Anti-B7-H3 antibody and application of combination of anti-B7-H3 antibody and anti-PD-1 antibody in preparation of cancer treatment drugs

Abstract

The invention discloses an anti-B7-H3 antibody and application of the anti-B7-H3 antibody and an anti-PD-1 antibody in preparation of cancer treatment drugs. According to the present invention, the anti-B7-H3 antibody and the anti-PD-1 antibody are combined to obtain the cancer treatment drug, and the drug can significantly improve the Hela cell killing efficiency of PBMCs in mice.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-B7-H3 antibody and its use in combination with an anti-PD-1 antibody in the preparation of cancer therapeutic drugs. Background Art

[0002] The B7-H3 protein is a key tumor antigen. Human B7-H3 molecules consist of two isoforms, 4IgB7-H3 and 2IgB7-H3, belonging to the immunoglobulin family. These two isoforms are structurally very similar, differing only in that 4IgB7-H3 has an additional IgC-IgV immunoglobulin domain in its extracellular region. Both 4IgB7-H3 and 2IgB7-H3 are highly expressed in various tumors and are potential targets for tumor immunotherapy.

[0003] Existing technologies use anti-B7-H3 antibody-drug conjugates (ADCs) to treat solid cancers such as lung cancer; however, their efficacy remains to be improved. Therefore, the unmet need for a drug that can significantly enhance the efficiency of PBMCs in killing HeLa cells remains a major challenge for drug developers. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an anti-B7-H3 antibody and its use in combination with an anti-PD-1 antibody in the preparation of cancer therapeutic drugs, which solves the problem that the existing technology uses anti-B7-H3 antibody-drug conjugates (ADCs) to treat solid cancers such as lung cancer, but has poor effects.

[0005] To achieve the above objectives, the technical solution of the present invention is achieved as follows: a B7-H3 antibody, comprising a HCDR1 region with an amino acid sequence as shown in SEQ ID NO: 1, a HCDR2 region with an amino acid sequence as shown in SEQ ID NO: 2, a HCDR3 region with an amino acid sequence as shown in SEQ ID NO: 3, a LCDR1 region with an amino acid sequence as shown in SEQ ID NO: 4, a LCDR2 region with an amino acid sequence as shown in SEQ ID NO: 5, and a LCDR3 region with an amino acid sequence as shown in SEQ ID NO: 6.

[0006] The application described in the above technical solution, wherein: it also includes a heavy chain VH region with an amino acid sequence as shown in SEQ ID NO:7; the amino acid sequence of the heavy chain VH region has at least 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:7.

[0007] The application described in the above technical solution, wherein: it also includes a VL region of a light chain whose amino acid sequence is shown in SEQ ID NO:8; the amino acid sequence of the VL region of the light chain has at least 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:8.

[0008] The application described in the above technical solution, wherein: it also includes a heavy chain with an amino acid sequence as shown in SEQ ID NO:9, and the amino acid sequence of the heavy chain has at least 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:9.

[0009] The application described in the above technical solution, wherein: it also includes a light chain with an amino acid sequence as shown in SEQ ID NO: 10, and the amino acid sequence of the light chain has at least 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO: 10.

[0010] Another technical solution of the present invention is achieved as follows: use of the above-mentioned B7-H3 antibody in combination with an anti-PD-1 antibody in the preparation of cancer therapeutic drugs.

[0011] The application described in the above technical solution, wherein: the anti-PD-1 antibody includes pembrolizumab injection.

[0012] The application described in the above technical solution, wherein: the dosage of the anti-B7-H3 antibody is 100 μg / kg to 10 mg / kg; the dosage of the anti-PD-1 antibody is 100 μg / kg to 10 mg / kg.

[0013] The application of the above technical solution, wherein: the cancer includes B7-H3 + cancer.

[0014] The application described in the above technical solution, wherein: the B7-H3+ cancer is at least one of cervical cancer, liver cancer, neuroblastoma, glioblastoma, melanoma, non-small cell lung cancer, breast cancer, pancreatic cancer, colorectal cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer and ovarian cancer; preferably cervical cancer.

[0015] Compared with the prior art, the present invention combines anti-B7-H3 antibodies with anti-PD-1 antibodies to obtain a cancer treatment drug, which can significantly improve the efficiency of PBMCs in killing Hela cells in mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1This is a bar graph showing the test results of the effects of anti-B7-H3 antibody alone, anti-PD-1 antibody alone, and anti-B7-H3 antibody combined with anti-PD-1 antibody on the efficiency of PBMCs killing Hela cells in vitro in Example 1 of the present invention. It is also a graph showing the first cycle performance test results of the long-lasting energy storage battery of the present invention. DETAILED DESCRIPTION

[0017] All technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0018] The terms "a" and "an" refer to one or to more than one (ie, to at least one) of the grammatical object of the article. As an example, "an element" refers to one element or more than one element.

[0019] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[0020] Portions of the antibodies or antibody fragments thereof of the present invention can exist in a variety of forms, wherein the antigen binding domain is expressed as part of a continuous polypeptide chain comprising, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen binding domain of the present invention comprises an antibody fragment. In another aspect, the antibody fragment of the present invention is an scFv. The exact amino acid sequence boundaries of a given CDR can be determined using any one or a combination of a number of well-known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme).

[0021] The term "anti-PD-1 antibody" refers to antibodies that specifically bind to PD-1. PD-1 (programmed death receptor-1) is a surface protein whose primary function is to inhibit T cell activity by binding to its ligand, PD-L1, expressed on some tumor cells, thereby helping tumor cells evade immune system attack. By using anti-PD-1 antibodies to specifically bind to PD-1, thereby blocking the immune escape signaling pathway mediated by the binding of PD-1 and PD-L1, tumor cells can effectively prevent them from evading immune surveillance and attack.

[0022] The term "identity" refers to the subunit sequence identity between two polymer molecules, such as two nucleic acid molecules (such as two DNA molecules or two RNA molecules), or between two polypeptide molecules. When a subunit position in both molecules is occupied by the same monomeric subunit, for example, if a position in two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of positions that are paired or homologous; for example, if half (e.g., 5 positions in a polymer that is 10 subunits in length) of the positions in the two sequences are homologous, then the two sequences are 50% homologous; if 90% of the positions (e.g., 9 out of 10) match or are homologous, then the two sequences are 90% homologous.

[0023] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise expressly limited, the term encompasses nucleic acids comprising known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly shown. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0024] The term "treating" refers to a subject treated with the applications described herein to achieve at least one positive therapeutic effect (e.g., a decrease in the number of cancer cells, a decrease in tumor size, a decrease in the rate of cancer cell infiltration into peripheral organs, or a decrease in the rate of tumor metastasis or tumor growth). The therapeutic approach that effectively treats a patient may vary depending on a variety of factors, such as the patient's disease state, age, weight, and the ability of the therapy to elicit an anti-cancer response in the subject.

[0025] As used herein, "treatment" includes any beneficial or desired effect associated with treatment."Treatment" does not necessarily indicate complete eradication or cure of the disease or condition, or its associated symptoms.

[0026] The term "cancer" is defined as a disease characterized by the rapid, uncontrolled growth of abnormal cells. Abnormal cells can form solid tumors or constitute blood malignancies. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system.

[0027] Herein, unless the context requires otherwise, the word "comprising" will be understood to mean the inclusion of the specified steps, or elements, or groups of steps or elements, but not the exclusion of any other steps, or elements, or groups of steps or elements. In some embodiments of the present invention, the terms "including," "having," "containing," and "comprising" are used synonymously.

[0028] "Embodiments": Reference throughout this specification to "some embodiments" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, various appearances of the foregoing phrase throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0029] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0030] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0031] The embodiments of the present invention provide an anti-B7-H3 antibody in combination with an anti-PD-1 antibody for the preparation of a cancer treatment drug; wherein the B7-H3 antibody comprises an HCDR1 region with an amino acid sequence as shown in SEQ ID NO: 1, an HCDR2 region with an amino acid sequence as shown in SEQ ID NO: 2, an HCDR3 region with an amino acid sequence as shown in SEQ ID NO: 3, a LCDR1 region with an amino acid sequence as shown in SEQ ID NO: 4, a LCDR2 region with an amino acid sequence as shown in SEQ ID NO: 5, and a LCDR3 region with an amino acid sequence as shown in SEQ ID NO: 6; and the anti-PD-1 antibody comprises pembrolizumab injection.

[0032] In a further embodiment, the B7-H3 antibody further comprises a heavy chain VH region having an amino acid sequence as shown in SEQ ID NO:7; the amino acid sequence of the heavy chain VH region is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:7.

[0033] In a further embodiment, the B7-H3 antibody further comprises a light chain VL region having an amino acid sequence as shown in SEQ ID NO:8; the amino acid sequence of the light chain VL region is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:8.

[0034] In a further embodiment, the B7-H3 antibody further comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO:9, wherein the amino acid sequence of the heavy chain is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:9.

[0035] In a further embodiment, the B7-H3 antibody further comprises a light chain having an amino acid sequence as shown in SEQ ID NO: 10, wherein the amino acid sequence of the light chain is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10.

[0036] Wherein, the cancer includes B7-H3 + Cancer; the B7-H3+ cancer is at least one of cervical cancer, liver cancer, neuroblastoma, glioblastoma, melanoma, non-small cell lung cancer, breast cancer, pancreatic cancer, colorectal cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer and ovarian cancer.

[0037] In a further implementation process, the dosage of the anti-B7-H3 antibody is 100 μg / kg to 10 mg / kg; the dosage of the anti-PD-1 antibody is 100 μg / kg to 10 mg / kg.

[0038] The following are specific embodiments

[0039] Example 1

[0040] To test the application of anti-B7-H3 antibody combined with anti-PD-1 antibody in killing target cells Hela cells (adult cervical cancer cells).

[0041] The specific detection process is as follows:

[0042] Day 0: thaw frozen PBMCs from healthy individuals (thaw methods are well known to those skilled in the art); prepare four groups of mixed cells as follows: 1×10 5 The PBMCs were mixed with 5 × 10 5 The two groups of mixed cells were mixed with Hela cells carrying luciferase reporter genes, and the mixed cell culture medium was used to resuspend the two groups of mixed cells. The mixed cell culture medium included: 1640 culture medium + 10% FBS;

[0043] To three of the mixed cell culture systems, 1 μg / mL of anti-2IgB7-H3 antibody (PBMC+antibody1 group), 1 μg / mL of anti-PD-1 antibody (pembrolizumab injection) (PBMC+antibody2 group), or 1 μg / mL of anti-2IgB7-H3 antibody and 1 μg / mL of anti-PD-1 antibody (PBMC+antibody1+2 group) were added respectively;

[0044] Synthesize and purify the anti-2IgB7-H3 antibody (methods known to those skilled in the art);

[0045] The amino acid sequence of the HCDR1 region of the anti-2IgB7-H3 antibody is shown in SEQ ID NO: 1:

[0046] GYSFSNYW (SEQ ID NO: 1)

[0047] The amino acid sequence of the HCDR2 region of the anti-2IgB7-H3 antibody is shown in SEQ ID NO: 2:

[0048] IYPGDSDT (SEQ ID NO: 2)

[0049] The amino acid sequence of the HCDR4 region of the anti-2IgB7-H3 antibody is shown in SEQ ID NO: 3:

[0050] ARSRGYPTNYMDV (SEQ ID NO: 3)

[0051] The amino acid sequence of the LCDR1 region of the anti-2IgB7-H3 antibody is shown in SEQ ID NO: 4:

[0052] SSNIGNNI (SEQ ID NO: 4)

[0053] The amino acid sequence of the LCDR2 region of the anti-2IgB7-H3 antibody is shown in SEQ ID NO: 5:

[0054] RER (SEQ ID NO: 5)

[0055] The amino acid sequence of the LCDR3 region of the anti-2IgB7-H3 antibody is shown in SEQ ID NO: 6:

[0056] ATWDDSLNSWV (SEQ ID NO: 6)

[0057] The amino acid sequence of the VH region of the anti-2IgB7-H3 antibody is shown in SEQ ID NO: 7:

[0058] QVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWIGWVKQMPGKGLEW MGIIYPGDSDTRYSPSFQGQVTSADKSISTAYLQWSSLKASDTAMYYCARSG YPTNYMDVWGRGTTVTVSS(SEQ ID NO:7)

[0059] The amino acid sequence of the VL region of the anti-2IgB7-H3 antibody is shown in SEQ ID NO: 8:

[0060] QAVLTQPPSVSGAPRQRVTISSCSGSSSNIGNNIVSWYQHLPGKAPRLLIYR ERQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDDDLNSWVFG GGTKLT(SEQ ID NO:8)

[0061] The amino acid sequence of the heavy chain of the anti-2IgB7-H3 antibody is shown in SEQ ID NO: 9:

[0062] QVQLVQSGAEVKKPGESLKISCKGSGYSFSNYWIGWVKQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTSADKSISTAYLQWSSLKASDTAMYYCARSGYPTNYMDVWGRGT TVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP PCPAPEFLGGGSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQID NO:9)

[0063] The amino acid sequence of the light chain of the anti-2IgB7-H3 antibody is shown in SEQ ID NO: 10:

[0064] QAVLTQPPSVSGAPRQRVTISSCSGSSSNIGNNIVSWYQHLPGKAPRLLIYRERQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCATWDDDLNSWVFGGGTKLTRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:10)

[0065] The four groups of mixed cells were inoculated into three wells of an ELISA plate, and the substrate was added after culturing for 2 hours. Chemiluminescence detection was performed using the ELISA plate to calculate the killing efficiency. The specific calculation method was as follows: the well containing only target Hela cells was used as the reference well, and its fluorescence value was the total fluorescence value of the original tumor cell count; the fluorescence value of the tumor remaining in each well after killing with different antibodies and their combinations was the residual fluorescence value, and the killing efficiency was calculated as follows: Killing efficiency (%) = (total fluorescence value - residual fluorescence value) / total fluorescence value × 100%. The killing results are shown in Figure 2. Figure 1 As shown;

[0066] Depend on Figure 1 It can be seen that compared with the use of anti-B7-H3 antibody or anti-PD-1 antibody alone, the combination of anti-B7-H3 antibody and anti-PD-1 antibody will significantly improve the efficiency of PBMCs in killing Hela liver cancer cells.

[0067] Example 2

[0068] 20 NKG mice (purchased from Saiye Bio) were subcutaneously injected with 2×10 6 HeLa tumor cells were grown in the mice and divided into 4 groups, with 5 tumor-bearing mice in each group.

[0069] On Day 0 and Day 7, anti-B7-H3 antibody, anti-PD-1 antibody, or anti-B7-H3 antibody combined with anti-PD-1 antibody were respectively administered via tail vein injection to three groups of tumor-bearing mice according to the administration method shown in Table 1 below.

[0070] Table 1 Grouping of 20 NKG mice

[0071] Group 1 5 control group Group 2 5 20 μg / anti-B7-H3 antibody Group 3 5 20 μg / anti-PD-1 antibody Group 4 5 20μg / anti-B7-H3 antibody + 20μg / anti-PD-1 antibody

[0072] Starting from the third day, the tumor growth was observed every day, the tumor size was recorded, and the tumor volume was calculated according to the following formula: V = ab 2 / 2 (V-volume, a-long diameter of tumor, b-short diameter of tumor). Tumor volume of each group (mm 3) changes are shown in Table 2 below (mean ± standard deviation).

[0073] Table 2 Changes in tumor volume of mice in each group on days 3, 6, 9, 12 and 15

[0074]

[0075] As shown in Table 2, compared with the use of anti-B7-H3 antibody or anti-PD-1 antibody alone, the combination of anti-B7-H3 antibody and anti-PD-1 antibody can significantly improve the efficiency of PBMCs in killing Hela cells in mice.

[0076] In summary, the present invention combines anti-B7-H3 antibodies with anti-PD-1 antibodies to obtain a cancer therapeutic drug, which can significantly improve the efficiency of PBMCs in killing Hela cells in mice.

[0077] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A B7-H3 antibody, characterized in that It includes a HCDR1 region with an amino acid sequence as shown in SEQ ID NO: 1, a HCDR2 region with an amino acid sequence as shown in SEQ ID NO: 2, a HCDR3 region with an amino acid sequence as shown in SEQ ID NO: 3, a LCDR1 region with an amino acid sequence as shown in SEQ ID NO: 4, a LCDR2 region with an amino acid sequence as shown in SEQ ID NO: 5, and a LCDR3 region with an amino acid sequence as shown in SEQ ID NO:

6.

2. The B7-H3 antibody according to claim 1, characterized in that Also included is a heavy chain VH region having an amino acid sequence as shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain VH region is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

7.

3. The B7-H3 antibody according to claim 2, characterized in that Also included is a light chain VL region having an amino acid sequence as shown in SEQ ID NO: 8; the amino acid sequence of the light chain VL region is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

8.

4. The B7-H3 antibody according to claim 3, characterized in that Also included is a heavy chain having an amino acid sequence as shown in SEQ ID NO:9, wherein the amino acid sequence of the heavy chain is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

9.

5. The B7-H3 antibody according to any one of claims 1 to 4, characterized in that Also included is a light chain having an amino acid sequence as shown in SEQ ID NO: 10, wherein the amino acid sequence of the light chain is at least 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:

10.

6. Use of the B7-H3 antibody according to any one of claims 1 to 5 in combination with an anti-PD-1 antibody in the preparation of a cancer therapeutic drug.

7. The use according to claim 6, characterized in that The anti-PD-1 antibody includes pembrolizumab injection.

8. The use according to claim 7, characterized in that The dosage of the anti-B7-H3 antibody is 100 μg / kg to 10 mg / kg; the dosage of the anti-PD-1 antibody is 100 μg / kg to 10 mg / kg.

9. The use according to any one of claims 6 to 8, characterized in that: The cancer includes B7-H3 + cancer.

10. The use according to claim 9, characterized in that The B7-H3+ cancer is at least one of cervical cancer, liver cancer, neuroblastoma, glioblastoma, melanoma, non-small cell lung cancer, breast cancer, pancreatic cancer, colorectal cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer and ovarian cancer.

Images