WO2018210279A1

WO2018210279A1 - Use of toll-like receptor agonist combined with immune effector cell

Info

Publication number: WO2018210279A1
Application number: PCT/CN2018/087162
Authority: WO
Inventors: 李宗海; 狄升蒙
Original assignee: 科济生物医药（上海）有限公司; 上海市肿瘤研究所
Priority date: 2017-05-16
Filing date: 2018-05-16
Publication date: 2018-11-22
Also published as: CN108853144A

Abstract

The present invention provides a use of a TOLL-like receptor agonist combined with an immune effector cell in preparing an anti-tumor drug, an anti-pathogen drug, or a drug for immunosuppression. The TOLL-like receptor agonist can enhance the effect of the immune effector cell. The present invention also provides a composition of the TOLL-like receptor agonist and the immune effector cell.

Description

Combination of TOLL-like receptor agonists and immune effector cells

The present application claims the priority of the two patents, which are hereby incorporated by reference.

Technical field

The invention belongs to the field of immunotherapy; relates to the combined use of immune effector cells and TOLL-like receptor agonists with targeted recognition of tumor antigens or pathogen antigens.

Background technique

In recent years, genetically engineered immune cells have been increasingly used in the field of cancer therapy or in the treatment of infectious diseases, such as chimeric antigen receptor-modified T cells (CAR-T cells), chimeric antigen receptor-modified NK cells (CAR-NK cells), TCR-modified T cells (TCR-T), and the like.

However, due to the complexity of the tumor microenvironment, in the treatment of tumors, especially solid tumors, immune effector cells usually do not exert anti-tumor effects in vivo.

Summary of the invention

The present invention aims to provide a use of a combination of a TOLL-like receptor agonist and an immune effector cell for the treatment of tumors, infectious diseases and other immune-related diseases. TOLL-like receptor agonists are capable of increasing the efficacy of immune effector cells.

In a first aspect of the invention, there is provided a composition comprising an immune effector cell and a receptor for recognizing a tumor antigen or a pathogen antigen, the immune effector cell The potentiator is a TOLL-like receptor agonist.

In particular, the present invention provides a composition comprising an immune effector cell having a receptor that targets a tumor antigen or a pathogen antigen, the immune effector cell being associated with expression of a tumor antigen or a pathogen antigen A combination of agents that increase the efficacy of the immune effector cells in the treatment of a subject of the disease, wherein:

(i) the receptor has an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain has an antigen binding domain that binds to a tumor antigen or a pathogen antigen associated with the disease;

(ii) The agent that increases the efficacy of immune effector cells is a TOLL-like receptor agonist.

In a preferred embodiment, the intracellular region of the receptor has a domain involved in eliciting activation of immune cells.

In a preferred embodiment, the TOLL-like receptor agonist is a Toll-like receptor 3 (TLR3) agonist.

In a preferred embodiment, the TLR3 agonist is selected from natural or synthetic double-stranded ribonucleic acids.

In a preferred embodiment, the TLR3 agonist is polyinosinic acid cytidine (poly(I:C)).

In a preferred embodiment, the TLR3 agonist is a mismatched double-stranded ribonucleic acid poly (I: C11-14U), exemplified by poly (I: C12U), poly (I: C13U).

In a preferred embodiment, the recognition of a tumor antigen or a pathogen antigen receptor comprises a chimeric antigen receptor (CAR), a T cell (antigen) receptor (TCR), a T cell fusion protein (TFP), or a T cell. Antigen coupler (TAC).

In a preferred embodiment, the chimeric antigen receptor comprises:

(i) an antibody that specifically binds to the antigen, a transmembrane region of CD28, a costimulatory signal domain of CD28, and a CD3 sputum cytoplasmic signaling domain;

(ii) an antibody that specifically binds to the antigen, a transmembrane region of CD28, a costimulatory signal domain of CD137, and a CD3 purine cytoplasmic signaling domain;

(iii) an antibody that specifically binds to the antigen, a transmembrane region of CD28, a costimulatory signal domain of CD28, a costimulatory signal domain of CD137, and a CD3 sputum cytoplasmic signaling domain.

In a preferred embodiment, the TFP comprises: (a) a TCR subunit comprising a TCR partial extracellular domain, a transmembrane domain, and a TCR intracellular domain, said intracellular structure The domain includes a stimulation signaling domain; (b) an antigen binding domain, the TCR subunit and the antigen binding domain are operably linked, and the extracellular, transmembrane and intracellular signal domains of the TCR subunit are derived CD3 epsilon or CD3 gamma, which integrates into the TCR expressed on T cells.

In a preferred embodiment, the TAC comprises: (a) an extracellular domain comprising: a single chain antibody having an antigen binding domain and binding to CD3; (b) a transmembrane region; c) an intracellular domain that links to protein kinase LCK.

In a preferred embodiment, the immune effector cells are selected from the group consisting of T cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, or bone marrow-derived phagocytic cells.

In a preferred embodiment, the immune cell is a T cell, and the intracellular domain of the receptor is capable of triggering activation of T cells.

In another preferred embodiment, the immune effector cells also express other exogenous proteins. Preferably, the exogenous protein is selected from the group consisting of FLT3L, interferon, interleukin, interleukin receptor, PD-1 inhibition. Agents (such as antibodies to PD-1), PD-L1 inhibitors (such as antibodies to PD-L1, soluble PD-1, or proteins containing soluble PD-1), or a combination thereof.

In another preferred embodiment, the composition further comprises an MDSC inhibitor, and/or an immunological checkpoint inhibitor.

In another preferred embodiment, the MDSC inhibitor is selected from the group consisting of an anti-Gr1 antibody, a cyclooxygenase-2 inhibitor, a prostaglandin-type stem cell factor inhibitor, a macrophage colony-stimulating factor inhibitor, a granule mononuclear A cell colony stimulating factor inhibitor, a vascular endothelial growth factor inhibitor, or a combination thereof.

In another preferred embodiment, the immunological checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor (such as an antibody to PD-1), a PD-L1 inhibitor (such as an antibody to PD-L1), and CTLA-4. An inhibitor (such as a CTLA-4 antibody), or a combination thereof.

In another preferred embodiment, the immune effector cell is a TCR knockdown or PD-1 knockout T cell.

In another preferred embodiment, the expression of the TCR gene or the PD-1 gene in the immune effector cell is silenced.

In another preferred embodiment, the "expression of the gene is silenced" means that the silenced gene is not expressed or underexpressed.

In another preferred embodiment, the "low expression" refers to a ratio of the expression level G1 of the immune effector cell to the corresponding gene expression level G0 of the normal T cell, that is, G1/G0 ≤ 0.5, preferably G1/G0. ≤ 0.3, more preferably ≤ 0.2, more preferably ≤ 0.1, most preferably 0.

In a second aspect of the invention, there is also provided the use of a combination of an immune effector cell and an immune effector cell synergist for the preparation of an antitumor or antipathogenic drug or a drug having low immunity, the immune effector cell having a target To a receptor that recognizes a tumor antigen or a pathogen antigen, the immune effector cell potentiator is a TOLL-like receptor agonist.

In particular, the present invention provides the use of a combination of a TOLL-like receptor agonist and an immune effector cell for the preparation of an anti-tumor drug or an anti-pathogenic drug or an anti-immune drug, the immune effector cell having a targeted recognition tumor antigen or A receptor for a pathogen antigen having an extracellular region, a transmembrane region, and an intracellular region, the extracellular region having an antigen binding domain that binds to the tumor antigen or the pathogen antigen.

In a preferred embodiment, the TOLL-like receptor agonist is a Toll-like receptor 3 (TLR3) agonist, preferably a natural or synthetic double-stranded ribonucleic acid.

In a preferred embodiment, the TLR3 agonist is a mismatched double-stranded ribonucleic acid, exemplified by poly (I: C12U), poly (I: C13U).

In a preferred embodiment, the amount of the mismatched double-stranded ribonucleic acid is sufficient to bind to Toll-like receptor 3 (TLR3) and reduce or eliminate proliferation of the tumor or other transformed cell in the subject.

In a preferred embodiment, the TOLL-like receptor agonist has a synergistic effect on the immune effector cells.

In a preferred embodiment, the TOLL-like receptor agonist and the immune effector cells are administered parenterally, preferably intravenously and intratumorally; thus the TOLL-like receptor agonist The immunological effector cells can be prepared for parenteral administration, preferably intravenous and intratumoral injection.

In a preferred embodiment, the TOLL-like receptor agonist and the immune effector cells may be administered in the same or different manner.

In a preferred embodiment, the method of administration is co-administering the TOLL-like receptor agonist and the immune effector cell, or administering the immune effector cell first, and then administering the TOLL-like receptor agonist; or The TOLL-like receptor agonist is administered and the immune effector cells are administered.

In a preferred embodiment, the method of administration is to first administer the immune effector cells and then to the TOLL-like receptor agonist.

In a preferred embodiment, the tumor antigen is selected from the group consisting of EGFRvIII, GPC3, CLD18A2, CD19, CD20, CD22, CD30, WT1, CLDN6, MUC1, BCMA, IL-11Ra, IL-13Ra, FAP.

In a preferred embodiment, the TOLL-like receptor agonist can be administered in an amount from 1 mg/kg to 4 mg/kg, preferably from 2 mg/kg to 3 mg/kg, more preferably from 2.08 mg/kg to 2.77 mg/ Kg.

In a preferred embodiment, the TOLL-like receptor agonist is administered in a number of 2 to 4 times, preferably 2-3 times.

In a preferred embodiment, the immunotherapeutic cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a mast cell or a bone marrow-derived phagocytic cell, preferably a T cell.

In a preferred embodiment, the immune effector cell is a T cell, and the intracellular domain of the receptor is capable of triggering activation of T cells.

In a preferred embodiment, the immune effector cells are selected from the group consisting of CAR-expressing T cells, TCR-expressing T cells, CAR-expressing NK cells, TFP-expressing T cells, TFP-expressing NK cells, TAC-expressing T cells. NK cells expressing TAC.

In a preferred embodiment, the chimeric antigen receptor (CAR) comprises:

In a preferred embodiment, the immune effector cells are also expressed with other proteins, preferably, antibodies such as PD1, PDL1, type I interferons, and the like.

In another preferred embodiment, the medicament further comprises an MDSC inhibitor or an immunological checkpoint inhibitor.

In a third aspect of the invention, there is also provided the use of a combination of an immune effector cell, an immune effector cell synergist and a lymphocyte scavenger for the preparation of a medicament against or against a pathogen or against immunity.

In a preferred embodiment, the lymphocyte scavenger is administered prior to administering the immune effector cells and the immune effector cell potentiator to a subject in need thereof.

In a preferred embodiment, the immune effector cell potentiator is a TOLL-like receptor agonist.

Specifically, the present invention also provides the use of a combination of an immune effector cell, a TOLL-like receptor agonist, and a lymphocyte scavenger in the preparation of an antitumor drug or an antipathogenic drug or an anti-immune drug, the immune effector cell a receptor having a target for recognizing a tumor antigen or a pathogen antigen, the receptor having an extracellular region, a transmembrane region, and an intracellular region, the extracellular region having an antigen binding domain that binds to the tumor antigen or the pathogen antigen .

In a preferred embodiment, the lymphocyte scavenger is administered prior to administration of the TOLL-like receptor agonist and immune effector cells.

In a preferred embodiment, the lymphocyte scavenger comprises a drug that clears lymphocytes or is subjected to a radiation treatment. In a preferred embodiment, the drug that clears lymphocytes includes, but is not limited to, fludarabine and/or cyclophosphamide.

In a preferred embodiment, administering a TOLL-like receptor agonist and an immune effector cell to a subject in need thereof comprises simultaneous or sequential administration; for example, first administering a TOLL-like receptor agonist to the immune effector cell, or The immune effector cells are administered with a TOLL-like receptor agonist; more preferably, the immune effector cells are first administered with a TOLL-like receptor agonist.

In a fourth aspect of the invention, there is also provided a method of treating a tumor or pathogen infection or immunodeficiency, the method comprising: administering a combination of an immune effector cell and an immune effector cell synergist to a subject in need thereof;

A combination of an immune effector cell, an immune effector cell synergist, and a lymphocyte scavenger is administered to a subject in need thereof, wherein the lymphocyte is administered to the subject in need thereof before administration of the immune effector cell and the immune effector cell synergist Cell scavenger.

In particular, the invention provides a method of treating a tumor or pathogen infection or low immunity, the method comprising:

A combination of an immune effector cell and a TOLL-like receptor agonist having a receptor targeting a tumor antigen or a pathogen antigen having an extracellular region, a transmembrane region, and the like is administered to a subject in need thereof An intracellular region having an antigen binding domain that binds to said tumor antigen or a pathogen antigen; or

A combination of immune effector cells, a TOLL-like receptor agonist, and a lymphocyte scavenger is administered to a subject in need thereof.

A fifth aspect of the invention provides a composition product, comprising:

(i) a first pharmaceutical composition comprising at least one immune effector cell potentiator comprising a TOLL-like receptor agonist; and a pharmaceutically acceptable carrier ;

(ii) a second pharmaceutical composition comprising an immune effector cell having a receptor that targets a tumor antigen or a pathogen antigen; and a pharmaceutically acceptable carrier;

(iii) a third pharmaceutical composition comprising an MDSC inhibitor or an immunological checkpoint inhibitor; and a pharmaceutically acceptable carrier.

In another preferred embodiment, the first pharmaceutical composition, the second pharmaceutical composition, and the third pharmaceutical composition are mixed or independently present.

In another preferred embodiment, the total content of the first pharmaceutical composition, the second pharmaceutical composition and the third pharmaceutical composition is from 70 to 100% by weight, preferably from 80 to 100% by weight, more preferably 90%. ～100% by weight, based on the total weight of the product of the composition.

In another preferred embodiment, the immune effector cells are administered in an amount of from 1 × 10 ^{6 to} 1 × 10 ¹² cells/kg, more preferably from 1 × 10 ^{7 to} 1 × 10 ¹⁰ cells/kg.

In another preferred embodiment, the composition product is a pharmaceutical composition for treating a tumor or pathogen infection or low immunity.

In another preferred embodiment, the dosage form of the composition includes an injectable dosage form, a topical pharmaceutical dosage form, and an oral dosage form.

In another preferred embodiment, the composition can be administered by subcutaneous injection, intravenous injection, intramuscular injection.

In another preferred embodiment, the oral dosage form comprises a tablet, a capsule, a film, and a granule.

In another preferred embodiment, the dosage form of the composition comprises a sustained release dosage form, and a non-slow release dosage form.

A sixth aspect of the invention provides the use of a composition product according to the fifth aspect of the invention for the preparation of an antitumor or antipathogenic drug or an anti-immune drug.

In another preferred embodiment, in the composition product, the immune effector cell synergist may be administered in an amount of 1 mg/kg to 4 mg/kg, preferably 2 mg/kg to 3 mg/kg, more preferably selected from the group consisting of 2.08 mg/kg-2.77 mg/kg.

In another preferred embodiment, in the composition product, the immune effector cells are administered in an amount of 1 × 10 ⁶ - 1 × 10 ¹² cells/kg, more preferably, 1 × 10 ⁷ - 1 × 10 ¹⁰ Cells / kg.

In another preferred embodiment, the MDSC inhibitor is administered in an amount of from 2 mg/kg to 5 mg/kg, preferably from 2 mg/kg to 4 mg/kg, in the composition product.

In another preferred embodiment, in the composition product, the number of administrations of the immune effector cell synergist is 2 to 4 times, preferably 2-3 times.

In another preferred embodiment, in the composition product, the number of administration of the immune effector cells is 2 to 4 times, preferably 2-3 times.

In another preferred embodiment, in the composition product, the number of administrations of the MDSC inhibitor is 2 to 4 times, preferably 2-3 times.

A seventh aspect of the present invention provides a method for improving the viability of an immune effector cell, comprising the steps of:

Administering (i) an immune effector cell; (ii) an immune effector cell potentiator; and/or (iii) an optional MDSC inhibitor or immunological checkpoint inhibitor, wherein the immune effector cell has a target A receptor that recognizes a tumor antigen or a pathogen antigen, the immune effector cell potentiator being a TOLL-like receptor agonist.

In another preferred embodiment, the subject comprises a human or a non-human mammal.

In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, monkeys.

In another preferred embodiment, the component (i), component (ii), component (iii) are applied simultaneously or sequentially.

In another preferred embodiment, (i) immune effector cells; (ii) immune effector cell potentiators; and (iii) optional MDSC inhibitors or immunological checkpoint inhibitors are administered as compared to administration of immune effector cells alone. The tumor inhibition rate is increased by ≥ 20%, preferably ≥ 30%.

In another preferred embodiment, (i) immune effector cells; (ii) immune effector cell potentiators; and (iii) optional MDSC inhibitors or immunological checkpoint inhibitors are administered as compared to administration of immune effector cells alone. The level of interferon release is increased by ≥ 20%, preferably ≥ 30%, more preferably ≥ 50%.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 is a schematic representation of the structure of the recombinant retroviral vector EGFRvIII-28Z.

Figure 2 is the efficiency of retrovirus-infected mouse T lymphocytes.

Figure 3 is a graph showing the in vitro killing function of EGFRvIII-28Z CAR-T cells.

Figure 4A is a graph showing the anti-tumor effect of EGFRvIII-28Z CAR-T cells and poly I:C in a mouse breast cancer model; Figure 4B shows EGFRvIII-28Z CAR-T cells and poly I:C in a mouse breast cancer model. Tumor weight after end of anti-tumor experiment in vivo; Figure 4C shows IFNγ level in peripheral blood of EGFRvIII-28Z CAR-T cells and poly I:C in vivo in anti-tumor experiments in mouse breast cancer model; Figure 4D shows anti-tumor The end of the experiment was the number of CAR-T cell copies in the spleen and tumor of the mouse breast cancer model.

Figure 5A is an in vivo anti-tumor effect of EGFRvIII-28Z CAR-T cells and poly I:C in a mouse colon cancer model; Figure 5B shows EGFRvIII-28Z CAR-T cells and poly I:C in a mouse colon cancer model Tumor weight after the end of the anti-tumor experiment; Figure 5C shows the IFNγ level of peripheral blood after the end of the anti-tumor experiment of EGFRvIII-28Z CAR-T cells and poly I:C in the mouse colon cancer model; Figure 5D shows the anti-tumor experiment The CAR-T cell copy number in the spleen and tumor of the mouse colon cancer model was terminated.

Figure 6 is a plasmid map of MSCV-EGFRvIII-28Z-FLT3L.

Figure 7 shows the effect of EGFRvIII-28Z-FLT3L CAR-T+poly I:C cells in a mouse colon cancer model.

Figure 8A shows the growth of colon cancer xenografts after type I interferon is blocked; Figure 8B shows the tumor weight of colon cancer xenografts after type I interferon is blocked.

Figure 9A shows the growth of breast cancer xenografts after type I interferon is blocked; Figure 9B shows the tumor weight of breast cancer xenografts after type I interferon is blocked.

Figure 10 shows a comparison of the effects of MDSC inhibitors, CAR T cells, and polyI:C on tumor volume over time in a mouse tumor model (Figure 10A), tumor body weight (Figure 10B), and tumor photo comparison (Figure 10C). ).

detailed description

To overcome the deficiencies in the prior art, the present inventors have conducted intensive studies providing (a) TLR agonists and (b) chimeric antigen receptors and (c) optional MDSC inhibitors and/or immunological checkpoints. The combination of inhibitors in the treatment of tumors, infectious diseases and other immune-related diseases has significantly improved immunity to the body and the ability to kill tumors or pathogens.

the term

As used herein, the terms "the agent that increases the efficacy of immune effector cells", "immune effector cell synergist" are used interchangeably and refer to a drug or formulation that enhances the efficacy of immune effector cells.

The technical terms used herein have the same or similar meanings as are conventionally understood by those skilled in the art. To facilitate an understanding of the invention, some terms are defined as follows.

When referring to measurable values such as the amount administered, etc., including ±20% of the specified value, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, Or in some cases ± 0.1% change, so such a change is suitable for carrying out the disclosed method.

The term "immune effector cells (also referred to herein as immune cells)" refers to cells involved in an immune response, for example, to promote an immune effector response. Examples of immune effector cells include T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytic cells. For example, the T cells may be T cells directly derived from peripheral blood, or may be subtypes of T cells, such as CD8+ T cells, CD4+ T cells, α/β T cells, γ/δT cells, and the like.

As used herein, "chimeric antigen receptor" or "CAR" refers to a group of polypeptides that, when administered in an immune effector cell, provide said cells with specificity for a target cell, typically a cancer cell, and have Intracellular signal production. CAR typically includes at least one extracellular antigen binding domain, a transmembrane domain (also known as a transmembrane domain), and a cytoplasmic signaling domain (also referred to herein as an "intracellular region"), which includes a stimulus derived from the definitions below. Functional signaling domains of sex molecules and/or costimulatory molecules. In certain aspects, the polypeptide groups are contiguous with each other. A polypeptide group includes a dimerization switch that can couple the polypeptides to each other in the presence of a dimerization molecule, for example, an antigen binding domain can be coupled to an intracellular signaling domain. In one aspect, the stimulatory molecule is an ζ chain that binds to a T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one aspect, the costimulatory molecule is selected from a costimulatory molecule described herein, such as 4-1BB (ie, CD137), CD27, and/or CD28. In one aspect, a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and a functional signaling domain comprising a costimulatory molecule and a functionality derived from a stimulatory molecule The intracellular signaling domain of the signaling domain. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and two functional signaling comprising one or more costimulatory molecules.

The term "signaling domain" refers to a functional portion of a protein that functions by transmitting information within a cell, either by generating a second messenger or by acting in response to such a messenger effector via a determined signal The conduction pathway regulates the activity of the cells.

The "T cell receptor (TCR)" is a characteristic marker on the surface of all T cells, and binds to CD3 by a non-covalent bond to form a TCR-CD3 complex. The role of TCR is to recognize antigens. TCR is a heterodimer composed of two different peptide chains, consisting of two peptide chains, α and β. Each peptide chain can be further divided into variable region (V region), constant region (C region), transmembrane. The region and the cytoplasmic region are several parts; it is characterized by a short cytoplasmic region. The TCR molecule belongs to the immunoglobulin superfamily, and its antigen specificity exists in the V region; the V region (Vα, Vβ) has three hypervariable regions CDR1, CDR2, and CDR3, among which the CDR3 mutation is the largest, which directly determines the TCR antigen. Binding specificity. When the TCR recognizes the MHC-antigen peptide complex, CDR1, CDR2 recognizes and binds to the side wall of the MHC molecule antigen binding groove, while CDR3 binds directly to the antigen peptide. TCR is divided into two categories: TCR1 and TCR2; TCR1 consists of two chains of γ and δ, and TCR2 consists of two chains of α and β. Any T cell expresses only one of TCR2 and TCR1.

The "T CELL FUSION PROTEIN (TFP)", P includes various polypeptide-derived recombinant polypeptides constituting a TCR, which can i) bind to a surface antigen on a target cell, and ii) and intact Other polypeptide interactions of the TCR complex are usually localized on the surface of T cells. TFP consists of an antigen binding domain consisting of a TCR subunit and a human or humanized antibody domain, wherein the TCR subunit comprises at least a portion of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular domain. a stimulating domain of an internal signal domain; the TCR TCR subunit is operably linked to the antibody domain, wherein the extracellular, transmembrane, and intracellular signal domains of the TCR subunit are derived from CD3 epsilon or CD3 gamma, and the TFP integration TCR expressed on T cells.

By "effective link" is meant a functional link between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of a heterologous nucleic acid sequence. For example, when the first nucleic acid sequence is located in a functional regulatory region of the second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if a promoter is required to affect transcription and expression of a coding sequence, then this promoter can be operably linked to the coding sequence. Linkable DNA sequences can be contiguous with each other, for example, two protein coding regions can be placed in the same reading frame.

The "T CELL ANTIGEN COUPLER (TAC)" includes three functional domains: a tumor targeting domain, including a single-chain antibody, a designed ankyrin repeat protein (DARPin). Or other targeting group 2; is an extracellular domain domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor closer to other TCR receptors; the transmembrane region and the intracellular region of the CD4 co-receptor, wherein The intracellular domain is linked to the protein kinase LCK, which catalyzes the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as an initial step in T cell activation.

The "Toll-like receptor" (TLR) refers to a member of a family of receptors that bind to a pathogen-associated molecular pattern (PAMP) and promote an immune response in a mammal. Ten mammalian TLRs are known, such as TLR1-10. The term "toll-like receptor agonist" (TLR agonist) refers to a molecule that interacts with a TLR and stimulates the activity of the receptor. A synthetic TLR agonist is a compound designed to interact with a TLR and stimulate the activity of the receptor. Examples of TLR agonists include TLR-7 agonists, TLR-3 agonists or TLR-9 agonists.

The "Toll-like receptor 3 (TLR3) is a member of the Toll-like receptor family, which is capable of specifically recognizing the double-stranded RNA of the virus and its analog Poly(I:C). The dsRNA has the ability to replicate in the virus. Two complementary strands of RNA formed during the cycle. Upon recognition, TLR3 induces activation of transcription factors such as NF-κB and interferon regulatory factor 3 (IRF3) to increase the type I signaling to other cells to increase their antiviral defense. Interferon production. Studies have shown that TLR3 can be expressed in a variety of immune cells including NK cells, macrophages, dendritic cells and T cells, while Poly(I:C) acts as an agonist of TLR3. It promotes the growth and antigen presentation of B cells. In addition, agonists of TLR3 can also promote the maturation of dendritic cells (DC) cells, which are dendritic cell maturation agents.

The term "poly(I:C))" (in this document, has the same meaning as Poly I: C, and pIC in the drawing also refers to Poly (I: C)), Chinese is a polymuscular liver, an analog of synthetic dsRNA, a double-stranded RNA molecule having a MW distribution of up to, for example, 3,600,000 Daltons. Poly(I:C), as an agonist of TLR3, promotes the growth and antigen presentation of B cells. Furthermore, in the absence of specific antigen stimulation, the TLR3 agonist Poly(I:C) can directly induce B cells to produce IgG1 kappa antibodies. Its anti-tumor effect mainly depends on CD8+ T cells and NK cells, and can coordinate the induction of DC cells, macrophages/neutrophils, NK cells and T cells and other immune cells to secrete type I interferon to promote anti-resistant Tumor effect.

The term "dendritic cell (DC)" is derived from progenitor cells in the bone marrow, which migrate as immature cells to peripheral tissues where they endocytose antigens and undergo complex maturation processes. Antigens are endocytosed via a number of surface molecules, including complement receptors (eg, CD11c/CD18) and endocytic receptors (eg, DEC-205, DC-SIGN, and Toll-like receptors). Immature DCs also receive a "danger signal" in the form of a pathogen-associated molecule such as bacterial cell wall lipopolysaccharide (LPS) during antigen acquisition, or inflammatory stimulation via cytokines such as IFN-[gamma]. The DC then migrates to the secondary lymphoid organs and matures into competent APCs. Receptors such as CD11c/CD18, DEC-205, DC-SIGN, and Toll-like receptors play key roles in Ag capture and presentation, and are predominantly expressed on DCs.

The term "dendritic cell maturation agent" refers to a substance that induces maturation of immature DC cells to competent APC, including a group selected from the group consisting of: a STING agonist; a TLR agonist such as heat inactivated or formalin. Treated BCG, preferably cell wall component of BCG, BCG-derived lip arabinose or BCG component; lipopolysaccharide (LPS) derived from E. coli; Picibanil (OK432) or inactivated Gram-positive or Gram-negative microorganism; imidazoquinoline compound, preferably imidazoquinolin-4-amine compound, especially 4-amino-2-ethoxymethyl-x-dimethyl-1H-imidazole [ 4,5-c]quinoline-1-ethanol or 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine, or a derivative thereof; synthetic double strand Polyribonucleotides, preferably poly I:C; natural double-stranded RNA or RNA viruses or RNA fragments, or synthetic analogs, or synthetic or natural nucleic acid molecules comprising unmethylated CpG motifs; Combination of cytokines, preferably tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), IL-1, IL-6, IL-12 or prostaglandin E6, CD40L, preferably recombinant CD40L or CD4 a fusion protein in the 0L domain; or a genetically engineered primary cell or cell line that expresses CD40L, or activated T lymphocytes such as T lymphocytes that physiologically upregulate CD40L expression; 2-Hydroxypropyl-β-cyclodextrin (HP-β) -CD) [Reference: Front Immunol. 2016 Oct 20; 7: 435. eCollection 2016], galectin (Gal)-9 [Reference J Immunol. 2005 Sep 1; 175(5): 2974-81].

The term "tumor" refers to a disease characterized by pathological hyperplasia of cells or tissues, and its subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, and does not induce or inhibit normal cell proliferation. A tumor can affect a variety of cells, tissues or organs including, but not limited to, selected from the group consisting of bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, Nerve tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, vaginal organs, or tissue or corresponding cells. Tumors include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). The tumor of the present invention may include, but is not limited to, leukemia (such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute granulocyte-monocytic leukemia, Acute monocytic leukemia, acute leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia Disease, heavy chain disease, solid tumors such as sarcoma and cancer (such as fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial sarcoma, lymphangisarcoma, angiosarcoma, lymphatic endothelial sarcoma, synovial vioma , mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland cancer, sebaceous gland cancer, papillary Cancer, papillary adenocarcinoma, cancer, bronchial carcinoma, medullary carcinoma, renal cell carcinoma, liver cancer, Nile tube cancer, choriocarcinoma, fine Tumor, embryonic carcinoma, nephroblastoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, Ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannomas, meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder carcinoma , kidney cancer, multiple myeloma. Preferably, the "tumor" includes, but is not limited to, pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, Kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma.

The term "tumor antigen" refers to a molecule (usually a protein, carbohydrate or lipid) that is expressed on the surface of a tumor cell, either in whole or in the form of a fragment (eg, MHC/peptide), and which is used for pharmacological treatment. The agent preferentially targets tumor cells. The tumor antigen may be a marker expressed by normal cells and cancer cells, such as a lineage marker such as CD19 on B cells; the tumor antigen may also be a cell surface molecule that is overexpressed in comparison with normal cells in cancer cells, for example, Normal cells are overexpressed by 1x overexpression, 2x overexpression, 3x or more; tumor antigens can also be cell surface molecules that are improperly synthesized in cancer cells, for example, molecules expressed on normal cells Than a molecule containing a deletion, addition or mutation; the tumor antigen can also be specifically expressed on the cell surface of the cancer cell, either in whole or in fragment form (eg, MHC/peptide), rather than on the surface of normal cells or expression. In certain embodiments, a CAR of the invention comprises a CAR comprising an antigen binding domain (eg, an antibody or antibody fragment) that binds to an MHC presenting peptide. Typically, peptides derived from endogenous proteins fill the pockets of class I molecules of the major histocompatibility complex (MHC) and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes. The MHC class I complex is constitutively expressed by all nucleated cells. In tumors, virus-specific and/or tumor-specific peptide/MHC complexes represent a unique type of cell surface target for immunotherapy.

Tumor antigens include, but are not limited to, thyroid stimulating hormone receptor (TSHR); CD171; CS-1 (CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1) Ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); Tn antigen (Tn Ag); CD19; CD20; CD 22; CD 30 CD 70; CD 138; CD 33; CD 44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit alpha (IL-13Ra); 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; Lysin; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4) , EGFRvII); neural cell adhesion molecule (NCAM) Carbonic anhydrase IX (CAIX); proteasome (Prosome, Macropain) subunit, beta type, 9 (LMP2); ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis Molecular (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer; TGS5; high molecular weight melanoma associated antigen (HMWMAA); o-acetyl GD2 nerve Glucosamine (OAcGD2); folate receptor; tumor vascular endothelial marker 1 (TEM1/CD248); tumor vascular endothelial marker 7 associated (TEM7R); claudin 6 (CLDN6), Claudin 18.2, Claudin 18.1; ASGPR1; CDH16 ; 5T4; 8H9; αvβ6 integrin; B cell mature antigen (BCMA); CA9; kappa light chain; CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA- A2; MAGE A1, MAGE3; KDR; Lambda; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; sputum protein; carcinoembryonic variant of tumor necrosis; G protein coupling Receptor C class 5, member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placental specific 1 (PLAC1) Hexosaccharide moiety of globoH glycoceramide (GloboH); breast differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (PANX3); G protein coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCRγ alternate reading frame protein (TARP); nephroblastoma Protein (WT1); ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin binding cell surface receptor 2 (Tie2) Melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-associated antigen 1; p53 mutant; human telomerase reverse transcriptase ( hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myeloma virus oncogene neuroblastoma-derived homolog (MYCN); Ras Member family C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen 3 (SART3) recognized by T cells; paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase-anchored protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); CD79a CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecular-like family member f ( CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); containing EGF-like module mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1). In a specific embodiment, the tumor antigen can be, but is not limited to, EGFRvIII, GPC3, CLD18A2, CD19, CD20, CD22, CD30, WT1, CLDN6, MUC1, BCMA, IL-11Ra, IL-13Ra, FAP.

The term "pathogen" refers to a protozoan capable of causing a disease, including: a virus, a bacterium, a fungus or a parasite. The "viral antigen" refers to a polypeptide expressed by a virus capable of inducing an immune response.

As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds an antigen. Antibodies can be polyclonal or monoclonal, multi-stranded or single-stranded, or intact immunoglobulins, and can be derived from natural or recombinant sources. The antibody can be a tetramer of immunoglobulin molecules.

The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (eg, by binding, steric hindrance, stabilization/destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, Linear antibodies, single domain antibodies such as sdAb (VL or VH), camelid VHH domain, multispecific antibodies formed by antibody fragments (eg, bivalent fragments comprising two Fab fragments joined by a disulfide bond in the hinge region) and An isolated CDR or other epitope binding fragment of an antibody. Antigen-binding fragments can also be incorporated into single domain antibodies, maximal antibodies, minibodies, Nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, Nature). Biotechnology 23: 1126-1136, 2005). The antigen-binding fragment can also be grafted to a polypeptide-based scaffold, such as type III fibronectin (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies).

The term "scFv" refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein said light and heavy chain variable regions are contiguous ( For example, via a synthetic linker such as a short flexible polypeptide linker), and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv can have the VL and VH variable regions in any order (eg, relative to the N-terminus and C-terminus of the polypeptide), and the scFv can include a VL-linker-VH or A VH-linker-VL can be included.

As used herein, the term "binding domain" or "antibody molecule" refers to a protein, eg, an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term "binding domain" or "antibody molecule" includes antibodies and antibody fragments. The antibody molecule may be a multispecific antibody molecule, for example, it comprises a plurality of immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence has binding specificity for the first epitope and a plurality of The di-immunoglobulin variable domain sequence has binding specificity for the second epitope. Multispecific antibody molecules can also be bispecific antibody molecules. Bispecific antibodies have specificity for no more than two antigens. A bispecific antibody molecule is characterized by having a binding specific bispecific antibody molecule to a first epitope characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and for a second epitope A second immunoglobulin variable domain sequence having binding specificity.

The term "antigen" or "Ag" refers to a molecule that elicits an immune response. The immune response can involve activation of the antibody-producing or cells with specific immunity or both. Those skilled in the art will appreciate that any macromolecule comprising virtually all proteins or peptides can serve as an antigen. In addition, the antigen can be derived from recombinant or genomic DNA. As the term is used herein, one of ordinary skill in the art will appreciate any DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response, thus encoding an "antigen." Furthermore, it will be understood by those skilled in the art that the antigen need not be encoded only by the full length nucleotide sequence of the gene. It will be apparent that the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in different combinations to encode a polypeptide that elicits a desired immune response. Moreover, those skilled in the art will appreciate that the antigen does not need to be encoded by a "gene" at all. It will be apparent that the antigen may be produced synthetically, or may be derived from a biological sample, or may be a macromolecule other than a polypeptide. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells or liquids with other biological components.

The term "anticancer effect" refers to a biological effect that can be manifested by a variety of means including, but not limited to, for example, a reduction in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, a decrease in cancer cell proliferation, and a cancer cell survival rate. Amelioration of various physiological symptoms associated with or associated with cancerous conditions. "Anticancer effects" also prevent the first appearance of cancer by peptides, polynucleotides, cells and antibodies. The term "anticancer effect" refers to a biological effect that can be manifested by various means including, but not limited to, for example, a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.

As the term is used herein, "derived from" means the relationship between the first and second molecules. It generally refers to the structural similarity between the first molecule and the second molecule and does not imply or include limitations on the process or source of the first molecule derived from the second molecule. For example, in the case of an intracellular signaling domain derived from a CD3 purine molecule, the intracellular signaling domain maintains a sufficient CD3ζ structure such that it has the desired function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include limitations on the specific process by which the intracellular signaling domain is produced. For example, it does not imply that in order to provide an intracellular signaling domain, it is necessary to start with a CD3ζ sequence and to delete an unwanted sequence, or to apply Mutation to obtain an intracellular signaling domain.

The phrase "disease associated with expression of a tumor antigen as described herein" includes, but is not limited to, a disease associated with expression of a tumor antigen as described herein or a condition associated with a cell expressing a tumor antigen as described herein, Included, for example, are proliferative diseases such as cancer or malignancy or precancerous conditions, such as myelodysplasia, myelodysplastic syndrome or pre-leukemia; or non-cancer-related indications associated with cells expressing tumor antigens as described herein. . In one aspect, the cancer associated with expression of a tumor antigen described herein is a blood cancer. In one aspect, the cancer associated with expression of a tumor antigen described herein is a solid cancer. Other diseases associated with the expression of tumor antigens described herein include, but are not limited to, for example, atypical and/or non-classical cancers, malignant tumors, precancerous conditions or hyperplasia associated with expression of a tumor antigen as described herein. Sexual disease. Non-cancer related indications associated with expression of a tumor antigen as described herein include, but are not limited to, for example, autoimmune diseases (eg, lupus), inflammatory conditions (allergy and asthma), and transplantation. Tumor antigen expression can be expressed by cells, or expressed at any time, encoding the mRNA of the tumor antigen. Tumor antigen-expressing cells can produce tumor antigen proteins (e.g., wild-type or mutant), and the tumor antigen proteins can be present at normal or reduced levels. Tumor antigen-expressing cells can produce detectable levels of tumor antigen protein at one point and subsequently produce a substantially undetectable tumor antigen protein.

The term "stimulation" refers to the binding of a stimulatory molecule (eg, a TCR/CD3 complex or CAR) to its cognate ligand (or a tumor antigen in the case of a CAR), thereby mediating signal transduction events (eg, However, it is not limited to the initial response induced via signal transduction of the TCR/CD3 complex or via signal transduction of a suitable NK receptor or CAR signaling domain. Stimulation can mediate altered expression of certain molecules.

The term "irritating molecule" refers to a molecule expressed by an immune cell that provides a cytoplasmic signaling sequence that modulates the activation of immune cells for at least some aspects of the immune cell signaling pathway in an irritating manner. In one aspect, the signal is a primary signal initiated by binding of, for example, a TCR/CD3 complex to a peptide-loaded MHC molecule, and which results in a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. The primary cytoplasmic signaling sequence (also referred to as "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM-containing cytoplasmic signaling sequences specifically for use in the present invention include, but are not limited to, those derived from CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcEpsilon R1b), CD3γ, CD3δ, CD3ε , CD79a, CD79b, DAP10 and DAP12. In a specific CAR of the invention, the intracellular signaling domain in any one or more of the CARs of the invention comprises an intracellular signaling sequence, such as a CD3-ζ primary Signaling sequence. In the specific CAR of the present invention, the primary signaling sequence of CD3-ζ is an equivalent residue derived from a human or non-human species such as mouse, rodent, monkey, donkey, etc. In sex CAR, the primary signaling sequence of CD3-ζ is an equivalent residue from a human or non-human species such as mouse, rodent, monkey, donkey, and the like.

The term "antigen presenting cell" or "APC" refers to an immune system cell, such as a helper cell (eg, B-cell, dendritic cell), which presents a foreign antigen complexed with a major histocompatibility complex (MHC) on its surface. Wait). T-cells can recognize these complexes using their T-cell receptor (TCR). APC processes antigen and presents it to T-cells.

The term "intracellular signaling domain" refers to an intracellular portion of a molecule. The intracellular signaling domain produces a signal that promotes the immune effector function of cells containing CAR, such as CART cells. Examples of immune effector functions in, for example, CART cells include cell lytic activity and helper activity, including secretion of cytokines. In one embodiment, the intracellular signaling domain can comprise a first order intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen dependent stimulation. In one embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals or antigen independent stimuli. For example, in the case of CART, the primary intracellular signaling domain may comprise a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain may comprise a cytoplasmic sequence from a co-receptor or a costimulatory molecule.

The first-level intracellular signaling domain can include a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAM include, but are not limited to, those derived from: CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcEpsilon R1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10 And DAP12.

The term "ζ" or alternatively "ζ chain", "CD3-ζ" or "TCRζ" is defined as a protein as provided by GenBan Acc. No. BAG36664.1 or from a non-human species (eg mouse, rodent, Equivalent residues of monkeys, baboons, etc., and "ζ stimulating domain" or alternatively "CD3-ζ stimulating domain" or "TCR-ζ stimulating domain" are defined as amino acid residues from the cytoplasmic domain of the ζ chain A base or a functional derivative thereof sufficient to functionally deliver the initiation signal required for T cell activation. In one aspect, the cytoplasmic domain of sputum comprises residues 52 to 164 of GenBank Acc. No. BAG36664.1 or from a non-human species (eg, mouse, rodent, monkey, ape, etc.) as a functional homolog thereof Equivalent residue of ).

The term "co-stimulatory molecule" refers to a homologous binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of a T cell, such as, but not limited to, proliferation. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that promotes an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) and 4- 1BB (CD137). Further examples of such costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1 CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and ligands that specifically bind to CD83.

The costimulatory intracellular signaling domain can be an intracellular portion of a costimulatory molecule. Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), and NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, antigen-related antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and ligands that specifically bind to CD83.

The intracellular signaling domain may comprise part or all of the native intracellular signaling domain, or a functional fragment or derivative thereof, of all cells within the molecule.

The term "4-1BB" refers to a member of the TNFR superfamily having an amino acid sequence as provided by GenBank Acc. No. AAA62478.2, or an equivalent residue from a non-human species such as a mouse, a rodent, a monkey, a donkey, or the like. And "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Acc. No.. AAA62478.2, or equivalents from non-human species such as mice, rodents, monkeys, baboons, etc. Residues. In one aspect, the "4-1BB costimulatory domain" is an equivalent residue from a human or from a non-human species such as a mouse, rodent, monkey, donkey, and the like.

The term "encoding" refers to the intrinsic property of a particular sequence of a nucleotide in a polynucleotide, such as a gene, cDNA, or mRNA, as a template for the synthesis of other polymers and macromolecules in a biological process, the polymer and macromolecules. A defined nucleotide sequence (eg, rRNA, tRNA, and mRNA) or a defined amino acid sequence and biological properties derived therefrom. Thus, if transcription and translation of mRNA corresponding to the gene produces a protein in a cell or other biological system, the gene, cDNA or RNA encodes the protein. A non-coding strand whose nucleotide sequence is identical to the mRNA sequence and which is usually provided in the sequence listing and a non-coding strand which is used as a template for the transcription gene or cDNA may be referred to as a protein or other encoding the gene or cDNA. product

Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate forms of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA may also include an intron to the extent that the nucleotide sequence encoding the protein may contain introns in some forms.

The term "effective amount" or "therapeutically effective amount" is used interchangeably herein and refers to an amount of a compound, formulation, substance or composition effective to achieve a particular biological result as described herein.

The term "expression" refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

The term "transfer vector" refers to a composition of matter that includes an isolated nucleic acid and that can be used to deliver the isolated nucleic acid to the interior of a cell. A wide variety of vectors are known in the art and include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses. Thus, the term "transfer vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids incorporated into recombinant polynucleotides, plasmids (eg, naked or contained in liposomes), and viruses (eg, lentiviruses, retroviruses, glands) Virus and adeno-associated virus).

In the context of the present invention, the abbreviations of the nucleobases normally present below are used. "A" means adenosine, "C" means cytosine, "G" means guanosine, "T" means thymidine, and "U" means uridine.

The "parenteral" administration of the term immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or intrasternal injection, intratumoral or infusion techniques. The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. Unless explicitly defined, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, and well-defined sequences. . In particular, degenerate codon substitutions can be made by generating sequences in which three positions of one or more selected (or all) codons are mixed bases and/or deoxyinosine residues Substituent substitution (Batzer et al, Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al, J. Biol. Chem. 260: 2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8: 91-98 ( 1994)).

The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by a peptide bond. The protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can include the sequence of the protein or peptide. A polypeptide includes any peptide or protein comprising two or more amino acids that are bonded to each other by a peptide bond. As used herein, the term refers to short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (which are also commonly referred to in the art as proteins, which are present in many Type). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, or a combination thereof.

As used herein, the term "treat, treatment, treating" refers to slowing or ameliorating a proliferative disorder by administration of one or more therapies (eg, one or more therapeutic agents, such as the CAR of the invention). The progression, severity and/or duration, or amelioration of one or more symptoms of the proliferative disorder (preferably one or more discernible symptoms). The term "treatment" can also refer to at least one measurable physical parameter, such as tumor growth, that ameliorates a proliferative disorder, and is not necessarily discernible by the patient. The term "treating" can also mean inhibiting the progression of a proliferative disorder physically, for example, by stabilizing a discernible symptom, by physiological means, for example, by stabilizing physical parameters, or both. The term "treating" can also mean reducing or stabilizing tumor size or cancer cell count.

The term "signal transduction pathway" refers to the biochemical relationship between a variety of signal transduction molecules that function in transducing a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecular and molecular complexes that are capable of receiving signals and transmitting signals across the cell membrane.

The term "subject" is meant to include living organisms (eg, mammals, humans) in which an immune response can be elicited.

The term "substantially purified" cells refers to cells that are substantially free of other cell types. A substantially purified cell refers to a cell that has been isolated from other cell types that are normally associated with its naturally occurring state. In some cases, a substantially purified population of cells refers to a homologous population of cells. In other instances, the term refers only to cells that are isolated from cells with which they are naturally associated in their natural state. In certain aspects, the cell is cultured in vitro. In other aspects, the cells are not cultured in vitro.

As used herein, the term "treatment" refers to treatment. The therapeutic effect is obtained by alleviating, inhibiting, alleviating or eradicating the disease state.

As used herein, the term "prevention" refers to the prophylactic or protective treatment of a disease or condition.

The term "transfected" or "transformed" or "transduced" refers to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. "Transfected" or "transformed" or "transduced" refers to the process by which an exogenous nucleic acid is transferred or introduced into a host cell. "Transfected" or "transformed" or "transduced" cells A cell that has been transfected, transformed or transduced with an exogenous nucleic acid, including cells of the primary subject and progeny thereof.

The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a binding partner (eg, tumor antigen) protein present in a sample, but the antibody or ligand does not substantially recognize or bind other molecules in the sample. .

Scope: Throughout the disclosure, various aspects of the invention may exist in a range. It should be understood that the description of the scope of the invention is only for the purpose of convenience and clarity and should not be construed as limiting the scope of the invention. Accordingly, the description of a range should be considered to disclose all possible sub-ranges and individual values within the range. For example, a description of the range, such as from 1 to 6, should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and within the range Individual values, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes a range having 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This does not apply regardless of the width of the range.

The term "FLT3L" may be human FLT3L, a type IV transmembrane protein consisting of 235 amino acid residues (NCBI Reference Sequence: NP_001191431.1), and the amino acid sequence is set forth in SEQ ID NO: 8; a polypeptide having at least 80% similarity to SEQ ID NO: 8, preferably a polypeptide having at least 85% or at least 90% similarity to SEQ ID NO: 8; said "FLT3L" may also be derived from other species, including but Not limited to rats, monkeys, and pigs. FLT3L may be naturally occurring, such as it may be isolated or purified from a mammal; it may also be artificially prepared, such as recombinant elements or FLT3L, according to conventional genetic engineering recombination techniques. Preferably, the invention may employ recombinant elements or FLT3L.

combination

The present invention provides a composition comprising an immune effector cell, the composition further comprising a TOLL-like receptor agonist having a receptor targeted to recognize a tumor antigen or a pathogen antigen, the TOLL-like receptor agonist The effect of the immune effector cells can be increased in the treatment of a subject, wherein: the receptor has an extracellular region, a transmembrane region, and an intracellular region, wherein the extracellular region has a tumor antigen or An antigen binding domain of a pathogen antigen that binds to a tumor antigen or a pathogen antigen associated with the disease. In a preferred embodiment, the compositions of the invention may further comprise an MDSC inhibitor or an immunological checkpoint inhibitor.

The "immune effector cells" may be derived from autologous cells or from allogeneic cells.

The receptor for recognizing a tumor antigen or a pathogen antigen includes, but is not limited to, a chimeric antigen receptor (CAR), a T cell (antigen) receptor (TCR), a T cell fusion protein (TFP), or a T cell antigen coupler. (TAC).

The "immune effector cells" may also express other exogenous proteins, for example, the exogenous protein may be, but not limited to, FLT3L, interferon, interleukin, PD-1 or PD-L1 inhibitor. The presence of these exogenous proteins enhances the ability of "immune effector cells" to fight tumors or against pathogenic microorganisms.

In a specific embodiment, the interferon comprises, but is not limited to, a type I interferon; the interleukin comprises, but is not limited to, IL-2, IL-12, IL-21, IL18; the interleukin receptor comprises but Not limited to receptors for IL-4; inhibitors of PD-1 or PD-L1 include, but are not limited to, soluble PD-1 or fusion peptides containing soluble PD-1 (see WO2017080377), anti-PD-1 or anti-PD-1 PD-L1 antibody.

The TOLL-like receptor agonist can be, but is not limited to, a Toll-like receptor 3 (TLR3) agonist. The TLR3 agonist may be a natural or synthetic double-stranded ribonucleic acid such as polyinosinic acid cytidine (poly(I:C)). The TLR3 agonist may also be a mismatched double-stranded ribonucleic acid poly (I: C11-14U), exemplified by poly (I: C12U), poly (I: C13U).

The chimeric antigen receptor may comprise all of the conventional chimeric antigen receptor structures, and the transmembrane region may optionally be a protein or protein fragment having a transmembrane function, and the intracellular region may also be selected according to the prior art. Domains that trigger activation of immune cells, including but not limited to CD3ζ, FcRγ (FCER1G), FcγRIIa, FcRβ (FcEpsilon R1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, DAP12, CD137, CD27, CD28, etc. Full length or active fragment (eg CN201580013987 for a description of the intracellular domain). For example, a chimeric antigen receptor can also have the following structures:

The TFP comprises: (a) a TCR subunit comprising a TCR partial extracellular domain, a transmembrane domain, and a TCR intracellular domain, the intracellular domain comprising a stimulation signaling domain (b) an antigen binding domain, the TCR subunit and the antigen binding domain being operably linked. In a particular embodiment, the extracellular, transmembrane and intracellular signal domains of the TCR subunit are derived from CD3 epsilon or CD3 gamma, which integrates into the TCR expressed on T cells.

The TAC comprises: (a) an extracellular domain comprising: a single chain antibody having an antigen binding domain and binding to CD3; (b) a transmembrane region; (c) an intracellular domain, The intracellular domain is linked to protein kinase LCK.

In the present invention, the immune cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a mast cell, or a bone marrow-derived phagocytic cell. In a preferred embodiment, the immune cell is a T cell, and the intracellular domain of the receptor is capable of triggering activation of T cells.

The invention also provides a composition comprising an immune effector cell expressing a chimeric antigen receptor and a pharmaceutically acceptable carrier or excipient, the composition further comprising a TLR agonist and/or a dendritic cell maturation promoter .

In another aspect, the invention provides a composition comprising an immune effector cell, in addition to comprising a TOLL-like receptor agonist, an MDSC inhibitor, an anti-PD-1 antibody, or an antibody against PD-L1.

The invention also provides kits comprising an immune effector cell expressing a chimeric antigen receptor, a TLR agonist and/or a dendritic cell maturation promoter, and instructions for how to administer the cell to an individual.

Method of administration

The invention also provides a method of treating, controlling, or preventing a tumor or pathogen infection, the method comprising administering to the subject a therapeutically effective amount of an immune effector cell and a TOLL-like receptor agonist. The immune effector cell has a receptor that specifically recognizes a tumor antigen or a pathogen antigen, and the TOLL-like receptor agonist can increase the efficacy of the immune effector cell in the treatment of the subject, wherein: the receptor Having an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain has an antigen binding domain that recognizes the tumor antigen or pathogen antigen, and the antigen binding domain binds to a tumor antigen or pathogen associated with the disease antigen.

In a specific embodiment, the immune effector cells and the TOLL-like receptor agonist may be administered simultaneously, or the immune effector cells may be administered first, then the TOLL-like receptor agonist may be administered, or the TOLL-like receptor agonist may be administered first. Give immune effector cells.

The TOLL-like receptor agonist can be, but is not limited to, a Toll-like receptor 3 (TLR3) agonist. The TLR3 agonist may be a natural or synthetic double-stranded ribonucleic acid such as polyinosinic acid cytidine (poly(I:C)). The TLR3 agonist may also be a mismatched double-stranded ribonucleic acid poly (I: C _11-14 U), exemplified by poly (I: C ₁₂ U), poly (I: C ₁₃ U).

In the present invention, the immune cells include, but are not limited to, any one or a combination of T cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, or bone marrow-derived phagocytic cells. In a preferred embodiment, the immune cell is a T cell, and the intracellular domain of the receptor is capable of triggering activation of T cells.

In another aspect, the invention provides a method of treating, managing, or preventing a tumor or a pathogen infection, the method comprising administering to the subject a therapeutically effective amount of an immune effector cell, a TOLL-like receptor agonist, and administering to the MDSC An inhibitor, an anti-PD-1 antibody, or an antibody against PD-L1.

Use in the preparation of a medicament for treating/preventing/inhibiting a tumor or a pathogen infection, or modulating immune tolerance

The invention also provides a method for treating a tumor, an infectious disease and other immune-related diseases in combination with a TOLL-like receptor agonist and an immune effector cell, the tumor including but not limited to: pancreatic cancer, liver cancer, lung cancer, gastric cancer, Head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, leukemia, myeloma, ovarian cancer, cervical cancer, ovarian cancer, cervical cancer or glioma; Pathogens include, but are not limited to, viruses, bacteria, fungi, protozoa or parasites; preferably, the viruses include: cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus or influenza virus.

In a preferred embodiment, the TOLL-like receptor agonist is a Toll-like receptor 3 (TLR3) agonist, preferably a natural or synthetic double-stranded ribonucleic acid. In a preferred embodiment, the TLR3 agonist is polyinosinic acid cytidine (poly(I:C)). The TLR3 agonist may also be a mismatched double-stranded ribonucleic acid, exemplified by, for example, poly(I:C12U), poly(I:C13U).

Those skilled in the art can determine the administration of the TOLL-like receptor agonist and the immune effector cells according to factors such as age, weight, sex, severity of the disease to be treated, and the like by the professional knowledge and prior art. Mode and dosage. In a specific embodiment, the TOLL-like receptor agonist and the immune effector cells may be administered in the same or different manner. For example, the method of administration is co-administering the TOLL-like receptor agonist and the immune effector cells; or administering the immune effector cells first, and then administering the TOLL-like receptor agonist; or administering the TOLL-like sample first. The receptor agonist is administered to the immune effector cell. In a preferred embodiment, the method of administration is to first administer the immune effector cells and then to the TOLL-like receptor agonist.

In a preferred embodiment, the TOLL-like receptor agonist can be administered in an amount from 1 mg/kg to 4 mg/kg, preferably from 2 mg/kg to 3 mg/kg, more preferably from 2.08 mg/kg to 2.77 mg/ Kg. In a preferred embodiment, the TOLL-like receptor agonist is administered in a number of 2 to 4 times, preferably 2-3 times.

In a preferred embodiment, the immune cells are selected from the group consisting of T cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells or bone marrow-derived phagocytic cells, preferably T cells. In a preferred embodiment, the immune cell is a T cell, and the intracellular domain of the receptor is capable of triggering activation of T cells.

In a preferred embodiment, the recognition of a tumor antigen or a pathogen antigen receptor comprises a chimeric antigen receptor (CAR), a T cell (antigen) receptor (TCR), a T cell fusion protein (TFP), or a T cell. Antigen coupler (TAC). In a preferred embodiment, the immune cells may be CAR-expressing T cells, TCR-expressing T cells, CAR-expressing NK cells, TFP-expressing T cells, TFP-expressing NK cells, TAC-expressing T cells, NK cells expressing TAC.

The chimeric antigen receptor (CAR) may be, but is not limited to, the following structures: (i) an antibody that specifically binds to the antigen, a transmembrane region of CD28, a costimulatory signal domain of CD28, and CD3ζ; or (ii) specific An antibody that binds to the antigen, a transmembrane region of CD28, a costimulatory signal domain of CD137, and CD3ζ; or (iii) an antibody that specifically binds to the antigen, a transmembrane region of CD28, a costimulatory signal domain of CD28 , the co-stimulatory signal domain of CD137 and CD3ζ.

In a preferred embodiment, the TFP comprises: (a) a TCR subunit comprising a TCR partial extracellular domain, a transmembrane domain, and a TCR intracellular domain, said intracellular structure The domain includes a stimulation signaling domain; (b) an antigen binding domain, the TCR subunit and the antigen binding domain being operably linked. In a preferred embodiment, the extracellular, transmembrane and intracellular signal domains of the TCR subunit are derived from CD3 epsilon or CD3 gamma, which integrates into the TCR expressed on T cells.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually prepared according to conventional conditions such as J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the manufacturer. The suggested conditions.

Exemplary antigen receptors of the present invention, including CAR, and methods for engineering and introducing a receptor into a cell, are described, for example, in Chinese Patent Application Publication No. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A. , CN105331585A, CN106397593A, CN106467573A, CN104140974A, International Patent Application Publication No. WO2017186121A1, WO2018006882A1, WO2015172339A8, WO2018018958A1.

Example 1. Preparation of CAR-T cells

In this example, the target of EGFRvIII-acting CAR-T cells was selected, and in order to more accurately verify the anti-tumor effect in mice, the selected signal peptide, transmembrane region, intracellular region and the like were mouse-derived. The method of preparation is operated according to conventional CAR-T cell preparation methods in the art.

1. Construction of retroviral vector

Mouse CD8α signal peptide (SEQ ID NO: 1), anti-EGFRvIII monoclonal antibody (SEQ ID NO: 2), CD8α gelatin region and transmembrane region (SEQ ID NO: 3), CD28 intracellular domain (SEQ ID) NO: 4), CD3 sputum intracellular domain (SEQ ID NO: 5) was ligated in turn, the EGFRvIII-28Z gene fragment was obtained by in vitro gene synthesis, and the retroviral vector MSCV- was replaced with Mlu I and Sal I double restriction sites. The IRES-GFP fragment in IRES-GFP obtained the recombinant vector MSCV-EGFRvIII-28Z. The order of combination of the constituent segments is as shown in FIG.

2. Retroviral preparation

The operation is as follows:

(1) 293T cells were seeded at a density of 4.5×106 in a culture dish, and CO2 was cultured overnight, and the medium was DMEM supplemented with fetal bovine serum;

(2) 9 μg of MSCV-EGFRvIII-28Z and 9 μg of pCL-Eco were dissolved in 800 μL of serum-free DMEM medium to obtain a plasmid mixture; 54 μg of PEI (1 μg/μl) was dissolved in 800 μl of serum-free DMEM medium to obtain PEI mixture. The solution was added to the PEI mixture and incubated at room temperature to obtain a transfection complex.

(4) 1.6 ml of the transfection complex was added dropwise to the culture dish containing the step (1), and the supernatant was collected to obtain.

3. Retrovirus-infected mouse spleen T lymphocytes

Take healthy Balb/c mice, remove the spleen after cervical dislocation, and prepare the spleen cells into a single cell suspension; add 200 ul of red blood cell lysate and use the mouse CD3+ T cell sorting kit (STEMCELL, #19851) The mouse spleen T lymphocytes were obtained by sorting.

Mouse CD3+ T lymphocytes were purified and added to Dynabeads Mouse T-activator CD3/CD28 in a ratio of 1:1, and cultured in RPMI 1640 complete medium for 24 hours.

Each well of a 48-well plate was added to 400 μl of retroNectin solution (5 μg/mL) overnight, and then retroNectn was discarded; the spleen T lymphocytes activated for 24 hours were inoculated into the plate, the number of cells per well was 1×106, and 1 ml of reverse transcription was added. The virus was supplemented to 2 mL, cultured and subcultured after centrifugation.

4. Expression of chimeric antigen receptors in T lymphocytes

After 3 days of infection, 4×105 T cells were taken, the cells were resuspended, Biotin-labeled EGFRvIII protein was added to a final concentration of 25 ug/ml on ice, and then washed; 50 μL PBS+2% FCS was added and resuspended, and 1 μL of PE-labeled streptavidin was added. After incubating on ice, the cells were resuspended by adding 400 μL of PBS + 2% FCS, and the results were flow-through. As shown in Fig. 2, the infection efficiency of EGFRvIII-28Z CART cells was 76.0%.

Example 2. In vitro toxicity of EGFRvIII-28Z CAR T lymphocytes

Mouse colon cancer cell line CT26 (purchased from American Type Culture Collection (ATCC)) and mouse pancreatic cancer cell line E0771 (obtained from Baylor College of Medicine) were used to prepare CT26 overexpressing EGFRvIII by conventional techniques in the art. - EGFRvIII and E0771-EGFRvIII.

The in vitro cytotoxicity of EGFRvIII-28Z CAR T lymphocytes to target cells was determined using a cytoTox 96 non-radioactive cytotoxicity assay kit (Promega, Madison, USA). The target cells were adjusted to a concentration of CT26, CT26-EGFRvIII, E0771, E0771-EGFRvIII of 2×105/mL, and 50 μL was inoculated into a 96-well plate; the target ratio was 0.3:1, 1:1, and 3:1 to the 96-well plate. Add CAR-T cells and control T cells (50 ul), set each control well according to the kit instructions, and set 5 replicate wells in each group; incubate for 18 hours. After the incubation, the cytotoxicity of EGFRvIII-28Z CAR T lymphocytes to target cells was determined according to the kit instructions. The results are shown in Figure 3.

The results showed that EGFRvIII-28Z CAR-T cells showed no significant toxicity to CT26 and E0771 cells with negative expression of EGFRvIII, but showed strong killing effect on CT26-EGFRvIII and E0771-EGFRvIII cells expressing EGFR-vIII target antigen. At 3:1, the EGFRvIII-28Z CAR-T cells killed 71% and 67.8% of the two cells, respectively; the uninfected T cells showed no significant killing activity on the target cells. There was a significant difference in the killing effect of EGFRvIII-28Z CAR T and uninfected T cells on EGFR-vIII target antigen positive cells. These results indicate that our constructed EGFRvIII-28Z CAR T lymphocytes can specifically kill tumor cells positive for EGFRvIII expression in vitro.

Example 3. Antitumor effect of EGFRvIII-28Z CAR-T combined with poly I:C in a mouse breast cancer model

Inoculate 1×106 E0771-EGFRvIII cells in 6-week-old female C57 mice (body weight 18g-24g, purchased from Shanghai Lingchang Biotechnology Co., Ltd.), the fourth pair of breast fat pads; tumor inoculation for 13 days, the tumor-bearing small The rats were divided into 4 groups, which were divided into UT cell group, UT+poly I:C group, EGFRvIII-28Z CAR-T cell group and EGFRvIII-28Z CAR-T+poly I:C group. The average tumor volume of each group was about 150mm ³ ; after grouping, 5.0×10 ⁶ EGFRvIII-28Z CAR-T cells were infused through the tail vein (day 0 of the injection day), while the UT cell group was used as a control, and the CAR-T cells were returned after the third and sixth cells. 50 μg of polyI:C was injected into the tumor, and the UT cell group was intratumorally injected with the same dose of physiological saline. The growth of the transplanted tumor was observed, and the results are shown in Fig. 4A.

The results showed that the reinfusion of 5×106 EGFRvIII-28Z inhibited the growth of E0771-EGFRvIII xenografts, while the combination of poly I:C inhibited the growth of E0771-EGFRvIII xenografts, indicating that poly I:C further Enhances the anti-tumor ability of CAR-T cells. Compared with the UT group, the tumor inhibition rate was 36.1% in the EGFRvIII-28Z CAR-T cell group on the 16th day, and 69.6% in the poly I:C group. The difference was significant (P<0.05). Increase by more than 30%.

When the tumor size of the control mice reached 2000 mm ³ , the experiment was sacrificed, and after the tumors were isolated, the tumor weight was determined. As a result, as shown in FIG. 4B , the tumor weight of the combined group of Poly: IC was decreased as compared with the administration of CAR-T alone. 77.8%

After the sacrificed mice, peripheral blood was taken for measurement of IFNγ levels, and as shown in Fig. 4C, spleen and tumor tissues were taken, and CAR-T cell copy number was measured, and the results are shown in Fig. 4D.

Example 4. Antitumor effect of EGFRvIII-28Z CAR-T in combination with poly I:C in a mouse colon cancer model

6-week-old female Balb/c mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were inoculated with 3×105 CT26-EGFRvIII cells subcutaneously after 3Gy gamma ray irradiation (ie, lymphocyte clearance); tumor inoculation for 13 days The tumor-bearing mice were divided into 4 groups, which were divided into UT cell group, UT+poly I:C group, EGFRvIII-28Z CAR-T cell group and EGFRvIII-28Z CAR-T+poly I:C group. The volume average was approximately 150 mm3; after grouping, 5.0×106 EGFRvIII-28Z CAR-T cells were infused through the tail vein, while the U T cell group was used as a control, and the intratumoral injection was performed on the 3rd and 6th days after CAR-T cell reinfusion. 50ug polyI: C. The therapeutic effect of GFRvIII-28Z CAR-T combined with poly I:C on CT26-EGFRvIII xenografts is shown in Figure 5.

The results showed that 5×106 CT26-EGFRvIII alone could significantly inhibit the growth of CT26-EGFRvIII xenografts; while combined with poly I:C, the inhibitory effect on the growth of CT26-EGFRvIII xenografts was more significant, indicating that poly I:C further Enhances the anti-tumor ability of CAR-T cells. Compared with the UT group, the tumor inhibition rate was 36.1% in the EGFRvIII-28Z CAR-T cell group on the 16th day, and 74.3% in the poly I:C group, the difference was significant (P<0.05, P<0.01). The tumor inhibition rate increased by more than 30%.

When the tumor size of the control mice reached 2000 mm ³ , the experiment was sacrificed, and after the tumors were isolated, the tumor weight was determined. As a result, as shown in FIG. 5B, the tumor weight of the combined group of Poly: IC was decreased as compared with the administration of CAR-T alone. 48.5%.

After the sacrificed mice, peripheral blood was taken for measurement of IFNγ levels, and as shown in Fig. 5C, spleens and tumor tissues were taken, and CAR-T cell copy number was measured, and the results are shown in Fig. 5D.

Example 5 Expression of FLT3L CAR-T cells in combination with Poly I:C

The mouse muFLT3L gene fragment (SEQ ID No: 6) was ligated with the gene sequence of EGFRvIII-28Z constructed in Example 1 by F2A (SEQ ID No: 7), ie, F2A, according to a conventional CAR-T cell preparation method. After the FLT3L gene was inserted into the CD3ζ fragment of the EGFR-28Z gene, the EGFRvIII-28Z-FLT3L retroviral vector MSCV-EGFRvIII-28Z-FLT3L was constructed. The plasmid map is shown in Figure 6. The mouse spleen T lymphocytes were infected to prepare the expression EGFRvIII- 28Z-FLT3L CAR T cells.

Referring to the procedure of Example 4, a mouse colon cancer model was constructed, and the tumor-bearing mice were divided into EGFRvIII-28Z CAR-T cells + poly I: C group and EGFRvIII-28Z-FLT3L CAR-T + poly I: C cell group.

The results are shown in Figure 7. In the Balb/c mouse CT26-EGFRvIII xenograft model, the tumor suppressive effect of the EGFRvIII-28Z-FLT3L CAR-T+poly I:C cell group was significantly better than that of the EGFRvIII-28Z CAR-T cell. +poly I: group C, EGFRvIII-28Z-FLT3L CAR-T+poly I:C combination treatment group inhibition rate reached 47.8%, 50.1% and 54.0 on the 7th, 10th and 14th day after CAR T cell infusion, respectively. %.

Example 6 Effect of Interferon Inhibitor on the Antitumor Effect of CAR-T+poly I:C

Referring to Examples 3 and 4, a mouse breast cancer model and a colon cancer model were constructed, respectively. The antibody using the interferon receptor alpha chain (clone number MAR1-5A3, purchased from Bioxcell) was used as a blocking agent to block the release of type I interferon.

The mouse intestinal cancer model was divided into UT group (injected saline), EGFRvIII-28Z CAR-T cells + poly I:C group, and EGFRvIII-28Z CAR-T cells + poly I:C group + interferon receptor alpha Chain sealer. On the 3rd and 6th day after CAR-T cell reinfusion, 50ug polyI:C was injected intratumorally, and on the 0th day and the 2nd day after administration of poly I:C or normal saline, 50ug of interferon receptor alpha chain was blocked by intratumoral injection. Agent. Tumor growth was measured and the results are shown in Figure 8A. When the tumor size of the control mice reached 2000 mm ³ , the experiment was sacrificed, and after the tumors were isolated, the tumor weight was measured, and the results are shown in Fig. 8B.

Mouse breast cancer models were divided into UT group (injected saline), EGFRvIII-28Z CAR-T cells + poly I:C group, and EGFRvIII-28Z CAR-T cells + poly I:C group + interferon receptor alpha Chain sealer. On the 3rd and 6th day after CAR-T cell reinfusion, 50ug polyI:C was injected intratumorally, and on the 0th day and the 2nd day after administration of poly I:C or normal saline, intratumoral injection of interferon receptor α chain blocker was given. . Tumor growth was measured and the results are shown in Figure 9A. When the tumor size of the control mice reached 2000 mm ³ , the experiment was sacrificed, and after the tumors were isolated, the tumor weight was measured, and the results are shown in Fig. 9B.

The above results show that after blocking type I interferon, administration of CAR-T cells and poly I:C causes a decrease in antitumor activity, that is, the presence of type I interferon will be beneficial to anti-tumor of CAR-T cells and poly I:C. effect.

Example 7 Effect of MDSC Inhibitor on the Antitumor Effect of CAR-T+poly I:C

Referring to Example 4, a mouse colon cancer model was constructed. The MDSC scavenger anti-Gr1 antibody (purchased from BioXell e, clone number RB6-8C5) was used to inhibit MDSC.

MDSC is a bone marrow-derived suppressor cell and is positive for CD11b+Gr1+. The anti-Gr1 antibody alone did not have significant antitumor activity compared to the UT group.

The mouse intestinal cancer model was divided into UT group (injected saline), UT+anti-Gr1 group (administered anti-Gr1 antibody and normal saline), CAR-T+poly I:C group (administered to EGFRvIII-28Z-CAR- T and poly I: C), CAR-T+anti-Gr1 group (administered EGFRvIII-28Z-CAR-T and anti-Gr1 antibodies), CAR-T+poly I: C+anti-Gr1 group (administered EGFRvIII-28Z) -CAR-T, poly I: C and anti-Gr1 antibodies).

On day 2 after CAR-T cell reinfusion, anti-Gr1 antibody was intraperitoneally injected into 10 mg/kg mice, followed by three injections per week for a total of two weeks. On the 3rd and 6th day after CAR-T cell reinfusion, 50ug polyI:C was injected into the tumor.

Tumor growth was measured and the results are shown in Figure 10A. When the tumor size of the control mice reached 2000 mm3, the experiment was sacrificed, and after the tumors were isolated, the tumor weight was measured, and the results are shown in Fig. 10B. The tumor was isolated after the tumor was isolated, and the results are shown in Fig. 10C.

Illustratively, the above examples select CAR-T cells that target EGFR, it being understood that selection of CAR-T cells acting on other targets also has the same effect, such as GPC3, CLD18A2, CD19, BCMA, and the like. The antibody to be used may be mouse anti-human or humanized, and the transmembrane domain and the intracellular domain may be different species depending on the purpose, such as human.

Illustratively, although the above examples employ CAR-T cells, the T cells can also express other cytokines that enhance CAR-T cell function, such as CAR-T cells co-expressed by CAR and type I interferons. CAR-T cells co-expressed by CAR and PD1.

Illustratively, although the above embodiment uses CAR-T cells, other immune cells, such as NK cells and NK-T cells, may also be selected, and specific subtypes of immune cells, such as γ/δT cells, may be specifically selected. Wait.

Illustratively, the above embodiment selects a CAR of a murine source, but its signal peptide, hinge region, transmembrane region, and the like may be selected from other species depending on the purpose. These include, but are not limited to, human signal peptides, hinge regions, transmembrane domains, intracellular regions. Antibodies can also be selected for murine anti- or humanized antibodies or whole human antibodies against different targets for different purposes.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims

A composition comprising an immune effector cell having a receptor that targets a tumor antigen or a pathogen antigen, the immune effector cell being associated with a subject having a disease associated with expression of a tumor antigen or a pathogen antigen Agents for increasing the efficacy of the immune effector cells in therapy are used in combination, wherein:

(i) the receptor has an extracellular region, a transmembrane region, and an intracellular region, wherein the extracellular region has an antigen binding domain that recognizes the tumor antigen or the pathogen antigen,

(ii) The agent that increases the efficacy of immune cells is a TOLL-like receptor agonist.
The composition of claim 1 wherein the TOLL-like receptor agonist is a Toll-like receptor 3 (TLR3) agonist.
The composition of claim 2 wherein said TLR3 agonist is selected from the group consisting of natural or synthetic double-stranded ribonucleic acids, preferably polyinosinic acid cytidine (poly(I:C)).
The composition according to any one of claims 1 to 3, wherein the receptor for recognizing a tumor antigen or a pathogen antigen comprises a chimeric antigen receptor (CAR), a T cell (antigen) receptor (TCR), and a T cell fusion. Protein (TFP), or T cell antigen coupler (TAC).
The composition of claim 4 wherein said chimeric antigen receptor comprises:

(i) an antibody that specifically binds to the antigen, a transmembrane region of CD28, a costimulatory signal domain of CD28, and a CD3 sputum cytoplasmic signaling domain;

(ii) an antibody that specifically binds to the antigen, a transmembrane region of CD28, a costimulatory signal domain of CD137, and a CD3 purine cytoplasmic signaling domain;

(iii) an antibody that specifically binds to the antigen, a transmembrane region of CD28, a costimulatory signal domain of CD28, a costimulatory signal domain of CD137, and a CD3 sputum cytoplasmic signaling domain.
The composition of claim 4 wherein said TFP comprises:

(a) a TCR subunit comprising:

a TCR partial extracellular domain, a transmembrane domain, and a TCR intracellular domain, the intracellular domain comprising a stimulation signaling domain;

(b) an antigen binding domain, said TCR subunit and said antigen binding domain being operably linked,

Wherein the extracellular, transmembrane and intracellular signal domains of the TCR subunit are derived from CD3 epsilon or CD3 gamma, and the TFP is integrated into the TCR expressed on T cells.
The composition of claim 4 wherein said TAC comprises:

(a) Extracellular domain: the extracellular domain comprises a single chain antibody having an antigen binding domain and binding to CD3;

(b) a transmembrane zone;

(c) an intracellular domain that links to protein kinase LCK.
The composition according to any one of claims 1 to 7, wherein said immune cells are selected from the group consisting of T cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells or bone marrow-derived phagocytosis. cell.
Use of a combination of a TOLL-like receptor agonist and an immune effector cell for the preparation of an anti-tumor drug or an anti-pathogenic drug or an anti-immunological drug, the immune effector cell having a receptor for targeting a tumor antigen or a pathogen antigen, The receptor has an extracellular region, a transmembrane region, and an intracellular region, the extracellular region having an antigen binding domain that binds to the tumor antigen or the pathogen antigen;

The receptor is preferably a chimeric antigen receptor (CAR), a T cell (antigen) receptor (TCR), a T cell fusion protein (TFP), or a T cell antigen coupler (TAC).
The use according to claim 9, wherein the TOLL-like receptor agonist and the immune effector cells are prepared for parenteral administration, preferably intravenous and intratumoral injection.
The use according to claim 9, wherein the immune cells are selected from the group consisting of T cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells or bone marrow-derived phagocytic cells.
The use according to any one of claims 9 to 11, characterized in that the immune cells select T cells expressing CAR, NK cells expressing CAR, or T cells expressing TCR.
The use according to any one of claims 9-11, wherein the TOLL-like receptor agonist is a Toll-like receptor 3 (TLR3) agonist, preferably a natural or synthetic double-stranded ribonucleic acid, More preferred is polyinosinate cytidine (poly(I:C)).
The composition according to claim 1, wherein said immune effector cells further express other exogenous proteins, preferably, said exogenous protein is selected from the group consisting of FLT3L, interferon, interleukin, interleukin Receptor, PD-1 inhibitor, PD-L1 inhibitor, or a combination thereof.
The composition of claim 1 further comprising an MDSC inhibitor, and/or an immunological checkpoint inhibitor.
The composition according to claim 15, wherein said MDSC inhibitor is selected from the group consisting of an anti-Gr1 antibody, a cyclooxygenase-2 inhibitor, a prostaglandin-type stem cell factor inhibitor, and a macrophage colony-stimulation stimulus. A factor inhibitor, a granulocyte monocyte colony stimulating factor inhibitor, a vascular endothelial growth factor inhibitor, or a combination thereof.
The composition of claim 15 wherein said immunological checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, or a combination thereof.
A composition product characterized by comprising:

(i) a first pharmaceutical composition comprising at least one immune effector cell potentiator comprising a TOLL-like receptor agonist; and a pharmaceutically acceptable carrier ;

(ii) a second pharmaceutical composition comprising an immune effector cell having a receptor that targets a tumor antigen or a pathogen antigen; and a pharmaceutically acceptable carrier;

(iii) a third pharmaceutical composition comprising an MDSC inhibitor or an immunological checkpoint inhibitor; and a pharmaceutically acceptable carrier.
Use of a composition according to claim 18 for the preparation of an anti-tumor or anti-pathogenic drug or an anti-immune drug.
A method for improving the viability of an immune effector cell, comprising the steps of:

Administering (i) an immune effector cell; (ii) an immune effector cell potentiator; and/or (iii) an optional MDSC inhibitor or immunological checkpoint inhibitor, wherein the immune effector cell has a target A receptor that recognizes a tumor antigen or a pathogen antigen, the immune effector cell potentiator being a TOLL-like receptor agonist.
The method of claim 20, wherein (i) immune effector cells; (ii) immune effector cell potentiators; and (iii) optional MDSC inhibitors are administered as compared to administration of immune effector cells alone Or the inhibition rate of the immunological checkpoint inhibitor is increased by ≥ 20%, preferably ≥ 30%.
The method of claim 20, wherein (i) immune effector cells; (ii) immune effector cell potentiators; and (iii) optional MDSC inhibitors are administered as compared to administration of immune effector cells alone Or the level of interferon release by the immunological checkpoint inhibitor is increased by > 20%, preferably by 30%, more preferably by > 50%.
Use of a combination of an immune effector cell, an immune effector cell synergist, and a lymphocyte scavenger for the preparation of a medicament against an antitumor or antipathogenic or anti-immune.
A method for treating tumor or pathogen infection or low immunity, the method comprising: administering a combination of an immune effector cell, an immune effector cell synergist and a lymphocyte scavenger to a subject in need thereof, wherein The immune effector cells have a receptor that targets a tumor antigen or a pathogen antigen, and the immune effector cell synergist is a TOLL-like receptor agonist.