CN117659196A

CN117659196A - CD 7-targeted single domain antibody, chimeric antigen receptor and application thereof

Info

Publication number: CN117659196A
Application number: CN202311699827.1A
Authority: CN
Inventors: 周丹; 何雨辰; 孙海; 汪远进; 黄智威; 李红梅
Original assignee: Chengdu Yousainuo Biotechnology Co ltd
Current assignee: Chengdu Yousainuo Biotechnology Co ltd
Priority date: 2023-12-12
Filing date: 2023-12-12
Publication date: 2024-03-08

Abstract

The invention relates to the technical field of immunotherapy, and provides a single domain antibody targeting CD7, a Chimeric Antigen Receptor (CAR) targeting CD7 constructed by utilizing the single domain antibody and an engineered immune effector cell containing the CAR. The invention also provides the use of a single domain antibody targeting CD7, a chimeric antigen receptor, and engineered immune effector cells comprising the CAR in the manufacture of a medicament for the diagnosis, prevention and/or treatment of a disease or disorder associated with CD7 expression.

Description

CD 7-targeted single domain antibody, chimeric antigen receptor and application thereof

Technical Field

The invention belongs to the field of biological medicine, and more particularly relates to a single domain antibody specifically targeting CD7, a chimeric antigen receptor targeting CD7 constructed by using the single domain antibody, an engineered immune effector cell containing the chimeric antigen receptor, and application of the engineered immune effector cell in preparation of medicines for diagnosing, preventing and/or treating diseases.

Background

T cell malignancies are a class of lymphoid system malignancies derived from T cells, typically expressing antigens characteristic of T cells. WHO classification classifies T cell tumors into T lymphocyte leukemia, T lymphoblastic lymphoma, and mature T cell tumors. At present, no very effective treatment means for T cell malignant tumor exists, the main treatment scheme is chemotherapy, hematopoietic stem cell transplantation is needed to be combined if necessary, but the cancer has high recurrence rate and death rate in patients. For example, T cell acute lymphoblastic leukemia (T-ALL), a hematological disease caused by abnormal proliferation of T lymphocytes, has rapid progress of disease course, can rapidly infiltrate into tissue organs such as lymph nodes, liver, spleen, central nervous system and testis in a short period of time, and requires timely and effective treatment, otherwise the disease condition of patients can be aggravated in a short period of time and even life-threatening. Acute T-lymphoblastic leukemia in adults and children has a 5-year survival rate of about 50% and 75%, respectively, when subjected to high-dose chemotherapy. T cell lymphoma is a malignant tumor of T cells, can develop in lymphoid tissues (such as lymph nodes and spleen) or outside lymphoid tissues (such as gastrointestinal tract, liver, nasal cavity, skin, etc.), and accounts for about 10% -15% of the incidence rate of non-Hodgkin's lymphoma, which is higher in China. Peripheral T cell lymphomas receive CHOP-based chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone) and have a progression-free survival rate of about 40% -50% consolidated by autologous hematopoietic stem cell transplantation. Wherein the early precursor T acute lymphoblastic leukemia has a poor subgroup prognosis and a median survival time of only 20 months. Acute Myeloid Leukemia (AML) is a very refractory invasive hematopoietic stem cell malignancy that is easily relapsed, and has low survival rates in the majority of all patients in elderly, a lower patient survival rate malignancy. The current method for treating T cell malignant tumor is chemotherapy, but has the problems of poor disease prognosis and high recurrence rate. Thus, there remains a broad and unmet need for therapies.

CD7 (T cell antigen 7), also known as GP40, TP41, LEU-9, is a 40kDa single-chain transmembrane glycoprotein molecule comprising 240 amino acids. CD7 is typically expressed in 85% of peripheral blood T cells and NK cells and their precursors, a costimulatory receptor protein that aids T cell activation and interactions with other immune subpopulation cells. CD7 is expressed in most of T cells, NK cells, marrow cells, T cell acute lymphoblastic leukemia/lymphoma, acute myelogenous leukemia and chronic myelogenous leukemia tumor cells, and especially is highly expressed in most of T cell acute lymphoblastic leukemia and part of T cell acute myelogenous leukemia tumor cells, so that the CD7 is expected to become a potential treatment target for treating leukemia, lymphoma and other malignant tumors of blood systems.

Single-domain antibodies (sdabs) are antibodies consisting of only the variable region amino acids of heavy chain antibodies, and thus, unlike traditional 4-chain antibodies, have molecular weights of only 12-15kDa, also known as nanobodies. Camelids and sharks produce antibodies naturally devoid of light chains, which are referred to as heavy chain-only antibodies, or simply heavy chain antibodies (hcabs), the antigen binding fragments in each arm of the camelid heavy chain antibodies have a single heavy chain variable domain, known as heavy chain single domain antibodies (VHHs). The single domain antibody has similar or higher specificity and affinity as the traditional antibody, and has the natural advantages of good solubility, high stability, strong penetrating power and wide binding epitope, so that the single domain antibody is focused in the field of immunotherapy and is also applied to the treatment of malignant tumors, autoimmune diseases, anti-infection and other diseases.

Chimeric antigen receptor (Chimeric Antigen Receptor, CAR) modified T cells are widely used as an immunotherapeutic strategy in the treatment of many solid tumors, hematological tumors, and especially lymphocytic tumors. Chimeric antigen receptors are recombinant polypeptide constructs whose representative structure consists of four parts, an extracellular antigen binding domain (typically a single chain antibody with antigen recognition function), a hinge region, a transmembrane domain, and an intracellular signaling domain. Classical chimeric antigen receptor structures are generally classified into the first generation (without costimulatory molecules), the second generation (comprising one costimulatory molecule) and the third generation (comprising two costimulatory molecules) depending on whether or not the intracellular signaling domain is added to the costimulatory molecule and the amount added. The second generation chimeric antigen receptor structural designs are most commonly used by date in the market and clinical research stages. The principle of the chimeric antigen receptor is that through genetic engineering modification, T cells express a receptor structure (for example, a single-chain antibody) capable of specifically recognizing tumor cell surface antigens, and after the receptor is specifically combined with the tumor cell surface antigens, immune co-stimulatory molecules and T cells at the downstream of the receptor are activated, so that the restriction of a Major Histocompatibility Complex (MHC) is broken through, the tumor cells are directly specifically recognized and killed, and the purpose of targeted removal of malignant tumors is realized. Because the realization of the recognition and killing function requires the use of antibodies with good binding activity and high binding epitope efficiency, one of the keys to the success or failure of the CAR-T cell therapy is to screen high-affinity antibodies with good specificity, strong binding force and effective binding epitope.

Thus, there is still a broad need to screen and develop improved CD 7-targeting single domain antibodies, CD 7-targeting chimeric antigen receptors constructed using the same, and engineered immune effector cells. In particular, stable CD 7-targeting single domain antibodies suitable for more effective and more efficient CAR-T cell therapies were developed.

On the other hand, since CD7 antigen is expressed on the surface of T cells and thus CAR-T cells themselves have CD7 expression, the phenomenon of self-phase killing of CAR-T cells occurs during the preparation of CD 7-targeted CAR-T cells, resulting in difficulty in vitro expansion of the harvested CAR-T cells. To solve the problem of difficulty in vitro expansion of CD 7-targeting CAR-T cells caused by this "suicide" phenomenon, current solutions include:

(1) The endogenous CD7 expression of T cells on the cell surface was blocked by CD7 blockers as disclosed in related art schemes of patent CN 110760007B. The patent constructs a CD7 blocking vector capable of blocking the expression of CD7 on the surface of T cells, and prevents the expression of CD7 on the surface of the T cells by trapping CD7 protein on an endoplasmic reticulum and a Golgi apparatus, thereby avoiding the suicide phenomenon of CD 7-targeted CAR-T cells.

(2) New CD7 binding epitopes were sought, as disclosed in the related art schemes of patent CN 114716564B. This patent constructs CAR-T cells using a specific segment of the extracellular domain of SECTM1 (amino acid sequence at positions 29-145) as the extracellular binding domain of a chimeric antigen receptor. It is described that, although cell proliferation is affected to some extent during the production of the CAR-T cells, it is possible to successfully produce a large amount of desired CAR-T cells.

(3) The endogenous CD7 molecule of T cells was knocked out by gene editing methods, as disclosed in related art schemes of patent application CN112300282 a. This approach knocks out CD7 genes in T cells by the CRISPR/Cas9 system in order to avoid mutual killing between CAR-T cells.

The CRISPR/Cas system is a fast, efficient gene editing system, where CRISPR/Cas9 is one of the most widely used CRISPR/Cas systems that is capable of modifying, replacing and inserting DNA sequences efficiently and accurately. The mechanism by which the CRISPR/Cas9 system performs gene editing includes: CRISPR arrays homologous to specific target sequences (protospacer sequences) are transcribed into a number of pre CRISPR RNA (pre-crrnas) which form complexes with smaller transactivations crRNA (tracrRNA) by base pairing. This complex can bind to Cas9 protein, resulting in cleavage of longer pre-crrnas by rnase III, separating into separate crRNA/tracrRNA complexes. When the crRNA/tracrRNA directs the Cas9 complex to the target sequence, the crRNA binds to the target sequence following the Protospacer Adjacent Motif (PAM), the target sequence unwinds and is cleaved by the nuclease domains (RuvC and HNH) of the Cas9 protein, causing a double strand break. The two repair mechanisms of non-homologous end joining (NHEJ) or Homologous Recombination (HR) of the cell are activated, thereby realizing the knockout, insertion or modification of the gene. By bioengineering crRNA and tracrrRNA, the crRNA and tracrrRNA are combined into a guide RNA molecule (gRNA) with functions of crRNA and tracrrRNA. The gRNA is a short (about 20 nt) guide sequence at the 5' end, complementarily matches the target DNA sequence, and determines the specificity of directed cleavage of Cas9 protein. A part of the sequence of the gRNA can be combined with the Cas nuclease, the other part of the sequence of the gRNA can be complementary with a part of the sequence of the target gene, and the Cas nuclease can form a single-chain or double-chain notch at a specific site of the target gene by virtue of the recognition effect of the gRNA, so that the editing of the target gene is realized.

In recent years, the CRISPR/Cas9 system-based efficient and accurate gene editing function is widely applied to gene editing of mammalian cells, which also promotes great progress in tumor immunotherapy, and particularly greatly promotes preparation and application of chimeric antigen receptor T cells.

Disclosure of Invention

The invention aims at providing a single domain antibody targeting CD7, a CAR-T cell constructed by the single domain antibody and application thereof. The targeted CD7 single-domain antibody has a natural single-chain structure, has the advantages of small molecular weight, high solubility, high stability, low immunogenicity, high tissue permeability and no need of additional folding and assembling steps or linker optimization modification, is a promising alternative scheme of scFv single-chain antibodies with larger molecular weight, and the CAR-T cells constructed by using the single-domain antibodies have remarkable tumor cell killing capacity.

According to a first aspect of the present disclosure, there is provided a single domain antibody targeting CD 7. The single domain antibody targeting CD7 provided by the invention comprises CDR1, CDR2 and CDR3 regions; wherein CDR1 comprises any of the amino acid sequences shown in SEQ ID NO. 1, 2 or 3, wherein CDR2 comprises any of the amino acid sequences shown in SEQ ID NO. 4, 5 or 6, and wherein CDR3 comprises any of the amino acid sequences shown in SEQ ID NO. 7, 8 or 9.

In some embodiments, the CD 7-targeting single domain antibodies provided herein comprise CDR1, CDR2, and CDR3 regions; wherein CDR1 is any one of the amino acid sequences shown as SEQ ID NO. 1, 2 or 3, wherein CDR2 is any one of the amino acid sequences shown as SEQ ID NO. 4, 5 or 6, wherein CDR3 is any one of the amino acid sequences shown as SEQ ID NO. 7, 8 or 9.

In some embodiments, the CD 7-targeting single domain antibodies provided herein comprise CDR1, CDR2, and CDR3 regions; wherein CDR1, CDR2 and CDR3 comprise the amino acid sequences as shown in table 1.

In some embodiments, the CD 7-targeting single domain antibodies provided herein comprise CDR1, CDR2, and CDR3 regions; wherein CDR1, CDR2 and CDR3 are the amino acid sequences as shown in table 1.

In some embodiments, the CD 7-targeting single domain antibodies provided herein comprise CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 2, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 3.

In some embodiments, the present invention provides a CD 7-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 4, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 5, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 6.

In some embodiments, the present invention provides a CD 7-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 comprises the amino acid sequence shown as SEQ ID NO. 7, wherein CDR2 comprises the amino acid sequence shown as SEQ ID NO. 8, wherein CDR3 comprises the amino acid sequence shown as SEQ ID NO. 9.

In some embodiments, the present invention provides a CD 7-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 2, and wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 3.

In some embodiments, the present invention provides a CD 7-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 4, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 5, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 6.

In some embodiments, a CD 7-targeting single domain antibody of the invention comprises CDR1, CDR2, and CDR3 regions; wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 7, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 8, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 9.

The technical scheme disclosed by the invention, wherein the specific amino acid sequence information shown in SEQ ID NO 1-9 is shown in Table 1.

TABLE 1

SEQ ID NO:	Amino acid sequence
		1	GFTFPNQE(CDR1)
2	VSPSGNLR(CDR2)
		3	ARDRAGNY(CDR3)
4	GYTSRRSC(CDR1)
		5	IYTGGSST(CDR2)
6	AAHSRILCPGVAAREYDY(CDR3)
		7	GFSVRWNC(CDR1)
8	IGVSDIA(CDR2)
		9	AARRNRYCPAAFGQSDFNY(CDR3)

The single domain antibody for targeting CD7 provided by the invention comprises CDR1, CDR2 and CDR3 regions; wherein the determination of CDR1, CDR2 and CDR3 is according to any one of IMGT numbering scheme, kabat numbering scheme, abM numbering scheme, chothia numbering scheme or Contact numbering scheme. The single domain antibody for targeting CD7 provided by the invention comprises CDR1, CDR2 and CDR3 regions; wherein the determination of CDR1, CDR2 and CDR3 is according to the IMGT numbering scheme.

The single domain antibodies or CDR regions thereof provided herein encompass CDRs defined by the numbering scheme described above or other known numbering schemes. For example, it will be appreciated by those skilled in the art that CDR regions that are identified by any numbering scheme, as long as they are identical to the CDR regions of the present invention, include the CDR regions of the present invention, are within the scope of the present invention.

The present invention provides a single domain antibody targeting CD7 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of the amino acid sequences shown in table 2 or comprising SEQ ID NOs 10-12.

In some embodiments, the present invention provides a single domain antibody targeting CD7 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO 10.

In some embodiments, the present invention provides a single domain antibody targeting CD7 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 11.

In some embodiments, the present invention provides a single domain antibody targeting CD7 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 12.

The single domain antibody for targeting CD7 provided by the invention comprises any amino acid sequence shown in a table 2 or SEQ ID NO. 10-12.

In some embodiments, the CD 7-targeting single domain antibodies provided herein comprise an amino acid sequence as set forth in SEQ ID NO. 10. In some embodiments, the CD 7-targeting single domain antibodies provided herein comprise an amino acid sequence as set forth in SEQ ID NO. 11. In some embodiments, the CD 7-targeting single domain antibodies provided herein comprise the amino acid sequence shown as SEQ ID NO. 12.

The single domain antibody targeting CD7 provided by the invention is an amino acid sequence which has at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with any one of the amino acid sequences shown in Table 2 or SEQ ID NO 10-12.

In some embodiments, the present invention provides a single domain antibody targeting CD7 that is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO 10.

In some embodiments, the present invention provides a single domain antibody targeting CD7 that is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 11.

In some embodiments, the CD 7-targeting single domain antibodies provided herein are amino acid sequences that have at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 12.

The single domain antibody for targeting CD7 provided by the invention is any amino acid sequence shown in table 2 or SEQ ID NO. 10-12.

In some embodiments, the CD 7-targeting single domain antibody provided by the present invention is an amino acid sequence as shown in SEQ ID NO. 10. In some embodiments, the CD 7-targeting single domain antibody provided by the present invention is an amino acid sequence as shown in SEQ ID NO. 11. In some embodiments, the CD 7-targeting single domain antibody provided by the present invention is an amino acid sequence as shown in SEQ ID NO. 12.

The technical scheme disclosed by the invention, wherein the specific amino acid sequence information shown in SEQ ID NO 10-12 is shown in Table 2.

TABLE 2

According to a second aspect of the present disclosure, there is provided a chimeric antigen receptor comprising a CD 7-targeting single domain antibody of the present invention.

In some embodiments, the chimeric antigen receptor provided herein comprises an extracellular antigen binding domain comprising a single domain antibody that targets CD7 as described herein.

In some embodiments, the chimeric antigen receptor provided herein can further comprise one or more of the following structures: a linker (e.g., a peptide linker), a signal peptide, a hinge region, a transmembrane domain, a costimulatory signaling domain, and an intracellular signaling domain.

In some embodiments, the present invention provides chimeric antigen receptors comprising:

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises a CD 7-targeting single domain antibody of the first aspect of the disclosure.

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises a CD 7-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions, wherein CDR1, CDR2, and CDR3 regions comprise CDR1, CDR2, and CDR3 regions of a CD 7-targeting single domain antibody according to the first aspect of the present disclosure.

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises a CD 7-targeting single domain antibody comprising CDR1, CDR2, and CDR3 regions, wherein the CDR1, CDR2, and CDR3 regions are the CDR1, CDR2, and CDR3 regions of a CD 7-targeting single domain antibody according to the first aspect of the present disclosure.

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises a single domain antibody that targets CD 7; wherein the single domain antibody comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of the amino acid sequences shown in SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12.

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises a single domain antibody that targets CD 7; wherein the single domain antibody comprises any amino acid sequence shown as SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12.

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises a single domain antibody that targets CD 7; wherein the single domain antibody is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of the amino acid sequences shown in SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12.

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen-binding domain comprises a single domain antibody that targets CD 7; wherein, the single domain antibody is any amino acid sequence shown as SEQ ID NO. 10, SEQ ID NO. 11 or SEQ ID NO. 12.

In some embodiments, the present invention provides chimeric antigen receptors wherein the transmembrane domain is derived from CD8 a, CD28, cd3ζ, cd3γ, cd3δ, cd3ε, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, tcra, tcrβ, tcrγ, tcrδ, tcrζ, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, PD-1, or any combination thereof.

In some embodiments, the present invention provides chimeric antigen receptors in which the transmembrane domain is derived from CD8 a. In some embodiments, the present invention provides chimeric antigen receptors in which the transmembrane domain comprises the amino acid sequence shown as SEQ ID NO. 15. In some embodiments, the present invention provides chimeric antigen receptors wherein the transmembrane domain is the amino acid sequence shown as SEQ ID NO. 15.

In some embodiments, the present invention provides chimeric antigen receptors in which the intracellular signaling domain comprises the primary intracellular signaling domain of an immune effector cell (e.g., T cell). In some embodiments, the intracellular signaling domain is derived from fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD22, CD79a, CD79b, CD66d, or any combination thereof.

In some embodiments, the present invention provides chimeric antigen receptors in which the intracellular signaling domain is derived from cd3ζ. In some embodiments, the present invention provides chimeric antigen receptors in which the intracellular signaling domain comprises the amino acid sequence shown as SEQ ID NO. 17. In some embodiments, the present invention provides chimeric antigen receptors in which the intracellular signaling domain is the amino acid sequence shown as SEQ ID NO. 17.

In some embodiments, the invention provides chimeric antigen receptors wherein the intracellular signaling domain further comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from an MHC class I molecule, BTLA, and Toll ligand receptor. In some embodiments, the costimulatory signaling domain is derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX 40), CD2, CD7, CD27, CD28, CD30, CD40, CD83, ICAM, 4-1BB (CD 137), CD276 (B7-H3), CD278 (ICOS), GITR, LIGHT, HVEM (light), BTLA, CD8 a, LFA-1, NKG2C, LAT, SLP-76, DAP10, PD-1, TRIM, ZAP70 ligand, or any combination thereof.

In some embodiments, the present invention provides chimeric antigen receptors wherein the costimulatory signaling domain is derived from 4-1BB (CD 137). In some embodiments, the present invention provides chimeric antigen receptors in which the costimulatory signaling domain comprises the amino acid sequence depicted as SEQ ID NO. 16. In some embodiments, the present invention provides chimeric antigen receptors in which the costimulatory signaling domain is the amino acid sequence depicted as SEQ ID NO. 16.

In some embodiments, the chimeric antigen receptor provided herein further comprises a hinge region located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

In some embodiments, the present invention provides chimeric antigen receptors wherein the hinge region is derived from CD8 a, CD28, CD137, igG4, or IgG1, or any combination thereof.

In some embodiments, the invention provides chimeric antigen receptors in which the hinge region is derived from CD8 a. In some embodiments, the present invention provides chimeric antigen receptors in which the hinge region comprises the amino acid sequence set forth in SEQ ID NO. 14. In some embodiments, the present invention provides chimeric antigen receptors wherein the hinge region is the amino acid sequence shown as SEQ ID NO. 14.

In some embodiments, the chimeric antigen receptor provided herein further comprises a signal peptide located at the N-terminus of the chimeric antigen receptor polypeptide.

In some embodiments, the present invention provides chimeric antigen receptors wherein the signal peptide is derived from HLA-A, CD8 a, CD4, CD33, CD137, GM-CSFR a, igG1, ig kappa, IL-2, or any combination thereof.

In some embodiments, the present invention provides chimeric antigen receptors wherein the signal peptide is derived from CD8 a.

In some embodiments, the present invention provides chimeric antigen receptors wherein the signal peptide is derived from HLA-A. In some embodiments, the present invention provides chimeric antigen receptors in which the signal peptide comprises the amino acid sequence shown as SEQ ID NO. 13. In some embodiments, the present invention provides chimeric antigen receptors wherein the signal peptide is the amino acid sequence shown as SEQ ID NO. 13.

The technical scheme disclosed by the invention, wherein the specific amino acid sequence information shown in SEQ ID NO 13-17 is shown in Table 3.

TABLE 3 Table 3

In some embodiments, the chimeric antigen receptor polypeptides provided herein comprise, in order from N-terminus to C-terminus: HLA-A signal peptide or CD8 a signal peptide, extracellular antigen binding domain, CD8 a hinge region, CD8 a transmembrane domain, costimulatory signal domain derived from 4-1BB (CD 137) and intracellular signaling domain derived from cd3ζ.

The chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to an amino acid sequence shown in Table 4 or comprising an amino acid sequence shown in SEQ ID NOS.18-20.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 18.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 19.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 20.

The chimeric antigen receptor provided by the invention comprises an amino acid sequence shown in table 4 or SEQ ID NO. 18-20.

In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 18. In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 19. In some embodiments, the chimeric antigen receptor provided by the invention comprises an amino acid sequence as set forth in SEQ ID NO. 20.

The chimeric antigen receptor provided by the invention is an amino acid sequence which has at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity with the amino acid sequence shown in Table 4 or with the amino acid sequences shown in SEQ ID NOS.18-20.

In some embodiments, the chimeric antigen receptor provided herein is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 18.

In some embodiments, the chimeric antigen receptor provided herein is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 19.

In some embodiments, the chimeric antigen receptor provided herein is an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to the amino acid sequence set forth in SEQ ID NO. 20.

The chimeric antigen receptor provided by the invention is an amino acid sequence shown in table 4 or SEQ ID NO. 18-20.

In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 18. In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 19. In some embodiments, the chimeric antigen receptor provided by the invention is an amino acid sequence as shown in SEQ ID NO. 20.

The technical scheme disclosed by the invention, wherein the specific amino acid sequence information shown in SEQ ID NO 18-20 is shown in Table 4.

TABLE 4 Table 4

According to a third aspect of the present disclosure there is provided an isolated nucleic acid encoding a chimeric antigen receptor according to the present invention.

In some embodiments, the nucleic acids provided herein are in the form of DNA or RNA. In some embodiments, the DNA provided by the present invention includes cDNA, genomic DNA, or synthetic DNA. In some embodiments, the DNA provided by the invention is single-stranded or double-stranded DNA. In some embodiments, the DNA provided by the invention is coding strand or non-coding strand DNA.

The isolated nucleic acids provided by the invention comprise the nucleic acid sequences shown in Table 5 or as shown in SEQ ID NOS.21-23.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 21.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 22.

In some embodiments, the isolated nucleic acids provided herein comprise a nucleic acid sequence as set forth in SEQ ID NO. 23.

The technical scheme disclosed by the invention, wherein specific nucleic acid sequence information shown in SEQ ID NOs 21-23 is shown in Table 5.

TABLE 5

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According to a fourth aspect of the present disclosure there is provided a vector comprising an isolated nucleic acid according to the present invention.

In some embodiments, the vector is suitable for replication and integration in eukaryotic cells, such as mammalian cells. In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentiviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof.

In some embodiments, the vector is comprised in a host cell.

In some embodiments, the host cell is a killer cell.

In some embodiments, the killer cell is a T cell or an NK cell.

In some embodiments, the NK cells are primary NK cells.

In some embodiments, the T cells are peripheral blood T lymphocytes, umbilical cord blood T lymphocytes.

According to a fifth aspect of the present disclosure, there is provided an engineered immune effector cell comprising a chimeric antigen receptor or an isolated nucleic acid of the present invention.

In some embodiments, the engineered immune effector cells provided herein may be selected from T cells, B cells, NK cells, macrophages, dendritic cells, immune effector cells differentiated from induced pluripotent stem cells (ipscs), or any combination thereof.

In some embodiments, the engineered immune effector cells provided herein are T cells. For example, the T cells may be selected from CD4+/CD8+ T cells, CD4+/CD8-T cells, CD4-/CD8+ T cells, CD4-/CD8-T cells, CD4+ helper T cells (e.g., th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, γδ -T cells, αβ -T cells, NKT cells, DNT cells (double negative T cells).

In some embodiments, the T cell is a CD4+/CD8-T cell, a CD4-/CD8+ T cell, a CD4+/CD8+ T cell, a CD4-/CD8-T cell, or a combination thereof.

In some embodiments, T cells produce cytokines such as IL-2, IFNγ, and/or TNF- α after expressing the chimeric antigen receptor and binding to target cells (e.g., CD7+ tumor cells). In some embodiments, cd8+ T cells lyse antigen-specific target cells after expression of the chimeric antigen receptor and binding to the target cells.

In some embodiments, the immune effector cell is an NK cell. In some embodiments, the immune effector cell may be an established cell line, such as NK-92 cells.

In some embodiments, the immune effector cells may be differentiated from stem cells, e.g., from ipscs.

In some embodiments, the immune effector cells provided herein have their endogenous CD7 gene knocked out, silenced or inhibited.

In some embodiments, the present invention provides T cells in which the endogenous CD7 gene is knocked out, silenced or inhibited.

In some embodiments, the invention provides an immune effector cell with endogenous CD7 gene is knocked out by a homing endonuclease system, a zinc finger nuclease (zinc finger nucleases, ZFNs) system, a transcription activator-like effector nuclease (transcription activator like effector nucleases, TALENs) system, or a regularly repeated short palindromic sequence cluster (clustered regularly interspaced short palindromic repeats, CRISPR) system.

In some embodiments, the endogenous CD7 gene of a T cell provided herein is knocked out by a homing endonuclease system, a Zinc Finger Nuclease (ZFNs) system, a transcription activator-like effector nuclease (TALENs) system, or a regularly repeated short palindromic sequence Cluster (CRISPR) system.

In some embodiments, the invention provides immune effector cells of endogenous CD7 gene is knocked out by CRISPR/Cas9 system. In some embodiments, the endogenous CD7 gene of the T cell provided herein is knocked out by the CRISPR/Cas9 system.

In some embodiments, the invention provides a method of preparing a T cell in which an endogenous CD7 gene is knocked out, comprising the steps of:

(1) Providing a human primary T cell;

(2) Introducing an agent into a human primary T cell, wherein the agent comprises a gRNA and a Cas9 nuclease, wherein the gRNA comprises two portions of a crRNA and a tracrRNA, and the crRNA comprises a nucleic acid sequence set forth in SEQ ID No. 24 or 25;

(3) Culturing and amplifying the T cells obtained in the step (2).

In some embodiments, the agent is in the form of a Ribonucleoprotein (RNP) complex.

In some embodiments, the introduction of the agent is by electrotransfection.

In some embodiments, the human primary T cells are peripheral blood-derived T cells or umbilical cord blood-derived T cells.

In some embodiments, the method of preparing a T cell in which the endogenous CD7 gene is knocked out further comprises activating the T cell prior to introducing the agent into the human primary T cell.

The CRISPR/Cas system can be delivered into cells by any method known in the art, for example, by directly applying the system to human cells by transfection with plasmids encoding Cas and gRNA, or using viral vectors. Adenovirus Vectors (AAV) are commonly selected for in vivo gene delivery in somatic gene therapy. CRISPR delivery by Cas and gRNA Ribonucleoprotein (RNP) complexes also showed efficient gene editing in human cells. Viral vectors for CRISPR/Cas systems include adenoviruses, lentiviruses, and phages; non-viral vectors include liposomes such as Lipofectamine ^TM Polymer, gold nanoparticles. Physical methods of delivery of CRISPR/Cas gene editing systems include electroporation, microinjection, and the like. Electroporation techniques allow chemicals, drugs, DNA, RNA, or proteins to be introduced into cells by applying an electric field to the cells to increase the permeability of the cell membrane. And electroporation has been shown to be a highly efficient, low toxicity delivery modality due to the low transduction efficiency of viral (including lentivirus, retrovirus or adenovirus) transduced T cells and their presence of toxicity associated with prolonged expression of CRISPR/CasFor use in the delivery of CRISPR/Cas systems.

The term "CRISPR system", "Cas system" or "CRISPR/Cas system" as used herein includes RNA-guided nucleases or other effector molecules and guide RNA molecules (gRNA). CRISPR/Cas systems direct nucleases or other effector molecules onto targeting sequences through guide RNAs to effect modification of nucleic acids. The CRISPR/Cas system can include a guide RNA molecule and a Cas protein, such as a Cas9 protein. The guide RNA molecule and Cas protein may form a ribonucleic acid protein (RNP) complex. The Cas protein has the function of cutting a DNA double strand, the gRNA plays a guiding role, under the condition that a Protospacer Adjacent Motif (PAM) exists, the Cas protein can reach different target positions under the guiding role of the gRNA, cut target genes, realize DNA double strand break, activate non-homologous end-connection (NHEJ) to achieve the purpose of gene knockout, or create sites into which exogenous donor DNA is possibly inserted through a Homologous Directional Repair (HDR) mechanism, so as to realize gene insertion. CRISPR/Cas systems include two classes, class 1 can use the effector of a complex formed by multiple Cas proteins to cleave an exogenous nucleic acid and class 2 can use a single large Cas protein for the effector of cleaving an exogenous nucleic acid. Class 1 can be classified as type I, type III and type IV, and class 2 can be classified as type II, type V and type VI. These 6 system types can be further divided into 19 subtypes. The Cas9 protein may be a corresponding Cas9 protein isolated from streptococcus pyogenes (SpCas 9), staphylococcus aureus (SaCas 9), streptococcus thermophilus (StCas 9), neisseria meningitidis (NmCas 9), new francisus (FnCas 9) or campylobacter jejuni (CjCas 9). In some embodiments, the Cas protein is a Cas variant derived from a wild-type Cas protein. An exemplary Cas variant may be a high fidelity Cas protein. In some embodiments, the Cas protein variant has at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 amino acid mutations from its wild-type sequence. In some embodiments, the Cas variant may be fused to another enzyme, such as an activation-induced cytidine deaminase (AID).

The term "gRNA" or "guide RNA" as used herein refers to a synthetic or recombinant polynucleotide that is specific for a targeting sequence and can direct Cas protein recognition and cleavage of a target DNA sequence. The gRNA molecule can comprise multiple domains, for example, two domains: (1) A domain sharing homology with the targeting sequence (e.g., capable of guiding Cas9 protein to the target); and (2) a domain that binds to Cas protein. In some embodiments, the gRNA comprises two or more domains (1) and (2) and may be referred to as an "extended gRNA," which will bind to two or more Cas proteins and to the targeting sequence at two or more different regions; in some embodiments, the gRNA molecule comprises a targeting domain and interacts with a Cas enzyme molecule, e.g., cas9, or with another RNA-guided endonuclease, e.g., cpf 1. In some embodiments, the gRNA molecule comprises a crRNA domain (including a targeting domain) and a tracrRNA domain. The portion corresponding to the crRNA is responsible for recognizing the targeting sequence and the portion corresponding to the tracrRNA is responsible for binding to Cas protein, e.g., cas9 protein. The guiding of nucleases is achieved by hybridization of one part of the gRNA (e.g. crRNA) to DNA and the other part (e.g. tracrRNA) to the corresponding nuclease or other effector molecule. By combining these two parts into a single RNA molecule, a single stranded gRNA or unidirectional guide gRNA (sgRNA) can be formed.

The two parts, crRNA and tracrRNA, are provided on separate nucleic acid molecules, which are themselves capable of binding, typically by means of hybridization, to form a gRNA molecule. The crRNA and tracrRNA may also be linked by a chemical linker that is not a nucleic acid. In some embodiments, the gRNA includes a spacer sequence and a scaffold sequence. The scaffold sequence may comprise a hairpin structure. In some embodiments, the scaffold sequence is located downstream of the spacer sequence. In some embodiments, the scaffold sequence comprises a tracr sequence. In some embodiments, the scaffold sequence comprises one tracr sequence and one tracr mate sequence. Scaffold sequences are well known in the art and are available from commercial sources as disclosed in US20140356958A and US11261439B 2. The gRNA may be in a two-part composition (e.g., separate crRNA and tracrRNA, which may be hybridized together) or a complex of only one part (e.g., a crRNA-tracrRNA complex, or sgRNA).

The term "proto-spacer sequence adjacent motif (PAM)" or "PAM-like motif" as used herein refers to 2 to 6 base pairs immediately downstream of the targeting sequence targeted by the Cas protein in the CRISPR system. PAM sequences may be located 3-4 nucleic acids downstream of the cleavage site. Cas proteins require PAM sequences to recognize target sequences. After pairing of the gRNA with the targeting sequence, the Cas protein mediates a double strand break of about 3-nt upstream of the PAM. The PAM sequence may be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NAG, NGN, NGT, NGTT, NGCG, NGAG, NGAN, NGNG, NGCN, NGTN, NNGRRT, NNNRRT, NGGNG, NNGRR (N), TTTV, TYCV, TATV, NNNNGATT, NNAGAAW, or NAAAA, where N is any nucleobase; r is a purine; y is pyrimidine; w is A or T. The PAM sequence of Cas9 may be NGG, e.g., cas variants may recognize modified PAM sequences, such as NGG, NGAN, NGNG, NGAG, NGCG, NNG, TTTN, YTN or YG.

According to a sixth aspect of the present disclosure, there is provided a pharmaceutical composition comprising a CD7 single domain antibody, chimeric antigen receptor, engineered immune effector cell according to the present invention, and one or more pharmaceutically acceptable excipients and/or carriers.

Excipients and/or carriers are also commonly referred to as adjuvants. Pharmaceutically acceptable excipients and/or carriers include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, anti-adherent agents, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, wetting agents, preservatives, emulsifiers, coating agents, isotonicity agents, absorption delaying agents, stabilizers and tonicity adjusting agents, diluents, solubilizers, emulsifiers, preservatives and/or adjuvants. Those skilled in the art will appreciate that pharmaceutically acceptable excipients and/or carriers are non-toxic or substantially non-toxic to recipients at the dosages and concentrations employed, are pharmacologically and/or physiologically compatible with the active ingredients of the present invention, e.g., do not affect their viability or efficacy. In some embodiments, the pharmaceutical composition may contain substances for improving, maintaining or retaining, for example, pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. The optimal pharmaceutical composition can be determined depending on the intended route of administration, the mode of delivery and the dosage required. Other examples of pharmaceutically acceptable excipients are described in Remington and Gennaro, remington's Pharmaceutical Sciences (18 th edition, 1990). Excipients and/or carriers that may be used in the present invention include, but are not limited to: water; brine; buffers such as neutral buffered saline or sulfate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol, etc.; glycerol; a protein; polypeptides or amino acids such as glycine and the like; an antioxidant; chelating agents such as EDTA or glutathione and the like; ethanol; a polyol, and one or more thereof.

In some embodiments, the pharmaceutical compositions of the present invention are provided in the form of a sterile formulation. Sterilization is achieved by filtration through sterile filtration membranes. In lyophilizing a composition, the method may be used to sterilize prior to or after lyophilization, reconstitution, dilution. Sterile formulations such as isotonic aqueous solutions, suspensions, emulsions, dispersions and the like. In some embodiments, the compositions for parenteral administration may be stored in lyophilized form or in solution. For example, by using physiological saline or an aqueous solution containing glucose and other auxiliary agents by conventional methods. Parenteral compositions are typically placed in a container having a sterile access port, such as an intravenous solution bag or vial having a needle-penetratable stopper. Alternatively, the composition may be selected for inhalation or delivery through the digestive tract (such as orally). The preparation of the pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will also be apparent to those skilled in the art, including formulations comprising antibodies or engineered immune effector cells in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible particles or porous beads, and depot injections, are also well known to those skilled in the art. Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, viscous compositions, solids, crystals, or as lyophilized powders. The formulation may be stored in a ready-to-use form or reconstituted for use in a treated form prior to administration.

The pharmaceutical composition may be used in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The pharmaceutical compositions of the invention may be injected, for example, orally, nasally, intravenously, intraperitoneally, intracerebrally (intraparenchymally), intracerebroventricular, intramuscularly, intraocularly, intraarterially, portal vein or intralesionally, and may also be administered by a sustained release system or by an implanted device. In some embodiments, administration is accomplished parenterally. Parenteral delivery methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

The therapeutically effective amount of the pharmaceutical composition comprising the CD 7-targeting single domain antibodies or engineered immune effector cells of the invention to be employed will depend, for example, on the extent of treatment and the goal. Those skilled in the art will appreciate that the appropriate dosage level for treatment will vary depending in part on the molecule delivered, the indication, the route of administration, and the patient's condition (body weight, body surface or organ size) and/or condition (age and general health). In certain embodiments, the clinician may titrate the dose and alter the route of administration to obtain the optimal therapeutic effect.

The frequency of administration will depend on the pharmacokinetic parameters of the CD 7-targeting single domain antibody or the engineered immune effector cells in the formulation used. The clinician typically administers the pharmaceutical composition until a dose is reached that achieves the desired effect. The pharmaceutical composition may thus be administered as a single dose, or over time as two or more doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion through an implanted device or catheter.

According to a seventh aspect of the present disclosure, the present invention provides the use of a CD 7-targeting single domain antibody, chimeric antigen receptor, engineered immune effector cell, or pharmaceutical composition as described previously, in the manufacture of a medicament for the diagnosis, prevention and/or treatment of a disease or disorder.

The invention also provides a method of treating a subject having a disease associated with CD7 expression comprising administering to the subject an effective amount of an engineered immune effector cell or pharmaceutical composition according to the invention. Accordingly, the present invention provides the use of a CD 7-targeting single domain antibody, chimeric antigen receptor, engineered immune effector cell, or pharmaceutical composition of the invention in the manufacture of a medicament for the diagnosis, prevention and/or treatment of a disease or disorder.

In some embodiments, the disease or disorder comprises a disease or disorder associated with CD7 expression.

In some embodiments, the disease or disorder associated with CD7 expression comprises lymphoma or leukemia.

In some embodiments, the disease or disorder comprises T cell lymphoma, T cell leukemia, B cell leukemia, or NK cell lymphoma.

In some embodiments, the disease or disorder comprises T cell non-hodgkin's lymphoma.

In some embodiments, the disease or disorder comprises T lymphoblastic lymphoma (T-LBL), peripheral T-cell lymphoma (PTCL), acute T-lymphoblastic leukemia (T-ALL), T-lymphoblastic leukemia, acute Myeloid Leukemia (AML), or Chronic Lymphoblastic Leukemia (CLL).

The single domain antibodies, nanobodies or heavy chain antibodies of the invention can be prepared using methods conventional in the art, such as phage display techniques well known in the art. Alternatively, the various antibodies of the invention may be expressed in other cell lines. Suitable mammalian host cells may be transformed with sequences encoding the various antibodies of the invention. Transformation may be performed using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of DNA into the nucleus, etc. Host mammalian cell lines useful for expression are well known in the art, for example, a variety of immortalized cell lines available from the American Type Culture Collection (ATCC) including, but not limited to, chinese Hamster Ovary (CHO) cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma (HepG 2) cells, and the like. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and producing antibodies with substantial CD7 binding properties.

Chimeric antigen receptors of the invention can be prepared using methods conventional in the art, see, for example, park et al, trends Bio technol.,29:550-557,2011; grupp et al, N Engl J med 368:1509-1518,2013; han et al, J.Hematol.Oncol.,6:47,2013.

As is well known to those skilled in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids can be made, all of which encode the chimeric antigen receptor of the present invention. Thus, where a particular amino acid sequence has been identified, one of skill in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence encoding the protein. Thus, the present invention also relates to polynucleotides which hybridize to the above polynucleotide sequences and which have at least 70%, preferably at least 80%, more preferably at least 90% identity between the two sequences. The invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention.

The full-length nucleic acid sequences of the various chimeric antigen receptors of the invention or fragments thereof can be obtained generally by PCR amplification, recombinant methods or synthetic methods. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.

The invention also relates to nucleic acid constructs, such as expression vectors and recombinant vectors, comprising the above-described nucleic acid sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein. Vectors typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a leader sequence encoding for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, a multiple linker region for inserting a nucleic acid encoding an antibody to be expressed, and selectable marker elements.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, polypeptides, etc.) to which the present invention relates include biomolecules that exist in isolated form. At present, it is entirely possible to obtain DNA sequences encoding the polypeptides of the invention (or fragments or derivatives thereof) by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can be introduced into the polypeptide sequences of the invention by chemical synthesis.

In some embodiments, the nucleic acid encoding the chimeric antigen receptor is operably linked to a constitutive promoter. Constitutive promoters allow for constitutive expression of a heterologous gene (also referred to as a transgene) in a host cell. Exemplary constitutive promoters of the invention include, but are not limited to, the Cytomegalovirus (CMV) promoter, the human elongation factor-1 alpha (hEF 1 alpha) promoter, the ubiquitin C (Ubic) promoter, the phosphoglycerate kinase (PGK) promoter, the simian virus 40 (SV 40) early promoter, and the chicken beta-actin coupled to CMV early enhancer (CAGG) promoter. The efficiency of such constitutive promoters in driving transgene expression has been widely compared in a number of studies. For example Michael C.Milone et al (Molecular Therapy,17 (8): 1453-1464 (2009)) compared the efficiencies of CMV, hEF 1. Alpha., ubic and PGK in driving chimeric antigen receptor expression in human primary T cells and concluded that the hEF 1. Alpha. Promoter not only induced the highest levels of transgene expression, but was also optimally maintained in CD4 and CD8 human T cells. In some embodiments, the nucleic acid encoding the chimeric antigen receptor is operably linked to the hef1α promoter.

In some embodiments, the nucleic acid encoding the chimeric antigen receptor is operably linked to an inducible promoter. Inducible promoters are among the regulatory promoters. Inducible promoters may be induced by one or more conditions, e.g., physical conditions, inducers, etc.

In some embodiments, the induction conditions induce expression of an endogenous gene in the engineered mammalian cell and/or in a subject receiving the pharmaceutical composition. In some embodiments, the induction conditions are selected from: inducer, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox status, tumor environment, and activation status of the engineered mammalian cells.

Viral vector techniques are well known in the art and are described in Sambrook et al Molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory Press, cold Spring harbor (2001) ([ molecular cloning: laboratory Manual ], cold Spring harbor Laboratory, new York Cold Spring harbor, 2001), and other virology and molecular biology manuals.

Many virus-based systems have been developed in the art for transferring genes into mammalian cells. The heterologous nucleic acid can be inserted into the vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated in vitro or ex vivo and delivered to engineered mammalian cells. Many retroviral systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art. In some embodiments, self-inactivating lentiviral vectors are used. For example, self-inactivating lentiviral vectors carrying an immunomodulatory agent (e.g., immune checkpoint inhibitor) coding sequence and/or self-inactivating lentiviral vectors carrying a chimeric antigen receptor may be packaged using protocols known in the art. In some embodiments, lentiviral vectors are used. The resulting lentiviral vector may be used to transduce mammalian cells (e.g., primary human T cells) using methods known in the art.

The nucleic acid may be cloned into a vector using any molecular cloning method known in the art, including, for example, using restriction endonuclease sites and one or more selection markers. In some embodiments, the nucleic acid is operably linked to a promoter. The prior art has disclosed a variety of promoters for gene expression in mammalian cells, and any promoter known in the art may be used in the present invention. Promoters can be further classified as constitutive or regulated, e.g., inducible.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ The process is carried out using procedures well known in the art. Another approach is to use MgCl ₂ . Alternatively, transformation may be performed by electroporation. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the nucleic acid molecule of the present invention. The medium used in the culture may be selected from various conventional media, such as a serum-containing medium or a serum-free medium, depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods.

The engineered immune effector cells of the invention are prepared by introducing a chimeric antigen receptor into an immune effector cell (e.g., T cell).

Nucleic acid sequences encoding chimeric antigen receptors can be introduced into immune effector cells using conventional methods known in the art (e.g., by transfection, transduction, transformation, etc.).

Methods for introducing vectors or isolated nucleic acids into immune effector cells are known in the art. The vectors described can be transferred into immune effector cells by physical, chemical or biological means.

Physical methods for introducing the vector into immune effector cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for preparing cells comprising vectors and/or exogenous nucleic acids are well known in the art and are described, for example, in Sambrook, J., fritsch, E.F. and Maniatis, T. (2001) Molecular Cloning: A Laboratory Manual Spring Harbor Laboratory Press, cold Spring harbor, and other virology and molecular biology manuals. In some embodiments, the vector is introduced into the immune effector cell by electroporation.

Biological methods for introducing vectors into immune effector cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian cells (e.g., human cells).

Chemical methods for introducing the carrier into immune effector cells include colloidal dispersion systems, including, for example, macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including, for example, oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome.

In some embodiments, the vector further comprises a selectable marker gene or reporter gene to select cells expressing the chimeric antigen receptor from a population of host cells transfected with the lentiviral vector. The selectable marker and the reporter gene may be flanked by appropriate regulatory sequences for expression in the host cell. For example, the vector may contain transcription and translation terminators, initiation sequences, and promoters for regulating expression of the nucleic acid sequences.

Reporter genes can be used to identify potentially transfected cells and to evaluate the function of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue and which encodes a polypeptide that expresses some readily detectable property, such as enzymatic activity. The expression of the reporter gene is determined at a suitable time after introduction of the DNA into the recipient cell. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein (see Kumiko Ui-Tei. FEBS Letters,479:79-82 (2000)). Suitable expression systems are known in the art and may be prepared or commercially available using known techniques. Other methods of confirming the presence of a nucleic acid encoding a CAR in an engineered immune effector cell include: molecular biological testing methods well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assay methods, such as detecting the presence or absence of a particular peptide; immunological methods such as ELISA.

In some embodiments, nucleic acid molecules encoding any of the CARs described herein can be prepared by conventional methods (e.g., in vitro transcription), and then introduced into immune effector cells by known methods such as mRNA electroporation (see Peter M rabinovich. Human Gene Therapy,17:1027-1035 (2006)).

In some embodiments, the transduced or transfected immune effector cells are propagated in vitro following introduction of the vector or isolated nucleic acid. In some embodiments, transduced or transfected immune effector cells are cultured to proliferate for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 14 days. In some embodiments, the transduced or transfected immune effector cells can be further evaluated or screened to select for engineered immune effector cells.

Interpretation of the terms

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

The term "sequence identity" as used herein refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at identical positions in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies with identical sequences have 100% identity. Those skilled in the art know that some algorithms can be used to determine sequence identity, such as Blast (Altschul et al (1997) Nucleic acids Res.25:3389-3402), blast2 (Altschul et al (1990) J.mol.biol.215:403-410), smith Waterman (Smith et al (1981) J.mol.biol.147:195-197), and Clustal W. In addition, sequence analysis software may be used to perform the determination, such as computer programs BLAST, and in particular BLASTP or TBLASTN, using default parameters.

The term "derived from" as used herein refers to a relationship between the two, generally referring to structural similarity between the two. For example, in the case of an intracellular signaling domain derived from cd3ζ, the intracellular signaling domain retains sufficient cd3ζ structure such that it has the desired function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include limitations on the particular process by which the intracellular signaling domain is generated, e.g., it does not mean that in order to provide the intracellular signaling domain, unwanted sequences must be started from the cd3ζ sequence and deleted, or mutations imposed, to reach the intracellular signaling domain.

The term "antibody" as used herein refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. Including, for example, monoclonal antibodies (including intact antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single chain molecules, and antibody fragments or synthetic polypeptides, particularly antigen-binding fragments, such as Fab, F (ab') 2, and Fv, bearing one or more CDR sequences, which are capable of exhibiting the desired biological activity. In some embodiments of the invention, the terms "immunoglobulin (Ig)" and "antibody" are used interchangeably.

"variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are typically the most variable parts of an antibody (relative to other antibodies of the same type) and contain antigen binding sites.

Heavy chain antibodies are antibodies derived from camelidae or cartilaginous fish organisms. In contrast to 4-chain antibodies, heavy-chain antibodies lack the light and heavy chain constant region 1 (CH 1), comprising only 2 heavy chains consisting of variable regions (VHH) linked to the constant region by a hinge-like structure and other constant regions. Each heavy chain of a camelidae heavy chain antibody comprises 1 variable region (VHH) and 2 constant regions (CH 2 and CH 3), and each heavy chain of a cartilaginous fish heavy chain antibody comprises 1 variable region and 5 constant regions (CH 1-CH 5). Antigen binding fragments of heavy chain antibodies include VHH and single chain heavy chain antibodies. Heavy chain antibodies can have CH2 and CH3 of human IgG Fc by fusion to the constant region of human IgG Fc.

The term "antigen" or "Ag" as used herein refers to a molecule that causes an immune response. The immune response may involve antibody production or activation of specific immunocompetent cells or both. The skilled artisan will appreciate that virtually any macromolecule, including all proteins or peptides, can act as an antigen. Furthermore, the antigen may be derived from recombinant or genomic DNA. The skilled artisan will appreciate that any DNA comprising a nucleotide sequence or portion of a nucleotide sequence encoding a protein that elicits an immune response, thus encodes an "antigen". The antigen may be synthetically produced or may be derived from a biological sample or may be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or fluids having other biological components.

The term "antigen-binding fragment" or "antibody fragment" as used herein refers to at least a portion of an intact antibody or recombinant variant thereof, typically comprising the antigen-binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments include, but are not limited to: fab, scFv, heavy chain variable region (VH) of an antibody, linear antibodies, single domain antibodies, nanobodies, natural ligands of an antigen or functional fragments thereof, and the like.

The term "functional variant" or "functional fragment" as used herein refers to a variant that substantially comprises the amino acid sequence of a parent but that contains at least one amino acid modification (e.g., substitution, deletion, or insertion) as compared to the parent amino acid sequence, provided that the variant retains the biological activity of the parent amino acid sequence. In some embodiments, the amino acid modification is a conservative modification.

Single domain antibodies (sdabs) may have the same or different sources, and have the same or different sizes. Exemplary sdabs include, but are not limited to, heavy chain variable domains (e.g., VHH) from heavy chain-only antibodies, binding molecules that are naturally devoid of light chains, single domains (e.g., VH or VL) derived from conventional 4-chain antibodies, human single domain antibodies produced by transgenic mice or rats expressing human heavy chain fragments, and engineering domains and single domain scaffolds that are not derived from antibodies. Any sdAb known in the art or disclosed by the invention, including the single domain antibodies disclosed herein, can be used to construct a CAR described herein. The sdAb may be derived from any species, including but not limited to mouse, rat, human, camel, llama, lamprey, shark, goat, rabbit, and cow. The single domain antibodies of the invention also include naturally occurring single domain antibody molecules from species other than camelidae and shark.

In the present invention, "single domain antibody", "single domain antibody targeting CD 7", "heavy chain single domain antibody", "VHH", "nanobody" are used interchangeably and refer to a single domain antibody that specifically recognizes and binds to CD 7. Typically, single domain antibodies contain three CDR regions and four FR regions. Single domain antibodies are the smallest functional antigen binding fragments. Typically, after an antibody is obtained which naturally lacks the light and heavy chain constant regions 1 (CH 1), the variable regions of the heavy chain of the antibody are cloned, and a single domain antibody consisting of only one heavy chain variable region is constructed.

The term "complementarity determining regions" or "CDRs" as used herein refers to sequences of amino acids within the variable regions of an antibody that confer antigen specificity and binding affinity. Generally, there are three CDRs (e.g., HCDR1, HCDR2, and HCDR 3) in each heavy chain variable region, and three CDRs (LCDR 1, LCDR2, and LCDR 3) in each light chain variable region.

The portion of the chimeric antigen receptor comprising the antibody or antibody fragment thereof can exist in a variety of forms, for example, wherein the antigen binding domain is expressed as part of a polypeptide chain (including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), or, for example, a human or humanized antibody), see Harlow et al, 1999,In:Using Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory Press,NY; harlow et al 1989,In:Antibodies:A Laboratory Manual,Cold Spring Harbor,New York; houston et al 1988,Proc.Natl.Acad.Sci.USA 85:5879-5883; bird et al 1988,Science 242:423-426.

An "antigen binding domain" refers to the portion of a chimeric antigen receptor that specifically binds to a target antigen, which can be used to direct T cells and/or other immune cells to a selected target using its antigen binding properties. The antigen binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but it need not comprise both variable regions. For example, fab fragments, fab' fragments, single Fv fragments such as scFv, single domain antibodies, and the like, which have antigen binding activity, can be included.

A "transmembrane domain" is an extracellular domain and an intracellular domain that are used to connect a chimeric antigen receptor. The transmembrane domain may be natural or synthetic, and may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains include tcrα, tcrβ, tcrγ, tcrδ, tcrζ, CD28, cd3ζ, cd3ε, cd3γ, cd3δ, CD45, CD4, CD5, cd8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD152, CD154, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, PD-1.

An "intracellular signaling domain" refers to a functional portion of a protein, i.e., a functional portion of an intracellular signaling domain sufficient to transduce an effector function signal. Intracellular signaling domains are responsible for intracellular primary signaling after antigen binding by the antigen binding region, leading to activation of immune cells and immune responses. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cells in which the CAR is expressed. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines. In one embodiment, the intracellular signaling domains comprised by the chimeric antigen receptor of the invention may be cytoplasmic sequences of T cell receptors and co-receptors that function together to elicit signaling upon antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences having the same or similar function. The intracellular signaling domain may contain a number of immunoreceptor tyrosine-activating motifs (Immunoreceptor Tyrosine-based Activation Motifs, ITAM). Non-limiting examples of intracellular signaling domains include, but are not limited to, fcrγ, fcrβ, TCR ζ, cd3γ, cd3δ, cd3ε, cd3ζ, CD5, CD22, CD79a, CD79b, CD278 (ICOS), fcεri, DAP10, DAP12, or CD66d.

"hinge region" refers to any oligopeptide or polypeptide used to connect a transmembrane domain and an antigen binding domain. In particular, the hinge region serves to provide greater flexibility and accessibility to the antigen binding domain. The hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region may be derived in whole or in part from a natural molecule, for example, in whole or in part from the extracellular region of CD8 or CD28, or in whole or in part from the antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence.

The different domains of the CAR may also be fused to each other via a peptide linker. Depending on the structural and/or functional characteristics of the single domain antibody and/or the various domains, each peptide linker in the CAR may have the same or different length and/or sequence. Each peptide linker can be independently selected and optimized by one skilled in the art. In some embodiments, the peptide linker consists of amino acids linked together by peptide bonds, wherein the amino acids are selected from the group consisting of 20 naturally occurring amino acids: glycine, alanine, valine, leucine, isoleucine, serine, cysteine, threonine, methionine, proline, phenylalanine, tyrosine, tryptophan, histidine, lysine, arginine, aspartic acid, glutamic acid, asparagine and glutamine. As will be appreciated by those skilled in the art, one or more of these amino acids may be glycosylated. In some embodiments, the peptide linker comprises flexible residues (e.g., glycine and serine) such that adjacent domains can move freely relative to each other. For example, glycine-serine duplex may be a suitable peptide linker.

The peptide linker may have any suitable length. In some embodiments, the peptide linker is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or more amino acids in length. In some embodiments, the peptide linker is no more than about 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or less amino acids in length. In some embodiments, the peptide linker is about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids in length.

The peptide linker may have a naturally occurring sequence or a non-naturally occurring sequence. For example, sequences derived from the hinge region of heavy chain-only antibodies may be used as linkers. See, e.g., WO1996/34103. In some embodiments, the peptide linker is a flexible linker. Exemplary flexible linkers include, but are not limited to, glycine polymer (G) n, glycine-serine polymer (e.g., (GS) n, (GSG) n, (GGGS) n, and (GGGGS) n, where n is an integer of at least 1), glycine-alanine polymer, alanine-serine polymer, and other flexible linkers known in the art.

The "costimulatory signaling domain" may be an intracellular functional signaling domain from a costimulatory molecule, comprising the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof. "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell. Non-limiting examples of co-stimulatory domains include, but are not limited to, the intracellular regions of the following proteins: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activating NK cell receptors, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX 40), CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CDS, ICAM, CD137 (4-1 BB), CD276 (B7-H3), CD278 (ICOS), GITR, BAFFR, LIGHT, HVEM (light), BTLA, kirs 2, SLAMF7, NKp80 (KLRF 1), NKp44, NKp30, NKp46, CD19, CD4, CD8 a, CD8 β, IL2rβ, IL2rγ, IL7rα, ITGA4, VLA1, IL2rβ; CD49a, ITGA4, IA4, CD49D, ITGA, VLA-6, CD49f, ITGAD, CD11D, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11B, ITGAX, CD c, ITGB1, CD29, ITGB2, ITGB7, NKG2D, NKG2C, TRANCE/RANKL, DNAM1 (CD 226), SLAMF4 (CD 244, 2B 4), CD84, CD96 (Tactive), CEACAM1, CRTAM, ly9 (CD 229), CD160 (BY 55), PSGL1, CD100 (SEMA 4D), CD69, SLAMF6 (NTB-A, ly 108), SLAM (SLAMF 1, CD150, IPO-3), BLAME (SLAMF 8), PLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, CD28-OX40, CD 28-4-BB, TRIAP 10, PD-1, ZAP70, or any combination thereof.

The "signal peptide" may be such that when the chimeric antigen receptor is expressed in a cell (e.g., a T cell), the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. In general, the core of the signal peptide may contain long hydrophobic amino acid segments that have a tendency to form a single α -helix. The signal peptide directs the transport and/or secretion of the translated protein across the membrane. At the end of the signal peptide, there is typically an amino acid segment that is recognized and cleaved by the signal peptidase. The signal peptidase may cleave during or after translocation to produce the free signal peptide and the mature protein. The free signal peptide is then digested by a specific protease. The signal peptide may also be referred to as a targeting signal, transit peptide, localization signal or signal sequence. For example, the signal sequence may be a co-translated or post-translated signal peptide.

By "immune effector cell" is meant an immune cell that can perform immune effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). For example, the immune effector cells may be T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, or immune cells derived from stem cells, such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, hematopoietic stem cells, or the like. When the immune effector cell is a T cell, the T cell may be any T cell, such as an in vitro cultured T cell, e.g., a primary T cell, or a T cell from an in vitro cultured T cell line, e.g., jurkat, supT1, etc., or a T cell obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. T cells may also be concentrated or purified. T cells may be at any stage of development, including, but not limited to, cd4+/cd8+ T cells, cd4+ helper T cells (e.g., th1 and Th2 cells), cd8+ T cells (e.g., cytotoxic T cells), CD4-/CD8-T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ -T cells, αβ -T cells, and the like.

The terms "patient", "subject", "individual", "subject" as used herein are used interchangeably and include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human.

"treatment" refers to the use of the methods of treatment of the invention to achieve at least one positive therapeutic effect (e.g., reduced number of cancer cells, reduced tumor volume, reduced rate of infiltration of cancer cells into peripheral organs, or reduced rate of tumor metastasis or tumor growth) in a subject. The therapeutic regimen effective to treat a patient can be adjusted according to a variety of factors, such as the disease state, age, weight, and ability of the patient to elicit an anti-cancer response in the subject.

Drawings

Figure 1A shows the rate of CAR expression on cd4+ T cells after knocking out the CD7 gene of T cells using the CRISPR/Cas9 system and preparing it into CAR-T cells for 3 days of culture.

Figure 1B shows the rate of CAR expression on cd8+ T cells after knocking out the CD7 gene of T cells using the CRISPR/Cas9 system and preparing it into CAR-T cells for 3 days of culture.

Figure 2 shows the knockout rate of each set of effector cells after T cell CD7 gene knockout using CRISPR/Cas9 system and prepared into CAR-T cells.

FIG. 3A shows T in CD4+ CAR-T cells after 10 days of culture of the prepared CAR-T cells _SCM +T _CM The proportion of the components is as follows.

FIG. 3B shows T in CD8+ CAR-T cells after 10 days of culture of the prepared CAR-T cells _SCM +T _CM The proportion of the components is as follows.

FIG. 4A shows the proportion of CD4+ CAR-T cells expressing Tim3 after 10 days of culture.

Fig. 4B shows the proportion of cd8+ CAR-T cells expressing Tim3 after 10 days of culture.

Figure 5 shows fold proliferation of total T cells after knocking out CD7 gene of T cells using CRISPR/Cas9 system and preparing it into CAR-T cells for 15 days of culture.

FIG. 6 shows the separate determination of various target cells: expression of CD7 antigen on the surface of Raji cells, SUP-T1 cells, jukat cells.

Fig. 7A shows the results of in vitro killing rate flow assay of Jurkat cells by each group of effector cells on day 1 when the effective target ratio (E: T) =1:3.

Fig. 7B shows the results of in vitro killing rate flow assay of Jurkat cells by each group of effector cells on day 3 when the effective target ratio (E: T) =1:3.

Fig. 7C shows the results of in vitro killing rate flow assay of Jurkat cells by each group of effector cells on day 1 when the effective target ratio (E: T) =1:9.

Fig. 7D shows the results of in vitro killing rate flow assay of Jurkat cells by each group of effector cells on day 3 when the effective target ratio (E: T) =1:9.

Fig. 8A shows the results of in vitro killing of SUP-T1 cells by each group of effector cells on day 1 when the effective target ratio (E: T) =1:3.

Fig. 8B shows the results of in vitro killing of SUP-T1 cells by each group of effector cells on day 3 when the effective target ratio (E: T) =1:3.

Fig. 8C shows the results of in vitro killing of SUP-T1 cells by each group of effector cells on day 1 when the effective target ratio (E: T) =1:9.

Figure 8D shows the results of in vitro killing of SUP-T1 cells by each group of effector cells on day 3 when the effective target ratio (E: T) =1:9.

Fig. 9A shows the results of in vitro killing rate flow assay of Raji cells by each group of effector cells on day 1 when the effective target ratio (E: T) =1:3.

Fig. 9B shows the results of in vitro killing rate flow assay of Raji cells by each group of effector cells on day 3 when the effective target ratio (E: T) =1:3.

Fig. 9C shows the results of in vitro killing rate flow assay of Raji cells by each group of effector cells on day 1 when the effective target ratio (E: T) =1:9.

Fig. 9D shows the results of in vitro killing rate flow assay of Raji cells by each group of effector cells on day 3 when the effective target ratio (E: T) =1:9.

Fig. 10A shows the results of IL-2 release experiments when effector cells of each group kill target cells (Jurkat) at an effective target ratio (E: T) =1:9.

Fig. 10B shows TNF- α release assay results when effector cells of each group kill target cells (Jurkat) at an effective target ratio (E: T) =1:9.

Fig. 10C shows the results of IFN- γ release experiments when effector cells of each group killed target cells (Jurkat) at an effective target ratio (E: T) =1:9.

Detailed Description

The invention is further illustrated by means of the following examples, which are not intended to limit the invention thereto. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

Example 1 preparation of CD 7-targeting Single Domain antibodies (VHHs)

(1) Animal immunity and immune response test

Adult healthy Bactrian camels were selected as subjects for immunization, and 10mL of blood was taken as negative serum control prior to immunization. CD7-His recombinant protein is used for continuously immunizing 3 times of Bactrian camels, B cells are stimulated by the immune process to express antigen-specific nanobodies, a small amount of peripheral blood separation serum is collected on the 7 th day after each immunization, and the immune titer is determined by indirect ELISA. The antibody titer obtained by detection is 1:512000, which shows that the immune effect is good and the preparation for library establishment is prepared.

(2) Construction of antibody phage libraries

1) Extraction and reverse transcription of RNA

Peripheral blood 200mL was collected 7 days after impact immunization, peripheral blood lymphocytes were isolated and total RNA was extracted with RNAiso Plus reagent, and the extracted RNA was reverse transcribed into cDNA by reverse transcription kit.

2) PCR amplification

Specific antibody fragments were amplified from the reverse transcribed cDNA using the PCR method. Taking cDNA as a template, obtaining VHH genes through two rounds of PCR amplification, cutting the PCR products after the first round of PCR amplification, and recovering and purifying 700bp VHH gene fragments; in the second round, the 700bp VHH gene is used as a template for amplification to obtain the VHH gene.

3) Enzyme cutting

And simultaneously enzyme-cutting the pMECS vector and the target gene fragment by using endonuclease, and connecting the VHH gene to the vector by using T4 DNA ligase after enzyme-cutting is completed, so as to construct the recombinant plasmid.

4) Electric conversion

The recombinant plasmid is purified and transformed into TG1 competent cells by electrotransformation, and the electrotransformation parameters are as follows: electroporation apparatus BIO-RAD, gene Pulser Xcell Total System, voltage 2.5KV, electric cuvette 2mm. Immediately after electric shock, 1mL of 2YT medium (preheated at 37 ℃) was added to the cuvette, the electric shock product was sucked out and the cuvette was washed with the 2YT medium to obtain 100mL of liquid in total, and resuscitated at 180rpm at 37℃for 45 minutes as resuscitated product, 100. Mu.L was taken out therefrom, and diluted to 10 in a gradient ^-3 And 10 ^-4 Post-plating was used to determine the number of pool transformants. The remaining resuscitated product was centrifuged and resuspended in 8mL of 2YT medium before plating. After culturing, a nanobody library is obtained. The pool capacity was determined and the insertion rate and pool diversity of the constructed pool was verified by colony PCR.

(3) Phage packaging

Phage library was inoculated into 2X 300mL 2YT+A (Amp) +G (Glu) medium (Amp: 100. Mu.g/mL, glu: 1%) until the initial OD600 was 0.1-0.2. Culturing at 37deg.C and 230rpm until OD600 is above 0.8. Helper phage M13KO7 (number of helper phages: number of bacteria=20:1) was added according to OD value, and mixed well, and after standing at 37℃for 30min, shaking at 180rpm at 37℃was continued for 30min. Next, the supernatant was discarded after centrifugation at 5000rpm for 10min, and the pellet was resuspended in an equal volume of 2YT+A (Amp) +K (Kan) medium (Amp: 100. Mu.g/mL, kan: 50. Mu.g/mL) and incubated at 30℃overnight at 220 rpm. After centrifuging overnight culture at 4℃and 10000rpm for 20min, collecting supernatant, transferring to a new centrifuge tube, centrifuging at 4℃and 10000rpm for 20min, and collecting supernatant. Adding PEG8000/NaCl solution with volume of 1/5 of the supernatant, mixing, ice-bath precipitating for at least 2 hr, centrifuging at 4deg.C and 10000rpm for 20min, and removing supernatant. The precipitate was suspended by adding 1mL of 1 XPBS, and then precipitated again by adding 1/5 of its volume of PEG8000/NaCl solution for 1h. After the secondary precipitation, the supernatant was removed by centrifugation at 12000rpm for 10min at 4℃and the precipitate was resuspended by adding 1 XPBS according to the amount of precipitate. Then adding 100% glycerol until the final concentration of the liquid is 50%, mixing, and preserving at-80 ℃. 10 mu L of phage library was diluted in gradient with 2YT medium from 10 ^-8 And 10 ^-9 10. Mu.L of the culture medium was added to 90. Mu.L of TG1 bacteria solution, and the mixture was gently mixed. The mixture was allowed to stand at 37℃for 15min, amp-resistant plates were applied, and incubated overnight at 37 ℃. Phage library titers were measured the next day.

(4) Phage selection

1) Cell panning

CD7 (5X 10) ⁶ Cell) cells were centrifuged at 500g for 5min, resuspended in 5% serum-PBS and the Cell pellet washed, repeated twice to wash CD7 cells. To the cell EP tube was added 500. Mu.L of OVA blocking solution and blocked with gentle shaking at 4℃for 1h. Phage library was diluted with 5% serum-PBS and 3% BSA until BSA concentration was 2%, blocked at 37℃and used as phage library dilutions. After blocking the CD7 cells and centrifuging, removing the supernatant, adding 2X 10 ¹¹ cfu phage library dilutions were centrifuged after 1h of gentle shaking at 4 ℃, unbound phage were removed, and cells were washed 6 times with 5% serum-PBS. 100. Mu.L of Gly-HCl eluent was added and enriched at 37℃for 8min, the specifically bound phage was eluted, the eluent was transferred to a sterile centrifuge tube and immediately neutralized with 10. Mu.L of Tris-HCl neutralization buffer to obtain elutriation eluate. 10. Mu.L was subjected to gradient dilution for determining titer, and the panning recovery was calculated.

2) Amplification of library after panning

The elutriation eluate was mixed with 20mL of e.coll TG1 culture at the early stage of logarithmic growth, left at 37 ℃ for 30min, then added with 1mL of 20% glucose, incubated at 220rpm for 30min, added with M13K07 phage and 4 μl Amp in the ratio of cell: phage=1:20, left at 37 ℃ for 30min, added with 20mL of 2yt medium, and incubated at 220rpm for 30min. The culture was centrifuged at 5000rpm for 10min at 4℃and the resulting cell pellet was resuspended in 50mL of 2 XYT+A+K liquid medium and cultured overnight with shaking at 250rpm at 30 ℃. The overnight culture was centrifuged at 10000rpm at 4℃for 20min, the supernatant was transferred to a new centrifuge tube and 1/5 volume of PEG/NaCl solution was added to the supernatant, and after mixing, it was allowed to stand at 4℃for at least 2h. After the completion of the standing, the mixture was centrifuged at 10000rpm at 4℃for 20 minutes, the supernatant was removed, the precipitate was resuspended in 1mL of PBS, and 1/5 of the PEG/NaCl solution was added thereto, followed by mixing and standing at 4℃for at least 1 hour. After completion of the standing, the mixture was centrifuged again, and the mixture was centrifuged at 12000rpm for 2 minutes at 4℃to remove the supernatant, and the precipitate was resuspended in 200. Mu.L of PBS to obtain an amplification product. Titer was determined for the next round of panning or analysis, with panning repeated three rounds.

(5) Monoclonal ELISA detection and specificity detection

96 monoclonal antibodies were randomly picked from the plate prepared from the eluent TG1 after the third round of screening, expanded to log phase, and infected with helper phage M13K07 to obtain recombinant phage supernatant, which was used as primary antibody, and nanoantibodies capable of specifically binding to CD7 protein were detected and identified by indirect ELISA, while positive clones were sequenced.

In order to detect the binding specificity of the nano antibody obtained by screening and CD7 protein, CD7-His protein and control proteins CD5-His, CD47-His, CD22-mFc and mFc are respectively coated on an ELISA plate, after blocking, monoclonal recombinant phage supernatant is used as primary antibody to be respectively incubated with the proteins, and the reactivity of the nano antibody and the proteins is detected by indirect ELISA.

Example 2 Gene editing of T cells

Grnas were designed to knock out the endogenous CD7 gene of T cells, which were respectively:

(1) gRNA-CD7-GP4: ACGGGGACAGTCGTGCAGTG (SEQ ID NO: 24), PAM sequence: GGG;

(2) gRNA-CD7-EC8: GGAGCAGGTGATGTTGACGG (SEQ ID NO: 25), PAM sequence: AGG.

crRNAs comprising gRNA-CD7-GP4 or gRNA-CD7-EC8 were synthesized separately, and crRNA-tracrRNA complexes were formulated separately. crRNA and tracrRNA were added to nucleic-Free Duplex Buffer (IDT) in a ratio of 1:1 in a total volume of 100. Mu.L. Annealing at 95℃for 5min, taking out, standing at room temperature, and after the temperature is restored to 20-25℃adding crRNA, tracrRNA complex, cas9 and 1. Mu.M electroporation enhancer (IDT, cat# 1075916) to Opti-MEM (Gibco, cat#A 4124802), respectively, to prepare a nucleated glycoprotein (RNP) complex in a total volume of 25. Mu.L. Standing for 5min at room temperature to obtain RNP complex for use.

T cells were activated and grouped, wherein T cells that were not electrotransferred for CD7 knockout were designated as NoEp groups, and the T cell groups for CD7 knockout were individually named according to the gRNA names used for each. Cells were centrifuged, the supernatant was discarded to retain the cell pellet, and fresh appropriate amounts of Opti-MEM buffer were added to resuspend the cell pellet. The cell suspension is centrifuged, the supernatant is discarded, a fresh proper amount of Opti-MEM buffer solution is added to resuspend the cell sediment, the prepared RNP complex is added, and then the mixture is gently blown and mixed evenly, and the mixture is added into an electrode cup. A Celetrix electrotometer was used and set to a voltage of 500V. After the electrotransformation is finished, the cells are transferred to a culture medium, kept stand for 30min in a culture box at 37 ℃, and then transferred to a culture dish for culture, so that the T cells with the CD7 gene knocked out are obtained.

Cell densities of two groups of T cells from which the CD7 gene was knocked out were adjusted to 400 cells/. Mu.L, plated at a volume of 500. Mu.L per well, and mixed at 37℃with 5% CO ₂ Culturing in an incubator for 3 days, and detecting CD7 knockout rate on CD4+, CD8+ T cells using PE anti-human CD7 Antibody (PE anti-human CD7 Antibody, biolegend, B351621). The T cell expansion was observed every three days after plating and fresh complete medium was supplemented, and after continuous culture for 8 days, the knocking out rate of CD7 gene (the percentage of CD7-T cells) on cd4+ and cd8+ T cells was detected using PE anti-human CD7 Antibody, biolegend, B351621, and the detection results showed that the knocking out rate of the NoEp group for CD7 gene, the knocking out rate of the T cell group for CD7 gene knockout using gRNA-CD7-GP4, and the knocking out rate of the T cell group for CD7 gene knockout using gRNA-CD7-EC8 were all greater than 90%.

EXAMPLE 3 construction of CD 7-targeting chimeric antigen receptor and immunocyte expression

(1) Construction of CD7-CAR

Each set of CD 7-targeting CAR nucleotide sequences (SEQ ID NO: 21-23) was designed and artificially synthesized, each set comprising the coding nucleotide sequences of the HLA-A signal peptide (SEQ ID NO: 13), the extracellular antigen binding domain of CD7-VHH (SEQ ID NO: 10-12), the CD8 a hinge region (SEQ ID NO: 14), the CD8 a transmembrane domain (SEQ ID NO: 15), the 4-1BB (CD 137) costimulatory signal domain (SEQ ID NO: 16) and the CD3 zeta intracellular signal transduction domain (SEQ ID NO: 17), for expression of the complete CD7-CAR polypeptide molecules (SEQ ID NO: 18-20) of each experimental set, designated as T2, T19 and T60, respectively. The nucleotide sequence of the CD7-CAR is inserted into the multiple cloning site of the lentiviral expression vector pK1 through homologous recombination to obtain the pK1-CD7-CAR, and the successful construction of the lentiviral expression vector sequence is confirmed through electrophoresis and sequencing results.

In addition, positive control CAR molecules were constructed in the same way, T6 and TH69 respectively, wherein T6 used VHH6 disclosed in patent CN110760007B (amino acid sequence SEQ ID NO:6 in the patent) as antigen binding domain; the amino acid sequence of the CAR molecule of T6 is shown as SEQ ID NO. 26 (marked as T6 group), and the nucleic acid sequence is shown as SEQ ID NO. 27. TH69 uses the documents "Baum W, steininger H, bair HJ, becker W, hansen-Hagge TE, kressel M, kremer E, kalden JR, gramatzki M.therapeutic with CD7 monoclonal antibody TH-69is highly effective for xenografted human T-cell ALL.Br JHaemato.1996Nov; 95 (2) TH-69 as antigen binding domain disclosed in "327-38", the amino acid sequence of the CAR molecule of TH69 is shown in SEQ ID NO. 28 (noted as group TH 69), and the nucleic acid sequence thereof is shown in SEQ ID NO. 29.

Amino acid sequence of positive control CAR molecule T6:

MLLLVTSLLLCELPHPAFLLIPMDVQLQESGGGLVQAGGSLRLSCAVSGYPYSSYCMGWFRQAPGKEREGVAAIDSDGRTRYADSVKGRFTISQDNAKNTLYLQMNRMKPEDTAMYYCAARFGPMGCVDLSTLSFGHWGQGTQVTVSITTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:26)

nucleic acid sequence of positive control CAR molecule T6:

ATGCTGCTGCTGGTGACCTCTCTGCTGCTCTGCGAACTGCCTCACCCAGCCTTTCTGCTGATCCCCATGGACGTGCAGCTGCAGGAAAGCGGAGGAGGACTGGTGCAGGCAGGAGGATCTCTGAGGCTGTCTTGCGCAGTGTCCGGATACCCCTACAGCAGCTACTGCATGGGTTGGTTCAGACAGGCCCCAGGAAAGGAGAGAGAGGGAGTGGCCGCTATCGATAGCGACGGAAGAACCAGATACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCCAGGACAACGCCAAGAACACCCTGTACCTGCAGATGAACCGGATGAAGCCCGAGGACACCGCCATGTACTATTGCGCCGCTCGCTTCGGACCTATGGGTTGCGTGGATCTGAGCACCCTGAGCTTTGGCCATTGGGGACAGGGAACCCAGGTCACCGTGTCCATCACTACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:27)

amino acid sequence of positive control CAR molecule TH 69:

MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYSKLPYTFGGGTKLEIKRGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLSCAASGLTFSSYAMSWVRQTPEKRLEWVASISSGGFTYYPDSVKGRFTISRDNARNILYLQMSSLRSEDTAMYYCARDEVRGYLDVWGAGTTVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:28)

nucleic acid sequence of positive control CAR molecule TH 69:

ATGGCTCTGCCCGTCACCGCTCTGCTGCTGCCTCTGGCTCTGCTGCTGCACGCCGCAAGACCCGACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAGCAATTATTTAAACTGGTATCAGCAGAAACCAGATGGAACTGTTAAACTCCTGATCTATTACACATCAAGTTTACACTCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCAGCAACCTGGAACCTGAAGATATTGCCACTTATTATTGTCAGCAGTATAGCAAGCTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGTGGAGGAGGAGGAAGCGGAGGAGGAGGATCTGGAGGAGGCGGGTCTGAGGTGCAGCTGGTCGAATCTGGAGGAGGACTGGTGAAGCCAGGAGGATCTCTGAAACTGAGTTGTGCCGCTTCAGGCCTGACCTTCTCAAGCTACGCCATGAGCTGGGTGCGACAGACACCTGAGAAGCGGCTGGAATGGGTCGCTAGCATCTCCTCTGGCGGGTTCACATACTATCCAGACTCCGTGAAAGGCAGATTTACTATCTCTCGGGATAACGCAAGAAATATTCTGTACCTGCAGATGAGTTCACTGAGGAGCGAGGACACCGCAATGTACTATTGTGCCAGGGACGAAGTGCGCGGCTATCTGGATGTCTGGGGAGCTGGCACTACCGTCACCGTCTCCAGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:29)

(2) Packaging of lentiviral vectors

293T cells were thawed and cultured in DMEM medium containing 10% FBS. After 2-3 generation cell expansion culture, the method is carried out according to 4 multiplied by 10 ⁴ Individual/cm ² Is seeded into a 2-layer cell factory. Plasmid transfection was performed 3 days after cell inoculation, 40mL of Opti-MEM was added to a sterile 50mL centrifuge tube for plasmid transfection, and then the viral packaging vector and viral envelope vector were added according to the ratio of pK1-CD7-CAR: pLP1: pLP2: pLP-VSVG=5:4:3:1, and 800. Mu.L of PEI transfection reagent was added, mixed immediately, and incubated at room temperature for 15min. The plasmid/vector/transfection reagent complex was then added drop-wise to a culture flask of 293T cells, after 24h the virus supernatant was collected into a 50mL centrifuge tube, centrifuged at 250g for 5min, filtered through a 0.45 μm filter, and the filtered supernatant was ultracentrifuged (25000 g,4 ℃ C., 3 h) to obtain concentrated CD7-CAR lentivirus. Centrifuging, discarding supernatant, re-suspending lentivirus with PBS pre-cooled at 4deg.C, packaging the re-suspended CD7-CAR lentivirus liquid, and storing at-80deg.C.

(3) Resuscitation and activation of T cells

Taking out and freezingCord blood was thawed in a water bath at 38 ℃. Transferring cord blood into 50mL centrifuge tube, adding appropriate amount of RPMI1640 culture medium, mixing, sampling, counting, centrifuging under 300g for 5min, collecting lower cell layer, and re-suspending with complete culture medium (RPMI 1640 culture medium containing 10% FBS, supplemented with glutamine, 2-mercaptoethanol, IL-7 (10 ng/mL), IL-15 (10 ng/mL), IL-2 (200U/mL)) to T lymphocyte density of 1×10 ⁶ Adding activated magnetic beads Anti-human CD3 Anti-ibody and Anti-human CD28 Anti-ibody according to the re-suspension volume, wherein the Anti-human CD3 Anti-ibody is used at a concentration of 0.15 mug/mL, the Anti-human CD28 Anti-ibody is used at a concentration of 0.625 mug/mL, and placing the cells into a container at 37 ℃ and 5% CO ₂ Culturing in an incubator.

(4) Sorting and purification of T cells

After T cells are activated for 2 days, 20 mu L of the mixture is evenly mixed and sampled, 10 mu L of the diluted fluorescent labeled antibody is added, the mixture is dyed for 10min, PBS is added for 10 times, the mixture is diluted and then is detected and counted by a flow cytometry, the densities of CD45+, CD3+, CD4+ and CD8+ T cells are recorded, and the expression condition of the activation markers CD69 and CD25 molecules is observed. Cell volumes were recorded and T cell population was confirmed. Transferring the cell suspension into a centrifuge tube for centrifugation (500 g, 5 min), and discarding the supernatant to collect the lower layer cells; after washing with MACS Buffer, the lower cells were collected after centrifugation (500 g, 5 min) and resuspended with the appropriate amount of MACS Buffer. The amounts of CD4+ magnetic beads (Miltenyi Biotec, 130-045-101) and CD8+ magnetic beads (Miltenyi Biotec, 130-045-201) used were calculated based on the cell amounts and the bead specifications, and the sorted T cells were obtained by sorting using CD4+ and CD8+ magnetic beads according to the bead specifications.

(5) Endogenous CD7 gene knockout and CD7-CAR lentiviral transduction of T cells

The endogenous CD7 gene of the T cell obtained in step (4) was knocked out using the gRNA-CD7-EC8 obtained in example 2. The T cell density after knockdown was adjusted to 400 cells/. Mu.L for plating, with a volume of 500. Mu.L per well. Culturing for 24 hr, adding CD7-CAR slow virus solution according to the number of cells per hole, mixing, and adding 5% CO at 37deg.C ₂ Culturing in an incubator. After 3 days of culture, the expression rate of the CD7-CAR molecules on the surface of the T cells and the proliferation of the T cells were detected. People labeled with FITCThe CD7 protein was tested for the expression rate of CAR in each experimental group and the results are shown in fig. 1A and 1B. CD7 knockout rates were tested for UnT and experimental groups using PE anti-human CD7 antibodies, and CD7 knockout cases for each group of effector cells are shown in fig. 2, demonstrating successful acquisition of CD 7-targeted CAR-T cells with endogenous CD7 knockouts. The proliferation of CAR-T cells was periodically examined from plating and fresh complete medium was supplemented and T cells were examined after 10 days of culture _SCM +T _CM Phenotype and proportion of Tim 3-expressing CAR-T cells, T _SCM +T _CM The results of the phenotype assay are shown in FIGS. 3A and 3B, and the results of Tim3 expression assay are shown in FIGS. 4A and 4B. Harvesting after continuous culture for 15 days for subsequent in vitro killing experiments. Meanwhile, the proliferation condition of the T cells is detected and counted, and the proliferation condition of the T cells is shown in figure 5, which shows that a large number of suicide of the CAR-T cells is successfully avoided, and the CD7 targeted CAR-T cells are prepared. The resulting CD 7-targeting CAR-T cells were constructed as T2 (SEQ ID NO: 18), T19 (SEQ ID NO: 19) and T60 (SEQ ID NO: 20), respectively, negative control T cell group UnT (non-transduced CAR), positive control CAR-T cell group as T6 (SEQ ID NO: 26) and TH69 (SEQ ID NO: 28).

Stem cell-like memory T cells (T _SCM ) Is a T cell subgroup with self-renewal and effector T cell generation capacity, has the characteristic of being capable of long-term survival in vivo, and plays a role in maintaining long-term anti-tumor capacity. Central memory T cell (T) _CM ) Is T _SCM The latter stage of differentiation is usually carried out by analysis of T _SCM +T _CM The proportion is used for judging the in vivo persistence and the therapeutic potential of effector T cells. The invention evaluates T by analyzing the percent sum of CD45RO-CD62L+ and CD45RO+CD62L+ T cells _SCM +T _CM The ratio of (CD 45RO: APC/Cyanine7 anti-human CD45RO, biolegend, B350148; CD62L: PE/Cyanine7 anti-human CD62L, biolegend, B373155) predicts a stronger persistence of CAR-T cells in vivo. Thus, if a higher proportion of T can be maintained in CAR-T cells _SCM +T _CM It would also be advantageous for use in vivo therapy.

The present invention was performed by analyzing Tim3 expressing T cells (Brilliant Violet 421 ^TM anti-human CD366, biolegend, B355191) percent of T cellsThe higher the proportion of Tim 3-expressing CAR-T cells, the lower the effector function of the CAR-T cells.

Example 4 validation of tumor cell killing Effect of CD 7-targeting CAR-T cells

(1) Determination of CD7 antigen expression on target cell surface

Raji (ATCC, CCL-86), SUP-T1 (ATCC, CRL-1942) and Jurkat cells (Chengdu Biotechnology Co., ltd.) were stained for 7min under light-shielding conditions as described in the specification of CD7 fluorescent-labeled Antibody (PE anti-human CD7 anti-body, biolegend, B327127), and then the cell surface CD7 antigen expression was detected by a flow cytometer, and the detection results are shown in FIG. 6. The results show that the CD7 antigen is not expressed on Raji cells and is highly expressed on Jurkat cells (CD 7 positive cells) and SUP-T1 cells (CD 7 positive cells).

(2) In vitro killing effect flow assay of CD 7-targeted CAR-T cells

The following target cells (9X 10) were added to each of the 24-well plates ⁵ Number/hole): jurkat cells, SUP-T1 cells, raji cells, and each set of CAR-T cells constructed in example 3 was added to a 24-well plate in accordance with the corresponding effector cell amount, based on the amount of effector cells that need to be added, based on the cell amount of CD8+ CAR-T cells in the CAR-T cells, with an effective target ratio (E: T) of 1:3 or 1:9. The negative control UnT without CAR transduction was also added with the same amount of UnT cells and target cells according to the corresponding effective target ratio. All groups were supplemented with complete medium to 500. Mu.L/well, the well plates were placed at 37℃in 5% CO ₂ Culturing in an incubator. The residual amount of target cells in each well was measured by flow cytometry after 1 day and 3 days of culture, respectively, and the killing rate was calculated. The detection results are shown in FIGS. 7A, 7B, 7C, 7D, 8A, 8B, 8C, 8D, 9A, 9B, 9C, and 9D.

The calculation formula is as follows: killing% = target cell reduction/target cell plating cell amount x 100%.

(3) Cytokine release assay for CD 7-targeted CAR-T cells

18h after the in vitro killing experiment of example 4, item (2), the CAR-T cell culture supernatants of each group were collected and passed through the CBA detection kit LEGENDplex ^TM HU Th Cytokine Panel (12-plex) w/FP V02, cat No. 741027, the release of cytokines (IL-2, TNF-. Alpha., IFN-. Gamma.) from each group of CAR-T cells was examined, and the results are shown in FIGS. 10A, 10B and 10C.

The specific implementation steps are as follows:

the CAR-T cell culture supernatants of each group were collected separately 18h after plating of the in vitro killing experiments. After the captured microspheres (Beads) were returned to room temperature, they were vortexed for 2min to mix well and formulated to 15 μl per sample for use. The 20xWash Buffer was returned to room temperature to dissolve the salt therein sufficiently, and it was prepared into 1xWash Buffer with ultrapure water for use. The standard was dissolved and mixed with 250. Mu.LAssay Buffer and subjected to gradient dilution, 4-fold dilution at a dilution ratio, 6 total dilutions to obtain 6 gradient concentration points, for establishing a cell supernatant standard curve. And adding 15uL of each of an Assay Buffer, a Beads and a standard substance into an EP tube, uniformly mixing, and preparing a standard substance hole. And adding 15 mu L of Assay Buffer, beads and each sample into the EP tube, and uniformly mixing to prepare sample holes. The standard wells and sample wells were shaken at 900rpm under light-protected conditions and incubated for 2h at room temperature. The sample is filtered in vacuum, 200 mu L of 1xWash buffer is added to each EP tube, and the mixture is stirred and mixed uniformly, and the washing is repeated once. Add 15. Mu.L of detection antibody and 15. Mu.L of Assay buffer to each EP tube, blow mix well, shake at 900rpm under dark conditions and incubate at room temperature for 1h under dark conditions. Then 15. Mu.L of streptavidin-phycoerythrin (SA-PE) was added to each EP tube, shaken at 900rpm under light-shielding conditions, and incubated at room temperature for 0.5h under light-shielding conditions to bind the capture microspheres (Beads), the target analyte, with biotinylated detection antibody, SA-PE. 200. Mu.L of 1xWash buffer was added to each EP tube to wash twice, shake mix well and repeat the wash once. 200. Mu.L of 1xWash buffer was added to each tube and tested in the dark. Each sample was mixed by shaking for 90s before detection, and the flow cytometer parameters were selected for FSC, SSC, PE and APC channels to detect the release of cytokines IL-2, TNF- α, IFN- γ, respectively.

Claims

1. A single domain antibody targeting CD7, wherein the single domain antibody comprises CDR1, CDR2, and CDR3;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 1, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 2, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 3;

wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 4, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 5, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 6; or (b)

Wherein CDR1 is the amino acid sequence shown as SEQ ID NO. 7, wherein CDR2 is the amino acid sequence shown as SEQ ID NO. 8, wherein CDR3 is the amino acid sequence shown as SEQ ID NO. 9.

2. The single domain antibody of claim 1, wherein the single domain antibody comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of the amino acid sequences set forth in SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 12.

3. The single domain antibody of claim 1, wherein the single domain antibody comprises any one of the amino acid sequences shown as SEQ ID No. 10, SEQ ID No. 11 or SEQ ID No. 12.

4. A chimeric antigen receptor comprising the CD 7-targeting single domain antibody of any one of claims 1-3.

5. A chimeric antigen receptor comprising

(a) An extracellular antigen-binding domain,

(b) A transmembrane domain, and

(c) An intracellular signaling domain;

wherein the extracellular antigen binding domain comprises the CD 7-targeting single domain antibody of any one of claims 1-3.

6. The chimeric antigen receptor according to claim 5, wherein the transmembrane domain is derived from CD8 a, CD28, cd3ζ, cd3γ, cd3δ, cd3ε, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, tcra, tcrβ, tcrγ, tcrδ, tcrζ, OX40, ICOS, LAG-3, 2B4, BTLA, CTLA-4, PD-1, or any combination thereof.

7. The chimeric antigen receptor according to claim 6, wherein the transmembrane domain is derived from CD8 a.

8. The chimeric antigen receptor according to claim 5, wherein the intracellular signaling domain is derived from fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD22, CD79a, CD79b, CD66d, or any combination thereof.

9. The chimeric antigen receptor according to claim 8, wherein the intracellular signaling domain is derived from cd3ζ.

10. The chimeric antigen receptor according to claim 8, wherein the intracellular signaling domain further comprises a costimulatory signaling domain, wherein the costimulatory signaling domain is derived from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD134 (OX 40), CD2, CD7, CD27, CD28, CD30, CD40, CD83, ICAM, 4-1BB (CD 137), CD276 (B7-H3), CD278 (ICOS), GITR, LIGHT, HVEM (light), BTLA, CD8 a, LFA-1, NKG2C, LAT, SLP-76, DAP10, PD-1, TRIM, ZAP70 ligand, or any combination thereof.

11. The chimeric antigen receptor according to claim 10, wherein the costimulatory signaling domain is derived from 4-1BB (CD 137).

12. The chimeric antigen receptor according to any one of claims 5-10, further comprising a hinge region between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain.

13. The chimeric antigen receptor according to claim 12, wherein the hinge region is derived from CD8 a, CD28, CD137, igG4, igG1, or any combination thereof.

14. The chimeric antigen receptor according to any one of claims 5-13, further comprising a signal peptide at the N-terminus of the chimeric antigen receptor polypeptide.

15. The chimeric antigen receptor according to claim 14, wherein the signal peptide is derived from HLA-A, CD8 a, CD4, CD33, CD137, GM-csfra, igG1, igκ, IL-2, or any combination thereof.

16. A chimeric antigen receptor comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identity to any one of the amino acid sequences set forth in SEQ ID No. 18, SEQ ID No. 19 or SEQ ID No. 20.

17. A chimeric antigen receptor comprising any one of the amino acid sequences shown as SEQ ID No. 18, SEQ ID No. 19 or SEQ ID No. 20.

18. An isolated nucleic acid comprising a nucleic acid sequence encoding the chimeric antigen receptor of any one of claims 4-17.

19. The isolated nucleic acid of claim 18, comprising any one of the nucleic acid sequences set forth in SEQ ID No. 21, SEQ ID No. 22 or SEQ ID No. 23.

20. A vector comprising the isolated nucleic acid of claim 18 or 19.

21. An engineered immune effector cell comprising the chimeric antigen receptor of any one of claims 4-17, the isolated nucleic acid of claim 18 or 19, or the vector of claim 20.

22. The engineered immune effector cell of claim 21, wherein the immune effector cell is selected from T cells, B cells, NK cells, NKT cells, DNT cells, macrophages, dendritic cells, immune effector cells differentiated from induced pluripotent stem cells, or any combination thereof.

23. A pharmaceutical composition comprising the CD 7-targeting single domain antibody of any one of claims 1-3, the chimeric antigen receptor of any one of claims 4-17, or the engineered immune effector cell of any one of claims 21-22, and one or more pharmaceutically acceptable excipients and/or carriers.

24. Use of a CD 7-targeting single domain antibody according to any one of claims 1-3, a chimeric antigen receptor according to any one of claims 4-17, or an engineered immune effector cell according to any one of claims 21-22, or a pharmaceutical composition according to claim 23, in the manufacture of a medicament for the diagnosis, prevention and/or treatment of a disease or disorder associated with CD7 expression.

25. The use according to claim 24, wherein the disease or disorder associated with CD7 expression comprises lymphoma or leukemia.

26. The use according to claim 24, wherein the disease or disorder associated with CD7 expression comprises T lymphoblastic lymphoma (T-LBL), peripheral T-cell lymphoma (PTCL), acute T-lymphoblastic leukemia (T-ALL), T-lymphoblastic leukemia, acute Myeloid Leukemia (AML), or Chronic Lymphoblastic Leukemia (CLL).