WO2022007784A1

WO2022007784A1 - Methods of reducing graft rejection of allogeneic cell therapy

Info

Publication number: WO2022007784A1
Application number: PCT/CN2021/104704
Authority: WO
Inventors: Zhongyuan TU; Yafeng Zhang; Shuai Yang; Shu Wu; Liang Tan
Original assignee: Nanjing Legend Biotech Co., Ltd.
Priority date: 2020-07-06
Filing date: 2021-07-06
Publication date: 2022-01-13

Abstract

Provided is a therapeutic immunoresponsive cell which comprises an antigen binding receptor directed toward a molecule that is expressed on an immune cell and related to the activation of the immune cell in an innate or adaptive immune response.

Description

METHODS OF REDUCING GRAFT REJECTION OF ALLOGENEIC CELL THERAPY

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

This application claims priority to International Patent Application No. PCT/CN2020/100406 filed on July 6, 2020.

The foregoing application, and all documents cited therein or during its prosecution ( “appln cited documents” ) and all documents cited or referenced herein (including without limitation all literature documents, patents, published patent applications cited herein) ( “herein cited documents” ) , and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. Any Genbank sequences mentioned in this disclosure are incorporated by reference with the Genbank sequence to be that of the earliest effective filing date of this disclosure.

Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SEQUENCE STATEMENT

The instant application contains a Sequence Listing, which has been submitted electronically and is hereby incorporated by reference in its entirety. Said ASCII copy, created June 28, 2021, is named L2-W20208WO Sequence listing. txt and is 43kb in size.

FIELD OF THE INVENTION

The disclosure relates to a genetically engineered immunoresponsive cell which expresses an antigen binding receptor (e.g., a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR) ) directed toward a molecule that is expressed on an immune cell and related to activation of an innate or adaptive immune response.

BACKGROUND OF THE INVENTION

Adoptive cellular therapy or adoptive cell transfer (ACT) is becoming an ever more important treatment paradigm, particularly in the treatment of cancer. ACT refers to the transfer of cells, most typically immune cells, into a patient. These cells may have originated from the same patient (autologous therapy) or from another individual (allogeneic therapy) . The goal of the therapy is to improve immune functionality and characteristics, and in cancer immunotherapy, to raise an immune response against the cancer. Although T cells are most often used for ACT, it is also applied using other immune cell types such as NK cells, γδ T cells, NKT cells, lymphocytes (e.g. tumor-infiltrating lymphocytes (TILs) ) , dendritic cells and myeloid cells (1-2) .

Ideally, the cells that are infused in the subject (or reinfused in case of autologous therapy) receiving the ACT will expand and persist in the subject. To this end, lymph depletion is often used as neoadjuvant therapy, to ensure there are no competing immune cells to repopulate the immune cell space. This is especially important for allogeneic therapy, as is the case with transplants, an immune response may be raised against non-self infused cells, both by the adaptive and the innate immune system. Sometimes myeloablation, high-dose chemotherapy that kills cells in the bone marrow, is also used. However, lymph depletion or myeloablation is quite drastic measures that often results in severe side effects because of their effect on the immune system. Accordingly, it would be advantageous to prevent or reduce the response of immune system against infused ACT cells, as this would increase the persistence of the ACT cells in vivo and increase the benefits of the therapy. Ideally, this would lead to reduced need for lymph depletion or myeloablative therapy, so that patients receiving ACT suffer less from side effects.

The immune system has developed elaborate and effective mechanisms to combat foreign agents. The immune system remains the most formidable barrier to allogeneic adoptive cellular therapy. Conversely, graft-versus-host disease (GvHD) may occur after allogenic therapies, in which the donor T cells recognize the patient’s cells as foreign, resulting in attack of the healthy tissue. This event is attributed to the T cell receptor (TCR) at the surface of the donor cells recognizing human leukocyte antigen (HLA) on the patient’s tissues as foreign and instructing the T cells to attack. Whereas many in the field have used gene-editing technologies such as transcription activator-like effector nucleases (TALENs) , zinc finger nucleases or CRISPR–Cas9 to eliminate the TCR gene from the genome of the donor CAR-T cell. Genome editing applications have increased in frequency as a result of the efficacy and ease of use of recent tools, e.g., CRISPR and TALEN systems. However, the genome edited cells may be unwantedly rejected by the host immune response. Accordingly, there exists a need for cells suitable for allogeneic transplantation that eliminate or reduce the likelihood of triggering unwanted recipient immune responses to allogeneic transplants of such cells. Many companies also knockout beta-2 microglobulin (B2M) to eliminated graft rejection that caused by T cells. While B2M knockout facilitates NK based graft rejection. So far, no promising data achieved for allogeneic αβT cell therapy products (3-6) . CD8 T cells and NK cells are the two major cytotoxic lymphocytes that carry out cell-mediated immunity and regulate other immune responses. Therefore, to remove the activated CD8 T cells, CD8 memory T cells and NK cells will decrease the graft rejection.

SUMMARY OF THE INVENTION

The present disclosure generally provides methods and compositions for reducing or eliminating allogeneic rejection of cells infused into a subject, without suppressing the entire host immune system. According to the invention, allogeneic cells may be infused into a subject together with immune cells engineered to comprise an antigen binding receptor, e.g., a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR) , directed toward an immune cell activation molecule that is expressed on an immune cell and related to the activation of the immune cell in an innate or adaptive immune response, and the engineered immune cells may target and kill activated host immune cells against the allogeneic cells and/or the engineered immune cells in the subject to some extent. Alternatively, certain immune cells like allogeneic therapeutic cells may be engineered to comprise an antigen binding receptor, e.g., a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR) , directed toward (i) an immune cell activation molecule that is expressed on an immune cell and related to the activation of the immune cell in an innate or adaptive immune response and (ii) a disease associated antigen, and, when infused into a subject, kill activated immune cells against the allogeneic therapeutic cells to some extent.

Accordingly, in a first aspect, the present disclosure provides an antigen binding receptor directed toward an immune cell activation molecule that is expressed on an immune cell and related to the activation of the immune cell in an innate or adaptive immune response. Also provided is an immuneresponsive cell which comprises the antigen binding receptor.

The immune cell activation molecule may be a protein expressed on an immune cell and stimulates, regulates and/or simply indicates the activation of an immune response. The immune cell here may be a neutrophil, an eosinophil, a basophil, a mast cell, a monocyte, a macrophage, a dendritic cell, a natural killer cell, or a lymphocyte. In certain embodiments, the immune cell is a dendritic cell, a natural killer cell, or a lymphocyte.

In certain embodiments, the immune cell activation molecule is a protein that is expressed on an immune cell and responsible for the activation, function and/or proliferation of the immune cell itself and/or another type of immune cell. Such immune cell activation molecules include, but not limited to, costimulatory proteins such as CD30, CD33, D28, CD137, CD7 and CD69 expressed on immune cells such as T cells, NK cells and/or B cells, and non-costimulatory proteins such as CD2 and CD8 expressed on T cells and/or NK cells, and CD70 expressed on activated T cells and/or NK cell as well as on dendritic cells (DC) with upregulated expression upon DC maturation. The immune cell activation molecule in certain embodiments is uniquely expressed on activated immune cells, but not on non-immune cells or resting immune cells. The immune cell activation molecule in certain embodiments is highly expressed on activated immune cells, but not expressed or expressed at a low level on non-immune cells or resting immune cells. In certain embodiments, the immune cell activation molecule is CD8, CD70, CD30 or CD33. In certain embodiments, the immune cell activation molecule is CD8. In certain embodiments, the immune cell activation molecule is CD70. In certain embodiments, the immune cell activation molecule is CD30. In certain embodiments, the immune cell activation molecule is CD33.

The antigen binding receptor may be a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain selective for the immune cell activation molecule. The antigen binding receptor may be a recombinant T cell receptor (TCR) complex comprising an extracellular antigen binding domain, selective for the immune cell activation molecule, fused to a TCR complex. The extracellular antigen binding domain may be an antibody or an antigen-binding portion thereof to the immune cell activation molecule, or other proteins binding to the immune cell activation molecule, such as a ligand of the immune cell activation molecule or a binding fragment thereof.

In certain embodiments, the CAR may comprise (a) an extracellular antigen binding domain selective for the immune cell activation molecule, (b) a transmembrane domain, and (c) an intracellular domain. In certain embodiments, the extracellular antigen binding domain may comprise an antibody or an antigen binding portion thereof to the immune cell activation molecule, e.g., a single chain Fv (scFv) or a single domain antibody (sdAb) against the immune cell activation molecule. In certain embodiments, the CAR is a monospecific tandem CAR, wherein the extracellular antigen binding domain may comprise more than one antibody or antigen binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that recognize the same epitope on an immune cell activation molecule. In certain embodiments, the CAR is a bispecific or multispecific tandem CAR, wherein the extracellular antigen binding domain comprises more than one antibody or antigen binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that recognize different epitopes on an immune cell activation molecule and/or more than one immune cell activation molecule of the disclosure. In certain embodiments, the extracellular antigen binding domain may comprise an anti-CD8 sdAb having e.g., the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the transmembrane domain may comprises CD8α transmembrane region or CD28 transmembrane region. In certain embodiments, the transmembrane domain may comprise a CD8α transmemrabe region having e.g., the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the intracellular domain may comprise at least one signaling domain selected from the group consisting of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In certain embodiments, the intracellular domain may comprise at least one costimulatory domains selected from the group consisting of CD28, 4-1BB (CD137) , CD27, OX40, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, CD83, and a ligand that specifically binds with CD83. In certain embodiments, the intracellular domain may comprise a CD3ζsignaling domain and a 4-1BB costimulatory domain. In certain embodiments, the intracellular domain may comprise a CD3ζ signaling domain and a CD28 costimulatory domain. In certain embodiments, the intracellular domain may comprises a 4-1BB intracellular domain and a CD3ζ signaling domain. The 4-1BB intracellular domain may comprise the amino acid sequence of SEQ ID NO: 15. The CD3ζ signaling domain may comprise the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the CAR may further comprise a CD8α hinge between the extracellular antigen binding domain and the transmembrane domain. The CD8α hinge may comprise the amino acid sequence of SEQ ID NO: 13. In certain embodiments, the CAR may further comprise a leader sequence at the N-terminus.

In certain embodiments, the recombinant TCR complex may comprise (i) a TCR complex comprising (a) a TCR chain selected from the group consisting of an alpha (α) chain, a beta (β) chain, a gamma (γ) chain and a delta (δ) chain of a T cell receptor, (b) an epsilon (ε) chain, a delta (δ) chain, and/or a gamma (γ) chain of CD3, and/or (c) a zeta (ζ) chain, and (ii) an extracellular antigen binding domain selective for the immune cell activation molecule, fused to any of the chains of the TCR complex. The extracellular antigen binding domain may comprise an antibody or antigen binding portion thereof, such as a scFv or a sdAb, to the immune cell activation molecule. In certain embodiments, the extracellular antigen binding domain comprises more than one antibody or antigen-binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that target the same epitope on an immune cell activation molecule. In certain embodiments, the extracellular antigen binding domain compeises more than one antibody or antigen-binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that target different epitopes on an immune cell activation molecule and/or more than one immune cell activation molecule of the disclosure. In certain embodiments, the extracellular antigen binding domain may comprise an anti-CD8 sdAb having e.g., the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the recombinant TCR complex may comprise a TCR-α chain, a TCR-β chain, a CD3γ chain, a CD3δ chain, two CD3ε chains and a ζ-chain, or part of the chains, wherein one or more of these chains are linked, with a linker or not, to an extracellular antigen binding domain selective for the immune cell activation molecule. In certain embodiments, the extracellular antigen binding domain is linked to the CD3ε chain. The CD3ε chain may comprise the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the extracellular antigen binding domain is linked to the CD3ε chain via a linker such as the one having the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the recombinant TCR complex may comprise a TCR-γ chain, a TCR-δ chain, a CD3γ chain, a CD3δ chain, two CD3ε chains and a ζ-chain, or part of the chains, wherein one or more of these chains are linked, with a linker or not, to an extracellular antigen binding domain selective for the immune cell activation molecule. In certain embodiments, the extracellular antigen binding domain is linked to the CD3ε chain. The CD3ε chain may comprise the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the extracellular antigen binding domain is linked to the CD3ε chain via a linker such as the one having the amino acid sequence of SEQ ID NO: 20.

In certain embodiments, the immuneresponsive cell or immune cell may be a lymphocyte, such as a T cell, or a NK cell, including, but not limited to, an αβ T cell, a γδ T cell, a NKT cell, an iNKT cell, and a NKT like cell. In certain embodiments, the γδ T cell is selected from the group consisting of a Vδ1 cell, a Vδ2 cell, a Vδ3 cell, a Vγ9Vδ2 T cell, a Vδ5 cell or the combination thereof.

In certain embodiments, the immuneresponsive cell is genetically engineered to express a low level of the immune cell activation molecule, or not express the immune cell activation molecule, by e.g., gene editing.

In certain embodiments, the immuneresponsive cell is engineered to express and secrete one or more cytokines at high levels when the immunoresponsive cell is activated and at low levels or not at all when the immunoresponsive cell is not activated. In certain embodiments, the cytokine is one or more selected from the group consisting of IL-7, IL-12, IL-15, and IL-18.

In a second aspect, the present disclosure provides a dual antigen binding receptor which comprises (i) a first antigen binding receptor directed toward an immune cell activation molecule as defined herein; and (ii) a second antigen binding receptor directed toward a disease associated antigen. Also provided is an immuneresponsive cell which comprises the dual antigen binding receptor.

The disease associated antigen may be a tumor associated antigen, such as CD19, CD20, CD22, CD4, CD24, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα) , mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, and HER-2. The tumor associated antigen, in certain embodiments, may be BCMA. The disease associated antigen may be an infectious disease associated antigen, such as a marker molecule on a pathogen, or a disease marker on an infected cell, e.g., CD4, HBsAg, LMP-1, and LMP2. The disease associated antigen may be an inflammatory disease associated antigen, such as IL17R, CD20, and CD6. The disease associated antigen in certain embodiments may be the immune cell activation molecule described herein. The disease associated antigen in certain embodiments may be CD8.

The first antigen binding receptor may be a chimeric antigen receptor (CAR) described herein comprising an extracellular antigen binding domain selective for the immune cell activation molecule, or a recombinant T cell receptor (TCR) complex comprising an extracellular antigen binding domain, selective for the immune cell activation molecule, fused to a TCR complex described herein. The second antigen binding receptor may be a chimeric antigen receptor (CAR) described herein comprising an extracellular antigen binding domain selective for the disease associated antigen, or a recombinant TCR complex comprising an extracellular antigen binding domain, selective for the disease associated antigen, fused to a TCR complex described herein. The extracellular antigen binding domain may comprise or consist of an antibody or an antigen-binding portion thereof to the immune cell activation molecule or the disease associated antigen, or other proteins binding to the immune cell activation molecule or the disease associated antigen, such as a ligand of the immune cell activation molecule or the disease associated antigen, or a binding fragment thereof. In certain embodiments, the immune cell activation molecule is CD70, CD30, CD33, D28, CD137, CD7, CD69, CD2 or CD8. In certain embodiments, the immune cell activation molecule is CD8. In certain embodiments, the disease associated antigen is BCMA.

In certain embodiments, the CAR comprises (a) an extracellular antigen binding domain selective for the immune cell activation molecule or the disease associated antigen, (b) a transmembrane domain, and (c) an intracellular domain. In certain embodiments, the extracellular antigen binding domain may comprise an antibody or an antigen-binding portion thereof to the immune cell activation molecule or the disease associated antigen, e.g., a scFv or an sdAb against the immune cell activation molecule or the disease associated antigen. In certain embodiments, the extracellular antigen binding domain specifically binds to a disease associated antigen such as a tumor associated antigen, an infectious disease associated antigen, or an inflammatory disease associated antigen. In certain embodiments, the extracellular antigen binding domain may specifically bind to CD19, CD20, CD22, CD4, CD24, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα) , mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, HER-2, CD4, HBsAg, LMP-1, LMP2, IL17R, CD20 and CD6. In certain embodiments, the extracellular antigen binding domain may specifically bind to BCMA. In certain embodiments, the extracellular antigen binding domain may specifically bind to an immune cell activation molecule such as CD70, CD30, CD33, D28, CD137, CD7, CD69, CD2 and CD8. In certain embodiments, the extracellular antigen binding domain specifically binds to CD8. In certain embodiments, the CAR is a monospecific tandem CAR, wherein the extracellular antigen binding domain may comprise more than one antibody or antigen binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that recognize the same epitope on the immune cell activation molecule or the disease associated antigen. In certain embodiments, the CAR is a bispecific or multispecific tandem CAR, wherein the extracellular antigen binding domain may comprise more than one antibody or antigen binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that recognize different epitopes on an immune cell activation molecule and/or more than one immune cell activation molecule of the disclosure, or recognize different epitopes on a disease associated antigen and/or more than one disease associated antigen. In certain embodiments, the transmembrane domain may comprises CD8α transmembrane region or CD28 transmembrane region. In certain embodiments, the transmembrane domain may comprise a CD8α transmemrabe region having e.g., the amino acid sequence of SEQ ID NO: 14. In certain embodiments, the intracellular domain may comprise at least one signaling domain selected from the group consisting of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In certain embodiments, the intracellular domain may comprise at least one costimulatory domains selected from the group consisting of CD28, 4-1BB (CD137) , CD27, OX40, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, CD83, and a ligand that specifically binds with CD83. In certain embodiments, the intracellular domain may comprise a CD3ζ signaling domain and a 4-1BB costimulatory domain. In certain embodiments, the intracellular domain may comprise a CD3ζ signaling domain and a CD28 costimulatory domain. In certain embodiments, the intracellular domain may comprises a 4-1BB intracellular domain and a CD3ζ signaling domain. The 4-1BB intracellular domain may comprise the amino acid sequence of SEQ ID NO: 15. The CD3ζ signaling domain may comprise the amino acid sequence of SEQ ID NO: 16. In certain embodiments, the CAR may further comprise a CD8α hinge, having e.g., the amino acid sequence of SEQ ID NO: 13, between the extracellular antigen binding domain and the transmembrane domain. In certain embodiments, the CAR may further comprise a leader sequence at the N-terminus.

In certain embodiments, the recombinant TCR complex comprises (i) a TCR complex comprising (a) a TCR chain selected from the group consisting of an alpha (α) chain, a beta (β) chain, a gamma (γ) chain and a delta (δ) chain of a T cell receptor, (b) an epsilon (ε) chain, a delta (δ) chain, and/or a gamma (γ) chain of CD3, and/or (c) a zeta (ζ) chain, and (ii) an extracellular antigen binding domain selective for the immune cell activation molecule or the disease associated antigen, fused to any of the chains of the TCR complex. The extracellular antigen binding domain may comprises an antibody or antigen binding portion thereof, such as a scFv or a sdAb, to the immune cell activation molecule or the disease associated antigen. In certain embodiments, the extracellular antigen binding domain compeises more than one antibody or antigen-binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that target the same epitope on an immune cell activation molecule or a disease associated antigen. In certain embodiments, the extracellular antigen binding domain compeises more than one antibody or antigen-binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that recognize different epitopes on an immune cell activation molecule and/or more than one immune cell activation molecule of the disclosure, or recognize different epitopes on a disease associated antigen and/or more than one disease associated antigen. In certain embodiments, the TCR complex may comprise a TCR-α chain, a TCR-β chain, a CD3γ chain, a CD3δ chain, two CD3ε chains and a ζ-chain, or part of these chains, wherein one or more of these chains are linked, with a linker or not, to an extracellular antigen binding domain selective for the immune cell activation molecule or the disease associated antigen. In certain embodiments, the extracellular antigen binding domain is linked to the CD3ε chain. The CD3ε chain may comprise the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the extracellular antigen binding domain is linked to the CD3ε chain via a linker having e.g., the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the TCR complex may comprise a TCR-γ chain, a TCR-δ chain, a CD3γ chain, a CD3δ chain, two CD3ε chains and a ζ-chain, or part of these chains, wherein one or more of these chains are linked, with a linker or not, to an extracellular antigen binding domain selective for the immune cell activation molecule or the disease associated antigen. In certain embodiments, the extracellular antigen binding domain is linked to the CD3ε chain. The CD3ε chain may comprise the amino acid sequence of SEQ ID NO: 21. In certain embodiments, the extracellular antigen binding domain is linked to the CD3ε chain via a linker such as the one having the amino acid sequence of SEQ ID NO: 20.

In certain embodiments, the dual antigen binding receptor is a dual CAR comprising (i) a first CAR comprising (a) an extracellular antigen binding domain selective for the immune cell activation molecule, (b) a transmembrane domain, and (c) an intracellular domain; and (ii) a second CAR comprising (a) an extracellular antigen binding domain selective for the disease associated antigen, (b) a transmembrane domain, and (c) an intracellular domain. In certain embodiments, the intracellular domain of each of the first CAR and the second CAR comprises a signaling domain and a costimulatory domain as described above. In certain embodiments, the intracellular domain of one of the first CAR and the second CAR comprises a signaling domain and a costimulatory domain as described above, and the intracellular domain of the other CAR comprises a costimulatory domain as described above.

In certain embodiments, the dual antigen binding receptor is a recombinant dual TCR complex which comprises (i) a first recombinant TCR complex comprising (a) an extracellular antigen binding domain selective for the immune cell activation molecule, fused to (b) a TCR complex; and (ii) a second recombinant TCR complex comprising (a) an extracellular antigen binding domain selective for the disease associated antigen, fused to (b) a TCR complex.

In certain emdodiments, the dual antigen binding receptor comprises (i) a CAR comprising (a) an extracellular antigen binding domain selective for the immune cell activation molecule, (b) a transmembrane domain, and (c) an intracellular domain; and (ii) a recombinant TCR complex comprising (a) an extracellular antigen binding domain selective for the disease associated antigen, fused to (b) a TCR complex. In certain emdodiments, the dual antigen binding receptor comprises (i) a recombinant TCR complex comprising (a) an extracellular antigen binding domain selective for the immune cell activation molecule, fused to (b) a TCR complex; and (ii) a CAR comprising (a) an extracellular antigen binding domain selective for the disease associated antigen, (b) a transmembrane domain, and (c) an intracellular domain.

In certain embodiments, the dual antigen binding receptor comprises (i) arecombinant TCR complex comprising (a) an extracellular antigen binding domain selective for CD8, fused to (b) a TCR complex; and (ii) a CAR comprising (a) an extracellular antigen binding domain selective for the disease associated antigen such as BCMA, (b) a transmembrane domain, and (c) an intracellular domain. In certain embodiments, the extracellular antigen binding domain in the TCR complex comprises an anti-CD8 sdAb having e.g., the amino acid sequence of SEQ ID NO: 19. In certain embodiments, the TCR complex may comprise a TCR-γ chain, a TCR-δ chain, a CD3γ chain, a CD3δ chain, two CD3ε chains and a ζ-chain, wherein the extracellular antigen binding domain is linked to the CD3ε chain. In certain embodiments, the extracellular antigen binding domain in the TCR is linked to a CD3ε chain having e.g., the amino acid sequence of SEQ ID NO: 21, via a linker having e.g., the amino acid sequence of SEQ ID NO: 20. In certain embodiments, the extracellular antigen binding domain in the CAR may comprise an anti-BCMA sdAb having e.g., the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the CAR may comprise a CD8α hinge, a CD8α transmembrane region, a 4-1BB intracellular domain and a CD3ζ signaling domain, having e.g., the amino acid sequences of SEQ ID NOs: 13, 14, 15 and 16, respectively.

The immuneresponsive cell may be a T cell, or a NK cell, including, but not limited to, an αβ T cell, a γδ T cell, a NKT cell, an iNKT cell, and a NKT like cell. The γδ T cell is selected from the group consisting of a Vγ9Vδ2 T cell, a Vδ1 T cell, a Vδ2 cell, a Vδ3 T cell, a Vδ5 cell, or the combination thereof.

In certain embodiments, the immuneresponsive cell is genetically engineered to express a low level of the immune cell activation molecule and the disease associated antigen (if any) , or not express the immune cell activation molecule or the disease associated antigen (if any) , by e.g, gene editing.

In a third aspect, the present disclosure provides a tandem antigen binding receptor which comprises an extracellular antigen binding domain comprising (i) a first antigen binding domain directed toward an immune cell activation molecule as defined herein, and (ii) a second antigen binding domain directed toward a disease associated antigen as defined herein. Also provided is an immuneresponsive cell which comprises the tandem antigen binding receptor. The disease associated antigen in certain embodiments may be the immune cell activation molecule described herein.

The tandem antigen binding receptor may be a tandem chimeric antigen receptor (CAR) which comprises (a) an extracellular antigen binding domain comprising (i) a first antigen binding domain, selective for the immune cell activation molecule, and (ii) a second antigen binding domain, selective for the disease associated antigen; (b) a transmembrane domain, and (c) an intracellular domain. The tandem antigen binding receptor may be a recombinant tandem T cell receptor (TCR) complex which comprises (a) an extracellular antigen binding domain comprising (i) a first antigen binding domain, selective for the selective for the immune cell activation molecule and (ii) a second antigen binding domain, selective for the disease associated antigen, fused to (b) a TCR complex described herein. The first and second antigen binding domains may each comprise an antibody or an antigen-binding portion thereof to the immune cell activation molecule or the disease associated antigen, or other proteins binding to the immune cell activation molecule or the disease associated antigen, such as a ligand of the immune cell activation molecule or the disease associated antigen or a binding fragment thereof.

In certain embodiments, the tandem CAR comprises (a) an extracellular antigen binding domain comprising (i) a first antigen binding domain selective for the immune cell activation molecule, and (ii) a second antigen binding domain selective for the disease associated antigen, (b) a transmembrane domain, and (c) an intracellular domain. In certain embodiments, the first antigen binding domain may comprise an antibody or an antigen-binding portion thereof to the immune cell activation molecule, e.g., a scFv or an sdAb to the immune cell activation molecule. In certain embodiments, the first antigen binding domain may comprise more than one antibody or antigen binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that target the same epitope on an immune cell activation molecule. In certain embodiments, the first antigen binding domain comprises more than one antibody or antigen binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that target different epitopes on an immune cell activation molecule and/or more than one immune cell activation molecule of the disclosure. In certain embodiments, the second antigen binding domain may comprise an antibody or an antigen-binding portion thereof to the disease associated antigen, e.g., a scFv or an sdAb to the disease associated antigen. In certain embodiments, the second antigen binding domain may comprise more than one antibody or antigen binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that target the same epitope on a disease associated antigen. In certain embodiments, the second antigen binding domain comprises more than one antibody or antigen binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that target different epitopes on a disease associated antigen and/or more than one disease associated antigen. In certain embodiments, the transmembrane domain may comprises CD8α transmembrane region or CD28 transmembrane region. In certain embodiments, the intracellular domain may comprise at least one signaling domain selected from the group consisting of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In certain embodiments, the intracellular domain may comprise at least one costimulatory domains selected from the group consisting of CD28, 4-1BB (CD137) , CD27, OX40, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, CD83, and a ligand that specifically binds with CD83. In certain embodiments, the intracellular domain may comprise a CD3ζ signaling domain and a 4-1BB costimulatory domain. In certain embodiments, the intracellular domain may comprise a CD3ζ signaling domain and a CD28 costimulatory domain. In certain embodiments, the CAR may further comprise a CD8α hinge between the antigen binding domain and the transmembrane domain. In certain embodiments, the CAR may further comprise a leader sequence at the N-terminus.

In certain embodiments, the recombinant tandem TCR complex comprises (i) aTCR complex comprising (a) a TCR chain selected from the group consisting of an alpha (α) chain, a beta (β) chain, a gamma (γ) chain and a delta (δ) chain of a T cell receptor, (b) an epsilon (ε) chain, a delta (δ) chain, and/or a gamma (γ) chain of CD3, and/or (c) a zeta (ζ) chain, and (ii) an extracellular antigen binding domain selective for both the immune cell activation molecule and the disease associated antigen defined herein, fused to any of the chains of the TCR complex. The extracellular antigen binding domain may comprise a first antigen binding domain selective for the immune cell activation molecule, and a second antigen binding domain selective for the disease associated antigen. In certain embodiments, the first antigen binding domain may comprise an antibody or an antigen-binding portion thereof, e.g., a scFv or an sdAb, to the immune cell activation molecule. In certain embodiments, the first antigen binding domain comprises more than one antibody or antigen binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that target the same epitope on the immune cell activation molecule. In certain embodiments, the first antigen binding domain comprises more than one antibody or antigen binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that target different epitopes on an immune cell activation molecule and/or more than one immune cell activation molecule. In certain embodiment, the second antigen binding domain may comprise an antibody or an antigen-binding portion thereof to the disease associated antigen, e.g., a scFv or an sdAb to the disease associated antigen. In certain embodiments, the second antigen binding domain comprises more than one antibody or antigen binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that target the same epitope on a disease associated antigen. In certain embodiments, the second antigen binding domain comprises more than one antibody or antigen binding portion thereof, e.g., two scFvs, two sdAbs, or one scFv plus one sdAb, that target different epitopes on a disease associated antigen and/or more than one disease associated antigen. In certain embodiments, the TCR complex may comprise a TCR-α chain, a TCR-β chain, a CD3γ chain, a CD3δ chain, two CD3ε chains and a ζ-chain, or part of these chains, wherein one or more of these chains are linked, with a linker or not, to the extracellular antigen binding domain. In certain embodiments, the extracellular antigen binding domain is linked to the CD3ε chain. In certain embodiments, the TCR complex may comprise a TCR-γ chain, a TCR-δ chain, a CD3γ chain, a CD3δ chain, two CD3ε chains and a ζ-chain, or part of these chains, wherein one or more of these chains are linked, with a linker or not, to the extracellular antigen binding domain. In certain embodiments, the extracellular antigen binding domain is linked to the CD3ε chain.

The immuneresponsive cell may be a T cell, or a NK cell, including, but not limited to, an αβ T cell, a γδ T cell, a NKT cell, an iNKT cell, and a NKT like cell. The γδ T cell is selected from the group consisting of a Vγ9Vδ2 T cell, a Vδ1 T cell, a Vδ2 cell, a Vδ3 T cell, a Vδ5 cell or the combination thereof.

In certain embodiments, the immuneresponsive cell is genetically engineered to express a low level of the immune cell activation molecule and the disease associated antigen (if any) , or not express the immune cell activation molecule or the disease associated antigen (if any) , by e.g., gene editing.

The present disclosure also provides a pharmaceutical composition which comprises an effective amount of the immuneresponsive cells disclosed herein, and a pharmaceutically acceptable carrier. Nucleic acids encoding the CARs or TCR complexes of the disclosure, as well as the expression vectors and host cells comprising the nucleic acids, are also provided herein. In certain embodiments, nucleic acids encoding the dual antigen binding receptors of the disclosure are provided, which may each encode two CARs, two TCRs, or alternatively a CAR and a TCR linked by a self-cleaving 2A peptide. The 2A self-cleaving peptide enables generation of separate peptide products, such as a CAR fragment and a TCR fragment, and includes, but not limited to, T2A, P2A, E2A and F2A. In one embodiment, a nucleic acid is provided for encoding an anti-BCMA CAR and an anti-CD8 TCR linked by P2A, more specifically for encoding an anti-BCMA sdAb, a CD8α hinge, a CD8α transmembrane region, a 4-1BB intracellular domain, a CD3ζ signaling domain, a P2A peptide, a leader peptide, an anti-CD8 sdAb, a linker, and a CD3ε chain. In one embodiment, the nucleic acid encodes a polypeptide having the amino acid sequence of SEQ ID NO: 11.

In an aspect, there is provided a method of making or modifying an immune cell to obtain the immuneresponsive cell of the disclosure, which comprises introducing into the cell a nucleic acid that encodes a CAR and/or a recombinant TCR complex against an immunce cell activiation molecule, or a CAR and/or a recombinant TCR complex against an immunce cell activiation molecule and a disease associated antigen of the disclosure. In certain embodiments, the method comprises introducing into the cell two nucleic acids, one encoding a CAR or a recombinant TCR complex targeting the immune cell activation molecule, the other encoding a CAR or a recombinant TCR complex targeting the disease associated antigen. In certain embodiments, the method comprises introducing into the cell a nucleic acid encoding two CARs, two TCRs, or alternatively a CAR and a TCR linked by a self-cleaving 2A peptide. In certain embodiments, the method comprises introducing into the cell a nucleic acid encoding a polypeptide of SEQ ID NO: 11.

With the antigen binding receptor directed toward the immune cell activation molecule that is expressed on a cell of the immune system and related to the activation of the immune cell in an innate or adaptive immune response, the immuneresponsive cells of the disclosure, when infused into a subject, may kill the activated immune cells, such as the DCs, T cells and NK cells, especially the effector cells, against the immuneresponsive cells, so as to reduce or eliminate graft rejection, without repressing the entire immune system. Therefore, the immuneresponsive cell of the disclosure, with the antigen binding receptor directed toward both the immune cell activation molecule and a disease associated antigen, may be used to treat or slow down the progression of diseases such as tumors, infectious disease and inflammatory diseases, with enhanced efficacy. In certain embodiments, the immuneresponsive cells of the disclosure are engineered to express a low level of the immune cell activation molecule and the disease associated antigen (if any) , or not to express the immune cell activation molecule or the disease associated antigen (if any) , to which the CARs or TCR complexes in the immuneresponsive cells target, such that these cells of the disclosure will not hurt one another, or with limited hurt.

In one aspect, the present disclosure provides a method for reducing or eliminating graft rejection to allogeneic cells in a subject, comprising administering to the subject the pharmaceutical composition of the disclosure comprising the immuneresponsive cells having antigen binding receptors directed toward the immune cell activation molecule, prior to or together with the administration of the allogeneic cells.

In certain embodiments, the immuneresponsive cells in the pharmaceutical composition are allogeneic.

In certain embodiments, the subject is human.

In one aspect, the present disclosure provides a method for treating a disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of the disclosure which comprises immuneresponsive cells having antigen binding receptors directed toward both the immune cell activation molecule and the disease associated antigen described herein.

In certain embodiments, the disease is tumor. In certain embodiments, the tumor is a hematological tumor or a solid tumor. In certain embodiments, the tumor is leukemia, lymphomas, or myeloma.

In certain embodiments, the disease is an infectious disease, caused by e.g., virus infection, bacterial infection or fungal infection. In certain embodiment, the disease is acquired immune deficiency syndrome (AIDS) .

In certain embodiments, the disease is an inflamatory disease. In certain embodiments, the disease is autoimmune disease, such as myasthenia gravis.

In certain embodiments, the subject is human.

The immuneresponsive cell of the disclosure, with the antigen binding receptor directed toward the immune cell activation molecule, may be used to treat or alleviate inflammatory diseases such as autoimmune diseases, as the systematic administration or regional delivery of such immuneresponsive cells may target and eliminate immune cells, e.g., activated B cells, responsible for the diseases.

Accordingly, the present disclosure may provide a method for treating or alleviating an inflammatory disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of the disclosure comprising immuneresponsive cells having antigen binding receptors directed toward the immune cell activation molecule.

In certain embodiments, the inflammatory disease is an autoimmune disease.

In certain embodiments, the immuneresponsive cells in the pharmaceutical composition is allogeneic.

In certain embodiments, the subject is human.

Other features and advantages of the instant disclosure will be apparent from the following detailed description and examples which should not be construed as limiting. The contents of all references, GenBank entries, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC) , such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art. 53 (c) EPC and Rule 28 (b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent (s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise.

It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises” , “comprised” , “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes” , “included” , “including” , and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

FIG. 1 depicts an immuneresponsive immune cell comprising a first chimeric antigen receptor targeting a tumor associate antigen to eliminate tumor cells and a second chimeric antigen receptor targeting an immune cell activation molecule to decrease allogeneic rejection.

FIG. 2 depicts an immuneresponsive immune cell comprising a tandem chimeric antigen receptor with a first antigen binding domain targeting a tumor associate antigen to eliminate tumor cells and a second antigen binding domain targeting an immune response activation molecule to decrease allogeneic rejection.

FIG. 3 depicts an immuneresponsive immune cell comprising a chimeric antigen receptor targeting a tumor associate antigen to eliminate tumor cells and an engineered T cell receptor (γδ T cell receptor, left; αβ T cell receptor, right) for targeting an immune response activation molecule to decrease allogeneic rejection.

FIG. 4 shows the transduction efficiency of BSF17 CAR-γδ T cells and BSF17-H8 CAR-γδ T cells.

FIG. 5A and 5B show cytotoxicity of BSF17 CAR-γδ T cells and BSF17-H8 CAR-γδ T cells against H929 (A) and CD8 ⁺ αβT cells (B) .

FIG. 6A-6F show TNF-α (A) , GM-CSF (B) and IFN-γ (C) production by BSF17 CAR-γδT cells and BSF17-H8 CAR-γδ T cells when co-cultured with tumor cell H929, and TNF-α (D) , GM-CSF (E) and IFN-γ (F) production by BSF17 CAR-γδ T cells and BSF17-H8 CAR-γδ T cells when co-cultured with CD8 ⁺ αβT cells.

FIG. 7A-7E show proliferation of CD4 ⁺ αβ T cells (A) , CD8 ⁺ αβ T cells (B) , NK cells (C) , total γδ T cells (D) and CAR-γδ T cells (E) in two-way MLR assays of BSF17 CAR-γδ T cells and BSF17-H8 CAR-γδ T cells co-cultured with allogeneic or autologous PBMCs.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure generally provides methods and compositions for preventing allogeneic rejection of cells infused into a subject, without suppressing the entire host immune system. According to the invention, allogeneic cells may be infused into a subject together with immune cells engineered to comprise an antigen binding receptor, e.g., a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR) , directed toward an immune cell activation molecule that is expressed on an immune cell and related to the activation of the immune cell in an innate or adaptive immune response, and the engineered immune cells may target and kill activated host immune cells against the allogeneic cells and the engineered immune cells in the subject. Alternatively, certain immune cell like allogeneic therapeutic cells may be engineered to comprise an antigen binding receptor, e.g., a chimeric antigen receptor (CAR) or a recombinant T cell receptor (TCR) , directed toward an immune cell activation molecule that is expressed on an immune cell and related to the activation of the immune cell in an innate or adaptive immune response, and, when infused into a subject, kills activated immune cells against the allogeneic cells in the subject.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

Provided herein are an immuneresponsive cell which comprises an antigen binding receptor directed toward an immune cell activation molecule; an immuneresponsive cell which comprises a dual antigen binding receptor comprising (i) a first antigen binding receptor directed toward an immune cell activation molecule; and (ii) a second antigen binding receptor directed toward a disease associated antigen; and an immuneresponsive cell which comprises a tandem antigen binding receptor comprising an extracellular antigen binding domain that comprises (i) a first antigen binding domain directed toward an immune cell activation molecule, and (ii) a second antigen binding domain directed toward a disease associated antigen. Also provided are the pharmaceutical compositions comprising the immuneresponsive cells of the disclosure, nucleic acids encoding the CARs or TCR complexes of the disclosure, expression vectors and host cells comprising the nucleic acids, methods for making the immuneresponsive cells of the disclosure, as well as methods of using the immuneresponsive cells of the disclosure for reducing allogeneic rejection of cells infused into a subject, for treating tumors or inflammatory diseases.

Immune Cell Activation Molecule

The immune cell activation molecule described herein refers to any molecule that is expressed on a cell of the immune system and related to the activation of the immune cell in an immune response. Such molecules may be required for stimulation and/or regulation of an immune response, or required for the activation, function and regulation of an immune cell in an immune response. Some molecules may be not responsible for the immune cell activation, function and/or regulation in an immune response, but just simply indicate the activation of an immune response. The immune cell here may include, but not limited to, a neutrophil, an eosinophil, a basophil, a mast cell, a monocyte, a macrophage, a dendritic cell, a natural killer cell, and a lymphocyte.

The immune cell activation molecule in certain embodiments is uniquely expressed on activated immune cells, but not on non-immune cells or resting immune cells. The immune cell activation molecule in certain embodiments is highly expressed on activated immune cells, but not expressed or expressed at a low level on non-immune cells or resting immune cells. The unique or high level expression of the immune cell activation molecules may help the immuneresponsive cells of the disclosure to precisely target and kill host immune cells against the immuneresponsive cells themselves or other host cells administered together with or later than the immuneresponsive cells. The unique or high level expression of the immune cell activation molecules may also help the immuneresponsive cells of the disclosure to precisely target and kill host immune cells causing inflammatory diseases.

In certain embodiments, the immune cell activation molecule may be a protein that is expressed on an immune cell and responsible for the activation, function and/or proliferation of the immune cell itself and/or another type of immune cell in an immune response. Such immune cell activation molecules include, but not limited to, costimulatory proteins such as CD30, CD33, D28, CD137, CD7, and CD69 expressed on immune cells such as T cells, NK cells and/or B cells, and non-costimulatory proteins such as CD2 and CD8 expressed on T cells and/or NK cells, and CD70 expressed on activated T cells and NK cells and dendritic cells (DC) with upregulated expression upon DC maturation. In certain embodiments, the immune cell activation molecule is CD8, CD70, CD30 or CD33. In certain embodiments, the immune cell activation molecular is CD8. In certain embodiments, the immune cell activation molecule is CD70. In certain embodiments, the immune cell activation molecule is CD30. In certain embodiments, the immune cell activation molecule is CD33.

CD70

CD70 is a member of the TNF family whose expression is upregulated on DCs upon maturation. CD70 is also expressed on activated T cells and NK cells. Its only receptor, CD27, is expressed on T cells and NK cells. The CD70-CD27 pathway promotes effector CD8 ⁺ T cells responses (by sustaining survival of CTLs) and influences polarization of CD4 ⁺ T cells, as it represents an alternative IL-12-independent pathway of Th1 priming and suppresses effector Th17 function. CD70 blockade prolonged survival of fully mismatched vascularized cardiac allografts in wild-type murine recipients, and in CD28-deficient mice induced long-term survival while significantly preventing the development of chronic allograft vasculopathy. CD70 blockade had little effect on CD4 ⁺ T cell function but prevented CD8 ⁺ T cell-mediated rejection, inhibited the proliferation and activation of effector CD8 ⁺ T cells, and diminished the expansion of effector and memory CD8 ⁺ T cells in vivo. Thus, the CD27-CD70 pathway is critical for CD28-independent effector/memory CD8 ⁺ alloreactive T cell activation in vivo. An exemplary amino acid sequence of CD70 is set forth in SEQ ID NO: 2.

CD33

CD33, also known as Siglec-3, has been found expressed at protein and nucleic acid levels in some alloantigen-activated T and NK cells. It was reported to work as an inhibitory receptor on NK cells, as the cross-linking of CD33 diminished the cytotoxic activity of NKL cells against K562 and P815 target cells (Hernández-Caselles T, et al. (2006) J Leukoc Biol. 79 (1) : 46-58) . An exemplary amino acid sequence of CD33 is set forth in SEQ ID NO: 9.

CD30

CD30, a member of the TNFR family, is expressed on activated T cells, NK cells, and B cells. Its ligand, CD30L (CD153) , is expressed on activated T cells, resting B cells, and macrophages. Early in vitro studies led to the classification of CD30 as a T-cell “costimulatory receptor” based on observations that immobilized CD30-specific mAb or CD30L-transfected cells enhance the proliferation of human T cells in response to suboptimal stimulation via the TCR. As CD30 is expressed on T cells rather late after in vitro activation, it is possible that CD30-CD30L interactions occurring relatively late after antigen encounter promote T-cell survival and/or establishment of strong memory responses. CD30 signaling regulates peripheral T-cell responses, controlling T-cell survival, and downregulating cytolytic capacity.

The role of the CD30-CD30L interaction in GvHD has been demonstrated in murine BMT models using a neutralizing anti-CD30L mAb, CD30-/-donor mice, and CD30L-/-recipient mice. Recipients receiving CD30-/-donor T cells had longer survival compared to those receiving WT T cells in an MHC class II disparate BMT model. Similarly, administration of a blocking anti-CD30L mAb improved survival in mice after receiving MHC disparate CD4 ⁺ T cells. Thus, blockade of the CD30-CD30L pathway represents a new approach for preventing CD4 ⁺ T cell–mediated GvHD. CD30-expressing lymphocytes were found in the intestine and skin of GvHD patients. CD30 expression on CD8 T cells and plasma level of soluble CD30 are increased in patients with aGvHD after HCT, suggesting CD30 may be a potential biomarker for GvHD in clinic. An exemplary amino acid sequence of CD30 is set forth in SEQ ID NO: 1.

CD28

CD28 (Cluster of Differentiation 28) is one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival. T cell stimulation through CD28 in addition to the T-cell receptor (TCR) can provide a potent signal for the production of various interleukins (IL-6 in particular) . IL-2-activated NK cells express CD28, although at lower levels than αβ T cells. In addition to IL-2, NK cells require CD28-mediated costimulatory signals for optimal proliferation. IL-2-activated NK cells produced enhanced levels of IFN-γ and TNFα in response to stimulation with anti-CD28 and PMA. An exemplary amino acid sequence of CD28 is set forth in SEQ ID NO: 3.

CD137

CD137 is a member of the tumor necrosis factor (TNF) receptor family, and is rapidly expressed on activated CD8 ⁺ T cells and on activated CD4 ⁺ T cells with lower levels. It is of interest to immunologists as a co-stimulatory immune checkpoint molecule. NK cells could be induced by IL-2 and IL-15 to express CD137 and ligation of CD137-stimulated NK cell proliferation and IFN-γ secretion. Importantly, CD137-stimulated NK cells promoted the expansion of activated T cells in vitro, demonstrating immunoregulatory or “helper” activity for CD8 ⁺ CTL. An exemplary amino acid sequence of CD137 is set forth in SEQ ID NO: 4.

CD7

CD7 is a transmembrane glycoprotein and a member of the immunoglobulin supergene family. It is expressed on human T and natural killer cells and on cells in the early stages of T-, B-, and myeloid cell differentiation. Its expression is augmented on activated alloimmune-responsive T cells. CD7 has been thought to be an attractive target for MAbs, offering the possibility of alloimmune-activated T-cell-specific depletion. The CD7 was found to be expressed at a significantly higher level on fresh NK cells than on IL- 2-activated NK cells. An exemplary amino acid sequence of CD7 is set forth in SEQ ID NO: 5.

CD2

CD2 (cluster of differentiation 2) is a cell adhesion molecule found on the surface of T cells and natural killer (NK) cells. The CD2 on T cells binds to LFA-3 (CD58) or CD48 expressed on a wide variety of cells. Antibodies to CD2 have been shown to induce T cell proliferation, but their function is dependent on TCR expression. CD2 ligation results in augmented tyrosine phosphorylation of PLCγ1 and Ca2 ⁺ mobilization. NK cells expressed high levels of CD2, which synergistically enhanced ERK and S6RP phosphorylation following CD16 ligation. Thus, CD2 co-stimulation was critical for the ability of NK cells to respond to antibody-coated target cells. An exemplary amino acid sequence of CD2 is set forth in SEQ ID NO: 6.

CD69

CD69 is a differentiation antigen expressed shortly after activation on T lymphocytes and other cells of hematopoietic origin, including natural killer (NK) cells. The function of CD69 on T lymphocytes acting as a costimulatory molecule in proliferation and lymphokine secretion is well established. NK cells express CD69 after activation by different stimuli such as phorbol 12-myristate 13-acetate (PMA) , interleukin (IL) -2, IL-12, interferon-α (IFN-α) or anti-CD16 monoclonal antibodies (mAbs) . An exemplary amino acid sequence of CD69 is set forth in SEQ ID NO: 7.

CD8

CD8 (cluster of differentiation 8) is a transmembrane glycoprotein that serves as a co-receptor for the T-cell receptor (TCR) . Along with the TCR, the CD8 co-receptor plays a role in T cell signaling and aiding with cytotoxic T cell antigen interactions. CD8 ⁺ T cells are recognized as T cytotoxic cells once they become activated and are generally classified as having a pre-defined cytotoxic role within the immune system. An exemplary amino acid sequence of CD8 is set forth in SEQ ID NO: 8. An exemplary immunoresponsive cell of the disclosure with an anti-CD8 TCR showed inhibitory effect on growth of NK cells and CD8 ⁺ T cells both allogeneic and autologous.

The immuneresponsive cells of the disclosure in certain embodiments are engineered to express a low level of the immune cell activation molecule, or not to express the immune cell activation molecule, to which the CARs or TCR complexes in the immuneresponsive cells target, by e.g., gene editing, such that these cells of the disclosure will not hurt one another.

The immuneresponsive cells with modified immune cell activation molecule expression can be obtained by any suitable means, including a knock out or knock down of the immune cell activation molecule. For example, the immuneresponsive cell can include a knock down of the immune cell activation molecule using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR) , transcription-activator like effector nuclease (TALEN) , or zinc finger endonuclease (ZFN) . These means for downregulating the expression of the immune cell activation molecule will be further described hereinafter in the “Allogeneic CAR and TCR effector cells” part.

Disease Associated Antigen

The disease associated antigen herein refers to any protein marker for a specific disease.

The disease associated antigen in certain embodiments is a tumor associated antigen, which is a cell surface molecule expressed on tumor cells. The tumor associated antigen may be selected, depending on the specific tumor to treat. The tumor associated antigen is preferably uniquely expressed on tumor cells, but not on non-tumor cells. Alternatively, the tumor associated antigen is preferably highly expressed on tumor cells, but expressed at a low level or not expressed at all on tumor cells.

In certain embodiments, the tumor associated antigen is, e.g., CD19, CD20, CD22, CD4, CD24, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, HBsAg, LMP-1, LMP2, folate receptor (FRα) , mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, HER-2 and combinations thereof

The disease associated antigen in certain embodiments is an infectious disease associated antigen, which may be a marker molecule on a pathogen, or a disease marker on an infected cell. For example, CD4 is the target protein for AIDS treatmenet. Other infectious disease associated antigens include, but not limited to HBsAg, LMP-1, and LMP2. The infectious disease associated antigen is preferably uniquely expressed on pathogens, including certain viruses, bacteria, and fungi, and/or on infected cells. Alternatively, the infectious disease associated antigen is preferably highly expressed on pathogens and/or infected cells, but expressed at a low level or not expressed at normal host cells.

The disease associated antigen in certain embodiments is an inflammatory disease associated antigen, which may be a marker on activated immune cells responsible for the inflammations. The inflammatory disease associated antigens includes, but not limited to, IL17R, CD20, and CD6. The infectious disease associated antigen is preferably uniquely expressed on, e.g., activated immune cells responsible for the unwanted inflammation. Alternatively, the inflammatory disease associated antigen is preferably highly expressed, e.g., activated immune cells responsible for the unwanted inflammation, but expressed at a low level or not expressed at other normal cells.

In certain embodiments, the disease associated antigen is the immune cell activation molecule described herein.

Chimeric Antigen Receptor

The immuneresponsive cells of the disclosure comprise CARs, including but not limited to what are referred to as first-generation, second-generation, third-generation, and “armored” CARs.

The term “chimeric antigen receptor” or “CAR” as used herein refers to an artificially constructed hybrid protein or polypeptide containing an antigen binding moiety (e.g., an antibody) linked to immune cell (e.g. T cell) signaling or activation domains. In certain embodiments, CARs are synthetic receptors that retarget T or NK cells to cell surface antigens, such as tumor surface antigens (Sadelain et al., (2003) Nat. Rev. Cancer 3 (l) : 35-45; Sadelain et al., (2013) Cancer Discovery 3 (4) : 388-398) . CARs can provide both antigen binding and immune cell activation functions onto an immune cell such as a T cell or a NK cell. CARs have the ability to redirect T-cell or NK cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition can give T-cells or NK cells expressing CARs the ability to recognize an antigen independent of antigen processing, thus bypassing a mechanism of tumor escape.

In certain embodiments, the chimeric receptor comprises an extracellular antigen binding domain specific for one or more antigens (such as an immune cell activation molecule and/or a tumor associated antigen) or epitopes (such as epitopes on an immune cell activation molecule and/or a tumor associated antigen) , a transmembrane domain, and an intracellular domain comprising a signaling domain of a T cell and/or co-stimulatory domains. “CAR-T” refers to a T cell that expresses a CAR. “CAR-NK” refers to a NK cell that expresses a CAR. “Anti-CD19 CAR” refers to a CAR having an extracellular binding domain specific for CD19, “anti-BCMA CAR” refers to a CAR having an extracellular binding domain specific for BCMA.

Several “generations” of CARs have been developed. First-generation CAR T-cells utilize an intracellular domain from the CD3ζ-chain of the TCR, which provides so called ‘signal 1, ’ and induces cytotoxicity against targeted cells. Engagement and signaling via the CD3ζchain is required for T-cell stimulation and proliferation but is not often sufficient for sustained proliferation and activity in the absence of a second signal or ‘signal 2. ’ Second-generation CARs were developed to enhance efficacy and persistence in vivo after reinfusion into a subject and contain an second costimulatory signaling domain (CD28 or 4-1BB) that functions to provide ‘signal 2’ to mitigate anergy and activation-induced cell death seen with first generation CAR T-cells. Third-generation CARs are further optimized by use of two distinct costimulatory domains in tandem, e.g., CD28/4-1BB/CD3ζ or CD28/OX-40/CD3ζ (see, e.g., Yeku et al., (2016) Biochem Soc Trans. 44 (2) : 412) . CARs have been further optimized or “armored” to secrete active cytokines or express costimulatory ligands that further improve efficacy and persistence. There are several variants of armored CAR T-cells, designed for example to express IL-12, CD40L, or 4-1BBL.

Another class of CARs is based on NK cells, γδ cell and NK cell receptors. NK cells have robust antitumor activity and many of their receptors can recognize various stress induced or overexpressed ligands on different tumors, thereby activating NK cell cytotoxicity. (Spear P. et al., 2013, NKG2D ligands as therapeutic targets. Cancer Immunity 13: 8) . NK receptor based CARs are engineered with intact extracellular binding domains of NK cell receptors fused to cytoplasmic activation and costimulatory domains. The NKG2D CAR has been shown to recognize myelomas, lymphomas, and ovarian cancers. (Gacerez et al., 2016, J. Cell Physiol. 231 (12) : 2590) .

Intracellular Signaling Domain

The intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (such as T cell) . In certain embodiments, the primary intracellular signaling domain is derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In certain embodiments, the primary intracellular signaling domain is derived from CD3ζ (i.e., “a CD3ζ intracellular signaling domain” ) . In certain embodiments, the intracellular signaling domain comprises an intracellular co-stimulatory sequence. In certain embodiments, the intracellular signaling domain comprises both a primary intracellular signaling domain (e.g., a CD3ζ intracellular signaling domain) and an intracellular co-stimulatory domain. In certain embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain but does not comprise an intracellular co-stimulatory domain. In certain embodiments, the intracellular signaling domain comprises an intracellular co-stimulatory sequence but does not comprise a primary intracellular signaling domain.

Co-stimulatory Domain

Co-stimulatory domain" (CSD) as used herein refers to the portion of the CAR which enhances the proliferation, survival and/or development of memory cells. The CARs of the disclosure may comprise one or more co-stimulatory domains. Each costimulatory domain comprises a costimulatory domain of any one or more of, for example, members of the TNFR superfamily, CD28, CD137 (4-lBB) , CD134 (OX40) , DaplO, CD27, CD2, CD5, ICAM-1, LFA-1 (CD1 la/CD18) , Lck, TNFR-I, TNFR-II, Fas, CD30, CD40 or combinations thereof. Further costimulatory domains used with the invention comprise one or more of: 2B4/CD244/SLAMF4, 4-1BB/TNFSF9/CD137, B7-1/CD80, B7-2/CD86, B7-H1/PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BAFF-R/TNFRSF13C, BAFF/BLyS/TNFSF13B, BLAME/SLAMF8, BTLA/CD272, CD100 (SEMA4D) , CD103, CD11a, CD11b, CD11c, CD11d, CD150, CD160 (BY55) , CD18, CD19, CD2, CD200, CD229/SLAMF3, CD27 Ligand/TNFSF7, CD27/TNFRSF7, CD28, CD29, CD2F-10/SLAMF9, CD30 Ligand/TNFSF8, CD30/TNFRSF8, CD300a/LMIR1, CD4, CD40 Ligand/TNFSF5, CD40/TNFRSF5, CD48/SLAMF2, CD49a, CD49D, CD49f, CD53, CD58/LFA-3, CD69, CD7, CD8α, CD8β, CD82/Kai-1, CD84/SLAMF5, CD90/Thy1, CD96, CDS, CEACAM1, CRACC/SLAMF7, CRTAM, CTLA-4, DAP12, Dectin-1/CLEC7A, DNAM1 (CD226) , DPPIV/CD26, DR3/TNFRSF25, EphB6, GADS, Gi24/VISTA/B7-H5, GITR Ligand/TNFSF18, GITR/TNFRSF18, HLA Class I, HLA-DR, HVEM/TNFRSF14, IA4, ICAM-1, ICOS/CD278, Ikaros, IL2Rβ, IL2Rγ, IL7Rα, Integrin α4/CD49d, Integrin α4β1, Integrin α4β7/LPAM-1, IPO-3, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, KIRDS2, LAG-3, LAT, LIGHT/TNFSF14, LTBR, Ly108, Ly9 (CD229) , lymphocyte function associated antigen-1 (LFA-1) , Lymphotoxin-α/TNF-β, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1) , NTB-A/SLAMF6, OX40 Ligand/TNFSF4, OX40/TNFRSF4, PAG/Cbp, PD-1, PDCD6, PD-L2/B7-DC, PSGL1, RELT/TNFRSF19L, SELPLG (CD162) , SLAM (SLAMF1) , SLAM/CD150, SLAMF4 (CD244) , SLAMF6 (NTB-A) , SLAMF7, SLP-76, TACI/TNFRSF13B, TCL1A, TCL1B, TIM-1/KIM-1/HAVCR, TIM-4, TL1A/TNFSF15, TNF RII/TNFRSF1B, TNF-α, TRANCE/RANKL, TSLP, TSLP R, VLA1, and VLA-6.

Antigen Binding Domain in CAR

CARs typically comprises an antibody or an antigen-binding portion thereof, such as a scFv or sdAb, as or at the extracellular antigen binding domain, to target cell surface antigens of target cells.

The term “antibody” as used herein in some instances refers to an immunoglobulin molecule that recognizes and specifically binds a target, through at least one antigen-binding site wherein the antigen-binding site is usually within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, single-chain Fv (scFv) antibodies, heavy chain antibodies (HCAbs) , light chain antibodies (LCAbs) , multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, fusion proteins comprising an antigen-binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen-binding site (e.g., dual variable domain immunoglobulin molecules) as long as the antibodies exhibit the desired biological activity. Antibodies also include, but are not limited to, mouse antibodies, chimeric antibodies, humanized antibodies, and human antibodies. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) , based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Unless expressly indicated otherwise, the term “antibody” as used herein include “antigen-binding portion” of the intact antibodies. An IgG is a glycoprotein which may comprise two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain may be comprised of a heavy chain variable region (abbreviated herein as V _H) and a heavy chain constant region. The heavy chain constant region may be comprised of three domains, C _H1, C _H2 and C _H3. Each light chain may be comprised of a light chain variable region (abbreviated herein as V _L) and a light chain constant region. The light chain constant region may be comprised of one domain, C _L. The V _H and V _L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR) , interspersed with regions that are more conserved, termed framework regions (FR) . Each V _H and V _L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term “antigen-binding portion” or “antigen-binding fragment” as used in connection with an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigen-binding portion” of an antibody include, but not limited to, (i) a Fab fragment, a monovalent fragment consisting of the V _L, V _H, C _L and C _H1 domains; (ii) a F (ab') ₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V _H and C _H1 domains; (iv) a Fv fragment consisting of the V _L and V _H domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546) , which consists of a V _H domain; (vi) an isolated complementarity determining region (CDR) ; and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, V _L and V _H, are coded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V _L and V _H regions pair to form monovalent molecules (known as single chain Fv (scFv) ; see e.g., Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883) . Such single chain antibodies are also intended to be encompassed within the term “antigen-binding portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

The term “antibody” also includes the heavy chain only antibody or the antigen-binding portion thereof. The term “heavy chain only antibody” or “HCAb” refers to a functional antibody, which comprises heavy chains, but lacks the light chains usually found in 4-chain immunoglobulins. The naturally occurring heavy chain only antibodies are found in e.g., camelids (such as camels, llamas, or alpacas) . Each camelid heavy chain only antibody contains a heavy chain variable region/domain, called V _HH domain, V _HH fragment or single chain antibody (sdAb) , and a heavy chain constant region. The V _HH functions to interact with an antigen. The V _HH contains three complementarity determining regions (CDRs) and four framework regions (FRs) , arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain constant region contains a hinge region, a CH2 domain and a CH3 domain. The lacking C _H1 domain is replaced with an extended hinge region. In a chimeric or humanized heavy chain only antibody, the heavy chain constant region may contain a typical IgG, such as IgG1, IgG2 or IgG4, constant region. The constant region may mediate the binding of the heavy chain only antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The “antigen binding portion” as used in connection with a heavy chain only antibody refers to one or more fragments of a heavy chain only antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of a heavy chain antibody can be performed by fragments of a full-length heavy chain only antibody. Examples of “antigen-binding portions of a heavy chain only antibody include, but not limited to, (i) an isolated complementarity determining region (CDR) ; (ii) a monovalent V _HH fragment; (iii) a bivalent fragment comprising two monovalent V _HH fragments; (iv) a monovalent fragment comprising a V _HH fragment linked to a partial heavy chain constant region, such as a V _HH domain linked to the CH2 domain, or CH2 and CH3 domains of a heavy chain constant region; (v) a bivalent fragment comprising two V _HH fragments each linked to a partial heavy chain constant region; (vi) multiple monovalent V _HH domains linked with or without linkers.

The term “single domain antibody” or “sdAb” refers to a single antigen-binding polypeptide comprising a single monomeric variable antibody domain having three complementary determining regions (CDRs) , which is capable of binding to an antigen without pairing with a corresponding CDR-containing polypeptide. In some cases, the single domain antibody is engineered from a camelid HCAb, and is also called the V _HH domain or fragment of the HCAb. The single domain antibody is a kind of antigen-binding portion of a heavy chain only antibody. The V _HHs may also be known as Nanobodies. Camelid sdAb is one of the smallest known antigen binding antibody fragments (see, e.g., Hamers-Casterman et al., (1993) Nature 363: 446-8; Greenberg et al., (1995) Nature 374: 168-73; Hassanzadeh-Ghassabeh et al., (2013) Nanomedicine (Lond) , 8: 1013-26) .

The antigen binding domains take many forms. Non-limiting examples include bispecific receptors (Zakaria Grada, et al. (2013) Molecular Therapy, 2 (7) : e105) , single domain VHH based CARs (De Meyer T, et al., (2014) Trends Biotechnol. 32 (5) : 263-70) , and “universal” CARs comprising avidin that binds to any antigen receptor that incorporates biotin (Huan Shi, et al. (2014) Molecular Cancer, 13: 219) .

The antigen binding domain can be made specific for any disease-associated target, including but not limited to tumor-associated antigens (TAAs) , infectious disease targets, and targets useful to treat or reduce autoimmunity and allograft rejection. In certain embodiments, the antigen binding domain is bispecific, targeting both the immune cell activation molecule and the disease associated antigen. Antigens have been identified in most of the human cancers, including Burkitt lymphoma, neuroblastoma, melanoma, osteosarcoma, renal cell carcinoma, breast cancer, prostate cancer, lung carcinoma, and colon cancer. TAA’s include, without limitation, CD19, CD20, CD22, CD24, , CD4, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, HBsAg, LMP-1, LMP2, folate receptor (FRα) , mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP and HER-2. TAAs further include neoantigens, peptide/MHC complexes, and HSP/peptide complexes. In certain embodiments, the antigen binding domain is specific to a defined tumor associated antigen, such as but not limited to FRα, CEA, 5T4, CA125, SM5-1 or CD71. In certain embodiments, the tumor associated antigen can be a tumor-specific peptide-MHC complex. In certain such embodiments, the peptide is a neoantigen. In other embodiments, the tumor associated antigen it a peptide-heat shock protein complex. In certain embodiments, targeting domains of CARs of the invention target tumor-associated antigens. In certain embodiments, the tumor-associated antigen is selected from: 707-AP, a biotinylated molecule, a-Actinin-4, abl-bcr alb-b3 (b2a2) , abl-bcr alb-b4 (b3a2) , adipophilin, AFP, AIM-2, Annexin II, ART-4, BAGE, BCMA, b-Catenin, bcr-abl, bcr-abl p190 (e1a2) , bcr-abl p210 (b2a2) , bcr-abl p210 (b3a2) , BING-4, CA-125, CAG-3, CAIX, CAMEL, Caspase-8, CD171, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v7/8, CD70, CD123, CD133, CDC27, CDK-4, CEA, CLCA2, CLL-1, CTAG1B, Cyp-B, DAM-10, DAM-6, DEK-CAN, DLL3, EGFR, EGFRvIII, EGP-2, EGP-40, ELF2, Ep-CAM, EphA2, EphA3, erb-B2, erb-B3, erb-B4, ES-ESO-1a, ETV6/AML, FAP, FBP, fetal acetylcholine receptor, FGF-5, FN, FR-α, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GD2, GD3, GnT-V, Gp100, gp75, GPC3, GPC-2, Her-2, HLA-A*0201-R170I, HMW-MAA, HSP70-2 M, HST-2 (FGF6) , HST-2/neu, hTERT, iCE, IL-11Rα, IL-13Rα2, KDR, KIAA0205, K-RAS, L1-cell adhesion molecule, LAGE-1, LDLR/FUT, Lewis Y, L1-CAM, MAGE-1, MAGE-10, MAGE-12, MAGE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A6, MAGE-B1, MAGE-B2, Malic enzyme, Mammaglobin-A, MART-1/Melan-A, MART-2, MC1R, M-CSF, mesothelin, MUC1, MUC16, MUC2, MUM-1, MUM-2, MUM-3, Myosin, NA88-A, Neo-PAP, NKG2D, NPM/ALK, N-RAS, NY-ESO-1, OA1, OGT, oncofetal antigen (h5T4) , OS-9, P polypeptide, P15, P53, PRAME, PSA, PSCA, PSMA, PTPRK, RAGE, ROR1, RU1, RU2, SART-1, SART-2, SART-3, SOX10, SSX-2, Survivin, Survivin-2B, SYT/SSX, TAG-72, TEL/AML1, TGFaRII, TGFbRII, TP1, TRAG-3, TRG, TRP-1, TRP-2, TRP-2/INT2, TRP-2-6b, Tyrosinase, VEGF-R2, WT1, α-folate receptor, and κ-light chain.

In certain embodiments, the antigen binding domain comprises a natural ligand of a tumor expressed protein or a tumor-binding fragment thereof. In certain embodiments, the antigen binding domain comprises a natural ligand of an immune cell activation molecule, or a molecular binding fragment thereof. For example, CD30L is the natural ligand of CD30, and CD27 naturally binds CD70.

Transmembrane Domain

“Transmembrane domain” (TMD) as used herein refers to the region of the CAR which crosses the plasma membrane. The transmembrane domain of the CAR of the disclosure is the transmembrane region of a transmembrane protein (for example Type I transmembrane proteins) , an artificial hydrophobic sequence or a combination thereof. Although the main function of the transmembrane is to anchor the CAR in the T cell membrane, in certain embodiments, the transmembrane domain influences CAR function. In certain embodiments, the transmembrane domain is from CD4, CD8α, CD28, or ICOS. Gueden et al. associated use of the ICOS transmembrane domain with increased CAR T cell persistence and overall anti-tumor efficacy (Guedan S. et al., (2018) JCI Insight. 3: 96976) . In an embodiment, the transmembrane domain comprises a hydrophobic α helix that spans the cell membrane. Other transmembrane domains will be apparent to those of skill in the art and may be used in connection with alternate embodiments of the invention. In certain embodiments, the transmembrane domain is a human transmembrane domain. In certain embodiments, the transmembrane domain comprises human CD8α transmembrane domain. In certain embodiments, the transmembrane domain comprises human CD28 transmembrane domain.

Hinge region

The CARs of the disclosure may comprise a hinge domain that is located between the extracellular antigen binding domain and the transmembrane domain. A hinge domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular domain relative to the transmembrane domain of the effector molecule can be used. The hinge domain may contain about 10-100 amino acids, e.g., about any one of 15-75 amino acids, 20-50 amino acids, or 30-60 amino acids. In certain embodiments, the hinge domain may be at least about any one of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 amino acids in length.

In certain embodiments, the hinge domain is a hinge domain of a naturally occurring protein. Hinge domains of any protein known in the art to comprise a hinge domain are compatible for use in the chimeric receptors described herein. In certain embodiments, the hinge domain is at least a portion of a hinge domain of a naturally occurring protein and confers flexibility to the chimeric receptor. In certain embodiments, the hinge domain is derived from CD8, such as CD8α. In certain embodiments, the hinge domain is a portion of the hinge domain of CD8α, e.g., a fragment containing at least 15 (e.g., 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8α. In certain embodiments, the hinge domain is derived from CD28.

Hinge domains of antibodies, such as an IgG, IgA, IgM, IgE, or IgD antibodies, are also compatible for use in the chimeric receptor systems described herein. In certain embodiments, the hinge domain is the hinge domain that joins the constant domains CH1 and CH2 of an antibody. In certain embodiments, the hinge domain is of an antibody and comprises the hinge domain of the antibody and one or more constant regions of the antibody. In certain embodiments, the hinge domain comprises the hinge domain of an antibody and the CH3 constant region of the antibody. In certain embodiments, the hinge domain comprises the hinge domain of an antibody and the CH2 and CH3 constant regions of the antibody. In certain embodiments, the antibody is an IgG, IgA, IgM, IgE, or IgD antibody. In certain embodiments, the antibody is an IgG antibody. In certain embodiments, the antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In certain embodiments, the hinge region comprises the hinge region and the CH2 and CH3 constant regions of an IgG1 antibody. In certain embodiments, the hinge region comprises the hinge region and the CH3 constant region of an IgG1 antibody.

Non-naturally occurring peptides may also be used as hinge domains for the chimeric antigen receptors described herein. In certain embodiments, the hinge domain between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain is a peptide linker, such as a (GxS) n linker, wherein x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

T Cell Receptor and T Cell Recepter Complex

The immuneresponsive cells of the disclosure may be also engineered to comprise a recombinant T cell receptor complex.

The term “T cell receptor, ” or “TCR, ” refers to a heterodimeric receptor composed of αβ or γδ chains that pair on the surface of a T cell. Each α, β, γ, and δ chain is composed of two Ig-like domains: a variable domain (V) that confers antigen recognition through the complementarity determining regions (CDR) , followed by a constant domain (C) that is anchored to cell membrane by a connecting peptide and a transmembrane (TM) region. Each of the V domains has three CDRs. These CDRs interact with a complex between an antigenic peptide bound to a protein encoded by the major histocompatibility complex (pMHC) (Davis and Bjorkman (1988) Nature, 334, 395-402; Davis et al. (1998) Annu Rev Immunol, 16, 523-544) .

The TCR alone is not able to mediate signal transduction due to its short cytoplasmic tail, and thus forms a complex with CD3 and ζ-chain accessory molecules. CD3 is composed of four distinct chains. In mammals, CD3 contains a CD3γ chain, a CD3δ chain, and two CD3ε chains. In certain embodiments, the TCR complex comprises (a) a TCR chain selected from the group consisting of an alpha (α) chain, a beta (β) chain, a gamma (γ) chain and a delta (δ) chain of a T cell receptor, (b) an epsilon (ε) chain, a delta (δ) chain, and/or a gamma (γ) chain of CD3, and/or (c) a zeta (ζ) chain.

The recombinant TCR complex of the disclosure may be prepared by linking an extracellular antigen binding domain to any chain of the TCR complex. In certain embodiments, the recombinant TCR complex may comprise a TCR-α chain, a TCR-β chain, a CD3γ chain, a CD3δ chain, two CD3ε chains and a ζ-chain, or part of the chains, wherein one or more of these chains are linked, with a linker or not, to an extracellular antigen binding domain selective for the immune cell activation molecule. In certain embodiments, the extracellular antigen binding domain is linked to the CD3ε chain. In certain embodiments, the TCR may comprise a TCR-γ chain, a TCR-δ chain, a CD3γ chain, a CD3δ chain, two CD3ε chains and a ζ-chain, or part of the chains, wherein one or more of these chains are linked, with a linker or not, to an extracellular antigen binding domain selective for the immune cell activation molecule. In certain embodiments, the extracellular antigen binding domain is linked to the CD3ε chain.

The linker may be the hinge domain defined above, or a peptide linker, such as a (GxS) n linker, wherein x and n, independently can be an integer between 3 and 12, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.

Tandem Antigen Binding Receptor

The tandem antigen binding receptor herein refers to a chimeric antigen receptor comprising more than one antigen binding domain in the extracellular antigen binding domain, or a recombinant T cell receptor complex comprising more than one antigen binding domain in the extracellular antigen binding domain. In contrast, the single CAR or TCR refers to a CAR or TCR that contains one antigen binding domain in the extracellular antigen binding domain.

The extracellular antigen binding domain in the tandem antigen binding receptor may be monospecific, comprising more than one antigen binding domains, e.g., more than one antibody or antigen-binding portion thereof, typically scFv and sdAb, that recognize an epitope on a molecule of interest (e.g., an immune cell activation molecule, a disease associated antigen) .

The extracellular antigen binding domain in the tandem antigen binding receptor may be bispecific or multispecific, comprising more than one antigen binding domains, e.g., more than one antibody or antigen-binding portion thereof, typically scFv and sdAb, that recognize different epitopes on a molecule of interest (e.g., an immune cell activation molecule, a disease associated antigen) , and/or more than one molecule of interest (e.g., more than one immune cell activation molecule, more than one disease associated antigen, or one immune cell activation molecule plus one disease associated antigen) .

Dual Antigen Binding Receptor

The dual antigen binding receptor herein refers to a combination of two CARs, two recombinant TCR complexes, or one CAR plus one recombinant TCR complex, of the disclosure. In certain embodiments, one CAR/TCR complex in the combination targets a molecule of interest, such as an immune cell activation molecule of the disclosure, and the other CAR/TCR complex in the combination targets another molecule of the interest, such as a disease associated antigen. In certain embodiments, one CAR/TCR complex in the combination targets a molecule of interest, such as an immune cell activation molecule of the disclosure, and the other CAR/TCR complex in the combination targets another molecule of the interest, such as another immune cell activation molecule of the disclosure.

In certain emdodiments, the dual antigen binding receptor comprises (i) a CAR comprising (a) an extracellular antigen binding domain selective for the immune cell activation molecule, (b) a transmembrane domain, and (c) an intracellular domain comprising a signaling domain and a costimulatory domain; and (ii) a recombinant TCR complex comprising (a) an extracellular antigen binding domain selective for the disease associated antigen, fused to (b) a TCR complex. In certain emdodiments, the dual antigen binding receptor comprises (i) a recombinant TCR complex comprising (a) an extracellular antigen binding domain selective for the immune cell activation molecule, fused to (b) a TCR complex; and (ii) a CAR comprising (a) an extracellular antigen binding domain selective for the disease associated antigen, (b) a transmembrane domain, and (c) an intracellular domain comprising a signaling domain and a costimulatory domain.

In certain embodiments, the dual antigen binding receptor comprises: (i) a chimeric antigen receptor comprising (a) an extracellular antigen binding domain which comprises an antigen binding domain specific to a first molecule, (b) a transmembrane domain, and (c) an intracellular domain; and (ii) a recombinant T cell receptor complex, comprising (a) an extracellular antigen binding domain which comprises an antigen binding domain specific to a second molecule, and (b) a T cell receptor complex comprising a TCR chain selected from the group consisting of an alpha (α) chain, a beta (β) chain, a gamma (γ) chain and a delta (δ) chain of a T cell receptor, an epsilon (ε) chain, a delta (δ) chain, and/or a gamma (γ) chain of CD3, and/or a zeta (ζ) chain, wherein the extracellular antigen binding domain specific to the second molecule is fused to any chain of the T cell receptor complex, wherein one of the first molecule and the second molecule is an immune cell activation molecule that is expressed on an immune cell and related to the activation of the immune cell in an immune response, and the other is a disease associated antigen. In certain embodiments, the immune cell activation molecule is CD70, CD30, CD33, D28, CD137, CD7, CD69, CD2 or CD8. In certain embodiments, the immune cell activation molecule is CD8. In certain embodiments, the disease associated antigen is a tumor associated antigen selected from the group consisting of CD19, CD20, CD22, CD4, CD24, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα) , mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, and HER-2, an infectious disease associated antigen selected from the group consisting of CD4, HBsAg, LMP-1, and LMP2, or an inflammatory disease associated antigen selected from the group consisting of IL17R, CD20, and CD6. In certain embodiments, the tumor associated antigen is BCMA. In certain embodiments, the dual antigen binding receptor comprises SEQ ID NO: 11.

In certain embodiments, the dual antigen binding receptor is a dual CAR. The dual CAR may be a combination of any two CARs, in which each of a first CAR and a second CAR may be a single CAR or a tandem CAR, i.e., single CAR/single CAR, single CAR/tandem CAR, or tandem CAR/tandem CAR. The levels of dual CAR T cell signaling may be regulated by manipulating the intracellular domains of each first and second CARs. For example, the intracellular domains of each of the first CAR and the second CAR may contain a co-stimulatory domain, such as CD28, 4-1ΒΒ (CD137) , ICOS, OX40 (CD134) , CD27, and/or DAP10, and/or a signaling domain from a Τ cell receptor, such as a signaling domain from a Τ cell receptor (e.g., CD3ζ) . For example, dual CAR of the present disclosure may include a first CAR and a second CAR each having an intracellular domain containing a co-stimulatory domain and a signaling domain from a Τ cell receptor. Thus, when the dual CAR bind antigens (e.g., bispecific) , the T cell signals may be transmitted through two signaling domains from a Τ cell receptor. The dual CAR of the present disclosure may also include a first CAR having an intracellular domain containing a co-stimulatory domain and a signaling domain from a Τ cell receptor and a second CAR having an intracellular domain containing a co-stimulatory domain. When either or both CARs bind to the antigen, the T cell signals may be transmitted through the signaling domain from a Τ cell receptor of the first CAR. All forms of CARs can be suitably used in the present invention, including but not limited to single CAR, tandem CAR, dual CAR, and the combinations thereof.

Cytokines

The immuneresponsive cells of the disclosure may be administered with cytokines in a subject, to enhance immune cell fitness and/or enhancing immune cell cytotoxicity. Alternatively, the immuneresponsive cells of the disclosure may be engineered to express and secrete one or more cytokines at high levels when the immunoresponsive cell is activated and at low levels or not at all when the immunoresponsive cell is not activated administered with cytokines in a subject.

Cytokines useful for enhancing immune cell fitness and/or enhancing immune cell cytotoxicity include, without limitation, IL-7, IL-12, IL-15, and IL-18.

Variants

In certain embodiments, amino acid sequence variants of the CARs, TCRs, especially the extracellular antigen binding domains in the CARs or TCRs are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the extracellular antigen binding domain, including the antibody or antigen-binding portion thereof contained in the extracellular antigen binding domain. Amino acid sequence variants of an antibody or an antigen binding portion thereof may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or antigen binding portion thereof, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody or antigen binding portion thereof. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

In certain embodiments, the antibody or antigen binding portion thereof comprising one or more amino acid substitutions, deletions, or insertions are provided. Sites of interest for mutational changes include the heavy and light chain variable regions such as CDRs and frameworks (FRs) . Amino acid substitutions may be introduced into the antibody or antigen binding portion thereof of interest and the products may be screened for a desired activity, e.g., retained/improved antigen binding or decreased immunogenicity. Accordingly, the invention encompasses antigen binding domains, CARs, and TCRs particularly disclosed herein as well as binding domains, CARs, and TCRs having at least 75%, at least 80%, at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%sequence identity to the amino acid sequences particularly disclosed herein. The terms “percent similarity, ” “percent identity, ” and “percent homology” when referring to a particular sequence are used as set forth in the University of Wisconsin GCG software program BestFit. Other algorithms may be used, e.g. BLAST, psiBLAST or TBLASTN (which use the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410) , FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448) ,

Particular amino acid sequence variants may differ from the specifically disclosed sequence by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 or 20-30 amino acids. In certain embodiments, a variant sequence may comprise the specifically disclosed sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues inserted, deleted or substituted. For example, 5, 10, 15, up to 20, up to 30 or up to 40 residues may be inserted, deleted or substituted.

In some preferred embodiments, a variant may differ from the specifically disclosed sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative substitutions. Conservative substitutions involve the replacement of an amino acid with a different amino acid having similar properties. For example, an aliphatic residue may be replaced by another aliphatic residue, a non-polar residue may be replaced by another non-polar residue, an acidic residue may be replaced by another acidic residue, a basic residue may be replaced by another basic residue, a polar residue may be replaced by another polar residue or an aromatic residue may be replaced by another aromatic residue. Conservative substitutions may, for example, be between amino acids within the following groups.

Conservative substitutions are shown in the Table below.

Amino acids may be grouped into different classes according to common side-chain properties: a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. acidic: Asp, Glu; d. basic: His, Lys, Arg; e. residues that influence chain orientation: Gly, Pro; aomatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

Nucleic Acids

An aspect of the invention provides a nucleic acid sequence of the invention, encoding any of the CARs, TCRs or any components thereof described herein (including functional portions and functional variants thereof) .

As used herein, the terms “polynucleotide” , “nucleotide” , and “nucleic acid” are intended to be synonymous with each other. It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed, e.g. codon optimization. Nucleic acids according to the invention may comprise DNA or RNA. They may be single stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.

The terms “variant” , “homologue” or “derivative” in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.

The nucleic acid sequences may be joined by a sequence allowing co-expression of the two or more nucleic acid sequences. For example, the construct may rearranged and comprise an internal promoter. There can be expression of all TCR complex components, using for example, an additional promoter, an internal ribosome entry sequence (IRES) sequence or a sequence encoding a cleavage site. The cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into the discrete proteins without the need for any external cleavage activity. Various self-cleaving sites are known, including the Foot-and Mouth disease virus (FMDV) and the 2A self-cleaving peptide. The co-expressing sequence may be an internal ribosome entry sequence (IRES) . The co-expressing sequence may be an internal promoter.

Vectors

In an aspect, the present disclosure provides a vector which comprises a nucleic acid sequence or nucleic acid construct of the disclosure.

Such a vector may be used to introduce the nucleic acid sequence (s) or nucleic acid construct (s) into a host cell so that it expresses one or more CARs or TCRs and, optionally, one or more other proteins of interest. The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon-based vector or synthetic mRNA.

Vectors derived from retroviruses, such as the lentivirus, are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene or transgenes and its propagation in daughter cells. The vector may be capable of transfecting or transducing a T cell or an NK cell. The present disclosure also provides vectors in which a nucleic acid of the present disclosure is inserted. The expression of natural or synthetic nucleic acids encoding a TCR or CAR and optionally inducible cytokine is typically achieved by operably linking a nucleic acid encoding the CAR or TCR polypeptide or portions thereof to one promoter and the cytokine expressing portion to another promoter, and incorporating the construct into an expression vector.

Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.

One example of a suitable CAR or TCR promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-1α (EF-1α) . However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumour virus (MMTV) , human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the haemoglobin promoter, and the creatine kinase promoter.

The vectors can be suitable for replication and integration in eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) , and in other virology and molecular biology manuals, see also, WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193) . In order to assess the expression of the CAR or TCR polypeptide, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or transduced through viral vectors.

Cells

The term “immuneresponsive cell” used herein refers to an engineered immune cell comprising a CAR and/or a recombinant TCR complex directed toward a specified antigen, which is activated to kill a target cell when the CAR or TCR complex binds the specified antigen on the target cell.

The cells used in the present disclosure as or for preparation of the immuneresponsive cells may be any immune cell that is useful in adoptive cell therapy, such as a T cell or a NK cell, including, but not limited to, an NKT cell, a γδ T cell and T regulatory cell. The cells may be allogeneic or autologous.

T cells or T lymphocytes are a type of lymphocyte that have a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells) , by the presence of a T-cell receptor (TCR) on the cell surface. There are various types of T cell, as summarized below. Cytotoxic T cells (TC cells, or CTLs) destroy virally infected cells and tumour cells, and are also implicated in transplant rejection. CTLs express the CD8 molecule at their surfaces.

These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8 ⁺ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis.

Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections. Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells) . Memory cells may be either CD4 ⁺ or CD8 ⁺. Memory T cells typically express the cell surface protein CD45RO. Regulatory T cells (Treg cells) , formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell- mediated immunity toward the end of an immune reaction and to suppress auto-reactive T cells that escaped the process of negative selection in the thymus.

Two major classes of CD4 ⁺ Treg cells have been described-naturally occurring Treg cells and adaptive Treg cells. Naturally occurring Treg cells (also known as CD4 ⁺CD25 ⁺FoxP3 ⁺ Treg cells) arise in the thymus and have been linked to interactions between developing T cells with both myeloid (CD11c ⁺) and plasmacytoid (CD123 ⁺) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a normal immune response.

Natural Killer Cells (or NK cells) are a type of cytolytic cell which form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner. NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes.

In certain embodiments, therapeutic cells of the disclosure comprise autologous cells engineered to express the CARs and/or TCRs of the disclosure. In certain embodiments, therapeutic cells of the disclosure comprise allogeneic cells engineered to express CARs and/or TCRs of the disclosure. Autologous cells may be advantageous in avoiding graft-versus-host disease (GVHD) due to CAR-or TCR-mediated recognition of recipient alloantigens. Also, the immune system of a recipient could attack the infused CAR-or TCR-bearing cells, causing rejection. In certain embdoiments, to prevent GVHD, and to reduce rejection, endogenous TCR is removed from allogeneic cells by genome editing.

Sources of Cells

Prior to expansion and genetic modification, a source of cells (e.g., immune effector cells, e.g., T cells or NK cells) is cells obtained from a subject. The term "subject" is intended to include living organisms in which an immune response can be elicited (e.g., mammals) . Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumours.

In one aspect, immune cells such as T cells or NK cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation.

A specific subpopulation of T cells, such as CD3 ⁺, CD28 ⁺, CD4 ⁺, CD8 ⁺, CD45RA+, and CD45RO ⁺T cells, can be further isolated by positive or negative selection techniques. For example, in one aspect, T cells are isolated by incubation with anti-CD3/anti-CD28-conjugated beads, such as

M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In one aspect, the time period is about 30 minutes. In a further aspect, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumour infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8 ⁺ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein) , subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention. In certain aspects, it may be desirable to perform the selection procedure and use the“unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.

Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4 ⁺ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD16, HLA-DR, and CD8. In certain aspects, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4 ⁺, CD25 ⁺, CD62Lhi, GITR ⁺, CD137, PD1, TIM3, LAG-3, CD150 and FoxP3+. Alternatively, in certain aspects, T regulatory cells are depleted by anti-CD25 conjugated beads or other similar method of selection.

The methods described herein can include, e.g., selection of a specific subpopulation of immune effector cells, e.g., T cells, that are a T regulatory cell-depleted population, CD25 ⁺ depleted cells, using, e.g., a negative selection technique, e.g., described herein. Preferably, the population of T regulatory depleted cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%of CD25 ⁺ cells.

A specific subpopulation of effector cells that specifically bind to a target antigen can be enriched for by positive selection techniques. For example, in certain embodiments, effector cells are enriched for by incubation with target antigen-conjugated beads for a time period sufficient for positive selection of the desired abTCR effector cells. In certain embodiments, the time period is about 30 minutes. In certain embodiments, the time period ranges from 30 minutes to 36 hours or longer (including all ranges between these values) . In certain embodiments, the time period is at least one, 2, 3, 4, 5, or 6 hours. In certain embodiments, the time period is 10 to 24 hours. In certain embodiments, the incubation time period is 24 hours. For isolation of effector cells present at low levels in the heterogeneous cell population, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate effector cells in any situation where there are few effector cells as compared to other cell types. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention.

T cells or NK cells for stimulation can also be frozen after a washing step. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20%DMSO and 8%human serum albumin, or culture media containing 10%Dextran 40 and 5%Dextrose, 20%Human Serum Albumin and 7.5%DMSO, or 31.25%Plasmalyte-A, 31.25%Dextrose 5%, 0.45%NaCl, 10%Dextran 40 and 5%Dextrose, 20%Human Serum Albumin, and 7.5%DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80℃ at a rate of 1℃ per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20℃ or in liquid nitrogen.

Allogeneic CAR and TCR effector cells

In embodiments described herein, the immune effector cell can be an allogeneic immune effector cell, e.g., T cell or NK cell. For example, the cell can be an allogeneic T cell, e.g., an allogeneic T cell lacking expression of endogenous T cell receptor (TCR) and/or human leukocyte antigen (HLA) , e.g., HLA class I and/or HLA class II.

A T cell lacking a functional endogenous TCR can be, e.g., engineered such that it does not express any functional TCR on its surface, engineered such that it does not express one or more subunits that comprise a functional TCR (e.g., engineered such that it does not express (or exhibits reduced expression) of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε and ζ-chain or engineered such that it produces very little functional TCR on its surface. Alternatively, the T cell can express a substantially impaired TCR, e.g., by expression of mutated or truncated forms of one or more of the subunits of the TCR. The term "substantially impaired TCR" means that this TCR will not elicit an adverse immune reaction in a host.

A T cell or NK cell described herein can be, e.g., engineered such that it does not express a functional HLA on its surface. For example, a cell described herein can be engineered such that cell surface HLA, e.g., HLA class I and/or HLA class II, is downregulated. In some aspects, downregulation of HLA may be accomplished by reducing or eliminating expression of beta-2 microglobulin (B2M) .

In certain embodiments, the cell can lack a functional TCR and a functional HLA, e.g., HLA class I and/or HLA class II. Modified cells that lack expression of a functional TCR and/or HLA can be obtained by any suitable means, including a knock out or knock down of one or more subunit of TCR or HLA. For example, the T cell or NK cell can include a knock down of TCR and/or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR) , transcription-activator like effector nuclease (TALEN) , or zinc finger endonuclease (ZFN) .

In certain embodiments, the allogeneic cell can be a cell which does not expresses or expresses at low levels an inhibitory molecule. For example, the cell can be a cell that does not express or expresses at low levels an inhibitory molecule, e.g., that can decrease the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5) , LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276) , B7-H4 (VTCN1) , HVEM (TNFRSF14 or CD270) , KIR, A2aR, MHC class I, MHC class II, Gal9, adenosine, and TGFR beta. Inhibition of an inhibitory molecule, e.g., by inhibition at the DNA, RNA or protein level, can optimize a CAR-expressing cell performance. In embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., a siRNA or shRNA, a clustered regularly interspaced short palindromic repeats (CRISPR) , a transcription-activator like effector nuclease (TALEN) , or a zinc finger endonuclease (ZFN) , e.g., as described herein, can be used.

In certain embodiments, endogenous TCR expression and/or HLA expression can be inhibited using siRNA or shRNA that targets a nucleic acid encoding a TCR and/or HLA, and/or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5) , LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276) , B7-H4 (VTCN1) , HVEM (TNFRSF14 or CD270) , KIR, A2aR, MHC class I, MHC class II, Gal9, adenosine, and TGFR beta) , in a T cell.

Expression of siRNA and shRNAs in immune cells can be achieved using any conventional expression system, e.g., such as a lentiviral expression system. Exemplary shRNAs that downregulate expression of components of the TCR are described, e.g., in US Publication No.: 2012/0321667. Exemplary siRNA and shRNA that downregulate expression of HLA class I and/or HLA class II genes are described, e.g., in U.S. publication No.: US 2007/0036773.

“CRISPR” as used herein refers to a set of clustered regularly interspaced short palindromic repeats, or a system comprising such a set of repeats. “Cas” , as used herein, refers to a CRISPR-associated protein. A “CRISPR/Cas” system refers to a system derived from CRISPR and Cas which can be used to silence or mutate a TCR and/or HLA gene, and/or an inhibitory molecule described herein (e.g., PD1, PD-L1, PD-L2, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5) , LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276) , B7-H4 (VTCNl) , HVEM (TNFRSF14 or CD270) , KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, and TGFR beta) .

Naturally-occurring CRISPR/Cas systems are found in approximately 40%of sequenced eubacteria genomes and 90%of sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity (Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845) .

Activation and Expansion of Immune Cells

T cells may be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

Generally, the T cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4 ⁺ T cells or CD8 ⁺ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., (1998) Transplant Proc. 30 (8) : 3975-3977; Haanen et al., (1999) J. Exp. Med. 190 (9) : 13191328; Garland et al., (1999) J. Immunol Meth. 227 (l-2) : 53-63) .

In certain embodiments, expansion can be performed using flasks or containers, or gas-permeable containers known by those of skill in the art and can proceed for 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days, about 7 days to about 14 days, about 8 days to about 14 days, about 9 days to about 14 days, about 10 days to about 14 days, about 11 days to about 14 days, about 12 days to about 14 days, or about 13 days to about 14 days. In certain embodiments, the second T cell expansion can proceed for about 14 days.

In certain embodiments, the expansion can be performed using non-specific T-cell receptor stimulation in the presence of interleukin-2 (IL-2) or interleukin-15 (IL-15) . The non-specific T-cell receptor stimulus can include, for example, an anti-CD3 antibody, such as about 30 ng/ml of OKT3, a mouse monoclonal anti-CD3 antibody (commercially available from Ortho-McNeil, Raritan, N.J. or Miltenyi Biotech, Auburn, Calif. ) or UHCT-1 (commercially available from BioLegend, San Diego, Calif., USA) . CAR-or TCR-expressing cells can be expanded in vitro by including one or more antigens, including antigenic portions thereof, such as epitope (s) , of a cancer or an immune cell activation molecule, which can be optionally expressed from a vector, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., 0.3 μM MART-1: 26-35 (27 L) or gpl 00: 209-217 (210M) , optionally in the presence of a T-cell growth factor, such as 300 IU/mL IL-2 or IL-15. Other suitable antigens may include, e.g., NY-ESO-1, TRP-1, TRP-2, tyrosinase cancer antigen, MAGE-A3, SSX-2, and VEGFR2, or antigenic portions thereof. CAR or TCR cells may also be rapidly expanded by re-stimulation with the same antigen (s) of the cancer or the immune cell activation molecule pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, the cells can be further stimulated with, e.g., example, irradiated, autologous lymphocytes or with irradiated HLA-A2 ⁺ allogeneic lymphocytes and IL-2. In certain embodiments, the stimulation occurs as part of the expansion. In certain embodiments, the expansion occurs in the presence of irradiated, autologous lymphocytes or with irradiated HLA-A2 ⁺ allogeneic lymphocytes and IL-2.

In certain embodiments, the cell culture medium comprises IL-2. In certain embodiments, the cell culture medium comprises about 1000 IU/mL, about 1500 IU/mL, about 2000 IU/mL, about 2500 IU/mL, about 3000 IU/mL, about 3500 IU/mL, about 4000 IU/mL, about 4500 IU/mL, about 5000 IU/mL, about 5500 IU/mL, about 6000 IU/mL, about 6500 IU/mL, about 7000 IU/mL, about 7500 IU/mL, or about 8000 IU/mL, or between 1000 and 2000 IU/mL, between 2000 and 3000 IU/mL, between 3000 and 4000 IU/mL, between 4000 and 5000 IU/mL, between 5000 and 6000 IU/mL, between 6000 and 7000 IU/mL, between 7000 and 8000 IU/mL, or between 8000 IU/mL of IL-2.

In certain embodiments, the cell culture medium comprises OKT3 antibody. In certain embodiments, the cell culture medium comprises about 0.1 ng/mL, about 0.5 ng/mL, about 1 ng/mL, about 2.5 ng/mL, about 5 ng/mL, about 7.5 ng/mL, about 10 ng/mL, about 15 ng/mL, about 20 ng/mL, about 25 ng/mL, about 30 ng/mL, about 35 ng/mL, about 40 ng/mL, about 50 ng/mL, about 60 ng/mL, about 70 ng/mL, about 80 ng/mL, about 90 ng/mL, about 100 ng/mL, about 200 ng/mL, about 500 ng/mL, about 1 μg/mL or between 0.1 ng/mL and 1 ng/mL, between 1 ng/mL and 5 ng/mL, between 5 ng/mL and 10 ng/mL, between 10 ng/mL and 20 ng/mL, between 20 ng/mL and 30 ng/mL, between 30 ng/mL and 40 ng/mL, between 40 ng/mL and 50 ng/mL, or between 50 ng/mL and 100 ng/mL of OKT3 antibody.

In certain embodiments, a combination of IL-2, IL-7, IL-15, IL-18 and/or IL-21 are employed as a combination during the expansion. In certain embodiments, IL-2, IL-7, IL-15, IL-18 and/or IL-21 as well as any combinations thereof can be included during the expansion. In certain embodiments, a combination of IL-2, IL-15, and IL-18 are employed as a combination during the expansion. In certain embodiments, IL-2, IL-7, and IL-18 as well as any combinations thereof can be included.

In certain embodiments, the expansion can be conducted in a supplemented cell culture medium comprising IL-2, OKT-3, and antigen-presenting feeder cells.

In certain embodiments, the expansion culture media comprises about 500 IU/mL of IL-15, about 400 IU/mL of IL-15, about 300 IU/mL of IL-15, about 200 IU/mL of IL-15, about 180 IU/mL of IL-15, about 160 IU/mL of IL-15, about 140 IU/mL of IL-15, about 120 IU/mL of IL-15, or about 100 IU/mL of IL-15, or about 500 IU/mL of IL-15 to about 100 IU/mL of IL-15, or about 400 IU/mL of IL-15 to about 100 IU/mL of IL-15 or about 300 IU/mL of IL-15 to about 100 IU/mL of IL-15 or about 200 IU/mL of IL-15, or about 180 IU/mL of IL-15.

In certain embodiments, the expansion culture media comprises about 20 IU/mL of IL-18, about 15 IU/mL of IL-18, about 12 IU/mL of IL-18, about 10 IU/mL of IL-18, about 5 IU/mL of IL-18, about 4 IU/mL of IL-18, about 3 IU/mL of IL-18, about 2 IU/mL of IL-18, about 1 IU/mL of IL-18, or about 0.5 IU/mL of IL-18, or about 20 IU/mL of IL-18 to about 0.5 IU/mL of IL-18, or about 15 IU/mL of IL-18 to about 0.5 IU/mL of IL-18, or about 12 IU/mL of IL-18 to about 0.5 IU/mL of IL-18, or about 10 IU/mL of IL-18 to about 0.5 IU/mL of IL-18, or about 5 IU/mL of IL-18 to about 1 IU/mL of IL-18, or about 2 IU/mL of IL-18. In certain embodiments, the cell culture medium comprises about 1 IU/mL of IL-18, or about 0.5 IU/mL of IL-18.

In certain embodiments, the expansion culture media comprises about 20 IU/mL of IL-21, about 15 IU/mL of IL-21, about 12 IU/mL of IL-21, about 10 IU/mL of IL-21, about 5 IU/mL of IL-21, about 4 IU/mL of IL-21, about 3 IU/mL of IL-21, about 2 IU/mL of IL-21, about 1 IU/mL of IL-21, or about 0.5 IU/mL of IL-21, or about 20 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, or about 15 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, or about 12 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, or about 10 IU/mL of IL-21 to about 0.5 IU/mL of IL-21, or about 5 IU/mL of IL-21 to about 1 IU/mL of IL-21, or about 2 IU/mL of IL-21. In certain embodiments, the cell culture medium comprises about 1 IU/mL of IL-21, or about 0.5 IU/mL of IL-21.

In certain embodiments the antigen-presenting feeder cells (APCs) are PBMCs. In an embodiment, the ratio of CAR-or TCR-expressing cells to PBMCs and/or antigen-presenting cells in the expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500, or between 1 to 50 and 1 to 300, or between 1 to 100 and 1 to 200.

In certain aspects, the primary stimulatory signal and the costimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in "cis" formation) or to separate surfaces (i.e., in "trans" formation) . Alternatively, one agent may be coupled to a surface and the other agent in solution. In one aspect, the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain aspects, both agents can be in solution. In one aspect, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present invention.

In one aspect, the two agents are immobilized on beads, either on the same bead, i.e., “cis, ” or to separate beads, i.e., “trans. ” By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the costimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In one aspect, a 1: 1 ratio of each antibody bound to the beads for CD4 ⁺ T cell expansion and T cell growth is used. In certain aspects of the present invention, a ratio of anti CD3: CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1: 1. In one particular aspect an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1: 1. In one aspect, the ratio of CD3: CD28 antibody bound to the beads ranges from 100: 1 to 1: 100 and all integer values there between. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3: CD28 is less than one. In certain aspects of the invention, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2: 1. In one particular aspect, a 1: 100 CD3: CD28 ratio of antibody bound to beads is used. In one aspect, a 1: 75 CD3: CD28 ratio of antibody bound to beads is used. In a further aspect, a 1: 50 CD3: CD28 ratio of antibody bound to beads is used. In one aspect, a 1: 30 CD3: CD28 ratio of antibody bound to beads is used. In one preferred aspect, a 1: 10 CD3: CD28 ratio of antibody bound to beads is used. In one aspect, a 1: 3 CD3: CD28 ratio of antibody bound to the beads is used. In yet one aspect, a 3: 1 CD3: CD28 ratio of antibody bound to the beads is used.

Ratios of particles to cells from 1: 500 to 500: 1 and any integer values in between may be used to stimulate T cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In certain aspects the ratio of cells to particles ranges from 1: 100 to 100: 1 and any integer values in-between and in further aspects the ratio comprises 1: 9 to 9: 1 and any integer values in between, can also be used to stimulate T cells. The ratio of anti-CD3-and anti-CD28-coupled particles to T cells that result in T cell stimulation can vary as noted above, however certain preferred values include 1: 100, 1: 50, 1: 40, 1: 30, 1: 20, 1: 10, 1: 9, 1: 8, 1: 7, 1: 6, 1: 5, 1: 4, 1: 3, 1: 2, 1: 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, and 15: 1 with one preferred ratio being at least 1: 1 particles per T cell. In one aspect, a ratio of particles to cells of 1: 1 or less is used. In one particular aspect, a preferred particle: cell ratio is 1: 5. In further aspects, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one aspect, the ratio of particles to cells is from 1: 1 to 10: 1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1: 1 to 1: 10 (based on cell counts on the day of addition) . In one particular aspect, the ratio of particles to cells is 1: 1 on the first day of stimulation and adjusted to 1: 5 on the third and fifth days of stimulation. In one aspect, particles are added on a daily or every other day basis to a final ratio of 1: 1 on the first day, and 1: 5 on the third and fifth days of stimulation. In one aspect, the ratio of particles to cells is 2: 1 on the first day of stimulation and adjusted to 1: 10 on the third and fifth days of stimulation. In one aspect, particles are added on a daily or every other day basis to a final ratio of 1: 1 on the first day, and 1: 10 on the third and fifth days of stimulation. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present invention. In particular, ratios will vary depending on particle size and on cell size and type. In one aspect, the most typical ratios for use are in the neighborhood of 1: 1, 2: 1 and 3: 1 on the first day.

In further aspects of the present invention, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative aspect, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further aspect, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

Preparation of CAR-and TCR-Expressing Cells

Viral-and non-viral-based genetic engineering tools can be used to generate CAR-T cells, CAR-NK cells, TCR-T cells, and TCR-NK cells, resulting in permanent or transient expression of therapeutic genes. Retrovirus-based gene delivery is a mature, well-characterized technology, which has been used to permanently integrate CARs into the host cell genome (Scholler J., et al. (2012) Sci. Transl. Med. 4: 132ra53; Rosenberg S.A. et al., (1990) N. Engl. J. Med. 323: 570–578) .

Non-viral DNA transfection methods can also be used. For example, Singh et al describes use of the Sleeping Beauty (SB) transposon system developed to engineer CAR T cells (Singh H., et al., (2008) Cancer Res. 68: 2961–2971) and is being used in clinical trials (see e.g., ClinicalTrials. gov: NCT00968760 and NCT01653717) . The same technology is applicable to engineer T-cells, NK cells and the like according to the disclosure.

Multiple SB enzymes have been used to deliver transgenes. Mátés describes a hyperactive transposase (SB100X) with approximately 100-fold enhancement in efficiency when compared to the first-generation transposase. SB100X supported 35-50%stable gene transfer in human CD34 (+) cells enriched in hematopoietic stem or progenitor cells (Mátés L. et al., (2009) Nat. Genet. 41: 753–761) and multiple transgenes can be delivered from multicistronic single plasmids (e.g., Thokala R. et al., (2016) PLoS ONE. 11: e0159477) or multiple plasmids (e.g., Hurton L.V. et al., (2016) Proc. Natl. Acad. Sci. USA. 113: E7788–E7797) . Such systems are used in the present invention.

Morita et al, describes the piggyBac transposon system to integrate larger transgenes (Morita D. et al., (2017) Methods Clin. Dev. 8: 131–140) . Nakazawa et al. describes use of the system to generate EBV-specific cytotoxic T-cells expressing HER2- specific chimeric antigen receptor (Nakazawa Y et al, (2011) Mol. Ther. 19: 2133–2143) . Manuri et al used the system to generate CD-19 specific T cells (Manuri P.V.R. et al., (2010) Hum. Gene Ther. 21: 427–437) .

Transposon technology is easy and economical. One potential drawback is the longer expansion protocols currently employed may result in T cell differentiation, impaired activity and poor persistence of the infused cells. Monjezi et al describe development minicircle vectors that minimize these difficulties through higher efficiency integrations (Monjezi R. et al., (2017) Leukemia. 31: 186–194) .

Pharmaceutical Compositions

The present disclosure also relates to a pharmaceutical composition containing an immuneresponsive cell expressing the CARs and/or TCRs, together with a pharmaceutically acceptable carrier, diluent or excipient, and optionally one or more further pharmaceutically active polypeptides and/or compounds. In certain embodiments, a pharmaceutical composition of the disclosure comprises a nucleic acid encoding the CAR and/or TCR, and a pharmaceutically acceptable carrier. Such a formulation may, for example, be in a form suitable for intravenous infusion.

As used herein, by “pharmaceutically acceptable” or “pharmacologically compatible” is meant a material that is not biologically or otherwise undesirable, e.g., the material may be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained. Pharmaceutically acceptable carriers or excipients have preferably met the required standards of toxicological and manufacturing testing and/or are included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug administration.

Other aspects of the invention provide the use of a population of the effector cells as described herein for the manufacture of a medicament for the treatment of a disease, such as cancer, and a method of treatment of a disease such as cancer may comprise administering a population of the cells as described herein to an individual in need thereof.

Following administration of the effector cells, the recipient individual may exhibit a cell mediated immune response against cancer cells in the recipient individual. This may have a beneficial effect on the cancer condition in the individual.

An individual suitable for treatment as described above may be a mammal, such as a rodent (e.g. a guinea pig, a hamster, a rat, a mouse) , murine (e.g. a mouse) , canine (e.g. a dog) , feline (e.g. a cat) , equine (e.g. a horse) , a primate, simian (e.g. a monkey or ape) , a monkey (e.g. marmoset, baboon) , an ape (e.g. gorilla, chimpanzee, orang-utan, gibbon) , or a human.

In preferred embodiments, the individual is a human. In other preferred embodiments, non-human mammals, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, primate, porcine, canine, or rabbit animals) may be employed.

Method of Treatment

The immuneresponsive cells of the disclosure, when comprising a CAR and/or a recombinant TCR complex targeting the immune cell activation molecule, may be used with other allogeneic cells to reduce or eliminate graft rejection of the allogeneic cells, by targeting and killing activated host immune cells against allogeneic cells.

Thus, in one aspect, the present disclosure provides a method for reducing graft rejection to allogeneic cells in a subject, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the immuneresponsive cells of the disclosure, prior to or together with the administration of the allogeneic cells.

The term “therapeutically effective amount” here refers to an amount of the immuneresponsive cells of the disclosure that is sufficient and effective to reduce or eliminate the graft rejection of the allogeneic cells of interest.

In certain embodiments, the subject is human.

The immuneresponsive cells of the disclosure, when comprising a CAR and/or a recombinant TCR complex targeting the immune cell activation molecule, may be used to treat or alleviate an inflammatory disease, by targeting and killing the activated immune cells responsible for the unnecessary and unwated inflammation.

Thus, in one aspect, the present disclosure provides a method for treating or alleviating an inflammatory disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the immuneresponsive cells of the disclosure. In certain embodiments, the inflammatory disease is an autoimmune disease, such as myasthenia gravis.

The term “therapeutically effective amount” here refers to an amount of the immuneresponsive cells of the disclosure that is sufficient and effective to “treat” an inflammatory disease, such as to reduce or eliminate unwanted and/or misdirected inflammations, relieve to some extent one or more of the symptoms associated with the inflammatory disease in a patient, and/or improve progression free survival of a patient.

The immuneresponsive cells of the disclosure, when comprising a CAR and/or a recombinant TCR complex targeting both (i) an immune cell activation molecule and (ii) a disease associated antigen, may be used to treat or alleviate a disease, with enhanced efficacy, by targeting and killing (i) the cells expressing the disease associated antigen and (ii) activated immune cells against the immuneresponsive cells. In such instances, it is not necessary to suppress the entire host immune system, i.e., measures like lymph depletion is not needed before CAR-and/or TCR-immune cell infusion.

Thus, in one aspect, the present disclosure provides a method for treating a disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the immuneresponsive cells of the disclosure.

In certain embodiments, the subject is human.

The term “therapeutically effective amount” here refers to an amount of the immuneresponsive cells of the disclosure that is effective to "treat" a disease or disorder in an individual. In the case of cancer, the therapeutically effective amount as disclosed herein can reduce the number of cancer cells; reduce the tumor size or weight; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the immuneresponsive cell can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. In certain embodiments, the therapeutically effective amount is a growth inhibitory amount. In certain embodiments, the therapeutically effective amount is an amount that improves progression free survival of a patient. In the case of infectious disease, such as viral infection, the therapeutically effective amount of the immunoresponsive cell as disclosed herein can reduce the number of cells infected by the pathogen; reduce the production or release of pathogen-derived antigens; inhibit (i.e., slow to some extent and preferably stop) spread of the pathogen to uninfected cells; and/or relieve to some extent one or more symptoms associated with the infection. In certain embodiments, the therapeutically effective amount is an amount that extends the survival of a patient.

Cells, including αβ T cells, γδ T cells, NK cells, and the like, expressing CARs or TCRs for use in the methods of the present disclosure may either be created ex vivo from a patient's own peripheral blood (autologous) , or in the setting of a hematopoietic stem cell transplant from donor peripheral blood (allogenic) , or peripheral blood from an unconnected donor (allogenic) . Alternatively, the cells may be derived from ex-vivo differentiation of inducible progenitor cells or embryonic progenitor cells to αβ T cells, γδ T cells, or NK cells. In these instances, T-cells expressing the CAR or TCR, are generated by introducing DNA or RNA coding for the CAR or TCR, by one of many means including transduction with a viral vector, transfection with DNA or RNA.

αβ T cells, γδ T cells, or NK cells expressing the CAR and/or TCR of the present disclosure may be used for the treatment of haematological cancers or solid tumors.

A method for the treatment of a disease relates to the therapeutic use of a vector or cell, including a αβ T cell, γδ T cell, or NK cell, of the disclosure. In this respect, the vector, or αβ T cell, or γδ T cell, or NK cell may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease. The method of the invention may cause or promote T-cell mediated killing of cancer cells. The vector, or αβ T cell, or γδ T cell, or NK cell according to the present invention may be administered to a patient with one or more additional therapeutic agents. The one or more additional therapeutic agents can be co-administered to the patient. By “co-administering” is meant administering one or more additional therapeutic agents and the vector, or T or NK cell of the present invention sufficiently close in time such that the vector, or T or NK cell can enhance the effect of one or more additional therapeutic agents, or vice versa. In this regard, the vectors or cells can be administered first and the one or more additional therapeutic agents can be administered second, or vice versa. Alternatively, the vectors or cells and the one or more additional therapeutic agents can be administered simultaneously. One co-administered therapeutic agent that may be useful is IL-2, as this is currently used in existing cell therapies to boost the activity of administered cells. However, IL-2 treatment is associated with toxicity and tolerability issues.

The effector cells are used to treat cancers and neoplastic diseases associated with a target antigen. Cancers and neoplastic diseases that may be treated using any of the methods described herein include tumours that are not vascularized, or not yet substantially vascularized, as well as vascularized tumours. The cancers may comprise non-solid tumours (such as hematological tumours, for example, leukemias and lymphomas) or may comprise solid tumours. Types of cancers to be treated with the effector cells of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumours, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumours/cancers and pediatric tumours/cancers are also included.

Hematologic cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia) , chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia) , polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms) , multiple myeloma, plasmacytoma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

Solid tumours are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumours can be benign or malignant. Different types of solid tumours are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas) . Examples of solid tumours, such as sarcomas and carcinomas, include adrenocortical carcinoma, cholangiocarcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, stomach cancer, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, thyroid cancer (e.g., medullary thyroid carcinoma and papillary thyroid carcinoma) , pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumour, cervical cancer (e.g., cervical carcinoma and pre-invasive cervical dysplasia) , colorectal cancer, cancer of the anus, anal canal, or anorectum, vaginal cancer, cancer of the vulva (e.g., squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, and fibrosarcoma) , penile cancer, oropharyngeal cancer, esophageal cancer, head cancers (e.g., squamous cell carcinoma) , neck cancers (e.g., squamous cell carcinoma) , testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, Leydig cell tumour, fibroma, fibroadenoma, adenomatoid tumours, and lipoma) , bladder carcinoma, kidney cancer, melanoma, cancer of the uterus (e.g., endometrial carcinoma) , urothelial cancers (e.g., squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma, ureter cancer, and urinary bladder cancer) , and CNS tumours (such as a glioma (such as brainstem glioma and mixed gliomas) , glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases) .

When “an immunologically effective amount, ” “an anti-tumour effective amount, ” “a tumour-inhibiting effective amount, ” or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumour size, extent of infection or metastasis, and condition of the patient (subject) . It can generally be stated that a pharmaceutical composition comprising the immuneresponsive cells described herein may be administered at a dosage of 10 ⁴ to 10 ⁹ cells/kg body weight, in some instances 10 ⁵ to 10 ⁶ cells/kg body weight, including all integer values within those ranges. Immunoresponsive cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988) .

Combination Therapies

An immuneresponsive cell of the disclosure described herein may be used in combination with other known agents and therapies. Administered "in combination" , as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject's affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery" . In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

An immuneresponsive cell expressing a CAR and/or a TCR described herein and at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially. For sequential administration, the CAR-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.

The cellular therapy of the disclosure and/or other therapeutic agents, procedures or modalities can be administered during periods of active disorder, or during a period of remission or less active disease. The cellular therapy can be administered before the other treatment, concurrently with the treatment, post-treatment, or during remission of the disorder.

When administered in combination, the therapy and the additional agent (e.g., second or third agent) , or all, can be administered in an amount or dose that is higher, lower or the same than the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain embodiments, the administered amount or dosage of the cell therapy, the additional agent (e.g., second or third agent) , or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy. In other embodiments, the amount or dosage of the cell therapy, the additional agent (e.g., second or third agent) , or all, that results in a desired effect (e.g., treatment of cancer) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%lower) than the amount or dosage of each agent used individually, e.g., as a monotherapy, required to achieve the same therapeutic effect.

In further aspects, the CAR or TCR-expressing cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation, peptide vaccine, such as that described in Izumoto et al. (2008) JNeurosurg 108: 963-971.

In certain instances, the immuneresponsive cells of the present invention are combined with other therapeutic agents, such as other anti-cancer agents, anti-allergic agents, anti-nausea agents (or anti-emetics) , pain relievers, cytoprotective agents, and combinations thereof.

Treatments can be evaluated, for example, by tumour regression, tumour weight or size shrinkage, time to progression, duration of survival, progression free survival, overall response rate, duration of response, quality of life, protein expression and/or activity. Approaches to determining efficacy of the therapy can be employed, including for example, measurement of response through radiological imaging.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined in the appended claims.

The present invention will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the invention in any way.

Examples

Example 1: Plasmid construction, virus preparation, titer evaluation

Dual antigen binding receptor systems (including dual CARs, dual TCRs, and CAR plus TCR system) , and tandem antigen binding receptor systems of the disclosure targeting the immune cell activation protein, such as, but not limited to, CD30, CD33, CD70, CD7, 4-1BB, CD28, CD69 and CD8, were designed as in FIG. 1 to FIG. 3. To generate viral particles comprising polynucleic acids encoding any of the systems disclosed herein, lentivirus packaging plasmid mixtures including pMDLg/pRRE (Addgene#11251) , pRSV- Rev (Addgene#11253) , and pMD2. G (Addgene#11259) were pre-mixed with a PLVX-EF1A (including target system) vector at a pre-optimized ratio with polyetherimide (PEI) , mixed properly, and incubated at room temperature for 5 minutes. The transfection mix was added dropwise to 293-T cells and mixed gently. Transfected 293-T cells were incubated overnight at 37℃ and 5%CO ₂. Twenty-four hours post-transfection, supernatants were collected and centrifuged at 4℃, 500 g for 10 min to remove any cellular debris. Centrifuged supernatants were filtered through a 0.45 μm PES filter to concentrate the viral supernatants post ultracentrifugation. After centrifugation, the supernatants were carefully discarded and the virus pellets were rinsed with pre-chilled DPBS. Viruses were aliquoted and stored at -80℃ before titer determination by functional transduction on a T cell line.

Specifically, the following examples are demonstrated with gamma/delta T cells transduced with viral particles encoding anti-BCMA CAR BSF17 (SEQ ID NO.: 10) , or anti-BCMA CAR armored with anti-CD8 TCR, named BSF17-H8 (SEQ ID NO.: 11) . These two T cells were designated as BSF17 CAR-γδ T cell and BSF17-H8 CAR-γδ T cell, respectively.

Notably, the lentiviral vector was modified using pLVX-Puro (Clontech#632164) by replacing the original promoter with human elongation factor 1α promoter (hEF1α) and by removing the puromycin resistance gene with EcoRI and BamHI by GenScript. PLVX-EF1A was further subject to the lentivirus packaging procedure described above.

Example 2: T cell isolation and activation

αβ T leukocytes are collected in R10 medium, then mixed with 0.9%NaCl solution at a 1: 1 (v/v) ratio. Lymphoprep medium of 3 mL in volume is added to a 15 mL centrifuge tube and slowly layered to form 6 mL of diluted lymphocyte mix. The lymphocyte mix is centrifuged at 800 g for 30 minutes without brakes at 20℃. Lymphocyte buffy coat is then collected with a 200 μL pipette. The harvested fraction is diluted at least 6 fold of 0.9%NaCl or R10 to reduce the density of the solution before further centrifugation at 250 g for 10 minutes at 20℃. The supernatant is aspirated completely, and 10 mL of R10 is added to the cell pellet. The mixture is further centrifuged at 250 g for 10 minutes at 20℃. The supernatant is then aspirated. Two milliliters R10 pre-warmed at 37℃ with 100 IU/mL IL-2 is added to the cell pellet, and the cell pellet is re-suspended gently. Cells are quantified and the PBMC sample is ready for experimentation. Human T cells are purified from PBMCs using Miltenyi Pan T cell isolation kit (Cat#130-096-535) .

The prepared αβ T cells are subsequently pre-activated for 48 hours with human T cell Activation/Expansion kit (Milteny#130-091-441) by using one loaded anti-Biotin MACSiBead Particle per two cells (bead-to-cell ratio 1: 2) .

γδ T cells were prepared by addition of 5 μM Zoledronate and 1000 IU/mL IL-2 to PBMCs and cultured for 14 days with periodical change of media supplemented with 1000 IU/mL IL-2. Alternatively, γδ T cells were isolated from PBMCs or umbilical cord blood (UCB) and then stimulated by anti-γδ TCR antibody and anti-CD3 (OKT3) followed by co-incubation of K562-based artificial antigen-presenting cells (aAPCs) at an 1: 2 ratio for at least 10 days.

Example3: T cell Transduction

αβ T cells Transfection

The pre-activated alpha/beta T cells are collected and re-suspended in 1640 medium containing 300 IU/mL IL-2. A lentiviral vector encoding the system of Example 1 is diluted to MOI=5 with the same medium and infected with 1E+06 activated T cells. The pre-activated T cells are transduced with lentivirus stock in the presence of 8 μg/ml polybrene with centrifugation at 1000 g, 32℃ for 1h. The transduced cells are then transferred to the cell culture incubator for transgene expression under suitable conditions. The following day, the transduced cells are centrifuged and replaced with fresh media. Cell density is measured every other day, and fresh media are added to continue the expansion.

γδ T cells transduction

Forty-eight hours post-activation, cells were transduced with lentiviral vectors encoding the system of Example 1 at an MOI of 5 with 5 pg/ml polybrene followed by replenishment of fresh media containing IL-2 (1000 IU/ml) the next day. Cells were cultured in AIM-V supplemented with IL-2 (1000 IU/ml) in a humidified chamber with periodical change of media as determined by the pH of the culture media for further expansion. Cells were harvested 10 days post-transduction and the total number, purity and transduction efficiency were determined. Optionally, cells were further enriched with a negative TCRγ/δ ⁺ T cell isolation kit (Miltenyi Biotec) before future applications or cryopreserved.

Example 4: Quantification of transgene expression

On day 3 and onwards (typically day 3, 7 and 14) post transduction, cells were evaluated for expression of the system of Example 1 by flow cytometry. An aliquot of cells was collected from the culture, washed, pelleted, and resuspended in diluted antibodies (eBioscience Anti-human TCR beta PE, anti-CAR Ab) at a dilution factor of 100 in PBS+0.5%FBS for 50-100 μl per sample. Resuspended cells were resuspended in about 50 to 100 μl of solution. Cell were incubated at 4℃ for 30 minutes. Viability dye eFluor780 or SYTOX Blue viability stain was also added according to manufacturer’s instructions. Post incubation, cells were washed twice in PBS and resuspended in 100 to 200 μl PBS for analysis. The mean fluorescence of the system was quantified by flow cytometry.

For anti-BCMA CAR-T staining, cells were stained with Alexa Fluor 488-labeled anti-mouse scFv antibodies (Genscript) . Flow cytometry analysis for all experiments was performed by using FlowJo (Tree Star, Inc. ) .

We determined the transduction efficiency of BSF17 CAR-γδT and BSF17-H8 CAR-γδT cells to be 54.9%and 28.6%, respectively, as shown in FIG. 4.

For anti-CD19 CAR-T staining, cells are stained with Alexa Fluor 488-labeled human CD19 protein (Genscript) . Flow cytometry analysis for all experiments is performed by using FlowJo (Tree Star, Inc. ) .

Example 5: In vitro killing and cytokine release

Cytotoxicity ofαβT cells with designed systems of Example 1, as well as their control αβ T cells are determined in a 20h co-culture assay, and cytotoxicity of γδ T cells with designed systems of Example 1, as well as their control γδ T cells were determined in a 20h co-culture assay. In the experiments, the effector cells were centrifugally collected, then diluted to the desired concentrations with 1640 phenol-red free medium (Invitrogen) with 2%heat inactivated FBS (Invitrogen) . The target cell, H929, exhibited high expression of target antigens BCMA. The target cell, Raji, exhibits high expression of target antigens CD19. In addition, CD8 ⁺ αβ T cells were used as a target cell to evaluate the potency of anti-CD8 armor. The effector cells were co-cultured at indicated effector to target ratios (E: T=1: 1, 0.5: 1, 0.25: 1, 0.125: 1 for H929 while E: T=0.5: 1, 0.25: 1, 0.125: 1 and 0.0625: 1 for CD8 ⁺ αβ T cells) at 37℃ for 20h in a 96 well plate. Additional wells contained assay buffer only (1640 phenol red-free medium plus 2%hiFBS) , target cell only (T) , effector cell only (E) and max release of target cell (1%solution of triton-X 100) , respectively. Each condition was performed in triplicates, and the cytotoxicity of effector cells was detected by LDH assay kit (Roche) . After completion of 20h co-culture, the assay plate was centrifuged, and supernatants were collected in a new 96-well plate. The supernatant plate was diluted with an equal volume of the LDH assay reagent according to the manufacture’s manual. The assay plate was incubated for about 30 min at 15℃～25℃. The absorbance of the plate was measured at 492 nm and 650 nm using Flexstation reader (Molecular Devices) and calculated as previously described.

We found that both anti-BCMA CAR-γδ T cells (BSF17 CAR-γδ T cells) and anti-CD8 TCR armored BSF17 CAR-γδ T cells (BSF17-H8 CAR-γδ T cells) were equally efficacaious againt tumor cell line H929 (FIG. 5A) . Furthermore, anti-CD8 TCR armored BSF17 CAR-γδ T cells (BSF17-H8 CAR-γδ T cells) showed specific and highly potent toxicity against CD8 ⁺ αβ T cells (FIG. 5B) . These results indicated that the strategy targeting immune-activating molecules, desmonstrated by CD8 here, can be effective at targeting both tumor and activated host immune cells examplied here by CD8 ⁺ αβ T cells.

Example 6: Cytokine Release

The supernatants of the cytotoxicity assay plate were collected for cytokine release analysis (Human IFN gamma kit, Cisbio, Cat#62HIFNGPEH, Human TNF alpha kit, Cisbio, Cat#62HTNFAPEH, and Human GM-CSF kit, Cisbio, Cat#62HGMCSFPEG) . The cell supernatant and a standard were dispensed directly into the assay plate for the cytokine detection utilizing

reagents. The antibodies labeled with the HTRF donor and acceptor were pre-mixed and added in a single dispensing step. The ELISA standard curve was generated using the 4 Parameter Logistic (4PL) curve. The standard curve regression enabled the accurate measurement of an unknown sample concentration across a wider range of concentrations than linear analysis, making it suitable for the analysis of biological systems such as cytokine release.

We demonstrated that TNF-α, GM-CSF and IFN-γ production were similar between BSF17 CAR-γδ T and BSF17-H8 CAR-γδ T cells when co-cultured with tumor cell H929 (FIGs. 6A, 6B and 6C) , especially at lower E: T ratios. It was worth noting that cytokine production, especially IFN-γ, were higher for BSF17-H8 CAR-γδ T cells when co-cultured with CD8 ⁺αβ T cells (FIGs. 6D, 6E and 6F) , owning to its specific toxicity against CD8 ⁺ αβ T cells.

Example 7: In vitro HvGD assay

HvGD (Host versus Graft disease) was assessed via a two-way mixed lymphocyte reaction (MLR) assay. Briefly, armored CAR-T cells (BSF17-H8 CAR-γδ T cells) , alongside unarmored CAR-T cells (BSF17 CAR-γδ T cells) and untransduced T cells were co-incubated with CFSE-labelled HLA-mismatched allogeneic or control autologous PBMCs for a period of 7 days. During this period of time, proliferations of CAR-γδ T cells of the disclosure (BSF17 CAR-γδ T cells, BSF17-H8 CAR-γδ T cells) or total γδ T cells alongside allogeneic or autologous T and NK cells were determined by flow cytometry.

First, we found that, the proliferation of CD8 ⁺ but not CD4 ⁺ T cells were suppressed to the minimal level when co-cultured with BSF17-H8 CAR-γδ T cells (FIGs. 7A and 7B) . Second, to our surprise, growth of NK cells, especially the NK cells from allogeneic PBMCs, was also drastically reduced when co-cultured with BSF17-H8 CAR-γδ T cells (FIG. 7C) . Last, BSF17-H8 CAR-γδ T cells proliferated the best under allogeneic setting when co-cultured with PBMCs (FIGs. 7D and 7E) .

Taken together, these results indicated that the addition of an armor targeting activated immune cells, as exampliefied here by the anti-CD8 armor, can help resist allogeneic-based host rejection via the elimination of responsible host immune cells, such as CD8 ⁺ T and NK cells. Such engineering strategy can help promot the persistence of “off-the-shelf” cell therapy products.

Example 8: In vivo efficacy

Anti-tumor activity of an exemplary armored anti-BCMA CAR-T cell is assessed in vivo in an RPMI-8226 xenograft model. Briefly, one million (1×10E6) RPMI-8226 cells stably expressing the firefly luciferase reporter are implanted subcutaneously/intravenously on day 0 in NOD/SCID IL-2RγCnull (NSG) mice. Thirteen days after inoculation, mice are engrafted with 4 × 10E6 PBMC to mimic the host immune system. The following day, mice are treated with intravenous injection of 1 × 10E6 armored CAR-alpha/beta T or gamma/delta T or mock T cells or phosphate-buffered saline (PBS) . Tumor progression is monitored by bioluminescent imaging (BLI) once a week. In addition, T cell proliferation is monitored via FACS analysis from plasma drawn from blood. Based on in vitro validation results shown above, it is expected that BSF17-H8 CAR-γδ T cells display similar anti-tumor efficacy to BSF17 CAR-γδ T cells while showing better in vivo persistence by resisting host immune rejection via targeting CD8 ⁺ cells.

Anti-tumor activity of an exemplary armored anti-CD19 CAR-T is assessed in vivo in an Raji xenograft model. Briefly, one million (1×10E6) Raji cells stably expressing the firefly luciferase reporter are implanted subcutaneously/intravenously on day 0 in NOD/SCID IL-2RγCnull (NSG) mice. Six days after inoculation, mice are engrafted with 4 × 10E6 PBMC to mimic the host immune system. The following day, mice are treated with intravenous injection of 1 × 10E6 armored CAR-alpha/beta T or gamma/delta T or mock T cells or phosphate-buffered saline (PBS) . Tumor progression was monitored by bioluminescent imaging (BLI) once a week. In addition, T cell proliferation is monitored via FACS analysis from plasma drawn from blood. Based on in vitro validation results shown above, it is expected that anti-CD8-armored CAR-γδ T cells display similar anti-tumor efficacy to unarmored CAR-γδ T cells while showing better in vivo persistence by resisting host immune rejection via targeting CD8 ⁺ cells.

SEQUENCE LISTING

CD30 (SEQ ID NO: 1)

CD70 (SEQ ID NO: 2)

CD28 (SEQ ID NO: 3)

CD137 (SEQ ID NO: 4)

CD7 (SEQ ID NO: 5)

CD2 (SEQ ID NO: 6)

CD69 (SEQ ID NO: 7)

CD8 (SEQ ID NO: 8)

CD33 (SEQ ID NO: 9)

Anti-BCMA CAR (BSF17, SEQ ID NO: 10)

Anti-BCMA CAR with anti-CD8 TCR (BSF17-H8, SEQ ID NO: 11)

Anti-BCMA sdAb (SEQ ID NO: 12)

CD8α Hinge (SEQ ID NO: 13)

CD8α transmembrane (SEQ ID NO: 14)

4-1BB intracellular domain (SEQ ID NO: 15)

CD3 zeta domain (SEQ ID NO: 16)

P2A (SEQ ID NO: 17)

CD3 leader peptide (SEQ ID NO: 18)

Anti-CD8 sdAb (SEQ ID NO: 19)

Linker (SEQ ID NO: 20)

CD3ε (SEQ ID NO: 21)

REFERENCES

1. Mo Dao, Melinda Mata, Leonie Alten, Aleksandra Nowicka, Sabrina Kuttruff, Sarah Mis sel, et al. Abstract 3588: ACTallo: A novel approach using gamma-delta T cells to allogeneic cellular therapy to treat cancer. Cancer Res (2018) 78: 3588

2. Shabnum Patel, Rachel A. Burga, Allison B. Powell, Elizabeth A. Chorvinsky, Nia Hoq, Sarah E. McCormack, et al. Front Oncol. (2019) ; 9: 196.

3. Sommer C, Boldajipour B, Kuo TC, Bentley T, Sutton J, Chen A, et al. Preclinical Evaluation of Allogeneic CAR T Cells Targeting BCMA for the Treatment of Multiple Myeloma. Mol Ther. (2019) 27 (6) : 1126-1138.

4. MacLeod DT, Antony J, Martin AJ, Moser RJ, Hekele A, Wetzel KJ, et al. Integration of a CD19 CAR into the TCR Alpha Chain Locus Streamlines Production of Allogeneic Gene-Edited CAR T Cells. Mol Ther. (2017) 25 (4) : 949-96.

5. Sheridan, C. Allogene and Celularity move CAR-T therapy offthe shelf. Nat Biotechnol (2018) 36, 375–377

6. Qasim, W. Allogeneic CAR T cell therapies for leukemia. Am J Hematol. 2019; 94: S50–S54

* * *

Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention.

Claims

A recombinant T cell receptor complex, comprising

(i) an extracellular antigen binding domain which comprises an antigen binding domain specific to an immune cell activation molecule that is expressed on an immune cell and related to the activation of the immune cell in an immune response, and

(ii) a T cell receptor complex comprising (a) a TCR chain selected from the group consisting of an alpha (α) chain, a beta (β) chain, a gamma (γ) chain and a delta (δ) chain of a T cell receptor, (b) an epsilon (ε) chain, a delta (δ) chain, and/or a gamma (γ) chain of CD3, and/or (c) a zeta (ζ) chain,

wherein the extracellular antigen binding domain is fused to any chain of the T cell receptor complex.
The recombinant T cell receptor complex according to claim 1, wherein the extracellular antigen binding domain further comprises an antigen binding domain specific to a disease associated antigen.
The recombinant T cell receptor complex according to claim 1 or 2, wherein the immune cell activation molecule is CD70, CD30, CD33, D28, CD137, CD7, CD69, CD2 or CD8.
The recombinant T cell receptor complex according to claim 2, wherein the disease associated antigen is a tumor associated antigen, an infectious disease associated antigen, or an inflammatory disease associated antigen.
The recombinant T cell receptor complex according to claim 4, wherein the disease associated antigen is a tumor associated antigen selected from the group consisting of CD19, CD20, CD22, CD4, CD24, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα) , mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, and HER-2, an infectious disease associated antigen selected from the group consisting of CD4, HBsAg, LMP-1, and LMP2, or an inflammatory disease associated antigen selected from the group consisting of IL17R, CD20, and CD6.
The recombinant T cell receptor complex according to any one of claims 1-5, wherein the antigen binding domain specific to the immune cell activation molecule comprises one or more antibodies or antigen-binding portions thereof, and/or the antigen binding domain specific to the disease associated antigen comprises one or more antibodies or antigen-binding portions thereof.
The recombinant T cell receptor complex according to any one of claims 1-6, wherein the T cell receptor complex comprises (i) a TCR alpha (α) chain, a TCR beta (β) chain, two CD3 epsilon (ε) chains, a CD3 delta (δ) chain, a CD3 gamma (γ) chain, and a zeta (ζ) chain, or (ii) a TCR gamma (γ) chain, a TCR delta (δ) chain, two CD3 epsilon (ε) chains, a CD3 delta (δ) chain, a CD3 gamma (γ) chain, and a zeta (ζ) chain.
An chimeric antigen receptor, comprising (a) an extracellular antigen binding domain which comprises an antigen binding domain specific to an immune cell activation molecule that is expressed on an immune cell and related to the activation of the immune cell in an immune response, (b) a transmembrane domain, and (c) an intracellular domain.
The chimeric antigen receptor according to claim 8, wherein the extracellular antigen binding domain further comprises an antigen binding domain specific to a disease associated antigen.
The chimeric antigen receptor according to claim 8 or 9, wherein the immune cell activation molecule is CD70, CD30, CD33, D28, CD137, CD7, CD69, CD2 or CD8.
The chimeric antigen receptor according to claim 9, wherein the disease associated antigen is a tumor associated antigen, an infectious disease associated antigen, or an inflammatory disease associated antigen.
The chimeric antigen receptor according to claim 11, wherein the disease associated antigen is a tumor associated antigen selected from the group consisting of CD19, CD20, CD22, CD4, CD24, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα) , mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, and HER-2, an infectious disease associated antigen selected from the group consisting of CD4, HBsAg, LMP-1, and LMP2, or an inflammatory disease associated antigen selected from the group consisting of IL17R, CD20, and CD6.
The chimeric antigen receptor according to any one of claims 8-12, wherein the antigen binding domain specific to the immune cell activation molecule comprises one or more antibodies or antigen-binding portions thereof, and/or the antigen binding domain specific to the disease associated antigen comprises one or more antibodies or antigen-binding portions thereof.
The chimeric antigen receptor according to any one of claims 8-13, wherein the transmembrane domain comprises CD8α transmembrane region or CD28 transmembrane region.
The chimeric antigen receptor according to any one of claims 8-14, wherein the intracellular domain comprise at least one signaling domain selected from the group consisting of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d, and/or at least one costimulatory domains selected from the group consisting of CD28, 4-1BB (CD137) , CD27, OX40, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, CD83, and a ligand that specifically binds with CD83.
The chimeric antigen receptor according to any one of claims 8-15, further comprising a CD8α hinge between the extracellular antigen binding domain and the transmembrane domain, and/or a leader sequence at the N-terminus.
A dual antigen binding receptor comprising

(i) a chimeric antigen receptor comprising (a) an extracellular antigen binding domain which comprises an antigen binding domain specific to a first molecule, (b) a transmembrane domain, and (c) an intracellular domain; and

(ii) a recombinant T cell receptor complex, comprising (a) an extracellular antigen binding domain which comprises an antigen binding domain specific to a second molecule, and (b) a T cell receptor complex comprising a TCR chain selected from the group consisting of an alpha (α) chain, a beta (β) chain, a gamma (γ) chain and a delta (δ) chain of a T cell receptor, an epsilon (ε) chain, a delta (δ) chain, and/or a gamma (γ) chain of CD3, and/or a zeta (ζ) chain, wherein the extracellular antigen binding domain specific to the second molecule is fused to any chain of the T cell receptor complex,

wherein one of the first molecule and the second molecule is an immune cell activation molecule that is expressed on an immune cell and related to the activation of the immune cell in an immune response, and the other is a disease associated antigen.
The dual antigen binding receptor according to claim 17, wherein the immune cell activation molecule is CD70, CD30, CD33, D28, CD137, CD7, CD69, CD2 or CD8.
The dual antigen binding receptor according to claim 18, wherein the immune cell activation molecule is CD8.
The dual antigen binding receptor according to any one of claim 17-19, wherein the disease associated antigen is a tumor associated antigen, an infectious disease associated antigen, or an inflammatory disease associated antigen.
The dual antigen binding receptor according to claim 20, wherein the disease associated antigen is a tumor associated antigen selected from the group consisting of CD19, CD20, CD22, CD4, CD24, CD38, CD123, CD228, CD138, BCMA, GPC3, CEA, folate receptor (FRα) , mesothelin, CD276, gp100, 5T4, GD2, EGFR, MUC-1, PSMA, EpCAM, MCSP, SM5-1, MICA, MICB, ULBP, and HER-2, an infectious disease associated antigen selected from the group consisting of CD4, HBsAg, LMP-1, and LMP2, or an inflammatory disease associated antigen selected from the group consisting of IL17R, CD20, and CD6.
The dual antigen binding receptor according to any one of claims 17-21, wherein the antigen binding domain specific to the immune cell activation molecule comprises one or more antibodies or antigen-binding portions thereof, and/or the antigen binding domain specific to the disease associated antigen comprises one or more antibodies or antigen-binding portions thereof.
The dual antigen binding receptor according to any one of claims 17-22, wherein the transmembrane domain comprises CD8α transmembrane region or CD28 transmembrane region.
The dual antigen binding receptor according to any one of claims 17-23, wherein the intracellular domain comprise at least one signaling domain selected from the group consisting of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d, and/or at least one costimulatory domains selected from the group consisting of CD28, 4-1BB (CD137) , CD27, OX40, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, CD83, and a ligand that specifically binds with CD83.
The dual antigen binding receptor according to any one of claims 17-24, wherein the chimeric antigen receptor further comprising a CD8α hinge between the extracellular antigen binding domain and the transmembrane domain, and/or a leader sequence at the N-terminus.
The dual antigen binding receptor according to any one of claims 17-25, wherein the T cell receptor complex comprises (i) a TCR alpha (α) chain, a TCR beta (β) chain, two CD3 epsilon (ε) chains, a CD3 delta (δ) chain, a CD3 gamma (γ) chain, and a zeta (ζ) chain, or (ii) a TCR gamma (γ) chain, a TCR delta (δ) chain, two CD3 epsilon (ε) chains, a CD3 delta (δ) chain, a CD3 gamma (γ) chain, and a zeta (ζ) chain.
A dual chimeric antigen receptor, comprising

(i) a first chimeric antigen receptor comprising (a) an extracellular antigen binding domain which comprises an antigen binding domain specific to an immune cell activation molecule that is expressed on an immune cell and related to the activation of the immune cell in an immune response, (b) a transmembrane domain, and (c) an intracellular domain; and

(ii) a second chimeric antigen receptor comprising (a) an extracellular antigen binding domain which comprises an antigen binding domain specific to a disease associated antigen, (b) a transmembrane domain, and (c) an intracellular domain.
The dual chimeric antigen receptor according to claim 27, wherein the immune cell activation molecule is CD70, CD30, CD33, D28, CD137, CD7, CD69, CD2 or CD8.
The dual chimeric antigen receptor according to claim 27 or 28, wherein the disease associated antigen is a tumor associated antigen, an infectious disease associated antigen, or an inflammatory disease associated antigen.
The dual chimeric antigen receptor according to any one of claims 27-29, wherein the antigen binding domain of the first chimeric antigen receptor comprises one or more antibodies or antigen-binding portions thereof, and/or the antigen binding domain of the second chimeric antigen receptor comprises one or more antibodies or antigen-binding portions thereof.
The dual chimeric antigen receptor according to any one of claims 27-30, wherein the transmembrane domain of each of the first and second chimeric antigen receptors comprises CD8α transmembrane region or CD28 transmembrane region.
The dual chimeric antigen receptor according to any one of claims 27-31, wherein the intracellular domain of one of the first and second chimeric antigen receptos comprise at least one signaling domain selected from the group consisting of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d, and at least one costimulatory domains selected from the group consisting of CD28, 4-1BB (CD137) , CD27, OX40, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1) , CD2, CD7, LIGHT, NKG2C, B7-H3, TNFRSF9, TNFRSF4, TNFRSF8, CD40LG, ITGB2, KLRC2, TNFRSF18, TNFRSF14, HAVCR1, LGALS9, CD83, and a ligand that specifically binds with CD83; and

the intracellular domain of the other of the first and second chimeric antigen receptos comprise at least one signaling domain selected from the group consisting of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d.
The dual chimeric antigen receptor according to any one of claims 27-32, further comprising a CD8α hinge between the extracellular antigen binding domain and the transmembrane domain, and/or a leader sequence at the N-terminus, in the first chimeric antigen receptor and/or the second chimeric antigen receptor.
A recombinant dual T cell receptor complex, comprising

(i) a first recombinant T cell receptor complex, comprising (a) an extracellular antigen binding domain which comprises an antigen binding domain specific to an immune cell activation molecule that is expressed on an immune cell and related to the activation of the immune cell in an immune response, and (b) a T cell receptor complex comprising a TCR chain selected from the group consisting of an alpha (α) chain, a beta (β) chain, a gamma (γ) chain and a delta (δ) chain of a T cell receptor, an epsilon (ε) chain, a delta (δ) chain, and/or a gamma (γ) chain of CD3, and/or a zeta (ζ) chain, wherein the extracellular antigen binding domain is fused to any chain of the T cell receptor complex; and

(ii) a second recombinant T cell receptor complex, comprising (a) an extracellular antigen binding domain which comprises an antigen binding domain specific to a disease associated antigen, and (b) a T cell receptor complex comprising a TCR chain selected from the group consisting of an alpha (α) chain, a beta (β) chain, a gamma (γ) chain and a delta (δ) chain of a T cell receptor, an epsilon (ε) chain, a delta (δ) chain, and/or a gamma (γ) chain of CD3, and/or a zeta (ζ) chain, wherein the extracellular antigen binding domain is fused to any chain of the T cell receptor complex.
The recombinant dual T cell receptor complex according to claim 34, wherein the immune cell activation molecule is CD70, CD30, CD33, D28, CD137, CD7, CD69, CD2 or CD8.
The recombinant dual T cell receptor complex according to claim 34 or 35, wherein the disease associated antigen is a tumor associated antigen, an infectious disease associated antigen, or an inflammatory disease associated antigen.
The recombinant dual T cell receptor complex according to any one of claims 34-36, wherein the antigen binding domain specific to the immune cell activation molecule comprises one or more antibodies or antigen-binding portions thereof, and/or the antigen binding domain specific to the disease associated antigen comprises one or more antibodies or antigen-binding portions thereof.
The recombinant dual T cell receptor complex according to any one of claims 34-37, wherein the T cell receptor complex in each of the first and second recombinant T cell receptor complexes comprises (i) a TCR alpha (α) chain, a TCR beta (β) chain, two CD3 epsilon (ε) chains, a CD3 delta (δ) chain, a CD3 gamma (γ) chain, and a zeta (ζ) chain, or (ii) a TCR gamma (γ) chain, a TCR delta (δ) chain, two CD3 epsilon (ε) chains, a CD3 delta (δ) chain, a CD3 gamma (γ) chain, and a zeta (ζ) chain.
An engineered immune cell, comprising a recombinant T cell receptor complex according to any one of claims 1 to 7, a chimeric antigen receptor according to any one of claims 8 to 16, a dual antigen binding receptor according to any one of claims 17 to 26, a dual chimeric antigen receptor according to any one of claims 27 to 33, and/or a recombinant dual T cell receptor complex according to any one of claims 34 to 38.
The engineered immune cell according to claim 39, which is a T cell or a natural killing cell.
The engineered immune cell according to claim 40, wherein the T cell is αβ T cell or γδ T cell.
The engineered immune cell according to any one of claims 39-41, which is engineered to express the immune cell activation molecule at a low level, or not express the immune cell activation molecule.
The engineered immune cell according to any one of claim 39-42, which is engineered to express one or more cytokines at high levels when the engineered cell is activated, and express such cytokines at low levels or not express such cytokines when the engineered immune cell is not activated, wherein the one or more cytokines are selected from the group consisting of IL-7, IL-12, IL-15, and IL-18.
A nucleic acid encoding a recombinant T cell receptor complex according to any one of claims 1 to 7, a chimeric antigen receptor according to any one of claims 8 to 16, a dual antigen binding receptor according to any one of claims 17 to 26, a dual chimeric antigen receptor according to any one of claims 27 to 33, or a recombinant dual T cell receptor complex according to any one of claims 34 to 38.
An expression vector comprising the nucleic acid according to claim 44.
A host cell comprising the nucleic acid according to claim 44, or the expression vector according to claim 45.
A pharmaceutical composition comprising an effective amount of the engineered immune cell according to any one of claims 39 to 43, the nucleic acid according to claim 44, or the expression vector according to claim 45, and a pharmaceutically acceptable carrier.
A method for treating a disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of the engineered immune cells which comprises a recombinant T cell receptor complex according to any one of claims 1 to 7, a chimeric antigen receptor according to any one of claims 8 to 16, a dual antigen binding receptor according to any one of claims 17 to 26, a dual chimeric antigen receptor according to any one of claims 27 to 33, and/or a recombinant dual T cell receptor complex according to any one of claims 34 to 38, and a pharmaceutically acceptable carrier.
The method according to claim 48, wherein the disease is a tumor, an infectious disease, or an inflammatory disease.
The method according to claim 49, wherein the tumor is a hematological tumor.
The method according to claim 50, wherein the hematological tumor is leukemia, lymphomas, or myeloma.
The method according to claim 49, wherein the infectious disease is acquired immune deficiency syndrome.
The method according to claim 49, wherein the inflammatory disease is an autoimmune disease.
The method according to claim 53, wherein the inflammatory disease is myasthenia gravis.
A method for reducing or eliminating graft rejection of allogeneic cells in a subject, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of engineered immune cells which comprise a recombinant T cell receptor complex according to any one of claims 1 to 7, a chimeric antigen receptor according to any one of claims 8 to 16, a dual antigen binding receptor according to any one of claims 17 to 26, a dual chimeric antigen receptor according to any one of claims 27 to 33, and/or a recombinant dual T cell receptor complex according to any one of claims 34 to 38, and a pharmaceutically acceptable carrier, prior to or together with administration of the allogeneic cells.
A kit for making the engineered immune cell according to any one of claims 39 to 43, comprising a container comprising the nucleic acid according to claim 44, the vector according to claim 45, or the host cell according to claim 46, and instructions for using the kit.
Use of the kit according to claim 56 to make an engineered immune cell according to any one of claims 39 to 43.