An anti-b7-h3 nanobody, a symmetric anti-b7-h3xcd3 bispecific antibody and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses an anti-B7-H3 nanobody, a symmetric anti-B7-H3*CD3 bispecific antibody and a preparation method and application thereof, and specifically discloses an anti-B7-H3 nanobody, wherein the nanobody comprises a complementarity determining region CDR, and the complementarity determining region CDR comprises a complementarity determining region CDR1, a complementarity determining region CDR2 and a complementarity determining region CDR3. A brand-new specific anti-human B7-H3 nanobody sequence is obtained by using a nanobody library and through phage display screening technology, the anti-B7-H3 nanobody has specific recognition and combination capacity for B7-H3, and is expected to be used as a therapeutic antibody for various B7-H3 molecule high-expression malignant tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-B7-H3 nanobody, a symmetrical anti-B7-H3×CD3 bispecific antibody, and a preparation method and application thereof. Background Art

[0002] B7-H3, also known as CD276, and PD-L1 (B7-H1), are members of the B7 family of immune checkpoint proteins. Their basic structure comprises a pair of IgV- and IgC-like immunoglobulin domains, a transmembrane region, and a highly diverse short cytoplasmic tail. Multiple immunostaining studies have shown that B7-H3 is overexpressed in tumor tissues, while expression is low in normal tissues. Different cancer types may have distinct B7-H3 localization patterns. Furthermore, B7-H3 expression has been detected in tumor-associated vasculature and fibroblasts in most colorectal cancer specimens. Targeting tumor vasculature has the potential to improve the penetration of immune effector cells into solid tumors, potentially overcoming a major obstacle to T-cell-based immunotherapy. In recent years, various cancer treatment strategies targeting this target have been developed, including monoclonal antibodies, bispecific antibodies (BsAbs), antibody-drug conjugates (ADCs), and chimeric antigen receptor T cells (CAR-T). Currently, there are two bispecific antibody products that are developing rapidly for B7-H3. They are Obrindatamab (targeting B7-H3 and CD3) developed by MacroGenics. format, patent number US20190002563A1) and Xencor's XmAb808 (targeting B7-H3 and CD28, Format).

[0003] Nanobodies, also known as variable domain heavy chain antibodies (VHH), are antigen-binding units derived from camel heavy chain antibodies (HCAbs). Although they lack light chains, nanobodies can still bind to antigens with high specificity and affinity. At the same time, nanobodies, due to their excellent stability and good solubility, can support drug development and inhaled administration. Their unique advantages of low immunogenicity risk and small size also allow for the development of penetrant therapies for solid tumors. They have gradually attracted the interest of researchers and are being designed to construct multi-target antibodies with improved binding and new functions. Sequence discovery of antigen-specific nanobodies can be achieved through screening of immune antibody libraries and synthetic antibody libraries. The preparation of synthetic nanoantibody libraries does not require animal immunization, and the mutation sites and overall diversity of the library can be artificially controlled to simulate the evolution of nanoantibodies. In recent years, they have been widely used in antibody discovery.

[0004] Since bispecific antibodies have two different antigen-binding arms that can bind to different antigen-binding regions, relatively flexible targeting strategies can be designed. CD3, as a key membrane antigen for T cells to recognize antigens and participate in activation responses, is a target direction that was entered earlier in the development of bispecific antibodies. Currently, the combination of CD3 and other tumor targets, namely T cell redirecting bispecific antibodies, is the mainstream of bispecific antibody research and development. Its anti-tumor mechanism of action is that the antibody molecule interacts with tumor cells and T cells at the same time, one binding arm binds to the tumor target antigen, and the other binding arm binds to the TCR-CD3 complex on the functional cell to form a receptor complex to activate T cells, shorten the physical distance between the target effector cells, form an immune synapse, and enable T cells to kill tumor cells in a targeted manner. Blincyto is the world's first FDA-approved bispecific antibody drug targeting CD19 and CD3, and is used to treat CD19-positive acute lymphoblastic leukemia. But a drug like Blincyto, which is assembled from antigen binding units, Bisspecific antibodies are rapidly cleared from plasma and require continuous infusion to maintain efficacy. If the bispecific antibody format incorporates an Fc (fragment crystallizable) region, the drug half-life can be extended through FcRn (the neonatal Fc receptor for IgG)-mediated endocytosis. However, given the association between the effector function of the Fc region and adverse safety events, further modification of the Fc region is necessary.

[0005] Anti-B7-H3 BsAbs are primarily conjugates of BITE-based molecules with anti-CD3 and anti-B7-H3 antibodies prepared by chemical coupling. BITE-based antibodies have a short half-life, while antibodies prepared by chemical coupling have a large molecular weight and strong immunogenicity, limiting their potential applications. Therefore, screening and preparing new anti-B7-H3-specific nanobodies and designing novel anti-B7-H3 × CD3 bispecific antibodies are of great practical significance. Summary of the Invention

[0006] To address the shortcomings of existing anti-B7-H3 antibody-related products and bispecific antibody products for solid tumor treatment, this paper provides a method for screening anti-B7-H3 antibodies and the design of a recombinant bispecific antibody. This invention utilizes phage display technology to screen synthetic nanoantibody libraries. The resulting BH1 nanoantibody sequences can be used in the development of T cell-redirecting bispecific antibody drugs, potentially achieving superior tumor therapeutic efficacy at lower doses.

[0007] The purpose of the first aspect of the present invention is to provide an anti-B7-H3 nanobody.

[0008] The second aspect of the present invention aims to provide an Fc fusion protein of an anti-B7-H3 nanobody.

[0009] The third aspect of the present invention is to provide an anti-B7-H3×CD3 bispecific antibody.

[0010] The fourth aspect of the present invention aims to provide a biomaterial related to the anti-B7-H3 nanobody of the first aspect of the present invention, the Fc fusion protein of the anti-B7-H3 nanobody of the second aspect of the present invention, or the bispecific antibody of the third aspect of the present invention.

[0011] The fifth aspect of the present invention aims to provide an antibody-drug conjugate.

[0012] The sixth aspect of the present invention aims to provide a chimeric antigen receptor.

[0013] The seventh aspect of the present invention aims to provide a chimeric antigen receptor immune cell.

[0014] The purpose of the eighth aspect of the present invention is to provide the use of the anti-B7-H3 nanobody of the first aspect of the present invention, the Fc fusion protein of the anti-B7-H3 nanobody of the second aspect of the present invention, the bispecific antibody of the third aspect of the present invention, the biomaterial of the fourth aspect of the present invention, the antibody-drug conjugate of the fifth aspect of the present invention, the chimeric antigen receptor of the sixth aspect of the present invention or the chimeric antigen receptor immune cell of the seventh aspect of the present invention.

[0015] The ninth aspect of the present application aims to provide a product.

[0016] To achieve the above-mentioned object, the technical solution adopted by the present application is:

[0017] The present application first screens specific nanobodies against human B7-H3 through phage display from a self-built synthetic nanobody library. The enriched phage is verified to identify a clone specifically binding to human B7-H3, named BH1. Further, the BH1 sequence is used to construct a B7-H3xCD3 bispecific antibody through molecular cloning technology, and the antibody crude product is obtained through a 293F mammalian expression system. The antibody sample is purified through affinity chromatography and size exclusion chromatography to obtain the antibody protein (purity > 95%) for subsequent characterization and pharmacodynamic verification. Antibody characterization includes SDS-PAGE verification of protein molecular weight and purity, thermal shift detection of protein melting temperature Tm, and flow cytometry analysis of specific binding of recombinant antibody to target effector cell surface antigen. Pharmacodynamic detection includes T cell activation and induction of killing by recombinant antibody. The VHH construction unit targeting B7H3 is an essential component of the present application, and in addition to the examples provided by the present application, it can also be used to construct different formats of monovalent or multivalent monoclonal antibodies or multispecific antibodies.

[0018] The first aspect of the present application provides an anti-B7-H3 nanobody, which comprises a complementarity determining region CDR, the complementarity determining region CDR comprising a complementarity determining region CDR1, a complementarity determining region CDR2 and a complementarity determining region CDR3, wherein,

[0019] the amino acid sequence of the complementarity determining region CDR1 is as shown in SEQ ID NO: 2 or an amino acid sequence modified by substitution, deletion or addition of one or more amino acids of the amino acid sequence as shown in SEQ ID NO: 2 and functionally same or similar to the amino acid sequence as shown in SEQ ID NO: 2;

[0020] the amino acid sequence of the complementarity determining region CDR2 is as shown in SEQ ID NO: 3 or an amino acid sequence modified by substitution, deletion or addition of one or more amino acids of the amino acid sequence as shown in SEQ ID NO: 3 and functionally same or similar to the amino acid sequence as shown in SEQ ID NO: 3;

[0021] the amino acid sequence of the complementarity determining region CDR3 is as shown in SEQ ID NO: 4 or an amino acid sequence modified by substitution, deletion or addition of one or more amino acids of the amino acid sequence as shown in SEQ ID NO: 4 and functionally same or similar to the amino acid sequence as shown in SEQ ID NO: 4.

[0022] In some embodiments of the present invention, the amino acid sequence of the anti-B7-H3 Nanobody is as shown in SEQ ID NO: 1 or an amino acid sequence modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 and having the same or similar functions.

[0023] The second aspect of the present invention provides an Fc fusion protein of an anti-B7-H3 nanobody, comprising the anti-B7-H3 nanobody of the first aspect of the present invention and an Fc segment.

[0024] In some embodiments of the present invention, the amino acid sequence of the Fc segment is as shown in RSDKTYTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 8) or an amino acid sequence modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 8 and having the same or similar functions.

[0025] In some embodiments of the present invention, the amino acid sequence of the Fc fusion protein of the anti-B7-H3 Nanobody is as shown in SEQ ID NO: 5 or an amino acid sequence modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 5 and having the same or similar functions.

[0026] The third aspect of the present invention provides an anti-B7-H3×CD3 bispecific antibody targeting B7-H3 and CD3. The bispecific antibody has a symmetrical structure and comprises the anti-B7-H3 nanobody of the first aspect of the present invention, a scFv targeting CD3, and an Fc segment.

[0027] In some embodiments of the present application, the amino acid sequence of the scFv targeting CD3 is set forth in DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKS (SEQ ID NO: 9) or a function-conserved or similar amino acid sequence modified from the amino acid sequence set forth in SEQ ID NO: 9 by substitution, deletion, or addition of one or more amino acids.

[0028] In some preferred embodiments, the Fc fragment is altered, e.g., mutated, to modify the properties of the bispecific antibody molecule of the application (e.g., to alter one or more of the following properties: Fc receptor binding, antibody glycosylation, effector cell function, or complement function). The bispecific antibodies provided herein comprise Fc variants with amino acid substitutions, deletions, or additions that alter effector function (e.g., reduce or eliminate). The Fc region of an antibody mediates several important effector functions, e.g., ADCC, ADCP, CDC, etc. Methods for altering effector function by substituting amino acid residues in the Fc region of an antibody to alter the affinity of the antibody for an effector ligand, such as FcyR or complement Clq, are known in the art (see, e.g., EP 388,151 Al; US 564,8260; US 562,4821; Natsume A et al., Cancer Res., 68:3863-3872, 2008; Idusogie EE et al., J. Immunol., 166:2571-2575, 2001; Lazar GA et al., PNAS, 103:4005-4010, 2006; Shields RL et al., JBC, 276:6591-6604, 2001; Stavenhagen JB et al., Cancer Res., 67:8882-8890, 2007; Stavenhagen JB et al., Advan. Enzyme. Regul., 48:152-164, 2008; Alegre ML et al., J. Immunol., 148:3461-3468, 1992; and Kaneko E et al., Biodrugs, 25:1-11, 2011). In some preferred embodiments of the application, the amino acid L235 (EU numbering) on the constant region of the antibody is modified to alter Fc receptor interactions, e.g., L234A or L235A. In other preferred embodiments, the amino acids 234 and 235 on the constant region of the antibody are both modified, e.g., L234A and L235A (L234A / L235A) (EU numbering).

[0029] In some embodiments of the present application, the amino acid of the Fc segment is as shown in SEQ ID NO: 10 or an amino acid sequence modified by substitution, deletion, or addition of one or more amino acids of the amino acid sequence shown in SEQ ID NO: 10 and functionally equivalent or similar.

[0030] In some embodiments of the present application, the bispecific antibody comprises, from N-terminus to C-terminus, an anti-B7-H3 nanobody-targeting CD3 scFv-Fc segment or a targeting CD3 scFv-Fc segment-anti-B7-H3 nanobody.

[0031] In some embodiments of the present application, the anti-B7-H3 nanobody is linked to the targeting CD3 scFv and the Fc segment via a linker peptide.

[0032] In some embodiments of the present application, the amino acid sequence of the linker peptide is (GGGGX)n, X comprises Ser or Ala, and n is a natural number from 1 to 5.

[0033] In some embodiments of the present application, the amino acid sequence of the bispecific antibody is as shown in SEQ ID NO: 6 or SEQ ID NO: 7 or an amino acid sequence modified by substitution, deletion, or addition of one or more amino acids of the amino acid sequence shown in SEQ ID NO: 6-7 and functionally equivalent or similar.

[0034] In a fourth aspect of the present application, a biological material related to the anti-B7-H3 nanobody of the first aspect of the present application, the Fc fusion protein of the anti-B7-H3 nanobody of the second aspect of the present application, or the bispecific antibody of the third aspect of the present application is provided, and the biological material is any one of a1) to a12):

[0035] a1) a nucleic acid molecule encoding the anti-B7-H3 nanobody of the first aspect of the present application, the Fc fusion protein of the anti-B7-H3 nanobody of the second aspect of the present application, or the bispecific antibody of the third aspect of the present application;

[0036] a2) an expression cassette containing the nucleic acid molecule of a1);

[0037] a3) a recombinant vector containing the nucleic acid molecule described in a1);

[0038] a4) a recombinant vector containing the expression cassette described in a2);

[0039] a5) a recombinant microorganism containing the nucleic acid molecule described in a1);

[0040] a6) a recombinant microorganism containing the expression cassette described in a2);

[0041] a7) a recombinant microorganism containing the recombinant vector described in a3);

[0042] a8) a recombinant microorganism containing the recombinant vector described in a4);

[0043] a9) a transgenic animal cell line containing the nucleic acid molecule described in a1);

[0044] a10) a transgenic animal cell line containing the expression cassette described in a2);

[0045] a11) a transgenic animal cell line containing the recombinant vector described in a3);

[0046] a12) A transgenic animal cell line containing the recombinant vector described in a4).

[0047] In some embodiments of the present invention, the transgenic animal cell line does not contain reproductive material.

[0048] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0049] In some embodiments of the present invention, the vector is independently selected from a non-pathogenic viral vector and a viral vector.

[0050] In some embodiments of the present invention, the viral vector comprises at least one of a lentiviral vector, an adenoviral vector, a baculoviral vector, a retroviral vector, a poxvirus vector, a Sendai virus vector, and a herpes simplex virus vector.

[0051] In some embodiments of the present invention, the non-viral vector comprises at least one of a plasmid vector, a cationic polymer vector, chitosan, polyethyleneimine, a nanoparticle vector, and a liposome.

[0052] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a phage, a cosmid, an F cosmid, or an artificial chromosome.

[0053] In some embodiments of the present invention, the plasmid vector may be any plasmid, and the viral vector may be any virus.

[0054] In some embodiments of the present application, the recombinant expression vector uses pET-28a as the original expression vector.

[0055] In some embodiments of the present application, the cell includes a prokaryotic cell, a eukaryotic cell; the cell is not a new plant or animal variety.

[0056] In some embodiments of the present application, the prokaryotic cell includes Escherichia coli, Streptomyces, Bacillus subtilis and other bacteria well known in the art that can be used to express the target protein.

[0057] In some embodiments of the present application, the eukaryotic cell includes at least one of a yeast cell, a mammalian cell, a plant cell and an insect cell.

[0058] In a fifth aspect of the present application, an antibody conjugate is provided, comprising at least one of the anti-B7-H3 nanobody of the first aspect of the present application, the Fc fusion protein of the anti-B7-H3 nanobody of the second aspect of the present application or the bispecific antibody of the third aspect of the present application; and a conjugate moiety.

[0059] In some embodiments of the present application, the conjugate moiety comprises at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme.

[0060] In some embodiments of the present application, the detectable label is selected from a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or any combination thereof.

[0061] In some embodiments of the present application, the conjugate is selected from a fluorescent substance, a chemiluminescent label, a colored substance, a radioisotope, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent or an enzyme capable of generating a detectable product, a radionuclide, a biological toxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a virus particle, a liposome, a nanomagnetic particle, a prodrug activating enzyme, a chemotherapeutic agent (e.g., cisplatin) or any form of nanoparticle, etc.

[0062] In a sixth aspect of the present application, a chimeric antigen receptor is provided, comprising an extracellular antigen binding domain, a spacer domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular antigen binding domain specifically binds to B7-H3 and / or CD3.

[0063] In some embodiments of the present application, the extracellular antigen binding domain is selected from the anti-B7-H3 nanobody of the first aspect of the present application, the Fc fusion protein of the anti-B7-H3 nanobody of the second aspect of the present application or the bispecific antibody of the third aspect of the present application.

[0064] In some embodiments of the application, the transmembrane domain comprises a transmembrane region of a protein selected from the group consisting of alpha, beta or delta chain of T cell receptor, CD3 epsilon, CD3 delta, CD4, CD5, CD8 alpha, CD137, CD152, CD154, PD1.

[0065] In some embodiments of the application, the intracellular signaling domain comprises a primary signaling domain and / or a costimulatory signaling domain.

[0066] In some embodiments of the application, the intracellular signaling domain comprises, in order from N-terminus to C-terminus, a costimulatory signaling domain and a primary signaling domain.

[0067] In some embodiments of the application, the intracellular signaling domain comprises a primary signaling domain and at least one costimulatory signaling domain.

[0068] In some embodiments of the application, the primary signaling domain comprises an immunoreceptor tyrosine-based activation motif.

[0069] In some embodiments of the application, the primary signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of CD3 delta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b or CD66d.

[0070] In some embodiments of the application, the costimulatory signaling domain comprises an intracellular signaling domain of a protein selected from the group consisting of CARD11, CD2, CD7, CD27, CD28, CD30, CD134, 4-1BB, CD150, CD270, CD278 or DAP10.

[0071] In some embodiments of the application, the chimeric antigen receptor further comprises a signal peptide at its N-terminus; preferably the signal peptide comprises a heavy chain signal peptide, a granulocyte-macrophage colony-stimulating factor receptor 2 signal peptide or a CD8 alpha signal peptide.

[0072] In some embodiments of the application, the spacer domain is located between the extracellular antigen binding domain and the transmembrane domain, the spacer domain comprises a hinge domain and / or CH2 and CH3 regions of an immunoglobulin; preferably the hinge domain comprises a hinge region of CD8 alpha, PD1, CD152 or CD154.

[0073] In a seventh aspect of the present application, there is provided a chimeric antigen receptor immune cell expressing the chimeric antigen receptor of the fifth aspect of the present application.

[0074] In some embodiments of the present invention, the immune cells include any one of T cells, immune cells differentiated from stem cells, monocytes, macrophages or NK cells.

[0075] The eighth aspect of the present invention provides the use of the anti-B7-H3 nanobody of the first aspect of the present invention, the Fc fusion protein of the anti-B7-H3 nanobody of the second aspect of the present invention, the bispecific antibody of the third aspect of the present invention, the biomaterial of the fourth aspect of the present invention, the antibody-drug conjugate of the fifth aspect of the present invention, the chimeric antigen receptor of the sixth aspect of the present invention, or the chimeric antigen receptor immune cell of the seventh aspect of the present invention in b1) or b2):

[0076] b1) Preparation of drugs or preparations for the prevention and / or treatment of tumors and immune-related diseases;

[0077] b2) Preparation of reagents or kits for detecting tumors and immune-related diseases.

[0078] In some embodiments of the present invention, the tumor comprises colon cancer, melanoma, breast cancer, ovarian cancer, hepatocellular carcinoma, non-small cell lung cancer, prostate cancer, pancreatic ductal adenocarcinoma, esophageal cancer, bladder cancer, endometrial cancer, or cervical cancer.

[0079] The tumor-treating drugs referred to in the present invention refer to drugs that have the function of inhibiting and / or treating tumors, which may include delaying tumor-related symptoms and / or reducing the severity of these symptoms, further including alleviating existing tumor-associated symptoms and preventing the occurrence of other symptoms, and also including reducing or preventing tumor metastasis, etc.

[0080] In some embodiments of the present invention, the immune-related disease includes a disease associated with abnormal expression of B7-H3.

[0081] The ninth aspect of the present invention provides a product comprising the anti-B7-H3 nanobody of the first aspect of the invention, the Fc fusion protein of the anti-B7-H3 nanobody of the second aspect of the invention, the bispecific antibody of the third aspect of the invention, the biomaterial of the fourth aspect of the invention, the antibody-drug conjugate of the fifth aspect of the invention, the chimeric antigen receptor of the sixth aspect of the invention, or the chimeric antigen receptor immune cell of the seventh aspect of the invention.

[0082] In some embodiments of the present invention, the product includes a reagent, a kit, a chip, or a drug.

[0083] The beneficial effects of the present invention are:

[0084] The present invention uses a nanobody synthetic library and obtains a new specific anti-human B7-H3 nanobody sequence through phage screening technology. The anti-B7-H3 nanobody has specific recognition and binding capabilities to B7-H3 and is expected to be used as a therapeutic antibody for various malignant tumors with high expression of B7-H3 molecules.

[0085] The present invention uses the screened anti-B7-H3 VHH sequence as the antigen binding unit to design and construct a T cell-directed bispecific antibody. In the construction of the bispecific antibody, the present invention uses the OKT3 scFv sequence as the anti-CD3 binding arm template of the bispecific antibody. The flow cytometry experiment verifies that the binding arms at both ends of the recombinant bispecific antibody can effectively bind to the specific antigen on the surface of the target effector cell in a dose-dependent manner. In addition, OKT3 is a T cell agonist antibody. The T cell activation verification experiment confirmed that the bispecific antibody and the recombinant OKT3 monoclonal antibody have similar T cell activation efficacy, but in the presence of target cells, that is, tumor cells that highly express B7-H3, the activation efficacy is significantly enhanced. The T cell activation and killing experimental data verified the T cell activation efficacy and colorectal cancer cell killing efficacy of the bispecific antibody (ID10), and compared with the control antibody, the drug efficacy dose was reduced by at least one order of magnitude.

[0086] The bispecific antibody provided by the present invention has a 2+2 symmetrical, Fab-free format, eliminating the need to consider light and heavy chain mispairing, thereby reducing the difficulty of protein purification. Characterization of the molecular weight and thermal stability of the recombinant antibodies reveals that the bispecific antibody constructed by recombining the VHH with the OKT3scFv-hFc backbone maintains the thermal stability of the Fc format while keeping the overall protein size of the recombinant bispecific antibody close to that of a traditional monoclonal antibody, thus avoiding the added difficulty of penetrating solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 The figure shows the reducing SDS-PAGE profile of the BH1 VHH prokaryotic expression and purification process, where lane 1 is the protein marker, lane 2 is the expression bacterial suspension after IPTG induction, lane 3 is the precipitate obtained by centrifugation after periplasmic protein extraction, lane 4 is the supernatant of periplasmic protein extraction, and lane 5 is the protein eluate purified by nickel gravity column.

[0088] Figure 2 This is the SDS-PAGE profile of the purified recombinant antibodies. Lane 1 is the protein marker, and the remaining lanes are the protein products of the various recombinant antibodies marked in the figure after purification. The left picture is non-reduced SDS-PAGE, and the right picture is reduced SDS-PAGE.

[0089] Figure 3Figure 1 shows the results of the binding ELISA of the recombinant antibody BH1-hFc to human 4lg B7-H3. The left panel shows the results of the ELISA binding of the recombinant antibody BH1-hFc to human B7-H3 at a single dose, with Milk / PBS as the negative control group. The right panel shows the dose-dependent ELISA binding data of the recombinant antibody BH1-hFc to human B7-H3, with 8H9-hFc as the positive control group.

[0090] Figure 4 Figure 2 shows the structural diagram of the recombinant bispecific antibody.

[0091] Figure 5 Figure 3 shows the size exclusion chromatogram of each recombinant antibody.

[0092] Figure 6 Figure 4 shows the protein melting curve of each recombinant antibody.

[0093] Figure 7 Figure 5 shows the dose-dependent binding curve of each recombinant antibody to the surface antigen of HCT116, with commercially available Isotype as the human IgG1 isotype control group and BH1-hFc as the positive control group.

[0094] Figure 8 Figure 6 shows the dose-dependent binding curve of each recombinant antibody to the surface antigen of Jurkat, with commercially available Isotype as the human IgG1 isotype control group and OKT3-hFc as the positive control group.

[0095] Figure 9 Figure 7 shows the upregulation of Jurkat cell surface CD69 by each recombinant antibody. The upper panel shows the expression of CD69 on the surface of Jurkat cells after co-culture of HCT116 and Jurkat cell lines (target-to-effector ratio of 1:10) and detection by flow cytometry. The lower panel shows the expression of CD69 on the surface of Jurkat cells treated with each recombinant antibody at a concentration of 10 nM, with or without co-culture with HCT116. NT refers to the group without target cells, and T refers to the group co-cultured with target effector cells.

[0096] Figure 10 Figure 8 shows the expression of downstream luciferase mediated by NFAT signaling in Jurkat-NFAT-Luci cells activated by each recombinant antibody. The upper panel shows the intracellular luciferase content detected by fluorescence emission after co-culture of HCT116 and Jurkat cell lines (target-to-effector ratio of 1:10). The lower panel shows the intracellular luciferase content of the reporter cell line treated with each recombinant antibody at a concentration of 10 nM, with or without co-culture with HCT116. NT refers to the group without target cells, and T refers to the group co-cultured with target effector cells. In the figure, ns represents no significant difference, ** represents p<0.01, and *** represents p<0.001.

[0097] Figure 11 To human peripheral blood mononuclear cells and HCT116 cell lines after co-culture (target ratio 1:20), by detecting the release of lactate dehydrogenase content of each group of lysed cells, the dose-dependent tumor cell killing curve was calculated. DETAILED DESCRIPTION

[0098] The content of the present application will be further described in detail by specific examples.

[0099] It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application.

[0100] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely. The specific conditions are not marked in the embodiments, which are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used are not marked by the manufacturer, which are all conventional products that can be purchased in the market.

[0101] The features and properties of the present application will be further described in detail in the following examples.

[0102] Example 1 Preparation of recombinant antibody BH1-hFc

[0103] 1. Phage solid-phase screening based on human 4Ig B7-H3

[0104] (1) Construction of 4Ig B7-H3 antigen extracellular domain eukaryotic expression vector

[0105] NheI / XhoI double enzyme digestion linearizes pcDNA3.1(+) vector, and at the same time, the vector (SinoBiological, item number HG11188-ACG) containing human B7-H3 gene is used as a template, a primer is designed to introduce a C-terminal hexa-histidine tag, a stop codon, an N-terminal interleukin 2 signal peptide and N, C-terminal vector homologous arm sequence, and a human B7-H3 extracellular domain gene fragment containing the vector homologous arm is amplified to construct a pcDNA3.1-B7H3-ECD-His tag eukaryotic expression plasmid by homologous recombination. The recombinant plasmid is transformed into DH5α competent bacteria by heat shock method, and positive bacterial plaques are selected by coating ampicillin-resistant solid medium plates for further sequencing verification. Select the correct clone bacterial plaque, use LB liquid medium added with ampicillin antibiotic for amplification, and extract the endotoxin-free plasmid pcDNA3.1-B7H3-ECD-His tag for transfection.

[0106] (2) 293F mammalian cell expression of 4Ig B7-H3 antigen extracellular domain

[0107] Dilute 300 μg polyethylenimine and 100 μg plasmid to be transfected to 5 mL each with serum-free medium OPTI-MEM, mix well, and incubate for 20 min. Then add the transfection mixture to 90 mL 293F cells with a density of about 2-3 x 106 / mL in a good growth state. 6 Put the cell flask after transfection in a 8% CO2, 37°C, 115 rpm incubator for suspension culture for 4 days.

[0108] (3) Purification of 4Ig B7-H3 antigen extracellular domain protein

[0109] On the fourth day after transfection, collect the cell supernatant by freezing centrifugation and filter with 0.22 μm. The whole process of nickel column purification is operated at low temperature on ice, and the specific purification process is as follows:

[0110] Mix the cell transfection supernatant filtrate and His-tag binding buffer (20 mM phosphate, 10 mM imidazole, pH = 7.4) in equal volume, and incubate on ice for 30 min. First, use 5-10 column volumes of 0.22 μm filtered water and His-tag binding buffer solution to equilibrate the Ni NTA beads packed gravity column. Take the filtered supernatant and load it onto the gravity column. After loading, use 5-10 column volumes of binding buffer to wash away the residual impurities in the filler. Finally, use 5-10 column volumes of His-tag elution buffer solution (20 mM phosphate, 500 mM imidazole, pH = 7.4) to elute the protein bound to the affinity filler, and collect the protein eluate.

[0111] The molecular weight of 4Ig B7-H3 antigen extracellular domain is about 70 kD, so use an ultrafiltration tube with a cutoff of 10 kD to change the protein in high concentration imidazole to PBS, concentrate to a final concentration > 2 mg / mL, and store temporarily at 4°C.

[0112] (4) Phage solid-phase screening

[0113] Coat the ELISA plate with human 4Ig B7-H3 protein (coat 5 μg in the first round, and gradually reduce to 2.5 μg and 1 μg in the last two rounds), and stand overnight at 4°C. The next day, discard the liquid in the overnight coated wells, and add 250 μL of 5% milk blocking solution. Meanwhile, pre-incubate the same volume of blocking agent with the library, and incubate on a horizontal shaker at room temperature for 1 h.

[0114] Conduct three rounds of screening:

[0115] In both the first and second rounds, after discarding the blocking buffer, the phage library, pre-incubated with blocking agent, was directly added to the antigen-coated wells at 100 μL / well and incubated on a horizontal shaker for 2 hours at room temperature. In the third round, the phage library was first added to the negative pool (not coated with 4Ig B7-H3 protein, all other conditions were the same) and incubated on a horizontal shaker for 1 hour at room temperature. The supernatant was then aspirated and added to the positive pool and incubated on a horizontal shaker for 2 hours at room temperature. Unbound phage from the positive pool were recovered and each well was washed 15 times with PBST (containing 0.1% Tween-20) and then PBS (Phosphate-Buffered Saline). The wash buffer was discarded, and 35 μL of 100 mM Glycine-HCl solution (pH = 2.5) was added to each well for elution at room temperature for 10 minutes. Elution was terminated by adding 15 μL of 1M Tris-HCl (pH = 7.5), and the phage library eluate was collected. Take half of the volume of phage eluate to infect the TG1 strain in the logarithmic growth phase and spread it on 2×YT solid medium. Collect the amplified bacterial library and inoculate it into 2×YT medium supplemented with ampicillin antibiotics to make the initial OD 600 = 0.1, 250 rpm, 37 °C and continue to culture until OD 600 Reach 0.5-0.6. Add 20-fold the number of helper phage M13K07 and incubate at 250 rpm and 37°C for 30 min. Add kanamycin to a final concentration of 50 μg / mL and IPTG to a final concentration of 0.2 mM and amplify overnight at 30°C and 250 rpm. The next day, centrifuge the overnight culture, collect the supernatant, add 1 / 4 volume of 20% PEG 8000 / 2.5M NaCl solution, mix thoroughly, and let it stand on ice for 30 min. After centrifugation, discard the supernatant and resuspend the pellet in 1 mL of PBS. Centrifuge the resuspension at 13,000 rpm and 4°C for 20 min. Collect the supernatant and add 1 / 4 volume of 20% PEG 8000 / 2.5M NaCl solution again. Mix thoroughly and let it stand on ice for 10 min. Centrifuge at 13,000 rpm and 4°C for 10 min, discard the supernatant, resuspend the pellet in 1 mL of PBS, and centrifuge at 13,000 rpm and 4°C for 2 min. The supernatant after centrifugation is the new phage library, which can be used for titer determination and a new round of screening.

[0116] 2. Monoclonal phage ELISA

[0117] The sample wells of the ELISA plate were coated with human 4Ig B7-H3, and the background control wells were coated with milk, at a concentration of 250 ng per well. The plates were incubated overnight at 4°C. The overnight coating buffer was discarded, and 200 μL of 3% BSA in PBS was added to each well for blocking. The plates were shaken on a horizontal shaker at room temperature for 1 hour. The blocking buffer was discarded, and each well was washed three times with PBST. Monoclonal phage amplified overnight were centrifuged, and the supernatant containing phage was collected. 130 μL of blocking agent was added to each well, followed by 70 μL of phage supernatant. The plates were incubated on a horizontal shaker at room temperature for 2 hours. The plate liquid was discarded, and the plates were washed five times with PBST. 50 μL of 0.4 μg / mL HRP-conjugated mouse M13 Bacteriophage antibody (Sino Biological, Cat. No. 11973-MM05T-H) was added to each well. The plates were shaken on a horizontal shaker at room temperature for 1 hour. The plate liquid was discarded, and the plates were washed five times with PBST. Add 50 μL of TMB colorimetric solution to each well and develop in the dark for 2–3 minutes. Then, add 50 μL of 1 M HCl solution to terminate the reaction. Detect absorbance at 450 nm using a microplate reader. Single clones with a clear binding signal in the sample wells but a weak Milk binding signal were sequenced. The sequence of the positive clone was identified, indicating a clone that specifically binds to human B7-H3 and was designated BH1.

[0118] 3. Prokaryotic expression and purification of BH1 nanobody

[0119] (1) Construction of expression plasmid

[0120] The pET-22b(+) vector was linearized using NdeI / XhoI double digestion. A synthetic gene with a C-terminal 6xHis tag was synthesized using BH1 as the VHH (variable domain of heavy chain antibody) building block. This gene served as a template for amplifying the BH1 VHH gene fragment containing the vector homology arms. The pET-22b(+)-BH1 VHH-His tag prokaryotic expression plasmid was constructed by homologous recombination. The recombinant plasmid was heat-shocked and transformed into a DH5α competent strain. Positive plaques were screened on ampicillin-resistant solid medium plates and further verified by sequencing. Correct colonies were selected, plasmids extracted, and heat-shocked into a BL21(DE3) competent strain. Plates were plated on ampicillin-resistant solid medium plates and cultured overnight. Single colonies that emerged overnight were picked and inoculated into LB liquid medium supplemented with ampicillin to amplify the expression strain.

[0121] (2) IPTG-induced prokaryotic expression

[0122] The BH1 VHH expression bacterial solution was inoculated into 1 L of LB liquid medium supplemented with ampicillin antibiotics and cultured at 37°C and 220 rpm. The OD value of the bacterial solution was monitored during the process. 600Value. Waiting for OD 600 After the value reaches 0.6-0.8, add IPTG to a final concentration of 0.5mM and incubate at 30℃ and 220rpm for 5h. After the incubation, centrifuge at 4500rpm for 15min to collect the bacterial precipitate. Add 4mL TES buffer (50mM Tris-HCl, 1mM EDTA, 20% sucrose, pH=8.0) per 1g of bacteria, resuspend the bacteria, and let it stand on ice for 30min. Centrifuge at 4500rpm for 1h, resuspend the precipitate with 5mM MgCl2 equal to the volume of TES used above, and let it stand on ice for 15min. Centrifuge at 4500rpm for 45min, collect the supernatant, and dialyze into His-tag binding buffer (20mM phosphate, 10mM imidazole, pH=7.4) in a chromatography cabinet at 4℃.

[0123] (3) BH1 VHH protein purification

[0124] The entire nickel column purification process was performed on ice at low temperature. The specific purification process was consistent with the purification of 4Ig B7-H3 antigen extracellular domain protein. SDS-PAGE gel electrophoresis verified the separation and purification effect of BH1 VHH protein ( Figure 1 Based on SDS-PAGE results, the molecular weight of BH1 VHH is approximately 25 kD. Using a 3 kD cutoff ultrafiltration tube, the protein solution in high-concentration imidazole was exchanged into PBS and concentrated to a final concentration >2 mg / mL. The protein was then snap-frozen in liquid nitrogen and stored at -80°C until ready for use.

[0125] The amino acid sequence of BH1 VHH is shown in SEQ ID NO: 1.

[0126] QVQLQESGGGLVQAGGSLRLSCAASGSISQIPSMGWYRQAPGKEREFVASINNGS ITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVKGVFGNWYIYWGQG TQVTVSS(SEQ ID NO:1);

[0127] CDR1 of BH1 VHH: GSISQIPSM (SEQ ID NO: 2);

[0128] CDR2 of BH1 VHH: EFVASINNGSITNY (SEQ ID NO: 3);

[0129] CDR3 of BH1 VHH: AVKGVFGNWYIY (SEQ ID NO: 4).

[0130] 4. Construction of recombinant antibody BH1-hFc expression plasmid and expression and purification in 293F mammalian system

[0131] (1) Construction of expression plasmid

[0132] EcoRV / BstEII double enzyme digestion linearizes the pFUSE-hIgG1-Fc2-His tag vector, and simultaneously amplifies the BH1 VHH gene fragment containing the vector homologous arm with a synthetic gene with a six-histidine tag at the C-terminus as a template, to construct a pFUSE-BH1-hFc-His tag eukaryotic expression plasmid by homologous recombination. The recombinant plasmid is transformed into a DH5α competent strain by heat shock method, and positive bacterial plaques are selected by coating a solid culture medium resistant to blasticidin, and further verified by sequencing. The correct clone bacterial plaque is selected, and the LB liquid medium added with blasticidin antibiotic is used for amplification, and the endotoxin-free plasmid pFUSE-BH1-hFc-His tag to be transfected is extracted.

[0133] (2) Expression of recombinant antibody BH1-hFc by 293F mammalian expression system

[0134] The serum-free medium OPTI-MEM is used to dilute 300 μg of polyethyleneimine and 100 μg of the plasmid to be transfected to 5 mL, respectively, mixed, and incubated for 20 min, and then the transfection mixed reagent is added to 90 mL of 293F cells with a growth state of about 2-3 x 10 6 After transfection, the cell shake flask is placed in an incubator at 8% CO2, 37°C, and 115 rpm for 4 days of suspension culture.

[0135] (3) Purification of recombinant antibody

[0136] On the 4th day after transfection, the cell supernatant is collected by freezing centrifugation, and 0.22 μm filtration. After filtration, the supernatant is mixed with an equal volume of His-tag binding buffer, and then incubated on ice for 30 min. The whole process of nickel column purification is operated at low temperature on ice, and the specific purification process is consistent with the operation of 4Ig B7-H3 antigen extracellular domain protein purification.

[0137] SDS-PAGE gel electrophoresis verifies the protein separation and purification effect Figure 2 ). According to the SDS-PAGE result, the molecular weight of BH1-hFc is about 80 kD. The protein in high-concentration imidazole is exchanged to PBS by using an ultrafiltration tube with a cut-off of 10 kD, concentrated to a final concentration of >2 mg / mL, and temporarily stored at 4°C.

[0138] The amino acid sequence of the recombinant antibody BH1-hFc is shown in SEQ ID NO: 5:

[0139] QVQLQESGGGLVQAGGSLRLSCAASGSISQIPSMGWYRQAPGKEREFVASINNGSITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVKGVFGNWYIYWGQGTQVTVSSRSDKTYTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (SEQ ID NO: 5).

[0140] 5. ELISA detection of recombinant antibody

[0141] (1) Single dose recombinant antibody ELISA

[0142] ELISA plate sample wells were coated with 250 ng of 4lg B7-H3 ectodomain protein, negative control wells were coated with 250 ng of Milk, and PBS was used as a background control. Each group was set up in duplicate, and the plates were incubated at 4°C overnight. The next day, the liquid in the ELISA plate was discarded, and 200 μL of 3% BSA was added for 2 h of incubation at room temperature on a horizontal shaker. After incubation, the liquid in the ELISA plate was discarded, and each well was washed 3 times with 200 μL of PBST. Recombinant antibody BH1-hFc was diluted to 50 nM using 1% BSA in PBS as the diluent, and 100 μL was added to each sample well and control well. The plate was incubated at room temperature on a horizontal shaker for 1 h. After incubation, the liquid was discarded, and each well was washed 5 times with 200 μL of PBST. Each well was then added with 50 μL of 5 μg / mL HRP-conjugated goat anti-human IgG-Fc antibody (Sino Biological, Cat. No. SSA001), and the plate was incubated at room temperature on a horizontal shaker for 1 h. The liquid was discarded, and each well was washed 5 times with 200 μL of PBST. The wash liquid was discarded, and the ELISA plate was spun dry. Each well was then added with 50 μL of TMB color developing solution, and the plate was developed in the dark for about 5 min. The reaction was stopped by adding 50 μL of 1 M HC1 solution, and the absorbance at 450 nM was detected using an ELISA reader.

[0143] (2) Dose-dependent ELISA of recombinant antibody

[0144] ELISA plate sample wells were coated with 250 ng of human 4Ig B7-H3 extracellular domain protein in duplicate, and the plates were incubated at 4°C overnight. The next day, the plate was discarded and each well was blocked with 200 μL of 5% milk at room temperature for 2 hours on a horizontal shaker. After incubation, the plate was discarded and each well was washed three times with 200 μL of PBST. PBS containing 1% milk was used as a diluent to dilute BH1-hFc and the positive antibody 8H9-hFc (i.e., the 8H9 scFv sequence that specifically binds to B7-H3, disclosed in "hmed M, Cheng M, Zhao Q, et al. J Biol Chem. 2015; 290(50): 30018-30029") to prepare a gradient concentration of 100nM, 33nM, 10nM, 3.3nM, 1nM, 0.33nM, 0.1nM, 0.03nM, and 0.01nM antibody solution. 100μL of antibody dilution was added to each well and incubated on a horizontal shaker at room temperature for 1 hour. The liquid was discarded and the cells were washed five times with 200μL of PBST solution. 50μL of HRP-conjugated goat anti-human IgG-Fc antibody (Sino Biological, Cat. No.: SSA001) was added to each well and incubated on a horizontal shaker at room temperature for 1 hour. Discard the liquid and wash each well five times with 200 μL PBST. Discard the wash solution, spin dry the plate, and add 50 μL TMB colorimetric solution to each well. Develop in the dark for approximately 5 minutes, then terminate the reaction by adding 50 μL 1 M HCl solution. Measure the absorbance at a wavelength of 450 nM using a microplate reader.

[0145] The data were processed using Prsim nonlinear fitting to obtain the half effective concentration (EC) of BH1-hFc and 8H9-hFc for 4Ig B7-H3 antigen binding. 50 ) were 0.32nM and 0.15nM ( Figure 3 ).

[0146] Example 2 Construction of recombinant bispecific antibody expression vector and expression and purification in 293F mammalian system

[0147] (1) Construction of expression plasmid

[0148] First, according to the company to synthesize gene amplification containing pFUSE-hIgG1-Fc2-His tag vector homologous arm OKT3 scFv fragment (has been disclosed in U.S. patent US-7635472-B2), by homologous recombination to construct pFUSE-OKT3-hFc-Histag plasmid, and then through the multi-point mutation to achieve Fc skeleton LALA mutation (L234A and L235A) to silence its effector function. On the basis of pFUSE-OKT3-hFc-LALA-His tag plasmid, by whole plasmid PCR technology in the N terminal of the original plasmid OKT3 scFv sequence or the C terminal of the Fc skeleton sequence open loop, by homologous recombination to access BH1 VHH base sequence, construct recombinant pFUSE-BH1-OKT3-hFc-LALA-His tag (corresponding to the expression of bispecific antibody BH1-OKT3-hFc named ID9) and pFUSE-OKT3-hFc-LALA-BH1-His tag expression plasmid (corresponding to the expression of bispecific antibody OKT3-hFc-BH1 named ID10). After Dpn I digestion of template plasmid, the recombination plasmid was transformed into DH5α competent strain by heat shock method, and the positive bacterial plaque was selected by coating the solid culture medium plate resistant to blasticidin, and further sequencing verification. Select the correct clone bacterial plaque, use the LB liquid medium added with blasticidin antibiotic for amplification, and extract the endotoxin-free transfection plasmid pFUSE-BH1-OKT3-hFc-LALA-His tag and pFUSE-OKT3-hFc-LALA-BH1-His tag.

[0149] The structural diagram of the recombinant bispecific antibody is shown in Figure 4

[0150] (2) 293F mammalian expression system for expressing recombinant antibodies ID9 and ID10

[0151] The specific process operation of 293F transfection expression is consistent with that of 4Ig B7-H3 extracellular domain protein transfection expression.

[0152] (3) Nickel column purification of recombinant bispecific antibody

[0153] The specific process operation of nickel column purification is consistent with that of 4Ig B7-H3 extracellular domain protein purification.

[0154] SDS-PAGE gel electrophoresis was used to verify the protein separation and purification effect. According to the SDS-PAGE result, the molecular weight of ID9 and ID10 was about 140 kD. The protein stored in high concentration imidazole was exchanged to PBS by using ultrafiltration tube with a cut-off of 30 kD, concentrated to a final concentration of >2 mg / mL, and temporarily stored at 4°C.

[0155] ​(4) Purification of recombinant bispecific antibodies by size exclusion chromatography

[0156] Operate the protein purifier and equilibrate the gel filtration column with two column volumes of 0.22 μm filtered water and PBS buffer, respectively. Centrifuge the antibody concentrate at 10,000 g for 10 min at 4°C. Load the sample in two 500 μL sample loops. Carefully draw approximately 250 μL each time using a 1 mL syringe. After removing any bubbles, slowly inject the sample into the sample loop. Set the program parameters as follows:

[0157] a. Flow rate: 0.5 mL / min; b. The liquid path passes through the column; c. The effluent flows out of the collection tube; d. The flow rate is controlled by 2 MPa column head pressure; e. The liquid path pauses after flowing through one column volume (24 mL); f. Perform the injection.

[0158] Observe the retention volume and number of elution peaks of each protein to confirm the purity of the target protein. Collect the effluent during the protein elution process, concentrate it using an ultrafiltration tube to a final concentration of >2 mg / mL, and temporarily store it at 4°C.

[0159] The size exclusion chromatograms of each recombinant bispecific antibody are shown in Figure 2. Figure 5 shown.

[0160] The amino acid sequence of the recombinant bispecific antibody ID9 is shown in SEQ ID NO: 6, and the amino acid sequence of the recombinant bispecific antibody ID10 is shown in SEQ ID NO: 7.

[0161] QVQLQESGGGLVQAGGSLRLSCAASGSISQIPSMGWYRQAPGKEREFVASINNGSITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVKGVFGNWYIYWGQGTQVTVSSGGGGSGGGGSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSRSDKTYTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH (SEQ ID NO: 6).

[0162] DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKSRSDKTYTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSQVQLQESGGGLVQAGGSLRLSCAASGSISQIPSMGWYRQAPGKEREFVASINNGSITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVKGVFGNWYIYWGQGTQVTVSSHHHHHH (SEQ ID NO: 7).

[0163] Thermal stability characterization of recombinant bispecific antibodies of Example 3

[0164] Dilute 5000x SYPRO Orange dye to 25x with PBS, and dilute 0.1 pg^L OKT3-hFc, ID9 and ID10 protein solutions with PBS. Add 45 pL of protein solution and control PBS to each well of a qPCR clear-well plate, then add 5 pL of 25x dye and mix, seal the plate. Centrifuge the plate briefly, then analyze using a quantitative fluorescent PCR machine with 485 nm excitation and 530 nm emission filters, set to continuously increase the temperature from 20.0 °C to 99.0 °C at a rate of 0.3 °C per second. After the experiment is complete, export the melting curve analysis.

[0165] The protein melting curves of each recombinant antibody are shown in Figure 6 OKT3-hFc, ID9 and ID10 have a melting temperature Tm of about 74 °C.

[0166] Example 4 Dose-dependent binding of recombinant bispecific antibody to target positive cell lines

[0167] Jurkat cells and HCT116 cells after digestion were suspended for cell counting and viability analysis, respectively, 10 6 single cells were aliquoted in 1.5 mL centrifuge tubes, 1500 rpm centrifugation for 5 min, and the supernatant was discarded. Each cell pellet was resuspended with 100 μL of PBS containing 10% FBS blocking solution, and incubated on ice for 30 min, 1500 rpm centrifugation for 5 min, and the supernatant was discarded. A PBS solution containing 3% BSA was used as an antibody diluent to prepare 1000 nM, 300 nM, 100 nM, 30 nM, 10 nM, 3 nM, 1 nM, 0.3 nM, 0.1 nM, 0.03 nM gradient concentration antibody solution. Each cell pellet was resuspended with the corresponding labeled concentration of antibody solution, and incubated on ice for 30 min, 1500 rpm centrifugation for 5 min. The supernatant was discarded. Each cell pellet was resuspended with 500 μL of PBS, 1500 rpm centrifugation for 5 min, and the supernatant was discarded. The cell pellet was washed twice with PBS. A PBS solution containing 3% BSA was used as a diluent to prepare a 5 μg / mL FITC F(ab')2 Goat anti-human IgG Fcγ Antibody (BioLegend, Cat. No. 398006) fluorescent antibody diluent. Each cell pellet was resuspended with 100 μL of fluorescent antibody diluent, incubated on ice for 30 min, 1500 rpm centrifugation for 5 min, and the supernatant was discarded. Each cell pellet was resuspended with 500 μL of PBS, 1500 rpm centrifugation for 5 min, and the cell pellet was washed twice with PBS. The FITC channel signal was detected by flow cytometry, and the data was processed jointly by Flowjo and Prism.

[0168] The results show that the recombinant bispecific antibody dose-dependently binds to the target positive cell line. The data was processed by Prism non-linear fitting, and the EC 50 of ID9, ID10 and BH1-hFc for HCT116 surface B7-H3 antigen binding were 35.7 nM, 216.6 nM and 33.8 nM Figure 7 , respectively, and the EC 50 of ID9, ID10 and OKT3-hFc for Jurkat surface CD3 antigen binding were 0.38 nM, 0.17 nM and 0.17 nM Figure 8 , respectively.

[0169] Example 5 Antibody-mediated T cell activation verification

[0170] HCT116 was inoculated in a 96-well cell plate, 2 x 10 4Cells were cultured in a 37°C incubator for 24 hours. RPMI1640 complete medium containing 10% FBS was used as an antibody diluent to prepare a gradient of antibody solutions with concentrations of 10nM, 1nM, 100pM, 10pM, 1pM, and 0.1pM. The old medium was discarded from the 96-well plate, and 50μL of the antibody diluent was added to each well. The plates were incubated at 37°C for 30 minutes. The pre-incubated 96-well plate was removed and 50μL of Jurkat cell suspension was inoculated into each well. 2×10 5 Cells were cultured for 24 hours at 37°C under 5% CO2. After 24 hours, the 96-well plate was removed, the cell suspension was aspirated as much as possible, and each well was rinsed by gently pipetting with PBS. The cell suspension and rinse solution were combined and centrifuged at 1500 rpm for 5 minutes. The cells were then collected and washed with 500 μL of PBS. The cells were centrifuged again at 1500 rpm for 5 minutes, and the supernatant was discarded. A 5 μg / mL FITC anti-CD69 antibody (Sino Biological, Cat. No. 11150-MM06-F) incubation solution was prepared using PBS containing 3% BSA as the diluent. 100 μL of the fluorescently labeled antibody dilution was added to each tube of cell pellet and resuspended. The cells were incubated on ice for 30 minutes. The cells were centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the cells were washed once with PBS. Finally, the cells were resuspended in 500 μL of PBS. The FITC channel signal was detected by flow cytometry, and the data were processed using FlowJo and Prism.

[0171] The data were processed by Prsim nonlinear fitting to obtain the ECs of T cell activation determined by ID9, ID10 and OKT3-hFc through the upregulation of T cell surface activation marker CD69. 50 0.15nM, 0.01nM and 0.07nM ( Figure 9 ). In addition, by comparing the expression of CD69 antigen on the surface of Jurkat cells in the highest concentration treatment groups under the conditions of co-culture with HCT116 or not, it can be found that when target cells are not present, the dual antibody group and the positive monoclonal antibody group have similar effects in activating T cells. However, when target cells are present, due to the dual-arm effect of ID10, the activation ability of ID10 for T cells is significantly enhanced. However, due to the steric interference of the BH1 and OKT3 binding domains, ID9 cannot bind to specific targets at the same time, and its activation ability for T cells is not as good as ID10 ( Figure 9 ).

[0172] Example 6: Jurkat NFAT luciferase reporter cell assay for T cell activation

[0173] HCT116 were inoculated in a 96-well plate with a white bottom and opaque surface, with 2 × 10 4cells / mL, 37°C incubator for 24h. RPMI1640 complete medium containing 10% FBS as antibody diluent to prepare 10nM, 1nM, 100pM, 10pM, 1pM, 0.1pM gradient concentration of antibody solution. 96-well plate discarded old culture medium, add 50μL antibody diluent 37°C incubation for 30min. Take out the pre-incubated 96-well plate, inoculate Jurkat-NFAT-Luciferase, 50μL per well, 2×10 5 cells / mL, 37°C incubator for 24h. RPMI1640 complete medium containing 10% FBS as antibody diluent to prepare 10nM, 1nM, 100pM, 10pM, 1pM, 0.1pM gradient concentration of antibody solution. 96-well plate discarded old culture medium, add 50μL antibody diluent 37°C incubation for 30min. Take out the pre-incubated 96-well plate, inoculate Jurkat-NFAT-Luciferase, 50μL per well, 2×10 TM cells / mL, 37°C incubator for 24h. RPMI1640 complete medium containing 10% FBS as antibody diluent to prepare 10nM, 1nM, 100pM, 10pM, 1pM, 0.1pM gradient concentration of antibody solution. 96-well plate discarded old culture medium, add 50μL antibody diluent 37°C incubation for 30min. Take out the pre-incubated 96-well plate, inoculate Jurkat-NFAT-Luciferase, 50μL per well, 2×10

[0174] Prsim nonlinear fitting process data, get ID9, ID10 and OKT3-hFc through luciferase reporter system determined T cell activation EC 50 in turn 0.57nM, 0.02nM and 0.25nM( Figure 10 ).

[0175] Example 7 T cell-mediated tumor cell killing verification

[0176] 96 cell hole plate inoculates HCT116, 1×10 4 cells / mL, 37°C incubator for 24h. RPMI1640 complete medium containing 10% FBS as antibody diluent to prepare 10nM, 1nM, 100pM, 10pM, 1pM, 0.1pM gradient concentration of antibody solution. 96-well plate discarded old culture medium, add 50μL antibody diluent 37°C incubation for 30min. Take out the pre-incubated 96-well plate, inoculate Jurkat-NFAT-Luciferase, 50μL per well, 2×10 6 cells / mL, 37°C incubator for 24h. RPMI1640 complete medium containing 10% FBS as antibody diluent to prepare 10nM, 1nM, 100pM, 10pM, 1pM, 0.1pM gradient concentration of antibody solution. 96-well plate discarded old culture medium, add 50μL antibody diluent 37°C incubation for 30min. Take out the pre-incubated 96-well plate, inoculate Jurkat-NFAT-Luciferase, 50μL per well, 2×10 5Effector cells were cultured in a 37°C incubator for 36 hours. At the designated 36-hour detection point, the cell culture plate was centrifuged at 400g for 5 minutes. 120 μL of the supernatant was removed and added to a new 96-well plate. 60 μL of freshly prepared LDH assay working solution was added to each well. The plate was incubated on a horizontal shaker at room temperature in the dark for 30 minutes. The absorbance was measured at 490 nm using a full-band microplate reader, with 600 nm as the reference wavelength. Assuming a 100% cell-killing efficacy of the 10 nM ID10 drug group, the dose-dependent cell-killing efficacy of each group was evaluated according to the cytotoxicity normalization formula.

[0177] The cytotoxicity normalization formula is as follows:

[0178]

[0179] The data were processed by Prsim nonlinear fitting to obtain the normalized cytotoxic EC values ​​of ID9, ID10 and OKT3-hFc. 50 35.9pM, 1.0pM and 40.8nM ( Figure 11 ).

[0180] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. An anti-B7-H3 nanobody, comprising complementary determining regions (CDRs), wherein the complementary determining regions (CDRs) comprise complementary determining regions (CDR1), complementary determining regions (CDR2), and complementary determining regions (CDR3), wherein: The amino acid sequence of the complementary determining region CDR1 is shown in SEQ ID NO: 2; The amino acid sequence of the complementary determining region CDR2 is shown in SEQ ID NO: 3; The amino acid sequence of the complementary determining region CDR3 is shown in SEQ ID NO:

4.

2. The anti-B7-H3 nanobody according to claim 1, characterized in that The amino acid sequence of the anti-B7-H3 nanobody is as shown in SEQ ID NO: 1 or an amino acid sequence modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 and having the same or similar function.

3. An anti-B7-H3 nanobody Fc fusion protein, comprising the anti-B7-H3 nanobody according to claim 1 or 2 and an Fc segment.

4. The nanobody Fc fusion protein according to claim 3, characterized in that The amino acid sequence of the Fc segment is as shown in SEQ ID NO: 8, or an amino acid sequence that is modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 8 and has the same or similar function.

5. The nanobody Fc fusion protein according to claim 3, characterized in that The amino acid sequence of the Fc fusion protein of the anti-B7-H3 nanobody is as shown in SEQ ID NO: 5, or an amino acid sequence that is modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 5 and has the same or similar function.

6. An anti-B7-H3×CD3 bispecific antibody targeting B7-H3 and CD3, characterized in that: The bispecific antibody has a symmetrical structure, comprising the anti-B7-H3 nanobody according to claim 1 or 2, a CD3-targeting scFv and an Fc segment; the amino acid sequence of the CD3-targeting scFv is shown in SEQ ID NO:

9.

7. The bispecific antibody according to claim 6, characterized in that The amino acids of the Fc segment are as shown in SEQ ID NO: 10 or an amino acid sequence modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 10 and having the same or similar functions.

8. The bispecific antibody according to claim 6, characterized in that The bispecific antibody comprises, from N-terminus to C-terminus, an anti-B7-H3 nanobody-a scFv-Fc segment targeting CD3 or a scFv-Fc segment targeting CD3-an anti-B7-H3 nanobody.

9. The bispecific antibody according to claim 8, characterized in that The anti-B7-H3 nanobody is linked to the CD3-targeting scFv and Fc segment via a connecting peptide.

10. The bispecific antibody according to claim 9, characterized in that The amino acid sequence of the connecting peptide is (GGGGX)n, X includes Ser or Ala, and n is a natural number of 1-5.

11. The bispecific antibody according to claim 10, characterized in that The amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 6 or SEQ ID NO:

7.

12. A biomaterial related to the anti-B7-H3 Nanobody according to claim 1 or 2, the Fc fusion protein of the anti-B7-H3 Nanobody according to any one of claims 3 to 5, or the bispecific antibody according to any one of claims 6 to 11, wherein the biomaterial is any one of a1) to a12): a1) a nucleic acid molecule encoding the anti-B7-H3 Nanobody according to claim 1 or 2, the Fc fusion protein of the anti-B7-H3 Nanobody according to any one of claims 3 to 5, or the bispecific antibody according to any one of claims 6 to 11; a2) an expression cassette containing the nucleic acid molecule described in a1); a3) a recombinant vector containing the nucleic acid molecule described in a1); a4) a recombinant vector containing the expression cassette described in a2); a5) a recombinant microorganism containing the nucleic acid molecule described in a1); a6) a recombinant microorganism containing the expression cassette described in a2); a7) a recombinant microorganism containing the recombinant vector described in a3); a8) a recombinant microorganism containing the recombinant vector described in a4); a9) a transgenic animal cell line containing the nucleic acid molecule described in a1); a10) a transgenic animal cell line containing the expression cassette described in a2); a11) a transgenic animal cell line containing the recombinant vector described in a3); a12) A transgenic animal cell line containing the recombinant vector described in a4).

13. An antibody-drug conjugate comprising at least one of the anti-B7-H3 nanobody according to claim 1 or 2, the Fc fusion protein of the anti-B7-H3 nanobody according to any one of claims 3 to 5, or the bispecific antibody according to any one of claims 6 to 11; and a conjugated moiety.

14. The antibody-drug conjugate according to claim 13, characterized in that: The coupling portion comprises at least one of a detectable label, a drug, a toxin, and a cytokine.

15. A chimeric antigen receptor comprising an extracellular antigen binding domain, a spacer domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen binding domain specifically binds to B7-H3 and / or CD3; The extracellular antigen binding domain is selected from the anti-B7-H3 nanobody according to claim 1 or 2, the Fc fusion protein of the anti-B7-H3 nanobody according to any one of claims 3 to 5, or the bispecific antibody according to any one of claims 6 to 11. 16 . A chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the chimeric antigen receptor of claim 15 .

17. The chimeric antigen receptor immune cell according to claim 16, characterized in that The immune cells include any one of T cells, monocytes, macrophages or NK cells.

18. Use of the anti-B7-H3 nanobody according to claim 1 or 2, the Fc fusion protein of the anti-B7-H3 nanobody according to any one of claims 3 to 5, the bispecific antibody according to any one of claims 6 to 11, the biomaterial according to claim 12, the antibody-drug conjugate according to any one of claims 13 to 14, the chimeric antigen receptor according to claim 15, or the chimeric antigen receptor immune cell according to any one of claims 16 to 17 in b1) or b2): b1) Preparation of drugs or preparations for the prevention and / or treatment of colorectal cancer; b2) Prepare reagents or kits for detecting colorectal cancer.

19. A product comprising the anti-B7-H3 nanobody of claim 1 or 2, the Fc fusion protein of the anti-B7-H3 nanobody of any one of claims 3 to 5, the bispecific antibody of any one of claims 6 to 11, the biomaterial of claim 12, the antibody-drug conjugate of any one of claims 13 to 14, the chimeric antigen receptor of claim 15, or the chimeric antigen receptor immune cell of any one of claims 16 to 17.

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