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WO2024152864A1 - 抗cd26抗体及其应用 - Google Patents

抗cd26抗体及其应用 Download PDF

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Publication number
WO2024152864A1
WO2024152864A1 PCT/CN2023/142580 CN2023142580W WO2024152864A1 WO 2024152864 A1 WO2024152864 A1 WO 2024152864A1 CN 2023142580 W CN2023142580 W CN 2023142580W WO 2024152864 A1 WO2024152864 A1 WO 2024152864A1
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antibody
seq
antigen
cells
binding fragment
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French (fr)
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马永
曹锫沛
葛晨楠
袁昊
张韬
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江苏众红生物工程创药研究院有限公司
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Publication of WO2024152864A1 publication Critical patent/WO2024152864A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]

Definitions

  • CD26 The expression level of CD26 on the surface of various tumor cells is significantly increased, for example, malignant mesothelioma, renal cancer, prostate cancer, lung cancer, etc.
  • CD26 is an important target (CD26/DPP4-a potential biomarker and target for cancer therapy, Pharmacology & Therapeutics (2019), 198:135-159).
  • Antibody-drug conjugate consists of three parts: antibody, linker, and cytotoxic small molecule. It uses the antibody's specific recognition of antigens to transmit toxin molecules to the tumor site and effectively release drug molecules, thereby achieving the therapeutic purpose of killing tumor cells. Antibody-drug conjugates have both the specific recognition of antibodies and the tumor-killing effect of toxin molecules. The main function of the antibody part is to bind to the target antigen, giving the drug a targeted characteristic. At present, 15 ADC drugs have been approved for marketing worldwide, of which the most are ADCs targeting HER2.
  • the antibody part of ADC should have high specificity, high affinity, low immunogenicity, high endocytosis, long half-life, etc.
  • ADC drugs targeting CD26 that have been marketed or entered the clinical stage, and the potential of CD26 as an ADC drug target needs to be further explored.
  • the first object of the present invention is to provide an anti-CD26 antibody or antigen-binding fragment having higher specificity, affinity and better internalization.
  • the antibody or antigen-binding fragment comprises a heavy chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to that shown in SEQ ID NO:8, and a light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to that shown in SEQ ID NO:9.
  • the antibody or antigen-binding fragment wherein one, two, three, four, five, six, seven, eight, nine or ten amino acids in the sequence shown in SEQ ID NO: 8 or SEQ ID NO: 9 are inserted, deleted or substituted.
  • the amino acid substitution is a conservative amino acid substitution.
  • the antibody or antigen-binding fragment comprises a heavy chain variable region shown in SEQ ID NO: 8 and a light chain variable region shown in SEQ ID NO: 9.
  • the present application relates to an antibody-drug conjugate targeting CD26, comprising the above-mentioned anti-CD26 antibody.
  • the present invention also relates to a vector comprising the nucleotide sequence and a host cell comprising the nucleic acid.
  • the present invention also relates to a method for producing an antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell and recovering the antibody or antigen-binding fragment from the culture.
  • the present invention also relates to a method for treating tumors, comprising administering an effective amount of the antibody or antigen-binding fragment to a patient; the tumor highly expresses CD26, including but not limited to renal cancer, mesothelioma, lung cancer, liver cancer, prostate cancer, etc.
  • the antibody or antigen-binding fragment is administered alone or in combination with other anticancer agents.
  • the anti-CD26 antibodies screened by the present invention have high specificity, affinity and binding force to CD26-positive tumor cells, and the full-length humanized antibody derived from mouse antibody No. 79 has higher affinity, binding force and internalization level to tumor cells than full-length humanized antibodies derived from other mouse antibodies, and is more suitable as the antibody part in the ADC molecule.
  • Figure 1 Flow cytometry analysis of antibody binding differences in mixed cell systems
  • Figure 2 Flow cytometry detection of changes in fluorescence intensity after antibody internalization in 786-0 cells
  • Figure 3 Flow cytometry detection of changes in fluorescence intensity after antibody internalization in PBMC cells
  • Figure 4 Flow cytometry detection of changes in fluorescence intensity after antibody internalization in different PBMC cells
  • VH and VL can be further subdivided into a complementarity determining region (CDR, also known as a hypervariable region) with high variability, and a more conservative framework region (FR).
  • CDR complementarity determining region
  • FR more conservative framework region
  • Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4.
  • HCDR1, HCDR2, and HCDR3 are the three heavy chain complementary determining regions
  • LCDR1, LCDR2, and LCDR3 are the three light chain complementary determining regions.
  • the positions of CDRs and framework regions can be determined using various numbering systems well known in the art, such as Kabat, Chothia, IMGT, etc., and IMGT is used in this application.
  • the heavy chain variable region (VH) and the light chain variable region (VL) are responsible for antigen recognition, especially
  • the complementarity determining regions (CDRs) are usually specific for different epitopes of the antigen, while the constant region is mainly responsible for effector functions.
  • humanized antibody refers to an antibody in which the CDR regions are derived from non-human (eg, mouse) antibody sequences and the rest are derived from human antibody sequences.
  • BiTE is a bispecific antibody, which stands for "bispecific T cell engager". It is composed of two single chain variable fragments (scFv) with antigen specificity connected by a linker, one of which targets tumor-associated surface antigens and the other targets CD3 on the surface of T cells.
  • Single chain variable fragments (scFv) also known as single chain antibodies, are composed of antibody heavy chain variable regions and light chain variable regions connected by a linker.
  • Linkers are amino acid sequences used to connect different protein fragments. There have been many related studies on the design and selection of linkers.
  • the linker connects a heavy chain variable region and a light chain variable region to form an scFv, and also connects two scFvs in series and allows them to rotate freely. It is generally composed of a GGGGS repeating structure (S represents serine and G represents glycine). Glycine has a small molecular weight and a short side chain, which can increase the flexibility of the side chain. The strong hydrophilicity of serine can increase the hydrophilicity of the peptide chain.
  • Optimizing the number of repeat structures can not only help the light chain and heavy chain in the scFv to associate in the correct conformation, but also promote the optimal interaction between T cells and target cells in the immune synapse by affecting the distance between the two scFvs (Zhou Chong et al. Research progress of bispecific single-chain antibody BiTE [J]. Journal of Biology, 2018”).
  • the linkers connecting the heavy chain variable region and the light chain variable region in scFv are commonly KESGSVSSEQLAQFRSLD, EGKSSGSGSESKST, GSTSGGGSGGGSGGGGSS (US 20180326032), GSTSGSGKPGSGEGSTKG (Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and CD22), etc.
  • the flexible linker connecting a heavy chain and a light chain to form an scFv is composed of three GGGGS repeating structures, and the flexible linker connecting two scFvs in series is GGGGS.
  • GGGGS the flexible linker connecting two scFvs in series
  • CD3 is an integral part of T cell signaling.
  • the T cells are activated, promoting CD8+T cells to directly secrete perforin and granzymes, and CD4+T cells to secrete cytokines to further recruit activated killer T cells, thereby killing tumor cells.
  • the CD3 antibody sequence can be selected from OKT-3, L2K, TR66, UCHT1, SP34, IORT3, Catumaxomab, Blinatumomab, Solitomab and other CD3 antibody sequences known in the art.
  • the CD3 antibody sequence in Solitomab is used, and those skilled in the art can select and determine from other known CD3 antibodies by conventional experimental methods and limited tests, without being limited to the specific CD3 antibody sequence in the specific embodiments of the present application.
  • ADC antibody-drug conjugate
  • the cell pellet was resuspended with PBS, centrifuged again and the supernatant was discarded, and the cell pellet was collected; the corresponding antibody Alexa Fluor 488 mouse anti-human IgG1 was used as the secondary antibody, the cell pellet was resuspended, and incubated at room temperature in the dark for about 30 minutes. After the incubation, it was washed twice with PBS, centrifuged and the supernatant was discarded, and the cell pellet was collected; the cell pellet was resuspended with about 200ul of PBS solution, and the CD26 positivity rate was detected and analyzed by flow cytometry within 1 hour.
  • the rCD26 expression vector was designed based on the CD26 cDNA sequence published in GenBank (GenBank accession number: NM_001935.3). A histidine tag was introduced at the 5' end of the codon, and the whole gene was synthesized and connected to the pCHO1.0 plasmid. The CHO-S host cells expressed and purified the rCD26 expression vector.
  • the prepared recombinant CD26 target protein was immunized into BALB/c female mice according to the general immunization procedure. For specific immunization conditions, please refer to the "Antibody Preparation and Use Experimental Guide”. The serum titer of the immunized mice was tracked by indirect ELISA, and the immunized mice with the highest serum titer were selected. Mouse spleen cells and mouse myeloma cells were fused.
  • Step 2 Cell fusion
  • mice were killed by removing their eyeballs and taking blood. The mice were then sacrificed by breaking the cervical vertebrae and immersed in 75% (v/v) alcohol for 10 minutes. The spleens were removed on a sterile operating table and placed on a cell sieve. The cells were fully ground and passed through the sieve. After washing several times by centrifugation with sterile 1640 culture medium (purchased from Gibco), the cells were resuspended to make a single cell suspension, counted, and set aside.
  • sterile 1640 culture medium purchased from Gibco
  • a female BALB/c mouse aged 2 to 3 weeks was killed by cervical vertebrae dissection and then immersed in 75% (v/v) alcohol for 10 minutes; the thymus was aseptically taken out on a cell sieve, ground, and sieved to obtain thymic cells, which were placed in the above-mentioned 20% 1640HAT culture medium containing macrophages for later use.
  • Select mouse myeloma cell line SP2/0 in the logarithmic growth phase collect and count. Take about 10 8 spleen cells and 2 ⁇ 10 7 SP2/0 cell lines and add them to the fusion tube, mix them, centrifuge at 1000rpm for 10 minutes, discard the supernatant, place the fusion tube on the palm of your hand and rub it back and forth gently to loosen the precipitate.
  • Step 3 Screening of anti-CD26 specific hybridoma cell lines
  • test plate Dilute the recombinant CD26 target protein to 1 ⁇ g/ml with CB coating buffer, coat the plate with 96-well ELISA enzyme labeling plate, 100 ⁇ l/well, coat overnight at 2-8°C, wash once and pat dry; block with PBST buffer containing 2% BSA (200 ⁇ l/well) at 37°C for 2 hours; pat dry and set aside.
  • Step 4 Activity evaluation of CD26 mouse monoclonal antibody (mouse anti-79)
  • Tumor cells were labeled with green fluorescent signals using the fluorescent dye Calcein-AM. Tumor cells were inoculated into U-shaped 96-well cell culture plates at a cell concentration of 6 ⁇ 10 5 cells/ml and 50 ⁇ l per well. Three replicate wells were set up in each group. 50 ul of anti-CD26 antibody with a final concentration of 100 ng/ml was added to the reaction wells of the corresponding sample wells, 50 ul of culture medium control was added to the blank control wells, and 50 ul of Triton X-100 with a final concentration of 1% was added to the positive control wells.
  • mice antibody No. 79 was 34.4% (the cell lysis rates of PBMC mediated by other mouse antibodies such as mouse antibodies No. 47, 76, 62, and 82 under the same conditions were 15.4%, 19.9%, 49.9%, and 30.8%, respectively), the cell lysis rate of PBMC mediated by mouse antibody No. 79 on the target cell NCI-H226 was 25.5% (the cell lysis rates of PBMC mediated by other mouse antibodies such as mouse antibodies No. 47, 51, 62, and 82 under the same conditions were 6.7%, 21%, 14.3%, and 17.5%, respectively), and mouse antibody No. 79 had killing activity against different CD26-positive cell lines.
  • the CD26 protein was biotinylated with EZ-link NHS-PEG12-Biotin; the biotinylated CD26 protein was immobilized with a SA biosensor; the protein was equilibrated in a 1 ⁇ Kinetics solution; the protein was combined with a solution of the antibody molecule to be detected, where the concentration of the molecule to be detected was 500 nM; the protein was dissociated in a 1 ⁇ Kinetics solution; and the data was analyzed using Fortebio Data Analysis 8.0 software to calculate the affinity constant value.
  • the affinity of 19G300 to CD26 protein is 1.14E-09M
  • the affinity of 19G300 to CD3 protein is 1.61E-09M.
  • 21G430-431 is a full-length humanized anti-CD26 antibody from mouse antibody No. 79, and the amino acid sequences of its heavy and light chain variable regions are 19G300-VH (SEQ ID NO: 8) and 19G300-VL (SEQ ID NO: 9); 21G428-429 is a full-length humanized anti-CD26 antibody from mouse antibody No.
  • the signal peptide sequence (SEQ ID NO: 14) was added upstream of 19G300-VH (SEQ ID NO: 8), 19G294-VH (SEQ ID NO: 17), and YS110-VH (SEQ ID NO: 18), and the human IgG1 heavy chain constant region (SEQ ID NO: 15) was added downstream to obtain the IgG1 heavy chain sequences of the full-length humanized anti-CD26 antibodies 21G430-431, 21G428-429, and YS110.
  • the codon preference was optimized to obtain the optimized gene sequence, and the AvrII restriction site and kozak sequence were introduced upstream, and the stop codon and BstZ17I restriction site were added downstream. After direct synthesis, it was constructed into the AvrII and BstZ17I restriction sites of the pCHO1.0 vector.
  • a signal peptide sequence (SEQ ID NO: 14) was added upstream of 19G300-VL (SEQ ID NO: 9), 19G294-VL (SEQ ID NO: 19), and YS110-VL (SEQ ID NO: 20), and a human kappa light chain constant region (SEQ ID NO: 16) was added downstream to obtain the IgG1 light chain sequences of full-length humanized anti-CD26 antibodies 21G430-431, 21G428-429, and YS110, which were optimized according to the codon preference of mammalian cells CHO to obtain the optimized gene sequence, and an EcoRV restriction site and a kozak sequence were introduced upstream, a stop codon and a PacI restriction site were added downstream, and the sequences were directly synthesized and constructed into the EcoRV and PacI restriction sites of the pZHK5.18 vector.
  • the heavy chain vector and the light chain vector were co-transfected into CHO-S cells. After double pressure screening with hygromycin and puromycin, a CHO cell line stably expressing the full-length antibody was obtained. The stably expressing CHO cell line was inoculated in Dynamis medium and cultured in batch feed at 37°C, 8% CO 2 , and 130 rpm.
  • the supernatant culture fluid was harvested, centrifuged at 12000rpm for 15min, and filtered through a 0.45 ⁇ m collection filter to obtain the treated culture fluid supernatant.
  • the purified product was purified using Cytiva MabSelect Sure affinity chromatography filler, and the purified product was adjusted to pH 5.5 and then refined using Cytiva SP HP cationic chromatography filler to obtain the target product, which was approximately 150kDa in size.
  • Collect PBMC cells and adjust the cell density to 6 ⁇ 10 5 /mL after counting. Take 50 ⁇ L of 786-0 and PBMC cells and add them to the EP tube as a mixed cell system. Add antibody diluted to a final concentration of 0.3 ug/mL, mix with the mixed cell system, and incubate at 4°C for 1 hour.
  • the test results show (Figure 1) that the full-length humanized anti-CD26 antibody 21G430-431 exhibited a significantly higher binding level in cancer cells 786-0 than the full-length humanized anti-CD26 antibody 21G428-429. Relatively speaking, 21G430-431 exhibited a binding level comparable to that of 21G428-429 in PBMC cells.
  • the full-length humanized antibodies 21G428-429 and 21G430-431 have better selectivity for recognizing the CD26 target on tumor cells than on PBMC cells, and preferentially bind to the CD26 target on tumor cells, and have a very low degree of binding to the CD26 target on PBMC cells, thereby avoiding killing normal cells.
  • the full-length humanized antibody 21G430-431 has a higher tumor cell binding ability than 21G428-429.
  • Antibody-drug conjugates are conjugated with antibodies and cytotoxic molecules, combining the specificity of antibodies to recognize target cells with the powerful tumor-killing effect of toxins.
  • the antibody part should have: (1) high tumor tissue specificity and strong affinity/binding force to ensure that ADC can efficiently recognize tumor cells and reduce off-target toxicity; (2) after binding to the target antigen, it can be effectively internalized into tumor cells and quickly release toxin molecules after entering the endosome or lysosome, thereby exerting the small molecule cytotoxic effect.
  • the antibody was fluorescently labeled according to the instructions of pHrodo iFL Green STP ester.
  • This fluorescent label is sensitive to pH and can significantly enhance fluorescence under low pH conditions.
  • the pH of the lysosomal environment is about 5.0, which is significantly lower than the pH value of 7.2 in the cytoplasmic matrix. Therefore, when the fluorescently labeled antibody enters the tumor cell and is engulfed by the lysosome, its fluorescence is significantly enhanced due to the significant change in pH from 7.4 to 5.0. Therefore, it is used to indicate whether the antibody is internalized into the lysosome.
  • Collect 786-0/PBMC cells count and adjust the cell density of both to 2 ⁇ 10 5 /mL. Take 50 ⁇ L of cells and add them to the EP tube as a mixed cell system. Add fluorescently labeled antibodies, dilute to a final concentration of 5ug/mL, mix with the mixed cell system, and incubate at 37°C for 24h. Take another group of cells without adding fluorescently labeled antibodies, mix before detection as 0h. Centrifuge the samples before detection to remove the serum-containing culture medium and replace it with PBS solution. Use flow cytometry to detect its average fluorescence intensity.
  • test results show (Figure 2) that the anti-CD26 full-length humanized antibody 21G430-431 exhibited a significantly higher internalization level in cancer cell 786-0 than the anti-CD26 full-length humanized antibody 21G428-429, indicating that 21G430-431 is more suitable for use as an antibody in ADC molecules than 21G428-429.
  • the test results show ( Figure 5, Figure 6) that the three anti-CD26 antibody-drug conjugates all exhibit a certain ability to kill target cells.
  • DT3C-conjugated 21G430-431 showed a killing ability superior to DT3C-conjugated 21G428-429, with a significant difference. This indicates that antibodies with higher cell affinity, binding force and stronger internalization levels show a higher level of killing of target cells after being conjugated to toxin molecules.
  • YS110 which has progressed to clinical phase II, was also used as a control antibody. It can be seen that 21G430-431 showed Stronger ADC effect than YS110, with significant difference.
  • the anti-CD26 antibody derived from mouse antibody No. 79 has high specificity, affinity, and binding force to CD26-positive tumor cells, and the full-length humanized antibody derived from mouse antibody No. 79 has higher affinity and binding force to tumor cells, higher tumor cell internalization level, and lower non-tumor cell internalization level than other full-length humanized antibodies derived from mouse antibodies, thereby showing better efficacy and safety in ADC molecules and can be used as the antibody part of ADC molecules.

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Abstract

与人CD26结合的抗体或抗原结合片段，及其在制备治疗高表达CD26肿瘤的药物中的应用。一种CD26鼠源抗体及抗原结合片段具有较高特异性、亲和力、结合力，对不同肿瘤细胞具有一定的杀伤活性。以该CD26鼠源抗体成功构建人源化双特异性抗体、全长人源化抗体、抗体偶联药物，较其他鼠抗来源的体具有更高对肿瘤细胞亲和力、结合较其他鼠抗来源的体具有更高对肿瘤细胞亲和力、结合，肿瘤细胞内化水平更高，非肿瘤细胞内化水平更低，在ADC分子中表现出了更好的药效和安全性。

Description

抗CD26抗体及其应用 技术领域
本发明涉及生物制药技术领域，具体涉及与人CD26结合的抗体或抗原结合片段，及其在制备治疗高表达CD26肿瘤的药物中的应用。
背景技术
CD26是一种多功能Ⅱ型跨膜糖蛋白，也可以以溶解形式存在于血浆中。CD26常以同源二聚体形式存在，其单体含766个氨基酸，相对分子质量约110kDa。CD26氨基酸残基从内向外分为5个部分：胞内区(1～6)、跨膜区(7～28)、高度糖基化区(29～323)、富含半胱氨酸区(324～551)和C端催化结构域(552～766)，其分子三维结构与功能密切相关。CD26(DPP4)抑制剂在临床上用于Ⅱ型糖尿病的治疗已有数十年的历史。CD26在多种肿瘤细胞表面的表达量明显升高，例如，恶性间皮瘤，肾癌，前列腺癌，肺癌等，对于这类型CD26表达量较高的肿瘤，CD26是一种具有重要价值的作用靶点(CD26/DPP4-a potential biomarker and target for cancer therapy,Pharmacology & Therapeutics(2019),198:135-159)。
目前以人CD26为靶点的靶向性抗癌药物研究进展最快的是Y’S Therapeutics公司的单克隆抗体YS110。YS110已经完成的临床二期研究结果显示，YS110具有较好的耐受性，但疾病控制率未达到预期，这与YS110在临床前研究中所表现出来的低活性相对应。
尽管在文献资料中有报道CD26的广泛分布，但YS110的使用仍然具有较好的安全性，足见其在临床使用中的安全性有较好的保障。在YS110临床试验的30例患者中，未出现患者死亡，所表现出来的副作用主要为，16.1％的患者有输液不良反应，9.7％的患者出现呃逆，6.5％的患者出现腹泻等，较严重的不良反应有2例间质性肺病，1例细菌性肺炎，1例肿瘤溶解综合征，1例血碳酸过多症和1例缺氧。(Phase 2 Study of YS110,a Recombinant Humanized Anti-CD26 Monoclonal Antibody,in Japanese Patients With Advanced Malignant Pleural Mesothelioma,JTO Clinincal and Research Reports(2021),2(6))
综上所述，尽管CD26靶点的安全性已在临床试验中得到验证，但现有的以CD26为治疗靶点的抗体对肿瘤治疗难以取得较好的治疗效果。
抗体偶联药物(Antibody-drug conjugate，简称ADC)，由抗体、连接子、细胞毒性小分子三部分组成，利用抗体对抗原的特异性识别，将毒素分子传输到肿瘤部位并有效释放药物分子，从而达到杀死肿瘤细胞的治疗目的。抗体偶联药物兼具抗体的特异性识别与毒素分子的肿瘤杀伤效果，其中抗体部分的主要功能是与目标抗原结合，使药物具备靶向性的特性。目前，全球已有15款ADC药物获批上市，其中靶向HER2的ADC最多。
一般来说，ADC的抗体部分应具备高特异性、高亲和力、低免疫原性、高内吞性、长半衰期等特性。目前尚无已上市或进入临床阶段的靶向CD26的ADC药物，CD26作为ADC药物靶点的潜力有待进一步发掘。
发明内容
为解决上述问题，本发明的第一个目的在于提供一种具有较高特异性、亲和力，且内吞更佳的抗CD26抗体或抗原结合片段。
本发明提供的与人CD26特异性结合的抗体或抗原结合片段，包含SEQ ID NO：1所示的HCDR1、SEQ ID NO：2所示的HCDR2、SEQ ID NO：3所示的HCDR3；以及包含SEQ ID NO：4所示的LCDR1、序列为YRS的LCDR2(Y、R、S分别代表酪氨酸、精氨酸、丝氨酸)和SEQ ID NO：5所示的LCDR3。
所述抗体或抗原结合片段，包含与SEQ ID NO：8所示至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的重链可变区，以及包含与SEQ ID NO：9所示至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的轻链可变区。
所述抗体或抗原结合片段，其中SEQ ID NO：8、SEQ ID NO：9所示序列的一个、两个、三个、四个、五个、六个、七个、八个、九个或十个氨基酸被插入、缺失或取代。所述氨基酸取代是保守氨基酸取代。
所述抗体或抗原结合片段，其包含SEQ ID NO：8所示的重链可变区和包含SEQ ID NO：9所示的轻链可变区。
本申请涉及靶向CD26的抗体偶联药物，含有上述抗CD26抗体。
本发明还涉及编码所述抗体或抗原结合片段的氨基酸序列的核苷酸序列。
本发明还涉及包含所述核苷酸序列的载体、包含所述核酸的宿主细胞。
本发明还涉及用于产生抗体或其抗原结合片段的方法，所述方法包括培养所述宿主细胞，从培养物中回收抗体或抗原结合片段。
本发明还涉及包含所述抗体或抗原结合片段的药物组合物，其还包含药学上可接受的赋形剂。
本发明还涉及一种治疗肿瘤的方法，其包括向患者给予有效量的所述抗体或抗原结合片段；所述肿瘤高表达CD26，包括但不限于肾癌、间皮瘤、肺癌、肝癌、前列腺癌等。所述抗体或抗原结合片段单独给予，或者与其它抗癌剂联合给予。
本申请相比现有技术具有如下优势：
本发明筛选的抗CD26抗体对CD26阳性肿瘤细胞具有较高的特异性、亲和力、结合力，且79号鼠抗来源的全长人源化抗体较其他鼠抗来源的全长人源化抗体具有更高的对肿瘤细胞的亲和力、结合力和内化水平，更适合作为ADC分子中抗体部分。
附图说明
图1：流式检测混合细胞体系中抗体结合力差异
图2：流式检测抗体在786-0细胞中内化后荧光强度变化
图3：流式检测抗体在PBMC细胞中内化后荧光强度变化
图4：流式检测抗体在不同PBMC细胞中内化后荧光强度变化
图5：UT16细胞中DT3C偶联的21G428-429，21G430-431和YS110诱导的细胞存活率(72h)
图6：786-0细胞中DT3C偶联的21G428-429，21G430-431和YS110诱导的细 胞存活率(72h)
具体实施方式
除非在本文中明确定义，否则本文使用的技术术语具有本领域普通技术人员通常理解的含义。
单数形式词语如“一个(a、an)”“所述(the)”包括它们对应的复数指示物。
术语“或”意指“和/或”，并可与“和/或”互换使用。
术语“CD26”又称二肽基肽酶(DPP4，dipeptidyl peptidase 4)，人CD26氨基酸序列能以登录号NP_001926.2在Genbank中找到，其cDNA序列能以登录号NM_001935.3在Genbank中找到。
术语“保守氨基酸取代”意指用新的氨基酸取代原始氨基酸，新的氨基酸基本上不改变抗体或片段的化学、物理和/或功能特性，例如其对CD26的结合亲和力。氨基酸的常见保守取代是本领域熟知的，例如Ala可被保守取代为Gly、Ser；Arg可被保守取代为Lys、His；Asn可被保守取代为Gln、His等。术语“亲和力”是指抗体与抗原之间相互作用的强度。抗体可变区通过非共价力与抗原相互作用；相互作用越多，亲和力越强。
术语“抗体”是指可以非共价地、可逆地且以特异性方式结合相应抗原的免疫球蛋白家族。例如，天然存在的IgG抗体是四聚体，其包含通过二硫键相互连接的至少两条重链和两条轻链。每条重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由三个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。VH和VL可以进一步细分为具有高变性的互补决定区(CDR，又称高变区)，以及更保守的框架区(FR)。每个VH和VL由三个CDR和四个FR组成，按照以下顺序从氨基末端到羧基末端排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。HCDR1、HCDR2、HCDR3为三个重链互补决定区，LCDR1、LCDR2、LCDR3为三个轻链互补决定区。CDR和框架区的位置可以使用各种本领域熟知的编号系统来确定，例如Kabat、Chothia、IMGT等，本申请采用IMGT确定。重链可变区(VH)及轻链可变区(VL)负责抗原识别，尤其是 其中的互补决定区(CDR)，通常对抗原的不同表位具有特异性。恒定区主要负责效应子功能。
术语“抗原结合片段”指抗体的抗原结合片段，即保留与抗原特异性结合能力的抗体片段，例如保留一个或多个CDR区的片段，包括但不限于Fab片段、FV片段、双抗体、线性抗体、单链抗体、纳米抗体、多特异性抗体等。
术语“单克隆抗体”意指基本上同质的抗体群体，即除了少量存在的可能天然发生的突变，群体中包含的抗体分子的氨基酸序列是相同的。相比之下，多克隆抗体通常包括在其可变结构域(特别是其互补决定区)中具有不同氨基酸序列的多种不同抗体。单克隆抗体可以通过本领域技术人员已知的方法获得。本公开文本采用杂交瘤技术获得单克隆抗体，该方法是获得单克隆抗体已知的众多方法之一。
术语“鼠源抗体”指仅包含大鼠或小鼠免疫球蛋白序列的的抗体。
术语“嵌合抗体”的可变区来自非人(例如，鼠)抗体序列，其余部分来自人抗体序列。
术语“人源化抗体”CDR区来自非人(例如，鼠)抗体序列，其余部分来自人抗体序列。
术语“双特异性抗体”，是拥有两种相同或不同的特异性抗原结合位点的抗体。
术语“BiTE”是一种双特异性抗体，全称“双特异性T细胞接合器”，由两个具有抗原特异性的单链可变片断(single chain variable fragment，scFv)通过接头连接而成，其中一个scFv靶向识别肿瘤相关表面抗原，另一个scFv靶向识别T细胞表面CD3。单链可变片断(scFv)，也称为单链抗体由抗体重链可变区和轻链可变区通过接头连接而成。“接头”是用于将不同蛋白片段连接起来的氨基酸序列，针对接头的设计和选择已有诸多相关研究。在BiTE中，接头连接一个重链可变区与一个轻链可变区组成scFv，也串联两个scFv并允许它们自由旋转，一般由GGGGS重复结构组成(S代表丝氨酸，G代表甘氨酸)，甘氨酸分子量小、侧链短可增加侧链的柔韧性，丝氨酸较强的亲水性可增加肽链的亲水性。优化重复结构的数目不仅可以帮助scFv中轻链与重链以正确构象缔合，并可以通过影响两个scFv之间的距离以促进T细胞和靶细胞在免疫突触中的最佳交互作用(周冲等. 双特异性单链抗体BiTE的研究进展[J].生物学杂志,2018”)。在scFv中连接重链可变区与轻链可变区的接头常用KESGSVSSEQLAQFRSLD、EGKSSGSGSESKST、GSTSGGGSGGGSGGGGSS(US 20180326032)、GSTSGSGKPGSGEGSTKG(Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and CD22)等。在本申请具体的实施例中，连接一个重链与一个轻链组成scFv的柔性接头由3个GGGGS重复结构组成，串联两个scFv的柔性接头为GGGGS，本领域技术人员可以依据已有技术的教导，通过常规实验方法和有限次试验从已知接头中选择确定，可以对两种柔性接头中GGGGS的重复数量进行优化，也可以采用除GGGGS外的其他柔性接头。
CD3是T细胞信号传导的组成部分。当BiTE分子同时与T细胞和肿瘤细胞结合时，T细胞被激活，促进CD8+T细胞直接分泌穿孔素和颗粒酶，CD4+T细胞分泌细胞因子进一步招募活化杀伤性T细胞，从而杀伤肿瘤细胞。CD3抗体序列可选自OKT-3、L2K、TR66、UCHT1、SP34、IORT3、Catumaxomab、Blinatumomab、Solitomab等本领域已知的CD3抗体序列。在本申请具体的实施例中采用Solitomab中CD3抗体序列，本领域技术人员可以通过常规实验方法和有限次试验从其他已知CD3抗体中选择确定，而不局限于本申请具体实施例中特定的CD3抗体序列。
术语“抗体偶联药物”(Antibody-drug conjugate，简称ADC))，将小分子毒素通过连接子连接到抗体上，抗体作为载体将小分子毒素靶向运输到目标细胞中。
术语“给予”(“administration”、“administering”)、“治疗”(“treating”和“treatment”)指外源性药剂、治疗剂、诊断剂与受试者的组织、器官、细胞或生物流体的接触。“治疗”指减缓或阻止疾病临床症状的发展，或者指缓解或改善至少一种物理参数，或者指预防疾病的进展。
实施例1 CD26阳性和CD26阴性细胞的筛选
用T75细胞培养瓶培养靶细胞，待细胞融合到80％以上时，胰蛋白酶消化 细胞并收集，用PBS清洗一次，血球计数板进行计数，分为5×105个细胞每份；用抗CD26单克隆抗体作为一抗，与靶细胞于室温共孵育约40min，孵育结束后，离心弃上清，细胞沉淀用PBS重悬，再次离心弃上清，收集细胞沉淀；再用相应的抗体Alexa Fluor 488鼠抗人IgG1做为二抗，重悬细胞沉淀，于室温避光孵育约30min，孵育结束后，用PBS清洗两次，离心弃上清，收集细胞沉淀；用约200ul的PBS溶液将细胞沉淀重悬，在1h内用流式细胞仪进行检测并分析CD26阳性率。
经流式细胞仪鉴定，CD26阳性的细胞株有786-0、OS-RC-2、UT16、NCI-H226，G401、A-375为CD26阴性细胞株，阳性率如表1所示。CD26阳性细胞株与参考文献一致(中国专利申请号CN200680034937.4，CD26/DPP4-a potential biomarker and target for cancer therapy,Pharmacology & Therapeutics(2019),198:135-159)。
表1 靶细胞的CD26阳性率
实施例2：抗人CD26杂交瘤细胞株的制备与筛选
步骤1：动物免疫
根据GenBank已公开的CD26的cDNA序列(GenBank登录号：NM_001935.3)设计rCD26表达载体，在密码子5’端引入组氨酸标签，进行全基因合成，连接到pCHO1.0质粒中，CHO-S宿主细胞表达、纯化。将制备的重组CD26靶蛋白按照一般免疫程序免疫BALB/c雌性小鼠。具体免疫情况参见《抗体制备与使用实验指南》。采用间接ELISA法跟踪免疫小鼠血清滴度，选取血清效价最高的免疫小 鼠，将小鼠脾细胞和小鼠骨髓瘤细胞进行融合实验。
步骤2：细胞融合
(1).脾脏细胞的制备
将免疫小鼠，摘眼球取血，经断颈椎处死后置于75％(v/v)的酒精中浸泡10分钟，于无菌操作台中取出其脾脏，置于细胞筛网中，充分研磨细胞，过筛网，用无菌1640培养基(购自Gibco公司)离心洗涤数次后，重悬细胞以制成单细胞悬液，并计数，备用。
(2).饲养细胞的制备
取8～10周龄的雌性BALB/c小鼠一只，摘眼球获取阴性血清，经断颈椎处死后置75％(v/v)酒精中浸泡10分钟；无菌揭开腹部皮肤，暴露腹膜，用注射器将约10mL 1640HT培养基(购自SIGMA公司)注入小鼠腹腔，轻轻按摩腹部并吹打数次。吸取含有巨噬细胞的培养基注入20％1640HAT培养基中备用；
取2～3周龄的雌性BALB/c小鼠一只，经断颈椎处死后置于75％(v/v)酒精中浸泡10分钟；无菌取胸腺于细胞筛网中，研磨，过筛网，获得胸腺细胞置于上述含有巨噬细胞的20％1640HAT培养基中，备用。
(3).细胞融合
选择处于对数生长期的小鼠骨髓瘤细胞株SP2/0，收集并计数。取约108个上述脾细胞与2×107个上述SP2/0细胞株加入融合管中混合，1000rpm离心10分钟后弃上清，将融合管置手掌上来回轻轻摩擦以使沉淀松散。60秒内先慢后快地加入1mL预热的PEG1450(聚乙二醇1450，购自SIGMA公司)，加入1640HT培养基30mL终止，1000rpm离心10分钟，去上清，轻轻摩擦使沉淀松散，加入步骤2所获得的20％的1640HAT培养基中。
将上述HAT培养基充分混匀后，以200μL/孔分装至96孔细胞培养板中，置37℃，5％CO2的细胞培养箱中培养。一周后用10％1640HT培养基替换20％1640HAT培养基，3天后取上清进行检测。
步骤3：抗CD26特异性杂交瘤细胞株筛选
(1).检测板的准备：用CB包被液稀释重组CD26靶蛋白至1μg/ml，包被96孔ELISA酶标板，100μl/孔，2～8℃包被过夜，洗涤一次拍干；含2％BSA的PBST缓冲液封闭(200μl/孔)，37℃封闭2小时；拍干，备用。
(2).阳性克隆的筛选：将待检细胞培养上清100μl/孔加入上述检测板中，于37℃作用30分钟后洗涤并拍干，加入100μl/孔稀释后HRP标记的羊抗鼠IgG，于37℃作用30分钟后洗涤并拍干，加入100μl/孔的TMB显色液，于37℃避光显色15分钟，每孔加入50μl的2M H2SO4终止反应，并于OD450处读取数值。阳性孔确定原则：OD450值/阴性对照值≥2.1。选取阳性克隆株进行细胞克隆化筛选。经过三至四轮的克隆化筛选后，单克隆细胞株阳性率100％即确定为稳定细胞株，对细胞株进行定株。
步骤4：CD26鼠单抗体(79号鼠抗)活性评价
肿瘤细胞采用荧光染料Calcein-AM标记上绿色荧光信号，肿瘤细胞以6×105cells/ml的细胞浓度，每孔50μl接种到U-型96孔细胞培养板中，每组设置3个复孔，在相应样品孔的反应孔中分别加入50ul终浓度为100ng/ml的抗CD26抗体，在空白对照孔中加入50ul培养基对照，在阳性对照孔中加入50ul终浓度为1％Triton X-100，37℃培养箱中孵育30min，再按E/T为10:1的比例加入浓度为6×106cells/ml的PBMC细胞(外周血单个核细胞)，将反应体系置37℃二氧化碳培养下孵育5h，反应结束后，离心取上清置于一块新的96孔板中，再次离心后，取80μl上清置于黑色96孔板中，用酶标仪在470nm激发光波长和515nm的发射波波长的条件下进行检测。
细胞裂解率公式＝(Vsample-Vvehicle control)/(VTritonX-100-Vvehicle control)×100％。其中Vsample为药物处理组在激发光波长和发射光波长条件下测得的荧光信号读数的平均值，Vvehicle control为空白对照组在激发光波长和发射光波长条件下测得的荧光信号读数的平均值，VTritonX-100为阳性对照组在激发光波长和发射光波长条件下测得的荧光信号读数的平均值。
表2 79号鼠抗介导的PBMC对靶细胞的细胞毒效应检测
从上述结果可知，在抗体浓度为100ng/ml，作用5h的条件下，79号鼠抗介导的PBMC对靶细胞786-0的细胞裂解率为48.8％(其他鼠抗如62、72、160、51、212号鼠抗在相同条件下介导的PBMC对靶细胞786-0的细胞裂解率分别为53.1％、8.6％、13.0％、12.3％、11.3％)，79号鼠抗介导的PBMC对靶细胞OS-RC-2 的细胞裂解率为34.4％(其他鼠抗如47、76、62、82号鼠抗在相同条件下介导的PBMC对靶细胞OS-RC-2的细胞裂解率分别为15.4％、19.9％、49.9％、30.8％)，79号鼠抗介导的PBMC对靶细胞NCI-H226的细胞裂解率为25.5％(其他鼠抗如47、51、62、82号鼠抗在相同条件下介导的PBMC对靶细胞NCI-H226的细胞裂解率分别为6.7％、21％、14.3％、17.5％)，79号鼠抗对不同的CD26阳性的细胞株具有杀伤活性。
实施例3：杂交瘤细胞株可变区序列测序筛选
步骤1.CD26杂交瘤细胞总RNA的提取
将杂交瘤细胞株传代至T75培养瓶中培养，至细胞汇合至90％左右消化、离心收集细胞，使用RNA提取试剂盒(购自Roche)对单克隆杂交瘤细胞株进行总RNA提取。然后分别以总RNA为模板，使用cDNA反转录试剂盒(购自Thermo)逆转录扩增其cDNA第一链，反应产物存放于-20℃，长期储存，需要储存于-70℃。
步骤2.PCR扩增重、轻链可变区基因
分别以杂交瘤细胞cDNA第一链为模板，在50μl反应体系中，分别加入cDNA1μl，10×PCR缓冲液5μl，上游及下游引物各1μl(25pmo1)，dNTP1μl，25mmol/L MgCl2 1μl，H2O 39μl，95℃预变性10min，加Taq酶1μl，进入温度循环，进行PCR扩增。反应条件为94℃。变性1min，58℃。退火1min，72℃延伸1.5min，共30个循环，然后72℃保温10min。取5μl PCR产物进行1.2％琼脂糖凝胶电泳。
步骤3.重链及轻链可变区基因的克隆和序列测定
按照pGM-T Fast连接试剂盒说明书(北京天根生化科技有限公司，VT207-02)，将重、轻链可变区基因分别与pGM-T载体连接，转化大肠杆菌Top10感受态细胞中，蓝白斑筛选，37℃培养12-16h。
将得到的白色菌落接种到含有终浓度为100uM的氨苄青霉素1-5ml LB培养基中，37℃摇床振荡3-4h后，PCR筛选插入正确序列克隆。同时将PCR筛选阳性克隆测序，测定结果通过计算机网络基因库IMGT比较分析获得抗体的重链、 轻链可变区、CDR区的氨基酸序列。
其中79号鼠抗包含SEQ ID NO：1所示的HCDR1、SEQ ID NO：2所示的HCDR2、SEQ ID NO：3所示的HCDR3；SEQ ID NO：4所示的LCDR1、序列为YRS的LCDR2和SEQ ID NO：5所示的LCDR3。其重链可变区如SEQ ID NO：6所示、轻链可变区如SEQ ID NO：7所示。
实施例4：人源化双特异性抗体的构建
79号鼠抗人源化后重轻链可变区氨基酸序列：19G300-VH(SEQ ID NO：8)、19G300-VL(SEQ ID NO：9)。
将上述人源化后的重轻链可变区通过一段连接短肽(SEQ ID NO：10)相连，下游添加连接短肽(SEQ ID NO：11)，并按照哺乳动物细胞CHO密码子偏爱性进行优化，得到优化后的CD26单链抗体基因序列(SEQ ID NO：12)，并在上游引入AvrII酶切位点和kozak序列，下游添加CD3单链抗体基因(SEQ ID NO：13)、终止密码子和BstZ17I酶切位点。得到人源化CD26-CD3 BiTE基因序列，直接合成并构建至pUC57质粒中，命名为pUC57-19G300。
扩增目的基因，1％琼脂糖电泳回收PCR产物，并用AvrII和BstZ17I双酶切PCR回收产物和pZHK2.0载体，T4连接酶连接双酶切产物至pZHK2.0载体中，并转化到Top10感受态细胞中，涂于含有卡那霉素抗性的LB平板中37℃过夜培养。第二天筛选阳性克隆菌，并测序比对，与预期序列完全一致，即得到了人源化CD26-CD3 BiTE双特异性抗体表达质粒。
将含有高表达CD26-CD3人源化BiTE双特异性抗体稳定表达哺乳动物细胞株接种于Dynamis培养基中，37℃，8％CO2，130rpm进行分批补料流加培养。收获上清培养液，12000rpm，15min低温离心收集上清，0.45μm收集滤膜过滤即得处理后培养液上清，进行层析纯化。得到目的产物大小均约为55kDa。
实施例5：人源化双特异性抗体与CD26蛋白、CD3蛋白的亲和力分析
1、与CD26蛋白的亲和力研究
首先将CD26蛋白用生物素EZ-link NHS-PEG12-Biotin进行生物素化；用SA生物传感器固定生物素化的CD26蛋白；置于1×Kinetics溶液中平衡；与待检测抗体分子溶液结合，待检测分子的浓度为500nM；置于1×Kinetics溶液中解离；用Fortebio Data Analysis 8.0软件进行数据分析，计算亲和力常数值。
表3人源化双特异性抗体19G300与CD26蛋白的亲和力
2、抗体与CD3蛋白的亲和力研究
首先将CD3蛋白用生物素EZ-link NHS-PEG12-Biotin进行生物素化；用SA生物传感器固定生物素化的CD3蛋白；置于1×Kinetics溶液中平衡；与人源化双特异性抗体溶液结合，人源化双特异性抗体的检测浓度为500nM；置于1×Kinetics溶液中解离；用Fortebio Data Analysis 8.0软件进行数据分析，计算亲和力常数值
表4人源化双特异性抗体19G300与CD3蛋白的亲和力
综上所述，19G300与CD26蛋白的亲和力为1.14E-09M，19G300与CD3蛋白的亲和力为1.61E-09M。
实施例6：全长人源化抗体的构建
21G430-431是来自79号鼠抗的全长人源化抗CD26抗体，其重轻链可变区氨基酸序列为19G300-VH(SEQ ID NO：8)、19G300-VL(SEQ ID NO：9)；21G428-429是来自62号鼠抗的全长人源化抗CD26抗体，其重轻链可变区氨基酸序列为19G294-VH(SEQ ID NO：17)、19G294-VL(SEQ ID NO：19)；YS110重轻链可变区氨基酸序列为YS110-VH(SEQ ID NO：18)、YS110-VL(SEQ ID NO：20)。
19G300-VH(SEQ ID NO：8)、19G294-VH(SEQ ID NO：17)、YS110-VH(SEQ ID NO：18)上游分别添加信号肽序列(SEQ ID NO：14)，下游分别添加人IgG1重链恒定区(SEQ ID NO：15)，得到全长人源化抗CD26抗体21G430-431、21G428-429、YS110的IgG1重链序列，按照哺乳动物细胞CHO 密码子偏爱性进行优化，得到优化后的基因序列，并在上游引入AvrII酶切位点和kozak序列，下游添加终止密码子和BstZ17I酶切位点，直接合成后构建至pCHO1.0载体的AvrII和BstZ17I酶切位点内。
19G300-VL(SEQ ID NO：9)、19G294-VL(SEQ ID NO：19)、YS110-VL(SEQ ID NO：20)上游添加信号肽序列(SEQ ID NO：14)，下游添加人kappa轻链恒定区(SEQ ID NO：16)，得到全长人源化抗CD26抗体21G430-431、21G428-429、YS110的IgG1轻链序列，按照哺乳动物细胞CHO密码子偏爱性进行优化，得到优化后的基因序列，并在上游引入EcoRV酶切位点和kozak序列，下游添加终止密码子和PacI酶切位点，直接合成后构建至pZHK5.18载体的EcoRV和PacI酶切位点内。
pCHO1.0载体来源Thermo公司，具有嘌呤霉素抗性基因，pZHK5.18载体为pCHO1.0替换嘌呤霉素抗性基因为潮霉素抗性基因改造而来。
重链载体与轻链载体共同电转入CHO-S细胞，经过潮霉素与嘌呤霉素双压筛选得到全长抗体稳定表达CHO细胞系，将稳定表达CHO细胞系接种于Dynamis培养基中，37℃，8％CO2，130rpm进行分批补料流加培养。
收获上清培养液，12000rpm，15min低温离心收集上清，0.45μm收集滤膜过滤即得处理后培养液上清。使用Cytiva MabSelect Sure亲和层析填料进行纯化，纯化产物调节pH至5.5后使用Cytiva SP HP阳离子层析填料进行精纯，得到目的产物大小均约为150kDa。
实施例6：全长人源化抗体在混合细胞体系中结合能力差异
收集786-0细胞，计数后调整细胞密度为6×105个/mL。收集PBMC细胞，计数后调整细胞密度为6×105个/mL。取786-0和PBMC细胞各50微升加入EP管中作为混合细胞体系。加入抗体稀释到终浓度0.3ug/mL，与混合细胞体系混匀，于4℃条件下孵育1h。
孵育结束后，离心收集细胞，用PBS洗涤两次后用PBS重悬。加入羊抗人IgG荧光二抗，于4℃条件下孵育1h。结束后用PBS清洗两次，用PBS重悬后，BD ACCURI C6流式细胞仪检测。
检测结果显示(图1)，抗CD26全长人源化抗体21G430-431在癌细胞786-0中表现出显著高于抗CD26全长人源化抗体21G428-429的结合水平，相对而言21G430-431在PBMC细胞中表现出与21G428-429相当的结合水平。一方面表明，全长人源化抗体21G428-429、21G430-431对肿瘤细胞上CD26靶点的识别比对PBMC细胞上的CD26靶点的识别具有更好的选择性，优先选择与肿瘤细胞上的CD26靶点结合，与PBMC细胞CD26靶点的结合程度极低，从而避免对正常细胞的杀伤。另一方面表明，全长人源化抗体21G430-431比21G428-429具有更高的肿瘤细胞结合力。
实施例7：全长人源化抗体内化水平差异比较
抗体偶联药物由抗体与细胞毒性分子偶联，兼具抗体的特异性识别靶细胞与毒素的强大的肿瘤杀伤效果。其中抗体部分应具备：(1)肿瘤组织特异性高、亲和力/结合力强，确保ADC高效识别肿瘤细胞，以减少脱靶毒性；(2)与靶抗原结合后可以被有效内吞进入肿瘤细胞，并在进入内涵体或溶酶体后快速释放出毒素分子，从而发挥小分子细胞毒作用。
为了检测抗体内化进入溶酶体的水平，按照pHrodo iFL Green STP ester说明书所述对抗体进行荧光标记。该荧光标记对pH敏感，能够在低pH条件下荧光显著增强。溶酶体环境的pH约为5.0，要显著低于细胞质基质中7.2的pH值。所以当荧光标记抗体进入肿瘤细胞并被溶酶体吞噬后，其荧光由于pH从7.4到5.0的显著变化而荧光显著增强。因而用来指示抗体是否内化进入溶酶体中。
收集786-0/PBMC细胞，计数后调整两者的细胞密度均为2×105个/mL。取细胞50微升加入EP管中作为混合细胞体系。加入荧光标记抗体，稀释到终浓度5ug/mL，与混合细胞体系混匀，于37℃条件下孵育24h。另取一组细胞不加入荧光标记的抗体，在检测前混匀作为0h。检测前对样品离心去除含血清的培养液，置换为PBS溶液。用流式细胞仪检测其平均荧光强度。
检测结果显示(图2)，抗CD26全长人源化抗体21G430-431在癌细胞786-0中表现出显著高于抗CD26全长人源化抗体21G428-429的内化水平。表明21G430-431较之21G428-429更适合用作ADC分子中抗体。
另外为了避免ADC分子发生脱靶效应，其在非效应细胞中具有低的内化水平有助于降低其发生副作用的风险。由图3可知，在PBMC细胞中抗CD26抗体均表现出较之癌细胞更低的内化水平，比较内化24小时后与0小时平均荧光强度差值，21G430-431在PBMC细胞中表现出略低于21G428-429的内化水平。
由图4可知，选择三个不同个体来源的PBMC进行内化实验，在不同个体的PBMC中，21G430-431均表现出比21G428-429更低的内化水平。
实施例8：抗CD26抗体偶联生物毒素后对癌细胞的杀伤
根据实施例7与实施例8可知，21G430-431表现出优于21G428-429的亲和力、结合力和内化水平，因而认为其具有成为ADC中抗体部分的潜力。因此在本例中采用DT3C(白喉毒素)这一能够与抗体Fc端结合并且内化后被细胞内蛋白酶水解释放出白喉毒素的蛋白作为工具。将之与抗CD26抗体21G430-431、21G428-429、YS110分别进行孵育构建简易抗体偶联药物，初步验证抗体作为偶联药物的毒性水平。
将DT3C与各抗体分子加入培养基中，制成40ug/mLDT3C与20ug/mL抗体的溶液。混匀后置于培养箱中孵育一小时。在孵育的时间，收集786-0细胞、UT16细胞，计数后调整细胞密度为5×104个/mL。将细胞以50微升每孔的量加入96孔板中。待40ug/mLDT3C与20ug/mL抗体的溶液孵育结束后，以50uL每孔的量加入孔板中，于37℃条件下继续孵育72h。孵育结束后，每孔中加入10uL的CCK8溶液，置于培养箱中孵育一小时，用酶标仪检测450nm处的吸光度。每个样品吸光度需要扣除不含细胞培养基中的本底信号。以不加抗体组作为对照组计算细胞的存活率。计算公式如下所示：(ASample-AMedium)/ABlank(其中ASample为加入样品孵育细胞孔450nm处吸光度，AMedium为纯培养液孔450nm处吸光度，ABlank为不含抗体溶液细胞孔450nm处吸光度)
检测结果显示(图5、图6)，三个抗CD26抗体偶联药物均表现出一定的对靶细胞细胞杀伤能力。DT3C偶联21G430-431表现出优于DT3C偶联21G428-429的杀伤能力，具有显著性的差异。这表明具有更高的细胞亲和力、结合力和更强的内化水平的抗体偶联毒素分子后表现出了对靶细胞更高的杀伤水平。在本例中，已经进展到临床II期的YS110也被用作对照抗体。可以看到21G430-431表现出 比YS110更强的ADC效应，且具有显著性的差异。
综合上述实施例的结果，79号鼠抗来源的抗CD26抗体对CD26阳性肿瘤细胞具有较高的特异性、亲和力、结合力，且79号鼠抗来源的全长人源化抗体较其他鼠抗来源的全长人源化抗体具有更高的对肿瘤细胞的亲和力、结合力、肿瘤细胞内化水平更高、非肿瘤细胞内化水平更低，从而在ADC分子中表现出了更好的药效和安全性，可作为ADC分子中的抗体部分。

Claims (10)

  1. 与人CD26特异性结合的抗体或抗原结合片段，包含SEQ ID NO：1所示的HCDR1、SEQ ID NO：2所示的HCDR2、SEQ ID NO：3所示的HCDR3；以及包含SEQ ID NO：4所示的LCDR1、序列为YRS的LCDR2和SEQ ID NO：5所示的LCDR3。
  2. 权利要求1所述抗体或抗原结合片段，包含与SEQ ID NO：8所示至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的重链可变区，以及包含与SEQ ID NO：9所示至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％相同的氨基酸序列的轻链可变区。
  3. 权利要求2所述抗体或抗原结合片段，其中SEQ ID NO：8、SEQ ID NO：9所示序列的一个、两个、三个、四个、五个、六个、七个、八个、九个或十个氨基酸被插入、缺失或取代，所述氨基酸取代是保守氨基酸取代。
  4. 权利要求2所述抗体或抗原结合片段，包含SEQ ID NO：8所示的重链可变区和包含SEQ ID NO：9所示的轻链可变区。
  5. 靶向CD26的抗体偶联药物，其包含上述权利要求1～4任一项所述的抗CD26抗体或抗原结合片段。
  6. 编码权利要求1～4任一项所述的抗体或抗原结合片段的氨基酸序列的核苷酸序列。
  7. 包含权利要求6所述核苷酸序列的载体。
  8. 包含权利要求7所述载体的宿主细胞。
  9. 包含权利要求1～4任一项所述抗体或抗原结合片段的药物组合物
  10. 一种治疗高表达CD26肿瘤的方法，其包括向患者给予有效量的权利要求1～4任一项所述的抗体或抗原结合片段，或权利要求6所述的抗体偶联药物，所述肿瘤高表达CD26，包括但不限于肾癌、间皮瘤、肺癌、肝癌、前列腺癌等。
PCT/CN2023/142580 2023-01-18 2023-12-28 抗cd26抗体及其应用 WO2024152864A1 (zh)

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