Anti-B7-H3 antibody and application thereof

Abstract

The invention relates to the field of biological medicine, in particular to an anti-B7-H3 antibody and application thereof.The anti-B7-H3 antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises CDR-H1 with the amino acid sequence shown as SEQ ID No.2, CDR-H2 with the amino acid sequence shown as SEQ ID No.3 and CDR-H3 with the amino acid sequence shown as SEQ ID No.4; the light chain variable region comprises a CDR-L1 of which the amino acid sequence is as shown in SEQ ID No.6, a CDR-L2 of which the amino acid sequence is as shown in SEQ ID No.7 and a CDR-L3 of which the amino acid sequence is as shown in SEQ ID No.8. The antibody obtained by the invention is relatively high in affinity with human and monkey B7-H3 proteins and relatively high in specificity; the compound has higher binding activity on cancer cells with different abundance, and the binding activity is higher than that of selected clinical or marketed drugs; high internalization is shown on cancer cells with different abundance.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly to an anti-B7-H3 antibody and its uses. Background Art

[0002] B7-H3 (CD276) is a type I transmembrane protein and belongs to the members of the B7 co-stimulatory and co-inhibitory family, and its ligand is not yet clear. The B7-H3 protein is encoded by the chromosome 15q24 gene and structurally consists of 316 amino acids including an extracellular domain, a transmembrane domain, and a short intracellular domain. The intracellular domain of the B7-H3 protein is very short and there is no known signal motif. In addition, due to exon duplication, the human B7-H3 protein contains one or two pairs of identical extracellular domains, resulting in two subtypes: 2IgB7-H3 consists of a pair of immunoglobulin variable region (IgV)-like and immunoglobulin constant region (IgC)-like extracellular domains; 4IgB7-H3 contains two pairs of identical IgV-like and IgC-like extracellular domains, which is also the main subtype in human cells.

[0003] B7-H3 is mainly expressed on the surfaces of non-immune cells such as fibroblasts, endothelial cells, osteoblasts, amniotic fluid stem cells, as well as on the surfaces of activated antigen-presenting cells, NK cells, etc. Research shows that B7-H3 is overexpressed in various tumor cells such as melanoma, leukemia, breast cancer, prostate cancer, etc., and its overexpression is closely related to factors such as the growth, metastasis, recurrence, and poor prognosis of malignant tumors. B7-H3 can down-regulate the immune response mediated by T helper type 1, inhibit the activation of CD4+ T cells, and inhibit the production of cytokines, thus possibly playing a role in promoting the immune escape of cancer cells.

[0004] Most current targeted therapies regard B7-H3 as a negative regulator of T cell-mediated immune responses in tumors, and specific blockade of B7-H3 can provide a new targeted therapy method similar to anti-CTLA-4 mAb therapy.

[0005] The human cancer immunotherapy strategies for B7-H3 mainly include: 1) Blocking B7-H3 with mAb to neutralize the inhibitory signals in T cells, NK cells, and other immune cells; 2) Fc receptors of NK cells and other immune cells participate in triggering B7-H3-specific ADCC to induce tumor cell death; 3) CD3 / B7-H3 bispecific antibody binds to B7-H3 expressed on tumors, crosslinks the CD3 part of the TCR complex, activates T cells in the tumor microenvironment, and causes tumor cell death; 4) Small molecule inhibitors can bind to specific regions of B7-H3, such as the FG loop of the IgV domain, inhibit the ligand-receptor interaction between tumor cells and immune cells, thereby blocking receptor signal transduction and restoring the effector functions of immune cells; 5) CAR-T cells recognize membrane B7-H3 and directly kill tumor cells. SUMMARY OF THE INVENTION

[0006] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide an anti-B7-H3 antibody and its uses to solve the problems in the prior art.

[0007] To achieve the above object and other related objects, the present invention provides an anti-B7-H3 antibody, the anti-B7-H3 antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDR-H1 having the amino acid sequence shown in SEQ ID No.2, CDR-H2 having the amino acid sequence shown in SEQ ID No.3, and CDR-H3 having the amino acid sequence shown in SEQ ID No.4; the light chain variable region comprising CDR-L1 having the amino acid sequence shown in SEQ ID No.6, CDR-L2 having the amino acid sequence shown in SEQ ID No.7, and CDR-L3 having the amino acid sequence shown in SEQ ID No.8.

[0008] The present invention also provides a recombinant protein, the recombinant protein comprising the anti-B7-H3 antibody described above and another polypeptide linked to the anti-B7-H3 antibody.

[0009] The present invention also provides a chimeric antigen receptor, the chimeric antigen receptor comprising an intracellular domain, a transmembrane domain, and an extracellular domain, the antigen-binding region of the extracellular domain comprising the anti-B7-H3 antibody described above or a fragment thereof.

[0010] The present invention also provides a chimeric antigen receptor immune cell, the immune cell expressing the chimeric antigen receptor described above. Recombinant immune cells generally refer to immune cells modified by genetic engineering means to enable them to more effectively recognize and attack specific pathogens or tumor cells. The immune cells can be NK cells or T cells.

[0011] The present invention also provides an antibody conjugate, the antibody conjugate comprising the B7-H3 antibody and an effector molecule, the antibody being conjugated to the effector molecule and preferably chemically conjugated.

[0012] The present invention also provides an isolated polynucleotide encoding the anti-B7-H3 antibody, the recombinant protein, the chimeric antigen receptor, or the antibody conjugate.

[0013] The present invention also provides a nucleic acid construct comprising the isolated polynucleotide.

[0014] The present invention also provides an engineered cell, which contains the nucleic acid construct or has the exogenous polynucleotide integrated into its genome.

[0015] The present invention also provides a method for preparing the anti-B7-H3 antibody, comprising the following steps: culturing the engineered cell to express the anti-B7-H3 antibody, and purifying and isolating the anti-B7-H3 antibody.

[0016] The present invention also provides the uses of the anti-B7-H3 antibody, the recombinant protein, the chimeric antigen receptor, the immune cell, the antibody conjugate, the isolated polynucleotide, the nucleic acid construct, and the engineered cell in the preparation or screening of therapeutic drugs.

[0017] As described above, the anti-B7-H3 antibody of the present invention and its uses have the following beneficial effects: 1. The obtained antibody has a relatively high affinity and specificity for human and monkey B7-H3 proteins; 2. The obtained antibody has a relatively high binding activity on cancer cells with different abundances, and is higher than the selected clinical or marketed drugs; 3. The obtained antibody shows relatively high internalization on cancer cells with different abundances; 4. It has an epitope different from the selected clinical or marketed drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Shows the expression of NP040.

[0019] Figure 2 Shows the SPR result of the affinity of NP040 of the present invention for human B7-H3 protein.

[0020] Figure 3 Shows the SPR result of the affinity of NP040 of the present invention for monkey B7-H3 protein.

[0021] Figures 4 to 8 Shows the binding result of NP040 to cells with different expression abundances.

[0022] Figures 9 to 11 Shows the affinity result of NP040 for recombinant human B7-H2, PD-L2, and B7-2 proteins.

[0023] Figure 12 and Figure 13 Shows the binding result of NP040 to B7-1 and PD-L1 positive cells.

[0024] Figures 14 to 18 Shown is the internalization result of NP040 on cells with different expression abundances. Detailed implementation manners

[0025] The present invention provides an anti-B7-H3 antibody, the anti-B7-H3 antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDR-H1 with the amino acid sequence shown in SEQ ID No.2, CDR-H2 with the amino acid sequence shown in SEQ ID No.3, and CDR-H3 with the amino acid sequence shown in SEQ ID No.4; the light chain variable region comprising CDR-L1 with the amino acid sequence shown in SEQ ID No.6, CDR-L2 with the amino acid sequence shown in SEQ ID No.7, and CDR-L3 with the amino acid sequence shown in SEQ ID No.8.

[0026] The numbering scheme and the definition scheme are to label the CDR and FR regions by the method of IMGT.

[0027] CDR (complementarity determining region) generally refers to the region in an antibody that can form complementarity with an antigenic determinant in terms of spatial structure. The variability in an antibody is usually not evenly distributed throughout the variable region of the whole antibody. The heavy chain variable region of a monoclonal antibody usually has 3 hypervariable regions (HVRs), and these regions can usually form complementarity with an antigenic determinant in terms of spatial structure, so the hypervariable region is also called the complementarity determining region (CDR), that is, the heavy chain variable region usually includes three complementarity determining regions, namely CDR-H1, CDR-H2, and CDR-H3.

[0028] In some embodiments of the present invention, the heavy chain variable region may further include a framework region, and the framework region may be located between the complementarity determining regions or at both ends of the complementarity determining regions. In some specific embodiments of the present invention, the sequence of the framework region is a human, rabbit, or murine monoclonal antibody variable region.

[0029] In some embodiments of the present invention, the heavy chain variable region further includes framework regions HCFR1 to HCFR4. The amino acid sequences of the framework regions HCFR1 to HCFR4 are selected from the sequences shown in any of SEQ ID Nos. 11 to 14.

[0030] Preferably, the amino acid sequence of HCFR1 is as shown in SEQ ID No.11: QVQLVQSGAEVKKPGASVKVSCKAS.

[0031] Preferably, the amino acid sequence of HCFR2 is as shown in SEQ ID No. 12: INWVRQAPGQGLEWIGN.

[0032] Preferably, the amino acid sequence of HCFR3 is as shown in SEQ ID No. 13: NYNQKFKDKATLTVDTSASTAYMELSSLRSEDTAVYYC.

[0033] Preferably, the amino acid sequence of HCFR4 is as shown in SEQ ID No. 14: WGQGTSVTVSS.

[0034] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 1.

[0035] In certain embodiments of the present invention, the nucleotide sequence of the heavy chain variable region of the anti-B7-H3 antibody is as shown in SEQ ID No. 19.

[0036] In certain embodiments of the present invention, the light chain variable region further includes framework regions LCFR1-LCFR4. The amino acid sequences of the framework regions LCFR1-LCFR4 are selected from the sequences shown in any of SEQ ID Nos. 15-18.

[0037] Preferably, the amino acid sequence of LCFR1 is as shown in SEQ ID No. 15: DIVMTQSPSTLSASVGDRVTITCKAS.

[0038] Preferably, the amino acid sequence of LCFR2 is as shown in SEQ ID No. 16: VAWYQQKPGKAPKILIH.

[0039] Preferably, the amino acid sequence of LCFR3 is as shown in SEQ ID No. 17: TRHTGVPDRFSGSGSGTDYTLTISSLQPEDFATYYC.

[0040] Preferably, the amino acid sequence of LCFR4 is as shown in SEQ ID No. 18: FGGGTKLEIKR.

[0041] Preferably, the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 5.

[0042] In certain embodiments of the present invention, the nucleotide sequence of the light chain variable region of the anti-B7-H3 antibody is as shown in SEQ ID No. 20.

[0043] The anti-B7-H3 antibody is designated as NP040. The amino acid sequence of the heavy chain variable region of the anti-B7-H3 antibody is as shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 5.

[0044] The anti-B7-H3 antibody is an antibody fragment or a full antibody.

[0045] The "antibody fragment" comprises a part of the full antibody, preferably comprising its antigen-binding region or variable region. For example, the antibody fragment is selected from nanobody (VHH), single-chain antibody (scFv), Fab, Fab', F(ab') or F(ab')2.

[0046] The "Fab" fragment comprises a complete light chain (VL + CL) as well as the heavy chain variable region (VH) and the first constant region (CH1). The Fab fragment is the part of the antibody molecule responsible for binding to the antigen.

[0047] One F(ab’) antibody fragment comprises a pair of Fab fragments, which are usually covalently linked near the carboxyl terminus by the hinge cysteines between them.

[0048] In certain embodiments of the present invention, the anti-B7-H3 antibody is a full antibody. The full antibody comprises a variable region and a constant region. The constant region comprises the heavy chain constant regions CH1, CH2, CH3 (when it is IgG, IgA, IgD) and / or CH4 (when it is IgM, IgE), and also comprises the light chain constant region (CL). The constant region can be a natural sequence constant region (such as a human natural sequence constant region) or an amino acid sequence variant thereof.

[0049] Preferably, the heavy chain constant region is an IgG1 constant region. Preferably, the amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 9.

[0050] Preferably, the light chain constant region is a κ-chain constant region. Preferably, the amino acid sequence of the light chain constant region is as shown in SEQ ID No. 10.

[0051] In certain embodiments of the present invention, the anti-B7-H3 antibody is a monoclonal antibody.

[0052] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the various antibodies that make up the population are identical, except for possible naturally occurring mutants that are present in trace amounts. Monoclonal antibodies are highly specific, i.e., they are directed against a single epitope on an antigen. In addition, unlike polyclonal antibody preparations that contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. In addition to their specificity, one advantage of monoclonal antibodies is that they can now be synthesized without contamination by other antibodies. The modifier "monoclonal" indicates the nature of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any particular method for producing the antibody.

[0053] The present invention also provides a recombinant protein, which contains the anti-B7-H3 antibody described above in the present invention and another polypeptide linked to the anti-B7-H3 antibody.

[0054] In certain embodiments of the present invention, the polypeptide is a tag sequence for assisting expression and / or purification. The tag sequence is selected from a Flag tag, a His tag, etc.

[0055] In certain embodiments of the present invention, the recombinant protein further includes a linker fragment that fuses the anti-B7-H3 antibody with the polypeptide.

[0056] The present invention also provides a chimeric antigen receptor, which includes an intracellular domain, a transmembrane domain, and an extracellular domain, and the antigen-binding region of the extracellular domain includes the anti-B7-H3 antibody described above or a fragment thereof.

[0057] "CAR" or "chimeric antigen receptor" refers to a fusion protein that includes an extracellular domain capable of binding an antigen, a transmembrane domain derived from a polypeptide different from the polypeptide from which the extracellular domain is derived, and at least one intracellular domain. "Chimeric antigen receptor" is sometimes also referred to as "chimeric receptor", "T-body" or "chimeric immunoreceptor (CIR)". "Extracellular domain capable of binding an antigen" refers to any oligopeptide or polypeptide that can bind a specific antigen. "Intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transmits signals in a cell to cause activation or inhibition of a biological process.

[0058] In certain embodiments of the present invention, the intracellular domain may include a signal transduction domain.

[0059] The signal transduction domain includes an immunoreceptor tyrosine-based activation motif. The immunoreceptor tyrosine-based activation motif may be selected from CD3ζ.

[0060] Preferably, the signal transduction domain further comprises a co-stimulatory molecule. For example, the co-stimulatory molecule can be selected from any one or a combination of at least two protein molecules such as 4-1BB, CD28, OX40, ICOS, DAP 10, etc. For another example, the sequence of 4-1BB can be referred to NM_001561, the sequence of CD28 can be referred to NM_006139, the sequence of OX40 can be referred to NM_003327, the sequence of ICOS can be referred to NM_012092, the sequence of CD3ζ can be referred to NM_198053, and the sequence of DAP 10 can be referred to NM_014266.

[0061] In a specific embodiment of the present invention, the intracellular domain sequentially comprises 4-1BB and CD3ζ from the N-terminus to the C-terminus.

[0062] In certain embodiments of the present invention, the transmembrane domain can be selected from any one or more transmembrane domains such as the CD8α transmembrane region, the CD28 transmembrane region, the DAP 10 transmembrane region, etc.

[0063] For another example, the sequence of CD8α can be referred to NM_001145873, the sequence of CD28 can be referred to NM_006139, and the sequence of DAP10 can be referred to NM_014266.

[0064] In certain embodiments of the present invention, the extracellular domain can include a signal peptide, an anti-B7-H3 antibody, and a hinge region.

[0065] In certain embodiments of the present invention, the signal peptide includes the CD8α signal peptide.

[0066] In certain embodiments of the present invention, the hinge region is selected from the CD8α hinge region.

[0067] In some specific embodiments of the present invention, the polypeptide sequentially comprises a CD8α signal peptide, an anti-B7-H3 antibody, a CD8α hinge region, a CD8α transmembrane region, a co-stimulatory molecule, and CD3ζ from the N-terminus to the C-terminus.

[0068] In some specific embodiments of the present invention, the polypeptide sequentially comprises a CD8α signal peptide, an anti-B7-H3 antibody, a CD8α hinge region, a CD8α transmembrane region, 4-1BB, and CD3ζ from the N-terminus to the C-terminus.

[0069] The present invention also provides a chimeric antigen receptor immune cell, and the immune cell expresses the foregoing chimeric antigen receptor. The recombinant immune cell generally refers to an immune cell modified by genetic engineering means so that it can more effectively recognize and attack specific pathogens or tumor cells. The immune cell can be an NK cell or a T cell.

[0070] The present invention also provides an antibody conjugate, which comprises the anti-B7-H3 antibody and an effector molecule. The antibody is conjugated to the effector molecule, preferably by chemical conjugation. Among them, the effector molecule is preferably a therapeutically active drug. In addition, the effector molecule can be one or more of a detectable label, a cytotoxin, a cytokine, an enzyme, a chemotherapeutic drug, a small molecule drug or a radionuclide.

[0071] The antibody of the present invention and the effector molecule can be conjugated through a coupling agent. Examples of the coupling agent can be any one or several of a non-selective coupling agent, a coupling agent using carboxyl groups, a peptide chain, and a coupling agent using disulfide bonds. The non-selective coupling agent is a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde, etc. The coupling agent using carboxyl groups can be any one or several of cis-aconitic anhydride coupling agents (such as cis-aconitic anhydride) and acylhydrazone coupling agents (the coupling site is acylhydrazone).

[0072] Certain residues on the antibody (such as Cys or Lys, etc.) are used to connect with various functional groups, including imaging reagents (such as chromophores and fluorophores), diagnostic reagents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers), and therapeutic agents. The antibody can be conjugated to a functional agent to form an antibody-functional agent conjugate. The functional agent (such as a drug, a detection reagent, a stabilizer) is conjugated (covalently linked) to the antibody. The functional agent can be directly or indirectly connected to the antibody through a linker.

[0073] The antibody can be conjugated with a drug to form an antibody-drug conjugate (ADC). Typically, the ADC contains a linker located between the drug and the antibody. The linker can be degradable or non-degradable. The degradable linker typically degrades easily in the intracellular environment. For example, the linker degrades at the target site, so that the drug is released from the antibody. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-based linkers that can be degraded by intracellular proteases (such as lysosomal proteases or endosomal proteases), or sugar linkers such as glucuronide-containing linkers that can be degraded by glucuronidase. The peptide-based linker can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (such as linkers that hydrolyze at a pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (such as disulfide bond linkers). The non-degradable linker typically releases the drug under the condition that the antibody is hydrolyzed by protease.

[0074] Prior to attachment to the antibody, the linker has reactive groups capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive groups. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodo-, bromo- or chloro-substituted); haloesters (e.g., iodo-, bromo- or chloro-substituted); halomethyl ketones (e.g., iodo-, bromo- or chloro-substituted), benzyl halides (e.g., iodo-, bromo- or chloro-substituted); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercuriomethyl) dioxane, and the counterion is acetate, chloride or nitrate; and polymethylene dimethyl thioether thiosulfonates. The linker can include, for example, a maleimide attached to the antibody through a thiobutanediimide.

[0075] The drug can be any cytotoxic, cell growth inhibitory or immunosuppressive drug. In certain embodiments, the linker attaches the antibody and the drug, and the drug has a functional group capable of bonding to the linker. For example, the drug can have an amino, carboxyl, thiol, hydroxyl, or keto group capable of bonding to the linker. In the case where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0076] Useful classes of drugs include, for example, anti-tubulin drugs, DNA minor groove binding reagents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, etc. In the present invention, the drug-linker can be used to form an ADC in a single step. In other embodiments, a bifunctional linker compound can be used to form an ADC in a two-step or multi-step process. For example, a cysteine residue reacts with the reactive portion of the linker in the first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0077] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently attached to the linker through a 1,3-dipolar cycloaddition between the azide and the alkynyl. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazines and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other conjugation strategies, such as those described in Bioconjugate Techniques, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for the selective reaction of the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of the complementary pair can be used for either the linker or the drug.

[0078] The present invention also provides an isolated polynucleotide encoding the anti-B7-H3 antibody, the recombinant protein, the chimeric antigen receptor, or the antibody conjugate.

[0079] In certain embodiments of the present invention, the polynucleotide comprises the nucleotide sequence shown in SEQ ID No. 19 or SEQ ID No. 20.

[0080] The present invention also provides a nucleic acid construct comprising the isolated polynucleotide.

[0081] The "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into a target cell or tissue. The nucleic acid construct can be various expression vectors, and the expression vectors include a vector backbone, i.e., an empty vector, and an expression frame.

[0082] The term "expression frame" refers to a sequence having the potential to encode a protein.

[0083] The types of expression vectors are not specifically limited. An expression vector refers to a nucleic acid molecule that allows the insertion of exogenous nucleotides without destroying the ability of the vector to replicate and / or integrate in a host cell. An expression vector may include a nucleic acid sequence that allows it to replicate in a host cell, such as an origin of replication. An expression vector may also include one or more selectable marker genes and other genetic factors. An expression vector is a vector that contains the necessary regulatory sequences to transcribe and translate one or more inserted genes. The expression vector is selected from eukaryotic expression vectors or prokaryotic expression vectors.

[0084] The prokaryotic expression vector is selected from Escherichia coli expression vectors, Bacillus subtilis expression vectors, or Streptomyces expression vectors.

[0085] The eukaryotic expression vector is selected from yeast expression vectors, insect expression vectors or mammalian expression vectors. The mammalian expression vector is a non-viral expression vector or a viral expression vector. The non-viral expression vector can be the pcDNA3.4 vector. The viral expression vector is selected from retroviral expression vectors, lentiviral expression vectors, adenoviral expression vectors, adeno-associated viral expression vectors. In a preferred embodiment, the eukaryotic expression vector is selected from retroviral expression vectors, and the retroviral expression vector can be stably expressed in cell lines. The retroviral vector is, for example, pMSCV.

[0086] The host cell is selected from eukaryotic host cells or prokaryotic host cells. Eukaryotic host cells are selected from fungi such as yeast, insects, birds, plants, Caenorhabditis elegans or nematodes, or mammalian host cells. Non-limiting examples of insect cells are Spodoptera frugiperda cells. Examples of yeast host cells are Saccharomyces cerevisiae, Kluyveromyces lactis or Yarrowia lipolytica. Examples of mammalian cells are COS cells, baby hamster kidney cells, mouse L cells, LNCaP cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, African green monkey cells, CV1 cells, Vero or Hep-2 cells. Examples of prokaryotic host cells include bacterial cells such as Escherichia coli, Streptomyces, Bacillus subtilis, Salmonella typhi or Mycobacterium.

[0087] Those skilled in the art can transfect the expression vector into the host cell according to methods well known in the art to obtain a cell containing the coding gene of the antibody of the present invention. For example, introducing the expression vector into eukaryotic cells can be carried out by calcium phosphate co-precipitation, electroporation, microinjection, liposome transfection or transfection using polyamine transfection reagents.

[0088] The present invention also provides an engineered cell, which contains the nucleic acid construct or the exogenous polynucleotide is integrated into the genome.

[0089] The present invention also provides a method for preparing the anti-B7-H3 antibody, comprising the following steps: culturing the engineered cell to express the anti-B7-H3 antibody, and purifying and isolating the anti-B7-H3 antibody.

[0090] The present invention also provides the uses of the anti-B7-H3 antibody, the recombinant protein, the chimeric antigen receptor, the immune cell, the antibody conjugate, the isolated polynucleotide, the nucleic acid construct, the engineered cell in the preparation or screening of therapeutic drugs.

[0091] The therapeutic drug can be a drug that targets the B7-H3 antigen, binds to or acts on the B7-H3 antigen, thereby treating indications.

[0092] In certain embodiments of the present invention, the therapeutic drug may be an anti-tumor drug. The anti-tumor drug may target the B7-H3 antigen functionally expressed on the surface of tumor cells, bind to or act on the B7-H3 antigen, thereby treating and / or preventing tumors. The tumors may be tumors with positive B7-H3 expression such as non-small cell lung cancer, pancreatic cancer, renal cell carcinoma, brain tumors, melanoma, leukemia, breast cancer, prostate cancer, etc.

[0093] The following specific examples illustrate the embodiments of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0094] Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments, rather than limiting the protection scope of the present invention; in the specification and claims of the present invention, unless otherwise clearly indicated in the text, the singular forms "a", "an" and "the" include plural forms.

[0095] When the embodiments give a numerical range, it should be understood that unless otherwise stated in the present invention, any value at both ends of each numerical range and any value between the two ends can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art of this technology. In addition to the specific methods, devices, and materials used in the embodiments, according to the knowledge of those skilled in the art of this technology and the description of the present invention, any methods, devices, and materials similar or equivalent to the methods, devices, and materials described in the embodiments of the present invention can also be used to implement the present invention. Example 1 Construction and Expression of Mouse Anti-Human B7-H3 Antibody

[0096] To generate an antibody against human B7-H3, high-affinity specific antibodies were obtained from a phage antibody library through a solid-phase screening method. Through the structural analysis of the B7-H3 protein and its antibody, and with the assistance of computer-aided design, a series of humanized sequences were obtained.

[0097] The VH (nucleotide sequence shown in SEQ ID NO:19) and VL (nucleotide sequence shown in SEQ ID NO:20) of NP040 were respectively constructed onto the pcDNA3.1 vector (Youbao Biotech, product number VT1001) containing the hIgG1 κ constant region (amino acid sequences shown in SEQ ID NO:9 and 10). VH and VL were co-transfected into 293F cells. After culturing for 3 - 5 days, the supernatant was collected and purified using a protein A column to obtain the antibody protein corresponding to NP040. The expression situation is as Figure 1 shown: The lane of 3.0 in the figure represents the gel image of NP040 under the reducing system, and it can be seen that the sizes are 58.2 & 25.6 kd, which is consistent with the theoretical molecular weight; the N lane is the gel image of NP040 under the non-reducing system; MK is the protein molecular weight marker.

[0098] NP040 VH amino acid sequence (SEQ ID NO:1) QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDTSASTAYMELSSLRSEDTAVYYCTRGGNYISFAMDYWGQGTSVTVSS NP040 HCDR1 amino acid sequence (SEQ ID NO:2) GYTFTSYW NP040 HCDR2 amino acid sequence (SEQ ID NO:3) IYPSDSYT NP040 HCDR3 amino acid sequence (SEQ ID NO:4) TRGGNYISFAMDY NP040 VL amino acid sequence (SEQ ID NO: 5) DIVMTQSPSTLSASVGDRVTITCKASQGVSTAVAWYQQKPGKAPKILIHWASTRHTGVPDRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYNTPYTFGGGTKLEIKR NP040 LCDR1 amino acid sequence (SEQ ID NO:6) QGVSTA NP040 LCDR1 amino acid sequence (SEQ ID NO:7) WAS NP040 LCDR1 amino acid sequence (SEQ ID NO:8) QQHYNTPYT Amino acid sequence of the hIgG1 constant region (SEQ ID NO: 9) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Amino acid sequence of the κ-chain constant region (SEQ ID NO: 10) TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC VH nucleotide sequence of NP040 (SEQ ID NO:19) CAAGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGCGCTAGCGTGAAGGTGTCCTGTAAAGCCTCTGGCTACACATTCACCAGCTATTGGATCAACTGGGTGCGGCAGGCCCCTGGCCAGGGCCTGGAATGGATCGGCAACATCTACCCTAGCGACAGCTACACCAACTACAACCAGAAGTTCAAGGATAAGGCTACACTGACCGTGGACACCTCCGCCTCTACAGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGATACCGCCGTGTACTACTGCACCAGAGGCGGAAATTACATCAGCTTTGCCATGGACTACTGGGGACAGGGCACATCTGTGACCGTTTCCAGC VL nucleotide sequence of NP040 (SEQ ID NO:20) GATATCGTGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTAAAGCTTCTCAGGGCGTGTCCACAGCCGTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCTAAGATCCTGATCCACTGGGCCTCTACAAGACACACCGGCGTCCCAGATAGATTCAGCGGCAGCGGCTCCGGAACAGACTACACCCTGACCATTAGCAGCCTGCAGCCTGAGGACTTCGCTACATACTACTGCCAGCAACACTACAACACCCCTTATACATTTGGAGGCGGCACCAAGCTGGAAATCAAGCGG Example 2 Detection of the affinity of NP040 antibody with different B7-H3 proteins

[0099] This example mainly shows the affinity of NP040 with recombinant human B7-H3 protein and recombinant monkey B7-H3 protein.

[0100] SPR (Optical Surface Plasmon Resonance) is a commonly used method for molecular interaction analysis. The principle is that the antigen or antibody to be detected flows over the surface of the sensing chip. If there are molecules in the sample that can interact with the biomolecular recognition membrane on the chip surface, it will cause a change in the refractive index of the membrane surface, ultimately leading to a change in the SPR angle. By detecting the change in the SPR angle, the affinity between NP040 antibody and human B7-H3 and monkey B7-H3 proteins is detected. The affinity results are shown in Figure 2 、 Figure 3 。The Ka between NP040 antibody and recombinant human B7-H3 protein is 2.30E+05 (1 / Ms), Kd is 2.25E-04 (1 / s), and KD is 9.76E-10 M. The Ka between NP040 antibody and recombinant monkey B7-H3 protein is 1.24E+05 (1 / Ms), Kd is 3.62E-04 (1 / s), and KD is 2.91E-09 M. The results show that NP040 has high affinity with recombinant human B7-H3 and recombinant monkey B7-H3 proteins respectively. Example 3 Detection of the cell binding activity of anti-B7-H3 antibody

[0101] This example mainly demonstrates the binding ability of the NP040 anti-B7-H3 antibody of the present invention and the commercial anti-B7-H3 antibody proteins ibruxolizumab, obinutuzumab, and DS-7300 to B7-H3 positive cells HCC827 (human non-small cell lung cancer cells), NCI-H146 (human small cell lung cancer cells), HT-29 (human colorectal cancer cells), MCF-7 (human breast cancer cells), and A549 (human non-small cell lung cancer cells) with different expression abundances.

[0102] Take 3×10 5 target cells with different abundances, add gradient-diluted anti-B7-H3 antibody proteins, incubate for 1 h, wash once with PBS, add anti-hFc-APC (purchased from Jackson immunology), and perform flow cytometry detection after washing once with PBS. The S curve is plotted as shown in Figures 4 to 8 . The results show that NP040 has high cell binding activity with HCC827 (human non-small cell lung cancer cells), NCI-H146 (human small cell lung cancer cells), HT-29 (human colorectal cancer cells), MCF-7 (human breast cancer cells), and A549 (human non-small cell lung cancer cells). Example 4 Detection of Non-specific Binding of Antibody to B7-H3 Homologous Proteins

[0103] This example mainly demonstrates the affinity of NP040 for recombinant human B7-H2, PD-L2, and B7-2 proteins, and the affinity for B7-1 positive cells CHOK1-B7-1 cells and PD-L1 positive cells CHOK1-PD-L1 cells.

[0104] 1) Coat the plate with NP040 at 3 μg / ml, add gradient-diluted recombinant human B7-H2 (Novoprotein, Cat. No.: C34K), PD-L2 (Novoprotein, Cat. No.: CW20), and B7-2 (Novoprotein, Cat. No.: C475) proteins, and detect the binding of the antibody to homologous proteins by ELISA. The results are shown in Figures 9 to 11 . The results show that there is no non-specific binding of NP040 to recombinant human B7-H2, PD-L2, and B7-2 proteins.

[0105] 2) Binding to B7-1 and PD-L1 positive cells Construct the full-length plasmids of B7-1 (UniProtKB, P33681) and PD-L1 (UniProtKB, Q9NZQ7) and transiently transfect them into CHOK1 cells, and detect their non-specific binding to NP040 by flow cytometry. The specific steps are as follows: Take 4×10 5CHOK1-B7-1 cells and CHOK1-PD-L1 cells were added with serially diluted NP040. After incubation for 1 h, they were washed 3 times with PBS, and anti-hFC-APC (purchased from Jackson immunology) was added, followed by flow cytometry analysis. The S curve was plotted as shown in Figures 12 to 13 , and the results showed that there was no non-specific binding between NP040 and B7-1 positive cells (CHOK1-B7-1 cells) or PD-L1 positive cells (CHOK1-PD-L1 cells). Example 5 Internalization Mediated by Anti-B7-H3 Antibody

[0106] This example mainly demonstrated the internalization effect of anti-B7-H3 antibody on positive cells with different expression abundances. The specific steps were as follows: The internalization reagent was conjugated to serially diluted antibodies using iQue® Human Antibody Internalization Reagent (Sartorius, CAT NO: 90564), and then incubated with HCC827 (human non-small cell lung cancer cells), NCI-H146 (human small cell lung cancer cells), HT-29 (human colorectal cancer cells), MCF-7 (human breast cancer cells), and A549 (human non-small cell lung cancer cells) for 2 h. The positive rate was detected by flow cytometry, and the calculated results are shown in Figures 14 to 18 .

[0107] The results showed that NP040 had a high internalization effect on HCC827 (human non-small cell lung cancer cells), NCI-H146 (human small cell lung cancer cells), HT-29 (human colorectal cancer cells), MCF-7 (human breast cancer cells), and A549 (human non-small cell lung cancer cells). Example 6 Detection of the Binding Epitope of Anti-B7-H3 Antibody

[0108] To verify the NP040 binding epitopes, five extracellular domains of the B7-H3 protein were recombinantly constructed and expressed respectively: Ig-like V-type 1 (uniprot: Q5ZPR3 POSITION 29-139), Ig-like C2-type 1 (uniprot: Q5ZPR3 POSITION 145-238), Ig-like V-type 2 (uniprot: Q5ZPR3 POSITION 243-357), Ig-like C2-type 2 (uniprot: Q5ZPR3 POSITION 363-456), Ig-like V-type 1 + Ig-like C2-type 2 (uniprot: Q5ZPR3 POSITION 29-139 / 363-456). The recombinant extracellular domain proteins were plated at 5 μg / ml, and gradient-diluted NP040 was added. The binding of the antibody to each extracellular domain protein was detected by ELISA, and the results are shown in Table 1. The results indicate that NP040 binds to different epitopes of the antigens bound by DS7300, epratuzumab, and obinutuzumab. NP040 mainly binds to the Ig-like V-type domain segment.

[0109] Table 1 Detection of NP040 Binding Epitopes

[0110] Note: "+++" represents strong cell binding ability, "++" represents medium cell binding ability, "+" represents low cell binding ability, and "-" represents no or very low affinity for cells.

[0111] The above embodiments are for illustrating the embodiments disclosed in the present invention and should not be construed as limitations on the present invention. In addition, various modifications listed herein and changes in the methods of the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, all obvious modifications to those skilled in the art as described above to obtain the invention should be included within the scope of the present invention.

Claims

1. An anti-B7-H3 antibody, characterized in that, The anti-B7-H3 antibody comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises CDR-H1 with an amino acid sequence as shown in SEQ ID No.2, CDR-H2 with an amino acid sequence as shown in SEQ ID No.3, and CDR-H3 with an amino acid sequence as shown in SEQ ID No.

4. The light chain variable region comprises CDR-L1 with an amino acid sequence as shown in SEQ ID No.6, CDR-L2 with an amino acid sequence as shown in SEQ ID No.7, and CDR-L3 with an amino acid sequence as shown in SEQ ID No.

8.

2. The anti-B7-H3 antibody according to claim 1, wherein The heavy chain variable region further comprises framework regions HCFR1 to HCFR4, and the amino acid sequences of the framework regions HCFR1 to HCFR4 are the sequences shown in SEQ ID Nos. 11 to 14 respectively, and / or the light chain variable region further comprises framework regions LCFR1 to LCFR4, and the amino acid sequences of the framework regions LCFR1 to LCFR4 are the sequences shown in SEQ ID Nos. 15 to 18 respectively.

3. The anti-B7-H3 antibody according to claim 1, wherein The amino acid sequence of the heavy chain variable region of the anti-B7-H3 antibody is as shown in SEQ ID No.1, and / or the amino acid sequence of the light chain variable region is as shown in SEQ ID No.

5.

4. The anti-B7-H3 antibody according to claim 1, wherein, The anti-B7-H3 antibody is an antibody fragment or a complete antibody. The complete antibody comprises a variable region and a constant region. The constant region comprises a heavy chain constant region and a light chain constant region. The heavy chain constant region is an IgG1 constant region, or the light chain constant region is a κ chain constant region.

5. The anti-B7-H3 antibody according to claim 4, wherein, The amino acid sequence of the heavy chain constant region is as shown in SEQ ID NO: 9, and / or the amino acid sequence of the light chain constant region is as shown in SEQ ID No.

10.

6. A recombinant protein, characterized in that, The recombinant protein contains the anti-B7-H3 antibody according to any one of claims 1 to 5 and another polypeptide linked to the anti-B7-H3 antibody.

7. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises an intracellular domain, a transmembrane domain and an extracellular domain. The antigen-binding region of the extracellular domain comprises the anti-B7-H3 antibody according to any one of claims 1 to 5 or a fragment thereof.

8. A chimeric antigen receptor immune cell, characterized in that, The immune cell expresses the chimeric antigen receptor according to claim 7.

9. An antibody conjugate, characterized in that, The antibody conjugate comprises the B7-H3 antibody according to any one of claims 1 to 5 and an effector molecule, and the antibody is conjugated to the effector molecule.

10. The antibody conjugate according to claim 9, wherein, The effector molecule is selected from one or more of a detectable label, a cytotoxin, a cytokine, an enzyme, a chemotherapeutic drug, a small molecule drug or a radionuclide.

11. An isolated polynucleotide, characterized in that, Encoding the anti-B7-H3 antibody according to any one of claims 1 to 5, the recombinant protein according to claim 6, the chimeric antigen receptor according to claim 7 or the antibody conjugate according to claim 9 or 10.

12. A nucleic acid construct, characterized in that, Containing the isolated polynucleotide according to claim 11.

13. An engineered cell, characterized in that, The engineered cell contains the nucleic acid construct according to claim 12 or the exogenous polynucleotide according to claim 11 is integrated into the genome.

14. Use of the anti-B7-H3 antibody according to any one of claims 1 to 5, the recombinant protein according to claim 6, the chimeric antigen receptor according to claim 7, the immune cell according to claim 8, the antibody conjugate according to claim 9 or 10, the isolated polynucleotide according to claim 11, the nucleic acid construct according to claim 12, and the engineered cell according to claim 13 in the preparation or screening of a therapeutic drug.

15. The use according to claim 14, wherein The therapeutic drug is a therapeutic drug for tumors with positive B7-H3 expression.

16. The use according to claim 15, characterized in that, The tumors are selected from non-small cell lung cancer, pancreatic cancer, renal cell carcinoma, brain tumor, melanoma, leukemia, breast cancer or prostate cancer.

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