WO2021218874A1 - 一种靶向人claudin和人PDL1蛋白的双特异抗体及其应用 - Google Patents

一种靶向人claudin和人PDL1蛋白的双特异抗体及其应用 Download PDF

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WO2021218874A1
WO2021218874A1 PCT/CN2021/089729 CN2021089729W WO2021218874A1 WO 2021218874 A1 WO2021218874 A1 WO 2021218874A1 CN 2021089729 W CN2021089729 W CN 2021089729W WO 2021218874 A1 WO2021218874 A1 WO 2021218874A1
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seq
antibody
human
bispecific antibody
cancer
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French (fr)
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屈向东
潘琴
金后聪
郑翰
都业杰
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启愈生物技术(上海)有限公司
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Priority to US17/921,378 priority Critical patent/US20230183352A1/en
Priority to KR1020227040995A priority patent/KR20230005276A/ko
Priority to JP2023507848A priority patent/JP7463000B2/ja
Priority to CN202180031534.9A priority patent/CN115461372A/zh
Priority to EP21796383.4A priority patent/EP4144759A4/en
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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 a bispecific antibody targeting human claudin and human PDL1 protein and its application, and belongs to the field of biomedicine.
  • Bispecific antibodies also known as bifunctional antibodies, can simultaneously recognize and bind to two different antigens and epitopes, and block two different signal pathways to exert their effects.
  • BsAb adds a specific antigen binding site, and shows the following advantages in treatment:
  • bispecific antibodies Mediates the killing of tumors by immune cells: An important mechanism of bispecific antibodies is to mediate the killing of immune cells.
  • Bispecific antibodies have two antigen-binding arms, one of which binds to the target antigen, and the other binds to the effector cell. Tag antigen binding, the latter can activate effector cells to target and kill tumor cells.
  • the dual-target signal blocking can play a unique or overlapping function to effectively prevent drug resistance: simultaneous binding of dual targets and blocking the dual-signal pathway is another important mechanism of bispecific antibodies.
  • Receptor tyrosine kinases RTKs
  • RTKs are the largest type of enzyme-linked receptors, which play an important regulatory role in cell proliferation, such as the Her family. RTKs are abnormally highly expressed on the surface of tumor cells, leading to malignant proliferation of tumor cells, so they are also important targets for tumor therapy.
  • Single-target monoclonal antibodies against RTKs have been widely used in tumor therapy. However, tumor cells can activate intracellular activation by switching signaling pathways or by homologous or heterodimers between members of the HER family The signal is immune to escape. Therefore, the use of bispecific antibody drugs to block two or more RTKs or their ligands at the same time can reduce the escape of tumor cells and improve the therapeutic effect.
  • the two antigen-binding arms of bispecific antibodies can bind to different antigens.
  • the two antigen-binding arms respectively bind to two antigens on the surface of cancer cells, which can effectively enhance antibody pairs.
  • the binding specificity and targeting of cancer cells can reduce side effects such as off-target;
  • BiTE Effectively reduce treatment costs: Taking BiTE as an example, compared with traditional antibodies, it has strong competitiveness in terms of tissue penetration, tumor cell killing efficiency, off-target rate and clinical indications, and has significant clinical advantages. Especially in terms of dosage, because its therapeutic effect can reach 100-1000 times that of ordinary antibodies, the lowest dosage can be 1/2000 of the original, which significantly reduces the cost of drug treatment. Compared with combination therapy, the cost of bispecific antibodies is also much lower than two single-drug combination therapy.
  • PD-1 (CD279) was first reported in 1992.
  • the human PD-1 encoding gene PDCD1 is located at 2q37.3, with a total length of 2097bp, composed of 6 exons, and the translation product is a PD-1 precursor consisting of 288 amino acids. Protein, the mature protein is obtained after cutting the signal peptide composed of the first 20 amino acids.
  • PD-1 includes the extracellular immunoglobulin variable region IgV domain, hydrophobic transmembrane domain and intracellular domain.
  • the N-terminal ITIM motif of the intracellular tail domain contains two phosphorylation sites, and the C-terminal is one ITSM motif.
  • PD-1 is a membrane protein that belongs to the CD28 immunoglobulin superfamily.
  • PD-1 has two ligands, namely PD-L1 (CD274, B7-H1) and PD-L2 (CD273, B7-DC) of the B7 protein family.
  • PD-L1 and PD-L2 are 40% identical . The difference between the two is mainly in the expression pattern.
  • PD-L1 is constitutively under-expressed in APCs, non-hematopoietic cells (such as vascular endothelial cells, pancreatic islet cells) and immune-privileged sites (such as placenta, testis, and eyes). Inflammatory cytokines such as Type I and type II interferons, TNF- ⁇ and VEGF can all induce the expression of PD-L1. PD-L2 is only expressed in activated macrophages and dendritic cells.
  • the ITSM motif of PD-1 undergoes tyrosine phosphorylation, which in turn leads to the dephosphorylation of downstream protein kinases Syk and PI3K, and inhibits downstream AKT, ERK and other pathways. Activation ultimately inhibits the transcription and translation of genes and cytokines required for T cell activation, and plays a role in negatively regulating T cell activity.
  • tumor cells and tumor microenvironment up-regulate the expression of PD-L1 and combine with PD-1 on the surface of tumor-specific CD8 + T cells to negatively regulate T cell activity and inhibit immune response.
  • Tumor cells can up-regulate the expression of PD-L1 through the following four ways: 1. Amplification of the gene encoding PD-L1 (9p24.1); 2. EGFR, MAPK, PI3K-Akt signaling pathway activation, HIF-1 transcription factor, etc. Up-regulate the expression of PD-L1 from the transcriptional level; 3. Induction of Epstein-Barr virus (EB virus-positive gastric cancer and nasopharyngeal carcinoma show high PD-L1 expression); 4. Regulation of epigenetics.
  • the stimulation of inflammatory factors such as interferon- ⁇ can also induce the expression of PD-L1 and PD-L2.
  • Inflammatory factors can induce other cells in the tumor microenvironment, including macrophages, dendritic cells, and stromal cells to express PD-L1 and PD-L2, and tumor-infiltrating T cells that can recognize tumor antigens can secrete interferon- ⁇ , and then Inducing the up-regulation of PD-L1 expression, this process is called "adaptive immune resistance", tumor cells can achieve self-protection through this mechanism.
  • PD-L1 and PD-L2 have been found in various solid tumors and hematological malignancies.
  • the expression of PD-Ls has a strong correlation with the poor prognosis of tumor cells, which proves to include esophageal cancer, stomach cancer, kidney cancer, ovarian cancer, bladder cancer, pancreatic cancer and melanoma.
  • Tight junction plays a key role in the flow of material between cells. It also maintains cell polarity by blocking the radial diffusion of membrane proteins and membrane lipids. It also participates in the recruitment of signal molecules that regulate cell proliferation, differentiation and movement. . Tight junctions are formed by Claudin (CLDN), and the Claudin family is composed of more than 20 protein molecules, all of which contain a four-pass transmembrane domain and similar amino acid sequences, but the tissue distribution has a certain specificity sex. CLDN plays a key role in regulating the selective permeation of the cell bypass. CLDN2 and CLDN15 participate in the formation of cation channels and cation pores, and CLDN4/7/10 participates in the formation of anion channels and pores.
  • CLDN2 and CLDN15 participate in the formation of cation channels and cation pores
  • CLDN4/7/10 participates in the formation of anion channels and pores.
  • CLDN1 and CLDN7 are down-regulated in invasive breast cancer, prostate cancer and esophageal cancer, while CLDN3/4 are found to be up-regulated in various cancers such as cervical cancer, colon cancer, esophageal cancer, and gastric cancer.
  • Sahin et al. found that in normal tissues, CLDN18 isoform 2 subtype (Claudin 18.2) is only expressed in epidermal cells after differentiation of the gastric mucosa, but not expressed in the area of gastric stem cells, but in primary gastric cancer and its metastases Abnormally high expression was found in all of them.
  • Claudin 18.2 high expression in pancreatic cancer, esophageal cancer and lung cancer. Because Claudin 18.2 is located on the surface of cell membranes, its biological functions and characteristics determine that it is an ideal therapeutic target. In recent years, monoclonal antibodies against this target have also appeared.
  • the purpose of the present invention is to provide a bispecific antibody targeting human claudin and human PDL1 protein and its application.
  • a bispecific antibody targeting human claudin 18.2 and human PDL1 protein including:
  • Anti-human claudin 18.2 antibody part and anti-PD-L1 antibody part are provided.
  • the bispecific antibody targeting human claudin18.2 and human PDL1 protein of the present invention has sequences such as: SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO : 63, SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 66.
  • the anti-human claudin18.2 antibody partially binds to the extracellular region of human claudin18.2 protein, and the sequence of the anti-human claudin18.2 antibody portion is as follows: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29,
  • bispecific antibody targeting human claudin18.2 and human PDL1 protein of the present invention anti-PD-L1 antibody, whose sequence is as SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO : 52, SEQ ID NO: 53 or SEQ ID NO: 54.
  • bispecific antibodies in the preparation of drugs for the treatment of cancer, infection or immunomodulatory diseases.
  • the cancer or tumor is selected from the group or site: colorectal, breast, ovary, pancreas, stomach, esophagus, prostate, kidney, cervix, bone marrow cancer, lymphoma, leukemia, thyroid, endometrial, uterus, bladder , Neuroendocrine, head and neck, liver, nasopharyngeal, testis, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel Cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.
  • a bispecific antibody targeting human claudin 18.2 and human PD-L1 protein is provided, and the bispecific antibody includes:
  • Anti-human claudin18.2 antibody part and anti-PD-L1 antibody part are included in Anti-human claudin18.2 antibody part and anti-PD-L1 antibody part.
  • the bispecific antibody has both the activity of binding to human claudin 18.2 and the activity of binding to human PD-L1 protein.
  • the complementarity determining region CDR of the anti-human claudin 18.2 antibody includes:
  • the 3 heavy chain CDRs and 3 light chain CDRs of the anti-human claudin18.2 antibody are selected from the following group:
  • the complementarity determining region CDR of the anti-human claudin18.2 antibody includes: HCDR1 shown in SEQ ID NO: 93, HCDR2 shown in SEQ ID NO: 94 and SEQ ID NO: 95 And LCDR1 shown in SEQ ID NO: 90, LCDR2 shown in SEQ ID NO: 91, and LCDR3 shown in SEQ ID NO: 92.
  • the anti-PD-L1 antibody is a single domain antibody.
  • the three complementarity determining region CDRs of the single domain antibody include: HCDR1 shown in SEQ ID NO: 69, HCDR2 shown in SEQ ID NO: 70 or 96, and HCDR2 shown in SEQ ID NO: 71. HCDR3 shown.
  • the three complementarity determining region CDRs of the single domain antibody include: HCDR1 shown in SEQ ID NO: 69, HCDR2 shown in SEQ ID NO: 70, and HCDR2 shown in SEQ ID NO: 71 HCDR3.
  • the three complementarity determining region CDRs of the single domain antibody include: HCDR1 shown in SEQ ID NO: 69, HCDR2 shown in SEQ ID NO: 96, and HCDR2 shown in SEQ ID NO: 71 HCDR3.
  • the bispecific antibody is a dimer composed of two monomers, and the monomer has a structure shown in formula I from the N-terminus to the C-terminus:
  • L1, L2 and L3 are each independently a key or joint element
  • VH represents the variable region of the heavy chain of the anti-human claudin 18.2 antibody
  • VL stands for the variable region of the light chain of the anti-human claudin18.2 antibody
  • CH stands for the heavy chain constant region of the anti-human claudin18.2 antibody
  • CL represents the constant region of the light chain of the anti-human claudin 18.2 antibody
  • VHH stands for anti-PD-L1 single domain antibody
  • each of L1 and L3 is a bond (such as a peptide bond).
  • the heavy chain variable region (VH) of the anti-human claudin18.2 antibody includes the HCDR1 shown in SEQ ID NO: 93, 75, 79, 83 or 87, and SEQ ID NO: 94, 76 HCDR2 shown in, 80, 84, or 88 and HCDR3 shown in SEQ ID NO: 95, 77, 81, 85, or 89.
  • the heavy chain variable region (VH) of the anti-human claudin 18.2 antibody also includes a humanized FR region.
  • the amino acid sequence of the heavy chain variable region (VH) of the anti-human claudin18.2 antibody is as SEQ ID NO: 31, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6.
  • SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 32, SEQ ID NO: 33 or SEQ ID NO: 34 are shown.
  • the light chain variable region (VL) of the anti-human claudin18.2 antibody includes LCDR1 shown in SEQ ID NO: 90 or 72, LCDR2 shown in SEQ ID NO: 91 or 73, and SEQ ID NO: 91 or 73.
  • the light chain variable region (VL) of the anti-human claudin 18.2 antibody also includes a humanized FR region.
  • the amino acid sequence of the light chain variable region (VL) of the anti-human claudin18.2 antibody is as SEQ ID NO: 29, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3. SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 28, or SEQ ID NO: 30 are shown.
  • the heavy chain constant region (CH) of the anti-human claudin 18.2 antibody is of human or murine origin.
  • the light chain constant region (CL) of the anti-human claudin 18.2 antibody is of human or murine origin.
  • the anti-PD-L1 single domain antibody includes HCDR1 shown in SEQ ID NO: 69, HCDR2 shown in SEQ ID NO: 70, and HCDR3 shown in SEQ ID NO: 71.
  • the anti-PD-L1 single domain antibody includes HCDR1 shown in SEQ ID NO: 69, HCDR2 shown in SEQ ID NO: 96, and HCDR3 shown in SEQ ID NO: 71.
  • the anti-PD-L1 single domain antibody also includes a humanized FR region.
  • amino acid sequence of the anti-PD-L1 single domain antibody is as SEQ ID NO: 51, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID Shown in NO:53 or SEQ ID NO:54.
  • amino acid sequence of the VL of the bispecific antibody is shown in SEQ ID NO: 29
  • amino acid sequence of VH is shown in SEQ ID NO: 31
  • anti-PD-L1 single domain antibody VHH
  • the amino acid sequence of SEQ ID NO: 51 is shown.
  • amino acid sequence of the VL of the bispecific antibody is shown in SEQ ID NO: 1
  • amino acid sequence of VH is shown in SEQ ID NO: 5
  • anti-PD-L1 single domain antibody VHH
  • the amino acid sequence of SEQ ID NO: 51 is shown.
  • amino acid sequence of the VL of the bispecific antibody is shown in SEQ ID NO: 16
  • amino acid sequence of VH is shown in SEQ ID NO: 17
  • anti-PD-L1 single domain antibody VHH
  • the amino acid sequence of SEQ ID NO: 51 is shown.
  • amino acid sequence of the VL of the bispecific antibody is shown in SEQ ID NO: 8
  • amino acid sequence of VH is shown in SEQ ID NO: 13
  • anti-PD-L1 single domain antibody VHH
  • the amino acid sequence of SEQ ID NO: 51 is shown.
  • amino acid sequence of the VL of the bispecific antibody is shown in SEQ ID NO: 21
  • amino acid sequence of VH is shown in SEQ ID NO: 26
  • anti-PD-L1 single domain antibody VHH
  • the amino acid sequence of SEQ ID NO: 51 is shown.
  • the amino acid sequence of the L chain (VL-L3-CL) of the bispecific antibody is as SEQ ID NO: 63, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 65 As shown; and the amino acid sequence of the H chain (VH-L1-CH-L2-VHH) of the bispecific antibody is as SEQ ID NO: 64, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 66 shown.
  • amino acid sequence of the L chain (VL-L3-CL) of the bispecific antibody is shown in SEQ ID NO: 63
  • amino acid sequence of the H chain (VH-L1-CH-L2-VHH) The sequence is shown in SEQ ID NO: 64.
  • amino acid sequence of the L chain (VL-L3-CL) of the bispecific antibody is shown in SEQ ID NO: 59
  • amino acid sequence of the H chain (VH-L1-CH-L2-VHH) The sequence is shown in SEQ ID NO: 60.
  • amino acid sequence of the L chain (VL-L3-CL) of the bispecific antibody is shown in SEQ ID NO: 61
  • amino acid sequence of the H chain (VH-L1-CH-L2-VHH) The sequence is shown in SEQ ID NO: 62.
  • amino acid sequence of the L chain (VL-L3-CL) of the bispecific antibody is shown in SEQ ID NO: 65
  • amino acid sequence of the H chain (VH-L1-CH-L2-VHH) The sequence is shown in SEQ ID NO: 66.
  • the bispecific antibody is a partially or fully humanized antibody.
  • the second aspect of the present invention provides an isolated polynucleotide encoding the bispecific antibody of the first aspect of the present invention.
  • the polynucleotide includes DNA, RNA or cDNA.
  • the third aspect of the present invention provides a vector containing the polynucleotide according to the second aspect of the present invention.
  • the expression vector is selected from the group consisting of plasmids and viral vectors.
  • the expression vector includes: bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, adeno-associated virus AAV, retrovirus, or other vectors.
  • the fourth aspect of the present invention provides a genetically engineered host cell, which contains the vector according to the third aspect of the present invention, or the polynucleotide according to the second aspect of the present invention is integrated into the genome.
  • the host cell includes a prokaryotic cell or a eukaryotic cell.
  • the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.
  • the fifth aspect of the present invention provides a method for preparing the bispecific antibody according to the first aspect of the present invention, including the steps:
  • step (ii) Purifying and/or separating the mixture obtained in step (i) to obtain the bispecific antibody.
  • the purification can be purified and separated by a protein A affinity column to obtain the target antibody.
  • the purity of the target antibody after purification and separation is greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, and preferably 100%.
  • the sixth aspect of the present invention provides a pharmaceutical composition, which contains:
  • the pharmaceutical composition also contains other drugs for treating cancer (or tumor), such as chemotherapy drugs.
  • the pharmaceutical composition is used to block the interaction between PD-1 and PD-L1 and simultaneously bind to claudin 18.2 protein.
  • the pharmaceutical composition is used to treat cancers (or tumors) expressing claudin18.2 protein (that is, claudin18.2 positive).
  • the pharmaceutical composition is in the form of injection.
  • an immunoconjugate comprising:
  • a coupling moiety selected from the group consisting of detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.
  • the eighth aspect of the present invention provides the application of the bispecific antibody of the first aspect of the present invention in the preparation of drugs for the treatment of cancer (or tumor), infection or immunomodulatory diseases.
  • the ninth aspect of the present invention provides the application of the bispecific antibody according to the first aspect of the present invention in the preparation of drugs for inhibiting tumor growth.
  • the cancer or tumor is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, stomach cancer, esophageal cancer, prostate cancer, kidney cancer, cervical cancer, bone marrow cancer, lymphoma, Leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell Skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.
  • the main beneficial effects of the present invention include: the present invention provides a bispecific antibody that simultaneously targets Claudin 18.2 and PD-L1, and the bispecific antibody molecule can efficiently target Claudin 18.2 and PD-L1.
  • the invention can improve the therapeutic effect of tumors expressing claudin 18.2.
  • the bispecific antibody can not only bind to human claudin18.2 protein, but also block the binding of PD-1/PD-L1. It can activate NK cells to kill tumor cells in innate immunity, and it can also be used in adaptive immunity. Promote the killing effect of killer T lymphocytes on tumors, thereby having a synergistic tumor killing effect.
  • the bispecific antibody has better anti-tumor efficacy than the anti-claudin18.2 antibody alone.
  • Figure 1 shows a schematic diagram of the structure of the anti-CLDN18.2/anti-PD-L1 bispecific antibody molecule.
  • Figure 2 shows the results of the first group of antibodies identified by ELISA in the activity identification of humanized Claudin 18.2 antibody.
  • Figure 3 shows the results of the second group of antibodies identified by ELISA for the activity identification of humanized Claudin 18.2 antibody.
  • Figure 4 shows the results of the third group of antibodies identified by ELISA for the activity of the humanized Claudin 18.2 antibody.
  • Figure 5 shows the results of the fourth group of antibodies identified by ELISA on the activity of the humanized Claudin 18.2 antibody.
  • Figure 6 shows the results of the fifth group of antibodies identified by ELISA for humanized Claudin 18.2 antibody activity.
  • Figure 7 shows the results of the first group of antibodies identified by FACS for the activity of the humanized Claudin 18.2 antibody.
  • Figure 8 shows the results of the second group of antibodies identified by FACS for the activity of the humanized Claudin 18.2 antibody.
  • Figure 9 shows the results of the third group of antibodies identified by FACS for the activity of the humanized Claudin 18.2 antibody.
  • Figure 10 shows the results of the fourth group of antibodies identified by FACS on the activity of the humanized Claudin 18.2 antibody.
  • Figure 11 shows the results of the fifth group of antibodies identified by FACS on the activity of the humanized Claudin 18.2 antibody.
  • Figure 12 shows the ADCC results of the humanized Claudin 18.2 antibody.
  • Figure 13 shows the CDC results of the humanized Claudin 18.2 antibody.
  • Figure 14 shows the in vivo efficacy results of the humanized Claudin 18.2 antibody in the immunodeficient mouse CB.17-SCID model.
  • Figure 15 shows the results of binding-ELISA for activity identification of humanized anti-PD-L1 single domain antibodies.
  • Figure 16 shows the result of Blocking-ELISA for the activity identification of humanized anti-PD-L1 single domain antibody.
  • Figure 17 shows the results of the expression and purification of the first antibody of the anti-CLDN18.2/anti-PD-L1 bispecific antibody.
  • Figure 18 shows the results of the expression and purification of the second antibody with anti-CLDN18.2/anti-PD-L1 bispecific antibody.
  • Figure 19 shows the results of the expression and purification of the third antibody of the anti-CLDN18.2/anti-PD-L1 bispecific antibody.
  • Figure 20 shows the results of the expression and purification of the fourth antibody of the anti-CLDN18.2/anti-PD-L1 bispecific antibody.
  • Figure 21 shows the result of binding-ELISA for activity identification of anti-CLDN18.2/anti-PD-L1 bispecific antibody.
  • Figure 22 shows the result of PD-L1/PD-1 blocking-ELISA for the activity identification of anti-CLDN18.2/anti-PD-L1 bispecific antibody.
  • Figure 23 shows the result of PD-L1/CD80 blocking ELISA for the activity of anti-CLDN18.2/anti-PD-L1 bispecific antibody.
  • Figure 24 shows the results of ELISA detection of CLDN18.2 activity in the activity identification of anti-CLDN18.2/anti-PD-L1 bispecific antibody.
  • Figure 25 shows the results of the identification of the functional activity of the anti-CLDN18.2/anti-PD-L1 bispecific antibody PD-L1 (the candidate molecule CHO14 in the mixed lymphocyte response MLR is dependent on the concentration of the cytokine IFN ⁇ produced after T cell activation.
  • Figure 26 shows the results of the identification of the functional activity of the anti-CLDN18.2/anti-PD-L1 bispecific antibody PD-L1 (the candidate molecule CHO14 in the mixed lymphocyte response MLR is dependent on the concentration of the cytokine IL-2 produced after T cell activation.
  • Figure 27 shows the results of the cell killing experiment mediated by the anti-CLDN18.2/anti-PD-L1 bispecific antibody PBMC.
  • Figure 28 shows the results of the in vivo efficacy evaluation of the anti-CLDN18.2/anti-PD-L1 bispecific antibody in the immune target humanized transgenic mouse C57BL/6-hPDL1 model MC38-hPDL1-mClaudin18.2.
  • Figure 29 shows the results of the in vivo efficacy evaluation of the anti-CLDN18.2/anti-PD-L1 bispecific antibody in the immune system humanized mouse PBMC engrafted-NCG model HCC827-hClaudin 18.2.
  • Figure 30 shows the in vivo synergistic evaluation of the anti-CLDN18.2/anti-PD-L1 bispecific antibody in the C57BL/6 model MC38-hPDL1-mClaudin 18.2.
  • Figure 31 shows the in vivo synergistic evaluation of the anti-CLDN18.2/anti-PD-L1 bispecific antibody in the C57BL/6 model MC38-hPDL1-mClaudin 18.2.
  • the inventors After extensive and in-depth research, the inventors obtained high specificity and high affinity anti-CLDN18.2 antibody and anti-PD-L1 single domain antibody through a large number of screenings, and carried out humanization and genetic recombination on this basis Thus, a bispecific antibody targeting both human Claudin 18.2 and human PD-L1 was obtained.
  • the bispecific antibody of the present invention can specifically bind to human Claudin 18.2 and human PD-L1 molecules, and kill human lung cancer cells that recombinantly express Claudin 18.2 and naturally express PD-L1.
  • the bispecific antibody of the present invention has a synergistic effect and exhibits superior anti-tumor activity than Claudin 18.2 monoclonal antibody and PD-L1 monoclonal antibody in a humanized mouse model. On this basis, the present invention has been completed.
  • bispecific antibody of the present invention As used herein, the terms "bispecific antibody of the present invention”, “biantibody of the present invention”, and “anti-claudin18.2/PD-L1 bispecific antibody” have the same meaning, and all refer to specific recognition and binding to claudin18. .2 and PD-L1 bispecific antibody.
  • antibody or "immunoglobulin” is a heterotetrameric glycoprotein of about 150,000 daltons with the same structural characteristics, which consists of two identical light chains (L) and two identical heavy chains. (H) Composition. Each light chain is connected to the heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes is different. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. There are two types of light chains, ⁇ (l) and ⁇ (k).
  • the light chain includes two domains or regions, a variable domain (VL) and a constant domain (CL).
  • the heavy chain includes four domains, a variable region of the heavy chain (VH) and three constant regions (CH1, CH2, and CH3, collectively referred to as CH).
  • the variable regions of the light chain (VL) and heavy chain (VH) both determine the binding recognition and specificity of the antigen.
  • the constant domain (CL) of the light chain and the constant domain (CH) of the heavy chain confer important biological properties such as antibody chain binding, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR).
  • the Fv fragment is the N-terminal part of the immunoglobulin Fab fragment and consists of the variable part of one light chain and one heavy chain.
  • the specificity of an antibody depends on the structural complementarity between the antibody binding site and the epitope.
  • the antibody binding site is composed of residues mainly derived from the hypervariable region or complementarity determining region (CDR). Occasionally, residues from non-hypervariable or framework regions (FR) affect the overall domain structure and thus the binding site.
  • Complementarity determining region or CDR refers to an amino acid sequence that collectively defines the binding affinity and specificity of the natural Fv region of the natural immunoglobulin binding site.
  • the light chain and heavy chain of immunoglobulins each have three CDRs, which are called LCDR1 (CDR1-L), LCDR2 (CDR2-L), LCDR3 (CDR3-L) and HCDR1 (CDR1-H), HCDR2 (CDR2). -H), HCDR3 (CDR3-H).
  • Conventional antibody antigen binding sites therefore include six CDRs, including a set of CDRs from each of the heavy and light chain v regions.
  • single domain antibody As used herein, the terms “single domain antibody”, “VHH” and “Nanobody” have the same meaning and refer to cloning the variable region of the heavy chain of an antibody to construct a Nanobody (VHH) consisting of only one heavy chain variable region. , It is the smallest antigen-binding fragment with complete functions. Usually, after obtaining an antibody with naturally missing light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.
  • VHH single domain antibody
  • N-body nanobody
  • variable means that certain parts of the variable region of an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, the variability is not evenly distributed throughout the variable regions of antibodies. It is concentrated in three segments called complementarity determining regions (CDR) or hypervariable regions in the variable regions of the light and heavy chains. The more conserved part of the variable region is called the framework region (FR).
  • CDR complementarity determining regions
  • FR framework region
  • the variable regions of the natural heavy chain and light chain each contain four FR regions, which are roughly in a ⁇ -sheet configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial ⁇ -sheet structure.
  • the CDRs in each chain are closely placed together through the FR region and form the antigen binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume I, pages 647-669 (1991)). Constant regions do not directly participate in the binding of antibodies to antigens, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.
  • FR framework region
  • the light chain and heavy chain of an immunoglobulin each have four FRs, which are called FR1-L, FR2-L, FR3-L, FR4-L and FR1-H, FR2-H, FR3-H, FR4-H, respectively.
  • the light chain variable domain can therefore be referred to as (FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-( FR4-L) and the heavy chain variable domain can therefore be expressed as (FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H) -(FR4-H).
  • the FR of the present invention is a human antibody FR or a derivative thereof, and the derivative of the human antibody FR is basically the same as the naturally-occurring human antibody FR, that is, the sequence identity reaches 85%, 90%, 95%, 96%. , 97%, 98% or 99%.
  • humanized antibody refers to an antibody molecule derived from a non-human antibody capable of binding a target antigen that has one or more complementarity determining regions (CDR) of non-human origin and is derived from human immunity
  • CDR complementarity determining regions
  • the framework region of a globulin molecule Generally, the framework residues in the human framework region will be replaced by the corresponding residues from the CDR donor antibody to change, and preferably improve, the binding of the antigen.
  • framework substitutions can be identified by methods known in the art, for example, by simulating the interaction of CDR and framework residues to identify framework residues that play an important role in antigen binding, and by sequence comparison to identify abnormal framework residues at specific positions. base.
  • human framework region is substantially the same as the framework region of a naturally occurring human antibody (about 85% or more, specifically 90%, 95%, 97%, 99% or 100% identity) Frame area.
  • linker refers to the insertion of an immunoglobulin domain to provide sufficient mobility for the light chain and heavy chain domains to fold to exchange one or more amino acid residues of the immunoglobulin with dual variable regions. base.
  • the present invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.
  • fragment refers to polypeptides that substantially retain the same biological function or activity as the antibody of the present invention.
  • the polypeptide fragments, derivatives or analogues of the present invention may be (i) polypeptides with one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide with a substitution group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that prolongs the half-life of the polypeptide, such as Polyethylene glycol) fused to the polypeptide, or (iv) additional amino acid sequence fused to the polypeptide sequence to form a polypeptide (such as a leader sequence or secretory sequence or a sequence used to purify the polypeptide or proprotein sequence, or with Fusion protein formed by 6His tag
  • the antibody of the present invention refers to a double antibody with claudin18.2 and PD-L1 protein binding activity.
  • the term also includes variant forms of polypeptides that have the same functions as the antibodies of the present invention and include the same CDR regions. These variants include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletion , Insertion and/or substitution, and adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal. For example, in the field, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding one or several amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA that can hybridize with the coding DNA of the antibody of the present invention under high or low stringency conditions
  • the encoded protein and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.
  • “conservative variants” refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids compared with the amino acid sequence of the antibody of the present invention.
  • the amino acids are replaced to form a polypeptide (especially the framework region is replaced by amino acids with similar or similar properties to form a polypeptide).
  • These conservative variant polypeptides are best produced according to Table A by performing amino acid substitutions.
  • substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the form of DNA includes cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the polynucleotide encoding the mature polypeptide of the present invention includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence (and optional additional coding sequence) and non-coding sequences of the mature polypeptide .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides that hybridize with the aforementioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences.
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90% or more, and more Fortunately, hybridization occurs when more than 95%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence or fragments of the antibody of the present invention can usually be obtained by PCR amplification method, recombinant method or artificial synthesis method.
  • One feasible method is to use artificial synthesis to synthesize relevant sequences, especially when the fragment length is short. Usually, by first synthesizing multiple small fragments, and then ligating to obtain fragments with very long sequences.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein.
  • the recombination method can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • the biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules that exist in an isolated form.
  • the DNA sequence encoding the protein (or fragment or derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention through chemical synthesis.
  • the present invention also relates to a vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells so that they can express proteins.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, and 293 cells.
  • Transformation of host cells with recombinant DNA can be performed by conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as Escherichia coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method.
  • the steps used are well known in the art.
  • Another method is to use MgCl 2 .
  • the transformation can also be carried out by electroporation.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the culture is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to a suitable cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • the recombinant polypeptide in the above method can be expressed in the cell or on the cell membrane, or secreted out of the cell. If necessary, the physical, chemical, and other characteristics can be used to separate and purify the recombinant protein through various separation methods. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibody of the present invention can be used alone, or can be combined or coupled with a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modified portion, or any combination of these substances.
  • Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or those capable of producing detectable products Enzyme.
  • Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclides; 2. Biotoxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nano magnetic particles; 8. Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. Chemotherapeutics ( For example, cisplatin) or any form of nanoparticles.
  • DTD DT-diaphorase
  • BPHL biphenyl hydrolase-like protein
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules combined with the antibodies or fragments of the present invention to form ⁇ conjugate.
  • the present invention provides a bispecific antibody targeting human claudin 18.2 and human PD-L1 protein, which comprises: an anti-human claudin 18.2 antibody part and an anti-PD-L1 antibody part.
  • the anti-human claudin 18.2 antibody part of the bispecific antibody of the present invention is obtained by humanizing a murine anti-claudin 18.2 antibody.
  • the heavy and light chain variable region germline genes with high homology to QP190191, QP192193, QP199200, QP201202, and QP207208 were selected as templates.
  • the CDRs of the murine antibody were respectively transplanted into the corresponding human template, and the order of formation was the variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Then select some important amino acid residues to make back mutation combinations.
  • the heavy chain variable region (VH) of the murine anti-claudin18.2 antibody has the following amino acid sequences, respectively, and the CDRs are shown in the underlined part of the sequence:
  • the light chain variable regions of the murine anti-claudin18.2 antibody respectively have the following amino acid sequences: CDRs are shown in the underlined part of the sequence:
  • the CDR (underlined) regions of the light and heavy chain variable regions of the above antibodies are listed in Table B.
  • the anti-human PD-L1 antibody part of the bispecific antibody of the present invention is obtained by humanization of an anti-PD-L1 single domain antibody (anti-PD-L1 nanobody).
  • an anti-PD-L1 single domain antibody anti-PD-L1 nanobody.
  • the heavy and light chain variable region germline genes with high homology to QP1162 and QP1166 were selected as templates, and the CDRs of single domain antibodies were separately selected.
  • Transplant into the corresponding human template such as IGHV3-23 germline and J-region IGHJ4*01
  • the variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 select some important amino acid residues to make back mutation combinations.
  • amino acid sequence of the anti-PD-L1 single domain antibody is shown below, and the CDR is shown in the underlined part of the sequence:
  • anti-PD-L1 single domain antibody and “anti-PD-L1 Nanobody” are used interchangeably, and both refer to the naturally deleted light chain, which contains only one heavy chain variable region (VHH) and two conventional The CH2 and CH3 regions, an antibody targeting the PD-L1 molecule.
  • VHH heavy chain variable region
  • affinity is theoretically defined by the balanced association between the intact antibody and the antigen.
  • the affinity of the bispecific antibody of the present invention can be evaluated or determined by the KD value (dissociation constant) (or other measurement methods), such as Bio-layer Interferometry (BLI), which is measured and determined using the FortebioRed96 instrument.
  • KD value dissociation constant
  • BLI Bio-layer Interferometry
  • the invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the aforementioned antibody or active fragment or fusion protein thereof, and a pharmaceutically acceptable carrier.
  • these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, preferably about 6-8, although the pH can be The nature of the formulated substance and the condition to be treated vary.
  • the formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention can be directly used to bind claudin 18.2 and/or PD-L1 protein molecules, and thus can be used to treat tumors. In addition, it can also be used in combination with other therapeutic agents.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned bispecific antibody (or conjugate thereof) of the present invention, and A pharmaceutically acceptable carrier or excipient.
  • Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, prepared by conventional methods with physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under aseptic conditions.
  • the dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms/kg body weight to about 50 mg/kg body weight per day.
  • the polypeptides of the present invention can also be used together with other therapeutic
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases not more than about 50 mg/kg body weight, Preferably the dosage is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the specific dosage should also consider factors such as the route of administration and the patient's health status, which are all within the skill range of a skilled physician.
  • the present invention also provides the application of the antibody of the present invention, which relates to the application in the preparation of drugs for the treatment of cancer (or tumor), infection or immunomodulatory diseases.
  • a preferred application is for the treatment of cancer (or tumor).
  • the present invention provides a bispecific antibody that simultaneously targets human Claudin 18.2 and human PD-L1.
  • the bispecific antibody molecule can efficiently target human Claudin 18.2 and human PD-L1.
  • the bispecific antibody of the present invention can improve the therapeutic effect of tumors expressing claudin 18.2.
  • This bispecific antibody can not only bind to human claudin18.2 protein, but also block the binding of PD-1/PD-L1. It can activate NK cells and kill tumor cells in the innate immunity, and it can also be The immune response can promote the killing effect of killer T lymphocytes on tumors. Therefore, the bispecific antibody has a better anti-tumor effect than the anti-claudin18.2 antibody alone.
  • the bispecific antibody of the present invention has a synergistic effect.
  • the experimental methods without specific conditions in the following embodiments are based on conventional conditions or conditions recommended by raw material or commodity manufacturers. Or biotechnology textbooks such as molecular cloning, laboratory manuals, Cold Spring Harbor Laboratory, contemporary molecular biology methods, cell biology and other experimental methods recorded.
  • the reagents that do not indicate the specific source are conventional reagents purchased through commercial channels.
  • Example 1 Humanization of anti-Claudin 18.2 hybridoma monoclonal antibody
  • the heavy and light chain variable region germline genes with high homology to QP190191, QP192193, QP199200, QP201202, and QP207208 were selected as templates.
  • the CDRs of the murine antibody were respectively transplanted into the corresponding human template, and the order of formation was the variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Then select some important amino acid residues to make back mutation combinations. The amino acid residues are identified and annotated by the Kabat numbering system.
  • DNAWorks (v3.2.2) (http://helixweb.nih.gov/dnaworks/) to design multiple primers to synthesize VH/VK containing gene fragments required for recombination: 5'-30bp signal peptide+VH/VK+30bp CH1/CL-3'.
  • TaKaRa's Primer STAR GXL DNA polymerase operating instructions using multiple primers designed above, two-step PCR amplification was performed to obtain VH/VK containing gene fragments required for recombination.
  • the expression vector pQD (with signal peptide and constant region gene (CH1-FC/CL) fragment) was constructed and digested.
  • the expression vector pQD (with Signal peptide and constant region gene (CH1-FC/CL) fragment).
  • the vector is digested with BsmBI, and the gum is recovered for later use. Recombinant construction of expression vector VH-CH1-FC-pQD/VK-CL-pQD.
  • variable region of the light and heavy chain uses the kappa light chain constant region CL (SEQ ID NO: 67), and the antibody heavy chain uses the human IgG1 constant region. (SEQ ID NO: 68):
  • Table 1 Humanized design light and heavy chain variable region sequences and protein expression numbers
  • the design and expression of the pre-humanized chimeric antibody and control antibody are shown in the following table.
  • the light chain of all antibodies adopts the kappa light chain constant region CL
  • the antibody heavy chain adopts the human IgG1 constant region:
  • IMAB362 is the control antibody, corresponding to the protein number QP024025
  • the 293E cell culture density is maintained between 0.2-3 ⁇ 10 6 /ml, and the maintenance phase medium (GIBCO Freestyle 293 expression medium) is used for culture.
  • the maintenance phase medium GIBCO Freestyle 293 expression medium
  • the day before transfection the cells to be transfected are centrifuged to change the medium, and the cell density is adjusted to 0.5 -0.8 ⁇ 10 6 /ml. On the day of transfection, the density of 293E cells was 1-1.5 ⁇ 10 6 /ml.
  • Prepare plasmid and transfection reagent PEI The amount of plasmid to be transfected is 100ug/100ml cells, and the mass ratio of PEI to plasmid is 2:1. Mix the plasmid and PEI, and let stand for 15 minutes, not more than 20 minutes.
  • the plasmid and PEI mixture was slowly added to the 293E cells, cultured in a shaker at 8% CO2, 120 rpm, 37° C., on the fifth day of transfection, the cell supernatant was collected by centrifugation at 4700 rpm in a horizontal centrifuge for 20 minutes.
  • Detection reagents skimmed milk powder (BD, 232100), PBS (shenggong, B548117-0500); HRP-anti human IgG (H+L) (jackson, 109-035-088); TMB (Luoyang Baiaotong Experimental Materials Center , C060201); Elisa plate (costa, 9018) 1 ⁇ PBS buffer: Weigh out NaCl 8.00g, KCl 0.20g, Na2HPO4 ⁇ 12H20 2.9g, KH2PO4 0.2g to 800mL ddH2O, and dissolve it in ddH2O, and dilute to 1L after being fully dissolved. Adjust the pH to 7.4 and sterilize at high temperature for later use.
  • Blocking solution Weigh 5g of milk powder into PBS. The blocking solution needs to be prepared for current use.
  • Stop solution (1mol/LH2SO4) Take 109mL of 98% concentrated H2SO4 and slowly drop it into 2000mL of ddH2O. TMB develops color at 37°C for 10 minutes, and places it in a shaker (120rpm), 100ul/hole;
  • Add substrate chromogenic solution add substrate chromogenic solution TMB according to the amount of 100 ⁇ L/well, place in a shaker, 200rpm, 35°C for 10min in the dark. Termination: After the color development is completed, 100 ⁇ L/well is quickly added to the termination solution to terminate the reaction. Detection: After centrifugation at 3500 rpm for 5 minutes, take 120ul of the supernatant and transfer it to the Elisa plate, measure the OD value of A450nm on the microplate reader, and use graphpad prism software to analyze the results: Figure 2, Figure 3, Figure 4, Figure 5 and Shown in Figure 6.
  • the EC50 and Max values in Table 3 and Figure 7 prove that the humanized mouse chimeric antibody QP190191 humanized antibodies QP14361435, QP14371433, and QP14371435 bind to CHOS-CLDN18.2 cells better than or equivalent to the murine chimeric antibody QP190191;
  • the EC50 and Max values in Table 7 and Figure 11 prove that the humanized antibodies of mouse chimeric antibody QP201202 QP14561454, QP14581453, and QP14581454 bind to CHOS-CLDN18.2 cells equivalent to the mouse chimeric antibody QP201202; Table 6 and Figure 10
  • the EC50 and Max values of the mouse chimeric antibody QP192193 prove that the humanized antibody QP14451440, QP14441441, and QP14451442 bind to CHOS-CLDN18.2 cells equivalent to the mouse chimeric antibody QP192193; the EC50 and Max values of Table 5 and Figure 9 It is proved that the humanized antibodies
  • FACS Flow cytometry fluorescence sorting technology
  • the FACS result shows The humanized Claudin 18.2 antibodies of the present invention all bind to CHOS-Claudin 18.2 cells, but do not bind to CHOS-Claudin 18.1 and CHOS cells, which proves that the humanized Claudin 18.2 antibody specifically binds to Claudin 18.2 but does not bind to Claudin 18.1.
  • HEK293-CLDN18.2 is digested with trypsin at 1000rpm for 5min. Change the fresh medium to spread a 96-well plate, 20,000 cells/well, and incubate overnight at 37°C with 5% CO2.
  • Reagents cell HEK293-hCLDN18.2-H11, complement: normal human serum complement (quidel, A113), antibody: QP024025 (IMAB362), QP14631461 was synthesized in this example.
  • Plating cells/medium Set the background control culture media background, that is, add only 40ul of medium per well, and set the maximum release group maximum LDH release, that is, after adding 40ul target cells, add 12ul lysis solution 45 minutes before the test.
  • Set the volume correction of the volume correction group that is, add 40ul of medium per well; set the LDH positive control group, that is, 1ul of the positive control, after vortexing, dilute 5000 times (5mL), and the diluent is PBS+1%BSA. It can be diluted in a 10-fold gradient.
  • Set the experimental group experimental that is, add 40ul/well target cell.
  • Add complement + antibody mix add 20% complement (diluted with cell culture medium), 40ul/well, both the control group and the experimental group should be added.
  • the CDC results are shown in FIG. 13, and the results show that both the humanized Claudin 18.2 antibody QP14611463 and the control antibody IMAB362 (QP024025) of the present invention can concentration-dependent complement-mediated killing of HEK293-Claudin 18.2 cells with high Claudin 18.2 expression.
  • mice Collect HEK293-hClaudin18.2 cells stably expressing human claudin18.2 (hClaudin18.2) in logarithmic growth phase, adjust the cell concentration to 5 ⁇ 10 7 /mL with PBS buffer, and inoculate 0.1mL( 1:1 Matrigel) cell suspension was placed under the skin of the right flank of CB-17 SCID mice. Observe the mice after inoculation and monitor the growth of tumors. On the day of inoculation, mice are divided into groups and administered for observation.
  • the molecules to be tested are humanized claudin18.2 antibodies QP14331437 and QP14611463, and the positive control antibody is IMAB362 (QP024025).
  • the heavy and light chain variable region germline genes with high homology to QP1162 and QP1166 were selected as templates, and the CDRs of single domain antibodies were separately selected.
  • VH containing the gene fragments required for recombination and digestion with BsmBI to recover the expression vector pQD (with signal peptide and constant region gene (FC) fragments) were added to DH5H competent cells at a ratio of 3:1, ice bath at 0°C for 30min, heat at 42°C Press for 90s, add 5 times volume of LB medium, incubate at 37°C for 45min, spread on LB-Amp plate, culture overnight at 37°C, pick a single clone and send it to sequencing to obtain each target clone.
  • FC constant region gene
  • the length of the humanized VHH is the same as the single domain antibody of the original camel, both are 126 aa.
  • the 293E cell culture density is maintained between 0.2-3 ⁇ 10 6 /ml, and the maintenance phase medium (GIBCO Freestyle 293 expression medium) is cultured.
  • the maintenance phase medium GIBCO Freestyle 293 expression medium
  • the day before transfection the cells to be transfected are centrifuged to change the medium, and the cell density is adjusted to 0.5- 0.8 ⁇ 106/ml. On the day of transfection, the density of 293E cells was 1-1.5 ⁇ 10 6 /ml.
  • Prepare plasmid and transfection reagent PEI The amount of plasmid to be transfected is 100ug/100ml cells, and the mass ratio of PEI to plasmid is 2:1. Mix the plasmid and PEI, and let stand for 15 minutes, not more than 20 minutes.
  • the plasmid and PEI mixture was slowly added to the 293E cells, cultured in a shaker at 8% CO 2 , 120 rpm, 37° C., on the fifth day of transfection, the cell supernatant was collected by centrifugation in a horizontal centrifuge at 4700 rpm for 20 minutes.
  • PBS 3times. Closed: 3% BSA 250ul/hole, RT 1h. Respectively incubate 2ug/ml Biotin QP004.3 (biotin-PDL1-FC) 1:4 dilution with different concentrations, RT for 1h. PBST 3times, PBS 3times. Incubate the secondary antibody: HRP-strepavidin (1:5000) 50ul/well, PBST 6times, PBS 3times. Color development: TMB 100ul/hole, color development for 10 minutes. 2M H2SO4 50ul/hole termination.
  • the results are shown in Figure 15 and Table 14.
  • the humanized antibody QP322 of Nanobody QP1162, and the humanized antibody QP325 of Nanobody QP1166 of the present invention can bind to PD-L1 protein, which are comparable to the pre-humanized Nanobody.
  • the experimental results are shown in Figure 16 and Table 15.
  • the humanized antibody QP322 of the Nanobody QP1162 of the present invention and the humanized antibody QP325 of the Nanobody QP1166 of the present invention can block the binding and binding of PD-L1 to the PD-1 protein.
  • the pre-Nanobody is comparable.
  • Table 15 Activity identification of humanized anti-PD-L1 single domain antibody (Blocking-ELISA)
  • Biacore T200 was used to determine the affinity of the molecule to be tested with the protein human PD-L1 and cynoPD-L1
  • the antigen information is as follows:
  • the experimental results show that the SPR affinity results show that the humanized anti-PD-L1 antibodies QP322 and QP325 both bind to human PD-L1 protein and monkey PD-L1 protein.
  • the humanized Nanobody QP322 and the Camel Nanobody QP1162 have similar binding affinity to human and monkey PD-L1 proteins, and the humanization was successful.
  • the humanized Nanobody QP322 and the heavy chain of anti-CLDN18.2 were respectively formed into a fusion protein through the connecting peptide (G 4 S) 4 , which was used to construct the anti-CLDN18.2/anti-PD-L1 double Specific antibodies.
  • the sequence and protein expression number of the anti-CLDN18.2/anti-PD-L1 bispecific antibody are as follows:
  • the culture density of 293E cells is maintained between 0.2-3 ⁇ 106/ml, and the medium (GIBCO Freestyle 293 expression medium) is cultured.
  • the medium GIBCO Freestyle 293 expression medium
  • Prepare plasmid and transfection reagent PEI The amount of plasmid to be transfected is 100ug/100ml cells, and the mass ratio of PEI to plasmid is 2:1. Mix the plasmid and PEI, and let stand for 15 minutes, not more than 20 minutes.
  • the plasmid and PEI mixture was slowly added to the 293E cells, cultured in a shaker at 8% CO2, 120 rpm, 37° C., on the fifth day of transfection, the cell supernatant was collected by centrifugation at 4700 rpm in a horizontal centrifuge for 20 minutes.
  • the 4 anti-CLDN18.2/anti-PD-L1 bispecific antibodies were purified by Protein A, purified by SEC, concentrated, and then tested by SEC for a series of evaluations of their purity.
  • the summary table is shown in the following table:
  • the SEC purity identification was completed by HPLC.
  • the four molecular maps in the table are as follows: Figure 17, Figure 18, Figure 19 and Figure 20.
  • the purification of QP3691433 is shown in Figure 17, the purification of QP3701440 is shown in Figure 18, the purification of QP3711461 is shown in Figure 19, and the purification of QP3721116 is shown in Figure 20.
  • the results show that the anti-Claudin 18.2/PD-L1 bispecific antibody has good transient expression yield and good SEC purity. After the protein is concentrated, its physical and chemical properties are stable.
  • Anti-CLDN18.2/anti-PD-L1 bispecific antibody activity identification (PD-L1/PD-1 blocking)
  • Anti-CLDN18.2/anti-PD-L1 bispecific antibody activity identification (PD-L1/CD80 blocking)
  • Detection reagents skimmed milk powder (BD, 232100), PBS (shenggong, B548117-0500); HRP-anti human IgG (H+L) (jackson, 109-035-088); TMB (Luoyang Baiaotong Experimental Materials Center , C060201); Elisa plate (costa, 9018) 1 ⁇ PBS buffer: Weigh out NaCl 8.00g, KCl 0.20g, Na2HPO4 ⁇ 12H20 2.9g, KH2PO4 0.2g to 800mL ddH2O, and dissolve it in ddH2O, and dilute to 1L after being fully dissolved. Adjust the pH to 7.4 and sterilize at high temperature for later use.
  • Blocking solution Weigh 5g of skimmed milk powder into PBS. The blocking solution needs to be prepared for current use.
  • Stop solution (1mol/LH2SO4) Take 109mL of 98% concentrated H2SO4 and slowly drop it into 2000mL of ddH2O. TMB develops color at 37°C for 10 minutes, and places it in a shaker (120rpm), 100ul/hole;
  • Add substrate chromogenic solution add substrate chromogenic solution TMB according to the amount of 100 ⁇ L/well, place in a shaker, 200rpm, 35°C for 10min in the dark. Termination: After the color development is completed, 100 ⁇ L/well is quickly added to the termination solution to terminate the reaction. Detection: After centrifugation at 3500 rpm for 5 minutes, take 120ul of the supernatant and transfer it to the Elisa plate, measure the OD value of A450nm on the microplate reader, and analyze the result using graphpad prism software.
  • DC (donor1) cells Resuscitate PBMC, separate monocytes with EasySep TM Human Monocyte Isolation Kit (Stemcell 19359), add rhGM-CSF (1000U/ml) and rhIL4 (500U/ml), culture cells at 37°C for 6 days to induce iDC Change the medium every 2-3 days and add rhGM-CSF (1000U/ml) and rhIL4 (500U/ml) at the same time; collect the cells by centrifugation at 300x g for 5 min, add rhGM-CSF (1000U/ml) and rhIL4 (500U/ml) ml) medium was resuspended, while adding LPS (1 ⁇ g/ml), the cells were cultured at 37°C for 1 day to induce mature DC; the cells were collected and counted for later use.
  • donor1 cells Resuscitate PBMC, separate monocytes with EasySep TM Human Monocyte Isolation Kit (Stemcell
  • T (donor2) cells Resuscitate PBMC, and isolate CD4+Tcell with EasySep TM Human CD4+T Cell Isolation Kit (Stemcell 17952).
  • Prepare the antibody Dilute the antibody (initial concentration 10ug/ml) with 6 concentrations of 1:5 in the medium.
  • DC cells T cells were mixed at a ratio of 1:10, antibodies of different concentrations were added, mixed culture, the expression of IL2 in the culture supernatant was measured on the second day, and the expression of IFNg in the culture supernatant was measured on the fifth day.
  • the candidate molecule CHO14 has a significant antibody concentration-dependent on the concentration of the cytokines IFN ⁇ and IL-2 produced by T cells after activation. Prove the biological function of the PD-L1 antibody in the candidate molecule CHO14, CHO14 can significantly promote the proliferation of T cells. As shown in Figure 25 and Figure 26.
  • HCC827-CLDN18.2 HCC827-CLDN18.2 is digested with trypsin at 1000rpm for 5min. Change the fresh medium to spread a 96-well plate, 20,000 cells/well, and incubate overnight at 37°C with 5% CO2.
  • Prepare the antibody Dilute the antibody (200nM-0.000512nM, 0) 10 concentrations in a 1:5 gradient with medium. Aspirate the target cell culture medium of the 96-well plate, add 70ul/well of the antibody of each concentration diluted above, and set up multiple wells for each concentration.
  • Target cell 50:1, add 70ul/well to the above well plate, and incubate at 37°C for 4 hours. Max lysis well add 15ul lysis buffer (1% Triton-X100), and incubate at 37°C for 10 min.
  • anti-Claudin 18.2 monoclonal antibody QP14611463 and anti-CLDN18.2/anti-PD-L1 bispecific antibody QP3711461 were both concentration-dependent PBMC-mediated killing of recombinant human lung cancer cells expressing Claudin 18.2 and naturally expressing PD-L1 HCC827-CLDN18.2.
  • the bispecific antibody QP3711461 kills EC50 better than the anti-Claudin 18.2 monoclonal antibody QP14611463.
  • mice Take logarithmic growth phase mouse colon cancer cell MC38-hPDL1(Tg)-mClaudin18.2(Tg) cells (this cell overexpresses human PDL1 and mouse Claudin 18.2, and knocks out mouse PDL1 ), the culture medium was removed and washed twice with PBS, and then inoculated subcutaneously into the right flanks of C57BL/6-hPDL1 mice. The inoculation amount was 5 ⁇ 10 5 /100 ⁇ L/mouse. Observe the mice after inoculation and monitor the growth of tumors.
  • the molecule to be tested is anti-CLDN18.2/anti-PD-L1 bispecific antibody QP3711461, and the dosages are 1.5mpk, 4mpk, 10mpk, BIW ⁇ 3, iv administration.
  • the average tumor volume of the PBS group (negative control group) reached 1033.97 mm 3
  • the average tumor volume of the QP3711461 (1.5mpk) group was 932.52 mm 3
  • TGI 12.19%
  • the average tumor volume of the QP3711461 (4mpk) group was 360.92 mm 3
  • TGI 61.81%
  • QP3711461 (10mpk) group average tumor volume is 294.50mm 3
  • TGI 69.53%
  • 4mpk group, 10mpk group and PBS group have statistically significant differences in tumor volume (t test, p ⁇ 0.01) .
  • Table 26 Humanized mouse PBMC engrafted-NCG model HCC827-hClaudin 18.2 in vivo tumor volume in the evaluation of efficacy
  • the molecule to be tested is anti-CLDN18.2/anti-PD-L1 bispecific antibody QP3711461, and the dosages are 4mpk, 10mpk, BIW ⁇ 3, iv administration; the control antibody molecule Tecentriq, the dosage is 5mpk, BIW ⁇ 3, iv Administration.
  • the average tumor volume of the PBS group negative control group
  • the average tumor volume of the QP1461371 (4mpk) group was 258.51 mm 3
  • TGI 80.22%
  • the average tumor volume of the QP1461371 (10mpk) group was 104.81 mm 3 .
  • mice Take logarithmic growth phase mouse colon cancer cell MC38-hPDL1(Tg)-mClaudin18.2(Tg) cells (this cell overexpresses human PDL1 and mouse Claudin 18.2, and knocks out mouse PDL1 ), the culture medium was removed and washed twice with PBS, and then inoculated subcutaneously into the right flanks of C57BL/6 mice. The inoculation amount was 5 ⁇ 10 5 /100 ⁇ L/mouse. Observe the mice after inoculation and monitor the growth of tumors. On the 7th day after inoculation, when the average tumor volume reaches about 90mm 3 , they are randomly divided into 4 groups according to the tumor volume, with 10 mice in each group. The grouping day was defined as D0 day, and the administration was started on D0 day.
  • the molecules to be tested are anti-CLDN18.2/anti-PD-L1 bispecific antibody QP3711461, mutant PD-L1 binding ability molecule QP30771461 (QP3711461- ⁇ PDL1 null) and mutant Claudin 18.2 binding ability molecule QP30891902 (QP3711461- ⁇ Claudin 18.2 null),
  • the dosage is 5mg/kg, Q2D ⁇ 6, ip. administration.
  • the average tumor volume of the PBS group reached 1262.27mm 3
  • the average tumor volume of the QP3711461 group was 532.87mm 3
  • TGI 62.24%%
  • the average tumor volume of the QP30771461 group was 1173.62mm 3
  • TGI 7.59%%
  • the average tumor volume in the QP30891902 group was 794.75mm 3
  • TGI 39.63%
  • the tumor volume between the QP30771461 group and the PBS group was significantly different (t test, p ⁇ 0.01), while the tumors in the mutant molecule QP30891902 group and the PBS group There were statistically significant differences in volume (t test, p ⁇ 0.05).
  • mice Take logarithmic growth phase mouse colon cancer cell MC38-hPDL1(Tg)-mClaudin18.2(Tg) cells (this cell overexpresses human PDL1 and mouse Claudin 18.2, and knocks out mouse PDL1 ), the culture medium was removed and washed twice with PBS, and then inoculated subcutaneously into the right flanks of C57BL/6 mice. The inoculation amount was 5 ⁇ 10 5 /100 ⁇ L/mouse. Observe the mice after inoculation and monitor the growth of tumors. On the 6th day after inoculation, they were randomly divided into 3 groups according to the tumor volume, with 15 mice in each group. The grouping day was defined as D0 day, and the administration was started on D0 day.
  • the molecules to be tested are anti-CLDN18.2/anti-PD-L1 bispecific antibody QP3711461, mutant PD-L1 binding ability molecule QP30771461 (QP3711461- ⁇ PDL1 null) and mutant Claudin 18.2 binding ability molecule QP30891902 (QP3711461- ⁇ Claudin 18.2 null),
  • the dosages were 5mg/kg (QP3711461 group), 5mg/kg+5mg/kg (QP30771461+QP30891902 combined administration group), Q3D ⁇ 6, iv. administration.
  • the average tumor volume of the PBS group reached 838.76 mm 3
  • the average tumor volume of the QP3711461 group was 313.63 mm 3
  • TGI 67.15%
  • the tumor volume between the QP3711461 group and the PBS group has a statistically significant difference (t test, p ⁇ 0.01), while the combined administration of the mutant molecule QP30771461+QP30891902 group and the PBS group have a statistically significant difference in tumor volume (t test, p ⁇ 0.05).

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Abstract

一种靶向人claudin和人PDL1蛋白的双特异抗体，其特征在于，包括：抗人claudin18.2的抗体部分和抗PD-L1抗体部分。该双特异性抗体在结合人claudin18.2蛋白的同时，也可以阻断PD-1/PD-L1的结合，既能在先天性免疫中起到激活NK细胞杀伤肿瘤细胞，又能在获得性免疫重起到促进杀伤性T淋巴细胞对肿瘤的杀伤作用。该双特异性抗体比单独使用抗claudin18.2抗体具有更好的抗肿瘤疗效。

Description

一种靶向人claudin和人PDL1蛋白的双特异抗体及其应用 技术领域
本发明涉及一种靶向人claudin和人PDL1蛋白的双特异抗体及其应用，属于生物医药领域。
背景技术
双特异性抗体(BsAb)又称双功能抗体，可同时识别和结合两种不同的抗原和表位，并阻断两种不同的信号通路以发挥其作用。BsAb与普通抗体相比增加了一个特异性抗原结合位点，在治疗方面表现出了以下优势：
介导免疫细胞对肿瘤的杀伤：双特异性抗体的一个重要作用机制是介导免疫细胞杀伤，双特异性抗体有两条抗原结合臂，其中一条与靶抗原结合，另一条与效应细胞上的标记抗原结合，后者可以激活效应细胞，使其靶向杀伤肿瘤细胞。
双靶点信号阻断，发挥独特的或重叠的功能，有效防止耐药：同时结合双靶点，阻断双信号通路是双特异性抗体的另一个重要作用机制。受体络氨酸激酶(receptor tyrosine kinase，RTKs)是最大的一类酶联受体，在细胞增殖过程中发挥重要的调节作用，如Her家族等。RTKs在肿瘤细胞表面异常高表达，导致肿瘤细胞恶性增生，因此也是肿瘤治疗的重要靶点。针对RTKs的单靶点单克隆抗体已在肿瘤治疗中得到广泛应用，但是，肿瘤细胞可以通过转换信号通路或通过HER家族成员自身或不同成员之间的同源或异源二聚体激活细胞内信号进行免疫逃逸。因此采用双特异性抗体药物同时阻断两个或多个RTKs或其配体，可以减少肿瘤细胞逃逸，提高治疗效果。
具备更强特异性、靶向性和降低脱靶毒性：利用双特异性抗体两个抗原结合臂可以结合不同抗原的特点，两个抗原结合臂分别结合癌细胞表面2种抗原，可以有效增强抗体对癌细胞的结合特异性和靶向性，降低脱靶等副作用；
有效降低治疗成本：以BiTE为例，与传统抗体相比在组织渗透率、杀伤肿瘤细胞效率、脱靶率和临床适应症等指标方面都具有较强的竞争力，临床优势显著。特别在使用剂量方面，由于其治疗效果可以达到普通抗体的100-1000倍，使用剂量最低可将为原来的1/2000，显著降低药物治疗成本。相对于组合疗法，双特异性抗体的成本也远远低于两个单药联合治疗。
PD-1(CD279)最早于1992年被报道，人PD-1编码基因PDCD1位于2q37.3，全长2097bp，由6个外显子组成，翻译产物为288个氨基酸组成的PD-1前体蛋白，剪切前20个氨基酸组成的信号肽后得到成熟蛋白质。PD-1包括胞外免疫球蛋白可变区IgV结构域，疏水跨膜结构域和胞内结构域，胞内尾部结构域N端ITIM基序包含2个磷酸化位点，C端则是一个ITSM基序。PD-1是膜蛋白，属于CD28免疫球蛋白超家族，主要表达在激活后的T 细胞表面，此外还在胸腺的CD4 -CD8 -T细胞、活化的NK细胞和单核细胞有低丰度表达。PD-1有2个配体，分别是B7蛋白家族的PD-L1(CD274，B7-H1)和PD-L2(CD273，B7-DC)，PD-L1和PD-L2氨基酸序列有40％相同。两者区别主要在于表达模式不同，PD-L1组成性的低表达于APCs、非造血细胞(如血管内皮细胞、胰岛细胞)和免疫豁免部位(如胎盘、睾丸和眼睛)，炎性细胞因子如I型和II型干扰素、TNF-α和VEGF等均可以诱导PD-L1的表达。PD-L2则只在被激活的巨噬细胞和树突细胞中有表达。PD-1与PD-L1在激活的T细胞结合后，PD-1的ITSM基序发生酪氨酸磷酸化，进而导致下游蛋白激酶Syk和PI3K的去磷酸化，抑制下游AKT、ERK等通路的活化，最终抑制T细胞活化所需基因及细胞因子的转录和翻译，发挥负向调控T细胞活性的作用。
在肿瘤细胞中，肿瘤细胞及肿瘤微环境通过上调PD-L1表达并与肿瘤特异的CD8 +T细胞表面的PD-1结合，负调控T细胞活性，抑制免疫反应。肿瘤细胞可以通过以下4种途径上调PD-L1表达：1.编码PD-L1的基因扩增(9p24.1)；2.EGFR、MAPK、PI3K-Akt信号通路激活，HIF-1转录因子等可以从转录水平上调PD-L1的表达；3.EB病毒的诱导(EB病毒阳性的胃癌和鼻咽癌表现为PD-L1高表达)；4.表观遗传学的调控。在肿瘤微环境中，interferon-γ等炎症因子的刺激同样可以诱导PD-L1和PD-L2的表达。炎症因子可以诱导肿瘤微环境中其他细胞，包括巨噬细胞、树突状细胞和基质细胞表达PD-L1和PD-L2，而能够识别肿瘤抗原的肿瘤浸润性T细胞能够分泌interferon-γ，进而诱导PD-L1表达上调，这一过程被称为“适应性免疫抵抗”，肿瘤细胞通过这一机制可以实现自我保护。有越来越多的证据表明肿瘤利用PD-1依赖的免疫抑制免疫逃避。在各种实体瘤和血液系统恶性肿瘤种均已经发现PD-L1和PD-L2的高表达。此外，PD-Ls的表达与肿瘤细胞的不良预后之间具有很强相关性，证明了包括食道癌、胃癌、肾癌、卵巢癌、膀胱癌、胰腺癌和黑色素瘤等。
在过去的几十年里，美国和许多发达国家的胃癌发病率和死亡率都大幅度下降。然而，起源于胃(GC)、食道或食管胃交界处癌(GEJ)仍然是一个主要的全球健康问题，特别是在低收入和中等收入国家。胃癌的全球发病率显示出广泛的地理差异，高发病率和低发病率区域之间的差异为15至20倍。在全球范围内，2018年估计有103万例胃癌导致了超过78万人死亡，使胃癌成为世界上第五大最常诊断的癌症和第三大癌症相关死亡原因，但是在西欧、澳大利亚和北美，胃癌是罕见的癌症之一。2019年，估计美国将有27510人被确诊，11140人死于这种疾病，在美国是第15大最常见的确诊癌症和第15大癌症相关死亡原因。胃癌的最高发病率发生在东亚、南美和中美洲以及东欧，其中东亚三国(中国、日本和韩国)的发病率特别高。在中国，胃癌是男性最常见的癌症，也是导致癌症相关死亡率的主要原因。紧密连接蛋白-18A2(Claudin18.2)在近年来被视为消化道肿瘤、特别是胃癌的肿瘤特异性抗原而愈发受到重视。
紧密连接(Tight junction，TJ)在细胞间物质流转中起到关键作用，还通过阻断膜蛋白和膜脂径向扩散维持细胞极性，此外还参与招募调控细胞增殖、分化和运动的信号分子。 紧密连接由紧密连接蛋白(Claudin，CLDN)形成，而紧密连接蛋白家族由超过20种蛋白分子组成，其成员均含有一个四次跨膜的结构域和相似的氨基酸序列，但组织分布具有一定特异性。CLDN在调控细胞旁路的选择性渗透中起到关键作用，CLDN2和CLDN15参与形成阳离子通道和阳离子孔隙，CLDN4/7/10则参与构成阴离子通道和孔隙。CLDN蛋白的差异性表达被认为与多种癌症相关联。CLDN1和CLDN7在侵入性乳腺癌、前列腺癌和食管癌中发生下调，而在宫颈癌、结肠癌、食管癌、胃癌等多种癌症中发现CLDN3/4发生不同程度上调。Sahin等发现在正常组织中，CLDN18的isoform 2亚型(Claudin18.2)只在胃黏膜的分化后表皮细胞中表达，在胃干细胞区域则未见表达，但在原发性胃癌及其转移灶中均发现异常高表达。在胰腺癌、食管癌和肺癌中也有Claudin18.2高表达的报道。由于Claudin18.2定位于细胞膜表面，其生物学功能和特征决定了它是一种理想的治疗靶点，而近年来针对该靶点也有单抗面世。
发明内容
本发明的目的在于提供一种靶向人claudin和人PDL1蛋白的双特异抗体及其应用。
本发明采用了如下技术方案：
一种靶向人claudin18.2和人PDL1蛋白的双特异抗体，包括：
抗人claudin18.2的抗体部分和抗PD-L1抗体部分。
进一步，本发明的靶向人claudin18.2和人PDL1蛋白的双特异抗体，序列如：SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65或SEQ ID NO:66所示。
进一步，本发明的靶向人claudin18.2和人PDL1蛋白的双特异抗体，所述抗人claudin18.2抗体部分结合人claudin18.2蛋白胞外区，抗人claudin18.2抗体部分的序列如：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33或SEQ ID NO:34所示。
进一步，本发明的靶向人claudin18.2和人PDL1蛋白的双特异抗体，抗PD-L1抗体，其序列如SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53或SEQ ID NO:54所示。
上述任一项所述的双特异抗体，在制备治疗癌症、感染或免疫调节疾病的药物中的应用。
上述任一项所述的双特异抗体，在制备抑制肿瘤生长的药物中的应用。
进一步，所述癌症或肿瘤选自下组或部位：结直肠、乳腺、卵巢、胰腺、胃、食管、 前列腺、肾、宫颈、骨髓癌、淋巴癌、白血病、甲状腺、子宫内膜、子宫、膀胱、神经内分泌、头部颈部、肝、鼻咽、睾丸、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，和骨髓增生异常综合症。
具体地，在本发明的第一方面，提供了一种靶向人claudin18.2和人PD-L1蛋白的双特异抗体，所述双特异性抗体包括：
抗人claudin18.2抗体部分和抗PD-L1抗体部分。
在另一优选例中，所述双特异性抗体既有结合人claudin18.2活性，又有结合人PD-L1蛋白的活性。
在另一优选例中，所述的抗人claudin18.2抗体的互补决定区CDR包括：
SEQ ID NO:93、75、79、83或87所示的HCDR1，
SEQ ID NO:94、76、80、84或88所示的HCDR2,和
SEQ ID NO:95、77、81、85或89所示的HCDR3；以及
SEQ ID NO:90或72所示的LCDR1，
SEQ ID NO:91或73所示的LCDR2,和
SEQ ID NO:92、74、78、82或86所示的LCDR3。
在另一优选例中，抗人claudin18.2抗体的3个重链CDR和3个轻链CDR选自下组：
(Z1)SEQ ID No:93、94、95所示的HCDR1、HCDR2和HCDR3；以及SEQ ID No:90、91、92、所示的LCDR1、LCDR2和LCDR3；
(Z2)SEQ ID No:75、76、77所示的HCDR1、HCDR2和HCDR3；以及SEQ ID No:72、73、74所示的LCDR1、LCDR2和LCDR3；
(Z3)SEQ ID No:79、80、81所示的HCDR1、HCDR2和HCDR3；以及SEQ ID No:72、73、78所示的LCDR1、LCDR2和LCDR3；
(Z4)SEQ ID No:83、84、85所示的HCDR1、HCDR2和HCDR3；以及SEQ ID No:72、73、82所示的LCDR1、LCDR2和LCDR3；
(Z5)SEQ ID No:87、88、89所示的HCDR1、HCDR2和HCDR3；以及SEQ ID No:72、73、86所示的LCDR1、LCDR2和LCDR3。
在另一优选例中，所述的抗人claudin18.2抗体的互补决定区CDR包括：SEQ ID NO:93所示的HCDR1、SEQ ID NO:94所示的HCDR2和SEQ ID NO:95所示的HCDR3，以及SEQ ID NO:90所示的LCDR1、SEQ ID NO:91所示的LCDR2和SEQ ID NO:92所示的LCDR3。
在另一优选例中，所述的抗PD-L1抗体为单域抗体。
在另一优选例中，所述的单域抗体的3个互补决定区CDR包括：SEQ ID NO:69所示的HCDR1、SEQ ID NO:70或96所示的HCDR2和SEQ ID NO:71所示的HCDR3。
在另一优选例中，所述的单域抗体的3个互补决定区CDR包括：SEQ ID NO:69所示 的HCDR1、SEQ ID NO:70所示的HCDR2和SEQ ID NO:71所示的HCDR3。
在另一优选例中，所述的单域抗体的3个互补决定区CDR包括：SEQ ID NO:69所示的HCDR1、SEQ ID NO:96所示的HCDR2和SEQ ID NO:71所示的HCDR3。
在另一优选例中,所述双特异性抗体为二个单体构成的二聚体，所述单体具有从N端到C端具有式I所示的结构：
Figure PCTCN2021089729-appb-000001
其中，
L1、L2和L3各自独立地为键或接头元件；
VH代表抗人claudin18.2抗体的重链可变区；
VL代表抗人claudin18.2抗体的轻链可变区；
CH代表抗人claudin18.2抗体的重链恒定区；
CL代表抗人claudin18.2抗体的轻链恒定区；
VHH代表抗PD-L1单域抗体；
“-”代表肽键；
“～”代表二硫键或共价键。在另一优选例中，所述的L1和L3各自为键(如肽键)。在另一优选例中，所述抗人claudin18.2抗体的重链可变区(VH)包括SEQ ID NO:93、75、79、83或87所示的HCDR1，SEQ ID NO:94、76、80、84或88所示的HCDR2和SEQ ID NO:95、77、81、85或89所示的HCDR3。
在另一优选例中，所述抗人claudin18.2抗体的重链可变区(VH)还包括人源化的FR区。
在另一优选例中，所述抗人claudin18.2抗体的重链可变区(VH)的氨基酸序列如SEQ ID NO:31、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:32、SEQ ID NO:33或SEQ ID NO:34所示。
在另一优选例中，所述抗人claudin18.2抗体的轻链可变区(VL)包括SEQ ID NO:90或72所示的LCDR1，SEQ ID NO:91或73所示的LCDR2和SEQ ID NO:92、74、78、82或86所示的LCDR3。
在另一优选例中，所述抗人claudin18.2抗体的轻链可变区(VL)还包括人源化的FR区。
在另一优选例中，所述抗人claudin18.2抗体的轻链可变区(VL)的氨基酸序列如SEQ ID NO:29、SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:28、或SEQ ID NO:30所示。
在另一优选例中，所述抗人claudin18.2抗体的重链恒定区(CH)为人源或鼠源的。
在另一优选例中，所述抗人claudin18.2抗体的轻链恒定区(CL)为人源或鼠源的。
在另一优选例中，所述抗PD-L1单域抗体(VHH)包括SEQ ID NO:69所示的HCDR1、SEQ ID NO:70所示的HCDR2和SEQ ID NO:71所示的HCDR3。
在另一优选例中，所述抗PD-L1单域抗体(VHH)包括SEQ ID NO:69所示的HCDR1、SEQ ID NO:96所示的HCDR2和SEQ ID NO:71所示的HCDR3。
在另一优选例中，所述抗PD-L1单域抗体(VHH)还包括人源化的FR区。
在另一优选例中，所述的抗PD-L1单域抗体(VHH)的氨基酸序列如SEQ ID NO:51、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:53或SEQ ID NO:54所示。
在另一优选例中，所述双特异抗体的VL的氨基酸序列如SEQ ID NO:29所示，VH的氨基酸序列如SEQ ID NO:31所示，以及抗PD-L1单域抗体(VHH)的氨基酸序列SEQ ID NO:51所示。
在另一优选例中，所述双特异抗体的VL的氨基酸序列如SEQ ID NO:1所示，VH的氨基酸序列如SEQ ID NO:5所示，以及抗PD-L1单域抗体(VHH)的氨基酸序列SEQ ID NO:51所示。
在另一优选例中，所述双特异抗体的VL的氨基酸序列如SEQ ID NO:16所示，VH的氨基酸序列如SEQ ID NO:17所示，以及抗PD-L1单域抗体(VHH)的氨基酸序列SEQ ID NO:51所示。
在另一优选例中，所述双特异抗体的VL的氨基酸序列如SEQ ID NO:8所示，VH的氨基酸序列如SEQ ID NO:13所示，以及抗PD-L1单域抗体(VHH)的氨基酸序列SEQ ID NO:51所示。
在另一优选例中，所述双特异抗体的VL的氨基酸序列如SEQ ID NO:21所示，VH的氨基酸序列如SEQ ID NO:26所示，以及抗PD-L1单域抗体(VHH)的氨基酸序列SEQ ID NO:51所示。
在另一优选例中，所述双特异抗体的L链(VL-L3-CL)的氨基酸序列如SEQ ID NO:63、SEQ ID NO:59、SEQ ID NO:61、或SEQ ID NO:65所示；以及所述双特异抗体的H链(VH-L1-CH-L2-VHH)的氨基酸序列如SEQ ID NO:64、SEQ ID NO:60、SEQ ID NO:62、或SEQ ID NO:66所示。
在另一优选例中，所述双特异抗体的L链(VL-L3-CL)的氨基酸序列如SEQ ID NO:63所示，以及H链(VH-L1-CH-L2-VHH)的氨基酸序列如SEQ ID NO:64所示。
在另一优选例中，所述双特异抗体的L链(VL-L3-CL)的氨基酸序列如SEQ ID NO:59所示，以及H链(VH-L1-CH-L2-VHH)的氨基酸序列如SEQ ID NO:60所示。
在另一优选例中，所述双特异抗体的L链(VL-L3-CL)的氨基酸序列如SEQ ID NO:61所示，以及H链(VH-L1-CH-L2-VHH)的氨基酸序列如SEQ ID NO:62所示。
在另一优选例中，所述双特异抗体的L链(VL-L3-CL)的氨基酸序列如SEQ ID NO:65 所示，以及H链(VH-L1-CH-L2-VHH)的氨基酸序列如SEQ ID NO:66所示。
在另一优选例中，所述双特异性抗体是部分或全人源化的抗体。
本发明的第二方面，提供了一种分离的多核苷酸，所述多核苷酸编码本发明第一方面所述的双特异性抗体。
在另一优选例中，所述的多核苷酸包括DNA、RNA或cDNA。
本发明的第三方面，提供了一种载体，所述载体含有本发明第二方面所述的多核苷酸。
在另一优选例中，所述的表达载体选自：质粒、病毒载体。
在另一优选例中，所述的表达载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、腺相关病毒AAV、逆转录病毒、或其他载体。
本发明的第四方面，提供了一种基因工程化的宿主细胞，所述宿主细胞含有本发明第三方面所述的载体，或基因组中整合有本发明第二方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞、哺乳动物细胞。
本发明的第五方面，提供了一种制备本发明第一方面所述的双特异性抗体的方法，包括步骤：
(i)在合适的条件下培养本发明第四方面所述的宿主细胞，从而获得含有本发明第一方面所述的双特异性抗体的混合物；
(ii)对步骤(i)中得到的混合物进行纯化和/或分离，从而获得所述的双特异性抗体。
在另一优选例中，所述纯化可以通过蛋白A亲和柱纯化分离获得目标抗体。
在另一优选例中，所述经过纯化分离后的目标抗体纯度大于95％，大于96％、大于97％、大于98％、大于99％，优选为100％。
本发明的第六方面，提供了一种药物组合物，所述药物组合物含有：
(a)本发明第一方面所述的双特异性抗体或第七方面所述的偶联物；和
(b)药学上可接受的载体。
在另一优选例中，所述的药物组合物中还含有治疗癌症(或肿瘤)的其他药物，如化疗药物。
在另一优选例中，所述的药物组合物用于阻断PD-1和PD-L1的相互作用，同时结合claudin18.2蛋白。
在另一优选例中，所述的药物组合物用于治疗表达claudin18.2蛋白(即claudin18.2阳性)的癌症(或肿瘤)。
在另一优选例中，所述的药物组合物为注射剂型。
本发明的第七方面，提供了一种免疫偶联物，所述免疫偶联物包括：
(a)本发明第一方面所述的双特异性抗体；和
(b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
本发明的第八方面，提供了本发明第一方面所述的双特异抗体在制备治疗癌症(或肿瘤)、感染或免疫调节疾病的药物中的应用。
本发明的第九方面，提供了本发明第一方面所述的双特异抗体，在制备抑制肿瘤生长的药物中的应用。
在另一优选例中，所述癌症或肿瘤选自下组：结直肠癌、乳腺癌、卵巢癌、胰腺癌、胃癌、食管癌、前列腺癌、肾癌、宫颈癌、骨髓癌、淋巴癌、白血病、甲状腺癌、子宫内膜癌、子宫癌、膀胱癌、神经内分泌癌、头部颈部癌、肝癌、鼻咽癌、睾丸癌、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，和骨髓增生异常综合症。
本发明的主要有益效果包括：本发明提供了同时靶向Claudin18.2和PD-L1的双特异性抗体，该双特异性抗体分子可以高效率靶向Claudin18.2和PD-L1。本发明能够提高表达claudin18.2肿瘤的治疗效果。该双特异性抗体在结合人claudin18.2蛋白的同时，也可以阻断PD-1/PD-L1的结合，既能在先天性免疫中激活NK细胞杀伤肿瘤细胞，又能在获得性免疫中促进杀伤性T淋巴细胞对肿瘤的杀伤作用，从而具有协同的杀肿瘤效果。该双特异性抗体比单独使用抗claudin18.2抗体具有更好的抗肿瘤疗效。
附图说明
图1显示了抗CLDN18.2/抗PD-L1双特异抗体分子的结构示意图。
图2显示了人源化Claudin18.2抗体活性鉴定采用ELISA鉴定的第一组抗体的结果。
图3显示了人源化Claudin18.2抗体活性鉴定采用ELISA鉴定的第二组抗体的结果。
图4显示了人源化Claudin18.2抗体活性采用ELISA鉴定的第三组抗体的结果。
图5显示了人源化Claudin18.2抗体活性采用ELISA鉴定的第四组抗体的结果。
图6显示了人源化Claudin18.2抗体活性采用ELISA鉴定的第五组抗体的结果。
图7显示了人源化Claudin18.2抗体活性采用FACS鉴定的第一组抗体的结果。
图8显示了人源化Claudin18.2抗体活性采用FACS鉴定的第二组抗体的结果。
图9显示了人源化Claudin18.2抗体活性采用FACS鉴定的第三组抗体的结果。
图10显示了人源化Claudin18.2抗体活性采用FACS鉴定的第四组抗体的结果。
图11显示了人源化Claudin18.2抗体活性采用FACS鉴定的第五组抗体的结果。
图12显示了人源化Claudin18.2抗体ADCC的结果。
图13显示了人源化Claudin18.2抗体CDC的结果。
图14显示了人源化Claudin18.2抗体在免疫缺陷小鼠CB.17-SCID模型中的体内药效结果。
图15显示了人源化抗PD-L1单域抗体活性鉴定binding-ELISA的结果。
图16显示了人源化抗PD-L1单域抗体活性鉴定Blocking-ELISA的结果。
图17显示了抗CLDN18.2/抗PD-L1双特异抗体表达纯化第一个抗体的结果。
图18显示了抗CLDN18.2/抗PD-L1双特异抗体表达纯化第二个抗体的结果。
图19显示了抗CLDN18.2/抗PD-L1双特异抗体表达纯化第三个抗体的结果。
图20显示了抗CLDN18.2/抗PD-L1双特异抗体表达纯化第四个抗体的结果。
图21显示了抗CLDN18.2/抗PD-L1双特异抗体活性鉴定binding-ELISA的结果。
图22显示了抗CLDN18.2/抗PD-L1双特异抗体活性鉴定PD-L1/PD-1 blocking-ELISA的结果。
图23显示了抗CLDN18.2/抗PD-L1双特异抗体活性鉴定PD-L1/CD80 blocking ELISA的结果。
图24显示了抗CLDN18.2/抗PD-L1双特异抗体活性鉴定中ELISA检测CLDN18.2活性的结果。
图25显示了抗CLDN18.2/抗PD-L1双特异抗体PD-L1功能活性鉴定(混合淋巴细胞反应MLR中候选分子CHO14对T细胞激活后产生的细胞因子IFNγ的浓度依赖结果。
图26显示了抗CLDN18.2/抗PD-L1双特异抗体PD-L1功能活性鉴定(混合淋巴细胞反应MLR中候选分子CHO14对T细胞激活后产生的细胞因子IL-2的浓度依赖结果。
图27显示了抗CLDN18.2/抗PD-L1双特异抗体PBMC介导的细胞杀伤实验的结果。
图28显示了抗CLDN18.2/抗PD-L1双特异抗体在免疫靶点人源化转基因小鼠C57BL/6-hPDL1模型MC38-hPDL1-mClaudin18.2中的体内药效评估的结果。
图29显示了抗CLDN18.2/抗PD-L1双特异抗体在免疫系统人源化小鼠PBMC engrafted-NCG模型HCC827-hClaudin18.2中的体内药效评估的结果。
图30显示了抗CLDN18.2/抗PD-L1双特异抗体在C57BL/6模型MC38-hPDL1-mClaudin18.2中的体内协同药效评估。
图31显示了抗CLDN18.2/抗PD-L1双特异抗体在C57BL/6模型MC38-hPDL1-mClaudin18.2中的体内协同药效评估。
具体实施方式
本发明人经过广泛而深入的研究，通过大量筛选，获得了高特异性和高亲和力的抗CLDN18.2抗体和抗PD-L1单域抗体，并在此基础上进行了人源化和基因重组，从而获得 了同时靶向人Claudin18.2和人PD-L1的双特异性抗体。体外实验证明，本发明的双特异性抗体可特异性结合人Claudin18.2和人PD-L1分子，并杀伤重组表达Claudin 18.2及天然表达PD-L1的人肺癌细胞。体内药效实验证明，本发明的双特异性抗体具有协同作用，在人源化的小鼠模型中表现出比Claudin18.2单抗和PD-L1单抗更优越的抗肿瘤活性。在此基础上，完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
如本文所用，术语“本发明的双特异性抗体”、“本发明的双抗”、“抗claudin18.2/PD-L1双特异性抗体”具有相同的含义，均指特异性识别和结合claudin18.2和PD-L1的双特异性抗体。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。存在两种类型的轻链，λ(l)和κ(k)。存在五种主要的重链种类(或同型)，其决定抗体分子的功能活性：IgM、IgD、IgG、IgA和IgE。每种链包含不同的序列结构域。轻链包括两个结构域或区，可变结构域(VL)和恒定结构域(CL)。重链包括四个结构域，重链可变区(VH)和三个恒定区(CH1、CH2和CH3，统称为CH)。轻链(VL)和重链(VH)的可变区都决定对抗原的结合识别和特异性。轻链的恒定结构域(CL)和重链的恒定区(CH)赋予重要的生物性质如抗体链结合、分泌、经胎盘的移动性、补体结合和与Fc受体(FcR)的结合。Fv片段是免疫球蛋白Fab片段的N-末端部分且由一条轻链和一条重链的可变部分组成。抗体的特异性取决于抗体结合位点和抗原决定区间的结构互补。抗体结合位点由主要来自高度可变区或互补决定区(CDR)的残基组成。偶尔，来自非高度可变或框架区(FR)的残基影响整体结构域结构且进而影响结合位点。互补决定区或CDR指共同限定结合亲和力和天然免疫球蛋白结合位点天然Fv区的特异性的氨基酸序列。免疫球蛋白的轻链和重链各具有三个CDR，分另称为LCDR1(CDR1-L)、LCDR2(CDR2-L)、LCDR3(CDR3-L)和HCDR1(CDR1-H)、HCDR2(CDR2-H)、HCDR3(CDR3-H)。常规抗体抗原结合位点因此包括六个CDR，包含来自每个重链和轻链v区的CDR集合。
如本文所用，术语“单域抗体”、“VHH”、“纳米抗体”具有相同的含义，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的纳米抗体(VHH)，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的纳米抗体(VHH)。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成 了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为框架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位(参见Kabat等,NIH Publ.No.91-3242,卷I，647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本文所用，术语“框架区”(FR)指插入CDR间的氨基酸序列，即指在单一物种中不同的免疫球蛋白间相对保守的免疫球蛋白的轻链和重链可变区的那些部分。免疫球蛋白的轻链和重链各具有四个FR，分别称为FR1-L、FR2-L、FR3-L、FR4-L和FR1-H、FR2-H、FR3-H、FR4-H。相应地，轻链可变结构域可因此称作(FR1-L)-(CDR1-L)-(FR2-L)-(CDR2-L)-(FR3-L)-(CDR3-L)-(FR4-L)且重链可变结构域可因此表示为(FR1-H)-(CDR1-H)-(FR2-H)-(CDR2-H)-(FR3-H)-(CDR3-H)-(FR4-H)。优选地，本发明的FR是人抗体FR或其衍生物，所述人抗体FR的衍生物与天然存在的人抗体FR基本相同，即序列同一性达到85％、90％、95％、96％、97％、98％或99％。
获知CDR的氨基酸序列，本领域的技术人员可轻易确定框架区FR1-L、FR2-L、FR3-L、FR4-L和/或FR1-H、FR2-H、FR3-H、FR4-H。
如本文所用，术语“人源化抗体”，是能够结合目标抗原的非人源抗体衍生的抗体分子，所述目标抗原具有非人源的一个或多个互补决定区(CDR)和来自人免疫球蛋白分子的框架区。通常人框架区中的框架残基将被来自CDR供体抗体的相应残基取代，以改变、优选能够改善抗原的结合。这些框架取代可以通过本领域公知的方法鉴定，例如通过模拟CDR和框架残基的相互作用，以鉴定对抗原结合起重要作用的框架残基，和通过序列对比以鉴定特定位置上异常的框架残基。可以使用本领域公知的多种技术使抗体人源化，例如CDR移植(EP 239,400、PCT公布文本WO 91/09967、美国专利号5,225,539、5,530,101和5,585,089)，镶饰或重塑(EP 592,106、EP 519,596、Padlan,Molecular Immunology28(4/5):489-498(1991)、Studnicka等人,Protein Engineering 7(6):805-814(1994)、Roguska.等人,Proc.Natl.Sci.USA 91:969-973(1994))，以及链替换(U.S.Pat.No.5,565,332)，其全部内容通过引用并入本文。
如本文所用，术语“人框架区”是与天然存在的人抗体的框架区基本相同的(约85％或更多，具体地90％、95％、97％、99％或100％同一性)框架区。
如本文所用，术语“接头”是指插入免疫球蛋白结构域中为轻链和重链的结构域提供足够的可动性以折叠成交换双重可变区免疫球蛋白的一个或多个氨基酸残基。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有claudin18.2和PD-L1蛋白结合活性的双抗。该术语还包括具有与本发明抗体相同功能的、包含相同CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。
所述多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
其中，“保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽(尤其是框架区被性质相似或相近的氨基酸所替换而形成多肽)。这些保守性变异多肽最好根据表A进行氨基酸替换而产生。
表A
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂,如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且,可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl 2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔,脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))；10.化疗剂(例如，顺铂)或任何形式的纳米颗粒等。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。
本发明的双特异性抗体
本发明提供了一种靶向人claudin18.2和人PD-L1蛋白的双特异抗体，其包括：抗人claudin18.2抗体部分和抗PD-L1抗体部分。
(1)抗人claudin18.2抗体部分
本发明的双特异性抗体中的抗人claudin18.2抗体部分是对鼠源的抗claudin18.2抗体进行人源化改造所得。通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选与QP190191、QP192193、QP199200、QP201202、QP207208同源性高的重轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次 序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。再选择一些重要的氨基酸残基做回复突变组合。
所述鼠源的抗claudin18.2抗体的重链可变区(VH)分别具有以下氨基酸序列，CDR如序列中带下划线的部分所示：
>QD208 SEQ ID NO.46
Figure PCTCN2021089729-appb-000002
>QP191 SEQ ID NO.38
Figure PCTCN2021089729-appb-000003
>QD193 SEQ ID NO.40
Figure PCTCN2021089729-appb-000004
>QD200 SEQ ID NO.42
Figure PCTCN2021089729-appb-000005
>QD202 SEQ ID NO.44
Figure PCTCN2021089729-appb-000006
所述鼠源的抗claudin18.2抗体的轻链可变区分别具有以下氨基酸序列：CDR如序列中带下划线的部分所示：
>QD207 SEQ ID NO:45
Figure PCTCN2021089729-appb-000007
>QD190 SEQ ID NO:37
Figure PCTCN2021089729-appb-000008
>QD192 SEQ ID NO:39
Figure PCTCN2021089729-appb-000009
>QD199 SEQ ID NO:41
Figure PCTCN2021089729-appb-000010
>QD201 SEQ ID NO:43
Figure PCTCN2021089729-appb-000011
Figure PCTCN2021089729-appb-000012
上述抗体的轻重链可变区的CDR(下划线部分)区列于表B。
表B
Figure PCTCN2021089729-appb-000013
(2)抗PD-L1抗体部分
本发明的双特异性抗体中的抗人PD-L1抗体部分是对抗PD-L1单域抗体(抗PD-L1纳米抗体)进行人源化改造所得。通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选与QP1162、QP1166同源性高的重轻链可变区种系基因作为模板，将单域抗体的CDR分别移植到相应的人源模板(如IGHV3-23 germline及J-region IGHJ4*01) 中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。再选择一些重要的氨基酸残基做回复突变组合。
所述抗PD-L1单域抗体的氨基酸序列如下所示，CDR如序列中带下划线的部分所示：
>QD1162 SEQ ID NO:55
Figure PCTCN2021089729-appb-000014
>QD1166 SEQ ID NO:56
Figure PCTCN2021089729-appb-000015
总结上述单域抗体的的CDR(下划线部分)区，如下表所示
Figure PCTCN2021089729-appb-000016
如本文所用，术语“抗PD-L1单域抗体”和“抗PD-L1纳米抗体”可互换使用，均指天然缺失轻链，只包含一个重链可变区(VHH)和两个常规的CH2与CH3区，靶向PD-L1分子的抗体。
如本文所用，术语“亲和力”理论上通过完整抗体和抗原间的平衡缔合来定义。本发明双特异性抗体的亲和力可以通过KD值(解离常数)(或其它测定方式)进行评估或测定，例如生物膜层干涉技术(Bio-layer interferometry BLI)，使用FortebioRed96仪器测量确定。
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：瘤内、腹膜内、静脉内、或局部给药。
本发明的药物组合物可直接用于结合claudin18.2和/或PD-L1蛋白分子，因而可用于治疗肿瘤。此外，还可与其他治疗剂联合使用。
本发明的药物组合物含有安全有效量(如0.001-99wt％,较佳地0.01-90wt％，更佳地 0.1-80wt％)的本发明上述的双特异性抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重-约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重-约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
应用
本发明还提供了本发明抗体的应用，涉及在制备治疗癌症(或肿瘤)、感染或免疫调节疾病的药物中的应用。一个优选的应用是用于治疗癌症(或肿瘤)。
本发明的主要优点包括：
(1)本发明提供了同时靶向人Claudin18.2和人PD-L1的双特异性抗体，该双特异性抗体分子可以高效率靶向人Claudin18.2和人PD-L1。
(2)本发明的双特异性抗体能够提高表达claudin18.2肿瘤的治疗效果。该双特异性抗体在结合人claudin18.2蛋白的同时，也可以阻断PD-1/PD-L1的结合，既能在先天性免疫中起到激活NK细胞杀伤肿瘤细胞，又能在获得性免疫重起到促进杀伤性T淋巴细胞对肿瘤的杀伤作用，因此该双特异性抗体比单独使用抗claudin18.2抗体具有更好的抗肿瘤疗效。
(3)本发明的双特异性抗体具有协同作用。
以下结合具体实施方式来详细说明本发明的技术方案。
下列实施方式中中未注明具体条件的实验方法，是按照常规条件，或按照原料或商品制造厂商所建议的条件。或者生物技术教科书如分子克隆，实验室手册，冷泉港实验室，当代分子生物学方法，细胞生物学等记载的实验方法进行。未注明具体来源的试剂，为通过商业途径购买的常规试剂。
实施例1：抗Claudin18.2杂交瘤单克隆抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选与QP190191、QP192193、QP199200、QP201202、QP207208同源性高的重轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为 FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。再选择一些重要的氨基酸残基做回复突变组合。其中氨基酸残基由Kabat编号系统确定并注释。
1.抗Claudin18.2抗体人源化分子克隆
设计引物PCR搭建各人源化抗体VH/VK基因片段，再与带信号肽及恒定区基因(CH1-FC/CL)片段的表达载体pQD进行同源重组，构建抗体全长表达载体VH-CH1-FC-pQD/VK-CL-pQD。
利用在线软件DNAWorks(v3.2.2)(http：//helixweb.nih.gov/dnaworks/)设计多条引物合成VH/VK含重组所需基因片段：5'-30bp信号肽+VH/VK+30bp CH1/CL-3'。按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，分两步PCR扩增得到VH/VK含重组所需基因片段。表达载体pQD(带信号肽及恒定区基因(CH1-FC/CL)片段)构建及酶切，利用限制性内切酶BsmBI，识别序列与酶切位点不同的特性设计构建表达载体pQD(带信号肽及恒定区基因(CH1-FC/CL)片段)。BsmBI酶切载体，切胶回收备用。重组构建表达载体VH-CH1-FC-pQD/VK-CL-pQD.VH/VK含重组所需基因片段与BsmBI酶切回收表达载体pQD按3：1比例分别加入DH5H感受态细胞中，0℃冰浴30min，42℃热击90s，加入5倍体积LB介质，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
各克隆人源化设计轻重链可变区序列及蛋白表达编号如下所示，此表中所有抗体轻链采用kappa轻链恒定区CL(SEQ ID NO：67)，抗体重链采用人IgG1恒定区(SEQ ID NO：68)：
表1：人源化设计轻重链可变区序列及蛋白表达编号
Figure PCTCN2021089729-appb-000017
Figure PCTCN2021089729-appb-000018
同时设计克隆表达人源化前嵌合抗体及对照抗体如下表所示，此表中所有抗体轻链采用kappa轻链恒定区CL，抗体重链采用人IgG1恒定区：
表2：人源化前嵌合抗体及对照抗体(IMAB362为对照抗体，对应蛋白编号QP024025)
Figure PCTCN2021089729-appb-000019
Figure PCTCN2021089729-appb-000020
2.抗Claudin18.2抗体人源化蛋白表达
293E细胞培养密度维持在0.2-3×10 6/ml之间，使用维护阶段培养基(GIBCO Freestyle 293 expression medium)进行培养，转染前一天将待转染细胞离心换液，调整细胞密度为0.5-0.8×10 6/ml。转染当天，293E细胞密度为1-1.5×10 6/ml。准备质粒和转染试剂PEI，需转染质粒量为100ug/100ml细胞，使用PEI和质粒的质量比为2：1。将质粒和PEI进行混匀，静置15min，不宜超过20min。将质粒和PEI混合物缓慢加入293E的细胞中，放入8％CO2，120rpm，37℃的摇床中培养，转染第五天，水平离心机4700rpm离心20min收集细胞上清。
3.抗Claudin18.2抗体人源化蛋白纯化
Protein A亲和层析纯化
用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后培养液上清过柱，上样40ml，流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速0.33ml/min；用洗脱液过柱，UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速1ml/min。样品收集完成后，用pH调节液将PAC-EP调至中性。
4.人源化Claudin18.2抗体活性鉴定(CHOS-CLDN18.2 cell-ELISA)
检测试剂：脱脂奶粉(BD，232100)，PBS(生工，B548117-0500)；HRP-anti human IgG(H+L)(jackson，109-035-088)；TMB(洛阳佰奥通实验材料中心，C060201)；Elisa板(costa，9018)1×PBS缓冲液：称取NaCl 8.00g、KCl 0.20g、Na2HPO4·12H20 2.9g、KH2PO4 0.2g至800mL ddH2O中溶解，充分溶解后定容至1L，调节pH至7.4，高温灭菌备用。或购买商品化10×、20×的PBS溶液稀释至1×PBS缓冲液使用。封闭液：称取5g的奶粉至PBS中，封闭液需现配现用。终止液(1mol/LH2SO4)：取109mL 98％的浓H2SO4缓慢滴加至2000mL ddH2O中。TMB 37℃显色10min，置于摇床中(120rpm)，100ul/孔；
实验步骤：种2E5/孔细胞CHOS-CLD18.2-16-2于U型板，冰PBS洗一遍，1200rpm，离心3min。VCD：1.21E6，实际铺165ul/孔；清洗结束后，按200μL/孔加入封闭液，冰上孵育1h。封闭结束后，1200rpm离心3min后，甩掉上清，孵育CLD18.2对照抗体14-1和样品，按照100ug/ml 1：2稀释比例，共稀释12个梯度，最后设置一个空白对照，按 照100ug/mL、33.33333333ug/mL、11.11111111ug/mL、3.703703704ug/mL、1.234567901ug/mL、0.411522634ug/mL、0.137174211ug/mL、0.045724737ug/mL、0.015241579ug/mL、0.005080526ug/mL、0.001694ug/mL、0ug/mL加入100ul/孔充分混匀后，冰上孵育2h，冰PBS洗3遍；加酶标抗体：孵育HRP-anti human IgG(H+L)抗体，按照1：10000稀释比例，100ul/孔，充分混匀后，冰上1h，冰PBS洗3遍。加底物显色液：按100μL/孔用量加底物显色液TMB，置于摇床中，200rpm，35℃避光显色10min。终止：显色完成后，按100μL/孔用量快速加入终止液终止反应。检测：3500rpm离心5min后，取120ul上清转移至Elisa板上，在酶标仪上，测其A450nm的OD值，使用graphpad prism软件分析结果：如图2、图3、图4、图5和图6所示。
实验结果如图2、图3、图4、图5和图6所示，证明本发明人源化Claudin 18.2抗体均结合CHOS-Claudin 18.2。
5.人源化Claudin18.2抗体活性鉴定(CHOS-CLDN18.2 FACS)
分别收集细胞CHOS，CHOS-CLDN18.2，2E5/孔，1000r离心5min，3％BSA/PBS buffer 200ul/孔封闭60min，4℃。抗体20ug/ml初始浓度，1：4稀释，4℃孵育60min。PBS洗2遍，孵育PE-anti-human FC(1：200)50ul/孔，4℃孵育30min，PBS洗3遍，PBS重悬，FACS结果如下：表3、表4、表5、表6、表7和图7、图8、图9、图10和图11所示。
其中表3及图7的EC50及Max值证明鼠源嵌合抗体QP190191的人源化抗体QP14361435,QP14371433,QP14371435对CHOS-CLDN18.2细胞的结合优于鼠源嵌合抗体QP190191或与之相当；表7及图11的EC50及Max值证明鼠源嵌合抗体QP201202的人源化抗体QP14561454，QP14581453,QP14581454对CHOS-CLDN18.2细胞的结合与鼠源嵌合抗体QP201202相当；表6及图10的EC50及Max值证明鼠源嵌合抗体QP192193的人源化抗体QP14451440，QP14441441,QP14451442对CHOS-CLDN18.2细胞的结合与鼠源嵌合抗体QP192193相当；表5及图9的EC50及Max值证明鼠源嵌合抗体QP199200的人源化抗体QP14491448，QP14501446,QP14501448对CHOS-CLDN18.2细胞的结合与鼠源嵌合抗体QP199200相当；表4及图8的EC50及Max值证明鼠源嵌合抗体QP207208的人源化抗体QP14631461，QP14641460,QP14641461,QP14641462,QP14651460对CHOS-CLDN18.2细胞的结合优于鼠源嵌合抗体QP207208或与之相当。
表3：人源化Claudin18.2抗体活性鉴定FACS的第一组抗体的结果
conc.(ug/ml) QP190191 QP14361435 QP14371433 QP14371435 QP024025
20 88700 183000 115000 134000 72300
5 148000 182000 174000 197000 105000
1.250 114000 141000 133000 145000 63700
0.313 54200 75900 65900 72000 51000
0.078 15700 23600 17700 18000 18200
0.020 8004 8525 10800 10200 6946
0.005 2840 3389 3726 4132 2829
0 729 713 754 732 732
  QP190191 QP14361435 QP14371433 QP14371435 QP024025
EC50 0.3539 0.4657 0.3599 0.3799 0.2702
max 148000 182000 174000 197000 88650
表4：人源化Claudin18.2抗体活性鉴定FACS的第二组抗体的结果
Figure PCTCN2021089729-appb-000021
表5：人源化Claudin18.2抗体活性鉴定FACS的第三组抗体的结果
Figure PCTCN2021089729-appb-000022
表6：人源化Claudin18.2抗体活性鉴定FACS的第四组抗体的结果
Figure PCTCN2021089729-appb-000023
Figure PCTCN2021089729-appb-000024
表7：人源化Claudin18.2抗体活性鉴定FACS的第五组抗体的结果
Figure PCTCN2021089729-appb-000025
6.人源化Claudin18.2抗体特异性鉴定
流式细胞荧光分选技术FACS鉴定本发明的人源化抗体结合Claudin18.2不结合Claudin18.1的特异性
分别收集细胞CHOS，CHOS-CLDN18.2，CHOS-CLDN18.1，2E5/孔，1000r离心5min，3％BSA/PBS buffer 200ul/孔封闭60min，4℃。抗体20ug/ml初始浓度，1：4稀释，4℃孵育60min。PBS洗2遍，孵育PE-anti-human FC(1：200)50ul/孔，4℃孵育30min，PBS洗3遍，PBS重悬，FACS读数的平均荧光值如表8所示，FACS结果显示本发明人源化Claudin 18.2抗体均结合CHOS-Claudin 18.2细胞，且不结合CHOS-Claudin 18.1和CHOS细胞，证明人源化Claudin 18.2抗体特异性结合Claudin 18.2而不结合Claudin 18.1。
表8：FACS读数的平均荧光值
Figure PCTCN2021089729-appb-000026
Figure PCTCN2021089729-appb-000027
7.人源化Claudin18.2抗体ADCC(抗体依赖性细胞介导的细胞毒作用)
准备target cell(HEK293-CLDN18.2)：HEK293-CLDN18.2用胰酶消化，1000rpm 5min。换新鲜培养基铺96孔板，20000cell/孔，37度5％CO2孵育过夜。准备抗体：用培养基1：5梯度稀释抗体(80ug/ml—0.000512ug/ml，0ug/ml)10个浓度。吸掉96孔板中的培养基，加入上面稀释各浓度的抗体70ul/孔，每个浓度设复孔。准备PBMC：将第一天复苏的PBMC离心，用培养基重悬，计数。按PBMC：Target cell＝50：1，加入上面的孔板中70ul/孔，37度孵育4小时。Max lysis well加入15ul lysis缓冲液(1％Triton-X100)，37度孵育10min。
准备LDH试剂：96孔板离心200g，5分钟，转移上清100ul/孔到一块新的透明的96孔板上。取出LDH细胞毒性检测试剂盒(cayman，10008882-480well)，配制反应液，加入100ul/孔，37温和震荡30分钟。吸收490nm读值，按照公式Con(ug/ml)％Maximal signal＝(Test-Control)/(Max-Control)–Con(0ug/ml)％Maximal signal分析数据。
实验结果如图12、表9和表10所示，显示本发明人源化Claudin 18.2抗体QP14331437,QP14401445,QP14611463，全人抗体QP11151116及对照抗体IMAB362(QP024025)均能浓度依赖的PBMC介导的杀伤高表达Claudin 18.2的 HEK293-Claudin 18.2细胞。
表9：490nm读值
conc.(ug/ml) 40 8 1.6 0.32 0.064 0.0128 0.00256 0.00051 0.0001 0 MAX
QP14331437 1.012 1.005 1.012 0.956 0.856 0.752 0.596 0.561 0.55 0.56 1.21
QP14401445 0.98 0.919 0.856 0.907 0.861 0.726 0.573 0.557 0.463 0.527 1.17
QP14611463 0.844 0.947 0.903 0.908 0.835 0.678 0.567 0.516 0.513 0.522 1.144
QP11151116 1.176 0.927 0.885 0.863 0.771 0.752 0.58 0.521 0.521 0.508 1.321
QP024025 0.794 0.79 0.791 0.777 0.675 0.561 0.487 0.479 0.448 0.465 1.141
Target+Ab 0.488 0.418 0.358 0.361 0.272 0.253 0.274 0.261 0.274 0.279  
表10：杀伤百分率
conc.(ug/ml) QP14331437 QP14401445 QP14611463 QP11151116 QP024025
40 52.32％ 47.80％ 28.62％ 75.45％ 21.57％
8 53.76％ 42.72％ 46.32％ 43.75％ 26.16％
1.6 56.36％ 37.77％ 43.37％ 41.22％ 30.02％
0.32 49.58％ 43.72％ 43.84％ 38.46％ 28.17％
0.064 41.55％ 42.09％ 39.27％ 32.36％ 21.98％
0.0128 31.27％ 28.52％ 23.43％ 31.27％ 11.04％
0.00256 13.30％ 10.80％ 10.15％ 11.56％ 1.49％
0.000512 10.46％ 10.03％ 5.65％ 6.19％ 1.70％
0.0001024 8.31％ -1.11％ 4.30％ 5.17％ -2.74％
0 9.02％ 5.43％ 4.88％ 3.36％ -1.33％
EC50 0.0162 0.0082 0.01148 0.00716 0.01562
8.人源化Claudin18.2抗体CDC(补体依赖的细胞毒性)
试剂：细胞HEK293-hCLDN18.2-H11，补体：normal human serum complement(quidel，A113)，抗体：QP024025(IMAB362)，QP14631461来自本实施例中合成。
实验步骤：
铺细胞/培养基：设置背景对照组culture media background，即只加入培养基40ul/孔，设置最大释放组maximum LDH release，即加入40ul靶细胞后，检测前45min加入12ul lysis solution。设置体积矫正组volume correction，即加入培养基40ul/孔；设置LDH阳性对照组，即1ul阳性对照涡旋后，进行5000倍(5mL)稀释，稀释液为PBS+1％BSA。可以以10倍梯度稀释。设置实验组experimental，即加入40ul/孔target cell。加入补体+抗体mix：加入20％补体(用细胞培养基稀释)，40ul/孔，对照组和实验组都要加入。37℃，5％CO2培养细胞，1h15min后，加入12ul/孔lysis solution至最大释放组和体积矫正组。继续37℃培养45min后，LDH检测试剂盒检测，490nm吸光值读数，应在加 入终止液stop solution后1h内完成读数。计算：实验组去除背景对照组，最大释放组去除体积矫正组，公式如下：Percent cytotoxicity＝100*OD490(experimental LDH release)/OD490(maximum LDH release)。
CDC结果如图13所示，结果显示本发明人源化Claudin 18.2抗体QP14611463及对照抗体IMAB362(QP024025)均能浓度依赖的补体介导的杀伤高表达Claudin 18.2的HEK293-Claudin 18.2细胞。
9.人源化Claudin18.2抗体在免疫缺陷小鼠CB.17-SCID模型HEK293-hClaudin18.2中的体内药效
实验方法：收集对数生长期稳定表达人claudin18.2(hClaudin18.2)的稳转细胞株HEK293-hClaudin18.2细胞，用PBS缓冲液调整细胞浓度为5×10 7/mL，接种0.1mL(1：1 Matrigel)细胞悬液于CB-17 SCID小鼠右侧胁腹部皮下。观察接种后小鼠并监测肿瘤的生长，在接种当天小鼠体重进行分组并给药观察。
实验结果：如图14和表11所示。
表11：肿瘤体积
Figure PCTCN2021089729-appb-000028
待测分子为人源化claudin18.2抗体QP14331437和QP14611463，阳性对照抗体为IMAB362(QP024025)。给药方案10mpk×10，q2d，i.v.给药。给药后第37天PBS组(阴性对照组)平均肿瘤体积达到1091.34mm3，QP14331437组平均肿瘤体积260.65mm 3，TGI＝76.12％，QP14611463组平均肿瘤体积225.01mm 3，TGI＝79.38％，IMAB362组平均肿瘤体积324.19mm 3，TGI＝70.29％；该三组与PBS组的肿瘤体积均有统计学意义极显著差异(t检验，p<0.01)。
该实验说明在HEK293-CLDN18.2模型中我们的人源化claudin18.2抗体均表现出优于对照抗体IMAB362的抗肿瘤能力的趋势。
实施例2：抗PD-L1单域抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选与QP1162、QP1166同源性高的重轻链可变区种系基因作为模板，将单域抗体的CDR分别移植到相应的人源模板IGHV3-23 germline及J-region IGHJ4 *01中，形成次序为 FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。再选择一些重要的氨基酸残基做回复突变组合。其中氨基酸残基由Kabat编号系统确定并注释。
1.抗PD-L1单域抗体人源化分子克隆
设计引物PCR搭建各人源化抗体VH基因片段，再与带信号肽及恒定区基因(FC)片段的表达载体pQD进行同源重组，构建抗体全长表达载体VH-FC-pQD。
利用在线软件DNAWorks(v3.2.2)(http：//helixweb.nih.gov/dnaworks/)设计多条引物合成VH/VK含重组所需基因片段：5'-30bp信号肽+VH+30bp FC-3'。按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，分两步PCR扩增得到VH/VK含重组所需基因片段。带信号肽及恒定区基因(FC)片段的表达载体pQD的构建及酶切，利用限制性内切酶，如BsmBI，识别序列与酶切位点不同的特性设计构建带信号肽及恒定区基因(FC)片段的表达载体pQD。BsmBI酶切载体，切胶回收备用。重组构建表达载体VH-FC-pQD。VH含重组所需基因片段与BsmBI酶切回收表达载体pQD(带信号肽及恒定区基因(FC)片段)按3：1比例分别加入DH5H感受态细胞中，0℃冰浴30min，42℃热击90s，加入5倍体积LB介质，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
各克隆人源化设计轻重链可变区序列及蛋白表达编号如下所示，此表中抗体在其C端融合人IgG1-FC恒定区：
表12：QP1162和QP1166人源化设计
Figure PCTCN2021089729-appb-000029
*人源化的VHH与原骆驼的单域抗体的长度一致，均为126个aa。
同时设计克隆表达人源化前嵌合抗体及对照抗体如下表所示：
表13：人源化前嵌合抗体及对照抗体
Figure PCTCN2021089729-appb-000030
2.抗PD-L1单域抗体人源化蛋白表达
293E细胞培养密度维持在0.2-3×10 6/ml之间，维护阶段培养基(GIBCO Freestyle  293 expression medium)进行培养，转染前一天将待转染细胞离心换液，调整细胞密度为0.5-0.8×106/ml。转染当天，293E细胞密度为1-1.5×10 6/ml。准备质粒和转染试剂PEI，需转染质粒量为100ug/100ml细胞，使用PEI和质粒的质量比为2：1。将质粒和PEI进行混匀，静置15min，不宜超过20min。将质粒和PEI混合物缓慢加入293E的细胞中，放入8％CO 2，120rpm，37℃的摇床中培养，转染第五天，水平离心机4700rpm离心20min收集细胞上清。
3.抗PD-L1单域抗体人源化蛋白纯化
Protein A亲和层析纯化
用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后培养液上清过柱，上样40ml，流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速0.33ml/min；用洗脱液过柱，UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速1ml/min。样品收集完成后，用pH调节液将PAC-EP调至中性。
4.人源化抗PD-L1单域抗体活性鉴定(Binding-ELISA)
包被抗体QP1162/QP320/QP321/QP322/QP1166/QP323/QP324/QP325 0.75ug/ml，QP11801181 1.5ug/ml 50ul/孔，4℃过夜。PBS 3times。封闭：3％BSA 250ul/孔，RT 1h。分别孵育2ug/ml Biotin QP004.3(biotin-PDL1-FC)1：4稀释不同浓度，RT 1h。PBST 3times，PBS 3times。孵育二抗：HRP-strepavidin(1：5000)50ul/孔，PBST 6times，PBS 3times。显色：TMB 100ul/孔，显色10min。2M H2SO4 50ul/孔终止。
结果如图15和表14所示，本发明纳米抗体QP1162的人源化抗体QP322,纳米抗体QP1166的人源化抗体QP325及均能结合PD-L1蛋白，与人源化前纳米抗体相当。
表14：人源化抗PD-L1单域抗体活性鉴定(Binding-ELISA)结果
Figure PCTCN2021089729-appb-000031
5.人源化抗PD-L1单域抗体活性鉴定(Blocking-ELISA)
6.包被蛋白QP1138(PD1-FC)2ug/ml 50ul/孔，4℃过夜。PBS 3times。封闭：3％BSA 250ul/孔，RT 1h。分别配制2ug/ml Biotin QP004.3(biotin-PDL1-FC)和不同浓度QP1120 15ug/ml，QP11801181 30ug/ml，1：3稀释，等体积混匀，RT 1h。PBST 3times，PBS 3times。孵育二抗：HRP-strepavidin(1：5000)50ul/孔，PBST 6times，PBS 3times。显色：TMB 100ul/孔，显色10min。2M H2SO4 50ul/孔终止。
实验结果如图16和表15所示，本发明纳米抗体QP1162的人源化抗体QP322,纳米抗体QP1166的人源化抗体QP325能阻断PD-L1与PD-1蛋白结合结合，与人源化前纳米抗体相当。
表15：人源化抗PD-L1单域抗体活性鉴定(Blocking-ELISA)
Figure PCTCN2021089729-appb-000032
7.人源化抗PD-L1单域抗体SPR鉴定亲和力
表面等离子体共振(SPR)检测亲和力
通过Biacore T200(GE)测定待检分子与蛋白人PD-L1及cynoPD-L1的亲和力
抗原信息如下：
表16：蛋白编号
蛋白编号 蛋白描述 货号
QPP09.1 PD-L1 Protein,Human,Recombinant(His Tag) SinoBiologic,10084-H08H
QPP10.1 PD-L1 Protein,Cynomolgus,Recombinant(His Tag) SinoBiologic,90251-C08H
表17：SPR亲和力结果
Figure PCTCN2021089729-appb-000033
Figure PCTCN2021089729-appb-000034
实验结果显示，SPR亲和力结果显示人源化抗PD-L1抗体QP322,QP325均结合人PD-L1蛋白及猴PD-L1蛋白。其中人源化纳米抗体QP322与骆驼纳米抗体QP1162对人及猴PD-L1蛋白结合亲和力相当，人源化成功。
实施例3：抗CLDN18.2/抗PD-L1双特异抗体
在本实施例中，将人源化后的纳米抗体QP322分别与抗CLDN18.2的重链通过连接肽(G 4S) 4形成融合蛋白，用于构建抗CLDN18.2/抗PD-L1双特异抗体。
设计的抗CLDN18.2/抗PD-L1双特异抗体分子，其形式如图1所示。
1.抗CLDN18.2/抗PD-L1双特异抗体分子克隆
设计引物PCR搭建各人源化抗体VH基因片段，再与表达载体pQD(带信号肽及恒定区基因片段)进行同源重组，构建抗体全长表达载体pQD。
抗CLDN18.2/抗PD-L1双特异抗体序列及蛋白表达编号如下所示：
表18：抗CLDN18.2/抗PD-L1双特异抗体序列及蛋白表达编号
Figure PCTCN2021089729-appb-000035
Figure PCTCN2021089729-appb-000036
2.抗CLDN18.2/抗PD-L1双特异抗体蛋白表达
293E细胞培养密度维持在0.2-3×106/ml之间，维护阶段培养基(GIBCO Freestyle 293 expression medium)进行培养，转染前一天待转染细胞离心换液，调整细胞密度为0.5-0.8×106/ml。转染当天，293E细胞密度为1-1.5×106/ml。准备质粒和转染试剂PEI，需转染质粒量为100ug/100ml细胞，使用PEI和质粒的质量比为2：1。将质粒和PEI进行混匀，静置15min，不宜超过20min。将质粒和PEI混合物缓慢加入293E的细胞中，放入8％CO2，120rpm，37℃的摇床中培养，转染第五天，水平离心机4700rpm离心20min收集细胞上清。
3.抗CLDN18.2/抗PD-L1双特异抗体表达纯化
Protein A亲和层析纯化
用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速1ml/min；将离心后培养液上清过柱，上样40ml，流速0.33ml/min；用平衡液过柱，至少3CV，实际体积20ml，确保最终仪器中流出的溶液pH和电导与平衡液一致，流速0.33ml/min；用洗脱液过柱，UV280上升至15mAU时开始收集洗脱峰(PAC-EP)，UV280下降至15mAU时停止收集，流速1ml/min。样品收集完成后，用pH调节液将PAC-EP调至中性。
4个抗CLDN18.2/抗PD-L1双特异抗体经过Protein A纯化，SEC纯化，浓缩，再跑SEC测其纯度一系列评估，总表如下表所示：
表19：SEC纯度
Figure PCTCN2021089729-appb-000037
SEC纯度鉴定由HPLC完成，表中4个分子图谱如下所示：如图17、图18、图19和图20所示。QP3691433如图17，QP3701440纯化如图18，QP3711461纯化如图19，QP3721116纯化如图20。结果显示抗Claudin 18.2/PD-L1双特异抗体瞬转表达产量不错，SEC纯度很好。蛋白经过浓缩后，理化性质稳定。
4.抗CLDN18.2/抗PD-L1双特异抗体活性鉴定(PD-L1 binding)
PD-L1 Binding ELISA：
包被抗体QP322 0.75ug/ml，QP11801181 1.5ug/ml，QP3691433，QP3701440，QP3711461，QP3721116 50ul/孔，4℃过夜。PBS 3times。封闭：3％BSA 250ul/孔，RT 1h。分别孵育1ug/ml Biotin QP004.3(biotin-PDL1-FC)1：5稀释不同浓度，RT 1h。PBST 6times，PBS 3times。孵育二抗：HRP-strepavidin(1：5000)50ul/孔，PBST 6times，PBS 3times。显色：TMB 100ul/孔，显色10min。2M H2SO4 50ul/孔终止。
结果如图21和表20所示。ELISA结果显示抗Claudin 18.2/PD-L1双特异抗体QP3691433,QP3701440,QP3711461,QP3721116中PD-L1纳米抗体融合在FC片段的C端及同型对照(QP322)中PD-L1纳米抗体融合在FC片段的N端，均结合人PD-L1蛋白，结合EC50相当。
表20：PD-L1 Binding ELISA
Figure PCTCN2021089729-appb-000038
5.抗CLDN18.2/抗PD-L1双特异抗体活性鉴定(PD-L1/PD-1 blocking)
PD-L1/PD-1 blocking ELISA
包被：抗体QP1138 2ug/ml 50ul/孔，4℃过夜。PBS 3times。封闭：3％BSA 250ul/孔，RT 1h。分别配制2ug/ml Biotin QP004.3(biotin-PDL1-FC)和不同浓度QP322 15ug/ml，QP11801181 30ug/ml，QP3691433，QP3701440，QP3711461，QP3721116 36ug/ml，1：3稀释，等体积混匀，RT 1h。PBST 3times，PBS 3times。孵育二抗：HRP-strepavidin(1：5000)50ul/孔，PBST 3times，PBS 3times。显色：TMB 100ul/孔，显色10min。2M H2SO4 50ul/孔终止。
结果如图22和表21所示。ELISA结果显示，抗Claudin 18.2/PD-L1双特异抗体QP3691433,QP3701440,QP3711461,QP3721116中PD-L1纳米抗体融合在FC的C端及同型对照(QP322)中PD-L1纳米抗体融合在FC的N端，均能阻断人PD-L1蛋白和PD-1蛋白的结合，抑制IC50相当。
表21：PD-L1/PD-1 blocking ELISA结果
Figure PCTCN2021089729-appb-000039
6.抗CLDN18.2/抗PD-L1双特异抗体活性鉴定(PD-L1/CD80 blocking)
PD-L1/CD80 blocking ELISA
包被CD80-FC 4ug/ml，4℃过夜。PBS洗3遍，5％脱脂奶粉封闭，RT 1h。孵育终浓度0.5ug/ml Biotin-QP004+CHO14(10ug/ml，1：5稀释)，RT 1h。PBST洗5遍。HRP-Strepavidin(1：5000)，PBST洗6遍，PBS洗3遍。TMB显色。注：CHO14蛋白为QP3711461用CHO细胞表达所得蛋白。
结果如图23和表22所示。ELISA结果显示本发明的抗Claudin 18.2/PD-L1双特异抗体CHO14(QP3711461)能阻断PD-L1和CD80的结合。
表22：PD-L1/CD80 blocking ELISA结果
Figure PCTCN2021089729-appb-000040
7.抗CLDN18.2/抗PD-L1双特异抗体活性鉴定(ELISA检测CLDN18.2活性)
检测试剂：脱脂奶粉(BD，232100)，PBS(生工，B548117-0500)；HRP-anti human IgG(H+L)(jackson，109-035-088)；TMB(洛阳佰奥通实验材料中心，C060201)；Elisa板(costa，9018)1×PBS缓冲液：称取NaCl 8.00g、KCl 0.20g、Na2HPO4·12H20 2.9g、KH2PO4 0.2g至800mL ddH2O中溶解，充分溶解后定容至1L，调节pH至7.4，高温灭菌备用。或购买商品化10×、20×的PBS溶液稀释至1×PBS缓冲液使用。封闭液：称取5g的脱脂奶粉至PBS中，封闭液需现配现用。终止液(1mol/LH2SO4)：取109mL 98％的浓H2SO4缓慢滴加至2000mL ddH2O中。TMB 37℃显色10min，置于摇床中(120rpm)，100ul/孔；
实验步骤：种2E5/孔细胞CHOS-CLD18.2-16-2于U型板，冰PBS洗一遍，1200rpm，离心3min；。VCD：1.21E6，实际铺165ul/孔；清洗结束后，按200μL/孔加入封闭液，冰上孵育1h。封闭结束后，1200rpm离心3min后，甩掉上清，孵育CHO14样品，按照100ug/ml 1：2稀释比例，共稀释12个梯度，最后设置一个空白对照，按照100ug/mL、33.33333333ug/mL、11.11111111ug/mL、3.703703704ug/mL、1.234567901ug/mL、0.411522634ug/mL、0.137174211ug/mL、0.045724737ug/mL、0.015241579ug/mL、0.005080526ug/mL、0.001694ug/mL、0ug/mL加入100ul/孔充分混匀后，冰上孵育2h，冰PBS洗3遍；加酶标抗体：孵育HRP-anti human IgG(H+L)抗体，按照1：10000稀释比例，100ul/孔，充分混匀后，冰上1h，冰PBS洗3遍。加底物显色液：按100μL/孔用量加底物显色液TMB，置于摇床中，200rpm，35℃避光显色10min。终止：显色完成后，按100μL/孔用量快速加入终止液终止反应。检测：3500rpm离心5min后，取120ul上清转移至Elisa板上，在酶标仪上，测其A450nm的OD值，使用graphpad prism软件分析结果。
CHO14与CHOS-CLDN18.2高表达细胞株的结合EC50为0.2653nM。证明候选分子CHO14结合Claudin18.2，结果如图24所示。
8.抗CLDN18.2/抗PD-L1双特异抗体PD-L1功能活性鉴定(混合淋巴细胞反应MLR)
准备DC(donor1)细胞：复苏PBMC，用EasySep TMHuman Monocyte Isolation Kit(Stemcell 19359)分离monocytes，加入rhGM-CSF(1000U/ml)和rhIL4(500U/ml)，37℃培养细胞6天诱导为iDC；每2-3天半换液，同时补充rhGM-CSF(1000U/ml)和rhIL4(500U/ml)；收集细胞300x g离心5min，用加入rhGM-CSF(1000U/ml)和rhIL4(500U/ml)的培养基重悬，同时加入LPS(1μg/ml)，37℃继续培养细胞1天诱导为成熟DC；收集细胞，计数备用。准备T(donor2)细胞：复苏PBMC，用EasySep TMHuman CD4+T Cell Isolation Kit(Stemcell 17952)分离CD4+Tcell。准备抗体：用培养基1：5梯度稀释抗体(初始浓度10ug/ml)6个浓度。将DC细胞：T细胞为1：10的比例混合，加入不同浓度的抗体，混合培养，第2天检测培养上清中IL2的表达，第5天检测培养上清中IFNg的表达。
在混合淋巴细胞反应实验中，候选分子CHO14对T细胞激活后产生的细胞因子IFNγ及IL-2的浓度有明显的抗体浓度依赖。证明候选分子CHO14中PD-L1抗体的生物学功能，CHO14能显著促进T细胞增殖。如图25和图26所示。
9.抗CLDN18.2/抗PD-L1双特异抗体PBMC介导的细胞杀伤实验
准备target cell(HCC827-CLDN18.2)：HCC827-CLDN18.2用胰酶消化，1000rpm 5min。换新鲜培养基铺96孔板，20000cell/孔，37度5％CO2孵育过夜。准备抗体：用培养基1：5梯度稀释抗体(200nM—0.000512nM，0)10个浓度。吸掉96孔板target cell的培养基，加入上面稀释各浓度的抗体70ul/孔，每个浓度设复孔。准备PBMC：将第一天复苏的PBMC离心，用培养基重悬，计数。按PBMC：Target cell＝50：1，加入上面的孔板中70ul/孔， 37度孵育4小时。Max lysis well加入15ul lysis buffer(1％Triton-X100)，37度孵育10min。
准备LDH reagents：96孔板离心200g，5分钟，转移上清100ul/孔到一块新的透明的96孔板上。取出LDH cytotoxcity assay kit(cayman，10008882-480well)，配制Reaction Solution，加入100ul/孔，37温和震荡30分钟。吸收490nm读值，按照公式Con(ug/ml)％Maximal signal＝(Test-Control)/(Max-Control)–Con(0ug/ml)％Maximal signal分析数据。
结果如图27、表23和表24所示。在ADCC实验中，抗Claudin 18.2单抗QP14611463及抗CLDN18.2/抗PD-L1双特异抗体QP3711461均呈浓度依赖性的PBMC介导的杀伤重组表达Claudin 18.2及天然表达PD-L1的人肺癌细胞HCC827-CLDN18.2。双特异抗体QP3711461杀伤EC50优于抗Claudin 18.2单抗QP14611463。
表23：490nm读值
Figure PCTCN2021089729-appb-000041
表24：杀伤百分率
Figure PCTCN2021089729-appb-000042
10.抗CLDN18.2/抗PD-L1双特异抗体在免疫靶点人源化转基因小鼠C57BL/6-hPDL1模型MC38-hPDL1-mClaudin18.2中的体内药效评估
实验方法：取对数生长期小鼠结肠癌细胞MC38-hPDL1(Tg)-mClaudin18.2(Tg)细胞(该细胞过表达人的PDL1和小鼠的Claudin18.2，同时敲除小鼠的PDL1)，去除培养液并 用PBS洗两次后接种于C57BL/6-hPDL1小鼠右侧胁腹部皮下，接种量：5×10 5/100μL/只。观察接种后小鼠并监测肿瘤的生长，接种后第8天，平均肿瘤体积达到82.85mm 3时，根据肿瘤体积随机分成4组，每组9只。分组当天定义为D0天，并于D0天开始给药。
实验结果：如图28和表25所示。
表25：肿瘤体积
Figure PCTCN2021089729-appb-000043
待测分子为抗CLDN18.2/抗PD-L1双特异抗体QP3711461，给药剂量分别为1.5mpk、4mpk、10mpk，BIW×3，i.v.给药。给药后第28天PBS组(阴性对照组)平均肿瘤体积达到1033.97mm 3，QP3711461(1.5mpk)组平均肿瘤体积932.52mm 3，TGI＝12.19％，QP3711461(4mpk)组平均肿瘤体积360.92mm 3，TGI＝61.81％，QP3711461(10mpk)组平均肿瘤体积294.50mm 3，TGI＝69.53％；4mpk组、10mpk组与PBS组的肿瘤体积均有统计学意义极显著差异(t检验，p<0.01)。
该实验说明在免疫靶点人源化转基因小鼠的MC38-hPDL1-mClaudin18.2模型中我们的抗CLDN18.2/抗PD-L1双特异抗体表现出优越的抗肿瘤能力。
11.抗CLDN18.2/抗PD-L1双特异抗体在免疫系统人源化小鼠PBMC engrafted-NCG模型HCC827-hClaudin18.2中的体内药效评估
实验方法：取对数生长期人肺腺癌稳转细胞株HCC827-hClaudin18.2细胞(该细胞天然高表达人PDL1，过表达人Claudin18.2)，去除培养液并用PBS洗两次后接种于NCG小鼠右侧胁腹部皮下，接种量：5×10 6/100μL/只。观察接种后小鼠并监测肿瘤的生长，接种后第7天，平均肿瘤体积达到约150mm 3时，接种人PBMC(5×10 6/100μL/只)。根据肿瘤体积随机分成4组，每组9只。分组当天定义为D0天，并于D0天开始给药。
实验结果：如图29和表26所示。
表26：免疫系统人源化小鼠PBMC engrafted-NCG模型HCC827-hClaudin18.2中的体内药效评估中肿瘤体积
Figure PCTCN2021089729-appb-000044
待测分子为抗CLDN18.2/抗PD-L1双特异抗体QP3711461，给药剂量分别为4mpk、10mpk，BIW×3，i.v.给药；对照抗体分子Tecentriq，给药剂量5mpk，BIW×3，i.v.给药。给药后第24天PBS组(阴性对照组)平均肿瘤体积达到1306.8mm 3，QP1461371(4mpk)组平均肿瘤体积258.51mm 3，TGI＝80.22％，QP1461371(10mpk)组平均肿瘤体积104.81mm 3，TGI＝91.98％，Tecentriq(5mpk)组平均肿瘤体积90.90mm 3，TGI＝93.04％；QP3711461(4mpk)组、QP3711461(10mpk)组和Tecentriq(5mpk)组与PBS组的肿瘤体积均有统计学意义极显著差异(t检验，p<0.01)。
该实验说明在免疫系统人源化小鼠的HCC827-hClaudin18.2模型中我们的抗CLDN18.2/抗PD-L1双特异抗体也表现出优越的抗肿瘤能力。
12.抗CLDN18.2/抗PD-L1双特异抗体在C57BL/6模型MC38-hPDL1-mClaudin18.2中的体内协同药效评估
实验方法：取对数生长期小鼠结肠癌细胞MC38-hPDL1(Tg)-mClaudin18.2(Tg)细胞(该细胞过表达人的PDL1和小鼠的Claudin18.2，同时敲除小鼠的PDL1)，去除培养液并用PBS洗两次后接种于C57BL/6小鼠右侧胁腹部皮下，接种量：5×10 5/100μL/只。观察接种后小鼠并监测肿瘤的生长，接种后第7天，平均肿瘤体积达到约90mm 3时，根据肿瘤体积随机分成4组，每组10只。分组当天定义为D0天，并于D0天开始给药。
实验结果：如图30和表27所示。
表27肿瘤体积
Figure PCTCN2021089729-appb-000045
Figure PCTCN2021089729-appb-000046
待测分子为抗CLDN18.2/抗PD-L1双特异抗体QP3711461、突变PD-L1结合能力的分子QP30771461(QP3711461-ΔPDL1 null)和突变Claudin 18.2结合能力的分子QP30891902(QP3711461-ΔClaudin 18.2 null)，给药剂量为5mg/kg，Q2D×6，ip.给药。给药后第13天PBS组(阴性对照组)平均肿瘤体积达到1262.27mm 3，QP3711461组平均肿瘤体积532.87mm 3，TGI＝62.24％％，QP30771461组平均肿瘤体积1173.62mm 3，TGI＝7.59％％，QP30891902组平均肿瘤体积794.75mm 3，TGI＝39.63％；QP30771461组与PBS组的肿瘤体积均有统计学意义极显著差异(t检验，p<0.01),而突变分子QP30891902组与PBS组的肿瘤体积均有统计学意义显著差异(t检验，p<0.05)。
该实验说明在C57BL/6小鼠的MC38-hPDL1-mClaudin18.2模型中抗CLDN18.2/抗PD-L1双特异抗体具有协同效应，表现出比Claudin18.2单抗和PD-L1单抗更优越的抗肿瘤活性。
13.抗CLDN18.2/抗PD-L1双特异抗体在C57BL/6模型MC38-hPDL1-mClaudin18.2中的体内协同药效评估
实验方法：取对数生长期小鼠结肠癌细胞MC38-hPDL1(Tg)-mClaudin18.2(Tg)细胞(该细胞过表达人的PDL1和小鼠的Claudin18.2，同时敲除小鼠的PDL1)，去除培养液并用PBS洗两次后接种于C57BL/6小鼠右侧胁腹部皮下，接种量：5×10 5/100μL/只。观察接种后小鼠并监测肿瘤的生长，接种后第6天根据肿瘤体积随机分成3组，每组15只。分组当天定义为D0天，并于D0天开始给药。
实验结果：如图31和表28所示。
表28肿瘤体积
Figure PCTCN2021089729-appb-000047
待测分子为抗CLDN18.2/抗PD-L1双特异抗体QP3711461、突变PD-L1结合能力的分子QP30771461(QP3711461-ΔPDL1 null)和突变Claudin 18.2结合能力的分子QP30891902(QP3711461-ΔClaudin 18.2 null)，给药剂量分别为5mg/kg(QP3711461组)、 5mg/kg+5mg/kg(QP30771461+QP30891902联合给药组)，Q3D×6，iv.给药。给药后第19天PBS组(阴性对照组)平均肿瘤体积达到838.76mm 3，QP3711461组平均肿瘤体积313.63mm 3，TGI＝67.15％，QP30771461+QP30891902联合给药组平均肿瘤体积478.61mm 3，TGI＝45.98％；QP3711461组与PBS组的肿瘤体积有统计学意义极显著差异(t检验，p<0.01),而突变分子联合给药QP30771461+QP30891902组与PBS组的肿瘤体积有统计学意义显著差异(t检验，p<0.05)。
该实验说明在C57BL/6小鼠的MC38-hPDL1-mClaudin18.2模型中抗CLDN18.2/抗PD-L1双特异抗体具有协同效应，表现出比Claudin18.2单抗和PD-L1单抗更优越的抗肿瘤活性(p＝0.052)。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (22)

  1. 一种靶向人claudin18.2和人PD-L1蛋白的双特异性抗体，其特征在于，所述双特异性抗体包括：
    抗人claudin18.2抗体部分和抗PD-L1抗体部分。
  2. 如权利要求1所述双特异性的抗体，其特征在于，所述的抗人claudin18.2抗体的互补决定区CDR包括：
    SEQ ID NO:93、75、79、83或87所示的HCDR1，
    SEQ ID NO:94、76、80、84或88所示的HCDR2,和
    SEQ ID NO:95、77、81、85或89所示的HCDR3；以及
    SEQ ID NO:90或72所示的LCDR1，
    SEQ ID NO:91或73所示的LCDR2,和
    SEQ ID NO:92、74、78、82或86所示的LCDR3。
  3. 如权利要求1所述的双特异性抗体，其特征在于，抗人claudin18.2抗体的3个重链CDR和3个轻链CDR选自下组：
    (Z1)SEQ ID No:93、94、95所示的HCDR1、HCDR2和HCDR3；以及SEQ ID No:90、91、92、所示的LCDR1、LCDR2和LCDR3；
    (Z2)SEQ ID No:75、76、77所示的HCDR1、HCDR2和HCDR3；以及SEQ ID No:72、73、74所示的LCDR1、LCDR2和LCDR3；
    (Z3)SEQ ID No:79、80、81所示的HCDR1、HCDR2和HCDR3；以及SEQ ID No:72、73、78所示的LCDR1、LCDR2和LCDR3；
    (Z4)SEQ ID No:83、84、85所示的HCDR1、HCDR2和HCDR3；以及SEQ ID No:72、73、82所示的LCDR1、LCDR2和LCDR3；
    (Z5)SEQ ID No:87、88、89所示的HCDR1、HCDR2和HCDR3；以及SEQ ID No:72、73、86所示的LCDR1、LCDR2和LCDR3。
  4. 如权利要求1所述的双特异性抗体，其特征在于，所述的抗PD-L1抗体为单域抗体。
  5. 如权利要求4所述的双特异性抗体，其特征在于，所述的单域抗体的3个互补决定区CDR包括：SEQ ID NO:69所示的HCDR1、SEQ ID NO:70或96所示的HCDR2和SEQ ID NO:71所示的HCDR3。
  6. 如权利要求1-5任一项所述的双特异性抗体，其特征在于，所述双特异性抗体为二个单体构成的二聚体，所述单体具有从N端到C端具有式I所示的结构：
    Figure PCTCN2021089729-appb-100001
    其中，
    L1、L2和L3各自独立地为键或接头元件；
    VH代表抗人claudin18.2抗体的重链可变区；
    VL代表抗人claudin18.2抗体的轻链可变区；
    CH代表抗人claudin18.2抗体的重链恒定区；
    CL代表抗人claudin18.2抗体的轻链恒定区；
    VHH代表抗PD-L1单域抗体；
    “-”代表肽键；
    “～”代表二硫键或共价键。
  7. 如权利要求1-6任一项所述的双特异性抗体，其特征在于，所述抗人claudin18.2抗体的重链可变区(VH)的氨基酸序列如SEQ ID NO:31、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:32、SEQ ID NO:33或SEQ ID NO:34所示。
  8. 如权利要求1-6任一项所述的双特异性抗体，其特征在于，所述抗人claudin18.2抗体的轻链可变区(VL)的氨基酸序列如SEQ ID NO:29、SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:28、或SEQ ID NO:30所示。
  9. 如权利要求4-6任一项所述的双特异性抗体，其特征在于，所述的抗PD-L1单域抗体(VHH)的氨基酸序列如SEQ ID NO:51、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:52、SEQ ID NO:53或SEQ ID NO:54所示。
  10. 如权利要求6所述的双特异性抗体，其特征在于，所述双特异抗体的L链(VL-L3-CL)的氨基酸序列如SEQ ID NO:63、SEQ ID NO:59、SEQ ID NO:61、或SEQ ID NO:65所示；以及所述双特异抗体的H链(VH-L1-CH-L2-VHH)的氨基酸序列如SEQ ID NO:64、SEQ ID NO:60、SEQ ID NO:62、或SEQ ID NO:66所示。
  11. 如权利要求1-10任一项所述的双特异性抗体，其特征在于，所述双特异性抗体是部分或全人源化的抗体。12.如权利要求1所述的靶向人claudin18.2和人PDL1蛋白的双特异抗体，其特征在于：
    序列如：SEQ ID NO:59、SEQ ID NO:60、SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65或SEQ ID NO:66所示。
  12. 如权利要求1所述的靶向人claudin18.2和人PDL1蛋白的双特异抗体，其特征在于：
    所述抗人claudin18.2抗体部分结合人claudin18.2蛋白胞外区，抗人claudin18.2抗体部分的序列如：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、 SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33或SEQ ID NO:34所示。
  13. 如权利要求1所述的靶向人claudin18.2和人PDL1蛋白的双特异抗体，其特征在于：抗PD-L1抗体，其序列如SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ ID NO:53或SEQ ID NO:54所示。
  14. 一种分离的多核苷酸，其特征在于，所述多核苷酸编码如权利要求1-14任一项所述的双特异性抗体。
  15. 一种载体，其特征在于，所述载体含有如权利要求15所述的多核苷酸。
  16. 一种基因工程化的宿主细胞，其特征在于，所述宿主细胞含有权利要求16所述的载体，或基因组中整合有权利要求15所述的多核苷酸。
  17. 一种制备如权利要求1-14任一项所述的双特异性抗体的方法，其特征在于，包括步骤：
    (i)在合适的条件下培养权利要求17所述的宿主细胞，从而获得含有所述的双特异性抗体的混合物；
    (ii)对步骤(i)中得到的混合物进行纯化和/或分离，从而获得所述的双特异性抗体。
  18. 一种药物组合物，其特征在于，所述药物组合物含有：
    (a)如权利要求1-14任一项所述的双特异性抗体；和
    (b)药学上可接受的载体。
  19. 一种免疫偶联物，其特征在于，所述免疫偶联物包括：
    (a)如权利要求1-14任一项所述的双特异性抗体；和
    (b)选自下组的偶联部分：可检测标记物、药物、毒素、细胞因子、放射性核素、或酶。
  20. 如权利要求1-14中任一项所述的双特异抗体，在制备治疗癌症(或肿瘤)、感染或免疫调节疾病的药物中的应用。
  21. 如权利要求1-14中任一项所述的双特异抗体在制备抑制肿瘤生长的药物中的应用。
  22. 如权利要求21或22任一项所述的应用，其特征在于：
    所述癌症或肿瘤选自下组：结直肠癌、乳腺癌、卵巢癌、胰腺癌、胃癌、食管癌、前列腺癌、肾癌、宫颈癌、骨髓癌、淋巴癌、白血病、甲状腺癌、子宫内膜癌、子宫癌、膀胱癌、神经内分泌癌、头部颈部癌、肝癌、鼻咽癌、睾丸癌、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，和骨髓增生异常综合症。
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