An anti-B7-H3 antibody and its use

Abstract

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

Description

Technical Field

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

[0002] B7-H3 (CD276) is a type I transmembrane protein and a member of the B7 immune co-stimulatory and co-inhibitory family. Its ligand remains unknown. The B7-H3 protein is encoded by a gene on chromosome 15q24 and consists of 316 amino acids encompassing an extracellular domain, a transmembrane domain, and a short intracellular domain. The intracellular domain of the B7-H3 protein is very short, with no known signaling motif. Furthermore, due to exon duplication, the human B7-H3 protein contains one or two identical extracellular domains, resulting in two isoforms: 2IgB7-H3, which consists of a pair of immunoglobulin variable (IgV)-like and immunoglobulin constant (IgC)-like extracellular domains; and 4IgB7-H3, which contains two identical pairs of IgV-like and IgC-like extracellular domains and is the predominant isoform in human cells.

[0003] B7-H3 is primarily expressed on the surfaces of non-immune cells such as fibroblasts, endothelial cells, osteoblasts, and amniotic fluid stem cells, as well as activated antigen-presenting cells and natural killer (NK) cells. Studies have shown that B7-H3 is overexpressed in a variety of tumor cells, including melanoma, leukemia, breast cancer, and prostate cancer, and its overexpression is closely associated with factors such as tumor growth, metastasis, recurrence, and poor prognosis. B7-H3 can downregulate T helper type 1-mediated immune responses, inhibit CD4+ T cell activation, and suppress cytokine production, potentially promoting immune escape in cancer cells.

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

[0005] B7-H3 human cancer immunotherapy strategies mainly include:

[0006] 1) Blocking B7-H3 with mAbs to neutralize inhibitory signals in T cells, NK cells, and other immune cells;

[0007] 2) Fc receptors of NK cells and other immune cells engage to trigger B7-H3-specific ADCC, inducing tumor cell death;

[0008] 3) The CD3 / B7-H3 bispecific antibody binds to tumor-expressed B7-H3, crosslinks the CD3 portion of the TCR complex, activates T cells in the tumor microenvironment, and leads to tumor cell death;

[0009] 4) Small molecule inhibitors can bind to specific regions of B7-H3, such as the FG loop of the IgV domain, inhibiting the ligand-receptor interaction between tumor cells and immune cells, thereby blocking receptor signaling and restoring the effector function of immune cells;

[0010] 5) CAR-T cells recognize membrane B7-H3 and directly kill tumor cells. Summary of the Invention

[0011] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide an anti-B7-H3 antibody and its use to solve the problems in the prior art.

[0012] To achieve the above objectives and other related objectives, the present invention provides an anti-B7-H3 antibody, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 with an amino acid sequence as shown in SEQ ID No. 2, a CDR-H2 with an amino acid sequence as shown in SEQ ID No. 3, and a CDR-H3 with an amino acid sequence as shown in SEQ ID No. 4; and the light chain variable region comprises a CDR-L1 with an amino acid sequence as shown in SEQ ID No. 6, a CDR-L2 with an amino acid sequence as shown in SEQ ID No. 7, and a CDR-L3 with an amino acid sequence as shown in SEQ ID No. 8.

[0013] The present invention also provides a recombinant protein comprising the aforementioned anti-B7-H3 antibody of the present invention and another polypeptide linked to the anti-B7-H3 antibody.

[0014] The present invention also provides a chimeric antigen receptor, which comprises an intracellular domain, a transmembrane domain and an extracellular domain, wherein the antigen binding region of the extracellular domain comprises the aforementioned anti-B7-H3 antibody or a fragment thereof.

[0015] The present invention also provides a chimeric antigen receptor immune cell, which expresses the aforementioned chimeric antigen receptor. Recombinant immune cells generally refer to immune cells modified by genetic engineering to enable them to more effectively recognize and attack specific pathogens or tumor cells. The immune cell can be a NK cell or a T cell.

[0016] The present invention also provides an antibody conjugate, comprising the B7-H3 antibody and an effector molecule, wherein the antibody and the effector molecule are coupled, preferably chemically coupled.

[0017] 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.

[0018] The present invention also provides a nucleic acid construct comprising the isolated polynucleotide.

[0019] The present invention also provides an engineered cell, wherein the engineered cell contains the nucleic acid construct or the exogenous polynucleotide is integrated into the genome.

[0020] The present invention also provides a method for preparing the anti-B7-H3 antibody, comprising the following steps: culturing the engineered cells to express the anti-B7-H3 antibody, and purifying and isolating the anti-B7-H3 antibody.

[0021] The present invention also provides 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 preparing or screening therapeutic drugs.

[0022] As described above, the anti-B7-H3 antibodies and uses thereof of the present invention have the following beneficial effects:

[0023] 1. The obtained antibodies have high affinity and specificity for human and monkey B7-H3 proteins;

[0024] 2. The obtained antibodies have high binding activity on cancer cells of different abundances, and are higher than the selected clinical or marketed drugs;

[0025] 3. The obtained antibodies showed high internalization on cancer cells with different abundances;

[0026] 4. Possessing epitopes that are different from those of the selected clinical or marketed drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Shown is the expression of NP040.

[0028] Figure 2 Shown are the SPR results of affinity between NP040 of the present invention and human B7-H3 protein.

[0029] Figure 3 Shown are the affinity SPR results of NP040 of the present invention and monkey B7-H3 protein.

[0030] Figures 4 to 8 Shown are the results of NP040 binding to cells with different expression abundances.

[0031] Figures 9 to 11 Shown are the affinity results of NP040 with recombinant human B7-H2, PD-L2, and B7-2 proteins.

[0032] Figure 12 and Figure 13Shown are the results of NP040 binding to B7-1 and PD-L1 positive cells.

[0033] Figures 14 to 18 Shown are the internalization results of NP040 in cells with different expression abundances. DETAILED DESCRIPTION

[0034] The present invention provides an anti-B7-H3 antibody, which comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 with an amino acid sequence as shown in SEQ ID No. 2, a CDR-H2 with an amino acid sequence as shown in SEQ ID No. 3, and a CDR-H3 with an amino acid sequence as shown in SEQ ID No. 4; and the light chain variable region comprises a CDR-L1 with an amino acid sequence as shown in SEQ ID No. 6, a CDR-L2 with an amino acid sequence as shown in SEQ ID No. 7, and a CDR-L3 with an amino acid sequence as shown in SEQ ID No. 8.

[0035] The numbering and definition schemes were based on the IMGT method for annotating CDR and FR regions.

[0036] CDRs (complementarity determining regions) generally refer to regions within an antibody that are spatially complementary to antigenic determinants. The variability within an antibody is typically not evenly distributed throughout its variable region. The heavy chain variable region of a monoclonal antibody typically has three hypervariable regions (HVRs). These regions are spatially complementary to antigenic determinants, and are therefore also known as complementarity determining regions (CDRs). The heavy chain variable region typically includes three CDRs: CDR-H1, CDR-H2, and CDR-H3.

[0037] In certain embodiments of the present invention, the heavy chain variable region may further include a framework region, which may be located between the complementarity determining regions or at both ends of the complementarity determining regions. In certain specific embodiments of the present invention, the sequence of the framework region is a human, rabbit, or mouse monoclonal antibody variable region.

[0038] In certain embodiments of the present invention, the heavy chain variable region further comprises framework regions HCFR1 to HCFR4, wherein the amino acid sequence of the framework regions HCFR1 to HCFR4 is selected from any of the sequences shown in SEQ ID Nos. 11 to 14.

[0039] Preferably, the amino acid sequence of HCFR1 is shown in SEQ ID No. 11:

[0040] QVQLVQSGAEVKKPGASVKVSCKAS.

[0041] Preferably, the amino acid sequence of HCFR2 is shown in SEQ ID No. 12:

[0042] INWVRQAPGQGLEWIGN.

[0043] Preferably, the amino acid sequence of HCFR3 is shown in SEQ ID No. 13:

[0044] NYNQKFKDKATLTVDTSASTAYMELSSLRSEDTAVYYC.

[0045] Preferably, the amino acid sequence of HCFR4 is shown in SEQ ID No. 14:

[0046] WGQGTSVTVSS.

[0047] Preferably, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 1.

[0048] In certain embodiments of the present invention, the nucleotide sequence of the heavy chain variable region of the anti-B7-H3 antibody is shown in SEQ ID No. 19.

[0049] In certain embodiments of the present invention, the light chain variable region further comprises framework regions LCFR1 to LCFR4, wherein the amino acid sequence of the framework regions LCFR1 to LCFR4 is selected from any of the sequences shown in SEQ ID Nos. 15 to 18.

[0050] Preferably, the amino acid sequence of LCFR1 is shown in SEQ ID No. 15:

[0051] DIVMTQSPSTLSASVGDRVTITCKAS.

[0052] Preferably, the amino acid sequence of LCFR2 is shown in SEQ ID No. 16:

[0053] VAWYQQKPGKAPKILIH.

[0054] Preferably, the amino acid sequence of the LCFR3 is shown in SEQ ID No. 17:

[0055] TRHTGVPDRFSGSGSGTDYTLTISSLQPEDFATYYC.

[0056] Preferably, the amino acid sequence of the LCFR4 is shown in SEQ ID No. 18:

[0057] FGGGTKLEIKR.

[0058] Preferably, the amino acid sequence of the light chain variable region is shown as SEQ ID No.5.

[0059] In certain embodiments of the present invention, the nucleotide sequence of the light chain variable region of the anti-B7-H3 antibody is shown as SEQ ID No. 20.

[0060] The anti-B7-H3 antibody is coded as NP040. The amino acid sequence of the heavy chain variable region of the anti-B7-H3 antibody is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 5.

[0061] The anti-B7-H3 antibody is an antibody fragment or a complete antibody.

[0062] The "antibody fragment" comprises a portion of an intact antibody, preferably comprising its antigen-binding region or variable region. For example, the antibody fragment is selected from a nanobody (VHH), a single-chain antibody (scFv), Fab, Fab', F(ab') or F(ab')2.

[0063] The "Fab" fragment includes 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.

[0064] An F(ab') antibody fragment comprises a pair of Fab fragments, which are typically covalently linked near the carboxyl termini via hinge cysteines between them.

[0065] In certain embodiments of the present invention, the anti-B7-H3 antibody is an intact antibody, comprising a variable region and a constant region, wherein the constant region comprises the heavy chain constant regions CH1, CH2, CH3 (for IgG, IgA, IgD) and / or CH4 (for IgM, IgE), and also comprises a light chain constant region (CL). The constant region may be a native sequence constant region (e.g., a human native sequence constant region) or an amino acid sequence variant thereof.

[0066] Preferably, the heavy chain constant region is an IgG1 constant region. Preferably, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 9.

[0067] Preferably, the light chain constant region is a κ chain constant region. Preferably, the amino acid sequence of the light chain constant region is shown in SEQ ID No. 10.

[0068] In certain embodiments of the present invention, the anti-B7-H3 antibody is a monoclonal antibody.

[0069] The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutants that are usually present in very small amounts. Monoclonal antibodies are highly specific, i.e., being directed against a single epitope on an antigen. Furthermore, unlike polyclonal antibody preparations that contain different antibodies directed against different determinative regions (epitopes), each monoclonal antibody is directed against a single determinative region on the 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 property of the antibody being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring any particular method for producing the antibody.

[0070] The present invention also provides a recombinant protein comprising the aforementioned anti-B7-H3 antibody of the present invention and another polypeptide linked to the anti-B7-H3 antibody.

[0071] In certain embodiments of the present invention, the polypeptide is a tag sequence for facilitating expression and / or purification, and the tag sequence is selected from a Flag tag, a His tag, and the like.

[0072] In certain embodiments of the present invention, the recombinant protein further comprises a linker fragment that fuses the anti-B7-H3 antibody and the polypeptide together.

[0073] The present invention also provides a chimeric antigen receptor, which comprises an intracellular domain, a transmembrane domain and an extracellular domain, wherein the antigen binding region of the extracellular domain comprises the aforementioned anti-B7-H3 antibody or a fragment thereof.

[0074] "CAR" or "chimeric antigen receptor" refers to a fusion protein comprising an extracellular domain capable of binding to 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 a "chimeric receptor," "T-body," or "chimeric immunoreceptor (CIR)." An "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide that can bind to a specific antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function in a cell as a domain that transmits signals to cause activation or inhibition of a biological process.

[0075] In certain embodiments of the present invention, the intracellular domain may include a signal transduction domain.

[0076] The signal transduction domain includes an immunoreceptor tyrosine-based activation motif. The immunoreceptor tyrosine-based activation motif can be selected from CD3ζ.

[0077] Preferably, the signal transduction domain further includes a costimulatory molecule. For example, the costimulatory molecule can be selected from any one of 4-1BB, CD28, OX40, ICOS, DAP 10 or a combination of at least two protein molecules. For another example, the sequence of 4-1BB can refer to NM_001561, the sequence of CD28 can refer to NM_006139, the sequence of OX40 can refer to NM_003327, the sequence of ICOS can refer to NM_012092, the sequence of CD3ζ can refer to NM_198053, and the sequence of DAP 10 can refer to NM_014266.

[0078] In a specific embodiment of the present invention, the intracellular domain includes 4-1BB and CD3 ζ in sequence from N-terminus to C-terminus.

[0079] In certain embodiments of the present invention, the transmembrane domain can be selected from any one or more transmembrane domains of CD8α transmembrane region, CD28 transmembrane region, DAP 10 transmembrane region, etc.

[0080] For another example, the sequence of CD8α can be referenced to NM_001145873, the sequence of CD28 can be referenced to NM_006139, and the sequence of DAP10 can be referenced to NM_014266.

[0081] In certain embodiments of the present invention, the extracellular domain may include a signal peptide, an anti-B7-H3 antibody, and a hinge region.

[0082] In certain embodiments of the present invention, the signal peptide comprises a CD8α signal peptide.

[0083] In certain embodiments of the present invention, the hinge region is selected from the CD8α hinge region.

[0084] In some specific embodiments of the present invention, the polypeptide includes, from N-terminus to C-terminus, a CD8α signal peptide, an anti-B7-H3 antibody, a CD8α hinge region, a CD8α transmembrane region, a co-stimulatory molecule, and CD3ζ.

[0085] In some specific embodiments of the present invention, the polypeptide includes, from N-terminus to C-terminus, a CD8α signal peptide, an anti-B7-H3 antibody, a CD8α hinge region, a CD8α transmembrane region, 4-1BB, and CD3ζ.

[0086] The present invention also provides a chimeric antigen receptor immune cell, which expresses the aforementioned chimeric antigen receptor. Recombinant immune cells generally refer to immune cells modified by genetic engineering to enable them to more effectively recognize and attack specific pathogens or tumor cells. The immune cell can be a NK cell or a T cell.

[0087] The present invention also provides an antibody conjugate comprising the anti-B7-H3 antibody and an effector molecule, wherein the antibody and the effector molecule are conjugated, preferably chemically coupled. The effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule can be one or more of a detectable marker, a cytotoxin, a cytokine, an enzyme, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0088] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent may include any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group may include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0089] Certain residues on antibodies (e.g., Cys or Lys) are used to attach a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. The functional agent (e.g., drug, detection reagent, stabilizer) is conjugated (covalently linked) to the antibody. The functional agent can be attached to the antibody directly or indirectly via a linker.

[0090] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). ADCs typically contain a linker positioned between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers typically readily degrade under intracellular conditions, for example, at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including peptidyl linkers degradable by intracellular proteases (e.g., lysosomal or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers degradable by glucuronidases. Peptidyl linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH below 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug when the antibody is hydrolyzed by proteases.

[0091] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0092] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In certain embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0093] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. In the present invention, drug-linkers can be used to form ADCs in a single step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, a cysteine ​​residue reacts with a reactive portion of a linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0094] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides 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 linking strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that, for selective reaction between 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 both the linker and the drug.

[0095] 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.

[0096] In certain embodiments of the present invention, the polynucleotide comprises a nucleotide sequence as shown in SEQ ID No. 19 or SEQ ID No. 20.

[0097] The present invention also provides a nucleic acid construct comprising the isolated polynucleotide.

[0098] The "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into target cells or tissues. The nucleic acid construct can be various expression vectors, and the expression vector includes a vector skeleton, i.e., an empty vector, and an expression frame.

[0099] The term "expression cassette" refers to a sequence that has the potential to encode a protein.

[0100] The type of expression vector is not specifically limited. An expression vector refers to a nucleic acid molecule that allows the insertion of exogenous nucleotides without disrupting the vector's ability 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 enable transcription and translation of an inserted gene or genes. The expression vector is selected from a eukaryotic expression vector or a prokaryotic expression vector.

[0101] The prokaryotic expression vector is selected from an Escherichia coli expression vector, a Bacillus subtilis expression vector or a Streptomyces expression vector.

[0102] The eukaryotic expression vector is selected from a yeast expression vector, an insect expression vector, or a mammalian expression vector. The mammalian expression vector is a non-viral expression vector or a viral expression vector. The non-viral expression vector may be a pcDNA3.4 vector. The viral expression vector is selected from a retroviral expression vector, a lentiviral expression vector, an adenoviral expression vector, and an adeno-associated viral expression vector. In a preferred embodiment, the eukaryotic expression vector is selected from a retroviral expression vector, which can be stably expressed in a cell line, and an example of a retroviral vector is pMSCV.

[0103] The host cell is selected from a eukaryotic host cell or a prokaryotic host cell. The eukaryotic host cell is selected from a fungus such as yeast, an insect, a bird, a plant, a nematode or a nematode or a mammalian host cell. A non-limiting example of an insect cell is a Spodoptera frugiperda cell. 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.

[0104] Those skilled in the art can transfect the expression vector into host cells according to methods well known in the art to obtain cells containing the gene encoding the antibody of the present invention. For example, the expression vector can be introduced into eukaryotic cells by calcium phosphate coprecipitation, electroporation, microinjection, lipofection, or transfection using a polyamine transfection reagent.

[0105] The present invention also provides an engineered cell, wherein the engineered cell contains the nucleic acid construct or the exogenous polynucleotide is integrated into the genome.

[0106] The present invention also provides a method for preparing the anti-B7-H3 antibody, comprising the following steps: culturing the engineered cells to express the anti-B7-H3 antibody, and purifying and isolating the anti-B7-H3 antibody.

[0107] The present invention also provides 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 preparing or screening therapeutic drugs.

[0108] The therapeutic drug can be a drug that uses the B7-H3 antigen as a target and binds to or acts on the B7-H3 antigen to treat the indicated disease.

[0109] In certain embodiments of the present invention, the therapeutic drug may be a tumor therapeutic drug. The tumor therapeutic drug may target the B7-H3 antigen functionally expressed on the surface of tumor cells, binding to or acting on the B7-H3 antigen to thereby treat and / or prevent tumors. The tumor may be a B7-H3-positive tumor, such as non-small cell lung cancer, pancreatic cancer, renal cell carcinoma, brain tumor, melanoma, leukemia, breast cancer, or prostate cancer.

[0110] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0111] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0112] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0113] Example 1 Construction and expression of mouse anti-human B7-H3 antibody

[0114] To generate antibodies against human B7-H3, we used solid-phase screening to obtain high-affinity, specific antibodies from a phage antibody library. We then conducted computer-assisted humanization design based on the structural analysis of the B7-H3 protein and its antibodies, resulting in a series of humanized sequences.

[0115] The VH (nucleotide sequence shown in SEQ ID NO: 19) and VL (nucleotide sequence shown in SEQ ID NO: 20) of NP040 were constructed into the pcDNA3.1 vector (Ubao Bio, product number VT1001) containing the hIgG1 κ constant region (amino acid sequence shown in SEQ ID NO: 9 and 10). VH and VL were co-transfected into 293F cells. After 3-5 days of culture, the supernatant was collected and purified using a protein A column to obtain the antibody protein corresponding to NP040. The expression status is shown in Figure 2. Figure 1 As shown: Lane 3.0 in the figure represents the gel image of NP040 under a reducing system, and the sizes are 58.2 & 25.6 kd, which are consistent with the theoretical molecular weight; Lane N is the gel image of NP040 under a non-reducing system; MK is a protein molecular weight marker.

[0116] NP040 VH amino acid sequence (SEQ ID NO: 1)

[0117] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWINWVRQAPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDTSASTAYMELSSLRSEDTAVYYCTRGGNYISFAMDYWGQGTSVTVSS

[0118] Amino acid sequence of NP040 HCDR1 (SEQ ID NO: 2)

[0119] GYTFTSYW

[0120] Amino acid sequence of NP040 HCDR2 (SEQ ID NO: 3)

[0121] IYPSDSYT

[0122] Amino acid sequence of NP040 HCDR3 (SEQ ID NO: 4)

[0123] TRGGNYISFAMDY

[0124] NP040 VL amino acid sequence (SEQ ID NO: 5)

[0125] DIVMTQSPSTLASVGDRVTITCKASQGVSTAVAWYQQKPGKAPKILIHWASTRHTGVPDRFSGSGSGTDYTLTISSLQPEDFATYYCQQHYNTPYTFGGGTKLEIKR

[0126] Amino acid sequence of NP040 LCDR1 (SEQ ID NO: 6)

[0127] QGVSTA

[0128] Amino acid sequence of NP040 LCDR1 (SEQ ID NO: 7)

[0129] WAS

[0130] Amino acid sequence of NP040 LCDR1 (SEQ ID NO:8)

[0131] QQHYNTPYT

[0132] hIgG1 constant region amino acid sequence (SEQ ID NO: 9)

[0133] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0134] κ chain constant region amino acid sequence (SEQ ID NO: 10)

[0135] TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0136] NP040 VH nucleotide sequence (SEQ ID NO: 19)

[0137] CAAGTGCAGCTGGTGCAGAGCGGCCGAGGTGAAAAAGCCCGGCGCTAGCGTGAAGGTGTCCTGTAAAGCCTCTGGCTACACATTCACCAGCTATTGGATCAACTGGGTGCGGCAGGCCCCTGGCCAGGGCCTGGAATGGATCGGCAACATCTACCCTAGCGACAGCTACACCAACTAC AACCAGAAGTTCAAGGATAAGGCTACACTGACCGTGGACACCTCCGCCTCTACAGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGATACCGCCGTGTACTACTGCACCAGAGGCGGAAATTACATCAGCTTTGCCATGGACTACTGGGGACAGGGCACATCTGTGACCGTTTCCAGC

[0138] NP040 VL nucleotide sequence (SEQ ID NO: 20)

[0139] GATATCGTGATGACCCAGAGCCCCAGCACCCTGAGCGCCAGCGTGGGCGACAGAGTGACCATCACCTGTAAAGCTTCTCAGGGCGTGTCCACAGCCGTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCTAAGATCCTGATCCACTGGGCCTCTACAAGA CACACCGGCGTCCCAGATAGATTCAGCGGCAGCGGCTCCGGAACAGACTACACCCTGACCATTAGCAGCCTGCAGCCTGAGGACTTCGCTACATACTACTGCCAGCAACACTACAACACCCCTTATACATTTGGAGGCGGCACCAAGCTGGAAATCAAGCGG

[0140] Example 2 Affinity Detection of NP040 Antibody with Different B7-H3 Proteins

[0141] This example mainly demonstrates the affinity of NP040 to recombinant human B7-H3 protein and recombinant monkey B7-H3 protein.

[0142] SPR (Surface Plasmon Resonance) is a commonly used method for molecular interaction analysis. The principle is that the antigen or antibody to be tested flows over the surface of the sensor chip. If there are molecules in the sample that can interact with the biomolecular recognition membrane on the chip surface, it will cause the refractive index of the membrane surface to change, and ultimately lead to a change in the SPR angle. By detecting the change in SPR angle, the affinity between the 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 of NP040 antibody binding to recombinant human B7-H3 protein was 2.30E+05 (1 / Ms), Kd was 2.25E-04 (1 / s), and KD was 9.76E-10M. The Ka of NP040 antibody binding to recombinant monkey B7-H3 protein was 1.24E+05 (1 / Ms), Kd was 3.62E-04 (1 / s), and KD was 2.91E-09M. These results demonstrate that NP040 has high affinity for both recombinant human and monkey B7-H3 proteins.

[0143] Example 3 Detection of Anti-B7-H3 Antibody Cell Binding Activity

[0144] 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 ibrutinib, 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.

[0145] Take 3×10 5 Target cells with different abundances were added with gradient dilutions of anti-B7-H3 antibody protein, incubated for 1 hour, washed once with PBS, and anti-hFc-APC (purchased from Jackson Immunology) was added. After washing once with PBS, the cells were detected by flow cytometry. The results were plotted as S curves. Figures 4 to 8 The results showed that NP040 had 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).

[0146] Example 4 Detection of non-specific binding between antibodies and B7-H3 homologous proteins

[0147] This example mainly demonstrates the affinity of NP040 to recombinant human B7-H2, PD-L2, and B7-2 proteins, as well as the affinity to B7-1-positive cells CHOK1-B7-1 cells and PD-L1-positive cells CHOK1-PD-L1 cells.

[0148] 1) NP040 was plated at 3ug / ml, and gradient dilutions of recombinant human B7-H2 (nearshore, Cat. No.: C34K), PD-L2 (nearshore, Cat. No.: CW20), and B7-2 (nearshore, Cat. No.: C475) proteins were added. The binding of the antibodies to the homologous proteins was detected by ELISA. The results are shown in the table. Figures 9 to 11 The results showed that NP040 did not have non-specific binding with recombinant human B7-H2, PD-L2, and B7-2 proteins.

[0149] 2) Binding to B7-1 and PD-L1 positive cells

[0150] Full-length plasmids of B7-1 (UniProtKB, P33681) and PD-L1 (UniProtKB, Q9NZQ7) were constructed and transiently transfected into CHOK1 cells. Their nonspecific binding to NP040 was detected by flow cytometry. The specific steps are as follows:

[0151] Take 4×10 5 CHOK1-B7-1 cells and CHOK1-PD-L1 cells were added with gradient dilutions of NP040, incubated for 1 hour, washed three times with PBS, and anti-hFC-APC (purchased from Jackson Immunology) was added and detected by flow cytometry. The results were plotted as S curves. Figures 12 to 13 The results showed that NP040 did not have nonspecific binding with B7-1 positive cells CHOK1-B7-1 cells and PD-L1 positive cells CHOK1-PD-L1 cells.

[0152] Example 5 Anti-B7-H3 Antibody-Mediated Internalization

[0153] This example mainly demonstrates the internalization effect of anti-B7-H3 antibodies on cells with different expression abundances. The specific steps are as follows: iQue® Human Antibody Internalization Reagent (Sartorius, CAT NO: 90564) was used to couple the internalization reagent to the serially diluted antibody 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) cells for 2 hours. The positive rate was then detected by flow cytometry. The results were calculated and shown in Figure 2. Figures 14 to 18 .

[0154] The results showed that NP040 had a high internalization effect in 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).

[0155] Example 6 Detection of Binding Epitopes of Anti-B7-H3 Antibodies

[0156] To verify the binding epitope of NP040, five B7-H3 extracellular domains were recombinantly expressed: 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), and Ig-like V-type 1 + Ig-like C2 type 2 (uniprot: Q5ZPR3 POSITION 29-139 / 363-456). Each recombinant extracellular domain protein was plated at 5 μg / ml, and serially diluted NP040 was added. The binding of the antibody to each extracellular domain protein was detected by ELISA. The results are shown in Table 1. The results showed that NP040 binds to different epitopes of antigens from DS7300, ibrutinib, and obinutuzumab, and NP040 mainly binds to the Ig-like V-domain segment.

[0157] Table 1 NP040 binding epitope detection

[0158]

[0159] Note: "+++" indicates strong cell binding ability, "++" indicates intermediate cell binding ability, "+" indicates low cell binding ability, and "-" indicates no or very low affinity with cells.

[0160] The above examples are intended to illustrate the embodiments disclosed herein and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and variations of the methods in the invention will be apparent 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, various modifications apparent 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 includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes CDR-H1 as shown in SEQ ID No.2, CDR-H2 as shown in SEQ ID No.3, and CDR-H3 as shown in SEQ ID No.4; the light chain variable region includes CDR-L1 as shown in SEQ ID No.6, CDR-L2 as shown in SEQ ID No.7, and CDR-L3 as shown in SEQ ID No.

8.

2. The anti-B7-H3 antibody according to claim 1, wherein The heavy chain variable region also includes framework regions HCFR1~HCFR4, and the amino acid sequences of the framework regions HCFR1~HCFR4 are the sequences shown in SEQ ID No.11~14, respectively, and / or the light chain variable region also includes framework regions LCFR1~LCFR4, and the amino acid sequences of the framework regions LCFR1~LCFR4 are the sequences shown in SEQ ID No.15~18, respectively.

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

5.

4. The anti-B7-H3 antibody according to claim 1, characterized in that The anti-B7-H3 antibody is an antibody fragment or a complete antibody, wherein the complete antibody comprises a variable region and a constant region, wherein the constant region comprises a heavy chain constant region and a light chain constant region, wherein 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, characterized in that The amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 9, and / or the amino acid sequence of the light chain constant region is shown in SEQ ID No.

10.

6. An isolated polynucleotide, characterized in that Encodes the anti-B7-H3 antibody according to any one of claims 1 to 5.

7. A nucleic acid construct, characterized in that Contains the isolated polynucleotide according to claim 6.

8. An engineered cell, characterized in that The engineered cell contains the nucleic acid construct of claim 7 or the exogenous polynucleotide of claim 6 integrated into its genome.

9. Use of the anti-B7-H3 antibody of any one of claims 1 to 5, the isolated polynucleotide of claim 6, the nucleic acid construct of claim 7, or the engineered cell of claim 8 in the preparation of a therapeutic drug, wherein the therapeutic drug is a drug for treating tumors that are positive for B7-H3 expression, and the tumor is selected from non-small cell lung cancer, small cell lung cancer, colorectal cancer, or breast cancer.

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