[00820] Antibodi eswere used as following: • Mouse anti-human HIDE1 antibodie (Biots em) were diluted to a concentration of 2 pg/ml in TTBS/5% BSA.

• The secondary antibody, Goat Anti Mouse-HRP, was diluted 1:20,000 in 5% millk/TTBS.

Analysis of the mAbs performance in FACS application

[00821] The performan ceof the mouse mAbs, anti-human HIDE1 for FACS application was tested using stabl cellse expressing the human HIDE1. Cell s(2x105) were staine dwith Fixable viability stain diluted 1:1000 in PBS, for 10 min at R.T followed by cells washing wit hPBS. The mAbs were then added to cells (lOpg/m ldiluted in FACS buffer) followed by staining wit hGoat anti mouse-PE (dilut ed1:75 in FACS buffer).

Results

[00822] The sera of the immunized mouse were analyzed by ELISA against the respective Fc fusion proteins (human HIDE1). The negative control was pre immune serum .

ELISA result indis cated binding of serum to the fusion proteins (data is not shown). 256WO 2017/009712 PCT/IB2016/001079 Analysis of the purified mouse mAbs anti human HIDE1 by WB

[00823] The performan cethe mouse mAbs, anti-human HIDE1 (Biotem) in WB application was tested using whol celle extract ofs HEK293 cell sexpressing human HIDE1 protein. As negative control, whol cele lextract ofs cells transfect edwith the empty vector were used.

[00824] As shown in Figure 22, All the Biotem mAbs: 33B4-2F7 (A), 36C1-2F6 (B), 39A7-39A10-3G8 (C) recognize the human HIDE1 prote inexpressed on HEK293 cells, band in size of ~30 kDa can be observed (lanes 2). This specific band is absence in the negative control cells (empty vector, lane 1) and in the cell sexpressing the mouse HIDE1 (lane 3).

Antibody 36C1-2F6 (B) shows the best performance.

Analysis of the purified mouse mAbs anti human HIDE1 by FACS

[00825] The performan ceof the purified momoclonal antibodie agais nst human HIDE1 Fc fusion protein, anti-human HIDE1 mAbs (Biotem) for FACS application was tested using HEK293 cel lexpressing human HIDE1 Flag. HEK293 cell stransfect edwith an empty vector were used as a negative control.

[00826] As shown in Figure 14, the binding of all three anti-human HIDE1 mAbs (A- C) to the HEK293 cell sexpressing human HIDE1 Flag prote in(green line) is higher than the binding of thes eantibodie tos the empty vector cell s(orange line) or higher than the binding of the IgGl isotype control to HEK293 cells expressing human HIDE1 Flag prote in(light blue line) or to the empty vector cells (red line).

[00827] Schematic representation of Biotem’s antibodie (33B4-2F7,s 36C1-2F6, 39A7-39) are shown in figures 81-87.

B. Generation and characterization of custom Abs against HIDE1 protein by Serotec

[00828] Generation of Fab’s against human and mouse HIDE1 protein Fab’s were raise dat AbD Serotec (Bio Rad, Germany) using Human Combinatori Antibodyal Library (HuCAL®) production service. The HuCAL® librar isy based on the human IgGl Fab format whic, hconsists of the first tw odomains of the antibody heavy chain and the complete light chain. 257WO 2017/009712 PCT/IB2016/001079 Study Design

[00829] Generation of Fab’s against HIDE1 was performe atd AbD Serote (Bioc Rad, Germany). Antibodi esagainst the human and mouse HIDE1 prote inwere raised using the HuCAL® phage librar usingy, 3 rounds of enrichment and counter selection against non- related human IgGl fusion prote infor the depletion of unspecific antibodie s.After the panning, the enriched antibody pool from the phage displa vecty or was subcloned into expression vector to determine the final Fab forma t.The selected Fab format is Fab-A-FH (Bivalent Fab bacteria alkal line phosphatase fusion antibody (FLAG®- and His-6-tags)).

[00830] The Antibodi eswere raise dusing the human HIDE1 Fc fusion protein, ECD of human HIDE1 fused to human IgGl (batch #295) and mouse HIDE1 Fc fusion protein, ECD of mouse HIDE1 fused to mouse IgG2a (batch #233).

Materials & Methods Reagents used in this study: 1. Stable pool of HEK293 cell sover expressing human HIDE1 flag protein. 2. Stable pool of HEK293 cell sover expressing mouse HIDE1 flag protein. 3. Stable pool of HEK293 cell sover expressing cyno HIDE1 protein. 4. Stable pool of HEK293 cell stransduced with the empty pMSCV vector.

. Stable pool of HEK293 cell stransduce withd the empty pCDNA3.1+ vector. 6. Stable pool of CHO-S cells over expressing human HIDE1 flag protein. 7. Stable pool of CHO-S cells transduce witd hthe empty pCDNA3.1+ vector. 8. HIDE1 (H:H) recombinan Fct fusion prote in-Human ECD of HIDE1 fused to the Fc domain of Human IgGl (Batch #280). 9. HIDE1 (M:M) recombinan Fct fusion prote in-mouse ECD of HIDE1 fused to the Fc domain of mouse IgG2a (Batch #233).

. H:H_Intema lrecombinan tFc fusion protei ncontrol Huma: n ECD of Internal control fused to the Fc domain of Human IgGl (Batch #279). 11. M:M_Intemal recombinan tFc fusion prote incontrol: mouse ECD of internal control fused to the Fc domain of mouse IgG2a (Batch #214). 258WO 2017/009712 PCT/IB2016/001079 12. Commercial rabbit pAb anti-human HIDE1, C19ORF38 (Sigma, cat #HPA012150) 13. Goat Ant iRabbit-HRP (Jackson, Cat # 111-035-003). 14. Ant iFab-HRP (Bio-Rad, Cat#STAR126P).

. Goat Ant iHuman IgG F(ab')2-PE (Jackson, cat# 109-116-097). 16. Goat anti-human-PE (Jackson, cat# 109-116-098). 17. Fixable viability stain 450 (BD Horizon cat #562247). 18. FACS buffer 0.5% BSA in PBS.

Methods: Anti human or anti mouse HIDE1 Fab’s generation

[00831] Fab’s generation at AbD Serotec included the follow ingsteps: 1. Antigen immobilization - immobilization of the antigen on a soli dsupport The . standard method uses covalent coupling to magnetic beads. 2. Phage displa yselection - panning - The HuCAL® library presented on phage particl esis incubated wit hthe immobilized antigen. Nonspecific antibodie ares removed by extensive washing and specific antibody phage are eluted by adding a reducing agent.

An E. coli culture is infected with eluted phage and helpe rphage to generat ean enriched antibody phage librar yfor the next panning round. Typicall y,three rounds of panning. 3. Subcloning into antibody expression vector - After panning, the enriched antibody DNA is isolated as a pool and subcloned into a Fab expression vector. E. coli are transforme witd hthe ligation mixture and plated on agar plates. Each growing colony represents a monoclonal antibody at thi sstage. 4. Primary screening - Colonie ares picked and grown in a 384-wel lmicrotit plateer .

Antibody expression is induced and the culture is lysed to releas thee antibody molecules. Culture ares screened for specific antigen binding by ELISA.

. Secondary screening - Flow cytometry (FACS) on transfect edcell susing bacterial lysate froms E.coli expression cultures. 259WO 2017/009712 PCT/IB2016/001079 6. Sequencing - Hit sfrom the primary screening experiment are sequenced to identify unique antibodies. 7. Expression and purification - The unique Fab’s are expressed and purified using one-step affinity chromatography. 8. Antibody OC - Purified Fab’s are tested by ELISA and by FACS on transfected cells.

Analysis of the Fab’s in Western blot (WB) application

[00832] Whole cell extract ofs HEK293 transfect edcells over expressing the human or mouse HIDE1 flag prote inor whol cele lextract ofs HEK293 cell stransdued with an empty vector were analyzed by WB using the custom Fab’s (AbD Serotec) described above at a final concentration of l-7pg/ml in 1%TTBS/BSA. The commercial pAb anti-human HIDE1 (Sigma, cat #HPA012150) was diluted 1:100 in TTBS/1% BSA.

[00833] Staining with the custom and commercial antibodies described above was followed by a secondary antibody Ant i Fab-HRP (Bio Rad) diluted 1:5000 in 5%Milk/TTBS and goat anti Rabbit IgG - Peroxidas (Jae ckson, cat no. 111-035-003) diluted 1:10,000 in TTBS/5% milk solution.

Analysis of the Fab’s performance in Flow Cytometry (FACS) application

[00834] The performan ceof the Fab’s, anti-human HIDE1 and anti-mouse HIDE1 for FACS application were tested using stabl cellse over expressing the human HIDE1 or mouse HIDE1 or on cyno HIDE1 an empty vector transduce celd ls. Cell s(2xl05) were staine dwith Fixable viability stain diluted 1:1000 in PBS, for 10 min at R.T followed by cells washing with PBS.

[00835] The Fab’s were then added to cell s(10 |Jg/ml diluted in FACS buffer) followed by staining wit hGoat Ant iHuman IgG F(ab')2-PE (dilut ed1:100 in FACS buffer).

Affinity (Avidity) measurements of purified antibodies

[00836] Affinity measurements were done by AbD Serote usingc the ForteBio Octe r RED384 instrument. 260WO 2017/009712 PCT/IB2016/001079

[00837] Antibody concentrations: bivalent Fab antibodies in Fab-A-FH format (Mw: 198 kDa): 9.9 ug/ml (50 nM), 1:2 dilution series. The model used to fit the data: (ForteBio Data Analysis software 8.2.0.7) • 1:1 interaction model • 2:1 interaction model for heterogeneous binding Epitope Binning

[00838] Epitop Binninge measurements were done by AbD Serotec using the ForteBio Octer RED384 instrument Antibodies. were grouped into diffrents bins by using the “in tandem” measurements.

Reformatting of the Fab’s into full length immunoglobulin

[00839] The conversion to human IgGl was done to 5 Fab’s by AbD Serotec (AbD25751, AbD25753, AbD25754, AbD25755, AbD25760).

Analysis of the reformatted Fab’s (full hlgGl Abs) performance in Flow Cytometry (FACS) application

[00840] The performance of the antibodies, anti-human HIDE1, for FACS application were tested using stabl cellse over expressing the human HIDE1, mouse HIDE1 or cyno HIDE1 or an empty vector transduce celd ls. Cells (5x105) were stained wit hFixable viability stai ndiluted 1:1000 in PBS, for 10 min at R.T followed by cell swashing with PBS.

[00841] The Abs were then added to cells (10ug/ml dilute ind FACS buffer) followed by staining with Goat Anti Human-PE (dilut ed1:100 in FACS buffer).

Affinity measurements of the reformatted Fab’s

[00842] The performan ceof the antibodies anti-hum, an HIDE1, for the affinity measurements (KD) using FACS application were tested using stabl celle sover expressing the Results

[00843] The panning was performe atd AbD Serotec using the follow ingproteins: 261WO 2017/009712 PCT/IB2016/001079

[00844] Panning 1: Human HIDE1 ECDhlgGl protei batcn h #280 - for generation of anti-human Fab’s.

[00845] Panning 2: Mouse HIDE1 ECDmIgG2A prote inbatch #233 - for generation of anti-mouse Fab’s.

[00846] Panning 3: Mouse HIDE1 ECDmIgG2a protei batcn h #233 (1st and 3rd panning round)/ Human HIDE1 ECDhlgGl prote inbatch #280 (2nd panning round) - for generation of cross reactive Fab’s.

[00847] Serotec screened -360 clones by ELISA on human HIDE1 ECDhlgGl protein batch #280 and mouse HIDE1 ECDmIgG2A protei batchn #233 and H:H_Intemal ECDhlgGl protei batcn h #279 as control for the human panning or mouse M:M_Intemal ECDmIgG2A prote inbatch #214 as control for the mouse panning.

[00848] In the human HIDE1 panning 122 clones were positive, in the mouse HIDE1 panning 61 clones were positive and in the mouse/human panning 55 clones were positive.

In secondary screening 88 positive clones were screened from panning 1, 33 positive clones were screened from panning 2 and 55 positive clones were screened from panning 3 by FACS on cell soverexpressi ngthe human HIDE1 and mouse HIDE1 and on empty vector transduce celd ls.

[00849] The secondary scree nresult edin 43 positive clones from panning 1,15 positive clones from panning 2 and 29 positive clones from panning 3.

Sequencing was done on 20 positive clones from panning 1 which resul tsin 16 unique Fab’s, on 15 positive clones from panning 2 whic hresult ins 9 unique Fab’s and on 25 positive clones from panning 3 which result ins 4 unique Fab’s.

[00850] The purified Fab’s were analyzed by ELISA against the respective Fc fusion protei (humann HIDE1 or mouse HIDE1). ELISA result indis cate dbinding of the purified Fab’s to the fusion proteins (data not shown).

[00851] The purified Fab’s were also analyzed by WB. The antibodie ares not applicable for WB (data is not shown). 262WO 2017/009712 PCT/IB2016/001079 Analysis of the purified Fab’s anti human HIDE1 and anti-mouse HIDE1 by FACS

[00852] The performan ceof the purified Serot’ecs Fab’s against human HIDE1 and mouse HIDE1, in FACS application were tested using HEK293 cel lover-expressin humang HIDE1 Flag or mouse HIDE1. HEK293 cell stransduce withd an empty vector were used as a negative control.

[00853] As shown in Figure 23 the binding of 12 out of 16 anti-human HIDE1 Fab’s (1-16) to the HEK293 cell sover-expressing human HIDE1 Flag protei (bluen line) is higher than the binding of these Fab’s to the empty vector cells (red line), the Fab’s didn’t bind to the HEK293 cells over-expressing mouse HIDE1 Flag protei (orangen line).

[00854] As shown in Figure 24 the binding of 8 out of 13 anti-mouse HIDE1 Fab’s (1- 13) to the HEK293 cell sover-expressing mouse HIDE1 Flag protei (orangen line) is higher than the binding of these Fab’s to the empty vector cells (red line), two Fab’s also bind to the HEK293 cells over-expressing human HIDE1 Flag protei (n blue line).

[00855] As shown in Figure 90 the binding of 4 out of 18 anti-human HIDE1 Fab’s (1-16) and anti-mouse HIDE1 Fab’s (4,5) to the HEK293 cell sover-expressing cyno HIDE1 protei (lowen panel)r is higher than the binding of thes Fabe ’s to the empty vector cell s (upper panel).

Affinity mesurements

[00856] The purified Fab’s were also measured for affinity by AbD Serotec. Table 19 and Table 20 summarize the obtained avidity for anti human HIDE1 and anti mouse HIDE1 Fab’s. 263WO 2017/009712 PCT/IB2016/001079 Table 19 Serot ecmeasurement ofs the kinetic rate constants and KO values for anti human HIDE1 antibodies.

Sample ID Kd Alternate designation |[Vs! (Antibody) [nMf L37E-02 AbD25747.1* 1.77E+06 7,7 il.06E-02 AbD25748.1* 7.32E+G6 1.4 j4.89E-03 AbD25749.1* 8.61+05 5.7 h.!3E-03 AbD25750.1* 8.19E+05 2.6 b.48E-05 AB-506 AbD25751.1 5.23E+04 1.0 {3.08E-03 AbD257S2.1* 5.90E+05 5.2 |4.45E-05 AB-507 AbD25753,l 6.02E+05 0.07 fe.09E-05 AB-508 AbD25754,l 6.09E+05 0.10 b.65E-05 AbD25755.1 0.02 AB-509 1.17E+06 ^.OlE-05 AbD25757.1 1.02E+O6 0.07 H.45E-04 AbD25758.1 1.29E+06 0.11 _1.41E±06 _1.45E04 AbD25759.1* 0.10 |1.72E-05 AB-510 AbD2576O.l 5.26E+05 0.03 K.18E-03 AbD25762.1 1.S7E+O5 7.5 k55E-05___ AbD25766,l__ 9.17E4-05 0,06______ ,1.028406 i5.72E-05 AbD25767.1 0.06 * heterogeneous binding Table 20 Serot ecmeasurement ofs the kinetic rate constants and KO values for anti mouse HIDE1 antibodies.

Sampie ؛O ka ko WWWWWWWWWWWI (Antibody) W؛1 |||||||||، b.8 AbD25?63.1 1.12E*06 8.78E-04 |1.3 AbD25764.1 1.43E+06 1.81E-03 AbD25765.1 l.OZEW 8.71E-04 0.9 ^0.17 AbD25766.1 3.4OEtOS 5.896-05 ^0.13 AbD25767.1 3.916+05 5.116-05 .........k9.......................

AbD25918.1 1.31+06 i.34E-03 i0.19 AbD2S921.1 7,56405 1.43E-04 b.50E-04 ,7____________ AbD25962.1 1.30406 264WO 2017/009712 PCT/IB2016/001079 Binning Epitope

[00857] The purified Fab’s were also subjected to epitope binning by AbD Serotec.

Figure 91, Figure 92, Figure 93 and Figure 94 summarize the binning result fors anti human HIDE1 and anti mouse HIDE1 Fab’s

[00858] Figure 91 shows that antibodie froms group 1 [ AbD25747, AbD25748, AbD25749, AbD25751 (AB-506), AbD25752, AbD25762, AbD25750, and AbD25760 (AB- 510)] share mostl they same epitope “A”. AbD25760 seems to bind to anoth erepitope B.

AbD25750 share properti ofes both epitope A and B, and is therefore grouped into the overlappi ngepitope A/B. Figure 92 shows that antibodie froms group 2 [Grou p2: AbD25757, AbD25759, AbD25766, and AbD25767] AbD25757 and AbD25759 can be probably grouped together in epitope C. AbD25766 and AbD25767 seem to bind to another epitope D. Figure 93 shows that all antibodie froms group 3 [ AbD25753 (AB-507), AbD25754 (AB-508), AbD25755 (AB-509), and AbD25758] probably share the same epitope E.

[00859] Figure 94 shows the binning for anti mouse HIDE1 Fab’s, AbD25763, AbD25765, AbD25918 and AbD25962 share the same epitope A. AbD25764 has an overlappi ngepitope part; ares derive dfrom epitope A of the above mentioned antibodie ands from epitope B of AbD25766 and AbD25767. AbD25766 and AbD25767 share the same unique epitope B. AbD25921 binds to another unique epitope C.

Analysis of the reformatted Fab’s (full hlgGl Abs) performance in Flow Cytometry (FACS)

[00860] The performan ceof the reformatte Serotecd ’s antibodie agais nst human HIDE1, in FACS application were tested using HEK293 cel lover-expressin humang HIDE1 Flag ,mouse HIDE1 or cyno HIDE1. HEK293 cell stransduced/ transfected wit han empty vector were used as a negative control

[00861] As shown in Figure 95. the binding of 5 out of 5 anti-human HIDE1 reformatte antibodied (1-5)s to the HEK293 cells over-expressing human HIDE1 Flag protein (orange line) is higher than the binding of thes reforme atte antibodied tos the empty vector cells (blue line), the reformatte antibodied didns ’t bind to the HEK293 cells over-expressi ng 265WO 2017/009712 PCT/IB2016/001079 mouse HIDE1 Flag prote in(light green line). Three out of five reformatte antibodied (3,4s and 5) are CR to the HEK293 cell sover-expressing cyno HIDE1 prote in(dark green line).

Affinity measurements of the reformatted Fab’s using Flow Cytometry (FACS)

[00862] The affinity The affinity measurement ofs the reformatte Serotd ’ecs Ab’s (full hlgGl Abs) against human HIDE1, using FACS application were tested using CHO-S cell over-expressing human HIDE1. CHO-S cells transduce witd han empty vector were used as a negative control.

[00863] As shown in Figure 96. the binding of 5 out of 5 anti-human HIDE1 reformatte Abd’s (1-5) to the CHO-S cell sover-expressin humang HIDE1 Flag prote in(circl e dots) is higher than the binding of thes reformattede Ab’s to the empty vector cells (squar e dots) Two. of the five antibodie (2s and 3) reaches saturati onat the concentrations that were measured. The Kd value and R square are represent ined the figure.

Conclusions and summary of anti HIDE1 custom Abs

[00864] The follow ingcustom antibodie generateds were validate andd used for furthe r target characterization:

[00865] Fab’s anti-human HIDE1; Serote (ID:c AB-326, AB-327, AB-328, AB-329, AB-331, AB-332, AB-333, AB-335, AB-336, AB-337, AB-338, AB-340, see Table 21) and reformatte Fabd’s anti-human HIDE1; Serotec (ID: AB-506, AB-507, AB-508, AB-509, AB- 510, see Table 21) validate ford FACS application on ectopi cexpression, with no cross reactivi tyto mouse HIDE1 protein, and with cross reactivi tyof four Fab’s (ID: AB-327, AB- 332, AB-333, AB-338, see Table 21) and three reformat tedantibodie s(ID: AB-508, AB- 509, AB-510, see Table 21) to cyno HIDE1 protein.

[00866] Fab’s anti-mouse anti-HIDE1 antibody; Serotec (ID: AB-341, AB-342, AB- 343, AB-344, AB-345, AB-354, AB-357, AB-359, see Table 22) validate ford FACS application on ectopi cexpression, wit hcross reactivi tyof two antibodie (ID:s AB-344, AB- 345) to human HIDE1 protein. 266WO 2017/009712 PCT/IB2016/001079 Table 21:Serote anti-humc an HIDE1 Fab’s and mAbs. iD 1__ 1.025748. Ser Fab__ A8-326 J___ ___________________________________ .A8.327.

A8-328 ؛ »Ser.Fab 25750.1 AS-329 ؛ «hNDEL-Ab025751.1Ser.Fsb AB-331 i «bNDEL-AbD25753.1Ser.Fab AS 332 : ahNDE 3.Ab025754 1Sec.fab AS 333 3 ahNDE1.A b025755. 1Ser.fab A8-335 !........ahNIDE 1.Ab025757, ...........................

A8-336 ؛ ahNne3.A20297S&.1Sec.Fab A8-337 sahNIDE 1A892 5755,1.8 A8-3.38 : shH0E*.A8D25780.1ser.Fab AB-340 : ahMDEk.AaD29782.Sex.Eab AS-SQS ؛ ahHtDE „A602I 5752 MigGL.Ser.mAb ؛ ahMtDE3 „AbD257S3.AigGL.Ser.mAb A8507 A8-508 ؛ shHIDEI_A8D257S4-algGLSer.mAb lahMe1a602s788MgGLSer.mab A8-509 A8-810 I ahMIDEL.A 8025780.NgG LSer.mAb Table 22: Serotec anti-human HIDE1 Fab’s. ) to { Mame SoSSSSSSSSSSSSSSSSOGSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSOSSSSSSSSSSSSOSSSSSSSSSSOSSSOSSSSSSGSSSSSS 1.48.348.48108140025763.86. 888428188025284 SeL.Esb.. .88-343 LamMr 86025785.1 Sex Esb. 1.88.34418025786-1 Ser Eab 1.88-345.468811 88025767 8.854 LmNE 1 ASIS.LSetEab [ $8-357 IomHDel A80559211 Ser ٠٠ 1.8.359 01802592.1 Ser Esh.

[00867] Five reformatte Fabd’s anti-human HIDE1; Serotec were characterize ford affinity. Two of the five antibodi es(AB-507 and AB-508) reache dsaturation at the concentrations that were measured. 267WO 2017/009712 PCT/IB2016/001079 EXAMPLE 11: ENDOGENOUS EXPRESSION OF HIDE! Endogenous expression of HIDE1 protein in human and mouse cell lines [99868j The aim of thi sstudy was to identify human and mouse cancer cell lines that endogenously express HIDE!.

MATERIALS AND METHODS MATERIALS • RNA extracti onwas performed with RNAeasy Mini Kit (Qiagen cat # 74014). • cDNA was produce dusing High Capacity cDNA Revers eTranscription Kit (Applied Biosystems cat#4368814).

• Specific primers for human and mouse were designed on separat exonse and detailed in Table T1 and Figure 25 • Human HIDE1 TaqMan probes: Hs01128129_ml, Hs01128131_ml ,Life technologies.

• Human housekeeping gene, hRPL19 TaqMan probe: Hs01577060, Life technologies.

• Mouse HIDE1 TaqMan probe: HOJEX12_CCAAZKY, custom made, Life technologies.

• Mouse housekeeping gene, mRPL19 TaqMan probe: Mm02601633_gl, Life technologies.

• Commercial cDNA panel I, Mouse, Biochain, Cat no. C8334501 • Commercial cDNA panel II, Mouse, Biochain, Cat no. C8334502 • Commercial Human and Mouse cel lline sfrom American Type Culture Collection (ATCC) are detailed in Table 23 and 24, respectively.

Table 23 Human cell lines Morplwloov HL-60 ATCC CCL-240 Leukemia Blood THP1 ATCC TIB-202 Monocyte Blood Bone KG-1 ATCC CCL-246 Macrophage marrow Jurkat ATCC TIB-152 TCell Blood 268WO 2017/009712 PCT/IB2016/001079 Bone NCI-H929 ATCC CRL-9068 B lymphocyte marrow A-704 ATCC HTB-45 Epithelial Kidney RPMI8226 ATCC CCL-155 B lymphocyte Blood NCI-H28 ATCC CRL-5820 Epithelial Lung U937 ECACC 85011440 Monocyte Blood Table 24. Mouse cel lline 4T1 CRL-2539 Epithelial Mammary gland Spindle-shaped+epithelial- like B16-F1 CRL-6323 Skin cells EL4 TIB-39 T-lymphoblast Blood E.G7-OVA CRL-2113 T-lymphoblast Blood YAC-1 TIB-160 T-lymphoblast Blood A20 TIB-208 B-lymphoblast Blood P815 TIB-64 mast cells Blood NIH/3T3 CRL-1658 fibroblast Embryo Sal/N CRL-2544 fibroblast Fibrosarcoma J774A.1 TIB-67 Macrophage Blood LL/2 CRL-1642 Epithelial Lung N h: \ tn־m :ן CRL-1887 B104-1-1 Glioblastoma RAW264.7 TIB-71 macrophage Blood P388D1 CCL-46 macrophage Blood KLN205 CRL-1453 Epithelia Lung CT26.WT CRL-2638 fibroblast Colon • Commercial polyclonal rabbit anti-Human HIDE1, Sigma cat HPA012150 • Custom polyclonal rabbit anti-mouse HIDE1 Genscript ID#, 488536 13 .

• Rabbit IgG whol molece ule, Jackson, cat # 011-000-003.

• Goat Anti Rabbit-HRP, Jackson, cat # 111-035-003.

• Donkey anti Rabbit-PE, Jackson cat # 711-116-152.

• Custom mouse monoclonal anti-human HIDE1,(BIOTEM# 33B4-2F7), 36C1-2F6.

• Custom mouse monoclonal anti-human HIDE1, Ab-364 (BIOTEM# 33B4-2F7- AF647) • Human IgGl Isotype control lot F1150528d-2695-. Alexa 647, BIOTEM • Mouse IgGl isotype control, Life technologie cats # MG100. 269WO 2017/009712 PCT/IB2016/001079 • Goat Anti Mouse-PE, Jackson, cat# 115-116-146.

• AB329, anti-human HIDE 1 F(ab’)2 AbD25751.1, Serotec • Ab333, anti-human HIDE1 F(ab’)2 AbD25755.1, Serotec • Ab332 anti-human HIDE 1 F(ab’)2 AbD25754.1, Serotec • Ab360 anti -mouse HIDE1 F(ab’)2 AbD25963.1, Serotec • Ab359 anti -mouse HIDE1 F(ab’)2 AbD25962.1, Serotec • Ab-345 anti -mouse HIDE1 F(ab’)2 AbD25767.1, Serotec • Non Relevant F(ab')2 control Ab: 307, AbD25909.1, Serotec • Goat Anti Human IgG F(ab')2-PE, Jackson, cat# 109-116-097 • VioBlue, Fixable viability stain 450, BD Bioscience, cat # 562247.

• Human Fc block, Trustai FcX,n Biolegend, cat#422302.

• Mouse BD Fc Block Rat, Anti-Mouse CD16/CD32, BD Pharmingen, cat.553142.

• Cell line Nucleofecto kitr V, Lonza # VACA-1003.

• Cell line Nucleofecto kitr T, Lonza # VACA-1002.

• On-TARGET plus human C190rf38 siRNA SMART pool L-023981-02, Dharmacon.

• On-TARGET plus Mouse AB124611 siRNA SMART pool L-063774-01, Dharmacon.

• On-TARGET plus non targeting (scrambled) siRNA SMART pool D-001810-01-05, Dharmacon.

Quantitate real time PCR (qRT-PCR)

[00869] RNA (l-5ug) extracti onof human and mouse cell line s(detailed above in Tables 23 and 24) was preforme accordd ing to manufactures protocols.

[00870] cDNA was prepared according to manufactures protocols (lug RNA diluted in 20ul cDNA mix reaction).

Two different approaches were used: qRT-PCR using SYBR green

[00871] cDNA, prepared as described above, diluted 1:10 (representi 25ngng RNA per reaction) in DDW was used as a templat fore qRT-PCR reactions using the SYBR Green I 270WO 2017/009712 PCT/IB2016/001079 assay (PE Applied Biosystem) with specific primers (liste ind Table 27). Detection was performe usingd the PE Applied Biosystem SDS 7000 or QuantStudi 12ko flex device.

[00872] The cycle in which the reactions reached a threshold level of fluorescen ce (Ct= Threshold Cycle,) was registered and was used to calculate the relati vetranscript quantity in the RT reactions. The relati vequantity was calculate usingd the equation Q=Efficiency A־Ct. The efficiency of the PCR reaction was calculate fromd a standard curve, created by using serial dilutions of mouse commercial cDNA mixed panels (Cat no.

C8334501 and C8334502, Biochain), and for human efficiency calculations, several cDNA from tissues and cel llines that were prepared in house, created for each primer set.

[00873] The resulting quantities were normalize tod quantities of a housekeeping gene (hHPRT1 ormHPRTl). gRT-PCR using TaaMan:

[00874] cDNA, prepared as described above, diluted 1:10 (representi 25ngng RNA per reaction) in DDW, was used as a template for qRT-PCR reactions, using a gene specific TaqMan probes (detailed in Material above).s Detection was performed using QuantStudi o 12k flex device.

[00875] The cycle in which the reactions achieved a threshol leveld of fluorescence (Ct= Threshold Cycle )was registered and was used to calculate the relati vetranscript quantity in the RT reactions.

[00876] The absolute gene quantity was calculate byd using the equation Q=2 A־ACt.

[00877] The resulting quantities were normalize tod quantities of human or mouse housekeeping gene, hRPL19 or mRPL19 relatively.

Protein expression detection by Western Blot (WB)

[00878] The expression of HIDE1 in human and mouse cel lline swas analyzed by WB using whol cele lextract (50-75s pg for the cancer cel llines, and 30pg for the over expressing cel lline and negative control cell line)

[00879] For human HIDE1 prote indetection: 271WO 2017/009712 PCT/IB2016/001079 • Commercial rabbit polyclonal anti-human HIDE1 pAb, Sigma, cat # HPA012150, diluted to 2pg/ml in 5% BSA/TBST follow byed secondary Ab goat anti-Rabbit - Peroxidas conjugate ed (Jackson, cat # 111-035-003), dilut ed1:20,000 in 5% milk TEST.

• Custom mouse monoclonal anti human HIDE1, 36C1-2F6, BIOTEM, diluted to 2pg/ml in 5% BSA/TBST followed by secondary Ab Goat Ant i Mouse-HRP (Jackson, cat# 115-035-146), diluted 1:20,000 in 5% milk TEST.

For mouse HIDE1 protei deten ction: • Custom rabbit polyclonal anti-mouse HIDE1 pAb (GenScript ,ID# 488536 13) diluted to 2pg/ml in 5% BSA/TBST follow byed a secondary Ab goat anti-rabbit - Peroxidas conjugae ted, diluted 1:20,000 in 5% milk TEST.

Protein expression analysis by Flow Cytometry (FACS)

[00880] The cel lsurfac eexpression of HIDE 1 protei wasn analyzed by FACS. Human or mouse cel llines were staine dwith VioBlue reagent diluted 1:1000 in PBS. Cells were incubated 10 min at R.T. and then washed twice with FACS buffer (0.5% BSA in PBS). Cell lines for endogenous prote inanalysis were pre-incubated with the Fc receptor blocking solutions listed above in materia sectl ion (2.5 ul/reacti onof human blocker and 0.5mg/ml of mouse blocker were used according to the manufactures procedures). To detect the human HIDE1 protein, cells were stained with a custom mouse monoclonal anti human HIDE1 mAbs (BIOTEM, detailed in materia secl tion above) diluted to a concentrati onof 10ug/ml or IgGl Isotype control at the same concentration followed by Goat anti mouse PE conjugated Ab. To detect the mouse HIDE1 protein, cell swere stained wit ha custom rabbit polyclonal anti-mouse HIDE1 pAb (GenScript, ID# 488536 13) diluted to a concentrati onof 10ug/ml or rabbit IgG whol mole ecul ase isotype controls at the same concentration followed by Donkey anti Rabbit-PE conjugated Ab diluted 1:100.

[00881] To detect the mouse HIDE1 protein, cells were staine dwith a custom rabbit polyclonal anti-mouse HIDE1 pAb (GenScript, ID# 488536 13) diluted to a concentrati onof 10ug/ml or rabbit IgG whol molee cule as isotypes control at the same concentration followed by Donkey anti Rabbit-PE conjugated Ab diluted 1:100. 272WO 2017/009712 PCT/IB2016/001079 HIDE1 knock down

[00882] Knock down of endogenous mouse or human HIDE1 was carrie dout by transient transfection of siRNA. Transfection of 50 pmol HIDE1 siRNA pool or scramble d siRNA performe byd electroporat usingion Amaxa nucleofect ordevice and Amaxa nucleofect orkit sas liste aboved and according to the manufacture procedur 48e. hours post transfection, cells were collect fored furth eranalysis by qRT-PCR and FACS. EL4 cell line was further tested by FACS 72 hours post transfection.

RESULTS ENDOGENOUS EXPRESSION OF HIDE1 IN HUMAN CELL LINE Endogenous expression of HIDE1 transcripts in human cell lines detected by qRT-PCR

[00883] In order to verify the presence of the HIDEI transcript in human cel llines (listed in Table 23), qRT-PCR was performe usingd a specific primers (Table 27 and Figure .) or using TaqMan probes as describe above in Material & Methods.

[00884] As shown in Figure 26 human HIDEI transcript is observed using TaqMan probe Hs01128131_ml wit hrelatively high level sin HL-60, KG1 and U937 cell lines. Lower transcript level is observed in THP1 cel lline, then A704 and NCI-H28 cell lines with lower transcript levels. NCI-H929 and RPMI8226 cel lline sshows low to no transcript Simil. ar result weres obtained using TaqMan probe HsOl 128129_ml and specific primers, SYBR green (data is, not shown).

Endogenous expression of HIDEI protei nsin human cell lines delected by WB

[00885] WB analysis for endogenous expression of HIDEI prote inwas earned out on various human cancer cel llines lysat esas detaile d,in Table 23 using commercial anti human HIDE! pAb (Sigma, HPA012150 ) or custom anti human HIDEI mAb (B1OTEM, 36C1- 2F6) as described in Material &s Methods above. As a positive control, whol cele lextract of stabl HEK293Te cell pool over -expressing HIDE! was used while cells transfected with an empty vector served as the negative control. 273WO 2017/009712 PCT/IB2016/001079

[00886] WB result rels ate tod positive and negative controls were as expected. WB result fors endogenous expression were inconclusive as multiple bands were detected using the pAb, and no signal was observed using the mAh (data is not shown).

Endogenous expression of HIDE! protei nsin human ceil lines detected by FACS

[00887] To verify the cell-surfac endogene ous expression of human H1DE1, various cell lines (detailed in Table 23) were tested as described in Materia &l Methods above. The cell lines were stained wit hthe custom monoclonal anti human-HIDEl mAbs: BIOTEM, 33B4-2F7, 36C1-2F6 (Figure 27, orang eline, upper or lower panels respectivel y),or with Isotype control (Figure 27, light blue )followed by a secondary goat anti mouse PE Ab. As an additional negative control, cells were stained with the secondary antibody only (Figure 27, red line). Analysis was perform edby FACS. As shown in Figure 27 binding of the BIOTEM 33B4-2F7 antibody was observed in three human cancer cel lline sU-937, THP-1 and HL-60 as compared to isotype control binding. Binding of the BIOTEM 36C1-2F6 antibody was observed only in U-937 cell line .No binding of either of the two BIOTEM antibodie wass observed inNCI-H929 and Jurkat cel llines.

[00888] Furthe analysisr for endogenous confirmation of human HIDE1 in THP1 and U937 cell lines, was done by testing various anti-human HIDE 1 F(ab’)2, produce byd Serotec.

[00889] Both cell line swere staine dwit hanti-human HIDE! custom F(ab’)2s (Serotec detaile, ind Materia &l Methods)(Figure 28, A, B), or with Non relevant control anti-human F(ab’)2, Ab307, (Figure 28, A, B light blue) followed by a secondary goat anti- human F(ab’)2 PE Ab. As an additional negative control, cells were staine dwit hthe secondary antibody only (Figure 28, A, B red line). Analysis was performed, by FACS. As shown in Figure 28, A, B, expression of human HIDE! in U937 human cel lline was observed by Ab329, Ab333 and AbD332 Serotec F(ab’)2 only as compared to isotype control expression. No expression of human HIDE! was observed in U937 cell line by using the othe F(abr ’)2. 274WO 2017/009712 PCT/IB2016/001079

[00890] No expression of human HIDE! was observed in THP-1 human ceil line by either of the various F(ab’)2 (data not shown).

Knock down of human HIDE1 by siRNA in human cancer ceil lines

[00891] In order to furth erconfirm endogenous expression of HIDE ! protei inn HL-60 and U937 cel llines, human HIDE! siRNA pool w;as used for knock down as described in Material & Methods. [00892[ 48 hours post siRNA transfection, cell swer eharvest edfor furth eranalysis by qRT-PCR or by FACS. As shown in Figure 29 human HIDE ! transcri levelpt in HL-60 (A) or U973 (B) cell stransfect edwit hhuman HIDE1 siRNA pool is significantl reducedy as compared to cells transfect edwith scramble siRNd A or untreated cell hues.

[00893] Further analysis of human HIDE! membrane expression in the same siRNA transfected cells was performed by FACS, As shown in Figure 30 membrane expressions of human HIDE1 prote inis reduce din cells transfect edwit hHIDE! siRNA (green) as compared to cell stransfect edwith scramble siRNAd (orange). The fold change (anti HIDE1 vs, Isotype control in )U937 cel lline is decreased from 17.7 fold to 7 fold .For HL-60 cell line the fold is changed from 5.2 fold to 3 fold.

[00894] Confirmation of HIDE 1 endogenous expression was also tested in THP1 cell s transfect edwith HIDE1 siRNA as describe ind M&M. section. Analysis of human H1DE1 membrane expression in the siRNA transfect edcell swas performed by FACS. As shown in Figure 31 membrane expressions of human HIDE! prote inis reduced, in cell stransfected with HIDE! siRNA (green) as compared to cells transfected with scramble siRNd A (orange). The fold change (anti HIDEl vs, Isotype control) in THP1 cel lline is decrease d from 3 fold to 1.9 fold.

[00895] Additional knock down experiment of human HIDE! was performed in, U937 cel lline .Confirmation of human HIDE ! membrane expression in 11937 cel lline was tested by using the positive Serotec F(ab’)2 (Ab329, Ab332 and Ab333 have already showed expression in U937 cell line in Figure 28). 275WO 2017/009712 PCT/IB2016/001079

[00896] As shown in Figure 32 (A, B, C) membrane expressions of human HIDE1 protei isn reduce din U937 cell stransfect edwit hHIDE1 siRNA (green) as compared to cell s transfected wit hscramble siRNd A (orange). The fold change (anti HIDE! vs, non-relevant F(ab’)2, Ab307) in U937 cel lline is decreased from 2.4 fold to 1.6 fold detected by Ab329, or from 1.9 fold to 1.3 fold detecte byd Ab332 or from 1.9 fold to 1.2 fold detecte byd Ab333.

ENDOGENOUS EXPRESSION OF HIDE1 IN MOUSE CELL LINES Endogenous expression of HIDE 1 transcripts in mouse cell lines detected by qRT- PCR

[00897] In order to verify the presence of the HIDE1 transcript in mouse cel llines (listed in Table 24), qRT-PCR experiment was performe usingd specific primers (Figure 25 and Table 27) or TaqMan probes as describe above in Material & Methods and in Figure 25.

[00898] As shown in Figure 33 mouse HIDE! transcript det, ected by specific primers, SYBR green (A) was observed in J774A. L EL4, YAC-1 and A20 cell lines, wit hrelativel y high transcript level, and low er transcript level sin the other tested cell lines. The presence of mouse HIDE1 transcr iptwas verified in additional cel llines using TaqMan probes (B).Transcript was observed in RAW264.7 and P388D1 cel llines but not in othe celr llines tested.

Endogenous expression of HIDE1 protein in mouse cell line detected by WB

[00899] WB analysis for endogenous expression of HIDE! prote inwas carrie dout on various mouse cancer cell lines as detailed in Table 24. Using GenScript anti mouse HIDE1 pAb, 488536_13 describe ind Materials & Methods above. As a positive control, whol celle extrac oft stable HEK293 cel lpool ectopical lyexpressing mouse HIDE! was used whil cellse transfect edwith an empty vector served as the negative control. WB resul tsrelat edto positive and. negative controls were as expected. WB result fors endogenous expression were inconclusive as multipl bandse were detected (data is not shown). 276WO 2017/009712 PCT/IB2016/001079 Endogenous expression of HIDE1 protein in mouse cell lines detected by FACS

[00900] To verify the cell-surface endogenous expression of mouse HIDE1 protein, various cel lline s(detailed in Table 24, were tested as described in Materia &l Methods above. Ilie mouse cel llines were stained wit ha rabbit anti mouse HIDE! pAb (Genescript, ID 488536 13) (Orange) or with the Isotype control (light blue) followed by Goat anti rabbit PE Ab (Jackson cat # 711-116-152). As additiona negatil ve control, cells were stained with the secondary antibody only and represent byed a red line .Analysis was performe byd FACS.

As shown in Figure 34 Higher binding of the anti-mouse-HIDEl antibody was observed in three mouse cel lline sYAC-1, EL4 and J77A4.1 as compared to isotyp controle binding ,with high background of the isotype control Ab on the J774A. 1 cell line. No binding was observed in B16-F1 and. Sal/N cell lines.

[00901] Furthe analysisr of mouse HIDE1 endogenous expression in EL4 and J774A.2 cell lines, was done by FACS using various F(ab’)2 produce byd Serotec.

[00902] Both cell line swere stained with anti-mouse HIDE! custom F(ab j2s produce byd Serote (Figurec 35, A, B, C), or with anti-human .Non relevant F(ab’)2,, Ab307, which used as a negative control (Figure 35, light blue) followed by a secondary goat anti-human F(ab’)2 PE Ab. As an additional negative control cells, were staine dwit hthe secondary antibody only (Figure 35, red Une). As shown m Figure 35, A, B expression of mouse HIDE! was observed, in EL4 mouse cell line by the followi F(abng ’)2: Ab345 (Figure 3SA, purpl line e), Ab360 and Ab359 (Figure 35B, yellow and blue line srespectively) as compare dto isotype control expression No. expression was observed, in EL4 cel lline by the othe F(abr ’)2.

[00903] Expression of mouse HIDE! was observed also in J774A.2 mouse cell line by the follow ingF(alf)2 as shown in Figure 35C: Ab360 and Ab35v (yellow and- blue lines respectively ) as compared to isotype control expression. No expression was observed in J774A.2 cel lline by the othe F(abr ’)2. 277WO 2017/009712 PCT/IB2016/001079 Knock down of mouse HIDE1 in mouse cell lines

[00904] In order to confirm endogenous expression of mouse HIDE1 protei inn EL4 cel lline, mouse HIDE1 siRNA pool was used for knock down experiment as described in Material & Methods.

[00905] 48 hours post siRNA transfection, cell swere harvest edfor further analysis by qRT-PCR and FACS. As shown in Figure 36 mouse HIDE1 transcript level in EL4 cells transfect edwit hmouse HIDE1 siRNA pool is significantl reducedy compared to cells transfect edwit hthe scramble siRNd A or untreated cell lines.

[00906] 48 or 72 hours post siRNA transfection, cells were harvest edand were analysed by FACS for membrane expression of mouse HIDE1. As shown in Figure 37 membrane expressions of mouse HIDE1 protei 48n hours post transfecti onis slightly lower in cells transfect edwith HIDE1 siRNA (green) as compared to cell stransfected with scramble siRNd A (orange). The fold change (anti HIDE1 vs. isotype control is )decreased from 7 fold to 4.4 fold. Expression in cells 72hr post siRNA transfecti onwas not decrease d followi FACSng staining.

[00907] 72 hours post siRNA transfecti onmembrane expression of mouse HIDE! was very׳ similar to the fold in cell stransfected wit hthe HIDE! siRNA (data not shown).

SUMMARY:

[00908] This example includes preliminary data on HIDE1 endogenous expression in cel llines both at the RNA level and the prote inlevel in human and mouse cel llines.

[00909] Various human cancer cel llines were teste byd qRT-PCR, WB and FACS for endogenous expression of HIDE 1.

[00910] Cell surfac eexpression was observed in HL-60, U937, and THP1 cel llines using the mouse monoclonal Ab (BIOTEM, 33B4-2F7) as shown in Figure 27 as wel las by using 3 anti-human F(ab’)2 (Serotec) ^Ab329, Ab333, Ab332), whic hshowed cel lsurface expression in U937 cell line as shown in Figure 28. These observations are in correlati toon RNA transcript level sas shown in Figure 26. Additional confirmation in HL-60 and U937 cel llines was done by knock down experiment confirming clear reduction in the RNA 278WO 2017/009712 PCT/IB2016/001079 transcript followin HIDg E1 siRNA transfection, as shown in Figure 29, as well as some reduction observed in the prote incel lsurface expression as shown in Figures 30 and 31.

However, WB result weres not conclusive to determine a clear band at the expected band size (data not shown).

[00911] Various mouse cel llines were tested by qRT-PCR, WB and FACS for endogenous expression of HIDE 1. In the transcript level, presence of HIDE 1 was observed in J774A.1, EL4, YAC-1 and A20 cel llines as well as in RAW264.7 and P388D1 cel llines as shown m Figure 32. Correlation between positive transcript expression and cell surface protei expressn ion was observed in J774A.1, EL4, YAC-1 as shown in Figures 33 and 34.

Additional confirmation in EL4 cel lline was done by knock down experiment confirming clear reduction in the RNA transcript followi HIDng E1 siRNA transfection, as shown in Figure 36. However, the reduction in the cell surfac eprotei asn measure by FACS was low under thi sexperimental conditions. Furthe knockr down experiment tos verily these observations are currently ongoing.

[00912] Tables 25 and 26 below indicate the summary of the findings described in thi s report, highlighting the cel llines showing correlati betwon een qPCR and FACS, confirmed by knock down. Further analyses are currently on going.

Table 25. Expression analysis on human cel llines WB %KD- %KD- Cell line qRT-PCR FACS St N F S old chanop from (CGEN-H1DE1 Ct ( ivlll V1I،111؛V 11 Vil / HSKG Ct) isUlype) Not U937 24.472 / 22.732 14 conclu 80 60 sive Not HL-60 24.994 / 25.726 4 conclu 70 40 sive NCIH92 X NT 31.581 ! 24.789 9 26.217/23.911 X KG1 NT Not THP1 29.113/24.311 3 conclu sive 279WO 2017/009712 PCT/IB2016/001079 A704 30.833/25.041 NT NT RPM182 34.585 / 25.158 NT NT 26 NCI- .911 / 24.972 NT NT H28 K562 NT NT X Jurkat NT X X HSKG- housekeeping gene, NT-N ot Tested, X-negative, KD-knockdown Table 26. Expression analysis on mouse cell lines %KD- %KD- Cell line qRT-PCR FACS WB RNA FAC^S (HIDE1 Ct/HSKG (fold change from ............. .......................... 4T1 32.16 / 19.63 NT X 31.68 / 19.16 X X B16-F1 EL4 23.32 / 19.16 8 X 72 27 YAC-1 24.42 / 19.82 8 X P815 28.76 / 19.56 NT X E.G7 26 /17.92 NT X N1H3T3 29.94 / 21.25 NT NT SAI/N 31.3 / 18.61 X X J774A.1 20.31 / 18.79 7 X A20 24.94 / 20 NT NT LL/2 37.962 / 18.618 NT NT Bl 04-1-1 38.5 / 20.204 NT NT RAW264. 27.204 / 20.645 NT NT 7 P388D1 28.82 8 / 19.483 NT NT IJ ndetermined/19.61 KLN205 NT NT 7 HSKG- housekeeping gene, NT-Not Tested, X-negative, KD-knockdown TABLE 27. Primers Sequences Of Mouse And Human HIDE1 And The House-Keeping Gene Mouse HPRT1 Prime rname Prime rsequence 200-774 hHIDEl F GCCCAGATTAACTTCGACAG 280WO 2017/009712 PCT/IB2016/001079 200-775 hHIDEl R CTGAGTGATCATCCAAGGTG 200-780 mHIDEl F TGGGCTCAGATCAACTTCAC 200-781 mHIDEl R CCACTGAGTCTTCCTGAGTC 200-824 hHPRTl F TGACACTGGCAAAACAATGCA 200-825 hHPRTl R GGTCCTTTTCACCAGCAAGCT 200-719 rnHPRT lF GCAGTACAGCCCCAAAATGG 200-720 mHPRTI_R TGCAGATTCAACTTGCGCTC EXAMPLE 12: HIDE1 EXPRESSION ON HUMAN PBMCs

[00913] To evaluat ethe expression of human HIDE1 on human peripheral mononuclear cell s(PBMCs) using 2 monoclonal antibodie fors HIDE1 by flow cytometry.

Material ands Methods

[00914] Isolation of PBMCs: Buffy coats were obtained from Stanfor Bloodd Bank from healthy human donors. PBMCs were isolated by Ficol gradientl separation, washed wit h medium ,and cryo-preserve ford futur eexperiments.

[00915] Antibodies: The follow ingantibodie weres used. Alexa flour 647 labeled mouse IgGl isotype control (lot. F1150528d-2695, BIOTEM), Alexa flour 647 labeled anti - human HIDE1 antibodies (BIOTEM, 33B4-2f7 and 36-C1-2F6). Brillia violent 510t labeled anti-CD3 (Biolegend, cat. 344828), PE-labeled anti-CD14 (Biolegend, cat .301806) and PercP-labeled anti-CD19 (Biolegend, cat .302228).

Flow cytometry analysis

[00916] PBMCs cells were thawed and stained with viability dye (BD Horizon; Cat# 562247, BD biosciences), washed and pre-block edwit hFc blocking soluti on(cat# 422302, Biolegend; 1:20) and/or human Ig Fc (Jackson; 200pg/ml; cat# 009-000-008) to avoid nonspecifi cbinding via Fc receptor Cells. swere then stained wit hanti-hHIDEl mAbs or mlgG lisotype control in the presence or absence of lymphocyt ores monocytes surface marker. All samples were run on a MACSQuant analyzer (Milteny i)and data was analyzed using Tree Star FlowJo software. Cell swere first gated for lymphocyt ores monocyte (FSC-A vs. SSC-A), and furth ergated for live cells before analyzed for HIDE1 expression by flow cytometry. 281WO 2017/009712 PCT/IB2016/001079 Results

[00917] Freshl thawy ed PBMCs from 2 donors were thawed and stained wit hviability dye, washed and pre-blocked with Fc blocking soluti onas described in material and methods.

HIDE1 expression was observed on CD 14+ cell sbut not on CD3+ cells (Figure 38) in 2 donors using 2 monoclonal antibodie agais nst HIDE1. The staining was in the range of 3 folds over isotype control No. expression on CD 19+ was observed (data not shown ).To avoid non-specific binding to Fc receptor the staining was repeated in the presence of addition human Fc fragments with 4 different donors. As shown in Figure 39 HIDE1 expression was observed in 4 different donors while no expression on lymphocyt wases observed (data not shown).

Summary

[00918] HIDE1 expression on human monocyte wass observed using 2 anti-human HIDE1 mAh in 4 different donors. The expression was exclude dto monocytes. No expression on T cell sor B cells was observed. Furthe experimr ent are ongoing in order to analyze the expression of HIDE1 on othe leukocyter populats ion such as neutrophil ands dendritic cells.

EXAMPLE 13: HIDE1 EX-VIVO RNA EXPRESSION Study A: Material & Methods Reagents used in this study 1. RNA extracti onwas performed with RNAeasy Mini Kit (Qiagen cat # 74014). 2. cDNA was produced using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems cat#4368814). 3. Mouse HIDE1 TaqMan probe: HOJEX12_CCAAZKY, custom made, Life technologies. 4. TaqMan probes for Housekeeping gene (HSKG) (Life technologies) mouse RPL19: Mm02601633_gl , mouse SDHA: Mm013526363_ml, mouse PBGD: Mm01143545_ml. 282WO 2017/009712 PCT/IB2016/001079 . ABI TaqMan Fast Advanced Master mix, part no. 4444557, Applied Biosystem 6. CT26.WT cell s,CRL-2638, ATCC 7. Ant iPDL-1 mlgGl, Inc production (YWW243.58.870, LOT #40315,) 8. mlgGl isotype control (MOPC-21), Biolegend.

Methods Animals:

[00919] 7wk old female BALB/c mice were purchased from ENVIGO laboratories and kept in specific pathogen-fre conditie ons at the Tel Aviv University animal facility.

Experiment respes cte instid tutional guidelines and were approved by the Tel Aviv University ethics committee Tumor model and mAh treatments:

[00920] The murine CT26, an N-nitroso-N methylurethane - induced undifferentiated colon carcinoma cel lline, was obtained from American Type Culture Collection (CRL- 2638). CT26 cell s(5x105) were injected subcutaneously at day 0 in the right flank of BALB/c mice (female ,7wk). Tumor growth was monitored twice a week using a caliper.

[00921] Treatments were given twice and started at day 8, when tumor area reache d 50mm2 and on day 12 post tumor inoculation. Treatments were injected intraperitonea at l lOmg/Kg (9 mice/treatment) of either anti PDL-1 mlgGl (YWW243.58.870, LOT #40315,) or mlgG lisotype control (MOPC-21).

Tissue samples:

[00922] Whole tumor and spleens tota RNAl was extracte usingd gentleMACSTM Octo Dissociat or(Miltenyi Biotec), and tissues were homogenized wit hM tubes (# 130-096-335).

[00923] For tumor singl ecel lsuspension, tumor weres enzymaticall digesy ted wit h lug/m lCollagenase D (Worthington Biomedical Corporations 0.5pg/ml) Deoxyribonuclea se I (Worthington Biomedical Corporations in RPMI) -1670 (Biological Industrie fors) Ihr in 370c. Remains of undigested tumor tissues were gently agitate dfor full a dissociation. 283WO 2017/009712 PCT/IB2016/001079 Cells isolation:

[00924] Thy 1.2+ T lymphocyt werees purified from tumor usings a CD90.2+ positive isolation kit (EasySep, #18751) and purified Thyl.2 + T lymphocyt andes the negative fraction cells were lysed in RET to extract tota RNA.l

[00925] CD1 lb+ cell swere purified from tumors using a CD1 lb+ positive isolation kit (EasySep, #18770) and purified CD1 lb+ cells and the negative fraction cells were lysed in RET to extrac totat RNAl Transcript expression Quantitative RT-PCR (qRT-PCR)

[00926] RNA (l-5ug) extracti onof mouse cells, derived from Ex-vivo experiments (346-MA-455 & 459-MA) was preforme accordid ng to manufactures protocols.

[0100] cDNA was prepared according to manufactures protocols (lug RNA diluted in 20ul cDNA mix reaction).

[00927] cDNA, prepared as described above, diluted 1:10 (representi 25ngng RNA per reaction), was used as a templat fore qRT-PCR reaction s,using a gene specific TaqMan probes (detailed in Reagents 3&4) Detection was perform edusing QuantStudi 12ko device.

[00928] The cycle in which the reactions achieved a threshol leveld of fluorescence (Ct= Threshold Cycle )was registered and was used to calculate the relati vetranscript quantity in the RT reactions.

[00929] The absolute quantity was calculate byd using the equation Q=2 A-Ct.

[00930] The resulting relati vequantities were normalized to a relative quantities of housekeeping gene, mRPL19, mSDHA and mPBGD. 284WO 2017/009712 PCT/IB2016/001079 Results Endogenous expression of HIDE 1 in mouse cells Endogenous expression of HIDE 1 in tumor derived cells (Ex vivo# 455)

[00931] In order to verify the presence of the HIDE! transcript in tumo rderive dcell, qRT-PCR was performe usingd a specific TaqMan probe as describe above in Material & Methods.

[00932] As shown in Figure 40, mouse HIDE! transcri ispt observed using TaqMan probe HOJEX12-CCAAZKY with relatively high level sin GDI lb+ subpopulati oncells , compared to tumor derive dcells and to CD1 lb- subpopulation.

Endogenous expression of HIDE 1 in tumor derived cells (Ex vivo# 459)

[00933] In order to verify the presence of the HIDE! transcript in tumor derive dcell, qRT-PCR was performed using, a specific TaqMan probe as describe above in Materia &l Methods.

[00934] As shown in Figures 41, 42 and 43 mouse HIDE! transcript is observed using TaqMan probe HOJEX12-CCAAZKY wit hrelatively high level sin (I) lib subpopulation (Figures 41, 42, and 43), spleen cell s(Figure 42) and in colon lamina, propri anda whol e colon cell s(Figure 42). Lower transcript level is observed in colon epithelia cells.

StudyB Material & Methods Reagents used in this study 1. RNA extracti onwas performed with RNAeasy Mini Kit (Qiagen cat # 74014). 2. cDNA was produced using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems cat#4368814). 3. Mouse HIDE1 TaqMan probe: HOJEX12_CCAAZKY, custom made, Life technologies. 4. TaqMan probes for Housekeeping gene (HSKG) (Life technologies) mouse RPL19: Mm02601633_gl, . ABI TaqMan Fast Advanced Master mix, part no. 4444557, Applied Biosystem 285WO 2017/009712 PCT/IB2016/001079 6. CT26.WT cell s,CRL-2638, ATCC 7. Ant iPDL-1 mlgGl, Inc production (YWW243.58.870, LOT #40315,) 8. mlgGl isotype control (MOPC-21), Biolegend.

Methods Animals:

[00935] 7wk old female BALB/c mice were purchased from ENVIGO laboratories and kept in specific pathogen-fre conditie ons at the Tel Aviv University animal facility.

Experiment respes cte instid tutional guidelines and were approved by the Tel Aviv University ethics committee Tumor model and mAh treatments:

[00936] The murine CT26, an N-nitroso-N methylurethane - induced undifferentiated colon carcinoma cel lline, was obtained from American Type Culture Collection (CRL- 2638). CT26 cell s(5x105) were injected subcutaneously at day 0 in the right flank of BALB/c mice (female ,7wk). Tumor growth was monitored twice a week using a caliper.

[00937] Treatments were given twice and started at day 8, when tumor area reache d 50mm2 and on day 12 post tumor inoculation. Treatments were injected intraperitonea at l lOmg/Kg (9 mice/treatment) of either anti PDL-1 mlgGl (YWW243.58.870, LOT #40315,) or mlgG lisotype control (MOPC-21).

Tissue samples:

[00938] Whole tumor and spleens tota RNAl was extracte usingd gentleMACSTM Octo Dissociat or(Miltenyi Biotec), and tissues were homogenized wit hM tubes (# 130-096-335).

[00939] For tumor singl ecel lsuspension, tumor weres enzymaticall digesy ted wit h lug/m lCollagenase D (Worthington Biomedical Corporations 0.5pg/ml) Deoxyribonuclea se I (Worthington Biomedical Corporations in RPMI) -1670 (Biological Industrie fors) Ihr in 370c. Remains of undigested tumor tissues were gently agitate dfor full a dissociation. 286WO 2017/009712 PCT/IB2016/001079 Cells isolation:

[00940] Thy 1.2+ T lymphocyt werees purified from tumor usings a CD90.2+ positive isolation kit (EasySep, #18751) and purified Thyl.2 + T lymphocyt andes the negative fraction cells were lysed in RET to extract tota RNA.l

[00941] CD1 lb+ cell swere purified from tumors using a CD1 lb+ positive isolation kit (EasySep, #18770) and purified CD1 lb+ cells and the negative fraction cells were lysed in RET to extrac totat RNAl Transcript expression Quantitative RT-PCR (qRT-PCR)

[00942] RNA (1-5 pg) extracti onof mouse cell s,derived from Ex-vivo experiments (346-MA-455 & 459-MA) was preforme accordid ng to manufactures protocols.

[00943] cDNA was prepared according to manufactures protocols (lug RNA diluted in 20ul cDNA mix reaction).

[00944] cDNA, prepared as described above, diluted 1:10 (representi 25ngng RNA per reaction), was used as a templat fore qRT-PCR reaction s,using a gene specific TaqMan probes (detailed in Reagents 3&4) Detection was perform edusing QuantStudi 12ko device.

[00945] The cycle in which the reactions achieved a threshol leveld of fluorescence (Ct= Threshold Cycle )was registered and was used to calculate the relati vetranscript quantity in the RT reactions.

[00946] The absolute quantity was calculate byd using the equation Q=2 A-Ct.

[00947] The resulting relati vequantities were normalized to a relative quantities of housekeeping gene, mRPL19. 287WO 2017/009712 PCT/IB2016/001079 Results Endogenous expression of HIDE 1 in intestine populations derived from C57 black mouse cells Endogenous expression of HIDE 1 in intestine populations derived from C57 black mouse cells (Ex vivo# 466)

[00948] In order to verify the presence of the HIDEl transcript in intestine population cells derive dfrom C57 black mouse ,qRT-PCR was performe usingd a specific TaqMan probe as describe above in Material & Methods.

[00949] As shown in Figure 44, mouse HIDEl transcri ispt observed using TaqMan probe HOJEX12-CCAAZKY with relative lyhigh level sin mesenter icand peripheral lymph nodes cells as, wel las in spleen and peyer patches cells compared to smal lintestine and colon cells.

EXAMPLE 14: HIDEl Tetramer Binding to Human T Cells

[00950] The aim of thes eexperiments was to evaluate binding of HIDEl tetramer to resting and activated human isolated primary CD4+ and CD8+ T cells, as well as TILs (Tumor Infiltrati Lymphocytesng isola) ted from human melanoma samples and propagat edin the presence of melanoma specific antigens and IL2.

MATERIALS AND METHODS BIOTINYLATED MONOMERIC PROTEINS:

[00951] Human HIDEl was produce asd a monomer, includin gthe extracellular domain (ECD), a biotinylati signaon l and a His-tag (batch #502). Following enzymatic biotinylat ionof thi sprote inperforme byd Genscript thi, sprotei (batchn #503) was used for tetramer production and binding studies. As negative and positive controls for binding studies, similar biotinylat monomed eric proteins of human EGFR (EGR-H82E7, Acrobiosystem ands) human B7H4 (B74-H8222, Acrobiosystem weres), used for tetrame r production, respectively. 288WO 2017/009712 PCT/IB2016/001079 STREPTAVIDIN-PE:

[00952] Streptavidi R-Phycn, oerythrin Conjugat e(SA-PE) - premium grade (Molecular probes S21388,, 1 mg/ml) was used for the production of tetramers. This streptavidin-PE is a high-purit7 product,y ensuring a uniform material with a constant molecular weight (300kDa).

PRODUCTION OF TETRAMERS:

[00953] Similar w eights of HIDE! (batch #503), EGFR and B7H4 were used for tetramer production. Tetramers were created by mixing the above proteins with SA-PE at a 4:1 mola rratio. SA-PE was added by gradual titrati intono the proteins in 15 portions. Afte r each portion was added, the mixture was incubated for 5 minutes. In the first five portions, a lower amount of SA-PE was added (1.5-fold less than the followi tenng portions), in order to saturat thee streptavidin sites. The entir proceduree was carrie dout at room temperature (SA- PE was kept on ice) in the dark, and tetramers were store afterwad rds at 4 degrees. Similar concentration (by weight per volume) of the different tetramers was used for the binding experiments.

PRIMARY T CELLS

[00954] In thi sseries of experiment cellss from two different donors, obtained from Astarte Biologies were used: • CD4+and CD8+from donor #147 • CD4+and CD8+from donor #186

[00955] Human primary cells (>95% purity), were thawed 24h prior to beginning of experiment Cell. swere thawed in RPMI complete medium (RPMI + 10% FBS + 1% Glutama x+ 1% Na-Pyruvat e+ 1% Pen-Strep) supplemented wit h300 U/ml of rhIL2 (Biolegend 509129). Cells were left to recover for 24 hours.

TILS

[00956] In thi sseries of experiment s,two different TILs from resecte metd astases of three melanoma patients were, used: • TIL-Mart1- HLA-A2-Marti specific • TIL-209- HLA-A2-gpl00 specific 289WO 2017/009712 PCT/IB2016/001079

[00957] Human TILs (>95% CD8+), were thawed 24h prior to beginning of experiment Cell. swere thawed in 12 ml of TIL medium (IMDM + 10% human serum + 1% Glutama x+ 1% Na-Pyruvat e+ 1% non-essential amino acids + 1% Pen-Strep) supplemented with 300 U/ml of rhIL2 (Biolegend 509129). Cell swere left to recove rfor 24 hours.

CELL LINES

[00958] The follow inghuman T cel llines were used: H9 (ATCC, HTB-176, clonal derivative of HUT-78), Jurkat (ATCC, TIB-152, human acute T cell leukemia) and U937 (ATCC, CRL-1593.2 monocyte).

[00959] Cells were culture ind complet media:e RPMI (Biological Industries Cat# 01- 100-1A) supplemented with 10% FBS (Biological Industrie Cats # 04-001-1A), 1% Glutamax (Life technologies Cat# 35050-038).

ASSAY CONDITIONS

[00960] After recovery, cell swere activated using a polyclonal activation of T cells, with 1 ug/ml of plat bounde anti CD3 antibody (BD-pharmingen clone Ucht-1, cat-555329), 2 ug/ml of anti CD28 ab (eBioscience clone CD28.2 cat16-0289-85)- and 300 U/ml of IL2.

Activation was carried out for 24h, 48h, 72h and 144h.

CELL STAINING

[00961] Cell swere harvest edand washed wit hPBS. Cells were stained at room temprature for 10 minutes wit hPBS supplemented wit h1/1000 of fixable viability stain efluor 450 (BD horizon cat-562247). After staining, cells were washed twice wit hPBS and staine dwith the Abs at the concentrations liste ind Table 9 for 30 minutes at room temperature (RT) in FACS buffer (PBS + 0.5% BSA + 2 mM EDTA + 0.05% Azide) and concentrations that are liste ind Table 28.

Table 28. Antibodies specifics and staining concentration used Antibodies Isotype Conjugated Manufacturer Catalog concentration Staining number concentration to (pg/ul) Anti-CD4 300506 0.4 mlgGl FITC Biolegend 4 ug/ml Anti-CD8 mlgGl FITC Biolegend 300906 0.15 1.5 ug/ml 290WO 2017/009712 PCT/IB2016/001079 PE 309804 0.2 Anti- mlgGl Biolegend 2 ug/ml CD137 Anti-PDl mlgGl APC-cy7 Biolegend 329922 0.2 4 ug/ml isotype mlgGl PE Biolegend 400112 0.4 2 ug/ml control isotype mlgGl APC-cy7 Biolegend 400128 0.2 4 ug/ml control

[00962] For tetramer binding, cells were incubated for 30-40 min at RT with the tetramer proteins and controls at 3pg/wel (50pl/wl ell=60pg/ml) in FACS buffer (PBS + 0.5% BSA + 2 mM EDTA).

[00963] After binding, cells were washed three times and re-suspended in FACS buffer for analysis.

RESULTS

[00964] The binding of HIDE1 tetrame tor various human cel llines was tested, as described in Material ands Methods (M&M). HIDE1 exhibited binding to H9 and Jurkat, at comparable level s(Figure 57), but not to U937 myeloid cel lline.

[00965] The binding of HIDE1 to activated primary human T cell sand TILs was evaluated next. T cell sfrom two different donors and TILs were left untreat ed(resting) or polyclonal stimulate ford various timepoints as described in M&M. Cel lactivation state was evaluated by detection of surface expression of CD 137 and PD-1 at each time point compare d to isotype control (FMO), as shown for activated CD8+, CD4+ T cells and TILs (Figure 58A-58C respectivel y).As expected, PD-1 and CD137 expression was elevated apon activation (Figure 58A-58C).

[00966] Results show binding of HIDE1 to activated CD4+ and CD8+ T cell sbetween 24 and 72 hours (Figure 59A-65C). The binding of HIDE1 was more prominent to activated CD4+ T cells at 72h reaching 3-fold increase in the Geo Mean compared to EGFR negative control. 291WO 2017/009712 PCT/IB2016/001079

[00967] In addition, HIDE1 tetrame bindingr to activated Marti and 209 TILs was detected at 24h after activation and, sustained to 72h of activation (Figure 60A-60C). On day 6 of activation, no binding of HIDE1 tetramers was detecte d,similarly to what was observed with resting TILs (Figure 60).

SUMMARY

[00968] The study presented here demonstrate thes binding of HIDE1 tetramers to human T cel llines, as well as, human CD4+ and CD8+ T cells and TILs, whic hwere polyclonal activated. Results points to expression of the putative molecular counterpa ofrt HIDE1 on thes targete cells. Binding of HIDE 1 tetramer was detected only upon activation of CD4+ and CD8+ cell s,reaching a peak at 72h post activation. This data suggests the expression of aHIDEl counterpa onrt T cells and its up-regulati onupon TCR-dependent T cel lactivation.

EXAMPLE 15: IN-VITRO IMMUNOMODULATORY ACTIVITIES OF HIDE1 ECD- IG ON MOUSE T CELLS Experiment A:

[00969] In thes experie ment thes immunomodulat oryactiviti esof the recombinan t fused prote inHIDEl-ECD-Ig was investigated on mouse T cel lactivation.

[00970] In order to evaluate the activit7 yof HIDE1 prote inon T cel lactivation, recombinan protet inwas produced comprising the mouse extracellul domainar (ECD) of the mouse HIDE ! fused to the Fc of mouse IgG2a (designated HIDE1-ECD Ig M:M, SEQ ID NO: 18). The effect of the Fc fused prote inco-immobilize witd hanti-CD3 on mouse CD4 T cel lfunctions, as manifested by activation markers and cytokine secrets ion was investigated.

Mouse IgG2a (clone MOPC-173; Biolegend or Cl. 18.4; BioXcell) was used as isotype control.

Materials and MethodsFc fusion protein and control Ig

[00971] The Fc fusion protein, HIDEl-ECD-l g(batche s#72 and #195, SEQ ID NO:18) were tested. Mouse IgG2a (clone MOPC-173; Biolegend or Cl.18.4; BioXcell) was used as isotype control. 292WO 2017/009712 PCT/IB2016/001079 Mouse CD4 T cell sisolation

[00972] Untouched CD4+CD25- T cell swere isolated from pools of spleens of BALB/C mice using a T cel lisolation Kit (Miltenyi Cat# 130-093-227) according to the manufacture’sr instructions. The purity obtained was >90%.

Activation of mouse CD4 T cells [00973[ Anti-mouse CD3-e mAb (clone 145-2C11; BD Biosciences) at 2pg/ml together wit hHIDEl-ECD-Ig protei (SEQn ID NO: 18) or contr olIg at various concentrati on (1, 3 or lOug/ml), were co-immobilize ford 3hr at 37°C, on 96-well flat bottom tissue culture plates (Sigma, Cat. # Z707910). Control Ig was added to each wel lin order to complet ae total protei concentratn ion of I2pg/ml per wel l.Well swere washed 3 times wit hPBS and plated wit h1x105 purified CD4+CD25- T cells per well and kept in a humidified, 5% CO2, 37°C incubator In. some experiments, solubl antie -CD28 (clone: 37.51; eBioscience ;Ipg/ml ) was added. Culture supernatan tswere collect edat the indicated times post stimulation and analyzed for mouse IFNy or IL-2 secretion by ELISA kit s(R&D Systems ).The effect of HIDEl-ECD-Ig prote in(SEQ ID NO: 18) on the expression of the activation marker CD69 on mouse CD4+ T cell swas analyzed by flow cytometry. Cells were stained 48h post stimulati onwith a cocktail of antibodie incls uding PerCP-anti-CD4 (clone G41.5; Biolegend), FITC or PE-anti-CD69 (clone H1.2F3; Biolegend), in the presence of anti - CD16/32 (clone 2.4g2; BD Biosciences) for blocking of Fcy-receptor Cellss. were evaluated using MACSQuant analyzer 9 (Miltenyi) and data analyzed using BD CellQuest or by MACSQuantify ™ Software. Data was analyzed using Excel or Prisn14 software.

RESULTS AND SUMMARY Effect of HIDE1-ECD Ig M:M on mouse CD4+ T cells function

[00974] Figure 45 shows in-vitro immunomodulat oryactivities of HIDEl-ECD-Ig M:M (SEQ ID NO: 18) on isolated mouse spleni cT cells (CD4+, >95%purity) stimulated with microplates co-immobilized with anti-CD3 (2pg/ml) togethe withr control Ig (mIgG2a) or HIDEl-ECD-Ig (SEQ ID NO: 18) (10 pg/ml) in the presence of solubl antie -CD28 (Ipg/ml). HIDEI-ECD-Ig (SEQ ID NO: 18) suppressed mouse CD4 T cel lactivation as manifeste dby reduction in TCR-induced cytokines (IL-2 and IFNy) secretion (Figures 45B- 293WO 2017/009712 PCT/IB2016/001079 45C). The magnitude of Ilie inhibito7ry effect of HIDEl-ECD-Ig (SEQ ID NO: 18) was in the range of 50-60%. Under similar conditions no, effect on CD69 expression was observed (Figure 45A).

[00975] HIDE1-ECD-Ig (SEQ ID NO: 18) inhibits T cell activation in a concentration- dependent manner when the reagent is co-immobilized with anti-CD3 on plates. Maximal inhibitory effect was observed at lOpg/m lof HIDEl-ECD-Ig (SEQ ID NO: 18).

[00976] Figure 45 present thes effect of various HIDEl-ECD-Ig (SEQ ID NO: 18) on mouse CD4 T cell activation. Plates were coated with anti-CD3 mAb (2pg/mL) in the presence of 10ug/ml HIDEl-Ig (batch #195) ybar is the mean of duplicate cultures, the error bars indicating the standard deviation. (Figures 45B-45C) Culture supernatants were collect edat 48 h post-stimulati andon mouse IL-2 and IFNy levels were analyzed by ELISA.

Results are shown as Mean ± Standard errors of duplicate samples. One experiment out of two performed is presented.

[00977] The resul tsdemonstrat thee inhibitory effect of HIDEl-ECD-Ig on mouse T cells activation mani, feste dby reduced cytokine secretion, and suppression of activation marker CD69 upregulatio Thisn. inhibition of T cel lactivation, supports the therapeuti c potential of immunoinhibitory HIDE1-ECD polypeptides and/or fusion proteins thereof according to at least some embodiments of the present invention in treating T cell-driven autoimmune diseases, such as rheumatoid arthri timults, iple sclerosi psoris, asis and inflammatory bowel disease, as well as for othe immr une relate dised ases and/or for reducing the undesirable immune activation that follows gene therapy.

Experiment B: Introduction

[00978] In order to evaluat ethe activit y'of HIDE! protei onn T cel lactivation, recombinan protet incomprising the extracellula domainr (ECD) of the mouse HIDE1 prote in fused to the Fc of mouse IgG2a (designated HIDEl-Fc) was used. Ure effect of the HIDE1- Fc fused proteins co-immobilize withd anti-CD3 on mouse CD4 T cel lfunctions, as manifeste dby activation markers and cytokines secretion, was investigated. 294WO 2017/009712 PCT/IB2016/001079 MATERIALS AND METHODS Table 29: Fc Fusion proteins and control IgG Teste dproteins Batch #/ Clone/Cat# ECD:Fc Vendor Comment mIgG2A MOPC-173 Biolegend Isotype control HIDEl-Fc #195 M:M ProBioGen Tested compound #72 HIDEl-Fc M:M Excellgene Tested compound B7H4-Ig 4206-B7-100 M:M R&D Systems Positive control

[00979] Mouse IgG2a (clone MOPC-173; Biolegend) was used as isotype control.

During assay set-up, B7H4-Ig (Cat. 4206-B7, R&D), a Fc fusion protei comprisn ing the extracellula domainr (ECD) of mouse B7H4 fused to the Fc of mouse IgG2a, was used as a positive control Table. 29, above.

Mouse CD4 T cells isolation

[00980] Untouched CD4+CD25- T cell swere isolated from pools of spleens of BALB/C mice using a T cel lisolation Kit (Miltenyi Cat# 130-093-227) according to the manufacture’sr instructions. The purit yobtained was >90%. Follow ingisolation, cells -wer e used either fresh or afte rfreezing (in 90% FCS, 10% DMSO) and thawing.

Activation of mouse CD4 T cells

[00981] Anti-mouse CD3-e mAb (clone 145-2C11; BD Biosciences) at 2pg/ml of PBS, alone or together wit hHIDEl-Fc prote inor control Ig at various ratios, was co- immobilized for 3hr at 37°C, on 96-well flat bottom tissue culture plates (Sigma, Cat. # Z707910). Well swere washed 3 times wit hPBS and plated wit hIxlO5 purified CD4+CD25- T cells per well in the presenc eof soluble anti-CD28 (clone: 37.51; eBioscience ;2pg/ml ) and kept in a humidified, 5% CO2, 37°C incubator Cult. ure supernatants were collect 48hed post stimulation and analyzed for mouse IFNy or IL-2 secretion by ELISA kit s(R&D Systems). ’Ilie effect of CGEN-Fc proteins on the expression of the activation marker CD69 on mouse CD4+ T cell swas analyzed by flow cytometry. Cell swere stained 48h post stimulation wit ha cocktai ofl antibodie includins gPerCP-anti-CD4 (clone G41.5; Biolegend), FITC or PE-anti-CD69 (clone H1.2F3; Biolegend), in the presence of anti- 295WO 2017/009712 PCT/IB2016/001079 CD16/32 (clone 2.4g2; BD Biosciences) for blocking of Fcy-receptor Cellss. were evaluated using MACSQuant analyzer 9 (Miltenyi) and data analyzed using BD CellQuest or by MACSQuantify TM Software. Data was analyzed using Excel or Prism 4software.

RESULTS Experimental system and B7H4-Ig effect on mouse T cell activity

[00982] We established a functional assay to evaluat ethe activity of tested protein on mouse CD4 T cell sfunction. The assay evaluates T cel lfunction follow ingactivation wit h plate-bound anti-CD3 mAb, co-immobilized wit htested Fc fusion proteins as, reporte d before for other immune checkpoi ntregulators such, as B7H4 and VISTA (Ref 1, 2). B7H4- Ig was used as a positive control in the assay (inhibitory ligand) while mouse IgG2a was used as a negative control Schem. ati cillustrati ofon the experiment alsystem, and the effect of B7H4-Ig on T cel lactivation are described in Figure 75.

Effect of HIDE 1 on mouse CD4 function

[00983] In order to evaluat ethe immunomodulat oryeffect of HIDE1 on mouse CD4+ cells,

[00984] HIDEl-Fc (batch #72 and #195) was co-immobilize withd anti-CD3, as described in material and methods. The effect of tw obatches of HIDEl-Fc on mouse CD4 T cel lactivation in, the presence of solubl anti-Ce D28, upon co-immobilizati onon microplate s with anti-CD3 was evaluate din two separat experie ments using fresh or thawed isolated CD4 T cells. (Figure 76 and Table 30, respectivel y).HIDEl-Fc (batch #72 and #195) inhibits freshly isolated mouse CD4+ CD25- T cel lactivation, as manifeste dby reduced CD69 expression (Figure 76A), and IFNy and IL-2 secretion (Figure 76B-C). The magnitude of the inhibitory effect on CD69 expression of batch #72 was reduced compar eto batch #195 at lOug/m l(-30% vs -11%, respectively) while both batches inhibit IL-2 and IFNy secretion in the range of 50-60% compar eto Control Ig (Figure 76). Table 30 summarize the dose response effect of HIDEl-Fc on freshly thawed CD4+ T cell function (CD69 expression or IFNy secretion). 296WO 2017/009712 PCT/IB2016/001079

[00985] No effect of HIDEl-Fc on T ceil sactivit ywas observed when 1 or 3ug/ml of HIDE1-Fc were used. Nevertheless, at lOug/ml, HIDEl-Fc (batch #72 but not #195) inhibits CD69 expression and IFNy secretion (Table 30).

Table 30. Tbe dose response inhibitory effect of HIDEl-Fc on mouse CD4+ T cell function.

% Inhibition vs. control Ig (48h post stimulation) Experiment lug/ml 3pg/ml 10ug/ml CD69 INFy CD69 INFy CD69 INFy batch# 72 -10% 4% 2% HIDEl-Fc 3% iiiiiiiii iiiiiiiii iiiiiiiii 195 17% 4% 1200 HIDEl-Fc 2196 SUMMARY

[00986] Two experiment weres performed wit hHIDEl-Fc, co-immobilized with anti- CDS, aiming at assessment of its effect on CD4 T cell sactivity. In the firs experimt ent, performe witd hfreshly isolated CD4 T cell s,both batches of HIDEl-Fc suppressed mouse CD4 T cel lactivation. In the second experiment s,performe witd hthawe dCD4 T cells, the two batched inhibite dT cel lactivit yfor different extent. Overall the, maximal inhibitory effect of HIDEl-Fc on cytokine secretion was observed at lOug/m land the magnitude of the inhibition was in the range of 50-60%.

EXAMPLE 16: EFFECT OF HIDE1-ECD FUSION PROTEIN ON HUMAN T CELLS ACTIVATED USING ANTI-CD3 AND ANTI-CD28 IN THE PRESENCE OF AUTOLOGOUS PBMCS MATERIALS AND METHODS

[00987] In thes eexperiments the effects of HIDE1 on human T cell swhich were activated using anti-CD3 and anti-CD28 in the presence of autologou PBMCSs was evaluated. 297WO 2017/009712 PCT/IB2016/001079

[00988] HIDE1 hECD-hlg fusion prote in(SEQ ID NO: 17, batch #48), composed of the ECD of human HIDE1 fused to the Fc of human IgGl bearing C220 to S mutation at the hinge, was produce atd ExcellGen e(Switzeland) by transient transfection in CHO-DG44 cells using Excellgene’s propriet aryvector system Cell. swere culture ford 10 days, followed by Protein A purification of cell har vest .The final produ ctwas formulated in 0.1M Glycine pH 6.

[00989] Expression vector used was Mammalian Expression Vector pTT5, in whic h HIDE1 gene is drive nby CMV promoter.

[00990] CD4+ Human T cel lIsolation Kit II is purchased from Miltenyi (Cat. #130- 094-131). hlgGl control (Synagis®) is obtained from Medimmune Inc. Anti-huma nCD3 Ab (OKT3, Cat# 16-0037) and anti-human CD28 Ab (clone CD28. 2; Cat# 16-0289) were purchased from eBioscience. Dynabeads M-450 Epoxy (Cat .# 140. 11) were purchased from Invitrogen. Buffy coats of human blood were obtained from LifeSource. Ficoll-Paque Plus (Cat. #17-1440-02), was purchased from GE Healthcare.

Isolation of PBMCs from buffy coat susing Ficoll separation

[00991] Total PBMCs were suspended in Ex-Vivo 20 medium ,and irradiate atd 3000rad. Naive CD4+ T cells were isolated from buffy coats of three healthy human donors’ blood using CD4+ Human T cell Isolation Kit II (Miltenyi) according to manufacturer’s instructions and co-cultured wit hirradiate autold ogous PBMCs at a ratio of 1:1 (1. 5x105 T cells wit h1. 5xl05 irradiate PBMCd s per well) The. cultures were activated wit hanti-CD3 (0. 5pg/ml) and anti-CD28 (0. 5 ug/ml) antibodies. HIDE1 hECD-hlg (SEQ ID NO: 17) or hlgGl control Ig (Synagis®) were added to the culture at the indicate dconcentrations. Afte r 24 hr in culture, cell swere pulse dwit hH3-thymidine. Cells were harvest edafter 72 hours in culture.

RESULTS

[00992] As shown in Figure 46, the addition of HIDE1 hECD-h!g_(SEQ ID NO: 17) to cultures of naive T cell sactivated by anti-CD3/anti-CD28 in the presence of irradiate d autologous PBMCs, resulted in a dose dependent inhibition of T cel lproliferation This. inhibition of T cel lactivation supports the therapeutic potential of immunoinhibitory HIDE1 298WO 2017/009712 PCT/IB2016/001079 based therapeutic agents according to at least some embodiments of the present invention, for treating T cell-driven autoimmune diseases, such as rheumatoid arthri tismult, iple sclerosi s, psorias andis inflammatory bowe ldisease, as well as for treating other immune related diseases and/or for reducing the undesirable immune activation that follows gene or cell therapy Essenti. all HIDE1y, based therapeutic agents that agonize HIDE1 should prevent or reduce the activation of T cells and the production of proinflammatory cytokines involved in the disease pathology of such conditions.

[00993] Figure 46 demonstrate inhibits ion of human T cel lproliferati induceon d by anti-CD3 and anti-CD28 in the presence of irradiate autologousd PBMCs by human HIDE1 ECD-Ig (SEQ ID NO: 17). Figure 46A shows averages of three donors tested. Figures 46B- 48D show the individual data of each donor. The control Ig is Synagis.

EXAMPLE 17: IN-VITRO IMMUNOMODULATORY ACTIVITIES OF HIDE1 ON HUMAN CYTOTOXIC T CELLS (CTLS)

[00994] The experiment describes ind thi sexample evaluated the effect of ectopic expression of human HIDE1 on different melanoma cel lline son thei abilitr yto activate CTLs (cytotoxic T lymphocytes and) serve as targets for killing by these cells.

MATERIALS & METHODS:

[00995] Three human melanoma cel lline swhic hpresent the MART-1 antigen in HLA-A2 context (SK-MEL-23, Mel-624 and Mel-624.38) were used as targets for CTLs.

Mel-888 whic hdoes not express HLA-A2, served as a negative control.

Ectopic expression of human HIDE1 on cytotoxic T lymphocytes (CTLs).

[00996] In order to express human HIDE1 in peripheral blood leukocyte (PEL) cultures, the cDNA encoding for HIDE1 was amplified using specific primers and cloned into an MSCV-based retrovi ralvector (pMSGVl). Verification of the cloning was done firs usingt restricti enzymon e digestion and subsequently by sequencing. Upon sequence confirmation, large amounts of the retroviral vector (Maxi-prep) were produced for subsequent use. 299WO 2017/009712 PCT/IB2016/001079

[00997] Peripheral blood leukocyte ofs healthy human donors were transduce witd h the retrovi ralconstructs encoding HIDE1 or with the retrovi ralvector encodis ng for NGFR1 or an empty vector, as a negative control Transduc. tion was carrie dout using a retronect in- based protocol; briefly, retroviral supernatant was produce ind 293GP cell s(a retrovir al packaging cel lline) follow ingtransfection with the retrovi ralvector and an amphotropic envelop gene (VSV-G). The retroviral supernata ntwas plated on retronectin-coate platdes prior to the transduction to enable the binding of virions to the plat e,and the PBLs were added to the plat fore 6 hours. After that the, cells were replenished in a new culture vessel.

An antibody against an intracellular FLAG sequence (cat. No 637309; Biolegend) was used in order to evaluate transduction efficiency. Expression of NGFR was detected wit h commercial anti-NGFR (Cat.No 345108; BioLegend). Rabbit IgG (Sigma Cat. No. 15006) was used as isotype control, and as secondary antibody we used APC-conjugated anti-rabbit IgG (Jackson, Cat. No. 711-136-152).

Ectopic expression of the F4 T cell receptor on cytotoxic T lymphocytes (CTLs)

[00998] In order to obtai neffector lymphocyt thates express the MART-1-specific F4 TCR, specifically recognizing MART-126-35-/HLA-A2 peptide-MHC complex, freshly isolated human PBLs previous lytransduce tod express either with HIDE1, NGFR or an empty vector were stimulated with PHA and cultured for 5-10 days, and subsequentl y transduce withd in vitro-transcri mRNbedA encoding both a and [3 chains from the MART-1- specific F4 TCR. The transduce lymd phocyt werees cultured in lymphocyt medie um (Bio target medium ,fetal bovine serum (10%), L Glutamine Penicillin/ Streptomicyn (100 units/ml), IL-2 300 IU), replenished every 2-3 days. F4 TCR expression levels were verified by FACS staining using a specific monoclonal antibody that recognizes the extra-cellular domain of the beta-chain from the transduce specd ific TCR (TCR-Vbl2-PE, (Cat.No IM2291; Beckman Coulter).

Cytokine secretion from HIDE1, NGFR or an empty vector and F4-TCR transduced lymphocytes upon co-culture with melanoma cells

[00999] PBLs expressing HIDE1 or NGFR along wit hF4-TCR were co-cultured wit h un-manipulated melanoma cells. 105 transduce PBLsd were co-cultured with 105 melanoma target cells for 16 hours. In order to assess the response of the effector CDS T cells to the 300WO 2017/009712 PCT/IB2016/001079 different tumor cel llines, cytokine secretion (IFN-y IL-2 and TNF-a) was measured by ELISA in culture supernatan ts(IFN-y (Cat.No DY285E), IL-2 (Cat.No DY202E), TNF-a (Cat.No DY210E) R&D SYSTEMS), diluted to be in the linear range of the ELISA assay.

Assessment of CTL activation

[001000] In order to analyze HIDE1 effect on T cel lactivation and CD137 surface expression in particular, cells were collected after 18 hr co-culture (E:T 1:1) and stained them with APC-anti-CD8a ,FITC-anti-137 (Biolegend) in FACS buffer made of PBS, 0.5% BSA, and 0.02% sodium azide. All samples were analyzed by FACS.

Results: General design of the experimental system

[001001] In the experimental system described herein (depicted in Figure 47), HIDE1 are over expressed on human PBLs which are next manipulated to express the MARTI- specific and HLA-A2 restricted F4 TCR. Over expressing cell sare then co-culture witd h HLA-A2 positive and HLA-A2 negative melanoma cel llines. The F4 TCR was recently used in clinical trial ins terminally-i melanll oma patients to specifically confer tumo recognir tion by autologous lymphocytes from periphera bloodl by using a retrovirus encoding the TCR (Morgan et al, 2006 Science, 314:126-129). The effect of HIDE1 expression on antigen- specific activation of CD8 T cell sby co-culture wit hcognate melanoma cells was assessed by cytokine secretion.

Over expression of HIDE1 on human PBLs

[001002] Human PBLs were transduce witd ha retroviral vector encoding the HIDE1 or an empty vector as negative control, as described in Materials & Methods. The level sof HIDE1 were assessed by flow cytometry at 48hrs afte rtransduction, and compared to cell s transduce withd an empty vector. Representativ datae showing HIDE1 expression detected by intra-cellula antir-FLAG staining is shown in Figure 48.

Over expression of F4 TCR on human PBLs

[001003] To perform functional assays with human CTLs, we used PBLs engineered to express the F4 TCR, which recognizes HLA-A2+/MART1+ melanoma cell s,as describe ind 301WO 2017/009712 PCT/IB2016/001079 Material &s Methods. Figure 49 shows representat dataive for F4 TCR expression obtained upon TCR transducti ofon leukocyte previouss lytransduce tod expres sthe different targets.

Effect of HIDE 1 expression on PBLs on IFNy secretion

[001004] HIDE1 or Empty-vector and F4-transduced PBLs were co-culture witd h melanoma cell lines. The level sof IFNy secretion were measured at 16-hours of co-cultur e.

The magnitude of inhibition of cytokine secretion due to over-expression is in the range of 90%. Representative data showing the effect of HIDE1 expressed on F4 expressing PBLs co- culture withd Mel-624, Mel-624.38 and SK-Mel23 on IFNy secretion is presented in Figure 50. Co-culture with the HLA-A2 negative cel lline Mel-888 which served as a negative control, caused only a minor activation dependent IFNy secretion from F4-transduce d lymphocytes PBLs. not expressing the F4 TCR (designated W/O) serve as an additional negative control. Altogether, two different PBL donors were transduce andd three co-culture experiment weres performed.

[001005] Figure 50 present thes resul tsof the expression of HIDE 1 on the F4 expressing PBLs causes a reduction of IFNy secretion upon co-culture with SK-MEL23, MEL-624 and MEL-624.38 in comparison to expression of an empty vector.

Effect of HIDE 1 on TNFa secretion

[001006] HIDE1 or an empty vector and F4-transduced PBLs were co-cultured with different melanoma cel llines. The level sof TNFa secretion were assessed at 16-hours of co- culture. The magnitude of inhibition of cytokine secretion due to HIDE1 expression ranged between 60 to 80%. Representativ datae showing the effect of HIDE1 expressed on F4 expressing PBLs co-cultured with Mel-624, Mel-624.38, SK-Mel23 and MEL-526 on TNFa secretion is presented in Figure 51. As expected, co-culture wit hthe HLA-A2 negative cell line Mel-888 which served as a negative control, caused only a minor activation dependent TNFa secretion from F4-transduced lymphocyte PBLss. not expressing the F4 TCR (designated W/O) serve as an additional negative control.

[001007] Figure 51 demonstrate thats expression of HIDE 1 on F4 expressing PBLs causes a reduction of secretion upon co-culture wit hSK-MEL23, MEL-624 and MEL- 624.38. 302WO 2017/009712 PCT/IB2016/001079 Effect of HIDE1 expression on PBL on expression of CD 137 activation marker

[001008] HIDE1 or an empty vector and F4-transduced PBLs were co-cultured with different melanoma cel llines. As shown in Figure 52, the expression levels of CD 137 were assessed at 16-hours of co-culture. The magnitude of inhibition in CD 137 expression due to HIDE1 expression was in the range of 80%.

[001009] Figure 52 demonstrate thats expression of HIDE1 on F4 expressing PBLs causes a reduction in the expression of CD137 (4-1BB) upon co-culture wit hSK-MEL23, MEL-624 and MEL-624.38.

Summary

[001010] Without wishing to be limite byd a singl ehypothesi thes, resul tspresented herei nindicate that overexpression on primary lymphocyt resules tsin reduced cytokine secretion by CTLs, suggesting that HIDE1 has an inhibitory effect on CTLs, furth er supporti ngthe therapeutic potential of the immunoinhibitory HIDE1-ECD polypeptides and/or fusion proteins thereof for agonizing HIDE1 inhibitory effect on immunity and treatment of autoimmune diseases.

EXAMPLE 18: EFFECT OF HIDE1 ON TCR TRANSGENIC PMEL-1 AND CD8+ T CELLS EFFECTOR FUNCTION UPON ANTIGEN-SPECIFIC ACTIVATION

[001011] In order to evaluat ethe immunomodulat oryeffect of HIDE1 on CD8+ T cell activation the, full length protei wasn ectopical lyexpressed on EL4 cell swhich were pulsed with gplOO25-33 peptide and then served as specific targe cellst for pre-activated pmel-1 transgenic CD8+ T cell s,expressing H-2Db/gpl0025-33. The effect of the HIDE1 on antigen- specific activity of pmel-1 CD8+ T cells was studied using several T cel lactivation readouts 303WO 2017/009712 PCT/IB2016/001079 Materials and Methods Cells.

[001012] EL4 (TIB-39, ATCC) cells were transduced to express mouse HIDE1 prote in using pMSCV retroviral vector encoding for HIDE1 codon-optimize cDNd A. Following selection with Puromycin (4pg/ml ,SIGMA), HIDE !-expressing cell swere generated. Mock- transduce EL4d cells were produce ind parallel and further used as control in all experiments described below Expressi. on of HIDE1 in EL4 cell swas confirmed applying staining wit h pAbs anti- mouse HIDE1 (GenScript, #488536 13), Detection was done using Donkey Ant i Rabbit PE conjugated (Jackson, cat #711-116-152 (1:100)), followed by FACS analysis. To ensure comparabl expresse ion levels of H2-Db in HIDE1- and empty vector-transduced cells, staining with anti-H2Db (Cat# 111508, Biolegends) antibody and subsequent FACS analysis were used. pmel-l/EL4 in vitro assay.

Primary Stimulation (day 0-4).

[001013] Spleens were harvest edfrom pmel-1 mice (6-12 weeks male or female).

Splenocyt eswere culture ind the presence of lug/ml of gplOO-peptide (human gpl0025-33 peptide, Sigma) and 25ng/ml of IL-2 (Cat# 589106, Biolegend) for 4 days in 6 well plat eat 1.5xl06 cells/we (Cat#ll 6570160, Greiner). Culture media was RPMI+10% fetal bovine serum and the followi supplemng ents from Gibco: 2-mercaptoethanol (dilution of 1:1000), Gluta-MAX, sodium pyruvate, penicillin/streptomyci andn, non-essential amino acids (all at a dilution of 1:100) (splenocytes medium). Cells were diluted 1:2 on the next day wit hfull culture media supplemented wit hgplOO-peptide (lug/ml) and IL2 (25ng/ml).

CD8+ purification and O.N resting (day 5).

[001014] Pmel-1 splenocytes were collected and CD8+ cell swere isolated using CD8a (ly-2) microbead (Miltenyi, 130-049-401). CD8+ pmel-1 cells were cultured in the presence 304WO 2017/009712 PCT/IB2016/001079 of 25ng/ml of IL-2 for ON in 6 wel lplat eat 1x106 cells/we inll splenocytes medium. The purity of CD8+ pmel-1 cell swas analyzed by FACS (>88% purity).

Secondary Stimulation: co-culture of pre-activated CD8+ pmel-1 with target cells (day 6)

[001015] Pmel-1 cultures were spun down and washed twice wit hfresh media. EL4 cells were pulse dwith gp-100 peptide (0.3, 1 ng/ml) for 1 hour at 37°C in serum free media.

Followi washingng , peptide-pulsed targets cells were over-night co-cultured wit h50,000 cell/wel pmel-l 1 CD8+ T cell sat an effector to targe (E:Tt ) rati oof 2:1. T cel lactivity was assessed based on detection of cytokines secretion (IFN-gamma, TNF-alfa, IL-2 and IL-10) in co-culture supernatants and by FACS analysi sof surfac eexpression of 41-BB (CD137) and CD25 (IL-2Ra).

Evaluation of pmel-1 T cell activation

[001016] 16-24 hours post co-culture, cells were stained wit hviability dye (BD Horizon; Cat# 562247, BD biosciences) follow byed surface staining wit hFITC-anti-CD8a (Cat #100706, Biolegend), PE-anti-CD137 (Cat #106106, Biolegend) and APC-anti-CD25 (Cat # 102012, Biolegend) in the presence of anti-CD16/32 (clone 2.4g2; Cat #101302, Biolegend) for blocking of Fcy-receptors All. samples were run on a MACSQuant analyzer (Miltenyi) and data was analyzed using Tree Star FlowJo software. Culture supernatants were collect edand analyzed for mouse Thl/Th2/Thl7 cytokine CBA kit (Cat# 560485, BD biosciences).

Results

[001017] In order to evaluat ethe immunomodulat oryeffect of HIDE1 on CD8+ pmel-1 T cel lfunction, the mouse protei wasn ectopical lyexpressed on EL4 cell swhich served as specific targe cellt sto pmel-1 CD8+ cells follow ingpulsing with gp-10025-33 peptide (H2-Db restricted peptide) Figure. 53A shows the expression levels of HIDE1 and MHC class I (H2- Db) on mock or HIDE1-overexpressin celg ls. Similar H2-Db expression level swere observe d for mock and HIDE1- transduce EL4d cells (variation of H2-Db expression of HIDE1- overexpressi cellng scompar eto mock cells in three different experiments: (-17) - (+23) %). 305WO 2017/009712 PCT/IB2016/001079 The folds of HIDE1 expression compare to mock cell svaries between 2.1 to 3.3 (in three different experiments), the relatively low expression drives form the fact that mock cell s endogeni cexpress HIDE1. The positive control for the assay is a clone of PDL1- overexpressing cell stogethe witr ha clone of mock cells. These clones were generated to overcome the obstacl esof unmatched H2-Db expression between PDLl-overexpressi cellsng to mock cells and low fold of PDL1 expression Figur. e 53B shows the expression level sof

[001018] PDL1 and MHC class I (H2-Db) on mock or PDLl-overexpressi clonesng .

Similar H2-Db expression level swere observed for mock and PDL1- transduce EL4d clones cells (variation of H2-Db expression of PDLl-overexpressi cellsng compar eto mock cell sin three different experiments: (-3) - (+20) %, experiment 349-023 excluded: use of polyclonal PDLl-overexpressi cellsng express 43% more H2-Db compar eto polyclonal mock cells. The folds of PDL1 expression compar eto mock clones cells varies between 9.5 to 25 (in four different experiments).

[001019] Figure 54 summarizes pmel-l/EL4 in vitro assay design. Targe tcells (mock, HIDE1 or PDL1-overexpressed) were pulsed with gplOO peptide and co-culture wit hpre- activated CD8+ pmel-1 and the effect of PDL1 and HIDE1 on mouse pmel-1 CD8+ T effector function, as manifested by activation markers and cytokine secretis on, was investigated.

[001020] In order to validate thi sassay for both targe test ting and functional Abs scree n for the target, the inhibitory effect of PDL1 on pmel-1 CD8+ cell sactivity and the reverse effect of blocking anti-PDLl (20pg/ml, YWW243.55.S70, Inc.) was assessed firs upont co- culture of pre-activated pmel-1 CD8+ cell swith gp100-pulsed EL4 cell swit hforced expression of PDL1 (2:1, E:T) - PDL1 did reduced pmel-1 CD8+ cells activity, thi s inhibition was also restored by anti-PDLl (CD137 expression is shown) (Figure 55B). Co- culturin withg gp100-pulse dEL4 cells with forced expression of HIDE1 (2:1, E:T) also resulted in reduced activit yof pmel-1 CD8+ T cel l(Figure 55C), as manifested by reduction in activation markers (CD 137 expression is shown) and by reduced cytokine secrets ion (IFNy secretion is shown ).Tables 31 and 32 summarizes the inhibition percent of HIDE 1 and PDL1 in all readouts checked and in four experiments under two gp-100 concentrations (0.3 and Ing/ml) 306WO 2017/009712 PCT/IB2016/001079 Table 31. Summary of HIDE1 inhibitor effey ct on pmel-1 CD8+ T cell activit yin various experiments HIDE1 overexpressing EL4 %CD137 % A GM GM + of Exp. # IFNy IL-10 TNFa CD137 CD25 H2D CD25+ b vs. to gplOO EV cone, 1 0.3 1 0.3 1 0.3 1 0.3 1 0.3 1 0.3 (ng/ml ) 349- 43 20 32 13 27 16 29 23 25 2% 4% 7% 019 % % % % % % % % % 17% 349- 17 ine ine 13 +12 3% 6% 4% 8% 8% 020 % rt rt 9% 3% % 6% % 349- 40 57 18 31 27 42 34 28 55 16 +14 8% 9% 022 % % % % % % % % % % % 349- 30 50 25 34 16 30 49 44 40 51 30 44 +23 023 % % % % % % % % % % % % % 307WO 2017/009712 PCT/IB2016/001079 Table 32. Summary of PDL1 inhibitor effecty on pmel-1 CD8+ T cel lactivit yin various experiments PDL1 overexpressing EL4 % CD 137 % AofH2Db GM GM + vs. to EV Exp. # IFNy IL-10 TNFa CD137 CD25 CD25+ gplOO cone. 1 0.3 1 0.3 1 0.3 1 0.3 1 0.3 1 0.3 (ng/ml ) 349- 67 67 37 20 59 46 48 25 019 8% 18 11 30 +14% % % % % % % % % % % % 349- -3% 28 15 11 21 37 21 020 55 14 13 40 86 35 % % % % % % % % % % % % 349- 58 74 36 46 53 66 15 39 58 71 30 35 +20% 022 % % % % % % % % % % % % 349- ine 13 11 11 10 ine 26 19 ine 14 6% 8% +43% 023 rt % % % % rt % % % rt 308WO 2017/009712 PCT/IB2016/001079 Summary

[001021] Four experiments were performe tod evaluat ethe proposed immuno- modulatory effect of ectopi cexpression of HIDE1 in target cell s,specific to TCR-transgenic pmel-1 CD8+ T cells. The inhibitory effect of HIDE1, as well as of PDL1 as positive control, on activation of Ag-specific CD8+ T cell s(pmel-1) followi co-culng ture wit htarge cellt s (EL4) pulse dwith gpl0025-33 peptide was, observed in 4 out of 4 experiments, as manifeste d by reduced cytokines secretion and by suppression of the activation-induced expression of activation markers CD137 and CD25. Both, for HIDE1 and PDL1, the inhibitor effecty was greater at a concentration of 0.3ng/ml gplOO. In the second experiment (349-020) magnitude of inhibition was lesse thanr the two other experiments, but since that was also the case for PDL1 and since trends of inhibition remain, we can assume that some technical faul t occurred in thi sexperiment. In the fourt experih ment (349-023) PDL1 over-expressing cell s express 43% more H2-Db vs. mock ,that could be the reason for the relatively low percent of inhibition by PDL1 in thi sexperiment. This assay design is used to test the abilit yof HIDE1- ECD polypeptides and/or fusion proteins thereof to agonise HIDE1 mediated inhibition.

EXAMPLE 19: EFFICACY OF HIDE1-ECD-IG IN MOUSE R-EAE MODEL OF MULTIPLE SCLEROSIS

[001022] Efficacy of HIDE 1-ECD fusion protei inn autoimmunit modely support s immunoinhibitory function of HIDE1, whic hwithout wishing to be limited by a single theory, might allow tumor cell sescape from immune surveillance. Activating immunoinhibitory HIDE1 prote inwit himmunoinhibitory antibodies wil bel efficient for treatment of MS and othe autoimmuner disorders

[001023] To investigate further the immunoinhibitory HIDE1 propertie ands he therapeutic potenti alof immunoinhibitory HIDE1 targeting antibodie whics h agonize HIDE1 activity for treatment of autoimmune diseases, HIDEl-ECD-Ig is was tested in a mouse model of Multiple Sclerosi Relapsis; ng Remitting Experimental Autoimmune Encephalomyeli tis(R-EAE). 309WO 2017/009712 PCT/IB2016/001079

[001024] Female SIL mice 6 weeks old were purchased from Harlan and maintained in the CCM facility for 1 week prior to beginning the experiment Mice. were randomly assigned into groups of 10 animals and primed wit h50 pg PLP139-151/CFA on day 0. Mice received 6 i.p. injections of 100ug/dose of immunoinhibitory HIDE1 targeting antibody, mIgG2a isotype control, or CTLA4-Ig (mouse ECD fused to mouse IgG2a Fc) as positive control .

Treatment begans at the time of disease induction (preventive mode )or at onset of disease remission (therapeuti mode)c and were given 3 times per week for at least 2 weeks. Mice were scored for disease symptom ons a 0-5 disease score scale :0, no abnormality; 1, limp tail 2,; limp tai land hind limb weakness; 3, hind limb paralysis; 4, hind limb paralysis and forelim weaknb ess; and 5, moribund.

[001025] On day 35, (during the peak of disease relapse) 5 mice of each group are assayed for DTH (delayed type hypersensitivity) respons toe disease inducing epitope (PLP139-151) and to relapse-associated myelin epitope (PLP178-191) via injection of lOpg of PLP139-151 in one ear and PLP178-191 into the opposite ear. The level of ear swellin g are assayed at 24 hours post challenge.

[001026] The result preses nted in Figure 56 show a pronounced decrease in disease severity of R-EAE-induced mice upon treatment with HIDEl-ECD-Ig Ig (SEQ ID NO: 18), in a therapeutic mode wit h100|Jg/dose at 3 times per week 2. The level of inhibition was similar to that of CTLA4-Ig. Similarl HIDy, El-ECD-Ig fusion proteins are anticipated to inhibi tDTH respons esto the disease inducing epitope (PLP139-151) and to relapse- associated epitope (PLP178-191) at day 35.

[001027] In a similar model, the dose dependency of the efficacy of HIDEl-ECD-Ig (SEQ ID NO: 18) as well as its mode of action in the PLP-induced R-EAE model is evaluated.

Disease is induced as described above and mice are treat edfrom onset of disease remission with 100, 30 or 10 ug/dose HI DEl-ECD-Ig (SEQ ID NO: 18) 3 times per week over two weeks. The level of disease seventy is evaluated and is anticipated to be reduced in a dose dependent manner.

[001028] HIDEl-ECD-Ig (SEQ ID NO: 18) fusion proteins are also anticipate tod inhibi tDTH respons esto spread epitopes PLP178-191 and MBP84-104 on days 45 and 76. 310WO 2017/009712 PCT/IB2016/001079 Furthermore recal, responsl esanalysis is carried out on day 45 and day 76 splenocytes and cervical lymph node cells, to PLP139-151, PLP178-191 and MBP84-104 in which proliferation as wel las cytokine secretion (IFNy, IL-4, IL-17, IL-10, TNFa, IL-6, GM-CSF, and other ares) analyzed .Without being bound by a singl ehypothesi modulats, ory effects on Thi Thi7 and/or Th2 cytokine ares anticipated follow ingtreatme ntwith HIDEl-ECD-Ig. In addition, CNS, draining (cervical lym) ph nodes and spleens of mice treate witd hHIDE1- ECD-Ig are evaluated for infiltrati ofon immune cells.

[001029] Figure 56 shows the therapeutic effect of HIDE 1-ECD-Ig (SEQ ID: 18) in the PLP139-151-induced R-EAE model in SIL mice. HIDEl-ECD-Ig (SEQ ID: 18) was administered in a therapeutic mode from the onset of disease remission, at 100 microg/mouse i.p. 3 times per week for two weeks. Therapeut iceffects on clinical symptom ares demonstrat ased reduction in Mean Clinical Score.

EXAMPLE 20: HIDE1 EXPRESSION IN HEALTHY PBLS AND IN AML

[001030] Surface expression of HIDE 1 by healthy PBLs was evaluated using whol e blood taken from 3 different healthy donors using anti-Hidel mAbs 33B4-2F7 and Ab 36C1- 2F6 as compared to staining wit hisotype control.

[001031] Prominen stait ning with anti HIDE1 mAb 33B4-2F7 was observe don monocytes (Figure 61). Neutrophi alsols showed positive stain, albite with a lower intensit y.

Essential lyno stain was detected on lymphocytes No. staining was observed in any of the populations using mAb 36C1-2F.

[001032] Similarly, prominent HIDE1 expression was detected on myeloid blast ins AML while lymphocyt werees essentiall negatiy ve (Figure 62). 311WO 2017/009712 PCT/IB2016/001079 EXAMPLE 21: EFFECT OF HIDE1 KNOCK DOWN (KD) AND ANTI-HIDE1 ANTIBODIES ON HUMAN T CELL FUNCTION Introduction

[001033] The aim of the presented functional assay was to evaluat ethe effect HIDE1 Knock down by siRNA or overexpression on T cel lfunction as well as the effect of anti - HIDE1 antibodies on T cel lfunction. In particular, to evaluat ethe immuno-modulatory effect of anti-HIDE1 antibodie thats can be used to target monocyte s,Tumor associate d macrophages (TAMs), or other myeloid cells and screen for various functional activities , including modulating the interaction between HIDE-1 and its putative receptor(s), modulation of HIDE1 level sor direc signalt ing and attenuati onof negative signaling. Such recombinan antibodiet mays be used as modulatory molecules to decrease or preven tHIDE1 from interacting with inhibitory receptor(s) on T cells or other cell sin the tumor microenvironmen theret, by releasing T cell sor othe functr ional cell sfrom HIDE1 check point (“break”)/suppressive signaling .

MATERIALS AND METHODS THP1 - poly clonal co-culture assay (Ab independent)

[001034] THP1 cell line expressing HIDE1 were knocked down (KD) by specific siRNA to HIDE1 or scramble (SCR)d control or PDL1. 24h post KD, cell swere treated wit h 500 units of IFNy for another 24h to induce PDL1 expression and cell maturation. Prior to entering into co-culture assay, levels of HIDE1 or PDL1 KD compared to control siRNA (SCR) were evaluated amongs tother markers as pan HLA-I/II or CD86 which were compared to their isotype control THP1. cell streate withd Mitomycin C (SIGMA; 50ug/ml for Ihr), were co-cultured at 1:1 ratio wit hCD3 T cells labeled with CFSE in 96 wel lflat bottom immobilized with lOOOng/ml CD3 (#555329 UCHT1) for 5 days. In order to assess the response of the effector CD3 T cells to the different THP1 KD effects, proliferati wason measured by the dilution of CFSE and cytokine secretion (IFN-y & TNF-a) was measured by TH-1/2/17 CBA kit (BD biosciences) in culture supernatants.

Effect of anti-HIDE1 antibodies in THP1 - poly clonal co-culture assay 312WO 2017/009712 PCT/IB2016/001079

[001035] THP1 cell line expressing HIDE1 were pre-treated wit h500 units of INFv for 24h. 24h post INFv treatment cell swere treated with mitomycin C (SIGMA; 50ug/ml for Ihr) and co-cultured with CFSE labeled CD3+ cells at 1:1 ratio in the presence of immobilize d anti-CD3 (lug/ml; UCHT-1). Anti-HIDEl blocking or agonist icmAb at 20ug/ml were added to each culture. Either no antibody or an isotype control antibody was used as a negative control Afte. r day 5, the effect of anti-HIDEl antibodie ons cytokine secretion was assessed from the supernatant using TH-1/2/17 CBA kit (BD biosciences).

Effect of anti-HIDEl antibodies in Mixed Lymphocyte Reaction (MLR)

[001036] A mixed lymphocyt reactie on was employed to demonstrate the effect of blocking the HIDE1 pathway to lymphocyte effector cells. T cells in the assay were tested for proliferation and cytokine secretion in the presence or absence of an anti-HIDEl human monoclonal antibodie (14s Abs with hIgG4 and 5 Abs with hlgGl backbone). Human CD3 T-cells were purified from PBMC derive dfrom Buffy coat susing negative selection isolati on kit s(EasySep™, STEMCELLS technologies) All. oreactivit dendrie tic cells in a tota volumel of 200 pl. Anti-HIDEl blocking or agonisti mAbc at 20ug/ml were added to each culture .

Either no antibody or an isotype control antibody was used as a negative control Aft. er day 5, the effect of anti-HIDEl antibodies on T cel lproliferation (CFSE dilution and) cytokine secretion (ELISA or TH1/2/17 CBA kits in) culture supernatants were assessed.

Effect of anti-HIDEl antibodies in CHOS-OKT3 co-culture assay

[001037] CFSE-labeled T cells were stimulate witd hstimulat cellsor (CHO cells expressing membrane-bound anti-CD3 mAb fragments). CHOS-stimulator cells expressing human HIDE1 and control stimulat cellsor (empty vector) treated wit hmitomyci nC (50 |Jg/ml for Ih) before co-cultured with CFSE-labeled human T cells at the ratio of 1:5. After 5 days at 37°C and 5.0% CO2, the effect of anti-HIDEl antibodie (20ug/ml)s on T cell proliferati (CFSEon dilution and) cytokine secretion (ELISA or TH1/2/17 CBA kits in) culture supernatants was assessed. All samples were acquired in MACSQuant analyzer (Miltenyi) and data was analyzed using FlowJo software (vl0.0.8). Culture supernatants were collect edand analyzed for cytokine secretion by CBA kit (Cat #560484, BD). 313WO 2017/009712 PCT/IB2016/001079 RESULTS Effect of HIDE1 Knock-Down in human THP-1 co-culture assay

[001038] THP1 cell swere knocked down (KD) by specific siRNA to HIDE1 or scrambled (SCR) control or PDL1. 24 post KD, cells were treate withd 500 units of IFNy for another 24h whic hinduced PDL1 expression and cel lmaturation. Knock down level sof HIDE1 and PDL1 were 73% and 61% respectively, sufficient for entering into assay.

Moreover, no variations in levels of HLA-I, HLA-II and CD86 compared to their isotype control suggesting KD doesn’t effect THP1-1 cel lstate (Figure 67B). Proliferation and cytokine secretion was assessed for THP1 co-cultured wit hCD3 T cells. Enhanced proliferation was detected for HIDE1 KD when compared to SCR, with a more prominent effect in CD4 T cells compared to CDS T cells. Moreover, robust IFNy and TNFa secretion was observed in HIDE1 KD cell scompared to SCR KD cell s(Figure 67C-D). In contrast , thi seffect was not observed for PDL1 in comparison to SCR.

Effect of anti-HIDEl antibodies on T cell function in THP-1 poly activation assay

[001039] IFNy-treate THP1d cell swere co-cultured wit hCD3+ cell sat 1:1 ratio in the presence of lug/m limmobilized anti-CD3 and the effect of anti-HIDEl antibodie ats 20ug/ml on cytokine secretion 5d post co-culture was tested (Figure 68A-B). Increase in IFNy and TNFa secretion was observed for CPA.12.006-H4, CPA.12.007-H4, CPA.12.0012-H4, Ab- 507 & AB-508 in comparison to HIDE1 isotype control.

Effect of anti-HIDEl antibodies on HIDE1 over-expression in CHOS-OKT3 assay

[001040] CHOS-OKT3 overexpressi HIDE1ng were co-cultured wit hCD3+ cell sat 1:1 rati oin the presence of immobilized anti-CD3 and the effect of anti-HIDEl antibodie ats 20ug/ml on cytokine secretion 5d post co-culture was tested (Figure 69A-B). Increase in IFNy secretion was observed for Ab-509, Ab-510 and CPA-12-008 in comparison to HIDE1 isotype control.

Effect of anti-HIDEl antibodies on T cell function in MLR assay

[001041] The effect of anti-HIDEl blocking or agonist icmAb in MLR assay was assessed 5 days post co-culture wit hallogeneic imDC. Either no antibody or HIDE1 isotype 314WO 2017/009712 PCT/IB2016/001079 control antibody was used as a negative control Afte. rday 5, the effect of anti-HIDEl antibodie ons T cell proliferation (CFSE dilution) gating on CD4 or CD8+ cell swas evaluated (Figure 70A-B).

SUMMARY AND CONCLUSIONS

[001042] Several anti-HIDEl antibodie augments ed T cell function in several functional assays.

In THP-1 co-cultur assay,e CPA.12.006-H4, CPA.12.007-H4, CPA.12.0012-H4, Ab-507 & AB-508 enhance IFNy & TNFa secretion levels in at least 1 donor (Figure 68). In CHOS- OKT3 assay, 3 anti-HIDEl Abs (Ab-509 and Ab-510, CPA.12.008-H4) enhance IFNy secretion in at leas t1 donor (Figure. 69). In MLR assay 4 antibodie (Ab-5s 06, Ab-507, Ab- 508 and Ab-509) enhance T cel lproliferation.

EXAMPLE 22: EFFECT OF HIDE1 ON HUMAN TUMOR-INFILTRATING LYMPHOCYTE (TIL) FUNCTIONAL ACTIVITY

[001043] Aim: To evaluate the effect of HIDE1 on function of human Tumor- infiltrati lympng hocyt (TIL)es upon co-culture wit hHIDE1-transduced target cells.

MATERIALS AND METHODS: Tumor cell lines

[001044] Mel-526 and Mel-624 cell lines are MART-1+, HLA-A2+ melanoma cell line.

All cel llines were cultured in complete medium consisting of DMEM supplemented wit h % FCS, 25 mmol/L HEPES, 2 mmol/L glutamine and, combined antibiotics (all from Invitrogen Life Technologies).

Cloning of peptide-specific tumor-infiltrating (TIL) lymphocytes

[001045] Tumor-infiltra tinglymphocyt (TIL)e micro-cultures were initiated and expanded from tumor specimens take nfrom resecte metd astases of two melanoma patients , as described (Uzana et al, JI 2012). Briefly, TILs were culture ind complete medium consisting of RPMI 1640 supplemented wit h10% heat-inactivated human AB serum ,6000 lU/ml human rIL-2 (rhIL-2; Chiron, Amsterdam The, Netherlands), 2mM L-glutamin e,1 315WO 2017/009712 PCT/IB2016/001079 mM sodium pyruvate, 25 mM HEPES, 50 mM 2-ME, and combined antibiotics (Invitrogen Life Technologies). On day 14 of TIL initiation, the lymphocyte weres washed wit hPBS, resuspended in PBS supplemented with 0.5% BSA, and stained with FITC-conjugated HLA- A*0201/MART-126-35 dextram er(Immudex, Copenhagen, Denmark) for 30 min at 4°C. The lymphocyt werees then incubated wit hallophycocyanin-conjugated mouse anti-human CDS (eBioscience) for an additional 30 min at 4°C. CD8+ lymphocytes, positivel staiy ned by the dextram er(CD8+/dextramer+ cells), were sorte byd a BD FACSAria (BD Biosciences) and direct lyclone dat one or two cells/we inll 96-well plates in the presence of ortho-anti-CD3 (30 ng/ml; eBioscience), rhIL-2 (6000 lU/ml ),and 4 Gy-irradiated allogenei PBMCsc as feeder cells. Five days later, rhIL-2 (6000 lU/ml) was added and renewed every 2d thereafter .

On day 14, the clones were assayed for IFNy secretion in a peptide-specific manner followi ng their co-incubation wit hMART-l26-35-pulsed T2 cells (peptides were commercial ly synthesized and purified [>95%] by reverse-phase HPLC) using commerciall availy able ELISA reagent s(R&D Systems, Minneapoli s,MN). The MART-126-35-reactive clones were furth erexpanded in a second-round exposure to ortho-anti-C (30D3 ng/ml )and (6000 lU/ml) rhIL-2 in the presenc eof 50-fold excess of irradiate feederd cells. A similar protoc wasol performe tod obtai na gpl00154_162 and gp 100209-217 reactive clones MAR. T-126-35 and gpl00154-162-reactive clones were derived from patient 412 whil gp!00e 209-217 -reactive clones were derived from patient 209. Follow ingTILs clones expansion the cells were stain wit h FITC or PE-conjugated HLA-A*0201/MART-l26-35,HLA-A*0201/gpl00154-162 orHLA- A*0201/gp 100209-217 dextramer and analyzed by FACS. The study was approved by the Institutional Review Board at Sharet Instit tute of Oncology, Hadassah Medical Organization, Israel, and all patients gave thei informr ed consent prior to initiation of melanoma and lymphocyte cel lcultures. The TILs, which were sent to Compugen LTD under service agreement, kept in liquid nitrogen (10-20xl06/vial) and thawed one day before co-culture with target cells.

Ectopic expression of hPDLl and hHIDEl on target cells Section A

[001046] Mel-526 and Mel-624 cells were transduced to express the human HIDE1 with a flag-tag in the C-terminus using, pMSGVl retrovir vectoral or human PD-L1, using a 316WO 2017/009712 PCT/IB2016/001079 pMSCV retrovir vector.al Expression of hPDLl and hHIDEl on Mel cells was confirmed by flow cytometry followi surfacng estaining with a specific antibody to hPDLl (Cat# 329708, Biolegend) and hHIDEl (in-house). Ilie expression level sof HLA-A2 in hPDLl and hHIDEl was compared to the level sin the mock transduce celld sby using anti-HLA-A2 antibody (Cat# 343306, Biolegend).

Section B

[001047] Mel-526 and Mel-624 cells were transduced to express the human HIDE! with a flag-tag in the C-terminus using, pMSGVl retroviral vector. Expression of hPDLl and hHIDEl was validated as indicated in section A follow ingsorti ngof the cell sfor higher HIDEl expression.

Purification of HIDEl over expressing cells (relevant for section B above)

[001048] To enlarge folds of expression of the transduced human HIDEl Mel-526 and Mel-624 cel llines, cell swere purified using anti-PE beads (Cat# 130-048-801, Milteniy,) follow ingstaining by PE anti-hHIDEl (Cat# 33B4-2F7, Biotem foll) owed by Goat anti - mouse PE (cat# 115-116-146, Jackson). The purified cell swere used in herei nassay.

Co-culture of TILs with target cells

[001049] One day prior to co-culture, TILs were thawed and cultured wit hfull IMDM media (cat# 01-052-1 A, Biological industries lt d(BI)) supplemented wit h10% human serum (Sigma, H3667), 1% glutamax (Life technologie 35050-03s, 8), 1% MEM eagle (cat# 01-340- IB, BI), 1% Sodium pyruvate (cat# 03-042-1B, BI), 1% PenStre p(cat# 03-031-1B, BI) in the presence of 300IU/ml of rhIL-2 (Cat# 589106, Biolegend). Targe tcells and TILs were harvest and co-cultured at effector to targe (E:T)t ratios of 1:1 (1x10A5 cells from each/well) or 3:1 (1x10A5 TIL : 3xl0A4 target cells/ well in) full IMDM media.

Flow cytometry

[001050] 16 hours post co-culture, cell swere stained wit hviability dye (BD Horizon; Cat# 562247, BD biosciences) followed by surface staining wit hFITC-anti-CD8a (Cat # 300906, Biolegend), PE-anti-CD137 (Cat #30984, Biolegend) and APC-anti־CD107a (Cat # 328620, Biolegend) in the presence of Fc blocking soluti on(cat# 422302, Biolegend). All 317WO 2017/009712 PCT/IB2016/001079 samples were run on a MACSQuant analyzer (Miltenyi) and data was analyzed using Tree Star FlowJo software. Cell swere firs gatedt for lymphocyt (FSC-Aes vs. SSC-A), followed by singlets gate (FSC-H vs. FSC-A), and further gated for live cells. CD137, CD107a surface expression on gated CD 8+ was analyzed. Culture supernatants were collect edand analyzed for mouse Thl/Th2/Thl cytoki7 ne CBA kit (Cat# 560485, BD biosciences).

RESULTS Section A

[001051] In order to investigate immune-modulatory effect of HIDE 1 gplOO or MART- 1 specific TILs were co-cultured with over expressing HIDE1 and HLA-A2+ target cells. The effect on T cells was assessed by analyzing activation markers surface expression and cytokine secretion Target. cell sHLA-A2 and HIDE1/PD-L1 expression was validate priord to experiment (Figus re 71). Over expressing HIDE1 cell sare compared to mock transduce d pMSGVl vector cells whil overe expressing PD-L1 cell sare compared to mock transduced pMSCV vector cells.

[001052] hPDLl transduce Meld -526 and Mel-624 cells were used as positive control in thi sassay. At the firs experit ment two ratios of E:T were tested, 1:1 and 1:3, as well as two target cells 526 and 624. The inhibitor effecy t of PDL1 was observed in co-culture of TILs with both target cells and was manifested in reduced IFNg secretion and surface expression of CD137. The inhibitory effect was most pronounced in co-culture of TILs with 624 cell s and so we did not proceed to test 526 cell sin the second experiment hPDL. l had an inhibitory effect on all TILs tested, gpl00154_162 (designated TIL-154), gpl00209-217 (designated TIL-209) and MART-126 35 (designated MART-1), thoug theh inhibitory effect by hPDLl was less pronounced wit hTIL 154 (Figure 72A,72C and Table 33). hHIDEl had no inhibitory effect but a stimulatory effect on TILs activity when co-cultured with Mel-526 and 624 Figure 72B,D and Table 34) wit hthe exception of one experiment in which a 15% reduction in CD107a was observed in co-culture of TILs 209/MART1 wit h526 in 3:1 E:T ratio. On the same experiment a 71% reduction in IFNg was observe din TIL MARTI :526 however the tota amoul nt of IFNg was extremel low.y 318WO 2017/009712 PCT/IB2016/001079 Table 33. Summary of hPDLl effect on TIL-209, TIL-154 and TIL-MART-1 activity.

TIL 209 (% of EV) TIL MARTI (% of EV) TIL 154 (% of EV) Cells Mel-526 Mel-624 Mel-526 Mel-624 Mel-526 Mel-624 Exp. Exp. Exp.

Exp.367-037 Exp.367-037 367- Exp.367-037 Exp.367-037 367- Exp.367-037 Exp.367-037 367- 038 038 038 ./ readout 3:1 3:1 3:1 3:1 3:1 3:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 1:1 -21% -53% -51% -29% -27% -39% -72% -60% -53% -21% -21% -50% -46% -32% GM CD137 18% -17% -35% -24% -20% -22% -25% -48% -32% -35% -23% -15% -35% -23% -22% GM CD107a 12% %CD137+CD107 -54% -24% -35% -25% -20% -40% -54% -60% -33% -26% -27% -40% -45% -22% 11% a+ -13% -51% -63% -55% -63% -89% N.D N.D -68% N.T N.T N.T N.T -30% IFNg (pg/ml) 42% -3% -100% -71% -27% -39% 0% -43% N.T N.T N.T N.T -16% TNFa (pg/ml) % 100% 100% Percentage in this table represent the A of the effect on TILs when comparing to co-culture with mock transduced cells, *overallpercentage is low. N.D not detected/amount lower than lOOpg/mL.

N. T not tested Table 34. Summary of HIDE 1 effect on TIL-209, TIL-154 and TIL-MART-1 activity.

TIL 209 (% of EV) TIL MARTI (% of EV) TIL 154 (% of EV) Cells/ Mel-526 Mel-624 Mel-526 Mel-624 Mel-526 Mel-624 Exp. Exp. Exp.

Exp.367-037 Exp.367-037 367- Exp.367-037 Exp.367-037 367- Exp.367-037 Exp.367-037 367- 038 038 038 /,readout 1:1 3:1 1:1 3:1 1:1 1:1 3:1 1:1 3:1 1:1 1:1 3:1 1:1 3:1 1:1 +12 +198 +78 +25 GM CD137 +164% +63% +38% +62% +98% +36% +26% +59% +96% +16% +12% % % % % +13 +10 +6% -15% +19% +21% +22% -15% +20% +22% +10% +9% -5% 0% +7% GM CD107a % % %CD137+CD107 +13 +15 +17% 0% +56% +38% +40% +28% +14% +25% +21% +16% 0% +3% +15% % % a+ +23 -71% +200 +68 IFNg (pg/ml) +129% +80% +38% +80% N.D N.D N.D N.T N.T N.T N.T * % % % +47 +100 +26 N.D N.D +66% +66% N.D N.D +66% +56% N.T N.T N.T N.T TNFa (pg/ml) % % % Percentage in this table represent the A of the effect on TILs when comparing to co-culture with mock transduced cells, *overall gMFI/pg values are low. N.D not detected/amount lower than lOOpg/mL N. T not tested Section B

[001053] In the experimental system described herein, gplOO or MART-1-reactive TILs were co-cultured with HLA-A2+ target cell s(624 and 526 Mels), which ectopical lyexpress HIDE1 and were sorte ford higher expression of HIDE1 (149 and 54 folds vs. empty vector), and the effect on TILs effector function compar eto mock-transduce celdls, as manifeste dby 319WO 2017/009712 PCT/IB2016/001079 activation markers and cytokines secretio wasn, investigated. Ceils were validate befored each experiment for their level sof targe aridt HLA-A2 expression and were found out to be within the tolerable limit of target expression (mor ethan 5 folds of expression) and of HLA- A2 level (differenti alexpression between mock and hHIDE1/hPDL1 transduce celd llines of ±30%) (Figure 73A-73B). The assay was validate byd using hPDLl transduce Mel-d 526 and Mel-624 cells as target s.At the firs repeat ttwo ratios of E:T were tested, 1:1 and 1:3, in which the inhibitory effect of PDL1 was greater with the 1:1 rati o(data not shown), and so thi srati owas used in the second repeat. hPDLl had an inhibitor effecty on all TILs tested , pl00154-162 (designated TIL-154), gp!00209-217 (designatedTIL-209) and MART-126 35 (designated MART-1), though the inhibitory' effect by hPDLl was more profound with Mel- 526 than with Mel-624 overexpressi cellng s(Figure 74A). hHIDEl had no inhibitory effect on TILs activit ywhen co-cultured with Mel-526 and 624 (Figure 74B). Tables 35 and 36 summarize sthe inhibition percent of hPDL l and hHIDEl in all readouts checked - for Mel- 624 and TIL 154 tw orepeats were made, in the first repea tgreate percer nt of inhibition by hPDLl were observed compar eto the second repeat, probably due to the low expression of HLA-A2 by hPDLl overexpressi cellsng in the firs repeat.t For all other treatment ones repea twas made.

Table 35. Summary of hPDLl inhibitory effect on TIL-209, TIL-154 and TIL-MART-1 activity.

TIL 209 TIL MARTI TIL 154 (% inhibition vs. EV) (% inhibition vs. EV) (% inhibition vs. EV) Cells / Mel-526 Mel-624 Mel-526 Mel-624 Mel-526 Mel-624 y Read-out 349-031 349-031 349-031 349-031 349-031 349-031 349-029 % 17% 58% 19% 23% 4% GM CD137 11% GM CD107a 15% -12% 48% 10% -10% 19% -24% %CD137+CD107a+ 17% -10% 66% 18% -7% 20% -13% IFNg (pg/ml) 15% 32% 72% 23% 1% 32% 8% 27% 41% 92% 14% 9% 48% -2% TNFa (pg/ml) IL-2 (pg/ml) 36% 21% NA 11% -2% 51% -2% 320WO 2017/009712 PCT/IB2016/001079 % A of HLA-A2 vs. to ±23% ±8% -29% ±23% ±8% ±23% ±8% EV [tolerabl liemit ±30%] Table 36. Summary of hHIDEl inhibitory effect on TIL-209, TIL-154 and TIL-MART-1 activity.

TIL 154 TIL 209 TIL MARTI (% inhibition vs. EV) (% inhibition vs. EV) (% inhibition vs. EV) Cells Mel-526 Mel-624 Mel-526 Mel-624 Mel-526 Mel-624 Read-out 349-031 349-031 349-031 349-031 349-031 349-031 349-029 GM CD137 -40% -15% inert -35% -48% -42% -36% -7% -3% -5% -9% GM CD107a -11% -18% -12% -5% -3% -8% %CD137+CD107a+ -18% -29% -10% -15% IFNg (pg/ml) -46% -18% -26% -44% -58% -35% -18% TNFa (pg/ml) -59% -15% -31% -53% -72% -49% -23% -27% -7% NA -5% -7% 5% IL-2 (pg/ml) inert % A of HLA-A2 vs. to EV ±3% -15% -16% ±3% -15% ±3% -15% [tolerabl liemi t±30%] EXAMPLE 23: HIDE1 ANTIBODY DISCOVERY Rationale and Objectives

[001054] This study was designed to isolate human antibodie wits hhigh affinity and specificity for the HIDE1 immuno-oncology target. This was achieved by panning a human antibody phage display librar agaiy nst a recombinant protei comprisn ing the human HIDE1 extracellula domainr (ECD) fused to human IgG, Fc (HIDE1-HH-1).

Protocols

[001055] Preparation of biotinylated HIDE1: HIDE1-HH-1 (batch #280) was biotinylat usinged a Sulfo-NHS-LC-Bioti kitn (Pierce). Free bioti nwas removed from the 321WO 2017/009712 PCT/IB2016/001079 reactions by dialysis against PBS pH 7.2, 0.01% Tween 20. Synagis hlgGl was also biotinylat toed use for depletion steps in panning experiments.

[001056] General method for ELISA: Biotinylated proteins were captured on the well ofs streptavidin-co ated96-well plates (Pierce). Antigen captured plat wele ls were blocke withd PBS-milk (PBS pH 7.4, 5% skim milk powder). Blocking buffer was removed and a primary antibody soluti onwas added and incubated for Ihr at room temperature (RT).

Plates were washed with PBS-T (PBS pH 7.4, 0.05% Tween20) followed by PBS. HRP- conjugated secondary antibody (dependent on the assay system) was added and incubated at RT for Ihr, and plate weres washed again. In some cases, a HRP-conjugated primary antibody (or other detection protei n)was used directl ELISAy. signals were developed using Sureblue TMB substrate (KPL Inc). Assay signal swere read on 96-wel lplate reader at absorbance 450 nm.

[001057] Phage display and Fab screening: A singl ecampaign was divided into four sub-campaigns with different washes (Table 37). Panning reactions were carried out in soluti onusing streptavidin-co atedmagnetic beads to capture the biotinylat antigeed ns. Beads were recovered using a magnetic rack (Promega).

[001058] Preparation of phage library for panning: All phage panning experiment useds the XOMA031 human Fab antibody phage displa ylibrary (XOMA Corporati on,Berkele y, CA) blocked with 5% skim milk powder.

[001059] Antigen coupling to streptavidin beads: For each sub-campaign ,biotinylat ed HIDE1-HH-1 was captured on Dynal streptavidin-co atedmagnetic beads (Life Technologies). Beads used for subtracti panningve were coupled with the ‘depletion’ antigen (biotinylated Synagis hlgGl).

[001060] Depletion of human IgG Fc and streptavidin bead binders from the phage library: It was necessar yto remove unwanted binders to streptavidin beads and the Fc region of HIDE1-HH-1 during the panning process. To achieve this, a phage aliquot was mixed wit h beads coupled to the Synagis hlgGl depletion antigen and incubated at RT for 30mins. Two depletion reactions were applie dto all sub-campaigns. The depletion beads were then discarded. 322WO 2017/009712 PCT/IB2016/001079

[001061] Phage panning: Four rounds of phage panning were performe ford each sub- campaign. For the firs round,t the blocked and depleted phage library was mixed wit h magnetic beads coupled to biotinylat HIDE1-Hed H-1 and incubated at RT for 1 hr. Non- specific phage were removed by washing with PBS-T and PBS at various stringency levels , depending on the specific panning sub-campaign (Table 37). After washing, bound phage were eluted by incubation with triethylamine (TEA) (EMD) and the eluate was neutralize byd adding Tris-HCl pH 8.0 (Teknova). Second and later rounds were conducted the same way, except that the rescued phage supernatant from the previous round was used in place of the phage library.

Table 37: Was king stringency used for phage panning against human HIDE1.

Sub- Washing Stringency campaign 2nd Rd 3 rd Rd 4th Rd 1st Rd 3 PBS-T washes 6 PBS-T 6 PBS-T washes 6 PBS-T washes A & 3 PBS washe s& 6 & 6 PBS washes & 6 PBS washes washes PBS washes 3 PBS-T washes 10 PBS-T 10 PBS-T 10 PBS-T B & 3 PBS washes & 10 washes & 10 washes & 10 washes PBS washes PBS washes PBS washes PBS-T washes 6 PBS-T 6 PBS-T washes 6 PBS-T washes C &5PBS washe s& 6 & 6 PBS washes & 6 PBS washes washes PBS washes PBS-T 10 PBS-T PBS-T washes 10 PBST D &5PBS washes & 10 washes & 10 washes & 10 PBS washes PBS washes PBS washes washes

[001062] Phage rescue: The phage eluate was infected into TGI E. coll, which transforme thed cells with the XOMA031 phagemid. Transformed cells were then spread on selective agar plates (carbenicilli n)and incubated overnight at 30°C.

[001063] The resulting E. coll lawns were scraped and re-suspended in liquid growt h media. A smal laliquot of re-suspended cell swas inoculate intd o a 50 mL culture (2YT wit h 2% glucose and carbenicilli andn) grown at 37°C unti lthe OD at 600 nm reached 0.5. This culture was infected with M13K07 helper phage (New England Biolabs) and spun down. The cel lpell etwas resuspended in 50 ml media (2YT with carbenicill andin kanamycin, which is selection antibiot foric M13K07). The culture was then maintained at 25°C to allow phage 323WO 2017/009712 PCT/IB2016/001079 packaging. An aliquot of the culture supernatant was carried over for a subsequent round of panning.

Binding screens using Fabs prepared in periplasmic extracts (PPEs) by ELISA and FACS

[001064] Fab PPE production: The XOMA031 library is based on phagemid constructs that also function as IPTG inducible Fab expression vectors. Elute dphage pools from panning rounds 3 and 4 were diluted and infected into TGI E. coll cell s(Lucigen) so that singl ecolonie weres generated when spread on an agar plat e.Individual clones were grown in 1 mL cultures (2YT wit hglucose and ampicillin) and protei expressn ion was induced by adding IPTG (Teknova). Expression cultures were incubated overnight at 25°C.

Fab proteins secrete intod the E. coll periplasm were then extract edfor analysis. Each plat eof samples also included duplicat ‘eblank PPE’ wells to serve as negative contro ls.These were created from non-inoculat edcultures process edthe same way as the Fab PPEs.

[001065] ELISA binding assays: PPEs from the sub-campaigns were tested for binding to the panning antigen (biotinylated HIDE1-HH-1) and negative control antigens, PVR- hlgGl Fc fusion and Synagis hIgG1. The ELISA followed the general protoc inol paragraph

[001056] (“General method for ELISA”). The primary antibody was replaced with a 50 pL aliquot of Fab or blank PPE and the secondary antibody was a HRP-conjugated anti-human F(ab')2 antibody (Jackson Immunoresearch). ELISA binding was expressed as the ratio of Fab PPE binding signal: blank PPE signal .Positive hit swere identified as those giving a ratio of at leas ttwo.

[001066] FACS screening of PPE: FACS analyses were conducted using adherent HEK-293T cells over-expressing the human HIDE1 (293T-hHIDEl). All analyses included negative control parental HEK-293T cells.

[001067] Reagent preparation and wash steps were carried out in FACS buffer (PBS with 1% BSA). Fab and blank PPEs were mixed wit han aliquot of cell s,incubated for Ihr at 4°C and then washed wit hFACS buffer. Cell swere then mixed wit han anti-C-myc primary antibody (Roche). After the same incubation and wash step cell swere stained wit han anti - mouse IgG Fc AlexaFlour-647 antibody (Jackson Immunoresearch). After a final incubation 324WO 2017/009712 PCT/IB2016/001079 and wash cells were fixed in 2% paraformaldehyde made up in FACS buffer .Samples were read on a FACS Calibur (BD Bioscience) or HTFC screening system (Intellicyt). Data were analyzed using FCS Express (De Novo Software, CA, USA) or F10J0 (De Novo Software, CA, USA). Results were expressed as the ratio of mean fluorescence intensity (MFI) of 293T-hHIDEl cells: MFI signal of 293T control cells (MFI ratio). Positive hit swere identified as those giving an MFI rati oof at least five.

SPR Kinetic Screen of 24 Anti-HIDEl Fab PPE

[001068] Preliminary Fab binding kinetics were determined using Fab PPEs produced as described in paragraph [001064] (“Fab PPE production”)[001064], Studies were conducte dusing Biacore 3000 and ProteO XPRn 36 instrument ats 22°C.

Measurement of fab concentration in PPE samples

[001069] A high densit yanti-human Fab antibody surface was prepared over one flow cel lof a CM5 chip using a Biacore Human Fab Capture Kit (GE Healthcare) according to manufacture’sr instructions. One surfac eof the CM5 chip was activated and blocked without coupling any anti-Fab antibody to act as a reference surface .Antibody immobilization was performe witd hHEPES-buffered saline, 0.005% polysorbate 20, pH 7.4 (HBS-P).

[001070] The mola rconcentrations of Fabs in PPE samples were quantitate usingd a Biacore 3000 instrument Each. Fab was diluted 10-fold and injected for 1 minute at 5pL/min over the Fab capture and reference surface sdescribed above. A standard human Fab at a known concentrati on(Bethyl Laboratori Inc.)es, was then injected over the anti-Fab surface under the same conditions as the Fab supernatants The. association slopes of each sensorgram from each Fab supernatant were fit against the association slope of the standard human Fab of known concentrati onusing CLAMP 3.40 software to estimat thee mola rconcentrations of each Fab in supernata nt(Table39).

Measurement of Fab binding kinetics

[001071] A high densit ygoat anti-human Fc antibody (Invitroge n)was immobilized by standard amine coupling over all lanes of a GLC chip using a ProteOn XPR 36 biosensor (Biorad ).This surfac ewas used to capture the recombinan HIDt E1- H:H fusion protein (GenScript Lot 393383-21, batch # 280) or a control Fc fusion protei (CD155,n Sino 325WO 2017/009712 PCT/IB2016/001079 Biologicals) in the vertical flow-cell direction at a concentration of ~0.5|Jg/mL for 90 seconds.

[001072] Each anti-HIDEl Fab PPE sampl ewas then injected at three concentrations (undiluted fab supernatant and two three-fold serial dilutions) in the horizonta direcl tion over the capture dfusion proteins The. dilutions were made using degassed PBS wit h0.05% Tween 20 and 0.01% BSA. PPEs were injected for two minutes follow byed 10 minutes of dissociati onat a flow rate of 50 pL/min. The starting concentrati onrange for each Fab is recorded in Table 39. The anti-human fc capture surface swere regenerated with two 30- second pulses of 146 mM phosphoric acid after each cycle.

[001073] The resulting sensorgram weres process edand double-referenced using a ProteOn version of Scrubber 2.0 (BioLogi cSoftware). Where appropriate the, sensorgram s were fit wit ha simple 1:1 kinetic binding model (with a term for mass transport using) ProteOn Scrubber 2.0. Sensorgram ands their respective kinetic fits are shown in Figures 77 and 78. Table 39 lists the resulting binding constants. Binding parameters for complex kinetics were not determined.

Re-formatting of fab hits and production as human IgG molecules

[001074] HIDE1 binding Fabs were converted to full length human IgGs by sub-cloning the relevant Vh and Vl domains into separat heavye and light chain expression vectors that already contained the appropriat heavye or light chain constant regions: pUNO3-H4 (human IgG4 heavy chain vector) pUNO3-H, K (human kappa light chain vector) or pUNO3-HL (human lambda light chain vector). The base pUNO3 vector was source dfrom Invivogen.

Matched heavy and light chain constructs were co-transfect intoed Expi293 cell susing Expifectamine and Opti-MEM (Life Technologies). Expression cultures were incubated at 37°C for a further six days and supernatants were harvest edby centrifugatio IgGsn. were purified from the supernatants using an AKTA Pure FPLC (GE Healthca reBio-Sciences) and HiTrap MabSelect Sure affinity columns (GE Healthca reBio-Sciences).

[001075] FACS screening of reformatte IgG4d antibodies The: cel llines describe ind paragraph [001066] (“FACS screening of PPE”) were mixed wit hFACS buffer containing the purified anti-HIDEl IgGs or isotype controls. Afte rincubation for Ihr at 4°C, cell swere 326WO 2017/009712 PCT/IB2016/001079 washed wit hFACS buffer and staine dwith an anti-human IgG Fab AlexaFluor-647 antibody (Jackson Immunoresearch) for 45 mins at 4°C. Cell swere washed again and fluorescence was detected using an Intellicyt HTFC screening system (Intellicyt). Data were analyzed using FCS Express (DeNovo) and plotte ford affinity calculations (KD) in GraphPad Prism (GraphPad Software, Inc.). Note that each anti-HIDEl antibody was tested over a titrati on range from 0-80 pg/mL.

RESULTS Fab PPE screening

[001076] Fab clones drawn from the four sub-campaigns were tested for binding by FACS and ELISA. Hit s(defined as described in paragraphs [001065] and [001066]; “ELISA binding assays” and “FACS screening of PPE”) were sequenced to eliminate redundant Fabs. General screening outcomes are presented in Table 38. The ELISA and FACS screening hit rate sfrom the 3rd Rd output rangeds from 30% to 50% and 15% to 19% respectively. A sub-set of Fabs had binding activity that correlate betwd een FACS and ELISA (35% - 57%, depending on the sub-campaign). Sequence diversi tyranged from 27% to 38%, which represent uniques HCDR-3 families. A HCDR-3 family contains Fabs with the same heavy chain CDR-3 sequence, but minor differences in the heavy or light chain framework regions.

[001077] The ELISA and FACS screening hit rates from the 4th Rd output rangeds from % to 36% and 7% to 16% respectively. A sub-set of Fabs had binding activity that correla tedbetween FACS and ELISA (23% - 75%, depending on the sub-campaign).

Sequence diversi tyranged from 18% to 41%.

[001078] The HCDR-3 familie sselected from different sub campaigns were highly redundant for both rounds 3 and 4. However, there were some additional low-frequency clones associated wit hspecific sub-campaigns or panning rounds Overall. the, ELISA and FACS hit rates decreased from the thir roundd to the fourt round.h This suggests that optimum phage enrichment occurred in round three. Nonetheles positis, ve hit sfrom the both rounds were included for the subsequen tstep. 327WO 2017/009712 PCT/IB2016/001079

[001079] A sub-se tof the ELISA and FACS positive hit swere subject to SPR kinetic screening. At least one member of each HCDR-3 family was included. Most Fabs displayed 1:1 kinetic binding (Table 39) to HIDE1-HH-1 (Figure 77) and minimal binding to the negative control protei (Figurn e 78).

[001080] The overal panningl and screening exercise (ELISA, FACS and SPR) yielded 14 Fabs that showed sufficient binding activit yto move forward into IgG reformatti ng.The identified Fabs include 12 HCDR-3 families, two of which have 2 members.

[001081] IgG reformatting and characterization: The Fab binders described in paragraphs [001076]-[001079] (“Fab PPE screening”) were cloned into expression vectors for production as human IgG4 molecules All. of these antibodie weres successfull expresy sed and purified. Thirteen were shown to bind the 293T-huHIDEl cel lline (Table 40). None of thes eantibodies were cross-react againsive t the mouse HIDE1 ortholog.

SUMMARY AND CONCLUSIONS

[001082] This example provides characterization data for a panel of human antibodies raised against the HIDE1 antigen by phage display. A phage panning campaign was conducte dusing an ECD-Fc fusion protein. Overall, 13 IgGs binding to human HIDE1 were identified. These are recommended for further characterizati ofon binding on cells lines that express HIDE1 endogenously, as well as in vitro functional assays

[001083] Table 38: Summary of ELISA and FACS screening of Fab PPEs. FACS result meas sure binding against the 293T-huHIDEl cel lline. ELISA result measures binding against recombinan HIDt E1HH-1. FACS/ELISA correlati referson to the proportion of Fabs that bound in both assays (FACS and ELISA) compared to the total number of Fabs showing binding in either assay. Sequence diversity refers to the number of different Fabs present among the analyzed binders (i.e. 30% would indicate 30 different Fab sequences found within a set of 100 HIDE1 binders).

Table 38: Summary of ELISA and FACS screening of Fab PPEs.

FACS/ELISA Sequence Sub-campaign FACS hit rate ELISA hit rate correlation diversity 3 rd Rd A 15% 31% 47% 38% 328WO 2017/009712 PCT/IB2016/001079 B 18% 50% 35% 27% C 17% 30% 57% 41% D 19% 38% 50% 33% 4th Rd A 7% 30% 23% 19% B 36% 32% 18% 11% C 11% 15% 75% 41% D 16% 25% 65% 38%

[001084] Table 39: Preliminary Fab binding kinetic sdetermine from PPE samples. The Fabs are listed in order of decreasing KD. Asteris ksindicate the values should be take nwit h some caution, wit han explanation in the comments column. Figure 78 shows the binding responses of the Fabs injected over the control fusion protein. Responses over the control protei showedn either no binding or a minimal response compared to the responses in Figure 77.

Table 39: Preliminary Fab binding kinetics determine from PPE samples.

Stock cone. ka kd Kd (M^sec1־) (sec1־) (M) (M) CPA ID Rmax CPA.12.011 B03 S400-01.E01* 9.41E-09 2.9E+05 1.9E-04 106 6.4E-10 CPA.12.OO3 A03 S397-01.A12 5.46E-08 2.5E+05 2.2E-04 110 9.1E-10 CPA.12.OO9 A10 S398-02.A04* 7.22E-09 1.4E+06 1.4E-03 105 1.0E-09 CPA.12.OO1 AOI S397-01.A04 1.79E-07 6.7E+05 8.1E-04 210 1.2E-09 CPA.12.OO7 AOS S398-01.E08 2.48E-08 2.1E+05 3.4E-04 142 1.6E-09 605 S401-01.E06 7.89E-08 1.6E+05 3.1E-04 114 1.9E-09 CPA.12.004 A04 S397-O1.DO6 2.54E-08 1.6E+06 3.3E-03 253 2.1E-09 CPA.12.OO5 A06 S397-01.H07 1.02E-07 4.4E+05 1.0E-03 203 2.3E-09 A09 S398-01.H06 1.68E-07 6.8E+05 1.8E-03 322 2.7E-09 Bll S404-01.E01 6.95E-08 4.1E+04 1.1E-04 441 2.8E-09 B10 S404-01.D11 2.85E-07 2.2E+06 7.4E-03 261 3.4E-09 CPA.12.OO6 A07 S398-O1.DO9 1.83E-07 6.8E+04 2.7E-04 36 3.9E-09 B02 S400-01.C02 6.82E-08 1.1E+05 4.5E-04 49 4.0E-09 B07 S403-01.D03 1.82E-08 9.1E+05 4.1E-03 87 4.5E-09 CPA.12.OO2 A02 S397-O1.AO8 1.61E-07 1.0E+05 8.3E-04 222 8.0E-09 A12 S399-O1.DO5 1.27E-07 2.1E+05 2.0E-03 181 9.6E-09 BOG S403-01.A12 1.41E-07 1.0E+05 1.0E-03 251 1.0E-08 All S399-O1.AO7 2.82E-07 5.9E+04 7.0E-04 163 1.2E-08 CPA.12.014 B12 5404-01. E06 1.28E-07 9.9E+04 3.7E-03 142 3.7E-08 329WO 2017/009712 PCT/IB2016/001079 AOS S397-01.H06 1.80E-08 B01 S399-O1.DO9 3.20E-07 B04 S401-01.A03 8.14E-08 BOS S404-01.A06 5.36E-08 CPA.12.012 B09 S404-01.C08 1.90E-07

[001085] Table 40: Overview of FACS binding result fors human IgG4 antibodies reformatte fromd Fab screening hits. FACS binding was teste agaid nst the 293T-huHIDEl (human) and 293T-moHIDEl (mouse) cell The. resul tsare expressed as a ‘Preliminary FACS Kd’ calculate fromd titration curves describe hereid n(“SPR Kinetic Screen of 24 Anti- HIDE1 Fab PPE”). IgGs from the same H-CDR3 family are indicated by the same color, either blue or green. CPA. 12.003 & CPA. 12.008 have identical heavy chain CDR-3 regions and minor differences in framework regions or other CDRs. The same holds for CPA. 12.004 & CPA. 12.013. N.B. not binding.

Table 40: Overview of FACS binding results for human IgG4 antibodies reformatted from Fab screening hits HEK293-human HEK293-mouse H-CDR-3 Family Antibody ID KD (nM) KD (nM) CPA.12.001 61 N.B.

CPA.12.002 6.4 N.B. ■■■■■ 18 N.B. green 1.4 N.B. blue CPA.12.005 4 N.B.

CPA.12.006 2.5 N.B.

CPA.12.007 14 N.B. caa 1.3 N.B. green CPA.12.009 1.7 N.B.

N.B. N.B.

CPA.12.011 CPA.12.012 1.3 N.B.

CPA CPA.12.014 13 N.B.

CPA.12.015 11 N.B. 330WO 2017/009712 PCT/IB2016/001079 EXAMPLE 24: HIDE1 RNA EXPRESSION IN PATIENT SAMPLES MATERIAL & METHODS Reagents

[001086] RNA extracti onwas performed wit hHigh Pure Paraffin Kit by JHU (Roche, cat# 03270289001).

[001087] cDNA was produced using High Capacity cDNA Revers eTranscription Kit by JHU (Applied Biosystem cat#4s 368814).

[001088] Custom Taqman PreAmp pool (Life Technologies, cat #29233051).

[001089] TaqMan® PreAmp Master Mix Kit x2 (Life Technologies, cat# 4384266).

[001090] HIDE1 TaqMan probes: Hs01128131_ml andHsO1128129_ml, Life technologies.

[001091] TaqMan probes for Housekeeping genes (HSKG) (Life technologi es)human GUSB: Hs99999908_ml, human GAPDH: Hs99999905_ml, human RPL19: Hs01577060_gH and human HPRT1: Hs02800695_ml.

[001092] TaqMan Custom Arrays, 96A TLDA, Life Technologies, cat #4342259.

METHODS Tumor Samples (The Vigorous Immune Microenvironment of Microsatellite Instable Colon Cancer Is Balanced by Multiple Counter-Inhibitory Checkpoints, Cancer Discov; (1); 43-51. ©2014 AACR)

[001093] Tumor tissues were collect edat the Johns Hopkins Hospit al(Baltimore, MD) from patients with primary sporadic colorec talcancer and free of prior chemotherapy.

Assessment of MSI was done using the length of a panel of microsatelli markerste in the tumor and a normal reference (either norma lmucosa or germline) by using fragment analysi s of PCR product labes led wit hfluorescent dyes. Fragment analysi sdetermined the expression level of the proteins in charge of maintaining the integrit ofy microsatel litracte ts. Differences in the length of two or more markers (the standard Bethesda panel uses five marker s)were 331WO 2017/009712 PCT/IB2016/001079 indicative of MSI status. 3 patients tested as MSI (microsatelli instteable positi) ve and 3 patients as MSS (microsatelli stateble); all patients’ details are indicated in Table 42.

Laser Capture Microdissection (LCM) and RNA extraction

[001094] FFPE and hematoxyli andn eosin-stain edtissue sections (5 pm) were used for the LCM procedure using the Leica LMD 7000 system For. each patient, tissue sections were microdissected from three defined areas (TIL, Invasive Front, and Strom a)and directly collect ined tissue lysi sbuffer for RNA extraction. RNA was isolated follow ingthe manufacture’sr instructions. RNA was converted to cDNA using the High-Capacit yRNA-to- cDNA Kit .The cDNA was used as undilute ord diluted 1:4, and a step of preamplification was performe usingd a pool of TaqMan probes and a preamplification master mix kit, followed by TaqMan qPCR.

Transcript expression Quantitative PCR (qPCR)

[001095] Preampliife cDNAd , prepared as described above, was used as a templat fore qPCR reactions, using a gene specific TaqMan probes (detailed in Reagents 5&6) Detection was performe usingd QuantStudio 12k device.

[001096] The cycle in which the reactions achieved a threshol leveld of fluorescence (Ct= Threshold Cycle )was registered and was used to calculate the relati vetranscript quantity in the PCR reactions.

[001097] The absolute quantity was calculate byd using the equation Q=2 A-Ct .

Samples were normalized by the average Ct of the housekeeping genes: hGUSB, hRPL19, hGAPDH and hHPRTl, set across all samples to 25.

[001098] Undetectable value, was assigned as 40 cycles. 332WO 2017/009712 PCT/IB2016/001079 RESULTS Endogenous expression of HIDE1 in MSS and MSI derived from colorectal cancer patients Endogenous expression of HIDE 1 in MSI and MSS derived from colorectal cancer patients tested by qPCR

[001099] In order to verify the presence of the HIDE! transcript in colorecta cancerl derived cell s,qRT-PCR was performed using a. specific TaqMan probes as describe above in Material & Methods. qRT-PCR was performed by using undilute andd diluted 1:4 cDNA samples.

[001100] As shown in Figure 79, Analysis indicating higher transcript expression in TIL, Invasive front and Stroma areas in MSI patients (3/3) compared to MSS patients and higher expression in strom anda IF areas compared to TIL area.

[001101] Table 41. Represent normalized Ct values of undiluted and diluted (1:4) samples. Human HIDE1 transcr iptwas observed using two specific TaqMan probes describes in M&M, in cel lderived from three areas (TIL, IF, Strom a)from MSI and MSS colorect al cancer patients. 333WO 2017/009712 PCT/IB2016/001079 Table 41: Transcri ptExpression M 43597 M1JX3398 ^0^5993 Prone IF STR .........TH.......... STR nj SJR TfL nm llit 18121ili ,$יז$ ?B.C-3525 26.29225 2684325 26VS8S 2381-25 24 K-U2S 2/.145S 268(0/5 253505 H؛Dtl 14-3111812Rr>i 30.36525 28.28825 KI 72675 829845 27 96235 29803/5 MA :: 22.57825641 GAFEF 218S92W 22.04825-38/ 22 7865 222325 238-3125 22.5935 2254975 2S7j§5 Ms 23.96-7^ ׳■■■■•' •H؛598SSS&5 סי■ 3 •׳ 24.7732S01S 24.33924571 28.52175025 K 23.1925 ?3 £2/5 23 13725 32.87779 23 9045 .1^525-532 2808225612 28x44524922 30.G8S5 39a*282S 29 7/25 23.7255 5PU9 29.498/5 A4tjevoeas .:^1 M^RTl 23J12b2^r 2Si«،24S<׳ 2898734985 23.7355 31.«95 24.72325 24 l»S/s 24.*05 fob #3753 mu s>79b W *3753 pTOte Hl FF S؟R TK t؛ STR TH. STS HIDE 39.50? 75 2550225 28 86735 2793125 258375 25 25625 27JO0/S 27 2005 1251^^1 _: 2576375 ?2 ?7525 31.51525 74 0295 27 50535 .13.34378 31.3S 299025 .......... :'"22713435"' 22.54235 2231323 18.983 22.6855 6AWJM 2257075 : 2239225 3246325 32 6025 ?4 52275 24.19525 21 7215 2358535 ?4.55975 2S 551 2430625 23S815 RPL19 R9)i177aw^M 27 S4r;5 2732625 3/.66125 28.5585 28.84325 '38.4823 2 26.0695 2S.S66.35 30371 MPRT1 jrd 25 25875 25.3S225 ?5.82925 28x42625 247175 25.07825 34.80375 36.99S 25.2815 Fyotmaiize tfu/vakte syt diksted ،BNAJ1:4} mu ^99,' mu *3928 A1U #3992 "Senef-fal-TA !« IF STR r*TR 7777iti17777 STR nm 11.6112SL(1.r>i 27 26825- 28.332 26.01925 25.653 26.02?5 ?4.3£-77S 37.058? 5 ?5.01325 2>.8r-sr* MND1 314-8225- FJA ?!a -1!8 FJA kA 2/ 9475 1H61؛ 18123^1 28.922 MA »655555^_ס<1 21.56225- 23 31335 25.452 248C-US 23 804/ 22.26225 22,35125 22 8995 ■:SF$L ;;;;;:24^18;;;;;; 25.19926 &US8 24.?9؟2s 2 *.215 IS 5815 >880875 2431225 259^65 spl 1* 28.228 2758275 50 ?64 26^345 29 18175- 7? ־4693, 29.352?5 28.0615 7:i4^?^2S77 29^8671; H NRT1 ״.^OvOGaSjTti 24.346 23.544 23^855 24.O9475 23.1025 r* '9475 772093^77 mu #37=13 mu t؛3756 JHU #3762 : T؛i Til FF STR TIL IF : STR :£ : STR 1Gene To^Mas suite f-f-315 28131^>1 933672$•: J 28.6035 28.828 38.78575 25744 24 98635 27.36475 2S.599 26.319 H^liZSl^.rol MH?tl 35.58 2_d5 KA 3938875 >615135 :7777MA7i77i 29./S6 23.252 WDH ...: ^$55559335 :ס<1< ?2 81925 ?24535 ?3 585 22.68675 2? 10T25- 22 48976 23.02 72 76? toss 23.453 &1JS8 ««8595$fl،J8jtvX 24.62425 24.6265 24.594 34.17175 24 09035 28.53775 23 ג3ר 23 96 #19 22 27425 27*8؟S 28.544/5 28.54 ?25 27,93/75 29,329 28,909 ?5 46225 253385 ?5292 28.63675 34.438 24.95925 25.30475 25263 HPRT1 *C/SlOtSISjcil L...^:?^:....J Table 42: Patient details ; MS; CRC u: :Age: Gender Bace Stage Sze(mra) : Histoto^y ؛ Kras SFiAF ؛ 45 0 : 3992 ; 23959 : 92 W 2 tisgts grade Adenoca. WT Mutated i 3958 F' : 79 w 2 72.-3 Low grade Adenoca. ; Rotated WT : F ; 3957 : 33347 :A 4 53.0 J:R:igh grade Adenoca, iwT WT : ؛ P.UD Age Gender Race Stage Site ؛mm{ : Histotogy Kra: BRXF ؛ ؛ MSSCRC ؛ 3756 ; 10164 ؛ 55 w 4 45.0 low grade AdenecB. : WT WT; ؛ : : ؛ 3762 ؛ 23393 73 w 4 54.5 Low. grade Ad:encta.: ؛ 3753 ؛ 5555 : 49 F AA 4 47.-3 Low grade Adenoca. wmuci no 334WO 2017/009712 PCT/IB2016/001079

[001102] The examples set forth above are provided to give those of ordinary skill in the art a complete disclosure and descripti onof how to make and use the embodiments of the compositions syst, ems and methods of the invention, and are not intended to limi tthe scope of what the inventors regard as thei inventir on. Modifications of the above-described modes for carrying out the invention that are obvious to persons of skil inl the art are intended to be within the scope of the followi claing ms. All patents and publications mentioned in the specificatio aren indicative of the level sof skil ofl those skilled in the art to which the invention pertains All. reference scited in thi sdisclosure are incorporat byed reference to the same extent as if each reference had been incorporated by reference in its entirety individually.

[001103] All headings and section designations are used for clarity and reference purposes only and are not to be considered limiting in any way. For example, thos ofe skill in the art will appreciate the usefulness of combining various aspects from different headings and sections as appropriat accorde ing to the spiri andt scope of the inventio ndescribed herein.

[001104] All references cited herei nare hereby incorporated by reference herei nin their entireti esand for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporat byed reference in its entirety for all purposes.

[001105] Many modifications and variations of thi sapplication can be made without departing from its spir itand scope, as will be apparent to thos skie ll edin the art. The specific embodiments and examples described herei nare offered by way of example only. 335256261/2

Claims (9)

CLAIMED:

1. An anti-HIDE1 antibody for use in treating cancer in a patient, the use comprising administering the anti-HIDE1 antibody to said patient, wherein said cancer is treated, and wherein the antibody stimulates the immune system by inhibiting the action of HIDE1.

2. An anti-HIDE1 antibody for use according to Claim 1, wherein said cancer is selected from the group consisting of Acute Myeloid Leukemia, Acute Myeloid Leukemia Induction Failure, Acute Lymphoblastic Leukemia, Diffuse Large B-cell Lymphoma, Malignant Lymphoma, Non-Hodgkin Lymphoma, Diffuse Large B-Cell Lymphoma, Glioblastoma multiforme, Mesothelioma, Thymoma, Testicular Germ Cell Tumors, Kidney renal clear cell carcinoma, Sarcoma, Brain Lower Grade Glioma, Chronic Lymphocytic Leukemia, Non-Hodgkin Lymphoma- Follicular Lymphoma, Uterine Carcinosarcoma, Pediatric Brain Tumors, Lung adenocarcinoma, Cervical squamous cell carcinoma, endocervical adenocarcinoma, Pancreatic adenocarcinoma, Skin Cutaneous Melanoma, Kidney renal papillary cell carcinoma, Liver hepatocellular carcinoma; Bladder Urothelial Carcinoma, Colon adenocarcinoma, Head and Neck squamous cell carcinoma, Lung squamous cell carcinoma, Rectum adenocarcinoma, and Stomach adenocarcinoma.

3. An anti-HIDE1 antibody for use according to Claim 1 or 2, wherein said cancer is a cancer having high immune infiltrate of myeloid cells expressing HIDE1.

4. An anti-HIDE1 antibody for use according to Claim 1, wherein the cancer is treated by restricting the pro-tumorigenic effects of the myeloid cells in the tumor microenvironment in the patient.

5. An anti-HIDE1 antibody for use according to Claim 1, wherein the cancer is treated by eliciting one or more of the following effects on immunity in the patient, wherein said effect is selected from the group consisting of: i) increases immune response, (ii) increases T cell activity, (iii) increases activation of ap and/or yd T cells, (iv) increases cytotoxic T cell activity, (v) increases NK and/or NKT cell activity, (vi) alleviates ap and/or yd T-cell suppression, (vii) increases pro-inflammatory cytokine secretion, (viii) increases IL-2 secretion; (ix) increases interferon-y production, (x) increases Th1 response, (xi) decrease Th2 response, (xii) decreases or eliminates cell number and/or activity of at least one of regulatory T cells (Tregs), myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xiii) reduces regulatory cell activity, and/or the activity of one or more of myeloid derived suppressor cells (MDSCs), iMCs, mesenchymal stromal cells, TIE2-expressing monocytes, (xiv) decreases or eliminates M2 macrophages, (xv) reduces M2 macrophage pro-tumorigenic activity, (xvi) decreases or eliminates N2 neutrophils, (xvii) reduces N2 neutrophils pro-tumorigenic activity, (xviii) reduces inhibition of T cell activation, (xix) reduces inhibition of CTL activation, (xx) reduces inhibition of NK 215256261/2 and/or NKT cell activation, (xxi) reverses ap and/or y6 T cell exhaustion, (xxii) increases ap and/or yd T cell response, (xxiii) increases activity of cytotoxic cells, (xxiv) stimulates antigen-specific memory responses, (xxv) elicits apoptosis or lysis of cancer cells, (xxvi) stimulates cytotoxic or cytostatic effect on cancer cells, (xxvii) induces direct killing of cancer cells, (xxviii) increases Th17 activity and (xxix) modulating myeloid cell polarization, (xxx) modulating myeloid cell shifting toward a pro-inflammatory response, (xxxi) shifting myeloid from M2 toward M1 phenotype, (xxxii) modulating myeloid cell in the TME to support anti­ cancer immune response, (xxxiii) restricting the pro-tumorigenic effects of the myeloid cells in the TME, (xxxiv) enhancing myeloid and lymphoid infiltration into the tumor cite thereby shifting the tumor into more immunogenic, (xxxv) induces complement dependent cytotoxicity and/or antibody dependent cell-mediated cytotoxicity.

6. An anti-HIDE1 antibody for use according to Claim 1, wherein the cancer is treated by depleting myeloid cells or other circulating tumor cells expressing HIDE1 from a patient or patient sample, by: (a) contacting said patient with an anti-HIDE1 antibody, wherein said anti-HIDE1 antibody binds to HIDE1 expressing cells, (b) identifying cells to which said anti-HIDE1 antibody has bound, and (c) removing said cells in step (b) from said patient.

7. An anti-HIDE1 antibody for use according to Claim 1, wherein the anti-HIDE1 antibody binds to an isolated HIDE1 polypeptide consisting of a HIDE1 polypeptide ECD domain having at least 95% identity to the ECD domain of an amino acid sequence selected from the group consisting of the sequences depicted in Figure 66.

8. An anti-HIDE1 antibody for use according to Claim 7, wherein said isolated HIDE1 polypeptide has at least 99% identity to an amino acid sequence selected from the group consisting of the sequences depicted in Figure 66.

9. An anti-HIDE1 antibody for use according to Claim 7, wherein said isolated HIDE1 polypeptide is selected from the group consisting of the sequences depicted in Figure 66. 216