WO2023160260A1 - Cd7-car-t细胞及其制备方法和应用 - Google Patents

Cd7-car-t细胞及其制备方法和应用 Download PDF

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WO2023160260A1
WO2023160260A1 PCT/CN2023/070315 CN2023070315W WO2023160260A1 WO 2023160260 A1 WO2023160260 A1 WO 2023160260A1 CN 2023070315 W CN2023070315 W CN 2023070315W WO 2023160260 A1 WO2023160260 A1 WO 2023160260A1
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cells
antibody
antigen
car
seq
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许中伟
张海燕
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先进生物(苏州)有限公司
许中伟
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    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to the technical field of biological immunotherapy, in particular to CD7-CAR-T cells and their preparation methods and applications.
  • Acute lymphocytic leukemia is a common malignant tumor of the blood system, mainly originating from the B lymphocyte line and T lymphocyte line. Primitive immature lymphocytes undergo multi-step specific gene damage to form tumor cells, which abnormally proliferate and accumulate in the bone marrow, and inhibit the hematopoietic function of normal bone marrow stem cells, leading to the occurrence of ALL. At the same time, these tumor cells can also infiltrate into extramedullary tissues (such as lymph nodes, meninges, liver, gonads, etc.), causing related tissue damage.
  • extramedullary tissues such as lymph nodes, meninges, liver, gonads, etc.
  • ALL can occur in all age groups, and the incidence rate of children and young adults is higher at 3.6/100,000, while the incidence rate of adult ALL is lower at 0.69/100,000.
  • Initial chemotherapy and targeted therapy can kill aggressive proliferating cells and cells with selective or acquired mutations, but drug-insensitive ALL cells still lead to disease relapse.
  • Bone marrow transplantation is an important treatment for ALL. Although allogeneic transplantation has a good curative effect on acute leukemia, most patients are limited by the lack of suitable marrow donors and the high cost of transplantation. Autologous bone marrow transplantation, as an alternative treatment of allogeneic transplantation, has developed rapidly in recent years.
  • T-ALL T Cell Acute Lymphocytic leukemia
  • T-ALL a type of ALL
  • Gene mutations in T-ALL are diverse and highly heterogeneous, including gene deletions, mutations, and chromosomal translocations. These abnormalities can cause various signaling pathways (such as MAPK and Jak/Stat, PI3K/Akt/mTOR, etc.) and/or cell cycle abnormalities. Studies have found that at least 170 potential oncogenic driver genes are related to the occurrence of T-ALL.
  • T-ALL mostly occurs in children and adolescents, but can also occur in adults. T-ALL accounts for 10%-15% of ALL patients in children and about 25% in adults. Compared with acute lymphoblastic leukemia (B-ALL), T-ALL is less sensitive to chemotherapy, has poorer effect, higher recurrence rate, lower remission rate and long-term survival rate. Predominant clones after relapse are often enriched in genes resistant to conventional chemotherapy drugs. Compared with children's T-ALL, the treatment effect of adult T-ALL is worse.
  • CAR-T chimeric antigen receptor T cell
  • CAR-T therapy targeting CD19 can achieve 90%-100% CR in B-ALL, and both autologous CAR-T and allogeneic CAR-T therapy can achieve a relapse-free survival of more than 5 years .
  • One of the key technologies of CAR-T therapy is to select a specific target expressed on tumor cells, such as tumor-associated antigen or tumor-specific antigen, and prepare CAR-T cells with specific antibodies to this antigen for treatment.
  • CD7 is a cell membrane single-chain glycoprotein with a molecular weight of 40kDa, which is mainly expressed in thymocytes and most peripheral blood T lymphocytes, and precedes the appearance of other T lineage antigens (CD1, CD2, CD3, CD4, CD5, CD8) , Also earlier than the TCR- ⁇ chain gene rearrangement.
  • CD7 antigen is also expressed in NK cells and some myeloid cells. Highly expressed on the surface of leukemia cells originating from the T lymphoid lineage. In addition to T-ALL, most T-cell lymphomas, NK and NKT lymphomas also express CD7, and the expression rate is 20-30% in acute myeloid leukemia (AML).
  • CD7 is an ideal target for T-ALL and CD7-positive tumors.
  • CD7-CAR-T cells Compared with the CAR-T treatment of B-ALL, the application of CD7-CAR-T cells in the targeted therapy of T-cell acute lymphoblastic leukemia and CD7-positive lymphoma still faces great technical problems, because the normal effect Both T cells and T cell tumors express the CD7 antigen, which will lead to the "cannibalism" effect of CD7-CAR-T cells. Therefore, it is difficult to successfully prepare CD7-CAR-T cells in vitro; CD7-CAR-T cells cannot be allowed to exist in vivo indefinitely for a long time to avoid serious side effects. At present, there are only a few studies on CD7-CAR-T technology.
  • the method of CRISPR gene editing is used to knock out the CD7 gene in T cells, but it is difficult to knock out the CD7 molecule 100% in practice and theory , while the clinical application risk of graft-versus-host disease (GVHD) remains.
  • GVHD graft-versus-host disease
  • the possible side effects of gene editing's natural "off-target effects” will also limit the widespread development of clinical applications.
  • the use of nano-body (nanobody) technology to reduce the volume of the antibody-binding region has certain advantages, but the disadvantage is that the reduced and single-armed antibody will lead to relatively weakened antigen-antibody binding and stability, resulting in CD7-CAR- Decreased T cell potency.
  • CD7 expression are an important force in maintaining normal cellular immunity and fighting against viral and bacterial infections in the body. Only the persistent lethality of CD7-CAR-T in vivo survival is emphasized. At the same time, it will cause side effects such as long-term infection, so rationally controlling the existence of CD7-CAR-T cells is also an important factor for excellent CAR-T design.
  • the inventor prepared and selected a unique anti-human CD7 monoclonal antibody sequence through in-depth research, as the antigen recognition element of the CD7-CAR-T construction of the present invention, and simultaneously applied the anti-CD7 antigen
  • the fusion structure of the antibody fragment and the endoplasmic reticulum (ER) localization signal enables the CD7 antigen molecule to be localized in the ER reticulum of T cells to block its expression on the surface of CAR-T cells, thereby eliminating CD7-CAR-T
  • the "cannibalism" effect of cells is beneficial to the preparation and production of CD7-CAR-T cells in vitro.
  • the present invention applies a suicide gene structure to the CAR-T structure, which can eliminate CAR-T cells when not needed, so as to ensure the safety of its application.
  • the present invention includes the following contents.
  • an antibody or antigen-binding fragment thereof which contains the heavy chain variable region of the complementarity determining regions CDR1, CDR2 and CDR3 having the amino acid sequence shown in SEQ ID NO.: 12-14; and
  • the light chain variable region of the complementarity determining regions CDR1, CDR2 and CDR3 having the amino acid sequence shown in SEQ ID NO.: 15-17.
  • the antibody has any one of the amino acid sequences shown in (I), (II) or (III):
  • amino acid sequence has antibody activity against CD7 antigen.
  • the antibody includes at least one of polyclonal antibody, monoclonal antibody, chimeric antibody, humanized antibody or bispecific antibody;
  • Antigen-binding fragments include at least one of Fab fragments, Fab', F(ab')2 fragments, single-chain variable fragments scFv, scFv-Fc fragments, or single-chain antibody ScAbs.
  • the second aspect of the present invention provides a CD7 blocking molecule, which comprises:
  • a chimeric antigen receptor comprising:
  • an antigen-binding domain that recognizes a CD7 antigen, wherein the antigen-binding domain includes the antibody or antigen-binding fragment thereof according to the first aspect;
  • it further includes a hinge region
  • the transmembrane domain is selected from: polypeptide CD28, NKp30, CDS, DAP10, 4-1BB, DAP12, CD3C, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64 , CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1, ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 ( KLRF1), CD160, CD19, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM , CD11b, ITGAX, CD11
  • the intracellular signaling domain is selected from the group consisting of: CD8, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , Fc ⁇ RI- ⁇ , Fc ⁇ RIII- ⁇ , Fc ⁇ RI ⁇ , Fc ⁇ RI ⁇ , DAP10, DAP12, CD32, CD79a, CD79b, CD28, CD3C, At least one of CD4, b2c, CD137(4-1BB), ICOS, CD27, CD28 ⁇ , CD80, NKp30, OX40 or any combination thereof.
  • an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the blocking molecule according to the second aspect, or according to the third aspect
  • the chimeric antigen receptor is provided.
  • the fifth aspect of the present invention provides a vector comprising the nucleic acid molecule according to the fourth aspect.
  • a sixth aspect of the present invention provides a host cell comprising the vector according to the fifth aspect.
  • the seventh aspect of the present invention provides the preparation method of the chimeric antigen receptor according to the third aspect, which comprises culturing the host cell according to the sixth aspect.
  • the eighth aspect of the present invention provides an immune effector cell expressing the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the blocking molecule according to the second aspect, or the blocking molecule according to the third aspect said chimeric antigen receptor;
  • the immune effector cells are selected from the group consisting of: leukocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, ⁇ T cells, ⁇ T cells , natural killer (NK) cells, natural killer T (NKT) cells, B cells, innate lymphoid cells (ILC), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTL), lymphokine-activated At least one of killer (LAK) cells, T lymphocytes, peripheral blood mononuclear cells, and hematopoietic stem cells.
  • NK natural killer
  • NKT natural killer T
  • B cells innate lymphoid cells
  • CIK cytokine-induced killer
  • CTL cytotoxic T lymphocytes
  • LAK lymphokine-activated At least one of killer (LAK) cells, T lymphocytes, peripheral blood mononuclear cells, and hematopoietic stem cells.
  • the ninth aspect of the present invention provides the use of a reagent in the preparation of a composition, medicament, preparation or kit for preventing and/or treating cancer or tumor, said reagent comprising: the antibody according to the first aspect of the present invention or an antigen-binding fragment thereof, or a blocking molecule according to the second aspect, or a chimeric antigen receptor according to the third aspect, or an immune effector cell according to the eighth aspect;
  • the cancer or tumor refers to a cancer or tumor related to CD7 expression, and preferably, the cancer or tumor is a hematological malignancy; further preferably, the hematological malignancy is a T cell-related tumor, so
  • the T cell-associated tumors include leukemia, lymphoma, and myeloma.
  • the tenth aspect of the present invention provides the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the blocking molecule according to the second aspect, or the chimeric antigen receptor according to the third aspect, Or the use of the immune effector cells according to the eighth aspect in combination with other drugs.
  • Other drugs include, but are not limited to: diagnostic, prophylactic and/or therapeutic agents.
  • the excellent technical effects of the present invention include but are not limited to: the antibody of the present invention and the CD7-CAR based on the antibody fragment have a very strong affinity with the CD7 antigen molecule, and the blocking molecule of the present invention can almost completely block the CD7 molecule in cells
  • the expression on the surface of CD7-CAR-T cells can effectively avoid the cannibalism of CD7-CAR-T cells without affecting the normal expansion of T cells.
  • the CD7-CAR-T cells of the present invention have a significant and specific killing effect on CD7-positive target cells, and provide useful CAR-T cells for the clinical application of cell therapy.
  • the present invention applies a suicide gene structure to the CAR-T structure, which can eliminate CAR-T cells when not needed, so as to ensure the safety of its application.
  • Figure 1 is the plasmid map of the third generation lentiviral vector pCDH-EF1(X6)-MCS-T2A-Puro.
  • Figure 2 is a schematic diagram of the molecular structure of CD7-Blocker.
  • Figure 3 is a schematic diagram of the molecular structure of CD7-CAR.
  • Fig. 4 is a flow cytometric detection diagram of the blocking effect of CD7-Blocker on CD7 molecules on the surface of Jurkat cells.
  • a to E correspond to the expression of CD7 molecules on the surface of Jurkat cells transduced with different virus MOI values, and M0, M2.5, M5, M10 and M15 represent MOIs of 0, 2.5, 5, 10 and 15, respectively.
  • Fig. 5 is a flow cytometric detection diagram of the blocking effect of CD7-Blocker on CD7 molecules on the surface of T cells.
  • Figure 6 is a flow cytometric detection chart of the positive rate of CD7 molecules and the positive rate of CAR on the surface of T cells.
  • Figure 7 is the CD7-Blocker-CAR-T cell expansion curve.
  • Figure 8 is the killing curve of CD7-Blocker-CAR-T cells co-cultured with positive target cells U87-CD7-eGFP.
  • the co-culture cell groups corresponding to the three curves are: single target cell U87-CD7-eGFP (a), control CD7-Blocker-T: U87-CD7-eGFP (b), CD7-Blocker-CAR-T: U87 - CD7-eGFP(c).
  • Figure 9 is the killing curve of CD7-Blocker-CAR-T cells co-cultured with negative target cells U87.
  • the co-culture cell groups corresponding to the three curves are: single control target cell U87 (b'), control CD7-Blocker-T: U87 (a'), CD7-Blocker-CAR-T: U87 (c').
  • Figure 10 shows the killing efficiency of CD7-Blocker-CAR-T on CD7-positive target cells U87-CD7-eGFP.
  • the heavy chain variable region and light chain variable region of an antibody usually include 3 complementarity determining regions CDR and 4 framework regions FR.
  • the complementarity determining regions are connected by the framework region, and when the antibody is recognized, the FR molecules are coiled so that the CDR molecules are close to each other.
  • the complementarity determining region is the binding site between the antibody or antigen-binding fragment and the antigen. Therefore, the sequence of the complementarity determining region determines the specificity of the antibody.
  • an antibody is a glycoprotein or an antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • a heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • a light chain comprises a light chain variable region (VL) and a light chain constant region (CL).
  • the variable regions of the heavy and light chains comprise framework regions (FRs) and complementarity determining regions (CDRs).
  • FRs framework regions
  • CDRs complementarity determining regions
  • the four FRs are relatively conserved, while the CDR regions (CDR1, CDR2 and CDR3) contain hypervariable regions.
  • Antigen-binding fragment refers to a polypeptide fragment, which comprises a part of an intact antibody, such as the antigen-binding or variable region of an intact antibody, and has the property of being able to specifically target CD7. Preferably, it contains at least one CDR of the antibody heavy chain variable region and/or the light chain variable region; also preferably, it may contain CDR1-3 of the heavy chain variable region and/or CDR1 of the light chain variable region -3.
  • Antigen-binding fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies, or expression by host cells containing the antigen-binding fragments.
  • the present invention provides the above CD7-targeting antibody or antigen-binding fragment thereof, which has good safety and targeting, and can specifically bind to the extracellular domain of human CD7, and will contain the antibody or its antigen-binding fragment.
  • the carrier of the coding sequence of the antigen-binding fragment is used to infect immune cells, and can obtain immune effector cells with significant killing ability to tumor cells expressing CD7, and the immune effector cells can be applied to treat or improve diseases related to CD7 expression, so as to provide It lays the foundation for the treatment of CD7-positive tumors.
  • sequences of heavy chain variable region CDR1, CDR2, CDR3 and light chain variable region CDR1, CDR2 and CDR3 of an antibody or antigen-binding fragment thereof can be randomly selected within the following range: The heavy chain variable region of the antigen complementarity determining region CDR1, CDR2 and CDR3 of the amino acid sequence shown in ID NO.:12-14; And have the antigen complementarity determining region CDR1, CDR2 of the amino acid sequence shown in SEQ ID NO.:15-17 and the light chain variable region of CDR3.
  • the antibody or its antigen-binding fragment has any one of the amino acid sequences shown in (I), (II) or (III): (I) heavy chain variable region shown in SEQ ID NO: 9 Amino acid sequence and the light chain variable region amino acid sequence shown in SEQ ID NO: 11; (II) have at least 90%, preferably at least 95%, and preferably at least 98% of the amino acid sequence shown in SEQ ID NO.: 9 and 11 %, most preferably an amino acid sequence of at least 99% homology; (III) an amino acid sequence obtained by modifying, substituting, deleting or adding one or more amino acids to the amino acid sequences shown in SEQ ID NO.:9 and 11, It should be noted that the above-mentioned homologous (sometimes referred to as "identity" herein) sequence will not change the binding properties of the antigen and the antibody, that is, the amino acid sequence selected from the above-mentioned still retains the activity of the antibody against the tumor surface antigen CD7.
  • the coding sequence of the heavy chain variable region is shown in SEQ ID NO: 22, and the coding sequence of the light chain variable region is shown in SEQ ID NO: 23.
  • the above-mentioned amino acid sequence in the present invention is a sequence obtained by expressing the coding sequence of the murine antibody through host codon preference modification.
  • modification by host codon bias refers to base substitution of base sequences according to degenerate codons in order to adapt to the expression needs of different hosts, and codon bias modification generally does not change the identity of the product protein or polypeptide. sequence.
  • the coding sequence of the murine antibody the coding sequence of the heavy chain variable region is shown in SEQ ID NO: 8
  • the coding sequence of the light chain variable region is shown in SEQ ID NO: 10.
  • the antibody includes at least one of monoclonal antibody, humanized antibody, chimeric antibody, and bispecific antibody; the antigen-binding fragment is Fab, F(ab'), F(ab')2 , Fd, single chain antibody scFv, disulfide-linked Fv (sdFv), or at least one of single domain antibody. Also preferably, the antibody or antigen-binding fragment thereof is humanized.
  • the antibody further includes an antibody constant region; also preferably, the antibody constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.
  • the heavy chain constant region of the antibody constant region is selected from any one of IgG1, IgG2, IgG3, IgG4 heavy chain constant region, preferably the heavy chain constant region of IgG4; the light chain constant region of the antibody constant region is The regions are either kappa or lambda.
  • Antibodies of the invention may comprise an Fc region derived from an IgG, such as IgGl, IgG2, IgG3 or IgG4.
  • monoclonal antibody refers to immunoglobulins obtained from a clone of cells, having identical structural and chemical properties, and specific for a single antigenic determinant .
  • Monoclonal antibodies differ from conventional polyclonal antibody preparations (which typically have different antibodies directed against different determinants) in that each monoclonal antibody is directed against a single determinant on the antigen.
  • monoclonal antibodies have the advantage that they are obtained in hybridoma or recombinantly engineered cell culture and are not contaminated with other immunoglobulins.
  • the modifier "monoclonal” indicates the identity of an antibody obtained from a homogeneous population of antibodies, but this should not be construed as requiring any particular or specific method for producing said antibody.
  • variant antibodies are also included within the scope of the present invention.
  • the sequence of the variant is not particularly limited, as long as it has binding properties targeting the CD7 antigen, or an antibody with increased affinity, other variants with such sequences can be obtained using methods known in the art, And all are included in the scope of the present invention.
  • the amino acid sequence of a polypeptide can be modified by those skilled in the art using recombinant methods and/or synthetic chemistry techniques for producing variant polypeptides. For example, amino acid substitutions can be used to obtain antibodies with further improved affinity. Alternatively, codon optimization of the nucleotide sequence can be used to increase translation efficiency in the expression system used to produce the antibody.
  • Such variant antibody sequences have 80% or more (i.e., 85%, 90%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to the sequences recited in the present invention sex. Said sequence identity is calculated with respect to the sequences recited in the present invention. Or when performing an optimal alignment, such as by the programs GAP or BESTFIT using default gap weights.
  • modification means that the amino acid modification does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such modifications include amino acid substitutions, additions and deletions. Preferably, residue positions that are not identical differ by conservative amino acid substitutions.
  • Antibodies of the invention may include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications, among others.
  • Conservative amino acid substitutions refer to the interchangeability of residues with similar side chains.
  • the groups of amino acids with aliphatic side chains are glycine, alanine, valine, leucine, and isoleucine; the groups of amino acids with aliphatic-hydroxyl side chains are serine and threonine;
  • the amino acid groups with side chains are asparagine and glutamine; the amino acid groups with aromatic side chains are phenylalanine, tyrosine and tryptophan;
  • the amino acid groups with basic side chains are lysine, arginine and histidine; and the groups of amino acids with sulfur-containing side chains are cysteine and methionine.
  • Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-tianmen Paragmate and Asparagine-Glutamine.
  • one or more amino acid residues in a CDR region of an antibody of the invention may be replaced with other amino acid residues from the same side chain family.
  • variable region modification is to mutate amino acid residues in the CDR1, CDR2 and/or CDR3 regions of the VH and/or VL to improve one or more binding properties (eg, affinity) of the antibody of interest.
  • Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis. Conservative modifications (as described above) are preferably introduced. Mutations may be amino acid substitutions, additions or deletions, but are preferably substitutions. Furthermore, typically no more than one, two, three, four or five residues are changed in the CDR regions.
  • the present invention provides a CD7 blocking molecule, which comprises the antibody or antigen-binding fragment thereof according to the present invention and an endoplasmic reticulum localization domain.
  • a CD7 blocking molecule which comprises the antibody or antigen-binding fragment thereof according to the present invention and an endoplasmic reticulum localization domain.
  • it has the amino acid sequence shown in SEQ ID NO.:18, and its coding sequence is shown in SEQ ID NO.:19.
  • the endoplasmic reticulum localization domain has the amino acid sequence shown in SEQ ID NO.:32, and its coding sequence is shown in SEQ ID NO.:31.
  • the endoplasmic reticulum localization domain contains an ER localization signal molecule, and the ER localization signal molecule has an amino acid sequence shown in SEQ ID NO.:28, and its coding sequence is shown in SEQ ID NO.:27.
  • the blocking molecule of the present invention can localize the antibody fragment of the present invention to the ER reticulum, and the CD7 antigen molecule is intercepted by the antibody fragment in the ER reticulum in the secretory pathway, so it cannot be normally positioned on the cell surface, thereby avoiding CD7-CAR- "Cannibalism" between T cells.
  • the linker connecting the antibody fragment of the present invention and the ER localization signal molecule is a myc sequence, which has the amino acid sequence shown in SEQ ID NO:33.
  • the present invention also provides an anti-human CD7 chimeric antigen receptor CAR, which comprises an antigen-binding domain capable of recognizing CD7 antigen (sometimes referred to herein as an "antigen recognition region"), a hinge region, and a transmembrane structure Domain (also sometimes referred to herein as “transmembrane region”) and intracellular signaling domain (also sometimes referred to herein as "intracellular region”), wherein the antigen recognition region includes the CD7 specific binding Antibodies or antigen-binding fragments thereof.
  • an antigen-binding domain capable of recognizing CD7 antigen
  • an antigen recognition region sometimes referred to herein as an "antigen recognition region”
  • transmembrane region also sometimes referred to herein as "transmembrane region”
  • intracellular signaling domain also sometimes referred to herein as “intracellular region”
  • an "antigen recognition region” may be monovalent or multivalent (eg, bivalent or trivalent).
  • Antigen binding regions may be monospecific or multispecific (eg bispecific). Bispecificity can be against CD7 and another antigen, or against two different epitopes of CD7.
  • the antigen recognition region is a single-chain antibody (monovalent or multivalent).
  • the single-chain antibody scFv includes a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region are connected by a Linker (linker).
  • the linking mode of scFv heavy chain and light chain is VH-Linker-VL or VL-Linker-VH.
  • the sequence of the Linker can be an existing linker sequence.
  • the CAR further includes a leader signal peptide sequence.
  • a signal peptide is a peptide sequence that targets a polypeptide to a desired location in a cell.
  • the signal peptide targets the polypeptide to the secretory pathway of the cell and will allow integration and anchoring of the polypeptide to the lipid bilayer.
  • the signal peptide is a membrane localized signal peptide.
  • the leader peptide sequence is derived from the leader peptide sequence of CD8; more preferably, the CD8 leader peptide sequence has the amino acid sequence shown in SEQ ID NO:36.
  • the "hinge region”, transmembrane region” and “intracellular region” herein can be selected from the sequences of the hinge region, transmembrane region and intracellular region in the existing known CAR-T technology.
  • the hinge region of the chimeric antigen receptor is located between the extracellular antigen-binding region and the transmembrane region. moving relative to each other.
  • the hinge region may be the hinge region of a naturally occurring protein or a portion thereof.
  • the hinge regions of antibodies (such as IgG, IgA, IgM, IgE or IgD antibodies) can also be used in the chimeric antigen receptors described herein. Non-naturally occurring peptides can also be used as the hinge region of the chimeric antigen receptors described herein.
  • the hinge region is a peptide linker.
  • the hinge region is derived from CD8 ⁇ .
  • the CD8 ⁇ hinge region has the amino acid sequence shown in SEQ ID NO: 38.
  • the transmembrane region of the chimeric antibody receptor can form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure capable of spanning the cellular phospholipid bilayer.
  • Transmembrane regions can be of natural or synthetic origin.
  • the transmembrane region can be derived from CD3 ⁇ , CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, T cell receptor alpha, beta or zeta chain.
  • the transmembrane region is derived from CD8 ⁇ .
  • the CD8 ⁇ transmembrane region has the amino acid sequence shown in SEQ ID NO:40.
  • the intracellular region of the chimeric antigen receptor comprises a signaling region and/or a co-stimulatory signaling region.
  • the number of signal transduction regions and/or co-stimulatory signal transduction regions can be one or more.
  • the intracellular signaling domain is responsible for the activation of at least one normal effector function of the immune effector cell expressing the chimeric antigen receptor.
  • the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines. While it is often possible to utilize the entire intracellular signaling domain, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of an intracellular signaling region is used, such truncated portion may be used in place of the intact chain as long as it transduces an effector function signal.
  • an intracellular signaling region includes any truncated form of an intracellular signaling region sufficient to transduce an effector function signal.
  • the signaling region is derived from at least one of CD3 ⁇ , FcR ⁇ (FCER1G), FcR ⁇ (Fc ⁇ Rib), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD137, CD79a, CD79b, and CD66d.
  • the intracellular region is derived from the intracellular region of human CD3 ⁇ .
  • the human CD3 ⁇ intracellular region has the amino acid sequence shown in SEQ ID NO:44.
  • costimulation domain may be the cytoplasmic portion of a costimulatory molecule.
  • co-stimulatory molecule refers to a cognate binding partner on an immune cell, such as a T cell, that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response by the immune cell, such as, but not limited to, proliferation and survival .
  • Co-stimulatory signaling regions can be derived from CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278,
  • the co-stimulatory signaling region is derived from 4-1BB.
  • the 4-1BB co-stimulatory signaling region comprises the amino acid sequence set forth in SEQ ID NO:42.
  • the nucleotide sequence of the CAR is shown in SEQ ID NO: 20, and its coding sequence is shown in SEQ ID NO: 21.
  • the chimeric antigen receptor CAR designed by the inventors further includes a "suicide switch" RQR8 molecule, which has SEQ ID NO: The amino acid sequence shown in 47, its coding sequence is shown in SEQ ID NO:48.
  • the RQR8 molecule is fused with the intracellular signaling domain CD3 ⁇ in the CD7-CAR structure with a self-cleaving T2A linking peptide.
  • the RQR8 molecule has two CD20 epitopes, and anti-CD20 rituximab (Rituximab) is used to target CD20 to activate antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated Cytotoxic effect (CDC), can induce T cell apoptosis.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-mediated Cytotoxic effect
  • rituximab for example, can eliminate CAR-T cells, thereby increasing the safety of CAR-T cell therapy.
  • the present invention provides an isolated nucleic acid encoding an antibody or antigen-binding fragment thereof, or a blocking molecule, or a chimeric antigen receptor as described above.
  • the present invention provides a vector comprising the isolated nucleic acid of the present invention.
  • a vector can be an expression vector or a cloning vector.
  • the vector is a viral vector.
  • Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof.
  • the present invention provides a host cell comprising the above-mentioned vector.
  • Suitable host cells for cloning or expressing DNA are prokaryotic cells, yeast cells or higher eukaryotic cells. Examples of commonly used prokaryotic host cells include Escherichia coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, mammalian cells, and the like.
  • the present invention provides a preparation method of chimeric antigen receptor CAR against human CD7, which comprises culturing the above-mentioned host cells.
  • the culture conditions of the preparation method are sufficient to enable the host cells to express the anti-human CD7 chimeric antigen receptor CAR.
  • the present invention provides an immune effector cell, which expresses the above-mentioned antibody specifically binding to CD7 or an antigen-binding fragment thereof, or a blocking molecule, or a chimeric antigen receptor CAR against human CD7.
  • immune effector cells are immune cells capable of performing immune effector functions.
  • the immune effector cells express at least FcyRIII and perform ADCC effector functions.
  • immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer
  • monocytes cytotoxic T cells
  • neutrophils neutrophils
  • eosinophils eosinophils.
  • the immune effector cells are selected from: at least one of immune cells cultured and differentiated from pluripotent stem cells or embryonic stem cells, T lymphocytes, NK cells, peripheral blood mononuclear cells (PBMC) and hematopoietic stem cells.
  • the immune effector cells are T lymphocytes (same as T cells).
  • T cells can be CD4+/CD8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-, or combinations thereof.
  • the T cell produces IL-2, IFN and/or TNF when expressing the chimeric antigen receptor and binding to the target cell.
  • a CD8+ T cell lyses an antigen-specific target cell when expressing a chimeric antigen receptor and binding to the target cell.
  • the present invention provides the preparation method of the immune effector cells, which includes infecting the immune effector cells with the isolated nucleic acid or the vector of the present invention.
  • the present invention produces genetically engineered immune effector cells by introducing chimeric antigen receptors into immune effector cells, such as T cells.
  • nucleic acid or vector into mammalian cells methods for introducing nucleic acid or vector into mammalian cells are known in the art, and the vector can be transferred into immune effector cells by physical, chemical or biological methods.
  • Physical methods for introducing vectors into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
  • Chemical means for introducing nucleic acids or vectors into immune effector cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, bundles and liposomes).
  • An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome (eg, an artificial membrane vesicle).
  • Biological methods for introducing nucleic acids or vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, eg human, cells.
  • the transduced or transfected immune effector cells are propagated ex vivo following introduction of the nucleic acid or vector.
  • the preparing further comprises further evaluating or screening the transduced or transfected immune effector cells to select engineered immune effector cells.
  • the present invention further provides a drug or pharmaceutical composition, which includes: the antibody specifically binding to CD7 or its antigen-binding fragment, the isolated nucleic acid, the carrier, the chimeric antigen receptor Body CAR, the isolated nucleic acid, the chimeric antigen receptor CAR prepared by the preparation method of the chimeric antigen receptor CAR, the immune effector cells and the preparation of the immune effector cells The method prepares at least one of the obtained immune effector cells.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
  • compositions can be prepared in the form of lyophilized formulations or aqueous solutions by admixing the active agent having the desired purity with optional pharmaceutically acceptable carriers.
  • a pharmaceutically acceptable carrier is nontoxic to recipients at the dosages and concentrations employed, and may include at least one of buffers, antioxidants, preservatives, isotonic agents, stabilizers and surfactants.
  • buffers nontoxic to recipients at the dosages and concentrations employed, and may include at least one of buffers, antioxidants, preservatives, isotonic agents, stabilizers and surfactants.
  • Pharmaceutical compositions can be rendered sterile by filtration through sterile filtration membranes.
  • the pharmaceutical composition may contain at least one additive of cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, growth inhibitors, and active agents as required for the particular indication being treated.
  • the specific addition amount of additives can be adjusted according to actual needs.
  • the present invention also provides the application of a reagent in the preparation of a drug or a pharmaceutical composition for treating or improving cancer, wherein the reagent is selected from: the antibody specifically binding to CD7 or an antigen-binding fragment thereof, the isolated Nucleic acid, the vector, the host cell, the anti-human CD7 chimeric antigen receptor CAR, the preparation method of the anti-human CD7 chimeric antigen receptor CAR, and the anti-human CD7 At least one of the immune effector cells prepared by the chimeric antigen receptor CAR, the immune effector cells and the preparation method of the immune effector cells.
  • the reagent is selected from: the antibody specifically binding to CD7 or an antigen-binding fragment thereof, the isolated Nucleic acid, the vector, the host cell, the anti-human CD7 chimeric antigen receptor CAR, the preparation method of the anti-human CD7 chimeric antigen receptor CAR, and the anti-human CD7 At least one of the immune effector cells prepared by the chimeric antigen receptor CAR, the
  • the treating or improving cancer refers to being able to stimulate or improve the immune function of cancer patients.
  • the cancer refers to a cancer associated with CD7 expression.
  • cancer related to CD7 expression refers to a disease directly or indirectly caused by abnormal expression of CD7, and generally refers to a disease caused by overexpression of CD7.
  • the cancer or tumor is a hematological malignancy.
  • the hematological malignancy is a T-cell-related tumor, and the T-cell-related tumor includes leukemia, lymphoma, and myeloma.
  • the present invention also provides a method for treating/preventing cancer, which includes the step of administering a therapeutically effective amount of a drug to a subject in need, wherein the drug includes: the antibody specifically binding to CD7 or its antigen Preparation of the binding fragment, the isolated nucleic acid, the carrier, the host cell, the chimeric antigen receptor CAR against human CD7, and the chimeric antigen receptor CAR against human CD7
  • the method is to prepare at least one of the obtained anti-human CD7 chimeric antigen receptor CAR, the immune effector cells and the preparation method of the immune effector cells.
  • subject and “patient” are used interchangeably herein to refer to any animal that may be in need of an antibody-related formulation or drug, treatment described herein.
  • Subjects and patients thus include, but are not limited to, primate (including humans), canine, feline, murine and other mammalian subjects.
  • the subject is a human.
  • treatment refers to both therapeutic treatment and prophylactic or preventive measures, the aim of which is to prevent or slow down (reduce) the progression of an undesired physiological change or disorder, such as an autoimmune disease.
  • beneficial or desired clinical outcomes include, but are not limited to, the following, whether detectable or not, including relief of symptoms, reduction in extent of disease, stabilization of disease state (i.e., not worsening), delay or slowing of disease progression, Amelioration or palliation as well as alleviation (whether partial or total) of a disease state.
  • Treatment also means prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • an effective amount means the amount of a drug or agent that elicits the biological or pharmaceutical response of a tissue, system, animal or human being sought, for example, by a researcher or clinician.
  • therapeutically effective amount means an amount that causes an improved treatment, cure, prevention, or alleviation of a disease, disorder, or side effect, or reduces the rate of progression of a disease or condition, as compared to a corresponding subject not receiving that amount. quantity.
  • the term also includes within its scope amounts effective to enhance normal physiological function.
  • an effective amount herein will vary depending on factors such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the subject being treated, etc., but can still be Routinely determined by those skilled in the art. Effective amounts of compounds of the present invention can be readily determined by those skilled in the art by routine methods known in the art.
  • the present invention also provides the antibody or its antigen-binding fragment according to the present invention, or the blocking molecule, or the chimeric antigen receptor, or the immune effector cell in combination with other drugs use.
  • said other drugs include diagnostic, prophylactic and/or therapeutic agents.
  • the other drug is a CD20-targeting antibody drug, which includes but is not limited to: rituximab, atezolizumab, ofatumumab, imomoumab wait.
  • This example is for the preparation of mouse monoclonal antibody against CD7 antigen.
  • polypeptides were designed and synthesized for the CD7 antigen to immunize BALB/c mice respectively. After cell fusion, primary screening, and secondary screening, a positive clone was obtained that simultaneously recognized the polypeptide BST001-2 and CD7 recombinant protein. The clone number of the positive hybridoma cell line is 5B5.
  • the amino acid sequence of the CD7 antigen precursor protein is shown in SEQ ID NO: 1, wherein amino acid residues 26-180 are the extracellular domain of the CD7 antigen.
  • the CD7 recombinant protein is a recombinant human CD7 protein (with a his tag, a product of Biopsies, the article number is 11028-H08H), and its amino acid sequence is the sequence of the extracellular domain of the CD7 antigen, as shown in SEQ ID NO: 2.
  • amino acid sequences of the five synthetic antigen polypeptides used to immunize mice correspond to the five amino acid residues in the extracellular domain of the CD7 antigen respectively, and the specific sequences are shown in Table 1:
  • the first amino acid at the N-terminal of BST001-2 and BST001-5 (bold in the table) is the added cysteine (Cys), and the C of polypeptide BST001-1, BST001-3 and BST001-4
  • the terminal amino acid itself is Cys.
  • the five polypeptides were coupled to the carrier protein KLH (Keyhole Limpet Hemocyanin, hemocyanin) through the N-terminal or C-terminal Cys residues, and used as immunogens to immunize a group of mice respectively. Tail blood detection and monoclonal antibody screening were performed on the immunized mice.
  • the mouse monoclonal antibody of the present invention is a monoclonal antibody that recognizes the polypeptide BST001-2 and the CD7 antigenic protein obtained by immunizing the polypeptide BST001-2 as an immunogen.
  • the following takes the polypeptide BST001-2 as an example to describe in detail.
  • BST001-2 polypeptide to coat the microtiter plate: add 100 ⁇ L of BST001-2 polypeptide (concentration 1 ⁇ g/mL) to each well, and react overnight at 4°C;
  • Table 2 shows the indirect ELISA evaluation results of mouse tail blood on the 14th day after immunization. It can be seen from the results that the antibody titers of the tail blood of the three mice all reached above 1:10000, and the 1# mouse was subsequently selected for cell fusion in the following step 1.2.
  • the negative control NC is 5% milk-PBS.
  • mice According to the ELISA evaluation results of mouse tail blood in Table 2, the splenocytes of 1# mice were selected for cell fusion with myeloma cell SP2/0 on the 21st day after immunization. On the 10th day after fusion, 564 monoclonal cells were picked and cultured in a 96-well plate. After 7 days of culture, the culture supernatant of the 564 monoclonal cells in the 96-well plate was evaluated according to the indirect ELISA method described in step 1.1. Screen hybridoma cell lines capable of secreting monoclonal antibodies that recognize CD7 antigen polypeptides. Seven positive clones were preliminarily selected.
  • step 1.1 According to the indirect ELISA method described in step 1.1, the 7 positive clones were further re-screened, and the results are shown in Table 3.
  • Negative control NC is 5% Milk-PBS
  • positive control PC is 1# mouse heart blood, diluted 1:500 for use.
  • NC negative control 5% Milk-PBS
  • PC positive control 1# mouse heart blood, diluted 1:500 for use.
  • step 1.1 Further adopt the indirect ELISA method described in step 1.1, replace the BST001-2 polypeptide with the aforementioned CD7 recombinant protein (with his tag) to coat the microtiter plate, and detect the cell supernatant of the 5B5 clone, and the results are shown in Table 5 .
  • Negative control NC is 5% Milk-PBS
  • positive control PC is His-tagged mouse monoclonal antibody against CD7 recombinant protein.
  • the 5B5 hybridoma cell line was cultured, the cells were collected, RNA was extracted, the cDNA sequence encoding the monoclonal antibody against CD7 was obtained by RT-PCR method, and then the variable regions of the heavy chain and light chain were cloned by PCR method and the PCR product was connected to On the T-vector, the sequences of the heavy chain variable region VH and the light chain variable region VL of the anti-CD7 monoclonal antibody were obtained by sequencing, and the sequences were further compared and confirmed through the Uniprot database.
  • the nucleotide sequence of the VH obtained is shown in SEQ ID NO: 8, and the amino acid sequence encoded by it is shown in SEQ ID NO: 9; the nucleotide sequence of the VL obtained is shown in SEQ ID NO: 10, and the amino acid sequence encoded by it is shown in SEQ ID NO: 10. The sequence is shown in SEQ ID NO: 11.
  • This example is for the construction of CD7-Blocker and CD7-CAR lentiviral expression vectors.
  • the CD7 blocking molecules CD7-Blocker and CD7-CAR were respectively constructed into the third-generation lentiviral expression plasmids by conventional technical means in the field.
  • the plasmid is pCDH-EF1(X6)-MCS-T2A-Puro, the map of which is shown in Figure 1, the vector linearization restriction site is XbaI and SalI, the DNA sequence of CD7-Blocker and CD7-CAR (including N terminal KOZAC sequence) was inserted between the two restriction sites.
  • the molecular structure of CD7-Blocker is shown in Figure 2.
  • the CD7-Blocker molecule is composed of CD8a signal peptide SP, (G4S)3 linker connecting VL and VH, anti-CD7 scFv, ER Retention Domain (endoplasmic reticulum localization domain) three In part, the front end of the SP is inserted with a KOZAK sequence to promote expression. Its full-length amino acid sequence is shown in SEQ ID NO: 18, and its full-length DNA sequence is shown in SEQ ID NO: 19 (including the N-terminal KOZAC sequence).
  • CD7-CAR The molecular structure of CD7-CAR is shown in Figure 3. It consists of CD8a signal peptide SP, (G4S)3 linker connecting VL and VH anti-CD7 scFv, CD8a hinge region, CD8a transmembrane region, and 4-1BB and CD3 ⁇ Two intracellular signaling domains constitute the main part of the CD7-CAR molecule, and then a cell "suicide switch" RQR8 molecule is fused with a self-cleaving T2A linking peptide, and a KOZAK sequence that promotes expression is also inserted at the front of the SP.
  • SEQ ID NO: 20 Its full-length amino acid sequence is shown in SEQ ID NO: 20, and its full-length DNA sequence is shown in SEQ ID NO: 21 (including the N-terminal KOZAC sequence).
  • the nucleotide coding sequences of VH and VL in the two molecular structures of CD7-Blocker and CD7-CAR are respectively shown in SEQ ID NO: 22 and SEQ ID NO: 23, which are derived from the mouse VH sequence (SEQ ID NO: 8) and VL sequence (SEQ ID NO: 10) were optimized according to human codons.
  • VL and VH are fused through a linker (G4S)3 to form anti-CD7 scFv.
  • CD7-Blocker is formed by connecting anti-CD7 scFv and ER localization signal KDEL through a linker (G4S)2, and (G4S)2-KDEL constitutes the ER Retention Domain (endoplasmic reticulum localization domain).
  • the ER Retention Domain can localize anti-CD7 scFv to the ER reticulum, and the CD7 antigen molecule is intercepted by the anti-CD7 scFv in the ER reticulum during the secretory pathway, so it cannot be normally located on the cell surface, thereby avoiding CD7-CAR-T cells "Cannibalism" among them.
  • the linker connecting the anti-CD7 scFv and the ER localization signal is the myc sequence EQKLISEEDL (SEQ ID NO: 33).
  • CD7-Blocker and CD7-CAR The sequence numbers corresponding to the amino acid and nucleotide sequences of each fragment in the molecular structure of CD7-Blocker and CD7-CAR are shown in Table 7, wherein SP is the signal peptide of CD8a, CD8H is the hinge region of CD8a, and CD8TM is the transmembrane region of CD8a. Both 4-1BB and CD3 ⁇ are intracellular signaling domains.
  • the present invention incorporates a "suicide switch" RQR8 molecule (SEQ ID NO: 47, SEQ ID NO: 47, NO:48), the RQR8 molecule is fused with the intracellular signaling domain CD3 ⁇ in the CD7-CAR structure with a T2A linking peptide with self-cleavage function.
  • the RQR8 molecule carries two CD20 epitopes, and the anti-CD20 rituximab (Rituximab) targets CD20 to activate antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC), can induce T cell apoptosis.
  • Rituximab can be used when necessary to eliminate CAR-T cells, thereby increasing the safety of CAR-T cell therapy.
  • Lentiviral packaging adopts the conventional four-plasmid system in the field, in which the three helper plasmids are pMDLg/pRRE, pRSV-Rev and pMD2.G.
  • 293T cells were used as lentiviral packaging cells.
  • the ratio of plasmid dosage for co-transfection of 293T cells carrying CD7-Blocker or CD7-CAR lentiviral expression plasmid with pMDLg/pRRE, pRSV-Rev and pMD2.G is 7.5:9:9:3.5; for T75 cell culture flask,
  • the dosages of the four plasmids were 7.5ug, 9ug, 9ug, and 3.5ug, respectively.
  • the amount (ug) of the transfection reagent PEI was 3 times the total amount of the four plasmids, and for the T75 culture flask, the amount of PEI was 87ug (1ug/ul, 87ul).
  • Collect the cell culture medium 48 hours after the co-transfection of the four plasmids into 293T take the supernatant after centrifugation (2000rpm, 15min), filter through a 0.45um filter, use ultracentrifugation (20000rpm, 2h) to concentrate the supernatant, and then use according to the dilution factor Resuspend the virus pellet in a corresponding volume of culture medium, aliquot and freeze at -80°C.
  • the titer of the lentivirus was directly measured using the lentiviral vector HIV P24 rapid detection card.
  • the lentivirus was serially diluted and then transfected into 293T cells. After 48 hours, the transfection efficiency was detected by flow cytometry, and the activity titer of the lentivirus was calculated.
  • This example is the identification of the affinity between Anti-CD7 scFv and CD7 antigen molecule.
  • CD7-CAR lentivirus to transduce 293T cells, the positive rate of CD7-CAR in 293T cells and the ratio of 293T cells combined with CD7 antigen protein were detected by flow cytometry, and the CD7-CAR and CD7 antigen protein in CD7-CAR-293T cells were calculated
  • the affinity ratio is used to express the affinity between Anti-CD7 scFv and CD7 antigen molecule.
  • the CD7 antigenic protein is the His-tagged recombinant human CD7 protein in Example 1. During flow cytometric detection, the CD7 antigenic protein is first incubated with CD7-CAR-293T cells, and then detected with a fluorescently labeled anti-His mouse monoclonal antibody. CD7 antigen protein bound to 293T cells.
  • This example is the blocking effect of CD7-Blocker on CD7 molecules on the surface of Jurkat cells.
  • the CD7-Blocker lentivirus was transfected into Jurkat cells, and the positive rate of CD7 molecules on the surface of Jurkat cells was detected by flow cytometry 4 days later.
  • the results are shown in Table 9 and Figure 4.
  • This example is the blocking effect of CD7-Blocker on CD7 molecules on the surface of T cells.
  • the cryopreserved PBMCs were resuscitated and then activated with CD3/CD28 antibody magnetic beads (activation time point is marked as D0), transfected with CD7-Blocker lentivirus (B-T experimental group) 1 day after activation (D1), and 2 days after activation (D2 ) transfected with CD7 CAR lentivirus.
  • the experiment was divided into four groups: non-transfected control group (NT group), only transfected with CD7-Blocker virus (B-T group), and co-transfected with CD7-Blocker and CD7 CAR virus on the basis of NT (B-CAR-T group). group), only CD7 CAR virus was transfected (CAR-T group).
  • the MOI value of virus transfection was 5.
  • the positive rate of CD7 antigen molecules on the cell surface was detected by flow cytometry on D6 after transfection (6 days after activation, 5 days after transfection with Blocker virus, and 4 days after transfection with CAR virus). The results are shown in Table 10 and Figure 5.
  • This example is the effect of CD7-Blocker on the expansion of T cells.
  • This example detected and compared the control T cells (NT group) that were not transfected with CD7-Blocker virus, the CD7-Blocker-T cells (B-T group) that were only transfected with CD7-Blocker lentivirus, and the CD7-Blocker-T cells that were only transfected with CD7- Four groups including CD7-CAR-T cells with CAR lentivirus (CAR-T group) and CD7-Blocker-CAR-T cells transfected with both CD7-Blocker virus and CD7-CAR virus (B-CAR-T group) In vitro expansion of T cells.
  • the time points of virus transfection of each group of cells are as in Example 4.
  • Cultivation time description The cryopreserved PBMCs were resuscitated and cultured for one day and then activated by adding CD3/CD28 antibody magnetic beads. The activation time point was marked as D0, and the time points of 1 day and 2 days after activation were respectively marked as D1 and D2, and so on.
  • the surface CD7 molecule positive rate and CAR positive rate of T cells in the four groups are shown in Table 11 and Figure 6, the cell expansion ratio is shown in Table 12, and the cell expansion curve is shown in Figure 7.
  • This example is an in vitro killing experiment of CD7-CAR-T cells on CD7-positive target cells.
  • a CD7-overexpressed U87-CD7-eGFP cell line was constructed using CD7-negative U87 cells, and CD7-CAR-T cells were analyzed by RTCA instrument Killing effect on CD7-positive target cells U87-CD7-eGFP.
  • the coding sequence of the CD7 antigen molecule used to construct the U87-CD7-eGFP cell line is the DNA coding sequence of the CD7 antigen precursor protein (SEQ ID NO: 51), and the amino acid sequence of the eGFP molecule used is shown in SEQ ID NO: 52 , its DNA coding sequence is shown in SEQ ID NO: 53, and the CD7 molecule and the eGFP molecule are connected by a self-cleaving linker peptide T2A (SEQ ID NO: 45, SEQ ID NO: 46).
  • the N-terminal of the CD7-T2A-eGFP structure was added with KOZAK sequence and inserted between the XbaI and SalI restriction sites of the lentiviral vector pCDH-EF1(X6)-MCS-T2A-Puro to construct a CD7 overexpression lentiviral vector.
  • the lentivirus CD7-T2A-eGFP was transduced into U87 cells according to conventional means, and eGFP was used as a screening and detection marker for transduced cells.
  • CD7-CAR-T cells can kill each other, the effector cells used in the killing experiment were CD7-Blocker-CAR-T cells (B-CAR-T for short) that blocked the surface expression of CD7 molecules by CD7-Blocker.
  • the positive rate of CAR was 45.79%, and the positive rate of surface CD7 molecule was 5.97%.
  • the control T cells in the killing experiment were CD7-Blocker-T (abbreviated as B-T) cells, and the positive rate of CD7 molecules on their surface was 8.35%.
  • the positive rate of CD7 surface molecule of unblocked T cells was 94.44%.
  • the killing experiment curves are shown in Figure 8 and Figure 9.
  • Time point 0.0 is the starting point of the target cell culture, and the effector cells are added to co-culture when the target cells are cultured for 28 hours.
  • the whole experiment lasted 96h.
  • Figure 8 shows the killing curves of two kinds of T cells co-cultured with positive target cells U87-CD7-eGFP. It can be seen that only B-CAR-T has a significant killing effect on CD7-positive target cells U87-CD7-eGFP.
  • Figure 9 shows the killing curves of two kinds of T cells co-cultured with negative target cells U87. It can be seen that B-CAR-T has only a slight killing effect on U87.
  • the cell index values at both ends of the early period of co-culture were intercepted to obtain the killing efficiency
  • the B-T group is co-cultured with control CD7-Blocker-T cells and U87-CD7-eGFP cells

Abstract

本发明公开一种CD7-CAR-T细胞及其制备方法和应用，其中CD7-CAR-T细胞包含靶向CD7抗原的抗体或其抗原结合片段，该抗体或其抗原结合片段含有具有SEQ ID NO.:12-14所示氨基酸序列的抗原互补决定区CDR1、CDR2和CDR3的重链可变区；和具有SEQ ID NO.:15-17所示氨基酸序列的抗原互补决定区CDR1、CDR2和CDR3的轻链可变区。本发明的抗体以及基于该抗体片段的CD7-CAR与CD7抗原分子具有极强的亲和力，同时本发明的阻断分子几乎能完全阻断CD7分子在细胞表面的表达，而基本不会影响T细胞的正常扩增，从而能够有效避免CD7-CAR-T细胞的自相残杀。此外，本发明的CD7-CAR-T细胞对CD7阳性靶细胞具有显著且特异的杀伤作用，为细胞治疗临床应用提供了有益的CAR-T细胞。

Description

CD7-CAR-T细胞及其制备方法和应用 技术领域
本发明涉及生物免疫治疗技术领域，具体地涉及CD7-CAR-T细胞及其制备方法和应用。
背景技术
急性淋巴细胞白血病(Acute Lymphocytic leukemia,ALL)是血液系统的常见恶性肿瘤，主要起源于B淋巴细胞系和T淋巴细胞系。原始样的未成熟淋巴细胞经过多步骤的特异基因损伤而形成肿瘤细胞，其在骨髓异常增生和聚集，并抑制正常骨髓干细胞的造血功能，导致ALL的发生。同时，这些肿瘤细胞也可浸润到骨髓外组织(如淋巴结、脑膜、肝脏、性腺等)，引起相关组织损伤。
ALL各年龄段均可发病，儿童和青年发病率较高，为3.6/10万，成人ALL发病率较低，为0.69/10万。初始化疗和靶向治疗可杀伤侵袭性的增殖细胞和选择性或获得性突变的细胞，但是对药物不敏感的ALL细胞仍会导致疾病复发。骨髓移植是ALL的重要治疗手段，异基因移植对急性白血病虽有很好的疗效，但多数患者缺乏适合的供髓者及高昂的移植费用，受到较大限制。自体骨髓移植作为异基因移植的一种替代治疗，在近些年内得到迅速的发展。目前虽已积累了不少临床资料，但由于各报告的疗效相差较远，至今对自体骨髓移植在急性白血病治疗中的地位尚有争议，在初次完全缓解期进行自体骨髓移植的长期无白血病存活率(LFS)从不足30％到70％以上不等。有的资料说明自体骨髓移植并不改善急性淋巴细胞白血病患者的LFS，但也有资料证明自体骨髓移植对初次完全缓解的急性淋巴细胞白血病的疗效与异基因骨髓移植接近，远优于单纯化疗。因此，由于现有治疗方法的不确定性和低有效性，人们需要继续探索新的、更好的治疗方法。
急性T淋巴细胞白血病(T Cell Acute Lymphocytic leukemia,T-ALL)是ALL的一种，是由T系前体细胞在骨髓和胸腺发生恶性转化以及克隆扩增引起的恶性肿瘤。T-ALL的基因突变是多样化的，异质性很强，包括基因缺失、突变、染色体易位等。这些异常可引起多种信号传导通路(如MAPK和Jak/Stat、PI3K/Akt/mTOR等)和/或细胞周期的异常。研究发 现，至少170多个潜在的致癌驱动基因与T-ALL的发生有关。
T-ALL多发生于儿童和青少年，也可发生于成年人。T-ALL占儿童ALL患者的比例为10％-15％，成人患者比例约为25％。相比于急性B淋巴细胞白血病(B-ALL)，T-ALL对化疗不敏感，效果差，复发率高，缓解率和远期生存率低。复发后的优势克隆往往富含对常规化疗药的耐药基因。相对于儿童T-ALL，成人T-ALL治疗效果更差，约50％成人患者在治疗缓解后一年左右复发，再次化疗的缓解率仅有30％-45％，最终只有40％的患者能长期生存，而且复发/难治型的T-ALL患者预后通常更差。
近年来，嵌合抗原受体T细胞(CAR-T)疗法在血液肿瘤的治疗中显示出了非常显著的临床疗效，为肿瘤治疗带来了新的曙光。如以CD19为靶点的CAR-T治疗在B-ALL中可以达到90％-100％的CR，无论是自体CAR-T和异体CAR-T的治疗均可达到5年以上的无复发生存状态。CAR-T治疗的关键技术之一就是选择一个肿瘤细胞上表达的特异性靶点，如肿瘤相关抗原或者肿瘤特异性抗原，以此抗原的特异性抗体为要件制备CAR-T细胞，进行治疗。
CD7是一种分子量为40kDa的细胞膜单链糖蛋白，主要表达在胸腺细胞、大部分外周血T淋巴细胞，且先于T系其它抗原(CD1、CD2、CD3、CD4、CD5、CD8)的出现，也早于TCR-β链基因重排。CD7抗原也表达于NK细胞和部分髓系细胞。在起源于T淋巴系的白血病细胞表面高表达。除了T-ALL，绝大部分T细胞淋巴瘤、NK和NKT淋巴瘤也表达CD7，在急性髓性白血病(AML)中也有20-30％的表达率。但是，动物实验表明，破坏了CD7分子的小鼠T祖细胞依然会产生正常的T细胞发育和体内平衡，基本没有引起T细胞效应功能的变化，提示CD7对T细胞的发育和功能不会产生关键性的影响。因此，CD7是T-ALL及CD7阳性肿瘤的一个理想靶点。
相较于B-ALL的CAR-T治疗，应用CD7-CAR-T细胞靶向治疗T细胞急性淋巴细胞白血病和CD7阳性的淋巴瘤等依然面临着很大的技术问题，这是因为正常的效应T细胞和T细胞肿瘤都表达CD7抗原，而会导致CD7-CAR-T细胞的“自相残杀”效应，因此，CD7-CAR-T细胞很难在体外制备成功；临床应用上，同时要考虑不能让CD7-CAR-T细胞在体内无限制的长期存在，以避免严重的副作用。目前仅有少数的CD7-CAR-T技术研究，为避免“自相残杀”效应，应用CRISPR基因编辑的方法敲除T细胞中的CD7基因，但实践和理论上均 难以100％敲除CD7分子，而存留移植物抗宿主病(GVHD)的临床应用风险。同时，基因编辑天然的“脱靶效应”可能带来的副作用，也会限制临床应用的广泛开展。而应用nano-body(纳米抗体)技术缩小抗体结合区的体积，虽有一定的优势，但缺点是缩小、单臂的抗体会导致抗原抗体的结合力和稳定性相对减弱，导致CD7-CAR-T细胞效能降低。此外，正常的T细胞、NK细胞等也有CD7表达，它们是体内维持正常细胞免疫、抗击病毒和细菌感染的重要力量，只强调CD7-CAR-T在体内存活的持久杀伤性，在消灭肿瘤的同时会造成长期的感染等副作用，因此合理管控CD7-CAR-T细胞的存在也是优秀的CAR-T设计的重要因素。
背景技术中的信息仅仅在于说明本发明的总体背景，不应视为承认或以任何形式暗示这些信息构成本领域一般技术人员所公知的现有技术。
发明内容
为解决现有技术中的技术问题，本发明人通过深入研究，制备、挑选了特有的抗人CD7单克隆抗体序列，作为本发明CD7-CAR-T构建的抗原识别元件，同时应用抗CD7抗原的抗体片段与内质网(ER)定位信号相融合的结构，使CD7抗原分子定位在T细胞的ER网腔，以阻断其在CAR-T细胞表面的表达，从而消除CD7-CAR-T细胞的“自相残杀”效应，以利于CD7-CAR-T细胞的体外制备和生产。此外，本发明在CAR-T结构上应用自杀性基因结构，可以在不需要时消除CAR-T细胞，以保障其应用的安全性。具体地，本发明包括以下内容。
本发明的第一方面，提供一种抗体或其抗原结合片段，其含有具有SEQ ID NO.:12-14所示氨基酸序列的抗原互补决定区CDR1、CDR2和CDR3的重链可变区；和
具有SEQ ID NO.:15-17所示氨基酸序列的抗原互补决定区CDR1、CDR2和CDR3的轻链可变区。
根据本发明所述的抗体或其抗原结合片段，优选地，所述抗体具有(I)、(II)或(III)所示的氨基酸序列中的任意一个氨基酸序列：
(I)SEQ ID NO：9所示的重链可变区氨基酸序列和SEQ ID NO：11所示的轻链可变区氨基酸序列；
(II)与SEQ ID NO.:9和11所示的氨基酸序列具有至少90％，优选至少95％，还优选至少98％，最优选至少99％同源性的氨基酸序列；
(III)与SEQ ID NO.:9和11所示的氨基酸序列经修饰、取代、缺失或添加一个或多于一个氨基酸获得的氨基酸序列；
其中，所述氨基酸序列具有针对CD7抗原的抗体的活性。
根据本发明所述的抗体或其抗原结合片段，优选地，其中所述抗体包括多克隆抗体、单克隆抗体、嵌合抗体、人源化抗体或双特异性抗体中的至少一种；所述抗原结合片段包括Fab片段、Fab’、F(ab’)2片段、单链可变片段scFv、scFv-Fc片段或单链抗体ScAb中的至少一种。
本发明的第二方面，提供一种CD7阻断分子，其包含：
a.根据第一方面所述的抗体或其抗原结合片段；和
b.内质网定位结构域。
本发明的第三方面，提供一种嵌合抗原受体，其包括：
1)识别CD7抗原的抗原结合结构域，其中所述抗原结合结构域包括根据第一方面所述的抗体或其抗原结合片段；
2)跨膜结构域；和
3)胞内信号传导结构域；
优选地，进一步包括铰链区；
优选地，进一步包括自杀开关分子；
优选地，进一步包括细胞内共刺激域；
优选地，所述跨膜结构域选自：多肽CD28、NKp30、CDS、DAP10、4-1BB、DAP12、CD3C、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、LFA-1、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、 DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、PAG/Cbp中的至少一种或其组合；
优选地，所述胞内信号传导结构域选自：CD8、CD3ζ、CD3δ、CD3γ、CD3ε、FcγRI-γ、FcγRIII-γ、FcεRIβ、FcεRIγ、DAP10、DAP12、CD32、CD79a、CD79b、CD28、CD3C、CD4、b2c、CD137(4-1BB)、ICOS、CD27、CD28δ、CD80、NKp30、OX40中的至少一种或其任何组合。
本发明的第四方面，提供一种分离的核酸分子，其编码根据本发明第一方面所述的抗体或其抗原结合片段，或根据第二方面所述的阻断分子，或根据第三方面所述的嵌合抗原受体。
本发明的第五方面，提供一种载体，其包含根据第四方面所述的核酸分子。
本发明的第六方面，提供一种宿主细胞，其包含根据第五方面所述的载体。
本发明的第七方面，提供根据第三方面所述的嵌合抗原受体的制备方法，其包括培养根据第六方面所述的宿主细胞。
本发明的第八方面，提供一种免疫效应细胞，其表达根据本发明第一方面所述的抗体或其抗原结合片段，或根据第二方面所述的阻断分子，或根据第三方面所述的嵌合抗原受体；
优选地，所述免疫效应细胞选自：白细胞、单核细胞、巨噬细胞、树突细胞、肥大细胞、嗜中性粒细胞、嗜碱性粒细胞、嗜酸性粒细胞、αβT细胞、γδT细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、B细胞、天然淋巴样细胞(ILC)、细胞因子诱导的杀伤(CIK)细胞、细胞毒性T淋巴细胞(CTL)、淋巴因子激活的杀伤(LAK)细胞、T淋巴细胞、外周血单个核细胞和造血干细胞中的至少一种。
本发明的第九方面，提供试剂在用于制备预防和/或治疗癌症或肿瘤的组合物、药物、制剂或试剂盒中的用途，所述试剂包括：根据本发明第一方面所述的抗体或其抗原结合片段，或根据第二方面所述的阻断分子，或根据第三方面所述的嵌合抗原受体，或根据第八方面所述的免疫效应细胞；
优选地，所述癌症或肿瘤是指CD7表达相关的癌症或肿瘤，还优选地，所述癌症或肿瘤是血液系统恶性肿瘤；进一步优选地，所述血液系统恶性肿瘤为T细胞相关肿瘤，所述T细胞相关肿瘤包括白血病和淋巴瘤、骨髓瘤。
本发明的第十方面，提供根据本发明第一方面所述的抗体或其抗原结合片段，或根据第二方面所述的阻断分子，或根据第三方面所述的嵌合抗原受体，或根据第八方面所述的免疫效应细胞在与其它药物联合用药中的用途。其它药物包括但不限于：诊断剂、预防剂和/或治疗剂。
本发明的优异技术效果包括但不限于：本发明的抗体以及基于该抗体片段的CD7-CAR与CD7抗原分子具有极强的亲和力，同时本发明的阻断分子几乎能完全阻断CD7分子在细胞表面的表达，而基本不会影响T细胞的正常扩增，从而能够有效避免CD7-CAR-T细胞的自相残杀。此外，本发明的CD7-CAR-T细胞对CD7阳性靶细胞具有显著且特异的杀伤作用，为细胞治疗临床应用提供了有益的CAR-T细胞。同时本发明在CAR-T结构上应用自杀性基因结构，可以在不需要时消除CAR-T细胞，以保障其应用的安全性。
附图说明
图1为第三代慢病毒载体pCDH-EF1(X6)-MCS-T2A-Puro的质粒图谱。
图2为CD7-Blocker分子结构示意图。
图3为CD7-CAR分子结构示意图。
图4为CD7-Blocker对Jurkat细胞表面CD7分子的阻断效应的流式检测图。其中，A至E分别对应不同的病毒MOI值转导的Jurkat细胞表面CD7分子的表达情况，M0、M2.5、M5、M10和M15分别表示MOI为0、2.5、5、10和15。
图5为CD7-Blocker对T细胞表面CD7分子的阻断效应流式检测图。
图6为T细胞表面CD7分子阳性率和CAR阳性率流式检测图。
图7为CD7-Blocker-CAR-T细胞扩增曲线。
图8为CD7-Blocker-CAR-T细胞与阳性靶细胞U87-CD7-eGFP共培养杀伤曲线。其中三条曲线分别对应的共培养细胞组为：单独的靶细胞U87-CD7-eGFP(a)、对照CD7-Blocker-T：U87-CD7-eGFP(b)、CD7-Blocker-CAR-T：U87-CD7-eGFP(c)。
图9为CD7-Blocker-CAR-T细胞与阴性靶细胞U87共培养杀伤曲线。其中三条曲线分别对应的共培养细胞组为：单独的对照靶细胞U87(b’)、对照CD7-Blocker-T：U87(a’)、CD7-Blocker-CAR-T：U87(c’)。
图10为CD7-Blocker-CAR-T对CD7阳性靶细胞U87-CD7-eGFP的杀伤效率。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为具体公开了该范围的上限和下限以及它们之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其它陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
抗体的重链可变区和轻链可变区通常包括3个互补决定区CDR和4个骨架区FR。互补决定区之间通过骨架区连接，在识别抗体时，FR分子卷曲使CDR分子相互靠近。互补决定区为抗体或抗原结合片段与抗原的结合部位，因此，互补决定区的序列决定了抗体的特异性。如本领域所理解，抗体是包含通过二硫键互连的至少两个重(H)链和两个轻(L)链的糖蛋白或其抗原结合部分。重链包含重链可变区(VH)和重链恒定区(CH)。轻链包含轻链可变区(VL)和轻链恒定区(CL)。重链和轻链的可变区包含框架区(FR)和互补决 定区(CDR)。四个FR是相对保守的，而CDR区域(CDR1、CDR2和CDR3)包含高变区。
本文中的“抗原结合片段”是指多肽片段，其包含完整抗体的一部分，诸如完整抗体的抗原结合区或可变区，并且具有能够特异性靶向CD7的特性。优选地，其含有抗体重链可变区和/或轻链可变区的至少一个CDR；还优选地，其可以含有重链可变区的CDR1-3和/或轻链可变区的CDR1-3。抗原结合片段可以通过多种技术制备，包括但不限于将完整的抗体蛋白水解消化，或由包含抗原结合片段的宿主细胞表达产生。
本发明提供了上述靶向CD7的抗体或其抗原结合片段，所述抗体或其抗原结合片段具有良好的安全性和靶向性，能够特异性结合人CD7的胞外域，将包含该抗体或其抗原结合片段的编码序列的载体，用于感染免疫细胞，能够获得对表达CD7的肿瘤细胞具有显著杀伤能力的免疫效应细胞，该免疫效应细胞能够应用于治疗或改善CD7表达相关的疾病，从而为CD7阳性肿瘤的治疗奠定基础。
在没有限定或理论约束的情况下，抗体或其抗原结合片段的重链可变区CDR1、CDR2、CDR3和轻链可变区CDR1、CDR2和CDR3的序列可在下述范围内随机选择：具有SEQ ID NO.:12-14所示氨基酸序列的抗原互补决定区CDR1、CDR2和CDR3的重链可变区；和具有SEQ ID NO.:15-17所示氨基酸序列的抗原互补决定区CDR1、CDR2和CDR3的轻链可变区。
本发明中，抗体或其抗原结合片段具有(I)、(II)或(III)所示的氨基酸序列中的任意一个氨基酸序列：(I)SEQ ID NO：9所示的重链可变区氨基酸序列和SEQ ID NO：11所示的轻链可变区氨基酸序列；(II)与SEQ ID NO.:9和11所示的氨基酸序列具有至少90％，优选至少95％，还优选至少98％，最优选至少99％同源性的氨基酸序列；(III)与SEQ ID NO.:9和11所示的氨基酸序列经修饰、取代、缺失或添加一个或多于一个氨基酸获得的氨基酸序列，需要说明的是上述同源性(本文有时也称为“同一性”)序列不会改变抗原与抗体结合特性，即选自上述的氨基酸序列依然保留针对肿瘤表面抗原CD7的抗体的活性。
优选地，所述重链可变区的编码序列如SEQ ID NO：22所示，所述轻链可变区的编码序列如SEQ ID NO：23所示。
优选地，本发明中的上述氨基酸序列是根据鼠源抗体的编码序列经宿主密码子偏好 性改造序列后通过表达得到的序列。本发明中，经宿主密码子偏好性改造是指为了适应于不同宿主表达的需要，根据简并密码子来对碱基序列进行碱基替换，密码子偏好性改造一般不改变产物蛋白或多肽的序列。所述鼠源抗体的编码序列中，其重链可变区的编码序列如SEQ ID NO：8所示，轻链可变区的编码序列如SEQ ID NO：10所示。
优选地，所述抗体包括单克隆抗体、人源化抗体、嵌合抗体、双特异性抗体中的至少一种；所述抗原结合片段为Fab、F(ab’)、F(ab’)2、Fd、单链抗体scFv、二硫键连接的Fv(sdFv)、或单域抗体中的至少一种。还优选地，所述抗体或其抗原结合片段是人源化的。
优选地，所述抗体还包括抗体恒定区；还优选地，所述抗体恒定区选自：IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中的任意一者的恒定区。
优选地，所述抗体恒定区的重链恒定区选自IgG1、IgG2、IgG3、IgG4中任意一者的重链恒定区，优选为IgG4的重链恒定区；所述抗体恒定区的轻链恒定区为κ或λ。
本发明的抗体可包含Fc区，所述Fc区来自IgG，例如IgG1、IgG2、IgG3或IgG4。
本文所用术语“单克隆抗体”，有时也称为“单抗”或mAb，其是指从一纯系细胞得到的免疫球蛋白，具有相同的结构和化学特性，对单一抗原决定簇有特异性。单克隆抗体与常规多克隆抗体制剂(通常是具有针对不同决定簇的不同抗体)不同，各单克隆抗体是针对抗原上的单个决定簇。除了它们的特异性外，单克隆抗体的好处还在于它们是通过杂交瘤或重组工程细胞培养获得，不会混杂有其它免疫球蛋白。修饰语“单克隆”表示了抗体的特性，是从均一的抗体群中获得的，但这不应被解释成需要用任何特殊或特定的方法来生产所述抗体。
变体抗体也都被包括在本发明的范围内。在本发明对变体的序列不特别限定，只要其具有靶向CD7抗原的结合特性，或具有提高的亲和力的抗体即可，具有这样序列的其它变体可以使用本领域已知的方法得到，并都包括在本发明的范围内。本领域技术人员利用用于生产变体多肽的重组方法和/或合成化学技术可以修改多肽的氨基酸序列。例如，可以使用氨基酸置换得到具有进一步提高的亲和力的抗体。可选地，可以使用核苷酸序列的密码子优化来提高在用于生产抗体的表达系统中的翻译效率。这样的变体抗体序列与在本发明中列举的序列具有80％或更高的(即，85％、90％、95％、96％、97％、98％、99％或更大)序列同一性。相对于在本发明中列举的序列，计算所述序列同一性。或进行 最佳比对时，如通过程序GAP或使用默认值间隙权重的BESTFIT。
本文所用术语“修饰”意指氨基酸修饰不会显著影响或改变含有该氨基酸序列的抗体的结合特征。此类修饰包括氨基酸的取代、添加和缺失。优选地，不相同的残基位置因保守氨基酸取代而不同。本发明的抗体可包括糖基化、乙酰化、磷酸化、酰胺化、通过已知的保护/封闭基团衍生化、蛋白水解切割或非天然发生的氨基酸修饰等。
保守氨基酸取代指的是具有类似侧链的残基的可互换性。例如，具有脂肪族侧链的氨基酸组为甘氨酸、丙氨酸、缬氨酸、亮氨酸和异亮氨酸；具有脂肪族-羟基侧链的氨基酸组为丝氨酸及苏氨酸；具有含酰胺侧链的氨基酸组为天冬酰胺和谷氨酰胺；具有芳香族侧链的氨基酸组为苯丙氨酸、酪氨酸和色氨酸；具有碱性侧链的氨基酸组为赖氨酸、精氨酸和组氨酸；以及具有含硫侧链的氨基酸组为半胱氨酸及甲硫氨酸。优选的保守氨基酸取代组为：缬氨酸-亮氨酸-异亮氨酸、苯丙氨酸-酪氨酸、赖氨酸-精氨酸、丙氨酸缬氨酸、谷氨酸-天门冬酸和天冬酰胺-谷氨酰胺。因此，可以用来自同一侧链家族的其它氨基酸残基替换本发明抗体CDR区中的一个或多个氨基酸残基。
另一类可能存在的可变区修饰是突变VH和/或VL的CDR1、CDR2和/或CDR3区中的氨基酸残基以改进目的抗体的一种或多种结合特性(例如亲和力)。可以通过定点诱变或PCR介导的诱变来导入突变。优选导入(如上所述的)保守修饰。突变可以是氨基酸的取代、添加或缺失，但优选为取代。此外，CDR区中残基变化通常不超过一个、两个、三个、四个或五个。
本发明提供了一种CD7阻断分子，其包含根据本发明所述的抗体或其抗原结合片段和内质网定位结构域。优选地，其具有SEQ ID NO.:18所示的氨基酸序列，其编码序列如SEQ ID NO.:19所示。还优选地，内质网定位结构域具有SEQ ID NO.:32所示的氨基酸序列，其编码序列如SEQ ID NO.:31所示。所述内质网定位结构域含有ER定位信号分子，所述ER定位信号分子具有SEQ ID NO.:28所示的氨基酸序列，其编码序列如SEQ ID NO.:27所示。
本发明的阻断分子能够将本发明的抗体片段定位到ER网腔，CD7抗原分子在分泌途径被ER网腔内的抗体片段拦截，因而不能正常定位到细胞表面，从而可避免CD7-CAR-T细胞之间的“自相残杀”。在另外的实施方案中，连接本发明抗体片段与ER定位 信号分子的接头为myc序列，其具有SEQ ID NO:33所示的氨基酸序列。
本发明还提供了一种抗人CD7的嵌合抗原受体CAR，所述CAR包含可识别CD7抗原的抗原结合结构域(本文有时也称为“抗原识别区”)、铰链区、跨膜结构域(本文有时也称为“跨膜区”)和胞内信号传导结构域(本文有时也称为“胞内区”)，其中所述抗原识别区包括本发明所述的特异性结合CD7的抗体或其抗原结合片段。
在没有限定的情况下，“抗原识别区”可以为单价的或多价的(如二价的或三价的)。抗原结合区可以是单特异性的或多特异性的(例如双特异性的)。双特异性可以是针对CD7和另一种抗原，也可以是针对CD7的两种不同表位。优选地，所述抗原识别区为单链抗体(一价或多价)。单链抗体scFv包括重链可变区和轻链可变区，重链可变区和轻链可变区通过Linker(接头)连接而成的抗体。
优选地，scFv重链和轻链的连接方式为VH-Linker-VL或VL-Linker-VH。在一些实施方案中，Linker的序列可选用现有的接头序列。还优选地，Linker的序列是(GGGGS)n，其中n＝1-6。进一步优选地，Linker的序列为GGGGSGGGGSGGGGS。
优选地，所述CAR还包括前导信号肽序列。一般而言，信号肽是使多肽靶向细胞中的所需部位的肽序列。在一些实施方案中，信号肽使多肽靶向细胞的分泌通路，并且将允许多肽整合和锚定至脂双层。在一些实施方案中，信号肽为膜定位信号肽。优选地，所述前导肽序列来源于CD8的前导肽序列；更优选地，所述CD8前导肽序列具有SEQ ID NO:36所示的氨基酸序列。
本文中的“铰链区”、“跨膜区”和“胞内区”均可选自现有已知的CAR-T技术中的铰链区、跨膜区和胞内区的序列。
嵌合抗原受体的铰链区位于胞外抗原结合区和跨膜区之间，铰链区是通常在蛋白质的两个域之间存在的氨基酸区段，并且可以允许蛋白质的柔性和两个域的彼此相对运动。铰链区可以是天然存在的蛋白质的铰链区或其部分。抗体(诸如IgG、IgA、IgM、IgE或IgD抗体)的铰链区也可用于本文所述的嵌合抗原受体。非天然存在的肽也可用作本文所述的嵌合抗原受体的铰链区。在一些实施方案中，铰链区是肽接头。优选地，所述铰链区来源于CD8α。还优选地，所述CD8α铰链区具有SEQ ID NO:38所示的氨基酸序列。
嵌合抗体受体的跨膜区可以形成α螺旋、多于一个α螺旋的复合物、β桶或能够跨域细 胞磷脂双层的任何其它稳定结构。跨膜区可以是天然或合成来源的。跨膜区可源自CD3ε、CD4、CD5、CD8α、CD9、CD16、CD22、CD28、CD33、CD37、CD45、CD64、CD80、CD86、CD134、CD137、CD154、T细胞受体的α、β或ζ链。优选地，所述跨膜区来源于CD8α。优选地，所述CD8α跨膜区具有SEQ ID NO:40所示的氨基酸序列。
优选地，嵌合抗原受体的胞内区包含信号传导区和/或共刺激信号传导区。信号传导区和/或共刺激信号传导区的个数均可以为一个或多个。
胞内信号传导区负责表达嵌合抗原受体的免疫效应细胞的至少一种正常效应子功能的活化。例如，T细胞的效应子功能可以是细胞裂解活性或辅助活性，包括细胞因子的分泌。虽然通常可以利用整个胞内信号传导区，但是在很多情况下，使用整个链是不必要的。就使用胞内信号传导区的截短部分而言，只要其转导效应子功能信号，就可以使用这种截短部分代替完整链。因此，胞内信号传导区包括足以转导效应子功能信号的胞内信号传导区的任何截短形式。在一些实施方案中，信号传导区来源于CD3ζ、FcRγ(FCER1G)、FcRβ(FcεRib)、CD3γ、CD3δ、CD3ε、CD5、CD22、CD137、CD79a、CD79b和CD66d中的至少一种。优选地，所述胞内区来源于人CD3ζ胞内区。进一步地，所述人CD3ζ胞内区具有SEQ ID NO:44所示的氨基酸序列。
在抗原特异性信号的刺激以外，很多免疫效应细胞还需要共刺激来促进细胞增殖、分化和存活，以及活化细胞的效应子功能。“共刺激信号传导区”可以是共刺激分子的胞质部分。术语“共刺激分子”是指免疫细胞(诸如T细胞)上的关联结合伴侣，该关联结合伴侣与共刺激配体特异性结合，从而由免疫细胞介导共刺激响应，诸如但不限于增殖和存活。共刺激信号传导区可源自CARD11，CD2，CD7，CD27，CD28，CD30，CD40，CD54，CD83，OX40，CD137，CD134，CD150，CD152，CD223，CD270，PD-L2，PD-L1，CD278，DAP10，LAT，NKD2C，SLP76，TRIM，FcεRIγ，MyD88和4-1BB中至少一种的胞内信号区。在一些实施方案中，共刺激信号传导区来源于4-1BB。在一些实施方案中，4-1BB共刺激信号传导区包含SEQ ID NO：42所示的氨基酸序列。
优选地，所述CAR的核苷酸序列如SEQ ID NO:20所示，其编码序列如SEQ ID NO:21所示。
为了解决CAR-T细胞治疗伴随的多种毒副作用，增加CAR-T细胞治疗的安全性，本 发明人设计的嵌合抗原受体CAR进一步包括“自杀开关”RQR8分子，其具有SEQ ID NO:47所示的氨基酸序列，其编码序列如SEQ ID NO:48所示。所述RQR8分子以具自剪切功能的T2A连接肽与CD7-CAR结构中的胞内信号传导结构域CD3ζ相融合。优选地，所述RQR8分子带有两个CD20抗原表位，使用抗CD20的利妥昔单抗(Rituximab)靶向CD20，激活抗体依赖性细胞介导的细胞毒作用(ADCC)和补体介导的细胞毒作用(CDC)，可诱导T细胞凋亡。在必要的时候利用例如利妥昔单抗，可实现对CAR-T细胞的消除，从而增加CAR-T细胞治疗的安全性。
本发明提供了一种分离的核酸，其编码如前所述的抗体或其抗原结合片段，或阻断分子，或嵌合抗原受体。
本发明提供了一种载体，其包括本发明所述的分离的核酸。载体可以为表达载体或克隆载体。在一些实施方案中，载体为病毒载体。病毒载体包括但不限于腺病毒载体、腺相关病毒载体、慢病毒载体、逆转录病毒载体、牛痘载体、单纯疱疹病毒载体及其衍生物。
本发明提供了一种宿主细胞，其包括上述的载体。用于克隆或表达DNA的合适宿主细胞是原核细胞、酵母细胞或高等真核细胞。常用的原核宿主细胞的例子包括大肠杆菌、枯草杆菌等。常用的真核宿主细胞包括酵母细胞、昆虫细胞、哺乳动物细胞等。
本发明提供了一种抗人CD7的嵌合抗原受体CAR的制备方法，其包括培养上述的宿主细胞。优选地，所述制备方法的培养条件足以使宿主细胞能够表达抗人CD7的嵌合抗原受体CAR。
本发明提供了一种免疫效应细胞，其表达上述的特异性结合CD7的抗体或其抗原结合片段，或阻断分子，或抗人CD7的嵌合抗原受体CAR。
本发明中，“免疫效应细胞”是可执行免疫效应功能的免疫细胞。在一些实施方案中，免疫效应细胞表达至少FcγRIII并执行ADCC效应子功能。介导ADCC的免疫效应细胞的实例包括外周血单个核细胞(PBMC)、天然杀伤(NK)细胞、单核细胞、细胞毒性T细胞、中性粒细胞和嗜酸性粒细胞。优选地，所述免疫效应细胞选自：由多能干细胞或胚胎干细胞培养分化的免疫细胞、T淋巴细胞、NK细胞、外周血单个核细胞(PBMC)和造血干细胞中的至少一种。更优选地，所述免疫效应细胞为T淋巴细胞(同T细胞)。在一些实施方案 中，T细胞可以为CD4+/CD8-、CD4-/CD8+、CD4+/CD8+、CD4-/CD8-或它们的组合。在一些实施方案中，T细胞在表达嵌合抗原受体并结合至靶细胞时产生IL-2、IFN和/或TNF。在一些实施方案中，CD8+T细胞在表达嵌合抗原受体并结合至靶细胞时裂解抗原特异性靶细胞。
本发明提供了所述的免疫效应细胞的制备方法，其包括采用本发明所述的分离的核酸或所述的载体感染免疫效应细胞。优选地，本发明通过将嵌合抗原受体引入免疫效应细胞(诸如T细胞)来制备经基因改造的免疫效应细胞。
需要说明的是，将核酸或载体引入哺乳动物细胞的方法是本领域已知的，所述载体可以通过物理、化学或生物方法转入免疫效应细胞。用于将载体引入免疫效应细胞的物理方法包括磷酸钙沉淀、脂质体转染、粒子轰击、显微注射、电穿孔等等。用于将核酸或载体引入免疫效应细胞的化学手段包括胶体分散体系，诸如大分子复合物、纳米胶囊、微球体、珠粒和基于脂质的体系(包括水包油乳液、胶束、混合胶束和脂质体)。用作体外递送媒介物的示例性胶体体系是脂质体(例如人工膜囊泡)。用于将核酸或载体引入免疫效应细胞的生物方法包括使用DNA和RNA载体。病毒载体已成为将基因插入哺乳动物，例如人细胞的最广泛使用的方法。在一些实施方案中，转导的或转染的免疫效应细胞在引入核酸或载体之后离体繁殖。
在一些实施方案中，所述制备还包括进一步评估或筛选转导的或转染的免疫效应细胞以选择经改造的免疫效应细胞。
本发明进一步提供了一种药物或药物组合物，其包括：所述的特异性结合CD7的抗体或其抗原结合片段、所述的分离的核酸、所述的载体、所述的嵌合抗原受体CAR、所述的分离的核酸、所述嵌合抗原受体CAR的制备方法制备获得的抗人CD7的嵌合抗原受体CAR、所述的免疫效应细胞以及所述的免疫效应细胞的制备方法制备获得的免疫效应细胞中的至少一种。
在一些实施方案中，所述药物组合物还包括药学上可接受的载体。
药物组合物可以通过使具有所需纯度的活性药剂与任选的药学上可接受的载剂混合以冻干制剂或水溶液的形式制备。药学上可接受的载剂在所用的剂量和浓度下对接受者是无毒的，可包括缓冲剂、抗氧化剂、防腐剂、等渗剂、稳定剂和表面活性剂中的至少一 种。此外，为了使药物组合物可用于体内施用，它们必须是无菌的。可以通过无菌过滤膜过滤使药物组合物无菌。
在一些实施方案中，药物组合物可以含有：细胞毒性剂、化学治疗剂、细胞因子、免疫抑制剂、生长抑制剂以及待治疗的具体适应症所需的活性药剂中的至少一种添加剂。添加剂的具体添加量可根据实际需要进行调整。
本发明还提供了试剂在制备用于治疗或改善癌症的药物或药物组合物中的应用，所述试剂选自：所述的特异性结合CD7的抗体或其抗原结合片段、所述的分离的核酸、所述的载体、所述的宿主细胞、所述的抗人CD7的嵌合抗原受体CAR、所述的抗人CD7的嵌合抗原受体CAR的制备方法制备获得的抗人CD7的嵌合抗原受体CAR、所述的免疫效应细胞以及所述的免疫效应细胞的制备方法制备获得的免疫效应细胞中的至少一种。
优选地，所述治疗或改善癌症是指能够激发或提高癌症患者的免疫功能。
优选地，所述癌症是指与CD7表达相关的癌症。
本文中“与CD7表达相关的癌症”是指由CD7表达异常所直接或间接导致的疾病，通常是指由CD7过表达所导致的疾病。优选地，所述癌症或肿瘤是血液系统恶性肿瘤。进一步优选地，所述血液系统恶性肿瘤为T细胞相关肿瘤，所述T细胞相关肿瘤包括白血病和淋巴瘤、骨髓瘤。
本发明还提供一种治疗/与预防癌症的方法，其包括向有需要的受试者施用治疗有效量的药物的步骤，其中所述药物包括：所述的特异性结合CD7的抗体或其抗原结合片段、所述的分离的核酸、所述的载体、所述的宿主细胞、所述的抗人CD7的嵌合抗原受体CAR、所述的抗人CD7的嵌合抗原受体CAR的制备方法制备获得的抗人CD7的嵌合抗原受体CAR、所述的免疫效应细胞以及所述的免疫效应细胞的制备方法制备获得的免疫效应细胞中的至少一种。
本文所用术语“受试者”和“患者”在本文中互换地用于指可能需要本文描述的抗体相关制剂或药物、治疗的任何动物。受试者和患者因此包括但不限于：灵长类动物(包括人类)、犬科动物、猫科动物、鼠和其它哺乳动物受试者。优选地，所述受试者是人类。
在本发明中，术语“治疗”是指治疗性治疗和预防性或防治性措施，其目的是预防或减缓(减少)不期望发生的生理改变或紊乱，例如自身免疫性疾病的进程。有益的或期望的临 床结果包括但不限于以下无论是可检测还是不可检测的结果，包括症状的缓解、疾病程度的减小、疾病状态的稳定(即不恶化)、疾病进展的延迟或减缓、疾病状态的改善或缓和以及减轻(无论是部分还是全部)。“治疗”还意指与不接受治疗时预期的生存期限相比所延长的生存期限。需要治疗的包括那些已经患有病症或紊乱的人，以及那些容易患有病症或紊乱的人，或者那些需要预防该病症或紊乱的人。
本文所用术语“有效量”表示引发例如研究者或临床医师所追求的组织、系统、动物或人的生物学或药学响应的药物或药剂的量。此外，术语“治疗有效量”表示与没有接受该量的相应受试者相比，引起疾病、病症或副作用的改进治疗、治愈、预防或减轻的量，或者使疾病或病况的进展速率降低的量。该术语在其范围内还包括有效增强正常生理功能的量。通常，本文中的有效量根据各种因素而变化，所述因素例如给定的药物或化合物、药学制剂、给药途径、疾病或病症的类型、被治疗的受试者等等，但仍然可以由本领域技术人员常规地确定。本发明的化合物的有效量可以由本领域技术人员通过本领域已知的常规方法容易地确定。
本发明还提供根据本发明所述的抗体或其抗原结合片段，或所述的阻断分子，或所述的嵌合抗原受体，或所述的免疫效应细胞在与其它药物联合用药中的用途。优选地，所述其它药物包括诊断剂、预防剂和/或治疗剂。进一步优选地，所述其它药物为靶向CD20抗体药物，所述靶向CD20抗体药物包括但不限于：利妥昔单抗、阿托珠单抗、奥法木单抗、替伊莫单抗等。
实施例1
本实施例为针对CD7抗原的小鼠单克隆抗体制备。
本实施例针对CD7抗原设计并合成5条多肽分别对BALB/c小鼠进行免疫，经过细胞融合和初筛、复筛，获得1株同时识别多肽BST001-2和CD7重组蛋白的阳性克隆，该阳性杂交瘤细胞株的克隆编号为5B5。
CD7抗原前体蛋白的氨基酸序列如SEQ ID NO：1所示，其中氨基酸残基26-180为CD7抗原胞外结构域。所述CD7重组蛋白为重组人CD7蛋白(带his标签，为百普赛斯产品，货号为11028-H08H)，其氨基酸序列为CD7抗原胞外结构域序列，如SEQ ID NO： 2所示。
用于免疫小鼠的5条合成的抗原多肽的氨基酸序列分别对应CD7抗原胞外结构域中的5段氨基酸残基，具体序列如表1所示：
表1. 5条合成多肽的氨基酸序列
多肽编号 多肽序列 对应序列表中的序列号
BST001-1 AQEVQQSPHC SEQ ID NO：3
BST001-2 CEDGVVPTTDRRFRGRID SEQ ID NO：4
BST001-3 TEEQSQGWHRC SEQ ID NO：5
BST001-4 HRLQLSDTGTYTC SEQ ID NO：6
BST001-5 CPPTGSALPD SEQ ID NO：7
5条多肽中，BST001-2和BST001-5的N端第一个氨基酸(表中粗体)为添加的半胱氨酸(Cys)，多肽BST001-1、BST001-3和BST001-4的C末端氨基酸本身为Cys。5条多肽分别通过N端或C端的Cys残基与载体蛋白KLH(Keyhole Limpet Hemocyanin，血蓝蛋白)偶联，作为免疫原分别免疫一组小鼠。对免疫小鼠进行尾血检测和单克隆抗体筛选。
本发明的小鼠单克隆抗体为以多肽BST001-2作为免疫原进行免疫获得的识别多肽BST001-2和CD7抗原蛋白的单克隆抗体，下面以多肽BST001-2为例具体描述。
1.1小鼠的免疫及免疫反应评价
使用BST001-2-KLH，分别与佐剂CFA和AD11.15混合，制备免疫原，免疫3只BALB/c小鼠，免疫后在第14天抽取小鼠尾血，使用间接ELISA方法进行血清中抗体效价的评价。
所述间接ELISA方法的具体操作步骤为：
(1)使用BST001-2多肽包被酶标板：每孔加入BST001-2多肽100μL(浓度1μg/mL)，4℃过夜反应；
(2)使用PBS溶液洗板3次，使用5％Milk-PBS在室温封闭1hr；
(3)使用PBS溶液洗板1次，加入使用5％milk-PBS溶液梯度稀释的小鼠尾血，室温反应1hr；
(4)使用PBS溶液洗板3次，并进行拍干后，加入1:2000稀释的HRP标记的羊抗小鼠IgG(Fc)二抗，室温反应1hr；
(5)PBS溶液洗板5次后，拍干，加入等体积的A液和B液，避光、室温条件下反 应20min；
(6)加入50μL终止液，混匀后在酶标仪上读取OD 450值。
免疫后第14天的小鼠尾血的间接ELISA评价结果见表2。从结果中看出，3只小鼠尾血抗体效价都到达了1:10000以上，后续选择1#小鼠进行下述步骤1.2的细胞融合。
表2.免疫后第14天小鼠尾血抗体效价ELISA评价
Figure PCTCN2023070315-appb-000001
注：阴性对照NC为5％milk-PBS。
1.2细胞融合及杂交瘤细胞株的筛选
根据表2中小鼠尾血的ELISA评价结果，于免疫后第21天选择1#小鼠的脾细胞与骨髓瘤细胞SP2/0进行细胞融合。融合后第10天挑取564个单克隆细胞在96孔板中进行培养，培养7天后，按照步骤1.1所描述的间接ELISA方法对96孔板中的564个单克隆细胞培养上清进行评价，筛选能够分泌识别CD7抗原多肽的单克隆抗体的杂交瘤细胞株。从中初步挑选了7个阳性克隆株。
按照步骤1.1所描述的间接ELISA方法对该7个阳性克隆株进一步进行了复筛实验,结果如表3所示。
表3.小鼠融合阳性克隆的ELISA复筛确认
Figure PCTCN2023070315-appb-000002
注：阴性对照NC为5％Milk-PBS；阳性对照PC为1#小鼠心脏血，1:500稀释使用。
从表3的复筛结果中，获得了识别BST001-2多肽的强阳性杂交瘤细胞株5B5。
1.3对强阳性杂交瘤细胞株5B5的再确认
采用步骤1.1所描述的间接ELISA方法，对该编号为5B5的强阳性杂交瘤克隆株的 扩大培养后的上清再次进行确认实验，结果如表4所示。
表4. 5B5阳性克隆的ELISA重复确认
5B5 5B5 NC NC PC PC
2.677 2.749 0.316 0.107 2.767 2.677
注：NC为阴性对照5％Milk-PBS；PC为阳性对照1#小鼠心脏血，1:500稀释使用。
进一步采用步骤1.1所描述的间接ELISA方法，以前述的CD7重组蛋白(带his标签)代替BST001-2多肽包被酶标板，对5B5克隆株的细胞上清进行检测，结果如表5所示。
表5. 5B5阳性克隆的竞争性ELISA检测
5B5 NC PC
2.782 0.047 2.636
2.759 0.043 2.629
注：阴性对照NC为5％Milk-PBS；阳性对照PC为抗CD7重组蛋白的His标签的小鼠单抗。
从上述表4和表5的结果可以看出，5B5克隆株的细胞上清与CD7的BST001-2多肽抗原和CD7重组蛋白抗原均产生强烈识别反应，5B5即为所需要的能产生抗CD7单克隆抗体的杂交瘤细胞株。
1.4杂交瘤细胞株5B5单克隆抗体可变区的克隆与测序
培养5B5杂交瘤细胞株，收集细胞，提取RNA，采用RT-PCR法获得编码抗CD7的单克隆抗体的cDNA序列，再通过PCR方法克隆重链和轻链的可变区并将PCR产物连接到T-载体上，测序获得抗CD7单抗的重链可变区VH和轻链可变区VL的序列，并进一步通过Uniprot数据库进行序列比对和确认。
所得VH的核苷酸序列如SEQ ID NO：8所示，其编码的氨基酸序列如SEQ ID NO：9所示；所得VL的核苷酸序列如SEQ ID NO：10所示，其编码的氨基酸序列如SEQ ID NO：11所示。
进一步对所得VH和VL的氨基酸序列进行分析，找出其中的互补决定区(CDR)，结果如表6所示。
表6.抗CD7的5B5单克隆抗体的VH和VL结构域的CDR分析
Figure PCTCN2023070315-appb-000003
Figure PCTCN2023070315-appb-000004
实施例2
本实施例为CD7-Blocker及CD7-CAR慢病毒表达载体的构建。
利用本领域的常规技术手段将CD7阻断分子CD7-Blocker及CD7-CAR分别构建到第三代慢病毒表达质粒中。所述质粒为pCDH-EF1(X6)-MCS-T2A-Puro，其图谱如图1所示，载体线性化酶切位点为XbaI和SalI，CD7-Blocker及CD7-CAR的DNA序列(含N端KOZAC序列)被插入该两酶切位点之间。CD7-Blocker的分子结构如图2所示，CD7-Blocker分子由CD8a信号肽SP、(G4S)3接头连接VL和VH组成的anti-CD7 scFv、ER Retention Domain(内质网定位结构域)三部分构成，SP前端插入了促进表达的KOZAK序列。其全长氨基酸序列如SEQ ID NO：18所示，其全长DNA序列如SEQ ID NO：19(含N端KOZAC序列)所示。
CD7-CAR的分子结构如图3所示，其由CD8a信号肽SP、(G4S)3接头连接VL和VH而成的anti-CD7 scFv、CD8a铰链区、CD8a跨膜区以及4-1BB和CD3ζ两个胞内信号传导结构域构成CD7-CAR分子的主体部分，其后以自剪切T2A连接肽融合一细胞“自杀开关”RQR8分子，SP前端也插入了促进表达的KOZAK序列。其全长氨基酸序列如SEQ ID NO:20所示，其全长DNA序列如SEQ ID NO：21(含N端KOZAC序列)所示。CD7-Blocker和CD7-CAR两种分子结构中的VH和VL的核苷酸编码序列，分别如SEQ ID NO：22和SEQ ID NO：23所示，是分别由鼠源的VH序列(SEQ ID NO：8)和VL序列(SEQ ID NO：10)按人源密码子优化而成的。VL与VH通过接头(G4S)3融合而成anti-CD7 scFv。
CD7-Blocker由anti-CD7 scFv与ER定位信号KDEL通过接头(G4S)2连接而成，(G4S)2-KDEL构成ER Retention Domain(内质网定位结构域)。ER Retention Domain能将anti-CD7 scFv定位到ER网腔，CD7抗原分子在分泌途径被ER网腔内的anti-CD7 scFv拦截，因而不能正常定位到细胞表面，从而可避免CD7-CAR-T细胞之间的“自相残杀”。 在另外的实施例中，连接anti-CD7 scFv与ER定位信号的接头为myc序列EQKLISEEDL(SEQ ID NO:33)。
CD7-Blocker及CD7-CAR分子结构中各片段的氨基酸和核苷酸序列所对应的序列编号如表7所示，其中SP为CD8a信号肽，CD8H为CD8a铰链区，CD8TM为CD8a跨膜区，4-1BB和CD3ζ均为胞内信号传导结构域。
表7.CD7-Blocker及CD7-CAR分子结构中各片段序列对应表
片段名称 氨基酸序列 核苷酸序列
anti-CD7 scFv的VH结构域 SEQ ID NO:9 SEQ ID NO:22
anti-CD7 scFv的VL结构域 SEQ ID NO:11 SEQ ID NO:23
KOZAK / SEQ ID NO:24
(G4S)3接头 SEQ ID NO:25 SEQ ID NO:26
ER定位信号KDEL SEQ ID NO:27 SEQ ID NO:28
(G4S)2接头 SEQ ID NO:29 SEQ ID NO:30
ER Retention Domain SEQ ID NO:31 SEQ ID NO:32
Myc接头 SEQ ID NO:33 SEQ ID NO:34
SP(CD8a信号肽) SEQ ID NO:35 SEQ ID NO:36
CD8H(CD8a铰链区) SEQ ID NO:37 SEQ ID NO:38
CD8TM(CD8a跨膜区) SEQ ID NO:39 SEQ ID NO:40
4-1BB SEQ ID NO:41 SEQ ID NO:42
CD3ζ SEQ ID NO:43 SEQ ID NO:44
T2A SEQ ID NO:45 SEQ ID NO:46
RQR8 SEQ ID NO:47 SEQ ID NO:48
CD20 Epitope SEQ ID NO:49 SEQ ID NO:50
CAR-T细胞治疗通常会伴随多种毒副作用，为了增加CAR-T细胞治疗的安全性，本发明在CD7-CAR分子结构里融入了“自杀开关”RQR8分子(SEQ ID NO:47，SEQ ID NO:48)，该RQR8分子以具自剪切功能的T2A连接肽与CD7-CAR结构中的胞内信号传导结构域CD3ζ相融合。该RQR8分子带有两个CD20抗原表位，使用抗CD20的利妥昔单抗(Rituximab)靶向CD20，激活抗体依赖性细胞介导的细胞毒作用(ADCC)和补体介导的细胞毒作用(CDC)，可诱导T细胞凋亡。在必要的时候利用利妥昔单抗，可实现对CAR-T细胞的消除，从而增加CAR-T细胞治疗的安全性。
慢病毒包装采用本领域的常规四质粒体系，其中三种辅助质粒为pMDLg/pRRE、pRSV-Rev和pMD2.G。采用293T细胞作为慢病毒包装细胞。携带CD7-Blocker或CD7-CAR的慢病毒表达质粒与pMDLg/pRRE、pRSV-Rev和pMD2.G的共转染293T细胞的 质粒用量比例为7.5:9:9:3.5；对于T75细胞培养瓶，则四种质粒用量分别为7.5ug、9ug、9ug、3.5ug。转染试剂PEI的用量(ug)为四种质粒总量的3倍，对于T75培养瓶，PEI用量为87ug(1ug/ul,87ul)。
四种质粒共转染293T后48小时收取细胞培养液，离心(2000rpm，15min)后取上清，经0.45um滤器过滤后，采用超速离心(20000rpm，2h)浓缩上清，然后根据稀释倍数用相应体积的培养基重悬病毒沉淀，分装，置于-80℃冻存。
对于CD7-Blocker慢病毒，采用慢病毒载体HIV P24快速检测卡直接测出慢病毒的滴度。对于CD7-CAR慢病毒的滴度测定，是将慢病毒进行系列梯度稀释后转染293T细胞，48h后流式检测转染效率，计算得出慢病毒的活性滴度。
实施例3
本实施例为Anti-CD7 scFv与CD7抗原分子的亲和力鉴定。
利用CD7-CAR慢病毒转导293T细胞，流式检测293T细胞中的CD7-CAR阳性率以及与CD7抗原蛋白结合的293T细胞比率，计算出CD7-CAR-293T细胞中CD7-CAR与CD7抗原蛋白的亲和率，用以表示Anti-CD7 scFv与CD7抗原分子的亲和力。
所述CD7抗原蛋白为实施例1中的带His标签的重组人CD7蛋白，流式检测时首先用CD7抗原蛋白与CD7-CAR-293T细胞孵育，再用荧光标记的抗His小鼠单抗检测与293T细胞结合的CD7抗原蛋白。
对CD7-CAR慢病毒转导的293T细胞先后进行了两次流式检测，结果如表8所示。
表8.Anti-CD7 scFv与CD7抗原分子的亲和力检测
Figure PCTCN2023070315-appb-000005
表8的结果显示，CD7-CAR与CD7抗原分子的平均亲和率99.51％，表明anti-CD7 scFv与CD7抗原分子具有极强的亲和力。
实施例4
本实施例为CD7-Blocker对Jurkat细胞表面CD7分子的阻断效果。
将CD7-Blocker慢病毒转导入Jurkat细胞，4天后通过流式细胞术检测Jurkat细胞表面CD7分子的阳性率，结果如表9和图4所示。未经导入CD7-Blocker的对照Jurkat细胞表面CD7分子阳性率为92.45％；在转导病毒MOI＝10时，Jurkat细胞表面CD7分子阳性率降为0.09％(见图4D)，此时D7 Blocker对Jurkat细胞表面CD7分子的阻断效率达到极限值99.90％。在MOI＝5时(见图4C)，阻断效率已接近极限，继续提高MOI值至15(见图4E)对阻断效率提高效应不明显。这些结果表明，CD7-Blocker几乎能完全阻断CD7分子在Jurkat细胞表面的表达。
表9.CD7-Blocker对Jurkat细胞表面CD7分子的阻断效率
转导MOI 0 2.5 5 10 15
CD7阳性率(％) 92.45 2.3 0.58 0.09 0.14
CD7阳性率流式图 图4A 图4B 图4C 图4D 图4E
CD7阻断效率(％)   97.5 99.37 99.90 99.89
实施例5
本实施例为CD7-Blocker对T细胞表面CD7分子的阻断效果。
将冻存的PBMC复苏后加CD3/CD28抗体磁珠激活(激活时间点记为D0)，激活后1天(D1)转染CD7-Blocker慢病毒(B-T实验组)，激活后2天(D2)转染CD7 CAR慢病毒。实验共分为四组：未转染对照组(NT组)，只转染CD7-Blocker病毒(B-T组)，在NT的基础上共转染CD7-Blocker和CD7 CAR病毒(B-CAR-T组)，只转染CD7 CAR病毒(CAR-T组)。病毒转染的MOI值均为5。转染后于D6(激活后6天、转染Blocker病毒后5天、转染CAR病毒后4天)流式检测细胞表面CD7抗原分子的阳性率，结果详见表10和图5。
可以看出，在本实施例实验条件下，CD7-Blocker(B-T组)对CD7分子的阻断效率达91.12％，CD7-CAR单独(CAR-T组)对CD7分子的细胞表面表达几乎不影响，CD7-Blocker&CD7-CAR(B-CAR-T组)对CD7分子的阻断效率达93.67％。
表10.CD7-Blocker对T细胞表面CD7分子的阻断效率
  NT B-T B-CAR-T CAR-T
CD7阳性率(％) 94.44 8.34 5.98 93.58
CD7阳性率流式图 图5A 图5B 图5C 图5C
CD7阻断效率(％)   91.12 93.67 0.91
实施例6
本实施例为CD7-Blocker对T细胞扩增的影响。
本实施例检测并比较了未转染CD7-Blocker病毒的对照T细胞(NT组)、只转染了CD7-Blocker慢病毒的CD7-Blocker-T细胞(B-T组)、只转染了CD7-CAR慢病毒的CD7-CAR-T细胞(CAR-T组)以及同时转染了CD7-Blocker病毒和CD7-CAR病毒的CD7-Blocker-CAR-T细胞(B-CAR-T组)等四组T细胞的体外扩增情况。各组细胞病毒转染时间点如实施例4。
培养时间说明：冻存的PBMC复苏培养一天后加入CD3/CD28抗体磁珠激活，激活时间点记为D0，激活后1天、2天的时间点分别记为D1、D2，以此类推。四组T细胞的表面CD7分子阳性率、CAR阳性率如表11及图6所示，细胞扩增倍数如表12所示，细胞扩增曲线如图7所示。
这些结果表明，与对照T细胞相比较，单独转染CD7-Blocker病毒的B-T以及共转染了CD7-Blocker病毒和CD7-CAR病毒的B-CAR-T细胞，都可以正常扩增，而只转染了CD7-CAR慢病毒的CAR-T在体外很难扩增。这种现象说明，阻断CD7分子后基本不会影响T细胞的正常扩增，而未经CD7分子阻断的CAR-T细胞表面由于表达CD7分子，会导致CD7-CAR-T细胞的自相残杀。
表11.CD7分子阳性率与CAR阳性率
Figure PCTCN2023070315-appb-000006
表12.细胞扩增倍数(相对于D2)
Day 2 6 7 9 12
NT 1.00 13.85 17.04 36.46 33.91
B-T 1.00 9.12 10.12 14.88 23.81
B-CAR-T 1.00 5.08 6.05 11.62 20.92
实施例7
本实施例为CD7-CAR-T细胞对CD7阳性靶细胞的体外杀伤实验。
为了进一步验证CD7-CAR-T细胞对CD7阳性靶细胞杀伤的特异性，利用CD7阴性的U87细胞构建了CD7过表达的U87-CD7-eGFP细胞株，利用RTCA仪器分析了CD7-CAR-T细胞对CD7阳性靶细胞U87-CD7-eGFP的杀伤作用。
构建U87-CD7-eGFP细胞株所采用的CD7抗原分子编码序列为CD7抗原前体蛋白的DNA编码序列(SEQ ID NO：51)，所采用的eGFP分子的氨基酸序列如SEQ ID NO:52所示，其DNA编码序列如SEQ ID NO:53所示，CD7分子与eGFP分子之间由自剪切连接肽T2A(SEQ ID NO:45，SEQ ID NO:46)连接。CD7-T2A-eGFP结构N端添加KOZAK序列后插入到慢病毒载体pCDH-EF1(X6)-MCS-T2A-Puro的XbaI和SalI两酶切位点之间，构建成CD7过表达慢病毒载体。按照常规手段将慢病毒CD7-T2A-eGFP转导入U87细胞,eGFP用作转导后细胞的筛选和检测标记。
由于CD7-CAR-T细胞具有自相残杀现象，杀伤实验的效应细胞采用的是经CD7-Blocker阻断了CD7分子表面表达的CD7-Blocker-CAR-T细胞(简称B-CAR-T)，其CAR阳性率为45.79％，表面CD7分子阳性率为5.97％。杀伤实验的对照T细胞为CD7-Blocker-T(简称B-T)细胞，其表面CD7分子阳性率为8.35％。未经阻断的T细胞CD7表面分子阳性率为94.44％。上述阳性率数据如实施例6中的表11和图6所示。
杀伤实验曲线如图8和图9所示。时间点0.0为靶细胞培养开始的起点，在靶细胞培养28h时加入效应细胞共培养，共培养效靶比为B-CAR-T(或对照B-T)：U87-CD7-eGFP(或对照U87)＝4：1。整个实验持续96h。
图8为两种T细胞与阳性性靶细胞U87-CD7-eGFP共培养的杀伤曲线，可以看出，只有B-CAR-T对CD7阳性靶细胞U87-CD7-eGFP具有显著的杀伤作用。图9为两种T细胞与阴性靶细胞U87共培养的杀伤曲线，可以看出，B-CAR-T对U87只有轻微的杀伤作用。这些结果表明，经CD7 Blocker阻断了CD7表面分子的CD7-CAR-T细胞对CD7阳性靶细胞具有显著且特异的杀伤作用，本发明为下一步的细胞治疗临床应用提供了有益的CAR-T细胞。
为了进一步分析B-CAR-T对靶细胞的杀伤效率，截取了共培养的早期时间段两端的 细胞指数值(Cell Index at:60:04:20&at:30:33:52)，得到的杀伤效率如图10所示，B-T组为对照CD7-Blocker-T细胞与U87-CD7-eGFP细胞共培养，BC-T组为CD7-Blocker-CAR-T细胞与U87-CD7-eGFP细胞共培养，可以看出，在E:T＝4:1时，与对照B-T细胞相比，B-CAR-T对CD7阳性靶细胞具有很高的杀伤效率。
尽管本发明已经参考示例性实施方案进行了描述，但应理解本发明不限于公开的示例性实施方案。在不背离本发明的范围或精神的情况下，可对本发明说明书的示例性实施方案做多种调整或变化。权利要求的范围应基于最宽的解释以涵盖所有修改和等同结构与功能。

Claims (10)

  1. 抗体或其抗原结合片段，其特征在于，其含有具有SEQ ID NO.:12-14所示氨基酸序列的抗原互补决定区CDR1、CDR2和CDR3的重链可变区；和
    具有SEQ ID NO.:15-17所示氨基酸序列的抗原互补决定区CDR1、CDR2和CDR3的轻链可变区。
  2. 根据权利要求1所述的抗体或其抗原结合片段，其特征在于，所述抗体具有(I)、(II)或(III)所示的氨基酸序列中的任意一个氨基酸序列：
    (I)SEQ ID NO：9所示的重链可变区氨基酸序列和SEQ ID NO：11所示的轻链可变区氨基酸序列；
    (II)与SEQ ID NO.:9和11所示的氨基酸序列具有至少90％，优选至少95％，还优选至少98％，最优选至少99％同源性的氨基酸序列；
    (III)与SEQ ID NO.:9和11所示的氨基酸序列经修饰、取代、缺失或添加一个或多于一个氨基酸获得的氨基酸序列；
    其中，所述氨基酸序列具有针对肿瘤表面抗原CD7的抗体的活性。
  3. 根据权利要求2所述的抗体或其抗原结合片段，其特征在于，其中所述抗体包括多克隆抗体、单克隆抗体、嵌合抗体、人源化抗体或双特异性抗体中的至少一种；所述抗原结合片段包括Fab片段、Fab’、F(ab’) 2片段、单链可变片段scFv、scFv-Fc片段或单链抗体ScAb中的至少一种。
  4. 一种CD7阻断分子，其特征在于，其包含：
    a.根据权利要求1-3中任一项所述的抗体或其抗原结合片段；和
    b.内质网定位结构域。
  5. 一种嵌合抗原受体，其特征在于，其包括：
    1)识别CD7抗原的抗原结合结构域，其中所述抗原结合结构域包括根据权利要求1-3任一项所述的抗体或其抗原结合片段；
    2)跨膜结构域；和
    3)胞内信号传导结构域；
    优选地，进一步包括铰链区；
    优选地，进一步包括自杀开关分子；
    优选地，进一步包括细胞内共刺激域；
    优选地，所述跨膜结构域选自：多肽CD28、NKp30、CDS、DAP10、4-1BB、DAP12、CD3C、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、KIRDS2、OX40、CD2、CD27、LFA-1、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、PAG/Cbp中的至少一种或其组合；
    优选地，所述胞内信号传导结构域选自：CD8、CD3ζ、CD3δ、CD3γ、CD3ε、FcγRI-γ、FcγRIII-γ、FcεRIβ、FcεRIγ、DAP10、DAP12、CD32、CD79a、CD79b、CD28、CD3C、CD4、b2c、CD137(4-1BB)、ICOS、CD27、CD28δ、CD80、NKp30、OX40中的至少一种或其组合。
  6. 一种分离的核酸分子，其特征在于，其编码根据权利要求1-3任一项所述的抗体或其抗原结合片段，或根据权利要求4所述的阻断分子，或根据权利要求5所述的嵌合抗原受体。
  7. 一种载体，其特征在于，其包含权利要求6所述的核酸分子。
  8. 一种宿主细胞，其特征在于，其包含根据权利要求7所述的载体。
  9. 一种免疫效应细胞，其特征在于，其表达根据权利要求1-3任一项所述的抗体或其抗原结合片段，或根据权利要求4所述的阻断分子，或根据权利要求5所述的嵌合抗原受体；
    优选地，所述免疫效应细胞选自：白细胞、单核细胞、巨噬细胞、树突细胞、肥大细胞、嗜中性粒细胞、嗜碱性粒细胞、嗜酸性粒细胞、αβT细胞、γδT细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、B细胞、天然淋巴样细胞(ILC)、细胞因子诱导的杀伤(CIK) 细胞、细胞毒性T淋巴细胞(CTL)、淋巴因子激活的杀伤(LAK)细胞、T淋巴细胞、外周血单个核细胞和造血干细胞中的至少一种。
  10. 试剂在用于制备预防和/或治疗癌症或肿瘤的药物中的用途，其特征在于，所述试剂包括：根据权利要求1-3任一项所述的抗体或其抗原结合片段，或根据权利要求4所述的阻断分子，或根据权利要求5所述的嵌合抗原受体，或根据权利要求6所述的核酸分子，或根据权利要求7所述的载体，或根据权利要求8所述的载体细胞，或根据权利要求9所述的免疫效应细胞；
    优选地，所述癌症或肿瘤是指CD7表达相关的癌症或肿瘤；
    还优选地，所述癌症或肿瘤是血液系统恶性肿瘤；
    进一步优选地，所述血液系统恶性肿瘤为T细胞相关肿瘤，所述T细胞相关肿瘤包括白血病、淋巴瘤、骨髓瘤；
    优选地，所述用途进一步包括：根据权利要求1-3任一项所述的抗体或其抗原结合片段，或根据权利要求4所述的阻断分子，或根据权利要求5所述的嵌合抗原受体，或根据权利要求9所述的免疫效应细胞在与其它药物联合用药中的用途；
    优选地，所述其它药物包括诊断剂、预防剂和/或治疗剂；
    还优选地，所述其它药物为靶向CD20抗体药物。
PCT/CN2023/070315 2022-02-28 2023-01-04 Cd7-car-t细胞及其制备方法和应用 WO2023160260A1 (zh)

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