WO2022242710A1 - 一种特异性识别baff-r的嵌合抗原受体分子及其应用 - Google Patents

一种特异性识别baff-r的嵌合抗原受体分子及其应用 Download PDF

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WO2022242710A1
WO2022242710A1 PCT/CN2022/093782 CN2022093782W WO2022242710A1 WO 2022242710 A1 WO2022242710 A1 WO 2022242710A1 CN 2022093782 W CN2022093782 W CN 2022093782W WO 2022242710 A1 WO2022242710 A1 WO 2022242710A1
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baff
seq
amino acid
domain
acid sequence
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French (fr)
Chinese (zh)
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张新敏
粘伟红
许传营
何峰
周清
张剑冰
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Guangzhou Yinnuolai Biotechnology Co Ltd
Shanghai Escugen Biotechnology Co Ltd
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Guangzhou Yinnuolai Biotechnology Co Ltd
Shanghai Escugen Biotechnology Co Ltd
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Priority to EP22804030.9A priority Critical patent/EP4342913B1/en
Priority to JP2023572071A priority patent/JP2024519943A/ja
Priority to US18/513,330 priority patent/US20250268939A1/en
Publication of WO2022242710A1 publication Critical patent/WO2022242710A1/zh
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    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
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    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
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    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4214—Receptors for cytokines
    • A61K40/4215—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
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    • C07K2317/00—Immunoglobulins specific features
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2319/00—Fusion polypeptide
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present application relates to an engineered specific antigen recognition receptor molecule and an engineered immune effector cell comprising the receptor molecule.
  • CAR-T Chimeric Antigen Receptor T-cell
  • T lymphocytes play a major role in the tumor immune response and have a strong ability to kill tumor cells, but their function is limited by MHC.
  • the single-chain antibody that recognizes the target antigen in a non-MHC-restricted manner is connected with the hinge region, transmembrane domain, and T cell activation motif to assemble a CAR structure, and the CAR structure is integrated into the CAR structure with the help of a virus and other vectors.
  • T cells and finally enable T cells to specifically bind to antigens associated with the surface of tumor cells and be activated, that is, CAR-T.
  • CAR-T directly kills tumor cells by releasing perforin and granzyme B; on the other hand, activated CAR-T further releases cytokines to recruit human endogenous immune cells to kill tumor cells, achieving the purpose of effectively inhibiting tumors ; At the same time, immune memory T cells can also be formed to obtain a specific long-term anti-tumor mechanism.
  • a chimeric antigen receptor consists of an extracellular domain, a transmembrane domain, and an activation-stimulatory domain.
  • the extracellular domain is composed of an antigen recognition domain and a connecting hinge region (Hinge).
  • the antigen recognition domain is the basis for CAR to specifically bind tumor antigens. Its main structure is scFv, which is usually composed of the light chain (VL) and heavy chain (VH) of monoclonal antibodies connected by polypeptides, retaining the specificity of the antibody to the antigen. sex and affinity.
  • the hinge region connects the scFv and the transmembrane domain, and the hinge region of most CARs is derived from the hinge of IgG or the extracellular region of CD8 ⁇ /CD28.
  • the transmembrane domain connects the extracellular domain of the CAR with the intracellular activation stimulation domain and anchors the receptor to the T cell membrane. Commonly used transmembrane domains include those derived from CD4, CD8 ⁇ , CD28 and CD3 ⁇ . membrane domain.
  • the activation stimulation domain consists of an intracellular costimulatory domain and a signal transduction domain, and the costimulatory domain is usually from the CD28 receptor family (CD28, ICOS) or tumor necrosis factor receptor family (4-1BB, OX40, CD27) , and the signal transduction domain is usually the T cell receptor TCR/CD3 ⁇ chain, containing immunoreceptor tyrosine-based activation motifs (immunoreceptor tyrosine-based activation motifs, ITAMs).
  • the activation stimulation domain plays a key role in the process of mediating T cell signal transmission until T cell activation and proliferation, and finally completes the killing of tumors.
  • B-cell malignancies are high-incidence malignancies, and there are about 300,000 new patients worldwide each year, with an annual growth rate of 5%-7%.
  • Chimeric antigen receptor T cells (CAR-T) have been widely used in the treatment of B-cell malignancies, and CAR-T cell therapy targeting CD19 has a good effect on refractory B-cell malignancies.
  • Response rates in patients with leukemia (ALL) and various non-Hodgkin's lymphoma subtypes are as high as 80%-90%. However, only about 40% to 50% of patients have long-term efficacy, and CD19 downregulation/negative relapse occurs in about 30% of patients.
  • B lymphocyte stimulator is a member of the tumor necrosis factor (TNF) superfamily, also known as TNF and apoptosis ligand-related leukocyte expression ligand 1 (TNF-and ApoL related leukocyte-expressed ligand 1, TALL-1), TNF homologue that activates apoptosis, nuclear factor- ⁇ B and c-Jun NH2-terminal kinase , THANK), tumor superfamily member 13B (TNF superfamily member 13B, TNFSF13B), B cell-activating factor (B cell-activating factor, BAFF) and zTNF4, which belong to type II transmembrane proteins, can specifically bind to B cells, B cell survival, proliferation, development and differentiation play a key role.
  • TNF tumor necrosis factor
  • BAFF B cell-activating factor
  • zTNF4 which belong to type II transmembrane proteins
  • BAFF has three receptors, namely BAFF receptor (BAFF receptor, BAFF-R), B cell maturation antigen (B cell maturation antigen, BCMA) and transmembrane activation, calcium regulation and cyclophilin ligand interaction factor (TNFR homology transmembrane activator and calcium modulator and cyclophilin ligand intergrator, TACI), are type III transmembrane proteins.
  • BAFF receptor BAFF receptor
  • BAFF-R B cell maturation antigen
  • BCMA B cell maturation antigen
  • TNFR homology transmembrane activator and calcium modulator and cyclophilin ligand intergrator, TACI type III transmembrane proteins.
  • BCMA and TACI can not only bind to BAFF, but also bind to another member of the TNF ligand family, a proliferation-inducing ligand (APRIL), and BAFF-R is the specific receptor of BAFF.
  • APRIL proliferation-inducing ligand
  • BAFF-R
  • BAFF-R B-cell activating factor receptor, B cell activating factor receptor
  • B cell activating factor receptor also known as tumor necrosis factor superfamily member 13C (tumor necrosis factor receptor superfamily member 13C, TNFRSF13C)
  • Tumor necrosis factor receptors TNFR
  • the published human BAFF-R amino acid sequence has a total length of 184 amino acids, of which amino acids 1-78 are the extracellular domain, amino acids 79-99 are the transmembrane domain, and amino acids 100-184 are the intracellular domain.
  • BAFF-R is mainly expressed in B cells, promotes the survival and proliferation of B cells by activating the NF- ⁇ B pathway, and is highly expressed on the surface of various B cell malignant tumor cells.
  • the imbalance of BAFF-BAFF-R signaling pathway can cause the immune imbalance of the body, including a series of diseases caused by abnormally high expression of BAFF-R in abnormal or normal cells, such as autoimmune diseases, graft-versus-host diseases and tumors.
  • This target covers a wide range of stages in the maturation of B cells, and can target all B cell malignancies except plasma cell lesions (multiple myeloma). It has a wide range of applications, and this target is not expressed in stem cells and B cells. progenitor cells, and therefore does not permanently harm the recovery and functional reconstitution of normal B cells, with a favorable safety profile.
  • This application provides a chimeric antigen receptor molecule (CAR molecule) that specifically recognizes BAFF-R and an engineered immune effector for diseases caused by the imbalance of the B cell BAFF-BAFF-R signaling pathway, such as B cell malignancies cells (such as chimeric antigen receptor T cells (CAR-T)).
  • CAR molecule chimeric antigen receptor molecule
  • B cell malignancies cells such as chimeric antigen receptor T cells (CAR-T)
  • Studies have proved that the chimeric antigen receptor T cells targeting BAFF-R of the present application have a strong killing effect on tumor cells and an in vivo tumor suppressing effect, and have potential for development.
  • this application relates to:
  • a chimeric antigen receptor molecule that specifically recognizes BAFF-R comprising a specific recognition domain and an activation stimulation domain, and a transmembrane structure between the specific recognition domain and the activation stimulation domain Domain, wherein, the specific recognition domain comprises selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 Amino acid sequences or amino acid sequences having about 70% or more sequence identity with them, for example having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity with them amino acid sequence.
  • the chimeric antigen receptor molecule that specifically recognizes BAFF-R according to item 1, the specific recognition domain comprising:
  • a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or an amino acid sequence having about 70% or more sequence identity therewith, For example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity therewith; and
  • a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or an amino acid sequence having about 70% or more sequence identity therewith, For example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto.
  • the present application relates to a chimeric antigen receptor molecule that specifically recognizes BAFF-R, comprising a specific recognition domain and an activation stimulation domain, and the specific recognition domain and activation stimulation domain
  • the transmembrane domain between the domains wherein the specific recognition domain comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises three complementarity determining regions CDR H1, CDR H2 and CDR H3, and the CDR H1 comprises the amino acid sequence shown in SEQ ID NO:6, the CDR H2 comprises the amino acid sequence shown in SEQ ID NO:7, and the CDR H3 comprises the amino acid sequence shown in SEQ ID NO:8
  • the amino acid sequence of; said light chain variable region comprises CDR L1, CDR L2 and CDR L3, and said said CDR L1 comprises the amino acid sequence shown in SEQ ID NO:10, and said CDR L2 comprises SEQ ID NO:11
  • the amino acid sequence shown, the CDR L3 comprises the amino acid sequence shown in SEQ ID NO:
  • CDR H1, CDR H2, CDR H3 and CDR L1, CDR L2, CDR L3 are according to Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991) , vols.1-3) determined.
  • the human antibody framework region is a human common framework region.
  • the human universal framework region comprises the framework region sequence of the kappa I subgroup ( ⁇ I subgroup) of VL and the framework region sequence of the III subgroup (VH III subgroup) of VH.
  • the framework region sequence is determined according to Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols.1-3).
  • the human universal framework region comprises 1-15, 1-10, 2-9, 3-8, 4-7 or 5-6 amino acid changes.
  • the chimeric antigen receptor molecule that specifically recognizes BAFF-R according to item 4, wherein the heavy chain variable region is connected to the light chain variable region (for example, connected by a peptide chain, such as A linker or linker consisting of (G)n(S)m, wherein n or m can be any integer between 1-10) forms scFv, and the scFv comprises the amino acid sequence shown in SEQ ID NO:4 Or an amino acid sequence having about 70% or more sequence identity therewith, such as an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity therewith.
  • a peptide chain such as A linker or linker consisting of (G)n(S)m, wherein n or m can be any integer between 1-10) forms scFv, and the scFv comprises the amino acid sequence shown in SEQ ID NO:4 Or an amino acid sequence having about 70% or more sequence identity therewith, such as an amino acid sequence having
  • the chimeric antigen receptor molecule that specifically recognizes BAFF-R according to any one of items 1-5, wherein the activation stimulating domain comprises a signal transduction comprising an immunoreceptor tyrosine activation motif domain.
  • the signal transduction domain is derived from CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ R1 ⁇ , CD79 ⁇ , CD79 ⁇ , Fc ⁇ RIIa , DAP10, or the intracellular region of a DAP12 molecule.
  • the signal transduction domain comprises the signal transduction domain of the intracellular region of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ R1 ⁇ , CD79 ⁇ , CD79 ⁇ , Fc ⁇ RIIa, DAP10 or DAP12 molecules, or retains the same variant of the function.
  • the signaling domain comprises the signaling domain of the intracellular region of CD3 ⁇ or Fc ⁇ RI ⁇ , or a variant that retains the same function thereof.
  • An amino acid sequence with sequence identity for example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity therewith.
  • the activation stimulating domain further comprises one, two or more co-stimulatory domains.
  • the costimulatory domain is derived from a costimulatory domain of the CD28 receptor family or the tumor necrosis factor receptor family.
  • the chimeric antigen receptor molecule specifically recognizing BAFF-R comprises intracellular regions of one or more of the following molecules: CD27, CD28, 4- 1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80(KLRF1), CD160 , CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-l , ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, IT
  • the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 18 or has more than about 70% of the sequence therewith
  • An amino acid sequence having identity for example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity therewith.
  • transmembrane domain comprises a transmembrane domain selected from the group of molecules or it retains the same Functional variants: TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40 , CD2, CD27, LFA-1, ICOS (CD278), 4-1BB, CTLA-4, GITR, CD40, BAFFR, LIGHTR, SLAMF7, NKp80, CD160, CD19, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA1, VLA1, CD49 ⁇ , ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103,
  • transmembrane domain comprises the transmembrane domain of CD8 ⁇ or a variant thereof retaining the same function.
  • the transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having about 70% or more sequence identity therewith, such as about 75%, 80%, 85% therewith %, 90%, 95%, 97%, 98%, or more than 99% sequence identity of amino acid sequences.
  • the chimeric antigen receptor molecule specifically recognizing BAFF-R according to any one of items 1-13, wherein the transmembrane domain is directly linked to the specific recognition domain.
  • the hinge region comprises an amino acid sequence selected from the group consisting of an Fc fragment of human CD8 ⁇ or an antibody or a functional equivalent, fragment or derivative thereof, a hinge region of human CD8 ⁇ or an antibody or a functional equivalent thereof substances, fragments or derivatives, CH2 regions of antibodies, CH3 regions of antibodies, artificial spacers and combinations thereof.
  • the hinge region comprises the hinge region amino acid sequence of IgG, IgD, CD8 ⁇ , or CD28, or variants thereof that retain the same function.
  • the chimeric antigen receptor molecule that specifically recognizes BAFF-R according to any one of items 1-16, further comprising a signal peptide selected from signal peptides of any secreted protein or membrane protein sequence.
  • the signal peptide sequence is the signal peptide of CD8 ⁇ .
  • the chimeric antigen receptor molecule that specifically recognizes BAFF-R according to any one of item 17, wherein the signal peptide sequence comprises the amino acid sequence as in SEQ ID NO: 2 or has more than about 70% thereof A polynucleotide sequence with sequence identity, for example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity therewith.
  • nucleic acid molecule according to item 19, which comprises a polynucleotide sequence as shown in SEQ ID NO: 3 or a polynucleotide sequence having more than about 70% sequence identity thereof, for example having about 75%, 80% %, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity of polynucleotide sequences.
  • the nucleic acid molecule comprises a nucleotide sequence obtained by codon-optimizing the nucleotide sequence in SEQ ID NO: 3 for a specific cell or tissue.
  • the coding sequence of the membrane localization signal peptide molecule comprises a polynucleotide sequence as shown in SEQ ID NO: 1 or a polynucleotide sequence having more than about 70% sequence identity thereof, for example having about Polynucleotide sequences having 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or greater sequence identity.
  • nucleic acid molecule according to item 21 which sequentially comprises the polynucleotide sequence described below from the 5' end to the 3' end or a polynucleotide sequence having about 70% or more sequence identity therewith, for example having Polynucleotide sequences with about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or more than 99% sequence identity: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO :13, SEQ ID NO:15, SEQ ID NO:17 and SEQ ID NO:19.
  • An engineered immune effector cell comprising a chimeric antigen receptor molecule that specifically recognizes BAFF-R as described in any one of items 1-18 or a chimeric antigen receptor molecule as described in any one of items 19-22. nucleic acid molecule.
  • An engineered T cell receptor for specific recognition of BAFF-R comprising: 11.
  • T cell receptor comprising an alpha chain variable region (V ⁇ ) and a beta chain variable region (V ⁇ ), wherein:
  • Said V ⁇ comprises an amino acid sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or an amino acid sequence having more than about 70% sequence identity with them, and said V ⁇ comprises an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:8 The amino acid sequence of ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or an amino acid sequence having more than about 70% sequence identity with them;
  • the V ⁇ comprises an amino acid sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or an amino acid sequence having more than about 70% sequence identity with them
  • the V ⁇ comprises an amino acid sequence selected from The amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or an amino acid sequence having more than 70% sequence identity with them.
  • An engineered immune effector cell comprising the T cell receptor according to item 25 or 26 and/or the nucleic acid molecule according to item 27.
  • a pharmaceutical composition comprising a therapeutically effective amount of a chimeric antigen receptor molecule specifically recognizing BAFF-R as described in any one of items 1-18 or a T cell as described in item 25 or 26 A receptor, a nucleic acid molecule according to any one of items 19-22 or item 27, or an engineered immune effector cell according to any one of items 23, 24, 28 or 29.
  • a method of treating or preventing a disease in a subject in need thereof comprising administering to the subject a pharmaceutical composition according to item 30, said disease being selected from the group consisting of B cell BAFF-BAFF-R signaling Diseases caused by pathway imbalances.
  • the chimeric antigen receptor molecule that specifically recognizes BAFF-R provided by this application can specifically recognize and bind to BAFF-R and activate downstream signaling pathways to cause, promote or enhance the immune response against BAFF-R, for example, in vitro Or specifically kill target cells expressing BAFF-R in vivo, and finally achieve the purpose of treating or preventing diseases caused by the imbalance of B cell BAFF-BAFF-R signaling pathway.
  • Fig. 1 is an example of the structure of the chimeric antigen receptor of BAFF-R of the present invention.
  • Fig. 2 is a plasmid map of an exemplary anti-BAFF-R chimeric antigen receptor lentiviral expression vector pCDH-EF1a-H90-11CAR-4-1BB-EGFRt.
  • Fig. 3 is the plasmid map of the lentiviral expression vector pCDH-EF1a-EGFRt-AT-Free (delete T2A) as a control.
  • Figure 4 is the result of flow cytometry detection of CAR positive rate using FITC-EGFRt antibody.
  • Figure 5 shows the results of flow cytometry detection of CAR positive rate using BAFF-R protein.
  • Figure 6 is a graph showing the results of H90-11 CAR-T cells specifically killing Nalm6-luciferase target cells.
  • Figures 7A-7B are the results of secretion of IL-2 ( Figure A) and IFN- ⁇ ( Figure B) in the supernatant after H90-11 CAR-T cells were co-cultured with Nalm6-luciferase target cells.
  • Figures 8A-8C show the anti-tumor effects of H90-11 CAR-T in mice.
  • Figure 8A is the in vivo imaging results of mice at different time points
  • Figure 8B is a graph of absolute luminescence in mice at different time points
  • Figure 8C is a graph of body weight changes in mice within 22 days .
  • this application designed a new type of chimeric antigen receptor molecule that recognizes BAFF-R for the BAFF-R protein specifically and highly expressed on the surface of B cells, and provided Nucleic acid molecules encoding the receptors, engineered immune effector cells comprising the receptors, pharmaceutical compositions, and uses thereof for treating or preventing diseases caused by imbalance of B cell BAFF-BAFF-R signaling pathway, such as B The purpose of cellular malignancies.
  • receptor refers to a biomacromolecule capable of binding to hormones, neurotransmitters, drugs, or intracellular signaling molecules and causing changes in cell functions. According to the location of receptors in the cell, receptors can be divided into two categories: cell membrane receptors and intracellular receptors. The receptor itself contains at least two active sites: one is the active site that recognizes and binds to the ligand (referred to as the "specific recognition domain” in this application); referred to as the "activation stimulation domain").
  • the activation-stimulating domain can only produce a response after the receptor binds to the ligand to form a binary complex and is allosteric, thereby initiating a series of biochemical reactions, and finally causing the effector cells where the receptor is located to produce biological effects.
  • the "activation stimulating domain” comprises a signal transduction domain, or comprises one or more signal transduction domains and one or more co-stimulatory domains.
  • a "signal transduction domain” provides a first signal for activating lymphocytes, such as activating T cells or NK cells, while a "co-stimulatory domain” provides a second signal for activating lymphocytes.
  • a transmembrane domain is further included between the antigen recognition region and the activation stimulation domain.
  • the antigen recognition region and the transmembrane domain are connected by a hinge region.
  • intracellular domain and “intracellular region” may be used interchangeably, and may refer to a domain of a receptor molecule that is located in the cell and plays a role in signal transduction after the receptor binds to a ligand.
  • chimeric antigen receptor is a class of engineered cell surface receptors, generally expressed on immune cells, which mediate the killing of engineered immune cells against specific target tumor cells or other diseased cells .
  • CAR also contains a specific recognition domain and an activation stimulation domain.
  • the specific recognition domain of CAR can specifically recognize the antigen, so it is also called the antigen recognition region.
  • the antigen recognition region of CAR is located outside the cell membrane.
  • the antigen recognition region is a single chain variable fragment (scFv) of Ig, a "scFv” comprising one heavy chain variable region (VH) and one light chain variable region (VL) of Ig.
  • the VL and VH regions are linked together by a peptide chain.
  • CH heavy chain
  • CL light chain
  • VL light chain variable region
  • VH variable region
  • framework region refers to the domains in naturally occurring immunoglobulins and the corresponding domains of synthetic (eg recombinant) binding proteins (eg humanized antibodies).
  • the basic structural unit of naturally occurring immunoglobulins, such as IgG, is a tetramer with two light chains and two heavy chains.
  • the amino-terminal (“N) portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminus ("C" portion) of each chain defines a constant region, with light chains having a single constant domain and heavy chains typically having three constant domains and a hinge region.
  • the naturally occurring light chain structure of an IgG molecule is N-VL-CL-C
  • the structure of an IgG heavy chain is N-VH-CH1-H-CH2-CH3-C (where H is the hinge region).
  • CH1, CH2 and CH3 are the components of the constant region of the heavy chain of the antibody
  • the CH3 region is involved in the binding of receptors on the cell membrane surface
  • CH2 is involved in the complement activation pathway and is the complement binding site
  • CH1 has the Ig allotype genetic marker.
  • variable region of IgG consists of complementarity determining regions (CDRs) and non-CDR segments called framework regions.
  • CDRs complementarity determining regions
  • framework regions non-CDR segments
  • the CDR contains residues that contact the antigen
  • the framework region is used to maintain the structure of the variable region and determine the position of the CDR loop.
  • the VL and VH domains have the following structure: N-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C.
  • the specific recognition domain is a polypeptide or protein ligand other than scFv, for example, modified interleukin-13 (IL-13) molecules can be used to prepare specific recognition domains, thereby obtaining IL-13R ⁇ 2-specific CAR for the treatment of glioblastoma.
  • IL-13 modified interleukin-13
  • single-chain variable fragment refers to a form of an antibody comprising the variable regions of only the heavy (VH) and light (VL) chains, defined by Linker peptide ligation.
  • scFv can be expressed as a single chain polypeptide.
  • the light and heavy chains can be in any order, eg, VH-linker-VL or VL-linker-VH, as long as the specificity of the scFv for the target antigen is preserved.
  • linkers can also be omitted.
  • linker refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any of the domains/regions of the CAR of the invention.
  • Linkers can be composed of flexible residues such as glycine and serine so that adjacent protein domains are free to move relative to each other. Longer linkers can be used when it is desired to ensure that two adjacent domains do not sterically interfere with each other.
  • Alternative linkers are known to those skilled in the art and may be used in conjunction with alternative embodiments of the invention.
  • antibody refers to an intact immunoglobulin or an FcRn-binding fragment having an Fc (fragment crystallizable) region or an Fc region (referred to herein as an "Fc fragment” or “Fc domain”).
  • Fc fragment fragment crystallizable region or an Fc region
  • Fc domain Monoclonal or polyclonal antigen-binding fragments. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen binding fragments include, inter alia, Fab, Fab', F(ab') 2 , Fv, dAb and complementarity determining region (CDR) fragments, single chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies and such A polypeptide comprising at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide.
  • the Fc domain comprising the CH2 and CH3 portions of the two heavy chains, can be produced by recombinant DNA techniques or by enzymatic (eg papain cleavage) or by chemical cleavage of intact antibodies.
  • antibody fragment refers to a fragment of a protein that comprises only a portion of an intact antibody, usually including the antigen-binding site of the intact antibody, and thus retains the ability to bind antigen.
  • antibody fragments described herein include, but are not limited to: (i) Fab fragments having VL, CL, VH, and CH1 domains; (ii) Fab' fragments, i.e., having one or more halves at the C-terminus of the CH1 domain; Fab fragments with cystine residues; (iii) Fd fragments with VH and CH1 domains; (iv) with VH and CH1 domains and one or more cysteine residues at the C-terminus of the CH1 domains (v) Fv fragments with VL and VH domains of antibody single arm; (vi) dAb fragments consisting of VH domains (Ward et al., Nature 341, 544-546 (1989)); ( vii) isolated CDR regions; (viii) F
  • the term "specifically binds" means that an antibody binds to a specific antigen and does not bind to other antigens other than the specific antigen.
  • the specific antigens may be one or more, and in some embodiments, the specific antigens contain the same or similar antigenic epitopes.
  • the specific binding has a binding affinity of at least 10 ⁇ 6 M contacts between the antibody and the antigen.
  • the antibody binds with an affinity of at least about 10 ⁇ 7 M, preferably 10 ⁇ 8 M, 10 ⁇ 9 M, 10 ⁇ 10 M, 10 ⁇ 11 M, or 10 ⁇ 12 M.
  • polynucleotide or “nucleic acid” and “nucleic acid molecule” are used interchangeably, including but not limited to DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA) , shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA and/or tRNA.
  • RNA complementary DNA
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • shRNA small hairpin RNA
  • snRNA small nuclear RNA
  • snoRNA short nucleolar RNA
  • miRNA miRNA
  • vector refers to a polynucleotide sequence (such as a foreign gene) that can be introduced into a host cell to transform the host and facilitate the expression (such as transcription and expression) of the introduced sequence. translation) carrier.
  • the vectors include plasmids, phages, viruses and the like.
  • the "signal transduction domain” generally includes immunoreceptor tyrosine-based activation motifs (immune-receptor tyrosine-based activation motifs, ITAM), the basic composition of which is: YXXL/V. Wherein Y is tyrosine, L/V refers to leucine or valine, and X can be any amino acid.
  • ITAM immunoreceptor tyrosine-based activation motifs
  • ITAM immunoreceptor tyrosine-based activation motifs
  • the "co-stimulatory domain” also known as “co-stimulatory signal domain” is mainly used to provide co-stimulatory signals to enhance the ability of immune cells, including, for example, enhancing the proliferation, survival and/or development of memory cells.
  • the "co-stimulatory domain” is selected from CD28, 4-1BB (CD137), OX40 (CD134) and the like.
  • transmembrane domain also known as “transmembrane region” refers to a thermodynamically stable protein structural region anchored in the cell membrane.
  • Transmembrane domains can be obtained from natural proteins, such as those derived from the T cell receptor (TCR).
  • TCR T cell receptor
  • the transmembrane domain is selected from the group consisting of transmembrane domains of CD4, CD8 ⁇ , CD28, and CD3 ⁇ .
  • the "hinge region” is a peptide chain connecting the antigen recognition region and the transmembrane domain, and is usually elastic.
  • the hinge region is derived from the hinge of IgG or the extracellular region of CD8 ⁇ /CD28.
  • the “hinge” of IgG refers to the region between the CH1 and CH2 functional regions of IgG, which usually contains a large amount of proline.
  • the "anti-BAFF-R chimeric antigen receptor” is one of the “receptors that specifically recognize BAFF-R". It specifically refers to a chimeric antigen receptor containing an anti-BAFF-R antibody heavy chain variable region and a light chain variable region in the antigen recognition region.
  • the chimeric antigen receptor can specifically recognize BAFF-R, and activate the downstream pathway of the cell where the receptor is located through signal transduction.
  • the anti-BAFF-R chimeric antigen receptor is located on the surface of immune cells selected from NK cells, macrophages, neutrophils, T cells, etc. After specifically recognizing BAFF-R, Activating the immune effector cells to produce immune effects such as humoral immunity, cellular immunity, and/or cytotoxicity; or activating the immune cells to further proliferate, etc.
  • immune effector cells refer to cells capable of achieving immune effects and immune responses against target antigens or target cells, such as immune killing effects and immune response effects, such as T cells and natural killer (NK) cells.
  • CAR-T is a chimeric antigen receptor T cell, which is a T cell expressing a chimeric antigen receptor molecule on the cell surface, which can recognize the target antigen on the cell surface.
  • CAR-T has been developed to the fourth generation.
  • the CAR molecule of the first-generation CAR-T is composed of the CD3 ⁇ chain or the signal transduction domain of Fc ⁇ RI ⁇ and the fusion of the antigen recognition region, and does not contain a co-stimulatory domain.
  • the first-generation CAR-T has limited proliferation ability in vivo and is prone to apoptosis.
  • the second-generation CAR adds a co-stimulatory domain, such as CD28 or 4-1BB (CD137).
  • CD28 has strong anti-tumor activity, and the advantage of 4-1BB is that it can prolong the survival time of T lymphocytes and maintain its anti-tumor effect.
  • the second-generation CAR has stronger proliferation ability than the first-generation CAR, and can secrete more cytokines and anti-apoptotic proteins.
  • the third-generation CAR-T can not only express two co-stimulatory signal molecules at the same time, but also secrete more IFN- ⁇ , which has higher anti-tumor cytotoxicity.
  • the fourth-generation CAR-T can also secrete specific cytokines (such as IL-12) in tumors, thereby changing the tumor microenvironment, and influencing and activating other immune cells to generate an immune response.
  • the "signal peptide” refers to a short peptide chain that guides the transfer of newly synthesized proteins to the secretory pathway, usually 5-30 amino acids in length.
  • the signal peptide is a membrane localization signal peptide, i.e., an amino acid sequence used to direct transmembrane transfer (localization) of a protein.
  • the signal peptide is located at the N-terminus of the amino acid sequence.
  • the coding sequence of the signal peptide is usually located after the initiation codon, which is an RNA region encoding a hydrophobic amino acid sequence. After the signal peptide guides the protein to complete its positioning, it is usually excised under the action of signal peptidase.
  • the "variant" of the protein or nucleic acid refers to a protein or nucleic acid that has the same function as compared with a specific protein or nucleic acid, but has one or more mutations in its sequence.
  • the "variant" of a certain protein refers to the mutation of one or more amino acids in the amino acid sequence of the certain protein through artificial or natural mutation, which is similar to the certain protein. Proteins having the same function and at least 70% sequence identity.
  • Identity of sequences as used herein refers to the degree of similarity between amino acid sequences or between nucleotide sequences as determined by sequence alignment software, such as BLAST.
  • the imbalance of BAFF-BAFF-R signaling pathway can cause various diseases, including autoimmune diseases, graft-versus-host diseases and tumors.
  • the neoplasms include all B-cell malignancies except plasma cell lesions (multiple myeloma), such as mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, Burkitt's Lymphoma, lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, etc.
  • the autoimmune diseases include, for example, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, Sjögren's syndrome, or autoimmune hemolytic anemia.
  • the term "effective amount” or “therapeutically effective amount” refers to the amount of a pharmaceutical composition comprising one or more peptides, proteins, nucleic acids or mutants, variants, analogs or derivatives thereof disclosed in the present application. amount, and the patient or subject receiving the "effective amount” or “therapeutically effective amount” of the pharmaceutical composition can obtain a reasonable benefit/risk ratio of medical treatment, thereby alleviating or preventing at least one or more of the diseases or conditions A variety of symptoms, to achieve the desired therapeutic or preventive effect.
  • the term “about” refers to the usual error range for the respective value readily known to those skilled in the art. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. As used herein, the term “about” when preceding a numerical value means within 10% above or below the numerical value. For example, “about 100” encompasses 90 and 110.
  • the present application provides a receptor that specifically recognizes BAFF-R.
  • the receptor that specifically recognizes BAFF-R is a chimeric antigen receptor (CAR).
  • the receptor that specifically recognizes BAFF-R is a chimeric antigen receptor (CAR)
  • CAR chimeric antigen receptor
  • the specific recognition domain refers to the part of the CAR that specifically binds the target antigen on the target cell.
  • the specific recognition domain comprises an antibody or a functional equivalent thereof or a fragment or derivative thereof, for example may comprise a full-length heavy chain, a Fab fragment, a single chain Fv (scFv) fragment, a bivalent Single-chain antibodies or diabodies, each specific for a target antigen.
  • the specific recognition domain comprises a T cell receptor (TCR) or an antigen recognition portion thereof, such as a single chain TCR (scTCR).
  • TCR T cell receptor
  • scTCR single chain TCR
  • the specific recognition domain comprises a sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: The amino acid sequence of 12 or an amino acid sequence having about 70% or more sequence identity with them, for example having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity with them identical amino acid sequences.
  • the specific recognition domain comprises:
  • a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or an amino acid sequence having about 70% or more sequence identity therewith, For example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity therewith; and
  • a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or an amino acid sequence having about 70% or more sequence identity therewith, For example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity thereto.
  • the heavy chain variable region comprises three complementarity determining regions, respectively CDR H1, CDR H2 and CDR H3, and the CDR H1, CDR H2 and CDR H3 comprise SEQ ID NO: 6,
  • the light chain variable region also includes 3 complementarity determining regions, respectively CDR L1, CDR L2 and CDR L3, and the CDR L1, CDR L2 and CDR L3 respectively comprise the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 or an amino acid sequence having about 70% or more sequence identity therewith, for example having about 75%, 80%, Amino acid sequences having 85%, 90%, 95%, 97%, 98%, or 99% or greater sequence identity.
  • the heavy chain variable region and the light chain variable region further comprise a framework region, preferably a human antibody framework region.
  • the heavy chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 5 or an amino acid sequence having about 70% or more sequence identity therewith, such as about 75%, 85%, 90%, 95%, 97%, 98%, or an amino acid sequence of more than 99% sequence identity; and the light chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 9 or has about 70% thereof An amino acid sequence with the above sequence identity, for example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity therewith.
  • the heavy chain variable region and the light chain variable region are connected by a linker to form scFv, and the scFv comprises the amino acid sequence shown in SEQ ID NO: 4 or has more than about 70% of it
  • An amino acid sequence with sequence identity for example, an amino acid sequence having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity therewith.
  • CAR usually includes a specific recognition domain, an activation stimulation domain, and a transmembrane domain between them.
  • an activation stimulation domain usually includes a specific recognition domain, an activation stimulation domain, and a transmembrane domain between them.
  • the difference mainly lies in the difference in the activation stimulation domain, including:
  • the second-generation CAR whose activation-stimulatory domain contains an intracellular signal transduction domain and a co-stimulatory domain.
  • the third-generation CAR whose activation stimulation domain contains an intracellular signal transduction domain and two co-stimulatory domains.
  • the CAR is a first-generation CAR in which the signal transduction domain may be cytoplasmic and may transduce effector function signals and direct the cell to perform its specialized functions.
  • signaling domains include, but are not limited to, the zeta chain of a T cell receptor or any homologue thereof (e.g., ⁇ chain, Fc ⁇ R1 ⁇ , ⁇ chain, MB1 (Ig ⁇ ) chain, B29 (Ig ⁇ ) chain, etc.) , CD3 polypeptides ( ⁇ , ⁇ , and ⁇ ), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and other molecules involved in T cell transduction, such as CD2, CD5 and CD28.
  • the zeta chain of a T cell receptor or any homologue thereof e.g., ⁇ chain, Fc ⁇ R1 ⁇ , ⁇ chain, MB1 (I
  • the intracellular signal transduction domain can be a human CD3 ⁇ chain, Fc ⁇ RIII, Fc ⁇ RI, the cytoplasmic tail of an Fc receptor, a cytoplasmic receptor with an immunoreceptor tyrosine activation motif (ITAM), or a combination thereof . Additional intracellular signaling domains will be apparent to those skilled in the art and may be used in conjunction with alternative embodiments of the invention.
  • the signal transduction domain comprised by the activation stimulation domain is a signal transduction domain comprising an immunoreceptor tyrosine activation motif.
  • the intracellular signaling domain comprises a signaling domain selected from one or more of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ R1 ⁇ , CD79 ⁇ , CD79 ⁇ , Fc ⁇ RIIa, Intracellular regions of DAP10 and DAP12 molecules, or variants that retain the same function.
  • the intracellular signal transduction domain comprises the intracellular region of CD3 ⁇ or Fc ⁇ RI ⁇ , or a variant retaining the same function thereof.
  • the intracellular signaling domain is derived from the intracellular region of CD3 ⁇ .
  • the intracellular signal transduction domain comprises an amino acid sequence as shown in SEQ ID NO: 20 or an amino acid sequence having more than about 70% sequence identity therewith, for example having about 75%, 80% , 85%, 90%, 95%, 97%, 98%, or 99% or more amino acid sequence identity.
  • the CAR is a second-generation CAR, wherein the signal transduction domain is the above-mentioned signal transduction domain, and the co-stimulatory domain further included is the following molecular intracellular Any of the regions: CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80(KLRF1), CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d , ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11
  • the CAR is a third-generation CAR, wherein the signal transduction domain is the above-mentioned signal transduction domain, and the co-stimulatory domain further included is the following molecular intracellular Any two, three or more of the regions: CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, LFA-1, LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, LIGHTR, SLAMF7, CD7, NKp80(KLRF1), CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA -6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, IT
  • the co-stimulatory domain is preferably the 4-1BB intracellular domain, the CD28 intracellular domain, the OX40 intracellular domain, or variants thereof that retain the same function.
  • the co-stimulatory domain comprises an intracellular region derived from 4-1BB.
  • the co-stimulatory domain comprises an amino acid sequence as shown in SEQ ID NO: 18 or an amino acid sequence having more than about 70% sequence identity therewith, such as about 75%, 80%, Amino acid sequences having 85%, 90%, 95%, 97%, 98%, or 99% or greater sequence identity.
  • the effector cells where the CAR is located can be appropriately activated when the CAR binds to the ligand. Signal.
  • the transmembrane domain between the specific recognition domain and the activation stimulation domain can be derived from any protein with a transmembrane domain (including type I, type II or type III transmembrane proteins). any) of the transmembrane sequence.
  • the transmembrane domain of the CAR of the present invention may also contain artificial hydrophobic sequences.
  • the transmembrane domain comprises a transmembrane domain selected from the group consisting of: TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1, ICOS (CD278), 4-1BB, CTLA-4, GITR, CD40, BAFFR, LIGHTR, SLAMF7 , NKp80, CD160, CD19, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA1, VLA1, CD49 ⁇ , ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX , CD11c, ITGB1, CD29, IT
  • the transmembrane domain may comprise one or more additional amino acids adjacent to the transmembrane domain, for example, one associated with the extracellular region of the protein from which the transmembrane domain is derived. or multiple amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids of the extracellular region) and/or with the intracellular One or more additional amino acids associated with a region (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids of an intracellular region).
  • the transmembrane domain is derived from the same protein as the signal transduction domain, co-stimulatory domain or hinge domain is derived from.
  • the transmembrane domain is not derived from the same protein as any other domain of the CAR is derived from.
  • the transmembrane domain comprises the transmembrane domain of CD8 ⁇ .
  • the transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having about 70% or more sequence identity therewith, such as about 75%, 80%, 85% therewith %, 90%, 95%, 97%, 98%, or more than 99% sequence identity of amino acid sequences.
  • the transmembrane domain is directly linked to the specific recognition domain.
  • the transmembrane domain and the specific recognition domain are connected by a hinge region.
  • the hinge region comprises an amino acid sequence selected from the group consisting of an Fc fragment of human CD8 ⁇ or an antibody or a functional equivalent, fragment or derivative thereof, a hinge region of human CD8 ⁇ or an antibody or a functional equivalent thereof substances, fragments or derivatives, CH2 region of antibody, CH3 region of antibody, artificial spacer sequence and their combination; preferably the hinge region amino acid sequence of IgG, IgD, CD8 ⁇ or CD28.
  • the hinge region comprises any one or more of: (i) an IgG4 hinge region, a CH2 region, and a CH3 region, (ii) an IgG4 hinge region, (iii) an IgG4 hinge region and CH2 region of (iv) CD8 ⁇ hinge region, (v) IgG1 hinge region, CH2 region and CH3 region, (vi) IgG1 hinge region, (vi) IgG1 hinge region and CH2 region, or ( vii) combinations thereof.
  • the hinge region is the hinge region of human CD8 ⁇ .
  • the hinge region comprises an amino acid sequence as shown in SEQ ID NO: 14 or an amino acid sequence having more than about 70% sequence identity thereto, such as about 75%, 80%, 85% therewith. %, 90%, 95%, 97%, 98%, or more than 99% sequence identity of amino acid sequences.
  • the CAR further comprises a signal peptide.
  • the signal peptide is located at the N-terminus of the protein and carries information about protein secretion, which participates in determining the secretion pathway and distribution of the protein.
  • proteins located in the nucleus, mitochondria, and cytoplasm, as well as free proteins in the cytoplasm and proteins secreted or finally anchored on the cell membrane usually have different signal peptides.
  • Those skilled in the art can select a signal peptide sequence suitable for a certain protein to function through software prediction and signal peptide database.
  • the signal peptide is selected from signal peptide sequences of any secreted protein or cell membrane protein, or variants thereof that retain the same function.
  • the signal peptide sequence is the signal peptide of CD8 ⁇ .
  • the signal peptide sequence comprises an amino acid sequence such as SEQ ID NO: 2 or a polynucleotide sequence having about 70% or more sequence identity therewith, for example having about 75%, 80%, 85% therewith , 90%, 95%, 97%, 98%, or more than 99% sequence identity of amino acid sequences.
  • the present application also provides an engineered T cell receptor (TCR) that specifically recognizes BAFF-R.
  • TCR engineered T cell receptor
  • MHC major histocompatibility complex
  • T cell receptors are heterodimers composed of two distinct subunits. Normally, 95% of T cell receptors are composed of ⁇ and ⁇ subunits, and the other 5% of receptors are composed of ⁇ and ⁇ subunits. Each subunit contains two extracellular domains: a variable region and a constant region.
  • variable region of each subunit contains at least three highly variable regions.
  • CDR3 is responsible for directly binding to the polypeptide presented by MHC
  • CDR1 of the ⁇ subunit and ⁇ subunit act on the N-terminal and C-terminal of the polypeptide respectively
  • CDR2 is involved in recognizing MHC.
  • the engineered T cell receptor comprises:
  • V ⁇ which comprises an amino acid sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or an amino acid sequence having more than about 70% sequence identity with them; and V ⁇ , which comprises an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:8 The amino acid sequence of ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or an amino acid sequence having more than about 70% sequence identity thereto; or
  • V ⁇ which comprises an amino acid sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or an amino acid sequence having more than about 70% sequence identity with them; and V ⁇ , which comprises an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:8; The amino acid sequence of ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or an amino acid sequence having more than about 70% sequence identity with them.
  • the engineered T cell receptor comprises:
  • V ⁇ which comprises an amino acid sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or an amino acid sequence having more than about 70% sequence identity with them; and V ⁇ , which comprises an amino acid sequence selected from SEQ ID NO: The amino acid sequence of ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or an amino acid sequence having more than about 70% sequence identity thereto; or
  • V ⁇ which comprises an amino acid sequence selected from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or an amino acid sequence having more than about 70% sequence identity with them; and V ⁇ , which comprises an amino acid sequence selected from SEQ ID NO:7, SEQ ID NO:8 The amino acid sequence of ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or an amino acid sequence having more than about 70% sequence identity with them.
  • the engineered TCR can specifically recognize BAFF-R presented by MHC, and bind to CD3 ⁇ / ⁇ dimer, CD3 ⁇ / ⁇ dimer and CD247 ⁇ / ⁇ or ⁇ / ⁇ dimer responsible for signal transduction, as well as in In some embodiments, co-activating downstream signaling pathways with co-receptors.
  • the present application also provides a nucleic acid molecule encoding a receptor that specifically recognizes BAFF-R or a fragment thereof.
  • the receptor that specifically recognizes BAFF-R is the aforementioned chimeric antigen receptor; in some implementations In the scheme, the receptor specifically recognizing BAFF-R is the aforementioned engineered T cell receptor.
  • the nucleic acid molecule comprises a polynucleotide sequence encoding the aforementioned chimeric antigen receptor or engineered T cell receptor or a fragment thereof.
  • the nucleic acid molecule can be DNA or RNA. In some embodiments, the nucleic acid molecule is linear, and in some embodiments, the nucleic acid molecule is circular.
  • the nucleic acid molecule is double stranded. In some embodiments, the nucleic acid molecule is single stranded. In some embodiments, the nucleic acid molecule is chemically synthesized. In some embodiments, the nucleic acid molecule comprises chemical modifications to render it more stable in the cell or in the animal body. In some embodiments, the nucleic acid molecule is synthesized by bacterial, fungal, or animal cells. In some embodiments, the nucleic acid molecule is a plasmid, viral vector, or oligonucleotide.
  • the polynucleotide sequence comprises a specific recognition domain encoding the receptor that specifically recognizes BAFF-R.
  • the specific recognition domain is a scFv.
  • the polynucleotide sequence comprises a DNA sequence as shown in SEQ ID NO: 3 and/or a DNA complementary thereto, or an RNA sequence corresponding or complementary thereto, or a DNA sequence similar to that described in this paragraph Or a polynucleotide sequence having about 70% or more sequence identity to an RNA sequence, such as having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity therewith polynucleotide sequence.
  • the "complementary" of nucleic acids means that two polynucleotides can hybridize under stringent conditions, and the stringent hybridization means that when the two polynucleotides hybridize, each base or each Nucleotides are all in accordance with the Watson-Crick base pairing principle, that is, A is paired with T or U, and C is paired with G or I.
  • the RNA "corresponding" to a DNA sequence refers to a ribonucleotide (RNA) sequence that is consistent with the sequence of bases in the DNA sequence and in which all T bases are replaced by U bases .
  • the nucleic acid molecule comprises a coding sequence for a membrane localization signal peptide molecule.
  • said wherein said membrane localization signal peptide is a membrane localization signal peptide of CD8 ⁇ .
  • the coding sequence of the membrane localization signal peptide molecule comprises the DNA sequence shown in SEQ ID NO: 1 and/or the DNA complementary thereto, or the RNA sequence corresponding or complementary thereto, Or a polynucleotide sequence having about 70% or more sequence identity with the DNA or RNA sequence described in this paragraph, for example having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or Polynucleotide sequences with more than 99% sequence identity.
  • the nucleic acid molecule comprises a sequence encoding a hinge region.
  • the encoding hinge region is the hinge region of CD8 ⁇ .
  • the sequence encoding the hinge region comprises a DNA sequence as shown in SEQ ID NO: 13 and/or DNA complementary thereto, or an RNA sequence corresponding or complementary thereto, or as described in this paragraph A polynucleotide sequence having about 70% or more sequence identity to a DNA or RNA sequence, for example having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more sequence identity therewith polynucleotide sequence.
  • the nucleic acid molecule comprises a sequence encoding a signaling domain.
  • the signaling domain is that of CD3 ⁇ .
  • the sequence encoding the signal transduction domain comprises a DNA sequence as shown in SEQ ID NO: 19 and/or DNA complementary thereto, or an RNA sequence corresponding thereto or complementary thereto, or with The DNA or RNA sequence described in this paragraph has a polynucleotide sequence with more than about 70% sequence identity, such as having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% with it A polynucleotide sequence having the above sequence identity.
  • the nucleic acid molecule comprises a sequence encoding a co-stimulatory domain.
  • the co-stimulatory domain is the signaling domain of 4-1BB.
  • the sequence encoding the signal transduction domain comprises a DNA sequence as shown in SEQ ID NO: 17 and/or DNA complementary thereto, or an RNA sequence corresponding thereto or complementary thereto, or with The DNA or RNA sequence described in this paragraph has a polynucleotide sequence with more than about 70% sequence identity, such as having about 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% with it A polynucleotide sequence having the above sequence identity.
  • the nucleic acid molecule comprises the aforementioned sequence encoding the co-stimulatory domain, the aforementioned sequence encoding the signal transduction domain, the aforementioned sequence encoding the hinge region, the aforementioned encoding sequence of the membrane localization signal peptide molecule, and the aforementioned encoding sequence.
  • Specific recognition domain sequence How to arrange the sequence contained in the aforementioned nucleic acid molecule to make the nucleic acid molecule, and which related components to regulate expression to be added, so that after the nucleic acid molecule is introduced into the cell, a complete receptor that recognizes the specific recognition of BAFF-R can be successfully synthesized is obvious to those skilled in the art.
  • the nucleic acid molecule sequentially comprises the polynucleotide sequence described below from the 5' end to the 3' end or a polynucleotide sequence having about 70% or more sequence identity therewith, for example having about Polynucleotide sequences with 75%, 80%, 85%, 90%, 95%, 97%, 98%, or more than 99% sequence identity: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO: 13. SEQ ID NO: 15, SEQ ID NO: 17 and SEQ ID NO: 19, wherein other sequences may be included between the polynucleotide sequences.
  • the present application also provides mixtures comprising a plurality of nucleic acid molecules.
  • the multiple nucleic acid molecules respectively comprise one or more of the following: the aforementioned sequence encoding co-stimulatory domain, the aforementioned sequence encoding signal transduction domain, the aforementioned sequence encoding hinge region, the aforementioned membrane localization signal peptide
  • the coding sequence of the molecule, the aforementioned coding-specific recognition domain sequence is co-introduced into cells, and then a complete receptor that specifically recognizes BAFF-R can be synthesized.
  • An engineered immune effector cell comprising the aforementioned receptor specifically recognizing BAFF-R or the aforementioned nucleic acid molecule.
  • the receptor that specifically recognizes BAFF-R is the aforementioned chimeric antigen receptor, and the engineered immune effector cells are CAR-T or CAR-NK cells.
  • the receptor specifically recognizing BAFF-R is the aforementioned engineered T cell receptor, and the engineered immune effector cells are TCR-T cells. In some embodiments, the receptor specifically recognizing BAFF-R is the aforementioned engineered T cell receptor, and the engineered immune effector cells are TCR-NK cells.
  • Virus particles, liposomes, lipid nanoparticles, pharmaceutical compositions are provided.
  • the present application also provides a viral plasmid, wherein the packaged viral vector comprises the aforementioned chimeric antigen receptor or engineered T cell receptor and/or encodes the chimeric antigen receptor or engineered T cell receptor body polynucleotide sequence.
  • the present application also provides liposome or lipid nanoparticle, the liposome or lipid nanoparticle comprising the receptor or its fragment specifically recognizing BAFF-R as described above or encoding the receptor or its Fragments of nucleic acid molecules.
  • the present application further provides a pharmaceutical composition, which comprises a therapeutically effective amount of one or more selected from the following: the aforementioned chimeric antigen receptor, the aforementioned T cell receptor, the aforementioned nucleic acid molecule, the aforementioned engineered immune Effector cells, aforementioned virus particles, liposomes, lipid nanoparticles.
  • the pharmaceutical composition further includes suitable excipients, carriers, and/or stabilizers.
  • Such acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, for example, buffers such as phosphate, citrate or acetate, usually at a pH of 5.0 to 8.0, optionally 6.0 to 7.0; salts to achieve isotonicity, such as sodium chloride, potassium chloride, etc.; antioxidants; preservatives; low molecular weight polypeptides; proteins; hydrophilic polymers, such as polysorbate Esters 80; amino acids, such as glycine; carbohydrates; chelating agents; sugars; and other standard ingredients known to those skilled in the art (Remington: The Science and Practice of Pharmacy, 22nd Ed., Loyd V. Allen et al. eds. Pharmaceutical Press (2012)).
  • the present application also discloses a method for treating or preventing a disease or symptom of a subject in need, the method comprising administering to the subject an effective amount of the aforementioned pharmaceutical composition, the disease being selected from B Diseases caused by an imbalance in the cellular BAFF-BAFF-R signaling pathway.
  • the method further comprises administering to the subject a second therapeutic agent, such as a monoclonal antibody capable of binding CD20 antigen, a monoclonal antibody capable of binding CD19, an immune checkpoint capable of binding, such as PD-1/PD- Monoclonal antibody against L1, or engineered immune effector cells targeting CD20, CD19, PD-L1, etc.
  • the disease is selected from any of the following: autoimmune disease, graft versus host disease, or tumor.
  • the disease is cancer.
  • the cancer is lymphoma, leukemia or myeloma.
  • the cancer is lymphoma.
  • the lymphoma is mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, or Burkitt's lymphoma.
  • the cancer is leukemia.
  • the leukemia is lymphoblastic leukemia, chronic lymphocytic leukemia or hairy cell leukemia.
  • the cancer is myeloma.
  • the myeloma is multiple myeloma.
  • the disease is an autoimmune disease.
  • the autoimmune disease is rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, glomerulonephritis, Sjögren's syndrome, or autoimmune hemolytic anemia.
  • the present application also provides a method for inhibiting cell proliferation.
  • the method comprises contacting the cells with a pharmaceutical composition as provided herein, thereby forming contacted cells.
  • the anti-BAFF-R receptor or its functional fragments are combined with the BAFF-R protein on the cells to inhibit cell proliferation.
  • the cells are lymphocytes.
  • the cells are B cells or cancer cells.
  • the cells are lymphoma cells.
  • treating or preventing a disease or condition refers to methods of obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results may include, but are not limited to: alleviation or amelioration of one or more symptoms or conditions; lessening the extent of a condition, disorder or disease; stabilizing the state of a condition, disorder or disease; preventing the development of a condition, disorder or disease; Preventing the spread of a condition, disorder or disease; delaying or slowing the progression of a condition, disorder or disease; delaying or slowing the onset of a condition, disorder or disease; ameliorating or palliation of a condition, disorder or disease state; Treating can also mean prolonging the survival of a subject beyond expected survival in the absence of treatment. Treating can also mean inhibiting the progression of a condition, disorder or disease, temporarily slowing the progression of a condition, disorder or disease, but in some cases it involves permanently stopping the progression of the condition, disorder or disease.
  • Example 1 Design of chimeric antigen receptors targeting BAFF-R
  • the present invention constructs an anti-BAFF-R chimeric antigen receptor (H90-11 CAR, which comprises the heavy chain variable region and light chain of the H90-11 mAb monoclonal antibody as shown in the US application publication: US 2021/0261676Al.
  • H90-11 CAR anti-BAFF-R chimeric antigen receptor
  • the chimeric antigen receptor includes a signal peptide sequence (Leader) of CD8 ⁇ , The single-chain antibody sequence (scFv) that specifically binds to the BAFF-R antigen, the hinge region (Hinge) and transmembrane domain sequence (Transmembrane) of human CD8 ⁇ , the 4-1BB co-stimulatory domain sequence and the CD3 ⁇ signaling domain sequence, specifically
  • the sequence of each part is as follows, and the specific sequence is shown in Table 1:
  • the polynucleotide sequence of human CD8 ⁇ molecular signal peptide (Leading signal) is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 2;
  • the polynucleotide sequence of the human source BAFF-R single-chain antibody (scFv) is shown in SEQ ID NO: 3, and the amino acid sequence is shown in SEQ ID NO: 4; wherein the heavy chain variable region (VH) of the scFv is SEQ ID NO: 5, the amino acid sequences of the three complementarity determining regions CDR H1, CDR H2, and CDR H3 contained in the VH are respectively: SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8;
  • the light chain variable region (VL) of scFv is SEQ ID NO: 9, and the amino acid sequences of the three complementary determining regions CDR L1, CDR L2, and CDR L3 contained in the VL are: SEQ ID NO: 10, SEQ ID NO : 11, SEQ ID NO: 12;
  • CD8Hinge The polynucleotide sequence of the human CD8 ⁇ hinge region (CD8Hinge) is shown in SEQ ID NO: 13, and the amino acid sequence is shown in SEQ ID NO: 14;
  • CD8TM human CD8 ⁇ transmembrane domain
  • amino acid sequence is shown in SEQ ID NO:16;
  • polynucleotide sequence of the human 4-1BB molecular intracellular region (4-1BB) is shown in SEQ ID NO: 17, and the amino acid sequence is shown in SEQ ID NO: 18;
  • CD3 ⁇ molecular intracellular region (CD3 ⁇ ) nucleotide sequence is shown in SEQ ID NO: 19, and the amino acid sequence is shown in SEQ ID NO: 20;
  • the anti-BAFF-R H90-11 CAR lentiviral expression vector pCDH-EF1a-H90-11CAR-4-1BB-EGFRt and the control EGFRt CAR lentiviral expression vector pCDH-EF1a-EGFRt-AT-Free(delete T2A) were both provided by Nanjing Synthesized and constructed by GenScript, the specific experimental steps are as follows:
  • the anti-BAFF-R scFv gene sequence was synthesized by Nanjing GenScript Company, and the gene-synthesized scFv sequence was inserted into the pCDH-EF1a-4-1BB-EGFRt vector ( Nanjing GenScript, order number: C046EEE310), obtained the anti-BAFF-R chimeric antigen receptor lentiviral expression vector pCDH-EF1a-H90-11CAR-4-1BB-EGFRt (Nanjing GenScript, order number: C9401FG210 ), the carrier map is shown in Figure 2.
  • pCDH-EF1a-T2A-EGFRt (provided by iCarTab) lentiviral expression vector, because there is an ATG start codon in the T2A linker, EGFRt cannot be expressed normally due to frameshift mutation, produced according to In-Fusion Snap assembly seamless cloning reagent According to the manufacturer’s instructions, T2A in the pCDH-EF1a-T2A-EGFRt vector was deleted to obtain the control EGFRt CAR lentiviral expression vector pCDH-EF1a-EGFRt-AT-Free (delete T2A) (constructed by Nanjing GenScript), vector map See Figure 3.
  • Embodiment 3 lentivirus packaging and titer determination
  • the concentrated lentivirus was added to the aforementioned 6-well plate in an amount of 50 ⁇ L, and polybrene (Sigma, catalog number: 107689-10G) with a final concentration of 6 ⁇ g/mL was added at the same time, and the 6-well plate was returned to 37°C, 5% In a CO2 incubator, culture was continued for 48 hours.
  • the cells in each well were washed with PBS, and then the genomic DNA was extracted using the MiniBEST Universal Genomic DNA Extraction Kit (Takara, catalog number: 9765), and the concentration of the extracted genomic DNA was determined using NanoDrop2000. According to the PCR reaction system shown in Table 2 below, the fluorescent quantitative PCR reaction master mix was prepared.
  • X-Vivo 15 medium containing 200IU/mL IL-2 (Beijing Yuance Pharmaceutical Co., Ltd., catalog number: 20150713B), 10ng/mL IL-7 (PrimeGene, Product number: 101-07), 5ng/mL IL-15 (PrimeGene, product number: 101-15) was resuspended and counted, the cell density was adjusted to 0.5-1 ⁇ 106 cells/mL, transferred to 6 -well plate, Continuously cultivated for 48 hours in a 37°C, 5% CO 2 incubator.
  • polybrene Sigma, product number: 107689-10G
  • X-Vivo 15 medium containing 200IU/mL IL-2, 10ng/mL IL-7, 5ng/mL IL- 15
  • FITC-Anti-EGFR antibody product number: IAB006A
  • H90-11 CAR-T T cells infected with pCDH-EFIa-H90-11CAR-4-1BB-EGFRt lentivirus
  • EGFRt CAR-T cells T cells infected with pCDH-EF1a-EGFRt-AT-Free (delete T2A) lentivirus
  • H90-11 CAR-T T cells infected with pCDH-EF1a-EGFRt-AT-Free (delete T2A) lentivirus
  • EGFRt CAR-T cells T cells infected with pCDH-EF1a-EGFRt-AT-Free (delete T2A) lentivirus
  • BAFF-R Liama IgG2b Fc Tag recombinant protein (Acro, product number: BAR-H5258) (final concentration: 10 ⁇ g/mL) was used to incubate with H90-11 CAR-T and EGFRt CAR-T cells respectively.
  • H90-11 CAR-T cells express both EGFRt protein and anti-BAFF-R CAR molecule, and the positive rate is basically the same; while EGFRt CAR-T cells only express EGFRt protein but not anti-BAFF-R CAR molecule, indicating that H90-11 Both CAR-T and EGFRt CAR-T cells were successfully prepared.
  • Nalm6-luciferase cells are acute lymphoblastic leukemia cells highly expressing human BAFF-R, which are cells overexpressing Luciferase (luciferase) obtained by lentiviral infection of Nalm6 cells (purchased from Genio Biotech) .
  • Luciferase Luciferase
  • the cytotoxicity of H90-11 CAR-T cells to Nalm6-luciferase target cells was detected with Luciferase detection kit (Promega, product number: E2610), and EGFRt CAR-T cells were used as a control.
  • the specific steps are as follows:
  • the acute lymphoblastic leukemia Nalm6-luciferase cell line was xenografted into an NSG mouse model to evaluate the in vivo activity of H90-11 CAR-T.
  • mice were grouped according to the live imaging results, and the mice were randomly divided into 2 groups, with 8 mice in each group, and 2.25 ⁇ 10 6 H90-11 CAR-T cells or EGFRt CAR-T cells were infused through the tail vein, every 7 D-luciferin (Shanghai Lechen, product number: BC219-10) was injected intraperitoneally into the mice on the first day, and the mice were anesthetized using a gas anesthesia machine to collect tumor imaging data in vivo, record the weight changes of the mice, and draw the survival curve of the mice .
  • D-luciferin Shanghai Lechen, product number: BC219-10
  • the small animal in vivo imaging equipment was used to conduct in vivo imaging observations.
  • the imaging results are shown in Figure 8A, and the absolute luminescence curve drawn is shown in Figure 8B.
  • the EGFRt Compared with the CAR-T control group, H90-11 CAR-T had a significant killing effect on tumor cells; after CAR-T cell infusion on day 8, compared with the EGFRt CAR-T infusion control group, H90-11 11
  • the increase in tumor burden absolute luminescence
  • the tumor burden decreased rapidly after the 15th day.
  • the body weight change curve of the drawn mice is shown in Figure 8C.
  • the body weight of mice infused with EGFRt CAR-T control cells was comparable to that of mice infused with H90-11 CAR-T cells; the body weight of mice was maintained slowly during the experiment growth, indicating that CAR-T cell infusion will not cause severe toxic reactions.

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