CD80 protein variants and CD80 fusion proteins

Abstract

The present invention provides a CD80 protein variant capable of binding to PD-L1, CTLA-4, and CD28, activating the activity of T lymphocytes, and inhibiting the growth and / or proliferation of tumors or tumor cells, and a fusion protein comprising said CD80 protein variant. Further provided are a nucleic acid encoding said fusion protein, a vector comprising said fusion protein, a cell comprising said nucleic acid or said vector, a method for producing said fusion protein, and applications of said fusion protein.

Description

[Technical field]

[0001] The present application relates to the biopharmaceutical field, and in particular to CD80 protein variants and fusion proteins comprising CD80 variants. [Background technology]

[0002] CD80, also known as B7, B7.1 or BB1, belongs to the immunoglobulin superfamily and is mainly expressed on activated B lymphocytes, activated T lymphocytes, macrophages, etc. CD80 receptors include CD28, CD152 (CTLA4) and PD-L1, and the cis interaction between PD-L1 and CD80 can inhibit the functions of PD-1 and CTLA-4, respectively, and the binding of CD28 to CD80 can generate a costimulatory signal and enhance the activation, function and survival of T cells against homologous antigens. Through these mechanisms, CD80 maintains the balance between T cell activation and inhibition and exerts an attacking effect on tumor cells.

[0003] Currently, single or dual antibodies targeting CTLA4 and PD-L1 against these mechanisms are being developed, but the lack of sufficient T cell costimulation in the tumor microenvironment may result in low response and drug resistance in patients. Given the important role of CD80 ligand in immune regulation, targeting CD80 ligand is always an attractive policy in cancer and autoimmunity. Summary of the Invention

[0004] The present application provides a fusion protein comprising CD80, which can bind to PD-L1, CTLA-4, CD28, activate the activity of T lymphocytes, and inhibit the growth and / or proliferation of tumors or tumor cells. The present application also relates to a nucleic acid encoding the fusion protein, a vector comprising the fusion protein, a cell comprising the nucleic acid or the vector, a method for producing the fusion protein, and an application of the fusion protein.

[0005] In one aspect, the present application provides a CD80 protein variant comprising a functionally active fragment of a CD80 protein or a variant thereof, said variant having a mutation at one or more amino acid sites compared to a corresponding functionally active fragment of a wild-type CD80, and said variant having a) binding to PD-L1; b) binding to CTLA-4; c) binding to CD28; d) Activation of T lymphocyte activity, and e) Inhibition of the growth and / or proliferation of tumors or tumor cells The present invention has one or more of the following characteristics:

[0006] In some embodiments, the PD-L1 is human PD-L1.

[0007] In some embodiments, the CTLA-4 is human CTLA-4.

[0008] In some embodiments, the CD28 is human CD28.

[0009] In some embodiments, the functionally active fragment of a CD80 protein comprises the extracellular domain (ECD) of a CD80 protein or a fragment thereof.

[0010] In some embodiments, the fragment of the extracellular domain of a CD80 protein comprises an IgV domain of a CD80 protein.

[0011] In some embodiments, the CD80 protein variant comprises an amino acid mutation at one or more amino acid residues selected from the group consisting of N55, N64, N152, N173, N177, N192, and N198, compared to the extracellular domain of a corresponding wild-type CD80 protein or a fragment thereof.

[0012] In some embodiments, the CD80 protein variant comprises: 1) N64, 2) N55 and N64, 3) N55, N64 and N152, 4) N55, N64 and N192, 5) N55, N64 and N198, 6) N55, N64, N152 and N198, 7) N55, N64, N152 and N192, 8) N55, N64, N152, N173, N177 and N198, 9) N55, N64, N152, N173 and N198, and 10) N55, N64, N152, N177 and N198 The present invention includes a mutation at any one of the amino acid positions selected from the group consisting of:

[0013] In some embodiments, the CD80 protein variant comprises an amino acid mutation at one or more amino acid residues selected from the group consisting of N55A / Q, N64A, N152A, N173Q, N177A, N192A and N198A / Q, compared to a corresponding functionally active fragment of a wild-type CD80 protein.

[0014] In some embodiments, the CD80 protein variant comprises: 1) N64A, 2) N55A and N64A, 3) N55A, N64A and N152A, 4) N55A, N64A and N192A, 5) N55A, N64A and N198A, 6) N55A, N64A, N152A and N198A, 7) N55A, N64A, N152A and N192A, 8) N55Q, N64A, N152A and N198Q, 9) N55Q, N64A, N152A, N173Q, N177A and N198Q, 10) N55Q, N64A, N152A, N173Q and N198Q, and 11) N55Q, N64A, N152A, N177A and N198Q It contains any one amino acid mutation selected from the group consisting of:

[0015] In some embodiments, the CD80 protein variant comprises at least one class I amino acid mutation, which can maintain or enhance the stability of the CD80 protein variant.

[0016] In some embodiments, the CD80 protein variant comprises a class I amino acid mutation at one or more of the following amino acid positions: N55, N64, N152, N192, and N198.

[0017] In some embodiments, the CD80 protein variant comprises: 1) N64, 2) N55 and N64, 3) N55, N64 and N152, 4) N55, N64 and N192, 5) N55, N64 and N198, 6) N55, N64, N152 and N198, and 7) N55, N64, N152 and N192 The present invention includes a class I amino acid mutation at any one of the amino acid positions selected from the group consisting of:

[0018] In some embodiments, the CD80 protein variant comprises one or more of the following class I amino acid mutations: N55A / Q, N64A, N152A, N192A, and N198A / Q.

[0019] In some embodiments, the CD80 protein variant comprises: 1) N64A, 2) N55A and N64A, 3) N55A, N64A and N152A, 4) N55A, N64A and N192A, 5) N55A, N64A and N198A, 6) N55A, N64A, N152A and N198A, 7) N55A, N64A, N152A and N192A, and 8) N55Q, N64A, N152A and N198Q The amino acid mutation comprises any one of class I amino acid mutations selected from the group consisting of:

[0020] In some embodiments, the CD80 protein variant comprises at least one class II amino acid mutation, wherein the class II amino acid mutation is capable of retaining the binding activity of the CD80 protein variant.

[0021] In some embodiments, the CD80 protein variant comprises a class II amino acid mutation at one or more of the following amino acid positions: N152, N192, and N198.

[0022] In some embodiments, the CD80 protein variant comprises one or more of the following class II amino acid mutations: N152A, N192A, and N198A / Q.

[0023] In some embodiments, the CD80 protein variant comprises a class II amino acid mutation at position N55 and / or N64.

[0024] In some embodiments, the CD80 protein variant comprises the N55A / Q and / or N64A class II amino acid mutations.

[0025] In some embodiments, the CD80 protein variant comprises at least one class III amino acid mutation, wherein the amino acid mutation is capable of retaining the affinity of the CD80 protein variant.

[0026] In some embodiments, the CD80 protein variant comprises a class III amino acid mutation at one or more of the following amino acid positions: N55, N64, N152, and N198.

[0027] In some embodiments, the CD80 protein variant comprises one or more of the following class III amino acid mutations: N55A / Q, N64A, N152A, and N198A.

[0028] In some embodiments, the CD80 protein variant comprises a class III amino acid mutation at position N192.

[0029] In some embodiments, the CD80 protein variant comprises the N192A class III amino acid mutation.

[0030] In some embodiments, the CD80 protein variant comprises at least one class IV amino acid mutation, which is capable of enhancing binding of the CD80 protein variant to CD28 and / or PD-L1.

[0031] In some embodiments, the CD80 protein variant comprises a class IV amino acid mutation at position N173 and / or N177.

[0032] In some embodiments, the CD80 protein variant comprises the N173Q and / or N177A class IV amino acid mutations.

[0033] In some embodiments, the numbering of the amino acid positions in the CD80 protein variant is determined with reference to the amino acid position numbering of SEQ ID NO:1.

[0034] In some embodiments, the CD80 protein variant comprises the amino acid sequence set forth in any one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.

[0035] In some embodiments, the CD80 protein variant is capable of binding to PD-L1, CD28 and / or CTLA-4.

[0036] In some embodiments, the sialic acid content of the CD80 protein variant is about 0.7 or greater.

[0037] In another embodiment, the present application provides a fusion protein comprising the above CD80 protein variant.

[0038] In some embodiments, the fusion protein further comprises an immunoglobulin Fc region or a variant thereof.

[0039] In some embodiments, the CD80 protein variant in the fusion protein is directly or indirectly linked to the immunoglobulin Fc region.

[0040] In some embodiments, the immunoglobulin Fc region in the fusion protein comprises an IgG Fc region.

[0041] In some embodiments, the IgG in the fusion protein is selected from the group consisting of IgG1 and IgG4.

[0042] In some embodiments, the CD80 protein variant in the fusion protein is located at the N-terminus or C-terminus of the immunoglobulin Fc region.

[0043] In some embodiments, the immunoglobulin Fc region in the fusion protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:25 and SEQ ID NO:26.

[0044] In some embodiments, the fusion protein comprises an amino acid sequence set forth in any one of SEQ ID NOs:27-37.

[0045] In another aspect, the present application further provides a polypeptide comprising said CD80 protein variant or said fusion protein.

[0046] In another aspect, the present application provides an isolated nucleic acid molecule or molecules encoding a CD80 protein variant described herein, a fusion protein described herein, or a polypeptide described herein.

[0047] In another aspect, the present application provides a vector comprising the nucleic acid molecule described herein.

[0048] In another aspect, the present application provides a host cell comprising a nucleic acid molecule described herein, or a vector described herein.

[0049] In another aspect, the present application provides a method of producing a CD80 protein variant described herein or a fusion protein described herein, comprising culturing a cell described herein under conditions that allow expression of the fusion protein.

[0050] In another aspect, the present application provides a pharmaceutical composition comprising a CD80 variant protein described herein, a fusion protein described herein, a polypeptide described herein, a nucleic acid molecule described herein, a vector described herein, and / or a host cell described herein, and optionally a pharma- ceutically acceptable carrier agent.

[0051] In another aspect, the present application provides a pharmaceutical combination comprising the above-mentioned CD80 protein variant and an immune checkpoint inhibitor.

[0052] In another aspect, the present application provides a pharmaceutical combination comprising the above-mentioned fusion protein and an immune checkpoint inhibitor.

[0053] In another aspect, the present application provides a pharmaceutical combination comprising the above-mentioned polypeptide and an immune checkpoint inhibitor.

[0054] In some embodiments, the immune checkpoints include PD-1, PD-L1, GITR, and / or CTLA-4.

[0055] In some embodiments, the immune checkpoint comprises one or more selected from the group consisting of a PD-1 antibody, a PD-L1 antibody, a CTLA-4 antibody, and a GITR antibody.

[0056] In another aspect, the present application provides a method of modulating an immune response in a subject, comprising administering a CD80 protein variant, said fusion protein, or said polypeptide described herein.

[0057] In some embodiments, the method of modulating an immune response in a subject comprises increasing an immune response.

[0058] In some embodiments, in the method of modulating an immune response in a subject, the CD80 protein variant, the fusion protein, or the polypeptide exhibits increased binding affinity to PD-L1 compared to a wild-type CD80 protein or a fragment thereof.

[0059] In some embodiments, in the method of modulating an immune response in a subject, the CD80 protein variant, the fusion protein or the polypeptide exhibits increased binding affinity to CD28 compared to a wild-type CD80 protein or a fragment thereof.

[0060] In some embodiments, in the method of modulating an immune response in a subject, the CD80 protein variant, the fusion protein or the polypeptide exhibits enhanced activation of T lymphocyte activity compared to a wild-type CD80 protein or a fragment thereof.

[0061] In another aspect, the present application provides the use of said CD80 protein variant, said CD80 fusion protein, said polypeptide, said nucleic acid molecule, said vector, said host cell, said pharmaceutical composition, or said pharmaceutical combination in the manufacture of a medicament for the prevention and / or treatment of a disease and / or condition.

[0062] In some embodiments, the disease and / or condition comprises a tumor.

[0063] In some embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0064] In some embodiments, the tumor comprises a tumor that responds to the proliferation of central memory T cells.

[0065] In some embodiments, the tumor in the above applications comprises malignant melanoma, breast cancer, gastric cancer, renal cancer, non-small cell lung cancer, colon cancer, rectal cancer, squamous cell carcinoma of the head and neck, liver cancer, renal cancer, mesothelioma bladder cancer, pancreatic cancer, ovarian cancer, endometrial cancer, and lymphoma.

[0066] In another aspect, the present application provides a method for preventing and / or treating a disease and / or condition, comprising administering to a subject in need thereof the CD80 protein variant, the fusion protein, the polypeptide, the nucleic acid molecule, the vector, the host cell, the pharmaceutical composition or the pharmaceutical combination.

[0067] In some embodiments, the disease and / or condition comprises a tumor.

[0068] In some embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0069] In some embodiments, the tumor comprises a tumor that responds to the proliferation of central memory T cells.

[0070] In another aspect, the present application provides said CD80 protein variant, said fusion protein, said polypeptide, said nucleic acid molecule, said vector, said host cell, said pharmaceutical composition or said pharmaceutical combination for the prevention and / or treatment of a disease and / or condition.

[0071] In some embodiments, the disease and / or condition comprises a tumor.

[0072] In some embodiments, the tumor comprises a solid tumor and / or a hematological tumor.

[0073] In some embodiments, the tumor comprises a tumor that responds to the proliferation of central memory T cells.

[0074] In another aspect, the present application provides a method for detecting a CD80-binding ligand in a biological sample, comprising contacting the biological sample with a binding reagent of a CD80 protein variant or fusion protein described herein.

[0075] In some embodiments, the binding ligand in the method comprises PD-L1, CD28 and / or CTLA-4.

[0076] In another aspect, the present application further provides a method for detecting the frequency and / or proliferation of central memory T cells in a subject, the method comprising administering the above-mentioned CD80 protein variant, the above-mentioned fusion protein, or the above-mentioned polypeptide.

[0077] In some embodiments, the central memory T cells are CD95+ and CD28+ cells.

[0078] In some embodiments, the central memory T cells are CD4+ central memory T cells and / or CD8+ central memory T cells.

[0079] In another aspect, the present application further provides a reagent kit comprising the CD80 protein variant, the fusion protein, the polypeptide, the nucleic acid molecule, the vector, the host cell, the pharmaceutical composition, or the pharmaceutical combination.

[0080] In some embodiments, the reagent kit is used to detect the frequency and / or proliferation of central memory T cells in a subject.

[0081] In some embodiments, the reagent kit is used to detect the presence and / or content of a CD80-binding ligand in a biological sample.

[0082] In another aspect, the present application further provides said CD80 protein variant, said fusion protein, said polypeptide, said nucleic acid molecule, said vector, said host cell, said pharmaceutical composition or said pharmaceutical combination for enhancing the proliferation or frequency of central memory T cells.

[0083] Those skilled in the art can easily discern other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As will be apparent to those skilled in the art, the contents of the present application may allow those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention of the present application. Accordingly, the drawings and descriptions in the present application are merely illustrative, not limiting. [Brief description of the drawings]

[0084] Specific features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by reference to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is given below. [Figure 1] FIG. 1 shows a schematic diagram of the CD80 fusion protein described in the present application. [Figure 2A-2B] 1 shows the results of detecting the binding activity of the CD80 fusion protein described in the present application to human PD-L1 protein. [Figure 3A-3D] 1 shows the results of detecting the binding activity of the CD80 fusion protein described in the present application to human CTLA-4 protein. [Figure 4A-4C] 1 shows the results of detecting the binding activity of the CD80 fusion protein described in the present application to human CD28 protein. [Diagram 5] 1 shows the results of detecting the binding activity of the CD80 fusion protein described in the present application to human CTLA-4 positive cells. [Figure 6] 1 shows the results of detecting the binding activity of the CD80 fusion protein described in the present application to human PD-L1 positive cells. [Figure 7]1 shows the results of detecting the binding activity of the CD80 fusion protein described in the present application to human CD28-positive cells. [Figure 8] 1 shows the results of detecting activation of T lymphocyte activity by the CD80 fusion protein described in the present application under aAPC / T mixed cell culture conditions. [Figure 9A-9C] 1 shows detection of activation of CD28 activity by the CD80 fusion proteins described herein. [Figure 10] 1 shows the results of antitumor activity of the CD80 fusion proteins described in this application in a CT26 subcutaneous tumor model. [Figure 11] 1 shows the results of the antitumor effect of combined administration of a CD80 fusion protein described in the present application and a Treg inhibitor in a CT26 subcutaneous tumor model. [Figure 12] 1 shows the results of the antitumor effect of combined administration of a CD80 fusion protein described in the present application and a PD-1 inhibitor in a CT26 subcutaneous tumor model. [Figure 13] 1 shows the results of the antitumor effect of combined administration of a CD80 fusion protein described in the present application and a CTLA-4 inhibitor in a CT26 subcutaneous tumor model. [Figure 14A-14B] 1 shows the results of induction of cytokine release by CD80 fusion proteins described in this application. [Figure 15] 1 shows the results of an analysis of the charge heterogeneity of the CD80 fusion proteins described in this application. [Figure 16] 1 shows the antitumor effect of CD80 fusion proteins described herein with different degrees of sialic acid modification. [Figure 17] The mean blood drug concentration-time curves for each group after a single intravenous injection of stock solutions of CD80-WT-G1 and CD80M28 into Sprague Dawley rats are shown (Mean + SD, n = 8). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0085] Hereinafter, embodiments of the present invention will be described with reference to specific examples, and those skilled in the art will easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0086] Definition of Terms In the present application, the term "CD80 protein variant" refers to a protein having an amino acid sequence that is modified compared to wild-type CD80. The wild-type CD80 generally refers to naturally occurring CD80 obtained from nature. In the present application, the wild-type CD80 may be human CD80. The variant generally refers to a substitution, deletion and / or addition of one or more amino acids in the sequence compared to the wild-type. In some cases, the CD80 protein variant may be the extracellular domain (ECD) of the CD80 protein, and furthermore, the CD80 protein variant may be a protein having one or more amino acid residue mutations based on the ECD, and in some cases, the CD80 protein variant may be the IgV domain of the CD80 protein, and furthermore, the CD80 protein variant may be a protein having one or more amino acid residue mutations based on the IgV domain. In some cases, the sequence number of the human CD80 in the GenBank database may be NP_005182.1.

[0087] In the present application, the term "extracellular domain (ECD) of a CD80 protein" generally refers to a polypeptide fragment located on the cell membrane surface expressing the CD80 protein. The CD80 protein is a transmembrane glycoprotein comprising two Ig-like extracellular domains, one transmembrane helix and one short cytoplasmic tail. The Ig-like extracellular domain comprises a single V-type and C2-type domain. The extracellular domain of the CD80 protein may comprise all or part of the polypeptide fragments of the two Ig-like extracellular domains. In the present application, the extracellular domain of the CD80 protein may refer to the extracellular domain of a human CD80 protein. For example, the extracellular domain of the CD80 protein described in the present application may be a protein domain comprising all or part of the amino acid sequence set forth in SEQ ID NO:1.

[0088] In the present application, the term "IgV domain of a CD80 protein" generally refers to a polypeptide fragment of a V-type domain located on the cell membrane surface expressing the CD80 protein. The IgV domain of the CD80 protein may comprise all or a part of the polypeptide fragment of the V-type domain. In the present application, the IgV domain of the CD80 protein may refer to the IgV domain of a human CD80 protein. For example, the IgV domain of a CD80 protein described in the present application may be a protein domain comprising all or a part of the amino acid sequence shown in SEQ ID NO:2.

[0089] In this application, the terms "PD-L1" and "PDL1" are used interchangeably and generally refer to the surface antigen cluster of differentiation 274 (CD274) or B7 homologous protein 1 (B7-H1), also known as PDCD1L1, PDCD1LG1, PDL1, which belongs to the tumor necrosis factor superfamily. "PD-L1" may be the complete PD-L1 and fragments thereof, as well as functional variants, isoforms, species homologs, derivatives, analogs, and at least one analog having a common epitope with PD-L1. PD-L1 may be a type I transmembrane glycoprotein consisting of 290 amino acid residues including an IgV-like domain, an IgC-like domain, a transmembrane hydrophobic domain, and a 30 amino acid intracellular tail. In this application, the PD-L1 may be human PD-L1.

[0090] In this application, the term "CTLA-4" generally refers to cytotoxic T-lymphocyte antigen-4, also called CTLA4, surface antigen cluster of differentiation 152 (CD152), which is a leukocyte differentiation antigen and an immunosuppressive receptor that shares a B7 molecular ligand with CD28 and belongs to the CD28 family. CTLA-4 includes an extracellular V-type domain, a transmembrane domain, and a cytoplasmic tail. "CTLA-4" may be the complete CTLA-4 and fragments thereof, or functional variants, isoforms, species homologs, derivatives, analogs, and at least one analog having a common epitope with CTLA-4. In this application, the CTLA-4 may be human CTLA-4.

[0091] In the present application, the term "CD28" generally refers to the surface antigen cluster of differentiation 28, which is a costimulatory molecule expressed on the surface of T lymphocytes and is a receptor for the CD80 and CD86 proteins that mediate costimulation of T cells and promote their survival, proliferation and cytokine production. "CD28" may be the complete CD28 and fragments thereof, or may be a functional variant, isoform, species homolog, derivative, analog, and at least one analog having a common epitope with CD28. In the present application, the CD28 may be human CD28.

[0092] In the present application, the term "amino acid mutation" generally refers to substitution, deletion, insertion and modification of amino acids. Any combination of substitution, deletion, insertion and modification can be used to achieve the final construct. In the present application, an amino acid mutation may be a replacement of at least one existing amino acid residue with another different amino acid residue (a replacement amino acid residue), and the replacement amino acid residue may be a "naturally occurring amino acid residue", and may be any of the following: alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (glutamine, G), and the like. n, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

[0093] In the present application, the above amino acid positions are confirmed according to the amino acid sequence shown in SEQ ID NO: 1 (the amino acid sequence of the extracellular domain of human CD80 (UniProtKB / Swiss-Prot:P33681.1 sequence), i.e., positions 35 to 243 of the UniProtKB / Swiss-Prot:P33681.1 sequence). That is, the number of the first amino acid in the amino acid sequence shown in SEQ ID NO: 1 is the first amino acid.

[0094] In the present application, the extracellular domain of the CD80 protein may comprise amino acids 35 to 243 of the UniProtKB / Swiss-Prot:P33681.1 sequence.

[0095] In the present application, the IgV domain of the CD80 protein may comprise amino acids 35 to 140 of the UniProtKB / Swiss-Prot:P33681.1 sequence.

[0096] In the present application, the term "class I amino acid mutation" generally refers to an amino acid mutation that affects the stability of the CD80 protein variant. The stability of the CD80 protein variant refers to the ability of the CD80 protein variant to maintain the structure and function of the CD80 protein substantially identical to that of the CD80 protein before the mutation, and the effect may be the maintenance, decrease, or increase of the stability of the CD80 protein variant. In the present application, the CD80 protein variant may be a mutation of an amino acid sequence based on the human CD80 protein. For example, in the present application, the class I amino acid mutation may include one or more amino acid mutations in all or a part of the AA35-AA242 fragment of human CD80, and in some cases, the one or more amino acid mutations may include an amino acid mutation at any one position selected from the group consisting of N55, N64, N152, N192, and N198.

[0097] In the present application, the term "class II amino acid mutation" generally refers to an amino acid mutation that affects the binding activity of the CD80 protein variant. The effect may be maintaining, decreasing, or increasing the binding activity of the CD80 protein variant. In the present application, the CD80 protein variant may be a mutation in an amino acid sequence based on the human CD80 protein. For example, in the present application, the class II amino acid mutation may include one or more amino acid mutations in all or part of the AA35-AA242 fragment of human CD80, and in some cases, the one or more amino acid mutations may include an amino acid mutation at any one position selected from the group consisting of N55, N64, N152, N192, and N198.

[0098] In this application, the term "binding activity" generally refers to the ability of two binding sites to bind to each other, said binding sites may be regions on a binding molecule that may include proteins, enzyme substrates, second messengers, hormones or allosteric modulators. In this application, binding activity may be the ability of a CD80 protein variant to bind to its ligand, and the binding may be reversible (transient and non-covalent), covalently reversible or irreversible.

[0099] In the present application, the term "class III amino acid mutation" generally refers to an amino acid mutation that affects the affinity of the CD80 protein variant. The effect may be maintaining, decreasing, or increasing the affinity of the CD80 protein variant. In the present application, the CD80 protein variant may be a mutation of an amino acid sequence based on the human CD80 protein. For example, in the present application, the class III amino acid mutation may include one or more amino acid mutations in all or part of the AA35-AA242 fragment of human CD80, and in some cases, the one or more amino acid mutations may include an amino acid mutation at any one position selected from the group consisting of N55, N64, N152, N192, and N198.

[0100] In this application, the term "affinity" generally refers to the specific affinity with which two or more substances can interact, and in some cases may be the affinity of a receptor for a ligand.

[0101] In the present application, the term "class IV amino acid mutation" generally refers to an amino acid mutation that affects the binding of the CD80 protein variant to CD28 and / or PD-L1. The effect may be a maintained, decreased, or increased binding of the CD80 protein variant to CD28 and / or PD-L1. In the present application, the CD80 protein variant may be a mutation in an amino acid sequence based on the human CD80 protein. For example, in the present application, the class III amino acid mutation may include one or more amino acid mutations in all or part of the AA35-AA242 fragment of human CD80, and in some cases, may include an amino acid mutation at any one position selected from the group consisting of N173 and / or N177.

[0102] In the present application, the term "fusion protein" generally refers to a macromolecule in which two or more different protein molecules are linked by chemical and / or genetic fusion methods. A fusion protein may be an expression product of two or more genes obtained by DNA recombinant technology, or a group of proteins that mediate the fusion of two or more cytoplasmic membranes. In the present application, a fusion protein may be a protein molecule having the above two domains, which is genetically linked at the genetic level to an immunoglobulin Fc region or a variant thereof and is expressed in eukaryotic or prokaryotic cells.

[0103] In this application, the term "immunoglobulin Fc region" generally refers to a crystallizable segment of an immunoglobulin, and may include the CH2 and CH3 domains or fragments thereof of immunoglobulins IgG, IgA and IgD, and the CH2, CH3 and CH4 domains or fragments thereof of IgM and IgE. For example, the IgG may include IgG1, IgG2, IgG3 or IgG4.

[0104] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms may be used to refer to amino acid polymers in which one or more amino acid residues are artificial synthetic chemical mimetics of their corresponding naturally occurring amino acids, and may be used to refer to naturally occurring amino acid polymers, those amino acid polymers containing modified residues, and non-naturally occurring amino acid polymers.

[0105] As used herein, the term "nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or analogs thereof of any length, either isolated from a natural environment or synthesized artificially.

[0106] In this application, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating in a suitable host and transferring an inserted nucleic acid molecule into and / or between cells (e.g., a host cell). The vector may include a vector primarily for inserting DNA or RNA into a cell, a vector primarily for replicating DNA or RNA, and a vector primarily for expressing transcription and / or translation of DNA or RNA. The vector may further include vectors having various of the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable cell. Generally, the vector can produce a desired expression product by culturing a suitable cell containing the vector. In this application, the vector may be a plasmid.

[0107] In the present application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, which may be a human patient. For example, a pharmaceutical composition described herein may comprise a CD80 variant protein described herein, a fusion protein described herein, a nucleic acid molecule described herein, a vector described herein, and / or a cell described herein, and optionally a pharma- ceutically acceptable carrier agent. Also, the pharmaceutical composition may further comprise an appropriate formulation of one or more (pharmaceutically effective) carrier agents, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. Acceptable components of the composition are preferably non-toxic to the recipient under the dosages and concentrations used. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0108] In this application, the term "pharmaceutical combination" generally refers to the simultaneous use of two or more drugs. In the pharmaceutical combination, different drug components may be mixed or separately arranged. In the pharmaceutical combination, different drug components may be arranged in the same container or in different containers. Different drug components in the pharmaceutical combination may be administered simultaneously or separately, for example, different drug components may be administered sequentially. In the pharmaceutical combination, the administration mode of different drug components may be administered in the same manner or in different manners.

[0109] As used herein, the term "pharmaceutically acceptable carrier" generally refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with administration of the drug and are generally safe, non-toxic, and biologically or otherwise undesirable.

[0110] In this application, the term "tumor" generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In this application, a tumor may be a solid tumor or a non-solid tumor. In some cases, a tumor may include a tumor that responds to the proliferation of central memory T cells. In some cases, a tumor may include a tumor that includes cells that are surface positive for CTLA-4.

[0111] In the present application, such proteins, polypeptides and / or amino acid sequences should also be understood to include variants or homologues having at least the same or similar function as the above proteins or polypeptides.

[0112] In the present application, the variant may be, for example, a protein or polypeptide in which one or more amino acids have been substituted, deleted or added in the amino acid sequence of the protein and / or polypeptide (e.g., CD80 protein variant). For example, the functional variant may include a protein or polypeptide that already has an amino acid modification by substitution, deletion and / or insertion of at least one, for example 1 to 30, 1 to 20 or 1 to 10, for example 1, 2, 3, 4 or 5 amino acids. The functional variant can basically retain the biological properties of the protein or polypeptide before modification (e.g., substitution, deletion or addition). For example, the functional variant can retain at least 60%, 70%, 80%, 90% or 100% of the biological activity (e.g., antigen binding ability) of the protein or polypeptide before modification. For example, the substitution may be a conservative substitution.

[0113] In the present application, the homologue may be a protein or polypeptide having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or polypeptide (e.g., a CD80 protein variant).

[0114] In this application, the above-mentioned homology generally refers to the similarity, similarity or relationship between two or more sequences. The "sequence homology percentage" can be calculated by the following method: comparing two aligned sequences in a comparison window, determining the number of positions in which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) exists in the two sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the size of the window), and multiplying the result by 100 to generate the sequence homology percentage. Alignment for determining sequence homology percentage can be achieved by various methods known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for alignment sequences, including any algorithms required to achieve maximum alignment within the full length sequences or within the region of the target sequence to be compared. The homology can also be measured by the FASTA and BLAST methods. For a description of the FASTA algorithm, see WR Pearson and DJ Lipman, "Improved Tools for Biological Sequence Comparison," Proc. Natl. Acad. Sci., 85:2444-2448, 1988, and DJ Lipman and WR Pearson, "Rapid and Sensitive Protein Similarity Searching," Science, 227:1435-1441, 1989. For a description of the BLAST algorithm, see S. Altschul, W. Gish, W. Miller, EW Myers and D. Lipman, "Basic Local Alignment Search Tool," Journal of Molecular Biology, 215:403-410, 1990.

[0115] In this application, the term "comprise" generally refers to the meaning of including, grouping together, containing or covering. In some cases, it also means "is" or "consists of."

[0116] In this application, the term "about" generally refers to a variation within 0.5% to 10% more or less than a specified value, for example, a variation within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% more or less than a specified value.

[0117] Detailed Description of the Invention CD80 protein variants In one aspect, the present application provides a CD80 protein variant, which may comprise a functionally active fragment of a CD80 protein or a variant thereof, said variant having mutations at one or more amino acid sites compared to a corresponding functionally active fragment of wild-type CD80.

[0118] In the present application, the CD80 protein variant may have one or more of the following properties: 1) binding to PD-L1; 2) binding to CTLA-4; 3) binding to CD28; 4) activation of T lymphocyte activity; and 5) inhibition of the growth and / or proliferation of tumors or tumor cells.

[0119] For example, the CD80 protein variants can bind to PD-L1 and CD28 proteins simultaneously. For example, the CD80 variants can bind to PD-L1, CD28 and CTLA-4 proteins simultaneously.

[0120] In the present application, the PD-L1 may be human PD-L1. In the present application, the CD28 may be human CD28. In the present application, the CTLA-4 may be human CTLA-4.

[0121] In the present application, the CD80 protein may be a human CD80 protein.

[0122] In the present application, the CD80 protein variant may comprise a functionally active fragment of CD80 or a variant thereof. For example, the functionally active fragment of CD80 may comprise the extracellular domain of the CD80 protein or a fragment thereof. For example, the fragment of the extracellular domain of the CD80 protein may comprise the IgV domain of CD80.

[0123] In the present application, the IgV domain of the wild-type CD80 protein may comprise the amino acid sequence shown in SEQ ID NO:2.

[0124] In the present application, the extracellular domain of the wild-type CD80 protein may comprise the amino acid sequence shown in SEQ ID NO:1.

[0125] In the present application, the mutant of the IgV domain of the CD80 protein may contain one or more amino acid mutations.

[0126] In the present application, the mutant IgV domain of the CD80 protein may comprise the amino acid sequence VIHVTKEVKEVATLSCGHNVSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNX1LSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKA. The mutant IgV domain of the CD80 protein may comprise an amino acid substitution at least in X1.

[0127] In the present application, the IgV domain variant of the CD80 protein may comprise an amino acid substitution at least in X1, compared to the IgV domain comprising the CD80 protein shown in SEQ ID NO: 2. For example, the amino acid in X1 may be substituted with A.

[0128] In the present application, the extracellular domain mutant of the CD80 protein may contain one or more amino acid mutations.

[0129] In the present application, the extracellular domain variant of the CD80 protein may comprise the amino acid sequence of VIHVTKEVKEVATLSCGHX1VSVEELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKX2RTIFDITNX3LSIVILALRPSDEGTYECVVLKYEKDAFKREHLAEVTLSVKADFPTPSISDFEIPTSNIRRIICSTSGGFPEPHLSWLENGEELNAIX4TTVSQDPETELYAVSSKLDFX5MTTX6HSFMCLIKYGHLRVX7QTFNWX8TTKQEHFPDNL. The extracellular domain variant of the CD80 protein may comprise amino acid substitutions at least at X1, X2, X3, X4, X5, X6, X7 and / or X8.

[0130] For example, the CD80 protein variant may comprise amino acid mutations at one or more positions in X1, X2, X3, X4, X5, X6, X7 and X8, where the amino acid at X1 may be substituted with A, the amino acid at X2 may be substituted with A or Q, the amino acid at X3 may be substituted with A, the amino acid at X4 may be substituted with A, the amino acid at X5 may be substituted with A or Q, the amino acid at X6 may be substituted with A, the amino acid at X7 may be substituted with A and the amino acid at X8 may be substituted with A or Q.

[0131] In the present application, the CD80 protein variant may comprise a class I amino acid mutation. For example, the class I amino acid mutation may preserve the stability of the CD80 protein variant.

[0132] In the present application, the CD80 protein variant comprising the above-mentioned class I amino acid mutation may comprise, compared to the extracellular domain of the CD80 protein shown in SEQ ID NO: 1, an amino acid substitution at least at X2, X3, X4, X7 and / or X8, wherein the amino acid at X2 may be substituted with A or Q, the amino acid at X3 may be substituted with A, the amino acid at X4 may be substituted with A, the amino acid at X7 may be substituted with A, and the amino acid at X8 may be substituted with A or Q.

[0133] For example, the class I amino acid mutation may include an amino acid mutation at position N64. In the present application, the class I amino acid mutation may include an amino acid mutation at positions N55 and N64. For example, the class I amino acid mutation may include an amino acid mutation at positions N55, N64, and N152. For example, the class I amino acid mutation may include an amino acid mutation at positions N55, N64, and N192. For example, the class I amino acid mutation may include an amino acid mutation at positions N55, N64, and N198. For example, the class I amino acid mutation may include an amino acid mutation at positions N55, N64, N152, and N198. For example, the class I amino acid mutation may include an amino acid mutation at positions N55, N64, N152, and N192.

[0134] For example, the class I amino acid mutation may include an amino acid mutation of N64A. For example, the class I amino acid mutation may include an amino acid mutation of N55A and N64A. For example, the class I amino acid mutation may include an amino acid mutation of N55A, N64A, and N152A. For example, the class I amino acid mutation may include an amino acid mutation of N55A, N64A, and N192A. For example, the class I amino acid mutation may include an amino acid mutation of N55A, N64A, and N198A. For example, the class I amino acid mutation may include an amino acid mutation of N55A, N64A, N152A, and N198A. For example, the class I amino acid mutation may include an amino acid mutation of N55A, N64A, N152A, and N192A. For example, the class I amino acid mutations may include the following amino acid mutations: N55Q, N64A, N152A, and N198Q.

[0135] In the present application, the CD80 variant may comprise a class II amino acid mutation, wherein the class II amino acid mutation can retain the binding activity of the CD80 variant.

[0136] In the present application, the CD80 protein variant comprising the above-mentioned class II amino acid mutations may comprise, compared to the extracellular domain comprising the CD80 protein shown in SEQ ID NO:1, an amino acid substitution at least at X4, X7 and / or X8, wherein the amino acid at X4 may be substituted with A, the amino acid at X7 may be substituted with A, and the amino acid at X8 may be substituted with A and Q.

[0137] For example, the class II amino acid mutations may include mutations at one or more of the following amino acid positions: N152, N192, N198. For example, the class II amino acid mutations may include one or more of the following amino acid mutations: N152A, N192A, and N198A / Q. For example, the class II amino acid mutations may further include amino acid mutations at positions N55 and / or N64. For example, the class II amino acid mutations may further include amino acid mutations at positions N55A / Q and / or N64A.

[0138] In the present application, the CD80 protein variant may comprise a class III amino acid mutation, wherein the class III amino acid mutation can retain the affinity of the CD80 protein variant.

[0139] In the present application, the CD80 protein variant comprising the above-mentioned class III amino acid mutation may comprise, compared to the extracellular domain comprising the CD80 protein shown in SEQ ID NO:1, an amino acid substitution at least at X2, X3, X4 and / or X8 of the extracellular domain variant of the CD80 protein, wherein the amino acid at X2 may be substituted with A or Q, the amino acid at X3 may be substituted with A, the amino acid at X4 may be substituted with A, and the amino acid at X8 may be substituted with A.

[0140] For example, the class III amino acid mutations may include amino acid mutations at one or more of positions N55, N64, N152, and N198. For example, the class III amino acid mutations may include one or more of amino acid mutations N55A / Q, N64A, N152A, and N198A. For example, the class III amino acid mutations may further include an amino acid mutation at position N192. For example, the class III amino acid mutations may further include an amino acid mutation at N192A.

[0141] In the present application, the CD80 protein variant may comprise a class IV amino acid mutation, wherein the class IV amino acid mutation can enhance binding of the CD80 protein variant to CD28 and / or PD-L1.

[0142] In the present application, the CD80 protein variant comprising the above-mentioned class IV amino acid mutation, compared to the extracellular domain comprising the CD80 protein shown in SEQ ID NO: 1, may comprise an amino acid substitution at least at X5 and / or X6, wherein the amino acid at X5 may be substituted with Q and the amino acid at X6 may be substituted with A.

[0143] For example, the class IV amino acid mutation may include amino acid mutations at positions N173 and / or N177. For example, the class IV amino acid mutation may include amino acid mutations at positions N173Q and / or N177A.

[0144] In the present application, compared to the amino acid sequence shown in SEQ ID NO: 2, the CD80 protein variant may comprise an amino acid mutation at position N64. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise an amino acid mutation at positions N64 and N55. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise an amino acid mutation at positions N55, N64 and N152. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise an amino acid mutation at positions N55, N64 and N192. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise an amino acid mutation at positions N55, N64 and N198. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise an amino acid mutation at positions N55, N64, N152 and N198. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise amino acid mutations at positions N55, N64, N152 and N192. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise amino acid mutations at positions N55, N64, N152, N173, N177 and N198. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise amino acid mutations at positions N55, N64, N152, N173 and N198. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise amino acid mutations at positions N55, N64, N152, N173 and N198.

[0145] In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise an amino acid mutation of N64A. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise an amino acid mutation of N55A and N64A. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise an amino acid mutation of N55A, N64A and N152A. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise an amino acid mutation of N55A, N64A and N192A. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise an amino acid mutation of N55A, N64A and N198A. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise an amino acid mutation of N55A, N64A and N152A. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise the following amino acid mutations: N55A, N64A, N152A, and N192A. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise the following amino acid mutations: N55Q, N64A, N152, and N198Q. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise the following amino acid mutations: N55Q, N64A, N152A, N173Q, N177A, and N198Q. In the present application, compared to the amino acid sequence shown in SEQ ID NO: 1, the CD80 protein variant may comprise the following amino acid mutations: N55Q, N64A, N152A, N173Q, and N198Q. In the present application, compared to the amino acid sequence shown in SEQ ID NO:1, said CD80 protein variant may comprise the following amino acid mutations: N55Q, N64A, N152A, N177A and N198Q.

[0146] In the present application, the CD80 protein variant may comprise the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.

[0147] In the present application, the sialic acid content of the CD80 protein variant may be about 0.7 or more, about 0.8 or more, about 0.9 or more, about 1.0 or more, about 1.1 or more, about 1.2 or more, about 1.3 or more, about 1.4 or more, about 1.5 or more, about 2 or more, about 3 or more, about 4 or more, about 5 or more, about 6 or more, about 7 or more, about 8 or more, about 9.5 or more, about 10 or more, about 11 or more, about 12 or more, about 13, or about 14 or more.

[0148] In the present application, the sialic acid content of the CD80 protein variant may be about 0.7 or more and about 15 or less, about 0.8 or more and about 15 or less, about 0.9 or more and about 15 or less, about 1.0 or more and about 15 or less, about 1.1 or more and about 15 or less, about 1.2 or more and about 15 or less, about 1.3 or more and about 15 or less, about 1.4 or more and about 15 or less, about 1.5 or more and about 15 or less, about 2 or more and about 15 or less, about 3 or more and about 15 or less, about 4 or more and about 15 or less, about 5 or more and about 15 or less, about 6 or more and about 15 or less, about 7 or more and about 15 or less, about 8 or more and about 15 or less, about 9 or more and about 15 or less, about 10 or more and about 15 or less, about 11 or more and about 15 or less, about 12 or more and about 15 or less, about 13 or more and about 15 or less, or about 14 or more and about 15 or less.

[0149] Fusion proteins In another aspect, the present application provides a fusion protein comprising a CD80 protein variant. In the present application, the fusion protein may further comprise another functional fragment. For example, the fusion protein may comprise the CD80 protein variant and an immunoglobulin Fc region or a variant thereof.

[0150] In the present application, the immunoglobulin Fc region may comprise an IgG Fc region, the IgG may be human IgG, and the human IgG may be human IgG1 and / or human IgG4. The variant may be all or a part of an amino acid fragment of an immunoglobulin Fc region. For example, the immunoglobulin Fc region may comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 25 or SEQ ID NO: 26. For example, the IgG1 Fc region may comprise the amino acid sequence shown in SEQ ID NO: 25. For example, the IgG4 Fc region may comprise the amino acid sequence shown in SEQ ID NO: 26.

[0151] In the present application, the CD80 protein variant and the immunoglobulin Fc region can be linked directly or indirectly. Indirect linking as used herein includes linking via a polypeptide linker.

[0152] In the present application, the polypeptide linker may comprise an amino acid sequence consisting of one or more amino acid residues, the amino acid residue may comprise proline or cysteine, the linker may link two binding fragments, the binding fragments may comprise homologous or different fragments. For example, the linker may link the CD80 protein variant and the immunoglobulin Fc region. For example, the linker may comprise an amino acid sequence of (GGGGS)n, where n may be any positive integer from 1 to 10. For example, n may be 1 to 4. For example, the linker may comprise an amino acid sequence of (GGGGS)2. For example, the linker may comprise an amino acid sequence of (GGGGS)3. For example, the linker may comprise an amino acid sequence of (GGGGS)4.

[0153] In the present application, the fusion protein may comprise a first polypeptide chain or a second polypeptide chain.

[0154] In the present application, the polypeptide fragment of the first polypeptide chain may be, in N-terminus to C-terminus order, CD80 protein variant-linker-immunoglobulin Fc region, wherein the CD80 protein variant can be linked to the linker, and the linker can be linked to the N-terminus of the immunoglobulin Fc region.

[0155] In the present application, the polypeptide fragment of the second polypeptide chain may be, in N-terminal to C-terminal order, CD80 protein variant-immunoglobulin Fc region, and the CD80 protein variant may be linked to the N-terminus of the immunoglobulin Fc region.

[0156] In the present application, the fusion protein may comprise two identical first polypeptide chains or may comprise two identical second polypeptide chains.

[0157] For example, the first polypeptide chain or the second polypeptide chain may comprise a CD80 protein variant sequence set forth in any one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24. For example, the first polypeptide chain or the second polypeptide chain may comprise an amino acid sequence set forth in any one of SEQ ID NOs:27-37.

[0158] In the present application, the fusion protein comprises the CD80 protein variant and another protein / polypeptide molecule, for example, the other protein / polypeptide molecule does not affect the function of the CD80 protein variant.

[0159] Nucleic acids, vectors, host cells and methods of production In another aspect, the present application further provides one or more isolated nucleic acid molecules capable of encoding the CD80 protein variants and fusion proteins thereof described herein, for example, each nucleic acid molecule in the one or more nucleic acid molecules may encode the entire CD80 protein variants and fusion proteins thereof, or a portion thereof.

[0160] The nucleic acid molecules described herein may be isolated. For example, they can be produced or synthesized by a method such as (1) in vitro amplification, for example, by polymerase chain reaction (PCR), (2) cloning and recombinant production, (3) purification, for example, by enzymatic cleavage and gel electrophoretic fractionation, or (4) synthesis, for example, chemical synthesis. For example, the isolated nucleic acid can be produced by amplification using polymerase chain reaction (PCR). In the present application, the nucleic acid encoding the CD80 protein variant and its fusion protein can be produced by various methods known in the art.

[0161] In another aspect, the present application provides one or more vectors comprising one or more nucleic acid molecules described herein. Each vector may comprise one or more of the nucleic acid molecules. The vector may further comprise other genes, such as marker genes, that allow the vector to be selected in a suitable host cell and under suitable conditions. The vector may further comprise various expression control elements, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. The vector may further comprise an origin of replication. The vector may further comprise, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used, for example, in genetic engineering.

[0162] In another aspect, the present application provides host cells that can include one or more nucleic acid molecules described herein and / or one or more vectors described herein. In some embodiments, each or each host cell can include one or more nucleic acid molecules or vectors described herein. In some embodiments, each or each host cell can include a plurality (e.g., two or more) or a plurality (e.g., two or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into the host cells, such as prokaryotic cells (e.g., bacterial cells), CHO cells, NS / 0 cells, HEK293 cells, or other eukaryotic cells, such as cells of plant origin, fungi, or yeast cells. The vectors described herein can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0163] In another aspect, the present application provides a method for producing the CD80 protein variants and fusion proteins thereof described herein. The method may comprise culturing a host cell described herein under conditions that allow expression of the CD80 protein variants and fusion proteins thereof, for example by using an appropriate medium, an appropriate temperature and incubation time, etc., and these methods are understood by those skilled in the art.

[0164] In some cases, the method may further comprise the step of collecting (e.g., isolating and / or purifying) the CD80 protein variants and fusion proteins thereof described herein. For example, affinity chromatography may be performed using protein G-agarose, protein A-agarose, or metal chelation, and the CD80 protein variants and fusion proteins thereof described herein may be purified and isolated by gel electrophoresis and / or high performance liquid chromatography, etc.

[0165] Pharmaceutical Compositions, Pharmaceutical Combinations, Applications In another aspect, the present application provides a pharmaceutical composition comprising the CD80 protein variants and fusion proteins thereof described herein, the nucleic acid molecules, the vectors, the host cells, and optionally a pharma- ceutically acceptable carrier agent.

[0166] For example, the pharma- ceutically acceptable carrier agent may include a buffer, an antioxidant, a preservative, a low molecular weight polypeptide, a protein, a hydrophilic polymer, an amino acid, a sugar, a chelating agent, a counterion, a metal complex, and / or a non-ionic surfactant.

[0167] In the present application, the pharmaceutical composition can be prepared for oral administration, intravenous administration, intramuscular administration, in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration or subcutaneous depot administration. The pharmaceutical composition can be used to inhibit tumor growth. For example, the pharmaceutical composition of the present application can inhibit or delay the onset or progression of the disease, reduce the size of the tumor (even essentially eradicate the tumor), and / or alleviate and / or stabilize the disease state.

[0168] The pharmaceutical compositions described herein may comprise a prophylactically and / or therapeutically effective amount of said CD80 protein variants and fusion proteins thereof, said prophylactically and / or therapeutically effective amount being the amount required to prevent and / or treat (at least partially treat) a disease or condition and / or any complications thereof in a subject suffering from or at risk of developing the disease or condition and / or any complications thereof.

[0169] In another aspect, the present application provides a pharmaceutical combination which may comprise the above CD80 protein variant and an immune checkpoint inhibitor.

[0170] In another aspect, the present application provides a pharmaceutical combination that may include the above-mentioned fusion protein and an immune checkpoint inhibitor.

[0171] In the present application, the immune checkpoints may include PD-1, PD-L1, GITR and / or CTLA-4.

[0172] In the present application, the above-mentioned immune checkpoint inhibitor may include one or more antibodies selected from the group consisting of PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, and GITR antibody.

[0173] In the present application, the PD-1 antibody may be selected from nivolumab, pembrolizumab, toripalimab, sintilimab, camrelizumab, and genosumab.

[0174] In the present application, the PD-L1 antibody may be selected from involumab, atezolizumab, embolizumab, adebelimab, sugemalimab, and titilimumab.

[0175] In the present application, the CTLA-4 antibody may be selected from ipilimumab, tremelimumab.

[0176] In the present application, the GITR antibody may be selected from MK-4166, TRX-518, INCAGN01876, BMS-986156, AMG228, IBI102, and ASP-1951.

[0177] The above antibody examples are merely illustrative, and such antibodies can be used in the present application as long as they can functionally achieve the effect of an inhibitor of the corresponding immune checkpoint.

[0178] In the pharmaceutical combinations described herein, the CD80 protein variant / fusion protein can be present in the same container as the immune checkpoint inhibitor. In the pharmaceutical combinations described herein, the CD80 protein variant / fusion protein can be present in a different container than the immune checkpoint inhibitor.

[0179] In the present application, the CD80 protein variant / fusion protein and the immune checkpoint inhibitor can be administered simultaneously. The simultaneous administration can be administered by mixing each component together or separately. It can be administered by the same mode, for example, administration into the same vein or other blood vessel, or by different modes, for example, administration intravenously and intratumorally at the same time.

[0180] In the present application, the CD80 protein variant / fusion protein and the immune checkpoint inhibitor may be administered sequentially. The order of administration may be such that the CD80 protein variant / fusion protein is administered before the immune checkpoint inhibitor, or the immune checkpoint inhibitor is administered before the CD80 protein variant / fusion protein. They may be administered in the same manner or in different manners. Each component may be administered once or in multiple doses. In some embodiments, the sequential administration may be administered at any time interval, including minutes, hours, days, weeks, months, or years. In some embodiments, the sequential administration refers to administration at any time interval of at least 2 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 6 weeks, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0181] In another aspect, the present application provides the use of the above CD80 protein variants and fusion proteins thereof in the manufacture of a medicament for preventing or treating a disease or condition.

[0182] In another aspect, the present application provides the above CD80 protein variants and fusion proteins thereof for the manufacture of a medicament for preventing or treating a disease or condition.

[0183] In another aspect, the present application provides a method for preventing or treating tumors, which may further include colorectal cancer, comprising administering to a subject in need thereof the CD80 protein variants and fusion proteins thereof described herein.

[0184] In the present application, the tumor may include a solid tumor. In the present application, the tumor may include a non-solid tumor. In the present application, the tumor may include one or more types selected from the group consisting of malignant melanoma, breast cancer, gastric cancer, renal cancer, non-small cell lung cancer, colon cancer, rectal cancer, head and neck squamous cell carcinoma, liver cancer, renal cancer, mesothelioma bladder cancer, pancreatic cancer, ovarian cancer, endometrial cancer, and lymphoma.

[0185] Without being limited to any theory, the following examples are merely intended to illustrate the present invention's variants, fusion proteins, methods of manufacture and uses, and are not intended to limit the scope of the present invention.

[0186] Working Example Example 1: Production of CD80 fusion proteins The CD80 fusion protein is obtained by linking the CD80 extracellular domain or a mutant thereof to the N-terminus of the Fc fragment of human IgG1 (amino acid sequence: SEQ ID NO:25), and the CD80 extracellular domain or a mutant thereof includes wild-type CD80 ECD (SEQ ID NO:1, the extracellular domain of wild-type CD80), M12 (SEQ ID NO:6), M13 (SEQ ID NO:7), M14 (SEQ ID NO:8), M15 (SEQ ID NO:9), M16 (SEQ ID NO:10), M18 (SEQ ID NO:11), M19 (SEQ ID NO:12), M20 (SEQ ID NO:13), M21 (SEQ ID NO:14), M22 (SEQ ID NO:15), M23 (SEQ ID NO:16), M24 (SEQ ID NO:17), M25 (SEQ ID NO:18), M26 (SEQ ID NO:19), M27 (SEQ ID NO:20), M28 (SEQ ID NO:21), M29 (SEQ ID NO:22), M30 (SEQ ID NO:23), and M31 (SEQ ID NO:24). CD80 protein IgV domain or variants thereof include wild type CD80 IgV (SEQ ID NO:2), M09 (SEQ ID NO:3), M10 (SEQ ID NO:4), M11 (SEQ ID NO:5).

[0187] The above CD80 extracellular domain or a mutant thereof was linked to the N-terminus of the Fc fragment of human IgG1 (amino acid sequence code: SEQ ID NO: 25), to obtain CD80 fusion proteins designated as CD80-WT, CD80M12, CD80M13, CD80M14, CD80M15, CD80M16, CD80M18, CD80M19, CD80M20, CD80M21, CD80M22, CD80M23, CD80M24, CD80M25, CD80M26, CD80M27, CD80M28, CD80M29, CD80M30, CD80M31, CD80-IgV, CD80M09, CD80M10, and CD80M11, respectively.

[0188] The specific structure of the CD80 fusion protein is shown in FIG.

[0189] Among them, CD80M12, CD80M11, CD80M14, CD80M15, CD80M16, CD80M26, CD80M27, CD80M28, CD80M29, CD80M30, CD80M31 are stably expressed, have moderate expression levels, and have good binding activity to PD-L1, CTLA-4 and / or CD28, and can retain affinity for PD-L1, CTLA-4 and / or CD28.

[0190] Example 2 Detection of the binding activity of CD80 fusion protein to human PD-L1, CTLA-4, and CD28 proteins (1) The binding activity of CD80 fusion proteins to PD-L1 was assessed by ELISA, using CD80-WT as a control.

[0191] ELISA strips were coated with PD-L1 (Human PD-L1 / B7-H1 / CD274 Protein (His Tag), Sino Biological) and left at 4℃ overnight. After washing with PBST, 10% fetal bovine serum was added and blocked at 37℃ for 1 hour. Different concentrations of CD80 fusion protein were added and reacted at 37℃ for 1 hour. After washing with PBST, horseradish peroxidase-labeled goat anti-human IgG Fc antibody (horseradish peroxidase (HRP) conjugate, affinity purified, Invitrogen) was added and reacted at 37℃ for 30 minutes. After washing five times with PBST, 100μL of TMB (eBioscience) was added to each well and left at room temperature (20±5℃) for 1~2 minutes in the dark. After that, 100μL of 2N TMB was added to each well. The substrate reaction was stopped by adding H2SO4 stop solution, and the OD value at 450 nm was read using a microplate reader to analyze the binding ability of CD80 fusion protein to PD-L1.

[0192] The results are shown in Figures 2A-2B, which showed that under high concentration conditions, the binding activity of the CD80 protein mutants described in the present application to PD-L1 was obviously stronger than that of CD80-WT.

[0193] (2) The binding activity of CD80 fusion proteins to CTLA-4 was assessed by ELISA, using CD80-WT as a control.

[0194] ELISA strips were coated with CTLA-4 (Human CTLA-4 / CD152 Protein, ACROBiosystems), left overnight at 4°C, washed with PBST, added 10% fetal bovine serum, blocked for 1 hour at 37°C, added different concentrations of CD80 fusion protein, reacted for 1 hour at 37°C, washed with PBST, added horseradish peroxidase-labeled goat anti-human IgG Fc antibody (horseradish peroxidase (HRP) conjugate, affinity purified, Invitrogen), reacted for 30 minutes at 37°C, washed five times with PBST, added 100 μL of TMB (eBioscience) to each well, left for 1-2 minutes in the dark at room temperature (20±5°C), and then added 100 μL of 2N PBST to each well. The substrate reaction was stopped by adding H2SO4 stop solution, and the OD value was read at 450 nm using a microplate reader to analyze the binding ability of CD80 fusion protein to CTLA-4.

[0195] The results are shown in Figures 3A to 3C. The results in Figure 3A showed that the binding activity of the remaining mutants to CTLA-4 was equivalent to that of CD80WT, except that CD80M09, CD80M10, CD80M11, CD80M19, and CD80M20 had significantly weaker binding compared to wild-type CD80-WT.

[0196] (3) The binding activity of CD80 fusion proteins to CD28 was assessed by ELISA using CD80-WT as a control.

[0197] ELISA strips were coated with CD28 (Human CD28, Sino Biological) and left at 4℃ overnight. After washing with PBST, 10% fetal bovine serum was added and blocked at 37℃ for 1 hour. Different concentrations of CD80 fusion protein were added and reacted at 37℃ for 1 hour. After washing with PBST, horseradish peroxidase-labeled goat anti-human IgG Fc antibody (horseradish peroxidase (HRP) conjugate, affinity purified, Invitrogen) was added and reacted at 37℃ for 30 minutes. After washing five times with PBST, 100μL of TMB (eBioscience) was added to each well and left at room temperature (20±5℃) in the dark for 1-2 minutes. After that, 100μL of 2N H2SO4 stop solution was added to each well to stop the substrate reaction. The OD value at 450nm was read using a microplate reader to analyze the binding ability of CD80 fusion protein to CD28.

[0198] The results are shown in Figures 4A to 4C and showed that the binding activities of CD80M11, CD80M23, CD80M25, CD80M27, CD80M29, CD80M30, and CD80M31 to CD28 were significantly stronger than that of CD80-WT, and the binding activities of the remaining fusion proteins were similar to that of CD80-WT.

[0199] Example 3 Detection of binding activity of CD80 fusion protein to human PD-L1, CTLA-4, and CD28 positive cells (1) Using CD80-WT as a control, binding of CD80 fusion proteins to CTLA-4 positive cells was assessed by FACS.

[0200] Collect CHOK1 cells overexpressing human CTLA-4 (CHOK1-CTLA-4), 5 × 10 per tube. 5The cells were added to 1.5 mL EP tubes, and different concentrations of CD80 fusion protein were added, incubated on ice in the dark for 30 min, washed with FACS washing solution, and then PE fluorescently labeled goat anti-human IgG Fc secondary antibody (Invitrogen) was added, incubated on ice in the dark for 30 min, washed twice with FACS washing solution, and 400 μL of 1% paraformaldehyde fixative (Solarbio) was added per tube to fix the cells. After uniform mixing, the PE fluorescence positivity was detected using a machine to analyze the binding ability of CD80 fusion protein to CTLA-4 positive cells.

[0201] The results are shown in FIG. 5 and indicated that CD80M28 and CD80-WT have similar binding ability to cell surface CTLA-4.

[0202] (2) Binding of CD80 fusion proteins to PD-L1-positive cells was assessed by FACS, using CD80-WT as a control.

[0203] Collect CHOK1 cells overexpressing human PD-L1 (CHOK1-PD-L1) and culture 5 × 10 cells per tube. 5 The cells were added to 1.5 mL EP tubes, and different concentrations of CD80 fusion protein were added, incubated on ice in the dark for 30 min, washed with FACS washing solution, and then PE fluorescently labeled Goat Anti-Human IgG Fc Secondary Antibody (Invitrogen) was added, incubated on ice in the dark for 30 min, washed twice with FACS washing solution, and 400 μL of 1% paraformaldehyde fixative (Solarbio) was added per tube to fix the cells. After uniform mixing, the PE fluorescence positive rate was detected using a machine to analyze the binding ability of CD80 fusion protein to PD-L1 positive cells.

[0204] The results are shown in Figure 6, which showed that the binding ability of CD80M28 to cell surface PD-L1 was stronger than that of CD80-WT.

[0205] (3) Using CD80-WT as a control, binding of CD80 fusion proteins to CD28-positive cells was assessed by FACS.

[0206] Collect CHOK1 cells overexpressing human CD28 (CHOK1-CD28), 5 x 10 per tube. 5 The cells were added to 1.5 mL EP tubes, and different concentrations of CD80 fusion protein were added, incubated on ice in the dark for 30 min, washed with FACS washing solution, and then PE fluorescently labeled Goat Anti-Human IgG Fc Secondary Antibody (Invitrogen) was added, incubated on ice in the dark for 30 min, washed twice with FACS washing solution, and 400 μL of 1% paraformaldehyde fixative (Solarbio) was added per tube to fix the cells. After uniform mixing, the PE fluorescence positivity was detected using a machine to analyze the binding ability of CD80 fusion protein to CD28 positive cells.

[0207] The results are shown in FIG. 7 and indicate that the binding ability of CD80M28 and cell surface CD28 is slightly stronger than that of CD80-WT.

[0208] Example 4 Detection of activation of T lymphocyte activity by CD80 fusion protein using aAPC / T mixed cell culture The activation of T lymphocyte activity by CD80 fusion proteins was evaluated using an experimental system in which CD80 fusion proteins CD80-Fc and IgG1-Fc proteins were used as controls, 293T cells overexpressing OKT3 scFv and CD64 (293T-OKT3-CD64) were used as antigen-presenting cells (artificial APCs, aAPCs), and CD4+ T lymphocytes were used as effector cells in a mixed culture of aAPC cells and CD4+ T lymphocytes.

[0209] Harvest 293T-OKT3-CD64 cells, 2.5 x 10 per well. 4The cells were added to a 96-well cell culture plate, and PBMC cells from healthy donors (MT-BIO) were isolated with a CD4+ T cell isolation kit (CD4+ T Cell Isolation Kit, human, Miltenyi Biotec) to obtain CD4+ T lymphocytes, with 5 × 10 per well. 4 The cells were added to a 96-well cell culture plate, and 1200 nM of CD80 fusion protein, IgG1-Fc protein (Human IgG1 Fc Protein, Tag Free, ACROBiosystems), and CD80 fusion protein CD80-Fc (Human B7-1 / CD80 Protein, Fc Tag, ACROBiosystems) were further added. After incubation in a 37°C incubator for 24 hours, the supernatant was collected and the IL-2 level in the cell supernatant was detected using an IL-2 ELISA detection reagent kit (HUMAN IL-2 ELISA KIT, ExCell Bio) to analyze the activation of T lymphocyte activity by the CD80 fusion protein in the aAPC / T cell mixed culture system.

[0210] The results are shown in FIG. 8, which shows that CD80-WT, CD80M26, CD80M27, and CD80M28 can activate lymphocytes in APC / T mixed cells, and the activation activity is similar to that of CD80 molecule.

[0211] Example 5: Evaluation of activation activity of the CD28 signal transduction pathway by the reporter gene method FCGR2B / TCR Activator / CHO (abbreviated as FCGR2B / CHO) cells were collected and cultured at 1.5 × 10 4 Cells / well were added to a 96-well white / Clear bottom plate (Thermo), and different concentrations of the fusion protein were added to the cells. 4Add CD28 Effector Reporter (CD28 effector) effector cells at 100 cells / well, place the 96-well plate in a 37°C, 5% CO2 incubator, and incubate for 5.5 h. Add 120 μL of luciferase detection reagent (Bright-LumiNova) to each well. TM Firefly luciferase reporter gene detection reagent kit (Biyuntian) was added and incubated at room temperature in the dark for 10 min. The relative fluorescence intensity of chemiluminescence was then detected using a multi-function microplate reader to analyze the CD28 activation activity mediated by the fusion protein in the FCGR2B / CHO cell + CD28 effector cell system.

[0212] The results are shown in Figures 9A to 9C, which show that all fusion proteins have CD28 activation activity, and the activity of CD80M28 is stronger than that of CD80-WT, CD80M12, CD80M14, CD80M16, CD80M26, CD80M30, and CD80M31, and is close to that of CD80-WT.

[0213] Example 6 Evaluation of antitumor activity of CD80 fusion protein using CT26 subcutaneous tumor model 5 × 10 5 CT26 cells were inoculated subcutaneously and resuspended in PBS (0.1 mL / mouse). Tumor growth was monitored periodically until the tumor volume reached an average of 76 mm. 3 When the tumors grew to 100 μg / mL, the mice were randomly divided into groups according to tumor size and mouse weight for administration. There were 5 groups with 6 mice in each group. Group G1 was CD80M28 2mg / kg, group G2 was CD80M28 0.5mg / kg, group G3 was CD80M28 0.1mg / kg, group G4 was CD80-WTG1 0.5mg / kg, and group G5 was the vehicle control group. The mice were intraperitoneally administered 3 times a week for a total of 3 times.

[0214] On day 22, the tumor volume in the vehicle control group was 3000 mm 3Because some mice were euthanized after 14 days, data from Day 22 was used as the basis for analysis. The vehicle control group (G5) had a mean tumor volume of 2189.79 mm on Day 22. 3 The CD80M28 2mg / kg TIW group had a mean tumor volume of 865.57mm on Day 22. 3 The relative tumor inhibition rate (TGI) was 60.47%, which was significantly different from the control group (p<0.05). The CD80M28 0.5mg / kg TIW group had a mean tumor volume of 877.01mm on Day 22. 3 The relative tumor inhibition rate (TGI) was 59.95%, which was significantly different from the control group (p<0.05). The CD80M28 0.1mg / kg TIW administration group had a mean tumor volume of 1507.25mm on Day 22. 3 The relative tumor inhibition rate (TGI) was 31.17%, which was not significantly different from the control group. The CD80-WTG1 0.5 mg / kg TIW group had a mean tumor volume of 1337.30 mm on Day 22. 3 The relative tumor inhibition rate (TGI) was 38.93%, which was not significantly different from the control group. The mice in each group had normal body weight, no abnormal symptoms, and were in good general condition.

[0215] The results, as shown in FIG. 10, showed that CD80M28 inhibited tumor growth in a dose-dependent manner, and its activity was significantly superior to that of wild-type CD80-WT under the same dose conditions.

[0216] Example 7 Evaluation of antitumor effect of combined administration of CD80 fusion protein and Treg inhibitor using CT26 subcutaneous transplant model Using anti-GITR antibody as an example, we investigated the synergistic antitumor effect of CD80 fusion protein and Treg inhibitor.

[0217] The anti-GITR antibody disclosed in WO2017096189A1 was combined with the fusion protein of the present application to study the synergistic effect of the two. Mouse colorectal cancer cells CT26 were inoculated subcutaneously in the right anterior flank of female PD1 / GITR double humanized mice, and tumors were 80 mm 3When the tumors had grown to about 100% of the tumor volume, the tumors were divided into six groups, each consisting of 5 mice, and administered with CD80-WT and CD80M28 intravenously three times a week, and anti-GITR antibody twice a week for 2 weeks. The tumor volume and body weight were measured every week, and the relationship between the changes in the body weight and tumor volume of the tumor-bearing mice and the administration time was recorded. Tumor growth inhibition rate TGI TV (%) was calculated and statistical analysis was performed.

[0218] As shown in Figure 11, the tumor growth inhibition rates of CD80M28, CD80-WT, anti-GITR antibody, CD80M28 + anti-GITR antibody, and CD80-WT + anti-GITR antibody were 86.43%, 64.57%, 69.98%, 89.06%, and 80.80%, respectively, and the tumor volumes of each treatment group were significantly lower than those of the solvent control group (p<0.05), showing significant anti-tumor effects and effectively inhibiting tumor growth. In addition, the tumor volume of the CD80M28 + anti-GITR antibody combination treatment group was the smallest of all treatment groups, and the tumor inhibition activity of the combination treatment group was superior to that of the single drug group.

[0219] Example 8 Evaluation of the antitumor effect of combined administration of fusion protein and anti-PD-1 antibody using a CT26 subcutaneous tumor model The synergistic effect of the CD80 fusion protein of the present application in combination with an anti-m-PD-1 antibody was studied. The anti-m-PD-1 antibody used in the study was purchased from Bioxcell, Inc., with the product number BP0146.

[0220] Mouse colorectal cancer cells, CT26, were inoculated subcutaneously into the right anterior flank of female Balb / c mice, and the average tumor volume was approximately 70.9 mm 6 days after inoculation. 3Using a random grouping method, the tumor-bearing mice were divided into 4 groups, including Group 1, a PBS group (hereinafter abbreviated as Group 1), Group 2, a CD80M28 (0.4 mg / kg) group (hereinafter abbreviated as Group 2), Group 3, an anti-mPD-1 antibody (5 mg / kg) group (hereinafter abbreviated as Group 3), and Group 4, an anti-mPD-1 antibody (5 mg / kg) + CD80M28 (0.4 mg / kg) group (hereinafter abbreviated as Group 4), with 6 mice in each group. The administration volume for each group was 10 mL / kg. Groups 1 and 2 were administered intravenously into the tail vein three times a week for one week, Group 3 was administered intraperitoneally twice a week for two weeks for a total of four times, and Group 4 was administered intraperitoneally with anti-mPD-1 antibody (5 mg / kg) twice a week for two weeks and CD80M28 (0.4 mg / kg) three times a week for one week for a total of seven administrations.

[0221] The results are shown in FIG. 12. On the 18th day after the start of administration, the tumor volume in Group 1 was 2264.23±514.76 mm 3 The tumor volume in Group 3 was 1109.87 ± 224.72 mm 3 The tumor volume inhibition rate (TGI) was 52.64%, which was not statistically different from the tumor volume in the control group (P>0.05). The tumor volume in Group 2 was 821.79±387.96 mm 3 The tumor volume inhibition rate (TGI) was 65.76%, which was statistically different from the control group (P<0.05). The tumor volume in Group 4 was 140.49±34.58 mm 3 The tumor volume inhibition rate (TGI) was 96.83%, which was significantly statistically different from that of the control group (P<0.01).

[0222] The antitumor activity of the combined administration group was significantly superior to the antitumor activity of each single agent group, and a significant synergistic effect was observed.

[0223] Example 9 Evaluation of antitumor effect by combined administration of fusion protein and anti-CTLA-4 antibody using MC38 subcutaneous transplant model The synergistic effect of the CD80 fusion protein of the present invention in combination with anti-m-CTLA-4 antibody was studied. The anti-m-CTLA-4 antibody used in the study was purchased from Bioxcell, Inc., product number BP0164. Mouse colorectal cancer cells MC38 were subcutaneously inoculated into the right anterior flank of female C57BL / 6 mice. Seven days after inoculation, the average tumor volume was about 78 mm. 3 Using a random grouping method, the tumor-bearing mice were divided into 4 groups, each of which had 6 mice, including Group 1 (PBS group; hereafter abbreviated as Group 1), Group 2 (CD80M28 (0.5 mg / kg) group; Group 3 (CTLA-4 (4 mg / kg) group; hereafter abbreviated as Group 3), and Group 4 (CTLA-4 (5 mg / kg) + CD80M28 (0.4 mg / kg) group; hereafter abbreviated as Group 4). CD80M28 was administered intravenously once, and CTLA-4 antibody was administered intraperitoneally twice a week for a total of 3 times.

[0224] On day 17 of the experiment, subcutaneous tumors disappeared in both animals in the anti-mCTLA-4 4mg / kg (G3) and CD80M28+Anti-mCTLA-4 (G4) combination administration groups. On day 24 of the experiment, subcutaneous tumors disappeared in two animals in the anti-mCTLA-4 4mg / kg (G3) group, with a tumor elimination rate of 33.3%, and subcutaneous tumors disappeared in five animals in the CD80M28+Anti-mCTLA-4 (G4) combination administration group, with a tumor elimination rate of 83.3%, showing better activity than the single drug group. The results are shown in Figure 13.

[0225] Example 10 Induction of cytokine release by CD80 fusion proteins Using the CD3 antibody hOKT3 and the CD28 antibody TGN1412 as controls, the ability of CD80 fusion proteins to induce cytokine release from PBMCs was evaluated by ELISA.

[0226] Various concentrations of CD80 fusion protein, CD3 antibody hOKT3 (Human CD3 (humanized OKT3) Antibody-Human IgG4 (GMP-grade), Sino Biological), CD28 antibody TGN1412 (self-produced), and IgG1-Fc protein (Human IgG1 Fc Protein, Tag Free, ACROBiosystems) were added to a 96-well cell culture plate, dried overnight at room temperature, washed twice with saline, and then 1 × 10 5 PBMC cells (Schbio Biotech) from healthy donors were added and incubated in a 37°C incubator for 24 h. The supernatants were then collected and the TNF-α and IL-6 levels in the supernatants were detected using a TNF-α ELISA detection kit (HUMAN TNF-α ELISA KIT, ExCell Bio) and an IL-6 ELISA detection kit (Human IL-6 Precoated ELISA Kit, DAKEWE), respectively, to analyze the ability of CD80 fusion proteins to induce cytokine release from PBMCs.

[0227] The results are shown in Figures 14A-14B, and indicate that CD80-WT and CD80M28 do not stimulate cytokine release and therefore have good safety.

[0228] Example 11 Analysis of charge heterogeneity of CD80 fusion proteins Taking CD80M26 and CD80M28 as examples, the charge heterogeneity of CD80 mutant fusion proteins was analyzed. The capillary was washed at room temperature, and a mixed standard sample solution and a detection sample solution were prepared. The CD80M28 solution contained 50 μL of 1% MC, 50 μL of 8.6 mM urea, 10 μL of 200 mM IDA, 7 μL of 500 mM arginine, 25 μL of 1 mg / mL CD80M28, 1.6 μL of ampholyte 3-10, and 6.4 μL of ampholyte 5-8. The preparation method of CD80-WT, CD80M26 solution was 50μL of 1%MC, 50μL of 8.6mM urea, 10μL of 200mM IDA, 7μL of 500mM arginine, 25μL of 1mg / mL CD80-WT and CD80M26, 1.2μL of ampholytes 3-10, 10.8μL of ampholytes 5-8, the sampling pressure was selected at 2bar, the sampling time was set at 100s, electrophoresis was performed, and a chromatogram was collected to achieve separation due to the difference in charge heterogeneity. Quantitative analysis of acid-base variants was performed using the area normalization method.

[0229] The results are shown in Figure 15. The results show that the charge heterogeneity of CD80M26 and CD80M28 is obviously smaller than that of CD80-WT. The quality of the charge heterogeneous protein is more uniformly controlled, and the drug formability is better.

[0230] Example 12 Analysis of sialic acid ratios in CD80M28 samples derived from different cell clones by HPLC-FLD detection CD80M28 was used as an example to detect the effect of sialic acid ratio. The content of sialic acid was detected and analyzed by HPLC-FLD. After exchanging the protein sample with PBS solution five times, the protein concentration was measured, and the protein was further diluted to 1mg / mL, and 4M acetic acid was added to the protein sample in a 1:1 ratio, mixed uniformly, and incubated at 80℃ for 2 hours. 50μL of DMB solution was added to the above system and incubated at 50℃ for 2.5 hours. Two common sialic acid Neu5Ac(NA) / Neu5Gc(NG) quantitative standards in glycoproteins were used to plot standard curves of different concentrations, and quantitative analysis of Neu5Ac(NA) / Neu5Gc(NG) was performed.

[0231] TIFF2025511150000001.tif34170

[0232] Example 13: Evaluation of the antitumor effect of CD80M28 modified with different degrees of sialic acid using a CT26 subcutaneous transplant model Mouse colorectal cancer cells, CT26, were inoculated subcutaneously into the right anterior flank of 20 female BABL / C mice, and tumors were grown to 80 mm 3 When the mice had grown to about this size, they were divided into seven groups of six mice per group: G1 (solvent control (PBS) group), G2 (CD80M28-15 group), G3 (CD80M28-1 group), G4 (CD80M28-12 group), G5 (CD80M28-99 group), G6 (CD80M28-5 group), and G7 (CD80-WT), and were treated once every three days for a total of three times.

[0233] In the vehicle control group, the mean tumor volume at the end of the study was 2906.96 mm 3 In the CD80M28-15 treatment group, the mean tumor volume at the end of the study was 1979.69 mm 3 The relative tumor inhibition rate (TGI) was 31.90%, and the mean tumor volume at the end of the study was 2536.42 mm in the CD80M28-1 treatment group. 3 The relative tumor inhibition rate (TGI) was 12.75%, and the mean tumor volume at the end of the study was 1683.28 mm in the CD80M28-12 treatment group. 3 The relative tumor inhibition rate (TGI) was 42.09%, and the mean tumor volume at the end of the study was 1612.37 mm in the CD80M28-9 treatment group. 3 The relative tumor inhibition rate (TGI) was 44.53%, and the mean tumor volume at the end of the study was 1940.70 mm in the CD80M28-5 treatment group. 3 The relative tumor inhibition rate (TGI) was 33.24%, and the mean tumor volume at the end of the experiment was 2805.13 mm in the CD80-WT treatment group. 3 The relative tumor inhibition rate (TGI) was 3.50%.

[0234] The results are shown in Figure 16. The results show that the tumor inhibition effect of CD80M28 with different sialic acid contents is superior to that of wild-type CD80-WT, and when the sialic acid content is greater than 0.76, the tumor inhibition rate of CD80 fusion protein is more significant. In particular, when the sialic acid content is greater than 1, the tumor inhibition rate of CD80 fusion protein is significantly improved. This indicates that the improvement of sialic acid content contributes to the improvement of the tumor inhibition effect of CD80 fusion protein.

[0235] Example 14 Pharmacokinetic Animal Experiments After a single intravenous injection of stock solutions of CD80-WT-G1 (CD80-WT) and CD80M28 into Sprague Dawley rats, the pharmacokinetic properties of the two test substances were detected by detecting the concentrations of the test substances in the serum, providing data support for subsequent studies.

[0236] Analysis method: Quantitative range: 50.0 to 3200 ng / mL

[0237] TIFF2025511150000002.tif39170

[0238] Sample Collection: A total of 224 samples were taken at 14 time points, including 0 min before administration, 5 min, 30 min, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 96 h, 120 h, 168 h, 240 h, and 336 h after administration.

[0239] TIFF2025511150000003.tif127170

[0240] After a single intravenous injection of 2 mg / kg stock solution of CD80-WT-G1 and 2 mg / kg stock solution of CD80M28, respectively, in Sprague Dawley rats, the C max , AUC (0-t) and AUC infThe K was 1.44, 26.3, and 23.8 times, respectively, the CL was 0.0396 times, and the MRT was 2.60 times. t-test analysis was performed using SPSS software, and the results showed that the K el , t 1 / 2 and T max There was no statistical difference except for the remaining parameters C max , AUC (0-t) , AUC inf , AUC extr , V d The results showed that there was a significant statistical difference in CL and MRT. The mean blood drug concentration-time curves of each group after a single intravenous injection of CD80-WT-G1 and CD80M28 stock solutions into Sprague Dawley rats are shown in Figure 17. The results showed that the exposure of CD80M28 in rats was higher, the clearance was slower, and the sustained exposure time in vivo was longer.

Claims

1. 1. A CD80 protein variant comprising: The present invention relates to a functionally active fragment of a CD80 protein or a variant thereof, the variant having a mutation at one or more amino acid sites compared to a corresponding functionally active fragment of a wild-type CD80, and the variant comprises: a) binding to PD-L1; b) binding to CTLA-4; c) binding to CD28; d) Activation of T lymphocyte activity, and e) Inhibition of the growth and / or proliferation of tumors or tumor cells The present invention has one or more of the following characteristics: CD80 protein variants.

2. The PD-L1 is human PD-L1, the CTLA-4 is human CTLA-4, and the CD28 is human CD28. A CD80 protein variant according to claim 1.

3. The functionally active fragment of a CD80 protein comprises the extracellular domain (ECD) of a CD80 protein or a fragment thereof. A CD80 protein variant according to claim 1 or 2.

4. the fragment of the extracellular domain of the CD80 protein comprises the IgV domain of the CD80 protein; A CD80 protein variant according to claim 3.

5. comprising an amino acid mutation at one or more amino acid residues selected from the group consisting of N55, N64, N152, N173, N177, N192 and N198, as compared to the extracellular domain of a corresponding wild-type CD80 protein or a fragment thereof; A CD80 protein variant according to any one of claims 1 to 4.

6. 1) N64, 2) N55 and N64, 3) N55, N64 and N152, 4) N55, N64 and N192, 5) N55, N64 and N198, 6) N55, N64, N152 and N198, 7) N55, N64, N152 and N192, 8) N55, N64, N152, N173, N177 and N198, 9) N55, N64, N152, N173, and N198; and 10) N55, N64, N152, N177 and N198 The present invention relates to a method for producing a mutated nucleotide sequence comprising the steps of: A CD80 protein variant according to any one of claims 1 to 5.

7. comprising one or more amino acid mutations selected from the group consisting of N55A / Q, N64A, N152A, N173Q, N177A, N192A and N198A / Q; A CD80 protein variant according to any one of claims 1 to 6.

8. 1) N64A, 2) N55A and N64A, 3) N55A, N64A and N152A, 4) N55A, N64A and N192A, 5) N55A, N64A and N198A, 6) N55A, N64A, N152A and N198A, 7) N55A, N64A, N152A and N192A, 8) N55Q, N64A, N152A and N198Q, 9) N55Q, N64A, N152A, N173Q, N177A and N198Q, 10) N55Q, N64A, N152A, N173Q and N198Q, and 11) N55Q, N64A, N152A, N177A and N198Q The present invention relates to a method for producing a nucleic acid sequence comprising the steps of: A CD80 protein variant according to any one of claims 1 to 7.

9. at least one class I amino acid mutation, which can maintain or enhance the stability of the CD80 protein variant; A CD80 protein variant according to any one of claims 1 to 8.

10. comprising a class I amino acid mutation at one or more of the following amino acid positions: N55, N64, N152, N192, and N198; A CD80 protein variant according to claim 9.

11. 1) N64, 2) N55 and N64, 3) N55, N64 and N152, 4) N55, N64 and N192, 5) N55, N64 and N198, 6) N55, N64, N152, and N198; and 7) N55, N64, N152 and N192 The present invention relates to a method for treating a mutated schizophrenia comprising administering to a patient ... A CD80 protein variant according to any one of claims 9 to 10.

12. comprising one or more class I amino acid mutations: N55A / Q, N64A, N152A, N192A, and N198A / Q; A CD80 protein variant according to any one of claims 9 to 11.

13. 1) N64A, 2) N55A and N64A, 3) N55A, N64A and N152A, 4) N55A, N64A and N192A, 5) N55A, N64A and N198A, 6) N55A, N64A, N152A and N198A, 7) N55A, N64A, N152A and N192A, and 8) N55Q, N64A, N152A and N198Q The present invention relates to a method for the preparation of a polypeptide comprising the steps of: A protein variant according to any one of claims 9 to 12.

14. at least one class II amino acid mutation, wherein said class II amino acid mutation is capable of retaining the binding activity of said CD80 protein variant. A CD80 protein variant according to any one of claims 1 to 13.

15. comprising a class II amino acid mutation at one or more of positions N152, N192, and N198; A CD80 protein variant according to claim 14.

16. comprising one or more class II amino acid mutations: N152A, N192A, and N198A / Q; A CD80 protein variant according to any one of claims 14 to 15.

17. comprising a class II amino acid mutation at positions N55 and / or N64; A CD80 protein variant according to any one of claims 14 to 16.

18. comprising N55A / Q and / or N64A class II amino acid mutations; A CD80 protein variant according to any one of claims 14 to 17.

19. at least one class III amino acid mutation, said amino acid mutation being capable of retaining the affinity of said CD80 protein variant; A CD80 protein variant according to any one of claims 1 to 18.

20. comprising a class III amino acid mutation at one or more of positions N55, N64, N152, and N198; 20. A CD80 protein variant according to claim 19.

21. comprising one or more class III amino acid mutations: N55A / Q, N64A, N152A, and N198A; A CD80 protein variant according to any one of claims 19 to 20.

22. containing a class III amino acid mutation at position N192, A CD80 protein variant according to any one of claims 19 to 21.

23. Containing the N192A class III amino acid mutation, A CD80 protein variant according to any one of claims 19 to 22.

24. at least one class IV amino acid mutation, said mutation being capable of enhancing binding of the CD80 protein variant to CD28 and / or PD-L1; A CD80 protein variant according to any one of claims 1 to 23.

25. comprising a class IV amino acid mutation at position N173 and / or N177; 25. A CD80 protein variant according to claim 24.

26. comprising the class IV amino acid mutations N173Q and / or N177A; A CD80 protein variant according to any one of claims 24 to 25.

27. The numbering of the amino acid positions is determined with reference to the numbering of the amino acid positions in SEQ ID NO:

1. A CD80 protein variant according to any one of claims 1 to 26.

28. The amino acid sequence of any one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24 is included. A CD80 protein variant according to any one of claims 1 to 27.

29. The sialic acid content is about 0.7 or more. A CD80 protein variant according to any one of claims 1 to 28.

30. A fusion protein comprising:

30. A method for treating a CD80 protein comprising administering to a subject a therapeutically effective amount of a CD80 protein variant according to any one of claims 1 to 29. Fusion proteins.

31. Further comprising an immunoglobulin Fc region or a variant thereof; The fusion protein of claim 30.

32. said CD80 protein variant is directly or indirectly linked to said immunoglobulin Fc region; 32. The fusion protein of claim 31.

33. The immunoglobulin Fc region comprises an IgG Fc region. A fusion protein according to any one of claims 31 to 32.

34. The IgG is selected from the group consisting of IgG1 and IgG4; The fusion protein of claim 33.

35. the CD80 protein variant is located at the N-terminus or C-terminus of the immunoglobulin Fc region; A fusion protein according to any one of claims 31 to 34.

36. The immunoglobulin Fc region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:25 and SEQ ID NO:

26. A fusion protein according to any one of claims 31 to 35.

37. Contains an amino acid sequence shown in any one of SEQ ID NOs: 27 to 37; A fusion protein according to any one of claims 30 to 36.

38. A polypeptide comprising: A CD80 protein variant according to any one of claims 1 to 29, or a fusion protein according to any one of claims 30 to 37. Polypeptides.

39. 1. An isolated nucleic acid molecule or molecules comprising: A gene encoding a CD80 protein variant according to any one of claims 1 to 29, a fusion protein according to any one of claims 30 to 37, or a polypeptide according to claim 38. Nucleic acid molecule.

40. A vector comprising:

40. The nucleic acid molecule of claim 39, vector.

41. A host cell comprising:

41. Comprising a nucleic acid molecule according to claim 39 or a vector according to claim 40. host cell.

42. A method for producing a CD80 protein variant according to any one of claims 1 to 29 or a fusion protein according to any one of claims 30 to 37, comprising the steps of: Culturing the cell of claim 41 under conditions that allow expression of the fusion protein. method.

43. 1. A pharmaceutical composition comprising:

20. A method for the preparation of a CD80 protein variant according to claim 1, a fusion protein according to claim 30, a polypeptide according to claim 38, a nucleic acid molecule according to claim 39, a vector according to claim 40 and / or a host cell according to claim 41, and optionally a pharma- ceutically acceptable carrier agent. Pharmaceutical compositions.

44. A pharmaceutical combination comprising: A method for the treatment of a disease comprising administering to a patient a therapeutically effective amount of a CD80 protein variant according to any one of claims 1 to 19, a fusion protein according to any one of claims 30 to 37, or a polypeptide according to claim 38, and an immune checkpoint inhibitor. Pharmaceutical combinations.

45. The immune checkpoints include PD-1, PD-L1, GITR, and / or CTLA-4.

45. A pharmaceutical combination according to claim 44.

46. The immune checkpoint inhibitor comprises one or more selected from the group consisting of PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, and GITR antibody; A pharmaceutical combination according to any one of claims 44 to 45.

47. 1. A method of modulating an immune response in a subject, comprising: Administering a CD80 protein variant according to any one of claims 1 to 29, a fusion protein according to any one of claims 30 to 37 or a polypeptide according to claim 38. method.

48. The method of modulating an immune response in a subject includes increasing an immune response.

42. The method of claim 41.

49. wherein the CD80 protein variant exhibits increased binding affinity to PD-L1 compared to a wild-type CD80 protein or a fragment thereof; 49. The method according to any one of claims 47 to 48.

50. wherein, compared to a wild-type CD80 protein or a fragment thereof, the CD80 protein variant exhibits increased binding affinity to CD28.

50. The method according to any one of claims 47 to 49.

51. wherein, compared to a wild-type CD80 protein or a fragment thereof, the CD80 protein variant exhibits enhanced activation of T lymphocyte activity.

51. The method according to any one of claims 47 to 50.

52. a CD80 protein variant according to any one of claims 1 to 29, a fusion protein according to any one of claims 30 to 37, a polypeptide according to claim 38, a nucleic acid molecule according to claim 39, a vector according to claim 40, a host cell according to claim 41, a pharmaceutical composition according to claim 43, or a pharmaceutical combination according to any one of claims 44 to 46 in the manufacture of a medicament for the prevention and / or treatment of a disease and / or condition. application.

53. The disease and / or condition includes a tumor.

53. The application of claim 52.

54. The tumor comprises a solid tumor and / or a hematological tumor.

54. The application of claim 53.

55. The tumor comprises a tumor that responds to the proliferation of central memory T cells.

55. The application according to any one of claims 53 to 54.

56. The tumor is one or more selected from the group consisting of malignant melanoma, breast cancer, gastric cancer, renal cancer, non-small cell lung cancer, colon cancer, rectal cancer, head and neck squamous cell carcinoma, liver cancer, kidney cancer, mesothelioma bladder cancer, pancreatic cancer, ovarian cancer, endometrial cancer and lymphoma; 56. The application according to any one of claims 53 to 55.

57. 1. A method for preventing and / or treating a disease and / or condition, comprising: Administering to a subject in need thereof a CD80 protein variant according to any one of claims 1 to 29, a fusion protein according to any one of claims 30 to 37, a polypeptide according to claim 38, a nucleic acid molecule according to claim 39, a vector according to claim 40, a host cell according to claim 41, a pharmaceutical composition according to claim 43, or a pharmaceutical combination according to any one of claims 44 to 46. method.

58. The disease and / or condition includes a tumor.

58. The method of claim 57.

59. The tumor comprises a solid tumor and / or a hematological tumor.

59. The method of claim 58.

60. The tumor comprises a tumor that responds to the proliferation of central memory T cells.

60. The method according to any one of claims 58 to 59.

61. The tumor is one or more selected from the group consisting of malignant melanoma, breast cancer, gastric cancer, renal cancer, non-small cell lung cancer, colon cancer, rectal cancer, head and neck squamous cell carcinoma, liver cancer, kidney cancer, mesothelioma bladder cancer, pancreatic cancer, ovarian cancer, endometrial cancer and lymphoma; 61. The method according to any one of claims 58 to 60.

62. For preventing and / or treating a disease and / or condition, A CD80 protein variant according to any one of claims 1 to 29, a fusion protein according to any one of claims 30 to 37, a polypeptide according to claim 38, a nucleic acid molecule according to claim 39, a vector according to claim 40, a host cell according to claim 41, a pharmaceutical composition according to claim 43, or a pharmaceutical combination according to any one of claims 44 to 46.

63. The disease and / or condition includes a tumor.

63. The CD80 protein variant, fusion protein, nucleic acid molecule, vector, host cell, pharmaceutical composition or pharmaceutical combination of claim 62.

64. The tumor comprises a solid tumor and / or a hematological tumor.

64. The CD80 protein variant, fusion protein, nucleic acid molecule, vector, host cell, pharmaceutical composition or pharmaceutical combination of claim 63.

65. The tumor comprises a tumor that responds to the proliferation of central memory T cells.

65. A CD80 protein variant, a fusion protein, a nucleic acid molecule, a vector, a host cell, a pharmaceutical composition or a pharmaceutical combination according to any one of claims 63 to 64.

66. The tumor is one or more selected from the group consisting of malignant melanoma, breast cancer, gastric cancer, renal cancer, non-small cell lung cancer, colon cancer, rectal cancer, head and neck squamous cell carcinoma, liver cancer, kidney cancer, mesothelioma bladder cancer, pancreatic cancer, ovarian cancer, endometrial cancer and lymphoma; 66. A CD80 protein variant, a fusion protein, a nucleic acid molecule, a vector, a host cell, a pharmaceutical composition or a pharmaceutical combination according to any one of claims 63 to 65.

67. 1. A method for detecting the frequency and / or proliferation of central memory T cells in a subject, comprising: Administering a CD80 protein variant according to any one of claims 1 to 29, a fusion protein according to any one of claims 30 to 37, or a polypeptide according to claim 38. method.

68. The central memory T cells are CD95+ and CD28+ cells.

68. The method of claim 67.

69. The central memory T cells are CD4+ central memory T cells and / or CD8+ central memory T cells, 68. The method of claim 67.

70. To enhance the proliferation or frequency of central memory T cells, A CD80 protein variant according to any one of claims 1 to 29, a fusion protein according to any one of claims 30 to 37, a polypeptide according to claim 38, a nucleic acid molecule according to claim 39, a vector according to claim 40, a host cell according to claim 41, a pharmaceutical composition according to claim 43, or a pharmaceutical combination according to any one of claims 44 to 46.

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