CN118109416A - Functional enhancement type engineering immune cell, preparation and application thereof - Google Patents

Abstract

The invention provides a functional enhancement type engineering immune cell, and preparation and application thereof. The invention obviously improves the activation degree of IL-7 signals of engineering immune cells and the resistance to tumor microenvironment by simultaneously up-regulating IL-7 channels and inhibiting TGF beta channels, and simultaneously reduces the toxic and side effects caused by the over-expression of IL-7. The engineered immune cells of the invention, particularly TIL, are useful for treating solid tumors.

Description

Functional enhancement type engineering immune cell, preparation and application thereof

Technical Field

The invention belongs to the field of tumor immunity and cell therapy, and in particular relates to a functional enhancement type engineering immune cell, and preparation and application thereof.

Background

T cells are an important class of lymphocytes involved in cellular immunity, and can specifically recognize and kill tumor cells with the aid of antigen presenting cells. However, tumor cells also hinder specific recognition of T cells by reducing or losing epitopes, constructing immunosuppressive microenvironments, evolving tumors of different heterogeneity (i.e., differences in genotype to phenotype between tumor cells of the same malignancy among different patients or within the same patient), and the like, thereby evading immune responses of the organism.

Chimeric antigen receptor T cell (CHIMERIC ANTIGEN receptor T cell, CAR-T) therapy has been developed to address this problem. Specifically, the CAR molecule is an artificially designed and constructed receptor molecule, consists of a signal peptide, an extracellular antigen binding domain, a hinge region, a transmembrane region, a co-stimulatory domain, an intracellular signaling domain and the like, and has the functions of specifically recognizing tumor surface antigens, activating T cell killing activity, stimulating T cell proliferation and the like. The CAR-T cells are obtained by collecting T cells of a tumor patient and artificially transducing genes of the CAR molecules, so that the self T cells of the patient express the CAR molecules. After being returned to the body of a patient, the CAR-T cells can specifically and efficiently identify and kill tumor cells through CAR molecules, so that the effect of treating cancers is achieved.

The concept of CAR-T therapy was first proposed in 1989, undergoing thirty years of development and multiple rounds of technological alternation. The first generation of CAR-T cells consist of only single-chain antibodies as extracellular antigen binding domains and cd3ζ as intracellular signaling domains, and cannot fully activate T cell activity, resulting in poor therapeutic effects. The second generation CAR-T cell introduces a co-stimulatory domain based on the first generation CAR-T cell, thereby improving the in vitro proliferation capacity and cytokine release level of the T cell. The third generation CAR-T cells are added with a co-stimulatory domain based on the second generation CAR-T cells, and although the killing activity of the T cells can be improved, excessive release of cytokines is possibly induced, and serious side effects are caused. Therefore, the new generation of CAR-T cells jointly express other auxiliary factors, such as intracellular domains which jointly express IL-12 or certain cytokine receptors, on the basis of the second generation of CAR-T cells, and the like, thereby contributing to the improvement of tumor killing activity and safety.

However, currently developed CAR-T cells suffer from low proliferation capacity, increased failure, low persistence, high toxic and side effects, and the like. Clinical researches show that in the treatment of solid cancers, anti-tumor drugs are 'disabled' due to the limitations of a plurality of factors such as inhibitory tumor immune microenvironment, poor immune cell survival ability and the like, so that tumor immune escape is caused. Therefore, by expressing the auxiliary factors, the immune cells can promote the tumor killing capacity, and simultaneously promote proliferation, prolong survival time and improve memory capacity, so that the immune cells are key to improving the therapeutic effect of the immune cells.

In view of the foregoing, there is a strong need in the art to develop more efficient, longer-lasting engineered immune cells against tumor cells that survive long-term in the body and resist tumor microenvironment inhibition.

Disclosure of Invention

The invention aims to provide an engineering immune cell (such as CAR-T cell and TIL cell) with higher efficiency and better therapeutic effect aiming at malignant tumor (especially solid tumor).

It is a further object of the present invention to provide an engineered immune cell (e.g., CAR-T cell, TIL cell) that modulates IL-7 and tgfβ signaling pathways, and methods of making and using the same.

In a first aspect of the invention, there is provided an engineered immune cell which is a T cell, a TIL cell, or an NK cell, and which has the following characteristics:

(a) The immune cells express exogenous IL-7 protein; and

(B) The immune cells express exogenous TGF-beta RII dominant repression mutant proteins or shRNA targeting endogenous TGF-beta RII transcripts.

In another preferred embodiment, the T cells comprise αβ T cells, γδ T cells, NKT cells, MAIT cells, or a combination thereof.

In another preferred embodiment, the engineered immune cell is selected from the group consisting of:

(i) Chimeric antigen receptor T cells (CAR-T cells);

(ii) Chimeric antigen receptor NK cells (CAR-NK cells);

(iii) Chimeric antigen receptor TIL cells (CAR-TIL cells).

In another preferred embodiment, the engineered immune cell is a TIL cell.

In another preferred embodiment, the engineered immune cells are T cells from a patient undergoing radiation and/or chemotherapy.

In another preferred embodiment, the engineered immune cells are autologous TIL cells from the patient undergoing radiation and/or chemotherapy.

In another preferred embodiment, the engineered immune cell is autologous or allogeneic.

In another preferred embodiment, the IL-7 protein and/or TGF-beta RII dominant-suppression mutant may be constitutively expressed or inducible expressed.

In another preferred embodiment, the IL-7 protein is membrane-bound or secreted.

In another preferred embodiment, the dominant tgfbetarii inhibiting mutant is membrane bound.

In another preferred embodiment, the immune cell expresses a Chimeric Antigen Receptor (CAR), wherein the CAR targets a surface marker of a tumor cell.

In another preferred embodiment, the CAR is constitutively expressed or inducible expressed.

In another preferred embodiment, the IL-7 protein has the structure shown in formula Z below:

A-H-TM-D (Z)

In the method, in the process of the invention,

A is IL-7 protein, or an active fragment thereof, or a mutant thereof;

H is the no or hinge region;

TM is an absent or transmembrane domain;

D is a non-or degradation domain (including wild-type, or mutant/modification thereof); the "-" is a connecting peptide or peptide bond.

In another preferred embodiment, the A is a wild-type or mutant IL-7 protein of a human or non-human mammal.

In another preferred embodiment, the amino acid sequence of A is shown in SEQ ID No. 1.

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

In another preferred embodiment, the H is the hinge region of the CD8 protein.

In another preferred embodiment, the H is the hinge region of an IgG protein Fc.

In another preferred embodiment, the amino acid sequence of H is shown in SEQ ID No. 2.

In another preferred embodiment, the TM is the transmembrane region ：CD28、CD3 epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、CD278、CD152、CD279、CD233、CD314、 of a protein selected from the group consisting of mutations/modifications thereof, or combinations thereof.

In another preferred embodiment, the TM is the transmembrane region of CD 8.

In another preferred embodiment, the amino acid sequence of the TM is shown in SEQ ID No. 3.

In another preferred embodiment, the D is an oxygen-dependent degradation domain (ODD) or a partial sequence thereof;

In another preferred embodiment, the amino acid sequence of the ODD is shown as SEQ ID No. 35.

In another preferred embodiment, the IL-7 protein and the TGF-beta RII dominant-suppression mutant have the structure shown in formula I:

Z0-Z1-Z2-D (I)

In the method, in the process of the invention,

One of Z0 and Z2 is IL-7 protein, and the other is a TGF-beta RII dominant inhibition mutant;

z1 is none, or a linking peptide;

d is a non-or degradation domain (including wild-type, or mutant/modification thereof);

The "-" is none, a linking peptide or a peptide bond.

In another preferred embodiment, Z0 is an IL-7 protein and Z2 is a TGF-beta RII dominant-suppression mutant.

In another preferred embodiment, the coding sequence for the IL-7 protein and the coding sequence for the TGF-beta RII dominant inhibitory mutant are linked by an Internal Ribosome Entry Site (IRES), the coding sequence for a 2A-linked peptide, or the coding sequence for a flexible linked peptide.

In another preferred embodiment, the linker peptide is selected from the group consisting of: 2A linker peptide, or a flexible linker peptide.

In another preferred embodiment, the 2A connecting peptide is P2A (SEQ ID No: 4), T2A (SEQ ID No: 5).

In another preferred embodiment, the flexible linker peptide is one or more G4S-containing linkers, such as 4 XGGGGS (SEQ ID No: 6).

In another preferred embodiment, the nucleotide sequence of IRES is shown in SEQ ID No. 7.

In another preferred embodiment, the amino acid sequence of the IL-7 protein (Z0) is shown in SEQ ID No. 1 or SEQ ID No. 8.

In another preferred embodiment, the amino acid sequence of said TGF-beta RII dominant-inhibiting mutant (Z2) is shown in SEQ ID No. 9 or SEQ ID No. 34.

In another preferred embodiment, the shRNA targeting the endogenous tgfbetarii transcript inhibits expression of the endogenous tgfbetarii transcript.

In another preferred embodiment, the TGF-beta RII dominant-repressing mutant protein may be replaced with an shRNA expression cassette targeting an endogenous TGF-beta RII transcript (SEQ ID No: 10).

In another preferred embodiment, the shRNA expression cassette consists of a promoter and a shRNA coding sequence.

In another preferred embodiment, the promoter of the shRNA expression cassette is the U6 promoter (SEQ ID No: 11).

In another preferred embodiment, the binding site of the shRNA is located within 703-738nt of the open reading frame of the TGF-beta RII gene (SEQ ID No: 12).

In another preferred example, the combined nucleotide sequence of the shRNA is shown as SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.15, SEQ ID No. 16 or SEQ ID No. 17.

In another preferred example, the nucleotide sequence of the shRNA expression frame is shown as SEQ ID No. 18, SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21 or SEQ ID No. 22.

In another preferred embodiment, the cell is a chimeric antigen receptor T cell (CAR-T cell) having one or more of the following characteristics:

(a) The CAR-T cells express exogenous IL-7 proteins;

(b) The CAR-T cells express exogenous tgfbetarii dominant inhibitory mutant proteins or shRNA targeting endogenous tgfbetarii transcripts; and

(C) Optionally, the CAR-T cell expresses a chimeric antigen receptor CAR.

In another preferred embodiment, the CAR-T cell induces expression of a CAR molecule and/or IL-7 and/or tgfbetarii dominant inhibitory mutant protein when the CAR-T cell is contacted with an inducer or inducing conditions.

In another preferred embodiment, in said CAR-T cell, the CAR, IL-7 and tgfbetarii dominant inhibitory mutant proteins are expressed in tandem.

In another preferred embodiment, in said CAR-T cell, the CAR, IL-7, tgfbetarii dominant inhibitory mutant protein and shRNA targeting endogenous tgfbetarii are each expressed independently.

In another preferred embodiment, the "activating" refers to binding of the CAR to a surface marker of a tumor cell.

In another preferred embodiment, the "tumor surface marker" refers to a specific antigen on the tumor surface.

In another preferred embodiment, said chimeric antigen receptor CAR is located at the cell membrane of said engineered immune cell.

In another preferred embodiment, said chimeric antigen receptor CAR is located at the cell membrane of said CAR-T cell.

In another preferred embodiment, the IL-7 protein and/or TGF-beta RII dominant-suppression mutant protein is localized to the cell membrane of the CAR-T cell.

In another preferred embodiment, the CAR targets a surface marker of a tumor cell, said marker selected from the group consisting of: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD271, GUCY2C, CD, CD133, CD44, CD166, CD276, ABCB5, ALDH1, mesothelin (MSLN), EGFR, GPC3, BCMA, erbB2, NKG2D ligands (ligands), LMP1, epCAM, VEGFR-1, lewis-Y, ROR1, claudin18.2, CEA, TAG-72, TROP2, CD147, CLDN6.

In another preferred embodiment, the CAR has the structure shown in formula II:

L-scFv1-H-TM-C-CD3ζ-D(II)

In the method, in the process of the invention,

L is a none or signal peptide sequence;

scFv1 is an antigen binding domain (e.g., an antibody or active fragment thereof) that targets a surface marker of a tumor cell;

H is the no or hinge region;

TM is a transmembrane domain;

c is a costimulatory signaling domain;

Cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ (including wild-type, or mutant/modification thereof);

d is a non-or degradation domain (including wild-type, or mutant/modification thereof);

the "-" is a connecting peptide or peptide bond.

In another preferred embodiment, the antigen binding domain (scFv 1) is selected from the group consisting of: single chain antibody scFv, single domain antibody VHH, extracellular domain of receptor protein, or a combination thereof.

In another preferred embodiment, the tumor cell surface marker is CD133 (SEQ ID No: 23) or CEA (SEQ ID No: 24).

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

In another preferred embodiment, the amino acid sequence of L is shown in SEQ ID No. 25.

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

In another preferred embodiment, the H is selected from the hinge region of IgG proteins Fc or CD8 of the following group.

In another preferred embodiment, the amino acid sequence of H is shown in SEQ ID No. 26 or SEQ ID No. 27.

In another preferred embodiment, the TM is selected from the group consisting of the transmembrane region ：CD28、CD3 epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、CD278、CD152、CD279、CD233、CD314、 of a protein of the following group, or a mutant/modification thereof, or a combination thereof.

In another preferred embodiment, the TM is the transmembrane region of CD 8.

In another preferred embodiment, the amino acid sequence of the TM is shown in SEQ ID No. 3.

In another preferred embodiment, the C is selected from the group consisting of costimulatory domain ：OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD1、Dap10、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、OX40L、 of proteins, or mutations/modifications thereof, or combinations thereof.

In another preferred embodiment, C is a costimulatory domain of 4-1BB origin.

In another preferred embodiment, the amino acid sequence of C is shown in SEQ ID No. 28.

In another preferred embodiment, the amino acid sequence of CD3 zeta is shown in SEQ ID No. 29.

In another preferred embodiment, the amino acid sequence of scFv1 is shown as SEQ ID NO. 30 or SEQ ID NO. 31 or SEQ ID NO. 32.

In another preferred embodiment, the CAR-T cell contains, in addition to the first CAR of formula II, a second CAR for a second antigen, the second CAR having the structure of formula III:

L-scFv2-H-TM-C-CD3ζ-D (III)

In the method, in the process of the invention,

L is a none or signal peptide sequence;

scFv2 is an antigen binding domain (e.g., an antibody or active fragment thereof) that targets a surface marker of a second tumor cell;

H is the no or hinge region;

TM is a transmembrane domain;

c is a costimulatory domain;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ or a mutant/modification thereof;

d is a non-or degradation domain (including wild-type, or mutant/modification thereof);

the "-" is a connecting peptide or peptide bond.

In another preferred embodiment, the antigen binding domain (scFv 2) is selected from the group consisting of: single chain antibody scFv, single domain antibody VHH, extracellular domain of receptor protein, or a combination thereof.

In another preferred embodiment, scFv2 in formula III targets a different antigen than scFv1 in formula II.

In another preferred embodiment, the scFv2 is an antibody single chain variable region or a single domain antibody sequence that targets a tumor antigen and active fragments thereof.

In another preferred embodiment, the scFv2 is an antibody single chain variable region or single domain antibody sequence and active fragments thereof that targets an antigen selected from the group consisting of: CD19, CD20, CD22, CD123, CD47, CD138, CD33, CD30, CD271, GUCY2C, CD, CD133, CD44, CD166, CD276, ABCB5, ALDH1, mesothelin (MSLN), EGFR, GPC3, BCMA, erbB2, NKG2D ligands (ligands), LMP1, epCAM, VEGFR-1, lewis-Y, ROR1, claudin18.2, TROP2, CEA, TAG-72, CD147, CLDN6, or combinations thereof.

In another preferred embodiment, the first CAR of formula II and the second CAR of formula III may be combined to form a CAR of formula IVa or IVb:

L-scFv1-scFv2-H-TM-C-CD3ζ-D (IVa)

L-scFv2-scFv1-H-TM-C-CD3ζ-D (IVb)

In the method, in the process of the invention,

L is a none or signal peptide sequence;

scFv1 is an antibody or active fragment thereof that targets a surface marker of a first tumor cell;

scFv2 is an antibody or active fragment thereof that targets a surface marker of a second tumor cell;

H is the no or hinge region;

TM is a transmembrane domain;

c is a costimulatory domain;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ or a mutant/modification thereof;

d is a non-or degradation domain (including wild-type, or mutant/modification thereof);

the "-" is a connecting peptide or peptide bond.

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

(A) Providing an immune cell to be modified; and

(B) The immune cell is engineered such that the immune cell expresses an exogenous IL-7 protein and an exogenous tgfbetarii dominant inhibitory mutant protein or shRNA targeting an endogenous tgfbetarii transcript, thereby obtaining an engineered immune cell according to the first aspect of the invention.

In another preferred embodiment, in step (B), it includes:

(B1) Introducing a first expression cassette that expresses IL-7 into the immune cell;

(B2) Introducing into said immune cell a second expression cassette that expresses a dominant tgfβrii suppression mutant or shRNA that targets an endogenous tgfβrii transcript;

wherein steps (B1) and (B2) may be performed in any order, or may be performed in combination.

In another preferred embodiment, the step (B1) may be performed before, after, simultaneously with, or alternatively with the step (B2).

In another preferred embodiment, said step (B) further comprises (B3) introducing a third expression cassette expressing a chimeric antigen receptor CAR into said immune cell; wherein steps (B1), (B2), and (B3) may be performed in any order, or combined.

In another preferred embodiment, the "combining" means combining two or three of the first, second, and optionally third expression cassettes into one expression cassette and introducing into immune cells.

In another preferred embodiment, there is provided a method of preparing said CAR-immune cell comprising the steps of:

(A) Providing an immune cell to be engineered;

(B) Modifying the immune cell so that the immune cell expresses the CAR molecule and the exogenous IL-7 protein and the exogenous tgfbetarii dominant inhibitory mutant protein or shRNA targeting endogenous tgfbetarii transcripts, thereby obtaining an engineered immune cell according to the first aspect of the invention.

In another preferred embodiment, the immune cells are T cells, TIL cells, or NK cells.

In another preferred embodiment, in step (B), it includes: introducing into the immune cell a first expression cassette that expresses IL-7, a second expression cassette that expresses a tgfbetarii dominant-repressing mutant or shRNA that targets an endogenous tgfbetarii transcript, and optionally a third expression cassette that expresses the CAR; the introduction steps may be performed in any order, or may be combined.

In another preferred embodiment, the transcription direction is the same direction (→→), opposite direction (→≡), or opposite direction (≡Σ) for any two of the first, second and third expression cassettes (for example, the first expression cassette and the second expression cassette).

In another preferred embodiment, the first expression cassette, the second expression cassette and the third expression cassette are on the same or different vectors.

In another preferred embodiment, the first expression cassette, the second expression cassette and the third expression cassette are located on the same vector.

In another preferred embodiment, when two or three of the CAR molecule, the exogenous IL-7 protein, and the exogenous tgfbetarii dominant-suppression mutant protein are expressed in tandem, a linker peptide or IRES element is also included between the two proteins.

In another preferred embodiment, the connecting peptide is P2A or T2A or 4 XGGGGS.

In another preferred embodiment, the vector is a viral vector, preferably the viral vector comprises the first, second and third expression cassettes in tandem.

In another preferred embodiment, the carrier is selected from the group consisting of: DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, retroviral vectors, transposons, other gene transfer systems, or combinations thereof.

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

In a third aspect of the invention there is provided a formulation comprising an engineered immune cell according to the first aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the formulation contains the CAR-T cells of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

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

In another preferred embodiment, the dosage form of the formulation comprises an injection.

In another preferred embodiment, the concentration of the engineered immune cells (e.g., CAR-T cells) in the formulation is 1 x10 ³-1×10⁸ cells/ml, preferably 1 x10 ⁴-1×10⁷ cells/ml.

In a fourth aspect of the invention there is provided the use of an engineered immune cell according to the first aspect of the invention for the preparation of a medicament or formulation for the prophylaxis and/or treatment of cancer.

In another preferred embodiment, there is provided the use of a CAR-T cell and a TIL cell according to the first aspect of the invention, for the preparation of a medicament or formulation for the prevention and/or treatment of cancer or tumour.

In another preferred embodiment, the formulation contains CAR-T cells and TIL cells, and a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the tumor comprises a solid tumor.

In another preferred embodiment, the tumor is selected from the group consisting of: colorectal cancer, gastric cancer, pancreatic cancer, esophageal cancer, breast cancer, ovarian cancer or liver cancer.

In a fifth aspect of the invention there is provided a kit for preparing an engineered immune cell according to the first aspect of the invention, the kit comprising a container, and within the container:

(1) A first nucleic acid sequence comprising a first expression cassette for expressing IL-7; and

(2) A second nucleic acid sequence comprising a second expression cassette for expressing a dominant tgfβrii suppression mutant or shRNA targeting an endogenous tgfβrii transcript.

In another preferred embodiment, there is provided a kit for preparing an engineered immune cell according to the first aspect of the invention, the kit comprising a container, and within the container:

(1) A first nucleic acid sequence comprising a first expression cassette for expressing IL-7;

(2) A second nucleic acid sequence comprising a second expression cassette for expressing a dominant tgfβrii suppression mutant or shRNA targeting an endogenous tgfβrii transcript; and

(3) Optionally a third nucleic acid sequence comprising a third expression cassette for expressing the CAR.

In another preferred embodiment, the first, second and third nucleic acid sequences are independent or linked.

In another preferred embodiment, the first, second and third nucleic acid sequences are located in the same or different containers.

In another preferred embodiment, the first, second and third nucleic acid sequences are located on the same or different vectors.

In another preferred embodiment, the first, second and third nucleic acid sequences are located in the same vector.

In another preferred embodiment, when two or three of said first, second and third nucleic acid sequences are located in the same vector, a coding sequence or IRES element for expression of the connecting peptide is also included between two adjacent expression cassettes.

In another preferred embodiment, the connecting peptide is P2A or T2A or 4 XGGGGS.

In another preferred embodiment, the vector is a viral vector, preferably the viral vector contains the first, second and third nucleic acid sequences in tandem.

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 the structural design of the cofactor.

FIG. 2 shows the structural design of CEA-targeting second and fourth generation CARs.

Figure 3 shows the structural design of CD133 targeted second and fourth generation CARs.

FIG. 4 shows the expression rate of each cofactor in flow-test enhanced TIL cells.

FIG. 5 shows the secretion levels of IL-7 in enhanced TIL cells.

Figure 6 shows the CAR molecule expression rate of flow-detected CEA CAR-T cells.

FIG. 7 shows the dnTGF. Beta. RII expression rate of flow-detected CEA CAR-T cells.

FIG. 8 shows IL-7 secretion level detection of CEA CAR-T cells.

Figure 9 shows CAR molecule expression rates for flow detection of CD133 CAR-T cells.

FIG. 10 shows the expression rate of dnTGF. Beta. RII in flow-detected CD133 CAR-T cells.

FIG. 11 shows detection of IL-7 secretion levels by CD133 CAR-T cells.

FIG. 12 shows the detection of the killing effect of enhanced TIL cells on liver cancer cells.

FIG. 13 shows IFN-gamma release levels from enhanced TIL cells.

FIG. 14 shows the detection of the killing effect of CEA CAR-T cells co-expressing IL-7 and dnTGF. Beta. RII on HT 55.

FIG. 15 shows the detection of the killing effect of SW620 by CD133 CAR-T cells co-expressing IL-7 and dnTGF βRII.

FIG. 16 shows the in vivo tumor-inhibiting effect of CD133 CAR-T cells co-expressing IL-7 and dnTGF βRII.

FIG. 17 shows the killing effect of CAR-T cells co-expressing IL-7 and dnTGF βRII fusion proteins on a variety of solid tumor cells.

FIG. 18 shows the in vitro proliferative capacity of CAR-T cells co-expressing IL-7 and dnTGF. Beta. RII fusion proteins.

Detailed Description

Through extensive and intensive research, the inventor performs a large number of screening, and regulates the IL-7 and TGF beta channels at the same time for the first time, namely up-regulates the IL-7 channel and inhibits the TGF beta channel, thereby remarkably improving the activation degree of IL-7 signals of engineering immune cells and remarkably improving the resistance to tumor microenvironment. Experiments show that the engineering immune cells (taking TIL and CAR-T cells as examples) not only remarkably improve the proliferation capacity of the immune cells, reduce failure and strengthen the resistance to immunosuppressive microenvironment, but also unexpectedly reduce side effects compared with the immune cells expressing IL-7 xCCL 19. The present invention has been completed on the basis of this finding.

The present invention will be described in detail with reference to TIL and CAR-T cells as representative examples. The engineered immune cells of the invention are not limited to the TIL and CAR-T cells described in the context, but have the same or similar technical features and benefits as the TIL and CAR-T cells described in the context. Specifically, when the immune cell expresses the chimeric antigen receptor CAR, the NK cell or TIL cell is equivalent to a T cell (i.e., the T cell may be replaced with an NK cell or a TIL cell).

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.

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 "administration" or "administration" (ADMINISTER OR ADMINISTRATION) refers to the introduction of a product of the invention into a subject using any of a variety of methods and delivery systems known to those of skill in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

Antibodies to

As used herein, 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.

"Antibody" also includes single domain antibodies (also known as nanobodies). A single domain antibody is an antibody in which a naturally deleted light chain exists in the peripheral blood of animals such as alpaca, camel, shark, etc., and which comprises only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. The VHH structure cloned and expressed alone has structural stability comparable to that of the original heavy chain antibody and binding activity to the antigen, the smallest unit known to bind the antigen of interest.

Antigen binding domains

As used herein, "antigen binding domain," "single chain antibody fragment" refers to Fab fragments, fab 'fragments, F (ab') ₂ fragments, single Fv fragments, or VHH fragments of single domain antibodies that have antigen binding activity. 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 an scFv (single-chain variable fragment). The single chain antibody is preferably an amino acid sequence encoded by a single nucleotide chain.

In addition, the immune cells of the invention may contain additional antibodies, preferably single chain antibodies, fv antibodies, or single domain antibodies, that specifically recognize antigens that are highly expressed by the tumor.

Chimeric Antigen Receptor (CAR)

As used herein, a chimeric immune antigen receptor (CHIMERIC ANTIGEN receptor, CAR) includes an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain. Extracellular domains include optional signal peptides and target-specific binding domains (also referred to as antigen binding domains). The intracellular domain includes a costimulatory domain and a cd3ζ chain moiety. When CAR is expressed in T cells, the extracellular segment recognizes a specific antigen, and then transduces the signal through the intracellular domain, causing activated proliferation of the cell, cytolytic toxicity, and secretion of cytokines such as IL-2 and IFN- γ, etc., affecting the tumor cells, causing the tumor cells to not grow, to be caused to die or otherwise be affected, and causing 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 the cd3ζ chain. Preferably, the antigen binding domain is fused to the intracellular domain of the combination of the 4-1BB signaling domain and the CD3 zeta signaling domain.

Chimeric antigen receptor T cells (CAR-T cells)

As used herein, the terms "CAR-T cell", "CAR-T cell of the invention" all refer to CAR-T cells of the first aspect of the invention.

CAR-T cells have the following advantages over other T cell-based therapies: (1) the course of action of CAR-T cells is not restricted by MHC; (2) In view of the fact that many tumor cells express the same tumor antigen, CAR gene construction for a certain tumor antigen can be widely utilized once completed; (3) The CAR can utilize not only tumor protein antigens but also glycolipid non-protein antigens, so that the target range of the tumor antigens is enlarged; (4) The use of autologous patient cells reduces the risk of rejection; (5) The CAR-T cells have an immunological memory function and can survive in vivo for a long time.

In the immune cells of the invention, constructs that activate the IL-7 pathway (construct), constructs that inhibit the tgfβ pathway, and CAR molecule constructs can be combined in a variety of different ways, representative ways including (but not limited to): 2A peptide, flexible linker peptide, IRES element, multiple promoters, co-transfection, co-transduction, and the like.

Chimeric antigen receptor NK cells (CAR-NK cells)

As used herein, the terms "CAR-NK cell", "CAR-NK cell of the invention" all refer to CAR-NK cells of the first aspect of the invention.

Natural Killer (NK) cells are a major class of immune effector cells that protect the body from viral infection and tumor cell invasion by non-antigen specific pathways. New functions may be obtained by engineered (genetically modified) NK cells, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.

CAR-NK cells also have advantages over autologous CAR-T cells, such as: (1) The perforin and the granzyme are released to directly kill tumor cells, and the perforin and granzyme have no killing effect on normal cells of the organism; (2) They release very small amounts of cytokines and thus reduce the risk of cytokine storms; (3) Is easy to expand and develop into a ready-made product in vitro. In addition, similar to CAR-T cell therapy.

TIL cells

In addition to CAR-T cell therapies, tumor-infiltrating lymphocyte (tumor infiltrating lymphocytes, TIL) therapies can also efficiently recognize and kill tumor cells, achieving the effect of cancer treatment.

TIL is an infiltrating immune cell isolated from tumor tissue that is responsive to tumor cells. These immune cells have the ability to recognize and attack tumor cells, and TIL cells are also currently used to treat tumor patients. Compared with other immunotherapy, such as CAR-T cell therapy, TCR-T cell therapy and PD-1/PD-L1 immune checkpoint inhibitor, TIL therapy has the advantages of stronger specificity, higher safety, low recurrence rate after treatment and the like. However, the number of TIL cells in tumor tissue is small and the activity is usually inhibited by immune microenvironment, and it is possible to obtain a desired antitumor effect by preparing a very large number of TIL cells during clinical treatment. Therefore, TIL therapy also needs to continue to be optimized.

In the present invention, novel TIL cells with significantly improved performance are unexpectedly obtained by simultaneously activating the IL-7 pathway and inhibiting the tgfβ pathway in TIL cells.

IL-7

Human interleukin 7 (interleukin-7, IL-7) is a pleiotropic cytokine with a broad immune response. For a long time, knowledge of IL-7 has focused on its effects on the growth, survival and differentiation of B cells and T cells. Exogenously injected IL-7 can enhance the antitumor immunity of the body in vivo. IL-7 has been attracting more attention as an immunomodulator because of its powerful immune effects, especially its functions of regulating T cell proliferation, maintaining intracellular homeostasis, enhancing T immune response, etc.

In the present invention, the therapeutic effect of engineered immune cells is enhanced by activating the IL-7 pathway. Representative methods of activating the IL-7 pathway include (but are not limited to): expression of secreted or membrane-bound IL-7.

In the present invention, IL-7 may be expressed in fusion with any structure capable of anchoring to the cell membrane, thereby achieving expression of membrane-bound IL-7.

In the present invention, the term "IL-7" includes wild-type and mutant IL-7 in humans and other mammals. It is to be understood that the term also includes IL-7 analogues or IL-7 derived proteins, as long as they have or retain the essential function of wild-type IL-7.

TGF-β

Transforming growth factor-beta (transforming growth factor-beta, TGF-beta) is a superfamily of proteins consisting of structurally related multifunctional cytokines, including TGF-beta 1/2/3 subtypes, activin, bone morphogenic proteins, and growth differentiation factors, among others. TGF-beta superfamily ligands regulate cellular function through 7 type I TGF-beta superfamily receptors (activin-like kinases-1 through 7) and 5 type II TGF-beta superfamily receptors (Tbeta RII, actRII, actRIIB, AMHRII and BMPRII), playing an important role in embryonic development, tissue repair, and skeletal muscle, cardiovascular, neural, endocrine, and immune system homeostasis.

TGF-beta is a tumor promoting factor capable of inducing epithelial-to-mesenchymal transition (EMT) of tumor cells, promoting tumor cell invasion and metastasis. In addition, TGF- β is a key regulator of T cell responses, plays an important role in regulating immune responses, and can regulate the function of almost every innate and adaptive immune cell, including dendritic cells, B cells, NK cells, innate lymphocytes, granulocytes, and the like. In late stages of the tumor, most tumor cells secrete TGF- β. Once the concentration of TGF- β is increased, it can block the differentiation of immature T cells toward Th1 cells and promote their transformation toward Treg subpopulations, while inhibiting the antigen presenting function of dendritic cells, resulting in immune escape of tumor cells.

In the present invention, representative ways of inhibiting tgfβ pathways include, but are not limited to: antibodies that overexpress a dominant inhibitory mutant of the TGF-beta receptor (dnTGF. Beta. RII) or TGF-beta, knock down the TGF-beta receptor by RNAi, and knock down the TGF-beta receptor by gene knockout (TGF-beta receptors include TGF-beta. RI and TGF-beta. RII). One representative way by RNAi is to use shRNA targeting endogenous TGF-beta RII transcripts.

It will be appreciated that in the present invention dnTGF βrii may be replaced by a tgfβr inhibitor (membrane bound/secreted), means for expression of an inhibitor/interfering gene of a related protein in the tgfβr signaling pathway, the receptor tgfβrii knocked out, knockdown of tgfβ in immune cells, or overexpressing its dominant inhibitory mutant dnTGF βrii, or overexpressing a membrane bound tgfβ antibody.

TGF beta RII dominant inhibitory mutant (dnTGF beta RII)

In the signaling pathway of tgfβ, tgfβ initiates the transmission of related signals by binding to tgfβri and tgfβrii. Specifically, after TGF beta binds to the extracellular domains of TGF beta RI and TGF beta RII, the TGF beta RI and the TGF beta RII can be made to approach each other in spatial position, and the intracellular domain of the TGF beta RII activates the intracellular domain of the TGF beta RI and is phosphorylated, so that the TGF beta RI shows kinase activity, and further the transcription factor Smad is phosphorylated and enters the cell nucleus to regulate the expression of the downstream gene. dnTGF beta RII is an intracellular domain deletion mutant of TGF beta RII that can still form complexes with TGF beta and TGF beta RI via the extracellular domain, but cannot phosphorylate the intracellular domain of TGF beta RI. Thus, overexpressed dnTGF βrii may compete for the ability of endogenous tgfβrii to bind to bound tgfβ and tgfβri or activate downstream Smad, thereby blocking tgfβ signaling.

In the invention, the receptor TGF beta RII of TGF beta is knocked down or knocked out in immune cells, or the dominant inhibition mutant dnTGF beta RII is overexpressed, so that the signal path of the endogenous TGF beta RII of the cells can be effectively blocked, the inhibition effect of TGF beta on the immune cells is reduced, the activity of the immune cells in the tumor microenvironment is maintained, and the tumor treatment effect is effectively improved.

Expression cassette

As used herein, "expression cassette" or "expression cassette of the invention" includes a first expression cassette, a second expression cassette, and a third expression cassette. The expression cassette of the invention is according to the fifth aspect of the invention, wherein the first expression cassette expresses an exogenous IL-7 protein, the second expression cassette expresses an exogenous tgfbetarii dominant-suppression mutant protein, and the optional third expression cassette expresses the CAR.

In the present invention, the IL-7 protein, TGF-beta RII dominant-suppression mutant protein, and CAR molecule may each be expressed constitutively or inducible independently.

In the case of induction expression, the first expression cassette expresses the IL-7 protein, the second expression cassette expresses the tgfbetarii dominant-suppression mutant protein, and the third expression cassette expresses the CAR molecule upon activation of the CAR-T cell by a corresponding inducer or a corresponding induction condition. Thus, in the absence of exposure to the corresponding inducer, the first expression cassette does not express the IL-7 protein, the second expression cassette does not express the TGF-beta RII dominant-suppression mutant protein, and the third expression cassette does not express the CAR molecule.

In one embodiment, the first, second and/or third expression cassette further comprises a promoter and/or terminator, respectively. The promoters of the first, second and third expression cassettes may be constitutive or inducible promoters.

Carrier body

The invention also provides vectors containing the expression cassettes of the invention. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer, as they allow for long-term, stable integration of transgenes into the cell genome and replication with replication of the daughter cell genome. Lentiviral vectors have advantages over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they transduce non-proliferating cells and have the advantage of low immunogenicity.

In general, the expression cassette or nucleic acid sequence of the invention may be linked downstream of the promoter by conventional procedures and incorporated into an expression vector. The vector may integrate into the eukaryotic cell genome and replicate accordingly. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

The expression vectors of the invention can also be used in standard gene delivery protocols for nucleic acid immunization and gene therapy. 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.

The expression cassette or nucleic acid sequence may be cloned into many types of vectors. For example, the expression cassette or nucleic acid sequence 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, and the like.

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 Molecular Cloning: A Laboratory Manual (Sambrook et al, cold Spring Harbor Laboratory, new York, 2001) and other manual of virology and molecular biology. 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 at least one replication origin that functions in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers (e.g., WO01/96584; WO01/29058; and U.S. Pat. No. 6,326,193).

Many viral-based systems have been developed and used for gene transduction of 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 one embodiment, a lentiviral vector is used. Many DNA virus systems are known in the art. In some embodiments, an adenovirus vector is used. Many adenoviral vectors are known in the art.

Additional promoter elements, such as enhancers, may regulate the frequency of transcription initiation. Typically, these elements 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 an element is inverted or moved relative to another element. 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 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 ebstein-Barr virus (EBV) immediate early promoter, the ruses sarcoma virus promoter, and human gene promoters such as but not limited to the actin promoter, the myosin promoter, the heme promoter, and the 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 can either initiate expression of the polynucleotide sequence linked to the inducible promoter when desired, or shut down expression when not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

The expression vector introduced into the cell may also contain either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of the expression cell from the transfected or infected cell population 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 gene and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable marker genes include, for example, antibiotic resistance genes, such as neomycin and the like.

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 (e.g., human T cell), 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, cationic complex transfection, 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, e.g., molecular Cloning: A Laboratory Manual (Sambrook et al, cold Spring Harbor Laboratory, new York, 2001). Preferred methods for introducing the polynucleotide into a host cell are liposome transfection and cationic complex polyethylenimine transfection.

Biological methods for introducing polynucleotides into host cells 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 (DELIVERY VEHICLE) 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. They may also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. The lipid is a lipid substance, which may be a naturally occurring or synthetic lipid. 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.

It will be appreciated that in the present invention, in addition to transduction with multiple lentiviruses, direct transfection of mRNA or plasmid, or by expression of artificial transcription factors, etc., may be used to express IL-7 in combination with TGF-beta RII dominant-repressing mutant molecules or shRNA targeting endogenous TGF-beta RII transcripts in immune cells such as T cells, TIL cells, etc.

Formulations

The invention provides a formulation comprising an engineered immune cell (e.g., CAR-T cell) according to the first aspect 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 immune cells (such as TIL cells or CAR-T cells) in the formulation is 1X 10 ³-1×10⁸ cells/ml, more preferably 1X 10 ⁴-1×10⁷ 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 provides therapeutic applications using cells (e.g., T cells and TIL cells, etc.) transduced with vectors (e.g., lentiviral vectors) comprising the expression cassettes of the invention. The transduced T cells or TIL cells can target the surface markers of tumor cells and express IL-7 and TGF-beta RII dominant inhibition mutant proteins, thereby synergistically and remarkably improving the killing efficiency of the tumor cells.

Accordingly, the present invention also provides a method of stimulating an immune response mediated by T cells or TIL cells targeted to a mammalian tumor cell population or tissue, comprising the steps of: administering the CAR-T cells of the invention or the enhanced TIL cells of the invention to a mammal.

In one embodiment, the invention provides 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 approach results in a very low probability of graft versus host response, and antigen is recognized by T cells in a non-MHC restricted manner. In addition, CAR-T can treat a variety of different 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 sustained control of tumors.

In one embodiment, the invention provides a class of cell therapies in which autologous TIL cells (or heterologous donors) are isolated from a patient, activated and genetically engineered to produce enhanced TIL cells, which are subsequently injected into the same patient. The method has the advantages that the occurrence probability of risks such as graft versus host reaction, cytokine storm, off-target, tumor cell immune escape and the like is extremely low, and the tumor cells are accurately identified by the TIL cells. Unlike antibody therapies, TIL cells are able to replicate in vivo, producing long-term persistence that can lead to sustained control of tumors.

In one embodiment, the CAR-T cells or enhanced TIL cells of the invention can undergo stable in vivo expansion and can last from months to years. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which the CAR-T cells can induce a specific immune response to highly expressing tumor cells of the antigen recognized by the CAR antigen binding domain.

Treatable cancers include tumors that are not vascularized or have not been substantially vascularized, as well as vascularized tumors. Types of cancers treated with the CARs of the invention include, but are not limited to: colorectal cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer, ovarian cancer, and the like.

In general, cells activated and expanded as described herein are useful in the treatment and prevention of diseases such as tumors. Accordingly, the invention provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T cell or enhanced TIL cell of the invention.

The CAR-T cells and enhanced TIL 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, IL-7, IL-15, IL-21 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.

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 factors such as the condition of the patient, and the type and severity of the patient's disease, or may be determined by clinical trials.

When referring to "therapeutically effective amount", "antitumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the 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 patient (subject). The pharmaceutical composition comprising T cells or enhanced TIL cells described herein may be administered at a dose of 10 ⁴ to 10 ¹¹ cells/kg body weight, preferably at a dose of 10 ⁵ to 10 ⁸ cells/kg body weight (including all whole values within the range). 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). The optimal dosage and treatment regimen 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, intranodal, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell or enhanced TIL cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell or enhanced TIL cell compositions of the invention are preferably administered by intravenous injection. The composition of T cells or enhanced TIL cells can be injected directly into a 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 (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 or enhanced TIL 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, from 1X 10 ⁵ to 1X 10 ¹² modified T cells or enhanced TIL cells of the invention can be administered to a patient by, for example, intravenous infusion, per treatment or per course of treatment.

The main advantages of the invention

(1) Simultaneously activating IL-7 pathway and inhibiting TGF beta pathway, and can be applied to various immune cells, such as TIL, CAR-T, TCR-T, NK and other immune cells.

(2) The invention activates IL-7 pathway and inhibits the modification of TGF beta pathway, and the amplitude of the improvement of TIL cell function is unexpectedly higher than that of T cell function. The CAR-TIL cells (or enhanced TIL cells) of the invention have a significantly more efficient ability to kill tumor cells.

(3) Unexpectedly, the magnitude of the functional enhancement resulting from the activation of the IL-7 pathway and inhibition of the tgfβ pathway strategy of the present invention is greater when T cells or TIL cells from patients undergoing radiotherapy and/or chemotherapy (which are often unsuitable for use in preparing engineered immune cells due to a higher degree of immune decline or depletion) are employed to prepare engineered immune cells than T cells from healthy donors.

(4) The CAR immune cell has great potential in the treatment of solid tumors, in particular to the treatment of colorectal cancer, ovarian cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer and the like.

(5) Compared with the CAR-T cells which only express dnTGF beta RII or TGF beta RII-IL-7RA fusion protein in a combined way, the invention can synergistically and further improve the proliferation capacity of the CAR-T cells, the release level of IFN-gamma and the killing effect on tumors.

(6) Compared with the CAR-T cell which jointly expresses IL-7 and CCL19, the engineered immune cell can improve the killing effect of the CAR-T cell on tumor cells which express high TGF beta in vitro and in vivo, and unexpectedly reduces toxic and side effects.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are for illustration of the invention only and are not intended to limit the scope of the invention. The experimental procedure, in which the specific conditions are not noted in the following examples, is generally followed by conventional conditions, such as, for example, molecular cloning: conditions described in the laboratory Manual (Sambrook et al, 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.

Reagents and materials used in the examples were obtained commercially unless otherwise indicated.

Materials and methods

Structural design of cofactor and CAR molecules

The structural design of each auxiliary factor is shown in figure 1.

(1) BW7TG is formed by sequentially connecting the following structures in series: IL-7, self-cleaving peptide P2A (abbreviated as P2A), TGF beta RII dominant inhibitory mutant (abbreviated as dnTGF beta RII).

(2) BW197-2 is composed of the following structures in series: IL-7, self-cleaving peptide T2A (abbreviated as T2A), CCL19, P2A, enhanced green fluorescent protein (abbreviated as eGFP).

(3) BWTG is composed of the following structures in series in sequence: dnTGF. Beta. RII.

The structural design of the CEA-targeted CAR molecule is shown in figure 2.

(1) BC001 is composed of the following structures in series: human CD8 signal peptide [ abbreviated as CD8 (SP) ], anti-human CEA single chain antibody (abbreviated as CEA scFv), human CD8 hinge region [ abbreviated as CD8 (hinge) ], human CD8 transmembrane domain [ abbreviated as CD8 (TM) ], human 4-1BB intracellular co-stimulatory domain [ abbreviated as 4-1BB (ID) ], human CD3 zeta intracellular signaling domain [ abbreviated as CD3 zeta (ID) ].

(2) BC010 is composed of the following structures in series: CD8 (SP), CEA scFv, CD8 (range), CD8 (TM), 4-1BB (ID), CD3 zeta (ID), self-cleaving peptide F2A (abbreviated as F2A), IL-7, T2A, CCL, and the like.

(3) BC011 is composed of the following structures in series: CD8 (SP), CEA scFv, CD8 (range), CD8 (TM), 4-1BB (ID), CD3 zeta (ID), F2A, IL-7, P2A, dnTGF beta RII.

The structural design of a CD 133-targeted CAR molecule is shown in figure 3.

(1) BW133-2 is composed of the following structures in series: CD8 (SP), CD133 scFv, human IgG hinge region [ abbreviated IgG (Fc) ], CD8 (TM), 4-1BB (ID), CD3 ζ (ID).

(2) BW133-12 is composed of the following structures in series in sequence: IL-7, T2A, CCL19, F2A, CD8 (SP), CD133 scFv, igG (Fc), CD8 (TM), 4-1BB (ID), CD3 zeta (ID).

(3) BW133-13 is composed of the following structures in series: IL-7, P2A, dnTGF. Beta. RII, F2A, CD8 (SP), CD133 scFv, igG (Fc), CD8 (TM), 4-1BB (ID), CD3 zeta (ID).

(4) BW133-11B is composed of the following structures in series in sequence: CD8 (SP), CD133 scFv, igG (Fc), CD8 (TM), 4-1BB (ID), CD3 ζ (ID), internal ribosome entry site (IRES for short), dnTGF βRII.

(5) BW133-6B is composed of the following structures in series in sequence: CD8 (SP), CD133 scFv, igG (Fc), CD8 (TM), 4-1BB (ID), CD3 ζ (ID), IRES, IL-7, T2A, CCL19.

(6) BW133-7B is composed of the following structures in series in sequence: CD8 (SP), CD133 scFv, igG (Fc), CD8 (TM), 4-1BB (ID), CD3 ζ (ID), IRES, IL-7, P2A, dnTGF. Beta. RII.

(7) BW133-7E is composed of the following structures in series in sequence: CD8 (SP), CD133 scFv, igG (Fc), CD8 (TM), 4-1BB (ID), CD3 ζ (ID), IRES, fusion proteins of IL-7 and dnTGF βRII (abbreviated as IL-7: dnTGFβRII).

The amino acid sequence of the relevant structure is as follows:

human IL-7(SEQ ID No:1)：

CD8 hinge 1(SEQ ID No:2)：

CD8 TM(SEQ ID No:3)：

P2A(SEQ ID No:4)：

T2A(SEQ ID No:5)：

4×GGGGS(SEQ ID No:6)：

IRES(SEQ ID No:7)：

mIL-7(SEQ ID No:8)：

TGF-beta RII dominant-suppression mutant 1 (SEQ ID No: 9):

TGFβRII mRNA(SEQ ID No:10)：

/>

U6 promoter nucleotide sequence (SEQ ID No: 11):

TGF-beta RII shRNA targeting-domain nucleotide sequence (SEQ ID No: 12):

TGF-beta RII shRNA binding site nucleotide sequence 1 (SEQ ID No: 13):

TGF-beta RII shRNA binding site nucleotide sequence 2 (SEQ ID No: 14):

TGF-beta RII shRNA binding site nucleotide sequence 3 (SEQ ID No: 15):

TGF-beta RII shRNA binding site nucleotide sequence 4 (SEQ ID No: 16):

TGF-beta RII shRNA binding site nucleotide sequence 5 (SEQ ID No: 17):

TGF-beta RII shRNA expression cassette nucleotide sequence 1 (SEQ ID No: 18):

TGF-beta RII shRNA expression cassette nucleotide sequence 2 (SEQ ID No: 19):

TGF-beta RII shRNA expression cassette nucleotide sequence 3 (SEQ ID No: 20):

TGF-beta RII shRNA expression cassette nucleotide sequence 4 (SEQ ID No: 21):

TGF-beta RII shRNA expression cassette nucleotide sequence 5 (SEQ ID No: 22):

CD133(SEQ ID No:23)：

CEA(SEQ ID No:24)：

CD8 signal peptide(SEQ ID No:25)：

/>

IgG hinge(SEQ ID No:26)：

CD8 hinge 2(SEQ ID No:27)：

4-1BB co-stimulatory domain (SEQ ID No: 28):

CD3 zeta intracellular domain (SEQ ID No: 29):

CD133 scFv-1(SEQ ID No:30)：

CD133 scFv-2(SEQ ID No.31)：

CEA scFv(SEQ ID No:32)：

IL7:dnTGFβRII(SEQ ID No:33)：

TGF-beta RII dominant-suppression mutant 2 (SEQ ID No: 34):

ODD(SEQ ID:35)：

EXAMPLE 1 preparation of lentiviral vectors

Each of the above-described target gene fragments was constructed in the lentiviral expression vector pCDH-EF 1. Alpha. -MCS, respectively, and each vector plasmid was mixed with lentiviral packaging plasmids pMDLg-pRRE, pRSV-Rev and pMD2.G, respectively, and transfected into 293T cells with Lipofectamine 3000 reagent according to the instructions. 6h after transfection was replaced with complete medium.

Virus supernatants were collected 48h and 72h after transfection, respectively, to obtain virus concentrates, designated BW7TG、BW197-2、BWTG、BC001、BC010、BC011、BW133-2、BW133-12、BW133-13、BW133-11B、BW133-6B、BW133-7B、BW133-7E., and the collected virus concentrates were stored at-80 ℃.

Finally, the active titer of the lentiviruses is detected by using Jurkat cells as a material.

Example 2 preparation and detection of enhanced TIL cells

(1) Preparation of enhanced TIL cells

The single cell suspension obtained after digestion treatment of tumor tissue is cultured in a culture medium containing anti-human CD3 antibody and IL-2 until TIL cells enter the logarithmic phase. The lentiviral vectors were added at MOI=10IU/ml, and the whole culture medium was changed to X-VIVO 15 72 hours after infection. The obtained enhanced TIL cells are named BW7TG TIL, BW197-2 TIL and BWTG TIL respectively along with the names of lentivirus, and the T cells without transduction lentivirus are named Ctrl TIL.

(2) Enhanced TIL cell transduction efficiency and IL-7 expression detection

Cells to be detected were washed twice with PBS and resuspended with FACS buffer. BV 421-labeled anti-human CD3 antibody and APC-labeled anti-human TGF beta RII antibody are added into a cell suspension to be detected according to an antibody specification, incubation is carried out at 4 ℃ for 60min, TIL cells which are not transfected by lentiviruses are used as negative controls, and dnTGF βRII and eGFP expression rates of the TIL cells are detected by a flow cytometer to characterize transduction efficiency. Flow jo software analysis was used.

As a result, as shown in FIG. 4, the expression rate of BW7TG in TIL cells was 67.0%, the expression rate of BWTG in TIL cells was 51.7%, and the expression rate of BW197-2 in TIL was 30.8%, indicating that dnTGF. Beta. RII was efficiently expressed in TIL cells.

TIL cells to be tested were each adjusted to 1X 10 ⁵ cells/well with X-VIVO 15 medium. After 96h incubation, the above co-cultured cell supernatants were centrifuged and aspirated, and the samples were diluted with 1% BSA in PBS. Simultaneously, the IL-7 standard is dissolved by ddH ₂ O, and the standard is diluted according to the recommended gradient multiple ratio. The standard and experimental samples were added separately to wells coated with IL-7 capture antibody, 100. Mu.l per well. After incubation for 1-3 h at room temperature, 1 Xwashing liquid was prepared and each well was washed 3 times with 300. Mu.l of washing liquid. After the liquid in the wells is dried by beating, 100 mu l of enzyme-labeled IL-7 detection antibody is added into each well, and the wells are incubated for 1 to 3 hours at room temperature. Each well was washed 3 times with 300. Mu.l of wash solution. After the wells were dried by pipetting, 100. Mu.l of chromogenic substrate was added. After incubation at room temperature for 20min, 50. Mu.l of stop solution was added to each well, the absorbance at 450nm was measured with a microplate reader and the IL-7 concentration was calculated.

As a result, as shown in FIG. 5, the secretion level of IL-7 by Ctrl TIL was 0.8.+ -. 0.2pg/ml, BW7TG TIL was 8604.6.+ -. 1003.8pg/ml, BWTG was 1.4.+ -. 0.1pg/ml, and BW197-2 was 1486.2.+ -. 242.3pg/ml, indicating that IL-7 was efficiently expressed and secreted in TIL cells.

Example 3 preparation and detection of CAR-T cells

(1) Preparation of CAR-T cells

Peripheral blood mononuclear cells (PERIPHERAL BLOOD MONONUCLEAR CELL, PBMCs) of healthy donors were resuscitated in AIM V medium and T cells were stimulated with anti-human CD3/CD28 antibodies. The cells were transduced with lentiviruses at MOI=5 IU/ml, respectively. On day 7 after transduction, the expression of the corresponding CAR molecule was detected, and cells were harvested on day 10 after transduction and stored in liquid nitrogen for later use. The obtained CAR-T cells are named BC001, BC010, BC011, BW133-2, BW133-12, BW133-13, BW133-11B, BW133-6B, BW133-7B, BW133-7E respectively, and the T cells without lentivirus transduction are named Ctrl T.

(2) Detection of expression of CAR molecules, dnTGF. Beta. RII and IL-7 in CEA CAR-T cells

Ctrl T, BC001, BC010 and BC011 cells to be detected were washed twice with PBS and resuspended with FACS buffer. PE-labeled CEA protein and BV 421-labeled anti-human CD3 antibody are added into a cell suspension to be detected according to the antibody instruction, and incubated at 4 ℃ for 60min. The expression rate of CEA CAR molecules of Ctrl T, BC001, BC010 and BC011 cells was detected with a flow cytometer.

As shown in fig. 6, the expression rates of BC001, BC010, and BC011 cell CAR molecules were 80.8%, 88.1%, and 86.5%, respectively.

Ctrl T and BC011 cells to be detected were washed twice with PBS and resuspended with FACS buffer. Adding BV421 marked anti-human CD3 antibody and APC marked anti-human TGF beta RII antibody into a cell suspension to be detected according to an antibody specification, incubating at 4 ℃ for 60min, taking Ctrl T cells which are not transfected by lentivirus as negative control, and detecting the dnTGF βRII expression rate of the Ctrl T and BC011 cells by using a flow cytometer. Flow jo software analysis was used.

The results are shown in FIG. 7, where the dnTGF. Beta. RII expression rate of BC011 cells is 34.2%, indicating successful expression of dnTGF. Beta. RII molecule by BC011 cells.

The T cells to be tested were individually adjusted to 1×10 ⁵/well with AIM V medium. After 96h incubation, the above co-cultured cell supernatants were centrifuged and aspirated, and the samples were diluted with 1% BSA in PBS. Simultaneously, the IL-7 standard is dissolved by ddH ₂ O, and the standard is diluted according to the recommended gradient multiple ratio. The standard and experimental samples were added separately to wells coated with IL-7 capture antibody, 100. Mu.l per well. After incubation for 1-3 h at room temperature, 1 Xwashing liquid was prepared and each well was washed 3 times with 300. Mu.l of washing liquid. After the liquid in the wells is dried by beating, 100 mu l of enzyme-labeled IL-7 detection antibody is added into each well, and the wells are incubated for 1 to 3 hours at room temperature. Each well was washed 3 times with 300. Mu.l of wash solution. After the wells were dried by pipetting, 100. Mu.l of chromogenic substrate was added. After incubation at room temperature for 20min, 50. Mu.l of stop solution was added to each well, the absorbance at 450nm was measured with a microplate reader and the IL-7 concentration was calculated.

As a result, as shown in FIG. 8, IL-7 secretion levels of Ctrl T and BC001 cells were 0.0.+ -. 0.0pg/ml and 0.4.+ -. 0.0pg/ml. In contrast, the IL-7 secretion levels of BC010 and BC011 cells were 301.5 + -46.9 pg/ml and 502.4+ -121.3 pg/ml, indicating that both the four-generation CAR structure was effective in secreting IL-7.

(3) Detection of expression of CAR molecules, dnTGF βrii and IL-7 in CD133 CAR-T cells

The expression of CD133 CAR molecules, dnTGF. Beta. RII and IL-7 in Ctrl T, BW133-2, BW133-12, BW133-13, BW133-11B, BW133-6B, BW133-7B, BW133-7E cells was examined as described above.

The expression rates of CD133 CAR molecules are shown in FIG. 9, with CAR molecule expression rates of BW133-2, BW133-12, BW133-13, BW133-11B, BW133-6B, BW133-7B, BW133-7E cells of 78.7%, 57.2%, 42.4%, 74.9%, 67.7%, 61.8% and 70.0%, respectively, indicating successful expression of the CAR molecules. The expression rates of dnTGF. Beta. RII are shown in FIG. 10, and dnTGF. Beta. RII expression rates of BW133-13, BW133-11B, BW133-7B, BW133-7E cells are 44.0%, 44.1%, 30.0% and 28.2%, indicating successful expression of dnTGF. Beta. RII molecules in these four-generation CARs.

The expression levels of IL-7 are shown in FIG. 11A, and the secretion levels of IL-7 by Ctrl T, BW133-2, BW133-12, BW133-13, BW133-6B, BW-7B are 6.0.+ -. 0.9pg/ml, 6.6.+ -. 0.2pg/ml, 7338.6.+ -. 166.3pg/ml, and 5244.9.+ -. 271.8pg/ml, respectively. The level of membrane bound IL-7 expression is shown in FIG. 11B, and the rate of membrane bound IL-7 expression in BW133-7E is 17.3%, indicating that IL-7 was successfully expressed in these four-generation CAR structures.

Example 4 target cell detection

(1) Culture conditions of target cells

The tumor cells used in this example were all reporter cell lines expressing mCherry fluorescent protein: colorectal cancer cells HT55-Luc-mCherry (DMEM Medium+10% foetal calf serum+100U/ml penicillin+100. Mu.g/ml streptomycin), SW620-Luc-mCherry (Leibovitz L-15 Medium+10% foetal calf serum+100U/ml penicillin+100. Mu.g/ml streptomycin), HCT 116-TGFbeta-Luc-mCherry and HT 29-TGFbeta-Luc-mCherry (McCoy's 5A Medium+10% foetal calf serum+100U/ml penicillin+100. Mu.g/ml streptomycin); gastric cancer cells HGC-27-Luc-mCherry, pancreatic cancer Capan-2-Luc-mCherry (McCoy's 5A medium+10% fetal bovine serum+100U/ml penicillin+100. Mu.g/ml streptomycin), and liver cancer cells SK-Hep1-Luc-mCherry (EMEM medium+10% fetal bovine serum+100U/ml penicillin+100. Mu.g/ml streptomycin).

(2) CEA expression detection of target cells

Cells to be detected were washed twice with PBS and resuspended with FACS buffer. The AF 700-labeled anti-CEA antibody was added to the cell suspension to be detected according to the antibody instructions and incubated at 4℃for 60min. Target cells incubated with the corresponding Isotype antibodies were used as negative controls and the CEA expression rate of the target cells was measured using a flow cytometer.

Cells to be detected were washed twice with PBS and resuspended with FACS buffer. PE-Cy 7-labeled anti-CD 133 antibodies were added to the cell suspension to be detected according to the antibody instructions and incubated at 4℃for 60min. Target cells incubated with the corresponding Isotype antibodies were used as negative controls and the CD133 expression rate of the target cells was measured using a flow cytometer. The results are shown in Table 1 using FlowJo software analysis.

TABLE 1 expression rates of CEA and CD133 in tumor cells

Example 5 enhanced TIL cell function Studies

(1) Enhanced TIL cell killing effect detection

The killing effect of the enhanced TIL cells was examined with SK-Hep 1-Luc-mCherry. Target cells were digested and counted, seeded at a density of 1×10 ⁴/well in 96-well plates and cocultured with TIL cells added at1×10 ⁴/well or 2×10 ⁴/well, depending on experimental requirements. On the other hand, to examine dnTGF. Beta. RII function, 10ng/ml TGF. Beta. Was added to the co-culture system, and the change of the target cell area with time was detected by an IncuCyte living cell analyzer. The smaller the target cell area, the higher the death degree of the target cells, and the better the T cell killing effect.

The results are shown in FIG. 12. The killing effect of BW197-2 TIL, BWTG TIL and BW7TG TIL groups is obviously better than that of Ctrl TIL control groups. As the number of target cell stimulations increased, the killing effect of BW197-2 was significantly reduced. BWTG TIL and BW7TG TIL cells remained strong killing on day 11 co-culture with target cells, but BW7TG TIL was significantly more killing than the other groups (P < 0.05) (fig. 12). This shows that activating IL-7 pathway and inhibiting TGF beta pathway simultaneously can raise the capacity of TIL cell to resist tumor micro environment immunosuppression obviously and raise the killing effect of TIL cell on tumor cell.

(2) Enhanced IFN-gamma release levels from TIL cells

The concentrations of effector cells and target cells were adjusted with medium and seeded in 96-well plates at a ratio of 1:1 to give a total cell number of 1×10 ⁵/well (i.e., effector cells and target cells cell numbers of 0.5×10 ⁵/well, respectively). Then 10ng/ml TGF-. Beta.1 was added and each group was replicated three times and incubated in a 5% CO ₂ incubator at 37 ℃. After 72 hours, the culture supernatant was collected and the concentration of IFN-. Gamma.in the supernatant was measured by ELISA.

The results are shown in FIG. 13. IFN-gamma release levels for Ctrl TIL, BW197-2 TIL, BWTG TIL, BW7TG TIL were 8417.8 + -1004.7 pg/ml, 10712.3 + -1521.3 pg/ml, 37561.7 + -3846.0 pg/ml, and 52859.2 + -4101.4 pg/ml, respectively, with the IFN-gamma release levels for BW7TG TIL being significantly higher than for the other groups (P < 0.05). This suggests that the combined expression of IL-7 and dnTGF. Beta. RII synergistically increases the level of TIL cell activation in tumor microenvironments with higher TGF. Beta. Concentrations.

EXAMPLE 6 functional Studies of CAR-T cells

(1) CEA CAR-T cell killing effect detection

According to the flow detection result of the target antigen, HT55-Luc-mCherry is selected for detecting the killing effect of the CAR-T cells. Target cells were digested and counted, seeded at a density of 1×10 ⁴/well in 96-well plates and co-cultured by adding T cells at 1×10 ⁴/well or 2×10 ⁴/well, depending on experimental requirements. On the other hand, to examine dnTGF. Beta. RII function, 10ng/ml of TGF. Beta. Was added to the co-culture system, and the change of the target cell area with time was detected by an IncuCyte living cell analyzer. The smaller the target cell area, the higher the death degree of the target cells, and the better the T cell killing effect.

The results are shown in FIG. 14. Although BC001 and BC010 had some killing ability against target cells, the area of target cells still increased significantly on day 5 of co-culture, indicating that the killing effect of BC001 and BC010 was inhibited by tgfβ. In contrast, CAR-T cell BC011 expressing IL-7 and dnTGF βrii was able to effectively and consistently inhibit target cell growth, with significantly stronger killing effect than the other groups (P < 0.05). This demonstrates that the combined expression of IL-7 and dnTGF. Beta. RII can effectively resist the inhibition of TGF beta to T cells and enhance the killing capacity of CAR-T cells to tumors.

(2) Detection of killing effect of CD133 CAR-T cells

According to the flow detection result of the target antigen, HCT116-Luc-mCherry is selected for detecting the killing effect of the CAR-T cells. Target cells were digested and counted, seeded at a density of 1×10 ⁴/well in 96-well plates and co-cultured by adding T cells at 1×10 ⁴/well or 2×10 ⁴/well, depending on experimental requirements. On the other hand, to examine dnTGF. Beta. RII function, 10ng/ml of TGF. Beta. Was added to the co-culture system, and long-term killing effect was detected using an IncuCyte living cell analyzer.

The results are shown in FIG. 15. Similar to the results for CEA CAR-T cells, BW133-13 group had significantly stronger killing effect on tumor cells than the other groups (P < 0.05). This result demonstrates that the combined expression of IL-7 and dnTGF. Beta. RII can effectively resist the inhibition of TGF beta to T cells and enhance the killing capacity of CAR-T to tumors.

(3) In vivo tumor inhibition experiments.

Target cells SW620 in the logarithmic growth phase and in good growth state were collected by the pancreatin digestion method, and after washing 1 time with physiological saline, the cell density was adjusted to 3X 10 ⁷/ml. 100 μl of cell suspension was subcutaneously injected near the underarm area on the right side of B-NDG mice, i.e., each mouse was inoculated with 3X 10 ⁶ target cells, and when the average tumor volume was 50-100 mm ³, CAR-T cells (1X 10 ⁷ /) and Ctrl T cells (1X 10 ⁷ /) were injected via the tail vein, respectively, and the tumor size was measured weekly by injecting the test subjects on day 0 of treatment.

The results are shown in FIG. 16. In the SW620 subcutaneous tumor model, after day 25 of CAR-T cell reinfusion, the BW133-13 group, which jointly expressed IL-7 and dnTGF βRII, had a tumor volume of 654.3.+ -. 255.8mm ³, significantly less than the BW133-2 control group (tumor volume of 1503.2.+ -. 365.4mm ³) (P < 0.05). Although there was a trend towards a reduction in tumor volume in BW133-12 group that co-expressed IL-7 and CCL19 on day 14 after CAR-T cell injection, mice developed significant side effects and died afterwards, suggesting that CAR-T cells present a certain safety risk.

The results show that the IL-7 pathway is activated and the TGF beta signal pathway is inhibited, so that the enhancement effect of IL-7 on T cells can be maintained, the toxic and side effects in vivo can be remarkably reduced unexpectedly, and the safety is better than that of the existing technology for jointly expressing IL-7 and CCL 19.

Example 7CAR-T cell indication study

(1) Detection of killing effect of CAR-T cells combined expressing IL-7 and dnTGF beta RII on various solid tumor cells

To further enhance the safety of CAR-T cells of IL-7 and dnTGF βrii, IL-7 was expressed in fusion with dnTGF βrii, allowing the anchoring of IL-7 to the membrane of T cells via dnTGF βrii, resulting in BW133-7E cells. Meanwhile, to investigate whether CAR-T cells that co-express IL-7 and dnTGF βrii are also suitable for the treatment of malignant solid tumors other than colorectal cancer. The killing effect of CAR-T cells was examined using HCT116-TGF beta-Luc-mCherry, HT-29-Luc-mCherry, HGC-27-Luc-mCherry, capan-2-Luc-mCherry cells. Target cells were digested and counted, inoculated into 96-well plates at a density of 1X 10 ⁴/well, and co-cultured by adding 1X 10 ⁴/well or 2X 10 ⁴/well of CAR-T cells (BW 133-2, BW133-6B, BW133-11B, BW 133-7E) according to experimental requirements. After TGF beta is added into the co-culture system, the long-time killing effect detection is carried out by using an Incucyte living cell analyzer.

The results are shown in FIG. 17. The killing effect of each experimental group is obviously better than that of a Ctrl T control group. With increasing number of target cell stimulations, CAR-T cells expressing IL-7 and dnTGF βrii fusion proteins were significantly more potent in killing HCT116-Luc-mCherry colorectal cancer cells (fig. 17A), HT-29-Luc-mCherry colorectal cancer cells (fig. 17B), capan-2-Luc-mCherry pancreatic cancer cells (fig. 17C) and HGC-27-Luc-mCherry gastric cancer cells (fig. 17D) than the other groups (P < 0.05).

Ctrl T, BW133-2 and BW133-7E cells were cultured in 96-well plates at different initial cell densities, and after 72h AO/PI staining was performed and the number of viable cells counted.

When the initial cell density was 1X 10 ⁵/ml to 2X 10 ⁵/ml, there was no significant difference in the cell expansion factor of the BW133-7E group from the BW133-2 group (FIGS. 18A and 18B). When the initial cell density was 5X 10 ⁵/ml, the amplification factor of BW133-2 group was 1.48.+ -. 0.13, and the amplification factor of BW133-7E group was 2.08.+ -. 0.19, which was significantly higher than that of BW133-2 group (P <0.05, FIG. 18C). The above results demonstrate that the cell proliferation capacity of CAR-T cells expressing IL-7 and dnTGF βrii fusion proteins can be self-regulated according to cell density. When aggregated in tumor lesions, the cell proliferation capacity can be significantly increased without uncontrolled proliferation at non-tumor sites.

Discussion of the invention

For the treatment of solid tumors, current immune cell therapies suffer from a number of drawbacks. For example, conventional second generation CAR-T cells survive in vivo for a shorter period of time and have a weaker expansion capacity. IL-7 is jointly expressed on the basis of the second-generation CAR-T cells, so that proliferation, survival and immunological memory of the T cells can be effectively promoted. Studies have shown that further co-expression of IL-7 and chemokines CCL19 or CCL21, while promoting infiltration and survival of CAR-T cells to tumor sites, have not been effective in resolving the immunosuppressive effects of the immunosuppressive microenvironment on immune cells in solid tumors.

On the one hand, the receptor for knocking down or knocking out TGF beta in immune cells or the dominant inhibition mutant (such as dnTGF beta RII) of the overexpression TGF beta receptor can effectively block the endogenous TGF beta signal path of the cells and reduce the inhibition effect of TGF beta on the immune cells, thereby maintaining the activity of the immune cells in tumor microenvironment. However, while these techniques can improve the killing effect of T cells in tumor microenvironments to some extent, they do not preserve the memory and persistence capabilities of CAR-T cells in vivo.

On the other hand, in order to solve the problems of short in vivo duration, poor memory capacity and the like of tumor microenvironment inhibition and engineered immune cells, it is studied to jointly express a fusion protein consisting of a TGF beta RII extracellular domain and an IL-7RA intracellular domain on the basis of CD19 CAR-T so as to reverse the inhibition signal of TGF beta into the activation signal of IL-7. However, the complete IL-7 activation signal requires IL-2RG and IL-7RA to form a complex and co-conduct. Thus, the fusion protein is only able to signal via the IL-7RA intracellular domain, the degree of activation is incomplete. Furthermore, since this technique requires the presence of TGF-beta to activate the IL-7 pathway of immune cells. Thus, it is difficult to maintain self-survival through the IL-7 pathway when the TGF-beta concentration is low in the environment in which the immune cells are located.

In the invention, by up-regulating IL-7 and simultaneously down-regulating TGF beta channel, the activation degree of IL-7 signal of the engineering immune cells is improved, and the resistance to tumor microenvironment is obviously provided, so that the IFN-gamma release level of the immune cells is obviously and synergistically improved, the killing effect on tumor cells is enhanced, and the curative effect of the engineering immune cells is synergistically improved. Experiments show that the engineering immune cells (taking CAR-T cells or TIL as an example) have the advantages of remarkably improved proliferation capacity of the immune cells, remarkably reduced failure and remarkably enhanced resistance to immunosuppressive environment. In the present invention, the risk of toxic side effects caused by the overexpression of IL-7 is also unexpectedly reduced by the optimized structure of IL-7 and dnTGF. Beta. RII.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Furthermore, 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 (10)

1. An engineered immune cell, wherein the engineered immune cell is a T cell, a TIL cell, or an NK cell, and the immune cell has the following characteristics:

(a) The immune cells express exogenous IL-7 protein; and

(B) The immune cells express exogenous TGF-beta RII dominant repression mutant proteins or shRNA targeting endogenous TGF-beta RII transcripts.

2. The engineered immune cell of claim 1, wherein the immune cell expresses a Chimeric Antigen Receptor (CAR), wherein the CAR targets a surface marker of a tumor cell.

3. The engineered immune cell of claim 1, wherein the IL-7 protein has the structure of formula Z:

A-H-TM-D(Z)

In the method, in the process of the invention,

A is IL-7 protein, or an active fragment thereof, or a mutant thereof;

H is the no or hinge region;

TM is an absent or transmembrane domain;

D is a non-or degradation domain (including wild-type, or mutant/modification thereof); the "-" is a connecting peptide or peptide bond.

4. An engineered immune cell according to claim 1, wherein the structures of said IL-7 protein and tgfbetarii dominant-suppression mutant are as shown in formula I:

Z0-Z1-Z2-D(I)

In the method, in the process of the invention,

One of Z0 and Z2 is IL-7 protein, and the other is a TGF-beta RII dominant inhibition mutant;

z1 is none, or a linking peptide;

d is a non-or degradation domain (including wild-type, or mutant/modification thereof);

The "-" is none, a linking peptide or a peptide bond.

5. The engineered immune cell of claim 2, wherein the CAR has the structure of formula II:

L-scFv1-H-TM-C-CD3ζ-D(II)

In the method, in the process of the invention,

L is a none or signal peptide sequence;

scFv1 is an antigen binding domain (e.g., an antibody or active fragment thereof) that targets a surface marker of a tumor cell;

H is the no or hinge region;

TM is a transmembrane domain;

c is a costimulatory signaling domain;

Cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ (including wild-type, or mutant/modification thereof);

d is a non-or degradation domain (including wild-type, or mutant/modification thereof);

the "-" is a connecting peptide or peptide bond.

6. The engineered immune cell of claim 5, wherein the CAR-T cell contains, in addition to a first CAR of formula II, a second CAR for a second antigen, the second CAR having the structure of formula III:

L-scFv2-H-TM-C-CD3ζ-D(III)

In the method, in the process of the invention,

L is a none or signal peptide sequence;

scFv2 is an antigen binding domain (e.g., an antibody or active fragment thereof) that targets a surface marker of a second tumor cell;

H is the no or hinge region;

TM is a transmembrane domain;

c is a costimulatory domain;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ or a mutant/modification thereof;

d is a non-or degradation domain (including wild-type, or mutant/modification thereof);

the "-" is a connecting peptide or peptide bond.

7. A method of preparing the engineered immune cell of claim 1, comprising the steps of:

(A) Providing an immune cell to be modified; and

(B) Engineering the immune cell such that the immune cell expresses an exogenous IL-7 protein and an exogenous tgfbetarii dominant inhibitory mutant protein or shRNA targeting an endogenous tgfbetarii transcript to obtain the engineered immune cell of claim 1.

8. A formulation comprising the engineered immune cell of claim 1, and a pharmaceutically acceptable carrier, diluent, or excipient.

9. Use of an engineered immune cell according to claim 1, for the preparation of a medicament or formulation for the prevention and/or treatment of cancer.

10. A kit for preparing an engineered immune cell of claim 1, comprising a container, and within the container:

(1) A first nucleic acid sequence comprising a first expression cassette for expressing IL-7; and

(2) A second nucleic acid sequence comprising a second expression cassette for expressing a dominant tgfβrii suppression mutant or shRNA targeting an endogenous tgfβrii transcript.