WO2022151851A1 - 靶向cd7的工程化免疫细胞、嵌合抗原受体、cd7阻断分子及应用 - Google Patents

靶向cd7的工程化免疫细胞、嵌合抗原受体、cd7阻断分子及应用 Download PDF

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WO2022151851A1
WO2022151851A1 PCT/CN2021/133817 CN2021133817W WO2022151851A1 WO 2022151851 A1 WO2022151851 A1 WO 2022151851A1 CN 2021133817 W CN2021133817 W CN 2021133817W WO 2022151851 A1 WO2022151851 A1 WO 2022151851A1
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cells
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engineered immune
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WO2022151851A8 (zh
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张鸿声
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上海雅科生物科技有限公司
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Priority to JP2023542493A priority patent/JP2024502632A/ja
Priority to CA3204370A priority patent/CA3204370A1/en
Priority to AU2021418636A priority patent/AU2021418636A1/en
Priority to KR1020237027583A priority patent/KR20230134133A/ko
Publication of WO2022151851A1 publication Critical patent/WO2022151851A1/zh
Priority to US18/351,466 priority patent/US20240075143A1/en
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Definitions

  • the present invention relates to the technical field of cellular immunotherapy, in particular, to an immunotherapy method targeting CD7, in particular to an engineered immune cell targeting CD7, a chimeric antigen receptor, a CD7 blocking molecule and an application thereof.
  • T-cell malignancies are a group of highly heterogeneous clonal growth and T-cell dysfunction diseases, mainly divided into T-cell lymphomas (T-cell lymphomas, TCLs) and T-cell leukemias, with mature and precursor subtypes type.
  • T-cell lymphomas TCLs
  • T-cell leukemias T-cell leukemias
  • This class of diseases represents an extremely malignant group of hematological cancers with high recurrence and mortality rates in children and adults for which there are currently no effective or targeted treatments.
  • T-ALL T-cell acute lymphoblastic leukemia
  • salvage chemotherapy regimens are only able to induce remission in 20% to 40% of cases.
  • T-cell malignancies In patients with relapsed or chemotherapy-refractory T-cell malignancies, treatment outcomes are poor, and effective and tolerable treatments are limited. For 10-50% of patients who achieve complete remission (CR) after salvage chemotherapy, the only treatment option remains allogeneic stem cell transplantation (ASCT). However, ASCT cure rates remain at 30% or less, and not all CR patients are eligible for transplantation. Other T-cell malignancies, including cutaneous and peripheral T-cell lymphomas (CTCL and PTCL, respectively), have lower initial response rates to chemotherapy, with progression-free survival rates remaining at 40–50% even in responding patients.
  • CTCL and PTCL cutaneous and peripheral T-cell lymphomas
  • T-cell malignancies such as T-ALL and T-NHL
  • AML acute myeloid leukemia
  • Chimeric antigen receptor T (CAR-T) cells are one of the most promising tumor immunotherapy approaches, capable of producing significant response rates in patients with B lymphocyte malignancies.
  • CAR-T cells targeting CD19 an antigen widely expressed in B-cell leukemias and lymphomas, have become the first licensed cancer T-cell therapy.
  • CD19CAR-T cells in the treatment of relapsed and refractory B-cell malignancies has led to broader application in other tumors. Given the similarities between B-lymphocyte and T-lymphocyte malignancies, extending CAR-T cell therapy to these diseases seems straightforward. However, CAR-T cell therapy for T-cell malignancies has proven difficult to develop and implement.
  • CD7 is a transmembrane glycoprotein, usually expressed in most peripheral blood T cells and NK cells (NK cells are natural killer cells, derived from bone marrow lymphoid stem cells, and their differentiation and development depend on the bone marrow and thymus microenvironment, mainly distributed in Bone marrow, peripheral blood, liver, spleen, lung and lymph nodes; NK cells are different from T and B cells and are a type of lymphocytes that can non-specifically kill tumor cells and virus-infected cells without prior sensitization) and their precursor cells. , as a costimulatory protein for helper T cell activation and interaction with other immune subsets.
  • NK cells are natural killer cells, derived from bone marrow lymphoid stem cells, and their differentiation and development depend on the bone marrow and thymus microenvironment, mainly distributed in Bone marrow, peripheral blood, liver, spleen, lung and lymph nodes; NK cells are different from T and B cells and are a type of lymph
  • CD7-deficient T cells exhibit largely undisturbed developmental, homeostatic, and protective functions. Since CD7 has no significant effect on peripheral blood T cell function, it is a promising target for CAR-T cell therapy.
  • CD7 has been evaluated as a target of monoclonal antibodies (mAbs) using immunotoxins to treat patients with T-cell malignancies. This monoclonal antibody conjugate did not produce severe CD7-related toxic side effects, but the antitumor response was not significant, which may be due to the limited activity of murine antibodies in patient therapy.
  • CD7-CAR-T cell therapy T cells expressing chimeric antigen receptor (CAR) are a promising approach for tumor immunotherapy. This targeted therapy shows great potential in achieving remission and even long-term relapse-free survival in patients with B-cell leukemia and lymphoma.
  • CD7-specific CAR-T cell therapy in preclinical models of T-cell malignancies. In all these studies, the expression of CD7-CAR on T cells resulted in severe suicide, resulting in the inability of CAR-T cells to expand in vitro.
  • CD7 antigen-specific CAR-T cells can cause severe suicide during the preparation process, resulting in ineffective expansion of CAR-T cells.
  • Two strategies exist to reduce this suicide phenomenon 1) use genome editing tools to knock out genes for CD7 target antigens; 2) prevent CD7 protein from being transported during intracellular expression by anchoring the CD7-binding domain to the endoplasmic reticulum to the cell surface.
  • CD7-CAR-T cells Both approaches effectively reduced CD7 expression on the cell surface and minimized suicide by targeting CD7-CAR-T cells.
  • the deletion of CD7 did not affect the proliferation and short-term effector function of CAR-T cells, enabling the expansion of functional CAR-T cells with high antitumor activity.
  • CD7-CAR-T cells After removal of CD7 from the cell surface, CD7-CAR-T cells exhibited strong antitumor activity against primary CD7-positive T-ALL and lymphomas in vitro and in vivo. CD7-CAR-T cells are also cytotoxic to peripheral blood CD7-positive T cells and NK cells, suggesting that these cell subsets will also be targets of CD7-CAR-T cells.
  • allogeneic CD7-CAR-T cells produced by healthy donors can be used for the treatment of relapsed and refractory T-cell malignancies to achieve the purpose of bridging patients with hematopoietic stem cell transplantation, or for the treatment of post-hematopoietic stem cell transplantation.
  • Recurrent T-cell malignancy If graft-versus-host side effects can be properly controlled, the advantage of allogeneic CD7-CAR-T cell therapy is that in addition to T cells from healthy donors, it can also produce a graft-versus-leukemia effect.
  • Using readily available allogeneic T cells or NK cells as delivery vehicles completely avoids the risk of genetically modifying malignant cells. Because these cellular products will be produced from healthy donors, this avoids the use of patient-derived T cells, which are often dysfunctional due to prolonged exposure to a suppressive tumor microenvironment or from prior intensive therapy.
  • CD7 is expressed at high density (about 60,000 mol/cell) on T cells, and CD7 is rapidly internalized even when bound by monovalent antibody fragments. Therefore, it is an ideal target antigen for immunotoxin-mediated therapy of T-cell tumors.
  • anti-CD7 immunotoxins are mainly composed of anti-CD7 monoclonal antibodies coupled to toxins.
  • An anti-CD7 immunotoxin using a mouse anti-human CD7 monoclonal antibody (WTI) conjugated to ricin A has been used to deplete tumor cells in vitro for autologous bone marrow transplantation in patients with T-cell malignancies.
  • WTI mouse anti-human CD7 monoclonal antibody
  • the immunotoxin DA7 is constructed by chemically linking mouse IgG2b anti-CD7 (3AlE) monoclonal antibody and deglycosylated ricin A chain. It has been used in SCID animal model to treat human T-ALL, suggesting that its Potential therapeutic effect on T-cell leukemia with poor prognosis. In a phase I clinical trial of DA7, objective clinical responses were achieved at its maximum tolerated dose, although limited by instability and vascular toxicity.
  • Anti-human CD7 monoclonal antibody TH69 has also been used to construct recombinant immunotoxin, which is a single-chain antibody fragment (scFv) linked to a truncated Pseudomonas exotoxin A fragment by genetic recombination.
  • scFv single-chain antibody fragment linked to a truncated Pseudomonas exotoxin A fragment by genetic recombination.
  • the CD52 gene was also knocked out in the reinfused CAR-T cells, making them resistant to alemtuzumab.
  • the CRISPR/Cas9 system has been used to knock out CD7 or knock down the T cell receptor alpha chain at the same time as CD7, a new gene editing system that can quickly and efficiently knock out target genes in T cells.
  • Intracellular protein expression blocking technology in many biochemical and immune systems, it is often difficult to determine the role of some specific molecules, but one approach is to inhibit gene expression and look for its functional effects.
  • Gene knockout is performed by homologous recombination or recently developed gene editing techniques such as Zince Finger nucleases, TALENs and CRISPR/Cas9. Although these techniques are very powerful, there are many shortcomings or technical difficulties in practice. Therefore, there is a great need to develop a technique for simple, effective and controllable removal of molecules.
  • intracellular antibodies can be used to target and inactivate important molecules in cells.
  • the antibody sequence can be anchored to three major intracellular domains: endoplasmic reticulum (ER)/Golgi, Cytoplasm and nucleus.
  • ER endoplasmic reticulum
  • scFv single chain antibodies
  • intracellular antibodies can be used to prevent HIV gp160 to gp130 processing in the endoplasmic reticulum; block the expression of the IL-2 receptor alpha chain; inhibit yeast, Xenopus oocytes and human Cytoplasmic enzymes in derived T cell lines and inhibition of viral activity in transgenic plants.
  • Research by Marasco and colleagues shows that single-chain antibodies can function in the ER.
  • Greenman et al. showed that intracellular single-chain antibodies can be used to prevent the expression of cell membrane proteins using a high-affinity monoclonal antibody that binds to the T lymphocyte surface antigen CD2 and an ER retention signal (KDEL).
  • the antigen recognition domain is an important part of the CAR structure and is responsible for binding to tumor cell surface antigens.
  • the antigen recognition domains of CD7-specific CAR-T cell therapy are mostly composed of variable regions of mouse or alpaca-derived antibodies. Once reinfused into the patient, these CAR-T cells carrying the xenogeneic antigen recognition domain can induce strong host cell and humoral responses, and the reinfused CAR-T cells are rapidly cleared by the host's immunity, which seriously affects the CAR-T cells in the body. persistence, eventually leading to refractory and relapse of the disease.
  • the present invention adopts natural human CD7 ligand as the antigen recognition domain of CAR. good curative effect.
  • the purpose of the present invention is to provide an engineered immune cell targeting CD7, a chimeric antigen receptor, a CD7 blocking molecule and its application.
  • a first aspect of the present invention provides an engineered immune cell comprising a polynucleotide sequence encoding a chimeric antigen receptor comprising an antigen recognition domain targeting CD7.
  • the chimeric antigen receptor further comprises a hinge transmembrane domain, an intracellular co-stimulatory domain and an intracellular primary stimulatory domain.
  • the hinge transmembrane domain in the chimeric antigen receptor is selected from the amino acid sequences of at least one of the following groups: ⁇ chain, ⁇ chain, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ chain derived from T cell receptors , CD4, CD5, CD8 ⁇ , CD8 ⁇ , CD9, CD16, CD22, CD28, CD32, CD33, CD34, CD35, CD37, CD45, CD64, CD80, CD86, CD137, ICOS, CD154, FAS, FGFR2B, OX40, or VEGFR2 amino acid sequence.
  • the hinge transmembrane domain in the chimeric antigen receptor is derived from CD8 ⁇ .
  • amino acid sequence of the hinge transmembrane domain CD8 ⁇ in the chimeric antigen receptor is shown in SEQ ID NO.1.
  • the intracellular costimulatory domain in the chimeric antigen receptor is selected from at least one of the following: CD2, CD4, CD5, CD8 ⁇ , CD8 ⁇ , CD27, CD28, CD30, CD40, 4-1BB ( CD137), ICOS, OX40, LIGHT (CD258) or NKG2C.
  • the intracellular costimulatory domain in the chimeric antigen receptor is derived from 4-1BB.
  • amino acid sequence of the intracellular costimulatory domain 4-1BB in the chimeric antigen receptor is shown in SEQ ID NO.2.
  • the hinge transmembrane domain and the intracellular costimulatory domain in the chimeric antigen receptor are derived from CD28.
  • amino acid sequence of the hinge transmembrane domain and the intracellular costimulatory domain CD28 in the chimeric antigen receptor is shown in SEQ ID NO.3.
  • the major intracellular stimulatory domain in the chimeric antigen receptor is selected from at least one of the following: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , FcR ⁇ , FcR ⁇ , CD5, CD66, CD22, CD79a or CD79b.
  • the major intracellular stimulatory domain in the chimeric antigen receptor is derived from CD3 ⁇ .
  • amino acid sequence of the major intracellular stimulation domain CD3 ⁇ in the chimeric antigen receptor is shown in SEQ ID NO.4.
  • the antigen recognition domain targeting CD7 in the chimeric antigen receptor is a part or all of the sequence of the extracellular domain of human CD7-L, or has at least the same sequence as the extracellular domain of human CD7-L. 90% sequence identity, the amino acid sequence of the human CD7-L extracellular domain is shown in SEQ ID NO.5.
  • the engineered immune cells further include a CD7 blocking molecule, the CD7 blocking molecule includes a CD7 binding domain and an intracellular anchoring domain, and the CD7 blocking molecule can prevent the transport of CD7 protein on the cell surface and Express.
  • the CD7 blocking molecule specifically prevents CD7 protein from being transported to the cell surface by connecting the intracellular anchoring domain with the CD7 binding domain.
  • the intracellular anchoring domain is the amino acid sequence of an endoplasmic reticulum retention domain, a Golgi retention domain or a proteasome localization domain.
  • the intracellular anchoring domain is an endoplasmic reticulum retention domain.
  • amino acid sequence of the endoplasmic reticulum retention domain of the intracellular anchoring domain is shown in SEQ ID NO.6 or SEQ ID NO.7.
  • the CD7 binding domain is a part or all of the sequence of the human CD7-L extracellular domain, or has at least 90% sequence identity with the human CD7-L extracellular domain.
  • the amino acid sequence of the source CD7-L extracellular domain is shown in SEQ ID NO.5.
  • the CD7 binding domain is the scFv of the anti-CD7 monoclonal antibody TH69.
  • the antigen recognition domain targeting CD7 in the chimeric antigen receptor is the scFv of the anti-CD7 monoclonal antibody TH69, or has at least 90% sequence with the scFv of the anti-CD7 monoclonal antibody TH69 Consistent, the amino acid sequence of the scFv of the anti-CD7 monoclonal antibody TH69 is shown in SEQ ID NO.8.
  • the engineered immune cells further include CD7 blocking molecules, and the CD7 blocking molecules can prevent the transport and expression of CD7 protein on the cell surface.
  • the CD7 blocking molecule prevents the transport of CD7 protein to the cell surface by connecting the intracellular anchoring domain with the CD7 binding domain.
  • the intracellular anchoring domain is the amino acid sequence of an endoplasmic reticulum retention domain, a Golgi retention domain or a proteasome localization domain.
  • the CD7 binding domain is a part or all of the sequence of the human CD7-L extracellular domain, or has at least 90% sequence identity with the human CD7-L extracellular domain.
  • the amino acid sequence of the source CD7-L extracellular domain is shown in SEQ ID NO.5.
  • the engineered immune cells remove the gene expression of CD7 through gene knockout technology.
  • the genome editing tool used in the knockout technology is TALENs or CRISPR/cas9.
  • the cells are T cells, ⁇ T cells, NK or NKT cells, and T cells differentiated from induced pluripotent stem cells (iPSCs), ⁇ T cells, NK or NKT cells.
  • iPSCs induced pluripotent stem cells
  • the second aspect of the present invention provides an antigen recognition domain targeting CD7 in a chimeric antigen receptor, wherein the sequence of the antigen recognition domain includes a partial or full sequence of the human CD7-L extracellular domain, or a human-derived CD7-L extracellular domain.
  • the CD7-L extracellular domain has at least 90% sequence identity; the amino acid sequence of the human CD7-L extracellular domain is shown in SEQ ID NO.5.
  • the third aspect of the present invention provides an application of the above-mentioned CD7-targeting antigen recognition domain in the preparation of an immunotoxin against CD7-positive hematological malignancies.
  • the fourth aspect of the present invention provides a nucleic acid molecule encoding the antigen recognition domain targeting CD7 in the above-mentioned chimeric antigen receptor.
  • the nucleic acid molecule encoding the antigen recognition domain targeting CD7 in the chimeric antigen receptor is CD7BB-002; the nucleotide sequence of CD7BB-002 is shown in SEQ ID NO.9.
  • the fifth aspect of the present invention provides a recombinant vector comprising the above-mentioned nucleic acid molecule encoding the antigen recognition domain targeting CD7 in the chimeric antigen receptor.
  • the vector is selected from retrovirus, lentivirus or transposon.
  • the sixth aspect of the present invention provides the use of the above-mentioned recombinant vector in preparing engineered immune cells.
  • the seventh aspect of the present invention provides a CD7 blocking molecule for preparing the above-mentioned engineered immune cells, wherein the CD7 binding domain of the CD7 blocking molecule includes a partial or full sequence of human CD7-L extracellular structure domain, or a protein with at least 90% sequence identity to the human CD7-L extracellular domain.
  • the eighth aspect of the present invention provides a nucleic acid sequence encoding the aforementioned CD7 blocking molecule.
  • the nucleic acid sequence of the CD7 blocking molecule is CD7-L-ER2.1; the nucleotide sequence of the CD7-L-ER2.1 is shown in SEQ ID NO.10.
  • the ninth aspect of the present invention also provides a CD7 blocking molecule for preparing the engineered immune cells described in the first aspect above, wherein the CD7 binding domain of the CD7 blocking molecule is the scFv of the anti-CD7 monoclonal antibody TH69 , or a protein with at least 90% sequence identity with the scFv of the anti-CD7 monoclonal antibody TH69.
  • the tenth aspect of the present invention provides a nucleic acid sequence encoding the CD7 blocking molecule of the ninth aspect.
  • the coding sequence of the nucleic acid molecule of the CD7 blocking molecule is TH69-ER2.1; the nucleotide sequence of the TH69-ER2.1 is shown in SEQ ID NO.11.
  • the eleventh aspect of the present invention provides a recombinant vector, comprising the nucleic acid sequence encoding the CD7 blocking molecule provided in the eighth or tenth aspect.
  • the vector is selected from retrovirus, lentivirus or transposon.
  • a twelfth aspect of the present invention provides a use of the recombinant vector provided in the eleventh aspect above in preparing engineered immune cells.
  • the thirteenth aspect of the present invention provides an antigen recognition domain that encodes both the CD7-targeting chimeric antigen receptor described in the second aspect and a CD7 blocking molecule (the CD7 blocking molecule provided in the seventh aspect or the ninth aspect). molecule) nucleic acid molecules.
  • the fourteenth aspect of the present invention provides a recombinant vector, comprising the nucleic acid molecule of the thirteenth aspect.
  • the vector is selected from retrovirus, lentivirus, and transposon.
  • the recombinant vector comprises the following sequence: CD7BB-BL4-002 or CD7BB-BL6-002, the schematic diagram of the vector sequence of the recombinant vector CD7BB-BL4-002 or CD7BB-BL6-002 is shown in Figure 5, respectively shown in A and B.
  • the recombinant vector CD7BB-BL4-002 is connected by CD7BB-002 and TH69-ER2.1 through T2A.
  • the schematic diagram of the sequence structure of the recombinant vector is shown in (1) and (2 of A in Figure 5 ). ), the amino acid sequence of the T2A is shown in SEQ ID NO.16.
  • the recombinant vector CD7BB-BL6-002 is connected by TH69BB-002 and CD7-L-ER2.1 through T2A, and the schematic diagram of the sequence structure of the recombinant vector is shown in Figure 5 in B (3) and As shown in (4), the amino acid sequence of the T2A is shown in SEQ ID NO.16.
  • the signal peptide of the carrier is derived from CD8 ⁇ .
  • the amino acid sequence of the CD8 ⁇ signal peptide of the vector is shown in SEQ ID NO.12.
  • the signal peptide of the carrier is derived from GM-CSF-R.
  • amino acid sequence of the GM-CSF-R signal peptide of the vector is shown in SEQ ID NO.13
  • the light chain and the heavy chain of the scFv of the vector are connected by a (GGGGS)3 linking peptide, and the amino acid sequence of the linking peptide is shown in SEQ ID NO.14.
  • the light chain and heavy chain of the scFv of the vector are connected by a Whitlow connecting peptide, and the amino acid sequence of the connecting peptide is shown in SEQ ID NO.15.
  • the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule encoding the CD7 blocking molecule in the vector have internal ribosome entry sites (Internal Ribosome Entry Site, IRES) or ribosomal codons A ribosomal codon skipping site is ligated.
  • IRES Internal Ribosome Entry Site
  • the internal ribosome entry site (Internal Ribosome Entry Site, IRES) of the vector is derived from encephalomyocarditis virus (Encephalomyocarditis virus, EMCV) or enterovirus (Enterovirus).
  • the ribosomal codon skipping site of the vector comprises a 2A self-cleaving peptide
  • the 2A self-cleaving peptide can be selected from foot-and-mouth disease virus F2A peptide (foot-and-mouth disease virus 2A peptide), vest rhinitis Virus E2A peptide (equine rhinitis A virus 2A peptide), porcine teschovirus-1 2A peptide or T2A peptide (thosea aigna virus 2A).
  • the 2A self-cleaving peptide of the carrier is derived from T2A.
  • the amino acid sequence of the T2A self-cleaving peptide of the carrier is shown in SEQ ID NO.16.
  • the 2A self-cleaving peptide of the carrier is derived from F2A.
  • amino acid sequence of the F2A self-cleaving peptide of the vector is shown in SEQ ID NO.17.
  • a fifteenth aspect of the present invention provides use of the recombinant vector described in the fourteenth aspect in preparing engineered immune cells.
  • a sixteenth aspect of the present invention provides a reagent combination, the combination comprising: (1) the recombinant vector described in the fifth aspect, and (2) the vector described in the eleventh aspect.
  • the seventeenth aspect of the present invention provides the use of the reagent combination described in the sixteenth aspect in the preparation of CAR-T or CAR-NK cells for the treatment of CD7-positive hematological malignancies.
  • An eighteenth aspect of the present invention provides a reagent combination, the combination comprising: (1) the vector described in the fifth aspect, and (2) a gene editing tool capable of knocking out the CD7 gene in cells.
  • the gene editing tool is TALENs or CRISPR/cas9.
  • the nineteenth aspect of the present invention provides an antigen recognition domain sequence targeting CD7 in a chimeric antigen receptor, the sequence being the scFv of the anti-CD7 monoclonal antibody TH69, or the scFv of the anti-CD7 monoclonal antibody TH69 Having at least 90% sequence identity, the amino acid sequence of the scFv of the anti-CD7 monoclonal antibody TH69 is shown in SEQ ID NO.8.
  • the twentieth aspect of the present invention provides a nucleic acid molecule encoding an antigen recognition domain sequence targeting CD7 in the chimeric antigen receptor of the nineteenth aspect.
  • nucleic acid molecule coding sequence of the twentieth aspect is TH69BB-002, and the nucleic acid molecule sequence of TH69BB-002 is shown in SEQ ID NO.18.
  • a twenty-first aspect of the present invention provides a recombinant vector comprising the sequence of the nucleic acid molecule of the twenty-first aspect.
  • the vector is selected from retrovirus, lentivirus, and transposon.
  • a twenty-second aspect of the present invention provides a combination of reagents, the combination comprising:
  • the twenty-third aspect of the present invention provides the use of the reagent combination according to the twenty-second aspect in the preparation of CAR-T or CAR-NK cells for the treatment of CD7-positive hematological malignancies.
  • the present invention Compared with the prior art, the present invention has the following beneficial effects: the present invention uses human-derived CD7-L to replace the antibody sequence as the antigen recognition domain of CD7-specific CAR-T cells, and uses human-derived CD7 in targeting CD7 CAR.
  • the advantage of -L as an antigen recognition domain is that it can prevent the cellular and humoral responses generated by the host, thereby achieving long-term survival and better efficacy of CAR-T cells in vivo after reinfusion.
  • CD7 is a transmembrane glycoprotein normally expressed on most peripheral T cells and NK cells and their precursors. Diseased T cells and NK cells themselves express CD7 at high density; CD7-deficient T cells exhibit largely undisturbed developmental, homeostatic, and protective functions; the effect of CD7 on peripheral blood T cell function is insignificant , so CD7 is a promising target for CAR-T cell therapy. Since both normal and diseased T cells themselves express CD7, two factors need to be considered when preparing chimeric antigen receptor (CAR) T cells: 1. Genetic modification of normal T cells, Make T cells express CD7-targeted chimeric antigen receptor (CAR-T) to kill CD7-positive diseased T cells; CD7 expression of T cells themselves.
  • CAR chimeric antigen receptor
  • the technical solution of the present invention not only genetically modifies normal T cells to make T cells express CD7-specific CAR, but also takes into account the blocking of CD7 expression inside normal T cells.
  • the applicant of the present invention has undergone a large number of experiments, constantly revised and verified the experimental parameters, and finally obtained the technical solution of the present invention.
  • the technical solution of the present invention can genetically modify normal T cells so that the T cells express CD7-specific CAR, and at the same time, normal T cells can be genetically modified to express CD7-specific CAR.
  • the CD7 expression of T cells was blocked, and unexpected technical effects were achieved.
  • Figure 1 shows the establishment of K562 and HeLa cell lines expressing CD7.
  • K562 and HeLa cell lines were transduced with lentiviral vectors carrying CD7 cDNA, and K562-CD7 and HeLa-CD7 were obtained by flow sorting.
  • the figure shows the expression of CD7 in K562 and HeLa cell lines by flow cytometry.
  • FIG. 2 is a schematic diagram of the carrier structure of CD7-CAR and CD7 blocking molecule.
  • a and B are two second-generation CD7-specific CARs. Among them, the antigen recognition region of A, CD7BB-002 is CD7-L; the antigen recognition region of B, TH69BB-002 is the scFv of monoclonal antibody TH69; the two CAR vectors have the same CD8a hinge transmembrane region, 4-1BB total.
  • the stimulatory domain and CD3 ⁇ are T-cell stimulatory domains.
  • the scFv of the monoclonal antibody TH69 adopts the CD8 ⁇ signal peptide, and the linking peptide of the light chain (VL) and heavy chain (VH) variable regions is (GGGGS)3.
  • C and D are two CD7 blocking molecules. Among them, the CD7 binding domain of C, CD7-L-ER2.1 is CD7-L; D, the CD7 binding domain of TH69-ER2.1 is the scFv of TH69, which uses the CD8 ⁇ signal peptide, its light chain (VL) and The linking peptide of the heavy chain (VH) variable region is (GGGGS)3; the two CD7 blocking molecules share the same ER retention domain.
  • FIG. 3 is a schematic diagram of the results of flow detection and in vitro killing experiments of CD7-CAR-T cells.
  • A. The expression of two CD7-targeting CARs, CD7BB-002 and TH69BB-002, on T cells was detected by flow cytometry;
  • B. The iCELLigence TM real-time cell analyzer (Agilent Biosciences, Inc.) was used to conduct in vitro killing experiments; "T cell “control” is an untransduced T cell control, "CD7BB-002” is a CAR-T cell expressing CD7BB-002, and "TH69BB-002" is a CAR-T cell expressing TH69BB-002.
  • the flow detection reagent of CD7-CAR-T cells is CD7-CAR-GREEN; the target cells used in the in vitro killing experiments are HeLa-CD7.
  • Figure 4 is a schematic diagram of the results of blocking the expression of CD7 on the cell membrane by using Intrablock TM CD7 expression blocking technology.
  • K562-CD7 cell lines (A) or T were transduced with lentiviral vectors CD7-L-ER2.1 and TH69-ER2.1 carrying CD7-binding ligands CD7-L or TH69scFV cDNA and ligating the ER retention domain cells (B), and subjected to flow cytometry of CD7.
  • the results showed that both CD7 expression blocking molecules could effectively reduce the expression of CD7 on the cell surface.
  • Figure 5 is a schematic diagram of the construction of a lentiviral vector targeting CD7 CAR using Intrablock TM CD7 expression blocking technology.
  • a and B are two CD7-CAR lentiviral vectors constructed using Intrablock TM CD7 expression blocking technology.
  • A CD7BB-BL4-002
  • (1) is a schematic diagram of a chimeric antigen receptor targeting CD7, and its antigen recognition region is CD7-L
  • (2) is a schematic diagram of a CD7 blocking molecule, which can block the expression of CD7
  • the acting CD7 binding domain is the scFv of the monoclonal antibody TH69
  • B CD7BB-BL6-002
  • (3) is a schematic diagram of the chimeric antigen receptor targeting CD7, and its antigen recognition region is the scFv of the monoclonal antibody TH69
  • (4) ) is a schematic diagram of the CD7 blocking molecule, the CD7-binding domain that blocks CD7 expression is CD7-L
  • the two CAR vectors have the same CD8 ⁇ hinge transmembrane region, 4-1BB costimulatory domain, CD3 ⁇ T cell stimulation domain and ER Endoplasmic reticulum retention domain.
  • the scFv of the monoclonal antibody TH69 uses the CD8 ⁇
  • FIG. 6 Using Intrablock TM CD7 expression blocking technology, CD7-CAR-T cells can overcome the suicide phenomenon and effectively kill target cells.
  • A Flow cytometry detects the expression of four different CARs targeting CD7 on T cells;
  • B Flow cytometry detects the expression of CD7 on the above four different CAR-T cells; the vectors used are: CD7BB -002, TH69BB-002 and CD7BB-BL4-002 and CD7BB-BL6-002 constructed with Intrablock TM CD7 expression blocking technology; dashed line is untransduced T cell control, solid line is transduced CAR targeting CD7 of T cells.
  • T cell control is an untransduced T cell control
  • CD7BB-002, TH69BB-002, CD7BB-BL4-002 and CD7BB-BL6-002 are all CAR-T cells targeting CD7, among which CD7BB-BL4-002 and CD7BB -BL6-002 is a CD7-CAR-T cell using Intrablock TM CD7 expression blocking technology.
  • the flow detection reagent of CD7-CAR-T cells is CD7-CAR-GREEN; the target cells used in the in vitro killing experiments are HeLa-CD7.
  • FIG. 7 The results of the in vivo tumor killing experiment of CD7-CAR-T cells using Intrablock TM CD7 expression blocking technology.
  • Female NSG mice were used, and tumor cells (luciferase-carrying tumor cell line, CCRF-CEM-Luc, 5x10E5/mouse, iv) were injected on D0 day.
  • A 1. Negative control group; 2 and 3. Reinfused T cell control group and CD7BB-BL4-002 CAR-T cell treatment group targeting CD7, respectively.
  • T cells or CD7BB-BL4-002 CAR-T cells (8x10E6/mouse) were injected intravenously, respectively, and then in vivo luciferase imaging was performed every 7 days.
  • B Survival curves of mice in the control group and CD7BB-BL4-002 CAR-T cell treatment group.
  • the present invention uses the extracellular domain of SECTM1 (K12) as the antigen recognition domain of CD7, and is used to develop CAR-T cell therapy and immunotoxin against CD7-positive hematological malignancies.
  • CD7-L is used instead of SECTM1 or K12.
  • the CD7-L gene was originally identified as the 5' end of the CD7 gene on human chromosome 17.
  • Human CD7-L protein is mainly expressed in spleen, prostate, testis, small intestine and peripheral blood leukocytes.
  • a feature of CD7-L is that it encodes a transmembrane protein with an extracellular domain similar to an immunoglobulin-like domain.
  • CD7-L was cloned in 2000 and found to be a binding protein for CD7.
  • CD7-L-Fc fusion protein of the extracellular domain of CD7-L (amino acids 1-145) with the Fc portion of human IgG1.
  • Flow cytometry experiments showed that the CD7-L-Fc fusion protein could detect high-level binding on human T cells and NK cells.
  • CD7-targeting antibodies blocked the binding of the CD7-L-Fc fusion protein to cells to varying degrees.
  • CD7-L-Fc fusion proteins can block the binding of these CD7 mAbs to CD7, indicating that CD7-L-Fc can bind to the CD7 receptor on cells.
  • the CD7-L-Fc fusion protein was radiolabeled and used in binding experiments to determine its affinity for Jurkat cells (a human T-cell leukemia cell line) or KG-1 cells (a human myeloid leukemia cell line), both of which Both express CD7.
  • the binding affinity (Ka) of CD7-L-Fc for human CD7 is approximately in the range of 1 ⁇ 10 8 M ⁇ 1 . Since CD7 is considered to be a good marker for T-cell malignancies, it has been studied to produce immunotoxins by conjugated anti-human CD7 monoclonal antibody to ricin or saponin.
  • this patent application uses the extracellular domain of CD7-L in the construction of CD7-targeting chimeric antigen receptors and CD7-blocking molecules for the development of CD7-CAR-T or CAR-NK cells for the treatment of T cells malignant tumor.
  • the present patent application also binds the extracellular region of CD7-L to toxins and uses these conjugates as immunotoxins against T-cell malignancies, which may be less immunogenic than antibody-based conjugates. Has a longer half-life.
  • Example 1 CAR-T cells using CD7-L as antigen recognition domain can effectively recognize CD7 tumor antigen
  • two second-generation CAR lentiviral vectors CD7BB-002 and TH69BB-002, using the scFv of CD7-L or CD7-specific monoclonal antibody TH69 as the antigen recognition region were constructed first ( Figure 2A, B).
  • CAR-T cells were obtained by transduction of CAR lentiviral vector, and the flow detection reagent (CD7-CAR-GREEN) of CD7-CAR-T cells produced by fusion protein of CD7 and reporter gene eGFP was used for flow detection ( Figure 3A) .
  • CD7-CAR-GREEN can be effectively used to detect the two CD7-targeting CAR-T cells, indicating that both CD7-L and the scFv of the monoclonal antibody TH69 can be used as the CD7-specific antigen recognition region of CAR-T cells. .
  • Embodiment 2 CAR-T cells targeting CD7 can effectively kill tumor cells expressing CD7 positive;
  • K562 and HeLa cell lines were first transduced with a lentiviral vector carrying CD7 cDNA, and K562-CD7 and HeLa-CD7 cell lines expressing CD7 were obtained by flow sorting (Fig. 1).
  • Two CD7-targeting CAR-T cells, CD7BB-002 and TH69BB-002, were obtained by CAR lentiviral vector transduction, and the iCELLigence TM real-time cell analyzer (Agilent Biosciences, Inc.) was used for in vitro killing experiments (Figure 3B).
  • Embodiment 3 Intrablock TM CD7 expression blocking technology can effectively block the expression of CD7 on the cell membrane;
  • a lentiviral vector, CD7-L-ER2.1 and TH69-ER2.1 (Fig. 2C, D).
  • K562-CD7 cell line or primary T cells were transduced by lentiviral vector, and the CD7 expression blocking technology was evaluated by flow cytometry.
  • the results showed that both TH69-ER2.1 and CD7-L-ER2.1 could effectively block the expression of CD7 on K562-CD7 cell line (Fig. 4A) or T cells (Fig. 4B), indicating that TH69-ER2.1 and Both CD7-L-ER2.1 can bind to CD7 in cells and retain it in the endoplasmic reticulum. Therefore, this Intrablock TM CD7 expression blocking technology can be used to block the expression of CD7 on cells and prevent the suicide phenomenon of CD7-targeted CAR-T cells.
  • Intrablock TM CD7 expression blocking technology can be used to overcome the suicide phenomenon of CD7-CAR-T cells and effectively kill CD7-positive target cells;
  • CD7BB-BL4-002 and CD7BB-BL6-002 were constructed, CD7BB-BL4-002 and CD7BB-BL6-002 ( Figure 5A, B). Since CD7 is expressed on the T cells used for CAR-T cell preparation, suicide will occur when CAR-T cells targeting CD7 are prepared, making the preparation of CAR-T cells difficult. As shown in Figure 6, both CD7-targeting CAR-T cells, CD7BB-002 and TH69BB-002, were effectively recognized by CD7-CAR-GREEN ( Figure 6A) and could effectively kill CD7-positive HeLa - CD7 target cells (Fig. 6D).
  • Example 5 Validation of the in vivo tumor-killing function of CD7-CAR-T cells using Intrablock TM CD7 expression blocking technology in animal models;
  • an in vivo tumor killing experiment was performed using CD7BB-BL4-002 CAR-T cells with Intrablock TM CD7 expression blocking technology (Fig. 7).
  • Female NSG mice aged 6-8 weeks were used, and the luciferase-carrying tumor cell line, CCRF-CEM-Luc, 5x10E5/mouse, iv, was injected into the tail vein on D0 day.
  • T cells re-infused T cell control group
  • CD7BB-BL4-002 CAR-T cells (8x10E6/mouse) CAR-T cell treatment group
  • In vivo mouse luciferase imaging observation was performed every 7 days after D0 day. The results prove that CD7BB-BL4-002 CAR-T cells using Intrablock TM CD7 expression blocking technology can effectively kill tumors in this mouse tumor model and prolong the survival period of mice in the CAR-T cell treatment group (such as A and B in Figure 7).
  • the present invention uses human-derived CD7-L to replace the antibody sequence as the antigen recognition domain of CD7-specific CAR-T cells, and uses human-derived CD7 in targeting CD7 CAR.
  • the advantage of -L as an antigen recognition domain is that it can prevent the cellular and humoral responses generated by the host, thereby achieving long-term survival and better efficacy of CAR-T cells in vivo after reinfusion.
  • CD7 is a transmembrane glycoprotein normally expressed on most peripheral T cells and NK cells and their precursors.
  • CD7-deficient T cells exhibit largely undisturbed developmental, homeostatic, and protective functions; the effect of CD7 on peripheral blood T cell function is insignificant , so CD7 is a promising target for CAR-T cell therapy. Since both normal and diseased T cells themselves express CD7, two factors need to be considered when preparing chimeric antigen receptor (CAR) T cells: 1. Genetic modification of normal T cells, Make T cells express CD7-targeting chimeric antigen receptor (CAR-T) to kill CD7-positive diseased T cells; CD7 expression of T cells themselves.
  • CAR chimeric antigen receptor
  • the technical solution of the present invention not only genetically modifies normal T cells to make T cells express CD7-specific CAR, but also takes into account the blocking of CD7 expression inside normal T cells.
  • the applicant of the present invention has undergone a large number of experiments, constantly revised and verified the experimental parameters, and finally obtained the technical solution of the present invention.
  • the technical solution of the present invention can genetically modify normal T cells so that the T cells express CD7-specific CAR, and at the same time, normal T cells can be genetically modified to express CD7-specific CAR.
  • the CD7 expression of T cells was blocked, and unexpected technical effects were achieved.

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Abstract

一种靶向CD7的工程化免疫细胞、嵌合抗原受体、CD7阻断分子及应用。利用人源CD7的天然配体取代抗体序列作为CD7特异性CAR-T或CAR-NK细胞的抗原识别域。在CD7特异性CAR中使用人源CD7作为抗原识别域的优点是可以防止宿主产生的细胞和体液反应，从而实现CAR-T细胞的长期持久性和更好的疗效。

Description

靶向CD7的工程化免疫细胞、嵌合抗原受体、CD7阻断分子及应用 技术领域
本发明涉及细胞免疫治疗技术领域，具体地，涉及靶向CD7的免疫治疗方法，尤其是一种靶向CD7的工程化免疫细胞、嵌合抗原受体、CD7阻断分子及应用。
背景技术
T细胞恶性肿瘤是一组异质性很强的克隆性生长和T细胞功能障碍性疾病，主要分为T细胞淋巴瘤(T-cell lymphomas，TCLs)和T细胞白血病，具有成熟和前体亚型。这类疾病代表了一类极其恶性的血液系统癌症，在儿童和成人中复发率和死亡率都很高，目前还没有有效或针对性的治疗方法。尽管现有技术中已经有多种的化疗方案，在患有T细胞急性淋巴细胞白血病(T-cell acute lymphoblastic leukemia，T-ALL)的病患中，只有不到50％的成人和75％的儿童能够存活5年以上。对于初次治疗后复发的患者，挽救性化疗方案只是对20％-40％的病例能够起到诱导缓解效果。在复发或化疗难治性T细胞恶性肿瘤患者中，治疗预后较差，而有效且可耐受的治疗方法有限。对于10-50％在挽救性化疗后获得完全缓解(CR)的患者，唯一的治疗选择仍然是异基因干细胞移植(allogeneic stem cell transplantation，ASCT)。然而，ASCT的治愈率仍保持在30％或更低，而且并非所有CR患者都有资格接受移植。其他T细胞恶性肿瘤，包括皮肤和外周T细胞淋巴瘤(分别为CTCL和PTCL)对化疗的初始反应率更低，即使在有反应的患者中，无进展生存率仍保持在40–50％。因此，尽管在治疗T细胞恶性肿瘤方面取得了进展，但是仍然需要新的、有针对性的治疗方案来改善预后，特别是对于复发和难治的患者。除了T细胞恶性肿瘤，如T-ALL和T-NHL，必须认识到还有其他CD7+血液恶性肿瘤仍然缺乏有效治疗和靶向治疗方案，如20％-30％的急性髓性白血病(AML)，以及绝大部分的NK和NKT淋巴瘤。
嵌合抗原受体T(CAR-T)细胞是最有前途的肿瘤免疫治疗方法之一，对B淋巴细胞恶性肿瘤患者能够产生显著的应答率。因此，以CD19(一种在B细胞白血病和淋巴瘤中广泛表达的抗原)为靶点的CAR-T细胞已成为首个获得许可的癌症T细胞疗法。CD19CAR-T细胞在治疗复发性和难治性B细胞恶性肿瘤方面的成功使其在其他肿瘤中的应用更加广泛。鉴于B淋巴细胞和T淋巴细胞恶性肿瘤之间的相似性， 将CAR-T细胞治疗扩展到这些疾病似乎很简单。然而，针对T细胞恶性肿瘤的CAR-T细胞治疗已被证明很难开发和实施。这主要是由于在工程化T细胞上同时表达靶抗原可导致CAR-T细胞在制备过程中自相残杀；同时，CAR-T细胞回输后正常外周血T细胞的清除可导致严重的免疫缺陷。
CD7是一种跨膜糖蛋白，通常表达在大多数外周血T细胞和NK细胞(NK细胞即自然杀伤细胞，来源于骨髓淋巴样干细胞，其分化发育依赖于骨髓及胸腺微环境，主要分布于骨髓、外周血、肝、脾、肺和淋巴结；NK细胞不同于T、B细胞，是一类无需预先致敏就能非特异性杀伤肿瘤细胞和病毒感染细胞的淋巴细胞)及其前体细胞上，作为一种辅助T细胞活化和与其他免疫亚群相互作用的共刺激蛋白。95％以上的淋巴母细胞性白血病和淋巴瘤以及部分外周T细胞淋巴瘤表达CD7。在小鼠动物模型中，缺乏CD7的T细胞在很大程度上表现出不受干扰的发育、稳态和保护功能。由于CD7对外周血T细胞功能没有显著的影响，因此它是一个很有前途的CAR-T细胞治疗靶标。CD7曾被作为单克隆抗体(mAb)的靶标采用免疫毒素治疗T细胞恶性肿瘤患者进行过评估。这种单克隆抗体的偶联物没有产生严重的CD7相关的毒副作用，但抗肿瘤反应不显著，这可能是由于鼠源抗体在患者治疗上的有限活性所致。
现有技术中，关于靶向CD7的治疗研究进展如下：
i)CD7-CAR-T细胞治疗；表达嵌合抗原受体(CAR)的T细胞是一种很有前途的肿瘤免疫治疗方法。这种靶向治疗在获得B细胞白血病和淋巴瘤患者缓解甚至长期无复发生存方面显示出巨大的潜力。最近已有几个研究小组报告了CD7特异性CAR-T细胞治疗在T细胞恶性肿瘤的临床前模型中的研究进展。在所有这些研究中，CD7-CAR在T细胞上的表达导致严重的自杀现象，造成CAR-T细胞无法在体外扩增。
靶标特异性的CAR在所转导T细胞上的表达可以造成因持续的配体结合引起的CAR受体激活，可导致转导T细胞的自杀和加速细胞的终末分化使其在体内不能长期存活。由于CD7是一种泛T细胞抗原，在大部分T细胞上均有表达。因此CD7抗原特异性的CAR-T细胞在制备过程中可产生严重的自杀现象，从而造成CAR-T细胞无法有效地扩增。现有两种策略来减少这种自杀现象：1)使用基因组编辑工具敲除CD7靶抗原的基因；2)通过CD7结合域在内质网锚定的方法阻止CD7蛋白在细胞内表达过程中转运到细胞表面。这两种方法都可以有效地降低CD7在细胞表面的表达，并最大限度地减少了靶向CD7-CAR-T细胞的自杀现象。重要的是，CD7的缺失并不影响CAR-T细胞的增殖和短期效应功能，使具有高抗肿瘤活性的功能性CAR-T 细胞得以扩增。
从细胞表面上去除CD7后，CD7-CAR-T细胞在体外和体内对原发性CD7阳性T-ALL和淋巴瘤具有较强的抗肿瘤活性。CD7-CAR-T细胞对外周血CD7阳性T细胞和NK细胞也具有杀伤性，这表明这些细胞亚群也将成为CD7-CAR-T细胞的靶点。
使用自体细胞进行过继性细胞治疗的一个风险是无意中将外周血的恶性肿瘤细胞也同时进行基因修饰。用CAR基因修饰的恶性T细胞，在某些情况下，允许它们进行基因编辑以降低目标分子的表达可能是一个真实存在的风险，因此需要在使用这些治疗的知情同意书中加以说明。然而，考虑到在制造过程中恶性肿瘤细胞的存活率和扩增性较差，这些不必要的恶性细胞基因修饰应该以较低的频率发生。此外，在缺乏基因编辑以降低靶抗原表达的情况下，表达靶抗原的恶性肿瘤细胞在转导后和输注到患者体内之前可能会被CAR-T细胞自相残杀而清除。同时，可以采用健康供者生产的异基因CD7-CAR-T细胞用于治疗复发性和难治性T细胞恶性肿瘤，以达到将患者桥接造血干细胞移植的目的，或用于治疗造血干细胞移植后复发的T细胞恶性肿瘤。如果移植物抗宿主的副作用能够得到适当的控制，异基因CD7-CAR-T细胞治疗的优势是除了可以由健康供者提供T细胞外，还可以产生移植物抗白血病的效应。利用现成的同种异体T细胞或NK细胞作为运载工具，可以完全避免基因修饰恶性细胞的风险。由于这些细胞产物将由健康的供者产生，这样可以避免使用患者来源的T细胞，这些T细胞往往由于长期处于抑制性肿瘤微环境或因经受过先前的强化治疗而功能低下。
ii)靶向CD7的免疫毒素；CD7在T细胞上以高密度(约60000mol/cell)表达，即使被单价抗体片段结合，CD7也被迅速内化。因此，它是免疫毒素介导治疗T细胞肿瘤的理想靶抗原。目前，抗CD7免疫毒素主要由抗CD7单克隆抗体与毒素偶联而成。采用鼠抗人CD7单克隆抗体(WTI)与蓖麻毒素A结合的抗CD7免疫毒素曾被用来在体外清除肿瘤细胞以用于T细胞恶性肿瘤患者的自体骨髓移植。免疫毒素DA7通过化学的方法连接小鼠IgG2b抗CD7(3AlE)单克隆抗体与去糖基化蓖麻毒蛋白A链而构建，曾被用于SCID动物模型中治疗人的T-ALL，提示其对预后不良的T细胞白血病具有潜在的治疗作用。在DA7的I期临床试验中，尽管受到不稳定性和血管毒性的限制，在其最大耐受剂量下实现了客观的临床反应。抗人CD7的单克隆抗体TH69也曾被用来构建重组免疫毒素，该抗体通过基因重组的方法将单链抗体片段(scFv)与截短的假单胞菌外毒素A片段相连。
iii)基因敲除；靶向基因编辑T细胞在过继免疫治疗的临床可行性已经得到了 很好的证明。使用锌指核酸酶对CD4阳性T细胞中的HIV共受体CCR5进行基因敲除，使这些细胞对HIV感染具有抵抗力，并使CCR5阴性的T细胞能够在HIV感染患者中植入并持续存在。采用TALENs对CD19CAR-T细胞中的T细胞受体(TCR)基因进行敲除，使第三方T细胞可以成功治疗B细胞白血病患者，这是“通用”现成T细胞产品的一个令人鼓舞的里程碑，极大地降低了移植物抗宿主病的风险。在此项研究中，CD52基因也在回输的CAR-T细胞中进行了敲除，从而使其对阿来单抗产生抗药性。最近，CRISPR/Cas9系统被用来敲除CD7或在敲除CD7的同时也敲除了T细胞受体α链，这一新的基因编辑系统可以快速有效地敲除T细胞中的靶基因。
iv)细胞内蛋白表达阻断技术；在许多生化和免疫系统中，通常很难判定某些特定分子的作用，但有一种方法是抑制基因表达并寻找其功能效应。通过同源重组或近年来发展的基因编辑技术，如Zince Finger核酸酶、TALENs和CRISPR/Cas9进行基因敲除。虽然这些技术十分强大，但在实践中有很多不足或技术上的困难。因此非常需要发展一种简单有效、可控地去除分子的技术。
已有大量研究表明采用细胞内抗体可以靶向灭活细胞内的重要分子。通过构建抗体的重链和轻链转染哺乳动物细胞，并对其先导序列进行简单的修改，就可以把抗体序列锚定在三个主要细胞内区域：内质网(ER)/高尔基体、胞浆和细胞核。最初的细胞内抗体研究使用全IgG，但近来的研究集中在Fab'和单链抗体(scFv)上。单链抗体分子很小(约30kDa)，通过短肽序列连接重链和轻链的可变区来构建。这些构建产物作为细胞内抗体有许多优点，主要是它们无需将两个独立的抗体链结合形成抗原结合位点。
到目前为止，已有研究表明细胞内抗体可用来防止HIV gp160到gp130在细胞内质网中的处理；阻断IL-2受体α链的表达；抑制酵母、非洲爪蟾卵母细胞及人源T细胞株中的胞浆酶和抑制转基因植物中的病毒活性。Marasco及其同事的研究表明，单链抗体可以在ER中起作用。Greenman等人的研究表明，细胞内单链抗体可以用来防止细胞膜蛋白的表达，他们采用的是一种可以结合T淋巴细胞表面抗原CD2的高亲和力单克隆抗体和一个ER滞留信号(KDEL)。
抗原识别域是CAR结构的重要组成部分，负责与肿瘤细胞表面抗原结合。目前，CD7特异性CAR-T细胞治疗的抗原识别域大多由小鼠或羊驼源性抗体的可变区组成。一旦回输至患者体内，这些携带异种抗原识别域的CAR-T细胞可诱导很强的宿主细胞和体液反应，回输的CAR-T细胞被宿主免疫迅速清除，严重影响CAR-T细胞在体内的持久性，最终导致疾病的难治和复发。本发明采用天然的人源CD7配体作为CAR 的抗原识别域，其优势在于，由此构建的靶向CD7-CAR-T不会诱导产生宿主的免疫反应，可以在体内长期存活，从而达到更好的疗效。
发明内容
针对现有技术中的缺陷，本发明的目的是提供一种靶向CD7的工程化免疫细胞、嵌合抗原受体、CD7阻断分子及应用。
本发明的目的是通过以下方案实现的：
本发明的第一方面提供一种工程化免疫细胞，其中包括编码嵌合抗原受体的多核苷酸序列，所述嵌合抗原受体包括靶向CD7的抗原识别域。
在另一优选例中，所述嵌合抗原受体还包括铰链跨膜域、细胞内共刺激域和细胞内主要刺激域。
在另一优选例中，所述嵌合抗原受体中铰链跨膜域选自以下至少一组的氨基酸序列：来源于T细胞受体的α链、β链,CD3δ,CD3ε,CD3γ,CD3ζ链,CD4,CD5,CD8α,CD8β,CD9,CD16,CD22,CD28,CD32,CD33,CD34,CD35,CD37,CD45,CD64,CD80,CD86,CD137,ICOS,CD154,FAS,FGFR2B,OX40或VEGFR2的氨基酸序列。
在另一优选例中，所述嵌合抗原受体中铰链跨膜域来源于CD8α。
在另一优选例中，所述嵌合抗原受体中铰链跨膜域CD8α的氨基酸序列如SEQ ID NO.1所示。
在另一优选例中，所述嵌合抗原受体中细胞内共刺激域选自以下至少一种：为CD2,CD4,CD5,CD8α,CD8β,CD27,CD28,CD30,CD40,4-1BB(CD137),ICOS，OX40,LIGHT(CD258)或NKG2C。
在另一优选例中，所述嵌合抗原受体中细胞内共刺激域来源于4-1BB。
在另一优选例中，所述嵌合抗原受体中细胞内共刺激域4-1BB的氨基酸序列如SEQ ID NO.2所示。
在另一优选例中，所述嵌合抗原受体中铰链跨膜域和细胞内共刺激域来源于CD28。
在另一优选例中，所述嵌合抗原受体中铰链跨膜域和细胞内共刺激域CD28的氨基酸序列如SEQ ID NO.3所示。
在另一优选例中，所述嵌合抗原受体中细胞内主要刺激域选自以下至少一种：CD3δ,CD3ε,CD3γ,CD3ζ,FcRβ,FcRγ,CD5,CD66,CD22,CD79a或CD79b。
在另一优选例中，所述嵌合抗原受体中细胞内主要刺激域来源于CD3ζ。
在另一优选例中，所述嵌合抗原受体中细胞内主要刺激域CD3ζ的氨基酸序列如SEQ  ID NO.4所示。
在另一优选例中，所述嵌合抗原受体中靶向CD7的抗原识别域为人源CD7-L细胞外结构域的部分或全部序列，或者与人源CD7-L细胞外结构域至少具有90％的序列一致性，所述人源CD7-L细胞外结构域的氨基酸序列如SEQ ID NO.5所示。
在另一优选例中，所述工程化免疫细胞内还包括CD7阻断分子，CD7阻断分子包括CD7结合域和胞内锚定域，CD7阻断分子能够阻止CD7蛋白在细胞表面的转运与表达。
在另一优选例中，所述CD7阻断分子具体为通过CD7结合域连接胞内锚定域阻止CD7蛋白向细胞表面转运。
在另一优选例中，所述胞内锚定域为内质网滞留域、高尔基体滞留域或蛋白酶体定位域的氨基酸序列。
在另一优选例中，所述胞内锚定域为内质网滞留域。
在另一优选例中，所述胞内锚定域内质网滞留域的氨基酸序列如SEQ ID NO.6或SEQ ID NO.7所示。
在另一优选例中，所述CD7结合域为人源CD7-L细胞外结构域的部分或全部序列，或者与人源CD7-L细胞外结构域至少具有90％的序列一致性，所述人源CD7-L细胞外结构域的氨基酸序列如SEQ ID NO.5所示。
在另一优选例中，所述CD7结合域为抗CD7的单克隆抗体TH69的scFv。
在另一优选例中，所述嵌合抗原受体中靶向CD7的抗原识别域为抗CD7的单克隆抗体TH69的scFv，或者与抗CD7的单克隆抗体TH69的scFv至少具有90％的序列一致性，所述抗CD7的单克隆抗体TH69的scFv的氨基酸序列如SEQ ID NO.8所示。
在另一优选例中，所述工程化免疫细胞内还包括CD7阻断分子，CD7阻断分子能够阻止CD7蛋白在细胞表面的转运与表达。
在另一优选例中，所述CD7阻断分子为通过CD7结合域连接胞内锚定域阻止CD7蛋白向细胞表面转运。
在另一优选例中，所述胞内锚定域为内质网滞留域、高尔基体滞留域或蛋白酶体定位域的氨基酸序列。
在另一优选例中，所述CD7结合域为人源CD7-L细胞外结构域的部分或全部序列，或者与人源CD7-L细胞外结构域至少具有90％的序列一致性，所述人源CD7-L细胞外结构域的氨基酸序列如SEQ ID NO.5所示。
在另一优选例中，所述工程化免疫细胞通过基因敲除技术去除CD7的基因表达。
在另一优选例中，所述敲除技术采用的基因组编辑工具为TALENs或CRISPR/cas9。
在另一优选例中，所述细胞为T细胞、γδT细胞、NK或NKT细胞及诱导多能干细胞(iPSC)分化的T细胞、γδT细胞、NK或NKT细胞。
本发明的第二方面提供一种嵌合抗原受体中靶向CD7的抗原识别域，所述抗原识别域的序列包括部分或全部序列的人源CD7-L细胞外结构域，或者与人源CD7-L细胞外结构域至少具有90％序列一致性；所述人源CD7-L细胞外结构域的氨基酸序列如SEQ ID NO.5所示。
本发明的第三方面提供一种上述所述的靶向CD7的抗原识别域在制备抗CD7阳性血液恶性肿瘤的免疫毒素中的应用。
本发明的第四方面提供一种编码上述所述的嵌合抗原受体中靶向CD7的抗原识别域的核酸分子。
在另一优选例中，所述编码嵌合抗原受体中靶向CD7的抗原识别域的核酸分子为CD7BB-002；所述CD7BB-002的核苷酸序列如SEQ ID NO.9所示。
本发明的第五方面提供一种重组载体，包含上述所述的编码嵌合抗原受体中靶向CD7的抗原识别域的核酸分子。
在另一优选例中，载体选自逆转录病毒、慢病毒或转座子。
本发明的第六方面提供一种上述所述的重组载体在制备工程化免疫细胞中的用途。
本发明的第七方面提供一种用于制备上述所述的工程化免疫细胞的CD7阻断分子，所述CD7阻断分子的CD7结合域包括部分或全部序列的人源CD7-L细胞外结构域，或与人源CD7-L细胞外结构域至少具有90％序列一致性的蛋白质。
本发明的第八方面提供一种编码上述所述的CD7阻断分子的核酸序列。
在另一优选例中，所述CD7阻断分子的核酸序列为CD7-L-ER2.1；所述CD7-L-ER2.1的核苷酸序列如SEQ ID NO.10所示。
本发明的第九方面还提供一种用于制备上述第一方面所述的工程化免疫细胞的CD7阻断分子，所述CD7阻断分子的CD7结合域为抗CD7的单克隆抗体TH69的scFv，或者与抗CD7的单克隆抗体TH69的scFv至少具有90％的序列一致性的蛋白质。
本发明的第十方面提供一种编码第九方面所述的CD7阻断分子的核酸序列。所述CD7阻断分子的核酸分子的编码序列为TH69-ER2.1；所述TH69-ER2.1的核苷酸序列如SEQ ID NO.11所示。
本发明的第十一方面提供一种重组载体，包括第八方面或第十方面提供的编码CD7阻断分子的核酸序列。
在另一优选例中，所述载体选自逆转录病毒、慢病毒或转座子。
本发明的第十二方面提供一种上述第十一方面提供的重组载体在制备工程化免疫细胞中的用途。
本发明的第十三方面提供一种编码同时含有第二方面所述的嵌合抗原受体中靶向CD7的抗原识别域以及CD7阻断分子(第七方面或第九方面提供的CD7阻断分子)的核酸分子。
本发明的第十四方面提供一种重组载体，包括第十三方面所述的核酸分子。
在另一优选例中，所述载体选自逆转录病毒、慢病毒、转座子。
在另一优选例中，所述重组载体包含如下序列：CD7BB-BL4-002或CD7BB-BL6-002，所述重组载体CD7BB-BL4-002或CD7BB-BL6-002的载体序列示意图分别如图5中A和B所示。
在另一优选例中，所述重组载体CD7BB-BL4-002由CD7BB-002和TH69-ER2.1通过T2A连接,所述重组载体的序列结构示意图如图5中A的(1)和(2)所示,所述T2A的氨基酸序列如SEQ ID NO.16所示。
在另一优选例中，所述重组载体CD7BB-BL6-002由TH69BB-002和CD7-L-ER2.1通过T2A连接,所述重组载体的序列结构示意图如图5中B的(3)和(4)所示,所述T2A的氨基酸序列如SEQ ID NO.16所示。
在另一优选例中，所述载体的信号肽来源于CD8α。
在另一优选例中，所述载体的CD8α信号肽的氨基酸序列如SEQ ID NO.12所示。
在另一优选例中，所述载体的信号肽来源于GM-CSF-R。
在另一优选例中，所述载体的GM-CSF-R信号肽的氨基酸序列如SEQ ID NO.13所示
在另一优选例中，所述载体的scFv的轻链和重链通过(GGGGS)3连接肽相连，所述连接肽的氨基酸序列如SEQ ID NO.14所示。
在另一优选例中，所述载体的scFv的轻链和重链通过Whitlow连接肽相连，所述连接肽的氨基酸序列如SEQ ID NO.15所示。
在另一优选例中，所述载体中编码嵌合抗原受体的核酸分子和编码CD7阻断分子的核酸分子由内部核糖体进入位点(Internal Ribosome Entry Site，IRES)or或核糖体密码子跳过位点(a ribosomal codon skipping site)连接。
在另一优选例中，所述载体的内部核糖体进入位点(Internal Ribosome Entry Site，IRES)来源于脑心肌炎病毒(Encephalomyocarditis virus，EMCV)或肠病毒(Enterovirus)。
在另一优选例中，所述载体的核糖体密码子跳过位点包含2A自裂解肽，2A自裂解 肽可以选自口蹄疫病毒F2A肽(foot-and-mouth disease virus 2A peptide)，马甲鼻炎病毒E2A肽(equine rhinitis A virus 2A peptide)，猪破伤风病毒P2A肽(porcine teschovirus-1 2A peptide)或T2A肽(thosea asigna virus 2A)。
在另一优选例中，所述载体的2A自裂解肽来源于T2A。
在另一优选例中，所述载体的T2A自裂解肽的氨基酸序列如SEQ ID NO.16所示。
在另一优选例中，所述载体的2A自裂解肽来源于F2A。
在另一优选例中，所述载体的F2A自裂解肽的氨基酸序列如SEQ ID NO.17所示。
本发明的第十五方面提供一种第十四方面所述的重组载体在制备工程化免疫细胞中的用途。
本发明的第十六方面提供一种试剂组合，所述组合包括：(1)上述第五方面所述的重组载体，以及(2)上述第十一方面所述的载体。
本发明的第十七方面提供第十六方面所述的试剂组合在制备治疗CD7阳性血液恶性肿瘤的CAR-T或CAR-NK细胞中的用途。
本发明的第十八方面提供一种试剂组合，所述组合包括：(1)上述第五方面所述的载体，以及(2)能够敲除细胞中的CD7基因的基因编辑工具。
在另一优选例中，所述基因编辑工具为TALENs或CRISPR/cas9。
本发明的第十九方面提供一种嵌合抗原受体中靶向CD7的抗原识别域序列，所述序列为抗CD7的单克隆抗体TH69的scFv，或者与抗CD7的单克隆抗体TH69的scFv至少具有90％的序列一致性，所述抗CD7的单克隆抗体TH69的scFv的氨基酸序列如SEQ ID NO.8所示。
本发明的第二十方面提供一种编码第十九方面所述的嵌合抗原受体中靶向CD7的抗原识别域序列的核酸分子。
在另一优选例中，第二十方面所述核酸分子编码序列为TH69BB-002，所述TH69BB-002的核酸分子序列如SEQ ID NO.18所示。
本发明的第二十一方面提供一种重组载体，包含第二十方面所述的核酸分子的序列。
在另一优选例中，载体选自逆转录病毒、慢病毒、转座子。
本发明的第二十二方面提供一种试剂组合，所述组合包括：
(1)第二十一方面所述的重组载体，
以及(2)选择以下组合中的一种：含有第八方面所述核酸序列的重组载体，或能够敲除细胞中的CD7基因的基因编辑工具。
本发明的第二十三方面提供一种第二十二方面所述的试剂组合在制备治疗CD7阳性 血液恶性肿瘤的CAR-T或CAR-NK细胞中的用途。
与现有技术相比，本发明具有如下的有益效果：本发明是利用人源CD7-L取代抗体序列作为CD7特异性CAR-T细胞的抗原识别域，在靶向CD7 CAR中使用人源CD7-L作为抗原识别域的优点是可以防止宿主产生的细胞和体液反应，从而实现回输后CAR-T细胞在体内的长期生存和更好的疗效。
CD7是一种跨膜糖蛋白，通常在大多数外周T细胞和NK细胞及其前体表达。病变的T细胞和NK细胞本身会高密度表达CD7；缺乏CD7的T细胞在很大程度上表现出不受干扰的发育、稳态和保护功能；由于CD7对外周血T细胞功能的影响不明显，因此CD7是一个很有前途的CAR-T细胞治疗靶点。由于正常和病变的T细胞本身都会表达CD7，所以在进行嵌合抗原受体(Chimeric Antigen Receptor,CAR)T细胞制备时，需要同时考虑两方面的因素：1、对正常T细胞进行基因修饰，使T细胞表达靶向CD7的嵌合抗原受体(CAR-T)，以杀伤CD7阳性的病变T细胞；2、为避免CAR-T细胞发生因互相识别产生的自杀现象，需要阻断CAR-T细胞本身的CD7表达。因此，本发明的技术方案既对正常T细胞进行基因修饰使T细胞表达CD7特异性CAR，同时又兼顾了对正常T细胞内部的CD7表达进行阻断。本发明的申请人经过大量的实验，不断修正验证实验参数，最终获得本发明之技术方案，本发明的技术方案能够对正常T细胞进行基因修饰使T细胞表达CD7特异性CAR，同时能够对正常T细胞的CD7表达进行阻断，实现了意想不到的技术效果。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为建立表达CD7的K562和HeLa细胞株。采用携带CD7cDNA的慢病毒载体转导K562和HeLa细胞株，并通过流式分选获得K562-CD7和HeLa-CD7。图示为流式检测CD7在K562和HeLa细胞株上的表达。
图2为CD7-CAR和CD7阻断分子的载体结构示意图。A和B为两种第二代CD7特异性CAR。其中，A、CD7BB-002的抗原识别区为CD7-L；B、TH69BB-002的抗原识别区为单克隆抗体TH69的scFv；两种CAR载体具有相同的CD8a铰链跨膜区，4-1BB共刺激域和CD3ζ为T细胞刺激域。单克隆抗体TH69的scFv采用了CD8α信号肽，其轻链(VL)和重链(VH)可变区的连接肽为(GGGGS)3。C和D为两种CD7阻断分子。其中，C、CD7-L-ER2.1的CD7结合域为CD7-L；D、TH69-ER2.1的CD7结合域 为TH69的scFv，其采用了CD8α信号肽，其轻链(VL)和重链(VH)可变区的连接肽为(GGGGS)3；两种CD7阻断分子具有相同的ER内质网滞留域。
图3为CD7-CAR-T细胞的流式检测和体外杀伤实验结果示意图。A、流式检测CD7BB-002和TH69BB-002两种靶向CD7的CAR在T细胞上的表达；B、采用iCELLigence TM实时细胞分析仪(Agilent Biosciences,Inc.)进行体外杀伤实验；“T cell control”为未转导的T细胞对照，“CD7BB-002”为表达CD7BB-002的CAR-T细胞，“TH69BB-002”为表达TH69BB-002的CAR-T细胞。结果表明这两种CAR-T细胞都能够靶向CD7进行肿瘤细胞杀伤。CD7-CAR-T细胞的流式检测试剂为CD7-CAR-GREEN；细胞体外杀伤实验采用的靶细胞为HeLa-CD7。
图4为采用Intrablock TM CD7表达阻断技术阻断CD7在细胞膜上的表达结果示意图。采用携带可与CD7结合的配体CD7-L或TH69scFV cDNA，并连接ER滞留域的慢病毒载体CD7-L-ER2.1和TH69-ER2.1转导K562-CD7细胞株(A)或T细胞(B)，并进行CD7的流式检测。结果表明这两种CD7表达阻断分子都能够有效减少CD7在细胞表面的表达。
图5为采用Intrablock TM CD7表达阻断技术构建靶向CD7 CAR的慢病毒载体结构示意图。A和B为两种采用Intrablock TM CD7表达阻断技术构建的CD7-CAR慢病毒载体。其中，A、CD7BB-BL4-002，(1)为靶向CD7的嵌合抗原受体示意图，其抗原识别区为CD7-L；(2)为CD7阻断分子示意图，起到CD7表达阻断作用的CD7结合域为单克隆抗体TH69的scFv；B、CD7BB-BL6-002，(3)为靶向CD7的嵌合抗原受体示意图，其抗原识别区为单克隆抗体TH69的scFv；(4)为CD7阻断分子示意图，起到CD7表达阻断作用的CD7结合域为CD7-L；两种CAR载体具有相同的CD8α铰链跨膜区，4-1BB共刺激域、CD3ζT细胞刺激域和ER内质网滞留域。单克隆抗体TH69的scFv采用了CD8α信号肽，其轻链和重链可变区的连接肽为(GGGGS)3。
图6、采用Intrablock TM CD7表达阻断技术后CD7-CAR-T细胞可以克服自杀现象有效地杀伤靶细胞。(a)中，A、流式检测靶向CD7的四种不同CAR在T细胞上的表达；B、流式检测CD7在上述四种不同CAR-T细胞上的表达；所用载体分别为：CD7BB-002、TH69BB-002和采用Intrablock TM CD7表达阻断技术构建的CD7BB-BL4-002和CD7BB-BL6-002；虚线为未转导的T细胞对照，实线为靶向CD7的CAR转导过的T细胞。(b)中，C、上述四种不同CAR-T细胞在体外培养的增殖观察；D、采用iCELLigence TM实时细胞分析仪(Agilent Biosciences,Inc.)进行体外杀伤实验。T cell control为未转导的T细胞对照，CD7BB-002、TH69BB-002、CD7BB-BL4-002 和CD7BB-BL6-002均为靶向CD7的CAR-T细胞，其中CD7BB-BL4-002和CD7BB-BL6-002为采用Intrablock TM CD7表达阻断技术的CD7-CAR-T细胞。CD7-CAR-T细胞的流式检测试剂为CD7-CAR-GREEN；细胞体外杀伤实验采用的靶细胞为HeLa-CD7。
图7、采用Intrablock TM CD7表达阻断技术的CD7-CAR-T细胞体内肿瘤杀伤实验结果图。采用雌性NSG小鼠，D0天注射肿瘤细胞(携带荧光素酶的肿瘤细胞株，CCRF-CEM-Luc,5x10E5/mouse,i.v.)。A、1.阴性对照组；2和3.分别为回输T细胞对照组和靶向CD7的CD7BB-BL4-002 CAR-T细胞治疗组。在注射肿瘤细胞后D3天，分别静脉注射T细胞或CD7BB-BL4-002 CAR-T细胞(8x10E6/mouse)，之后每7天进行小鼠活体荧光素酶成像观察。B、回输T细胞对照组(control group)和CD7BB-BL4-002 CAR-T细胞治疗组(treatment group)小鼠的生存曲线。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变化和改进。这些都属于本发明的保护范围。
本发明将SECTM1(K12)的胞外域作为CD7的抗原识别域，用于开发针对CD7阳性血液恶性肿瘤的CAR-T细胞治疗和免疫毒素。在此专利申请中，使用CD7-L代替SECTM1或K12。CD7-L基因最初被鉴定为在人类17号染色体上CD7基因的5′端。人类CD7-L蛋白主要表达于脾脏、前列腺、睾丸、小肠和外周血白细胞,CD7-L的一个特点是编码一种跨膜蛋白，细胞外结构域类似于免疫球蛋白样的结构域。CD7-L于2000年被克隆，并发现它是CD7的结合蛋白。
为了确定CD7-L蛋白的结合作用，之前的研究采用了CD7-L的细胞外域(氨基酸1-145)与人IgG1的Fc部分的融合蛋白。流式细胞术实验表明，CD7-L-Fc融合蛋白可在人源T细胞和NK细胞上检测到高水平结合。几种靶向CD7的抗体不同程度地阻断了CD7-L-Fc融合蛋白与细胞的结合。反之亦然，CD7-L-Fc融合蛋白可以阻断这些CD7单克隆抗体与CD7的结合，表明CD7-L-Fc可与细胞上的CD7受体结合。将CD7-L-Fc融合蛋白进行放射标记并用于结合实验，以确定其与Jurkat细胞(人类T细胞白血病细胞系)或KG-1细胞(人类粒细胞白血病细胞系)的亲和力，这两种细胞都表达CD7。CD7-L-Fc对人CD7的结合亲和力(Ka)约在1x10 8M -1范围 内。由于CD7被认为是T细胞恶性肿瘤的良好标记物，已有研究通过抗人CD7单克隆抗体偶联蓖麻毒素或皂甙制造免疫毒素。
因此，本专利申请在构建靶向CD7的嵌合抗原受体及其CD7的阻断分子中使用CD7-L的胞外结构域，用于发展CD7-CAR-T或CAR-NK细胞治疗T细胞恶性肿瘤。同时，本专利申请还将CD7-L的胞外区域与毒素相结合，并将这些结合物用作抗T细胞恶性肿瘤的免疫毒素，因其免疫原性低于基于抗体的结合物，因此可能具有更长的半衰期。
实施例1、采用CD7-L为抗原识别域的CAR-T细胞可以有效地识别CD7肿瘤抗原；
本实施例首先构建了以CD7-L或CD7特异性单克隆抗体TH69的scFv为抗原识别区的两种第二代CAR慢病毒载体，CD7BB-002和TH69BB-002(图2A、B)。通过CAR慢病毒载体转导获得CAR-T细胞，并采用CD7和报告基因eGFP融合蛋白生产的CD7-CAR-T细胞的流式检测试剂(CD7-CAR-GREEN)进行流式检测(图3A)。结果证明CD7-CAR-GREEN可以有效地用来检测这两种靶向CD7的CAR-T细胞，说明CD7-L与单克隆抗体TH69的scFv均可作为CAR-T细胞的CD7特异性抗原识别区。
实施例2、靶向CD7的CAR-T细胞可以有效地杀伤表达CD7阳性的肿瘤细胞；
本实施例首先采用携带CD7cDNA的慢病毒载体转导K562和HeLa细胞株，并通过流式分选获得表达CD7的K562-CD7和HeLa-CD7细胞株(图1)。通过CAR慢病毒载体转导获得CD7BB-002和TH69BB-002两种靶向CD7的CAR-T细胞，并采用iCELLigence TM实时细胞分析仪(Agilent Biosciences,Inc.)进行体外杀伤实验(图3B)。结果证明采用CD7-L为抗原识别区的CD7BB-002与采用单克隆抗体TH69scFv为抗原识别区的TH69BB-002均可有效地识别CD7抗原，并在CD7阳性的HeLa-CD7靶细胞杀伤上具有同等效果。
实施例3、Intrablock TM CD7表达阻断技术可以有效地阻断CD7在细胞膜上的表达；
本实施例首先构建了以CD7-L或CD7特异性单克隆抗体TH69的scFv为CD7结合域，并连接ER滞留域的慢病毒载体，CD7-L-ER2.1和TH69-ER2.1(图2C、D)。通过慢病毒载体转导K562-CD7细胞株或原代T细胞，并进行流式检测对CD7表达阻断技术进行评估。结果表明TH69-ER2.1和CD7-L-ER2.1均可有效地阻断CD7在K562-CD7细胞株(图4A)或T细胞(图4B)上的表达，说明TH69-ER2.1和CD7-L-ER2.1均可在细胞内与CD7结合并将其滞留在内质网。因此，这一Intrablock TM CD7表达阻断技术可用来阻断CD7 在细胞上的表达、防止靶向CD7 CAR-T细胞的自杀现象。
实施例4、Intrablock TM CD7表达阻断技术可用来克服CD7-CAR-T细胞的自杀现象和有效地杀伤CD7阳性的靶细胞；
本实施例首先构建了具有Intrablock TM CD7表达阻断功能，同时靶向CD7的CAR慢病毒载体，CD7BB-BL4-002和CD7BB-BL6-002(图5A、B)。由于用作CAR-T细胞制备的T细胞上本身就表达CD7，所以在靶向CD7的CAR-T细胞制备时会发生自杀现象，造成CAR-T细胞的制备困难。如图6所示，靶向CD7的两种CAR-T细胞，CD7BB-002和TH69BB-002均可有效地被CD7-CAR-GREEN所识别(图6A)，并能有效地杀伤CD7阳性的HeLa-CD7靶细胞(图6D)。但是，这两种靶向CD7的CAR-T细胞在体外培养过程中均会发生严重的自杀现象，造成CAR-T细胞在体外扩增和制备上的困难(图6C)。采用实施例3所描述的Intrablock TM CD7表达阻断技术，进行靶向CD7的CD7BB-BL4-002和CD7BB-BL6-002慢病毒载体构建，并用来制备CAR-T细胞。这两种采用Intrablock TM CD7表达阻断技术制备的CAR-T细胞，CD7BB-BL4-002和CD7BB-BL6-002均可有效地被CD7-CAR-GREEN所识别(图6A)、保持HeLa-CD7靶细胞的杀伤能力(图6D)，同时能够阻断CD7的表达(图6B)、克服在CAR-T细胞制备中的自杀现象(图6C)，使靶向CD7特异性CAR-T细胞的体外扩增和制备成为可能。
实施例5、在动物模型上验证采用Intrablock TM CD7表达阻断技术的CD7-CAR-T细胞在体内的肿瘤杀伤功能；
本实施例采用Intrablock TM CD7表达阻断技术的CD7BB-BL4-002 CAR-T细胞进行了体内肿瘤杀伤实验(图7)。采用6-8周的雌性NSG小鼠，D0天尾静脉注射携带荧光素酶的肿瘤细胞株，CCRF-CEM-Luc,5x10E5/mouse,i.v.。在注射肿瘤细胞后D3天，分别静脉注射T细胞(回输T细胞对照组)或CD7BB-BL4-002 CAR-T细胞(8x10E6/mouse)(CAR-T细胞治疗组)。D0天后每7天进行小鼠活体荧光素酶成像观察。结果证明采用Intrablock TM CD7表达阻断技术的CD7BB-BL4-002 CAR-T细胞可以有效地在这一小鼠肿瘤模型上杀伤肿瘤，并延长了CAR-T细胞治疗组小鼠的生存期(如图7中A、B所示)。
与现有技术相比，本发明具有如下的有益效果：本发明是利用人源CD7-L取代抗体序列作为CD7特异性CAR-T细胞的抗原识别域，在靶向CD7 CAR中使用人源CD7-L作为抗原识别域的优点是可以防止宿主产生的细胞和体液反应，从而实现回输后CAR-T细胞在体内的长期生存和更好的疗效。CD7是一种跨膜糖蛋白，通常在 大多数外周T细胞和NK细胞及其前体表达。病变的T细胞和NK细胞本身会高密度表达CD7；缺乏CD7的T细胞在很大程度上表现出不受干扰的发育、稳态和保护功能；由于CD7对外周血T细胞功能的影响不明显，因此CD7是一个很有前途的CAR-T细胞治疗靶点。由于正常和病变的T细胞本身都会表达CD7，所以在进行嵌合抗原受体(Chimeric Antigen Receptor,CAR)T细胞制备时，需要同时考虑两方面的因素：1、对正常T细胞进行基因修饰，使T细胞表达靶向CD7的嵌合抗原受体(CAR-T)，以杀伤CD7阳性的病变T细胞；2、为避免CAR-T细胞发生因相互识别产生的自杀现象，需要阻断CAR-T细胞本身的CD7表达。因此，本发明的技术方案既对正常T细胞进行基因修饰使T细胞表达CD7特异性CAR，同时又兼顾了对正常T细胞内部的CD7表达进行阻断。本发明的申请人经过大量的实验，不断修正验证实验参数，最终获得本发明之技术方案，本发明的技术方案能够对正常T细胞进行基因修饰使T细胞表达CD7特异性CAR，同时能够对正常T细胞的CD7表达进行阻断，实现了意想不到的技术效果。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变化或修改，这并不影响本发明的实质内容。Intrablock系商标标识，也不构成对本发明技术方案的任何限制或缩限。在不冲突的情况下，本申请的实施例和实施例中的特征可以任意相互组合。

Claims (52)

  1. 一种工程化免疫细胞，其中包括编码嵌合抗原受体的多核苷酸序列，其特征是，所述嵌合抗原受体包括靶向CD7的抗原识别域。
  2. 如权利要求1所述的工程化免疫细胞，其特征是，所述嵌合抗原受体还包括铰链跨膜域、细胞内共刺激域和细胞内主要刺激域。
  3. 如权利要求2所述的工程化免疫细胞，其特征是，所述嵌合抗原受体中铰链跨膜域包括选自以下组合中的至少一种氨基酸序列：来源于T细胞受体的α链、β链，CD3δ,CD3ε,CD3γ,CD3ζ链,CD4,CD5,CD8α,CD8β,CD9,CD16,CD22,CD28,CD32,CD33,CD34,CD35,CD37,CD45,CD64,CD80,CD86,CD137,ICOS,CD154,FAS,FGFR2B,OX40或VEGFR2的氨基酸序列。
  4. 如权利要求2所述的工程化免疫细胞，其特征是，所述嵌合抗原受体中细胞内共刺激域包括选自以下组中的至少一种：为CD2,CD4,CD5,CD8α,CD8β,CD27,CD28,CD30,CD40,4-1BB(CD137),ICOS，OX40,LIGHT(CD258)或NKG2C。
  5. 如权利要求2所述的工程化免疫细胞，其特征是，所述嵌合抗原受体中细胞内主要刺激域包括选自以下组合中的至少一种：CD3δ,CD3ε,CD3γ,CD3ζ,FcRβ,FcRγ,CD5,CD66d,CD22,CD79a或CD79b。
  6. 如权利要求1所述的工程化免疫细胞，其特征是，所述嵌合抗原受体中靶向CD7的抗原识别域为人源CD7-L细胞外结构域的部分或全部序列，或者与人源CD7-L细胞外结构域至少具有90％的序列一致性，所述人源CD7-L细胞外结构域的氨基酸序列如SEQ ID NO.5所示。
  7. 如权利要求6所述的工程化免疫细胞，其特征是，所述工程化免疫细胞内还包括CD7阻断分子，CD7阻断分子能够阻止CD7蛋白在细胞表面的转运与表达。
  8. 如权利要求7所述的工程化免疫细胞，其特征是，所述CD7阻断分子具体为通过CD7结合域连接胞内锚定域阻止CD7蛋白向细胞表面转运。
  9. 如权利要求8所述的工程化免疫细胞，其特征在于，所述胞内锚定域为内质网滞留域、高尔基体滞留域或蛋白酶体定位域的氨基酸序列。
  10. 如权利要求8所述的工程化免疫细胞，其特征是，所述CD7结合域为人源CD7-L细胞外结构域的部分或全部序列，或者与人源CD7-L细胞外结构域至少具有90％的序列一致性的蛋白质，所述人源CD7-L细胞外结构域的氨基酸序列如SEQ ID NO.5所示。
  11. 如权利要求8所述的工程化免疫细胞，其特征是，所述CD7结合域为抗CD7的 单克隆抗体TH69的scFv。
  12. 如权利要求1所述的工程化免疫细胞，其特征是，所述嵌合抗原受体中靶向CD7的抗原识别域为抗CD7的单克隆抗体TH69的scFv，或者与抗CD7的单克隆抗体TH69的scFv至少具有90％的序列一致性，所述抗CD7的单克隆抗体TH69的scFv的氨基酸序列如SEQ ID NO.8所示。
  13. 如权利要求12所述的工程化免疫细胞，其特征是，所述工程化免疫细胞内还包括CD7阻断分子，CD7阻断分子能够阻止CD7蛋白在细胞表面的转运与表达。
  14. 如权利要求12所述的工程化免疫细胞，其特征是，所述CD7阻断分子为通过CD7结合域连接胞内锚定域阻止CD7蛋白向细胞表面转运。
  15. 如权利要求14所述的工程化免疫细胞，其特征是，所述胞内锚定域为内质网滞留域、高尔基体滞留域或蛋白酶体定位域的氨基酸序列。
  16. 如权利要求14所述的工程化免疫细胞，其特征是，所述CD7结合域为人源CD7-L细胞外结构域的部分或全部序列，或者与人源CD7-L细胞外结构域至少具有90％的序列一致性，所述人源CD7-L细胞外结构域的氨基酸序列如SEQ ID NO.5所示。
  17. 如权利要求6或12所述的工程化免疫细胞，其特征在于，所述工程化免疫细胞通过基因敲除技术去除CD7的基因表达。
  18. 如权利要求17所述的工程化免疫细胞，其特征是，所述敲除技术采用的基因组编辑工具为TALENs或CRISPR/cas9。
  19. 如权利要求1-16、18任一项所述的工程化免疫细胞，其特征是，所述细胞为T细胞、γδT细胞、NK或NKT细胞、及诱导多能干细胞分化的T细胞、γδT细胞、NK或NKT细胞。
  20. 一种嵌合抗原受体中靶向CD7的抗原识别域，其特征是，所述抗原识别域的序列包括部分或全部序列的人源CD7-L细胞外结构域，或者与人源CD7-L细胞外结构域至少具有90％序列一致性；所述人源CD7-L细胞外结构域的氨基酸序列如SEQ ID NO.5所示。
  21. 一种如权利要求20所述的靶向CD7的抗原识别域在制备抗CD7阳性血液恶性肿瘤的免疫毒素中的应用。
  22. 一种编码如权利要求20所述的嵌合抗原受体中靶向CD7的抗原识别域的核酸分子。
  23. 根据权利要求22所述的编码靶向CD7的抗原识别域的核酸分子，其特征是，所述核酸分子编码序列为CD7BB-002，所述CD7BB-002的核酸分子序列如SEQ ID NO.9 所示。
  24. 一种重组载体，其特征是，包含如权利要求22所述的编码嵌合抗原受体中靶向CD7的抗原识别域的核酸分子。
  25. 如权利要求24所述的重组载体，其特征是，载体选自逆转录病毒、慢病毒或转座子。
  26. 一种如权利要求24所述的重组载体在制备工程化免疫细胞中的用途。
  27. 一种用于制备权利要求1-16任一项所述的工程化免疫细胞的CD7阻断分子，其特征是，所述CD7阻断分子的CD7结合域包括部分或全部序列的人源CD7-L细胞外结构域，或与人源CD7-L细胞外结构域至少具有90％序列一致性的蛋白质。
  28. 一种编码权利要求27所述的CD7阻断分子的核酸序列。
  29. 如权利要求28所述的编码CD7阻断分子的核酸序列，其特征是，所述核酸序列为CD7-L-ER2.1，所述CD7-L-ER2.1的核酸分子序列如SEQ ID NO.10所示。
  30. 一种用于制备权利要求1-16任一项所述的工程化免疫细胞的CD7阻断分子，其特征是，所述CD7阻断分子的CD7结合域为抗CD7的单克隆抗体TH69的scFv，或者与抗CD7的单克隆抗体TH69的scFv至少具有90％的序列一致性。
  31. 一种编码权利要求30所述的CD7阻断分子的核酸序列，其特征是，所述CD7阻断分子的核酸分子的编码序列为TH69-ER2.1，所述TH69-ER2.1的核酸分子序列如SEQ ID NO.11所示。
  32. 一种重组载体，其特征是，包括如权利要求29或31所述的核酸序列。
  33. 根据权利要求32所述的重组载体，其特征是，所述载体选自逆转录病毒、慢病毒或转座子。
  34. 一种如权利要求32所述的重组载体在制备工程化免疫细胞中的用途。
  35. 一种核酸分子，其特征在于，包括：
    (1)编码含有如权利要求20所述的抗原识别域序列的核酸序列；
    以及(2)编码如权利要求27或权利要求30所述的CD7阻断分子的核酸序列。
  36. 一种重组载体，其特征在于，包括如权利要求35所述的核酸分子。
  37. 根据权利要求36所述的重组载体，其特征是，所述载体选自逆转录病毒、慢病毒、转座子。
  38. 如权利要求36所述的重组载体，其特征是，所述载体中编码嵌合抗原受体的核酸分子和编码CD7阻断分子的核酸分子由内部核糖体进入位点或核糖体密码子跳过位点连接。
  39. 如权利要求36所述的重组载体，其特征是，所述载体的内部核糖体进入位点来源于脑心肌炎病毒或肠病毒。
  40. 如权利要求36所述的重组载体，其特征是，所述载体的核糖体密码子跳过位点包含2A自裂解肽，2A自裂解肽可以选自口蹄疫病毒F2A肽，马甲鼻炎病毒E2A肽，猪破伤风病毒P2A肽或T2A肽。
  41. 一种如权利要求36所述的重组载体在制备工程化免疫细胞中的用途。
  42. 一种试剂组合，其特征在于，所述组合包括：(1)如权利要求24所述的重组载体，以及(2)如权利要求32所述的重组载体。
  43. 一种如权利要求42所述的试剂组合在制备治疗CD7阳性血液恶性肿瘤的CAR-T或CAR-NK细胞中的用途。
  44. 一种试剂组合，其特征是，所述组合包括：(1)如权利要求24所述的重组载体，以及(2)能够敲除细胞中的CD7基因的基因编辑工具。
  45. 如权利要求44所述的试剂组合，其特征是，所述基因编辑工具为TALENs或CRISPR/cas9。
  46. 一种嵌合抗原受体中靶向CD7的抗原识别域序列，其特征在于，所述序列为抗CD7的单克隆抗体TH69的scFv，或者与抗CD7的单克隆抗体TH69的scFv至少具有90％的序列一致性，所述抗CD7的单克隆抗体TH69的scFv的氨基酸序列如SEQ ID NO.8所示。
  47. 一种编码如权利要求46所述的嵌合抗原受体中靶向CD7的抗原识别域序列的核酸分子。
  48. 如权利要求47所述的核酸分子，其特征是，所述核酸分子编码序列为TH69BB-002，所述TH69BB-002的核酸分子序列如SEQ ID NO.18所示。
  49. 一种重组载体，其特征在于，包含如权利要求47所述的核酸分子的序列。
  50. 如权利要求49所述的重组载体，其特征是，载体选自逆转录病毒、慢病毒、转座子。
  51. 一种试剂组合，其特征在于，所述组合包括：
    (1)如权利要求49所述的重组载体，
    以及(2)选择以下组合中的一种：含有权利要求29所述核酸序列的重组载体，或能够敲除细胞中的CD7基因的基因编辑工具。
  52. 一种如权利要求51所述的试剂组合在制备治疗CD7阳性血液恶性肿瘤的CAR-T或CAR-NK细胞中的用途。
PCT/CN2021/133817 2021-01-12 2021-11-29 靶向cd7的工程化免疫细胞、嵌合抗原受体、cd7阻断分子及应用 WO2022151851A1 (zh)

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JP2023542493A JP2024502632A (ja) 2021-01-12 2021-11-29 Ｃｄ７を標的とする改変免疫細胞、キメラ抗原受容体、ｃｄ７ブロッキング分子およびその応用
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116003631A (zh) * 2022-11-15 2023-04-25 江苏元晟生物科技有限公司 靶向cd7的人源化抗体及其应用
WO2023133595A2 (en) 2022-01-10 2023-07-13 Sana Biotechnology, Inc. Methods of ex vivo dosing and administration of lipid particles or viral vectors and related systems and uses
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024119157A1 (en) 2022-12-02 2024-06-06 Sana Biotechnology, Inc. Lipid particles with cofusogens and methods of producing and using the same

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115786271A (zh) * 2021-01-12 2023-03-14 上海雅科生物科技有限公司 靶向cd7的工程化免疫细胞、嵌合抗原受体、cd7阻断分子及应用
CN114716564B (zh) * 2021-12-20 2023-02-28 四川大学华西医院 基于sectm1构建的嵌合抗原受体免疫细胞制备及其应用
CN114560943B (zh) * 2022-02-28 2022-12-16 先进生物(苏州)有限公司 Cd7-car-t细胞及其制备方法和应用
CN114685662B (zh) * 2022-03-30 2022-12-27 河北森朗生物科技有限公司 抗cd7纳米抗体、衍生物及其在肿瘤治疗中的应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107249602A (zh) * 2015-02-27 2017-10-13 美商生物细胞基因治疗有限公司 靶向血液恶性肿瘤之嵌合抗原受体（car）、其组合物及使用方法
US20180148506A1 (en) * 2016-11-22 2018-05-31 National University Of Singapore Blockade of cd7 expression and chimeric antigen receptors for immunotherapy of t-cell malignancies
US20190144522A1 (en) * 2016-06-06 2019-05-16 St. Jude Children's Research Hospital Anti-cd7 chimeric antigen receptor and method of use thereof
CN110760007A (zh) * 2019-11-21 2020-02-07 博生吉医药科技(苏州)有限公司 Cd7-car-t细胞及其制备和应用
WO2020102589A1 (en) * 2018-11-14 2020-05-22 Medisix Therapeutics Pte Ltd. Two-gene vectors for generating car-t cells and uses thereof
CN113234682A (zh) * 2021-01-12 2021-08-10 上海雅科生物科技有限公司 靶向cd7的工程化免疫细胞、嵌合抗原受体、cd7阻断分子及应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201622044D0 (en) * 2016-12-22 2017-02-08 Ucl Business Plc T cell-targeted T cells
WO2018231871A1 (en) * 2017-06-12 2018-12-20 Emory University T-cell antigen targeted chimeric antigen receptor (car) and uses in cell therapies

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107249602A (zh) * 2015-02-27 2017-10-13 美商生物细胞基因治疗有限公司 靶向血液恶性肿瘤之嵌合抗原受体（car）、其组合物及使用方法
US20190144522A1 (en) * 2016-06-06 2019-05-16 St. Jude Children's Research Hospital Anti-cd7 chimeric antigen receptor and method of use thereof
US20180148506A1 (en) * 2016-11-22 2018-05-31 National University Of Singapore Blockade of cd7 expression and chimeric antigen receptors for immunotherapy of t-cell malignancies
WO2020102589A1 (en) * 2018-11-14 2020-05-22 Medisix Therapeutics Pte Ltd. Two-gene vectors for generating car-t cells and uses thereof
CN110760007A (zh) * 2019-11-21 2020-02-07 博生吉医药科技(苏州)有限公司 Cd7-car-t细胞及其制备和应用
CN113234682A (zh) * 2021-01-12 2021-08-10 上海雅科生物科技有限公司 靶向cd7的工程化免疫细胞、嵌合抗原受体、cd7阻断分子及应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BADE-DÖDING CHRISTINA; GÖTTMANN WIEBKE; BAIGGER ANJA; FARREN MATTHEW; LEE KELVIN P.; BLASCZYK RAINER; HUYTON TREVOR : "Autocrine GM-CSF transcription in the leukemic progenitor cell line KG1a is mediated by the transcription factor ETS1 and is negatively regulated through SECTM1 mediated ligation of CD7", BIOCHIMICA ET BIOPHYSICA ACTA, ELSEVIER, AMSTERDAM, NL, vol. 1840, no. 3, 6 November 2013 (2013-11-06), AMSTERDAM, NL , pages 1004 - 1013, XP028817239, ISSN: 0304-4165, DOI: 10.1016/j.bbagen.2013.10.043 *
GOMES-SILVA DIOGO; SRINIVASAN MADHUWANTI; SHARMA SANDHYA; LEE CIARAN M.; WAGNER DIMITRIOS L.; DAVIS TIMOTHY H.; ROUCE RAYNE H.; BA: "CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 130, no. 3, 20 July 2017 (2017-07-20), US , pages 285 - 296, XP086677144, ISSN: 0006-4971, DOI: 10.1182/blood-2017-01-761320 *
LYMAN S D ET AL: "Identification of CD7 as a cognate of the human K12 (SECTM1) protein.", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 275, no. 5, 4 February 2000 (2000-02-04), US , pages 3431 - 3437, XP002156210, ISSN: 0021-9258, DOI: 10.1074/jbc.275.5.3431 *
SHI JUMEI, ET AL.: "Uses of Natural Killer Cells in Treating Hematologic Tumors", SHANGHAI MEDICAL JOURNAL, vol. 33, no. 9, 31 December 2010 (2010-12-31), pages 830 - 833, XP055951548 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023133595A2 (en) 2022-01-10 2023-07-13 Sana Biotechnology, Inc. Methods of ex vivo dosing and administration of lipid particles or viral vectors and related systems and uses
WO2024040194A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
WO2024040195A1 (en) 2022-08-17 2024-02-22 Capstan Therapeutics, Inc. Conditioning for in vivo immune cell engineering
CN116003631A (zh) * 2022-11-15 2023-04-25 江苏元晟生物科技有限公司 靶向cd7的人源化抗体及其应用
CN116003631B (zh) * 2022-11-15 2024-01-26 江苏元晟生物科技有限公司 靶向cd7的人源化抗体及其应用
WO2024119157A1 (en) 2022-12-02 2024-06-06 Sana Biotechnology, Inc. Lipid particles with cofusogens and methods of producing and using the same

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