WO2022105893A1 - 一种cd7-car-t细胞的制备方法及应用 - Google Patents

一种cd7-car-t细胞的制备方法及应用 Download PDF

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WO2022105893A1
WO2022105893A1 PCT/CN2021/131917 CN2021131917W WO2022105893A1 WO 2022105893 A1 WO2022105893 A1 WO 2022105893A1 CN 2021131917 W CN2021131917 W CN 2021131917W WO 2022105893 A1 WO2022105893 A1 WO 2022105893A1
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cells
car
cell
another preferred
viral vector
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PCT/CN2021/131917
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French (fr)
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杨林
汪敏
王宇
潘桂芳
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博生吉医药科技(苏州)有限公司
博生吉安科细胞技术有限公司
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Priority to US18/253,819 priority Critical patent/US20240016842A1/en
Priority to EP21894040.1A priority patent/EP4249599A1/en
Publication of WO2022105893A1 publication Critical patent/WO2022105893A1/zh

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Definitions

  • the invention relates to the field of biotechnology, and more particularly to a preparation method and application of CD7-CAR-T cells.
  • T-cell-derived malignancies represent a typical hematological malignancy with high recurrence and mortality rates in both children and adults, and there is currently no very effective treatment.
  • T-cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous hematological malignancy that accounts for approximately 25% of adult acute lymphoblastic leukemia cases and approximately 15% of childhood acute lymphoblastic leukemia cases.
  • the clinical symptoms of T-ALL mainly include infection, anemia, fever, abnormal bleeding, etc.
  • T-ALL progresses rapidly and can rapidly infiltrate into lymph nodes, liver, spleen, central nervous system and testis and other tissues and organs in a short period of time.
  • T-ALL T-cell acute lymphoblastic leukemia
  • allo-HSCT allogeneic hematopoietic stem cell transplantation
  • molecular targeted therapy usually does not prevent disease recurrence after treatment.
  • salvage chemotherapy regimens is only about 20-40%.
  • hematopoietic stem cell transplantation may be the only curative option at present, it is also associated with life-threatening graft-versus-host disease.
  • CAR-T chimeric antigen receptor T cell
  • CAR-T therapy targets and eliminates malignant tumors in a major histocompatibility complex-independent manner by genetically modifying a patient's T lymphocytes.
  • One of the keys to the effective application of this technology is to select an appropriate target to construct the corresponding CAR molecule.
  • the optimal target antigen should be expressed only on tumor cells, not on normal cells, or it can also be expressed on cells that have clinical response plans after the transient loss of normal cells.
  • CD7 The CD7 molecule is highly expressed on most T-cell hematological tumor cells.
  • CD7 is a transmembrane glycoprotein expressed by T, NK cells and their precursor cells and stimulates T cell activation upon binding to its ligand K12/SECTM1.
  • the CD7 molecule does not appear to play a critical role in T cell development or function, since disruption of the CD7 molecule in the progenitors of murine T cells does not affect normal T cell development and homeostasis, and has no effect on T cell effector function. influences.
  • CD7 has been used as an antibody target for the treatment of T-cell lymphoma, and patients did not experience serious adverse reactions after targeting CD7.
  • the CD7 molecule may be a very suitable therapeutic target for T-cell hematological tumors.
  • targeting T-cell acute lymphoblastic leukemia with CD7-CAR-T cells still faces great problems, because both normal effector T cells and T-cell tumors express CD7 antigen, which can lead to the development of CD7-CAR-T cells. Cannibalism, CD7-CAR-T cells are difficult to prepare successfully in vitro.
  • the purpose of the present invention is to provide a preparation method and application of CD7-CAR-T cells.
  • the present invention provides a method for preparing CD7-CAR-T cells based on humanized CD7 nanobody sequences.
  • the present invention also provides the results of in vitro killing of CD7-positive tumor cells by CD7-CART cells constructed with PA007CD and PA107CD vectors.
  • a method for preparing CD7-CAR-T cells comprising the steps of:
  • the first viral vector for expressing CD7 blocking molecule is introduced into the activator of T cells.
  • the interval between the introduction of the first viral vector and the introduction of the second viral vector is 36-60 h, preferably, the interval is 42-54 h, and more preferably, the interval is 46-50 h.
  • step (iii) comprise step:
  • step (b) the step of removing the activator added in step (ii) is further included.
  • step (b) the first viral vector and the activator in the first transfection mixture are removed.
  • step (iv) including steps:
  • the first viral vector and the second viral vector are lentiviral vectors.
  • the co-cultivation is carried out at 36-38°C.
  • step (a) the number ratio (multiplicity of infection) of the first viral vector to T cells is 1:0.05-0.4.
  • step (c) the number ratio (multiplicity of infection) of the second viral vector to T cells is 1:0.5-4.
  • the T cells are washed with a buffer to remove viral vectors.
  • the sample is peripheral blood mononuclear cells.
  • step (ii) an activator is used to carry out the sorting and activation.
  • the activator is magnetic beads targeting CD3 and CD28.
  • the magnetic beads are Medini Transact (which is easy to remove, degrade, and has a good activation effect).
  • Medini Transact is the magnetic beads targeting CD3 and CD28 used in the examples.
  • the ratio of the T cells to the activator is 1 ⁇ 10 6 : 5-20ul.
  • the magnetic beads targeting CD3/CD28 are incubated with T cells for 12-36h, preferably, 18-30h, more preferably, 22-26h, and then used to express CD7 blocking
  • the molecule's first viral vector is introduced into the activated T cells.
  • step (iv) it also includes steps:
  • the antigen binding domain of the CD7-CAR includes one or more CD7 nanobodies.
  • amino acid sequence of the CD7 Nanobody is shown in SEQ ID NO: 13.
  • the CAR further comprises an ICOS-derived transmembrane region and/or an ICOS-derived intracellular co-stimulatory region.
  • the CAR comprises an ICOS-derived intracellular costimulatory region and a 4-1BB-derived intracellular costimulatory region.
  • each "-" is independently a linking peptide or peptide bond
  • L is the signal peptide sequence
  • VHH is an antigen binding domain comprising one or two CD7 Nanobodies
  • H is the hinge region
  • TM is the transmembrane domain
  • C is a costimulatory signal molecule
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ .
  • the structure of the CAR is shown in Figure 1 .
  • the L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.
  • the L is a CD8-derived signal peptide.
  • amino acid sequence of L is shown in SEQ ID NO:4.
  • the VHH includes two CD7 Nanobodies, preferably, the two CD7 Nanobodies are connected by a connecting peptide, more preferably, the amino acid sequence of the connecting peptide is as SEQ ID NO:8 shown.
  • nucleotide sequence of the connecting peptide is shown in SEQ ID NO:7.
  • the CD7 Nanobodies include humanized Nanobodies and camel-derived Nanobodies, preferably humanized Nanobodies.
  • the VHH is a humanized CD7 nanobody.
  • the H is a hinge region derived from a protein selected from the group consisting of Fc, CD8, CD28, CD137, or a combination thereof.
  • the H is an Fc fragment, preferably, the Fc fragment is a human IgG4 Fc fragment.
  • amino acid sequence of the Fc fragment is shown in SEQ ID NO: 14.
  • the TM is a transmembrane region of a protein selected from the group consisting of: ICOS, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80 , CD86, CD134, CD137, CD154, or a combination thereof.
  • the TM is a transmembrane region derived from ICOS.
  • amino acid sequence of the ICOS-derived transmembrane region is shown in SEQ ID NO: 15.
  • the C is a costimulatory signal molecule selected from the following group of proteins: ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137) , PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.
  • proteins ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137) , PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.
  • the C is a costimulatory signal molecule derived from ICOS and/or 4-1BB.
  • amino acid sequence of the costimulatory signal molecule derived from ICOS is shown in SEQ ID NO: 16.
  • amino acid sequence of the costimulatory signal molecule derived from 4-1BB is shown in SEQ ID NO: 17.
  • amino acid sequence of CD3 ⁇ is shown in SEQ ID NO:18.
  • the CAR (preferably C-terminal) further comprises a cell suicide element.
  • the CAR is linked to the cell suicide element through a self-cleaving element.
  • the cell suicide element is linked to the CD3 ⁇ of the CAR through T2A.
  • the cell suicide element is selected from the group consisting of HSV-TK, iCasp9, ⁇ CD20, mTMPK, ⁇ CD19, RQR8, EGFRt, or a combination thereof.
  • the cell suicide element is tEGFR.
  • amino acid sequence of the tEGFR is shown in SEQ ID NO: 20.
  • amino acid sequence of the T2A is shown in SEQ ID NO: 19.
  • the CAR is PA107CD CAR (namely, the CD7 CAR named PA3-17 in the Chinese patent application with application number 201911150678.7).
  • amino acid sequence of the CAR is shown in SEQ ID NO: 12.
  • the CD7 blocking molecule comprises one or more CD7 nanobodies and endoplasmic reticulum retention sequences.
  • CD7 blocking molecule In another preferred example, the structure of the CD7 blocking molecule is shown in the following formula II:
  • each "-" is independently a linking peptide or peptide bond
  • L' is the signal peptide sequence
  • VHH' is the binding region comprising two CD7 Nanobodies
  • ER is the endoplasmic reticulum retention sequence.
  • amino acid sequence of L' is shown in SEQ ID NO:4.
  • nucleotide sequence of L' is shown in SEQ ID NO: 3.
  • the two CD7 Nanobodies of the VHH' are connected by a connecting peptide, more preferably, the connecting peptide is shown in SEQ ID NO: 8.
  • amino acid sequence of the VHH' is shown in SEQ ID NO:6.
  • nucleotide sequence of the VHH' is shown in SEQ ID NO:5.
  • the VHH' is a camel-derived nanobody.
  • amino acid sequence of the ER is shown in SEQ ID NO: 10.
  • nucleotide sequence of the ER is shown in SEQ ID NO:9.
  • amino acid sequence of the CD7 blocking molecule is shown in SEQ ID NO:2.
  • nucleotide sequence of the CD7 blocking molecule is shown in SEQ ID NO: 1.
  • a CD7-CAR-T cell prepared by the method of the first aspect of the present invention is provided.
  • the CD7-CAR-T cells at least 80%, preferably 90%, more preferably 95%, and optimally 98% of the endogenous CD7 expression is blocked.
  • a preparation comprising the CD7-CAR-T cells of the second aspect of the present invention and a pharmaceutically acceptable carrier.
  • the preparation is a liquid preparation.
  • the dosage form of the preparation is an injection.
  • the concentration of the engineered immune cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml ml.
  • a method for treating a disease comprising administering an appropriate amount of the CD7-CAR-T cells described in the second aspect of the present invention or the preparation described in the third aspect of the present invention to a subject in need of treatment .
  • the disease is cancer or tumor.
  • Figure 1 shows the carrier structure diagram of the CD7-CAR and CD blocking molecules of the present invention.
  • Figure 2 shows a flow chart of the preparation of the CD7-CART of the present invention.
  • Figure 3 shows the detection of CD7 molecule expression on the surface of T cells (using the CD7 antibody of the FITC channel to detect the expression of the CD7 molecule), the transfection efficiency of CD7-CART (using the CD3 antibody of the PE channel to detect the expression of T cells, and the use of the FC antibody of the APC channel) Detection of CAR expression) flow chart.
  • Figure 4 shows the cytotoxicity of CD7-CAR-T cells against CD7-overexpressing 293T cells.
  • Figure 5 shows the cytotoxicity of CD7-CAR-T cells against acute lymphoblastic leukemia cells (CEM) that naturally express CD7.
  • CEM acute lymphoblastic leukemia cells
  • Figure 6 shows cytokine secretion after CD7-CAR-T cells were incubated with 293T-CD7 cells.
  • Figure 7 shows the cytokine secretion after incubation of CD7-CAR-T cells with CEM cells.
  • the inventors unexpectedly discovered a preparation method and application of CD7-CAR-T cells for the first time.
  • the method of the present invention improves the transfection efficiency and enhances the cytotoxicity of CAR-T cells by adjusting the transfection sequence and transfection time of the CD7 blocking molecule-expressing lentivirus and the CD7-CAR-expressing lentivirus.
  • the present invention has been completed on this basis.
  • the present invention is to provide a method for preparing CD7-CAR-T cells based on humanized CD7 nanobody sequences. Since the preparation process of CD7-CAR-T cells involves the transfection of two lentiviruses, one is One is the PA007CD virus that anchors the CD7 antigen expressed on the surface of T cells in the endoplasmic reticulum or Golgi apparatus, and the other is the PA107CD lentivirus that can express CD7-CAR on the surface of T cells. The inventors found that the order in which the two lentiviruses are used, the time interval of transfection and other factors will directly affect the quality of the final CART cells.
  • the present invention describes the CD7-CAR that transfects PA007CD lentivirus 24 hours after T cell sorting and activation, washes and removes PA007CD lentivirus and activator after 48 hours, and then cleans and removes PA107CD lentivirus 24 hours after transfection of PA107CD lentivirus -T preparation method.
  • the CD7-CAR-T cells prepared by this method have higher CAR transfection efficiency and better cell viability. It has been proved by in vitro functional experiments that the CD7-CAR-T cells prepared in this application can specifically kill CD7-positive cells. target cells and secrete higher specific cytokines.
  • administration refers to the physical introduction of a product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or Other routes of parenteral administration, such as by injection or infusion.
  • antibody shall include, but is not limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigens thereof combined part.
  • Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region contains three constant domains, CH1, CH2 and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region contains one constant domain, CL.
  • VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL contains three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • amino acids in this article are identified by the international single English letter, and the corresponding three English letter abbreviations of the amino acid names are: Ala(A), Arg(R), Asn(N), Asp(D), Cys (C), Gln(Q), Glu(E), Gly(G), His(H), I1e(I), Leu(L), Lys(K), Met(M), Phe(F), Pro (P), Ser(S), Thr(T), Trp(W), Tyr(Y), Val(V).
  • CD7 blocking molecule PA007CD
  • CD7-CAR PA107CD
  • the design of CARs has gone through the following process: the first-generation CAR has only one intracellular signal component, CD3 ⁇ or Fc ⁇ RI molecule. Since there is only one activation domain in the cell, it can only cause transient T cell proliferation and less cytokine secretion. , and can not provide long-term T cell proliferation signal and sustained anti-tumor effect in vivo, so it has not achieved good clinical efficacy.
  • the second-generation CARs introduce a costimulatory molecule on the basis of the original structure, such as CD28, 4-1BB, OX40, and ICOS. Compared with the first-generation CARs, the function is greatly improved, which further strengthens the persistence of CAR-T cells and promotes tumor cells. destructive ability. On the basis of second-generation CARs, some new immune costimulatory molecules such as CD27 and CD134 are connected in series to develop into third- and fourth-generation CARs.
  • the chimeric antigen receptor (CAR) of the present invention includes an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes target-specific binding elements (also referred to as antigen binding domains).
  • the intracellular domain includes the costimulatory signaling region and the zeta chain portion.
  • a costimulatory signaling region refers to a portion of an intracellular domain that includes a costimulatory molecule.
  • Costimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, that are required for effective lymphocyte responses to antigens.
  • a linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.
  • the term "linker” generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain.
  • the linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.
  • the extracellular domain of the CAR provided by the present invention includes an antigen binding domain targeting CD7.
  • the CAR of the present invention when expressed in T cells, is capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, it affects tumor cells, causing the tumor cells to not grow, being driven to die, or otherwise being affected, and resulting in a reduction or elimination of the patient's tumor burden.
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the zeta chain.
  • the antigen binding domain is fused to the intracellular domain in combination with the ICOS and/or 4-1BB signaling domain, and the CD3 ⁇ signaling domain.
  • antigen binding domain refers to a Fab fragment, a Fab' fragment, an F(ab') 2 fragment, or a single Fv fragment having antigen-binding activity.
  • the antigen binding domain comprises one or more Nanobodies that specifically recognize CD7.
  • the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.
  • the transmembrane domain naturally associated with one of the domains in the CAR is used.
  • transmembrane domains may be selected, or modified by amino acid substitutions, to avoid binding such domains to transmembrane domains of the same or different surface membrane proteins, thereby minimizing interaction with receptor complexes interactions with other members.
  • the intracellular domain in the CAR of the present invention includes the signaling domain of ICOS and/or 4-1BB and the signaling domain of CD3 ⁇ .
  • the present invention also provides CAR-T cells targeting CD7. Unless otherwise specified, the expression of CD7 on the surface of the CAR-T cells of the present invention is blocked. blocked by the molecule.
  • the CAR-T cells in the present invention all have a suicide gene switch, which can effectively remove CAR-T cells in the body under the action of exogenous drugs. , blocking unknown or uncontrollable long-term toxicity to ensure patient safety.
  • the suicide gene switch used in the present invention can be HSV-TK, iCasp9, CD20, mTMPK and the like.
  • HSV-TK, iCasp9 and CD20 have the same ability to clear T cells, but iCasp9 and CD20 clear faster, and HSV-TK clears slower.
  • the iCasp9 suicide gene contains the FKBP12-F36V domain, which can be linked to caspase 9 via a flexible linker, which does not contain a recruitment domain.
  • FKBP12-F36V contains a FKBP domain with phenylalanine instead of valine at the 36th amino acid residue position. With high selectivity and sub-nanomolar affinity, it can bind dimerized into ligands such as other inert small molecules AP1903. When the small molecule is added, it can promote its dimerization, thereby inducing cell apoptosis, but it has no effect on normal cells that do not carry the suicide gene.
  • Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to include the gene, or by using standard technology to isolate directly from cells and tissues that contain the gene. Alternatively, the gene of interest can be produced synthetically.
  • the present invention also provides vectors into which the expression cassettes of the present invention are inserted.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools to achieve long-term gene transfer because they allow long-term, stable integration of the transgene and its proliferation in daughter cells.
  • Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia virus because they can transduce non-proliferating cells such as hepatocytes. They also have the advantage of low immunogenicity.
  • an expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector.
  • the vector is suitable for replication and integration in eukaryotic cells.
  • Typical cloning vectors contain transcriptional and translational terminators, initial sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence.
  • the expression constructs of the present invention can also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, eg, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are hereby incorporated by reference in their entirety.
  • the present invention provides gene therapy vectors.
  • the nucleic acid can be cloned into many types of vectors.
  • the nucleic acid can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • expression vectors can be provided to cells in the form of viral vectors.
  • Viral vector techniques are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO01/96584; WO01/29058; and U.S. Patent No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to subject cells in vivo or ex vivo.
  • Many retroviral systems are known in the art.
  • adenoviral vectors are used.
  • Many adenoviral vectors are known in the art.
  • lentiviral vectors are used.
  • promoter elements can regulate the frequency of transcription initiation. Typically, these are located in a region of 30-110 bp upstream of the initiation site, although it has recently been shown that many promoters also contain functional elements downstream of the initiation site.
  • the spacing between promoter elements is often flexible so that promoter function is maintained when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50 bp before activity begins to decline.
  • individual elements appear to act cooperatively or independently to initiate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is elongation growth factor-1 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences can also be used, including but not limited to the simian virus 40 (SV40) early promoter, the mouse breast cancer virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russell sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter , myosin promoter, heme promoter and creatine kinase promoter.
  • the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention.
  • an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off expression when expression is not desired.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • the expression vector introduced into the cells may also contain either or both of a selectable marker gene or a reporter gene to facilitate the search for the transfected or infected cell population from the viral vector Identification and selection of expressing cells.
  • the selectable marker can be carried on a single piece of DNA and used in co-transfection procedures. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.
  • Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is clearly indicated by some readily detectable property such as enzymatic activity. After the DNA has been introduced into the recipient cells, the expression of the reporter gene is measured at an appropriate time.
  • Suitable reporter genes can include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (eg, Ui-Tei et al., 2000 FEBS Letters 479:79). -82).
  • Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. Typically, constructs with a minimum of 5 flanking regions showing the highest levels of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to assess the ability of an agent to modulate promoter-driven transcription.
  • an expression vector can be readily introduced into a host cell, eg, mammalian, bacterial, yeast or insect cells, by any method known in the art.
  • a host cell eg, mammalian, bacterial, yeast or insect cells
  • an expression vector can be transferred into a host cell by physical, chemical or biological means.
  • Physical methods of introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, eg, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). The preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.
  • Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors.
  • Viral vectors especially retroviral vectors, have become the most widely used method of inserting genes into mammalian, eg, human cells.
  • Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, among others. See, eg, US Patent Nos. 5,350,674 and 5,585,362.
  • colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids plastid.
  • lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and lipids plastid.
  • Exemplary colloidal systems for use as in vitro and in vivo delivery vehicles are liposomes (eg, artificial membrane vesicles).
  • exemplary delivery vehicles are liposomes.
  • lipid formulations is contemplated to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo).
  • nucleic acid can be associated with a lipid.
  • Nucleic acids associated with lipids can be encapsulated into the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, attached via linker molecules associated with both liposomes and oligonucleotides to liposomes, entrapped in liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, associated with lipids, contained in lipids as a suspension, contained in micelles or Complex with micelles, or otherwise associated with lipids.
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may exist in bilayer structures, as micelles or have a "collapsed" structure.
  • Lipids are fatty substances, which can be naturally occurring or synthetic lipids.
  • lipids include lipid droplets, which occur naturally in the cytoplasm as well as in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes.
  • the vector is a lentiviral vector.
  • the present invention provides a CAR-T cell containing the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a liquid formulation.
  • the formulation is an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, more preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine ; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, etc.
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • preservatives e.g, aluminum hydroxide
  • the present invention includes therapeutic applications of cells (eg, T cells) transduced with lentiviral vectors (LVs) encoding the expression cassettes of the present invention.
  • the transduced T cells can target the tumor cell marker CD7, synergistically activate T cells, and cause T cell immune responses, thereby significantly improving their killing efficiency against tumor cells.
  • the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, comprising the steps of: administering to the mammal a CAR-T cell of the present invention.
  • the present invention includes a type of cell therapy wherein a patient's autologous T cells (or a heterologous donor) are isolated, activated and genetically engineered to produce CAR-T cells, and subsequently infused into the same patient.
  • a patient's autologous T cells or a heterologous donor
  • CAR-T can treat all cancers that express this antigen.
  • CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.
  • the CAR-T cells of the invention can undergo robust in vivo T cell expansion for extended amounts of time.
  • a CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR.
  • anti-CD7 CAR-T cells elicit specific immune responses against CD7-expressing cells.
  • Cancers that can be treated include tumors that are not vascularized or substantially not vascularized, as well as tumors that are vascularized. Cancers may include non-solid tumors (such as hematological tumors, eg, leukemias and lymphomas) or may include solid tumors. Cancer types treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemic or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Also includes adult tumors/cancers and pediatric tumors/cancers.
  • Hematological cancers are cancers of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastoid, promyelocytic, myelomonocytic type) , monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (myeloid) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non- Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • acute leukemias such
  • Solid tumors are abnormal masses of tissue that typically do not contain cysts or areas of fluid. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell type that forms them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer.
  • the CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunization and/or in vivo therapy of mammals.
  • the mammal is a human.
  • CAR-modified cells are isolated from mammals (preferably human) and genetically modified (ie, transduced or transfected in vitro) with vectors expressing the CARs disclosed herein.
  • CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefit.
  • the mammalian recipient can be human, and the CAR-modified cells can be autologous to the recipient.
  • the cells may be allogeneic, syngeneic or xenogeneic with respect to the recipient.
  • the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.
  • the present invention provides methods of treating tumors comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the present invention.
  • the CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical compositions of the present invention may include a target cell population as described herein in association with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelates Adjuvants such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • the compositions of the present invention are preferably formulated for intravenous administration.
  • compositions of the present invention can be administered in a manner appropriate to the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease - although appropriate doses may be determined by clinical trials.
  • the precise amount of the composition of the invention to be administered can be determined by a physician, taking into account the patient (subject). ) individual differences in age, weight, tumor size, degree of infection or metastasis, and condition. It may generally be indicated that the pharmaceutical compositions comprising the T cells described herein may be administered at a dose of 104 to 109 cells/kg body weight, preferably 105 to 106 cells/kg body weight (including all integers within those ranges). value) application. The T cell composition can also be administered multiple times at these doses.
  • Cells can be administered by using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • Optimal dosages and treatment regimens for a particular patient can be readily determined by those skilled in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection.
  • the T cell composition of the present invention is preferably administered by i.v. injection.
  • the composition of T cells can be injected directly into tumors, lymph nodes or the site of infection.
  • cells activated and expanded using the methods described herein, or other methods known in the art to expand T cells to therapeutic levels are combined with any number of relevant therapeutic modalities (eg, previously , concurrently or subsequently) to a patient in a form of treatment including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or elfazizumab therapy for psoriasis patients or other treatments for PML patients.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or elfazizumab therapy for psoriasis patients or other treatments for PML patients.
  • the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutics.
  • the cellular composition of the invention is administered in combination with (eg, before, concurrently or after) bone marrow transplantation, using chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an infusion of expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • the dosage of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be carried out according to art-accepted practice. Typically, 1 x 106 to 1 x 1010 modified T cells of the invention (eg, CAR-T20 cells) can be administered to a patient per treatment or per course of treatment, eg, by intravenous infusion .
  • the CD7-CAR-T cells prepared by the method of the present invention have higher CAR transfection efficiency and better cell viability.
  • the CD7-CAR-T cells prepared by the method of the present invention can specifically kill CD7-positive target cells and secrete high specific cytokines.
  • the CD7 blocking molecule is constructed using conventional technical means in the art, the structure is shown in Figure 1, and the amino acid sequence is shown in SEQ ID NO: 2.
  • the above CD7 blocking molecule is inserted into a lentiviral vector, and then a CD7 blocking lentivirus, namely PA007CD, is prepared by lentiviral packaging technology.
  • CD7-CAR is constructed by conventional technical means in the art, the structure is shown in Figure 1, and the amino acid sequence is shown in SEQ ID NO: 12.
  • the above CD7-CAR was inserted into the lentiviral vector, and then the CD7-CAR lentivirus, namely PA107CD, was prepared by lentiviral packaging technology.
  • CD7-CAR-T Since more than 90% of normal human T cells express CD7 antigen, it is necessary to block the CD7 antigen on the surface of T cells in the process of preparing CD7-CAR-T cells, otherwise CD7-CAR-T will recognize itself Suicide caused by the antigen CD7.
  • T cells were transfected with CD7 blocking lentivirus (PA007CD), so that CD7 expressed by T cells was blocked in the endoplasmic reticulum or Golgi apparatus and could not be presented to the surface of T cells, and then CD7-CAR lentivirus (PA107CD) was used for transfection. T cells were transfected to prepare CD7-CAR-T cells.
  • CD7 blocking lentivirus PA007CD
  • PA107CD CD7-CAR lentivirus
  • PBMCs mononuclear cells
  • T cells were enriched from mononuclear cells using CD4/CD8 dual magnetic beads
  • an activator Transact
  • CD7-CAR-T cells During the preparation of CD7-CAR-T cells, the expression rate of CD7 on the surface of T cells and the proportion of CAR-positive cells (transfection efficiency) are important indicators of CD7-CAR-T cells. After first transfecting PA007CD virus, it is necessary to block the expression of CD7 molecules on the surface of T cells, and then transfecting PA107CD can successfully prepare CD7-CAR-T cells. For the three batches of CD7-CAR-T prepared by this method, the cell CD7 molecule expression and transfection efficiency statistics at the time of sample collection are shown in Table 2.
  • An important method to detect the biological efficacy of CD7-CAR-T cells is to detect the killing ability of CAR-T cells to target cells after co-incubation of CD7-CAR-T cells with CD7-positive target cells.
  • the CD7-overexpressing 293T cells (293T-CD7) were constructed as target cells to evaluate the in vitro killing function of CD7-CAR-T cells.
  • LZ012019122101, ZL012019123001, and ZZY12020010701 prepared in Example 2 three groups of CD7-CAR-T were used for killing experiments, and the effect-target ratios were 20:1, 10:1, and 5:1, respectively.
  • 293T cells were CD7-negative target cells.
  • CD7-CAR-T cells have a significant specific killing effect on 293T-CD7 cells (CD7 positive), but not on 293T cells (CD7 negative). It has specific killing, indicating that the CD7-CAR-T cells prepared in Example 2 can produce specific cytotoxicity to CD7-positive 293T-CD7 cells in vitro, but have no specific cytotoxicity to CD7-negative 293T cells.
  • CD7-CAR-T cells Cytotoxicity of CD7-CAR-T cells against acute lymphoblastic leukemia cells (CEM) naturally expressing CD7
  • the CD7-CAR-T cells prepared in Example 2 were used for in vitro killing experiments on acute lymphoblastic leukemia cells (CEM), and the effector-target ratios were 1:1, 1:10, 1:50, and 1:100, respectively. T cells are control effector cells.
  • CEM acute lymphoblastic leukemia cells
  • the release of cytokines after co-incubation of CD7-CAR-T cells and CEM cells was further analyzed.
  • the cell group was used as a control group to detect the secretion of cytokines IL-2, IFN- ⁇ and Granzyme B.
  • the main content of the present invention is to use a novel transfection method to successfully prepare CD7-CAR-T cells with high transfection efficiency and better cell activity, and through a series of in vitro functional experiments to prove that the prepared CD7-CAR-T cells -CAR-T cells have significant and specific killing of CD7-positive tumor cells.

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Abstract

提供了一种CD7-CAR-T细胞的制备方法及应用，所述方法包括(i)提供一含T细胞的待处理样本，(ii)分选并活化所述样本中包含的T细胞，从而得到激活的T细胞，(iii)将用于表达CD7阻断分子的第一病毒载体导入到所述激活的T细胞，从而得到CD7被阻断的T细胞，和(iv)将用于表达CD7-CAR的第二病毒载体导入到所述CD7被阻断的T细胞，从而得到CD7-CAR-T细胞；调整表达CD7阻断分子的慢病毒和表达CD7-CAR的慢病毒的转染顺序和转染时间，提高转染效率，增强CAR-T细胞的细胞毒性。

Description

一种CD7-CAR-T细胞的制备方法及应用 技术领域
本发明涉及生物技术领域，更具体地涉及一种CD7-CAR-T细胞的制备方法及应用。
背景技术
T细胞来源的恶性肿瘤代表着一种典型的血液系统恶性肿瘤，在儿童和成人患者中都具有很高的复发率和死亡率，目前尚无非常有效的治疗手段。T细胞急性淋巴细胞白血病(T-ALL)是一种高度异质性的血液系统恶性肿瘤，大约占成人急性淋巴细胞白血病病例的25％和儿童急性淋巴细胞白血病病例的15％左右。T-ALL在临床上的症状主要有感染、贫血、发热、异常出血等，T-ALL进展很快，可以在短期内快速浸润到淋巴结、肝、脾、中枢神经系统和睾丸等组织器官，如果没有及时有效的治疗方法，患者发病后很快就会死亡。目前，对于T细胞急性淋巴细胞白血病(T-ALL)的治疗策略主要包括强化化疗，异基因造血干细胞移植(allo-HSCT)，抗病毒治疗和分子靶向治疗。但是，强化化疗通常不能阻止疾病治疗后的复发。对于那些在初次治疗后复发的患者，补救性化疗方案的缓解率大约只有20-40％。另外，虽然造血干细胞移植可能是目前唯一可以治愈的方案，但是也会伴有移植物抗宿主病带来的生命危险。
近年来，嵌合抗原受体T细胞(CAR-T)疗法显示出非常显著的临床疗效，作为一种新型的过继性免疫治疗技术，被用于治疗多种实体肿瘤和血液肿瘤，其中疗效最显著的是针对B细胞来源的淋巴细胞白血病和淋巴瘤的治疗。CAR-T疗法是通过基因修饰患者的T淋巴细胞，以主要组织相容性复合物非依赖性的方式靶向和消除恶性肿瘤。有效应用该技术的关键之一是选择一个合适的靶点来构建对应的CAR分子。最佳的靶抗原应该只在肿瘤细胞表达，不在正常细胞表达，或者也可以表达在那种正常细胞短暂缺失后，临床上有应对方案的细胞上。
CD7分子在大多数的T细胞血液肿瘤细胞上是高表达的。CD7是由T、NK细胞和它们的前体细胞表达的跨膜糖蛋白，结合其配体K12/SECTM1后刺激T细胞活化。然而，CD7分子似乎未在T细胞发育或功能中起关键作用，因为破坏鼠T细胞的祖细胞中的CD7分子后，不影响正常T细胞发育和体内平衡，并且对T细胞效应功能不会有影响。重要的是，CD7曾作为抗体靶标用于治疗T细胞淋巴瘤，并且靶向CD7后患者未出现严重的不良反应。因此，CD7分子可能是一个针对T细胞血液肿瘤的非常合适的治疗靶点。但是用CD7-CAR-T细胞靶向治疗T细胞急性淋巴细胞白血病依然面临着很大的问题，这是因为正常的效应T细胞和T细胞肿瘤都表达CD7抗原会导致CD7-CAR-T细胞的自相残杀，CD7-CAR-T细胞很难在 体外制备成功。
因此，本领域迫切需要开发新的CD7-CAR-T细胞的制备方法。
发明内容
本发明的目的在于提供一种CD7-CAR-T细胞的制备方法及应用。
具体地，本发明提供了一种基于人源化CD7纳米抗体序列的CD7-CAR-T细胞制备方法。
本发明还提供了用PA007CD和PA107CD载体构建的CD7-CART细胞对CD7阳性肿瘤细胞的体外杀伤结果。
在本发明的第一方面，提供了一种制备CD7-CAR-T细胞的方法，包括步骤：
(i)提供一含T细胞的待处理样本，
(ii)分选并活化所述样本中包含的T细胞，从而得到激活的T细胞，
(iii)将用于表达CD7阻断分子的第一病毒载体导入到所述激活的T细胞，从而得到CD7被阻断的T细胞，和
(iv)将用于表达CD7-CAR的第二病毒载体导入到所述CD7被阻断的T细胞，从而得到所述CD7-CAR-T细胞，
其中，所述T细胞与激活剂共孵育12-36h后，较佳地，18-30h，更佳地，22-26h，将用于表达CD7阻断分子的第一病毒载体导入到所述激活的T细胞。
在另一优选例中，所述的导入第一病毒载体与导入第二病毒载体之间间隔36-60h，较佳地，间隔42-54h，更佳地，间隔46-50h。
在另一优选例中，在步骤(iii)中，包括步骤：
(a)在适合转染的条件下，将用于表达CD7阻断分子的第一病毒载体与所述激活的T细胞共培养36-60h，较佳地，42-54h，更佳地，46-50h，从而得到第一转染混合液；
(b)去除所述第一转染混合液中的第一病毒载体，从而得到CD7被阻断的T细胞。
在另一优选例中，在步骤(b)中，还包括去除步骤(ii)中所添加的激活剂的步骤。
在另一优选例中，在步骤(b)中，去除所述第一转染混合液中的第一病毒载体和激活剂。
在另一优选例中，在步骤(iv)中，包括步骤：
(c)在适合转染的条件下，将用于表达CD7-CAR的第二病毒载体与所述CD7被阻断的T细胞共培养12-36h，较佳地，18-30h，更佳地，22-26h，从而得到第二转染混合液；
(d)去除所述第二转染混合液中的第二病毒载体，从而得到所述CD7-CAR-T细 胞。
在另一优选例中，所述的第一病毒载体和第二病毒载体为慢病毒载体。
在另一优选例中，所述的共培养在36-38℃的条件下进行。
在另一优选例中，在步骤(a)中，所述的第一病毒载体与T细胞的数量比(感染复数)为1:0.05-0.4。
在另一优选例中，在步骤(c)中，所述的第二病毒载体与T细胞的数量比(感染复数)为1:0.5-4。
在另一优选例中，利用缓冲液清洗所述T细胞，从而去除病毒载体。
在另一优选例中，所述的样本为外周血单个核细胞。
在另一优选例中，在步骤(ii)中，利用激活剂进行所述的分选和活化。
在另一优选例中，所述的激活剂为靶向CD3和CD28的磁珠。
在另一优选例中，所述磁珠为美天妮Transact(具有易去除可降解且激活效果好的特点)。美天妮Transact即为实施例中使用的靶向CD3和CD28的磁珠。
在另一优选例中，所述的T细胞与激活剂比例为1×10 6个：5-20ul。
在另一优选例中，将所述靶向CD3/CD28的磁珠与T细胞孵育12-36h，较佳地，18-30h，更佳地，22-26h后，将用于表达CD7阻断分子的第一病毒载体导入到所述激活的T细胞。
在另一优选例中，在步骤(iv)之后，还包括步骤：
(v)在适合T细胞扩增的条件下，对所述CD7-CAR-T细胞进行扩大培养。
在另一优选例中，所述的CD7-CAR的抗原结合结构域包括一个或多个CD7纳米抗体。
在另一优选例中，所述的CD7纳米抗体的氨基酸序列如SEQ ID NO:13所示。
在另一优选例中，所述的CAR还包含ICOS来源的跨膜区和/或ICOS来源的胞内共刺激区。
在另一优选例中，所述的CAR包含ICOS来源的胞内共刺激区和4-1BB来源的胞内共刺激区。
在另一优选例中，所述CAR的结构如下式I所示：
L-VHH-H-TM-C-CD3ζ    (I)
式中，
各“-”独立地为连接肽或肽键；
L为信号肽序列；
VHH为包含一个或两个CD7纳米抗体的抗原结合结构域；
H为铰链区；
TM为跨膜结构域；
C为共刺激信号分子；
CD3ζ为源于CD3ζ的胞浆信号传导序列。
在另一优选例中，所述CAR的结构如图1所示。
在另一优选例中，所述的L为选自下组的蛋白的信号肽：CD8、CD28、GM-CSF、CD4、CD137、或其组合。
在另一优选例中，所述L为CD8来源的信号肽。
在另一优选例中，所述L的氨基酸序列如SEQ ID NO:4所示。
在另一优选例中，所述的VHH包括两个CD7纳米抗体，较佳地，所述的两个CD7纳米抗体通过连接肽相连，更佳地，所述的连接肽的氨基酸序列如SEQ ID NO:8所示。
在另一优选例中，所述连接肽的核苷酸序列如SEQ ID NO:7所示。
在另一优选例中，所述的CD7纳米抗体包括人源化的纳米抗体和骆驼源纳米抗体，优选为人源化的纳米抗体。
在另一优选例中，所述的VHH为一个人源化的CD7纳米抗体。
在另一优选例中，所述的H为来源于选自下组的蛋白的铰链区：Fc、CD8、CD28、CD137、或其组合。
在另一优选例中，所述的H为Fc片段，优选地，所述的Fc片段为人IgG4Fc片段。
在另一优选例中，所述的Fc片段的氨基酸序列如SEQ ID NO:14所示。
在另一优选例中，所述的TM为选自下组的蛋白的跨膜区：ICOS、CD28、CD3epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、或其组合。
在另一优选例中，所述的TM为ICOS来源的跨膜区。
在另一优选例中，所述ICOS来源的跨膜区的氨基酸序列如SEQ ID NO:15所示。
在另一优选例中，所述的C为选自下组的蛋白的共刺激信号分子：ICOS、OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD1、Dap10、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、TLR2、或其组合。
在另一优选例中，所述的C为ICOS和/或4-1BB来源的共刺激信号分子。
在另一优选例中，所述ICOS来源的共刺激信号分子的氨基酸序列如SEQ ID NO:16所示。
在另一优选例中，所述4-1BB来源的共刺激信号分子的氨基酸序列如SEQ ID NO:17所示。
在另一优选例中，所述CD3ζ的氨基酸序列如SEQ ID NO:18所示。
在另一优选例中，所述CAR(优选地为C端)还包括细胞自杀元件。
在另一优选例中，所述的CAR与细胞自杀元件通过自剪切元件相连接。
在另一优选例中，所述细胞自杀元件与所述CAR的CD3ζ通过T2A连接。
在另一优选例中，所述的细胞自杀元件选自下组：HSV-TK、iCasp9、ΔCD20、mTMPK、ΔCD19、RQR8、EGFRt、或其组合。
在另一优选例中，所述细胞自杀元件为tEGFR。
在另一优选例中，所述tEGFR的氨基酸序列如SEQ ID NO:20所示。
在另一优选例中，所述T2A的氨基酸序列如SEQ ID NO:19所示。
在另一优选例中，所述CAR为PA107CD CAR(即申请号201911150678.7的中国专利申请中的名称为PA3-17的CD7CAR)。
在另一优选例中，所述CAR的氨基酸序列如SEQ ID NO:12所示。
在另一优选例中，所述CD7阻断分子包括一个或多个CD7纳米抗体和内质网滞留序列。
在另一优选例中，所述CD7阻断分子的结构如下式II所示：
L’-VHH’-ER    (II)
式中，
各“-”独立地为连接肽或肽键；
L’为信号肽序列；
VHH’为包含两个CD7纳米抗体的结合区；
ER为内质网滞留序列。
在另一优选例中，所述L’的氨基酸序列如SEQ ID NO:4所示。
在另一优选例中，所述L’的核苷酸序列如SEQ ID NO:3所示。
在另一优选例中，所述VHH’的两个CD7纳米抗体通过连接肽相连，更佳地，所述的连接肽如SEQ ID NO:8所示。
在另一优选例中，所述VHH’的氨基酸序列如SEQ ID NO:6所示。
在另一优选例中，所述VHH’的核苷酸序列如SEQ ID NO:5所示。
在另一优选例中，所述VHH’为骆驼源的纳米抗体。
在另一优选例中，所述ER的氨基酸序列如SEQ ID NO:10所示。
在另一优选例中，所述ER的核苷酸序列如SEQ ID NO:9所示。
在另一优选例中，所述CD7阻断分子的氨基酸序列如SEQ ID NO:2所示。
在另一优选例中，所述CD7阻断分子的核苷酸序列如SEQ ID NO:1所示。
在本发明的第二方面，提供了一种本发明第一方面所述方法制备的CD7-CAR-T细胞。
在另一优选例中，所述CD7-CAR-T细胞中，至少80％，较佳地90％，更佳地95％， 最佳地98％的内源性CD7表达被阻断。
在本发明的第三方面，提供了一种制剂，所述制剂含有本发明第二方面所述的CD7-CAR-T细胞，以及药学上可接受的载体。
在另一优选例中，所述制剂为液态制剂。
在另一优选例中，所述制剂的剂型为注射剂。
在另一优选例中，所述制剂中所述工程化的免疫细胞的浓度为1×10 3-1×10 8个细胞/ml，较佳地1×10 4-1×10 7个细胞/ml。
在本发明的第四方面，提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明第二方面所述的CD7-CAR-T细胞或本发明第三方面所述的制剂。
在另一优选例中，所述疾病为癌症或肿瘤。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了本发明的CD7-CAR和CD阻断分子的载体结构图。
图2显示了本发明的CD7-CART的制备流程图。
图3显示了检测T细胞表面的CD7分子表达(使用FITC通道的CD7抗体检测CD7分子表达)、CD7-CART的转染效率(使用PE通道的CD3抗体检测T细胞表达，使用APC通道的FC抗体检测CAR的表达)的流式图。
图4显示了CD7-CAR-T细胞对过表达CD7的293T细胞的细胞毒性。
图5显示了CD7-CAR-T细胞对天然表达CD7的急性淋巴白血病细胞(CEM)的细胞毒性。
图6显示了CD7-CAR-T细胞与293T-CD7细胞孵育后细胞因子分泌情况。
图7显示了CD7-CAR-T细胞与CEM细胞孵育后细胞因子分泌情况。
具体实施方式
本发明人经过广泛而深入地研究，首次意外地发现一种CD7-CAR-T细胞的制备方法及应用。具体地，本发明的方法通过调整表达CD7阻断分子的慢病毒和表达CD7-CAR的慢病毒的转染顺序和转染时间，提高了转染效率，增强了CAR-T细胞的细胞毒性。在此基础上完成了本发明。
具体地，本发明是要提供一种基于人源化CD7纳米抗体序列构建的 CD7-CAR-T细胞制备方法，由于CD7-CAR-T细胞的制备过程中涉及两种慢病毒的转染，一种是将表达在T细胞表面的CD7抗原锚定在细胞内质网或高尔基体中的PA007CD病毒，另一种是可以在T细胞表面表达CD7-CAR的PA107CD慢病毒。发明人发现两种慢病毒使用的先后顺序，转染的时间间隔等因素会直接影响最终CART细胞的质量。本发明阐述了在T细胞分选活化后24小时转染PA007CD慢病毒，48小时后清洗去除PA007CD慢病毒及激活剂，然后转染PA107CD慢病毒24小时后再次清洗去除PA107CD慢病毒的CD7-CAR-T制备方法。使用此种方法制备的CD7-CAR-T细胞具有较高的CAR转染效率，较好的细胞活率，通过体外功能实验证明，本申请制备的CD7-CAR-T细胞能特异性杀伤CD7阳性的靶细胞并分泌较高的特异性细胞因子。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。
术语“给予”是指使用本领域技术人员已知的各种方法和递送系统中的任一种将本发明的产品物理引入受试者，包括静脉内，肌内，皮下，腹膜内，脊髓或其它肠胃外给药途径，例如通过注射或输注。
术语“抗体”(Ab)应包括但不限于免疫球蛋白，其特异性结合抗原并包含通过二硫键互连的至少两条重(H)链和两条轻(L)链，或其抗原结合部分。每条H链包含重链可变区(本文缩写为VH)和重链恒定区。重链恒定区包含三个恒定结构域CH1、CH2和CH3。每条轻链包含轻链可变区(本文缩写为VL)和轻链恒定区。轻链恒定区包含一个恒定结构域CL。VH和VL区可以进一步细分为称为互补决定区(CDR)的高变区，其散布有更保守的称为框架区(FR)的区域。每个VH和VL包含三个CDR和四个FR，从氨基末端到羧基末端按照以下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。
应理解，本文中氨基酸名称采用国际通用的单英文字母标识，与其相对应的氨基酸名称三英文字母简写分别是：Ala(A)、Arg(R)、Asn(N)、Asp(D)、Cys(C)、Gln(Q)、Glu(E)、Gly(G)、His(H)、I1e(I)、Leu(L)、Lys(K)、Met(M)、Phe(F)、Pro(P)、Ser(S)、Thr(T)、Trp(W)、Tyr(Y)、Val(V)。
本申请的序列表中提供了CD7阻断分子(PA007CD)、CD7-CAR(PA107CD)及其元件的序列，各个序列所代表的含义如下表1所示。
表1 序列表中各个序列的名称
序列 名称
SEQ ID NO:1 CD7阻断分子碱基序列
SEQ ID NO:2 CD7阻断分子氨基酸序列
SEQ ID NO:3 sp(信号肽)碱基序列
SEQ ID NO:4 sp(信号肽)氨基酸序列
SEQ ID NO:5 CD7纳米抗体(VHH6)的碱基序列
SEQ ID NO:6 CD7纳米抗体(VHH6)的氨基酸序列
SEQ ID NO:7 (G4S)4碱基序列
SEQ ID NO:8 (G4S)4氨基酸序列
SEQ ID NO:9 ER碱基序列
SEQ ID NO:10 ER氨基酸序列
SEQ ID NO:11 PA107CD碱基序列
SEQ ID NO:12 PA107CD氨基酸序列
SEQ ID NO:13 HuVHH6(CD7纳米抗体人源化序列)氨基酸序列
SEQ ID NO:14 Fc(人IgG4Fc序列，铰链区)氨基酸序列
SEQ ID NO:15 ICOS-tran(ICOS跨膜区序列)氨基酸序列
SEQ ID NO:16 ICOS(ICOS胞内区序列)氨基酸序列
SEQ ID NO:17 41BB(41BB胞内区序列)氨基酸序列
SEQ ID NO:18 CD3ζ氨基酸序列
SEQ ID NO:19 T2A氨基酸序列
SEQ ID NO:20 tEGFR(截短的EGFR序列)氨基酸序列
嵌合抗原受体(CAR)
CARs的设计经历了以下过程：第一代CAR只有一个胞内信号组份CD3ζ或者FcγRI分子，由于胞内只有一个活化结构域，因此它只能引起短暂的T细胞增殖和较少的细胞因子分泌，而并不能提供长时间的T细胞增殖信号和持续的体内抗肿瘤效应，所以并没有取得很好地临床疗效。第二代CARs在原有结构基础上引入一个共刺激分子，如CD28、4-1BB、OX40、ICOS，与一代CARs相比功能有很大提高，进一步加强CAR-T细胞的持续性和对肿瘤细胞的杀伤能力。在二代CARs基础上串联一些新的免疫共刺激分子如CD27、CD134，发展成为三代和四代CARs。
本发明的嵌合抗原受体(CAR)包括细胞外结构域、跨膜结构域、和细胞内结构域。胞外结构域包括靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激信号传导区和ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所 需要的细胞表面分子，而不是抗原受体或它们的配体。
在CAR的胞外结构域和跨膜结构域之间，或在CAR的胞浆结构域和跨膜结构域之间，可并入接头。如本文所用的，术语“接头”通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸，优选地2至100个氨基酸和最优选地3至50个氨基酸。
在本发明的一个较佳的实施方式中，本发明提供的CAR的胞外结构域包括靶向CD7的抗原结合结构域。本发明的CAR当在T细胞中表达时，能够基于抗原结合特异性进行抗原识别。当其结合其关联抗原时，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和ζ链中的一个或多个的细胞内结构域融合。优选地，抗原结合结构域与ICOS和/或4-1BB信号传导结构域、和CD3ζ信号结构域组合的细胞内结构域融合。
如本文所用，“抗原结合结构域”是指具有抗原结合活性的Fab片段，Fab’片段，F(ab’) 2片段，或单一Fv片段。作为本发明的优选方式，所述抗原结合结构域包含一个或多个特异性识别CD7的纳米抗体。
对于绞链区和跨膜区(跨膜结构域)，CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中，使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中，可选择跨膜结构域，或通过氨基酸置换进行修饰，以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构域，从而最小化与受体复合物的其他成员的相互作用。
本发明的CAR中的胞内结构域包括ICOS和/或4-1BB的信号传导结构域和CD3ζ的信号传导结构域。
本发明还提供了靶向CD7的CAR-T细胞，除非特别说明，本发明的CAR-T细胞表面的CD7表达是被阻断的，较佳地，被本发明第二方面所述的CD7阻断分子所阻断。
自杀基因开关
为进一步控制CAR-T细胞非肿瘤靶向和细胞因子释放综合征等不良，本发明中的CART细胞皆带有自杀基因开关，在外源性药物的作用下，可以有效清除体内的CAR-T细胞，阻断未知的或不可控的远期毒性，以保证患者的安全。
本发明中所用自杀基因开关可以为HSV-TK、iCasp9、CD20、mTMPK等。比较而言，HSV-TK、iCasp9和CD20对T细胞的清除能力等同，但是iCasp9和CD20的清除较迅速，HSV-TK清楚速度较慢。
iCasp9自杀基因包含FKBP12-F36V结构域，可通过柔性接头连接半胱氨酸天冬氨酸蛋白酶9，后者不含募集结构域。FKBP12-F36V包含一个FKBP结构域， 在第36个氨基酸残基位点上苯丙氨酸替代了缬氨酸。它具有高选择性和亚纳摩尔亲和力，能够结合二聚合成配基，如其他惰性小分子AP1903。当加入小分子后，能够促使其二聚话，从而诱导细胞的凋亡，而对未携带自杀基因的正常细胞无效用。
载体
编码期望分子的核酸序列可利用在本领域中已知的重组方法获得，诸如例如通过从表达基因的细胞中筛选文库，通过从已知包括该基因的载体中得到该基因，或通过利用标准的技术，从包含该基因的细胞和组织中直接分离。可选地，感兴趣的基因可被合成生产。
本发明也提供了其中插入本发明的表达盒的载体。源于逆转录病毒诸如慢病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因长期、稳定的整合并且其在子细胞中增殖。慢病毒载体具有超过源自致癌逆转录病毒诸如鼠科白血病病毒的载体的优点，因为它们可转导非增殖的细胞，诸如肝细胞。它们也具有低免疫原性的优点。
简单概括，通常可操作地连接本发明的表达盒或核酸序列至启动子，并将其并入表达载体。该载体适合于复制和整合真核细胞。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、初始序列和启动子。
本发明的表达构建体也可利用标准的基因传递方案，用于核酸免疫和基因疗法。基因传递的方法在本领域中是已知的。见例如美国专利号5,399,346、5,580,859、5,589,466，在此通过引用全文并入。在另一个实施方式中，本发明提供了基因疗法载体。
该核酸可被克隆入许多类型的载体。例如，该核酸可被克隆入如此载体，其包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。特定的感兴趣载体包括表达载体、复制载体、探针产生载体和测序载体。
进一步地，表达载体可以以病毒载体形式提供给细胞。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记(例如，WO01/96584；WO01/29058；和美国专利号6,326,193)。
已经开发许多基于病毒的系统，用于将基因转移入哺乳动物细胞。例如，逆转录病毒提供了用于基因传递系统的方便的平台。可利用在本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分 离和传递至体内或离体的对象细胞。许多逆转录病毒系统在本领域中是已知的。在一些实施方式中，使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一个实施方式中，使用慢病毒载体。
额外的启动子元件，例如增强子，可以调节转录开始的频率。通常地，这些位于起始位点上游的30-110bp区域中，尽管最近已经显示许多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔经常是柔性的，以便当元件相对于另一个被倒置或移动时，保持启动子功能。在胸苷激酶(tk)启动子中，启动子元件之间的间隔可被增加隔开50bp，活性才开始下降。取决于启动子，表现出单个元件可合作或独立地起作用，以起动转录。
合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关，其能够当这样的表达是期望的时，打开可操作地连接诱导型启动子的多核苷酸序列的表达，或当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
为了评估CAR多肽或其部分的表达，被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面，可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表达。有用的可选择标记包括例如抗生素抗性基因，诸如neo等等。
报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。通常地，报道基因为以下基因：其不存在于受体有机体或组织或由受体有机体或组织进行表达，并且其编码多肽，该多肽的表达由一些可容易检测的性质例如酶活性清楚表示。在DNA已经被引入受体细胞后，报道基因的表达在合适的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白的基因(例如，Ui-Tei等，2000FEBS Letters479:79-82)。合适的表达系统是公知的并可利用已知技术制备或从商 业上获得。通常，显示最高水平的报道基因表达的具有最少5个侧翼区的构建体被鉴定为启动子。这样的启动子区可被连接至报道基因并用于评价试剂调节启动子-驱动转录的能力。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。在表达载体的内容中，载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。生产包括载体和/或外源核酸的细胞的方法在本领域中是公知的。见例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)。将多核苷酸引入宿主细胞的优选方法为磷酸钙转染。
将感兴趣的多核苷酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体，特别是逆转录病毒载体，已经成为最广泛使用的将基因插入哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。见例如美国专利号5,350,674和5,585,362。
将多核苷酸引入宿主细胞的化学手段包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体。用作体外和体内传递工具(del ivery vehicle)的示例性胶体系统为脂质体(例如，人造膜囊)。
在使用非病毒传递系统的情况下，示例性传递工具为脂质体。考虑使用脂质制剂，以将核酸引入宿主细胞(体外、离体(ex vivo)或体内)。在另一方面，该核酸可与脂质相关联。与脂质相关联的核酸可被封装入脂质体的水性内部中，散布在脂质体的脂双层内，经与脂质体和寡核苷酸两者都相关联的连接分子附接至脂质体，陷入脂质体，与脂质体复合，分散在包含脂质的溶液中，与脂质混合，与脂质联合，作为悬浮液包含在脂质中，包含在胶束中或与胶束复合，或以其他方式与脂质相关联。与组合物相关联的脂质、脂质/DNA或脂质/表达载体不限于溶液中的任何具体结构。例如，它们可存在于双分子层结构中，作为胶束或具有“坍缩的(collapsed)”结构。它们也可简单地被散布在溶液中，可能形成大小或形状不均一的聚集体。脂质为脂肪物质，其可为天然发生或合成的脂质。例如，脂质包括脂肪小滴，其天然发生在细胞质以及包含长链脂肪族烃和它们的衍生物诸如脂肪酸、醇类、胺类、氨基醇类和醛类的该类化合物中。
在本发明的一个优选地实施方式中，所述载体为慢病毒载体。
制剂
本发明提供了一种含有本发明第一方面所述的CAR-T细胞，以及药学上可接受的载体、稀释剂或赋形剂。在一个实施方式中，所述制剂为液态制剂。优选地，所述制剂为注射剂。优选地，所述制剂中所述CAR-T细胞的浓度为1×10 3-1×10 8个细胞/ml，更优地1×10 4-1×10 7个细胞/ml。
在一个实施方式中，所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的制剂优选配制用于静脉内施用。
治疗性应用
本发明包括用编码本发明表达盒的慢病毒载体(LV)转导的细胞(例如，T细胞)进行的治疗性应用。转导的T细胞可靶向肿瘤细胞的标志物CD7，协同激活T细胞，引起T细胞免疫应答，从而显著提高其对肿瘤细胞的杀伤效率。
因此，本发明也提供了刺激对哺乳动物的靶细胞群或组织的T细胞-介导的免疫应答的方法，其包括以下步骤：给哺乳动物施用本发明的CAR-T细胞。
在一个实施方式中，本发明包括一类细胞疗法，分离病人自体T细胞(或者异源供体)，激活并进行基因改造产生CAR-T细胞，随后注入同一病人体内。这种方式患移植物抗宿主病概率极低，抗原被T细胞以无MHC限制方式识别。此外，一种CAR-T就可以治疗表达该抗原的所有癌症。不像抗体疗法，CAR-T细胞能够体内复制，产生可导致持续肿瘤控制的长期持久性。
在一个实施方式中，本发明的CAR-T细胞可经历稳固的体内T细胞扩展并可持续延长的时间量。另外，CAR介导的免疫应答可为过继免疫疗法步骤的一部分，其中CAR-修饰T细胞诱导对CAR中的抗原结合结构域特异性的免疫应答。例如，抗CD7的CAR-T细胞引起抗表达CD7的细胞的特异性免疫应答。
尽管本文公开的数据具体公开了包括CD7纳米抗体、Fc片段、跨膜区、和ICOS胞内区、4-1BB和CD3ζ信号传导结构域的慢病毒载体，但本发明应被解释为包括对构建体组成部分中的每一个的任何数量的变化。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤，以及血管化的肿瘤。癌症可包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)或可包括实体瘤。用本发明的CAR治疗的癌症类型包括但不限于癌、胚细胞瘤和肉瘤，和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤，例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病，包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨 髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌、卵巢癌。
本发明的CAR-修饰T细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地，哺乳动物为人。
对于离体免疫，以下中的至少一项在将细胞施用进入哺乳动物前在体外发生：i)扩增细胞，ii)将编码CAR的核酸引入细胞，和/或iii)冷冻保存细胞。
离体程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞从哺乳动物(优选人)中分离并用表达本文公开的CAR的载体进行基因修饰(即，体外转导或转染)。CAR-修饰的细胞可被施用给哺乳动物接受者，以提供治疗益处。哺乳动物接受者可为人，和CAR-修饰的细胞可相对于接受者为自体的。可选地，细胞可相对于接受者为同种异基因的、同基因的(syngeneic)或异种的。
除了就离体免疫而言使用基于细胞的疫苗之外，本发明也提供了体内免疫以引起针对患者中抗原的免疫应答的组合物和方法。
本发明提供了治疗肿瘤的方法，其包括施用给需要其的对象治疗有效量的本发明的CAR-修饰的T细胞。
本发明的CAR-修饰的T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-17或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞群，与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的组合物优选配制用于静脉内施用。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症、和患者疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量” 或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的T细胞的药物组合物可以以10 4至10 9个细胞/kg体重的剂量，优选10 5至10 6个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，NewEng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
对象组合物的施用可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。在一个实施方式中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过i.v.注射施用。T细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将T细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄法珠单抗治疗或对PML患者的其他治疗。在进一步的实施方式中，本发明的T细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506，抗体或其他免疫治疗剂。在进一步的实施方式中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方式中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方式中，在移植后，对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方式中，扩展的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×10 6个至1×10 10个本发明经修饰的T细胞(如，CAR-T20细胞)，通过例如静脉回输的方式，施用于患者。
本发明的主要优点包括：
(a)本发明方法制备的CD7-CAR-T细胞具有较高的CAR转染效率，较好的细胞活率。
(b)本发明方法制备的CD7-CAR-T细胞能特异性杀伤CD7阳性的靶细胞并分泌较高的特异性细胞因子。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1
CD7-CAR和CD7阻断分子的制备
利用本领域的常规技术手段构建CD7阻断分子，结构如图1所示，氨基酸序列如SEQ ID NO:2所示。将上述CD7阻断分子插入到慢病毒载体中，然后通过慢病毒包装技术，制备CD7阻断慢病毒，即PA007CD。
利用本领域的常规技术手段构建CD7-CAR，结构如图1所示，氨基酸序列如SEQ ID NO:12所示。将上述CD7-CAR插入到慢病毒载体中，然后通过慢病毒包装技术，制备CD7-CAR慢病毒，即PA107CD。
实施例2
CD7-CAR-T细胞的制备
由于人正常T细胞中有90％以上表达CD7抗原，所以在制备CD7-CAR-T细胞的过程中需要将T细胞表面的CD7抗原阻断在胞内，否则会出现CD7-CAR-T识别自身抗原CD7而造成的自杀现象。
使用CD7阻断慢病毒(PA007CD)转染T细胞，使得T细胞表达的CD7被阻断在内质网或高尔基体中无法呈递到T细胞表面，然后再使用CD7-CAR慢病毒(PA107CD)进行T细胞转染，制备出CD7-CAR-T细胞。
在此实施例中，使用来源于供者的单采血样，使用Ficoll采用密度梯度离心方法得到单个核细胞(PBMC),再使用CD4/CD8双磁珠从单个核细胞中富集出T细胞，在得到T细胞后使用激活剂(Transact)对T细胞进行活化，在活化后的第二天(活化24小时后)根据前期的验证结果选用合适的PA007CD病毒数目进行T细胞的转染，具体比例为MOI(感染复数)＝0.05-0.4，在此MOI的条件下T细胞表面CD7分子可以被完全阻断且对后期的PA107CD病毒转染影响较小，在PA007CD慢病毒转染48h后离心去除残留激活剂和PA007CD慢病毒后再加入PA107CD慢病毒进行转染，PA107CD转染时的用量也是根据前期的验证结果选用了合适的病毒数目，具体比例为MOI(感染复数)＝0.5-4，此MOI的选择保证了转染效率，转染24h后离心去除残留PA107CD慢病毒并开始CAR-T细胞的扩增培养。 制备流程图如图2所示。
实施例3
T细胞表面的CD7分子表达、CD7-CART的转染效率
在CD7-CAR-T细胞的制备过程中，T细胞表面CD7的表达率，CAR阳性细胞的比例(转染效率)是CD7-CAR-T细胞的重要考察指标。在先转染PA007CD病毒后需阻断T细胞表面的CD7分子表达，接着转染PA107CD就可以成功制备出CD7-CAR-T细胞。使用此方法制备的3批CD7-CAR-T，收样时细胞CD7分子表达及转染效率统计如表2所示。
表2 细胞表面CD7分子表达及转染效率
细胞批号 LZ012019122101 ZL012019123001 ZZY12020010701
CD7分子表达 0.20 1.05 0.99
转染效率 37.80 18.86 11.56
流式图如图3所示。
结果显示，在转染PA007CD病毒后T细胞表面的CD7分子已接近被完全阻断，表达量在1％左右，此时转染PA107CD慢病毒制备CD7-CAR-T减少细胞的自杀现象，可以有效的保证CD7CAR-T细胞的活率，显著提高细胞的制备成功率。
实施例4
CD7-CAR-T细胞对过表达CD7的293T细胞的细胞毒性
一个重要地检测CD7-CAR-T细胞生物学效力的方法是检测CD7-CAR-T细胞与CD7阳性靶细胞共孵育后，CAR-T细胞对靶细胞的杀伤能力。
构建了CD7过表达的293T细胞(293T-CD7)作为靶细胞，评估CD7-CAR-T细胞的体外杀伤功能。利用实施例2制备的LZ012019122101、ZL012019123001、ZZY12020010701，三组CD7-CAR-T进行的杀伤实验，效靶比分别为20:1，10:1,5:1。293T细胞为CD7阴性靶细胞。
结果如图4所示，通过下面3组细胞的杀伤结果可以看出CD7-CAR-T细胞对293T-CD7细胞(CD7阳性)具有显著的特异性杀伤作用，而对293T细胞(CD7阴性)不具有特异性杀伤，说明实施例2制备的CD7-CAR-T细胞在体外可以对过表达CD7阳性的293T-CD7细胞产生特异的细胞毒性，而对CD7阴性的293T细胞不具有特异的细胞毒性。
实施例5
CD7-CAR-T细胞对天然表达CD7的急性淋巴白血病细胞(CEM)的细胞毒性
利用实施例2制备出的CD7-CAR-T细胞对急性淋巴白血病细胞(CEM)进行 体外杀伤实验，效靶比分别为1:1,1:10,1:50,1:100。T细胞为对照效应细胞。
结果如图5所示，与对照的T细胞相比，CD7-CART细胞对CEM都具有显著的特异性杀伤作用，说明实施例2制备的CD7-CART细胞在体外对CD7阳性的CEM细胞具有显著的细胞毒性。
实施例6
CD7-CAR-T细胞与293T-CD7细胞孵育后细胞因子分泌情况
分析了CD7-CAR-T细胞与293T-CD7细胞孵育后细胞因子的释放情况，选择效应细胞与靶细胞共孵育效靶比为1:1组的杀伤上清,检测细胞因子IL-2,IFN-γ，Granzyme B的分泌。
结果如图6所示，CD7-CAR-T细胞与293T共孵育后基本不会有细胞因子的产生，而CD7-CAR-T细胞与293T-CD7细胞孵育后，有明显的细胞因子释放升高。说明制备的CD7-CAR-T细胞可以CD7阳性的293T-CD7细胞特异性激活。
实施例7
CD7-CAR-T细胞与CEM细胞孵育后细胞因子分泌情况
进一步分析了CD7-CAR-T细胞与CEM细胞共孵育后细胞因子的释放情况，CD7-CAR-T细胞与靶细胞孵育的效靶比分别为1:10,1:50,1:100,T细胞组作为对照组，检测细胞因子IL-2,IFN-γ，Granzyme B的分泌。
结果如图7所示，T细胞与靶细胞共孵育后基本不会有细胞因子的产生，而CD7-CAR-T细胞与CEM细胞孵育后，有明显的细胞因子释放升高。进一步证明了制备的CD7-CAR-T细胞可以被CD7阳性肿瘤细胞特异性激活。
小结：本发明的主要内容是用一种新型的转染方法成功制备出转染效率较高，细胞活性较好的CD7-CAR-T细胞，并通过一系列的体外功能实验证明了制备的CD7-CAR-T细胞对CD7阳性肿瘤细胞具有显著的特异的杀伤。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种制备CD7-CAR-T细胞的方法，其特征在于，包括步骤：
    (i)提供一含T细胞的待处理样本，
    (ii)分选并活化所述样本中包含的T细胞，从而得到激活的T细胞，
    (iii)将用于表达CD7阻断分子的第一病毒载体导入到所述激活的T细胞，从而得到CD7被阻断的T细胞，和
    (iv)将用于表达CD7-CAR的第二病毒载体导入到所述CD7被阻断的T细胞，从而得到所述CD7-CAR-T细胞，
    其中，所述T细胞与激活剂共孵育12-36h后，较佳地，18-30h，更佳地，22-26h，将用于表达CD7阻断分子的第一病毒载体导入到所述激活的T细胞。
  2. 如权利要求1所述的方法，其特征在于，在步骤(iii)中，包括步骤：
    (a)在适合转染的条件下，将用于表达CD7阻断分子的第一病毒载体与所述激活的T细胞共培养36-60h，较佳地，42-54h，更佳地，46-50h，从而得到第一转染混合液；
    (b)去除所述第一转染混合液中的第一病毒载体，从而得到CD7被阻断的T细胞。
  3. 如权利要求2所述的方法，其特征在于，在步骤(b)中，还包括去除步骤(ii)中所添加的激活剂的步骤。
  4. 如权利要求1所述的方法，其特征在于，在步骤(iv)中，包括步骤：
    (c)在适合转染的条件下，将用于表达CD7-CAR的第二病毒载体与所述CD7被阻断的T细胞共培养12-36h，较佳地，18-30h，更佳地，22-26h，从而得到第二转染混合液；
    (d)去除所述第二转染混合液中的第二病毒载体，从而得到所述CD7-CAR-T细胞。
  5. 如权利要求2所述的方法，其特征在于，在步骤(a)中，所述的第一病毒载体与T细胞的数量比(感染复数)为1:0.05-0.4。
  6. 如权利要求4所述的方法，其特征在于，在步骤(c)中，所述的第二病毒载体与T细胞的数量比(感染复数)为1:0.5-4。
  7. 如权利要求1所述的方法，其特征在于，在步骤(iv)之后，还包括步骤：
    (v)在适合T细胞扩增的条件下，对所述CD7-CAR-T细胞进行扩大培养。
  8. 如权利要求1所述的方法，其特征在于，所述CD7阻断分子包括一个或多个CD7纳米抗体和内质网滞留序列。
  9. 一种权利要求1所述方法制备的CD7-CAR-T细胞。
  10. 一种制剂，其特征在于，所述制剂含有权利要求9所述的CD7-CAR-T细胞，以及药学上可接受的载体。
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