WO2022210487A1

WO2022210487A1 - Method for producing immunocyte expressing receptor specific to antigen

Info

Publication number: WO2022210487A1
Application number: PCT/JP2022/014872
Authority: WO
Inventors: いづみ槇; 幸子岡本; 舞子杉崎; 泰典天石; 佳典田中; 圭一朗三原
Original assignee: タカラバイオ株式会社; 学校法人藤田学園
Priority date: 2021-03-29
Filing date: 2022-03-28
Publication date: 2022-10-06
Also published as: JPWO2022210487A1

Abstract

The present invention provides a method for producing an immunocyte that expresses a receptor specific to an antigen which is present in both an immunocyte and a target cell, and the like, said method being characterized by comprising, in no particular order: (a) a step for introducing a nucleic acid that encodes the receptor specific to the antigen into an immunocyte; (b) a step for culturing the immunocyte in a culture medium that contains a kinase inhibitor; and (c) a step for inhibiting expression of the antigen in the immunocyte.

Description

Method for producing immune cells expressing antigen-specific receptors

The present invention provides a method for producing immune cells expressing receptors specific to antigens present in both immune cells and target cells, and a method for producing immune cells that express antigen-specific receptors present in both immune cells and target cells, It relates to a method for producing a pharmaceutical composition comprising the step of obtaining immune cells that express the receptor.

As a therapeutic strategy for tumors, a nucleic acid encoding a chimeric antigen receptor (CAR) that binds to a specific antigen present on the surface of tumor cells, or a T cell receptor (TCR) gene that recognizes tumor cells is used. By introducing it into any T cell, it can be expected to produce T cells that target the specific antigen. Here, T cells into which a nucleic acid encoding CAR has been introduced are called CAR-T cells, and T cells into which a TCR gene has been introduced are called TCR-T cells.

However, if the specific antigen is present not only on the surface of tumor cells but also on the surface of T cells, CAR-T cells or TCR-T cells will attack themselves, so CAR-T cells or There is a problem that TCR-T cells cannot be produced efficiently.

To address this problem, a method was recently developed to inactivate genes involved in antigen expression or presentation present on both the surface of T cells and the surface of target cells, followed by expression of CAR. (Patent Document 1).

Patent No. 6673838

In the technique described in Patent Document 1, gene inactivation is performed by modifying the genome of CAR-T cells using endonucleases such as CRISPR and TALEN, so the inactivation is irreversible. Therefore, there remains a need for new methods to eliminate or reduce the presence of antigen on the surface of T cells.

The present inventors have made intensive studies to solve the above problems, and found that immune cells into which nucleic acids encoding antigen-specific receptors present in both immune cells and target cells have been introduced into immune cells The inventors have found that the antigen can be produced by suppressing the expression of the antigen in the introduced immune cells with siRNA and culturing the immune cells in a medium containing a kinase inhibitor, thereby completing the present invention.

That is, if the present invention is outlined,
[1] A method for producing immune cells that express antigen-specific receptors present in both immune cells and target cells, the method comprising the following steps in random order:
(a) introducing into an immune cell a nucleic acid encoding a receptor specific for said antigen;
(b) culturing immune cells in a medium containing a kinase inhibitor; and (c) suppressing expression of said antigen in immune cells;
[2] The method of [1], wherein the immune cells are T cells or NK cells;
[3] The method of [1], wherein the antigen is present on both the surface of normal immune cells and the surface of target cells.
[4] the method of [1], wherein the antigen is CD38;
[5] The method of [1], wherein the receptor is a chimeric antigen receptor or a T cell receptor;
[6] The method of [1], wherein the kinase inhibitor is a tyrosine kinase inhibitor;
[7] The method of [1], wherein the kinase inhibitor is Dasatinib;
[8] The method of [1], wherein step (c) is performed by knocking down a gene encoding an antigen;
[9] The method of [8], wherein siRNA that suppresses antigen expression is used;
[10] The step (c) is performed by introducing a nucleic acid encoding an siRNA that suppresses the expression of the antigen into the immune cells, and the introduction of the siRNA into the immune cells is performed at the same time as the step (a) [9 ],
[11] A method for producing a pharmaceutical composition, comprising the step of obtaining immune cells expressing antigen-specific receptors present on both immune cells and target cells by the method of [1];
Regarding.

According to the present invention, the production of immune cells expressing antigen-specific receptors present on both immune cells and target cells, and the methods for producing antigen-specific receptors present on both immune cells and target cells Methods of making pharmaceutical compositions comprising immune cells expressing immune cells are provided. The immune cells obtained by the production method of the present invention have a low expression rate of exhaustion markers, a high naive cell rate, and high cytotoxic activity.

It is a figure which shows the preparation procedures of CAR used in an Example. It is a figure which shows the structure of CAR used in an Example. FIG. 3 is a diagram showing the expression level of the CD38 gene with respect to the virus copy number. FIG. 3 is a diagram showing the positive rate of PD-1 expression with respect to virus copy number. FIG. 4 shows cell counts and cell viability. FIG. 3 is a diagram showing the positive rate of PD-1 expression with respect to virus copy number. FIG. 10 is a diagram showing the TIM-3 expression positive rate with respect to the virus copy number. FIG. 4 is a diagram showing the positive rate of LAG-3 expression with respect to virus copy number. FIG. 10 is a graph showing the naive cell rate with respect to virus copy number. FIG. 4 is a diagram showing the cell number of Daudi cells;

As used herein, the term "immune cells" refers to cells in general that are involved in immune function in vivo, including neutrophils, macrophages, lymphocytes <B cells and T cells>, natural killer (NK) cells, plasma A cell etc. are illustrated. Of these, T cells and NK cells are preferred for the present invention. The term "immune cells" as used herein also includes "precursor cells of immune cells" that have the ability to differentiate into the aforementioned immune cells.

"T cells" are also called T lymphocytes, and mean cells derived from the thymus among lymphocytes involved in immune responses. T cells include helper T cells, suppressor T cells, regulatory T cells, cytotoxic T cells (CTLs), naive T cells, memory T cells, αβ T cells expressing α and β chain TCRs, γ chains and γδ T cells expressing TCRs of the δ chain. In addition, "cells capable of differentiating into T cells" and "T cells or cell populations containing cells capable of differentiating into T cells" can also be used in the present invention. "Cells that can differentiate into T cells" are not particularly limited as long as they are cells that differentiate into T cells in vivo or by artificial stimulation. Included are progenitor cells, T-cell progenitor cells, and the like. The "cell population containing T cells or cells capable of differentiating into T cells" includes blood (peripheral blood, umbilical cord blood, etc.), bone marrow fluid, and peripheral blood collected, isolated, purified, and induced therefrom. Cell populations including nuclear cells (PBMC), hematopoietic cells, hematopoietic stem cells, cord blood mononuclear cells and the like are exemplified. Various cell populations derived from blood lineage cells containing T cells can also be used in the present invention. These cells may be activated in vivo or ex vivo by cytokines such as anti-CD3 antibodies and IL-2. These cells can be either collected from a living body or obtained through in vitro culture, for example, a T cell population obtained from a living body as it is or cryopreserved.

NK cells are cytotoxic cells differentiated from hematopoietic stem cells via lymphoid stem cells, and express CD16, CD56, and CD57 on their surface. NK cells recognize cells that do not express MHC class I antigens and cells bound with antibody molecules as non-self, and directly damage them. NK cells can be prepared in vitro, for example, using PBMC as a starting material and culturing them in the presence of OK432 or other inducers.

The immune cells obtained by the present invention are provided with receptors having specificity for antigens known to be commonly expressed by target cells and immune cells or present on the surface of said immune cells. The phrase "known to be present" refers to the presence of an antigen in vivo, particularly on the surface of immune cells in the blood or on the surface of immune cells cultured in vitro. It means that it is not always found. In any event, the use of the methods of the present invention results in reduced antigen expression in immune cells, thereby preventing immune cells expressing the receptor from attacking themselves.

As used herein, the term "target cell" means a cell that is desired to be reduced or eliminated in a patient, and is exemplified by tumor cells and pathogen-infected cells. Although not particularly limited, it is preferably a tumor cell, more preferably a B-cell leukemia cell, or a solid tumor cell.

As used herein, the term "antigen" means a biomolecule such as a protein or an immunological fragment thereof, and is a substance that binds to an antibody or an antigen receptor on an immune cell and induces an immune response. General term. When an antigen exists on the surface of a cell, the cell will be removed from the body by the action of antibodies and lymphocytes. Normally, antigens originate from foreign pathogens such as bacteria and viruses, and heterologous proteins that enter the body through artificial injections. an immune response occurs.

In the present invention, antigens recognized by receptors encoded by nucleic acids introduced into immune cells are present in both immune cells and target cells. Furthermore, in one aspect of the present invention, the antigen is assumed to exist on the surface of normal immune cells. Examples of antigens include CD38, CD4 and CD7, preferably CD38.

The CD38 protein is a marker for HIV infection, leukemia, myeloma, solid tumors, type II diabetes, and bone metabolism, as well as several other genetically determined conditions. In particular, CD38 protein is used as a prognostic marker for leukemia (Ibrahim, S. et al. 2001, Blood 98: 181-186).

As used herein, the term "receptor" refers to an antigen-specific receptor. Preferably, it is a receptor capable of endowing immune cells with the property of recognizing and/or damaging target cells. Examples of such receptors include CAR and TCR.

CAR is a fusion protein comprising an antigen-binding extracellular domain (hereinafter referred to as an antigen-binding domain), a transmembrane domain derived from a polypeptide different from the antigen-binding domain, and at least one intracellular domain. . CARs are sometimes called "chimeric receptors," "T-bodies," and "chimeric immune receptors (CIRs)."

As used herein, the term "domain" refers to a region within a polypeptide that folds into a specific structure independently of other regions.

"Antigen-binding domain" means any oligopeptide or polypeptide that can bind to an antigen. Examples of antigen-binding domains include antigen-binding sites derived from antibodies, such as Fab' fragments, Fab fragments, Fv fragments, etc., but single-chain variable region fragments (scFv) are preferred for the present invention. . scFv refers to an antibody-derived single-chain polypeptide that retains antigen-binding ability. Examples thereof include polypeptides formed by recombinant DNA technology, in which the Fv region of an immunoglobulin heavy chain (H chain) and the Fv region of a light chain (L chain) are linked via a spacer sequence. Various methods for producing scFv are known, US Pat. No. 4,694,778, Science, vol. 242, pp. 423-442 (1988), Nature, vol. 334, pp. 54454 (1989). , Science, Vol. 242, pp. 1038-1041 (1988).

"Intracellular domain" means any oligopeptide or polypeptide known to function as a domain that transmits a signal that activates or inhibits a biological process within a cell.

The structure of a typical CAR consists of an scFv, a transmembrane domain, and an intracellular domain that activates cells. Transmembrane domains derived from the TCR complex CD3ζ, CD28, CD8α, etc. are known. As the intracellular domain, the intracellular domain of CD3ζ is preferably used. A CAR with such a configuration is called a first generation CAR. CAR-expressing T cells (CAR-T cells) directly recognize surface antigens of tumor cells independently of the expression of major histocompatibility antigen class I on tumor cells, and simultaneously activate T cells themselves. , can efficiently kill tumor cells.

With the aim of enhancing the ability of first-generation CARs to activate T cells, second-generation CARs linked to the intracellular domains of T-cell co-stimulatory molecules have been developed. As co-stimulatory molecules for T cells, CD28, the intracellular domain of CD137 (4-1BB) or CD134 (OX40), which is a tumor necrosis factor (TNF) receptor superfamily, the intracellular domain of interleukin receptors and modifications thereof glucocorticoid-induced tumor necrosis factor receptor (GITR) intracellular domain and the like are preferably used. As a further improvement, third-generation CARs in which the intracellular domains of these co-stimulatory molecules are linked in tandem have also been developed, and many CAR molecules targeting various tumor antigens have been reported. The nucleic acid constructs of the invention can contain any CAR-encoding sequence as the desired gene sequence.

As used herein, TCR is responsible for the antigen recognition function of T cells, and is composed of polypeptides such as α-chain, β-chain, γ-chain, and δ-chain. Among them, heterodimers of TCRα chain (TCRα) and TCRβ chain (TCRβ) or heterodimers of γ chain and δ chain are used as auxiliary molecules in the CD3 complex (γ, δ, ε, ζ ), together with CD4 or CD8, etc., form the TCR.

TCR recognizes peptides (antigen epitopes) presented by target cells (phagocytic cells, virus-infected cells, cancer cells, etc.) via the major histocompatibility complex (MHC) in vivo, and target cells Responsible for the function of making an immune response against Human MHC is also referred to as the human histocompatibility leukocyte antigen (HLA) system. HLA is classified into class I and class II. Class I includes HLA-A, B, C, E, F, G, H, and J. Class II includes HLA-DR, DQ, and DP. included. HLA, like TCR, is also formed by a complex of α and β chains, and presents antigenic epitopes on the cell surface via these chains. HLA class I is present in almost all cells in the body and presents antigenic epitopes approximately nine amino acid residues long. For example, when a virus-infected cell presents a virus-derived antigen epitope via HLA, T cells (such as CTL) having a TCR specific to the antigen epitope are activated, resulting in an immune response of the body (e.g. , killing of virus-infected cells) are brought about, and biological defense is performed.

TCRs are usually able to make an immune response only by binding to a complex formed by a specific type of HLA and an antigenic epitope. This restriction is referred to as "HLA-restricted". As used herein, the term HLA (or MHC)-restricted refers to TCRs, as well as T cells expressing such TCRs, or antigen-HLA complexes presented by various peptides. , cells and the like. Binding of the TCR to the antigen-epitope-HLA complex results in an immune response, exemplified by the secretion of cytokines (interferon-γ, TNF-α, IL-2, etc.) by CD8+ T cells. Binding of TCRs to antigen-epitope-HLA complexes can be measured indirectly by measuring secretion of the cytokines described above. Further, by detecting the binding of TCR to a complex of HLA (or a peptide fragment thereof) having a label such as green fluorescent protein (GFP) and an antigen epitope, the binding of TCR and antigen-HLA can be detected. It is directly measurable. Alternatively, known optical detection means utilizing the effect of light emission and extinction due to the proximity of two or more molecules can also be used.

(1) Method for producing immune cells of the present invention (a) Step of introducing a nucleic acid encoding an antigen-specific receptor into immune cells The method for producing immune cells of the present invention comprises antigen-specific receptors. into immune cells. The process is typically performed ex vivo.

The method of the present invention can use immune cells derived from mammals, such as humans, or immune cells derived from non-human mammals such as monkeys, mice, rats, pigs, cows, and dogs. The immune cells used in the method of the present invention are not particularly limited, and any immune cells can be used. For example, cells collected, isolated, purified, or induced from blood (peripheral blood, umbilical cord blood, etc.), bodily fluids such as bone marrow, or tissues or organs can be used. In the present invention, it is particularly preferable to use peripheral blood mononuclear cells (PBMC), T cells, T cell progenitor cells (hematopoietic stem cells, lymphocyte progenitor cells, etc.), or cell populations containing these. T cells include CD8 positive T cells, CD4 positive T cells, regulatory T cells, cytotoxic T cells, or tumor infiltrating lymphocytes. Furthermore, NK cells and their progenitor cells are also suitable as hosts for expressing antigen-specific receptors. Cell populations containing T cells and T cell progenitor cells include PBMCs. The above-mentioned cells may be those collected from living organisms, those obtained by expanding culture thereof, or those established as cell lines. When it is desired to transplant cells expressing a receptor specific to the manufactured antigen or cells differentiated from such cells into living organisms, the nucleic acid is transferred to the living organism itself or cells collected from the same kind of living organism. It is preferable to introduce

Introduction of nucleic acids encoding antigen-specific receptors into immune cells can be carried out by known methods. For example, liposomes and WO 96/10038, WO 97/18185, WO 97/25329, WO 97/30170 and WO 97/31934 (any (also incorporated herein by reference) can be used to introduce nucleic acids encoding antigen-specific receptors into immune cells using condensing agents such as cationic lipids. can. In addition, nucleic acids encoding antigen-specific receptors can be introduced into immune cells by calcium phosphate transfection, DEAE-dextran, electroporation, particle bombardment.

Nucleic acids encoding antigen-specific receptors can be loaded into appropriate vectors and introduced into immune cells. As vectors, plasmid vectors (including episomal vectors), viral vectors (retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, etc.) and other known vectors can be used. When a nucleic acid encoding an antigen-specific receptor is loaded into a vector, an expression control sequence (promoter, terminator, enhancer, etc.) that functions in the introduced immune cells may be added.

Viral vectors can infect cells and introduce nucleic acids retained in the viral genome. Methods for producing viral vectors as infectious particles are also well known to those skilled in the art. For example, when using a retroviral vector (including a lentiviral vector), suitable packaging cells are selected based on the LTR sequence and packaging signal sequence possessed by the vector, and used to prepare retroviral particles. can be prepared and carried out. For example, PG13 (ATCC CRL-10686), PA317 (ATCC CRL-9078), GP + E-86 and GP + envAm-12 (US Patent No. 5,278,056), Psi-Crip [Proceedings of the National Academy of Sciences, Volume 85, 6460-6464 (1988)]. Retroviral particles can also be produced using 293 cells and 293T cells, which have high transfection efficiency. Retroviral vectors based on many types of retroviruses and packaging cells that can be used for packaging the vectors are widely available commercially from various companies. Also, many reagents and kits for producing adenovirus vectors and AAV vectors are commercially available.

According to the present invention, retroviral vectors and lentiviral vectors are suitable for introducing nucleic acids encoding antigen-specific receptors into immune cells. Methods for viral transduction are well known in the art (Waither et al. (2000) Viral Vectors for Gene Transfer. Drugs. 60(2):249-271). Integrating viral vectors allow stable integration of polynucleotides into the cellular genome and long-term expression of antigen-specific receptors.

In the step of introducing a nucleic acid encoding an antigen-specific receptor into an immune cell, a functional substance that improves the introduction efficiency can be used [e.g., International Publication No. 95/26200, International Publication No. 00/ 01836 pamphlet (both incorporated herein by reference)]. Substances that improve transduction efficiency include substances that have activity to bind to viral vectors, such as fibronectin or fibronectin fragments. Preferably, a fibronectin fragment having a heparin-binding site, such as a fragment commercially available as RetroNectin (registered trademark, CH-296, manufactured by Takara Bio Inc.) can be used. In addition, polybrene, fibroblast growth factor, type V collagen, polylysine, or DEAE-dextran, which are synthetic polycations that have the effect of improving the efficiency of retroviral cell infection, can be used.

In a preferred embodiment of the present invention, the functional substance is immobilized on an appropriate solid phase, such as a container (plate, petri dish, flask, bag, etc.) or a carrier (microbeads, etc.) used for cell culture. can be used in

Nucleic acids introduced into immune cells using retroviral vectors are integrated into the chromosomes of the immune cells. Similarly, methods for integrating nucleic acids onto chromosomes include methods using transposons (PiggyBac, etc.) and methods using genome editing technology (CRISPR/Cas9, TALEN, etc.).

(b) Step of culturing immune cells in a medium containing a kinase inhibitor The method for producing immune cells of the present invention is characterized by including a step of culturing immune cells in a medium containing a kinase inhibitor. do.

The conditions for culturing immune cells in the medium containing the kinase inhibitor are not particularly limited, and conditions commonly used for cell culture can be used. For example, culture can be performed using plates, flasks, cell culture bags, large culture tanks, etc. under conditions such as 37° C. and 5% CO ₂ . In addition, operations such as adding fresh medium to the cell culture solution to dilute it at appropriate time intervals, exchanging the medium, and exchanging the cell culture equipment can be performed. The medium used for culture is not particularly limited, either, and a serum-containing medium, serum-free medium, xeno-free medium, or the like having a general composition may be used.

In the present invention, the term "kinase inhibitor" refers to a drug that inhibits kinase activity, and inhibits kinases such as low-molecular-weight compounds, polypeptides, proteins, nucleic acids (siRNA, miRNA, aptamers, etc.), and other macromolecular compounds. Including drugs. As kinase inhibitors according to the invention, use of tyrosine kinase inhibitors (TKIs) is suitable.

A tyrosine kinase is an enzyme that phosphorylates the hydroxyl groups on the side chains of tyrosine residues in proteins, and is a type of protein kinase. A protein kinase transfers phosphate from ATP to a substrate protein and phosphorylates it, thereby changing the three-dimensional structure of the substrate protein and regulating its activity, thereby regulating cell functions. Serine/threonine kinases have long been known as representative intracellular protein kinases, but various tyrosine kinases have been reported since it was discovered that retroviral oncogene products act as tyrosine kinases. . Since these tyrosine kinases were cell growth factor receptors and oncogene products, they are the subject of research on cell signaling, cell proliferation and canceration.

In the present invention, tyrosine kinase inhibitors are exemplified by dasatinib, imatinib, nilotinib, sunitinib, pazopanib, quizartinib, crenolanib or sorafenib, preferably dasatinib. Dasatinib is an antineoplastic agent (anti-cancer agent) developed by Bristol-Myers Squibb as a tyrosine kinase inhibitor, a molecularly targeted therapeutic drug that targets multiple tyrosine kinases including BCR-ABL. be.

The concentration of the tyrosine kinase inhibitor added to the medium used in this step is, for example, 5-200 nM, preferably 15-100 nM, more preferably 40-60 nM. In addition, the tyrosine kinase inhibitor is added after the step of introducing the nucleic acid encoding the antigen-specific receptor (after the last introduction when introducing multiple times), for example, within 3 days, preferably 2 days. It is carried out within days, more preferably within 24 hours. Alternatively, a tyrosine kinase inhibitor may be added immediately after introduction of a nucleic acid encoding an antigen-specific receptor into cells and culture may be initiated. The culture time in the presence of the tyrosine kinase inhibitor is, for example, 1 to 15 days, preferably 2 to 10 days, more preferably 3 to 5 days. It is a matter of course to determine an appropriate culture time depending on the type. Also, the concentration of the tyrosine kinase inhibitor may be changed as appropriate during the culture time. Furthermore, after culturing the cells in the presence of the tyrosine kinase inhibitor, the cells may be cultured in medium without the tyrosine kinase inhibitor.

(c) Step of Suppressing Antigen Expression in Immune Cells The method for producing immune cells of the present invention is characterized by including a step of suppressing antigen expression in immune cells.

As used herein, the term "suppression of expression" refers to suppression of final production of a polypeptide by preventing transcription and/or translation from a gene encoding a polypeptide. This means that the volume will decrease. Therefore, even if the transcription reaction from the gene encoding the polypeptide is not suppressed, it is included in "suppression of expression" if the transcription product (mRNA) is rapidly degraded and the production of the polypeptide is suppressed. A state in which the expression is suppressed is a state in which the expression level is reduced by 20% or more, 40% or more, 60% or more, or 80% or more compared to the case where the expression is not suppressed, or a state in which the expression level is reduced by 100%, that is, completely suppressed state.

According to a preferred embodiment of the present invention, the above "suppression of expression" is performed by knocking down the antigen gene using siRNA. Knockdown of an antigen gene by siRNA is performed by introducing into cells a nucleic acid encoding the siRNA, that is, a siRNA-producing sequence. An example of an siRNA generating sequence is a sequence from which RNA is transcribed that forms at least one stem-loop structure and is capable of inducing RNA interference in mammalian cells. RNA interference in the present invention aims to selectively suppress the expression of specific endogenous genes that cells naturally express by introducing nucleic acids containing siRNA-producing sequences. RNA interference is induced by siRNA annealed with an RNA molecule homologous to and complementary to the base sequence of mRNA transcribed from a gene whose expression is desired to be suppressed (hereinafter referred to as a target gene). .

The siRNA-generating sequence used in the present invention includes, for example, a sequence (sense sequence) homologous to a region of the mRNA transcribed from the target gene and a complementary sequence (antisense sequence) arranged in series. are placed in A single RNA strand transcribed from this siRNA-producing sequence forms a double-stranded structure by annealing the sense sequence and the antisense sequence within the molecule, and the formed double-stranded RNA portion is used as the stem region. A stem-loop structure is formed with any sequence located between the sequence and the antisense sequence as the loop region. In cells, siRNA is produced from this stem region by the action of RNase III (Dicer). From the viewpoint of interferon response suppression in mammalian cells, the chain length of the portion corresponding to the stem region in the siRNA generating sequence is, for example, 13 to 29 bases, preferably 15 to 25 bases, more preferably 19 to 25 bases. . The loop region may have any sequence, and a sequence of 1 to 30 nucleotides is exemplified, preferably a sequence of 1 to 25 nucleotides, more preferably 5 to 22 nucleotides.

Here, the siRNA produced in cells according to the present invention is composed of RNA with a sequence that is homologous to the specific base sequence of the mRNA transcribed from the target gene and RNA with a sequence that is complementary to each other. It does not need to be completely homologous or complementary to the specific base sequence of mRNA. siRNA consisting of substantially homologous RNA and substantially complementary sequence RNA may be used as long as the function of suppressing the expression of the target gene is exhibited. In addition, the siRNA generating sequence used in the present invention transcribes one type of siRNA targeting one type of gene, and also transcribes a plurality of siRNAs corresponding to the base sequences of different regions of one type of target gene. or transcribe a plurality of siRNAs corresponding to a plurality of target genes. For example, the number of siRNAs generated from the siRNA-generating sequences used in the present invention is 1-10, 1-6, 1-4, multiple or several. The present invention relates to the siRNA-generating sequences of the base sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9, and nucleic acids in which either or both of these sequences are arranged so that siRNA is expressed in cells, which are described in the following examples. Provide constructs.

Introduction of a nucleic acid containing an siRNA-producing sequence into an immune cell can be performed in the same manner as for nucleic acids encoding antigen-specific receptors. Although the present invention is not particularly limited, from the viewpoint of stably suppressing the expression of antigens in immune cells, retroviral vectors and lentiviral vectors that have the property of integrating introduced nucleic acids into chromosomes are used in cells. It is desirable to load nucleic acid containing an siRNA-producing sequence in such a configuration that siRNA is expressed therein and then introduce it into immune cells. The timing of introducing the nucleic acid containing the siRNA generating sequence into the immune cells may be before, at the same time as, or after the step (a) of introducing the nucleic acid encoding the antigen-specific receptor. In one aspect of the present invention, introduction of a nucleic acid containing an siRNA-generating sequence into an immune cell is performed at the same time as step (a). A vector carrying a nucleic acid encoding a specific receptor may be simultaneously introduced into an immune cell, or both the nucleic acid containing the siRNA-generating sequence and the nucleic acid encoding the antigen-specific receptor are carried in the same vector. , may be introduced into immune cells. That is, step (a) and step (c) may be performed in one step.

The steps (a) to (c) may be performed in any order, or two or more of the steps (a) to (c) may be performed simultaneously.

(2) Method for producing the pharmaceutical composition of the present invention The present invention includes the step of obtaining immune cells expressing antigen-specific receptors present on both immune cells and target cells by the method of (1). , a method of making a pharmaceutical composition.

The present invention provides a pharmaceutical composition containing, as an active ingredient, immune cells that express antigen-specific receptors present on both immune cells and target cells. The pharmaceutical composition may further contain suitable excipients. Such excipients include, for example, pharmaceutically acceptable excipients, various cell culture media, isotonic saline, and the like. The disease to which the pharmaceutical composition is administered is not particularly limited as long as it is a disease that shows sensitivity to the immune cells. Examples include cancer [blood cancer (leukemia), solid tumor, etc.], inflammatory disease /autoimmune diseases (asthma, eczema), infectious diseases caused by viruses such as hepatitis, influenza, HIV, bacteria, and fungi, such as tuberculosis, MRSA, VRE, and deep mycosis. A pharmaceutical composition containing, as an active ingredient, an immune cell expressing a receptor that binds to an antigen possessed by cells whose reduction or elimination is desired in the aforementioned diseases, that is, a tumor antigen, a viral antigen, a bacterial antigen, or the like, is used to treat these diseases. administered for treatment. In addition, the pharmaceutical composition of the present invention can also be used for bone marrow transplantation, prevention of infectious diseases after irradiation, donor lymphocyte transfusion for the purpose of remission of recurrent leukemia, and the like. The pharmaceutical compositions of the present invention can be administered by, but not limited to, parenteral administration, such as injection or infusion, intradermally, intramuscularly, subcutaneously, intraperitoneally, intranasally, intraarterially, intravenously, intratumorally, or It can be administered into afferent lymphatics and the like.

Although the present invention will be described in more detail with reference to examples below, the present invention is not limited only to the following examples.

Example 1 Preparation of Plasmid First, pMS3-MC described in International Publication No. 2013/051718 was prepared. pMS3-MC has, in order from the 5′ end, MMLV (Moloney murine leukemia virus)-derived 5′ LTR (long terminal repeat), MMLV-derived SD (splice donor) sequence, MMLV-derived ψ (packaging signal) sequence, It is a retroviral vector plasmid having an SA (splice acceptor) sequence derived from the human EF1α gene and a 3'LTR derived from MMLV, and the U3 region of the 3'LTR is replaced with a sequence derived from MSCV (mouse stem cell virus).

Next, based on the description of Patent 5328018 and literature (J Immunother. 2009 Sep;32(7):737-43), a DNA fragment denoted as antiCD38-CAR in Fig. 1 was synthesized. This DNA fragment contains a Kozak sequence (SEQ ID NO: 1), which is said to have the highest translation efficiency, at the 5' end, a CD8α signal peptide (amino acid sequence: SEQ ID NO: 2), and an anti-CD38 monoclonal antibody that binds to the cancer antigen CD38. VL (amino acid sequence: SEQ ID NO: 3), linker sequence (amino acid sequence: SEQ ID NO: 4), VH of anti-CD38 monoclonal antibody (amino acid sequence: SEQ ID NO: 5), CD28 domain (CD28-derived polypeptide containing transmembrane domain; It encodes one molecule of CAR containing amino acid sequence: SEQ ID NO: 6) and CD3ζ intracellular domain (amino acid sequence: SEQ ID NO: 7) in order from the N-terminus. This DNA fragment was inserted into pMS3-MC to generate pMS3-CD38-CAR.

In FIG. 1, the Kozak sequence is "K", the CD8α signal peptide is "SP", the VL of the anti-CD38 monoclonal antibody is "anti-CD38 VL", the linker sequence is "L", and the VH of the anti-CD38 monoclonal antibody is "anti -CD38 VH", CD28 domain as "CD28", CD3ζ intracellular domain as "CD3ζ", terminal repeat as "LTR", splice donor sequence as "SD", splice acceptor sequence as "SA", packaging signal sequence is displayed as "ψ".

FIG. 2 shows a plasmid prepared by modifying pMS3-CD38-CAR. That is, sequences that generate two types of siRNA against the CD38 gene (SEQ ID NO: 8 and SEQ ID NO: 9) are inserted between the ψ sequence and SA sequence of pMS3-CD38-CAR to prepare pMS3-CD38-siRNA-CAR. did. Furthermore, the sequences encoding the anti-CD38 monoclonal antibodies VL and VH of pMS3-CD38-CAR were converted to VL and anti-CEA monoclonal antibodies that bind to the cancer antigen CEA (carcinoembryonic antigen), respectively, based on the description of Japanese Patent Application No. 2020-164927. pMS3-CEA-CAR was created by replacing the VH-encoding sequence. In addition, based on the description in International Publication No. 2016/127257 and literature (Nat Med. 2018 Mar;24(3)352-359), interleukin 2 receptor β chain is inserted between the CD28 domain and the CD3ζ intracellular domain. A portion of the intracellular domain (IL2Rβ) (amino acid sequence: SEQ ID NO: 10) is inserted, and the amino acid sequence (LHMQ) in the CD3ζ intracellular domain is replaced with a STAT3 binding motif (YRHQ) required for activation of STAT3 signaling. pMS3-CD38-JS-CAR and pMS3-CD38-JS-siRNA-CAR were prepared by doing so.

In Figure 2, a portion of the intracellular domain of the interleukin-2 receptor β chain (IL2Rβ) is indicated as "IL2Rβ". In addition, the CD3ζ intracellular domain (amino acid sequence: SEQ ID NO: 11) having the STAT3 binding motif (YRHQ) is indicated as "CD3ζ (YRHQ)".

Example 2 Preparation of Retrovirus Solution Escherichia coli HST08 was transformed with each of the plasmids prepared in Example 1 to obtain transformants. Plasmid DNAs possessed by these transformants were each purified using NucleoBond Xtra Midi (manufactured by Macharei Nagel) and subjected to the following procedures as DNAs for transfection. 293T cells were transfected with the prepared DNA for transfection and pGP vector and pE-eco vector contained in Retrovirus Packaging Kit Eco (manufactured by Takara Bio Inc.). This operation was performed according to the product protocol of the kit. A supernatant containing the ecotropic virus was obtained from each of the obtained transduced cells and filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore). This supernatant was used to infect PG13 cells (ATCC CRL-10686) with ecotropic virus by a method using polybrene. The culture supernatant of the obtained cells was collected and filtered through a 0.45 μm filter to prepare each retrovirus solution, which was used in subsequent examples.

Example 3 Suppression of CD38 gene expression by siRNA Retronectin (Registered Trademark, manufactured by Takara Bio Inc.) was infected twice by a standard method to prepare PBMCs expressing each CAR. Furthermore, PBMCs were cultured under conditions of 37° C., 95% humidity and 5% CO ₂ . Here, the composition of the medium is LymphoONE T-Cell Expansion Xeno containing 200 IU/mL Proleukin (IL-2) (manufactured by Nipro) and 0.6% (v/v) AB serum (manufactured by Access Biologicals). - Free Medium (manufactured by Takara Bio Inc.). Cells were collected 7 days after the second virus infection, and total RNA was extracted and treated with DNase I using NucleoSpin RNA Plus (manufactured by Mach Liner Gel). Using the obtained total RNA as a template, cDNA was synthesized using PrimeScript RT reagent Kit (Perfect Real Time) (manufactured by Takara Bio Inc.). Furthermore, using this cDNA as a template, real-time PCR was performed using TB Green Premix Ex Taq II (manufactured by Takara Bio Inc.) and a CD38 gene amplification primer set (SEQ ID NO: 12 and SEQ ID NO: 13) to measure the gene expression level. . In addition, the relative expression level of the CD38 gene was calculated by measuring the expression level of the GAPDH gene, which is a housekeeping gene, using the primer set shown in SEQ ID NO: 14 and SEQ ID NO: 15. Furthermore, genomic DNA was extracted from the cells 7 days after the second virus infection using SimplePreagent for DNA (manufactured by Takara Bio Inc.), Provirus Copy Number Detection Primer Set, Human (manufactured by Takara Bio Inc.) and Cycleave PCR Core Kit. (manufactured by Takara Bio Inc.) was used to measure the retrovirus copy number integrated into the genome.

By calculating the relative value of CD38 gene expression in CD38-CAR-introduced cells and CD38-siRNA-CAR-introduced cells when the virus copy number is around 1, the inhibitory effect of siRNA on CD38 gene expression was evaluated. The results are shown in FIG. In FIG. 3, the vertical axis indicates the relative value of the expression level of the CD38 gene in the CD38-siRNA-CAR-introduced cells when the expression level of the CD38 gene in the CD38-CAR-introduced cells is set to 100. As shown in FIG. 3, CD38 gene expression was suppressed in CD38-siRNA-CAR-transfected cells compared to CD38-CAR-transfected cells.

Example 4 Increase in Exhaustion Marker by CD38-CAR Expression A standard method using retronectin, in which retroviral solutions for expressing CAR prepared in Example 2 were added to PBMCs isolated from human peripheral blood at various dilutions. to generate PBMCs expressing each CAR. By the same procedure as in Example 3, the number of retroviruses integrated into the genome of cells 3 days after the second virus infection was measured.

In addition, the cells were stained by adding PerCP/Cyanine5.5-labeled anti-Human CD279 (PD-1) antibody (manufactured by BioLegend) to the cells 3 days after the second virus infection. Using a flow cytometer, the percentage of cells positive for the exhaustion marker PD-1 was determined for the stained cells. FIG. 4 shows the ratio of PD-1 positive cells to the copy numbers of CD38-CAR and CEA-CAR retroviruses. As shown in FIG. 4, the proportion of cells expressing the exhaustion marker PD-1 was increased in CD38-CAR-transfected cells compared to non-transfected cells (NGMC) and CEA-CAR-transfected cells.

Example 5 Increase in Cell Number and Cell Viability by siRNA and Kinase Inhibitor Retronectin was applied to PBMCs isolated from human peripheral blood at various dilutions of each CAR-expressing retrovirus solution prepared in Example 2. Each CAR-expressing PBMC was generated by two rounds of infection using standard methods. After the second virus infection, each cell was divided into two halves, Dasatinib (manufactured by Cell Signaling Technology) was added to one half at a concentration of 50 nM, and Dasatinib was not added to the other half, and cultured for 4 days. The retrovirus copy number was measured in the same manner as in Example 3, and the cell number and cell viability were measured.

Figure 5 shows the cell number and cell viability in CAR-introduced cells with a virus copy number of 1.5 to 1.8 copies/cell. As shown in FIG. 5, under Dasatinib-free conditions, CD38-CAR-transfected cells and CD38-siRNA-CAR-transfected cells decreased in cell number and cell viability compared to NGMC. At this time, the number of CD38-siRNA-CAR-introduced cells was greater than the number of CD38-CAR-introduced cells. On the other hand, addition of Dasatinib restored the cell number and cell viability of CD38-CAR-introduced cells and CD38-siRNA-CAR-introduced cells to the same level as NGMC.

Example 6 Reduction of Exhaustion Markers by siRNA and Kinase Inhibitor PBMCs isolated from human peripheral blood were diluted with various dilutions of each CAR-expressing retrovirus solution prepared in Example 2, and retronectin was used as a standard method. method to generate PBMCs expressing each CAR. In some conditions, Dasatinib (manufactured by Cell Signaling Technology) was added at a concentration of 50 nM after the second virus infection, and Dasatinib was removed 4 days later. In the same manner as in Example 3, retrovirus copy numbers integrated into the genome of cells cultured for 15 days after retrovirus infection were measured. In addition, PE-labeled anti-Human CD279 (PD-1) antibody (manufactured by Becton Dickinson) and APC-Cy7-labeled anti-Human CD366 (Tim-3) antibody (manufactured by BioLegend) were added to cells cultured for 15 days after retrovirus infection. , and PerCP/Cyanine 5.5-labeled anti-Human CD223 (LAG-3) antibody (BioLegend) to stain the cells. A flow cytometer was used to determine the percentage of cells positive for each exhaustion marker after staining.

Figure 6 shows the ratio of PD-1 positive cells to the virus copy number, Figure 7 shows the ratio of Tim-3 positive cells to the virus copy number, and Figure 8 shows the ratio of LAG-3 positive cells to the virus copy number. As shown in each figure, the positive rate of each exhaustion marker was decreased in cells expressing siRNA against CD38 compared to cells not expressing siRNA. In addition, addition of Dasatinib further decreased the positive rate of exhaustion markers. That is, the positive rate of exhaustion markers decreased most under the condition that Dasatinib was added to cells expressing siRNA against CD38.

Example 7 Increase in Naive Rate by siRNA and Kinase Inhibitor PBMCs isolated from human peripheral blood were added with various dilutions of each CAR-expressing retrovirus solution prepared in Example 2, and retronectin-based standard method to generate PBMCs expressing each CAR. Furthermore, under certain conditions, Dasatinib was added at a concentration of 50 nM after the second virus infection, and a biotin-labeled anti-mouse IgG antibody (manufactured by Jackson ImmunoResearch) was added to cells cultured for 15 days after retrovirus infection. , streptavidin-APC (manufactured by BioLegend), PerCP/Cyanine5.5-labeled anti-Human CD8 antibody (manufactured by Beckman Coulter), FITC-labeled anti-Human CD197 (CCR7) antibody (manufactured by BioLegend), and PE-labeled anti-Human CD45RA Cells were stained by adding an antibody (BioLegend). Using a flow cytometer, the percentage of cells positive for CCR7 and CD45RA among cells positive for APC among PerCP/Cyanine5.5-positive cells was measured for the stained cells. That is, the proportion of CAR-positive cells among CD8-positive cells and naive cells was measured.

Figure 9 shows the ratio of naive cells to virus copy number. As shown in FIG. 9, addition of Dasatinib to CD38-CAR-transfected cells resulted in more naive cells. Furthermore, the condition in which Dasatinib was added to the CD38-siRNA-CAR-introduced cells resulted in containing the largest number of naive cells.

Example 8 Maintenance of cytotoxic activity by siRNA and kinase inhibitor PBMCs isolated from human peripheral blood were diluted with various dilutions of each CAR-expressing retrovirus solution prepared in Example 2, and retronectin was used as a standard method. Each CAR-expressing PBMC was prepared by infecting each CAR twice. After the second virus infection, each cell was divided into two halves, one of which was added with Dasatinib at a concentration of 50 nM, and the other of which was cultured for 7 days without Dasatinib. Seven days after the second virus infection, the cells were co-cultured with CD38-positive Daudi cells (National Institute of Biomedical Innovation, Health and Nutrition JCRB9071), and the number of Daudi cells was measured every 3 to 4 days. In addition, Daudi cells were added during cell number determination.

Fig. 10 shows the number of Daudi cells versus the number of days of co-culture. Until the 17th day, proliferation of Daudi cells was suppressed under all conditions except NGMC. However, on day 21, Daudi cells proliferated only in CD38-JS-CAR-introduced cells without the addition of Dasatinib. This result indicates that the cytotoxic activity of CAR-expressing cells is maintained by suppressing CD38 expression with siRNA and/or by adding Dasatinib.

According to the present invention, the production of immune cells expressing antigen-specific receptors present on both immune cells and target cells, and the methods for producing antigen-specific receptors present on both immune cells and target cells Methods of making pharmaceutical compositions comprising immune cells expressing immune cells are provided. The method of the present invention is particularly useful for medical applications.

SEQ ID NO:1: Kozak sequence
SEQ ID NO:2: CD8 alpha signal peptide
SEQ ID NO:3: anti-CD38 VL sequence
SEQ ID NO:4: linker sequence
SEQ ID NO:5: anti-CD38 VH sequence
SEQ ID NO:6: CD28 domains
SEQ ID NO:7: CD3 zeta intracellular domain
SEQ ID NO:8: CD38_siRNA_2
SEQ ID NO:9: CD38_siRNA_3
SEQ ID NO:10: IL2R beta domain
SEQ ID NO:11: CD3 zeta intracellular domain with STAT3-binding motif (YRHQ)
SEQ ID NO:12: CD38-Fw primer
SEQ ID NO:13: CD38-Rv primer
SEQ ID NO:14: GAPDH-Fw primer
SEQ ID NO:15: GAPDH-Rv primer

Claims

A method for producing immune cells expressing antigen-specific receptors present on both immune cells and target cells, the method comprising the steps of, in random order:
(a) introducing into an immune cell a nucleic acid encoding a receptor specific for said antigen;
(b) culturing immune cells in a medium containing a kinase inhibitor; and (c) suppressing expression of said antigen in immune cells.
The method according to claim 1, wherein the immune cells are T cells or NK cells.
The method according to claim 1, wherein the antigen is present on both the surface of normal immune cells and the surface of target cells.
The method according to claim 1, wherein the antigen is CD38.
The method according to claim 1, wherein the receptor is a chimeric antigen receptor or a T cell receptor.
The method according to claim 1, wherein the kinase inhibitor is a tyrosine kinase inhibitor.
The method of claim 1, wherein the kinase inhibitor is Dasatinib.
The method according to claim 1, wherein step (c) is performed by knocking down a gene encoding an antigen.
The method according to claim 8, wherein siRNA that suppresses antigen expression is used.
wherein step (c) is performed by introducing a nucleic acid encoding an siRNA that suppresses antigen expression into immune cells, and the introduction of the nucleic acid encoding the siRNA into immune cells is performed at the same time as step (a). Item 9. The method of Item 9.
A method for producing a pharmaceutical composition, comprising the step of obtaining immune cells expressing antigen-specific receptors present on both immune cells and target cells by the method according to claim 1.