WO2024114546A1 - Anticorps monoclonal anti-fgfr4, lymphocytes t porteurs de récepteur antigénique chimérique ciblant fgfr4 et leur utilisation - Google Patents

Anticorps monoclonal anti-fgfr4, lymphocytes t porteurs de récepteur antigénique chimérique ciblant fgfr4 et leur utilisation Download PDF

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WO2024114546A1
WO2024114546A1 PCT/CN2023/134156 CN2023134156W WO2024114546A1 WO 2024114546 A1 WO2024114546 A1 WO 2024114546A1 CN 2023134156 W CN2023134156 W CN 2023134156W WO 2024114546 A1 WO2024114546 A1 WO 2024114546A1
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fgfr4
chimeric antigen
antigen receptor
variable region
chain variable
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万晓春
鄢德洪
刘婉
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中国科学院深圳先进技术研究院
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Definitions

  • the present invention belongs to the technical field of CAR-T cell preparation, and specifically relates to anti-FGFR4 monoclonal antibodies, chimeric antigen receptor T cells targeting FGFR4, and applications thereof.
  • liver cancer According to the International Agency for Research on Cancer (IARC), the mortality rate of liver cancer (10.2%) ranks second among malignant tumors, second only to lung cancer in total male cases. China is a country with a high incidence of liver cancer.
  • small molecule therapeutic drugs can be used to treat primary liver cancer with distant metastasis that cannot be operated on, such as the multikinase inhibitor sorafenib, but the prognosis is still poor, with a 5-year survival rate of only 3-11%.
  • phase III clinical trials of targeted treatments for liver cancer have also failed, which is largely attributed to the inability of drugs to fully exert their anti-tumor effects in the lesions in the late stage of cancer and the high tumor recurrence rate in the surrounding inflamed liver. Therefore, studying tumor cell immunotherapy is conducive to finding new strategies for effective treatment of liver cancer.
  • Chimeric antigen receptor modified T cells can specifically recognize and kill cancer cells expressing specific antigens, and can proliferate and activate in vivo to obtain a specific anti-tumor long-term mechanism. It is considered to be the only technology that has the potential to completely cure cancer. CAR-T technology has achieved great success in the treatment of blood tumors represented by acute B lymphocytic leukemia, but its efficacy in treating various solid tumors such as liver cancer is far from satisfactory.
  • Fibroblast growth factor receptor FGFR belongs to the receptor tyrosine kinase family (RTKs) and consists of 5 members, including FGFR1, FGFR2, FGFR3, FGFR4 and FGFR5 (also known as FGFRL1).
  • Fibroblast growth factors act on their receptors FGFRs to initiate signal cascades such as MAPK and STAT3 downstream of FGFRs, playing an important role in many physiological processes, such as embryogenesis, wound repair, and angiogenesis.
  • FGFs Fibroblast growth factors
  • MAPK and STAT3 downstream of FGFRs
  • FGFR4 is a potential therapeutic target for such tumors.
  • Overactivation of the FGF19/FGFR4 pathway can induce the formation of liver tumor cells, and knocking out FGFR4 can effectively block the tumorigenic effect mediated by FGF19/FGFR4.
  • FGFR4 is significantly negatively correlated with prognosis and overall survival rate.
  • CAR-T cell immunotherapy targeting FGFR4 may have a better prognosis for this group of liver cancer patients induced by FGF19/FGFR4.
  • the object of the present invention is to provide anti-FGFR4 monoclonal antibodies, chimeric antigen receptor T cells targeting FGFR4 and applications thereof.
  • the first aspect of the present invention provides an anti-FGFR4 monoclonal antibody, which comprises a light chain and a heavy chain, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.3.
  • a second aspect of the present invention provides an anti-FGFR4 single-chain antibody, which comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of the light chain variable region is shown in SEQ ID NO.5, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.3.
  • the anti-FGFR4 single-chain antibody further comprises a linker between the light chain variable region and the heavy chain variable region.
  • the third aspect of the present invention provides a nucleotide sequence encoding the anti-FGFR4 monoclonal antibody or the anti-FGFR4 single-chain antibody;
  • nucleotide sequence of the light chain variable region is shown as SEQ ID NO.4, and the nucleotide sequence of the heavy chain variable region is shown as SEQ ID NO.2;
  • nucleotide sequence encoding the anti-FGFR4 monoclonal antibody is as shown in SEQ ID NO.1;
  • the nucleotide sequence encoding the anti-FGFR4 single-chain antibody is as shown in SEQ ID NO.6.
  • the fourth aspect of the present invention provides the use of the anti-FGFR4 monoclonal antibody or the anti-FGFR4 single-chain antibody in the preparation of a drug for preventing and/or treating tumors;
  • the tumor includes hematological tumors and solid tumors
  • the tumor is liver cancer.
  • the fifth aspect of the present invention provides a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the anti-FGFR4 single-chain antibody;
  • the chimeric antigen receptor comprises, from N-terminus to C-terminus, the anti-FGFR4 single-chain antibody of claim 2 or 3, an extracellular hinge region, a transmembrane domain, a co-stimulatory domain and an activation domain.
  • extracellular hinge region is selected from the CD8 hinge region
  • the transmembrane domain is the CD8 transmembrane region
  • the co-stimulatory domain is 41BB;
  • the activation domain is CD3zeta.
  • the sixth aspect of the present invention provides a nucleotide sequence encoding the chimeric antigen receptor.
  • the seventh aspect of the present invention provides an mRNA transcribed from the nucleotide sequence of the chimeric antigen receptor.
  • the eighth aspect of the present invention provides an expression vector comprising a nucleotide sequence encoding the chimeric antigen receptor;
  • the expression vector is selected from a lentiviral vector, a retroviral vector or an adenoviral vector.
  • a ninth aspect of the present invention provides a chimeric antigen receptor T cell targeting FGFR4, which is a T cell whose cell surface is modified by the chimeric antigen receptor.
  • the tenth aspect of the present invention provides a method for preparing a chimeric antigen receptor T cell targeting FGFR4, the preparation method comprising: transfecting the mRNA transcribed from the nucleotide sequence of the chimeric antigen receptor, or the expression vector comprising the nucleotide sequence encoding the chimeric antigen receptor into T cells.
  • the eleventh aspect of the present invention provides a pharmaceutical composition, comprising the expression vector comprising the nucleotide sequence encoding the chimeric antigen receptor, or the chimeric antigen receptor T cell targeting FGFR4.
  • the twelfth aspect of the present invention provides the chimeric antigen receptor T cell targeting FGFR4, or the use of the pharmaceutical composition in the preparation of a drug for preventing and/or treating tumors;
  • the tumor includes hematological tumors and solid tumors
  • the tumor is liver cancer.
  • the present invention provides an anti-FGFR4 monoclonal antibody and an anti-FGFR4 single-chain antibody, which have unique antibody variable region sequences, high biological activity and efficient and specific targeting. Further provided is a chimeric antigen receptor CAR-FGFR4 targeting a variety of solid tumor-specific targets FGFR4 represented by liver cancer, wherein CAR-FGFR4 comprises an extracellular single-chain antibody variable region gene (ScFv) segment that specifically recognizes FGFR4, an extracellular hinge region, a transmembrane domain, a co-stimulatory domain, and an activation domain.
  • ScFv single-chain antibody variable region gene
  • CAR-FGFR4 is expressed on human killer T cells using genetic engineering technology to prepare FGFR4-CAR-T, and FGFR4-CAR-T is re-infused into the patient's body, which can efficiently and specifically kill cancer cells and can be used to treat a variety of cancers such as liver cancer.
  • FGFR4 monoclonal antibody 6G12F5 can specifically recognize FGFR4 on the surface of Jurkat cells. Black: isotype control; blue: 6G12F5; red: commercial antibody (Biolegend, 324305).
  • FIG. 3 Schematic diagram of the construction of the CAR-FGFR4BBzeta lentiviral vector. It uses the HIV NL4-3 clone as the backbone and EF1alpha as the promoter, and includes the anti-FGFR4ScFv segment, CD8 hinge segment, CD8 transmembrane region, and the intracellular 41BB and CD3zeta signaling pathway segments.
  • FIG. 4 Results of the test of effective transduction of Jurkat cells by FGFR4-CAR lentivirus. UTD, Jurkat cells were infected with the supernatant of untransfected cells as a negative control.
  • Figure 5 Flow chart of the production process of FGFR4-CAR-T.
  • FIG. 6 FGFR4-CAR-T targeted killing of T cells.
  • the figure shows the activity status of target cells Huh7 in the co-culture system.
  • CAR-T can target and kill tumor cells in vivo.
  • A) CAR-T can target and kill blood tumor cells in vivo. On days 0, 6, 10, and 15 after CAR-T cells were injected into tumor-bearing mice, mice were imaged by the small animal imager IVIS Lumina XR to monitor tumor growth and confirm the anti-tumor activity of CAR-T. It can be seen that over time, the target cells (carrying luciferase) were cleared by CAR-T cells. The figure shows the clearance of tumor cells on the 10th day after CAR-T cell injection.
  • B) FGFR4-CAR-T can target and kill liver cancer cell lines in vivo.
  • the hybridoma method (first proposed by Kohler et al., Nature, 256: 495 (1975)) was used.
  • Female BALB/c mice (6 weeks old) were immunized with FGFR4-Fc protein antigen (provided by Shenzhen Zhongke Aishen Pharmaceutical Co., Ltd., amino acid sequence and preparation method reference patent 201610067931.2).
  • Freund's complete adjuvant was used to emulsify the antigen for the first immunization.
  • Freund's incomplete adjuvant was used to emulsify the antigen and injected subcutaneously at 5 to 6 points. The amount of antigen injected per mouse was ⁇ 100 ⁇ g.
  • mice Ten days after the third immunization, a small amount of blood was collected from the mice by tail clipping for serum titer ELISA detection. Mice with high antibody titer (>1:100000) were selected for the fourth booster immunization, and the antigen protein was injected intraperitoneally, with 100 ⁇ g injected per mouse. 3 to 5 days after the fourth immunization, the mice were killed and their spleen cells were fused with SP2/0 cells, and stable hybridoma cells were obtained by culture in HAT medium.
  • Hybridoma cells that can secrete FGFR4 antibodies were screened by ELISA, subcloned by limiting dilution, and monoclonal hybridoma cell lines that can secrete FGFR4 antibodies were screened by ELISA.
  • the cells were expanded step by step and cryopreserved in liquid nitrogen for seed preservation.
  • mice Female BALB/c mice (8 weeks old) were intraperitoneally injected with Freund's incomplete adjuvant, 0.5 ml per mouse, and hybridoma cells in the logarithmic growth phase were intraperitoneally injected 3 to 5 days later, 1 to 5 ⁇ 10 5 cells (0.5 ml) per mouse were injected. The mice were killed 11 days after the injection of hybridoma cells to obtain ascites. Centrifuge at 3000rpm, 4°C for 10min, remove the precipitate, dilute the ascites with 10 times the volume of 1 ⁇ PB solution, mix well and filter through a 0.45 ⁇ m filter.
  • Protein G Protein G Sepharose 4 Fast Flow, GE Healthcare
  • purified FGFR4 antibody protein was run on SDS-PAGE (load amount 5.4 ⁇ g) and stained with Coomassie Brilliant Blue.
  • Monoclonal antibody hybridoma cells in the logarithmic growth phase were harvested, lysed by TRIZOL for RNA extraction, and cDNA was obtained after reverse transcription.
  • the heavy and light chain variable regions were amplified and obtained, and the non-functional VK gene was removed and cloned into the pMD18-T vector for sequencing.
  • the sequencing results were compared with the IMGT/V-QUEST database for further analysis.
  • the nucleotide sequence of the light chain variable region is SEQ ID NO.4:
  • the nucleotide sequence of anti-FGFR4 single-chain antibody (ScFv) is SEQ ID NO.6:
  • FGFR4 monoclonal antibody 6G12F5 can bind to FGFR4 protein on the surface of Jurkat membrane cells and is applied to flow cytometry detection ( Figure 2).
  • the chimeric antigen receptor targeting FGFR4 provided by the present invention comprises, from N-terminus to C-terminus, the anti-FGFR4 single-chain antibody in Example 1, an extracellular hinge region, a transmembrane domain, a costimulatory domain and an activation domain.
  • the extracellular hinge region is selected from the CD8 hinge region, the transmembrane domain is the CD8 transmembrane region, the co-stimulatory domain is 41BB, and the activation domain is CD3zeta.
  • This embodiment further provides the design of a chimeric antigen receptor (CAR-FGFR4) lentiviral expression plasmid targeting FGFR4.
  • CAR-FGFR4 lentiviral expression plasmid uses EF1alpha as a promoter and contains a ScFv segment that binds to FGFR4, a CD8 hinge segment, a CD8 transmembrane region, and a 41BB and CD3zeta signaling pathway segment located in T cells ( Figure 3).
  • the vector of the CAR-FGFR4 lentiviral plasmid comes from the NL4-3 clone of HIV. The internal structure of HIV has been destroyed to the greatest extent to remove its pathogenicity, so the vector only retains a part of the conserved region of HIV.
  • This example further constructs a CAR-FGFR4 lentiviral vector by lentiviral packaging, and the viral packaging uses the vesicular stomatitis virus glycoprotein (VSV-G) capsid.
  • VSV-G vesicular stomatitis virus glycoprotein
  • a third-generation lentiviral four-plasmid packaging system is used to co-transfect four plasmids into HEK293T cells to produce viral vectors.
  • all plasmids have been converted to kanamycin resistance.
  • the VSV-G capsid will assist the viral vector in adhering to the cell membrane and maintain the infectivity of the lentivirus.
  • the specific operation is: co-transfect HEK293T cells with CAR-FGFR4 expression plasmid (pWPXLd-CAR-FGFR4), packaging plasmid (pMDLg/pRRE&pRSV-Rev) and VSV-G envelope plasmid (pMD2.G), and collect the virus supernatant after 48 hours. Take 0.2ml of viral supernatant to infect Jurkat cells, harvest the cells two days later, stain with protein L, and use flow cytometry to detect the expression level of FGFR4-CAR on the membrane surface. The untransfected cell supernatant was used to infect Jurkat cells as a negative control (UTD).
  • CAR-FGFR4 expression plasmid pWPXLd-CAR-FGFR4 expression plasmid
  • packaging plasmid pMDLg/pRRE&pRSV-Rev
  • VSV-G envelope plasmid pMD2.G
  • the virus supernatant was collected to infect Jurkat cells, and the FGFR4-positive cells were as high as 58.6% by flow cytometry (Figure 4). This shows that the prepared CAR-FGFR4 lentiviral vector has a considerable transduction efficiency.
  • the preparation of CAR-T cells targeting FGFR4 can be performed by transfecting T cells using lentiviral vectors, retroviral vectors, adenoviral vectors, etc. expressing chimeric antigen receptors.
  • transfection methods such as liposomes and calcium phosphate can be used.
  • the preparation of FGFR4-CAR-T cells using the above-constructed CAR-FGFR4 lentiviral vector is described below.
  • the FGFR4-CAR-T cell preparation process is shown in Figure 5, which specifically includes the following steps:
  • cytotoxic lymphocytes from the patient's peripheral blood by centrifugal sedimentation and cell size screening
  • Physical methods such as ELISA and magnetic bead-targeted cell surface antigen-specific screening methods were used to screen out T cell populations with high activity and proliferation in the patient's peripheral blood;
  • the separated killer lymphocytes are specifically expanded in vitro.
  • the specific culture method is to activate T cells using magnetic beads coated with anti-CD3/anti-CD28 antibodies.
  • the cell preparation adopts a GMP-grade fully enclosed cell culture system and clinical-grade cell culture fluid. The cell preparation cycle is about 2 weeks. CD3 + T lymphocytes are obtained.
  • FGFR4-CAR-T After the in vitro expansion of FGFR4-CAR-T is completed, it is subjected to efficacy experiments (e.g., tumor killing, expansion, cytokine secretion ability, etc.) and safety experiments (e.g., endotoxin, allergen, bacteria/fungus/mycoplasma, etc.).
  • efficacy experiments e.g., tumor killing, expansion, cytokine secretion ability, etc.
  • safety experiments e.g., endotoxin, allergen, bacteria/fungus/mycoplasma, etc.
  • the cultured FGFR4-CAR-T is frozen in a cell freezing solution dedicated for reinfusion for the patient's reinfusion.
  • FGFR4-CAR-T cells can target and kill target cells

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Abstract

La présente invention concerne un anticorps monoclonal anti-FGFR4. La séquence d'acides aminés d'une région variable de chaîne légère de l'anticorps monoclonal anti-FGFR4 est telle que représentée dans SEQ ID NO. 5, et la séquence d'acides aminés d'une région variable de chaîne lourde est telle que représentée dans SEQ ID NO. 3. L'invention concerne également un anticorps à chaîne unique anti-FGFR4 et un récepteur antigénique chimérique (CAR) de l'anticorps à chaîne unique anti-FGFR4, et un lymphocyte T portant CAR ciblant FGFR4 (FGFR4-CAR-T) est préparé. FGFR4-CAR-T peut détruire de manière spécifique des cellules cancéreuses, lorsqu'il est administré à un patient, et peut être ainsi utilisé pour traiter divers cancers tels que le cancer du foie.
PCT/CN2023/134156 2022-11-29 2023-11-24 Anticorps monoclonal anti-fgfr4, lymphocytes t porteurs de récepteur antigénique chimérique ciblant fgfr4 et leur utilisation WO2024114546A1 (fr)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100169992A1 (en) * 2006-11-03 2010-07-01 Johannes Bange Fgfr4 antibodies
CN102149730A (zh) * 2008-07-08 2011-08-10 赛诺菲-安万特 Fgf-r4受体特异性拮抗剂
WO2014105849A1 (fr) * 2012-12-28 2014-07-03 Xoma (Us) Llc Anticorps spécifiques contre le fgfr4 et méthodes d'utilisation
WO2017049296A1 (fr) * 2015-09-20 2017-03-23 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Anticorps monoclonaux spécifiques du récepteur de facteur de croissance des fibroblastes 4 (fgfr4) et leurs procédés d'utilisation
CN111315777A (zh) * 2017-08-16 2020-06-19 埃克兹瑞斯公司 抗-fgfr4的单克隆抗体
CN111471109A (zh) * 2020-04-08 2020-07-31 中国科学院深圳先进技术研究院 一种抗成纤维细胞生长因子受体4单克隆抗体及其制备方法和用途
CN112812182A (zh) * 2019-11-15 2021-05-18 深圳宾德生物技术有限公司 一种靶向fgfr4的单链抗体、嵌合抗原受体、嵌合抗原受体t细胞及其制备方法和应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100169992A1 (en) * 2006-11-03 2010-07-01 Johannes Bange Fgfr4 antibodies
CN102149730A (zh) * 2008-07-08 2011-08-10 赛诺菲-安万特 Fgf-r4受体特异性拮抗剂
WO2014105849A1 (fr) * 2012-12-28 2014-07-03 Xoma (Us) Llc Anticorps spécifiques contre le fgfr4 et méthodes d'utilisation
WO2017049296A1 (fr) * 2015-09-20 2017-03-23 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Anticorps monoclonaux spécifiques du récepteur de facteur de croissance des fibroblastes 4 (fgfr4) et leurs procédés d'utilisation
CN111315777A (zh) * 2017-08-16 2020-06-19 埃克兹瑞斯公司 抗-fgfr4的单克隆抗体
CN112812182A (zh) * 2019-11-15 2021-05-18 深圳宾德生物技术有限公司 一种靶向fgfr4的单链抗体、嵌合抗原受体、嵌合抗原受体t细胞及其制备方法和应用
CN111471109A (zh) * 2020-04-08 2020-07-31 中国科学院深圳先进技术研究院 一种抗成纤维细胞生长因子受体4单克隆抗体及其制备方法和用途

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ALIJAJ NAGJIE, MOUTEL SANDRINE; GOUVEIA ZELIA L.; GRAY MAXIM; ROVERI MAURIZIO; DZHUMASHEV DZHANGAR; WEBER FLORIAN; MEIER GIANMARCO: "Novel FGFR4-Targeting Single-Domain Antibodies for Multiple Targeted Therapies against Rhabdomyosarcoma", CANCERS, MDPI AG, CH, vol. 12, no. 11, 10 November 2020 (2020-11-10), CH , pages 3313, XP093175501, ISSN: 2072-6694, DOI: 10.3390/cancers12113313 *

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