antibody to anticlaudin 18a2 and its use

专利摘要:
An anti-claudin 18a2 antibody and an immune effector cell targeting claudin 18a2 is provided in the present invention. Methods are also provided to induce cell death and treat tumors.
公开号:BR112019000327A2
申请号:R112019000327
申请日:2017-07-10
公开日:2019-08-13
发明作者:Jiang Hua；Wang Huamao；Yang Linlin；Wang Peng；Shi Zhimin；Li Zonghai
申请人:Carsgen Therapeutics Co Ltd；Shanghai Cancer Inst；
IPC主号:

专利说明:

“ANTIBODY FOR ANTICLAUDIN 18A2 AND USE OF THE SAME” CROSSED REFERENCE [0001] This application claims the benefit of document n ^Q CN201610536449.9 filed on July 8, 2016, and of PCT International Patent Application n ^Q PCT / CN2017 / 082024 , filed on April 26, 2017, and its entire disclosures are incorporated by reference in this document.
FIELD OF TECHNIQUE [0002] The present invention belongs to the field of immunology and, in particular, the invention relates to antibodies against claudin 18A2 and their uses.
BACKGROUND [0003] The chimeric antigen receptor (CAR) is an artificial 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 domain of a cell. immune response.
[0004] Gastric cancer is one of the cancers with the highest incidence rate in the world. According to statistics from the WHO Cancer Control Project, there are 7 million patients who die from cancer every year in the world, and 700,000 of them die from gastric cancer. Compared to conventional gastric cancer treatment regimens, antibody-based treatment regimens have wide application prospects, due to their high specificity and low side effects.
[0005] Claudin 18 (CLD18) is an intrinsic membrane protein located at the tight junction of the epithelium and endothelium, with a molecular weight of approximately 27.9 KD. The GenBank registration number is splicing variant 1 (CLD18A1, CLD18.1): NP_057453, NM016369, and splicing variant 2 (CLD18A2, CLD18.2): NM_001002026, NP_001002026. Figure 1A shows an identity comparison between claudin 18A2 (SEQ ID NO: 55) and claudin 18A1 (SEQ ID NO: 57). In normal cells, CLD18A1 is selectively expressed in the lung and stomach epithelium, while CLD18A2 is slightly expressed in normal gastric epithelial short-lived cells. However, in tumor cells, CLD18A2 is strongly expressed in several types of cancer.
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For example, 75% of gastric cancer patients have high expression of CLD18A2, 50% of pancreatic cancer patients have high expression of CLD18A2 and 30% of esophageal cancer patients have high expression of CLD18A2, which is also highly expressed in cancer of lung. For this reason, it is of great significance, for the treatment and detection of cancer, to find antibodies that bind to CLD18A2 with higher specificity, without binding to CLD18A1.
[0006] Type I interferons contain IFNa protein (a class of identical proteins encoded by 13 human genes from IFNA1 to IFNA13), ΙΡΝβ (encoded by a single human and mouse IFNB1 gene) and other less studied interferons. Studies have shown that type I interferons have anticancer effects on some tumors, probably due to their immune stimulating functions. However, the systematic administration of type I interferons can have immunosuppressive effects (FE Major depression during interferon-α treatment: vulnerability and prevention. Dialogues Clin. Neurosci. 11, pages 417 to 425 (2009)) with important undesirable events, in that the most common among them are fatigue, anorexia, hepatotoxicity, flu-like symptoms and severe depression (Kreutzer, K., Bonnekoh, B., Franke, I., Ulrich, J. & Gollnick, H. Sarcoidosis, myasthenia gravis And previous ischaemic optic neuropathy: severe side effects of adjuvant interferon-a therapy in malignant melanoma . J. Dtsch. Dermatol. Ges. 2, pages 689 to 694 (in German) (2004)), and such serious side effects severely limit that application.
SUMMARY OF THE INVENTION [0007] The present invention overcomes the problems mentioned above and has additional advantages.
[0008] In accordance with one aspect of the present invention, the present invention provides an antibody that specifically binds to claudin 18A2, characterized by the fact that the antibody comprises a heavy chain CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 31,32, 33, 37, 38, 39, 43, 44, 45, 49, 50, 51, 83, 84, 85, or a variant thereof, and / or a light chain CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 34, 35, 36, 40, 41,42, 46, 47, 48, 52, 53, 54, or a
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3/117 your variant.
[0009] In some embodiments, the antibody of the invention is selected from the group consisting of (a) an antibody comprising a heavy chain variable region, wherein the heavy chain variable region has CDR1 comprising an amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 43 or SEQ ID NO: 49, CDR2 comprising an amino acid sequence 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 or SEQ ID NO: 85, CDR3 comprising an amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45 or SEQ ID NO: 51; (b) an antibody comprising a light chain variable region, wherein the light chain variable region has CDR1 which comprises an amino acid sequence of SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46 or SEQ ID NO: 52, CDR2 comprising an amino acid sequence of SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47 or SEQ ID NO: 53, CDR3 comprising an amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48 or SEQ ID NO: 54; (c) an antibody comprising (a) a heavy chain variable region of said antibody and (b) a light chain variable region of said antibody; (d) an antibody, which recognizes the same antigenic determining site as that of the antibody of any of (a) to (c).
[0010] In some embodiments, the CDR1, CDR2 and CDR3 regions of the antibody heavy chain variable region are SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33; 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, respectively; and / or the CDR1, CDR2 and CDR3 regions of the antibody light chain variable region are SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36; or SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42; or SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48; or SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, respectively.
[0011] In some embodiments, the antibody comprises a variable region of heavy chain and a variable region of light chain, in
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4/117 that the heavy chain variable region has an amino acid sequence of SEQ ID NO: 3, 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 or SEQ ID NO: 29; and the light chain variable region has an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 21 or SEQ ID NO: 25. In in some embodiments, the antibody is an antibody that has a heavy chain variable region of SEQ ID NO: 3 and a light chain variable region of SEQ ID NO: 1; an antibody that has a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 5; an antibody that has a heavy chain variable region of SEQ ID NO: 11 and a light chain variable region of SEQ ID NO: 9; an antibody that has a heavy chain variable region of SEQ ID NO: 15 and a light chain variable region of SEQ ID NO: 13; an antibody having a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 1; an antibody that has a heavy chain variable region of SEQ ID NO: 19 and a light chain variable region of SEQ ID NO: 1; an antibody that has a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 21; an antibody having a heavy chain variable region of SEQ ID NO: 27 and a light chain variable region of SEQ ID NO: 25; or an antibody that has a SEQ ID NO: 29 heavy chain variable region and a SEQ ID NO: 25 light chain variable region. In some embodiments, the antibody is an antibody that has a SEQ heavy chain variable region ID NO: 3 and a light chain variable region of SEQ ID NO: 1; an antibody having a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 1; an antibody that has a heavy chain variable region of SEQ ID NO: 19 and a light chain variable region of SEQ ID NO: 1; an antibody that has a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 21; an antibody having a heavy chain variable region of SEQ ID NO: 27 and a light chain variable region of SEQ ID NO: 25; or an antibody that has a heavy chain variable region of SEQ ID NO: 29 and a region
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5/117 light chain variable of SEQ ID NO: 25. In some embodiments, the antibody is a humanized antibody, a chimeric antibody or a fully humanized antibody; or the antibody is a monoclonal antibody; or the antibody is a single chain antibody or a domain antibody. In some embodiments, the antibody is a humanized antibody selected from the group consisting of an antibody that has a heavy chain variable region of SEQ ID NO: 27 and a light chain variable region of SEQ ID NO: 25; an antibody that has a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 21; an antibody that has a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 25. In some embodiments, the antibody is selected from the group consisting of an antibody that has a heavy chain of SEQ ID NO: 63 and a light chain of SEQ ID NO: 65; an antibody that has a light chain of SEQ ID NO: 61 and a heavy chain of SEQ ID NO: 59; and an antibody that has a heavy chain of SEQ ID NO: 67 and a light chain of SEQ ID NO: 65.
[0012] According to one aspect of the invention, the invention provides a nucleic acid that encodes the antibody, as mentioned above. According to another aspect of the invention, the invention provides an expression vector that comprises nucleic acid. In accordance with another aspect of the present invention, the present invention provides a host cell that comprises the expression vector of the present invention or that has the nucleic acid of the present invention integrated into its genome.
[0013] According to one aspect of the invention, the invention provides the use of an antibody, according to the invention, to prepare a target drug, drug conjugate and antibody or multifunctional antibody that targets tumor cells that express claudin 18A2; or to prepare a reagent to diagnose a tumor that expresses 18A2 claudin; or to prepare a chimeric antigen receptor-modified immune cell. In some embodiments, the tumor that expresses claudin 18A2 includes: gastric cancer, pancreatic cancer, esophageal cancer, lung cancer.
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In accordance with one aspect of the invention, the invention provides a chimeric antigen receptor comprising an antibody of the invention, wherein the chimeric antigen receptor comprises components linked, in sequence, below: an antibody of the invention , a transmembrane region and an intracellular signal region. In some embodiments, the intracellular signal region is selected from the group consisting of: an intracellular signal region sequence of ΟΟ3ζ, FcsRly, CD27, CD28, CD137, CD134, MyD88, CD40, or a combination thereof; or the transmembrane region comprises a transmembrane region of CD8 or CD28. In some embodiments, the chimeric antigen receptor comprises, linked in sequence, an antibody, a transmembrane region and an intracellular signaling region: an antibody of the invention, CD8 and ΟΟ3ζ; an antibody of the invention, CD8, CD137 and ΟΟ3ζ; or an antibody of the invention, a transmembrane region of CD28 molecule, an intracellular signal region of CD28 molecule, and ΟΟ3ζ; or an antibody of the invention, a transmembrane region of CD28 molecule, an intracellular signal region of CD28, CD137 and CD3C molecule.
[0015] In accordance with another aspect of the invention, the invention provides a nucleic acid that encodes the chimeric antigen receptor. According to another aspect of the invention, the invention provides an expression vector that comprises the nucleic acid of the invention. According to another aspect of the invention, the invention provides a virus that comprises the vector of the invention.
[0016] In accordance with an aspect of the present invention, the present invention provides uses of a chimeric antigen receptor, nucleic acid, expression vector or virus of the present invention to prepare immune cells modified by chimeric antigen receptor that target cells tumor that express claudin 18A2. In some embodiments, the tumor that expresses claudin 18A2 includes: gastric cancer, pancreatic cancer, esophageal cancer, lung cancer.
[0017] In accordance with an aspect of the present invention, the present invention provides an immune cell modified by a chimeric antigen receptor transduced with a nucleic acid, expression vector or HIV virus.
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7/117 the present invention; or which has a chimeric antigen receptor of the present invention expressed on the surface. In some embodiments, the immune cell is: a T lymphocyte, NK cell or NKT lymphocyte. In some embodiments, the immune cell additionally carries a coding sequence for an exogenous cytokine; or additionally expresses another chimeric antigen receptor that does not contain ΟΟ3ζ, but contains an intracellular signal domain of CD28, an intracellular signal domain of CD137 or a combination of both; or additionally expresses a chemokine receptor (preferably, said chemokine receptor comprises: CCR); or further expresses a siRNA that can reduce the expression of PD-1 or a protein that can block PD-L1; or endogenous PD-1, in the immune cell, is eliminated by gene editing techniques; or additionally express a safety switch.
[0018] In accordance with an aspect of the present invention, the present invention provides uses of the chimeric antigen receptor-modified immune cell to produce a tumor inhibitory drug, wherein the tumor is a tumor that expresses claudin 18A2; preferably, the tumor includes: gastric cancer, pancreatic cancer, esophageal cancer and lung cancer.
[0019] In accordance with an aspect of the present invention, the present invention provides a multifunctional immunoconjugate comprising an antibody of the present invention; and a functional molecule attached to it; and the functional molecule is selected from the group consisting of: a molecule that targets a surface marker on a tumor, a tumor inhibiting molecule, a molecule that targets a surface marker on an immune cell or a detectable identification. In some embodiments, the molecule that targets an immune cell surface marker is an antibody that binds to a T cell surface marker, which forms a bifunctional antibody with the antibody of the invention in which the T cell is enveloped. . In accordance with another aspect of the invention, the invention provides a nucleic acid that encodes said multifunctional immunoconjugate and uses it to prepare anti-tumor drugs. In some embodiments, the nucleic acid encoding the multifunctional immunoconjugate is used to prepare a reagent to diagnose a tumor that expresses 18A2 claudin. In some embodiments, the nucleic acid encoding the immunoconjugate
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Multifunctional 8/117 is used to prepare immune cells modified by a chimeric antigen receptor. In some embodiments, immune cells include: a T lymphocyte, an NK cell or an NKT lymphocyte.
[0020] In accordance with an aspect of the invention, the invention provides a pharmaceutical composition comprising an antibody of the invention or a nucleic acid encoding the antibody. According to one aspect of the invention, the invention provides a pharmaceutical composition that comprises an immunoconjugate of the invention or a nucleic acid encoding the conjugate. According to one aspect of the invention, the invention provides a pharmaceutical composition comprising a chimeric antigen receptor of the invention or a nucleic acid encoding the chimeric antigen receptor. According to one aspect of the invention, the invention provides a pharmaceutical composition comprising an immune cell modified by the chimeric antigen receptor of the invention. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier or excipient.
[0021] According to one aspect of the invention, a kit is provided in the invention comprising a container and a pharmaceutical composition of the invention in the container; or a container and an antibody of the invention or a nucleic acid encoding the antibody in the container; or the immunoconjugate of the present invention or a nucleic acid encoding the conjugate or chimeric antigen receptor of the present invention or a nucleic acid encoding the chimeric antigen receptor; or the chimeric antigen receptor-modified immune cell of the invention.
[0022] In accordance with an aspect of the present invention, 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 peptide 18A2 claudin; and the antigen binding unit does not bind significantly to an 18A1 claudin peptide. In accordance with another aspect of the present invention, the present invention provides an antigen binding unit comprising a light chain CDR and a heavy chain CDR, wherein the
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9/117 antigen binding unit specifically binds to an 18A2 claudin peptide; and the antigen binding unit, compared to a reference antigen binding unit, exhibits less non-specific binding to the 18A1 claudin peptide. In some embodiments, the reference antigen binding unit comprises a light chain amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 88 and / or a heavy chain amino acid sequence of SEQ ID NO: 87 or SEQ ID NO: 89. In some embodiments, the 18A2 claudin peptide comprises an amino acid sequence of SEQ ID NO: 55. In some embodiments, the 18A1 claudin peptide comprises an amino acid sequence of SEQ ID NO: 57. In some embodiments , the non-specific binding of the antigen binding unit to the claudin peptide 18A1 does not exceed 20% of the specific binding to the claudin peptide 18A2. In some embodiments, binding specificity is determined by flow cytometry. In some embodiments, the specificity of binding is determined by FACS. In some embodiments, the specificity of binding is determined by ELISA. In some embodiments, the antigen binding unit binds to the claudin peptide 18A2 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. In some embodiments, 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 have respectively 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 HCDR1, HCDR2 and HCDR3 have respectively 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: 50, SEQ ID NO: 51, SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85. In some embodiments, 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. In some
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10/117 modalities, LCDR2 comprises 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. In some modalities, 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. In some embodiments, HCDR1 comprises an amino acid sequence selected from from the group consisting of SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 43 and SEQ ID NO: 49. In some embodiments, 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. In some embodiments, HCDR3 comprises a sequence amino acid selected from the group consisting of SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45 and SEQ ID NO: 51. In some embodiments, the antigen binding unit geno is scFv, Fv, Fab or (Fab) 2.
[0023] In accordance with an aspect of the invention, 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; said heavy chain CDR comprises HCDR1, HCDR2 and HCDR3; LCDR1, LCDR2 and LCDR3 respectively comprise an amino acid sequence that is 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; and HCDR1, HCDR2 and HCDR3 respectively comprise an amino acid sequence that is 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: 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: 85. In some embodiments, the light chain CDR comprises LCDR1, LCDR2 and LCDR3; the heavy chain CDR comprises HCDR1, HCDR2 and HCDR3; LCDR1, LCDR2 and LCDR3 respectively have an amino acid sequence selected from the group
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11/117 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 HCDR1, HCDR2 and HCDR3 have respectively 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: 50, SEQ ID NO: 51, SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85. In some embodiments, LCDR1 comprises an amino acid sequence that is 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. In some embodiments, LCDR2 comprises an amino acid sequence that is at least 80% identical to 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. In some embodiments, LCDR3 comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the r of the group consisting of SEQ ID NO: 35, SEQ ID NO: 42, SEQ ID NO: 48 and SEQ ID NO: 54. In some embodiments, HCDR1 comprises an amino acid sequence that is at least 80% identical to a 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. In some embodiments, HCDR2 comprises an amino acid sequence that is 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. In some embodiments, HCDR3 comprises an amino acid sequence that is 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. In some embodiments, the antigen binding unit is a monoclonal antibody, a humanized antibody, an antibody that immersion, a multivalent antibody or a chimeric antigen receptor. In some embodiments, the antigen binding unit is scFv, Fv, Fab or (Fab) 2.
[0024] According to one aspect of the invention, the invention provides a
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12/117 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. According to one aspect of the invention, the invention provides a composition comprising an antigen binding unit or a chimeric antigen receptor of the invention. In some embodiments, the composition comprises a type I interferon. According to one aspect of the invention, the invention provides an isolated nucleic acid that encodes an antigen binding unit or chimeric antigen receptor of the invention and, optionally, an interferon of the type I. According to one aspect of the invention, the invention provides a vector comprising a nucleic acid of the invention.
[0025] According to one aspect of the invention, the invention provides a host cell that expresses an antigen binding unit or chimeric antigen receptor of the invention and, optionally, a type I interferon. According to one aspect of the invention, the invention provides a host cell comprising a nucleic acid encoding the antigen binding unit or chimeric antigen receptor of the invention and, optionally, a type I interferon. In some embodiments, the host cell is an immune response cell . In some embodiments, the host cell is a T cell, natural killer cell, cytotoxic T lymphocyte, natural killer T cell, DNT cell and / or regulatory T cell. In some embodiments, the host cell is an NK92 cell.
[0026] In some embodiments, the host cell is cytotoxic to a cell that comprises a claudin 18A2 peptide that comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the host cell lacks significant cytotoxicity for a cell that comprises an 18A1 claudin peptide, while it does not comprise an 18A2 claudin peptide, and the 18A1 claudin peptide comprises an amino acid sequence of SEQ ID NO: 57, and the 18A2 claudin peptide comprises the amino acid sequence of SEQ ID NO: 55.
[0027] In accordance with one aspect of the invention, the invention provides a method for producing an antigen binding unit or chimeric antigen receptor or composition of the invention, which includes: culturing a cell
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13/117 host of the invention under suitable conditions and obtain the product expressed by the host cell.
[0028] In accordance with one aspect of the invention, the invention provides a method for inducing the death of a cell comprising an 18A2 claudin peptide, which includes bringing the cell into contact with an antigen binding unit, an antigen receptor chimeric, a composition or a host cell of the invention. In some embodiments, the cell is brought into contact with the antigen binding unit, the chimeric antigen receptor, the composition or the host cell in vitro. In some embodiments, the cell is brought into contact with the antigen binding unit, the chimeric antigen receptor, the composition or the host cell in vivo. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a solid tumor cell. In some embodiments, the cell is selected from the group consisting of: a gastric cancer cell, esophageal cancer cell, intestinal cancer cell, pancreatic cancer cell, nephroblastoma cell, lung cancer cell, cancer cell ovarian, cervical cancer cell, rectal cancer cell, liver cancer cell, head and neck cancer cell, chronic myeloid leukemia cell and gallbladder cancer cell.
[0029] According to one aspect of the invention, the invention provides a method for treating a tumor in an individual in need of it, the method including administering to the individual an effective amount of an antigen binding unit, a receptor of chimeric antigen, a composition or a host cell of the invention. In some embodiments, the tumor is a solid tumor. In some embodiments, 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 cancer of gallbladder. In some embodiments, the method additionally includes administering an additional therapeutic agent to the individual. In some embodiments, the additional therapeutic agent is at least one selected from the group consisting of epirubicin, oxaliplatin and 5-fluorouracil.
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INCORPORATION BY REFERENCE [0030] All publications, patents and patent applications mentioned in this specification are incorporated by reference, as if each of the publications, patents or each patent application were incorporated into reference title.
DESCRIPTION OF THE DRAWINGS [0031] The drawings additionally illustrate innovative features revealed in this specification. The features and advantages of the present disclosure will be better understood from the description below of the attached drawings. It should be understood, however, that the drawings are intended only to illustrate the specific modalities of the application of principles disclosed in this document, and are not intended to limit the scope of the appended claims.
[0032] Figure 1A shows the comparison of identity between claudin 18A2 (SEQ ID NO: 55) and claudin 18A1 (SEQ ID NO: 57); Figure 1B shows the binding of hybridoma supernatants 2B1, 3E12, 4A11 and 8E5 to HEK293 cells stably transfected with human CLD18A2 and CLD18A1, as determined by flow cytometry.
[0033] Figure 2 shows a sequence alignment of 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 ID NO: 13).
[0034] Figure 3 shows the relative binding affinity of murine anti-2B1, 8E5 ScFv, after fused to the human IgG1 Fc portion, to HEK293 cells stably transfected with human CLD18A2.
[0035] Figure 4 shows the relative binding of 2B1-N52D and 2B1-S54A of projected murine anti-2B1 antibody, after fusing to the human IgG1 Fc portion, to HEK293 cells stably transfected with human CLD18A2.
[0036] Figure 5 shows the relative binding affinity of humanized hu2B1-S54A, after being fused to the human IgG1 Fc portion,
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15/117 to HEK293 cells stably transfected with human CLD18A2.
[0037] Figure 6 shows the relative binding affinity of humanized hu8E5, after fusing to the human IgG 1 Fc portion, to HEK293 cells stably transfected with human CLD18A2.
[0038] Figure 7 shows the relative binding affinity of humanized hu8E5-2l designed to HEK293 cells stably transfected with human CLD18A2.
[0039] Figure 8A compares the CDC effects of the humanized antibodies hu2B1-S54A, hu8E5-2l and the known chimeric antibody ch-163E12 (see CN103509110A) in HEK293 cells transfected with CLD18A2; and Figure 8B compares the CDC results of the humanized antibodies hu2B1-S54A, hu8E5-2l and ch-163E12 in HEK293 cells transfected with CLD18A1.
[0040] Figure 9 compares the ADCC effects of the humanized antibodies hu2B1-S54A, hu8E5-2l and the chimeric antibodies ch-163E12, ch-175D10 (see CN103509110A).
[0041] Figure 10 compares the destruction activities of hu8E5-2l and ch-175D10 in mice.
[0042] Figure 11 compares, in vitro, T cell destruction activities of hu8E5-28Z, hu8E5-BBZ and hu8E5-28BBZ in different cell lines.
[0043] Figure 12 compares, in vitro, T cell destruction activities of hu8E5-28Z, hu8E5-2l-28Z and hu2B1-hs54A in different cell lines.
[0044] Figure 13 is a comparison chart of the effect of CLDN18A2-CAR T on tumor volume over time, in a subcutaneous xenograft model of PDX mice with gastric cancer (Figure 13A) and a comparison chart of tumor photographs (Figure 13B).
[0045] Figure 14 shows results of hu8E5-28Z and hu8E5-2l-28Z cytokine secretion assay.
[0046] Figure 15 is a graph comparing the effect of CLDN18A2-CAR T on tumor volume, over time, in a mouse subcutaneous xenograft model with gastric cancer BGC-823-A2 (Figure 15A), a tumor weight comparison chart (Figure 15B) and a
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16/117 tumor photograph comparison chart (Figure 15C).
[0047] Figure 16 shows the infiltration of CLDN18A2-CAR T tumor.
[0048] Figure 17A shows the secretion of cell molecules after IFN coexpression; Figure 17B is a comparison chart of antitumor activities of CAR-T cells containing IFN and free of IFN in subcutaneous xenografts of a PDX model of gastric cancer; and Figure 17C is a graph comparing the number of viable cells in the peripheral blood of a mouse on days 5, 7 and 10 of CAR-T return cells.
[0049] Figure 18A and Figure 18B are plasmid maps for the construction of CAR NK cells.
[0050] Figure 19 shows the determination of the positive rate of hu8E5-2l-28Z CAR-NK92 and hu8E5-28BBZ CAR-NK92.
[0051] Figure 20 is a graph showing the cytotoxicity of hu8E5-2l-28Z CAR-NK92.
[0052] Figure 21 is a graph showing the cytotoxicity of hu8E5-28BBZ CAR-NK92.
MODE FOR CARRYING OUT THE INVENTION [0053] The detailed description below reveals the modalities disclosed in this document in detail. It should be understood that the description is not intended to be limited to the particular modalities disclosed in this document, which can be varied. It will be understood by a knowledgeable person that the present revelation can be broadly modified or varied, and all modifications are within the scope and spirit of the revelation. Each modality may be combined arbitrarily with any other modality, unless stated otherwise.
[0054] Certain modalities disclosed in this document are intended to cover a range of values, and certain aspects of the invention can be described using a range. Unless stated otherwise, it should be understood that a range of values or the description of a scope is for the purpose of simplicity and convenience, and the scope of the invention should not be understood to be strictly limited by the scope of the invention. For this reason, the description of a scope should be interpreted as being specifically described as all possible sub-ranges and
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17/117 all possible specific numerical points within the range, since these sub-ranges and numeric points are explicitly mentioned in this document. For example, the description of the range from 1 to 6 should be considered to specifically reveal sub-ranges from 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the numeric points within these ranges, such as 1,2, 3, 4, 5 and 6. Regardless of the range of declared values, the above principles are equally applicable. When a track is described, the track includes end points of the track.
[0055] When referring to measurable values, such as quantity, temporary duration, etc., the term about should mean including a change of ± 20% or, in some cases, ± 10% or, in some cases, ± 5 % or, in some cases, ± 1% or, in some cases, ± 0.1% of the specified value.
[0056] As used in this document, the terms activate and activation can be used interchangeably and, as well as others among its grammatical forms, can refer to a process by which a cell transitions from a quiescent state to a state active. The process may include a response to an antigen, migration and / or a phenotypic or genotypic change in the active state of functionality. For example, the term activation can refer to a process by which immune cells are gradually activated. For example, T cells may require at least two signals to be fully activated. The first signal can occur after TCR is bound by the MHC antigen complex, while the second signal can occur by conjugating costimulatory molecules (see costimulators listed in Table 1). Anti-CD3 can stimulate the first signal in vitro, and anti-CD28 can stimulate the second signal. For example, the projected T cells can be activated by the express CAR. As used herein, immune cell activation may refer to a condition that has been sufficiently stimulated to induce detectable cell proliferation, cytokine production and / or detectable effector function.
[0057] The term co-stimulatory molecule, as used herein, refers to a homologous binding partner in an immune cell, such as a T cell, which specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response , such as,
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18/117 but without limitation, proliferation. A co-stimulatory molecule is a molecule on the cell surface other than an antigen receptor or its ligand that promotes an effective immune response. Co-stimulatory molecules include, but are not limited to, class I molecules of MHC, BTLA and Toll ligand receptors, and 0X40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) and 4-1 BB (CD137). Examples of costimulatory molecules include, but are not limited to, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8a, CD8p, IL2Rp , IL2Ry, IL7Ra, 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 and a linker that specifically binds to CD83.
[0058] As used herein, a 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 upward or downward regulation of key molecules.
[0059] The term antigen binding unit, as used herein, refers to an immunoglobulin molecule and an immunologically active portion of an immune molecule, that is, a molecule that contains an antigen binding site that binds specifically to an antigen (immune response). The term antigen binding unit also includes immunoglobulin molecules of various species, which include invertebrates and vertebrates. The simplest naturally occurring antibody (e.g., IgG) structurally comprises four polypeptide chains, two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Immunoglobulins represent a large family of molecules that include several types of molecules, such as IgD, IgG, IgA, IgM and IgE. The term immunoglobulin molecule includes, for example, a hybrid or altered antibody and fragments thereof. It has been shown that the antigen-binding function of an antibody can be performed by fragments of an antibody from
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11/197 naturally occurring. Such fragments are collectively called the antigen combination unit. The term antigen binding unit also includes any molecular structure that contains a polypeptide chain that has a specific shape that conforms to an epitope and recognizes an epitope, in which one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. Examples of such an antigen binding unit include a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains, a divalent fragment comprising two Fab fragments joined by a disulfide bridge in the hinge region (fragment of F (ab) 2); Fd fragment consisting of the VH and CH1 domains, Fv fragment consisting of the VL and VH domains of a single arm of an antibody; dAb fragment consisting of the VH domain (Ward et al., Nature, 341: pages 544 to 546, 1989); and isolated complementarity determining regions (CDRs) or any fusion protein comprising such antigen binding units.
[0060] The term antibody, as used herein, includes an intact antibody and any antigen-binding fragments (i.e., antigen-binding portions) or their unique chains. A naturally occurring antibody is a glycoprotein comprising at least two heavy chains (H) and two light chains (L) joined by means of a disulfide bond. Each heavy chain consists of a variable heavy chain 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 variable light chain region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of a CL domain. The VH and VL regions can be further subdivided into regions of high variability, called complementarity determining regions (CDRs) that are spaced through more conserved regions called the framework region (ER). Each VH and V L consists of three CDRs and four FRs arranged in the following order, from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy chain and the light chain contain a binding domain that interacts with the antigen. The constant region of an antibody can mediate
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20/117 binding of immunoglobulin to host factors or tissues, which includes several cells of the immune system (for example, effector cells) and the first component (C1q) of the classic complement system.
[0061] The term scFv refers to a fusion protein that comprises at least one antibody fragment that comprises a variable region of a light chain and at least one antibody fragment that comprises a variable region of a heavy chain, wherein the said light chain and the variable regions of the heavy chain are contiguous (for example, by means of a synthetic linker, such as a short flexible polypeptide linker), and can be expressed as a single chain polypeptide, and in which scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv can have the VL and VH variable regions in any order (for example, with respect to the N-terminal and C-terminal of the polypeptide), and the scFv can include a VH linker of VL, or a VH-binding VL can be included.
[0062] As used in this document, the terms complementarity determining region and CDR refer to an amino acid sequence in the variable region of the antibody that confers antigen specificity and binding affinity. In general, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in the light chain variable region.
[0063] An antigen binding unit specifically binds to an antigen or is immunoreactive with the antigen, if the antigen binding unit binds to the antigen with greater affinity than binding to other reference antigens, which includes polypeptides or other substances.
[0064] The term humanized, as used herein, is used for a non-human antibody (such as that of a rodent or primate), it is a hybrid immunoglobulin, immunoglobulin chain or a fragment thereof comprising a minimal sequence derived from a non-human immunoglobulin. In most cases, the humanized antibody is a human immunoglobulin (receptor antibody), in which residues from the complementarity-determining regions (CDRs) of the recipient are replaced by CDR residues from non-human species (donor antibody), such as mice , rats, rabbits or primates that have
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11/21 specificities, affinities and desired performances. In some cases, residues in the human immunoglobulin Fv (FR) framework region are replaced by corresponding non-human residues. In addition, a humanized antibody may comprise residues that are present in the recipient antibody, as long as they are not in the introduced CDR or framework sequences. These modifications are made to further improve and optimize the performance of the antibody and to minimize immunogenicity when introduced into a human body. In some examples, a humanized antibody will comprise substantially all, at least one, typically two variable domains, where all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the FR regions are regions of a human immunoglobulin sequence. A humanized antibody can also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin constant region. In some embodiments, a humanized antibody may include a mutation, such as a mutation introduced by site-directed or random mutagenesis in vitro or by somatic mutation in vivo.
[0065] The term immunoglobulin or Ig, as used in this document, can refer to a class of proteins that can function as antibodies. Antibodies expressed by B cells are sometimes called chimeric antigen receptors or antigen receptors. Five members included in the class 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 pathogenic antigens, can be recognized by the CAR.
[0066] As used herein, the term isolated refers to the separation of cellular components or other components in which polynucleotides, peptides, polypeptides, proteins, antibodies or fragments thereof are, in general, associated in a natural state. As will be understood by a knowledgeable person, it is necessary to isolate polynucleotides, peptides, polypeptides, proteins, antibodies that are not naturally occurring or
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22/117 its fragments, in order to distinguish them from naturally occurring counterparts. In addition, a polynucleotide peptide, polypeptide, a protein, a concentrated, isolated or diluted antibody, or a fragment thereof, can be distinguished from its naturally occurring counterpart, since the concentration or quantity of a molecule by volume is greater (concentrated) or lower (diluted) than the concentration of its naturally occurring counterpart. The degree of enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured, in relation to another potential interference present in the source mixture. In some embodiments, the greatest extent of enrichment is preferred for the technical solutions of the present invention. For this reason, for example, 2-fold enrichment is preferred, 10-fold enrichment is more preferred, 100-fold enrichment is more preferred, 1000-fold enrichment is even more preferred. Separate materials can also be provided by artificial assembly methods, such as recombinant expression or chemical synthesis.
[0067] As used in this document, antigen refers to a substance that is recognized and specifically bound by an antigen binding unit. Antigens can include peptides, proteins, glycoproteins, polysaccharides, lipids, their portions and combinations. Exemplary antigens without limitation include tumor antigens or pathogenic antigens. Antigen can also refer to a molecule that elicits an immune response. This immune response may involve the production of antibodies or the activation of specific immunologically competent cells, or both. A knowledgeable person will observe that any macromolecule, which includes virtually all proteins or all peptides, can serve as an antigen.
[0068] The terms polypeptide, peptide and protein are used interchangeably in this document to refer to a polymer of amino acids of any length. The polymer can be linear, cyclic or branched, can comprise modified amino acids, particularly conservatively modified amino acids, and can be disrupted by non-amino acids. The term also includes polymers of modified amino acids, such as a modified amino acid polymer
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23/117 through sulfonation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, prenylation, racemization, selenoylation, transfer RNA-mediated amino addition (such as argination, ubiquitination) or any other manipulation, such as conjugation with an identified component. As used herein, the term amino acid refers to synthetic, natural and / or unnatural amino acids, which includes glycine and D or L optical isomers, as well as amino acid and peptidomimetic analogs. A polypeptide or sequence of amino acids derived from a specific protein refers to the source of the polypeptide. The term also encompasses polypeptides expressed by a specified nucleic acid sequence.
[0069] The term amino acid modification (or modified amino acid) includes amino acid substitutions, insertions and / or deletions in a polypeptide sequence. As used herein, amino acid substitution or substitution means the replacement of an amino acid at a particular position in a polypeptide sequence of origin with another amino acid. For example, the R94K substitution means that arginine at position 94 is replaced by lysine. For the same substitution, in the same position, in the original polypeptide sequence, it can also be represented by 94K, that is, substitution of position 94 by lysine. For the purpose of this document, multiple substitutions are typically separated by bars. For example, R94K / L78V refers to a double variant that comprises the substitutions R94K and L78V. As used herein, amino acid insertion or insertion means the addition of an amino acid at a particular position in the parent polypeptide sequence. For example, insert 94 indicates an insertion at position 94. As used herein, amino acid deletion or deletion means the removal of an amino acid at a particular position in the sequence of origin polypeptides. For example, R94- indicates the deletion of arginine at position 94.
[0070] The term conservative modification or conservative sequence modification, as used herein, means an amino acid modification that does not significantly affect or alter the desired activities or properties of a peptide containing the sequence
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24/117 amino acids. Such conservative modifications include amino acid substitutions, insertions and deletions. Modifications can be introduced into the antibody of the invention by means of standard techniques known in the art, such as site-directed mutagenesis and PCR-directed mutagenesis. Conservative amino acid substitutions are substitutions in which amino acid residues are replaced by amino acid residues that have similar side chains. A family of amino acid residues that have similar side chains has been defined in the art. These families include amino acids that contain basic side chains (for example, lysine, arginine, histidine), acidic side chains (for example, aspartic acid, glutamic acid), uncharged polar side chains (for example, glycine, asparagine, serine, threonine , tyrosine, cysteine, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For that reason, one or more amino acid residues, in the CDR regions or framework regions of the antibody of the invention, can be replaced by other amino acid residues with similar side chains.
[0071] The term autologous, as used in this document, and others among its grammatical forms, can refer to substances from the same source. For example, a sample (for example, a cell) can be removed, processed and administered to the same individual (for example, a patient) at a later time. The autologous process is different from the allogeneic process, in which the donor and the recipient are different individuals.
[0072] As used in this document, xenograft and others among its grammatical forms may include any procedure in which a recipient and a donor are of different species, and cells, tissues or organs are transplanted, implanted or infused into the recipient. The transplantation of cells, organs and / or tissues, described in this document, can be used as xenografts in humans. Xenografts include, but are not limited to, vascularized xenografts, partially vascularized xenografts, non-vascularized xenografts, xenogenic dressings, xenogenic bandages and xenogenic structures.
[0073] As used in this document, allograft and other
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25/117 among its grammatical forms (for example, allogeneic transplantation) can include any procedure in which a recipient and a donor are of the same species, but different individuals, and cells, tissues or organs are transplanted, implanted or infused into the recipient. The transplantation of cells, organs and / or tissues, as described in this document, can be used as allografts in humans. Allografts include, but are not limited to, vascularized allografts, partially vascularized allografts, non-vascularized allografts, allogeneic dressings, allogeneic bandages and allogeneic structures.
[0074] As used in this document, autologous transplantation and others among its grammatical forms (for example, autologous transplantation) may include any procedure in which a recipient and a donor are from the same individual, and the cells, tissues or organs are transplanted, implanted or infused into the recipient. The transplantation of cells, organs and / or tissues described in this document can be used as autografts in humans. Autografts include, but are not limited to, autologous vascular transplantation, partial autologous vascular transplantation, non-vascularized autologous transplantation, autologous dressings, autologous bandages and autologous structures.
[0075] The term chimeric antigen receptor or CAR, as used herein, refers to a projected molecule that can be expressed by an immune cell that includes, but is not limited to, T cell or NK cell. CAR is expressed in T cells and redirects T cells to induce specific destruction of target cells with a specificity determined by the artificial receptor. The extracellular binding domain of CAR can be derived from a murine, humanized or fully humanized monoclonal antibody.
[0076] The term epitope, as used in this document, and others among its grammatical forms, can refer to a portion of an antigen that can be recognized by an antibody, a B cell, a T cell or a projected cell. For example, an epitope can be a tumor epitope or an epitope of a pathogen recognized by a TCR. Multiple epitopes within an antigen can also be recognized. Epitopes can also mutate.
[0077] Cell line or cell culture means a cell
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26/117 of bacteria, plant, insect or superior eukaryotic cell grown or maintained in vitro. The progeny of a cell may not be identical (in morphology, genotype or phenotype) to the maternal cell.
[0078] The term projected, as used in this document, and others among its grammatical forms can refer to one or more changes in a nucleic acid, such as a nucleic acid within the genome of an organism. The projected term can refer to changes, additions and / or deletions of genes. Projected cells can also refer to cells that have genes that are added, deleted and / or changed. Projected cells can also refer to cells that express CAR.
[0079] The term transfection, as used in this document, refers to the introduction of an exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by several means known in the art, including calcium phosphate DNA coprecipitation, DEAE-dextran-mediated transfection, polyamine-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection and biological.
[0080] The term stable transfection or stable transfection refers to the introduction and integration of exogenous nucleic acids, DNAs or RNAs into the genome of a transfected cell. The term stable transfectant refers to a cell that has exogenous nucleic acids stably integrated into genomic DNA.
[0081] As used herein, the terms nucleic acid molecule encoding, encoding DNA sequence and encoding DNA refer to the order or sequence of deoxyribonucleotides along a deoxyribonucleotide chain. The order of these deoxyribonucleotides determines the order of amino acids along the polypeptide (protein) chain. For that reason, a sequence of nucleic acids encodes a sequence of amino acids.
[0082] The term individual, as used herein, refers to any animal, such as a mammal or a marsupial. Subjects of the invention include, but are not limited to, humans, non-human primates (for example, rhesus monkeys or other types of monkeys), mice, pigs, horses, donkeys, cows, sheep, rats and chickens.
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27/117 all kinds.
[0083] The term peripheral blood lymphocytes (PBL) and others among their grammatical forms, as used in this document, can refer to lymphocytes circulating in the blood (for example, peripheral blood). Peripheral blood lymphocytes can refer to lymphocytes that are not restricted to organs. Peripheral blood lymphocytes can comprise T cells, NK cells, B cells or any of their combinations.
[0084] The term immune response cell (or immunoreactive cell, immune effector cell or immune cell), as used herein, can 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. Examples of immune cells include, but are not limited to, T cells, such as α / β T cells and γ / δ T cells, B cells, natural killer cells (NK), natural killer T cells (NKT), breast cells and cells bone marrow-derived phagocytic cells, their precursor cells and their progenies.
[0085] The term T cell and others among its grammatical forms, as used in this document, can refer to T cells from any source. For example, the T cell can be a primary T cell, such as an autologous T cell or the like. The T cell can also be human or non-human.
[0086] The term T cell activation or T cell activation, as used in this document, and others among its grammatical forms, can refer to the status of a T cell that is sufficiently stimulated to induce detectable cell proliferation, production detectable cytokine and / or effector function. In some embodiments, complete T cell activation can be similar to triggering T cell cytotoxicity. T cell activation can be measured using various assays known in the art. The assay can be an ELISA to measure cytokine secretion, ELISPOT, a flow cytometry assay to measure intracellular cytokine expression (CD107), a flow cytometry assay to measure proliferation, and a cytotoxicity assay to determine the target cell elimination (51 Cr release assay). In the assay, controls (non-engineered cells) are typically used to compare with engineered cells (CART) in order to
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28/117 to determine the relative activation of projected cells, compared to controls. In addition, the assay can be performed by comparison with the projected cells that are incubated or that are in contact with the 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.
[0087] The term sequence, as used in this document, and others among its grammatical forms, when used in reference to a sequence of nucleotides, 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 that has 2 to 1,000,000 or bad nucleotides (or any of those integers or above), for example, about 100 to about 10,000 nucleotides or about 200 to about 500 nucleotides in length.
[0088] The term effective amount, as used in this document, refers to an amount that provides therapeutic or prophylactic benefits.
[0089] The term expression vector, as used herein, refers to a vector that comprises a recombinant polynucleotide that comprises an expression regulation sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; and 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 (for example, naked or contained in liposomes), and viruses (for example, lentiviruses, retroviruses, adenoviruses and adeno-associated viruses).
[0090] The term lentivirus, as used in this document, refers to the genus of retrovirus family. Retroviruses are unique, in retroviruses, in their ability to infect cells that do not divide; they can deliver large amounts of genetic information to host cell DNAs, so they are one of the most efficient methods of gene delivery vectors. HIV, SIV and FIV are examples of lentiviruses. The
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29/117 vectors derived from lentiviruses provide a means of achieving significant levels of gene transfer in vivo.
[0091] The term operably linked, as used herein, refers to a functional link between a regulatory sequence and a heterologous nucleic acid sequence, which results in the expression of the latter. For example, when the first nucleic acid sequence is functionally associated with the second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence, if the promoter affects the transcription or expression of the coding sequence. Typically, the operably linked DNA sequences are contiguous and, where necessary, two protein coding regions are linked in the same reading frame.
[0092] The term promoter, as used in this document, is defined as a DNA sequence that is recognized by the synthetic machinery or the synthetic machinery introduced that is required to initiate the specific transcription of a polynucleotide sequence.
[0093] The term vector, as used herein, is a composition comprising an isolated nucleic acid and which is used to deliver an isolated nucleic acid to the interior of a cell. Various vectors are known in the art, which include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. For this reason, the term vector includes plasmids or replication viruses autonomously. The term should also be interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids to cells, such as polylysine compounds, liposomes and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.
[0094] A host cell includes an individual cell or cell culture that may 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, the progeny may not necessarily be identical to the original mother cell, for example, in the properties
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30/117 morphological or genomic DNA or total DNA. Host cells include cells that are transfected in vivo with the vectors of the invention. Host cell can refer to a prokaryotic cell, a eukaryotic cell or a cell line that is grown as a single cell entity that can be, or that has been used as a receptor for recombinant vectors or other transfer polynucleotides, and includes cells from progenies that have been transfected.
[0095] The term sequence identity, as used in this document, determines identity in percentage by comparing the two best compatible sequences over a comparison window (for example, at least 20 positions), in which the polynucleotide portions or of the polypeptide sequence, in the comparison window, may comprise the addition or deletion (i.e., gap), for example, 20% or less of ranges (for example, 5 to 15%, or 10 to 12%) compared to a reference sequence (which contains no additions or deletions) for the two best compatible sequences. The percentage is usually calculated by determining the number of positions in which the nucleotides or amino acid residues are the same, in both sequences, to produce the number of positions correctly matched. The percentage of sequence identity can be obtained by dividing the number of positions with correct compatibility by the total number of positions in the reference sequence (that is, the window size) and multiplying the result by 100.
[0096] The term disease or condition or disorder, as used herein, refers to any change or disorder that impairs or interferes with the normal function of a cell, tissue or organ. For example, the term disease includes, but is not limited to, a tumor, a pathogen infection, autoimmune disease, T-cell dysfunction disease, or a defect in immune tolerance (for example, transplant rejection).
[0097] The term exogenous, as used in this document, refers to a nucleic acid molecule, or polypeptide, that is not endogenously expressed in a cell, or in which its level of expression is insufficient to achieve function overexpression. For this reason, exogenous includes recombinant nucleic acid molecules, or polypeptides, expressed in a cell, such as nucleic acid molecules, and
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11/31 polypeptides, exogenous, heterologous and overexpressed.
[0098] The term regulation, as used in this document, refers to a positive or negative change. Regulation example includes 1%, 2%, 10%, 25%, 50%, 75% or 100% variation.
[0099] As used in this document, the term treatment refers to a clinical intervention in an attempt to alter an individual or treat a disease caused by 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, relieving symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the progression of the disease, improving or improving the condition, relieving or improving the prognosis.
[00100] The term constitutive expression, as used in this document, refers to expression under all physiological conditions.
[00101] The term inducible expression, as used in this document, refers to expression under certain conditions, such as when a T cell binds to an antigen. A person skilled in the art will know how to perform conventional induced expression.
[00102] In some embodiments, an antigen binding unit, comprising a light chain CDR and a heavy chain CDR, is provided herein, wherein the antigen binding unit specifically binds to the claudin peptide 18A2 ; and wherein the antigen binding unit does not bind significantly to the 18A1 claudin peptide.
[00103] In some embodiments, an antigen binding unit, comprising a light chain CDR and a heavy chain CDR, is provided herein, where the antigen binding unit specifically binds to the claudin peptide 18A2 ; and where the antigen binding unit shows less non-specific binding to the 18A1 claudin peptide, as compared to a reference antigen binding unit.
[00104] In some embodiments, an antigen binding unit described herein comprises a light chain CDR. The light chain CDR can be a determining region of complementarity of
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32/117 an antigen binding unit. The light chain CDR may comprise a contiguous sequence of amino acid residues, or two or more contiguous sequences of amino acid residues spaced by non-complementarity determining regions, such as framework regions. In some embodiments, the light chain CDR comprises two or more light chain CDRs, which may be called CDR-1, light chain CDR-2, and the like. In some embodiments, the light chain CDR comprises three light chain CDRs, which can be called light chain CDR-1 (LCDR1), light chain CDR-2 (LCDR2) and light chain CDR-3 (LCDR3) , respectively. In some embodiments, a set of CDRs present in a common light chain can be collectively referred to as a light chain CDR.
[00105] In some embodiments, an antigen binding unit described herein comprises a heavy chain CDR. The heavy chain CDR can be a complementary determining region of an antigen binding unit. The heavy chain CDR may comprise a contiguous sequence of amino acid residues, or two or more contiguous sequences of amino acid residues spaced by non-complementarity determining regions, such as framework regions. In some embodiments, the heavy chain CDR comprises two or more heavy chain CDRs, which may be called CDR-1, heavy chain CDR-2, and the like. In some embodiments, the heavy chain CDR comprises three heavy chain CDRs, which may be called heavy chain CDR-1 (HCDR1), heavy chain CDR-2 (HCDR2) and heavy chain CDR-3 (HCDR3), respectively. In some embodiments, a set of CDRs present in a common heavy chain can be collectively referred to as a heavy chain CDR.
[00106] In some embodiments, an antigen binding unit, comprising a light chain CDR and a heavy chain CDR, is provided herein, 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:
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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 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 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: 85.
[00107] In some embodiments, an antigen binding unit, comprising a light chain CDR and a heavy chain CDR, is provided herein, wherein the light chain CDR comprises LCDR1, LCDR2 and LCDR3; and the heavy chain CDR comprises HCDR1, HCDR2 and HCDR3; where the LCDR1, LCDR2 and LCDR3 amino acid sequences are at least 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% or 99.9% identical to a sequence amino acid selected from the following: 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 where the amino acid sequences of HCDR1, HCDR2 and HCDR3 are at least 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% or 99.9% identical to a 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: 50, SEQ ID NO: 51, SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85.
[00108] In some embodiments, in the antigen binding unit, in this document, the light chain CDR comprises LCDR1, LCDR2 and LCDR3; and the heavy chain CDR comprises HCDR1, HCDR2 and HCDR3; wherein LCDR1, LCDR2 and LCDR3 have, respectively, 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
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NO: 53 and SEQ ID NO: 54; and wherein said HCDR1, HCDR2 and HCDR3 have, respectively, 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: 50, SEQ ID NO: 51, SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85.
[00109] In some embodiments, an antigen binding unit is provided, in this document, in which the LCDR1 comprises amino acid sequences that are at least 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% or 99.9% 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. In some embodiments, an antigen binding unit is provided herein, where LCDR2 comprises amino acid sequences that are at least 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% or 99.9% identical to 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.
[00110] In some embodiments, an antigen binding unit is provided, in this document, in which the LCDR3 comprises amino acid sequences that are at least 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% or 99.9% 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. In some embodiments, an antigen binding unit is provided herein, where HCDR1 comprises amino acid sequences that are at least 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% or 99.9% identical 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 In some embodiments, an antigen binding unit is provided in this document, where HCDR2 comprises sequences of amino acids that are
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35/117 at least 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% or 99.9% 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. In some embodiments, an antigen binding unit is provided herein, where HCDR3 comprises amino acid sequences that are at least 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% or 99.9% 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.
[00111] In some embodiments, an antigen binding unit of the invention binds to claudin 18A2 or to a claudin 18A2 peptide. The term claudin 18A2 or claudin 18A2 peptide (CLD18.2, CLD18A2, CLDN18A2, CLDN18.2, Claudina18.2 or Claudina18A2), in this document, can also refer to an optimized form of codon, inter-species homolog, orthologist, homolog , truncated form, fragmented form, mutated form or any other known form derived from a known 18A2 claudin sequence, such as a post-translational modification variant. In some embodiments, claudin 18A2 or claudin 18A2 peptide is a peptide with GenBank registration number NP_001002026 (mRNA: NM_001002026). In some embodiments, 18A2 claudin or 18A2 claudin peptide is a peptide comprising the amino acid sequence of SEQ ID NO: 55.
[00112] In some embodiments, the antigen binding unit of the invention does not bind significantly to the 18A1 claudin peptide. The term claudin 18A1 or claudin 18A1 peptide (CLD18A1, CLD18.1, CLDN18A1, CLDN18.1, Claudina18.1 or Claudina18A1), in this document, can also refer to an optimized form of codon, inter-species homolog, orthologist, homolog , truncated form, fragmented form, mutated form or any other form derived from a known 18A1 claudin sequence, such as a post-translational modification variant. In some embodiments, claudin 18A1 or the peptide from
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36/117 claudin 18A1 is a peptide with GenBank registration number NP_057453 (mRNA: NM_016369). In some embodiments, claudin 18A1 or claudin 18A1 peptide is a peptide that comprises an amino acid sequence of SEQ ID NO: 57.
[00113] 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. Compared to other reference antigens or reference peptides, a given heavy chain CDR, light chain CDR or a combination of them, provides a given binding pocket with greater affinity and / or specificity to claudin 18A2.
[00114] The binding of the antigen binding unit to the 18A2 claudin peptide can be characterized or expressed by any method known in the art. For example, binding can be characterized by binding affinity, which can be the resistance of the interaction between the antigen binding unit and the antigen. The binding affinity can be determined by any method known in the art, such as in vitro binding assays. For example, when tested in an in vitro binding assay, using cells expressing 18A2 claudin, the binding affinity of the antigen binding unit disclosed herein can be determined. The binding affinity of the tested antigen binding unit can be expressed as Kd, which is the equilibrium dissociation constant between the antibody and its respective antigen. In some cases, the antigen binding unit disclosed in this document specifically binds to claudin 18A2, with a Kd variation of about 10 μΜ to about 1 mM. For example, the antigen binding unit can specifically bind to claudin 18A2 with a Kd of less than about 10 μΜ, 1 μΜ, 0.1 μΜ, 10 nM, 1 nM, 0.1 nM, 10 pM, 1 pM, 0.1 pM, 10 fM, 1 fM or less than 0.1 fM.
[00115] In some embodiments, the antigen binding unit does not exhibit significant binding to a reference peptide. In some instances, the level of binding of the antigen binding unit to the reference peptide is not higher than 20% of the binding level of the antigen binding unit to claudin 18A2. For example, the connection level can be 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7% , 6%, 5%,
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4%, 3%, 2%, 1% or less than 1% of the binding level of the antigen binding unit to 18A2 claudin. In some embodiments, the antigen binding unit in this document binds to claudin 18A2 at a level that is 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more than 10 times the level of binding to the reference peptide. In some embodiments, a reference peptide is 18A1 claudin peptide. In some embodiments, the reference peptide is a peptide that comprises an amino acid sequence of SEQ ID NO: 57. In some embodiments, the reference peptide is a peptide of SEQ ID NO: 57.
[00116] In some embodiments, compared to a reference antigen binding unit, the antigen binding unit, in this document, exhibits less non-specific binding to a reference peptide. In some embodiments, the level of non-specific binding of the antigen binding unit in this document to the reference peptide is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% less than that of the reference antigen binding unit to the reference peptide. In some embodiments, the level of non-specific binding of the antigen binding unit in this document to the reference peptide is 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more than 10 times less than that of the reference antigen binding unit to the reference peptide. In some embodiments, the reference antigen binding unit comprises a light chain of SEQ ID NO: 86 or SEQ ID NO: 88 and / or a heavy chain amino acid sequence of SEQ ID NO: 87 or SEQ ID NO: 89 In some embodiments, the reference antigen binding unit comprises an amino acid sequence of SEQ ID NO: 86. In some embodiments, the reference antigen binding unit comprises an amino acid sequence of SEQ ID NO: 87. In in some embodiments, the reference antigen binding unit comprises an amino acid sequence of SEQ ID NO: 88. In some embodiments, the reference antigen binding unit comprises an amino acid sequence of SEQ ID NO: 89. In some embodiments , a reference peptide is 18A1 claudin peptide. In some embodiments, the reference peptide is a peptide that comprises a sequence of
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38/117 amino acids of SEQ ID NO: 57. In some embodiments, the reference peptide is a peptide of SEQ ID NO: 57.
[00117] In some embodiments, the antigen binding unit is cytotoxic to a cell comprising 18A2 claudin peptide that comprises an amino acid sequence of SEQ ID NO: 55. The level of cytotoxicity is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or 45%, 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.
[00118] In some embodiments, the antigen binding unit lacks significant cytotoxicity for a cell comprising 18A1 claudin peptide, but which does not comprise 18A2 claudin peptide, wherein the 18A1 claudin peptide comprises a sequence of SEQ amino acids ID NO: 57, and the claudin peptide 18A2 comprises an amino acid sequence of SEQ ID NO: 55. In some embodiments, the level of cytotoxicity is not higher than 10%, 5%, 4%, 3%, 2 % or 1%.
[00119] In some embodiments, an antibody that specifically binds to claudin 18A2 is provided in the present document, characterized by the fact that the antibody comprises a heavy chain CDR that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31,32, 33, 37, 38, 39, 43 of 44, 45, 49, 50, 51, 83, 84, 85 or a variant thereof and / or a light chain CDR comprising a sequence of amino acids selected from the group consisting of SEQ ID NO: 34, 35, 36, 40, 41.42, 46, 47, 48, 52, 53, 54, or a variant thereof.
[00120] In some embodiments, an antibody is provided herein which is selected from the group consisting of (a) an antibody comprising a heavy chain variable region, wherein the heavy chain variable region has CDR1 comprising an amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 43 or SEQ ID NO: 49, CDR2 comprising an amino acid sequence 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 or SEQ ID NO: 85 and CDR3 comprising an amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45 or SEQ ID NO: 51; (b) an antibody comprising a light chain variable region, in
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39/117 that the light chain variable region has CDR1 which comprises an amino acid sequence of SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46 or SEQ ID NO: 52, CDR2 which comprises a sequence of amino acids of SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47 or SEQ ID NO: 53 and CDR3 comprising an amino acid sequence of SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48 or SEQ ID NO: 54; (c) an antibody comprising (a) a heavy chain variable region of said antibody and (b) a light chain variable region of said antibody; and (d) an antibody, which recognizes the same antigenic determining site as that of the antibody of any of (a) to (c). In some embodiments, an antibody is provided herein, in which the regions of CDR1, CDR2 and CDR3 of the variable region of the heavy chain of the antibody are SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33; 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, respectively; and / or the CDR1, CDR2 and CDR3 regions of the antibody light chain variable region are SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36; or SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42; or SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48; or SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, respectively.
[00121] In some embodiments, the amino acid sequences of the CDR1, CDR2 and CDR3 of the antibody heavy chain of the invention are selected from the group consisting of the amino acid sequences shown in the following Table, or their variants:
TABLE 1
HCDR1 HCDR2 HCDR3 THE 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 Ç 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 AND 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
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G SEQ ID NO: 49 SEQ ID NO: 85 SEQ ID NO: 51
[00122] In some embodiments, an antibody is provided herein, in which the amino acid sequences of CDR1, CDR2 and CDR3 light chain are selected from the group consisting of the amino acid sequences in the following Table, or their variants:
TABLE 2
LCDR1 LCDR2 LCDR3 THE 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 Ç 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
[00123] In some embodiments, an antibody of the invention comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 3, 7, 11, 15, 17, 19, 23, 27, 29 or its variants, and / or a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 1, 5, 9, 13, 21, 25 or a variant thereof. In some embodiments, 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 a variant thereof. In some embodiments, 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. In some embodiments, 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 its variant. 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 its variant. In some preferred embodiments, the heavy chain CDR1, CDR2 and CDR3 amino acid sequences are selected from the Table below.
TABLE 3
HCDR1 HCDR2 HCDR3 AND 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
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41/117 [00124] In some embodiments, an antibody of the invention comprises a variable region of heavy chain comprising an amino acid sequence selected from SEQ ID NO: 17, 19, or variants thereof. In some embodiments, 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 a variant thereof. In some embodiments, 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 its variant.
[00125] In some embodiments, an antibody of the invention or a functional fragment thereof, comprises a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 23, 27, 29, or a variant thereof, and / or a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 21, 25, or a variant thereof. In some embodiments, 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 a variant thereof. In some embodiments, 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 a variant thereof. In some embodiments, 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 a variant thereof.
[00126] In some embodiments, an antigen or antibody binding unit of the invention is additionally attached or fused to another functional molecule. Consequently, the invention also encompasses formed multifunctional immunoconjugates.
[00127] Bonded or casted are used interchangeably in this document. These terms mean that two or more chemical elements or components are joined by any means that includes chemical conjugation or recombinant methods. In-frame fusion ”means that two or more reading phases are joined in order to maintain the correct reading phase of the original open reading phase (ORF), to form a contiguous and longer ORF. For this reason, the resulting recombinant fusion protein is a single protein that contains two or more fragments that correspond to the polypeptide encoded by the original ORF (these fragments are not
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42/117 usually so connected in a natural state). The reading phases are contiguous throughout the fusion fragment, however, the fragments can be physically or spatially separated, for example, by an in-frame joining sequence (for example, flexion).
[00128] The functional molecule is, for example, used for the diagnosis or treatment of a tumor.
[00129] The term tumor as used in this document, refers to a disease characterized by pathological hyperplasia of cells or tissues, and 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. Tumors can affect a variety of cells, tissues or organs, including but not limited to, 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, testicles, team, thyroid, trachea, urethra, ureter, uterus, vaginal organs or corresponding tissue or cells. Tumors include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant plasma cell tumors). The tumor of the present invention can include, but is not limited to, leukemia (such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute monocytic-granulocytic 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, lymphangiosarcoma, angiosarcoma, lymphatic endothelial sarcoma, synovial vioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, cancer of the breast, cancer of the prostate, ovarian cancer , basal cell carcinoma, adenocarcinoma, sweat gland cancer , sebaceous gland cancer, papillary cancer, papillary adenocarcinoma, carcinoma, bronchial carcinoma, medullary carcinoma, renal cell carcinoma, liver cancer, gallbladder cancer,
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43/117 choriocarcinoma, thin tumor, embryonic carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannomas, meningiomas, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder carcinoma, kidney cancer, multiple myeloma. The tumor preferably includes, but is not limited to, pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, squamous cell carcinoma of the head and neck, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma.
[00130] The functional molecule includes, for example, a tumor antigen, such as a tumor specific antigen (TSA) or a tumor associated antigen (TAA). TSA is unique to tumor cells and does not occur in other cells in the body. The antigen associated with TAA is not unique to tumor cells, but is expressed in normal cells under conditions in which the state of immune tolerance to the antigen cannot be induced. The expression of an antigen in a tumor can occur under conditions that allow the immune system to respond to the antigen. When the immune system is immature and unable to respond, TAA may be an antigen that is expressed on normal cells during fetal development, or they may be antigens that are normally present, at very low levels, in normal cells, but are expressed at a higher level in tumor cells.
[00131] Examples without limitation of TSA or TAA antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel17), tyrosinase, TRP-1, TRP-2 and multicentric antigens tumor specific, 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; single 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, EBVA antigens, and human papillomavirus (HPV) E6 and E7 antigens, etc. Other antigens
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44/117 large, protein-based include 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-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, 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, M0V18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 Mac-2 C-related protein Cyclophilin, TAAL6, TAG72, TLP and TPS.
[00132] In some embodiments, the tumor antigen includes, but is not limited to, prostate specific membrane antigen (PSMA), carcinoembryogenic antigen (CEA), IL13Ralfa, HER-2, CD19, NY-ESO-1, HIV-1 Gag, Lewis Y, MART-1, gp100, tyrosinase, WT-I, hTERT, mesothelin, EGFR, EGFRvIII, phosphatidylinositol 3, EphA2, HER3, EpCAM, MUC1, MUC16, Folate receptor, CLDN6, CD30, CD138, ASGPR1, CDH16, GD2, 5T4, 8H9, ανβ6 integrin, mature B cell antigen (BCMA), B7-H3, B7-H6, CAIX, CA9, CD20, CD22, Kappa κ light chain, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD171, CSPG4, EGP2, EGP40, ERBB3, ERBB4, ErbB3 / 4, FAP, FAR, FBP, embryonic AchR, GD2, GD3, HLA-AI MAGE A1, MAGE3, HLA-A2, IL11 Ra, KDR, Lambda, MCSP, NCAM, NKG2D ligand, PRAME, PSCA, PSC1, ROR1, Sp17, SURVIVIN, TAG72, TEM1, TEM8, VEGRR2, HMW - MAA, VEGF receptor and / or fibronectin, tenascin or carcinoembryonic variants of tumor necrotic regions.
[00133] In some embodiments, the functional molecule is an interferon. In some embodiments, interferon is a type I interferon.
[00134] The term type I interferon, as used herein, includes IFNa, ΙΡΝβ, IFN-ε, IFN-κ, IFN-ω and the like. All type I interferons bind to specific cell surface receptors (so-called IFN-α / β receptors) that consist of two strands of IFNARI and IFNAR2. In some embodiments, the term type I interferon, as used in this document, is IFNa or ΙΡΝβ. In some embodiments, the term type I interferon, as used in this document, is ΙΡΝβ. In some embodiments, type I interferon, as used herein, includes human, mouse, or synthetic type I interferon. In some modalities, the term interferon α,
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45/117 as used herein, can be a polypeptide that has the sequence shown in NCBI aaa52724.1 or aaa52716.1 or aaa52725.1, or a polypeptide, in which its sequence has at least 85% identity for those sequences . In some embodiments, the term interferon β (INF-β), as used in this document, can be a protein that has at least 85% identity to NCBI aac41702.1 or np_002167.1 or aah96152.1 p41273 or NP 001552, or a fragment that serves as a tumor necrosis factor (TNF) ligand. In some modalities, interferon β is human β interferon. In some embodiments, interferon β has an amino acid sequence of SEQ ID NO: 92.
[00135] In some embodiments, type I interferon may be naturally occurring, for example, isolated or purified from a mammal; or it can be artificially prepared, for example, recombinant components or type I interferon can be produced according to conventional genetic engineering recombination techniques. Preferably, type I recombinant elements or interferons can be used in the present invention.
[00136] Amino acid sequences formed based on the type I interferon polypeptide sequence, by substitution, deletion or addition of one or more amino acid residues, are also included in the present invention. Proper amino acid substitution is a technique well known in the art, which can be readily implanted and ensures that the biological activities of a resulting molecule will not be altered. Based on these techniques, a knowledgeable person will observe that, in general, the alteration of a single amino acid, in a non-essential region of a polypeptide, does not substantially alter biological activities.
[00137] Polypeptides modified according to the type I interferon polypeptide sequence can also be used in the present invention. For example, a polypeptide modified to promote its half-life, metabolism and / or potency effectiveness can be used. That is, any variation that does not affect the biological activities of a polypeptide can be used in the present invention.
[00138] Biologically active fragments of type I interferon polypeptide can be used in the present invention. As used in
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46/117 In this document, the meaning of a biologically active fragment refers to a polypeptide that, as part of a whole polypeptide, still retains all or part of the entire polypeptide's function. Typically, the biologically active fragment retains at least 50% of the activities of the entire polypeptide. Under more preferential conditions, the active fragment is capable of retaining 60%, 70%, 80%, 90%, 95%, 99% or 100% of the activities of the entire polypeptide.
[00139] In another aspect, the invention provides a chimeric antigen receptor that comprises an extracellular antigen binding unit, as described herein, a transmembrane domain and an intracellular domain. The term Chimeric Antigen Receptor (CAR), as used herein, refers to a tumor antigen binding domain fused to an intracellular signal transduction domain that activates T cells. Typically, the extracellular binding domain CAR is derived from a mouse or humanized or human monoclonal antibody.
[00140] A chimeric antigen receptor typically comprises an extracellular antigen binding region or antigen binding unit. In some embodiments, the extracellular antigen binding unit is an antigen binding unit, as described in the present document above.
[00141] In some embodiments, the extracellular antigen binding region may be entirely human. In other examples, the extracellular antigen binding region can be humanized. In other examples, the extracellular antigen binding region may be murine, or the chimera in the extracellular antigen binding region consists of amino acid sequences derived from at least two different animals. In some embodiments, the extracellular antigen-binding region may be non-human.
[00142] A variety of antigen binding regions can be designed. Examples without limitation include single chain variable fragments (scFv) derived from antibodies, fragments of antigen binding regions (Fabs) selected from libraries, single domain fragments, or natural ligands that bind to their cognate receptors. In some embodiments, the extracellular antigen-binding region can
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47/117 comprise scFv, Fab or natural ligand, as well as any of its derivatives. An extracellular antigen binding region may refer to a molecule other than an intact antibody, which may comprise a portion of an intact antibody and may bind to an antigen to which the intact antibody binds. Examples of antibody fragments can include, but are not limited to, Fv, Fab, Fab ', Fab'-SH, Ffabjz; bifunctional antibodies, linear antibodies; single chain antibody molecules (for example, scFv); and multispecific antibodies formed from the antibody fragments.
[00143] An extracellular antigen binding region, such as a scFv, Fab or natural ligand, can be a 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 with a known variable region sequence. The extracellular antigen binding region can be obtained from the antibody sequences from available mouse hybridomas. Alternatively, extracellular antigen binding regions can be obtained from tumor cells or primary cells, such as tumor-infiltrating lymphocytes (TIL) through total external cut sequencing.
[00144] In some embodiments, 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. In many examples, binding specificity can be determined by light chain CDRs and heavy chain CDRs. Compared to other reference antigens, a combination of a given heavy chain CDR and light chain CDR can provide a given binding pocket with greater affinity and / or specificity to an antigen.
[00145] In certain aspects of any of the embodiments disclosed herein, the extracellular antigen binding region, such as scFv, may comprise an antigen-specific light chain CDR. The light chain CDR can be a region for determining the complementarity of an antigen binding unit, such as scFv light chain from a CAR. Light chain CDRs can comprise a contiguous sequence of amino acid residues, or two or more sequences
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48/117 contiguous amino acid residues separated by non-complementarity determining regions (for example, framework regions). In some embodiments, a light chain CDR may comprise two or more light chain CDRs, which may be called light chain CDR-1, CDR-2, and the like. In some embodiments, light chain CDRs may comprise three light chain CDRs, which may be called light chain CDR-1, light chain CDR-2 and light chain CDR-3, respectively. In some instances, a set of CDRs present in a common light chain can be collectively called a light chain CDR.
[00146] In certain aspects of any of the embodiments disclosed herein, the extracellular antigen binding region, such as scFv, may comprise an antigen-specific heavy chain CDR. The heavy chain CDR can be a complementary determining region of an antigen binding unit, such as scFv heavy chain. Heavy chain CDRs can comprise a contiguous sequence of amino acid residues, or two or more contiguous sequences of amino acid residues separated by non-complementarity determining regions (for example, framework regions). In some embodiments, a heavy chain CDR may comprise two or more heavy chain CDRs, which may be called CDR-1, heavy chain CDR-2, and the like. In some embodiments, heavy chain CDRs may comprise three heavy chain CDRs, which may be called heavy chain CDR-1, heavy chain CDR2 and heavy chain CDR3, respectively. In some examples, a set of CDRs present in a common heavy chain can be collectively called a heavy chain CDR.
[00147] The extracellular antigen binding region can be modified in several ways through genetic engineering. In some embodiments, the extracellular antigen binding region can be mutated so that the extracellular antigen binding region can be selected so that it has a higher affinity for its target. In some embodiments, the affinity of the extracellular antigen-binding region for its target can be optimized for targets expressed at a low level in normal tissues. This optimization can be carried out to minimize toxicities
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49/117 potentials. In other examples, clones of an extracellular antigen binding region, with a higher affinity for the membrane-bound form of a target, may be preferable through the counterpart in a soluble form. Such modifications can be made, since different levels of soluble forms of a target can also be detected and their targeting can cause unwanted toxicity.
[00148] In some embodiments, the extracellular antigen binding region comprises a hinge or spacer region. The terms "hinge" and "spacer region" can be used interchangeably. The hinge can be considered as a part of a CAR to render flexibility for the extracellular antigen binding region. In some embodiments, the hinge can be used to detect CAR on the surface of a cell, especially when antibodies that detect the extracellular antigen binding region are ineffective or available. For example, it may be necessary to optimize the length of the hinge derived from an immunoglobulin, which depends on the epitope site on the target that the extracellular antigen binding region targets.
[00149] In some embodiments, the hinge may not belong to an immunoglobulin, but to another molecule, such as the native hinge of a CD8a molecule. The CD8a hinge may contain cysteine and proline residues known to play a role in the interaction of CD8 coeceptor and MHC molecule. Cysteine and proline residues can affect CAR performance.
[00150] The CAR hinge can be adjustable in size. This morphology of the immune synapse between an immune response cell and a target cell also defines the distance that cannot be functionally crossed by a CAR, due to the distal membrane epitope on a target molecule on the cell surface, that is, the distance synaptic cannot reach an approximation that a signal can be conducted, even with the use of a short hinged CAR. Similarly, for the target epitope of a CAR's membrane proximal antigen, signal outputs can only be seen in the context of a long hinged CAR. The hinge can be adjusted, which depends on the extracellular antigen binding region used. The hinge can be any length.
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50/117 [00151] The transmembrane domain can anchor a CAR in the plasma membrane of a cell. The natural transmembrane portion of CD28 can be used for a CAR. In other examples, the natural transmembrane portion of CD8a can also be used in a CAR. CD8 can be a protein that has at least 85, 90, 95, 96, 97, 98, 99 or 100% identity with the NCBI reference number: NP_001759, or a fragment of yours that has stimulating activities. A CD8 nucleic acid molecule can be a polynucleotide that encodes a CD8 polypeptide and, in some examples, the transmembrane region can be a natural transmembrane portion of CD28. CD28 can be a protein that has at least 85, 90, 95, 96, 97, 98, 99 or 100% identity with the NCBI reference number: NP_006130, or a fragment of yours that has stimulating activities. A CD28 nucleic acid molecule can be a polynucleotide that encodes a CD28 polypeptide. In some embodiments, the transmembrane portion may comprise a region of CD8a.
[00152] The intracellular signaling region of a CAR may be responsible for activating at least one of the effector functions of an immune response cell in which a CAR has been placed. CAR can induce T cell effector functions, for example, the effector function is a cytolytic activity or auxiliary activity, which includes secretion of cytokines. For this reason, the term intracellular signaling region refers to a portion of protein that transduces the signals of effector function and directs the cell to perform a specific function. The entire intracellular signaling region can, in general, be used, however, 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 effector function signals.
[00153] 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 binding the receptor to the target, as well as any derivatives or their variant strings and any of the strings
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51/117 synthetic of those strings that have the same functionality.
[00154] In some embodiments, the intracellular signaling region may contain a known signal motif for an immunoreceptor tyrosine activation motif (ITAM). Examples of ITAMs that contain cytoplasmic signaling sequences include those derived from ΤΟΡζ, FcRy, FcRp, CD3y, CD3, CD3s, CD5, CD22, CD79a, CD79b and CD66d. However, in a preferred embodiment, the intracellular signal domain is derived from a Οϋ3ζ chain.
[00155] An example of a T cell signaling domain that contains one or more ITAM motifs is the Οϋ3ζ domain, also known as the T cell receptor Τ3ζ chain or CD247. This domain is a part of the T cell receptor CD3 complex and plays an important role in the recognition of binding antigen of various intracellular signal transduction pathways for the primary effector activation of T cells. As used herein, CD3ζ is refers primarily to human CD3ζ and its isoforms, as known from the entry in the Swissprot P20963 database, which includes proteins that have substantially identical sequences. As a part of a chimeric antigen receptor, it is reiterated that the entire célula3ζ T cell receptor chain is not required, and that any derivative of the signal domain comprising T3ζ T cell receptor chain is suitable, which includes any of its functional equivalents.
[00156] The intracellular signaling domain can be selected from any of the domains in Table 1. In some modalities, the domain can be modified so that the identity with the reference domain can vary from about 50% to about 100%. Any domain in Table 1 can be modified, so that the modified form can comprise about 50, 60, 70, 80, 90, 95, 96, 97, 98, 99 or even about 100% identity.
[00157] The intracellular signaling region of CAR may additionally comprise one or more co-stimulatory domains. The intracellular signaling region can comprise a single co-accumulator domain, such as a ζ chain (first generation of CAR) or it and CD28 or 4-1 BB (second generation of CAR). In other examples, the intracellular signaling region can comprise two co-stimulatory domains, such as CD28 / OX40 or
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11/117
CD28 / 4-1 BB (third generation).
[00158] Along with intracellular signaling domains, such as CD8, these co-stimulatory domains can generate activation downstream of the kinase pathway, thereby supporting gene transcription and functional cellular responses. This CAR co-stimulatory domain can activate the GD28 (phosphatidylinositol-4,5-diphosphate 3-kinase) or 4-1BB / OX40 (TNF receptor-associated factor adapter protein) pathways, as well as proximal signaling protein associated with activation of MAPK and Akt.
[00159] In some examples, the signals generated by a CAR can be combined with an auxiliary or co-stimulator signal. For co-stimulatory signaling domains, chimeric antigen receptor-like complexes can be designed to contain several possible co-stimulating signal domains. As is well known in the art, in virgin T cells, binding of T cell receptors alone is not sufficient to induce complete activation of T cells in cytotoxic T cells. A second co-stimulatory signal is required for complete productive T cell activation. Several receptors have been reported to provide co-stimulus for T cell activation, which includes, but is not limited to, GD28, 0X40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1BBL, MyD88 and 4 -1 BB. The signal transduction paths used by these costimulatory molecules can act synergistically with the primary T cell receptor activation signal. The signals provided by these co-stimulating signaling regions can act synergistically with primary effect activation signals derived from one or more ITAM motifs (for example, the CD3zeta signal transduction domain) and can meet the requirements for T cell activation.
[00160] In some embodiments, the addition of a co-stimulatory domain to a complex similar to the chimeric antigen receptor can enhance the effectiveness and durability of projected cells. In other embodiments, the T cell signaling domain and the co-stimulatory domain are fused together to form a signaling region.
TABLE 4. COESTIMULATOR DOMAIN
Gene marker Abbreviation Name
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CD27 CD27; T14; S152; Tp55;TNFRSF7; S152. LPFS2 CD27 molecule CD28 Tp44 antigen; CD28; CD28 CD28 molecule TNFRSF9 ILA; 4-1 BB; CD137; CDw137 Tumor necrosis factor receptor superfamily member 9 TNFRSF4 0X40; ACT35; CD134; IMD16;TXGP1L Tumor necrosis factor receptor superfamily member 4 TNFRSF8 CD30; Ki-1; D1S166E Tumor necrosis factor receptor superfamily member 8 CD40LG IGM; IMD3; TRAP; gp39; CD154;CD40L; HIGM1; T-BAM;TNFSF5; hCD40L CD40 binder ICOS AILIM; CD278; CVID1 Inducible T cell co-stimulator ITGB2 LAD; CD18; MF17; MFI7;LCAMB; LFA-1; MAC-1 Integrin β2 (Complement component 3 and 4 receptor subunits) CD2 T11; SRBC; LFA-2 CD2 molecule CD7 GP40; TP41; Tp40; LEU-9 CD7 molecule KLRC2 NKG2C; CD159c; NKG2-C Killer cell lectin-like receptor subfamily C, member 2 TNFRSF18 AITR; GITR; CD357; GITR-D Tumor necrosis factor 18 superfamily member TNFRSF14 TR2; TIE; HVEA; HVEM;CD270; LIGHTR Tumor necrosis factor receptor superfamily member 14 HAVCR1 TIM; KIM1; TIM1; CD365;HAVCR; KIM-1; TIM-1; TIMD1;TIMD-1; HAVCR-1 Hepatitis A virus cell receptor 1 LGALS9 HUAT; LGALS9A, Galectin-9 Lectin, which binds to the soluble galactoside, 9 CD83 BL11; HB15 CD83 molecule
[00161] The chimeric antigen receptor binds to the target antigen When T cell activation is measured in vitro or ex vivo, the target antigen can be obtained or isolated from various sources. The target antigen, as used herein, is an antigen or an immunological epitope in a
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54/117 antigen that is critical in mammals for immune recognition and, ultimately, elimination or control of pathogenic factors or disease states. Immune recognition can be a cell and / or a body fluid. In the case of intracellular pathogens and cancer, immune recognition can be, for example, a T lymphocyte reaction.
[00162] In some embodiments, the target antigen comprises an antigen associated with a precancerous or proliferative state. Target antigens can also be associated with or caused by cancer. For example, in some embodiments, a chimeric antigen receptor of the invention recognizes and binds to a tumor antigen comprising TSA and TAA, as described in the present document above.
[00163] In some embodiments, when a chimeric antigen receptor in this document is present on a cell's plasma membrane, binds to its target and is activated, the cell expressing the chimeric antigen receptor may produce cytotoxicity for a cell that carries the target. For example, in some embodiments, the chimeric antigen receptor is present in a cytotoxic cell, such as an NK cell or a cytotoxic T cell, and, when activated by a target, the toxicity of the cytotoxic cell to the target cell can be increased. In some embodiments, the chimeric antigen receptors herein may enhance the effect of immunoreactive cells on cells that express claudin 18A2, such as tumor cells. In some embodiments, compared to a cell that does not express a chimeric antigen receptor, in this document, the cytotoxic effect of a cell that expresses a chimeric antigen receptor, described in this document, on cells that express claudin 18A2 increases by at least 10%, at least 15.%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 1 time, at least 1.5 times at least 2 times, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times or at least 10 times.
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55/117 [00164] In some embodiments, when a chimeric antigen receptor in this document is present in the plasma membrane of a cell, it binds to its target and a chimeric antigen receptor is activated in this document, it does not induce significant cytotoxicity in cells that comprise 18A1 claudin peptide, but not in the 18A2 claudin peptide. In some embodiments, the level of cytotoxicity is no more than 10%, 5%, 4%, 3%, 2% or 1%.
[00165] A transgene that encodes a receptor or a CAR that binds to a target antigen can be incorporated into a cell. For example, a transgene can be incorporated into an immune response cell, such as a T cell. When inserted into a cell, the transgene can be a complementary DNA fragment (cDNA) that is a copy of messenger RNA (mRNA); or the gene itself (with or without introns) located in the original region of your genomic DNA.
[00166] A nucleic acid encoding a transgene sequence, such as DNA, can be randomly inserted into a cell's chromosome. Random integration can be produced by any method that introduces a nucleic acid, such as DNA, into a cell. For example, the method may include, but is not limited to, electroporation, ultrasound, use of a gene gun, lipofection, calcium phosphate transfection, use of dendrimers, microinjection and use of virus vectors that include adenovirus, AAV and retroviral vectors and / or type II ribozyme.
[00167] The DNA encoding the transgene can also be designed to include a reporter gene, so that the presence of the transgene or its expression product can be detected by activating 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.
[00168] CAR expression can be verified by expression assays, such as qPCR or by measuring the RNA level. The level of expression can also indicate the number of copies. For example, if the level of expression is very high, it may indicate that more than one copy of a CAR is integrated into the genome. Alternatively, the high expression may indicate that the transgene is integrated in a high transcribed region, such as close to a
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56/117 highly expressed promoter. Expression can also be verified by measuring protein levels, for example, by Western blotting.
[00169] In some embodiments, an immune response cell of the invention may comprise one or more transgenes. Said one or more transgenes can express a CAR protein that recognizes and binds to at least one epitope on an antigen or binds to a mutant epitope on the antigen. CAR can be a functional CAR. In some embodiments, the immune response cells of the invention may comprise one or more CARs, or they may comprise a single CAR and a secondary designed receptor.
[00170] In some embodiments, the transgene can encode a suicide gene. As evidenced by many effective treatments for cancer patients, CAR immune response cells can cause tumor regression, albeit with toxicity. In some embodiments, when the target antigen is shared in normal tissues and tumor cells, CAR immune response cells may not be able to distinguish between tumors and normal tissues (target / off-target toxicity). In other cases, a systemic immune system dysfunction, called cytokine release syndrome (CRS), can occur. CRS may comprise a systemic inflammatory response syndrome or a cytokine storm, which may be a consequence of the rapid expansion of CAR immune response cells in vivo. CRS is a condition characterized by fever and hypotension, which can lead to multiple organ failure. In most cases, toxicity is associated with the in vivo expansion of infused CAR immune response cells, which can cause general dysfunction of the immune system, as well as high levels of pro-inflammatory cytokine release, such as TNFa and IL- 6. Suicidal genes can induce the elimination of CAR immunoreactive cells. The suicide gene can be any gene that induces apoptosis in immunoreactive CAR cells. A suicide gene can be encoded in the viral vector, along with the antigen-binding receptor. The coding of the suicide gene allows for the relief or total abortion of toxicity caused by the in vivo expansion of the CAR immune response cells infused under specific conditions.
[00171] In some modalities, CAR immunoreactive cells, for an antigen, which are present in normal tissues can be
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57/117 produced so that they transiently express the CAR, for example, after electroporation of the mRNA encoding the receptor. In addition, in the case of severe target toxicity, CAR immunoreactive cells can be substantially eliminated in an effort to further strengthen CAR immunoreactive cells including a safety switch.
[00172] In some embodiments, the CAR coding vector can be combined, for example, with an inducible caspase-9 gene (activated by a dimeric chimeric inducer) or a truncated form of EGF R receptor (activated by the monoclonal antibody Cetuximab) or safety switch RQR8.
[00173] One or more transgenes used in this document may be of different species. For example, one or more transgenes can comprise a human gene, a mouse gene, a mouse gene, a porcine gene, a bovine gene, a dog gene, a cat gene, a monkey gene, a gene chimpanzee, or any of its combinations. For example, a transgene can be from a human that has a human genetic sequence. One or more transgenes can comprise a human gene. In some cases, one or more transgenes are not adenoviral genes.
[00174] As described above, a transgene can be inserted into the genome of an immunoreactive cell at random or at a specific site. For example, 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 anywhere in the genome. For example, a transgene can encode its own promoter or it can be inserted into a position controlled by the internal promoter Alternatively, 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 disrupt a gene, such as an endogenous immune checkpoint.
[00175] In some modalities, more than one copy of a transgene can be inserted in multiple random sites, within the genome. For example, multiple copies can be inserted at random sites in the genome. This can
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58/117 result in an increase in overall expression, compared to random insertion of the transgene for once. Alternatively, one copy of the transgene can be inserted into one gene, and another copy of the transgene can be inserted into a different gene. The transgene can be targeted so that it can be inserted at a specific site in the genome of an immunoreactive cell.
[00176] In some embodiments, a polynucleic acid comprising a sequence encoding an antigen-binding receptor may take the form of a plasmid vector. The plasmid vector can comprise a promoter. In some cases, the prosecutor may be constitutive. In some embodiments, the prosecutor is inducible. The promoter can be, or can be derived from, CMV, U6, MND or EF1a. In some embodiments, the promoter may be adjacent to the CAR sequence. In some embodiments, the plasmid vector further comprises a splice acceptor. In some embodiments, the splice acceptor may be adjacent to a CAR sequence. The promoter sequence can be a PKG or MND promoter. The MND promoter may be a synthetic promoter comprising the U3 region of MoMuLV LTR modified with myeloproliferative virus sarcoma enhancer.
[00177] In some embodiments, a polynucleic acid encoding a receptor of interest can be designed to be delivered to a cell by non-viral techniques. In some cases, polynucleic acid may be a GMP-compatible reagent.
[00178] The expression of a polynucleic acid that encodes a receptor that binds to an antigen or a CAR can be controlled by one or more promoters. Promoters can be ubiquitous, constitutive (promoters without restriction, which allow continuous transcription of related genes), tissue-specific promoters or inducible promoters. The expression of an inserted transgene adjacent to or near a promoter can be modulated. For example, a transgene can be inserted near or beside a ubiquitous promoter. Some ubiquitous promoters can be CAGGS promoter, hCMV promoter, PGK promoter, SV40 promoter or ROSA26 promoter.
[00179] The promoters can be endogenous or exogenous. For example, one or more transgenes can be inserted adjacent or close to the endogenous or exogenous ROSA26 promoter. In addition, the promoter may be specific for immunoreactive cells. For example, one or more transgenes
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59/117 can be inserted adjacent or close to the porcine ROSA26 promoter.
[00180] Tissue-specific promoters or cell-specific promoters can be used to control the site of expression. For example, one or more transgenes can be inserted close to a specific tissue promoter. The tissue specific promoter can 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.
[00181] Inducible promoters can also be used. These inducible promoters can be turned on and off by adding or removing an inductor, if necessary. The inducible promoter is contemplated to be, but not limited to, Lac, tac, trc, trp, araBAD, phoA, recA, proU, cst-1, theta, cadA, nar, PL, cspA, T7, VHB, Mx e / or Trex.
[00182] The term inducible promoter, as used in this document, is a controlled promoter, which does not express or underexpress a gene operably linked to it, before the desired conditions are reached, and expresses, or expresses at a high level, a gene operably linked to it when the desired conditions are achieved. For example, in some embodiments, an inducible promoter of the present application does not express or underexpress a gene operably linked to it under normal or hyperoxic conditions in a cell and, in response to a reduced oxygen content in the cell, a gene operably linked to it it is expressed or overexpressed under hypoxic conditions. In some embodiments, an inducible promoter used herein includes hypoxia-inducible transcription factor-1a (HIF-1a). In some embodiments, the term inducible promoter, as used herein, refers to an immune cell inducible promoter that does not express or underexpress a gene operably linked to it, before an immune response cell comes into contact with an antigen , or when the immune response cell is not activated, whereas, only when the immune response cell comes into contact with the antigen or the immune response cell is activated, the promoter triggers the gene operably linked to expression at a high level or high expression, under conditions such as hypoxia. In some embodiments, the immune cell-inducible promoter comprises an NFAT (active T cell nuclear factor) promoter.
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60/117 [00183] As used herein, an NFAT-type promoter refers to a class of promoters that regulate the expression of a gene operably linked to it, based on NFAT-binding activity.
[00184] 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.
[00185] 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, since calmodulin (CaM) activates serine / threonine phosphatase (CN) calcineurin. The activated CN quickly dephosphorylates the serine-rich region (SRR) and SP is repeated at the amino terminus of NFAT protein, which results in a conformational change that exposes the nuclear localization signals that lead to the entry of NFAT into the nucleus.
[00186] Based on the role of NFAT in the transcriptional expression of cytokines during T cell activation, it can be used to modulate the immune cell-inducible promoters described herein, thereby expressing, or expressing at high levels, an operably gene linked to it, when the immune response cells come into contact with the antigen and are activated.
[00187] A nucleic acid of the invention can comprise any suitable nucleotide sequence encoding the NFAT-type promoter (or one of its functional parts or one of its variants). As used herein, an NFAT-type promoter refers to one or more NFAT response elements linked to the minimum promoter of any gene expressed by a T cell. Preferably, the minimum promoter of the gene expressed by T cells is the smallest IL promoter -2 human. The NFAT response element can include, for example, NFAT1, NFAT2, NFAT3 and / or NFAT4 response elements. In some embodiments, an NFAT-type promoter, as described in this document, includes more than one reason for binding to NFAT. For example, the NFAT-type promoter can include 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NFAT-binding motifs. In some modalities, the
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61/117 NFAT-type promoter includes up to 12 reasons for binding to NFAT. In some embodiments, the NFAT-type promoter may be a promoter consisting of a plurality of NFAT-binding motifs in series, with a promoter, such as IL2 minimal promoter. In some embodiments, the NFAT-type promoter described in this document understands his reasons for connecting to NFAT, named (NFAT) e. For the sake of convenience, (NFAT) is also called NFAT6. In some embodiments, NFAT6 also represents 6 repeated NFAT binding motifs (SEQ ID NO: 94) in the NFAT-type promoter.
[00188] In addition, transgenic sequences may also include transcriptional or translational regulatory sequences, such as promoters, enhancers, isolators, internal ribosome entry sites, sequences encoding 2A peptides and / or polyadenylation signals, although not essential for expression.
[00189] In some embodiments, the transgene encodes a receptor or CAR that binds to the antigen, in which the transgene is inserted into a safe port, so that the antigen-binding receptor is expressed. In some embodiments, the transgene is inserted into the PD1 and / or CTLA-4 locus. In other cases, the transgene is delivered as a lentivirus to cells for random insertion, while a specific PD1 or CTLA-4 nuclease can be supplied as mRNA. In some embodiments, the transgene is delivered by a viral vector system, such as retrovirus, AAV, or adenovirus, and mRNA that encodes a specific safe harbor nuclease (for example, AAVS1, CCR5, albumin or HPRT). The cells can also be treated with mRNA encoding PD1 and / or CTLA-4 specific nucleases. In some embodiments, the polynucleotide encoding the CAR is provided by a delivery system, along with an mRNA encoding a specific HPRT nuclease and a specific PD1 or CTLA-4 nuclease. CARs that can be used with the methods and compositions disclosed in this document can include all types of these chimeric proteins.
[00190] In some embodiments, a transgene can be introduced into an immunoreactive cell using a retroviral vector (γ-retroviral or lentiviral vector). For example, a transgene that encodes a CAR or any receptor that binds an antigen, or a variant thereof, or a fragment
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62/117 yours, can be cloned into a retroviral vector and can be triggered from an endogenous promoter, a long-term retroviral terminal repeat, or a type-specific target cell promoter. Non-viral vectors can also be used. Non-viral vector delivery systems can include a DNA plasmid, a naked nucleic acid and a nucleic acid complexed with a delivery vehicle, such as a liposome or poloxamer.
[00191] Many virus-based systems have been developed to transfer genes to mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and compressed into retroviral particles using techniques known in the art. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer, as they allow long-term stable integration of the transgene and its propagation in daughter cells. Lentiviral vectors have additional advantages over retrovirus-derived vectors, such as murine leukemia virus, as they can transduce cells that do not proliferate. They also have additional advantages of low immunogenicity. An advantage of adenoviral vectors is that they do not fuse with the genome of a target cell, thereby avoiding events related to negative integration.
[00192] The cells can be transfected with a transgene that encodes the antigen-binding receptor. The concentration of a transgene can vary from about 100 picograms to about 50 micrograms. In some embodiments, the amount of nucleic acid (for example, ssDNA, dsDNA or RNA) introduced into a cell can be changed to optimize the transfection efficiency and / or cell viability. For example, 1 microgram of dsDNA can be added to each cell sample for electroporation. In some embodiments, the amount of nucleic acid (for example, double-stranded DNA) required for optimal transfection efficiency and / or cell viability varies, depending on the type of cell. In some embodiments, the amount of nucleic acid (eg, dsDNA) used for each sample can directly correspond to the transfection efficiency and / or cell viability, for example, a range of transfection concentrations. The vector-encoded transgene can be integrated into a cell's genome. In some
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63/117 modalities, the transgene encoded by the vector is integrated forward. In other cases, the vector-encoded transgene is integrated in reverse.
[00193] The vector is delivered to an individual patient typically by systemic administration (eg, intravenous, intraperitoneal, intramuscular, subcutaneous or intracranial infusion) or topical application, as described below. Alternatively, the vector can be delivered ex vivo to a cell, such as a cell removed from an individual patient (for example, lymphocytes, T cells, bone marrow aspirate, tissue biopsy), and then the cells in which the vector is embedded are typically selected and implanted in a patient. The cells can be expanded before or after selection.
[00194] The immunoreactive cells suitable for expression of a receptor that binds to an antigen can be cells that are autologous or non-autologous for an individual in need.
[00195] An adequate source of immune response cells can be obtained from the individual. In some cases, T cells can be obtained. T cells can be obtained from a variety of sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, team tissue, and tissues from infected sites, ascites, pleural effusion, spleen tissue and tumor tissue . In some cases, T cells can be obtained from blood collected from the subject, using various techniques known to a knowledgeable person, such as Ficoll ™ separation. In some embodiments, an individual's circulating blood cells are obtained by apheresis. Apheresis products typically contain lymphocytes, which include T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and platelets. In some embodiments, cells collected by apheresis can be washed to remove plasma fractions and placed in a buffer or medium suitable for subsequent processing steps.
[00196] Alternatively, the cells can be derived from a healthy donor, a patient diagnosed with cancer or a patient diagnosed with an infection. In some embodiments, the cells may be a part of a population of mixed cells, with different phenotypic characteristics. Cell lines can also be obtained from
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64/117 from T cells transformed according to the previously described methods. The cells can also be obtained from a cell therapy library. The modified cells that are resistant to immunosuppressive therapy can be obtained by any of the methods described in this document. It is also possible to select an appropriate cell population before modification. The projected cell population can also be selected after modification. The projected cells can be used for autologous transplantation. Alternatively, the cells can be used for allogeneic transplantation. In some embodiments, the cells are administered to a sample to identify the same patient from a target sequence associated with cancer. In other examples, the cells are administered to a patient other than a patient, whose sample is used to identify a target sequence associated with cancer.
[00197] In some embodiments, suitable primary cells include peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL) and other subpopulations of blood cells, 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. In some embodiments, the cell can be any immune cell, which includes 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 another type of T cell. T cells can also include memory T cells, memory T stem cells or effector T cells. You can also select T cells from a large population, for example, select T cells from whole blood. T cells can also be expanded from a large population. T cells may also be prone to specific populations and phenotypes. For example, the T cell may be prone to a phenotype comprising CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+) and / or IL-7Ra ( +). Suitable cells can have one or more of the following markers: CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+) and / or IL-7Ra (+ ). Suitable cells also include stem cells, such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells and
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65/117 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 an individual (for example, a patient) to be treated.
[00198] The therapeutically effective amount of cells required in a patient may vary, depending on the viability of the cells and the efficiency with which the cells are genetically modified (for example, the efficiency with which the transgene is integrated with one or more cells, or the level of expression of the protein encoded by the transgene). In some embodiments, the product (for example, duplication) of cell viability, after genetic modification, and the efficiency of transgene integration may correspond to a therapeutic amount of cells that can be used for administration to an individual. In some embodiments, an increase in cell viability, after genetic modification, may correspond to a reduction in the essential amount of effective cells to be administered to a patient. In some embodiments, an increase in the efficiency of integrating a transgene into one or more cells may correspond to a reduction in the amount of necessary cells administered to a patient for effective treatment. In some embodiments, determining the number of cells needed for effective treatment may include determining functions associated with changes in cells over time. In some embodiments, determining the number of cells needed for effective treatment may include determining functions that correspond to changes in the efficiency of integrating a transgene into one or more cells, according to a time-dependent variable (for example, time cell culture, electroporation time, cell stimulation time). In some embodiments, the therapeutically effective cells may be a population of cells that comprise about 30% to about 100% of the expression of an antigen-binding receptor on the cell surface. In some embodiments, therapeutically effective cells, as measured by flow cytometry, can express about 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% or more of about 99 , 9% of the
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66/117 binding to the antigen on the cell surface.
[00199] According to one aspect of the invention, the invention also encompasses a nucleic acid that encodes the antigen-binding receptor. The invention also relates to variants of the above polynucleotides that encode a polypeptide or fragment, analog and derivatives of the polypeptide that have the same amino acid sequence as the invention.
[00200] The present invention also provides a vector comprising the above nucleic acid that encodes the receptor protein that binds to the antigen expressed on the surface of an immune response cell.
[00201] The invention also includes viruses that comprise the vectors described above. The virus of the present invention includes an infectious virus, after compaction, and also includes a virus to be compacted to contain components necessary for it to be compacted into an infectious virus. Other viruses known in the art that can be used to transduce exogenous genes into immune response cells and their corresponding plasmid vectors can also be used in the present invention.
[00202] In another aspect, a host cell is provided herein, which comprises an antigen binding unit or a chimeric antigen receptor, as described in this document, and optionally type I interferon. In another aspect, a cell host is provided herein which comprises a nucleic acid encoding an antigen binding unit or the chimeric antigen receptor described herein and, optionally, type I interferon.
[00203] In some embodiments, the host cell is an immune response cell. In some embodiments, 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. In some embodiments, the host cell is an NK92 cell.
[00204] In some embodiments, an expression construct can be included in an immune response cell of the invention, and the elements are linked in sequence as follows: antibody, CD28 co-stimulatory signal domain, Οϋ3ζ, as well as expression unit of type I interferon and NFAT6 inversely linked to the elements mentioned above. Preferably, the antibody and the signal domain
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67/117 CD28 co-stimulators are joined by a CD8a transmembrane region and a CD8a hinge region.
[00205] In some embodiments, NFAT (nuclear factor activated T cells) plays an important role in the transcriptional expression of cytokines during T cell activation. Based on this consideration, the inventors placed the IFN-beta coding sequence under regulation of NFAT6 promoter, so that IFN-beta can be expressed at a high level only when CAR-T cells come into contact with the antigen to induce T cell activation.
[00206] The NFAT6 promoter is a promoter obtained by combining six NFAT binding positions and a minimal IL2 promoter (Hooijberg E, Bakker AQ, Ruizendaal JJ, Spits H. NFAT-controlled expression of GFP permits visualization and Isolation of antigen-stimulated primary human Tcells.Blood 12 July 2000; 96 (2): pages 459 to 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, Yu Z, Zheng Z, Yang S, Restifo NP, Rosenberg SA, Morgan RA. Improving adoptive T cell therapy by targeting and controlling IL-12 expression to the tumor environment. Mol Ther. Apr. 2011; 19 ( 4): pages 751 to 759).
[00207] 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. Conventional nucleic acid transduction methods in the art, which include viral and non-viral transduction methods, can be used in the present invention.
[00208] The immune response cell of the present invention can additionally carry an exogenous cytokine coding sequence, which includes, but is not limited to, IL-12, IL-15 or IL-21 and the like. These cytokines have additional immunomodulatory or antitumor activity, enhance the function of effector T cells and activated NK cells, or directly exert antitumor effects. In this way, a knowledgeable person will observe that the use of these cytokines will help the immune response cells to function better.
[00209] The immune response cell of the present invention can also express an antigen-binding receptor other than the binding receptor
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68/117 to the antigen described above.
[00210] The immune response cells of the invention can also express a chemokine receptor; and chemokine receptors include, but are not limited to, CCR2. A skilled person will observe that the CCR2 chemokine receptor can compete with CCR2 binding in vivo, which is advantageous for blocking tumor metastasis.
[00211] The immune response cells of the present invention can also express siRNA, which can reduce the expression of PD-1 or a protein that can block PD-L1. A knowledgeable person will note that competitive blocking of the interaction of PD-L1 with its PD-1 receptor facilitates the recovery of anti-tumor T cell responses, thereby inhibiting tumor growth.
[00212] The immune response cells of the present invention can also express a safe switch; and, preferably, the secure switch includes: ICaspase-9, EGFR or Truncated RQR8.
[00213] In some embodiments, the immune response cells of the invention do not express a co-stimulatory ligand, such as 4-1BBL [00214] Consequently, in another aspect, a method for producing an antigen binding unit or a chimeric antigen receptor described in this document, or a composition comprising them, is provided herein, which comprises growing a host cell described in this document under suitable conditions. In some embodiments, the method includes isolating and obtaining an expression product from the host cell.
[00215] In another aspect, a composition is provided herein that comprises an antigen binding unit, a chimeric antigen receptor or a nucleic acid described herein. In some embodiments, the composition is a pharmaceutical composition that comprises the antigen binding unit, the chimeric antigen receptor or the nucleic acid. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
[00216] In another aspect, a pharmaceutical composition is provided in this document, which comprises a host cell described in
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69/117 this document and a pharmaceutically acceptable carrier.
[00217] The term pharmaceutically acceptable means that when a molecule and composition are properly administered to an animal or a human, they do not produce adverse, allergic or other unfavorable reactions.
[00218] In some embodiments, the composition comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, such as those described in document No. ^Q US20140271820 and / or a pharmaceutically acceptable salt or its analogue. In some embodiments, the therapeutic agent includes, but is not limited to, a mitotic inhibitor (vinca alkaloid), which includes vincristine, vinblastine, vindesine and novibine (TM) (vinorelbine, 5 'dehydro-hydrogen sulfide); topoisomerase I inhibitors, such as camptothecin compounds, which include Camptosar ™ (irinotecan HCL), Hicanthin ™ (topotecan HCL), and other compounds derived from camptothecin and the like; a podophyllotoxin derivative, such as etoposide, teniposide and midozozo; an alkylating agent cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphamide, carmustine, busulfan, chlorambucil, briquetazine, uracil mustard, cloprophene and dacarbazine; antimetabolites, which include cytarabine, 5-fluorouracil, methotrexate, guanidine, azathioprine and procarbazine; antibiotics, which include, but are not limited to, doxorubicin, bleomycin, dactiomycin, daunorubicin, mycine, mitomycin, sarcoma C and daunorubicin; as well as other chemotherapy drugs, which include, but are not limited to, anti-tumor antibodies, dacarbazine, cytidine, amushakang, melphalan, ifosfamide and mitoxantrone. In some embodiments, the additional therapeutic agent is selected from one or more of epirubicin, oxaliplatin and 5-fluorouracil. In some embodiments, the additional therapeutic agent includes, but is not limited to, an anti-angiogenic agent, which includes anti-VEGF antibodies (which include chimeric and humanized antibodies, anti-VEGF aptamers and antisense oligonucleotides), and other angiogenesis inhibitors, such as angiostatin , endostatin, interferon, interleukin 1 (which includes α and β), interleukin 12, retinoic acid and tissue inhibitors of metalloproteinases-1 and -2, and the like.
[00219] Specific examples of some substances that can
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70/117 be used as pharmaceutically acceptable carriers or its components are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and methyl cellulose; tragacanth gum powder; malt; gelatine; baby powder; 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 Tween®; wetting agents, such as sodium lauryl sulfate dye; flavoring agent; tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; and phosphate buffer.
[00220] The pharmaceutical composition described herein can comprise one or more pharmaceutically acceptable salts. Pharmaceutically acceptable salt refers to a salt that retains the desired biological activities of the parent compound and has no adverse toxicological effect (see, for example, Berge, SM, et al., 1977, J. Pharm. Sci. 66: pages 1 to 19). Examples of such salts include acid addition salts and base addition salts.
[00221] 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, phosphoric acid and the like, and non-toxic organic acid derivatives, such as aliphatic monocarboxylic acids and dicarboxylic acid, a phenyl substituted alkanoic acid, a hydroxyalkanoic acid, an aromatic acid, an aliphatic or aromatic sufonic acid. Base addition salts include salts derived from alkaline earth metals (such as sodium, potassium, magnesium, calcium, etc.), as well as salts derived from non-toxic organic amines, such as Ν, Ν'-dibenzylethylenediamine, N-methylglycosamine, glucosamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.
[00222] The pharmaceutical composition described in this document can also comprise an antioxidant. Examples of antioxidants include, but are not limited to, water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium hydrogen sulfate, metabisulfide
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71/117 sodium, sodium sulfide, etc .; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisol (BHA), butylated hydroxytoluene (BHT), lecithin, propylgalate, alpha-tocopherol, etc .; and metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.
[00223] The composition of the present invention can be formulated in various dosage forms, as needed, and can be administered by a doctor, according to factors such as type of patient, age, body weight, general condition of the disease and method of administration, and the like, in a beneficial dose, to a patient. The mode of administration can be, for example, parenteral administration (for example, injection) or other treatment.
[00224] Parenteral administration of an immunogenic composition includes, for example, subeutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal infusion or injection techniques.
[00225] Formulations comprising a population of immunoreactive cells administered to an individual comprise a plurality of immunoreactive cells effective to treat and / or prevent a particular indication or disease. For this reason, a therapeutically effective population of immunoreactive cells can be administered to an individual. Typically, a formulation comprising from about 1 x 10 ⁴ to about 1 x 10 ¹⁰ immunoreactive cells is administered. In most cases, 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 x 10 ⁷ immunoreactive cells. However, depending on the location, source, identity, extent and severity of a cancer, the age and physical condition of an individual being treated, and the like, the amount of immunoreactive CAR cells administered to the individual will vary. within a wide range. The doctor will finalize an appropriate dose to be used.
[00226] In some embodiments, a chimeric antigen receptor is used to stimulate an immune cell-mediated immune response. For example, a T cell-mediated immune response is an immune response that involves T cell activation. Specific antigen-activated cytotoxic T cells are able to induce apoptosis in target cells that exhibit an exogenous epitope on the surface, such as cancer cells that exhibit
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72/117 tumor antigens. In other embodiments, a chimeric antigen receptor is used to provide anti-tumor immunity in a mammal. Individuals will develop antitumor immunity due to T cell-mediated immune responses.
[00227] In certain examples, a method for treating an individual who has cancer may involve administering one or more immune response cells of the invention to an individual in need of treatment. The immune response cell can bind to a tumor target molecule and induce cancer cell death. As also mentioned above, the invention also provides a method for treating pathogenic infections in an individual, which comprises administering to a subject a therapeutically effective amount of immune response cells of the invention.
[00228] The frequency of administration of the immunoreactive cells of the present invention will depend on factors including the treated disease, 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 per week, once every eight days, once every nine days, once every ten days, once a week, or twice a month. As described herein, the immune response cells of the present application can be administered not only in a therapeutically effective amount, which is less than that of a similar immune response cell, without expressing exogenous type I interferon, but can also be administered at a lower frequency to achieve at least similar therapeutic effects and, preferably, more pronounced, since the immune response cells of the present application had improved viability.
[00229] In some modalities, the compositions can be isotonic, that is, they can have the same osmotic pressure as blood and tears. The desired isotonicity of the compositions of the present invention can be achieved with the use of sodium chloride or other pharmaceutically acceptable agents, such as glucose, boric acid, sodium tartrate, propylene glycol or other organic or inorganic solutes. If desired, the viscosity of the composition can be maintained at a selected level using
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73/117 of a pharmaceutically acceptable thickening agent. Such 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 vehicle and other additives will depend on the exact route of administration and the properties of the particular dosage form, such as a liquid dosage form.
[00230] The invention also provides kits that comprise an antigen binding unit, a chimeric antigen receptor, a nucleic acid or an immune response cell, as described herein. In some embodiments, a kit may include a therapeutic or prophylactic composition that comprises an effective amount of an antigen binding unit, a chimeric antigen receptor, a nucleic acid or an immune response cell described herein in one or more forms dosing unit. In some embodiments, the kit comprises a sterile container that can contain a therapeutic or prophylactic composition; and such a container can be a cartridge, ampoule, bottle, vial, tube, bag, blister pack, or other suitable container form known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil or other materials suitable for retaining the drug. In some embodiments, the kit comprises an antigen binding unit, a chimeric antigen receptor, a nucleic acid or an immune response cell, as described in this document, and an instruction for administering the antigen binding unit, the receptor chimeric antigen, nucleic acid or cellular immune response described herein to an individual. The instruction, in general, includes methods to treat or prevent cancer or tumors using the antigen binding units, the chimeric antigen receptors, nucleic acids or immune response cells described herein. In some embodiments, the kit comprises host cells, as described herein, and can comprise from about 1 x 10 ⁴ cells to about 1 x 10 ⁶ cells. In some embodiments, the kit may comprise at least about 1 x 10 ⁶ cells, at least about 1 x 10 ⁶ cells, at least about 1 x 10 cells, at least about 4 x 10 ⁷ cells, at least about 5 x 10 cells
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74/117 cells, at least about 6 x 10 ⁷ cells, at least about 6 x 10 ⁷ cells, 8 x 10 ⁷ cells, at least about 9 x 10 ⁷ cells, at least about 1 x 10 ⁸ cells at least about 2 x 10 ⁸ 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 cells, at least about 8 x 10 ⁸ cells, at least about 9 x 10 cells, at least about 1 x 10 ⁹ cells, at least about 2 x 10 cells, at least about 3 x 10 ⁹ cells, at least about 4 x 10 cells, at least about 5 x 10 ⁹ cells, at least about 6 x 10 cells, at least about 8 x 10 ⁹ cells, at least about 9 x ¹⁰ cells, at least about 1 x 10 ¹⁰ cells, at least about 2 x 10 cells, at least about 3 x 10 ¹⁰ cells, at least about 4 x ¹⁰ cells, at least about 5 x 10 ¹⁰ cells, at least about 6 x 10 cells, at least at least about 9 x 10 ¹⁰ cells, at least about 9 x 10 ¹⁰ cells, at least about 1 x 10 ¹¹ cells, at least about 2 x 10 ¹¹ cells, at least about 3 x 10 ¹¹ cells, at least about d and 4 x 10 ¹¹ cells, at least about 5 x 10 ¹¹ cells, at least about 8 x 10 ¹¹ cells, at least about 9 x 10 ¹¹ cells or at least about 1 x 10 ¹² cells. For example, approximately 5 x 10 ¹⁰ cells can be included in the kit. In another example, the kit can include 3 x 10 ⁶ cells; and the cells can be expanded to about 5 x 10 ¹⁰ cells and administered to an individual.
[00231] In some embodiments, the kit may include allogeneic cells.
In some embodiments, the kit may include cells that may contain genomic modifications. In some embodiments, the kit may comprise ready-to-use cells. In some embodiments, the kit may include cells that can be expanded for clinical use. In some cases, the kit may contain content for research purposes.
[00232] In some modalities, the instruction includes at least one among: a description of a therapeutic agent; a dosage and administration regimen to treat or prevent a tumor or one of its symptoms; preventive measures, warnings, contraindications, excessive information, adverse reactions, animal pharmacology, clinical research and / or citations. The instruction can be printed directly on the container (if any), or as an identification on the container, or as a separate paper, a brochure, a
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75/117 card or a leaflet in the container. In some embodiments, the instruction provides a method for administering an immune response cell of the invention to treat or prevent a tumor. In some cases, the instruction provides a method for administering an immunoreactive cell of the invention before, after, or simultaneously with the administration of a chemotherapeutic agent.
[00233] In another aspect, a method for inducing the death of a cell comprising 18A2 claudin peptide is provided herein, the method comprising: contacting the cell with an antigen binding unit described herein , a chimeric antigen receptor described herein, compositions described herein, or a host cell described herein. In some modalities, contact is in vitro contact. In some modalities, contact is in vivo contact.
[00234] In some embodiments, 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 tumor cell, as described in this document. Particular examples of such cells may include, but are not limited to, leukemia cells (e.g., acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute granulocytic-monocytic leukemia, acute monocytic leukemia, acute leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma cells (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia disease cells, heavy chain disease cells, solid tumors, such as sarcoma and cancer cells ( such as fibrosarcoma, mucino sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial sarcoma, lymphangiosarcoma, angiosarcoma, lymphatic endothelial sarcoma, synovial vioma, mesothelioma, Ewing's tumor, cancerous tumor, cancer of the sarcomas, sarcoma, cancer , ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cancer, bronchial carcinoma, medullary carcinoma, kidney cell carcinoma, liver cancer, gallbladder cancer, choriocarcinoma, carcinoma, semino embryonic, nephroblastoma, cancer
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76/117 cervical, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwanno , meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer cells, gallbladder cancer cells, kidney cancer cells, multiple myeloma cells, and the like. In some embodiments, 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, an colon cancer cell, a rectal cancer cell, a liver cancer cell, head and neck cancer cells, a chronic myeloid leukemia cell and a gallbladder cancer cell.
[00235] In another aspect, a method for treating a tumor in an individual in need is provided herein, the method comprising administering to the individual an effective amount of an antigen binding unit, an antigen receptor chimeric, composition, vector or host cell described in this document.
[00236] In some embodiments, the tumor includes, but is not limited to, a 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, testicles, team, thyroid, trachea, urethra, ureter, uterus, vaginal organs. In some embodiments, the tumor includes, but is not limited to, leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute granulocytic-monocytic leukemia, acute monocytic leukemia, acute leukemia, chronic leukemia, myeloid leukemia chronic, 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, mucino sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial sarcoma, lymphangiosarcoma, angiosarcoma, lymphatic endothelial sarcoma, synovial vioma,
Petition 870190015344, of 02/14/2019, p. 82/151 / 117 mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary cancer, papillary adenocarcinoma, cancer, bronchial carcinoma, medullary carcinoma, renal cell carcinoma, liver cancer, bile tubule cancer, choriocarcinoma, seminoma, embryonic carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular cancer , lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannomas, meningiomas, melanoma, neuroblastomas, neuroblastomas, neuroblastomas esophageal cancer, gallbladder cancer, kidney cancer, multiple myeloma. In some embodiments, 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 cancer of gallbladder.
[00237] In some embodiments, the immunoreactive cells can be administered to an individual, wherein the immunoreactive cells that can be administered can be from about 1 to about 35 days old. For example, the cells administered may have 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 even about 40 days old. The age of CAR immunoreactive cells can be calculated from the stimulus time. The age of the immunoreactive cells can be calculated from the time of blood collection. The age of immunoreactive cells can be calculated from the time of transduction. In some embodiments, the immunoreactive cells that can be administered to an individual are about 10 to about 14 or about 20 days old. In some embodiments, the age of an immunoreactive cell can be determined by the length of telomeres. For example, a young immune response cell may have a longer telomere than that of depleted or old immunoreactive cells. ” Without being bound by a particular theory, it is believed that immunoreactive cells lose an estimated telomere length of about 0.8 kb per week
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78/117 in the culture, and a young immunoreactive cell culture may have a longer telomere than an immunoreactive cell of about 44 days, of about 1.4 kb. Without being bound by a particular theory, it is believed that a longer telomere length may be associated with a positive objective clinical response in a patient and with the persistence of cells in vivo.
[00238] The cells (for example, projected cells or projected primary T cells) can be functional, before, after and / or during transplantation. For example, transplanted cells can function at least 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, 6, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90 or 100 days after the transplant. 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. In some embodiments, the transplanted cells may function during the patient's life.
[00239] In addition, transplanted cells can function at 100% of the function normally expected. The transplanted cells can also exert 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 even fence
100% of its normally expected function.
[00240] The transplanted cells can also perform more than 100% of their normally expected function. For example, transplanted cells can exert about 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000 or even about 5,000% of its normally expected function.
[00241] The transplant can be any type of transplant. The position of the site may include, but is not limited to, subhepatic cavity space, subsplenic cavity space, subcapsular space, intestinal or gastric submucosa, vascular segment of the small intestine, venous cavity, testicles, brain, spleen or cornea. For example, the transplant can be a subcapsular transplant. The transplant can also be a transplant
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79/117 intramuscular. The transplant can be a portal venal transplant.
[00242] Transplant rejection can be improved after treatment with the immune response cells of the present invention, as compared to the situation when one or more wild-type cells are transplanted into a recipient. For example, transplant rejection can be a hyperacute rejection. Transplant rejection can also be an acute rejection. Other types of rejection can include chronic rejection. Transplant rejection can also be cell-mediated rejection or T-cell mediated rejection. Transplant rejection can also be natural killer cell-mediated rejection.
[00243] The improvement in transplantation may refer to the relief of hyperacute rejection, which may include reduction, relief or decrease of adverse effects or symptoms. The transplant can refer to the adoptive transplant of cellular products.
[00244] Another sign of successful transplantation may be the number of days for which the recipient does not need immunosuppressive therapy. For example, after providing the immune response cells of the invention, 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 successful transplantation. This may also indicate that the transplanted cells, tissues and / or organs are not rejected.
[00245] In some cases, the recipient does not require immunosuppressive therapy for at least 1 day. The recipient may not require immunosuppressive therapy 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. In addition, the recipient may not require immunosuppressive therapy for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.
[00246] Another sign of successful transplants may be the reduced number of days for which a recipient needs immunosuppressive therapy. For example, after the treatment provided in this document, the recipient may require reduced immunosuppressive therapy for
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80/117 at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days. This can indicate successful transplantation. This may also indicate that there is neither rejection of the transplanted cells, tissues and / or organs, or there is only minimal rejection.
[00247] For example, a recipient may require reduced immunosuppressive therapy for at least 1 day. The recipient may also require reduced immunosuppressive therapy for at least 7 days. The recipient may require reduced immunosuppressive therapy for at least 14 days. The recipient requires reduced immunosuppressive therapy for at least 21 days. The recipient requires reduced immunosuppressive therapy for at least 28 days. The recipient requires reduced immunosuppressive therapy for at least 60 days. In addition, the recipient may require reduced immunosuppressive therapy for at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more years.
[00248] A reduced immunosuppressive therapy may refer to less demand on immunosuppressive therapy, as compared to the situation when one or more wild-type cells are transplanted into a recipient.
[00249] Immunosuppressive therapy can include any treatment that inhibits the immune system. Immunosuppressive therapy can facilitate the relief, reduction or elimination of transplant rejection in patients. For example, immunosuppressants can be used before, during and / or after transplantation, which includes MMF (Mofetil mycophenolate, from Cellcept), ATG (anti-thymocyte globulin), anti-CD154 (CD4OL), anti-CD40 (2C10), immunosuppressive drugs , anti-IL-6R antibodies (tocilizumab, Actemra), anti-IL-6 antibodies (sarilumab, oloquizumab), CTLA4-lg (Abatacept / Orencia), anti-IL-6 antibodies (ASKP1240, CCFZ533X2201)), amphetamine (Campath ), anti-CD20 (rituximab), bevacizumab (LEA29Y), sirolimus (Rapimune), everolimus, tacrolimus (Prograf), Zemegabe, Zemilect, Remicade, cyclosporine, deoxygenin, soluble complement receptor 1, venom snake, anti-C5 antibody / Soliris), methylprednisolone, FTY720, everolimus, leflunomide, anti-IL-2R-Ab, rapamycin, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody and anti-CD122 antibody. In addition, one or more immunosuppressive agents / drugs can be used together or in combination
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81/117 sequence. One or more immunosuppressive agents / drugs can be used to induce therapy or to maintain therapy. The same or different drugs can be used in the induction and maintenance phases. In some cases, daclizumab (Zenapax) can be used for induction therapy, and Tacrolimus (Prograf) and Sirolimus (Rapimune) can be used to maintain treatment. Non-pharmacological regimens can also be used to achieve immunosuppression, which includes, but is not limited to, whole-body irradiation, thymic irradiation and total and / or partial splenectomy. These techniques can also be used in combination with one or more immunosuppressive drugs.
[00250] In some embodiments, an antigen binding unit, a chimeric antigen receptor, a composition, a vector or a host cell described herein can be administered in combination with another therapeutic agent. In some embodiments, the additional therapeutic agent is a chemotherapeutic agent, such as those described in document n ^Q US20140271820. Chemotherapeutic agents that can be used in combination with the immune response cells of the invention include, but are not limited to, mitotic inhibitors (vinca alkaloids), which include vincristine, vinblastine, vindesine and novibine (TM) (vinorelbine), 5-sulfide '-hydro-hydrogen); topoisomerase I inhibitors, such as camptothecin compounds, which include Camptosar ™ (Irinotecan HCL), Hicatin ™ (topotecan HCL), and other compounds derived from camptothecin and their analogs; podophyllotoxin derivatives, such as etoposide, teniposide and midozozo; cisplatin of alkylating agents, cyclophosphamide, nitrogen mustard, trimethylene thiophosphamide, nitrogen mustard, busulfan, chlorambucil, briquetazine, uracil mustard, cloprophene and dacarbazine; antimetabolites, which include cytarabine, 5-fluorouracil, methotrexate, anthraquinone, azathioprine and procarbazine; antibiotics that include, but are not limited to, doxorubicin, bleomycin, dactiomycin, daunorubicin, mycine, mitomycin, sarcoma C and daunorubicin; and other chemotherapeutic drugs, which include, but are not limited to, anti-tumor antibodies, dacarbazine, cytidine, amushakang, melphalan, ifosfamide and mitoxantrone. In some embodiments, the additional therapeutic agent is selected from one or more of epirubicin, oxaliplatin and 5-fluorouracil.
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82/117 [00251] In some embodiments, chemotherapeutic agents that can be used in combination with the immune response cells of the invention that include, but are not limited to, an anti-angiogenic agent, which include anti-VEGF antibodies (which include chimeric antibodies and humanized, anti-VEGF aptamers and antisense oligonucleotides), and other angiogenesis inhibitors, such as angiostatin, endostatin, interferon, interleukin 1 (including α and β), interleukin 12, retinoic acid and metalloproteinase-1 and - tissue inhibitors 2, and the like.
EXAMPLE [00252] The invention is further illustrated below in conjunction with specific embodiments. It should be understood that the examples are intended to demonstrate the invention, while they are not intended to limit the scope of the invention. Experimental methods, in the examples below, where specific conditions are not specified, are usually prepared according to conventional conditions, such as the conditions described in J. Sambrook et al., Molecular Cloning Experimental Guide, Third Edition, Science Press , 2002, or according to the conditions suggested by the manufacturer.
[00253] In the following examples of the invention, when the antigen-binding receptor or CAR is constructed, the co-stimulatory signal domain of CD28 is abbreviated to 28; CD3ζ is abbreviated as Z; 4-1 BB or CD137 is abbreviated as BB. For example, a chimeric antigen receptor constructed by an scFv with a code of 8E5-2I and CD3ζ as well as co-stimulating signal domains CD28 as an intracellular signal domain, can be termed as 8E5-2I-28Z. CARs for different antigens are constructed as such.
EXAMPLE 1. PRODUCTION AND CHARACTERIZATION OF MOUSE ANTIBODIES AGAINST CLD18A2 [00254] The antibody fragments were obtained using standard biological protocols. Briefly, 8-week-old Balb / c mice were immunized with a eukaryotic expression vector that contains the entire human CLD18A2 sequence (NCBI reference sequence: NM_001002026.2). The spleen of the immunized mouse was removed, and a monoclonal antibody was obtained using a biological scheme
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83/117 conventional in the art.
[00255] The individual's cells were selected for monoclonal anti-CLD18A2 antibody by flow cytometry, and HEK293 cells (HEK-CLD18A2) that stably express human CLD18A2 were used for primary selection by flow cytometry, using an Instrument HT Guava easyCyte ™ System. The binding of the antibody to the human CLD18A1 and CLD18A2 transformers was then compared by flow cytometry. The instrument used was the HT Guava easyCyte ™ System.
[00256] After multiple rounds of hybridoma preparation and selection, the inventors found several antibodies with relatively ideal binding properties. Figure 1B shows an example of the binding of hybridoma supernatants 2B1, 3E12, 4A11, 8E5 to HEK293 stably transfected with human CLD18A2 or CLD18A1, as determined by flow cytometry. As shown in Figure 1B, after two rounds of subcloning, most subclones of antibodies 2B1,3E12, 4A11 and 8E5 specifically bound to human CLD18A2, but not human CLD18A1, and mean fluorescence intensities differed by more than 5 times.
[00257] The hybridoma cell strain that secretes monoclonal antibody was cultured, and the total RNA was extracted from the cell pellet, according to the instructions of the TRIzol® Plus RNA purification kit (Invitrogen, 12183-555). The cDNA was reverse transcribed using total RNA as a template, according to the RNA kit instructions for high capacity cDNA (Invitrogen, 4387406). The cDNA was used as a template, and the 5'-FuII RACE kit (TAKARA, D315) and primers from the antibody constant region were used for amplification. The PCR products were separated into 1.5% agarose gel, and the DNA fragments were purified and recovered. The sequencing results were as follows:
TABLE 5. SEQUENCING RESULTS
Sequence ofamino acids Nucleotide sequence 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
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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
[00258] The antibody sequences have been aligned, and the results are shown in Figure 2.
EXAMPLE 2. CONSTRUCTION OF SCFV_FC FUSION ANTIBODY OF ANTICLAUDIN 18A2 AND ITS TRANSIENT EXPRESSION IN EUCARIOTIC CELLS [00259] For fragments of VH and VL of 2B1, 8E5, a flexible amino acid GGGGSGGGGSGGGS was used as an ID in the following: to constitute scFv; an appropriate restriction site and protective bases were introduced upstream into the VH, and an appropriate restriction site and protective bases were introduced downstream into the VL; and digested and ligated into a eukaryotic expression vector (see vector pH or vector pK used in document No. ⁵ CN101602808). The 293fectin ™ transfection reagent (Invitrogen, 12347-019) was used for transient transfection, the supernatant was collected and subjected to affinity purification, and the antibody obtained was analyzed qualitatively and quantitatively by SDS PAGE.
[00260] The binding of 18A2 scFv_Fc anticlaudin fusion antibody to HEK293 cells stably transfected with CLD18A2 was determined by flow cytometry. The experimental data were analyzed using the HT GraphPad Prism and Guava easyCyte ™ System Instrument, as described in Example 1, to obtain the EC50 value. Figure 3 shows the relative binding affinity of scFvs of monoclonal antibody 2B1, 8E5, after fusing to the human IgG1 Fc portion, 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.
EXAMPLE 3. PREPARATION OF ANTICLAUDIN 18A2 ANTIBODY VARIABLES [00261] Antibody 2B1 was subjected to site-directed mutagenesis by means of the PCR bridge. The mutations were introduced at position 52 or
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85/117 (N-glycosylation site) on antibody 2B1 heavy chain to prepare two 2B1 mutants 2B1-N52D (VH amino acid sequence: SEQ ID NO: 17; nucleotide sequence: SEQ ID NO: 18) and 2B1- S54A (VH amino acid sequence: SEQ ID NO: 19; nucleotide sequence: SEQ ID NO: 20).
[00262] The amino acid sequences and nucleotide sequences of the 2B1-N52D and 2B1-S54A light chains are identical to the corresponding 2B1 sequences.
[00263] The expression vectors of the ScFv_Fc form of the two mutants were constructed as described in Example 2 and according to the procedure of Example 2, and the experimental data were analyzed using the HT Graph Pad Prism and Guava easyCyte ™ System Instrument , to obtain EC50.
[00264] Figure 4 shows the relative binding affinity of 2B1-N52D and 2B1-S54A, after fusing to the human IgG1 Fc portion, to HEK293 cells stably transfected with human CLD18A2. It can be seen that the EC50 value of 2B1-N52D is 6.11 nM, and the EC50 value of 2B1-S54A is 3.85 nM.
EXAMPLE 4. PREPARATION OF 2B1-S54A HUMANIZED ANTIBODY [00265] The sequences of 6 antibody light and heavy chain CDRs were determined according to the Kabat, Chothia and IMGT Naming schemes. Using sequence similarity alignment, the antibody sequence with the highest similarity for 2B1-S54A was selected as the antibody model. In this example, IGHV1-46 * 01 in the IMGT database was selected as an antibody model for hu2B1-S54A heavy chain. IGKV4-1 * 01 was used as an antibody model for hu2B1-S54A light chain. The light and heavy chain CDR regions of 2B1-S54A were replaced with CDR regions of the antibody model.
[00266] Determination of reverse mutation sites: (1) Align a projected 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 loop structure of the antibody, or amino acids that affect the binding of variable regions
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86/117 of the light and heavy chains, and if so, these regions are relatively conserved regions. (3) Check for any potential post-translational modification sites in humanized antibodies, such as deamidation sites (Asn-Gly), isomerization sites (Asp-Gly), methionine exposed to the surface, N-glycosylation site (Asn -X-Ser / Thr, X is not proline). (4) There are six potential reverse mutation sites in the humanized antibody heavy chain (hu2B1-S54A), namely, M48I, V68A, M70L, R72A, T74K, T91S, respectively. There is a potential reverse mutation site on the humanized antibody light chain (hu2B1-S54A), L84V.
[00267] Expression and purification of humanized antibodies: (1) A nucleotide sequence has been designed and synthesized based on the humanized antibody amino acid sequence (hu2B1-S54A). A sequence of light chain nucleotides (SEQ ID NO: 62) was synthesized; and a sequence of heavy chain nucleotides (SEQ ID NO: 60) was synthesized. (2) The synthetic antibody nucleotide sequence, which includes the signal peptide, the antibody variable region and the constant region, is inserted into a mammalian cell expression vector to construct antibody expression vectors that contain the chain heavy and light chain, respectively, sequenced and identified.
[00268] The hu2B1 -S54A heavy chain amino acid sequence is shown in SEQ ID NO: 59; and the nucleotide sequence is shown in SEQ ID NO: 60. The amino acid sequence of light chain hu2B1-S54A is shown in SEQ ID NO: 61; and the nucleotide sequence is shown in SEQ ID NO: 62. The amino acid sequence of the hu2B1 -S54A heavy chain variable region is shown in SEQ ID NO: 23, the nucleotide sequence is shown in SEQ ID NO: 24; and the light chain variable region amino acid sequence is shown in SEQ ID NO: 21, and the nucleoside sequence is shown from SEQ ID NO: 22.
[00269] 293F cells were transiently transfected by 293Fectin, and the HCDR of hu2B1-S54A and the same LCDR sequence were expressed.
[00270] The binding activity assay was performed as described in Example 2, and the experimental data were analyzed using HEK293 cells (HEK-CLD18A2) which stably express human CLD18A2,
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HT System Instrument GraphPad Prism and Guava easyCyte ™ to obtain EC50, and the results are shown in Figure 5. The relative binding affinity value of the hF2B1-S54A scFv EC50, after fusing to the Fc portion of human lgG1, the HEK293 cells stably transfected with human CLD18A2 was 18.59 nM, which indicates that hu2B1 -S54A also exhibited good binding to HEK-CLD18A2.
EXAMPLE 5. PREPARATION AND OPTIMIZATION OF HUMANIZED MONOCLONAL ANTIBODY 8E5 [00271] After the procedure of Example 4, 8E5 was humanized, while the N-glycosylation site, in monoclonal antibody 8E5, was removed by point mutation of S62A, to obtain humanized antibody hu8E5 (or hu8E5-S62A). The specific method is described as follows:
(1) IGHV4-30 * 03 was selected as the 8E5 heavy chain antibody model, and IGKV4-1 * 01 was selected as the 8E5 light chain antibody model. The CDR of 8E5 antibody light chain or heavy chain regions are replaced by the CDR regions of the antibody model.
(2) Reverse mutation sites are determined, there are six potential reverse mutation sites in the humanized antibody heavy chain (hu8E5), namely, G27Y, G45K, L46M, I49M, V68I, V72R, A97T, respectively. There is a potential reverse mutation site on the humanized antibody light chain (hu8E5), L84V.
(3) The nucleotide sequences were designed, the light chain nucleotide sequence was synthesized, and a heavy chain nucleotide sequence was synthesized, based on the humanized antibody amino acid sequence hu8E5.
[00272] The hu8E5-S62A heavy chain amino acid sequence is shown in SEQ ID NO: 67; and the nucleotide sequence is shown in SEQ ID NO: 68. The hu8E5-S62A light chain amino acid sequence is shown in SEQ ID NO: 65; and the nucleotide sequence is shown in SEQ ID NO: 66. The hu8E5-S62A heavy chain variable region amino acid sequence is shown in SEQ ID NO: 27, the nucleotide sequence is shown in SEQ ID NO: 28; the hu8E5-S62A light chain variable region amino acid sequence is shown in SEQ ID NO: 25, the nucleotide sequence is shown in SEQ ID NO: 26; HCDR2 in
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88/117 hu8E5-S62A is different from that in 8E5, the sequence of which is shown in SEQ ID NO: 85; and the other HCDRs and LCDR are the same as 8E5.
[00273] (4) The synthetic antibody nucleotide sequence that includes 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 a heavy chain and a light chain, respectively, sequenced and identified. 293F cells were transiently transfected by 293Fectin and expressed.
[00274] (5) Binding activity was tested, and experimental data were analyzed using human HEK293 (HEK-CLD18A2) CLD18A2 cells that are stably expressed, HT GraphPad Prism System Instrument and Guava easyCyte ™, and the The results are shown in Figure 6. It is shown that the relative binding affinity of the hu8E5 scFv, after fusing to the human IgG1 Fc portion, to HEK293 cells stably transfected with human CLD18A2 was 107 nM.
[00275] The 3D model of hu8E5 was stabilized by Discovery studio, and the sites of potential aggregation were analyzed. It has been found that 12 ^to 93 and ^the heavy chain of valine tend to cause aggregation of antibodies, which in turn affects the stability of the antibody. By analyzing the point mutation, it was found that when the two sites mutated to I, the antibody was more stable. The results of the molecular sieve showed that, after these two sites mutated to I (hu8E5-2l), the proportion of the monomeric form in the scFv_Fc fusion antibody increased from the initial 74% (hu8E5) to 87% (hu8E5-2l).
[00276] The hu8E5-2l heavy chain amino acid sequence is shown in SEQ ID NO: 63; and the nucleotide sequence is shown in SEQ ID NO: 64. The hu8E5-2l light chain amino acid sequence is shown in SEQ ID NO: 65; and the nucleotide sequence is shown in SEQ ID NO: 66. HCDR2 in hu8E5-2l is different from that in 8E5, but it is identical to that in hu8E5, and its sequence is shown in SEQ ID NO: 85; other HCDRs and LCDRs are the same as 8E5.
[00277] The humanized antibody hu8E5-2l mutant hu8E5 was constructed as described in Example 3. The experimental data were analyzed using HEK293 cells (HEK-CLD18A2) that express
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89/117 stably human CLD18A2, HT System Instrument GraphPad Prism and Guava easyCyte ™, and the results are shown in Figure 7. The relative binding affinity, EC50 value of the hF8E5-2l scFv, after fused to the Fc portion of human IgG1, to the HEK293 cell stably transfected with human CLD18A2, was 9.22 nM. Compared to the antibody of origin 8E5, the affinity of mutant hu8E5-2l increased 5-fold.
EXAMPLE 6. FUNCTIONAL TEST IN VITRO AND FUNCTIONAL TEST IN VIVO OF HUMANIZED ANTIBODY HU2B1-S54A AND HUMANIZED ANTIBODY HU8E5-2I [00278] The humanized antibody hu2B1-S54A (light chain sequence: SEQ ID NO: 62, heavy chain sequence SEQ ID NO: 60); humanized antibody hu8E5-2l (light chain sequence: SEQ ID NO: 66, heavy chain sequence: SEQ ID NO: 64) were cloned into a eukaryotic expression vector by standard methods known to a skilled person. The 293F cells, in the logarithmic growth phase, were transiently transfected with the 293fectin ™ transfection reagent (Invitrogen, 12347-019), and the culture supernatant was collected and subjected to affinity purification. The antibodies obtained were analyzed qualitatively and quantitatively by SDS PAGE. The eukaryotic expression vector above uses the vector pH or vector pK used in document n ^Q CN101602808B.
1. Complement Dependent Cytotoxicity (CDC) [00279] Blood was collected from healthy volunteers and serum was prepared by centrifugation. A CCK-8 cell proliferation toxicity assay kit (Dojindo, # CK04) was used. HEK293 cells stably transfected with CLD18A2 or CLD18A1 were used as target cells. The cells were washed twice, resuspended in complete medium at a density of 1 x 10 ⁵ cells / ml, seeded in a 96-well culture plate, at 100 µl per well, and grown overnight at 37 ° C. The next day, the antibody was added to each well, at a final concentration of 20 pg / ml, and incubated for 30 min., At 37 ° C, in an incubator. Then, the serum, in a final concentration of 10%, was added and incubated at 37 ° C, for 1.5 hours. 10 ul of CCK-8 solution was added to each well, incubated at 37 ° C, for 3.5 h (adjusted, as appropriate), and the absorbance at 450 nm was measured with a microplate reader. The experiment was divided into six groups, and the
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90/117 duplicate wells were adjusted, as shown in the following Table.
TABLE 6. GROUPING
Experimental materials Experiment well Lysis well Antibody Control well Complet ent Control well Cell to Control Well Blank sample cell cell Sample well in white 0 CLD18A2 cell 1 * 10 ⁶ / ml 100 ul 100 ul 100 ul 100 ul 100 ul 0 0 DMEM -50 ul 0 -50 ul -25 ul 0 50 ul 100 ul Antibody Ch-163E1 2 60 ug / ml 25 ul Lisa from 10 ul 25 ul 0 0 25 ul 0 Complete (serum) 40% 25 ul 25 ul 25 ul 00 Completely inactive 25 ulSolutionCCK-8 10 ul 10 ul 10 ul 10 ul 10 ul 10 ul 10 ul
The percentage of lysis is calculated as follows:
Percentage of lysis = (cell control well - experiment well) / cell control well - (cell control well - antibody control well) / cell control well.
[00280] Figure 8A compares the CDC effects of the humanized antibodies hu2B1-S54A, hu8E5-2l and the chimeric antibody ch-163E12 on HEK293 cells transfected with CLD18A2. The results of the experiment showed that when the concentration of hu2B1-S54A and hu8E5-2l was 20pg / ml, the effects of CDC against HEK293-CLD18A2 were 79.88% and 82.65%, respectively, and the effects of CDC of ch-163E12, under the same conditions
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91/117 reaction, was less than 55%. Figure 8B compares the CDC results of the humanized antibodies hu2B1-S54A, hu8E5-2l and the chimeric antibody ch-163E12 in HEK293 cells transfected with CLD18A1. The results showed that ch-163E12 also had some destruction in cells that express 18A1, and hu2B1-S54A and hu8E5-2l did not kill cells that express 18A1.
[00281] 2. Antibody-dependent cytotoxicity activity (ADCC) [00282] The ADCC activity of the humanized antibody, the claudin 18A2 antibody, was measured by a lactic dehydrogenase (LDH) release assay, using a kit cytoTox 96 non-radioactive cytotoxicity assay test (Promega, Madison, USA). Human peripheral blood mononuclear cells (PBMC) were purified from whole blood with citrate by standard separation with Ficoll-paque and resuspended in complete medium (RPMI-1640, Gibco culture) supplemented with 10% fetal bovine serum (FBS , Gibco) at a density of 8 x 10 ⁶ cells / ml. HEK293 cells stably transfected with CLD18A2 were used as target cells. The cells were washed twice and resuspended in a complete culture at a density of 2 x 10 ⁵ cells / ml. PBMCs were incubated with an antibody at a final concentration of 20 µg / ml for 30 minutes, at 37 ° C, and then 50 μΙ of antibody and effector cells were added in 50 μΙ of target cells in an effector to 50: 1.20: 1, 10: 1 target (total 1 χ 10 ⁴ of target cells). After incubated for 4 hours at 37 ° C, the cells were centrifuged, and 50 μΙ of cell free supernatant sample was collected, transferred to a flat bottom 96-well plate and the assay was performed. The percentage of lysis was calculated as follows: (sample release spontaneous release of target - spontaneous release of effector cells) / (maximum release - spontaneous release of target) * 100; where spontaneous release of target is fluorescence in wells that contain only target cells, spontaneous release of effector cells is fluorescence in wells that contain only effector cells, and maximum release is fluorescence in wells that contain only target cells that were treated with lysis buffer.
[00283] Figure 9 compares the ADCC effects of the humanized antibodies hu2B1-S54A, hu8E5-2l and the known chimeric antibodies
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92/117 ch-163E12, ch-175D10 (see document ^Q CN103509110A). The experimental results showed that the humanized antibodies hu2B1 -S54A and hu8E5-2l, at an antibody concentration of 20 pg / ml and an effector-to-target ratio of 50: 1.20: 1 and 10: 1, significantly exhibited ADCC effects. greater than ch-175D10 and ch-163E12, and the effects of ADCC against HEK293-CLD18A2 at an effector to target ratio of 50: 1 were 62.84% and 72.88%, respectively. Although the ADCC effects of ch-163E12 and ch-175D10 against HEK293-CLD18A2, under the same reaction conditions, were only 33.39% and 43.74%. The antibody of the present invention significantly exhibits better deadly effects than ch-163E12 and ch-175D10.
[00284] 3. In vivo experiment in mice [00285] Establishment of a PDX model of gastric cancer: Tumors with size of about 3 mm x 3 mm x 3 mm were inoculated subcutaneously in the right ankle of BALB / c hairless mice . The tumor cell inoculation day was recorded as day D0, the tumor volume was measured at D27 from the tumor inoculation, and the mice were randomly divided into 5 groups. The specific groups are as follows: (1) PBS control group (phosphate buffer); (2) hu8E5-2l 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) hu8E5-2l antibody (40 mg / kg) + EOF treatment group (1.25 mg / kg epirubicin + 3.25 mg / kg oxaliplatin + 56.25 mg / kg 5-fluorouracil); (5) ch175D10 antibody (40 mg / kg) + EOF treatment group (1.25 mg / kg epirubicin + 3.25 mg / kg oxaliplatin + 56.25 mg / kg 5-fluorouracil). Dosage: EOF was administered once a week, for 2 weeks; hu8E5-2l and ch175D10 antibodies were administered 3 times a week for 2 weeks.
[00286] The results are shown in Figure 10. The tumor was inoculated for D56 days, the EOF was injected twice, the antibody was injected 6 times, and the mice were sacrificed by cervical dislocation. Compared to the PBS control group, tumor inhibition rates were: 21.13% in the hu8E5-2l monoclonal antibody group, 47.89% in the EOF + PBS treatment group, 81.69% in the EOF treatment group + hu8E5-2l mAb, and 71.83% in the EOF treatment group + 175D10 mAb, respectively. Starting
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93/117 of tumor weighting, the EOF + mAb hu8E5-2l treatment group was statistically different from the EOF + PBS group, P = 0.033; while the treatment group EOF + mAb 175D10 was not statistically different from the group EOF + PBS, P = 0.097.
EXAMPLE 7. CONSTRUCTION OF HUMANIZED ANTIBODY CHEMICAL ANTIGEN RECEPTOR PLASMIDE (CAR PLASMIDE)
1. CONSTRUCTION OF HUMANIZED ANTIBODY HU8E5 CHEMICAL ANTIGEN RECEPTOR PLASMIDE [00287] Using PRRLSIN-cPPT.EF-1a as a vector, lentiviral plasmids encoding the second and third generation of chimeric human antibody antigen receptors hu8E5 were built, which include PRRLSIN-cPPT.EF-1a-hu8E5-28Z, PRRLSIN-cPPT.EF-1a-hu8E5-BBZ and PRRLSIN-cPPT.EF-1a-hu8E5-28BBZ.
[00288] The Hu8E5-28Z mainly includes (from 5 'to 3' final): CD8a signal peptide coding sequence (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 the intracellular signaling domain (SEQ ID NO: 76) as well as an intracellular segment ξ3ξ of CD3 (SEQ ID NO: 78).
[00289] hu8E5-BBZ mainly includes (from 5 'to 3' final): CD8a signal peptide coding sequence (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), CD8 transmembrane region (SEQ ID NO: 80), CD137 intracellular signaling domain (SEQ ID NO: 82) and Ξ3ξ (SEQ ID NO: 78).
[00290] The hu8E5-28BBZ mainly includes (from 5 'to 3' final): CD8a signal peptide coding sequence (SEQ ID NO: 70), hu8E5-scFV, CD8 hinge (SEQ ID NO: 72), transmembrane region of CD28 (SEQ ID NO: 74) and intracellular segment (SEQ ID NO: 76), intracellular signaling domain CD137 (SEQ ID NO: 82) and ΟΟ3ξ (SEQ ID NO: 78).
2. CONSTRUCTION OF HU8E5-2l CHEMICAL ANTIGEN RECEPTOR PLASMIDE FROM HUMANIZED ANTIBODY [00291] Using PRRLSIN-cPPT.EF-1a as a vector, plasmid
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94/117 lentiviral PRRLSIN-cPPT.EF-1a-hu8E5-2l-28Z encoding the second generation of hu8E5 humanized antibody chimeric antigen receptors was constructed.
[00292] The hu8E5-2l-28Z mainly includes (from 5 'to 3' final): CD8a signal peptide coding sequence (SEQ ID NO: 70), hu8E5-2l scFV (VH: SEQ ID NO: 29 , VL: SEQ ID NO: 25, Linker: SEQ ID NO: 93), CD8 hinge (SEQ ID NO: 72), CD28 transmembrane region (SEQ ID NO: 74), and the intracellular signaling domain (SEQ ID NO: 76) and the 33ξ CD3 intracellular signaling domain (SEQ ID NO: 78).
3. HU2B1-S54A CHEMICAL ANTIGEN RECEPTOR PLASMID CONSTRUCTION OF HUMANIZED ANTIBODY [00293] Using PRRLSIN-cPPT.EF-1a as a vector, lentiviral plasmid PRRLSIN-cPPT.EF-1a-huABZ 281 encodes the second generation of humanized antibody chimeric antigen receptors hu2B1-S54A has been constructed.
[00294] The hu2B1-S54A-28Z mainly includes (from 5 'end to 3' end): CD8a signal peptide coding sequence (SEQ ID NO: 70), hu2B1-S54A scFV (VH: SEQ ID NO: 23; VL: SEQ ID NO: 21; Connector: SEQ ID NO: 93), CD8 hinge (SEQ ID NO: 72), CD28 transmembrane region (SEQ ID NO: 74) and intracellular segment (SEQ ID NO: 76) ), and CD3 intracellular CD3Z (SEQ ID NO: 78).
EXAMPLE 8 DETERMINATION OF DEGREE AND COMPRESSION LENTIVIRAL [00295] 293T cells cultured for 6 ^to 10 for ^the passage were seeded in a density 5x10 ⁶ virus in a 10 cm culture plate and cultured overnight at 37 ° C, 5% CO2 for transfection, and the medium was DMEM, which contains 10% fetal bovine serum (Gibico).
[00296] The target gene plasmid PRRLSIN-cPPT.EF-1a-EGFP (Simulated) and the different CAR plasmids prepared in Example 9 (5.4 pg) and the pRsv-REV compaction plasmid (6.2 pg) , RRE-PMDLg (6.2 pg) and Vsvg (2.4 pg) were dissolved in 800 pL of blank sample DMEM medium; 60 pg of PEI (1 pg / pl) was dissolved in 800 pl of serum-free DMEM medium and mixed for 5 min. at room temperature.
[00297] The plasmid mixture was added to the PEI mixture, and
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95/117 mixed for 20 min. at room temperature to form a transfection complex; 1.6 ml of the transfection complex was added in drops and a 10 cm culture plate containing 11 ml of DMEM medium; after 4 to 5 hours, 10% FBS DMEM medium was used to change the medium for the transfected 293T cells, and incubated at 37 ° C for 72 h. The virus supernatant was collected and concentrated, and the titration was determined. The number of cells with a positive rate of 5-20% was preferred, and the titration (U / mL) was calculated as = cell number x positive rate / virus volume.
[00298] By concentration, virus titrations were:
[00299] hu8E5-28Z: 2.3x10 ⁷ U / ml;
[00300] hu8E5-BBZ: 6.65x10 ⁷ U / ml;
[00301] hu8E5-28BBZ: 6.67x10 ⁷ U / ml;
[00302] hu8E5-2l-28Z: 1.54x10 ⁸ U / ml;
[00303] hu2B1-S54A-28Z: 1.14x10 ⁸ U / ml.
EXAMPLE 9. TEST OF C-TOTOXICITY OF CAR-T CELLS AND T-LYMPHOCYTES TRANSDUCTED IN LENTIVIRAL
1. LENTIVIRUS-INFECTED T-LYMPHOCYTES [00304] (1) The lymphocyte culture medium was added at a density of about 1x10 ⁶ / ml for culture, and magnetic microspheres (Invitrogen) coated with anti-CD3 and CD28 and IL antibodies -2 recombinant human with a final concentration of 300 U / mL were added according to a magnetic microsphere: 1: 1 cell ratio for stimulation and culture for 48 h;
[00305] (2) 24-well plates coated with retronectin: 380 pl of 5 pg / ml retronectin solution (PBS) were added to each well and, after incubation at 4 ° C overnight, the retronectin solution ( PBS), in a 24-well plate, was discharged and washed twice with 1 ml of PBS;
[00306] (3) The cells were seeded in a 24-well plate coated with retronectin. The number of cells per well was 3χ10 ⁵ , and the volume of the culture solution was 600 μΙ. The concentrated lentivirus was added to the PBMC cells at MOI = 10, centrifuged at 32 ° C for 40 min. and transferred to a cell culture incubator;
[00307] (4) Expansion culture: The infected cells passed every other day at a density of 5 χ 10 ⁵ / mL, and the recombinant human IL-2
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96/117 was supplemented in the lymphocyte culture solution at a final concentration of 300 U / mL.
2. EXPRESSION OF T-LYMPHOCYTE CHEMICAL ANTIGEN RECEPTOR [308] (1) On the 7th day of culture of lentivirus-infected T lymphocytes, 1 x 10 ⁶ T cells were obtained, aliquoted in a 2 ml centrifuge tube, centrifuged at 4 ° C, 5,000 rpm, for 5 min., the supernatant was discarded, and the PBS was washed twice.
[309] (2) To the control cells, 50 µl of PE-SA antibody (1: 200 dilution) was added and incubated for 45 min. on ice, washed twice with PBS (2% NBS), and resuspended as a control; the cells in the test group + 50 μΙ of 1: 50 of antibody F (ab ') 2, goat anti-human IgG diluted in biotin, incubated on ice for 45 min .; washed twice with PBS (2% NBS).
[310] (3) 50 μΙ of PE-SA antibody (1: 200 dilution) was added and incubated for 45 min. on ice; 2 ml of PBS (2% NBS) was added to resuspend the cells, and the supernatant was discarded by centrifugation.
[311] The proportion of CAR positive T cells was detected by flow cytometry. The positive infection rates of three CAR T cells, hu8E5-28Z, hu8E5-BBZ and hu8E5-28BBZ and simulated control cells were 52.1%, 47.8%, 44.6% and 71.7%, respectively.
3. CAR T-CELL CITOTOXICITY ASSAY TARGETING CLD18A2 [312] (1) Target cells: 75 μΙ_ of 2x10 ⁵ / mL 293T-A1 cells, 293T-A2 cells, adenocarcinoma cell line gastric AGS, AGS-A2, gastric cancer cell lines BGC-823 and BGC-823-A2 cells were inoculated respectively. The AGS gastric adenocarcinoma cell line, BGC-823 gastric cancer cell line was purchased from the ATCC cell bank, 293T-A1 cells, 293T-A2 cells, AGS-A2, BGC-823-A2 cells were constructed with reference to document ^{Q Q} CN101602808B, among which the 293T-A2, AGS-A2 cells and the BGC-823-A2 cells were positive cells for CLD18A2, and the remainder were CLD18A2 negative cells.
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97/117 [313] (2) Effector cells: Simulated T and CAR cells expressing different chimeric antigen receptors were added in an effector to target ratio of 3: 1, 1: 1 or 1: 3;
[314] (3) 4 duplicate wells were defined for each group, and the average of 4 replicate wells was obtained. The detection time was 18 h.
[315] Each experimental group and each control group are as follows:
[316] Each experimental group: each target cell + CAR T that expresses different chimeric antigen receptors;
[317] Control group 1: maximum LDH release from target cells;
[318] Control group 2: spontaneous release of LDH from target cells;
[319] Control group 3: spontaneous release of LDH from effector cells;
[320] (4) Detection method: The CytoTox 96 non-radioactive cytotoxicity test kit (Promega) was used. The CytoTox 96® assay quantitatively measures lactic dehydrogenase (LDH), in particular, with reference to the CytoTox 96 non-radioactive cytotoxicity assay kit instructions.
[321] (5) The calculation formula for cytotoxicity is:
[322] Cytotoxicity% = (Experimental group - Control group 2 - Control group 3) / (Control group 1 - Control group 2) * 100% [323] The results of cell destruction, in different effector ratios for target, are shown in Figure 11 and Figure 12. The results showed that the CAR-T products showed good destruction effects in 293T-A2, AGS-A2 and BGC-823-A2 cells with positive expression for CLD18A2. Among them, hu8E5-28Z and hu8E5-2l-28Z cells can destroy more than 60% of AGS-A2 cells in a 3: 1 effector-to-target ratio, and the destruction effects on BGC-823 cells -A2 can reach more than 90%. At the same time, the results also showed that the destruction effects of each CAR T cell (except Hu2B1-S54A-28Z), in cells with negative expression of CLD18A2, were not obvious.
EXAMPLE 10. CLD18A2 CAR-T CELL ACTIVITY
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98/117 [324] 1) Antitumor treatment experiments of untransfected T cells (UTD) and hu8E5-2l-28Z T cells in subcutaneously transplanted tumors in PDX gastric cancer were observed.
[325] 2) Establishment of a gastric cancer PDX model: a gastric cancer PDX tumor of approximately 2x2x2 mm was inoculated in the right auxiliary area of the female 6 to 8 week old NOD / SCID mice, and the day of tumor cell inoculation was recorded as DO day.
[326] 3) Experimental group: Tumors were inoculated for 15 days, and NOD-SCID mice were randomly divided into 3 groups, 7 in each group, non-transfected cell group and hu8E5-2l-28Z T cell group .
[327] 4) Adoptive T-cell transfer: 100 mg / kg cyclophosphamide was injected intraperitoneally when a tumor volume was 30 mm ³ , and 1.0 x 10 ⁷ CAR-T cells were infused through the tail vein, 24 hours after injection, while groups of non-transfected T cells were used as controls. The growth of subcutaneous xenografts from PDX gastric cancer was observed and measured. The results of the experiment are shown in Figures 13A and 13B. On day D32, after the injection of CAR T, the mice were sacrificed by cervical dislocation. Compared to the UTD group, the anti-tumor effect of the hu8E5-2l-28Z treatment group was significant, and the inhibition rate was 79.2%. From the weight of the tumor, the hu8E5-2l-28Z treatment group was statistically different from the UTD group, P = 0.01.
EXAMPLE 11. CLD18A2 IN VITRO CAR-T CELL-INDUCED CYTOKINE RELEASE TEST [328] To verify that hu8E5-28Z and hu8E5-2l-28Z T cells can be efficiently activated under target cell stimulation, hu8E5-28Z and hu8E5-2l-28Z T cell cytokine secretions were examined after coincubation with target cells.
[329] Cytokines released by transfected T cells (Simulated), hu8E5-28Z and hu8E5-2l-28Z T cells were detected respectively. Both T cells above good growth within 1 to 2 weeks after infection with the lentivirus were collected, inoculated into a 24 well plate in 5χ10 ^4/200 ul (amount of positive cell), and 5x10 ^4/200 æl / 24 wells
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99/117 target cells were inoculated at an effector-to-target ratio of 1: 1. 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-culture. The sandwich ELISA method was used to detect IL2, IFN-γ and TNF-α in the supernatant released during the coculture of CAR T lymphocytes with target cells.
[330] The results of the experiment are shown in Figure 14. The results showed that when hu8E5-28Z, hu8E5-2l-28Z were incubated with CLD18A2 positive 293T-A2, AGS-A2 and BGC-823-A2 cells, the cytokine secretion of IL-2, IFN-γ and TNF-α was activated, whereas in the simulated control group, the secretion of these cytokines cannot be activated and there were significant differences; when hu8E5-2l-28Z was incubated with 293T-A1, AGS and BGC-823 cells with negative expression of CLD18A2, the cytokine secretion of IL-2, IFN-γ and TNF-α cannot be activated, and in the group simulated control, the secretion of the above cytokines cannot be activated either. The above experimental results indicate that cells with positive expression of CLD18A2 can effectively activate hu8E5-2l-28Z CAR T cells.
EXAMPLE 12. IN VIVO DESTRUCTION ACTIVITY OF CLD18A2 CAR-T CELLS [325] The anti-tumor treatment experiments on untransfected t cells (Simulated), hu8E5-28Z and hu8E5-2l-28Z T cells in subcutaneous cell xenografts of BGC-823-A2 were determined.
[326] 1) Inoculation of subcutaneous xenografts of BGC-823-A2: BGC-823-A2 cells collected in logarithmic growth phase and growth well were adjusted to a density of 2.5x10 ⁷ / mL using physiological saline , and a volume of the cell suspension (200 μΙ_, 5 x ^{10 6} / animal) was injected subcutaneously into the right side of the mouse. The tumor cell inoculation day was recorded as day 0.
[327] 2) Experiment groups: On the 1st day of tumor inoculation, the tumor volume of BGC-823-A2 was measured, and the NOD-SCID mice were randomly divided into 3 groups, 6 mice from each group. The groups were the untransfected T cell, hu8E5-28Z T cell group and hu8E5-2l-28Z T cell group, respectively.
[328] 3) Adoptive transfer of T cells: 100 mg / kg of
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100/117 cyclophosphamide were injected intraperitoneally, when the tumor volume was 100 to 150 mm ³ (day 11), and 1x10 ⁷ T cells from CAR (simulated cells, T cells from hu8E5-28Z or T cells from hu8E5-2l- 28Z) were infused through the tail vein, 24 hours after injection, and the non-transfected T cell group (sham group) was used as a control to observe the growth of subcutaneous xenografts.
[329] The results of animal experiments are shown in Figure 15. The results showed that on the 17th day of treatment of hu8E5-28Z and hu8E5-2l-28Z CAR T cells, tumor inhibition rates of BGC xenografts -823-A2 were 81.3% and 89.2%, respectively; and there were significant differences in therapeutic effects on BGC-823-A2 xenografts between the hu8E5-28Z, hu8E5-2l-28Z treatment group and the simulated control group. After the 17th day of treatment, the mice were sacrificed, and the tumors were removed and weighed. The average tumor weight of BGC-823-A2 transplanted tumors in the hu8E5-28Z and hu8E5-2l-28Z treatment groups was 0.1 and 0.06 g, respectively, while the average tumor weight in the simulated control group was 0.53 g, and there were significant differences between the CAR T cell treatment group and the simulated control group, and the P values were 0.0013 and <0.0001, respectively.
EXAMPLE 13. EFFECT OF CLD18A2 CAR-T CELLS ON TUMOR INFILTRATION IN VIVO [330] According to the animal model of BGC-823-A2 subcutaneous xenograft established in Example 12, 17 days after the simulation, hu8E5-28Z and hu8E5-2l-28Z cells returned, tumor tissues were obtained and CD3 + cells were detected by histochemistry.
[331] The results are shown in Figure 16. Almost no T cell infiltration was observed around the tumor tissue in the simulated T cell group, almost no T cell infiltration was observed around the tumor tissue in the cell group Simulated T; in the hu8E5-28Z and hu8E5-2l-28Z cell groups, infiltration of CD3 + T cells can be seen at the edge of the tumor tissue; and more T cell infiltration can be seen at the edge of the tumor tissue in the hu8E5-2l-28Z treatment group.
EXAMPLE 14. PREPARATION OF CAR-T CELLS THAT COEXPRESS IFN
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101/117 [332] According to the procedure of examples 7 to 9, a hu8E5-28Z-IFNb CAR plasmid that expresses IFNb cytokine was constructed based on hu8E5-28Z, and the plasmid hu8E5-2l-28Z-IFN CAR that can express IFNb cytokine was built on the basis of hu8E5-2l-28Z CAR, which was compacted and infected with lentivirus, so that hu8E5-28Z-IFNb CAR T cells that co-express IFNb (also identified as hu8E5-28Z & IFNB) and cells T hu8E5-2l-28Z-IFN CAR that coexpress IFNb (also identified as hu8E5-2l-28Z & IFNB) were obtained. Hu8E5-28Z-IFNb CAR is encoded by the nucleotide sequence of SEQ ID NO: 90; and hu8E5-2l-28Z-IFN CAR is encoded by the nucleotide sequence of SEQ ID NO: 91.
[333] According to the procedure of Example 11, the in vitro induction of cytokine release assay was performed. The results are shown in Figure 17A, and the presence of IFN resulted in an increase in IFN-γ cytokine secretion, when hu8E5-28Z CAR T cells were matched with the target cells.
[334] Antitumor treatment experiments for untransfected T cells (UTD), hu8E5-28Z T cells and hu8E5-2l-28Z-IFN T cells in subcutaneous xenografts of a PDX model of gastric cancer. A gastric cancer PDX tumor of about 2x2x2 mm was inoculated subcutaneously in the right armpit of a 6 to 8 week old female, from NOD / SCID mice, and the tumor cell inoculation day was recorded as DO day. On day D15 of tumor inoculation, the NOD-SCID mice were randomly divided into 3 groups, 7 in each group, untransfected T cell group, hu8E5-28Z T cell group and hu8E5-2l- T cell group. 28Z-IFN. When the tumor volume was 30 mm ³ , 100 mg / kg cyclophosphamide was injected intraperitoneally, and 1.0 x 10 ⁷ cells of CAR-T (hu8E5-28Z T cells or hu8E5-2l-28Z-IFN T cells) were injected. infused through the tail vein, 24 hours after injection. At the same time, the non-transfected T cell group was used as a control. The growth of subcutaneous xenografts from PDX gastric cancer was observed and measured. The results were shown in Figure 17B, and in one of the 7 mice, in the group treated with hu8E5-2l-28Z-IFN, the tumor regressed completely.
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102/117 [335] CAR-T cells were determined for in vivo survival using the PDX model described above. Peripheral blood was collected from the saphenous vein of mice at D5, D7 and D10 days after the infusion of CAR-T, and the CAR-T cells (blank sample T cells (simulated), hu8E5-28Z T cells) or hu8E5-2l-28Z-IFN T cells) were detected for in vivo survival. The results were shown in Figure 17C, and the amount of T cell survival in the hu8E5-2l-28Z-IFN T cell treated group was significantly greater than that of the hu8E5-28Z -28Z T cell treatment group.
EXAMPLE 15. CAR-NK CELL BUILDING [336] As shown in the plasmid maps shown in Figures 18A and 18B, using PRRLSIN-cPPT.EF-1a as a vector, lentiviral plasmids encoding the chimeric antigen receptors of humanized antibody hu8E5 were constructed, which include PRRLSIN-cPPT.EF-1a-hu8E5-28BBZ and PRRLSIN-cPPT.EF-1a-hu8E5-2l-28Z. The hu8E5-28BBZ sequence consists of a CD8a signal peptide (SEQ ID NO: 70), hu8E5-scFV, CD8 hinge (SEQ ID NO: 72), a CD28 transmembrane region (SEQ ID NO: 74) and a intracellular segment (SEQ ID NO: 76).), intracellular signaling domain segment CD137 (SEQ ID NO: 82) and ΟΟ3ξ (SEQ ID NO: 78); and the hu8E5-2l-28Z sequence consists of CD8a signal peptide (SEQ ID NO: 70), hu8E5-2l scFV, CD8 hinge (SEQ ID NO: 72), CD28 transmembrane region (SEQ ID NO: 74 ) and intracellular signaling domain CD28 (SEQ ID NO: 76) and intracellular segment ΟΟ3ξ of CD3 (SEQ ID NO: 78).
1. PREPARATION OF NK-92 POSITIVE CELL LINE FOR CAR [337] 24-well plates coated with retronectin: 380 µl of 5 pg / ml retronectin solution (PBS) were added to each well, and incubated at 4 ° C for in the evening. The cells were seeded on the 24-well plate coated with retronectin. The number of cells per well was 5χ10 ⁵ , and the volume of the culture solution was 500 pi;
[338] Concentrated lentivirus was added to NK92 cells at MOI = 30, centrifuged at 32 ° C for 90 min. and transferred to a cell culture incubator;
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103/117 [339] Expansion culture: Infected cells passed every other day at a density of 5 χ 10 ⁵ / mL, and recombinant human IL-2 was supplemented in the lymphocyte culture solution at a final concentration of 500 U / ml.
2. EXPRESSION OF NK-92 CELL CHEMICAL ANTIGEN RECEIVER [340] (1) On the 7th day of culture of NK92 cells infected with lentivirus, 1 χ 10 ⁶ cells were obtained, aliquoted in a 2-liter centrifuge tube. ml;
[341] (2) To the control cells, 50 µl of PE-SA antibody (1: 200 dilution) was added and incubated on ice and resuspended as a control; cells in the + 50 μΙ 1: 50 F (ab ') 2 antibody, goat anti-human IgG diluted in biotin, incubated on ice for 45 min; washed twice with PBS (2% NBS); 50 μΙ of PE-SA antibody (1: 200 dilution) was added and incubated on ice;
[342] (3) 2 ml of PBS (2% NBS) was added to resuspend the cells, and the supernatant was discarded by centrifugation at 4 ° C; 500 μΙ of PBS (2% NBS) was added and transferred to a flow tube. The PE channel was detected by flow cytometry to determine the proportion of NK92 cells positive for CAR. The results are shown in Figure 19.
[343] Cytotoxicity assay: Target cells: 10 ⁴ cells from AGS, AGS-A2, BGC-823, BGC-823-A2 were inoculated into 96-well plates, respectively; effector cells: NK92 and CAR NK92 cells were added in an effector to target ratio of 6: 1.3: 1 or 1.5: 1; 5 duplicate wells were defined for each group, and the average of 5 replicate wells was obtained. The detection time was 4 hours. Each experimental group and each control group are as follows:
[344] Each experimental group: each target cell + effector cells above; Control group 1: maximum LDH release from target cells; Control group 2: spontaneous release of LDH from target cells; and Control group 3: spontaneous release of LDH from effector cells.
[345] Detection method: The CytoTox 96 non-radioactive cytotoxicity test kit (Promega) was used, in particular, with reference to
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104/117 CytoTox 96 non-radioactive cytotoxicity assay kit instructions. The calculation formula for cytotoxicity is:
[346] Cytotoxicity% = (Experimental group - Control group 2 - Control group 3) I (Control group 1 - Control group 2) * 100% [347] The results are shown in Figures 20 and 21, where hu8E5-21-28Z are hu8E5-2l-28Z CAR-NK92 cells and hu8E5-28BBZ are hu8E5-28BBZ CAR-NK92 cells. The results showed that hu8E5 CAR-NK92 cells had significant in vitro destruction activities against cells that overexpress CLDN18A2 and almost no deadly toxicity against CLDN18A2 negative cells.
TABLE 7. SEQUENCES USED IN THIS DOCUMENT
SEQID: Sequence 1 divmtqspssltvtagekvtmsckssqsllnsgnqknyltwyqqkpgqppklliywastresgvpdrftgsgsgtdftltissvqaedlavyycqndysypltfgagtklelkr 2 GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAAT CAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAA CTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATACAGGCAGTGGATCTGGAACAGAI I I CACTCTCACCATCAGCAGTGTGCAG GCTGAAGACCTGGCAG I I IATTACTGTCAGAATGATTATAGTTATCCGCTCA CGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG 3 qvqlqqsgaelarpgasvkmsckasgytftsytmhwvkqrpgqglewigyinpssgytnynqkfkdkatltad kssstaymqlssltsedsavyycariyygnsfaywgqgttvtvss 4 CAGGTCCAGCTGCAGCAGTCTGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCI I IACTAGCTACACGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCAGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAATCTACTATGGTAACTCG I I I GCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 5 QIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPYTFGGGTKLEIK
Petition 870190015344, of 02/14/2019, p. 110/151
105/117
R 6 CAAATTGTTCTCACCCAGTCTCCAG CAATCATGTCTG CATCTCCAG G G G AG AAGGTCACCATGACCTGCAGTGCCAGCTCAAGTATAAGTTACATGCACTGG TACCAGCAGAAGCCAGGCACCTCCCCCAAAAGATGGAI I IATGACACATCC AAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGAC CTCTTATTCTCTCACAATCAG G CATG CAG CTG AGG AAG ATG CTG CCACTTAT TACTGCCATCAGCGGAGTAGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGG 7 QVQLQQSGPELVKPGALVKISCKASGYTFTSYDINWVKQRPGQGLEWIGWIYPGDGSTKYNEKFKGKATLTADKSSSTAYMQLSSLTSENSAVYFCARGGYRYDEAM DYWGQGTTVTVSS 8 CAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCI I I AGTGAAGATATCCTGCAAGGCTTCTGGTTACACCTTCACAAGCTACGATATA AACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGAGTGATTTATCCTGGAGATGGTAGTACTAAGTACAATGAGAAATTCAAGGGCAAGGC CACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAG CCTGACTTCTGAGAACTCTGCAGTCTAI I I CTGTGCAAGAGGGGCTGTACGACGAGGCTATGGACTACTGGGGTCAAGGGACCACGGTCACCGTCT CCTCA 9 divmtqspsslsvsagekvtmsckssqsllnsgnqknylawyqqkpgqppklliygastresgvpdrftgsgsgtdftltissvqaedlavyycqndhsypltfgagtklelkr 10 GACATTGTGATGACACAGTCTCCATCCTCCCTGAGTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAAACTGTTGATCTACGGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACCGAI I I CACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAG I I IATTACTGTCAGAATGATCATAGTTATCCGCTCA CGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG 11 qiqlvqsgpelkkpgetvkisckasgytftnygmnwvkqapgkglkwmgwintntgeptyaeefkgrfafslet sastaylqi η η I knedtatyfcarfsyg nsfaywgqgttvtvss 12 CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAG ATCTCCTG CAAG G CTTCTG G GTATACCTTCACAAACTATG G AATGAACTGGGTGAAGCAGGCTCCAGGAAAGGGI I IAAAGTGGATGGGCTGGAT
Petition 870190015344, of 02/14/2019, p. 111/151
106/117
AAACACCAACACTGGAGAGCCAACATATGCTGAAGAGTTCAAGGGACGGTTTGCCTTCTCI I I GGAAACCI 01 GCCAGCACI GCCIAI I I GCAGATCAACAAC CTCAAAAATGAGGACACGGCTACATAI I I CTGTGCTAGATTCTCTTATGGTA ACTCC I I GCTTACTGGGGCCAAGGGACCACGGTCACCGT 13 divmtqspssltvtpgekvtmtckssqslfnsgnqknyltwyqqrpgqppkmliywastresgvpdrftgsgsgtdftltissvqaedlavfycqnaysfpytfgggtkleikr 14 GACATTGTGATGACACAGTCTCCATCCTCCCTGACTGTGACACCAGGAGAG AAGGTCACTATGACCTGCAAGTCCAGTCAGAGTTTGTTTAATAGTGGAAATC AAAAGAACTACTTGACCTGGTACCAACAGAGGCTGATGATCTCAGGCAGTGGATCTGGAACAGAI I I CACTCTCACCATCAGCAGTGTGCAGG CTGAAGACCTGGCAG I I I I I I I AC I G I CAGAAI GCI IAI AG I I I I CCGTACAC GTTCGGAGGGGGACCAAGCTGGAAATAAA 15 dvqlqesgpdlvkpsqslsltctvtgysitsgynwhwirqfpgnkmewmgyihytgstnynpslrsrisitrdtsknqfflqlnsvttddtatyyctriyngnsfpywgqgtsvtvss 16 GATGTGCAACTTCAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACI I I CACTCACCTGCACTGTCACTGGCTACTCCATCACCAGTGGTTATAACTGGCACTGGATCCGGCAG I I I CCAGGAAACAAAATGGAATGGATGGGCTACATACACTACACTGGTAGCACTAATTACAACCCATCTCTCAGAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACCACTGATGACACAGCCACATATTACTGTACAAGAATCTACAATGGTAACTCI I I I CCTTACTGGGGCCAAGGAACCTCAGTCACCGTCTCCTCA 17 qvqlqqsgaelarpgasvkmsckasgytftsytmhwvkqrpgqglewigyidpssgytnynqkfkdkatltad kssstaymqlssltsedsavyycariyygnsfaywgqgttvtvss 18 CAGGTCCAGCTGCAGCAGTCTGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCI I IACTAGCTACACGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTGACCCTAGCAGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAATCTACTATGGTAACTCG I I I GCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 19 qvqlqqsgaelarpgasvkmsckasgytftsytmhwvkqrpgqglewigyinpasgytnynqkfkdkatltad kssstaymqlssltsedsavyycariyygnsfaywgqgttvtvss
Petition 870190015344, of 02/14/2019, p. 112/151
107/117
20 CAGGTCCAGCTGCAGCAGTCTGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCI I IACTAGCTACACGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTGCCAGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAATCTACTATGGTAACTCG I I I GCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA 21 divmtqspdslavslgeratinckssqsllnsgnqknyltwyqqkpgqppklliywastresgvpdrfsgsgsgtdftltisslqaedvavyycqndysypltfgggtkveikr 22 GACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGGGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGCTGAACAGCGGCAACCAGAAGAACTACCTGACCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCCAGCACCCGGGAGAGCGGCGTGCCCGACCGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGAACGACTACAGCTACcCTCTGACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGCGG 23 qvqlvqsgaevkkpgasvkvsckasgytftsytmhwvrqapgqglewmgyinpasgytnynqkfkdrvtmt rdtststaymelsslrsedtavyycariyygnsfaywgqgtlvtvss 24 CAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGCCAGCGTGAAGGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACACCATGCACTGGGTGCGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCTACATCAACCCCGCCAGCGGCTACACCAACTACAACCAGAAGTTCAAGGACCGGGTGACCATGACCCGGGACACCAGCACCAGCACCGCCTACATGGAGCTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGCGCCCGGATCTACTACGGCAACAGCTTCGCCTACTGGGGCCAGGGCACCCTGGTGACCGTGA GCAGC 25 divmtqspdslavslgeratinckssqslfnsgnqknyltwyqqkpgqppklliywastresgvpdrfsgsgsgtdftltisslqaedvavyycqnaysfpytfgggtkleikr 26 GACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGGGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGTTCAACAGCGGCAACCAGAAGAACTACCTGACCTGGTACCAGCAGAAGCCCGGCCAGCCCCCCAAGCTGCTGATCTACTGGGCCAGCACCCGGGAGAGCGGCGTGCCCGACCGGTTCAGCGGCAGCGGCAGCGGCACCGACTTCACCCTGACCATCAGCAGC
Petition 870190015344, of 02/14/2019, p. 113/151
108/117
CTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCAGAACGCCTACAGCTT→TACACCTTCGGCGGCGGCACCAAGCTGGAGATCAAGCGG 27 qvqlqesgpglvkpsqtlsltctvsggsissgynwhwirqppgkglewigyihytgstnynpalrsrvtisvdtsknqfslklssvtaadtavyycariyngnsfpywgqgttvtvss 28 CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGTGAAGCCCAGCCAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAGCGGCTACAACTGGCACTGGATCCGGCAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCTACATCCACTACACCGGCAGCACCAACTACAACCCCGCCCTGCGGAGCCGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCGTGTACTACTGCGCCCGGATCTACAACGGCAACAGCTTCCCCTACTGGGGCCAGGGCACCACCGTGACCGTG AGCAGC 29 QVQLQESGPGLIKPSQTLSLTCTVSGGSISSGYNWHWIRQPPGKGLEWIGYIHYTGSTNYNPALRSRVTISVDTSKNQFSLKLSSVTAADTAIYYCARIYNGNSFPYWGQGTTVTVSS 30 CAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGATCAAGCCCAGCCAGACCCTGAGCCTGACCTGCACCGTGAGCGGCGGCAGCATCAGCAGCGGCTACAACTGGCACTGGATCCGGCAGCCCCCCGGCAAGGGCCTGGAGTGGATCGGCTACATCCACTACACCGGCAGCACCAACTACAACCCCGCCCTGCGGAGCCGGGTGACCATCAGCGTGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCCGCCGACACCGCCATCTACTACTGCGCCCGGATCTACAACGGCAACAGCTTCCCCTACTGGGGCCAGGGCACCACCGTGACCGTG AGCAGC 31 SERTYR THRMETHIS 32 TYR ILE ASN PRO SER SER GLY TYR THR ASN TYR ASN GLN LYS PHE LYSASP 33 ILE TYR TYR GLY ASN SER PHE ALA TYR 34 LYS SER SER GLN SER LEU LEU ASN SER GLY ASN GLN LYS ASN TYRLEU THR 35 TRP ALA SER THR ARG GLU SER 36 GLN ASN ASP TYR SER TYR PRO LEU THR 37 SYDIN 38 WIYPGDGSTKYNEKFKG
Petition 870190015344, of 02/14/2019, p. 114/151
11/117
39 GGYRYDEAMDY 40 SASSSISYMH 41 DTSKLAS 42 HQRSSYPYT 43 ASN TYRGLYMET ASN 44 TRP ILE ASN THR ASN THR GLY GLU PRO THR TYR ALA GLU GLU PHE LYSGLY 45 PHE SER TYR GLY ASN SER PHE ALA TYR 46 LYS SER SER GLN SER LEU LEU ASN SER GLY ASN GLN LYS ASN TYRLEU ALA 47 GLY ALA SER THR ARG GLU SER 48 GLN ASN ASP HIS SER TYR PRO LEU THR 49 SER GLY TYR ASN TRP HIS 50 TYR ILE HIS TYR THR GLY SER THR ASN TYR ASN PRO SER LEU ARG SER 51 ILE TYR ASN GLY ASN SER PHE PRO TYR 52 LYS SER SER GLN SER LEU PHE ASN SER GLY ASN GLN LYS ASN TYRLEU THR 53 TRP ALA SER THR ARG GLU SER 54 GLN ASN ALA TYR SER PHE PRO TYR THR 55 MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV 56 atggccgtgactgcctgtcagggcttggggttcgtggtttcactgattgggattgcgggcatcattgctgccacctg catggaccagtggagcacccaagacttgtacaacaaccccgtaacagctgttttcaactaccaggggctgtgg cgctcctgtgtccgagagagctctggcttcaccgagtgccggggctacttcaccctgctggggctgccagccatg ctgcaggcagtgcgagccctgatgatcgtaggcatcgtcctgggtgccattggcctcctggtatccatctttgccct gaaatgcatccgcattggcagcatggaggactctgccaaagccaacatgacactgacctccgggatcatgttc attgtctcaggtctttgtgcaattgctggagtgtctgtgtttgccaacatgctggtgactaacttctggatgtccacagc taacatgtacaccggcatgggtgggatggtgcagactgttcagaccaggtacacatttggtgcggctctgttcgtg ggctgggtcgctggaggcctcacactaattgggggtgtgatgatgtgcatcgcctgccggggcctggcaccag aagaaaccaactacaaagccgtttcttatcatgcctcaggccacagtgttgcctacaagcctggaggcttcaag
Petition 870190015344, of 02/14/2019, p. 115/151
110/117
gccagcactggctttgggtccaacaccaaaaacaagaagatatacgatggaggtgcccgcacagaggacgaggtacaatcttatccttccaagcacgactatgtgtaa 57 MSI 1 1 CQVVAFLLSILGLAGCIAATGMDMWSTQDLYDNPVTSVFQYEGLWRSCVRQSSGFTECRPYFTILGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLCAIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPS KHDYV 58 atgtccaccaccacatgccaagtggtggcgttcctcctgtccatcctggggctggccggctgcatcgcggccac cgggatggacatgtggagcacccaggacctgtacgacaaccccgtcacctccgtgttccagtacgaagggct ctggaggagctgcgtgaggcagagttcaggcttcaccgaatgcaggccctatttcaccatcctgggacttccag ccatgctgcaggcagtgcgagccctgatgatcgtaggcatcgtcctgggtgccattggcctcctggtatccatcttt gccctgaaatgcatccgcattggcagcatggaggactctgccaaagccaacatgacactgacctccgggatc atgttcattgtctcaggtctttgtgcaattgctggagtgtctgtgtttgccaacatgctggtgactaacttctggatgtcc acagctaacatgtacaccggcatgggtgggatggtgcagactgttcagaccaggtacacatttggtgcggctct gttcgtgggctgggtcgctggaggcctcacactaattgggggtgtgatgatgtgcatcgcctgccggggcctggc accagaagaaaccaactacaaagccgtttcttatcatgcctcaggccacagtgttgcctacaagcctggaggct tcaaggccagcactggctttgggtccaacaccaaaaacaagaagatatacgatggaggtgcccgcacagag gacgaggtacaatcttatccttccaagcacgactatgtgtaa 59 qvqlvqsgaevkkpgasvkvsckasgytftsytmhwvrqapgqglewmgyinpasgytnynqkfkdrvtmt rdtststaymelsslrsedtavyycariyygnsfaywgqgtlvtvssastkgpsvfplapsskstsggtaalgclvk dyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkt htcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqy nstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfy psdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg k 60 caggtgcagctggtgcagagcggcgccgaggtgaagaagcccggcgccagcgtgaaggtgagctgcaag gccagcggctacaccttcaccagctacaccatgcactgggtgcggcaggcccccggccagggcctggagtg gatgggctacatcaaccccgccagcggctacaccaactacaaccagaagttcaaggaccgggtgaccatga cccgggacaccagcaccagcaccgcctacatggagctgagcagcctgcggagcgaggacaccgccgtgta ctactgcgcccggatctactacggcaacagcttcgcctactggggccagggcaccctggtgaccgtgagcagc gctagcaccaaaggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggcc ctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcg gcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctcca
Petition 870190015344, of 02/14/2019, p. 116/151
111/117
gcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaa agttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggacc gtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggt ggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgc caagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcacc aggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaa accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagct gaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtggga gagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctc tatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagg ctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 61 divmtqspdslavslgeratinckssqsllnsgnqknyltwyqqkpgqppklliywastresgvpdrfsgsgsgt dftltisslqaedvavyycqndysypltfgggtkveikrtvaapsvfifppsdeqlksgtvsnstkstvsn 62 gacatcgtgatgacccagagccccgacagcctggccgtgagcctgggcgagcgggccaccatcaactgca agagcagccagagcctgctgaacagcggcaaccagaagaactacctgacctggtaccagcagaagcccg gccagccccccaagctgctgatctactgggccagcacccgggagagcggcgtgcccgaccggttcagcggc agcggcagcggcaccgacttcaccctgaccatcagcagcctgcaggccgaggacgtggccgtgtactactgc cagaacgactacagctaccccctgaccttcggcggcggcaccaaggtggagatcaagcggacggtggctgc accatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataac ttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagt gtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagac tacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttc aacaggggagagtgt 63 Qvqlqesgpglikpsqtlsltctvsggsissgynwhwirqppgkglewigyihytgstnynpalrsrvtisvdtskn qfslklssvtaadtaiyycariyngnsfpywgqgttvtvssastkgpsvfplapsskstsggtaalgclvkdyfpep vtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppc papellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvv svltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiave wesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 64 caggtgcagctgcaggagagcggccccggcctgatcaagcccagccagaccctgagcctgacctgcaccgtgagcggcggcagcatcagcagcggctacaactggcactggatccggcagccccccggcaagggcctggagtggatcggctacatccactacaccggcagcaccaactacaaccccgccctgcggagccgggtgaccatca
Petition 870190015344, of 02/14/2019, p. 117/151
112/117
gcgtggacaccagcaagaaccagttcagcctgaagctgagcagcgtgaccgccgccgacaccgccatctac tactgcgcccggatctacaacggcaacagcttcccctactggggccagggcaccaccgtgaccgtgagcagc gctagcaccaaaggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggcc ctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcg gcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctcca gcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaa agttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggacc gtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggt ggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgc caagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcacc aggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaa accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagct gaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtggga gagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgg actccgacggctccttcttcctc tatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagg ctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 65 divmtqspdslavslgeratinckssqslfnsgnqknyltwyqqkpgqppklliywastresgvpdrfsgsgsgt dftltisslqaedvavyycqnaysfpytfgggtkleikrtvaapsvfifppsdeqlksgtvsnkstvsn 66 gacatcgtgatgacccagagccccgacagcctggccgtgagcctgggcgagcgggccaccatcaactgca agagcagccagagcctgttcaacagcggcaaccagaagaactacctgacctggtaccagcagaagcccgg ccagccccccaagctgctgatctactgggccagcacccgggagagcggcgtgcccgaccggttcagcggca gcggcagcggcaccgacttcaccctgaccatcagcagcctgcaggccgaggacgtggccgtgtactactgcc agaacgcctacagcttcccctacaccttcggcggcggcaccaagctggagatcaagcggacggtggctgca ccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataactt ctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgt cacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagacta cgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt 67 qvqlqesgpglvkpsqtlsltctvsggsissgynwhwirqppgkglewigyihytgstnynpalrsrvtisvdtskn qfslklssvtaadtaiyycariyngnsfpywgqgttvtvssastkgpsvfplapsskstsggtaalgclvkdyfpep vtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkthtcppc papellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvv
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svltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 68 caggtgcagctgcaggagagcggccccggcctgatcaagcccagccagaccctgagcctgacctgcaccgt gagcggcggcagcatcagcagcggctacaactggcactggatccggcagccccccggcaagggcctgga gtggatcggctacatccactacaccggcagcaccaactacaaccccgccctgcggagccgggtgaccatca gcgtggacaccagcaagaaccagttcagcctgaagctgagcagcgtgaccgccgccgacaccgccatctac tactgcgcccggatctacaacggcaacagcttcccctactggggccagggcaccaccgtgaccgtgagcagc gctagcaccaaaggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggcc ctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcg gcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctcca gcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaa agttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggacc gtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggt ggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgc caagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcacc aggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctc ccagcccccatcgagaaa accatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagct gaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtggga gagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctc tatagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagg ctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa 69 malpvtalllplalllhaarp 70 atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg 71 Tttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacd 72 accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgat 73 Fwvlvvvggvlacysllvtvafiifwv 74 ttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtggcctttattattttctgggtg 75 Rskrsrllhsdymnmtprrpgptrkhyqpyapprdfaayrs 76 aggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgccgccccgggccaacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctcc 77 rvkfsrsadapayqqgqnqlynelnlgrreeydvldkrrgrdpemggkpqrrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr
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78 agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagc tcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatgggggga aagccgcagagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggag gcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc 79 lyiwaplagtcgvlllslvit 80 Atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcacc 81 Krgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcel 82 aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaagaaggaggatgtgaactg 83 YIDPSSGYTNYNQKFKD 84 YINPASGYTNYNQKFKD 85 yihytgstnynpalrs 86 Divmtqspssltvtagekvtmsckssqsllnsgnqknyltwyqqkpgqppklliywastresgvpdrftgsgsgt dftltissvqaedlavyycqndysypltfgagtklelkrtvaapsvfifpqdekstnstvsnk 87 Qiqlvqsgpelkkpgetvkisckasgytftnygmnwvkqapgkglkwmgwintntgeptyaeefkgrfafslet sastaylqinnlknedtatyfcarlgfgnamdywgqgtsvtvssastkgpsvfplapsskstsggtaalgclvkdy fpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdktht cppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqyns tyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfyps diavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 88 Divmtqspssltvtagekvtmsckssqsllnsgnqknyltwyqqkpgqppklliywastresgvpdrftgsgsgt dftltissvqaedlavyycqndysypftfgsgtkleikrtvaapsvfifppsdeqlksgtvsnkstvsn 89 Qvqlqqpgaelvrpgasvklsckasgytftsywinwvkqrpgqglewigniypsdsytnynqkfkdkatltvdk ssstaymqlssptsedsavyyctrswrgnsfdywgqgttltvssastkgpsvfplapsskstsggtaalgclvkd yfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkscdkth tcppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqyn styrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfyp sdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk 90 atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgcaggtgcagctgcaggagagcggccccggcctggtgaagcccagccagaccctgagcctgacctgcaccgtgagcggcggc
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115/117 agcatcagcagcggctacaactggcactggatccggcagccccccggcaagggcctggagtggatcggcta catccactacaccggcagcaccaactacaaccccgccctgcggagccgggtgaccatcagcgtggacacc agcaagaaccagttcagcctgaagctgagcagcgtgaccgccgccgacaccgccgtgtactactgcgcccg gatctacaacggcaacagcttcccctactggggccagggcaccaccgtgaccgtgagcagcggtggaggcg gttcaggcggaggtggttctggcggtggcggatcggacatcgtgatgacccagagccccgacagcctggccg tgagcctgggcgagcgggccaccatcaactgcaagagcagccagagcctgttcaacagcggcaaccagaa gaactacctgacctggtaccagcagaagcccggccagccccccaagctgctgatctactgggccagcaccc gggagagcggcgtgcccgaccggttcagcggcagcggcagcggcaccgacttcaccctgaccatcagcag cctgcaggccgaggacgtggccgtgtactactgccagaacgcctacagcttcccctacaccttcggcggcggc accaagctggagatcaagcggaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgc gtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggg gctggacttcgcctgtgatttttgggtgctggtggtggttggtggagtcctggcttgctatagcttgctagtaacagtg gcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgc cgccccgggccaacccgcaagcattaccagccctatgccccaccacgcg acttcgcagcctatcgctccaga gtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctca atctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaa gccgcagagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggc ctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctca gtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaggtcgacaatcaac ctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgcttt aatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgag gagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggc attgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgc ctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgac gtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctc aatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagac gagtcggatctccctttgggccgcctccccgcctggaattcgctagcctcgagctcacacaa aaaaccaacac acagatgtaatgaaaataaagatattttattgcggccgctttagtttcggaggtaacctgtaagtctgttaatgaagt aaaagttccttaggatttccactctgactatggtccaggcacagtgactgtactccttggccttcaggtaatgcaga atcctcccataatatcttttcaggtgcagactgctcatgagttttcccctggtgaaatcttctttctccagtttttcttccag gactgtcttcagatggtttatctgatgatagacattagccaggaggttctcaacaatagtctcattccagccagtgct agatgaatcttgtctgaaaatagcaaagatgttctggagcatctcatagatggtcaatgcggcgtcctccttctgg aactgctgcagctgcttaatctcctcagggatgtcaaagttcatcctgtccttgaggcagtattcaagcctcccattc
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aattgccacaggagcttctgacactgaaaattgctgcttctttgtaggaatccaagcaagttgtagctcatggaaa gagctgtagtggagaagcacaacaggagagcaatttggaggagacacttgttggtcatggtggcgaccggta gcgctaggtcatatgcaggagttgaggttactgtgagtagtgattaaagagagtgatagggaactcttgaacaa gagatgcaatttatactgttaattctggaaaaatattatgggggtgtcaaaatgtcccgggacaattgacgccttct gtatgaaacagtttttcctccacgccttctgtatgaaacagtttttcctccacgccttctgtatgaaacagtttttcctcc gtcgaggacaattgacgccttctgtatgaaacagtttttcctccacgccttctgtatgaaacagtttttcctccacgcc ttctgtatgaaacagtttttcctcc 91 atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccgcaggtgcagct gcaggagagcggccccggcctgatcaagcccagccagaccctgagcctgacctgcaccgtgagcggcggc agcatcagcagcggctacaactggcactggatccggcagccccccggcaagggcctggagtggatcggcta catccactacaccggcagcaccaactacaaccccgccctgcggagccgggtgaccatcagcgtggacacc agcaagaaccagttcagcctgaagctgagcagcgtgaccgccgccgacaccgccatctactactgcgcccg gatctacaacggcaacagcttcccctactggggccagggcaccaccgtgaccgtgagcagcggtggaggcg gttcaggcggaggtggttctggcggtggcggatcggacatcgtgatgacccagagccccgacagcctggccg tgagcctgggcgagcgggccaccatcaactgcaagagcagccagagcctgttcaacagcggcaaccagaa gaactacctgacctggtaccagcagaagcccggccagccccccaagctgctgatctactgggccagcaccc gggagagcggcgtgcccgaccggttcagcggcagcggcagcggcaccgacttcaccctgaccatcagcag cctgcaggccgaggacgtggccgtgtactactgccagaacgcctacagcttcccctacaccttcggcggcggc accaagctggagatcaagcggaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgc gtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggg gctggacttcgcctgtgatttttgggtgctggtggtggttggtggagtcctggcttgctatagcttg ctagtaacagtg gcctttattattttctgggtgaggagtaagaggagcaggctcctgcacagtgactacatgaacatgactccccgc cgccccgggccaacccgcaagcattaccagccctatgccccaccacgcgacttcgcagcctatcgctccaga gtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctca atctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaa gccgcagagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggc ctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctca gtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctaggtcgacaatcaac ctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgcttt aatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgag gagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggc attgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgc ctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaagctgac
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gtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctc aatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagac gagtcggatctccctttgggccgcctccccgcctggaattcgctagcctcgagctcacacaaaaaaccaacac acagatgtaatgaaaataaagatattttattgcggccgctttagtttcggaggtaacctgtaagtctgttaatgaagt aaaagttccttaggatttccactctgactatggtccaggcacagtgactgtactccttggccttcaggtaatgcaga atcctcccataatatcttttcaggtgcagactgctcatgagttttcccctggtgaaatcttctttctccagtttttcttccag gactgtcttcagatggtttatctgatgatagacattagccaggaggttctcaacaatagtctcattccagccagtgct agatgaatcttgtctgaaaatagcaaagatgttctggagcatctcatagatggtcaatgcggcgtcctccttctgg aactgctgcagctgcttaatctcctcagggatgtcaaagttcatcctgtccttgaggcagtattcaagcctcccattc aattgccacaggagcttctgacactgaaaattgctgcttctttgtaggaatccaagcaagttgtagctcatggaaa gagctgtagtggagaagcacaacaggagagcaatttggaggagacacttgttggtcatggtggcgaccggta gcgctaggtcatatgcaggagttgaggttactgtgagtagtgattaaagagagtgatagggaactcttgaacaa gagatgcaatttatactgttaattctggaaaaatattatgggggtgtcaaaatgtcccgggacaatt gacgccttct gtatgaaacagtttttcctccacgccttctgtatgaaacagtttttcctccacgccttctgtatgaaacagtttttcctcc gtcgaggacaattgacgccttctgtatgaaacagtttttcctccacgccttctgtatgaaacctctttga 92 MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN 93 GGGGSGGGGSGGGGS 94 Acgccttctgtatgaaacagtttttcctccacgccttctgtatgaaacagtttttcctccacgccttctgtatgaaacagtttttcctccgtcgaggacaattgacgccttctgtatgaaacagtttttcctccacgccttctgtatgaaacagtttttcctccacgccttctgtatgaaacagtttttcctcc
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权利要求:
Claims (85)
[1]
1. Antibody that specifically binds to claudin 18A2, CHARACTERIZED by the fact that the antibody comprises a heavy chain CDR that comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 31,32, 33, 37, 38, 39, 43, 44, 45, 49, 50, 51.83, 84, 85, or a variant thereof, and / or a light chain CDR comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 34, 35, 36, 40, 41,42, 46, 47, 48, 52, 53, 54, or a variant thereof.
[2]
2. Antibody, according to claim 1, CHARACTERIZED by the fact that the antibody is selected from the group consisting of:
(a) an antibody comprising a heavy chain variable region, wherein the heavy chain variable region has CDR1 comprising an amino acid sequence of SEQ ID NO: 31, SEQ ID NO: 37, SEQ ID NO: 43 or SEQ ID NO: 49, CDR2 comprising an amino acid sequence 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 or SEQ ID NO: 85, CDR3 comprising an amino acid sequence of SEQ ID NO: 33, SEQ ID NO: 39, SEQ ID NO: 45 or SEQ ID NO: 51;
(b) an antibody comprising a light chain variable region, wherein the light chain variable region has CDR1 which comprises an amino acid sequence of SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46 or SEQ ID NO: 52, CDR2 comprising an amino acid sequence of SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47 or SEQ ID NO: 53, CDR3 comprising an amino acid sequence of SEQ ID NO: 36 , SEQ ID NO: 42, SEQ ID NO: 48 or SEQ ID NO: 54;
(c) an antibody comprising (a) a heavy chain variable region of said antibody and (b) a light chain variable region of said antibody;
(d) an antibody, which recognizes the same antigenic determining site as that of the antibody of any of (a) through (c).
[3]
3. Antibody, according to claim 2, CHARACTERIZED by the fact that the regions of CDR1, CDR2 and CDR3 of the variable region of chain
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2/15 heavy of the antibody are SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33; 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, respectively; and / or the CDR1, CDR2 and CDR3 regions of the antibody light chain variable region are SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36; or SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42; or SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48; or SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, respectively.
[4]
4. Antibody according to claim 3, CHARACTERIZED by the fact that the antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region has an amino acid sequence of SEQ ID NO: 3, 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 or SEQ ID NO: 29; and the light chain variable region has an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 21 or SEQ ID NO: 25.
[5]
5. Antibody according to claim 4, CHARACTERIZED by the fact that the antibody is:
an antibody having a heavy chain variable region of SEQ ID NO: 3 and a light chain variable region of SEQ ID NO: 1;
an antibody that has a heavy chain variable region of SEQ ID NO: 7 and a light chain variable region of SEQ ID NO: 5;
an antibody that has a heavy chain variable region of SEQ ID NO: 11 and a light chain variable region of SEQ ID NO: 9;
an antibody that has a heavy chain variable region of SEQ ID NO: 15 and a light chain variable region of SEQ ID NO: 13;
an antibody having a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 1;
an antibody that has a heavy chain variable region of SEQ ID NO: 19 and a light chain variable region of SEQ ID NO: 1;
an antibody that has a heavy chain variable region of
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SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 21;
an antibody having a heavy chain variable region of SEQ ID NO: 27 and a light chain variable region of SEQ ID NO: 25; or an antibody that has a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 25.
[6]
6. Antibody, according to claim 5, CHARACTERIZED by the fact that the antibody is:
an antibody having a heavy chain variable region of SEQ ID NO: 3 and a light chain variable region of SEQ ID NO: 1; or an antibody that has a heavy chain variable region of SEQ ID NO: 17 and a light chain variable region of SEQ ID NO: 1; or an antibody that has a heavy chain variable region of SEQ ID NO: 19 and a light chain variable region of SEQ ID NO: 1; or an antibody that has a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 21; or an antibody that has a heavy chain variable region of SEQ ID NO: 27 and a light chain variable region of SEQ ID NO: 25; or an antibody that has a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 25.
[7]
Antibody according to any one of claims 1 to 6, CHARACTERIZED by the fact that the antibody is a humanized antibody, a chimeric antibody or a fully humanized antibody; or the antibody is a monoclonal antibody; or the antibody is a single chain antibody or a domain antibody.
[8]
8. Antibody according to claim 7, CHARACTERIZED by the fact that the antibody is a humanized antibody selected from the group consisting of:
an antibody having a heavy chain variable region of SEQ ID NO: 27 and a light chain variable region of SEQ ID NO: 25;
an antibody that has a heavy chain variable region of SEQ ID NO: 23 and a light chain variable region of SEQ ID NO: 21;
an antibody that has a heavy chain variable region of SEQ ID NO: 29 and a light chain variable region of SEQ ID NO: 25.
[9]
9. Antibody according to claim 8,
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CHARACTERIZED by the fact that the antibody is selected from the group consisting of:
(a) an antibody that has a heavy chain of SEQ ID NO: 63 and a light chain of SEQ ID NO: 65;
(b) an antibody that has a light chain of SEQ ID NO: 61 and a heavy chain of SEQ ID NO: 59; and (c) an antibody that has a heavy chain of SEQ ID NO: 67 and a light chain of SEQ ID NO: 65.
[10]
10. Nucleic acid, CHARACTERIZED by the fact that it encodes the antibody as defined in any one of claims 1 to 9.
[11]
11. Expression vector, CHARACTERIZED by the fact that it comprises the nucleic acid as defined in claim 10.
[12]
12. Host cell, CHARACTERIZED by the fact that it comprises the expression vector as defined in claim 11, or that it has the nucleic acid, as defined in claim 10, integrated into its genome.
[13]
13. Use of an antibody, as defined in any one of claims 1 to 9, CHARACTERIZED by the fact that it is to prepare a target drug, drug-antibody conjugate or multifunctional antibody that specifically targets tumor cells that express claudin 18A2 ; or to prepare a reagent to diagnose a tumor that expresses 18A2 claudin; or to prepare a chimeric antigen receptor-modified immune cell.
[14]
14. Use according to claim 13, CHARACTERIZED by the fact that the tumor that expresses claudin 18A2 includes: gastric cancer, pancreatic cancer, esophageal cancer, lung cancer.
[15]
15. Chimeric antigen receptor, CHARACTERIZED by the fact that it comprises an antibody as defined in any of claims 1 to 9, wherein the chimeric antigen receptor comprises components linked in sequence below: an antibody as defined in any of the claims 1 to 9, a transmembrane region and an intracellular signal region.
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[16]
16. Chimeric antigen receptor, according to claim 15, CHARACTERIZED by the fact that the intracellular signal region is selected from the group consisting of: an intracellular signal region sequence of Οϋ3 CD, FcsRly, CD27, CD28, CD137, CD134, MyD88, CD40, or a combination thereof; or the transmembrane region comprises the transmembrane region of CD8 or CD28.
[17]
17. Chimeric antigen receptor according to claim 16, CHARACTERIZED by the fact that the chimeric antigen receptor comprises, linked in sequence, an antibody, a transmembrane region and an intracellular signaling region, as follows:
an antibody, as defined in any an of claims 1 to 9, CD8 and Οϋ3ζ;an antibody, as defined in any an of claims 1 to 9, CD8, CD137 and Οϋ3ζ; or an antibody, as defined in any an of
claims 1 to 9, a transmembrane region of CD28 molecule, an intracellular signal region of CD28 molecule, and Οϋ3ζ; or an antibody, as defined in any one of claims 1 to 9, a transmembrane region of CD28 molecule, an intracellular signal region of CD28 molecule, CD137 and Οϋ3ζ.
[18]
18. Nucleic acid, CHARACTERIZED by the fact that it encodes the chimeric antigen receptor as defined in any one of claims 15 to 17.
[19]
19. Expression vector, CHARACTERIZED by the fact that it comprises nucleic acid as defined in claim 18.
[20]
20. Virus, CHARACTERIZED by the fact that it comprises the vector as defined in claim 19.
[21]
21. Use of the chimeric antigen receptor, as defined in any one of claims 15 to 17, CHARACTERIZED by the fact that the nucleic acid, as defined in claim 18, the expression vector, as defined in claim 19, or the virus, as defined in claim 20, are for preparing chimeric antigen receptor-modified immune cells that target cells
Petition 870190015344, of 02/14/2019, p. 128/151
6/15 tumor expressing 18A2 claudin.
[22]
22. Use, according to claim 21, CHARACTERIZED by the fact that the tumor that expresses claudin 18A2 includes: gastric cancer, pancreatic cancer, esophageal cancer, lung cancer.
[23]
23. Immune cell modified by chimeric antigen receptor, CHARACTERIZED by the fact that it is transduced with nucleic acid, as defined in claim 18, the expression vector, as defined in claim 19, or the virus, as defined in claim 20; or which has the chimeric antigen receptor, as defined in any one of claims 15 to 17, expressed on the surface;
preferably, the immune cell is: a T lymphocyte, an NK cell or an NKT lymphocyte.
[24]
24. Immune cell, according to claim 23, CHARACTERIZED by the fact that the immune cell additionally carries a coding sequence for an exogenous cytokine; or additionally expresses another chimeric antigen receptor that does not contain ΟΟ3ζ, but contains an intracellular signal domain of CD28, an intracellular signal domain of CD137, or a combination of both; or additionally expresses a chemokine receptor; preferably, said chemokine receptor comprises: CCR); or additionally expresses a siRNA that can reduce the expression of PD-1, or a protein that can block PD-L1; or endogenous PD-1 in the immune cell is eliminated by means of gene editing techniques; or additionally expresses safe switching.
[25]
25. Use of the chimeric antigen receptor-modified immune cell, as defined in any one of claims 23 to 24, CHARACTERIZED by the fact that it is to produce a tumor inhibitory drug, wherein the tumor is a tumor that expresses claudin 18A2; preferably, the tumor includes gastric cancer, pancreatic cancer, esophageal cancer, lung cancer.
[26]
26. Multifunctional immunoconjugate, CHARACTERIZED by the fact that it comprises:
an antibody, as defined in any one of claims 1 to 9; and
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7/15 a functional molecule attached to it; and the functional molecule is selected from the group consisting of: a molecule that targets a surface marker on a tumor, a tumor inhibiting molecule, a molecule that targets a surface marker on an immune cell or a detectable identification.
[27]
27. Multifunctional immunoconjugate according to claim 26, CHARACTERIZED by the fact that the molecule that targets an immune cell surface marker is an antibody that binds to a T cell surface marker, which forms an antibody bifunctional with the antibody as defined in any one of claims 1 to 9, in which the T cell is involved.
[28]
28. Nucleic acid, CHARACTERIZED by the fact that it encodes the multifunctional immunoconjugate as defined in any of claims 26 to 27.
[29]
29. Use of the multifunctional immunoconjugate, as defined in any of claims 26 to 27, CHARACTERIZED by the fact that it is for preparing antitumor drugs; or prepare a reagent to diagnose a tumor that expresses 18A2 claudin; or preparing immune cells modified by a chimeric antigen receptor; preferably, immune cells include: a T lymphocyte, an NK cell or an NKT lymphocyte.
[30]
30. Pharmaceutical composition, CHARACTERIZED by the fact that it comprises, the then antibody as defined in any one of claims 1 to 9, or a nucleic acid encoding the antibody; or the immunoconjugate as defined in any one of claims 26 to 27, or a nucleic acid encoding the conjugate; or the chimeric antigen receptor as defined in any one of claims 15 to 17, or a nucleic acid encoding the chimeric antigen receptor; or the chimeric antigen receptor-modified immune cell as defined in any one of claims 23 to 24.
[31]
31. Kit, CHARACTERIZED by the fact that it comprises:
a container and the pharmaceutical composition as defined in
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8/15 claim 30 in the container; or a container and the antibody as defined in any one of claims 1 to 9, or a nucleic acid encoding the antibody in the container; or the immunoconjugate as defined in any one of claims 26 to 27, or a nucleic acid encoding the conjugate; or the chimeric antigen receptor as defined in any one of claims 15 to 17, or a nucleic acid encoding the chimeric antigen receptor; or the chimeric antigen receptor-modified immune cell as defined in any one of claims 23 to 24.
[32]
32. Antigen binding unit, CHARACTERIZED by the fact that it comprises a light chain CDR and a heavy chain CDR, in which the antigen binding unit specifically binds to an 18A2 claudin peptide; and the antigen binding unit does not bind significantly to an 18A1 claudin peptide.
[33]
33. Antigen binding unit, CHARACTERIZED by the fact that it comprises a light chain CDR and a heavy chain CDR, in which the antigen binding unit specifically binds to an 18A2 claudin peptide; and the antigen binding unit, compared to a reference antigen binding unit, exhibits less non-specific binding to an 18A1 claudin peptide.
[34]
34. Antigen binding unit according to claim 32 or 33, characterized by the fact that the referenced antigen binding unit comprises a light chain amino acid sequence of SEQ ID NO: 86 or SEQ ID NO: 88 and / or a heavy chain amino acid sequence of SEQ ID NO: 87 or SEQ ID NO: 89.
[35]
35. Antigen binding unit according to claim 32 or 33, characterized in that the claudin peptide 18A2 comprises an amino acid sequence of SEQ ID NO: 55.
[36]
36. Antigen binding unit according to claim 32 or 33, characterized in that the claudin peptide 18A1 comprises an amino acid sequence of SEQ ID NO: 57.
[37]
37. Antigen binding unit according to claim 32 or 33, CHARACTERIZED by the fact that the non-specific binding of the antigen binding unit to the claudin peptide 18A1 does not exceed 20%
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9/15 of the specific binding to the 18A2 claudin peptide.
[38]
38. Antigen binding unit according to claim 32 or 33, characterized by the fact that the binding specificity is determined by flow cytometry.
[39]
39. Antigen binding unit according to claim 32 or 33, CHARACTERIZED by the fact that the binding specificity is determined by FACS.
[40]
40. Antigen binding unit according to claim 32 or 33, CHARACTERIZED by the fact that the binding specificity is determined by ELISA.
[41]
41. Antigen binding unit according to claim 32 or 33, characterized by the fact that the antigen binding unit binds to the claudin peptide 18A2 with an EC50 of less than about 100 nM.
[42]
42. Antigen binding unit according to claim 32 or 33, characterized by the fact that the antigen binding unit is a monoclonal antibody, a humanized antibody, a chimeric antibody, a multivalent antibody or a chimeric antigen receptor .
[43]
43. Antigen binding unit according to claim 32 or 33, characterized by the fact that the light chain CDR comprises LCDR1, LCDR2 and LCDR3; and the heavy chain CDR comprises HCDR1, HCDR2 and HCDR3;
wherein 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 HCDR1, HCDR2 and HCDR3, respectively, 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: 50, SEQ ID NO: 51, SEQ ID NO: 83 , SEQ ID NO: 84 and SEQ ID NO: 85.
[44]
44. Antigen binding unit, according to claim 32 or 33, CHARACTERIZED by the fact that the LCDR1 comprises a
Petition 870190015344, of 02/14/2019, p. 132/151
10/15 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.
[45]
45. Antigen binding unit according to claim 32 or 33, CHARACTERIZED by the fact that LCDR2 comprises 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.
[46]
46. Antigen binding unit according to claim 32 or 33, CHARACTERIZED by the fact that 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.
[47]
47. Antigen binding unit according to claim 32 or 33, CHARACTERIZED by the fact that 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.
[48]
48. Antigen binding unit according to claim 32 or 33, CHARACTERIZED by the fact that 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.
[49]
49. Antigen binding unit according to claim 32 or 33, CHARACTERIZED by the fact that 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.
[50]
50. Antigen binding unit according to claim 32 or 33, characterized by the fact that the antigen binding unit is scFv, Fv, Fab or (Fab) 2.
[51]
51. Antigen binding unit, CHARACTERIZED by the fact that it comprises a light chain CDR and a heavy chain CDR, wherein said light chain CDR comprises LCDR1, LCDR2 and LCDR3; said heavy chain CDR comprises HCDR1, HCDR2 and HCDR3; LCDR1, LCDR2 and LCDR3, respectively, comprise an amino acid sequence that is 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:
Petition 870190015344, of 02/14/2019, p. 133/151
11/15
46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54; and HCDR1, HCDR2 and HCDR3, respectively, comprise an amino acid sequence that is 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: 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: 85.
[52]
52. Antigen binding unit according to claim
51, FEATURED by the fact that the light chain CDR comprises LCDR1, LCDR2 and LCDR3; the heavy chain CDR comprises HCDR1, HCDR2 and HCDR3; 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 HCDR1, HCDR2 and HCDR3. respectively, 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: 50, SEQ ID NO: 51, SEQ ID NO: 83, SEQ ID NO: 84 and SEQ ID NO: 85.
[53]
53. Antigen binding unit according to claim 51, CHARACTERIZED by the fact that LCDR1 comprises an amino acid sequence that is 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.
[54]
54. Antigen binding unit according to claim 51, CHARACTERIZED by the fact that LCDR2 comprises an amino acid sequence that is at least 80% identical to 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.
[55]
55. Antigen binding unit according to claim 51, CHARACTERIZED by the fact that LCDR3 comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO : 35,
Petition 870190015344, of 02/14/2019, p. 134/151
12/15
SEQ ID NO: 42, SEQ ID NO: 48 and SEQ ID NO: 54.
[56]
56. Antigen binding unit according to claim 51, CHARACTERIZED by the fact that HCDR1 comprises an amino acid sequence that is at least 80% identical 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.
[57]
57. Antigen binding unit according to claim 51, CHARACTERIZED by the fact that HCDR2 comprises an amino acid sequence that is 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.
[58]
58. Antigen binding unit according to claim 51, CHARACTERIZED by the fact that HCDR3 comprises an amino acid sequence that is 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.
[59]
59. Antigen binding unit according to claim 51, characterized by the fact that the antigen binding unit is a monoclonal antibody, a humanized antibody, a chimeric antibody, a multivalent antibody or a chimeric antigen receptor.
[60]
60. Antigen binding unit according to claim 51, CHARACTERIZED by the fact that the antigen binding unit is scFv, Fv, Fab or (Fab) 2.
[61]
61. Chimeric antigen receptor, CHARACTERIZED by the fact that it comprises an extracellular antigen binding unit, a transmembrane domain and an intracellular domain, wherein the extracellular antigen binding unit comprises the antigen binding unit as defined in any one claims 32 to 60.
[62]
62. Composition, CHARACTERIZED by the fact that it comprises the antigen binding unit as defined in any of claims 32 to 60, or the chimeric antigen receptor as defined in claim 61.
[63]
63. The composition of claim 62,
Petition 870190015344, of 02/14/2019, p. 135/151
13/15
CHARACTERIZED by the fact that it comprises a Type I interferon.
[64]
64. Isolated nucleic acid, CHARACTERIZED by the fact that it encodes the antigen binding unit, as defined in any of claims 32 to 60, or the chimeric antigen receptor, as defined in claim 61.
[65]
65. Isolated nucleic acid according to claim 64, CHARACTERIZED by the fact that the nucleic acid encodes a type I interferon.
[66]
66. Vector, CHARACTERIZED by the fact that it comprises a nucleic acid encoding the antigen binding unit, as defined in any of claims 32 to 60, or the chimeric antigen receptor as defined in claim 61, and, optionally, a type I interferon.
[67]
67. Host cell, CHARACTERIZED by the fact that it expresses the antigen binding unit, as defined in any of claims 32 to 60, or the chimeric antigen receptor, as defined in claim 61, and, optionally, an interferon of the type I
[68]
68. Host cell, CHARACTERIZED by the fact that it comprises a nucleic acid encoding the antigen binding unit, as defined in any of claims 32 to 60, or the chimeric antigen receptor as defined in claim 61, and, optionally, a type I interferon.
[69]
69. Host cell according to claim 67, CHARACTERIZED by the fact that the host cell is an immune response cell.
[70]
70. Host cell according to claim 67, CHARACTERIZED by the fact that the host cell is a T cell, natural killer cell, a cytotoxic T lymphocyte, a natural killer T cell, DNT cell and / or regulatory T cell.
[71]
71. Host cell according to claim 67,
CHARACTERIZED by the fact that the host cell is an NK92 cell.
[72]
72. Host cell according to claim 67,
CHARACTERIZED by the fact that the host cell is cytotoxic to a cell comprising an 18A2 claudin peptide, and the claudin peptide
Petition 870190015344, of 02/14/2019, p. 136/151
14/15
18A2 comprises the amino acid sequence of SEQ ID NO: 55.
[73]
73. Host cell according to claim 67, CHARACTERIZED by the fact that the host cell does not have significant cytotoxicity to a cell that comprises an 18A1 claudin peptide while it does not comprise an 18A2 claudin peptide, the 18A1 claudin peptide comprises a amino acid sequence of SEQ ID NO: 57, and the claudin peptide 18A2 comprises the amino acid sequence of SEQ ID NO: 55.
[74]
74. Method, CHARACTERIZED by the fact that it is to produce the antigen binding unit as defined in any of claims 32 to 60, or the chimeric antigen receptor as defined in claim 61, or the composition as defined in any of the claims 62 to 63, which includes: culturing the host cell as defined in any of claims 67 to 73, under suitable conditions and obtaining the product expressed by the host cell.
[75]
75. Method, CHARACTERIZED by the fact that it is to induce the death of a cell comprising an 18A2 claudin peptide, which includes putting the cell in contact with the antigen binding unit as defined in any of claims 32 to 60, the chimeric antigen receptor, as defined in claim 61, the composition, as defined in any of claims 62 to 63, or the host cell, as defined in any of claims 67 to 73.
[76]
76. Method according to claim 75, CHARACTERIZED by the fact that the cell is brought into contact with the antigen binding unit, the chimeric antigen receptor, the composition or host cell in vitro.
[77]
77. Method according to claim 75, CHARACTERIZED by the fact that the cell is brought into contact with the antigen binding unit, the chimeric antigen receptor, the composition or host cell in vivo.
[78]
78. Method according to claim 75, CHARACTERIZED by the fact that the cell is a cancer cell.
[79]
79. Method according to claim 75, CHARACTERIZED by the fact that the cell is a solid tumor cell.
Petition 870190015344, of 02/14/2019, p. 137/151
15/15
[80]
80. Method according to claim 75, CHARACTERIZED by the fact that the cell is selected from the group consisting of: a gastric cancer cell, esophageal cancer cell, intestinal cancer cell, pancreatic cancer cell, cell nephroblastoma cell, lung cancer cell, ovarian cancer cell, cervical cancer cell, rectal cancer cell, liver cancer cell, head and neck cancer cell, chronic myeloid leukemia cell and vesicle cancer cell biliary.
[81]
81. Method, CHARACTERIZED by the fact that it is to treat a tumor in an individual in need, which includes administering to the individual, an effective amount of the antigen binding unit, as defined in any of claims 32 to 60, the recipient chimeric antigen as defined in claim 61, the composition as defined in any of claims 62 to 63, the vector as defined in claim 66, and / or the host cell as defined in any of claims 67 to 73 .
[82]
82. Method according to claim 81, CHARACTERIZED by the fact that the tumor is a solid tumor.
[83]
83. Method according to claim 81, CHARACTERIZED by the fact that 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.
[84]
84. The method of claim 81, the method being CHARACTERIZED in that it additionally includes administering to the individual an additional therapeutic agent.
[85]
85. Method according to claim 84, CHARACTERIZED by the fact that the additional therapeutic agent is at least one selected from the group consisting of epirubicin, oxaliplatin and 5-fluorouracil.

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法律状态:
2021-10-05| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2022-01-04| B25A| Requested transfer of rights approved|Owner name: CAFA THERAPEUTICS LIMITED (IE) |

优先权:

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申请号 | 申请日 | 专利标题

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CN201610536449|2016-07-08|

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PCT/CN2017/082024|WO2017186121A1|2016-04-26|2017-04-26|Method for improving function of immune response cell|

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PCT/CN2017/092381|WO2018006882A1|2016-07-08|2017-07-10|Antibody for anti-claudin 18a2 and use thereof|

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