WO2023273762A1 - Épitope conformationnel spatial induisant une rétention efficace de cd3 dans des cellules et son utilisation - Google Patents

Épitope conformationnel spatial induisant une rétention efficace de cd3 dans des cellules et son utilisation Download PDF

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WO2023273762A1
WO2023273762A1 PCT/CN2022/096017 CN2022096017W WO2023273762A1 WO 2023273762 A1 WO2023273762 A1 WO 2023273762A1 CN 2022096017 W CN2022096017 W CN 2022096017W WO 2023273762 A1 WO2023273762 A1 WO 2023273762A1
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antibody
antigen
cells
sequence
seq
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李俊
张鹏潮
张银航
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苏州方德门达新药开发有限公司
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Definitions

  • the invention relates to a spatial conformation epitope that mediates effective retention of CD3 ⁇ in cells, a polypeptide or an antibody specifically combined with it, and applications thereof.
  • chimeric antigen receptor T cell Chimeric antigen receptor-T, CAR-T
  • CAR-T chimeric antigen receptor T cell
  • the chimeric antigen receptor (CAR) expressed by CAR-T cells generally includes an extracellular antigen-binding domain, a transmembrane domain and an intracellular signaling domain.
  • CAR-T cells are transduced and expanded by the patient's own T cells through the CAR gene, and then reinfused back into the patient.
  • CAR-T cells can effectively recognize tumor antigens and induce specific anti-tumor immune responses without being restricted by the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • the preparation and reinfusion strategies of the above two CAR-T drugs are to collect the patient's own peripheral blood, isolate T cells, integrate the coding gene of CAR into the genome of T cells through a viral vector, and then expand and cultivate them. infused into the patient.
  • This fully individualized CAR-T cell production not only has a long production cycle and high cost, but also brings many uncertainties to the reinfusion therapy. For example, the failure of CAR-T cell production due to insufficient number or malfunction of the isolated T cells, the rapid progression of the patient's disease during cell production and the loss of the therapeutic window, etc. Therefore, a common expectation in the field of CAR-T is ready-to-use (off-the-shelf) allogeneic (i.e. universal) CAR-T cells from healthy donors to effectively avoid or solve the above-mentioned production and treatment-related problems , and greatly reduce production costs.
  • GVHD graft-versus-host disease
  • the first aspect of the present invention provides an epitope of CD3 ⁇ , and the epitope is an epitope peptide.
  • the epitope mediates intracellular retention of CD3 ⁇ .
  • the epitope comprises glutamic acid at position 56, histidine at position 61, asparagine at position 62, arginine at position 101, lysine at position 104 and Aspartic acid at position 107.
  • the epitope further comprises glycine at position 54, serine at position 55, glutamine at position 60, lysine at position 64 and aspartic acid at position 69 of SEQ ID NO:1.
  • the epitope comprises amino acids 56, 60-65, 69-71, 101, 104, 107 of SEQ ID NO:1.
  • the epitope comprises amino acids 56, 60-65, 69-71, 101-107 of SEQ ID NO: 1.
  • the epitope comprises amino acids 56, 60-71, 101-107 of SEQ ID NO: 1.
  • the epitope comprises amino acids 56-65, 69-71, 101-107 of SEQ ID NO: 1.
  • the epitope comprises amino acids 56-71, 101-107 of SEQ ID NO: 1.
  • the epitope comprises amino acids 56-107 of SEQ ID NO: 1.
  • the epitope comprises amino acids 54-107 of SEQ ID NO: 1.
  • the epitope is a steric epitope, the amino acids of the epitope forming a steric conformation that binds the antibody or antigen-binding fragment thereof.
  • the epitope is represented by the following formula (I):
  • each X in X 1 -X 55 and X 108 -X 207 is independently any amino acid or none, and each of the other Xs is independently any amino acid.
  • the epitope is represented by the following formula (II):
  • each X in X 1 -X 53 and X 108 -X 207 is independently any amino acid or none, and each of the other Xs is independently any amino acid.
  • the epitope peptide is represented by the following formula (III):
  • each X is independently any amino acid.
  • the epitope peptide is represented by the following formula (IV):
  • each X is independently any amino acid.
  • the epitope is a fragment of SEQ ID NO: 1 comprising amino acids 56, 61, 62, 101, 104 and 107, and optionally amino acids 54, 55, 60, 64 and 69 .
  • the epitope is a fragment of SEQ ID NO: 1 comprising amino acids 56, 60-65, 69-71, 101, 104, 107.
  • the epitope is a fragment of SEQ ID NO: 1 comprising amino acids 56, 60-65, 69-71, 101-107.
  • the epitope is a fragment of SEQ ID NO: 1 comprising amino acids 56, 60-71, 101-107.
  • the epitope is a fragment of SEQ ID NO: 1 comprising amino acids 56-65, 69-71, 101-107.
  • the epitope is a fragment of SEQ ID NO: 1 comprising amino acids 56-71, 101-107.
  • the epitope is a fragment of SEQ ID NO: 1 comprising amino acids 56-107.
  • the epitope is a fragment of SEQ ID NO: 1 comprising amino acids 54-107.
  • the fragments are at least 1 amino acid, at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 6 amino acids, at least 7 amino acids, at least 8 amino acids in length. amino acids, at least 9 amino acids, at least 10 amino acids, at least 11 amino acids, at least 12 amino acids, at least 13 amino acids, at least 14 amino acids, at least 15 amino acids, at least 16 amino acids, at least 52 amino acids, at least 54 amino acids.
  • glutamic acid at position 56, histidine at position 61, asparagine at position 62, arginine at position 101, lysine at position 104 and aspartic acid at position 107 in the epitope acid, and optionally Glycine 54, Serine 55, Glutamine 60, Lysine 64, and Aspartate 69 are bound to the antibody or antigen-binding fragment thereof.
  • the present invention also provides an anti-CD3 ⁇ antibody or an antigen-binding fragment thereof that binds an epitope of CD3 ⁇ , or a variant having at least 90% sequence identity to said anti-CD3 ⁇ antibody or an antigen-binding fragment thereof and retaining its epitope-binding activity , the epitope is located in the 54-107th position of SEQ ID NO:1, preferably, the epitope comprises glutamic acid at position 56, histidine at position 61, asparagine at position 62 of SEQ ID NO:1 , 101 arginine, 104 lysine and 107 aspartic acid.
  • the epitope is as described in the first aspect of the present invention.
  • the anti-CD3 ⁇ antibody is a monoclonal antibody.
  • the anti-CD3 ⁇ antibody is a neutralizing antibody.
  • the anti-CD3 ⁇ antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region
  • the heavy chain variable region has the following HCDRs:
  • HCDR1 GYTFISYT
  • HCDR2 X 1 NPRSGYT, wherein X 1 is any amino acid
  • HCDR3 AX 2 X 3 X 4 YYDYX 5 X 6 FAY, wherein X 2 , X 3 , X 4 , X 5 , X 6 are any amino acids,
  • the light chain variable region has the following LCDRs:
  • LCDR1 SSVSY
  • LCDR2 DTS
  • LCDR3 QQWSSX 7 PPT, where X 7 is any amino acid.
  • X is alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, threonine , Glycine, Asparagine, Glutamine, Serine, Tyrosine, or Cysteine.
  • X is I or T.
  • X is lysine, arginine or histidine.
  • X2 is K or R.
  • X is glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, valine, or isoleucine.
  • X3 is T or S.
  • X is glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, alanine, valine, leucine, iso Leucine, proline, phenylalanine, methionine, or tryptophan.
  • X4 is G or A.
  • X is aspartic acid, glutamic acid, lysine, arginine, histidine, tyrosine, phenylalanine, or tryptophan.
  • X 5 is D or H.
  • X is glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, alanine, valine, leucine, iso Leucine, proline, phenylalanine, methionine, or tryptophan.
  • X 6 is G or A.
  • X is glycine, asparagine, glutamine, serine, threonine, tyrosine or cysteine.
  • X7 is Q or N.
  • the heavy chain variable region has the following HCDRs:
  • HCDR1 as shown in SEQ ID NO:2: GYTFISYT,
  • HCDR2 as shown in SEQ ID NO:3: TNPRSGYT,
  • HCDR3 as shown in SEQ ID NO:4: AKTGYYDYHAFAY.
  • the light chain variable region has the following LCDRs:
  • LCDR3 as shown in SEQ ID NO:7: QQWSSQPPT.
  • the heavy chain variable region has the sequence set forth as residues 659-778 of SEQ ID NO: 16 or a sequence having at least 90% sequence identity thereto.
  • the light chain variable region has the sequence set forth as residues 538-643 of SEQ ID NO: 16 or a sequence having at least 90% sequence identity thereto.
  • the antibody has the sequence set forth as residues 538-778 of SEQ ID NO: 16 or a sequence having at least 90% sequence identity thereto.
  • the antibody or antigen-binding fragment thereof is a single chain antibody.
  • the antibody or antigen-binding fragment thereof further comprises a localization sequence and an optional signal peptide.
  • the positioning sequence is selected from an endoplasmic reticulum retention sequence, a Golgi retention sequence, an E3 ubiquitin ligase binding sequence, a proteasome targeting sequence or a lysosome targeting sequence; preferably, the The endoplasmic reticulum retention sequence comprises or consists of any one of SEQ ID NOs: 10-15, wherein X is any amino acid.
  • the signal peptide is shown in residues 516-537 of SEQ ID NO:16.
  • the present invention also provides a kit comprising a polypeptide having the sequence of the epitope described in the first aspect herein and the antibody or antigen-binding fragment or variant thereof described herein.
  • the polypeptide has the sequence shown in amino acids 54-107 or 56-107 of SEQ ID NO:1.
  • the kit further includes reagents for detecting CD3 ⁇ by antigen-antibody reaction, such as coating solution, blocking solution, secondary antibody, chromogenic solution, and stop solution.
  • reagents for detecting CD3 ⁇ by antigen-antibody reaction such as coating solution, blocking solution, secondary antibody, chromogenic solution, and stop solution.
  • the present invention also provides nucleic acid molecules having a coding sequence for an epitope described herein, an antibody or antigen-binding fragment thereof, or its complement.
  • the invention also provides nucleic acid constructs comprising nucleic acid molecules of the invention.
  • the nucleic acid construct is a cloning vector, an integrating vector or an expression vector.
  • the invention also provides lentiviruses comprising the nucleic acid constructs of the invention.
  • the invention also provides cells, preferably engineered T cells, expressing the antibodies or antigen-binding fragments thereof described herein.
  • the cells contain the nucleic acid molecules, nucleic acid constructs and/or lentiviruses described herein.
  • the antibody or antigen-binding fragment thereof down-regulates expression of the TCR/CD3 complex in the cell.
  • the antibody or antigen-binding fragment thereof comprises a localization sequence and optionally a signal peptide.
  • the positioning sequence is selected from an endoplasmic reticulum retention sequence, a Golgi retention sequence, an E3 ubiquitin ligase binding sequence, a proteasome targeting sequence or a lysosome targeting sequence; preferably, the The endoplasmic reticulum retention sequence comprises or consists of any one of SEQ ID NOs: 10-15, wherein X is any amino acid.
  • the signal peptide is shown in residues 516-537 of SEQ ID NO:16.
  • the cells are T cells expressing a therapeutic protein.
  • the therapeutic protein is a chimeric antigen receptor.
  • the T cells contain the expression cassette of the therapeutic protein and the expression cassette of the antibody or antigen-binding fragment thereof, or the coding sequence of the therapeutic protein and the antibody or its The coding sequences of the antigen-binding fragments are in the same expression frame.
  • the coding sequence of the therapeutic protein and the coding sequence of the antibody or antigen-binding fragment thereof are in the same expression frame, the coding sequence of the therapeutic protein and the coding sequence of the antibody or antigen-binding fragment thereof comprise Linker sequences that enable expression of multiple cistrons on a single vector.
  • the linker sequence is a coding sequence for a 2A peptide.
  • the 2A peptides include F2A, P2A or T2A peptides.
  • the chimeric antigen receptor contains from the N-terminus to the C-terminus: a single-chain antibody against a tumor antigen, a hinge region, a transmembrane region, and an intracellular region.
  • the chimeric antigen receptor specifically binds to one or more of the following tumor antigens: EGFRvIII, mesothelin, GD2, Tn antigen, sTn antigen, Tn-O - Glycopeptide, sTn-O-glycopeptide, PSMA, CD97, TAG72, CD44v6, CEA, EPCAM, KIT, IL-13Ra2, leguman, GD3, CD171, IL-11Ra, PSCA, MAD-CT-1, MAD-CT -2, VEGFR2, LewisY, CD24, PDGFR- ⁇ , SSEA-4, folate receptor ⁇ , ERBB, Her2/neu, MUC1, EGFR, NCAM, ephrin B2, CAIX, LMP2, sLe, HMWMAA, o-acetyl Genes - GD2, folate receptor beta, TEM1/CD248, TEM7R, FAP, pod protein, HPV E6 or E7
  • the hinge region is selected from a CD8 ⁇ hinge region, an IgG1 Fc CH2CH3 hinge region, an IgD hinge region, a CD28 extracellular hinge region, an IgG4 Fc CH2CH3 hinge region, and a CD4 extracellular hinge region.
  • the transmembrane region is selected from the group consisting of CD28 transmembrane region, CD8 transmembrane region, CD3 ⁇ transmembrane region, CD134 transmembrane region, CD137 transmembrane region, ICOS transmembrane region and DAP10 transmembrane region one or more of.
  • the intracellular region is selected from one or more intracellular regions of CD28, CD134/OX40, CD137/4-1BB, LCK, ICOS, DAP10, CD3 ⁇ and Fc310.
  • the chimeric antigen receptor comprises a signal peptide.
  • the anti-tumor antigen single chain antibody is an anti-CD19 single chain antibody.
  • the hinge region is a CD8 ⁇ hinge region.
  • the transmembrane region is a CD8 transmembrane region.
  • the intracellular region comprises a 4-1BB intracellular region and a human CD3 ⁇ intracellular region.
  • the light chain variable region sequence of the anti-CD19 single chain antibody comprises or consists of amino acids 23-129 of SEQ ID NO:16.
  • the heavy chain variable region sequence of the anti-CD19 single chain antibody comprises or consists of amino acids 148-267 of SEQ ID NO:16.
  • the sequence of the hinge region comprises or consists of amino acids 268-312 of SEQ ID NO:16.
  • the sequence of the transmembrane region comprises or consists of amino acids 313-336 of SEQ ID NO:16.
  • the 4-1BB intracellular region comprises or consists of amino acids 337-378 of SEQ ID NO:16; the CD3 ⁇ intracellular region comprises amino acids 379-490 of SEQ ID NO:16 or consisting of it.
  • the signal peptide of the chimeric antigen receptor comprises or consists of amino acid residues 1-22 of SEQ ID NO:16.
  • composition comprising the antibody or antigen-binding fragment thereof described herein and optionally the therapeutic protein, nucleic acid molecule, nucleic acid construct, lentivirus or cell described herein, and pharmaceutically acceptable excipients .
  • the present invention provides the use of the epitope of the present invention in the preparation of CD3 ⁇ -specific antibodies or antigen-binding fragments thereof or cells expressing the antibodies or antigen-binding fragments thereof.
  • the CD3 ⁇ -specific antibody or antigen-binding fragment thereof mediates efficient intracellular retention of CD3 ⁇ .
  • the present invention provides a method for expressing cells of CD3 ⁇ -specific antibody or antigen-binding fragment thereof, comprising: immunizing an animal with the epitope peptide described herein or a recombinant antigen comprising the epitope peptide, so that the splenocytes of the animal and the myeloma Cell fusion to obtain cells expressing antibodies or antigen-binding fragments thereof; or, using the epitope peptides described herein or recombinant antigens containing the epitope peptides to immunize animals, using the nucleic acid of the animal's antibody-secreting B cells as a template to amplify
  • the gene sequence of the heavy chain and/or light chain variable region of the antibody is obtained by increasing, and the antibody or its antigen-binding fragment is recombinantly expressed in the cell, and the cell of the antibody or its antigen-binding fragment is obtained.
  • the animal is mouse, rat, sheep, rabbit, dog, monkey.
  • the present invention also provides a method for preparing an antibody, comprising: culturing the cells expressing the CD3 ⁇ -specific antibody or antigen-binding fragment thereof prepared by the method herein and optionally collecting the antibody.
  • the present invention provides antibodies or antigen-binding fragments thereof or coding sequences thereof, nucleic acid molecules, nucleic acid constructs or lentiviruses described herein, and optionally therapeutic proteins in the preparation of chimeric antigen receptor T cells or T cells for cancer therapy Applications.
  • Figure 1 The effect of polypeptides containing different anti-CD3 antibody sequences on the TCR and CD3 protein levels on the surface of CAR-T cells.
  • the inventors used the CD3 ⁇ complex protein as an immunogen to prepare a series of monoclonal antibodies that recognize CD3 ⁇ ; and then used the detection of the expression level of the TCR/CD3 complex on the surface of T cells to screen out the most effective down-regulation of T cells through endoplasmic reticulum retention.
  • epitope is a sequence or result that exists on the surface of an antigen and determines the specificity of the antigen, also known as an antigenic determinant.
  • An epitope peptide is one or more peptide segments consisting of such sequences.
  • Antigens specifically bind to corresponding antibodies or cells through epitopes.
  • the "spatial conformation epitope” mentioned herein refers to the spatial conformation formed by the amino acids of the epitope in the absence of a linear continuous sequence for binding to an antibody or its antigen-binding fragment. The conformational epitope bound by the antibody requires at least 5 amino acids, and the number of amino acids is usually 5-17 amino acids (Nevagi R J, et al. 2018).
  • a single protein antigen molecule has many different epitopes, including linear epitopes (that is, the epitopes are composed of linearly continuous amino acids) and spatial conformational epitopes (that is, the amino acids that make up such epitopes are not completely linear and continuous). Therefore, epitope is the target structure recognized by immune cells, and it is also the basis for the specificity of immune response. Its nature, number and spatial configuration determine the specificity of antigen.
  • the spatial conformation epitope of CD3 ⁇ provided by the present invention has been verified by experiments to effectively down-regulate the expression of the TCR/CD3 complex on the surface of T cells.
  • the sequence of CD3 ⁇ is shown in SEQ ID NO:1.
  • the epitope of CD3 ⁇ of the present invention is located in the 54th-107th position of SEQ ID NO:1.
  • the epitope comprises glutamic acid at position 56, histidine at position 61, asparagine at position 62, arginine at position 101, lysine at position 104 and aspartic acid at position 107 of SEQ ID NO:1.
  • the epitope may also comprise glycine at position 54, serine at position 55, glutamine at position 60, lysine at position 64 and aspartic acid at position 69 of SEQ ID NO:1. These above-mentioned amino acids bind to the antibody or its antigen-binding fragment.
  • the epitope is a polypeptide whose amino acid sequence is shown in formula (I): X 1 X 2 ... X 55 EX 57 ... X 60 HNX 63 ... X 100 RX 102 X 103 KX 105 X 106 DX 108 ... X 207 (I), wherein, each X in X 1 -X 55 and X 108 -X 207 is independently any amino acid or none, and each of the remaining Xs is independently any amino acid.
  • the amino acid sequence of the epitope is shown in the following formula (II): X 1 X 2 ... X 53 GSEX 57 X 58 X 59 QHNX 63 KX 65 ...
  • each X in X 1 -X 53 and X 108 -X 207 is independently any amino acid or nothing, and each of the remaining Xs is independently any amino acid.
  • the epitope is a fragment of SEQ ID NO: 1 comprising amino acids 56, 61, 62, 101, 104, and 107, and optionally amino acids 54, 55, 60, 64, and 69.
  • the fragment may be one or more fragments comprising the above amino acids. Wherein any one of the plurality of fragments comprises any two of the above amino acids and the sequence therebetween, and the group composed of the plurality of fragments comprises all of the above amino acids.
  • the length of the fragment can be any number of amino acids, for example, 1-207 amino acids in length.
  • the epitope is a fragment of SEQ ID NO: 1 comprising amino acids 56-107 or 54-107.
  • the present invention also provides an anti-CD3 ⁇ antibody or an antigen-binding fragment thereof, which binds to the epitope described herein, the epitope comprising glutamic acid at position 56 and histidine at position 61 of SEQ ID NO:1 , 62 asparagine, 101 arginine, 104 lysine and 107 aspartic acid.
  • the antibodies are preferably monoclonal antibodies and/or neutralizing antibodies.
  • antibody refers to a polypeptide comprising immunoglobulin sequence elements sufficient to allow it to specifically bind an antigen or epitope. Specific binding refers to the reaction between an antibody or antigen-binding fragment thereof and the antigen it is directed against.
  • an antibody that specifically binds to an antigen refers to an antibody that is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Binds the antigen with an affinity (KD) of 10 ⁇ 8 M, 10 ⁇ 9 M or 10 ⁇ 10 M or less.
  • the antibodies described herein are preferably monoclonal antibodies.
  • each heavy chain comprises at least four domains: an amino-terminal variable domain VH, a constant domain CH1, a constant domain CH2, and a carboxy-terminal constant domain CH3.
  • Each light chain comprises two domains: an amino-terminal variable domain VL and a carboxy-terminal constant domain CL.
  • Each variable domain comprises three "complementarity determining regions": CDR1, CDR2, and CDR3, and four "framework" regions: FR1, FR2, FR3, and FR4.
  • "Antigen-binding fragment” means a fragment of an antibody that specifically binds to a target antigen.
  • Antibodies or antigen-binding fragments thereof include Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, Fv fragments, disulfide bonded Fv fragments, single chain antibodies (scFv), isolated CDRs Or CDR group, polypeptide-Fc fusion, single domain antibody, camel antibody, masking antibody, small module immune drug, bifunctional antibody, nanobody, Humabody antibody.
  • the heavy chain variable region of an anti-CD3 ⁇ antibody of the invention or an antigen-binding fragment thereof has the following HCDRs: HCDR1: GYTFISYT, HCDR2: X 1 NPRSGYT (SEQ ID NO: 19), wherein X 1 is any Amino acid, HCDR3: AX 2 X 3 X 4 YYDYX 5 X 6 FAY (SEQ ID NO: 20), wherein X 2 , X 3 , X 4 , X 5 , X 6 are any amino acids.
  • the light chain variable region of an exemplary anti-CD3 ⁇ antibody or antigen-binding fragment thereof has the following LCDRs: LCDR1: SSVSY, LCDR2: DTS, LCDR3: QQWSSX7PPT (SEQ ID NO: 21 ), where X7 is any amino acid.
  • X1 is alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, threonine, glycine, asparagine, glutamine Amide, serine, tyrosine or cysteine, preferably I or T ;
  • X2 is lysine, arginine or histidine, preferably K or R;
  • X3 is glycine, asparagine, gluten Aminoamide, serine, threonine, tyrosine, cysteine, valine or isoleucine, preferably T or S;
  • X4 is glycine, asparagine, glutamine, serine, threonine acid, tyrosine, cysteine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine or tryptophan, preferably G or A ;
  • X5
  • the heavy chain variable region has the sequence set forth as residues 659-778 of SEQ ID NO: 16 or a sequence having at least 90% sequence identity thereto. In one or more embodiments, the light chain variable region has the sequence set forth as residues 538-643 of SEQ ID NO: 16 or a sequence having at least 90% sequence identity thereto.
  • a single-chain antibody refers to an antibody fragment that has the ability to bind to an antigen and is composed of an antibody light chain variable region (VL region) amino acid sequence and a heavy chain variable region (VH region) amino acid sequence connected by a hinge.
  • a linker or hinge is a polypeptide fragment connecting different proteins or polypeptides, the purpose of which is to keep the connected proteins or polypeptides in their respective spatial conformations, so as to maintain the function or activity of the proteins or polypeptides.
  • Exemplary linkers include G and/or S-containing linkers.
  • an antibody or antigen-binding fragment thereof includes a mutant having at least 70% sequence identity to a reference sequence and retaining the antigen-binding activity of said antibody or antigen-binding fragment.
  • Said mutants include: having at least 70%, at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with the reference sequence and retaining the biological activity of the reference sequence amino acid sequence. Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp. Mutants also include amino acid sequences having one or several mutations (insertions, deletions or substitutions) in said amino acid sequence while still retaining the biological activity of the reference sequence.
  • the number of mutations usually refers to within 1-50, such as 1-20, 1-10, 1-8, 1-5 or 1-3. Substitutions are preferably conservative substitutions.
  • the mutation can occur in the CDR region or in the FR region, as long as the biological activity of the reference sequence remains after the mutation.
  • conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein or polypeptide.
  • amino acids with similar or similar properties include, for example, families of amino acid residues with similar side chains, which families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains, chain amino acids (such as aspartic acid, glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine amino acids), amino acids with non-polar side chains (e.g.
  • Antibodies are bound to localization sequences that direct them to subcellular structures.
  • the positioning sequence can be located at the N-terminal or C-terminal of the polypeptide.
  • the localization sequence includes an endoplasmic reticulum retention sequence, a Golgi apparatus retention sequence, an E3 ubiquitin ligase binding sequence, a proteasome localization sequence, or a lysosome localization sequence.
  • the endoplasmic reticulum retention sequence suitable for use in the present invention may be an endoplasmic reticulum retention sequence well known in the art.
  • An endoplasmic reticulum retention sequence may be located at the C-terminus of an adjacent polypeptide.
  • the positioning sequence and its adjacent polypeptide can be connected directly, or can be connected through a linker sequence well known in the art, such as the linker sequence containing G and S mentioned above.
  • exemplary endoplasmic reticulum retention sequences include or consist of any of SEQ ID NOs: 10-15, wherein X is any amino acid.
  • An antibody or antigen-binding fragment thereof described herein may also include a signal peptide.
  • the signal peptide may be a membrane protein signal peptide, such as a signal peptide of an antibody heavy chain, a signal peptide of an antibody light chain, a CD8 signal peptide, a CD28 signal peptide, and a CD4 signal peptide.
  • An exemplary signal peptide used herein is shown as residues 516-537 of SEQ ID NO:16.
  • the present invention includes a method for preparing hybridoma cells and using the hybridoma cells to prepare antibodies, comprising: immunizing animals with the epitope of the present invention or a recombinant antigen comprising the epitope peptide, and making the spleen cells of the animal and the myeloma cells Fusing, obtaining cells expressing the antibody or antigen-binding fragment thereof, and optionally culturing the fused cells.
  • the method for recombinantly expressing the epitope peptide or the recombinant antigen comprising the epitope peptide can be any method for recombinantly expressing the polypeptide in the art, for example, the coding sequence of the epitope peptide is introduced into the cell through the expression vector and then expressed, and the method described below is passed.
  • the method for constructing expression vectors to express antibodies from cells is similar.
  • the antibodies, preferably monoclonal antibodies, described herein can be obtained by culturing the hybridoma cells.
  • the present invention also includes a method for obtaining antibody-expressing cells using single-cell sequencing methods and using the cells to prepare antibodies, including: immunizing animals with the epitope peptides described herein or recombinant antigens containing the epitope peptides, and secreting
  • the nucleic acid of the B cell of the antibody is used as a template to amplify the gene sequence of the heavy chain and/or light chain variable region of the antibody, and recombinantly express the antibody or its antigen-binding fragment in the cell to obtain the expressed antibody or its antigen-binding fragment cells, and optionally cultured cells.
  • amplifying and obtaining the gene sequence of the heavy chain and/or light chain variable region of the antibody includes, for example, isolating antigen-specific B cells from tissues (such as spleen) or peripheral blood, and secreting B cells from a single antibody by single-cell PCR technology.
  • the antibody heavy and light chain variable region genes are amplified in cells. After the heavy chain and light chain variable region genes are linked into an antibody or its antigen-binding fragment (such as scFv), it can be transformed into cells (such as Escherichia coli or yeast) for expression using conventional methods to obtain the antibody or its antigen-binding fragment. Methods of chemical synthesis are well known in the art.
  • the method of expression from cells by constructing an expression vector is as follows.
  • the invention also provides cells, such as engineered T cells, that express the antibodies described herein, or antigen-binding fragments thereof.
  • Therapeutic polypeptides can be further expressed in the engineered T cells.
  • these T cells were effective in avoiding graft-versus-host reactions.
  • the T cells prepared by the method of the present invention avoid the exhaustion of CD8+ T cells caused by the simultaneous activation of the TCR/CD3 complex and CAR, thereby improving the therapeutic effect.
  • therapeutic protein or “therapeutic polypeptide” described herein refers to molecules that exert corresponding therapeutic activity after being expressed in cells (especially T cells), which may be natural or isolated from the natural environment, or manufactured Substances, including but not limited to: proteins or polypeptides derived from viruses, bacteria, plants, animals, small molecular active peptides, antigens or fragments thereof (such as antigenic epitopes, antigenic determinants), antibodies or fragments thereof (such as heavy chain , light chain, Fab, Fv, scFv), antigen receptor (such as chimeric antigen receptor CAR), fusion protein or polypeptide, etc.
  • the present invention also provides a CAR-T cell comprising a chimeric antigen receptor (CAR) targeting a tumor antigen of interest and a polypeptide of the antibody or antigen-binding fragment thereof described herein.
  • CAR chimeric antigen receptor
  • the present invention also provides a CAR-T cell, comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) targeting a tumor antigen of interest and a nucleic acid molecule of an antibody or an antigen-binding fragment thereof.
  • suitable T cells may be various T cells well known in the art, especially various T cells routinely used in cellular immunotherapy, including but not limited to peripheral blood T lymphocytes, cytotoxic killer T cells, helper T cells Cells, suppressor/regulatory T cells, ⁇ T cells, cytokine-induced killer cells, tumor infiltrating lymphocytes, etc., and any one or more mixtures of the above cells.
  • CAR-T cells refer to T cells expressing at least a chimeric antigen receptor.
  • chimeric antigen receptor has a well-known meaning in the art. It is an artificially engineered receptor capable of anchoring specific molecules (such as antibodies) that recognize tumor cell surface antigens on immune cells (such as T cells) , allowing immune cells to recognize tumor antigens and kill tumor cells.
  • Chimeric antigen receptors suitable for use herein can be various CARs known in the art.
  • a CAR comprises a polypeptide that binds a tumor antigen, a hinge region, a transmembrane region, and one or more intracellular signaling regions. Common tumor antigens of interest and their specific molecules are known in the art.
  • the tumor antigen-binding polypeptide of the present invention is a single-chain antibody that specifically binds a tumor antigen.
  • the tumor antigen of interest is CD19 and the single chain antibody of interest is a single chain antibody that specifically binds CD19.
  • the anti-CD19 single chain antibody is derived from FMC63.
  • the light chain variable region sequence of an anti-CD19 single-chain antibody comprises SEQ ID NO: 16 amino acids 23-129 or consists of it;
  • the heavy chain variable region sequence of an anti-CD19 single-chain antibody comprises SEQ ID NO: 16 amino acids 148-267 or consist thereof, wherein the heavy chain variable region and the light chain variable region are connected by a linker sequence containing G and S.
  • hinge region a region containing at least one part contained in the CAR suitable for the present invention, such as the hinge region, transmembrane region, intracellular signal region and optional signal peptide can be the hinge region, transmembrane region and intracellular signal area.
  • the sequence of the hinge region of human CD8 ⁇ comprises or consists of amino acid residues 268-312 of SEQ ID NO: 16; the amino acid sequence of the transmembrane region comprises or consists of amino acid residues 313-336 of SEQ ID NO: 16 Composition;
  • the amino acid sequence of intracellular signal region can be as shown in SEQ ID NO:16 the 337th-490th amino acid residue;
  • Exemplary signal peptide amino acid sequence can comprise SEQ ID NO:16 the 1st-22th amino acid residue or consists of it.
  • the CAR contains an optional anti-tumor antigen single-chain antibody, a hinge region, a transmembrane region, and one or more intracellular regions from the N-terminus to the C-terminus.
  • the amino acid sequence of an exemplary chimeric antigen receptor comprises or consists of amino acid residues 23-490 of SEQ ID NO: 16, or comprises or consists of amino acid residues of SEQ ID NO: 16.
  • the above-mentioned parts that form the chimeric antigen receptor herein can be directly interacted with each other. or can be linked by a linker sequence well known in the art, such as a linker sequence comprising G and S.
  • a linker sequence such as a linker sequence comprising G and S.
  • the therapeutic protein of the present invention and the antibody can be connected directly, or can be connected through a linker sequence, such as a linker sequence containing G and S.
  • the therapeutic protein and the antibody also contain linking sequences capable of expressing multiple polycistrons on a single vector, such as 2A peptides, including F2A, P2A, T2A peptides and the like.
  • the amino acid sequence of the 2A peptide comprises or consists of amino acids 494-515 of SEQ ID NO:16.
  • the 2A peptide can also be connected to the polypeptides on both sides by conventional G and S-containing linkers.
  • the invention includes nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof of the invention.
  • a nucleic acid molecule of the invention may be in the form of DNA or RNA.
  • Forms of DNA include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be either the coding strand or the non-coding strand.
  • the present invention also includes degenerate variants of nucleic acid molecules encoding polypeptides or proteins, that is, nucleic acid molecules that encode the same amino acid sequence but differ in nucleotide sequence.
  • the nucleic acid molecule of the present invention may be the coding sequence of a therapeutic protein such as CAR and the coding sequence of an antibody or an antigen-binding fragment thereof, or an expression cassette of a therapeutic protein and the protein or polypeptide.
  • the coding sequence refers to the nucleic acid sequence that directly determines the amino acid sequence of its protein product (such as CAR, single-chain antibody, hinge region, transmembrane region, intracellular signal region, or the antibody or antigen-binding fragment thereof described herein, etc.) part.
  • the boundaries of the coding sequence are usually determined by the ribosome binding site (for prokaryotic cells) immediately upstream of the 5' open reading frame of the mRNA and the transcription termination sequence immediately downstream of the 3' open reading frame of the mRNA.
  • a coding sequence may include, but is not limited to, DNA, cDNA, and recombinant nucleic acid sequences.
  • the expression cassette refers to the complete elements required to express the gene of interest, including the promoter, gene coding sequence and PolyA tailing signal sequence.
  • the nucleic acid molecules described herein can be two independent nucleic acid molecules, respectively containing the coding sequence of the therapeutic protein and the coding sequence of the antibody, such as the expression cassette of the therapeutic protein and the expression cassette of the polypeptide;
  • the coding sequence of the protein and the coding sequence of the antibody can be connected into one nucleic acid molecule through a linker, such as the coding sequence of the therapeutic protein and the coding sequence of the antibody are in the same expression frame, or the two expression frames are connected into the same nucleic acid molecule through a suitable linker. nucleic acid molecule.
  • the nucleic acid molecule of the present invention is a nucleic acid molecule in which the coding sequence of the therapeutic protein and the coding sequence of the polypeptide are in the same expression frame, which contains a promoter, a nucleic acid sequence encoding the therapeutic protein and polypeptide, and PolyA tailed signal.
  • the nucleic acid molecule further comprises a coding sequence for an optional endoplasmic reticulum retention sequence.
  • the coding sequence or expression cassette is integrated into the genome of the T cell.
  • the T cells described herein have stably integrated into their genomes expression cassettes encoding the therapeutic proteins and antibodies described herein.
  • the nucleic acid molecules described herein can generally be obtained by PCR amplification.
  • primers can be designed according to the nucleotide sequence disclosed herein, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art can be used as a template, related sequences were amplified. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.
  • the nucleic acid molecules described herein can also be directly synthesized.
  • nucleic acid constructs for expression by cells involve nucleic acid constructs.
  • the nucleic acid constructs herein comprise the nucleic acid molecules described herein, and one or more regulatory sequences operably linked to these sequences.
  • the nucleic acid molecules of the invention can be manipulated in a variety of ways to ensure expression of the antibody or therapeutic protein. Before inserting the nucleic acid construct into the vector, the nucleic acid construct can be manipulated according to the differences or requirements of the expression vector. Techniques for altering the sequence of nucleic acid molecules using recombinant DNA methods are known in the art.
  • the regulatory sequence may be a suitable promoter sequence.
  • the promoter sequence is usually operably linked to the coding sequence of the protein to be expressed.
  • the promoter can be any nucleotide sequence that shows transcriptional activity in the host cell of choice, including mutated, truncated, and hybrid promoters, and can be derived from an extracellular sequence that encodes either homologous or heterologous to the host cell. Or intracellular polypeptide gene acquisition.
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription.
  • a terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.
  • the regulatory sequence may also be a suitable leader sequence, an untranslated region of an mRNA important for translation by the host cell.
  • a leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.
  • the regulatory sequences may also be an origin of replication functional in at least one organism, convenient restriction enzyme sites and one or more selectable markers.
  • the invention utilizes a lentiviral vector comprising an origin of replication, a 3'LTR, a 5'LTR, a nucleic acid molecule as described herein, and optionally a selectable marker .
  • the nucleic acid construct is a vector.
  • the vector can be a cloning vector, an expression vector, or a homologous recombination vector.
  • the nucleic acid molecules of the invention can be cloned into many types of vectors, eg, plasmids, phagemids, phage derivatives, animal viruses and cosmids.
  • Cloning vectors can be used to provide the coding sequences of the therapeutic proteins and polypeptides of the present invention, such as a nucleic acid molecule comprising the coding sequences of the therapeutic proteins and polypeptides.
  • Expression vectors can be provided to cells as viral vectors.
  • nucleic acid molecule of the invention is typically achieved by operably linking the nucleic acid molecule of the invention to a promoter, and incorporating the construct into an expression vector.
  • Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • Homologous recombination vectors are used to integrate the expression cassettes described herein into the host genome.
  • the expression vector introduced into the cell may also contain a selectable marker gene or reporter gene to facilitate the identification and selection of expressing cells from the population of cells sought to be transfected or infected by the viral vector.
  • selectable markers can be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.
  • Vectors can be readily introduced into host cells, eg, mammalian, bacterial, yeast or insect cells, by any method known in the art.
  • expression vectors can be transferred into host cells by physical, chemical or biological means.
  • Physical methods for introducing nucleic acid molecules into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
  • Biological methods for introducing nucleic acid molecules of interest into host cells include the use of DNA and RNA vectors.
  • Chemical means of introducing nucleic acid molecules into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipid body.
  • Biological methods for introducing nucleic acid molecules into host cells include the use of viral vectors, such as vectors derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, especially retroviral vectors.
  • the selected gene can be inserted into a vector and packaged into a retroviral particle, such as a lentiviral particle, using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo.
  • Reagents for lentiviral packaging are well known in the art, for example, conventional lentiviral vector systems include pRsv-REV, pMDlg-pRRE, pMD2G and objective interfering plasmids.
  • the lentiviral vector pWPXL is used. Therefore, in certain embodiments, the present invention also provides a lentivirus for activating T cells, the virus comprising the retroviral vector described herein and the corresponding packaging genes, such as gag, pol, vsvg and/or rev.
  • a host cell contains, expresses and/or secretes an antibody or antigen-binding fragment thereof and optionally a therapeutic polypeptide described herein.
  • a cell contains or contains, expresses, or secretes a molecule such as a polypeptide
  • "contains” means that the molecule is contained in or on the surface of the cell;
  • expression means that the cell produces the molecule "secretion” means that the cell secretes the expressed molecule out of the cell.
  • Host cells include not only T cells that are ultimately used for the purpose of disease treatment, but also various cells used in the process of producing CAR-T cells, such as E. coli cells, for example, providing the coding sequence of the protein of the present invention or providing the carrier described.
  • a CAR-T cell stably expressing an antibody described herein.
  • compositions which contains the antibody or its antigen-binding fragment or T cell described herein and pharmaceutically acceptable auxiliary materials.
  • pharmaceutically acceptable excipients refer to carriers, diluents and/or excipients that are pharmacologically and/or physiologically compatible with the subject and the active ingredient, including but not limited to: pH regulators, topical Active agents, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers and preservatives. More specifically, suitable pharmaceutically acceptable adjuvants may be adjuvants commonly used in the art for administration of antibodies or antigen-binding fragments thereof or T cells (such as CAR-T cells).
  • the pharmaceutical composition contains a therapeutically effective amount of an antibody or antigen-binding fragment thereof or T cells.
  • a therapeutically effective amount refers to a dose that can achieve treatment, prevention, alleviation and/or alleviation of a disease or condition in a subject.
  • the therapeutically effective dose can be determined according to factors such as the patient's age, sex, disease and its severity, and other physical conditions of the patient.
  • a subject or a patient generally refers to a mammal, especially a human.
  • kits for detecting CD3 ⁇ comprising a polypeptide and the antibody or antigen-binding fragment thereof described herein, wherein the sequence of the polypeptide is the sequence of the epitope described herein.
  • the kit further includes reagents for detecting CD3 ⁇ by antigen-antibody reaction, such as coating solution, blocking solution, secondary antibody, chromogenic solution, and stop solution.
  • kits for vector transfection comprising the nucleic acid construct described herein.
  • the kit may also contain various reagents suitable for transfecting the nucleic acid construct into cells, and optionally instructions to guide those skilled in the art to transfect the recombinant expression vector into cells.
  • the invention also includes an antibody or cell therapy wherein T cells are genetically modified to express the therapeutic proteins and antibodies described herein, and the antibody or T cells are administered to a subject.
  • administered antibodies or CAR-T cells are able to kill the recipient's tumor cells.
  • the anti-tumor immune response induced by CAR-T cells can be active or passive immune response.
  • the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-T cells induce an immune response specific for the antigen-binding portion of the CAR.
  • the diseases suitable for treatment with the CAR, polypeptides, their coding sequences, nucleic acid constructs, expression vectors, viruses or CAR-T cells of the present invention are related to the tumor antigen single-chain antibody contained in the CAR. Therefore, the diseases described herein include various types of cancers related to the aforementioned tumor antigens, including solid tumors and blood tumors, such as adenocarcinoma, lung cancer, colon cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, gastric cancer , cholangiocarcinoma, gallbladder cancer, esophageal cancer, pancreatic cancer, and prostate cancer, as well as leukemias and lymphomas, such as B-cell lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myeloid leukemia etc.
  • solid tumors and blood tumors such as adenocarcinoma, lung cancer,
  • the diseases that can be treated by using the CARs, polypeptides, their coding sequences, nucleic acid constructs, expression vectors, viruses or CAR-T cells described herein comprising anti-CD19 single chain antibodies are preferably CD19-mediated diseases; such as acute/chronic B-lineage lymphocytic leukemia, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma, etc.
  • Antibodies, antigen-binding fragments thereof, or T cells of the invention may be administered alone or as pharmaceutical compositions.
  • the antibodies, antigen-binding fragments thereof, or cells of the invention can be administered in a manner suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by various factors, such as the patient's condition, and the type and severity of the patient's disease.
  • Administration of the compositions may be by any convenient means, including by injection, infusion, implantation or transplantation.
  • the compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection or intraperitoneally.
  • an antibody, antigen-binding fragment thereof, or T cell of the invention is administered to a patient by intradermal or subcutaneous injection.
  • the antibody, antigen-binding fragment thereof or T cell composition of the invention is administered preferably by intravenous injection.
  • Compositions of T cells can be injected directly into tumors, lymph nodes or sites of infection.
  • antibodies, antigen-binding fragments thereof, or T cells or compositions thereof of the invention may be combined with other therapies known in the art.
  • Such therapies include, but are not limited to, chemotherapy, radiation therapy, and immunosuppressants.
  • treatment may be combined with radiotherapy or chemotherapy agents known in the art to treat tumor antigen-mediated diseases.
  • anti-tumor effect refers to a biological effect that can be represented by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer.
  • the present invention adopts CD3 ⁇ to prepare hybridoma to obtain antibody 2B4, and takes the gene sequence of anti-CD19 antibody as an example, searches human CD8 ⁇ hinge region, human CD8 transmembrane region, 4-1BB intracellular region, human CD3 ⁇ cell region from NCBI GenBank database Sequence information such as the inner region and anti-CD3 single-chain antibody, the whole gene synthesis of the chimeric antigen receptor and the gene fragment of the antibody 2B4scFV are inserted into the lentiviral vector.
  • the recombinant plasmid packs the virus in 293T cells, infects T cells, and makes T cells express the chimeric antigen receptor.
  • the method for realizing the transformation of T lymphocytes modified by the chimeric antigen receptor gene of the present invention is based on the lentivirus transformation method.
  • the transformed nucleic acid is expressed through transcription and translation.
  • the proportions of TCR+ and CD3+ populations on the cell surface of the CAR-T cells prepared by the present invention were greatly reduced, being 9.9% and 25.5% respectively.
  • An epitope peptide of CD3 ⁇ comprising amino acids 56, 61, 62, 101, 104 and 107 of SEQ ID NO:1,
  • said epitope peptide is a steric epitope peptide
  • the epitope peptide also comprises amino acids 54, 55, 60, 64 and 69 of SEQ ID NO: 1,
  • the epitope peptide comprises amino acids 56-107 of SEQ ID NO: 1, or comprises amino acids 54-107 of SEQ ID NO: 1.
  • each X in X 1 -X 55 and X 108 -X 207 is independently any amino acid or none, and each of the remaining Xs is independently any amino acid
  • the epitope peptide is represented by the following formula (III):
  • X54 and X55 are each independently any amino acid or none, and the remaining X are each independently any amino acid, or
  • the epitope peptide is represented by the following formula (IV):
  • X is each independently any amino acid
  • the epitope peptide is a fragment of SEQ ID NO: 1 comprising amino acids 56, 61, 62, 101, 104 and 107, and optionally amino acids 54, 55, 60, 64 and 69, or,
  • the epitope peptide is a fragment of SEQ ID NO: 1 comprising amino acids 56-107 or a fragment of SEQ ID NO: 1 comprising amino acids 54-107.
  • a method for preparing cells expressing CD3 ⁇ -specific antibodies or antigen-binding fragments thereof comprising:
  • the animals are mice, rats, sheep, rabbits, dogs, monkeys.
  • a method for preparing an antibody or its binding fragment comprising: culturing the cells prepared by the method of item 4 and optionally collecting the antibody or its binding fragment.
  • the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has the following HCDRs:
  • HCDR1 as shown in SEQ ID NO:2: GYTFISYT,
  • HCDR2 as shown in SEQ ID NO:3: TNPRSGYT,
  • HCDR3 as shown in SEQ ID NO:4: AKTGYYDYHAFAY,
  • the light chain variable region has the following LCDRs:
  • the antibody or antigen-binding fragment thereof further comprises a localization sequence; more preferably, the localization sequence is selected from an endoplasmic reticulum retention sequence, a Golgi apparatus retention sequence, an E3 ubiquitin ligase binding sequence, a proteasome localization sequence or Lysosomal targeting sequence.
  • the localization sequence is selected from an endoplasmic reticulum retention sequence, a Golgi apparatus retention sequence, an E3 ubiquitin ligase binding sequence, a proteasome localization sequence or Lysosomal targeting sequence.
  • a cell comprising:
  • a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of item 6, the nucleic acid molecule of item 7 and/or the cell of item 8, and pharmaceutically acceptable excipients.
  • the antibody or antigen-binding fragment thereof described in item 6, the nucleic acid molecule described in item 7, and/or the cell described in item 8 are used in the preparation of chimeric antigen receptor T cells, T cells for cancer treatment, and specific detection of CD3 ⁇ Application in reagents or vaccines.
  • CD3 ⁇ complex protein (Cat.No.CDG-H52W6, ACRO) as the immunization antigen, add complete Freund's adjuvant at a ratio of 1:1 for the initial immunization, mix well until fully emulsified, and use conventional subcutaneous injection for 6-8 weeks Inject 100 ⁇ l subcutaneously into Balb/c mice at the age of 12, and repeat booster immunization every two weeks, a total of 2 times. The booster immunization is added with incomplete Freund's adjuvant at a ratio of 1:1. Level. Intrasplenic injection was used for the last immunization, and the dose was the same as before. The mice were sacrificed 3 days later, and the spleen was taken out. The spleen single cell suspension was obtained by conventional methods, and the cell viability was detected to be >90%.
  • the remaining anti-CD3 antibody sequences are from Example 1 and have been humanized. All relevant amino acid sequences are codon-optimized on the website https://www.thermofisher.com/order/geneartgenes to ensure that it is more suitable for expression in human cells without changing the encoded amino acid sequence.
  • anti-CD19-scFv gene human CD8 hinge region gene, human CD8 transmembrane region gene, 4-1BB intracellular region gene, human CD3 ⁇ intracellular region, F2A and CD3-scFv gene,
  • the anti-CD3-scFv gene and endoplasmic reticulum retention sequence (AEKDEL) are connected to form complete gene sequence information.
  • the amino acid sequence comprising the signal peptide is shown in residues 1-22 and residues 516-537 of SEQ ID NO:16.
  • the nucleotide sequence of the CAR molecule was seamlessly cloned into the BamHI/EcoRI site of the lentiviral plasmid pWPXL (Addgene), and transformed into competent Escherichia coli (Stbl3).
  • Antisense sequence CCAGTCAATCTTTCACAAATTTTG (SEQ ID NO: 18).
  • the plasmid was extracted and purified using Qiagen’s plasmid purification kit, and the purified plasmid was transfected into 293T cells by the calcium phosphate method for lentiviral packaging experiments (Molecular Therapy-Methods & Clinical Development, 2016, 3: 16017 ), thus preparing the following lentiviral vectors: control (CAR19-F2A-GFP), sample control 1 (CAR19-F2A-hun291scfv-AEKDEL), sample control 2 (CAR19-F2A-OKT3scfv-AEKDEL), sample 1 (CAR19 -F2A-1B3-scfv-AEKDEL), sample 2 (CAR19-F2A-2B4-scfv-AEKDEL), sample 3 (CAR19-F2A-6B8-scfv-AEKDEL), sample 4 (CAR19-F2A-6D2-scfv-AEKDEL
  • CD3+T cells are activated by Dynabeads CD3/CD28 (Life Technology) for 24 hours The proportion of CD25+CD69+T cells was detected by flow cytometry.
  • Count and collect 5.0E+05 cells wash the cells twice and resuspend in 100 ul buffer containing 4% BSA. Add 8ul of anti-human TCR or CD3 antibody to each tube of cells, vortex and mix well, and incubate at 4°C for 30 minutes. After staining and incubation, wash the cells repeatedly, dilute the fluorescently labeled anti-CAR19 antibody Protein L 500x, resuspend the cells, 200ul per tube, vortex and mix, and incubate at 4°C for 30 minutes.
  • the cells were washed repeatedly, resuspended in 500 ul of buffer containing 4% BSA, 4 ul of 7AAD dye was added to each tube, vortexed and incubated for 10 minutes at room temperature in the dark. Finally, the samples were transferred to a flow tube, and the CAR19 transfection efficiency and the TCR or CD3 surface level of the CAR19+ T cell population were detected on a Calibr flow cytometer.
  • the widely used anti-human CD3 murine antibody (OKT3) was evaluated.
  • OKT3 anti-human CD3 murine antibody
  • it was humanized and tested at the cell level.
  • the results are shown in Figure 1 and Table 1, the expression of polypeptides containing humanized OKT3 antibody sequences can down-regulate the activity of TCR/CD3 complexes on the cell surface, and in transduction positive cells (CAR+), the TCR- and CD3-populations
  • the proportions are 62.00% and 52.60% respectively.
  • the proportion of TCR/CD3+ population is still high, which will bring great challenges to the purification of CAR-T cells and also bring great risks to clinical application.
  • Peptides containing antibody 2B4 sequences can most effectively down-regulate the level of TCR/CD3 complexes on the cell surface, and up to 91.40% of CAR+ cells do not express surface TCR; up to 89.59% of CAR+ cells do not express surface CD3 ( Figure 1 and Table 1 ).
  • the hum291 clone with high amino acid variable region sequence homology with the 2B4 clone did not show the same excellent ability to down-regulate the TCR/CD3 complex on the cell surface as 2B4 (the proportion of the TCR- and CD3-populations of hum291 82.1% and 50.2%, respectively).
  • the lentivirus-transfected T cells in each group were cultured in vitro until the 8th day, and the TCR-dependent T cell activation antibody OKT3 was added to the final concentrations of 50ng/ml, 100ng/ml, 150ng/ml, and 200ng/ml, respectively.
  • CD3 ⁇ mutant plasmids (1 ⁇ g) and CD3 ⁇ plasmids (1 ⁇ g) were co-transfected into 293T cells respectively, and the expression of intracellular CD3 molecules was detected according to the method in Example 5 after 48 hours of transfection.
  • CD3 is a transmembrane protein found on T cells. There are four subtypes, namely CD3 ⁇ , CD3 ⁇ , CD3 ⁇ and CD3 ⁇ .
  • CD3 ⁇ /CD3 ⁇ exist in the form of heterodimerization. Antibodies to the CD3 ⁇ /CD3 ⁇ complex recognize only this complex, but do not bind CD3 ⁇ or CD3 ⁇ monomers.
  • CD3 ⁇ mutant plasmid 3 CD3 ⁇ mutant plasmid 5
  • CD3 ⁇ mutant plasmid 6 CD3 ⁇ mutant plasmid 13
  • CD3 ⁇ mutant plasmid 14 and CD3 ⁇ mutant plasmid 15 could not effectively bind to the 2B4 antibody.
  • the 2B4 antibody and the OKT3 antibody exhibited different epitope binding abilities to the CD3 ⁇ protein (Table 2 and Figure 3).
  • Our experiments have confirmed that 2B4 recognizes CD3 as a spatial epitope, which partially overlaps with the recognition epitope of OKT3.
  • the spatial epitope recognized by 2B4 must contain at least 56 glutamic acid, 61 histidine, 62 asparagine, 101 Arginine at position 104, lysine at position 104 and aspartic acid at position 107.
  • the spatial epitope recognized by 2B4 further includes 5 amino acids of glycine at position 54, serine at position 55, glutamine at position 60, lysine at position 64, and aspartic acid at position 69.
  • CD3 ⁇ mutation 9 N65A +++ +++ CD3 ⁇ mutation 10 D69A ++ +++ CD3 ⁇ mutation 11 E70A +++ +++ CD3 ⁇ mutation 12 D71A +++ +++ CD3 ⁇ mutation 13 R101A - - CD3 ⁇ mutation 14 K104A - + CD3 ⁇ mutation 15 D107A - +++

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Abstract

La présente invention concerne un épitope conformationnel spatial induisant une rétention efficace de CD3 dans des cellules et son utilisation. Plus particulièrement, la présente invention concerne un peptide épitope de CD3ε, un anticorps se liant de manière spécifique à celui-ci ou un fragment de liaison à l'antigène de celui-ci, et son utilisation. La présente invention peut retenir de manière efficace des molécules contenant CD3ε dans des cellules, ce qui permet de réduire la réaction du greffon contre l'hôte.
PCT/CN2022/096017 2021-06-30 2022-05-30 Épitope conformationnel spatial induisant une rétention efficace de cd3 dans des cellules et son utilisation WO2023273762A1 (fr)

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