Fully human antibodies targeting b7h3, chimeric antigen receptors and related products and medical uses thereof

Abstract

The application discloses a full human source antibody targeting B7H3, a chimeric antigen receptor and related products and medical purposes, and provides a brand-new full human source monoclonal antibody AHF23050 targeting B7H3, wherein the antibody AHF23050 can specifically combine with cells expressing B7H3, and the application further provides a chimeric antigen receptor, a CAR-T cell, a pharmaceutical composition, a pharmaceutical preparation, an immunoconjugate, a detection reagent and the like containing the antibody AHF23050, and the application is verified through experiments to find that the CAR-T cell can efficiently and specifically kill tumor cells expressing B7H3, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to fully human antibodies targeting B7H3, chimeric antigen receptors and related products and pharmaceutical uses. Background Technology

[0002] B7H3 (CD276) belongs to the B7 superfamily and is a transmembrane glycoprotein. Its extracellular domain has two structures: a monovalent 2Ig-B7-H3 and a bivalent 4Ig-B7-H3 composed of two repeating units. Related studies have shown that B7H3, through interaction with a receptor of unknown structure, can inhibit T cell proliferation and cytokine release. Although the receptor for B7H3 is unknown, recent reports on the negative regulation of tumor immunity by B7H3 and its receptor have been increasing. Tumor cells express B7H3, enabling them to circumvent CD8. + T-cell immune surveillance has been studied, and related research has shown that B7H3 gene knockout mice or the use of anti-B7H3 antibodies can significantly inhibit tumor growth. This inhibitory effect depends on CD8. + The above studies have shown that B7H3 can serve as an effective therapeutic target for tumor immunotherapy, considering the functions of T and NK cells.

[0003] Furthermore, the non-immunological effects of B7H3 have attracted increasing attention from researchers. B7H3 plays a non-immune role not only in various diseases and functions such as bone differentiation, inflammatory responses, and metabolism, but also in tumor cells, where it can function independently by regulating tumor cell biological signals without relying on immune effects. B7H3 can directly participate in tumor progression, chemotherapeutic drug sensitivity, and glycolysis in various tumor cells through multiple signaling pathways, including PI3K / Akt, Jak / STAT, NF-κB-p65 / MAPK-p38, and Ras / Raf / MEK signaling. B7H3 can be detected and aberrantly expressed in tumor tissues of patients with various cancer types and predicts poor prognosis; therefore, B7H3 can serve as a candidate cancer therapeutic target. It is evident that further development of anti-B7H3 monoclonal antibodies for the treatment of B7H3-positive diseases has significant scientific and clinical value. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide the art with a fully human antibody targeting B7H3, a chimeric antigen receptor and related products and pharmaceutical uses.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A first aspect of the present invention provides a monoclonal antibody targeting B7H3.

[0007] Furthermore, the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-3 and the light chain variable region comprises LCDR1-3; wherein HCDR1-3 is HCDR1-3 in the heavy chain variable region as shown in SEQ ID NO:4; and wherein LCDR1-3 is LCDR1-3 in the light chain variable region as shown in SEQ ID NO:9.

[0008] Further, the amino acid sequences of HCDR1-3 are as shown in SEQ ID NO:1-3 or have at least 70% homology with SEQ ID NO:1-3, respectively; optionally, the amino acid sequences of LCDR1-3 are as shown in SEQ ID NO:6-8 or have at least 70% homology with SEQ ID NO:6-8, respectively; optionally, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:4 or has at least 70% homology with SEQ ID NO:4; optionally, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO:9 or has at least 70% homology with SEQ ID NO:9.

[0009] In this invention, antibody sequences that have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with the amino acid sequences corresponding to the heavy chain variable region, the light chain variable region, HCDR1-3, and / or LCDR1-3 of the monoclonal antibody provided by this invention are all within the scope of protection of this invention.

[0010] The amino acid sequences of HCDR1-3 and LCDR1-3 corresponding to the monoclonal antibodies described in this invention are not limited to the amino acid sequences shown above. The amino acid sequences or nucleotide sequences corresponding to HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 obtained by defining CDR1, CDR2, and CDR3 in the heavy chain variable region and light chain variable region described above in this invention using any CDR numbering scheme are all within the protection scope of this invention.

[0011] In some implementations, the CDR numbering scheme includes, but is not limited to, any one or any combination of two or more of the following: IMGT numbering scheme, Chothia numbering scheme, Kabat numbering scheme, Contact numbering scheme, Martin (enhanced Chothia) numbering scheme, AbM numbering scheme, and Aho numbering scheme.

[0012] A second aspect of the present invention provides a bispecific antibody.

[0013] Furthermore, the bispecific antibody comprises the monoclonal antibody described in the first aspect of the present invention; optionally, the bispecific antibody further comprises a second antibody that specifically binds to other antigens.

[0014] In this invention, the bispecific antibody refers to an antibody having two distinct antigen-binding regions defined by different antibody sequences. This can be understood as binding to different targets, but also includes binding to different epitopes of a single target. It includes full-length bispecific antibodies and their antigen-binding fragments. Bispecific antibodies may contain additional modifications, such as non-naturally occurring amino acids, mutations in the Fc region, and mutations at glycosylation sites. Bispecific antibodies also include post-translational modified antibodies, fusion proteins containing antibody antigenic determinants, and immunoglobulin molecules containing any other modifications to antigen recognition sites, provided these antibodies exhibit the desired biological activity; all such antibodies fall within the scope of the bispecific antibody category.

[0015] A third aspect of the present invention provides a chimeric antigen receptor that targets B7H3.

[0016] Furthermore, the chimeric antigen receptor comprises the monoclonal antibody described in the first aspect of the present invention; optionally, the chimeric antigen receptor further comprises a transmembrane domain, an intracellular signal transduction domain, a hinge region, and a co-stimulatory signal domain; optionally, the chimeric antigen receptor further comprises any one or more of a signal peptide, a self-splitting peptide, a safety switch, an immunomodulatory molecule, or a cytokine.

[0017] Optionally, the transmembrane domain is selected from any of the following molecules: CD8, CD28, 4-1BB, CD34, CD3ε, PD-1, IgG1, IgG4, OX40, IL-2 receptor, IL-7 receptor, IL-11 receptor; Optionally, the intracellular signal transduction domain is selected from any of the following molecules: CD3ζ, CD3γ, CD3δ, CD3ε, FcRγ, FcRβ, TCRζ, CD4, CD5, CD8, CD21, CD22, CD79a, CD79b, CD278, Fcε. RI, DAP10, DAP12, CD66d; Optionally, the hinge region is selected from the hinge regions of any one of the following molecules: CD8, CD28, CD34, 4-1BB, OX40, CD3ε, IgG1, IgG4, PD-1, IL-2 receptor, IL-7 receptor, IL-11 receptor; Optionally, the co-stimulatory signaling domain is selected from the co-stimulatory signaling domains of any one of the following molecules: CD28, 4-1BB, CD19, CD4, CD27, ICOS, CD8α, CD8β, BAFFR, HVEM, LIGHT, KIRDS2, SLAMF7, NKp30, NKp46, CD40, CDS, ICAM-1, B7-H3, OX40, DR3, GITR, CD30, TIM1, CD2, CD7, CD226; Optionally, the signal peptide is selected from the signal peptides of any one of the following molecules: α and β chains of T cell receptors, CD3ζ, CD3ε, CD16, CD22, CD33, CD4, CD5, CD8, CD9, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, GITR, GM-CSF; Optionally, the self-splitting peptide is selected from any one of the following self-splitting peptides: T2A, P2A, E2A, F2A; Optionally, the safety switch is selected from any one of the following safety switches: tEGFR, iCaspase-9, RQR8; Optionally, the immunomodulatory molecule or cytokine is selected from any one of the following immunomodulatory molecules or cytokines: B7.1, CCL19, CCL21, CD40L, CD137L, GITRL, GM-CSF, IL-12, IL-2, IL-15, IL-18, IL-21, LEC, OX40L;

[0018] Optionally, the chimeric antigen receptor is formed by sequentially connecting a signal peptide, the heavy chain variable region of the monoclonal antibody described in the first aspect of the present invention, a linker, the light chain variable region of the monoclonal antibody described in the first aspect of the present invention, a CD8 hinge region and a transmembrane domain, a CD28 co-stimulatory signaling domain, a CD3ζ intracellular signal transduction domain, a T2A self-cleaving peptide, and a tEGFR safety switch.

[0019] Optionally, the Linker is (G4S)3; Optionally, the amino acid sequence of the signal peptide is as shown in SEQ ID NO:11; Optionally, the amino acid sequence of the Linker is as shown in SEQ ID NO:15; Optionally, the amino acid sequences of the CD8 hinge region and transmembrane domain are as shown in SEQ ID NO:19; Optionally, the amino acid sequence of the CD28 co-stimulatory signaling domain is as shown in SEQ ID NO:21; Optionally, the amino acid sequence of the CD3ζ intracellular signal transduction domain is as shown in SEQ ID NO:23; Optionally, the amino acid sequence of the T2A self-cleaving peptide is as shown in SEQ ID NO:25; Optionally, the amino acid sequence of the tEGFR safety switch is as shown in SEQ ID NO:27; Optionally, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:29.

[0020] A fourth aspect of the present invention provides a nucleic acid molecule.

[0021] Further, the nucleic acid molecule comprises a nucleotide sequence encoding the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, or the chimeric antigen receptor of the third aspect of the present invention; optionally, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody of the first aspect of the present invention is shown in SEQ ID NO:14; optionally, the nucleotide sequence of the light chain variable region of the monoclonal antibody of the first aspect of the present invention is shown in SEQ ID NO:18; optionally, the nucleotide sequence of the signal peptide in the chimeric antigen receptor of the third aspect of the present invention is shown in SEQ ID NO:12; optionally, the nucleotide sequence of the linker in the chimeric antigen receptor of the third aspect of the present invention is shown in SEQ ID NO:16; optionally, the nucleotide sequence of the CD8 hinge region and transmembrane domain in the chimeric antigen receptor of the third aspect of the present invention is shown in SEQ ID NO:20; optionally, the nucleotide sequence of the CD28 co-stimulatory signaling domain in the chimeric antigen receptor of the third aspect of the present invention is shown in SEQ ID NO:22; optionally, the nucleotide sequence of the CD3ζ intracellular signal transduction domain in the chimeric antigen receptor of the third aspect of the present invention is shown in SEQ ID NO:14. As shown in NO:24; Optionally, the nucleotide sequence of the T2A self-cleaving peptide in the chimeric antigen receptor of the third aspect of the present invention is shown in SEQ ID NO:26; Optionally, the nucleotide sequence of the tEGFR safety switch in the chimeric antigen receptor of the third aspect of the present invention is shown in SEQ ID NO:28; Optionally, the nucleotide sequence of the chimeric antigen receptor of the third aspect of the present invention is shown in SEQ ID NO:30.

[0022] A fifth aspect of the present invention provides an expression carrier.

[0023] Furthermore, the expression vector comprises the nucleic acid molecule described in the fourth aspect of the present invention; optionally, the vector is a DNA vector, RNA vector, plasmid, transposon vector, CRISPR / Cas9 vector, or a virus-derived vector; optionally, the virus-derived vector is a lentiviral vector, adenovirus vector, adeno-associated virus vector, retroviral vector, poxvirus vector, or herpesvirus vector.

[0024] In some embodiments, the expression vector described in this invention refers to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a plasmid, which is a circular double-stranded DNA loop into which additional DNA fragments can be linked. Another type of vector is a viral vector, in which additional DNA fragments can be linked to a viral genome.

[0025] A sixth aspect of the present invention provides a recombinant host cell.

[0026] Furthermore, the recombinant host cell comprises the expression vector described in the fifth aspect of the present invention; optionally, the host cell is a eukaryotic cell and / or a prokaryotic cell; optionally, the host cell is an immune cell; optionally, the immune cell is a T cell, B cell, NK cell, iNKT cell, CTL cell, dendritic cell, myeloid cell, monocyte, macrophage or any combination thereof.

[0027] In some embodiments, the present invention does not particularly limit the specific type of host cell. Any host cell capable of carrying the expression vector described in the fifth aspect of the present invention is within the scope of protection of the present invention. For example, the host cell includes, but is not limited to: T cells, B cells, NK cells, iNKT cells, CTL cells, dendritic cells, myeloid cells, monocytes, macrophages or any combination thereof, CHO-K1, DUkXB11, Pro-5, CHO-S, NSO, SP2 / 0-Ag14, YB2 / 0 or CHO-3E7, etc.

[0028] The seventh aspect of the present invention provides any of the following products:

[0029] (1) A diagnostic conjugate comprising the monoclonal antibody described in the first aspect of the present invention and / or the bispecific antibody described in the second aspect of the present invention;

[0030] (2) A detection reagent comprising the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, and / or the diagnostic conjugate;

[0031] (3) A detection product comprising the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the diagnostic conjugate and / or the detection reagent;

[0032] (4) A therapeutic conjugate comprising the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, and / or the recombinant host cell of the sixth aspect of the present invention;

[0033] (5) A pharmaceutical composition comprising the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the recombinant host cell of the sixth aspect of the present invention, and / or the therapeutic conjugate thereof;

[0034] (6) A pharmaceutical preparation comprising the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the recombinant host cell of the sixth aspect of the present invention, the therapeutic conjugate and / or the pharmaceutical composition;

[0035] (7) A kit containing a therapeutically effective amount of the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the recombinant host cell of the sixth aspect of the present invention, the therapeutic conjugate, the pharmaceutical composition and / or the pharmaceutical preparation;

[0036] Optionally, the diagnostic conjugate is a complex formed by the direct or indirect binding of the monoclonal antibody described in the first aspect of the present invention or the bispecific antibody described in the second aspect of the present invention to the diagnostic agent; optionally, the diagnostic agent is a radionuclide, a contrast agent, and / or a photosensitizing diagnostic agent; optionally, the radionuclide includes gamma-emitting isotopes, beta-emitting isotopes, and / or positron-emitting isotopes; optionally, the contrast agent includes iodine compounds, barium compounds, gallium compounds, and / or thallium compounds; optionally, the photosensitizing diagnostic agent includes chemiluminescent compounds and / or bioluminescent compounds; in some embodiments, specific examples of the radionuclide include, but are not limited to: 110 In、 111 In、 177 Lu、 18 F, 52 Fe、 62 Cu、 64 Cu、 67 Cu、 67 Ga、 68 Ga、 86 Y、 90 Y、 89 Zr、 94 mTc, 94 Tc,99 mTc, 120 I, 123 I, 124 I, 125 I, 131 I, 154-158 Gd, 32 P, 11 C, 13 N, 15 O, 186 Re, 188 Re, 51 Mn, 52 mMn, 55 Co, 72 As, 75 Br, 76 Br, 82 mRb, 83 Sr, 51 Cr, 57 Co, 58 Co, 59 Fe, 75 Se, 97 Ru, 114 mIn, 169 Yb, 197 Hg and / or 201Tl; In some embodiments, the contrast agent is a radioactive contrast agent, which may be a paramagnetic ion, including but not limited to: chromium (III), manganese (II), iron (III), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III), erbium (III), lanthanum (III), gold (III), lead (II) and / or bismuth (III); In some embodiments, the contrast agent is an ultrasound enhancer (e.g., liposome); In some embodiments, the contrast agent is a radiopaque substance of iodine compound, barium compound, gallium compound and / or thallium compound. The radiopaque substances include, but are not limited to: barium, diatrizoate, ethyl iodized oil, gallium citrate, iocarboxylic acid, iodicalcic acid, iodamicin, cholanic acid, iosalicylic acid, iogulamide, iohexol, iopamidol, iopannic acid, ioprocyic acid, iosifaric acid, ioseriic acid, iosulfonamide meglumine, iodophthalic acid, iotitic acid, iotalamic acid, iodochroic acid, iodoxazone, hydroxydiatrizoate, iopoise, meglumine, methyldiatrizoate, methyldiatrizoate, proiodophorone and / or thallium chloride; in some embodiments, the photosensitizing diagnostic agent includes chemiluminescent compounds and / or bioluminescent compounds, the bioluminescent compounds including, but not limited to: fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescein, luciferin, luciferase and / or jellyfish luminescent protein. The chemiluminescent compounds are: luminol, isoluminol, aromatic acridine esters, imidazoles, acridine salts and / or oxalates;

[0037] Optionally, the detection product is a test kit, a test strip, or a detection chip;

[0038] Optionally, the therapeutic conjugate is a monoclonal antibody as described in the first aspect of the present invention, a bispecific antibody as described in the second aspect of the present invention, or a complex formed by recombinant host cells directly or indirectly binding to a therapeutic agent as described in the sixth aspect of the present invention; optionally, the therapeutic agent is a radionuclide, an immunomodulator, a hormone, a hormone antagonist, an oligonucleotide, an enzyme, an enzyme inhibitor, a photosensitizing agent, a cytotoxic agent, and / or angiogenesis inhibitor.

[0039] Optionally, the medicine box is packaged in a container and includes a label affixed to or packaged in the container, the label describing the contents of the container and providing instructions and / or descriptions regarding the use of the contents of the container to treat and / or prevent B7H3-positive related diseases; optionally, the medicine box may also include a second therapeutic agent, the second therapeutic agent being a medicine capable of being used to treat and / or prevent (or as adjunctive treatment and / or prevention) B7H3-positive related diseases (the present invention does not particularly limit the specific type of the second therapeutic agent).

[0040] The eighth aspect of the present invention provides any of the following methods:

[0041] (1) A method for producing the monoclonal antibody of the first aspect of the present invention or the bispecific antibody of the second aspect of the present invention, the method comprising the following steps: culturing the recombinant host cell of the sixth aspect of the present invention, and isolating the monoclonal antibody of the first aspect of the present invention or the bispecific antibody of the second aspect of the present invention from the recombinant host cell culture product;

[0042] (2) A method for preparing the recombinant host cell according to the sixth aspect of the present invention, the method comprising the following steps: introducing the expression vector according to the fifth aspect of the present invention into a host cell to obtain the recombinant host cell according to the sixth aspect of the present invention;

[0043] (3) A method for detecting B7H3 protein in a test sample for non-diagnostic and non-therapeutic purposes, the method comprising the following steps: contacting the test sample with the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the diagnostic conjugate described in the seventh aspect of the present invention, a detection reagent and / or a detection product, and detecting the formation of B7H3 protein immune complex with the antibody;

[0044] (4) A method for inhibiting the activity of B7H3 protein in vitro, the method comprising the following steps: contacting the sample to be tested with the monoclonal antibody described in the first aspect of the present invention and / or the bispecific antibody described in the second aspect of the present invention.

[0045] Furthermore, the present invention also provides a method for diagnosing whether a subject has a B7H3-related disease, the method comprising the following steps: contacting a test sample derived from the subject with the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the diagnostic conjugate described in the seventh aspect of the present invention, a detection reagent and / or a detection product, detecting the formation of antibody immune complexes, and determining, based on this result, whether the subject has a B7H3-related disease or the risk of the subject having a B7H3-related disease.

[0046] Furthermore, the present invention provides a method for treating and / or preventing B7H3-related diseases in subjects in need, the method comprising the steps of administering to the subject in need an effective amount of the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the recombinant host cell of the sixth aspect of the present invention, the therapeutic conjugate of the seventh aspect of the present invention, the pharmaceutical composition of the seventh aspect of the present invention, and / or the pharmaceutical preparation of the seventh aspect of the present invention.

[0047] In this invention, the B7H3-positive related diseases include any diseases associated with B7H3 expression, including but not limited to: ovarian cancer, prostate cancer, liver cancer, lung cancer, kidney cancer, breast cancer, colorectal cancer, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, bladder cancer, osteosarcoma, glioma, or myeloid leukemia.

[0048] In this invention, the subject refers to any animal, preferably a mammal, including but not limited to: humans, higher primates, domestic and farm animals, as well as zoo animals, racing animals, or pets, such as horses, pigs, cattle, dogs, cats, and ferrets. In a specific embodiment of this invention, the subject is preferably a human.

[0049] In this invention, administration refers to a method of giving a certain dose of a compound (e.g., a monoclonal antibody, bispecific antibody, or recombinant host cell as described in this invention) or a composition (e.g., a therapeutic conjugate, pharmaceutical composition, or pharmaceutical preparation as described in this invention) to a subject in need.

[0050] In some embodiments, the present invention does not particularly limit the method of administration, which includes, but is not limited to: intralesional, intramuscular, intravenous, intradermal, percutaneous, intraarticular, intraperitoneal, intracranial, intra-articular, intraprostatic, intrapleural, intratracheal, intranasal, intrarectal, superficial, subcutaneous, subconjunctival, intrabladder, transmucosal, intraperitoneal, intraumbilical, intraocular, oral, local, by inhalation, by injection, by infusion, by continuous infusion, by catheter, by irrigation, as a cream, or as a lipid composition. The specific method of administration and the dosage form of the pharmaceutical preparation can vary depending on a variety of factors (e.g., the compound or composition applied and the severity of the disease, condition, or symptom being treated).

[0051] The ninth aspect of the present invention provides any of the following applications:

[0052] (1) The use of the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, and / or the nucleic acid molecule described in the fourth aspect of the present invention in the preparation of a diagnostic conjugate for detecting B7H3 protein;

[0053] (2) The use of the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, and / or the diagnostic conjugate described in the seventh aspect of the present invention in the preparation of a detection reagent for detecting B7H3 protein;

[0054] (3) The use of the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, the diagnostic conjugate described in the seventh aspect of the present invention, and / or the detection reagent described in the seventh aspect of the present invention in the preparation of a detection product for detecting B7H3 protein;

[0055] (4) The use of the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, the diagnostic conjugate described in the seventh aspect of the present invention, the detection reagent described in the seventh aspect of the present invention, and / or the detection product described in the seventh aspect of the present invention in the detection of B7H3 protein for non-diagnostic and non-therapeutic purposes;

[0056] (5) The use of the monoclonal antibody described in the first aspect of the present invention, the bispecific antibody described in the second aspect of the present invention, the nucleic acid molecule described in the fourth aspect of the present invention, the diagnostic conjugate described in the seventh aspect of the present invention, the detection reagent described in the seventh aspect of the present invention, and / or the detection product described in the seventh aspect of the present invention in the preparation of diagnostic products for diagnosing or assisting in the diagnosis of B7H3-positive related diseases;

[0057] (6) The use of the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the chimeric antigen receptor of the third aspect of the present invention, the nucleic acid molecule of the fourth aspect of the present invention, the expression vector of the fifth aspect of the present invention, and the recombinant host cell of the sixth aspect of the present invention in the preparation of therapeutic conjugates for the treatment and / or prevention of B7H3-positive related diseases;

[0058] (7) The use of the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the chimeric antigen receptor of the third aspect of the present invention, the nucleic acid molecule of the fourth aspect of the present invention, the expression vector of the fifth aspect of the present invention, the recombinant host cell of the sixth aspect of the present invention, and / or the therapeutic conjugate of the seventh aspect of the present invention in the preparation of a pharmaceutical composition for treating and / or preventing B7H3-positive related diseases;

[0059] (8) The use of the monoclonal antibody of the first aspect of the present invention, the bispecific antibody of the second aspect of the present invention, the chimeric antigen receptor of the third aspect of the present invention, the nucleic acid molecule of the fourth aspect of the present invention, the expression vector of the fifth aspect of the present invention, the recombinant host cell of the sixth aspect of the present invention, the therapeutic conjugate of the seventh aspect of the present invention and / or the pharmaceutical composition of the seventh aspect of the present invention in the preparation of a pharmaceutical formulation for the treatment and / or prevention of B7H3-positive related diseases;

[0060] Optionally, the B7H3-positive associated diseases are ovarian cancer, prostate cancer, liver cancer, lung cancer, kidney cancer, breast cancer, colorectal cancer, esophageal cancer, oral cancer, gastric cancer, pancreatic cancer, endometrial cancer, bladder cancer, osteosarcoma, glioma, or myeloid leukemia.

[0061] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0062] This invention provides a novel fully human monoclonal antibody, AHF23050, targeting B7H3. The antibody AHF23050 specifically binds to cells expressing B7H3. This invention also provides a chimeric antigen receptor comprising the antibody AHF23050, CAR-T cells, a pharmaceutical composition, a pharmaceutical formulation, an immunoconjugate, and a detection reagent. Experimental verification shows that the CAR-T cells can efficiently and specifically kill tumor cells expressing B7H3, demonstrating broad prospects for clinical application. Attached Figure Description

[0063] Figure 1 Flow cytometry was used to detect B7H3 expression in prostate cancer cells. Figure A shows prostate cancer cells DU145, and Figure B shows prostate cancer cells DU145. B7H3 KO ;

[0064] Figure 2 Flow cytometry was used to detect antibody specificity. The images show AB plots of DU145 prostate cancer cells and CD plots of DU145 prostate cancer cells. B7H3 KO ;

[0065] Figure 3 Antibody-mediated NK cell ADCC killing effect;

[0066] Figure 4 Preparation and detection of fully human B7H3 CAR-T cells, where Figure A: Flow cytometry of CAR-T positivity rate, and Figure B: Statistical graph of CAR-T positivity rate;

[0067] Figure 5 The tumor sphere assay was used to detect the killing effect of CAR-T cells prepared based on the fully human B7H3 antibody described in this application on tumor cells. Among them, A: ovarian cancer cells SKOV3, B: prostate cancer cells DU145, and C: lung cancer cells A549.

[0068] Figure 6 RTCA detection is based on CAR-T inhibitors prepared from the fully human B7H3 antibody described in this application to target B7H3. +The killing effect of tumor cells, in which: A: ovarian cancer cells SKOV3, B: prostate cancer cells DU145, C: lung cancer cells A549, D: lung cancer cells H1299. Detailed Implementation

[0069] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. Unless otherwise specified, the reagents, biological materials, etc., used in the following embodiments are commercially available.

[0070] Example 1: Monoclonal antibody AHF23050 targeting B7H3

[0071] This invention constructs a fully human monoclonal antibody, AHF23050, specifically targeting B7H3. The sequence information of the AHF23050 antibody is shown in Table 1 below. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:4, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:9. Furthermore, this embodiment experimentally demonstrates that the AHF23050 antibody has good affinity for B7H3.

[0072] Table 1. Sequence of AHF23050 antibody

[0073]

[0074]

[0075] Example 2: Flow cytometry detection of the specificity of antibody AHF23050

[0076] 1. Flow cytometry detection of B7H3 expression in prostate cancer cells

[0077] This embodiment first uses flow cytometry to detect B7H3 expression in prostate cancer cells, and then uses antibody AHF23050 with DU145 and DU145. B7H3 KO Flow cytometry analysis was performed after co-incubation of cells. The specific experimental steps are as follows:

[0078] (1) Collect DU145 and DU145 samples from prostate cancer. B7H3 KO Cells were co-incubated with antibody AHF23050 (1:200) at 4°C for 30 min;

[0079] (2) Add PBS to wash, centrifuge and discard the supernatant, resuspend the cell pellet in 300 μL PBS, and filter into a flow cytometer.

[0080] (3) Flow cytometry testing.

[0081] 2. Flow cytometry for detecting antibody specificity

[0082] To determine whether the B7H3 antibody AHF23050 used could specifically bind to the B7H3 antigen expressed by cells, this example involved serially diluting ① antibody AHF23050 and ② commercially available B7H3 antibody (Enoblituzumab MGA271) with DU145 and DU145, respectively. B7H3 KO Flow cytometry analysis was performed after co-incubation of cells. The specific experimental steps are as follows:

[0083] (1) Selected cells DU145 and DU145 B7H3 KO 2-5×10 5 / Sample;

[0084] (2) Antibodies selected: ① Antibody AHF23050; ② Commercial B7H3 antibody (Enoblituzumab MGA271);

[0085] (3) Antibody gradient: 0, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 5 μg / mL (antibody dilution buffer: PBS containing 2% FBS), incubate at 37℃ for 30 min, add 1 mL PBS to wash, centrifuge, and discard as much supernatant as possible;

[0086] (4) Secondary antibody: AF647-IgG (1:500), incubated at 4℃ for 30 min, washed with 1 mL PBS, centrifuged, discarded supernatant, resuspended in 300 μL PBS containing 0.5% BSA, and detected by flow cytometry (10,000 target cells were collected).

[0087] 3. Experimental Results

[0088] Flow cytometry analysis of prostate cancer cells DU145 and DU145 B7H3 KO The expression status of B7H3 and the corresponding results are as follows Figure 1 As shown, the results indicate that B7H3 is highly expressed in DU145 cells. B7H3 KO It is not expressed in Chinese.

[0089] Results of flow cytometry detection of antibody specificity, such as Figure 2 As shown, the results indicate that, compared to DU145 B7H3 KO In comparison, antibody AHF23050 exhibits a highly specific binding ability to DU145 expressing B7H3, which is superior to commercially available antibodies.

[0090] Example 3: The fully human B7H3 antibody AHF23050 can kill DU145 cells through ADCC effect.

[0091] 1. Experimental Methods

[0092] This embodiment uses an RTCA assay to detect the killing effect of NK cells treated with antibody AHF23050 on DU145 cells. The specific experimental steps are as follows:

[0093] (1) Collect prostate cancer DU145 cells and seed them into RTCA plates at a density of 20,000 cells per well;

[0094] (2) When the cell index of tumor cells reaches approximately 1, add 50 μL of antibody (final concentration 5 μg / mL), ① antibody AHF23050; ② commercial B7H3 antibody (Enoblituzumab MGA271); ③ IgG1 isotype control (InVivoMAb MOPC-21); add 50 μL of culture medium to the remaining wells and incubate the antibody at 37℃ for 1 h;

[0095] (3) Add 50 μL of NK cell suspension at a 1:1 effector-target ratio and continue to culture and observe for 2-3 days;

[0096] (4) The RTCA instrument automatically records the cell growth index. After 2-3 days, RTCA is stopped and a curve reflecting the cell growth index is automatically generated.

[0097] 2. Experimental Results

[0098] The results are as follows Figure 3 As shown, the results indicate that at an effector-to-target ratio of 1:1, in DU145 cells, the combination of NK cells and the antibody significantly enhanced the tumor-killing effect of NK cells compared to the NK cell-only group. Furthermore, the effect was essentially equivalent to that of the commercially available antibody. These results demonstrate that the fully human B7H3 antibody AHF23050 can kill DU145 cells through ADCC.

[0099] Example 4: Preparation and Detection of Fully Human B7H3 CAR-T Cells

[0100] 1. Experimental Methods

[0101] A CAR expression plasmid was constructed using the sequence of the antibody AHF23050, and the virus was packaged. The collected retroviral fluid was used to infect T cells to prepare CAR-T cells, named AHF23050.CAR-T. Cells were harvested 48 hours after infection and flow cytometry was used to detect the positive rate. The coding sequence of the fully human chimeric antigen receptor (CAR) targeting B7H3 is shown in Table 2 below. Specific experimental steps are as follows:

[0102] (1) The constructed plasmid was packaged as a retrovirus, and the viral supernatant was harvested at 48h and 72h respectively. After centrifugation and filtration, the supernatant was aliquoted into 1.5mL EP tubes and stored at -80℃.

[0103] (2) After T cell activation, administer 0.5 × 10⁻⁶ cells. 6 / mL was dispensed into 1.5mL EP tubes, 1mL of virus solution (AHF23050.CAR-T) was added to resuspend the cells, and then the cells were slowly added into 24-well plates pre-coated with Retronectin. The plates were centrifuged at 30℃ and 1500g for 2 hours. After centrifugation, the virus solution was discarded, complete culture medium was added, and the plates were placed in an incubator for incubation.

[0104] (3) Take 0.2×10 after 48 hours. 6 To detect the CAR positivity rate in cells, the PE-protein L and FITC-CD3 flow cytometry antibodies were diluted to a final concentration of 1 μg / μL, and 50 μL / sample was added to the cells. The cells were incubated at 4°C in the dark for 30 min, washed with 1 mL of PBS, and centrifuged to discard the supernatant.

[0105] (4) Resuspend the cell pellet in 300 μL PBS, filter it into a flow cytometer tube, and perform flow cytometer analysis.

[0106] 2. Experimental Results

[0107] The results are as follows Figure 4 As shown, the results indicate that the positivity rate of CAR-T cells remained stable between 70% and 90% compared with uninfected T cells. These results demonstrate that this embodiment successfully prepared CAR-T cells targeting B7H3.

[0108] Table 2. Coding sequences of fully human chimeric antigen receptors (CARs) targeting B7H3.

[0109]

[0110]

[0111]

[0112] Example 5: Tumor spheroid assay to detect the killing effect of CAR-T cells prepared based on fully human B7H3 antibody (AHF23050) on tumor cells.

[0113] 1. Experimental Methods

[0114] To verify the killing ability of the CAR-T cells against B7H3-positive tumors, this embodiment observed the killing effect of AHF23050 CAR-T cells on ovarian cancer SKOV3, prostate cancer DU145, and lung cancer A549 cells that highly express B7H3 and exhibit GFP fluorescence through a 3D tumor experiment. The specific experimental steps are as follows:

[0115] (1) Add 0.15g agarose to 10mL of DMEM and autoclave at 121℃ for 20 minutes;

[0116] (2) Add 50 μL of hot agarose solution to each well of the 96-well plate and cool to room temperature;

[0117] (3) 200 μL of tumor cell suspension with GFP fluorescence was seeded into 96-well plates covered with agarose, at a cell density of 1.2 × 10⁻⁶ cells / well. 4 Cells / mL, further cultured for 3-4 days;

[0118] (4) Observe the size of the tumor sphere under a microscope. When the diameter is about 300 μm, take a picture and record it.

[0119] (5) Collect effector cells (AHF23050.CAR-T), stain CAR-T effector cells with DIL cell membrane dye (1:500), stain at room temperature in the dark for 20 min, wash twice with 10 mL PBS, and discard the supernatant;

[0120] (6) Resuspend the precipitate in the effector cell culture medium, count the cells, and add 50 μL of effector cell suspension at the initial cell-to-target ratio of 1:1.

[0121] (7) Take photos and record observations under a fluorescence microscope.

[0122] 2. Experimental Results

[0123] The results are as follows Figure 5 As shown, the results indicate that, compared with T cells, the presence of CAR significantly enhances the killing ability of T cells against solid tumors (ovarian cancer, prostate cancer, and liver cancer). These results demonstrate that CAR-T cells prepared based on the fully human B7H3 antibody (AHF23050) have a good killing effect on tumor cells.

[0124] Example 6: RTCA detection of CAR-T against B7H3 + Killing effect of tumor cells

[0125] 1. Experimental Methods

[0126] To verify the killing ability of CAR-T cells against B7H3-positive tumors, the killing effect of AHF23050 CAR-T cells on tumor cells highly expressing B7H3 (ovarian cancer SKOV3, prostate cancer DU145, lung cancer A549, and lung cancer H1299 cells, all highly expressing B7H3) at an effector-target ratio of 2:1 was detected by RTCA. The specific experimental steps are as follows:

[0127] (1) Collect tumor cells and seed them into RTCA plates at a rate of 20,000 cells per well;

[0128] (2) When the cell index of tumor cells reaches approximately 1, add the corresponding AHF23050 CAR-T cells;

[0129] (3) The RTCA instrument automatically records the cell growth index;

[0130] (4) After 2-3 days, stop RTCA and automatically generate a curve of the cell growth index.

[0131] 2. Experimental Results

[0132] The results are as follows Figure 6 As shown, the results indicate that the presence of CAR significantly enhances the killing ability of T cells against solid tumors compared to T cells. These results further demonstrate that CAR-T cells prepared based on the fully human B7H3 antibody (AHF23050) have a good killing effect on tumor cells.

Claims

1. A monoclonal antibody targeting B7H3, characterized in that, The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1-3 and the light chain variable region comprises LCDR1-3. The amino acid sequences of HCDR1-3 are shown in SEQ ID NO:1-3, respectively; The amino acid sequences of LCDR1-3 are shown in SEQ ID NO:6-8, respectively.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

4.

3. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO:

9.

4. A chimeric antigen receptor targeting B7H3, characterized in that, The chimeric antigen receptor comprises the monoclonal antibody according to any one of claims 1-3; The monoclonal antibody is obtained by sequentially connecting the heavy chain variable region, the linker, and the light chain variable region of the monoclonal antibody as described in claim 1. The chimeric antigen receptor also includes a hinge region and a transmembrane domain, an intracellular signal transduction domain, and a co-stimulatory signal domain. The chimeric antigen receptor also includes a signal peptide, a self-splitting peptide, and a safety switch; The hinge region and transmembrane structural domain are CD8 hinge region and transmembrane structural domain; The intracellular signal transduction domain is the CD3ζ intracellular signal transduction domain; The co-stimulation signal structure domain is the CD28 co-stimulation signal structure domain; The self-cleaving peptide is a T2A self-cleaving peptide; The safety switch is a tEGFR safety switch.

5. The chimeric antigen receptor according to claim 4, characterized in that, The chimeric antigen receptor is formed by sequentially connecting a signal peptide, the heavy chain variable region of the monoclonal antibody as described in claim 1, a linker, the light chain variable region of the monoclonal antibody as described in claim 1, a CD8 hinge region and transmembrane domain, a CD28 co-stimulatory signaling domain, a CD3ζ intracellular signal transduction domain, a T2A self-cleaving peptide, and a tEGFR safety switch.

6. The chimeric antigen receptor according to claim 5, characterized in that, The Linker is (G4S)3; The amino acid sequence of the signal peptide is shown in SEQ ID NO:11; The amino acid sequence of the Linker is shown in SEQ ID NO:15; The amino acid sequences of the CD8 hinge region and transmembrane domain are shown in SEQ ID NO:19; The amino acid sequence of the CD28 co-stimulatory signaling domain is shown in SEQ ID NO:21; The amino acid sequence of the CD3ζ intracellular signal transduction domain is shown in SEQ ID NO:23; The amino acid sequence of the T2A self-cleaving peptide is shown in SEQ ID NO:25; The amino acid sequence of the tEGFR safety switch is shown in SEQ ID NO:

27.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecule comprises a nucleotide sequence encoding a monoclonal antibody as described in any one of claims 1-3 or a chimeric antigen receptor as described in any one of claims 4-6.

8. The nucleic acid molecule according to claim 7, characterized in that, The nucleotide sequence of the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO:

14.

9. The nucleic acid molecule according to claim 7, characterized in that, The nucleotide sequence of the light chain variable region of the monoclonal antibody is shown in SEQ ID NO:

18.

10. The nucleic acid molecule according to claim 7, characterized in that, The nucleotide sequence of the signal peptide in the chimeric antigen receptor is shown in SEQ ID NO:12; The nucleotide sequence of the Linker in the chimeric antigen receptor is shown in SEQ ID NO:16; The nucleotide sequences of the CD8 hinge region and transmembrane domain in the chimeric antigen receptor are shown in SEQ ID NO:

20. The nucleotide sequence of the CD28 co-stimulatory signaling domain in the chimeric antigen receptor is shown in SEQ ID NO:22; The nucleotide sequence of the CD3ζ intracellular signal transduction domain in the chimeric antigen receptor is shown in SEQ ID NO:

24. The nucleotide sequence of the T2A self-cleaving peptide in the chimeric antigen receptor is shown in SEQ ID NO:26; The nucleotide sequence of the tEGFR safety switch in the chimeric antigen receptor is shown in SEQ ID NO:

28.

11. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule according to any one of claims 7-10.

12. The expression vector according to claim 11, characterized in that, The vector is a DNA vector, an RNA vector, or a viral vector.

13. The expression vector according to claim 12, characterized in that, The vector is a plasmid.

14. The expression vector according to claim 12, characterized in that, The vector is a transposon vector or a CRISPR / Cas9 vector.

15. The expression vector according to claim 12, characterized in that, The vector from which the virus originates is a lentiviral vector, adenovirus vector, adeno-associated virus vector, retroviral vector, poxvirus vector, or herpesvirus vector.

16. A recombinant host cell, characterized in that, The recombinant host cell comprises the expression vector according to any one of claims 11-15.

17. The recombinant host cell according to claim 16, characterized in that, The host cells are immune cells; The immune cells are T cells, NK cells, or iNKT cells; The expression vector contained in the recombinant host cell encodes the chimeric antigen receptor as described in any one of claims 4-6.

18. A diagnostic conjugate, characterized in that, The diagnostic conjugate comprises the monoclonal antibody according to any one of claims 1-3; The diagnostic conjugate is a complex formed by the direct or indirect binding of any one of the monoclonal antibodies of claims 1-3 to a diagnostic agent; The diagnostic agent is a radionuclide, a contrast agent, and / or a photosensitizing diagnostic agent; The diagnostic conjugate is used to diagnose diseases associated with B7H3 positivity. The diseases associated with B7H3 positivity are ovarian cancer, prostate cancer, liver cancer, or lung cancer.

19. A detection reagent, characterized in that, The detection reagent comprises the monoclonal antibody of any one of claims 1-3 and / or the diagnostic conjugate of claim 18.

20. A testing product, characterized in that, The detection product comprises the monoclonal antibody of any one of claims 1-3, the diagnostic conjugate of claim 18, and / or the detection reagent of claim 19.

21. The testing product according to claim 20, characterized in that, The testing products are test kits, test strips, or test chips.

22. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the monoclonal antibody of any one of claims 1-3 and / or the recombinant host cell of claim 16 or 17.

23. A pharmaceutical preparation, characterized in that, The pharmaceutical formulation comprises the monoclonal antibody of any one of claims 1-3, the recombinant host cell of claim 16 or 17, and / or the pharmaceutical composition of claim 22.

24. A medicine box, characterized in that, The kit contains a therapeutically effective amount of any one of claims 1-3, the recombinant host cell of claim 16 or 17, the pharmaceutical composition of claim 22, and / or the pharmaceutical formulation of claim 23.

25. The medicine box according to claim 24, characterized in that, The medicine box is packaged in a container and also includes a label affixed to or packaged in the container, which describes the contents of the container and provides instructions and / or instructions on using the contents of the container to treat B7H3-positive related diseases. The diseases associated with B7H3 positivity are ovarian cancer, prostate cancer, liver cancer, or lung cancer.

26. A method for producing the monoclonal antibody according to any one of claims 1-3, characterized in that, The method includes the following steps: culturing the recombinant host cell according to claim 16, and isolating the monoclonal antibody according to any one of claims 1-3 from the recombinant host cell culture product.

27. A method for preparing the recombinant host cell according to claim 16 or 17, characterized in that, The method includes the following steps: introducing the expression vector of any one of claims 11-15 into a host cell to obtain the recombinant host cell.

28. A method for detecting B7H3 protein in a sample for non-diagnostic and non-therapeutic purposes, characterized in that, The method includes the following steps: contacting the sample to be tested with the monoclonal antibody of any one of claims 1-3, the diagnostic conjugate of claim 18, the detection reagent of claim 19, and / or the detection product of claim 20 or 21, and detecting the formation of the B7H3 protein immune complex with the antibody.

29. The use of the monoclonal antibody according to any one of claims 1-3 in the preparation of a diagnostic conjugate for detecting B7H3 protein; The diagnostic conjugate is used to diagnose diseases associated with B7H3 positivity. The diseases associated with B7H3 positivity are ovarian cancer, prostate cancer, liver cancer, or lung cancer.

30. The use of the monoclonal antibody of any one of claims 1-3 and / or the diagnostic conjugate of claim 18 in the preparation of a detection reagent for detecting B7H3 protein.

31. The use of the monoclonal antibody of any one of claims 1-3, the diagnostic conjugate of claim 18, and / or the detection reagent of claim 19 in the preparation of a detection product for detecting B7H3 protein.

32. The use of the monoclonal antibody of any one of claims 1-3, the diagnostic conjugate of claim 18, the detection reagent of claim 19, and / or the detection product of claim 20 or 21 in the detection of B7H3 protein for non-diagnostic and non-therapeutic purposes.

33. The use of the monoclonal antibody of any one of claims 1-3, the diagnostic conjugate of claim 18, the detection reagent of claim 19, and / or the detection product of claim 20 or 21 in the preparation of diagnostic products for the diagnosis or auxiliary diagnosis of B7H3-related diseases; The diseases associated with B7H3 positivity are ovarian cancer, prostate cancer, liver cancer, or lung cancer.

34. The use of the monoclonal antibody of any one of claims 1-3 and / or the recombinant host cell of claim 16 or 17 in the preparation of a pharmaceutical composition for treating B7H3-positive related diseases; The diseases associated with B7H3 positivity are ovarian cancer, prostate cancer, liver cancer, or lung cancer.

35. The use of the monoclonal antibody of any one of claims 1-3, the recombinant host cell of claim 16 or 17, and / or the pharmaceutical composition of claim 22 in the preparation of a pharmaceutical formulation for treating B7H3-positive related diseases; The diseases associated with B7H3 positivity are ovarian cancer, prostate cancer, liver cancer, or lung cancer.

Images