WO2024083213A1

WO2024083213A1 - Universal car-t cell targeting cd7

Info

Publication number: WO2024083213A1
Application number: PCT/CN2023/125577
Authority: WO
Inventors: 赵阳兵; 刘晓军; 朱庚振
Original assignee: 上海优替济生生物医药有限公司
Priority date: 2022-10-20
Filing date: 2023-10-20
Publication date: 2024-04-25

Abstract

The present invention relates to a modified T cell, a preparation method therefor and a use thereof. The expression of endogenous TRAC, B2M and CD7 genes in the T cell is inhibited, and the T cell expresses a chimeric antigen receptor targeting CD7. The modified T cell can be used to treat diseases associated with CD7 expression.

Description

Universal CAR-T cells targeting CD7

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to PCT application No. PCT/CN2022/126382, filed on October 20, 2022, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to the field of immunotherapy, and in particular, to universal CAR-T cells targeting CD7.

Background technique

Acute T-cell lymphoblastic leukemia (T-ALL) and T-cell lymphoblastic lymphoma (T-LBL) are highly aggressive T-lineage malignancies. Many patients will still relapse after chemotherapy or even allogeneic hematopoietic stem cell transplantation, and clinical needs are urgently needed. CD7 is highly expressed on the surface of T-ALL/T-LBL cells and is a potential therapeutic target. However, the preparation of CD7 CAR-T will face many challenges, including: it is difficult to separate normal T cells from the peripheral blood of such patients, especially when there are a large number of malignant T cells; CAR-T cells targeting CD7 also express CD7 themselves, and the proliferation of CAR-T is limited due to the "self-killing mechanism".

Summary of the invention

The present invention provides a universal CAR-T cell targeting CD7. The universal CAR-T cell targeting CD7 of the present invention can avoid allogeneic rejection and cannibalism, and also has a strong killing effect.

One aspect of the present invention provides a modified T cell, in which the expression of endogenous TRAC, B2M and CD7 genes is inhibited, and the T cell expresses a chimeric antigen receptor targeting CD7, wherein the chimeric antigen receptor includes a binding domain that specifically binds to CD7, a transmembrane domain and an intracellular signaling domain.

In some embodiments, the binding domain that specifically binds to CD7 comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3; wherein,

LCDR1 has an amino acid sequence selected from SEQ ID NO: 113-125;

LCDR2 has an amino acid sequence selected from SEQ ID NO: 126-137;

LCDR3 has an amino acid sequence selected from SEQ ID NO: 138-150;

HCDR1 has an amino acid sequence selected from SEQ ID NO: 87-94;

HCDR2 has an amino acid sequence selected from SEQ ID NO:95-102; and

HCDR3 has an amino acid sequence selected from SEQ ID NO:103-112.

In some embodiments, the binding domain that specifically binds to CD7 is a light chain variable region and a heavy chain variable region. comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 selected from the group consisting of:

(a) LCDR1 having the amino acid sequence set forth in SEQ ID NO:113, LCDR2 having the amino acid sequence set forth in SEQ ID NO:126, LCDR3 having the amino acid sequence set forth in SEQ ID NO:138, HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, HCDR2 having the amino acid sequence set forth in SEQ ID NO:95, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:103;

(b) LCDR1 having the amino acid sequence set forth in SEQ ID NO:114, LCDR2 having the amino acid sequence set forth in SEQ ID NO:127, LCDR3 having the amino acid sequence set forth in SEQ ID NO:139, HCDR1 having the amino acid sequence set forth in SEQ ID NO:88, HCDR2 having the amino acid sequence set forth in SEQ ID NO:96, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:104;

(c) LCDR1 having the amino acid sequence set forth in SEQ ID NO:115, LCDR2 having the amino acid sequence set forth in SEQ ID NO:128, LCDR3 having the amino acid sequence set forth in SEQ ID NO:140, HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, HCDR2 having the amino acid sequence set forth in SEQ ID NO:95, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:105;

(d) LCDR1 having the amino acid sequence set forth in SEQ ID NO:116, LCDR2 having the amino acid sequence set forth in SEQ ID NO:129, LCDR3 having the amino acid sequence set forth in SEQ ID NO:141, HCDR1 having the amino acid sequence set forth in SEQ ID NO:89, HCDR2 having the amino acid sequence set forth in SEQ ID NO:97, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:106;

(e) LCDR1 having the amino acid sequence set forth in SEQ ID NO: 117, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 130, LCDR3 having the amino acid sequence set forth in SEQ ID NO: 142, HCDR1 having the amino acid sequence set forth in SEQ ID NO: 90, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 98, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 107;

(f) LCDR1 having the amino acid sequence set forth in SEQ ID NO:118, LCDR2 having the amino acid sequence set forth in SEQ ID NO:131, LCDR3 having the amino acid sequence set forth in SEQ ID NO:143, HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, HCDR2 having the amino acid sequence set forth in SEQ ID NO:95, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:103;

(g) LCDR1 having the amino acid sequence set forth in SEQ ID NO:119, LCDR2 having the amino acid sequence set forth in SEQ ID NO:132, LCDR3 having the amino acid sequence set forth in SEQ ID NO:144, HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, HCDR2 having the amino acid sequence set forth in SEQ ID NO:95, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:103;

(h) LCDR1 having the amino acid sequence set forth in SEQ ID NO:120, LCDR2 having the amino acid sequence set forth in SEQ ID NO:133, LCDR3 having the amino acid sequence set forth in SEQ ID NO:145, HCDR1 having the amino acid sequence set forth in SEQ ID NO:91, HCDR2 having the amino acid sequence set forth in SEQ ID NO:99 and HCDR3 having the amino acid sequence set forth in SEQ ID NO:108;

(i) a LCDR1 having an amino acid sequence as shown in SEQ ID NO: 121, a LCDR2 having an amino acid sequence as shown in SEQ ID NO: 134, a LCDR3 having an amino acid sequence as shown in SEQ ID NO: 146, a HCDR1 having an amino acid sequence as shown in SEQ ID NO: 87, a HCDR2 having an amino acid sequence as shown in SEQ ID NO: 95, and a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 103;

(j) LCDR1 having the amino acid sequence set forth in SEQ ID NO: 122, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 135, LCDR3 having the amino acid sequence set forth in SEQ ID NO: 147, HCDR1 having the amino acid sequence set forth in SEQ ID NO: 87, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 95, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 109;

(k) LCDR1 having the amino acid sequence set forth in SEQ ID NO:123, LCDR2 having the amino acid sequence set forth in SEQ ID NO:136, LCDR3 having the amino acid sequence set forth in SEQ ID NO:148, HCDR1 having the amino acid sequence set forth in SEQ ID NO:92, HCDR2 having the amino acid sequence set forth in SEQ ID NO:100 and HCDR3 having the amino acid sequence set forth in SEQ ID NO:110;

(l) a LCDR1 having an amino acid sequence as set forth in SEQ ID NO:124, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO:137, a LCDR3 having an amino acid sequence as set forth in SEQ ID NO:149, a HCDR1 having an amino acid sequence as set forth in SEQ ID NO:93, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO:101 and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO:111; and

(m) LCDR1 having the amino acid sequence shown as SEQ ID NO:125, LCDR2 having the amino acid sequence shown as SEQ ID NO:137, LCDR3 having the amino acid sequence shown as SEQ ID NO:150, HCDR1 having the amino acid sequence shown as SEQ ID NO:94, HCDR2 having the amino acid sequence shown as SEQ ID NO:102 and HCDR3 having the amino acid sequence shown as SEQ ID NO:112.

In some embodiments, the binding domain that specifically binds to CD7 comprises a light chain variable region and a heavy chain variable region selected from the group consisting of:

(a) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:35 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:36;

(b) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:40 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:41;

(c) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:44 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:45;

(d) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:48 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:49;

(e) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:52 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:53;

(f) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:56 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:57;

(g) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:60 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:61;

(h) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:64 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:65;

(i) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 68 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 69;

(j) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:72 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:73;

(k) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:76 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:77;

(l) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:80 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:81; and

(m) comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO:84 and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:85.

In some embodiments, the binding domain that specifically binds to CD7 is a scFv.

In some embodiments, the transmembrane domain is a CD8 transmembrane domain.

In some embodiments, the intracellular signaling domain includes a 4-1BB co-stimulatory domain and a CD3 zeta intracellular domain.

In some embodiments, the chimeric antigen receptor further comprises a hinge region between the binding domain and the transmembrane domain.

In some embodiments, the hinge region is a CD8 hinge region.

In some embodiments, the chimeric antigen receptor targeting CD7 comprises an amino acid sequence selected from SEQ ID NO: 37, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86.

In some embodiments, the chimeric antigen receptor targeting CD7 further comprises a signal peptide.

In some embodiments, the signal peptide comprised by the chimeric antigen receptor targeting CD7 is a CD8 signal peptide.

In some embodiments, the nucleic acid sequence encoding the chimeric antigen receptor is inserted into the TRAC locus of the T cell.

In some embodiments, the nucleic acid sequence encoding the chimeric antigen receptor is operably linked to a PGK promoter or an endogenous TRAC promoter of the T cell.

In some embodiments, the modified T cells further express a fusion protein of a lymphocyte-antigen presenting cell co-stimulatory factor, wherein the fusion protein comprises a first domain that activates antigen presenting cells (APCs) and a second domain that activates immune effector cells, wherein

The first domain comprises: (i) a ligand that binds to an activating receptor of the APC, or a receptor-binding fragment thereof, or (ii) an antibody that binds to an activating receptor of the APC, or an antigen-binding fragment thereof; and

The second domain includes: (i) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (ii) a co-stimulatory ligand of the immune effector cell, or a receptor binding fragment thereof, or (iii) an antibody that binds to the co-stimulatory receptor of the immune effector cell, or an antigen binding fragment thereof.

In some embodiments, the activating receptor of the APC is CD40.

In some embodiments, the first domain is an anti-CD40 antibody or an antigen-binding fragment thereof.

In some embodiments, the first domain is a scFv.

In some embodiments, the immune effector cells are T cells.

In some embodiments, the second domain comprises the intracellular domain of the co-stimulatory receptor.

In some embodiments, the co-stimulatory receptor is CD28.

In some embodiments, the second domain further comprises a transmembrane domain of the co-stimulatory receptor.

In some embodiments, the first domain and the second domain are connected by a CD28 hinge region.

In some embodiments, the lymphocyte-antigen presenting cell co-stimulatory factor fusion protein includes an amino acid sequence as shown in SEQ ID NO:161.

In some embodiments, the lymphocyte-antigen presenting cell co-stimulatory factor fusion protein further comprises a signal peptide.

In some embodiments, the signal peptide contained in the lymphocyte-antigen presenting cell co-stimulatory factor fusion protein is a CD8 signal peptide.

In some embodiments, the nucleic acid sequence encoding the lymphocyte-antigen presenting cell co-stimulatory factor fusion protein and the nucleic acid sequence encoding the chimeric antigen receptor targeting CD7 are in the same expression frame or in separate expression frames.

In some embodiments, endogenous TRAC, B2M and CD7 genes are knocked out in the T cells.

Another aspect of the present invention provides a method for producing a modified T cell, the method comprising:

(a) inhibiting the expression of endogenous TRAC, B2M and CD7 genes in the T cells; and

(b) introducing a polynucleotide sequence comprising a nucleic acid sequence encoding a chimeric antigen receptor targeting CD7 into the T cell, so that the T cell expresses the chimeric antigen receptor, wherein the chimeric antigen receptor targeting CD7 comprises a binding domain that specifically binds to CD7, a transmembrane domain, and an intracellular signaling domain.

LCDR1 has an amino acid sequence selected from SEQ ID NO: 113-125;

LCDR2 has an amino acid sequence selected from SEQ ID NO: 126-137;

LCDR3 has an amino acid sequence selected from SEQ ID NO: 138-150;

HCDR1 has an amino acid sequence selected from SEQ ID NO: 87-94;

HCDR2 has an amino acid sequence selected from SEQ ID NO:95-102; and

HCDR3 has an amino acid sequence selected from SEQ ID NO:103-112.

In some embodiments, the light chain variable region and heavy chain variable region of the binding domain that specifically binds to CD7 include LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 selected from the group consisting of:

(b) LCDR1 having the amino acid sequence shown in SEQ ID NO: 114, and LCDR2 having the amino acid sequence shown in SEQ ID NO: 127 a LCDR2 having the amino acid sequence shown in SEQ ID NO: 139, a LCDR3 having the amino acid sequence shown in SEQ ID NO: 139, a HCDR1 having the amino acid sequence shown in SEQ ID NO: 88, a HCDR2 having the amino acid sequence shown in SEQ ID NO: 96, and a HCDR3 having the amino acid sequence shown in SEQ ID NO: 104;

(i) LCDR1 having the amino acid sequence set forth in SEQ ID NO:121, LCDR2 having the amino acid sequence set forth in SEQ ID NO:134, LCDR3 having the amino acid sequence set forth in SEQ ID NO:146, HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, HCDR2 having the amino acid sequence set forth in SEQ ID NO:95 and HCDR3 having the amino acid sequence set forth in SEQ ID NO:103;

(k) LCDR1 having the amino acid sequence of SEQ ID NO: 123, LCDR2 having the amino acid sequence of SEQ ID NO: 136, LCDR3 having the amino acid sequence of SEQ ID NO: 148, LCDR4 having the amino acid sequence of SEQ ID NO: 150, a HCDR1 having the amino acid sequence set forth in SEQ ID NO:92, a HCDR2 having the amino acid sequence set forth in SEQ ID NO:100, and a HCDR3 having the amino acid sequence set forth in SEQ ID NO:110;

(i) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:68 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:69;

(1) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 80 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 81; and

In some embodiments, the transmembrane domain is a CD8 transmembrane domain.

In some embodiments, the hinge region is a CD8 hinge region.

In some embodiments, step (a) comprises knocking out endogenous TRAC, B2M and CD7 genes in the T cells.

In some embodiments, step (a) comprises introducing CRISPR-Cas protein and gRNA targeting endogenous TRAC, B2M and CD7 genes in the T cell into the T cell to produce cuts in the endogenous TRAC, B2M and CD7 genes of the T cell and disrupt their expression.

In some embodiments, the gRNA targeting the TRAC gene comprises a guide sequence selected from SEQ ID NO:23 and SEQ ID NO:24.

In some embodiments, the gRNA targeting the B2M gene comprises a guide sequence selected from SEQ ID NO:15 and SEQ ID NO:22.

In some embodiments, the gRNA targeting the CD7 gene comprises a guide sequence as shown in SEQ ID NO:25.

In some embodiments, the gRNAs targeting TRAC, B2M, and CD7 genes respectively comprise guide sequences selected from the group consisting of:

The gRNA targeting the TRAC gene comprises the guide sequence shown in SEQ ID NO: 23, the gRNA targeting the B2M gene comprises the guide sequence shown in SEQ ID NO: 15, and the gRNA targeting the CD7 gene comprises the guide sequence shown in SEQ ID NO: 25; and

The gRNA targeting the TRAC gene comprises the guide sequence shown as SEQ ID NO:24, the gRNA targeting the B2M gene comprises the guide sequence shown as SEQ ID NO:15, and the gRNA targeting the CD7 gene comprises the guide sequence shown as SEQ ID NO:25.

In some embodiments, the polynucleotide sequence comprises homology arms flanking the nucleic acid sequence encoding the chimeric antigen receptor, which are homologous to the upstream and downstream sequences of the TRAC gene cleavage site, respectively, so that the nucleic acid sequence encoding the chimeric antigen receptor is inserted into the TRAC locus by homologous recombination.

In some embodiments, the nucleic acid sequence encoding the chimeric antigen receptor is inserted into the TRAC locus at a position operably linked to the TRAC endogenous promoter.

In some embodiments, the polynucleotide sequence comprises a PGK promoter operably linked to a nucleic acid sequence encoding a chimeric antigen receptor.

In some embodiments, the polynucleotide sequence is contained in a viral vector.

In some embodiments, the viral vector is an AAV vector.

In some embodiments, the method further comprises:

(c) introducing a polynucleotide sequence comprising a nucleic acid sequence encoding a fusion protein of a lymphocyte-antigen presenting cell co-stimulatory factor into the T cell,

The fusion protein of lymphocyte-antigen presenting cell co-stimulatory factor comprises a first domain for activating antigen presenting cells (APC) and a second domain for activating immune effector cells, wherein

In some embodiments, the activating receptor of the APC is CD40.

In some embodiments, the first domain is a scFv.

In some embodiments, the immune effector cells are T cells.

In some embodiments, the co-stimulatory receptor is CD28.

In some embodiments, the nucleic acid sequence encoding the lymphocyte-antigen presenting cell co-stimulatory factor fusion protein and the nucleic acid sequence encoding the chimeric antigen receptor are in the same expression frame or in separate expression frames.

Another aspect of the present invention also provides modified T cells produced by the above method.

Another aspect of the present invention provides a pharmaceutical composition, which includes any of the aforementioned modified T cells, or modified T cells produced according to any of the aforementioned methods, and a pharmaceutically acceptable carrier.

Another aspect of the present invention provides a method for treating a disease associated with CD7 expression, comprising administering a therapeutically effective amount of any one of the aforementioned modified T cells or the aforementioned pharmaceutical composition to a subject in need thereof.

Another aspect of the present invention also provides use of any of the aforementioned modified T cells, or modified T cells produced according to any of the aforementioned methods, in the preparation of a medicament for treating a disease associated with CD7 expression.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. pDA-T7-B2M gRNA plasmid map. A is the plasmid map containing B2M gRNA-1, and B is the sequence nucleotides of the promoter, gRNA guide sequence, gRNA backbone sequence, and termination signal contained in the plasmid.

Figure 2. Flow cytometry analysis of CRISPR/Cas9/B2M gRNA gene knockout in T cells and screening results of 22 candidate gRNAs.

Figure 3. Flow cytometry analysis and result summary of simultaneous knockout of TRAC/B2M/CD7 genes in T cells using CRISPR/Cas9 technology and different gRNA combinations. The results showed that the gene knockout effect of TRAC gRNA3.4 was better than that of TRAC gRNA19.6, and the gene knockout effect of B2M gRNA-11 was better than that of B2M gRNA-22.

Figure 4. Readout of anti-human CD7-Fc monoclonal phage ELISA in three 96-well plates.

Figure 5. Schematic diagram of the structures of three AAV vectors: R196H1A, R3H1A and R3PGK.

Figure 6. Technical scheme for preparing TRAC/B2M/CD7 triKO CD7 CAR-T by simultaneous knockout of TRAC/B2M/CD7 genes and infection with three AAVs: R196H1A, R3H1A and R3PGK.

Figure 7. Flow cytometry analysis of the efficiency of TCR and B2M knockout after simultaneous knockout of TRAC/B2M/CD7 genes in T cells and infection with three AAVs, R196H1A, R3H1A and R3PGK, for 4 days.

Figure 8. Flow cytometry analysis of the efficiency of TCR and CD7 knockout after simultaneous knockout of TRAC/B2M/CD7 genes in T cells and infection with three AAVs, R196H1A, R3H1A and R3PGK, for 4 days.

Figure 9. Four days after simultaneous knockout of TRAC/B2M/CD7 genes in T cells and infection with three AAVs, R196H1A, R3H1A and R3PGK, the expression of CD7H1CAR was analyzed by flow cytometry using CD7-Fc recombinant protein.

Figure 10. Four days after simultaneous knockout of TRAC/B2M/CD7 genes in T cells and infection with three AAVs, R196H1A, R3H1A and R3PGK, the expression of LACO was analyzed by flow cytometry using CD40-Fc recombinant protein.

Figure 11. Flow cytometry analysis of the efficiency of TCR and B2M knockout after simultaneous knockout of TRAC/B2M/CD7 genes in T cells and infection with three AAVs, R196H1A, R3H1A and R3PGK, 8 days later.

Figure 12. Flow cytometry analysis of the efficiency of TCR and CD7 knockout after simultaneous knockout of TRAC/B2M/CD7 genes in T cells and infection with three AAVs, R196H1A, R3H1A and R3PGK, 8 days later.

Figure 13. 8 days after simultaneous knockout of TRAC/B2M/CD7 genes in T cells and infection with three AAVs, R196H1A, R3H1A and R3PGK, the expression of CD7H1CAR was analyzed by flow cytometry using CD7-Fc recombinant protein.

Figure 14. Simultaneous knockout of TRAC/B2M/CD7 genes in T cells and R196H1A, R3H1A and R3PGK Eight days after infection with the three AAVs, the expression of LACO was analyzed by flow cytometry using CD40-Fc recombinant protein.

Figure 15. Statistical analysis of the number of cells proliferating and expanding during in vitro culture of triKO CD7 CAR-T cells and control T cells prepared by infection with three AAV viruses, R196H1A, R3H1A and R3PGK, at different MOIs.

Figure 16. The killing effect of triKO CD7 CAR-T cells and control T cells prepared by infecting three AAV viruses with different MOIs, R196H1A, R3H1A and R3PGK, and co-incubated with CD7-positive CCRF-CEM tumor target cell line in vitro at E:T=2:1, inCucyte analysis results.

Figure 17. TriKO CD7 CAR-T cells and control T cells prepared by infection with three AAV viruses R196H1A, R3H1A and R3PGK at different MOIs were co-incubated with CD7-positive CCRF-CEM tumor target cell line in vitro at E:T=0.5:1. InCucyte analysis results.

Figure 18. TriKO CD7 CAR-T cells and control T cells prepared by infection with three AAV viruses R196H1A, R3H1A and R3PGK at different MOIs were co-incubated with CD7-positive CCRF-CEM tumor target cell line in vitro at E:T=0.2:1. InCucyte analysis results.

Detailed ways

The present invention prepares a universal CD7 CAR-T by collecting T cells from healthy donors for gene editing to avoid allogeneic rejection and cannibalism. The present invention uses CRISPR/Cas9 technology to simultaneously knock out the TCR, B2M (Beta-2 microglobulin) and CD7 genes of T cells, and at the same time as the gene knockout, adds AAV encoding CD7 CAR and LACO co-stimulatory molecules of CD7H1.BBZ.A40C2828 as donor DNA fragments, integrates them into the genome position of TRAC, and uses the endogenous promoter of TRAC or the exogenous PGK promoter to start the expression of CD7 CAR and LACO co-stimulatory molecules, thereby preparing a universal CAR-T targeting CD7 molecules.

The experimental results showed that CRISPR/cas9 technology successfully and efficiently knocked out TCR, B2M and CD7 genes in T cells, and AAV as a donor DNA fragment mediated site-specific integration also achieved high efficiency, and CD7 CAR and LACO molecules were successfully expressed on the surface of T cell membranes. The results of in vitro killing experiments co-incubated with CD7-positive tumor target cell lines showed that the triKO CD7 CAR-T cells with TCR/B2M/CD7 triple knockout prepared in this way had a very strong killing effect.

Unless otherwise indicated, the terms used in the present invention have the meanings generally understood in the art and can be understood by referring to standard textbooks, reference books, and documents known to those skilled in the art. All publications mentioned herein are incorporated herein by reference in their entirety.

It should be understood that the specific methods and materials described in the embodiments of the present invention are only for the purpose of describing specific embodiments and are not restrictive. Any methods and materials similar to or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention. The explanation of the relevant theory or mechanism in the present invention is only used to help understand the invention and should not be regarded as a limitation on the scheme protected by the present invention.

The term "comprising" or other terms with similar meanings as "including", "containing" or "having" as used herein should be understood to include the listed elements but not to exclude the existence of other elements. These terms also include the situation that they are composed of only the listed elements. The term "consisting of..." means that it is composed of only the listed elements. “Consisting essentially of…” means that elements that have no significant effect on the involved solution are not excluded.

Unless otherwise specifically stated, "a", "an" or "the" herein include both singular and plural forms. The meaning of the term "at least one" or "at least one" or other similar expressions is equivalent to the meaning of "one or more" or "one or more".

The numerical ranges described herein, such as temperature ranges, time ranges, composition or concentration ranges, or other numerical ranges, include the end values, all intermediate ranges, and sub-ranges (e.g., the range between a certain intermediate value and a certain end value) within the range. Furthermore, any intermediate ranges, sub-ranges, and all individual values described in the numerical range may be excluded from the numerical range.

The terms “about” or “approximately” used herein mean a range of ±10% of the numerical value.

The “and/or” described herein should be understood as any one element or any combination of several elements connected by this term.

In the present invention, unless otherwise indicated, nucleic acid sequences are generally described from the 5' end to the 3' end, and amino acid sequences are generally described from the N-terminus to the C-terminus.

The present invention provides a CAR-T cell targeting CD7, which expresses a chimeric antigen receptor targeting CD7. The endogenous TRAC and B2M genes of the CAR-T cell are suppressed, and it will not cause graft-versus-host disease (GVHD) when it is re-infused into an allogeneic subject; the endogenous CD7 gene of the CAR-T cell is suppressed, so that it will not have a killing effect on itself.

"T cell" herein, i.e. T lymphocyte, may include thymocytes, natural T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. The T cell may be a helper T cell (Th), a cytotoxic T cell (CTL), a memory T cell, a regulatory T cell, a natural killer T cell, a CD4+/CD8+T cell, a CD4-/CD8-T cell or any other T lymphocyte subtype. In some cases, the modified T cell is a human T cell. Before amplification and genetic modification of the T cell of the present application, a cell source may be obtained from a subject, such as a patient, by various non-limiting methods. T cells may be obtained from many non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue of infection site, ascites, pleural effusion, spleen tissue and tumor. In some cases, any number of T cell lines available and known to those skilled in the art may be used. In other cases, the T cells can be derived from healthy donors, from patients diagnosed with cancer, or obtained from patients diagnosed with infection. In other cases, the T cells are part of a cell population, such as a mixed population of cells with different phenotypic properties. In some embodiments, the T cells are autologous T cells for the subject in need of treatment, and in other embodiments, the T cells are allogeneic T cells for the subject in need of treatment.

The term "endogenous" refers to a nucleotide sequence that occurs naturally in a cell prior to being modified as described herein.

The TRAC gene encodes the TCR alpha chain, one of the components of the TCR complex. Disruption of the TRAC gene results in loss of TCR function and renders engineered T cells non-alloreactive and suitable for allogeneic transplantation, thereby minimizing the risk of graft-versus-host disease.

The B2M gene encodes beta-2 microglobulin, the light chain of the MHC class I molecule. The β2M gene encodes the common (invariant) component of the major histocompatibility complex (MHC) I complex. Disruption of the β2M gene can prevent host anti-therapeutic allogeneic T cell responses. Knocking out the TRAC gene and the β2M gene will result in the production of cells that are Allogeneic T cells for cell therapy.

The term "CD7" refers to cluster of differentiation 7 which is well known in the art.

The term "inhibit" refers to reducing or eliminating the expression of a gene. The inhibition will result in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in the level of the gene product. Methods for measuring the level of the gene product are inhibited by those skilled in the art, and measurements can be made before and after using a means of inhibiting gene expression to assess the effect of gene inhibition.

In the present invention, gene expression can be reduced (i.e., knocked down) by means such as antisense RNA, siRNA, shRNA, etc.; gene expression can also be eliminated (i.e., knocked out) by gene editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or CRISPR-Cas systems and methods thereof.

The term "knockdown" as used herein refers to a detectable decrease (e.g., detected by antibodies or flow cytometry) in the expression of a target gene or corresponding protein in a cell after a measure to inhibit gene expression is taken, compared to the expression of the target gene or corresponding protein in the cell before the measure to inhibit gene expression is taken.

The term "knockout" may also be referred to herein as "disruption" or "inactivation". The disrupted gene does not express (e.g., not expressed on the cell surface) a detectable level (e.g., detected by antibodies or flow cytometry) of the protein encoded by the gene. In some embodiments, the term "knockout" includes deleting all or part of the target gene sequence.

The CRISPR-Cas system is a naturally occurring defense mechanism in prokaryotes that has been used as an RNA-guided DNA targeting platform for gene editing. The CRISPR-Cas system includes a Cas nuclease and two non-coding RNAs (crRNA and tracrRNA), which can form a CRISPR-Cas complex that is guided to the target sequence by hybridization of a guide sequence of about 20 nt contained in the crRNA with a target sequence in the target gene, and then the Cas protein cuts the DNA at a specific position near the PAM site, resulting in a double-strand break (DSB). Many types and species of CRISPR-Cas systems have been described (see, for example, Koonin et al., 2017, Curr. Opin. Microbiol., 37: 67-78).

In the present invention, the Cas proteins that can be used include but are not limited to Cas9, Cas12, etc., and their orthologs or variants. Cas9 proteins include but are not limited to Cas9 proteins from Streptococcus pyogenes (SpCas9) or Cas9 proteins from Staphylococcus aureus (SaCas9).

The term "gRNA" refers to a guide RNA that can form a complex with the Cas protein and can guide the Cas protein to the target sequence for cutting. The gRNA may include crRNA and tracrRNA. The crRNA contains a guide sequence that can be paired with the target sequence through base complementation. The gRNA can be a bimolecular gRNA or a single molecule gRNA (sgRNA).

Cas protein can be introduced into cells in the form of protein or in the form of its encoding nucleic acid sequence (e.g., mRNA or cDNA). Nucleic acid encoding Cas protein can be contained in a plasmid or viral vector and introduced into cells, for example, by transfection. Cas protein or nucleic acid encoding Cas protein can be directly delivered into cells by electroporation, liposomes, microinjection, etc.

gRNA can be introduced into cells by any method suitable for introducing RNA into cells. For example, gRNA can be introduced into cells in the form of isolated RNA. Any in vitro transcription system known in the art can be used to prepare isolated gRNA by in vitro transcription, and then gRNA is introduced into cells by electroporation. gRNA can also be introduced into cells by a vector comprising a sequence encoding gRNA and a promoter. The vector can be a viral vector or a plasmid, and the mode of introduction into cells can be transfection.

The term "target gene" as used herein refers to a gene whose gene expression is to be reduced or eliminated.

The term "target sequence" refers to a nucleotide sequence contained in the target gene, which is complementary to the guide sequence contained in the gRNA through base pairing, thereby guiding the CRISPR-Cas complex to cut the target gene. A target gene can contain multiple target sequences. By designing gRNAs that complementarily pair with different target sequences, the CRISPR-Cas system can cut at different locations of the same target gene.

The term "guide sequence" refers to a sequence that is complementary to a target sequence contained in a target gene through base pairing, which is contained in crRNA. The CRISPR-Cas complex is positioned to the target sequence for cutting by hybridization of the guide sequence and the target sequence.

The degree of complementarity between the guide sequence and the target sequence in the target gene can be at least 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or 100%. In some embodiments, the guide sequence is 100% complementary to the target sequence in the target gene. In some embodiments, the guide sequence may contain up to 5, up to 4, up to 3, up to 2, or up to 1 mismatch with the target sequence in the target gene. The length of the guide sequence and/or the target sequence may be about 15 to about 25 nucleotides. In some embodiments, the length of the guide sequence and/or the target sequence may be about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides. In some embodiments, the guide sequence included in the gRNA for targeting the B2M gene may be selected from SEQ ID NO: 1-22. In some embodiments, the guide sequence included in the gRNA for targeting the B2M gene is SEQ ID NO: 15 or SEQ ID NO: 21. The guide sequence included in the gRNA for targeting the TRAC gene can be selected from SEQ ID NO: 23 or SEQ ID NO: 24. The guide sequence included in the gRNA for targeting the CD7 gene can be SEQ ID NO: 25.

The three endogenous genes TRAC, B2M and CD7 in the modified T cells of the present invention can be knocked out simultaneously or separately. For example, one or more different CRISPR-Cas systems can be used to knock out TRAC, B2M and CD7, and the one or more different CRISPR-Cas systems respectively include different Cas proteins and corresponding gRNAs. One, two or three of TRAC, B2M and CD7 can be knocked out using one of the CRISPR-Cas systems, and the remaining genes can be knocked out using other CRISPR-Cas systems. The gRNAs targeting different genes can be located on the same or different vectors, and can be operably connected to the same or different promoters, respectively.

In some embodiments, three endogenous genes TRAC, B2M and CD7 are knocked out simultaneously. For example, gRNA targeting TRAC, gRNA targeting B2M and gRNA targeting CD7 are used together with Cas protein to make the CRISPR-Cas system cut three endogenous genes TRAC, B2M and CD7 simultaneously.

The DSB caused by CRISPR-Cas system cutting at the target site can be repaired by non-homologous end joining (NHEJ) or homology-directed recombination (HDR), resulting in insertions or deletions in the target gene, thereby achieving destruction and knockout of the target gene.

NHEJ is highly active in most cell types, including non-dividing cells, and usually results in the removal or addition of one to several hundred nucleotides at the site of DSB. The resulting insertions and deletions (indels) can destroy the coding or non-coding regions of the gene (depending on the design of the gRNA). Alternatively, HDR uses a donor repair template to repair DSB with high fidelity. The donor repair template contains homology arms that are homologous to the sequences on both sides of the DSB site on the target sequence, and the sequence between the homology arms is inserted into the target sequence by homologous recombination. HDR is only effective in dividing cells.

In some embodiments, knockout of endogenous TRAC in T cells by CRISPR-Cas system can be achieved by Repaired by NHEJ or HDR. In some embodiments, knockout of endogenous B2M in T cells by the CRISPR-Cas system can be repaired by NHEJ or HDR. In some embodiments, knockout of endogenous CD7 in T cells by the CRISPR-Cas system can be repaired by NHEJ or HDR.

The donor repair template comprises homology arms homologous to the sequences on both sides of the DSB site on the target sequence. The length of the homology arms can be about 100bp, about 200bp, about 300bp, about 400bp, about 500bp, about 600bp, about 700bp, about 800bp, about 900bp, about 1000bp or longer.

In some embodiments, the knockout of endogenous TRAC in T cells is repaired by HDR through the CRISPR-Cas system. The donor repair template used has homology arms that are homologous to the sequences on both sides of the DSB site of the endogenous TRAC gene, B2M gene or CD7 gene in T cells. In some embodiments, the sequence of the 5' homology arm of the donor repair template is shown in SEQ ID NO: 172, and the sequence of the 3' homology arm is shown in SEQ ID NO: 173. In some embodiments, the sequence of the 5' homology arm of the donor repair template is shown in SEQ ID NO: 174, and the sequence of the 3' homology arm is shown in SEQ ID NO: 175.

The donor repair template can be introduced into T cells in the form of naked nucleic acid or can be contained in a vector. In some embodiments, the donor repair template is introduced into T cells by a vector. The term "vector" is used herein to refer to a nucleic acid molecule that can transfer or transport another nucleic acid molecule. The transferred nucleic acid is generally connected to a carrier nucleic acid molecule, such as inserted into a carrier nucleic acid molecule. Illustrative examples of vectors include, but are not limited to, plasmid vectors, viral vectors, autonomous replication sequences, and transposable elements. Examples of viral vectors include, but are not limited to, adeno-associated viruses (AAV), adenoviruses, retroviruses (including slow viruses), herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomavirus vectors, etc. In some embodiments, the vector is an AAV vector.

The term "CAR-T cell" refers to a T cell expressing a chimeric antigen receptor (CAR), which can specifically recognize and kill cells expressing a target antigen recognized by the chimeric antigen receptor.

The term "chimeric antigen receptor" or "CAR" refers to an artificial T cell surface receptor that is modified to be expressed on immune effector cells and specifically binds to an antigen. A chimeric antigen receptor generally includes an extracellular binding domain, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the chimeric antigen receptor may further include a hinge region between the binding domain and the transmembrane domain.

The term "binding domain" refers to a domain that can specifically bind to a target antigen. The binding domain may comprise an antibody or a ligand or a fragment thereof that can specifically bind to a target antigen.

The term "antibody" refers to a polypeptide molecule that can specifically recognize and/or neutralize a specific antigen. For example, an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof. The term "antibody" includes monoclonal antibodies, antigen-binding or antibody derivatives, including but not limited to human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, single-chain antibodies (e.g., scFv), and antibody fragments that bind to antigens (e.g., Fab, Fab' and (Fab)2 fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antibody fragments and derivatives thereof that bind to antigens described herein. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may be composed of a light chain variable region (VL) and a light chain constant region. The VH and VL regions may be further distinguished into hypervariable regions called complementary determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In this article, the three heavy chain complementary determining regions are referred to as HCDR1, HCDR2 and HCDR3, and the four heavy chain framework regions are referred to as HFR1, HFR2, HFR3 and HFR4; the three light chain complementary determining regions are referred to as LCDR1, LCDR2 and LCDR3, and the four light chain framework regions are referred to as LFR1, LFR2, LFR3 and LFR4. The variable regions (VH and VL) of the heavy and light chains form antigen binding sites, respectively. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of a CDR can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al., (1989) Nature 342: 878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, one skilled in the art will readily identify the CDRs defined according to each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art (for example, see Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). The antibodies of the present invention can use any of these numbering systems to define CDRs, although it is preferred to use the Clothia numbering system to define CDRs.

The term "antigen-binding fragment" refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to a given antigen. Antigen-binding fragments can be obtained by any suitable technique, such as proteolytic digestion of intact antibodies or recombinant DNA technology. Examples of the term "antigen-binding fragment" include (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) Fab' fragments, which are essentially Fab with a portion of the hinge region; (iii) F(ab') ₂ fragments, which are bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region; (iv) Fd fragments consisting of VH and CH1 domains; (v) Fv fragments consisting of the VL and VH domains of a single arm of an antibody; (vi) single-chain Fv fragments (scFv, also called single-chain antibodies), in which the VL and VH regions are paired to form a single protein chain of a monovalent molecule); (vii) disulfide-stabilized Fv fragments (dsFv), which are Fvs with artificially engineered intermolecular disulfide bonds that stabilize the VH-VL pair; (viii) single-domain antibodies (sdAbs) consisting only of the heavy chain variable region, etc.

In some embodiments, the binding domain comprised by the CAR of the present invention is an anti-CD7 antibody or an antigen-binding fragment thereof.

In some embodiments, the anti-CD7 antibody comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3; wherein LCDR1 has an amino acid sequence selected from SEQ ID NO:113-125; LCDR2 has an amino acid sequence selected from SEQ ID NO:126-137; LCDR3 has an amino acid sequence selected from SEQ ID NO:138-150; HCDR1 has an amino acid sequence selected from SEQ ID NO:87-94; HCDR2 has an amino acid sequence selected from SEQ ID NO:95-102; and HCDR3 has an amino acid sequence selected from SEQ ID NO:103-112.

In some embodiments, the light chain variable region and the heavy chain variable region of the anti-CD7 antibody include LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 selected from the group consisting of:

(a) LCDR1 having the amino acid sequence shown in SEQ ID NO: 113, having the amino acid sequence shown in SEQ ID NO: 126 a LCDR2 having an amino acid sequence as shown in SEQ ID NO: 138, a LCDR3 having an amino acid sequence as shown in SEQ ID NO: 138, a HCDR1 having an amino acid sequence as shown in SEQ ID NO: 87, a HCDR2 having an amino acid sequence as shown in SEQ ID NO: 95, and a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 103;

(j) LCDR1 having the amino acid sequence of SEQ ID NO: 122, LCDR2 having the amino acid sequence of SEQ ID NO: 135, LCDR3 having the amino acid sequence of SEQ ID NO: 147, LCDR4 having the amino acid sequence of SEQ ID NO: 150, a HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, a HCDR2 having the amino acid sequence set forth in SEQ ID NO:95, and a HCDR3 having the amino acid sequence set forth in SEQ ID NO:109;

In some embodiments, the anti-CD7 antibody comprises a light chain variable region and a heavy chain variable region selected from the group consisting of:

(k) a light chain variable region comprising the amino acid sequence of SEQ ID NO: 76 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 77 A heavy chain variable region having the amino acid sequence shown;

In some embodiments, the binding domain in the CAR of the present invention is a single-chain antibody (scFv), which comprises an antibody light chain and an antibody heavy chain connected in series. In some embodiments, in the single-chain antibody, the C-terminus of the antibody light chain is connected to the N-terminus of the antibody heavy chain. In some embodiments, in the single-chain antibody, the C-terminus of the antibody heavy chain is connected to the N-terminus of the antibody light chain. In some embodiments, in the single-chain antibody, the light chain and the heavy chain are connected by a joint. In some embodiments, the joint between the antibody light chain and the heavy chain has a sequence as shown in SEQ ID NO: 177 or SEQ ID NO: 178.

In some embodiments, the anti-CD7 scFv comprises an amino acid sequence as shown in SEQ ID NO:34, 39, 43, 47, 51, 55, 59, 63, 67, 71, 75, 79 or 83.

The term "transmembrane domain" refers to a domain in CAR that passes through the cell membrane, which is connected to the intracellular signal transduction domain and plays a role in transmitting signals. Transmembrane domains from the following proteins can be used in CAR: α, β or ζ chain of T cell receptor, CD28, CD3e, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154. In some embodiments, the CAR of the present invention includes a CD8 transmembrane domain. In some embodiments, the CD8 transmembrane domain includes an amino acid sequence as shown in SEQ ID NO: 155.

The term "signaling domain" is a part that conducts the information of CAR binding to the target antigen to the inside of the immune effector cell to cause effector cell function, such as activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors into target cells bound to CAR, or other cell responses caused by antigen binding to the extracellular CAR domain. The effector function of T cells can be, for example, cytolytic activity or help or activity, including the secretion of cytokines. Although the entire intracellular signaling domain can generally be used, it should be understood that any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal can be used. In some embodiments, the CAR of the present invention comprises a signaling domain having an immunoreceptor tyrosine activation motif (ITAM), examples of which include signaling domains derived from primary signaling domains FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d. In some embodiments, the CAR of the present invention includes a CD3ζ intracellular signaling domain. In some embodiments, the CD3ζ intracellular signaling domain comprises an amino acid sequence as shown in SEQ ID NO:159.

In some embodiments, the intracellular portion of the CAR of the present invention may further include a costimulatory domain. The term "costimulatory domain" refers to the intracellular domain of an immune costimulatory molecule, which is a cell surface molecule required for the effective response of lymphocytes to antigens. In some embodiments of the present invention, CAR may include a costimulatory domain derived from a protein selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM and ZAP70. In some embodiments, 4-1BB is included in the CAR of the present invention. In some embodiments, the 4-1BB co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:157.

The term "hinge region" refers to the connecting region between the binding domain and the transmembrane domain. In some embodiments of the present invention, the CAR may include a hinge region or a combination thereof derived from a protein selected from the group consisting of CD8, CD28, IgG, 4-1BB, CD4, CD27, CD7, PD-1, and CH2CH3. In some embodiments, the CAR of the present invention includes a CD8 hinge region. In some embodiments, the CD8 hinge region includes an amino acid sequence as shown in SEQ ID NO: 153.

In some embodiments, the CAR of the present invention may also include a signal peptide. The term "signal peptide" refers to a leader sequence located at the amino terminus (N-terminus) of a polypeptide, which directs the polypeptide to the endoplasmic reticulum during or after translation. In some embodiments of the present invention, the signal peptide may include a signal peptide or a combination thereof derived from a protein selected from the group consisting of CD8, 4-1BB, GM-CSF, CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, and CD66d. In some embodiments, the CAR of the present invention includes a CD8 signal peptide. In some embodiments, the CD8 hinge region includes an amino acid sequence as shown in SEQ ID NO: 151.

In some embodiments, the CD7 targeting CAR of the present invention comprises an amino acid sequence as shown in SEQ ID NO:37, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82 or 86.

The term "specific binding" means that the binding domain of the chimeric antigen receptor of the present invention does not or substantially does not cross-react with any polypeptide other than the target antigen. The degree of specificity can be determined by immunological techniques, including but not limited to immunoblotting, immunoaffinity chromatography, flow cytometry, etc.

In some embodiments, the modified T cells of the present invention also express lymphocyte-antigen presenting cell co-stimulator (LACO-Stim). Regarding LACO-Stim, see CN115052902A, which is incorporated herein by reference in its entirety. LACO-Stim is a fusion protein, the expression of which can not only promote the proliferation and activation of immune effector cells (e.g., T cells), but also stimulate the maturation and epitope diffusion activity of antigen presenting cells.

The LACO-Stim comprises a first domain that activates antigen presenting cells (APCs) and a second domain that activates immune effector cells, wherein

In some embodiments, the C-terminus of the first domain is connected to the N-terminus of the second domain. In some embodiments, the N-terminus of the first domain is connected to the C-terminus of the second domain.

In some embodiments, the first domain and the second domain are connected by a linker. The linker can be a flexible linker or a rigid linker. In some embodiments, the linker is a CD28 hinge region. In some embodiments, the CD28 hinge region comprises an amino acid sequence as shown in SEQ ID NO: 164.

APC refers to any cell that displays one or more antigens on its surface, for example, in conjunction with one or more major histocompatibility complex (MHC) proteins. The MHC/antigen complex can be used by T cells to bind to the antigen using their T cell receptors. (TCRs) recognize and elicit an immune response. APCs include, for example, dendritic cells (DCs), macrophages, monocytes, myeloid-derived suppressor cells, certain B cells, T cells, and Langerhans cells.

"Activating receptor" refers to a membrane protein expressed on APC, which, after binding to a ligand or antibody, can trigger a signal to promote the migration, differentiation, proliferation and/or activation of APC. APC activating receptors include, for example, CD40, CD80, CD86, CD91, DEC-205 and DC-SIGN. In some embodiments, the APC activating receptor is CD40.

A "ligand" of a receptor refers to a molecule that selectively binds to a receptor. In some embodiments, the ligand is a polypeptide. A "receptor binding fragment" of a ligand refers to a fragment of a ligand that retains its receptor binding ability.

In some embodiments, the first domain comprises an antibody that binds to an activating receptor of an APC, or an antigen binding fragment thereof. In some embodiments, the first domain comprises an anti-CD40 antibody. In some embodiments, the antibody comprised by the first domain is a scFv, such as an anti-CD40 scFv. In some embodiments, the anti-CD40 scFv comprises an amino acid sequence as shown in SEQ ID NO: 163.

"Immune effector cell" refers to a cell with hematopoietic origin and plays a direct role in the immune response to a target (such as a pathogen, a cancer cell or a foreign substance). Immune effector cells include T cells, B cells, natural killer (NK) cells, NKT cells, macrophages, granulocytes, neutrophils, eosinophils, mast cells and basophils. In some embodiments, the second domain of the activation immune effector cell of the fusion protein provided by the present invention includes a co-stimulatory receptor of the immune effector cell. In some embodiments, the immune effector cell is a T cell, a NK cell, a NKT cell, a macrophage, a neutrophil or a granulocyte. In some embodiments, the immune effector cell is a T cell.

"Stimulation" of immune effector cells refers to the primary response induced by the binding of stimulatory molecules to their cognate ligands, thereby mediating signal transduction events in immune effector cells, which can change the expression of certain genes and/or the reorganization of cytoskeletal structures, etc. "Stimulatory molecules" of immune effector cells refer to a molecule on immune effector cells that, after binding to cognate ligands usually present on APCs, can mediate signal transduction to promote the maturation, differentiation, proliferation and/or activation of immune effector cells.

As used in the present invention and as understood in the art, "costimulatory signal" refers to the signal from co-stimulatory receptors (such as CD28 or 4-1BB), which is combined with primary signals (such as TCR/CD3) to promote the best clonal expansion, differentiation and effector function of immune effector cells (such as T cells).As used in the present invention and as understood in the art, "costimulatory receptor" of immune effector cells refers to the molecule of the co-stimulatory response of immune effector cells specifically combined with "co-stimulatory ligand" on immune effector cells, and the co-stimulatory response is such as enhancing the activation or proliferation of immune effector cells.The co-stimulatory receptors of immune effector cells include but are not limited to CD28, 4-1BB, ICOS, CD27, OX40, DAP10, CD30, 2B4, CD2, LIGHT, GITR, TLR, DR3 and CD43.The "functional fragment" of co-stimulatory receptors is a fragment of co-stimulatory receptors, which retains the function of co-stimulatory receptors mediating co-stimulatory signals and stimulating immune effector cells.

In some embodiments, the co-stimulatory receptor is CD28. In some embodiments, the second domain comprises a CD28 polypeptide or a functional fragment thereof. In some embodiments, the second domain comprises an intracellular domain of CD28. In some embodiments, the intracellular domain of CD28 comprises an amino acid sequence as shown in SEQ ID NO: 166. In some embodiments, the second domain further comprises a transmembrane domain of a co-stimulatory receptor, such as a CD28 transmembrane domain. In some embodiments, the CD28 transmembrane domain comprises an amino acid sequence as shown in SEQ ID NO: 165.

In some embodiments, the LACO-Stim of the present invention may further comprise a signal peptide. In some embodiments of the present invention, the signal peptide of LACO-Stim may comprise a signal peptide or a combination thereof derived from a protein selected from the group consisting of CD8, 4-1BB, GM-CSF, CD3γ, CD3δ, CD3ε, CD22, CD79a, CD79b, and CD66d. In some embodiments, the LACO-Stim of the present invention comprises a CD8 signal peptide. In some embodiments, the CD8 hinge region comprises an amino acid sequence as shown in SEQ ID NO: 151.

In some embodiments, the LACO-Stim comprises an amino acid sequence as shown in SEQ ID NO:161.

Nucleic acids encoding chimeric antigen receptors targeting CD7 and/or nucleic acids encoding LACO-Stim can be introduced into T cells by methods known to those skilled in the art. For example, nucleic acids encoding chimeric antigen receptors can be introduced into T cells via vectors. Non-limiting examples of methods for introducing nucleic acids encoding chimeric antigen receptors into cells include: liposome transfection, calcium phosphate transfection, microinjection, electroporation, cell extrusion, sonication, protoplast fusion, puncture infection, gene gun, magnetic transfection, viral vector transduction, plasmid vector transfection, etc.

The nucleic acid encoding the chimeric antigen receptor targeting CD7 and the nucleic acid encoding LACO-Stim can be located in the same expression frame in T cells, and their expression is driven by the same promoter, or the two can be located in different expression frames, and their expression is driven by their respective promoters. When they are located in different expression frames, the promoters they use can be the same or different. The chimeric antigen receptor targeting CD7 and LACO-Stim can be expressed as independent proteins in T cells, or they can be expressed as a fusion protein of the two. The chimeric antigen receptor targeting CD7 and LACO-Stim can be connected by a linker. The linker can be a cleavable linker, such as a 2A linker (self-cleaving peptide), such as P2A, T2A or F2A. In some embodiments, F2A includes an amino acid sequence as shown in SEQ ID NO: 179.

The nucleic acid encoding the chimeric antigen receptor targeting CD7 and the nucleic acid encoding LACO-Stim can be introduced into T cells through the same vector or through different vectors. When the two are contained in the same vector, they can be located in the same expression frame and driven by the same promoter, or the two can be located in different expression frames and driven by their respective promoters. When they are located in different expression frames, the promoters used by them can be the same or different.

The term "expression cassette" refers to a nucleic acid sequence that contains the complete elements required to express a gene, including the coding sequence of the gene and the coding sequence of regulatory sequences sufficient to direct the transcription of the gene. The expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment.

In some embodiments, nucleic acid encoding a chimeric antigen receptor targeting CD7 and/or nucleic acid encoding LACO-Stim is introduced into T cells via a vector. The vector includes, but is not limited to, a plasmid vector, a viral vector, an autonomously replicating sequence, and a transposable element. In some embodiments, a viral vector, such as an adeno-associated virus (AAV) vector, an adenoviral vector, a retrovirus (including a lentivirus) vector, a herpes virus (eg, herpes simplex virus) vector, a poxvirus vector, a baculovirus vector, or a papillomavirus vector, is used to introduce nucleic acid encoding a chimeric antigen receptor into T cells. In some embodiments, an AAV vector is used to introduce a chimeric antigen receptor targeting CD7 into T cells.

AAV vectors are replication-deficient vectors that can infect both dividing and non-dividing cells and can integrate their genomes into the host cell genome. Recombinant AAV (rAAV) includes at least transgenes and regulatory sequences, as well as 5' and 3' AAV inverted terminal repeats (ITRs). AAV has multiple serotypes. In some embodiments, rAAV can include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Separate ITR and capsid sequences of AAV11, AAV 12, AAV13, AAV 14, AAV15 or AAV16. In some embodiments, chimeric AAV vectors may be used, i.e., the ITR sequences and capsid sequences are derived from different AAV serotypes.

The vector may generally contain regulatory elements, such as replication origin, promoter, enhancer, translation initiation signal, post-transcriptional regulatory elements, polyadenylation sequence and/or 5' and 3' untranslated regions, etc. These regulatory elements interact with host cell proteins to perform transcription and translation. The selection and use of different regulatory elements are well known to those skilled in the art.

The term "promoter" refers to a recognition site of a polynucleotide (DNA or RNA) to which RNA polymerase binds. A promoter is usually operably linked to a gene to be expressed to drive the expression of the gene. In the present invention, the expression of a chimeric antigen receptor targeting CD7 and/or LACO-Stim in T cells can be driven by an endogenous promoter of the T cells, or by a promoter that is exogenous to the T cells.

In some embodiments, nucleic acid encoding a chimeric antigen receptor targeting CD7 and/or nucleic acid encoding LACO-Stim can be inserted into a position operably connected to an endogenous promoter of a T cell, thereby driving its expression through the endogenous promoter. A polynucleotide comprising a nucleic acid encoding a chimeric antigen receptor targeting CD7 and/or a nucleic acid encoding LACO-Stim can be designed to insert a nucleic acid encoding a chimeric antigen receptor into a position operably connected to an endogenous promoter of a T cell. For example, nucleic acid encoding a chimeric antigen receptor targeting CD7 and/or nucleic acid encoding LACO-Stim can have homology arms homologous to endogenous gene sequences of T cells on both sides, thereby being integrated into a position operably connected to an endogenous promoter of a T cell by homology directed recombination (HDR). In some embodiments, the endogenous promoter can be a TRAC promoter, a B2M promoter, or a CD7 promoter endogenous to a T cell.

In some embodiments, nucleic acid encoding a chimeric antigen receptor targeting CD7 and/or nucleic acid encoding LACO-Stim can be operably linked to a promoter exogenous to T cells to drive its expression by the exogenous promoter. For example, nucleic acid encoding a chimeric antigen receptor targeting CD7 and/or nucleic acid encoding LACO-Stim can be included in a vector and introduced into T cells together with an exogenous promoter operably linked thereto. The promoters that are exogenous to T cells include, but are not limited to, cytomegalovirus (CMV) immediate early promoter, simian virus 40 (SV40) promoter, Moloney murine leukemia virus (MoMLV) LTR promoter, herpes simplex virus (HSV) (thymidine kinase) promoter, H5, P7.5 and P11 promoters from vaccinia virus, short elongation factor 1-α (EF1a-short) promoter, long elongation factor 1-α (EF1a-long) promoter, ubiquitin C promoter (UBC) promoter, phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer/chicken β-actin (CAG) promoter, β-actin promoter, etc. In some embodiments, the promoter that is exogenous to T cells is phosphoglycerate kinase-1 (PGK) promoter. In some embodiments, the PGK promoter comprises a sequence as shown in SEQ ID NO: 176.

The term "operably linked" means that a regulatory sequence (such as a promoter) is placed at an appropriate position relative to a coding sequence so that the regulatory sequence (such as a promoter) can direct the production of a polypeptide encoded by the coding sequence.

The term "endogenous" refers to a nucleic acid sequence or amino acid sequence that occurs naturally within a T cell prior to the introduction of a modification described herein.

The term "exogenous" refers to a nucleic acid sequence or amino acid sequence that is not naturally present or expressed in T cells.

The polynucleotide comprising a nucleic acid encoding a chimeric antigen receptor targeting CD7 and/or a nucleic acid encoding LACO-Stim can be designed to insert the nucleic acid encoding a chimeric antigen receptor targeting CD7 and/or a nucleic acid encoding LACO-Stim into The target gene site in the T cell, such as the safe harbor locus. In the present invention, the nucleic acid encoding the chimeric antigen receptor targeting CD7 and/or the nucleic acid encoding LACO-Stim can be inserted into the TRAC, B2M or CD7 locus in the T cell to destroy the TRAC, B2M or CD7 gene and express the chimeric antigen receptor. This site-specific insertion can be achieved by, for example, homologous directed recombination. In some embodiments, the nucleic acid encoding the chimeric antigen receptor targeting CD7 and/or the nucleic acid encoding LACO-Stim can be included in the donor repair template of the CRISPR/Cas system, and the nucleic acid encoding the chimeric antigen receptor targeting CD7 and/or the nucleic acid encoding LACO-Stim is located between the two homologous arms of the donor repair template, and is inserted near the Cas protein cutting position by homologous directed recombination to destroy the TRAC, B2M or CD7 genes in the T cell and express the chimeric antigen receptor and/or LACO-Stim. In some embodiments, nucleic acid encoding a chimeric antigen receptor targeting CD7 and/or nucleic acid encoding LACO-Stim is contained in a donor repair template of a CRISPR/Cas system targeting the TRAC gene and inserted into the endogenous TRAC gene of the T cell.

In some embodiments, a donor repair template comprising a nucleic acid encoding a chimeric antigen receptor targeting CD7 and/or a nucleic acid encoding LACO-Stim is contained in a vector, such as an AAV vector, and introduced into T cells as part of the vector.

In some embodiments, the modified T cells of the present invention can be used as drugs for treating diseases associated with the expression of CD7, which can be formulated as pharmaceutical compositions.

Diseases associated with CD7 expression include non-solid tumors (such as hematological tumors, eg, leukemias and lymphomas) and solid tumors. Hematological tumors are cancers of the blood or bone marrow and include, but are not limited to, acute leukemias (such as acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), acute myeloid leukemia and myeloblastic, promyelocytic, myelo-monocytic, monocytic and erythroleukemias), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma (indolent and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, myelodysplastic syndrome, hairy cell leukemia, Burkitt lymphoma, diffuse large cell lymphoma, mantle cell lymphoma, T-lymphoblastic lymphoma (T-LBL), early pre-T lymphoblastic leukemia (ETP-ALL), extranodal NK/T-cell lymphoma, small lymphocytic lymphoma (SLL) and myelodysplasia. Solid tumor is the abnormal mass of the tissue that does not usually comprise cyst or liquid zone, and it can be benign or malignant.Dissimilar solid tumors are named with the cell type that forms them (such as sarcoma, cancer and lymphoma).The example of solid tumor includes but is not limited to fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, pancreatic cancer, ovarian cancer, peritoneum, omentum and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, melanoma, kidney cancer, laryngeal cancer, soft tissue cancer, gastric cancer, testicular cancer, colon cancer, esophageal cancer, cervical cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer, pleural cancer, nasal cancer, middle ear cancer, oral cancer, vulvar cancer, thyroid cancer and ureteral cancer. In some embodiments, the disease associated with CD7 expression is acute T-cell lymphoblastic leukemia (T-ALL) or T-lymphoblastic lymphoma.

The terms "subject" and "patient" are used interchangeably herein. The term "subject" as used herein refers to any organism to which the antibodies or antigen-binding fragments thereof of the invention can be administered, e.g., for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, such as chimpanzees and other simians, and humans). The subject can be a mammal, particularly Human beings include females (women) or males (men), and include newborns, infants, teenagers, young people, adults or the elderly, and further include various races and ethnicities. In some examples, a subject refers to an individual in need of diagnosis, treatment or prevention of a disease or condition, and the subject may suffer from the disease or condition, or has the risk of suffering from the disease or condition.

The term "treatment" as used herein refers to providing a beneficial or desired clinical outcome to a disease, such as eliminating the disease, alleviating symptoms, reducing the extent of the disease, stabilizing, improving or alleviating the state of the disease, or slowing down the progression of the disease. Measurement of treatment outcomes can be based on, for example, the results of physical examinations, pathological tests, and/or diagnostic tests known in the art. Treatment can also refer to extending the survival period compared to the expected survival period when the subject does not receive treatment. Treatment can also refer to reducing the incidence or morbidity of the disease, or its recurrence, compared to the disease that would occur if the measure was not taken. Clinically, this treatment can also be referred to as prevention.

The terms "pharmaceutical composition" and "pharmaceutical preparation" of the present invention can be used interchangeably. The pharmaceutical composition may contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" used herein refers to any carrier contained in a pharmaceutical composition as an inactive ingredient, which gives the pharmaceutical composition an appearance and properties suitable for administration. Pharmaceutically acceptable carriers have substantially no long-term or permanent adverse effects when administered to a subject, such as stabilizers, diluents, additives, adjuvants, excipients, etc. "Pharmaceutically acceptable carriers" should be a pharmaceutically inert material, substantially without biological activity, and constitute the main part of the preparation.

The pharmaceutical composition of the present invention can be formulated into various modes of administration according to known techniques. For example, see Remington, The Science and Practice of Pharmacy (9th Ed. 1995). When manufacturing a pharmaceutical composition, the active agent is usually mixed with a pharmaceutically acceptable carrier or the like. Of course, the pharmaceutically acceptable carrier must be acceptable, i.e., compatible with any other ingredients in the formulation, and must be harmless to the subject. Pharmaceutically acceptable carriers may include, but are not limited to, buffers, excipients, stabilizers, preservatives, wetting agents, surfactants, emulsifiers, or combinations thereof. Examples of buffers include, but are not limited to, acetic acid, citric acid, histidine, boric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, Tris buffer, HEPPSO, HEPES, neutral buffered saline, phosphate buffered saline, and the like.

The pharmaceutical composition of the present invention can be administered in any manner suitable for the disease to be treated (or prevented) and the subject. In certain embodiments, the mode of administration may include, but is not limited to, parenteral or non-parenteral routes, including oral, sublingual, oral, percutaneous, rectal, vaginal, intradermal, intranasal or parenteral routes, such as intravenous injection (i.v.), intraperitoneal, intradermal, subcutaneous, intramuscular, intracranial, intrathecal, intratumoral, percutaneous, intramucosal, intraarticular, intrathecal, intrathecal, intrahepatic, intraneural or intracranial injection or infusion. The pharmaceutical composition can be directly injected into a tumor, lymph node, tissue, organ or site of infection.

Suitable oral dosage forms include, but are not limited to, tablets, capsules, powders, pills, granules, suspensions, solutions or preconcentrates of solutions, emulsions or preconcentrates of emulsions. Pharmaceutically acceptable carriers that can be used for oral dosage forms include water, ethylene glycol, oil, alcohol, flavoring agents, preservatives, colorants, etc. Carriers such as starch, sugar, microcrystalline cellulose, diluents, fillers, lubricants, granulating agents, lubricants, binders, stabilizers, disintegrants, etc. can be used to prepare oral solid preparations, such as powders, capsules or tablets.

Dosage forms suitable for parenteral administration include, but are not limited to, sterile liquid preparations such as isotonic aqueous solutions, emulsions, suspensions, dispersions or viscous compositions, which may be buffered to a desired pH. Parenteral dosage forms may be ready for use or may be dry products ready to be dissolved or suspended in a pharmaceutically acceptable carrier. Parenteral dosage forms may be sterile formulations or may be administered to a subject. The test subject can be disinfected before taking. Pharmaceutically acceptable carriers that can be used to provide parenteral dosage forms include, but are not limited to, water for injection; aqueous carriers, such as, but not limited to, sodium chloride injection, Ringer's injection, and glucose injection. Water-soluble carriers, such as, but not limited to, ethanol, polyethylene glycol, and polypropylene glycol; non-aqueous carriers, such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate; and solubilizers, such as cyclodextrins.

The modified T cells or pharmaceutical compositions of the present invention are administered to a subject in a therapeutically effective amount. The terms "therapeutically effective amount" and "effective amount" used herein are used interchangeably and refer to an amount that is effective at the necessary dosage and time period to achieve the desired therapeutic effect. The therapeutically effective amount may vary according to different factors, such as disease state, age, sex, and individual weight, and the ability of a treatment method or combination of treatment methods to induce a desired response in an individual. An effective amount may refer to an amount that causes a detectable change in biological or chemical activity. Detectable changes may be detected and/or further quantified by personnel in related fields. In addition, an "effective amount" may specify an amount that maintains a desired physiological state, i.e., reduces or prevents a significant decline and/or promotes improvement in a condition.

The amount and frequency of dosing will be determined by factors such as the subject's condition (such as age, weight, sex, and the subject's response to the drug) and the type and severity of the subject's disease, although appropriate dosages may be determined by clinical trials.

In the present invention, the effective amount of modified T cells may be, for example, 5×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 5×10 ⁷ , 1×10 ⁸ , 2×10 8 , 5×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ or 5×10 ⁹ cells administered in a ^single administration.

The modified T cells or pharmaceutical compositions of the present invention can be administered once or twice a day; or once every 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, once every 1, 2, 3, 4, 5 or 6 weeks, or once every 1, 2, 3, 4, 5 or 6 months or longer. The pharmaceutical composition can also be administered several times a week (such as 1, 2, 3, 4 or 5 times) or several times a month (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times). For example, in a 5-times-a-week regimen, the pharmaceutical composition can be administered once a day for 5 consecutive days, and then rest for two consecutive days.

The modified T cells or pharmaceutical compositions of the present invention can be used in combination with other drugs and treatment methods, such as other anti-tumor drugs, chemotherapy or radiotherapy. "Combined use" as mentioned here refers to the administration of two (or more) different drugs and/or therapies to the subject during the treatment process. The two or more drugs and/or therapies in the combination can be administered by different routes and schemes. Two or more drugs and/or treatment methods can be administered to the subject simultaneously or sequentially. In some embodiments, when the administration of the second drug or therapy begins, the administration of one drug or therapy is still in progress, so there is overlap in administration. Such a scheme may be referred to as "simultaneous" herein. When administered simultaneously, two or more drugs and/or therapies can be formulated into a single dosage form together, or formulated into two or more independent dosage forms respectively.

Example

Unless otherwise specified, the methods and materials of the examples described below are conventional products that can be purchased from the market.

Example 1: In vitro transcription (IVT) preparation and purification of gRNA

1. Design 22 B2M gRNAs, each containing a guide sequence as shown in SEQ ID NOs: 1-22. Construct plasmids pDA-T7-B2M gRNA for in vitro transcription of these B2M gRNAs, respectively. Take the plasmid containing B2M gRNA-1 as an example, and refer to Figure 1 (i.e., pDAB2M gRNA-M1). Linearize the 22 pDA-T7-B2M gRNA plasmids by Hind III restriction endonuclease.

2. Purify the linearized vector using a PCR cleanup kit (Qiagen) and elute with RNase-free water.

3. DNA concentration was measured by Nanodrop and checked by running an agarose DNA gel.

4. In vitro transcription (IVT) was performed according to the manufacturer's standard operating procedure (Thermofisher, Cat. No. AMB13345). Briefly, 1 μg of template DNA, ATP buffer, UTP buffer, CTP buffer, GTP buffer, 10X reaction buffer, T7 enzyme and RNase-free H ₂ O were added to a 0.2 mL PCR tube in a volume of 20 ul and incubated at 37°C for 4 hours.

5. After 4 hours, add 2 μl of DNase I to each reaction and react at 37°C for 15 minutes.

6. Purify IVT gRNA using RNasy kit (Qiagen).

7. The concentration of the purified gRNA was measured by Nanodrop and checked by PAGE gel.

Example 2: CCRF-CEM-GFP tumor cells and T cell culture

The CCRF-CEM-GFP tumor cell line was cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% double antibody, and passaged approximately every 2-3 days. Primary CD3+T cells isolated and purified from PBMC were activated with anti-CD3/CD28 Dynabeads (Thermofisher, catalog number: 402031) and cultured in R10 medium (RPMI-1640 basal medium with 10% fetal bovine serum, 1% double antibody, 1% HEPES, 1% sodium pyruvate, 1% Glutamax and 1% non-essential amino acids (NEAA) added). On day 4, the magnetic beads were removed from the T cells and continued to be cultured for subsequent electroporation of Cas9 protein/gRNA complexes and AAV virus infection.

Example 3: Testing B2M gRNA gene knockout efficiency

1. Collect T cells activated with anti-CD3/CD28 Dynabeads on day 4 and remove the beads, and wash them three times with Opti-MEM medium.

2. Resuspend the cell pellet with Opti-MEM medium and adjust the cell concentration to 6×10e ⁷ /ml.

3. 7.5 μg of each B2M gRNA obtained in Example 1 and 15 μg of TrueCut ^™ Cas9 Protein v2 protein (Thermofisher, catalog number: A36499) were mixed evenly in a 1.5 ml EP tube and incubated at room temperature for 10 minutes. After 10 minutes, 100 μl of T cells were added to the Cas9 protein/gRNA mixture, gently mixed, and immediately electroporated.

4. Set the parameters on the BTX ECM 830 machine: 360 voltage, 1 millisecond.

5. Add 100 μl of cells mixed with Cas9 protein/gRNA into the BTX electroporation cup and tap gently to avoid bubble.

6. Perform electroporation, then transfer the electroporated T cells into 1 ml of pre-warmed culture medium, mix well, and then place in a 37 degree incubator for further culture.

7. On the 4th day after electroporation, take about 0.5× ^10e6 cells and analyze them by anti-B2M flow cytometry staining to detect the gene knockout efficiency of B2M.

The results are shown in Figure 2. B2M gRNA-15 and gRNA-22 have the highest gene knockout efficiencies, reaching 79.1% and 80.2%, respectively, and can be used in subsequent experiments.

Example 4: TriKO T cell preparation condition test

2. Resuspend the cell pellet with Opti-MEM medium and adjust the cell concentration to 6×10e7/ml.

3. 6 μg of each of different combinations of TRAC gRNA/B2M gRNA/CD7 gRNA (all obtained by in vitro transcription according to the method described in Example 1) and 24 μg of TrueCut ^™ Cas9 Protein v2 protein (Thermofisher, catalog number: A36499) were mixed evenly in a 1.5 ml EP tube and incubated at room temperature for 10 minutes. After 10 minutes, 100 μl of T cells were added to the Cas9 protein/gRNA mixture, gently mixed, and immediately electroporated.

4. Set the parameters on the BTX ECM 830 machine: 360 voltage, 1 millisecond.

5. Add 100 μl of cells mixed with Cas9 protein/gRNA into the BTX electroporation cuvette and tap gently to avoid bubbles.

6. Perform electroporation, then transfer the electroporated T cells into 1 ml of pre-warmed culture medium, gently mix evenly, and then place in a 37 degree incubator for further culture.

7. Detect the gene knockout efficiency of TCR/CD7/B2M on the 4th day after electroporation.

The results are shown in Figure 3. For TCR knockout, the knockout efficiency of TRAC gRNA3.4 is higher than that of TRAC gRNA19.6, and for B2M knockout, the knockout efficiency of B2M gRNA-15 is higher than that of B2M gRNA-22. For TRAC/B2M/CD7, the best gRNA combination for simultaneous knockout of the three genes is TRAC gRNA3.4/B2M gRNA-15/CD7 gRNA-11.

Example 5: Anti-CD7 Antibodies and Chimeric Antigen Receptors Targeting CD7

1. Infect the logarithmic phase TG1 library culture with freshly thawed M13K07 helper phage at a multiplicity of infection of 20:1 (phage-to-cell ratio) and induce the expression of the fully human antibody phage display library with IPTG overnight.

2. Purify the phage library by PEG/NaCl precipitation and determine the phage titer. Store the phage at 4°C and proceed to scFv selection as soon as possible.

3. Selection of CD7-specific scFv-phage. For the first round of selection, 20 μg/ml CD7-6His protein was dissolved in 1× PBS and coated on Maxisorp plates and incubated overnight at 4°C. (For subsequent rounds, reduce The protein concentration was 2 μg/ml in the second round of biopanning and 0.5 μg/ml in the third round of biopanning for more stringent selection).

4. Wash three times with PBS, add blocking buffer (5% milk + 1% BSA in 1× PBS) to each well and incubate at RT for 2 hours.

5. Discard the blocking buffer, add the phage solution, seal with sealing film, and incubate with gentle shaking for 2 hours.

6. In the first round of screening, wash 10 times with PBST. In the following rounds, increase the stringency of washing by increasing the number of washing cycles (20 times in the second round and 30 times in the third round).

7. Elute the antigen-bound scFv phages. Add 1 ml of acidic elution buffer (pH 2.2), incubate for about 8 minutes, then use a pipette to pipette the eluted phages into about 100 μl of neutralization buffer and place in a new PP tube.

8. Inoculate 15 mL of the logarithmic phase TG1 culture (OD600 = 0.5) and the eluted phage solution in a new 50 mL test tube. Incubate at 37°C, let stand for 30 minutes, shake for 30 minutes. Then culture on a 15 cm plate containing 2xYT-GA agar.

9. Incubate the plates at 30°C overnight and harvest the bacteria for subsequent screening.

10. After three rounds of selection, select hundreds of positive colonies for monoclonal phage ELISA (mpELISA) screening.

11.mpELISA screening. Phage supernatants produced by single bacteria were incubated with pre-blocked Maxisorp plates coated with 2μg/ml CD7-6His protein. After washing three times, 100μl/well of HRP-conjugated anti-M13 antibody diluted 1:5000 in blocking buffer (5% milk + 1% BSA in 1× PBS) was added and incubated for 60 minutes at RT. After washing the plate 5 times with PBST, 100μl/well of TMB substrate solution was added and incubated for 10-30 minutes until blue color appeared. The reaction was terminated by adding 50μl/well of stop solution (2N H2SO4). The absorbance at 450nm was read on a microplate reader.

12. Positive clones were selected based on the ELISA results and used as templates for PCR cloning of scFv sequences (forward primer sequence: tgcagctggcacgacaggtttc (SEQ ID NO: 30), reverse primer sequence: cgtcagactgtagcacgtt (SEQ ID NO: 31)). The PCR products were then sequenced using Sanger sequencing (forward primer sequence: aacaattgaattcaggagga (SEQ ID NO: 32), reverse primer sequence: cctcctaagaagcgtagtc (SEQ ID NO: 33)).

13. The CDR regions of scFv were analyzed using the abysis website (http://abysis.org/) using the Chothia numbering system.

The results are shown in Figure 4. After three rounds of bio-panning, 288 colonies were selected for culture and phage supernatant was produced, which was tested for binding to CD7-Fc protein. The positive clones are highlighted in gray. Thirteen anti-CD7 scFv antibodies were screened, namely H1, H4, H6, H8, H9, H10, H12, H13, H17, H18, H5, H7, and H15.

These scFvs were used to construct chimeric antigen receptors targeting CD7, which included, from N-terminus to C-terminus: CD8 signal peptide, anti-CD7 scFv, CD8 hinge region, CD8 transmembrane domain, 4-1BB co-stimulatory domain, CD3ζ intracellular signaling domain. The corresponding chimeric antigen receptors were CD7H1.BBZ, CD7H4.BBZ, CD7H6.BBZ, CD7H8.BBZ, CD7H9.BBZ, CD7H10.BBZ, CD7H12.BBZ, CD7H13.BBZ, CD7H17.BBZ, CD7H18.BBZ, CD7H5.BBZ, CD7H7.BBZ, CD7H15.BBZ.

Example 6: Construction of LACO-Stim fusion protein A40C2828

A40C2828 was used as a lymphocyte-antigen presenting cell co-stimulator (LACO-Stim) to co-express with a chimeric antigen receptor targeting CD7. A40C2828 is a fusion protein that includes the CD40-targeting scFv A40C, the CD28 hinge region, the CD28 transmembrane domain, and the CD28 intracellular signaling domain from the N-terminus to the C-terminus.

Example 7: AAV plasmid construction

As shown in Figure 5, for the TRAC gRNA19.6 gene knock-in site, the R196H1A plasmid (pAAV-HA gRNA19.6-CD7H1.BBZ-F2A-A40C2828) was designed, which contained AAV ITR elements, homology arm sequences on both sides of the TRAC gRNA19.6 site, P2A elements, CD7H1.BBZ-F2A-A40C2828 gene expression frame, BGH plus A tail signal, etc. For the TRAC gRNA3.4 gene knock-in site, the R3H1A plasmid (pAAV-HA gRNA3.4-CD7H1.BBZ-F2A-A40C2828) was designed, which contained AAV ITR elements, homology arm sequences on both sides of the TRAC gRNA3.4 site, P2A elements, CD7H1.BBZ-F2A-A40C2828 gene expression frame, BGH plus A tail signal, etc. For the knock-in site of TRAC gRNA3.4 gene, R3PGK plasmid (pAAV-HA gRNA3.4-PGK-CD7H1.BBZ-F2A-A40C2828) was also designed, which contains AAV ITR elements, homology arm sequences on both sides of the TRAC gRNA3.4 site, PGK promoter elements, CD7H1.BBZ-F2A-A40C2828 gene expression frame, BGH plus A tail signal, etc. R196H1A and R3H1A use the endogenous promoter sequence of TRAC gene, while R3PGK uses the exogenous promoter sequence of PGK.

Example 8: Preparation of TriKO CD7 CAR-T cells

3. Mix 6 μg of different combinations of TRAC gRNA/B2M gRNA/CD7 gRNA and 24 μg of TrueCut ^TM Cas9 Protein v2 (Thermofisher, catalog number: A36499) in a 1.5 ml EP tube and incubate at room temperature for 10 minutes. After 10 minutes, take 100 μl of T cells and add them to the Cas9 protein/gRNA mixture, mix gently, and immediately perform electroporation.

4. Set the parameters on the BTX ECM 830 machine: 360 voltage, 1 millisecond.

6. Perform electroporation, then transfer the electroporated T cells to 450 μl of preheated culture medium, mix well, and divide into 5 wells of a 48-well plate with a volume of 100 μl per well. Then add the corresponding volume of AAV virus (Guangzhou Paizhen Biotechnology Co., Ltd.) to the electroporated T cells according to different MOIs (Figure 6), mix gently, and then place in a 37-degree incubator for continued culture.

7. After culturing at 37 degrees for 2 hours, add 1 ml of R10 culture medium to each well and continue culturing in a 37 degree incubator.

8. The gene knockout efficiency of TCR/CD7/B2M and the expression efficiency of CAR were detected on the 4th and 8th days after electroporation, respectively.

The results showed that on the fourth day after electroporation, the three genes TCR/B2M/CD7 were all efficiently knocked out (Figures 7 and 8). At the same time, the expression of CD7 CAR and A40C2828 (LACO-stim) was also detected (Figures 9 and 10). Moreover, the efficiency of gene knockout, the expression of CD7 CAR and the expression of A40C2828 were positively correlated with the MOI value of AAV addition.

On the 8th day after electroporation, the above test was repeated, and the results showed that the knockout efficiency of the three genes TCR/B2M/CD7 remained unchanged (Figures 11 and 12). At the same time, the expression of CD7 CAR and A40C2828 (LACO-stim) also remained constant (Figures 13 and 14).

As shown in the flow cytometry analysis of Figure 10, the proportion of CD7 positive cells becomes 0%. It can be seen that the TriKO CD7 CAR-T cells prepared in this way can completely eliminate CD7 positive T cells.

The cell growth curve showed that the higher the MOI of AAV addition, the higher the CAR positivity rate, and the easier it was to reduce the growth rate of CAR-T cells ( FIG. 15 ).

Example 9: In vitro cytotoxicity assay of TriKO CD7 CAR-T cells

1. 12 hours before the co-culture of TriKO CD7 CAR-T cells prepared in Example 8 and tumor target cell cytotoxicity experiments, CCRF-CEM-GFP cells were inoculated into a flat-bottom 96-well plate pre-coated with Poly-L-Ornithine (Sigma-Aldrich, Catalog No.: P4957), with 10,000 cells/100 μl per well.

2. After 12 hours, when CCRF-CEM-GFP cells are fully attached to the wall, the various TriKO CD7 CAR-T cells prepared in Example 8 are diluted to an appropriate cell density and then co-incubated with tumor cells at different effector-target ratios (e.g., E:T = 2:1, 0.5:1, and 0.2:1).

3. Place the 96-well plate into the InCucyte S3 machine and set the scanning parameters.

4. After 4 days of scanning, analyze the total green fluorescence cumulative intensity (GCU xμm ² /well) to calculate tumor cell killing Injury efficiency.

The results showed that the killing effect of the three AAV-prepared TriKO CD7 CAR-T cells on CD7-positive tumor target cells CCRF-CEM was very strong, and the target cells could be effectively killed under the conditions of E:T ratio＝2:1 and E:T ratio＝0.5:1, and even E:T ratio＝0.2:1 (Figure 16, Figure 17 and Figure 18). Compared with the three AAV-prepared TriKO CD7 CAR-T cells, the killing effect of R3H1A was stronger.

Specific sequences used in Examples 1-9:

SEQ ID NO:26 pDA-T7-B2M gRNA 1 (pDA B2M gRNA-M1)

SEQ ID NO:27 T7 promoter

taatacgactcactatag

SEQ ID NO:28 gRNA backbone

SEQ ID NO:29 Termination signal

TTTTTTT

SEQ ID NO:30 Forward primer for PCR cloning

tgcagctggcacgacaggtttc

SEQ ID NO:31 Reverse primer for PCR cloning

cgtcagactgtagcacgtt

SEQ ID NO:32 Forward primer for Sanger sequencing

aacaattgaattcaggagga

SEQ ID NO:33 Reverse primer for Sanger sequencing

cctcctaagaagcgtagtc

SEQ ID NO:34 anti-CD7 scFv H1, amino acid sequence

SEQ ID NO:35 anti-CD7 scFv H1, VL, amino acid sequence

SEQ ID NO:36 anti-CD7 scFv H1, VH, amino acid sequence

SEQ ID NO:37 CD7H1.BBZ, amino acid sequence

SEQ ID NO:38 CD7H1.BBZ, nucleotide sequence

SEQ ID NO:39 anti-CD7 scFv H4, amino acid sequence

SEQ ID NO:40 anti-CD7 scFv H4, VL, amino acid sequence

SEQ ID NO:41 anti-CD7 scFv H4, VH, amino acid sequence

SEQ ID NO:42 CD7H4.BBZ, amino acid sequence

SEQ ID NO:43 anti-CD7 scFv H6, amino acid sequence

SEQ ID NO:44 anti-CD7 scFv H6, VL, amino acid sequence

SEQ ID NO:45 anti-CD7 scFv H6, VH, amino acid sequence

SEQ ID NO:46 CD7H6.BBZ, amino acid sequence

SEQ ID NO:47 anti-CD7 scFv H8, amino acid sequence

SEQ ID NO:48 anti-CD7 scFv H8, VL, amino acid sequence

SEQ ID NO:49 anti-CD7 scFv H8, VH, amino acid sequence

SEQ ID NO:50 CD7H8.BBZ, amino acid sequence

SEQ ID NO:51 anti-CD7 scFv H9, amino acid sequence

SEQ ID NO:52 anti-CD7 scFv H9, VL, amino acid sequence

SEQ ID NO:53 anti-CD7 scFv H9, VH, amino acid sequence

SEQ ID NO:54 CD7H9.BBZ, amino acid sequence

SEQ ID NO:55 anti-CD7 scFv H10, amino acid sequence

SEQ ID NO:56 anti-CD7 scFv H10, VL, amino acid sequence

SEQ ID NO:57 anti-CD7 scFv H10, VH, amino acid sequence

SEQ ID NO:58 CD7H10.BBZ, amino acid sequence

SEQ ID NO:59 anti-CD7 scFv H12, amino acid sequence

SEQ ID NO:60 anti-CD7 scFv H12, VL, amino acid sequence

SEQ ID NO:61 anti-CD7 scFv H12, VH, amino acid sequence

SEQ ID NO:62 CD7H12.BBZ, amino acid sequence

SEQ ID NO:63 anti-CD7 scFv H13, amino acid sequence

SEQ ID NO:64 anti-CD7 scFv H13, VL, amino acid sequence

SEQ ID NO:65 anti-CD7 scFv H13, VH, amino acid sequence

SEQ ID NO:66 CD7H13.BBZ, amino acid sequence

SEQ ID NO:67 anti-CD7 scFv H17, amino acid sequence

SEQ ID NO:68 anti-CD7 scFv H17, VL, amino acid sequence

SEQ ID NO:69 anti-CD7 scFv H17, VH, amino acid sequence

SEQ ID NO:70 CD7H17.BBZ, amino acid sequence

SEQ ID NO:71 anti-CD7 scFv H18, amino acid sequence

SEQ ID NO:72 anti-CD7 scFv H18, VL, amino acid sequence

SEQ ID NO:73 anti-CD7 scFv H18, VH, amino acid sequence

SEQ ID NO:74 CD7H18.BBZ, amino acid sequence

SEQ ID NO:75 anti-CD7 scFv H5, amino acid sequence

SEQ ID NO:76 anti-CD7 scFv H5, VL, amino acid sequence

SEQ ID NO:77 anti-CD7 scFv H5, VH, amino acid sequence

SEQ ID NO:78 CD7H5.BBZ, amino acid sequence

SEQ ID NO:79 anti-CD7 scFv H7, amino acid sequence

SEQ ID NO:80 anti-CD7 scFv H7, VL, amino acid sequence

SEQ ID NO:81 anti-CD7 scFv H7, VH, amino acid sequence

SEQ ID NO:82 CD7H7.BBZ, amino acid sequence

SEQ ID NO:83 anti-CD7 scFv H15, amino acid sequence

SEQ ID NO:84 anti-CD7 scFv H15, VL, amino acid sequence

SEQ ID NO:85 anti-CD7 scFv H15, VH, amino acid sequence

SEQ ID NO:86 CD7H15.BBZ, amino acid sequence

SEQ ID NO:151 CD8 signal peptide, amino acid sequence

MALPVTALLLPLALLLHAARP

SEQ ID NO:152 CD8 signal peptide, nucleotide sequence

atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccg

SEQ ID NO:153 CD8 hinge region, amino acid sequence

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

SEQ ID NO:154 CD8 hinge region, nucleotide sequence

SEQ ID NO:155 CD8 transmembrane domain, amino acid sequence

IYIWAPLAGTCGVLLLSLVITLYC

SEQ ID NO:156 CD8 transmembrane domain, nucleotide sequence

atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc

SEQ ID NO:157 4-1BB co-stimulatory domain, amino acid sequence

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

SEQ ID NO:158 4-1BB co-stimulatory domain, nucleotide sequence

SEQ ID NO:159 CD3-zeta, amino acid sequence

SEQ ID NO:160 CD3-zeta, nucleotide sequence

SEQ ID NO:161 A40C2828, amino acid sequence

SEQ ID NO:162 A40C2828, nucleotide sequence

SEQ ID NO:163 Anti-CD40 scFv A40, amino acid sequence

SEQ ID NO: 164 CD28 hinge region, amino acid sequence

IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

SEQ ID NO:165 CD28 transmembrane domain, amino acid sequence

FWVLVVVGGVLACYSLLVTVAFIIFWV

SEQ ID NO: 166 CD28 intracellular domain, amino acid sequence

RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

SEQ ID NO:167 R196H1A (pAAV-HA gRNA19.6-CD7H1.BBZ-F2A-A40C2828)

SEQ ID NO:168 R3H1A (pAAV-HA gRNA3.4-CD7H1.BBZ-F2A-A40C2828)

SEQ ID NO:169 R3PGK (pAAV-HA gRNA3.4-PGK-CD7H1.BBZ-F2A-A40C2828)

SEQ ID NO:170 CD7H1.BBZ-F2A-A40C2828, amino acid sequence

SEQ ID NO:171 CD7H1.BBZ-F2A-A40C2828, nucleotide sequence

SEQ ID NO: 172 Homologous arm gRNA3.4-left (5')

SEQ ID NO: 173 Homologous arm gRNA3.4-right side (3')

SEQ ID NO: 174 Homologous arm gRNA19.6-left (5')

SEQ ID NO: 175 Homologous arm gRNA19.6-right side (3')

SEQ ID NO: 176 PGK promoter

SEQ ID NO: 177 Linker 1

GGGGSGGGGSGGGGS

SEQ ID NO: 178 Linker 2

GGGSGGGGSGGGGS

SEQ ID NO:179 F2A

VKQTLNFDLLKLAGDVESNPGP

Claims

A modified T cell, wherein the expression of endogenous TRAC, B2M and CD7 genes in the T cell is inhibited, and the T cell expresses a chimeric antigen receptor targeting CD7, wherein the chimeric antigen receptor includes a binding domain that specifically binds to CD7, a transmembrane domain and an intracellular signaling domain.
The modified T cell of claim 1, wherein the binding domain that specifically binds to CD7 comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3; wherein,

LCDR1 has an amino acid sequence selected from SEQ ID NO: 113-125;

LCDR2 has an amino acid sequence selected from SEQ ID NO: 126-137;

LCDR3 has an amino acid sequence selected from SEQ ID NO: 138-150;

HCDR1 has an amino acid sequence selected from SEQ ID NO: 87-94;

HCDR2 has an amino acid sequence selected from SEQ ID NO:95-102; and

HCDR3 has an amino acid sequence selected from SEQ ID NO:103-112.
The modified T cell of claim 2, wherein the light chain variable region and heavy chain variable region of the binding domain that specifically binds to CD7 include LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 selected from the group consisting of:

(a) LCDR1 having the amino acid sequence set forth in SEQ ID NO:113, LCDR2 having the amino acid sequence set forth in SEQ ID NO:126, LCDR3 having the amino acid sequence set forth in SEQ ID NO:138, HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, HCDR2 having the amino acid sequence set forth in SEQ ID NO:95, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:103;

(b) LCDR1 having the amino acid sequence set forth in SEQ ID NO:114, LCDR2 having the amino acid sequence set forth in SEQ ID NO:127, LCDR3 having the amino acid sequence set forth in SEQ ID NO:139, HCDR1 having the amino acid sequence set forth in SEQ ID NO:88, HCDR2 having the amino acid sequence set forth in SEQ ID NO:96, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:104;

(c) LCDR1 having the amino acid sequence set forth in SEQ ID NO:115, LCDR2 having the amino acid sequence set forth in SEQ ID NO:128, LCDR3 having the amino acid sequence set forth in SEQ ID NO:140, HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, HCDR2 having the amino acid sequence set forth in SEQ ID NO:95, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:105;

(d) LCDR1 having the amino acid sequence set forth in SEQ ID NO:116, LCDR2 having the amino acid sequence set forth in SEQ ID NO:129, LCDR3 having the amino acid sequence set forth in SEQ ID NO:141, HCDR1 having the amino acid sequence set forth in SEQ ID NO:89, HCDR2 having the amino acid sequence set forth in SEQ ID NO:97, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:106;

(e) LCDR1 having the amino acid sequence of SEQ ID NO: 117, LCDR2 having the amino acid sequence of SEQ ID NO: 130, LCDR3 having the amino acid sequence of SEQ ID NO: 142, LCDR4 having the amino acid sequence of SEQ ID NO: 154, a HCDR1 having the amino acid sequence set forth in SEQ ID NO:90, a HCDR2 having the amino acid sequence set forth in SEQ ID NO:98, and a HCDR3 having the amino acid sequence set forth in SEQ ID NO:107;

(f) LCDR1 having the amino acid sequence set forth in SEQ ID NO:118, LCDR2 having the amino acid sequence set forth in SEQ ID NO:131, LCDR3 having the amino acid sequence set forth in SEQ ID NO:143, HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, HCDR2 having the amino acid sequence set forth in SEQ ID NO:95, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:103;

(g) LCDR1 having the amino acid sequence set forth in SEQ ID NO:119, LCDR2 having the amino acid sequence set forth in SEQ ID NO:132, LCDR3 having the amino acid sequence set forth in SEQ ID NO:144, HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, HCDR2 having the amino acid sequence set forth in SEQ ID NO:95, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:103;

(h) LCDR1 having the amino acid sequence set forth in SEQ ID NO:120, LCDR2 having the amino acid sequence set forth in SEQ ID NO:133, LCDR3 having the amino acid sequence set forth in SEQ ID NO:145, HCDR1 having the amino acid sequence set forth in SEQ ID NO:91, HCDR2 having the amino acid sequence set forth in SEQ ID NO:99 and HCDR3 having the amino acid sequence set forth in SEQ ID NO:108;

(i) LCDR1 having the amino acid sequence set forth in SEQ ID NO:121, LCDR2 having the amino acid sequence set forth in SEQ ID NO:134, LCDR3 having the amino acid sequence set forth in SEQ ID NO:146, HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, HCDR2 having the amino acid sequence set forth in SEQ ID NO:95 and HCDR3 having the amino acid sequence set forth in SEQ ID NO:103;

(j) LCDR1 having the amino acid sequence set forth in SEQ ID NO: 122, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 135, LCDR3 having the amino acid sequence set forth in SEQ ID NO: 147, HCDR1 having the amino acid sequence set forth in SEQ ID NO: 87, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 95, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 109;

(k) LCDR1 having the amino acid sequence set forth in SEQ ID NO:123, LCDR2 having the amino acid sequence set forth in SEQ ID NO:136, LCDR3 having the amino acid sequence set forth in SEQ ID NO:148, HCDR1 having the amino acid sequence set forth in SEQ ID NO:92, HCDR2 having the amino acid sequence set forth in SEQ ID NO:100 and HCDR3 having the amino acid sequence set forth in SEQ ID NO:110;

(l) a LCDR1 having an amino acid sequence as set forth in SEQ ID NO:124, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO:137, a LCDR3 having an amino acid sequence as set forth in SEQ ID NO:149, a HCDR1 having an amino acid sequence as set forth in SEQ ID NO:93, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO:101 and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO:111; and

(m) LCDR1 having the amino acid sequence shown as SEQ ID NO:125, LCDR2 having the amino acid sequence shown as SEQ ID NO:137, LCDR3 having the amino acid sequence shown as SEQ ID NO:150, HCDR1 having the amino acid sequence shown as SEQ ID NO:94, HCDR2 having the amino acid sequence shown as SEQ ID NO:102 and HCDR3 having the amino acid sequence shown as SEQ ID NO:112.
The modified T cell of claim 2, wherein the binding domain that specifically binds to CD7 comprises a light chain variable region and a heavy chain variable region selected from the group consisting of:

(a) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 35 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 36 A heavy chain variable region having the amino acid sequence shown;

(b) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:40 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:41;

(c) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:44 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:45;

(d) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:48 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:49;

(e) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:52 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:53;

(f) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:56 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:57;

(g) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:60 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:61;

(h) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:64 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:65;

(i) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:68 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:69;

(j) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:72 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:73;

(k) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:76 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:77;

(l) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:80 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:81; and

(m) comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO:84 and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:85.
The modified T cell according to any one of claims 1 to 4, wherein the binding domain that specifically binds to CD7 is a scFv.
The modified T cell according to any one of claims 1 to 5, wherein the transmembrane domain is a CD8 transmembrane domain.
The modified T cell of any one of claims 1 to 6, wherein the intracellular signaling domain comprises a 4-1BB co-stimulatory domain and a CD3ζ intracellular domain.
The modified T cell according to any one of claims 1 to 7, wherein the chimeric antigen receptor further comprises a hinge region between the binding domain and the transmembrane domain.
The modified T cell of claim 8, wherein the hinge region is a CD8 hinge region.
The modified T cell according to any one of claims 1 to 9, wherein the chimeric antigen targeting CD7 The receptor comprises an amino acid sequence selected from SEQ ID NO: 37, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86.
The modified T cell according to any one of claims 1 to 10, wherein the chimeric antigen receptor targeting CD7 further comprises a signal peptide.
The modified T cell of claim 11, wherein the signal peptide comprised by the chimeric antigen receptor targeting CD7 is a CD8 signal peptide.
The modified T cell of any one of claims 1 to 12, wherein the nucleic acid sequence encoding the chimeric antigen receptor is inserted into the TRAC locus of the T cell.
The modified T cell of claim 13, wherein the nucleic acid sequence encoding the chimeric antigen receptor is operably linked to the PGK promoter or the endogenous TRAC promoter of the T cell.
The modified T cell according to any one of claims 1 to 14, wherein the T cell further expresses a fusion protein of a lymphocyte-antigen presenting cell co-stimulatory factor, wherein the fusion protein comprises a first domain that activates an antigen presenting cell (APC) and a second domain that activates an immune effector cell, wherein

The first domain comprises: (i) a ligand that binds to an activating receptor of the APC, or a receptor-binding fragment thereof, or (ii) an antibody that binds to an activating receptor of the APC, or an antigen-binding fragment thereof; and

The second domain includes: (i) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (ii) a co-stimulatory ligand of the immune effector cell, or a receptor binding fragment thereof, or (iii) an antibody that binds to the co-stimulatory receptor of the immune effector cell, or an antigen binding fragment thereof.
The modified T cell of claim 15, wherein the activation receptor of the APC is CD40.
The modified T cell of claim 15, wherein the first domain is an anti-CD40 antibody or an antigen-binding fragment thereof.
The modified T cell of claim 17, wherein the first domain is a scFv.
The modified T cell according to any one of claims 15 to 18, wherein the immune effector cell is a T cell.
The modified T cell of any one of claims 15 to 19, wherein the second domain comprises the intracellular domain of the co-stimulatory receptor.
The modified T cell according to any one of claims 15 to 20, wherein the co-stimulatory receptor is CD28.
The modified T cell of claim 20 or 21, wherein the second domain further comprises a transmembrane domain of the co-stimulatory receptor.
The modified T cell of any one of claims 15 to 22, wherein the first domain and the second domain are connected by a CD28 hinge region.
The modified T cell as described in any one of claims 15-23, wherein the lymphocyte-antigen presenting cell co-stimulatory factor fusion protein comprises the amino acid sequence shown in SEQ ID NO:161.
The modified T cell according to any one of claims 15 to 24, wherein the lymphocyte-antigen presenting cell co-stimulatory factor fusion protein further comprises a signal peptide.
The modified T cell according to claim 25, wherein the signal peptide contained in the fusion protein of lymphocyte-antigen presenting cell co-stimulatory factor is a CD8 signal peptide.
The modified T cell according to any one of claims 15 to 26, wherein the nucleic acid sequence encoding the fusion protein of the lymphocyte-antigen presenting cell co-stimulatory factor and the nucleic acid sequence encoding the chimeric antigen receptor targeting CD7 are in the same expression frame or in separate expression frames.
The modified T cell according to any one of claims 1 to 27, wherein the endogenous TRAC, B2M and CD7 genes in the T cell are knocked out.
A method of producing a modified T cell, the method comprising:

(a) inhibiting the expression of endogenous TRAC, B2M and CD7 genes in the T cells; and

(b) introducing a polynucleotide sequence comprising a nucleic acid sequence encoding a chimeric antigen receptor targeting CD7 into the T cell, so that the T cell expresses the chimeric antigen receptor, wherein the chimeric antigen receptor targeting CD7 comprises a binding domain that specifically binds to CD7, a transmembrane domain, and an intracellular signaling domain.
The method of claim 29, wherein the binding domain that specifically binds to CD7 comprises a light chain variable region and a heavy chain variable region, the light chain variable region comprises LCDR1, LCDR2 and LCDR3, and the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3; wherein,

LCDR1 has an amino acid sequence selected from SEQ ID NO: 113-125;

LCDR2 has an amino acid sequence selected from SEQ ID NO: 126-137;

LCDR3 has an amino acid sequence selected from SEQ ID NO: 138-150;

HCDR1 has an amino acid sequence selected from SEQ ID NO: 87-94;

HCDR2 has an amino acid sequence selected from SEQ ID NO:95-102; and

HCDR3 has an amino acid sequence selected from SEQ ID NO:103-112.
The method of claim 30, wherein the light chain variable region and heavy chain variable region of the binding domain that specifically binds to CD7 include LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 selected from the group consisting of:

(a) LCDR1 having the amino acid sequence set forth in SEQ ID NO:113, LCDR2 having the amino acid sequence set forth in SEQ ID NO:126, LCDR3 having the amino acid sequence set forth in SEQ ID NO:138, HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, HCDR2 having the amino acid sequence set forth in SEQ ID NO:95, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:103;

(b) LCDR1 having the amino acid sequence set forth in SEQ ID NO:114, LCDR2 having the amino acid sequence set forth in SEQ ID NO:127, LCDR3 having the amino acid sequence set forth in SEQ ID NO:139, HCDR1 having the amino acid sequence set forth in SEQ ID NO:88, HCDR2 having the amino acid sequence set forth in SEQ ID NO:96, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:104;

(c) a LCDR1 having an amino acid sequence as set forth in SEQ ID NO: 115, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO: 128, a LCDR3 having an amino acid sequence as set forth in SEQ ID NO: 140, a HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 87, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 95, and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO: 105;

(d) LCDR1 having the amino acid sequence set forth in SEQ ID NO:116, LCDR2 having the amino acid sequence set forth in SEQ ID NO:129, LCDR3 having the amino acid sequence set forth in SEQ ID NO:141, HCDR1 having the amino acid sequence set forth in SEQ ID NO:89, HCDR2 having the amino acid sequence set forth in SEQ ID NO:97, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:106;

(e) LCDR1 having the amino acid sequence set forth in SEQ ID NO: 117, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 130, LCDR3 having the amino acid sequence set forth in SEQ ID NO: 142, HCDR1 having the amino acid sequence set forth in SEQ ID NO: 90, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 98, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 107;

(f) LCDR1 having the amino acid sequence set forth in SEQ ID NO:118, LCDR2 having the amino acid sequence set forth in SEQ ID NO:131, LCDR3 having the amino acid sequence set forth in SEQ ID NO:143, HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, HCDR2 having the amino acid sequence set forth in SEQ ID NO:95, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:103;

(g) LCDR1 having the amino acid sequence set forth in SEQ ID NO:119, LCDR2 having the amino acid sequence set forth in SEQ ID NO:132, LCDR3 having the amino acid sequence set forth in SEQ ID NO:144, HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, HCDR2 having the amino acid sequence set forth in SEQ ID NO:95, and HCDR3 having the amino acid sequence set forth in SEQ ID NO:103;

(h) LCDR1 having the amino acid sequence set forth in SEQ ID NO:120, LCDR2 having the amino acid sequence set forth in SEQ ID NO:133, LCDR3 having the amino acid sequence set forth in SEQ ID NO:145, HCDR1 having the amino acid sequence set forth in SEQ ID NO:91, HCDR2 having the amino acid sequence set forth in SEQ ID NO:99 and HCDR3 having the amino acid sequence set forth in SEQ ID NO:108;

(i) LCDR1 having the amino acid sequence set forth in SEQ ID NO:121, LCDR2 having the amino acid sequence set forth in SEQ ID NO:134, LCDR3 having the amino acid sequence set forth in SEQ ID NO:146, HCDR1 having the amino acid sequence set forth in SEQ ID NO:87, HCDR2 having the amino acid sequence set forth in SEQ ID NO:95 and HCDR3 having the amino acid sequence set forth in SEQ ID NO:103;

(j) LCDR1 having the amino acid sequence set forth in SEQ ID NO: 122, LCDR2 having the amino acid sequence set forth in SEQ ID NO: 135, LCDR3 having the amino acid sequence set forth in SEQ ID NO: 147, HCDR1 having the amino acid sequence set forth in SEQ ID NO: 87, HCDR2 having the amino acid sequence set forth in SEQ ID NO: 95, and HCDR3 having the amino acid sequence set forth in SEQ ID NO: 109;

(k) LCDR1 having the amino acid sequence set forth in SEQ ID NO:123, LCDR2 having the amino acid sequence set forth in SEQ ID NO:136, LCDR3 having the amino acid sequence set forth in SEQ ID NO:148, HCDR1 having the amino acid sequence set forth in SEQ ID NO:92, HCDR2 having the amino acid sequence set forth in SEQ ID NO:100 and HCDR3 having the amino acid sequence set forth in SEQ ID NO:110;

(l) a LCDR1 having an amino acid sequence as set forth in SEQ ID NO:124, a LCDR2 having an amino acid sequence as set forth in SEQ ID NO:137, a LCDR3 having an amino acid sequence as set forth in SEQ ID NO:149, a HCDR1 having an amino acid sequence as set forth in SEQ ID NO:93, a HCDR2 having an amino acid sequence as set forth in SEQ ID NO:101 and a HCDR3 having an amino acid sequence as set forth in SEQ ID NO:111; and

(m) LCDR1 having the amino acid sequence shown in SEQ ID NO: 125, having the amino acid sequence shown in SEQ ID NO: 137 The invention further comprises a LCDR2 having an amino acid sequence as shown in SEQ ID NO: 150, a LCDR3 having an amino acid sequence as shown in SEQ ID NO: 154, a HCDR1 having an amino acid sequence as shown in SEQ ID NO: 94, a HCDR2 having an amino acid sequence as shown in SEQ ID NO: 102, and a HCDR3 having an amino acid sequence as shown in SEQ ID NO: 112.
The method of claim 30, wherein the binding domain that specifically binds to CD7 comprises a light chain variable region and a heavy chain variable region selected from the group consisting of:

(a) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:35 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:36;

(b) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:40 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:41;

(c) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:44 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:45;

(d) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:48 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:49;

(e) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:52 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:53;

(f) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:56 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:57;

(g) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:60 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:61;

(h) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:64 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:65;

(i) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:68 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:69;

(j) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:72 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:73;

(k) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:76 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:77;

(l) a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:80 and a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:81; and

(m) comprises a light chain variable region having an amino acid sequence as shown in SEQ ID NO:84 and a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO:85.
The method according to any one of claims 29 to 32, wherein the binding domain that specifically binds to CD7 is a scFv.
The method according to any one of claims 29 to 33, wherein the transmembrane domain is a CD8 transmembrane domain.
The method of any one of claims 29 to 34, wherein the intracellular signaling domain comprises 4-1BB Co-stimulatory domain and CD3ζ intracellular domain.
The method of any one of claims 29 to 35, wherein the chimeric antigen receptor further comprises a hinge region between the binding domain and the transmembrane domain.
The method of claim 36, wherein the hinge region is the CD8 hinge region.
A method as described in any one of claims 29-37, wherein the chimeric antigen receptor targeting CD7 comprises an amino acid sequence selected from SEQ ID NO: 37, 42, 46, 50, 54, 58, 62, 66, 70, 74, 78, 82, 86.
The method of any one of claims 29 to 38, wherein the chimeric antigen receptor targeting CD7 further comprises a signal peptide.
The method of claim 39, wherein the signal peptide comprised by the chimeric antigen receptor targeting CD7 is a CD8 signal peptide.
The method according to any one of claims 29 to 40, wherein step (a) comprises knocking out endogenous TRAC, B2M and CD7 genes in the T cells.
The method of claim 41, wherein step (a) comprises introducing CRISPR-Cas protein and gRNA targeting endogenous TRAC, B2M and CD7 genes in the T cells into the T cells to produce cuts in the endogenous TRAC, B2M and CD7 genes of the T cells and disrupt their expression.
A method as claimed in claim 42, wherein the gRNA targeting the TRAC gene comprises a guiding sequence selected from SEQ ID NO:23 and SEQ ID NO:24.
A method as described in claim 42 or 43, wherein the gRNA targeting the B2M gene comprises a guiding sequence selected from SEQ ID NO:15 and SEQ ID NO:22.
A method as described in any one of claims 42-44, wherein the gRNA targeting the CD7 gene comprises a guide sequence as shown in SEQ ID NO:25.
The method of claim 42, wherein the gRNAs targeting TRAC, B2M and CD7 genes respectively comprise guide sequences selected from the group consisting of:

The gRNA targeting the TRAC gene comprises the guide sequence shown in SEQ ID NO: 23, the gRNA targeting the B2M gene comprises the guide sequence shown in SEQ ID NO: 15, and the gRNA targeting the CD7 gene comprises the guide sequence shown in SEQ ID NO: 25; and

The gRNA targeting the TRAC gene comprises the guide sequence shown as SEQ ID NO:24, the gRNA targeting the B2M gene comprises the guide sequence shown as SEQ ID NO:15, and the gRNA targeting the CD7 gene comprises the guide sequence shown as SEQ ID NO:25.
The method according to any one of claims 29 to 46, wherein the polynucleotide sequence comprises homology arms flanking the nucleic acid sequence encoding the chimeric antigen receptor and homologous to the upstream and downstream sequences of the TRAC gene cleavage site, respectively, so that the nucleic acid sequence encoding the chimeric antigen receptor is inserted into the TRAC locus by homologous recombination.
The method of claim 47, wherein the nucleic acid sequence encoding the chimeric antigen receptor is inserted into the TRAC locus at a position operably linked to the TRAC endogenous promoter.
The method according to any one of claims 29 to 47, wherein the polynucleotide sequence comprises a chimeric The nucleic acid sequence of the antigen receptor is operably linked to the PGK promoter.
The method of any one of claims 29 to 49, wherein the polynucleotide sequence is contained in a viral vector.
The method of claim 50, wherein the viral vector is an AAV vector.
The method according to any one of claims 29 to 51, further comprising:

(c) introducing a polynucleotide sequence comprising a nucleic acid sequence encoding a fusion protein of a lymphocyte-antigen presenting cell co-stimulatory factor into the T cell,

The fusion protein of lymphocyte-antigen presenting cell co-stimulatory factor comprises a first domain for activating antigen presenting cells (APC) and a second domain for activating immune effector cells, wherein

The first domain comprises: (i) a ligand that binds to an activating receptor of the APC, or a receptor-binding fragment thereof, or (ii) an antibody that binds to an activating receptor of the APC, or an antigen-binding fragment thereof; and

The second domain includes: (i) a co-stimulatory receptor of the immune effector cell, or a functional fragment thereof, (ii) a co-stimulatory ligand of the immune effector cell, or a receptor binding fragment thereof, or (iii) an antibody that binds to the co-stimulatory receptor of the immune effector cell, or an antigen binding fragment thereof.
The method of claim 52, wherein the activating receptor of the APC is CD40.
The method of claim 52, wherein the first domain is an anti-CD40 antibody or an antigen-binding fragment thereof.
The method of claim 54, wherein the first domain is a scFv.
The method of any one of claims 52 to 55, wherein the immune effector cells are T cells.
The method of any one of claims 52-56, wherein the second domain comprises the intracellular domain of the co-stimulatory receptor.
The method of any one of claims 52-57, wherein the co-stimulatory receptor is CD28.
The method of claim 57 or 58, wherein the second domain further comprises a transmembrane domain of the co-stimulatory receptor.
The method of any one of claims 52-59, wherein the first domain and the second domain are connected by a CD28 hinge region.
A method as described in any one of claims 52-60, wherein the lymphocyte-antigen presenting cell co-stimulatory factor fusion protein comprises an amino acid sequence as shown in SEQ ID NO:161.
The method according to any one of claims 52 to 61, wherein the lymphocyte-antigen presenting cell co-stimulatory factor fusion protein further comprises a signal peptide.
The method of claim 62, wherein the signal peptide contained in the fusion protein of lymphocyte-antigen presenting cell co-stimulatory factor is a CD8 signal peptide.
The method according to any one of claims 52 to 63, wherein the nucleic acid sequence encoding the fusion protein of the lymphocyte-antigen presenting cell co-stimulatory factor and the nucleic acid sequence encoding the chimeric antigen receptor are in the same expression frame or in separate expression frames.
A pharmaceutical composition comprising the modified T cell according to any one of claims 1 to 28, or the modified T cell produced according to the method according to any one of claims 29 to 64, and a pharmaceutically acceptable carrier.
A method for treating a disease associated with CD7 expression, comprising administering a therapeutically effective amount of the modified T cells according to any one of claims 1 to 28 or the pharmaceutical composition according to claim 65 to a subject in need thereof.
Use of the modified T cells according to any one of claims 1 to 28, or the modified T cells produced according to the method according to any one of claims 29 to 64, in the preparation of a medicament for treating a disease associated with CD7 expression.