Antibody specifically binding to GPC3, and preparation method therefor and use thereof

Abstract

Provided are an antibody specifically binding to GPC3 and a use thereof, and specifically provided are a GPC3 antibody or an antigen binding fragment thereof, a multi-specific antigen binding molecule, a chimeric antigen receptor, an immune effector cell, a polynucleotide encoding the GPC3 antibody, a nucleic acid construct comprising the polynucleotide, an expression vector comprising the nucleic acid construct, a preparation method therefor, a transformed cell, etc. The antibody specifically binding to GPC3 as provided by the present invention overcomes the defects of high immunogenicity, poor solubility, easy adhesion and aggregation, easy degradation by protease, etc., of common complete antibodies, is more efficient and more potential in tumor immunotherapy, and can be used for diagnosing and treating cancers.

Description

Antibodies specifically binding to GPC3 and preparation methods and applications thereof Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an antibody or antigen-binding fragment thereof that can specifically bind to human glypican 3 (GPC3). The present invention also relates to a method for preparing the antibody and its use. Background Art

[0002] Human glypican 3 (GPC3) is a membrane protein highly expressed in various cancers, including hepatocellular carcinoma and melanoma. GPC3 consists of two subunits: a C-terminal subunit of approximately 30 kDa and an amino-terminal subunit of approximately 40 kDa. The carboxyl-terminal subunit contains two heparan sulfate (HS) chains. GPC3 protein is anchored to the cell membrane via its carboxyl terminus, linked to a glycosylphosphatidylinositol (GPI) anchor. Cell surface GPC3 protein promotes liver cancer growth by participating in the Wnt signaling pathway by forming a complex with Wnt signaling molecules. GPC3 protein can also regulate cell growth and development through the Hedgehog signaling pathway.

[0003] Hepatocellular carcinoma (HCC) is currently one of the most common malignant tumors. Over 110,000 people die from HCC annually in my country, accounting for 45% of all HCC deaths worldwide. Studies have found that GPC3 is highly expressed in HCC tissues but not in normal liver cells. Clinical testing of HCC cases using the monoclonal antibody IG12 has revealed GPC3 expression in over 70% of HCC cases. Studies have shown that elevated GPC3 expression is significantly associated with poor 5-year survival and adverse prognosis in HCC patients.

[0004] Given its high expression in various cancers, including hepatocellular carcinoma, GPC3 is considered a potential target for tumor immunotherapy. Numerous monoclonal antibodies that specifically bind to GPC3 have been discovered. Compared to current standard therapies for hepatocellular carcinoma, such as sorafenib, lenvatinib, and regorafenib, anti-GPC3 therapies hold great promise.

[0005] Currently, most intact antibodies suffer from drawbacks such as high immunogenicity, poor solubility, easy adhesion and aggregation, and susceptibility to protease degradation, which limit their application in blocking GPC3 and inhibiting tumor growth and migration. Therefore, it is crucial to develop antibodies that can efficiently enter tumors and maintain high affinity and specificity for GPC3 even in the acidic, hypoxic environment of tumors. This will effectively improve immune function in the tumor microenvironment, inhibit tumor development and metastasis, and enhance the effectiveness of anti-tumor treatment.

[0006] Nanobodies are a type of antibody that naturally lacks light chains and only has the variable region of the heavy chain. Their molecular weight is only 15kD, about one-tenth that of a typical antibody, and they are therefore also called single-domain antibodies or VHH antibodies. Nanobodies are the smallest fully functional antigen-binding units currently available. They have far greater penetration into high-density solid tumors and brain tissue than regular antibodies, effectively reaching tumor sites and exerting therapeutic effects. Nanobodies are also easy to modify, facilitating the subsequent development of antibodies with higher affinity and specificity.

[0007] The development of nanoantibodies that can efficiently enter tumors and highly specifically recognize GPC3 will effectively improve anti-tumor efficacy and has important practical significance. Summary of the Invention

[0008] The present invention addresses the deficiencies of the prior art by providing antibodies or antigen-binding fragments thereof that specifically bind to GPC3. The antibodies specifically binding to GPC3 provided herein can recognize and bind to human GPC3 and exhibit strong GPC3 neutralization activity. The present invention also provides polypeptides specifically binding to GPC3, chimeric antigen receptors, immune effector cells, nucleic acid fragments, vectors, host cells, recombinant viral vectors, pharmaceutical compositions, preparation methods, pharmaceutical uses, and disease treatment methods.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention screened two antibodies targeting human GPC3 using a natural phage antibody library. Both of these antibodies can recognize human antibodies that bind to human GPC3 and have the activity to neutralize the GPC3 protein. The antibodies against human GPC3 protein described in the present invention exert the activity of GPC3 neutralizing antibodies and can be used as standalone antibody drugs or in the preparation of CART cells to exert anti-tumor effects.

[0011] A first aspect of the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to GPC3, wherein the antibody or antigen-binding fragment comprises CDR1, CDR2, and CDR3, and the CDR1, CDR2, and CDR3 respectively comprise HCDR1, HCDR2, and HCDR3 selected from the VHH domain of any one of SEQ ID NOs: 1 to 5.

[0012] In some embodiments, each HCDR1, HCDR2, and HCDR3 is identified according to the IMGT numbering system, the Kabat numbering system, or the Chothia numbering system;

[0013] Optionally, the HCDR1 has an amino acid sequence as shown in SEQ ID NO: 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50 or 53;

[0014] Optionally, the HCDR2 has an amino acid sequence as shown in SEQ ID NO: 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51 or 54;

[0015] Optionally, the HCDR3 has an amino acid sequence as shown in SEQ ID NO: 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52 or 55;

[0016] Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 11, 12 and 13, respectively; or

[0017] Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 14, 15 and 16, respectively; or

[0018] Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 17, 18 and 19, respectively; or

[0019] Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 20, 21 and 22, respectively; or

[0020] Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 23, 24 and 25, respectively; or

[0021] Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 26, 27 and 28, respectively; or

[0022] Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 29, 30 and 31, respectively; or

[0023] Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 32, 33 and 34, respectively; or

[0024] Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 35, 36 and 37, respectively; or

[0025] Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 41, 42 and 43, respectively; or

[0026] Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 44, 45 and 46, respectively; or

[0027] Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 47, 48 and 49, respectively; or

[0028] Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 50, 51 and 52, respectively; or

[0029] Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 53, 54 and 55, respectively.

[0030] According to an embodiment of the present invention, the CDR1, CDR2 and / or CDR3 comprise an amino acid sequence having at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations on the HCDR1, HCDR2 and / or HCDR3; the mutations are selected from insertions, deletions and / or substitutions, and the substitutions are preferably substitutions of conservative amino acids.

[0031] According to an embodiment of the invention, said CDR1, CDR2 and / or CDR3 comprise a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to said HCDR1, HCDR2 and / or HCDR3.

[0032] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof comprises a Nanobody comprising the CDR1, CDR2 and CDR3.

[0033] According to an embodiment of the present invention, the Nanobody comprises a sequence as shown in any one of SEQ ID NOs: 1 to 5;

[0034] Optionally, the Nanobody comprises a sequence that has at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations compared to the sequence shown in any one of SEQ ID NOs: 1 to 5, said mutations being chosen from insertions, deletions and / or substitutions, said substitutions being preferably substitutions of conservative amino acids;

[0035] Optionally, the Nanobody comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in any one of SEQ ID NOs: 1 to 5.

[0036] Preferably, the Nanobody comprises the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 or a mutated sequence thereof.

[0037] According to an embodiment of the present invention, the Nanobody comprises a polypeptide formed by the fusion of at least two sequences selected from the sequences shown in any one of SEQ ID NOs: 1 to 5 or mutant sequences thereof.

[0038] Preferably, the Nanobody comprises a polypeptide formed by a fusion of the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 or mutant sequences thereof, for example, the fusion is formed by connecting via a linker, the linker being, for example, an amino acid sequence as shown in SEQ ID NO: 64, the encoding nucleic acid sequence of which is shown in SEQ ID NO: 63. More preferably, the Nanobody is an amino acid sequence as shown in SEQ ID NO: 70, the encoding nucleic acid sequence of which is shown in SEQ ID NO: 69.

[0039] According to an embodiment of the present invention, the Nanobody comprises the FR region in the VHH domain shown in any one of SEQ ID NOs: 1 to 5;

[0040] Alternatively, the Nanobody comprises a sequence having at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations compared to the FR region of the VHH domain represented by any one of SEQ ID NOs: 1 to 5, said mutations being chosen from insertions, deletions and / or substitutions, said substitutions being preferably substitutions of conservative amino acids;

[0041] Optionally, the Nanobody comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the FR region of the VHH domain represented by any one of SEQ ID NOs: 1 to 5.

[0042] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof is: (1) a chimeric antibody or a fragment thereof; (2) a humanized antibody or a fragment thereof; or (3) a fully human antibody or a fragment thereof;

[0043] Optionally, the antibody or antigen-binding fragment thereof comprises or does not comprise an antibody heavy chain constant region; preferably, the antigen-binding fragment is Fv;

[0044] Optionally, the antibody heavy chain constant region is selected from human, alpaca, mouse, rat, rabbit or sheep;

[0045] Optionally, the antibody heavy chain constant region is selected from IgG, IgM, IgA, IgE or IgD, and the IgG is selected from IgG1, IgG2, IgG3 or IgG4;

[0046] Optionally, the antibody heavy chain constant region is selected from IgG, IgM, IgA, IgE or IgD, and the IgG is selected from IgG1, IgG2, IgG3 or IgG4; preferably, selected from IgG1 or IgG4; more preferably, selected from human IgG1 or human IgG4;

[0047] Optionally, the heavy chain constant region is selected from the Fc region, CH3 region or complete heavy chain constant region, preferably a human Fc region; preferably, the antibody or antigen-binding fragment thereof is a heavy chain antibody.

[0048] According to an embodiment of the present invention, the antibody or antigen-binding fragment thereof is further coupled to a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic drug or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent marker, a chemiluminescent marker, an ultrasound contrast agent and a photosensitizer.

[0049] The second aspect of the present invention provides a polypeptide comprising the aforementioned antibody or antigen-binding fragment thereof, preferably, the polypeptide is further linked to other functional molecules, and the other functional molecules are selected from one or more of the following: a signal peptide, a protein tag or other antigen-binding molecules or cytokines;

[0050] Preferably, the other antigen-binding molecule specifically binds to an antigen other than GPC3 or binds to a GPC3 epitope different from that of the aforementioned antibody or antigen-binding fragment thereof;

[0051] Preferably, the antigen other than GPC3 is selected from the group consisting of: CD3, preferably CD3ε; CD16, preferably CD16A; CD19; GPC3 type II receptor; NKG2D; CD40; 4-1BB; CD137 or CD19; EGFR; EGFRvIII; mesothelin; HER2; EphA2; Her3; EpCAM; MUC1; MUC16; CEA; Claudin18.2; folate receptor; Claudin6; WT1; NY-ESO-1; MAGE3; ASGPR1 or CDH16;

[0052] Preferably, the other antigen-binding molecule is an antibody or antigen-binding fragment;

[0053] Preferably, the polypeptide is a multispecific antigen-binding molecule, such as a bispecific, trispecific or tetraspecific. More preferably, the multispecific antigen-binding molecule may be bivalent, tetravalent or hexavalent.

[0054] The third aspect of the present invention provides a chimeric antigen receptor (CAR), which comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen binding domain comprises the aforementioned antibody or antigen-binding fragment thereof.

[0055] The fourth aspect of the present invention provides an immune effector cell, which expresses the aforementioned chimeric antigen receptor or contains a nucleic acid fragment encoding the aforementioned chimeric antigen receptor; preferably, the immune effector cell is selected from T cells, NK cells, NKT cells, DNT cells, monocytes, macrophages, dendritic cells or mast cells, and the T cells are preferably selected from cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell.

[0056] The fifth aspect of the present invention provides an isolated nucleic acid fragment, which encodes the aforementioned antibody or antigen-binding fragment thereof, the aforementioned polypeptide or the aforementioned chimeric antigen receptor.

[0057] In some embodiments, the isolated nucleic acid fragment comprises a nucleic acid sequence as shown in any one of SEQ ID NOs: 6-10, 69.

[0058] The sixth aspect of the present invention provides a vector comprising the aforementioned isolated nucleic acid fragment; preferably, the vector is an antibody heavy chain expression vector AbVec-hIgG1 WT.

[0059] The seventh aspect of the present invention provides a host cell, wherein the host cell comprises the aforementioned vector;

[0060] Preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and / or Bacillus subtilis or other cells suitable for preparing antibodies or antigen-binding fragments, or multispecific antibodies; most preferably, the host cell is a 293 cell or a CHO cell.

[0061] The eighth aspect of the present invention provides a method for preparing the aforementioned antibody or antigen-binding fragment thereof or the aforementioned polypeptide, wherein:

[0062] The method comprises culturing the aforementioned host cells and isolating the antibodies, antigen-binding fragments or polypeptides expressed by the cells.

[0063] The ninth aspect of the present invention provides a method for preparing the aforementioned chimeric antigen receptor, wherein:

[0064] The method comprises introducing a nucleic acid fragment encoding the aforementioned chimeric antigen receptor (CAR) into the immune effector cell. Optionally, the method further comprises activating the immune effector cell to express the aforementioned chimeric antigen receptor (CAR).

[0065] The aforementioned antibodies or antigen-binding fragments thereof of the present invention can function in the form of proteins, or in the form of mRNA, or by inserting target genes expressing the aforementioned antibodies or antigen-binding fragments thereof into different viral vectors.

[0066] Therefore, the tenth aspect of the present invention provides a recombinant viral vector comprising the aforementioned isolated nucleic acid fragment.

[0067] Preferably, the backbone of the recombinant viral vector is derived from a modified or transformed vaccinia virus Tiantan strain, vaccinia virus New York strain, vaccinia virus Copenhagen strain, vaccinia virus Canary strain, vaccinia virus Ankara strain, adenovirus vector, adeno-associated virus vector, herpes simplex virus vector, varicella-zoster virus (VZV) vector, respiratory syncytial virus (RSV), Semliki forest virus (Semliki forest virus) vector, or adenovirus vectors. virus, SFV), Epstein-Barr virus, cytomegalovirus, human herpesvirus-6, smallpox virus, vaccinia virus, molluscum contagiosum virus, canker sore virus, reovirus, rotavirus, enterovirus, Seneca virus, poliovirus, coxsackievirus, rhinovirus, hepatitis A virus, foot-and-mouth disease virus, togavirus, alphavirus, Semliki Forest virus, Eastern equine encephalitis virus, Sindbis virus, rubella virus, coronavirus, flavivirus, hepatitis C virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley fever virus, yellow fever virus, West Nile virus, Zika virus, dengue virus, Ebola virus, Marburg virus , arenavirus, Lassa fever virus, lymphocytic choriomeningitis virus, Pichinde virus, Junin virus, Machupo virus, hantavirus, Rift Valley fever virus, paramyxovirus, human parainfluenza virus, mumps virus, simian virus 5, measles virus, vesicular stomatitis virus, rabies virus, respiratory syncytial virus, orthomyxovirus, influenza A virus, influenza B virus, influenza C virus, hepatitis D virus, simian immunodeficiency virus, human immunodeficiency virus type 1 and human immunodeficiency virus type 2, Rous sarcoma virus, human T-cell leukemia virus type 1, simian foamy virus, hepatitis B virus, hepatitis E virus, human papillomavirus, or polyomavirus.

[0068] In an embodiment of the present invention, the recombinant viral vector is a recombinant lentivirus, preferably a GPC3 nanobody chimeric receptor lentivirus or a GPC3 nanobody T cell engager (BiTE) lentivirus. Lentiviral vectors are viral vector systems modified from human immunodeficiency virus (HIV-1 virus). In the present invention, lentiviral vectors can be purchased commercially. For example, lentiviral vectors can be synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0069] Preferably, the recombinant lentivirus comprises a polynucleotide as shown in any one of SEQ ID NOs: 6 to 10, and 69.

[0070] In a preferred embodiment, the recombinant lentiviral vector of the present invention is a GPC3 nanobody chimeric receptor lentivirus. Wherein, the spacer domain of the GPC3 nanobody chimeric receptor is the CD8α transmembrane domain, the amino acid sequence of which is shown in SEQ ID NO: 62, and the encoding nucleic acid sequence thereof is shown in SEQ ID NO: 61; the costimulatory signaling domain of the GPC3 nanobody chimeric receptor is the 4-1BB signaling domain, the amino acid sequence of which is shown in SEQ ID NO: 66, and the encoding nucleic acid sequence thereof is shown in SEQ ID NO: 65; the intracellular signaling domain of the GPC3 nanobody chimeric receptor is the CD3ζ chain signaling domain, the amino acid sequence of which is shown in SEQ ID NO: 68, and the encoding nucleic acid sequence thereof is shown in SEQ ID NO: 67.

[0071] In another preferred embodiment, the recombinant lentiviral vector of the present invention is a GPC3 nanobody T cell engager (BiTE) lentivirus, wherein the CD3-activating antibody clone of the BiTE is OKT3, whose amino acid sequence is shown in SEQ ID NO: 74 and nucleotide sequence is shown in SEQ ID NO: 73.

[0072] The recombinant lentiviral vector of the present invention can be prepared by cloning the aforementioned isolated nucleic acid fragment into a blank lentiviral expression plasmid, such as pXW-EF1α-MCS. As a preferred example, the recombinant lentiviral vector can be prepared by a method comprising the following steps:

[0073] (1) Synthesize the nucleic acid fragment of the target gene;

[0074] (2) cloning the nucleic acid fragment synthesized in step (1) into a blank lentiviral expression plasmid (e.g., pXW-EF1α-MCS) to obtain a lentiviral expression plasmid carrying the target gene;

[0075] (3) The lentiviral expression plasmid obtained in step (2) as well as the backbone plasmid and envelope plasmid are co-transfected into virus production cells (e.g., HEK293T cells), and lentiviral particles are packaged and concentrated by centrifugation to obtain a lentiviral concentrate.

[0076] The eleventh aspect of the present invention provides a pharmaceutical composition, wherein:

[0077] The pharmaceutical composition comprises the aforementioned antibody or antigen-binding fragment, the aforementioned polypeptide, the aforementioned immune effector cell, the aforementioned isolated nucleic acid fragment, the aforementioned vector or the product prepared according to the aforementioned method or the aforementioned recombinant viral vector; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; optionally, the pharmaceutical composition further comprises an additional anti-tumor agent.

[0078] A twelfth aspect of the present invention provides a use of the aforementioned antibody or antigen-binding fragment, the aforementioned polypeptide, the aforementioned immune effector cell, the aforementioned isolated nucleic acid fragment, the aforementioned vector, the product obtained by the aforementioned method, the aforementioned recombinant viral vector, or the aforementioned drug combination in the preparation of a medicament for inhibiting the growth and / or proliferation of tumors or tumor cells;

[0079] Preferably, the tumor is selected from B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, glioma, high-grade glioma, blastoma neuroblastoma, osteosarcoma, plasmacytoma, histiocytoma, pancreatic cancer, breast cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer, gastric cancer, liver cancer (hepatocellular carcinoma or cholangiocarcinoma), colon cancer, rectal cancer, esophageal cancer, large intestine cancer, hematopoietic system cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell carcinoma, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma or blood oncogenic disease.

[0080] Preferably, the recombinant lentiviral vector can be used alone, or the recombinant lentiviral vector can be used in combination with T cells, CAR-T, iNKT, NK, K, macrophages, CAR-iNKT, CAR-NK or CAR-macrophages.

[0081] A thirteenth aspect of the present invention provides a method for inhibiting the growth and / or proliferation of a tumor or tumor cell, comprising administering to a patient in need thereof the aforementioned antibody or antigen-binding fragment, the aforementioned polypeptide, the aforementioned immune effector cell, the aforementioned isolated nucleic acid fragment, the aforementioned vector, the product prepared by the aforementioned method, the aforementioned recombinant viral vector, or the aforementioned drug combination;

[0082] Preferably, the tumor is selected from B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, glioma, high-grade glioma, blastoma neuroblastoma, osteosarcoma, plasmacytoma, histiocytoma, pancreatic cancer, breast cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer, gastric cancer, liver cancer (hepatocellular carcinoma or cholangiocarcinoma), colon cancer, rectal cancer, esophageal cancer, large intestine cancer, hematopoietic system cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell carcinoma, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma or blood oncogenic disease.

[0083] The fourteenth aspect of the present invention provides a kit comprising the aforementioned antibody or antigen-binding fragment, the aforementioned polypeptide, the aforementioned immune effector cell, the aforementioned nucleic acid fragment, the aforementioned vector or the product prepared by the aforementioned method, the aforementioned recombinant viral vector or the aforementioned drug combination; and instructions for use.

[0084] The beneficial effects of the present invention are as follows: the present invention provides nanobodies that can specifically bind to human GPC3, and the nanobodies have high affinity. For example, the nanobodies numbered VI006-3 and VI006-4 have high affinity, which are 2.39×10 -9 M and 2.12×10 -9 M. Through the identification of the in vitro biological functions of antibodies VI006-1, VI006-2, VI006-3, VI006-4, and VI006-5 that specifically bind to human GPC3, it was found that these antibodies all have strong neutralizing activity against GPC3, and can be used to prepare therapeutic drugs such as CAR-T or monoclonal antibodies for diseases with high GPC3 expression, which has very good prospects. In addition, compared with intact antibodies (such as the antibody Codrituzumab (GC33)), the nanobodies of the present invention have the advantages of stronger penetration and can efficiently reach tumor sites.

[0085] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here.

[0086] BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0088] Figure 1 shows the results of the first round of PCR amplification of the VHH fragment in Example 1 of the present invention. As can be seen from Figure 1, a 750 bp fragment was obtained in the first round of amplification.

[0089] Figure 2 shows the results of the second round of PCR amplification of the VHH fragment in Example 1 of the present invention. As can be seen from Figure 2, a fragment of about 400 bp was obtained in the second round of amplification.

[0090] Figure 3 shows the diversity analysis results of the phage display library in Example 1. Sequencing results show that the empty rate and antibody duplication rate of the phage display library are no more than 10%, and the library capacity of the E. coli library is 3.38E8.

[0091] FIG4 is a map of the expression vector Cloning vector AbVec-hIgG1 (GenBank: FJ475055.1) in Example 3 of the present invention.

[0092] Figures 5A to 5E are the FACS results of the purified candidate Nanobodies VI006-1, VI006-2, VI006-3, VI006-4, and VI006-5 in Example 4 of the present invention, respectively, and the recombinant cell lines overexpressing GPC3 and the blank parental cell line. The results of Figures 5A to 5E show that the candidate clones can bind to the cell line overexpressing GPC3 with high affinity.

[0093] Figures 6A to 6D are the affinity test results of the candidate Nanobodies VI006-3 and VI006-4 in Example 4 of the present invention. The results of Figures 6A to 6D show that both candidate antibody clones can bind to the GPC3 protein with high affinity.

[0094] Figure 7 is the in vitro mediated killing test results of the candidate Nanobody VI006-3 in Example 5 of the present invention. The results show that the candidate antibody clone can mediate high killing activity against liver cancer cells.

[0095] FIG8 is a diagram of the lentiviral vector GPC3 nanobody chimeric receptor and the GPC3 nanobody T cell engager (BiTE) in Example 6 of the present invention.

[0096] Figure 9 is the FACS detection result of the lentivirus-carried GPC3 nanobody chimeric receptor in Example 8 of the present invention. The results show that the GPC3 nanobody chimeric receptor prepared by the candidate clone is highly expressed on the surface of T cells.

[0097] Figure 10A shows the results of an in vitro mediated killing test using a GPC3 nanobody chimeric receptor. Figure 10B shows the results of an in vitro mediated killing test using a GPC3 nanobody T cell engager (BiTE). The results demonstrate that the nanobody clones of the present invention, prepared with GPC3 nanobody chimeric receptor T cells and GPC3 nanobody T cell engager (BiTE) T cells, can mediate high killing activity against liver cancer cells.

[0098] Best Mode for Carrying Out the Invention

[0099] The following examples are only used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0100] The following description of the present application is only for the purpose of illustrating various embodiments of the present application. Therefore, the specific modifications discussed herein should not be construed as limiting the scope of the application. A person skilled in the art can easily derive multiple equivalents, variations, and modifications without departing from the scope of the present application, and it should be understood that such equivalent embodiments are included within the scope of the present invention. All documents cited in this application, including publications, patents, and patent applications, are incorporated by reference in their entirety.

[0101] Definitions and Explanations of Terms

[0102] The term "monospecific" as used herein refers to having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.

[0103] The term "multispecific" herein refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. Thus, terms such as "bispecific," "trispecific," and "tetraspecific" refer to the number of different epitopes to which an antibody / antigen-binding molecule can bind.

[0104] The term "antigen-binding fragment" herein refers to one or more antibody fragments that retain the ability to specifically bind to a target antigen. The antigen-binding function of an antibody can be performed by a fragment of a full-length antibody. An antibody fragment can be a Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, nanobody, aptamer, or domain antibody.

[0105] The term "chimeric" antibody herein refers to an antibody that has variable sequences derived from an immunoglobulin from one source organism (e.g., rat or mouse) and constant regions derived from an immunoglobulin from a different organism (e.g., human). Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719): 1202-7; Oi et al., 1986, BioTechniques 4: 214-221; Gillies et al., 1985 J Immunol Methods 125: 191-202; each of which is incorporated herein by reference.

[0106] As used herein, the term "nanoantibody" refers to a natural heavy chain antibody lacking a light chain. Cloning its variable region can obtain a single-domain antibody consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy chain of heavy chain antibody), which is the smallest functional antigen-binding fragment. For a further description of VHHs and Nanobodies, reference is made to the review article by Muyldermans (2001, Reviews in Molecular Biotechnology 74: 277-302), and to the following patent applications mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 of the Free University of Bruxelles; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1 134 231 and WO 02 / 48193 of Unilever; WO 97 / 49805, WO 01 / 21817, WO 01 / 23696 of Vlaams Instituut voor Biotechnologie (VIB) 03 / 035694, WO 03 / 054016 and WO 03 / 055527 to Algonomics NV and Ablynx NV; WO 03 / 050531 to the National Research Council of Canada; WO 01 / 90190 to the Institute of Antibodies (= EP 1433793); and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825, and further published patent applications of Ablynx NV. Reference is also made to the additional prior art mentioned in these applications, in particular to the list of references mentioned on pages 41-43 of international application WO 06 / 040153, said list and references being incorporated herein by reference.

[0107] As described in these references, Nanobodies (in particular VHH sequences and partially humanized Nanobodies) can be characterized, inter alia, by the presence of one or more "signature residues" in one or more framework sequences. Further descriptions of Nanobodies, including humanization and / or camelization of Nanobodies, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multivalent constructs (including some non-limiting examples of linker sequences) and different modifications that increase the half-life of Nanobodies and their formulations, can be found in, for example, WO 08 / 101985 and WO 08 / 142164. For a further general description of Nanobodies, reference is made to the prior art cited herein, for example as described in WO 08 / 020079 (page 16).

[0108] Nanobodies comprise immunoglobulin domains of four "framework regions", referred to in the art and below as "framework region 1" or "FRI", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", respectively, wherein the framework regions are separated by three "complementarity determining regions" or "CDRs", referred to in the art and below as "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3", respectively. Thus, the general structure or sequence of a Nanobody (VHH) can be represented as follows:

[0109] FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0110] The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

[0111] The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0112] The term "IMGT numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883).

[0113] The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase homology with the sequence of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (e.g., variable region FR and / or constant region) comes from a human immunoglobulin (recipient antibody). Humanized antibodies generally retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, ability to increase immune cell activity, ability to enhance immune response, etc.

[0114] The term "fully human antibody" herein refers to an antibody having a variable region in which both FR and CDR are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, "fully human antibodies" herein are not intended to include antibodies in which the CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been transplanted to human framework sequences.

[0115] The term "conservative amino acids" herein generally refers to amino acids that belong to the same class or have similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, main chain conformation, and rigidity). The following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0116] 1) Alanine (A), serine (S), threonine (T);

[0117] 2) Aspartic acid (D), glutamic acid (E);

[0118] 3) Asparagine (N), glutamine (Q);

[0119] 4) Arginine (R), Lysine (K), Histidine (H);

[0120] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0121] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0122] In the present invention, "sequence identity" between two polypeptide sequences generally indicates the percentage of identical amino acids between the sequences. "Sequence identity" indicates the percentage of amino acids substituted with the same amino acid. Methods for evaluating the degree of sequence identity between amino acids or nucleotides are known to those skilled in the art. For example, amino acid sequence identity is generally measured using sequence analysis software. For example, the BLAST program of the NCBI database can be used to determine identity.

[0123] As used herein, "T cells" include CD4+ T cells, CD8+ T cells, T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells, and suppressor T cells.

[0124] "Specific" binding, when referring to a ligand / receptor, antibody / antigen or other binding pair, refers to the determination of the presence or absence of a binding reaction between a protein, e.g., a Nanobody of the invention and a GPC3 protein, within a heterogeneous population of proteins and / or other biological agents. Thus, under the specified conditions, a specific ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample. Example

[0125] Experimental materials and sources

[0126] 1. The experimental animals were alpacas (Vicugna pacos), purchased from Suzhou Aikonde Biotechnology Co., Ltd.

[0127] 2. Strains and plasmid vectors:

[0128] Escherichia coli SS320 competent cells were purchased from Suzhou Aikangde Biotechnology Co., Ltd.; pComF phage display vector was purchased from Suzhou Aikangde Biotechnology Co., Ltd.

[0129] 3. Enzymes and reagents:

[0130] Sfli endonuclease (NEB, USA);

[0131] PrimeScript TM II 1st Strand cDNA Synthesis Kit, T4 DNA ligase, DNA fragment recovery kit (TaKaRa, Japan);

[0132] High-fidelity Mix (Suzhou Xinhai Biotechnology Co., Ltd.);

[0133] Sodium chloride, potassium chloride (Shanghai Aladdin Biochemical Technology Co., Ltd.).

[0134] 4. Instruments:

[0135] Eppendorf Multiporator electroporator (VWR, USA);

[0136] Desktop high-speed refrigerated centrifuge H1650R (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.);

[0137] Electric constant temperature incubator DNP-9052 (Shanghai Jinghong Experimental Equipment Co., Ltd.);

[0138] Constant temperature shaking incubator DZ-85A (Changzhou Langyue Instrument Manufacturing Co., Ltd.);

[0139] Clean bench SW-CJ-1FD (Suzhou Antai Air Technology Co., Ltd.);

[0140] ABI2720 PCR instrument (Applied Biosystems, USA);

[0141] Biosafety cabinet HR40-IIA2 (Haier Group) Attune Nxt flow cytometer (ThermoFisher, USA);

[0142] Thermo 3111 CO2 incubator (ThermoFisher, USA).

[0143] Example 1 Alpaca immunization and phage display library construction

[0144] 1.1 Alpaca immunization and ELISA immune titer detection

[0145] 200 μg of GPC3 recombinant protein was diluted to 1 mL with PBS and emulsified with 1 mL of complete Freund's adjuvant for 10 minutes. Two alpacas (1# and 2#) were immunized subcutaneously at multiple sites. Immunizations were repeated every 21 days. Ten days after the final immunization, 5 mL of peripheral blood was collected. The centrifuge tube containing the blood sample was placed in a 37°C incubator for 1 hour, then transferred to 4°C overnight. The serum fraction was transferred to a new sterile centrifuge tube and centrifuged at 5000 rpm for 20 minutes. After separating the upper serum layer, GPC3 was coated onto a 96-well ELISA plate. After serial dilution of the serum, the immune titer was determined by ELISA. As shown in Table 1, the separated serum was subjected to limiting dilution according to the dilution series shown in Table 1 and assayed by ELISA on a 96-well plate pre-coated with the antigen. The results are shown in Table 1. After five immunizations, antibodies against GPC3 with relatively high titers were obtained. According to the ELISA test results of the immune titer, 150 mL of peripheral blood was collected from the immune batches of alpacas with higher immune titers for the construction of phage display libraries.

[0146] Table 1 ELISA titer of alpaca serum after immunization with GPC3 recombinant protein

[0147] 1.2 PBMC isolation and VHH antibody fragment cloning

[0148] 150 mL of peripheral blood was collected from alpacas 10 days after the last immunization, and peripheral blood mononuclear cells (PBMCs) were separated using lymphocyte separation medium. TM cDNA was prepared by reverse transcription using the II 1st Strand cDNA Synthesis Kit. A first round of PCR was performed using the cDNA as a template. The results of the first round of VHH fragment PCR are shown in Figure 1, yielding a single DNA fragment of approximately 750 bp. The approximately 750 bp fragment was recovered by gel excision and its concentration was determined using a NanoDrop flow cytometer. This fragment served as the template for the second round of PCR. The results of the second round of PCR electrophoresis, shown in Figure 2, yielded a single DNA fragment band of approximately 400 bp. A VHH fragment with a molecular weight of approximately 400 bp was isolated. The VHH PCR product was recovered using a gel recovery kit, and its concentration was determined using a NanoDrop flow cytometer.

[0149] 1.3 Construction of antibody phage display library

[0150] The pComF vector and the VHH PCR product obtained above were digested separately with the restriction endonuclease Sfil. The digested pComF vector and VHH fragment were then ligated using T4 ligase. The pre-chilled ligation product was added to electrocompetent SS320 cells and electroporated at 2500V for 5ms to transfer the cloning vector into the electrocompetent SS320 cells. After electroporation, 15 mL of the bacterial suspension was collected for phage rescue. Twenty μL of the bacterial suspension was diluted with 2×YT medium and spread onto an LB plate containing ampicillin. The plate was incubated at 37°C overnight. The next day, the plate was removed and the number of colonies generated from each ligation was counted to calculate the library capacity. Twenty single clones from the plate were simultaneously transferred to 2×YT medium containing ampicillin and incubated at 37°C with shaking for approximately 6-8 hours. The bacterial suspension was sequenced and the library diversity was calculated. The results are shown in Table 2. Twenty single clones were randomly selected for sequencing to analyze the diversity of the constructed phage display library. Sequencing analysis revealed significant variability among the monoclonal sequences, demonstrating good library diversity. Some samples in this batch were not monoclonal, resulting in overlapping peaks, but all were antibody sequences (as shown in Figure 3). Comparison of the sequencing results revealed that the empty rate and antibody duplication rate of the phage display library were no higher than 10%, and the E. coli library had a capacity of 3.38E8.

[0151] Table 2 Phage display library magnetic bead panning enrichment results

[0152] Example 2: Selection of specific nanobodies from phage library

[0153] 2.1 Phage library preparation and precipitation

[0154] The electroporated product in Example 1 was diluted with 2×YT medium and added with a medium containing 100 μg / mL ampicillin and placed in a constant temperature shaker. The culture was continued until the OD 600nm According to M13KO7 volume = 10 × volume × OD 600 ×5×10 8M13KO7 helper phage was added to a titer of 1 / M13KO7. The cells were centrifuged at 6000 rpm for 10 minutes, resuspended in 2×YT-AK medium, and cultured overnight at 200 rpm at 25°C. The next day, the culture was centrifuged at 10000 rpm for 15 minutes, the pellet discarded, and the supernatant transferred to a new centrifuge tube. 1 / 5 the volume of PEG / NaCl was added to the tube, mixed, and incubated at 4°C for 2 hours. The supernatant of the precipitated phage was centrifuged at 10000 rpm at 4°C for 30 minutes. The supernatant was discarded, and the pellet (phage) in each 50 mL centrifuge tube was resuspended in 1 mL of sterile PBS. The resuspended phage was transferred to a 1.5 mL EP tube and centrifuged at 12000 g at 4°C for 5 minutes. The supernatant was transferred to a new 1.5 mL EP tube, 250 μL of PEG / NaCl was added to each tube, mixed, and incubated at 4°C for 10 minutes.

[0155] Centrifuge at 12,000 g for 10 minutes, discard the supernatant, and resuspend in 1 mL of PBS. Centrifuge at 12,000 g for 5 minutes, transfer the supernatant, and continue centrifugation at 12,000 g for another 5 minutes. Transfer the supernatant to a new 1.5 mL EP tube to obtain the original phage library.

[0156] Take 10 μL and put it into 90 μL 2×YT medium, record it as 10 -1 , then dilute 10 times to 10 -9 , take 10 -7 , 10 -8 , 10 -9 Three gradient 20 μL diluted samples were added to 200 μL of pre-prepared OD 600 0.5% ER2738, mix well and place in 37℃ water bath, let it stand for 10min, and then 8 μl was applied to one LB-AMP solid plate, incubated at 37°C overnight, and the number of plaques was counted the next day to determine the titer.

[0157] 2.2 Panning of phage display library and ELISA

[0158] Panning was performed using a recombinant cell line overexpressing GPC3 (created by Suzhou Aikonde Biotechnology Co., Ltd.). The phage display library was first incubated with blank cells to wash away nonspecifically bound phage. The phage display library was then incubated with the recombinant cell line overexpressing GPC3. Recombinant phage bound to the target antigen were eluted using TEA (triethanolamine solution) and amplified. After 3-4 rounds of panning (enrichment results are shown in Table 2), single clones were selected for sequencing.

[0159] Aliquot 2×YT-Amp medium into 96-well deep-well plates, 500 μL per well, pick single clones on the output plate, and culture at 37°C, 225 rpm until OD 600Until 0.5. In the last two wells H11 and H12, no clones were selected, and only culture medium was placed as blank control. At the same time, the antigen was coated on the ELISA plate with CBS at a concentration of 1μg / mL, 100μL / well, 37℃, and coated for 2h. Take another 96-well deep-well plate, fill it with 2×YT-A culture medium, 500μL per well, and use a spray gun to draw OD 600 10 μL of the 0.5% bacterial solution was added to a newly aliquoted 96-well plate and cultured at 37°C and 225 rpm overnight. This was the bacterial solution for sample sequencing.

[0160] To OD 600 Add M13KO7 helper phage to the 0.5% bacterial solution, mix well, and place at 37°C for 15 minutes. The volume of M13KO7 helper phage added is calculated as follows: 10 × bacterial solution volume × bacterial solution OD 600 ×5×10 8 / M13KO7 helper phage titer. Place the infected bacterial solution on a shaker and incubate at 37°C, 225 rpm for 45 minutes. Place the bacterial solution in a centrifuge and centrifuge at 4000 rpm for 10 minutes. Discard the supernatant and resuspend with 2×YT-AK medium, 800 μL per well, return to the shaker and incubate overnight at 30°C, 210 rpm. At the same time, shake off the antigen from the ELISA plate, wash three times with PBST solution, and block with 3% MPBS (250 μL / well) at 4°C overnight; and additionally block a blank plate as BLANK. On the next day, the 96-well deep-well plate was placed in a centrifuge and centrifuged at 4000 rpm for 10 minutes. The milk in the ELISA plate was discarded and the plate was washed four times with 200 μL PBST. 50 μL PBST was first added to each well, followed by 50 μL of the centrifuged phage supernatant, and the plates were incubated at 4°C for 1 hour. The supernatant was discarded and the plates were washed five times with PBST. HRP-Anti M13 secondary antibody was diluted with PBST, 100 μL per well, and incubated at 4°C for 45 minutes. The secondary antibody was then washed away, the plates were washed five times with PBST, and TMB color development was performed at room temperature for 10 minutes. The plates were terminated with hydrochloric acid and tested. The test results are shown in Table 3.

[0161] Table 3 Results of ELISA detection after phage panning and enrichment

[0162] Based on the ELISA test results in Table 3, the control plate was blocked with 3% MPBS blocking buffer. Clones with an S / N ratio of 4 or higher were selected for sequencing, and antibody sequences were analyzed using the website wwww.imgt.org. After multiple rounds of screening, five clones were selected, and their sequences are shown in Table 4.

[0163] Table 4 VHH antibody sequence information

[0164] The nucleic acid sequences thereof are shown in SEQ ID NOs: 6-10.

[0165] The CDR region information was determined using the IMGT numbering system, the Kabat numbering system, or the Chothia numbering system. The results are shown in Table 5.

[0166] Table 5 VHH antibody CDR region information

[0167] 2.3 Amplification, transient infection, and detection of overlap PCR products

[0168] In the first round of PCR, the PCR reaction system was prepared according to the NuHi power mix (Xinhai Biotechnology, Catalog No. NH9303) instructions to amplify CMV, VHH, and Fc. In the second round of PCR, the reaction system was prepared according to the NuHi power mix (Xinhai Biotechnology, Catalog No. NH9303) instructions to ligate CMV, VHH, and Fc. The overlap PCR product was purified using the TaKaRa DNA Fragment Recovery Kit (TakaRa, Catalog No. 9761) and the concentration was determined using a NanoDrop assay. Eukaryotic transfection experiments were then performed.

[0169] Example 3 Expression and purification of candidate nanobodies and control antibodies

[0170] Based on the ELISA test results of the candidate antibodies, positive clones were selected, and the obtained VHH antibody sequences were gene synthesized and subcloned into the expression vector Cloning vector AbVec-hIgG1 (GenBank: FJ475055.1) ( Figure 4 ).

[0171] After the vector was verified by sequencing, the Qiagen plasmid extraction kit was used to prepare the endotoxin plasmid for use. Add 130μg Lenti-hIgG1-Fc2 (commissioned by Suzhou Aikonde Biotechnology Co., Ltd.) to 2mL PBS, mix thoroughly, add 400μL LV Transm transfection reagent (commissioned by Suzhou Aikonde Biotechnology Co., Ltd.), pipet and mix, and let stand at room temperature for 10 minutes. Add the above DNA / LV Transm complex to 50mL 293F cells and mix thoroughly. Place the cells in a 37°C, 5% CO2 incubator and culture at 130rpm for 6 to 8 hours, then add 50mL fresh FreeStyle TM 293 medium and return the cells to the incubator for continued culture. After 7 days of continuous culture, the supernatant was collected by centrifugation, filtered through a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube. The antibody was purified using a Protein A column.

[0172] Example 4 Affinity detection of candidate antibodies and FACS detection of their binding to target protein

[0173] 4.1 FACS detection of binding of candidate antibodies to target proteins

[0174] Recombinant cells overexpressing GPC3 and blank parental cells were revived from liquid nitrogen and serially passaged until the logarithmic growth phase. The candidate antibodies were serially diluted and incubated with recombinant cells overexpressing GPC3 and blank cells for 1 hour; the cells were washed three times with PBS; PE-Anti-Human IgG (Biolegend, Cat. No. 409304) was added and incubated in the dark at room temperature for 1 hour; the well plate was washed three times with PBS; after resuspending the cells in 500 μL PBS, FACS analysis was performed to determine the binding of the candidate antibodies to the target membrane protein. Figures 5A to 5E show the FACS analysis results of the candidate single-domain antibodies. The results showed that the candidate antibodies had a high affinity for the recombinant cell line overexpressing GPC3 and no affinity for the blank parental cell line. In particular, VI006-3 and VI006-4 still had a binding ratio close to 100% at a concentration of 2 μg / mL, demonstrating their high affinity and high specificity.

[0175] 4.2 Affinity testing of candidate antibodies VI006-3 and VI006-4

[0176] use R2 protein analysis platform (Sartorius) and Biacore TM T200 (GE Healthcare) candidate antibodies VI006-3 and VI006-4 were tested for affinity. The results are shown in Figures 6A to 6D. R2 protein analysis platform (Forebio R2 detection), the affinity coefficients of antibodies VI006-3 and VI006-4 are KD=9.030*10 -8 M and KD = 3.75*10 -8 M; and using Biacore TM On the T200 platform, the affinity coefficients of antibodies VI006-3 and VI006-4 are KD = 2.39*10 -9 M and KD = 2.12*10 -7 M, all showed higher affinity.

[0177] Example 5 Functional testing of candidate nanobodies

[0178] The present invention verifies the in vitro killing experiment of the liver cancer cell line HepG2 (carrying a reporter gene encoding luciferase) mediated by the candidate antibody VI006-3.

[0179] Specifically, tumor cells were plated in a 96-well plate (1E4 cells / well) and incubated at 37°C, 5% CO2 for 24 hours. The medium was then removed and the candidate antibody VI006-3 of the present invention and the control antibody codrituzumab (GC33) (GC33 (generic name: codrituzumab) used in this example is a recombinant humanized IgG1 monoclonal antibody that binds to human GPC3 with high affinity (WO2006 / 006693)) were added at a dose of 2 μg / mL. Activated CD16 T cells were added to each well, with effector cell to HepG2 cell ratios of 2:1 and 4:1. A blank control group and a nonspecific killing group (CD16 T cells alone) were also established. After continuing to culture at 37°C, 5% CO2 for 24 hours, the supernatant was removed and 50 μL of 1x cell lysis buffer (Promega, Cat. No. E1531) was added to each well. After shaking and incubating at room temperature for 30 minutes, 30 μL of luciferase assay substrate (Promega, Cat. No. E151A) was added to each well and the color was developed for 30 seconds. The fluorescence value was detected by the fluorescence analyzer ( Navigator Microplate Luminometer). The specific killing data were calculated using the values ​​of the non-specific killing group with only CD16 T cells as the control.

[0180] The results are shown in Figure 7. For liver cancer cell lines, the candidate nanoantibody VI006-3 of the present invention can mediate high killing activity of CD16T cells in vitro, and has similar killing activity compared with the control antibody GC33, indicating that the anti-GPC3 nanoantibody of the present invention has the activity of mediating tumor killing in vitro.

[0181] Unless otherwise specified, all reagents and instruments used in this example were purchased from Promega.

[0182] Example 6 Construction of Lentivirus Expression Plasmid

[0183] The construction of the GPC3 nanobody chimeric receptor lentiviral vector is as follows:

[0184] The nucleic acid sequences shown in SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 8, SEQ ID NO: 61, SEQ ID NO: 65, and SEQ ID NO: 67 were synthesized by Shanghai Qingke Biotechnology Co., Ltd. and cloned into the blank lentiviral expression plasmid pXW-EF1α-MCS to obtain the pXW-EF1α-VI006-3-4-1BB-CD3Z recombinant lentiviral expression plasmid. The plasmid map is shown in Figure 8a.

[0185] The nucleic acid sequences shown in SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 9, SEQ ID NO: 61, SEQ ID NO: 65, and SEQ ID NO: 67 were synthesized by Shanghai Qingke Biotechnology Co., Ltd. and cloned into the blank lentiviral expression plasmid pXW-EF1α-MCS to obtain the pXW-EF1α-VI006-4-4-1BB-CD3Z recombinant lentiviral expression plasmid. The plasmid map is shown in FIG8 b.

[0186] The nucleic acid sequences shown in SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 69, SEQ ID NO: 61, SEQ ID NO: 65, and SEQ ID NO: 67 were synthesized by Shanghai Qingke Biotechnology Co., Ltd. and cloned into the blank lentiviral expression plasmid pXW-EF1α-MCS to obtain the pXW-EF1α-VI006-3-4-4-1BB-CD3Z recombinant lentiviral expression plasmid. The plasmid map is shown in Figure 8 c; wherein, VI006-3-4 represents a sequence formed by connecting VI006-3 and a variant of VI006-3-4 with a linker as shown in SEQ ID NO: 64; the amino acid sequence of VI006-3-4 is shown in SEQ ID NO: 70, and the encoding nucleic acid is shown in SEQ ID NO: 69.

[0187] The nucleic acid sequences shown in SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 71, SEQ ID NO: 61, SEQ ID NO: 65, and SEQ ID NO: 67 were synthesized by Shanghai Qingke Biotechnology Co., Ltd. and cloned into the blank lentiviral expression plasmid pXW-EF1α-MCS to obtain the pXW-EF1α-GC33-4-1BB-CD3Z recombinant lentiviral expression plasmid. The plasmid map is shown in Figure 8d.

[0188] The nucleotide sequence of the promoter EF-1α of the above-mentioned plasmid pXW-EF1α-MCS is shown in SEQ ID NO: 56; the nucleotide sequence of the signal peptide is shown in SEQ ID NO: 57, and the amino acid sequence is shown in SEQ ID NO: 58; the nucleotide sequence of the FLAG tag is shown in SEQ ID NO: 59, and the amino acid sequence is shown in SEQ ID NO: 60.

[0189] The spacer domain of the GPC3 nanobody chimeric receptor involved above is selected from the CD8α transmembrane structure region (nucleotide sequence as shown in SEQ ID NO: 61, amino acid sequence as shown in SEQ ID NO: 62); the co-stimulatory signal transduction domain of the GPC3 nanobody chimeric receptor is selected from the 4-1BB signal transduction domain (nucleotide sequence as shown in SEQ ID NO: 65, amino acid sequence as shown in SEQ ID NO: 66); the intracellular signal transduction domain of the GPC3 nanobody chimeric receptor is selected from the CD3ξ chain signal transduction domain (nucleotide sequence as shown in SEQ ID NO: 67, amino acid sequence as shown in SEQ ID NO: 68).

[0190] The GPC3 nanobody T cell engager (BiTE) lentiviral vector was constructed as follows:

[0191] The nucleic acid sequences shown in SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 8, and SEQ ID NO: 73 were synthesized by Shanghai Qingke Biotechnology Co., Ltd. and cloned into the blank lentiviral expression plasmid pXW-EF1α-MCS to obtain the pXW-EF1α-VI006-3-BiTE recombinant lentiviral expression plasmid (the nucleotide sequence of VI006-3-BiTE is shown in SEQ ID NO: 77, and the amino acid sequence is shown in SEQ ID NO: 78). The plasmid map is shown in Figure 8e.

[0192] The nucleic acid sequences shown in SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 9, and SEQ ID NO: 73 were synthesized by Shanghai Qingke Biotechnology Co., Ltd. and cloned into the blank lentiviral expression plasmid pXW-EF1α-MCS to obtain the pXW-EF1α-VI006-4-BiTE recombinant lentiviral expression plasmid (the nucleotide sequence of VI006-4-BiTE is shown in SEQ ID NO: 79, and the amino acid sequence is shown in SEQ ID NO: 80). The plasmid map is shown in FIG8f .

[0193] The nucleic acid sequences shown in SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 72, and SEQ ID NO: 73 were synthesized by Shanghai Qingke Biotechnology Co., Ltd. and cloned into the blank lentiviral expression plasmid pXW-EF1α-MCS to obtain the pXW-EF1α-GC33-BiTE recombinant lentiviral expression plasmid (the nucleotide sequence of GC33-BiTE is shown in SEQ ID NO: 75, and the amino acid sequence is shown in SEQ ID NO: 76). The plasmid map is shown in Figure 8g.

[0194] The CD3 activating antibody clone of the aforementioned GPC3 nanobody T cell engager (BiTE) is OKT3 (nucleotide sequence shown in SEQ ID NO: 73, amino acid sequence shown in SEQ ID NO: 74).

[0195] Example 7 Packaging, concentration and titer determination of lentivirus

[0196] 7.1 Lentivirus Packaging

[0197] HEK293T cell treatment: 24 h before transfection, HEK293T cells in the logarithmic growth phase were collected and seeded into 10 cm cell culture dishes (6-8×10 6 The cells were grown in 10 mL of complete DMEM medium and cultured in a 37° C., 5% CO 2 cell culture incubator for 18 to 24 hours. Plasmid transfection was performed when the cell density reached 70 to 90%.

[0198] HEK293T cell transfection: Add 1 mL of basal DMEM medium to a 15 mL centrifuge tube and prepare a transfection mix at a mass ratio of lentiviral expression plasmid: packaging plasmid: envelope plasmid = 1:3:1, for a total plasmid volume of 15 μg / dish. Add 30 μL of TurboFect transfection reagent at a ratio of plasmid (μg): transfection reagent (μL) = 1:2. Incubate at room temperature for 15-20 minutes, then add to a dish containing HEK293T cells. Incubate in a 37°C, 5% CO2 incubator for 48 hours. Collect the viral supernatant and centrifuge at 1000 × g at 4°C for 10 minutes. Discard the pellet and collect the viral supernatant.

[0199] 7.2 Concentration of Lentivirus

[0200] Filter the collected viral supernatant through a 0.45 μm filter and add 1 / 3 the volume of the original supernatant to the Lenti-X Lentivirus Concentration Reagent. Mix by inversion several times. Incubate overnight at 4°C. Centrifuge at 2000 × g for 45 minutes at 4°C. A white precipitate will appear at the bottom of the tube, representing the concentrated viral particles. Carefully discard the supernatant and resuspend the white precipitate in 1 / 50–1 / 100 volume of blank RPMI-1640 medium. Aliquot and freeze at -80°C until needed.

[0201] 7.3 Lentiviral titer determination

[0202] Jurkat T cells were cultured at a rate of 1×10 5Inoculate cells / well in a 96-well U-bottom plate and dilute the collected lentiviral concentrate in 10-fold increments. Add 100 μL of the virus dilution to the corresponding well, add the infection-promoting reagent protamine sulfate and adjust the concentration to 10 μg / mL. Centrifuge at 1000 × g at 32°C for 90 minutes. After overnight incubation, replace with fresh RPMI1640 complete medium and continue incubation for 48 hours. Detect the proportion of fluorescence-positive cells by flow cytometry. Calculate the virus titer using the following formula:

[0203] Virus titer (TU / mL) = 1 × 10 5 × the ratio of fluorescence-positive cells / 100 × 1000 × the corresponding dilution factor.

[0204] Example 8 GPC3 Nanobody Chimeric Receptor Expression Detection

[0205] The lentivirus expression plasmid of Example 6 and the lentivirus vector preparation method of Example 7 carrying the target gene (MOI=5) were added to a 1×10 6 In a 48-well flat-bottom plate containing pre-activated peripheral blood mononuclear cells, add the infection-promoting reagent protamine sulfate and adjust the working concentration to 10 μg / mL. Centrifuge at 1000×g at 32°C for 90 minutes. After overnight culture, replace with fresh T cell growth medium and continue culture. Add fresh T cell growth medium every 2 to 3 days and adjust the cell density to 0.5 to 2×10 6 Cells / mL. 6-7 days after infection, remove the immunomagnetic beads that activate T cells, continue culturing and expanding the genetically engineered T cells, and allow the cells to rest (9-14 days after removing the beads) before conducting subsequent functional experiments. After completion, the cells were collected and eluted once with FACS buffer. Add 2 μg / mL of the flow cytometry antibody PE-anti-DYKDDDDK and incubate at room temperature in the dark for 20 minutes. After completion, the cells were eluted twice with FACS buffer, resuspended and mixed thoroughly in 300 μL of FACS buffer, and then the expression of the CAR molecule was detected using a flow cytometer.

[0206] The results are shown in Figure 9. Flow cytometry results showed that the GPC3 nanobody chimeric receptor was highly expressed on the T cell membrane. Under the same infection MOI conditions, the positive rate of the VI006-3 nanobody chimeric receptor was 67%, which was higher than the control GC33 chimeric receptor 57.2%, showing a significant expression advantage.

[0207] Example 9: Killing of Tumor Cells by GPC3 Nanobody Chimeric Receptor and GPC3 Nanobody T Cell Engager (BiTE) Carried by Lentiviral Vector

[0208] Tumor cell killing efficiency is determined by measuring the amount of LDH released from damaged cell membranes to determine the number of dead cells. Lactate dehydrogenase (LDH) is an enzyme that is stably present in the cell cytoplasm and, under normal conditions, is confined to the cell interior. When cells are stimulated to die, the plasma membrane ruptures and LDH is rapidly released outside the cell (in experiments, into the cell culture medium). Therefore, it is one of the most widely used markers in cytotoxicity studies.

[0209] 1×10 4 Huh7 (human hepatoma cells) were seeded into 96-well flat-bottom plates with 100 μL of culture medium per well and cultured in a 37°C, 5% CO2 cell culture incubator for 18 hours. The next day, the target cells were divided into five groups. The experimental groups were treated with GPC3 chimeric receptor T cells carrying VI006-3-4-1BB-CD3Z, VI006-4-4-1BB-CD3Z, VI006-3-4-4-1BB-CD3Z, and GC33-4-1BB-CD3Z, while the control group was left untreated. The ratio of target cells to effector cells was 1:1, and the cells were cultured for another 20 hours. After the co-culture, the LDH release of the target cells was detected using a microplate reader (main wavelength 450 nm, reference wavelength 630 nm).

[0210] The results are shown in Figure 10A. T cells expressing the GPC3 nanobody chimeric receptor demonstrated highly effective cytotoxicity against Huh7 tumor cells. Under a 1:1 effector cell:target cell ratio, the VI006-4-CART demonstrated superior cytotoxicity compared to the control GC33-CART. Furthermore, the VI006-3-4-CART, created by fusing VI006-3 and VI006-4, further enhanced its cytotoxicity, with LDH secretion reaching 1.8 times higher than the control GC33-CART.

[0211] 1×10 4 Huh7 (human hepatoma cell) cells were seeded into 96-well flat-bottom plates with 100 μL of culture medium per well and cultured in a 37°C, 5% CO2 cell culture incubator for 18 hours. The next day, the target cells were divided into four groups. T cells loaded with VI006-3-BiTE, VI006-4BiTE, and GC33-BiTE were added to the experimental groups, while the control group received no treatment. The target cell to effector cell ratio was 1:1, and the cells were cultured for another 20 hours. After the co-culture, the LDH release of the target cells was measured using a microplate reader (main wavelength 450nm, reference wavelength 630nm).

[0212] The results are shown in Figure 10B. T cells expressing the GPC3 nanobody BiTE were highly effective in killing Huh7 tumor cells. At a 1:1 effector cell:target cell ratio, the VI006-4-BiTE-T cells demonstrated a 1.2-fold greater cytotoxicity than the control GC33-BiTE-T cells.

[0213] The above embodiments are exemplary and should not be construed as limiting the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to GPC3, wherein: The antibody or antigen-binding fragment thereof comprises CDR1, CDR2 and CDR3, wherein the CDR1, CDR2 and CDR3 respectively comprise HCDR1, HCDR2 and HCDR3 selected from the VHH domain shown in any one of SEQ ID NOs: 1 to 5.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein The HCDR1, HCDR2 and HCDR3 are determined according to the IMGT numbering system, the Kabat numbering system or the Chothia numbering system; Optionally, the HCDR1 has an amino acid sequence as shown in SEQ ID NO: 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50 or 53; Optionally, the HCDR2 has an amino acid sequence as shown in SEQ ID NO: 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51 or 54; Optionally, the HCDR3 has an amino acid sequence as shown in SEQ ID NO: 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52 or 55; Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 11, 12 and 13, respectively; or Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 14, 15 and 16, respectively; or Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 17, 18 and 19, respectively; or Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 20, 21 and 22, respectively; or Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 23, 24 and 25, respectively; or Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 26, 27 and 28, respectively; or Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 29, 30 and 31, respectively; or Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 32, 33 and 34, respectively; or Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 35, 36 and 37, respectively; or Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 41, 42 and 43, respectively; or Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 44, 45 and 46, respectively; or Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 47, 48 and 49, respectively; or Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 50, 51 and 52, respectively; or Preferably, the HCDR1, HCDR2 and HCDR3 have the amino acid sequences shown in SEQ ID NOs: 53, 54 and 55, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The CDR1, CDR2 and / or CDR3 comprise an amino acid sequence having at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations on the HCDR1, HCDR2 and / or HCDR3; the mutations are selected from insertions, deletions and / or substitutions, and the substitutions are preferably substitutions of conservative amino acids.

4. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein The CDR1, CDR2 and / or CDR3 comprise a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the HCDR1, HCDR2 and / or HCDR3.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein The antibody or antigen-binding fragment thereof comprises a nanobody comprising the CDR1, CDR2 and CDR3.

6. The antibody or antigen-binding fragment thereof according to claim 5, wherein The nanobody comprises a sequence as shown in any one of SEQ ID NOs: 1 to 5; Optionally, the Nanobody comprises a sequence that has at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations compared to the sequence shown in any one of SEQ ID NOs: 1 to 5, said mutations being chosen from insertions, deletions and / or substitutions, said substitutions being preferably substitutions of conservative amino acids; Optionally, the Nanobody comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence shown in any one of SEQ ID NOs: 1 to 5; Preferably, the Nanobody comprises the sequence shown by SEQ ID NO: 3 or SEQ ID NO: 4 or a mutant sequence thereof; or, The nanobody comprises a polypeptide formed by fusion of at least two sequences selected from any one of SEQ ID NOs: 1 to 5 or mutant sequences thereof; Preferably, the Nanobody comprises a polypeptide formed by a fusion of the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 or mutant sequences thereof; more preferably, the Nanobody comprises the amino acid sequence shown in SEQ ID NO:

70.

7. The antibody or antigen-binding fragment thereof according to claim 5 or 6, wherein The nanobody comprises the FR region in the VHH domain shown in any one of SEQ ID NOs: 1 to 5; Alternatively, the Nanobody comprises a sequence having at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutations compared to the FR region of the VHH domain represented by any one of SEQ ID NOs: 1 to 5, said mutations being chosen from insertions, deletions and / or substitutions, said substitutions being preferably substitutions of conservative amino acids; Optionally, the Nanobody comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the FR region of the VHH domain represented by any one of SEQ ID NOs: 1 to 5.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, wherein The antibodies or antigen-binding fragments thereof are: (1) chimeric antibodies or fragments thereof; (2) a humanized antibody or a fragment thereof; or (3) a fully human antibody or a fragment thereof; Optionally, the antibody or antigen-binding fragment thereof comprises or does not comprise an antibody heavy chain constant region; Preferably, the antigen-binding fragment is Fv; Optionally, the antibody heavy chain constant region is selected from human, alpaca, mouse, rat, rabbit or sheep; Optionally, the antibody heavy chain constant region is selected from IgG, IgM, IgA, IgE or IgD, and the IgG is selected from IgG1, IgG2, IgG3 or IgG4; preferably, selected from IgG1 or IgG4; more preferably, selected from human IgG1 or human IgG4; Optionally, the heavy chain constant region is selected from the Fc region, CH3 region or complete heavy chain constant region, preferably a human Fc region; preferably, the antibody or antigen-binding fragment thereof is a heavy chain antibody.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein The antibody or antigen-binding fragment thereof is further coupled to a therapeutic agent or a tracer; preferably, the therapeutic agent is selected from a radioisotope, a chemotherapeutic drug or an immunomodulator, and the tracer is selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a chemiluminescent label, an ultrasound contrast agent and a photosensitizer.

10. A polypeptide, wherein The polypeptide comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, preferably, the polypeptide is further linked to other functional molecules, and the other functional molecules are selected from one or more of the following: a signal peptide, a protein tag or other antigen-binding molecules or cytokines; Preferably, the other antigen-binding molecule specifically binds to an antigen other than GPC3 or binds to a GPC3 epitope different from the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9; Preferably, the antigen other than GPC3 is selected from the group consisting of: CD3, preferably CD3ε; CD16, preferably CD16A; CD19; GPC3 type II receptor; NKG2D; CD40; 4-1BB; CD137 or CD19; EGFR; EGFRvIII; mesothelin; HER2; EphA2; Her3; EpCAM; MUC1; MUC16; CEA; Claudin18.2; folate receptor; Claudin6; WT1; NY-ESO-1; MAGE3; ASGPR1 or CDH16; Preferably, the other antigen-binding molecule is an antibody or antigen-binding fragment; Preferably, the polypeptide is a multispecific antigen-binding molecule, such as a bispecific, trispecific or tetraspecific, more preferably, the multispecific antigen-binding molecule is bivalent, tetravalent or hexavalent.

11. A chimeric antigen receptor, wherein The chimeric antigen receptor comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

12. An immune effector cell, wherein: The immune effector cell expresses the chimeric antigen receptor of claim 11 or comprises a nucleic acid fragment encoding the chimeric antigen receptor of claim 11; preferably, the immune effector cell is selected from T cells, NK cells, NKT cells, DNT cells, monocytes, macrophages, dendritic cells or mast cells, and the T cell is preferably selected from cytotoxic T cells, regulatory T cells or helper T cells; preferably, the immune effector cell is an autologous immune effector cell or an allogeneic immune effector cell.

13. An isolated nucleic acid fragment, wherein The nucleic acid fragment encodes the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the polypeptide according to claim 10, or the chimeric antigen receptor according to claim 11; Preferably, the isolated nucleic acid fragment comprises a polynucleotide as shown in any one of SEQ ID NOs: 6 to 10.

14. A carrier, wherein The vector comprises the isolated nucleic acid fragment according to claim 13; preferably, the vector is the antibody heavy chain expression vector AbVec-hIgG1 WT.

15. A host cell, wherein The host cell comprises the vector of claim 14; Preferably, the host cell is a eukaryotic cell or a prokaryotic cell; more preferably, the host cell is derived from a mammalian cell, a yeast cell, an insect cell, Escherichia coli and / or Bacillus subtilis or other cells suitable for preparing antibodies or antigen-binding fragments, or multispecific antibodies; most preferably, the host cell is a 293 cell or a CHO cell.

16. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 or the polypeptide according to claim 10, wherein: The method comprises culturing the host cell of claim 15 and isolating the antibody, antigen-binding fragment or polypeptide expressed by the cell.

17. A method for preparing the chimeric antigen receptor according to claim 11, wherein: The method comprises introducing a nucleic acid segment encoding the chimeric antigen receptor of claim 11 into the immune effector cell of claim 12. Optionally, the method further comprises initiating the immune effector cell to express the chimeric antigen receptor of claim 11.

18. A recombinant viral vector, wherein: The recombinant viral vector comprises the isolated nucleic acid segment of claim 13; Preferably, the backbone of the recombinant viral vector is derived from a modified or transformed vaccinia virus Tiantan strain, vaccinia virus New York strain, vaccinia virus Copenhagen strain, vaccinia virus Canary strain, vaccinia virus Ankara strain, adenovirus vector, adeno-associated virus vector, herpes simplex virus vector, varicella-zoster virus (VZV) vector, respiratory syncytial virus (RSV), Semliki forest virus (Semliki forest virus) vector, or adenovirus vectors. virus, SFV), Epstein-Barr virus, cytomegalovirus, human herpesvirus-6, smallpox virus, vaccinia virus, molluscum contagiosum virus, canker sore virus, reovirus, rotavirus, enterovirus, Seneca virus, poliovirus, coxsackievirus, rhinovirus, hepatitis A virus, foot-and-mouth disease virus, togavirus, alphavirus, Semliki Forest virus, Eastern equine encephalitis virus, Sindbis virus, rubella virus, coronavirus, flavivirus, hepatitis C virus, Japanese encephalitis virus, St. Louis encephalitis virus, Murray Valley fever virus, yellow fever virus, West Nile virus, Zika virus, dengue virus, Ebola virus, Marburg virus , arenavirus, Lassa fever virus, lymphocytic choriomeningitis virus, Pichinde virus, Junin virus, Machupo virus, Hantavirus, Rift Valley fever virus, paramyxovirus, human parainfluenza virus, mumps virus, simian virus 5, measles virus, vesicular stomatitis virus, rabies virus, respiratory syncytial virus, orthomyxovirus, influenza A virus, influenza B virus, influenza C virus, hepatitis D virus, simian immunodeficiency virus, human immunodeficiency virus type 1 and human immunodeficiency virus type 2, Rous sarcoma virus, human T-cell leukemia virus type 1, simian foamy virus, hepatitis B virus, hepatitis E virus, human papillomavirus, or polyomavirus; More preferably, the recombinant viral vector is a recombinant lentivirus, and even more preferably, the recombinant lentivirus is a nanobody chimeric receptor lentivirus or a nanobody T cell engager lentivirus; further preferably, the recombinant lentivirus comprises a polynucleotide as shown in any one of SEQ ID NOs: 6 to 10, 69.

19. A pharmaceutical composition, wherein The pharmaceutical composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the polypeptide according to claim 10, the immune effector cell according to claim 12, the isolated nucleic acid fragment according to claim 13, the vector according to claim 14, the product prepared according to the method of claim 16 or 17, or the recombinant viral vector according to claim 18; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or adjuvant; optionally, the pharmaceutical composition further comprises an additional anti-tumor agent.

20. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the polypeptide according to claim 10, the immune effector cell according to claim 12, the isolated nucleic acid fragment according to claim 13, the vector according to claim 14, the product prepared by the method according to claim 16 or 17, the recombinant viral vector according to claim 18, or the pharmaceutical combination according to claim 19 in the preparation of a medicament for inhibiting the growth and / or proliferation of a tumor or tumor cell; Preferably, the tumor is selected from B-cell lymphoma, T-cell lymphoma, melanoma, prostate cancer, renal cell carcinoma, sarcoma, glioma, high-grade glioma, blastoma neuroblastoma, osteosarcoma, plasmacytoma, histiocytoma, pancreatic cancer, breast cancer, lung cancer such as small cell lung cancer and non-small cell lung cancer, gastric cancer, liver cancer (hepatocellular carcinoma or cholangiocarcinoma), colon cancer, rectal cancer, esophageal cancer, large intestine cancer, hematopoietic system cancer, testicular cancer, cervical cancer, ovarian cancer, bladder cancer, squamous cell carcinoma, adenocarcinoma, AIDS-related lymphoma, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, head and neck squamous cell carcinoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma or blood oncogenic disease; Optionally, the recombinant lentiviral vector is used alone, or the recombinant lentiviral vector is used in combination with T cells, CAR-T, iNKT, NK, K, macrophages, CAR-iNKT, CAR-NK or CAR-macrophages.

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