Engineered immune cells targeting CD7, chimeric antigen receptors, CD7 blocking molecules and applications

Abstract

The present invention relates to an engineered immune cell targeting CD7, a chimeric antigen receptor, a CD7 blocking molecule and applications thereof. The present invention uses the natural ligand of human CD7 to replace the antibody sequence as the antigen recognition domain of CD7-specific CAR-T or CAR-NK cells. The advantage of using human CD7 as the antigen recognition domain in CD7-specific CARs is that it can prevent cellular and humoral responses generated by the host, thereby achieving long-term persistence and better efficacy of CAR-T cells.

Description

[0001] This application is a divisional application of the application with the application number 202110036169.2, the application date of January 12, 2021, and the invention title of "Engineered Immune Cells Targeting CD7, Chimeric Antigen Receptors, CD7 Blocking Molecules and Applications". Technical Field

[0002] The present invention relates to the field of cellular immunotherapy technology. Specifically, it relates to an immunotherapy method targeting CD7, and in particular to an engineered immune cell targeting CD7, a chimeric antigen receptor, a CD7 blocking molecule and their applications. Background Art

[0003] T-cell malignancies are a highly heterogeneous group of clonal growth and T-cell dysfunctional diseases, mainly divided into T-cell lymphomas (TCLs) and T-cell leukemias, with mature and precursor subtypes. These diseases represent a class of extremely malignant hematological cancers with high recurrence and mortality rates in both children and adults, and there is currently no effective or targeted treatment method. Although there are already various chemotherapy regimens in the prior art, in patients with T-cell acute lymphoblastic leukemia (T-ALL), only less than 50% of adults and 75% of children can survive for more than 5 years. For patients who relapse after initial treatment, salvage chemotherapy regimens can only induce remission in 20%-40% of cases. In patients with relapsed or chemotherapy-refractory T-cell malignancies, the treatment prognosis is poor, and there are limited effective and tolerable treatment methods. For 10%-50% of patients who achieve complete remission (CR) after salvage chemotherapy, the only treatment option remains allogeneic stem cell transplantation (ASCT). However, the cure rate of ASCT remains at 30% or lower, 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 even lower initial response rates to chemotherapy. Even in patients who respond, the progression-free survival rate remains at 40-50%. Therefore, although progress has been made in the treatment of T-cell malignancies, there is still a need for new and targeted treatment regimens to improve the prognosis, especially for relapsed and refractory patients. In addition to T-cell malignancies such as T-ALL and T-NHL, it must be recognized that there are other CD7+ hematological malignancies that still lack effective treatment and targeted treatment regimens, such as 20%-30% of acute myeloid leukemia (AML), and the vast majority of NK and NKT lymphomas.

[0004] Chimeric antigen receptor T (CAR-T) cells are one of the most promising approaches for cancer immunotherapy and can generate significant response rates in patients with B-lymphocyte malignancies. Therefore, CAR-T cells targeting CD19, an antigen widely expressed in B-cell leukemia and lymphoma, have become the first licensed cancer T-cell therapy. The success of CD19 CAR-T cells in treating relapsed and refractory B-cell malignancies has led to their expanded use in other tumors. Given the similarities between B-lymphocyte and T-lymphocyte malignancies, extending CAR-T cell therapy to these diseases might seem straightforward. However, CAR-T cell therapy for T-cell malignancies has proven difficult to develop and implement. This is mainly because the co-expression of the target antigen on engineered T cells can lead to fratricide of CAR-T cells during preparation; meanwhile, the depletion of normal peripheral blood T cells after CAR-T cell infusion can result in severe immunodeficiency.

[0005] CD7 is a transmembrane glycoprotein that is normally expressed on most peripheral blood T cells and NK cells (NK cells, namely natural killer cells, are derived from bone marrow lymphoid stem cells, and their differentiation and development depend on the bone marrow and thymic microenvironments. They are mainly distributed in the bone marrow, peripheral blood, liver, spleen, lungs, and lymph nodes; NK cells are different from T and B cells and are a type of lymphocyte that can non-specifically kill tumor cells and virus-infected cells without prior sensitization) and their precursors, and acts as a co-stimulatory protein for T-cell activation and interaction with other immune subsets. More than 95% of lymphoblastic leukemia and lymphoma, as well as some peripheral T-cell lymphomas, express CD7. In murine animal models, T cells lacking CD7 largely exhibit undisturbed development, homeostasis, and protective functions. Since CD7 has no significant impact on the function of peripheral blood T cells, it is a promising target for CAR-T cell therapy. CD7 has been evaluated as a target for monoclonal antibodies (mAbs) in the treatment of patients with T-cell malignancies using immunotoxins. The conjugate of this monoclonal antibody did not produce severe CD7-related toxic side effects, but the anti-tumor response was not significant, which might be due to the limited activity of the murine antibody in patient treatment.

[0006] In the prior art, the research progress on the treatment targeting CD7 is as follows:

[0007] i) CD7-CAR-T cell therapy; T cells expressing chimeric antigen receptor (CAR) are a promising approach for cancer immunotherapy. This targeted therapy has shown great potential in achieving remission and even long-term relapse-free survival in patients with B-cell leukemia and lymphoma. Recently, several research groups have reported progress in preclinical models of T-cell malignancies using CD7-specific CAR-T cell therapy. In all these studies, expression of CD7-CAR on T cells led to severe suicide, preventing CAR-T cells from expanding in vitro.

[0008] Expression of target-specific CAR on transduced T cells can cause CAR receptor activation due to continuous ligand binding, leading to suicide of transduced T cells and accelerated terminal differentiation of cells, making them unable to survive long term in vivo. Since CD7 is a pan-T cell antigen expressed on most T cells, CD7 antigen-specific CAR-T cells can exhibit severe suicide during preparation, preventing effective expansion of CAR-T cells. There are two strategies to reduce this suicide: 1) using genome editing tools to knockout the gene for the CD7 target antigen; 2) preventing the CD7 protein from being transported to the cell surface during intracellular expression by anchoring the CD7 binding domain in the endoplasmic reticulum. Both methods can effectively reduce CD7 expression on the cell surface and minimize suicide of targeted CD7-CAR-T cells. Importantly, deletion of CD7 does not affect the proliferation and short-term effector functions of CAR-T cells, allowing expansion of functional CAR-T cells with high anti-tumor activity.

[0009] After removing CD7 from the cell surface, CD7-CAR-T cells have strong anti-tumor activity against primary CD7-positive T-ALL and lymphoma in vitro and in vivo. CD7-CAR-T cells are also cytotoxic to peripheral blood CD7-positive T cells and NK cells, indicating that these cell subsets will also be targets of CD7-CAR-T cells.

[0010] One risk of adoptive cell therapy using autologous cells is the inadvertent genetic modification of malignant cells in peripheral blood. Malignant T cells modified with the CAR gene may, in some cases, allow them to be genetically edited to reduce the expression of target molecules, which may be a real risk and thus needs to be stated in the informed consent for using these treatments. However, considering the poor survival and expansion of malignant cells during the manufacturing process, these unnecessary genetic modifications of malignant cells should occur at a relatively low frequency. In addition, in the absence of genetic editing to reduce the expression of the target antigen, malignant tumor cells expressing the target antigen may be eliminated by fratricide of CAR-T cells after transduction and before infusion into the patient. At the same time, allogeneic CD7-CAR-T cells produced from healthy donors can be used to treat relapsed and refractory T-cell malignancies to bridge the patient to hematopoietic stem cell transplantation or to treat T-cell malignancies that recur after hematopoietic stem cell transplantation. If the side effects of graft-versus-host can be properly controlled, the advantage of allogeneic CD7-CAR-T cell therapy is that in addition to T cells being provided by healthy donors, it can also produce a graft-versus-leukemia effect. Using off-the-shelf allogeneic T cells or NK cells as carriers can completely avoid the risk of genetically modifying malignant cells. Since these cell products will be produced by healthy donors, the use of patient-derived T cells, which are often dysfunctional due to long-term exposure to an inhibitory tumor microenvironment or previous intensive treatment, can be avoided.

[0011] ii) Immunotoxins targeting CD7; CD7 is expressed on T cells at a high density (about 60,000 molecules / cell), and even when bound by a monovalent antibody fragment, CD7 is rapidly internalized. Therefore, it is an ideal target antigen for immunotoxin-mediated treatment of T-cell tumors. Currently, anti-CD7 immunotoxins are mainly composed of anti-CD7 monoclonal antibodies conjugated with toxins. An anti-CD7 immunotoxin composed of a murine anti-human CD7 monoclonal antibody (WTI) conjugated with ricin A has been used to eliminate tumor cells in vitro for autologous bone marrow transplantation in patients with T-cell malignancies. The immunotoxin DA7 was constructed by chemically linking a murine IgG2b anti-CD7 (3AlE) monoclonal antibody with deglycosylated ricin A chain and has been used to treat human T-ALL in a SCID animal model, suggesting its potential therapeutic effect on T-cell leukemia with poor prognosis. In the phase I clinical trial of DA7, although limited by instability and vascular toxicity, an objective clinical response was achieved at its maximum tolerated dose. The monoclonal antibody TH69 against human CD7 has also been used to construct a recombinant immunotoxin, which links a single-chain antibody fragment (scFv) with a truncated fragment of Pseudomonas exotoxin A by genetic recombination.

[0012] iii) Gene knockout; The clinical feasibility of targeted gene-edited T cells in adoptive immunotherapy has been well demonstrated. Gene knockout of the HIV co-receptor CCR5 in CD4+ T cells using zinc finger nucleases renders these cells resistant to HIV infection and enables CCR5-negative T cells to engraft and persist in HIV-infected patients. T cell receptor (TCR) gene knockout in CD19 CAR-T cells using TALENs enables the successful treatment of B cell leukemia patients with third-party T cells, which is an encouraging milestone for "universal" off-the-shelf T cell products and greatly reduces the risk of graft-versus-host disease. In this study, the CD52 gene was also knocked out in the infused CAR-T cells to render them resistant to alemtuzumab. Recently, the CRISPR / Cas9 system has been used to knockout CD7 or knockout both CD7 and the T cell receptor alpha chain simultaneously. This new gene editing system can rapidly and efficiently knockout target genes in T cells.

[0013] iv) Intracellular protein expression blocking technology; In many biochemical and immune systems, it is often difficult to determine the role of certain specific molecules. One approach is to inhibit gene expression and look for its functional effects. Gene knockout is achieved through homologous recombination or recently developed gene editing technologies such as Zince Finger nucleases, TALENs, and CRISPR / Cas9. Although these technologies are very powerful, there are many deficiencies or technical difficulties in practice. Therefore, there is a great need to develop a simple, effective, and controllable technology for removing molecules.

[0014] Numerous studies have shown that intracellular antibodies can be used to target and inactivate important molecules within cells. By constructing the heavy and light chains of an antibody and transfecting mammalian cells, and making simple modifications to its leader sequence, the antibody sequence can be anchored in three major intracellular compartments: the endoplasmic reticulum (ER) / Golgi apparatus, the cytoplasm, and the nucleus. Initial intracellular antibody studies used whole IgG, but recent studies have focused on Fab' and single-chain antibodies (scFv). Single-chain antibody molecules are very small (about 30 kDa) and are constructed by linking the variable regions of the heavy and light chains with a short peptide sequence. These constructs have many advantages as intracellular antibodies, mainly that they do not require the association of two separate antibody chains to form an antigen-binding site.

[0015] So far, studies have shown that intracellular antibodies can be used to prevent the processing of HIV gp160 to gp130 in the endoplasmic reticulum of cells; block the expression of the IL-2 receptor α chain; inhibit cytoplasmic enzymes in yeast, Xenopus oocytes, and human T cell lines, and inhibit virus activity in transgenic plants. The research by Marasco and his colleagues showed that single-chain antibodies can function in the ER. The research by Greenman et al. showed that intracellular single-chain antibodies can be used to prevent the expression of cell membrane proteins. They used a high-affinity monoclonal antibody that binds to the surface antigen CD2 of T lymphocytes and an ER retention signal (KDEL).

[0016] The antigen recognition domain is an important part of the CAR structure and is responsible for binding to the surface antigen of tumor cells. Currently, the antigen recognition domains of CD7-specific CAR-T cell therapies mostly consist of the variable regions of mouse or alpaca-derived antibodies. Once infused into patients, these CAR-T cells carrying xenogeneic antigen recognition domains can induce strong host cellular and humoral responses, and the infused CAR-T cells are rapidly cleared by the host immune system, severely affecting the persistence of CAR-T cells in the body and ultimately leading to the intractability and recurrence of the disease. The present invention uses the natural human CD7 ligand as the antigen recognition domain of the CAR. Its advantage is that the constructed CD7-targeted CAR-T will not induce host immune responses and can survive in the body for a long time, thus achieving better therapeutic effects. Summary of the Invention

[0017] Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide an engineered immune cell targeting CD7, a chimeric antigen receptor, a CD7 blocking molecule, and their applications.

[0018] The purpose of the present invention is achieved through the following solutions:

[0019] In a first aspect of the present invention, there is provided an engineered immune cell, which includes a polynucleotide sequence encoding a chimeric antigen receptor, and the chimeric antigen receptor includes an antigen recognition domain targeting CD7.

[0020] In another preferred embodiment, the chimeric antigen receptor further includes a hinge transmembrane domain, an intracellular co-stimulatory domain, and an intracellular primary stimulatory domain.

[0021] In another preferred embodiment, the hinge transmembrane domain in the chimeric antigen receptor is selected from the amino acid sequences of at least one of the following groups: the α-chain, β-chain of the T cell receptor, CD3δ, CD3ε, CD3γ, CD3ζ chain, CD4, CD5, CD8α, CD8β, CD9, CD16, CD22, CD28, CD32, CD33, CD34, CD35, CD37, CD45, CD64, CD80, CD86, CD137, ICOS, CD154, FAS, FGFR2B, OX40 or VEGFR2.

[0022] In another preferred embodiment, the hinge transmembrane domain in the chimeric antigen receptor is derived from CD8α.

[0023] In another preferred embodiment, the amino acid sequence of the hinge transmembrane domain CD8α in the chimeric antigen receptor is as shown in SEQ ID NO.1.

[0024] In another preferred embodiment, the intracellular co-stimulatory domain in the chimeric antigen receptor is selected from at least one of the following: CD2, CD4, CD5, CD8α, CD8β, CD27, CD28, CD30, CD40, 4-1BB (CD137), ICOS, OX40, LIGHT (CD258) or NKG2C.

[0025] In another preferred embodiment, the intracellular co-stimulatory domain in the chimeric antigen receptor is derived from 4-1BB.

[0026] In another preferred embodiment, the amino acid sequence of the intracellular co-stimulatory domain 4-1BB in the chimeric antigen receptor is as shown in SEQ ID NO.2.

[0027] In another preferred embodiment, the hinge transmembrane domain and the intracellular co-stimulatory domain in the chimeric antigen receptor are derived from CD28.

[0028] In another preferred embodiment, the amino acid sequence of the hinge transmembrane domain and the intracellular co-stimulatory domain CD28 in the chimeric antigen receptor is as shown in SEQ ID NO.3.

[0029] In another preferred embodiment, the intracellular primary stimulatory domain in the chimeric antigen receptor is selected from at least one of the following: CD3δ, CD3ε, CD3γ, CD3ζ, FcRβ, FcRγ, CD5, CD66, CD22, CD79a or CD79b.

[0030] In another preferred embodiment, the intracellular primary stimulatory domain in the chimeric antigen receptor is derived from CD3ζ.

[0031] In another preferred example, the amino acid sequence of the intracellular major stimulating domain CD3ζ in the chimeric antigen receptor is as shown in SEQ ID NO.4.

[0032] In another preferred example, the antigen recognition domain targeting CD7 in the chimeric antigen receptor is a partial or full sequence of the extracellular domain of human CD7-L, or has at least 90% sequence identity with the extracellular domain of human CD7-L, and the amino acid sequence of the extracellular domain of human CD7-L is as shown in SEQ ID NO.5.

[0033] In another preferred example, the engineered immune cells further include a CD7 blocking molecule, which includes a CD7 binding domain and an intracellular anchoring domain, and the CD7 blocking molecule can prevent the transport and expression of CD7 protein on the cell surface.

[0034] In another preferred example, the CD7 blocking molecule specifically blocks the transport of CD7 protein to the cell surface by connecting the intracellular anchoring domain through the CD7 binding domain.

[0035] In another preferred example, the amino acid sequence of the intracellular anchoring domain is an endoplasmic reticulum retention domain, a Golgi retention domain, or a proteasome localization domain.

[0036] In another preferred example, the intracellular anchoring domain is an endoplasmic reticulum retention domain.

[0037] In another preferred example, the amino acid sequence of the endoplasmic reticulum retention domain of the intracellular anchoring domain is as shown in SEQ ID NO.6 or SEQ ID NO.7.

[0038] In another preferred example, the CD7 binding domain is a partial or full sequence of the extracellular domain of human CD7-L, or has at least 90% sequence identity with the extracellular domain of human CD7-L, and the amino acid sequence of the extracellular domain of human CD7-L is as shown in SEQ ID NO.5.

[0039] In another preferred example, the CD7 binding domain is the scFv of the anti-CD7 monoclonal antibody TH69.

[0040] In another preferred example, 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 identity with the scFv of the anti-CD7 monoclonal antibody TH69, and the amino acid sequence of the scFv of the anti-CD7 monoclonal antibody TH69 is as shown in SEQ ID NO.8.

[0041] In another preferred example, the engineered immune cells further include a CD7 blocking molecule, which can prevent the transport and expression of CD7 protein on the cell surface.

[0042] In another preferred example, the CD7 blocking molecule is to prevent the transport of CD7 protein to the cell surface by connecting an intracellular anchoring domain through a CD7 binding domain.

[0043] In another preferred example, the intracellular anchoring domain is an amino acid sequence of an endoplasmic reticulum retention domain, a Golgi retention domain or a proteasome targeting domain.

[0044] In another preferred example, the CD7 binding domain is a partial or full sequence of the extracellular domain of human CD7-L, or has at least 90% sequence identity with the extracellular domain of human CD7-L, and the amino acid sequence of the extracellular domain of human CD7-L is shown as SEQ ID NO.5.

[0045] In another preferred example, the engineered immune cells remove the gene expression of CD7 by gene knockout technology.

[0046] In another preferred example, the genome editing tool used in the knockout technology is TALENs or CRISPR / cas9.

[0047] In another preferred example, the cells are T cells, γδ T cells, NK or NKT cells, and T cells, γδ T cells, NK or NKT cells differentiated from induced pluripotent stem cells (iPSCs).

[0048] The second aspect of the present invention provides an antigen recognition domain targeting CD7 in a chimeric antigen receptor, and the sequence of the antigen recognition domain includes a partial or full sequence of the extracellular domain of human CD7-L, or has at least 90% sequence identity with the extracellular domain of human CD7-L; the amino acid sequence of the extracellular domain of human CD7-L is shown as SEQ ID NO.5.

[0049] The third aspect of the present invention provides an application of the antigen recognition domain targeting CD7 as described above in the preparation of an immunotoxin against CD7-positive hematological malignancies.

[0050] The fourth aspect of the present invention provides a nucleic acid molecule encoding the antigen recognition domain targeting CD7 in the chimeric antigen receptor as described above.

[0051] In another preferred example, 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 as SEQ ID NO.9.

[0052] The fifth aspect of the present invention provides a recombinant vector comprising the nucleic acid molecule encoding the antigen recognition domain targeting CD7 in the chimeric antigen receptor described above.

[0053] In another preferred embodiment, the vector is selected from retroviruses, lentiviruses or transposons.

[0054] The sixth aspect of the present invention provides the use of the recombinant vector described above in the preparation of engineered immune cells.

[0055] The seventh aspect of the present invention provides a CD7 blocking molecule for the preparation of the engineered immune cells described above, wherein the CD7 binding domain of the CD7 blocking molecule comprises the extracellular domain of human CD7-L with partial or all of its sequences, or a protein having at least 90% sequence identity with the extracellular domain of human CD7-L.

[0056] The eighth aspect of the present invention provides a nucleic acid sequence encoding the CD7 blocking molecule described above.

[0057] In another preferred embodiment, the nucleic acid sequence of the CD7 blocking molecule is CD7-L-ER2.1; the nucleotide sequence of CD7-L-ER2.1 is shown as SEQ ID NO.10.

[0058] The ninth aspect of the present invention further provides a CD7 blocking molecule for the preparation of 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 having at least 90% sequence identity with the scFv of the anti-CD7 monoclonal antibody TH69.

[0059] The tenth aspect of the present invention provides a nucleic acid sequence encoding the CD7 blocking molecule described in the ninth aspect. The coding sequence of the nucleic acid molecule of the CD7 blocking molecule is TH69-ER2.1; the nucleotide sequence of TH69-ER2.1 is shown as SEQ ID NO.11.

[0060] 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 aspect or the tenth aspect.

[0061] In another preferred embodiment, the vector is selected from retroviruses, lentiviruses or transposons.

[0062] The twelfth aspect of the present invention provides the use of the recombinant vector provided in the eleventh aspect above in the preparation of engineered immune cells.

[0063] The thirteenth aspect of the present invention provides a nucleic acid molecule encoding a chimeric antigen receptor that simultaneously contains an antigen recognition domain targeting CD7 described in the second aspect and a CD7 blocking molecule (the CD7 blocking molecule provided in the seventh aspect or the ninth aspect).

[0064] The fourteenth aspect of the present invention provides a recombinant vector comprising the nucleic acid molecule described in the thirteenth aspect.

[0065] In another preferred example, the vector is selected from retroviruses, lentiviruses, and transposons.

[0066] In another preferred example, the recombinant vector contains the following sequences: CD7BB-BL4-002 or CD7BB-BL6-002. The schematic diagrams of the vector sequences of the recombinant vectors CD7BB-BL4-002 or CD7BB-BL6-002 are respectively as Figure 5 shown as A and B in

[0067] In another preferred example, the recombinant vector CD7BB-BL4-002 is formed by ligating CD7BB-002 and TH69-ER2.1 through T2A. The schematic diagram of the sequence structure of the recombinant vector is as Figure 5 shown as (1) and (2) of A in

[0068] In another preferred example, the recombinant vector CD7BB-BL6-002 is formed by ligating TH69BB-002 and CD7-L-ER2.1 through T2A. The schematic diagram of the sequence structure of the recombinant vector is as Figure 5 shown as (3) and (4) of B in

[0069] In another preferred example, the signal peptide of the vector is derived from CD8α.

[0070] In another preferred example, the amino acid sequence of the CD8α signal peptide of the vector is as shown in SEQ ID NO.12.

[0071] In another preferred example, the signal peptide of the vector is derived from GM-CSF-R.

[0072] In another preferred example, the amino acid sequence of the GM-CSF-R signal peptide of the vector is as shown in SEQ ID NO.13

[0073] In another preferred example, the light chain and heavy chain of the scFv of the vector are connected by a (GGGGS)3 linker peptide, and the amino acid sequence of the linker peptide is as shown in SEQ ID NO.14.

[0074] In another preferred embodiment, the light chain and heavy chain of the scFv of the vector are linked by a Whitlow linker peptide, and the amino acid sequence of the linker peptide is as shown in SEQ ID NO.15.

[0075] In another preferred embodiment, the nucleic acid molecule encoding the chimeric antigen receptor and the nucleic acid molecule encoding the CD7 blocking molecule in the vector are linked by an Internal Ribosome Entry Site (IRES) or a ribosomal codon skipping site.

[0076] In another preferred embodiment, the Internal Ribosome Entry Site (IRES) of the vector is derived from Encephalomyocarditis virus (EMCV) or Enterovirus.

[0077] In another preferred embodiment, the ribosomal codon skipping site of the vector comprises a 2A self-cleaving peptide, and the 2A self-cleaving peptide can be selected from the foot-and-mouth disease virus 2A peptide, equine rhinitis A virus 2A peptide, porcine teschovirus-1 2A peptide or thosea asigna virus 2A (T2A).

[0078] In another preferred embodiment, the 2A self-cleaving peptide of the vector is derived from T2A.

[0079] In another preferred embodiment, the amino acid sequence of the T2A self-cleaving peptide of the vector is as shown in SEQ ID NO.16.

[0080] In another preferred embodiment, the 2A self-cleaving peptide of the vector is derived from F2A.

[0081] In another preferred embodiment, the amino acid sequence of the F2A self-cleaving peptide of the vector is as shown in SEQ ID NO.17.

[0082] The fifteenth aspect of the present invention provides the use of the recombinant vector described in the fourteenth aspect in the preparation of engineered immune cells.

[0083] The sixteenth aspect of the present invention provides a reagent combination, which comprises: (1) the recombinant vector described in the fifth aspect above, and (2) the vector described in the eleventh aspect above.

[0084] 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 treating CD7-positive hematological malignancies.

[0085] The eighteenth aspect of the present invention provides a reagent combination, which comprises: (1) the vector described in the fifth aspect above, and (2) a gene editing tool capable of knocking out the CD7 gene in cells.

[0086] In another preferred example, the gene editing tool is TALENs or CRISPR / cas9.

[0087] The nineteenth aspect of the present invention provides an antigen recognition domain sequence targeting CD7 in a chimeric antigen receptor, which is the scFv of the anti-CD7 monoclonal antibody TH69, or has at least 90% sequence identity with the scFv of the anti-CD7 monoclonal antibody TH69, and the amino acid sequence of the scFv of the anti-CD7 monoclonal antibody TH69 is shown in SEQ ID NO.8.

[0088] The twentieth aspect of the present invention provides a nucleic acid molecule encoding the antigen recognition domain sequence targeting CD7 in the chimeric antigen receptor described in the nineteenth aspect.

[0089] In another preferred example, the nucleic acid molecule encoding sequence described in the twentieth aspect is TH69BB-002, and the nucleic acid molecule sequence of TH69BB-002 is shown in SEQ ID NO.18.

[0090] The twenty-first aspect of the present invention provides a recombinant vector comprising the sequence of the nucleic acid molecule described in the twentieth aspect.

[0091] In another preferred example, the vector is selected from retroviruses, lentiviruses, and transposons.

[0092] The twenty-second aspect of the present invention provides a reagent combination, which comprises:

[0093] (1) the recombinant vector described in the twenty-first aspect,

[0094] and (2) one of the following combinations: a recombinant vector containing the nucleic acid sequence described in the eighth aspect, or a gene editing tool capable of knocking out the CD7 gene in cells.

[0095] The twenty-third aspect of the present invention provides the use of the reagent combination described in the twenty-second aspect in the preparation of CAR-T or CAR-NK cells for treating CD7-positive hematological malignancies.

[0096] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses a human-derived CD7-L substituted antibody sequence as the antigen recognition domain of CD7-specific CAR-T cells. The advantage of using human-derived CD7-L as the antigen recognition domain in targeted CD7 CAR is that it can prevent host cell and humoral responses, thereby achieving long-term survival and better efficacy of CAR-T cells in vivo after reinfusion.

[0097] CD7 is a transmembrane glycoprotein that is usually expressed in most peripheral T cells, NK cells, and their precursors. Lesion T cells and NK cells themselves highly express CD7; T cells lacking CD7 largely exhibit undisturbed development, homeostasis, and protective functions; since the effect of CD7 on peripheral blood T cell function is not obvious, CD7 is a promising CAR-T cell therapy target. Since both normal and lesion T cells themselves express CD7, when preparing chimeric antigen receptor (CAR) T cells, two aspects need to be considered simultaneously: 1. Genetically modify normal T cells to make them express chimeric antigen receptors (CAR-T) targeting CD7 to kill CD7-positive lesion T cells; 2. To avoid the suicide phenomenon caused by mutual recognition of CAR-T cells, it is necessary to block the CD7 expression of CAR-T cells themselves. Therefore, the technical solution of the present invention both genetically modifies normal T cells to make them express CD7-specific CAR and takes into account blocking the CD7 expression inside normal T cells. The applicant of the present invention has conducted a large number of experiments, continuously corrected and verified 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 to make them express CD7-specific CAR, and at the same time can block the CD7 expression of normal T cells, achieving unexpected technical effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] By reading the detailed description of the non-limiting embodiments with reference to the following drawings, other features, objects, and advantages of the present invention will become more apparent:

[0099] Figure 1 To establish K562 and HeLa cell lines expressing CD7. K562 and HeLa cell lines were transduced with a lentiviral vector carrying CD7 cDNA, and K562-CD7 and HeLa-CD7 were obtained by flow sorting. The figure shows the flow cytometry detection of CD7 expression on K562 and HeLa cell lines.

[0100] Figure 2Schematic diagram of the vector structures of CD7-CAR and CD7 blocking molecules. A and B are two second-generation CD7-specific CARs. Among them, for A, the antigen recognition region of CD7BB-002 is CD7-L; for B, the antigen recognition region of TH69BB-002 is the scFv of monoclonal antibody TH69; the two CAR vectors have the same CD8a hinge transmembrane region, 4-1BB co-stimulatory domain, and CD3ζ as the T cell stimulation domain. The scFv of monoclonal antibody TH69 uses the CD8α signal peptide, and the linker peptide between the variable regions of its light chain (VL) and heavy chain (VH) is (GGGGS)3. C and D are two CD7 blocking molecules. Among them, for C, the CD7 binding domain of CD7-L-ER2.1 is CD7-L; for D, the CD7 binding domain of TH69-ER2.1 is the scFv of TH69, which uses the CD8α signal peptide, and the linker peptide between the variable regions of its light chain (VL) and heavy chain (VH) is (GGGGS)3; the two CD7 blocking molecules have the same ER endoplasmic reticulum retention domain.

[0101] Figure 3 Schematic diagram of the flow cytometry detection and in vitro killing experiment results of CD7-CAR-T cells. A. Flow cytometry detection of the expression of two CD7-targeting CARs, CD7BB-002 and TH69BB-002, on T cells; B. Use the iCELLigence TM Real-time cell analyzer (Agilent Biosciences, Inc.) for in vitro killing experiments; "T cell control" is an untransduced T cell control, "CD7BB-002" is CAR-T cells expressing CD7BB-002, and "TH69BB-002" is CAR-T cells expressing TH69BB-002. The results show that both of these two CAR-T cells can target CD7 to kill tumor cells. The flow cytometry detection reagent for CD7-CAR-T cells is CD7-CAR-GREEN; the target cells used in the in vitro cell killing experiment are HeLa-CD7.

[0102] Figure 4 For using Intrablock TM Schematic diagram of the results of blocking the expression of CD7 on the cell membrane using the Intrablock CD7 expression blocking technology. Lentiviral vectors CD7-L-ER2.1 and TH69-ER2.1 carrying the ligand CD7-L or TH69 scFV cDNA that can bind to CD7 and linked with the ER retention domain are used to transduce the K562-CD7 cell line (A) or T cells (B), and flow cytometry detection of CD7 is performed. The results show that both of these two CD7 expression blocking molecules can effectively reduce the expression of CD7 on the cell surface.

[0103] Figure 5 For using IntrablockTM Schematic diagram of the lentiviral vector structure of CD7-CAR constructed by CD7 expression blocking technology. A and B are two types using Intrablock TM CD7-CAR lentiviral vectors constructed by CD7 expression blocking technology. Among them, A, CD7BB-BL4-002, (1) is a schematic diagram of the chimeric antigen receptor targeting CD7, and its antigen recognition region is CD7-L; (2) is a schematic diagram of the CD7 blocking molecule, and the CD7 binding domain that plays the role of CD7 expression blocking 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, and the CD7 binding domain that plays the role of CD7 expression blocking is CD7-L; the two CAR vectors have the same CD8α hinge transmembrane region, 4-1BB co-stimulatory domain, CD3ζ T cell stimulation domain and ER endoplasmic reticulum retention domain. The scFv of the monoclonal antibody TH69 uses the CD8α signal peptide, and the linker peptide between the light chain and the heavy chain variable regions is (GGGGS)3.

[0104] Figure 6 , After using Intrablock TM CD7-CAR-T cells can overcome the suicide phenomenon and effectively kill target cells by CD7 expression blocking technology. In (a), A, Flow cytometry was used to detect the expression of four different CARs targeting CD7 on T cells; B, Flow cytometry was used to detect the expression of CD7 on the above four different CAR-T cells; the vectors used were: CD7BB-002, TH69BB-002 and CD7BB-BL4-002 and CD7BB-BL6-002 constructed by CD7 expression blocking technology using Intrablock TM ; The dotted line is the control of untransduced T cells, and the solid line is the T cells transduced with the CAR targeting CD7. In (b), C, Proliferation observation of the above four different CAR-T cells in vitro culture; D, Using iCELLigence TM Real-time cell analyzer (Agilent Biosciences, Inc.) was used to perform in vitro killing experiments. T cell control is the control of untransduced T cells, 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 are constructed by CD7 expression blocking technology using Intrablock TMCD7-CAR-T cells using CD7 expression blocking technology. The flow cytometry detection reagent for CD7-CAR-T cells is CD7-CAR-GREEN; the target cells used in the in vitro cytotoxicity assay are HeLa-CD7.

[0105] Figure 7 , using Intrablock TM Results of in vivo tumor killing experiment of CD7-CAR-T cells using CD7 expression blocking technology. Female NSG mice were used. On day D0, tumor cells (tumor cell line carrying luciferase, CCRF-CEM-Luc, 5x10E5 / mouse, i.v.) were injected. A, 1. Negative control group; 2 and 3. T cell infusion control group and CD7BB-BL4-002 CAR-T cell treatment group targeting CD7, respectively. On day D3 after injecting tumor cells, T cells or CD7BB-BL4-002 CAR-T cells (8x10E6 / mouse) were injected intravenously, and then in vivo luciferase imaging of mice was observed every 7 days. B, Survival curves of mice in the T cell infusion control group (control group) and CD7BB-BL4-002 CAR-T cell treatment group (treatment group). Detailed implementation manners

[0106] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several changes and improvements can still be made. These all belong to the protection scope of the present invention.

[0107] The present invention uses the extracellular domain of SECTM1 (K12) as the antigen recognition domain of CD7 for the development of CAR-T cell therapy and immunotoxin against CD7-positive hematological malignancies. In this patent application, CD7-L is used instead of SECTM1 or K12. The CD7-L gene was initially identified at the 5′ end of the CD7 gene on human chromosome 17. Human CD7-L protein is mainly expressed in the spleen, prostate, testis, small intestine and peripheral blood leukocytes. One feature of CD7-L is that it encodes a transmembrane protein, and the extracellular domain is similar to an immunoglobulin-like domain. CD7-L was cloned in 2000 and was found to be a binding protein of CD7.

[0108] To determine the binding of CD7-L protein, previous studies used a fusion protein of the extracellular domain of CD7-L (amino acids 1-145) and the Fc portion of human IgG1. Flow cytometry experiments showed that the CD7-L-Fc fusion protein could be detected with high levels of binding on human T cells and NK cells. Several anti-CD7 antibodies blocked the binding of the CD7-L-Fc fusion protein to cells to varying degrees. Conversely, the CD7-L-Fc fusion protein could block the binding of these anti-CD7 monoclonal antibodies to CD7, indicating that CD7-L-Fc could 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 (human T cell leukemia cell line) or KG-1 cells (human granulocytic leukemia cell line), both of which express CD7. The binding affinity (Ka) of CD7-L-Fc for human CD7 was approximately in the range of 1x10 8 M -1 range. Since CD7 is considered a good marker for T cell malignancies, studies have been conducted to produce immunotoxins by conjugating anti-human CD7 monoclonal antibodies with ricin toxin or saponin.

[0109] Therefore, this patent application uses the extracellular domain of CD7-L in the construction of chimeric antigen receptors targeting CD7 and their CD7-blocking molecules for the development of CD7-CAR-T or CAR-NK cell therapies for T cell malignancies. At the same time, this patent application also combines the extracellular region of CD7-L with toxins and uses these conjugates as immunotoxins against T cell malignancies, which may have a longer half-life due to their lower immunogenicity than antibody-based conjugates.

[0110] Example 1: CAR-T cells using CD7-L as the antigen recognition domain can effectively recognize the CD7 tumor antigen;

[0111] In this example, two second-generation CAR lentiviral vectors, CD7BB-002 and TH69BB-002 ( Figure 2 A, B), were first constructed with the scFv of CD7-L or the anti-CD7 monoclonal antibody TH69 as the antigen recognition regions. CAR-T cells were obtained by transduction with the CAR lentiviral vectors, and flow cytometry was performed using a flow cytometry reagent for CD7-CAR-T cells produced by fusing CD7 and the reporter gene eGFP (CD7-CAR-GREEN) ( Figure 3 A). The results demonstrated that CD7-CAR-GREEN could be effectively used to detect these two types of CAR-T cells targeting CD7, indicating that both CD7-L and the scFv of the monoclonal antibody TH69 could serve as CD7-specific antigen recognition regions for CAR-T cells.

[0112] Example 2. CAR-T cells targeting CD7 can effectively kill tumor cells expressing CD7 positive;

[0113] In this example, lentiviral vectors carrying CD7 cDNA were first used to transduce K562 and HeLa cell lines, and K562-CD7 and HeLa-CD7 cell lines expressing CD7 were obtained by flow sorting ( Figure 1 ). Two types of CAR-T cells targeting CD7, CD7BB-002 and TH69BB-002, were obtained by transduction with CAR lentiviral vectors, and an in vitro killing experiment was performed using an iCELLigence TM real-time cell analyzer (Agilent Biosciences, Inc.) ( Figure 3 B). The results proved that both CD7BB-002 with CD7-L as the antigen recognition region and TH69BB-002 with the monoclonal antibody TH69 scFv as the antigen recognition region could effectively recognize the CD7 antigen and had the same effect on killing HeLa-CD7 target cells positive for CD7.

[0114] Example 3. Intrablock TM CD7 expression blocking technology can effectively block the expression of CD7 on the cell membrane;

[0115] In this example, lentiviral vectors with CD7-L or the scFv of the CD7-specific monoclonal antibody TH69 as the CD7 binding domain and linked to the ER retention domain, CD7-L-ER2.1 and TH69-ER2.1, were first constructed ( Figure 2 C, D). K562-CD7 cell lines or primary T cells were transduced with lentiviral vectors, and flow cytometry was performed to evaluate the CD7 expression blocking technology. The results showed that both TH69-ER2.1 and CD7-L-ER2.1 could effectively block the expression of CD7 on the K562-CD7 cell line ( Figure 4 A) or T cells ( Figure 4 B), indicating that both TH69-ER2.1 and CD7-L-ER2.1 could bind to CD7 intracellularly 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.

[0116] Example 4. 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;

[0117] In this example, a CAR lentiviral vector, CD7BB-BL4-002 and CD7BB-BL6-002, with Intrablock TM CD7 expression blocking function and targeting CD7 was first constructed ( Figure 5 A, B). Since T cells used for the preparation of CAR-T cells inherently express CD7, suicidal phenomenon occurs during the preparation of CAR-T cells targeting CD7, making it difficult to prepare CAR-T cells. As Figure 6 shown, two types of CAR-T cells targeting CD7, CD7BB-002 and TH69BB-002, can both be effectively recognized by CD7-CAR-GREEN ( Figure 6 A), and can effectively kill CD7-positive HeLa-CD7 target cells ( Figure 6 D). However, serious suicidal phenomenon occurs during the in vitro culture process of these two types of CAR-T cells targeting CD7, resulting in difficulties in the in vitro expansion and preparation of CAR-T cells ( Figure 6 C). The Intrablock TM CD7 expression blocking technology described in Example 3 was used to construct CD7BB-BL4-002 and CD7BB-BL6-002 lentiviral vectors targeting CD7 and used to prepare CAR-T cells. These two types of CAR-T cells prepared using the Intrablock TM CD7 expression blocking technology, CD7BB-BL4-002 and CD7BB-BL6-002, can both be effectively recognized by CD7-CAR-GREEN ( Figure 6 A), maintain the killing ability of HeLa-CD7 target cells ( Figure 6 D), while being able to block the expression of CD7 ( Figure 6 B), overcome the suicidal phenomenon in the preparation of CAR-T cells ( Figure 6 C), making it possible to expand and prepare CD7-specific CAR-T cells in vitro.

[0118] Example 5: Verify the tumor killing function in vivo of CD7-CAR-T cells using Intrablock TM CD7 expression blocking technology in an animal model;

[0119] In this example, an in vivo tumor killing experiment was conducted using CD7BB-BL4-002 CAR-T cells with Intrablock TM CD7 expression blocking technology ( Figure 7)。 Female NSG mice at 6 - 8 weeks old were used. On day D0, the tumor cell line CCRF-CEM-Luc carrying luciferase was injected into the tail vein at a dose of 5x10E5 / mouse, i.v. On day D3 after injecting the tumor cells, T cells (T cell reinfusion control group) or CD7BB-BL4-002 CAR-T cells (8x10E6 / mouse) (CAR-T cell treatment group) were injected intravenously respectively. After day D0, in vivo luciferase imaging of mice was performed every 7 days. The results demonstrated that CD7BB-BL4-002 CAR-T cells using the Intrablock TM CD7 expression blocking technology could effectively kill tumors in this mouse tumor model and prolonged the survival time of mice in the CAR-T cell treatment group (as shown in A and B of Figure 7 ).

[0120] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses the human CD7-L substituted antibody sequence as the antigen recognition domain of CD7-specific CAR-T cells. The advantage of using human CD7-L as the antigen recognition domain in the targeted CD7 CAR is that it can prevent cellular and humoral responses generated by the host, thereby achieving the long-term survival and better efficacy of CAR-T cells in vivo after reinfusion. CD7 is a transmembrane glycoprotein that is usually expressed in most peripheral T cells and NK cells and their precursors. Diseased T cells and NK cells themselves express CD7 at high density; T cells lacking CD7 largely exhibit undisturbed development, homeostasis, and protective functions; since the effect of CD7 on the function of peripheral blood T cells is not obvious, CD7 is a promising CAR-T cell therapy target. Since both normal and diseased T cells themselves express CD7, when preparing chimeric antigen receptor (CAR) T cells, two aspects need to be considered simultaneously: 1. Genetically modify normal T cells to make T cells express chimeric antigen receptors targeting CD7 (CAR-T) to kill CD7-positive diseased T cells; 2. To avoid the suicide phenomenon caused by mutual recognition of CAR-T cells, it is necessary to block the CD7 expression of CAR-T cells themselves. Therefore, 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 blocking the CD7 expression inside normal T cells. The applicant of the present invention has conducted a large number of experiments, continuously corrected and verified 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 to make T cells express CD7-specific CAR, and at the same time can block the CD7 expression of normal T cells, achieving unexpected technical effects.

[0121] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various changes or modifications within the scope of the claims, which does not affect the essence of the present invention. Intrablock is a trademark identification and does not constitute any limitation or restriction on the technical solution of the present invention. Without conflict, the embodiments of the present application and the features in the embodiments can be combined with each other arbitrarily.

Claims

1. An engineered immune cell comprising a polynucleotide sequence encoding a chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises an antigen recognition domain targeting CD7; the antigen recognition domain targeting CD7 in the chimeric antigen receptor is the entire sequence of the extracellular domain of human CD7-L; the amino acid sequence of the extracellular domain of human CD7-L is as shown in SEQ ID NO.5; The chimeric antigen receptor further comprises a hinge transmembrane domain, an intracellular co-stimulatory domain and an intracellular primary stimulatory domain; The engineered immune cell further comprises a CD7 blocking molecule, and the CD7 blocking molecule can prevent the transport and expression of CD7 protein on the cell surface.

2. The engineered immune cells according to claim 1, characterized in that, The hinge transmembrane domain in the chimeric antigen receptor comprises at least one amino acid sequence selected from the following combinations: derived from the chain, β chain, CD3δ, CD3ε, CD3γ, CD3ζ chain, CD4, CD5, CD8α, CD8β, CD9, CD16, CD22, CD28, CD32, CD33, CD34, CD35, CD37, CD45, CD64, CD80, CD86, CD137, ICOS, CD154, FAS, FGFR2B, OX40 or VEGFR2 amino acid sequence.

3. The engineered immune cell according to claim 1, wherein The intracellular co-stimulatory domain in the chimeric antigen receptor comprises at least one selected from the following group: CD2, CD4, CD5, CD8α, CD8β, CD27, CD28, CD30, CD40, 4-1BB (CD137), ICOS, OX40, LIGHT (CD258) or NKG2C.

4. The engineered immune cell according to claim 1, characterized in that, The intracellular primary stimulatory domain in the chimeric antigen receptor comprises at least one selected from the following combinations: CD3δ, CD3ε, CD3γ, CD3ζ, FcRβ, FcRγ, CD5, CD66d, CD22, CD79a or CD79b.

5. The engineered immune cell according to claim 1, characterized in that, The CD7 blocking molecule specifically prevents the transport of CD7 protein to the cell surface by connecting an intracellular anchoring domain through a CD7 binding domain.

6. The engineered immune cell according to claim 5, wherein, The intracellular anchoring domain is the amino acid sequence of an endoplasmic reticulum retention domain, a Golgi retention domain or a proteasome localization domain.

7. The engineered immune cell according to claim 5, characterized in that, The CD7 binding domain is a partial or entire sequence of the extracellular domain of human CD7-L, and the amino acid sequence of the extracellular domain of human CD7-L is as shown in SEQ ID NO.

5.

8. The engineered immune cell according to claim 5, wherein, The CD7 binding domain is the scFv of the anti-CD7 monoclonal antibody TH69, and the amino acid sequence of the scFv of the anti-CD7 monoclonal antibody TH69 is as shown in SEQ ID NO.

8.

9. The engineered immune cell according to claim 1, characterized in that, The engineered immune cell removes the gene expression of CD7 through gene knockout technology.

10. The engineered immune cell according to claim 9, wherein, The genome editing tool used in the knockout technology is TALENs or CRISPR / Cas9.

11. The engineered immune cells according to any one of claims 1-10, characterized in that, The cell is a T cell or an NK cell.

12. The engineered immune cell according to claim 11, wherein, The T cell includes γδT or NKT cell.

13. The engineered immune cells according to any one of claims 1-10, characterized in that, The cell is a T cell differentiated from induced pluripotent stem cells or an NK cell differentiated from induced pluripotent stem cells.

14. The engineered immune cell according to claim 13, wherein, The T cell differentiated from induced pluripotent stem cells includes a γδT cell differentiated from induced pluripotent stem cells or an NKT cell differentiated from induced pluripotent stem cells.

Images