Anti-B7-H3 nano antibody, symmetric anti-B7-H3*CD3 bispecific antibody as well as preparation method and application of symmetric anti-B7-H3*CD3 bispecific antibody

Abstract

The invention belongs to the technical field of biological medicine, discloses an anti-B7-H3 nano antibody, a symmetric anti-B7-H3 * CD3 bispecific antibody and a preparation method and application thereof, and particularly discloses the anti-B7-H3 nano antibody which comprises a complementary determining region CDR, and the complementary determining region CDR comprises a complementary determining region CDR1, a complementary determining region CDR2 and a complementary determining region CDR3. According to the invention, a nano antibody library is used, a brand new specific anti-human B7-H3 nano antibody sequence is obtained through a phage display screening technology, and the anti-B7-H3 nano antibody has specific recognition and binding capacity to B7-H3 and is expected to be used as a therapeutic antibody for various malignant tumors with high expression of B7-H3 molecules.

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 immune checkpoint protein B7 family. Its basic structure includes a pair of IgV-like and IgC-like immunoglobulin domains, a transmembrane region, and a highly diverse short cytoplasmic tail. Multiple immunostaining results show that B7-H3 is overexpressed in tumor tissues, while its expression is low in normal tissues. Different cancer types may have different B7-H3 localization spectra. In addition, B7-H3 expression has also been detected in tumor-associated vasculature and fibroblasts in most colorectal cancer specimens. Targeting tumor blood vessels is expected to improve the penetration of immune effector cells into solid tumors, thereby potentially overcoming the main obstacle of T cell-based immunotherapy. In recent years, various cancer treatment strategies targeting this target have been developed one after another, such as monoclonal antibodies, bispecific antibodies (Bispecific antibody, BsAb, referred to as "double antibody"), antibody-drug conjugates (antibody-drug conjugate, ADC), and chimeric antigen receptor T cells (Chimeric antigen receptor T cell, CAR-T). Currently, there are two bispecific antibody products that are developing rapidly against B7-H3, namely, 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 of heavy chain antibody (VHH), are antigen-binding units derived from camel heavy chain antibodies HCAbs (heavy-chain antibodies). Although they lack light chains, nanobodies can still bind antigens with high specificity and affinity. At the same time, nanobodies can support drug development and inhalation administration due to their excellent stability and good solubility, and their unique advantages of low immunogenicity risk and small size can also be considered for the development of penetration therapy for solid tumors. They have gradually attracted the interest of researchers and are designed to construct multi-target antibodies with improved binding and new functions. The sequence discovery of antigen-specific nanobodies can be screened through immune antibody libraries and synthetic antibody libraries. Among them, the preparation process 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 an earlier target direction 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 functional cells to form a receptor complex to activate T cells, shorten the physical distance between 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 quickly cleared from plasma and require continuous infusion to maintain efficacy. If the bispecific antibody format is designed to incorporate the Fc (fragmentcrystallizable) region, the half-life of the drug can be extended through the FcRn (The neonatal Fc receptor for IgG, neonatal receptor)-mediated endocytosis recycling mechanism. At the same time, based on the consideration that the effector function of the Fc region is related to adverse safety events, further modification of the Fc region is required.

[0005] Anti-B7-H3 BsAbs are mainly conjugates of BITE structural molecules and anti-CD3 antibodies and anti-B7-H3 antibodies prepared by chemical coupling methods. BITE structural antibodies have a short half-life, while antibodies prepared by chemical coupling methods have large molecular weights and strong immunogenicity. Both types of structural antibodies have limitations in subsequent applications. Therefore, it is of great practical significance to screen and prepare new anti-B7-H3 specific nanoantibodies and design and construct new anti-B7-H3×CD3 bispecific antibodies. Summary of the invention

[0006] In view of the deficiencies of anti-B7-H3 antibody related products and bispecific antibody products for solid tumor treatment in the prior art, an anti-B7-H3 antibody screening method and the design of a recombinant bispecific antibody are provided. The present invention adopts phage display screening of synthetic nanoantibody library technology, and the obtained BH1 nanoantibody sequence can be used for the development of T cell redirection bispecific antibody drugs, which is expected to achieve better tumor treatment efficacy at a low dose.

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

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

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

[0010] The purpose of the fourth aspect of the present invention is 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 invention aims to provide a product.

[0016] In order to achieve the above object, the technical solution adopted by the present invention is:

[0017] The present invention first uses a self-built synthetic nanoantibody library to perform phage display to screen specific nanoantibodies against human B7-H3. The enriched phage is verified, and a clone that specifically binds to human B7-H3 is identified and named BH1. Furthermore, the BH1 sequence is used to construct a B7-H3×CD3 bispecific antibody through molecular cloning technology, and the crude antibody product is obtained through the 293F mammalian expression system. The antibody sample solution is purified by affinity chromatography and size exclusion chromatography in two steps to obtain antibody protein (purity>95%) for subsequent characterization and efficacy verification. Antibody characterization includes SDS-PAGE verification of protein molecular weight and purity, thermal thift detection of protein melting temperature Tm, and flow cytometry analysis of the specific binding of recombinant antibodies to target cell surface antigens. Efficacy testing includes the activation and induced killing of T cells by recombinant antibodies. The VHH building block targeting B7H3 is an essential component of the present invention. In addition to the embodiments provided by the present invention, it can also be used to construct monovalent or multivalent monoclonal antibodies or multispecific antibodies in different formats.

[0018] In a first aspect of the present invention, an anti-B7-H3 nanobody is provided, wherein the nanobody comprises a complementary determining region CDR, wherein the complementary determining region CDR comprises a complementary determining region CDR1, a complementary determining region CDR2 and a complementary determining region CDR3, wherein:

[0019] The amino acid sequence of the complementary determining region CDR1 is as shown in SEQ ID NO: 2 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: 2 and has the same or similar function;

[0020] The amino acid sequence of the complementary determining region CDR2 is as shown in SEQ ID NO: 3 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: 3 and has the same or similar function;

[0021] The amino acid sequence of the complementary determining region CDR3 is as shown in SEQ ID NO: 4 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: 4 and has the same or similar functions.

[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 that is modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 and has the same or similar function.

[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 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 functions.

[0026] The third aspect of the present invention provides an anti-B7-H3×CD3 bispecific antibody targeting B7-H3 and CD3, wherein the bispecific antibody has a symmetrical structure, comprising 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 invention, the amino acid sequence of the scFv targeting CD3 is as shown in DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKS (SEQ ID NO: 9) 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: 9 and having the same or similar functions.

[0028] In some preferred embodiments, the Fc fragment is altered, for example, mutated, to modify the properties of the bispecific antibody molecule of the present invention (e.g., to change one or more of the following properties: Fc receptor binding, antibody glycosylation, effector cell function or complement function). The bispecific antibodies provided by the present invention comprise Fc variants with amino acid substitutions, deletions or additions having altered effector functions (e.g., reduced or eliminated). The Fc region of an antibody mediates several important effector functions, such as ADCC, ADCP, CDC, etc. Methods for changing the affinity of an antibody for an effector ligand (such as FcγR or complement C1q) and thereby altering effector function by replacing amino acid residues in the Fc region of an antibody are known in the art (see, e.g., EP 388,151 A1; U.S. Pat. No. 5,647,260; U.S. Pat. No. 5,627,4821; 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., Cancer Res., 68:8882-8890, 2007). 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 present invention, the amino acid L235 (EU numbering) on ​​the antibody constant region is modified to change the Fc receptor interaction, such as L234A or L235A. In other preferred embodiments, the amino acids 234 and 235 on the antibody constant region are modified simultaneously, such as L234A and L235A (L234A / L235A) (EU numbering).

[0029] In some embodiments of the present invention, the amino acids of the Fc segment are as shown in RSDKTYTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (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.

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

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

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

[0033] In some embodiments of the present invention, 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 that is modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO:6-7 and has the same or similar functions.

[0034] In a fourth aspect of the present invention, 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 is provided, wherein the biomaterial 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 invention, the Fc fusion protein of the anti-B7-H3 Nanobody of the second aspect of the invention, or the bispecific antibody of the third aspect of the invention;

[0036] a2) an expression cassette containing the nucleic acid molecule described in 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 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 includes 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 bacteriophage, a cosmid, a F cosmid, or an artificial chromosome.

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

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

[0055] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new species of plants or animals.

[0056] In some embodiments of the present invention, the prokaryotic cells include Escherichia coli, Streptomyces, Bacillus subtilis and other bacteria well known in the art that can be used to express target proteins.

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

[0058] The fifth aspect of the present invention provides an antibody-drug conjugate, comprising at least one 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, or the bispecific antibody of the third aspect of the present invention; and a conjugated portion.

[0059] In some embodiments of the present invention, the conjugated moiety comprises at least one of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

[0060] In some embodiments of the present invention, the detectable marker is selected from a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or any combination thereof.

[0061] In some embodiments of the present invention, the conjugate is selected from: fluorescent substances, chemiluminescent markers, colored substances, radioactive isotopes, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents or enzymes capable of producing detectable products, radionuclides, biological toxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activated enzymes, chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0062] The sixth aspect of the present invention provides 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.

[0063] In some embodiments of the present invention, the extracellular antigen binding domain is selected from 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.

[0064] In some embodiments of the present invention, the transmembrane domain comprises a transmembrane region selected from the following proteins: α, β or δ chain of T cell receptor, CD3ε, CD3δ, CD4, CD5, CD8α, CD137, CD152, CD154, PD1.

[0065] In some embodiments of the invention, the intracellular signaling domain comprises a primary signaling domain and / or a co-stimulatory signaling domain.

[0066] In some embodiments of the present invention, the intracellular signaling domain comprises a co-stimulatory signaling domain and a primary signaling domain in sequence from the N-terminus to the C-terminus.

[0067] In some embodiments of the invention, the intracellular signaling domain comprises a primary signaling domain and at least one co-stimulatory signaling domain.

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

[0069] In some embodiments of the invention, the primary signaling domain comprises an intracellular signaling domain selected from the group consisting of CD3δ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b or CD66d.

[0070] In some embodiments of the invention, the co-stimulatory signaling domain comprises an intracellular signaling domain 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 present invention, 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 CD8α signal peptide.

[0072] In some embodiments of the present invention, the spacer domain is located between the extracellular antigen binding domain and the transmembrane domain, and the spacer domain comprises a hinge domain and / or the CH2 and CH3 regions of an immunoglobulin; preferably, the hinge domain comprises the hinge region of CD8α, PD1, CD152 or CD154.

[0073] The seventh aspect of the present invention provides a chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the chimeric antigen receptor of the fifth aspect of the present invention.

[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 preventing and / or treating 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, including 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.

[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 nano-antibody synthetic library and obtains a new specific anti-human B7-H3 nano-antibody sequence through phage screening technology. The anti-B7-H3 nano-antibody 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 anti-B7-H3 VHH sequence obtained by screening as an 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, and verifies through flow cytometry 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 belongs to a T cell agonist antibody. The T cell activation verification experiment confirms that the bispecific antibody and the recombinant OKT3 monoclonal antibody have similar T cell activation efficacy, but in the presence of target cells, i.e., tumor cells that highly express B7-H3, the activation efficacy is significantly enhanced. The T cell activation and killing experimental data of the bispecific antibody (ID10) verified the T cell activation efficacy and colorectal cancer cell killing efficacy, and compared with the control antibody, the drug efficacy dose was reduced by at least 1 order of magnitude.

[0086] The format of the bispecific antibody provided by the present invention is a 2+2 symmetrical antibody format without Fab, and there is no need to consider light and heavy chain mispairing, which reduces the difficulty of protein purification. From the characterization of the molecular weight and thermal stability of the recombinant antibody, it can be seen that the bispecific antibody recombined with VHH and OKT3scFv-hFc skeleton not only maintains the thermal stability of the Fc format, but also makes the overall protein size of the recombinant bispecific antibody similar to that of the traditional monoclonal antibody, avoiding adding difficulty to the penetration treatment of solid tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 The figure is the reducing SDS-PAGE profile of the BH1 VHH prokaryotic expression and purification process, wherein 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 periplasmic protein extraction supernatant, and lane 5 is the protein eluate purified by a nickel gravity column.

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

[0089] Figure 3This is an ELISA test for the binding of recombinant antibody BH1-hFc to human 4Ig B7-H3. The left figure is the Elisa binding result of recombinant antibody BH1-hFc and human B7-H3 at a single dose, and Milk / PBS is used as the negative control group. The right figure is the dose-dependent Elisa binding data of recombinant antibody BH1-hFc and human B7-H3, and 8H9-hFc is the positive control group.

[0090] Figure 4 Schematic diagram of the structure of the recombinant bispecific antibody.

[0091] Figure 5 The size exclusion chromatograms of each recombinant antibody are shown in Figure 2 .

[0092] Figure 6 The protein melting curves of the recombinant antibodies are shown in Figure 2 .

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

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

[0095] Fig. 9 Comparison of the upregulation of CD69 on the surface of Jurkat cells by recombinant antibody activation. The upper figure shows the abundance of CD69 antigen expression on the surface of Jurkat cells detected by flow cytometry after co-culture of HCT116 and Jurkat cell lines (target-effect ratio 1:10). The lower figure shows the comparison of CD69 antigen expression on the surface of Jurkat cells when treated with the highest concentration of 10 nM of each recombinant antibody detected by flow cytometry under the conditions of co-culture with or without HCT116. NT refers to the group without target cells, and T refers to the group co-cultured with target-effect cells.

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

[0097] Fig.11 After co-culture of human peripheral blood mononuclear cells and HCT116 cell line (target-effect ratio 1:20), the dose-dependent tumor cell killing curve was calculated by detecting the content of lactate dehydrogenase released by the lysed cells in each group. DETAILED DESCRIPTION

[0098] The present invention is further described in detail below through specific examples.

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

[0100] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0101] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[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] The pcDNA3.1(+) vector was linearized by double digestion with NheⅠ / XhoI. At the same time, the vector containing the human B7-H3 gene (SinoBiological, catalog number HG11188-ACG) was used as a template to design primers to introduce the C-terminal hexahistidine tag, stop codon, N-terminal interleukin 2 signal peptide and vector homology arm sequences at both N and C ends. The human B7-H3 extracellular domain gene fragment containing the vector homology arms was amplified, and the pcDNA3.1-B7H3-ECD-His tag eukaryotic expression plasmid was constructed by homologous recombination. The recombinant plasmid was transformed into the DH5α competent strain by heat shock method, and the positive plaques were screened on ampicillin-resistant solid culture medium plates, and further sequenced for verification. The correct cloned plaques were selected and amplified using LB liquid culture medium supplemented with ampicillin antibiotics to extract the endotoxin-free transfection plasmid pcDNA3.1-B7H3-ECD-His tag.

[0106] (2) 293F mammalian cells expressing 4Ig B7-H3 antigen extracellular domain

[0107] Dilute the transfection reagent 300 μg of polyethyleneimine and 100 μg of the plasmid to be transfected to 5 mL each with serum-free medium OPTI-MEM, mix well, incubate for 20 min, and then add the transfection mixed reagent to 90 mL of cells with good growth and a density of about 2-3×10 6 After transfection, the cells were placed in a shaker in an incubator at 8% CO2, 37°C, and 115 rpm for 4 days of suspension culture.

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

[0109] On the 4th day after transfection, the cell supernatant was collected by refrigerated centrifugation and filtered at 0.22 μm. The entire nickel column purification process was performed on ice at low temperature. The specific purification process is as follows:

[0110] The cell transfection supernatant filtrate and His-tag binding buffer (20mM phosphate, 10mM imidazole, pH = 7.4) were mixed in equal volumes and incubated on ice for 30 minutes. The Ni NTA beads were loaded into the gravity column with 5 to 10 column volumes of 0.22μm filtered water and His-tag binding buffer solution. The filtered supernatant was added to the gravity column for sample loading. After the sample loading, the residual impurities in the filler were washed with 5 to 10 column volumes of binding buffer. Finally, the protein bound to the affinity filler was eluted with 5 to 10 column volumes of His-tag elution buffer (20mM phosphate, 500mM imidazole, pH = 7.4), and the protein eluate was collected.

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

[0112] (4) Phage solid phase screening

[0113] Human 4Ig B7-H3 protein was coated on the ELISA plate (5 μg in the first round, and gradually reduced to 2.5 μg and 1 μg in the next two rounds) and left overnight at 4°C. The next day, the liquid in the overnight coated wells was discarded, 250 μL of 5% Milk blocking solution was added, and the same volume of blocking agent as the library was pre-incubated with the library, and incubated on a horizontal shaker at room temperature for 1 hour.

[0114] Three rounds of screening were conducted:

[0115] In the first and second rounds, the phage library pre-incubated with the blocking agent was directly added to the antigen-coated wells after the blocking solution was discarded, 100 μL / well, and incubated on a horizontal shaker at room temperature for 2 hours. In the third round, the phage library was first added to the negative pool (not coated with 4Ig B7-H3 protein, other conditions were the same), incubated on a horizontal shaker at room temperature for 1 hour, and then the supernatant was aspirated and added to the positive pool, and incubated on a horizontal shaker at room temperature for 2 hours. The unbound phages in the positive pool were recovered, and PBST (containing 0.1% Tween-20) and PBS (Phosphate-Buffered Saline) were added to each well in turn for 15 times. The washing solution was discarded, 35 μL 100mM Glycine-HCl solution (pH=2.5) was added to each well for elution at room temperature for 10 minutes, and then 15 μL 1MTris-HCl (pH=7.5) was added to terminate the elution, and the phage library eluate was collected. Take half of the phage eluate to infect the TG1 strain in the logarithmic growth phase and apply it to 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 times the number of helper phage M13K07, and culture at 250rpm and 37℃ for 30min. Add kanamycin with a final concentration of 50μg / mL and IPTG with a final concentration of 0.2mM, and amplify overnight at 30℃ and 250rpm. The next day, centrifuge the overnight bacterial solution, collect the supernatant, add 1 / 4 volume of 20% PEG 8000 / 2.5M NaCl solution, mix well and let stand on ice for 30min. After centrifugation, discard the supernatant, resuspend the precipitate with 1mL PBS, and centrifuge the resuspension at 4℃ and 13000rpm for 20min. After collecting the supernatant, add 1 / 4 volume of 20% PEG8000 / 2.5M NaCl solution again, mix well and let stand on ice for 10min. Centrifuge at 13000rpm and 4℃ for 10min, discard the supernatant, add 1mL PBS to resuspend the precipitate, and centrifuge at 4℃ and 13000rpm for 2min. 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, 250ng per well, and left to stand overnight at 4°C. The overnight coating solution was discarded, and 200μL of PBS solution containing 3% BSA was added to each well for blocking, and the wells were shaken on a horizontal shaker at room temperature for 1h. The blocking solution was discarded, and PBST was added to each well for washing 3 times. The monoclonal phages amplified overnight were centrifuged, and the supernatant containing phages was collected. After adding 130μL of blocking agent to each well, 70μL of supernatant containing phages was added, and the wells were shaken on a horizontal shaker at room temperature for 2h. The well plate liquid was discarded and washed 5 times with PBST. 50μL of 0.4μg / mL HRP-coupled mouse M13Bacteriophage antibody (Sino Biological, Cat. No.: 11973-MM05T-H) was added to each well, and the wells were shaken on a horizontal shaker at room temperature for 1h. The well plate liquid was discarded and washed 5 times with PBST. Add 50μL TMB colorimetric solution to each well, color for 2-3 minutes in the dark, then add 50μL 1M HCl solution to terminate the reaction, and detect the absorbance at 450nm with an ELISA reader. The single clones with obvious binding signals in the sample wells and weak Milk binding signals were sent for sequencing, and the positive clone sequence was identified, that is, a clone that specifically binds to human B7-H3 was obtained, named BH1.

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

[0119] (1) Construction of expression plasmid

[0120] The pET-22b(+) vector was linearized by double digestion with NdeⅠ / XhoⅠ. At the same time, BH1 was used as the VHH (variable domain of heavy chain antibody, heavy chain variable region single domain antibody / nanoantibody) construction unit to synthesize a synthetic gene with a hexahistidine tag at the C-terminus and used as a template to amplify the BH1 VHH gene fragment containing the vector homology arm. The pET-22b(+)-BH1 VHH-His tag prokaryotic expression plasmid was constructed by homologous recombination. The recombinant plasmid was transformed into the DH5α competent strain by heat shock method, and the positive plaques were screened on ampicillin-resistant solid culture medium plates, and further sequenced for verification. The correct clone plaque was selected, the plasmid was extracted, and the heat shock method was used to transform it into the BL21(DE3) competent strain, and the ampicillin-resistant solid culture medium plates were coated for overnight culture. Pick the overnight single clone, inoculate it into LB liquid culture medium supplemented with ampicillin antibiotics, and amplify the expression strain.

[0121] (2) IPTG-induced prokaryotic expression

[0122] The BH1 VHH expression bacterial solution was inoculated into 1L LB liquid medium supplemented with ampicillin antibiotics and cultured at 37°C and 220rpm. The OD 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 culture at 30℃ and 220rpm for 5h. After the culture is completed, centrifuge at 4500rpm for 15min to collect the bacterial precipitate. Add 4mL TES buffer (50mM Tris-HCl, 1mM EDTA, 20% sucrose, pH=8.0) for every 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 of the same volume as the TES used above, and let it stand on ice for 15min. Centrifuge at 4500rpm for 45min, collect the supernatant, and dialyze it into His-tag binding buffer (20mM phosphate, 10mM imidazole, pH=7.4) in a 4℃ chromatography cabinet.

[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 4Ig B7-H3 antigen extracellular domain protein purification operation. SDS-PAGE gel electrophoresis verified the separation and purification effect of BH1 VHH protein ( Figure 1 ). According to the SDS-PAGE results, the molecular weight of BH1 VHH is about 25kD. Use an ultrafiltration tube with a cutoff of 3kD to replace the protein in high-concentration imidazole with PBS, concentrate to a final concentration of >2mg / mL, freeze in liquid nitrogen, and store in a -80℃ refrigerator for later 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] The pFUSE-hIgG1-Fc2-His tag vector was linearized by EcoRV / BstEII double digestion. At the same time, the synthetic gene with a hexahistidine tag at the C-terminus was used as a template to amplify the BH1 VHH gene fragment containing the vector homology arm, and the pFUSE-BH1-hFc-His tag eukaryotic expression plasmid was constructed by homologous recombination. The recombinant plasmid was transformed into the DH5α competent strain by heat shock method, and the positive plaques were screened by coating the bleomycin-resistant solid culture medium plate, and further sequenced for verification. The correct cloned plaques were selected and amplified using LB liquid culture medium supplemented with bleomycin antibiotics to extract the endotoxin-free plasmid pFUSE-BH1-hFc-His tag to be transfected.

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

[0134] Dilute the transfection reagent 300 μg of polyethyleneimine and 100 μg of the plasmid to be transfected to 5 mL each with serum-free medium OPTI-MEM, mix well, incubate for 20 min, and then add the transfection mixed reagent to 90 mL of cells with good growth and a density of about 2-3×10 6 After transfection, the cells were placed in a shaker in an incubator at 8% CO2, 37°C, and 115 rpm for 4 days of suspension culture.

[0135] (3) Recombinant antibody purification

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

[0137] SDS-PAGE gel electrophoresis verifies protein separation and purification effect ( Figure 2 ). According to the SDS-PAGE results, the molecular weight of BH1-hFc is about 80kD. Use an ultrafiltration tube with a cutoff of 10kD to replace the protein in high-concentration imidazole with PBS, concentrate to a final concentration of >2mg / mL, and temporarily store at 4°C.

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

[0139] Question VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH(SEQ ID NO:5).

[0140] 5. ELISA detection of recombinant antibodies

[0141] (1) Single-dose recombinant antibody ELISA

[0142] The sample wells of the ELISA plate were coated with 250ng 4Ig B7-H3 extracellular domain protein, the negative control wells were coated with 250ng Milk, and PBS was used as the background control group. Each group was set up in parallel and left to stand overnight at 4°C. The next day, the liquid in the ELISA plate was discarded, 200μL 3% BSA was added, and the plate was blocked on a horizontal shaker at room temperature for 2h. After the incubation, the liquid in the ELISA plate was discarded, and each well was washed 3 times with 200μL PBST. PBS containing 1% BSA was used as a diluent to dilute the recombinant antibody BH1-hFc to 50nM, added to all sample wells and control wells, 100μL / well, and incubated on a horizontal shaker at room temperature for 1h. After the incubation, the liquid was discarded, and each well was washed 5 times with 200μL PBST. 50μL 5μg / mL 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 1h. Discard the liquid, wash each well with 200 μL PBST 5 times. Discard the washing solution, spin dry the ELISA plate, add 50 μL TMB colorimetric solution to each well, color for about 5 minutes in the dark, add 50 μL 1M HCl solution to terminate the reaction, and detect the absorbance at a wavelength of 450 nM with an ELISA reader.

[0143] (2) Recombinant antibody dose-dependent ELISA

[0144] The sample wells of the ELISA plate were coated with 250ng of human 4Ig B7-H3 extracellular domain protein, and duplicate wells were set up in parallel for each group and left to stand overnight at 4°C. The next day, the liquid in the ELISA plate was discarded, and each well was blocked with 200μL 5% Milk at room temperature for 2h and placed on a horizontal shaker. After the incubation, the liquid in the ELISA plate was discarded, and each well was washed 3 times with 200μL PBST. PBS containing 1% Milk was used as a diluent to dilute BH1-hFc and positive antibody 8H9-hFc (i.e., 8H9 scFv sequence that can specifically bind to B7-H3, which has been disclosed in "hmed M, Cheng M, Zhao Q, et al. J Biol Chem. 2015; 290(50): 30018-30029.") to prepare gradient concentration antibody solutions of 100nM, 33nM, 10nM, 3.3nM, 1nM, 0.33nM, 0.1nM, 0.03nM, and 0.01nM. 100μL of antibody diluent was added to each well and incubated on a horizontal shaker at room temperature for 1h. The liquid was discarded and washed 5 times with 200μL PBST solution. 50μL of HRP-conjugated goat anti-human IgG-Fc antibody (Sino Biological, catalog number: SSA001) was added to each well and incubated on a horizontal shaker at room temperature for 1h. Discard the liquid, wash each well with 200 μL PBST 5 times. Discard the washing solution, spin dry the ELISA plate, add 50 μL TMB colorimetric solution to each well, color for about 5 minutes in the dark, add 50 μL 1M HCl solution to terminate the reaction, and detect the absorbance at a wavelength of 450 nM with an ELISA 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 respectively ( 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, the OKT3scFv fragment containing the homologous arms of the pFUSE-hIgG1-Fc2-His tag vector was amplified based on the company's synthetic gene (disclosed in U.S. Patent US-7635472-B2), and the pFUSE-OKT3-hFc-Histag plasmid was constructed by homologous recombination. Then, the Fc backbone LALA mutation (L234A and L235A) was achieved through multiple point mutations to silence its effector function. Based on the pFUSE-OKT3-hFc-LALA-His tag plasmid, the N-terminus of the original plasmid OKT3 scFv sequence or the C-terminus of the Fc backbone sequence was opened by whole plasmid PCR technology, and the BH1 VHH base sequence was inserted by homologous recombination to construct the recombinant pFUSE-BH1-OKT3-hFc-LALA-His tag (the corresponding bispecific antibody BH1-OKT3-hFc was named ID9) and pFUSE-OKT3-hFc-LALA-BH1-His tag expression plasmid (the corresponding bispecific antibody OKT3-hFc-BH1 was named ID10). After the template plasmid was digested with DpnⅠ, the recombinant plasmid was transformed into the DH5α competent strain by heat shock method, and the positive plaques were screened by coating the bleomycin resistance solid culture medium plate, and further sequenced for verification. The correct cloned plaques were selected and amplified using LB liquid medium supplemented with bleomycin antibiotics to extract the endotoxin-free transfection plasmids pFUSE-BH1-OKT3-hFc-LALA-His tag and pFUSE-OKT3-hFc-LALA-BH1-His tag.

[0149] Schematic diagram of the structure of the recombinant bispecific antibody Figure 4 shown.

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

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

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

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

[0154] SDS-PAGE gel electrophoresis verifies the protein separation and purification effect. According to the SDS-PAGE results, the molecular weight of ID9 and ID10 is about 140kD. Use an ultrafiltration tube with a cutoff of 30kD to replace the protein in high-concentration imidazole with PBS, concentrate to a final concentration of >2mg / mL, and temporarily store at 4°C.

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

[0156] Operate the protein purifier, equilibrate the gel filtration column with 2 column volumes of 0.22 μm filtered water and PBS buffer, centrifuge the antibody concentrate at 4°C and 10000 g for 10 min, load the sample twice with a 500 μL sample loop, carefully draw about 250 μL each time with a 1 mL syringe, remove bubbles and slowly inject 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 from 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. Execute inject.

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

[0159] The size exclusion chromatograms of each recombinant bispecific antibody are shown in 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] (SEQ ID NO:7).

[0163] Example 3 Characterization of thermal stability of recombinant bispecific antibodies

[0164] Dilute 5000×SYPRO Orange dye to 25× with PBS, and dilute 0.1μg / μL OKT3-hFc, ID9 and ID10 protein solutions with PBS. In the qPCR transparent well plate, add 45μL protein solution and control PBS to each well, then add 5μL 25× dye and mix well, and seal the plate. After instant centrifugation in a microplate centrifuge, use a quantitative fluorescence PCR instrument with 485nm excitation light and 530nm emission light filter to detect, and set the temperature to continuously increase from 20.0℃ to 99.0℃ at a rate of 0.3℃ per second. After the experiment is completed, export the melting curve analysis.

[0165] The protein melting curves of each recombinant antibody are shown in Figure 6 As shown, the melting temperatures Tm of OKT3-hFc, ID9 and ID10 were approximately 74°C.

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

[0167] Jurkat cells and digested HCT116 cell suspensions were taken for cell counting and activity analysis, respectively, and 10 6 Single cells were collected and centrifuged at 1500rpm for 5min, and the supernatant was discarded. Each tube of cell pellet was resuspended with 100μL PBS blocking solution containing 10% FBS, allowed to stand on ice for 30min, centrifuged at 1500rpm for 5min, and the supernatant was discarded. PBS solution containing 3% BSA was used as an antibody diluent to prepare 1000nM, 300nM, 100nM, 30nM, 10nM, 3nM, 1nM, 0.3nM, 0.1nM, and 0.03nM gradient antibody solutions. Each tube of cell pellet was resuspended with the corresponding marker antibody solution with a concentration gradient, allowed to stand on ice for 30min, and centrifuged at 1500rpm for 5min. The supernatant was discarded. Each tube of cell pellet was resuspended with 500μL PBS, centrifuged at 1500rpm for 5min, and the supernatant was discarded. The cell pellet was washed with PBS twice. PBS solution containing 3% BSA was used as a diluent to prepare 5μg / mL FITC F(ab')2Goat anti-humanIgG FcγAntibody (BioLegend, Cat. No. 398006) fluorescent antibody diluent. 100μL fluorescent antibody diluent was added to each tube of cell pellet to resuspend, stand on ice for 30min, centrifuge at 1500rpm for 5min, and discard the supernatant. Each tube of cell pellet was resuspended with 500μL PBS, centrifuged at 1500rpm for 5min, and the cell pellet was washed twice with PBS. FITC channel signals were detected by flow cytometry, and data were processed jointly by Flowjo and Prism.

[0168] The results showed that the recombinant bispecific antibody bound to the target positive cell line in a dose-dependent manner. The data were processed using Prsim nonlinear fitting to obtain the EC values ​​of ID9, ID10 and BH1-hFc for the binding of B7-H3 antigen on the surface of HCT116. 50 , 35.7nM, 216.6nM and 33.8nM respectively ( Figure 7 ), while ID9, ID10 and OKT3-hFc have no significant effect on the binding of CD3 antigen on the surface of Jurkat to EC 50 0.38nM, 0.17nM and 0.17nM respectively ( Figure 8 ).

[0169] Example 5 Validation of Antibody-Mediated T Cell Activation

[0170] HCT116 cells were seeded in 96-well plates, with 2×10 4cells and 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 concentration of 10nM, 1nM, 100pM, 10pM, 1pM, and 0.1pM antibody solution. Discard the old medium from the 96-well plate, add 50μL of antibody diluent to each well, and incubate at 37°C for 30 minutes. Take out the pre-incubated 96-well plate, inoculate 50μL of Jurkat cell suspension to each well, and 2×10 5 Cells were cultured for 24 hours at 37°C and 5% CO2. After 24 hours, the 96-well plate was removed, the cell solution was aspirated as much as possible, and PBS was added to gently rinse each sample well. The cell solution and the rinse solution were combined, and the cells were collected after centrifugation at 1500rpm for 5 minutes, and the cells were resuspended and washed with 500μL PBS. The cells were centrifuged at 1500rpm for 5 minutes again, and the supernatant was discarded. A 5μg / mL FITC Anti-CD69Antibody (Sino Biological, Catalog No. 11150-MM06-F) incubation solution was prepared with a PBS solution containing 3% BSA as a diluent. 100μL of fluorescent labeled antibody diluent was added to each tube of cell pellet to resuspend the cells and incubate on ice for 30 minutes. Centrifuge at 1500rpm for 5 minutes, discard the supernatant, wash once with PBS, and finally resuspend with 500μL PBS. The FITC channel signal was detected by flow cytometry, and the data was processed jointly by Flowjo and Prism.

[0171] The data were processed by Prsim nonlinear fitting to obtain the T cell activation ECs 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 ( Fig. 9 ). In addition, by comparing the expression of CD69 antigen on the surface of Jurkat cells in the highest concentration of each drug treatment group 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, but 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 that of ID10 ( Fig. 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, and 2 × 10 4cells and 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 concentration of 10nM, 1nM, 100pM, 10pM, 1pM, and 0.1pM antibody solution. Discard the old medium from the 96-well plate, add 50μL of antibody diluent and incubate at 37°C for 30 minutes. Take out the pre-incubated 96-well plate and inoculate Jurkat-NFAT-Luciferase, 50μL per well, 2×10 5 Cells were cultured at 37℃ for 5.5h. The plate was removed and placed at room temperature for 30min to allow the plate to equilibrate to room temperature. After centrifugation at 4500rpm for 5min, 50μL of supernatant was removed from each well. An equal volume of Stable-Lite PBS was added to each well to balance the remaining 50μL of cell solution. TM Luciferase Assay reagent (Vazyme, Cat. No. DD1202) was added and incubated on a shaker at room temperature for 5 min to fully lyse the cells. The fluorescence was measured using a microplate chemiluminescence detector.

[0174] The data were processed by Prsim nonlinear fitting to obtain the EC of T cell activation determined by ID9, ID10 and OKT3-hFc using the luciferase reporter system. 50 0.57nM, 0.02nM and 0.25nM respectively ( Fig.10 ).

[0175] Example 7 Validation of T cell-mediated tumor cell killing

[0176] HCT116 cells were seeded in a 96-well plate, with 1×10 4 Cells were cultured in a 37°C incubator for 24 h. RPMI1640 medium containing 1% FBS was used as an antibody diluent to prepare a gradient antibody solution of 10nM, 1nM, 100pM, 10pM, 1pM, 100fm, 10fm, and 1fm. The old medium was discarded from the 96-well plate and 100μL of antibody diluent was added. The density of unactivated hPBMc cells was adjusted to 2×10 using RPMI1640 medium containing 1% FBS. 6 cells / mL, 100 μL was inoculated into each well, that is, 2×10 5Effector cells were cultured in a 37°C incubator for 36 hours. At the predetermined 36-hour detection point, the cell culture plate was centrifuged at 400g for 5 minutes, 120 μL of supernatant was taken and added to a new 96-well plate, 60 μL of freshly prepared LDH detection working solution was added to each well, and the plate was placed on a horizontal shaker and incubated at room temperature in the dark for 30 minutes. The absorbance at 490 nm was measured with a full-band microplate reader, and 600 nm was used as the reference wavelength. Assuming that the cell killing efficacy of the 10nM ID10 drug experimental group is 100%, the dose-dependent cell killing efficacy of each group was evaluated according to the cytotoxicity normalization calculation 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 respectively ( Fig.11 ).

[0180] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An anti-B7-H3 nanobody, comprising a complementary determining region CDR, wherein the complementary determining region CDR comprises a complementary determining region CDR1, a complementary determining region CDR2 and a complementary determining region CDR3, wherein: The amino acid sequence of the complementary determining region CDR1 is as shown in SEQ ID NO: 2 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: 2 and has the same or similar function; The amino acid sequence of the complementary determining region CDR2 is as shown in SEQ ID NO: 3 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: 3 and has the same or similar function; The amino acid sequence of the complementary determining region CDR3 is as shown in SEQ ID NO: 4 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: 4 and has the same or similar functions.

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 that is modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 1 and has 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; Preferably, 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 one or more amino acids substitution, deletion or addition to the amino acid sequence shown in SEQ ID NO: 8 and has the same or similar function; preferably, 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 one or more amino acids substitution, deletion or addition to the amino acid sequence shown in SEQ ID NO: 5 and has the same or similar function.

4. 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; Preferably, the amino acid sequence of the CD3-targeting scFv is as shown in SEQ ID NO: 9 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: 9 and has the same or similar functions; Preferably, the amino acids in 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; Preferably, 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; Preferably, the anti-B7-H3 nanobody is linked to the scFv and Fc segment targeting CD3 via a connecting peptide; Preferably, the amino acid sequence of the connecting peptide is (GGGGX)n, X comprises Ser or Ala, and n is a natural number of 1 to 5; Preferably, 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 that is modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NOs:6 to 7 and has the same or similar functions.

5. 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 claim 3, or the bispecific antibody according to claim 4, 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 claim 3, or the bispecific antibody according to claim 4; 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).

6. 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 claim 3, or the bispecific antibody according to claim 4; and a conjugated moiety; Preferably, the conjugated moiety comprises at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

7. 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; Preferably, 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 claim 3, or the bispecific antibody according to claim 4.

8. A chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the chimeric antigen receptor of claim 7; Preferably, the immune cells include any one of T cells, immune cells differentiated from stem cells, monocytes, macrophages or NK cells.

9. 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 claim 3, the bispecific antibody according to claim 4, the biomaterial according to claim 5, the antibody-drug conjugate according to claim 6, the chimeric antigen receptor according to claim 7, or the chimeric antigen receptor immune cell according to claim 8 in b1) or b2): b1) Preparation of drugs or preparations for preventing and / or treating tumors and immune-related diseases; b2) Preparation of reagents or kits for detecting tumors and immune-related diseases.

10. A product comprising the anti-B7-H3 nanobody according to claim 1 or 2, the Fc fusion protein of the anti-B7-H3 nanobody according to claim 3, the bispecific antibody according to claim 4, the biomaterial according to claim 5, the antibody-drug conjugate according to claim 6, the chimeric antigen receptor according to claim 7, or the chimeric antigen receptor immune cell according to claim 8.

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