WO2022257937A1 - Method and composition for cellular immunotherapy - Google Patents

Abstract

The present invention provides a method and a composition for cellular immunotherapy. Specifically, the present invention provides a method for treating diseases associated with CD7 expression, comprising administering to a subject a first dose of engineered immune cells containing a chimeric antigen receptor that specifically binds to CD7, the first dose being 0.1x106 to 1x108 CAR+ cells/kg, wherein the subject receives a lymphodepletion therapy, prior to administration of the first dose, the lymphodepletion therapy comprising cyclophosphamide, fludarabine, etoposide, melphalan, or a combination thereof.

Description

Methods and compositions for cellular immunotherapy technical field

The invention belongs to the field of immunotherapy. More specifically, the present invention relates to methods and compositions for treating diseases, especially diseases associated with CD7 expression, such as T-ALL or T-LBL, using cells expressing chimeric antigen receptors targeting CD7.

Background technique

Adoptive cell therapy has been increasingly used to treat disease. For example, the use of immune cells expressing recombinant receptors specific to the disease to be treated (such as CAR-T cells, TCR-T cells, CAR-NK cells, etc.) to treat cancer or tumors has been proven to have good efficacy and control side effects. The present invention provides methods, compositions and articles of manufacture for administering or repeatedly administering CAR-T cells to a subject. The methods of the invention can provide improved or longer lasting responses or therapeutic effects with less risk of toxicity or other side effects.

Description of drawings

Figure 1 shows an exemplary administration scheme for UCAR-T cells of the present invention.

Figure 2 shows the copy number of CAR in peripheral blood of responders (a) and non-responders (b) detected by qPCR.

Figure 3 shows the number of UCAR-T cells in the peripheral blood of responders (a) and non-responders (b) detected by flow cytometry.

Figure 4 shows the levels of IL-6, IL-2 and IFN-γ in responders (a1, b1, c1) and non-responders (a2, b2, c2) detected by flow cytometry.

Figure 5 shows the copy number of CAR in the peripheral blood of subjects who received multiple doses, where the arrows show the timing of administering the second dose.

Figure 6 shows the amplification and persistence of UCAR-T in the peripheral blood of the subjects detected by qPCR (a) and flow cytometry (b).

Contents of the invention

The present invention relates to methods for treating diseases, such as cancers and tumors associated with CD7 expression, comprising administering to a subject engineered immune cells expressing a chimeric antigen receptor that specifically binds CD7 antigens and induces an immune response. The methods provided by the present invention have characteristics, such as administration timing, administration dose, treatment regimen, etc., which can provide improved or longer-lasting response or curative effect and lower risk of toxicity or other side effects. The present invention also provides compositions and products that can be used in the above treatment methods.

I Cell Therapy with Engineered Immune Cells

In a first aspect, the present invention provides a method for treating a disease associated with CD7 expression comprising administering to a subject a first dose of engineered immune cells comprising cells that specifically bind to CD7 said first dose is 0.1×10 ⁶ to 1×10 ⁸ CAR+ cells/kg, wherein prior to administering said first dose, said subject is subjected to a lymphodepletion regimen comprising Cyclophosphamide, fludarabine, etoposide, melphalan, or a combination thereof.

A application plan

As used herein, the term "dose" refers to the total amount of engineered immune cells administered to a subject within a course of treatment. The dose can be a dose calculated on a weight basis, for example, the amount administered to a subject calculated on the basis of the subject's body weight, expressed in mg/kg or cell number/kg, etc.; it can also be expressed in terms of body surface area (BSA ) based on the calculated dose, for example, the amount administered to the subject calculated on the basis of the patient's surface area, expressed in mg/ ^m2 or cell number/ ^m2 , etc.; Quantitatively calculated doses are expressed in number of cells. In the present invention, administration of a given dose of engineered immune cells encompasses administration as a single transient and/or single uninterrupted administration (e.g., as a single injection or as a single continuous infusion) of a single composition A given amount of cells also encompasses administering the given amount of cells in divided doses in multiple compositions over a period of not more than 3 days. In the latter case, the sum of the divided doses administered from multiple times is considered a single dose. Thus, in some embodiments, a given dose of engineered immune cells is administered or initiated at a single time point within a course of treatment, either as a single or sequential administration; or, as multiple injections or infusions at A given dose of engineered immune cells is divided into multiple sub-doses for administration within a period not exceeding 3 days, for example, once a day or once every other day, or multiple times a day.

As used herein, the term "first dose" is used to describe the timing of administering a given dose of engineered immune cells as defined herein, ie, the dose administered in the first course of treatment. This dose may be the only dose during the course of treatment, or it may be followed by one or more additional doses (ie, subsequent doses). Accordingly, the methods of the invention may further comprise administering to the subject at least one subsequent dose of engineered immune cells comprising a chimeric antigen receptor that specifically binds CD7.

Where the method of the invention comprises multiple doses, the first dose and subsequent doses may be the same or different. In one embodiment, subsequent doses may be higher or lower than the first dose. For example, the subsequent dose is 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times or more higher than the first dose, or 2 times, 3 times lower than the first dose times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times or more times. Whether to administer subsequent doses to the subject and the specific administration regimen will be determined by the doctor according to the specific conditions of the subject, including but not limited to the subject's age, sex, weight, overall health, disease severity, previous therapy received , the response to the previous dose of the same course of treatment, the response to the previous course of treatment, the combination of drug use, the degree of toxicity, complications, cancer metastasis, etc., and the engineered immune system that the doctor thinks will affect the determination of suitable administration to the subject Any other factor in the amount of cells.

^In one embodiment, the first or subsequent dose is selected from ^0.1x106 to ^1x108 CAR+ cells/kg, 0.5x106 to ^1x108 CAR+ cells/kg, ^1x106 to ^1x108 CAR+ cells/kg , ^2x106 to ^1x108 CAR+ cells/kg, ^3x106 to ^1x108 CAR+ cells/kg, ^4x106 to ^1x108 CAR+ cells/kg, ^5x106 to ^1x108 CAR+ cells/kg, ^6x106 to ^1x108 CAR+ cells/kg, 7x10 ⁶ to 1x10 ⁸ CAR+ cells/kg, 8x10 ⁶ to 1x10 ⁸ CAR+ cells/kg, 9x10 ⁶ to 1x10 ⁸ CAR+ cells/kg, 1x10 ⁷ to 1x10 ⁸ CAR+ cells/kg, ^1.5x107 to ^1x108 CAR+ cells/kg, ^1.8x107 to ^1x108 CAR+ cells/kg, ^2x107 to ^1x108 CAR+ cells/kg, ^3x107 to ^1x108 CAR+ cells/kg, ^4x107 to 1x108 CAR+ cells/kg ⁸ CAR+ cells/kg, 5x10 ⁷ to 1x10 ⁸ CAR+ cells/kg, 6x10 ⁷ to 1x10 ⁸ CAR+ cells/kg, 7x10 ⁷ to 1x10 ⁸ CAR+ cells/kg, or 8x10 ⁷ to 1x10 ⁸ CAR+ cells/kg kg. In one embodiment, the first or subsequent dose is selected from ^0.1x106 to ^1x108 CAR+ cells/kg, 0.5x106 to ^5x107 CAR+ cells/kg, ^1x106 to ^{5x107 CAR} + cells/kg , ^2x106 to ^5x107 CAR+ cells/kg, 3x106 to ^5x107 CAR+ cells/kg, ^4x106 to ^5x107 CAR+ cells/kg, ^5x106 to ^{5x107 CAR} + cells/kg, ^5x106 to ^4x107 CAR+ cells/kg, or 5x10 ⁶ to 3x10 ⁷ CAR+ cells/kg. In a preferred embodiment, the first dose or subsequent dose is selected from 1x10 ⁶ to 5x10 ⁷ CAR+ cells/kg, or 5x10 ⁶ to 3x10 ⁷ CAR+ cells/kg, such as 3x10 ⁶ CAR+ cells/kg. kg, 4x10 ⁶ CAR+ cells/kg, 5x10 ⁶ CAR+ cells/kg, 6x10 ⁶ CAR+ cells/kg, 7x10 ⁶ CAR+ cells/kg, 8x10 ⁶ CAR+ cells/kg, 9x10 ⁶ CAR+ cells/kg, 1x10 ⁷ CAR+ cells/kg, 1.5x10 ⁷ CAR+ cells/kg, 1.8x10 ⁷ CAR+ cells/kg, 2x10 ⁷ CAR+ cells/kg, 2.5x10 ⁷ CAR+ cells/kg, 3x10 ⁷ CAR+ cells/kg , 4x10 ⁷ CAR+ cells/kg, or 5x10 ⁷ CAR+ cells/kg.

In one embodiment, the number of CAR+ cells administered in said first or subsequent dose amounts to about ^5x106 to ^10x109 CAR+ cells, ^7.5x106 to ^5x109 CAR+ cells, ^1x107 to ^5x109 CAR+ cells , ^2.5x107 to ^5x109 CAR+ cells, ^5x107 to ^5x109 CAR+ cells, ^7.5x107 to ^5x109 CAR+ cells, ^1x108 to ^5x109 CAR+ cells, about ^2x108 to ^4x109 CAR+ cells, or Approximately 2x10 ⁸ to 3x10 ⁹ CAR+ cells.

In one embodiment, one or more doses, eg, 2, 3, 4, 5, 6 or more doses, may be administered to a subject. Where multiple doses are administered, at least 5-90 days (e.g., at least 5-80, 5-70, 5-60, 5-50, 5-40, 5-30 or 5-25 days), a subsequent dose (e.g., a second dose) of engineered immune cells is administered at a time point, e.g., on day 5, day 7, day 9, day 12, Administer subsequent doses of engineered immunization on day 15, day 18, day 22, day 25, day 30, day 40, day 50, day 60, day 70, day 80 or day 90 cell.

In one embodiment, a given dose is administered in divided doses, eg, a first dose or a subsequent dose. A given dose may be administered to a subject in the same or different divided doses over 2 or 3 days. For example, 25% of the divided dose on the first day and the remaining 75% of the divided dose on the second day; or 50% of the divided dose on the first day and the remaining 50% of the divided dose on the second day Dosage; or 10% of the divided dose on the first day, 30% of the divided dose on the second day, and 60% of the divided dose on the third day.

In some embodiments, the subject's tumor burden is stabilized or reduced following administration of the first dose of engineered immune cells. Preferably, one or more subsequent doses are administered after the tumor burden has been stabilized or reduced with the first dose, but before an adaptive host immune response (ie, immune rejection of the CAR-T cells by the body) has developed. Under such conditions, subsequent doses can safely and effectively provide immune surveillance, eradicate residual tumor cells, or prevent proliferation or metastasis of residual tumor cells. Thus, in some embodiments, the subsequent dose is a disease consolidating dose.

As used herein, the term "tumor burden" includes, but is not limited to, tumor volume or degree of differentiation, or type of metastasis, stage, and/or appearance and disappearance of common complications in advanced cancers, and/or appearance or expression of tumor markers Changes in levels, and/or likelihood or incidence of toxic outcomes (e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or host immune response against administered cells) in the subject . In some embodiments, tumor size is measured by PET (Positron Emission Computed Tomography) and CT (Computed Tomography) scales.

Tumor markers refer to substances that are characteristically present in malignant tumor cells, or abnormally produced by malignant tumor cells, or produced by the host in response to tumor stimulation, and can reflect tumor occurrence and development, and monitor tumor response to treatment. A class of substances. Tumor markers exist in the tissues, body fluids and excreta of tumor patients, and can be detected by immunological, biological and chemical methods, including alpha-fetoprotein (AFP), CA125, CA15-3, squamous cell carcinoma antigen ( SCC), soluble fragment of cytokeratin 19 (CYFRA21-1), carcinoembryonic antigen (CEA), CA199, CA724, etc.

In one embodiment, the subsequent dose of engineered immune cells is administered when the subject after receiving the first dose of engineered immune cells:

(i) the subject has a serum level of a factor indicative of cytokine release syndrome (CRS) that is multiple times less than about 10 times less, about 25 times less than the level in the subject immediately prior to administration of said first dose, and / or about 50 times smaller;

(ii) have not demonstrated grade 3 or higher neurotoxicity;

(iii) neurotoxicity or CRS levels are reduced compared to peak levels of neurotoxicity or CRS levels following administration of the first dose of engineered immune cells; or

(iv) the subject does not exhibit a detectable immune response (eg, a humoral or cell-mediated immune response) against the CAR expressed by the first dose of engineered immune cells.

In another embodiment, after the subject receives the first dose of engineered immune cells, the subject is regularly administered subsequent doses of engineered immune cells, for example, every 5 weeks, 6 weeks, 7 weeks, 8 weeks , 9 weeks, or 10 weeks to administer subsequent doses of the engineered immune cells to the subject. In this embodiment, the subject receives a total of 3, 4, 5, 6, 7, 8, 9 or 10 doses of engineered immune cells. In a preferred embodiment, the subject receives a lymphodepletion regimen prior to each subsequent dose.

In one embodiment, a detectable immune response refers to a specific immune response to specific antigens and cells that can be detected by any known method. For example, a specific type of immune response can be detected by performing ELISPOT, ELISA or antibody detection (eg, by flow cytometry) on the subject's serum to detect the presence of antibodies that specifically bind to cell surface antigens.

In one embodiment, the dose of engineered immune cells administered to the subject each time, and the interval between multiple doses are determined by the doctor according to the specific conditions of the subject, including but not limited to the age of the subject , gender, weight, overall health, disease severity, previous therapy received, response to the previous dose of the same course of treatment, response to previous course of treatment, combined drug use, degree of toxicity, complications, cancer metastasis, etc., and any other factors that, in the opinion of the physician, will affect the determination of the amount of engineered immune cells suitable for administration to the subject.

B Lymphocyte Depletion Protocol

In all embodiments, the methods of the invention comprise subjecting the subject to a lymphodepleting regimen prior to the first dose. In some embodiments, when the subject receives a subsequent dose, the method comprises administering a lymphodepletion regimen prior to administering the subsequent dose to the subject (i.e., between administering the first dose and the subsequent dose, again Subjects were subjected to a lymphodepletion regimen). In other embodiments, no additional lymphodepletion regimen is administered to the subject prior to administration of the subsequent dose (i.e., the subject receives a lymphodepletion regimen only prior to the first dose, and the subsequent dose is administered directly when the subsequent dose is administered). engineer immune cells without additional lymphodepletion). The present invention also provides a composition for removing lymphocytes.

In one embodiment, the lymphodepletion regimen or composition for lymphodepletion comprises cyclophosphamide, fludarabine, etoposide, melphalan, or combinations thereof. In one embodiment, the lymphodepletion regimen or composition for lymphodepletion comprises cyclophosphamide, fludarabine and etoposide. In one embodiment, the lymphodepletion regimen or composition for lymphodepletion comprises cyclophosphamide, fludarabine and melphalan.

In certain embodiments, the lymphodepletion regimen or composition for lymphocyte depletion comprises a dose of about 10-60 mg/m ² /day, 10-50 mg/m ² /day, 15-40 mg/m ² /day , or 15-35mg/m ² /day, such as about 10mg/m ² /day, 15mg/m ² /day, 20mg/m ² /day, 25mg/m ² /day, 30mg/m ² /day, 35mg Fludarabine/m ² /day, 40mg/m ² /day, 45mg/m ² /day, 50mg/m ² /day 55mg/m ² /day, or 65mg/m ² /day; and/or dose is about 100-700 mg/m ² /day, 150-650 mg/m ² /day, 200-600 mg/m ² /day, 250-600 mg/m ² /day, or 300-600 mg/m ² /day, for example About 100mg/m ² /day, 150mg/m ² /day, 200mg/m ² /day, 250mg/m ² /day, 300mg/m ² /day, 325mg/m ² /day, 350mg/m ² /day, 375mg/m ² /day, 400mg/m ² /day, 425mg/m ² /day, 450mg/m ² /day, 475mg/m ² /day, 500mg/m ² /day, 550mg/m ² /day, 600mg /m ² /day, 650mg/m ² /day, or 700mg/m ² /day of cyclophosphamide; and/or a dose of about 50-150mg/day, 50-125mg/day, 50-100mg/day, for example Etoposide at about 50 mg/day, 60 mg/day, 70 mg/day, 80 mg/day, 90 mg/day, 100 mg/day, 125 mg/day, or 150 mg/day; and/or at a dose of about 50-150 mg/m ² /day, 50-125mg/m ² /day, or 50-100mg/m ² /day, such as about 50mg/m ² /day, 60mg/m ² /day, 70mg/m ² /day, 80mg/m ² /day, 90 mg/m ² /day, 100 mg/m ² /day, 125 mg/m ² /day, or 150 mg/m ² /day of melphalan.

In certain embodiments, the total dose of fludarabine administered is about 30-250 mg/m ² , 50-200 mg/m ² , 60-180 mg/m ² , 75-175 mg/m ² , or 75- 100mg/m ² , for example about 30mg/m ² , 50mg/m ² , 60mg/m ² , 75mg/m ² , 90mg/m ² , 100mg/m ² , 125mg/m ² , 150mg/m ² , 175mg/m 2 m ² , 180 mg/m ² , 200 mg/m ² , 225 mg/m ² , or 250 mg/m ² . In certain embodiments, the total dose of cyclophosphamide administered is about 300-3500 mg/m ² , 500-3000 mg/m ² , 750-2500 mg/m ² , 1000-2500 mg/m ² , or 1000-2000 mg/m 2 m ² , for example about 300 mg/m ² , 500 mg/m ² , 750 mg/m ² , 1000 mg/m ² , 1100 mg/m ² , 1200 mg/m ² , 1300 mg/m ² , 1400 mg/m ² , 1500 mg/m ² , 1600mg/m ² , 1700mg/m ² , 1800mg/m ² , 1900mg/m ² , 2000mg/m ² , 2250mg/m ² , 2500mg/m ² , 2750mg/m ² , 3000mg/m ² , 3250mg/m ² , or 3500 mg/m ² . In certain embodiments, the total dose of etoposide administered is about 150-750 mg, 200-700 mg, 250-600 mg, or 300-500 mg, such as about 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg , 500mg, 550mg, 600mg, 650mg, 700mg, or 750mg. In certain embodiments, the total dose of melphalan administered is about 50-750 mg/m ² , 50-600 mg/m ² , 50-500 mg/m ² , 50-400 mg/m ² , or 50-300 mg/m ² , for example about 50 mg/m ² , 75 mg/m ² , 100 mg/m ² , 200 mg/m ² , 300 mg/m ² , 400 mg/m ² , 500 mg/m ² , 600 mg/m ² , or 700 mg/m ² .

The time of administration of the engineered immune cells (e.g., the time of administration of the first dose or subsequent doses) is designated as day 0 (D0), so for example, D-2 refers to the second day before the administration of the engineered immune cells, and D2 is Refers to day 2 after administration of engineered immune cells. In certain embodiments, administration begins at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days prior to administration of the engineered immune cells (e.g., first dose or subsequent doses) Lymphodepletion regimens, including cyclophosphamide, fludarabine, etoposide, and/or melphalan. Preferably, administration of a lymphodepleting regimen consisting of cyclophosphamide, fludarabine, etoposide is initiated 7 (ie D-7), 8, 9, 10, 11 or 12 days prior to administration of the first or subsequent dose and/or melphalan.

In certain embodiments, the lymphodepletion regimen (comprising cyclophosphamide, fludarabine, etoposide, and/or melphalan) is administered intermittently or continuously for 1, 2, 3, 4, 5, 6, or 7 days. For example, in some embodiments, cyclophosphamide is administered daily for 2, 3, 4, or 5 consecutive days; fludarabine is administered daily for 3, 4, or 5 consecutive days; etopol is administered daily Glycosides were administered continuously for 3 days, 4 days or 5 days; melphalan was administered daily for 1 day or continuously applied for 2 days or 3 days.

In certain embodiments, cyclophosphamide, fludarabine, etoposide, and/or melphalan may be administered on the same day or on different days. In certain embodiments, cyclophosphamide, fludarabine, etoposide, and/or melphalan may be administered simultaneously or sequentially. Cyclophosphamide, fludarabine, etoposide and/or melphalan may be administered by any suitable route, eg, by intravenous injection or intravenous infusion.

C Subject Screening and Assessment

In one embodiment, the subject has received one or more, such as 2, 3, 4, 5, 6, 7 or 8, prior to receiving the treatment method of the present invention. previous treatment. Such prior treatment includes, but is not limited to, stem cell transplantation, radiation therapy, chemotherapy, antibody therapy, small molecule targeted therapy, or other engineered immune cell therapy, etc.

Cell kinetics and cytokine kinetics in the subject are assessed following administration of the first dose or subsequent doses. For example, quantitative PCR or flow cytometry can be used to assess the amount of chimeric antigen receptor-expressing cells (ie, CAR+ cells) in the subject's blood or organs or tissues, thereby evaluating the effect of engineered immune cells in the subject. proliferation and persistence. In one embodiment, the release levels of cytokines associated with cytokine release syndrome (CRS), such as IL6, IL2, IFN-γ, etc., are also assessed by flow cytometry.

Following administration of the first dose or subsequent doses, the subject's disease status is assessed to assess its response to UCAR-T cells. For example, the incidence and grade of immune effector cell-associated neurotoxicity (ICANS) and cytokine release syndrome (CRS) were assessed according to the American Society for Transplantation and Cellular Therapy (ASTCT) criteria, as shown in Tables 1 and 2. Graft versus host disease (GvHD) was also assessed according to National Comprehensive Cancer Network (NCCN) criteria.

Table 1. Exemplary Grading Criteria for ICANS

Table 2. Exemplary Grading Criteria for CRS

D indications

In one embodiment, diseases associated with CD7 expression include CD7-positive hematological tumors (eg, leukemias and lymphomas) and solid tumors. Hematological neoplasms are cancers of the blood or bone marrow, including but not limited to acute leukemias such as acute lymphoblastic leukemia (ALL, eg T-ALL, NK-ALL), acute myeloid leukemia (AML), acute myelogenous leukemia and myeloid cellular, promyelocytic, myelomonocytic, monocytic, and erythroleukemia; chronic leukemia, such as chronic myelogenous leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia; non-Hodgkin's lymphoma ( indolent and high-grade forms), such as T-lymphoblastic lymphoma (T-LBL), peripheral T-cell lymphoma (PTCL), extranodal NK/T-cell lymphoma, γδT-cell lymphoma; T-cell large granular lymphoma Leukemia (T-LGL), Polycythemia Vera, Lymphoma, Hodgkin Lymphoma, Angioimmunoblastic T-Cell Lymphoma (AITL), Anaplastic Large Cell Lymphoma (ALCL), Multiple Myeloma, Val Denstrom's macroglobulinemia, myelodysplastic syndrome, hairy cell leukemia, Burkitt's lymphoma, diffuse large cell lymphoma, mantle cell lymphoma, early pro-T lymphoblastic leukemia (ETP-ALL) ), small lymphocytic lymphoma (SLL), and myelodysplasia. Solid tumors are abnormal masses of tissue that usually do not contain cysts or areas of fluid, which can be benign or malignant. The different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, pancreatic, ovarian, peritoneal, omental, and mesenteric, pharyngeal, prostate, rectal, renal, skin, Small intestine cancer, melanoma, kidney cancer, laryngeal cancer, soft tissue cancer, stomach cancer, testicular cancer, colon cancer, esophagus cancer, cervical cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, anal cancer , eye cancer, intrahepatic cholangiocarcinoma, joint cancer, neck cancer, gallbladder cancer, pleural cancer, nasal cancer, middle ear cancer, oral cancer, vulvar cancer, thyroid cancer and ureter cancer.

In one embodiment, the disease associated with CD7 expression is preferably selected from CD7-positive acute lymphoblastic leukemia (ALL, such as T-ALL, NK-ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia, chronic Lymphocytic leukemia, chronic myelogenous leukemia, T-cell large granular lymphocytic leukemia (T-LGL), non-Hodgkin's lymphoma (such as T-lymphoblastic lymphoma (T-LBL), peripheral T-cell lymphoma (PTCL) ), extranodal NK/T-cell lymphoma, γδT-cell lymphoma) and early pro-T lymphoblastic leukemia (ETP-ALL).

II Chimeric Antigen Receptor

The engineered immune cells provided by the present invention comprise chimeric antigen receptors that specifically bind CD7 antigen.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes an antigen-binding region (such as an antibody or antigen-binding portion thereof), a transmembrane domain, any The selected co-stimulatory domain and the primary signal transduction domain are connected by linkers. CARs are able to exploit the antigen-binding properties of antibodies to redirect the specificity and reactivity of T cells and other immune cells to a target of choice in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition confers on CAR-expressing immune cells the ability to recognize antigens independently of antigen processing, thus bypassing major mechanisms of tumor escape.

In one embodiment, the CAR expressed by the engineered immune cells provided by the present invention comprises an antigen-binding region targeting CD7, a transmembrane domain and an intracellular signaling region, and the intracellular signaling region comprises a co-stimulatory domain and Primary signaling domain. In a preferred embodiment, the intracellular signaling region of the chimeric antigen receptor of the present invention further comprises a γc chain or its intracellular region. Still more preferably, in one embodiment, the intracellular signaling region consists of a co-stimulatory domain, a primary signaling domain, and a γc chain or an intracellular region thereof; that is, the intracellular domain of a chimeric antigen receptor Except for costimulatory domain, primary signal transduction domain, and γc chain or its intracellular region, the signal transduction domain does not contain other structures with signal transduction function.

As used herein, "antigen-binding region" refers to any structure or functional variant thereof that can bind to an antigen. The antigen binding region can be an antibody or an antigen binding portion thereof. As used herein, the term "antibody" has the broadest meaning understood by those skilled in the art and includes monoclonal antibodies (including whole antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (such as bispecific antibodies) ), and antibody fragments or synthetic polypeptides carrying one or more CDR sequences capable of exhibiting the desired biological activity. Antibodies of the present invention also include recombinant antibodies, human antibodies, humanized antibodies, murine antibodies, chimeric antibodies, and antigen-binding portions thereof. "Antibody fragment" or "antigen-binding portion" refers to a portion of an intact antibody, generally comprising the antigen-binding site of the intact antibody and thus retaining the ability to bind antigen. Examples of antibody fragments in the present invention include, but are not limited to: Fab, Fab', F(ab') ₂ , Fd fragment, Fd', Fv fragment, scFv, disulfide-linked Fv (sdFv), antibody heavy Chain variable (VH) or light chain variable (VL), linear antibodies, "diabodies" with two antigen binding sites, single domain antibodies, Nanobodies, natural ligands for said antigens or Functional fragments etc. Accordingly, an "antibody" of the invention encompasses antibody fragments or antigen-binding portions as defined above.

Thus, in one embodiment, the CD7-targeting antigen binding region of the invention is selected from the group consisting of IgG, Fab, Fab', F(ab') ₂ , Fd, Fd', Fv, scFv, sdFv, linear antibody, single structure Domain antibodies, Nanobodies and diabodies are preferably selected from Fab, scFv, single domain antibodies and Nanobodies, most preferably scFv. In the present invention, the antigen binding domain may be monovalent or bivalent, and may be a monospecific, bispecific or multispecific antibody.

"Fab" refers to either of two identical fragments of an immunoglobulin molecule produced after cleavage by papain, consisting of the complete light chain and the N-terminal portion of the heavy chain linked by a disulfide bond, wherein the N-terminal portion of the heavy chain includes Heavy chain variable region and CH1. Compared with intact IgG, Fab has no Fc fragment, has higher fluidity and tissue penetration ability, and can monovalently bind antigen without mediating antibody effect.

"Single-chain antibody" and "scFv" are used interchangeably herein, and refer to an antibody formed by linking an antibody heavy chain variable region (VH) and a light chain variable region (VL) through a linker. The optimal length and/or amino acid composition of the linker can be determined as desired. The length of the linker can significantly affect the variable domain folding and interaction of scFv. In fact, if shorter linkers (eg, between 5-10 amino acids) are used, intrachain folding can be prevented. For selection of linker size and composition, see, e.g., Hollinger et al., 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448; U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794; and PCT Publication No. WO2006/020258 and WO2007/024715 are hereby incorporated by reference in their entirety. Commonly used linkers such as GSTSGSGKPGSGEGSTKG (SEQ ID NO: 43), GGGGSGGGGSGGGGS (SEQ ID NO: 44). A scFv may comprise VH and VL linked in any order, eg VH-linker-VL or VL-linker-VH.

"Single domain antibody" or "sdAb" refers to an antibody naturally devoid of light chains, which contains only a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, also known as "heavy chain Antibody".

"Nanobody" or "Nb" refers to the VHH structure cloned and expressed separately, which has the same structural stability and antigen-binding activity as the original heavy chain antibody, and is currently the smallest unit known to bind the target antigen .

The term "functional variant" or "functional fragment" refers to a variant comprising essentially the amino acid sequence of a parent but containing at least one amino acid modification (i.e. substitution, deletion or insertion) compared to the parent amino acid sequence, provided that the Such variants retain the biological activity of the parent amino acid sequence. In one embodiment, the amino acid modification is preferably a conservative modification.

As used herein, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment comprising the amino acid sequence. These conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into chimeric antigen receptors of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid, ), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.

Thus, a "functional variant" or "functional fragment" has at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% of the parent amino acid sequence. %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, And retain the biological activity of the parent amino acid, such as binding activity.

As used herein, the term "sequence identity" means the degree to which two (nucleotide or amino acid) sequences in an alignment have the same residue at the same position, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Therefore, two copies of the exact same sequence have 100% identity. Those skilled in the art will recognize that several algorithms can be used to determine sequence identity using standard parameters, 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 one embodiment, the CD7-targeted antigen binding region comprised by the chimeric antigen receptor of the present invention is an anti-CD7 antibody or its antigen receptor, which comprises the CDRs shown in SEQ ID NO: 1, 2, and 3 -L1, CDR-L2 and CDR-L3, and CDR-H1, CDR-H2 and CDR-H3 as shown in SEQ ID NO: 4, 5 and 6. Preferably, the antibody targeting CD7 of the present invention comprises at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 98%, The light chain variable region with 99% or 100% sequence identity and at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, Heavy chain variable regions of 98%, 99% or 100% sequence identity. More preferably, the chimeric antigen receptor of the present invention comprises an anti-CD7 antibody, the amino acid sequence of which is shown in SEQ ID NO:9.

As used herein, the term "transmembrane domain" refers to a polypeptide capable of expressing a chimeric antigen receptor on the surface of an immune cell (such as a lymphocyte, NK cell or NKT cell) and directing a cellular response of the immune cell against a target cell structure. Transmembrane domains can be natural or synthetic and can be derived from any membrane-bound or transmembrane protein. The transmembrane domain is capable of signaling when the chimeric receptor polypeptide binds to the target antigen. Transmembrane domains particularly suitable for use in the present invention may be derived from, for example, TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and functional fragments thereof. Alternatively, the transmembrane domain may be synthetic and may comprise predominantly hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from human CD8α chain, which has at least 70%, preferably at least 80%, of the amino acid sequence shown in SEQ ID NO:19 or the nucleotide sequence shown in SEQ ID NO:20 , more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

In one embodiment, the chimeric antigen receptor of the present invention may further comprise a hinge region located between the antigen binding region and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to an antigen binding region. Specifically, the hinge region is used to provide greater flexibility and accessibility to the antigen binding region. 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, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be an entirely synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a part of the hinge region of a CD8α chain, CD28, FcγRIIIα receptor, IgG4 or IgG1, more preferably a CD8α, CD28 or IgG4 hinge, which is identical to SEQ ID NO: 33, 35 or 37. The amino acid sequence shown or have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the nucleotide sequence shown in SEQ ID NO: 34, 36 or 38 sequence identity.

As used herein, the term "primary signaling domain" refers to the portion of a protein that transduces effector function signals and directs the cell to perform a given function. The primary signaling domain is responsible for intracellular primary signaling following antigen binding at the antigen binding region, resulting in activation of immune cells and immune responses. In other words, the primary signaling domain is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed. For example, the effector function of a T cell can be cytolytic activity or helper activity, including secretion of cytokines.

In one embodiment, the primary signaling domain comprised by the chimeric antigen receptors of the invention may be the cytoplasmic sequence of a T cell receptor and a co-receptor, which act together to initiate primary signaling following antigen receptor binding , and any derivatives or variants of these sequences and any synthetic sequences having the same or similar function. The primary signaling domain can contain many immunoreceptor tyrosine-based activation motifs (Immunoreceptor Tyrosine-based Activation Motifs, ITAM). Non-limiting examples of primary signaling domains of the invention include, but are not limited to, those derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a preferred embodiment, the primary signal transduction domain of the CAR of the present invention may comprise a CD3ζ signaling domain, which is identical to the amino acid sequence shown in SEQ ID NO: 27 or the amino acid sequence shown in SEQ ID NO: 28. The nucleotide sequences have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

In one embodiment, a chimeric antigen receptor of the invention comprises one or more co-stimulatory domains. A co-stimulatory domain may be an intracellular functional signaling domain from a co-stimulatory molecule comprising the entire intracellular portion of said co-stimulatory molecule, or a functional fragment thereof. A "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (eg, proliferation) of the T cell. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Non-limiting examples of co-stimulatory domains of the invention include, but are not limited to, intracellular regions derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134(OX40), CD137(4-1BB), CD270(HVEM), CD272(BTLA), CD276(B7-H3), CD278(ICOS ), CD357(GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP70.

In a preferred embodiment, the co-stimulatory domain comprises one or more intracellular regions of a protein selected from DAP10, DAP12, CD27, CD28, CD134, 4-1BB or CD278. For example, in one embodiment, the co-stimulatory domain comprises the intracellular region of 4-1BB. In one embodiment, the co-stimulatory domain comprises the intracellular region of CD28. In one embodiment, the co-stimulatory domain comprises the intracellular region of 4-1BB and the intracellular region of CD28.

In one embodiment, the intracellular region of 4-1BB has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO: 25 % sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% with the nucleotide sequence shown in SEQ ID NO:26 sequence identity. In one embodiment, the intracellular region of CD28 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO:23. Sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence with the nucleotide sequence shown in SEQ ID NO:24 identity.

In one embodiment, in addition to the co-stimulatory domain and cellular primary signaling domain for signal transduction, the chimeric antigen receptor of the present invention may also comprise a γc chain or its intracellular region to enhance signal transduction.

In a more preferred embodiment, the intracellular signaling region (i.e., the structure for signaling) of the chimeric antigen receptor of the present invention consists of a costimulatory domain, a primary signaling domain, and a γc chain or The intracellular domain consists of these three signaling structures. This means that in this embodiment the chimeric antigen receptor does not comprise a fourth signaling structure such as the signaling region of other cytokines such as IL-2Ra, IL2Ra, IL2Rb, IL4Ra, IL7Ra, IL9Ra, IL15Ra, IL21Ra and other intracellular regions.

In one embodiment, the γc chain that can be used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity. In one embodiment, the γc chain intracellular region that can be used in the present invention has at least 70%, preferably at least 80%, of the amino acid sequence shown in SEQ ID NO:42 or the nucleotide sequence shown in SEQ ID NO:41, More preferably at least 90%, 95%, 97% or 99% or 100% sequence identity. Preferably, the γc chain of the present invention is shown in SEQ ID NO: 40, and its intracellular region is shown in SEQ ID NO: 42.

In one embodiment, the CAR of the invention may also comprise a signal peptide such that when it is expressed in a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. The core of the signal peptide may contain a long stretch of hydrophobic amino acids with a propensity to form a single α-helix. At the end of the signal peptide, there is usually a stretch of amino acids that is recognized and cleaved by the signal peptidase. The signal peptidase can cleave during translocation or after completion to generate a free signal peptide and mature protein. Then, the free signal peptide is digested by specific proteases. Signal peptides that can be used in the present invention are well known to those skilled in the art, for example, signal peptides derived from CD8α, IgG1, GM-CSFRα, and the like.

In one embodiment, the CAR of the present invention may also include a switch structure to regulate the expression time of the CAR. For example, the switch structure can be in the form of a dimerization domain, which causes a conformational change through binding with its corresponding ligand, exposing the extracellular antigen-binding region, allowing it to bind to the targeted antigen, thereby activating the signal transduction pathway. Alternatively, a switch structure can also be used to link the antigen-binding region and the signaling domain separately, and only when the switch structures are associated with each other (for example, in the presence of an inducing compound), the antigen-binding region and the signaling domain can be linked by dimerization together, thereby activating the signaling pathway. The switch structure can also be in the form of a masking peptide. The masking peptide can mask the extracellular antigen-binding region and prevent it from binding to the targeted antigen. When the masking peptide is cleaved by, for example, protease, the extracellular antigen-binding region can be exposed, making it a "normal" CAR structure . Various switch configurations known to those skilled in the art can be used in the present invention.

In one embodiment, the CAR of the present invention may also contain a suicide gene, that is, to express a cell death signal that can be induced by an exogenous substance, so as to eliminate CAR cells when necessary (eg, when severe toxic side effects occur). For example, suicide genes can be in the form of inserted epitopes, such as CD20 epitopes, RQR8, etc., and when necessary, CAR cells can be eliminated by adding antibodies or reagents targeting these epitopes. The suicide gene can also be herpes simplex virus thymidine kinase (HSV-TK), which causes cell death induced by ganciclovir treatment. The suicide gene can also be iCaspase-9, and the dimerization of iCaspase-9 can be induced by chemically inducing drugs such as AP1903 and AP20187, thereby activating the downstream Caspase3 molecule and leading to cell apoptosis. Various suicide genes known to those skilled in the art can be used in the present invention.

III Engineered Immune Cells

A. Immune cells

As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). For example, immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells. In one embodiment, the immune cells are derived from stem cells, such as adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells, among others. Preferably, the immune cells are T cells. The T cells may be any T cells, such as T cells cultured in vitro, such as primary T cells, or T cells from T cell lines cultured in vitro, such as Jurkat, SupT1, etc., or T cells 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 a site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be enriched or purified. T cells can be of any type and at any developmental stage, including, but not limited to, CD4+/CD8+ double positive 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, etc. In a preferred embodiment, the immune cells are human T cells. T cells can be obtained from the blood of a subject using a variety of techniques known to those of skill in the art, such as Ficoll separation. In the present invention, immune cells are engineered to express chimeric antigen receptor polypeptides.

In one embodiment, the engineered immune cells administered to a subject comprise multiple cell populations or subtypes (eg, CD4+ and CD8+ cells or subtypes). For example, engineered immune cells can comprise a ratio of CD4+ and CD8+ cells that is between 1:5 and 5:1, between 1:3 and 3:1, or 1:5 (CD4+ cells:CD8+ cells) Between 2 and 2:1, such as 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1: 1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5 or 1:5.

B. Suppression or silencing of endogenous genes

In one embodiment, the expression of endogenous CD7, at least one TCR/CD3 gene and at least one MHC-II related gene of the engineered immune cells provided by the present invention is suppressed or silenced.

In one embodiment, at least one TCR/CD3 gene is selected from: TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ, preferably TRAC or TRBC.

In one embodiment, the at least one MHC class II-associated gene includes the MHC class II gene itself, as well as genes that interact with or regulate the expression of the MHC class II gene. For example, at least one MHC class II related gene is selected from: HLA-DPA, HLA-DQ, HLA-DRA, RFX5, RFXAP, RFXANK and CIITA, preferably selected from RFX5, RFXAP, RFXANK and CIITA.

In one embodiment, the endogenous MHC-class I genes (eg, HLA-A, HLA-B, HLA-C, B2M, etc.) in the CAR-T cells are functional. In another embodiment, the expression of endogenous MHC-class I genes in CAR-T cells is also suppressed or silenced.

In one embodiment, in addition to CD7, MHC-II-related genes and TCR/CD3 genes, the engineered immune cells of the present invention may also contain at least one gene selected from the group whose expression is suppressed or silenced: CD52, GR, dCK and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3.

Methods for inhibiting gene expression or gene silencing are well known to those skilled in the art, including but not limited to, for example, mediating DNA fragmentation by meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in the CRISPR system, or by Antisense oligonucleotides, RNAi, shRNA and other technologies inactivate genes.

C. Expression of NK inhibitory molecules

In one embodiment, in order to inhibit the killing of CAR-T cells by NK cells in the patient, the engineered immune cells further express NK inhibitory molecules, and the NK inhibitory molecules comprise one or more NK inhibitory ligands, Transmembrane domain and co-stimulatory domain. For example, the NK inhibitory molecule comprises one or two NK inhibitory ligands, a transmembrane domain and a co-stimulatory domain.

In one embodiment, the NK inhibitory molecule does not comprise a primary signaling domain. In another embodiment, the NK inhibitory molecule further comprises a primary signaling domain.

The definitions of the transmembrane domain, co-stimulatory domain and primary signaling domain contained in NK inhibitory molecules are the same as those contained in the "Chimeric Antigen Receptor" section above The domains are defined the same.

In one embodiment, the NK inhibitory ligand is an antibody targeting an NK inhibitory receptor selected from the group consisting of NKG2/CD94 components (e.g. NKG2A, NKG2B, CD94); killer cell Ig Ig-like receptor (KIR) family members (eg, KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, and KIR3DL3); leukocyte Ig-like receptor (LIR) family members (eg, LIR1, LIR2, LIR3, LIR5, and LIR8); NK cell receptor protein 1 (NKR-P1) family members (eg, NKR-P1B and NKR-P1D); immune checkpoint receptors (eg, PD-1, TIGIT, and CD96, TIM3, LAG3); carcinoembryonic antigen-associated cells Adhesion molecule 1 (CEACAM1); sialic acid-binding immunoglobulin-like lectin (SIGLEC) family members (eg, SIGLEC7 and SIGLEC9); leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Ly49 family members (eg, Ly49A, Ly49C, Ly49F, Ly49G1 and Ly49G4) and killer lectin-like receptor G1 (KLRG1). Preferably, the NK inhibitory receptors are preferably selected from NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, CEACAM1, LAIR1, NKR- P1B, NKR-P1D, PD-1, TIGIT, CD96, TIM3, LAG3, SIGLEC7, SIGLEC9, Ly49A, Ly49C, Ly49F, Ly49G1, Ly49G4 and KLRG1. More preferably, the NK inhibitory receptor is selected from NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, KIR2DL1, KIR2DL2/3, KIR3DL1, CEACAM1, LAIR1 and KLRG1. Still more preferably, the NK inhibitory receptor is selected from NKG2A, NKG2B, LIR1, KIR2DL1, KIR2DL2/3, KIR3DL1, CEACAM1, LAIR1 and KLRG1.

In one embodiment, the NK inhibitory ligand is an antibody targeting the NK inhibitory receptor, said antibody being a whole antibody, Fab, Fab', F(ab')2, Fv fragment, scFv antibody fragment, linear antibody , sdAb or nanobody.

In a preferred embodiment, the NK inhibitory ligand is an antibody targeting NKG2A. More preferably, the antibody targeting NKG2A comprises CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NO: 10, 11, and 12, and as shown in SEQ ID NO: 13, 14 and 15 CDR-H1, CDR-H2 and CDR-H3 are indicated. More preferably, the antibody targeting NKG2A comprises at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% of the amino acid sequence shown in SEQ ID NO: 16 A light chain variable region sequence of sequence identity and at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 17 identity of the heavy chain variable region sequence. More preferably, the amino acid sequence of the antibody targeting NKG2A is shown in SEQ ID NO: 18. Other antibodies targeting NKG2A known in the art can also be used in the present invention, such as Z270 (available from Immunotech, France), Z199 (available from Beckman Coulter, USA), 20D5 (available from BD Biosciences Pharmingen, USA) , P25 (available from Moretta et al, Univ. Genova, Italy) and the like.

IV Compositions and Articles

The engineered immune cells provided by the present invention are generally administered to the subject in the form of a pharmaceutical composition, the pharmaceutical composition comprising the above-defined engineered immune cells as an active agent, and one or more pharmaceutically acceptable excipients Formulations.

As used herein, the term "pharmaceutically acceptable excipient" means pharmacologically and/or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any adverse desired local or systemic effect), which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesive agents, glidants, antioxidants, flavoring agents, coloring agents, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity regulators . The selection of suitable excipients is known to those skilled in the art for the preparation of the desired pharmaceutical compositions of the present invention. In general, the selection of suitable excipients depends inter alia on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.

In a specific embodiment, the excipients include one or more selected from the group consisting of preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzeth Ammonium chloride, phenol, butanol or benzyl alcohol, alkylparabens (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, 3 - Pentanol, m-cresol; buffers such as phosphate, citrate and other organic acids; antioxidants such as ascorbic acid and methionine; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; Sugars and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG).

The composition may also contain multiple active ingredients, which are useful for a particular indication, disease or condition to be prevented or treated with engineered immune cells, where the individual activities do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for their intended purpose. Accordingly, in some embodiments, the pharmaceutical composition further comprises other active ingredients in addition to engineered immune cells, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, Doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.

Active ingredients (e.g., engineered immune cells and/or other active ingredients) can be embedded in microcapsules, colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions middle. In certain embodiments, pharmaceutical compositions are formulated as inclusion complexes, or as liposomes. Liposomes can be used to target active ingredients (e.g., engineered T cells or NK cells) to specific tissues.

In some aspects, the pharmaceutical compositions may employ time-release, delayed-release, and sustained-release delivery systems such that delivery of the composition occurs prior to sensitization of the site to be treated, and with sufficient time to cause sensitization. Many types of release delivery systems are known.

The pharmaceutical composition may be administered by any suitable means, such as by infusion, by injection such as intravenous or subcutaneous injection, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, Intrachoroidal, anterior chamber, subconjunctival, subconjunctival, subfascial, retrobulbar, peribulbar, or posterior scleral delivery. In some embodiments, by parenteral, intrapulmonary and intranasal administration and, if desired for local treatment, intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration.

In some embodiments, the compositions are sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions or viscous compositions, which in some aspects can be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. In another aspect, viscous compositions can be formulated in an appropriate viscosity range to provide a longer contact time with a particular tissue. Liquid or viscous compositions can comprise a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.

In some embodiments, the present invention also provides an article of manufacture and/or a kit comprising a unit dose of CD7-targeting allogeneic chimeric antigen receptor (CAR)-T cells and instructions. , the unit dose comprising about 0.1×10 ⁶ to about 1×10 ⁸ CAR+ cells. In some embodiments, the instructions specify specific instructions for administering the cell therapy, such as dosage, timing, selection and/or identification of subjects for administration and conditions of administration. In some embodiments, the article of manufacture and/or kit further comprises a composition for lymphodepletion, and optionally further comprises instructions for administering a lymphodepletion regimen.

An article of manufacture and/or a kit of the invention may comprise a container and a label or instructions on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, flexible cell infusion bags, and the like. The container can be formed from various materials such as glass or plastic. In some embodiments, the container contains the composition itself or in combination with another composition that is effective for the treatment, prevention and/or diagnosis of a condition. In some embodiments, the container has a sterile access. Exemplary containers include bags of intravenous solutions, vials (including those having a stopper pierceable by an injection needle), or bottles or vials for oral administration of the agent.

On the one hand, the treatment scheme provided by the present invention not only improves the efficacy of CAR-T cells, but also achieves effective stabilization and even cure of CD7-positive tumors (such as T-ALL, T-LBL, etc.), and greatly reduces the CAR-T As for the side effects of the cells, GvHD and ICANS basically do not occur, and the CRS is also controlled at grade 2 or lower. On the other hand, the present invention also provides a pretreatment composition that can effectively remove lymphocytes and improve the curative effect of CAR-T cells, which includes cyclophosphamide, fludarabine and etoposide, or includes cyclophosphamide, Fludarabine and melphalan.

specific implementation plan

Example 1. Construction and preparation of universal UCAR-T cells

Peripheral blood T cells were obtained from healthy donors, and after activation, the TCR/CD3 components (specifically TRAC gene), CD7 gene and MHC-II related genes (specifically RFX5) were knocked out with the CRISPR/Cas9 system to obtain TCR/CD7/RFX5 triple knockout tKO-T cells.

The coding sequences of the following proteins were synthesized and cloned into the MSCV vector sequentially: CD8α signal peptide (SEQ ID NO: 31), anti-CD7 scFv (SEQ ID NO: 9), CD8α hinge region (SEQ ID NO: 33), CD8α span Membrane region (SEQ ID NO: 19), 4-1BB co-stimulatory domain (SEQ ID NO: 25), CD3ζ primary signaling domain (SEQ ID NO: 27), γ chain intracellular region (SEQ ID NO: 42 ), T2A, anti-NKG2A scFv (SEQ ID NO: 18), IgG4 hinge region (SEQ ID NO: 37), CD28 transmembrane region (SEQ ID NO: 21), CD28 costimulatory domain (SEQ ID NO: 23) , obtain the target plasmid containing CD7-CAR and NK inhibitory molecules, and confirm the correct insertion of the target sequence by sequencing. The plasmid and packaging plasmid were co-transfected into 293T cells to obtain lentiviral vector.

The above-mentioned tKO-T cells were transduced with the lentiviral vector containing the target plasmid to obtain universal UCAR-T cells targeting CD7. After the general-purpose UCAR-T cells are expanded in large quantities, they are mixed with the infusion medium to form an infusion solution, which is cryopreserved in a separate flexible frozen cell infusion bag. Before use, the infusion solution is resuscitated by warming and then administered to subjects in need.

Example 2. Universal UCAR-T cells targeting CD7 to treat cancer patients

Figure 1 shows an exemplary administration scheme for UCAR-T cells of the present invention. The time point of administering the first dose of UCAR-T cells was set as D0.

14 days to 8 days (D-14 to D-8) before the administration of UCAR-T cells, the subjects meeting the inclusion criteria were screened. 7 days to 1 day (D-7 to D-1) before the administration of UCAR-T cells, use the FCV regimen (fludarabine (total dose range about 30mg-250mg/m ² ) + cyclophosphamide (total dose range about 300mg-3500mg/m ² ) + etoposide (total dose range about 150-750mg)) or FCM regimen (fludarabine (total dose range about 30mg-250mg/m ² ) + cyclophosphamide (total Subjects with CD7-positive tumors in the range of about 300mg-3500mg/m ² ) + melphalan (total dose range of about 50-750mg/m ² )) are pretreated to eliminate lymphocytes.

On D0, the subject is administered a first dose of CD7-targeted universal UCAR-T cells.

From about day 1 to about day 90 after the infusion (D1 to D90), the subject's response to administration of the first dose of UCAR-T cells is assessed. The expansion and persistence of CAR-T cells in the peripheral blood and bone marrow of treated subjects are determined by techniques such as flow cytometry or qPCR. Positive tumor minimal residual disease (MRD) or loss of CAR-T persistence suggested subsequent administration of UCAR-T cells. In general, subsequent doses (eg, second doses) are administered between days 5-90, preferably days 5-60, after administration of the first dose. In another embodiment, after the subject receives the first dose of the engineered immune cells, the subject may be administered a subsequent dose of the engineered immune cells on a regular basis, for example, every 5 weeks, 6 weeks, 7 weeks, Subsequent doses of engineered immune cells are administered to the subject at 8 weeks, 9 weeks, or 10 weeks. In this embodiment, the subject receives a total of 3, 4, 5, 6, 7, 8, 9 or 10 doses of engineered immune cells.

Before the subsequent administration of UCAR-T cells, based on factors such as tumor burden, degree of myelosuppression, CRS response, recovery of the patient's own T lymphocytes, etc., it was determined whether the subjects received secondary pretreatment of lymphocyte depletion. Where a second pretreatment is received, subsequent doses are administered to the subject within 2-14 days after completion of the second pretreatment.

The size of the follow-up dose is subject-specific and is determined based on tumor burden, expansion and persistence of UCAR-T cells, degree of CRS, degree of neurotoxicity, degree of GvHD, recovery from bone marrow toxicity of the subject, etc. The expansion and persistence of UCAR-T cells in the peripheral blood and bone marrow of treated subjects are determined by techniques such as flow cytometry or qPCR. Multiple doses of universal UCAR-T cells may be administered to some subjects until MRD negativity is achieved.

Following administration of the first dose or subsequent doses, the subject's disease status is assessed to assess its response to UCAR-T cells. Specifically, in addition to evaluating the incidence and grade of immune effector cell-associated neurotoxicity (ICANS) and cytokine release syndrome (CRS) according to the American Society for Transplantation and Cell Therapy (ASTCT) criteria, it was also evaluated according to the Lugano2014 version of the Lymphoma Treatment Response Criteria. The efficacy of CAR-T cells against relapsed/refractory T-cell lymphoblastic lymphoma (T-LBL) (Table 3), and the evaluation of CAR-T cells against relapsed/refractory Efficacy of T-cell acute lymphoblastic leukemia (Table 4).

Table 3. Exemplary Response Evaluation Criteria for Relapsed/Refractory T-LBL

Table 4. Exemplary Response Evaluation Criteria for Relapsed/Refractory T-ALL

Example 3. Treatment of Adult Subjects with CD7+ Relapsed/Refractory T-ALL or T-LBL with Universal UCAR-T Cells Targeting CD7

The target prepared in Example 1 was administered to 8 adult subjects with CD7+ relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) or T-cell lymphoblastic lymphoma/leukemia (T-LBL). Universal UCAR-T cells towards CD7.

The age range of the subjects was 26 to 66 years old, with a mean age of 45 years. The subjects had previously received severe treatment, the median of the previous treatment lines was 4.5, ranging from 2 to 8 lines; the median time from the latest recurrence was 2.34 months, ranging from 0.53 to 5.53 months; two of them were tested The patients were relapsed after receiving hematopoietic stem cell transplantation; 2 subjects were accompanied by high-risk gene damage, one of which was TEL-ABL and IDH2 high-risk gene damage, and the other was BCOR and EZH2 high-risk gene damage. The median percentage of blasts in bone marrow before preconditioning was 29%, with a range from 0% to 95%. Among the 8 subjects, 7 cases were accompanied by bone marrow lesions, and 1 case was a simple extramedullary recurrence; 4 cases were accompanied by extramedullary lesions, including 1 case with central nervous system leukemia, and the CNSL grade was 3. The demographic and baseline characteristics of the subjects are shown in Table 5.

Table 5. Demographics and Baseline Characteristics

The pretreatment regimens and UCAR-T cell administration regimens of the 8 subjects and their response to CAR-T cell therapy are shown in Table 6, where the response results include efficacy evaluation, and whether there is severe CRS and neurotoxicity. Of the 8 subjects, 1 died before evaluable results were available. Of the remaining 7 evaluable subjects, 5 were observed to respond to treatment, with an overall ORR of 71.4% (5/7), of which 4 subjects achieved MRD-, a ratio of 80% (4/5 ). In addition, among the 7 evaluable subjects, 6 subjects had bone marrow lesions, and all of them obtained bone marrow morphology CR/CRi; among them, 5 cases obtained bone marrow MRD-, and 1 case obtained MRD+. It is worth noting that all subjects did not observe any grade of ICANS, and only 1-2 grade CRS occurred, but did not produce 3 grade or more severe CRS, which shows that the dosage regimen of the present invention is effective in treating adult patients. Safety on the patient side is very good.

The results in Table 6 show that, using the dosage regimen described in the present invention, subjects with morphological disease were administered a first dose of UCAR-T cells, and higher doses were administered as needed after the tumor burden stabilized or had decreased or progressed. Or an equivalent subsequent dose, which minimizes toxicity and maximizes efficacy without serious risk of CRS or neurotoxicity. In some cases, some subjects were non-responsive (NR) to the first dose or even had progressive disease (PD) (see subjects 2 and 6), thus failing to achieve stable or stable tumor burden levels. However, no serious risk of CRS or neurotoxicity was observed in these subjects. This suggests that even if some subjects did not respond to the first or second dose, the risk of severe CRS or neurotoxicity after readministration of subsequent doses is low.

In order to further evaluate the expansion and persistence of UCAR-T cells in vivo after the administration of the first dose and subsequent doses, the expansion and persistence of UCAR-T cells in peripheral blood were regularly detected by qPCR and flow cytometry, The results are shown in Figure 2 (qPCR) and Figure 3 (flow cytometry). As can be seen, expansion of UCAR-T cells was detected in all subjects, and both responders (Fig. 2a and Fig. 3a) and non-responders (Fig. -Amplification peak was reached around day 21. In addition, the levels of IL-6, IL-2 and IFN-γ were also detected by flow cytometry, and the results are shown in FIG. 4 . It can be seen that after receiving UCAR-T cell therapy, all three cytokines were observed to be elevated in subjects who achieved CR/CRi, while in non-responding subjects, the levels of cytokines were lower. Rapidly decreased (subject No. 2) or remained at a low level (subject No. 6).

Example 4. Treatment of Pediatric Subjects with CD7+ Relapsed/Refractory T-ALL or T-LBL with Universal UCAR-T Cells Targeting CD7

The target prepared in Example 1 was administered to 2 child subjects with CD7+ relapsed/refractory T-cell acute lymphoblastic leukemia (T-ALL) or T-cell lymphoblastic lymphoma/leukemia (T-LBL). Universal UCAR-T cells towards CD7.

Both subjects had primary refractory diseases, and the proportions of baseline bone marrow blasts were 8.5% and 14.7%, respectively. One of the subjects had T-LBL with myeloid expression. The administration regimens of the two subjects and their response results to CAR-T cell therapy are shown in Table 7, where the response results include efficacy evaluation, and whether there is severe CRS and neurotoxicity. After treatment, 1 subject achieved PR, 1 subject had no response, and the ORR was 50%. No ICANS of any grade was observed in any of the 2 subjects, and grade 1 CRS occurred in only 1 subject, which indicates that the dosage regimen of the present invention is very safe in treating pediatric patients.

Example 5. Repeat Administration Regimen for Treating Subjects with CD7+ Relapsed/Refractory T-ALL or T-LBL

Subject 1 had relapsed/refractory CD7+ T-cell acute lymphoblastic leukemia. Before administering UCAR-T cells, the patients received the lymphodepleting preconditioning regimen of the FCV regimen, including fludarabine 30 mg/m ² /day x 3 days (D-7, D-6, D-5), ring Phosphoramide 500 mg/m ² /day x 2 days (D-6, D-5) and etoposide 100 mg/day x 3 days (D-5, D-4, D-3). Then, the subject was given a single injection of the first dose (about 5×10 ⁶ CAR+ T cells/kg) of UCAR-T cells on D0. After administration of the first dose, subjects were observed to have a Grade 2 CRS response on day 2 without neurotoxicity. In addition, the expansion level of UCAR-T cells was detected by qPCR, and it was found that although the level of UCAR-T cells was slightly increased at D3 and reached a peak at D5 (the CRS reaction had disappeared at this time), but immediately after D7 A substantial decrease in the copy number of UCAR-T cells was detected and was lower than the level before administration ( FIG. 5 ). The second dose (1×10 ⁷ CAR+ T cells/kg) was administered on D7, and it was observed that the subject had a grade 2 CRS reaction but no neurotoxicity, and the number of UCAR-T cells reached the second peak on D14. The subject eventually achieved CR (MRD-).

Subject No. 2 suffered from relapsed/refractory CD7+ T-cell acute lymphoblastic leukemia, and tumor lesions occurred in both bone marrow and extramedullary. Before the administration of UCAR-T cells, the patients received the lymphodepletion preconditioning regimen of the FCV regimen, including fludarabine 30mg/ ^m2 /day x 3 days (D-5, D-4, D-3), ring Phosphoramide 300 mg/m ² /day x 3 days (D-5, D-4, D-3) and etoposide 100 mg/day x 3 days (D-5, D-4, D-3). Then, the subject was given a single injection of the first dose (about 1×10 ⁷ CAR+ T cells/kg) of UCAR-T cells on D0. After the first dose, no subjects were observed to experience CRS or neurotoxicity. In addition, the expansion level of UCAR-T cells was detected by qPCR, and it was found that the expansion level of UCAR-T cells was not obvious and decreased on D14 (Figure 5), so it was decided to administer the second dose. Before administering the second dose, the subject received a second FCV regimen of lymphodepletion pretreatment, including fludarabine 30 mg/m ² /day x 5 days (D15, D16, D17, D18, D19), Cyclophosphamide 300 mg/m ² /day x 5 days (D15, D16, D17, D18, D19) and etoposide 100 mg/day x 5 days (D15, D16, D17, D18, D19). When the second dose (1×10 ⁷ CAR+ T cells/kg) was administered on D22, no CRS or neurotoxicity was observed in the subject, and the number of UCAR-T cells reached its peak on D28 (Figure 5). The subject's bone marrow lesions eventually achieved MRD-, but the extramedullary lesions did not achieve remission.

The above results show that after the infusion of the first dose of UCAR-T cells, in the absence of severe CRS and neurotoxicity, if the expansion of UCAR-T cells in vivo is not obvious or the number continues to decline, administration of subsequent doses will be beneficial. Aids in disease relief.

Example 6. Treatment of CD7+ relapsed/refractory T-cell lymphoma with universal UCAR-T cells targeting CD7

Subject No. 11 was a male patient with relapsed/refractory CD7+ peripheral T-cell lymphoma, non-specific (PTCL, NOS), and both bone marrow and extramedullary lesions were present. Before administering UCAR-T cells, the patient received a lymphodepleting preconditioning regimen of FCV regimen, including fludarabine 25 mg/ ^m2 /day x 5 days (D-7, D-6, D-5, D- 4. D-3), cyclophosphamide 500mg/m ² /day x 5 days (D-7, D-6, D-5, D-4, D-3) and etoposide 100mg/day x 5 days (D-7, D-6, D-5, D-4, D-3). Then, the subject was given a single injection of the first dose (about 2×10 ⁷ CAR+ T cells/kg) of UCAR-T cells on D0. After administration, subjects were observed to have a grade 1 CRS response on D9, but no neurotoxicity occurred. The expansion level of UCAR-T cells in peripheral blood was detected by qPCR and flow cytometry, and it was found that the expansion was rapid and reached a peak around D14 (Figure 6). The subject's bone marrow lesions finally achieved CR, and the extramedullary lesions achieved CR.

Subject No. 12 was a female patient with relapsed/refractory CD7+ extranodal NK/T-cell lymphoma, nasal type (NKTL), and both bone marrow lesions and extramedullary lesions were present. Prior to the administration of UCAR-T cells, the patient received a lymphodepleting preconditioning regimen of the FCV regimen, including fludarabine 30 mg/m ² /day x 5 days (D-7, D-6, D-5, D- 4. D-3), cyclophosphamide 300mg/m ² /day x 5 days (D-7, D-6, D-5, D-4, D-3) and etoposide 100mg/day x 5 days (D-7, D-6, D-5, D-4, D-3). Then, the subject was given a single injection of the first dose (about 2×10 ⁷ CAR+ T cells/kg) of UCAR-T cells on D0. After administration, the subject did not develop CRS and neurotoxicity. The expansion level of UCAR-T cells in peripheral blood was detected by qPCR and flow cytometry, and it was found that the expansion was rapid and reached a peak around D14 (Figure 6). The subject's bone marrow lesions finally achieved CR, and the extramedullary lesions achieved PR.

Embodiment 7. Statistics of adverse reactions of patients

The adverse reactions of a total of 12 subjects in the aforementioned examples were counted, including CRS, ICANS, GvHD and other adverse events, wherein CRS and ICANS were evaluated according to the American Society for Transplantation and Cell Therapy (ASTCT) standards, and GvHD was evaluated according to the American National Comprehensive Cancer Other adverse events were evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE 5.0). The statistical results are shown in Table 8 below.

Table 8. Adverse Reaction Statistics

Adverse reactions	Number of subjects
CRS Level 1	2
CRS Level 2	7
ICANS	0
GvHD	0
Dizziness	4
Headache	6
nausea	8
stomach ache	6
diarrhea	5
oral mucositis	8
fever	11
chills	10
sepsis	2
abnormal liver function	8

The above results show that the administration regimen of UCAR-T targeting CD7 in the present invention can greatly reduce the risk of adverse events while obtaining good curative effect, and the safety is very good. It is worth noting that in all treated subjects, ICANS and GvHD were not observed at all, and the proportion of CRS occurred was low and most of them were of mild grade, and these were the results of cellular immunotherapy, especially CAR-T cells. The most frequently occurring adverse reactions during treatment.

It should be noted that the above are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Those skilled in the art understand that any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (42)

1. A method for treating a disease associated with CD7 expression, comprising administering to a subject a first dose of engineered immune cells comprising a chimeric antigen receptor specifically binding to CD7, said The first dose is 0.1×10 ⁶ to 1×10 ⁸ CAR+ cells/kg, wherein the subject is subjected to a lymphodepleting regimen including cyclophosphamide, fluda Labine, etoposide, melphalan, or a combination thereof.
2. The method of claim 1, wherein the first dose is 5× ^{10 6} to 10× ^{10 9} CAR+ cells.
3. The method of claim 1, further comprising administering to the subject at least one subsequent dose of the engineered immune cells expressing a chimeric antigen receptor targeting CD7.
4. The method of claim 3, when the subject has the following characteristics after receiving the first dose of engineered immune cells, administering the subsequent dose of engineered immune cells:

   (i) the subject has a serum level of a factor indicative of cytokine release syndrome (CRS) that is multiple times less than about 10 times less, about 25 times less than the level in the subject immediately prior to administration of said first dose, and / or about 50 times smaller;

   (ii) have not demonstrated grade 3 or higher neurotoxicity;

   (iii) neurotoxicity or CRS levels are reduced compared to peak levels of neurotoxicity or CRS levels following administration of the first dose of engineered immune cells; or

   (iv) the subject does not exhibit a detectable immune response against the CAR expressed by the first dose of engineered immune cells.
5. The method of claim 3, wherein said subsequent dose is higher or lower than said first dose.
6. 3. The method of claim 3, wherein the subsequent dose is administered at a time point of at least 5-90 days after the administration of the first dose.
7. The method of claim 3, wherein the method further comprises administering a lymphodepletion regimen prior to administering the subsequent dose to the subject.
8. The method of claim 3, wherein no additional lymphodepletion regimen is administered prior to administering the subsequent dose to the subject.
9. The method according to claim 3, after the subject receives the first dose of engineered immune cells, the subject is regularly administered a subsequent dose of engineered immune cells.
10. The method of claim 9, administering subsequent doses of the engineered immune cells to the subject every 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks or 10 weeks.
11. The method of claim 9, wherein the subject receives a lymphodepletion regimen prior to each subsequent dose.
12. The method of any one of claims 1-11, wherein the lymphodepletion regimen comprises cyclophosphamide, fludarabine and etoposide, or comprises cyclophosphamide, fludarabine and melphalan.
13. The method of claim 12, wherein the dose of fludarabine is 10-60 mg/m ² /day, 10-50 mg/m ² /day, 15-40 mg/m ² /day, or 15-35 mg/m ² /day; the dose of cyclophosphamide is 100-700mg/m ² /day, 150-650mg/m ² /day, 200-600mg/m ² /day, 250-600mg/m ² /day, or 300-600mg/m 2 /day m ² /day; the dose of etoposide is 50-150mg/day, 50-125mg/day, 50-100mg/day; the dose of melphalan is 50-150mg/m ² /day, 50-125mg/m ² , or 50-100 mg/m ² .
14. The method of any one of claims 1-13, starting to administer lymphoid at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days before administering the engineered immune cells Cell Clearance Protocol.
15. The method according to any one of claims 1-14, wherein the lymphocyte depletion regimen is administered intermittently or continuously for 1, 2, 3, 4, 5, 6 or 7 days.
16. The method according to any one of claims 1-15, wherein the lymphocyte depletion regimen comprises administration of cyclophosphamide every day for 2 days, 3 days, 4 days or 5 days in a row; fludarabine is used for 3 days in a row. 1, 4 or 5 days; etoposide administered daily for 3, 4 or 5 consecutive days; or melphalan administered daily for 1 day or 2 or 3 consecutive days.
17. The method of claim 1, the subject has received one or more previous treatments before receiving the treatment method of the present invention, and the previous treatments include stem cell transplantation, radiotherapy, chemotherapy, antibody therapy, Small molecule targeted therapy, or other engineered immune cell therapy.
18. The method of any one of claims 1-17, wherein the chimeric antigen receptor comprises a CD7 antigen binding region, a transmembrane domain, and an intracellular signaling region, the intracellular signaling region comprising a co-stimulatory domain and primary signaling domains.
19. The method of claim 18, wherein the intracellular signaling region further comprises a γc chain or an intracellular region thereof.
20. The method of claim 19, wherein the CD7 antigen binding region comprises CDR-L1, CDR-L2 and CDR-L3 shown in SEQ ID NO: 1, 2, and 3, and shown in SEQ ID NO: 4, CDR-H1, CDR-H2 and CDR-H3 shown in 5 and 6.
21. The method of claim 19, wherein the transmembrane domain is derived from TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5 , CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, or CD154.
22. The method of claim 19, wherein the co-stimulatory domain is selected from the intracellular region of the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134(OX40), CD137(4-1BB), CD270(HVEM), CD272(BTLA), CD276(B7-H3), CD278(ICOS), CD357(GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP70.
23. The method of claim 19, wherein the primary signaling domain is derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, or CD66d.
24. The method of claim 19, wherein the γc chain has at least 70% sequence identity with SEQ ID NO:42; the intracellular region of the γc chain has at least 70% sequence identity with SEQ ID NO:44.
25. The method of any one of claims 1-24, wherein the expression of endogenous CD7, at least one TCR/CD3 gene and at least one MHC-II related gene of the engineered immune cells is suppressed or silenced.
26. The method of claim 25, wherein said at least one TCR/CD3 gene is selected from: TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ; said at least one MHC-II related gene is selected from: HLA- DPA, HLA-DQ, HLA-DRA, RFX5, RFXAP, RFXANK, and CIITA.
27. The method of claim 26, wherein the expression of endogenous CD7, TRAC and RFX5 of the engineered immune cells is inhibited or silenced.
28. The method according to any one of claims 1-27, wherein the engineered immune cells further express NK inhibitory molecules comprising one or more NK inhibitory ligands, transmembrane domains and co- Stimulatory domain.
29. 28. The method of claim 28, wherein the NK inhibitory molecule does not comprise a primary signaling domain.
30. The method of claim 28, wherein the NK inhibitory ligand is an antibody targeting an NK inhibitory receptor selected from the group consisting of NKG2A, NKG2B, CD94, LIR1, LIR2, LIR3, LIR5, LIR8, KIR2DL1, KIR2DL2/3, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, CEACAM1, LAIR1, NKR-P1B, NKR-P1D, PD-1, TIGIT, CD96, TIM3, LAG3, SIGLEC7, SIGLEC9, Ly49A, Ly49C, Ly49F, Ly49G1, Ly49G4 and KLRG1.
31. The method of claim 30, wherein the NK inhibitory ligand is an antibody targeting NKG2A.
32. The method of any one of claims 1-31, wherein the engineered immune cells are T cells, macrophages, dendritic cells, monocytes, NK cells or NKT cells.
33. The method according to any one of claims 1-32, the disease associated with CD7 expression comprises CD7-positive hematological tumors and solid tumors, preferably selected from CD7-positive acute lymphoblastic leukemia (ALL, such as T-ALL, NK-ALL), acute myeloid leukemia (AML), chronic myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, T-cell large granular lymphocytic leukemia (T-LGL), non-Hodgkin lymphoma (eg lymphoblastic lymphoma (T-LBL), peripheral T-cell lymphoma (PTCL), extranodal NK/T-cell lymphoma, γδT-cell lymphoma) and early pro-T lymphoblastic leukemia (ETP-ALL).
34. The method of any one of claims 1-33, wherein the engineered immune cells are administered parenterally.
35. A composition for depleting lymphocytes, comprising cyclophosphamide, fludarabine, etoposide, melphalan or a combination thereof.
36. The composition of claim 35 comprising cyclophosphamide, fludarabine and etoposide, or comprising cyclophosphamide, fludarabine and melphalan.
37. The composition of claim 35, wherein the cyclophosphamide, fludarabine, etoposide and/or melphalan are administered simultaneously or sequentially.
38. The composition of claim 35, wherein the dose of fludarabine is 10-60 mg/m ² /day, 10-50 mg/m ² /day, 15-40 mg/m ² /day, or 15-35 mg/m ² /day; the dose of cyclophosphamide is 100-700mg/m ² /day, 150-650mg/m ² /day, 200-600mg/m ² /day, 250-600mg/m ² /day, or 300-600mg /m ² /day; the dosage of etoposide is 50-150mg/day, 50-125mg/day, 50-100mg/day; the dosage of melphalan is 50-150mg/m ² /day, 50-125mg/m ² , Or 50-100 mg/m ² .
39. An article comprising:

    -- at least one sealable container comprising a unit dose of CD7-targeted allogeneic chimeric antigen receptor (CAR)-T cells comprising about ^0.1x106 to about ^1x108 CAR+ cells; and

    --manual.
40. 39. The article of manufacture of claim 39, further comprising a composition for depleting lymphocytes.
41. 40. The article of manufacture of claim 40, wherein the composition comprises cyclophosphamide, fludarabine and etoposide, or comprises cyclophosphamide, fludarabine and melphalan.
42. 39. The article of manufacture of claim 39, wherein said container is a flexible cell infusion bag.

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