WO2018006882A1 - 抗密蛋白18a2的抗体及其应用 - Google Patents

抗密蛋白18a2的抗体及其应用 Download PDF

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WO2018006882A1
WO2018006882A1 PCT/CN2017/092381 CN2017092381W WO2018006882A1 WO 2018006882 A1 WO2018006882 A1 WO 2018006882A1 CN 2017092381 W CN2017092381 W CN 2017092381W WO 2018006882 A1 WO2018006882 A1 WO 2018006882A1
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seq
antibody
cells
cell
amino acid
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PCT/CN2017/092381
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English (en)
French (fr)
Inventor
王鹏
蒋华
杨琳琳
石志敏
王华茂
李宗海
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科济生物医药（上海）有限公司
上海市肿瘤研究所
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Priority claimed from PCT/CN2017/082024 external-priority patent/WO2017186121A1/zh
Priority to IL264144A priority Critical patent/IL264144B2/en
Priority to KR1020197003874A priority patent/KR20190038564A/ko
Priority to JP2019521178A priority patent/JP2019531084A/ja
Priority to EP17823694.9A priority patent/EP3483182B1/en
Priority to RU2019101430A priority patent/RU2793445C2/ru
Application filed by 科济生物医药（上海）有限公司, 上海市肿瘤研究所 filed Critical 科济生物医药（上海）有限公司
Priority to BR112019000327A priority patent/BR112019000327A8/pt
Priority to SG11201900171QA priority patent/SG11201900171QA/en
Priority to US16/316,331 priority patent/US11111295B2/en
Priority to CN201780042611.4A priority patent/CN109790222B/zh
Priority to AU2017294276A priority patent/AU2017294276A1/en
Priority to CA3030257A priority patent/CA3030257A1/en
Publication of WO2018006882A1 publication Critical patent/WO2018006882A1/zh
Priority to US17/395,223 priority patent/US20220185880A1/en

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Definitions

  • the present invention is in the field of immunology, and more particularly, the invention relates to antibodies against the albumin 18A2 and uses thereof.
  • Chimeric antigen receptor is an artificially recombinant receptor that usually contains the antigen recognition domain of a monoclonal antibody located in the extracellular region, a transmembrane region, and an intracellular activation signal structure of an immune response cell. Domain composition.
  • Gastric cancer is one of the most common cancers worldwide. According to the statistics of the World Health Organization's Cancer Control Project, there are 7 million patients who die of cancer every year in the world, and 700,000 of them die of gastric cancer. Compared with conventional gastric cancer treatment regimens, antibody-based treatment regimens have far-reaching application prospects due to their high specificity and low side effects.
  • Claudin 18 is an intrinsic membrane protein located in the tight junction of the epithelium and the endothelium with a molecular weight of approximately 27.9 KD.
  • GenBank accession number is splice variant 1 (CLD18A1, CLD18.1): NP_057453, NM016369, and splice variant 2 (CLD18A2, CLD18.2): NM_001002026, NP_001002026.
  • Figure 1A shows a comparison of the identity of claudin 18A2 (SEQ ID NO: 55) and claudin 18A1 (SEQ ID NO: 57).
  • CLD18A1 is selectively expressed in the epithelium of the lung and stomach, while CLD18A2 is slightly expressed in normal gastric epithelial short-lived cells, but in tumor cells, CLD18A2 is strongly expressed in various cancer types. For example, 75% of patients with gastric cancer have high expression of CLD18A2, 50% of patients with pancreatic cancer have high expression of CLD18A2, and 30% of patients with esophageal cancer have high expression of CLD18A2, and are also highly expressed in lung cancer. Therefore, finding antibodies that bind CLD18A2 with higher specificity without binding to CLD18A1 is of great significance for the treatment and detection of cancer.
  • Type I interferons contain IFN ⁇ protein (a class of identical proteins encoded by 13 human genes from IFNA1 to IFNA13), IFN ⁇ (encoded by a single individual and mouse gene IFNB1), and other less studied interferons. Studies have shown that type I interferons have anticancer effects on some tumors, probably due to their immune stimulating function. However, systemic administration of type I interferons may have immunosuppressive effects (Lotrich, FEMajor depression during interferon- ⁇ treatment: vulnerability and prevention. Dialogues Clin. Neurosci. 11 , 417-425 (2009)) with major adverse events.
  • the present invention overcomes the aforementioned problems and has additional advantages.
  • the present invention provides an antibody which specifically binds to claudin 18A2, characterized in that the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31, 32, 33, 37, 38, 39, 43 a heavy chain CDR of 44, 45, 49, 50, 51, 83, 84, 85 or a variant thereof, and/or selected from the group consisting of amino acid sequences SEQ ID NO: 34, 35, 36, 40, 41, 42, 46, 47, 48, 52, 53, 54 or a variant thereof light chain CDR.
  • the antibody of the invention is selected from the group consisting of (a) an antibody comprising a heavy chain variable region comprising SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: CDR1 of the amino acid sequence of 43 or SEQ ID NO: 49 comprising SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 83, SEQ ID
  • the CDR2 of the amino acid sequence of NO: 84 or one of SEQ ID NO: 85 comprises the amino acid sequence shown by one of SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45 or SEQ ID NO: 51 a CDR3;
  • the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of the antibody are SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 37, respectively.
  • the antibody comprises a heavy chain variable region and a light chain variable region having SEQ ID NO:3, or SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO
  • the antibody is an antibody having the heavy chain variable region set forth in SEQ ID NO: 3, the light chain variable region set forth in SEQ ID NO: 1; having the heavy chain set forth in SEQ ID NO: a variable region, an antibody of the light chain variable region of SEQ ID NO: 5; an antibody having a heavy chain variable region of SEQ ID NO: 11, a light chain variable region of SEQ ID NO: 9; having SEQ ID NO: a heavy chain variable region of 15, an antibody of the light chain variable region of SEQ ID NO: 13; having the heavy chain variable region of SEQ ID NO: 17, and the light chain of SEQ ID NO:
  • the antibody is an antibody having the heavy chain variable region set forth in SEQ ID NO: 3, the light chain variable region set forth in SEQ ID NO: 1, or the heavy chain set forth in SEQ ID NO: a variable region, an antibody of the light chain variable region of SEQ ID NO: 1; or an antibody having a heavy chain variable region of SEQ ID NO: 19, a light chain variable region of SEQ ID NO: 1;
  • the antibody is a humanized antibody, a chimeric antibody or a fully human antibody; or the antibody is a monoclonal antibody; or the antibody is a single chain antibody or a domain antibody.
  • the antibody is a humanized antibody selected from the group consisting of an antibody having a heavy chain variable region set forth in SEQ ID NO:27 and a light chain variable region set forth in SEQ ID NO:25; An antibody having the heavy chain variable region of SEQ ID NO: 23 and the light chain variable region of SEQ ID NO: 21; having the heavy chain variable region of SEQ ID NO: 29 and SEQ ID NO: An antibody of the light chain variable region shown in 25.
  • the invention provides a nucleic acid encoding the aforementioned antibody.
  • the invention provides an expression vector comprising the nucleic acid.
  • the present invention provides a host cell comprising the expression vector or genome of the present invention in which the nucleic acid of the present invention is integrated.
  • the invention provides the use of an antibody according to the invention for the preparation of a targeted drug, antibody drug conjugate or multifunctional antibody which specifically targets tumor cells expressing claudin 18A2 Or for the preparation of a reagent for diagnosing a tumor, the tumor expresses claudin 18A2; or is used to prepare a chimeric antigen receptor-modified immune cell.
  • the tumor expressing claudin 18A2 comprises: gastric cancer, pancreatic cancer, esophageal cancer, lung cancer.
  • the invention provides a chimeric antigen receptor comprising an antibody of the invention comprising a sequence of linked antibodies: an antibody, a transmembrane region and an intracellular signal region of the invention.
  • the intracellular signal region is selected from the group consisting of: CD3 ⁇ , Fc ⁇ RI ⁇ , CD27, CD28, CD137, CD134, MyD88, an intracellular signal sequence of CD40, or a combination thereof; or the transmembrane region comprises Transmembrane region of CD8 or CD28.
  • the chimeric antigen receptor comprises the following sequentially linked antibodies, transmembrane regions and intracellular signaling regions: antibodies of the invention, CD8 and CD3 ⁇ ; antibodies of the invention, CD8, CD137 and CD3 ⁇ Or an antibody of the invention, a transmembrane region of a CD28 molecule, an intracellular signal region of a CD28 molecule, and CD3 ⁇ ; or an antibody of the invention, a transmembrane region of a CD28 molecule, an intracellular signal region of a CD28 molecule, CD137 and CD3 ⁇ .
  • the invention provides a nucleic acid encoding the chimeric antigen receptor. According to another aspect of the invention, the invention provides an expression vector comprising the nucleic acid of the invention. According to another aspect of the invention, the invention provides a virus comprising a vector of the invention.
  • the present invention provides a chimeric antigen receptor, nucleic acid, expression vector or virus of the present invention for preparing chimeric antigen receptor-modified immune cells targeting tumor cells expressing claudin 18A2 the use of.
  • the tumor expressing claudin 18A2 comprises: stomach Cancer, pancreatic cancer, esophageal cancer, lung cancer.
  • the present invention provides a chimeric antigen receptor-modified immune cell transduced with the nucleic acid, expression vector or virus of the present invention; or a surface thereof for expressing the chimeric antigen of the present invention body.
  • the immune cell is: a T lymphocyte, an NK cell, or an NKT lymphocyte.
  • the immune cell further carries a coding sequence for a foreign cytokine; or it also expresses another chimeric antigen receptor that does not contain CD3 ⁇ but contains an intracellular signal of CD28 a domain, an intracellular signal domain of CD137, or a combination of the two; or a chemokine receptor thereof; preferably, said chemokine receptor comprises: CCR; or it is also expressed to reduce PD -1 expressed siRNA or a protein that blocks PD-L1; or endogenous PD-1 in its cells is knocked out by gene editing techniques; or it also expresses a safety switch.
  • the present invention provides use of the chimeric antigen receptor-modified immune cell for producing a tumor suppressing drug, wherein the tumor is a tumor expressing claudin 18A2; preferably, The tumor includes: gastric cancer, pancreatic cancer, esophageal cancer, and lung cancer.
  • the present invention provides a multifunctional immunoconjugate comprising the antibody of the present invention; and a functional molecule linked thereto; the functional molecule selected from the group consisting of: targeting a tumor surface A molecule of a marker, a molecule that inhibits a tumor, a molecule that targets a surface marker of an immune cell, or a detectable label.
  • the molecule that targets a surface marker of an immune cell is an antibody that binds to a T cell surface marker that forms a bifunctional antibody with T cell involvement with the antibody of the invention.
  • the invention provides nucleic acids encoding said multifunctional immunoconjugates and their use for the preparation of anti-tumor drugs.
  • the nucleic acid encoding the multifunctional immunoconjugate is used to prepare a reagent for diagnosing a tumor that expresses claudin 18A2. In some embodiments, the nucleic acid encoding the multifunctional immunoconjugate is used to prepare chimeric antigen receptor modified immune cells.
  • the immune cells comprise: T lymphocytes, NK cells, or NKT lymphocytes.
  • the invention provides a pharmaceutical composition comprising an antibody of the invention or a nucleic acid encoding the antibody.
  • the invention provides a pharmaceutical composition comprising an immunoconjugate of the invention or a nucleic acid encoding the conjugate.
  • the invention provides a pharmaceutical composition comprising a chimeric antigen receptor of the invention or a nucleic acid encoding the chimeric antigen receptor.
  • the invention provides a pharmaceutical composition comprising a chimeric antigen receptor modified immune cell of the invention.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient.
  • kits comprising a container, and a pharmaceutical composition of the invention in a container; or a container, and an antibody of the invention or a nucleic acid encoding the antibody in the container;
  • the immunoconjugate of the present invention or a nucleic acid encoding the same; or the chimeric antigen receptor of the present invention or a nucleic acid encoding the chimeric antigen receptor; or the chimeric antigen of the present invention Body modified immune cells.
  • the invention provides an antigen binding unit comprising a light chain CDR and a heavy chain CDR, wherein the antigen binding unit specifically binds to a claudin 18A2 peptide; and wherein the antigen binding unit does not significantly bind Protein 18A1 peptide.
  • the present invention provides an antigen binding unit comprising a light chain CDR and a heavy chain CDR, wherein the antigen binding unit specifically binds to a claudin 18A2 peptide; and wherein the antigen binding unit and a reference antigen The binding unit showed less non-specific binding to the claudin 18A1 peptide compared to the binding unit.
  • the reference antigen binding unit comprises the light chain of SEQ ID NO:86 or SEQ ID NO:88 and/or the heavy chain amino acid sequence of SEQ ID NO:87 or SEQ ID NO:89.
  • the clathrin 18A2 peptide comprises the amino acid sequence of SEQ ID NO:55.
  • the clathrin 18A1 peptide comprises the amino acid sequence of SEQ ID NO:57.
  • the non-specific binding of the antigen binding unit to the claudin 18A1 peptide does not exceed 20% of its specific binding to the claudin 18A2 peptide.
  • the binding specificity is determined by flow cytometry.
  • the binding specificity is determined by FACS. In some embodiments, the binding specificity is determined by ELISA. In some embodiments, the antigen binding unit binds to the claudin 18A2 peptide with an EC50 of less than about 100 nM. In some embodiments, the antigen binding unit is a monoclonal antibody, a humanized antibody, a chimeric antibody, a multivalent antibody, or a chimeric antigen receptor.
  • the light chain CDR comprises LCDR1, LCDR2 and LCDR3; and the heavy chain CDR comprises HCDR1, HCDR2 and HCDR3; wherein the LCDR1, LCDR2 and LCDR3 respectively have an amino acid sequence selected from the group consisting of: SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54; and wherein said HCDR1, HCDR2 and HCDR3 each have an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 34
  • the LCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:46, and SEQ ID NO:52.
  • the LCDR2 An amino acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 41, SEQ ID NO: 47, and SEQ ID NO: 53.
  • the LCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:35, SEQ ID NO:42, SEQ ID NO:48, and SEQ ID NO:54.
  • the HCDR1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, and SEQ ID NO:49.
  • the HCDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 83, SEQ ID NO : 84 and SEQ ID NO:85.
  • the HCDR3 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, and SEQ ID NO:51.
  • the antigen binding unit is scFv, Fv, Fab or (Fab)2.
  • the invention provides an antigen binding unit comprising a light chain CDR and a heavy chain CDR, wherein said light chain CDR comprises LCDR1, LCDR2 and LCDR3; and said heavy chain CDR comprises HCDR1, HCDR2 and HCDR3;
  • the LCDR1, LCDR2 and LCDR3 comprise an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54;
  • the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO:
  • the light chain CDR comprises LCDR1, LCDR2 and LCDR3; and the heavy chain CDR comprises HCDR1, HCDR2 and HCDR3; the LCDR1, LCDR2 and LCDR3 respectively have an amino acid sequence selected from the group consisting of: SEQ ID NO :34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48 SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54; and the HCDR1, HCDR2 and HCDR3 each have an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO:
  • the LCDR1 comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46, and SEQ ID NO: 52 .
  • the LCDR2 comprises an amino acid sequence selected from the group consisting of The amino acid sequence having at least 80% identity: SEQ ID NO: 34, SEQ ID NO: 41, SEQ ID NO: 47, and SEQ ID NO: 53.
  • the LCDR3 comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO: 48, and SEQ ID NO: 54 .
  • the HCDR1 comprises an amino acid sequence comprising at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 43 and SEQ ID NO: 49.
  • the HCDR2 comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID NO: 50 SEQ ID NO:83, SEQ ID NO:84 and SEQ ID NO:85.
  • the HCDR3 comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45, and SEQ ID NO: 51 .
  • the antigen binding unit is a monoclonal antibody, a humanized antibody, a chimeric antibody, a multivalent antibody, or a chimeric antigen receptor.
  • the antigen binding unit is scFv, Fv, Fab or (Fab)2.
  • the invention provides a chimeric antigen receptor comprising an extracellular antigen binding unit, a transmembrane domain and an intracellular domain, wherein the extracellular antigen binding unit comprises an antigen binding unit of the invention.
  • the invention provides a composition comprising an antigen binding unit or a chimeric antigen receptor of the invention.
  • the composition comprises a Type I interferon.
  • the invention provides an isolated nucleic acid encoding an antigen binding unit or chimeric antigen receptor of the invention, and optionally a type I interferon.
  • the invention provides a vector comprising a nucleic acid of the invention.
  • the invention provides a host cell which expresses an antigen binding unit or chimeric antigen receptor of the invention, and optionally a type I interferon.
  • the invention provides a host cell comprising a nucleic acid binding unit or chimeric antigen receptor of the invention, and optionally a nucleic acid of type I interferon.
  • the host cell is an immune response cell.
  • the host cell is a T cell, a natural killer cell, a cytotoxic T lymphocyte, a natural killer T cell, a DNT cell, and/or a regulatory T cell.
  • the host cell is an NK92 cell.
  • the host cell is cytotoxic to a cell comprising a claudin 18A2 peptide comprising the amino acid sequence of SEQ ID NO:55. In some embodiments, the host cell does not have significant cytotoxicity to a cell comprising a claudin 18A1 peptide that does not comprise a claudin 18A2 peptide, the clathrin 18A1 peptide comprising the amino acid sequence of SEQ ID NO: 57, and The clathrin 18A2 peptide comprises the amino acid sequence of SEQ ID NO:55.
  • the invention provides for the production of an antigen binding unit or chimeric antigen of the invention
  • a method of a body or composition comprising: culturing a host cell of the invention under suitable conditions and obtaining an expression product of said host cell.
  • the invention provides a method of inducing cell death comprising a claudin 18A2 peptide comprising contacting the cell with an antigen binding unit, chimeric antigen receptor, composition or host cell of the invention.
  • the cell is contacted with the antigen binding unit, the chimeric antigen receptor, the composition or host cell in vitro.
  • the cell is contacted with the antigen binding unit, the chimeric antigen receptor, the composition or host cell in vivo.
  • the cell is a cancer cell.
  • the cell is a solid tumor cell.
  • the cell is selected from the group consisting of: gastric cancer cells, esophageal cancer cells, intestinal cancer cells, pancreatic cancer cells, nephroblastoma cells, lung cancer cells, ovarian cancer cells, colon cancer cells, rectal cancer cells, liver cancer cells , head and neck cancer cells, chronic myeloid leukemia cells and gallbladder cancer cells.
  • the invention provides a method of treating a tumor in an individual in need thereof, the method comprising administering to the individual an effective amount of an antigen binding unit, chimeric antigen receptor, composition or host cell of the invention .
  • the tumor is a solid tumor.
  • the tumor is gastric cancer, esophageal cancer, intestinal cancer, pancreatic cancer, nephroblastoma, lung cancer, ovarian cancer, colon cancer, rectal cancer, liver cancer, head and neck cancer, chronic myelogenous leukemia, or gallbladder cancer.
  • the method further comprises administering to the individual an additional therapeutic agent.
  • the additional therapeutic agent is at least one selected from the group consisting of epirubicin, oxaliplatin, and 5-fluorouracil.
  • Figure 1A shows the identity comparison of claudin 18A2 (SEQ ID NO: 55) and claudin 18A1 (SEQ ID NO: 57);
  • Figure 1B shows hybridoma supernatants 2B1, 3E12 as determined by flow cytometry, Binding of 4A11, and 8E5 to HEK293 cells stably transfected with human CLD18A2 and CLD18A1 cells.
  • Figure 2 shows murine anti-2B1 (heavy chain variable region SEQ ID NO: 3, light chain variable region SEQ ID NO: 1), 3E12 (heavy chain variable region SEQ ID NO: 7, light chain variable region SEQ ID NO: 5), 4A11 (heavy chain variable region SEQ ID NO: 11, light chain variable region SEQ ID NO: 9), 8E5 (heavy chain variable region SEQ ID NO: 15, light chain variable region SEQ Sequence alignment of ID NO: 13).
  • Figure 3 shows the binding affinity of the mouse anti-2B1, 8E5 ScFv to the HEK293 cells stably transfected with human CLD18A2 after chimeric with the human IgG1 Fc portion.
  • Figure 4 shows the binding relative affinities of the engineered murine anti-2B1 antibody 2B1-N52D, 2B1-S54A, and human IgG1 Fc, after binding to HEK293 cells stably transfected with human CLD18A2.
  • Figure 5 shows the binding affinity of humanized hu2B1-S54A to HEK293 cells stably transfected with human CLD18A2 after chimeric with human IgG1 Fc.
  • Figure 6 shows the binding affinity of humanized hu8E5 to human HELD3 cells stably transfected with human CLD18A2 after chimeric with human IgGl Fc.
  • Figure 7 shows the binding relative affinities of engineered humanized hu8E5-2I to HEK293 cells stably transfected with human CLD18A2.
  • Figure 8A compares the CDC effect of the humanized antibodies hu2B1-S54A, hu8E5-2I and the known chimeric antibody ch-163E12 (see CN103509110A) on HEK293 cells transfected with CLD18A2;
  • Figure 8B compares the humanized antibody hu2B1- CDC results of S54A, hu8E5-2I and ch-163E12 against HEK293 cells transfected with CLD18A1.
  • Figure 9 compares the ADCC effects of the humanized antibodies hu2B1-S54A, hu8E5-2I and the chimeric antibodies ch-163E12, ch-175D10 (see CN103509110A).
  • Figure 10 compares the killing activity of hu8E5-2I and ch-175D10 in mice.
  • Figure 11 compares the in vitro killing activity of hu8E5-28Z, hu8E5-BBZ and hu8E5-28BBZ T cells on different cell lines.
  • Figure 12 compares the in vitro killing activity of hu8E5-28Z, hu8E5-2I-28Z, and hu2B1-hs54A T cells against different cell lines.
  • Figure 13 depicts a comparison of the effect of CLDN18A2-CAR T on tumor volume over time in a subcutaneous xenograft model of gastric cancer PDX mice (Figure 13A) and a comparison of tumor photographs ( Figure 13B).
  • Figure 14 shows the results of secretion assay of cytokines of hu8E5-28Z and hu8E5-2I-28Z.
  • Figure 15 depicts a comparison of the effect of CLDN18A2-CAR T on tumor volume over time in a subcutaneous xenograft model of gastric cancer BGC-823-A2 in mice (Figure 15A), tumor weight comparison (Figure 15B), and tumor photo comparison (Fig. 15C).
  • Figure 16 shows the tumor infiltration of CLDN18A2-CAR T.
  • Figure 17A shows the secretion of cellular molecules after co-expression of IFN
  • Figure 17B is a comparison of anti-tumor activity of CAR-T cells containing IFN and no IFN in subcutaneous xenografts of gastric cancer PDX
  • Figure 17C is mouse peripheral blood Comparison of the number of viable cells in the 5, 7 and 10 days of returning CAR-T cells.
  • Figure 18A and Figure 18B are plasmid maps for the construction of CAR-NK cells.
  • Figure 19 shows the determination of the positive rate of hu8E5-2I-28Z CAR-NK92 and hu8E5-28BBZ CAR-NK92.
  • Figure 20 is a graph showing the cytotoxicity of hu8E5-2I-28Z CAR-NK92 cytotoxicity.
  • Figure 21 is a graph showing the cytotoxicity of hu8E5-28BBZ CAR-NK92 cytotoxicity.
  • activated and “activated” are used interchangeably herein and they, as well as other grammatical forms thereof, may refer to the process by which a cell transitions from a quiescent state to an active state.
  • the process can include a response to a phenotypic or genetic change in the antigen, migration, and/or functional activity state.
  • activation can refer to the process by which immune cells are gradually activated.
  • T cells may require at least two signals to be fully activated. The first signal can occur after the TCR is bound by the antigen-MHC complex, while the second signal can occur by the conjugation of costimulatory molecules (see co-stimulatory molecules listed in Table 1).
  • anti-CD3 can simulate the first
  • the signal, anti-CD28 can simulate the second signal.
  • engineered T cells can be activated by the expressed CAR.
  • immune cell activation or activation may refer to a state that has been sufficiently stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function.
  • co-stimulatory molecule refers to a homologous binding partner on an immune cell, such as a T cell, that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response, such as, but not limited to, proliferation.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that promotes an effective immune response.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) and 4-1BB (CD137).
  • costimulatory molecules include, but are not limited to, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1 , CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, C
  • co-stimulatory signal refers to a signal that, in combination with a first signal, such as TCR/CD3, results in T cell proliferation and/or up- or down-regulation of key molecules.
  • antigen binding unit refers to an immunoglobulin molecule and an immunologically active portion of an immune molecule, ie, a molecule containing an antigen binding site that specifically binds to an antigen ("immune response").
  • immunoglobulin molecules of various species including invertebrates and vertebrates. Structurally, the simplest naturally occurring antibody (eg, IgG) comprises four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Immunoglobulins represent a large family of molecules including several types of molecules, such as IgD, IgG, IgA, IgM, and IgE.
  • immunoglobulin molecule includes, for example, hybrid antibodies or altered antibodies and fragments thereof. It has been shown that the antigen binding function of antibodies can be carried out by fragments of naturally occurring antibodies. These fragments are collectively referred to as "antigen combining units.” Also included in the term “antigen binding unit” is any polypeptide chain-containing molecular structure having a specific shape that conforms to an epitope and recognizes an epitope, wherein one or more non-covalent binding interactions stabilize the structure between the molecule and the epitope Complex.
  • antigen-binding unit examples include a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains, A bivalent fragment (F(ab)2 fragment) comprising two Fab fragments joined by a disulfide bridge on the hinge region; an Fd fragment consisting of a VH and CH1 domain, by the one-armed VL and VH domains of the antibody A Fv fragment consisting of; a dAb fragment consisting of a VH domain (Ward et al, Nature, 341: 544-546, 1989); and an isolated complementarity determining region (CDR) or any fusion protein comprising such an antigen binding unit.
  • Fab fragment a monovalent fragment consisting of VL, VH, CL, and CH1 domains
  • F(ab)2 fragment comprising two Fab fragments joined by a disulfide bridge on the hinge region
  • Fd fragment consisting of a VH and CH1 domain, by the one-armed VL and VH domains of
  • antibody as used herein includes intact antibodies and any antigen-binding fragments (ie, "antigen-binding portions") or single chains thereof.
  • a naturally occurring "antibody” is a glycoprotein comprising at least 2 heavy (H) chains and 2 light (L) chains joined by a disulfide bond.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain CL.
  • VH and VL regions can be further subdivided into regions of high variability called complementarity determining regions (CDRs) separated by a more conserved region called the framework region (ER).
  • CDRs complementarity determining regions
  • ER framework region
  • Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with the antigen.
  • the constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.
  • scFv refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein said light and heavy chain variable regions are contiguous (for example, via a synthetic linker such as a short flexible polypeptide linker), and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker such as a short flexible polypeptide linker
  • an scFv can have the VL and VH variable regions in any order (eg, relative to the N-terminus and C-terminus of the polypeptide), and the scFv can include a VL-linker-VH or A VH-linker-VL can be included.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in the light chain variable region.
  • An antigen binding unit “specifically binds" to an antigen or is “immunoreactive” with an antigen if the antigen binding unit binds to the antigen with greater affinity or affinity than binding to other reference antigens, including polypeptides or other substances. ".
  • humanized for use in a non-human antibody, such as a rodent or primate, is a hybrid immunoglobulin, immunoglobulin chain or the like comprising a minimal sequence derived from a non-human immunoglobulin Fragment.
  • the humanized antibody is a human immunoglobulin (receptor antibody) in which the complement is derived from the receptor.
  • the residues of the decision region (CDR) are replaced by residues from the CDRs of a non-human species (donor antibody) having the desired specificity, affinity and properties, such as mice, rats, rabbits or primates.
  • the Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • a humanized antibody may comprise residues that are not present in the recipient antibody or in the introduced CDR or framework sequences. These modifications are made to further improve and optimize antibody performance and minimize immunogenicity when introduced into the human body.
  • a humanized antibody will comprise substantially all of at least one, typically two variable domains, wherein all or substantially all of the CDR regions correspond to non-human immunoglobulins, and all or substantially All FR regions are regions of human immunoglobulin sequences.
  • a humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin.
  • a "humanized antibody” can include a mutation, such as a mutation introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo.
  • immunoglobulin may refer to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as chimeric antigen receptors or antigen receptors. The five members included in such proteins are IgA, IgG, IgM, IgD and IgE, with IgG being the most common circulating antibody. It is the most potent immunoglobulin in agglutination, complement fixation and other antibody reactions and is important in protecting against bacteria and viruses. For example, tumor cell antigens (or “tumor antigens”) or pathogen antigens can be identified by CAR.
  • tumor cell antigens or "tumor antigens”
  • pathogen antigens can be identified by CAR.
  • isolated refers to separation from cellular components or other components in which polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof are generally associated in a natural state.
  • non-naturally occurring polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof need not be “isolated” to distinguish them from naturally occurring counterparts.
  • a "concentrated”, “isolated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody or fragment thereof may be distinguished from its naturally occurring counterpart, since the concentration or amount per molecule is greater (“concentrated ”) or less (“diluted") the concentration of its naturally occurring counterpart.
  • the degree of enrichment can be measured on an absolute basis, such as the weight per solution volume, or can be measured relative to another potential interferent present in the source mixture.
  • the technical solution of the present invention preferably has a higher degree of enrichment.
  • 2-fold enrichment more preferably 10-fold enrichment, more preferably 100-fold enrichment, more preferably 1000-fold enrichment is preferred.
  • "Separated” materials can also be provided by artificial assembly methods, such as by chemical synthesis or recombinant expression.
  • antigen refers to a substance that is recognized and specifically bound by an antigen binding unit.
  • Antigens can include peptides, proteins, glycoproteins, polysaccharides, and lipids, portions thereof, and combinations thereof.
  • Non-limiting exemplary antigens include tumor antigens or pathogen antigens.
  • Antigen can also refer to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immunologically-competent cells, or both. Those skilled in the art will appreciate that any macromolecule, including virtually all proteins or peptides, Both can be used as antigens.
  • polypeptide peptide and protein are used interchangeably herein to refer to a polymer of amino acids of any length.
  • the polymer may be linear, cyclic or branched, it may comprise modified amino acids, particularly conservatively modified amino acids, and it may be interrupted by non-amino acids.
  • modified amino acid polymers such as those which have been subjected to sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, elimination.
  • amino acid refers to natural and/or non-natural or synthetic amino acids, including glycine and D or L optical isomers, as well as amino acid analogs and peptidomimetics.
  • a polypeptide or amino acid sequence "derived from" a specified protein refers to the source of the polypeptide. The term also encompasses polypeptides expressed by a specified nucleic acid sequence.
  • amino acid modification includes amino acid substitutions, insertions and/or deletions in a polypeptide sequence.
  • amino acid substitution or “substitution” herein is meant the replacement of an amino acid at a particular position in the parent polypeptide sequence with another amino acid.
  • substituting R94K means that the arginine at position 94 is replaced by lysine.
  • substitution at the same position in the parent polypeptide sequence it can also be represented by 94K, i.e., replacing the 94 position with lysine.
  • multiple substitutions are typically separated by slashes.
  • R94K/L78V refers to a double variant comprising the substitutions R94K and L78V.
  • amino acid insertion or “insertion” means the addition of an amino acid at a particular position in the parent polypeptide sequence.
  • insert -94 indicates an insertion at 94 bits.
  • amino acid deletion or “deletion” means the removal of an amino acid at a particular position in the parent polypeptide sequence.
  • R94- indicates deletion of arginine at position 94.
  • conservative modification or “conservative sequence modification” as used herein means an amino acid modification that does not significantly affect or alter the desired activity or property of a peptide containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions, and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced with amino acid residues having similar side chains. A family of amino acid residues having similar side chains has been defined in the art.
  • amino acids containing basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged acute side chains eg, , glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan
  • non-polar side chains eg, alanine, valine, leucine, isoleucine
  • Acid, proline, phenylalanine, methionine ⁇ -branched side chains (eg, threonine, valine, isoleucine)
  • aromatic side chains eg, tyrosine, benzene
  • a sample eg, a cell
  • administered e.g, a patient
  • the autologous process differs from the allogeneic process in which the donor and recipient are different individuals.
  • xenograft and other grammatical forms thereof can include any procedure in which a recipient, a donor, and a donor are different species, transplanting, implanting, or infusing a cell, tissue, or organ into a recipient. Transplantation of cells, organs and/or tissues described herein can be used for xenografting into humans.
  • Xenografts include, but are not limited to, vascularized xenografts, partially vascularized xenografts, non-vascularized xenografts, xenogeneic dressings, xenogenic bandages, and xenogenic structures.
  • allogeneic transplantation may include transplantation of cells, tissues or organs in which the recipient and donor are the same species but different individuals, implanted. Or infusion to any of the recipients. Transplantation of cells, organs and/or tissues described herein can be used for allogeneic transplantation into humans. Allografts include, but are not limited to, vascularized allografts, partially vascularized allografts, non-vascularized allografts, allogeneic dressings, allogeneic bandages, and allogeneic structures.
  • autologous transplantation and other forms of grammar thereof (eg, autologous transplantation) may include transplantation, implantation or infusion of a cell, tissue or organ into a recipient in which the recipient and donor are the same individual. Any program in . Transplantation of cells, organs and/or tissues described herein can be used for autologous transplantation into the human body. Autologous transplantation includes, but is not limited to, vascular autologous transplantation, partial vascular autologous transplantation, non-vascularized autografts, autologous dressings, autologous bandages, and autologous structures.
  • chimeric antigen receptor or "CAR” as used herein refers to an engineered molecule that can be expressed by immune cells including, but not limited to, T cells or NK cells. CAR is expressed in T cells and T cells can be redirected to induce specific killing of target cells by a human receptor.
  • the extracellular binding domain of CAR can be derived from a murine, humanized or fully human monoclonal antibody.
  • epitope and its grammatical other forms as used herein may refer to a portion of an antigen that can be recognized by an antibody, B cell, T cell, or engineered cell.
  • an epitope can be a tumor epitope or a pathogen epitope recognized by a TCR. Multiple epitopes within the antigen can also be identified. Epitopes can also be mutated.
  • Cell line or “cell culture” means a bacterium, plant, insect or higher eukaryotic cell that is grown or maintained in vitro.
  • the progeny of the cell may not be identical (in morphology, genotype or phenotype) to the maternal cell.
  • engineered and its grammatical other forms as used herein may refer to one or more changes in a nucleic acid, such as a nucleic acid within the genome of an organism.
  • engineered can refer to alterations, additions, and/or deletions of genes.
  • Engineered cells can also refer to cells having genes that are added, deleted, and/or altered. Engineering cells are also available Refers to cells that express CAR.
  • transfection refers to the introduction of an exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by a variety of means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran mediated transfection, polyamine transduce transfection, electroporation, microinjection, Liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.
  • stable transfection or “stable transfection” refers to the introduction and integration of exogenous nucleic acids, DNA or RNA into the genome of a transfected cell.
  • stable transfectant refers to a cell that stably integrates foreign DNA into genomic DNA.
  • nucleic acid molecule encoding refers to the order or sequence of deoxyribonucleotides along a deoxyribonucleic acid strand. The order of these deoxyribonucleotides determines the order of the amino acids along the polypeptide (protein) chain. Thus, the nucleic acid sequence encodes an amino acid sequence.
  • the term "individual” as used herein refers to any animal, such as a mammal or marsupial. Individuals of the invention include, but are not limited to, humans, non-human primates (e.g., rhesus or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and poultry of any kind.
  • non-human primates e.g., rhesus or other types of macaques
  • mice pigs, horses, donkeys, cows, sheep, rats, and poultry of any kind.
  • peripheral blood lymphocytes and other grammatical forms thereof as used herein may refer to lymphocytes circulating in blood (eg, peripheral blood).
  • Peripheral blood lymphocytes can refer to lymphocytes that are not limited to organs.
  • Peripheral blood lymphocytes can comprise T cells, NK cells, B cells, or any combination thereof.
  • immune response cell may refer to a cell that can elicit an immune response.
  • An immune response cell can also refer to a cell of the lymphoid or myeloid lineage.
  • immune cells include, but are not limited to, T cells, such as alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytic cells, Respective precursor cells and their progeny.
  • T cell and its grammatical other forms as used herein may refer to T cells of any origin.
  • the T cell can be a primary T cell such as an autologous T cell or the like.
  • T cells can also be human or non-human.
  • T cell activation or “T cell trigger” and other grammatical forms thereof as used herein may refer to a T cell that is sufficiently stimulated to induce detectable cell proliferation, cytokine production, and/or detectable effector function. status. In some embodiments, "complete T cell activation” can be similar to triggering cytotoxicity of T cells.
  • T cell activation can be measured using various assays known in the art.
  • the assay can be an ELISA for measuring cytokine secretion, ELISPOT, a flow cytometry assay for measuring intracellular cytokine expression (CD107), a flow cytometry assay for measuring proliferation, and for determining target cell elimination. Cytotoxicity assay (51Cr release assay).
  • the assay typically uses a control (non-engineered cell) and Engineered cells (CART) were compared to determine the relative activation of engineered cells compared to controls. Furthermore, the assay can be compared to engineered cells that are incubated or contacted with target cells that do not express the target antigen. For example, the comparison can be a comparison with CD19-CART cells incubated with target cells that do not express CD19.
  • sequence and its grammatical other forms as used herein, when used in reference to a nucleotide sequence, may include DNA or RNA, and may be single-stranded or double-stranded.
  • the nucleic acid sequence can be mutated.
  • the nucleic acid sequence can be of any length, for example, a nucleic acid having a length of from 2 to 1,000,000 or more nucleotides (or any integer value therebetween or above), for example, from about 100 to about 10,000 nucleotides in length or from about 200 to about 500. Nucleotides.
  • an effective amount refers to an amount that provides a therapeutic or prophylactic benefit.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed.
  • the expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by host cells or in vitro expression systems.
  • Expression vectors include those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).
  • lentivirus refers to the genus of the family Retroviridae. Retroviruses are unique in retroviruses in their ability to infect non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are all examples of lentiviruses. Vectors derived from lentivirus provide a means to achieve significant levels of gene transfer in vivo.
  • operably linked refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter.
  • first nucleic acid sequence when the first nucleic acid sequence is functionally related to the second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects transcription or expression of the coding sequence.
  • the operably linked DNA sequences are contiguous and, where necessary, ligated two protein coding regions in the same reading frame.
  • promoter is defined as a DNA sequence that is recognized by the synthetic machinery or the introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence.
  • vector is a composition comprising an isolated nucleic acid and which can be used to deliver an isolated nucleic acid to the interior of a cell.
  • vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses.
  • vector includes autonomously replicating plasmids or viruses.
  • the term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.
  • a "host cell” includes an individual cell or cell culture that can be or has been an acceptor of a target vector.
  • Host cells include the progeny of a single host cell. Due to natural, accidental or intentional mutations, progeny may not necessarily be identical to the original parental cell, for example, in morphological or genomic DNA or total DNA complement.
  • Host cells include cells that are transfected in vivo with the vectors of the invention.
  • "Host cell” may refer to a prokaryotic cell, a eukaryotic cell, or a cell line cultured as a single cell entity that may or has been used as a recipient of a recombinant vector or other transfer polynucleotide, and includes progeny progenitor cells that have been transfected.
  • sequence identity determines the percent identity by comparing two best matched sequences over a comparison window (eg, at least 20 positions), wherein portions of the polynucleotide or polypeptide sequence in the comparison window can comprise Addition or deletion (ie gap), for example 20% or less gap (eg 5 to 15%, or 10 to 12) compared to the reference sequence (which does not contain additions or deletions) for the best matched two sequences %).
  • a comparison window eg, at least 20 positions
  • portions of the polynucleotide or polypeptide sequence in the comparison window can comprise Addition or deletion (ie gap), for example 20% or less gap (eg 5 to 15%, or 10 to 12) compared to the reference sequence (which does not contain additions or deletions) for the best matched two sequences %).
  • the percentage is usually calculated by determining the number of positions in which the same nucleic acid base or amino acid residue occurs in both sequences to produce the number of correctly matched positions, dividing the number of correctly matched positions by the total number of positions in the reference sequence ( That is, the window size), and multiply the result by 100 to produce a percentage of sequence identity.
  • disease refers to any alteration or disorder that impairs or interferes with the normal function of a cell, tissue or organ.
  • disease includes, but is not limited to, a tumor, a pathogen infection, an autoimmune disease, a T cell dysfunction disease, or a defect in immune tolerance (eg, transplant rejection).
  • exogenous refers to a nucleic acid molecule or polypeptide that is not endogenously expressed in a cell, or that has a level of expression insufficient to achieve overexpression.
  • exogenous includes recombinant nucleic acid molecules or polypeptides expressed in a cell, such as exogenous, heterologous and overexpressed nucleic acid molecules and polypeptides.
  • modulation refers to a positive or negative change. Modification examples include 1%, 2%, 10%, 25%, 50%, 75%, or 100% variation.
  • treatment refers to a clinical intervention in an attempt to alter a disease caused by an individual or a cell, both prophylactically and in a clinical pathological process.
  • Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the progression of the disease, improving or ameliorating the condition, alleviating or improving the prognosis.
  • induced expression refers to expression under conditions such as when T cells bind to an antigen.
  • T cells bind to an antigen.
  • One skilled in the art how to perform conventional "induced expression”.
  • antigen binding units comprising a light chain CDR and a heavy chain CDR, wherein the antigen binding unit specifically binds to a claudin 18A2 peptide; and wherein the antigen binding unit The claudin 18A1 peptide was not significantly bound.
  • antigen binding units comprising a light chain CDR and a heavy chain CDR, wherein the antigen binding unit specifically binds to a claudin 18A2 peptide; and wherein the antigen binding unit is associated with a reference antigen binding unit This shows less non-specific binding to the claudin 18A1 peptide.
  • an antigen binding unit described herein comprises a light chain CDR.
  • the light chain CDR can be a complementarity determining region of an antigen binding unit.
  • the light chain CDRs may comprise a contiguous sequence of amino acid residues, or two or more contiguous sequence of amino acid residues flanked by, and optionally flanking, non-complementarity determining regions, such as framework regions.
  • the light chain CDR comprises two or more light chain CDRs, which may be referred to as light chain CDR-1, CDR-2, and the like.
  • the light chain CDR comprises three light chain CDRs, which may be referred to as light chain CDR-1 (LCDR1), light chain CDR-2 (LCDR2), and light chain CDR-3 (LCDR3), respectively.
  • LCDR1 light chain CDR-1
  • LCDR2 light chain CDR-2
  • LCDR3 light chain CDR-3
  • a set of CDRs present on a common light chain can be collectively referred to as a light chain CDR.
  • an antigen binding unit described herein comprises a heavy chain CDR.
  • the heavy chain CDR can be a complementarity determining region of an antigen binding unit.
  • the heavy chain CDRs may comprise a contiguous sequence of amino acid residues, or two or more contiguous sequence of amino acid residues separated by non-complementarity determining regions, such as framework regions, and optionally flanking.
  • the heavy chain CDR comprises two or more heavy chain CDRs, which may be referred to as heavy chain CDR-1, CDR-2, and the like.
  • the heavy chain CDR comprises three heavy chain CDRs, which may be referred to as heavy chain CDR-1 (HCDR1), heavy chain CDR-2 (HCDR2), and heavy chain CDR-3 (HCDR3), respectively.
  • HCDR1 heavy chain CDR-1
  • HCDR2 heavy chain CDR-2
  • HCDR3 heavy chain CDR-3
  • a set of CDRs present on a common heavy chain can be collectively referred to as a heavy chain CDR.
  • antigen binding units comprising a light chain CDR and a heavy chain CDR, wherein the light chain CDR comprises LCDR1, LCDR2 and LCDR3; and the heavy chain CDR comprises HCDR1, HCDR2 and HCDR3;
  • the amino acid sequences of LCDR1, LCDR2 and LCDR3 are at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54;
  • the amino acid sequences of HCDR1, HCDR2 and HCDR3 are at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO:
  • antigen binding units comprising a light chain CDR and a heavy chain CDR, wherein the light chain CDR comprises LCDR1, LCDR2 and LCDR3; and the heavy chain CDR comprises HCDR1, HCDR2 and HCDR3; wherein the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, and the amino acid sequence selected from the group consisting of 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% Or 99.9% identity: SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO:
  • the light chain CDR comprises LCDR1, LCDR2 and LCDR3; and the heavy chain CDR comprises HCDR1, HCDR2 and HCDR3; wherein the LCDR1, LCDR2 and LCDR3 respectively have a selected from The following amino acid sequences: SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 46, SEQ ID NO And SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54; SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO: 34, SEQ ID NO: 35,
  • the LCDR1 comprises at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83% with an amino acid sequence selected from the group consisting of , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5 Amino acid sequences of %, or 99.9% identity: SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46, and SEQ ID NO: 52.
  • the LCDR2 comprises at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83% with an amino acid sequence selected from the group consisting of , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5 %, or 99.9% identity ammonia
  • Base acid sequence SEQ ID NO: 34, SEQ ID NO: 41, SEQ ID NO: 47, and SEQ ID NO: 53.
  • antigen binding units wherein the LCDR3 comprises at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83% with an amino acid sequence selected from the group consisting of , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5 Amino acid sequences of %, or 99.9% identity: SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO: 48, and SEQ ID NO: 54.
  • antigen binding units wherein the HCDR1 comprises at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83 comprising an amino acid sequence selected from the group consisting of %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, Amino acid sequences of 99.5%, or 99.9% identity: SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 43 and SEQ ID NO: 49.
  • antigen binding units wherein the HCDR2 comprises at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83% with an amino acid sequence selected from the group consisting of , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5 Amino acid sequences of %, or 99.9% identity: SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID NO: 50, SEQ ID NO: 83, SEQ ID NO: 84, and SEQ ID NO :85.
  • antigen binding units wherein the HCDR3 comprises at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83% with an amino acid sequence selected from the group consisting of , 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5 Amino acid sequences of %, or 99.9% identity: SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45, and SEQ ID NO: 51.
  • an antigen binding unit of the invention binds to a claudin 18A2 or a claudin 18A2 peptide.
  • the term "Minolin 18A2" or “Minolin 18A2 peptide” herein may also refer to a homologue, direct line of the known claudin 18A2 sequence.
  • the claudin 18A2 or claudin 18A2 peptide is a peptide having the GenBank accession number NP_001002026 (mRNA: NM_001002026). In some embodiments, the claudin 18A2 or claudin 18A2 peptide is a peptide comprising the amino acid sequence of SEQ ID NO:55.
  • the antigen binding unit of the invention does not significantly bind to the claudin 18A1 peptide.
  • the term "Minolin 18A1" or “Minolin 18A1 peptide” (CLD18A1, CLD18.1, CLDN18A1, CLDN18.1, Claudin18.1 or Claudin18A1) herein may also refer to a homologue of the known claudin 18A1 sequence, An ortholog, an interspecies homolog, a codon optimized form, a truncated form, a fragmented form, a mutated form, or any other known derivative form, such as a post-translational modification variant.
  • the claudin 18A1 or claudin 18A1 peptide is a peptide having the GenBank accession number NP_057453 (mRNA: NM_016369). In some embodiments, the claudin 18A1 or claudin 18A1 peptide is a peptide comprising the amino acid sequence of SEQ ID NO:57.
  • Binding specificity can be determined by complementarity determining regions or CDRs, such as light chain CDRs or heavy chain CDRs. In many cases, binding specificity is determined by light chain CDRs and heavy chain CDRs. A given heavy chain CDR, light chain CDR, or combination thereof, provides a given binding pocket with greater affinity and/or specificity to claudin 18A2 than other reference antigens or reference peptides.
  • Binding of the antigen binding unit to the claudin 18A2 peptide can be characterized or expressed by any method known in the art.
  • binding can be characterized by binding affinity, which can be the strength of the interaction between the antigen binding unit and the antigen.
  • Binding affinity can be determined by any method known in the art, such as in vitro binding assays. For example, when assayed in an in vitro binding assay using cells expressing claudin 18A2, the binding affinity of the antigen binding units disclosed herein can be determined.
  • the binding affinity of the test antigen binding unit can be expressed by Kd, which is the equilibrium dissociation constant between the antibody and its respective antigen.
  • the antigen binding units disclosed herein specifically bind to claudin 18A2, with Kd ranging from about 10 [mu]M to about 1 mM.
  • the antigen binding unit can specifically bind to clathrin 18A2 with a Kd of less than about 10 ⁇ M, 1 ⁇ M, 0.1 ⁇ M, 10 nM, 1 nM, 0.1 nM, 10 pM, 1 pM, 0.1 pM, 10 fM, 1 fM, or less than 0.1 fM.
  • the antigen binding unit does not exhibit significant binding to a reference peptide.
  • the level of binding of the antigen binding unit to the reference peptide does not exceed 20% of the level of binding of the antigen binding unit to claudin 18A2.
  • the binding level may be 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10 of the binding level of the antigen binding unit to the claudin 18A2. %, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less than 1%.
  • the antigen binding unit herein binds to claudin 18A2 at a level that is 1 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold greater than its binding level to the reference peptide. , 9 times, 10 times or more than 10 times.
  • the reference peptide or reference peptide is a claudin 18A1 peptide.
  • the reference peptide or reference peptide is a peptide comprising the amino acid sequence of SEQ ID NO:57.
  • the reference peptide or reference peptide is the peptide of SEQ ID NO:57.
  • the antigen binding unit herein exhibits less non-specific binding to a reference peptide than a reference antigen binding unit.
  • the antigen binding unit herein is 5%, 10%, 15%, 20%, 25%, 30% lower than the non-specific binding level of the reference antigen binding unit to the reference peptide, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.
  • the non-specific binding level of the antigen binding unit to the reference peptide herein is one, two, three, four, five, six, seven, and eight times lower than the binding level of the reference antigen binding unit. Times, 9 times, 10 times or more than 10 times.
  • the reference antigen binding unit comprises the light chain of SEQ ID NO:86 or SEQ ID NO:88 and/or the heavy chain amino acid sequence of SEQ ID NO:87 or SEQ ID NO:89. In some embodiments, the reference antigen binding unit comprises the amino acid sequence of SEQ ID NO:86. In some embodiments, the reference antigen binding unit comprises the amino acid sequence of SEQ ID NO:87. In some embodiments, the reference antigen binding unit comprises the amino acid sequence of SEQ ID NO:88. In some embodiments, the reference antigen binding unit comprises the amino acid sequence of SEQ ID NO:89. In some embodiments, the reference peptide or reference peptide is a claudin 18A1 peptide. In some embodiments, the reference peptide or reference peptide is a peptide comprising the amino acid sequence of SEQ ID NO:57. In some embodiments, the reference peptide or reference peptide is the peptide of SEQ ID NO:57.
  • the antigen binding unit is cytotoxic to a cell comprising a claudin 18A2 peptide comprising the amino acid sequence of SEQ ID NO:55.
  • the cytotoxicity level is at least 5% when the ratio of the antigen binding unit to the target cell is 20:1, 10:1, 5:1, 3:1, 2.5:1, 1:1 or 1:3. , 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%.
  • the antigen binding unit does not have significant cytotoxicity to a cell comprising a claudin 18A1 peptide comprising no amino acid sequence of SEQ ID NO: 57, and The claudin 18A2 peptide comprises the amino acid sequence of SEQ ID NO:55.
  • the cytotoxicity level is no greater than 10%, 5%, 4%, 3%, 2%, or 1%.
  • antibodies that specifically bind to claudin 18A2 characterized in that the antibody comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31, 32, 33, 37, 38, 39, 43, 44 a heavy chain CDR of 45, 49, 50, 51, 83, 84, 85 or a variant thereof, and/or selected from the group consisting of amino acid sequences SEQ ID NO: 34, 35, 36, 40, 41, 42, 46, 47, 48, 52, 53, 54 or a variant thereof light chain CDR.
  • antibodies selected from the group consisting of (a) an antibody comprising a heavy chain variable region comprising SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID CDR1 of the amino acid sequence of NO:43 or one of SEQ ID NO:49 comprising SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:50, SEQ ID NO:83, CDR2 of the amino acid sequence set forth in SEQ ID NO: 84 or SEQ ID NO: 85, comprising one of SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45 or SEQ ID NO: 51 a CDR3 of an amino acid sequence; (b) an antibody comprising a light chain variable region comprising SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46 or CDR1 of the amino acid sequence shown by one of SEQ ID NO: 52, CDR2 comprising the amino acid sequence shown by one of SEQ ID NO: 35, SEQ ID NO: 41
  • antibodies wherein the CDR1, CDR2, and CDR3 regions of the heavy chain variable region of the antibody are SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, respectively; or SEQ ID NO :37, SEQ ID NO: 38, SEQ ID NO: 39; or SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45; or SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO :51; or SEQ ID NO: 31, SEQ ID NO: 83, SEQ ID NO: 33; or SEQ ID NO: 31, SEQ ID NO: 84, SEQ ID NO: 33; or SEQ ID NO: 49, SEQ ID NO 85, SEQ ID NO: 51; and/or the CDR1, CDR2, CDR3 regions of the light chain variable region of the antibody are SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, respectively; NO: 40, SEQ ID NO: 41, SEQ ID NO: 42;
  • amino acid sequences of the CDR1, CDR2, and CDR3 of the heavy chain of the antibodies of the invention are selected from the group consisting of the amino acid sequences set forth in the following table or variants thereof:
  • HCDR1 HCDR2 HCDR3 A SEQ ID NO: 31 SEQ ID NO:32 SEQ ID NO:33 B SEQ ID NO:37 SEQ ID NO:38 SEQ ID NO:39 C SEQ ID NO:43 SEQ ID NO: 44 SEQ ID NO:45 D SEQ ID NO:49 SEQ ID NO: 50 SEQ ID NO: 51 E SEQ ID NO: 31 SEQ ID NO:83 SEQ ID NO:33 F SEQ ID NO: 31 SEQ ID NO:84 SEQ ID NO:33 G SEQ ID NO:49 SEQ ID NO 85 SEQ ID NO: 51
  • antibodies wherein the amino acid sequences of the light chain CDR1, CDR2 and CDR3 are selected from the group consisting of the amino acid sequences in the following table or variants thereof:
  • an antibody of the invention comprises a heavy chain variable region selected from the group consisting of amino acid sequences of SEQ ID NO: 3, 7, 11, 15, 17, 19, 23, 27, 29, or variants thereof, and/or selected A light chain variable region of the amino acid sequence of SEQ ID NO: 1, 5, 9, 13, 21, 25 or a variant thereof.
  • the heavy chain variable region is SEQ ID NO: 3 or a variant thereof and the light chain variable region is SEQ ID NO: 1 or variant thereof.
  • the heavy chain variable region is SEQ ID NO: 7 or a variant thereof and the light chain variable region is SEQ ID NO: 5 or a variant thereof.
  • the heavy chain variable region is SEQ ID NO: 11 or a variant thereof and the light chain variable region is SEQ ID NO: 9 or variant thereof. In some embodiments, the heavy chain variable region is SEQ ID NO: 15 or a variant thereof and the light chain variable region is SEQ ID NO: 13 or variant thereof. In some preferred embodiments, the amino acid sequences of the heavy chain CDR1, CDR2 and CDR3 are selected from the table below.
  • an antibody of the invention comprises a heavy chain variable region selected from the group consisting of amino acid sequences of SEQ ID NO: 17, 19, or variants thereof.
  • the heavy chain variable region is SEQ ID NO: 17 or a variant thereof and the light chain variable region is SEQ ID NO: 1 or variant thereof.
  • the heavy chain variable region is SEQ ID NO: 19 or a variant thereof and the light chain variable region is SEQ ID NO: 1 or a variant thereof.
  • an antibody of the invention or a functional fragment thereof comprises a heavy chain variable region selected from the amino acid sequence of SEQ ID NO: 23, 27, 29 or variants thereof, and/or is selected from the group consisting of the amino acid sequence SEQ ID NO: a light chain variable region of 21, 25, or a variant thereof.
  • the heavy chain variable region is SEQ ID NO: 23 or a variant thereof and the light chain variable region is SEQ ID NO: 21 or variant thereof.
  • the heavy chain variable region is SEQ ID NO: 27 or a variant thereof and the light chain variable region is SEQ ID NO: 25 or variant thereof.
  • the heavy chain variable region is SEQ ID NO: 29 or a variant thereof and the light chain variable region is SEQ ID NO: 25 or variant thereof.
  • an antigen binding unit or antibody of the invention is also linked to another functional molecule Or fusion. Accordingly, the invention also encompasses multifunctional immunoconjugates so formed.
  • In-frame fusion refers to the joining of two or more ORFs in a manner that maintains the correct reading frame of the original open reading frame (ORF) to form a contiguous longer ORF.
  • ORF open reading frame
  • the resulting recombinant fusion protein is a single protein containing two or more fragments corresponding to the polypeptide encoded by the original ORF (these fragments are usually not so ligated in the natural state).
  • the reading frames are thus contiguous throughout the fusion fragment, the fragments may be physically or spatially separated by, for example, an in-frame joining sequence (eg, "flexon").
  • the functional molecule is for example used for the diagnosis or treatment of a tumor.
  • tumor refers to a disease characterized by pathological hyperplasia of cells or tissues, and its subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, and does not induce or inhibit normal cell proliferation.
  • a tumor can affect a variety of cells, tissues or organs including, but not limited to, selected from the group consisting of bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, Nerve tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, vaginal organs, or tissue or corresponding cells.
  • Tumors include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).
  • the tumor of the present invention may include, but is not limited to, leukemia (such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute granulocyte-monocytic leukemia, Acute monocytic leukemia, acute leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia Disease, heavy chain disease, solid tumors such as sarcoma and cancer (such as fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial sarcoma, lymphangisarcoma, angiosarcoma, lymphatic endothelial sarcoma
  • the "tumor” includes, but is not limited to, pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, Kidney cancer, white Blood disease, multiple myeloma, ovarian cancer, cervical cancer and glioma.
  • the functional molecule includes, for example, a tumor antigen, such as a tumor specific antigen (TSA) or a tumor associated antigen (TAA).
  • TSA tumor specific antigen
  • TAA tumor associated antigen
  • TAA-associated antigen is not unique to tumor cells, but is expressed on normal cells under conditions in which the immune tolerance state to the antigen cannot be induced. Expression of the antigen on the tumor can occur under conditions that enable the immune system to respond to the antigen.
  • TAA may be antigens expressed on normal cells during fetal development, or they may be antigens that are normally present at very low levels on normal cells but are expressed at higher levels on tumor cells. .
  • TSA or TAA antigens include the following: differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2, and tumor-specific multicenter antigens Such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutant tumor suppressor genes such as p53, Ras, HER-2/neu Unique tumor antigens caused by chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein Barr virus antigen EBVA and human papilloma Virus (HPV) antigens E6 and E7, etc.
  • differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel17),
  • the "tumor antigen” includes, but is not limited to, prostate specific membrane antigen (PSMA), carcinoembryonic antigen (CEA), IL13Ralpha, HER-2, CD19, NY-ESO-1, HIV- 1Gag, Lewis Y, MART-1, gp100, tyrosinase, WT-I, hTERT, mesothelin, EGFR, EGFRvIII, phosphatidylinositol 3, EphA2, HER3, EpCAM, MUC1, MUC16, folic acid Receptors, CLDN6, CD30, CD138, ASGPR1, CDH16, GD2, 5T4, 8H9, ⁇ v ⁇ 6 integrin, B cell mature antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD20, CD22, ⁇ light chain (kappa light chain), CD33, CD38, CD44, CD44v6, CD44v7/8, CD
  • PSMA
  • the functional molecule is an interferon.
  • the interferon is a type I interferon.
  • type I interferon as used herein includes IFN ⁇ , IFN ⁇ , and IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ and the like. All type I interferons bind to specific cell surface receptors (so-called IFN-[alpha]/[beta] receptors) consisting of two strands of IFNARl and IFNAR2. In some embodiments, the term “type I interferon” as used herein is IFN[alpha] or IFN[beta]. In some embodiments, the term “type I interferon” as used herein is IFN[beta]. In some embodiments, a Type I interferon as used herein includes a human, mouse, or synthetic Type I interferon.
  • the term "interferon alpha" as used herein may be a polypeptide having the sequence shown in NCBI aaa52724.1 or aaa52716.1 or aaa52725.1, or the sequence has at least 85% identity to these sequences. Peptide.
  • the term "interferon beta" (INF-[beta]) as used herein may be a protein having at least 85% identity to NCBI aac41702.1 or np_002167.1 or aah96152.1p41273 or NP 001552, or A fragment having the function of a tumor necrosis factor (TNF) ligand.
  • the interferon beta is human interferon beta.
  • the interferon beta has the amino acid sequence of SEQ ID NO:92.
  • the Type I interferon may be naturally occurring, such as may be isolated or purified from a mammal; or may be artificially prepared, such as may be produced according to conventional genetic engineering recombination techniques.
  • Component or type I interferon Preferably, the present invention may employ recombinant elements or type I interferons.
  • Amino acid sequences formed by substitution, deletion or addition of one or more amino acid residues are also included in the present invention on the basis of the type I interferon polypeptide sequence. Proper replacement of amino acids is a technique well known in the art that can be readily implemented and ensures that the biological activity of the resulting molecule is not altered. These techniques have taught one in the art that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity.
  • modified or modified polypeptides can also be used in the present invention, for example, modified to promote half-life, effectiveness, metabolism, and/or potency of the polypeptide. Or improved polypeptide. That is, any variation that does not affect the biological activity of the polypeptide can be used in the present invention.
  • the biologically active fragments of the type I interferon polypeptide can be used in the present invention.
  • the meaning of the biologically active fragment means as a polypeptide which remains as part of the full length polypeptide. Full or partial function of a full length polypeptide.
  • the biologically active fragment retains at least 50% of the activity of the full length polypeptide. Under more preferred conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full length polypeptide.
  • the invention provides a chimeric antigen receptor comprising an extracellular antigen binding unit, a transmembrane domain, and an intracellular domain as described herein.
  • Chimeric Antigen Receptor refers to a tumor antigen binding domain fused to an intracellular signal transduction domain that activates T cells.
  • the extracellular binding domain of CAR is derived from a mouse or humanized or human monoclonal antibody.
  • a chimeric antigen receptor typically comprises an extracellular antigen binding region or antigen binding unit.
  • the extracellular antigen binding unit is an antigen binding unit as described herein before.
  • the extracellular antigen binding region can be fully human. In other cases, the extracellular antigen binding region can be humanized. In other instances, the extracellular antigen binding region can be murine or the chimera in the extracellular antigen binding region consists of amino acid sequences from at least two different animals. In some embodiments, the extracellular antigen binding region can be non-human.
  • antigen binding regions can be designed. Non-limiting examples include single-chain variable fragments (scFv) derived from antibodies, fragment antigen binding regions (Fabs) selected from libraries, single domain fragments, or natural ligands that bind to their cognate receptors.
  • the extracellular antigen binding region can comprise an scFv, Fab, or natural ligand, as well as any derivatives thereof.
  • An extracellular antigen binding region can refer to a molecule other than an intact antibody, which can comprise a portion of an intact antibody and can bind to an antigen to which the intact antibody binds.
  • antibody fragments can include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; bifunctional antibodies, linear antibodies; single-chain antibody molecules (eg, scFv); and formed from antibody fragments Multispecific antibodies.
  • An extracellular antigen binding region such as a scFv, Fab or natural ligand, can be part of a CAR that determines antigen specificity.
  • the extracellular antigen binding region can bind to any complementary target.
  • the extracellular antigen binding region can be derived from an antibody of known variable region sequence.
  • the extracellular antigen binding region can be obtained from antibody sequences obtained from available mouse hybridomas.
  • extracellular antigen binding regions can be obtained from whole-out cleavage of tumor cells or primary cells, such as tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the binding specificity of the extracellular antigen binding region can be determined by a complementarity determining region or CDR, such as a light chain CDR or a heavy chain CDR.
  • CDR complementarity determining region
  • binding specificity can be determined by light chain CDRs and heavy chain CDRs.
  • a given combination of heavy chain CDRs and light chain CDRs can provide a given binding pocket that can confer greater affinity and/or specificity to the antigen than other reference antigens.
  • the extracellular antigen binding region can comprise a light chain CDR specific for the antigen.
  • the light chain CDR can be an antigen binding unit such as a CAR scFv light
  • the complementary region of the chain may comprise contiguous amino acid residue sequences, or two or more contiguous sequence of amino acid residues separated by non-complementarity determining regions (eg, framework regions).
  • a light chain CDR can comprise two or more light chain CDRs, which can be referred to as a light chain CDR-1, CDR-2, and the like.
  • the light chain CDRs can comprise three light chain CDRs, which can be referred to as light chain CDR-1, light chain CDR-2 and light chain CDR-3, respectively.
  • a set of CDRs present on a common light chain can be collectively referred to as a light chain CDR.
  • the extracellular antigen binding region can comprise a heavy chain CDR that is specific for the antigen.
  • the heavy chain CDRs can be heavy chain complementarity determining regions of antigen binding units such as scFv.
  • the heavy chain CDRs may comprise a contiguous sequence of amino acid residues, or a contiguous sequence of two or more amino acid residues separated by a non-complementarity determining region (eg, a framework region).
  • the heavy chain CDRs can comprise two or more heavy chain CDRs, which can be referred to as heavy chain CDR-1, CDR-2, and the like.
  • the heavy chain CDRs can comprise three heavy chain CDRs, which can be referred to as heavy chain CDR-1, heavy chain CDR-2 and heavy chain CDR-3, respectively.
  • a set of CDRs present on a common heavy chain can be collectively referred to as a heavy chain CDR.
  • the extracellular antigen binding region can be modified in various ways by using genetic engineering.
  • the extracellular antigen binding region can be mutated such that the extracellular antigen binding region can be selected to have a higher affinity for its target.
  • the affinity of the extracellular antigen binding region for its target can be optimized for targets that can be expressed at low levels on normal tissues. This optimization can be done to minimize potential toxicity.
  • clones of the extracellular antigen binding region with a higher affinity for the membrane-bound form of the target may be preferred over the counterpart of its soluble form. This modification can be made because different levels of soluble forms of the target can also be detected and their targeting can cause undesirable toxicity.
  • the extracellular antigen binding region comprises a hinge or spacer.
  • the terms hinge and spacer are used interchangeably.
  • the hinge can be considered as part of a CAR for providing flexibility to the extracellular antigen binding region.
  • the hinge can be used to detect CAR on the cell surface of a cell, particularly when detecting antibodies to the extracellular antigen binding region are ineffective or available.
  • the length of the hinge derived from an immunoglobulin may need to be optimized, depending on the location of the extracellular antigen binding region that targets the epitope on the target.
  • the hinge may not belong to an immunoglobulin, but to another molecule, such as the native hinge of a CD8 alpha molecule.
  • the CD8 alpha hinge may contain cysteine and proline residues known to play a role in the interaction of the CD8 co-receptor and the MHC molecule. The cysteine and proline residues can affect the performance of the CAR.
  • the CAR hinge can be adjustable in size. This is the immune synapse between the immune response cell and the target cell
  • the morphology also defines the distance that cannot be functionally bridged by the CAR due to the distal epitope of the membrane on the target molecule on the cell surface, ie the use of a short hinge CAR does not allow the synaptic distance to reach an approximation of the signal's ability to conduct.
  • the membrane proximal CAR target epitope was only observed for signal output in the context of a long hinged CAR.
  • the hinge can be adjusted depending on the extracellular antigen binding region used.
  • the hinge can be of any length.
  • the transmembrane domain can anchor the CAR to the plasma membrane of the cell.
  • the natural transmembrane portion of CD28 can be used for CAR.
  • the natural transmembrane portion of CD8 ⁇ can also be used in the CAR.
  • CD8 may be a protein having at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to the NCBI reference number: NP_001759 or a fragment thereof having stimulatory activity.
  • a “CD8 nucleic acid molecule” may be a polynucleotide encoding a CD8 polypeptide, and in some cases, the transmembrane region may be a natural transmembrane portion of CD28, and “CD28” may refer to NCBI reference number: NP_006130 or its stimulating activity.
  • a fragment has a protein of at least 85, 90, 95, 96, 97, 98, 99 or 100% identity.
  • a "CD28 nucleic acid molecule” can be a polynucleotide encoding a CD28 polypeptide.
  • the transmembrane portion can comprise a CD8 alpha region.
  • the (fine) intracellular signaling region of CAR may be responsible for activating at least one of the effector functions of the immune response cells into which the CAR has been placed.
  • CAR can induce effector functions of T cells, for example, the effector function is cytolytic activity or helper activity, including secretion of cytokines.
  • intracellular signaling region refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specific function. Although the entire intracellular signaling region can generally be used, in many cases it is not necessary to use the entire chain of the signal domain. In some embodiments, a truncated portion of an intracellular signaling region is used. In some embodiments, the term intracellular signaling region is therefore intended to include any truncated portion of an intracellular signaling region sufficient to transduce an effector function signal.
  • Preferred examples of signal domains for use in CAR may include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction after target-receptor binding, as well as any derivatives thereof or Variant sequences and any synthetic sequences of these sequences that have the same functionality.
  • TCRs T cell receptors
  • co-receptors that act synergistically to initiate signal transduction after target-receptor binding
  • the intracellular signaling region can contain a known signal motif for an immunoreceptor tyrosine activation motif (ITAM).
  • ITAMs containing cytoplasmic signaling sequences include those derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, and CD66d.
  • the intracellular signal domain is derived from a CD3 ⁇ chain.
  • T cell signaling domain containing one or more ITAM motifs is the CD3 ⁇ domain, also known as the T cell receptor T3 ⁇ chain or CD247.
  • This domain is part of the T cell receptor-CD3 complex and combines antigen recognition of several intracellular signal transduction pathways with major effector activation of T cells. makes an important impact.
  • CD3 ⁇ primarily refers to human CD3 ⁇ and its isoforms, as known from the Swissprot entry P20963, including proteins having substantially the same sequence.
  • the full T cell receptor T3 ⁇ chain is not required and that any derivative of the signal domain comprising the T cell receptor T3 ⁇ chain is suitable, including any functional equivalent thereof. .
  • the intracellular signaling domain can be selected from any one of the domains of Table 1.
  • the domain can be modified such that identity to the reference domain can range from about 50% to about 100%.
  • Any of the domains of Table 1 can be modified such that the modified form can comprise about 50, 60, 70, 80, 90, 95, 96, 97, 98, 99 or up to about 100% identity.
  • the intracellular signaling region of CAR may further comprise one or more costimulatory domains.
  • the intracellular signaling region may comprise a single costimulatory domain, such as an ⁇ chain (first generation CAR) or it is with CD28 or 4-1BB (second generation CAR).
  • the intracellular signaling region can comprise two costimulatory domains, such as CD28/OX40 or CD28/4-1BB (third generation).
  • CD28 phosphatidylinositol-4,5-diphosphate 3-kinase
  • 4-1BB/OX40 TNF-receptor-associated factor adapter protein
  • signals generated by the CAR may be combined with an auxiliary or costimulatory signal.
  • costimulatory signaling domains chimeric antigen receptor-like complexes can be designed to contain several possible costimulatory signal domains.
  • costimulatory signaling domains chimeric antigen receptor-like complexes can be designed to contain several possible costimulatory signal domains.
  • the individual ligation of T cell receptors is not sufficient to induce complete activation of T cells into cytotoxic T cells.
  • a second co-stimulatory signal is required for complete productive T cell activation.
  • receptors have been reported to provide co-stimulation for T cell activation including, but not limited to, CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/CD18), 4-1BBL, MyD88, and 4- 1BB.
  • the signaling pathways used by these costimulatory molecules work synergistically with the primary T cell receptor activation signal.
  • the signals provided by these costimulatory signaling regions can act synergistically with primary effect activation signals derived from one or more ITAM motifs (eg, the CD3zeta signal transduction domain) and can fulfill the requirements for T cell activation.
  • the addition of a costimulatory domain to a chimeric antigen receptor-like complex can enhance the efficacy and durability of engineered cells.
  • the T cell signal domain and the costimulatory domain are fused to each other to form a signaling region.
  • the chimeric antigen receptor binds to the target antigen.
  • the target antigen can be obtained or isolated from various sources.
  • a target antigen as used herein is an antigenic epitope on an antigen or antigen that is critical in mammals for immune recognition and ultimately elimination or control of pathogenic factors or disease states.
  • the immune recognition can be a cell and/or a body fluid. In the case of intracellular pathogens and cancer, the immune recognition can be, for example, a T lymphocyte reaction.
  • the target antigen comprises an antigen associated with a pre-cancerous or proliferative state.
  • Target antigen may also Associated with or caused by cancer.
  • a chimeric antigen receptor of the invention recognizes and binds to a tumor antigen comprising TSA and TAA as described herein before.
  • a chimeric antigen receptor herein is present on the plasma membrane of a cell, and when bound to its target and activated, the cell expressing the chimeric antigen receptor can produce a cell carrying the target Cytotoxicity.
  • the chimeric antigen receptor is present on a cytotoxic cell, such as an NK cell or a cytotoxic T cell, and, when activated by a target, increases the toxicity of the cytotoxic cell to the target cell .
  • the chimeric antigen receptors herein increase the effect of immunoreactive cells on cells expressing claudin 18A2, such as tumor cells.
  • a cell expressing a chimeric antigen receptor described herein increases the cytotoxic effect on cells expressing claudin 18A2 by at least 10%, at least 15 compared to a cell that does not express a chimeric antigen receptor herein.
  • a chimeric antigen receptor herein when present on the plasma membrane of a cell, and when bound to its target and activated, does not induce significant in cells comprising the claudin 18A1 peptide but not the claudin 18A2 peptide. Cytotoxicity. In some embodiments, the cytotoxicity level is no greater than 10%, 5%, 4%, 3%, 2%, or 1%.
  • a transgene encoding a receptor or a CAR that binds to an antigen can be incorporated into the cell.
  • a transgene can be incorporated into an immune response cell, such as a T cell.
  • the transgene can be a complementary DNA (cDNA) fragment that is a copy of messenger RNA (mRNA); or the gene itself (with or without introns) located in the original region of its genomic DNA.
  • cDNA complementary DNA
  • mRNA messenger RNA
  • a nucleic acid encoding a transgene sequence, such as DNA can be randomly inserted into the chromosome of the cell. Random integration can be produced by any method that introduces a nucleic acid, such as DNA, into a cell.
  • the method can include, but is not limited to, electroporation, ultrasound, use of a gene gun, lipofection, calcium phosphate transfection, use of dendrimers, microinjection, and use of viruses including adenovirus, AAV, and retroviral vectors.
  • Vector, and/or type II ribozyme can be produced by any method that introduces a nucleic acid, such as DNA, into a cell.
  • the method can include, but is not limited to, electroporation, ultrasound, use of a gene gun, lipofection, calcium phosphate transfection, use of dendrimers, microinjection, and use of viruses including adenovirus, AAV, and retroviral vectors.
  • Vector, and/or type II ribozyme
  • the DNA encoding the transgene can also be designed to include a reporter gene such that the presence of the transgene or its expression product can be detected by activation of the reporter gene. Any reporter gene can be used, such as those described above.
  • the cells containing the transgene can be selected by selecting cells in the cell culture in which the reporter gene has been activated.
  • Expression of CAR can be verified by expression assays such as qPCR or by measuring the level of RNA.
  • the level of expression can also indicate the number of copies. For example, if the level of expression is very high, this may indicate that more than one copy of the CAR is integrated into the genome. Alternatively, high expression may indicate that the transgene is integrated in a high transcribed region, such as near a highly expressed promoter. Expression can also be verified by measuring protein levels, for example by Western blotting.
  • an immune response cell of the invention may comprise one or more transgenes.
  • the one or more transgenes can express a CAR protein that recognizes and binds to at least one epitope on the antigen or binds to a mutant epitope on the antigen.
  • CAR can be a functional CAR.
  • the immune response cells of the invention may comprise one or more CARs, or they may comprise a single CAR and a secondary engineered receptor.
  • the transgene can encode a suicide gene.
  • CAR immune response cells cause tumor regression but can be associated with toxicity.
  • the target antigen when the target antigen is shared in normal tissues and tumor cells, the CAR immune response cells may not be able to distinguish between tumors and normal tissues ("target/off-target toxicity").
  • a systemic disturbance of the immune system called cytokine release syndrome (CRS)
  • CRS may comprise a systemic inflammatory response syndrome or a cytokine storm, which may be a consequence of rapid expansion of the CAR immune response cells in vivo.
  • CRS is a condition characterized by fever and hypotension, which can lead to multiple organ failure.
  • the toxicity is associated with in vivo expansion of infused CAR immune response cells, which can cause an overall disturbance of the immune system, as well as release high levels of pro-inflammatory cytokines such as TNF[alpha] and IL-6.
  • Suicide genes can induce the elimination of CAR immunoreactive cells.
  • the suicide gene may be any gene that induces apoptosis in the CAR immunoreactive cells.
  • a suicide gene can be encoded in the viral vector together with the antigen-binding receptor. The coding of the suicide gene allows for the mitigation or complete abortion of the toxicity caused by in vivo expansion of the infused CAR immune response cells under specific conditions.
  • CAR immunoreactive cells that are present in antigens of normal tissues can be produced such that they transiently express CAR, eg, after electroporating the mRNA encoding the receptor.
  • a major effort to further strengthen CAR immunoreactive cells by including a safety switch can substantially eliminate CAR immunoreactive cells in the case of severe target toxicity.
  • the CAR-encoding vector can be associated with, for example, an inducible caspase-9 gene (activated by a dimeric chemical inducer) or a truncated form of EGF receptor R (by the monoclonal antibody Cetuximab) Monoclonal activation) or RQR8 safety switch combination.
  • transgenes used herein may be from different species.
  • one or more transgenes may comprise a human gene, a mouse gene, a rat gene, a pig gene, a bovine gene, a dog gene, a cat gene, a monkey base. Factor, chimpanzee gene or any combination thereof.
  • a transgene can be from a human having a human genetic sequence.
  • One or more transgenes may comprise a human gene. In some cases, one or more of the transgenes are not adenoviral genes.
  • the transgene can be inserted into the genome of the immunoreactive cell in a random or site-specific manner.
  • a transgene can be inserted into a random site in the genome of an immune cell.
  • These transgenes can be functional, for example, fully functional when inserted into any part of the genome.
  • a transgene can encode its own promoter or can be inserted into a position controlled by its internal promoter.
  • the transgene can be inserted into a gene, such as an intron of a gene or an exon, promoter or non-coding region of a gene.
  • a transgene can be inserted to insert a disruptive gene, such as an endogenous immune checkpoint.
  • more than one copy of the transgene can be inserted into multiple random sites within the genome. For example, multiple copies can be inserted into random sites in the genome. This may result in an increase in overall expression compared to random insertion of the transgene once.
  • a copy of the transgene can be inserted into the gene and another copy of the transgene can be inserted into a different gene.
  • the transgene can be targeted such that it can be inserted into a specific site in the genome of the immunoreactive cell.
  • a polynucleic acid comprising a receptor sequence encoding an antigen binding agent can take the form of a plasmid vector.
  • the plasmid vector may comprise a promoter. In some cases, the promoter can be constitutive. In some embodiments, the promoter is inducible. The promoter may be or may be derived from CMV, U6, MND or EF1a. In some embodiments, the promoter can be adjacent to the CAR sequence. In some embodiments, the plasmid vector further comprises a splice acceptor. In some embodiments, the splice acceptor can be adjacent to the CAR sequence.
  • the promoter sequence can be a PKG or MND promoter.
  • the MND promoter may be a synthetic promoter of the U3 region of the MoMuLV LTR modified with myeloproliferative sarcoma virus enhancer.
  • a polynucleic acid encoding a receptor of interest can be designed to be delivered to a cell by non-viral techniques.
  • the polynucleic acid can be a Good Manufacturing Practice (GMP) compatible reagent.
  • GMP Good Manufacturing Practice
  • Promoters can be ubiquitous, constitutive (unrestricted promoters, allowing for continuous transcription of related genes), tissue-specific promoters or inducible promoters. Expression of a transgene inserted adjacent to or proximate to the promoter can be modulated. For example, a transgene can be inserted near or beside a ubiquitous promoter.
  • Some ubiquitous promoters may be the CAGGS promoter, the hCMV promoter, the PGK promoter, the SV40 promoter or the ROSA26 promoter.
  • Promoters can be endogenous or exogenous.
  • one or more transgenes can be inserted adjacent to or proximate to the endogenous or exogenous ROSA26 promoter.
  • the promoter may be a special immunoreactive cell opposite sex.
  • one or more transgenes can be inserted adjacent to or proximate to the porcine ROSA26 promoter.
  • Tissue-specific promoters or cell-specific promoters can be used to control the location of expression.
  • one or more transgenes can be inserted into proximity or proximity of a tissue-specific promoter.
  • Tissue-specific promoters may be FABP promoter, Lck promoter, CamKII promoter, CD19 promoter, keratin promoter, albumin promoter, aP2 promoter, insulin promoter, MCK promoter, MyHC promoter, WAP Promoter, or Col2A promoter.
  • Inducible promoters can also be used. These inducible promoters can be turned on and off by adding or removing an inducer if necessary.
  • the inducible promoter is contemplated to be, but not limited to, Lac, tac, trc, trp, araBAD, phoA, recA, proU, cst-1, tetA, cadA, nar, PL, cspA, T7, VHB, Mx, and/or Trex.
  • inducible promoter is a controlled promoter which does not express or underexpress a gene operably linked thereto before the desired condition is reached, and is achieved under the expected conditions.
  • a gene that is operably linked to it is expressed or expressed at a high level.
  • an inducible promoter of the present application does not express or underexpress a gene operably linked thereto under normal or high oxygen content conditions in a cell, and in response to a reduced oxygen content in the cell, A gene that is operably linked thereto is expressed or overexpressed under hypoxic conditions.
  • an inducible promoter for use herein includes Hypoxia-Inducible Transcription factor-1 ⁇ (HIF-1 ⁇ ).
  • the term "inducible promoter” as used herein refers to an "immune cell-inducible promoter” that does not express or underexpresses an immune response cell prior to its exposure to the antigen or when the immune response cell is not activated.
  • the "immune cell-inducible promoter” comprises a NFAT (activated T cell nuclear factor) type promoter.
  • NFAT-type promoter refers to a class of promoters that regulate the expression of a gene to which they are operably linked based on NFAT binding activity.
  • NFAT is a general term for a family of transcription factors that play an important role in immune responses. One or more members of the NFAT family are expressed in most cells of the immune system. NFAT is also involved in the development of the heart, skeletal muscle and nervous system.
  • the NFAT transcription factor family consists of five members, NFAT1, NFAT2, NFAT3, NFAT4, and NFAT5.
  • NFAT1 to NFAT4 are regulated by calcium signals.
  • Calcium signaling is critical for NFAT activation because calmodulin (CaM) activates serine/threonine phosphatase calcineurin (CN).
  • CN serine/threonine phosphatase calcineurin
  • CN serine/threonine phosphatase calcineurin
  • CN serine/threonine phosphatase calcineurin
  • Activated CN rapidly dephosphorylates the serine-rich region (SRR) and SP repeats at the amino terminus of the NFAT protein.
  • SRR serine-rich region
  • SP repeats at the amino terminus of the NFAT protein.
  • the conformational change causes the nuclear localization signal to be exposed, resulting in the NFAT input into the nucleus
  • NFAT in the transcriptional expression of cytokines during T cell activation, which can be used to modulate the immune cell-inducible promoters described herein, thereby expressing or expressing high levels of expression when the immune response cells are exposed to antigen activation.
  • a nucleic acid of the invention may comprise any suitable nucleotide sequence encoding a NFAT type promoter (or a functional part or a functional variant thereof).
  • NFAT-type promoter refers to one or more NFAT response elements linked to the minimal promoter of any gene expressed by a T cell.
  • the minimal promoter of the gene expressed by T cells is the smallest human IL-2 promoter.
  • the NFAT response element can include, for example, NFAT1, NFAT2, NFAT3, and/or NFAT4 response elements.
  • more than one NFAT binding motif can be included in a "NFAT-type promoter" as described herein.
  • the "NFAT-type promoter” can include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NFAT binding motifs.
  • the "NFAT-type promoter” includes up to 12 NFAT binding motifs.
  • the "NFAT-type promoter” can be a promoter consisting of a plurality of the NFAT-binding motifs in series with a promoter, such as the IL2 minimal promoter.
  • the NFAT type promoters described herein comprise six NFAT binding motifs, designated (NFAT) 6 .
  • the (NFAT) 6 is also referred to as NFAT6.
  • the NFAT6 also represents a 6-repeat NFAT binding motif (SEQ ID NO: 94) in the NFAT-type promoter.
  • the transgenic sequences may also include transcriptional or translational regulatory sequences, such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.
  • transcriptional or translational regulatory sequences such as promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.
  • the transgene encodes a receptor or CAR that binds to the antigen, wherein the transgene is inserted into a safe harbor such that the antigen-binding receptor is expressed.
  • the transgene is inserted into the PD1 and/or CTLA-4 locus.
  • the transgene is delivered as a lentivirus to the cells for random insertion, while a PD1- or CTLA-4 specific nuclease can be provided as mRNA.
  • the transgene is delivered by a viral vector system such as retrovirus, AAV or adenovirus, and mRNA encoding a nuclease specific for safe harbor (eg, AAVS1, CCR5, albumin, or HPRT). Cells can also be treated with mRNA encoding PD1 and/or CTLA-4 specific nucleases.
  • the polynucleotide encoding the CAR is provided by a viral delivery system with an mRNA encoding a HPRT-specific nuclease and a PD1- or CTLA-4 specific nuclease. CARs that can be used with the methods and compositions disclosed herein can include all types of these chimeric proteins.
  • a retroviral vector (gamma-retroviral or lentiviral vector) can be used.
  • the transgene is introduced into an immunoreactive cell.
  • a transgene encoding a CAR or any receptor that binds an antigen, or a variant or fragment thereof can be cloned into a retroviral vector and can be derived from an endogenous promoter, a retroviral long terminal repeat, or a target Cell type-specific promoter drive.
  • Non-viral vectors can also be used.
  • Non-viral vector delivery systems can include DNA plasmids, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes or poloxamers.
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of the transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from retroviruses such as murine leukemia virus because they can transduce non-proliferating cells. They also have the added advantage of low immunogenicity.
  • An advantage of adenoviral vectors is that they do not fuse into the genome of the target cell, thereby bypassing negative integration-related events.
  • the cells can be transfected with a transgene encoding the antigen-binding receptor.
  • the transgenic concentration can range from about 100 picograms to about 50 micrograms.
  • the amount of nucleic acid (eg, ssDNA, dsDNA, or RNA) introduced into the cell can be altered to optimize transfection efficiency and/or cell viability. For example, 1 microgram of dsDNA can be added to each cell sample for electroporation.
  • the amount of nucleic acid (eg, double stranded DNA) required for optimal transfection efficiency and/or cell viability varies depending on the cell type.
  • the amount of nucleic acid (eg, dsDNA) used for each sample can directly correspond to transfection efficiency and/or cell viability. For example, a range of transfection concentrations.
  • the transgene encoded by the vector can be integrated into the genome of the cell. In some embodiments, the transgene encoded by the vector is forward integrated. In other cases, the reverse integration of the transgene encoded by the vector.
  • the vector by administration to an individual patient is typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or topical application, as described below.
  • the vector can be delivered ex vivo to the cells, such as cells removed from an individual patient (eg, lymphocytes, T cells, bone marrow aspirate, tissue biopsy), and then typically after re-selecting the cells into which the vector is incorporated Implanted in a patient. Cells can be expanded before or after selection.
  • Suitable immunoreactive cells for expression of a receptor that binds to an antigen may be cells that are autologous or non-autologous to the individual in need thereof.
  • a suitable source of immune response cells can be obtained from the individual.
  • T cells can be obtained.
  • the T cells can be obtained from a number of sources, including PBMC, bone marrow, lymph node tissue, cord blood, thymus tissue, and tissues from infected sites, ascites, pleural effusion, spleen tissue, and tumors.
  • cells from circulating blood of an individual are obtained by apheresis.
  • Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • cells collected by apheresis collection can be washed to remove plasma fractions and placed in a suitable buffer or medium for subsequent processing steps.
  • cells can be derived from a healthy donor, from a patient diagnosed with cancer, or a patient diagnosed with an infection.
  • the cells can be part of a mixed cell population with different phenotypic characteristics.
  • Cell lines can also be obtained from transformed T cells according to the methods previously described.
  • Cells can also be obtained from a cell therapy library.
  • Modified cells that are resistant to immunosuppressive therapy can be obtained by any of the methods described herein. It is also possible to select a suitable cell population prior to modification.
  • the engineered cell population can also be selected after modification.
  • Engineered cells can be used for autologous transplantation.
  • the cells can be used for allogeneic transplantation.
  • the cells are administered to a sample for identification of the same patient of a cancer associated target sequence. In other instances, the cells are administered to a patient different from the patient whose sample is used to identify the cancer-related target sequence.
  • suitable primary cells include peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL), and other blood cell subpopulations such as, but not limited to, T cells, natural killer cells, monocytes, Natural killer T cells, monocyte precursor cells, hematopoietic stem cells or non-pluripotent stem cells.
  • the cell can be any immune cell, including any T cell such as a tumor infiltrating cell (TIL), such as a CD3+ T cell, a CD4+ T cell, a CD8+ T cell, or any other type of T cell.
  • T cells can also include memory T cells, memory stem T cells, or effector T cells.
  • T cells can also be expanded from a large population.
  • T cells may also be inclined to specific populations and phenotypes.
  • a T cell can be tilted to a phenotype comprising CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+), and/or IL-7R ⁇ (+).
  • Suitable cells may be selected from one or more of the following list: CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+) and/or IL-7R ⁇ (+).
  • Suitable cells also include stem cells such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells.
  • stem cells such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, and mesenchymal stem cells.
  • Suitable cells can comprise any number of primary cells, such as human cells, non-human cells, and/or mouse cells.
  • Suitable cells can be progenitor cells.
  • Suitable cells can be derived from a subject (eg, a patient) to be treated.
  • the amount of therapeutically effective cells required in a patient can vary depending on the viability of the cells and the efficiency with which the cells are genetically modified (eg, the efficiency with which the transgene is integrated into one or more cells, or the level of expression of the protein encoded by the transgene) ).
  • the product of cell viability after genetic modification (eg, For example, doubling) and the efficiency of transgene integration can correspond to the therapeutic amount of cells available for administration to a subject.
  • an increase in cell viability after genetic modification may correspond to a reduction in the amount of essential cells effective to administer the treatment to the patient.
  • an increase in the efficiency of integration of the transgene into one or more cells can correspond to a reduction in the number of cells necessary to administer a therapeutically effective in a patient.
  • determining the amount of therapeutically effective cells required can include determining a function associated with changes in cells over time.
  • determining the amount of cells that are required to be therapeutically effective can include determining a function corresponding to a change in efficiency of integrating the transgene into one or more cells according to a time-dependent variable (eg, cell culture time, electroporation time, Cell stimulation time).
  • the therapeutically effective cell can be a population of cells comprising about 30% to about 100% of the expression of a receptor that binds to the antigen on the surface of the cell.
  • the therapeutically effective cells can express about 30%, 35%, 40%, 45%, 50%, 55%, 60 of the antigen-binding receptor on the cell surface as measured by flow cytometry. %, 65%, 70%, 75% 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9 % or more than about 99.9%.
  • the invention also encompasses a nucleic acid encoding the antigen-binding receptor.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • the present invention also provides a vector comprising the above nucleic acid encoding a receptor protein that binds to an antigen expressed on the surface of an immune response cell.
  • the invention also includes viruses comprising the vectors described above.
  • the virus of the present invention includes a packaged infectious virus, and also includes a virus to be packaged containing components necessary for packaging as an infectious virus.
  • Other viruses known in the art that can be used to transduce foreign genes into immune response cells and their corresponding plasmid vectors can also be used in the present invention.
  • a host cell comprising an antigen binding unit or chimeric antigen receptor as described herein, and optionally a type I interferon.
  • a host cell comprising a nucleic acid encoding an antigen binding unit or chimeric antigen receptor described herein, and optionally a type I interferon.
  • the host cell is an immune response cell.
  • the immune response cell is a T cell, a natural killer cell, a cytotoxic T lymphocyte, a natural killer T cell, a DNT cell, and/or a regulatory T cell.
  • the host cell is an NK92 cell.
  • an expression construct can be included in an immune response cell of the invention, wherein the expression construct has elements that are sequentially linked as follows: antibody, CD28 costimulatory signal domain, CD3 ⁇ , and in opposition to the aforementioned elements Linked NFAT6, type I interferon expression unit.
  • the antibody is The CD28 costimulatory signal domains are joined by a CD8 alpha transmembrane region and a CD8 alpha hinge region.
  • the NFAT nuclear factor of activated T cells
  • the NFAT plays an important role in the transcriptional expression of cytokines during T cell activation.
  • the inventors placed the IFN-beta coding sequence under the regulation of the NFAT6 promoter, so that IFN-beta can be expressed at a high level only when the CAR-T cells contact the antigen to induce T cell activation.
  • the NFAT6 promoter is a promoter composed of a combination of six NFAT binding positions and a minimal promoter of IL2 (Hooijberg E, Bakker AQ, Ruizendaal JJ, Spits H. NFAT-controlled expression of GFP permits visualization and Isolation of antigen-stimulated primary human Tcells. Blood. 2000 Jul 15; 96(2): 459-66), which can be used to regulate the expression of cytokines such as IL12 in T lymphocytes such as TCR-T (Zhang L, Kerkar SP, YuZ, Zheng Z, Yang S, Restifo NP, Rosenberg SA, Morgan RA. Immunity adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment. Mol Ther. 2011 Apr; 19(4): 751-9).
  • the immune response cell of the present invention is transduced with a construct capable of expressing an antigen-binding receptor and an exogenous type I interferon, or an expression vector, or a virus comprising the plasmid.
  • a construct capable of expressing an antigen-binding receptor and an exogenous type I interferon or an expression vector, or a virus comprising the plasmid.
  • Conventional nucleic acid transduction methods including non-viral and viral transduction methods, can be used in the present invention.
  • the immune response cell of the present invention may further carry a coding sequence of a foreign cytokine; the cytokine includes, but not limited to, IL-12, IL-15 or IL-21 and the like.
  • cytokine includes, but not limited to, IL-12, IL-15 or IL-21 and the like.
  • These cytokines have further immunomodulatory or anti-tumor activity, enhance the function of effector T cells and activated NK cells, or directly exert anti-tumor effects.
  • cytokines will help the immune response cells to function better.
  • the immune response cell of the present invention may also express another antigen-binding receptor other than the antigen-binding receptor described above.
  • the immune response cells of the invention may also express a chemokine receptor; the chemokine receptors include, but are not limited to, CCR2. Those skilled in the art will appreciate that the CCR2 chemokine receptors may allow CCR2 binding in vivo to compete with it, which is advantageous for blocking tumor metastasis.
  • the immune response cells of the present invention can also express siRNA that reduces PD-1 expression or a protein that blocks PD-L1.
  • siRNA that reduces PD-1 expression
  • the immune response cells of the present invention may also express a safety switch; preferably, the safety switch comprises: iCaspase-9, Truncated EGFR or RQR8.
  • the immune response cells of the invention do not express a costimulatory ligand such as 4-1BBL.
  • a method of producing an antigen binding unit or chimeric antigen receptor described herein, or a composition comprising the same comprising culturing a host cell described herein under suitable conditions .
  • the method comprises isolating and obtaining an expression product of the host cell.
  • composition comprising an antigen binding unit, chimeric antigen receptor, or nucleic acid described herein.
  • the composition is a pharmaceutical composition comprising the antigen binding unit, a chimeric antigen receptor, or a nucleic acid.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
  • composition comprising a host cell as described herein and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that when the molecular body and composition are suitably administered to an animal or a human, they do not produce an adverse, allergic or other untoward reaction.
  • the composition comprises another therapeutic agent.
  • the additional therapeutic agent is a chemotherapeutic agent, such as those described in US20140271820 and/or a pharmaceutically acceptable salt or analog thereof.
  • the therapeutic agent includes, but is not limited to, a mitotic inhibitor (vinca alkaloid), including vincristine, vinblastine, vindesine, and novibin (TM) (vinorelbine, 5' - dehydro sulfide); topoisomerase I inhibitors such as camptothecin compound, including Camptosar TM (irinotecan HCL), Hycamtin TM (topotecan HCL) derived from camptothecin and its analogues Other compounds; podophyllotoxin derivatives such as etoposide, teniposide and midozozoz; alkylating agents cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, camo Statin, bus
  • TM novibin
  • the additional therapeutic agent is selected from one or more of epirubicin, oxaliplatin, and 5-fluorouracil.
  • the additional therapeutic agent includes, but is not limited to, an anti-angiogenic agent, including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides), and other blood vessels Inhibitors such as angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta) interleukin 12, tissue inhibitors of retinoic acid and metalloproteinases-1 and -2, and the like occur.
  • anti-VEGF antibodies including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides
  • Inhibitors such as angiostatin, endostatin, interferon, interleukin 1 (including alpha and beta) interleukin 12, tissue inhibitors of retinoic acid and metalloproteinases-1 and -2, and the like
  • sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof such as carboxymethyl fibers Sodium, ethyl cellulose and methyl cellulose; western yellow gum powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Wetting agents, such as sodium lauryl sulfate; colorants; flavoring agents; compressed tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers
  • compositions described herein may comprise one or more pharmaceutically acceptable salts.
  • “Pharmaceutically acceptable salt” refers to a salt that retains the desired biological activity of the parent compound and does not produce any adverse toxicological effects (see, for example, Berge, SM, et al. 1977, J. Pharm. Sci. 66 :1-19). Examples of such salts include acid addition salts and base addition salts.
  • Acid addition salts include salts derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, and derived from non-toxic organic acids such as aliphatic monocarboxylic acids and A salt of a dicarboxylic acid, a phenyl-substituted alkanoic acid, a hydroxyalkanoic acid, an aromatic acid, an aliphatic or an aromatic sulfonic acid.
  • non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like
  • non-toxic organic acids such as aliphatic monocarboxylic acids and A salt of a dicarboxylic acid, a phenyl-substituted alkanoic acid, a hydroxyalkanoic acid, an aromatic
  • Base addition salts include salts derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, as well as salts derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucosamine. Glucosamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.
  • antioxidants include, but are not limited to, water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, Phosphoric acid, etc.
  • water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, sodium metabisulfite, sodium sulfite, etc.
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • composition of the present invention can be formulated into various dosage forms as needed, and can be administered by a physician in accordance with factors such as patient type, age, body weight, and general disease condition, mode of administration, and the like.
  • the mode of administration can be, for example, parenteral administration (e.g., injection) or other treatment.
  • parenteral administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection or infusion techniques.
  • Formulations comprising an immunoreactive cell population administered to an individual comprise a plurality of immunoreactive cells effective to treat and/or prevent a particular indication or disease.
  • a therapeutically effective population of immunoreactive cells can be administered to an individual.
  • administration contain from about 1 ⁇ 10 4 to about 1 ⁇ 10 10 cells reactive immunization formulation.
  • the formulation will comprise from about 1 x 10 5 to about 1 x 10 9 immunoreactive cells, from about 5 x 10 5 to about 5 x 10 8 immunoreactive cells, or about 1 x 10 6 to About 1 ⁇ 10 7 immunoreactive cells.
  • the number of CAR immunoreactive cells administered to the individual will vary from wide range. The doctor will finalize the appropriate dose to use.
  • a chimeric antigen receptor is used to stimulate an immune cell mediated immune response.
  • a T cell mediated immune response is an immune response involving T cell activation.
  • Activated antigen-specific cytotoxic T cells are capable of inducing apoptosis in target cells that exhibit a foreign antigenic epitope on the surface, such as cancer cells that display tumor antigens.
  • the chimeric antigen receptor is used to provide anti-tumor immunity in a mammal. Subjects will develop anti-tumor immunity due to T cell-mediated immune responses.
  • a method of treating a subject having cancer can involve administering one or more immune response cells of the invention to a subject in need of treatment.
  • the immune response cell binds to a tumor target molecule and induces cancer cell death.
  • the invention also provides a method of treating a pathogen infection in an individual comprising administering to the individual a therapeutically effective amount of an immune response cell of the invention.
  • the frequency of administration of the immunoreactive cells of the present invention will depend on factors including the disease being treated, the elements of the particular immunoreactive cells, and the mode of administration. For example, it can be administered 4 times, 3 times, 2 times a day, once a day, every other day, every three days, every four days, every five days, every six days, once a week, once every eight days, every time. Dosing once every nine days, every ten days, once a week, or twice a month.
  • the immune response cells of the present application have improved viability, they can be administered not only in a therapeutically effective amount that is lower than an immune response cell that is similar but does not express exogenous type I interferon, and can Administration at a lower frequency to achieve at least a similar, and preferably more pronounced, effect.
  • the compositions may be isotonic, ie they may have the same osmotic pressure as blood and tears.
  • the desired isotonicity of the compositions of the present invention can be achieved using sodium chloride or other pharmaceutically acceptable agents such as glucose, boric acid, sodium tartrate, propylene glycol or other inorganic or organic solutes.
  • the viscosity of the composition can be maintained at a selected level using a pharmaceutically acceptable thickening agent.
  • Suitable thickeners include, for example, methylcellulose, xanthan gum, carboxymethylcellulose, hydroxypropylcellulose, carbomer, and the like. The preferred concentration of thickener will depend on the reagent selected. It will be apparent that the choice of suitable carrier and other additives will depend on the exact route of administration and the nature of the particular formulation, such as a liquid dosage form.
  • kits comprising an antigen binding unit, chimeric antigen receptor, nucleic acid or immune response cell as described herein.
  • a kit can include a therapeutic or prophylactic composition comprising an effective amount of an antigen binding unit, chimeric antigen receptor, nucleic acid, or immune response cell described herein comprising one or more unit dosage forms.
  • the kit comprises a sterile container that can contain a therapeutic or prophylactic composition; such a container can be a cartridge, ampule, bottle, vial, tube, bag, blister pack, or other suitable as is known in the art.
  • Container form Such containers may be made of plastic, glass, laminated paper, metal foil or other materials suitable for holding the drug.
  • the kit comprises an antigen binding unit, chimeric antigen receptor, nucleic acid or immune response cell as described herein, and an antigen binding unit, chimeric antigen receptor, nucleic acid or immunization described herein.
  • the instructions for administering the individual to the individual in response to the response generally include methods of treating or preventing cancer or tumors using the antigen binding units, chimeric antigen receptors, nucleic acids or immune response cells described herein.
  • the kits comprise a host cell as described herein and can include from about 1 x 10 4 cells to about 1 x 10 6 cells.
  • the kit can include at least about 1 x 10 5 cells, at least about 1 x 10 6 cells, at least about 1 x 10 7 cells, at least about 4 x 10 7 cells, at least about 5 x 10 7 cells, at least about 6 ⁇ 10 7 cells, at least about 6 ⁇ 10 7 cells, 8 ⁇ 10 7 cells, at least about 9 ⁇ 10 7 cells, at least about 1 ⁇ 10 8 cells, at least about 2 x 108 cells, at least about 3 x 10 8 cells, at least about 4 x 10 8 cells, at least about 5 x 10 8 cells, at least about 6 x 10 8 cells, at least about 6 x 10 8 cells, At least about 8 x 10 8 cells, at least about 9 x 10 8 cells, at least about 1 x 10 9 cells, at least about 2 x 10 9 cells, at least about 3 x 10 9 cells, at least about 4 x 10 9 cells, at least about 5 ⁇ 10 9 cells, at least about 6 ⁇ 10 9 cells, at least about 8 ⁇ 10 9 cells, at least about 9
  • the kit can include allogeneic cells.
  • a kit can include cells that can include genomic modifications.
  • the kit can comprise "off the shelf" cells.
  • the kit can include cells that can be expanded for clinical use. In some cases, the kit may contain content for research purposes.
  • the instructions include at least one of: a description of a therapeutic agent; a dosage regimen and administration for treating or preventing a tumor or a symptom thereof; preventive measures, warnings, contraindications, excessive information, adverse reactions, animals Pharmacology, clinical studies, and/or citations. Instructions can be printed directly on the container (if any), or as a label on the container, or as a separate paper, booklet, card or folder in the container or in the container. In some embodiments, the instructions provide for the administration of the invention as described A method for treating or preventing a tumor by an responsive cell. In some cases, the instructions provide methods of administering an immunoreactive cell of the invention before, after or simultaneously with the administration of a chemotherapeutic agent.
  • a method of inducing cell death comprising a claudin 18A2 peptide, the method comprising: ligating the cell with an antigen binding unit described herein, a chimeric antigen receptor described herein, The compositions described herein, or host cells described herein, are contacted.
  • the contacting is in vitro contact. In some embodiments, the contacting is in vivo contact.
  • the cell is a tumor cell. In some embodiments, the cell is a solid tumor cell. In some embodiments, the cell is a cancer or a cell of a tumor as described herein. Specific examples of such cells may include, but are not limited to, leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute single) Nuclear cell leukemia, acute leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera) cells, lymphoma (Hodgkin's disease, non-Hodgkin's disease) cells, primary macroglobulin blood Diseased cells, heavy chain disease cells, solid tumors such as sarcoma and cancer cells (eg fibrosarcoma, mucinous sarcoma, liposarcom
  • the cell is a gastric cancer cell, an esophageal cancer cell, an intestinal cancer cell, a pancreatic cancer cell, a nephroblastoma cell, a lung cancer cell, an ovarian cancer cell, a colon cancer cell, a rectal cancer cell, a liver cancer cell, a head and neck Cancer cells, chronic myeloid leukemia cells and gallbladder cancer cells.
  • provided herein is a method of treating a tumor in an individual in need thereof, the method comprising administering to the individual an effective amount of an antigen binding unit, chimeric antigen receptor, composition, vector described herein Or host cells.
  • the tumor includes, but is not limited to, selected from the group consisting of bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue , ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, Tumors of the urethra, ureter, urethra, uterus, and vaginal organs.
  • bladder selected from the group consisting of bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, nervous tissue , ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, Tumors of the
  • the tumor includes, but is not limited to, leukemia (eg, acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, Acute monocytic leukemia, acute leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia Disease, heavy chain disease, solid tumors such as sarcoma and cancer (such as fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial sarcoma, lymphangisarcoma, angiosarcoma, lymphatic endothelial sarcoma,
  • the tumor is gastric cancer, esophageal cancer, intestinal cancer, pancreatic cancer, nephroblastoma, lung cancer, ovarian cancer, colon cancer, rectal cancer, liver cancer, head and neck cancer, chronic myelogenous leukemia, or gallbladder cancer.
  • the subject can administer immunoreactive cells, wherein the immunoreactive cells that can be administered can be from about 1 to about 35 days of age.
  • the cells administered may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or up to about 40 days.
  • the age of CAR immunoreactive cells can be calculated from the time of stimulation.
  • the age of the immunoreactive cells can be calculated from the time of blood collection.
  • the age of the immunoreactive cells can be calculated from the time of transduction.
  • the immunoreactive cells that can be administered to the subject are from about 10 to about 14 or about 20 days of age.
  • the "age" of an immunoreactive cell can be determined by the telomere length.
  • a "young" immune response cell can have a longer telomere length than "depleted” or "old” immunoreactive cells.
  • immunoreactive cells lose an estimated telomere length of about 0.8 kb per week in culture, and young immunoreactive cell cultures can have longer than about 44 days of immunoreactive cells.
  • About 1.4 kb of telomeres it is believed that a longer telomere length can be associated with a positive objective clinical response in a patient and persistence of cells in vivo.
  • Cells can be functional before, after, and/or during transplantation.
  • the transplanted cells can be at least about 1, 2, 3, 4, 5, 6, 7, after transplantation. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 6, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90 or 100 days work.
  • the transplanted cells can function at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after transplantation.
  • the transplanted cells can function at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 years after transplantation.
  • the transplanted cells can function during the life of the recipient.
  • transplanted cells can function at 100% of their normal expected function.
  • the transplanted cells can also perform their normal expected functions of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, , 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 , 96, 97, 98, or up to about 100% of the functionality.
  • Transplanted cells can also perform more than 100% of their normal intended function.
  • the transplanted cells can function as approximately 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 as normal expected functions. Or up to about 5,000% of the functionality.
  • Porting can be done by any type of transplant.
  • Topography may include, but is not limited to, subhepatic sac space, subsplenic sac space, subcapsular space, omentum, gastric or intestinal submucosa, small intestinal vascular segment, venous sac, testis, brain, spleen, or cornea.
  • the transplant can be a subcapsular transplant.
  • Transplantation can also be intramuscular transplantation.
  • the transplant can be a portal vein transplant.
  • transplant rejection can be improved after treatment with the immune response cells of the present invention as compared to when one or more wild type cells are transplanted to the recipient.
  • transplant rejection can be a hyperacute rejection.
  • Transplant rejection can also be an acute rejection.
  • Other types of rejection may include chronic rejection.
  • Transplant rejection can also be cell-mediated rejection or T cell-mediated rejection.
  • Transplant rejection can also be a natural killer cell mediated rejection.
  • Improving transplantation may mean alleviating hyperacute rejection, which may include reducing, alleviating or reducing adverse effects or symptoms.
  • Transplantation can refer to adoptive transplantation of cellular products.
  • Another indication of successful transplantation may be the number of days the recipient does not need immunosuppressive therapy.
  • the recipient may not require at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days of immunosuppressive therapy. This can indicate that the transplant was successful. This can also indicate that the transplanted cells, tissues and/or organs are not repelled.
  • the recipient does not require immunosuppressive therapy for at least 1 day.
  • the recipient does not need to be immune Inhibition treatment for at least 7 days.
  • the recipient does not require immunosuppressive therapy for at least 14 days.
  • the recipient does not require immunosuppressive therapy for at least 21 days.
  • the recipient does not require immunosuppressive therapy for at least 28 days.
  • the recipient does not require immunosuppressive therapy for at least 60 days.
  • the recipient may not require at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years of immunosuppressive therapy.
  • Another sign of successful transplants may be the number of days that recipients need to reduce their immunosuppressive therapy. For example, after the treatment provided herein, the recipient may require at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days of reduced immunosuppressive therapy. This can indicate that the transplant was successful. This may also indicate that there is no or only minimal rejection of the transplanted cells, tissues and/or organs.
  • a recipient may require at least 1 day of reduced immunosuppressive therapy.
  • Recipients may also require at least 7 days of reduced immunosuppressive therapy.
  • the recipient may require at least 14 days of reduced immunosuppressive therapy.
  • Recipients require at least 21 days of reduced immunosuppressive therapy.
  • Recipients require at least 28 days of reduced immunosuppressive therapy.
  • Recipients require at least 60 days of reduced immunosuppressive therapy.
  • the recipient may require at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years of reduced immunosuppressive therapy.
  • Reduced immunosuppressive therapy can refer to less immunosuppressive therapy as compared to the immunosuppressive therapy required when transplanting one or more wild-type cells into a recipient.
  • Immunosuppressive therapy can include any treatment that inhibits the immune system. Immunosuppressive therapy can help alleviate, reduce or eliminate transplant rejection in patients.
  • immunosuppressants can be used before, during, and/or after transplantation, including MMF (Cellcept), ATG (anti-thymocyte globulin), anti-CD154 (CD4OL), anti-CD40 (2C10) , immunosuppressive drugs, anti-IL-6R antibodies (tocilizumab, Actemra), anti-IL-6 antibodies (sarilumab, olokizumab), CTLA4-Ig (Abatacept/Orencia), anti-IL-6 antibodies (ASKP1240, CCFZ533X2201) ), amphetamine (Campath), anti-CD20 (rituximab), bevacizumab (LEA29Y), sirolimus (Rapimune), everolimus, tacrolimus (Prograf), Zele-napax, Silimict, Remicade,
  • one or more immunosuppressive agents/drugs may be used together or sequentially.
  • One or more immunosuppressive agents/drugs can be used to induce therapy or to maintain treatment.
  • the same or different drugs can be used in the induction and maintenance phases.
  • daclizumab Zenapax
  • tacrolimus Prograf
  • sirolimus Rostune
  • Non-pharmacological regimens can also be used to achieve immunosuppression, including but not limited to whole body irradiation, thymic irradiation, and total and/or partial splenectomy. These technologies are also available Used in combination with one or more immunosuppressive drugs.
  • an antigen binding unit, chimeric antigen receptor, composition, vector or host cell described herein can be administered in combination with another therapeutic agent.
  • the additional therapeutic agent is a chemotherapeutic agent, such as those described in US20140271820.
  • Chemotherapeutic agents that can be used in conjunction with the immune response cells of the invention include, but are not limited to, mitotic inhibitors (vinca alkaloids), including vincristine, vinblastine, vindesine, and novibin (TM) (vinorelbine) , 5'-dehydro sulfide); topoisomerase I inhibitors such as camptothecin compound, including Camptosar TM (irinotecan HCL), Hycamtin TM (topotecan HCL) and derived from camptothecin Other compounds of its analogs; podophyllotoxin derivatives such as etoposide, teniposide and midozozoz; alkylating agents cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide , carmustine, busulfan, chlorambucil, briquetazine, uracil mustard, cloprofen and dacarbazine; antimetabolites, including c
  • chemotherapeutic agents that can be used in conjunction with the immune response cells of the invention include, but are not limited to, anti-angiogenic agents, including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligos) Nucleotide) and other angiogenesis inhibitors such as angiostatin, endostatin, interferon, interleukin-1 (including alpha and beta) interleukin 12, retinoic acid and metalloproteinase-1 and -2 tissue inhibition Agent.
  • anti-angiogenic agents including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligos) Nucleotide) and other angiogenesis inhibitors such as angiostatin, endostatin, interferon, interleukin-1 (including alpha and beta) interleukin 12, retinoic acid and metalloproteinase-1 and -2 tissue inhibition Agent.
  • the CD28 costimulatory signal domain is abbreviated as 28; CD3 ⁇ is abbreviated as Z; 4-1BB or CD137 is abbreviated as BB.
  • a chimeric antigen receptor constructed by the scFv and CD3 ⁇ and CD28 co-stimulatory signal domains of the code 8E5-2I as an intracellular signal domain can be referred to as 8E5-2I-28Z.
  • the construction of CAR for different antigens is in this way.
  • Antibody fragments were obtained using standard biological protocols. Briefly, 8-week old Balb/c mice were immunized with a eukaryotic expression vector containing the human CLD18A2 full-length sequence (NCBI Reference Sequence: NM_001002026.2). The spleen of the immunized mouse is removed, and a monoclonal antibody is obtained using a biological scheme conventional in the art.
  • Figure 1B shows an example of the binding of hybridoma supernatants 2B1, 3E12, 4A11, 8E5 to HEK293 cells stably transfected with human CLD18A2 or CLD18A1 as determined by flow cytometry.
  • FIG. 1B after two rounds of subcloning, most of the antibodies 2B1, 3E12, 4A11, and 8E5 were subcloned specifically to bind human CLD18A2 but not human CLD18A1, and the average fluorescence intensity differed by more than 5 times.
  • RNA from cell pellets The Plus RNA Purification kit (Invitrogen, 12183-555) instructions extract total RNA from cell pellets.
  • the cDNA was reverse transcribed according to the instructions using total RNA as a template and High capacity RNA to cDNA kit (Invitrogen, 4387406).
  • the cDNA was used as a template and amplified using the constant region primer of the antibody using a 5'-Full RACE kit (TAKARA, D315).
  • the PCR product was separated by 1.5% agarose gel electrophoresis, and the DNA fragment was purified and recovered. The results were as follows:
  • the flexible amino acid GGGGSGGGGSGGGGS (SEQ ID NO: 93) was introduced as the linker to constitute the scFv; the appropriate restriction sites and protective bases were introduced upstream of the VH, and appropriate primers were introduced downstream of the VL.
  • the restriction enzyme site and the protection base are digested and ligated into the eukaryotic expression vector (see the vector pH or vector pK used in CN101602808).
  • 293fectin TM Transfection reagent Invitrogen, 12347-019
  • transient transfection culture supernatants were collected and affinity purified, quantitative and qualitative analysis of the antibody obtained by SDSPAGE.
  • Binding of the anti-milin 18A2 scFv_Fc fusion antibody to HEK293 cells stably transfected with CLD18A2 was determined by flow cytometry.
  • Figure 3 shows the binding relative affinities of monoclonal antibodies 2B1, 8E5 scFv to human IgGl Fc, and to HEK293 cells stably transfected with human CLD18A2. It can be seen that the EC50 value of 2B1 is 3.56 nM, and the EC50 value of 8E5 is 49.19 nM.
  • the 2B1 mutant 2B1- was prepared by site-directed mutagenesis of antibody 2B1 by mutation in the heavy chain of antibody 2B1 or site 54 (N-glycosylation site).
  • N52D VH amino acid sequence: SEQ ID NO: 17; nucleotide sequence: SEQ ID NO: 18
  • 2B1-S54A VH amino acid sequence: SEQ ID NO: 19; nucleotide sequence: SEQ ID NO: 20.
  • amino acid sequence and nucleotide sequence of the light chains of 2B1-N52D and 2B1-S54A are the same as the corresponding sequences of 2B1.
  • the CDRs of the three antibody CDRs regions of Kabat, Chothia and IMGT were combined to determine the sequence of the six CDRs regions of the antibody light and heavy chains.
  • the antibody strain with the highest similarity to 2B1-S54A was selected as the antibody template.
  • IGHV1-46*01 in the IMGT database was selected as the antibody template of the hu2B1-S54A heavy chain.
  • IGKV4-1*01 was used as an antibody template for the hu2B1-S54A light chain.
  • the light and heavy chain CDR regions of 2B1-S54A were replaced with the CDR regions of the antibody template.
  • Identify the site of the back mutation (1) Align the designed humanized antibody with the starting antibody and check which amino acids in the antibody framework region are different. (2) Check whether these different amino acids are amino acids that support the structure of the antibody loop or amino acids that affect the binding of the variable regions of the light and heavy chains, and if so, these regions are relatively conserved regions. (3) Check whether there are some potential post-translational modification sites in humanized antibodies, such as deamidation site (Asn-Gly), isomerization site (Asp-Gly), surface exposed methionine , N glycosylation site (Asn-X-Ser/Thr, X is not proline).
  • hu2B1-S54A There are six potential reversion sites in the heavy chain of humanized antibody (hu2B1-S54A), which are M48I, V68A, M70L, R72A, T74K, T91S.
  • the light chain of the humanized antibody (hu2B1-S54A) has a potential reversion site and is L84V.
  • the nucleotide sequence was designed and synthesized based on the amino acid sequence of the humanized antibody (hu2B1-S54A).
  • a light chain nucleotide sequence (SEQ ID NO: 62) was synthesized; a heavy chain nucleotide sequence (SEQ ID NO: 60) was synthesized.
  • the synthetic antibody nucleotide sequence including the signal peptide, the variable region of the antibody, and the constant region are inserted into a mammalian cell expression vector to construct an antibody expression vector containing the heavy chain and the light chain, respectively, and identified by sequencing.
  • the heavy chain amino acid sequence of hu2B1-S54A is set forth in SEQ ID NO: 59; the nucleotide sequence is set forth in SEQ ID NO:60.
  • the amino acid sequence of the hu2B1-S54A light chain is set forth in SEQ ID NO: 61; the nucleotide sequence is set forth in SEQ ID NO:62.
  • the hu2B1-S54A heavy chain variable region amino acid sequence is set forth in SEQ ID NO: 23, the nucleotide sequence is set forth in SEQ ID NO: 24, and the light chain variable region amino acid sequence is set forth in SEQ ID NO: 21, nucleoside The acid sequence is shown in SEQ ID NO:22.
  • nucleotide sequence is designed and a light chain nucleotide sequence and a heavy chain nucleotide sequence are synthesized.
  • the heavy chain amino acid sequence of hu8E5-S62A is set forth in SEQ ID NO: 67; the nucleotide sequence is set forth in SEQ ID NO:68.
  • the amino acid sequence of the hu8E5-S62A light chain is set forth in SEQ ID NO: 65; the nucleotide sequence is set forth in SEQ ID NO:66.
  • the heavy chain variable region amino acid sequence of hu8E5-S62A is set forth in SEQ ID NO:27, the nucleotide sequence is set forth as SEQ ID NO:28, and the light chain variable region amino acid sequence of hu8E5-S62A is SEQ ID NO:25 As shown, the nucleotide sequence is set forth in SEQ ID NO:26; the HCDR2 in hu8E5-S62A is different from 8E5, the sequence of which is set forth in SEQ ID NO:85; the other HCDRs and LCDR are the same as 8E5.
  • the synthetic antibody nucleotide sequence including a signal peptide, a variable region of the antibody, and a constant region are inserted into a mammalian cell expression vector to construct an antibody expression vector containing a heavy chain and a light chain, respectively, and identified by sequencing. Transiently transfected into 293F cells by 293Fectin and expressed.
  • the 3D model of hu8E5 was established by Discovery Studio software, and the potential aggregation sites were analyzed. It was found that the 12th and 93rd valine of the heavy chain tend to cause aggregation of antibodies, which in turn affects the stability of the antibody. By performing point mutation analysis, it was found that the two sites were mutated to I, and the antibody was more stable. The results of the molecular sieve showed that after mutating these two sites to I (hu8E5-2I), the proportion of the monomeric form in the scFv_Fc fusion antibody was increased from the original 74% (hu8E5) to 87% (hu8E5-2I).
  • the heavy chain amino acid sequence of hu8E5-2I is set forth in SEQ ID NO: 63; the nucleotide sequence is set forth in SEQ ID NO:64.
  • the amino acid sequence of the hu8E5-2I light chain is set forth in SEQ ID NO: 65; the nucleotide sequence is set forth in SEQ ID NO:66.
  • HCDR2 in hu8E5-2I is different from 8E5, but is identical to hu8E5, and its sequence is shown in SEQ ID NO: 85; other HCDRs and LCDR are the same as 8E5.
  • the mutant hu8E5-2I of the humanized antibody hu8E5 was constructed as described in Example 3. Stably expressing human CLD18A2 using HEK293 cells (HEK-CLD18A2), using the Guava easyCyte TM HT System instrument, analysis of experimental data using GraphPad Prism, the results shown in Figure 7, after the scFv to human IgG1Fc hu8E5-2I portion fitted with stabilizer The binding affinity of HEK293 cells transfected with human CLD18A2 with an EC50 value of 9.22 nM. The mutant hu8E5-2I has a 5-fold increase in affinity compared to the parent antibody 8E5.
  • Example 6 In vitro functional assay and in vivo functional assay of humanized antibody hu2B1-S54A and humanized antibody hu8E5-2I
  • Humanized antibody hu2B1-S54A (light chain sequence: SEQ ID NO: 62, heavy chain sequence: SEQ ID NO: 60); humanized antibody hu8E5-2I by standard methods known to those skilled in the art (Light chain sequence: SEQ ID NO: 66, heavy chain sequence: SEQ ID NO: 64) was cloned into a eukaryotic expression vector. Affinity purification using 293fectin TM Transfection reagent (Invitrogen, 12347-019 ) were transiently transfected 293F cells of logarithmic growth phase, and culture supernatants were collected. The obtained antibodies were quantitatively and qualitatively analyzed by SDS PAGE.
  • the above eukaryotic expression vector uses the vector pH or vector pK used in CN101602808B.
  • Serum was prepared by drawing blood from healthy volunteers and centrifuging. A CCK-8 cell proliferation-toxicity test kit (Dojindo, #CK04) was used. HEK293 cells stably transfected with CLD18A2 or CLD18A1 were used as target cells. The cells were washed twice and resuspended in complete medium at a density of 1 x 10 5 cells/ml. 100 ⁇ l per well was seeded in a 96-well culture plate and cultured overnight at 37 °C. The next day, the final concentration of antibody added to each well was 20 ⁇ g/ml. Incubate for 30 min at 37 ° C incubator. Then, serum at a final concentration of 10% was added and incubated at 37 ° C for 1.5 hours.
  • Percent lysis (cell control well - experimental well) / cell control well - (cell control well - antibody control well) / cell control well.
  • Figure 8A is a comparison of CDC results of humanized antibodies hu2B1-S54A, hu8E5-2I and chimeric antibody ch-163E12 against HEK293 cells transfected with CLD18A2.
  • the experimental results showed that the CDC effects of hu2B1-S54A and hu8E5-2I against HEK293-CLD18A2 were 79.88% and 82.65%, respectively, and the CDC effect of ch-163E12 under the same reaction conditions was lower than 55. %.
  • Figure 8B is a comparison of the CDC results of humanized antibodies hu2B1-S54A, hu8E5-2I and chimeric antibody ch-163E12 against HEK293 cells transfected with CLD18A1.
  • the results of the experiment show that ch-163E12 also has a certain effect on cells expressing 18A1.
  • ADCC Antibody-dependent cellular toxicity
  • the ADCC activity of the humanized antibody claudin 18A2 antibody was measured by a lactate dehydrogenase (LDH) release assay using a cytoTox 96 non-radioactive cytotoxicity assay kit (Promega, Madison, USA).
  • LDH lactate dehydrogenase
  • Human peripheral blood mononuclear cells (PBMC) were purified from citrated whole blood by standard Ficoll-paque separation and resuspended in complete medium supplemented with 10% fetal bovine serum (FBS, Gibco) (RPMI-1640 culture) In the Gibco, the density was 8 ⁇ 10 6 cells/ml.
  • FBS fetal bovine serum
  • the cells were washed twice and resuspended in complete culture at a density of 2 ⁇ 10 5 cells/ml. Incubate PBMC with antibody at a final concentration of 20 ug/ml for 30 minutes at 37 ° C, then add 50 ⁇ l of antibody and effector cells to 50 ⁇ l of target cells according to a 50:1, 20:1, 10:1 ratio. 10 4 target cells) After incubating for 4 hours at 37 ° C, the cells were centrifuged, 50 ⁇ l of cell-free supernatant samples were collected, transferred to a flat-bottomed 96-well plate, and assayed.
  • Percentage of lysis was calculated as follows: (sample release - target spontaneous Release-effector cell spontaneous release) / (maximum release - target spontaneous release) * 100; wherein the spontaneous release of the target is fluorescence in the well containing only the target cell, and the spontaneous release of the effector cell is fluorescence in the well containing only the effector cell, and Maximum release is in wells containing target cells that have been treated with lysis buffer Light.
  • Figure 9 is a comparison of ADCC results for the humanized antibodies hu2B1-S54A, hu8E5-2I and the known chimeric antibodies ch-163E12 and ch-175D10 (see CN103509110A).
  • the results showed that the humanized antibodies hu2B1-S54A and hu8E5-2I had a significantly higher ADCC effect than ch-175D10 at an antibody concentration of 20 ⁇ g/ml and a target ratio of 50:1, 20:1 and 10:1.
  • the ADCC effect of ch-163E12 on the target ratio of 50:1 against HEK293-CLD18A2 was 62.84% and 72.88%, respectively.
  • a tumor mass of about 3 mm ⁇ 3 mm ⁇ 3 mm was inoculated subcutaneously into the right axilla of BALB/c nude mice. On the day of tumor cell inoculation, D0 days, tumors were inoculated for D27 days, and tumor volume was measured and randomly divided into 5 groups.
  • the specific groups are as follows: (1) PBS (phosphate buffer) control group; (2) hu8E5-2I antibody treatment group (40 mg/kg); (3) EOF treatment group (E is epirubicin: 1.25 mg/kg) O is oxaliplatin: 3.25 mg/kg; F is 5-fluorouracil: 56.25 mg/kg) + PBS; (4) hu8E5-2I antibody (40 mg/kg) + EOF treatment group (1.25 mg/kg tenderness) Bixing+3.25mg/kg oxaliplatin +56.25mg/kg 5-fluorouracil); (5)ch175D10 antibody (40mg/kg)+EOF treatment group (1.25mg/kg epirubicin +3.25mg/kg Saliplatin + 56.25mg / kg 5-fluorouracil). Dosage: EOF was administered once a week for 2 weeks; hu8E5-2I and ch175D10 antibodies were administered 3 times per week for 2 weeks.
  • the results are shown in Fig. 10.
  • the tumor was inoculated for D56 days, the EOF was injected twice, and the antibody was injected 6 times, and the mice were sacrificed by neck.
  • the tumor inhibition rates were 21.13% in the hu8E5-2I monoclonal antibody group, 47.89% in the EOF+PBS treatment group, 81.69% in the EOF+mAb hu8E5-2I treatment group, and 71.83% in the EOF+mAb 175D10 treatment group.
  • a lentiviral plasmid encoding the second and third generation chimeric antigen receptors of the humanized antibody hu8E5 was constructed using PRRLSIN-cPPT.EF-1 ⁇ as a vector, including PRRLSIN-cPPT.EF-1 ⁇ -hu8E5-28Z, PRRLSIN-cPPT .EF-1 ⁇ -hu8E5-BBZ and PRRLSIN-cPPT.EF-1 ⁇ -hu8E5-28BBZ.
  • Hu8E5-28Z mainly includes (from 5' to 3' end): CD8 ⁇ signal peptide (SEQ ID NO: 70), hu8E5 scFV (VH: SEQ ID NO: 27, VL: SEQ ID NO: 25, Linker: SEQ ID NO: 93), CD8 hinge (SEQ ID NO: 72), CD28 transmembrane region (SEQ ID NO: 74) and intracellular signaling domain (SEQ ID NO: 76), and intracellular CD3 of CD3 (SEQ. ID NO: 78) The coding sequence.
  • hu8E5-BBZ mainly includes (from 5' to 3' end): CD8 ⁇ signal peptide (SEQ ID NO: 70), hu8E5 scFV (VH: SEQ ID NO: 27, VL: SEQ ID NO: 25, Linker: SEQ ID NO:93), CD8 hinge (SEQ ID NO:72) and CD8 transmembrane region (SEQ ID NO:80), CD137 intracellular signaling domain (SEQ ID NO:82), and CD3 ⁇ (SEQ ID NO: 78) The coding sequence.
  • hu8E5-28BBZ mainly includes (from 5' to 3' end): CD8 ⁇ signal peptide (SEQ ID NO: 70), hu8E5-scFV, CD8hinge (SEQ ID NO: 72), CD28 transmembrane region (SEQ ID NO: 74) and the coding sequence of the intracellular domain (SEQ ID NO: 76), the CD137 intracellular signaling domain (SEQ ID NO: 82), and the CD3 (SEQ ID NO: 78).
  • the lentiviral plasmid PRRLSIN-cPPT.EF-1 ⁇ -hu8E5-2I-28Z expressing the second-generation chimeric antigen receptor of the humanized antibody hu8E5-2I was constructed using PRRLSIN-cPPT.EF-1 ⁇ as a vector.
  • hu8E5-2I-28Z mainly includes (arranged from the 5' end to the 3' end): CD8 ⁇ signal peptide (SEQ ID NO: 70), hu8E5-2I scFV (VH: SEQ ID NO: 29, VL: SEQ ID NO: 25, Linker: SEQ ID NO: 93), CD8hinge (SEQ ID NO: 72), CD28 transmembrane region (SEQ ID NO: 74) and intracellular signaling domain (SEQ ID NO: 76), and intracellular signals of CD3
  • the coding sequence for the transduction domain CD3 SEQ ID NO: 78).
  • the lentiviral plasmid PRRLSIN-cPPT.EF-1 ⁇ -hu2B1-S54A-28Z expressing the second-generation chimeric antigen receptor of the humanized antibody hu2B1-S54A was constructed using PRRLSIN-cPPT.EF-1 ⁇ as a vector.
  • hu2B1-S54A-28Z mainly includes (arranged from the 5' end to the 3' end): CD8 ⁇ signal peptide (SEQ ID NO: 70), hu2B1-S54A scFV (VH: SEQ ID NO: 23; VL: SEQ ID NO: 21; Linker: SEQ ID NO: 93), CD8 hinge (SEQ ID NO: 72), CD28 transmembrane region (SEQ ID NO: 74) and intracellular region (SEQ ID NO: 76), and intracellular CD3Z of CD3 ( The coding sequence of SEQ ID NO: 78).
  • Example 8 lentiviral packaging and titer determination
  • 293T cells cultured to the 6th to 10th passages were inoculated at a virus density of 5 ⁇ 10 6 in a 10 cm culture dish, and cultured at 37 ° C, 5% CO 2 overnight for transfection, and the medium was containing 10% fetal bovine serum ( Gibico) DMEM.
  • the target gene plasmid PRRLSIN-cPPT.EF-1 ⁇ -EGFP (Mock) and the different CAR plasmids prepared in Example 9 were 5.4 ⁇ g and the packaging plasmid pRsv-REV 6.2 ⁇ g, RRE-PMDLg 6.2 ⁇ g, and Vsvg 2.4 ⁇ g were dissolved in 800 ⁇ L.
  • the concentrated virus titers are:
  • hu8E5-28Z 2.3 ⁇ 10 7 U/ml
  • hu8E5-BBZ 6.65 ⁇ 10 7 U/ml
  • hu8E5-2I-28Z 1.54 ⁇ 10 8 U/ml
  • hu2B1-S54A-28Z 1.14 ⁇ 10 8 U/ml.
  • Example 9 Determination of cytotoxicity of lentiviral transduced T lymphocytes and CAR-T cells
  • lymphocyte culture medium at a density of about 1 ⁇ 10 6 /mL, and add magnetic beads (Invitrogen) and final concentration simultaneously coated with anti-CD3 and CD28 antibodies according to the magnetic bead: cell ratio of 1:1. 300 U/mL recombinant human IL-2 was stimulated for 48 h;
  • Retronectin coated 24-well plates 380 ⁇ l of 5 ⁇ g/ml retronectin solution (PBS) was added to each well, and after overnight incubation at 4 ° C, the retronectin solution (PBS) in a 24-well plate was discarded, and washed twice with 1 ml of PBS;
  • Expansion culture The infected cells were passaged every other day at a density of 5 ⁇ 10 5 /mL, and a recombinant human IL-2 having a final concentration of 300 U/mL was supplemented in the lymphocyte culture solution.
  • T lymphocytes On the 7th day of culture, 1 ⁇ 10 6 T cells were taken, aliquoted in a 2 ml centrifuge tube, centrifuged at 4 ° C, 5000 rpm for 5 min, the supernatant was discarded, and PBS was washed twice.
  • Control cells were incubated with 50 ⁇ l of PE-SA (1:200 dilution) antibody for 45 min on ice, washed twice with PBS (2% NBS), and resuspended as a control; cells in the test group +50 ⁇ l 1:50 diluted biotin -Goat anti human IgG, F(ab')2 antibody, incubated on ice for 45 min; washed twice with PBS (2% NBS).
  • Target cells 75 ⁇ L of 2 ⁇ 10 5 /mL of 293T-A1 cells, 293T-A2 cells, gastric adenocarcinoma cell line AGS, AGS-A2, gastric cancer cell lines BGC-823, and BGC-823-A2 cells were inoculated separately.
  • Gastric adenocarcinoma cell line AGS, gastric cancer cell line BGC-823 were purchased from ATCC cell bank, 293T-A1 cells, 293T-A2 cells, AGS-A2, BGC-823-A2 cells were constructed with reference to CN101602808B, among which 293T- A2 cells, AGS-A2, and BGC-823-A2 cells were CLD18A2-positive cells, and the rest were CLD18A2-negative cells.
  • Each group is provided with 4 duplicate wells, and the average of 4 replicate wells is taken.
  • the detection time is 18h.
  • Each experimental group each target cell + CAR T expressing different chimeric antigen receptors
  • Control group 1 maximum release of LDH from target cells
  • Control group 2 spontaneous release of LDH from target cells
  • Control group 3 effector cells spontaneously release LDH
  • CytoTox 96 non-radioactive cytotoxicity test kit (Promega) was used. CytoTox The assay quantitatively measures lactate dehydrogenase (LDH). Refer specifically to the instructions for the CytoTox 96 non-radioactive cytotoxicity test kit.
  • the cell killing results of different effect ratios are shown in Fig. 11 and Fig. 12.
  • the results showed that the CAR-T products showed good killing effects on 293T-A2, AGS-A2 and BGC-823-A2 cells positive for CLD18A2 expression.
  • hu8E5-28Z and hu8E5-2I-28Z cells can kill more than 60% of AGS-A2 cells at a target-to-target ratio of 3:1, and the killing of BGC-823-A2 cells can reach more than 90%.
  • the results also showed that the killing effect of each CAR T cell (except Hu2B1-S54A-28Z) on cells with negative expression of CLD18A2 was not obvious.
  • Example 11 In vitro induction of cytokine release assay by CLD18A2 CAR-T cells
  • the cytokines released by T cells (Mock), hu8E5-28Z and hu8E5-2I-28Z T cells transfected with no load were detected, respectively.
  • the above two T cells with good growth status within 1-2 weeks after lentivirus infection were collected, inoculated with 5 ⁇ 10 4 /200 ⁇ L (positive cell number) in 24-well plates, and inoculated with 5 ⁇ 10 4 according to the effective target ratio of 1:1. /200 ⁇ L/24 well target cells.
  • Target cells include 293T-A1, 293T-A2, AGS, AGS-A2, BGC-823, and BGC-823-A2 cells.
  • the supernatant was collected after 24 hours of co-cultivation.
  • the sandwich ELISA method was used to detect IL2, IFN- ⁇ and TNF- ⁇ released during the co-culture of CAR T lymphocytes with target cells in the supernatant.
  • Example 12 in vivo killing activity of CLD18A2 CAR-T cells
  • BGC-823-A2 cells collected in logarithmic growth phase and grown well were adjusted to a density of 2.5 ⁇ 10 7 /mL using physiological saline, and the volume of the injected cell suspension was 200 ⁇ L ( 5 ⁇ 10 6 /only) subcutaneously in the right temporal part of the mouse. The day of tumor cell inoculation is recorded as day 0.
  • Adoptive transfer of T cells 100 mg/kg of cyclophosphamide was intraperitoneally injected at a tumor volume of 100-150 mm 3 (Day 11), and 1 ⁇ 10 7 CAR T cells were infused through the tail vein 24 hours after injection (Mock) Cells, hu8E5-28Z T cells or hu8E5-2I-28Z T cells), and untransfected T cell group (Mock group) as a control, observed to measure the growth of subcutaneous xenografts.
  • the average tumor weight of the transplanted tumors of BGC-823-A2 in the hu8E5-28Z and hu8E5-2I-28Z treatment groups were 0.1 and 0.06 g, respectively, while the average tumor of the Mock control group was The weight was 0.53g, there was a significant difference between the CAR T cell treatment group and the Mock control group, and the P values were 0.0013 and ⁇ 0.0001, respectively.
  • Example 12 Referring to the animal model of BGC-823-A2 cell subcutaneous xenograft established in Example 12, the Mock, hu8E5-28Z, and hu8E5-2I-28Z cells were returned for 17 days, and the tumor tissues were taken and the CD3+ cells were detected by histochemistry.
  • IFNb cytokines were constructed on the basis of hu8E5-28Z with reference to the procedures of Examples 7-9.
  • the plasmid of hu8E5-28Z-IFNb CAR was constructed on the basis of hu8E5-2I-28Z CAR.
  • the hu8E5-2I-28Z-IFN CAR plasmid expressing IFNb cytokine was constructed and co-expressed with IFNb by lentiviral packaging and infection.
  • hu8E5-28Z-IFNb CAR T cells also labeled as hu8E5-28Z & IFNB
  • hu8E5-2I-28Z-IFN CAR T cells also labeled as hu8E5-2I-28Z & IFNB
  • hu8E5-28Z-IFNb CAR is encoded by the nucleotide sequence of SEQ ID NO: 90
  • hu8E5-2I-28Z-IFN CAR is encoded by the nucleotide sequence of SEQ ID NO:91.
  • the in vitro induced cytokine release assay was carried out in accordance with the procedure of Example 11, and as shown in Fig. 17A, the presence of IFN resulted in an increase in IFN- ⁇ cytokine secretion when hu8E5-28Z CAR T cells were co-incubated with target cells.
  • a tumor volume of 30 mm 3 100 mg/kg of cyclophosphamide was intraperitoneally injected, and 1.0 ⁇ 10 7 CAR-T cells (hu8E5-28Z T cells or hu8E5-2I-28Z-IFN T) were infused through the tail vein 24 hours after the injection. Cells), while the untransfected T cell group was used as a control. Observe and measure the growth of subcutaneous xenografts of gastric cancer PDX. As a result, as shown in Fig. 17B, in one of the 7 mice of the hu8E5-2I-28Z-IFN-treated group, the tumor completely resolved.
  • CAR-T cells The in vivo survival of CAR-T cells was determined using the PDX model described above. Peripheral blood was taken from the saphenous vein of mice after detection of D5, D7 and D10 after CAR-T cell infusion, and CAR-T cells were detected. Survival of T cells (Mock), hu8E5-28Z T cells or hu8E5-2I-28Z-IFN T cells) in vivo, as shown in Figure 17C, T cell survival in the hu8E5-2I-28Z-IFN T cell treated group The number was significantly higher than that of the hu8E5-28Z-28Z T cell treatment group.
  • a lentiviral plasmid expressing the chimeric antigen receptor of the humanized antibody hu8E5, including PRRLSIN-cPPT.EF-1 ⁇ - was constructed using PRRLSIN-cPPT.EF-1 ⁇ as a vector. hu8E5-28BBZ, PRRLSIN-cPPT.EF-1 ⁇ -hu8E5-2I-28Z.
  • the hu8E5-28BBZ sequence consists of CD8 ⁇ signal peptide (SEQ ID NO: 70), hu8E5-scFV, CD8hinge (SEQ ID NO: 72), CD28 transmembrane region (SEQ ID NO: 74) and intracellular segment (SEQ ID NO: 76) , CD137 intracellular signaling domain segment (SEQ ID NO: 82) and CD3 ⁇ (SEQ ID NO: 78); hu8E5-2I-28Z sequence from CD8 ⁇ signal peptide (SEQ ID NO: 70), hu8E5-2I scFV, CD8hinge (SEQ ID NO: 72), The CD28 transmembrane region (SEQ ID NO: 74) and the CD28 intracellular signaling domain region (SEQ ID NO: 76) and the intracellular segment CD3 (CD ID NO: 78) of CD3.
  • Retronectin coated 24 well plates 380 ⁇ l of 5 ⁇ g/ml retronectin solution (PBS) was added to each well, incubated overnight at 4 degrees, and the cells were seeded in a 24-well plate coated with retronectin, the number of cells per well was 5 ⁇ 105.
  • the volume of the culture solution is 500 ⁇ l;
  • Amplification culture The infected cells were passaged every other day at a density of 5 ⁇ 10 5 /mL, and a recombinant human IL-2 having a final concentration of 500 U/mL was supplemented in the lymphocyte culture solution.
  • Control cells were incubated with 50 ⁇ l of PE-SA (1:200 dilution) antibody on ice, resuspended as a control; test group +50 ⁇ l 1:50 diluted biotin-Goat anti human IgG, F(ab')2 The antibody was incubated on ice for 45 min; washed twice with PBS (2% NBS); 50 ⁇ l of PE-SA (1:200 dilution) antibody was added to incubated on ice;
  • Cytotoxicity assay target cells: 10 ⁇ 4 AGS, AGS-A2, BGC-823, and BGC-823-A2 cells were seeded in 96well plates, respectively; effector cells: target ratio 6:1, 3:1, 1.5: 1 NK-92 and CAR NK-92 cells were added; 5 replicate wells were set in each group, and the average of 5 replicate wells was taken. The detection time is 4h.
  • Each experimental group and each control group were as follows: each experimental group: each target cell + the above effector cells; the control group 1: the target cell released the largest LDH; the control group 2: the target cell spontaneously released LDH; the control group 3: the effector cell spontaneous release LDH.
  • CytoTox 96 non-radioactive cytotoxicity test kit (Promega) was used. Refer specifically to the instructions for the CytoTox 96 non-radioactive cytotoxicity test kit.
  • the cytotoxicity calculation formula is:

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Abstract

本发明提供了抗密蛋白18A2的抗体以及靶向密蛋白18A2的免疫效应细胞。还提供了用于诱导细胞死亡和治疗肿瘤的方法。

Description

抗密蛋白18A2的抗体及其应用
交叉引用
本申请要求2016年7月8日提交的中国专利申请201610536449.9、2017年4月26日提交的PCT国际申请PCT/CN2017/082024的权益，该申请通过引用将其全部内容并入本文。
技术领域
本发明属于免疫学领域，更具体地，本发明涉及抗密蛋白18A2的抗体及其应用。
背景技术
嵌合抗原受体(Chimeric antigen receptor，CAR)是一种人工重组受体，通常含有位于胞外区的单克隆抗体的抗原识别结构域、跨膜区、及免疫应答细胞的胞内激活信号结构域组成。
胃癌在全世界范围内是发病率最高的癌症之一，据世界卫生组织癌控项目的统计数据，全球每年死于癌症的患者高达700万，其中死于胃癌的患者占了70万例。与常规的胃癌治疗方案相比，基于抗体的治疗方案由于具有高特异性和低副作用，具有深远的应用前景。
密蛋白18(Claudin 18，CLD18)是位于上皮和内皮的紧密连接中的内在膜蛋白，分子量约为27.9KD。GenBank登记号为剪接变体1(CLD18A1、CLD18.1)：NP_057453、NM016369，以及剪接变体2(CLD18A2、CLD18.2)：NM_001002026、NP_001002026。图1A显示了密蛋白18A2(SEQ ID NO:55)和密蛋白18A1(SEQ ID NO:57)的同一性比较。在正常细胞中，CLD18A1在肺和胃的上皮中选择性表达，而CLD18A2则在正常胃上皮短寿细胞中呈现微量表达，但在肿瘤细胞中，CLD18A2在多种癌症类型中均呈现强烈表达，如75％的胃癌患者高表达CLD18A2，50％的胰腺癌患者高表达CLD18A2，30％的食管癌患者高表达CLD18A2，在肺癌等中也有高表达。因此，找到以更高的特异性结合CLD18A2而不结合CLD18A1的抗体，对癌症的治疗和检测具有重要的意义。
I型干扰素包含IFNα蛋白(一类从IFNA1到IFNA13共13个人基因编码的同一性蛋白)，IFNβ(由单一个人和小鼠基因IFNB1编码)以及其他研究较少的干扰素。 已有研究表明，I型干扰素对一些肿瘤有抗癌作用，可能归因于它们的免疫刺激功能。但是，I型干扰素的全身用药可能发生免疫抑制作用(Lotrich，F.E.Major depression during interferon-αtreatment:vulnerability and prevention.Dialogues Clin.Neurosci.11，417-425(2009))，并伴有重大的不良事件，最常见的有乏力、厌食、肝毒性、流感样症状和严重的抑郁症(Kreutzer，K.，Bonnekoh，B.，Franke，I.，Ulrich，J.& Gollnick，H.Sarcoidosis，myasthenia gravis and anterior ischaemic optic neuropathy:severe side effects of adjuvant interferon-α therapy in malignant melanoma？.J.Dtsch.Dermatol.Ges.2，689-694(in German)(2004))，这类严重的毒副作用严重限制了其应用。
发明内容
本发明克服了前述问题，并且具有额外的优势。
根据本发明的一个方面，本发明提供了可特异性地结合密蛋白18A2的抗体，其特征在于，该抗体包含选自氨基酸序列SEQ ID NO:31，32，33，37，38，39，43，44，45，49，50，51，83，84，85或其变体的重链CDR，和/或选自氨基酸序列SEQ ID NO：34，35，36，40，41，42，46，47，48，52，53，54，或其变体的轻链CDR。
在一些实施方案中，本发明的抗体选自(a)抗体，其包含重链可变区，所述重链可变区具有包含SEQ ID NO:31、SEQ ID NO:37、SEQ ID NO:43或SEQ ID NO:49之一所示的氨基酸序列的CDR1、包含SEQ ID NO:32、SEQ ID NO:38、SEQ ID NO:44、SEQ ID NO:50、SEQ ID NO:83、SEQ ID NO:84或SEQ ID NO:85之一所示的氨基酸序列的CDR2、包含SEQ ID NO:33、SEQ ID NO:39、SEQ ID NO:45或SEQ ID NO:51之一所示的氨基酸序列的CDR3；(b)抗体，其包含轻链可变区，所述轻链可变区具有包含SEQ ID NO:34、SEQ ID NO:40、SEQ ID NO:46或SEQ ID NO:52之一所示的氨基酸序列的CDR1、包含SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:47或SEQ ID NO:53之一所示的氨基酸序列的CDR2、包含SEQ ID NO:36、SEQ ID NO:42、SEQ ID NO:48或SEQ ID NO:54之一所示氨基酸序列的CDR3；(c)抗体，包含(a)所述抗体的重链可变区及(b)所述抗体的轻链可变区；(d)抗体，识别与(a)～(c)中任一项所述的抗体所识别的抗原决定部位相同的抗原决定部位。
在一些实施方案中，所述抗体的重链可变区的CDR1、CDR2、CDR3区分别为SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33；或者SEQ ID NO:37、 SEQ ID NO:38、SEQ ID NO:39；或者SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45；或者SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51；或者SEQ ID NO:31、SEQ ID NO:83、SEQ ID NO:33；或者SEQ ID NO:31、SEQ ID NO:84、SEQ ID NO:33；或者SEQ ID NO:49、SEQ ID NO 85、SEQ ID NO:51；和/或所述抗体的轻链可变区的CDR1、CDR2、CDR3区分别为SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36；或者SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42；或者SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48；或者SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54。
在一些实施方案中，抗体包括重链可变区和轻链可变区，其具有SEQ ID NO:3、或SEQ ID NO:7、SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:23、SEQ ID NO:27或SEQ ID NO:29之一所示氨基酸序列的重链可变区；SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:9、SEQ ID NO:13、SEQ ID NO:21或SEQ ID NO:25之一所示氨基酸序列的轻链可变区。在一些实施方案中，所述抗体是具有SEQ ID NO:3所示重链可变区，SEQ ID NO:1所示轻链可变区的抗体；具有SEQ ID NO:7所示重链可变区，SEQ ID NO:5所示轻链可变区的抗体；具有SEQ ID NO:11所示重链可变区，SEQ ID NO:9所示轻链可变区的抗体；具有SEQ ID NO:15所示重链可变区，SEQ ID NO:13所示轻链可变区的抗体；具有SEQ ID NO:17所示重链可变区，SEQ ID NO:1所示轻链可变区的抗体；具有SEQ ID NO:19所示重链可变区，SEQ ID NO:1所示轻链可变区的抗体；具有SEQ ID NO:23所示重链可变区，SEQ ID NO:21所示轻链可变区的抗体；具有SEQ ID NO:27所示重链可变区，SEQ ID NO:25所示轻链可变区的抗体；或具有SEQ ID NO:29所示重链可变区，SEQ ID NO:25所示轻链可变区的抗体。在一些实施方案中，所述抗体是具有SEQ ID NO:3所示重链可变区，SEQ ID NO:1所示轻链可变区的抗体；或具有SEQ ID NO:17所示重链可变区，SEQ ID NO:1所示轻链可变区的抗体；或具有SEQ ID NO:19所示重链可变区，SEQ ID NO:1所示轻链可变区的抗体；或具有SEQ ID NO:23所示重链可变区，SEQ ID NO:21所示轻链可变区的抗体；或具有SEQ ID NO:27所示重链可变区，SEQ ID NO:25所示轻链可变区的抗体；或具有SEQ ID NO:29所示重链可变区，SEQ ID NO:25所示轻链可变区的抗体。在一些实施方案中，所述的抗体是人源化的抗体，嵌合抗体或全人抗体；或者所述抗体是单克隆抗体；或者所述抗体为单链抗体或结构域抗体。在 一些实施方案中，所述的抗体是人源化的抗体，选自：具有SEQ ID NO:27所示的重链可变区和SEQ ID NO:25所示的轻链可变区的抗体；具有SEQ ID NO:23所示的重链可变区和SEQ ID NO:21所示的轻链可变区的抗体；具有SEQ ID NO:29所示的重链可变区和SEQ ID NO:25所示轻链可变区的抗体。在一些实施方案中，所述抗体选自具有SEQ ID NO:63所示的重链和SEQ ID NO:65所示的轻链的抗体；具有SEQ ID NO:61所示的轻链和SEQ ID NO:59所示的重链的抗体；以及具有SEQ ID NO:67所示的重链和SEQ ID NO:65所示的轻链的抗体。
根据本发明的一个方面，本发明提供了编码前述抗体的核酸。根据本发明的另一个方面，本发明提供了包含所述核酸的表达载体。根据本发明的另一个方面，本发明提供了一种宿主细胞，其包含本发明所述的表达载体或基因组中整合有本发明所述的核酸。
根据本发明的一个方面，本发明提供了本发明所述的抗体的用途，其用于制备特异性靶向表达密蛋白18A2的肿瘤细胞的靶向性药物，抗体药物偶联物或多功能抗体；或用于制备诊断肿瘤的试剂，该肿瘤表达密蛋白18A2；或用于制备嵌合抗原受体修饰的免疫细胞。在一些实施方案中，所述表达密蛋白18A2的肿瘤包括：胃癌，胰腺癌，食管癌，肺癌。
根据本发明的一个方面，本发明提供了包含本发明抗体的嵌合抗原受体，述的嵌合抗原受体包含顺序连接的：本发明的抗体、跨膜区和胞内信号区。在一些实施方案中，所述的胞内信号区选自：CD3ζ，FcεRIγ，CD27，CD28，CD137，CD134，MyD88，CD40的胞内信号区序列，或其组合；或所述的跨膜区包含CD8或CD28的跨膜区。在一些实施方案中，所述的嵌合抗原受体包括如下的顺序连接的抗体，跨膜区和胞内信号区：本发明的抗体、CD8和CD3ζ；本发明的抗体、CD8、CD137和CD3ζ；或本发明的抗体、CD28分子的跨膜区、CD28分子的胞内信号区和CD3ζ；或本发明的抗体、CD28分子的跨膜区、CD28分子的胞内信号区、CD137和CD3ζ。
根据本发明的另一个方面，本发明提供了编码所述嵌合抗原受体的核酸。根据本发明的另一个方面，本发明提供了表达载体，其包含本发明所述的核酸。根据本发明的另一个方面，本发明提供了一种病毒，其包含本发明所述的载体。
根据本发明的一个方面，本发明提供了本发明所述的嵌合抗原受体、核酸、表达载体或者病毒用于制备靶向表达密蛋白18A2的肿瘤细胞的嵌合抗原受体修饰的免疫细胞的用途。在一些实施方案中，所述的表达密蛋白18A2的肿瘤包括：胃 癌，胰腺癌，食管癌，肺癌。
根据本发明的一个方面，本发明提供了嵌合抗原受体修饰的免疫细胞，其转导有本发明所述的核酸、表达载体或病毒；或其表面表达本发明所述的嵌合抗原受体。在一些实施方案中，所述的免疫细胞为：T淋巴细胞、NK细胞或者NKT淋巴细胞。在一些实施方案中，所述的免疫细胞其还携带外源的细胞因子的编码序列；或其还表达另一种嵌合抗原受体，该受体不含有CD3ζ，但含有CD28的胞内信号结构域、CD137的胞内信号结构域或者这两者的组合；或其还表达趋化因子受体；较佳地，所述的趋化因子受体包括：CCR；或其还表达能降低PD-1表达的siRNA或者阻断PD-L1的蛋白；或其细胞中内源性的PD-1被基因编辑技术敲除；或其还表达安全开关。
根据本发明的一个方面，本发明提供了所述嵌合抗原受体修饰的免疫细胞的用途，其用于制备抑制肿瘤的药物，所述的肿瘤是表达密蛋白18A2的肿瘤；较佳地，所述的肿瘤包括：胃癌，胰腺癌，食管癌，肺癌。
根据本发明的一个方面，本发明提供了一种多功能免疫缀合物，包括本发明所述的抗体；以及与之连接的功能性分子；所述的功能性分子选自：靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子或可检测标记物。在一些实施方案中，所述的靶向免疫细胞的表面标志物的分子是结合T细胞表面标志物的抗体，其与本发明所述的抗体形成T细胞参与的双功能抗体。根据本发明的另一个方面，本发明提供了编码所述的多功能免疫缀合物的核酸及其用于制备抗肿瘤药物的用途。在一些实施方案中，所述编码所述的多功能免疫缀合物的核酸用于制备诊断肿瘤的试剂，该肿瘤表达密蛋白18A2。在一些实施方案中，所述编码所述的多功能免疫缀合物的核酸用于制备嵌合抗原受体修饰的免疫细胞。在一些实施方案中，所述免疫细胞包括：T淋巴细胞、NK细胞或者NKT淋巴细胞。
根据本发明的一个方面，本发明提供了一种药物组合物，其包含本发明的抗体或编码所述抗体的核酸。根据本发明的一个方面，本发明提供了一种药物组合物，其包含本发明的免疫缀合物或编码所述缀合物的核酸。根据本发明的一个方面，本发明提供了一种药物组合物，其包含本发明的嵌合抗原受体或编码所述嵌合抗原受体的核酸。根据本发明的一个方面，本发明提供了一种药物组合物，其包含本发明的嵌合抗原受体修饰的免疫细胞。在一些实施方案中，所述药物组合物包含药学上可接受的载体或赋形剂。
根据本发明的一个方面，本发明提供了一种药盒，其包括容器，以及位于容器中的本发明药物组合物；或容器，以及位于容器中的本发明抗体或编码该抗体的核酸；或本发明所述的免疫辍合物或编码该辍合物的核酸；或本发明所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或本发明所述的嵌合抗原受体修饰的免疫细胞。
根据本发明的一个方面，本发明提供了包含轻链CDR和重链CDR的抗原结合单元，其中所述抗原结合单元特异性地结合密蛋白18A2肽；并且其中所述抗原结合单元不显著结合密蛋白18A1肽。根据本发明的另一个方面，本发明提供了包含轻链CDR和重链CDR的抗原结合单元，其中所述抗原结合单元特异性地结合密蛋白18A2肽；并且其中所述抗原结合单元与参考抗原结合单元相比，显示更少的与密蛋白18A1肽的非特异性结合。在一些实施方案中，所述所述参考抗原结合单元包含SEQ ID NO:86或SEQ ID NO:88的轻链和/或SEQ ID NO:87或SEQ ID NO:89的重链氨基酸序列。在一些实施方案中，所述密蛋白18A2肽包含SEQ ID NO:55的氨基酸序列。在一些实施方案中，所述密蛋白18A1肽包含SEQ ID NO:57的氨基酸序列。在一些实施方案中，所述抗原结合单元与所述密蛋白18A1肽的非特异性结合不超过其与所述密蛋白18A2肽的特异性结合的20％。在一些实施方案中，所述结合特异性通过流式细胞术测定。在一些实施方案中，所述结合特异性通过FACS测定。在一些实施方案中，所述结合特异性通过ELISA测定。在一些实施方案中，所述抗原结合单元与所述密蛋白18A2肽结合的EC50低于约100nM。在一些实施方案中，所述抗原结合单元是单克隆抗体、人源化抗体、嵌合抗体、多价抗体或嵌合抗原受体。在一些实施方案中，所述轻链CDR包含LCDR1、LCDR2和LCDR3；并且所述重链CDR包含HCDR1、HCDR2和HCDR3；其中所述LCDR1、LCDR2和LCDR3分别具有选自以下的氨基酸序列：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:53和SEQ ID NO:54；以及其中所述HCDR1、HCDR2和HCDR3分别具有选自以下的氨基酸序列：SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85。在一些实施方案中，所述LCDR1包含选自以下的氨基酸序列：SEQ ID NO:34、SEQ ID NO:40、SEQ ID NO:46和SEQ ID NO:52。在一些实施方案中，所述LCDR2 包含选自以下的氨基酸序列：SEQ ID NO:34、SEQ ID NO:41、SEQ ID NO:47和SEQ ID NO:53。在一些实施方案中，所述LCDR3包含选自以下的氨基酸序列：SEQ ID NO:35、SEQ ID NO:42、SEQ ID NO:48和SEQ ID NO:54。在一些实施方案中，所述HCDR1包含选自以下的氨基酸序列：SEQ ID NO:31、SEQ ID NO:37、SEQ ID NO:43和SEQ ID NO:49。在一些实施方案中，所述HCDR2包含选自以下的氨基酸序列：SEQ ID NO:32、SEQ ID NO:38、SEQ ID NO:44、SEQ ID NO:50、SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85。在一些实施方案中，所述HCDR3包含选自以下的氨基酸序列：SEQ ID NO:33、SEQ ID NO:39、SEQ ID NO:45和SEQ ID NO:51。在一些实施方案中，所述抗原结合单元是scFv、Fv、Fab或(Fab)2。
根据本发明的一个方面，本发明提供包含轻链CDR和重链CDR的抗原结合单元，其中所述轻链CDR包含LCDR1、LCDR2和LCDR3；并且所述重链CDR包含HCDR1、HCDR2和HCDR3；其中所述LCDR1、LCDR2和LCDR3包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:53和SEQ ID NO:54；以及其中所述HCDR1、HCDR2和HCDR3包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85。在一些实施方案中，所述轻链CDR包含LCDR1、LCDR2和LCDR3；并且所述重链CDR包含HCDR1、HCDR2和HCDR3；所述LCDR1、LCDR2和LCDR3分别具有选自以下的氨基酸序列：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:53和SEQ ID NO:54；以及所述HCDR1、HCDR2和HCDR3分别具有选自以下的氨基酸序列：SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85。在一些实施方案中，所述LCDR1包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:34、SEQ ID NO:40、SEQ ID NO:46和SEQ ID NO:52。在一些实施方案中，所述LCDR2包含与选自以下的氨基酸序 列具有至少80％同一性的氨基酸序列：SEQ ID NO:34、SEQ ID NO:41、SEQ ID NO:47和SEQ ID NO:53。在一些实施方案中，所述LCDR3包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:35、SEQ ID NO:42、SEQ ID NO:48和SEQ ID NO:54。在一些实施方案中，所述HCDR1包含包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:31、SEQ ID NO:37、SEQ ID NO:43和SEQ ID NO:49。在一些实施方案中，所述HCDR2包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:32、SEQ ID NO:38、SEQ ID NO:44、SEQ ID NO:50、SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85。在一些实施方案中，所述HCDR3包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:33、SEQ ID NO:39、SEQ ID NO:45和SEQ ID NO:51。在一些实施方案中，所述抗原结合单元是单克隆抗体、人源化抗体、嵌合抗体、多价抗体或嵌合抗原受体。在一些实施方案中，所述抗原结合单元是scFv、Fv、Fab或(Fab)2。
根据本发明的一个方面，本发明提供嵌合抗原受体，其包含胞外抗原结合单元、跨膜域和胞内域，其中所述胞外抗原结合单元包含本发明的抗原结合单元。根据本发明的一个方面，本发明提供包含本发明抗原结合单元或嵌合抗原受体的组合物。在一些实施方案中，所述组合物包含I型干扰素。根据本发明的一个方面，本发明提供分离的核酸，其编码本发明的抗原结合单元或嵌合抗原受体、以及任选的I型干扰素。根据本发明的一个方面，本发明提供载体，其包含本发明的核酸。
根据本发明的一个方面，本发明提供宿主细胞，其表达本发明的抗原结合单元或嵌合抗原受体，以及任选的I型干扰素。根据本发明的一个方面，本发明提供宿主细胞，其包含本发明抗原结合单元或嵌合抗原受体、以及任选的I型干扰素的核酸。在一些实施方案中，所述宿主细胞是免疫应答细胞。在一些实施方案中，所述宿主细胞是T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、和/或调节性T细胞。在一些实施方案中，所述宿主细胞是NK92细胞。
在一些实施方案中，所述宿主细胞对包含密蛋白18A2肽的细胞具有细胞毒性，所述密蛋白18A2肽包含SEQ ID NO:55的氨基酸序列。在一些实施方案中，所述宿主细胞对包含密蛋白18A1肽但不包含密蛋白18A2肽的细胞不具有显著的细胞毒性，所述密蛋白18A1肽包含SEQ ID NO:57的氨基酸序列，且所述密蛋白18A2肽包含SEQ ID NO:55的氨基酸序列。
根据本发明的一个方面，本发明提供生成本发明的抗原结合单元或嵌合抗原受 体或组合物的方法，包括：在适合的条件下培养本发明的宿主细胞，并获得所述宿主细胞的表达产物。
根据本发明的一个方面，本发明提供诱导包含密蛋白18A2肽的细胞死亡的方法，包括使所述细胞与本发明的抗原结合单元、嵌合抗原受体、组合物或宿主细胞接触。在一些实施方案中，使所述细胞与所述抗原结合单元、所述嵌合抗原受体、所述组合物或宿主细胞在体外接触。在一些实施方案中，使所述细胞与所述抗原结合单元、所述嵌合抗原受体、所述组合物或宿主细胞在体内接触。在一些实施方案中，所述细胞是癌细胞。在一些实施方案中，所述细胞是实体瘤细胞。在一些实施方案中，所述细胞选自：胃癌细胞、食道癌细胞、肠癌细胞、胰腺癌细胞、肾母细胞瘤细胞、肺癌细胞、卵巢癌细胞、结肠癌细胞、直肠癌细胞、肝癌细胞、头颈癌细胞、慢性骨髓性白血病细胞和胆囊癌细胞。
根据本发明的一个方面，本发明提供治疗有需要的个体的肿瘤的方法，所述方法包括向所述个体给予有效量的本发明的抗原结合单元、嵌合抗原受体、组合物或宿主细胞。在一些实施方案中，所述肿瘤是实体瘤。在一些实施方案中，所述肿瘤是胃癌、食道癌、肠癌、胰腺癌、肾母瘤、肺癌、卵巢癌、结肠癌、直肠癌、肝癌、头颈癌、慢性骨髓性白血病或胆囊癌。在一些实施方案中，所述方法还包括向所述个体给予额外的治疗剂。在一些实施方案中，所述额外的治疗剂是选自以下的至少一种：表柔比星、奥沙利铂和5-氟尿嘧啶。
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图1A显示了密蛋白18A2(SEQ ID NO:55)和密蛋白18A1(SEQ ID NO:57)的同一性比较；图1B显示了通过流式细胞术测定的杂交瘤上清液2B1、3E12、4A11、和8E5与稳定转染了人CLD18A2及CLD18A1细胞的结合的HEK293细胞的结合。
图2显示了鼠抗2B1(重链可变区SEQ ID NO:3，轻链可变区SEQ ID NO:1)，3E12(重链可变区SEQ ID NO:7，轻链可变区SEQ ID NO:5)，4A11(重链可变区SEQ ID NO:11，轻链可变区SEQ ID NO:9)，8E5(重链可变区SEQ ID NO:15，轻链可变区SEQ ID NO:13)的序列比对。
图3显示了鼠抗2B1、8E5的ScFv与人IgG1Fc部分嵌合以后，与稳定转染了人CLD18A2的HEK293细胞的结合相对亲和力。
图4显示了改造过的鼠抗2B1抗体2B1-N52D，2B1-S54A与人IgG1Fc部分嵌合以后，与稳定转染了人CLD18A2的HEK293细胞的结合相对亲和力。
图5显示了人源化的hu2B1-S54A与人IgG1Fc部分嵌合以后，与稳定转染了人CLD18A2的HEK293细胞的结合相对亲和力。
图6显示了人源化的hu8E5与人IgG1Fc部分嵌合以后，与稳定转染了人CLD18A2的HEK293细胞的结合相对亲和力。
图7显示了改造过的人源化的hu8E5-2I与稳定转染了人CLD18A2的HEK293细胞的结合相对亲和力。
图8A比较了人源化抗体hu2B1-S54A、hu8E5-2I与已知嵌合抗体ch-163E12(参见CN103509110A)对转染了CLD18A2的HEK293细胞的CDC效应；图8B是比较人源化抗体hu2B1-S54A、hu8E5-2I和ch-163E12对转染了CLD18A1的HEK293细胞的CDC结果。
图9比较了人源化抗体hu2B1-S54A、hu8E5-2I与嵌合抗体ch-163E12、ch-175D10(参见CN103509110A)的ADCC效应。
图10比较了hu8E5-2I和ch-175D10的小鼠体内杀伤活性。
图11比较了hu8E5-28Z、hu8E5-BBZ以及hu8E5-28BBZ T细胞对不同细胞系的体外杀伤活性。
图12比较了hu8E5-28Z、hu8E5-2I-28Z、hu2B1-hs54A T细胞对不同细胞系的体外杀伤活性。
图13描述了CLDN18A2-CAR T体内治疗胃癌PDX小鼠皮下移植瘤模型中随时间对肿瘤体积影响的对比图(图13A)以及肿瘤照片对比图(图13B)。
图14为hu8E5-28Z和hu8E5-2I-28Z的细胞因子的分泌测定结果。
图15描述了CLDN18A2-CAR T体内治疗胃癌BGC-823-A2小鼠皮下移植瘤模型中随时间对肿瘤体积影响的对比图(图15A)、肿瘤重量对比图(图15B)以及肿瘤照片对比图(图15C)。
图16显示了CLDN18A2-CAR T的肿瘤浸润情况。
图17A显示了共表达IFN后的细胞分子分泌情况；图17B为含有IFN和不含IFN的CAR-T细胞在胃癌PDX模型皮下移植瘤中的抗肿瘤活性对比图；图17C为小鼠外周血中在回输CAR-T细胞5天、7天和10天的存活细胞数对比图。
图18A和图18B为构建CAR-NK细胞的质粒图。
图19为hu8E5-2I-28Z CAR-NK92和hu8E5-28BBZ CAR-NK92的阳性率测定。
图20为hu8E5-2I-28Z CAR-NK92细胞毒性的细胞毒性图。
图21为hu8E5-28BBZ CAR-NK92细胞毒性的细胞毒性图。
具体实施方式
以下具体说明详尽地展示了本文所公开的实施方案。应当理解，本说明书并非意欲仅限于此处所公开的具体的实施方案，而是可以发生改变。本领域技术人员将理解，本说明书中所公开的内容可以有多种改变或变化，而均涵盖于所公开的范围和原则之内。除非另有说明，每个实施方案均可与任何其他实施方案任意组合。
本文所公开的某些实施方案包含了数值范围，并且本发明的某些方面可采用范围的方式描述。除非另有说明，应当理解数值范围或者以范围描述的方式仅是出于简洁、便利的目的，并不应当认为是对本发明的范围的严格限定。因此，采用范围方式的描述应当被认为具体地公开了所有可能的子范围以及在该范围内的所有可能的具体数值点，正如这些子范围和数值点在本文中已经明确写出。例如，从1至6的范围的描述应当被认为具体公开了从1至3、1至4、1至5、2至4、2至6、3至6等的子范围，以及在这些范围内的具体的数值点，例如1、2、3、4、5、6。不论所述数值的宽窄，上述原则均同等适用。当采用范围描述时，该范围包括范围的端点。
当涉及可测量值比如量、暂时持续时间等时，术语"约”是指包括指定值的±20％、或在某些情况下±10％、或在某些情况下±5％、或在某些情况下±1％、或在某些情况下±0.1％的变化。
本文所用的术语“激活”和“活化”可互换使用，它们以及其语法上的其他形式可以指细胞从静止状态转变为活性状态的过程。该过程可以包括对抗原、迁移和/或功能活性状态的表型或遗传变化的响应。例如，术语“激活”可以指免疫细胞逐步活化的过程。例如，T细胞可能需要至少两个信号才能完全激活。第一信号可以在由抗原-MHC复合物接合TCR之后发生，而第二信号可以通过共刺激分子(参见表1中所列举的共刺激分子)的接合发生。在体外，抗CD3可以模拟所述第一 信号，抗CD28可以模拟所述第二信号。例如，工程化T细胞可以被表达的CAR激活。本文所用的免疫细胞激活或活化可以指已经被充分刺激以诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的状态。
本文所用的术语“共刺激性分子”是指免疫细胞如T细胞上的同源结合配偶体，其特异性地结合共刺激配体，从而介导共刺激反应，比如但不限于增殖。共刺激性分子为除了抗原受体或其配体之外的细胞表面分子，其促进有效的免疫应答。共刺激性分子包括但不限于MHCI类分子，BTLA和Toll配体受体，以及OX40、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)和4-1BB(CD137)。共刺激性分子的实例包括但不限于：CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a，以及特异性地结合CD83的配体。。
如本文所用的“共刺激信号”是指与第一信号，例如TCR/CD3结合，组合导致T细胞增殖和/或关键分子的上调或下调的信号。
本文所用的术语“抗原结合单元”是指免疫球蛋白分子和免疫分子的免疫活性部分，即含有与抗原特异性结合(“免疫反应”)的抗原结合位点的分子。术语“抗原结合单元”中还包括各种物种来源的免疫球蛋白分子，包括无脊椎动物和脊椎动物。结构上，最简单的天然存在的抗体(例如IgG)包含四条多肽链，通过二硫键相互连接的两条重(H)链和两条轻链(L)链。免疫球蛋白代表包括几种类型的分子的一大家族的分子，如IgD、IgG、IgA、IgM和IgE。术语“免疫球蛋白分子”包括例如杂交抗体或改变的抗体及其片段。已经显示抗体的抗原结合功能可以通过天然存在的抗体的片段进行。这些片段统称为“抗原结合单元”。术语“抗原结合单元”中还包括具有与表位吻合并识别表位的特定形状的任何含多肽链的分子结构，其中一个或多个非共价结合相互作用稳定分子结构和表位之间的复合物。所述抗原结合单元的实例包括Fab片段，由VL、VH、CL和CH1结构域组成的单价片段， 包含通过铰链区上的二硫桥连接的两个Fab片段的双价片段(F(ab)2片段)；由VH和CH1结构域组成的Fd片段，由抗体的单臂的VL和VH结构域组成的Fv片段；由VH结构域组成的dAb片段(Ward等人，Nature,341:544-546,1989)；和分离的互补决定区(CDR)或包含这样的抗原结合单元的任何融合蛋白。
术语“抗体”在本文中包括完整的抗体和任何抗原结合片段(即，“抗原结合部分”)或其单链。天然存在的“抗体”是包含通过二硫键链间连接的至少2个重(H)链和2个轻(L)链的糖蛋白。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为称为互补决定区(CDR)的具有高可变性的区域，其间隔以更保守的称为框架区(ER)的区域。每一个VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q))的结合。
术语“scFv”是指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非指定，否则如正如本文中使用的那样，scFv可以以任何顺序(例如相对于多肽的N-末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。
如本文中所用，术语“互补决定区”和“CDR”是指抗体可变区内赋予抗原特异性和结合亲和力的氨基酸的序列。一般而言，在每一个重链可变区中存在3个CDR(HCDR1、HCDR2、HCDR3)并且在轻链可变区中存在3个CDR(LCDR1、LCDR2、LCDR3)。
如果抗原结合单元以与其它参考抗原(包括多肽或其他物质)结合相比更大亲和力或亲合力结合抗原，则所述抗原结合单元与抗原“特异性结合”或与抗原是“免疫反应性的”。
本文所用的术语“人源化”用于非人抗体，例如啮齿动物或灵长类动物等，是含有来源于非人免疫球蛋白的最小序列的杂合免疫球蛋白，免疫球蛋白链或其片段。在大多数情况下，人源化抗体是人免疫球蛋白(受体抗体)，其中来自受体的互补 决定区(CDR)的残基被来自具有所需的特异性、亲和力和性能的非人物种(供体抗体)的CDR的残基替代，例如小鼠、大鼠、兔子或灵长类动物。在一些情况下，人免疫球蛋白的Fv框架区(FR)残基被相应的非人残基代替。此外，人源化抗体可以包含在受体抗体中也不存在于导入的CDR或框架序列中的残基。进行这些修饰以进一步改进和优化抗体性能并且当引入人体时使免疫原性最小化。在一些实例中，人源化抗体将包含基本上所有的至少一个、通常为两个可变结构域，其中所有或基本上所有的CDR区对应于非人免疫球蛋白的，并且所有或基本上所有的FR区域是人免疫球蛋白序列的区域。人源化抗体还可以包含至少一部分免疫球蛋白恒定区(Fc)，通常为人免疫球蛋白的恒定区。在一些实施方案中，“人源化抗体”可包括突变，例如通过体外随机或定点诱变或通过体内体细胞突变引入的突变。
本文所用的术语“免疫球蛋白”或“Ig”可以指作为抗体起作用的一类蛋白质。由B细胞表达的抗体有时称为嵌合抗原受体或抗原受体。包括在这类蛋白质中的五个成员是IgA、IgG、IgM、IgD和IgE，其中IgG是最常见的循环抗体。它是凝集、补体固定和其他抗体反应中最有效的免疫球蛋白，在防御细菌和病毒方面是重要的。例如，可以通过CAR识别肿瘤细胞抗原(或“肿瘤抗原”)或病原体抗原。
如本文所用，术语“分离的”是指与细胞成分或其他成分相分离，在这些成分中，多核苷酸、肽、多肽、蛋白质、抗体或其片段在自然状态通常是相关联的。如本领域技术人员将理解，非天然存在的多核苷酸、肽、多肽、蛋白质、抗体或其片段不需要“分离”以将其与天然存在的对应物区分开。此外，“浓缩”、“分离”或“稀释”的多核苷酸、肽、多肽、蛋白质、抗体或其片段可与其天然存在的对应物区分开，因为每体积分子的浓度或数量大于(“浓缩”)或小于(“稀释”)其天然存在的配对物的浓度。富集程度可以以绝对基础测量，例如每溶液体积的重量，或者可以相对于存在于源混合物中的另一潜在的干扰物测量。在一些实施方案中，本发明的技术方案优选的富集程度更高。因此，例如，优选2倍富集、更优选10倍富集、更优选100倍富集、更优选1000倍富集。也可以通过人工组装的方法，例如通过化学合成或重组表达，从而提供“分离的”物质。
本文所用的“抗原”是指被抗原结合单元识别并特异性结合的物质。抗原可以包括肽、蛋白质、糖蛋白、多糖和脂质，其部分及其组合。非限制性示例性抗原包括肿瘤抗原或病原体抗原。“抗原”也可以指引发免疫反应的分子。这种免疫反应可能涉及抗体产生或特定免疫活性细胞(immunologically-competent cells)的活化，或两者兼有。本领域技术人员将理解，任何大分子，包括实际上所有的蛋白质或肽， 都可以作为抗原。
术语“多肽”、“肽”和“蛋白质”在本文中可互换使用以指任何长度的氨基酸的聚合物。聚合物可以是直链、环状或支链的，它可以包含修饰的氨基酸，特别是保守修饰的氨基酸，并且它可以被非氨基酸中断。该术语还包括改性的氨基酸聚合物例如已经通过硫酸化、糖基化、脂化、乙酰化、磷酸化、碘化、甲基化、氧化、蛋白水解加工、异戊二烯化、外消旋化、硒酰化、转移-RNA介导的氨基加成如精氨酸化、泛在化、或任何其他操作如与标记组分缀合等改性的氨基酸聚合物。如本文所用，术语“氨基酸”是指天然和/或非天然或合成氨基酸，包括甘氨酸以及D或L光学异构体，以及氨基酸类似物和肽模拟物。“衍生自”指定的蛋白质的多肽或氨基酸序列是指多肽的来源。该术语还包括由指定的核酸序列表达的多肽。
术语“氨基酸修饰”(或“修饰的氨基酸”)包括在多肽序列中的氨基酸取代、插入和/或缺失。本文中的“氨基酸取代”或“取代”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸。例如，取代R94K指94位的精氨酸被赖氨酸替换。对于亲本多肽序列上述同一位置上的相同取代，还可以用94K表示，即用赖氨酸取代94位。出于本文的目的，通常通过斜杠分隔多个取代。例如，R94K/L78V指包含取代R94K和L78V的双变体。本文中使用的“氨基酸插入”或“插入”意指在亲本多肽序列中的特定位置添加氨基酸。例如，插入-94表示在94位的插入。本文中使用的“氨基酸缺失”或“缺失”意指去除亲本多肽序列中特定位置上的氨基酸。例如R94-表示删除94位的精氨酸。
本文中使用的术语“保守修饰”或“保守序列修饰”意指不显著影响或改变含有所述氨基酸序列的肽的所需活性或特性的氨基酸修饰。此类保守修饰包括氨基酸取代、插入和缺失。可通过本领域已知的标准技术将修饰导入本发明的抗体中，例如定点诱变和PCR介导的诱变。保守的氨基酸取代是用具有相似侧链的氨基酸残基替换氨基酸残基的取代。本领域已经定义了具有相似侧链的氨基酸残基家族。这些家族包括含碱性侧链的氨基酸(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电的急性侧链(例如，甘氨酸、天冬酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因而，可以用其他相似侧链的氨基酸残基替换本发明抗体的CDR区中或框架区中的一个或多个氨基酸残基。
本文使用的术语“自体”及其语法上的其他形式可以指来自相同的本源。例如，样品(例如，细胞)可以被去除、处理并在稍后的时间给予相同的个体(例如，患者)。自体过程与其中供体和受体是不同个体的同种异体过程不同。
本文使用的“异种移植”及其语法上的其他形式可以包括其中接受者和供体是不同的物种的将细胞、组织或器官移植、植入或输注到受体中的任何程序。本文所述的细胞、器官和/或组织的移植可用于异种移植入人中。异种移植包括但不限于血管化异种移植物、部分血管化异种移植物、非血管化异种移植、异种敷料、异种绷带和异种结构等。
本文使用的“同种异体移植”及其语法上的其他形式(例如，同种异体的移植)可以包括其中受体和供体是同一物种但不同个体的将细胞、组织或器官移植，植入或输注到接受者中的任何程序。本文所述的细胞、器官和/或组织的移植可以用于同种异体移植入人体。同种异体移植包括但不限于血管化同种异体移植、部分血管化的同种异体移植、无血管化的同种异体移植、同种异体敷料、同种异体绷带、和同种异体结构。
本文所用的“自体移植”及其语法上的其他形式(例如，自体的移植)可以包括其中接受者和供体是相同的个体的将细胞、组织或器官移植、植入或输注到接受者中的任何程序。本文所述的细胞、器官和/或组织的移植可用于自体移植入人体。自体移植包括但不限于血管化自体移植、部分血管化自体移植、非血管化自体移植、自体敷料、自体绷带和自体结构。
本文所用的术语“嵌合抗原受体”或“CAR”是指可以由包括但不限于T细胞或NK细胞的免疫细胞表达的工程化分子。CAR在T细胞中表达并且可以重定向T细胞以诱导以由人造受体决定的特异性杀死靶细胞。CAR的细胞外结合结构域可以衍生自鼠、人源化或完全人单克隆抗体。
本文所用的术语“表位”及其语法上的其他形式可以指可被抗体、B细胞、T细胞或工程细胞识别的部分抗原。例如，表位可以是被TCR识别的肿瘤表位或病原体表位。也可以识别抗原内的多个表位。表位也可以突变。
“细胞系”或“细胞培养物”表示在体外生长或维持的细菌、植物、昆虫或更高真核细胞。细胞的后代与母体细胞可能不完全相同(在形态上，基因型或表型上)。
本文所用的术语“工程化”及其语法上的其他形式可以指核酸的一个或多个改变，例如生物体基因组内的核酸。术语“工程化”可以指基因的改变、添加和/或缺失。工程细胞还可以指具有加入、缺失和/或改变的基因的细胞。工程细胞也可 以指表达CAR的细胞。
本文所用的术语“转染”是指将外源核酸引入真核细胞。转染可以通过本领域已知的各种手段来实现，包括磷酸钙-DNA共沉淀、DEAE-葡聚糖介导的转染、聚凝胺介导的转染、电穿孔、显微注射、脂质体融合、脂质转染、原生质体融合、逆转录病毒感染和生物弹道技术(biolistics)。
术语“稳定转染”或“稳定地转染”是指将外源核酸、DNA或RNA引入和整合到转染细胞的基因组中。术语“稳定转染体”(stable transfectant)是指将外来DNA稳定地整合到基因组DNA中的细胞。
如本文所用，术语“核酸分子编码”、“编码DNA序列”和“编码DNA”是指沿着脱氧核糖核酸链的脱氧核糖核苷酸的顺序或顺序。这些脱氧核糖核苷酸的顺序决定了沿着多肽(蛋白质)链的氨基酸的顺序。因此，核酸序列编码氨基酸序列。
本文所用的术语“个体”是指任何动物，例如哺乳动物或有袋动物。本发明的个体包括但不限于人类、非人类灵长类动物(例如恒河猴或其他类型的猕猴)、小鼠、猪、马、驴、牛、绵羊、大鼠和任何种类的家禽。
本文所用的术语“外周血淋巴细胞”(PBL)及其语法上的其他形式可以指在血液(例如外周血)中循环的淋巴细胞。外周血淋巴细胞可以指不局限于器官的淋巴细胞。外周血淋巴细胞可以包含T细胞、NK细胞、B细胞或其任何组合。
本文所用的术语“免疫应答细胞”(或“免疫反应性细胞”、“免疫效应细胞”或“免疫细胞”)可以指可以引发免疫应答的细胞。免疫应答细胞还可以指淋巴或骨髓谱系的细胞。免疫细胞的实例包括但不限于T细胞，例如α/βT细胞和γ/δT细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和骨髓源性吞噬细胞、它们各自的前体细胞及其后代。
本文所用的术语“T细胞”及其语法上的其他形式可以指任何来源的T细胞。例如，T细胞可以是原代T细胞例如自体T细胞等。T细胞也可以是人或非人的。
本文所用的术语“T细胞活化”或“T细胞触发”及其语法上的其他形式可以指被充分刺激以诱导可检测的细胞增殖、细胞因子产生和/或可检测的效应物功能的T细胞的状态。在一些实施方案中，“完全T细胞活化”可以类似于触发T细胞的细胞毒性。可以使用本领域已知的各种测定来测量T细胞活化。所述测定可以是测量细胞因子分泌的ELISA、ELISPOT、用于测量细胞内细胞因子表达的流式细胞术测定(CD107)、用于测量增殖的流式细胞术测定、和用于确定靶细胞消除的细胞毒性测定(51Cr释放测定)。所述测定通常使用对照(非工程细胞)与 工程细胞(CART)进行比较，以确定与对照相比，工程细胞的相对激活。此外，所述测定可以与未表达靶抗原的靶细胞孵育或接触的工程细胞进行比较。例如，所述比较可以是与不表达CD19的靶细胞孵育的CD19-CART细胞进行的比较。
当用于指核苷酸序列时，本文所用的术语“序列”及其语法上的其他形式可以包括DNA或RNA，并且可以是单链或双链。核酸序列可以突变。核酸序列可以具有任何长度，例如长度为2至1,000,000或更个核苷酸(或其间或之上的任何整数值)核酸，例如长度为约100至约10,000个核苷酸或约200至约500个核苷酸。
本文所用的术语“有效量”是指提供治疗或预防益处的量。
本文所用的术语“表达载体”是指包含重组多核苷酸的载体，其包含与待表达的核苷酸序列有效连接的表达调控序列。表达载体包含用于表达的足够的顺式作用元件(cis-acting elements)；用于表达的其它元件可以由宿主细胞或体外表达系统提供。表达载体包括本领域所有已知的那些，例如粘粒、质粒(例如裸露或包含在脂质体中的)和病毒(例如，慢病毒、逆转录病毒、腺病毒和腺相关病毒)。
本文所用的术语“慢病毒”是指逆转录病毒科的属。逆转录病毒在能够感染非分裂细胞方面是逆转录病毒中独特的；它们可以将大量的遗传信息递送到宿主细胞的DNA中，因此它们是基因递送载体最有效的方法之一。HIV、SIV和FIV都是慢病毒的实例。源自慢病毒的载体提供了在体内实现显著水平的基因转移的手段。
本文所用的术语“可操作地连接”是指在调控序列和异源核酸序列之间的功能性连接，该连接导致后者的表达。例如，当第一核酸序列与第二核酸序列成功能关系时，第一核酸序列与第二核酸序列可操作地连接。例如，如果启动子影响编码序列的转录或表达，则启动子可操作地连接到编码序列。通常，可操作地连接的DNA序列是连续的，并且在必要时在相同的阅读框中连接两个蛋白质编码区。
本文使用的术语“启动子”定义为由启动多核苷酸序列的特异性转录所需的细胞的合成机制或引入的合成机制识别的DNA序列。
本文使用的术语“载体”是包含分离的核酸并可用于将分离的核酸递送至细胞内部的组合物。在本领域中已知许多载体，包括但不限于线性多核苷酸、与离子或两亲化合物相关的多核苷酸、质粒和病毒。因此，术语“载体”包括自主复制的质粒或病毒。该术语还应被解释为包括促进核酸转移到细胞中的非质粒和非病毒化合物，例如聚赖氨酸化合物、脂质体等。病毒载体的实例包括但不限于腺病毒载体、腺相关病毒载体、逆转录病毒载体等。
“宿主细胞”包括可以是或已经是目标载体的接受体的个体细胞或细胞培养物。 宿主细胞包括单个宿主细胞的后代。由于自然、意外或有意为之的突变，后代可能例如在形态学或基因组DNA或总DNA补体上不一定与原始亲本细胞完全相同。宿主细胞包括用本发明的载体体内转染的细胞。“宿主细胞”可以指原核细胞、真核细胞或作为单细胞实体培养的细胞系，其可以或已经用作重组载体或其他转移多核苷酸的受体，并且包括已经转染的后代原始细胞。
本文使用的术语序列“同一性”通过在比较窗口(例如至少20个位置)上比较两个经最佳匹配的序列来确定同一性百分比，其中比较窗口中多核苷酸或多肽序列的部分可以包含添加或缺失(即间隙)，例如对于最佳匹配的两个序列而言与参考序列(其不包含添加或缺失)相比20％或更少的间隙(例如5至15％、或10至12％)。通常通过确定在两个序列中发生相同的核酸碱基或氨基酸残基的位置的数目来计算百分比，以产生正确匹配的位置的数目，将正确匹配位置的数目除以参考序列中的位置总数(即窗口大小)，并将结果乘以100，以产生序列同一性的百分比。
本文所用的术语“疾病”或“病症”或“紊乱”等是指任何损害或干扰细胞、组织或器官的正常功能的改变或失调。例如，所述的“疾病”包括但不限于：肿瘤、病原体感染、自身免疫性疾病、T细胞功能障碍性疾病、或免疫耐受能力缺陷(如移植排斥)。
本文所用的术语“外源性”指的是一个核酸分子或多肽，其在细胞内没有内源性表达，或表达水平不足以实现过表达时具有的功能。因而，“外源性”包括在细胞内表达的重组核酸分子或多肽，如外源性、异源性和过表达的核酸分子和多肽。
本文所用的术语“调节”是指正向或负向改变。调节范例包括1％、2％、10％、25％、50％、75％、或100％变化。
本文所用的术语“治疗”是指在试图改变个人或处理细胞引起的的疾病过程中的临床干预，既可以进行预防也可以在临床病理过程干预。治疗效果包括但不限于，防止疾病的发生或复发、减轻症状、减少任何疾病直接或间接的病理后果、防止转移、减慢疾病的进展速度、改善或缓解病情、缓解或改善预后等。
本文所用的术语“组成性表达”是指在所有的生理条件下的表达。
本文所用的术语“诱导表达”是指在一定条件下的表达，所述的条件例如T细胞与抗原发生结合的时候。本领域技术人员如何进行常规的“诱导表达”。
在一些实施方案中，本文提供了包含轻链CDR和重链CDR的抗原结合单元，其中所述抗原结合单元特异性地结合密蛋白18A2肽；并且其中所述抗原结合单元 不显著结合密蛋白18A1肽。
在一些实施方案中，本文提供了包含轻链CDR和重链CDR的抗原结合单元，其中所述抗原结合单元特异性地结合密蛋白18A2肽；并且其中所述抗原结合单元与参考抗原结合单元相比，显示更少的与密蛋白18A1肽的非特异性结合。
在一些实施方案中，本文所述的抗原结合单元包括轻链CDR。轻链CDR可以是抗原结合单元的互补决定区。轻链CDR可以包含连续的氨基酸残基序列，或两个或更多个由非互补决定区域例如框架区域分隔开并且可选择地侧翼的连续的氨基酸残基序列。在一些实施方案中，轻链CDR包含两个或多个轻链CDR，其可以称为轻链CDR-1、CDR-2等。在一些实施方案中，轻链CDR包含三个轻链CDR，其可分别称为轻链CDR-1(LCDR1)、轻链CDR-2(LCDR2)和轻链CDR-3(LCDR3)。在一些实施方案中，存在于共同的轻链上的一组CDR可统称为轻链CDR。
在一些实施方案中，本文所述的抗原结合单元包括重链CDR。重链CDR可以是抗原结合单元的互补决定区。重链CDR可以包含连续的氨基酸残基序列，或两个或更多个由非互补决定区域例如框架区域分隔开并且可选择地侧翼的连续的氨基酸残基序列。在一些实施方案中，重链CDR包含两个或多个重链CDR，其可以称为重链CDR-1、CDR-2等。在一些实施方案中，重链CDR包含三个重链CDR，其可分别称为重链CDR-1(HCDR1)、重链CDR-2(HCDR2)和重链CDR-3(HCDR3)。在一些实施方案中，存在于共同的重链上的一组CDR可统称为重链CDR。
在一些实施方案中，本文提供了包含轻链CDR和重链CDR的抗原结合单元，其中所述轻链CDR包含LCDR1、LCDR2和LCDR3；并且所述重链CDR包含HCDR1、HCDR2和HCDR3；其中所述LCDR1、LCDR2和LCDR3的氨基酸序列与选自以下的氨基酸序列具有至少80％的同一性：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:53和SEQ ID NO:54；以及其中所述HCDR1、HCDR2和HCDR3的氨基酸序列与选自以下的氨基酸序列具有至少80％的同一性：SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85。
在一些实施方案中，本文提供了包含轻链CDR和重链CDR的抗原结合单元，其中所述轻链CDR包含LCDR1、LCDR2和LCDR3；并且所述重链CDR包含 HCDR1、HCDR2和HCDR3；其中所述LCDR1、LCDR2和LCDR3的氨基酸序列与选自以下的氨基酸序列具有至少60％、65％、70％、75％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、或99.9％的同一性：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:53和SEQ ID NO:54；以及其中所述HCDR1、HCDR2和HCDR3的氨基酸序列与选自以下的氨基酸序列具有至少60％、65％、70％、75％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、或99.9％的同一性：SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85。
在一些实施方案中，本文的抗原结合单元中，所述轻链CDR包含LCDR1、LCDR2和LCDR3；并且所述重链CDR包含HCDR1、HCDR2和HCDR3；其中所述LCDR1、LCDR2和LCDR3分别具有选自以下的氨基酸序列：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:53和SEQ ID NO:54；以及其中所述HCDR1、HCDR2和HCDR3分别具有选自以下的氨基酸序列：SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85。
在一些实施方案中，本文提供了抗原结合单元，其中所述LCDR1包含与选自以下的氨基酸序列具有至少60％、65％、70％、75％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、或99.9％同一性的氨基酸序列：SEQ ID NO:34、SEQ ID NO:40、SEQ ID NO:46和SEQ ID NO:52。在一些实施方案中，本文提供了抗原结合单元，其中所述LCDR2包含与选自以下的氨基酸序列具有至少60％、65％、70％、75％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、或99.9％同一性的氨 基酸序列：SEQ ID NO:34、SEQ ID NO:41、SEQ ID NO:47和SEQ ID NO:53。
在一些实施方案中，本文提供了抗原结合单元，其中所述LCDR3包含与选自以下的氨基酸序列具有至少60％、65％、70％、75％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、或99.9％同一性的氨基酸序列：SEQ ID NO:35、SEQ ID NO:42、SEQ ID NO:48和SEQ ID NO:54。在一些实施方案中，本文提供了抗原结合单元，其中所述HCDR1包含包含与选自以下的氨基酸序列具有至少60％、65％、70％、75％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、或99.9％同一性的氨基酸序列：SEQ ID NO:31、SEQ ID NO:37、SEQ ID NO:43和SEQ ID NO:49。在一些实施方案中，本文提供了抗原结合单元，其中所述HCDR2包含与选自以下的氨基酸序列具有至少60％、65％、70％、75％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、或99.9％同一性的氨基酸序列：SEQ ID NO:32、SEQ ID NO:38、SEQ ID NO:44、SEQ ID NO:50、SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85。在一些实施方案中，本文提供了抗原结合单元，其中所述HCDR3包含与选自以下的氨基酸序列具有至少60％、65％、70％、75％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、或99.9％同一性的氨基酸序列：SEQ ID NO:33、SEQ ID NO:39、SEQ ID NO:45和SEQ ID NO:51。
在一些实施方案中，本发明的抗原结合单元结合至密蛋白18A2或密蛋白18A2肽。本文中的术语“密蛋白18A2”或“密蛋白18A2肽”(CLD18.2、CLD18A2、CLDN18A2、CLDN18.2、Claudin18.2或Claudin18A2)也可以指已知密蛋白18A2序列的同源物、直系同源物、种间同源物、密码子优化形式、截短形式、片段化形式、突变形式或任何其它已知衍生形式，例如翻译后修饰变体。在一些实施方案中，所述密蛋白18A2或密蛋白18A2肽是具有GenBank登记号NP_001002026的肽(mRNA:NM_001002026)。在一些实施方案中，所述密蛋白18A2或密蛋白18A2肽是包含SEQ ID NO:55的氨基酸序列的肽。
在一些实施方案中，本发明的抗原结合单元不显著结合密蛋白18A1肽。本文中的术语“密蛋白18A1”或“密蛋白18A1肽”(CLD18A1、CLD18.1、CLDN18A1、CLDN18.1、Claudin18.1或Claudin18A1)也可以指已知密蛋白18A1序列的同源物、 直系同源物、种间同源物、密码子优化形式、截短形式、片段化形式、突变形式或任何其它已知衍生形式，例如翻译后修饰变体。在一些实施方案中，所述密蛋白18A1或密蛋白18A1肽是具有GenBank登记号NP_057453的肽(mRNA:NM_016369)。在一些实施方案中，所述密蛋白18A1或密蛋白18A1肽是包含SEQ ID NO:57的氨基酸序列的肽。
结合特异性可以通过互补决定区或CDR，如轻链CDR或重链CDR来确定。在许多情况下，通过轻链CDR和重链CDR确定结合特异性。与其他参考抗原或参考肽相比，给定的重链CDR、轻链CDR或其组合提供与密蛋白18A2具有更大亲和力和/或特异性的给定结合口袋。
抗原结合单元与与密蛋白18A2肽的结合可通过本领域已知的任何方法表征或表达。例如，结合可以通过结合亲和力来表征，其可以是抗原结合单元和抗原之间的相互作用的强度。结合亲和力可以通过本领域已知的任何方法测定，例如体外结合测定。例如，当使用表达密蛋白18A2的细胞的体外结合测定中测定时，可以确定本文公开的抗原结合单元的结合亲和力。受试抗原结合单元的结合亲和力可以用Kd表示，Kd是抗体与其各自抗原之间的平衡解离常数。在一些情况下，本文公开的抗原结合单元特异地结合密蛋白18A2，Kd在约10μM至约1mM的范围内。例如，抗原结合单元可以以小于约10μM、1μM、0.1μM、10nM、1nM、0.1nM、10pM、1pM、0.1pM、10fM、1fM、或少于0.1fM的Kd特异性结合密蛋白18A2。
在一些实施方案中，抗原结合单元不显示与参考肽的显著结合。在一些实例中，抗原结合单元与参考肽的结合水平不超过所述抗原结合单元与密蛋白18A2的结合水平的20％。例如，结合水平可以是所述抗原结合单元与密蛋白18A2的结合水平的20％、19％、18％、17％、16％、15％、14％、13％、12％、11％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％或小于1％。在一些实施方案中，本文的抗原结合单元与密蛋白18A2的结合水平是其与参考肽的结合水平的1倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍或多于10倍。在一些实施方案中，参考肽或参考肽是密蛋白18A1肽。在一些实施方案中，参考肽或参考肽是包含SEQ ID NO:57的氨基酸序列的肽。在一些实施方案中，参考肽或参考肽是SEQ ID NO:57的肽。
在一些实施方案中，与参考抗原结合单元相比，本文的抗原结合单元显示出对参考肽的非特异性结合较少。在一些实施方案中，本文的抗原结合单元比参考抗原结合单元与参考肽的非特异性结合水平低5％、10％、15％、20％、25％、30％、 35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或100％。在一些实施方案中，本文的抗原结合单元与参考肽的非特异性结合水平比参考抗原结合单元的结合水平低1倍、2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍或多于10倍。在一些实施方案中，所述参考抗原结合单元包含SEQ ID NO:86或SEQ ID NO:88的轻链和/或SEQ ID NO:87或SEQ ID NO:89的重链氨基酸序列。在一些实施方案中，参考抗原结合单元包含SEQ ID NO：86的氨基酸序列。在一些实施方案中，参考抗原结合单元包含SEQ ID NO：87的氨基酸序列。在一些实施方案中，参考抗原结合单元包含SEQ ID NO：88的氨基酸序列。在一些实施方案中，参考抗原结合单元包含SEQ ID NO：89的氨基酸序列。在一些实施方案中，参考肽或参考肽是密蛋白18A1肽。在一些实施方案中，参考肽或参考肽是包含SEQ ID NO:57的氨基酸序列的肽。在一些实施方案中，参考肽或参考肽是SEQ ID NO:57的肽。
在一些实施方案中，所述抗原结合单元对包含密蛋白18A2肽的细胞具有细胞毒性，所述密蛋白18A2肽包含SEQ ID NO:55的氨基酸序列。所述细胞毒性水平当在所述抗原结合单元与靶细胞的比例为20:1、10:1、5:1、3:1、2.5:1、1:1或1:3时为至少5％、10％、15％、20％、25％、30％、35％、40％或45％。
在一些实施方案中，所述抗原结合单元对包含密蛋白18A1肽但不包含密蛋白18A2肽的细胞不具有显著的细胞毒性，所述密蛋白18A1肽包含SEQ ID NO:57的氨基酸序列，且所述密蛋白18A2肽包含SEQ ID NO:55的氨基酸序列。在一些实施方案中，所述细胞毒性水平不高于10％、5％、4％、3％、2％或1％。
在一些实施方案中，本文提供了可特异性地结合密蛋白18A2的抗体，其特征在于，该抗体包含选自氨基酸序列SEQ ID NO:31，32，33，37，38，39，43，44，45，49，50，51，83，84，85或其变体的重链CDR，和/或选自氨基酸序列SEQ ID NO：34，35，36，40，41，42，46，47，48，52，53，54，或其变体的轻链CDR。
在一些实施方案中，本文提供了抗体，其选自(a)抗体，其包含重链可变区，所述重链可变区具有包含SEQ ID NO:31、SEQ ID NO:37、SEQ ID NO:43或SEQ ID NO:49之一所示的氨基酸序列的CDR1、包含SEQ ID NO:32、SEQ ID NO:38、SEQ ID NO:44、SEQ ID NO:50、SEQ ID NO:83、SEQ ID NO:84或SEQ ID NO:85之一所示的氨基酸序列的CDR2、包含SEQ ID NO:33、SEQ ID NO:39、SEQ ID NO:45或SEQ ID NO:51之一所示的氨基酸序列的CDR3；(b)抗体，其包含轻链可变区，所述轻链可变区具有包含SEQ ID NO:34、SEQ ID NO:40、SEQ ID NO:46或 SEQ ID NO:52之一所示的氨基酸序列的CDR1、包含SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:47或SEQ ID NO:53之一所示的氨基酸序列的CDR2、包含SEQ ID NO:36、SEQ ID NO:42、SEQ ID NO:48或SEQ ID NO:54之一所示氨基酸序列的CDR3；(c)抗体，包含(a)所述抗体的重链可变区及(b)所述抗体的轻链可变区；(d)抗体，识别与(a)～(c)中任一项所述的抗体所识别的抗原决定部位相同的抗原决定部位。在一些实施方案中，本文提供了抗体，该抗体的重链可变区的CDR1、CDR2、CDR3区分别为SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33；或者SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39；或者SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45；或者SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51；或者SEQ ID NO:31、SEQ ID NO:83、SEQ ID NO:33；或者SEQ ID NO:31、SEQ ID NO:84、SEQ ID NO:33；或者SEQ ID NO:49、SEQ ID NO 85、SEQ ID NO:51；和/或该抗体的轻链可变区的CDR1、CDR2、CDR3区分别为SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36；或者SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42；或者SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48；或者SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54。
在一些实施方案中，本发明的抗体的重链的CDR1、CDR2和CDR3的氨基酸序列选自下表中的氨基酸序列或其变体的组中：
表1
  HCDR1 HCDR2 HCDR3
A SEQ ID NO:31 SEQ ID NO:32 SEQ ID NO:33
B SEQ ID NO:37 SEQ ID NO:38 SEQ ID NO:39
C SEQ ID NO:43 SEQ ID NO:44 SEQ ID NO:45
D SEQ ID NO:49 SEQ ID NO:50 SEQ ID NO:51
E SEQ ID NO:31 SEQ ID NO:83 SEQ ID NO:33
F SEQ ID NO:31 SEQ ID NO:84 SEQ ID NO:33
G SEQ ID NO:49 SEQ ID NO 85 SEQ ID NO:51
在一些实施方案中，本文提供了抗体，其轻链CDR1、CDR2和CDR3的氨基酸序列选自下表中的氨基酸序列或其变体的组中：
表2
  LCDR1 LCDR2 LCDR3
A SEQ ID NO:34 SEQ ID NO:35 SEQ ID NO:36
B SEQ ID NO:40 SEQ ID NO:41 SEQ ID NO:42
C SEQ ID NO:46 SEQ ID NO:47 SEQ ID NO:48
D SEQ ID NO:52 SEQ ID NO:53 SEQ ID NO:54
在一些实施方案中，本发明抗体包含选自氨基酸序列SEQ ID NO:3、7、11、15、17、19、23、27、29或其变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：1、5、9、13、21、25或其变体的轻链可变区。在一些实施方案中，所述重链可变区为SEQ ID NO:3或其变体并且所述轻链可变区为SEQ ID NO:1或其变体。在一些实施方案中，所述重链可变区为SEQ ID NO:7或其变体并且所述轻链可变区为SEQ ID NO:5或其变体。在一些实施方案中，所述重链可变区为SEQ ID NO:11或其变体并且所述轻链可变区为SEQ ID NO:9或其变体。在一些实施方案中，所述重链可变区为SEQ ID NO:15或其变体并且所述轻链可变区为SEQ ID NO:13或其变体。在一些优选的实施方案中，重链CDR1、CDR2和CDR3的氨基酸序列选自下表。
表3
  HCDR1 HCDR2 HCDR3
E SEQ ID NO:31 SEQ ID NO:83 SEQ ID NO:33
F SEQ ID NO:31 SEQ ID NO:84 SEQ ID NO:33
G SEQ ID NO:49 SEQ ID NO 85 SEQ ID NO:51
在一些实施方案中，本发明抗体包含选自氨基酸序列SEQ ID NO:17、19、或其变体的重链可变区。在一些实施方案中，所述重链可变区为SEQ ID NO:17或其变体并且所述轻链可变区为SEQ ID NO:1或其变体。在一些实施方案中，所述重链可变区为SEQ ID NO:19或其变体并且所述轻链可变区为SEQ ID NO:1或其变体。
在一些实施方案中，本发明抗体或其功能性片段包含选自氨基酸序列SEQ ID NO:23、27、29或其变体的重链可变区，和/或选自氨基酸序列SEQ ID NO：21，25，或其变体的轻链可变区。在一些实施方案中，所述重链可变区为SEQ ID NO:23或其变体并且所述轻链可变区为SEQ ID NO:21或其变体。在一些实施方案中，所述重链可变区为SEQ ID NO:27或其变体并且所述轻链可变区为SEQ ID NO:25或其变体。在一些实施方案中，所述重链可变区为SEQ ID NO:29或其变体并且所述轻链可变区为SEQ ID NO:25或其变体。
在一些实施方案中，本发明的抗原结合单元或抗体还与另外的功能性分子连接 或者融合。因此，本发明也包括如此形成的多功能免疫缀合物。
“连接”或“融合”在本文中可互换使用。这些术语是指通过包括化学缀合或重组方法的任何手段将两个以上化学元件或组件连接在一起。“框内融合”是指以维持原始开放阅读框(ORF)的正确阅读框的方式连接两个或更多个ORF以形成连续的较长的ORF。因此，所得到的重组融合蛋白是含有两个或更多个片段的单一蛋白质，这些片段对应于由原始ORF编码的多肽(这些片段在自然状态通常不是如此连接)。尽管阅读框因此在整个融合片段中是连续的，但这些片段可以在物理上或空间上通过例如框内连接序列(例如“flexon”)分开。
所述的功能性分子例如用于诊断或者治疗肿瘤。
本文所用的术语“肿瘤”指的是一种以细胞或组织的病理性增生为特征的疾病，及其随后的迁移或侵袭其他组织或器官。肿瘤生长通常是不受控制的和进行性的，不诱导或抑制正常细胞增殖。肿瘤可影响多种细胞、组织或器官，包括但不限于选自膀胱、骨、脑、乳腺、软骨、神经胶质细胞、食管、输卵管、胆囊、心脏、肠、肾、肝、肺、淋巴结、神经组织、卵巢、胰腺、前列腺、骨骼肌、皮肤、脊髓、脾、胃、睾丸、胸腺、甲状腺、气管、尿道、输尿管、尿道、子宫、阴道器官，或组织或相应的细胞。肿瘤包括癌症，如肉瘤，癌，或浆细胞瘤(浆细胞的恶性肿瘤)。本发明所述的肿瘤，可包括，但不限于白血病(如急性白血病、急性淋巴细胞白血病、急性髓细胞性白血病，急性粒细胞白血病，急性早幼粒细胞白血病、急性粒-单核细胞白血病、急性单核细胞白血病、急性白血病、慢性白血病、慢性粒细胞白血病、慢性淋巴细胞白血病、真性红细胞增多症)，淋巴瘤(霍奇金病、非霍奇金病)、原发性巨球蛋白血症，重链病，实体瘤如肉瘤和癌症(如纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、骨肉瘤、脊索瘤、内皮肉瘤、淋巴管肉瘤、血管肉瘤、淋巴管内皮肉瘤，滑膜vioma，间皮瘤，尤文氏瘤、平滑肌肉瘤、横纹肌肉瘤、结肠癌、胰腺癌、乳腺癌、卵巢癌、前列腺癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、癌、支气管癌、髓样癌、肾细胞癌、肝癌，尼罗河管癌，绒癌、精原细胞瘤、胚胎癌、肾母细胞瘤、宫颈癌、子宫癌、睾丸癌、肺癌、小细胞肺癌、膀胱癌、上皮癌、胶质瘤、星形细胞瘤、髓母细胞瘤，颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤，听神经瘤，少突胶质瘤、神经鞘瘤、脑膜瘤、黑色素瘤、神经母细胞瘤、视网膜母细胞瘤)、食管癌、胆囊癌、肾癌、多发性骨髓瘤。较佳地，所述的“肿瘤”包括但不限于：胰腺癌、肝癌、肺癌、胃癌、食管癌、头颈部鳞状细胞癌、前列腺癌、结肠癌、乳腺癌、淋巴瘤、胆囊癌、肾癌、白 血病、多发性骨髓瘤、卵巢癌、宫颈癌和胶质瘤。
所述功能性分子例如包括肿瘤抗原，例如肿瘤特异性抗原(TSA)或肿瘤相关抗原(TAA)。TSA是肿瘤细胞独特的，不发生在体内的其他细胞上。TAA相关抗原不是肿瘤细胞独有的，而是在不能诱导对抗原的免疫耐受状态的条件下在正常细胞上表达。抗原在肿瘤上的表达可以在使得免疫系统能够对抗原作出反应的条件下发生。当免疫系统不成熟并且不能应答时，TAA可以是在胎儿发育期间在正常细胞上表达的抗原，或者它们可以是通常以正常细胞上极低水平存在但在肿瘤细胞上以更高水平表达的抗原。
TSA或TAA抗原的非限制性实例包括以下：分化抗原如MART-1/MelanA(MART-I)、gp100(Pmel17)、酪氨酸酶、TRP-1、TRP-2和肿瘤特异性多中心抗原如MAGE-1、MAGE-3、BAGE、GAGE-1、GAGE-2、p15；过表达的胚胎抗原如CEA；过表达的癌基因和突变的肿瘤抑制基因如p53、Ras、HER-2/neu；由染色体易位引起的独特的肿瘤抗原，例如BCR-ABL、E2A-PRL、H4-RET、IGH-IGK、和MYL-RAR；以及病毒抗原，如爱泼斯坦巴尔病毒抗原EBVA和人乳头瘤病毒(HPV)抗原E6和E7等。其他大的、基于蛋白质的抗原包括TSP-180、MAGE-4、MAGE-5、MAGE-6、RAGE、NY-ESO、p185erbB2、p180erbB-3、c-met、nm-23H1、PSA、TAG-72、CA 19-9、CA 72-4、CAM 17.1、NuMa、K-ras、β-联蛋白、CDK4、Mum-1、p 15、p 16、43-9F、5T4、791Tgp72、甲胎蛋白、beta-HCG、BCA225、BTAA、CA 125、CA 15-3\CA 27.29\BCAA、CA 195、CA 242、CA-50、CAM43、CD68\P1、CO-029、FGF-5、G250、Ga733\EpCAM、HTgp-175、M344、MA-50、MG7-Ag、MOV18、NB/70K、NY-CO-1、RCAS1、SDCCAG16、TA-90\Mac-2结合蛋白\亲环蛋白C相关蛋白、TAAL6、TAG72、TLP、以及TPS。
在一些实施方案中，所述的“肿瘤抗原”包括但不限于：前列腺特异性膜抗原(PSMA)、癌胚抗原(CEA)、IL13Ralpha、HER-2、CD19、NY-ESO-1、HIV-1Gag、Lewis Y、MART-1、gp100、酪氨酸酶、WT-I、hTERT、间皮素、EGFR、EGFRvIII、磷脂酰肌醇蛋白聚糖3、EphA2、HER3、EpCAM、MUC1、MUC16、叶酸受体、CLDN6、CD30、CD138、ASGPR1、CDH16、GD2、5T4、8H9、αvβ6整合素、B细胞成熟抗原(BCMA)、B7-H3、B7-H6、CAIX、CA9、CD20、CD22、κ轻链(kappa light chain)、CD33、CD38、CD44、CD44v6、CD44v7/8、CD70、CD123、CD171、CSPG4、EGP2、EGP40、ERBB3、ERBB4、ErbB3/4、FAP、FAR、FBP、胚胎型AchR、GD2、GD3、HLA-AI MAGE A1、MAGE3、HLA-A2、IL11Ra、 KDR、Lambda、MCSP、NCAM、NKG2D配体、PRAME、PSCA、PSC1、ROR1、Sp17、SURVIVIN、TAG72、TEM1、TEM8、VEGRR2、HMW-MAA、VEGF受体、和/或纤连蛋白、腱生蛋白或肿瘤坏死区的癌胚变体。
在一些实施方案中，所述功能性分子是干扰素。在一些实施方案中，所述干扰素是I型干扰素。
本文所用的术语“I型干扰素”包括IFNα、IFNβ、以及IFN-ε、IFN-κ以及IFN-ω等。所有的I型干扰素均与由IFNAR1和IFNAR2两条链组成的特定的细胞表面受体(即所谓的IFN-α/β受体)结合。在一些实施方案中，本文所用的术语“I型干扰素”为IFNα或IFNβ。在一些实施方案中，本文所用的术语“I型干扰素”为IFNβ。在一些实施方案中，本文所用I型干扰素包括人类、小鼠、或者合成的I型干扰素。在一些实施方案中，本文所用的术语“干扰素α”可以是具有NCBI aaa52724.1或aaa52716.1或aaa52725.1所示序列的多肽，或者是序列与这些序列具有至少有85％的同一性的多肽。在一些实施方案中，本文所用的术语“干扰素β”(INF-β)可以是具有NCBI aac41702.1或np_002167.1或aah96152.1p41273或NP 001552至少有85％同一性的蛋白，或者是其具有肿瘤坏死因子(TNF)配体功能的片段。在一些实施方案中，所述干扰素β是人干扰素β。在一些实施方案中，所述干扰素β具有SEQ ID NO:92的氨基酸序列。
在一些实施方案中，所述的I型干扰素可以是天然存在的，比如其可被分离或纯化自哺乳动物；也可以是人工制备的，比如可以根据常规的基因工程重组技术来生产重组各元件或I型干扰素。优选的，本发明可采用重组的各元件或I型干扰素。
在所述I型干扰素多肽序列的基础上，经过一个或多个氨基酸残基的取代、缺失或添加而形成的氨基酸序列也包括在本发明中。适当替换氨基酸是本领域公知的技术，所述技术可以很容易地被实施并且确保不改变所得分子的生物活性。这些技术使本领域人员认识到，一般来说，在一种多肽的非必要区域改变单个氨基酸基本上不会改变生物活性。
在所述I型干扰素多肽序列的基础上，经修饰或改良的多肽也可以应用到本发明中，比如，可采用为了促进其半衰期、有效性、代谢、和/或多肽的效力而加以修饰或改良的多肽。也就是说，任何不影响多肽的生物活性的变化形式都可用于本发明中。
所述I型干扰素的多肽的生物活性片段都可以应用到本发明中。在这里，所述的生物活性片段的含义是指作为一种多肽，其作为全长多肽的一部分，仍然能保持 全长的多肽的全部或部分功能。通常情况下，所述的生物活性片段至少保持50％的全长多肽的活性。在更优选的条件下，所述活性片段能够保持全长多肽的60％、70％、80％、90％、95％、99％、或100％的活性。
在另一个方面，本发明提供了嵌合抗原受体，其包含本文所述的胞外抗原结合单元、跨膜域和胞内域。本文所用的术语“嵌合抗原受体(Chimeric Antigen Receptor，CAR)”指一种融合到细胞内信号转导域的肿瘤抗原结合结构域，能激活T细胞。常见地，CAR的胞外结合结构域来源于小鼠或人源化或人的单克隆抗体。
嵌合抗原受体通常包含胞外抗原结合区或者抗原结合单元。在一些实施方案中，所述胞外抗原结合单元是本文如前所述的抗原结合单元。
在一些实施方案中，胞外抗原结合区可以是完全人的。在其他情况下，胞外抗原结合区域可以被人源化。在其他情况下，胞外抗原结合区可以是鼠源的，或者所述胞外抗原结合区中的嵌合体由来自至少两种不同动物的氨基酸序列组成。在一些实施方案中，所述胞外抗原结合区可以是非人的。
可以设计多种抗原结合区域。非限制性实例包括衍生自抗体的单链可变片段(scFv)、选自文库的片段抗原结合区(Fab)、单结构域片段或与接合其同源受体的自然配体。在一些实施方案中，胞外抗原结合区域可以包含scFv、Fab或天然配体，以及它们的任何衍生物。胞外抗原结合区可以指除完整抗体之外的分子，其可以包含完整抗体的一部分并且可以与完整抗体所结合的抗原结合。抗体片段的实例可以包括但不限于Fv、Fab、Fab'、Fab'-SH、F(ab')2；双功能抗体、线性抗体；单链抗体分子(例如scFv)；和由抗体片段形成的多特异性抗体。
胞外抗原结合区，例如scFv、Fab或天然配体，可以是确定抗原特异性的CAR的一部分。胞外抗原结合区可以结合任何互补靶。胞外抗原结合区可以衍生自已知可变区序列的抗体。胞外抗原结合区可以从获自可获得的小鼠杂交瘤的抗体序列中得到。或者，可以从肿瘤细胞或原代细胞例如肿瘤浸润淋巴细胞(TIL)的全外切割测序获得胞外抗原结合区。
在一些实施方案中，胞外抗原结合区的结合特异性可以通过互补决定区或CDR，如轻链CDR或重链CDR来确定。在许多情况下，结合特异性可以通过轻链CDR和重链CDR来确定。与其他参考抗原相比，给定的重链CDR和轻链CDR的组合可以提供给定的结合袋，其可以赋予抗原更大的亲和力和/或特异性。
在本文公开的任何实施方案的某些方面，胞外抗原结合区，例如scFv可以包含对抗原特异性的轻链CDR。轻链CDR可以是抗原结合单元例如CAR的scFv轻 链的互补决定区。轻链CDR可以包含连续的氨基酸残基序列，或由非互补决定区(例如框架区)隔开的两个或更多个连续的氨基酸残基序列，。在一些实施方案中，轻链CDR可以包含两个或更多个轻链CDR，其可以被称为轻链CDR-1，CDR-2等。在一些实施方案中，轻链CDR可以包含三个轻链CDR，其可分别称为轻链CDR-1，轻链CDR-2和轻链CDR-3。在一些实例中，存在于普通轻链上的一组CDR可统称为轻链CDR。
在本文公开的任何实施方案的某些方面，胞外抗原结合区，例如scFv可以包含对抗原特异的重链CDR。重链CDR可以是抗原结合单元例如scFv的重链互补决定区。重链CDR可以包含氨基酸残基的连续序列，或由非互补决定区(例如框架区)隔开的两个或更多个氨基酸残基的连续序列。在一些实施方案中，重链CDR可以包含两个或更多个重链CDR，其可以称为重链CDR-1，CDR-2等。在一些实施方案中，重链CDR可以包含三个重链CDR，其可分别称为重链CDR-1，重链CDR-2和重链CDR-3。在一些实施方案中，存在于共同重链上的一组CDR可统称为重链CDR。
通过使用基因工程，可以以各种方式修饰胞外抗原结合区。在一些实施方案中，可以突变胞外抗原结合区域，从而可以选择胞外抗原结合区域以对其靶标具有更高的亲和力。在一些实施方案中，胞外抗原结合区域对其靶标的亲和力可针对可在正常组织上以低水平表达的靶标进行优化。可以进行此优化，以尽量减少潜在的毒性。在其他情况下，对靶标的膜结合形式具有更高亲和力的胞外抗原结合区域的克隆可以优于其可溶形式的对应物。可以进行这种修饰，因为也可以检测到不同水平的可溶形式的靶标，并且它们的靶向可引起不期望的毒性。
在一些实施方案中，胞外抗原结合区域包括铰链或间隔区。术语铰链和间隔区可以互换使用。铰链可以被认为是用于向胞外抗原结合区提供柔性的CAR的一部分。在一些实施方案中，铰链可用于检测细胞的细胞表面上的CAR，特别是当检测胞外抗原结合区的抗体不起作用或可用时。例如，衍生自免疫球蛋白的铰链的长度可能需要优化，这取决于胞外抗原结合区域靶向靶上的表位的位置。
在一些实施方案中，铰链可能不属于免疫球蛋白，而是属于另一种分子，如CD8α分子的天然铰链。CD8α铰链可以含有已知在CD8辅助受体和MHC分子的相互作用中起作用的半胱氨酸和脯氨酸残基。所述半胱氨酸和脯氨酸残基可影响所述CAR的性能。
CAR铰链可以是尺寸可调的。免疫应答细胞和靶细胞之间的免疫突触的这种 形貌还限定了由于细胞表面靶分子上的膜远端表位而不能由CAR进行功能桥接的距离，即使用短铰链CAR也不能使突触距离达到信号能够传导的近似值。同样，膜近端CAR靶抗原表位仅在长铰链CAR的背景下观察到信号输出。可以根据所使用的胞外抗原结合区域来调节铰链。铰链可以是任何长度的。
跨膜结构域可以将CAR锚定在细胞的质膜上。CD28的天然跨膜部分可用于CAR。在其他情况下，也可以在CAR中使用CD8α的天然跨膜部分。“CD8”可以是与NCBI参考号：NP_001759或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD8核酸分子”可以是编码CD8多肽的多核苷酸，在某些情况下，跨膜区可以是CD28的天然跨膜部分，“CD28”可以指与NCBI参考号：NP_006130或其具有刺激活性的片段具有至少85、90、95、96、97、98、99或100％同一性的蛋白质。“CD28核酸分子”可以是编码CD28多肽的多核苷酸。在一些实施方案中，跨膜部分可以包含CD8α区域。
CAR的(细)胞内信号传导区域可以负责活化CAR已经置于其中的免疫应答细胞的效应子功能中的至少一种。CAR可以诱导T细胞的效应子功能，例如，所述效应子功能是细胞溶解活性或辅助活性，包括细胞因子的分泌。因此，术语细胞内信号传导区域是指转导效应子功能信号并引导细胞进行特异功能的蛋白质部分。虽然通常可以使用整个细胞内信号传导区域，但是在许多情况下，不必使用信号结构域的整个链。在一些实施方案中，使用细胞内信号传导区的截短部分。在一些实施方案中，术语细胞内信号传导区域因此意在包括足以转导效应子功能信号的细胞内信号传导区的任何截短部分。
在CAR中使用的信号结构域的优选实例可以包括T细胞受体(TCR)的细胞质序列和协同作用以在靶-受体结合之后启动信号转导的共同受体，以及它们的任何衍生物或变体序列和这些序列的具有相同功能性的任何合成序列。
在一些实施方案中，所述细胞内信号传导区域可以含有已知的免疫受体酪氨酸激活基序(ITAM)的信号基序。含有细胞质信号传导序列的ITAM的实例包括衍生自TCRζ、FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d的那些。然而，在优选的实施方案中，细胞内信号结构域衍生自CD3ζ链。
含有一个或多个ITAM基序的T细胞信号结构域的实例是CD3ζ结构域，也称为T细胞受体T3ζ链或CD247。该结构域是T细胞受体-CD3复合物的一部分，并且在将几种细胞内信号转导途径的抗原识别与T细胞的主效应激活相结合方面 起重要作用。如本文所用，CD3ζ主要是指人类CD3ζ及其同种型，如从Swissprot条目P20963所知的，包括具有基本相同序列的蛋白质。作为嵌合抗原受体的一部分，再次重申，不需要全T细胞受体T3ζ链，并且其包含T细胞受体T3ζ链的信号结构域的任何衍生物都是合适的，包括其任何功能等同物。
细胞内信号传导结构域可以选自表1的任何一个结构域。在一些实施方案中，可以修饰结构域，使得与参考结构域的同一性可以为约50％至约100％。可以修饰表1的任何一个结构域，使得修饰形式可以包含约50、60、70、80、90、95、96、97、98、99或至多约100％的同一性。
CAR的细胞内信号传导区可以进一步包含一个或多个共刺激结构域。细胞内信号传导区可以包含单个共刺激结构域，例如ζ链(第一代CAR)或其与CD28或4-1BB(第二代CAR)。在其他实例中，细胞内信号传导区可以包含两个共刺激结构域，例如CD28/OX40或CD28/4-1BB(第三代)。
与细胞内信号结构域如CD8一起，这些共刺激结构域可以产生激酶途径的下游激活，从而支持基因转录和功能性细胞反应。CAR的共刺激结构域可以激活与CD28(磷脂酰肌醇-4,5-二磷酸3-激酶)或4-1BB/OX40(TNF-受体相关因子衔接蛋白)途径以及MAPK和Akt激活相关的近端信号蛋白。
在某些情况下，通过CAR产生的信号可能与辅助或共刺激信号相结合。对于共刺激信号结构域，嵌合抗原受体样复合物可被设计成包含若干可能的共刺激信号结构域。如本领域众所周知的，在幼稚T细胞中，T细胞受体的单独接合不足以诱导T细胞的完全活化为细胞毒性T细胞。完整的生产性T细胞激活需要第二共刺激信号。已经报道对T细胞活化提供共刺激的几种受体，包括但不限于CD28、OX40、CD27、CD2、CD5、ICAM-1、LFA-1(CD11a/CD18)、4-1BBL、MyD88和4-1BB。这些共刺激分子使用的信号传导途径均能与主T细胞受体激活信号协同作用。这些共刺激信号传导区域提供的信号可以与源自一个或多个ITAM基序(例如CD3zeta信号转导域)的主效应激活信号协同作用，并且可以完成T细胞激活的要求。
在一些实施方案中，向嵌合抗原受体样复合物添加共刺激结构域可以增强工程细胞的功效和耐久性。在另一些实施方案中，T细胞信号结构域和共刺激结构域彼此融合从而构成信号传导区。
表4.共刺激结构域
Figure PCTCN2017092381-appb-000001
嵌合抗原受体结合靶抗原。当在体外或离体测定T细胞活化时，可以从各种来源获得或分离靶抗原。本文使用的靶抗原是抗原或抗原上的免疫表位，其在哺乳动物中对于免疫识别和最终消除或控制致病因素或疾病状态中至关重要。免疫识别可以是细胞和/或体液。在细胞内病原体和癌症的情况下，免疫识别可以是例如T淋巴细胞反应。
在一些实施方案中，靶抗原包括与癌前或增生状态相关的抗原。靶抗原也可能 与癌症相关或起因于癌症。例如，在一些实施方案中，本发明的嵌合抗原受体识别并结合包括本文前述的TSA和TAA的肿瘤抗原。
在一些实施方案中，本文的嵌合抗原受体当存在于细胞的质膜上，并且当与其靶标结合并激活时，表达所述嵌合抗原受体的细胞可对携带所述靶标的细胞产生细胞毒性。例如，在一些实施方案中，所述嵌合抗原受体存在于细胞毒性细胞上，如NK细胞或者细胞毒性T细胞，并且在被靶标激活时，可以增加所述细胞毒性细胞对靶细胞的毒性。在一些实施方案中，本文的嵌合抗原受体可增加免疫反应性细胞对表达密蛋白18A2的细胞如肿瘤细胞的作用。在一些实施方案中，与不表达本文的嵌合抗原受体的细胞相比，表达本文所述嵌合抗原受体的细胞对表达密蛋白18A2的细胞的细胞毒性作用提高至少10％、至少15％、至少20％、至少25％、至少30％、至少35％、至少40％、至少45％、至少50％、至少55％、至少60％、至少65％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少1倍、至少1.5倍、至少2倍、至少2.5倍、至少3倍、至少3.5倍、至少4倍、至少4.5倍、至少5倍、至少6倍、至少7倍、至少8倍、至少9倍、或者至少10倍。
在一些实施方案中，本文的嵌合抗原受体当存在于细胞的质膜上，并且当与其靶标结合并激活时，对包含密蛋白18A1肽但不包含密蛋白18A2肽的细胞中不诱导显著的细胞毒性。在一些实施方案中，所述细胞毒性水平不高于10％、5％、4％、3％、2％或1％。
可以将编码目标结合抗原的受体或CAR的转基因并入细胞中。例如，可将转基因并入免疫应答细胞，例如T细胞。当插入细胞时，转基因可以是互补DNA(cDNA)片段，其是信使RNA(mRNA)的拷贝；或者位于其基因组DNA原始区域的基因本身(含或不含内含子)。
编码转基因序列的核酸如DNA可以随机插入细胞的染色体。随机整合可以由将核酸(例如DNA)引入细胞的任何方法产生。例如，该方法可以包括但不限于电穿孔、超声、使用基因枪、脂转染、磷酸钙转染、使用树枝状大分子、显微注射和使用包括腺病毒、AAV和逆转录病毒载体的病毒载体、和/或II型核酶。
编码转基因的DNA也可以设计成包括报告基因，从而可以通过报告基因的活化检测转基因或其表达产物的存在。可以使用任何报道基因，例如上述那些。通过在细胞培养物中选择其中报道基因已经被活化的细胞，可以选择含有转基因的细胞。
CAR的表达可以通过表达测定法，例如qPCR或通过测量RNA的水平来验证。 表达水平也可以指示拷贝数。例如，如果表达水平非常高，这可以表明CAR的多于一个拷贝被整合到基因组中。或者，高表达可以指示转基因整合在高转录区域中，例如高度表达的启动子附近。也可以通过测量蛋白质水平来验证表达，例如通过Western印迹。
在一些实施方案中，本发明的免疫应答细胞可以包含一种或多种转基因。所述一种或多种转基因可以表达CAR蛋白，而该CAR蛋白识别并结合抗原上的至少一个表位或结合抗原上的突变表位。CAR可以是功能性CAR。在一些实施方案中本发明的免疫应答细胞可以包含一种或多种CAR，或者其可以包含单一CAR和二次工程化受体。
在一些实施方案中，转基因可编码自杀基因。如癌症患者的许多有效治疗所证明的，CAR免疫应答细胞使得肿瘤消退但可伴随毒性。在一些实施方案中，当靶标抗原在正常组织和肿瘤细胞中共享时，CAR免疫应答细胞可能不能区分肿瘤和正常组织(“在靶/脱靶毒性”)。在另一些情况下，可以发生免疫系统的全身扰动，称为细胞因子释放综合征(CRS)。所述CRS可以包含全身炎症反应综合征或细胞因子风暴，这可能是CAR免疫应答细胞体内快速膨胀的后果。CRS是以发热和低血压为特征的病症，严重者可导致多器官功能衰竭。在大多数情况下，所述毒性与输注的CAR免疫应答细胞的体内扩增相关，其可引起免疫系统的整体扰动，以及释放高水平的促炎细胞因子，例如TNFα和IL-6。自杀基因可以诱导消除CAR免疫反应性细胞。自杀基因可以是在所述CAR免疫反应性细胞中诱导细胞凋亡的任何基因。自杀基因可以与所述结合抗原的受体一起编码在病毒载体内。编码自杀基因使得在特定的情况下可以缓解或者彻底中止由输注的CAR免疫应答细胞在体内扩增引起的毒性。
在一些实施方案中，可以产生存在于正常组织的抗原的CAR免疫反应性细胞，使得它们瞬时表达CAR，例如在电穿孔编码受体的mRNA之后。此外，通过包括安全开关来进一步加强CAR免疫反应性细胞的重大努力，在严重的在靶毒性的情况下，可以大大消除CAR免疫反应性细胞。
在一些实施方案中，编码CAR的载体可以与诸如可诱导的半胱天冬酶-9基因(由二聚化学诱导剂激活)或截短形式的EGF受体R(由单克隆抗体西妥昔单抗激活)或RQR8的安全开关组合。
本文所用的一个或多个转基因可以来自不同的物种。例如，一个或多个转基因可以包含人基因、小鼠基因、大鼠基因、猪基因、牛基因、狗基因、猫基因、猴基 因、黑猩猩基因或其任何组合。例如，转基因可以来自具有人类遗传序列的人。一个或多个转基因可以包含人基因。在某些情况下，一个或多个转基因不是腺病毒基因。
如上所述，转基因可以以随机或位点特异性方式插入免疫反应性细胞的基因组中。例如，可以将转基因插入到免疫细胞的基因组中的随机位点。这些转基因可以是功能性的，例如，在插入到基因组中的任何地方中时是完全功能性的。例如，转基因可以编码其自身的启动子，或者可以插入其内部启动子控制的位置。或者，可以将转基因插入基因，例如基因的内含子或基因的外显子、启动子或非编码区。可以插入转基因使得插入破坏基因，例如内源性免疫检查点。
在一些实施方案中，一个以上拷贝的转基因可以插入到基因组内的多个随机位点。例如，可将多个拷贝插入基因组中的随机位点。与转基因随机插入一次相比，这可能导致整体表达增加。或者，转基因的拷贝可以插入到基因中，转基因的另一拷贝可以插入到不同的基因中。可以靶向转基因，使其可以插入到免疫反应性细胞的基因组中的特定位点。
在一些实施方案中，包含编码结合抗原的受体序列的多核酸可以采取质粒载体的形式。质粒载体可以包含启动子。在某些情况下，启动子可以是组成型的。在一些实施方案中，启动子是可诱导的。启动子可以是或可以衍生自CMV、U6、MND或EF1a。在一些实施方案中，启动子可以与CAR序列相邻。在一些实施方案中，质粒载体还包含剪接受体。在一些实施方案中，剪接受体可以与CAR序列相邻。启动子序列可以是PKG或MND启动子。MND启动子可以是含有骨髓增生性肉瘤病毒增强子修饰的MoMuLV LTR的U3区域的合成启动子。
在一些实施方案中，可以设计编码目标受体的多核酸，以通过非病毒技术递送至细胞。在某些情况下，多核酸可以是良好的制造规范(GMP)兼容试剂。
编码目标结合抗原的受体或CAR的多核酸的表达可以由一种或多种启动子控制。启动子可以是普遍存在的，组成型(不受限制的启动子，允许相关基因的连续转录)，组织特异性启动子或诱导型启动子。可以调节插入邻近或接近启动子的转基因的表达。例如，转基因可插入到普遍存在的启动子附近或旁边。一些普遍存在的启动子可以是CAGGS启动子、hCMV启动子、PGK启动子、SV40启动子或ROSA26启动子。
启动子可以是内源的或外源的。例如，可以将一个或多个转基因插入到内源或外源ROSA26启动子的邻近或接近处。此外，启动子可以是免疫反应性细胞的特 异性。例如，一个或多个转基因可以插入猪ROSA26启动子的邻近或接近。
组织特异性启动子或细胞特异性启动子可用于控制表达的位置。例如，可以将一个或多个转基因插入组织特异性启动子的邻近或接近。组织特异性启动子可以是FABP启动子、Lck启动子、CamKII启动子、CD19启动子、角蛋白启动子、白蛋白启动子、aP2启动子、胰岛素启动子、MCK启动子、MyHC启动子、WAP启动子、或Col2A启动子。
也可以使用诱导型启动子。如果需要，可以通过添加或除去诱导剂来开启和关闭这些诱导型启动子。预期诱导型启动子可以是但不限于Lac、tac、trc、trp、araBAD、phoA、recA、proU、cst-1、tetA、cadA、nar、PL、cspA、T7、VHB、Mx和/或Trex。
本文所用的术语“诱导型启动子”是一种受控的启动子，其在所期待的条件未达成前不表达或者低表达与其可操作地连接的基因，而在所期待的条件达成的情况下表达或者高水平表达与其可操作地连接的基因。例如，在一些实施方中，本申请的诱导型启动子在细胞中的正常或高氧含量的条件下不表达或者低表达与其可操作地连接的基因，而响应于细胞中降低的氧含量，在缺氧条件下表达或者高表达与其可操作地连接的基因。在一些实施方案中，本文所用的诱导型启动子包括低氧可诱导的转录因子-1α(Hypoxia-Inducible Transcription factor-1α,HIF-1α)。在在一些实施方案中，本文所用的术语“诱导型启动子”是指“免疫细胞诱导型启动子”，其在免疫应答细胞接触抗原之前或免疫应答细胞未被激活之时不表达或者低表达与其可操作地连接的基因，而仅在免疫应答细胞接触抗原或免疫应答细胞被激活时，才会驱动与其操作性连接的基因发生高水平表达或者在缺氧等条件下表达。在一些实施方案中，所述的“免疫细胞诱导型启动子”包括NFAT(活化T细胞核因子)型启动子。
本文所用的“NFAT型启动子”是指基于NFAT结合活性进行调控与其可操作地连接的基因的表达的一类启动子。
NFAT是一个在免疫反应中具有重要作用的转录因子家族的统称。NFAT家族的一个或多个成员在免疫系统的大多数细胞中表达。NFAT也参与心脏、骨骼肌和神经系统的发育。
NFAT转录因子家族由五个成员NFAT1、NFAT2、NFAT3、NFAT4和NFAT5组成。NFAT1至NFAT4受钙信号调节。钙信号对NFAT激活至关重要，因为钙调蛋白(CaM)激活丝氨酸/苏氨酸磷酸酶钙调神经磷酸酶(CN)。活化的CN使NFAT蛋白氨基末端的丝氨酸丰富区域(SRR)和SP重复序列快速脱磷酸化，导 致构象变化，暴露核定位信号，导致NFAT输入核中。
基于NFAT在T细胞活化过程中细胞因子的转录表达所起的作用，其可用于调控本文所述的免疫细胞诱导型启动子，从而在免疫应答细胞接触抗原活化时，表达或者高水平表达与其可操作地连接的基因。
本发明的核酸可以包含编码NFAT型启动子(或其功能部分或功能变体)的任何合适的核苷酸序列。本文所用的“NFAT型启动子”是指与T细胞表达的任何基因的最小启动子连接的一个或多个NFAT应答元件。优选地，由T细胞表达的基因的最小启动子是最小的人IL-2启动子。NFAT应答元件可以包括例如NFAT1、NFAT2、NFAT3和/或NFAT4应答元件。在一些实施方案中，本文所述的“NFAT型启动子”中可包括多于1个NFAT结合基序。例如，所述“NFAT型启动子”可包括2、3、4、5、6、7、8、9、10或更多个NFAT结合基序。在一些实施方案中，所述的“NFAT型启动子”中包括多达12个NFAT结合基序。在一些实施方案中，所述“NFAT型启动子”可以是多个所述NFAT结合基序与启动子如IL2最小启动子串联所组成的启动子。在一些实施方案中，本文所述的NFAT型启动子包括6个NFAT结合基序，记作(NFAT)6。出于便利的目的，所述(NFAT)6也记作NFAT6。在一些实施方案中，所述NFAT6也表示在所述NFAT型启动子中的6个重复的NFAT结合基序(SEQ ID NO:94)。
此外，尽管不是表达必需的，但转基因序列还可以包括转录或翻译调控序列，例如启动子、增强子、绝缘体、内部核糖体进入位点、编码2A肽和/或多腺苷酸化信号的序列。
在一些实施方案中，转基因编码目标结合抗原的受体或CAR，其中将转基因插入安全港，使得表达所述结合抗原的受体。在一些实施方案中，将转基因插入PD1和/或CTLA-4基因座。在其他情况下，将转基因以慢病毒递送至细胞随机插入，而PD1-或CTLA-4特异性核酸酶可作为mRNA提供。在一些实施方案中，转基因通过病毒载体系统如逆转录病毒、AAV或腺病毒以及编码对于安全港特异的核酸酶(例如AAVS1、CCR5、白蛋白或HPRT)的mRNA递送。也可以用编码PD1和/或CTLA-4特异性核酸酶的mRNA处理细胞。在一些实施方案中，编码CAR的多核苷酸通过病毒递送系统与编码HPRT特异性核酸酶和PD1-或CTLA-4特异性核酸酶的mRNA一起提供。可以与本文公开的方法和组合物一起使用的CAR可以包括所有类型的这些嵌合蛋白。
在一些实施方案中，可以使用逆转录病毒载体(γ-逆转录病毒或慢病毒载体) 将转基因导入免疫反应性细胞。例如，编码CAR的转基因或结合抗原的任何受体或其变体或片段可被克隆到逆转录病毒载体中，并且可以由其内源性启动子、逆转录病毒长末端重复序列、或对靶细胞类型特异性的启动子驱动。也可以使用非病毒载体。非病毒载体递送系统可以包括DNA质粒、裸核酸和与递送载体如脂质体或泊洛沙姆复合的核酸。
已经开发了许多基于病毒的系统用于将基因转移到哺乳动物细胞中。例如，逆转录病毒为基因递送系统提供了便利的平台。可以使用本领域已知的技术将所选择的基因插入载体并包装在逆转录病毒颗粒中。衍生自逆转录病毒如慢病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因的长期稳定整合及其在子细胞中的繁殖。慢病毒载体与衍生自逆转录病毒如鼠类白血病病毒的载体相比具有附加优点，因为它们可以转导非增殖细胞。它们还具有低免疫原性的附加优点。腺病毒载体的优点是它们不融合到靶细胞的基因组中，从而绕过负面的整合相关事件。
可以用编码所述结合抗原的受体的转基因转染细胞。转基因浓度可以为约100皮克至约50微克。在一些实施方案中，可以改变引入细胞的核酸(例如，ssDNA、dsDNA或RNA)的量以优化转染效率和/或细胞活力。例如，可以向每个细胞样品中加入1微克dsDNA用于电穿孔。在一些实施方案中，最佳转染效率和/或细胞活力所需的核酸(例如，双链DNA)的量根据细胞类型而不同。在一些实施方案中，用于每个样品的核酸(例如，dsDNA)的量可以直接对应于转染效率和/或细胞活力。例如，一系列转染浓度。由载体编码的转基因可以整合到细胞基因组中。在一些实施方案中，由载体编码的转基因前向整合。在其他情况下，由载体编码的转基因的反向整合。
通常通过全身给药(例如静脉内、腹膜内、肌内、皮下或颅内输注)或局部应用，通过给予个体患者体内递送载体，如下所述。或者，载体可以离体递送到细胞，例如从个体患者(例如，淋巴细胞、T细胞、骨髓抽吸物、组织活检)移出的细胞，然后通常在选择并入了该载体的细胞后将细胞再植入患者体内。在选择之前或之后，可以扩增细胞。
用于表达结合抗原的受体的合适的免疫反应性细胞可以是对于有需要的个体是自体的或非自体的细胞。
可以从个体获得合适的免疫应答细胞的来源。在某些情况下，可以获得T细胞。所述T细胞可以从许多来源获得，包括PBMC、骨髓、淋巴结组织、脐带血、胸腺组织和来自感染部位、腹水、胸腔积液、脾组织和肿瘤的组织。在某些情况下， 可以使用任何数量的本领域技术人员已知的技术，例如FicollTM分离，从自所述个体收集的血液获得T细胞。在一些实施方案中，通过单采血获得来自个体的循环血液的细胞。单采制品通常含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核白细胞、红细胞和血小板。在一些实施方案中，可以洗涤通过单采采集收集的细胞以除去血浆级分并将细胞置于合适的缓冲液或培养基中用于随后的加工步骤。
或者，可以从健康供体，来自诊断患有癌症的患者或诊断为感染的患者衍生细胞。在一些实施方案中，细胞可以是具有不同表型特征的混合细胞群体的一部分。还可以根据前述方法从转化的T细胞获得细胞系。还可以从细胞治疗库获得细胞。可以通过本文所述的任何方法获得对免疫抑制治疗有抗性的修饰细胞。还可以在修饰前选择合适的细胞群。修饰后也可以选择工程细胞群。工程细胞可用于自体移植。或者，细胞可用于同种异体移植。在一些实施方案中，将细胞施用于样品用于鉴定癌症相关靶序列的同一患者。在其他情况下，将细胞施用于不同于其样本用于鉴定癌症相关靶序列的患者的患者。
在一些实施方案中，合适的原代细胞包括外周血单核细胞(PBMC)、外周血淋巴细胞(PBL)和其它血液细胞亚群，例如但不限于T细胞、天然杀伤细胞、单核细胞、天然杀伤剂T细胞、单核细胞前体细胞、造血干细胞或非多能干细胞。在一些实施方案中，细胞可以是任何免疫细胞，包括任何T细胞如肿瘤浸润细胞(TIL)，如CD3+T细胞、CD4+T细胞、CD8+T细胞或任何其他类型的T细胞。T细胞还可以包括记忆T细胞、记忆干T细胞或效应T细胞。也可以从大量群体中选择T细胞，例如从全血中选择T细胞。T细胞也可以从大量群体中扩增。T细胞也可能倾向于特定种群和表型。例如，T细胞可以倾斜于表型包含CD45RO(-)、CCR7(+)、CD45RA(+)、CD62L(+)、CD27(+)、CD28(+)和/或IL-7Rα(+)。合适的细胞可以选自以下列表中的一种或多种标志物：CD45RO(-)、CCR7(+)、CD45RA(+)、CD62L(+)、CD27(+)、CD28(+)和/或IL-7Rα(+)。合适的细胞还包括干细胞，例如，例如胚胎干细胞、诱导的多能干细胞、造血干细胞、神经元干细胞和间充质干细胞。合适的细胞可以包含任何数量的原代细胞，例如人细胞、非人细胞和/或小鼠细胞。合适的细胞可以是祖细胞。合适的细胞可以衍生自要治疗的受试者(例如，患者)。
患者中需要的治疗有效的细胞的量可以根据细胞的存活力和细胞被遗传修饰的效率而变化(例如，转基因被整合到一个或多个细胞中的效率，或者由转基因编码的蛋白质的表达水平)。在一些实施方案中，遗传修饰后细胞存活力的产物(例 如，倍增)和转基因整合的效率可以对应于可用于给予受试者的细胞的治疗量。在一些实施方案中，遗传修饰后细胞存活力的增加可能对应于给予治疗对患者有效的必需细胞量的减少。在一些实施方案中，转基因整合到一个或多个细胞中的效率的增加可以对应于给予在患者中治疗有效的必需的细胞数量的减少。在一些实施方案中，确定所需的治疗有效的细胞的量可以包括确定与细胞随时间变化相关的功能。在一些实施方案中，确定需要治疗有效的细胞的量可以包括确定与根据时间相关变量将转基因整合到一个或多个细胞中的效率变化相对应的功能(例如，细胞培养时间、电穿孔时间、细胞刺激时间)。在一些实施方案中，治疗有效的细胞可以是细胞群，其包含在细胞表面上约30％至约100％的结合抗原的受体的表达。在一些实施方案中，通过流式细胞术测量，治疗有效的细胞可以在细胞表面上表达所述结合抗原的受体约30％、35％、40％、45％、50％、55％、60％、65％、70％、75％80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、99.5％、99.9％或超过约99.9％。
根据本发明的一个方面，本发明也包括编码所述结合抗原的受体的核酸。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。
本发明还提供了包含上述编码表达于免疫应答细胞表面的结合抗原的受体蛋白的核酸的载体。
本发明还包括包含上述载体的病毒。本发明的病毒包括包装后的具有感染力的病毒，也包括包含包装为具有感染力的病毒所必需成分的待包装的病毒。本领域内已知的其它可用于将外源基因转导入免疫应答细胞的病毒及其对应的质粒载体也可用于本发明。
在另一个方面，本文提供了宿主细胞，其包含本文所述的抗原结合单元或嵌合抗原受体、以及任选的I型干扰素。在另一个方面，本文提供了宿主细胞，其包含编码本文所述的抗原结合单元或嵌合抗原受体、以及任选的I型干扰素的核酸。
在一些实施方案中，所述宿主细胞是免疫应答细胞。在一些实施方案中，所述免疫应答细胞是T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、和/或调节性T细胞。在一些实施方案中，所述宿主细胞是NK92细胞。
在一些实施方案中，本发明的免疫应答细胞中可以包括表达构建物，该表达构建物中存在按如下方式顺序连接的元件：抗体、CD28共刺激信号结构域、CD3ζ，以及与前述元件反向连接的NFAT6、I型干扰素表达单元。较佳地，所述的抗体与 CD28共刺激信号结构域之间通过CD8α跨膜区和CD8α铰链区相连。
在一些实施方案中，活化T细胞核因子NFAT(Nuclear factor of activated T cells)在T细胞活化过程中细胞因子的转录表达起着重要的作用。基于这样考虑，本发明人将IFN-beta编码序列置于NFAT6启动子的调控之下，这样只有当CAR-T细胞接触抗原引发T细胞活化，IFN-beta才能高水平表达。
NFAT6启动子是利用6个NFAT的结合位子与IL2的最小启动子(minimal promoter)串联在一起组成的启动子(Hooijberg E,Bakker AQ,Ruizendaal JJ,Spits H.NFAT-controlled expression of GFP permits visualization and isolation of antigen-stimulated primary human Tcells.Blood.2000Jul 15；96(2):459-66)，可用于调节细胞因子如IL12等在T淋巴细胞如TCR-T中的表达(Zhang L,Kerkar SP,YuZ,Zheng Z,Yang S,Restifo NP,Rosenberg SA，Morgan RA.Improving adoptive T cell therapy by targeting and controlling IL-12expression to the tumor environment.Mol Ther.2011 Apr；19(4):751-9)。
本发明的免疫应答细胞，其被转导有能表达结合抗原的受体和外源性I型干扰素的构建物，或表达载体，或包含该质粒的病毒。本领域常规的核酸转导方法，包括非病毒和病毒的转导方法都可以用于本发明。
本发明所述的免疫应答细胞还可以进一步携带外源的细胞因子的编码序列；所述的细胞因子包括但不限于：IL-12，IL-15或IL-21等。这些细胞因子具有进一步的免疫调节或抗肿瘤的活性，能增强效应T细胞及活化的NK细胞的功能，或直接发挥抗肿瘤作用。因此，本领域技术人员可以理解，这些细胞因子的运用有助于所述的免疫应答细胞更好地发挥作用。
本发明所述的免疫应答细胞还可以表达除了上述结合抗原的受体以外的另一种结合抗原的受体。
本发明所述的免疫应答细胞还可以表达趋化因子受体；所述的趋化因子受体包括但不限于CCR2。本领域技术人员可以理解，所述的CCR2趋化因子受体可以使得体内的CCR2与之竞争性结合，对于阻断肿瘤的转移是有利的。
本发明所述的免疫应答细胞还可以表达能降低PD-1表达的siRNA或者阻断PD-L1的蛋白。本领域技术人员可以理解，竞争性阻断PD-L1与其受体PD-1的相互作用，有利于恢复抗肿瘤T细胞反应，从而抑制肿瘤生长。
本发明所述的免疫应答细胞还可以表达安全开关；较佳地，所述的安全开关包括：iCaspase-9，Truancated EGFR或RQR8。
在一些实施方案中，本发明的免疫应答细胞不表达诸如4-1BBL这类的共刺激配体。
因此，在另一方面，本文还提供了一种生成本文所述的抗原结合单元或嵌合抗原受体或者包含它们的组合物的方法，其包括在适合的条件下培养本文所述的宿主细胞。在一些实施方案中，所述方法包括分离并获得所述宿主细胞的表达产物。
在另一方面，本文提供了一种组合物，其包含本文所述的抗原结合单元、嵌合抗原受体、或核酸。在一些实施方案中，所述组合物是包含所述抗原结合单元、嵌合抗原受体或核酸的药物组合物。在一些实施方案中，所述药物组合物还包含药学可接受的载体。
在另一方面，本文提供了一种药物组合物，其包含本文所述的宿主细胞和药学可接受的载体。
术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。
在一些实施方案中，所述组合物包含另一治疗剂。在一些实施方案中，所述另一治疗剂是化疗剂，如US20140271820中记载的那些和/或其药学上可接受的盐或类似物。在一些实施方案中，所述治疗剂包括但不限于有丝分裂抑制剂(长春花生物碱)，包括长春新碱、长春花碱、长春地辛和诺维宾(TM)(长春瑞滨，5'-去氢硫化氢)；拓扑异构酶I抑制剂，例如喜树碱化合物，包括CamptosarTM(伊立替康HCL)、HycamtinTM(托泊替康HCL)和衍生自喜树碱及其类似物的其它化合物；鬼臼毒素衍生物，例如依托泊苷、替尼泊苷和米多昔佐兹；烷基化剂顺铂、环磷酰胺、氮芥、三亚甲基硫代磷酰胺、卡莫司汀、白消安、苯丁酸氮芥、布列喹嗪、尿嘧啶芥末、氯洛芬和达卡巴嗪；抗代谢物，包括阿糖胞苷、5-氟尿嘧啶、甲氨蝶呤、巯嘌呤、硫唑嘌呤和丙卡巴肼；抗生素，包括但不限于多柔比星、博来霉素、更生霉素、柔红霉素、霉素霉素、丝裂霉素、肉瘤霉素C和道诺霉素；以及其它化疗药物，包括但不限于抗肿瘤抗体、达卡巴嗪、氮胞苷、阿姆沙康、美法仑、异环磷酰胺和米托蒽醌。在一些实施方案中，所述另外的治疗剂选自表柔比星、奥沙利铂和5-氟尿嘧啶中的一种或多种。在一些实施方案中，所述另外的治疗剂包括但不限于抗血管生成剂，包括抗VEGF抗体(包括人源化和嵌合抗体、抗VEGF适体和反义寡核苷酸)以及其他血管发生抑制剂，例如血管抑素、内皮抑制素、干扰素、白细胞介素1(包括α和β)白介素12、视黄酸和金属蛋白酶-1和-2的组织抑制剂等。
可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如
Figure PCTCN2017092381-appb-000002
润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；和磷酸盐缓冲液等。
本文所述的药物组合物可包含一种或多种药学可接受的盐。“药学可接受的盐”指这样一种盐，其保留亲本化合物的期望生物学活性且不产生任何不利的毒物学效果(参见例如，Berge,S.M.,等人1977,J.Pharm.Sci.66:1-19)。此类盐的例子包括酸加成盐和碱加成盐。
酸加成盐包括衍生自无毒无机酸，诸如盐酸、硝酸、磷酸、硫酸、氢溴酸、氢碘酸、亚磷酸等的盐，以及衍生自无毒有机酸，诸如脂肪族单羧酸和二羧酸、苯基取代的链烷酸、羟基链烷酸、芳香族酸、脂肪族和芳香族磺酸等的盐。碱加成盐包括衍生自碱土金属(诸如钠、钾、镁、钙等)的盐，以及衍生自无毒有机胺的盐，诸如N,N'-二苄乙二胺、N-甲基葡糖胺、氯普鲁卡因、胆碱、二乙醇胺、乙二胺、普鲁卡因等。
本文所述的药物组合物还可包含抗氧化剂。抗氧化剂的实例包括但不限于：水溶性抗氧化剂，诸如抗坏血酸、盐酸半胱氨酸、硫酸氢钠、焦亚硫酸钠、亚硫酸钠等；油溶性抗氧化剂，诸如抗坏血酸棕榈酸酯、丁基化羟基茴香醚(BHA)、丁基化羟基甲苯(BHT)，卵磷脂、没食子酸丙酯、α-生育酚等；和金属螯合剂，诸如柠檬酸、乙二胺四乙酸(EDTA)、山梨醇、酒石酸、磷酸等。
本发明的组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用肠胃外给药(如注射)或其它治疗方式。
免疫原性组合物的“肠胃外”施用包括例如皮下(s.c.)、静脉内(i.v.)、肌内(i.m.)或胸骨内注射或输注技术。
给予个体的包含免疫反应性细胞群体的制剂包含有效治疗和/或预防特定适应症或疾病的多个免疫反应性细胞。因此，可以向个体施用免疫反应性细胞的治疗有效群体。通常，施用包含约1×104至约1×1010个免疫反应性细胞的制剂。在大多数情况下，制剂将包含约1×105至约1×109个免疫反应性细胞、约5×105至约5 ×108个免疫反应性细胞、或约1×106至约1×107个免疫反应性细胞。然而，根据癌症的位置、来源、身份、程度和严重程度、待治疗的个体的年龄和身体状况等，对个体施用的CAR免疫反应性细胞的数量将在宽的范围之间变化。医生将最终确定要使用的适当剂量。
在一些实施方案中，使用嵌合抗原受体来刺激免疫细胞介导的免疫应答。例如，T细胞介导的免疫应答是涉及T细胞活化的免疫应答。活化的抗原特异性细胞毒性T细胞能够在表面上显示外源抗原表位的靶细胞中诱导细胞凋亡，例如显示肿瘤抗原的癌细胞。在另一些实施方案中，使用嵌合抗原受体在哺乳动物中提供抗肿瘤免疫。由于T细胞介导的免疫应答，受试者将产生抗肿瘤免疫。
在某些情况下，治疗患有癌症的受试者的方法可以涉及向需要治疗的受试者施用一种或多种本发明所述的免疫应答细胞。所述免疫应答细胞可结合肿瘤靶分子并诱导癌细胞死亡。如前文所述，本发明还提供治疗个体中的病原体感染的方法，包括向所述个体施用治疗有效量的本发明的免疫应答细胞。
本发明的免疫反应性细胞的给药频率将根据包括所治疗疾病的因素、特定免疫反应性细胞的元件和给药方式。例如可以每日给药4次、3次、2次或每日一次、每隔一天、每三天、每四天、每五天、每六天一次、每周一次、每八天一次、每九天一次、每十天、每周一次、或者每月两次给药。如本文所述，由于本申请的免疫应答细胞具有改善的活力，从而可以不仅以与类似的但不表达外源性I型干扰素的免疫应答细胞更低的治疗有效的量给药，并且可以以更低的频率给药，以获得至少类似、并且优选更加显著的疗效。
在一些实施方案中，组合物可以是等渗的，即它们可以具有与血液和泪液相同的渗透压。本发明组合物的期望等渗性可以使用氯化钠或其它药学上可接受的试剂如葡萄糖、硼酸、酒石酸钠、丙二醇或其它无机或有机溶质来实现。如果需要，组合物的粘度可以使用药学上可接受的增稠剂维持在选定的水平。合适的增稠剂包括，例如，甲基纤维素、黄原胶、羧甲基纤维素、羟丙基纤维素、卡波姆等。增稠剂的优选浓度将取决于所选择的试剂。显然，合适的载体和其它添加剂的选择将取决于确切的给药途径和特定剂型的性质，例如液体剂型。
本发明还提供了包含本文所述的抗原结合单元、嵌合抗原受体、核酸或免疫应答细胞的试剂盒。在一些实施方案中，试剂盒可以包括含有有效量的包含一种或多种单位剂型的本文所述的抗原结合单元、嵌合抗原受体、核酸或免疫应答细胞的治疗或预防组合物。在一些实施方案中，试剂盒包含可含有治疗或预防性组合物的无 菌容器；这样的容器可以是盒、安瓿、瓶、小瓶、管、袋、泡罩包装或本领域已知的其它合适的容器形式。这种容器可以由塑料、玻璃、层压纸、金属箔或其他适合于保持药物的材料制成。在一些实施方案中，所述试剂盒包含本文所述的抗原结合单元、嵌合抗原受体、核酸或免疫应答细胞，以及将本文所述的抗原结合单元、嵌合抗原受体、核酸或免疫应答细胞给予个体的说明书。说明书中通常包含使用本文所述的抗原结合单元、嵌合抗原受体、核酸或免疫应答细胞来治疗或预防癌症或肿瘤的方法。在一些实施方案中，试剂盒包含本文所述的宿主细胞，并且可以包括约1×104个细胞至约1×106个细胞。在一些实施方案中，试剂盒可以包括至少约1×105个细胞，至少约1×106个细胞，至少约1×107个细胞，至少约4×107个细胞，至少约5×107个细胞，至少约6×107个细胞，至少约6×107个细胞，8×107个细胞，至少约9×107个细胞，至少约1×108个细胞，至少约2×108个细胞，至少约3×108个细胞，至少约4×108个细胞，至少约5×108个细胞，至少约6×108个细胞，至少约6×108细胞，至少约8×108个细胞，至少约9×108细胞，至少约1×109个细胞，至少约2×109个细胞，至少约3×109个细胞，至少约4×109个细胞，至少约5×109个细胞，至少约6×109个细胞，至少约8×109个细胞，至少约9×109个细胞，至少约1×1010个细胞，至少约2×1010个细胞，至少约3×1010个细胞，至少约4×1010个细胞，至少约5×1010个细胞，至少约6×1010个细胞，至少为ab至少约9×1010个细胞，至少约9×1010个细胞，至少约1×1011个细胞，至少约2×1011个细胞，至少约3×1011个细胞，至少约4×1011个细胞，至少约5×1011个细胞，至少约8×1011个细胞，至少约9×1011个细胞，或至少约1×1012个细胞。例如，可以在试剂盒中包括大约5×1010个细胞。在另一个实例中，试剂盒可以包括3×106个细胞；细胞可以扩增至约5×1010个细胞并施用于受试者。
在一些实施方案中，试剂盒可以包括同种异体细胞。在一些实施方案中，试剂盒可以包括可以包含基因组修饰的细胞。在一些实施方案中，试剂盒可以包含“现成的”细胞。在一些实施方案中，试剂盒可以包括可以扩展用于临床使用的细胞。在某些情况下，试剂盒可能包含用于研究目的的内容物。
在一些实施方案中，说明书包括以下中的至少一个：治疗剂的描述；用于治疗或预防肿瘤或其症状的剂量方案和给药；预防措施、警示、禁忌症、过量信息、不良反应、动物药理学、临床研究、和/或引用文献。说明书可以直接打印在容器上(如果有的话)，或作为容器上的标签，或作为容器内或容器中提供的单独的纸张、小册子、卡片或文件夹打印。在一些实施方案中，说明书提供施用本发明所述的免 疫应答细胞用于治疗或预防肿瘤的方法。在某些情况下，说明书提供了施用化学治疗剂之前、之后或同时给予本发明的免疫反应性细胞的方法。
在另一方面，本文还提供了一种诱导包含密蛋白18A2肽的细胞死亡的方法，所述方法包括使所述细胞与本文所述的抗原结合单元、本文所述的嵌合抗原受体、本文所述的组合物、或本文所述的宿主细胞接触。在一些实施方案中，所述接触是在体外接触。在一些实施方案中，所述接触是在体内接触。
在一些实施方案中，所述细胞是肿瘤细胞。在一些实施方案中，所述细胞是实体瘤细胞。在一些实施方案中，所述细胞是如本文所述的癌症或者肿瘤的细胞。这些细胞的具体实例可包括但不限于：白血病(如急性白血病、急性淋巴细胞白血病、急性髓细胞性白血病，急性粒细胞白血病，急性早幼粒细胞白血病、急性粒-单核细胞白血病、急性单核细胞白血病、急性白血病、慢性白血病、慢性粒细胞白血病、慢性淋巴细胞白血病、真性红细胞增多症)细胞，淋巴瘤(霍奇金病、非霍奇金病)细胞、原发性巨球蛋白血症细胞，重链病细胞，实体瘤如肉瘤和癌症细胞(如纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、骨肉瘤、脊索瘤、内皮肉瘤、淋巴管肉瘤、血管肉瘤、淋巴管内皮肉瘤，滑膜vioma，间皮瘤，尤文氏瘤、平滑肌肉瘤、横纹肌肉瘤、结肠癌、胰腺癌、乳腺癌、卵巢癌、前列腺癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、癌、支气管癌、髓样癌、肾细胞癌、肝癌，尼罗河管癌，绒癌、精原细胞瘤、胚胎癌、肾母细胞瘤、宫颈癌、子宫癌、睾丸癌、肺癌、小细胞肺癌、膀胱癌、上皮癌、胶质瘤、星形细胞瘤、髓母细胞瘤，颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤，听神经瘤，少突胶质瘤、神经鞘瘤、脑膜瘤、黑色素瘤、神经母细胞瘤、视网膜母细胞瘤)、食管癌细胞、胆囊癌细胞、肾癌细胞、多发性骨髓瘤细胞等。在一些实施方案中，所述细胞是胃癌细胞、食道癌细胞、肠癌细胞、胰腺癌细胞、肾母细胞瘤细胞、肺癌细胞、卵巢癌细胞、结肠癌细胞、直肠癌细胞、肝癌细胞、头颈癌细胞、慢性骨髓性白血病细胞和胆囊癌细胞。
在另一方面，本文提供了一种治疗有需要的个体的肿瘤的方法，所述方法包括向所述个体给予有效量的本文所述的抗原结合单元、嵌合抗原受体、组合物、载体或者宿主细胞。
在一些实施方案中，所述肿瘤包括但不限于选自膀胱、骨、脑、乳腺、软骨、神经胶质细胞、食管、输卵管、胆囊、心脏、肠、肾、肝、肺、淋巴结、神经组织、卵巢、胰腺、前列腺、骨骼肌、皮肤、脊髓、脾、胃、睾丸、胸腺、甲状腺、气管、 尿道、输尿管、尿道、子宫、阴道器官的肿瘤。在一些实施方案中，所述肿瘤包括但不限于白血病(如急性白血病、急性淋巴细胞白血病、急性髓细胞性白血病，急性粒细胞白血病，急性早幼粒细胞白血病、急性粒-单核细胞白血病、急性单核细胞白血病、急性白血病、慢性白血病、慢性粒细胞白血病、慢性淋巴细胞白血病、真性红细胞增多症)，淋巴瘤(霍奇金病、非霍奇金病)、原发性巨球蛋白血症，重链病，实体瘤如肉瘤和癌症(如纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、骨肉瘤、脊索瘤、内皮肉瘤、淋巴管肉瘤、血管肉瘤、淋巴管内皮肉瘤，滑膜vioma，间皮瘤，尤文氏瘤、平滑肌肉瘤、横纹肌肉瘤、结肠癌、胰腺癌、乳腺癌、卵巢癌、前列腺癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、癌、支气管癌、髓样癌、肾细胞癌、肝癌，尼罗河管癌，绒癌、精原细胞瘤、胚胎癌、肾母细胞瘤、宫颈癌、子宫癌、睾丸癌、肺癌、小细胞肺癌、膀胱癌、上皮癌、胶质瘤、星形细胞瘤、髓母细胞瘤，颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤，听神经瘤，少突胶质瘤、神经鞘瘤、脑膜瘤、黑色素瘤、神经母细胞瘤、视网膜母细胞瘤)、食管癌、胆囊癌、肾癌、多发性骨髓瘤。在一些实施方案中，所述肿瘤是胃癌、食道癌、肠癌、胰腺癌、肾母瘤、肺癌、卵巢癌、结肠癌、直肠癌、肝癌、头颈癌、慢性骨髓性白血病或胆囊癌。
在一些实施方案中，受试者可以给予免疫反应性细胞，其中可以施用的免疫反应性细胞可以是约1至约35天龄。例如，所施用的细胞可以是1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34或最多约40天。可以从刺激时起计算CAR免疫反应性细胞的年龄。可以从血液采集的时间开始计算免疫反应性细胞的年龄。可以从转导时起计算免疫反应性细胞的年龄。在一些实施方案中，可以给予受试者的免疫反应性细胞为约10至约14或约20天龄。在一些实施方案中，免疫反应性细胞的“年龄”可以通过端粒长度来确定。例如，“年轻”的免疫反应细胞可以具有比“耗尽”或“老”的免疫反应性细胞更长的端粒长度。不受特定理论的束缚，可以认为免疫反应性细胞在培养物中每周丢失约0.8kb的估计端粒长度，并且年轻的免疫反应性细胞培养物可以具有比约44天的免疫反应性细胞长约1.4kb的端粒。不受特定理论的束缚，人们认为更长的端粒长度可以与患者中的阳性客观临床反应和体内细胞的持久性相关联。
在移植之前、之后和/或期间，细胞(例如，工程细胞或工程化的原代T细胞)可以是功能性的。例如，移植的细胞可以是在移植后至少约1、2、3、4、5、6、7、 8、9、10、11、12、13、14、15、16、17、18，19、20、21、22、23、24、25、6、27、28、29、30、40、50、60、70、80、90或100天起作用。移植细胞可以在移植后至少约1、2、3、4、5、6、7、8、9、10、11或12个月起作用。移植细胞可以在移植后至少约1、2、3、4、5、6、7、8、9、10、15、20、25或30年起作用。在一些实施方案中，移植细胞可以在接受者的寿命期间起作用。
此外，移植细胞可以以其正常预期功能的100％起作用。移植细胞还可以发挥其正常预期功能约1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、或高达约100％的功能。
移植细胞也可以发挥其正常预期功能的超过100％的作用。例如，移植的细胞可以起到正常预期功能的约110、120、130、140、150、160、170、180、190、200、250、300、400、500、600、700、800、900、1000或至多约5000％的功能。
移植可以通过任何类型的移植。局部可以包括但不限于肝下囊空间、脾囊下空间、肾囊下空间、网膜、胃或肠粘膜下层、小肠血管节段、静脉囊、睾丸、脑、脾或角膜。例如，移植可以是囊下移植。移植也可以是肌内移植。移植可以是门静脉移植。
与当一个或多个野生型细胞被移植到接受者之时相比，采用本发明的免疫应答细胞治疗之后可以改善移植排斥反应。例如，移植排斥可以是超急性排斥反应。移植排斥也可以是急性排斥反应。其他类型的排斥可能包括慢性排斥反应。移植排斥也可以是细胞介导的排斥反应或T细胞介导的排斥反应。移植排斥也可以是自然杀伤细胞介导的排斥反应。
改善移植可能意味着减轻超急性排斥反应，其可以包括减少、减轻或降低不良作用或症状。移植可以指细胞产品的过继性移植。
移植成功的另一个迹象可以是接受者不需要免疫抑制治疗的天数。例如，在提供本发明的免疫应答细胞之后，接受者可以不要求至少约1、2、3、4、5、6、7、8、9、10或更多天的免疫抑制治疗。这可以表明移植成功。这也可以表明移植的细胞，组织和/或器官没有排斥。
在某些情况下，接受者不需要免疫抑制治疗至少1天。接受者也可不需要免疫 抑制治疗至少7天。接受者不需要免疫抑制治疗至少14天。接受者不需要免疫抑制治疗至少21天。接受者不需要免疫抑制治疗至少28天。接受者不需要免疫抑制治疗至少60天。此外，接受者可能不需要至少1、2、3、4、5、6、7、8、9、10或更多年的免疫抑制治疗。
移植成功的另一个迹象可能就是接受者需要减少的免疫抑制治疗的天数。例如，在本文提供的所述治疗之后，接受者可能需要至少1、2、3、4、5、6、7、8、9、10或更多天的减少的免疫抑制治疗。这可以表明移植成功。这也可以表明移植的细胞、组织和/或器官没有或仅有很小的排斥。
例如，接受者可能需要至少1天的减少的免疫抑制治疗。接受者也可能需要至少7天的减少的免疫抑制治疗。接受者可能需要至少14天的减少的免疫抑制治疗。接受者需要至少21天的减少的免疫抑制治疗。接受者需要至少28天的减少的免疫抑制治疗。接受者需要至少60天的减少的免疫抑制治疗。此外，接受者可能需要至少1、2、3、4、5、6、7、8、9、10或更多年的减少的免疫抑制治疗。
减少的免疫抑制治疗可以指与当将一种或多种野生型细胞移植到接受者中时所需的免疫抑制治疗相比而言较少的免疫抑制治疗。
免疫抑制治疗可以包括抑制免疫系统的任何治疗。免疫抑制治疗可以帮助缓解、减少或消除患者的移植排斥反应。例如，免疫抑制剂可以在移植之前、期间和/或之后使用，包括MMF(霉酚酸酯(Cellcept))、ATG(抗胸腺细胞球蛋白)、抗-CD154(CD4OL)、抗-CD40(2C10)、的免疫抑制药物、抗-IL-6R抗体(tocilizumab、Actemra)、抗-IL-6抗体(sarilumab、olokizumab)、CTLA4-Ig(Abatacept/Orencia)、抗-IL-6抗体(ASKP1240、CCFZ533X2201)、安非他明(Campath)、抗CD20(利妥昔单抗)、贝伐单抗(LEA29Y)、西罗莫司(Rapimune)、依维莫司、他克莫司(Prograf)、达替珠单抗(Ze-napax)、巴利昔单抗(Similect)、英夫利昔单抗(Remicade)、环孢菌素、脱氧精蛋白、可溶性补体受体1、眼镜蛇毒素、抗C5抗体(eculizumab/Soliris)、甲基泼尼松龙、FTY720、依维莫司、来氟米特、抗IL-2R-Ab、雷帕霉素、抗CXCR3抗体、抗ICOS抗体、抗OX40抗体和抗CD122抗体。此外，一种或多种免疫抑制剂/药物可一起使用或依次使用。一种或多种免疫抑制剂/药物可用于诱导治疗或维持治疗。诱导和维持阶段可以使用相同或不同的药物。在某些情况下，daclizumab(Zenapax)可用于诱导治疗，他克莫司(Prograf)和西罗莫司(Rapimune)可用于维持治疗。还可以使用非药物方案来实现免疫抑制，包括但不限于全身照射，胸腺照射和全部和/或部分脾切除术。这些技术也可 以与一种或多种免疫抑制药组合使用。
在一些实施方案中，本文所述的抗原结合单元、嵌合抗原受体、组合物、载体或者宿主细胞可以与另一治疗剂联合给药。在一些实施方案中，所述另一治疗剂是化疗剂，如US20140271820中记载的那些。可以与本发明的免疫应答细胞联合应用的化疗药物包括但不限于有丝分裂抑制剂(长春花生物碱)，包括长春新碱、长春花碱、长春地辛和诺维宾(TM)(长春瑞滨，5'-去氢硫化氢)；拓扑异构酶I抑制剂，例如喜树碱化合物，包括CamptosarTM(伊立替康HCL)、HycamtinTM(托泊替康HCL)和衍生自喜树碱及其类似物的其它化合物；鬼臼毒素衍生物，例如依托泊苷、替尼泊苷和米多昔佐兹；烷基化剂顺铂、环磷酰胺、氮芥、三亚甲基硫代磷酰胺、卡莫司汀、白消安、苯丁酸氮芥、布列喹嗪、尿嘧啶芥末、氯洛芬和达卡巴嗪；抗代谢物，包括阿糖胞苷、5-氟尿嘧啶、甲氨蝶呤、巯嘌呤、硫唑嘌呤和丙卡巴肼；抗生素，包括但不限于多柔比星、博来霉素、更生霉素、柔红霉素、霉素霉素、丝裂霉素、肉瘤霉素C和道诺霉素；以及其它化疗药物，包括但不限于抗肿瘤抗体、达卡巴嗪、氮胞苷、阿姆沙康、美法仑、异环磷酰胺和米托蒽醌。在一些实施方案中，所述额外的治疗剂选自表柔比星、奥沙利铂和5-氟尿嘧啶中的一种或多种。
在一些实施方案中，可以与本发明的免疫应答细胞联合应用的化疗药物包括但不限于抗血管生成剂，包括抗VEGF抗体(包括人源化和嵌合抗体、抗VEGF适体和反义寡核苷酸)以及其他血管发生抑制剂，例如血管抑素、内皮抑制素、干扰素、白细胞介素1(包括α和β)白介素12、视黄酸和金属蛋白酶-1和-2的组织抑制剂。
实施例
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
在本发明以下实施例中，当构建所述的结合抗原的受体或CAR时，CD28共刺激信号结构域简称为28；CD3ζ简称为Z；4-1BB或CD137简称为BB。例如，将代码为8E5-2I的scFv与CD3ζ以及CD28共刺激信号结构域作为胞内信号结构域所构建的嵌合抗原受体可以记作8E5-2I-28Z。针对不同抗原的CAR的构建均是 如此。
实施例1、产生针对CLD18A2的小鼠抗体及表征
用标准生物学方案，来获得抗体片段。简言之，用包含人CLD18A2全长序(NCBI Reference Sequence:NM_001002026.2)的真核表达载体免疫8周龄Balb/c小鼠。摘除免疫后的小鼠的脾脏，使用本领域常规的生物学方案，获得单克隆抗体。
通过流式细胞术对单个细胞筛选抗CLD18A2单克隆抗体，使用稳定表达人CLD18A2的HEK293细胞(HEK-CLD18A2)，使用Guava easyCyteTM HT System仪器，通过流式细胞术评估进行初步筛选后，再通过流式细胞数比较抗体和人CLD18A1及CLD18A2转化体的结合。使用的仪器为Guava easyCyteTM HT System。
经过了多轮杂交瘤的制备和筛选，发明人找到若干结合性能较为理想的抗体。图1B显示了通过流式细胞术测定的杂交瘤上清液2B1，3E12，4A11，8E5与稳定转染了人CLD18A2或CLD18A1的HEK293细胞的结合实例。如图1B所示，经过两轮亚克隆以后，抗体2B1，3E12，4A11，8E5大部分亚克隆特异性的结合人CLD18A2但不结合人CLD18A1，平均荧光强度相差5倍以上。
培养分泌单克隆抗体的杂交瘤细胞株，按照
Figure PCTCN2017092381-appb-000003
 Plus RNA Purification kit(Invitrogen，12183-555)说明书，从细胞沉淀中提取总RNA。以总RNA做为模板，使用High capacity RNA to cDNA kit(Invitrogen，4387406)，按照说明书逆转录合成cDNA。以cDNA为模板，使用5’-Full RACE kit(TAKARA，D315)，使用抗体的恒定区引物扩增。PCR产物经1.5％琼脂糖凝胶电泳分离后，纯化回收DNA片段，测序后结果如下：
表5.测序结果
  氨基酸序列 核苷酸序列
2B1 VL SEQ ID NO:1 SEQ ID NO:2
2B1 VH SEQ ID NO:3 SEQ ID NO:4
3E12 VL SEQ ID NO:5 SEQ ID NO:6
3E12 VH SEQ ID NO:7 SEQ ID NO:8
4A11 VH SEQ ID NO:9 SEQ ID NO:10
4A11VL SEQ ID NO:11 SEQ ID NO:12
8E5 VL SEQ ID NO:13 SEQ ID NO:14
8E5 VH SEQ ID NO:15 SEQ ID NO:16
对抗体序列进行比对，结果如图2所示。
实施例2、抗密蛋白18A2scFv_Fc融合抗体的构建及其在真核细胞中的瞬转表达
分别针对2B1，8E5的VH和VL片段，引入柔性氨基酸GGGGSGGGGSGGGGS (SEQ ID NO:93)作为的linker组成scFv；在VH上游引入合适的酶切位点和保护碱基，在VL的下游引入合适的酶切位点和保护碱基，酶切后连接入真核表达载体(参见CN101602808所采用的载体pH或载体pK)。采用293fectinTMTransfection reagent(Invitrogen，12347-019)瞬时转染，收集培养上清并进行亲和纯化，通过SDSPAGE对得到的抗体进行定量和定性分析。
通过流式细胞术测定抗密蛋白18A2scFv_Fc融合抗体与稳定转染了CLD18A2的HEK293细胞的结合。参照实施例1所述，使用Guava easyCyteTM HT System仪器，使用GraphPad Prism分析实验数据，得到EC50。图3显示了单克隆抗体2B1，8E5的ScFv与人IgG1Fc部分嵌合以后与稳定转染了人CLD18A2的HEK293细胞的结合相对亲和力。可见，2B1的EC50值为3.56nM，8E5的EC50值为49.19nM。
实施例3、生产和制备抗密蛋白18A2的抗体的变体
采用搭桥PCR的方法，对抗体2B1进行定点突变，在抗体2B1的重链上52号位点或者54号位点(N糖基化位点)进行突变，制备了两个2B1的突变体2B1-N52D(VH氨基酸序列：SEQ ID NO:17；核苷酸序列：SEQ ID NO:18)和2B1-S54A(VH氨基酸序列：SEQ ID NO:19；核苷酸序列：SEQ ID NO:20)。
2B1-N52D以及2B1-S54A的轻链的氨基酸序列及核苷酸序列同2B1的相应序列。
参照实施例2所述，构建这两个突变体的ScFv_Fc形式表达载体，参照实施例2的操作，参照实施例1的操作，使用Guava easyCyteTM HT System仪器，使用GraphPad Prism分析实验数据，得到EC50。
结果如图4所示，2B1-N52D和2B1-S54A的ScFv与人IgG1Fc部分嵌合以后与稳定转染了人CLD18A2的HEK293细胞的结合相对亲和力。可见，2B1-N52D的EC50值为6.11nM，2B1-S54A的EC50值为3.85nM。
实施例4、2B1-S54A的人源化抗体的制备
综合Kabat、Chothia以及IMGT三种抗体CDRs区的命名方案，确定抗体轻链与重链的六个CDRs区序列。通过序列相似性比对，选取与2B1-S54A相似度最高的抗体种系作为抗体模板，在本实施例中，选取IMGT数据库中IGHV1-46*01做为hu2B1-S54A重链的抗体模板。IGKV4-1*01做为hu2B1-S54A轻链的抗体模板。将2B1-S54A的轻链和重链CDR区替换掉抗体模板的CDR区。
确定回复突变的位点：(1)比对设计好的人源化抗体与出发抗体，检查抗体framework区中哪些氨基酸不同。(2)检查这些不同的氨基酸是否是支持抗体loop结构的氨基酸或者是影响轻重链可变区结合的氨基酸，如果是，这些区域就是相对保守的区域。(3)检查人源化抗体中是否有一些潜在的翻译后修饰位点，如脱酰胺化位点(Asn-Gly)，异构化位点(Asp-Gly)，表面暴露的甲硫氨酸，N糖基化位点(Asn-X-Ser/Thr，X不是脯氨酸)。(4)人源化抗体(hu2B1-S54A)中重链有六个潜在回复突变位点，分别是M48I，V68A，M70L，R72A，T74K，T91S。人源化抗体(hu2B1-S54A)中轻链有一个潜在回复突变位点，是L84V。
表达和纯化人源化的抗体：(1)根据人源化的抗体(hu2B1-S54A)的氨基酸序列，设计核苷酸序列并合成。合成轻链核苷酸序列(SEQ ID NO:62)；合成重链核苷酸序列(SEQ ID NO:60)。(2)把合成的抗体核苷酸序列包括信号肽，抗体的可变区，恒定区插入到哺乳动物细胞表达载体中从而分别构建出含有重链和轻链的抗体表达载体，测序鉴定。
hu2B1-S54A的重链氨基酸序列如SEQ ID NO:59所示；核苷酸序列如SEQ ID NO:60所示。hu2B1-S54A轻链的氨基酸序列如SEQ ID NO:61所示；核苷酸序列如SEQ ID NO:62所示。hu2B1-S54A重链可变区氨基酸序列如SEQ ID NO:23所示，核苷酸序列如SEQ ID NO:24所示；轻链可变区氨基酸序列如SEQ ID NO:21所示，核苷酸序列如SEQ ID NO:22所示。
通过293Fectin瞬时转染至293F细胞中并表达hu2B1-S54A的HCDR以及LCDR相同序列。
参照实施例2所述进行结合活性测定，使用稳定表达人CLD18A2的HEK293细胞(HEK-CLD18A2)，使用Guava easyCyteTM HT System仪器，使用GraphPad Prism分析实验数据，得到EC50，结果如图5所示，说明hu2B1-S54A的scFv与人IgG1Fc部分嵌合以后与稳定转染了人CLD18A2的HEK293细胞的结合相对亲和力EC50值为18.59nM，说明hu2B1-S54A与HEK-CLD18A2也具有较好的结合。
实施例5、单克隆抗体8E5的人源化抗体的制备及优化
参照实施例4的操作，对8E5进行人源化，同时通过S62A的点突变去掉了单克隆抗体8E5中N糖基化位点，以获得人源化抗体hu8E5(或者hu8E5-S62A)。具体方法描述如下：
(1)选取IGHV4-30*03作为8E5重链的抗体模板，选取IGKV4-1*01作为8E5 轻链的抗体模板。将8E5抗体的轻链或者重链CDR区替换掉抗体模板的CDR区。
(2)确定回复突变的位点，人源化抗体(hu8E5)中重链有六个潜在回复突变位点，分别是G27Y、G45K、L46M、I49M、V68I、V72R、A97T。人源化抗体(hu8E5)中轻链有一个潜在回复突变位点，是L84V。
(3)根据人源化的抗体hu8E5的氨基酸序列，设计核苷酸序列并合成轻链核苷酸序列、重链核苷酸序列。
hu8E5-S62A的重链氨基酸序列如SEQ ID NO:67所示；核苷酸序列如SEQ ID NO:68所示。hu8E5-S62A轻链的氨基酸序列如SEQ ID NO:65所示；核苷酸序列如SEQ ID NO:66所示。hu8E5-S62A的重链可变区氨基酸序列如SEQ ID NO:27所示，核苷酸序列如SEQ ID NO:28所示；hu8E5-S62A的轻链可变区氨基酸序列如SEQ ID NO:25所示，核苷酸序列如SEQ ID NO:26所示；hu8E5-S62A中HCDR2不同于8E5，其序列如SEQ ID NO:85所示；其它HCDR以及LCDR同8E5。
(4)把合成的抗体核苷酸序列包括信号肽，抗体的可变区，恒定区插入到哺乳动物细胞表达载体中从而分别构建出含有重链和轻链的抗体表达载体，测序鉴定。通过293Fectin瞬时转染至293F细胞中并表达。
(5)测定结合活性，使用稳定表达人CLD18A2的HEK293细胞(HEK-CLD18A2)，使用Guava easyCyteTM HT System仪器，使用GraphPad Prism分析实验数据，结果如图6所示，显示了hu8E5的scFv与人IgG1Fc部分嵌合以后与稳定转染了人CLD18A2的HEK293细胞的结合相对亲和力为107nM。
通过Discovery studio软件建立hu8E5的3D模型，分析其中的潜在的聚集位点，发现重链第12位和第93位的缬氨酸倾向于导致抗体的聚集，进而影响抗体的稳定性。通过进行点突变的分析，发现将这两个位点突变成I，抗体更稳定。分子筛的结果显示将这两个位点突变成I(hu8E5-2I)以后，scFv_Fc融合抗体中单体形式的比例由原来的74％(hu8E5)提高到87％(hu8E5-2I)。
hu8E5-2I的重链氨基酸序列如SEQ ID NO:63所示；核苷酸序列如SEQ ID NO:64所示。hu8E5-2I轻链的氨基酸序列如SEQ ID NO:65所示；核苷酸序列如SEQ ID NO:66所示。hu8E5-2I中HCDR2不同于8E5，但与hu8E5相同，其序列如SEQ ID NO:85所示；其它HCDR以及LCDR同8E5。
参照实施例3所述构建人源化抗体hu8E5的突变体hu8E5-2I。使用稳定表达人CLD18A2的HEK293细胞(HEK-CLD18A2)，使用Guava easyCyteTM HT System仪器，使用GraphPad Prism分析实验数据，结果如图7所示，hu8E5-2I的scFv与 人IgG1Fc部分嵌合以后与稳定转染了人CLD18A2的HEK293细胞的结合相对亲和力，其EC50值为9.22nM。突变体hu8E5-2I与亲本抗体8E5相比亲和力提高了5倍。
实施例6、人源化抗体hu2B1-S54A与人源化抗体hu8E5-2I的体外功能实验和体内功能实验
通过本领域技术人员已知的标准方法，将人源化的抗体hu2B1-S54A(轻链序列：SEQ ID NO:62，重链序列：SEQ ID NO:60)；人源化的抗体hu8E5-2I(轻链序列：SEQ ID NO:66，重链序列：SEQ ID NO:64)克隆至真核表达载体中。采用293fectinTM Transfection reagent(Invitrogen，12347-019)瞬时转染对数生长期的293F细胞，收集培养上清并进行亲和纯化。通过SDS PAGE对得到的抗体进行定量和定性分析。上述真核表达载体采用CN101602808B所采用的载体pH或载体pK。
1、补体依赖性细胞毒性(CDC)
通过从健康志愿者中抽血，离心制备血清。使用CCK-8细胞增殖-毒性检测试剂盒(Dojindo，#CK04)。以稳定转染了CLD18A2或CLD18A1的HEK293细胞作为靶细胞。洗涤细胞两次，重悬在完全培养基中，密度为1×105细胞/ml。每孔100μl接种于96孔培养板，37℃培养过夜。第二天，每孔加入抗体终浓度为20μg/ml。37℃培养箱孵育30min。然后加入终浓度10％的血清37℃孵育1.5小时。向每孔中加入10ul CCK-8溶液，37℃孵育3.5h(视情况调整)，用酶标仪测定在450nm处的吸光度。实验共分为六组，并设置复孔，具体分组如下表所示。
表6.分组表
Figure PCTCN2017092381-appb-000004
如下计算裂解的百分比：
裂解百分比＝(细胞对照孔-实验孔)/细胞对照孔-(细胞对照孔-抗体对照孔)/细胞对照孔。
图8A是比较人源化抗体hu2B1-S54A、hu8E5-2I和嵌合抗体ch-163E12对转染了CLD18A2的HEK293细胞的CDC结果。实验结果显示，hu2B1-S54A、hu8E5-2I在抗体浓度为20μg/ml时，针对HEK293-CLD18A2的CDC效应分别为79.88％和82.65％，而ch-163E12在同样反应条件下的CDC效应低于55％。图8B是比较人源化抗体hu2B1-S54A、hu8E5-2I和嵌合抗体ch-163E12对转染了CLD18A1的HEK293细胞的CDC结果，实验结果显示，ch-163E12对表达18A1的细胞也具有一定的杀伤，hu2B1-S54A、hu8E5-2I对表达18A1的细胞没有杀伤。
2、抗体依赖性细胞的毒性(ADCC)活性
使用cytoTox 96非放射活性的细胞毒性测定试剂盒(Promega，Madison，USA)，通过乳酸脱氢酶(LDH)释放测定测量人源化抗体密蛋白18A2抗体的ADCC活性。通过标准的Ficoll-paque分离，从柠檬酸化的全血中纯化人外周血单核细胞(PBMC)，重悬在补充了10％胎牛血清(FBS，Gibco)的完全培养基(RPMI-1640培养基(Gibco)中，密度为8×106细胞/ml。使用稳定转染了CLD18A2的HEK293细胞作为靶细胞。洗涤细胞两次，重悬在完全培养中，密度为2×105细胞/ml。将PBMC与终浓度为20ug/ml的抗体37℃孵育30分钟，然后按照50:1，20:1，10:1的效靶比，将50ul抗体和效应细胞加入50μl靶细胞(共计1×104靶细胞)。在37℃孵育4小时后，离心细胞，收集50μl无细胞上清液样品，转移至平底的96孔板中，并测定。如下计算裂解的百分比：(样品释放-靶自发释放-效应细胞自发释放)/(最大释放-靶自发释放)*100；其中靶自发释放是仅含有靶细胞的孔中的荧光，效应细胞自发释放是仅含有效应细胞的孔中的荧光，而最大释放是含有已用裂解缓冲液处理靶细胞的孔中的荧光。
图9是比较人源化抗体hu2B1-S54A、hu8E5-2I和已知嵌合抗体ch-163E12和ch-175D10(参见CN103509110A)的ADCC结果。实验结果显示，人源化抗体hu2B1-S54A、hu8E5-2I在抗体浓度为20μg/ml，效靶比为50:1、20:1和10:1时，其ADCC效应显著优于ch-175D10和ch-163E12，在效靶比为50:1针对HEK293-CLD18A2的ADCC效应分别为62.84％和72.88％。而ch-163E12和ch-175D10在同样反应条件下针对HEK293-CLD18A2的ADCC效应仅为33.39％和43.74％，本发明的抗体具有显著优于ch-163E12和ch-175D10的杀伤作用。
3、小鼠体内实验
胃癌PDX模型的建立：接种约3mm×3mm×3mm大小的瘤块于BALB/c裸鼠右侧腋部皮下。在肿瘤细胞接种之日记为D0天，肿瘤接种D27天，测量肿瘤体积，随机进行分为5组。具体分组如下：(1)PBS(磷酸盐缓冲液)对照组；(2)hu8E5-2I抗体治疗组(40mg/kg)；(3)EOF治疗组(E即表柔比星：1.25mg/kg；O即奥沙利铂：3.25mg/kg；F即5-氟尿嘧啶：56.25mg/kg)+PBS；(4)hu8E5-2I抗体(40mg/kg)+EOF治疗组(1.25mg/kg表柔比星+3.25mg/kg奥沙利铂+56.25mg/kg 5-氟尿嘧啶)；(5)ch175D10抗体(40mg/kg)+EOF治疗组(1.25mg/kg表柔比星+3.25mg/kg奥沙利铂+56.25mg/kg 5-氟尿嘧啶)。给药剂量：EOF每周给予1次，持续2周；hu8E5-2I以及ch175D10抗体每周分别给药3次，持续2周。
结果如图10所示，肿瘤接种D56天，EOF注射2次，抗体注射6次，将小鼠引颈处死。与PBS对照组相比较，抑瘤率分别为：hu8E5-2I单抗组21.13％，EOF+PBS治疗组47.89％，EOF+mAb hu8E5-2I治疗组81.69％，EOF+mAb 175D10治疗组71.83％。从肿瘤称重来看，EOF+mAb hu8E5-2I治疗组与EOF+PBS组相比较P＝0.033，具有统计学差异，而EOF+mAb 175D10治疗组与EOF+PBS组相比较P＝0.097，没有统计学差异。
实施例7、人源化抗体嵌合抗原受体质粒(CAR质粒)的构建
1.人源化抗体hu8E5嵌合抗原受体质粒的构建
以PRRLSIN-cPPT.EF-1α为载体，构建了表达人源化抗体hu8E5的二代、三代嵌合抗原受体的慢病毒质粒，包括PRRLSIN-cPPT.EF-1α-hu8E5-28Z、PRRLSIN-cPPT.EF-1α-hu8E5-BBZ以及PRRLSIN-cPPT.EF-1α-hu8E5-28BBZ。
Hu8E5-28Z主要包括(从5’端到3’端依次排列)：CD8α信号肽(SEQ ID NO:70)、hu8E5 scFV(VH：SEQ ID NO:27，VL：SEQ ID NO:25，Linker：SEQ ID NO:93)、CD8 hinge(SEQ ID NO：72)、CD28跨膜区(SEQID NO:74)和胞内信号传导结构域(SEQ ID NO:76)以及CD3的胞内段CD3ξ(SEQ ID NO:78)的编码序列。
hu8E5-BBZ主要包括(从5’端到3’端依次排列)：CD8α信号肽(SEQ ID NO:70)、hu8E5 scFV(VH：SEQ ID NO:27，VL：SEQ ID NO:25，Linker：SEQ ID NO:93)、CD8 hinge(SEQ ID NO:72)和CD8跨膜区(SEQ ID NO:80)、CD137胞内信号传导结构域(SEQ ID NO:82)以及CD3ξ(SEQ ID NO:78)的编码序列。
hu8E5-28BBZ主要包括(从5’端到3’端依次排列)：CD8α信号肽(SEQ ID NO:70)、hu8E5-scFV、CD8hinge(SEQ ID NO:72)、CD28跨膜区(SEQID NO:74)和胞内段(SEQ ID NO:76)、CD137胞内信号传导结构域(SEQ ID NO:82)以及CD3ξ(SEQ ID NO:78)的编码序列。
2.人源化抗体hu8E5-2I嵌合抗原受体质粒的构建
以PRRLSIN-cPPT.EF-1α为载体，构建了表达人源化抗体hu8E5-2I的二代嵌合抗原受体的慢病毒质粒PRRLSIN-cPPT.EF-1α-hu8E5-2I-28Z。
hu8E5-2I-28Z主要包括(从5’端到3’端依次排列)：CD8α信号肽(SEQ ID NO:70)、hu8E5-2I scFV(VH：SEQ ID NO:29，VL：SEQ ID NO:25，Linker：SEQ ID NO:93)、CD8hinge(SEQ ID NO:72)、CD28跨膜区(SEQID NO:74)和胞内信号传导结构域(SEQ ID NO:76)以及CD3的胞内信号传导结构域CD3ξ(SEQ ID NO:78)的编码序列。
3.人源化抗体hu2B1-S54A嵌合抗原受体质粒的构建
以PRRLSIN-cPPT.EF-1α为载体，构建了表达人源化抗体hu2B1-S54A的二代嵌合抗原受体的慢病毒质粒PRRLSIN-cPPT.EF-1α-hu2B1-S54A-28Z。
hu2B1-S54A-28Z主要包括(从5’端到3’端依次排列)：CD8α信号肽(SEQ ID NO:70)、hu2B1-S54A scFV(VH:SEQ ID NO:23；VL:SEQ ID NO:21；Linker:SEQ ID NO:93)、CD8 hinge(SEQ ID NO:72)、CD28跨膜区(SEQID NO:74)和胞内区(SEQ ID NO:76)以及CD3的胞内段CD3Z(SEQ ID NO:78)的编码序列。
实施例8、慢病毒包装及滴度测定
以5×106的病毒密度接种培养至第6～10代的293T细胞于10cm培养皿中，37℃，5％CO2培养过夜准备用于转染，培养基为含10％胎牛血清(Gibico)的DMEM。
分别将目的基因质粒PRRLSIN-cPPT.EF-1α-EGFP(Mock)及实施例9制备的不同的CAR质粒5.4μg与包装质粒pRsv-REV 6.2μg、RRE-PMDLg 6.2μg、Vsvg 2.4μg溶入800μL空白DMEM培养液；将60μg PEI(1μg/μl)溶解于800μl的无血清DMEM培养液中，混匀室温孵育5min。
将质粒混合液加入PEI混合液中，混匀室温孵育20min，形成转染复合物；将转染复合物1.6ml滴加入含11ml DMEM培养基的10cm培养皿中；4-5h小时后，用10％FBS的DMEM培基给转染的293T细胞换液，37℃孵育72h，收集病毒液 上清，浓缩后进行滴度测定，以阳性率为5～20％的细胞数为宜，计算滴度(U/mL)＝细胞数目×阳性率/病毒体积。
浓缩后的病毒滴度分别为：
hu8E5-28Z：2.3×107U/ml；
hu8E5-BBZ：6.65×107U/ml；
hu8E5-28BBZ:6.67×107U/ml；
hu8E5-2I-28Z:1.54×108U/ml；
hu2B1-S54A-28Z:1.14×108U/ml。
实施例9、慢病毒转导T淋巴细胞及CAR-T细胞的细胞毒性测定
1、慢病毒感染的T淋巴细胞
(1)以约1×106/mL密度加入淋巴细胞培养基液培养，并按照磁珠:细胞比例为1:1加入同时包被有抗CD3和CD28抗体的磁珠(Invitrogen)和终浓度300U/mL的重组人IL-2刺激培养48h；
(2)Retronectin包被24孔板：每孔加入380μl 5μg/ml的retronectin溶液(PBS)，4℃孵育过夜后弃去24孔板中的retronectin溶液(PBS)，1ml PBS洗两次；
(3)将细胞接种于包被了retronectin的24孔板内，每孔细胞数目3×105，培养液体积600μl；按照MOI＝10在PBMCs细胞中加入浓缩后的慢病毒，32℃离心40min后，转移至细胞培养箱中；
(4)扩增培养：感染后的细胞每隔一天采用5×105/mL的密度进行传代，同时在淋巴细胞培养液中补加终浓度300U/mL的重组人IL-2。
2、T淋巴细胞嵌合抗原受体表达
(1)慢病毒感染的T淋巴细胞在培养第7天，取1×106的T细胞，二等分于2ml离心管内，4℃，5000rpm，离心5min，弃上清，PBS洗两次。
(2)对照组细胞加入50μl PE-SA(1:200稀释)抗体冰上孵育45min，PBS(2％NBS)洗两次，重悬后作为对照；检测组细胞+50μl 1：50稀释的biotin-Goat anti human IgG，F(ab’)2抗体，冰上孵育45min；PBS(2％NBS)洗两次。
(3)加入50μl PE-SA(1:200稀释)抗体冰上孵育45min；加入2ml PBS(2％NBS)重悬细胞，离心后弃上清。
(4)流式细胞仪检测CAR阳性的T细胞比例。hu8E5-28Z、hu8E5-BBZ以及hu8E5-28BBZ三种CAR T细胞以及Mock对照细胞的感染阳性率分别为52.1％、 47.8％、44.6％以及71.7％。
3、靶向CLD18A2CAR T细胞的细胞毒性测定
(1)靶细胞：分别接种75μL 2×105/mL的293T-A1细胞、293T-A2细胞、胃腺癌细胞系AGS、AGS-A2、胃癌细胞系BGC-823、BGC-823-A2细胞。胃腺癌细胞系AGS、胃癌细胞系BGC-823购自ATCC细胞库，293T-A1细胞、293T-A2细胞、AGS-A2、BGC-823-A2细胞参照CN101602808B进行细胞系的构建，其中，293T-A2细胞、AGS-A2、BGC-823-A2细胞为CLD18A2阳性细胞，其余为CLD18A2阴性细胞。
(2)效应细胞：按效靶比3:1、1:1或1:3加T-Mock及表达不同嵌合抗原受体的CAR T细胞；
(3)各组均设4个复孔，取4个复孔的平均值。检测时间为第18h。
其中各实验组和各对照组如下：
各实验组：各靶细胞+表达不同嵌合抗原受体的CAR T；
对照组1：靶细胞最大释放LDH；
对照组2：靶细胞自发释放LDH；
对照组3：效应细胞自发释放LDH；
(4)检测方法：采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)进行。CytoTox 
Figure PCTCN2017092381-appb-000005
检测定量地测量乳酸脱氢酶(LDH)。具体参照CytoTox 96非放射性细胞毒性检测试剂盒说明书。
(5)细胞毒性计算公式为：
Figure PCTCN2017092381-appb-000006
不同效靶比的细胞杀伤结果如图11和图12所示，结果显示CAR-T产品对CLD18A2表达阳性的293T-A2、AGS-A2和BGC-823-A2细胞均显示出良好的杀伤效果。其中，hu8E5-28Z和hu8E5-2I-28Z胞在效靶比3:1的时候，可以杀死60％以上的AGS-A2细胞杀伤对BGC-823-A2细胞的杀伤则可以达到90％以上。同时结果还显示各CAR T细胞(Hu2B1-S54A-28Z除外)对CLD18A2表达阴性的细胞的杀伤作用不明显。
实施例10、CLD18A2CAR-T细胞体内活性
观察未转染的T细胞(UTD)、hu8E5-2I-28Z T细胞对胃癌PDX模型皮下移植 瘤的抗肿瘤治疗实验。
1)胃癌PDX模型的建立：接种约2×2×2mm大小的胃癌PDX瘤块于6-8周龄的雌性NOD/SCID小鼠的右侧腋部皮下，在肿瘤细胞接种之日记为D0天。
2)实验分组：肿瘤接种D15天，将NOD-SCID小鼠随机分为3组，每组7只，分为未转染的T细胞组和hu8E5-2I-28Z T细胞组。
3)过继转移T细胞：在肿瘤体积为30mm3时，腹腔注射100mg/kg的环磷酰胺，注射24小时后通过尾静脉输注1.0×107CAR-T细胞，同时以未转染的T细胞组作为对照。观察测量胃癌PDX皮下移植瘤的生长。实验结果如图13A和13B所示，CAR T注射第D32天，将小鼠引颈处死，与UTD组相比，hu8E5-2I-28Z治疗组抑瘤效果显著，抑制率为79.2％。从肿瘤称重来看，hu8E5-2I-28Z治疗组与UTD组相比较P＝0.01，具有统计学差异。
实施例11、CLD18A2CAR-T细胞体外诱导细胞因子释放试验
为了验证构建的hu8E5-28Z、hu8E5-2I-28Z T细胞在靶细胞刺激下能够被有效激活，我们检测了在与靶细胞共孵育后，hu8E5-28Z、hu8E5-2I-28Z T细胞的细胞因子的分泌。
分别检测转染空载后的T细胞(Mock)、hu8E5-28Z和hu8E5-2I-28Z T细胞释放的细胞因子。收集慢病毒感染后1-2周内生长状态良好的上述两种T细胞，接种5×104/200μL(阳性细胞数)于24孔板，按效靶比1:1分别接种5×104/200μL/24孔靶细胞。靶细胞包括293T-A1、293T-A2、AGS、AGS-A2、BGC-823以及BGC-823-A2细胞。共培养24小时后收集上清。采用夹心ELISA的方法检测上清中CAR T淋巴细胞与靶细胞共同培养过程中释放的IL2、IFN-γ以及TNF-α。
实验结果如图14所示，结果显示hu8E5-28Z、hu8E5-2I-28Z与CLD18A2表达阳性的293T-A2、AGS-A2和BGC-823-A2细胞共孵育时，可以激活IL-2、IFN-育和TNF-α细胞因子的分泌，而Mock对照组则不能激活上述细胞因子的分泌，且均有显著性差异；hu8E5-2I-28Z与CLD18A2表达阴性的293T-A1、AGS和BGC-823细胞共孵育时，不能激活IL-2、IFN-γ和TNF-α细胞因子的分泌，Mock对照组也不能激活上述细胞因子的分泌。上述实验结果表明CLD18A2表达阳性的细胞可以有效激活hu8E5-2I-28Z CAR T细胞。
实施例12、CLD18A2CAR-T细胞的体内杀伤活性
测定未转染的T细胞(Mock)、hu8E5-28Z和hu8E5-2I-28Z T细胞对BGC-823-A2细胞皮下移植瘤的抗肿瘤治疗实验。
1)BGC-823-A2皮下移植瘤的接种：收集处于对数生长期并且生长状态良好的BGC-823-A2细胞使用生理盐水调整密度为2.5×107/mL，注射细胞悬液体积200μL(5×106/只)于小鼠右侧腋部皮下。在肿瘤细胞接种之日记为第0天。
2)实验分组：肿瘤接种第11天，测量BGC-823-A2移植瘤体积，将NOD-SCID小鼠随机分为3组，每组6只。分别为未转染的T细胞组、hu8E5-28Z T细胞、hu8E5-2I-28Z T细胞组。
3)过继转移T细胞：在肿瘤体积为100-150mm3时(第11天)，腹腔注射100mg/kg的环磷酰胺，注射24小时后通过尾静脉输注1×107CAR T细胞(Mock细胞、hu8E5-28Z T细胞或hu8E5-2I-28Z T细胞)，同时以未转染的T细胞组(Mock组)作为对照，观察测量皮下移植瘤的生长。
动物实验结果如图15所示，结果显示在hu8E5-28Z、hu8E5-2I-28Z CAR T细胞治疗的第17天，对BGC-823-A2移植瘤的抑瘤率分别为81.3％和89.2％；且hu8E5-28Z、hu8E5-2I-28Z治疗组与Mock对照组对BGC-823-A2移植瘤的治疗效果存在显著性差异。治疗第17天后处死小鼠取出移植瘤并称重，hu8E5-28Z、hu8E5-2I-28Z治疗组BGC-823-A2移植瘤的平均瘤重分别为0.1和0.06g，而Mock对照组的平均瘤重为0.53g，CAR T细胞治疗组与Mock对照组之间存在显著性差异，P值分别为0.0013、<0.0001。
实施例13、CLD18A2CAR-T细胞在体内对肿瘤浸润的影响
参照实施例12中建立的BGC-823-A2细胞皮下移植瘤动物模型，回输Mock、hu8E5-28Z、hu8E5-2I-28Z细胞17天后，取肿瘤组织，组化检测CD3+细胞。
结果如图16所示，Mock T细胞在肿瘤组织周围几乎观察不到T细胞的浸润，Mock T细胞在肿瘤组织周围几乎观察不到T细胞的浸润，hu8E5-28Z、hu8E5-2I-28Z细胞在肿瘤组织的边缘可以看到CD3+T细胞的浸润，且hu8E5-2I-28Z治疗组的肿瘤组织边缘可以看到更多的T细胞浸润。
实施例14、共表达IFN的CAR-T细胞的制备
参照实施例7-9的操作，在hu8E5-28Z的基础上构建了表达IFNb细胞因子的 hu8E5-28Z-IFNb CAR的质粒，在hu8E5-2I-28Z CAR的基础上构建了可以表达IFNb细胞因子的hu8E5-2I-28Z-IFN CAR的质粒，经慢病毒包装和感染，得到共表达IFNb的hu8E5-28Z-IFNb CAR T细胞(又可标记为hu8E5-28Z&IFNB)和hu8E5-2I-28Z-IFN CAR T细胞(又可标记为hu8E5-2I-28Z&IFNB)。其中hu8E5-28Z-IFNb CAR由SEQ ID NO:90的核苷酸序列编码；hu8E5-2I-28Z-IFN CAR由SEQ ID NO:91的核苷酸序列编码。
参照实施例11的操作，进行体外诱导细胞因子释放试验，结果如图17A所示，IFN的存在导致hu8E5-28Z CAR T细胞与靶细胞共孵育时IFN-γ细胞因子分泌增多
观察未转染的T细胞(UTD)、hu8E5-28Z T细胞和hu8E5-2I-28Z-IFN T细胞对胃癌PDX模型皮下移植瘤的抗肿瘤治疗实验。接种约2×2×2mm大小的胃癌PDX瘤块于6-8周龄的雌性NOD/SCID小鼠的右侧腋部皮下，在肿瘤细胞接种之日记为D0天，肿瘤接种D15天，将NOD-SCID小鼠随机分为3组，每组7只，分为未转染的T细胞组、hu8E5-28Z T细胞组和hu8E5-2I-28Z-IFN T细胞组。在肿瘤体积为30mm3时，腹腔注射100mg/kg的环磷酰胺，注射24小时后通过尾静脉输注1.0×107CAR-T细胞(hu8E5-28Z T细胞或hu8E5-2I-28Z-IFN T细胞)，同时以未转染的T细胞组作为对照。观察测量胃癌PDX皮下移植瘤的生长。结果如图17B所示，hu8E5-2I-28Z-IFN治疗组7只小鼠有1只小鼠肿瘤完全消退。
采用上述的PDX模型，测定CAR-T细胞的体内存活情况，分别在CAR-T细胞输注后的D5，D7和D10天，经小鼠隐静脉抽取外周血，检测CAR-T细胞(空载T细胞(Mock)，hu8E5-28Z T细胞或hu8E5-2I-28Z-IFN T细胞)在体内的存活情况，结果如图17C所示，hu8E5-2I-28Z-IFN T细胞治疗组的T细胞存活数量明显多于hu8E5-28Z-28Z T细胞治疗组。
实施例15、CAR-NK细胞的构建
如图18A和18B所示的质粒图谱，以PRRLSIN-cPPT.EF-1α为载体，构建了表达人源化抗体hu8E5的嵌合抗原受体的慢病毒质粒，包括PRRLSIN-cPPT.EF-1α-hu8E5-28BBZ、PRRLSIN-cPPT.EF-1α-hu8E5-2I-28Z。hu8E5-28BBZ序列由CD8α信号肽(SEQ ID NO:70)、hu8E5-scFV、CD8hinge(SEQ ID NO:72)、CD28跨膜区(SEQID NO:74)和胞内段(SEQ ID NO:76)、CD137胞内信号传导结构域段(SEQ ID NO:82)以及CD3ξ(SEQ ID NO:78)组成；hu8E5-2I-28Z序列由CD8α信号肽(SEQ ID NO:70)、hu8E5-2I scFV、CD8hinge(SEQ ID NO:72)、 CD28跨膜区(SEQID NO:74)和CD28胞内信号传导结构域区(SEQ ID NO:76)以及CD3的胞内段CD3ξ(SEQ ID NO:78)组成。
1、CAR阳性的NK-92细胞系的制备
1)Retronectin包被24孔板：每孔加入380μl 5μg/ml的retronectin溶液(PBS)，4度孵育过夜，将细胞接种于包被了retronectin的24孔板内，每孔细胞数目5×105，培养液体积500μl；
2)按照MOI＝30在NK92细胞中加入浓缩后的慢病毒，32度离心90min转移至细胞培养箱中；
3)扩增培养：感染后的细胞每隔一天采用5×10^5/mL的密度进行传代，同时在淋巴细胞培养液中补加终浓度500U/mL的重组人IL-2。
2、NK-92细胞嵌合抗原受体表达
1)慢病毒感染的NK92细胞在培养第7天，取1×10^6的细胞，二等分于离心管内；
2)对照组细胞加入50μl PE-SA(1:200稀释)抗体冰上孵育，重悬后作为对照；检测组细胞+50μl 1:50稀释的biotin-Goat anti human IgG，F(ab’)2抗体，冰上孵育45min；PBS(2％NBS)洗两次；加入50μl PE-SA(1:200稀释)抗体冰上孵育；
3)加入2ml PBS(2％NBS)重悬细胞，4℃离心后弃上清；加入500μl PBS(2％NBS)，转移至流式管中。流式细胞仪检测PE通道，确定CAR阳性的NK92细胞比例。结果如图19所示。
细胞毒性测定：靶细胞：分别接种10^4的AGS、AGS-A2、BGC-823、BGC-823-A2细胞于96well板；效应细胞：按效靶比6:1、3:1、1.5:1加NK-92及CAR NK-92细胞；各组均设5个复孔，取5个复孔的平均值。检测时间为第4h。其中各实验组和各对照组如下：各实验组：各靶细胞+上述效应细胞；对照组1：靶细胞最大释放LDH；对照组2：靶细胞自发释放LDH；对照组3：效应细胞自发释放LDH。
检测方法：采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)进行。具体参照CytoTox 96非放射性细胞毒性检测试剂盒说明书。细胞毒性计算公式为：
Figure PCTCN2017092381-appb-000007
结果如图20和21所示，图中，hu8E5-21-28Z为hu8E5-2I-28Z CAR-NK92细胞，hu8E5-28BBZ为hu8E5-28BBZ CAR-NK92细胞。结果显示，hu8E5CAR-NK92细胞对过表达CLDN18A2的细胞有显著的体外杀伤活性，对CLDN18A2阴性的细胞几乎没有杀伤毒性。
表7.本文中所用的序列
Figure PCTCN2017092381-appb-000008
Figure PCTCN2017092381-appb-000009
Figure PCTCN2017092381-appb-000010
Figure PCTCN2017092381-appb-000011
Figure PCTCN2017092381-appb-000012
Figure PCTCN2017092381-appb-000013
Figure PCTCN2017092381-appb-000014
Figure PCTCN2017092381-appb-000015
Figure PCTCN2017092381-appb-000016
Figure PCTCN2017092381-appb-000017
Figure PCTCN2017092381-appb-000018
Figure PCTCN2017092381-appb-000019
Figure PCTCN2017092381-appb-000020
Figure PCTCN2017092381-appb-000021

Claims (85)

  1. 可特异性地结合密蛋白18A2的抗体，其特征在于，该抗体包含选自氨基酸序列SEQ ID NO:31，32，33，37，38，39，43，44，45，49，50，51，83，84，85或其变体的重链CDR，和/或选自氨基酸序列SEQ ID NO：34，35，36，40，41，42，46，47，48，52，53，54，或其变体的轻链CDR。
  2. 如权利要求1所述的抗体，其特征在于，该抗体选自：
    (a)抗体，其包含重链可变区，所述重链可变区具有包含SEQ ID NO:31、SEQ ID NO:37、SEQ ID NO:43或SEQ ID NO:49之一所示的氨基酸序列的CDR1、包含SEQ ID NO:32、SEQ ID NO:38、SEQ ID NO:44、SEQ ID NO:50、SEQ ID NO:83、SEQ ID NO:84或SEQ ID NO:85之一所示的氨基酸序列的CDR2、包含SEQ ID NO:33、SEQ ID NO:39、SEQ ID NO:45或SEQ ID NO:51之一所示的氨基酸序列的CDR3；
    (b)抗体，其包含轻链可变区，所述轻链可变区具有包含SEQ ID NO:34、SEQ ID NO:40、SEQ ID NO:46或SEQ ID NO:52之一所示的氨基酸序列的CDR1、包含SEQ ID NO:35、SEQ ID NO:41、SEQ ID NO:47或SEQ ID NO:53之一所示的氨基酸序列的CDR2、包含SEQ ID NO:36、SEQ ID NO:42、SEQ ID NO:48或SEQ ID NO:54之一所示氨基酸序列的CDR3；
    (c)抗体，包含(a)所述抗体的重链可变区及(b)所述抗体的轻链可变区；
    (d)抗体，识别与(a)～(c)中任一项所述的抗体所识别的抗原决定部位相同的抗原决定部位。
  3. 如权利要求2所述的抗体，其特征在于，
    该抗体的重链可变区的CDR1、CDR2、CDR3区分别为SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33；或者SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39；或者SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45；或者SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51；或者SEQ ID NO:31、SEQ ID NO:83、SEQ ID NO:33；或者SEQ ID NO:31、SEQ ID NO:84、SEQ ID NO:33；或者SEQ ID NO:49、SEQ ID NO 85、SEQ ID NO:51；和/或
    该抗体的轻链可变区的CDR1、CDR2、CDR3区分别为SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36；或者SEQ ID NO:40、SEQ ID NO:41、SEQ  ID NO:42；或者SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48；或者SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54。
  4. 如权利要求3所述的抗体，其特征在于，该抗体包括重链可变区和轻链可变区，其具有SEQ ID NO:3、或SEQ ID NO:7、SEQ ID NO:11、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:23、SEQ ID NO:27或SEQ ID NO:29之一所示氨基酸序列的重链可变区；SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:9、SEQ ID NO:13、SEQ ID NO:21或SEQ ID NO:25之一所示氨基酸序列的轻链可变区。
  5. 如权利要求4所述的抗体，其特征在于，该抗体是：
    具有SEQ ID NO:3所示重链可变区，SEQ ID NO:1所示轻链可变区的抗体；
    具有SEQ ID NO:7所示重链可变区，SEQ ID NO:5所示轻链可变区的抗体；
    具有SEQ ID NO:11所示重链可变区，SEQ ID NO:9所示轻链可变区的抗体；
    具有SEQ ID NO:15所示重链可变区，SEQ ID NO:13所示轻链可变区的抗体；
    具有SEQ ID NO:17所示重链可变区，SEQ ID NO:1所示轻链可变区的抗体；
    具有SEQ ID NO:19所示重链可变区，SEQ ID NO:1所示轻链可变区的抗体；
    具有SEQ ID NO:23所示重链可变区，SEQ ID NO:21所示轻链可变区的抗体；
    具有SEQ ID NO:27所示重链可变区，SEQ ID NO:25所示轻链可变区的抗体；或
    具有SEQ ID NO:29所示重链可变区，SEQ ID NO:25所示轻链可变区的抗体。
  6. 如权利要求5所述的抗体，其特征在于，该抗体是：
    具有SEQ ID NO:3所示重链可变区，SEQ ID NO:1所示轻链可变区的抗体；或
    具有SEQ ID NO:17所示重链可变区，SEQ ID NO:1所示轻链可变区的抗体；或
    具有SEQ ID NO:19所示重链可变区，SEQ ID NO:1所示轻链可变区的抗体；或
    具有SEQ ID NO:23所示重链可变区，SEQ ID NO:21所示轻链可变区的抗体；或
    具有SEQ ID NO:27所示重链可变区，SEQ ID NO:25所示轻链可变区的抗体；或
    具有SEQ ID NO:29所示重链可变区，SEQ ID NO:25所示轻链可变区的抗体。
  7. 如权利要求1-6任一所述的抗体，其特征在于，所述的抗体是人源化的抗体，嵌合抗体或全人抗体；或者所述抗体是单克隆抗体；或者所述抗体为单链抗体或结构域抗体。
  8. 如权利要求7所述的抗体，其特征在于，所述的抗体是人源化的抗体，选自：
    具有SEQ ID NO:27所示的重链可变区和SEQ ID NO:25所示的轻链可变区的抗体；
    具有SEQ ID NO:23所示的重链可变区和SEQ ID NO:21所示的轻链可变区的抗体；
    具有SEQ ID NO:29所示的重链可变区和SEQ ID NO:25所示轻链可变区的抗体。
  9. 如权利要求8所述的抗体，其特征在于，所述的抗体选自：
    (a)具有SEQ ID NO:63所示的重链和SEQ ID NO:65所示的轻链的抗体；
    (b)具有SEQ ID NO:61所示的轻链和SEQ ID NO:59所示的重链的抗体；
    (c)具有SEQ ID NO:67所示的重链和SEQ ID NO:65所示的轻链的抗体。
  10. 编码权利要求1-9任一所述的抗体的核酸。
  11. 一种表达载体，其包含权利要求10所述的核酸。
  12. 一种宿主细胞，其包含权利要求11所述的表达载体或基因组中整合有权利要求10所述的核酸。
  13. 权利要求1-9任一所述的抗体的用途，用于制备特异性靶向表达密蛋白18A2的肿瘤细胞的靶向性药物，抗体药物偶联物或多功能抗体；或
    用于制备诊断肿瘤的试剂，该肿瘤表达密蛋白18A2；或
    用于制备嵌合抗原受体修饰的免疫细胞。
  14. 如权利要求13所述的用途，其特征在于，表达密蛋白18A2的肿瘤包括：胃癌，胰腺癌，食管癌，肺癌。
  15. 包含权利要求1-9任一所述的抗体的嵌合抗原受体，其特征在于，所述的嵌合抗原受体包含顺序连接的：权利要求1-9任一所述的抗体、跨膜区和胞内信号区。
  16. 如权利要求15所述的嵌合抗原受体，所述的胞内信号区选自：CD3ζ，FcεRIγ，CD27，CD28，CD137，CD134，MyD88，CD40的胞内信号区序列，或其组合；或所述的跨膜区包含CD8或CD28的跨膜区。
  17. 如权利要求16所述的嵌合抗原受体，其特征在于，所述的嵌合抗原受体包括如下的顺序连接的抗体，跨膜区和胞内信号区：
    权利要求1-9任一所述的抗体、CD8和CD3ζ；
    权利要求1-9任一所述的抗体、CD8、CD137和CD3ζ；或
    权利要求1-9任一所述的抗体、CD28分子的跨膜区、CD28分子的胞内信号区和CD3ζ；或
    权利要求1-9任一所述的抗体、CD28分子的跨膜区、CD28分子的胞内信号区、CD137和CD3ζ。
  18. 编码权利要求15-17任一所述的嵌合抗原受体的核酸。
  19. 一种表达载体，其特征在于，其包含权利要求18所述的核酸。
  20. 一种病毒，其特征在于，所述的病毒包含权利要求19所述载体。
  21. 权利要求15-17任一所述的嵌合抗原受体、或权利要求18所述的核酸、或权利要求19所述的表达载体、或权利要求20所述的病毒的用途，用于制备靶向表达密蛋白18A2的肿瘤细胞的嵌合抗原受体修饰的免疫细胞。
  22. 如权利要求21所述的用途，其特征在于，所述的表达密蛋白18A2的肿瘤包括：胃癌，胰腺癌，食管癌，肺癌。
  23. 一种嵌合抗原受体修饰的免疫细胞，其特征在于，其转导有权利要求18所述的核酸，或权利要求19所述的表达载体或权利要求20所述的病毒；或
    其表面表达权利要求15-17任一所述的嵌合抗原受体；
    较佳地，所述的免疫细胞为：T淋巴细胞、NK细胞或者NKT淋巴细胞。
  24. 如权利要求23所述的免疫细胞，其特征在于，其还携带外源的细胞因子的编码序列；或
    其还表达另一种嵌合抗原受体，该受体不含有CD3ζ，但含有CD28的胞内信号结构域、CD137的胞内信号结构域或者这两者的组合；或
    其还表达趋化因子受体；较佳地，所述的趋化因子受体包括：CCR；或
    其还表达能降低PD-1表达的siRNA或者阻断PD-L1的蛋白；或其细胞中内源性的PD-1被基因编辑技术敲除；或
    其还表达安全开关。
  25. 权利要求23-24任一所述的嵌合抗原受体修饰的免疫细胞的用途，其特征在于，用于制备抑制肿瘤的药物，所述的肿瘤是表达密蛋白18A2的肿瘤；较佳地，所述的肿瘤包括：胃癌，胰腺癌，食管癌，肺癌。
  26. 一种多功能免疫辍合物，其特征在于，所述的多功能免疫辍合物包括：
    权利要求1-9任一所述的抗体；以及
    与之连接的功能性分子；所述的功能性分子选自：靶向肿瘤表面标志物的分子，抑制肿瘤的分子，靶向免疫细胞的表面标志物的分子或可检测标记物。
  27. 如权利要求26所述的多功能免疫辍合物，其特征在于，所述的靶向免疫细胞的表面标志物的分子是结合T细胞表面标志物的抗体，其与权利要求1-9任一所述的抗体形成T细胞参与的双功能抗体。
  28. 编码权利要求26-27任一所述的多功能免疫辍合物的核酸。
  29. 权利要求26-27任一所述的多功能免疫辍合物的用途，用于制备抗肿瘤药物，或
    用于制备诊断肿瘤的试剂，该肿瘤表达密蛋白18A2；或
    用于制备嵌合抗原受体修饰的免疫细胞；较佳地，所述免疫细胞包括：T淋巴细胞、NK细胞或者NKT淋巴细胞。
  30. 药物组合物，其特征在于，其包括：权利要求1-9任一所述的抗体或编码该抗体的核酸；或权利要求26-27任一所述的免疫辍合物或编码该辍合物的核酸；或权利要求15-17任一所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或权利要求23-24任一所述的嵌合抗原受体修饰的免疫细胞；以及药学上可接受的载体或赋形剂。
  31. 药盒，其特征在于，其包括：
    容器，以及位于容器中的权利要求30所述的药物组合物；或
    容器，以及位于容器中的权利要求1-9任一所述的抗体或编码该抗体的核酸；或权利要求26-27任一所述的免疫辍合物或编码该辍合物的核酸；或权利要求15-17任一所述的嵌合抗原受体或编码该嵌合抗原受体的核酸；或权利要求23-24任一所述的嵌合抗原受体修饰的免疫细胞。
  32. 包含轻链CDR和重链CDR的抗原结合单元，其中所述抗原结合单元特异性地结合密蛋白18A2肽；并且其中所述抗原结合单元不显著结合密蛋白18A1肽。
  33. 包含轻链CDR和重链CDR的抗原结合单元，其中所述抗原结合单元特异性地结合密蛋白18A2肽；并且其中所述抗原结合单元与参考抗原结合单元相比，显示更少的与密蛋白18A1肽的非特异性结合。
  34. 权利要求32或33的抗原结合单元，其中所述参考抗原结合单元包含SEQ ID NO:86或SEQ ID NO:88的轻链和/或SEQ ID NO:87或SEQ ID NO:89的重链氨基酸序列。
  35. 权利要求32或33的抗原结合单元，其中所述密蛋白18A2肽包含SEQ ID NO:55的氨基酸序列。
  36. 权利要求32或33的抗原结合单元，其中所述密蛋白18A1肽包含SEQ ID NO:57的氨基酸序列。
  37. 权利要求32或33的抗原结合单元，其中所述抗原结合单元与所述密蛋白18A1肽的非特异性结合不超过其与所述密蛋白18A2肽的特异性结合的20％。
  38. 权利要求32或33的抗原结合单元，其中所述结合特异性通过流式细胞术测定。
  39. 权利要求32或33的抗原结合单元，其中所述结合特异性通过FACS测定。
  40. 权利要求32或33的抗原结合单元，其中所述结合特异性通过ELISA测定。
  41. 权利要求32或33的抗原结合单元，其中所述抗原结合单元与所述密蛋白18A2肽结合的EC50低于约100nM。
  42. 权利要求32或33的抗原结合单元，其中所述抗原结合单元是单克隆抗体、人源化抗体、嵌合抗体、多价抗体或嵌合抗原受体。
  43. 权利要求32或33的抗原结合单元，其中所述轻链CDR包含LCDR1、LCDR2和LCDR3；并且所述重链CDR包含HCDR1、HCDR2和HCDR3；
    其中所述LCDR1、LCDR2和LCDR3分别具有选自以下的氨基酸序列：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:53和SEQ ID NO:54；以及
    其中所述HCDR1、HCDR2和HCDR3分别具有选自以下的氨基酸序列：SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:49、SEQ  ID NO:50、SEQ ID NO:51、SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85。
  44. 权利要求32或33的抗原结合单元，其中所述LCDR1包含选自以下的氨基酸序列：SEQ ID NO:34、SEQ ID NO:40、SEQ ID NO:46和SEQ ID NO:52。
  45. 权利要求32或33的抗原结合单元，其中所述LCDR2包含选自以下的氨基酸序列：SEQ ID NO:34、SEQ ID NO:41、SEQ ID NO:47和SEQ ID NO:53。
  46. 权利要求32或33的抗原结合单元，其中所述LCDR3包含选自以下的氨基酸序列：SEQ ID NO:35、SEQ ID NO:42、SEQ ID NO:48和SEQ ID NO:54。
  47. 权利要求32或33的抗原结合单元，其中所述HCDR1包含选自以下的氨基酸序列：SEQ ID NO:31、SEQ ID NO:37、SEQ ID NO:43和SEQ ID NO:49。
  48. 权利要求32或33的抗原结合单元，其中所述HCDR2包含选自以下的氨基酸序列：SEQ ID NO:32、SEQ ID NO:38、SEQ ID NO:44、SEQ ID NO:50、SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85。
  49. 权利要求32或33的抗原结合单元，其中所述HCDR3包含选自以下的氨基酸序列：SEQ ID NO:33、SEQ ID NO:39、SEQ ID NO:45和SEQ ID NO:51。
  50. 权利要求32或33的抗原结合单元，其中所述抗原结合单元是scFv、Fv、Fab或(Fab)2。
  51. 包含轻链CDR和重链CDR的抗原结合单元，其中所述轻链CDR包含LCDR1、LCDR2和LCDR3；并且所述重链CDR包含HCDR1、HCDR2和HCDR3；其中所述LCDR1、LCDR2和LCDR3包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:53和SEQ ID NO:54；以及其中所述HCDR1、HCDR2和HCDR3包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO: 49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85。
  52. 权利要求51的抗原结合单元，其中所述轻链CDR包含LCDR1、LCDR2和LCDR3；并且所述重链CDR包含HCDR1、HCDR2和HCDR3；
    其中所述LCDR1、LCDR2和LCDR3分别具有选自以下的氨基酸序列：SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQ ID NO:52、SEQ ID NO:53和SEQ ID NO:54；以及
    其中所述HCDR1、HCDR2和HCDR3分别具有选自以下的氨基酸序列：SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85。
  53. 权利要求51的抗原结合单元，其中所述LCDR1包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:34、SEQ ID NO:40、SEQ ID NO:46和SEQ ID NO:52。
  54. 权利要求51的抗原结合单元，其中所述LCDR2包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:34、SEQ ID NO:41、SEQ ID NO:47和SEQ ID NO:53。
  55. 权利要求51的抗原结合单元，其中所述LCDR3包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:35、SEQ ID NO:42、SEQ ID NO:48和SEQ ID NO:54。
  56. 权利要求51的抗原结合单元，其中所述HCDR1包含包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:31、SEQ ID NO:37、SEQ ID NO:43和SEQ ID NO:49。
  57. 权利要求51的抗原结合单元，其中所述HCDR2包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:32、SEQ ID NO:38、SEQ ID NO:44、SEQ ID NO:50、SEQ ID NO:83、SEQ ID NO:84和SEQ ID NO:85。
  58. 权利要求51的抗原结合单元，其中所述HCDR3包含与选自以下的氨基酸序列具有至少80％同一性的氨基酸序列：SEQ ID NO:33、SEQ ID NO:39、SEQ ID NO:45和SEQ ID NO:51。
  59. 权利要求51的抗原结合单元，其中所述抗原结合单元是单克隆抗体、人源化抗体、嵌合抗体、多价抗体或嵌合抗原受体。
  60. 权利要求51的抗原结合单元，其中所述抗原结合单元是scFv、Fv、Fab或(Fab)2。
  61. 嵌合抗原受体，其包含胞外抗原结合单元、跨膜域和胞内域，其中所述胞外抗原结合单元包含权利要求32至60之一的抗原结合单元。
  62. 组合物，其包含权利要求32-60之一的抗原结合单元或权利要求61嵌合抗原受体。
  63. 权利要求62的组合物，其包含I型干扰素。
  64. 分离的核酸，其编码权利要求32-60之一的抗原结合单元或权利要求61嵌合抗原受体。
  65. 权利要求64的分离的核酸，其编码I型干扰素。
  66. 载体，其包含编码权利要求32-60之一的抗原结合单元或权利要求61嵌合抗原受体、以及任选的I型干扰素的核酸。
  67. 宿主细胞，其表达权利要求32-60之一的抗原结合单元或权利要求61嵌合抗原受体、以及任选的I型干扰素。
  68. 宿主细胞，其包含编码权利要求32-60之一的抗原结合单元或权利要求61嵌合抗原受体、以及任选的I型干扰素的核酸。
  69. 权利要求67的宿主细胞，其中所述宿主细胞是免疫应答细胞。
  70. 权利要求67的宿主细胞，其中所述宿主细胞是T细胞、自然杀伤细胞、细胞毒性T淋巴细胞、自然杀伤T细胞、DNT细胞、和/或调节性T细胞。
  71. 权利要求67的宿主细胞，其中所述宿主细胞是NK92细胞。
  72. 权利要求67的宿主细胞，其中所述宿主细胞对包含密蛋白18A2肽的细胞具有细胞毒性，所述密蛋白18A2肽包含SEQ ID NO:55的氨基酸序列。
  73. 权利要求67的宿主细胞，其中所述宿主细胞对包含密蛋白18A1肽但不包含密蛋白18A2肽的细胞不具有显著的细胞毒性，所述密蛋白18A1肽包含SEQ ID NO:57的氨基酸序列，且所述密蛋白18A2肽包含SEQ ID NO:55的氨基酸序列。
  74. 生成权利要求32-60之一的抗原结合单元或权利要求61嵌合抗原受体或权利要求62-63之一的组合物的方法，包括：在适合的条件下培养权利要求67-73的宿主细胞，并获得所述宿主细胞的表达产物。
  75. 诱导包含密蛋白18A2肽的细胞死亡的方法，包括使所述细胞与权利要求32-60之一的抗原结合单元、权利要求61嵌合抗原受体、权利要求62-63之一的组合物或权利要求67-73之一的宿主细胞接触。
  76. 权利要求75的方法，其中使所述细胞与所述抗原结合单元、所述嵌合抗原受体、所述组合物或宿主细胞在体外接触。
  77. 权利要求75的方法，其中使所述细胞与所述抗原结合单元、所述嵌合抗原受体、所述组合物或宿主细胞在体内接触。
  78. 权利要求75的方法，其中所述细胞是癌细胞。
  79. 权利要求75的方法，其中所述细胞是实体瘤细胞。
  80. 权利要求75的方法，其中所述细胞选自：胃癌细胞、食道癌细胞、肠癌细胞、胰腺癌细胞、肾母细胞瘤细胞、肺癌细胞、卵巢癌细胞、结肠癌细胞、直肠癌细胞、肝癌细胞、头颈癌细胞、慢性骨髓性白血病细胞和胆囊癌细胞。
  81. 治疗有需要的个体的肿瘤的方法，所述方法包括向所述个体给予有效量的权利要求32-60之一的抗原结合单元、权利要求61嵌合抗原受体、权利要求62-63之一的组合物、权利要求66的载体和/或权利要求67-73之一的宿主细胞。
  82. 权利要求81的方法，其中所述肿瘤是实体瘤。
  83. 权利要求81的方法，其中所述肿瘤是胃癌、食道癌、肠癌、胰腺癌、肾母瘤、肺癌、卵巢癌、结肠癌、直肠癌、肝癌、头颈癌、慢性骨髓性白血病或胆囊癌。
  84. 权利要求81的方法，还包括向所述个体给予额外的治疗剂。
  85. 权利要求84的方法，其中所述额外的治疗剂是选自以下的至少一种：表柔比星、奥沙利铂和5-氟尿嘧啶。
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