WO2017028374A1

WO2017028374A1 - Construct, genetically modified lymphocyte, preparation method and usage thereof

Info

Publication number: WO2017028374A1
Application number: PCT/CN2015/092730
Authority: WO
Inventors: Yongchao YAN; Yanling Liu; Yilin ZHU; Siyi Chen
Original assignee: Beijing Marino Biotechnology Pty Ltd.
Priority date: 2015-08-20
Filing date: 2015-10-23
Publication date: 2017-02-23
Also published as: CN106467906B; CN106467906A

Abstract

A T cell, a retrovirus, a genetically modified lymphocyte, a construct, a method of preparing the T cell, a therapeutic composition for treating cancer, a method for treating cancer, and method of improving the activity of a lymphocyte carrying a chimeric antigen receptor are provided. The T cell expresses a functional 4-1BB and a chimeric antigen receptor, the chimeric antigen receptor comprises: an ectodomain comprising a single-chain antibody binding to an antigen CD19, a transmembrane domain linked to the ectodomain and embedded in a cellular membrane of the T cell, and an endodomain linked to the transmembrane domain, comprising intracellular domain of CD28 or 4-1BB andζ-chain of CD3.

Description

Construct, Genetically Modified Lymphocyte, PreparationMethod And Usage Thereof

RELATED APPLICATIONS

This application claims priority and benefits of Chinese Patent Application No. 201510516475.0, filed with State Intellectual Property Office on August 20, 2015, the entire content of which is incorporated herein by reference.

Field

The present invention relates to biotechnology, especially to a T cell, a retrovirus, a genetically modified lymphocyte, a construct, a method of preparing the T cell, a therapeutic composition for treating cancer, a method for treating cancer, and method of improving the activity of a lymphocyte carrying a chimeric antigen receptor are provided.

Background

Adoptive T cell therapy is the ex vivo activation, expansion, and subsequent administration of T cells into a patient. Some transferred T cells are able to migrate the tumor site and directly lyse tumor cells. T cells properly stimulated ex vivo may have the capacity to induce significant therapeutic effects in preclinical models of established cancer and against melanoma in clinical trials.

Summary

The aim of the present invention is to solve at least one of the technical problems of the prior art. The present invention is based on the following findings of the present inventor:

Despite these promising results, adoptive T cell therapies with tumor infiltrating lymphocytes (TILs) are typically not effective in inhibiting tumor growth in cancer patients, due to the insufficient number of tumor-reactive CTLs and immune inhibitory mechanisms in the tumor microenvironment. Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) targeting CD19 have elicited dramatic antitumor responses in some patients with B cell lymphomas and leukemia, but a considerable portion of these patients did not respond to the therapy. Thus, there is a need to improve the efficacy of CAR T cell therapy.

As a result, the present disclosure provide a construct carrying a sequence encoding a full length, functional immune costimulatory molecule and a chimeric antigen receptor, and a genetically modified lymphocyte generated by introducing the construct. The construct and genetically modified lymphocyte may be used in the adoptive T cell therapies, which may significantly improve a survival of the T cell, improve the anti-tumor effect of the T cell, especially may show significant therapeutic effect in treating hemopoietic malignancies or lymphoma and leukemia.

In one aspect of present disclosure, a T cell is provided, and according to embodiments of present disclosure, the T cell expresses: functional 4-1BB； and a chimeric antigen receptor, in which the chimeric antigen receptor comprises: an ectodomain comprising a heavy chain variable region and a light chain variable region of a single-chain antibody binding to an antigen CD19； a transmembrane domain linked to the ectodomain and embedded in a cellular membrane of the T cell； and an endodomain linked to the transmembrane domain, comprising intracellular domain of CD28 and ζ-chain of CD3. According to embodiments of present disclosure, the genetically modified lymphocyte may be used in the adoptive T cell therapies, which may significantly improve a survival of the T cell, improve the anti-tumor effect of the T cell, especially may show significant therapeutic effect in treating hemopoietic malignancies or lymphoma and leukemia.

In another aspect of present disclosure, a retrovirus is provided, and according to embodiments of present disclosure, the retrovirus may comprises a nucleic acid molecule encoding: a full length 4-1BB comprising a sequence of SEQ ID NO:1.

and a CD19-specific chimeric antigen receptor comprising a sequence of SEQ ID NO : 2 .

According to embodiments of present disclosure, the genetically modified lymphocyte generated by introducing the retrovirus may be used in the adoptive T cell therapies, which may significantly improve a survival of the T cell, improve the anti-tumor effect of theT cell, especially may show significant therapeutic effect in treating hemopoietic malignancies or lymphoma and leukemia.

In still another aspect, a retrovirus comprising a nucleic acid molecule comprising a sequence of SEQ ID NO : 3 or 4 is provided, in which SEQ ID NO: 3 shows nucleic acid sequence of human CD19-targeted CAR-IRES-4-1BB.

and SEQ ID NO ： 4 shows nucleic acid of human CD19-targeted CAR-IRES-OX40.

According to embodiments of present disclosure, the retrovirus may be introduced into a lymphocyte, and the genetically modified lymphocyte generated by introducing the retrovirus may be used in the adoptive T cell therapies, which may significantly improve a survival of the T cell, improve the anti-tumor effect of the T cell, especially may show significant therapeutic effect in treating hemopoietic mali-gnancies or lymphoma and leukemia.

In still another aspect of present disclosure, a genetically modified lymphocyte expressing a functional immune costimulatory molecule and a chimeric antigen receptor is provided. It was surprisingly found by the inventor that the survival of a lymphocyte expressing a functional immune costimulatory molecule and a chimeric antigen receptor is significantly improved. And the present inventive lymphocyte may shows more significantly improved killing activity on tumor, especially hemopoietic malignancies or lymphoma and leukemia.

According to embodiments of present disclosure, the above mentioned genetically modified lymphocyte may possess at least one of the following additional features:

According to embodiments of present disclosure, the chimeric antigen receptor comprises: an ectodomain capable of binding to an antigen specifically； a transmembrane domain； and an endodomain comprising an intracellular domain of an immune costimulatory molecule. With the presence of the chimeric antigen receptor, the genetically modified lymphocyte may show targeted killing activity on a tumor cell expressing the antigen.

According to embodiments of present disclosure, the antigen is a molecule expressed on a tumor cell. Then the targeted killing activity of the genetically modified lymphocyte may be further improved.

According to embodiments of present disclosure, the ectodomain comprising a heavy chain variable region linked to a light chain variable region of an antibody binding to the antigen. The specific binding of the antibody to the antigen may further improve the targeted location and targeted killing activity on the tumor cell expressing the antigen.

According to embodiments of present disclosure, the antibody is a single-chain antibody. Then the targeted killing activity of the genetically modified lymphocyte may be further improved.

According to embodiments of present disclosure, the antigen is CD19. Then the targeted killing activity of the genetically modified lymphocyte may be further improved.

According to embodiments of present disclosure, the functional immune costimulatory molecules is selected independently from a group consisting of 4-1BB, OX-40, CD40L, CD27, CD30, CD28 and variants thereof. Then the targeted killing activity of the genetically modified lymphocyte may be further improved, and the survival of the genetically modified lymphocyte may be further improved.

According to embodiments of present disclosure, the intracellular domain of the immune costimulatory molecule is derived from at least one selected from a group consisting of 4-1BB, OX-40, CD40L, CD27, CD30, CD28 and variants thereof. Then the targeted killing activity of the genetically modified lymphocyte may be further improved, and the survival of the genetically modified lymphocyte may be further improved.

According to embodiments of present disclosure, the functional immune costimulatory molecule is a full length 4-1BB, OX40 or CD40L, and the intracellular domain of the immune costimulatory molecule is the intracellular domain of CD28 or 4-1BB. Then the targeted killing activity of the genetically modified lymphocyte may be further improved, and the survival of the genetically modified lymphocyte may be further improved.

According to embodiments of present disclosure, the lymphocyte is a CD3+ T lymphocyte. According to embodiments of present disclosure, the targeted killing activity of the genetically modified CD3+ T lymphocyte is further improved, and the survival of the genetically modified lymphocyte is further improved.

According to embodiments of present disclosure, the lymphocyte is a nature killer (NK) cell. According to embodiments of present disclosure, the targeted killing activity of the genetically modified NK cell is further improved, and the survival of the genetically modified lymphocyte is further improved.

According to embodiments of present disclosure, the lymphocyte is a nature killer T (NKT) cell. According to embodiments of present disclosure, the targeted killing activity of the genetically modified NKT cell may be further improved, and the survival of the genetically modified lymphocyte may be further improved.

In still another aspect, a construct is provided, and the construct may comprises a first nucleic acid molecule encoding a functional immune costimulatory molecule； and a second nucleic acid molecule encoding a chimeric antigen receptor, in which the functional immune costimulatory molecule and the chimeric antigen receptor is defined above. According to embodiments of present disclosure, the construct may be introduced into a lymphocyte, and the genetically modified lymphocyte generated by introducing the construct may be used in the adoptive T cell therapies, which may significantly improve a survival of the T cell, improve the anti-tumor effect of the T cell, especially may show significant therapeutic effect in treating hemopoietic malignancies or lymphoma and leukemia.

According to embodiments of present disclosure, the construct may further comprises an internal ribosome entry site (IRES) provided between the first nucleic acid molecule and the second nucleic acid molecule and comprising a nucleic acid sequence of SEQ ID NO: 5.

Then the functional immune costimulatory molecule and the chimeric antigen receptor may be expressed in a non-fusion form. According to an embodiment of the present invention, the introduction of an internal ribosome entry site sequence effectively guarantee the functional immune costimulatory molecules and chimeric antigen receptor biological effects. So the survivability of lymphocytes in the tumor greatly improve, and anti-tumor effect is much more stronger.

According to embodiments of present disclosure, the construct may further comprises a third nucleic acid molecule provided between the first nucleic acid molecule and second nucleic acid molecule, the third nucleic acid molecule encoding a cleavable linker peptide in the lymphocyte. Then the functional immune costimulatory molecule and the chimeric antigen receptor may be expressed in a fusion form, and the generated fusion protein may be further cut by cellular enzyme, and both functional immune costimulatory molecule and the chimeric antigen receptor may be generated. According to an embodiment of the present invention, the introduction of an a cleavable linker peptide sequence effectively guarantee the functional immune costimulatory molecules and chimeric antigen receptor biological effects. So the survivability of lymphocytes in the tumor greatly improves, and anti-tumor effect is much more stronger.

According to embodiments of present disclosure, the cleavable linker peptide comprises a sequence of SEQ ID NO: 6.

GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 6) .

The cleavable linker peptide is a 2A peptide derived from the foot-and-mouth disease virus (FMDV) , a member of the picornavirus. The introduction of a cleavable peptide between the functional immune costimulatory molecules and chimeric antigen receptor make the functional immune costimulatory molecules and chimeric antigen receptor in a non-fusion form in lymphocyte membrane. According to an embodiment of the present invention, the cleavable peptide introduced herein is to ensure the function of immune co-stimulatory molecules and chimeric antigen receptor biological effects, such that the survivability rate of lymphocytes in the tumor is greatly improved, more tumor killing effect of is much more stronger.

According to embodiments of present disclosure, the construct may further comprise a first promoter operably linked to the first nucleic acid molecule； and a second promoter operably linked to the second nucleic acid molecule. According to an embodiment of the present invention, the first and second promoter is introduced, so that the first nucleic acid molecule and second nucleic acid molecules are independently expressed and the functional immune costimulatory molecules and chimeric antigen receptor was in non-fusion form. The functional immune costimulatory molecules and chimeric antigen receptor expression makes the survivability rate of lymphocytes in the tumor greatly improved, and anti-tumor effect more significant.

According to embodiments of present disclosure, each of the first and second promoters is selected independently from a group consisting of CMV, EF-1, RSV, and LTR promoters.

According to embodiments of present disclosure, the construct may further comprise that the construct is a non-pathogenic virus. According to an embodiment of the present invention, the promoters have a high transcription efficiency, thus a functional immune co-stimulatory molecules and chimeric antigen receptors are efficiently expressed. So that the survivability of lymphocytes in the tumor is greatly improved , anti-tumor effect is more significant.

According to embodiments of present disclosure, the construct may further comprise that the construct is a virus selected from a retrovirus, lentivirus and adenovirus. According to an embodiment of the present invention, the above-described construct is with a wide range of viral infections, which can transduce either terminally differentiated cells, but also the cells in division, either integrate into the host chromosome, or be free with the host chromosome, to achieve an efficient infection efficiency. Thus, the expression of functional immune costimulatory molecules and chimeric antigen receptor is greatly improved in lymphocytes. And the survivability of lymphocytes in the tumor is greatly improved, killing tumor effect is more significant.

In still another aspect of present disclosure, a method of preparing the T cell above mentioned or the genetically modified lymphocyte above mentioned is also provided, and the method may comprises introducing the construct above mentioned, or the retrovirus above mentioned into a lymphocyte or T lymphocyte cell. The successful introduction of the above lymphocytes or T lymphocytes with the construct or retrovirus will allow lymphocytes or T lymphocytes to express functional immune costimulatory molecules and chimeric antigen receptors. So that the lymphocyte or T lymphocyte survival in a cancer patient has been greatly improved and the killing tumor effect is much more stronger.

In still another aspect of present disclosure, a therapeutic composition for treating cancer, comprising: the construct above mentioned, or the retrovirus above mentioned or the genetically modified lymphocyte mentioned above. According to embodiments of present disclosure, the cancer comprises hematopoietic malignancies. The therapeutic composition of any of the above composition can make the functional immune costimulatory molecule and chimeric antigen receptor highly expressed in transgenic lymphocytes or T lymphocytes. So that in vitro lymphocyte or T lymphocyte proliferation and survivablity have been greatly improved and the anti-tumor effect is much more stronger.

A method for treating cancer, comprising: administrating the construct above mentioned, or the retrovirus above mentioned or the genetically modified lymphocyte mentioned above to a patient in need of such treatment, wherein the chimeric antigen receptor binding to an antigen of the cancer specifically is also provided.

According to embodiments of present disclosure, the method may comprises isolating a lymphocyte from the patient； introducing the construct above mentioned, or the retrovirus above mentioned or the genetically modified lymphocyte mentioned above into the lymphocyte, and expressing the functional immune costimulatory molecule and the chimeric antigen receptor in the lymphocyte to obtain the genetically modified lymphocyte； and administrating the genetically modified lymphocyte to the patient. According to an embodiment of the present invention, the method for treating cancer is more efficient. The lymphocyte or T lymphocyte survivablity in tumor patient has been greatly improved and the killing tumor effect is much more stronger.

In still another aspect of present disclosure, a method of improving the activity of a lymphocyte carrying a chimeric antigen receptor, comprising: expressing a functional immune costimulatory molecule in the lymphocyte, wherein the functional immune costimulatory molecule, the lymphocyte and the chimeric antigen receptor is defined above, and the activity of the lymphocyte is at least one selected from a group consisting of in vitro proliferation of the lymphocyte, survival of the lymphocyte in a tumor patient and cytolytic activity of the lymphocyte in the tumor patients. According to an embodiment of the present invention, the functional immune costimulatory molecules above up-regulate the anti-apoptotic gene, thus preventing the activation-induced apoptosis. So that the lymphocytes in vitro proliferation, the survival of the lymphocyte in a tumor patient and the cytolytic activity of the lymphocyte in the tumor patient have been greatly improved. The anti-tumor effect is much more stronger.

More aspects and advantages will be described below, at least a part thereof will be clear in the following description accompanying the figures as attached, and/or be obvious for a person normally skilled in the art from embodiments described herein after.

Brief Description of the Figures

The aforementioned features and advantages of the invention as well as additional features and advantages thereof will be more clearly understood hereafter as a result of a detailed description of the following embodiments when taken conjunction with the drawings, wherein:

Fig. 1 shows a result of Example 2.

Fig. 2 shows a map of a retrovirus of Example 3.

Fig. 3 shows a result of Example 4.

Fig. 4 shows a result of Example 5.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The aforementioned features and advantages of the invention as well as additional features and advantages thereof will be more clearly understood hereafter as a result of a detailed description of the following embodiments when taken conjunction with the drawings.

The embodiments described herein with reference to drawings are explanatory, illustrative, and used to generally understand the present invention. The embodiments shall not be construed to limit the scope of the present invention. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions.

T cell or genetically modified lymphocyte

The present invention provided a T cell or genetically modified lymphocyte, which expresses: functional immune costimulatory molecules； and chimeric antigen receptors. Wherein the chimeric antigen receptor comprises: an ectodomain, comprising a heavy chain variable region linked to a light chain variable region of a single-chain antibody binding to an antigen CD19； a transmembrane domain linked to the ectodomain and embedded in a cellular membrane of the T cell or genetically modified lymphocyte； and an endodomain linked to the transmembrane domain, comprising intracellular domain of immune costimulatory molecules and ζ-chain of CD3.

Functional immune costimulatory molecules can make Bcl-xl of T cell or genetically modified lymphocyte up-regulated, and the expression product of Bcl-xl has the function of transmembrane potential stabilization and membranous permeability decrease, which can resist cell apoptosis in the end. As a result, survivability of T cell or genetically modified lymphocyte in tumour is increased dramatically.

Ectodomain antibody of chimeric antigen receptor is a single-chain antibody. The chimeric antigen receptor provided by the present invention will make the targeted killing activity of the genetically modified lymphocyte be further improved.

Ectodomain of chimeric antigen receptor binds to an antigen CD19, thus the genetically modified lymphocyte have targeted killing activity to cells expressing the antigen CD19. As there is stronger specific binding function between antigen and antibody, the targeted killing activity of the genetically modified lymphocyte to tumour cells expressing antigen CD19 will be further improved.

The said functional immune costimulatory molecules and intracellular domain of the immune costimulatory molecule are selected independently from a group consisting of 4-1BB, OX-40, CD40L, CD27, CD30, CD28 and variants thereof. According to the present invention, the functional immune costimulatory molecules is a full length 4-1BB, OX40 or CD40L, and the intracellular domain of the immune costimulatory molecule is the intracellular domain of CD28 or 4-1BB. The said molecules have positive regulation and cellular immunologic stimulation function. The functional immune costimulatory molecules and intracellular domain of the immune costimulatory molecule may be selected from the same or different immune costimulatory molecules. The intracellular domain of the immune costimulatory molecule and functional immune costimulatory molecules have positive regulation effect and improve the cellular immunologic response, which can further improve the genetically modified lymphocyte’s ability of expansion in vitro and survival in vivo, making the targeted killing activity to tumour cells stronger.

The lymphocyte of the present invention is CD3+ lymphocyte or nature killer (NK) cell. CD3+lymphocyte is a T lymphocyte. NK is one kind of immune cell, non-specifically recognizing a target cell. NK T lymphocyte is a T lymphocyte subset having T lymphocyte markers and NK receptors. The said lymphocyte expresses functional immune costimulatory molecules and chimeric antigen receptors, which made the targeted killing activity of the said lymphocyte cellular immunity to tumour cell stronger.

Retrovirus or construct

The construct presented by the present invention comprises a first nucleic acid molecule encoding a functional immune costimulatory molecule； and a second nucleic acid molecule encoding a chimeric antigen receptor. The first and second nucleic acid molecules are configured to express the chimeric antigen receptor and the functional immune costimulatory molecule in a non-fusion form in the lymphocyte. The lymphocyte configured with the first and second nucleic acid molecules has stronger ability of expansion in vitro and survival in vivo, which made the targeted killing activity to tumour cells stronger.

As for the said chimeric antigen receptor and the functional immune costimulatory molecule in a non-fusion form in the lymphocyte, inventor gained those through the following steps:

An internal ribosome entry site (IRES) is provided between the first nucleic acid molecule and the second nucleic acid molecule. Internal ribosome entry site is usually in 5’ untranslated region (UTR) of RNA viral genome, thus one viral protein translation may not rely on 5’ cap sequence and the other protein translation usually begin near 5’ cap sequence. Two proteins’ , before and after IRES, expressions are usually proportional. Internal ribosome entry site make the first nucleic acid molecule and the second nucleic acid molecule translation independently, then the functional immune costimulatory molecule and the chimeric antigen receptor may be expressed in a non-fusion form and play their role respectively. Internal ribosome entry site may ensure the biological function of the functional immune costimulatory molecule and the chimeric antigen receptor effectively to increase the survivability of lymphocyte in tumour microenvironments and the targeted killing activity to tumour cells.

A third nucleic acid molecule is provided between the first nucleic acid molecule and second nucleic acid molecule. The third nucleic acid molecule encodes a cleavable linker peptide in the lymphocyte. The cleavable linker peptide comprises a sequence of SEQ ID NO: 6. SEQ ID NO: 6 is a 2A peptide derived from the foot-and-mouth disease virus (FMDV) , a member of the picornavirus. The third nucleic acid molecule make the functional immune costimulatory molecule and the chimeric antigen receptor be expressed in a self-cleaving fusion form on the lymphocyte membrane to ensure the biological function of both the functional immune costimulatory molecule and the chimeric antigen receptor. As a result, the survivability of lymphocyte in tumour microenvironments and the targeted killing activity to tumour cell are improved.

First promoter can be operably linked to the first nucleic acid molecule. And second promoter can be operably linked to the second nucleic acid molecule. In the present invention, the first promoter and the second promoter may be chosen from CMV, EF-1, RSV, and LTR promoter respectively. The promoters are all efficient to ensure the functional immune costimulatory molecule and the chimeric antigen receptor be efficiently expressed respectively, which increase the survivability of lymphocyte in tumour and the targeted killing activity to tumour cells.

The construct according the present invention is a non-pathogenic virus, selected from a retrovirus, lentivirus and adenovirus. The infection scope of the said construct is abroad. Lentivirus may infect terminally differentiated cell. Retroviruse may infect cells in division stage. Lentivirus and retroviruse may be integrated into chromosome of the host. Adenovirus may also dissociate from chromosome of host. Thus efficiency of infection is broad spectrum and effective, which make the functional immune costimulatory molecule and the chimeric antigen receptor be expressed effectively in the lymphocyte. As a result, the survivability of lymphocyte in tumour and the targeted killing activity to tumour cells are improved.

Method of preparing the genetically modified lymphocyte

The present invention provided a method of preparing genetically modified lymphocyte, which may comprises introducing the construct above mentioned, or the retrovirus above mentioned into a lymphocyte or T lymphocyte cell. Gene delivery may be chosen from electroporation or virus transduction of a host cell. Introducing the construct or retrovirus into the lymphocyte or T lymphocyte cell can make the functional immune costimulatory molecule and the chimeric antigen receptor be expressed efficiently in the lymphocyte or T lymphocyte cell. As a result, the survivability of lymphocyte or T lymphocyte cell in tumour surroundings and the targeted killing activity to tumour cells are improved.

Therapeutic composition for treating cancer

The present invention provided a therapeutic composition for treating cancer, comprising the construct, the retrovirus, T lymphocyte or the genetically modified lymphocyte.

Providing the mentioned composition to patients may be used in biocompatible solution or acceptable pharmacy carrier. As a prepared therapeutic composition, it may be suspended or dissolved into medically or physiologically acceptable carrier, such as saline, isotonic saline solution or other obvious formula in the prior art. Suitable carrier depends on administration route mostly. Other isotonic sterility injection with or without water and sterility suspension liquid with or without water are also a medically acceptable carrier.

Enough virus carrier is introduced into a target T cell and provide enough transgenes to coexpress 4-1BB or OX40 and particular CD19 chimeric antigen receptor. Dosage mainly depends on treatment state, age, weight and patient’s condition to make patient variation.

Method for expression 4-1BB or OX40 and particular CD19 chimeric antigen receptor is part of combined treatment. Those virus carriers and antitumor T cells using to adoptive immunity treatment can be solely or combined used with other cancer treatment methods. In suitable condition, a treatment invention includes using one or many pharmacotherapy.

The said cancer comprises hematopoietic malignancies. The effective expression of the functional immune costimulatory molecule and the chimeric antigen receptor in the lymphocyte or T lymphocyte cell make the survivability of the lymphocyte or T lymphocyte cell in vitro and in hematopoietic malignancies and the targeted killing activity to hematopoietic malignancies is further improved.

Method for treating cancer

According to embodiments of present disclosure, the method may comprises isolating a lymphocyte from the patient； introducing the construct above mentioned, or the retrovirus above mentioned or the genetically modified lymphocyte mentioned above into the lymphocyte, and expressing the functional immune costimulatory molecule and the chimeric antigen receptor in the lymphocyte to obtain the genetically modified lymphocyte； and administrating the genetically modified lymphocyte to the patient. According to an embodiment of the present invention, the method for treating cancer is more efficient. The lymphocyte or T lymphocyte survivablity in tumor patient is greatly improved and the killing tumor effect is much more stronger.

Method of improving the activity of a lymphocyte

The present invention also provided a method of improving the activity of a lymphocyte, carrying a chimeric antigen receptor, comprising: expressing a functional immune costimulatory molecule in the lymphocyte. According to the present invention, the activity of the lymphocyte is at least one selected from a group consisting of in vitro proliferation of the lymphocyte, survival of the lymphocyte in a tumor patient and cytolytic activity of the lymphocyte in the tumor patients. Functional immune costimulatory molecule is expressed in the lymphocyte, and anti-apoptosis genes are up regulated in the lymphocyte, such as Bcl-xl, to prevent activated induced cell apoptosis, which make lymphocyte’s ability of expansion in vitro and survivability in a cancer patient increased dramatically and the targeted killing activity to tumor cells further improved.

EXAMPLES

Example 1

Cell lines and basic experimental techniques used in the examples of the present invention are described below:

Cell lines

The following cell lines were used: Raji and Daudi (CD19+ Burkitt lymphoma cell lines) , Jurkat cells (CD19-immortalized human T lymphocyte cell line) , and K562 (NK target cell) . All cells were obtained from ATCC (American Type Culture Collection) and maintained in culture in RPMI 1640 (Gibco-BRL, San Francisco, CA, USA) supplemented with 10％fetal bovine serum and 2 ml of L-glutamine.

Transduction of peripheral blood mononuclear cell (PBMC)

PBMCs were transduced with an anti-CD19 CAR retroviral vector pMIP-CD19 CAR to stably express anti-CD19 CAR and GFP. Retroviruses were produced by transfecting the Phoenix packaging cell line with a DNA mixture containing 2.5μg env (pVSV-G； Clontech, BD Biosciences) and 3.5μg retroviral vector. The specific process was as follows: 2.5μg env and 3.5 μg retroviral vector vortex mixture was put into the Phoenix culture medium. Phoenix with DNA mixture was cultured for 8 hours and washed with PBS once. Then Phoenix was cultured with RPMI 1640 for 48 hours. Supernatant with retroviruses was recovered， filtered and diluted 1: 2 in RPMI 1640 medium.

The transduction of peripheral blood mononuclear cell (PBMC) with retroviruses was carried out as follows: abandoning the original medium of PBMC (5.0 × 10⁵ PBMCs in every well) cultured in 24-well plates (Costar, Corning, NY) , adding 200μl retroviral supernatant and 6μg/ml polybrene per well, centrifuging at 1800 rpm, 30℃ for 90 min. Finally, 300μl of complete RPMI medium was added, and cells were cultured at 37℃ in 5％CO₂ atmosphere.

Cytokine production

2-7 days after transduction, non-transduced or transduced T cells (1 × 10⁶ cells per well) were co-cultured in 24-well plates with CD19+ Burkitt lymphoma cells in various effector and tumor cell ratios (E: T) . Culture supernatants were collected after 24, 48, and 72 h of culture to measure the production of interferon-γ (IFNγ) using specific enzyme-linked immunosorbent assays (R&D Systems, Inc., Minneapolis, MN, USA)

Briefly, 100 μL of cytokine standard (eg. IFNγ) or culture medium sample were added to well. After incubation for 2 hours at room temperature, solution of each well was abandoned and washed for a total of four times using the wash buffer (400 μL) . After the last wash, 200 μL of enzyme-linked anti-cytokine antibody were added to each well. After 2 hours of incubation at room temperature, 200 μL of Substrate Solution were added to each well. After incubation for 30 minutes at room temperature, 50 μL of Stop Solution were added to each well. The optical density of each well was determined within 30 minutes, using a microplate reader set at 450 nm. Chromium release assay

4 hours ⁵¹Cr-release assays were used to evaluate the cytotoxic activity of the anti-CD19 CAR-transduced T lymphocytes.

The specific process was as follows: Target tumor cells were labeled with ⁵¹Cr for 1 h at 37℃, washed with RPMI medium supplemented with 10％FCS, and resuspended in the same medium at a concentration of 1 × 10⁵ cells/mL. Transduced T cells were added to tumor cells at varying effector to target cell (E: T) ratios in 96-well tissue culture plates in a final volume of 200μL, and incubated for 4h at 37℃. Thereafter, 30μL of supernatant from each well was analyzed using Lumaplate-96 microplates (Packard Bioscience) by a Top Count NXT microplate scintillation counter (Packard Bioscience) . Effector cell number in all assays was calculated based on the total number of T cells. The labeled target cells tested included Raji and Daudi (CD19⁺) , and Jurkat (CD19^–) .

Example 2 4-1BB activation enhancing the antitumor activity of antigen-specific cytotoxic lymphocyte (CTLs)

Inventors tested the effect of 4-1BB activation on CTLs. 5 × 10⁵ B16-OVA tumor cells were injected s. c. into the flank of C57BL/6 syngenic mice. On day 4 after tumor inoculation, mice were treated i. v. with 2×10⁶ OT-1 lymphocytes, obtained from OT1 transgenic mice spleens and activated ex vivo for 48 h with 0.1μg/mL of SIINFEKL peptide, and with 150μg of the agonistic rat anti-mouse anti-CD137 mAb (binding and activation 4-1BB ) (clone 3H3) (BioXCell) intraperitoneally. Groups of mice were also treated i. v. with 2×10⁶ OT-1 lymphocytes, or the agonistic rat anti-mouse anti-CD137 mAb (150μg) intraperitoneally alone. Blank control group was injected with PBS. Tumor growth was then monitored.

As shown in Figure 1， ordinate represents tumor size. Adoptive transfer of 2 × 10⁶ activated OT-1 CTLs to mice s. c. inoculated with 5×10⁵ tumor cells and given no antibody (PBS instead) or control antibody (IgG) showed rapid tumor growth. Treatment with 100μg of the agonist anti-CD137 mAb alone also showed no apparent therapeutic impact. In contrast, combined treatment with activated OT1 CTL and anti-CD137 mAb resulted in the regression of established tumors. (P <0.01, OT-I vs. OT-1+α4-1BB) (n＝5) .

Example 3 construction of a vector that co-expresses 4-1BB or OX40 and anti –CD19 CAR The cassette encoding the anti-human CD19 single-chain antibody, the CD28 endodomain, and the ζ-chain of the T cell receptor complex was cloned into the pMIG retroviral vector to generate the pMIG-CD19 CAR retroviral vector (RV-CD19 CAR) . Xbal /NotI and NotI/Xhol were used during construction. RV-CD19-CAR was constructed with digestion， ligation， screening and positive vector amplification. A cassette containing the IRES and human 4-1BB or OX40 were cloned into the pMIG-CD19 CAR retroviral vector, or a third nucleic acid molecule that encoding a cleavable peptide linked with human 4-1BB or OX40 was cloned into the pMIG-CD19 CAR retroviral vector, or a different promoter, such as CMV， EF-1， RSV or LTR operably linked to human 4-1BB or OX40 was cloned into the pMIG-CD19 CAR retroviral vector to generate the retroviral vector RV-CD19-CAR/4-1BB that

coexpresses anti-CD19-CAR and 4-1BB or RV-CD19-CAR/OX40 that coexpresses

anti-CD19-CAR and OX40 (RV-CD19-CAR/4-1BB or RV-CD19-CAR/OX40) .

As shown in Figure 2, 2A showed the pMIG retroviral vector schematic diagram containing IRES、human 4-1BB or OX40 and anti-CD19 CAR. 2B showed the pMIG retroviral vector schematic diagram containing CMV promoter, human 4-1BB or OX40 and anti-CD19 CAR.

Example 4 Enhanced cytolytic activity of human T cells that were transduced to co-express 4-1BB and anti-CD19 CAR

Human peripheral blood mononuclear cells (PBMC) were obtained from anonymous donor blood samples and isolated using gradient centrifugation on Ficoll-Hypaque. T-lymphocytes were activated with Dynabeads T-activator CD3/CD28 (Invitrogen, Carlsbad, CA) in RPMI Medium 1640 (Invitrogen Gibco Cat. no. 12633-012) with 2 mM L-Glutamin, 10％heat-inactivated fetal calf serum (FCS) (Sigma-Aldrich Co. ) and 100 U/ml penicillin/streptomycin in 72 hour incubation in 5％CO₂ at 37℃. Cells were then washed during magnetic bead removal, and then transduced with retroviral vectors in plates coated with recombinant fibronectin fragment (FN CH-296； Retronectin) . T cells were transduced with a retroviral vector expressing anti-CD19 CAR and 4-1BB (RV-CD19-CAR/4-1BB) , a retroviral vector expressing CD19 CAR (RV-CD19 CAR) , a retroviral vector expressing 4-1BB (RV-4-1BB) , or blank control vector (RV-blank) , respectively. The transduction was descripted in example 1 . The transduced T cells were cultured and expanded in RPMI-1640 with rhuIL-2 (10 ng/ml； R&D Systems) for 7-10 days before the functional experiments. Inventors measured the cytotoxic activity of the transfected T cells against CD19+and CD19-tumor cell lines using standard 4 hours ⁵¹Cr-release assays as shown in Example 1. The effector to target cell (E: T) ratio is 10: 1.

As shown in Figure 3, human T cells that were transduced to co-express 4-1BB and anti-CD19 CAR were more potent than T cells that were transduced to express CD19-CAR or 4-1BB alone in killing CD19+ lymphoma cells (Raji) . Human T cells that were transduced to express 4-1BB alone had no apparent cytolytic activities against CD19+ lymphoma cells. CD19-CAR-transduced T cells or CD19-CAR/4-1BB-transduced T cells had no significant cytolytic activities against CD19^-Jurkat. Mock-transduced T cells (control T cell) showed no significant cytotoxic activity against any of these target cell lines.

Example 5 Enhanced cytokine production and more viable human T cells that were transduced to co-express 4-1BB and CD19 CAR

Human T cells that were activated with Dynabeads T-activator CD3/CD28, transduced with a retroviral vector as descripted in example 1and expanded ex vivo for 10 days were assessed for their cytokine production and cell viability using ELISA assays and Trypan Blue dye exclusion, and ELISA assays was descripted in example 1.

As shown in Figure 4, 4A shows that human T cells transduced with RV-CD19-CAR/4-1BB or RV-4-1BB produced higher levels of IFNgamma than T cells transduced with RV-CD19-CAR or control RV-blank (P <0.05； RV-CD19-CAR/4-1BB vs. RV-CD19-CAR) . 4B shows that the viable cell percentage of the human T cells transduced with RV-CD19-CAR/4-1BB or RV-4-1BB was significantly higher than the human T cells transduced with RV-CD19-CAR or RV-blank (P <0.05； RV-CD19-CAR/4-1BB vs. RV-CD19-CAR) . The results also indicated that the in vitro proliferation and anti-apoptotic ability of human T cells transduced with RV-CD19-CAR/4-1BB or RV-4-1BB was stronger than human T cells transduced with RV-CD19-CAR or control RV-blank.

Example 6 Enhanced cytolytic activity, enhanced cytokine production and more viable human T cells that were transduced to co-express OX-40 and anti-CD19 CAR

Inventors also examined the cytolytic activity, cytokine production and live cell percentage of the human T cells transduced with RV-CD19-CAR/OX-40 as described in example 4 and 5. The results was similar to example 4 and 5 which indicated that human T cells that were transduced to co-express OX-40 and anti-CD19 CAR were more potent in cytolytic activity than T cells that were transduced to express CD19-CAR or full length OX-40 alone, and human T cells that were transduced to co-express OX-40 and anti-CD19 CAR or express full length OX-40 produced more cytokine than T cells that were transduced to express anti-CD19 CAR. Aslo, the living cell percentage of the human T cells transduced with RV-CD19-CAR/OX-40 or solely express overall length OX-40 was significantly higher than the human T cells transduced with RV-CD19-CAR or RV-blank (P <0.05； RV-CD19-CAR/OX-40 vs. RV-CD19-CAR) . The results also indicated that the in vitro proliferation and anti-apoptotic ability of human T cells transduced with RV-CD19-CAR/OX-40 was obviously stronger than human T cells transduced with RV-CD19-CAR or control RV-blank.

To sum up, a T cell co-expressing functional immune costimulatory molecules and chimeric antigen receptors will be more efficiently expanded and survive in tumor microenvironments. Moreover, the T cell co-expressing functional immune costimulatory molecules and chimeric antigen receptors is more potent and efficient in killing antigen-expressing tumor cells, such as lymphoma cells.

It will be apparent to those skilled in the art that variations and modifications of the present invention may be made without departing from the scope or spirit of the present invention. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

A T cell, expressing:

functional 4-1BB； and

a chimeric antigen receptor, wherein

the chimeric antigen receptor comprises:

an ectodomain comprising a heavy chain variable region and a light chain variable region of a single-chain antibody binding to an antigen CD19；

a transmembrane domain linked to the ectodomain and embedded in a cellular membrane of the T cell； and

an endodomain linked to the transmembrane domain, comprising intracellular domain of CD28 and ζ-chain of CD3.
A retrovirus comprising a nucleic acid molecule encoding:

a full length 4-1BB comprising a sequence of SEQ ID NO : 1； and

a CD19-specific chimeric antigen receptor comprising a sequence of SEQ ID NO: 2.
A retrovirus comprising a nucleic acid molecule comprising a sequence of SEQ ID NO : 3 or 4.
A genetically modified lymphocyte, expressing:

a functional immune costimulatory molecule； and

a chimeric antigen receptor.
The genetically modified lymphocyte of claim 4, wherein the chimeric antigen receptor comprises:

an ectodomain capable of binding to an antigen specifically；

a transmembrane domain； and

an endodomain comprising an intracellular domain of an immune costimulatory molecule.
The genetically modified lymphocyte of claim 5, wherein the antigen is a molecule expressed on a tumor cell.
The genetically modified lymphocyte of claim 6, wherein the ectodomain comprising a heavy chain variable region linked to a light chain variable region of an antibody binding to the antigen.
The genetically modified lymphocyte of claim 7, wherein the antibody is a single-chain antibody.
The genetically modified lymphocyte of claim 8, wherein the antigen is CD19.
The genetically modified lymphocyte of claim 4, wherein the functional immune costimulatory molecules is selected independently from a group consisting of 4-1BB, OX-40, CD40L, CD27, CD30, CD28 and variants thereof.
The genetically modified lymphocyte of claim 5, wherein the intracellular domain of the immune costimulatory molecule is derived from at least one selected from a group consisting of 4-1BB, OX-40, CD40L, CD27, CD30, CD28 and variants thereof.
The genetically modified lymphocyte of claim 10 or 11, wherein the functional immune costimulatory molecule is a full length 4-1BB, OX40 or CD40L, and the intracellular domain of the immune costimulatory molecule is the intracellular domain of CD28 or 4-1BB.
The genetically modified lymphocyte of claim 4, wherein the lymphocyte is a CD3+ T lymphocyte.
The genetically modified lymphocyte of claim 13, wherein the lymphocyte is a nature killer cell.
The genetically modified lymphocyte of claim 14, wherein the lymphocyte is a nature killer T cell.
A construct, comprising:

a first nucleic acid molecule encoding a functional immune costimulatory molecule； and

a second nucleic acid molecule encoding a chimeric antigen receptor, wherein

the functional immune costimulatory molecule and the chimeric antigen receptor is defined in any one of claims 4 to 15.
The construct of claim 16, wherein the first and second nucleic acid molecules are configured to express the chimeric antigen receptor and the functional immune costimulatory molecule in a non-fusion form in the lymphocyte defined in any one of claims 4 to 15.
The construct of claim 17, further comprising:

an internal ribosome entry site provided between the first nucleic acid molecule and the second nucleic acid molecule and comprising a nucleic acid sequence of SEQ ID NO: 5.
The construct of claim 16, further comprising:

a third nucleic acid molecule provided between the first nucleic acid molecule and second nucleic acid molecule, the third nucleic acid molecule encoding a cleavable linker peptide in the lymphocyte.
The construct of claim 19, wherein the cleavable linker peptide comprises a sequence of SEQ ID NO: 6.
The construct of claim 16, further comprising:

a first promoter operably linked to the first nucleic acid molecule； and

a second promoter operably linked to the second nucleic acid molecule.
The construct of claim 21, wherein each of the first and second promoters is selected independently from a group consisting of CMV, EF-1, RSV, and LTR promoters.
The construct of claim 16, wherein the construct is a non-pathogenic virus.
The construct of claim 23, wherein the construct is a virus selected from a retrovirus, lentivirus, and adenovirus.
A method of preparing the T cell of claim 1 or the genetically modified lymphocyte of any one of claims 4 to 15, comprising:

introducing the construct of any one of claims 16 to 24, or the retrovirus of claim 2 or 3 into a lymphocyte or T lymphocyte.
A therapeutic composition for treating cancer, comprising:

the construct of any one of claims 16 to 24, the retrovirus of claim 2 or 3, the T cell of claim 1 or the genetically modified lymphocyte of any one of claims 4 to 15.
The therapeutic composition of claim 26, wherein the caner comprises hematopoietic malignancies.
A method for treating cancer, comprising:

administrating the construct of any one of claims 16 to 24, the retrovirus of claim 2 or 3, the T cell of claim 1 or the genetically modified lymphocyte of any one of claims 4 to 15 to a patient in need of such treatment, wherein

the chimeric antigen receptor binding to an antigen of the cancer specifically.
The method of claim 28, comprising:

isolating a lymphocyte from the patient；

introducing the construct of any one of claims 16 to 24, or the retrovirus of claim 2 or 3 into the lymphocyte, and coexpressing the functional immune costimulatory molecule and the chimeric antigen receptor in the lymphocyte to obtain the genetically modified lymphocyte； and

administrating the genetically modified lymphocyte to the patient.
A method of improving the activity of a lymphocyte carrying a chimeric antigen receptor, comprising:

coexpressing a functional immune costimulatory molecule in the lymphocyte, wherein

the functional immune costimulatory molecule, the lymphocyte and the chimeric antigen receptor is defined in any one of claims 4 to 15, and the activity of the lymphocyte is at least one selected from a group consisting of in vitro proliferation of the lymphocyte, survival of the lymphocyte in a tumor patient and cytolytic activity of the lymphocyte in the tumor patients.