WO2023025315A1 - Anticorps anti-b7-h3, son procédé de préparation et son utilisation - Google Patents

Anticorps anti-b7-h3, son procédé de préparation et son utilisation Download PDF

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WO2023025315A1
WO2023025315A1 PCT/CN2022/115318 CN2022115318W WO2023025315A1 WO 2023025315 A1 WO2023025315 A1 WO 2023025315A1 CN 2022115318 W CN2022115318 W CN 2022115318W WO 2023025315 A1 WO2023025315 A1 WO 2023025315A1
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antibody
seq
amino acid
heavy chain
light chain
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Chinese (zh)
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魏海涛
宁婷婷
李亚男
董国良
李秀兰
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上海祥耀生物科技有限责任公司
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    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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Definitions

  • the invention relates to the technical field of antibodies, in particular to an anti-B7-H3 antibody, its preparation method and application.
  • Immune checkpoints refer to some inhibitory signaling pathways in the immune system.
  • the body In the case of normal anti-tumor immune response, the body maintains a balance between co-stimulatory signals and co-inhibitory signals, and maintains immunity by regulating the intensity of the autoimmune response. tolerance.
  • the immune checkpoint signaling pathway When the body is invaded by tumors, it usually blocks the immune checkpoint signaling pathway to inhibit autoimmunity and provide opportunities for tumor cell growth and escape.
  • tumor microenvironment and tumor immune escape mechanism it was found that negative B7 family molecules were abnormally expressed in various tumor tissues and tumor infiltrating immune cells, which were an important part of tumor microenvironment and involved in tumor immune escape.
  • B7-H3 also known as CD276, was first cloned from the cDNA library of human dendritic cells by Andrei I.Chapoval et al. in 2001, and has 20–27% homology with the B7 immunoglobulin superfamily at the amino acid level , a member of the immunoglobulin superfamily B7 (Chapoval, Ni et al.2001).
  • B7-H3 protein belongs to type I transmembrane protein, including a signal peptide, a C-terminal immunoglobulin constant region (IgC) and N-terminal variable region (IgV), a transmembrane region and an intracellular region (Vigdorovich, Ramagopal et al. 2013).
  • the B7-H3 protein has two variant splicing variants.
  • the extracellular segment of variant 1 is composed of four immunoglobulin domains of IgV-IgC-IgV-IgC, called 4Ig-B7-H3, and the extracellular segment of variant 2 is composed of IgV -IgC consists of 2 immunoglobulin domains, called 2Ig-B7-H3.
  • B7-H3 is low expressed in healthy tissues, but highly expressed in a large number of malignant tumors. Studies have shown that B7-H3 can play a role in esophageal cancer, melanoma, colorectal cancer, adenocarcinoma, ovarian cancer, non-small cell lung cancer, kidney cancer, gastric cancer, bladder cancer, glioblastoma multiforme and osteosarcoma, etc. Highly expressed in cancer (Tang, Zhao et al.2019, Tang, Liu et al.2020). B7-H3 is not only expressed on tumor cells, but also highly expressed on tumor neovascular endothelial cells, and is a very broad-spectrum tumor marker antigen. High expression of B7-H3 protein can promote cancer progression and is associated with poor prognosis of patients. Therefore, inhibiting the expression of B7-H3 protein has a potential effect on the treatment of cancer.
  • the present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the present invention proposes an anti-B7-H3 antibody capable of binding to B7-H3 with high specificity.
  • the present invention also proposes recombinant proteins, pharmaceutical compositions, polynucleotides, recombinant plasmids and isolated cells related to the above antibodies.
  • the present invention also proposes a preparation method of the above-mentioned antibody.
  • the present invention also proposes the application of the above antibody in the preparation of anticancer drugs.
  • the present invention also proposes the application of the above-mentioned antibody in the preparation of an antibody detection kit.
  • the antibody comprises a heavy chain variable region and/or a light chain variable region:
  • the heavy chain variable region comprises: represented by SEQ ID NO:1
  • the heavy chain complementarity determining region HCDR1 composed of the amino acid sequence
  • the heavy chain complementarity determining region HCDR2 composed of the amino acid sequence shown in SEQ ID NO:2
  • the heavy chain complementarity determining region HCDR3 composed of the amino acid sequence shown in SEQ ID NO:3
  • the light chain variable region which comprises: the light chain complementarity determining region LCDR1 composed of the amino acid sequence shown in SEQ ID NO: 4, the light chain complementarity determining region LCDR2 composed of the WAS amino acid sequence, composed of SEQ ID NO:
  • the light chain complementarity determining region LCDR3 composed of the amino acid sequence shown in 5.
  • the antibody according to the embodiment of the present invention has at least the following beneficial effects: the antibody of the present invention has (1) good binding activity and strong binding ability to B7-H3 protein; (2) cross-reactivity with monkey B7-H3 It has cross-reactivity, which is beneficial to carry out verification experiments and facilitate the subsequent development of products with therapeutic use; (3) has ADCC activity.
  • antibody-dependent cell-mediated cytotoxicity is a cell-mediated immune defense mechanism, which is the elimination of pathogenic target cells bound to specific antibodies by immune cells. mode of action.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the antibody is a murine antibody, a chimeric antibody or a human antibody.
  • the heavy chain variable region is selected from such as SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO The amino acid sequence shown in: 22;
  • the light chain variable region is selected from such as SEQ ID NO: 7, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 or SEQ ID The amino acid sequence shown in NO:27.
  • the antibody comprises a heavy chain constant region and/or a light chain constant region: the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 10; the light chain constant region comprises Amino acid sequence as shown in SEQ ID NO:12.
  • the antibody comprises a heavy chain and/or a light chain: the heavy chain is selected from the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:46 and SEQ ID NO:47; The light chain is selected from the amino acid sequences shown in SEQ ID NO: 15, SEQ ID NO: 48 and SEQ ID NO: 49.
  • the antibody comprises any one of the following properties i to v:
  • the antibody can specifically bind to B7-H3;
  • the antibody has antibody-dependent cell-mediated cytotoxicity
  • the antibody specifically binds to human 4Ig-B7-H3 and cross-reacts with monkey 4Ig-B7-H3;
  • the antibody has reduced or aglycosylated or hypofucosylated.
  • the above antibody sequences have the best binding activity, reaction specificity, species cross-reactivity and ADCC activity. It can be proved that the antibody of the present invention has a strong binding force to the B7-H3 protein through the experiments of the examples; it has the characteristic of species cross-reactivity, which is helpful for the toxicity analysis of the antibody in monkeys, and is conducive to carrying out verification tests in related animals. It is beneficial to the application and development of subsequent therapeutic purposes; it has strong ADCC activity and is more suitable for the subsequent development of antibody drugs.
  • the amino acid positions of the Fc region are modified to enhance Fc ⁇ R binding.
  • the Fc region has a stronger Fc ⁇ RIII binding ability, thereby exerting a stronger antibody-mediated cytotoxicity (ADCC) effect.
  • ADCC antibody-mediated cytotoxicity
  • the recombinant protein, pharmaceutical composition, polynucleotide, vector or isolated cell according to the embodiment of the second aspect of the present invention: the recombinant protein comprises the above-mentioned antibody; the pharmaceutical composition comprises the above-mentioned antibody or the above-mentioned recombinant protein; the multinuclear
  • the nucleotide contains the nucleotide sequence encoding the above-mentioned antibody or recombinant protein; the vector contains the above-mentioned polynucleotide; and the isolated cell produces the above-mentioned antibody.
  • the recombinant protein further comprises a tag sequence to assist expression and/or purification.
  • the recombinant protein is a double antibody, which also includes antibodies capable of binding to other target proteins. Further, the double antibody also includes antibodies that specifically bind to different epitopes of B7-H3.
  • the pharmaceutical composition further includes the above-mentioned double antibody.
  • the pharmaceutical composition further includes an ADC drug containing the above-mentioned antibody.
  • the ADC drug also includes a linker and a toxic molecule.
  • the pharmaceutical composition further includes pharmaceutically available excipients.
  • the preparation method according to the embodiment of the third aspect of the present invention includes the following steps: culturing the above-mentioned isolated cells, and recovering the antibody from the culture.
  • the specific preparation method is as follows: the heavy chain variable region sequence encoding the above-mentioned anti-B7-H3 antibody is cloned into recombinant plasmid 1 containing the IgG1 heavy chain constant region amino acid sequence, and the light chain variable region sequence is cloned into In the recombinant plasmid 2 containing the amino acid sequence of the Kappa light chain constant region, the recombinant plasmid 1 and the recombinant plasmid 2 were simultaneously transfected into cells and cultured, and the anti-B7-H3 antibody was recovered from the culture.
  • the antibody can be applied to the preparation of anticancer drugs and/or antibody detection kits.
  • the anticancer drug is mainly used for preventing or treating cancer, wherein the cancer is breast cancer, endometrial cancer, ovarian cancer, lung cancer, gastric cancer, prostate cancer, kidney cancer, Cancer of the liver, pancreas, colorectum, esophagus, bladder, cervix, blood, lymphoma, or malignant melanoma.
  • anti-cancer drugs also include antibodies targeting other targets, such as: CD3, BCMA, CD38, etc. to construct bispecific antibodies, and develop various methods for regulating tumor cells.
  • the antibody of the present invention can also be coupled to other types of molecules, such as toxins, nucleic acid molecules, etc., and the antibody can specifically bring the coupled molecules into the body of tumor cells, thereby regulating the effect of tumor cells.
  • the B7-H3-specific antibody or antigen-binding fragment can be labeled for use in the method or other methods known to those skilled in the art.
  • the antibodies or antigen-binding fragments thereof of the present invention can be radiolabeled, fluorescently labeled, epitope tags, biotin, chromophore labels, ECL labels, enzymes, ruthenium, 111In-DOTA, 111In-di Ethylenetriaminepentaacetic acid (DTPA), horseradish peroxidase, alkaline phosphatase and beta-galactosidase, or polyhistidine or similar such labels known in the art.
  • DTPA 111In-DOTA
  • DTPA 111In-di Ethylenetriaminepentaacetic acid
  • horseradish peroxidase alkaline phosphatase and beta-galactosidase
  • polyhistidine or similar such labels known in the art.
  • the antibody can also be developed into a method for detecting the expression level of B7-H3 on the surface of tissue cells by means of immunization.
  • the antibody can also be used for detecting the presence of B7-H3 in a biological sample such as blood or serum, for quantitative analysis of the amount of B7-H3 in a biological sample such as blood or serum, for diagnosing B7-H3 expressing cancer, for To determine a method of treating a subject with cancer, or for monitoring the progression of a B7-H3 expressing cancer in a subject, and the like.
  • the term "antibody” specifically includes antibodies in a narrow sense, and also includes “chimeric” antibodies and antibody fragments, wherein part of the heavy chain and/or light chain corresponds to an antibody derived from a specific species or belonging to a specific antibody type or subclass.
  • the sequence is identical or homologous, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody type or subclass, so long as it specifically binds the target antigen and/or exhibits the desired Biological activity (US Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
  • antibody in a narrow sense refers to a type of glycosyl-containing globulin that is secreted by antigens entering the body to stimulate B cells to differentiate and proliferate into plasma cells, which can specifically bind to corresponding antigens and produce immune effects.
  • World Health Organization held a meeting and collectively referred to globulins with antibody activity and chemical structure similar to antibodies as immunoglobulins. Modern immunology believes that antibodies and immunoglobulins are equivalent concepts, but antibodies focus on the description of their biological activities, while immunoglobulins focus on their chemical structures.
  • the basic structure of immunoglobulin consists of four peptide chains: two heavy chains (Heavy chain, H chain) and two light chains (Light chain, L chain), the light chain and the heavy chain are connected by disulfide bonds to form a symmetrical tetrapeptide Chain molecules become monomers of immunoglobulin molecules, which are the basic structure of all immunoglobulins.
  • Each heavy and light chain is divided into amino-terminus (N-terminus) and carboxy-terminus (C-terminus).
  • variable region V region
  • C region C-terminal amino acid
  • HVR hypervariable region
  • framework region Framework region
  • HVR1, HVR2 and HVR3 of the heavy chain or light chain respectively.
  • the hypervariable region is the binding site of the antibody and the antigen, and is called the complementarity-determining region (CDR). Therefore, HVR1, HVR2, and HVR3 of the heavy chain or light chain are also called CDR1, CDR2, and CDR3.
  • the term "diabodies”, that is, "bispecific" antibodies refers to an antibody, generally a monoclonal antibody, that has the binding properties of at least two different antigenic epitopes.
  • the epitopes are from the same antigen.
  • the epitopes are from two different antigens.
  • Methods of making bispecific antibodies are known in the art. For example, bispecific antibodies can be produced recombinantly by co-expressing two immunoglobulin heavy/light chain pairs. See, eg, Milstein et al., Nature 305:537-39 (1983). Alternatively, bispecific antibodies can be prepared using chemical linkage. See, eg, Brennan et al., Science 229:81 (1985).
  • Bispecific antibodies include bispecific antibody fragments (eg, Hollinger et al., Proc. Natl. Acad. Sci. U.S.A. 90:6444-48 (1993), Gruber et al., J. Immunol. 152:5368 (1994)).
  • humanized antibody refers to a form of antibody that contains sequences derived from non-human (eg, murine) antibodies as well as human antibodies.
  • the antibody is a chimeric antibody containing minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies will comprise substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, all or substantially all of which The FR regions are those of human immunoglobulin sequences.
  • the humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. See, e.g., Cabilly U.S. Patent No. 4,816,567; Queen et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033; Oxford University Press) 1996).
  • Fc immunoglobulin constant region
  • Fig. 1 shows the binding force of chimeric antibody 27B4 and MDA-MB-231 in Example 3 of the present invention
  • Figure 2 shows the ADCC activity of chimeric antibody 27B4 in Example 3 of the present invention
  • Figure 3 shows the binding ability of different 27B4 humanized scFv antibodies to B7-H3 antigen in Example 4 of the present invention
  • Fig. 4 shows the binding force of different Whole IgG forms 27B4 humanized antibody B7-H3 antigen in Example 4 of the present invention
  • Figure 5 shows the ADCC activities of different Whole IgG forms of 27B4 humanized antibodies in Example 4 of the present invention.
  • Fig. 6 shows the binding activity of 27B4 humanized antibody h27B4-H4-L3 (27B4Z02 molecule) in Example 4 of the present invention to B7-H32Ig and 4Ig proteins.
  • Fig. 7 shows the activity of binding human and monkey B7-H3 proteins of the 27B4 humanized antibody h27B4-H4-L3 (27B4Z02 molecule) in Example 4 of the present invention.
  • the human 4Ig-B7-H3 antigen used for screening and detection is a commercial product (Acro, product number B7B-H52E7), and the sequence is as follows:
  • the human 2Ig-B7-H3 antigen used for screening and detection is a commercial product (Acro, product number B73-H52E2), and the sequence is as follows:
  • the macaque 4Ig-B7-H3 antigen used for screening and detection is a commercial product (Acro, product number B73-C52Ha), and the sequence is as follows:
  • mouse 2Ig-B7-H3 antigen used for screening and detection is a commercial product (Acro, product number B73-M52H4), and the sequence is as follows:
  • the horizontal line part is the extracellular region of B7-H3; the italic part is the linker; the black bold part is the His-tag tag.
  • the CDS region encoding human B7-H3 variant 1 (4Ig-B7-H3) was obtained from the NCBI database, and the full-length gene sequence was routinely synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. NotI was constructed into the lentiviral overexpression vector pCDH-EF1-T2A-copGFP, and the sequence was verified to be correct by sequencing, that is, the plasmid pCDH-EF1-h4Ig-B7-H3-T2A-copGFP was successfully constructed.
  • HEK293 cells in the logarithmic growth phase (derived from ATCC) in a good growth state, inoculate 8E6 cells in a 10 cm culture dish, add 10% fetal bovine serum to DMEM, and culture in a 37°C, 5% CO 2 incubator , when the cell fusion rate reaches about 70% to 80%, transfection.
  • the three plasmids psPAX2, pMD2.G and pCDH-EF1-h4Ig-B7-H3-T2A-copGFP were co-transfected using the transfection reagent PEI.
  • the virus liquid was collected 48 hours and 72 hours after transfection, filtered through a 0.45 ⁇ M syringe filter, infected with the target cell CHO-S (derived from ATCC) at an MOI of 10, and cultured in RPMI-1640 plus 10% fetal bovine serum. The medium was changed 48 hours after virus infection. The positive rate of human 4Ig-B7-H3 expression was tested by flow cytometry, and the positive rate was over 95%. The 293-h4Ig-B7-H3-copGFP stably transfected cell line was successfully constructed.
  • mice of SPF grade BALB/C strain purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.
  • the immune antigen was His-tagged human recombinant 4Ig-B7-H3 antigen (Acro, catalog number B7B- H52E7)
  • the antigen was emulsified with an equal volume of adjuvant.
  • the antigen was emulsified with complete Freund's adjuvant (Sigma, product number F5881), and injected subcutaneously at multiple points on the back of the neck of the mouse, and each mouse was immunized with 50 ⁇ g of the antigen.
  • the antigen was emulsified with Freund's incomplete adjuvant (Sigma, product number F5602) for immunization, and each mouse was immunized with 25 ⁇ g of the antigen.
  • Freund's incomplete adjuvant Sigma, product number F5602
  • each mouse was immunized with 25 ⁇ g of the antigen.
  • blood was collected from the orbit of the mouse, and the titer of the mouse immune serum was detected by ELISA according to the method of Examples 2-4.
  • the pre-immune serum was used as a negative control.
  • the immune serum was diluted 1:100, it was then serially diluted to 11 concentrations by 3 times, and the serum dilution corresponding to the OD value of 3 times the negative control was used as its titer.
  • Select the mouse with the highest titer and the serum titer tends to be stable after two booster immunizations.
  • 25 ⁇ g recombinant human 4Ig-B7-H3 antigen is injected intraperitoneally for a booster immunization.
  • the antigen does not need adjuvant emulsification , the buffer is PBS.
  • flow cytometry Flow Cytometry, FACS detection was carried out according to the method in Example 2-5, and the binding force between the supernatant and the overstable cells 293-h4Ig-B7-H3-copGFP was detected.
  • Positive hybridoma cells were monoclonalized by the limiting dilution method, and then detected by ELISA and FACS.
  • the FACS method simultaneously detected the samples and the overstable cells 293-h4Ig-B7-H3-copGFP and endogenous expression cells MDA-MB For the binding status of -231, select positive monoclonal wells and repeat monoclonalization and positive clone screening and identification once.
  • Hybridoma cells were inoculated into a 250mL cell culture flask at 2.5 ⁇ 10 5 /mL, and the culture volume was 50mL. When the cell viability dropped to 30%, the supernatant containing the antibody was harvested by centrifugation, and filtered with a 0.45 ⁇ M filter. Mouse monoclonal antibodies were purified in Protein G medium and replaced by dialysis into PBS pH7.2 buffer. Absorbance was measured by Nanodrop to determine antibody concentration and purity, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining.
  • B7-H3 antigen human 4Ig-B7-H3 antigen, Acro, Cat. No. B7B-H52E7 or human 2Ig-B7-H3, Acro, Cat. B73-C52Ha or mouse 2Ig-B7-H3, Acro, Cat. No. B73-M52H4
  • PBS PBS containing 2% skim milk powder
  • the antibody produced by a hybridoma cell 27B4 has the highest binding activity to human 4Ig-B7-H3 antigen and human 2Ig-B7-H3 antigen, and has cross-species reactivity, and can simultaneously bind macaque 4Ig-B7- H3. This cross-reactivity facilitates the application of the antibody in animal experiments.
  • RNAiso Plus RNAiso Plus
  • cDNA was obtained by reverse transcription with 5'RACE method (SMARTer RACE5'/3'Kit, Clontech, product number 634859).
  • Adapter as an upstream primer
  • CL and CH1 as downstream primers for antibody light and heavy chains respectively
  • ExTaq PCR 94°C, 3min; 94°C, 30s, 55°C, 30s, 72°C, 45s, 32cycle; 72°C, 5min
  • Tiangen universal DNA reagent After the cassettes were recovered and purified, they were ligated with the pMD18-T vector (Takara, Cat. No.
  • the ligation products were transformed into TG-1 Escherichia coli competent cells for sequencing.
  • VBASE2 http://www.vbase2.org/vbscAb.php
  • the screened antibody is referred to as 27B4.
  • the amino acid sequence of the 27B4 heavy chain variable region includes heavy chain complementarity determining regions HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2) and HCDR3 (SEQ ID NO: 3); the light chain variable region includes Light chain complementarity determining regions LCDR1 (SEQ ID NO: 4), LCDR2 (WAS) and LCDR3 (SEQ ID NO: 5).
  • Amino acid sequence of 27B4VH of 27B4 heavy chain variable region SEQ ID NO:6.
  • the amino acid sequence hIgG1 (AWK57454.1) of the ⁇ heavy chain constant region of the antibody was obtained from NCBI, and mutations L235V, F243L, R292P, Y300L and P396L were introduced to enhance the ADCC effect (antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity cytotoxicity), and at the N-terminal of the sequence, Kozak sequence (5'-GCCACCATGG-3'), signal peptide sequence and multiple cloning site region sequence were sequentially added, and eukaryotic codon optimization was performed to obtain the base sequence, in which the cloning site The dot region contains restriction sites KpnI (5'-GGTACC-3') and MfeI (5'-CAATTG-3'), and the routine synthetic gene sequence of Sangon Bioengineering (Shanghai) Co., Ltd. was constructed into the pTT5 vector (Biovector ), the eukaryotic heavy chain expression vector pTT5-hIgG
  • Optimized hIgG1 base sequence SEQ ID NO: 11.
  • the amino acid sequence HL (CAR58102.1) of the ⁇ light chain constant region of the antibody was obtained from NCBI, and the Kozak sequence (5'-GCCACCATGG-3'), the signal peptide sequence and the sequence of the multiple cloning site region were sequentially added to the N-terminal of the sequence.
  • the base sequence was obtained by codon optimization for eukaryotic expression, in which the cloning site region contains restriction sites KpnI (5'-GGTACC-3') and MfeI (5'-CAATTG-3'), in Sangon Bioengineering (Shanghai ) Co., Ltd. routinely synthesized the gene sequence into the multiple cloning site of the pTT5 vector to obtain the eukaryotic heavy chain expression vector pTT5-HL containing the antibody light chain constant region sequence.
  • Amino acid sequence of constant region of kappa light chain SEQ ID NO:12.
  • the base sequence of the constant region of the ⁇ light chain SEQ ID NO: 13.
  • the Kozak sequence is a nucleic acid sequence located behind the cap structure at the 5' end of eukaryotic mRNA, which can bind to translation initiation factors to mediate translation initiation of mRNA containing the 5' cap structure.
  • the heavy chain and light chain variable region sequences of 27B4 were routinely synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., and cloned into pTT5-hIgG1 and pTT5-HL vector plasmids by KpnI and MfeI, respectively, to obtain the heavy chain expression plasmid pTT5- 27B4-VH-hIgG1 and light chain expression plasmid pTT5-27B4-VL-HL.
  • PEI co-transfects light chain expression plasmid pTT5-27B4-VL-HL and heavy chain expression plasmid pTT5-27B4 - 25 ⁇ g each of VH-hIgG1.
  • the cell supernatant on the 7th day after transfection was collected, centrifuged and filtered using a 0.45 ⁇ M filter, the protein A medium was used to purify the antibody, and the antibody was replaced by dialysis into PBS pH7.2 buffer.
  • Absorbance was measured by Nanodrop to determine antibody concentration and purity, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining.
  • the binding activity of the chimeric antibody xw.M0883B9.27B4 obtained in Example 3-3 to the B7-H3 antigen was examined by FACS method.
  • the positive reference antibody is MGA-271 (NCT02475213).
  • PBMC peripheral blood mononuclear cell
  • ADCC medium RPMI 1640 containing 1% FBS
  • 40 ⁇ m cell strainer BD Biosciences, Ltd.
  • Collect the MDA-MB-231 cells in the logarithmic growth phase in good growth state wash the cells once with ADCC medium, filter with a 40 ⁇ m cell strainer, count the cells and adjust the cell density to 2x 10 5 /mL, as the target cells.
  • Experimental group experimental well-medium background control well
  • Target cell spontaneous target cell spontaneous LDH release well-medium background control well
  • Effector cell spontaneous effector cell spontaneous LDH release well-medium background control well
  • Maximum LDH release from target cells maximum LDH release pores from target cells - volume correction pores.
  • the mouse anti-human 27B4 monoclonal antibody was humanized according to the so-called CDR grafting method. Briefly, the VH and VK base sequences of the 27B4 antibody were analyzed using the IMGT/V-QUEST tool (http://www.imgt.org/IMGT_vquest/input), and the CDR region sequences of the antibody light chain and heavy chain were determined. Use the IgBlast tool (https://www.ncbi.nlm.nih.gov/igblast/) to analyze the amino acid sequence of the 27B4 antibody to obtain the closest human germline VH and VK sequences of the 27B4 antibody.
  • IMGT/V-QUEST tool http://www.imgt.org/IMGT_vquest/input
  • IgBlast tool https://www.ncbi.nlm.nih.gov/igblast/
  • the CDRs of the 27B4 antibody are grafted into the framework regions of the selected VH and VK human germline sequences, and this sequence is the humanized antibody sequence. Analyze the human germline VH of the 27B4 antibody VH, select 5 different sequences, graft the CDRs of the 27B4 antibody VH into the framework regions of these 5 sequences, and obtain 5 heavy chain variable regions 27B4-H1, 27B4 -H2, 27B4-H3, 27B4-H4 and 27B4-H5. The same method was used to carry out CDR grafting on the light chain, and five light chain variable region sequences 27B4-L1, 27B4-L2, 27B4-L3, 27B4-L4 and 27B4-L5 were obtained.
  • amino acid sequences of 27B4-H1, 27B4-H2, 27B4-H3, 27B4-H4 and 27B4-H5 are respectively denoted as SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 21 and SEQ ID NO: 22.
  • amino acid sequences of 27B4-L1, 27B4-L2, 27B4-L3, 27B4-L4 and 27B4-L5 are respectively denoted as SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 26 and SEQ ID NO: 27.
  • the base sequences of 27B4-H1, 27B4-H2, 27B4-H3, 27B4-H4 and 27B4-H5 are recorded as SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 32.
  • the base sequences of 27B4-L1, 27B4-L2, 27B4-L3, 27B4-L4 and 27B4-L5 are shown in the sequence list SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36 and SEQ ID NO: 37.
  • the humanized scFv 27B4 antibody obtained through the combination of 27B4-H1-F, linker and 27B4-L1-F is named "scFv-27B4-H1-L1", and the humanized scFv 27B4 antibody obtained through the combination of 27B4-H1-F, linker and 27B4-L2-
  • the humanized scFv 27B4 antibody obtained by combining F is named "scFv-27B4-H1-L2”
  • the humanized scFv 27B4 antibody obtained by combining 27B4-H1-F, linker and 27B4-L3-F is named "scFv -27B4-H1-L3”
  • the humanized scFv 27B4 antibody obtained through the combination of 27B4-H1-F, linker and 27B4-L4-F is named "scFv-27B4-H1-L4"
  • the humanized scFv 27B4 antibody is named "scFv-27B4-H3-L3", and the humanized scFv 27B4 antibody obtained by combining 27B4-H3-F, linker and 27B4-L4-F is named “scFv-27B4-H3 -L4", will be obtained by combining 27B4-H3-F, linker and 27B4-L5-F
  • the humanized scFv 27B4 antibody was named "scFv-27B4-H3-L5", and the humanized scFv 27B4 antibody obtained by combining 27B4-H4-F, linker and 27B4-L1-F was named "scFv-27B4- H4-L1", the humanized scFv 27B4 antibody obtained through the combination of 27B4-H4-F, linker and 27B4-L2-F is named "scFv-27B4-H4-L2", and
  • the humanized scFv 27B4 antibody obtained by combining 27B4-H5-F, linker and 27B4-L1-F is named "scFv-27B4-H5-L1", and will be obtained by combining 27B4-H5-F, linker and 27B4-L2-F
  • the combined humanized scFv 27B4 antibody was named "scFv-27B4-H5-L2”
  • the humanized scFv 27B4 antibody obtained by combining 27B4-H5-F, linker and 27B4-L3-F was named "scFv- 27B4-H5-L3”
  • the humanized scFv 27B4 antibody obtained through the combination of 27B4-H5-F, linker and 27B4-L4-F is named "scFv-27B4-H5-L4"
  • 25 scFv single-chain antibodies constructed in Example 4-2 with humanized sequences of 27B4 were expressed and purified.
  • the centrifuged sample on the column wash the column with 25mL washing buffer containing 10mM and 20mM imidazole in turn, and finally wash the column with 5mL washing buffer containing 250mM imidazole, and collect the eluate.
  • Antibodies were exchanged by dialysis into PBS pH 7.2 buffer. Absorbance was measured by Nanodrop to determine antibody concentration and purity, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining
  • amino acid sequences of scFv-27B4-H2-L2, scFv-27B4-H2-L3, scFv-27B4-H4-L2 and scFv-27B4-H4-L3 are SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41.
  • the base sequences of scFv-27B4-H2-L2, scFv-27B4-H2-L3, scFv-27B4-H4-L2 and scFv-27B4-H4-L3 are SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45.
  • the heavy chain humanized sequence 27B4-H2 and 27B4-H4 were conventionally synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., and cloned into pTT5-hIgG1 through KpnI and MfeI respectively, and the light chain humanized sequence 27B4- L2 and 27B4-L3 were routinely synthesized at Sangon Bioengineering (Shanghai) Co., Ltd., and cloned into the pTT5-HL vector plasmid by KpnI and MfeI, respectively, to obtain heavy chain expression plasmids pTT5-27B4-H2-hIgG1, pTT5-27B4- H4-hIgG1 and light chain expression plasmids pTT5-27B4-L2-HL, pTT5-27B4-L3-HL.
  • the 293F cells in the logarithmic growth phase in good growth state were inoculated into 250 mL cell culture flasks and cultured in 50 mL medium, and 25 ⁇ g of each light and heavy chain expression plasmids were co-transfected with PEI.
  • the cell supernatant on the 7th day of culture after transfection was collected, centrifuged and filtered using a 0.45 ⁇ M filter, the protein A medium was used to purify the antibody, and the antibody was replaced by dialysis into PBS pH7.2 buffer.
  • Absorbance was measured by Nanodrop to determine antibody concentration and purity, and purity was checked by sodium dodecyl sulfate gel electrophoresis and Coomassie staining.
  • the antibody obtained by the combination of pTT5-27B4-H2-hIgG1 and pTT5-27B4-L2-HL was named "h27B4-H2-L2", and the antibody obtained by the combination of pTT5-27B4-H2-hIgG1 and pTT5-27B4-L3-HL
  • the combined antibody was named "h27B4-H2-L3”
  • the antibody obtained by combining pTT5-27B4-H4-hIgG1 and pTT5-27B4-L2-HL was named "h27B4-H4-L2”
  • the antibody obtained from the combination of 27B4-H4-hIgG1 and pTT5-27B4-L3-HL was named "h27B4-H4-L3".
  • the heavy chain amino acid sequences of h27B4-H2 and h27B4-H4 SEQ ID NO: 46, SEQ ID NO: 47.
  • Heavy chain base sequences of h27B4-H2 and h27B4-H4 SEQ ID NO: 50, SEQ ID NO: 51.
  • the light chain base sequences of h27B4-L2 and h27B4-L3 SEQ ID NO: 52, SEQ ID NO: 53.
  • the ADCC activity of the whole IgG form 27B4 human antibody was detected according to the method of Example 3-5.
  • four Whole IgG forms of 27B4 humanized antibodies h27B4-H2-L2, h27B4-H4-L3, h27B4-H2-L3, and h27B4-H4-L2 (identified in the figure as zw.M0883B9.27B4Z01, zw .M0883B9.27B4Z02, zw.M0883B9.27B4Z03, zw.M0883B9.27B4Z04) all have ADCC activity, which is comparable to the ADCC activity of the 27B4 antibody before humanization.
  • the results are shown in Figure 5.
  • the molecule zw.M0883B9.27B4Z02 (h27B4-H4-L3, referred to as molecule Z02) had the highest activity, and the cross-reactivity of molecule Z02 was detected according to the method of Example 2-4. Further binding EC50 and special cross-binding experiments on the Z02 molecule proved that the molecule can simultaneously recognize human 4Ig-B7-H3 antigen and human 2Ig-B7-H3 antigen, and has the ability to bind to monkey 4Ig-B7-H3 protein. Reactivity facilitates the use of antibodies in animal experiments. The results are shown in Figures 6 and 7.
  • the anti-B7-H3 antibodies prepared in the present invention have high B7-H3 antigen binding activity and ADCC activity, and can be applied in the fields of cancer treatment and the like.

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Abstract

La présente invention concerne un anticorps anti-B7-H3, son procédé de préparation et son utilisation. L'anticorps anti-B7-H3 présente une activité de liaison relativement élevée, une réactivité hétérospécifique et une activité ADCC, et peut être utilisé pour traiter des maladies telles que le cancer.
PCT/CN2022/115318 2021-08-27 2022-08-26 Anticorps anti-b7-h3, son procédé de préparation et son utilisation WO2023025315A1 (fr)

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