Anti-human HHLA2 nano antibody and application thereof

Abstract

The invention relates to an anti-human HHLA2 nano antibody and application thereof, a variable region of the nano antibody comprises CDR1, CDR2 and CDR3, an amino acid sequence of the CDR1 comprises any one of sequences as shown in SEQ ID NO: 29-36 or a mutation thereof, an amino acid sequence of the CDR2 comprises any one of sequences as shown in SEQ ID NO: 37-46 or a mutation thereof, an amino acid sequence of the CDR3 comprises any one of sequences as shown in SEQ ID NO: 47-56 or a mutation thereof, and the variable region of the nano antibody comprises CDR1, CDR2 and CDR3. The mutation is the substitution, insertion or deletion of 1, 2, 3 or 4 amino acids in the amino acid sequence. The nano antibody disclosed by the invention can be specifically combined with tumor cells expressing human HHLA2, and the killing effect of immune cells on the tumor cells can be improved by blocking a signal channel between the HHLA2 and KIR3DL3.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to an anti-human HHLA2 nano antibody and an application thereof. Background Art

[0002] HHLA2 (human endogenous retro virus-H long terminal repeat-associating 2), the gene was discovered when searching for the long terminal repeat sequence (LTR) of human retrovirus (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 a variety of tumor tissues, and is only expressed in small amounts in epithelial cells of the intestine, kidney, gallbladder, and breast, and epithelial cells of placental trophoblastic tissue. HHLA2 is closely related to the growth, metastasis, pathological grade, and prognosis of a variety of tumors, such as 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 through interaction with TMIGD2, and enhance T cell proliferation and cytokine production through AKT-dependent signaling cascades; on the other hand, HHLA2 can recruit SHP-1 and SHP-2 through interaction with KIR3DL3 on T / NK immune cells to attenuate downstream Vav1, ERK1 / 2, AKT, and NF-κB signals, thereby showing immunosuppressive activity (International Immunopharmacology, 2023, 121: 110403).

[0004] Cancer immunotherapy enhances the body's immunity to tumors by reducing immune escape. Compared with traditional chemotherapy, immunotherapy has the advantages of less drug dosage, wider range of action, and fewer side effects. For example, immune checkpoint inhibitors such as anti-PD1 and PD-L1 have achieved great success in clinical practice and have shown excellent anti-tumor activity. However, the above-mentioned immune checkpoint inhibitors have a low patient response rate in clinical practice (about 30%). This is due to the complex comprehensive mechanism of the tumor microenvironment. The presence of multiple immunosuppressive molecules, inhibitory cells, and special tumor tissue structures jointly block the occurrence of normal immune responses. If only a single immune checkpoint is blocked, it often cannot achieve the desired therapeutic effect. 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 (especially antibody drugs) that block the HHLA2 / KIR3DL3 pathway can inhibit tumor development and metastasis, and can produce synergistic effects when combined with PD-(L)1 monoclonal antibodies. Therefore, anti-tumor therapies targeting HHLA2 / KIR3DL3 are currently attracting much attention (Cancer Immunology Research, 2021, 9(2):156-169; Science Immunology, 2021, 6(61):eabf9792).

[0005] There are antibodies in the peripheral blood of animals such as alpacas and camels that are naturally missing light chains and are composed only of heavy chains. The heavy chain contains two constant regions (CH2 and CH3), a hinge region and a variable heavy chain domain (VHH, i.e., antigen binding region). The relative molecular mass of the variable region of the VHH heavy chain is about 15KDa, which is only 1 / 10 of that of conventional antibodies. The molecular height and diameter are both at the nanometer level, which can penetrate the blood-brain barrier, so it is called a nanobody (Nanobody, Nb). Nanobodies are usually highly soluble, stable (not easy to aggregate, and resistant to denaturing conditions such as high temperature, strong acid, and strong alkali), have high affinity, low immunogenicity (more than 80% homology with human antibodies), are easy to engineer, and are suitable for various prokaryotic and eukaryotic expression systems. They are widely used in the development of antibody drugs, detection reagents, etc. (Annual review of biochemistry, 2013, 82: 775-797; Analytical and Bioanalytical Chemistry, 2019, 411: 1703-1713). At present, most of the publicly reported antibodies acting on the HHLA2 / KIR3DL3 signaling pathway are hybridoma / mouse / human monoclonal antibodies (CN114729052A; CN112153977A; WO2023138579A1; US20210198366A1). Among them, the only one in clinical research is the fully human monoclonal antibody HBM1020 (ClinicalTrials.gov ID: NCT05824663) developed by Platinum Medicine based on its self-developed H2L2 transgenic mouse platform. There has been no report on the progress of the development of nano-antibody drugs targeting HHLA2. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention aims to provide an anti-human HHLA2 nanobody and its application.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In the first aspect, the present invention provides an anti-human HHLA2 nanobody, the variable region of the nanobody includes CDR1, CDR2 and CDR3, the amino acid sequence of the 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 the CDR2 includes any one of the sequences shown in SEQ ID NO:37-SEQ ID NO:46 or a mutation thereof, the amino acid sequence of the CDR3 includes any one of the sequences shown in SEQ ID NO:47-SEQ ID NO:56 or a mutation thereof, and 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 nano-antibodies disclosed in the present 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 pathways. Therefore, the nano-antibodies disclosed in the present invention have the potential to be used in the development of drugs or detection reagents for treating tumors.

[0038] In the present application, the mutations may include mutations designed by antibody screening and engineering during antibody preparation or application, such as mutations of variable region (especially CDRs) post-transcriptional modification (PTMs) sites, including potential deamidation sites (NG, NS, NH, etc.), potential aspartate isomerization sites (DG, DP, DS, etc.), potential N-glycosylation sites (N-{P}S / T) and potential oxidation sensitive sites (M, W), etc. The mutations may also include mutations of potential sites that affect antibody aggregation, saccharification, solubility, viscosity, expression, activity (affinity, etc.), purity, isoelectric point, Tm, fragmentation, PK, immunogenicity, etc.

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

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

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

[0042] Preferably, the amino acid sequence of the 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 among R2I / Y / V / L / K / E / T / S, F14E / R / S / T in the amino acid sequence shown in SEQ ID NO:47.

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

[0045] Those skilled in the art should understand the meaning of the above-mentioned mutations, for example, D13Q / E means that the 13th amino acid D in the amino acid sequence as shown in SEQ ID NO:39 is mutated to Q or E, and 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, 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 the present invention, the amino acid sequences of the CDRs listed above are all represented according to the Kabat numbering system based on sequence variability (Kabat EA. Sequences of proteins of immunological interest [M]. USDepartment of Health and Human Services, Public Health Service, National Institutes of Health, 1991). It is well known to those skilled in the art that the CDRs of antibodies can be represented by a variety of numbering systems in the art (e.g., IMGT, Chothia, Honegger, AbM, and Contact, etc.). Although the CDRs sequence range claimed in the present invention is based on the sequence represented by the Kabat numbering system, the amino acid sequences defined according to other CDRs numbering schemes should also fall within the scope of protection of the present invention.

[0060] Preferably, the variable region in the Nanobody also includes 4 framework regions.

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

[0062] In the present application, single-domain antibodies (nanoantibodies) refer to VHH structures isolated or cloned from heavy chain antibodies, which are the smallest units known to bind to target antigens.

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

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

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

[0066] The mutants include mutations that enhance / weaken the effector function of the heavy chain crystallizable region fragment, mutations that extend or shorten the half-life of the heavy chain crystallizable region fragment, etc., which are well known to professionals in the field.

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

[0068] Alternatively, it is selected from any one of murine IgG1 or a mutant thereof, IgG2a or a mutant thereof, IgG2b or a mutant thereof, and IgG3 or a mutant thereof.

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

[0070] Preferably, the heavy chain crystallizable region fragment is derived from humans, mice, rats, alpacas, goats, rabbits or horses.

[0071] Preferably, the amino acid sequence of the variable region in the Nanobody comprises the sequence shown in any one of SEQ ID NO: 15 to 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 the present invention, the amino acid sequences of the CDRs listed above are all represented according to the Kabat numbering system based on sequence variability (Kabat EA. Sequences of proteins of immunological interest [M]. USDepartment of Health and Human Services, Public Health Service, National Institutes of Health, 1991). It is well known to those skilled in the art that the CDRs of antibodies can be represented by a variety of numbering systems in the art (e.g., IMGT, Chothia, Honegger, AbM, and Contact, etc.). Although the CDRs sequence range claimed in the present invention is based on the sequence represented by the Kabat numbering system, the amino acid sequences defined according to other CDRs numbering schemes should also fall within the scope of protection of the present invention.

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

[0131] The preparation of the nucleic acid can use conventional molecular biological techniques in the art, such as obtaining the nucleic acid encoding the anti-human HHLA2 nanobody by molecular cloning technology, or obtaining the nucleic acid by codon optimization and artificial full sequence synthesis. It is well known to those skilled in the art that the base sequence encoding the amino acid sequence of the anti-human HHLA2 nanobody can form a homologue of a polynucleotide by appropriately introducing substitution, deletion or insertion. The homologue of the polynucleotide in the present invention can be obtained by replacing, deleting or inserting one or more bases of the gene encoding the human HHLA2 nanobody within the range of maintaining the antibody activity.

[0132] In a third aspect, the present invention provides an expression vector, wherein the expression vector contains the nucleic acid molecule described in the second aspect.

[0133] The expression vector can be obtained by conventional molecular biological techniques in the art, that is, the nucleic acid described in the present application is inserted into various expression vectors by means of enzyme ligation or homologous recombination.

[0134] In a fourth aspect, the present invention provides a host cell, wherein the host cell contains the nucleic acid molecule described in the second aspect or the expression vector described in the third aspect.

[0135] Host cells that can be used in the present invention include prokaryotic cells 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 cell or CHO cell (expressing heavy chain antibodies).

[0136] In a fifth aspect, the present invention provides a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the anti-human HHLA2 nanobody described in the first aspect.

[0137] In a sixth aspect, the present invention provides a genetically modified cell, wherein the genetically modified cell contains 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 the multispecific antibody comprises the anti-human HHLA2 nanobody described in the first aspect.

[0141] In the present invention, the bispecific antibody or multispecific antibody comprises the anti-human HHLA2 nanobody described in the first aspect of the present invention; and an antibody having another antigen binding property functionally linked to the antibody. The other antibody includes but is not limited to antibodies or antigen binding fragments that can bind 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, Siglec10, etc.

[0142] In an eighth aspect, the present invention provides a conjugate comprising the anti-human HHLA2 nanobody described in the first aspect.

[0143] In the present invention, the conjugate is formed by coupling the nanobody of the present invention to a functional agent.

[0144] Functional agents can be cytotoxic agents such as chemotherapeutic agents, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant or animal origin or fragments thereof), or radioactive isotopes (i.e., radioconjugates), antibiotics, nucleolytic enzymes, or any combination thereof. Chemotherapeutic agents can be used to produce immunoconjugates, for example, methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes, and / or fragments thereof, such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof. Enzymatically active toxins and fragments thereof that can be used include, for example, diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, saponaria officinalis toxin, schizonepeta tenuifolia ... officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecenes. Any suitable radionucleotide or radioactive agent known or available in the art can be used to produce radioconjugated antibodies.

[0145] The immunoconjugate may also be a complex formed by a Nanobody of the invention coupled directly or indirectly to a detectable moiety.

[0146] Detectable moieties include, but are not limited to, enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals, and nonradioactive paramagnetic metal ions.

[0147] Fluorescent labeling compounds that can be used in the present invention include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine.

[0148] Chemiluminescent labeling compounds useful in the present invention include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

[0149] Bioluminescent compounds useful in the present invention include luciferin, luciferase, and aequorin.

[0150] Enzymes useful in the present invention include horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase.

[0151] The labeling for detection and / or analysis and / or diagnostic purposes depends on the specific detection / analysis / diagnosis technique and / or method used, such as immunohistochemical staining of (tissue) samples, flow cytometry, laser scanning cytometry detection, fluorescent immunoassay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), bioassay (e.g. phagocytosis assay), Western blot application, etc. Suitable labels for detection / analysis / diagnosis techniques and / or methods known in the art are well known to 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 composition of the present invention also includes a pharmaceutically acceptable carrier, which includes but is not limited to any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the Food and Drug Administration and can be used in humans or animals, and has no side effects on the composition of the pharmaceutical composition.

[0154] The pharmaceutical composition must be sterile and stable under production and storage conditions. The pharmaceutical composition can be formulated into solutions, microemulsions, liposomes or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using a surfactant, suitable fluidity can be maintained. In many cases, it is preferred to include an isotonic agent in the composition, for example, sugars, polyols such as mannitol, sorbitol or sodium chloride. By including an agent that delays absorption in the composition, for example, monostearate and gelatin, the extended absorption of the injectable composition can be caused.

[0155] The pharmaceutical composition can be administered orally, by injection, nasally, transdermally or by mucosal administration. The pharmaceutical composition can also include other anti-tumor antibodies as active ingredients.

[0156] The pharmaceutical compositions may include, but are not limited to, liquid, frozen and lyophilized compositions.

[0157] The pharmaceutical composition can 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 the composition to achieve the desired diagnosis or treatment result. The administration regimen can also be a single injection or multiple injections, or adjusted. 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, the combination with other drugs or treatments, the disease or condition to be detected and / or treated, and the health status and previous medical history of the subject to be treated and are reasonably adjusted. 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 a use of the anti-human HHLA2 nanobody according to the first aspect, the nucleic acid molecule according to the second aspect, the expression vector according to the third aspect, 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] In an eleventh aspect, 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 genetically 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 products that detect antigen-antibody binding using enzyme-linked immunosorbent assay, immunofluorescence assay, radioimmunoassay, luminescent immunoassay, colloidal gold immunochromatography, agglutination assay, and immunoturbidimetry.

[0162] Specific examples of the product include a kit. The kit also includes a solid phase carrier, and the antibody is fixed to the solid phase carrier (such as a multi-well plate, a cover glass, a microbead) or exists in a free state.

[0163] Furthermore, the kit also includes:

[0164] A detectable portion that can be linked to the antibody, which can be detachably present in the kit; and / or a substrate corresponding to the detectable portion; and / or enzyme-linked immunosorbent assay reagents (including but not limited to: coating (buffer) solution, washing (buffer) solution, blocking solution, fixing solution, stopping solution, color developing solution); and / or instructions for use of the method for detecting human HHLA2.

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

[0166] The cancers include, but are not limited to, melanoma, lung cancer, breast cancer, pancreatic cancer, Hodgkin's lymphoma, bladder cancer, gastric 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 the anti-human HHLA2 nanobody fragment described in the first aspect, the chimeric antigen receptor described in the fifth aspect, the genetically modified cell described in the sixth aspect, the bi / multi-specific antibody described in the seventh aspect, the conjugate described in the eighth aspect, or the pharmaceutical composition described in the ninth aspect.

[0168] Preferably, the contacting further comprises ELISA, western blot, flow cytometry or immunofluorescence detection.

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

[0170] The present invention provides a nano antibody that can specifically bind to human HHLA2, which has good anti-tumor activity. The nano antibody disclosed in the present invention can specifically bind to tumor cells expressing human HHLA2, and can improve the killing effect of immune cells on tumor cells by blocking the HHLA2 and KIR3DL3 signaling pathways. Therefore, the nano antibody disclosed in the present invention has the potential to be used in the development of drugs or detection reagents for treating tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0171] Figure 1 Figure 1 is a graph showing the binding (ELISA) results of some of the HHLA2 antibodies described in the present application to human HHLA2, wherein Figure a is a binding curve of the candidate parent Ab01-63G4 and its 13 humanized or engineered mutants to human HHLA2, and Figure b is a binding curve of the candidate parent Ab01-63G4 and its 5 humanized or engineered mutants to human HHLA2.

[0172] Figure 2 Figure 1 is a result graph of specific binding of some HHLA2 antibodies described in the present application to human / cynomolgus monkey HHLA2 (FACS), wherein Figure a is the fluorescence signal of different concentrations of candidate parent Ab01-63M2 binding to K562 cells overexpressing human HHLA2, Figure b is the fluorescence signal of different concentrations of positive control antibody 2C4 binding to K562 cells overexpressing human HHLA2, Figure c is the fluorescence signal of different concentrations of candidate parent Ab01-63M2 binding to HEK293 cells overexpressing cynomolgus monkey HHLA2, and Figure d is the fluorescence signal of different concentrations of positive control antibody 2C4 binding to K562 cells overexpressing human HHLA2. Figure 2C4 shows the fluorescence signal when it binds to HEK293 cells overexpressing cynomolgus monkey HHLA2. Figure e shows the binding curve of candidate parent 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 Ab01-63M2 on HEK293 cells overexpressing cynomolgus monkey HHLA2. Figure h shows the binding curve of positive control antibody 2C4 on HEK293 cells overexpressing cynomolgus monkey HHLA2.

[0173] Figure 3 These are the result graphs of some of the HHLA2 antibodies described in the present application binding to HCC827 tumor cells (FACS), wherein Figure a is the fluorescence signal when different concentrations of candidate parent Ab01-63M2 bind to HCC827 (human non-small cell lung cancer cells, highly expressing HHLA2) cells, Figure b is the fluorescence signal when different concentrations of positive control antibody 2C4 bind to HCC827 cells, and Figure c is the binding curve of candidate parent Ab01-63M2 and positive control antibody 2C4 on HCC827 cells.

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

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

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

[0177] Figure 7 These are result graphs showing the anti-tumor effects of some HHLA2 antibodies described in this application in the NCG mouse / NK92MI / K562-hHHLA2 CDX model, wherein Figure a is a schematic diagram of the experimental scheme, Figure b is the tumor growth curves of different groups, and Figure c is the weight change curves of mice in different groups. DETAILED DESCRIPTION

[0178] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0179] If no specific techniques or conditions are indicated in the examples, all are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents used or those without manufacturer indication are conventional products that can be purchased through regular channels.

[0180] Sequence Listing

[0181]

[0182] Example 1

[0183] Screening of Nanoantibody Molecules against Human HHLA2 by Phage Display

[0184] Alpacas were immunized with an emulsified 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 for serum before and after each immunization. Blood was collected about one week after the fourth immunization and the antibody titer against the immunization antigen in the peripheral serum after each immunization was tested. If the antibody titer reached 10 5 If the immunization is successful, 50 mL of peripheral blood can be collected from the alpaca to separate peripheral blood mononuclear cells (PBMC) for immune library construction.

[0185] The RNA in PBMC (PBMC in 50mL alpaca peripheral blood) was extracted using the Trizol method, and the concentration and gel electrophoresis were detected to analyze the quality of total RNA. All the extracted RNA was divided into two parts, and reverse transcribed with oligo T and Random primers to obtain cDNA. The cDNA obtained by reverse transcription with different primers was used as a substrate for two rounds of PCR to obtain a diversified PCR product of the variable region fragment of the alpaca heavy chain antibody, namely the VHH DNA sequence. The PCR purified product of the diversified VHH DNA sequence was digested with restriction endonucleases SpeI (Takara, 1086A) and SacI (Takara, 1078A), and then connected and inserted into the phagemid pComb3XSS vector with T4 ligase (NEB, M0202L). The recombinant phagemid was transformed into TG1 Escherichia coli by electroporation and plated for overnight culture, and the bacteria were collected, which was the bacterial library; the diversified phages in the purified bacterial library were amplified, which was the phage library, and used for subsequent screening.

[0186] The alpaca-derived phage display nanoantibody library was screened using the immunotube method. The selected phage display library had a capacity of 2×10 9 The target protein was coated on the immunotube at a concentration of 20 μg / mL and three rounds of enrichment screening were performed. The enrichment degree of each round of screening is shown in Table 1:

[0187] Table 1

[0188] Number of screening rounds Input valence Screening titer Control titer Enrichment 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 panning, 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, the sequence is derived from patent CN112153977A, wherein the VH sequence is shown in SEQ ID NO: 57, and the VL sequence is shown in SEQ ID NO: 58) solution can be used for rotation elution at room temperature for 30 minutes. The third round of phage elution solution was used to infect Escherichia coli TG1, and after serial dilution and plating, it was placed in a 32°C incubator overnight. After picking monoclonal colonies to amplify phages, ELISA verification was performed (the coating was human HHLA2), and the corresponding backup positive clone TG1 colonies were taken for nanobody gene sequencing. Through sequence analysis, 136 different anti-human HHLA2 nanobody molecules were initially obtained.

[0190] SEQ ID NO:57:

[0191] EVHLQQSGPELEKPGVSVKISCKASGFSFTDYNMNWVKQSSGKSLEWIGNIDPYYGRINYNQKFKGKATLSVDKSSSTAYMHLKSLTSEDSAVYYCATGAYTSGYSWFAYWGQGTLVTVSS.

[0192] SEQ ID NO:58:

[0193] ENVLTQSPGIMAASLGEKVTMTCSASSSVSSSYLHWYQQRSGASPKPLIHRTSNLASGVPARFSGSGSGTSYSLTISSVEAEDDATYYCQQWSGYPFTFGSGTKLEIK.

[0194] Example 2

[0195] Antibody expression and purification

[0196] The nucleotide sequence of the nanobody molecule obtained in Synthesis Example 1 was codon-optimized and cloned into the eukaryotic expression vector pcDNA3.1-hG1Fc or pcDNA3.1-mG2aFc designed and modified in this laboratory (the Fc region of human IgG1 or mouse IgG2a has been inserted into the vector). The expression plasmid was prepared using Tiangen endotoxin-free plasmid extraction kit and transfected into ExpiCHO cells using ExpiFectamine CHO Transfection kit (Gbico, A29130). The cell culture shake flask was then placed in a shaker 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 filler (Tian Di Renhe, SA015025). The target antibody was eluted with 0.1M pH 3.5 glycine solution, and then concentrated and replaced with PBS buffer using an ultrafiltration tube (Shenzhen Avidity, MCP030C41). After the concentration was determined by A280 and the purity was detected by protein electrophoresis, it was packaged and frozen for later use. Table 2 shows the transient expression data 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, and Ab01-63Ghm28) after being treated at 37°C for 0, 3, and 7 days are as follows: Figure 4 As shown (Figure a is reduced electrophoresis; Figure d is non-reduced electrophoresis), it can be seen that the purity and stability of the candidate parent Ab01-63G4 and its three humanized or engineered mutants (Ab01-63Ghm25, Ab01-63Ghm26, Ab01-63Ghm28) are good.

[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 known sequences in the prior art.

[0200] Example 3

[0201] ELISA screening of antibodies that bind to human HHLA2

[0202] 0.5 μg / mL human HHLA2 protein (Acro Biosystems, B77-H52H5) was coated on an Immuno transparent standard plate (ThermoFisher, 468667), 100 μL per well, overnight at 4°C; after washing with PBST, 1% BSA in PBS blocking solution was added, 200 μL per well, and blocked at 37°C for 1 hour; different concentrations of the antibody protein prepared in Example 2 were added and incubated at 37°C for 1 hour; 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 incubated at 37°C for 30 minutes; washed with PBST 5 times; 50 μL TMB (Solerbo, PR1200) was added to each well, and placed in the dark at room temperature (20±5°C) for 10 minutes, and then 50 μL of The substrate reaction was terminated with ELISA stop solution (Solebol, C1058), and the OD value was read at 450 nm by an ELISA 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, Ab01-110M1, etc. can bind to human HHLA2 through ELISA verification. Among them, the ELISA binding curves of some HHLA2 antibodies to human HHLA2 are as follows: 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 with human HHLA2.

[0204] Example 4

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

[0206] Antibody binding experiments at the cellular level were performed using K562 cell lines (Punosai), K562 cell lines overexpressing human HHLA2 (K562-hHHLA2, Kehui Intelligent Medicine), HEK293 cell lines (Zhuhai Kairui), HEK293 cell lines overexpressing cynomolgus monkey HHLA2 (HEK293-cyHHLA2, Kehui Intelligent Medicine), and HCC827 cell lines overexpressing human HHLA2 (Punosai). Various cells were collected using 1×PBS, centrifuged at 300g for 5 minutes, and resuspended in ice-cold blocking buffer (PBS + 5% FBS), and the cell density was adjusted to 10 6 / mL; add 50 μL of cells and 50 μL of serially diluted antibodies to be tested to each well of a 96-well V-bottom plate (Corning, 3894), incubate on ice for 2 hours in the dark; add 200 μL of ice-cold wash buffer ((PBS + 0.1% BSA), centrifuge at 300 g for 5 min, discard the supernatant carefully, repeat once; resuspend the cells in a 500-fold diluted fluorescent secondary antibody (APC anti-human IgG Fc, BioLegend, 409306; or Alexa Fluor 647-AffiniPure Goat Anti-Mouse IgGFc, Jackson, 115-605-164) in blocking buffer (PBS + 5% FBS), incubate on ice in the dark for 30 min; add 200 μL ice-cold wash buffer ((PBS + 0.1% BSA), centrifuge at 300g for 5 min, discard the supernatant, repeat once, resuspend the cells in 100 μL PBS, and place on ice; transfer the sample to a flow detection tube, and use a flow cytometer (Wellgrow EasyCell 206A1) to detect the corresponding fluorescence signal.

[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 were verified by FACS to be able to specifically bind to human / cynomolgus monkey HHLA2. Among them, the results of some HHLA2 antibodies specifically binding to human / cynomolgus monkey HHLA2 (FACS) are shown in Figure 2 As shown, the results of some HHLA2 antibodies binding to HCC827 tumor cells (FACS) are shown in Figure 3 As shown, it can be seen that both the candidate parent Ab01-63M2 and the positive control antibody 2C4 can bind to human / cynomolgus monkey HHLA2 at the cellular level, and can 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 Corresponding to the results of HHLA2 antibody binding to HCC827 tumor cells (FACS), the data in Table 3 show that the binding ability of candidate parent Ab01-63M2 to 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 block HHLA2 / KIR3DL3 binding

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

[0214] Table 4

[0215] serial number Group Name 1 K562-hHHLA2+hKIR3DL3-Jurkat+test antibody+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 5 cells / mL, and transferred to a white opaque bottom 96-well plate (ThermoFisher, 15042) at 25 μL / well. An equal volume of serially diluted antibodies or cell culture medium (RPMI1640+10% FBS+1% P / S) was added and pre-incubated for 30 min. The cell density of hKIR3DL3-Jurkat (Jiman Biotechnology; overexpressing human KIR3DL3 protein) was adjusted to 1×10 6 cells / ml, 50 μL / well was added to the 96-well plate in the previous step; 25 μL Anti-CD3 epsilon antibody OKT3 (Jiman Biotechnology, GM-51478AB-100) was added to a final concentration of 0.2 μg / mL, and incubated for 30 min; after incubation in a 37°C carbon dioxide incubator for 16 h, the fluorescence signal was determined using the GM One-Step Luciferase Reporter Gene Assay Kit (Jiman Biotechnology, GM-040403B).

[0217] The results of the Luciferase reporter gene test 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 could block the HHLA2 / KIR3DL3 signaling pathway. Among them, the results of some HHLA2 antibodies blocking HHLA2 / KIR3DL3 are shown in the figure below. Figure 5As shown, it can be seen that the candidate antibodies such as Ab01-7M2, Ab01-43M2, Ab01-63M2, Ab01-79M2, Ab01-84M2 have better or equivalent ability to block HHLA2 / KIR3DL3 than the positive control antibody 2C4.

[0218] Example 6

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

[0220] Set up 5 processing as shown below:

[0221] Table 5

[0222]

[0223] K562 or K562-hHHLA2 cells were collected and the density was adjusted to 4 × 10 5 cells / mL, 50 μL was added to each well of a V-shaped / conical-bottom 96-well plate, and then 50 μL / well of the serially diluted antibody to be tested was added and pre-incubated for 30 min; effector cells NK92MI cells (Pricella, CL-0533; KIR3DL3 positive effector cells) were collected and the density was adjusted to 2×10 cells / mL with N500 medium. 5 cells / mL, added to the cells or culture medium in the previous step at 100 μL / well, centrifuged at 250 g for 3 min, and incubated at 37 °C for 6 h; centrifuged at 250 g for 5 min at room temperature, aspirated 50 μL of the supernatant into a 96-well plate, and used the LDH cytotoxicity detection 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 could significantly enhance the killing effect of NK92MI cells on target cells K562-hHHLA2. Among them, the results of some HHLA2 antibodies promoting NK92-MI killing of target cells are shown in the figure. Figure 6 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 the HHLA2 antibodies 2C4 and Ab01-63M2 promoting NK92-MI to kill target cells (K562 overexpressing human HHLA2). From the data in Table 6, it can be seen that the ability of the candidate parent Ab01-63M2 and the positive control antibody 2C4 to promote NK92-MI to kill K562-hHHLA2 is equivalent.

[0228] Example 7

[0229] Engineering of candidate antibodies

[0230] It is well known to those skilled in the art that alpaca nanobodies generally have low immunogenicity because their germline genes are highly homologous to human germline genes, so their humanization process is relatively simple, usually through conventional CDRs transplantation (if the antigen binding ability is reduced after humanization, the corresponding differential sites can be tried for back mutation). Optionally, retaining the four conservative amino acids on the nanobody FR backbone is generally beneficial to improving the hydrophilicity and solubility of the humanized nanobody molecule. Specifically, if the candidate antibody molecule Ab01-63M2 (SEQ ID NO: 7) or Ab01-63G4 (SEQ ID NO: 6) is humanized, the IMGT online database can be used to compare and select the human germline gene IGHV3_23+IGHJ5 with the highest homology to the germline gene of the candidate nanobody variable region (SEQ ID NO: 20) as the framework, and the preliminary humanized nanobody sequence (SEQ ID NO: 21) can be obtained by transplanting CDRs and replacing the 4 conservative amino acids on the FR framework (Kabat numbering: V37F, G44E, L45R, W47G). Further sequence analysis of PTM modification sites can be performed on SEQ ID NO:21 to mutate N74T, eliminating the aspartic acid isomerism and asparagine deamidation motif "D73N74S75" in the FR3 region. Secondly, several amino acid mutations can be replaced at the high-risk site of aspartic acid isomerism D55 and the risk site of aspartic acid isomerism D62 in CDR2 of SEQ ID NO:21; further based on three-dimensional structure prediction analysis, several amino acid substitution mutations can be optimized for the positive charge patch "R99R100" and the hydrophobic patch "F112F113F114" in CDR3 to obtain an antibody molecule with better drugability. 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 extend or shorten its half-life.

[0231] The drugability optimization of antibody molecules can generally be reflected in the improvement of one or more aspects such as improved antibody stability, optimized affinity (or increased or decreased), increased yield, improved solubility, and improved anti-tumor activity. After testing and comparing the stability, affinity, yield, purity, activity, etc. of a series of mutants of Ab01-63M2 or Ab01-63G4 after engineering modification, 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 to human HHLA2 are shown in Figure 1 As shown in 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, Ab01-63Ghm28) after constant temperature treatment at 37°C for 0, 3, and 7 days and their ELISA binding curves with human HHLA2 are shown in Figure 4 As shown. It can be seen that the three mutants Ab01-63Ghm25, Ab01-63Ghm26, and Ab01-63Ghm28 after humanization or engineering have similar affinities and improved stability compared with the candidate parent Ab01-63G4.

[0233] Example 8

[0234] NCG mouse / NK92MI / K562-hHHLA2 CDX model to determine the anti-tumor efficacy of candidate antibodies

[0235] The K562-hHHLA2 CDX tumor model was established using NCG mice for in vivo pharmacodynamic studies. The specific method is as follows: K562-hHHLA2 cells were revived and expanded according to experimental requirements. 7 cells / mouse for tumor cell inoculation. When the average tumor volume of mice was 100-200mm 3 The drug was administered in groups every 3 days, with a total of 3 tail vein administrations, 20 mpk / IV / Q3D. NK92MI cells were injected intratumorally 2 hours after antibody administration, with 2×10 cells injected each time. 6 After the start of drug administration, the animal body weight and tumor volume were measured every three days. The tumor volume was calculated as follows: tumor volume (mm3 ) = 0.5 × tumor long diameter × tumor short diameter 2 The experiment was terminated on the 10th day after administration, and all mice were euthanized.

[0236] In vivo antitumor efficacy of NCG / K562-hHHLA2 CDX tumor model can be found in Figure 7 Specifically, the average tumor volume of mice in the positive antibody (2C4, variable region sequence as shown in SEQ ID NO: 57-58, derived from the published patent CN112153977A) control group was 757.5 mm on the 10th day after administration. 3 The tumor inhibition rate TGI was 49.58%; the average tumor volume of the candidate antibody Ab01-63G4 treatment group was 223.0 mm on the 10th day after administration. 3 There were significant differences compared with the solvent control group and the positive antibody control group (p<0.05), the tumor inhibition rate TGI was 85.16%, and Ab01-63G4 (20mpk / IV / Q3D) had significant efficacy in the K562-hHHLA2NCG CDX model, which was better than the positive control 2C4.

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

[0238] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to 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 further describe various possible combinations.

Claims

1. A nanobody against human HHLA2, characterized in that: The variable region of the Nanobody includes CDR1, CDR2 and CDR3, 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, 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, and the mutation is a substitution, insertion or deletion of 1, 2, 3 or 4 amino acids in the amino acid sequence.

2. The anti-human HHLA2 nanobody according to claim 1, characterized in that The amino acid sequence of the CDR1 is the sequence shown in SEQ ID NO:29 or SEQ ID NO:31; Preferably, the amino acid sequence of the CDR2 is the sequence shown in SEQ ID NO: 37 or SEQ ID NO: 38; Preferably, the amino acid sequence of the CDR3 is any one of SEQ ID NO:48 to SEQ ID NO:50; Preferably, the nanobody comprises a single domain antibody, a VHH-Fc antibody, a bispecific antibody or a multispecific antibody; Preferably, the Nanobody comprises a variable region, a heavy chain crystallizable region fragment or a mutant thereof; Preferably, the heavy chain crystallizable region fragment is selected from any one of human IgG1 or a mutant thereof, IgG2 or a mutant thereof, IgG3 or a mutant thereof, IgG4 or a mutant thereof; Alternatively, any one selected from murine IgG1 or a mutant thereof, IgG2a or a mutant thereof, IgG2b or a mutant thereof, IgG3 or a mutant thereof; Preferably, the heavy chain crystallizable region fragment is selected from any one of human IgG1 or its mutant, mouse IgG2a or its mutant; Preferably, the amino acid sequence of the variable region in the Nanobody comprises the sequence shown in any one of SEQ ID NO: 15 to SEQ ID NO: 28; Preferably, the amino acid sequence of the VHH-Fc antibody comprises any one of SEQ ID NO:1-SEQ ID NO:

14.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the anti-human HHLA2 nanobody according to claim 1 or 2.

4. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 3.

5. A host cell, characterized in that The host cell contains the nucleic acid molecule according to claim 3 or the expression vector according to claim 4.

6. A chimeric antigen receptor, characterized in that The chimeric antigen receptor contains the anti-human HHLA2 nanobody according to claim 1 or 2.

7. A genetically modified cell, characterized in that The genetically modified cell contains the chimeric antigen receptor of claim 6.

8. A bispecific antibody or a multispecific antibody, characterized in that: The bispecific antibody or multispecific antibody contains the anti-human HHLA2 nanobody according to claim 1 or 2.

9. A conjugate, characterized in that: The conjugate contains the anti-human HHLA2 nanobody according to claim 1 or 2.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the anti-human HHLA2 nanobody according to claim 1 or 2.

11. Use of the anti-human HHLA2 nanobody according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4, the bi- / multi-specific antibody according to claim 8, or the conjugate according to claim 9 in the preparation of a product for detecting human HHLA2 protein.

12. Use of the anti-human HHLA2 nanobody according to claim 1 or 2, the nucleic acid molecule according to claim 3, the expression vector according to claim 4, the chimeric antigen receptor according to claim 6, the genetically modified cell according to claim 7, the bi / multi-specific antibody according to claim 8, the conjugate according to claim 9, or the pharmaceutical composition according to claim 10 in the preparation of a drug for treating and / or preventing cancer.

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