Multifunctional fusion protein and its use

Abstract

The present invention relates to a fusion protein and a plurality of treatment methods, wherein the plurality of treatment methods comprise administering a therapeutically effective amount of the fusion protein to a patient in need thereof.

Description

Technical Field

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 789,212, filed on January 7, 2019, the contents of which are incorporated herein by reference in their entirety. Background Art

[0002] Multiple pathways regulate the survival of infected and cancerous cells, as well as the activation or inhibition of cells of the immune system such as T cells and natural killer (NK) cells. One pathway that provides co-stimulatory and inhibitory second signals to T cells is represented by programmed death 1 (PD-1, also known as CD279) receptor and its ligands, PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273). PD-1 is a member of the CD28 / CTL4 family that is expressed on activated T cells but not on resting T cells (Nishimura et al. (1996) Int. Immunol. 8: 773). Binding of PD-1 to its ligand mediates an inhibitory signal that leads to reduced cytokine production and decreased T cell survival (Nishimura et al. (1999) Immunity 11:141; Nishimura et al. (2001) Science 291:319; Chemnitz et al. (2004) J. Immunol. 173:945).

[0003] Viral macrophage inflammatory protein-II (vMIP-II) is a chemokine encoded by human herpesvirus 8 (HHV-8) that interacts with CC and CXC chemokine receptors, including CCR5 and CXCR4. vMIP-II's inhibition of HIV-1 entry is mediated through CCR3, CCR5, and CXCR4, the HIV-1 receptors required for HIV-1 entry into target cells.

[0004] A complex interplay of positive and negative signals regulates T cell activation and the maintenance of T cell effector function. Members of the TNF ligand / TNF receptor superfamily, bridging cells of the immune system, and cells from other organ systems play crucial roles in this signaling matrix. In this process, TNF superfamily members contribute to homeostasis and pathogenesis by influencing cell survival and death, cell differentiation, and inflammation.

[0005] There remains a need for improved therapies for treating diseases such as cancer. The present invention addresses this need. Summary of the Invention

[0006] As described herein, the present invention relates to a fusion protein having multi-point molecular attachment capabilities and methods of use thereof.

[0007] One aspect of the present invention includes a fusion protein comprising component A and / or component B. Component A comprises component Y, component Z2, and component Z3. Component B comprises component X', component Z2', and component Z3'.

[0008] Another aspect of the present invention includes a fusion protein comprising component A, component B, and component C. Component A comprises component Y, component Z2, and component Z3. Component B comprises component X', component Z2', and component Z3'. Component C comprises component X and component C' L Component Y includes at least a portion of PD-1, component Z2 and component Z2' include the CH2 domain of human Fc, and component Z3 and component Z3' include the CH3 domain of human Fc. Component X' and component X include at least a portion of vMIP-II, and component C L ' includes at least one CH1 domain of human IgG1κ.

[0009] Yet another aspect of the present invention includes a method for producing a fusion protein, comprising administering to cells a first nucleic acid encoding human PD-1-hFcA, a second nucleic acid encoding vMIPII-CH'-hFcB, and a third nucleic acid encoding vMIPII-CL'.

[0010] Yet another aspect of the present invention includes a fusion protein comprising the amino acid sequences shown in SEQ ID NO: 14, SEQ ID NO: 49 and SEQ ID NO: 57.

[0011] In another aspect, the present invention includes a pharmaceutical composition comprising a pharmaceutically acceptable carrier and any one of the fusion proteins described herein.

[0012] In another aspect, the present invention includes a method of treating a proliferative disease in a patient, comprising administering to a patient in need of such treatment a therapeutically effective amount of any of the fusion proteins described herein.

[0013] In various embodiments of the above aspects or any other aspect of the invention described herein, component B further comprises component Z1 '. In certain embodiments, component A further comprises component Z1.

[0014] In certain embodiments, the fusion protein further comprises component C, wherein component C comprises component X and component C. L '.

[0015] In certain embodiments, the fusion protein further comprises component D, wherein component D comprises component Q and component C. L .

[0016] In certain embodiments, component Y comprises a ligand domain, a receptor domain, a scFv domain, or a lipocalin domain. In certain embodiments, component Y comprises at least a portion of PD-1, CD112R, CD113, or MHC-I polypeptide-related sequence A (MICA).

[0017] In certain embodiments, the fusion protein binds to PD-L1 or PD-L2.

[0018] In certain embodiments, component X' comprises a virus-derived peptide, a ligand-derived peptide, a receptor-derived peptide, or an HTS-selected peptide.

[0019] In certain embodiments, component X' comprises at least a portion of vMIP-II.

[0020] In certain embodiments, component X' comprises V1 or V1Δ.

[0021] In certain embodiments, component X comprises a virus-derived peptide, a ligand-derived peptide, a receptor-derived peptide, or a HTS-selected peptide. In certain embodiments, component X comprises V1 or V1Δ.

[0022] In certain embodiments, the fusion protein binds to CXCR4.

[0023] In some embodiments, component Y and component Z2 are connected by a hinge. In some embodiments, component Z1' and component Z2' are connected by a hinge.

[0024] In certain embodiments, component X' and component Z1' are linked by a linker. L Connected by a linker. In certain embodiments, component Q and component C L Connected via a linker.

[0025] In certain embodiments, the fusion protein binds to a receptor or ligand on an immune cell. In certain embodiments, the receptor is an Fc receptor.

[0026] In certain embodiments, X' comprises at least a portion of PD-1, TIGIT, CD96, CD112R, CD113, CD155, CD111, CD112, MHC-I polypeptide-related sequence A (MICA), NKG2A (CD94), MICB, ULBP1-5, TIM-3, CD226, NECL2, CRTAM, CD80, CTLA-4, KIR2DL1 / 2 / 3, or CD48.

[0027] In certain embodiments, component Y includes at least a portion of PD-1, TIGIT, CD96, CD112R, CD113, CD155, CD111, CD112, MHC-I polypeptide-related sequence A (MICA), NKG2A (CD94), MICB, ULBP1-5, TIM-3, CD226, NECL2, CRTAM, CD80, CTLA-4, KIR2DL1 / 2 / 3, or CD48.

[0028] In certain embodiments, component X includes at least a portion of PD-1, TIGIT, CD96, CD112R, CD113, CD155, CD111, CD112, MHC-I polypeptide-related sequence A (MICA), NKG2A (CD94), MICB, ULBP1-5, TIM-3, CD226, NECL2, CRTAM, CD80, CTLA-4, KIR2DL1 / 2 / 3, or CD48.

[0029] In certain embodiments, component Q includes at least a portion of PD-1, TIGIT, CD96, CD112R, CD113, CD155, CD111, CD112, MHC-I polypeptide-related sequence A (MICA), NKG2A (CD94), MICB, ULBP1-5, TIM-3, CD226, NECL2, CRTAM, CD80, CTLA-4, KIR2DL1 / 2 / 3, or CD48.

[0030] In certain embodiments, component A comprises the amino acid sequence shown in any one of SEQ ID NOs: 14-22.

[0031] In certain embodiments, component B comprises the amino acid sequence set forth in any one of SEQ ID NOs: 32 to 35. In certain embodiments, component B comprises the amino acid sequence set forth in any one of SEQ ID NOs: 49 to 55.

[0032] In certain embodiments, component A comprises the amino acid sequence set forth in any one of SEQ ID NOs: 14-22, and component B comprises the amino acid sequence set forth in any one of SEQ ID NOs: 32-35.

[0033] In certain embodiments, component C comprises the amino acid sequence set forth in any one of SEQ ID NOs: 57-63.

[0034] In certain embodiments, component B comprises the amino acid sequence set forth in any one of SEQ ID NOs: 49-55, and component C comprises the amino acid sequence set forth in any one of SEQ ID NOs: 57-63.

[0035] In certain embodiments, the fusion protein is capable of binding to (i) PD-L1 or PD-L2, (ii) CXCR4, and (iii) an Fc receptor or ligand on an immune cell.

[0036] In certain embodiments, component A comprises the amino acid sequence set forth in SEQ ID NO:29, and component B comprises the amino acid sequence set forth in SEQ ID NO:30.

[0037] In certain embodiments, component Y comprises the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0038] In certain embodiments, component Z2 comprises the amino acid sequence set forth in SEQ ID NO:12 and / or component Z3 comprises the amino acid sequence set forth in SEQ ID NO:13.

[0039] In certain embodiments, component A comprises the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO: 15.

[0040] In certain embodiments, component B comprises the amino acid sequence shown in SEQ ID NO:49.

[0041] In certain embodiments, component X' comprises the amino acid sequence set forth in SEQ ID NO: 37. In certain embodiments, component X comprises the amino acid sequence set forth in SEQ ID NO: 37.

[0042] In certain embodiments, component Z2' comprises the amino acid sequence set forth in SEQ ID NO: 12 and / or component Z3' comprises the amino acid sequence set forth in SEQ ID NO: 48.

[0043] In certain embodiments, component C L' includes the amino acid sequence shown in SEQ ID NO: 64.

[0044] In certain embodiments, component B comprises the amino acid sequence shown in SEQ ID NO:53.

[0045] In certain embodiments, component C comprises the amino acid sequence shown in SEQ ID NO:61.

[0046] In certain embodiments, component A comprises the amino acid sequence shown in SEQ ID NOs: 14, component B comprises the amino acid sequence shown in SEQ ID NOs: 53, and component C comprises the amino acid sequence shown in SEQ ID NOs: 61.

[0047] In certain embodiments, the method includes wherein the first nucleic acid encodes the amino acid sequence set forth in SEQ ID NO: 14, and / or the second nucleic acid encodes the amino acid sequence set forth in SEQ ID NO: 49, and / or the third nucleic acid encodes the amino acid sequence set forth in SEQ ID NO: 57.

[0048] In certain embodiments, the proliferative disease is cancer. In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, melanoma, glioblastoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), multiple myeloma, or colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The following detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, presently preferred embodiments are shown in the accompanying drawings. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the accompanying drawings.

[0050] Figure 1A and Figure 1B is a schematic diagram of some embodiments of a fusion protein platform.

[0051] Figure 2 is a schematic diagram of some embodiments of a fusion protein platform. The figure illustrates a fusion protein targeting the natural killer (NK) cell:tumor cell interface.

[0052] Figure 3Schematic diagram of a fusion protein that binds to CXCR4, PD-L1, and FcγRIIIa.

[0053] Figure 4 Schematic diagram of the interaction between a fusion protein that binds to CXCR4, PD-L1, and FcγRIIIa and a cancer cell and NK cell.

[0054] Figure 5 Schematic diagram of the possible interactions of a fusion protein that binds to CXCR4, PD-L1, and FcγRIIIa with various cell types.

[0055] Figure 6 is a schematic diagram of the possible interactions of various cell types with various fusion proteins.

[0056] Figure 7 is a schematic diagram of the possible interactions of various cell types with various fusion proteins.

[0057] Figure 8 Schematic diagram of the possible interactions of tumor-associated macrophages (M1) with various fusion proteins.

[0058] Figure 9 It is a Coomassie blue stained (reduced) SDS-PAGE gel of hPD-1-FcA / hFcB (SEQ ID NO: 14 plus SEQ ID NO: 36).

[0059] Figure 10 It is a Coomassie blue stained SDS-PAGE gel of hFcA / hFcB (SEQ ID NO: 23 plus SEQ ID NO: 36).

[0060] Figure 11 It is a Coomassie blue stained SDS-PAGE gel of hMICA-FcA / hFcB (SEQ ID NO: 22 plus SEQ ID NO: 36).

[0061] Figure 12Coomassie blue-stained (reducing) SDS-PAGE gel of hFcA / hTIGIT-FcB (SEQ ID NO: 14 plus SEQ ID NO: 36), hFcA / hCD155-FcB (SEQ ID NO: 23 plus SEQ ID NO: 32), hFcA / hFcB (SEQ ID NO: 23 plus SEQ ID NO: 36), and hMICA-FcA / hTIGIT-FcB (SEQ ID NO: 22 plus SEQ ID NO: 33).

[0062] Figure 13 It is a Coomassie blue stained SDS-PAGE gel of hCD112R-FcA / hFcB (SEQ ID NO: 16 plus SEQ ID NO: 36).

[0063] Figure 14 It is a Coomassie blue stained SDS-PAGE gel of HA-PD-1-FcA / hCD113-FcB (SEQ ID NO: 15 plus SEQ ID NO: 35).

[0064] Figure 15 Coomassie blue-stained SDS-PAGE gel of hFcA / hCD113-FcB (SEQ ID NO: 23 plus SEQ ID NO: 33) and PD-1-hFcA / hCD113-FcB (SEQ ID NO: 14 plus SEQ ID NO: 35).

[0065] Figure 16 It is a Coomassie blue stained SDS-PAGE gel of hFcA / hCD155-FcB (SEQ ID NO: 23 plus SEQ ID NO: 32).

[0066] Figure 17 Coomassie blue-stained SDS-PAGE gel of hFcA / TIM-3-hFcB (SEQ ID NO: 23 plus SEQ ID NO: 34) and PD-1-hFcA / TIM-3-hFcB (SEQ ID NO: 14 plus SEQ ID NO: 34).

[0067] Figure 18hFcA / hTIGIT-FcB (SEQ ID NO: 23 plus SEQ ID NO: 33) was shown to bind to cells expressing human CD155.

[0068] Figure 19 hFcA / hTIGIT-FcB (SEQ ID NO: 23 plus SEQ ID NO: 33) was shown to bind to cells expressing human CD112.

[0069] Figure 20 hFcA / hTIGIT-FcB (SEQ ID NO: 23 plus SEQ ID NO: 33) was shown to bind to cells expressing human TIGIT.

[0070] Figure 21 hFcA / hTIGIT-FcB (SEQ ID NO: 23 plus SEQ ID NO: 33) and hFcA / hCD155-FcB (SEQ ID NO: 23 plus SEQ ID NO: 32) were shown to bind to cells expressing CD16 (FcγRIIIa).

[0071] Figure 22 Fusion proteins separated and reduced (R) on SDS-PAGE, Coomassie-stained gels are shown. As depicted, 'A' refers to hFcA / hFcB (SEQ ID NO:23 plus SEQ ID NO:36), 'B' refers to PD-1-hFcA / FcB (SEQ ID NO:14 plus SEQ ID NO:36), 'C' refers to hFcA / VpI-CH'-FcB / VpI-CL (SEQ ID NO:23 plus SEQ ID NO:53 and SEQ ID NO:61), and 'D' refers to PD-1-hFcA / VpI-CH'-FcB / VpI-CL (SEQ ID NO:14 plus SEQ ID NO:53 and SEQ ID NO:61).

[0072] Figures 23A to 23D is a series of Western blots. Western blot analysis was performed on protein-A purified fusion proteins from conditioned medium of Expi-CHO cells transfected with the expression construct, as shown in FIG. Figure 22The observed bands were consistent with those expected for PD-1-hFcA (SEQ ID NO: 14) and vMIPII-CL' (SEQ ID NO: 57); 125 kDa for the non-reduced (NR) sample and 60 kDa and 20 kDa for the reduced (R) sample, respectively. Western blots were probed with ( FIG23A ) anti-PD-1 antibody, ( FIG23B ) anti-IgGκ light chain antibody, ( FIG23C ) anti-IgG heavy chain antibody, and ( FIG23D ) anti-vMIP-II antibody.

[0073] Figure 24 The fusion protein was shown to bind to the B16 melanoma cell line, which expresses PD-L1 and CXCR4 or CXCR7 on its cell surface. The fusion proteins tested were (in order from top to bottom): hFcA / hFcB (SEQ ID NO: 23 plus SEQ ID NO: 36), PD-1-hFcA / FcB (SEQ ID NO: 14 plus SEQ ID NO: 36), hFcA / V1Δmut-CH'-FcB / V1Δmut-CL' (SEQ ID NO: 23 plus SEQ ID NO: 52 and SEQ ID NO: 60), hFcA / V1Δ-CH'-FcB / V1Δ-CL' (SEQ ID NO: 23 plus SEQ ID NO: 51 and SEQ ID NO: 59), PD-1-hFcA / V1Δmut-CH'-FcB / V1Δmut-CL' (SEQ ID NO: 14 plus SEQ ID NO: 52 and SEQ ID NO: 60), and PD-1-hFcA / V1Δ-CH'-FcB / V1Δ-CL' (SEQ ID NO: 14 plus SEQ ID NO: 52 and SEQ ID NO: 60). NO: 51 and SEQ ID NO: 59).

[0074] Figure 25A transwell assay measuring the inhibitory effect of the fusion protein on melanoma cell migration is shown. Representative images of a single transwell migration assay of migrating B16-F10 cells treated with 100 ng / mL of CXCL12 and the fusion protein are shown. The fusion proteins tested were (from top to bottom): hFcA / hFcB (SEQ ID NO: 23 plus SEQ ID NO: 36), PD-1-hFcA / FcB (SEQ ID NO: 14 plus SEQ ID NO: 36), hFcA / V1Δmut-CH'-FcB / V1Δmut-CL' (SEQ ID NO: 23 plus SEQ ID NO: 52 and SEQ ID NO: 60), hFcA / V1Δ-CH'-FcB / V1Δ-CL' (SEQ ID NO: 23 plus SEQ ID NO: 51 and SEQ ID NO: 59), PD-1-hFcA / V1Δmut-CH'-FcB / V1Δmut-CL' (SEQ ID NO: 14 plus SEQ ID NO: 52 and SEQ ID NO: 60), and PD-1-hFcA / V1Δ-CH'-FcB / V1Δ-CL' (SEQ ID NO: 14 plus SEQ ID NO: 52 and SEQ ID NO: 60). NO: 51 and SEQ ID NO: 59). The analysis was performed by staining with 2% crystal violet.

[0075] FIG. 26A to FIG. 26B The results show that the fusion protein (FP) PD1-hFcA / v1Δ-CH'-hFcB / v1Δ-CL' (SEQ ID NO: 14 plus SEQ ID NO: 51 and SEQ ID NO: 59) can inhibit tumor growth in a B16F10 melanoma subcutaneous model. C57BL / 6 mice were subcutaneously inoculated with 1×10 5 After 100 μl (10 μg / mL) of FP in PBS (mouse R, mouse 2L, and mouse 2R) or PBS alone (mouse X or mouse L) were used for treatment on days 13, 14, 15, 16, and 21. Figure 26A The changes in tumor size over time in each mouse were detected and plotted. Figure 26B Representative images of one mouse from the PBS-only treatment group (top) and the PD1-hFcA / v1Δ-CH′-hFcB / v1Δ-CL′(FP) treatment group (bottom) are shown.

[0076] Figure 27This study demonstrates the enhancement of NK cell-mediated ADCC by the addition of multifunctional fusion proteins. SKOV-3 ovarian cells (target cells (T)) were seeded at 3000 cells / well in a 96-well plate, allowed to adhere, and labeled with a red fluorescent cytoplasmic probe (CellTracker Red CMTPX). SKOV-3 cells were then labeled with a green fluorescent caspase-3 reagent. CD16.NK-92 cells (V158 variant, effector cells (E)) were added to the wells at an E:T ratio of 5:1, along with 25 mg / ml of various fusion proteins or no protein. The number of double-positive (red + green) fluorescent cells was determined using the Incucyte Live Cell Analysis System. Results shown represent the 20-hour time point. Fusion proteins tested: high affinity (HA)-PD-1-hFcA / FcB (SEQ ID NO: 15 plus SEQ ID NO: 36), hFcA / hCD113-FcB (SEQ ID NO: 23 plus SEQ ID NO: 35), and high affinity (HA)-PD-1-hFcA / hCD113-FcB (SEQ ID NO: 15 plus SEQ ID NO: 35). DETAILED DESCRIPTION

[0077] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used to test the practice of the present invention, preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0078] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0079] The articles "a" and "an" are used herein to refer to one or more than one (ie, to at least one) of the grammatical object of the article. For example, "an element" refers to one element or more than one element.

[0080] As used herein, "about" when referring to a measurable value such as an amount, a duration, etc., is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and more preferably ±0.1% of the specified value, as such variations are suitable for performing the disclosed methods.

[0081] As used herein, "activation" refers to a state of a T cell that has been sufficiently stimulated to induce one or more of cytokine production, detectable effector function, and cell proliferation. The term "activated T cell" refers to a T cell that displays one or more of these activation characteristics, etc.

[0082] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from a natural or recombinant source, and can be an immunoreactive portion of an intact immunoglobulin. Antibodies are typically tetramers of immunoglobulin molecules, but can also exist as higher order multimers of such tetramers. Antibodies of the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies (scFv) and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0083] The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigen-determining variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0084] As used herein, an "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. Gamma (γ), mu (μ), delta (Δ), alpha (α), and epsilon (ε) heavy chains refer to the five major antibody heavy chain isotypes.

[0085] As used herein, an "antibody light chain" refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0086] As used herein, the term "synthetic antibody" refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a phage as described herein. The term should also be interpreted as referring to an antibody produced by synthesizing a DNA molecule encoding the antibody, and said DNA molecule expresses RNA encoding an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA, RNA or amino acid sequence has been obtained using techniques for synthesizing nucleic acids or amino acid sequences that are available and well known in the art.

[0087] As used herein, the term "antigen" or "Ag" is defined as a molecule that induces an immune response or binds to an immune recognition moiety, such as an antibody and a T-cell receptor. This immune response can involve the production of antibodies, the activation of specific immunocompetent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Furthermore, antigens can be derived from recombinant DNA or genomic DNA. Those skilled in the art will appreciate that any DNA, including a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response, encodes the term "antigen" as used herein. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. Clearly, the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and such nucleotide sequences can be arranged in various combinations to elicit the desired immune response. Furthermore, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. Clearly, an antigen can be produced, synthesized, or derived from a biological sample. Such biological samples can include, but are not limited to, a tissue sample, a tumor sample, a cell, or a biological fluid.

[0088] As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested as a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with the cancer condition. An "anti-tumor effect" can also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention to prevent the appearance of a tumor in the first place.

[0089] According to the present invention, the term "autoantigen" refers to any self-antigen that is recognized as foreign by the immune system. Autoantigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins, including cell surface receptors.

[0090] As used herein, the term "autoimmune disease" is defined as a condition caused by an autoimmune response. An autoimmune disease is the result of an inappropriate and excessive response to a self-antigen. Examples of autoimmune diseases include, but are not limited to, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, diabetes mellitus (type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatism, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, and the like.

[0091] As used herein, the term "autologous" refers to any material originating from the same individual that is subsequently reintroduced into that individual.

[0092] "Allogeneic" refers to a transplant derived from a different animal of the same species.

[0093] "Xenogeneic" refers to a transplant derived from an animal of a different species.

[0094] As used herein, the term "cancer" is defined as a disease characterized by the rapid and uncontrolled proliferation and / or accumulation of abnormal cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. In certain embodiments, the cancer is medullary thyroid carcinoma.

[0095] The term "cleavage" refers to the breaking of a covalent bond, such as in the backbone of a nucleic acid molecule. Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of phosphodiester bonds. Both single-strand cleavage and double-strand cleavage are possible. Double-strand cleavage can occur as a result of two distinct single-strand cleavage events. DNA cleavage can result in the production of blunt ends or staggered ends. In certain embodiments, fusion polypeptides can be used to target cleavage of double-stranded DNA.

[0096] As used herein, the term "conservative sequence modification" is intended to refer to amino acid modifications that do not significantly affect or alter the binding properties of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR region of an antibody can be substituted with other amino acid residues from the same side chain family, and the altered ability of the antibody to bind to the antigen can be tested using the functional assays described herein.

[0097] A "disease" is a health state in an animal in which the animal is unable to maintain homeostasis. If the disease is not ameliorated, the animal's health continues to deteriorate. In contrast, a "disorder" in an animal is a health state in which the animal is able to maintain homeostasis, but the animal's health is less than it would be in the absence of the disease. A disorder does not necessarily lead to a further decline in the animal's health if left untreated.

[0098] "Effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition as described herein that is effective to achieve a specific biological result or provide a therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any suitable method in the art.

[0099] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, that serves as a template for the synthesis of other polymers and macromolecules in biological processes. These polymers and macromolecules have a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the resulting biological properties. Thus, if a gene is transcribed into mRNA, and the mRNA corresponding to the gene is translated in a cell or other biological system to produce a protein, then the gene encodes the protein. The coding strand, whose nucleotide sequence is identical to the mRNA sequence and is typically provided in a sequence listing, and the non-coding strand, which serves as a template for transcription of a gene or cDNA, can both be said to encode a protein or other product of the gene or cDNA.

[0100] As used herein, "endogenous" refers to any material that originates from or is produced internally within an organism, cell, tissue, or system.

[0101] As used herein, the term "exogenous" refers to any material that is introduced from or originates outside an organism, cell, tissue, or system.

[0102] As used herein, the term "expand" refers to an increase in number, such as an increase in the number of T cells. In one embodiment, the number of T cells expanded ex vivo is increased relative to the number originally present in the culture. In another embodiment, the number of T cells expanded ex vivo is increased relative to other cell types in the culture. As used herein, the term "ex vivo" refers to cells that have been removed from a living organism (e.g., a human) and propagated outside of the organism (e.g., in a culture dish, test tube, or bioreactor).

[0103] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its regulatory elements, such as a promoter.

[0104] An "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other expression elements may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) that incorporate a recombinant polynucleotide.

[0105] As used herein, "homologous" refers to the identity of the subunit sequence between two polymeric molecules, such as two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or two polypeptide molecules. When a subunit position in both molecules is occupied by the same monomeric subunit; for example, if a position in each of the two DNA molecules is occupied by adenine, then they are homologous at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half (e.g., five positions in a ten-subunit-long polymer) of the positions in the two sequences are homologous, then the two sequences have 50% homology; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences have 90% homology.

[0106] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that include minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some cases, residues in the Fv framework region (FR) of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, a humanized antibody will comprise substantially all of at least one, typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are FR regions of a human immunoglobulin sequence. The humanized antibody preferably also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988, Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

[0107] "Fully human" refers to an immunoglobulin, such as an antibody, in which the entire molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody.

[0108] As used herein, "identity" refers to the subunit sequence identity between two polymeric molecules, particularly between two amino acid molecules, for example, between two polypeptide molecules. Two amino acid sequences are identical when they have the same residue at the same position; for example, if one position in each of the two polypeptide molecules is occupied by arginine, then they are identical at that position. The identity or degree to which two amino acid sequences have the same residue at the same position in an alignment is usually expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of positions that match or are identical; for example, if half (e.g., five positions in a ten amino acid long polymer) of the positions in the two sequences are identical, then the two sequences are 50% identical; if 90% of the positions (e.g., 9 out of 10) are matched or homologous, then the two sequences are 90% identical.

[0109] As used herein, the term "immunoglobulin" or "Ig" is defined as a class of proteins that act as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgG, IgM, IgD, IgA, and IgE. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced by most subjects in primary immune responses. It is the most effective immunoglobulin in agglutination, complement fixation, and other antibody reactions, and is important in defending against bacteria and viruses. IgD is an immunoglobulin with no known antibody function, but can act as an antigen receptor. IgA is the main antibody found in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgE is an immunoglobulin that mediates immediate hypersensitivity reactions by causing mediators released from mast cells and basophils upon exposure to allergens.

[0110] As used herein, the term "immune response" is defined as a cellular reaction to an antigen when lymphocytes recognize the antigen molecule as foreign and induce the formation of antibodies and / or activate lymphocytes to remove the antigen.

[0111] As used herein, the term "lipocalin" is defined as a class of proteins that transport hydrophobic molecules such as steroids, bilins, retinoids, and lipids in their natural environment. They share limited regions of sequence homology and a common tertiary structural architecture consisting of an eight-stranded antiparallel β-barrel with a repeating +1 topology containing an internal ligand binding site. Lipocalins are found in Gram-negative bacteria, vertebrate cells, invertebrate cells, and plants, and are involved in many biological processes, including immune responses, pheromone transport, bioprostaglandin synthesis, retinoid binding, and cancer cell interactions. Lipocalins can be modified in many of the same ways as antibodies to alter or enhance their binding properties, for example, to bind to a cell surface molecule and thereby block, enhance, or otherwise alter its functional properties.

[0112] As used herein, "industructional material" includes a publication, a recording, a diagram, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present invention. The instructional material of the kit of the present invention can, for example, be attached to a container containing the nucleic acids, peptides, and / or compositions of the present invention, or shipped together with a container containing the nucleic acids, peptides, and / or compositions. Alternatively, the instructional material can be shipped separately from the container so that the recipient can use the instructional material and the compound together.

[0113] "Isolated" means altered or removed from its natural state. For example, a nucleic acid or peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or can exist in a non-natural environment, for example, a host cell.

[0114] As used herein, "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can transfer large amounts of genetic information into the host cell's DNA, making them one of the most effective gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide a means to achieve significant levels of gene transfer in vivo.

[0115] As used herein, the term "modified" refers to an altered state or structure of a molecule or cell of the invention. Molecules can be modified in a variety of ways, including chemical, structural, and functional modifications. Cells can be modified by introducing nucleic acids.

[0116] As used herein, the term "modulate" refers to mediating a detectable increase or decrease in the level of a response in a subject compared to the level of the subject's response in the absence of the treatment or compound, and / or compared to the level of response in an otherwise identical but untreated subject. The term includes perturbing and / or influencing a natural signal or response, thereby mediating a beneficial therapeutic response in a subject, preferably a human.

[0117] In the context of the present invention, the following abbreviations are used for common nucleic acid bases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0118] Unless otherwise indicated, "a nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase "a nucleotide sequence encoding a protein or an RNA" may also include multiple introns, to the extent that a nucleotide sequence encoding a protein may include one or more introns in certain versions.

[0119] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence, resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, when necessary to link two protein coding regions, in the same reading frame.

[0120] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to refer to an abnormal expression level of a tumor antigen in cells from a diseased area, such as a solid tumor within a particular tissue or organ of a patient, relative to the expression level in normal cells from that tissue or organ. Patients with solid tumors or hematological tumors characterized by overexpression of a tumor antigen can be identified by standard assays known in the art.

[0121] "Parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection, or infusion techniques.

[0122] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, nucleic acid and polynucleotide are used interchangeably herein. It is well known to those skilled in the art that nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides." The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including, but not limited to, recombinant means, i.e., using conventional cloning techniques and PCR. TM etc., as well as cloning nucleic acid sequences from a recombinant library or a cell genome by synthetic means.

[0123] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must include at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a protein sequence or peptide sequence. Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the terms refer to both short chains, which are also commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, for example, which are commonly referred to in the art as proteins, which have many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0124] As used herein, the terms "fusion protein" and "chimeric protein" are used interchangeably and refer to a compound composed of two or more polypeptides. In some embodiments, the two or more polypeptides are covalently linked. In further embodiments, the two or more polypeptides are covalently linked via a peptide bond, a linker, or a disulfide bond. Fusion proteins can be produced by a variety of methods well known to those skilled in the art, the most common of which is by introducing a vector comprising a nucleic acid sequence encoding or specifying the amino acid sequence of a fusion protein into a cell. Additional amino acids or polypeptides can be incorporated into a fusion protein to induce additional functional properties, such as stability, half-life, multimerization, and ease of purification. An example of such an element is a polypeptide linker.

[0125] As used herein, the term "promoter" is defined as a DNA sequence recognized by the cell's synthetic machinery or introduced synthetic machinery to initiate specific transcription of a polynucleotide sequence.

[0126] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product to which the promoter / regulatory sequence is operably linked. In some cases, this sequence may be a core promoter sequence, while in other cases, the sequence may also include an enhancer sequence and other regulatory elements required for expression of the gene product. For example, the promoter / regulatory sequence may be a sequence that expresses a gene product in a tissue-specific manner.

[0127] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0128] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell essentially only when an inducer corresponding to the promoter is present in the cell.

[0129] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene, causes the gene product to be produced in a cell substantially only when the cell is a cell of the tissue type corresponding to the promoter.

[0130] "Signal transduction pathway" refers to the biochemical relationships between multiple signal transduction molecules that play a role in transmitting a signal from one part of a cell to another. The phrase "cell surface receptor" includes molecules and complexes of molecules that are capable of receiving a signal and transmitting the signal across the plasma membrane of a cell.

[0131] The term "specifically binds," as used herein with respect to an antibody, refers to an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to antigens from one or more species. However, this cross-species reactivity, in itself, does not change the antibody's specific classification. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, this cross-reactivity, in itself, does not change the antibody's specific classification. In some cases, the terms "specifically bind" or "specifically binds" may be used to refer to the interaction of an antibody, a protein, or a peptide with a second chemical substance to indicate that the interaction is dependent on a specific structure (e.g., an antigenic determinant or epitope) present on the chemical species. For example, an antibody recognizes and binds to a specific protein structure, rather than proteins in general. If an antibody is specific for epitope "A," the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody.

[0132] The term "subject" is intended to include living organisms (e.g., mammals) in which an immune response can be elicited. A "subject" or "patient," as used herein, can be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, and murine mammals. Preferably, the subject is human.

[0133] As used herein, a "substantially purified" cell is a cell that is substantially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cell types that are normally associated with its naturally occurring state. In some cases, a substantially purified cell population refers to a homogenous population of cells. In other cases, the term refers only to cells that have been separated from the cells with which they are naturally associated in nature. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0134] "Target site" or "target sequence" refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule can specifically bind under conditions sufficient for binding to occur.

[0135] As used herein, the term "treat" refers to treatment and / or prevention, and a therapeutic effect achieved by inhibiting, alleviating or eradicating a disease state.

[0136] As used herein, the term "transfected" or "transformed" or "transduced" refers to a process by which an exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include the primary subject cell and its progeny.

[0137] The term "transgene" refers to genetic material that has been or will be artificially inserted into the genome of an animal, particularly a mammal, and more particularly a mammalian cell of a living animal.

[0138] "Treating" a disease, as the term is used herein, means reducing the frequency or severity of at least one sign or symptom of a disease or condition experienced by a subject.

[0139] As used herein, the phrase "under transcriptional control" or "operably linked" means that the promoter is in the correct location and orientation relative to the polynucleotide to control transcription initiation by RNA polymerase and expression of the polynucleotide.

[0140] A "vector" is a composition of matter that includes an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes an autonomously replicating plasmid or a virus. The term should also be interpreted to include non-plasmid and non-viral compounds that promote the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. An example of a plasmid vector is an attachment-type vector, in which self-replication is driven or enhanced by regulatory elements derived from a virus, such as Epstein-Barr virus and BK virus. Examples of viral vectors include, but are not limited to, Sendai virus vectors, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, etc.

[0141] Range: Throughout this disclosure, various aspects of the invention may be presented in range format. It should be understood that descriptions in range format are merely for convenience and brevity and should not be construed as inflexible limitations on the scope of the invention. Therefore, descriptions of ranges should be considered to have explicitly disclosed all possible subranges and individual values ​​within the ranges. For example, a description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within the ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0142] When any amino acid sequence consists of Where an accession number is specifically mentioned, the sequence is partially and / or entirely incorporated herein by reference. Information associated with the accession number, such as identification of signal peptides, extracellular domains, transmembrane domains, promoter sequences and translation initiation, is also partially and / or entirely incorporated herein by reference.

[0143] As contemplated herein with respect to the disclosed compositions of matter and methods, in one aspect, embodiments of the present invention comprise the components and / or steps disclosed herein. In another aspect, embodiments of the present invention consist essentially of the components and / or steps disclosed herein. In yet another aspect, embodiments of the present invention consist of the components and / or steps disclosed therein.

[0144] describe:

[0145] A fusion protein is provided. In some exemplary embodiments, the first component of the fusion protein blocks a chemokine receptor, such as CXCR4 and / or CXCR7 (through the binding of vMIPII or V1 peptide or its derivatives in the fusion protein), and this blockade is used to fix tumor cells and interfere with their migration, invasion, metastasis, and other tumorigenic properties; the second component of the fusion protein blocks a checkpoint inhibitor on the tumor cells, such as PD-L1 and / or PD-L2 (through the binding of PD1 or its derivatives in the fusion protein), and this blockade is used to interfere with the inhibition of a tumor-directed immune effector cell, such as NK cell; the third component of the fusion protein triggers an activating receptor on the same immune effector cell, such as Fc γ RIIIa receptor (via Fc γ or its derivatives in fusion proteins), which drives NK cell activation and promotes ADCC and ADCP.

[0146] In some exemplary embodiments, one component of the fusion protein blocks a checkpoint inhibitor on a tumor or other cell, and the other two components each trigger a different activating receptor on an immune effector cell. In some embodiments, when the checkpoint inhibitor is on a tumor cell, the fusion protein molecule bridges an immune effector cell and a target tumor cell. In addition, the three interactions of the fusion protein, the combination of checkpoint inhibition pathway blocking and activation receptor triggering, are used to functionally enhance each other, and all three synergistically drive the activation of the immune effector cell, for example, NK cell. In a preferred embodiment, the checkpoint inhibitor blocked by the fusion protein is composed of PD-L1, PD-L2, CD113, CD112, CD155 or CD111, and the two activating receptors jointly triggered by the fusion protein are Fc on NK cells. γ RIIIa receptor and 4-1BB.

[0147] Fusion Protein:

[0148] Providing a fusion protein comprising component A and / or component B; wherein component A comprises component Y, component Z2 and component Z3; and

[0149] Wherein component B comprises component X', component Z2' and component Z3'. In some embodiments, component B further comprises component Z1'. Therefore, in some embodiments, component B comprises component X', component Z1', component Z2' and component Z3'.

[0150] In some embodiments, component A further comprises component Z1. Thus, in some embodiments, component A comprises component Y, component Z1, component Z2, and component Z3.

[0151] In some embodiments, the fusion protein further comprises component C, wherein component C comprises component X and component C. L '. In a further embodiment, component C L ' includes at least a portion of an immunoglobulin light chain. In some embodiments, the fusion protein further includes component D, wherein component D includes component Q and component C L In a further embodiment, component C L Includes at least a portion of an immunoglobulin light chain.

[0152] In some embodiments, component A further comprises a leader sequence. In further embodiments, the leader sequence is a human albumin leader sequence.

[0153] In some embodiments, component B further comprises a leader sequence. In further embodiments, the leader sequence is a human albumin leader sequence.

[0154] In some embodiments, component Y comprises a ligand domain, a receptor domain, a scFv domain, or a lipocalin domain. In further embodiments, component Y comprises at least a portion of PD-1, TIGIT, CD96, CD112R, CD113, CD155, CD111, CD112, MHC-I polypeptide-related sequence A (MICA), NKG2A (CD94), MICB, ULBP1-5, TIM-3, CD226, NECL2, CRTAM, CD80, CTLA-4, KIR2DL1 / 2 / 3, or CD48. In further embodiments, the fusion protein binds to PD-L1 or PD-L2.

[0155] In some embodiments, component Z1 comprises a domain of an immunoglobulin, a TNF superfamily member, a TNF-L superfamily member, transferrin, a transferrin receptor, human serum albumin, or a lipocalin. In some embodiments, the immunoglobulin domain is a CH1 domain. In some embodiments, the immunoglobulin is IgG. In further embodiments, the immunoglobulin is IgE.

[0156] In some embodiments, component Z2 comprises an immunoglobulin domain, a TNF superfamily member, a TNF-L superfamily member, transferrin, a transferrin receptor, human serum albumin, or a lipocalin. In some embodiments, the immunoglobulin domain is a CH2 domain. In some embodiments, the immunoglobulin is IgG. In further embodiments, the immunoglobulin is IgE.

[0157] In some embodiments, component Z3 comprises an immunoglobulin domain, a TNF superfamily member, a TNF-L superfamily member, transferrin, a transferrin receptor, human serum albumin, or a lipocalin. In some embodiments, an immunoglobulin domain is a CH3 domain. In some embodiments, the immunoglobulin is IgG. In further embodiments, the immunoglobulin is IgE.

[0158] In some embodiments, component Z l The invention also provides a method for preparing a polypeptide comprising a domain of an immunoglobulin, a TNF superfamily member, a TNF-L superfamily member, a transferrin, a transferrin receptor, a human serum albumin, or a lipocalin. In some embodiments, the domain of an immunoglobulin is a CH1 domain. In some embodiments, the immunoglobulin is an IgG. In further embodiments, the immunoglobulin is an IgE.

[0159] In some embodiments, component Z2′ comprises an immunoglobulin domain, a TNF superfamily member, a TNF-L superfamily member, transferrin, a transferrin receptor, human serum albumin, or a lipocalin. In some embodiments, an immunoglobulin domain is a CH2 domain. In some embodiments, the immunoglobulin is IgG. In further embodiments, the immunoglobulin is IgE.

[0160] In some embodiments, component Z3' comprises an immunoglobulin domain, a TNF superfamily member, a TNF-L superfamily member, transferrin, a transferrin receptor, human serum albumin, or a lipocalin. In some embodiments, an immunoglobulin domain is a CH3 domain. In some embodiments, the immunoglobulin is IgG. In further embodiments, the immunoglobulin is IgE.

[0161] In some embodiments, component X' comprises a virus-derived peptide, a ligand-derived peptide, a receptor-derived peptide, or a peptide selected by high-throughput screening (HTS). In some embodiments, component X' comprises a peptide sequence that binds to a chemokine receptor, a cytokine receptor, a counterreceptor for a functional ligand, an integrin, a ligand, or a portion of a membrane signaling complex. In some embodiments, component X' comprises at least a portion of PD-1, TIGIT, CD96, CD112R, CD113, CD155, CD111, CD112, MHC-I polypeptide-related sequence A (MICA), NKG2A (CD94), MICB, ULBP1-5, TIM-3, CD226, NECL2, CRTAM, CD80, CTLA-4, KIR2DL1 / 2 / 3, or CD48. In further embodiments, component X' comprises at least a portion of vMIP-II. In further embodiments, component X' comprises a V1 or V1Δ polypeptide. In still further embodiments, the fusion protein binds to CXCR4.

[0162] In some embodiments, component Q includes at least a portion of PD-1, TIGIT, CD96, CD112R, CD113, CD155, CD111, CD112, MHC-I polypeptide-related sequence A (MICA), NKG2A (CD94), MICB, ULBP1-5, TIM-3, CD226, NECL2, CRTAM, CD80, CTLA-4, KIR2DL1 / 2 / 3, or CD48.

[0163] In some embodiments, component X comprises a virus-derived peptide, a ligand-derived peptide, a receptor-derived peptide, or an HTS-selected peptide. In further embodiments, component X comprises a V1 or V1Δ polypeptide. In further embodiments, the fusion protein binds to CXCR4.

[0164] In some embodiments, component Z1' and component Z2' are connected by a hinge, such as an IgG hinge. In some embodiments, component Z1' and component Z2' are connected by a hinge, such as an IgG hinge. In some embodiments, component X' and component Z1' are connected by a linker. In some embodiments, component X and component C L Connected via a linker.

[0165] In some embodiments, the fusion protein comprises component A and Fc. In further embodiments, the fusion protein comprises component A and human FcB (hFcB).

[0166] In some embodiments, the fusion protein comprises component B and Fc. In further embodiments, the fusion protein comprises component B and human FcA (hFcA).

[0167] In some embodiments, component A and component B are covalently linked. In some embodiments, the covalent link is via a disulfide bond or via a linker.

[0168] In some embodiments, component A and component B are not covalently linked. In some embodiments, component A and component B are held together by knobs-into-holes interactions. In some embodiments, component A and component B include knobs-into-hole mutations and are covalently linked by disulfide bonds. In some embodiments, component A includes mutations Y349C and T366W, and component B includes mutations D356C, T366S, L368A, and Y407V ("knobs-into-holes" mutations). The positions of mutations and alterations in these component chains are defined by the Kabat numbering convention (Johnson, G and Wu, TT, (2001) Nucleic Acids Res, 28(1), 214-18).

[0169] In some embodiments, the fusion protein comprises component A and a domain of an immunoglobulin. In some embodiments, the immunoglobulin domain is an Fc domain.

[0170] In some embodiments, the fusion protein comprises component B and a domain of an immunoglobulin. In some embodiments, the immunoglobulin domain is an Fc domain.

[0171] In some embodiments, component B and component C are covalently linked. In some embodiments, said covalent link is via a disulfide bond.

[0172] In some embodiments, component B and component C are not covalently linked.

[0173] In some embodiments, component A and component D are covalently linked. In some embodiments, the covalent link is through a disulfide bond.

[0174] In some embodiments, component A and component D are not covalently linked.

[0175] In some embodiments, the fusion protein binds to a receptor or ligand on an immune cell.

[0176] In some embodiments, the receptor is an Fc receptor.

[0177] Also provided is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the fusion protein of any one of the aforementioned embodiments.

[0178] Also provided is a method of treating a proliferative disease in a patient, comprising administering a therapeutically effective amount of a fusion protein of any one of the preceding embodiments to a patient in need of such treatment. In some embodiments, the proliferative disease is cancer. In further embodiments, the cancer is a solid tumor. In still further embodiments, the cancer is pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, melanoma, glioblastoma, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), multiple myeloma, colon cancer, lung cancer, liver cancer, or any solid or liquid tumor type.

[0179] The present invention provides novel fusion proteins that can be used to treat proliferative diseases, such as cancer. On cells expressing a receptor or ligand for component Y and a receptor or ligand for component X', the fusion protein of the present invention can block one or both of the receptors or ligands. Therefore, on cells co-expressing a receptor or ligand for component Y and a receptor or ligand for component X', the fusion protein of the present invention can cause death, fixation and clearance of tumor cells. In addition, on cells expressing a receptor or ligand for component Z3 or component Z3', for example, on a natural killer (NK) cell, the fusion protein of the present invention can trigger the receptor or ligand and can cause activation of the cell. Therefore, the fusion protein of the present invention can mediate its activity by spanning two adjacent cells. In addition, the fusion protein of the present invention can be combined with three or more different molecules on the cell. In some embodiments, the fusion protein can treat a disease, such as cancer, by causing the inhibition or reduction of certain cells, or the activation or increase of certain cells.

[0180] Component A:

[0181] Component A includes component Y, component Z2 and component Z3.

[0182] Component Y:

[0183] In some embodiments, component Y comprises a ligand domain, a receptor domain, a scFv domain, or a lipocalin domain. In further embodiments, component Y comprises at least a portion of PD-1, TIGIT, CD96, CD112R, CD113, CD155, CD111, CD112, MHC-I polypeptide-related sequence A (MICA), NKG2A (CD94), MICB, ULBP1-5, TIM-3, CD226, NECL2, CRTAM, CD80, CTLA-4, KIR2DL1 / 2 / 3, or CD48.

[0184] An exemplary sequence of component Y comprises or consists of:

[0185] GWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQ

[0186] (SEQ ID NO: 1) human PD-1 extracellular domain, Accession number NM_005018.3.

[0187] An exemplary sequence of component Y comprises or consists of:

[0188] GWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQ

[0189] (SEQ ID NO: 2) High affinity human PD-1 extracellular domain.

[0190] An exemplary sequence of component Y comprises or consists of:

[0191] MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP

[0192] (SEQ ID NO: 3) human TIGIT extracellular domain, Accession number NM_173799.4.

[0193] An exemplary sequence of component Y comprises or consists of:

[0194] VWEKTVNTEENVYATLGSDVNLTCQTQTVGFFVQMQWSKVTNKIDLIAVYHPQYGFYCAYGRPCESLVTFTETPENGSKWTLHLRNMSCSVSGRYECMLVLYPEGIQTKIYNLLIQTHVTADEW NSNHTIEIEINQTLEIPCFQNSSSKISSEFTYAWSVENSSTDSWVLLSKGIKEDNGTQETLISQNHLISNSTLLKDRVKLGTDYRLHLSPVQIFDDGRKFSCHIRVGPNKILRSSTTVKVFAKPE IPVIVENNSTDVLVERRFTCLLKNVFPKANITWFIDGSFLHDEKEGIYITNEERKGKDGFLELKSVLTRVHSNKPAQSDNLTIWCMALSPVPGNKVWNISSEKITFLLGSEISSTDPPLSVTES TLDTQPSPASSVSPARYPATSSVVTLVDVSALRPNTTPQPSNSSMTTRGFNYPWTSSGTDTKKSVSRIPSETYSSSSGAGSTLHDNVFTSTARAFSEVPTTANGSTKTNHVHITGIVVNKPKDGM

[0195] (SEQ ID NO: 4) human CD96 extracellular domain, Accession number NM_198196.2.

[0196] An exemplary sequence of component Y comprises or consists of:

[0197] MGHRTLVLPWVLLTLLCVTAGTPEVWVQVRMEATELSSFTIRCGFLGSGSISLVTVSWGGPNGAGGTTLAVLHPERGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEGSWEACGSLPPSSDPGLSAPPTPAPILRAD

[0198] (SEQ ID NO: 5) human CD112R extracellular domain, Accession number NM_024070.3.

[0199] An exemplary sequence of component Y comprises or consists of:

[0200] EEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQDSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQGTWQKVIQVVQSDSFEAAVPS NSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQYTLFVA

[0201] (SEQ ID NO: 6) CD226 extracellular domain, Accession number NM_006566.3.

[0202] An exemplary sequence of component Y comprises or consists of:

[0203] QNLFTKDVTVIEGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLKDSRFQLLNFSSSELKVSLTNVSISDEGRYFCQLYTDPPQESYTTITVLVPPRNLMIDIQKDTAVEGEEIEVNCTAMASKPATTIRWFKGNTELKGKSEVEEWSDMYTVTSQLMLKV HKEDDGVPVICQVEHPAVTGNLQTQRYLEVQYKPQVHIQMTYPLQGLTREGDALELTCEAIGKPQPVMVTWVRVDDEMPQHAVLSGPNLFINNLNKTDNGTYRCEASNIVGKAHSDYMLYVYDPPTTIPPPTTTTTTTTTTTTTILTIITDSRAGEEGSIRAVDH

[0204] (SEQ ID NO: 6) human NECL2 extracellular domain, Accession number NM_014333.3.

[0205] An exemplary sequence of component Y comprises or consists of:

[0206] PIIVEPHVTAVWGKNVSLKCLIEVNETITQISWEKIHGKSSQTVAVHHPQYGFSVQGEYQGRVLFKNYSLNDATITLHNIGFSDSGKYICKAVTFPLGNAQSSTTVTVLVEPTVSLIKGPDSLIDGGNETVAAICIAATGKPVAHIDWEGDLGEMESTTTSFPNETATIIS QYKLFPTRFARGRRITCVVKHPALEKDIRYSFILDIQYAPEVSVTGYDGNWFVGRKGVNLKCNADANPPPFKSVWSRLDGQWPDGLLASDNTLHFVHPLTFNYSGVYICKVTNSLGQRSDQKVIYISDPPTTTTLQPTIQWHPSTADIEDLATEPKKLPFPLSTLATIKDD

[0207] (SEQ ID NO: 8) human CD113 extracellular domain, Accession number NM_015480.3.

[0208] An exemplary sequence of component Y comprises or consists of:

[0209] AEPHSLRYNLTVLSWDGSVQSGFLTEVHLDGQPFLRCDRQKCRAKPQGQWAEDVLGNKTWDRETRDLTGNGKDLRMTLAHIKDQKEGLHSLQEIRVCEIHEDNSTRSSQHFYYDGELFLSQNLETKEWTMPQSSRAQTLAMN VRNFLKEDAMKTKTHYHAMHADCLQELRRYLKSGVVLRRTVPPMVNVTRSEASEGNITVTCRASGFYPWNITLSWRQDGVSLSHDTQQWGDVLPDGNGTYQTWVATRICQGEEQRFTCYMEHSGNHSTHPVPSGKVLVLQSHW

[0210] (SEQ ID NO: 9) human MICA (MHC-I polypeptide-related sequence A) extracellular domain, Accession number NM_000247.3.

[0211] Preamble sequence:

[0212] An exemplary sequence of a leader sequence includes or consists of:

[0213] MKWVTFISLLFLFSSAYS

[0214] (SEQ ID NO: 10) Human albumin leader sequence.

[0215] Hinge:

[0216] An exemplary sequence of hinges comprises or consists of:

[0217] EPKSSDKTHTCPPCPAPELLGG

[0218] (SEQ ID NO: 11) Human IgG hinge.

[0219] Component Z2:

[0220] An exemplary sequence of component Z2 comprises or consists of:

[0221] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAK

[0222] (SEQ ID NO: 12) IgG1.

[0223] Component Z3:

[0224] An exemplary sequence of component Z3 comprises or consists of:

[0225] GQPREPQVCTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0226] (SEQ ID NO: 13) IgG1.

[0227] An exemplary sequence of component A comprises or consists of:

[0228] Human PD-1-hFcA:

[0229]

[0230] (SEQ ID NO: 14)

[0231] Italic - human albumin leader sequence

[0232] -Human PD-1 extracellular domain; Y domain

[0233] Bold - IgG1 hinge region

[0234] -IgG1Z2 domain

[0235] -IgG1Z3 domain

[0236] Another exemplary sequence of component A comprises or consists of:

[0237] High-affinity human PD-1-hFcA:

[0238]

[0239]

[0240] (SEQ ID NO: 15)

[0241] Italic - human albumin leader sequence

[0242] -High affinity human PD-1 extracellular domain; Y domain

[0243] Bold - IgG1 hinge region

[0244] -IgG1Z2 domain

[0245] -IgG1Z3 domain

[0246] Another exemplary sequence of component A comprises or consists of:

[0247] Human CD112R-hFcA:

[0248]

[0249] (SEQ ID NO: 16)

[0250] Italic - human albumin leader sequence

[0251] -Human CD112R extracellular domain; Y domain

[0252] Bold - IgG1 hinge region

[0253] -IgG1Z2 domain

[0254] -IgG1Z3 domain

[0255] Another exemplary sequence of component A comprises or consists of:

[0256] Human TIGIT-hFcA:

[0257]

[0258] (SEQ ID NO: 17)

[0259] Italic - human albumin leader sequence

[0260] -High-affinity human TIGIT extracellular domain; Y domain

[0261] Bold - IgG1 hinge region

[0262] -IgG1Z2 domain

[0263] -IgG1Z3 domain

[0264] Another exemplary sequence of component A comprises or consists of:

[0265] Human CD96-hFcA:

[0266]

[0267]

[0268] (SEQ ID NO: 18)

[0269] Italic - human albumin leader sequence

[0270] -Human CD96 extracellular domain; Y domain

[0271] Bold - IgG1 hinge region

[0272] -IgG1Z2 domain

[0273] -IgG1Z3 domain

[0274] Another exemplary sequence of component A comprises or consists of:

[0275] Human CD226-hFcA:

[0276]

[0277]

[0278] (SEQ ID NO: 19)

[0279] Italic - human albumin leader sequence

[0280] -Human CD226 extracellular domain; Y domain

[0281] Bold - IgG1 hinge region

[0282] -IgG1Z2 domain

[0283] -IgG1Z3 domain

[0284] Another exemplary sequence of component A comprises or consists of:

[0285] Human NECL2-hFcA:

[0286]

[0287] (SEQ ID NO: 20)

[0288] Italic - human albumin leader sequence

[0289] -Human NECL2 extracellular domain; Y domain

[0290] Bold - IgG1 hinge region

[0291] -IgG1Z2 domain

[0292] -IgG1Z3 domain

[0293] Another exemplary sequence of component A comprises or consists of:

[0294] Human CD113-hFcA:

[0295]

[0296] (SEQ ID NO: 21)

[0297] Italic - human albumin leader sequence

[0298] -Human CD113 extracellular domain; Y domain

[0299] Bold - IgG1 hinge region

[0300] -IgG1Z2 domain

[0301] -IgG1Z3 domain

[0302] Another exemplary sequence of component A comprises or consists of:

[0303] Human MICA-hFcA (MHC-I polypeptide-related sequence A):

[0304]

[0305] (SEQ ID NO: 22)

[0306] Italic - human albumin leader sequence

[0307] -Human MICA extracellular domain; Y domain

[0308] Bold - IgG1 hinge region

[0309] -IgG1Z2 domain

[0310] -IgG1Z3 domain

[0311] Human FcA:

[0312]

[0313]

[0314] (SEQ ID NO: 23)

[0315] Italic - human albumin leader sequence

[0316] Bold - IgG1 hinge region

[0317] -IgG1Z2 domain

[0318] -IgG1Z3 domain

[0319] Component B:

[0320] Component B includes X', component Z2', and component Z3'. In some embodiments, component X' includes a viral-derived peptide, a ligand-derived peptide, a receptor-derived peptide, or an HTS-selected peptide. In some embodiments, component X' includes at least a portion of PD-1, TIGIT, CD96, CD112R, CD113, CD155, CD111, CD112, MHC-I polypeptide-related sequence A (MICA), NKG2A (CD94), MICB, ULBP1-5, TIM-3, CD226, NECL2, CRTAM, CD80, CTLA-4, KIR2DL1 / 2 / 3, or CD48.

[0321] Component X':

[0322] An exemplary sequence of component X' comprises or consists of:

[0323] WPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNMEVTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLRVEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGRPPAQITWHS DLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKPQLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPFAVAQGAQLLIRPVDKPINTTLICNVTNALGARQAELTVQVKEGPPSEHSGMSRN

[0324] (SEQ ID NO: 24) human CD155 (Polio virus receptor, PVR) extracellular domain, Accession number NM_006505.5.

[0325] An exemplary sequence of component X' comprises or consists of:

[0326] MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIP

[0327] (SEQ ID NO: 25) human TIGIT (T cell immunoreceptor with Ig and ITIM domains), Accession number NM_173799.4.

[0328] An exemplary sequence of component X' comprises or consists of:

[0329] LEDGYKVEVGKNAYLPCSYTLPTSGTLVPMCWGKGFCPWSQCTNELLRTDERNVTYQKSSRYQLKGDLNKGDVSLIIKNVTLDDHGTYCCRIQFPGLMNDKKLELKLDIKAAKVTPAQTAHGDSTTASPRTLTTERNGSETQTLVTLHNNNGTKISTWADEIKDSGETIR

[0330] (SEQ ID NO: 26) mouse Tim-3 extracellular domain, Accession number NM_134250.2. Mouse TIM-3 binds well to human galactin-9.

[0331] An exemplary sequence of component X' comprises or consists of:

[0332] PIIVEPHVTAVWGKNVSLKCLIEVNETITQISWEKIHGKSSQTVAVHHPQYGFSVQGEYQGRVLFKNYSLNDATITLHNIGFSDSGKYICKAVTFPLGNAQSSTTVTVLVEPTVSLIKGPDSLIDGGNETVAAICIAATGKPVAHIDWEGDLGEMESTTTSFPNETATIIS QYKLFPTRFARGRRITCVVKHPALEKDIRYSFILDIQYAPEVSVTGYDGNWFVGRKGVNLKCNADANPPPFKSVWSRLDGQWPDGLLASDNTLHFVHPLTFNYSGVYICKVTNSLGQRSDQKVIYISDPPTTTTLQPTIQWHPSTADIEDLATEPKKLPFPLSTLATIKDD

[0333] (SEQ ID NO: 27) human CD113 extracellular domain, Accession number NM_015480.3.

[0334] Preamble sequence:

[0335] An exemplary sequence of a leader sequence includes or consists of:

[0336] MKWVTFISLLFLFSSAYS

[0337] (SEQ ID NO: 10) Human albumin leader sequence.

[0338] Hinge:

[0339] An exemplary sequence of hinges comprises or consists of:

[0340] EPKSSDKTHTCPPCPAPELLGG

[0341] (SEQ ID NO: 11) Human IgG hinge.

[0342] Component Z2:

[0343] An exemplary sequence of component Z2 comprises or consists of:

[0344] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAK

[0345] (SEQ ID NO: 12) IgG1.

[0346] Component Z3:

[0347] An exemplary sequence of component Z3 comprises or consists of:

[0348] GQPREPQVCTLPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0349] (SEQ ID NO: 13) IgG1.

[0350] An exemplary sequence of component B comprises or consists of:

[0351] Human CD155-hFcB:

[0352]

[0353] (SEQ ID NO: 32)

[0354] Italic - human albumin leader sequence

[0355] -Human CD155 (Polio virus receptor, PVR) extracellular domain; X' domain

[0356] Bold - IgG1 hinge region

[0357] -IgG1Z'2 domain

[0358] -IgG1Z'3 domain

[0359] An exemplary sequence of component B comprises or consists of:

[0360] Human TIGIT-hFcB:

[0361]

[0362] (SEQ ID NO: 33)

[0363] Italic - human albumin leader sequence

[0364] - Human TIGIT (T cell immunoreceptor with Ig and ITIM domains) extracellular domain; X' domain

[0365] Bold - IgG1 hinge region

[0366] -IgG1Z'2 domain

[0367] -IgG1Z'3 domain

[0368] An exemplary sequence of component B comprises or consists of:

[0369] Mouse TIM-3-hFcB:

[0370]

[0371]

[0372] (SEQ ID NO: 34)

[0373] Italic - human albumin leader sequence

[0374] - Mouse TIM-3 extracellular domain; X' domain

[0375] Bold - IgG1 hinge region

[0376] -IgG1Z'2 domain

[0377] -IgG1Z'3 domain

[0378] Another exemplary sequence of component B comprises or consists of:

[0379] Human CD113-hFcB:

[0380]

[0381] (SEQ ID NO: 35)

[0382] Italic - human albumin leader sequence

[0383] -Human CD113 extracellular domain; X' domain

[0384] Bold - IgG1 hinge region

[0385] -IgG1Z'2 domain

[0386] -IgG1Z'3 domain

[0387] Human FcB:

[0388]

[0389] (SEQ ID NO: 36)

[0390] Italic - human albumin leader sequence

[0391] Bold - IgG1 hinge region

[0392] -IgG1Z'2 domain

[0393] -IgG1Z'3 domain

[0394] Component B includes component Z1':

[0395] In some embodiments, component B further comprises component Z1 '. Thus, in some embodiments, component B comprises component X', component Z1 ', component Z2', and component Z3'.

[0396] In some embodiments, component X' comprises a viral-derived peptide, a ligand-derived peptide, a receptor-derived peptide, or an HTS-selected peptide. In some embodiments, component X' comprises at least a portion of PD-1, TIGIT, CD96, CD112R, CD113, CD155, CD111, CD112, MHC-I polypeptide-related sequence A (MICA), NKG2A (CD94), MICB, ULBP1-5, TIM-3, CD226, NECL2, CRTAM, CD80, CTLA-4, KIR2DL1 / 2 / 3, or CD48. In further embodiments, component X' comprises at least a portion of CD155, TIGIT, TIM-3, or CD113.

[0397] Component X':

[0398] An exemplary sequence of component X' comprises or consists of:

[0399] LGASWHRPDKCCLGYQKRPLPQVLLSSWYPTSQLCSKPGVIFLTKRGRQVCADKSKDWVKKLMQQLPVTAR

[0400] (SEQ ID NO: 37) vMIPII, Accession number YP_001129362.

[0401] An exemplary sequence of component X' comprises or consists of:

[0402] LGASWHRPDKCCLGYQKRPLP

[0403] (SEQ ID NO: 38) V1

[0404] An exemplary sequence of component X' comprises or consists of:

[0405] LGASWHRPDKCALGYQKRPLP

[0406] (SEQ ID NO: 39) V1Δ

[0407] An exemplary sequence of component X' comprises or consists of:

[0408] LGASWHRPDACALGYQKRPLP

[0409] (SEQ ID NO: 40) V1Δmut

[0410] An exemplary sequence of component X' comprises or consists of:

[0411] LGASWHRPDKCCLGYQKRPLPQVLLSSWYPTSQL

[0412] (SEQ ID NO: 41) Vp1

[0413] An exemplary sequence of component X' comprises or consists of:

[0414] LGASWHRPDKCALGYQKRPLPQVLLSSWYPTSQL

[0415] (SEQ ID NO: 42) Vp1Δ

[0416] An exemplary sequence of component X' comprises or consists of:

[0417] LGASHRPDACALGYQKRPLPQVLLSSWYPTSQL

[0418] (SEQ ID NO: 43) Vp1Δmut

[0419] Preamble sequence:

[0420] An exemplary sequence of a leader sequence includes or consists of:

[0421] MKWVTFISLLFLFSSAYS

[0422] (SEQ ID NO: 10) Human albumin leader sequence.

[0423] Component Z1'

[0424] An exemplary sequence of component Z1' comprises or consists of:

[0425] SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVX

[0426] (SEQ ID NO: 45)

[0427] Hinge:

[0428] An exemplary sequence of hinges comprises or consists of:

[0429] EPKSSDKTHTCPPCPAPELLGG

[0430] (SEQ ID NO: 11) Human IgG hinge.

[0431] Component Z2'

[0432] An exemplary sequence of component Z2' comprises or consists of:

[0433] PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAK

[0434] (SEQ ID NO: 12)

[0435] Component Z3'

[0436] An exemplary sequence of component Z3' comprises or consists of:

[0437] GQPREPQVYTLPPSRCELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK

[0438] (SEQ ID NO: 48)

[0439] An exemplary sequence of component B comprises or consists of:

[0440] vMIPII-CH'-hFcB:

[0441]

[0442]

[0443] (SEQ ID NO: 49)

[0444] Italic - human albumin leader sequence

[0445] -vMIPII

[0446] -Amino acid differences between V1, V1Δ and V1Δmut

[0447] -CH'

[0448] Bold - IgG1 hinge region

[0449] -IgG1Z'2 domain

[0450] -IgG1Z'3 domain

[0451] An exemplary sequence of component B comprises or consists of:

[0452] V1-CH'-hFcB:

[0453]

[0454] (SEQ ID NO: 50)

[0455] Italic - human albumin leader sequence

[0456] -V1 (V1 is the first 21 amino acids of VMIPII)

[0457] -Amino acid differences between V1, V1Δ and V1Δmut

[0458] -CH'

[0459] Bold - IgG1 hinge region

[0460] -IgG1Z'2 domain

[0461] -IgG1Z'3 domain

[0462] An exemplary sequence of component B comprises or consists of:

[0463] V1Δ-CH'-hFcB:

[0464]

[0465] (SEQ ID NO: 51)

[0466] Italic - human albumin leader sequence

[0467] -V1Δ (V1Δ is a C to A mutation at amino acid 11 of V1, which increases the dimerization of two V1 peptides)

[0468] -Amino acid differences between V1, V1Δ and V1Δmut

[0469] -CH'

[0470] Bold - IgG1 hinge region

[0471] -IgG1Z'2 domain

[0472] -IgG1Z'3 domain

[0473] An exemplary sequence of component B comprises or consists of:

[0474] V1Δmut-CH'-hFcB

[0475]

[0476] (SEQ ID NO: 52)

[0477] Italic - human albumin leader sequence

[0478] -V1Δmut (V1Δmut has the same C to A mutation at amino acid 11 as V1, but adds a K to A mutation at amino acid 9 to prevent receptor binding)

[0479] -Amino acid differences between V1, V1Δ and V1Δmut

[0480] -CH'

[0481] Bold - IgG1 hinge region

[0482] -IgG1Z'2 domain

[0483] -IgG1Z'3 domain

[0484] An exemplary sequence of component B comprises or consists of:

[0485] Vp1-CH'-hFcB:

[0486]

[0487]

[0488] (SEQ ID NO: 53)

[0489] Italic - human albumin leader sequence

[0490] -Vp1 (Vp1 is the first 34 amino acids of vMIPII)

[0491] -Amino acid differences between V1, V1Δ and V1Δmut

[0492] -CH'

[0493] Bold - IgG1 hinge region

[0494] -IgG1Z'2 domain

[0495] -IgG1Z'3 domain

[0496] An exemplary sequence of component B comprises or consists of:

[0497] Vp1Δ-CH'-hFcB:

[0498]

[0499] (SEQ ID NO: 54)

[0500] Italic - human albumin leader sequence

[0501] -Vp1Δ

[0502] -Amino acid differences between V1, V1Δ and V1Δmut

[0503] -CH'

[0504] Bold - IgG1 hinge region

[0505] -IgG1Z'2 domain

[0506] -IgG1Z'3 domain

[0507] Vp1Δmut-CH'-hFcB:

[0508]

[0509] (SEQ ID NO: 55)

[0510] Italic - human albumin leader sequence

[0511] -Vp1Δmut

[0512] -Amino acid differences between V1, V1Δ and V1Δmut

[0513] -CH'

[0514] Bold - IgG1 hinge region

[0515] -IgG1Z'2 domain

[0516] -IgG1Z'3 domain

[0517] Component C

[0518] In some embodiments, the fusion protein further comprises component C, wherein component C comprises component X and component C. L '.

[0519] Component X:

[0520] An exemplary sequence of component X comprises or consists of:

[0521] LGASWHRPDKCCLGYQKRPLPQVLLSSWYPTSQLCSKPGVIFLTKRGRQVCADKSKDWVKKLMQQLPVTAR

[0522] (SEQ ID NO: 37) vMIPII, Accession number YP_001129362.

[0523] An exemplary sequence of component X comprises or consists of:

[0524] LGASWHRPDKCCLGYQKRPLP

[0525] (SEQ ID NO: 38) V1

[0526] An exemplary sequence of component X comprises or consists of:

[0527] LGASWHRPDKCALGYQKRPLP

[0528] (SEQ ID NO: 39) V1Δ

[0529] An exemplary sequence of component X comprises or consists of:

[0530] LGASWHRPDACALGYQKRPLP

[0531] (SEQ ID NO: 40) V1Δmut

[0532] An exemplary sequence of component X comprises or consists of:

[0533] LGASWHRPDKCCLGYQKRPLPQVLLSSWYPTSQL

[0534] (SEQ ID NO: 41) Vp1

[0535] An exemplary sequence of component X comprises or consists of:

[0536] LGASWHRPDKCALGYQKRPLPQVLLSSWYPTSQL

[0537] (SEQ ID NO: 42) Vp1Δ

[0538] An exemplary sequence of component X comprises or consists of:

[0539] LGASHRPDACALGYQKRPLPQVLLSSWYPTSQL

[0540] (SEQ ID NO: 43) Vp1Δmut

[0541] In some embodiments, component X and component X' are the same. In further embodiments, component X and component X' are different.

[0542] Preamble sequence:

[0543] An exemplary sequence of a leader sequence includes or consists of:

[0544] MKWVTFISLLFLFSSAYS

[0545] (SEQ ID NO: 10) Human albumin leader sequence.

[0546] Component C L ':

[0547] Component C L An exemplary sequence of ' comprises or consists of:

[0548] KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0549] (SEQ ID NO: 64)

[0550] An exemplary sequence of component C comprises or consists of:

[0551] vMIPII-CL':

[0552]

[0553]

[0554] (SEQ ID NO: 57)

[0555] Italic - human albumin leader sequence

[0556] -vMIPII

[0557] -Amino acid differences between V1, V1Δ and V1Δmut

[0558] -CL'

[0559] An exemplary sequence of component C comprises or consists of:

[0560] V1-CL':

[0561]

[0562] (SEQ ID NO: 58)

[0563] Italic - human albumin leader sequence

[0564] -V1

[0565] -Amino acid differences between V1, V1Δ and V1Δmut

[0566] -CL'

[0567] An exemplary sequence of component C comprises or consists of:

[0568] V1Δ-CL':

[0569]

[0570]

[0571] (SEQ ID NO: 59)

[0572] Italic - human albumin leader sequence

[0573] -V1Δ

[0574] -Amino acid differences between V1, V1Δ and V1Δmut

[0575] -CL'

[0576] An exemplary sequence of component C comprises or consists of:

[0577] V1Δmut-CL':

[0578]

[0579] (SEQ ID NO: 60)

[0580] Italic - human albumin leader sequence

[0581] -V1Δmut

[0582] -Amino acid differences between V1, V1Δ and V1Δmut

[0583] -CL'

[0584] An exemplary sequence of component C comprises or consists of:

[0585] Vp1-CL':

[0586]

[0587]

[0588] (SEQ ID NO: 61)

[0589] Italic - human albumin leader sequence

[0590] -Vp1

[0591] -Amino acid differences between V1, V1Δ and V1Δmut

[0592] -CL'

[0593] An exemplary sequence of component C comprises or consists of:

[0594] Vp1Δ-CL':

[0595]

[0596] (SEQ ID NO: 62)

[0597] Italic - human albumin leader sequence

[0598] -Vp1Δ

[0599] -Amino acid differences between V1, V1Δ and V1Δmut

[0600] -CL'

[0601] An exemplary sequence of component C comprises or consists of:

[0602] Vp1Δmut-CL':

[0603]

[0604]

[0605] (SEQ ID NO: 63)

[0606] Italic - human albumin leader sequence

[0607] -Vp1Δmut

[0608] -Amino acid differences between V1, V1Δ and V1Δmut

[0609] -CL'

[0610] Component D

[0611] In some embodiments, the fusion protein further comprises component D, wherein component D comprises component Q and component C. L In some embodiments, component Q includes at least a portion of PD-1, TIGIT, CD96, CD112R, CD113, CD155, CD111, CD112, MHC-I polypeptide-related sequence A (MICA), NKG2A (CD94), MICB, ULBP1-5, TIM-3, CD226, NECL2, CRTAM, CD80, CTLA-4, KIR2DL1 / 2 / 3, or CD48.

[0612] An exemplary sequence of component Q comprises or consists of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, component Q and component Y are the same. In further embodiments, component Q and component Y are different.

[0613] Component C L :

[0614] Component C L An exemplary sequence of comprises or consists of:

[0615] KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0616] (SEQ ID NO: 64)

[0617] Configuration of fusion protein:

[0618] In some embodiments of the fusion proteins of the present invention, when prepared by recombinant methods described elsewhere herein, the coding sequences of the components of component A are fused together in frame, directly or through a linker. As used herein, the term "directly" refers to the fusion of two components without a peptide linker between them (i.e., in an expression construct, the codons encoding component Y, component Z2, and component Z3 are consecutive). As used herein, "in-frame fusion" refers to the expression of the fused coding sequences to produce a fusion protein that includes all of the polypeptide components, for example, in some embodiments, component A includes all of the polypeptide components of component Y, component Z2, and component Z3 in frame.

[0619] In some embodiments of fusion protein of the present invention, when prepared by the recombinant method described elsewhere herein, the coding sequence of the component of component B is fused together directly or through a linker frame. In some embodiments of the expression construct of component B, the codons encoding component X', component Z2 and component Z3' are continuous. In a further embodiment, in the expression construct of component B, the codons encoding component X', component Z1', component Z2 and component Z3' are continuous. In certain embodiments, component B includes all polypeptide components of component X', component Z2 and component Z3' in frame. In a further embodiment, component B includes all polypeptide components of component X', component Z1', component Z2' and component Z3' in frame.

[0620] In some embodiments of the fusion proteins of the present invention, when produced by recombinant methods described elsewhere herein, the coding sequences of the components of component C are fused together in frame, directly or through a linker. In some embodiments of the expression construct of component C, the coding sequences of components X and C are L In some embodiments, component C includes component X and component C in the framework. L 'All polypeptide components.

[0621] In some embodiments, any component A and any component B can be mixed and matched with each other. In some exemplary embodiments of the fusion protein of the present invention, component A and component B are as shown in Table 1. In some embodiments, component A and component B from Table 1 can be mixed and matched with each other and selected with additional component A and component B. The additional options can also be mixed and matched with each other. Additional selections of component A or component B may include at least a portion of PD-1, TIGIT, CD96, CD112R, CD113, CD155, CD111, CD112, MHC-I polypeptide-associated sequence A (MICA), NKG2A (CD94), MICB, ULBP1-5, TIM-3, CD226, NECL2, CRTAM, CD80, CTLA-4, KIR2DL1 / 2 / 3, or CD48.

[0622] In some embodiments, the fusion protein comprises component A and hFcB. In some embodiments, the fusion protein comprises component B and hFcA.

[0623] In some exemplary embodiments, one component of the fusion protein blocks an inhibitory receptor, while the other two components of the fusion protein each trigger a different activating receptor. In a preferred embodiment, these three receptors are co-located on the surface of the same immune effector cell, such as a NK cell, and the three interactions of the fusion protein, including a combination of inhibitory receptor blocking and activating receptor triggering, are used to reinforce each other, and all three synergistically drive NK cell activation.

[0624] In other exemplary embodiments, two components of the fusion protein each block a different inhibitory receptor, and another component of the fusion protein triggers an activating receptor. In a preferred embodiment, these three receptors are co-located on the surface of the same immune effector cell, such as a NK cell, and the three interactions of the fusion protein, including a combination of inhibitory receptor blocking and activating receptor triggering, are used to reinforce each other, and all three synergistically drive NK cell activation.

[0625] In other exemplary embodiments, the three components of the fusion protein each trigger an activating receptor. In a preferred embodiment, these three receptors are co-located on the surface of the same immune effector cell, such as a NK cell, and the three interactions of the fusion protein are all triggered by activating receptors, thereby enhancing each other's functions, and all three components synergistically drive NK cell activation.

[0626] For NK cells, the activating receptor in the above embodiment may be the FcγRIIIa receptor that drives NK cell activation and promotes antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).

[0627] Table 1 - Exemplary Components A and B

[0628]

[0629]

[0630] In some exemplary embodiments, the fusion protein includes component B and component C. In some embodiments, any component B and any component C can be mixed and matched with each other. In some embodiments, component B and component C are as shown in Table 2. In some embodiments, each row of Table 2 shows the pairing of component B and component C in a fusion protein.

[0631] Table 2

[0632] Component B Component C vMIPII-CH'-hFcB (SEQ ID NO: 49) vMIPII-CL' (SEQ ID NO: 57) V1-CH'-hFcB (SEQ ID NO: 50) V1-CL' (SEQ ID NO: 58) V1Δ-CH'-hFcB (SEQ ID NO: 51) V1Δ-CL' (SEQ ID NO: 59) V1Δmut-CH'-hFcB (SEQ ID NO: 52) V1Δmut-CL' (SEQ ID NO: 60) Vp1-CH'-hFcB (SEQ ID NO: 53) Vp1-CL' (SEQ ID NO: 61) Vp1Δ-CH'-hFcB (SEQ ID NO: 54) Vp1Δ-CL′ (SEQ ID NO: 62) Vp1Δmut-CH'-hFcB (SEQ ID NO: 55) Vp1Δmut-CL' (SEQ ID NO: 63)

[0633] In some embodiments, component A and component B are connected by disulfide-stabilized knob-into-hole interactions (KiH S-S ) are combined together. In some embodiments, component A includes mutations Y349C and T366W (e.g., SEQ ID NO: 29), and component B includes mutations D356C, T366S, L368A, and Y407V (e.g., SEQ ID NO: 30) ("knobs-into-holes" mutations), achieving forced dimerization of components A and B. Components Z1, Z2, and Z3 are based on the polypeptide amino acid backbones of the CH1, CH2, and CH3 domains of human IgG1, respectively. The mutations and altered positions within these component chains are defined by the Kabat numbering convention (Johnson, G, and Wu, TT, (2001) Nucleic Acids Res, 28(1), 214-18) and are based on the wild-type human IgG1 sequence.

[0634] Wild-type human IgG1 (CH1, CH2, and CH3 domains) (starting from amino acid number 118):

[0635]

[0636] (SEQ ID NO: 28)

[0637] -IgG1Z1 domain

[0638] Bold - IgG1 hinge region

[0639] -IgG1Z2 domain

[0640] -IgG1Z3 domain

[0641] In some embodiments, component A comprises or consists of the following sequence (Y349C and T366W):

[0642]

[0643]

[0644] (SEQ ID NO: 29)

[0645] - IgG1Z1 domain (SEQ ID NO: 67) (see, e.g., Figure 1a)

[0646] Bold - IgG1 hinge region (SEQ ID NO: 68)

[0647] S* - Compared to the wild type, this mutation is added to the hinge region to eliminate an unpaired cysteine ​​(C) that could potentially bind abnormally to another cysteine.

[0648] -IgG1Z2 domain (SEQ ID NO: 69)

[0649] - Add mutations to reduce C1q binding (see e.g. Figure 1a)

[0650] -IgG1 Z3 domain (SEQ ID NO: 44)

[0651] - Add mutations to increase FcRn binding (see e.g. Figure 1a)

[0652] In some embodiments, component B comprises or consists of the following sequence (D356C, T366S, L368A, and Y407V):

[0653]

[0654] (SEQ ID NO: 30)

[0655] -IgG1Z1' domain

[0656] The components shown in Tables 1 and 2 are intended to be exemplary and not limiting.

[0657] In some exemplary embodiments, one component of the fusion protein blocks a receptor on a tumor cell that contributes to the tumorigenicity and / or metastatic potential of the tumor cell; a second component of the fusion protein blocks a checkpoint inhibitor on the tumor cell; and a third component of the fusion protein triggers an activating receptor on an immune effector cell. Without wishing to be bound by theory, the fusion protein is used to molecularly bridge an immune effector cell and a target tumor cell, and the second and third components of the fusion protein mutually reinforce each other by blocking and triggering a combination of a checkpoint inhibitor on an immune effector cell, for example, an activating receptor on a NK cell, and together synergistically drive activation of the immune effector cell.

[0658] In a preferred embodiment, the first component of the fusion protein blocks chemokine receptors, such as CXCR4 and / or CXCR7 (through the binding of the vMIPII peptide or V1 peptide or its derivatives in the fusion protein), and this blockade is used to immobilize tumor cells and interfere with their migration, invasion, metastasis and other tumorigenic properties; the second component of the fusion protein blocks a checkpoint inhibitor on the tumor cells, such as PD-L1 and / or PD-L2 (through the binding of PD1 or its derivatives in the fusion protein), and this blockade is used to interfere with the inhibition of a tumor-directed immune effector cell, such as NK cell; the third component of the fusion protein triggers an activating receptor on the same immune effector cell, such as Fc γ RIIIa receptor (via Fc γ or its derivatives in a fusion protein), which drives NK cell activation and promotes ADCC and ADCP. An advantageous feature of the embodiment is that the same fusion protein also synergistically regulates other immune cells besides NK cells that have a net anti-tumor effect (see Figure 5 ).

[0659] In some exemplary embodiments, one component of the fusion protein blocks a checkpoint inhibitor on a tumor or other cell, while the other two components each trigger a different activating receptor on an immune effector cell. When the checkpoint inhibitor is located on a tumor cell, the fusion protein actually acts as a molecule bridging the gap between an immune effector cell and a target tumor cell. In addition, the three interactions of the fusion protein, namely the combination of checkpoint inhibition pathway blocking and activation receptor triggering, functionally enhance each other, and all three interactions together drive the activation of the immune effector cell, such as a NK cell. In a preferred embodiment, the checkpoint inhibitor blocked by the fusion protein is composed of PD-L1, PD-L2, CD113, CD112, CD155 or CD111, and the two activating receptors jointly triggered by the fusion protein are Fc on NK cells. γ RIIIa receptor and 4-1BB (see Figure 6 ).

[0660] In some exemplary embodiments, one component of the fusion protein blocks a checkpoint inhibitor on a tumor or other cell; a second component of the fusion protein blocks a co-inhibitory receptor of the same or different checkpoint inhibitor on an immune effector cell; and a third component of the fusion protein triggers an activating receptor on an immune effector cell. When the checkpoint inhibitor is located on a tumor cell, the fusion protein actually acts as a molecule bridging the role of the immune effector cell and a target tumor cell, and the three interactions of the fusion protein, that is, the combination of checkpoint inhibition pathway blocking and activation receptor triggering, are used to functionally enhance each other, and all three synergistically drive the activation of the immune effector cell, for example, NK cell. In a preferred embodiment, the checkpoint inhibitor blocked by the first component of the fusion protein is composed of PD-L1, PD-L2, CD113, CD112, CD155 or CD111; the co-inhibitory receptor on the NK cell blocked by the fusion protein is composed of PD-1, TIGIT, CD96 or CD112R; and the activating receptor triggered by the fusion protein is Fc γ RIIIa receptor, the same as that on NK cells (see Figure 7 ).

[0661] In some exemplary embodiments, one component of the fusion protein blocks a "don't eat me" inhibitory receptor on a tumor-associated M1 macrophage, thereby releasing its anti-tumor phagocytic and other activities; the other two components of the fusion protein each trigger a different activating receptor on the tumor-associated macrophage. The three interactions of the fusion protein, namely the combination of blocking a macrophage inhibitory pathway and triggering a separate activating receptor on the macrophage, are used to functionally enhance each other. These three interactions together drive the activation of tumor-associated macrophages and promote their anti-tumor function. In a preferred embodiment, the "don't eat me" receptor blocked by the first component of the fusion protein is SIRPα; the activating receptors triggered by the fusion protein are CD40 and Fc on the same macrophage. γ RIIIa receptors (see Figure 8 , left picture).

[0662] A "don't eat me" inhibitory receptor on a tumor-associated M1 macrophage, thereby releasing its anti-tumor phagocytic and other activities; a second component of the fusion protein blocks a unique inhibitory receptor on the macrophage; a third component of the fusion protein triggers an activating receptor on the tumor-associated macrophage. The three interactions of the fusion protein, combined with blocking two macrophage inhibitory pathways and triggering activating receptors on macrophages, are used to functionally enhance each other, and all three interactions together drive the activation and / or anti-tumor effector function of tumor-associated macrophages. In a preferred embodiment, the "don't eat me" receptor blocked by the first component of the fusion protein is SIRPα; the inhibitory receptor blocked by the second component of the fusion protein is PD-1; the activating receptor triggered by the fusion protein is Fc γ RIIIa receptors (see Figure 8 , right).

[0663] Preferred embodiments of the fusion proteins of the present invention include a cytokine or a portion or derivative thereof, which can be incorporated into component A, component B, component C, and / or component D. These include cytokines widely known to those skilled in the art, which are classified into many different categories, such as interleukins, tumor necrosis factors, interferons, colony-stimulating factors, etc.; have been assigned various functions, and have a range of activation or inhibition properties, such as adaptive immunity, pro-inflammatory signaling, anti-inflammatory signaling, stem cell regulation and differentiation, chemotaxis, phagocytosis, cytotoxicity, and antiviral effects; and are associated with a range of immune and non-immune cell targets, such as B cells, T cells, NK cells, macrophages / monocytes, dendritic cells, bone marrow stromal cells, stem cells, fibroblasts, endothelial cells, and epithelial cells. Preferred embodiments include cytokines associated with adaptive immunity (e.g., IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, GM-CSF); pro-inflammatory signaling [e.g., IL-1 family (IL-1, IL-18, IL-33, IL-36); IL-6 family (IL-6, IL-11, IL-31, CNTF, CT-1, LIF, OPN, OSM); TNFα family (TNFα, TNFβ, BAFF, APRIL); IL-17 family (IL-17A-F, IL-2 5); type I IFN family (IFNα, IFNβ, IFNκ, limitin); type II IFN family (IFNγ); type III IFN family (IFNλ1 / IL-29), IFNλ2 / IL-28A, IFNλ3 / IL-28B)]; and anti-inflammatory signaling [IL-12 family (IL-12, IL-23, IL-27, IL-35); and IL-10 family (IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, IL-28, IL-29)]. See Turner, Mark D. et al.: "Cytokines and chemokines: at the crossroads of cell signaling and inflammatory diseases," Biochimica et Biophysica Acta 1843 (2014) 2563-2582.

[0664] vMIP-II:

[0665] In some embodiments, components of the fusion proteins of the present invention may include vMIP-II or a variant thereof. Viral macrophage inflammatory protein-II (vMIP-II) is a chemokine that interacts with CC and CXC chemokine receptors, including CCR5 and CXCR4. CCR5 and CXCR4 are the primary coreceptors required for human immunodeficiency virus 1 (HIV-1) entry into cells. CXCR4 is also found on cancer cells, such as tumor cells. vMIP-II, a chemokine encoded by human herpesvirus 8 (HHV-8) (Moore, P.S. et al., Science, 274:1739-1744, 1996), exhibits multiple interactions with CC and CXC chemokine receptors and inhibits HIV-1 entry mediated by CCR3, CCR5, and CXCR4. See U.S. Patent Publication No. 2003 / 0220482, the entire contents of which are incorporated herein by reference. vMIP-II also binds to CXCR7, which, like CXCR4, is implicated in tumorigenesis. V1 (amino acids (aa) 1-21 of vMIP-II) and its related DV1 (D-amino acid isomer) exhibit antagonistic activity against CXCR4 and CXCR7, but not CCR5.

[0666] vMIP inhibits the binding of the natural ligand CXCL12. In some embodiments, Δ represents a mutation at amino acid 12, which results in dimerization between the two peptides of component B and component C. In some embodiments, Δmut represents a mutation at amino acid 10, which prevents peptide binding and serves as a negative control.

[0667] PD-1:

[0668] In some embodiments, the components of the fusion protein of the present invention may include PD-1 or a variant thereof. PD-1 (programmed cell death protein 1), also known as CD279, is a protein located on the cell surface that plays a role in regulating the immune system's response to cells. PD-1 downregulates the immune system by inhibiting T cell inflammatory activity and promotes self-tolerance. Without wishing to be bound by theory, PD-1 plays the role of an immune checkpoint through at least two mechanisms. PD-1 promotes apoptosis of antigen-specific T cells in lymph nodes. PD-1 can also reduce apoptosis of regulatory T cells. PD-1 binds to ligands PD-L1 and PD-L2, which are members of the B7 family. PD-L1 and PD-L2 are expressed on the surface of some tumor cells. PD-L1 expressed on tumor cells binds to PD-1 on effector T cells and NK cells and inhibits their function. Therefore, PD-L1 expressed on tumor cells inhibits the anti-tumor activity of effector T cells.

[0669] In some embodiments, the PD-1 variant of the fusion protein of the present invention is a high-affinity PD-1. Native PD-1 has a relatively low affinity for its ligands PD-L1 and PD-L2. Higher-affinity variants of the PD-1 extracellular domain will act as stronger competitive antagonists of its ligands. The PD-1 contact residues between human PD-1 and PD-L1 are mutated and then evaluated for binding. The high-affinity PD-1 described herein has 10 mutated amino acids, resulting in an affinity enhancement of more than 10,000-fold for PD-L1. For more detailed information, see Mauth et al., PNAS, 112(47): 6506-6514, 2015.

[0670] CD112R:

[0671] In some embodiments, the components of the fusion protein of the present invention may include CD112R or its variants. CD112R is expressed on T cells and NK cells and inhibits activation reactions. CD112 is widely expressed on antigen-presenting cells and tumor cells and is a ligand for CD112R. CD112R competes with CD226, a co-inhibitory receptor, for binding to CD112. Without wishing to be bound by theory, disrupting the CD112R-CD112 interaction can increase T cell responses. Human CD112R comprises a single extracellular IgV domain. The CD112R fusion protein variants described herein are composed of the entire extracellular domain of human CD112R connected to the hinge, CH2 and CH3 domains of human IgG1. CD112R and its variants can bind to its ligand CD112 and act as a competitive antagonist of natural CD112R on NK cells and T cells, thereby preventing inhibitory signal transduction.

[0672] CD113:

[0673] In some embodiments, the components of the fusion protein of the present invention may include CD113 or a variant thereof. CD113, also known as poliovirus receptor-related 3 (PVRL3) or nectin-3, is a member of the immunoglobulin superfamily and constitutes part of the adhesion junction. CD113 has been shown to interact with MLLT4, PARD3 and PTPRM, but is not limited to this. In addition, CD113 binds to TIGIT, CD111, CD112, CD155 and itself. The CD113 fusion protein variant described herein is composed of the entire extracellular domain of natural human CD113 connected to the hinge, CH2 and CH3 domains of human IgG1. Fusion proteins containing CD113 can act as competitive antagonists of natural TIGIT on NK cells and T cells to prevent inhibitory signal transduction. Alternatively, CD113 fusion proteins can bind to CD112, CD155, and / or CD111 and prevent their binding to inhibitory receptors CD112R, TIGIT, and CD96, respectively, thereby restoring NK cell and T cell cytotoxicity and cytokine production.

[0674] MICA:

[0675] In some embodiments, components of the fusion proteins of the present invention may include MHC class I polypeptide-related sequence A (MICA) or variants thereof. MICA is a cell surface glycoprotein encoded by the MICA gene located within the MHC locus. MICA does not associate with β2-microglobulin, nor does it bind peptides like traditional MHC class I molecules. Without wishing to be bound by theory, MICA may function as a stress-induced ligand for the NKG2D receptor. MICA is widely recognized by NK cells, γΔT cells, and CD8+ αβT cells that express NKG2D on their cell surfaces. Effector cytolytic responses by T cells and NK cells against MICA-expressing tumor cells are initiated by NKG2D-MICA binding. In some embodiments, MICA variants consist of the entire MICA extracellular domain linked to the hinge, CH2, and CH3 domains of human IgG1. MICA triggering NKG2D signaling on NK cells and cytotoxic T cells is an important mediator of anti-tumor activity.

[0676] CD155:

[0677] In some embodiments, the components of the fusion protein of the present invention may include CD155 or a variant thereof. CD155 is a type I transmembrane glycoprotein in the immunoglobulin superfamily. In humans, CD155 is encoded by the poliovirus receptor (PVR) gene. CD155 is involved in the establishment of intercellular adhesion junctions between epithelial cells. The external domain of CD155 mediates the binding of cells to the extracellular matrix molecule vitronectin, while its intracellular domain interacts with the dynamin light chain Tctex-1 / DYNLT1. In addition, CD155 binds to the NK cell inhibitory receptors TIGIT and CD96, limiting the cytotoxicity of NK cells, and the activating receptor CD226 (DNAM-1). Fusion proteins comprising the CD155 extracellular domain can bind to TIGIT or CD96 on NK and T cells and act as a competitive antagonist of endogenous CD155 expressed by tumor cells or antigen presenting cells (APC). Fusion proteins containing CD155 can also bind to the co-stimulatory receptor CD226 and induce NK cell-mediated lysis of tumor targets.

[0678] TIGIT:

[0679] In some embodiments, the components of the fusion proteins of the present invention may include TIGIT or a variant thereof. In some embodiments, TIGIT is mouse TIGIT. In further embodiments, TIGIT is human TIGIT. In some embodiments, the TIGIT variant includes a complete TIGIT extracellular IgV-like domain connected to the hinge, CH2, and CH3 domains of human IgG1.

[0680] TIGIT, also known as T cell immunoreceptor with Ig and ITIM domains, is an immunoreceptor present on certain T cells and NK cells. TIGIT is also known as WUCAM or Vstm3. TIGIT binds with high affinity to CD155 (PVR) on cells such as dendritic cells (DCs) and macrophages, and binds with lower affinity to CD112 (PVRL2). TIGIT is a checkpoint inhibitor and is overexpressed on tumor antigen-specific (TA-specific) CD8+ T cells and CD8+ tumor infiltrating lymphocytes (TILs) from individuals with cancer, such as melanoma. Without wishing to be bound by theory, blockade of TIGIT can lead to increased cell proliferation, cytokine production, and degranulation of tumor antigen-specific CD8+ T cells and TIL CD8+ T cells. Fusion proteins containing TIGIT may bind to its receptors CD155 (PVR) and CD112 (PVRL2) and prevent their interaction with TIGIT on NK cells. Disrupting the interaction between TIGIT and its ligands on cancer cells will restore NK cell cytotoxic activity and cytokine production.

[0681] TIM-3:

[0682] In some embodiments, components of the fusion protein of the present invention may include TIM-3 or a variant thereof. T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), also known as hepatitis A virus cellular receptor 2 (HAVCR2), is a protein that is encoded by the HAVCR2 gene in humans. HAVCR2 is a cell surface molecule expressed on IFNγ-producing CD4+ Th1 and CD8+ Tc1 cells. TIM-3 expression has also been detected in Th17 cells, regulatory T cells, and innate immune cells (dendritic cells, NK cells, monocytes). TIM-3 is an immune checkpoint and mediates T cell exhaustion. Without wishing to be bound by theory, TIM-3 is upregulated in tumor infiltrating lymphocytes (TILs) of several cancers, including but not limited to lung cancer, gastric cancer, head and neck cancer, schwannoma, melanoma, and follicular B-cell non-Hodgkin's lymphoma. In some embodiments, TIM-3 variants include the entire TIM-3 extracellular domain connected to the hinge, CH2, and CH3 domains of human IgG1, including the N-terminal IgV-like domain. Fusion proteins containing TIM-3 can bind to its natural ligands galectin-9, Ceacam-1, and phosphatidylserine, and prevent them from interacting with endogenous TIM-3 expressed on NK cells, T cells, and APCs, reversing T cell exhaustion and restoring NK cell cytotoxicity and cytokine production.

[0683] Tumor cell receptor target:

[0684] In some embodiments, the fusion protein or one or more components thereof bind to a tumor cell receptor target. In some embodiments, the fusion protein or one or more components thereof prevent the binding of a ligand to a tumor cell receptor target. In some embodiments, the tumor cell receptor targets include, but are not limited to, chemokine receptors, notch receptors, immune checkpoint inhibitors, and tumor vasculature ligands and receptors. In some embodiments, chemokine receptors include CXCR4, CCR10, or CCR7. In some embodiments, immune checkpoint inhibitors include PD-L1 or PD-L2. In some embodiments, tumor vasculature targets include ανβ3, ανβ5, CD13 (aminopeptidase N), a target of an NGF motif peptide, or Tie2 (receptor for angiopoietin-2).

[0685] Immune cell receptor targets:

[0686] In some embodiments, the immune cell receptor target is CD40. In some embodiments, the fusion protein or one or more components thereof include an agonist CD40 scFv. In further embodiments, the immune cell receptor target is SIRPα. In some embodiments, the fusion protein or one or more components thereof include an antagonist SIRPα peptide. In some embodiments, the immune cell receptor target is 4-1BB. In some embodiments, the fusion protein or one or more components thereof include an agonist 4-1BB scFv or an agonist 4-1BB peptide.

[0687] In some embodiments, the immune cell receptor target is CD96. In some embodiments, the components of the fusion protein of the present invention may include CD96 or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of CD96.

[0688] In some embodiments, the components of the fusion proteins of the present invention may include CD226 or a variant thereof. In some embodiments, the components of the fusion proteins of the present invention may include an antagonist or agonist of CD226.

[0689] In some embodiments, the components of the fusion protein of the present invention may include TIM-3 or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of TIM-3.

[0690] In some embodiments, the immune cell receptor target is CD111. In some embodiments, the components of the fusion protein of the present invention may include CD111 or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of CD111.

[0691] In some embodiments, the immune cell receptor target is CD112. In some embodiments, the components of the fusion protein of the present invention may include CD112 or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of CD112.

[0692] In some embodiments, the immune cell receptor target is CD113. In some embodiments, the components of the fusion protein of the present invention may include CD113 or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of CD113.

[0693] In some embodiments, the immune cell receptor target is CD115. In some embodiments, the components of the fusion protein of the present invention may include CD115 or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of CD115.

[0694] In some embodiments, the immune cell receptor target is TIGIT. In some embodiments, the components of the fusion protein of the present invention may include TIGIT or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of TIGIT.

[0695] In some embodiments, the immune cell receptor target is KIR2DL1 / 2 / or 3 or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include KIR2DL1 / 2 / or 3 or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of KIR2DL1 / 2 / or 3.

[0696] In some embodiments, the immune cell receptor target is HLA-C. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of HLA-C. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of HLA-C.

[0697] In some embodiments, the immune cell receptor target is NKG2A (CD94) or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include NKG2A (CD94) or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of NKG2A (CD94) or a variant thereof.

[0698] In some embodiments, the immune cell receptor target is HLA-E. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of HLA-E. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of HLA-E.

[0699] In some embodiments, the immune cell receptor target is 2B4 or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include 2B4 or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of 2B4 or a variant thereof.

[0700] In some embodiments, the immune cell receptor target is CD48 or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of CD48. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of CD48 or a variant thereof.

[0701] In some embodiments, the immune cell receptor target is NKG2D or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include NKG2D or a variant thereof. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of NKG2D or a variant thereof.

[0702] In some embodiments, the immune cell receptor target is MICA / B or ULBP1 or variants thereof. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of MICA / B or ULBP1. In some embodiments, the components of the fusion protein of the present invention may include an antagonist or agonist of MICA / B or ULBP1 or variants thereof.

[0703] In any of the aforementioned embodiments, the immune cell is a NK cell, a T cell, a dendritic cell (DC), an antigen-presenting cell (APC), a macrophage, or a tumor-associated macrophage (M1).

[0704] Immune cell receptors or ligands:

[0705] In some embodiments, the fusion protein or one or more components thereof binds to an immune cell receptor or ligand. In some embodiments, binding to an immune cell receptor or ligand results in NK cell activation (cytokine production (IFNγ and TNF), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP)). In some embodiments, the receptor is an Fc receptor. In some embodiments, binding to an Fc receptor results in NK cell activation (ADCC and ADCP). In some embodiments, the Fc receptor is an Fc γ 、Fc ∈ 、Fc α 、Fcμ or Fc δ Receptor. In some embodiments, the immune cell receptor or ligand is a member of the TNF superfamily or its receptor, a member of the TNF-L superfamily or its receptor, transferrin or its receptor, human serum albumin or its receptor, or a member of the lipocalin structural family or its receptor.

[0706] Linker

[0707] In some embodiments, the components of the fusion protein of the present invention may optionally be connected via a peptide linker. The residue of the linker may be selected from naturally occurring amino acids, non-naturally occurring amino acids, and modified amino acids. The linker typically connects the carboxyl terminus of the first component to the amino terminus of the second component. The linker can alter the distance between the two structural components of the fusion protein, as well as alter the flexibility of this region. The linker may comprise any number of amino acids. 55, 56, 57, 58, 59, 60 or more amino acids. In some embodiments, the linker can be composed of 3 to 60 amino acid residues, 3 to 40 amino acid residues, 3 to 30 amino acid residues, 3 to 24 amino acid residues, 3 to 18 amino acid residues, or 3 to 15 amino acid residues. The linker can include, for example, a repeating sub-sequence of 2, 3, 4, 5 or more amino acid residues, including repeats of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more sub-sequences.

[0708] The linker can be a naturally occurring sequence or a designed sequence. Peptide linkers that can be used in the fusion proteins of the present invention include, but are not limited to, glycine linkers, glycine-rich linkers, serine-glycine linkers, and the like. Glycine-rich linkers comprise at least about 50% glycine, and preferably at least about 60% glycine. In one embodiment, the linker comprises the amino acid sequence Gly-Ser, or repeats thereof. See, e.g., Houston et al., Methods in Enzymology, 203:46-88 (1991). In another embodiment, the linker comprises the amino acid sequence Gly-Lys, or repeats thereof. See, e.g., Whitler et al., Protein Eng, 6:989 (1993). In another embodiment, the linker comprises the amino acid sequence Gly-Gly-Ser, or repeats thereof. In another embodiment, the linker comprises the amino acid sequence Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 29) or repeats thereof. In certain specific embodiments, the linker comprises the amino acid sequence Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser (SEQ ID NO: 30). In certain embodiments, the linker comprises 2 to 12 repeats of Gly-Gly-Ser or Gly-Gly-Gly-Ser or Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 29). Examples of peptide linkers are described in U.S. Patent No. 6,541,219. In one embodiment, the linker may comprise the sequence GDPLVTAASVLEFGGSGGGSEGGGSEGGGSEGGGSDI (SEQ ID NO: 31).

[0709] Linkers can be used to separate the two components of a fusion protein to achieve proper folding of the components, to reduce potential steric issues, and / or to facilitate optimal receptor binding. Those skilled in the art are familiar with the design and selection of peptide linkers. For example, see Robinson et al., 1998, Proc. Natl. Acad. Sci. USA 95: 5929-5934. Automated programs can also be used for peptide linker design (e.g., Crasto et al., 2000, Protein Engineering 13: 309-312).

[0710] Optional additional elements:

[0711] In addition to component A, component B, component C, and / or component D, the fusion protein may optionally include additional elements. Such additional elements may include, but are not limited to, an initiator methionine, a signal peptide, an antigenic polypeptide, a trimerization domain, a higher-order multimerization domain, and a purification tag, such as His-6. An exemplary purification tag is ASHHHHHHM (SEQ ID NO: 46). In one embodiment, the fusion protein of the present invention includes an optional trimerization domain.

[0712] The fusion proteins of the present invention optionally include a signal peptide. As will be appreciated by those skilled in the art, the signal peptide may vary depending on the needs of the user, the expression system, and other factors. Signal peptides are well known in the art, and any desired signal peptide may be used, including those identified / predicted by publicly available signal peptide recognition software known to those skilled in the art.

[0713] In some embodiments, the fusion proteins of the invention include a hinge region that allows for flexibility between the components. See Lobner et al. (2016) Immunol. Reviews 270:113-131. In some embodiments, component Y and component Z2 are connected by a hinge, such as an IgG hinge. In some embodiments, component Z1' and component Z2' are connected by a hinge, such as an IgG hinge.

[0714] In some embodiments, the N-linked glycan is attached to Asn on component A and / or component B. In some embodiments, the N-linked glycan is attached to Asn297 on component A and / or component B. Without wishing to be bound by theory, this may promote FcR binding and may promote structural integrity and thermal stability of the fusion protein. See Arnold et al. (2007) Annu Rev Immunol 25:21-50.

[0715] In some embodiments, component A and / or component B comprises a K to A mutation. In some embodiments, component A and / or component B comprises a K322A mutation. Without wishing to be bound by theory, this may reduce Clq binding and complement-mediated lysis. See Idusogi et al. (2000) J. Immunol 164:4178-4184.

[0716] In some embodiments, component A and component B include knobs-into-holes mutations. In some embodiments, component A includes mutations Y349C and T366W, and component B includes mutations D356C, T366S, L368A, and Y407V ("knobs-into-holes" mutations). Without wishing to be bound by theory, this may promote heterodimerization rather than homodimerization. See Mo et al. (1998) Nature Biotech. 16:677-681.

[0717] In some embodiments, component A and / or component B include mutations that enhance binding to the neonatal Fc receptor (FcRn). In some embodiments, component A and / or component B include mutations M428L and N434S. Without wishing to be bound by theory, these may enhance binding to neonatal Fc receptors (FcRn) on various cells and extend serum half-life. See Guo and Ivison (2011) mAbs 3:422-430.

[0718] Trimerization domain:

[0719] Trimerization domains are well known in the art. Non-limiting examples of trimerization domains suitable as a heterologous trimerization domain in the fusion protein of the present invention include: the GCN4 leucine zipper (Habery et al., 1993, "Switching between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants," Science 262(5138): 1401-7); a 35 amino acid sequence from pulmonary surfactant protein (Hope et al., 1994, "Parallel three-stranded α-helical bundles at the nucleation site of triple helix formation in collagen," FEBS letters 344(2-3): 191-5); a short, repeated heptapeptide sequence from collagen (McClinden et al., 2000, "α-helical coiled coil oligomerization domains are nearly ubiquitous in the collagen superfamily," J Biol Chem. 278(43): 42200-7. Epub 2012 August 14, 2003); and the bacteriophage T4 fibrin "foldon" (see, e.g., Miroshnikov et al., 1998, "Engineered trimeric fibrin based on bacteriophage T4 adhesin," Protein Eng. 11(4):329-32). Exemplary trimerization domains are also disclosed in U.S. Patent Nos. 6,911,205 and 8,147,843 and U.S. Patent Publication No. 2010 / 0136032. An exemplary trimerization sequence is the T4 "foldon" having the following sequence: GYIPEAPRDGQAYVRKRGEWVLLSTFL (SEQ ID NO: 47). Another exemplary trimerization domain is from thrombospondin-1 and has the sequence: VTTLQDSIRKVTEENKELANELRR (SEQ ID NO: 56).

[0720] Modification:

[0721] The present invention includes variants of the fusion proteins described herein. While variants are generally expected to exhibit enhanced binding to a given molecule, in some embodiments, variants may be designed to exhibit slightly reduced activity compared to other fusion proteins of the invention, for example where attenuation of activity is intentional. Furthermore, variants or derivatives may be generated with altered multimerization properties.

[0722] Preferably, the variants or derivatives of the fusion proteins of the present invention maintain the hydrophobicity / hydrophilicity of the amino acid sequence.

[0723] In additional embodiments, the fusion protein of the invention is SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 32, 33, 34, 35, 49, 50, 51, 52, 53, 54, 55, 65, 66, 67, 68, 69, 70, or 71. In one embodiment, a variant of the fusion protein of the invention will have at least 80% or greater sequence identity or homology to SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 32, 33, 34, 35, 49, 50, 51, 52, 53, 54, 55, 65, 66, 67, 68, 69, 70, or 71, as those terms are understood in the art, and more preferably at least 80% or greater sequence identity or homology to SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 32, 33, 34, 35, 49, 50, 51, 52, 53, 54, 55, 65, 66, 67, 68, 69, 70, or 71. NO: 14, 15, 16, 17, 18, 19, 20, 21, 22, 32, 33, 34, 35, 49, 50, 51, 52, 53, 54, 55, 65, 66, 67, 68, 69, 70 or 71 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% sequence identity.

[0724] The present invention also provides chemical modifications of the fusion proteins of the present invention. Non-limiting examples of such modifications may include, but are not limited to, aliphatic esters or amides at the carboxyl terminus or aliphatic esters or amides of residues containing a carboxyl side chain, O-acyl derivatives containing hydroxyl residues, and amino-terminal amino acids or residues containing an amino group, such as lysine or arginine.

[0725] Other derivatives of fusion protein of the present invention include incorporating non-natural amino acid residues, or phosphorylated amino acid residues, such as phosphotyrosine, phosphoserine or phosphothreonine residues. Other potential modifications include sulfonation, biotinylation, or adding other parts, particularly parts where the molecular shape is similar to a phosphate group.

[0726] Derivatives also include polypeptides modified by glycosylation. These can be prepared by modifying the glycosylation pattern during synthesis and processing in various alternative eukaryotic host expression systems, or during further processing steps. Methods for producing glycosylation modifications include exposing the fusion protein to glycosylases derived from cells that typically perform such processing, such as mammalian glycosylases. Alternatively, deglycosylases can be used to remove carbohydrates attached during production in eukaryotic expression systems. Furthermore, the coding sequence can be modified to add glycosylation sites or to delete or disable glycosylation sites. Furthermore, if glycosylation is not desired, the protein can be produced in a prokaryotic host expression system.

[0727] Variants and / or derivatives of the fusion proteins of the present invention can be prepared by chemical synthesis or by modifying the nucleic acid encoding the intact receptor using site-directed mutagenesis (Gilman et al., Gene 8:81 (1979); Roberts et al., Nature 328:731 (1987)) or Innis (Ed.), 1990, PCR Protocols: A Guide to Methods and Applications, Academic Press, New York, NY) or polymerase chain reaction methods (PCR; Saiki et al., Science 239:487 (1988)), as exemplified by Dougherty et al., Nucleic Acids Res. 19:2471 (1991)).

[0728] In additional embodiments, the fusion proteins of the present invention may further include one or more additional polypeptide domains to facilitate protein purification, increase expression of the recombinant protein, or increase the solubility of the recombinant protein. Such purification / expression / solubility-enhancing domains include, but are not limited to, metal chelating peptides, such as histidine-tryptophan modules that allow purification on immobilized metals (Porras, 1992, Protein Expr Purif 3-0.26328 1), protein A domains that allow purification on immobilized immunoglobulins, and domains used in the FLAGS extension / affinity purification system (Immunex, Seattle, Washington). Including a cleavable linker sequence, such as factor Xa or enterokinase (Invitrogen, San Diego, CA), between the purification domain and the fusion of component A and component B helps facilitate purification.

[0729] Fusion expression vectors include pGEX (Pharmacia, Piscataway, NJ), pMAL (New England Biolabs, Beverly Hills, MA) and pRITS (Pharmacia, Piscataway, NJ) fused to glutathione S transferase (GST), maltose B binding protein or protein A, respectively, and linked to the target recombinant protein. EBV, BKV and other episomal expression vectors (Invitrogen) can also be used. In addition, retroviral and lentiviral expression vectors can also be used. In addition, any of a variety of in vivo expression systems designed for high-level expression of recombinant proteins in vivo can be used to produce the fusion proteins specified herein.

[0730] As described above, the fusion protein of the present invention may contain a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and / or secretion of the fusion protein can be increased by using a heterologous signal sequence. The signal sequence is typically characterized by a hydrophobic amino acid core, which is typically cleaved from the mature protein during the secretion process in one or more cleavage events. Such signal peptides contain processing sites that allow the signal sequence to be cleaved from the mature protein as the mature protein passes through the secretory pathway. Therefore, the present invention relates to polypeptides having a signal sequence as described, as well as polypeptides in which the signal sequence has been proteolytically cleaved (i.e., cleavage products).

[0731] In order to enhance stability and / or reactivity, the fusion protein of the present invention can also be modified to incorporate one or more polymorphisms in the amino acid sequence produced by natural allelic variation. In addition, D-amino acids, non-natural amino acids or non-amino acid analogs can be replaced or added to produce modified fusion proteins within the scope of the present invention.

[0732] The amino acid sequence of the present invention can be produced by expressing a nucleotide sequence encoding the same in a suitable expression system.

[0733] In addition, or alternatively, the fusion protein itself can be synthesized using chemical methods to synthesize all or part of the desired amino acid sequence. For example, the polypeptide can be synthesized using solid-phase techniques, cleaved from the resin, and purified by preparative high-performance liquid chromatography (e.g., Creighton (1983) Proteins: Structure and Molecular Principles, W.H. Freeman & Co., New York, NY). The composition of the synthesized polypeptide can be confirmed by amino acid analysis or sequencing (e.g., Edman degradation procedure). In addition, the amino acid sequence of the fusion protein of the present invention, or any portion thereof, can be altered during direct synthesis and / or combined using chemical methods with sequences from other subunits, or any portion thereof, to produce variant polypeptides.

[0734] Assays for detecting biological activity of any homologue, derivative or variant of any fusion protein of the invention are well known in the art.

[0735] Activity and efficacy

[0736] In one embodiment, the fusion proteins of the present invention reduce or prevent tumor cells from migrating, infiltrating adjacent tissues, and / or metastasizing to distant sites, effectively immobilizing the cells. In another embodiment, the fusion proteins of the present invention reduce or prevent tumor cells from escaping phagocytosis by phagocytes, such as macrophages, while also promoting apoptosis and / or immune destruction of the tumor cells. In other embodiments, the fusion proteins of the present invention reduce or prevent tumor cells from escaping phagocytosis by a phagocyte while promoting apoptosis of adjacent tumor cells. Thus, the fusion proteins of the present invention promote tumor cell destruction through any of a variety of mechanisms.

[0737] PD-1 ligands or receptors are expressed on a wide range of tumor cells, such as solid tumor cells. Therefore, in one embodiment, the present invention provides a method for treating a proliferative disease by administering a therapeutically effective amount of a fusion protein of the present invention to a subject diagnosed with a proliferative disease.

[0738] The fusion proteins according to the present invention can be administered to individuals (eg, mammals, including animals and humans) suffering from a cell proliferative disease, such as cancer, and malignant and benign tumors. In one embodiment of the present invention, the individual being treated is a human.

[0739] The fusion protein is believed to be effective against a variety of tumor types, including but not limited to: ovarian cancer; cervical cancer; breast cancer; prostate cancer; testicular cancer, lung cancer, kidney cancer; colorectal cancer; skin cancer; brain cancer; and leukemias, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia.

[0740] More specifically, cancers that can be treated by the compounds, compositions and methods of the present invention include, but are not limited to, the following:

[0741] Cardiac cancers, including, for example, sarcomas, e.g., angiosarcoma, fibrosarcoma, rhabdomyosarcoma, and liposarcoma; myxoma; rhabdomyomas; fibromas; lipomas and teratomas;

[0742] Lung cancer, including, for example, bronchogenic carcinoma, e.g., squamous cell carcinoma, undifferentiated small cell carcinoma, undifferentiated large cell carcinoma, and adenocarcinoma; alveolar and bronchiolar carcinoma; bronchial adenoma; sarcoma; lymphoma; enchondroma, hamartoma; and mesothelioma;

[0743] Gastrointestinal cancers, including, for example, esophageal cancer, such as squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, and lymphoma; gastric cancer, such as malignant tumor, lymphoma, and leiomyosarcoma; pancreatic cancer, such as ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, and vipoma; small intestinal cancer, such as adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, and fibroma; colorectal cancer, such as adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, and leiomyoma;

[0744] Genitourinary tract cancers, including, for example, renal cancer, such as adenocarcinoma, Wilm's tumor (Nephroblastoma), lymphoma, and leukemia; bladder cancer and urethral cancer, such as squamous cell carcinoma, transitional cell carcinoma, and adenocarcinoma; prostate cancer, such as adenocarcinoma and sarcoma; testicular cancer, such as seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor, and lipoma;

[0745] Liver cancer, including, for example, liver cancer, e.g., hepatocellular carcinoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hepatocellular adenoma; and hemangioma;

[0746] Bone cancer, including, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochrondroma (osteocartilaginous exostoses), exochondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma, and giant cell tumor;

[0747] Nervous system cancers, including, for example, skull cancers, such as osteomas, hemangiomas, granulomas, xanthomas, and osteitis deformans; meningeal cancers, such as meningiomas, meningiosarcomas, and gliomatosis; brain cancers, such as astrocytomas, medulloblastomas, gliomas, ependymomas, germ cell tumors (pinealomas), glioblastomas multiforme, oligodendrogliomas, schwannomas, retinoblastomas, and congenital tumors; and spinal cord cancers, such as neurofibromas, meningiomas, gliomas, and sarcomas;

[0748] Gynecological cancers, including, for example, uterine cancer, such as endometrial cancer; cervical cancer, such as cervical carcinoma, and precancerous cervical dysplasia; ovarian cancer, such as ovarian cancer, including serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified malignant tumor, granulosa thecal cell tumor, Sertoli Leydig cell tumor, dysgerminoma, and malignant teratoma; vulvar cancer, such as squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, and melanoma; vaginal cancer, such as clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma, and embryonal rhabdomyosarcoma; and fallopian tube cancer, such as malignant tumor;

[0749] Hematologic cancers, including, for example, cancers of the blood, such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma, and myelodysplastic syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma (malignant lymphoma), and Waldenstrom's macroglobulinemia ( macroglobulinemia), angioimmunoblastic T-cell lymphoma (AITL), chronic lymphocytic leukemia (CLL), acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia; Naegeli leukemia; plasma cell leukemia; plasma cell leukemia; promyelocyticleukemia; Rieder cell leukemia leukemia; Schilling's leukemia; stem cell leukemia; subleukemic leukemia; acute promyelocytic leukemia; adult T-cell leukemia; aleukemic leukemia; aleukocythemic leukemia; basophilic leukemia; blast cell leukemia; bovine leukemia; chronic myelocytic leukemia; leukemia cutis; embryonal leukemia; undifferentiated cell leukemia; eosinophilic leukemia; Gross' leukemia; hairy-cell leukemia; hemoblastic leukemia leukemia); hemocytoblastic leukemia; histiocytic leukemia; stem cell leukemia; acute monocytic leukemia; leukopenic leukemia;Lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, and microgranulocyte leukemia;

[0750] Skin cancers, including, for example, malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, hemangioma, dermatofibroma, keloids, psoriasis; and

[0751] Adrenal cancer, including, for example, neuroblastoma.

[0752] More specific examples of such cancers include kidney cancer or renal cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), cervical cancer, ovarian cancer, prostate cancer, liver cancer, bladder cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer, including gastrointestinal cancer, gastrointestinal stromal tumors (GIST), pancreatic cancer, head and neck cancer, glioblastoma, retinoblastoma, astrocytoma, cystic tumor, adenoblastoma, hepatoma, hematological malignancies, including non-Hodgkin's lymphoma (NHL), and leukemia. multiple myeloma and acute hematologic malignancies, endometrial or uterine cancer, endometriosis, fibrosarcoma, salivary gland cancer, vulval cancer, thyroid cancer, esophageal cancer, liver cancer, anal cancer, penile cancer, nasopharyngeal cancer, laryngeal cancer, Kaposi's sarcoma, melanoma, skin cancer, neurilemmoma, oligodendroglioma, neuroblastoma, rhabdomyosarcoma, osteosarcoma, leiomyosarcoma, urinary tract cancer, thyroid cancer, Wilm's tumor, and B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma, small lymphocytic (SL) NHL, intermediate-grade / follicular NHL, and follicular NHL). NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom macroglobulinemia; chronic lymphocytic leukemia (CLL);Acute lymphoblastic leukemia (ALL); acute myelogenous leukemia (AML); hairy-cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel proliferation associated with phakomatoses, edema (e.g., edema associated with brain tumors), and Meigs' syndrome. As used herein, "tumor" refers to all tumor cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0753] Cancer may be a solid tumor, which may or may not be metastatic. Cancer may also occur in the form of a diffuse tissue, such as in leukemia. Thus, the term "tumor cell" as provided herein includes cells afflicted by any of the above diseases.

[0754] In a preferred embodiment, the cancer is a solid tumor. In a preferred embodiment, the cancer is one of pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, melanoma and glioblastoma.

[0755] In another embodiment, the cancer is a hematological cancer. In a preferred embodiment, the hematological cancer is one of acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML).

[0756] Pharmaceutical composition and dosage regimen:

[0757] Administration of the compositions of the present invention is typically parenteral, by subcutaneous, intravenous, intramuscular or intraperitoneal injection, or by infusion or by any other acceptable systemic method. In a preferred embodiment, administration is by subcutaneous injection. In another preferred embodiment, administration is by intravenous infusion, which can typically be performed over a period of about 1 to 5 hours. In addition, there are a variety of oral administration methods for the administration of therapeutic proteins, which methods can be applied to the therapeutic fusion proteins of the present invention.

[0758] Typically, the therapeutic dose is titrated upward from a low level to optimize safety and efficacy. Typically, the daily dose will fall within the range of about 0.01 to 20 mg of protein per kilogram of body weight. Typically, the dosage range is about 0.1 to 5 mg of protein per kilogram of body weight. Various modifications or derivatives can be made to the fusion protein, for example, by adding polyethylene glycol chains (PEGylation) to affect its pharmacokinetic and / or pharmacodynamic properties.

[0759] In order to administer the fusion protein by a route other than parenteral administration, it may be necessary to coat the protein with a material, or to co-administer a material with the protein, to prevent its inactivation. For example, the protein can be administered in an incomplete adjuvant, co-administered with an enzyme inhibitor, or administered in liposomes. Enzyme inhibitors include trypsin inhibitors, diisopropylfluorophosphate (DEP), and trasylol. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Streihan et al., 1984, J. Neuroimmunol. 7:27).

[0760] Although the compositions of the present invention can be administered in the form of simple solutions, they are more typically used in combination with other materials, such as carriers, preferably pharmaceutically acceptable carriers. Useful pharmaceutically acceptable carriers can be any compatible, non-toxic substance suitable for delivering the compositions of the present invention to a patient. Carriers can include sterile water, alcohol, fats, waxes, and inert solids. Pharmaceutically acceptable adjuvants (buffers, dispersants) can also be incorporated into the pharmaceutical composition. In general, compositions that can be used for parenteral administration are well known. For example, Remington's Pharmaceutical Science, 17th ed. (Mac Publishing Company, Easton, Pennsylvania, 1990). Alternatively, the compositions of the present invention can be introduced into a patient's body via an implantable drug delivery system (Erquhart et al., 1984, Ann. Rev. Pharmacol. Toxicol. 24:199).

[0761] Therapeutic preparations can be administered in many conventional dosage forms. Preparations generally include at least one active ingredient and one or more pharmaceutically acceptable carriers. Preparations may include those suitable for oral, rectal, nasal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.

[0762] The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. See, for example, Gilman et al. (ed.) (1990), Pharmacological Basis of Therapy, 8th ed., Pergamon Press; and Remington's Pharmaceutical Science, supra, Easton, Pennsylvania; Avis et al. (ed.) (1993), Pharmaceutical Dosage Forms: Parenteral Use; Decker, NY, Lieberman et al. (ed.) (1990), Pharmaceutical Dosage Forms: Tablets, Decker, NY; and Lieberman et al. (ed.) (1990), Pharmaceutical Dosage Forms: Dispersion Systems, Decker, NY

[0763] In additional embodiments, the present invention contemplates administering the fusion protein by gene therapy, for example, administering an isolated nucleic acid encoding a fusion protein of interest. The protein building blocks of the fusion protein of the present invention (e.g., component A and component B) have been fully characterized in terms of the nucleic acid sequence encoding the protein and the amino acid sequence of the protein produced therefrom. It is within the capabilities of those skilled in the art to engineer such isolated nucleic acids by recombinant DNA methods. For the purpose of maximizing the yield of recombinant protein in a specific cell background, codon optimization is also within the capabilities of those skilled in the art. Administration of an isolated nucleic acid encoding the fusion protein is included in the expression "administering a therapeutically effective amount of the fusion protein of the present invention." Gene therapy methods are well known in the art. See, for example, WO96 / 07321, which discloses the use of gene therapy methods to produce intracellular antibodies. Gene therapy methods have also been successfully demonstrated in human patients. See, for example, Baumgartner et al., 1998, Circulation 97:12, 1114-1123, and more recently, Fish, 2007, "A gene therapy approach for treating autoimmune diseases," Immun. Res. 18:15-26; and U.S. Patent No. 7,378,089, both of which are incorporated herein by reference. See also Bainbridge et al., 2008, "Effects of gene therapy on visual function in Leber's congenital amaurosis," N Engl Med 358:2231-2239; and Maguire et al., 2008, "Safety and efficacy of gene transfer for Leber's congenital amaurosis," N Engl J Med 358:2240-8. There are two main methods for introducing a nucleic acid encoding a fusion protein (optionally contained in a vector) into a patient's cells: in vivo and ex vivo. For in vivo delivery, nucleic acid is injected directly into the patient's body, usually at the site where the fusion protein is needed. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are then administered to the patient directly or, for example, encapsulated in a porous membrane implanted in the patient's body (see, for example, U.S. Patents 4,892,538 and 5,283,187). There are a variety of techniques for introducing nucleic acids into living cells. The technology varies depending on whether the nucleic acid is transferred to cultured cells in vitro or transferred to the body in the cells of the target host. Technologies suitable for transferring nucleic acids into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, etc. Commonly used in vitro gene delivery vectors are retroviral vectors and lentiviral vectors.

[0764] Preferred in vivo nucleic acid transfer techniques include transfection using viral vectors, such as adenovirus, herpes simplex virus, adeno-associated virus, lipid-based systems (for example, useful lipids for lipid-mediated gene transfer are DOTMA, DOPE, and DC-Cho), naked DNA, and transposon-based expression systems. For a review of currently known gene marking and gene therapy protocols, see Anderson et al., Science 256:808-813 (1992). See also WO 93 / 25673 and references cited therein.

[0765] "Gene therapy" includes conventional gene therapy that achieves a lasting effect through a single treatment, as well as the administration of gene therapy agents, which involves one-time or repeated administration of a therapeutically effective DNA or mRNA. Oligonucleotides can be modified to enhance their absorption, for example, by replacing their negatively charged phosphodiester groups with uncharged groups. The fusion proteins of the present invention can be delivered using gene therapy methods, for example, locally, intrathecally, or systemically in a tumor bed (tumor bed) (for example, by vectors that selectively target specific tissue types, for example, tissue-specific adeno-associated viral vectors). In some embodiments, genes encoding any fusion protein of the present invention can be used to transfect primary cells (e.g., lymphocytes or stem cells) from an individual ex vivo, after which the transfected cells are returned to the individual's body.

[0766] "Treating" or "treatment" refers to therapeutic treatment, wherein the purpose is to prevent or slow down (alleviate) the target pathological condition or disorder. A subject is successfully "treated" when, after receiving a therapeutic amount of the fusion protein of the present invention according to the methods of the present invention, the subject shows a reduction or absence of one or more observable and / or detectable signs and symptoms of a specific disease. For example, for cancer, the number of cancer cells is reduced or the cancer cells are absent; the size of the tumor is reduced; tumor metastasis is inhibited (i.e., slowed to some extent and preferably stopped); tumor growth is inhibited to some extent; one or more symptoms associated with a specific cancer are alleviated to some extent and / or the duration of the alleviation is prolonged; morbidity and mortality are reduced, and quality of life issues are improved. The patient may also feel a reduction in the signs or symptoms of the disease. Treatment can achieve a complete response, defined as the disappearance of all signs of cancer, or a partial response, wherein the size of the tumor is reduced, preferably by more than 50%, more preferably by 75%. If the patient experiences stable disease, the patient is also considered to be receiving treatment. These parameters for assessing successful treatment and improvement of the disease can be easily detected by routine procedures familiar to physicians with appropriate skills in the art.

[0767] In the case of treating cancer, the fusion protein of the present invention can be optionally administered to a patient in combination with other chemotherapeutic agents. Suitable chemotherapeutic agents include, for example, alkylating agents such as thiotepa and cyclophosphamide. TM); alkyl sulfonates, such as butyl dimethanesulfonate, improsiilfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trishydroxymethylmelamine; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, and mechlorethamine hydrochloride. oxide hydrochloride), melphalan, novembicin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas (e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine);Antibiotics, such as aclacinomycins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, tinomycin), daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid acid), nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorbamycin antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine;Pyrimidine analogs (e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU); androgens (e.g., calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone); antiadreners (e.g., aminoglutethimide, mitotane, trilostane); folic acid supplements (e.g., frolinic acid); acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.R; TMrazoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel (TAXOL.R TM , Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE.R TM, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C C); mitoxantrone; vincristine; vinorelbine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0768] Other chemotherapeutic agents also include antihormonal agents used to modulate or inhibit hormonal effects on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

[0769] Other tumor cytotoxic agents that can be used in combination with the fusion proteins of the present invention are themselves fusion proteins. Examples of tumor cytotoxic fusion proteins are CTLA-4-FasL and Fn14-TRAIL, which act as cis loop-back proteins by generating an autoapoptotic signaling loop on the surface of tumor cells. See U.S. Patent Nos. 7,569,663, 8,329,657, and 8,039,437, each of which is incorporated by reference in its entirety. In turn, those cis-loop proteins comprising TRAIL components can be co-administered with chemotherapeutic agents that can sensitize tumor cells to TRAIL and overcome TRAIL resistance, such as proteasome inhibitors and histone deacetylase (HDAC) inhibitors, cycloheximide, imatinib mesylate and other protein tyrosine kinase inhibitors, 17-allylamino-17-demethoxygeldanamycin, arsenic trioxide and X-linked inhibitors of apoptosis protein small molecule antagonists, as well as any pharmaceutically acceptable salts, acids or derivatives thereof.

[0770] Additional information regarding methods of treating cancer is provided in US Patent No. 7,285,522, which is incorporated by reference in its entirety.

[0771] The following non-limiting examples illustrate the practice of the present invention. The present invention should not be construed as being limited to the compositions and methods described herein, but should be construed to encompass other compositions and methods. Those skilled in the art will appreciate that other compositions and methods may be used to perform the procedures described herein.

[0772] Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, all of which are within the capabilities of the skilled artisan. Such techniques are fully explained in the literature, for example, in "Molecular Cloning: A Laboratory Manual," Fourth Edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gette, 1984); "Animal Cell Culture" (Frisney, 2010); "Enzymatic Methods" and "Handbook of Experimental Immunology" (Will, 1997); "Gene Transfer Vectors in Mammalian Cells" (Miller and Carlos, 1987); "A Short Protocol in Molecular Biology" (Aussubel, 2002); "Polymerase Chain Reaction: Principles, Applications, and Troubleshooting" (Babar, 2011); and "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the present invention and, therefore, can be considered in making and practicing the present invention. Particularly useful techniques for specific embodiments are discussed in the following sections.

[0773] Experimental Examples

[0774] The present invention is further described in detail below with reference to experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise indicated. Therefore, the present invention should in no way be construed as limited to the following examples, but rather should be construed to include any and all variations that become apparent as a result of the teachings provided herein.

[0775] Without further explanation, it is believed that one of ordinary skill in the art can utilize the foregoing description and the following illustrative examples to prepare and utilize the compounds of the present invention and to implement the claimed methods. Therefore, the following working examples specifically point out preferred embodiments of the present invention and should not be construed as limiting the remainder of the disclosure in any way.

[0776] Example 1 - Fusion Protein Binding

[0777] Fusion protein constructs and expression

[0778] Expression plasmid constructs for these fusion proteins were generated using de novo gene fragments (ThermoFisher Scientific) for each desired ligand / receptor component, linked to synthetic gene fragments for the hinge, CH2, and CH3 domains of the A or B chain (i.e., the Z2, Z3, Z2', or Z3' domains). The components were spliced ​​together by polymerase chain reaction (PCR) and cloned into the expression plasmid pCEP4, an EBV episomal expression vector (originally developed in the Tykocinski laboratory) that replicates extrachromosomally at high copy number, using primers containing terminal restriction sites. During gene synthesis, the DNA fragments were codon-optimized for expression in Chinese hamster ovary suspension (CHO-S) cells. The A-chain construct and the B-chain construct were transiently co-transfected into ExpiCHO-S shake flask cultures using TransIT-Pro reagent (MIRUS) and cultured at 37°C for 24 hours, followed by 8 to 10 days at 32°C to generate the total number of days of fusion protein. Proteins were purified from the conditioned culture supernatant by mixing with protein A agarose resin at 6°C overnight, then collected and eluted with a non-denaturing neutral pH elution buffer (PIERCE). The size and integrity of each fusion protein were verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

[0779] Fusion protein binding studies

[0780] The expression of human CD155, T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), CD112 and CD16 (Fc γRIIIa) and cloned into the expression plasmid pcDNA3.1+ (ThermoFisher Scientific). Each expression construct was transfected into CHO-S cells using Lipofectamine 3000 (ThermoFisher) and stable transfectants selected by adding G418. Ligand expression was detected by flow cytometry after immunostaining with an appropriate fluorochrome-conjugated anti-ligand antibody (black line) or an appropriate fluorochrome-conjugated isotype control antibody (grey line and solid). Data were analyzed using FCSalyzer software. For fusion protein binding studies, fusion proteins were added to untransfected CHO-S cells as controls (grey line and solid) or CHO-S transfectants expressing the corresponding ligands and incubated at 6°C for 1 hour, followed by washing and immunoblotting using Cy5-conjugated anti-human IgG, Fc γ specific antibodies (black line) and analyzed by flow cytometry. Figures 18 to 20 For CD16 binding studies, fusion proteins containing TIGIT or CD155 were cultured with untransfected CHO-S cells as controls (grey lines and solid circles) or CHO-S transfectants expressing CD16 and detected by immunostaining with APC-conjugated antibodies or PE-conjugated antibodies recognizing TIGIT or CD155, respectively (black lines). Figure 21 .

[0781] Example 2 - Fusion protein binding assay and migration assay

[0782] Protein gel analysis

[0783] The reduced (R) or non-reduced (NR) fusion proteins were separated on 12% SDS-PAGE gels and stained in PageBlue protein staining solution (ThermoFisher Scientific) for 12 hours. Figure 22 .

[0784] Immunoblot analysis

[0785] The fusion proteins were then quantified using a Pierce BCA protein assay kit (Thermo Fisher) and loaded onto a 12% SDS-PAGE gel. Proteins were electrotransferred to Immobilon-P membranes (EMDMillipore, Billerica, MA) and incubated for 12 hours in Odyssey blocking buffer (Licor, Lincoln, NE) with (A) Abs against PD1 and (B) Abs against the primary IgG light chain. The corresponding secondary antibodies were used at a 1:10,000 dilution (Santa Cruz). Immunoblots were scanned using an Odyssey infrared imaging system (LI-COR Biosciences, model #9120). See Figure 23.

[0786] Fusion protein binding studies

[0787] CXCR4 / 7 and PD1+ melanoma cell lines, (A) YUMMER1.7 cells or (B) B16 cells, were incubated with the indicated fusion proteins at 6°C for 1 hour, washed, and immunostained with Cy5-conjugated anti-human IgG, Fcγ-specific antibodies and analyzed by flow cytometry (solid black line). Control samples not incubated with fusion proteins (solid gray line) were shown. Figure 24 shown.

[0788] Transwell assay

[0789] Transwell inserts were inserted into 24-well companion plates (Corning, catalog #353504). The cell suspension was seeded in the top invasion chamber at a rate of 25,000 cells / chamber. DME containing 100 ng / mL of CXCL12 was added to each bottom well as a chemoattractant. The samples were incubated at 37°C for 24 hours to allow cell migration. Non-invading cells on the top surface of the insert were removed using a cotton swab, and cells that migrated to the lower surface of the support were stained with 2% crystal violet. See Figure 25 .

[0790] Example 3 - TriTouch-101 induces melanoma regression

[0791] TriTouch-101 PD1hFcA*vMIPIICH-hFcB*CL(FP) induced tumor growth inhibition in vivo. The B16F10 melanoma subcutaneous model was used to demonstrate that PD1hFcA*vMIPIICH-hFcB*CL(FP) induced melanoma regression. Briefly, BL16 mice were subcutaneously inoculated with 1×10 5On days 13, 14, 15, 16, and 21 after inoculation of B16F10 cells, 100 μl (10 μg / mL) of FP (mice R, 2L, and 2R) or PBS (mice X or L) were used for 5 treatments. Figure 26A ). Figure 26B Representative images of one mouse from each treatment group are depicted in FIG (X top and 2R bottom). These in vivo data showing that TriTouch-101 induces melanoma regression are consistent with previous data showing that this fusion protein inhibits melanoma cells in a migration assay ( Figure 25 ).

[0792] Example 4 - NK Regulates TriTouch Protein Species

[0793] Enhanced ADCC by adding multifunctional fusion proteins Figure 27 ). SKOV-3 ovarian cells were seeded at 3,000 cells / well in a 96-well plate, allowed to adhere, and labeled with CellTracker Red CMTPX reagent. SKOV-3 cells were then labeled with a green fluorescent Caspase-3 reagent. CD16.NK-92 cells (V158 variant) were added to the wells at a 5:1 E:T ratio with various fusion proteins at 25 mg / ml or without protein, and analyzed using the Incucyte Live Cell Analysis System to measure the number of fluorescent double-positive (red + green) cells. The results shown depict the 20-hour time point ( Figure 27 ).

[0794] Other embodiments

[0795] The recitation of a list of elements in any definition of a variable herein includes defining the variable as any single element or combination (or subcombination) of the listed elements. The recitation of an embodiment herein includes the embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0796] The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with reference to certain specific embodiments, it is apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations. Sequence Listing <110> Thomas Jefferson University Mark L. Tykocinski Matthew C. Weber Carmela Romeo Smith <120> Multifunctional fusion protein and its use <130> 205961-7028WO1(00150) <150> 62 / 789,212 <151> 2019-01-07 <160> 69 <170> PatentIn version 3.5 <210> 1 <211> 144 <212> PRT <213> Homo sapiens <400> 1 Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe 1 5 10 15 Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr 20 25 30 Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg 35 40 45 Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp<9001897>50 55 60<00> Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro 65 70 75 80 Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp 85 90 95 Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln 100 105 110 Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala 115 120 125 Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln 130 135 140 <210> 2 <211> 144 <212> PRT <213> Homo sapiens <400> 2 Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe 1 5 10 15 Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr 20 25 30 Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe His Val Val Trp His Arg 35 40 45 Glu Ser Pro Ser Gly Gln Thr Asp Thr Leu Ala Ala Phe Pro Glu Asp '' 50 55 60 Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro 65 70 75 80 Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp 85 90 95 Ser Gly Thr Tyr Val Cys Gly Val Ile Ser Leu Ala Pro Lys Ile Gln 100 105 110 Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala 115 120 125 Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln 130 135 140 <210> 3 <211> 120 <212> PRT <213> Homo sapiens <400> 3 Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile Ser Ala Glu Lys 1 5 10 15 Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr Ala Gln 20 25 30 Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala Ile Cys 35 40 45 Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp Arg Val 50 55 60 Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn 65 70 75 80 Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95 Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110 His Gly Ala Arg Phe Gln Ile Pro 115 120 <210> 4 <211> 498 <212> PRT <213> Homo sapiens <400> 4 Val Trp Glu Lys Thr Val Asn Thr Glu Glu Asn Val Tyr Ala Thr Leu 1 5 10 15 Gly Ser Asp Val Asn Leu Thr Cys Gln Thr Gln Thr Val Gly Phe Phe 20 25 30 Val Gln Met Gln Trp Ser Lys Val Thr Asn Lys Ile Asp Leu Ile Ala 35 40 45 Val Tyr His Pro Gln Tyr Gly Phe Tyr Cys Ala Tyr Gly Arg Pro Cys 50 55 60 Glu Ser Leu Val Thr Phe Thr Glu Thr Pro Glu Asn Gly Ser Lys Trp 65 70 75 80 Thr Leu His Leu Arg Asn Met Ser Cys Ser Val Ser Gly Arg Tyr Glu 85 90 95 Cys Met Leu Val Leu Tyr Pro Glu Gly Ile Gln Thr Lys Ile Tyr Asn 100 105 110 Leu Leu Ile Gln Thr His Val Thr Ala Asp Glu Trp Asn Ser Asn His 115 120 125 Thr Ile Glu Ile Glu Ile Asn Gln Thr Leu Glu Ile Pro Cys Phe Gln 130 135 140 Asn Ser Ser Ser Lys Ile Ser Ser Glu Phe Thr Tyr Ala Trp Ser Val 145 150 155 160 Glu Asn Ser Ser Thr Asp Ser Trp Val Leu Leu Ser Lys Gly Ile Lys 165 170 175 Glu Asp Asn Gly Thr Gln Glu Thr Leu Ile Ser Gln Asn His Leu Ile 180 185 190 Ser Asn Ser Thr Leu Leu Lys Asp Arg Val Lys Leu Gly Thr Asp Tyr 195 200 205 Arg Leu His Leu Ser Pro Val Gln Ile Phe Asp Asp Gly Arg Lys Phe 210 215 220 Ser Cys His Ile Arg Val Gly Pro Asn Lys Ile Leu Arg Ser Ser Thr 225 230 235 240 Thr Val Lys Val Phe Ala Lys Pro Glu Ile Pro Val Ile Val Glu Asn 245 250 255 Asn Ser Thr Asp Val Leu Val Glu Arg Arg Phe Thr Cys Leu Leu Lys 260 265 270 Asn Val Phe Pro Lys Ala Asn Ile Thr Trp Phe Ile Asp Gly Ser Phe 275 280 285 Leu His Asp Glu Lys Glu Gly Ile Tyr Ile Thr Asn Glu Glu Arg Lys 290 295 300 Gly Lys Asp Gly Phe Leu Glu Leu Lys Ser Val Leu Thr Arg Val His 305 310 315 320 Ser Asn Lys Pro Ala Gln Ser Asp Asn Leu Thr Ile Trp Cys Met Ala 325 330 335 Leu Ser Pro Val Pro Gly Asn Lys Val Trp Asn Ile Ser Ser Glu Lys 340 345 350 Ile Thr Phe Leu Leu Gly Ser Glu Ile Ser Ser Thr Asp Pro Pro Leu 355 360 365 Ser Val Thr Glu Ser Thr Leu Asp Thr Gln Pro Ser Pro Ala Ser Ser 370 375 380 Val Ser Pro Ala Arg Tyr Pro Ala Thr Ser Ser Val Thr Leu Val Asp 385 390 395 400 Val Ser Ala Leu Arg Pro Asn Thr Thr Pro Gln Pro Ser Asn Ser Ser 405 410 415 Met Thr Thr Arg Gly Phe Asn Tyr Pro Trp Thr Ser Ser Gly Thr Asp 420 425 430 Thr Lys Lys Ser Val Ser Arg Ile Pro Ser Glu Thr Tyr Ser Ser Ser 435 440 445 Pro Ser Gly Ala Gly Ser Thr Leu His Asp Asn Val Phe Thr Ser Thr 450 455 460 Ala Arg Ala Phe Ser Glu Val Pro Thr Thr Ala Asn Gly Ser Thr Lys 465 470 475 480 Thr Asn His Val His Ile Thr Gly Ile Val Val Asn Lys Pro Lys Asp 485 490 495 Gly Met <210> 5 <211> 151 <212> PRT[[ID=二十一]] [[ID=二十二]]<213> Homo sapiens[[ID=二十三]] [[ID=二十四]]<400> 5[[ID=二十五]] [[ID=二十六]]Met Gly His Arg Thr Leu Val Leu Pro Trp Val Leu Leu Thr Leu Cys[[ID=二十七]] [[ID=二十八]]1 5 10 15[[ID=二十九]] [[ID=三十]]Val Thr Ala Gly Thr Pro Glu Val Trp Val Gln Val Arg Met Glu Ala[[ID=三十一]] [[ID=三十二]]20 25 30[[ID=三十三]] [[ID=三十四]]Thr Glu Leu Ser Ser Phe Thr Ile Arg Cys Gly Phe Leu Gly Ser Gly[[ID=三十五]] [[ID=三十六]]35 40 45[[ID=三十七]] [[ID=三十八]]Ser Ile Ser Leu Val Thr Val Ser Trp Gly Gly Pro Asn Gly Ala Gly[[ID=三十九]] [[ID=四十]]50 55 60[[ID=四十一]] [[ID=四十二]]Gly Thr Thr Leu Ala Val Leu His Pro Glu Arg Gly Ile Arg Gln Trp[[ID=四十三]] [[ID=四十四]]65 70 75 80[[ID=四十五]] It should be noted that in the above translation, the Chinese names for the tags are just for the purpose of showing the mapping. In actual patent translations, the original tags are usually used directly without translation of the tag names themselves. Also, the text "智人" is the Chinese name for "Homo sapiens" which is the scientific name for modern humans.Ala Pro Ala Arg Gln Ala Arg Trp Glu Thr Gln Ser Ser Ile Ser Leu 85 90 95 Ile Leu Glu Gly Ser Gly Ala Ser Ser Pro Cys Ala Asn Thr Thr Phe 100 105 110 Cys Cys Lys Phe Ala Ser Phe Pro Glu Gly Ser Trp Glu Ala Cys Gly 115 120 125 Ser Leu Pro Pro Ser Ser Asp Pro Gly Leu Ser Ala Pro Pro Thr Pro 130 135 140 Ala Pro Ile Leu Arg Ala Asp 145 150 <210> 6 <211> 236 <212> PRT <213> Homo sapiens <400> 6 Glu Glu Val Leu Trp His Thr Ser Val Pro Phe Ala Glu Asn Met Ser 1 5 10 15 Leu Glu Cys Val Tyr Pro Ser Met Gly Ile Leu Thr Gln Val Glu Trp 20 25 30 Phe Lys Ile Gly Thr Gln Gln Asp Ser Ile Ala Ile Phe Ser Pro Thr 35 40 45 His Gly Met Val Ile Arg Lys Pro Tyr Ala Glu Arg Val Tyr Phe Leu 50 55 60 Asn Ser Thr Met Ala Ser Asn Asn Met Thr Leu Phe Phe Arg Asn Ala 65 70 75 80 Ser Glu Asp Asp Val Gly Tyr Tyr Ser Cys Ser Leu Tyr Thr Tyr Pro 85 90 95 Gln Gly Thr Trp Gln Lys Val Ile Gln Val Val Gln Ser Asp Ser Phe 100 105 110 Glu Ala Ala Val Pro Ser Asn Ser His Ile Val Ser Glu Pro Gly Lys 115 120 125 Asn Val Thr Leu Thr Cys Gln Pro Gln Met Thr Trp Pro Val Gln Ala 130 135 140 Val Arg Trp Glu Lys Ile Gln Pro Arg Gln Ile Asp Leu Leu Thr Tyr 145 150 155 160 Cys Asn Leu Val His Gly Arg Asn Phe Thr Ser Lys Phe Pro Arg Gln 165 170 175 Ile Val Ser Asn Cys Ser His Gly Arg Trp Ser Val Ile Val Ile Pro 180 185 190 Asp Val Thr Val Ser Asp Ser Gly Leu Tyr Arg Cys Tyr Leu Gln Ala 195 200 205 Ser Ala Gly Glu Asn Glu Thr Phe Val Met Arg Leu Thr Val Ala Glu 210 215 220 Gly Lys Thr Asp Asn Gln Tyr Thr Leu Phe Val Ala 225 230 235 <210> 7 <211> 330 <212> PRT <213> Homo sapiens <400> 7 Gln Asn Leu Phe Thr Lys Asp Val Thr Val Ile Glu Gly Glu Val Ala 1 5 10 15 Thr Ile Ser Cys Gln Val Asn Lys Ser Asp Asp Ser Val Ile Gln Leu 20 25 30 Leu Asn Pro Asn Arg Gln Thr Ile Tyr Phe Arg Asp Phe Arg Pro Leu 35 40 45 Lys Asp Ser Arg Phe Gln Leu Leu Asn Phe Ser Ser Ser Glu Leu Lys 50 55 60 Val Ser Leu Thr Asn Val Ser Ile Ser Asp Glu Gly Arg Tyr Phe Cys 65 70 75 80 Gln Leu Tyr Thr Asp Pro Pro Gln Glu Ser Tyr Thr Thr Ile Thr Val 85 90 95 Leu Val Pro Pro Arg Asn Leu Met Ile Asp Ile Gln Lys Asp Thr Ala 100 105 110 Val Glu Gly Glu Glu Ile Glu Val Asn Cys Thr Ala Met Ala Ser Lys 115 120 125 Pro Ala Thr Thr Ile Arg Trp Phe Lys Gly Asn Thr Glu Leu Lys Gly 130 135 140 Lys Ser Glu Val Glu Glu Trp Ser Asp Met Tyr Thr Val Thr Ser Gln 145 150 155 160 Leu Met Leu Lys Val His Lys Glu Asp Asp Gly Val Pro Val Ile Cys 165 170 175 Gln Val Glu His Pro Ala Val Thr Gly Asn Leu Gln Thr Gln Arg Tyr 180 185 190 Leu Glu Val Gln Tyr Lys Pro Gln Val His Ile Gln Met Thr Tyr Pro 195 200 205 Leu Gln Gly Leu Thr Arg Glu Gly Asp Ala Leu Glu Leu Thr Cys Glu 210 215 220 Ala Ile Gly Lys Pro Gln Pro Val Met Val Thr Trp Val Arg Val Asp 225 230 235 240 Asp Glu Met Pro Gln His Ala Val Leu Ser Gly Pro Asn Leu Phe Ile 245 250 255 Asn Asn Leu Asn Lys Thr Asp Asn Gly Thr Tyr Arg Cys Glu Ala Ser 260 265 270 Asn Ile Val Gly Lys Ala His Ser Asp Tyr Met Leu Tyr Val Tyr Asp 275 280 285 Pro Pro Thr Thr Ile Pro Pro Pro Thr Thr Thr Thr Thr Thr Thr Thr 290 295 300 Thr Thr Thr Thr Thr Ile Leu Thr Ile Ile Thr Asp Ser Arg Ala Gly 305 310 315 320 Glu Glu Gly Ser Ile Arg Ala Val Asp His 325 330 <210> 8 <211> 342 <212> PRT <213> Homo sapiens <400> 8 Pro Ile Ile Val Glu Pro His Val Thr Ala Val Trp Gly Lys Asn Val 1 5 10 15 Ser Leu Lys Cys Leu Ile Glu Val Asn Glu Thr Ile Thr Gln Ile Ser 20 25 30 Trp Glu Lys Ile His Gly Lys Ser Ser Gln Thr Val Ala Val His His 35 40 45 Pro Gln Tyr Gly Phe Ser Val Gln Gly Glu Tyr Gln Gly Arg Val Leu 50 55 60 Phe Lys Asn Tyr Ser Leu Asn Asp Ala Thr Ile Thr Leu His Asn Ile 65 70 75 80 Gly Phe Ser Asp Ser Gly Lys Tyr Ile Cys Lys Ala Val Thr Phe Pro 85 90 95 Leu Gly Asn Ala Gln Ser Ser Thr Thr Val Thr Val Leu Val Glu Pro 100 105 110 Thr Val Ser Leu Ile Lys Gly Pro Asp Ser Leu Ile Asp Gly Gly Asn 115 120 125 Glu Thr Val Ala Ala Ile Cys Ile Ala Ala Thr Gly Lys Pro Val Ala 130 135 140 His Ile Asp Trp Glu Gly Asp Leu Gly Glu Met Glu Ser Thr Thr Thr 145 150 155 160 Ser Phe Pro Asn Glu Thr Ala Thr Ile Ile Ser Gln Tyr Lys Leu Phe 165 170 175 Pro Thr Arg Phe Ala Arg Gly Arg Arg Ile Thr Cys Val Val Lys His 180 185 190 Pro Ala Leu Glu Lys Asp Ile Arg Tyr Ser Phe Ile Leu Asp Ile Gln 195 200 205 Tyr Ala Pro Glu Val Ser Val Thr Gly Tyr Asp Gly Asn Trp Phe Val 210 215 220 Gly Arg Lys Gly Val Asn Leu Lys Cys Asn Ala Asp Ala Asn Pro Pro 225 230 235 240 Pro Phe Lys Ser Val Trp Ser Arg Leu Asp Gly Gln Trp Pro Asp Gly 245 250 255 Leu Leu Ala Ser Asp Asn Thr Leu His Phe Val His Pro Leu Thr Phe 260 265 270 Asn Tyr Ser Gly Val Tyr Ile Cys Lys Val Thr Asn Ser Leu Gly Gln 275 280 285 Arg Ser Asp Gln Lys Val Ile Tyr Ile Ser Asp Pro Pro Thr Thr Thr 290 295 300 Thr Leu Gln Pro Thr Ile Gln Trp His Pro Ser Thr Ala Asp Ile Glu 305 310 315 320 Asp Leu Ala Thr Glu Pro Lys Lys Leu Pro Phe Pro Leu Ser Thr Leu 325 330 335 Ala Thr Ile Lys Asp Asp 340 <210> 9 <211> 285 <212> PRT <213> Homo sapiens <400> 9 Ala Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp 1 5 10 15 Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His Leu Asp Gly Gln 20 25 30 Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly 35 40 45 Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr 50 55 60 Arg Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His 65 70 75 80 Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val 85 90 95 Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr 100 105 110 Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Lys Glu Trp 115 120 125 Thr Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg 130 135 140 Asn Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala 145 150 155 160 Met His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Lys Ser Gly 165 170 175 Val Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser 180 185 190 Glu Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Gly Phe 195 200 205 Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp Gly Val Ser Leu 210 215 220 Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly 225 230 235 240 Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln Gly Glu Glu Gln 245 250 255 Arg Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro 260 265 270 Val Pro Ser Gly Lys Val Leu Val Leu Gln Ser His Trp 275 280 285 <210> 10 <211> 18 <212> PRT <213> Homo sapiens <400> 10 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser <210> 11 <211> 22 <212> PRT <213> Homo sapiens <400> 11 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly 20 <210> 12 <211> 103 It should be noted that in the original text, there is a tag " " which seems to be an incorrect encoding. It is likely a typo and should probably be " ". Also, " " should likely be " ". The above translation has made the best effort based on the provided text while keeping these potential errors as they are in the original. <212> PRT <213> Homo sapiens <400> 12 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 1 5 10 15 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 20 25 30 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 35 40 45 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 50 55 60 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 65 70 75 80 Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 85 90 95 Lys Thr Ile Ser Lys Ala Lys 100 <210> 13 <211> 107 <212> PRT <213> Homo sapiens <400> 13 Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe<00022620 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 [[ID=I4]]Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 14 <211> 396 <212> PRT<L <213> Artificial Sequence <220> <223> Human PD-1-hFcA <400> 14 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Ala Ser Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn 20 25 30 Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn 35 40 45 Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu 50 55 60 Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala 65 70 75 80 Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val 85 90 95 Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala 100 105 110 Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala 115 120 125 Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr 130 135 140 Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg 145 150 155 160 Pro Ala Gly Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 165 170 175 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 180 185 190 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 195 200 205 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 210 215 220 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 225 230 235 240 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 245 250 255 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val 260 265 270 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 275 280 285 Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg 290 295 300 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly 305 310 315 320 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 325 330 335 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 340 345 350 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 355 360 365 Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His 370 375 380 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 385 390 395 <210> 15 <211> 396 <212> PRT <213> Synthetic sequence <220> <223> High affinity human PD-1-hFcA <400> 15 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Ala Ser Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn 20 25 30 Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn 35 40 45 Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe His Val 50 55 60 Val Trp His Arg Glu Ser Pro Ser Gly Gln Thr Asp Thr Leu Ala Ala 65 70 75 80 Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val 85 90 95 Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala 100 105 110 Arg Arg Asn Asp Ser Gly Thr Tyr Val Cys Gly Val Ile Ser Leu Ala 115 120 125 Pro Lys Ile Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr 130 135 140 Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg 145 150 155 160 Pro Ala Gly Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 165 170 175 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 180 185 190 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 195 200 205 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 210 215 220 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 225 230 235 240 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 245 250 255 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val 260 265 270 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 275 280 285 Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg 290 295 300 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly 305 310 315 320 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 325 330 335 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 340 345 350 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 355 360 365 Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His 370 375 380 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 385 390 395 <210> 16 <211> 403 <212> PRT <213> Synthetic Sequence <220> <223> Human CD112R-hFcA <400> 16 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Ala Ser Met Gly His Arg Thr Leu Val Leu Pro Trp Val Leu 20 25 30 Leu Thr Leu Cys Val Thr Ala Gly Thr Pro Glu Val Trp Val Gln Val 35 40 45 Arg Met Glu Ala Thr Glu Leu Ser Ser Phe Thr Ile Arg Cys Gly Phe 50 55 60 Leu Gly Ser Gly Ser Ile Ser Leu Val Thr Val Ser Trp Gly Gly Pro 65 70 75 80 Asn Gly Ala Gly Gly Thr Thr Leu Ala Val Leu His Pro Glu Arg Gly 85 90 95 Ile Arg Gln Trp Ala Pro Ala Arg Gln Ala Arg Trp Glu Thr Gln Ser 100 105 110 Ser Ile Ser Leu Ile Leu Glu Gly Ser Gly Ala Ser Ser Pro Cys Ala 115 120 125 Asn Thr Thr Phe Cys Cys Lys Phe Ala Ser Phe Pro Glu Gly Ser Trp 130 135 140 Glu Ala Cys Gly Ser Leu Pro Pro Ser Ser Asp Pro Gly Leu Ser Ala 145 150 155 160 Pro Pro Thr Pro Ala Pro Ile Leu Arg Ala Asp Glu Pro Lys Ser Ser 165 170 175 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 180 185 190 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 195 200 205 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 210 215 220 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 225 230 235 240 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 245 250 255 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 260 265 270 Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 275 280 285 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 290 295 300 Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 305 310 315 320 Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 325 330 335 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 340 345 350 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 355 360 365 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 370 375 380 His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 385 390 395 400 Pro Gly Lys <210> 17 <211> 370 <212> PRT <213> Artificial Sequence <220> <223> Human TIGIT-hFcA <400> 17 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15<00​​​​Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser Thr Thr 35 40 45 Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu Leu Ala 50 55 60 Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe Lys Asp 65 70 75 80 Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr 85 90 95 Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp 100 105 110 Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val 115 120 125 Ala Glu His Gly Ala Arg Phe Gln Ile Pro Glu Pro Lys Ser Ser Asp 130 135 140 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 145 150 155 160 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 165 170 175 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 180 185 190 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 195 200 205 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 210 215 220 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 225 230 235 240 Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 245 250 255 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys 260 265 270 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 275 280 285 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 290 295 300 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 305 310 315 320 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 325 330 335 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His 340 345 350 Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 355 360 365 Gly Lys 370 <210> 18 <211> 748 <212> PRT <213> Artificial Sequence <220> <223> Human CD96-hFcA <400> 18 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Val Trp Glu Lys Thr Val Asn Thr Glu Glu Asn Val Tyr Ala 20 25 30 Thr Leu Gly Ser Asp Val Asn Leu Thr Cys Gln Thr Gln Thr Val Gly 35 40 45​​​​​​​​​​​​​​​​​Tyr Glu Cys Met Leu Val Leu Tyr Pro Glu Gly Ile Gln Thr Lys Ile 115 120 125 Tyr Asn Leu Leu Ile Gln Thr His Val Thr Ala Asp Glu Trp Asn Ser 130 135 140 Asn His Thr Ile Glu Ile Glu Ile Asn Gln Thr Leu Glu Ile Pro Cys 145 150 155 160 Phe Gln Asn Ser Ser Ser Lys Ile Ser Ser Glu Phe Thr Tyr Ala Trp 165 170 175 Ser Val Glu Asn Ser Ser Thr Asp Ser Trp Val Leu Leu Ser Lys Gly 180 185 190 Ile Lys Glu Asp Asn Gly Thr Gln Glu Thr Leu Ile Ser Gln Asn His 195 200 205 Leu Ile Ser Asn Ser Thr Leu Leu Lys Asp Arg Val Lys Leu Gly Thr 210 215 220 Asp Tyr Arg Leu His Leu Ser Pro Val Gln Ile Phe Asp Asp Gly Arg 225 230 235 240 Lys Phe Ser Cys His Ile Arg Val Gly Pro Asn Lys Ile Leu Arg Ser 245 250 255 Ser Thr Thr Val Lys Val Phe Ala Lys Pro Glu Ile Pro Val Ile Val 260 265 270 Glu Asn Asn Ser Thr Asp Val Leu Val Glu Arg Arg Phe Thr Cys Leu 275 280 285 Leu Lys Asn Val Phe Pro Lys Ala Asn Ile Thr Trp Phe Ile Asp Gly 290 295 300 Ser Phe Leu His Asp Glu Lys Glu Gly Ile Tyr Ile Thr Asn Glu Glu 305 310 315 320 Arg Lys Gly Lys Asp Gly Phe Leu Glu Leu Lys Ser Val Leu Thr Arg 325 330 335 Val His Ser Asn Lys Pro Ala Gln Ser Asp Asn Leu Thr Ile Trp Cys 340 345 350 Met Ala Leu Ser Pro Val Pro Gly Asn Lys Val Trp Asn Ile Ser Ser 355 360 365 Glu Lys Ile Thr Phe Leu Leu Gly Ser Glu Ile Ser Ser Thr Asp Pro 370 375 380 Pro Leu Ser Val Thr Glu Ser Thr Leu Asp Thr Gln Pro Ser Pro Ala 385 390 395 400 Ser Ser Val Ser Pro Ala Arg Tyr Pro Ala Thr Ser Ser Val Thr Leu 405 410 415 Val Asp Val Ser Ala Leu Arg Pro Asn Thr Thr Pro Gln Pro Ser Asn 420 425 430 Ser Ser Met Thr Thr Arg Gly Phe Asn Tyr Pro Trp Thr Ser Ser Gly 435 440 445 Thr Asp Thr Lys Lys Ser Val Ser Arg Ile Pro Ser Glu Thr Tyr Ser 450 455 460 Ser Ser Pro Ser Gly Ala Gly Ser Thr Leu His Asp Asn Val Phe Thr 465 470 475 480 Ser Thr Ala Arg Ala Phe Ser Glu Val Pro Thr Thr Ala Asn Gly Ser 485 490 495 Thr Lys Thr Asn His Val His Ile Thr Gly Ile Val Val Asn Lys Pro 500 505 510 Lys Asp Gly Met Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 515 520 525 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 530 535 540 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 545 550 555 560 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 565 570 575 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 580 585 590 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 595 600 605 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val 610 615 620 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 625 630 635 640 Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg 645 650 655 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly 660 665 670 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 675 680 685 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 690 695 700 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 705 710 715 720 Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His 725 730 735 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 740 745 <210> 19 <211> 486 <212> PRT <213> artificial sequence <220> <223> Human CD226-hFcA <400> 19 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Glu Glu Val Leu Trp His Thr Ser Val Pro Phe Ala Glu Asn 20 25 30 Met Ser Leu Glu Cys Val Tyr Pro Ser Met Gly Ile Leu Thr Gln Val 35 40 45 Glu Trp Phe Lys Ile Gly Thr Gln Gln Asp Ser Ile Ala Ile Phe Ser 50 55 60 Pro Thr His Gly Met Val Ile Arg Lys Pro Tyr Ala Glu Arg Val Tyr 65 70 75 80 Phe Leu Asn Ser Thr Met Ala Ser Asn Asn Met Thr Leu Phe Phe Arg 85 90 95 Asn Ala Ser Glu Asp Asp Val Gly Tyr Tyr Ser Cys Ser Leu Tyr Thr 100 105 110 Tyr Pro Gln Gly Thr Trp Gln Lys Val Ile Gln Val Val Gln Ser Asp 115 120 125 Ser Phe Glu Ala Ala Val Pro Ser Asn Ser His Ile Val Ser Glu Pro 130 135 140 Gly Lys Asn Val Thr Leu Thr Cys Gln Pro Gln Met Thr Trp Pro Val 145 150 155 160 Gln Ala Val Arg Trp Glu Lys Ile Gln Pro Arg Gln Ile Asp Leu Leu 165 170 175 Thr Tyr Cys Asn Leu Val His Gly Arg Asn Phe Thr Ser Lys Phe Pro 180 185 190 Arg Gln Ile Val Ser Asn Cys Ser His Gly Arg Trp Ser Val Ile Val 195 200 205 Ile Pro Asp Val Thr Val Ser Asp Ser Gly Leu Tyr Arg Cys Tyr Leu 210 215 220 Gln Ala Ser Ala Gly Glu Asn Glu Thr Phe Val Met Arg Leu Thr Val 225 230 235 240 Ala Glu Gly Lys Thr Asp Asn Gln Tyr Thr Leu Phe Val Ala Glu Pro 245 250 255 Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 260 265 270 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 275 280 285 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 290 295 300 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 305 310 315 320 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 325 330 335 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 340 345 350 Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro 355 360 365 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 370 375 380 Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 385 390 395 400 Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 405 410 415 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 420 425 430 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 435 440 445 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 450 455 460 Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu 465 470 475 480 Serum Leu Serum Pro Gly Lys 485 <210> 20 <211> 580 <212> PRT <213> artificial sequence <220> <223> Human NECL2-hFcA <400> 20 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Gln Asn Leu Phe Thr Lys Asp Val Thr Val Ile Glu Gly Glu 20 25 30 Val Ala Thr Ile Ser Cys Gln Val Asn Lys Ser Asp Asp Ser Val Ile 35 40 45 Gln Leu Leu Asn Pro Asn Arg Gln Thr Ile Tyr Phe Arg Asp Phe Arg 50 55 60 Pro Leu Lys Asp Ser Arg Phe Gln Leu Leu Asn Phe Ser Ser Ser Glu 65 70 75 80 Leu Lys Val Ser Leu Thr Asn Val Ser Ile Ser Asp Glu Gly Arg Tyr 85 90 95 Phe Cys Gln Leu Tyr Thr Asp Pro Pro Gln Glu Ser Tyr Thr Thr Ile 100 105 110 Thr Val Leu Val Pro Pro Arg Asn Leu Met Ile Asp Ile Gln Lys Asp 115 120 125 Thr Ala Val Glu Gly Glu Glu Ile Glu Val Asn Cys Thr Ala Met Ala 130 135 140 Ser Lys Pro Ala Thr Thr Ile Arg Trp Phe Lys Gly Asn Thr Glu Leu 145 150 155 160 Lys Gly Lys Ser Glu Val Glu Glu Trp Ser Asp Met Tyr Thr Val Thr 165 170 175 Ser Gln Leu Met Leu Lys Val His Lys Glu Asp Asp Gly Val Pro Val 180 185 190 Ile Cys Gln Val Glu His Pro Ala Val Thr Gly Asn Leu Gln Thr Gln 195 200 205 Arg Tyr Leu Glu Val Gln Tyr Lys Pro Gln Val His Ile Gln Met Thr 210 215 220 Tyr Pro Leu Gln Gly Leu Thr Arg Glu Gly Asp Ala Leu Glu Leu Thr 225 230 235 240 Cys Glu Ala Ile Gly Lys Pro Gln Pro Val Met Val Thr Trp Val Arg 245 250 255 Val Asp Asp Glu Met Pro Gln His Ala Val Leu Ser Gly Pro Asn Leu 260 265 270 Phe Ile Asn Asn Leu Asn Lys Thr Asp Asn Gly Thr Tyr Arg Cys Glu 275 280 285 Ala Ser Asn Ile Val Gly Lys Ala His Ser Asp Tyr Met Leu Tyr Val 290 295 300 Tyr Asp Pro Pro Thr Thr Ile Pro Pro Pro Thr Thr Thr Thr Thr Thr 305 310 315 320 Thr Thr Thr Thr Thr Thr Thr Ile Leu Thr Ile Ile Thr Asp Ser Arg 325 330 335 Ala Gly Glu Glu Gly Ser Ile Arg Ala Val Asp His Glu Pro Lys Ser 340 345 350 Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 355 360 365 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 370 375 380 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 385 390 395 400 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 405 410 415 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 420 425 430 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 435 440 445 Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro 450 455 460 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 465 470 475 480 Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val 485 490 495 Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 500 505 510 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 515 520 525 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 530 535 540 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 545 550 555 560 Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu 565 570 575 Ser Pro Gly Lys 580 <210> 21 <211> 594 <212> PRT <213> Artificial Sequence <220> <223> Human CD113-hFcA <400> 21 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Ala Ser Pro Ile Ile Val Glu Pro His Val Thr Ala Val Trp 20 25 30 Gly Lys Asn Val Ser Leu Lys Cys Leu Ile Glu Val Asn Glu Thr Ile 35 40 45 ​​​​​​​​​​​​​​​​Val Thr Phe Pro Leu Gly Asn Ala Gln Ser Ser Thr Thr Val Thr Val 115 120 125 Leu Val Glu Pro Thr Val Ser Leu Ile Lys Gly Pro Asp Ser Leu Ile 130 135 140 Asp Gly Gly Asn Glu Thr Val Ala Ala Ile Cys Ile Ala Ala Thr Gly 145 150 155 160 Lys Pro Val Ala His Ile Asp Trp Glu Gly Asp Leu Gly Glu Met Glu 165 170 175 Ser Thr Thr Thr Ser Phe Pro Asn Glu Thr Ala Thr Ile Ile Ser Gln 180 185 190 Tyr Lys Leu Phe Pro Thr Arg Phe Ala Arg Gly Arg Arg Ile Thr Cys 195 200 205 Val Val Lys His Pro Ala Leu Glu Lys Asp Ile Arg Tyr Ser Phe Ile 210 215 220 Leu Asp Ile Gln Tyr Ala Pro Glu Val Ser Val Thr Gly Tyr Asp Gly 225 230 235 240 Asn Trp Phe Val Gly Arg Lys Gly Val Asn Leu Lys Cys Asn Ala Asp 245 250 255 Ala Asn Pro Pro Pro Phe Lys Ser Val Trp Ser Arg Leu Asp Gly Gln 260 265 270 Trp Pro Asp Gly Leu Leu Ala Ser Asp Asn Thr Leu His Phe Val His 275 280 285 Pro Leu Thr Phe Asn Tyr Ser Gly Val Tyr Ile Cys Lys Val Thr Asn 290 295 300 Ser Leu Gly Gln Arg Ser Asp Gln Lys Val Ile Tyr Ile Ser Asp Pro 305 310 315 320 Pro Thr Thr Thr Thr Leu Gln Pro Thr Ile Gln Trp His Pro Ser Thr 325 330 335 Ala Asp Ile Glu Asp Leu Ala Thr Glu Pro Lys Lys Leu Pro Phe Pro 340 345 350 Leu Ser Thr Leu Ala Thr Ile Lys Asp Asp Glu Pro Lys Ser Ser Asp 355 360 365 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 370 375 380 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 385 390 395 400 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 405 410 415 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 420 425 430 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 435 440 445 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 450 455 460 Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 465 470 475 480 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys 485 490 495 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 500 505 510 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 515 520 525 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 530 535 540 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 545 550 555 560 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His 565 570 575 Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 580 585 590 Gly Lys <210> 22 <211> 537 <212> PRT <213> Artificial sequence <220> <223> Human MICA-hFcA <400> 22 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Ala Ser Ala Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val 20 25 30 Leu Ser Trp Asp Gly Ser Val Gln Ser Gly Phe Leu Thr Glu Val His 35 40 45 Leu Asp Gly Gln Pro Phe Leu Arg Cys Asp Arg Gln Lys Cys Arg Ala 50 55 60 Lys Pro Gln Gly Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp 65 70 75 80 Asp Arg Glu Thr Arg Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met 85 90 95 Thr Leu Ala His Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln 100 105 110 Glu Ile Arg Val Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser 115 120 125 Gln His Phe Tyr Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu 130 135 140 Thr Lys Glu Trp Thr Met Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala 145 150 155 160 Met Asn Val Arg Asn Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr 165 170 175 His Tyr His Ala Met His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr 180 185 190 Leu Lys Ser Gly Val Val Leu Arg Arg Thr Val Pro Pro Met Val Asn 195 200 205 Val Thr Arg Ser Glu Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg 210 215 220 Ala Ser Gly Phe Tyr Pro Trp Asn Ile Thr Leu Ser Trp Arg Gln Asp 225 230 235 240 Gly Val Ser Leu Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro 245 250 255 Asp Gly Asn Gly Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Gln 260 265 270 Gly Glu Glu Gln Arg Phe Thr Cys Tyr Met Glu His Ser Gly Asn His 275 280 285 Ser Thr His Pro Val Pro Ser Gly Lys Val Leu Val Leu Gln Ser His 290 295 300 Trp Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro 305 310 315 320 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 325 330 335 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 340 345 350 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 355 360 365 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 370 375 380 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 385 390 395 400 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys 405 410 415 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 420 425 430 Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu 435 440 445 Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro 450 455 460 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 465 470 475 480 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 485 490 495 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 500 505 510 Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln 515 520 525 Lys Ser Leu Ser Leu Ser Pro Gly Lys 530 535 <210> 23 <211> 252 <212> PRT <213> artificial sequence <220> <223> HumansFcA <400> 23 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Ala Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 20 25 30 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 35 40 45 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 50 55 60 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 65 70 75 80 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 85 90 95 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 100 105 110 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val 115 120 125 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 130 135 140 Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg 145 150 155 160 Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly 165 170 175 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 180 185 190 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 195 200 205 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 210 215 220 Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His 225 230 235 240 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 245 250 <210> 24 <211> 323 <212> PRT <213> Homo sapiens <400> 24 Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln Ala Pro Thr Gln 1 5 10 15 Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro Cys Tyr Leu Gln 20 25 30 Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu Thr Trp Ala Arg 35 40 45 His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln Thr Gln Gly Pro 50 55 60 Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala Ala Arg Leu Gly 65 70 75 80 Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly Leu Arg Val Glu 85 90 95 Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe Pro Gln Gly Ser 100 105 110 Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys Pro Gln Asn Thr 115 120 125 Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro Val Pro Met Ala 130 135 140 Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln Ile Thr Trp His 145 150 155 160 Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val Pro Gly Phe Leu 165 170 175 Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu Val Pro Ser Ser 180 185 190 Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu His Glu Ser Phe 195 200 205 Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val Tyr Tyr Pro Pro 210 215 220 Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr Leu Gly Gln Asn 225 230 235 240 Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro Glu Pro Thr Gly 245 250 255 Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro Phe Ala Val Ala 260 265 270 Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys Pro Ile Asn Thr 275 280 285 Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala Arg Gln Ala Glu 290 295 300 Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu His Ser Gly Met 305 310 315 320 Ser Arg Asn <210> 25 <211> 120 <212> PRT <213> Homo sapiens <400> 2​​​​​​​​​​​​​​ 50 55 60 Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser Leu Thr Val Asn 65 70 75 80 Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr Pro Asp Gly Thr 85 90 95 Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser Ser Val Ala Glu 100 105 110 His Gly Ala Arg Phe Gln Ile Pro 115 120 <210> 26 <211> 17​​​​​​​​​​​​​​​​​​​​​​​​​​​Thr Leu Asp Asp His Gly Thr Tyr Cys Cys Arg Ile Gln Phe Pro Gly 85 90 95 Leu Met Asn Asp Lys Lys Leu Glu Leu Lys Leu Asp Ile Lys Ala Ala 100 105 110 Lys Val Thr Pro Ala Gln Thr Ala His Gly Asp Ser Thr Thr Ala Ser / 115 120 125 Pro Arg Thr Leu Thr Thr Glu Arg Asn Gly Ser Glu Thr Gln Thr Leu 130 135 140 Val Thr Leu His Asn Asn Asn Gly Thr Lys Ile Ser Thr Trp Ala Asp 145 150 155 160 Glu Ile Lys Asp Ser Gly Glu Thr Ile Arg 165 170 <210> 27 <211> 342 <212> PRT <213> Homo sapiens <400> 27 Pro Ile Ile Val Glu Pro His Val Thr Ala Val Trp Gly Lys Asn Val 1 5 10 15 Ser Leu Lys Cys Leu Ile Glu Val Asn Glu Thr Ile Thr Gln Ile Ser 20 25 30 Trp Glu Lys Ile His Gly Lys Ser Ser Gln Thr Val Ala Val His His 35 40 45 Pro Gln Tyr Gly Phe Ser Val Gln Gly Glu Tyr Gln Gly Arg Val Leu 50 55 60 Phe Lys Asn Tyr Ser Leu Asn Asp Ala Thr Ile Thr Leu His Asn Ile 65 70 75 80 Gly Phe Ser Asp Ser Gly Lys Tyr Ile Cys Lys Ala Val Thr Phe Pro 85 90 95 Leu Gly Asn Ala Gln Ser Ser Thr Thr Val Thr Val Leu Val Glu Pro 100 105 110 Thr Val Ser Leu Ile Lys Gly Pro Asp Ser Leu Ile Asp Gly Gly Asn 115 120 125 Glu Thr Val Ala Ala Ile Cys Ile Ala Ala Thr Gly Lys Pro Val Ala 130 135 140 His Ile Asp Trp Glu Gly Asp Leu Gly Glu Met Glu Ser Thr Thr Thr 145 150 155 160 Ser Phe Pro Asn Glu Thr Ala Thr Ile Ile Ser Gln Tyr Lys Leu Phe 165 170 175 Pro Thr Arg Phe Ala Arg Gly Arg Arg Ile Thr Cys Val Val Lys His 180 185 190 Pro Ala Leu Glu Lys Asp Ile Arg Tyr Ser Phe Ile Leu Asp Ile Gln 195 200 205 Tyr Ala Pro Glu Val Ser Val Thr Gly Tyr Asp Gly Asn Trp Phe Val 210 215 220 Gly Arg Lys Gly Val Asn Leu Lys Cys Asn Ala Asp Ala Asn Pro Pro 225 230 235 240 Pro Phe Lys Ser Val Trp Ser Arg Leu Asp Gly Gln Trp Pro Asp Gly 245 250 255 Leu Leu Ala Ser Asp Asn Thr Leu His Phe Val His Pro Leu Thr Phe 260 265 270 Asn Tyr Ser Gly Val Tyr Ile Cys Lys Val Thr Asn Ser Leu Gly Gln 275 280 285 Arg Ser Asp Gln Lys Val Ile Tyr Ile Ser Asp Pro Pro Thr Thr Thr 290 295 300 Thr Leu Gln Pro Thr Ile Gln Trp His Pro Ser Thr Ala Asp Ile Glu 305 310 315 320 Asp Leu Ala Thr Glu Pro Lys Lys Leu Pro Phe Pro Leu Ser Thr Leu 325 330 335 Ala Thr Ile Lys Asp Asp 340 <210> 28 <211> 330 <212> PRT <213> Homo sapiens <400> 28 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 29 <211> 330 <212> PRT <213> Synthetic sequence <220> <223> Component A Y349C and T366W <400> 29 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn Ser Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 30 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Component B D356C, T366S, L368A and Y407V <400> 30 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 31 <211> 37 <212> PRT <213> Artificial Sequence <220> <223> Linker <400> 31 Gly Asp Pro Leu Val Thr Ala Ala Ser Val Leu Glu Phe Gly Gly Ser 1 5 10 15 Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly 20 25 30 Gly Gly Ser Asp Ile 35 <210> 32 <211> 575 <212> PRT <213> Artificial Sequence <220> <223> Human CD155-hFcB <400> 32 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Ala Ser Trp Pro Pro Pro Gly Thr Gly Asp Val Val Val Gln 20 25 30 Ala Pro Thr Gln Val Pro Gly Phe Leu Gly Asp Ser Val Thr Leu Pro 35 40 45 Cys Tyr Leu Gln Val Pro Asn Met Glu Val Thr His Val Ser Gln Leu 50 55 60 Thr Trp Ala Arg His Gly Glu Ser Gly Ser Met Ala Val Phe His Gln 65 70 75 80 Thr Gln Gly Pro Ser Tyr Ser Glu Ser Lys Arg Leu Glu Phe Val Ala 85 90 95 Ala Arg Leu Gly Ala Glu Leu Arg Asn Ala Ser Leu Arg Met Phe Gly 100 105 110 Leu Arg Val Glu Asp Glu Gly Asn Tyr Thr Cys Leu Phe Val Thr Phe [[ID=5 Pro Gln Gly Ser Arg Ser Val Asp Ile Trp Leu Arg Val Leu Ala Lys 130 135 140 Pro Gln Asn Thr Ala Glu Val Gln Lys Val Gln Leu Thr Gly Glu Pro 145 150 155 160 Val Pro Met Ala Arg Cys Val Ser Thr Gly Gly Arg Pro Pro Ala Gln 165 170 175 Ile Thr Trp His Ser Asp Leu Gly Gly Met Pro Asn Thr Ser Gln Val 180 185 190 Pro Gly Phe Leu Ser Gly Thr Val Thr Val Thr Ser Leu Trp Ile Leu 195 200 205 Val Pro Ser Ser Gln Val Asp Gly Lys Asn Val Thr Cys Lys Val Glu 210 215 220 His Glu Ser Phe Glu Lys Pro Gln Leu Leu Thr Val Asn Leu Thr Val 225 230 235 240 Tyr Tyr Pro Pro Glu Val Ser Ile Ser Gly Tyr Asp Asn Asn Trp Tyr 245 250 255 Leu Gly Gln Asn Glu Ala Thr Leu Thr Cys Asp Ala Arg Ser Asn Pro 260 265 270 Glu Pro Thr Gly Tyr Asn Trp Ser Thr Thr Met Gly Pro Leu Pro Pro 275 280 285 Phe Ala Val Ala Gln Gly Ala Gln Leu Leu Ile Arg Pro Val Asp Lys 290 295 300 Pro Ile Asn Thr Thr Leu Ile Cys Asn Val Thr Asn Ala Leu Gly Ala 305 310 315 320 Arg Gln Ala Glu Leu Thr Val Gln Val Lys Glu Gly Pro Pro Ser Glu 325 330 335 His Ser Gly Met Ser Arg Asn Glu Pro Lys Ser Ser Asp Lys Thr His 340 345 350 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 355 360 365 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 370 375 380 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 385 390 395 400 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 405 410 415 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 420 425 430 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 435 440 445 Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 450 455 460 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 465 470 475 480 Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala 485 490 495 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 500 505 510 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 515 520 525 Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg 530 535 540 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu 545 550 555 560 His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 565 570 575 <210> 33 <211> 372 <212> PRT <213> Synthetic Sequence <220> <223> Human TIGIT-hFcB <400> 33 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Ala Ser Met Met Thr Gly Thr Ile Glu Thr Thr Gly Asn Ile 20 25 30 Ser Ala Glu Lys Gly Gly Ser Ile Ile Leu Gln Cys His Leu Ser Ser 35 40 45 Thr Thr Ala Gln Val Thr Gln Val Asn Trp Glu Gln Gln Asp Gln Leu 50 55 60 Leu Ala Ile Cys Asn Ala Asp Leu Gly Trp His Ile Ser Pro Ser Phe 65 70 75 80 Lys Asp Arg Val Ala Pro Gly Pro Gly Leu Gly Leu Thr Leu Gln Ser 85 90 95 Leu Thr Val Asn Asp Thr Gly Glu Tyr Phe Cys Ile Tyr His Thr Tyr 100 105 110 Pro Asp Gly Thr Tyr Thr Gly Arg Ile Phe Leu Glu Val Leu Glu Ser 115 120 125 Ser Val Ala Glu His Gly Ala Arg Phe Gln Ile Pro Glu Pro Lys Ser 130 135 140 Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 145 150 155 160 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 165 170 175 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 180 185 190 His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 195 200 205 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 210 215 220 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 225 230 235 240 Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro 245 250 255 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 260 265 270 Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val 275 280 285 Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 290 295 300 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 305 310 315 320 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr 325 330 335 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 340 345 350 Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu 355 360 365 Ser Pro Gly Lys 370 <210> 34 <211> 422 <212> PRT <213> Artificial Sequence <220> <223> Mouse TIM-3-hFcB <400> 34 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15<00^{3390}>Tyr Ser Ala Ser Leu Glu Asp Gly Tyr Lys Val Glu Val Gly Lys Asn 20 25 30 Ala Tyr Leu Pro Cys Ser Tyr Thr Leu Pro Thr Ser Gly Thr Leu Val 35 40 45 Pro Met Cys Trp Gly Lys Gly Phe Cys Pro Trp Ser Gln Cys Thr Asn 50 55 60 Glu Leu Leu Arg Thr Asp Glu Arg Asn Val Thr Tyr Gln Lys Ser Ser 65 70 75 80 Arg Tyr Gln Leu Lys Gly Asp Leu Asn Lys Gly Asp Val Ser Leu Ile 85 90 95 Ile Lys Asn Val Thr Leu Asp Asp His Gly Thr Tyr Cys Cys Arg Ile 100 105 110 Gln Phe Pro Gly Leu Met Asn Asp Lys Lys Leu Glu Leu Lys Leu Asp 115 120 125 Ile Lys Ala Ala Lys Val Thr Pro Ala Gln Thr Ala His Gly Asp Ser 130 135 140 Thr Thr Ala Ser Pro Arg Thr Leu Thr Thr Glu Arg Asn Gly Ser Glu 145 150 155 160 Thr Gln Thr Leu Val Thr Leu His Asn Asn Asn Gly Thr Lys Ile Ser 165 170 175 Thr Trp Ala Asp Glu Ile Lys Asp Ser Gly Glu Thr Ile Arg Glu Pro 180 185 190 Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 195 200 205 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 210 215 220 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 225 230 235 240 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 245 250 255 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 260 265 270 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 275 280 285 Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro 290 295 300 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 305 310 315 320 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn 325 330 335 Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile 340 345 350 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 355 360 365 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys 370 375 380 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 385 390 395 400 Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu 405 410 415 Ser Leu Ser Pro Gly Lys 420 <210> 35 <211> 594 <212> PRT <213> Artificial Sequence <220> <223> Human CD113-hFcB <400> 35 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Ala Ser Pro Ile Ile Val Glu Pro His Val Thr Ala Val Trp 20 25 30 Gly Lys Asn Val Ser Leu Lys Cys Leu Ile Glu Val Asn Glu Thr Ile 35 40 45 Thr Gln Ile Ser Trp Glu Lys Ile His Gly Lys Ser Ser Gln Thr Val 50 55 60 Ala Val His His Pro Gln Tyr Gly Phe Ser Val Gln Gly Glu Tyr Gln 65 70 75 80 Gly Arg Val Leu Phe Lys Asn Tyr Ser Leu Asn Asp Ala Thr Ile Thr 85 90 95 Leu His Asn Ile Gly Phe Ser Asp Ser Gly Lys Tyr Ile Cys Lys Ala 100 105 110 Val Thr Phe Pro Leu Gly Asn Ala Gln Ser Ser Thr Thr Val Thr Val 115 120 125 Leu Val Glu Pro Thr Val Ser Leu Ile Lys Gly Pro Asp Ser Leu Ile 130 135 140 Asp Gly Gly Asn Glu Thr Val Ala Ala Ile Cys Ile Ala Ala Thr Gly 145 150 155 160 Lys Pro Val Ala His Ile Asp Trp Glu Gly Asp Leu Gly Glu Met Glu 165 170 175 Ser Thr Thr Thr Ser Phe Pro Asn Glu Thr Ala Thr Ile Ile Ser Gln 180 185 190 Tyr Lys Leu Phe Pro Thr Arg Phe Ala Arg Gly Arg Arg Ile Thr Cys 195 200 205 Val Val Lys His Pro Ala Leu Glu Lys Asp Ile Arg Tyr Ser Phe Ile 210 215 220 Leu Asp Ile Gln Tyr Ala Pro Glu Val Ser Val Thr Gly Tyr Asp Gly 225 230 235 240 Asn Trp Phe Val Gly Arg Lys Gly Val Asn Leu Lys Cys Asn Ala Asp 245 250 255 Ala Asn Pro Pro Pro Phe Lys Ser Val Trp Ser Arg Leu Asp Gly Gln 260 265 270 Trp Pro Asp Gly Leu Leu Ala Ser Asp Asn Thr Leu His Phe Val His 275 280 285 Pro Leu Thr Phe Asn Tyr Ser Gly Val Tyr Ile Cys Lys Val Thr Asn 290 295 300 Ser Leu Gly Gln Arg Ser Asp Gln Lys Val Ile Tyr Ile Ser Asp Pro 305 310 315 320 Pro Thr Thr Thr Thr Leu Gln Pro Thr Ile Gln Trp His Pro Ser Thr 325 330 335 Ala Asp Ile Glu Asp Leu Ala Thr Glu Pro Lys Lys Leu Pro Phe Pro 340 345 350 Leu Ser Thr Leu Ala Thr Ile Lys Asp Asp Glu Pro Lys Ser Ser Asp 355 360 365 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 370 375 380 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 385 390 395 400 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 405 410 415 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 420 425 430 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 435 440 445 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 450 455 460 Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 465 470 475 480 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 485 490 495 Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu 500 505 510 Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 515 520 525 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 530 535 540 Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 545 550 555 560 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His 565 570 575 Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 580 585 590 Gly Light <210> 36 <211> 252 <212> PRT <213> artificial sequence <220> <223> Human FcB <400> 36 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Ala Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro 20 25 30 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 35 40 45 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 50 55 60 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 65 70 75 80 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 85 90 95 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 100 105 110 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Ala Val 115 120 125 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 130 135 140 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 145 150 155 160 Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly 165 170 175 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 180 185 190 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 195 200 205 Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 210 215 220 Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His 225 230 235 240 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 245 250 <210> 37 <211> 71 <212> PRT <213> Human gammaherpesvirus 8 <400> 37 Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu Gly Tyr Gln 1 5 10 15 Lys Arg Pro Leu Pro Gln Val Leu Leu Ser Ser Trp Tyr Pro Thr Ser 20 25 30 Gln Leu Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Arg Gly Arg 35 40 45 Gln Val Cys Ala Asp Lys Ser Lys Asp Trp Val Lys Lys Leu Met Gln 50 55 60 Gln Leu Pro Val Thr Ala Arg 65 70 <210> 38 <211> 21 <212> PRT <213> Human gammaherpesvirus 8 <400> 38 Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu Gly Tyr Gln 1 5 10 15[[ID=-33]] Lys Arg Pro Leu Pro 20[[ID=-37]] <210> 39 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> V1δ <400> 39 Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Ala Leu Gly Tyr Gln 1 5 10 15 Lys Arg Pro Leu Pro ​​<210> 40 <211> twenty one <212> PRT <213> Artificial sequence <220> <223> V1δ mut <400> 40 Leu Gly Ala Ser Trp His Arg Pro Asp Ala Cys Ala Leu Gly Tyr Gln 1 5 10 15 Lys Arg Pro Leu Pro 20 <210> 41 <211> 34 <212> PRT <213> human gammaherpesvirus-8 <400> 41 Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu Gly Tyr Gln 1 5 10 15 Lys Arg Pro Leu Pro Gln Val Leu Leu Ser Ser Trp Tyr Pro Thr Ser 20 25 30 Gln Leu <210> 42 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Vp1δ <400> 42 Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Ala Leu Gly Tyr Gln 1 5 10 15 Lys Arg Pro Leu Pro Gln Val Leu Leu Ser Ser Trp Tyr Pro Thr Ser 20 25 30 Gln Leu <210> 43 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Vp1δ mut <400> 43 Leu Gly Ala Ser Trp His Arg Pro Asp Ala Cys Ala Leu Gly Tyr Gln 1 5 10 15 Lys Arg Pro Leu Pro Gln Val Leu Leu Ser Ser Trp Tyr Pro Thr Ser 20 25 30 Gln Leu <210> 44 <211> 107 <212> PRT <213> Artificial sequence <220> <223> IgG1 Z3 domain <400> 44 Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn Ser Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 45 <211> 100 <212> PRT <213> Artificial sequence <220> <223> Example sequence for the best part of component Z1 <220> <221> misc_feature <222> (100)..(100) <223> Xaa can be any naturally occurring amino acid <400> 45 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 1 5 10 15 Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 20 25 30 Asp Tyr Phe Pro Glu Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 35 40 45 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 50 55 60 Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 65 70 75 80 Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp 85 90 95 Lys Lys Val Xaa 100 <210> 46 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Exemplary purification tags <400> 46 Ala Ser His His His His His Met 1 5 <210> 47 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Exemplary trimerization sequences <400> 47 Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg Lys 1 5 10 15 Arg Gly Glu Trp Val Leu Leu Ser Thr Phe Leu 20 25 <210> 48 <211> 107 <212> PRT <213> Artificial sequence <220> <223> Exemplary sequences for the optimal portion of component Z3 <400> 48 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 49 <211> 421 <212> PRT <213> Artificial sequence <220> <223> vMIPII-CH-hFcB <220> <221> misc_feature <222> (189)..(189) <223> Xaa can be any naturally occurring amino acid <400> 49 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu Gly 20 25 30 Tyr Gln Lys Arg Pro Leu Pro Gln Val Leu Leu Ser Ser Trp Tyr Pro 35 40 45 Thr Ser Gln Leu Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Arg 50 55 60 Gly Arg Gln Val Cys Ala Asp Lys Ser Lys Asp Trp Val Lys Lys Leu 65 70 75 80 Met Gln Gln Leu Pro Val Thr Ala Arg Ser Ser Ala Ser Thr Lys Gly 85 90 95 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 100 105 110 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Val Thr 115 120 125 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 130 135 140 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 145 150 155 160 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn 165 170 175 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Xaa Glu Pro Lys 180 185 190 Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 195 200 205 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 210 215 220 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 225 230 235 240 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 245 250 255 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 260 265 270 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 275 280 285 Asn Gly Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala 290 295 300 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 305 310 315 320 Gln Val Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln 325 330 335 Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 340 345 350 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 355 360 365 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu 370 375 380 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 385 390 395 400 Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser 405 410 415 Leu Ser Pro Gly Lys 420 <210> 50 <211> 371 <212> PRT <213> Artificial sequence <220> <223> V1-CH-hFcB <220> <221> misc_feature <222> (139)..(139) <223> Xaa can be any naturally occurring amino acid <400> 50 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu Gly 20 25 30 Tyr Gln Lys Arg Pro Leu Pro Ser Ser Ala Ser Thr Lys Gly Pro Ser 35 40 45 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 50 55 60 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Val Thr Val Ser 65 70 75 80 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 85 90 95 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 100 105 110 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 115 120 125 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Xaa Glu Pro Lys Ser Ser 130 135 140 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 145 150 155 160 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 165 170 175 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 180 185 190 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 195 200 205 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 210 215 220 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 225 230 235 240 Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 245 250 255 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 260 265 270 Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser 275 280 285 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 290 295 300 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 305 310 315 320 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 325 330 335 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 340 345 350 His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 355 360 365 Pro Gly Lys 370 <210> 51 <211> 371 <212> PRT <213> Artificial sequence <220> <223> V1δ-CH-hFcB <220> <221> misc_feature <222> (139)..(139) <223> Xaa can be any naturally occurring amino acid <400> 51 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Ala Leu Gly 20 25 30 Tyr Gln Lys Arg Pro Leu Pro Ser Ser Ala Ser Thr Lys Gly Pro Ser 35 40 45 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 50 55 60 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Val Thr Val Ser 65 70 75 80 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 85 90 95 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 100 105 110 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 115 120 125 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Xaa Glu Pro Lys Ser Ser 130 135 140 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 145 150 155 160 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 165 170 175 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 180 185 190 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 195 200 205 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 210 215 220 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 225 230 235 240 Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 245 250 255 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 260 265 270 Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser 275 280 285 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 290 295 300 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 305 310 315 320 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 325 330 335 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 340 345 350 His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 355 360 365 Pro Gly Lys 370 <210> 52 <211> 371 <212> PRT <213> Artificial sequence <220> <223> V1δ mut-CH-hFcB <220> <221> misc_feature <222> (139)..(139) <223> Xaa can be any naturally occurring amino acid <400> 52 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Leu Gly Ala Ser Trp His Arg Pro Asp Ala Cys Ala Leu Gly 20 25 30 Tyr Gln Lys Arg Pro Leu Pro Ser Ser Ala Ser Thr Lys Gly Pro Ser 35 40 45 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 50 55 60 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Val Thr Val Ser 65 70 75 80 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 85 90 95 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 100 105 110 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 115 120 125 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Xaa Glu Pro Lys Ser Ser 130 135 140 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 145 150 155 160 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 165 170 175 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 180 185 190 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 195 200 205 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 210 215 220 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 225 230 235 240 Lys Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 245 250 255 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 260 265 270 Tyr Thr Leu Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser 275 280 285 Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 290 295 300 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 305 310 315 320 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val 325 330 335 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 340 345 350 His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 355 360 365 Pro Gly Lys 370 <210> 53 <211> 384 <212> PRT <213> Artificial sequence <220> <223> Vp1-CH-hFcB <220> <221> misc_feature <222> (152)..(152) <223> Xaa can be any naturally occurring amino acid <400> 53 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu Gly 20 25 30 Tyr Gln Lys Arg Pro Leu Pro Gln Val Leu Leu Ser Ser Trp Tyr Pro 35 40 45 Thr Ser Gln Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 50 55 60 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 65 70 75 80 Cys Leu Val Lys Asp Tyr Phe Pro Glu Val Thr Val Ser Trp Asn Ser 85 90 95 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 100 105 110 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 115 120 125 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 130 135 140 Thr Lys Val Asp Lys Lys Val Xaa Glu Pro Lys Ser Ser Asp Lys Thr 145 150 155 160 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 165 170 175 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 180 185 190 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 195 200 205 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 210 215 220 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 225 230 235 240 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 245 250 255 Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 260 265 270 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 275 280 285 Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 290 295 300 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 305 310 315 320 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 325 330 335 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 340 345 350 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala 355 360 365 Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <210> 54 <211> 384 <212> PRT <213> Artificial sequence <220> <223> Vp1δ-CH-hFcB <220> <221> misc_feature <222> (152)..(152) <223> Xaa can be any naturally occurring amino acid <400> 54 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Ala Leu Gly 20 25 30 Tyr Gln Lys Arg Pro Leu Pro Gln Val Leu Leu Ser Ser Trp Tyr Pro 35 40 45 Thr Ser Gln Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 50 55 60 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 65 70 75 80 Cys Leu Val Lys Asp Tyr Phe Pro Glu Val Thr Val Ser Trp Asn Ser 85 90 95 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 100 105 110 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 115 120 125 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 130 135 140 Thr Lys Val Asp Lys Lys Val Xaa Glu Pro Lys Ser Ser Asp Lys Thr 145 150 155 160 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 165 170 175 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 180 185 190 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 195 200 205 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 210 215 220 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 225 230 235 240 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 245 250 255 Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 260 265 270 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 275 280 285 Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 290 295 300 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 305 310 315 320 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 325 330 335 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 340 345 350 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala 355 360 365 Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <210> 55 <211> 384 <212> PRT <213> Artificial sequence <220> <223> Vp1δ mut-CH-hFcB <220> <221> misc_feature <222> (152)..(152) <223> Xaa can be any naturally occurring amino acid <400> 55 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Leu Gly Ala Ser Trp His Arg Pro Asp Ala Cys Ala Leu Gly 20 25 30 Tyr Gln Lys Arg Pro Leu Pro Gln Val Leu Leu Ser Ser Trp Tyr Pro 35 40 45 Thr Ser Gln Leu Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 50 55 60 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 65 70 75 80 Cys Leu Val Lys Asp Tyr Phe Pro Glu Val Thr Val Ser Trp Asn Ser 85 90 95 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 100 105 110 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 115 120 125 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 130 135 140 Thr Lys Val Asp Lys Lys Val Xaa Glu Pro Lys Ser Ser Asp Lys Thr 145 150 155 160 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 165 170 175 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 180 185 190 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 195 200 205 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 210 215 220 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 225 230 235 240 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 245 250 255 Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 260 265 270 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 275 280 285 Pro Pro Ser Arg Cys Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys 290 295 300 Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 305 310 315 320 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 325 330 335 Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser 340 345 350 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala 355 360 365 Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380 <210> 56 <211> twenty four <212> PRT <213> Artificial sequence <220> <223> Exemplary trimerization domains <400> 56 Val Thr Thr Leu Gln Asp Ser Ile Arg Lys Val Thr Glu Glu Asn Lys 1 5 10 15 Glu Leu Ala Asn Glu Leu Arg Arg 20 <210> 57 <211> 197 <212> PRT <213> Artificial sequence <220> <223> vMIPII-CL Best Part <400> 57 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu Gly 20 25 30 Tyr Gln Lys Arg Pro Leu Pro Gln Val Leu Leu Ser Ser Trp Tyr Pro 35 40 45 Thr Ser Gln Leu Cys Ser Lys Pro Gly Val Ile Phe Leu Thr Lys Arg 50 55 60 Gly Arg Gln Val Cys Ala Asp Lys Ser Lys Asp Trp Val Lys Lys Leu 65 70 75 80 Met Gln Gln Leu Pro Val Thr Ala Arg Lys Arg Thr Val Ala Ala Pro 85 90 95 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 100 105 110 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 115 120 125 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 130 135 140 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 145 150 155 160 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 165 170 175 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 180 185 190 Asn Arg Gly Glu Cys 195 <210> 58 <211> 147 <212> PRT <213> Artificial Sequence <220> <223> Best Part of V1-CL <400> 58 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu Gly 20 25 30 Tyr Gln Lys Arg Pro Leu Pro Lys Arg Thr Val Ala Ala Pro Ser Val 35 40 45 Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 50 55 60 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 65 70 75 80 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 85 90 95 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 100 105 110 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 115 120 125 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 130 135 140 Gly Glu Cys 145 <210> 59 <211> 147 <212> PRT <213> artificial sequence <220> <223> V1δ-CL best part <400> 59 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Ala Leu Gly 20 25 30 Tyr Gln Lys Arg Pro Leu Pro Lys Arg Thr Val Ala Ala Pro Ser Val 35 40 45 Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 50 55 60 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 65 70 75 80 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 85 90 95 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 100 105 110 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 115 120 125 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 130 135 140 Gly Glu Cys 145 <210> 60 <211> 147 <212> PRT <213> artificial sequence <220> <223> V1δ mut-CL best part <400> 60 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Leu Gly Ala Ser Trp His Arg Pro Asp Ala Cys Ala Leu Gly 20 25 30 Tyr Gln Lys Arg Pro Leu Pro Lys Arg Thr Val Ala Ala Pro Ser Val 35 40 45 Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser 50 55 60 Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln 65 70 75 80 Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val 85 90 95 Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu 100 105 110 Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu 115 120 125 Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg 130 135 140 Gly Glu Cys 145 <210> 61 <211> 160 <212> PRT <213> artificial sequence <220> <223> Vp1-CL best part <400> 61 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu Gly 20 25 30 Tyr Gln Lys Arg Pro Leu Pro Gln Val Leu Leu Ser Ser Trp Tyr Pro 35 40 45 Thr Ser Gln Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 50 55 60 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 65 70 75 80 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 85 90 95 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 100 105 110 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 115 120 125 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 130 135 140 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 145 150 155 160 <210> 62 <211> 160 <212> PRT <213> artificial sequence <220> <223> Vp1δ-CL best part <400> 62 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Ala Leu Gly 20 25 30 Tyr Gln Lys Arg Pro Leu Pro Gln Val Leu Leu Ser Ser Trp Tyr Pro 35 40 45 Thr Ser Gln Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 50 55 60 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 65 70 75 80 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 85 90 95 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 100 105 110 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 115 120 125 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 130 135 140 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 145 150 155 160 <210> 63 <211> 160 <212> PRT <213> artificial sequence <220> <223> Vp1δ mut-CL best part <400> 63 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Leu Gly Ala Ser Trp His Arg Pro Asp Ala Cys Ala Leu Gly 20 25 30 Tyr Gln Lys Arg Pro Leu Pro Gln Val Leu Leu Ser Ser Trp Tyr Pro 35 40 45 Thr Ser Gln Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 50 55 60 Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 65 70 75 80 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val 85 90 95 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln 100 105 110 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser 115 120 125 Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His 130 135 140 Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 145 150 155 160 <210> 64 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Optimal portion for component CL or exemplary sequence of component CL <400> 64 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 1 5 10 15 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 20 25 30 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 35 40 45 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 50 55 60 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 65 70 75 80 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 85 90 95 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 65 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Linker <400> 65 Gly Gly Gly Gly Ser 1 5 <210> 66 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Linker <400> 66 Gly Gly Gly Ser Gly Gly Gly Ser 1 5 <210> 67 <211> 98 <212> PRT <213> Artificial sequence <220> <223> IgG1 Z1 domain <400> 67 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val <210> 68 <211> twenty two <212> PRT <213> Artificial sequence <220> <223> IgG1 hinge region <400> 68 Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly 20 <210> 69 <211> 103 <212> PRT <213> Artificial sequence <220> <223> IgG1 Z2 domain <400> 69 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 1 5 10 15 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 20 25 30 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 35 40 45 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 50 55 60 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 65 70 75 80 Glu Tyr Lys Cys Ala Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 85 90 95 Light Thr Ile Ser Light Ala Light 100

Claims

1. A fusion protein, characterized in that: The fusion protein comprises component A, component B and component C; wherein component A is composed of the amino acid sequence shown in SEQ ID NO: 14, component B is composed of the amino acid sequence shown in SEQ ID NO: 51, and component C is composed of the amino acid sequence shown in SEQ ID NO: 59, and Component A is linked to component B, and component B is linked to component C via a disulfide bond; The fusion protein is capable of binding to (i) PD-L1, (ii) CXCR4, and (iii) Fc receptor.

2. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises a pharmaceutically acceptable carrier and the fusion protein according to claim 1.

3. Use of the fusion protein according to claim 1 for preparing a drug for treating melanoma.

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