CN116648457A

CN116648457A - Membrane fusion proteins and their use in immune cells

Info

Publication number: CN116648457A
Application number: CN202180088001.4A
Authority: CN
Inventors: 董琦; 江鹏斐; 沈连军; 曹卫
Original assignee: Genxi Biotechnology Shanghai Co ltd
Current assignee: Genxi Biotechnology Shanghai Co ltd
Priority date: 2020-12-31
Filing date: 2021-12-30
Publication date: 2023-08-25
Also published as: TW202233662A; WO2022143928A1

Abstract

Chimeric antigen receptor immune cells expressing interleukin-15 and uses thereof. Specifically, a gene recombinant chimeric antigen receptor immune cell is provided, the cell surface of which expresses interleukin-15 mutant and IL-15Ra fusion protein, and experiments show that the mutant immune cell obviously improves the survival of the immune cell, promotes the expansion of the immune cell, reduces the inhibition of TME on the immune cell and enhances the persistence and the anti-tumor activity of the CAR-T cell under the condition of continuous antigen stimulation.

Description

Membrane fusion proteins and their use in immune cells

Technical Field

The invention relates to the field of immune cell therapy, in particular to an engineering immune cell which co-expresses an interleukin-15 mutant and IL-15 Ralpha fusion protein and targets solid tumors and hematological tumors and application thereof.

Background

Immune cell therapy is a brand-new drug development model. The use of genetic engineering to engineer human immune cells into active drugs has made significant progress in the treatment of B cell malignancies.

A variety of immunotherapies have been developed, including LAK, DC, CIK, DC-CIK, TCR-T, CAR-T, NK, CAR-NK, and the like. Common CAR-T cell immunotherapy includes autologous and universal CAR-T therapies, wherein autologous CAR-T therapy uses patient's immune cells, and allogeneic immunotherapy uses allogeneic subject's CAR-T cells, and the desired immune cells are cultured in vitro, genetically edited and expanded and then returned to the patient, and these cells recognize tumor cells in a non-MHC restricted manner, do not require antigen presentation mechanisms to recognize tumor antigens, and reduce tumor cell escape through down-regulation of MHC and antigen presentation mediated immunity. Aiming at a tumor-associated antigen Target (TAA), a cell surface Chimeric Antibody Receptor (CAR) molecule is designed to specifically identify the antigen target expressed by tumor cells, and the tumor cells are directly attacked and killed by cytokines such as interferon, perforin and the like, so that the aim of treating or relieving diseases is fulfilled.

Currently, immunotherapy has prominent efficacy in hematological tumors, which is not ideal in solid tumors. The main reasons include three aspects, namely a safe tumor specific antigen target, heterogeneity of solid tumors, existence of complex tumor cell microenvironment and other factors, including immunosuppressive cell Tregs, MDSCs and the like, and existence of immunosuppressive factors, which are unfavorable for survival of immune cells, so that the immunotherapeutic effect is inhibited. Mesothelin is a differentiation antigen present on normal mesothelial cells, is highly expressed in tumors such as mesothelioma, lung cancer, pancreatic cancer, breast cancer, ovarian cancer and the like, is limited in mesothelial cells of normal tissues such as normal pleura, pericardium and peritoneum, and is minimally expressed on the surface of epithelial cells of trachea, ovary, testis, tonsil and fallopian tube. Thus mesothelin can be used as an effective immunotherapeutic target, and immunotherapeutic strategies are currently designed against MSLN, mainly including antibody therapies, immunotoxins, and chimeric antigen receptor T cell therapies. The antibody medicines comprise Amatuximab (MORAb-009), anetumab Ravtansine (BAY 94-9343), DMOT4039A, MDX-1204 and the like, but the curative effect of the antibody medicines on solid tumors is not ideal, and the existing treatment technology is required to be perfected, so that the curative effect is enhanced, and the risk in the treatment process is reduced.

In view of the foregoing, there is a need in the art to further develop more effective, durable, safe immunotherapies targeting solid tumors.

Disclosure of Invention

The invention aims at providing a chimeric antigen receptor immune cell expressing interleukin-15 and application thereof.

In a first aspect of the invention, there is provided a Chimeric Antigen Receptor (CAR) construct having the structure shown in formula I or II,

X-A-E (I)

E-A-X (II)

in the method, in the process of the invention,

each "-" is independently a connecting peptide or peptide bond;

x is a CAR targeting a tumor antigen;

a is a self-shearing element;

e is an IL-15/IL-15Rα complex.

In another preferred embodiment, the CAR construct has the structure shown in formula I.

In another preferred embodiment, the IL-15/IL-15Rα complex comprises IL-15 and IL-15Rα.

In another preferred embodiment, the IL-15 and IL-15Rα are of human origin.

In another preferred embodiment, the IL-15 and IL-15Rα are linked by a linking peptide.

In another preferred embodiment, the IL-15/IL-15Rα complex further comprises a signal peptide element.

In another preferred embodiment, the structure of the IL-15/IL-15Rα complex is shown in formula III,

L’-M-I-R (III)

in the method, in the process of the invention,

each "-" is independently a connecting peptide or peptide bond;

l' is none or a signal peptide;

M is IL-15 or a mutant thereof;

i is a flexible joint;

r is IL-15Rα.

In another preferred embodiment, the IL-15 mutant has IL-15 biological activity.

In another preferred embodiment, the amino acid sequence of IL-15 is set forth in SEQ ID NO. 1.

In another preferred embodiment, the amino acid sequence of the IL-15 mutant (IL-15N 72D) is as set forth in SEQ ID NO. 2.

In another preferred embodiment, the IL-15Rα is a complete IL-15Rα element.

In another preferred embodiment, the IL-15Rα comprises a transmembrane region and an intracellular region.

In another preferred embodiment, the IL-15Rα comprises, in order from the N-terminus to the C-terminus, the sushi domain (active fragment binding to IL-15) extracellular region, transmembrane region and intracellular region.

In another preferred embodiment, the amino acid sequence of IL-15Rα is as set forth in SEQ ID NO. 3.

In another preferred embodiment, the flexible linker is a linker peptide, preferably having an amino acid sequence as set forth in SEQ ID NO. 4 (SGGGSGGGGSGGGGSGGGGSGGGSLQ).

In another preferred embodiment, the L' is a signal peptide derived from IgE, IL-2.

In another preferred embodiment, the amino acid sequence of the IL-15/IL-15Rα complex is as shown in SEQ ID No. 5.

In another preferred embodiment, the amino acid sequence of the IL-15 mutant/IL-15 Rα complex is as shown in SEQ ID NO. 6.

In another preferred embodiment, the self-shearing element comprises T2A, P a.

In another preferred embodiment, the structure of X (CAR) is as shown in formula IV,

L-scFv-H-TM-C-CD3ζ (IV)

each "-" is independently a connecting peptide or peptide bond;

l is none or a signal peptide;

scFv are antibody single chain variable regions that target tumor antigens;

h is a hinge-free region;

TM is a transmembrane domain;

c is a costimulatory signaling molecule;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ;

in another preferred embodiment, the tumor antigen is selected from the group consisting of: mesothelin, claudin18.2, MUC1, GPC3, PSCA, her2, CD19, or combinations thereof.

In another preferred embodiment, the tumor antigen is mesothelin.

In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of: CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

In another preferred embodiment, L is a CD 8-derived signal peptide.

In another preferred embodiment, the H is a hinge region of a protein selected from the group consisting of: CD8, CD28, CD137, or a combination thereof.

In another preferred embodiment, the H is a CD8 derived hinge region.

In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: ICOS, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, MUC1-Tn, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or combinations thereof.

In another preferred embodiment, the TM is a CD8 or CD28 derived transmembrane region.

In another preferred embodiment, C is a costimulatory signaling molecule of a protein selected from the group consisting of: ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD 137), PD1, dap10, CDs, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), NKG2D, GITR, TLR2, or combinations thereof.

In another preferred embodiment, said C is a CD28 derived costimulatory signaling molecule.

In another preferred embodiment, the amino acid sequence of X is as shown in SEQ ID NO. 8.

In another preferred embodiment, the amino acid sequence of the CAR construct is set forth in SEQ ID No. 7.

In a second aspect of the invention there is provided a nucleic acid molecule encoding a CAR construct according to the first aspect of the invention, or,

the nucleic acid molecule comprises a first nucleic acid molecule encoding a tumor antigen-targeted CAR and a second nucleic acid molecule encoding an IL-15/IL-15 ra complex, wherein the tumor antigen-targeted CAR and IL-15/IL-15 ra complex are defined as described above.

In another preferred embodiment, the first nucleic acid molecule and the second nucleic acid molecule may be in tandem or may exist independently.

In a third aspect of the invention there is provided a vector comprising a nucleic acid molecule according to the second aspect of the invention.

In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV), retroviral vectors, transposons, or combinations thereof.

In another preferred embodiment, the carrier is selected from the group consisting of: plasmid and viral vector.

In another preferred embodiment, the vector is in the form of a viral particle.

In another preferred embodiment, the vector is a lentiviral vector.

In a fourth aspect of the invention there is provided a host cell comprising a vector according to the third aspect of the invention or a nucleic acid molecule according to the second aspect of the invention or expressing a CAR construct according to the first aspect of the invention, exogenous to the vector or chromosome.

In another preferred embodiment, the host cell comprises a eukaryotic cell and a prokaryotic cell.

In another preferred embodiment, the host cell comprises E.coli.

In a fifth aspect of the invention there is provided an engineered immune cell expressing a CAR construct according to the first aspect of the invention, or

The immune cells express tumor antigen-targeted CARs and IL-15/IL-15 ra complexes, wherein the tumor antigen-targeted CARs and IL-15/IL-15 ra complexes are defined as described above.

In another preferred embodiment, the tumor antigen targeted CAR and IL-15/IL-15 ra complex are independently expressed on the cell membrane of the immune cell.

In another preferred embodiment, the cell is an isolated cell and/or the cell is a genetically engineered cell.

In another preferred embodiment, the immune cells are derived from a human or non-human mammal (e.g., a mouse).

In another preferred embodiment, the cells comprise T cells, NK cells.

In another preferred example, the engineered immune cell may be a chimeric antigen receptor T cell (CAR-T cell) or a chimeric antigen receptor NK cell (CAR-NK cell).

In a sixth aspect of the invention, there is provided a formulation comprising a CAR construct according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or an immune cell according to the fifth aspect of the invention, and a pharmaceutically acceptable carrier.

In another preferred embodiment, the formulation is a liquid formulation.

In another preferred embodiment, the formulation is in the form of an injection.

In another preferred embodiment, the concentration of said CAR-T cells in said formulation is 1X 10 ³ -1×10 ⁸ Individual cells/ml, preferably 1X 10 ⁴ -1×10 ⁷ Individual cells/ml.

In another preferred embodiment, the formulation further comprises a second active ingredient that is anti-tumor, preferably comprising a second antibody, or a chemotherapeutic agent.

In another preferred embodiment, the chemotherapeutic agent is selected from the group consisting of: docetaxel, carboplatin, or a combination thereof.

In a seventh aspect of the invention there is provided the use of a CAR construct according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or an immune cell according to the fifth aspect of the invention, or a formulation according to the sixth aspect of the invention, for the manufacture of a medicament or formulation for the prophylaxis and/or treatment of cancer or tumour.

In another preferred embodiment, the tumor is selected from the group consisting of: hematological tumors, solid tumors, or combinations thereof.

In another preferred embodiment, the hematological neoplasm is selected from the group consisting of: acute Myelogenous Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or combinations thereof.

In another preferred embodiment, the solid tumor is selected from the group consisting of: gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer, or a combination thereof.

In another preferred embodiment, the tumor is a mesothelin-positive tumor, preferably a mesothelin-high expressing tumor.

In an eighth aspect of the invention there is provided a kit for preparing a host cell according to the fourth aspect of the invention, the kit comprising a container, and within the container a nucleic acid molecule according to the second aspect of the invention, or a vector according to the third aspect of the invention.

In a ninth aspect of the invention, there is provided a method of preparing an engineered immune cell according to the fifth aspect of the invention, the method comprising the steps of:

(a) Providing an immune cell to be engineered; and

(b) Transduction of the nucleic acid molecule according to the second aspect of the invention or the vector according to the third aspect of the invention into said immune cell, thereby obtaining said engineered immune cell.

In another preferred embodiment, the engineered immune cell is a CAR-T cell or a CAR-NK cell.

In another preferred embodiment, the method further comprises the step of performing functional and validity assays on the obtained engineered immune cells.

In a tenth aspect of the invention there is provided a method of treating a disease comprising administering to a subject in need thereof an appropriate amount of a vector according to the third aspect of the invention, an immune cell according to the fifth aspect of the invention, or a formulation according to the sixth aspect of the invention.

In another preferred embodiment, the disease is cancer or tumor.

In an eleventh aspect of the invention, there is provided an IL-15/IL-15Rα complex, the structure of the IL-15/IL-15Rα complex being as shown in formula III,

L’-M-I-R (III)

in the method, in the process of the invention,

each "-" is independently a connecting peptide or peptide bond;

l' is none or a signal peptide;

m is IL-15 or a mutant thereof;

i is a flexible joint;

r is IL-15Rα, and R is IL-15Rα,

wherein the IL-15Rα comprises a transmembrane region and an intracellular region.

In a twelfth aspect of the invention, there is provided the use of an IL-15/IL-15 ra complex according to the eleventh aspect of the invention for the preparation of a formulation for enhancing persistence of CAR-T cells and/or enhancing cytotoxicity of CAR-T cells.

In another preferred embodiment, the formulation is for CAR-T cell based adoptive immunotherapy.

In another preferred embodiment, the enhancing the persistence of the CAR-T cell is enhancing the sustained killing of the tumor cell by the CAR-T cell.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows a schematic diagram of the mechanism of action of the membrane fusion protein of the present invention.

Figure 2A shows a schematic representation of a CAR structure employed in the present invention.

FIG. 2B shows a schematic representation of huIL-15/IL-15 mutant complexes.

FIG. 2C shows the structure of huIL-15Rα of huIL-15/IL-15 mutant complexes.

FIG. 3A shows the amino acid sequence of the MESO-E1m1 gene.

FIG. 3B shows the amino acid sequence of the E1m1 gene.

Figure 4 shows a positive rate assay for CAR-T cells.

FIG. 5A shows the rate of CAR-T cell lysis of target cells in a co-culture system of Meso-1, meso-E1 and Meso-E1m1 CAR-T cells with ovarian cancer cells OVCAR3 in vitro.

Figure 5B shows cytokine secretion by CAR-T cells after co-culture with ovarian cancer OVCAR3 in vitro.

FIG. 6A shows the rate of CAR-T cell lysis for target cells in an in vitro co-culture system of Meso-1, meso-E1 and Meso-E1m1 CAR-T cells with triple negative breast cancer MDA-MB-231-MESO cells. .

Figure 6B shows cytokine secretion by CAR-T cells after co-culturing CAR-T cells with triple negative breast cancer MDA-MB-231-MESO cells in vitro.

FIG. 7A shows the in vivo efficacy experiments of Meso-E1 and Meso-E1m1 CAR-T-1, HCC70 (breast cancer cells) tumor volume change curves.

FIG. 7B shows the in vivo efficacy experiments-1 of Meso-E1 and Meso-E1m1 CAR-T cells, animal body weight change curves.

FIG. 8A shows the tumor volume change curves of Meso-1 and Meso-E1m1 CAR-T in vivo efficacy experiment-2, HCC70 (breast cancer cells).

FIG. 8B shows in vivo efficacy experiments-2 of Meso-1 and Meso-E1m1 CAR-T, changes in animal body weight.

FIG. 8C shows tumor volume changes in each group of individual mice for Meso-1 and Meso-E1m1 CAR-T in vivo efficacy experiments-2.

Fig. 9A shows a multiple round killing experimental design.

FIGS. 9B and 9C show comparison of target cell lysis and CAR-T cell expansion after multiple rounds of killing with ovarian cancer cells OVCAR3, respectively, of the in vitro co-culture systems Meso-1, meso-E1 and Meso-E1m1 CAR-T cells.

FIGS. 9D and 9E show comparison of target cell lysis and CAR-T cell expansion after multiple rounds of stimulation of in vitro co-culture systems Meso-1, meso-E1 and Meso-E1m1 CAR-T cells with triple negative breast cancer MDA-MB-231-MESO cells, respectively.

FIG. 10A shows the lysis rate of target cells after multiple rounds of stimulation with various concentrations of TGF- β1 added in vitro co-culture systems, meso-1 and Meso-E1m1 CAR-T cells, respectively, with ovarian cancer cells OVCAR 3.

FIG. 10B shows the expansion curve of the CAR-T cells after multiple rounds of stimulation with various concentrations of TGF- β1 added to the in vitro co-culture system, meso-1 and Meso-E1m1 CAR-T cells, respectively, with ovarian cancer cells OVCAR 3.

FIG. 10C shows detection of apoptosis of Meso-1 and Meso-E1m1 CAR-T cells after a second round of multi-round killing with ovarian cancer cells OVCAR3, respectively, by addition of different concentrations of TGF- β1 in vitro co-culture systems.

FIG. 11 shows the proliferation of PBNK cells at 48 and 72 hours after co-culture of no CAR-T with PBNK.

Detailed Description

The inventor has studied extensively and deeply, find a kind of gene recombination chimeric antigen receptor immune cell and its application unexpectedly for the first time, said chimeric antigen receptor immune cell surface expresses super interleukin-15, namely interleukin-15 mutant and IL-15Ra fusion protein, the in vitro experiment shows that this mutant can obviously strengthen proliferation ability, survivability of immune cell, lasting killing power and promote NK cell to expand; in vivo experiments show that the mutant has lasting antitumor activity. The present invention has been completed on the basis of this finding.

Terminology

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in the present application, each of the following terms shall have the meanings given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" may refer to a value or composition that is within an acceptable error of a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or measured.

The term "administering" refers to physically introducing a product of the application into a subject using any of a variety of methods and delivery systems known to those of skill in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, e.g., by injection or infusion.

The term "antibody" (Ab) shall include, but is not limited to, an immunoglobulin that specifically binds an antigen and comprises at least two heavy (H) chains and two light (L) chains, or antigen binding portions thereof, interconnected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. VH and VL regions can be further subdivided into regions of hypervariability termed Complementarity Determining Regions (CDRs) interspersed with regions that are more conserved termed Framework Regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

It should be understood that, in this document, the amino acid names are identified by international single english letters, and the amino acid names corresponding to the amino acid names are abbreviated as "three english letters: ala (A), arg (R), asn (N), asp (D), cys (C), gln (Q), glu (E), gly (G), his (H), I1E (I), leu (L), lys (K), met (M), phe (F), pro (P), ser (S), thr (T), trp (W), tyr (Y), val (V).

The terms "chimeric antigen receptor", "chimeric membrane antigen receptor", "membrane fusion protein", and "recombinant membrane fusion protein" are used interchangeably and each represents an IL-15/IL-15Rα complex constructed in accordance with the present invention.

IL-15

IL-15 is a cytokine that can stimulate immune cell growth. IL-15-based drug development has long focused on soluble forms of IL-15 engineering. There are literature and patent reports focusing on the development of soluble IL-15 drugs, mainly with four structures of RL1, ALT803, HRP008 and P22339.

RL1 is a complex of IL-15 and Sushi domain of IL-15 receptor alpha, which are linked by a Linker. ALT803 is a complex of two IL-15N72D mutants with the Sushi domain/Fc fusion protein of dimeric IL-15 receptor alpha, i.e.IL-15 (N72D): IL-15RαSu/Fc. HRP00018 is a complex formed by linking IL-15/Fc fusion protein to the Sushi domain/Fc fusion protein of IL-15 receptor alpha via the Knob-into-Hole format. P22339 is that IL-15 (L52C) mutant and IL-15Rα Sushi domain (S40C)/Fc complex form disulfide bond at mutation site, and are connected by Knob-into-Hole form, so as to stabilize the structure of the complex.

In summary, the four structures overcome the defect of short half-life of recombinant IL-15, obviously prolong the half-life in vivo and improve the biological activity of the recombinant IL-15, such as promoting proliferation of CD8+ memory T cells, NKT and NK cells. However, the traditional Chinese medicine composition still has the problems of toxic and side effects and short half-life, and needs to be administered for many times in clinical use, so that the clinical practicability of the traditional Chinese medicine composition is limited.

IL-15/IL-15Rα complexes

The present invention provides an "IL-15/IL-15 ra complex" comprising IL-15 or a mutant thereof, and an intact IL-15 ra comprising a transmembrane region and an intracellular region, which can be used to enhance persistence of CAR-T cells and/or enhance cytotoxicity of CAR-T cells.

In a preferred embodiment, the IL-15/IL-15Rα complex of the invention may comprise an IL-15N72D/IL-15Rα complex, may comprise a signal peptide (IgE, IL-2, etc.), and may be co-expressed with the CAR basic structure.

The IL-15 (N72D) -IL-15Rα structure of the invention is a complex expressed on the surface of immunotherapeutic cells. IL-15 and IL-15Rα are linked by a linker, wherein the IL-15Rα structure refers to an intact IL-15Rα molecule including an extracellular region (containing sushi domain), a transmembrane region, and an intracellular region. IL-15Rα can activate downstream signal pathway signals such as JAK1/JAK3 and Stat3/Stat5 by recruiting IL-2Rβ and IL-2 Ryc chains expressed on the surface of immune therapy cells to achieve the purposes of promoting proliferation of immune therapy cells and inhibiting apoptosis of immune therapy cells, thereby obtaining better therapeutic effects. Meanwhile, the compound is limited on the surface of immune therapy cells, so that the immune suppression effect possibly caused by activating proliferation (such as Treg) of immune suppression type T cells, NK and NKT cells is avoided, and systemic or systemic toxic and side effects caused by overactivating the T cells, NK and NKT cells can also be avoided.

Chimeric Antigen Receptor (CAR)

The present invention illustratively employs the Meso-CAR basic structure, i.e., the mesothelin-targeted CAR structure, for construction of CAR-T cells expressing the IL-15/IL-15 ra complex.

In a preferred embodiment, the IL-15/IL-15Rα complex is attached to the C-terminus of the basic CAR structure by a self-shearing element and is expressed on the membrane of the CAR-T cell after shearing.

In a preferred embodiment, the Meso-CAR is made up of a signal peptide, a Mesothelin single chain variable region, CD28/4-1bb/ICOS, CD3 zeta in tandem.

In particular, the Chimeric Antigen Receptor (CAR) of the invention includes an extracellular domain, a transmembrane domain, and an intracellular domain. Extracellular domains include target-specific binding elements (also referred to as antigen binding domains). The intracellular domain includes a costimulatory signaling region and a zeta chain moiety. A costimulatory signaling region refers to a portion of an intracellular domain that comprises a costimulatory molecule. Costimulatory molecules are cell surface molecules that are required for the efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands. In a preferred embodiment, the CAR of the invention comprises a CD 28-derived costimulatory signaling molecule.

The linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to an extracellular domain or cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids.

In a preferred embodiment of the invention, the extracellular domain of the CAR provided by the invention comprises an antigen binding domain that targets Meso. The CARs of the invention, when expressed in T cells, are capable of antigen recognition based on antigen binding specificity. When it binds to its cognate antigen, affects tumor cells, causes tumor cells to not grow, to be caused to die or to be otherwise affected, and causes the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and zeta chain.

As used herein, "antigen binding domain" and "single chain antibody fragment" refer to Fab fragments, fab 'fragments, F (ab') ₂ Fragments, or single Fv fragments. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant regions, and have a minimal antibody fragment of the entire antigen binding site. Generally, fv antibodies also comprise a polypeptide linker between the VH and VL domains, and are capable of forming the structures required for antigen binding. The antigen binding domain is typically a scFv (single-chain variable fragment). The size of scFv is typically 1/6 of that of an intact antibody. The single chain antibody is preferably an amino acid sequence encoded by a single nucleotide chain. As a preferred mode of the invention, the scFv comprises an antibody, preferably a humanized single chain antibody, which specifically recognizes Meso.

For hinge and transmembrane regions (transmembrane domains), the CAR may be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a transmembrane domain is used that naturally associates with one of the domains in the CAR. In some examples, the transmembrane domain may be selected, or modified by amino acid substitutions, to avoid binding such domain to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interactions with other members of the receptor complex.

Sequence(s)

The sequences related in the sequence table of the application are as follows:

carrier body

Nucleic acid sequences encoding a desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by obtaining the gene from vectors known to include the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced synthetically.

The application also provides vectors into which the expression cassettes of the application are inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of transgenes and their proliferation in daughter cells. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.

In brief summary, the expression cassette or nucleic acid sequence of the invention is typically operably linked to a promoter and incorporated into an expression vector. The vector is suitable for replication and integration of eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

The expression constructs of the invention may also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety. In another embodiment, the invention provides a gene therapy vector.

The nucleic acid may be cloned into many types of vectors. For example, the nucleic acid may be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific vectors of interest include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

Further, the expression vector may be provided to the cell in the form of a viral vector. Viral vector techniques are well known in the art and are described, for example, in Sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York) and other virology and molecular biology manuals. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors include an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).

Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus may then be isolated and delivered to a subject cell in vivo or ex vivo. 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 one embodiment, a lentiviral vector is used.

Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these are located in the 30-110bp region upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible so as to maintain promoter function when the elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased by 50bp before the activity begins to decrease. Depending on the promoter, it appears that individual elements may act cooperatively or independently to initiate transcription.

One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is extended growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV 40) early promoter, the mouse mammary carcinoma virus (MMTV), the Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, the MoMuLV promoter, the avian leukemia virus promoter, the ebustan-balr (Epstein-Barr) virus immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that is capable of switching on expression of a polynucleotide sequence operably linked to the inducible promoter when such expression is desired, or switching off expression when expression is undesired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

To assess expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cell may also comprise either or both a selectable marker gene or a reporter gene to facilitate identification and selection of the expressing cell from a population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

The reporter gene is used to identify potentially transfected cells and to evaluate the functionality of the regulatory sequences. Typically, the reporter gene is the following gene: which is not present in or expressed by the recipient organism or tissue and which encodes a polypeptide whose expression is clearly indicated by some readily detectable property, such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is assayed at the appropriate time. Suitable reporter genes may include genes encoding luciferases, beta-galactosidases, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., ui-Tei et al 2000FEBS Letters479:79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available. Typically, constructs with a minimum of 5 flanking regions that show the highest level of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to evaluate agents for their ability to regulate promoter-driven transcription.

Methods for introducing genes into cells and expressing genes into cells are known in the art. In the context of expression vectors, the vector may be readily introduced into a host cell, e.g., a mammalian, bacterial, yeast or insect cell, by any method known in the art. For example, the expression vector may be transferred into the host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York). A preferred method of introducing the polynucleotide into a host cell is calcium phosphate transfection.

Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method of inserting genes into mammalian, e.g., human, cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. patent nos. 5,350,674 and 5,585,362.

Chemical means for introducing the polynucleotide into a host cell include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro and in vivo delivery tool is a liposome (e.g., an artificial membrane vesicle).

In the case of non-viral delivery systems, an exemplary delivery means is a liposome. Lipid formulations are contemplated for introducing nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid can be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated into the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution comprising the lipid, mixed with the lipid, associated with the lipid, contained in the lipid as a suspension, contained in or complexed with the micelle, or otherwise associated with the lipid. The lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles or have a "collapsed" structure. They may also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which naturally occur in the cytoplasm as well as in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

In a preferred embodiment of the invention, the vector is a lentiviral vector.

Formulations

The invention provides a cell comprising a CAR-T cell of the invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of said CAR-T cells in said formulation is 1 x 10 ³ -1×10 ⁸ Individual cells/ml, more preferably 1X 10 ⁴ -1×10 ⁷ Individual cells/ml.

In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, or the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.

Therapeutic applications

The invention includes therapeutic applications with cells (e.g., T cells) transduced with Lentiviral Vectors (LV) encoding the expression cassettes of the invention. The transduced T cells can target a tumor cell marker Meso, and synergistically activate the T cells to cause T cell immune response, so that the killing efficiency of the transduced T cells on the tumor cells is remarkably improved.

Accordingly, the present invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal comprising the steps of: administering the CAR-T cells of the invention to a mammal.

In one embodiment, the invention includes a class of cell therapies in which autologous T cells (or heterologous donors) from a patient are isolated, activated and genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. This way the probability of graft versus host disease is very low and the antigen is recognized by T cells in a non-MHC restricted manner. Furthermore, a CAR-T can treat all cancers that express this antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to persistent tumor control.

In one embodiment, the CAR-T cells of the invention can undergo robust in vivo T cell expansion and can last for an extended amount of time. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which the CAR-modified T cells induce an immune response specific for an antigen binding domain in the CAR. For example, CAR-T cells that are resistant to Meso elicit a specific immune response against Meso-positive cells.

Although the data disclosed herein specifically disclose lentiviral vectors comprising anti-Meso scFv, hinge and CD28 transmembrane and intracellular regions, and CD3 zeta signaling domains, the invention should be construed to include any number of changes to each of the construct components.

Treatable cancers include tumors that are not vascularized or have not been substantially vascularized, as well as vascularized tumors. Cancers may include non-solid tumors (such as hematological tumors, e.g., leukemia and lymphoma) or may include solid tumors. Types of cancers treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas and sarcomas, and certain leukemia or lymphoid malignancies, benign and malignant tumors, such as sarcomas, carcinomas and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included.

Hematological cancers are cancers of the blood or bone marrow. Examples of hematologic (or hematogenic) cancers include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, granulo-monocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelogenous (myelogenous) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas, hodgkin's disease, non-hodgkin's lymphomas (indolent and high grade forms), multiple myelomas, waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Solid tumors are abnormal masses of tissue that do not normally contain cysts or fluid areas. Solid tumors may be benign or malignant. Different types of solid tumors are named for the cell type that they are formed of (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma mesothelioma, lymphoid malignancies, pancreatic carcinoma ovarian cancer.

In a preferred embodiment, the cancer treatable is a Meso positive tumor.

The CAR-modified T cells of the invention can also be used as a vaccine type for ex vivo immunization and/or in vivo therapy of mammals. Preferably, the mammal is a human.

For ex vivo immunization, at least one of the following occurs in vitro prior to administration of the cells into a mammal: i) Expanding the cells, ii) introducing nucleic acid encoding the CAR into the cells, and/or iii) cryopreserving the cells.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with vectors expressing the CARs disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient can be a human, and the CAR-modified cells can be autologous with respect to the recipient. Alternatively, the cell may be allogeneic, syngeneic (syngeneic) or xenogeneic with respect to the recipient.

In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

The invention provides a method of treating a tumor comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.

The CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the invention may comprise a target cell population as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present invention are preferably formulated for intravenous administration.

The pharmaceutical composition of the present invention may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease-although the appropriate dosage may be determined by clinical trials.

When referring to an "immunologically effective amount", "antitumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences of the condition of the patient (subject). It can be generally stated that: pharmaceutical compositions comprising T cells described herein may be administered at 10 ⁴ To 10 ⁹ A dose of individual cells/kg body weight, preferably 10 ⁵ To 10 ⁶ Individual cells/kg body weight doses (including all integer values within those ranges) are administered. T cell compositions may also be administered multiple times at these doses. Cells can be administered by using injection techniques well known in immunotherapy (see, e.g., rosenberg et al, new Eng. J. Of Med.319:1676, 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject compositions may be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intradesmally, intraspinal, intramuscularly, by intravenous (i.v.) injection or intraperitoneally. In one embodiment, the T cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by i.v. injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination (e.g., before, simultaneously with, or after) any number of relevant therapeutic modalities, including, but not limited to, treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or ertapelizumab therapy for psoriasis patients or other therapy for PML patients. In a further embodiment, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressives such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to a patient in combination (e.g., before, simultaneously or after) with bone marrow transplantation, using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives injection of expanded immune cells of the invention after transplantation. In an additional embodiment, the expanded cells are administered pre-operatively or post-operatively.

The dose of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio administered to humans may be carried out according to accepted practices in the art. Typically, each treatment or each course of treatment,can be 1×10 ⁶ Up to 1X 10 ¹⁰ The modified T cells of the invention (e.g., CAR-T cells) are administered to a patient by, for example, intravenous infusion.

The main advantages of the invention include:

(a) The CAR structure of the CAR-T cell comprises the basic structure of the CAR and the IL-15/IL-15 Ralpha complex or the IL-15 mutant/IL-15 Ralpha complex, and the basic structure and the IL-15/IL-15 Ralpha complex respectively play roles without mutual interference.

(b) The in vivo experimental result shows that the immune cell Meso-E1m1 can obviously enhance the cytotoxicity and durability of the CAR-T cell, and is obviously superior to the Meso-E1.

(c) In vitro experiment results show that compared with Meso-1 CAR-T, the immune cell Meso-E1m1 of the invention obviously improves the survival rate of immune cells, promotes the expansion of the immune cells, reduces the inhibition of TGF-beta 1 on the immune cells and enhances the cytotoxicity and durability of the CAR-T cells under the condition of continuous co-culture of target cells and TGF-beta 1.

(d) Compared with the Meso-1 CAR-T, the immune cell Meso-E1m1 of the invention obviously reduces the secretion of inflammatory factor TNF-alpha in-vitro instant killing experiments.

(e) The immune cells of the invention can obviously enhance NK cell expansion.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Example 1 isolation of PBMC and expanded T cells from donor blood

Mononuclear cells were isolated from peripheral blood, density gradient centrifuged using Ficoll and enriched for T cells (EasySep human T cell enrichment kit, stemcell Technologies), activated culture and expansion using conjugated anti-CD3/CD28 magnetic beadsT cell proliferation, cell culture System Using x-vivo 15 (5% FBS,300IU/mL rhIL-2), cell culture at 37℃and 5% CO ₂ The incubator is used for continuous culture.

Example 2 cell culture and construction

Cell lines expressing MSLN were obtained from ATCC: OVCAR3 (human ovarian cancer cell line, ATCC HTB-161), HCC70 (human breast cancer cell line), MDA-MB-231 (human breast cancer cell line), wherein MDA-MB-231MESO transfers MSLN antigen into MDA-MB231 cells by lentivirus, and then a stable cell line with high expression of MSLN is obtained after monoclonal screening. The above cells were prepared and cultured according to ATCC guidelines.

Example 3 CAR structural design and transduction

This example illustrates the construction of CAR-T cells expressing the IL-15/IL-15 ra complex using the Meso-CAR basic structure, i.e., the mesothelin-targeted CAR structure.

The present example constructs second and fourth generation CARs with the structure shown in table 1.

The core structure of the CAR included a CD8 extracellular signal peptide, a P4scFv (scFv specifically targeting mesothelin), a CD 8-derived hinge region and a CD8/CD28 transmembrane region, and 3 Meso-CARs were constructed using a CD28 intracellular segment co-stimulatory signal.

TABLE 1

Naming the name	Co-stimulus signal	Intracellular activation region	Enhancers
Meso-1	CD28	3ζ	N/A
Meso-E1	CD28	3ζ	IL-15/IL-15Rα
Meso-E1m1	CD28	3ζ	IL-15 mutant/IL-15 Rα

The 3 Meso-CAR genes were cloned into FUW lentiviral vectors, respectively, and transferred into 293T cells with lentiviral packaging plasmids pmd2.G (adedge, plasmid # 12259) and psPAX2 (adedge, plasmid # 12260) using PEI transfection reagents, expressing the vectors, respectively, and the 48 and 72 hour viruses were collected, respectively, and after super-concentration, activated T cells were infected.

The results show that lentiviral vectors were successfully constructed using the three Meso-CAR genes.

Example 4 CAR-T cell preparation-lentiviral infection, detection of CAR positive rate and proliferation in vitro

Isolated and purified primary T cells were individually added to the lentiviruses concentrated in example 3 48 hours after activation, CAR positive rates were detected 72 hours after cell infection, scFv and IL-15 ra expression were detected using biotin-labeled MSLN antigen as primary antibody, APC-Streptavidin (BD) as secondary antibody, and anti-IL-15 ra antibody, respectively.

The flow detection result is shown in FIG. 4

T cells were placed at 37℃after transfection, 5% CO ₂ Continuous culture in incubator, fluid infusion every other day, harvesting cells on day 10, freezing, counting, calculating proliferation rate in vitro, and no obvious difference between proliferation of E1 CAR-T cells and E1m1 CAR-T cells.

Example 5 in vitro killing experiments and cytokine detection

In vitro killing experiments were performed on T cells harvested in example 4. By RTCA method, 1X10 ⁴ And (3) paving target cells on a 96-well RTCA plate, culturing for 18 hours, co-culturing CAR T and target cells OVCAR3 according to the ratio of 1:1,1:3 and 1:6, continuously culturing for 1-2 days, recording the growth condition of the target cells in real time, detecting the survival rate of the target cells, and calculating the killing efficiency of the CAR-T cells. After continuous culture for 1-2 days, RTCA plates were removed and the co-culture supernatants were centrifuged and frozen at-20 ℃.

As shown in FIG. 5A, there was significant killing of the Meso-1, meso-E1 and Meso-E1m1 CAR-T cells in co-culture with the OVCAR3 target cells, and there was no significant difference in killing between the three, and NT (Non-transduced T cell) did not significantly kill tumor cells when co-cultured with tumor cells (E: T=1:1, 1:3, 1:6), respectively.

According to the recommended method, the coculture supernatant was assayed using a humanTh1/Th2Cytokine kit II (BD, cat.551809), the coculture supernatant was thawed, the mixture was prepared mixed capture beads and the Human Th1/Th 2-II PE Detection, incubated with the sample or standard for 3 hours in the absence of light, and after incubation, 300g was centrifuged for 5min to discard the supernatant. 100 μl of washing buffer was added for resuspension, shaking for 5min, and detection on a flow cytometer. Data analysis was performed using FCAP Array v.3 software. The results are shown in FIG. 5B, and there is no obvious difference in IFN-gamma content in the supernatant of co-culture of Meso-1, meso-E1 and Meso-E1m1 CAR-T cells with OVCAR3 target cells; co-culture supernatant of Meso-E1 and Meso-E1m1 CAR-T cells and OVCAR3 target cells, TNF-alpha content was significantly lower than that of Meso-1.

As shown in FIG. 6A, there was no significant killing of the Meso-1, meso-E1 and Meso-E1m1CAR-T cells in co-culture with MDA-MB-231-MESO target cells, and no significant difference in killing between the three, and NT (Non-transduced T cell) did not significantly kill tumor cells when co-cultured with tumor cells (E: T=3:1, 1:1, 1:3), respectively. Co-culture supernatant cytokines were assayed using a HumanTh1/Th2 Cytokine kit II (BD) and as shown in FIG. 6B, the supernatant was co-cultured with Meso-E1m1CAR-T cells and MDA-MB-231-MESO target cells, with a significantly lower TNF- α content than Meso-1.

EXAMPLE 6 in vivo efficacy study-1

NOD mice were selected, 5E6 hcc70 cells were subcutaneously injected, tumor burden was continuously detected, tumors were allowed to grow at high speed, groups of 2-3 mice each were grouped, 200uL DPBS/mouse was injected tail vein after the grouping, 5E6 E1/E1m 1-CAR-T/mouse was injected, 1 st day after CAR-T cell injection, small amounts of mouse blood were taken to detect the number of surviving CAR-T cells in vivo, blood samples were taken weekly thereafter, CAR-T cell phenotypes were detected, and subcutaneous tumor size was detected twice weekly.

The results are shown in FIG. 7A, where mice injected with Meso-E1 CAR-T cells and Meso-E1m1CAR-T cells showed a more pronounced tumor volume reduction at D10; animal tumor volumes were observed continuously at D17, D21, D24, with the tumor volumes of animals in group E1 rising and the tumor volumes in group E1m1 falling continuously.

Body weight of mice as shown in fig. 7B, body weight of the mice in the Meso-E1m1 group was not substantially changed after CAR-T cell injection.

EXAMPLE 7 in vivo efficacy study-2

NOD mice were selected, 5E6 HCC70 cells were subcutaneously injected, tumor burden was continuously detected, when tumors were growing at high speed, the mice were grouped, 2-3 mice per group were injected by tail vein injection 200uL DPBS/mouse one day after the grouping, two groups of CAR-T were high (indicated by HD) low dose (indicated by LD), each mouse was injected at 5E6 or 2E6, respectively, CAR-T was Meso-1/Meso-E1m1, and on day 1 after CAR-T cell injection, a small amount of mouse blood was taken to detect the in vivo survival number of CAR-T cells, and thereafter blood was taken once a week to detect various phenotypes of CAR-T cells twice a week to detect subcutaneous tumor size.

As shown in fig. 8A, mice injected with Meso-1 CAR-T cells and Meso-E1m1 CAR-T cells exhibited a more pronounced tumor volume decrease at the beginning of D10; the tumor volume of animals is continuously observed at D17, D21 and D24, the tumor volume of the high-dose and low-dose Meso-1 animals is increased, the tumor volume of the high-dose E1m1 animals is not increased, the tumor volume of the low-dose E1m1 animals is recurrent at the beginning of the D40, and compared with the Meso-1 animals, the tumor volume is obviously small.

As shown in fig. 8B, the body weight of each group of mice did not change significantly.

As shown in fig. 8C, after CAR-T cell injection, the tumor volume of individual mice in each group changed, and the tumor volume of animals in the high dose Meso-E1m1 group had no recurrence compared to that in the Meso-1 group, and the tumor volume was significantly smaller than that in the Meso-1 group.

EXAMPLE 8 in vitro efficacy Studies-multiple rounds of killing

In vitro multiple killing experiments were performed on T cells harvested in example 4. The experimental method is shown schematically (FIG. 9A), and CAR-T cells and target cells are combined according to a ratio of 1:3 co-incubating, continuously culturing for 2-3 days, observing the growth condition of target cells under a microscope, taking half of the T cells to detect the phenotype and the cell number of the T cells when one of the CAR-T cells is used for completely lysing the target cells, and using pancreatin to digest the target cells, counting the target cells as a target cell residual value, and calculating a formula of the killing efficiency of the CAR-T cells: killing efficiency of CAR-T cells = (tumor only group-experimental group)/tumor only group = 100%, the remaining half of cells continued to co-culture with new target cells, continuing to kill as described above.

The results are shown in FIG. 9B, with each set of CAR-T and OVCAR3 co-cultures (E: T=1:3), the Meso-E1/E1m1 showed significant killing advantage after the third round of continuous culture, as shown in FIG. 9C, the Meso-E1/E1m1 after each round of continuous killing, the CAR-T cell values were calculated based on the CAR positive rate and the T cell count, and the results showed that the Meso-E1/E1m1 was significantly more continuously amplified than the Meso-1 CAR-T, with no significant difference between the Meso-E1/E1m 1.

Results as shown in figure 9D, each group of CAR-T showed significant killing advantage after the fourth round of continuous culture with MDA-MB231-Meso (E: t=1:3) co-culture. As shown in FIG. 9E, it was shown that after each round of continuous killing, the CAR-T cell values were calculated based on the CAR positive rate and the T cell count, and the results showed that there was significantly more continuous expansion of Meso-E1/E1m1 than Meso-1 CAR-T, with no significant difference between Meso-E1/E1m 1.

EXAMPLE 9 in vitro efficacy study-multiple rounds of killing in the Presence of TGF-. Beta.s

In vitro multiple killing experiments were performed on T cells harvested in example 4. The experimental protocol is shown in fig. 9A. CAR-T cells were combined with target cells at 1:5 co-incubating, adding different concentrations of recombinant TGF-beta 1 in the culture process, supplementing once every two days, continuously culturing for 2-3 days, recording the growth condition of target cells in real time, taking half of T cells to detect the phenotype of the T cells when one of the CAR-T cells is used for completely lysing the target cells, counting after the target cells are digested by pancreatin, obtaining the killing efficiency= (control group-experimental group)/100% of the control group, wherein the control group is a tumor only group, and continuously killing the rest half of the cells by the method.

Results as shown in fig. 10A, the Meso-E1m1 showed significant killing advantage after the second (R2), four-wheel (R4) continuous culture under co-culture of each group CAR-T with OVCAR3 (E: t=1:5). Table 1 shows the number of Meso-1, meso-E1m1 CAR-T cells calculated after each round of killing based on the cell positive rate, T cell count and dilution factor. FIG. 10B is a graph of CAR-T cell expansion plotted according to tables 2 and 3, showing that Meso-E1m1 expands significantly more than Meso-1 on the fourth round of continuous killing; in the second round of continuous killing under the condition of adding 1.25, 2.5 and 5ng/mL TGF beta 1, the expansion of the CAR-T cells is obviously 17 times, 19 times and 15 times more than that of the Meso-1; the continuous killing of the Meso-E1m1 under the culture condition of adding 1.25ng/mL TGF beta 1 has the obvious capability of killing target cells, which is obviously better than that of the Meso-1, and the expansion of the CAR-T cells is obviously better than that of the Meso-1 CAR-T under the same culture condition, and experimental results show that the Meso-E1m1 CAR-T can effectively reduce the proliferation and activation capability of the TGF beta to inhibit T cells, thereby reducing the immunosuppressive effect of the TGF beta.

TABLE 2 Meso-1

TABLE 3 Meso-E1m1

EXAMPLE 10 Annexin V staining to detect apoptosis

In vitro killing experiments were performed on T cells harvested in example 4, with CAR-T cells and target cells (MDA-MB 231-Meso) at a ratio of 1:2, after continuous culture for 3 days, the CAR-T cells are moved to fresh target cells, and after continuous culture for 3 days, the apoptosis of the CAR-T cells is detected, and the two groups of TGF-beta 1 groups with no addition/addition of the concentration shown in the figure are respectively arranged in the culture process.

As shown in FIG. 10C, the ratio of Annexin V to 7-AAD dian cells is 60% at the highest, the activity rate of the Meso-E1m1 CAR-T cells is not obviously changed when different concentrations of TGF-beta are added, the apoptosis cells are increased along with the concentration of TGF-beta 1 in the Meso-1 CAR group under the condition of TGF-beta 1 culture, the early apoptosis and the late apoptosis are obviously increased, and the experimental result shows that the Meso-E1m1 CAR-T can effectively reduce the capability of the TGF beta to promote T cell apoptosis, thereby reducing the immunosuppressive effect of the TGF beta.

EXAMPLE 11 Co-incubation of CAR-T cells with PBNK cells

T cells harvested in example 4 were compared to autologous PBNK cells cultured in vitro at a ratio of 1:1 proportion co-culture, firstly using CFSE to mark PBNK cells, respectively detecting the number of PBNK cells for 48 hours and 96 hours, adopting a flow detection method to detect FITC in a culture system ⁺ And (3) cells.

CFSE labeling method: after centrifugation of PBNK cells, the cells were resuspended to 1E6/mL with PBS, 4uM CFSE was added as a final concentration, incubated at room temperature for 2 minutes, and the reaction was terminated by adding medium.

Autologous PBNK is derived from monocytes of the same Donor of T cells, and is separated and purified by using anti-CD56microbeads, and a specific method is shown in protocols of Meitian and Tnet.

The experimental results are shown in FIG. 11, E1 CAR-T cells and E1m1 CAR-T cells are co-cultured with PBNK cells for 48-72 hours, and compared with NT cells, the E1 and E1m1 CAR-T cells can obviously promote the expansion of the PBNK cells.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

A Chimeric Antigen Receptor (CAR) construct characterized in that the CAR construct has the structure shown in formula I or II,

X-A-E (I)

E-A-X (II)

in the method, in the process of the application,

each "-" is independently a connecting peptide or peptide bond;

x is a CAR targeting a tumor antigen;

a is a self-shearing element;

e is an IL-15/IL-15Rα complex.
The CAR construct of claim 1 wherein the IL-15/IL-15Rα complex has the structure shown in formula III,

L’-M-I-R (III)

in the method, in the process of the application,

each "-" is independently a connecting peptide or peptide bond;

l' is none or a signal peptide;

m is IL-15 or a mutant thereof;

i is a flexible joint;

r is IL-15Rα.
The CAR construct of claim 2, wherein the IL-15 mutant is IL-15N72D having the amino acid sequence set forth in SEQ ID No.: 2.
The CAR construct of claim 2, wherein the IL-15 ra is a complete IL-15 ra element having the amino acid sequence set forth in SEQ ID No.: 3.
The CAR construct of claim 2, wherein the amino acid sequence of the IL-15/IL-15 ra complex is as set forth in SEQ ID No.:5 or 6.
The CAR construct of claim 1, wherein the structure of X (CAR) is represented by formula IV,

L-scFv-H-TM-C-CD3ζ (IV)

each "-" is independently a connecting peptide or peptide bond;

l is none or a signal peptide;

scFv are antibody single chain variable regions that target tumor antigens;

h is a hinge-free region;

TM is a transmembrane domain;

c is a costimulatory signaling molecule;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ.
A nucleic acid molecule encoding the Chimeric Antigen Receptor (CAR) construct according to claim 1, or,

the nucleic acid molecules comprise a first nucleic acid molecule encoding a CAR targeting a tumor antigen and a second nucleic acid molecule encoding an IL-15/IL-15 ra complex.
A vector comprising the nucleic acid molecule of claim 7.
An engineered immune cell expressing the CAR construct of claim 1, or

The immune cells express a CAR and an IL-15/IL-15 ra complex that target tumor antigens.
The immune cell of claim 9, wherein the tumor antigen-targeted CAR and IL-15/IL-15 ra complex are independently expressed on the cell membrane of the immune cell.
A formulation comprising the chimeric antigen receptor construct of claim 1, the nucleic acid molecule of claim 3, the vector of claim 4, or the immune cell of claim 5, and a pharmaceutically acceptable carrier.
Use of the chimeric antigen receptor construct of claim 1, the nucleic acid molecule of claim 7, the vector of claim 8, or the immune cell of claim 9, or the formulation of claim 11 for the preparation of a medicament or formulation for the prevention and/or treatment of cancer or tumor.
A method of making the engineered immune cell of claim 9, the method comprising the steps of:

(a) Providing an immune cell to be engineered; and

(b) Introducing the nucleic acid molecule of claim 7 or the vector of claim 8 into the immune cell, thereby obtaining the engineered immune cell.
Use of an IL-15/IL-15Rα complex for the preparation of a formulation for enhancing persistence of and/or cytotoxicity of CAR-T cells,

Wherein the structure of the IL-15/IL-15Rα complex is shown in the following formula III,

L’-M-I-R (III)

in the method, in the process of the invention,

each "-" is independently a connecting peptide or peptide bond;

l' is none or a signal peptide;

m is IL-15 or a mutant thereof;

i is a flexible joint;

r is IL-15Rα, and R is IL-15Rα,

wherein the IL-15Rα comprises a transmembrane region and an intracellular region.
A method of treating a disease, comprising administering to a subject in need thereof an appropriate amount of the vector of claim 8, the immune cell of claim 9, or the formulation of claim 11, wherein the disease is cancer or tumor.