WO2021004400A1 - Complexe anticorps-récepteur cd3 exprimant une cellule immunitaire et son utilisation - Google Patents

Complexe anticorps-récepteur cd3 exprimant une cellule immunitaire et son utilisation Download PDF

Info

Publication number
WO2021004400A1
WO2021004400A1 PCT/CN2020/100231 CN2020100231W WO2021004400A1 WO 2021004400 A1 WO2021004400 A1 WO 2021004400A1 CN 2020100231 W CN2020100231 W CN 2020100231W WO 2021004400 A1 WO2021004400 A1 WO 2021004400A1
Authority
WO
WIPO (PCT)
Prior art keywords
domain
immune cell
cells
cell according
modified immune
Prior art date
Application number
PCT/CN2020/100231
Other languages
English (en)
Chinese (zh)
Inventor
王文博
郭佩佩
冯爱华
林彦妮
Original Assignee
苏州克睿基因生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 苏州克睿基因生物科技有限公司 filed Critical 苏州克睿基因生物科技有限公司
Priority to CN202080003009.1A priority Critical patent/CN112204135A/zh
Publication of WO2021004400A1 publication Critical patent/WO2021004400A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • This application relates to the field of biomedicine, and specifically relates to an immune cell expressing a CD3 antibody receptor complex, which may be independent of TCR expression.
  • CAR-T chimeric antigen receptor T cell
  • the present application provides a modified immune cell which contains a CD3 antibody receptor complex and does not express T cell receptor (TCR).
  • the CD3 antibody receptor complex described in this application can be expressed in a TCR-independent form. And can be recognized by common CD3 antibodies.
  • the modified immune cells described in this application can be activated and secrete cytokines.
  • the modified immune cells described in this application can also be combined with anti-CD3 and anti-CD19 bispecific antibodies to kill tumor cells.
  • the present application provides a modified immune cell comprising a CD3 antibody receptor complex, the CD3 antibody receptor complex comprising a first CD3 recombinant protein and a second CD3 recombinant protein, wherein the first CD3
  • the recombinant protein comprises: (1) a first extracellular domain, the first extracellular domain comprising an extracellular domain derived from the CD3 epsilon domain, (2) a first transmembrane domain, (3) a first intracellular domain;
  • the second CD3 recombinant protein includes: (1) a second extracellular domain, the second extracellular domain comprising an extracellular domain derived from any one of CD3 gamma domain and CD3 delta domain, (2) second transmembrane Domain, (3) the second intracellular domain, and it does not express T cell receptor (TCR).
  • TCR T cell receptor
  • the modified immune cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes , White blood cells and/or peripheral blood mononuclear cells.
  • NK cells natural killer cells
  • macrophages include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes , White blood cells and/or peripheral blood mononuclear cells.
  • the extracellular domain of the CD3 epsilon domain comprises the amino acid sequence shown in SEQ ID NO:1.
  • the second extracellular domain comprises an extracellular domain derived from the CD3 gamma domain.
  • the extracellular domain of the CD3 gamma domain comprises an amino acid sequence as shown in SEQ ID NO: 2.
  • the second extracellular domain comprises an extracellular domain derived from the CD3 delta domain.
  • the extracellular domain of the CD3 delta domain comprises the amino acid sequence shown in SEQ ID NO:4.
  • the first transmembrane domain and the second transmembrane domain are the same or different.
  • the transmembrane domain does not comprise a transmembrane domain derived from CD3
  • the transmembrane domain comprises a transmembrane domain derived from any one protein selected from the group consisting of CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154 and SLAM.
  • any one protein selected from the group consisting of CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3 ⁇ , CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30,
  • the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:7.
  • the first intracellular domain and the second intracellular domain are the same or different.
  • At least one of the first intracellular domain and the second intracellular domain comprises a costimulatory domain and/or a signal transduction domain.
  • the costimulatory domain contained in the first intracellular domain and the costimulatory domain contained in the second intracellular domain are the same or different.
  • the costimulatory domain comprises a costimulatory domain derived from any one or more proteins selected from the following group: CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3 , SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML , CD244, CD100, ICOS, CD83 ligand, CD40 and MyD88.
  • any one or more proteins selected from the following group: CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3 , SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10
  • the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 8.
  • the signal transduction domain contained in the first intracellular domain and the signal transduction domain contained in the second intracellular domain are the same or different.
  • the signal transduction domain comprises at least one immunoreceptor tyrosine activation motif (ITAM).
  • ITAM immunoreceptor tyrosine activation motif
  • the signal transduction domain comprises a signal transduction domain derived from any one or more proteins selected from the group consisting of CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi sarcoma herpes virus (HSKV), DAP10 and DAP-12.
  • EBV Epstein-Barr virus
  • HSKV Kaposi sarcoma herpes virus
  • the signal transduction domain comprises an amino acid sequence as shown in SEQ ID NO: 9.
  • the extracellular domain and the transmembrane domain further comprise a hinge region.
  • the hinge region comprises a hinge region derived from any one or more proteins selected from the group consisting of CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, and PD-1.
  • the hinge region comprises the amino acid sequence shown in any one of SEQ ID NO: 6.
  • the first CD3 recombinant protein includes an extracellular domain derived from the epsilon domain of CD3, a transmembrane region derived from CD28, an intracellular domain derived from CD28, and an intracellular domain derived from CD3 zeta. In some embodiments, the first CD3 recombinant protein includes the amino acid sequence shown in SEQ ID NO: 10.
  • the second CD3 recombinant protein includes an extracellular domain derived from CD3 gamma domain, a transmembrane region derived from CD28, and an intracellular domain derived from CD3 gamma.
  • the second CD3 recombinant protein includes the amino acid sequence shown in SEQ ID NO: 11.
  • the second CD3 recombinant protein includes an extracellular domain derived from the CD3 gamma domain, a transmembrane region derived from CD28, and the peptide fragment.
  • the second CD3 recombinant protein includes the amino acid sequence shown in SEQ ID NO: 12.
  • the modified immune cell further comprises a third CD3 recombinant protein, the third CD3 recombinant protein comprising: (1) a third extracellular domain, the third extracellular domain comprising The extracellular domain of CD3 gamma domain or CD3 delta domain, (2) the third transmembrane domain, and (3) the third intracellular domain.
  • the third extracellular domain is the same or different from the second extracellular domain.
  • the third transmembrane domain is the same as or different from the first transmembrane domain and/or the second transmembrane domain.
  • the third intracellular domain is the same as or different from the first intracellular domain and/or the second intracellular domain.
  • the expression and/or activity of the major histocompatibility complex (MHC) of the modified immune cell is down-regulated.
  • the MHC complex includes B2M.
  • the modified immune cell comprises a chimeric antigen receptor (CAR) and/or a chimeric autoantibody receptor (CAAR).
  • CAR chimeric antigen receptor
  • CAAR chimeric autoantibody receptor
  • the present application provides a pharmaceutical composition, which comprises the modified immune cell and a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition includes antibodies.
  • the antibody can recognize and/or bind to the CD3 antibody receptor complex.
  • the antibody includes a bispecific antibody.
  • the bispecific antibody is derived from the immune cell.
  • the bispecific antibody can recognize and/or bind to receptors on the surface of target cells.
  • the target cell is a tumor cell.
  • the receptor on the target cell surface is selected from one of the following group: CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56, CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2/HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125, CTLA-4, GITR, BTLA, TGFBR1, TGFBR2, T
  • the present application provides a nucleic acid molecule that encodes the CD3 antibody receptor complex in the modified immune cell.
  • the present application provides a vector, which contains the nucleic acid molecule.
  • the vector is a viral vector.
  • the vector is a lentiviral vector.
  • the present application provides a cell, which contains the nucleic acid molecule and/or the vector.
  • the present application provides the use of the modified immune cells and/or the pharmaceutical composition in the preparation of medicines for the treatment of tumors.
  • the tumor includes solid tumors and non-solid tumors.
  • the tumor is selected from the group consisting of lymphoma, leukemia, and multiple myeloma.
  • the present application provides a method of treating tumors, the method comprising administering the modified immune cells and the pharmaceutical composition to a subject in need.
  • the tumor includes solid tumors and non-solid tumors.
  • the tumor is selected from the group consisting of lymphoma, leukemia, and multiple myeloma. .
  • Figure 1 shows a schematic diagram of the modified immune cells described in this application combined with bispecific antibodies to kill target cells.
  • FIG. 2 shows that the modified immune cells described in this application express CD3 antibody receptor complexes.
  • Figure 3 shows that the CD3 antibody receptor complex can be recognized by the CD3 antibody UCHT1 in TCR KO human primary T cells.
  • Figure 4 shows that the CD3 antibody receptor complex can be recognized by the CD3 antibody HIT3a in TCR KO human primary T cells.
  • Figure 5 shows that the CD3 antibody receptor complex cannot be recognized by the CD3 antibody SP34-2 in TCR KO human primary T cells
  • Figure 6 shows that the CD3 antibody receptor complex can be recognized by the CD3 antibody OKT3 in TCR KO human primary T cells.
  • Figure 7 shows that the modified immune cells described in this application are activated by the CD3 antibody to express the cell activation tag CD137.
  • Figure 8 shows that the modified immune cells described in this application are activated by tumor cells mediated by anti-CD3 and anti-CD19 bispecific antibodies.
  • Figure 9 shows that the modified immune cells described in this application kill tumor cells through anti-CD3 and anti-CD19 bispecific antibodies.
  • CD3 antibody usually refers to an antibody that can specifically recognize CD3 subunits (for example, CD3 epsilon, CD3 gamma, CD3 delta, or the above-mentioned complexes).
  • the antibody may be an antibody that can only recognize CD3.
  • CD3 antibody receptor complex generally refers to a receptor that can be recognized by a CD3 antibody, which may include at least two (e.g., three) CD3 subunits (e.g., CD3 epsilon and CD3 delta, Or CD3 epsilon and CD3 gamma).
  • the CD3 antibody receptor complex is composed of at least 2 (for example, 3, 4, 5, 6 or more) CD3 recombinant proteins, and the CD3 recombinant protein may include a CD3 subunit (for example, CD3 epsilon , CD3 gamma and/or CD3 delta) extracellular domain, transmembrane domain and intracellular domain.
  • CD3 epsilon and CD3 gamma or CD3 delta ectodomain is close to the natural conformation of CD3 epsilon heterodimer in TCR complex, including the epitope recognized by most CD3 antibodies such as UCHT1 and OKT3.
  • the conformation formed by the CD3 epsilon extracellular domain alone does not include the binding epitope of most CD3 antibody clones.
  • first CD3 recombinant protein generally refers to a recombinant protein containing an extracellular domain, a transmembrane domain, and an intracellular domain derived from CD3 (eg, CD3 epsilon).
  • the CD3 antibody receptor complex may include one or more (for example, 2, 3, 4 or more) of the first CD3 recombinant protein.
  • first extracellular domain generally refers to the extracellular domain part of the first CD3 recombinant protein, and the first extracellular domain may include the extracellular domain derived from the CD3 epsilon domain.
  • second CD3 recombinant protein generally refers to a recombinant protein containing an extracellular domain, a transmembrane domain, and an intracellular domain derived from CD3 (for example, CD3 delta and or CD3 gamma).
  • the CD3 antibody receptor complex may include one or more (for example, 2, 3, 4 or more) second CD3 recombinant protein.
  • second extracellular domain generally refers to the extracellular domain part of the second CD3 recombinant protein, and the second extracellular domain may include an extracellular domain derived from CD3 delta and or CD3 gamma domain.
  • third CD3 recombinant protein generally refers to a recombinant protein containing an extracellular domain, a transmembrane domain, and an intracellular domain derived from CD3 (for example, CD3 delta and or CD3 epsilon).
  • the CD3 antibody receptor complex may include one or more (for example, two, three, four or more) third CD3 recombinant proteins.
  • third extracellular domain generally refers to the extracellular domain part of the third CD3 recombinant protein, and the third extracellular domain may include an extracellular domain derived from the CD3 delta and or CD3 epsilon domains.
  • the third extracellular domain and the second extracellular domain may be derived from the same or different CD3 subunit (for example, CD3 delta and or CD3 gamma).
  • an antibody generally refers to a polypeptide molecule that can specifically recognize and/or neutralize a specific antigen.
  • an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds, and includes any molecule comprising an antigen binding portion thereof.
  • the term “antibody” includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (e.g., dAb), single chain antibodies (e.g., scFv), And antibody fragments that bind to the antigen (for example, Fab, Fab', and (Fab)2 fragments).
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described in this application.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region.
  • VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDR), which are interspersed in more conserved regions called framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • transmembrane domain generally refers to a sequence of cell surface proteins that spans the cell membrane, which may include a hydrophobic alpha helix.
  • the transmembrane domain can be connected to the intracellular signal transduction domain and play a role in transmitting signals.
  • the transmembrane domain can be derived from any type I, type II or type III transmembrane protein.
  • the transmembrane domain may not include a transmembrane protein derived from CD3 (for example, CD3 epsilon, CD3 gamma, and/or CD3 delta).
  • immunoreceptor tyrosine activation motif usually refers to a conserved sequence composed of more than ten amino acids, which often appears in the intracellular of transmembrane proteins of certain cells of the immune system Area. TAM is an important transduction signal of immune cells. Therefore, they often appear in the intracellular regions of important cell signaling molecules, such as the CD3 and ⁇ chains in the T cell receptor complex, the CD79 ⁇ and ⁇ chains in the B cell receptor complex, and certain Fc receptors.
  • the tyrosine residues on the intracellular ITAM will be phosphorylated, and the phosphorylated ITAM can bind to free proteins in the envelope with the SH2 domain to make immune cells
  • the signal is transmitted to the downstream signal molecule.
  • CAR Chimeric Antigen Receptor
  • CAR-T chimeric antigen receptor T cells
  • antigen for example, tumor-specific antigen and/or tumor-associated antigen binding domain
  • transmembrane domain for example, tumor-specific antigen and/or tumor-associated antigen binding domain
  • costimulatory domain for example, tumor-associated antigen binding domain
  • Intracellular signal domain for example, tumor-associated antigen binding domain
  • the CAR can be combined with the T cell receptor activation intracellular domain based on the specificity of the antigen (eg CD70) of the antibody.
  • Genetically modified T cells expressing CAR can specifically recognize and eliminate malignant cells expressing target antigens.
  • CAAR chimeric autoantibody receptor
  • CAAR can direct genetically modified immune cells expressing CAAR to attack B cells expressing antibodies capable of recognizing the antigen.
  • bispecific antibody generally refers to an antibody that has binding sites for two different antigens within a single antibody molecule.
  • one of the antigens can be CD3.
  • peptide generally refers to a polypeptide consisting of at least two (for example, four, five, six or more) amino acids. It can include all molecules with amino functionality and acid functionality and including naturally occurring amino acid polymers, including natural amino acids and artificial amino acids.
  • costimulatory domain generally refers to an intracellular domain that can provide immune costimulatory molecules, which are cell surface molecules required for effective response of lymphocytes to antigens.
  • hinge region generally refers to the junction region between the extracellular domain (for example, the CD3 extracellular domain) and the transmembrane region.
  • the term "signal transduction domain” generally refers to a domain located inside a cell capable of transducing signals.
  • the intracellular signal transduction domain can transmit signals into the cell.
  • a signal transduction domain is any continuous amino acid sequence used to direct protein targeting.
  • the signaling domain can be derived from CD3 ⁇ .
  • CD3 ⁇ can form a T cell receptor-CD3 complex with T cell receptor subunits and CD3-gamma, -delta, and -epsilon.
  • CD3 ⁇ contains three ITAM motifs, and the ITAM sequence mediates the activation of TCR intracellular signals.
  • the zeta chain is a receptor-activated protein tyrosine kinase substrate.
  • CD3 ⁇ plays a key role in antigen recognition and TCR signal transduction.
  • the term "pharmaceutically acceptable adjuvant” generally refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient.
  • Such formulations may conventionally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents.
  • Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans.
  • Other contemplated carriers, excipients, and/or additives that can be used in the formulations described herein include: for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, Protein excipients (such as serum albumin, gelatin, casein), salt-forming counterions (such as sodium), etc.
  • immune cell generally refers to a cell that participates in an immune response, such as promoting an immune effector response.
  • immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, mast cells, granulocytes, monocytes, lymphocytes, and macrophages.
  • NK natural killer
  • the term also includes engineered immune cells, such as immune cells that have been genetically modified by adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.
  • the "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, and is used to transfer the inserted nucleic acid molecule into and/or between host cells.
  • the vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of DNA or RNA transcription and/or translation.
  • the carrier also includes a carrier having multiple functions described above.
  • the vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product.
  • the vector can cover additional features besides the transgene insert sequence and main chain: promoter, genetic marker, antibiotic resistance, reporter gene, targeting sequence, protein purification tag.
  • Vectors called expression vectors are specifically used to express transgenes in target cells, and usually have control sequences.
  • the vector described in this application may be an expression vector, including viral vectors (lentiviral vectors and/or retroviral vectors), phage vectors, phagemids, cosmids, cosmid, artificial chromosomes such as yeast artificial chromosomes (YAC), Bacterial artificial chromosome (BAC) or P1 derived artificial chromosome (PAC) and/or plasmid.
  • viral vectors lentiviral vectors and/or retroviral vectors
  • phage vectors phagemids
  • cosmids cosmid
  • artificial chromosomes such as yeast artificial chromosomes (YAC), Bacterial artificial chromosome (BAC) or P1 derived artificial chromos
  • treatment generally refers to: (i) preventing the disease, disorder, or condition from appearing in patients who may be susceptible to the disease, disorder, and/or condition but have not yet been diagnosed with the disease; (ii) inhibiting the disease , Disease or condition, that is, curb its development; and (iii) alleviate the disease, disease, or condition, that is, make the disease, condition, and/or condition and/or symptoms associated with the disease, condition, and/or condition Subside.
  • MHC major histocompatibility complex
  • MHC mainly includes class I MHC molecules and class II MHC molecules.
  • Class I MHC molecules can span the membranes of almost all cells in the organism, while Class II molecules are usually found on immune cells.
  • class I MHC molecules are also called class I major histocompatibility complexes, which are heterodimeric glycoproteins composed of two peptide chains connected by non-covalent bonds; one of them is called a heavy chain and the structure is polymorphic , The other is the light chain or called ⁇ 2 microglobulin (B2M).
  • B2M microglobulin
  • class I MHC molecules present polypeptides that are not self-proteins degraded in the cell, thereby activating the immune system.
  • Human Class I MHC molecules are divided into classic HLA molecules (HLA-A, HLA-B, HLA-C) and non-classical HLA molecules (HLA-E, HLA-G, HLA-F).
  • the modified immune cell may not express active MHC, and the "not expressing active MHC" may include that the expressed MHC loses the activity of activating the immune system and/or the cell surface lacks class I MHC Molecule (e.g., missing HLA-A/B/C/E/F/G).
  • B2M or the corresponding heavy chain gene can be edited to delete MHC class I molecules on the cell surface.
  • B2M usually refers to ⁇ -2 microglobulin, usually refers to the light chain of MHC class I molecules, and therefore is an indispensable part of the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • B2M is encoded by the b2m gene located on chromosome 15, while other MHC genes exist as clusters on chromosome 6.
  • the human B2M protein has 119 amino acids (see UniProt database code P61769).
  • B2M is necessary for the presentation of class I MHC molecules on the cell surface and the stability of the polypeptide binding groove.
  • Class I MHC molecules exist on the surface of all nucleated cells in the human body. MHC mismatches can cause immune rejection and result in the destruction of the graft. Knockout of the B2M gene to remove the class I MHC molecules on the cell surface can prevent mismatches. .
  • CD3 generally refers to a CD3 protein multi-subunit complex, which is composed of 6 different polypeptide chains (subunits).
  • the polypeptide chain of CD3 can include one CD3 gamma ( ⁇ ) chain, one CD3 gamma ( ⁇ ) chain, two CD3 epsilon ( ⁇ ) chains, and two CD3 zeta ( ⁇ ) chains.
  • CD3 refers to any natural CD3 from any vertebrate source, including mammals, such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. small Rats and rats).
  • mammals such as primates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. small Rats and rats).
  • the term encompasses "full-length” and unprocessed CD3 proteins as well as any form of protein or one or more CD3 chains (polypeptides) (e.g. mature polypeptides) derived from processing in the cell.
  • the term also encompasses naturally occurring variants and isoforms of CD3, such as splice variants or allelic variants.
  • CD3 epsilon refers to any natural CD3 from any vertebrate source, including mammals, such as primates (such as humans). ), non-human primates (such as cynomolgus monkeys) and rodents (such as mice and rats).
  • CD3 epsilon covers “full length” and unprocessed CD3 epsilon, “CD3 gamma”, “CD3 gamma” and/or “CD3 zeta”, respectively "Protein and any form of CD3 chain (polypeptide) derived from processing in the cell (e.g. mature polypeptide).
  • the term also encompasses naturally occurring variants and isoforms of the CD3 chain, such as splice variants or allelic variants.
  • An exemplary CD3delta amino acid sequence can be found in UniProt database accession number P04234, an exemplary CD3 epsilon amino acid sequence can be seen in UniProt database accession number P07766, and an exemplary CD3 gamma amino acid sequence can be seen in UniProt database accession number P09693.
  • T cell receptor generally refers to a transmembrane protein complex that participates in the activation of T cells after recognizing an antigen.
  • TCR is a heterodimer formed by two different protein subunits. In humans, 95% of T cells express one alpha ( ⁇ ) chain and one beta ( ⁇ ) chain. The remaining 5% express gamma ( ⁇ ) and delta ( ⁇ ) chains. Each chain of a TCR molecule can contain two extramembrane domains: a variable region and a constant region.
  • variable region can bind to the polypeptide/major histocompatibility complex, and the variable regions of both the ⁇ chain and the ⁇ chain contain three complementarity determining regions (CDRs), which are responsible for the recognition of the antigen/MHC complex.
  • CDRs complementarity determining regions
  • the constant region is close to the cell membrane and connected to the transmembrane region.
  • the extracellular constant domains of the two heterologous subunits of TCR both contain short binding sequences with cysteine residues. A disulfide bond is formed between the cysteine residues to bind the two heterologous TCR subunits together.
  • the TCR transmembrane domain contains positively charged amino acids and is responsible for binding to CD3 molecules.
  • the intracellular region of TCR is very short and has no active domain.
  • TCR can recognize processed polypeptide fragments that bind to MHC molecules. Because recognition requires the presentation of MHC molecules, it is also called MHC restriction. When the donor and recipient have different MHC molecules, TCR can recognize the difference in MHC and cause the activation and expansion of T cells, which may cause graft-versus-host disease (GvHD). Knockout of the TRAC gene can remove the expression of the TCR ⁇ chain, thereby removing the TCR complex from the surface of the T cell, and thus can prevent the TCR from recognizing allogeneic antigens and causing graft-versus-host disease.
  • GvHD graft-versus-host disease
  • the CD3 multi-subunit complex and TCR can form a functional complex through non-covalent bonds, which is called the TCR-CD3 complex.
  • the CD3 epsilon molecule forms a heterodimer with CD3 gamma and CD3 delta
  • CD3 zeta forms a homodimer with itself.
  • a TCR-CD3 complex includes a CD epsilon:delta heterodimer, a CD epsilon:gamma heterodimer and a CD zeta:zeta homodimer and a TCR alpha:beta heterodimer .
  • CD3 gamma, CD3 delta, and CD3 epsilon molecules are very related immunoglobulin superfamily membrane proteins, all of which contain a single extracellular immunoglobulin domain, while the extracellular domain of CD3zeta is very short.
  • the intracellular regions of CD3 gamma, CD3 delta and CD3 epsilon molecules all contain a single conserved region immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • the CD3 zeta chain contains three ITAM motifs.
  • a single TCR-CD3 complex contains 10 ITAM motifs, and these ITAM motifs determine the degree of TCR activation.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • proteins are used interchangeably and generally refer to polymers of amino acids of any length.
  • the polymer can be linear or branched, it can contain modified amino acids, and can be interrupted by non-amino acids. These terms also cover amino acid polymers that have been modified. These modifications may include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as binding to a labeling component).
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and D and L optical isomers, as well as amino acid analogs and peptidomimetics.
  • polynucleotide used interchangeably, and generally refer to nucleosides of any length.
  • the polymerized form of acid such as deoxyribonucleotides or ribonucleotides, or their analogs.
  • the polynucleotide can have any three-dimensional structure and can perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of genes or gene fragments, multiple loci (one loci) defined according to linkage analysis, exons, introns, messenger RNA (mRNA), Transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence Of isolated DNA, isolated RNA of any sequence, nucleic acid probes, and primers.
  • mRNA messenger RNA
  • Transfer RNA transfer RNA
  • ribosomal RNA short interfering RNA
  • shRNA short hairpin RNA
  • miRNA micro-RNA
  • ribozyme ribozyme
  • cDNA recombinant polynucleotide
  • branched polynucleotide plasmid
  • vector any sequence
  • a polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be performed before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with labeled components.
  • the application may also include functional variants, derivatives, analogs, homologs and fragments thereof.
  • the term "functional variant” refers to a polypeptide that has substantially the same amino acid sequence as or is encoded by substantially the same nucleotide sequence and is capable of having one or more activities of the naturally occurring sequence.
  • a variant of any given sequence means that the specific sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide essentially retains at least one A sequence of endogenous functions.
  • the variant sequence can be obtained by the addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in the naturally-occurring protein and/or polynucleotide, as long as the The original functional activity is sufficient.
  • the term "derivative” generally refers to any substitution, variation, modification, substitution, deletion, and deletion of one (or more) amino acid residues of the self/pair sequence in the polypeptide or polynucleotide of the present application. /Or addition, as long as the resulting polypeptide or polynucleotide substantially retains at least one endogenous function.
  • analog generally refers to a polypeptide or polynucleotide, including any mimic of a polypeptide or polynucleotide, that is, possessing at least one endogenous function of the polypeptide or polynucleotide mimicked by the mimic Chemical compounds.
  • amino acid substitutions can be made, for example, at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 20) amino acid substitutions can be made, as long as the modified sequence basically remains as required The activity or ability.
  • Amino acid substitutions may include the use of non-naturally occurring analogs.
  • the protein or polypeptide used in the present application may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins.
  • Intentional amino acid substitutions can be made based on the polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or similarity of the amphoteric properties of the residues, as long as the endogenous function is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with a similar hydrophilicity value without an electrical head group include day Paraffin, glutamine, serine, threonine and tyrosine.
  • homolog generally refers to an amino acid sequence or a nucleotide sequence that has certain homology with the wild-type amino acid sequence and the wild-type nucleotide sequence.
  • the term “homology” can be equated with sequence "identity”.
  • the homologous sequence may include an amino acid sequence that may be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence .
  • the homologue will contain the same active site as the subject amino acid sequence, etc.
  • homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), and homology can also be expressed in terms of sequence identity.
  • any one of the SEQ ID NO of the mentioned amino acid sequence or the nucleotide sequence has the percent identity sequence refers to the sequence having the percentage identity over the entire length of the mentioned SEQ ID NO the sequence of.
  • sequence alignment can be performed, which can be performed in various ways known to those skilled in the art, for example, using BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for the alignment, including any algorithms needed to achieve optimal alignment among the full-length sequences being compared.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the T cells used in this application are universal T cells that express a CD3 antibody receptor and undergo gene knockout.
  • the CD3 antibody receptor is composed of two recombinant CD3 protein molecules.
  • the T cell expresses a CD3 antibody receptor that can be recognized by bispecific antibodies.
  • the difference from similar chimeric antibody receptor therapy is that the CD3 antibody receptor of the present application can be recognized by bispecific antibodies, and The expression does not depend on the TCR complex and can be directly expressed on the surface of T cells independently.
  • a modified lymphocyte the lymphocyte lacks TCR molecules, the lymphocyte expresses the CD3 antibody receptor complex, and the CD3 antibody receptor complex contains at least two CD3 recombinant proteins; wherein the first CD3 recombinant protein contains :
  • the second CD3 recombinant protein contains:
  • the intracellular domain is composed of an intracellular costimulatory signal transduction domain and an intracellular signal transduction domain, or the intracellular domain is a peptide fragment of at least two amino acids;
  • the first CD3 recombinant protein and the second At least one intracellular domain in the CD3 recombinant protein is composed of an intracellular costimulatory signal transduction domain and an intracellular signal transduction domain.
  • lymphocyte of technical solution 1 wherein the lymphocyte is T cell, B cell, NK cell or macrophage.
  • lymphocyte of technical solution 2 wherein the lymphocyte is a T cell.
  • the extracellular domain comprises the extracellular domain of CD3 epsilon shown in SEQ ID No:1, or a variant thereof.
  • the lymphocyte of technical solution 4 wherein a.
  • the extracellular domain is the extracellular domain of CD3 epsilon shown in SEQ ID No:1.
  • the extracellular domain comprises the extracellular domain of the CD3 gamma domain.
  • the extracellular domain comprises the extracellular domain of CD3 gamma shown in SEQ ID No: 2, or a variant thereof.
  • the extracellular domain is the extracellular domain of CD3 gamma shown in SEQ ID No: 2.
  • the extracellular domain comprises the extracellular domain of the CD3 delta domain, or a variant thereof.
  • the extracellular domain comprises the extracellular domain of CD3 delta shown in SEQ ID No: 4, or a variant thereof.
  • transmembrane domains b and e comprise at least one of the following: CD8 ⁇ transmembrane domain, CD28 transmembrane domain, 4-1BB transmembrane domain, CD4 transmembrane domain Membrane domain, transmembrane domain of CD27, transmembrane domain of CD7, transmembrane domain of PD-1, transmembrane domain of TRAC, and transmembrane domain of TRBC.
  • transmembrane domain of CD28 comprises the transmembrane domain of CD28 shown in SEQ ID No: 7, or a variant thereof.
  • the intracellular costimulatory signal transduction domain comprises at least one of the following: CD28, 4-1BB, CD40L, TIM1, CD226, DR3, SLAM, ICOS, OX40, NKG2D , 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD27, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT and DAP12 in the costimulatory signal transduction regions and the combination of costimulatory molecules.
  • the lymphocyte of technical solution 14, wherein the intracellular costimulatory signal transduction domain comprises the costimulatory signal transduction region of CD28 shown in SEQ ID No: 8, or a variant thereof.
  • the lymphocyte of technical solution 14, wherein the intracellular costimulatory signal transduction domain comprises the costimulatory signal transduction region of 4-1BB, or a variant thereof.
  • the intracellular signal transduction domain comprises at least one of the following: CD3zeta activation region, CD3delta activation region, CD3gamma activation region, FceRI ⁇ activation region, FceRI activation region, immunoglobulin alpha (Iga) activation area, Igbeta activation area, bovine leukemia virus gp30 activation area, Ep-stein-Barr virus (EBV) LMP2A activation area, simian immunodeficiencyvirus PBj14 Nef activation area, HSKV activation area, DAP-12 activation area, at least including An ITAM (tyrosine activation motif) domain, and a domain formed by combining the above domains.
  • Iga immunoglobulin alpha
  • Igbeta activation area bovine leukemia virus gp30 activation area
  • Ep-stein-Barr virus (EBV) LMP2A activation area Ep-stein-Barr virus (EBV) LMP2A activation area
  • the lymphocyte of technical solution 17, wherein the CD3 zeta activation region comprises the sequence shown in SEQ ID No: 9, or a variant thereof.
  • CD3 recombinant protein further has a hinge region between the extracellular domain and the transmembrane region.
  • the lymphocyte of technical solution 19, wherein the hinge region comprises at least one of the following: CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7 and PD-1 hinge region.
  • lymphocyte according to any one of the preceding technical solutions, wherein the intracellular domain is a peptide fragment of at least two amino acids.
  • the first CD3 recombinant protein is selected from one of the following recombinant proteins: CD3 ⁇ extracellular domain-CD8 ⁇ hinge region-CD8 ⁇ transmembrane region-4-1BB costimulation Signaling area-CD3 ⁇ activation area, CD3 epsilon extracellular domain-CD8 ⁇ transmembrane area-4-1BB costimulatory signaling area-CD3 ⁇ activation area, CD3 epsilon extracellular domain-CD28 hinge area-CD28 transmembrane area-CD28 costimulatory signaling area- CD3 ⁇ activation region, and CD3 ⁇ extracellular domain-CD28 transmembrane region-CD28 co-stimulatory signal transduction region-CD3 ⁇ activation region.
  • the lymphocyte of technical solution 22, wherein the first CD3 recombinant protein has the sequence shown in SEQ ID No: 10, or a variant thereof.
  • the second CD3 recombinant protein is selected from one of the following recombinant proteins: CD3 ⁇ extracellular region-CD8 ⁇ hinge region-CD8 ⁇ transmembrane region-CD3 ⁇ costimulatory signal Conduction region, CD3 ⁇ extracellular region-CD8 ⁇ transmembrane region-CD3 ⁇ costimulatory signaling region, CD3 ⁇ extracellular region-CD28 hinge region-CD28 transmembrane region-CD3 ⁇ costimulatory signaling region, and CD3 ⁇ extracellular region-CD28 transmembrane Region-CD3 ⁇ costimulatory signal transduction region, CD3 ⁇ extracellular region-CD28 hinge region-CD28 transmembrane region-peptide, wherein the peptide can be a peptide of at least 2, 4 or at least 6 amino acids.
  • the lymphocyte of technical solution 24, wherein the second CD3 recombinant protein has the sequence shown in SEQ ID No: 11 and SEQ ID No: 12, or a variant thereof.
  • the second CD3 recombinant protein is selected from one of the following recombinant proteins: CD3 ⁇ extracellular region-CD8 ⁇ hinge region-CD8 ⁇ transmembrane region-CD3 ⁇ Co-stimulatory signal transduction zone, CD3 ⁇ extracellular zone-CD8 ⁇ transmembrane zone-CD3 ⁇ costimulatory signal transduction zone, CD3 ⁇ extracellular zone-CD28 hinge zone-CD28 transmembrane zone-CD3 ⁇ costimulatory signal transduction zone, and CD3 ⁇ extracellular zone- CD28 transmembrane zone-CD3 ⁇ costimulatory signal transduction zone.
  • the intracellular domain of the first CD3 recombinant protein is composed of an intracellular costimulatory signal transduction domain and an intracellular signal transduction domain.
  • lymphocyte according to any one of the preceding technical solutions, wherein the intracellular domain of the second CD3 recombinant protein is composed of an intracellular costimulatory signal transduction domain and an intracellular signal transduction domain.
  • the CD3 antibody receptor complex comprises three CD3 recombinant proteins
  • the first CD3 recombinant protein comprises:
  • the second CD3 recombinant protein contains:
  • the third type of CD3 recombinant protein includes:
  • the intracellular domain is composed of an intracellular costimulatory signal transduction domain and an intracellular signal transduction domain, or the intracellular domain is a peptide fragment of at least two amino acids; among them, at least one of the three CD3 recombinant proteins
  • the intracellular domain consists of an intracellular costimulatory signal transduction domain and an intracellular signal transduction domain.
  • a pharmaceutical composition comprising the lymphocyte of any one of technical solutions 1-30, and a bispecific antibody, which can bind to the CD3 antibody receptor complex on the surface of the lymphocyte and can simultaneously Binding to receptors on the surface of target cells.
  • the pharmaceutical composition according to technical solution 31 or 32, wherein the bispecific antibody can bind to the CD3 antibody receptor complex on the surface of lymphocytes and can simultaneously bind to the CD19 receptor on the surface of target cells.
  • lymphocytes are T cells, B cells, NK cells or macrophages.
  • the drug further includes a bispecific antibody that can bind to the CD3 antibody receptor complex on the surface of lymphocytes and can simultaneously bind to receptors on the surface of target cells.
  • a method of treating a disease comprising injecting into a subject:
  • An effective amount of bispecific antibody that can bind to the CD3 antibody receptor complex on the surface of lymphocytes and can simultaneously bind to receptors on the surface of target cells.
  • the receptor of the target cell is selected from one of the following: CD19, CD20, CD22, CD123, CD33, BCMA, IL13R alpha, PSMA, EGFR, HER2, Mesothelin, and Claudin18 .2.
  • the present application provides a modified immune cell, which may comprise a CD3 antibody receptor complex, the CD3 antibody receptor complex comprising a first CD3 recombinant protein and a second CD3 recombinant protein.
  • the first CD3 recombinant protein may comprise (1) a first extracellular domain, (2) a first transmembrane domain, and (3) a first intracellular domain;
  • the second CD3 recombinant protein may Contains (1) a second extracellular domain, (2) a second transmembrane domain, and (3) a second intracellular domain.
  • the first extracellular domain may include an extracellular domain derived from the CD3 epsilon domain, for example, the first extracellular domain may include an amino acid sequence as shown in SEQ ID NO:1.
  • the first extracellular domain may comprise at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the second extracellular domain may include an extracellular domain derived from the CD3 gamma domain, for example, the second extracellular domain may include an amino acid sequence as shown in SEQ ID NO: 2.
  • the second extracellular domain may comprise an amino acid sequence that is at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the second extracellular domain may include an extracellular domain derived from the CD3 delta domain, for example, the second extracellular domain may include an amino acid sequence as shown in SEQ ID NO:4.
  • the second extracellular domain may comprise an amino acid sequence that is at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the CD3 recombinant protein (for example, the first CD3 recombinant protein and/or the second CD3 recombinant protein) may comprise a transmembrane domain (for example, the first and/or second transmembrane domain ).
  • the transmembrane domain (for example, the first transmembrane domain and/or the second transmembrane domain) may be derived from any type I transmembrane protein as long as it is not the transmembrane domain of CD3.
  • the exemplary transmembrane domains may include, but are not limited to, transmembrane domains derived from lower histones: CD8, CD28 , 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, ⁇ chain of T cell receptor, CD3 ⁇ , CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM , DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, SLAM, and any other independent Transmembrane domain expressed in TCR, or a mutant of the above transmembrane domain.
  • transmembrane domains derived from lower histones: CD8, CD28 , 4-1BB, CD4, CD27, CD7, PD
  • the transmembrane domain (eg, the first transmembrane domain and/or the second transmembrane domain) may be a transmembrane domain derived from human CD28.
  • the first transmembrane domain may include the amino acid sequence shown in SEQ ID NO:7.
  • the first transmembrane domain may comprise at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in SEQ ID NO: 7. , 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the CD3 recombinant protein may comprise an intracellular domain (for example, a first intracellular domain and/or a second intracellular domain).
  • the intracellular domain e.g., the first intracellular domain and/or the second intracellular domain
  • the intracellular domain may include an intracellular costimulatory domain and/or an intracellular signaling domain.
  • the intracellular signal transduction domain may include a domain with at least one ITAM motif.
  • Exemplary signal transduction domains can be derived from signal transduction domains selected from the following group, including but not limited to CD3zeta, CD3delta, CD3gamma, CD3 ⁇ , CD79a, CD79b, FceRI ⁇ , FceRI ⁇ , Fc ⁇ RIIa, bovine leukemia virus gp30 activation region, Epstein-Barr virus (EBV) LMP2A, simian immunodeficiency virus PBj14Nef, Kaposi sarcoma herpes virus (HSKV), DAP10 and DAP-12, and variants of the above.
  • EBV Epstein-Barr virus
  • HSKV Kaposi sarcoma herpes virus
  • the signal transduction domain may be a signal transduction domain derived from the intracellular domain of CD3zeta.
  • the signal transduction domain from the intracellular domain of CD3zeta may include the signal transduction domain shown in SEQ ID NO: 9.
  • the signal transduction domain from the intracellular domain of CD3zeta may comprise at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, 93%, at least 85%, 90%, 91%, 92%, 93%) of the amino acid sequence shown in SEQ ID NO: 9 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the costimulatory domain may include, but is not limited to, the following groups: CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligand, CD40 and MyD88 co-stimulation Co-stimulatory molecules composed of signal transduction regions and their combinations.
  • the costimulatory domain may be a costimulatory domain derived from the intracellular domain of human CD28.
  • the costimulatory domain of the human CD28 intracellular domain may include the costimulatory domain shown in SEQ ID NO: 8.
  • the costimulatory domain of the human CD28 intracellular domain may comprise at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, at least 80%) of the amino acid sequence shown in SEQ ID NO: 8 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the intracellular domain may include the costimulatory domain from the above-mentioned human CD28 intracellular domain and the above-mentioned signal from the CD3zeta intracellular domain Conduction domain.
  • the intracellular domain may comprise an intracellular domain derived from CD3 gamma.
  • the intracellular domain of CD3 gamma may include an amino acid sequence as shown in SEQ ID NO: 3.
  • the intracellular domain of the CD3 gamma may comprise at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 94%, at least 85%, 90%, 91%, 92%, 94%, etc.) of the amino acid sequence shown in SEQ ID NO: 3. 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the intracellular domain (for example, the first intracellular domain and/or the second intracellular domain) may comprise at least two (for example, at least four, at least six or more ) A peptide of amino acids.
  • the intracellular domain may be a peptide fragment of at least two (eg, at least four, at least six or more) amino acids.
  • the peptide fragment may include the amino acid sequence shown in SEQ ID NO:19.
  • the first CD3 recombinant protein and the second CD3 recombinant protein except for the extracellular region, other parts (for example, transmembrane domain, intracellular domain) may be the same or different.
  • the first transmembrane domain of the first CD3 recombinant protein and the second transmembrane domain of the second CD3 recombinant protein may be the same or different.
  • the first intracellular domain of the first CD3 recombinant protein and the second CD3 recombinant protein The two intracellular domains can be the same or different.
  • the signal transduction domain of the first intracellular domain and the signal transduction domain of the second intracellular domain may be the same or different.
  • the costimulatory domain of the first intracellular domain and the costimulatory domain of the second intracellular domain may be the same or different.
  • at least one of the first intracellular domain and the second intracellular domain (for example, at least two, three, four or more) comprises a costimulatory domain and/or signal transduction The domain, or, as long as it can provide sufficient signal transduction for antibody stimulation to activate the engineered immune cells.
  • the intracellular domain may be an intracellular domain derived from CD3 gamma and/or CD3delta.
  • the intracellular domain may include any one of SEQ ID NO: 3 and 5.
  • the intracellular domain may comprise at least 80% (e.g., at least 85%, 90%, 91%, 92%, 93%, at least 85%, 90%, 91%, 92%, 93%) of the amino acid sequence shown in any one of SEQ ID NO: 3 and 5. 94%, 95%, 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the first intracellular domain of the first CD3 recombinant protein may include a costimulatory domain and a signal transduction domain
  • An intracellular domain can be a peptide fragment of at least two (for example, at least four, at least six or more) amino acids.
  • the first intracellular domain of the second CD3 recombinant protein may include a costimulatory domain and a signal transduction domain.
  • the second CD3 recombinant protein is the first intracellular domain.
  • An intracellular domain can be a peptide fragment of at least two (for example, at least four, at least six or more) amino acids.
  • the CD3 recombinant protein may further include a hinge region.
  • the hinge region may be between the extracellular domain and the transmembrane domain.
  • the hinge region may comprise a hinge region derived from any one or more proteins selected from the group consisting of CD8 ⁇ , CD28, 4-1BB, CD4, CD27, CD7, and PD-1.
  • the hinge region may include an amino acid sequence as shown in any one of SEQ ID NO: 6.
  • the hinge region may comprise at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in any one of SEQ ID NO: 6 , 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the first CD3 recombinant protein may be derived from the extracellular domain of CD3 epsilon domain, the transmembrane region derived from CD28, the intracellular domain derived from CD28, and the intracellular domain derived from CD3 zeta.
  • the first CD3 recombinant protein may include the amino acid sequence shown in SEQ ID NO: 10.
  • the first CD3 recombinant protein may comprise at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in SEQ ID NO: 10 , 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the second CD3 recombinant protein may include an extracellular domain derived from CD3 gamma domain, a transmembrane region derived from CD28, and an intracellular domain derived from CD3 gamma.
  • the second CD3 recombinant protein may include the amino acid sequence shown in SEQ ID NO: 11.
  • the second CD3 recombinant protein may comprise at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in SEQ ID NO: 11. , 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the second CD3 recombinant protein may include an extracellular domain derived from the CD3 gamma domain, a transmembrane region derived from CD28, and the peptide fragment.
  • the second CD3 recombinant protein may include the amino acid sequence shown in SEQ ID NO: 12.
  • the second CD3 recombinant protein may comprise at least 80% (for example, at least 85%, 90%, 91%, 92%, 93%, 94%, 95%) of the amino acid sequence shown in SEQ ID NO: 12 , 96%, 97%, 98%, 99% or higher) sequence homology of amino acid sequences.
  • the CD3 antibody receptor complex may also include one or more (for example, two, three, four or more) first recombinant proteins.
  • the CD3 antibody receptor complex may also include one or more (for example, two, three, four or more) second recombinant proteins.
  • the CD3 antibody receptor complex may further include a third recombinant protein, and the third recombinant protein may include (1) a third extracellular domain, the third extracellular domain comprising a source An extracellular domain selected from any of CD3 gamma domain or CD3 delta domain, (2) the third transmembrane domain, and (3) the third intracellular domain.
  • the third transmembrane domain may be within the range of the transmembrane domain described above, and may be the same as or different from the first transmembrane domain and/or the second transmembrane domain.
  • the third intracellular domain may be within the scope of the intracellular domain described above, and may be the same as or different from the first intracellular domain and/or the second intracellular domain.
  • the intracellular domain of at least one (for example, at least two, three, four or more) CD3 recombinant protein in the CD3 antibody receptor complex contains a costimulatory domain and/or a signal transduction domain Or, as long as it can provide sufficient signal transduction for antibody stimulation to activate the modified immune cells.
  • the present application provides a modified immune cell.
  • the immune cells may include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, and/or peripheral blood mononuclear cells .
  • the immune cells may include T lymphocytes.
  • the T lymphocytes may include thymocytes, natural T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes.
  • the T cell may be a helper T cell (Th), for example, a helper T cell 1 (Th1) or a helper T cell 2 (Th2) cell.
  • the T lymphocytes may be CD4 + helper T cells (HTL; CD4 + T cells), cytotoxic T cells (CTL; CD8 + T cells), tumor infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + / CD8 + T cells, CD4 - / CD8 - T cells or any other subtypes of T lymphocytes.
  • the modified T cells are human T cells.
  • T cells can be obtained from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some cases, any number of T cell lines available and known to those skilled in the art can be used. In other cases, the cells may be derived from a healthy donor, from a patient diagnosed with cancer, or obtained from a patient diagnosed with an infection. In other cases, the cell is part of a mixed population of cells with different phenotypic characteristics.
  • the immune cells may include B cells.
  • the B cells may include effector B cells (plasma cells) and memory B cells.
  • the B cells may include B2 cells, B1 cells, marginal zone B cells, follicular B cells, and regulatory B cells.
  • the immune cells may include macrophages.
  • the B cells may include type I macrophages (M1) and type II macrophages (such as M2a, M2B, M2c).
  • the immune cells may include NK cells.
  • the NK cells may include CD56bright and CD56dim.
  • the NK cells may include NK1 and NK2.
  • the NK cells may include A-NK and NA-NK.
  • the immune cells may include white blood cells.
  • White blood cells usually refer to a kind of nucleated blood cells that have active mobility and can migrate from inside blood vessels to outside blood vessels, or from tissues outside blood vessels into blood vessels.
  • white blood cells can also be present in the lymphatic system, spleen, tonsils, and other tissues of the body.
  • the white blood cells may include granulocytes (such as neutrophils, eosinophils, basophils), agranulocytes (such as lymphocytes, monocytes, macrophages, phagocytes, Mast cells).
  • the immune cells may include lymphocytes, and the lymphocytes may include any monocytes, non-phagocytic leukocytes found in blood, lymph and lymphatic tissues, for example, B lymphocytes, T lymphocytes, Natural killer (NK) cells.
  • lymphocytes may include any monocytes, non-phagocytic leukocytes found in blood, lymph and lymphatic tissues, for example, B lymphocytes, T lymphocytes, Natural killer (NK) cells.
  • the immune cells may include peripheral blood mononuclear cells, which may include any cell having a mononucleus in peripheral blood.
  • the peripheral blood mononuclear cells may include T cells, B cells, NK cells, lymphocytes, monocytes, and dendritic cells.
  • the immune cells may include macrophages.
  • Macrophages are a kind of material that can swallow and digest cell debris, microorganisms, cancer cells, and all other substances that lack the surface markers expressed on the surface of normal cells. This process is called phagocytosis. Macrophages are found in almost all tissues, looking for possible pathogens through the movement of amoeba. In addition to playing an important role in the non-specific natural immune response, they can also help initiate adaptive immunity by recruiting other immune cell types, such as lymphocytes.
  • TCR T cell receptor
  • the non-expression of T cell receptor (TCR) may include down-regulation of the expression and/or activity of T cell receptor (TCR).
  • TCR T cell receptor
  • the down-regulation may include not expressing active TCR, not expressing endogenous TCR, not expressing exogenous TCR, not including TCR structure, including inactivated TCR, and/or missing TCR.
  • the expression and/or activity of the T cell receptor alpha constant region protein and/or the T cell receptor beta constant region protein in the immune cell can be down-regulated.
  • the down-regulation may include down-regulating the expression and/or activity of the nucleic acid molecule encoding the cell receptor alpha constant region protein and/or the T cell receptor beta constant region protein; and/or, including down-regulating the The expression and/or activity of cell receptor alpha constant region protein and/or T cell receptor beta constant region protein.
  • the expression of the CD3 antibody receptor complex may not depend on the expression of TCR.
  • the expression and/or activity of the MHC complex in the immune cell is down-regulated.
  • the down-regulation may include down-regulating the expression and/or activity of the nucleic acid molecule encoding the cellular MHC complex; and/or, including down-regulating the expression and/or activity of the cellular MHC complex protein.
  • the down-regulation can be achieved by knockout, knockdown, gene mutation, gene deletion, gene silencing, or any combination of the above to make the TCR of the immune cell and/ Or the expression and/or activity of the MHC complex is down-regulated.
  • one or more substances selected from the following group can be administered to the immune cells to achieve down-regulation: antisense RNA, siRNA, shRNA, CRISPR/Cas system, RNA editing system such as RNA adenosine deaminase (ADAR) , RNA-guided endonuclease, zinc finger nuclease (ZFN), Mega-TAL nuclease, transcription activator-like effector nuclease (TALEN), meganuclease (Meganuclease), base editing, CRISPR interference, And, zinc finger protein (Zinc finger) gene repressor and/or transcription activator-like effector (TALE) gene repressor mediated transcriptional inhibition.
  • RNA editing system such as RNA adenosine deaminase (ADAR) , RNA-guided endonuclease, zinc finger nuclease (ZFN), Mega-TAL nuclease, transcription activator-like effector nucle
  • the down-regulation may include administering to the immune effector cell a guide RNA targeting the exon portion of the nucleic acid molecule (e.g., a nucleic acid molecule encoding the MHC complex of the cell).
  • a guide RNA targeting the nucleic acid molecule encoding the B2M can use the guide RNA in the prior art, and the full text of WO2019/011118 is incorporated herein by reference.
  • the modified immune cells described in this application may also include chimeric antigen receptors (CAR) and/or chimeric autoantibody receptors (CAAR).
  • CAR chimeric antigen receptors
  • CAAR chimeric autoantibody receptors
  • This application provides a method for preparing modified lymphocytes, which may include the following steps: 1. Obtain peripheral blood T cells from healthy donors; 2. Use magnetic beads loaded with CD3 and CD28 antibodies to activate T cells; 3. T After cell activation, use lentivirus to transfer the chimeric antibody receptor gene into T cells; 4. Remove magnetic beads; 5. Use gene editing technology to knock out the important genes TRAC and B2M that produce immune rejection; 6. Continue to culture and Harvest the cells.
  • the TRAC and B2M genes in lymphocytes are inactivated, which can effectively reduce the immune rejection of lymphocytes in allogeneic cell therapy, and at the same time express two recombinant CD3 surface proteins in lymphocytes, which are co-expressed
  • the CD3 surface protein is the CD3 antibody receptor, which enables the engineered lymphocytes to bind bispecific antibodies.
  • the method for preparing modified lymphocytes includes: (i) preparing lymphocytes with surface deletion of class I MHC molecules and TCR molecules; (ii) expressing two recombinant CD3 surface proteins on the surface of lymphocytes.
  • the present application provides a vector that can be used to transfer the isolated nucleic acid molecule encoding the CD3 antibody receptor complex into cells.
  • the vector may be selected from one or more of plasmids, retroviral vectors and lentiviral vectors.
  • the vector may also contain other genes, such as a marker gene that allows selection of the vector in a suitable host cell and under suitable conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host.
  • control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually includes 5'non-transcribed sequences and 5'and 3'non-translated sequences involved in transcription and translation initiation, such as TATA box, plus Cap sequence, CAAT sequence, etc.
  • the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcriptional control functionally linked to the nucleic acid.
  • One or more nucleic acid molecules described in this application can be operably linked to the expression control element.
  • Nucleic acid non-viral delivery methods include lipofection, nuclear transfection, microinjection, gene gun, viral particles, liposomes, immunoliposomes, polycations or lipid nucleic acid conjugates, naked DNA, artificial virions And reagents to enhance DNA uptake.
  • RNA or DNA virus-based systems can be used to deliver nucleic acids.
  • the virus can be targeted to specific cells in the body to effectively load the virus payload into the nucleus.
  • the viral vector may be directly administered to the patient (in vivo) or may be in an indirect form, for example, the virus is used to treat cells in vitro, and then the treated cells are administered to the patient (ex vivo).
  • Retroviral vectors can include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus vectors for gene transfer.
  • retroviruses, lentiviruses, and adeno-associated viruses can be used to transfer genes into the host genome to allow long-term expression of the inserted genes.
  • Lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce higher viral titers.
  • the lentiviral vector may include a long terminal repeat sequence 5'LTR and a truncated 3'LTR, RRE, rev response element (cPPT), central termination sequence (CTS) and/or post-translational regulatory element (WPRE).
  • the molecule can be constructed on a lentiviral vector by digestion with BamHI and SalI.
  • the present application provides a pharmaceutical composition.
  • the pharmaceutical composition may include the modified immune cells described in this application, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable adjuvant generally refers to any and all solvents, dispersion media, coatings, antibacterial agents that are compatible with the administration of immune cells and/or cell populations of this application. Agents and antifungal agents, isotonic agents and absorption delay agents. Unless it is incompatible with the immune cells of the application and/or the cell population of the application, any conventional medium or reagent can be considered for use in the pharmaceutical composition of the application.
  • the pharmaceutical composition described in this application may include an antibody.
  • the antibody and the modified immune cell may be present in the same or different container.
  • the antibody can be administered before, during or after the administration of the modified immune cells.
  • the antibody may be a bispecific antibody.
  • the bispecific antibody can simultaneously bind to two targets, and the two targets can be on the same target protein or different target proteins.
  • T cell adapter is a relatively special bispecific antibody.
  • the T cell adaptor may include two linked scFvs, one end targeting CD3 on the surface of T cells, and the other end targeting receptors on the surface of target cells (eg, tumor cells), thereby mediating T cells to kill tumor cells.
  • the receptors on the target cell surface may include, but are not limited to, CD19, CD2, CD3, CD4, CD5, CD7, CD8, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD38, CD40, CD44V6, CD47, CD52, CD56 , CD57, CD58, CD79b, CD80, CD86, CD81, CD123, CD133, CD137, CD151, CD171, CD276, CLL1, B7H4, BCMA, VEGFR-2, EGFR, GPC3, PMSA, CEACAM6, c-Met, EGFRvIII, ErbB2 /HER2, ErbB3, HER-2, HER3, ErbB4/HER-4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, Flt1, KDR, Flt4, Flt3, CEA, CA125 , CTLA-4, GITR, BTLA, TGFBR1, TGFBR2,
  • the basic principle of this combination therapy is that bispecific antibodies are responsible for finding the target and guiding T cells to the target cells, while the modified immune cells are responsible for the powerful killing of target cells.
  • TCR In primary T cells, TCR is not knocked out, and the bispecific antibody can mediate the killing of target cells by control T cells through the naturally expressed TCR-CD3 complex.
  • the CD3 antibody receptor As shown in Figure 1, among the universal CD3 antibody receptor T cells, the CD3 antibody receptor is expressed on the surface of the modified immune cells.
  • Bispecific antibodies can simultaneously activate the co-activation signal and CD3 zeta activation signal through the CD3 antibody receptor to promote the killing of tumor cells and the proliferation of T cells.
  • the advantage of the combined use is that cell therapy can be combined with bispecific antibodies for multiple targets, and it is easy to control, making the therapy safer.
  • the bispecific antibody may be derived from the engineered immune cells described in this application.
  • the bispecific antibody may include a bispecific antibody secreted by the engineered immune cells described in this application
  • the recurrence after the use of CAR-T cells includes target protein negative recurrence and target protein positive recurrence.
  • the positive recurrence of the target protein is mainly caused by the failure of CAR-T cells after reinfusion.
  • This therapy can achieve continuous use of bispecific antibodies after cell regression, using chimeric antibody receptor T cells and the body's own T cells to suppress tumor recurrence. For patients with poor physical conditions in the late stage, they can also achieve good therapeutic effects through flexible adjustments on the basis of ensuring safety, and increase the scope of application for patients.
  • the application also provides the use of the modified immune cells and/or the pharmaceutical composition in the preparation of medicines for the treatment of tumors.
  • this application also provides a method for treating tumors, the method comprising administering the modified immune cells and the pharmaceutical composition to a subject in need.
  • the subject can first receive certain chemotherapy pretreatment, and then the transformed lymphocytes and the bispecific antibody are infused into the subject simultaneously or sequentially. Take cell transfusion as an example. After the cells are transfused, the bispecific antibodies are transfused in different doses. If the patient has side effects, adjust the input dose of the bispecific antibody.
  • the transformed lymphocytes and double antibodies can be re-infused until remission or serious side effects appear.
  • the modified lymphocytes of the present application can kill cancer cells, such as blood cancer and solid tumor cancer cells, after binding bispecific antibodies.
  • the tumor may include non-solid tumors, including but not limited to leukemia, lymphoma, and/or multiple myeloma, and solid tumors, including but not limited to lung cancer, gastric cancer, esophageal cancer, colon Cancer, breast cancer, ovarian cancer, bladder cancer, renal cell carcinoma, prostate cancer, melanoma, head and neck tumors, glioma and soft tissue sarcoma, etc.
  • non-solid tumors including but not limited to leukemia, lymphoma, and/or multiple myeloma
  • solid tumors including but not limited to lung cancer, gastric cancer, esophageal cancer, colon Cancer, breast cancer, ovarian cancer, bladder cancer, renal cell carcinoma, prostate cancer, melanoma, head and neck tumors, glioma and soft tissue sarcoma, etc.
  • the tumor may include lymphoma.
  • CG-UST-1 consists of two parts, which are the tandem of two CD3 recombinant protein genes.
  • the first CD3 recombinant protein contains CD3 epsilon extracellular domain, CD28 transmembrane region, CD28 intracellular domain and CD3 zeta intracellular domain (SEQ ID NO: 10).
  • the second CD3 recombinant protein includes CD3 gamma extracellular domain, CD28 transmembrane region, and CD3 gamma intracellular domain (SEQ ID NO: 11).
  • the two CD3 recombinant proteins are connected by the linker T2A gene (SEQ ID NO. 16).
  • the CG-UST-1 gene sequence was synthesized by Nanjing GenScript and cloned into pUC57 vector (Nanjing GenScript). When synthesizing genes, add specific restriction endonuclease sites: BamH1 and Sal1 at both ends of the gene.
  • the recombinant plasmid of gene synthesis was digested with restriction enzymes BamH1 (NEB; R3136S) and Sal1 (NEB; R3138S), and the gene fragments were separated by agarose gel electrophoresis and then purified by gel recovery (QIAGEN; 28706). Determine the concentration of recovered gene fragments.
  • T4 DNA ligase (NEB; M0202S) was used to ligate the synthesized gene sequence to the BamH1-Sal1 site of the lentiviral vector (Addgene; catalog number: 12252).
  • the cloned lentiviral vector is called: pL-CG-UST-1.
  • the lentiviral vector plasmid was sequenced and verified, and the recombinant plasmid sequencing primers were: Lenti-For (TCAAGCCTCAGACAGTGGTTC; SEQ ID NO: 17) and Lenti-Rev (CCTCATAAAGAGACAGCAACCAGG; SEQ ID NO: 18).
  • the plasmid construction of CG-UST-2 and CG-UST-3 uses the same construction process.
  • the constructed lentiviral vector plasmids are called pL-CG-UST-2 and pL-CG-UST-3 respectively.
  • the lentiviral vector plasmid constructed above was retransformed into E. coli.
  • Use Qiagen HiSpeed Plasmid Maxi Kit (Cat. No. 12662) to perform plasmid extraction according to the experimental procedure provided by the kit. After the plasmid was extracted, Nanodrop (Thermo Fisher Scientific) was used to detect the plasmid concentration and the DNA agarose gel was used to detect the supercoiled plasmid content.
  • inoculate 293T cells inoculate cells according to about 15-16 ⁇ 10 6 cells/T175 flask (cultured in 35-40 ml medium).
  • plasmid transfection Before transfection, the medium needs to be replaced with a medium with 10% FBS but no double antibody.
  • Viral vector plasmid 18 ⁇ g, psPAX2 plasmid (Addgene; article number: 12260): 9 ⁇ g, pMD2.G plasmid (Addgene; article number: 12259): 18 ⁇ g.
  • transfection reagent complex prepares 100 ⁇ l Lipofectamine 2000 (invitrogen; 11668-019) to 1.5ml Opti-MEM, mix well after adding, and let stand at room temperature for 5 minutes; then add the plasmid complex to the transfection reagent complex After mixing, let it stand for 25 minutes; finally add the transfection complex to the cell culture medium and shake gently.
  • PMBCs purchased from Miaotong Biotechnology
  • the concentrated lentivirus was added to the T cell culture flask to transfect the T cells.
  • CRISPR/Cas9 was used to knock out TCR and B2M in T cells to construct universal T cells.
  • the gRNA sequence and operation procedure used are carried out with reference to Example 3 of patent WO2019/011118.
  • Detect the expression of CD3 antibody receptor complex in cells by fluorescent antibody staining and flow cytometry The basic steps are as follows: centrifuge to collect a certain volume of modified T cells cultured, and use Flag antibody (BioLegend; 637309) and APC- TCR antibody (BioLegend; 306718) stained the cells, incubated in the dark for 30 minutes, washed once with PBS, resuspended in an appropriate volume of PBS, and finally used flow cytometry to detect the expression and expression of CD3 antibody receptor complex in T cells Expression in TCR-negative cells.
  • the expression efficiencies of the CD3 antibody receptor complex in TCR-negative T cells were: CG-UST-1: 30.2%, CG-UST-2: 25.0% and CG-UST-3: 65.6 %(figure 2).
  • the expression efficiency is similar to the corresponding non-knockout group, so the CD3 antibody receptor complex can be independently expressed on human T cells in a TCR-independent manner.
  • T cells expressing CG-UST-1 and CG-UST-2 can be recognized by the CD3 antibody HIT3a clone (see Figure 4), but not by the clone SP34-2 (see Figure 5) ). Further, the cells were stained with SP34-2 and OKT3 antibodies at the same time, and it was found that these cells can be recognized by the OKT3 antibody (see Figure 6). However, T cells expressing CG-UST-3 containing the extramembrane domain of CD3 epsilon alone cannot be recognized by common CD3 antibody clones (see Figure 3 to Figure 6).
  • the CD3 antibody receptor can be expressed in TCR knockout primary T cells in a TCR-independent form.
  • T cells co-expressing the CD3 antibody receptor complex of CD3 epsilon and CD3 gamma extracellular domain can be recognized by conventional CD3 antibodies, including CD3 antibodies UCHT1, HIT3a and OKT3.
  • Coated tablet Dilute OKT3 antibody with PBS to 0.25 ⁇ g/ml. The diluted antibody was added to a 96-well plate at 100 ⁇ l per well, and incubated at 37°C for 3 hours. After the incubation is complete, wash the plate with 1 ⁇ PBS to remove the PBS.
  • T cells (2) Activate T cells. Adjust the T cell density of different groups to 1 ⁇ 10 6 /ml. Then the cells were seeded into the plate according to 100 ⁇ l per well and incubated at 37°C for 24 hours.
  • cytokine secretion Take the supernatant of the above cells, transfer to a new 96-well plate, and use an ELISA kit (Thermo Fisher Scientific; Catalog No. 88-7316) to detect the secretion of IFN- ⁇ cytokines from T cells. Plate preparation and supernatant cytokine detection were performed in accordance with the procedures provided by the kit.
  • OKT3 antibody stimulated general antibody receptor T cells expressing CG-UST-1 and CG-UST-2 to secrete 341.28 and 248.76 pg/ml IFN- ⁇ , respectively. See Table 1. Therefore, the coated CD3 antibody can stimulate universal T cells expressing the CD3 antibody receptor complex CG-UST-1 and CG-UST-2 to secrete the cytokine IFN- ⁇ .
  • OKT3 antibody stimulates universal CD3 antibody receptor T cells to secrete cytokine IFN- ⁇ (pg/ml)
  • the anti-CD3 anti-CD19 bispecific antibody was purchased from Invivogen (Cat. No.: bimab-hcd19cd3).
  • the double antibody can simultaneously bind CD3 epsilon and CD19, and mediate T cells to kill target cells expressing CD19.
  • the CD3 antibody clone number used in this double antibody is L2K-07.
  • the cells are co-cultured first.
  • the steps are as follows: adjust the T cell concentration of different groups to 1 ⁇ 10 6 /ml, and then plant the T cells into a 96-well plate according to 100 ⁇ l per well. Place the seeded T cells temporarily at 37°C and incubate; adjust the concentration of Raji cells expressing CD19 (Cell Bank of the Chinese Academy of Sciences) to 1 ⁇ 10 6 /ml, and then seed the Raji cells to 96 cells containing T cells according to 100 ⁇ l per well Orifice plate species. The final ratio of T cells to tumor cells is 1:1.
  • the anti-CD3 anti-CD19 bispecific antibody was added to the cells.
  • the final concentration of the double antibody is 50ng/ml. After mixing thoroughly, centrifuge at 500 rpm for 3 minutes. The cells were incubated in a 37°C incubator for 24 hours.
  • CD19 negative cells are T cells.
  • CD137 fluorescent antibody eBioscience, 11-0199-42
  • TCR antibody TCR antibody
  • FIG. 8 By detecting the cell membrane activation label CD137, it is found that Raji cells with double antibodies can activate universal T cells expressing CD3 antibody receptors CG-UST-1 and CG-UST-2, as shown in Figure 8.
  • the positive rates of CD137 were 7.15% and 6.47%, respectively.
  • Example 5 After the incubation was completed, the supernatant was taken, as in Example 5, and the ELISA experiment was performed.
  • the results show that the anti-CD3 and anti-CD19 bispecific antibodies can mediate the secretion of the cytokine IFN- ⁇ from the universal CD3 antibody receptor T cells, as shown in Table 2.
  • TCR knockout the double antibody combined with tumor cells stimulated the general antibody receptor T cells expressing CG-UST-1 and CG-UST-2 to secrete 287.639 and 286.512 pg/ml IFN- ⁇ , respectively.
  • Double antibodies mediated tumor cells to stimulate universal CD3 chimeric antibody receptor T cells to secrete cytokine IFN- ⁇ (pg/ml)

Abstract

L'invention concerne une cellule immunitaire modifiée. La cellule immunitaire modifiée n'exprime pas un récepteur des lymphocytes T (TCR) et contient un complexe anticorps-récepteur CD3. La présente invention concerne en outre une composition pharmaceutique contenant la cellule immunitaire modifiée et un anticorps bispécifique, et l'utilisation de la composition pharmaceutique dans la préparation d'un médicament.
PCT/CN2020/100231 2019-07-06 2020-07-03 Complexe anticorps-récepteur cd3 exprimant une cellule immunitaire et son utilisation WO2021004400A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202080003009.1A CN112204135A (zh) 2019-07-06 2020-07-03 一种表达cd3抗体受体复合物的免疫细胞及其用途

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910607136 2019-07-06
CN201910607136.1 2019-07-06

Publications (1)

Publication Number Publication Date
WO2021004400A1 true WO2021004400A1 (fr) 2021-01-14

Family

ID=74115032

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/100231 WO2021004400A1 (fr) 2019-07-06 2020-07-03 Complexe anticorps-récepteur cd3 exprimant une cellule immunitaire et son utilisation

Country Status (1)

Country Link
WO (1) WO2021004400A1 (fr)

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103509122A (zh) * 2013-09-23 2014-01-15 武汉友芝友生物制药有限公司 一种cd3抗原及其制备方法和用途
WO2014140248A1 (fr) * 2013-03-15 2014-09-18 Amgen Research (Munich) Gmbh Molécules liantes pour bcma et cd3
CN104114578A (zh) * 2011-11-15 2014-10-22 安进研发（慕尼黑）股份有限公司 Bcma和cd3的结合分子
CN105949325A (zh) * 2016-07-08 2016-09-21 重庆精准生物技术有限公司 包含cd27胞内结构域的嵌合抗原受体、慢病毒载体及其应用
CN107090044A (zh) * 2010-04-01 2017-08-25 安进研发（慕尼黑）股份有限公司 跨物种特异性PSMAxCD3双特异性单链抗体
CN107404881A (zh) * 2014-09-19 2017-11-28 希望之城公司 用于过继性t细胞疗法的中央记忆t细胞
CN107849148A (zh) * 2015-05-21 2018-03-27 哈普恩治疗公司 三特异性结合蛋白质及使用方法
WO2018200496A1 (fr) * 2017-04-24 2018-11-01 Kite Pharma, Inc. Domaines de liaison à l'antigène humanisés dirigés contre les cd19 et leurs procédés d'utilisation
WO2019032927A1 (fr) * 2017-08-09 2019-02-14 Juno Therapeutics, Inc. Procédés de production de compositions de cellules génétiquement modifiées et compositions associées

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107090044A (zh) * 2010-04-01 2017-08-25 安进研发（慕尼黑）股份有限公司 跨物种特异性PSMAxCD3双特异性单链抗体
CN104114578A (zh) * 2011-11-15 2014-10-22 安进研发（慕尼黑）股份有限公司 Bcma和cd3的结合分子
WO2014140248A1 (fr) * 2013-03-15 2014-09-18 Amgen Research (Munich) Gmbh Molécules liantes pour bcma et cd3
CN103509122A (zh) * 2013-09-23 2014-01-15 武汉友芝友生物制药有限公司 一种cd3抗原及其制备方法和用途
CN107404881A (zh) * 2014-09-19 2017-11-28 希望之城公司 用于过继性t细胞疗法的中央记忆t细胞
CN107849148A (zh) * 2015-05-21 2018-03-27 哈普恩治疗公司 三特异性结合蛋白质及使用方法
CN105949325A (zh) * 2016-07-08 2016-09-21 重庆精准生物技术有限公司 包含cd27胞内结构域的嵌合抗原受体、慢病毒载体及其应用
WO2018200496A1 (fr) * 2017-04-24 2018-11-01 Kite Pharma, Inc. Domaines de liaison à l'antigène humanisés dirigés contre les cd19 et leurs procédés d'utilisation
WO2019032927A1 (fr) * 2017-08-09 2019-02-14 Juno Therapeutics, Inc. Procédés de production de compositions de cellules génétiquement modifiées et compositions associées

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
M. MALISSEN ET AL.: "Altered T cell development in mice with a targeted mutation of the CD3‐epsilon gene", THEEMBOJOURNAL, vol. 14, no. 19, 2 October 1995 (1995-10-02), XP055772395, DOI: 20200910083056A *

Similar Documents

Publication Publication Date Title
US20190375815A1 (en) Treatment of cancer using chimeric t cell receptor proteins having multiple specificities
JP2022153456A (ja) 導入遺伝子の遺伝子タグおよびその使用方法
US20220025001A1 (en) Nucleic acid constructs for co-expression of chimeric antigen receptor and transcription factor, cells containing and therapeutic use thereof
JP2023534928A (ja) 改変免疫細胞及びその使用
WO2017027392A1 (fr) Traitement du cancer à l'aide des protéines de récepteur cd3 chimères
AU2015357526A1 (en) Chimeric antigen receptors targeting B-cell maturation antigen and uses thereof
US11739297B2 (en) Method of increasing tumor killing activity of macrophages or monocytes comprising chimeric antigen receptor
JP2022523749A (ja) Ｔｃｒ融合タンパク質およびｔｃｒ融合タンパク質を発現する細胞
EP4148125A1 (fr) Cellule immunitaire modifiée destinée à une allotransplantation
EP4019538A1 (fr) Reprogrammation de cellules immunitaires par une intégration ciblée de transgènes de récepteur d'antigène chimérique déficient en zêta
CN112204135A (zh) 一种表达cd3抗体受体复合物的免疫细胞及其用途
EP4194472A1 (fr) Récepteur antigénique chimérique comprenant un nouveau domaine de co-stimulation et utilisation associée
US20240024475A1 (en) Antigen-Binding Protein Targeting CD70 and Use Thereof
WO2021004400A1 (fr) Complexe anticorps-récepteur cd3 exprimant une cellule immunitaire et son utilisation
JP2023540023A (ja) Ｃｅａを認識するキメラ抗原受容体（ｃａｒ）発現細胞
AU2021334107A1 (en) A chimeric antigen receptor construct encoding a checkpoint inhibitory molecule and an immune stimulatory cytokine and car-expressing cells recognizing CD44v6
WO2023035947A1 (fr) Cellule immunitaire modifiée et son utilisation
WO2023025009A1 (fr) Cellule immunitaire modifiée et son utilisation
US20220315931A1 (en) Immune cells expressing receptor specific to class i mhc molecule and interfering rna for beta2 microglobulin gene
WO2023083003A1 (fr) Cellule immunitaire modifiée et son utilisation
WO2023051361A1 (fr) Cellule immunitaire modifiée et son utilisation
WO2023071811A1 (fr) Cellule immunitaire modifiée et son utilisation
WO2023016284A1 (fr) Cellule immunitaire modifiée et son utilisation
CN115785279A (zh) 包含新型共刺激结构域的嵌合抗原受体及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20836685

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 20836685

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 24/05/2022)