WO2016008405A1 - 靶向cld18a2的免疫效应细胞及其制备方法和应用 - Google Patents
靶向cld18a2的免疫效应细胞及其制备方法和应用 Download PDFInfo
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- WO2016008405A1 WO2016008405A1 PCT/CN2015/084023 CN2015084023W WO2016008405A1 WO 2016008405 A1 WO2016008405 A1 WO 2016008405A1 CN 2015084023 W CN2015084023 W CN 2015084023W WO 2016008405 A1 WO2016008405 A1 WO 2016008405A1
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- chimeric antigen
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Definitions
- CLD18A1 is selectively expressed in normal lung and gastric epithelium, while CLD18A2 expression is limited to short-lived cells differentiated from gastric epithelium and not expressed in gastric stem cells; and, existing studies have shown that CLD18A2 is expressed in many tumor cells. .
- the intracellular signal region can be selected from the group consisting of CD3 ⁇ , Fc ⁇ RI ⁇ , CD28, CD137, the intracellular signal region of the CD134 protein, and combinations thereof.
- the CD3 molecule consists of five subunits, of which the CD3 ⁇ subunit (also known as CD3zeta, abbreviated as Z) contains three ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex.
- CD3 ⁇ Z is a truncated CD3 ⁇ sequence that does not have an ITAM motif and is generally constructed as a negative control in the practice of the present invention.
- Fc ⁇ RI ⁇ is mainly distributed on the surface of mast cells and basophils, which contains an ITAM motif similar in structure, distribution and function to CD3 ⁇ .
- the present invention also provides genetically modified T lymphocytes which are transduced with the nucleic acid of the present invention or which are transduced with the above-described recombinant plasmid containing the nucleic acid of the present invention, or a virus comprising the same.
- Conventional nucleic acid transduction methods including non-viral and viral transduction methods, can be used in the present invention.
- Non-viral based transduction methods include electroporation and transposon methods.
- the Nucleofector nuclear transfection device developed by Amaxa can directly introduce foreign genes into the nucleus to obtain efficient transduction of the target gene.
- the inventors have repeatedly studied and analyzed several scfv antibodies recognizing CLD18A2, abbreviated as 125, 163 and 175.
- the processed sample was affinity-purified by protein A (purchased from GE) affinity column to finally obtain purified single-chain antibody-Fc fusion protein scFv-125-Fc (referred to as scFv-125), scFv-163-Fc (abbreviation) scFv-163), scFv-175-Fc (referred to as scFv-175), the identification results are shown in Figure 3.
- the cells were digested with 10 mM EDTA, and the cells were collected by centrifugation at 200 g ⁇ 5 min.
- the cells were resuspended in 1% of calf serum-containing phosphate buffer (NBS PBS) at a concentration of 1 ⁇ 10 6 to 1 ⁇ 10 7 /mL, and added to a flow-type dedicated tube in an amount of 100 ul / tube.
- NBS PBS calf serum-containing phosphate buffer
- the single-chain antibody scFv-125 not only binds to CLD18A1 but also to CLD18A2 stably expressing 293T cells (Fig. 5), indicating that the single-chain antibody lacks binding specificity for CLD18A2.
- the single-chain antibodies ScFv-163 and scFv-175 specifically recognize CLD8A2 stably expressing 293T, but not 293T cells stably expressing CLD18A1, indicating that these two single-chain antibodies can specifically recognize CLD18A2.
- these two single-chain antibodies can also specifically recognize BGC-823 or NCI-N87 cell lines stably transfected with CLD18A2, but not BGC-823 or NCI-N87 cells that have not been transfected with CLD18A2.
- Table 1 illustrates the order of attachment of the various portions of the chimeric antigen receptor of the present invention, which can also be seen in Figure 2.
- Chimeric antigen receptor Extracellular binding region transmembrane region - intracellular signal region 1 - intracellular signal region 2etc CLD18A2- ⁇ Z scFv(CLD18A2)-CD8-CD3 ⁇ zeta (negative control) CLD18A2-163-Z scFv(CLD18A2-163)-CD8-CD3zeta CLD18A2-175-Z scFv(CLD18A2-175)-CD8-CD3zeta CLD18A2-163-28BBZ scFv(CLD18A2-163)-CD28a-CD28b-CD137 (ie: 4-1BB)-CD3zeta CLD18A2-175-28BBZ scFv(CLD18A2-163)-CD28a-CD28b-CD137-CD3zeta
- nucleic acid sequences of the anti-CLD18A2 chimeric antigen receptor protein other than scFv were respectively subjected to PCR using the sequence SEQ ID NO: 18, 21 disclosed in Patent Application No. 201310164725.X. Way to get.
- the eGFP-F2A sequence was obtained by PCR amplification of the primer pair (SEQ ID NO: 11, 12) using the plasmid of SEQ ID NO: 18 contained in Patent Application No. 201310164725.X as a template.
- CD8-CD3 ⁇ (Z) and CD28a-CD28b-CD137-CD3 ⁇ (28BBZ) Acquisition of CD8-CD3 ⁇ (Z) and CD28a-CD28b-CD137-CD3 ⁇ (28BBZ): scFv(GPC3)-CD8-CD3 ⁇ ( SEQ ID NO:18 in patent application 201310164725.X) and scFv(GPC3)-CD28a, respectively -CD28b-CD137-CD3 ⁇ (SEQ ID NO: 21 in Patent Application 201310164725.X) is a template, and PCR amplification is performed using a primer pair (SEQ ID NO: 13, 14) to obtain CD8-CD3 ⁇ (Z) and CD28a-, respectively. CD28b-CD137-CD3 ⁇ (28BBZ) fragment.
- SEQ ID NO: 21 in 201310164725.X corresponds to the sequence of SEQ ID NO: 24 in the present invention.
- elongation factor-1 ⁇ elongation factor-1 ⁇ , EF-1 ⁇
- eGFP enhanced green fluorescent protein
- F2A ribosomal skipping sequence
- This example constructs a lentiviral expression vector in which eGFP linked to F2A is co-expressed with a specific CAR, collectively referred to as pWPT-eGFP-F2A-CAR (Fig. 1).
- the target gene eGFP-F2A-CAR obtained in the above step 2 (see 1 (2) in Example 3, the element after F2A is abbreviated as CAR) was digested by MluI and SalI restriction enzymes, and ligated into the same double
- the pWPT vector was digested to construct a lentiviral vector expressing each chimeric antigen receptor.
- the successfully constructed vector can be prepared for lentiviral packaging after MluI and SalI digestion and sequencing.
- eGFP-F2A-CAR is transcribed into one mRNA, but is finally translated into two peptide chains, eGFP and anti-CLD18A2 chimeric antigen receptor, in which the anti-CLD18A2 chimeric antigen receptor will be localized under the guidance of CD8 ⁇ signal peptide.
- CD8 ⁇ signal peptide On the cell membrane.
- HEK-293T cells (ATCC: CRL-11268) cultured to the 6th to 10th passages were inoculated at a density of 6 ⁇ 10 6 in a 10 cm culture dish, and cultured overnight at 37 ° C, 5% CO 2 for transfection.
- the medium was DMEM (purchased from PAA) containing 10% fetal bovine serum (purchased from PAA), and the next day, the culture medium was changed to serum-free DMEM about 2 hours before transfection.
- 293T cells were seeded at 1 ⁇ 10 5 /mL in 96-well culture plates, 100 ⁇ L/well, 37 ° C, 5% CO 2 , and the culture was DMEM containing 10% fetal bovine serum.
- 50 ⁇ L/well of the culture supernatant was discarded, 50 ⁇ L/well of the above fresh culture solution was added, and polybrene was added at a final concentration of 6 ⁇ g/mL, and incubated at 37 ° C, 5% CO 2 for 30 min.
- the titer (U/mL) positive rate ⁇ dilution factor ⁇ 100 ⁇ 10 4 was calculated.
- the titer of the above virus containing the mock empty vector control and each eGFP-F2A-CAR packaged by the calcium phosphate transfection method was at a level of about 0.5 to 2 ⁇ 10 6 U/mL, and the virus titer was measured after concentration. It is approximately 2 x 10 7 U/mL.
- Example 4 recombinant lentivirus infection of CTL cells
- Human peripheral blood mononuclear cells (provided by Shanghai Blood Center) were obtained from peripheral blood of healthy people by density gradient centrifugation. Peripheral blood mononuclear cells were obtained by CTL cell magnetic beads (purchased from Stem Cell Technologies) negative sorting method to obtain CTL. The sorted CTL cells were subjected to flow cytometry to detect the purity of CTL cells, and the positive rate of CTL cells was ⁇ 95%.
- the Quantum 007 lymphocyte culture medium (purchased from PAA) was added at a density of about 1 ⁇ 10 6 /mL, and magnetic beads coated with anti-CD3 and CD28 antibodies were added at a cell:magnetic bead ratio of 1:1 (Invitrogen)
- the company and the recombinant human IL-2 (purchased from Shanghai Huaxin Biotech Co., Ltd.) with a final concentration of 100 U/mL were stimulated for 24 hours.
- CTL cells were then infected with the above recombinant lentivirus at MOI ⁇ 5. Every second day after infection the cells by density 5 ⁇ 10 5 / mL are subcultured while lymphocyte culture medium supplemented with a final concentration of 100U / mL of recombinant human IL-2.
- Infected CTL cells were tested for expression of different chimeric antigen receptors by flow cytometry on day 8 of culture. Since eGFP was co-expressed with CAR, positive cells detecting eGFP were positive cells expressing chimeric antigen receptors.
- the positive rate of virus-infected CTL cells expressing different chimeric antigen receptors using uninfected T lymphocytes as a negative control is shown in Table 3. The positive rate results indicate that a certain positive rate of CAR + CTL cells can be obtained by a method of lentiviral infection.
- CTL cells were subcultured, counted, and supplemented with IL-2 at a cell density of 5 ⁇ 10 5 /ml, respectively, after infection with a virus packed with different chimeric antigen receptors.
- the final concentration was 100U/ml), about 20-40 times amplification on the 11th day of culture, indicating that CTL cells expressing different chimeric antigen receptors can be expanded in vitro, providing for subsequent in vitro toxicity tests and in vivo experiments. Guarantee.
- Example 5 In vitro toxic effect test of chimeric antigen receptor cells
- the 293T and gastric cancer cell lines shown in Table 4 were used as target cells, and the effector cells were FACS for 12 days of in vitro culture as demonstrated in Example 4.
- the positive cells expressing chimeric antigen receptor expression were recorded as chimeric antigen receptor positive (
- the CTL of CAR + ) has a target ratio of 3:1, 1:1 and 1:3, respectively, and the target cell number is 10000/well, corresponding to effector cells according to different target-to-target ratios. Five replicate wells were set for each group, and the average of five replicate wells was taken. The detection time is 18h.
- Control group 1 The maximum release of LDH from target cells
- Control group 2 The target cells spontaneously release LDH
- Control group 3 Effector cells spontaneously released LDH.
- CytoTox 96 non-radioactive cytotoxicity test kit (Promega) was used. This method is based on the colorimetric detection method and can replace the 51 Cr release method. CytoTox The assay quantitatively measures lactate dehydrogenase (LDH). LDH is a stable cytoplasmic enzyme that is released when cells are lysed and released in much the same way as 51 Cr is released in radioactive analysis. The released LDH medium supernatant can be detected by a 30 minute coupled enzyme reaction in which LDH converts a tetrazolium salt (INT) to red formazan (Formazan). The amount of red product produced is directly proportional to the number of cells lysed. Refer specifically to the instructions for the CytoTox 96 non-radioactive cytotoxicity test kit.
- INT tetrazolium salt
- Formmazan red formazan
- the cytotoxicity calculation formula is:
- the CTL expressing the chimeric antigen receptor (fused to express single-chain antibody 163 or 175) CLD18A2-Z CAR + and the CTL of CLD18A2-28BBZ CAR + of the present invention highly express CLD18A2.
- 293T cells had significant killing effects, but did not kill 293T cells that highly expressed CLD18A1, indicating that they can selectively kill CLD18A2 cells.
- the CTL expressing the chimeric antigen receptor CLD18A2-Z CAR + and the CTL of CLD18A2-28BBZ CAR + of the present invention can also significantly kill the two gastric cancer cell lines BGC-823 and NCI-N87 which highly express CLD18A2 ( See Table 4 and Table 5), and the effect of the target-target gradient-dependent effect-target ratio is higher, and the cytotoxic effect is stronger, but there is no specific cytotoxic effect on BGC-823 and NCI-N87 which do not express CLD18A2.
- the data of the potency ratio dependence further showed the specific cytotoxic effect of the CTL expressing the anti-CLD18A2 chimeric antigen receptor of the present invention on gastric cancer cells highly expressing CLD18A2.
- CTLs transfected with a virus containing a mock plasmid (empty plasmid vector pWPT-eGFP carrying CLD18A2-CAR) as a blank control showed very cytotoxic effects on the above three cell lines highly expressing CLD18A2. low.
- the cytotoxicity of the cell line highly expressing CLD18A2 and the cytotoxicity data of the CTL expressing the anti-CLD18A2 chimeric antigen receptor of the present invention showed very significant differences.
- CLD18A2 antigen is a tight junction protein, and it is not possible to be contacted by CAR T cells and trigger the killing of the corresponding target cells. know.
- CAR T cells since the spatial conformation of proteins is involved in the whole body, many monoclonal antibodies evolve into single-chain antibodies, which often lose antigen binding activity or specificity. Fortunately, the inventors have found that two single chain antibodies (163 and 175) retain the antigen binding specificity of the monoclonal antibodies. Further studies have shown that CAR T cells composed of these two single-chain antibodies retain a selective killing effect on CLD18A2-positive cells. The results of the present invention indicate that CLD18A2 can indeed be a target for CAR T cell therapy; CAR T cells against CLD18A2 are a new candidate for tumor therapy.
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Abstract
Description
嵌合抗原受体 | 胞外结合区-跨膜区-胞内信号区1-胞内信号区2etc |
CLD18A2-δZ | scFv(CLD18A2)-CD8-CD3δzeta(阴性对照) |
CLD18A2-163-Z | scFv(CLD18A2-163)-CD8-CD3zeta |
CLD18A2-175-Z | scFv(CLD18A2-175)-CD8-CD3zeta |
CLD18A2-163-28BBZ | scFv(CLD18A2-163)-CD28a-CD28b-CD137(即:4-1BB)-CD3zeta |
CLD18A2-175-28BBZ | scFv(CLD18A2-163)-CD28a-CD28b-CD137-CD3zeta |
转染有下列CAR的CTL细胞 | CTL细胞eGFP阳性率 |
Mock(空载体对照) | 56% |
融合表达163单链抗体的CLD18A2-Z | 51% |
融合表达163单链抗体的CLD18A2-28BBZ | 54% |
融合表达175单链抗体的CLD18A2-Z | 52% |
融合表达175单链抗体的CLD18A2-28BBZ | 55% |
Claims (17)
- 表达于免疫效应细胞表面的嵌合抗原受体,其特征在于,所述的嵌合抗原受体包含顺序连接的:胞外结合区,跨膜区和胞内信号区,其中所述胞外结合区包含特异性识别CLD18A2的蛋白。
- 如权利要求1所述的嵌合抗原受体,其特征在于,所述的特异性识别CLD18A2的蛋白是抗体或配体;较佳地,所述的抗体是单链抗体或结构域抗体。
- 如权利要求1所述的嵌合抗原受体,其特征在于,所述的跨膜区是包含CD8或CD28的跨膜区和铰链区的序列。
- 如权利要求1所述的嵌合抗原受体,其特征在于,所述的胞内信号区选自:CD3ζ,FcεRIγ,CD27,CD28,CD137,CD134的胞内信号区序列,或其组合。
- 如权利要求1所述的嵌合抗原受体,其特征在于,所述的嵌合抗原受体包括如下的顺序连接的胞外结合区,跨膜区和胞内信号区:特异性识别CLD18A2的单链抗体、CD8和CD3ζ;特异性识别CLD18A2的单链抗体、CD8、CD137和CD3ζ;特异性识别CLD18A2的单链抗体、CD28分子的跨膜区、CD28分子的胞内信号区和CD3ζ;或特异性识别CLD18A2的单链抗体、CD28分子的跨膜区、CD28分子的胞内信号区、CD137和CD3ζ。
- 如权利要求1所述的嵌合抗原受体,其特征在于,所述的嵌合抗原受体具有SEQ ID NO:19~22任一所述的氨基酸序列。
- 如权利要求1所述的嵌合抗原受体,其特征在于,所述的免疫效应细胞包括:T淋巴细胞,NK细胞或NKT细胞。
- 编码权利要求1-7任一所述的嵌合抗原受体的核酸。
- 如权利要求8所述的核酸,其特征在于,所述的核酸具有SEQ ID NO:15~18任一所述的核苷酸序列。
- 一种表达载体,其特征在于,其包含权利要求8-9任一所述的核酸。
- 如权利要求10所述的表达载体,其特征在于,所述的表达载体来源于慢病毒质粒pWPT。
- 一种病毒,其特征在于,所述的病毒包含权利要求10或11所述载体。
- 权利要求1-7任一所述的嵌合抗原受体、或权利要求8或9所述的核酸、或权利要求10或11所述的表达载体、或权利要求12所述的病毒的用途,用于制备靶向CLD18A2的基因修饰的免疫效应细胞。
- 一种基因修饰的免疫效应细胞,其特征在于,其转导有权利要求8或9所述的核酸,或权利要求10或11所述的表达载体或权利要求12所述的病毒。
- 一种基因修饰的免疫效应细胞,其特征在于,其表面表达一种嵌合抗原受体,所述嵌合抗原受体的氨基酸序列选自SEQ ID NO:19-22任一所述的氨基酸序列。
- 如权利要求14或15所述的基因修饰的免疫效应细胞,其特征在于,所述的免疫效应细胞包括:T淋巴细胞,NK细胞或NKT细胞。
- 权利要求14-16任一所述的基因修饰的免疫效应细胞的用途,其特征在于,用于制备抑制肿瘤的药物,所述的肿瘤是CLD18A2阳性的肿瘤。
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DK15821331.4T DK3170842T3 (da) | 2014-07-17 | 2015-07-15 | Immunologisk effektorcelle af målrettet cld18a2, fremstillingsfremgangsmåde og anvendelse deraf |
KR1020177003178A KR101950747B1 (ko) | 2014-07-17 | 2015-07-15 | Cld18a2를 표적으로 하는 면역 작동 세포 및 그 제조방법과 용도 |
US15/326,974 US10377822B2 (en) | 2014-07-17 | 2015-07-15 | Immunologic effector cell of targeted CLD18A2, and preparation method and use thereof |
PL15821331T PL3170842T3 (pl) | 2014-07-17 | 2015-07-15 | Odpornościowa komórka efektorowa skierowana przeciwko CLD18A2, sposób jej otrzymywania i zastosowanie |
JP2017502187A JP2017522024A (ja) | 2014-07-17 | 2015-07-15 | Cld18a2標的免疫エフェクター細胞及びその調製方法と使用 |
ES15821331T ES2746555T3 (es) | 2014-07-17 | 2015-07-15 | Célula efectora inmunológica de CLD18A2 dirigido, y método de preparación y uso de la misma |
EP15821331.4A EP3170842B1 (en) | 2014-07-17 | 2015-07-15 | Immunologic effector cell of targeted cld18a2, and preparation method and use thereof |
US16/448,053 US11198729B2 (en) | 2014-07-17 | 2019-06-21 | Immunologic effector cell of targeted CLD18A2, and preparation method and use thereof |
US17/522,284 US20220363751A1 (en) | 2014-07-17 | 2021-11-09 | Immunologic effector cell of targeted cld18a2, and preparation method and use thereof |
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007059997A1 (en) * | 2005-11-24 | 2007-05-31 | Ganymed Pharmaceuticals Ag | Monoclonal antibodies against claudin-18 for treatment of cancer |
WO2013051718A1 (ja) * | 2011-10-07 | 2013-04-11 | 国立大学法人三重大学 | キメラ抗原受容体 |
CN103483453A (zh) * | 2012-06-12 | 2014-01-01 | 上海吴孟超医学科技基金会 | 结合egfr家族蛋白的嵌合抗原受体、其组合物及用途 |
CN103820393A (zh) * | 2014-02-24 | 2014-05-28 | 中国人民解放军总医院 | 工程化cd20靶向性的nkt细胞及其制备方法和应用 |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0362371A4 (en) | 1988-04-15 | 1990-10-24 | Protein Design Labs, Inc. | Il-2 receptor-specific chimeric antibodies |
IL162181A (en) | 1988-12-28 | 2006-04-10 | Pdl Biopharma Inc | A method of producing humanized immunoglubulin, and polynucleotides encoding the same |
SG48759A1 (en) | 1990-01-12 | 2002-07-23 | Abgenix Inc | Generation of xenogenic antibodies |
AU675661B2 (en) | 1992-07-24 | 1997-02-13 | Abgenix, Inc. | Generation of xenogeneic antibodies |
EP1978033A3 (en) | 1995-04-27 | 2008-12-24 | Amgen Fremont Inc. | Human antibodies derived from immunized xenomice |
DE102004024617A1 (de) * | 2004-05-18 | 2005-12-29 | Ganymed Pharmaceuticals Ag | Differentiell in Tumoren exprimierte Genprodukte und deren Verwendung |
US8793895B2 (en) | 2006-02-10 | 2014-08-05 | Praxair Technology, Inc. | Lyophilization system and method |
LT2618835T (lt) * | 2010-09-20 | 2017-10-10 | Biontech Cell & Gene Therapies Gmbh | Antigenui specifiniai t ląstelių receptoriai ir t ląstelių epitopai |
WO2014075697A1 (en) | 2012-11-13 | 2014-05-22 | Biontech Ag | Agents for treatment of claudin expressing cancer diseases |
JP6499079B2 (ja) * | 2012-11-13 | 2019-04-10 | バイオエヌテック アーゲーBioNTech AG | クローディンを発現するガン疾患を処置するための剤 |
CN107460201A (zh) | 2013-05-08 | 2017-12-12 | 科济生物医药(上海)有限公司 | 编码gpc‑3嵌合抗原受体蛋白的核酸及表达gpc‑3嵌合抗原受体蛋白的t淋巴细胞 |
ES2699753T3 (es) * | 2014-01-29 | 2019-02-12 | Biontech Ag | Mimótopos peptídicos de claudina 18.2 y sus usos |
CN105315375B (zh) | 2014-07-17 | 2021-04-23 | 恺兴生命科技(上海)有限公司 | 靶向cld18a2的t淋巴细胞及其制备方法和应用 |
WO2016180468A1 (en) | 2015-05-11 | 2016-11-17 | Biontech Cell & Gene Therapies Gmbh | Claudin-18.2-specific immunoreceptors and t cell epitopes |
-
2014
- 2014-07-17 CN CN201410341504.XA patent/CN105315375B/zh active Active
- 2014-07-17 CN CN202110389401.0A patent/CN112979828A/zh active Pending
-
2015
- 2015-07-15 EP EP15821331.4A patent/EP3170842B1/en not_active Revoked
- 2015-07-15 PT PT15821331T patent/PT3170842T/pt unknown
- 2015-07-15 PL PL15821331T patent/PL3170842T3/pl unknown
- 2015-07-15 KR KR1020177003178A patent/KR101950747B1/ko active IP Right Grant
- 2015-07-15 WO PCT/CN2015/084023 patent/WO2016008405A1/zh active Application Filing
- 2015-07-15 ES ES15821331T patent/ES2746555T3/es active Active
- 2015-07-15 JP JP2017502187A patent/JP2017522024A/ja active Pending
- 2015-07-15 DK DK15821331.4T patent/DK3170842T3/da active
- 2015-07-15 US US15/326,974 patent/US10377822B2/en active Active
-
2019
- 2019-02-27 JP JP2019034550A patent/JP6664528B2/ja not_active Expired - Fee Related
- 2019-06-21 US US16/448,053 patent/US11198729B2/en active Active
-
2021
- 2021-11-09 US US17/522,284 patent/US20220363751A1/en active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007059997A1 (en) * | 2005-11-24 | 2007-05-31 | Ganymed Pharmaceuticals Ag | Monoclonal antibodies against claudin-18 for treatment of cancer |
WO2013051718A1 (ja) * | 2011-10-07 | 2013-04-11 | 国立大学法人三重大学 | キメラ抗原受容体 |
CN103483453A (zh) * | 2012-06-12 | 2014-01-01 | 上海吴孟超医学科技基金会 | 结合egfr家族蛋白的嵌合抗原受体、其组合物及用途 |
CN103820393A (zh) * | 2014-02-24 | 2014-05-28 | 中国人民解放军总医院 | 工程化cd20靶向性的nkt细胞及其制备方法和应用 |
Non-Patent Citations (1)
Title |
---|
See also references of EP3170842A4 * |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11198729B2 (en) | 2014-07-17 | 2021-12-14 | Cafa Therapeutics Limited | Immunologic effector cell of targeted CLD18A2, and preparation method and use thereof |
US10377822B2 (en) | 2014-07-17 | 2019-08-13 | Carsgen Therapeutics Ltd. | Immunologic effector cell of targeted CLD18A2, and preparation method and use thereof |
EP3170842B1 (en) | 2014-07-17 | 2019-09-04 | Carsgen Therapeutics Limited | Immunologic effector cell of targeted cld18a2, and preparation method and use thereof |
US11713346B2 (en) * | 2015-05-11 | 2023-08-01 | Biontech Cell & Gene Therapies Gmbh | Claudin-18.2-specific immunoreceptors and T cell epitopes |
US20180282389A1 (en) * | 2015-05-11 | 2018-10-04 | Biontech Cell & Gene Therapies Gmbh | Claudin-18.2-specific immunoreceptors and t cell epitopes |
CN107523549A (zh) * | 2016-06-20 | 2017-12-29 | 上海细胞治疗研究院 | 一种高效稳定表达激活型抗体的car‑t细胞及其用途 |
US11111295B2 (en) | 2016-07-08 | 2021-09-07 | Cafa Therapeutics Limited | Antibody for anti-claudin 18A2 and use thereof |
EP3483182A4 (en) * | 2016-07-08 | 2020-07-29 | Carsgen Therapeutics Co., Ltd. | ANTIBODIES FOR ANTI-CLAUDIN-18A2 AND USE THEREOF |
IL264144B1 (en) * | 2016-07-08 | 2023-07-01 | Carsgen Therapeutics Co Ltd | Antibody 18a2 against claudin and its use |
JP2019531084A (ja) * | 2016-07-08 | 2019-10-31 | カースゲン セラピューティクス カンパニー リミテッドCarsgen Therapeutics Co., Ltd. | 抗クローディンタンパク質18a2の抗体及びその応用 |
CN109206524B (zh) * | 2018-09-25 | 2022-04-05 | 山东兴瑞生物科技有限公司 | 抗Claudin18A2嵌合抗原受体、其修饰的T细胞及T细胞制备方法和用途 |
CN109206524A (zh) * | 2018-09-25 | 2019-01-15 | 山东兴瑞生物科技有限公司 | 抗Claudin18A2嵌合抗原受体、其修饰的T细胞及T细胞制备方法和用途 |
CN114106183B (zh) * | 2019-01-15 | 2023-06-23 | 浙江道尔生物科技有限公司 | 抗cld18a2纳米抗体及其应用 |
CN114106183A (zh) * | 2019-01-15 | 2022-03-01 | 浙江道尔生物科技有限公司 | 抗cld18a2纳米抗体及其应用 |
CN114127109A (zh) * | 2019-05-30 | 2022-03-01 | 山东博安生物技术股份有限公司 | 靶向Claudin18.2的抗体或嵌合抗原受体 |
CN114630840A (zh) * | 2019-08-20 | 2022-06-14 | 苏州创胜医药集团有限公司 | 新型抗cldn18.2抗体 |
WO2021032157A1 (en) * | 2019-08-20 | 2021-02-25 | Mabspace Biosciences (Suzhou) Co., Limited | Novel anti-cldn18.2 antibodies |
CN111440245A (zh) * | 2020-04-10 | 2020-07-24 | 青岛麦迪赛斯医疗技术有限公司 | 一种用于靶向治疗实体瘤的嵌合抗原受体t淋巴细胞 |
CN111440245B (zh) * | 2020-04-10 | 2022-05-24 | 青岛麦迪赛斯医疗技术有限公司 | 一种用于靶向治疗实体瘤的嵌合抗原受体t淋巴细胞 |
WO2022028623A1 (zh) | 2020-08-07 | 2022-02-10 | 佧珐药业有限公司 | 工程化改造的细胞以及工程化改造细胞的方法 |
WO2022122709A1 (en) | 2020-12-07 | 2022-06-16 | Sotio Biotech A.S. | Antibody-drug conjugates based on humanized cldn18.2 antibodies |
CN113354739B (zh) * | 2021-01-11 | 2022-08-23 | 上海莱馥医疗科技有限公司 | 一种靶向表达Claudin18.2细胞的嵌合抗原受体及其应用 |
CN113354739A (zh) * | 2021-01-11 | 2021-09-07 | 上海莱馥医疗科技有限公司 | 一种靶向表达Claudin18.2细胞的嵌合抗原受体及其应用 |
WO2023274303A1 (zh) | 2021-06-29 | 2023-01-05 | 科济生物医药(上海)有限公司 | 调控细胞生理活动的嵌合多肽 |
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KR20170023181A (ko) | 2017-03-02 |
KR101950747B1 (ko) | 2019-02-21 |
EP3170842A1 (en) | 2017-05-24 |
US20170204177A1 (en) | 2017-07-20 |
US20220363751A1 (en) | 2022-11-17 |
PL3170842T3 (pl) | 2020-01-31 |
JP6664528B2 (ja) | 2020-03-13 |
CN105315375B (zh) | 2021-04-23 |
EP3170842A4 (en) | 2017-12-27 |
US10377822B2 (en) | 2019-08-13 |
JP2017522024A (ja) | 2017-08-10 |
JP2019122380A (ja) | 2019-07-25 |
CN105315375A (zh) | 2016-02-10 |
PT3170842T (pt) | 2019-09-30 |
US11198729B2 (en) | 2021-12-14 |
CN112979828A (zh) | 2021-06-18 |
US20200172613A1 (en) | 2020-06-04 |
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DK3170842T3 (da) | 2019-10-07 |
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