Nanobodies against human hhma2 and uses thereof

Abstract

The application relates to a kind of nanobodies against human HHLA2 and its application, the variable region of the nanobody includes CDR1, CDR2 and CDR3, the amino acid sequence of the CDR1 includes any one of the sequence shown in SEQ ID NO:29-36 or its mutation, the amino acid sequence of CDR2 includes any one of the sequence shown in SEQ ID NO:37-46 or its mutation, the amino acid sequence of CDR3 includes any one of the sequence shown in SEQ ID NO:47-56 or its mutation, the mutation is the replacement, insertion or deletion of 1, 2, 3 or 4 amino acids in amino acid sequence.The disclosed nanobody can be specifically combined with tumor cells expressing human HHLA2, and can improve the killing effect of immune cells on tumor cells by blocking the HHLA2 and KIR3DL3 signal pathway.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an anti-human HHLA2 nanobody and its application. Background Technology

[0002] HHLA2 (human endogenous retrovirus-H long terminal repeat-associating2) was discovered during the search for human retrovirus long terminal repeat (LTR) sequences (Genomics, 1999, 59(3):255-263). HHLA2, also known as B7-H7 or B7y, is a newly discovered B7 family molecule. Currently, there are 10 members of the B7 family that have been identified: CD80 (B7-1), CD86 (B7-2), B7-H1 (PD-L1, CD274), B7-DC (CD273 or PD-L2), B7-H2 (CD275), B7-H3 (CD276), B7-H4 (B7S1, B7x or VTCN1), B7-H5 (Vista, GI24 or PD-1H), B7-H6 (NCR3LG1), and B7-H7 (HHLA2 or B7y) (Frontiers in Immunology, 2020, 11:681).

[0003] HHLA2 is mainly distributed and highly expressed in various tumor tissues, with only small amounts expressed in epithelial cells of the intestine, kidney, gallbladder, and breast, as well as placental trophoblastic tissue. HHLA2 is closely related to the growth, metastasis, pathological grade, and prognosis of various tumors, including pancreatic cancer, lung cancer, breast cancer, osteosarcoma, colorectal cancer, and renal cancer. On the one hand, HHLA2 can co-stimulate T cells in the context of TCR-mediated activation by interacting with TMIGD2, enhancing T cell proliferation and cytokine production through an AKT-dependent signaling cascade. On the other hand, HHLA2 can recruit SHP-1 and SHP-2 by interacting with KIR3DL3 on T / NK immune cells, thereby attenuating downstream Vav1, ERK1 / 2, AKT, and NF-κB signaling, thus exhibiting immunosuppressive activity (International Immunopharmacology, 2023, 121: 110403).

[0004] Cancer immunotherapy enhances the body's immunity against tumors by reducing immune escape. Compared with traditional chemotherapy, immunotherapy has advantages such as lower drug dosage, broader range of action, and fewer side effects. For example, anti-PD1 and anti-PD-L1 immune checkpoint inhibitors have achieved great success in clinical practice, demonstrating excellent anti-tumor activity. However, these immune checkpoint inhibitors have a low patient response rate (approximately 30%) in clinical practice. This is due to the complex and integrated mechanisms of the tumor microenvironment, where multiple immunosuppressive molecules, suppressor cells, and unique tumor tissue structures collectively block the occurrence of normal immune responses. Blocking only a single immune checkpoint often fails to achieve ideal therapeutic effects. Existing preclinical studies have shown that the distribution of HHLA2 in tumor tissues is independent of PD-L1, and HHLA2 can promote tumor immune escape. Drugs that block the HHLA2 / KIR3DL3 pathway (especially antibody drugs) can inhibit tumor development and metastasis, and can produce a synergistic effect when used in combination with PD-(L)1 monoclonal antibodies. Therefore, anti-tumor therapy targeting HHLA2 / KIR3DL3 is currently receiving much attention (Cancerimmunology research, 2021, 9(2): 156-169; Science immunology, 2021, 6(61): eabf9792).

[0005] Animals such as alpacas and camels possess naturally occurring antibodies in their peripheral blood that lack light chains and consist only of heavy chains. These heavy chains contain two constant regions (CH2 and CH3), a hinge region, and a variable heavy chain domain (VHH, the antigen-binding region). The VHH heavy chain variable domain has a relative molecular mass of approximately 15 kDa, only 1 / 10 that of conventional antibodies, and its molecular height and diameter are both at the nanometer level, allowing it to penetrate the blood-brain barrier; therefore, it is called a nanobody (Nb). Nanobodies are typically highly soluble, highly stable (not easily aggregated, and resistant to denaturing conditions such as high temperature, strong acid, and strong alkali), highly affinity, and have low immunogenicity (homology to human antibodies exceeds 80%). They are easy to engineer and are suitable for various prokaryotic and eukaryotic expression systems, and are widely used in the development of antibody drugs and detection reagents (Annual review of biochemistry, 2013, 82:775-797; Analytical and Bioanalytical Chemistry, 2019, 411:1703–1713). Currently, most publicly reported antibodies targeting the HHLA2 / KIR3DL3 signaling pathway are hybridoma / mouse / human monoclonal antibodies (CN114729052A; CN112153977A; WO2023138579A1; US20210198366A1). Among them, only PlatinumPharma's fully human monoclonal antibody HBM1020 (ClinicalTrials.gov ID: NCT05824663), developed based on its self-developed H2L2 transgenic mouse platform, is in the clinical research stage. There are no reports on the development progress of nanobody drugs targeting HHLA2. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an anti-human HHLA2 nanobody and its applications.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a nanobody against human HHLA2, wherein the variable region of the nanobody includes CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 includes any one of the sequences shown in SEQ ID NO:29-SEQ ID NO:36 or a mutation thereof, the amino acid sequence of CDR2 includes any one of the sequences shown in SEQ ID NO:37-SEQ ID NO:46 or a mutation thereof, and the amino acid sequence of CDR3 includes any one of the sequences shown in SEQ ID NO:47-SEQ ID NO:56 or a mutation thereof, wherein the mutation is a substitution, insertion or deletion of 1, 2, 3 or 4 amino acids in the amino acid sequence.

[0009] SEQ ID NO:29: YYVIG.

[0010] SEQ ID NO:30: STPMY.

[0011] SEQ ID NO:31: DYSIG.

[0012] SEQ ID NO:32: NYLMG.

[0013] SEQ ID NO:33: FNVMG.

[0014] SEQ ID NO:34: FTAAG.

[0015] SEQ ID NO:35: FSAAG.

[0016] SEQ ID NO:36: DYAIG.

[0017] SEQ ID NO:37: CISSSTGSTYSADSVKG.

[0018] SEQ ID NO:38: CISSSTGSTYSAQSVKG.

[0019] SEQ ID NO:39: CISSSDGSTYSADSVKG.

[0020] SEQ ID NO:40: IMAPGGYEDIADSVRG.

[0021] SEQ ID NO:41: CISSTHGTTYYTDSVKD.

[0022] SEQ ID NO:42: AINSAGYSTVYAESVKG.

[0023] SEQ ID NO:43: LISSGGTANYADSVKG.

[0024] SEQ ID NO:44:VIANGGHTNYADSVKG.

[0025] SEQ ID NO:45:VITGGGHTNYADSVKG.

[0026] SEQ ID NO:46: CITSSTNRTYYANSVKG.

[0027] SEQ ID NO:47:RRSPGIEGWCALPFFFGS.

[0028] SEQ ID NO:48:RRSPGIEGWCALPEFFGS.

[0029] SEQ ID NO:49: RISPGIEGWCALPFFFGS.

[0030] SEQ ID NO:50: RYSPGIEGWCALPFFFGS.

[0031] SEQ ID NO:51: LNS.

[0032] SEQ ID NO:52: GLDGLACHRRDSGSRYRTQLYDY.

[0033] SEQ ID NO:53: RYDRWHDY.

[0034] SEQ ID NO:54: TTPVGQY.

[0035] SEQ ID NO:55: PPAFGS.

[0036] SEQ ID NO:56: EPILCVLWSAGGAMDY.

[0037] The nanobodies disclosed in this invention can specifically bind to tumor cells expressing human HHLA2, and can enhance the killing effect of immune cells on tumor cells by blocking the HHLA2 and KIR3DL3 signaling pathway. Therefore, the nanobodies disclosed in this invention have the potential to be used in the development of drugs or diagnostic reagents for treating tumors.

[0038] In this application, the mutations may include mutations designed during antibody screening and engineering modification in the antibody preparation or application process. For example, mutations may be made on potential post-translational modifications (PTMs) sites in variable regions (especially CDRs), including potential asparagine deamidation sites (NG, NS, NH, etc.), potential aspartic acid isomer sites (DG, DP, DS, etc.), potential N-glycosylation sites (N-{P}S / T), and potential oxidation-sensitive sites (M, W). The mutations may also include mutations on potential sites that affect antibody aggregation, glycosylation, solubility, viscosity, expression level, activity (affinity, etc.), purity, isoelectric point, Tm, fragmentation, PK, immunogenicity, and other properties.

[0039] Preferably, the mutation of CDR1 is a substitution of 2 or 1 amino acid in the amino acid sequence shown in SEQ ID NO:36, namely D1Y / G / N / S / T / R or S3A / V / G / S / T / W / Y.

[0040] Preferably, the amino acid sequence of CDR1 is the sequence shown in SEQ ID NO:29 or SEQ ID NO:31.

[0041] Preferably, the mutation of CDR2 is a substitution of 2 or 1 amino acid in the amino acid sequence shown in SEQ ID NO:39, with D6T / E / V / R / L / A / I and D13Q / E.

[0042] Preferably, the amino acid sequence of CDR2 is the sequence shown in SEQ ID NO:37 or SEQ ID NO:38.

[0043] Preferably, the mutation of the heavy chain variable region CDR3 is a substitution of 2 or 1 amino acid in the amino acid sequence R2I / Y / V / L / K / E / T / S or F14E / R / S / T as shown in SEQ ID NO:47.

[0044] Preferably, the amino acid sequence of CDR3 is any one of the sequences shown in SEQ ID NO:48-SEQ ID NO:50.

[0045] Those skilled in the art should understand the meaning of the above-described mutations. For example, D13Q / E means that the 13th amino acid D in the amino acid sequence shown in SEQ ID NO:39 is mutated to Q or E. Other amino acid substitutions include D1Y / G / N / S / T / R, S3A / V / G / S / T / W / Y, D6T / E / V / R / L / A / I, R2I / Y / V / L / K / E / T / S, and F14E / R / S / T, etc., and so on.

[0046] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the anti-human HHLA2 nanobody are the sequences shown in SEQ ID NO:29, SEQ ID NO:37 and SEQ ID NO:47, respectively.

[0047] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the anti-human HHLA2 nanobody are the sequences shown in SEQ ID NO:29, SEQ ID NO:37 and SEQ ID NO:48, respectively.

[0048] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the anti-human HHLA2 nanobody are the sequences shown in SEQ ID NO:29, SEQ ID NO:38 and SEQ ID NO:47, respectively.

[0049] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the anti-human HHLA2 nanobody are the sequences shown in SEQ ID NO:29, SEQ ID NO:37 and SEQ ID NO:49, respectively.

[0050] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the anti-human HHLA2 nanobody are the sequences shown in SEQ ID NO:29, SEQ ID NO:37 and SEQ ID NO:50, respectively.

[0051] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the anti-human HHLA2 nanobody are the sequences shown in SEQ ID NO:29, SEQ ID NO:39 and SEQ ID NO:47, respectively.

[0052] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the anti-human HHLA2 nanobody are the sequences shown in SEQ ID NO:30, SEQ ID NO:40 and SEQ ID NO:51, respectively.

[0053] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the anti-human HHLA2 nanobody are the sequences shown in SEQ ID NO:31, SEQ ID NO:41 and SEQ ID NO:52, respectively.

[0054] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the anti-human HHLA2 nanobody are the sequences shown in SEQ ID NO:32, SEQ ID NO:42 and SEQ ID NO:53, respectively.

[0055] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the anti-human HHLA2 nanobody are the sequences shown in SEQ ID NO:33, SEQ ID NO:43 and SEQ ID NO:54, respectively.

[0056] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the anti-human HHLA2 nanobody are the sequences shown in SEQ ID NO:34, SEQ ID NO:44 and SEQ ID NO:55, respectively.

[0057] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the anti-human HHLA2 nanobody are the sequences shown in SEQ ID NO:35, SEQ ID NO:45 and SEQ ID NO:55, respectively.

[0058] Preferably, the amino acid sequences of CDR1, CDR2 and CDR3 in the anti-human HHLA2 nanobody are the sequences shown in SEQ ID NO:36, SEQ ID NO:46 and SEQ ID NO:56, respectively.

[0059] In this invention, the amino acid sequences of the CDRs listed above are represented according to the Kabat numbering system based on sequence variability (Kabat E A. Sequences of proteins of immunological interest [M]. USDapartment of Health and Human Services, Public Health Service, National Institutes of Health, 1991). Those skilled in the art will know that various numbering systems can be used to represent antibody CDRs (e.g., IMGT, Chothia, Honegger, AbM, and Contact). Although the range of CDR sequences claimed in this invention is based on the sequences represented by the Kabat numbering system, amino acid sequences defined according to other CDR numbering schemes should also fall within the scope of protection of this invention.

[0060] Preferably, the variable region in the nanobody further includes four framework regions.

[0061] Preferably, the nanobody includes a single-domain antibody, a VHH-Fc antibody, a bispecific antibody, or a multispecific antibody.

[0062] In this application, a single-domain antibody (nanobody) refers to a VHH structure isolated or cloned from a heavy chain antibody, which is the smallest known unit capable of binding to a target antigen.

[0063] In this application, heavy chain antibody (VHH-Fc antibody) refers to an antibody containing a heavy chain variable region (VHH), a hinge region (or containing a linker, such as a repeating "GGGGS" amino acid sequence or a variant thereof), a CH2 constant region, and a CH3 constant region, and is usually a homodimer.

[0064] In this application, the terms "bispecific antibody" and "multispecific antibody" refer to antibodies that have two or more specific epitopes, i.e., "bispecific antibody" or "multispecific antibody" has two or more single variable regions, and therefore can bind to different targets or different epitopes of the same target.

[0065] Preferably, the nanobody includes a variable region, a heavy chain crystallizable region fragment, or a mutant thereof.

[0066] The mutants include mutations that enhance / weaken the function of fragments in the crystallizable region of heavy chains, mutations that prolong or shorten the half-life of fragments in the crystallizable region of heavy chains, etc., which are well known to those skilled in the art.

[0067] Preferably, the crystallizable region of the heavy chain is selected from any one of human IgG1 or its mutant, IgG2 or its mutant, IgG3 or its mutant, and IgG4 or its mutant;

[0068] Alternatively, it may be selected from any one of mouse IgG1 or its mutants, IgG2a or its mutants, IgG2b or its mutants, or IgG3 or its mutants.

[0069] Preferably, the crystallizable region of the heavy chain is selected from any one of human IgG1 or its mutants, mouse IgG2a or its mutants.

[0070] Preferably, the source of the heavy chain crystallizable region fragment includes humans, mice, rats, alpacas, goats, rabbits, or horses.

[0071] Preferably, the amino acid sequence of the variable region in the nanobody includes any of the sequences shown in SEQ ID NO:15-SEQ ID NO:28.

[0072] SEQ ID NO:15:

[0073] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSTGSTYSADSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTLVTVSS.

[0074] SEQ ID NO:16:

[0075] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSTGSTYSADSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARRSPGIEGWCALPEFFGSWGQGTLVTVSS.

[0076] SEQ ID NO:17:

[0077] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSTGSTYSAQSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTLVTVSS.

[0078] SEQ ID NO:18:

[0079] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSTGSTYSADSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARISPGIEGWCALPFFFGSWGQGTLVTVSS。

[0080] SEQ ID NO:19:

[0081] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSTGSTYSADSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARYSPGIEGWCALPFFFGSWGQGTLVTVSS。

[0082] SEQ ID NO:20:

[0083] DVQLQESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSDGSTYSADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTQVTVSS。

[0084] SEQ ID NO:21:

[0085] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSDGSTYSADSVKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTLVTVSS。

[0086] SEQ ID NO:22:

[0087] DVQLQESGGGLVQPGGSLRLSCAASGFTFSSTPMYWYRQAPGKERDFVAIMAPGGYEDIADSVRGRFSISRDNGNNTMYLQMNSLKPEDTALYYCRALNSWGQGTQVTVSS。

[0088] SEQ ID NO:23:

[0089] DVQLQESGGGLVQAGGSLRLSCAASGFPFDDYSIGWFRQAPGKAREGISCISSTHGTTYYTDSVKDRFTITRDNAKNTVYLQMNNLKPEDAAVYYCAAGLDGLACHRRDSGSRYRTQLYDYWGQGTQVTVSS。

[0090] SEQ ID NO:24:

[0091] DVQLQESGGGLVQPGGSLRLSCAASGFTFSNYLMGWYRQAPGKERELVAAINSAGYSTVYAESVKGRFTTSRDNAKSMVYLQMNSLKAEDTAVYYCNARYDRWHDYWGQGTQVTVSS。

[0092] SEQ ID NO:25:

[0093] DVQLQESGGGLVQPGGSLRLSCAASGSRFSFNVMGWHRQAPGKQRELVALISSGGTANYADSVKGRFTISRDNAGNTMYLQMNTLKPEDTAVYYCTATTPVGQYWGQGTQVTVSS。

[0094] SEQ ID NO:26:

[0095] DVQLQESGGGLVQPGGSLKLSCTASRRIFTFTAAGWYRQAPGQQREFVAVIANGGHTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTPPAFGSWGPGTQVTVSS。

[0096] SEQ ID NO:27:

[0097] DVQLQESGGGLVQPGGSLRLSCTASTRIFTFSAAGWYRQAPGKQREFVAVITGGGHTNYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCNTPPAFGSWGPGTQVTVSS。

[0098] SEQ ID NO:28:

[0099] DVQLQESGGGLVQTGGSLRLSCAAPGFTLDDYAIGWFRQAPGKERERISCITSSTNRTYYANSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCAAEPILCVLWSAGGAMDYWGKGTQVTVSS。

[0100] Preferably, the amino acid sequence of the VHH-Fc antibody comprises the sequence shown in any one of SEQ ID NO:1-SEQ ID NO:14.

[0101] SEQ ID NO:1:

[0102] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSTGSTYSADSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTLVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0103] SEQ ID NO:2:

[0104] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSTGSTYSADSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARRSPGIEGWCALPEFFGSWGQGTLVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0105] SEQ ID NO:3:

[0106] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSTGSTYSAQSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTLVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0107] SEQ ID NO:4:

[0108] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSTGSTYSADSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARISPGIEGWCALPFFFGSWGQGTLVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0109] SEQ ID NO:5:

[0110] EVQLLESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSTGSTYSADSVKGRFTISRDTSKNTVYLQMNSLRAEDTAVYYCAARYSPGIEGWCALPFFFGSWGQGTLVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0111] SEQ ID NO:6:

[0112] DVQLQESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSDGSTYSADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTQVTVSSGGGGSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0113] SEQ ID NO:7:

[0114] DVQLQESGGGLVQPGGSLRLSCAASGFTLDYYVIGWFRQAPGKEREGVSCISSSDGSTYSADSVKGRFTISRDNAENTVYLQMNSLKPEDTAVYYCAARRSPGIEGWCALPFFFGSWGQGTQVTVSSGGGGSGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK。

[0115] SEQ ID NO:8:

[0116] DVQLQESGGGLVQPGGSLRLSCAASGFTFSSTPMYWYRQAPGKERDFVAIMAPGGYEDIADSVRGRFSISRDNGNNTMYLQMNSLKPEDTALYYCRALNSWGQGTQVTVSSGGGGSGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPI QHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK。

[0117] SEQ ID NO:9:

[0118] DVQLQESGGGLVQAGGSLRLSCAASGFPFDDYSIGWFRQAPGKAREGISCISSTHGTTYYTDSVKDRFTITRDNAKNTVYLQMNNLKPEDAAVYYCAAGLDGLACHRRDSGSRYRTQLYDYWGQGTQVTVSSGGGGSGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK。

[0119] SEQ ID NO:10:

[0120] DVQLQESGGGLVQPGGSLRLSCAASGFTFSNYLMGWYRQAPGKERELVAAINSAGYSTVYAESVKGRFTTSRDNAKSMVYLQMNSLKAEDTAVYYCNARYDRWHDYWGQGTQVTVSSGGGGSGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK。

[0121] SEQ ID NO:11:

[0122] DVQLQESGGGLVQPGGSLRLSCAASGSRFSFNVMGWHRQAPGKQRELVALISSGGTANYADSVKGRFTISRDNAGNTMYLQMNTLKPEDTAVYYCTATTPVGQYWGQGTQVTVSSGGGGSGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK。

[0123] SEQ ID NO:12:

[0124] DVQLQESGGGLVQPGGSLKLSCTASRRIFTFTAAGWYRQAPGQQREFVAVIANGGHTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNTPPAFGSWGPGTQVTVSSGGGGSGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSR TPGK。

[0125] SEQ ID NO:13:

[0126] DVQLQESGGGLVQPGGSLRLSCTASTRIFTFSAAGWYRQAPGKQREFVAVITGGGHTNYADSVKGRFTISRDNAKDTVYLQMNSLKPEDTAVYYCNTPPAFGSWGPGTQVTVSSGGGGSGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK。

[0127] SEQ ID NO:14:

[0128] DVQLQESGGGLVQTGGSLRLSCAAPGFTLDDYAIGWFRQAPGKERERISCITSSTNRTYYANSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCAAEPILCVLWSAGGAMDYWGKGTQVTVSSGGGGSGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVS EDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK.

[0129] In this invention, the amino acid sequences of the CDRs listed above are represented according to the Kabat numbering system based on sequence variability (Kabat E A. Sequences of proteins of immunological interest [M]. USDapartment of Health and Human Services, Public Health Service, National Institutes of Health, 1991). Those skilled in the art will know that various numbering systems can be used to represent antibody CDRs (e.g., IMGT, Chothia, Honegger, AbM, and Contact). Although the range of CDR sequences claimed in this invention is based on the sequences represented by the Kabat numbering system, amino acid sequences defined according to other CDR numbering schemes should also fall within the scope of protection of this invention.

[0130] In a second aspect, the present invention provides a nucleic acid molecule that encodes the anti-human HHLA2 nanobody described in the first aspect.

[0131] The nucleic acid can be prepared using conventional molecular biology techniques in the art, such as obtaining the nucleic acid encoding the above-mentioned anti-human HHLA2 nanobody through molecular cloning, or obtaining the above-mentioned nucleic acid through codon optimization and artificial full-sequence synthesis. Those skilled in the art know that the base sequence encoding the above-mentioned anti-human HHLA2 nanobody can be transformed into a polynucleotide homologue by appropriately introducing substitutions, deletions, or insertions. In this invention, the polynucleotide homologue can be obtained by substituting, deleting, or inserting one or more bases encoding the human HHLA2 nanobody gene within the range of maintaining antibody activity.

[0132] Thirdly, the present invention provides an expression vector containing the nucleic acid molecule described in the second aspect.

[0133] The expression vector can be obtained using conventional molecular biology techniques in the field, that is, the nucleic acid described in this application is constructed by inserting it into various expression vectors through methods such as enzyme digestion and ligation or homologous recombination.

[0134] Fourthly, the present invention provides a host cell containing the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect.

[0135] The host cells that can be used in this invention include prokaryotic and / or eukaryotic cells. Commonly used prokaryotic host cells include *Escherichia coli* (such as TG1, HB2151, BL21(DE3), Rosseta(DE3) etc.), *Agrobacterium*, *Bacillus subtilis*, etc.; commonly used eukaryotic host cells include yeast cells, insect cells, human embryonic kidney epithelial cells (HEK293, 293F, 293T, etc.), hamster ovary cells (CHOS, CHO-K1, etc.), etc. Preferably, the prokaryotic host cell is *Escherichia coli* BL21(DE3) or Rosseta(DE3) (expressing nanobodies), and the eukaryotic host cell is HEK293 cells or CHO cells (expressing heavy chain antibodies).

[0136] Fifthly, the present invention provides a chimeric antigen receptor comprising the anti-human HHLA2 nanobody described in the first aspect.

[0137] In a sixth aspect, the present invention provides a genetically modified cell containing the chimeric antigen receptor described in the fifth aspect.

[0138] Preferably, the genetically modified cells are human immune cells.

[0139] Preferably, the human immune cells include T cells, NK cells, or macrophages.

[0140] In a seventh aspect, the present invention provides a bispecific antibody or a multispecific antibody, wherein the bispecific antibody or multispecific antibody contains the anti-human HHLA2 nanobody described in the first aspect.

[0141] In this invention, the bispecific or multispecific antibody comprises the anti-human HHLA2 nanobody described in the first aspect of this invention; and an antibody functionally linked to the antibody having another antigen-binding property. The other antibody includes, but is not limited to, antibodies or antigen-binding fragments capable of binding to targets / antigens such as PD1, PD-L1, VEGF, VEGFR1, VEGFR2, HER2, EGFR, CD73, Trop2, Trop1, Nectin-4, MSLN, TIGIT, CD3, CD28, CD80, LIV-1, GLP-1, c-Met, LILRB4, LILRB2, GD2, APOE4, MICA, Claudin18.2, CD20, AXL, CD47, CD19, 4-1BB, MUC16, and Siglec10.

[0142] Eighthly, the present invention provides a conjugate containing the anti-human HHLA2 nanobody described in the first aspect.

[0143] In this invention, the conjugate is formed by conjugating the nanobody of this invention onto a functional agent.

[0144] Functional agents can be cytotoxic agents such as chemotherapeutic agents, toxins (e.g., enzymatically active toxins or fragments thereof derived from bacteria, fungi, plants, or animals), or radioisotopes (i.e., radiocoupled substances), antibiotics, lysozymes, or any combination thereof. Chemotherapeutic agents can be used to produce immunocoupled substances such as methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents, enzymes, and / or fragments thereof, such as lysozymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins derived from bacteria, fungi, plants, or animals, including fragments and / or variants thereof. Usable enzymatically active toxins and their fragments include, for example, diphtheria A chain, non-bound active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleuritesfordii protein, dianthin protein, Phytolacca americana protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, and saponaria. Inhibitors of *Officinalis*, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes. Any suitable radionucleotide or radioactive reagent known or available in the art may be used to generate radiocoupled antibodies.

[0145] The immunoconjugate may also be a complex formed by directly or indirectly conjugating the nanobody of the present invention to the detectable portion.

[0146] The detectable components include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals, and non-radioactive paramagnetic metal ions.

[0147] Fluorescent labeling compounds that can be used in this invention include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, phthalaldehyde, and fluorescein.

[0148] Chemiluminescent labeling compounds that can be used in this invention include luminol, isoluminol, aromatic acridine esters, imidazoles, acridine salts, and oxalates.

[0149] Bioluminescent compounds that can be used in this invention include luciferin, luciferase, and jellyfish luminescent protein.

[0150] Enzymes that can be used in this invention include horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucosylamylase.

[0151] Labelling for detection and / or analysis and / or diagnostic purposes depends on the specific detection / analysis / diagnostic techniques and / or methods used, such as immunohistochemical staining (tissue) samples, flow cytometry, laser scanning cytometry, fluorescence immunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), bioassays (e.g., phagocytosis assays), Western blotting applications, etc. Suitable labeling for detection / analysis / diagnostic techniques and / or methods known in the art is well understood by those skilled in the art.

[0152] In a ninth aspect, the present invention provides a pharmaceutical composition comprising the anti-human HHLA2 nanobody described in the first aspect.

[0153] The pharmaceutical compositions of the present invention also include pharmaceutically acceptable carriers, including but not limited to any adjuvants, carriers, excipients, gliding agents, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers that have been approved by the Food and Drug Administration for use in humans or animals and have no side effects on the composition of the pharmaceutical composition.

[0154] The pharmaceutical composition must generally be sterile and stable under manufacturing and storage conditions. The pharmaceutical composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate flowability can be maintained, for example, by using a coating such as lecithin, in the case of dispersions by maintaining the desired particle size, and by using surfactants. In many cases, it is preferable to include an isotonic agent in the composition, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride. Prolonged absorption of the injectable composition can be achieved by including agents that delay absorption, such as monostearate and gelatin.

[0155] The pharmaceutical composition may be administered orally, by injection, by nasal administration, by transdermal administration, or by mucosal administration. The pharmaceutical composition may also include other antitumor antibodies as active ingredients.

[0156] The pharmaceutical composition may include, but is not limited to, liquid, freeze-dried, and lyophilized compositions.

[0157] The pharmaceutical composition described herein may also be administered in combination with other pharmaceutical agents.

[0158] The dosage level of the pharmaceutical composition can be adjusted according to the amount of composition required to achieve the desired diagnostic or therapeutic outcome. The administration regimen can be a single injection or multiple injections, or may be adjusted accordingly. The selected dosage level and regimen depend on various factors, including the activity and stability of the pharmaceutical composition, the formulation, the route of administration, combination with other drugs or treatments, the disease or condition to be detected and / or treated, and the health status and prior medical history of the subject to be treated, and can be rationally adjusted accordingly. The therapeutically effective dose of the pharmaceutical composition can be initially estimated in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs, and / or non-human primates.

[0159] In a tenth aspect, the present invention provides the use of the anti-human HHLA2 nanobody according to the first aspect, or the nucleic acid molecule according to the second aspect, or the expression vector according to the third aspect, or the bi / multi-specific antibody according to the seventh aspect, or the conjugate according to the eighth aspect, in the preparation of a product for detecting human HHLA2 protein.

[0160] Eleventhly, the present invention provides a product for detecting human HHLA2 protein, the product comprising the anti-human HHLA2 nanobody described in the first aspect, or the chimeric antigen receptor described in the fifth aspect, or the gene-modified cell described in the sixth aspect, or the bispecific antibody or multispecific antibody described in the seventh aspect, or the conjugate described in the eighth aspect, or the pharmaceutical composition described in the ninth aspect.

[0161] The products include those that utilize enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, radioimmunoassay, luminescent immunoassay, colloidal gold immunochromatography, agglutination, and immunoturbidimetry to detect antigen-antibody binding.

[0162] Specific examples of the product include a kit. The kit also includes a solid-phase support, on which the antibody is immobilized (e.g., a multi-well plate, coverslip, microbeads) or exists in free form.

[0163] Furthermore, the kit also includes:

[0164] The detectable portion that can be linked to the antibody, the detectable portion being detachably present in the kit; and / or a substrate corresponding to the detectable portion; and / or enzyme-linked immunosorbent assay (ELISA) reagents (including but not limited to: coating (buffer), washing (buffer), blocking solution, fixative, stop solution, chromogenic solution); and / or instructions for use describing the method for detecting human HHLA2.

[0165] In a twelfth aspect, the present invention provides the use of the anti-human HHLA2 nanobody according to the first aspect, or the nucleic acid molecule according to the second aspect, or the expression vector according to the third aspect, or the chimeric antigen receptor according to the fifth aspect, or the gene-modified cell according to the sixth aspect, or the bi / multispecific antibody according to the seventh aspect, or the conjugate according to the eighth aspect, or the pharmaceutical composition according to the ninth aspect in the preparation of a medicament for treating and / or preventing cancer.

[0166] The cancers mentioned include, but are not limited to, melanoma, lung cancer, breast cancer, pancreatic cancer, Hodgkin's lymphoma, bladder cancer, stomach cancer, head and neck cancer, kidney cancer, prostate cancer, colorectal cancer, ovarian cancer, and blood cancer.

[0167] In a thirteenth aspect, the present invention provides a method for detecting human HHLA2 protein for non-diagnostic purposes, the method comprising contacting a sample with an anti-human HHLA2 nanobody fragment as described in the first aspect, or a chimeric antigen receptor as described in the fifth aspect, or a genetically modified cell as described in the sixth aspect, or a bi / multispecific antibody as described in the seventh aspect, or a conjugate as described in the eighth aspect, or a pharmaceutical composition as described in the ninth aspect.

[0168] Preferably, the contact process further includes ELISA, Western blot, flow cytometry, or immunofluorescence assay.

[0169] Compared with the prior art, the present invention has the following beneficial effects:

[0170] This invention provides a nanobody that specifically binds to human HHLA2 and exhibits good anti-tumor activity. The nanobody disclosed in this invention can specifically bind to tumor cells expressing human HHLA2 and can enhance the killing effect of immune cells on tumor cells by blocking the HHLA2-KIR3DL3 signaling pathway. Therefore, the nanobody disclosed in this invention has the potential to be used in the development of drugs or diagnostic reagents for treating tumors. Attached Figure Description

[0171] Figure 1 Figure 1 shows the binding results (ELISA) of the HHLA2 antibody described in this application with human HHLA2. Figure 2a shows the binding curves of the candidate parent Ab01-63G4 and its 13 humanized or engineered mutants with human HHLA2. Figure 2b shows the binding curves of the candidate parent Ab01-63G4 and its 5 humanized or engineered mutants with human HHLA2.

[0172] Figure 2 The figures shown are the results of specific binding of the HHLA2 antibody described in this application to human / cynomolgus monkey HHLA2 (FACS). Figure a shows the fluorescence signal of different concentrations of candidate parent antibody Ab01-63M2 bound to K562 cells overexpressing human HHLA2; Figure b shows the fluorescence signal of different concentrations of positive control antibody 2C4 bound to K562 cells overexpressing human HHLA2; Figure c shows the fluorescence signal of different concentrations of candidate parent antibody Ab01-63M2 bound to HEK293 cells overexpressing cynomolgus monkey HHLA2; and Figure d shows the fluorescence signal of different concentrations of positive control antibody. The fluorescence signal of 2C4 binding to HEK293 cells overexpressing cynomolgus monkey HHLA2 is shown in Figure e, which shows the binding curve of candidate parent antibody Ab01-63M2 on K562 cells overexpressing human HHLA2; Figure f shows the binding curve of positive control antibody 2C4 on K562 cells overexpressing human HHLA2; Figure g shows the binding curve of candidate parent antibody Ab01-63M2 on HEK293 cells overexpressing cynomolgus monkey HHLA2; and Figure h shows the binding curve of positive control antibody 2C4 on HEK293 cells overexpressing cynomolgus monkey HHLA2.

[0173] Figure 3 Figure 1 shows the results of HCC827 tumor cells (FACS) bound to the HHLA2 antibody described in this application. Figure 2a shows the fluorescence signal of HCC827 (human non-small cell lung cancer cells, highly expressing HHLA2) cells when different concentrations of candidate parent antibody Ab01-63M2 are bound to HCC827 cells. Figure 3b shows the fluorescence signal of HCC827 cells when different concentrations of positive control antibody 2C4 are bound to HCC827 cells. Figure 4c shows the binding curves of candidate parent antibody Ab01-63M2 and positive control antibody 2C4 on HCC827 cells.

[0174] Figure 4 Figure 1 shows the SDS-PAGE and ELISA results of some of the HHLA2 antibodies described in this application. Figure 2a shows the SDS-PAGE reduction electrophoresis results of the candidate parent Ab01-63G4 and its three humanized or engineered mutants after being treated at 37°C for 0, 3, and 7 days. Figure 3b shows the binding curves of the candidate parent Ab01-63G4 with human HHLA2 after being treated at 37°C for 0, 3, and 7 days. Figure 3c shows the binding curves of the mutant Ab01-63Ghm25 after being treated at 37°C for 0, 3, and 7 days. Figure 3 shows the binding curves with human HHLA2 after 0, 3, and 7 days. Figure d shows the SDS-PAGE non-reducing electrophoresis results of the candidate parent Ab01-63G4 and its three humanized or engineered mutants after being treated at 37℃ for 0, 3, and 7 days. Figure e shows the binding curves of mutant Ab01-63Ghm26 with human HHLA2 after being treated at 37℃ for 0, 3, and 7 days. Figure f shows the binding curves of mutant Ab01-63Ghm28 with human HHLA2 after being treated at 37℃ for 0, 3, and 7 days.

[0175] Figure 5 This is a graph showing the results of partial HHLA2 antibody blocking HHLA2 / KIR3DL3 as described in this application.

[0176] Figure 6 This is a diagram showing the results of the HHLA2 antibody described in this application promoting NK92-MI to kill target cells.

[0177] Figure 7 The figures show the results of the anti-tumor effects of the partial HHLA2 antibody described in this application in the NCG mouse / NK92MI / K562-hHHLA2 CDX model. Figure a is a schematic diagram of the experimental protocol, Figure b is the tumor growth curve of different groups, and Figure c is the body weight change curve of mice in different groups. Detailed Implementation

[0178] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0179] Unless otherwise specified, all techniques or conditions used in the embodiments were performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. All reagents used, or those without manufacturer information, were conventional products that could be obtained through legitimate channels.

[0180] sequence list

[0181]

[0182] Example 1

[0183] Phage display for screening nanobody molecules against human HHLA2

[0184] Alpacas were immunized with an emulsion mixture of 500 μg human HHLA2 protein (Acro Biosystems, B77-H52H5) and 250 μL Freund's complete adjuvant, once every two weeks, for a total of four immunizations. Peripheral blood was collected before and after each immunization to obtain serum. Approximately one week after the fourth immunization, blood was collected again to simultaneously measure the antibody titer against the immune antigen in the peripheral serum after each immunization. If the antibody titer reached 10... 5 If immunization is successful, 50 mL of alpaca peripheral blood can be collected to separate peripheral blood mononuclear cells (PBMCs) for use in immunization library construction.

[0185] RNA was extracted from PBMCs (50 mL of alpaca peripheral blood) using the Trizol method, and the concentration and quality of total RNA were analyzed by gel electrophoresis. All extracted RNA was divided into two aliquots, and cDNA was obtained by reverse transcription using oligo T and Random primers, respectively. The cDNA obtained by reverse transcription with different primers was used as substrate for two rounds of PCR to obtain diverse PCR products of the variable region fragment of alpaca heavy chain antibody, i.e., VHH DNA sequences. The purified PCR products of the diverse VHH DNA sequences were digested with restriction endonucleases SpeI (Takara, 1086A) and SacI (Takara, 1078A), and then ligated into the pComb3XSS phage vector using T4 ligase (NEB, M0202L). The recombinant phage was transformed into TG1 E. coli by electroporation, plated overnight, and the bacterial cells were collected to form a bacterial library. The diverse phages in the bacterial library were amplified and purified to form a phage library, which was used for subsequent screening.

[0186] An immunotube method was used to screen alpaca-derived phage-displaying nanobody libraries. The selected phage display libraries had a capacity of 2 × 10⁻⁶. 9 The target protein was coated onto immunotubes at a concentration of 20 μg / mL, and three rounds of enrichment screening were performed. The enrichment levels in each round are shown in Table 1.

[0187] Table 1

[0188] Number of filtering rounds Input valence Screening valence Comparative valence enrichment level Difference multiple Round 1 <![CDATA[1.0×10 12 pfu]]> <![CDATA[2.0×10 9 pfu / mL]]> <![CDATA[1.0×10 8 pfu / mL]]> <![CDATA[2.0×10 -3 ]]> 20 Round 2 <![CDATA[2.5×10 12 pfu]]> <![CDATA[3.6×10 10 pfu / mL]]> <![CDATA[4.8×10 7 pfu / mL]]> <![CDATA[1.44×10 -2 ]]> 750 Round 3 <![CDATA[8.0×10 12 pfu]]> <![CDATA[6.4×10 11 pfu / mL]]> <![CDATA[2.8×10 8 pfu / mL]]> <![CDATA[8.0×10 -2 ]]> 2285

[0189] In the third round of phage washing, 0.25 mg / mL Trypsin solution, or 50 μg / mL KIR3DL3 (Acro Biosystems, KI3-H5259) solution, or 5 μg / mL positive control 2C4 antibody (prepared by Kehui Zhiyao, sequence derived from patent CN112153977A, where the VH sequence is shown in SEQ ID NO:57 and the VL sequence is shown in SEQ ID NO:58) solution can be eluted at room temperature for 30 min by rotation. *E. coli* TG1 was infected with the third round of phage elution buffer, serially diluted and plated, and incubated overnight at 32°C. Single colonies were picked and amplified for phage verification using ELISA (coating material: human HHLA2). Corresponding backup positive clones of TG1 colonies were used for nanobody gene sequencing. Sequence analysis initially yielded 136 different anti-human HHLA2 nanobody molecules.

[0190] SEQ ID NO:57:

[0191] EVHLQQSGPELEKPGVSVKISCKASGFSFTDYNMNWVKQSSGKSLEWIGNIDPYYGRINYNQKFKGKATLSVDKSSSTAYMHLKSLTSEDSAVYYCATGAYTSGYSWFAYWGQGTLVTVSS.

[0192] SEQ ID NO:58:

[0193] ENVLTQSPGIMAASLGEKVTMTCSASSSVSSSYLHWYQQRSGASPKPLIHRTSNLASGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPFTFGSGTKLEIK.

[0194] Example 2

[0195] Antibody expression and purification

[0196] The nanobody molecules obtained in Example 1 were synthesized with codon-optimized nucleotide sequences and cloned into the eukaryotic expression vectors pcDNA3.1-hG1Fc or pcDNA3.1-mG2aFc designed and modified in our laboratory (the Fc region of human IgG1 or mouse IgG2a has been inserted into the vectors). Expression plasmids were prepared using the Tiangen endotoxin-free plasmid extraction kit and transfected into ExpiCHO cells using the ExpiFectamine CHO Transfection kit (Gbico, A29130). Cell culture flasks were then placed in shakers and cultured at 37°C, 8% CO2, and 100 rpm for 7 days. The cell culture supernatant containing the target protein was collected by centrifugation, and the target antibody was purified by affinity chromatography using rProtein A Beads 4FF packing material (Tiandi Renhe, SA015025). The target antibody was eluted with 0.1M pH 3.5 glycine solution, then concentrated using ultrafiltration tubes (Shenzhen Avidite, MCP030C41) and transferred to PBS buffer. The concentration was determined by A280 assay, and the purity was verified by protein electrophoresis. The resulting products were then aliquoted and frozen for later use. Table 2 shows the transient expression levels of some antibodies. Among them, the SDS-PAGE electrophoresis results of the candidate parent Ab01-63G4 and its three humanized or engineered mutants (Ab01-63Ghm25, Ab01-63Ghm26, Ab01-63Ghm28) after being treated at 37℃ for 0, 3, and 7 days are as follows: Figure 4 As shown in Figure a (reduction electrophoresis; non-reduction electrophoresis), it can be seen that the candidate parent Ab01-63G4 and its three humanized or engineered mutants (Ab01-63Ghm25, Ab01-63Ghm26, Ab01-63Ghm28) have good purity and stability.

[0197] Table 2

[0198] Antibody name SEQ ID NO: Expression volume (mL) Expression level (mg / L) Ab01-63Ghm25 1 25 119 Ab01-63Ghm28 2 25 169 Ab01-63Ghm26 3 25 64 Ab01-63Ghm35 4 25 29 Ab01-63Ghm36 5 25 52 Ab01-63G4 6 150 201 Ab01-63M2 7 25 145 Ab01-7M2 8 25 253 Ab01-84M2 9 25 183 Ab01-91M2 10 25 132 Ab01-121M1 11 25 348 Ab01-122M1 12 25 206 Ab01-126M1 13 25 137 Ab01-110M1 14 25 137 Ab01-10M2 - 25 163 Ab01-13M2 - 25 206 Ab01-28M2 - 25 170 Ab01-43M2 - 25 222 Ab01-51M2 - 25 165 Ab01-63M2 - 25 145 Ab01-73M2 - 25 187 Ab01-79M2 - 25 203 Ab01-85M2 - 25 124 Ab01-87M2 - 25 187 Ab01-101M2 - 25 117 Ab01-102M2 - 25 122

[0199] Among them, Ab01-10M2 to Ab01-102M2 are antibodies with sequences known in the prior art.

[0200] Example 3

[0201] ELISA screening for antibodies that can bind to human HHLA2

[0202] 0.5 μg / mL human HHLA2 protein (Acro Biosystems, B77-H52H5) was coated onto Immuno clear standard plates (ThermoFisher, 468667), 100 μL per well, and incubated overnight at 4°C. After washing with PBST, 200 μL of PBS blocking buffer containing 1% BSA was added to each well, and the plates were blocked at 37°C for 1 h. Different concentrations of the antibody protein prepared in Example 2 were added, and the plates were incubated at 37°C for 1 h. After washing with PBST, horseradish peroxidase-labeled goat anti-human IgG-Fc secondary antibody (Goat anti-Human IgG FcSecondary Antibody, HRP; Invitrogen, A18817) was added, and the plates were incubated at 37°C for 30 min. The plates were washed 5 times with PBST. 50 μL of TMB (Solepro, PR1200) was added to each well, and the plates were incubated at room temperature (20±5°C) in the dark for 10 min. Then, 50 μL of TMB was added to each well. The ELISA stop solution (Solepro, C1058) was used to terminate the substrate reaction. The OD value was read at 450 nm using a microplate reader to analyze the binding ability of the antibody to human HHLA2.

[0203] Candidate antibody molecules Ab01-63M2, Ab01-7M2, Ab01-84M2, Ab01-91M2, Ab01-121M1, Ab01-122M1, Ab01-126M1, and Ab01-110M1, among others, were validated by ELISA to bind to human HHLA2. The ELISA binding curves of some HHLA2 antibodies to human HHLA2 are shown below. Figure 1 (a, b) and Figure 4 As shown in (b, c, e, f), it can be seen that the candidate parent Ab01-63G4 and its three humanized or engineered mutants (Ab01-63Ghm25, Ab01-63Ghm26, Ab01-63Ghm28) have a high affinity for human HHLA2.

[0204] Example 4

[0205] FACS screening for antibodies that specifically bind to human / cynomolgus monkey HHLA2

[0206] Antibody binding assays at the cellular level were performed using the K562 cell line (Pronos), the K562 cell line overexpressing human HHLA2 (K562-hHHLA2, Kehui Zhizhi), the HEK293 cell line (Zhuhai Kairui), the HEK293 cell line overexpressing cynomolgus monkey HHLA2 (HEK293-cyHHLA2, Kehui Zhizhi), and the HCC827 cell line highly expressing human HHLA2 (Pronos). Cells were collected using 1×PBS, centrifuged at 300g for 5 min, and resuspended on ice with pre-chilled blocking buffer (PBS + 5% FBS) to adjust the cell density to 10-1. 6 / mL; Add 50 μL of cells and 50 μL of serially diluted test antibody to each well of a 96-well V plate (Corning, 3894), and incubate on ice in the dark for 2 hours; Add 200 μL of pre-chilled wash buffer (PBS + 0.1% BSA), centrifuge at 300g for 5 min, carefully discard the supernatant, and repeat once; Resuspend the cells in a solution containing 500-fold diluted fluorescent secondary antibody (APC anti-human IgG Fc, BioLegend, 409306; or Alexa) Cells were incubated for 30 min on ice in blocking buffer (PBS + 5% FBS) with 647-Affini Pure Goat Anti-Mouse IgG Fc (Jackson, 115-605-164) in the dark. Then, 200 μL of pre-chilled wash buffer (PBS + 0.1% BSA) was added, and the cells were centrifuged at 300 g for 5 min. The supernatant was discarded, and this process was repeated once. Cells were resuspended in 100 μL of PBS and placed on ice. Samples were transferred to flow cytometry tubes, and the corresponding fluorescence signals were detected using a flow cytometer (Wellgrow EasyCell 206A1).

[0207] Positive control antibody 2C4 and candidate antibody molecules Ab01-63M2, Ab01-7M2, Ab01-84M2, Ab01-91M2, Ab01-121M1, Ab01-122M1, Ab01-126M1, and Ab01-110M1 all specifically bound to human / cynomolgus monkey HHLA2, as verified by FACS. The results of FACS for specific binding of some HHLA2 antibodies to human / cynomolgus monkey HHLA2 are as follows: Figure 2 As shown, the results of partial HHLA2 antibody binding to HCC827 tumor cells (FACS) are as follows: Figure 3 As shown, it can be seen that both the candidate parent antibody Ab01-63M2 and the positive control antibody 2C4 can bind to human / cynomolgus monkey HHLA2 at the cellular level, and can also bind to the human non-small cell lung cancer cell line HCC827 that highly expresses HHLA2.

[0208] Table 3

[0209] Ab01-63M2 2C4 <![CDATA[EC 50 (nM)]]> 0.043 0.281

[0210] EC in Table 3 50 The results of HHLA2 antibody binding to HCC827 tumor cells (FACS) are shown in Table 3. The data show that the binding ability of candidate parent antibody Ab01-63M2 to the tumor cell line HCC827 is slightly better than that of positive control antibody 2C4.

[0211] Example 5

[0212] Luciferase reporter gene assay to screen for antibodies that can block HHLA2 / KIR3DL3 binding.

[0213] Set up 4 processes as shown in the table below:

[0214] Table 4

[0215] serial number Group Name 1 K562-hHHLA2+hKIR3DL3-Jurkat+antibody to be tested+OKT3 2 K562-hHHLA2+hKIR3DL3-Jurkat+cell culture medium+OKT3 3 K562+hKIR3DL3-Jurkat+cell culture medium+OKT3 4 K562+hKIR3DL3-Jurkat+ cell culture medium

[0216] Adjust the density of K562 or K562-hHHLA2 cells to 4 × 10⁻⁶. 5 Cells / mL were transferred at 25 μL / well to a white opaque 96-well plate (Thermo Fisher, 15042), and an equal volume of serially diluted test antibody or cell culture medium (RPMI 1640 + 10% FBS + 1% P / S) was added. Pre-incubation was performed for 30 min. The hKIR3DL3-Jurkat (Jiman Biotechnology; overexpressing human KIR3DL3 protein) cell density was adjusted to 1 × 10⁻⁶ cells / mL. 6 Add 50 μL of the cells / ml sample to each well of the 96-well plate from the previous step; add 25 μL of Anti-CD3 epsilon antibody OKT3 (Jiman Biotechnology, GM-51478AB-100) to a final concentration of 0.2 μg / mL, and incubate for 30 min; after incubation at 37℃ for 16 h in a CO2 incubator, use the GMOne-Step Luciferase Reporter Gene Detection Kit (Jiman Biotechnology, GM-040403B) to measure the fluorescence signal.

[0217] Luciferase reporter gene assay results showed that the positive control 2C4 and candidate antibody molecules Ab01-63M2, Ab01-7M2, Ab01-84M2, Ab01-91M2, Ab01-121M1, Ab01-122M1, Ab01-126M1, and Ab01-110M1 all blocked the HHLA2 / KIR3DL3 signaling pathway. Among these, the results of some HHLA2 antibodies blocking HHLA2 / KIR3DL3 are as follows... Figure 5As shown, it can be seen that candidate antibodies such as Ab01-7M2, Ab01-43M2, Ab01-63M2, Ab01-79M2, and Ab01-84M2 have better or comparable ability to block HHLA2 / KIR3DL3 compared with the positive control antibody 2C4.

[0218] Example 6

[0219] LDH assay for the activation effect of anti-HHLA2 antibody on NK92MI cells

[0220] Set up 5 processes as shown in the table below:

[0221] Table 5

[0222]

[0223] K562 or K562-hHHLA2 cells were collected and their density was adjusted to 4 × 10⁶ cells / year using N500 serum-free medium (Dakeway, 6113031). 5 Add 50 μL of the assay solution per well to a V-shaped / conical bottom 96-well plate, followed by 50 μL of serially diluted test antibody per well. Pre-incubate for 30 min. Collect effector cells NK92MI cells (Pricella, CL-0533; KIR3DL3 positive effector cells) and adjust the density to 2 × 10⁶ cells / mL using N500 medium. 5 Add 100 μL of the supernatant to the cells or culture medium from the previous step at 100 μL / well, centrifuge at 250 g for 3 min, and incubate at 37 °C for 6 h; centrifuge at 250 g at room temperature for 5 min, and transfer 50 μL of the supernatant to a 96-well plate. Use the LDH cytotoxicity assay kit (Roche, 11644793001) to determine the cell killing results.

[0224] The results of LDH cell killing showed that the positive control 2C4 and candidate antibody molecules Ab01-63M2, Ab01-7M2, Ab01-84M2, Ab01-91M2, Ab01-121M1, Ab01-122M1, Ab01-126M1, and Ab01-110M1 all significantly enhanced the killing effect of NK92MI cells on target cells K562-hHHLA2. Among them, some HHLA2 antibodies promoted the killing of target cells by NK92-MI cells as shown in the following figures. Figure 6 As shown.

[0225] Table 6

[0226] 2C4 Ab01-63M2 <![CDATA[EC 50 (nM)]]> 0.1922 0.1251

[0227] EC in Table 6 50 Corresponding Figure 6The results of HHLA2 antibody 2C4 and Ab01-63M2 promoting NK92-MI killing of target cells (K562 cells overexpressing human HHLA2) are shown in Table 6. As can be seen from the data, the candidate parent antibody Ab01-63M2 and the positive control antibody 2C4 have comparable abilities to promote NK92-MI killing of K562-hHHLA2.

[0228] Example 7

[0229] Engineering of candidate antibodies

[0230] As is well known to those skilled in the art, alpaca nanobodies generally have low immunogenicity. Because their germline genes are highly homologous to human germline genes, their humanization process is relatively simple, usually involving conventional CDR transplantation (if antigen binding affinity decreases after humanization, reversion mutations can be attempted at the corresponding differential sites). Optionally, retaining the four conserved amino acids on the nanobody FR backbone is generally beneficial for improving the hydrophilicity and solubility of humanized nanobody molecules. Specifically, for the humanization of candidate antibody molecules Ab01-63M2 (SEQ ID NO:7) or Ab01-63G4 (SEQ ID NO:6), the human germline gene IGHV3_23+IGHJ5 with the highest germline gene homology to the candidate nanobody variable region (SEQ ID NO:20) can be selected as the backbone using the IMGT online database for comparison. After transplantation of CDRs and replacement of 4 conserved amino acids on the FR backbone (Kabat number: V37F, G44E, L45R, W47G), a preliminary humanized nanobody sequence (SEQ ID NO:21) can be obtained. Further sequence analysis of PTM modification sites can lead to the N74T mutation in SEQ ID NO:21, eliminating the aspartic acid isomerism and asparagine deamidation motif "D73N74S75" in the FR3 region. Secondly, several amino acid mutations can be performed on the high-risk aspartic acid isomerism sites D55 and D62 in CDR2 of SEQ ID NO:21. Further, based on three-dimensional structure prediction analysis, several amino acid mutations can be performed on the "R99R100" positively charged patch and the "F112F113F114" hydrophobic patch in CDR3 to optimize the antibody molecule and obtain a druggable antibody molecule. Optionally, as is well known to those skilled in the art, corresponding engineering modifications to the antibody Fc can silence or enhance its Fc effector function, or prolong or shorten its half-life.

[0231] Optimization of the druggability of antibody molecules can generally be reflected in improvements in one or more aspects, such as increased antibody stability, optimized affinity (either increased or decreased), increased yield, improved solubility, and enhanced antitumor activity. After testing and comparing the stability, affinity, yield, purity, and activity of a series of engineered mutants of Ab01-63M2 or Ab01-63G4, the preferred antibody molecules are Ab01-63Ghm25 (SEQ ID NO:1), Ab01-63Ghm28 (SEQ ID NO:2), Ab01-63Ghm26 (SEQ ID NO:3), Ab01-63Ghm35 (SEQ ID NO:4), and Ab01-63Ghm36 (SEQ ID NO:5).

[0232] The ELISA binding curves of candidate parent Ab01-63G4 and its humanized or engineered mutants with human HHLA2 are shown below. Figure 1 As shown in Figures a and b; the SDS-PAGE electrophoresis results of the candidate parent Ab01-63G4 and its three humanized or engineered mutants (Ab01-63Ghm25, Ab01-63Ghm26, and Ab01-63Ghm28) after being treated at 37°C for 0, 3, and 7 days, and their ELISA binding curves with human HHLA2 are shown in Figures a and b. Figure 4 As shown in the figure, it can be seen that the three mutants Ab01-63Ghm25, Ab01-63Ghm26, and Ab01-63Ghm28, after humanization or engineering modification, have similar affinity and improved stability compared with the candidate parent Ab01-63G4.

[0233] Example 8

[0234] The antitumor efficacy of candidate antibodies was determined using an NCG mouse / NK92MI / K562-hHHLA2 CDX model.

[0235] A K562-hHHLA2 CDX tumor model was established using NCG mice for in vivo pharmacodynamic studies. The specific methods are as follows: K562-hHHLA2 cells were revived and expanded according to experimental requirements, and cultured at a rate of 1×10⁻⁶ cells / mL. 7 Tumor cells were inoculated into mice. When the average tumor volume in mice was 100-200 mm², tumor cells were introduced. 3 The drug was administered in groups, with a dosing cycle of once every 3 days, for a total of 3 tail vein administrations. The administration method was 20 mpk / IV / Q3D. Two hours after antibody administration, NK92MI cells were injected intratumorally, with each cell injection dose being 2 × 10⁻⁶. 6 cells. After starting administration, animal body weight and tumor volume were measured every three days. Tumor volume was calculated as: tumor volume (mm²).3 = 0.5 × tumor long diameter × tumor short diameter 2 The experiment ended on day 10 after drug administration, and all mice were subsequently euthanized.

[0236] For the in vivo antitumor effect of the NCG / K562-hHHLA2 CDX tumor model, please refer to [reference needed]. Figure 7 Specifically, in the positive antibody (2C4, variable region sequence as shown in SEQ ID NO:57-58, derived from the published patent CN112153977A) control group mice, the average tumor volume was 757.5 mm on day 10 after drug administration. 3 The tumor inhibition rate (TGI) was 49.58%; the mean tumor volume in the candidate antibody Ab01-63G4 treatment group was 223.0 mm on day 10 after administration. 3 Compared with the solvent control group and the positive antibody control group, there were significant differences (p<0.05). The tumor inhibition rate (TGI) was 85.16%. Ab01-63G4 (20mpk / IV / Q3D) showed significant efficacy in the K562-hHHLA2NCG CDX model, which was superior to the positive control 2C4.

[0237] The applicant declares that this invention illustrates an anti-human HHLA2 nanobody and its application through the above embodiments, but this invention is not limited to the above embodiments, that is, it does not mean that this invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials of the product, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0238] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0239] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A nanobody against human HHLA2, characterized in that, The variable region of the nanobody includes CDR1, CDR2 and CDR3, wherein the amino acid sequence of CDR1 of the nanobody is the sequence shown in SEQ ID NO:29, the amino acid sequence of CDR2 is the sequence shown in SEQ ID NO:39, and the amino acid sequence of CDR3 is the sequence shown in SEQ ID NO:

47. Alternatively, the amino acid sequence of CDR1 of the nanobody is the sequence shown in SEQ ID NO:29, the amino acid sequence of CDR2 is the sequence shown in SEQ ID NO:37, and the amino acid sequence of CDR3 is the sequence shown in SEQ ID NO:

47. Alternatively, the amino acid sequence of CDR1 of the nanobody is the sequence shown in SEQ ID NO:29, the amino acid sequence of CDR2 is the sequence shown in SEQ ID NO:38, and the amino acid sequence of CDR3 is the sequence shown in SEQ ID NO:

47. Alternatively, the amino acid sequence of CDR1 of the nanobody is the sequence shown in SEQ ID NO:29, the amino acid sequence of CDR2 is the sequence shown in SEQ ID NO:37, and the amino acid sequence of CDR3 is the sequence shown in SEQ ID NO:

48.

2. The anti-human HHLA2 nanobody according to claim 1, characterized in that, The nanobody includes a variable region, a heavy chain crystallizable region fragment, or a mutant thereof.

3. The anti-human HHLA2 nanobody according to claim 2, characterized in that, The crystallizable region of the heavy chain is selected from any one of human IgG1 or its mutant, IgG2 or its mutant, IgG3 or its mutant, and IgG4 or its mutant; Alternatively, it may be selected from any one of mouse IgG1 or its mutants, IgG2a or its mutants, IgG2b or its mutants, or IgG3 or its mutants.

4. The anti-human HHLA2 nanobody according to claim 3, characterized in that, The crystallizable region of the heavy chain is selected from any one of human IgG1 or its mutants, or mouse IgG2a or its mutants.

5. The anti-human HHLA2 nanobody according to claim 1, characterized in that, The amino acid sequence of the variable region in the nanobody includes any of the sequences shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or SEQ ID NO:

20.

6. The anti-human HHLA2 nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody includes any one of the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6 or SEQ ID NO:

7.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-human HHLA2 nanobody according to any one of claims 1-6.

8. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 7.

9. A host cell, characterized in that, The host cell contains the nucleic acid molecule of claim 7 or the expression vector of claim 8.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains the anti-human HHLA2 nanobody according to any one of claims 1-6.

11. The use of the anti-human HHLA2 nanobody according to any one of claims 1-6, the nucleic acid molecule according to claim 7, or the expression vector according to claim 8 in the preparation of products for detecting human HHLA2 protein.

12. The use of the anti-human HHLA2 nanobody according to any one of claims 1-6, or the nucleic acid molecule according to claim 7, or the expression vector according to claim 8, or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating leukemia; The amino acid sequence of CDR1 of the anti-human HHLA2 nanobody is shown in SEQ ID NO:29, the amino acid sequence of CDR2 is shown in SEQ ID NO:39, and the amino acid sequence of CDR3 is shown in SEQ ID NO:47.

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