JP2022548866A - Compositions and methods for TCR reprogramming using fusion proteins - Google Patents

Abstract

Provided herein are recombinant nucleic acids encoding T-cell receptor (TCR) fusion proteins (TFPs) and TCR constant domains, engineered T-cells expressing the encoded molecules, and therapeutic agents for the treatment of diseases, including cancer. Provide instructions for its use. As used herein, fusion proteins of TCR subunits, including CD3 epsilon, CD3 gamma, CD3 delta, TCR gamma, TCR delta, TCR alpha, and TCR beta chains, with binding domains specific for cell surface antigens, including fusion proteins of existing We describe engineered T cells that have the potential to overcome the limitations of the approach. [Selection drawing] Fig. 3

Description

Cross-reference to related applications
[0001] This application claims the benefit of U.S. Provisional Application No. 62/899,563, filed September 12, 2019, and U.S. Provisional Application No. 62/971,682, filed February 7, 2020. , each of which is incorporated herein by reference in its entirety.

[0002] Most patients with hematological malignancies or end-stage solid tumors are incurable with standard therapy. Additionally, conventional treatment options often have serious side effects. Numerous attempts have been made to induce a patient's immune system to reject cancer cells, methods collectively referred to as cancer immunotherapy. However, several obstacles make achieving clinical efficacy rather difficult. Hundreds of so-called tumor antigens have been identified, but these are often autologous and thus may target cancer immunotherapy to healthy tissues or are poorly immunogenic. In addition, cancer cells use multiple mechanisms to either make themselves invisible or to counter the initiation and propagation of immune attack by cancer immunotherapy.

[0003] Chimeric antigen receptor (CAR)-modified autologous T-cell therapy, which utilizes a method of directing genetically engineered T cells to the appropriate cell surface molecules of cancer cells, has recently been used with this therapy. Developments have shown promising results in the treatment of B-cell malignancies using the action of the immune system (see, eg, Sadelain et al., Cancer Discovery 3:388-398 (2013)). Clinical results with CD19-specific CAR T cells (termed CTL019) showed complete remission in patients with chronic lymphocytic leukemia (CLL) as well as childhood acute lymphocytic leukemia (ALL) (e.g., Kalos et al. ., Sci Transl Med 3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368:1509-1518 (2013)). An alternative approach is to use T cell receptor (TCR) α and β chains selected against tumor-associated peptide antigens to genetically engineer autologous T cells. These TCR chains form a complete TCR complex and present the TCR to T cells for a second defined specificity. Engineered autologous T cells expressing NY-ESO-1-specific TCR α and β chains have yielded promising results in patients with synovial cancer.

[0004] Successful treatment of patients with modified T cells requires, in addition to the ability of genetically modified T cells expressing a CAR or a second TCR, to recognize and destroy their respective target cells in vitro/ex vivo. The T cells are required to be capable of , potent activation, proliferation, persistence over time and, in the case of recurrent disease, to enable a "memory" response. The high and manipulable clinical efficacy of CAR T cells is currently limited to CD19-positive B-cell malignancies and patients with HLA-A2 phenotype, NY-ESO-1-peptide-expressing synovial sarcoma It is

[0005] It is clear that there is a need for improved genetically engineered T-cells for broader action against a variety of human malignancies.
[0006] The present specification includes fusion proteins of TCR subunits including CD3 ε chain, CD3 γ chain, CD3 δ chain, TCR γ chain, TCR δ chain, TCR α chain, and TCR β chain and binding domains specific for cell surface antigens. describe engineered T cells that have the potential to overcome the limitations of existing approaches. In addition, the engineered T cells can functionally disrupt endogenous TCRs (eg, TCRα, β, or both). The modified T cells may have the ability to kill target cells more efficiently than CAR, but may release similar or lower levels of inflammatory cytokines than CAR. Since such elevated levels of cytokines are associated with the dose-limiting toxicity of adoptive CAR-T therapy, this engineered T cell and method of use may represent an advantage of such cells compared to CAR.

[0007] Provided herein are modified T cells comprising a T cell receptor (TCR) fusion protein (TFP) and a TCR constant domain, methods of making modified T cells, and methods of use thereof for the treatment of disease. . The TCR constant domains described herein can be TCRα constant domains, TCRβ constant domains, TCRα and TCRβ constant domains, TCRγ constant domains, TCRδ constant domains, or TCRγ and TCR constant domains. A TFP as described herein can comprise a TCR subunit and an antibody or fragment thereof. A TFP as described herein can comprise a TCR subunit and a binding ligand or fragment thereof. TCR subunits can be derived from any TCR chain, such as the TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ε, CD3δ, or CD3γ. Antibodies or fragments thereof can be murine, human, or humanized.

[0008] In one aspect, the present disclosure provides a T-cell receptor (TCR) fusion protein ( TFP) and a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a TCRγ constant domain or a TCRδ constant domain, or encodes a TCRγ constant domain and a TCRδ constant domain wherein the TCR subunit and antibody are operably linked, and TFP is operably integrated into the TCR complex when expressed in a modified T cell comprising functional disruption of the endogenous TCR I will provide a.

[0009] In another aspect, the present disclosure provides a T cell receptor comprising at least a portion of a TCR extracellular domain, a TCR subunit comprising a transmembrane domain, and a binding ligand or fragment thereof capable of binding to an antibody or fragment thereof. a sequence encoding a body (TCR) fusion protein (TFP) and a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRγ constant domain or a TCRδ constant domain, or a TCRγ constant domain and a TCRδ constant domain wherein the TCR subunit and binding ligand or fragment thereof are operably linked and TFP is functionally associated with the TCR complex when expressed in a modified T cell comprising functional disruption of the endogenous TCR A recombinant nucleic acid is provided that is incorporated into a.

[0010] In some embodiments, the TCR subunit comprises an intracellular domain of TCRα, TCRβ, TCRγ, or TCRδ, or a stimulatory domain from a CD3ε, CD3γ, or CD3δ intracellular signaling domain. further includes In some embodiments, the TCR constant domain is the TCRdelta constant domain. In some embodiments, the TCRδ constant domain comprises SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 243, or SEQ ID NO: 265, functional fragments thereof, or amino acid sequences thereof with at least one but no more than 20 modifications. include. In some embodiments, the intracellular domain is that of TCRγ. In some embodiments, the TCRdelta constant domain-encoding sequence further encodes a second antigen or ligand binding domain that is operably linked to the TCRdelta constant domain-encoding sequence. In some embodiments, the second antigen or ligand binding domain is the same as or different from the antigen or ligand binding domain of TFP. In some embodiments, the second antigen- or ligand-binding domain is operably linked via a linker to the sequence encoding the TCRdelta constant domain. In some embodiments, the TCR constant domain is the TCRγ constant domain. In some embodiments, the TCRγ constant domain comprises SEQ ID NO:21 or SEQ ID NO:155, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications. In some embodiments, the intracellular domain is the intracellular domain of TCRdelta. In some embodiments, the TCRγ constant domain-encoding sequence further encodes a second antigen or ligand binding domain that is operably linked to the TCRγ constant domain-encoding sequence. In some embodiments, the second antigen or ligand binding domain is the same as or different from the antigen or ligand binding domain of TFP. In some embodiments, the second antigen- or ligand-binding domain is operably linked via a linker to the sequence encoding the TCRγ constant domain.

[0011] In some embodiments, the recombinant nucleic acid comprises sequences encoding a TCRγ constant domain and a TCRδ constant domain. In some embodiments, the TCRγ constant domain comprises SEQ ID NO:21 or SEQ ID NO:155, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications. In some embodiments, the sequence encoding the TCRγ constant domain further encodes the TCRγ variable domain, thereby encoding the entire TCRγ domain. In some embodiments, the entire TCRγ domain is γ9 or γ4. In some embodiments, the entire TCRγ domain comprises SEQ ID NO:255, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications. In some embodiments, the TCRδ constant domain comprises SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 243, or SEQ ID NO: 265, functional fragments thereof, or amino acid sequences thereof with at least one but no more than 20 modifications. include. In some embodiments, a sequence encoding a TCRdelta constant domain further encodes a TCRdelta variable domain, thereby encoding the entire TCRdelta domain. In some embodiments, the entire TCR delta domain is delta 2 or delta 1. In some embodiments, the entire TCRδ constant domain comprises SEQ ID NO:256, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications. In some embodiments, the intracellular signaling domain is CD3ε, CD3γ, or CD3δ. In some embodiments, the intracellular signaling domain is CD3ε. In some embodiments, the recombinant nucleic acid further comprises at least one leader sequence and at least one linker. In some embodiments, the recombinant nucleic acid further comprises a portion of the TCRα constant domain, a portion of the TCRβ domain, or both. In some embodiments, the sequence is, from 5′ to 3′, a first leader sequence, an antigen binding domain sequence, a linker, a TRDC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRGC gene. Contains arrays. In some embodiments, the sequences are, in the 5′-3′ direction, a first leader sequence, a TRDC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker sequence, and a TRGC. Contains gene sequences. In some embodiments, the sequence is, in the 5′-3′ direction, a first leader sequence, an antigen binding domain sequence, a first linker sequence, a TRDC gene sequence, a cleavable linker, a second leader sequence, It includes a second antigen binding domain sequence, a second linker sequence, and a TRGC gene sequence. In some embodiments, the sequences are, in the 5′-3′ direction, a first leader sequence, a TRDC gene sequence, a first cleavable linker sequence, a second leader sequence, a TRGC gene sequence, a second It includes a cleavable linker sequence, a third leader sequence, an antigen binding domain sequence, a linker sequence, and a CD3ε gene sequence. In some embodiments, the sequence is, in the 5′-3′ direction, a first leader sequence, a first antigen binding domain sequence, a first linker sequence, a TRDC gene sequence or fragment thereof, a TRAC gene sequence or Fragments, cleavable linker sequences, second leader sequences, second antigen binding domain sequences, second linker sequences, TRGC gene sequences or fragments thereof, and TRBC gene sequences or fragments thereof.

[0012] In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:1. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:2. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:3. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:4. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:5. In some embodiments, the sequence encodes the sequence of SEQ ID NO:242. In some embodiments, the sequence encodes the sequence of SEQ ID NO:244. In some embodiments, the sequence encodes the sequence of SEQ ID NO:245. In some embodiments, the sequence encodes the sequence of SEQ ID NO:246. In some embodiments, the sequence encodes the sequence of SEQ ID NO:248. In some embodiments, the sequence encodes the sequence of SEQ ID NO:250. In some embodiments, the sequence encodes the sequence of SEQ ID NO:252. In some embodiments, the sequence encodes the sequence of SEQ ID NO:257. In some embodiments, the sequence encodes the sequence of SEQ ID NO:263. In some embodiments, the sequence encodes the sequence of SEQ ID NO:264.

[0013] In some embodiments, the binding ligand can bind to the Fc domain of an antibody. In some embodiments, the binding ligand can selectively bind to an IgG1 antibody. In some embodiments, the binding ligand can specifically bind to an IgG4 antibody. In some embodiments, the antibody or fragment thereof binds to a cell surface antigen. In some embodiments, the antibody or fragment thereof is murine, human, or humanized. In some embodiments, the antibody or fragment thereof binds to a cell surface antigen on the surface of tumor cells. In some embodiments, the binding ligand is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. Including body. In some embodiments, binding ligands do not include antibodies or fragments thereof. In some embodiments, the binding ligand comprises a CD16 polypeptide or fragment thereof. In some embodiments, the binding ligand comprises a CD16 binding polypeptide. In some embodiments, the binding ligand is human or humanized. In some embodiments, the recombinant nucleic acid further comprises a nucleic acid sequence encoding an antibody or fragment thereof that can be bound by a binding ligand. In some embodiments, the antibody or fragment thereof is secretable from the cell.

[0014] In another aspect, the present disclosure provides a TCR subunit comprising at least a portion of a TCR extracellular domain and a transmembrane domain and a ligand or fragment thereof that binds to a receptor or polypeptide expressed on the surface of a cell. a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising an antigen binding domain comprising; and a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a TCRγ constant domain comprises a sequence that is a TCRδ constant domain, or a sequence that encodes a TCRγ constant domain and a TCRδ constant domain, wherein the TCR subunit and antigen binding domain are operably linked and comprise functional disruption of the endogenous TCR A recombinant nucleic acid is provided that, when expressed in a modified T cell, functionally integrates TFP into the TCR complex.

[0015] In some embodiments, the TCR subunit is a TCRα, TCRβ, TCRγ, or TCRδ intracellular domain, or an intracellular domain comprising a stimulatory domain from a CD3ε, CD3γ, or CD3δ intracellular signaling domain. further includes In some embodiments, the recombinant nucleic acid further comprises at least a subsequence encoding a TCRα constant domain, a subsequence encoding a TCRβ constant domain, or a subsequence of both the TCRα and TCRβ constant domains. In some embodiments the antigen binding domain comprises a ligand. In some embodiments, the ligand binds to a receptor on the cell. In some embodiments, the ligand binds to a polypeptide expressed on the surface of the cell. In some embodiments, the receptor or polypeptide expressed on the surface of the cell comprises a stress response receptor or polypeptide. In some embodiments, the receptor or polypeptide expressed on the surface of the cell is an MHC class I-associated glycoprotein. In some embodiments, the MHC class I-associated glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4, and combinations thereof. In some embodiments, the antigen binding domain is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. Contains mers. In some embodiments, the antigen binding domain comprises a monomer or dimer of the ligand or fragment thereof. In some embodiments, the ligand or fragment thereof is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or It is decamer. In some embodiments, the ligand or fragment thereof is monomeric or dimeric. In some embodiments the antigen binding domain does not comprise an antibody or fragment thereof. In some embodiments, the antigen binding domain does not contain a variable region. In some embodiments, the antigen binding domain does not contain CDRs. In some embodiments, the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or fragment thereof. In some embodiments, the TCR constant domain is incorporated into a functional TCR complex upon expression in T cells. In some embodiments, the TCR constant domain is incorporated into the same functional TCR complex that incorporates TFP when expressed in T cells. In some embodiments, the sequence encoding the TFP and the sequence encoding the TCR constant domain(s) are contained within the same nucleic acid molecule. In some embodiments, the encoded TFP and the encoded TCR constant domain are operably linked by a first linker sequence. In some embodiments, the first linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A, eg, T2A or P2A cleavage site. In some embodiments, the sequence encoding the TFP and the sequence encoding the TCR constant domain(s) are contained within different nucleic acid molecules. In some embodiments, the TCR subunit of TFP and the antibody domain, antigen binding domain, or binding ligand, or fragment thereof are operably linked by a second linker sequence. In some embodiments, the second linker sequence comprises (G ₄ S) _n , where n=1-4. In some embodiments, the transmembrane domain is a TCR transmembrane domain from CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRδ, or TCRγ. In some embodiments, the intracellular domain is derived from CD3ε only, CD3γ only, CD3δ only, TCRα only, TCRβ, TCRγ, or TCRδ. In some embodiments, the TCR subunit comprises (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain, (i), (ii) , and (iii) are from the same TCR subunit. In some embodiments, the TCR extracellular domain is a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, a functional fragment thereof, or at least one but no more than 20 amino acids thereof modified It includes the extracellular domain of a protein or part thereof selected from the group consisting of sequences. In some embodiments, the TCR subunit comprises (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain of the TCR gamma or TCR delta chain. In some embodiments, the TCR extracellular domain comprises the extracellular portion of a TCRγ or TCRδ chain constant domain, a functional fragment thereof, or an amino acid sequence thereof having at least one but no more than 20 modifications. In some embodiments, the TCR subunit comprises (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain, with the delta constant domain or fragment thereof. have or contain them. In some embodiments, the delta constant domain comprises the sequence of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243, or SEQ ID NO:265, a functional fragment thereof, or at least one but no more than 20 amino acids thereof modified has an array. In some embodiments, the TCR subunit comprising (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain is a γ constant domain, or including it. In some embodiments, the gamma constant domain has the sequence of SEQ ID NO:21 or SEQ ID NO:155, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications. In some embodiments, the extracellular domain of TFP does not include a γ- or δ-chain variable domain. In some embodiments, the TCR subunit comprises a stimulatory domain of a protein selected from the intracellular signaling domains of CD3ε, CD3γ, or CD3δ, or an amino acid sequence having at least one modification thereto. Including internal domains. In some embodiments, the TCR subunit of TFP comprises the extracellular, transmembrane, and intracellular domains of CD3ε. In some embodiments, the TCR subunit of CD3ε comprises the sequence of SEQ ID NO:258, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications.

[0016] In some embodiments, the TFP, the TCRγ constant domain, the TCRδ constant domain, and any combination thereof functionally interacts with an endogenous TCR complex and/or at least one endogenous TCR polypeptide. can do. In some embodiments, the TCR constant domain is a TCRγ constant domain and the TFP is functionally integrated into a TCR complex comprising endogenous subunits of TCRδ, CD3ε, CD3γ, CD3δ, or combinations thereof; The TCR constant domain is a TCRdelta constant domain, and TFP is functionally integrated into a TCR complex comprising endogenous subunits of TCRγ, CD3ε, CD3γ, CD3δ, or combinations thereof, or the TCR constant domain is a TC
The Rγ and TCRδ constant domains that functionally integrate TFP into the TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or combinations thereof. In some embodiments, the at least one but no more than 20 modifications thereto comprise modifications of amino acids that mediate cell signaling or modifications of amino acids that are phosphorylated in response to ligand binding to TFP. In some embodiments the antibody is an antibody fragment. In some embodiments, the antibody fragment is a scFv, single domain antibody domain, VH domain, or VL domain. In some embodiments, the antigen binding domain is an anti-CD19 binding domain, an anti-B cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, an anti-79b binding domain , anti-HER2 binding domain, anti-PMSA binding domain, anti-MUC16 binding domain, anti-CD22 binding domain, anti-PD-L1 binding domain, anti-BAFF or BAFF receptor binding domain, anti-nectin-4 binding domain, anti-TROP-2 binding domain , an anti-GPC3 binding domain, and an anti-ROR-1 binding domain. In some embodiments, the anti-MSLN binding domain comprises CDR1 of SEQ ID NO:60, CDR2 of SEQ ID NO:61, and CDR3 of SEQ ID NO:62. In some embodiments, the anti-MSLN binding domain comprises CDR1 of SEQ ID NO:63, CDR2 of SEQ ID NO:64, and CDR3 of SEQ ID NO:65. In some embodiments, the anti-MSLN binding domain comprises CDR1 of SEQ ID NO:66, CDR2 of SEQ ID NO:67, and CDR3 of SEQ ID NO:68. In some embodiments, the anti-MSLN binding domain comprises a sequence having at least about 80% sequence identity to the sequence of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71. In some embodiments, the anti-CD19 binding domain comprises CDR1 of SEQ ID NO:73, CDR2 of SEQ ID NO:75, and CDR3 of SEQ ID NO:77. In some embodiments, the anti-CD19 binding domain comprises CDR1 of SEQ ID NO:79, CDR2 of SEQ ID NO:81, and CDR3 of SEQ ID NO:83. In some embodiments, the anti-CD19 binding domain comprises a sequence having at least about 80% sequence identity to the sequences of SEQ ID NO:85 and/or SEQ ID NO:87. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCRα transmembrane domain. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCRβ transmembrane domain. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCRα transmembrane domain and a sequence encoding a TCRβ transmembrane domain.

[0017] In another aspect, the disclosure provides a TCR subunit comprising at least a portion of the extracellular domain of mouse TCRα or mouse TCRβ and a transmembrane domain of mouse TCRα or mouse TCRβ, and an antibody or antibody thereof comprising an antigen binding domain. a sequence encoding a T cell receptor (TCR) fusion protein (TFP) and a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a mouse TCRα constant domain or a mouse TCRβ A modification comprising a sequence that is a constant domain, or that encodes a murine TCRα constant domain and a murine TCRβ constant domain, wherein the TCR subunit and antibody are operably linked, and which comprises functional disruption of the endogenous TCR A recombinant nucleic acid is provided that, when expressed in a T cell, functionally integrates TFP into the TCR complex.

[0018] In another aspect, the present disclosure provides a TCR subunit comprising at least a portion of the extracellular domain of mouse TCRα or mouse TCRβ and a transmembrane domain of mouse TCRα or mouse TCRβ and a binding capable of binding to an antibody or fragment thereof. a sequence encoding a T cell receptor (TCR) fusion protein (TFP), comprising a ligand or a fragment thereof, and a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a mouse TCRα constant domain or a sequence that is a mouse TCRβ constant domain, or a sequence encoding a mouse TCRα constant domain and a mouse TCRβ constant domain, wherein the TCR subunit and the binding ligand or fragment thereof are operably linked and endogenous TCR A recombinant nucleic acid is provided that functionally integrates TFP into the TCR complex when expressed in a modified T cell comprising a functional disruption of TFP.

[0019] In some embodiments, the TCR subunit comprises the intracellular domain of mouse TCRα or mouse TCRβ. In some embodiments, the TCR constant domain is T
CRα constant domain. In some embodiments, the TCRα constant domain has SEQ ID NO: 17, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO: 207, functional fragments thereof, or at least one but no more than 20 modifications including its amino acid sequence. In some embodiments, the TCRα constant domain comprises a mouse TCRα constant domain. In some embodiments, the mouse TCRα constant domain comprises amino acids 2-137 of the mouse TCRα constant domain. In some embodiments, the mouse TCRα constant domain comprises amino acids 2-137 of SEQ ID NO:146. In some embodiments, the mouse TCRα constant domain comprises the sequence of SEQ ID NO:207. In some embodiments, the mouse TCRα constant domain comprises amino acids 82-137 of SEQ ID NO:146. In some embodiments, the mouse TCRα constant domain comprises the sequence of SEQ ID NO:17. In some embodiments, the intracellular domain is that of TCRβ. In some embodiments, the TCRα constant domain-encoding sequence further encodes a second antigen or ligand binding domain that is operably linked to the TCRα constant domain-encoding sequence. In some embodiments, the second antigen or ligand binding domain is the same as or different from the antigen or ligand binding domain of TFP. In some embodiments, the second antigen- or ligand-binding domain is operably linked via a linker to the sequence encoding the TCRα constant domain. In some embodiments, the TCR constant domain is the TCRβ constant domain. In some embodiments, the TCRβ constant domain has SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO: 209, functional fragments thereof, or at least one but no more than 20 modifications including its amino acid sequence. In some embodiments, the TCRβ constant domain comprises a mouse TCRβ constant domain. In some embodiments, the mouse TCRβ constant domain comprises amino acids 2-173 of the mouse TCRβ constant domain. In some embodiments, the mouse TCRβ constant domain comprises amino acids 2-173 of SEQ ID NO:152. In some embodiments, the mouse TCRβ constant domain comprises SEQ ID NO:209. In some embodiments, the TCRβ constant domain comprises amino acids 123-173 of SEQ ID NO:152. In some embodiments, the TCRβ constant domain comprises SEQ ID NO:18. In some embodiments, the intracellular domain is that of TCRα. In some embodiments, the sequence encoding the TCRβ constant domain further encodes a second antigen or ligand binding domain operably linked to the sequence encoding the TCRβ constant domain. In some embodiments, the second antigen or ligand binding domain is the same as or different from the antigen or ligand binding domain of TFP. In some embodiments, the second antigen- or ligand-binding domain is operably linked via a linker to the sequence encoding the TCRβ constant domain. In some embodiments, the recombinant nucleic acid comprises sequences encoding TCRα and TCRβ constant domains. In some embodiments, the TCRα constant domain has SEQ ID NO: 17, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO: 207, functional fragments thereof, or at least one but no more than 20 modifications including its amino acid sequence. In some embodiments, the TCRβ constant domain has SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO: 209, functional fragments thereof, or at least one but no more than 20 modifications including its amino acid sequence. In some embodiments, the intracellular signaling domain is CD3ε, CD3γ, or CD3δ. In some embodiments, the intracellular signaling domain is CD3ε.

[0020] In some embodiments, the sequence is, from 5' to 3', a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and TRBC gene sequences. In some embodiments, the sequences are, from 5′ to 3′, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene. Contains arrays. In some embodiments, the sequence is, in the 5' to 3' direction, a first leader sequence, T
A RAC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker, and a TRBC gene sequence. In some embodiments, the sequence is, from 5′ to 3′, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain. Includes sequences, linkers, and TRBC gene sequences. In some embodiments, the sequences are, in the 5′-3′ direction, a first leader sequence, a TRAC gene sequence, a first cleavable linker sequence, a second leader sequence, a TRBC gene sequence, a second It includes a cleavable linker sequence, a third leader sequence, an antigen binding domain sequence, a linker sequence, and a CD3ε gene sequence. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:10. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:204. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:206. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:210. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:211. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:217. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:218. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:219. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:220. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:259. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:261. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:262. In some embodiments, the recombinant nucleic acid further comprises at least one leader sequence and at least one linker. In some embodiments, a binding ligand can bind to the Fc domain of an antibody. In some embodiments, the binding ligand can selectively bind to an IgG1 antibody. In some embodiments, the binding ligand can specifically bind to an IgG4 antibody. In some embodiments, the antibody or fragment thereof binds to a cell surface antigen. In some embodiments, the antibody or fragment thereof is murine, human, or humanized. In some embodiments, the antibody or fragment thereof binds to a cell surface antigen on the surface of tumor cells. In some embodiments, the binding ligand is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. Including body. In some embodiments, binding ligands do not include antibodies or fragments thereof. In some embodiments, the binding ligand comprises a CD16 polypeptide or fragment thereof. In some embodiments, the binding ligand comprises a CD16 binding polypeptide. In some embodiments, the binding ligand is human or humanized. In some embodiments, the recombinant nucleic acid further comprises a nucleic acid sequence encoding an antibody or fragment thereof that can be bound by a binding ligand. In some embodiments, the antibody or fragment thereof is secretable from the cell.

[0021] In another aspect, the present disclosure provides a TCR subunit comprising at least a portion of an extracellular domain of mouse TCRα or mouse TCRβ and a transmembrane domain of mouse TCRα or mouse TCRβ and a receptor expressed on the surface of a cell. or an antigen binding domain comprising a ligand or fragment thereof that binds to the polypeptide; wherein the TCR constant domain comprises a sequence that is a mouse TCRα constant domain or a mouse TCRβ constant domain, or a sequence encoding a mouse TCRα constant domain and a mouse TCRβ constant domain, wherein the TCR subunits and antigen-binding domains are functionally A recombinant nucleic acid is provided that is linked and that, when expressed in a modified T cell containing a functional disruption of the endogenous TCR, functionally integrates TFP into the TCR complex.

[0022] In some embodiments, the TCR subunit comprises the intracellular domain of mouse TCRα or mouse TCRβ. In some embodiments, the extracellular domain comprises an extracellular portion of a TCRα or TCRβ constant domain, or fragment thereof. In some embodiments, the recombinant nucleic acid further comprises at least a subsequence encoding a TCRγ constant domain, a subsequence encoding a TCRδ constant domain, or a subsequence of both the TCRγ and TCRδ constant domains. In some embodiments the antigen binding domain comprises a ligand. In some embodiments, the ligand binds to a receptor on the cell. In some embodiments, the ligand binds to a polypeptide expressed on the surface of the cell. In some embodiments, the receptor or polypeptide expressed on the surface of the cell comprises a stress response receptor or polypeptide. In some embodiments, the receptor or polypeptide expressed on the surface of the cell is an MHC class I-associated glycoprotein. In some embodiments, the MHC class I-associated glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4, and combinations thereof. In some embodiments, the antigen binding domain is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. Contains mers. In some embodiments, the antigen binding domain comprises a monomer or dimer of the ligand or fragment thereof. In some embodiments, the ligand or fragment thereof is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or It is a decamer. In some embodiments, the ligand or fragment thereof is monomeric or dimeric. In some embodiments the antigen binding domain does not comprise an antibody or fragment thereof. In some embodiments, the antigen binding domain does not contain a variable region. In some embodiments, the antigen binding domain does not contain CDRs. In some embodiments, the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or fragment thereof. In some embodiments, the TCR constant domain is incorporated into a functional TCR complex upon expression in T cells. In some embodiments, the TCR constant domain is incorporated into the same functional TCR complex that incorporates TFP when expressed in T cells. In some embodiments, the sequence encoding the TFP and the sequence encoding the TCR constant domain(s) are contained within the same nucleic acid molecule. In some embodiments, the TFP and TCR constant domains are operably linked by a first linker sequence. In some embodiments, the first linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A, eg, T2A or P2A cleavage site. In some embodiments, the sequence encoding the TFP and the sequence encoding the TCR constant domain(s) are contained within different nucleic acid molecules. In some embodiments, the TCR subunit of TFP and the antibody domain, antigen binding domain, or binding ligand or fragment thereof are operably linked by a second linker sequence. In some embodiments, the second linker sequence comprises (G ₄ S) _n , where n=1-4. In some embodiments, the transmembrane domain is a TCRα or TCRβ, eg, the TCR transmembrane domain from murine TCRα or TCRβ. In some embodiments, the TCR subunits comprise (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain of TCRα or TCRβ. In some embodiments, the TCR extracellular domain is a TCRα or TCRβ chain, e.g., an extracellular portion of a mouse TCRα or TCRβ chain constant domain, a functional fragment thereof, or at least one but no more than 20 modifications. including its amino acid sequence having In some embodiments, the TCR subunit is a TCR α or TCR β chain, such as the transmembrane domain of a murine TCR α or TCR β chain, a functional fragment thereof, or an amino acid sequence thereof having at least one but no more than 20 modifications. containing a transmembrane domain. In some embodiments, the TCR subunit comprises a TCR α or TCR β chain, eg, the TCR intracellular domain of a murine TCR α or TCR β chain, or an amino acid sequence having at least one modification thereto. In some embodiments, the TCR subunit comprises (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain and is an α constant domain, or or contain it. In some embodiments, the α constant domain comprises the sequence of SEQ ID NO: 17, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO: 207, functional fragments thereof, or at least one but no more than 20 modifications and its amino acid sequence has In some embodiments, the encoded TCR comprising (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain is a β constant domain. , or containing it. In some embodiments, the beta constant domain comprises the sequence of SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO: 209, functional fragments thereof, or at least one but no more than 20 modifications and its amino acid sequence has In some embodiments, the extracellular domain of the TCR subunit does not include an α or β chain variable domain. In some embodiments, the TCR subunit comprises a stimulatory domain of a protein selected from the intracellular signaling domains of CD3ε, CD3γ, or CD3δ, or an amino acid sequence having at least one modification thereto. Including internal domains. In some embodiments, the TCR subunit of TFP comprises the extracellular, transmembrane, and intracellular domains of CD3ε. In some embodiments, the TCR subunit of CD3ε comprises the sequence of SEQ ID NO:258, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications. In some embodiments, TFP, TCRα constant domain, TCRβ domain, and any combination thereof are capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide .

[0023] In some embodiments, the TCR constant domain is a TCRα constant domain, and TFP is functionally integrated into a TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or combinations thereof.

[0024] TCR constant domain is TCRβ constant domain and TFP is functionally integrated into a TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or combinations thereof; TFP is functionally integrated into the TCR complex comprising endogenous subunits of the constant domain and the TCRβ constant domain, CD3ε, CD3γ, CD3δ, or combinations thereof.

[0025] In some embodiments, at least one but no more than 20 modifications thereto are modifications of amino acids that mediate cell signaling or modifications of amino acids that are phosphorylated in response to ligand binding to TFP. including. In some embodiments the antibody is an antibody fragment. In some embodiments, the antibody fragment is a scFv, single domain antibody domain, VH domain, or VL domain. In some embodiments, the antigen binding domain is an anti-CD19 binding domain, an anti-B cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, an anti-79b binding domain , anti-HER2 binding domain, anti-PMSA binding domain, anti-MUC16 binding domain, anti-CD22 binding domain, anti-PD-L1 binding domain, anti-BAFF or BAFF receptor binding domain, anti-nectin-4 binding domain, anti-TROP-2 binding domain , an anti-GPC3 binding domain, and an anti-ROR-1 binding domain. In some embodiments, the anti-MSLN binding domain comprises CDR1 of SEQ ID NO:60, CDR2 of SEQ ID NO:61, and CDR3 of SEQ ID NO:62. In some embodiments, the anti-MSLN binding domain comprises CDR1 of SEQ ID NO:63, CDR2 of SEQ ID NO:64, and CDR3 of SEQ ID NO:65. In some embodiments, the anti-MSLN binding domain comprises CDR1 of SEQ ID NO:66, CDR2 of SEQ ID NO:67, and CDR3 of SEQ ID NO:68. In some embodiments, the anti-MSLN binding domain comprises a sequence having at least about 80% sequence identity to the sequence of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71. In some embodiments, the anti-CD19 binding domain comprises CDR1 of SEQ ID NO:73, CDR2 of SEQ ID NO:75, and CDR3 of SEQ ID NO:77. In some embodiments, the anti-CD19 binding domain comprises CDR1 of SEQ ID NO:79, CDR2 of SEQ ID NO:81, and CDR3 of SEQ ID NO:83. In some embodiments, the anti-CD19 binding domain comprises a sequence having at least about 80% sequence identity to the sequences of SEQ ID NO:85 and/or SEQ ID NO:87.

[0026] In some embodiments, the nucleic acid is selected from the group consisting of DNA and RNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is circRNA. In some embodiments, a recombinant nucleic acid comprises a nucleic acid analogue that is absent from the coding sequence of the recombinant nucleic acid. In some embodiments, the nucleic acid analogue is 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T- deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2 '-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA) modification, locked nucleic acid (LNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1′,5′-anhydrohexitol nucleic acid (HNA), morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, and 2′-fluoro N3-P5′-phospho selected from the group consisting of roamidites; In some embodiments, the recombinant nucleic acid further comprises a leader sequence. In some embodiments, the recombinant nucleic acid further comprises a promoter sequence. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some embodiments, the recombinant nucleic acid further comprises a 3'UTR sequence. In some embodiments, the nucleic acid is an isolated or non-naturally occurring nucleic acid. In some embodiments, the nucleic acid is an in vitro transcribed nucleic acid. In another aspect, the disclosure provides a vector comprising the recombinant nucleic acid. In some embodiments, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV), Rous sarcoma virus (RSV) vectors, or retroviral vectors. be. In some embodiments the vector is an AAV6 vector. In some embodiments the method further comprises a promoter. In some embodiments, the vector is an in vitro transcribed vector.

[0027] In another aspect, the disclosure provides a modified T cell comprising a recombinant nucleic acid or vector, the modified T cell comprising functional disruption of an endogenous TCR.
[0028] In another aspect, the present disclosure provides a modified T cell comprising a TFP-encoding sequence of a nucleic acid or a TFP encoded by a sequence of a TFP-encoding nucleic acid, wherein the modified T cell is endogenous TCR including functional disruption of

[0029] In another aspect, the disclosure provides a modified allogeneic T cell comprising a TFP encoded by a sequence encoding a TFP or a sequence of a nucleic acid encoding a TFP.
[0030] In some embodiments, the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRγ constant domain, a TCRδ constant domain, or a TCRγ and a TCRδ constant domain. In some embodiments, the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein said TCR constant domain is a TCRα constant domain, a TCRβ constant domain, or a TCRα constant domain and a TCRβ constant domain. In some embodiments, the TCR constant domain, e.g., TCRα constant domain, TCRβ constant domain, or TCRα and TCRβ constant domains, is a mouse TCRγ constant domain, e.g., mouse TCRα constant domain, mouse TCRβ constant domain, or mouse The TCRα constant domain and the mouse TCRβ constant domain. In some embodiments, the endogenous TCR that is functionally disrupted is an endogenous TCRα chain, an endogenous TCRβ chain, or an endogenous TCRα chain and an endogenous TCRβ chain. In some embodiments, endogenous TCRs that are functionally disrupted have reduced binding to MHC-peptide complexes compared to those of unmodified control T cells. In some embodiments, the functional disruption is disruption of the gene encoding the endogenous TCR. In some embodiments, disruption of the gene encoding the endogenous TCR is removal of the sequence of the gene encoding the endogenous TCR from the genome of the T cell. In some embodiments, the T cells are CD4 cells, CD8 cells, naive T cells, memory stem T cells, central memory T cells, double negative T cells, effector memory T cells, effector T cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, α/β T cells, γ/δ T cells, natural killer (NK) cells, natural killer T (NKT) cells, hematopoietic stem cells, and many Human T cells selected from potential stem cells. In some embodiments, the T cells are CD8 ⁺ T cells or CD4 ⁺ T cells. In some embodiments, the T cells are allogeneic T cells. In some embodiments, the method encodes an inhibitory molecule comprising a first polypeptide comprising at least a portion of the inhibitory molecule associated with a second polypeptide comprising a positive signal from an intracellular signaling domain. further comprising a nucleic acid that In some embodiments, the inhibitory molecule comprises a first polypeptide comprising at least a portion of PD1 and a second polypeptide comprising a co-stimulatory domain and a primary signaling domain.

[0031] In another aspect, the present disclosure provides pharmaceutical compositions comprising engineered T cells and a pharmaceutically acceptable carrier.
[0032] In another aspect, the present disclosure provides a method of making a modified T cell, comprising disrupting an endogenous TCR gene encoding a TCRα chain, a TCRβ chain, or a TCRα and TCRβ chain, thereby producing a T cell comprising a functional disruption of the endogenous TCR gene; and transducing the T cell comprising a functional disruption of the endogenous TCR gene with a recombinant nucleic acid or vector.

[0033] In some embodiments, the method includes delivering a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets an endogenous gene encoding a TCRα chain, a TCRβ chain, or a TCRα chain and a TCRβ chain to a T cell. Further includes disruption including transducing.

[0034] In another aspect, the present disclosure provides a method of making a modified T cell, comprising transducing a T cell comprising a functional disruption of an endogenous TCR gene with a recombinant nucleic acid or vector. including doing

[0035] In some embodiments, the T cell comprising a functional disruption of an endogenous TCR gene comprises a functional disruption of an endogenous TCR gene encoding a TCRα chain, a TCRβ chain, or a TCRα chain and a TCRβ chain. These are T cells.

[0036] In some embodiments, the T cells are human T cells.
[0037] In some embodiments, T cells comprising a functional disruption of an endogenous TCR gene have reduced binding to MHC-peptide complexes compared to those of unmodified control T cells.

[0038] In some embodiments, the nuclease is a meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR/Cas nuclease, or megaTAL nuclease.

[0039] In some embodiments, the sequence contained in the recombinant nucleic acid or vector is inserted into the endogenous TCR subunit gene at the cleavage site such that the insertion of the sequence into this endogenous TCR subunit gene results in an endogenous TCR. Subunits are functionally destroyed.

[0040] In some embodiments, the nuclease is a meganuclease.
[0041] In some embodiments, the meganuclease comprises a first subunit and a second subunit, wherein the first subunit binds to the first recognition half-site of the recognition sequence and the second The subunit binds to the second recognition half-site of the recognition sequence.

[0042] In some embodiments, the meganuclease is a single-stranded meganuclease comprising a linker, the linker covalently joining the first subunit and the second subunit.

[0043] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition.
[0044] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising (a) a modified T cell produced according to the methods described herein; and (b) administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0045] In another aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising: (a) a modified T cell produced according to the methods described herein; and (b) administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0046] In some embodiments, the engineered T cells are allogeneic T cells. In some embodiments, less cytokines are released in the subject compared to subjects administered an effective amount of unmodified control T cells. In some embodiments, less cytokine is released in a subject compared to a subject administered an effective amount of modified T cells comprising a recombinant nucleic acid or vector. In some embodiments, the method comprises administering the pharmaceutical composition in combination with an agent that increases the effectiveness of the pharmaceutical composition. In some embodiments, the method comprises
This includes administering the pharmaceutical composition in combination with an agent that ameliorates one or more side effects associated with the pharmaceutical composition. In some embodiments, the cancer is solid tumor, lymphoma, or leukemia. In some embodiments, the cancer is from the group consisting of renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney cancer, and gastric cancer selected. In some embodiments, less cytokines are released in a subject compared to a subject administered an effective amount of autologous T cells expressing TFP as described herein. In some embodiments, the method does not induce graft-versus-host disease. In some embodiments, the subject has a reduced risk of developing graft-versus-host disease compared to a subject administered an effective amount of autologous T cells expressing TFP described herein.

[0047] In another aspect, the disclosure provides a recombinant nucleic acid, vector, modified T cell, or pharmaceutical composition for use as a medicament or in the preparation of a medicament.
Inclusion by reference
[0048] All publications, patents, and patent applications referred to in this specification are as if each individual publication, patent, or patent application was specifically and individually incorporated herein by reference. , incorporated herein by reference.

[0049] A series of graphs showing surface expression of CD3 (SK7) versus TCRαβ (IP26) in TRA-edited (left) and TRB-edited (right) cells. Wild-type Jurkat cells were edited with either the TRAC or TRBC genes to prevent surface expression of TRA or TRB. Cells negative for CD3 and TCRαβ were purified using magnetic cell sorting (MACS). Gates are drawn on the plots to visualize CD3 and TCRαβ negative-negative cell populations, and the proportion of cells in each quadrant is indicated in the four corners. [0050] A series of graphs showing surface expression of CD3 (SK7) versus TCRαβ (IP26) in TRA ^−/− (left) and TRB ^−/− (right) Jurkat cells. The first panel from the left is the non-transduced negative control. The second panel shows TRB ^{-/- cells transduced with TCRβ full-} length TFP. The third and fourth panels show TRA ^-/- and TRB ^-/- transduced with the TCRγδ TFP construct. [0051] FIG. 1 shows various strategies for generating allogeneic TFP T cells. [0052] FIG. 3 is a schematic showing elements of constructs used to generate allogeneic TFP T cells. [0053] Figure 10 is a series of graphs showing the surface expression of CD3 versus TCRαβ in TCRγδ TFP construct transduced and wild-type and TRAC-edited non-transduced T cells after TRAC locus editing. The graph depicts cell populations prior to MACS purification. [0054] Figure 10 is a series of graphs showing surface expression of CD3 versus TCRαβ in T cells transduced with TCRγδ TFP constructs after TRAC locus editing versus wild-type and TRAC-edited non-transduced T cells. The graph illustrates cell populations after MACS purification. [0055] Figure 3 is a series of graphs showing surface expression of allogeneic TFP-transduced T cells (TFP ⁺ ). The left panel shows the TFP ⁺ fraction of T cells transduced with the TCRγδ TFP construct. The right panel shows the TFP ⁺ ratio of T cells transduced with the TCRβ full-length positive control construct compared to non-transduced control cells. [0056] Figure 10 is a series of graphs showing the surface expression of CD4 versus CD8 populations in T cells transduced with TCRγδ TFP constructs compared to T cells transduced with TCRβ full-length positive control constructs. [0057] Figure 10 is a series of graphs showing surface expression of CD45RA vs. CCR7 populations of CD4 or CD8 T cells transduced with TCRγδ TFP constructs compared to T cells transduced with TCRβ full-length positive control constructs. [0058] FIG. 2 is two graphs showing Luc-Cyto analysis of allogeneic T effector cells cultured with tumor target cells at 3:1, 1:1, or 1:3, and 1:9 ratios. . Targeted Nalm-6 (CD19 positive) cells are shown in the left panel and CD19 negative cells (K562 cells) are shown in the right panel. The x-axis represents the percentage of tumor cell lysis. [0059] FIG. 10 is four graphs showing cytokine secretion (in pg/mL) of supernatants collected from the Nalm-6 co-culture assay of FIG. Each graph shows cytokine secretion of GM-CSF, IFNγ, IL2, and TNFα. [0060] FIG. 13 is a schematic showing the TFP construct in TRAC or TRBC edited cells and whether expression of this construct reconstitutes the TCR. Figure 12 shows that mouse TCRα or TCRβ TFP constructs can reconstitute the TCR, and human TCRγ or TCRδ TFP constructs can reconstitute the TCR. [0061] Figure 1 shows cell surface expression of human TCRβ TFP constructs. FIG. 13C is a schematic diagram showing the construct depicted in FIG. 13B. Human anti-CD19 TCRβ TFP and human anti-CD19 TCRβ (constant domain) TFP are shown. Cell surface expression of human TCRβ TFP constructs is shown. FIG. 13A shows CD3 vs. TCRαβ surface expression in TRBC knockout Jurkat cells transduced with the constructs of FIG. 13A. (i) SSC-A vs. CD19; (ii) CD3 vs. TCRαβ before purification and (i) SSC-A vs. CD19 after purification in TRAC knockout T cells transduced with the indicated constructs. (iii) CD4 vs. CD8; and (iv) CD45RA vs. CCR7 surface expression. TRAC was not knocked out in cells transduced with anti-CD19-CD3ε. [0063] From left to right, Luc of TRAC knockout T cells transduced with the indicated constructs cultured with tumor target cells at 3:1, 1:1, or 1:3 and 1:9 ratios. - A series of graphs showing the Cyto analysis. TRAC was not knocked out in cells transduced with anti-CD19-CD3ε. Targeted Nalm-6 (CD19 positive) cells are shown in the upper panel and CD19 negative cells (K562 cells) are shown in the lower panel. The x-axis represents the percentage of tumor cell lysis. [0064] FIG. 15 is a series of four graphs showing cytokine secretion (in pg/mL) of supernatants collected from the Nalm-6 co-culture assay of FIG. Allogeneic T effector cells were cultured with tumor target cells at 3:1, 1:1, or 1:3, and 1:9 ratios from left to right. Each graph shows cytokine secretion of GM-CSF, IFNγ, IL2, and TNFα. [0065] (Left to right) A series of graphs showing GM-CSF and IFNγ secretion (in pg/mL) of TRAC knockout T cells transduced with the indicated constructs without or with HLA-mismatched dendritic cells. is. TRAC was not knocked out in cells transduced with anti-CD19-CD3ε. [0066] Figure 10 is a series of graphs showing tumor burden as measured by luminescence in Nalm6-luc tumor mouse model mice injected with TRAC knockout T cells transduced with the indicated constructs. TRAC was not knocked out in cells transduced with anti-CD19-CD3ε. FIG. 4 is a series of graphs showing tumor burden as measured by luminescence in Nalm6-luc tumor mouse model mice injected with TRAC knockout T cells transduced with the indicated constructs. TRAC was not knocked out in cells transduced with anti-CD19-CD3ε. FIG. 4 is a series of graphs showing tumor burden as measured by luminescence in Nalm6-luc tumor mouse model mice injected with TRAC knockout T cells transduced with the indicated constructs. TRAC was not knocked out in cells transduced with anti-CD19-CD3ε. [0067] FIG. 18 shows infiltration of CD7 ⁺ cells into the liver of non-tumor-bearing mice from the in vivo assay shown in FIG. Immunohistochemistry of CD7 expression in mouse liver is shown along with surface expression of CD45RA versus CD7 in mouse liver. [0068] From left to right, series showing Luc-Cyto analysis of TRAC knockout T cells transduced with the indicated constructs cultured with tumor target cells at a 3:1 ratio, a 1:1 ratio, or a 1:3 ratio. is a graph of Targeted Nalm-6 (CD19 positive) cells are shown in the left panel and CD19 negative cells (K562 cells) are shown in the right panel. The x-axis represents the percentage of tumor cell lysis. [0069] FIG. 20 is a series of four graphs showing cytokine secretion (in pg/mL) of supernatants collected from the Nalm-6 co-culture assay of FIG. Allogeneic T effector cells were cultured with tumor target cells in 3:1, 1:1, or 1:3 ratios from left to right. Each graph shows cytokine secretion of GM-CSF, IFNγ, IL2, and TNFα. [0070] From left to right, Luc-Cyto analysis of TRAC knockout T cells transduced with the indicated constructs cultured with tumor target cells at 3:1, 1:1, or 0.1:1 ratios. 3 is a series of graphs showing; Targeted Nalm-6 (CD19 positive) cells are shown in the left panel and CD19 negative cells (K562 cells) are shown in the right panel. The x-axis represents the percentage of tumor cell lysis. [0071] FIG. 20 is a series of four graphs showing cytokine secretion (in pg/mL) of supernatants collected from the Nalm-6 co-culture assay of FIG. Allogeneic T effector cells were cultured with tumor target cells at 3:1, 1:1, or 0.1:1 ratios from left to right. Each graph shows cytokine secretion of GM-CSF, IFNγ, IL2, and TNFα.

[0072] In some embodiments, disclosed herein are TCR subunits comprising (a)(i) (1) at least a portion of a TCR extracellular domain, and (2) a transmembrane domain. (ii) an antibody comprising an antigen binding domain; and (b) a sequence encoding a TCR constant domain(s), wherein , the TCR constant domain comprises a sequence that is a TCRγ constant domain or a TCRδ constant domain, or a sequence encoding a TCRγ constant domain and a TCRδ constant domain, wherein the TCR subunit and the antibody are operably linked and endogenous A recombinant nucleic acid that, when expressed in a modified T cell containing a functional disruption of the sexual TCR, functionally integrates TFP into the TCR complex.

[0073] In some embodiments, disclosed herein are (a)(i) (1) at least a portion of a TCR extracellular domain, (2) a TCR subunit comprising a transmembrane domain, and (ii) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising a binding ligand or fragment thereof capable of binding to an antibody or fragment thereof; and (b) a sequence encoding a TCR constant domain. wherein the TCR constant domain comprises a sequence that is a TCRγ constant domain or a TCRδ constant domain, or a sequence encoding a TCRγ constant domain and a TCRδ constant domain, wherein the TCR subunit and the binding ligand or fragment thereof are operably linked. and is a recombinant nucleic acid that, when expressed in a modified T cell containing a functional disruption of the endogenous TCR, functionally integrates TFP into the TCR complex.

[0074] In some embodiments, disclosed herein are TCR subunits comprising (a)(i) (1) at least a portion of a TCR extracellular domain, and (2) a transmembrane domain and (ii) an antigen-binding domain comprising a ligand or fragment thereof that binds to a receptor or polypeptide expressed on the surface of a cell; (b) a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a TCRγ constant domain or a TCRδ constant domain, or comprises a sequence encoding a TCRγ constant domain and a TCRδ constant domain; , a TCR subunit and an antigen-binding domain are operably linked, and TFP is functionally incorporated into the TCR complex when expressed in a modified T cell containing a functional disruption of the endogenous TCR. .

[0075] In some embodiments, disclosed herein are (a)(i) (1) at least a portion of the extracellular domain of mouse TCRα or mouse TCRβ, and (2) mouse TCRα or a sequence encoding a T-cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising the transmembrane domain of mouse TCRβ; (ii) an antibody or fragment thereof comprising an antigen-binding domain; a sequence encoding a TCR constant domain(s), wherein the TCR constant domain is a mouse TCRα constant domain or a mouse TCRβ constant domain, or a sequence encoding a mouse TCRα constant domain and a mouse TCRβ constant domain wherein the TCR subunit and the antibody are operably linked and are recombinant nucleic acids that, when expressed in a modified T cell containing functional disruption of the endogenous TCR, functionally integrate the TFP into the TCR complex.

[0076] Disclosed herein are (a)(i) (1) at least a portion of the extracellular domain of mouse TCRα or mouse TCRβ, and (2) the transmembrane domain of mouse TCRα or mouse TCRβ. (ii) a sequence encoding a T-cell receptor (TCR) fusion protein (TFP) comprising a binding ligand or fragment thereof capable of binding to an antibody or fragment thereof; and (b) a TCR constant domain ( a sequence encoding a TCR constant domain, wherein the TCR constant domain is a mouse TCRα constant domain or a mouse TCRβ constant domain, or a sequence encoding a mouse TCRα constant domain and a mouse TCRβ constant domain, wherein the TCR sub The unit and binding ligand or fragment thereof are recombinant nucleic acids that are operably linked and that, when expressed in a modified T cell containing functional disruption of the endogenous TCR, functionally integrate TFP into the TCR complex.

[0077] In some embodiments, disclosed herein are (a)(i) (1) at least a portion of the TCR extracellular domain, (2) the transmembrane domain, and (3) TCRα , TCRβ, TCRγ, or TCRδ, or a TCR subunit comprising a stimulatory domain from the intracellular signaling domains of CD3ε, CD3γ, CD3δ; and (ii) a human or humanized antibody comprising an antigen binding domain. and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRγ constant domain, a TCRδ constant domain, or A recombinant nucleic acid comprising a sequence that is a TCRδ constant domain and a TCRγ constant domain, wherein the TCR subunit and the antibody are operably linked, and which, when expressed in a T cell, TFP is operably integrated into the TCR complex be.

[0078] In some embodiments, disclosed herein are (a)(i) (1) at least a portion of the TCR extracellular domain, (2) the transmembrane domain, and (3) TCRα , TCRβ, TCRγ, or TCRδ, or a TCR subunit comprising a stimulatory domain from the intracellular signaling domains of CD3ε, CD3γ, CD3δ; (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRγ constant domain, a TCRδ constant domain, or comprises a sequence that is a TCRγ constant domain and a TCRδ constant domain, wherein the TCR subunit and binding ligand or fragment thereof are operably linked, and when expressed in a T cell, TFP is functionally incorporated into the TCR complex , is a recombinant nucleic acid.

[0079] In some embodiments, disclosed herein are (a)(i) (1) at least a portion of the TCR extracellular domain, (2) the transmembrane domain, and (3) TCRα , TCRβ, TCRγ, or TCRδ, or a TCR subunit comprising a stimulatory domain from the intracellular signaling domains of CD3ε, CD3γ, CD3δ; a sequence encoding a T-cell receptor (TCR) fusion protein (TFP), comprising a ligand or fragment thereof that binds to contains a sequence that is a TCRγ constant domain, a TCRδ constant domain, or a TCRγ constant domain and a TCRδ constant domain, wherein the TCR subunit and the antigen domain are operably linked, and when expressed in a T cell, TFP binds to the TCR complex It is a recombinant nucleic acid that is functionally integrated into the body.

[0080] In some embodiments, disclosed herein are (a)(i) (1) at least a portion of the TCR extracellular domain, (2) the transmembrane domain, and (3) TCRα , TCRβ, TCRγ, or TCRδ, or a TCR subunit comprising a stimulatory domain from the intracellular signaling domains of CD3ε, CD3γ, CD3δ; and (ii) a human or humanized antibody comprising an antigen binding domain. and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRα constant domain, a TCRβ constant domain, or The TCRα and TCRβ constant domains, the TCR subunits and the antibody are recombinant nucleic acids that are operably linked and that, when expressed in T cells, TFP is operably integrated into the TCR complex.

[0081] In some embodiments, disclosed herein are (a)(i) (1) at least a portion of the TCR extracellular domain, (2) the transmembrane domain, and (3) TCRα , TCRβ, TCRγ, or TCRδ, or a TCR subunit comprising a stimulatory domain from the intracellular signaling domains of CD3ε, CD3γ, CD3δ; and (b) a sequence encoding a TCR constant domain, the TCR constant domains being a TCRα constant domain, a TCRβ constant domain, or TCRα and TCRβ constant domains, wherein the TCR subunit and antibody are operably linked, and a recombinant nucleic acid that, when expressed in a T cell, operably integrates TFP into the TCR complex.

[0082] In some embodiments, disclosed herein are (a)(i) (1) at least a portion of the TCR extracellular domain, (2) the transmembrane domain, and (3) TCRα , TCRβ, TCRγ, or TCRδ, or a TCR subunit comprising a stimulatory domain from the intracellular signaling domains of CD3ε, CD3γ, CD3δ; (b) a sequence encoding a TCR constant domain, comprising a sequence encoding a T-cell receptor (TCR) fusion protein (TFP), comprising an antigen domain comprising a ligand or fragment thereof that binds to is a TCRα constant domain, a TCRβ constant domain, or a TCRα constant domain and a TCRβ constant domain, the TCR subunit and the antibody are operably linked, and TFP is functionally linked to the TCR complex when expressed in a T cell. integrated, recombinant nucleic acid.

[0083] In some embodiments, disclosed herein are vectors comprising the recombinant nucleic acids disclosed herein.
[0084] In some embodiments, disclosed herein is a modified T cell comprising a recombinant nucleic acid disclosed herein or a vector disclosed herein, wherein the modified T cell The cell contains a functional disruption of the endogenous TCR.

[0085] In some embodiments, disclosed herein is a TFP-encoding sequence of a nucleic acid disclosed herein, or encoded by a sequence of a nucleic acid disclosed herein. A modified T cell comprising TFP, said modified T cell comprising a functional disruption of the endogenous TCR.

[0086] In some embodiments, disclosed herein is a sequence encoding a TFP disclosed herein, or a TFP encoded by a sequence of a nucleic acid disclosed herein. Engineered allogeneic T cells comprising:

[0087] In some embodiments, disclosed herein are pharmaceutical compositions comprising (a) the engineered T cells of the present disclosure and (b) a pharmaceutically acceptable carrier.
[0088] In some embodiments, disclosed herein is a method of making a modified T cell of the present disclosure, comprising: (a) a TCRα chain, a TCRβ chain, or a TCRα chain; disrupting the endogenous TCR gene encoding the TCR beta chain, thereby generating a T cell comprising a functional disruption of the endogenous TCR gene; and (b) a T cell comprising a functional disruption of the endogenous TCR gene. includes transducing a recombinant nucleic acid of the disclosure or a vector disclosed herein.

[0089] In some embodiments, disclosed herein is a method of making a modified T cell of the present disclosure, comprising a T cell comprising a functional disruption of an endogenous TCR gene. includes transducing a recombinant nucleic acid disclosed herein or a vector disclosed herein.

[0090] In some embodiments, disclosed herein is a method of treating cancer in a subject in need thereof, wherein the method comprises a therapeutically effective amount of to the subject.

[0091] In some embodiments, disclosed herein is a method of treating cancer in a subject in need thereof, comprising (a) a method disclosed herein and (b) a pharmaceutically acceptable carrier to the subject.

specific term
[0092] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0093] "a" and "an" refer to one or more (ie, at least one) of the grammatical objects of the article. By way of example, "element" means one element or more than one element.

[0094] As used herein, "about" means less than 1%, or 1, 2, 3, 4, 5, 1, 2, 3, 4, 5, depending on the circumstances and what is known or knowable to those skilled in the art. An increase or decrease of more than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, or 30% can be meant.

[0095] As used herein, a "subject"(s) or "individual" refers to a mammal, such as a human or non-human mammal, such as domestic, agricultural, or wild animals, as well as birds and It can include, but is not limited to, aquatic animals and the like. A "patient" is a subject having or at risk of developing a disease, disorder, or condition or otherwise in need of the compositions and methods provided herein.

[0096] As used herein, "treating" or "treatment" refers to any indication of success in treating or ameliorating a disease or condition. Treating may include, for example, reducing, delaying, or alleviating the severity of one or more symptoms of a disease or condition, or the symptoms of a disease, disorder, disorder, or adverse condition may include It may also include reducing the frequency of manifestations in patients. As used herein, "treating or preventing" may also be used herein to refer to methods that result in some degree of treatment or amelioration of a disease or condition, and complete prevention of the condition. It contemplates a range of results for that purpose, including but not limited to.

[0097] As used herein, "prevent" refers to the prevention of a disease or condition, such as tumor formation, in a patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with a method of the present disclosure and does not subsequently develop a tumor or other form of cancer, the individual will be free from the disease for at least a period of time. been prevented throughout.

[0098] As used herein, a "therapeutically effective amount" is an amount sufficient to confer a beneficial effect or otherwise reduce adverse non-beneficial events in an individual to whom the composition is administered. It is the amount of the composition or its active ingredient. By "therapeutically effective dose" herein is meant a dose administered to produce one or more desired or desirable (e.g., beneficial) effects, such administration over a given period of time. One or more times. The exact dose will depend on the therapeutic goals and can be ascertained by those skilled in the art using known techniques (eg, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999), and Pickar, Dosage Calculations (1999)).

[0099] As used herein, "T-cell receptor (TCR) fusion protein" or "TFP" includes recombinant polypeptides derived from a variety of polypeptides comprising a TCR, which TCR generally comprises It is capable of i) binding to surface antigens on target cells and ii) interacting with other polypeptide components of intact TCR complexes when normally co-localized within or on the surface of T cells.

[0100] The term "stimulation" refers to the primary response induced by the binding of a stimulatory domain or molecule (e.g., TCR/CD3 complex) to its cognate ligand, thereby mediating a signaling event. , this signaling event includes, but is not limited to, signaling through the TCR/CD3 complex. Stimulation can mediate changes in the expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.

[0101] The term "stimulatory molecule" or "stimulatory domain" refers to a primary cytoplasmic signaling sequence that stimulates primary activation of the TCR complex for at least some aspects of the T cell signaling pathway. Refers to a molecule or part thereof expressed by a T cell that provides the (s). In one aspect, the primary signal is initiated, for example, by the binding of peptide-loaded MHC molecules to the TCR/CD3 complex, resulting in mediation of T cell responses including, but not limited to, proliferation, activation, differentiation, and the like. . A stimulatory primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") may contain a signaling motif known as an immunoreceptor tyrosine activation motif, or "ITAM." Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present invention include: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD278 (also referred to as “ICOS”); and those derived from CD66d.

[0102] The term "antigen-presenting cell" or "APC" refers to accessory cells (e.g., B cells, dendritic cells, etc.) that present foreign antigens in complex with the major histocompatibility complex (MHC) on their surface. Refers to immune system cells such as T cells can recognize these complexes using the T cell receptor (TCR). APCs process antigens and present them to T cells.

[0103] "Major histocompatibility complex (MHC) molecules are typically bound by the TCR as part of a peptide:MHC complex. MHC molecules can be MHC class I or II molecules. The complex may be present on the surface of antigen-presenting cells such as dendritic cells or B cells, or any other cell, including cancer cells, or immobilized, e.g., by coating on beads or plates In some cases.

[0104] The human leukocyte antigen system (HLA) encodes the human major histocompatibility complex (MHC) and includes HLA class I antigens (A, B, and C) and HLA class II antigens (DP, DQ, and DR ) is the name of the gene complex containing HLA alleles A, B, and C present peptides derived primarily from intracellular proteins, eg, proteins expressed intracellularly.

[0105] Upon T cell expression in vivo, T cells undergo a positive selection step that ensures recognition of self MHC, followed by a negative selection step that eliminates T cells that bind too tightly to MHC presenting self antigens. take the steps of As a result, a particular T cell and the TCRs it expresses recognize only peptides presented by a particular type of MHC molecule, ie those encoded by a particular HLA allele. This is known as HLA restriction.

[0106] One HLA allele of interest is HLA-A ^* 0201, which is expressed in the majority (>50%) of the Caucasian population. Thus, TCRs that bind to WT1 peptides presented by MHC encoded by (i.e., restricted by ^{) HLA-A* 0201} suggest that immunotherapies utilizing such TCRs would be effective in the Caucasian population. It is advantageous because it is suitable for most treatments.

[0107] Other HLA-A alleles of interest are HLA-A ^* 0101, HLA-A ^* 2402, and HLA-A ^* 0301.
[0108] Broadly expressed HLA-B alleles of interest are HLA-B ^* 3501, HLA-B ^* 0702, and HLA-B ^* 3502.

[0109] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. Intracellular signaling domains generate signals that promote immune effector functions of TFP-containing cells, eg, engineered TT cells. For example, immune effector functions in modified TT cells include cytolytic activity and T helper cell activity, including secretion of cytokines. In certain embodiments, an intracellular signaling domain may comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules involved in primary or antigen-dependent stimulation. In certain embodiments, the intracellular signaling domain may comprise a co-stimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules involved in costimulatory signals or antigen-independent stimulation.

[0110] A primary intracellular signaling domain may comprise an ITAM ("immunoreceptor tyrosine activation motif"). Examples of primary cytoplasmic signaling sequences comprising ITAMs include, but are not limited to, those derived from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d DAP10 and DAP12. not.

[0111] The term "co-stimulatory molecule" refers to allogeneic ligands in T cells that specifically bind to co-stimulatory ligands and thereby mediate co-stimulatory responses such as, but not limited to, proliferation by T cells. Refers to a binding partner. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Costimulatory molecules include MHC class 1 molecules, BTLA, and Toll ligand receptors, as well as OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), and 4-1BB (CD137). include, but are not limited to. A costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. Costimulatory molecules may appear in the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. There is Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, Examples include ligands that specifically bind to LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, CD83, and the like. An intracellular signaling domain may comprise the entire intracellular portion of the molecule from which it is derived or the entire naturally occurring intracellular signaling domain, or a functional fragment thereof. The term "4-1BB" refers to the TNFR superfamily of amino acid sequences assigned GenBank Accession Number AAA62478.2 or equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc. and "4-1BB co-stimulatory domain" refers to amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or from non-human species such as mouse, rodent, monkey, ape, etc. Defined as equivalent residues.

[0112] As used herein, the term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds an antigen. Antibodies may be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof, and may be derived from natural or recombinant sources.

[0113] The term "antibody fragment" refers to at least a portion of an antibody, or a recombinant variant thereof, which comprises the antigen-binding domain, i.e., the antigen-determining variable region of an intact antibody, and which contains a target, e.g., an antigen and its sufficient to confer recognition and specific binding of the antibody fragment to a defined epitope. Examples of antibody fragments include Fab, Fab′, F(ab′) ₂ and Fv fragments, single chain (sc) Fv (“scFv”) antibody fragments, linear antibodies, single domain antibodies (V _L or _VH ), camelid _VHH domains, and multispecific antibodies formed from antibody fragments.

[0114] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising the variable region of a light chain and at least one antibody fragment comprising the variable region of a heavy chain, wherein the light and heavy chains are The variable regions of are contiguously linked via short flexible polypeptide linkers and can be expressed as a single polypeptide chain, with the scFv retaining the specificity of the intact antibody from which it was derived.

[0115] A "heavy chain variable region" or " _VH " with respect to an antibody refers to a fragment of the heavy chain comprising the three CDRs interspersed between contiguous stretches known as the framework regions, which are generally , are more highly conserved than the CDRs and form the scaffold that supports the CDRs. Camelidae " _VHH " domains are heavy chains comprising a single variable antibody domain.

[0116] Unless otherwise specified, scFv as used herein has the V _L and V _H variable regions in any order, e.g., with respect to the N-terminus and C-terminus of the polypeptide. Alternatively, the scFv may comprise V _L -linker-V _H or may comprise V _H -linker-V _L .

[0117] Some of the TFP compositions of this disclosure, including antibodies or antibody fragments thereof, may exist in a variety of forms, wherein the antigen-binding domains include, for example, single domain antibody fragments (sdAbs), murine, humanized, or expressed as part of a continuous polypeptide chain, including single-chain antibodies (scFv) derived from human antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, N.W. Y. , Harlow et al. , 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.M. Y. , Houston et al. , 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883, Bird et al. , 1988, Science 242:423-426). In one aspect, the antigen-binding domain of the TFP compositions of this disclosure comprises an antibody fragment. In a further aspect, TFPs include antibody fragments comprising scFvs or sdAbs.

[0118] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as an antibody expressed by a bacteriophage or yeast expression system. The term also refers to an antibody produced by the synthesis of an antibody-encoding DNA molecule that expresses the antibody protein or amino acid sequence that specifies the antibody, which DNA or amino acid sequence is known in the art should be construed to mean an antibody obtained using recombinant DNA or amino acid sequence techniques available and well known in the United States.

[0119] The term "antigen" or "Ag" refers to a molecule capable of being specifically bound by an antibody or otherwise eliciting an immune response. This immune response may involve either antibody production or activation of specific immunologically competent cells, or both.

[0120] Those skilled in the art will appreciate that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Additionally, antigens can be derived from recombinant or genomic DNA. Accordingly, those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response encodes an "antigen" as the term is used herein. let's be Furthermore, those skilled in the art will appreciate that the antigen need not be encoded by the full-length nucleotide sequence of the gene alone. The disclosure includes, but is not limited to, the use of partial nucleotide sequences of multiple genes, arranged in various combinations to encode polypeptides that elicit the desired immune response. It is self-evident that there are Moreover, those skilled in the art will appreciate that an antigen need not be encoded by a "gene" at all. It will be appreciated that antigens may be produced synthetically, or derived from biological samples, or may be macromolecules other than polypeptides. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or liquids containing other biological components.

[0121] As used herein, the term "CD19" is an antigenic determinant detectable on B-cell leukemic progenitor cells, other malignant B-cells, and most cells of normal B-cell lineage. Refers to cluster 19 proteins.

[0122] As used herein, the term "BCMA" refers to B-cell maturation antigen, also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), a cluster of differentiation 269 protein (CD269) , is the protein encoded by the TNFRSF17 gene in humans. TNFRSF17 is a cell surface receptor of the TNF receptor superfamily that recognizes B-cell activating factor (BAFF) (see, eg, Laabi et al., EMBO 11(11):3897-904 (1992)). This receptor is expressed on mature B lymphocytes and may be important for B cell development and autoimmune responses.

[0123] As used herein, the term "CD16" (also known as FcγRIII) refers to a differentiated group found on the surface of natural killer cells, neutrophilic polymorphonuclear leukocytes, monocytes, and macrophages. refers to molecules. CD16 has been identified as the Fc receptors FcγRIIIa (CD16a) and FcγRIIIb (CD16b) involved in signal transduction. CD16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC).

[0124] As used herein, "NKG2D" refers to a transmembrane protein belonging to the CD94/NKG2 family of C-type lectin-like receptors. In humans, NKG2D is expressed by NK cells, γδ T cells, and CD8 ⁺ αβ T cells. NKG2D recognizes inducible self-proteins of the MIC and RAET1/ULBP families that appear on the surface of stressed, malignantly transformed, and infected cells.

[0125] Mesothelin (MSLN) refers to a tumor differentiation antigen normally present on the mesothelial cells lining the pleura, peritoneum, and pericardium. Mesothelin is overexpressed in several human tumors, including mesothelioma, ovarian adenocarcinoma, and pancreatic adenocarcinoma.

[0126] The tyrosine protein kinase transmembrane receptor ROR1, also known as neurotrophic tyrosine kinase receptor-related 1 (NTRKR1), is a member of the receptor tyrosine kinase-like orphan receptor (ROR) family. It is involved in cancer metastasis.

[0127] The term "MUC16," also known as "mucin 16, cell surface associated" or "ovarian cancer-associated tumor marker CA125," has an amino-terminal extracellular domain, a large tandem repeat domain, and a short cytoplasmic domain. It is a membrane-tethered mucin containing a transmembrane domain. The product of this gene is used as a marker for various cancers, with increased expression levels leading to worse outcomes.

[0128] The term "CD22," also known as sialic acid-binding Ig-like lectin 2, SIGLEC-2, T-cell surface antigen leu-14, and the B-cell receptor CD22, mediates B-cell/B-cell interactions. It is a protein believed to be involved in the localization of B cells within lymphoid tissue and has been implicated in diseases including refractory hematologic cancers and hairy cell leukemia. A fully human anti-CD22 monoclonal antibody (“M971”) suitable for use in the methods disclosed herein is described, for example, in Xiao et al. , MAbs. 2009 May-Jun;1(3):297-303.

[0129] Programmed cell death protein 1 (also known as "PD-1") and CD279 (differentiation antigen group 279) suppress the inflammatory activity of T cells, thereby down-regulating the immune system and promoting self-tolerance. are proteins on the surface of cells that play a role in controlling the immune system's response to cells in the human body. It prevents autoimmune diseases, but may also stop the immune system from killing cancer cells. PD-1 is an immune checkpoint and prevents autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T cells in lymph nodes. Second, it reduces apoptosis of regulatory T cells (anti-inflammatory, suppressive T cells). PD-1 is a cell surface receptor belonging to the immunoglobulin superfamily and is expressed on T cells and pro-B cells. PD-1 binds two ligands, PD-L1 and PD-L2.

[0130] Programmed Death Ligand 1 ("PD-L1") is a 40 kDa type 1 transmembrane protein that is induced during certain events such as pregnancy, tissue allografts, autoimmune diseases, and other diseases such as hepatitis. It is speculated to play an important role in suppressing the adaptive capacity of the immune system. The adaptive immune system normally responds to antigens associated with activation of the immune system by exogenous or endogenous danger signals. Next, clonal expansion of antigen-specific CD8 ⁺ T cells and/or CD4 ⁺ helper cells is expanded. Binding of PD-L1 to the inhibitory checkpoint molecule PD-1 is inhibitory based on interaction with phosphatases (SHP-1 or SHP-2) via immunoreceptor tyrosine-dependent switch motif (ITSM) motifs. Transmits signals. This reduces the proliferation of antigen-specific T cells in lymph nodes while simultaneously reducing apoptosis of regulatory T cells (anti-inflammatory, suppressive T cells), which is mediated by the downregulation of the gene Bcl-2.

[0131] The "CD79α" and "CD79β" genes encode proteins that make up the B-lymphocyte antigen receptor, a multimeric complex containing the antigen-specific component, surface immunoglobulin (Ig). Surface Ig non-covalently associates with two other proteins, Ig-α and Ig-β (encoded by CD79α and its paralog CD79β, respectively), which are required for B-cell antigen receptor expression and function. Functional disruption of this complex can lead to, for example, human B-cell chronic lymphocytic leukemia.

[0132] B-cell activating factor or "BAFF" is a cytokine that belongs to the tumor necrosis factor (TNF) ligand family. This cytokine is a ligand for the receptors TNFRSF13B/TACI, TNFRSF17/BCMA, and TNFRSF13C/BAFF-R. This cytokine is expressed in cells of the B-cell lineage and functions as a potent B-cell activator. It has also been shown to play an important role in B-cell proliferation and differentiation.

[0133] The term "anti-tumor effect" refers to, for example, a decrease in tumor volume, a decrease in tumor cell number, a decrease in metastasis number, an increase in life expectancy, a decrease in tumor cell proliferation, a decrease in tumor cell viability, or a Refers to biological effects that can be achieved by a variety of methods including, but not limited to, amelioration of various physiological symptoms associated with cancer pathology. An "anti-tumor effect" can also be manifested by the ability of the disclosed peptides, polynucleotides, cells and antibodies to prevent the development of tumors in the first place.

[0134] The term "autologous" refers to any substance that originates from the same individual and is later reintroduced into that individual.
[0135] The term "allogeneic" or alternatively "allogeneic" refers to any substance derived from a different animal of the same species or a different patient as the individual into which the substance is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may be genetically distinct enough to interact antigenically.

[0136] The term "xenogeneic" refers to a graft derived from an animal of a different species.
[0137] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can be locally distributed or spread through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colon cancer, kidney cancer, liver cancer, malignant brain cancer, lymphoma, leukemia, Examples include, but are not limited to, lung cancer.

[0138] The term "encodes" is defined as the intrinsic properties of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, in the synthesis of other polymers and macromolecules in biological processes. It refers to serving as a template that has either a defined nucleotide sequence (eg, rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties that result from it. Thus, a gene, cDNA, or RNA encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and usually shown in the sequence listing, and the non-coding strand, which serves as a template for transcription of the gene or cDNA, are defined as proteins or other products of that gene or cDNA. can be referred to as encoding

[0139] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Reference to a nucleotide sequence encoding a protein or RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some forms contain one or more introns.

[0140] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of an amount effective to achieve a particular biological or therapeutic effect. refers to the amount of a compound, formulation, material, or composition described in .

[0141] The term "endogenous" refers to any substance derived from or produced within an organism, cell, tissue, or system.
[0142] The term "exogenous" refers to any substance introduced or produced from outside an organism, cell, tissue, or system.

[0143] The term "expression" refers to the transcription and/or translation of a specific nucleotide sequence directed by a promoter.
[0144] The term "functional disruption" refers to a specific (e.g., target) nucleic acid (e.g., gene, RNA transcript) that interferes with normal expression and/or behavior within a cell. Refers to a physical or biochemical alteration of the encoded protein). In one embodiment, functional disruption refers to modification of a gene by gene editing methods. In one embodiment, functional disruption prevents expression of a target gene (eg, an endogenous gene).

[0145] The term "transcription vector" is a composition that contains an isolated nucleic acid and that can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transcription vector" includes self-replicating plasmids or viruses. The term should also be taken to further include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral transcription vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0146] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression may be supplied by the host cell or in an in vitro expression system. Expression vectors include cosmids, plasmids (e.g., naked plasmids, or those contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides. ), and all known in the art.

[0147] The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they are capable of infecting non-dividing cells, and are the most efficient of the gene delivery vectors, as they can deliver substantial amounts of genetic information into the host cell's DNA. It is one of the effective methods. HIV, SIV, and FIV are all examples of lentiviruses.

[0148] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including, inter alia, Milone et al. , Mol. Ther. 17(8):1453-1464 (2009). Other examples of clinically available lentiviral vectors include, but are not limited to, the LENTIVECTOR™ gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen, and the like. Non-clinical versions of lentiviral vectors are also available and known to those skilled in the art.

[0149] The term "homology" or "identity" refers to the subunit identity between two polymer molecules, e.g., between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. Refers to sequence identity. If a subunit position in both of the two sequences is occupied by the same monomeric subunit, e.g. if each position in two DNA molecules is occupied by adenine, then the molecules are homologous or are identical. The homology between two sequences is a direct function of the number of matching or homologous positions; Two sequences are 50% homologous and two sequences are 90% homologous if 90% of the positions (eg, 9 out of 10) are matched or homologous.

[0150] "Humanized" forms of non-human (e.g., murine) antibodies include chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab′, F(ab′) ₂ , or other antigen-binding subsequence of an antibody). Predominantly humanized antibodies and antibody fragments thereof are derived from a non-human species (donor antibody), e.g. mouse, rat, or a human immunoglobulin (recipient antibody or antibody fragment) substituted with residues from rabbit CDRs. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies/antibody fragments may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. Such modifications can further refine and optimize antibody or antibody fragment performance. Generally, a humanized antibody, or antibody fragment thereof, comprises substantially all of at least one, and usually two, variable domains in which all or substantially all of the CDR regions are those of a non-human immunoglobulin. and all or a substantial portion of the FR regions are of human immunoglobulin sequences. The humanized antibody or antibody fragment also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details see Jones
et al. , Nature, 321:522-525, 1986; Reichmann et al. , Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol. , 2:593-596, 1992.

[0151] "Human" or "fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, which is of human origin in its entirety or consists of an amino acid sequence identical to a human antibody or immunoglobulin. point to

[0152] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that occurs naturally in a living animal is not "isolated", whereas the same nucleic acid or peptide that is partially or completely separated from the coexisting materials in its natural state is "isolated". It is a "thing". An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment, such as a host cell.

[0153] In the context of this disclosure, the following abbreviations are used for commonly occurring nucleobases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0154] The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding properties of an antibody or antibody fragment comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment of this disclosure by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g. threonine, valine, isoleucine), and aromatic side chains (e.g. , tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the TFPs of the present disclosure can be replaced with other amino acid residues from the same side chain family, and the altered TFPs undergo functional assays as described herein. can be tested using

[0155] The terms "operably linked" or "transcriptional control" refer to functional linkages between a regulatory sequence and a heterologous nucleic acid sequence that effect expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. DNA sequences that are operably linked may be adjacent to each other and, for example, in the same reading frame when required for the joining of two protein coding regions.

[0156] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal Including injection, intratumoral, or infusion methods.

[0157] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a particular nucleic acid sequence includes conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences thereof as well as the designated sequence. Arrays are also implicitly included. Specifically, degenerate codon substitutions are made by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues. can be obtained (Batzer
et al. , Nucleic Acid Res. 19:5081 (1991), Ohtsuka et al. , J. Biol. Chem. 260:2605-2608 (1985), and Rossolini et al. , Mol. Cell. Probes
8:91-98 (1994)).

[0158] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound made up of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up the sequence of a protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids joined together by peptide bonds. As used herein, the term includes, for example, short chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, and proteins, commonly referred to in the art, which come in many varieties. Refers to both long chains. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, Fusion proteins are specifically included. Polypeptides include naturally occurring peptides, recombinant peptides, or combinations thereof.

[0159] The term "promoter" refers to a DNA sequence recognized by the cellular transcriptional machinery or introduced synthetic machinery required to initiate the specific transcription of a polynucleotide sequence.

[0160] The term "promoter/regulatory sequence" refers to a nucleic acid sequence required for the expression of a gene product to which it is operably linked. In some cases, this sequence may be the core promoter sequence, and in other cases, this sequence may include enhancer sequences and other regulatory elements required for expression of the gene product. A promoter/regulatory sequence may, for example, direct expression of a gene product in a tissue-specific manner.

[0161] The term "constitutive" promoter means that, when operably linked to a polynucleotide encoding or specifying a gene product, the gene product is Refers to a nucleotide sequence produced within.

[0162] The term "inducible" promoter means that when operably linked to a polynucleotide encoding or specifying a gene product, the promoter is substantially Generally refers to a nucleotide sequence that causes a cell to produce its gene product.

[0163] The term "tissue-specific" promoter means that, when operably linked to a polynucleotide encoding or specifying a gene product, the cell is of the tissue type corresponding to that promoter only if the cell Refers to a nucleotide sequence that, in effect, directs the production of its gene product within a cell.

[0164] The terms "linker" and "flexible polypeptide linker" used in the context of scFv refer to glycine residues used alone or in combination to link the variable heavy and light chain regions together. It refers to a peptide linker consisting of amino acids such as amino acids and/or serine residues. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser) _n , where n is a positive integer of 1 or greater. For example, n=1, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9, and n=10. In one embodiment, flexible polypeptide linkers include, but are not limited to (Gly ₄ Ser) ₄ or (Gly ₄ Ser) ₃ . In another embodiment, the linker comprises multiple repeats of (Gly ₂ Ser), (GlySer), or (Gly ₃ Ser). Also included within the scope of the present disclosure are linkers as described in WO2012/138475 (incorporated herein by reference). Optionally, the linker sequence comprises (G ₄ S) _n , where n=2-5. Optionally, the linker sequence comprises (G ₄ S) _n , where n=1-3.

[0165] As used herein, the 5' cap (also referred to as RNA cap, RNA7-methylguanosine cap, or RNA m7G cap) refers to the "head" or cap of eukaryotic messenger RNA immediately after transcription initiation. A modified guanine nucleotide added to the 5' end. The 5' cap consists of a terminal group linked to the first transcribed nucleotide. Its presence is essential for ribosomal recognition and protection from RNases. Cap addition is linked to transcription and occurs co-transcriptionally so that each influences the other. Shortly after transcription initiation, the 5' end of synthesized mRNA is bound by a cap synthesis complex associated with RNA polymerase. This enzyme complex catalyzes the chemical reactions required to cap mRNAs. Synthesis proceeds as a multistep biochemical reaction. The cap portion can be modified to modulate mRNA function, such as translational stability or efficiency.

[0166] As used herein, "in vitro transcribed RNA" means in
It refers to RNA synthesized in vitro, preferably mRNA. Generally, in vitro transcribed RNA is produced from an in vitro transcription vector. An in vitro transcription vector contains a template that is used to produce in vitro transcribed RNA.

[0167] As used herein, "poly(A)" is a series of adenosines attached to mRNA by polyadenylation. In preferred embodiments of constructs for transient expression, the polyA is 50-5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(A) sequences can be chemically or enzymatically modified to modulate mRNA function, such as translational localization, stability, or efficiency.

[0168] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety or modified variant thereof to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at their 3' ends. The 3′ poly(A) tail is a long sequence of adenine nucleotides (often hundreds) that is added to the pre-mRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, poly(A
) tails are added. The poly(A) tail and proteins attached to it serve to protect the mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, nuclear export of mRNA, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but can also occur later in the cytoplasm. After transcription is terminated, the mRNA strand is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA in the vicinity of the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the open 3' end of the cleavage site.

[0169] As used herein, "transient" refers to the expression of a non-integrated transgene for hours, days, or weeks, the duration of which is is shorter than the duration of expression of the gene when integrated into the genome or housed within a stable plasmid replicon of the host cell.

[0170] The term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in the transmission of a signal from one part of a cell to another part of the cell. The expression "cell surface receptor" includes molecules and complexes of molecules that can receive signals and transmit signals across cell membranes.

[0171] The term "subject" is intended to include organisms (eg, mammals, humans) capable of eliciting an immune response.
[0172] The term "substantially purified" cell refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell that has been separated from other cell types with which it is ordinarily associated in its natural state. In some cases, a substantially purified population of cells refers to a homogeneous cell population. In other cases, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[0173] As used herein, the term "therapeutic" means treatment. A therapeutic effect is achieved by reduction, suppression, amelioration, or eradication of the condition.
[0174] As used herein, the term "prevention" means prophylactic or protective treatment of a disease or condition.

[0175] In the context of this disclosure, "tumor antigen" or "hyperproliferative disease antigen" or "antigen associated with a hyperproliferative disease" refers to an antigen common to a particular hyperproliferative disease. In certain aspects, the hyperproliferative disease antigens of the disclosure are primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, NHL, leukemia, uterine cancer, cervical cancer, bladder cancer , renal cancer, and adenocarcinoma such as breast, prostate, ovarian, and pancreatic cancers, including but not limited to cancer.

[0176] The terms "transfected" or "transformed" or "transduced" refer to a process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid. The cells include primary subject cells and their passages.

[0177] The term "specifically binds" refers to recognizing and binding to a cognate binding partner (e.g., CD19) present in a sample, but not necessarily substantially recognizing other molecules in the sample. It refers to an antibody, antibody fragment, or specific ligand that does not bind.

[0178] As used herein, the term "meganuclease" refers to an endonuclease that binds double-stranded DNA with a recognition sequence greater than 12 base pairs. Preferably, the recognition sequence of the meganuclease of the present disclosure is 22 base pairs. A meganuclease may be an endonuclease derived from I-Crel, which is modified, for example, in terms of DNA binding specificity, DNA cleavage activity, DNA binding affinity, or dimerization properties compared to native I-Crel. It may also refer to engineered variants of I-Crel that have been modified. Methods for making such modified variants of I-Crel are known in the art (eg WO2007/047859). As used herein, a meganuclease may be doubled as a heterodimer or as a "single-chain meganuclease" in which a pair of DNA-binding domains are attached to a single polypeptide using a peptide linker. Binds to main-stranded DNA. The term "homing endonuclease" is synonymous with the term "meganuclease". The meganucleases of the present disclosure are substantially non-toxic when expressed in cells, particularly human T cells, such that the cells can be transfected and maintained at 37° C. using the methods described herein. No adverse effects on cell viability or significant reduction in meganuclease cleavage activity when measured.

[0179] As used herein, the term "single-chain meganuclease" refers to a polypeptide comprising a pair of nuclease subunits joined by a linker. The construction of a single-stranded meganuclease is N-terminal subunit-linker-C-terminal subunit. These two meganuclease subunits are generally not identical in amino acid sequence and recognize non-identical DNA sequences. Single-stranded meganucleases therefore usually cleave pseudo-palindromic or non-palindromic recognition sequences. Single-chain meganucleases are sometimes referred to as "single-chain heterodimers" or "single-chain heterodimeric meganucleases" although they are not actually dimers. For clarity, unless otherwise specified, the term "meganuclease" may refer to a dimeric meganuclease or a single-stranded meganuclease.

[0180] As used herein, the term "TALEN" refers to an endonuclease containing a DNA binding domain comprising 16-22 TAL domain repeats fused to any portion of the Fok1 nuclease domain. .

[0181] As used herein, the term "small TALENs" refers to 16-22 TAL domains fused in any orientation to any catalytically active portion of the nuclease domain of the I-Tev1 homing endonuclease. Refers to an endonuclease containing a DNA-binding domain with repeats.

[0182] As used herein, the term "CRISPR" refers to a caspase, including a caspase, such as Cas9, and a guide RNA that directs the caspase's DNA cleavage by hybridizing to a recognition site on genomic DNA. refers to the endonuclease of the system.

[0183] As used herein, the term "megaTAL" refers to a single-stranded nuclease comprising a transcription activator-like effector (TALE) DNA-binding domain and a modified sequence-specific homing endonuclease. Point.

[0184] As used herein, the terms "T cell receptor" and "T cell receptor complex" generally refer to molecules found on the surface of T cells that are involved in antigen recognition. Used synonymously. In 95% of T cells, the TCR contains a heterodimer consisting of TCRα and TCRβ chains, whereas 5% of T cells have a TCR consisting of TCRγ and TCRδ chains. TCRs further include one or more of CD3ε, CD3γ, and CD3δ. In some embodiments, the TCR comprises CD3ε. In some embodiments, the TCR comprises CD3γ. In some embodiments, the TCR comprises CD3delta. In some embodiments, the TCR comprises CD3ζ. The association of the TCR with antigens, eg antigen and MHC, results in activation of T cells through a series of biological events mediated by relevant enzymes, co-receptors and specialized accessory molecules. In some embodiments, the constant domain of human TCRα has the sequence of SEQ ID NO:142. In some embodiments, the constant domain of human TCRα has an IgC domain with the sequence of SEQ ID NO: 143, a transmembrane domain with the sequence of SEQ ID NO: 144, and an intracellular domain with the sequence of SS. In some embodiments, the mouse TCRα constant domain has the sequence of SEQ ID NO:147. In some embodiments, the constant domain of mouse TCRα has a transmembrane domain with the sequence of SEQ ID NO: 144 and an intracellular domain with the sequence of SS. In some embodiments, the constant domain of human TCRβ has the sequence of SEQ ID NO:148. In some embodiments, the constant domain of human TCRβ has an IgC domain with the sequence of SEQ ID NO:149, a transmembrane domain with the sequence of SEQ ID NO:150, and an intracellular domain with the sequence of SEQ ID NO:151. In some embodiments, the mouse TCRβ constant domain has the sequence of SEQ ID NO:152. In some embodiments, the constant domain of mouse TCRβ has a transmembrane domain having the sequence of SEQ ID NO:152 and an intracellular domain having the sequence of SEQ ID NO:153. In some embodiments, the constant domain of human TCRδ has the sequence of SEQ ID NO:243. In some embodiments, the constant domain of human TCRδ has an IgC domain with the sequence of SEQ ID NO:265, a transmembrane domain with the sequence of SEQ ID NO:159, and an intracellular domain with the sequence of L. In some embodiments, the constant domain of human TCRγ has the sequence of SEQ ID NO:21. In some embodiments, the constant domain of human TCRγ has an IgC domain with the sequence of SEQ ID NO:155, a transmembrane domain with the sequence of SEQ ID NO:156, and an intracellular domain with the sequence of SEQ ID NO:157.

[0185] In some embodiments, the human CD3ε has the sequence of SEQ ID NO:258. In some embodiments, the human CD3ε comprises an extracellular domain having the sequence of SEQ ID NO: 126, a transmembrane domain having the sequence of SEQ ID NO: 127, and an intracellular domain having the sequence of SEQ ID NO: 128, e.g. have a domain. In some embodiments, the human CD3delta has the sequence of SEQ ID NO:136. In some embodiments, the human CD3delta has an extracellular domain having the sequence of SEQ ID NO: 138, a transmembrane domain having the sequence of SEQ ID NO: 139, and an intracellular domain having the sequence of SEQ ID NO: 140, e.g. have a domain. In some embodiments, the human CD3γ has the sequence of SEQ ID NO:130. In some embodiments, the human CD3γ has an extracellular domain having the sequence of SEQ ID NO: 132, a transmembrane domain having the sequence of SEQ ID NO: 133, and an intracellular domain having the sequence of SEQ ID NO: 134, e.g. have a domain.

[0186] Ranges: Throughout this disclosure, various aspects of the disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, a description of a range such as 1 to 6 includes subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, as well as individual numbers within that range, For example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6 should also be considered specifically disclosed. As another example, a range such as 95-99% identity includes 95%, 96%, 97%, 98%, or 99% identity; %, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the width of the range.

[0187] Provided herein is a modified T cell comprising a T cell receptor (TCR) fusion protein (TFP) and a TCR constant domain, which also has a functionally disrupted endogenous TCR subunit. Compositions and methods of use for treating diseases such as cancer are provided. As used herein, "T-cell receptor (TCR) fusion protein" or "TFP" includes recombinant polypeptides derived from a variety of polypeptides containing TCRs, which TCRs generally i) target It is capable of binding cell surface antigens and ii) interacting with other polypeptide components of intact TCR complexes when normally co-localized within or on the surface of T cells. As shown herein, TFP offers significant advantages over chimeric antigen receptors. The term "chimeric antigen receptor" or alternatively "CAR" includes an extracellular antigen binding domain, e.g. in the form of a single domain antibody or scFv, a transmembrane domain, and a functional signaling derived from a stimulatory molecule as defined below. Refers to a recombinant polypeptide comprising a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") comprising the domain. In general, the central intracellular signaling domain of CAR is derived from the CD3 zeta chain, which is normally present in association with the TCR complex. This CD3ζ signaling domain can be fused with one or more functional signaling domains from at least one co-stimulatory molecule such as 4-1BB (ie CD137), CD27, and/or CD28.

T cell receptor (TCR) fusion protein (TFP)
[0188] The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises
Constructs comprising a binding domain, such as an antibody or antibody fragment, ligand, or ligand binding protein, wherein the sequence of the binding domain is contiguous and in the same reading frame with the nucleic acid sequence encoding the TCR subunit or portion thereof encompasses The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises an antibody fragment that specifically binds CD19, such as human CD19, the sequence of the antibody fragment comprising a TCR subunit or a portion thereof Constructs that are contiguous and in the same reading frame with the nucleic acid sequences encoding The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises an antibody fragment that specifically binds mesothelin, such as human mesothelin, wherein the sequence of the antibody fragment comprises a TCR subunit or a portion thereof It includes constructs that are contiguous and in the same reading frame as the encoding nucleic acid sequence. The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises an antibody fragment that specifically binds to MUC16, such as human MUC16, the sequence of the antibody fragment comprising a TCR subunit or a portion thereof Constructs that are contiguous and in the same reading frame with the nucleic acid sequences encoding The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises an antibody fragment that specifically binds CD20, such as human CD20, wherein the sequence of the antibody fragment is a TCR subunit or a portion thereof Constructs that are contiguous and in the same reading frame with the nucleic acid sequences encoding The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises an antibody fragment that specifically binds CD70, such as human CD70, wherein the sequence of the antibody fragment is a TCR subunit or a portion thereof Constructs that are contiguous and in the same reading frame with the nucleic acid sequences encoding The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises an antibody fragment that specifically binds CD79B, such as human CD79B, wherein the sequence of the antibody fragment is a TCR subunit or a portion thereof Constructs that are contiguous and in the same reading frame with the nucleic acid sequences encoding The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises an antibody fragment that specifically binds HER2, such as human HER2, wherein the sequence of the antibody fragment comprises a TCR subunit or a portion thereof Constructs that are contiguous and in the same reading frame with the nucleic acid sequences encoding The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises an antibody fragment that specifically binds to PSMA, such as human PSMA, wherein the sequence of the antibody fragment comprises a TCR subunit or a portion thereof Constructs that are contiguous and in the same reading frame with the nucleic acid sequences encoding The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises an antibody fragment that specifically binds BCMA, such as human BCMA, wherein the sequence of the antibody fragment comprises a TCR subunit or a portion thereof Constructs that are contiguous and in the same reading frame with the nucleic acid sequences encoding The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises an antibody fragment that specifically binds to ROR1, such as human ROR1, wherein the sequence of the antibody fragment is a TCR subunit or a portion thereof Constructs that are contiguous and in the same reading frame with the nucleic acid sequences encoding The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises an antibody fragment that specifically binds CD22, such as human CD22, wherein the sequence of the antibody fragment is a TCR subunit or a portion thereof Constructs that are contiguous and in the same reading frame with the nucleic acid sequences encoding The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises an antibody fragment that specifically binds to GPC3, such as human GPC3, the sequence of the antibody fragment comprising a TCR subunit or a portion thereof Constructs that are contiguous and in the same reading frame with the nucleic acid sequences encoding The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises an antibody fragment that specifically binds to Nectin-4, such as human Nectin-4, wherein the sequence of the antibody fragment comprises a TCR subunit or Constructs that are contiguous and in the same reading frame with the nucleic acid sequence encoding a portion thereof are included. The present disclosure is a recombinant DNA construct encoding a TFP, wherein the TFP comprises an antibody fragment that specifically binds Trop-2, such as human Trop-2, wherein the sequence of the antibody fragment comprises a TCR subunit or a construct that is contiguous and in the same reading frame with the nucleic acid sequence encoding a portion thereof. TFPs provided herein associate with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR subunits to form a functional TCR complex. can be formed.

[0189] In one aspect, the TFPs of this disclosure comprise a target-specific binding member otherwise referred to as an antigen binding domain. The selection of moieties depends on the type and number of target antigens that determine the surface of the target cell. For example, an antigen binding domain can be selected to recognize a target antigen that acts as a cell surface marker for target cells associated with a particular disease state. Thus, examples of cell surface markers that can serve as target antigens for the antigen-binding domains of TFPs of the present disclosure include viral, bacterial, and parasitic infections, autoimmune diseases, and cancer diseases (e.g., malignant tumor diseases). related to.

[0190] In one aspect, TFP-mediated T cell responses can be directed to the antigen of interest by engineering the antigen binding domain into a TFP that specifically binds to the desired antigen.

[0191] Antigen-binding domains include, but are not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, as well as heavy chain variable domains ( _VH ), light chain variable domains ( _VL ), and camelid Any domain that binds an antigen, including but not limited to functional fragments thereof, including single domain antibodies such as the heavy chain variable domain (V _HH ) of Nanobodies from the family, as well as functioning as antigen binding domains in the art. It can be any domain that binds to alternative scaffolds that are known to do so, such as recombinant fibronectin domains, anticalins, DARPINs, and the like. Similarly, natural or synthetic ligands that specifically recognize and bind target antigens can be used as antigen-binding domains for TFP. In some cases, antigen binding domains derived from the same species for which the TFP is ultimately used are beneficial. For example, for human use, it may be beneficial for the antigen-binding domain of TFP to comprise human or humanized residues relative to the antigen-binding domain of an antibody or antibody fragment.

[0192] Thus, in one aspect, the antigen-binding domain comprises a humanized or human antibody, or antibody fragment, or a murine antibody or antibody fragment. In one embodiment, the humanized or human anti-TAA binding domain is a humanized or human anti-TAA binding domain described herein light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and one or more (eg, all three) of light chain complementarity determining region 3 (LC CDR3), and/or a humanized or human anti-CD19 binding domain as described herein. one or more (e.g., all three) of heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of For example, a humanized or human anti-TAA domain comprising one or more, such as all three LC CDRs and one or more, such as all three HC CDRs. In one embodiment, the humanized or human anti-CD19 binding domain is the heavy chain complementarity determining region 1 (HC CDR1) of the humanized or human anti-TAA binding domain described herein, the heavy chain complementarity determining region 2 (HC CDR2), and one or more (e.g., all three) of heavy chain complementarity determining region 3 (HC CDR3), e.g., a humanized or human anti-TAA binding domain, each of which is described herein It has two variable heavy chain regions comprising HC CDR1, HC CDR2, and HC CDR3 as described in . In one embodiment, the humanized or human anti-TAA binding domain comprises a humanized or human light chain variable region as described herein and/or a humanized or human heavy chain variable region as described herein. include. In one embodiment, the humanized or human anti-TAA binding domain comprises a humanized heavy chain variable region as described herein, e.g., at least two humanized or human heavy chain variable regions as described herein . In one embodiment, the anti-TAA binding domain is a scFv comprising light and heavy chains of the amino acid sequences provided herein. In certain embodiments, the anti-TAA binding domain (e.g., scFv) has at least one, two, or three modifications (e.g., substitutions) to the light chain variable region amino acid sequence provided herein. , or a light chain variable region comprising an amino acid sequence having no more than 10 modifications (eg, substitutions), or a sequence having 95-99% identity to the amino acid sequences provided herein, and/or Amino acid sequences having at least 1, 2, or 3 modifications (e.g., substitutions), but no more than 30, 20, or 10 modifications (e.g., substitutions) in the heavy chain variable region amino acid sequence provided herein; Heavy chain variable regions comprising sequences having 95-99% identity to the amino acid sequences provided herein are included. In one embodiment, the humanized or human anti-TAA binding domain is a scFv and a light chain variable region comprising the amino acid sequences described herein is attached via a linker, e.g. linked to a heavy chain variable region comprising the amino acid sequences described herein. In one embodiment, the humanized anti-TAA binding domain comprises a (Gly ₄ -Ser) _n linker, where n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4. The scFv light chain variable region and heavy chain variable region may be arranged, for example, in either light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region. Optionally, the linker sequence includes a long linker (LL) sequence. Optionally, the long linker sequence comprises (G ₄ S) _n (where n=2-4). Optionally, the linker sequence includes a short linker (SL) sequence. Optionally, the short linker sequence comprises (G ₄ S) _n (where n=1-3).

[0193] In some embodiments, the antigen binding domain comprises the light chain CDR1 of SEQ ID NO:73, CDR2 of SEQ ID NO:75, and CDR3 of SEQ ID NO:77 and the heavy chain CDR1 of SEQ ID NO:79, CDR2 of SEQ ID NO:81 , and CDR3 of SEQ ID NO:83, humanized or human antibodies or fragments thereof, or murine antibodies or fragments thereof. In some embodiments, the anti-CD19 antibody is a murine scFv. In some embodiments, the anti-CD-19 antibody comprises VL of SEQ ID NO:85 and VH of SEQ ID NO:87.

[0194] In some embodiments, the antigen binding domain comprises CDR1 of SEQ ID NO:60, CDR2 of SEQ ID NO:61, and CDR3 of SEQ ID NO:62, or CDR1 of SEQ ID NO:63, CDR2 of SEQ ID NO:64, and SEQ ID NO:64. 65 CDR3s, or an anti-mesothelin humanized or human single domain antibody or antibody fragment having CDR1 of SEQ ID NO:66, CDR2 of SEQ ID NO:67, and CDR3 of SEQ ID NO:68. In some embodiments, the anti-mesothelin antibody has a variable domain of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71.

[0195] In some embodiments, the antigen binding domain comprises CDR1 of SEQ ID NO:88, CDR2 of SEQ ID NO:89, and CDR3 of SEQ ID NO:90, or CDR1 of SEQ ID NO:92, CDR2 of SEQ ID NO:93, and SEQ ID NO:93. CDR3 of SEQ ID NO: 94, or CDR1 of SEQ ID NO: 96, CDR2 of SEQ ID NO: 97, and CDR3 of SEQ ID NO: 98, or CDR1 of SEQ ID NO: 100, CDR2 of SEQ ID NO: 101, and CDR3 of SEQ ID NO: 102, CDR1 of SEQ ID NO: 104, CDR2 of SEQ ID NO: 105 and CDR3 of SEQ ID NO: 106, or CDRl of SEQ ID NO: 108, CDR2 of SEQ ID NO: 109, and CDR3 of SEQ ID NO: 110, or CDRl of SEQ ID NO: 112, CDR2 of SEQ ID NO: 113, and SEQ ID NO: 114 or CDR1 of SEQ ID NO: 116, CDR2 of SEQ ID NO: 117, and CDR3 of SEQ ID NO: 118, or CDR1 of SEQ ID NO: 120, CDR2 of SEQ ID NO: 121, and CDR3 of SEQ ID NO: 122. or human single domain antibodies or antibody fragments.

[0196] In some embodiments, non-human antibodies are humanized, in which certain sequences or regions of the antibody are made to increase similarity to naturally occurring antibodies or fragments thereof in humans. Qualified. In one aspect, the antigen binding domain is humanized.

[0197] Humanized antibodies can be made using a variety of techniques known in the art, such techniques including CDR grafting (e.g., each of which is incorporated herein by reference in its entirety). (see European Patent EP 239,400, International Publication No. WO 91/09967, and U.S. Patent Nos. 5,225,539, 5,530,101, and 5,585,089) , veneering or resurfacing (e.g. EP 592,106 and EP 519,596, Padlan, 1991, Molecular Immunology, 28(4/5), each of which is incorporated herein by reference in its entirety). :489-498, Studnicka et al., 1994, Protein Engineering, 7(6):805-814, and Roguska et al., 1994, PNAS, 91:969-973), strand shuffling (e.g., whole See U.S. Pat. No. 5,565,332, which is hereby incorporated by reference in its entirety), for example, U.S. Patent Application Publication No. US2005/0042664, U.S. Patent Applications, each of which is incorporated herein by reference in its entirety. Publication No. US2005/0048617, US Patent No. 6,407,213, US Patent No. 5,766,886, International Publication No. WO9317105, Tan et al. , J. Immunol. , 169:1119-25 (2002), Caldas et al. , Protein Eng. , 13(5):353-60 (2000), Morea et al. , Methods, 20(3):267-79 (2000); Baca et al. , J. Biol. Chem. , 272(16):10678-84 (1997), Roguska et al. , Protein Eng. , 9(10):895-904 (1996), Couto et al. , Cancer Res. , 55(23 Supp):5973s-5977s (1995), Couto et al. , Cancer Res. , 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10 (1994), and Pedersen et al. , J. Mol. Biol. , 235(3):959-73 (1994). Often, framework residues in framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, eg, improve, antigen binding. Such framework substitutions are identified by methods well known in the art, e.g., modeling the interaction of CDRs with framework residues to identify framework residues important for antigen binding and sequence comparison. and by identifying unique framework residues at particular positions (e.g., Queen et al., U.S. Pat. No. 5,585,089, and Riechmann et al., which are incorporated herein by reference in their entirety). al., 1988, Nature, 332:323).

[0198] A humanized antibody or antibody fragment has one or more amino acid residues remaining from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues and are typically obtained from an "import" variable domain. As provided herein, a humanized antibody or antibody fragment comprises one or more CDRs and framework regions from a non-human immunoglobulin molecule, wherein the amino acid residues comprising the framework are either completely or mostly Derived from the human germline. Many techniques for humanizing antibodies or antibody fragments are well known in the art and are described by Winter and co-workers (Jones et al., Nature
321:522-525 (1986), Riechmann et al. , Nature 332:323-327 (1988), Verhoeyen et al. , Science 239:1534-1536 (1988)), replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences, i. EP 239,400, PCT Publication No. WO 91/09967, and U.S. Pat. 101, 5,585,089 and 6,548,640). In such humanized antibodies and antibody fragments, intact human variable domains are significantly less substituted by corresponding sequences from non-human species. Humanized antibodies are often human antibodies in which some CDR residues and sometimes some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. . Humanization of antibodies and antibody fragments can be achieved by veneering or resurfacing (EP 592,106, EP 519,596, Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., Protein Engineering, 7 (6):805-814 (1994)); and Roguska et al. , Proc. Natl. Acad. Sci. USA, 91:969-973 (1994)), and chain shuffle (US Pat. No. 5,565,332), the entire contents of which are incorporated herein by reference.

[0199] The choice of human variable domains (both light and heavy chains) for use in making humanized antibodies is to reduce antigenicity. The sequence of the variable domain of the rodent antibody is screened against the entire library of known human variable domain sequences according to the so-called "best fit" method. The human sequence that most closely resembles the rodent one is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol Biol., 196:901 (1987)). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (eg, Nicholson et al. Mol. Immun. 34(16-17):1157- 1165 (1997), Carter et al., Proc. Natl. Acad. Sci. In some embodiments, the framework regions, eg, all four framework regions of the heavy chain variable region are derived from the V _H 4-4-59 germline sequence. In one embodiment, the framework regions may include, eg, 1, 2, 3, 4, or 5 modifications, eg, substitutions, from amino acids in the corresponding mouse sequence. In one embodiment, the framework regions, eg, all four framework regions of the light chain variable region are derived from the VK3-1.25 germline sequence. In one embodiment, the framework regions may include, eg, 1, 2, 3, 4, or 5 modifications, eg, substitutions, from amino acids in the corresponding mouse sequence.

[0200] In some embodiments, portions of the TFP compositions of this disclosure, including antibody fragments, are humanized with retention of high affinity for the target antigen and other favorable biological properties. According to one aspect of the present disclosure, humanized antibodies and antibody fragments are produced by a process of analysis of parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of the residues in the functionality of the candidate immunoglobulin sequence, e.g., analysis of residues that influence the ability of the candidate immunoglobulin to bind its target antigen. . Thus, FR residues from the recipient and import sequences can be selected and combined to achieve a desired antibody or antibody fragment property, eg, increased affinity for the target antigen. In general, the CDR residues are directly and most responsible for influencing antigen binding.

[0201] A humanized antibody or antibody fragment can retain similar antigen specificity as the original antibody, eg, in the present disclosure, the ability to bind human tumor-associated antigen (TAA). In some embodiments, a humanized antibody or antibody fragment can improve the affinity and/or specificity of binding, eg, to human CD19, human BCMA, or another tumor-associated antigen.

[0202] In one aspect, the binding domain is characterized by a particular functional feature or property of the antibody or antibody fragment. For example, in one aspect, a portion of a TFP composition of the present disclosure that includes an antigen binding domain specifically binds human CD19. In one aspect, the antigen binding domain is for human CD19 as described in Nicholson et al. Mol. Immun. 34(16-17):1157-1165 (1997). In one aspect, the disclosure relates to an antigen binding domain comprising an antibody or antibody fragment, wherein the antibody binding domain specifically binds to a CD19 or BCMA protein or fragment thereof, wherein the antibody or antibody fragment is described herein. A variable light chain and/or a variable heavy chain comprising the amino acid sequence provided in In certain aspects, the scFv is flanked by and in the same reading frame as the leader sequence.

[0203] In one aspect, the anti-tumor-associated antigen binding domain is a fragment, eg, a single chain variable fragment (scFv). In one aspect, the anti-TAA binding domain is an Fv, Fab, (Fab') ₂ , or bifunctional (e.g., bispecific) hybrid antibody (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)). In one aspect, the antibodies and fragments thereof of the present disclosure bind to CD19 protein with wild-type or enhanced affinity. In another aspect, the anti-TAA binding domain comprises a single domain antibody (sdAb or VHH).

[0204] Also, methods for obtaining antibody antigen binding domains specific for target antigens (e.g., CD19, BCMA, or any of the target antigens described elsewhere herein for targeting of fusion moiety binding domains) is provided herein, and the method provides an amino acid sequence variant of a _VH domain by addition, deletion, substitution, or insertion of one or more amino acids in the amino acid sequence of the _VH domain described herein. providing a _VH domain, optionally combining the _VH domain so provided with one or more _VL domains, and one or more of this _VH domain or _VH / _VL to identify specific binding members or antibody antigen-binding domains that are specific for the target antigen of interest (e.g., MSLN, CD79B, etc.) and optionally have one or more desired properties including.

[0205] Optionally, _VH domains and scFvs can be produced according to methods known in the art (eg, Bird et al., (1988) Science 242:423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). scFv molecules can be made by joining _VH and _VL regions using a flexible polypeptide linker. scFv molecules include linkers optimized for length and/or amino acid composition (eg, Ser-Gly linkers). The length of this linker can greatly affect how the variable regions of the scFv fold and interact. In fact, intrachain folding is prevented when short polypeptide linkers are used (eg 5-10 amino acids). Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. Optionally, the linker sequence includes one linker sequence. Optionally, the long linker sequence comprises (G ₄ S) _n (where n=2-4). Optionally, the linker sequence comprises (G ₄ S) _n , where n=1-3. For examples of linker placement and size, see, eg, Hollinger et al. 1993 Proc
Natl Acad. Sci. U.S.A. S. A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005/0100543, 2005/0175606, 2007/0014794, and PCT Publication Nos. WO2006/020258 and WO2007/024715.

[0206] The scFv may comprise a linker of about 10, 11, 12, 13, 14, 15, or more than 15 residues between its _VL and _VH regions. A linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises the amino acids glycine and serine. In another embodiment, the linker sequence comprises repeats of a glycine and serine set such as (Gly ₄ Ser) _n (where n is a positive integer greater than or equal to 1). In one embodiment, the linker can be ( _Gly4Ser ) ₄ or ( _Gly4Ser ) ₃ . Variation in linker length can retain or enhance activity, resulting in superior efficacy in activity assays. Optionally, the linker sequence comprises (G ₄ S) _n , where n=2-4. Optionally, the linker sequence comprises (G ₄ S) _n , where n=1-3.

Stability and Mutation
[0207] Stability of tumor-associated antigen binding domains, such as scFv molecules (e.g., soluble scFv), is assessed relative to biophysical properties (e.g., thermal stability) of conventional control scFv molecules or full-length antibodies. be able to. In one embodiment, the humanized or human scFv is about 0.1°C, about 0.25°C, about 0.5°C, about 0.75°C, about 1°C lower than the parental scFv in a described assay. , about 1.25° C., about 1.5° C., about 1.75° C., about 2° C., about 2.5° C., about 3° C., about 3.5° C., about 4° C., about 4.5° C., about 5°C, about 5.5°C, about 6°C, about 6.5°C, about 7°C, about 7.5°C, about 8°C, about 8.5°C, about 9°C, about 9.5°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, or about 15°C higher thermal stability.

[0208] Improved thermostability of anti-TAA binding domains, such as scFv, continues to
Conferred over the AA-TFP construct, it leads to improved therapeutic properties of the anti-TAA TFP construct. The thermostability of a binding domain, such as a scFv or sdAb, can be improved by at least about 2°C or 3°C compared to conventional antibodies. In one embodiment, the binding domain has a 1° C. improvement in thermal stability compared to conventional antibodies. In another embodiment, the binding domain has a 2°C improvement in thermal stability compared to a conventional antibody. In another embodiment, the scFv is thermostable at 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C or 15°C improvement. A comparison can be made, for example, between a scFv molecule disclosed herein and the scFv molecule or Fab fragment of the antibody from which the _VH and _VL of the scFv are derived. Thermal stability can be measured using methods known in the art. For example, _TM can be measured in one embodiment. _Methods for measuring TM and other methods for determining protein stability are detailed below.

[0209] Mutations in antibody sequences (generated via humanization or direct mutagenesis of soluble scFv) alter the stability of antibodies or fragments thereof and improve the overall stability of antibody and TFP constructs. Measurements such as T _M , temperature denaturation, and temperature aggregation are used to compare the stability of humanized antibodies or fragments thereof to murine antibodies or fragments thereof. In one embodiment, the binding domain, eg scFv or sdAb, comprises at least one mutation resulting from the humanization process, whereby the mutant scFv confers improved stability to the anti-TAA TFP construct. In another embodiment, the anti-TAA binding domain, eg scFv or sdAb, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations resulting from the humanization process, thereby Mutant scFv or sdAb confer improved stability to TAA-TFP constructs.

[0210] In one aspect, the antigen-binding domain of TFP comprises an amino acid sequence that is homologous to an amino acid sequence of an antigen-binding domain described herein, wherein the antigen-binding domain comprises an anti-tumor-associated antigen described herein. It retains the desired functional properties of the antibody fragment. In one specific aspect, the TFP compositions of this disclosure comprise antibody fragments. In a further aspect, the antibody fragment comprises scFv.

[0211] In various embodiments, the antigen-binding domain of TFP is in one or both variable regions (e.g., _VH and/or _VL ), such as in one or more CDR regions, and/or in one or more Altered by modifying one or more amino acids within the framework region. In one specific aspect, the TFP compositions of this disclosure comprise antibody fragments. In a further aspect, the antibody fragment comprises scFv.

[0212] It will be appreciated by those skilled in the art that the antibodies or antibody fragments of this disclosure can be further modified such that they differ with respect to amino acid sequence (eg, from wild-type) but not with respect to the desired activity. For example, additional nucleotide substitutions can be made into the protein that result in amino acid substitutions at "non-essential" amino acid residues. For example, a nonessential amino acid residue in a molecule can be replaced with another amino acid residue from the same side chain family. In another embodiment, a chain of amino acids may be replaced with a structurally similar chain that differs in the order and/or composition of side chain family members. For example, conservative substitutions can be made in which amino acid residues are replaced with amino acid residues having similar side chains.

[0213] Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid , glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan ), β-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).

[0214] Percent identity, in the context of two or more nucleic acid or polypeptide sequences, refers to the identity of the two or more sequences. When comparing and aligning maximal matches over comparison zones or designated regions, as determined using any of the following sequence comparison algorithms or using manual alignment and visual inspection, two the array is
Amino acid residues or nucleotides that are identical are represented by a specified percentage (e.g., 60% identity, optionally 70%, 71%, 72%, over a specified region or, if not specified, over the entire sequence). , 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity). is. optionally over a region that is at least about 50 nucleotides (or 10 amino acids) long, or more preferably a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200, or more amino acids) long There is identity throughout.

[0215] For sequence comparison, typically one sequence is used as a reference sequence, and test sequences are compared to the reference sequence. When utilizing a sequence comparison algorithm, test and reference sequences are entered into a computer, partial coordinates are designated, if necessary, and sequence algorithm program parameters are designated. You can use default program parameters, or you can designate different parameters. The sequence comparison algorithm then calculates the % sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Sequence alignment methods for comparison are well known in the art. Optimal alignments for sequence comparison are described, for example, in Smith and Waterman, (1970) Adv. Appl. Math. 2:482c by the homology algorithm of Needleman and Wunsch, (1970) J. Am. Mol. Biol. 48:443 by the homology alignment algorithm of Pearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or Manual alignment and visual inspection (see, eg, Brent et al., (2003) Current Protocols in Molecular Biology) can be performed. Two examples of algorithms suitable for determining % sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990)J. Mol. Biol. 215:403-410. Software for performing BLAST analyzes is published by the National Center for Biotechnology Information.

[0216] In one aspect, the present disclosure contemplates amino acid sequence modifications of the starting antibody or fragment (eg, scFv) to produce functionally equivalent molecules. For example, the binding domain comprised in the TFP, e.g., the _VH or _VL of the scFv, is at least about 70%, 71%, 72%, 73% of the starting _VH or _VL framework region of the anti-CD19 binding domain, e.g., the scFv. , 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. The present disclosure contemplates modification of the entire TFP construct, eg modification of the amino acid sequence of one or more of the various domains of the TFP construct to generate functionally equivalent molecules. The TFP construct is at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83% of the starting TFP construct. %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% Modifications can be made to preserve identity.

extracellular domain
[0217] The extracellular domain may be derived from either natural or recombinant sources. When the source is natural, this domain may be derived from any protein, but in particular a domain other than a membrane-bound or transmembrane protein. In one aspect, the extracellular domain can be associated with the transmembrane domain. Extracellular domains of particular use in this disclosure are, for example, the α-chain, β-chain, or ζ-chain of the T-cell receptor, or CD3ε, CD3γ, or CD3δ, or in another embodiment, CD28, CD45, CD4, It may comprise at least the extracellular region(s) of CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the extracellular domain is a TCR extracellular domain. Optionally, the TCR extracellular domain comprises a TCRα chain, a TCRβ chain, a TCRγ chain, a TCRδ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, functional fragments thereof, and at least one but no more than 20 modifications. the extracellular domain of a protein or a portion thereof selected from the group consisting of amino acid sequences thereof having

[0218] In some embodiments, the TCR extracellular domain comprises the extracellular domain of a TCRα chain, a TCRβ chain, a TCRδ chain, or a TCRγ chain or a portion thereof. In some embodiments, the TCR extracellular domain comprises the IgC domain of a TCRα, TCRβ, TCRδ, or TCRγ chain.

[0219] In some embodiments, the extracellular domain is 5, 6, 7, 8, 9, 10, 11, 12, 13 of the extracellular domain of a TCRα chain, TCRβ chain, TCRδ chain, or TCRγ chain. , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 , 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more contiguous amino acid residues, or at least. In some embodiments, the extracellular domain is at least about 50%, 55%, 60%, 65%, Sequences with 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity are included. In some embodiments, the extracellular domain has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acid truncations of TCRα, TCRβ, TCRδ, or TCRγ It contains the sequence encoding the extracellular domain of the chain.

[02220] In some embodiments, the extracellular domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, Contains or at least contains 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more contiguous amino acid residues. In some embodiments, the extracellular domain is at least about 50%, 55%, 60%, 65%, 70%, 75% relative to the sequence encoding the IgC domain of TCRα, TCRβ, TCRδ, or TCRγ , 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity. In some embodiments, the extracellular domain has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 13, 14, 12, 13, 14, 12, 13, 23, 24, 25, or more amino acid truncations of TCRα, TCRβ, TCRδ, or TCRγ. contains an array that encodes the

[0221] In some embodiments, the extracellular domain is 5, 6, 7, 8, 9, 10, 11, 12, 13 of the extracellular domain of a CD3εTCR subunit, a CD3γTCR subunit, or a CD3δTCR subunit. , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 , 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, or more contiguous amino acid residues, or at least. In some embodiments, the extracellular domain is at least about 50%, 55%, 60%, 65%, Sequences with 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity are included. In some embodiments, the extracellular domain has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 13, 14, 12, 13, 14, 12, 13, 23, 24, 25, or more amino acid truncations. Contains the sequence encoding the extracellular domain of the unit.

[0222] The extracellular domain can be a TCR extracellular domain. The TCR extracellular domain can be derived from the TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ε TCR subunit, CD3γ TCR subunit, or CD3δ TCR subunit. The extracellular domain can be a full-length TCR extracellular domain or a fragment (eg, functional fragment) thereof. The extracellular domain may comprise the variable domain of the TCRα, TCRβ, TCRγ, or TCRδ chains. The extracellular domain may comprise the variable and constant domains of the TCRα, TCRβ, TCRγ, or TCRδ chains. In some cases, the extracellular domain may be free of variable domains.

[0223] The extracellular domain may comprise the constant domain of a TCRα, TCRβ, TCRγ, or TCRδ chain. The extracellular domain may comprise the full-length constant domain of the TCRα, TCRβ, TCRγ, or TCRδ chains. The extracellular domain may comprise a fragment (eg, functional fragment) of the full-length constant domain of the TCRα, TCRβ, TCRγ, or TCRδ chains. For example, the extracellular domain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, It may contain 65, 70, 75, 80, 85, 90, 95, 100, 150, or more amino acid residues.

[0224] The TCRα, TCRβ, TCRγ, or TCRδ chains described herein can be derived from a variety of species. The TCR chain can be a murine or human TCR chain. For example, the extracellular domain can comprise the constant domain of mouse TCRα chain, mouse TCRβ chain, human TCRγ chain, or human TCRδ chain.

transmembrane domain
[0225] In general, a TFP sequence includes an extracellular domain and a transmembrane domain encoded by a single genomic sequence. In alternative embodiments, TFPs can be designed to contain a transmembrane domain that is heterologous to the extracellular domain of TFP. A transmembrane domain includes one or more additional amino acids that flank the transmembrane region, e.g., one or more amino acids associated with the extracellular region of the protein from which the transmembrane is derived (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids), and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane was derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids). Optionally, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60, or more amino acids of the extracellular region. Optionally, the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60, or more amino acids of the intracellular region. In one aspect, the transmembrane domain is associated with one of the other domains of the TFP used. Optionally, transmembrane domains are selected or modified by amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins, e.g. Minimize interaction with other members. In one aspect, the transmembrane domain is capable of homodimerization with another TFP on the TFP-T cell surface. In different aspects, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interaction with the binding domains of natural binding partners present in the same TFP.

[0226] The transmembrane domain may be derived from either natural or recombinant sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect, the transmembrane domain can signal to the intracellular domain(s) whenever TFP is bound to a target. In some cases, the TCR integrated subunit is TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, TCRζ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22 , CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. It contains a transmembrane domain that contains a transmembrane domain.

[0227] In some embodiments, the transmembrane domain is at least about 5-6 transmembrane domains of a TCRα chain, a TCRβ chain, a TCRγ chain, a TCRδ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, or a CD3δ TCR subunit. , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, or at least comprise or more contiguous amino acid residues. In some embodiments, the transmembrane domain is at least about Sequences with 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity are included. In some embodiments, the transmembrane domain has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 including sequences encoding the transmembrane domain of the TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ε TCR subunit, CD3γ TCR subunit, or CD3δ TCR subunit with truncation of or more amino acids.

[0228] Optionally, the transmembrane domain can be attached to the extracellular region of TFP, eg, the antigen-binding domain of TFP, via a hinge, eg, a hinge derived from a human protein. For example, in one embodiment the hinge can be a human immunoglobulin (Ig) hinge, such as an IgG4 hinge, or a CD8a hinge.

Linker
[0229] Optionally, the linkage is formed between the binding member and the TCR extracellular domain of TFP by a short oligopeptide or polypeptide linker of 2-10 amino acids in length. Glycine-serine doublets make particularly suitable linkers. In some cases, the linker can be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more in length. For example, in one aspect, the linker has the amino acid sequence GGGGSGGGGS, or the sequence (GGGGS
) _x where X is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more. In some embodiments, X is two. In some embodiments, X is four. In some embodiments, the linker is encoded by the nucleotide sequence GGTGGCGGAGGTTCTGGAGGTGGGAGGTTCC.

cytoplasmic domain
[0230] The cytoplasmic domain of TFP can include an intracellular domain. In some embodiments, the intracellular domain is derived from CD3γ, CD3δ, CD3ε, TCRα, TCRβ, TCRγ, or TCRδ. In some embodiments, the intracellular domain comprises a signaling domain when the TFP comprises a CD3 gamma, delta, or epsilon polypeptide. The TCRα, TCRβ, TCRγ, and TCRδ subunits generally have short (eg, 1-19 amino acids long) intracellular domains and are generally absent from the signaling domain. The intracellular signaling domain is generally involved in the activation of at least one normal effector function of immune cells into which TFP has been introduced. The intracellular domains of TCRα, TCRβ, TCRγ, and TCRδ do not have signaling domains, but recruit proteins with primary intracellular signaling domains described herein that function as intracellular signaling domains, such as CD3ζ. be able to. The term "effector function" refers to a specific function of a cell. Effector functions of T cells can be, for example, cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces effector function signals to induce the cell to perform a particular function. Although the entire intracellular signaling domain is usually available, in many cases it is not necessary to use the entire chain. If truncated portions of the intracellular signaling domain are used, they may be used in place of the intact chain as long as such truncated portions transmit effector function signals. Accordingly, the term intracellular signaling domain is intended to include any truncated portion of an intracellular signaling domain sufficient to transmit an effector function signal.

[0231] Examples of intracellular domains for use in TFPs of the present disclosure include cytoplasmic sequences of T-cell receptors (TCRs) and co-receptors that can act in concert to initiate signaling after antigen receptor binding. , and any derivative or variant of that sequence, and any recombinant sequence having the same functional ability. In some embodiments, the intracellular domain comprises the intracellular domain of a TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ε TCR subunit, CD3γ TCR subunit, or CD3δ TCR subunit. In some embodiments, the intracellular domain is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, of the intracellular domain of the TCRα chain, TCRβ chain, TCRγ chain, or TCRδ chain Contains or at least contains 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more contiguous amino acid residues. In some embodiments, the intracellular domain is at least about 50%, 55%, 60%, 65%, Sequences with 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity are included. In some embodiments, the transmembrane domain has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 Including sequences encoding intracellular domains of TCRα, TCRβ, TCRγ, or TCRδ chains with truncations of amino acids or more.

[0232] In some embodiments, the intracellular domain is 5, 6, 7, 8, 9, 10, 11, 12, 13 of the intracellular domain of a CD3εTCR subunit, a CD3γTCR subunit, or a CD3δTCR subunit. , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62, or at least comprise or more contiguous amino acid residues. In some embodiments, the intracellular domain is the CD3εTCR subunit, C
at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% to sequences encoding the D3γTCR subunit or the intracellular domain of the CD3δTCR subunit %, 98%, 99% or greater sequence identity. In some embodiments, the intracellular domain has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acid truncations. It contains the sequence encoding the intracellular domain of the unit.

[0233] It is known that the signal generated by the TCR alone is insufficient for full activation of naive T cells, requiring secondary and/or co-stimulatory signals. Thus, T cell activation consists of two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary intracellular signaling sequences) and antigen-independent It may be said to be mediated by those that act to produce secondary or co-stimulatory signals (secondary cytoplasmic domains, eg co-stimulatory domains).

[0234] Primary signaling domains regulate the primary activation of the TCR complex in either a stimulatory or an inhibitory manner. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine activation motifs (ITAMs).

[0235] Examples of ITAMs containing primary intracellular signaling domains that are particularly useful in this disclosure include those of CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. be done. In one embodiment, a TFP of the present disclosure comprises an intracellular signaling domain, eg, the primary signaling domain of CD3ε. In one embodiment, the primary signaling domain comprises a modified ITAM domain, eg, a mutant ITAM domain that has altered (eg, increased or decreased) activity compared to the native ITAM domain. In one embodiment, the primary signaling domain comprises a primary intracellular signaling domain comprising a modified ITAM, eg, a primary intracellular signaling domain comprising an optimized and/or truncated ITAM. In one embodiment, the primary signaling domain comprises 1, 2, 3, 4 or more ITAM motifs.

[0236] The intracellular signaling domain of TFP may comprise a CD3 signaling domain, e.g., CD3ε, CD3δ, CD3γ, or CD3ζ alone, or any other desired cellular signaling domain useful in the context of the TFPs of the present disclosure. It may be combined with an internal signaling domain(s). For example, the intracellular signaling domain of TFP can include a CD3 epsilon chain portion and a co-stimulatory signaling domain. A co-stimulatory signaling domain refers to the portion of TFP that contains the intracellular domain of the co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for the efficient response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, Examples include ligands that specifically bind to B7-H3 and CD83. For example, CD27 co-stimulation has been demonstrated to enhance human TFP-T cell proliferation, effector function, and survival in vitro, and to increase human T cell persistence and anti-tumor activity in vivo. (Song et al. Blood. 2012; 119(3):696-706).

[0237] The intracellular signaling sequences within the cytoplasmic portion of the TFPs of the present disclosure can be linked together in random or directed order. Intracellular signaling sequences are optionally linked by short oligo- or polypeptide linkers, eg, 2-10 amino acids in length (eg, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids). A bond between may be formed.

[0238] In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid such as alanine, glycine can be used as a suitable linker.

[0239] In one aspect, the TFP-expressing cells described herein are exposed to a second TFP, e.g., a different antigen binding domain, e.g. can further include a second TFP comprising In one embodiment, if the TFP-expressing cell comprises two or more different TFPs, the antigen binding domains of the different TFPs may be such that each antigen binding domain does not interact with each other. For example, cells expressing the first and second TFPs can have the antigen binding domain of the first TFP, e.g., as a fragment that does not form an association with the antigen binding domain of the second TFP, e.g., scFv, For example, the antigen binding domain of the second TFP is VHH.

[0240] In another aspect, the TFP-expressing cells described herein can further express another agent, eg, an agent that enhances the activity of engineered T cells. For example, in one embodiment, the agent can be an agent that inhibits inhibitory molecules. Suppressive molecules, such as PD1, can, in some embodiments, reduce the ability of engineered T cells to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFRβ. In one embodiment, an agent that inhibits an inhibitory molecule is a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that supplies a positive signal to a cell, e.g. Contains molecules. In one embodiment, the agent is a first polypeptide, inhibitory molecules such as PD1, LAG3, CTLA4, CD160, BTLA, LAIR1, TIM3, 2B4, and TIGIT, or fragments of any thereof (e.g., at least a portion of the extracellular domain of any of these) and an intracellular signaling domain described herein (e.g., a costimulatory domain (e.g., 4-1BB, CD27, or CD28) and/or a second polypeptide that is a primary signaling domain (eg, a CD3zeta signaling domain as described herein). In one embodiment, the agent is a PD1 first polypeptide or fragment thereof (e.g., at least a portion of the extracellular domain of PD1) and an intracellular signaling domain described herein (e.g., and a second polypeptide that is a CD28 signaling domain as described herein and/or a CD3ζ signaling domain as described herein). PD1 is an inhibitory member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, and BTLA. PD-1 is expressed on activated B cells, T cells, and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). PD-L1 and PD-L2, two ligands for PD1, have been shown to downregulate T cell activation upon binding to PD1 (Freeman et al. 2000 J Exp Med 192:1027- 34, Latchman et al. 2001 Nat Immunol 2:261-8, Carter et al. 2002 Eur J Immunol 32:634-43). PD-L1 is abundant in human cancers (Dong et al. 2003 J Mol Med 81:281-7, Blank et al. 2005 Cancer Immunol. Immunother 54:307-314, Konishi et al. 2004 Clin Cancer Res. 10:5094). Immunosuppression can be reversed by inhibiting the local interaction between PD1 and PD-L1.

[0241] In one embodiment, the agent comprises an extracellular domain (ECD) of an inhibitory molecule, e.g., programmed death 1 (PD1) fused with a transmembrane domain and optionally an intracellular signaling domain such as 41BB and CD3zeta. (also referred to herein as PD1 TFP). In one embodiment, PD1 TFPs improve T cell persistence when used in combination with the anti-TAA TFPs described herein. In one embodiment, the TFP is a PD1 TFP comprising the extracellular domain of PD1. Alternatively, a TFP is provided comprising an antibody or antibody fragment such as an scFv that specifically binds programmed death ligand 1 (PD-L1) or programmed death ligand 2 (PD-L2).

[0242] In another aspect, the disclosure provides a population of TFP-expressing T cells, eg, TFP-T cells. In some embodiments, the population of TFP-expressing T cells comprises a mixture of cells expressing different TFPs. For example, in one embodiment, the population of TFP-T cells is expressed by a first cell expressing TFP with a binding domain described herein and a different anti-TAA binding domain, such as a first cell and a second cell expressing TFP having a binding domain described herein that is different from the binding domain within TFP. As another example, the population of TFP-expressing cells includes a first cell expressing TFP comprising a first binding domain, e.g., as described herein, and a target other than the binding domain of the first cell ( and a second cell that expresses a TFP that includes an antigen binding domain (eg, another tumor-associated antigen).

[0243] In another aspect, the present disclosure provides a method for combining a population of cells, wherein at least one cell in the population expresses a TFP having a domain described herein, with another agent, e.g., the activity of a modified T cell. and a second cell expressing a potentiating agent. For example, in one embodiment, the agent can be an agent that inhibits inhibitory molecules. Suppressive molecules, such as, in some embodiments, can reduce the ability of engineered T cells to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFRβ. In one embodiment, an agent that inhibits an inhibitory molecule is a first polypeptide, e.g., an inhibitory molecule, a second polypeptide that provides a positive signal to a cell, e.g., an intracellular signaling domain described herein. including in association with

[0244] Disclosed herein are methods for making in vitro transcribed RNA encoding TFP. The disclosure also includes RNA constructs encoding TFP that can be directly transfected into cells. The method of producing mRNA used for transfection involves in vitro transcription (IVT) of a template with specially designed primers, followed by the addition of poly A, 3′ and 5′ untranslated sequences (“UTR”), Constructs can be produced that include a 5′ cap and/or an internal ribosome entry site (IRES), the nucleic acid to be expressed, and a poly A tail, typically 50-2000 bases in length. RNA so produced can efficiently transfect different types of cells. In one aspect, the template comprises a sequence for TFP.

[0245] In one aspect, the anti-TAA TFP is encoded by a messenger RNA (mRNA). In one aspect, mRNA encoding anti-TAA TFP is introduced into T cells to produce TFP-T cells. In one embodiment, the in vitro transcribed RNA
TFP can be introduced into cells in the form of transient transfection. RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. DNA of interest from any source can be converted directly into a template by PCR for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The DNA source can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequences, or any other suitable DNA source. A preferred template for in vitro transcription is the TFP of the present disclosure. In one embodiment, the DNA used for PCR contains an open reading frame. The DNA may be derived from naturally occurring DNA sequences from the genome of the organism. In one embodiment, a nucleic acid may include part or all of the 5' and/or 3' untranslated regions (UTRs). Nucleic acids may contain exons and introns. In one embodiment, the DNA used for PCR is a human nucleic acid sequence. In another embodiment, the DNA used for PCR is a human nucleic acid sequence containing 5' and 3' UTRs. Alternatively, the DNA may be an artificial DNA sequence not normally expressed in naturally occurring organisms. An exemplary artificial DNA sequence is one comprising portions of a gene ligated together to form an open reading frame encoding a fusion protein. The pieces of DNA that are ligated together can be from a single organism or multiple organisms.

[0246] PCR is used to generate a template for in vitro transcription of the mRNA used for transfection. Methods for performing PCR are well known in the art. Primers used for PCR are designed to have regions that are substantially complementary to regions of the DNA used as templates for PCR. As used herein, "substantially complementary" means that most or all of the bases in the primer sequences are complementary, or one or more bases are non-complementary or mismatched. A sequence of nucleotides. Substantially complementary sequences can anneal or hybridize to the intended DNA target under the annealing conditions used for PCR. A primer can be designed to be complementary to substantially any portion of the DNA template. For example, primers can be designed to amplify a portion of the nucleic acid that is normally transcribed in the cell (open reading frame), including the 5' and 3' UTRs. Primers can also be designed to amplify a portion of the nucleic acid encoding a particular domain of interest. In one embodiment, primers are designed to amplify the coding region of a human cDNA, including all or part of the 5' and 3' UTRs. Primers useful for PCR can be made by synthetic methods well known in the art. A "forward primer" is a primer that contains a region of nucleotides that are substantially complementary to nucleotides on the DNA template upstream of the DNA sequence to be amplified. As used herein, "upstream" refers to the 5' position of the DNA sequence to be amplified, relative to the coding strand. A "reverse primer" is a primer that contains a region of nucleotides substantially complementary to a double-stranded DNA template downstream of the DNA sequence to be amplified. As used herein, "downstream" refers to the 3' position of the amplified DNA sequence relative to the coding strand.

[0247] Any DNA polymerase useful for PCR can be used in the methods disclosed herein. Reagents and polymerases are commercially available from several sources.

[0248] Chemical structures with the ability to enhance stability and/or translation efficiency can also be used. Preferably the RNA has a 5' and a 3' UTR. In one embodiment, the 5'UTR is 1-3000 nucleotides in length. The length of the 5' and 3' UTR sequences added to the coding region can be varied by various methods, including designing primers for PCR that anneal to separate regions of the UTR, It is not limited to this. Using this approach, one skilled in the art can alter the length of the 5' and 3' UTRs required to achieve optimal translational efficiency after transfection of transcribed RNA.

[0249] The 5' and 3' UTRs can be the naturally occurring endogenous 5' and 3' UTRs of the nucleic acid of interest. Alternatively, UTR sequences not inherent in the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by some other modification of the template. The use of UTR sequences that are not native to the nucleic acid of interest can be useful in altering RNA stability and/or translational efficiency. For example, it is known that AU-rich elements in 3'UTR sequences can reduce mRNA stability. Thus, the 3'UTR can be selected or designed to enhance the stability of the transcribed RNA based on UTR properties that are well known in the art.

[0250] In one embodiment, the 5'UTR may comprise a Kozak sequence of the endogenous nucleic acid. Alternatively, if a 5'UTR not endogenous to the nucleic acid of interest is added by PCR as described above, the addition of the 5'UTR sequence can redesign the consensus Kozak sequence. Kozak sequences can increase the translation efficiency of some RNA transcripts, but do not appear to be required for all RNAs to allow efficient translation. It is known in the art that many mRNAs require Kozak sequences. In other embodiments, the 5'UTR is that of an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogues can be used in the 3' or 5'UTR to prevent exonucleolytic degradation of mRNA.

[0251] To allow synthesis of RNA from a DNA template without the need for gene cloning, a transcriptional promoter must be attached to the DNA template upstream of the sequence to be transcribed. A sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer so that the RNA polymerase promoter is incorporated into the PCR product upstream of the open reading frame to be transcribed. In one preferred embodiment, the promoter is the T7 polymerase promoter as described elsewhere herein. Other useful promoters include, but are not limited to, the T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3, and SP6 promoters are known in the art.

[0252] In some embodiments, the mRNA has both a 5' terminal cap and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability in cells. . On circular DNA templates, such as plasmid DNA, RNA polymerase produces long concatemer products that are not suitable for expression in eukaryotic cells. Transcription of linear plasmid DNA at the end of the 3'UTR yields normal-sized mRNA, which is not efficient for eukaryotic transfection, even though it is post-transcriptionally polyadenylated.

[0253] In a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the final base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 ( 1985), Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0254] A conventional method for incorporating poly A/T stretches into a DNA template is molecular cloning. However, polyA/T sequences incorporated into plasmid DNA can cause plasmid instability, which is why deletions and other abnormalities are frequently contaminating plasmid DNA templates obtained from bacterial cells. This is the reason why. As such, cloning procedures are not only laborious and time-consuming, but they are often unreliable. For these reasons, methods that allow the construction of DNA templates with poly A/T 3' stretches without cloning are highly desirable.

[0255] The poly A/T segment of the transcribed DNA template is obtained by using a reverse primer that contains a poly T tail, such as a 100T tail (which can be 50-5000T in size) during PCR, or after PCR. It can be produced by any other method including, but not limited to, ligation or in vitro recombination. Poly(A) tails also provide stability to RNA and reduce its degradation. In general, poly(A) tail length is positively correlated with the stability of transcribed RNA. In one embodiment, the poly(A) tail is 100-5000 adenosines.

[0256] The poly(A) tails of RNA are fused to poly(A) polymerases such as E. coli after in vitro transcription. Further extension can be achieved, such as by use of E. coli poly A polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides increases RNA translation efficiency by about 2-fold. Additionally, different chemical groups can be attached to the 3' end to enhance mRNA stability. Such binding moieties may include modified/artificial nucleotides, aptamers, and other compounds. For example, ATP analogs can be incorporated into poly(A) tails using poly(A) polymerase. ATP analogs can further enhance RNA stability.

[0257] The 5' cap also confers stability to the RNA molecule. In some embodiments, the RNA produced by the methods disclosed herein includes a 5' cap. The 5′ cap is attached using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001) , Stepinski, et al., RNA, 7:1468-95 (2001), Elango, et al., Biochim.Biophys.Res.Commun., 330:958-966 (2005)).

[0258] The RNA produced by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA to facilitate translation initiation. Any solute suitable for cell electroporation may be included, which may contain factors that promote cell permeability and viability, such as sugars, peptides, lipids, proteins, antioxidants, detergents, and the like.

[0259] RNA can be isolated by any of several different methods, e.g., electroporation (Amaxa Nucleofector®-II (Amaxa Biosystems, Cologne, Germany)), ECM 830 (BTX) (Harvard Instruments, Boston , Mass.), or Gene Pulser® II (BioRad, Denver, Colo.), Multiporator® (Eppendort, Hamburg Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide Commercially available methods including, but not limited to, biolistic particle delivery systems such as mediated transfection or "gene guns" can be used to introduce target cells (e.g., Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (see 2001).

Recombinant Nucleic Acids Encoding TFP and TCR Constant Domains
[0260] In some embodiments, disclosed herein is a recombinant nucleic acid molecule comprising a sequence encoding a T-cell receptor (TCR) fusion protein (TFP). A TFP may comprise a TCR subunit that includes at least a portion of the TCR extracellular domain. A TCR subunit may further comprise a transmembrane domain. A TCR subunit may further comprise an intracellular domain, including a stimulatory domain from the intracellular domain of TCRγ, TCRδ, TCRα, or TCRβ, or the intracellular signaling domain of CD3ε, CD3γ, CD3δ. TFPs can further include antibodies (eg, human, humanized, or murine antibodies) that contain antigen-binding domains. The recombinant nucleic acid molecule can further comprise a sequence encoding a TCR constant domain, the TCR constant domains being TCRα constant domain, TCRβ constant domain, TCRα and TCRβ constant domains, TCRγ constant domain, TCRδ constant domain, or the TCRγ constant domain and the TCRδ constant domain. A TCR subunit and an antibody can be operably linked. TFP can be functionally incorporated into the TCR complex (eg, the endogenous TCR complex) when expressed in T cells.

[0261] The constant domain may comprise that of a TCRα chain, a TCRβ chain, a TCRγ chain, or a TCRδ chain. The constant domain may comprise the full-length constant domain of a TCRα, TCRβ, TCRγ, or TCRδ chain. Constant domains may comprise fragments (eg, functional fragments) of the full-length constant domain of the TCRα, TCRβ, TCRγ, or TCRδ chains. For example, the constant domain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 constant domains of a TCRα chain, a TCRβ chain, a TCRγ chain, or a TCRδ chain. , 70, 75, 80, 85, 90, 95, 100, 150, or more amino acid residues. Sequences encoding TCR constant domains may further encode transmembrane domains and/or intracellular regions of TCRα, TCRβ, TCRγ, or TCRδ chains. A sequence encoding a TCR constant domain can encode a full-length constant region of a TCRα, TCRβ, TCRγ, or TCRδ chain. A constant region of a TCR chain may comprise a constant domain, a transmembrane domain, and an intracellular region. The constant region of a TCR chain can also exclude the transmembrane domain and intracellular region of the TCRα, TCRβ, TCRγ, or TCRδ chains.

[0262] The TCRα, TCRβ, TCRγ, or TCRδ chains described herein can be derived from a variety of species. The TCR chain can be a murine or human TCR chain. For example, the constant domain may comprise that of a murine or human TCRα, TCRβ, TCRγ, or TCRδ chain.

[0263] The constant domains may comprise truncations, additions or substitutions of the sequences of the constant domains described herein. For example, the constant domains are: at least about 5, 10, 15, 20, 25, 30, 35, 40, 45 of SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 243 or SEQ ID NO: 265; It may comprise truncated constant domains as described herein having 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues. For example, the constant domains are: at least about 5, 10, 15, 20, 25, 30, 35, 40, 45 of SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 243 or SEQ ID NO: 265; Sequences with 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, or more additional amino acid residues may be included. For example, the constant domains are: at least about 5, 10, 15, 20, 25, 30, 35, 40, 45 of SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 243 or SEQ ID NO: 265; Sequences with 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, or more amino acid substitutions may be included. The constant domains are SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 243, or SEQ ID NO: 265, or fragments thereof. The constant domains are SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 243, or SEQ ID NO: 265 , or more modifications, mutations, or deletions. The constant domains are SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 243, or SEQ ID NO: 265 up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 modification, mutation, or deletion. The constant domains are SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, SEQ ID NO: 148, at least about 40%, 45%, 50%, 55%, 60%, relative to the sequence of SEQ ID NO: 149, SEQ ID NO: 152, SEQ ID NO: 155, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 243, or SEQ ID NO: 265; Sequences having 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity may be included.

[0264] A mouse TCRα constant domain may comprise positions 2-137 of SEQ ID NO:146. Mouse TCRα constant domains may comprise truncations, additions or substitutions of the constant domain sequences described herein. For example, the constant domains are at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 2-137 of SEQ ID NO: 146, It may comprise truncated constant domains as described herein having 85, 90, 95, 100, 150 or more amino acid residues. For example, the constant domains are at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 2-137 of SEQ ID NO: 146, Sequences with 85, 90, 95, 100, 150, or more additional amino acid residues may be included. For example, the constant domains are at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 2-137 of SEQ ID NO: 146, Sequences with 85, 90, 95, 100, 150, or more amino acid substitutions may be included. The constant domain may comprise the sequence of positions 2-137 of SEQ ID NO:146 or a fragment thereof. The constant domain comprises at least 1, 2, 3, 4, 5, 6, 7, 8,
It may contain 9, 10 or more modifications, mutations or deletions. The constant domains are up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 of the sequence from positions 2 to 137 of SEQ ID NO: 146 , 3, 2, or 1 modification, mutation, or deletion. The constant domain is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, Sequences having 90%, 95%, 99% or 100% sequence identity may be included.

[0265] A mouse TCRβ constant domain may comprise positions 2-173 of SEQ ID NO:152. Mouse TCRβ constant domains may comprise truncations, additions or substitutions of the constant domain sequences described herein. For example, the constant domains are at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 2-173 of SEQ ID NO: 152, It may comprise truncated constant domains as described herein having 85, 90, 95, 100, 150 or more amino acid residues. For example, the constant domains are at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 2-173 of SEQ ID NO: 152, Sequences with 85, 90, 95, 100, 150, or more additional amino acid residues may be included. For example, the constant domains are at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 2-173 of SEQ ID NO: 152, Sequences with 85, 90, 95, 100, 150, or more amino acid substitutions may be included. The constant domain may comprise the sequence of positions 2-173 of SEQ ID NO:152 or a fragment thereof. The constant domain has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modifications, mutations, or deletions in the sequence from positions 2 to 173 of SEQ ID NO: 152. can contain. The constant domains are up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, It may contain 4, 3, 2, or 1 modifications, mutations, or deletions. The constant domain is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, Sequences having 90%, 95%, 99% or 100% sequence identity may be included.

[0266] Optionally, the TCR constant domain is a TCRdelta constant domain. The TCRdelta constant domain may comprise SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243, or SEQ ID NO:265, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications. In some embodiments, the TCRdelta constant domain may comprise SEQ ID NO:243. TCRδ constant domains may comprise truncations, additions or substitutions of the constant domain sequences described herein. For example, the constant domain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 of SEQ ID NO:243. , 100, 150, or more amino acid residues, as described herein. For example, the constant domain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 of SEQ ID NO:243. , 100, 150, or more additional amino acid residues. For example, the constant domain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 of SEQ ID NO:243. , 100, 150, or more amino acid substitutions. The constant domain may comprise the sequence of SEQ ID NO:243 or a fragment thereof. The constant domain may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:243. The constant domains are up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 of the sequence of SEQ ID NO:243 , or may contain a single modification, mutation, or deletion. The constant domain is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% relative to the sequence of SEQ ID NO:243 , 99% or 100% sequence identity.

[0267] The TCRdelta constant domain may comprise SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243, or SEQ ID NO:265, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications. Optionally, the sequence encoding the TCRdelta constant domain further encodes a TCRdelta variable domain, thereby encoding the entire TCRdelta domain. All TCR delta domains can be delta 2 or delta 1. The entire TCRδ constant domain may comprise SEQ ID NO:256, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications.

[0268] All TCRdelta domains may comprise truncations, additions or substitutions of the sequences of the constant domains described herein. For example, the delta domain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 of SEQ ID NO:256. , 100, 150, or more amino acid residues, as described herein. For example, the delta domain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 of SEQ ID NO:256. , 100, 150, or more additional amino acid residues. For example, the delta domain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 of SEQ ID NO:256. , 100, 150, or more amino acid substitutions. The delta domain may comprise the sequence of SEQ ID NO:256 or a fragment thereof. The delta domain can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modifications, mutations, or deletions of the sequence of SEQ ID NO:256. The delta domain is up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 of the sequence of SEQ ID NO:256 , or may contain a single modification, mutation, or deletion. The delta domain is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% relative to the sequence of SEQ ID NO:256 , 99% or 100% sequence identity.

[0269] The TCRγ constant domain may comprise SEQ ID NO:21. TCRγ constant domains may comprise truncations, additions or substitutions of the constant domain sequences described herein. For example, the constant domain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 of SEQ ID NO:21 , 100, 150, or more amino acid residues, as described herein. For example, the constant domain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 of SEQ ID NO:21 , 100, 150, or more additional amino acid residues. For example, the constant domain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 of SEQ ID NO:21 , 100, 150, or more amino acid substitutions. The constant domain may comprise the sequence of SEQ ID NO:21 or a fragment thereof. The constant domain may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modifications, mutations or deletions of the sequence of SEQ ID NO:21. The constant domains are up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 of the sequence of SEQ ID NO:21 , or may contain a single modification, mutation, or deletion. The constant domain is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% relative to the sequence of SEQ ID NO:243 , 99% or 100% sequence identity.

[0270] The TCRγ constant domain can comprise SEQ ID NO:21 or SEQ ID NO:155, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications. Optionally, the sequence encoding the TCRγ constant domain further encodes a TCRγ variable domain, thereby encoding the entire TCRγ domain. The entire TCRγ domain can be γ9 or γ4. An entire TCRγ domain can comprise SEQ ID NO:255, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications.

[0271] All TCRγ domains have the sequence truncations, additions, or additions of the constant domain sequences described herein.
or may contain substitutions. For example, the gamma domain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 of SEQ ID NO:255. , 100, 150, or more amino acid residues, as described herein. For example, the gamma domain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 of SEQ ID NO:255. , 100, 150, or more additional amino acid residues. For example, the gamma domain is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 of SEQ ID NO:255. , 100, 150, or more amino acid substitutions. A gamma domain may comprise the sequence of SEQ ID NO:255 or a fragment thereof. A gamma domain may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more modifications, mutations, or gammas of the sequence of SEQ ID NO:255. The gamma domain is up to 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 of the sequence of SEQ ID NO:255 , or may contain a single modification, mutation, or deletion. The gamma domain is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% relative to the sequence of SEQ ID NO:255 , 99% or 100% sequence identity.

[0272] TCR beta chain (Homo sapiens):
ＶＥＤＬＮＫＶＦＰＰＥＶＡＶＦＥＰＳＥＡＥＩＳＨＴＱＫＡＴＬＶＣＬＡＴＧＦＦＰＤＨＶＥＬＳＷＷＶＮＧＫＥＶＨＳＧＶＳＴＤＰＱＰＬＫＥＱＰＡＬＮＤＳＲＹＣＬＳＳＲＬＲＶＳＡＴＦＷＱＮＰＲＮＨＦＲＣＱＶＱＦＹＧＬＳＥＮＤＥＷＴＱＤＲＡＫＰＶＴＱＩＶＳＡＥＡＷＧＲＡＤＣＧＦＴＳＶＳＹＱＱＧＶＬＳＡＴＩＬＹＥＩＬＬＧＫＡＴＬＹＡＶＬＶＳＡＬＶＬＭＡＭＶＫＲＫＤＦ（配列番号１６）。

[0273] The canonical sequence of the constant region of the mouse TCR beta chain is as follows:
ＥＤＬＲＮＶＴＰＰＫＶＳＬＦＥＰＳＫＡＥＩＡＮＫＱＫＡＴＬＶＣＬＡＲＧＦＦＰＤＨＶＥＬＳＷＷＶＮＧＫＥＶＨＳＧＶＳＴＤＰＱＡＹＫＥＳＮＹＳＹＣＬＳＳＲＬＲＶＳＡＴＦＷＨＮＰＲＮＨＦＲＣＱＶＱＦＨＧＬＳＥＥＤＫＷＰＥＧＳＰＫＰＶＴＱＮＩＳＡＥＡＷＧＲＡＤＣＧＩＴＳＡＳＹＱＱＧＶＬＳＡＴＩＬＹＥＩＬＬＧＫＡＴＬＹＡＶＬＶＳＴＬＶＶＭＡＭＶＫＲＫＮＳ（配列番号１５２）。

[0274] TCRα constant region (mouse) (or [mm]TRAC(82-137)):
ATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 17).

[0275] The canonical sequence of the constant (mTRAC) region of the mouse TCRα chain is as follows:
XIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 146).

[0276] TCRβ constant region (mouse) (or [mm] TRBC1(123-173)): GRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO: 18).

[0277] The canonical sequence of the constant region of the mouse TCR beta chain is as follows:
ＥＤＬＲＮＶＴＰＰＫＶＳＬＦＥＰＳＫＡＥＩＡＮＫＱＫＡＴＬＶＣＬＡＲＧＦＦＰＤＨＶＥＬＳＷＷＶＮＧＫＥＶＨＳＧＶＳＴＤＰＱＡＹＫＥＳＮＹＳＹＣＬＳＳＲＬＲＶＳＡＴＦＷＨＮＰＲＮＨＦＲＣＱＶＱＦＨＧＬＳＥＥＤＫＷＰＥＧＳＰＫＰＶＴＱＮＩＳＡＥＡＷＧＲＡＤＣＧＩＴＳＡＳＹＱＱＧＶＬＳＡＴＩＬＹＥＩＬＬＧＫＡＴＬＹＡＶＬＶＳＴＬＶＶＭＡＭＶＫＲＫＮＳ（配列番号１５２）。

[0278] TCR beta chain (Homo sapiens):
PVDSGVTQTPKHLITATGQRVTLRCSPRSGDLSVSWYQQS
ＬＤＱＧＬＱＦＬＩＱＹＹＮＧＥＥＲＡＫＧＮＩＬＥＲＦＳＡＱＱＦＰＤＬＨＳＥＬＮＬＳＳＬＥＬＧＤＳＡＬＹＦＣＡＳＳＰＲＴＧＬＮＴＥＡＦＦＧＱＧＴＲＬＴＶＶＥＤＬＮＫＶＦＰＰＥＶＡＶＦＥＰＳＥＡＥＩＳＨＴＱＫＡＴＬＶＣＬＡＴＧＦＦＰＤＨＶＥＬＳＷＷＶＮＧＫＥＶＨＳＧＶＳＴＤＰＱＰＬＫＥＱＰＡＬＮＤＳＲＹＣＬＳＳＲＬＲＶＳＡＴＦＷＱＮＰＲＮＨＦＲＣＱＶＱＦＹＧＬＳＥＮＤＥＷＴＱＤＲＡＫＰＶＴＱＩＶＳＡＥＡＷＧＲＡＤＣＧＦＴＳＶＳＹＱＱＧＶＬＳＡＴＩＬＹＥＩＬＬＧＫＡＴＬＹＡＶＬＶＳＡＬＶＬＭＡＭＶＫＲＫＤＦ（配列番号１９）。

[0279] TCRdelta constant region version 1 (Homo sapiens):
SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKL0 (SEQ ID NO: 2).

[0280] ＴＣＲγ定常領域（ホモサピエンス）（又は［ｈｓ］ＴＲＧＣ（１－１７３））：ＤＫＱＬＤＡＤＶＳＰＫＰＴＩＦＬＰＳＩＡＥＴＫＬＱＫＡＧＴＹＬＣＬＬＥＫＦＦＰＤＶＩＫＩＨＷＱＥＫＫＳＮＴＩＬＧＳＱＥＧＮＴＭＫＴＮＤＴＹＭＫＦＳＷＬＴＶＰＥＫＳＬＤＫＥＨＲＣＩＶＲＨＥＮＮＫＮＧＶＤＱＥＩＩＦＰＰＩＫＴＤＶＩＴＭＤＰＫＤＮＣＳＫＤＡＮＤＴＬＬＬＱＬＴＮＴＳＡＹＹＭＹＬＬＬＬＬＫＳＶＶＹＦＡＩＩＴＣＣＬＬＲＲＴＡＦＣＣＮＧＥＫＳ（配列番号２１）。

[0281] TCRdelta constant region version 2 (Homo sapiens):
SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAV (SEQ ID NO:NFLL2TAK).

[0282] Optionally, the TCR constant domain is a TCRdelta constant domain. A sequence encoding a TCRδ constant domain may further encode a second antigen or ligand binding domain operably linked to the sequence encoding the TCRδ constant domain. The second antigen-binding domain or ligand-binding domain can be the same as or different from the antigen-binding domain or ligand-binding domain of TFP.

[0283] Optionally, the TCR constant domain is a TCRγ constant domain. A sequence encoding a TCRγ constant domain may further encode a second antigen or ligand binding domain operably linked to the sequence encoding the TCRγ constant domain. The second antigen-binding domain or ligand-binding domain can be the same as or different from the antigen-binding domain or ligand-binding domain of TFP.

[0284] Optionally, the recombinant nucleic acid comprises sequences encoding a TCRγ constant domain and a TCRδ constant domain. The TCRγ constant domain may comprise SEQ ID NO: 21 or SEQ ID NO: 155, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications. A sequence encoding a TCRγ constant domain may additionally encode a TCRγ variable domain, thus encoding the entire TCRγ domain. The TCRγ domain can be γ9 or γ4. All TCRγ domains include SEQ ID NO:255, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications. The TCRdelta constant domain may comprise SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243, or SEQ ID NO:265, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications. A sequence encoding a TCRdelta constant domain may additionally encode a TCRdelta variable domain, thus encoding the entire TCRdelta domain. The TCR delta domain can be delta 2 or delta 1. A full TCRδ domain can comprise SEQ ID NO:256, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications.

[0285] In some cases, the TCR constant domain is incorporated into a functional TCR complex when expressed in a T cell. In some cases, the TCR constant domain is incorporated into the same functional TCR complex that incorporates TFP when expressed in T cells. In some instances, the TFP-encoding sequence and the TCR constant domain-encoding sequence are contained within the same nucleic acid molecule. Optionally, the TFP-encoding sequence and the TCR constant domain-encoding sequence are contained within different nucleic acid molecules. This sequence may further encode a cleavage site (eg, a protease cleavage site) between the encoded TFP and the TCR constant domain. A cleavage site can be a protease cleavage site. Cleavage sites can be self-cleaving peptides such as the T2A, P2A, E2A, or F2A cleavage sites. A cleavage site may comprise the sequence of SEQ ID NO:23.

[0286] T2A cleavage site: EGRGSLLTCGDVEENPGP (SEQ ID NO:23).
[0287] The TCR subunit and constant domain of TFP may comprise sequences from the same TCR chain or from different TCR chains. In some cases, the TCR subunit and constant domain of TFP are derived from different TCR chains. For example, a TCR subunit includes (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, wherein the TCR extracellular domain, transmembrane domain, and intracellular domain is derived from the TCRα chain and the constant domain may comprise that of the TCRβ chain. In another example, a TCR subunit comprises (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, wherein the TCR extracellular domain, transmembrane domain, and The intracellular domain is derived from the TCRβ chain and the constant domain may comprise that of the TCRα chain. In another example, a TCR subunit comprises (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, wherein the TCR extracellular domain, transmembrane domain, and The intracellular domain is derived from the TCRγ chain and the constant domain may comprise that of the TCRδ chain. In yet another example, a TCR subunit comprises (1) at least a portion of a TCR extracellular domain, (2) a transmembrane domain, and (3) an intracellular domain, wherein the TCR extracellular domain, transmembrane domain, and the intracellular domain is derived from the TCR delta chain, and the constant domain may comprise that of the TCR gamma chain.

[0288] Optionally, the TCR subunit and the antibody domain, antigen domain, or binding ligand, or fragment thereof, are operably linked by a linker sequence. Optionally, the linker sequence comprises (G ₄ S) _n , where n=1-4.

[0289] Optionally, the transmembrane domain is a TCR transmembrane domain from CD3ε, CD3γ, CD3δ, TCRγ, TCRδ, TCRα, or TCRβ. Optionally, the intracellular domain is derived from CD3ε only, CD3γ only, CD3δ only, TCRγ only, TCRδ only, TCRα only, or TCRβ only.

[0290] Optionally, the TCR subunit comprises (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain, (i), (ii) , and (iii) are derived from the same TCR subunit.

[0291] Optionally, the TCR extracellular domain comprises a TCRα chain, a TCRβ chain, a TCRγ chain, a TCRδ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, functional fragments thereof, and at least one but no more than 20 the extracellular domain of a protein or a portion thereof selected from the group consisting of amino acid sequences thereof having modifications of

[0292] Optionally, the TCR subunit is a TCRα chain, a TCRβ chain, a TCRζ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28 , CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. Contains a transmembrane domain.

[0293] Optionally, the TCR subunit comprises the TCR intracellular domain of the TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, or fragment thereof. Optionally, the TCR subunit comprises a stimulatory domain of a protein selected from the intracellular signaling domains of CD3ε, CD3γ, or CD3δ, or an intracellular domain comprising an amino acid sequence having at least one modification thereto. .

[0294] In some cases, a TCR subunit can comprise (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain of a TCR gamma or TCR delta chain. The TCR extracellular domain may comprise the extracellular portion of the TCRγ or TCRδ chain constant domain, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. In some embodiments, the TCR subunit comprising (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain is a delta constant domain or fragment thereof, e.g. , is or comprises a delta constant domain as described herein. A delta constant domain can have the sequence of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:243, or SEQ ID NO:265, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications. In some embodiments, the TCR subunit comprising (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain is a γ constant domain, e.g., herein is or comprises a gamma constant domain as described in the literature. The gamma constant domain can have the sequence of SEQ ID NO:21 or SEQ ID NO:155, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications. The extracellular domain of TFP may be free of the γ- or δ-chain variable domains.

[0295] The TCR subunit of TFP can include the extracellular, transmembrane, and intracellular domains of CD3ε, CD3γ, or CD3δ. In some embodiments, the recombinant nucleic acid comprises a TFP that includes the extracellular, transmembrane, and intracellular domains of CD3ε, CD3γ, or CD3δ and the constant domains of TCRβ and TCRα. In some embodiments, the recombinant nucleic acid comprises a TFP that includes the extracellular, transmembrane, and intracellular domains of CD3ε and the constant domains of TCRγ and TCRδ. In some embodiments, the recombinant nucleic acid comprises extracellular, transmembrane, and intracellular domains of CD3ε and TFPs, including full-length TCFγ and full-length TCRδ. In some embodiments, the TCR subunit of TFP comprises CD3ε. The TCR subunit of CD3ε can comprise the sequence of SEQ ID NO:258, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications.

Optionally, the TCR subunit comprising at least part of the extracellular domain of mouse TCRα or mouse TCRβ and the transmembrane domain of mouse TCRα or mouse TCRβ is or comprises a TCRα constant domain or a TCRβ constant domain. A TCR subunit may comprise the intracellular domain of mouse TCRα or mouse TCRβ. The TCR constant domain may be a TCRα constant domain, such as the TCRα constant domain described herein. The TCRα constant domain can comprise SEQ ID NO: 17, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO: 207, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications. A sequence encoding a TCRα constant domain may further encode a second antigen or ligand binding domain operably linked to the sequence encoding the TCRα constant domain. The second antigen-binding domain or ligand-binding domain can be the same as or different from the antigen-binding domain or ligand-binding domain of TFP. The TCRα constant domain may comprise a mouse TCRα constant domain. The mouse TCRα constant domain may comprise amino acids 2-137 of the mouse TCRα constant domain. A mouse TCRα constant domain may comprise amino acids 2-137 of SEQ ID NO:146. A mouse TCRα constant domain may comprise the sequence of SEQ ID NO:207. A mouse TCRα constant domain may comprise amino acids 82-137 of SEQ ID NO:146. Mouse TCRα constant domain comprises the sequence of SEQ ID NO:17. The TCR constant domain may be a TCRβ constant domain, such as the TCRβ constant domain described herein. The TCRβ constant domain can comprise SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO: 209, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications. A sequence encoding a TCRβ constant domain may further encode a second antigen or ligand binding domain operably linked to the sequence encoding the TCRβ constant domain. The second antigen-binding domain or ligand-binding domain can be the same as or different from the antigen-binding domain or ligand-binding domain of TFP. The TCRβ constant domain may comprise a murine TCRβ constant domain. The mouse TCRβ constant domain may comprise amino acids 2-173 of the mouse TCRβ constant domain. A mouse TCRβ constant domain may comprise amino acids 2-173 of SEQ ID NO:152. A mouse TCRβ constant domain may comprise SEQ ID NO:209. A TCRβ constant domain may comprise amino acids 123-173 of SEQ ID NO:152. A TCRβ constant domain may comprise SEQ ID NO:18.

[0296] A recombinant nucleic acid may comprise sequences encoding a TCRα constant domain and a TCRβ constant domain. The TCRα constant domain can comprise SEQ ID NO: 17, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO: 207, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications. The TCRβ constant domain may comprise SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO: 209, functional fragments thereof, and amino acid sequences thereof with at least one but no more than 20 modifications. The intracellular signaling domain can be CD3ε, CD3γ, or CD3δ. The intracellular signaling domain can be CD3ε.

[0297] The sequences encoding the TCR constant domains are, from 5' to 3', a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and It may contain a TRBC gene sequence. The sequences encoding the TCR constant domains are, from 5′ to 3′, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence and a TRBC gene sequence. can include The sequences encoding the TCR constant domains are, from 5′ to 3′, a first leader sequence, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker, and a TRBC gene sequence. can include The sequences encoding the TCR constant domains are, from 5′ to 3′, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence. , linkers, and TRBC gene sequences. The sequences encoding the TCR constant domains are, in 5′-3′ orientation, a first leader sequence, a TRAC gene sequence, a first cleavable linker sequence, a second leader sequence, a TRBC gene sequence, a second cleavage. Possible linker sequences, third leader sequences, antigen binding domain sequences, linker sequences, and CD3ε gene sequences may be included.

[0298] As described herein, at least one but no more than 20 modifications to the sequences described herein are modifications of amino acids that mediate cell signaling or respond to ligand binding to TFP. may include modifications of amino acids that are phosphorylated at

[0299] Optionally, the TCR subunit comprises a stimulatory domain of a protein selected from the functional signaling domain of 4-1BB and/or the functional signaling domain of CD3zeta, or at least one modification thereto. an intracellular domain comprising an amino acid sequence having

[0300] Optionally, the recombinant nucleic acid further comprises a sequence encoding a co-stimulatory domain. Optionally, the co-stimulatory domain is OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137), and at least one but Include a functional signaling domain of a protein selected from the group consisting of amino acid sequences thereof having 20 or fewer modifications.

[0301] Optionally, the TCR subunit is CD3ζ TCR subunit, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, TCRζ chain, Fcε receptor 1 chain, Fcε receptor 2 chain, Fcγ receptor 1 chain, Fcγ Receptor 2a chain, Fcγ receptor 2b1 chain, Fcγ receptor 2b2 chain, Fcγ receptor 3a chain, Fcγ receptor 3b chain, Fcβ receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, CD79b, CD89
, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. including the ITAM of the TCR subunit. In some cases, the ITAM replaces the ITAM of CD3γ, CD3δ, or CD3ε. Optionally, the ITAM is selected from the group consisting of a CD3ζ TCR subunit, a CD3ε TCR subunit, a CD3γ TCR subunit, and a CD3δ TCR subunit; Replaces another ITAM that is selected.

[0302]
In some cases, TFP, the TCRγ constant domain, the TCRδ constant domain, and any combination thereof are capable of functionally interacting with the endogenous TCR complex and/or at least one endogenous TCR polypeptide. Optionally, (a) the TCR constant domain is a TCRγ constant domain and the TFP is functionally integrated into a TCR complex comprising endogenous subunits of TCRδ, CD3ε, CD3γ, CD3δ, or a combination thereof; (b) the TCR constant domain is a TCRδ constant domain and the TFP is functionally integrated into a TCR complex comprising endogenous subunits of TCRγ, CD3ε, CD3γ, CD3δ, or a combination thereof; or (c) The TCR constant domains are the TCRγ constant domain and the TCRδ constant domain, and TFP is functionally integrated into the TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or combinations thereof.

[0303] Optionally, the at least one but no more than 20 modifications thereto include modifications of amino acids that mediate cell signaling or modifications of amino acids that are phosphorylated in response to ligand binding to TFP.

[0304] The antibody or antigen binding domain may be an antibody fragment. Antibodies or antigen-binding domains can be murine, human, or humanized. In some cases, the human or humanized antibody is an antibody fragment. Optionally, the antibody fragment is a scFv, single domain antibody domain, VH domain, or VL domain. Optionally, the human or humanized antibody comprising the antigen binding domain is an anti-CD19 binding domain, an anti-B cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD22 binding domain, an anti-PD-1 is selected from the group consisting of a binding domain, an anti-BAFF or BAFF receptor binding domain, and an anti-ROR-1 binding domain.

[0305] The antigen binding domains described herein include anti-CD19 binding domains, anti-B cell maturation antigen (BCMA) binding domains, anti-mesothelin (MSLN) binding domains, anti-CD20 binding domains, anti-CD70 binding domains, anti-79b binding domain, anti-HER2 binding domain, anti-PMSA binding domain, anti-MUC16 binding domain, anti-CD22 binding domain, anti-PD-L1 binding domain, anti-BAFF or BAFF receptor binding domain, anti-nectin-4 binding domain, anti-TROP-2 It can be selected from the group consisting of binding domains, anti-GPC3 binding domains, and anti-ROR-1 binding domains.

[0306] Optionally, the nucleic acid is selected from the group consisting of DNA and RNA. In some cases, the nucleic acid is mRNA. Optionally, a recombinant nucleic acid comprises a nucleic acid analogue, which is not present in the coding sequence of the recombinant nucleic acid. In some cases, the nucleic acid analog is 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2 '-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O -DMAP), TO-dimethylaminoethyloxyethyl (2′-O-DMAEOE), 2′-ON-methylacetamide (2′-O-NMA) modification, locked nucleic acid (LNA), ethylene nucleic acid ( ENA), peptide nucleic acids (PNA), 1′,5′-anhydrohexitol nucleic acids (HNA), morpholinos, methylphosphonate nucleotides, thiolphosphonate nucleotides, and 2′-fluoro N3-P5′-phosphoramidites. selected from the group consisting of

[0307] Optionally, the recombinant nucleic acid further comprises a leader sequence. Optionally, the recombinant nucleic acid further comprises a promoter sequence. Optionally, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. Optionally, the recombinant nucleic acid further comprises a 3'UTR sequence. Optionally, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. Optionally, the nucleic acid is an in vitro transcribed nucleic acid.

[0308] Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRα transmembrane domain. Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRβ transmembrane domain. Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRα transmembrane domain and a sequence encoding a TCRβ transmembrane domain. Alternatively, the recombinant nucleic acid comprises a sequence encoding a TCRγ domain or a TCRδ domain, such as a transmembrane domain.

[0309] In some embodiments, disclosed herein are recombinant nucleic acids comprising sequences encoding a T-cell receptor (TCR) fusion protein (TFP). A TFP may comprise a TCR subunit comprising at least a portion of the TCR extracellular domain. A TCR subunit may further comprise a transmembrane domain. TCR subunits may further comprise intracellular domains of TCRα, TCRβ, TCRγ, TCRδ chains, or fragments thereof. Optionally, the TCR subunit comprises an intracellular domain of TCRα, TCRβ, TCRγ, or TCRδ, or an intracellular domain comprising a stimulatory domain from a CD3ε, CD3γ, or CD3δ intracellular signaling domain. A TFP may further comprise a binding ligand or fragment thereof capable of binding to an antibody or fragment thereof. A recombinant nucleic acid molecule can comprise a sequence encoding a TCR constant domain, which TCR constant domain is a TCRα constant domain, a TCRβ constant domain, a TCRα and a TCRβ constant domain, a TCRγ constant domain, a TCRδ constant domain, or The TCRγ and TCRδ constant domains, which operably link the TCR subunits and binding ligands or fragments thereof, and which, when expressed in T cells, functionally integrate TFP into the TCR complex. Optionally, the binding ligand can bind to the Fc domain of the antibody. In some cases, the binding ligand can selectively bind the IgG1 antibody. In some cases, the binding ligand can specifically bind the IgG1 antibody. Optionally, the antibody or fragment thereof binds to a cell surface antigen. Optionally, the antibody or fragment thereof binds to a cell surface antigen on the surface of tumor cells. In some cases, the binding ligand comprises a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer . Optionally, the binding ligand does not include an antibody or fragment thereof. Optionally, the binding ligand comprises a CD16 polypeptide or fragment thereof. Optionally, the binding ligand comprises a CD16 binding polypeptide. Optionally, the binding ligand is human or humanized. Optionally, the recombinant nucleic acid further comprises a nucleic acid sequence encoding an antibody or fragment thereof that can be bound by a binding ligand. In some cases, the antibody or fragment thereof can be secreted from the cell.

[0310] In some cases, the TCR constant domain is incorporated into a functional TCR complex when expressed in a T cell. In some cases, the TCR constant domain is incorporated into the same functional TCR complex that incorporates TFP when expressed in T cells. In some instances, the TFP-encoding sequence and the TCR constant domain-encoding sequence are contained within the same nucleic acid molecule. Optionally, the TFP-encoding sequence and the TCR constant domain-encoding sequence are contained within different nucleic acid molecules.

[0311] Optionally, the TCR subunit and the antibody domain, antigen domain, or binding ligand, or fragment thereof, are operably linked by a linker sequence. Optionally, the linker sequence comprises (G ₄ S) _n , where n=1-4.

[0312] Optionally, the transmembrane domain is a TCR transmembrane domain from CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRγ, or TCRδ. Optionally, the intracellular domain is derived from CD3ε only, CD3γ only, CD3δ only, TCRα only, or TCRβ only.

[0313] Optionally, the TCR subunit comprises (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain, (i), (ii) , and (iii) are from the same TCR subunit.

[0314] Optionally, the TCR extracellular domain comprises a TCRα chain, a TCRβ chain, a TCRγ chain, a TCRδ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, functional fragments thereof, and at least one but no more than 20 the extracellular domain of a protein or a portion thereof selected from the group consisting of amino acid sequences thereof having modifications of

[0315] Optionally, the TCR subunit is a TCRα chain, a TCRβ chain, a TCRζ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28 , CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. Contains a transmembrane domain.

[0316] Optionally, the TCR subunit comprises a stimulatory domain of a protein selected from the intracellular signaling domains of CD3ε, CD3γ, or CD3δ, or an amino acid sequence having at least one modification thereto. Including internal domains.

[0317] Optionally, the TCR subunit comprises a stimulatory domain of a protein selected from the functional signaling domain of 4-1BB and/or the functional signaling domain of CD3zeta, or at least one modification thereto. an intracellular domain comprising an amino acid sequence having

[0318] Optionally, the recombinant nucleic acid further comprises a sequence encoding a co-stimulatory domain. Optionally, the co-stimulatory domain is OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137), and at least one but Include a functional signaling domain of a protein selected from the group consisting of amino acid sequences thereof having 20 or fewer modifications.

[0319] Optionally, the TCR subunit is CD3ζ TCR subunit, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, TCRζ chain, Fcε receptor 1 chain, Fcε receptor 2 chain, Fcγ receptor 1 chain, Fcγ Receptor 2a chain, Fcγ receptor 2b1 chain, Fcγ receptor 2b2 chain, Fcγ receptor 3a chain, Fcγ receptor 3b chain, Fcβ receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, Immunoreceptor tyrosine activation of a protein selected from the group consisting of CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. Includes ITAMs of TCR subunits containing motifs (ITAMs) or portions thereof. In some cases, the ITAM replaces the ITAM of CD3γ, CD3δ, or CD3ε. Optionally, the ITAM is selected from the group consisting of a CD3 zeta TCR subunit, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, and a CD3 delta TCR subunit, and from the group consisting of a CD3 zeta TCR subunit, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, and a CD3 delta TCR subunit. Replaces another ITAM that is selected.

[0320] In some cases, the TFP, TCRγ constant domain, TCRδ constant domain, TCRα constant domain, TCRβ constant domain, and any combination thereof are associated with an endogenous TCR complex and/or at least one endogenous TCR polypeptide. They can interact functionally. Optionally, (a) the TCR constant domain is a TCRγ constant domain and the TFP is functionally integrated into a TCR complex comprising endogenous subunits of TCRβ, CD3ε, CD3γ, CD3δ, or a combination thereof; (b) the TCR constant domain is a TCRδ constant domain and the TFP is functionally integrated into a TCR complex comprising endogenous subunits of TCRα, CD3ε, CD3γ, CD3δ, or combinations thereof; or (c) the TCR the constant domains are a TCRγ constant domain and a TCRδ constant domain, and the TFP is functionally integrated into a TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or a combination thereof; or (d) the TCR constant domain is the TCRα constant domain and TFP is functionally integrated into the TCR complex comprising endogenous subunits of TCRβ, CD3ε, CD3γ, CD3δ, or combinations thereof; or (e) the TCR constant domain is the TCRβ constant domain domain that functionally integrates TFP into the TCR complex comprising endogenous subunits of TCRα, CD3ε, CD3γ, CD3δ, or combinations thereof.

[0321] Optionally, the above at least one but no more than 20 modifications to it are modifications of amino acids that mediate cell signaling or modifications of amino acids that are phosphorylated in response to ligand binding to TFP. including.

[0322] Optionally, the human or humanized antibody is an antibody fragment. Optionally, the antibody fragment is a scFv, single domain antibody domain (sdAb), VH domain, or VL domain. Optionally, the human or humanized antibody comprising the antigen binding domain is an anti-CD19 binding domain, an anti-B cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD22 binding domain, an anti-PD-1 selected from the group consisting of a binding domain, an anti-PD-L1 binding domain, an IL 13Rα2 binding domain, an anti-BAFF or BAFFR binding domain, and an anti-ROR-1 binding domain.

[0323] Optionally, the nucleic acid is selected from the group consisting of DNA and RNA. In some cases, the nucleic acid is mRNA. Optionally, a recombinant nucleic acid comprises a nucleic acid analogue, which is not present in the coding sequence of the recombinant nucleic acid. In some cases, the nucleic acid analogue is 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2 '-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O -DMAP), TO-dimethylaminoethyloxyethyl (2′-O-DMAEOE), 2′-ON-methylacetamide (2′-O-NMA) modification, locked nucleic acid (LNA), ethylene nucleic acid ( ENA), peptide nucleic acids (PNA), 1′,5′-anhydrohexitol nucleic acids (HNA), morpholinos, methylphosphonate nucleotides, thiolphosphonate nucleotides, and 2′-fluoro N3-P5′-phosphoramidites. selected from the group consisting of

[0324] Optionally, the recombinant nucleic acid further comprises a leader sequence. Optionally, the recombinant nucleic acid further comprises a promoter sequence. Optionally, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. Optionally, the recombinant nucleic acid further comprises a 3'UTR sequence. Optionally, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. Optionally, the nucleic acid is an in vitro transcribed nucleic acid.

[0325] Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRα transmembrane domain. Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRβ transmembrane domain. Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRα transmembrane domain and a sequence encoding a TCRβ transmembrane domain. Alternatively, the recombinant nucleic acid comprises a sequence encoding a TCRγ domain or a TCRδ domain, such as a transmembrane domain.

[0326] In some embodiments, disclosed herein is a recombinant nucleic acid comprising a sequence encoding a T-cell receptor (TCR) fusion protein (TFP). A TFP may contain a TCR subunit. A TCR subunit may comprise at least a portion of the TCR extracellular domain. A TCR subunit may further comprise a transmembrane domain. A TCR subunit may comprise the intracellular domain of TCRα, TCRβ, TCRγ, or TCRδ, or may comprise an intracellular domain comprising a stimulatory domain from the intracellular signaling domain of CD3ε, CD3γ, or CD3δ. A TFP may comprise an antigenic domain comprising a ligand or fragment thereof that binds to a receptor or polypeptide expressed on the surface of a cell. The recombinant nucleic acid molecule can comprise a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRα constant domain, a TCRβ constant domain, a TCRα constant domain and a TCRβ constant domain, a TCRγ constant domain, a TCRδ constant domain, or a TCRγ The constant domain and the TCRdelta constant domain, the TCR subunit and the antigen domain are operably linked, and a recombinant nucleic acid that, when expressed in T cells, operably integrates TFP into the TCR complex. Optionally, the antigen domain includes a ligand. In some cases, the ligand binds to a receptor on the cell. In some cases, the ligand binds to a polypeptide expressed on the surface of the cell. Optionally, the receptor or polypeptide expressed on the surface of the cell comprises a stress response receptor or polypeptide. In some cases, the receptor or polypeptide expressed on the surface of the cell is an MHC class I-associated glycoprotein. Optionally, the MHC class I-associated glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4, and combinations thereof. Optionally, the antigen domain comprises a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. . Optionally, the antigen domain comprises a monomer or dimer of the ligand or fragment thereof. Optionally, the ligand or fragment thereof is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. including. Optionally, the ligand or fragment thereof is monomeric or dimeric. Optionally, the antigen domain does not include an antibody or fragment thereof. In some cases, the antigen domain does not include variable regions. Optionally, the antigen domain does not contain CDRs. Optionally, the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or fragment thereof.

[0327] In some cases, the TCR constant domain is incorporated into a functional TCR complex when expressed in a T cell. In some cases, the TCR constant domain is incorporated into the same functional TCR complex that incorporates TFP when expressed in T cells. In some instances, the TFP-encoding sequence and the TCR constant domain-encoding sequence are contained within the same nucleic acid molecule. Optionally, the TFP-encoding sequence and the TCR constant domain-encoding sequence are contained within different nucleic acid molecules.

[0328] Optionally, the TCR subunit and the antibody domain, antigen domain, or binding ligand, or fragment thereof, are operably linked by a linker sequence. Optionally, the linker sequence comprises (G4S) _n , where n=1-4.

[0329] Optionally, the transmembrane domain is a TCR transmembrane domain from CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRδ, or TCRγ. Optionally, the intracellular domain is derived from CD3ε only, CD3γ only, CD3δ only, TCRα only, TCRβ only, TCRδ only, or TCRγ only.

[0330] Optionally, the TCR subunit comprises (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain, (i), (ii) , and (iii) are from the same TCR subunit.

[0331] Optionally, the TCR extracellular domain comprises a TCRα chain, a TCRβ chain, a TCRδ chain, a TCRγ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, functional fragments thereof, and at least one but no more than 20 the extracellular domain of a protein or a portion thereof selected from the group consisting of amino acid sequences thereof having modifications of

[0332] Optionally, the TCR subunit is a TCRα chain, a TCRβ chain, a TCRδ chain, a TCRγ chain, a TCRζ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16 , CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. contains a transmembrane domain containing the transmembrane domain of

[0333] Optionally, the TCR subunit comprises the TCR intracellular domain of TCRα, TCRβ, TCRγ, or TCRδ. Optionally, the TCR subunit comprises a stimulatory domain of a protein selected from the intracellular signaling domains of CD3ε, CD3γ, or CD3δ, or a TCR intracellular domain comprising an amino acid sequence having at least one modification thereto. include.

[0334] Optionally, the TCR subunit comprises a stimulatory domain of a protein selected from the functional signaling domain of 4-1BB and/or the functional signaling domain of CD3zeta, or at least one modification thereto. an intracellular domain comprising an amino acid sequence having

[0335] Optionally, the recombinant nucleic acid further comprises a sequence encoding a co-stimulatory domain. Optionally, the co-stimulatory domain is OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137), and at least one but Include a functional signaling domain of a protein selected from the group consisting of amino acid sequences thereof having 20 or fewer modifications.

[0336] Optionally, the TCR subunit is a CD3ζ TCR subunit, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, a TCR zeta chain, Fcε receptor 1 chain, Fcε receptor 2 chain, Fcγ receptor 1 chain, Fcγ Receptor 2a chain, Fcγ receptor 2b1 chain, Fcγ receptor 2b2 chain, Fcγ receptor 3a chain, Fcγ receptor 3b chain, Fcβ receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, Immunoreceptor tyrosine activation of a protein selected from the group consisting of CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. ITAMs of TCR subunits containing motifs (ITAMs) or portions thereof. In some cases, the ITAM replaces the ITAM of CD3γ, CD3δ, or CD3ε. Optionally, the ITAM is selected from the group consisting of a CD3ζ TCR subunit, a CD3ε TCR subunit, a CD3γ TCR subunit, and a CD3δ TCR subunit; Replaces another ITAM that is selected.

[0337] Optionally, the TFP, TCRγ constant domain, TCRδ constant domain, TCRα constant domain, TCRβ constant domain, and any combination thereof are associated with an endogenous TCR complex and/or at least one endogenous TCR polypeptide. They can interact functionally. Optionally, (a) the TCR constant domain is a TCRγ constant domain and the TFP is functionally integrated into a TCR complex comprising endogenous subunits of TCRβ, CD3ε, CD3γ, CD3δ, or a combination thereof; (b) the TCR constant domain is a TCRδ constant domain, and the TFP is functionally integrated into a TCR complex comprising endogenous subunits of TCRγ, CD3ε, CD3γ, CD3δ, or combinations thereof; or (c) the TCR the constant domains are a TCRγ constant domain and a TCRδ constant domain, and the TFP is functionally integrated into a TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or a combination thereof; or (d) the TCR constant domain is the TCRα constant domain and TFP is functionally integrated into the TCR complex comprising endogenous subunits of TCRβ, CD3ε, CD3γ, CD3δ, or combinations thereof, or (e) the TCR constant domain is the TCRβ constant domain domain that functionally integrates TFP into the TCR complex comprising endogenous subunits of TCRα, CD3ε, CD3γ, CD3δ, or combinations thereof.

[0338] Optionally, the at least one but no more than 20 modifications thereto include modifications of amino acids that mediate cell signaling or modifications of amino acids that are phosphorylated in response to ligand binding to TFP.

[0339] Optionally, the human or humanized antibody is an antibody fragment. Optionally, the antibody fragment is a scFv, single domain antibody domain, VH domain, or VL domain. Optionally, the human or humanized antibody comprising the antigen binding domain is an anti-CD19 binding domain, an anti-CD20 binding domain, an anti-mesothelin binding domain, an anti-PMSA binding domain, an anti-CD70 binding domain, an anti-CD79b binding domain, an anti-MUC16 binding domain. domain, anti-B cell maturation antigen (BCMA) binding domain, anti-mesothelin (MSLN) binding domain, anti-IL13Rα2 binding domain, anti-CD22 binding domain, anti-BAFF or BAFFR binding domain, anti-PD-1 binding domain, anti-PD-L1 binding domain, and anti-ROR-1 binding domain.

[0340] Optionally, the nucleic acid is selected from the group consisting of DNA and RNA. In some cases, the nucleic acid is mRNA. Optionally, a recombinant nucleic acid comprises a nucleic acid analogue, which is not in the coding sequence of the recombinant nucleic acid. In some cases, the nucleic acid analogue is 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2 '-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O -DMAP), TO-dimethylaminoethyloxyethyl (2′-O-DMAEOE), 2′-ON-methylacetamide (2′-O-NMA) modification, locked nucleic acid (LNA), ethylene nucleic acid ( ENA), peptide nucleic acids (PNA), 1′,5′-anhydrohexitol nucleic acids (HNA), morpholinos, methylphosphonate nucleotides, thiolphosphonate nucleotides, and 2′-fluoro N3-P5′-phosphoramidites. selected from the group consisting of

[0341] Optionally, the recombinant nucleic acid further comprises a leader sequence. Optionally, the recombinant nucleic acid further comprises a promoter sequence. Optionally, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. Optionally, the recombinant nucleic acid further comprises a 3'UTR sequence. Optionally, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. Optionally, the nucleic acid is an in vitro transcribed nucleic acid.

[0342] Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRα transmembrane domain. Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRβ transmembrane domain. Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRα transmembrane domain and a sequence encoding a TCRβ transmembrane domain. Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRγ transmembrane domain. Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRdelta transmembrane domain. Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRγ transmembrane domain and a sequence encoding a TCRδ transmembrane domain.

[0343] In some embodiments, further disclosed herein are vectors comprising the recombinant nucleic acids disclosed herein. Optionally, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV), Rous sarcoma virus (RSV) vectors, or retroviral vectors. Optionally, the vector is an AAV6 vector. Optionally, the vector further comprises a promoter. Optionally, the vector is an in vitro transcribed vector.

[0344] A nucleic acid sequence encoding a desired molecule can be obtained using recombinant methods known in the art, for example, by screening libraries of cells expressing the gene using standard techniques. , by deriving the gene from a vector known to contain the gene, or by isolating directly from cells and tissues containing the gene. Alternatively, the gene of interest can be made synthetically rather than cloned.

[0345] The disclosure also provides vectors into which the DNA of the disclosure is inserted. Retroviral-derived vectors, such as lentiviruses, are suitable tools for achieving long-term gene transfer, as they allow long-term stable integration of the transgene and its propagation to daughter cells. Lentiviral vectors have another advantage over vectors derived from oncoretroviruses such as murine leukemia virus in that they can transduce non-proliferating cells such as hepatocytes. They also have the additional advantage of being less immunogenic.

[0346] In another embodiment, the vector comprising a nucleic acid encoding a desired TFP of this disclosure is an adenoviral vector (A5/35). In another embodiment, expression of a nucleic acid encoding TFP can be achieved using transposons such as Sleeping Beauty, Crisper, CAS9, and zinc finger nucleases. See below, June et al. 2009 Nature Reviews Immunology. See 9.10:704-716.

[0347] The expression constructs of this disclosure can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art (e.g., US Pat. Nos. 5,399,346, 5,580,859, 5,589, which are incorporated herein by reference in their entirety). , 466). In another embodiment, the disclosure provides gene therapy vectors.

[0348] Nucleic acids can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0349] Furthermore, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and can be found, for example, in Sambrook et al. , 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor
Press, NY), and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Suitable vectors generally contain an origin of replication that is functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO01/96584, WO01/ 29058, and U.S. Pat. No. 6,326,193).

[0350] Several viral-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus is then isolated and in
It can be delivered to the subject's cells either in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Several adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0351] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Usually it is located in a region 30-110 bp upstream of the start site, although some promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible so that promoter function is maintained when the elements are flipped or moved relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to a distance of 50 bp, after which activity begins to decline. Depending on the promoter, individual elements may function cooperatively or independently to activate transcription.

[0352] An example of a promoter capable of expressing the TFP transgene in mammalian T cells is the EF1a promoter. The native EF1a promoter drives expression of the α subunit of the elongation factor 1 complex, which is involved in the enzymatic delivery of aminoacyl-tRNAs to the ribosome. The EF1a promoter is widely used in mammalian expression plasmids and has been shown to be effective in driving TFP expression from transgenes cloned into lentiviral vectors (eg, Milone et al. , Mol.Ther.17(8):1453-1464 (2009)). Another example of a 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 to which it is operably linked. However, other constitutive promoter sequences can also be used, including the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and actin promoter, myosin promoter, elongation factor 1a promoter, hemoglobin promoter, and creatine kinase promoter, including but not limited to human Gene promoters include, but are not limited to. Furthermore, the disclosure should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of this disclosure. The use of an inducible promoter provides a molecular switch that can turn on the expression of the polynucleotide sequence to which it is operably linked when such expression is desired and off when such expression is not desired. be done. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline-regulated promoters.

[0353] The expression vector introduced into the cells can also be used to assess the expression of the TFP polypeptide or a portion thereof by identifying and expressing expressing cells from a cell population to be transfected or infected via the viral vector. Either or both of a selectable marker gene or a reporter gene to facilitate selection can be included. In other embodiments, selectable markers can be carried on separate pieces of DNA and used in co-transfection procedures. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0354] Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and encodes a polypeptide whose expression is elicited by some readily detectable property, such as enzymatic activity. Gene. Reporter gene expression is assessed at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (eg, Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or obtained commercially. In general, constructs with minimal 5' flanking regions that exhibit the highest level of expression of the reporter gene are identified as promoters. Such promoter regions can be linked to a reporter gene and used to assess the ability of agents to modulate promoter-driven transcription.

[0355] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological means.

[0356] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and/or exogenous nucleic acids are well known in the art. For example, Sambrook et al. , 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY. A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0357] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for gene insertion into mammalian, eg human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, adeno-associated viruses, and the like (e.g., US Pat. Nos. 5,350,674 and 5,585,362). (see No.).

[0358] Chemical means for introducing polynucleotides into host cells include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles and liposomes. of colloidal dispersions. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane vesicle). Other state-of-the-art targeted nucleic acid delivery methods are available, such as delivery of polynucleotides in targeted nanoparticles or other suitable submicron-sized delivery systems.

[0359] When utilizing a non-viral delivery system, exemplary delivery vehicles are liposomes. The use of lipid formulations for introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo) is contemplated. In another aspect, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated within the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, or attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide. may be encapsulated in a liposome, may be complexed with a liposome, may be dispersed in a solution containing a lipid, may be mixed with a lipid, or may be complexed with a lipid. Often, it may be contained as a suspension in lipids, contained in or complexed with micelles, or otherwise associated with lipids. Lipid, lipid/DNA, or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as micelles, or in a "collapsed" structure. They may also simply be scattered in solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, lipids include classes of compounds containing lipid droplets naturally occurring in the cytoplasm as well as long-chain aliphatic hydrocarbons such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes and their derivatives.

[0360] Lipids suitable for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine (“DMPC”) is available from Sigma, St. Louis, MO, dicetyl phosphate ("DCP") is available from K&K Laboratories (Plainview, N.Y.), cholesterol ("Choi") is available from Calbiochem-Behring. and dimyristylphosphatidylglycerol (“DMPG”) and other lipids are available from Avanti Polar Lipids, Inc.; (Birmingham, Ala.). Lipid stock solutions in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform is used as the sole solvent because chloroform evaporates more readily than methanol. "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vehicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. It forms spontaneously when phospholipids are suspended in an excess of aqueous solution. Lipid components form closed structures after undergoing self-rearrangement, entrapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions that have structures in solution that differ from normal vesicular structures are also included. For example, lipids can have a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0361] The presence of a recombinant DNA sequence in a host cell is determined by whatever method is used to introduce exogenous nucleic acid into the host cell, or otherwise to expose the cell to the inhibitors of this disclosure. Various assays can be performed for confirmation. Such assays include, for example, "molecular biological" assays well known to those skilled in the art such as Southern and Northern blotting, RT-PCR, and PCR, immunological means (ELISA and Western blot) or the methods of the present disclosure. Assays described herein for identification of agents within scope include "biochemical" assays such as detecting the presence or absence of a particular peptide.

[0362] The disclosure further provides a vector comprising a nucleic acid molecule encoding a TFP. In one aspect, TFP vectors can be directly transduced into cells, eg, T cells. In one aspect, vectors include cloning or expression vectors, such as one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double microchromosomes), retroviral and lentiviral vector constructs. The vectors are not limited to these. In one aspect, the vector is capable of expressing the TFP construct in mammalian T cells. In one aspect, the mammalian T cells are human T cells.

[0363] In one aspect, the present disclosure provides (a)(i) (1) at least a portion of the TCR extracellular domain, (2) a transmembrane domain, and (3) a TCRα, TCRβ, TCRγ, or TCRδ cellular A T cell receptor ( (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRγ constant domain, a TCRδ constant domain, or a TCRγ constant domain and a TCRδ constant domain; wherein the TCR subunit and antibody are operably linked, and TFP is operably integrated into the TCR complex when expressed in a modified T cell comprising functional disruption of the endogenous TCR. I will provide a.

[0364] In another aspect, the present disclosure provides a (ii) a binding ligand or fragment thereof capable of binding to the antibody or fragment thereof; (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRγ constant domain, a TCRδ constant domain, or a TCRγ constant domain and a sequence which is a TCRdelta constant domain, wherein the TCR subunit and binding ligand or fragment thereof are operably linked, and which, when expressed in a modified T cell containing a functional disruption of the endogenous TCR, TFP binds to the TCR complex A recombinant nucleic acid is provided that is functionally integrated into the body.

[0365] In some embodiments, the sequence encoding the antigen- or ligand-binding domain is operably linked to the sequence encoding the delta constant domain. In some embodiments, the intracellular domain is that of TCRγ. In some embodiments, the antigen- or ligand-binding domain-encoding sequence is operably linked to the gamma constant domain-encoding sequence. In some embodiments, the intracellular domain is the intracellular domain of TCRdelta. In some embodiments, the antigen- or ligand-binding domain encoding sequence is operably linked to both the TCRδ constant domain or fragment thereof encoding sequence and the TCRγ constant domain or fragment thereof encoding sequence. ing. In some embodiments, the intracellular signaling domain is CD3ε, CD3γ, or CD3δ. In some embodiments, the intracellular signaling domain is CD3ε. In some embodiments, the recombinant nucleic acid further comprises at least one leader sequence and at least one linker. In some embodiments, the recombinant nucleic acid further comprises a portion of the TCRα constant domain, a portion of the TCRβ domain, or both. In some embodiments, the sequence is, from 5′ to 3′, a first leader sequence, an antigen binding domain sequence, a linker, a TRDC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRGC gene. Contains arrays. In some embodiments, the sequences are, in the 5′-3′ direction, a first leader sequence, a TRDC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker sequence, and a TRGC. Contains gene sequences. In some embodiments, the sequence is, in the 5′-3′ direction, a first leader sequence, an antigen binding domain sequence, a first linker sequence, a TRDC gene sequence, a cleavable linker, a second leader sequence, It includes a second antigen binding domain sequence, a second linker sequence, and a TRGC gene sequence. In some embodiments, the sequences are, in the 5′-3′ direction, a first leader sequence, a TRDC gene sequence, a first cleavable linker sequence, a second leader sequence, a TRGC gene sequence, a second It includes a cleavable linker sequence, a third leader sequence, an antigen binding domain sequence, a linker sequence, and a CD3ε gene sequence. In some embodiments, the sequence is, in the 5′-3′ direction, a first leader sequence, a first antigen binding domain sequence, a first linker sequence, a TRDC gene sequence or fragment thereof, a TRAC gene sequence or Fragments, cleavable linker sequences, second leader sequences, second antigen binding domain sequences, second linker sequences, TRGC gene sequences or fragments thereof, and TRBC gene sequences or fragments thereof. In some embodiments, the sequence encodes the polypeptide set forth in SEQ ID NO:1. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:2. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:3. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:4. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:5. In some embodiments, a binding ligand can bind to the Fc domain of an antibody. In some embodiments, the binding ligand can selectively bind to an IgG1 antibody. In some embodiments, the binding ligand can specifically bind to an IgG4 antibody. In some embodiments, the antibody or fragment thereof binds to a cell surface antigen. In some embodiments, the antibody or fragment thereof is murine, human, or humanized. In some embodiments, the antibody or fragment thereof binds to a cell surface antigen on the surface of tumor cells. In some embodiments, the binding ligand is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. Including body. In some embodiments, binding ligands do not include antibodies or fragments thereof. In some embodiments, the binding ligand comprises a CD16 polypeptide or fragment thereof. In some embodiments, the binding ligand comprises a CD16 binding polypeptide. In some embodiments, the binding ligand is human or humanized. In some embodiments, the recombinant nucleic acid further comprises a nucleic acid sequence encoding an antibody or fragment thereof that can be bound by a binding ligand. In some embodiments, the antibody or fragment thereof is secretable from the cell.

[0366] In another aspect, provided herein are (a)(i) (1) at least a portion of the TCR extracellular domain, (2) the transmembrane domain, and (3) TCRα, TCRβ (ii) a receptor expressed on the surface of a cell; or an antigen binding domain comprising a ligand or fragment thereof that binds to the polypeptide; and (b) a sequence encoding a TCR constant domain, wherein , the TCR constant domain comprises a sequence that is a TCRγ constant domain, a TCRδ constant domain, or a TCRγ constant domain and a TCRδ constant domain, wherein the TCR subunit and the antigen-binding domain are operably linked and endogenous TCR A recombinant nucleic acid that, when expressed in a modified T cell containing a functional disruption, functionally integrates TFP into the TCR complex.

[0367] In some embodiments, the recombinant nucleic acid further comprises at least a subsequence encoding a TCRα constant domain, a subsequence encoding a TCRβ constant domain, or a subsequence of both the TCRα and TCRβ constant domains. In some embodiments the antigen binding domain comprises a ligand. In some embodiments, the ligand binds to a receptor on the cell. In some embodiments, the ligand binds to a polypeptide expressed on the surface of the cell. In some embodiments, the receptor or polypeptide expressed on the surface of the cell comprises a stress response receptor or polypeptide. In some embodiments, the receptor or polypeptide expressed on the surface of the cell is an MHC class I-associated glycoprotein. In some embodiments, the MHC class I-associated glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4, and combinations thereof. In some embodiments, the antigen binding domain is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. Contains mers. In some embodiments, the antigen binding domain comprises a monomer or dimer of the ligand or fragment thereof. In some embodiments, the ligand or fragment thereof is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or It is decamer. In some embodiments, the ligand or fragment thereof is monomeric or dimeric. In some embodiments the antigen binding domain does not comprise an antibody or fragment thereof. In some embodiments, the antigen binding domain does not contain a variable region. In some embodiments, the antigen binding domain does not contain CDRs. In some embodiments, the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or fragment thereof. In some embodiments, the TCR constant domain is incorporated into a functional TCR complex upon expression in T cells. In some embodiments, the TCR constant domain is incorporated into the same functional TCR complex that incorporates TFP when expressed in T cells. In some embodiments, the TFP-encoding sequence and the TCR constant domain-encoding sequence are contained within the same nucleic acid molecule. In some embodiments, the TFP-encoding sequence and the TCR constant domain-encoding sequence are contained within different nucleic acid molecules. In some embodiments, the TCR subunit and antibody domain, antigen binding domain, or binding ligand, or fragment thereof are operably linked by a linker sequence. In some embodiments, the linker sequence comprises (G ₄ S) _n , where n=1-4. In some embodiments, the transmembrane domain is a TCR transmembrane domain from CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRδ, or TCRγ. In some embodiments, the encoded intracellular domain is derived from CD3ε only, CD3γ only, CD3δ only, TCRα only, TCRβ, TCRγ, or TCRδ. In some embodiments, the encoded TCR subunit comprises (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain, (i) At least two of (ii) and (iii) are from the same TCR subunit. In some embodiments, the TCR extracellular domain comprises a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and at least one but no more than 20 amino acid modifications thereof. It includes the extracellular domain of a protein or part thereof selected from the group consisting of sequences. In some embodiments, the TCR extracellular domain comprises a TCRγ or TCRδ chain constant domain or a portion thereof. In some embodiments, the TCR subunit is TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, TCRζ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16 , CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. contains a transmembrane domain containing the transmembrane domain of In some embodiments, the TCR subunit comprises a stimulatory domain of a protein selected from the intracellular signaling domains of CD3ε, CD3γ, or CD3δ, or an amino acid sequence having at least one modification thereto. Including internal domains. In some embodiments, the TCR subunit has a stimulatory domain of a protein selected from the functional signaling domain of 4-1BB and/or the functional signaling domain of CD3ζ, or at least one modification thereto. further includes an intracellular domain comprising an amino acid sequence having

[0368] In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a costimulatory domain. In some embodiments, the co-stimulatory domain is OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137), and at least A functional signaling domain of a protein selected from the group consisting of amino acid sequences thereof having one but no more than 20 modifications. In some embodiments, the TCR subunit is CD3ζ TCR subunit, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, TCRζ chain, Fcε receptor 1 chain, Fcε receptor 2 chain, Fcγ receptor 1 chain, Fcγ Receptor 2a chain, Fcγ receptor 2b1 chain, Fcγ receptor 2b2 chain, Fcγ receptor 3a chain, Fcγ receptor 3b chain, Fcβ receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, Immunoreceptor tyrosine activation of a protein selected from the group consisting of CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. ITAMs of TCR subunits containing motifs (ITAMs) or portions thereof. In some embodiments, the ITAM replaces the ITAM of CD3γ, CD3δ, or CD3ε. In some embodiments, the ITAM is selected from the group consisting of a CD3ζ TCR subunit, a CD3ε TCR subunit, a CD3γ TCR subunit, and a CD3δ TCR subunit, wherein: Replaces another ITAM selected from the group consisting of: In some embodiments, the TFP, TCRγ constant domain, TCRδ constant domain, and any combination thereof are capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide. can.

[0369] In some embodiments, the TCR constant domain is a TCRγ constant domain and TFP is functionally integrated into a TCR complex comprising endogenous subunits of TCRδ, CD3ε, CD3γ, CD3δ, or combinations thereof. or the TCR constant domain is a TCRdelta constant domain and the TFP is functionally integrated into a TCR complex comprising endogenous subunits of TCRgamma, CD3epsilon, CD3gamma, CD3delta, or a combination thereof; The constant domains are the TCRγ constant domain and the TCRδ constant domain, and TFP is functionally integrated into the TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or combinations thereof.

[0370] In some embodiments, at least one but no more than 20 modifications thereto are modifications of amino acids that mediate cell signaling or modifications of amino acids that are phosphorylated in response to ligand binding to TFP. including. In some embodiments the antibody is an antibody fragment. In some embodiments, the antibody fragment is a scFv, single domain antibody domain, VH domain, or VL domain. In some embodiments, the antigen binding domain is an anti-CD19 binding domain, an anti-B cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, an anti-79b binding domain , anti-HER2 binding domain, anti-PMSA binding domain, anti-MUC16 binding domain, anti-CD22 binding domain, anti-PD-L1 binding domain, anti-BAFF or BAFF receptor binding domain, and anti-ROR-1 binding domain be able to. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCRα transmembrane domain. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCRβ transmembrane domain. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCRα transmembrane domain and a sequence encoding a TCRβ transmembrane domain.

[0371] In another aspect, the present disclosure provides a a TCR subunit comprising an intracellular domain, or an intracellular domain comprising a stimulating domain from the intracellular signaling domain of CD3ε, CD3γ, or CD3δ; and (ii) an antibody or fragment thereof comprising an antigen binding domain. a sequence encoding a cell receptor (TCR) fusion protein (TFP); and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRα constant domain or a TCRβ constant domain, or a TCRα constant domain and a TCRβ. TCR subunits and antibodies are operably linked, and TFP is functionally incorporated into the TCR complex when expressed in a modified T cell containing a functional disruption of the endogenous TCR. , which provides recombinant nucleic acids.

[0372] In another aspect, the present disclosure provides a (ii) a binding ligand or fragment thereof capable of binding to the antibody or fragment thereof; (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRα constant domain or a TCRβ constant domain, or a TCRα constant domain and a sequence which is the TCRβ constant domain, wherein the TCR subunit and binding ligand or fragment thereof are operably linked, and which, when expressed in a modified T cell containing a functional disruption of the endogenous TCR, TFP binds to the TCR complex A recombinant nucleic acid is provided that is functionally integrated into the body.

[0373] In some embodiments, a sequence encoding an antigen- or ligand-binding domain is operably linked to a sequence encoding an alpha constant domain. In some embodiments, the intracellular domain is that of TCRβ. In some embodiments, the antigen- or ligand-binding domain-encoding sequence is operably linked to the beta constant domain-encoding sequence. In some embodiments, the intracellular domain is that of TCRα. In some embodiments, the sequences are, from 5′ to 3′, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene. Contains arrays. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:10. In some embodiments, the recombinant nucleic acid further comprises at least one leader sequence and at least one linker. In some embodiments, a binding ligand can bind to the Fc domain of an antibody. In some embodiments, the binding ligand can selectively bind to an IgG1 antibody. In some embodiments, the binding ligand can specifically bind to an IgG4 antibody. In some embodiments, the antibody or fragment thereof binds to a cell surface antigen. In some embodiments, the antibody or fragment thereof is murine, human, or humanized. In some embodiments, the antibody or fragment thereof binds to a cell surface antigen on the surface of tumor cells. In some embodiments, the binding ligand is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. Including body. In some embodiments, binding ligands do not include antibodies or fragments thereof. In some embodiments, the binding ligand comprises a CD16 polypeptide or fragment thereof. In some embodiments, the binding ligand comprises a CD16 binding polypeptide. In some embodiments, the binding ligand is human or humanized. In some embodiments, the recombinant nucleic acid further comprises a nucleic acid sequence encoding an antibody or fragment thereof that can be bound by a binding ligand. In some embodiments, the antibody or fragment thereof is secretable from the cell.

[0374] In another aspect, the present disclosure provides a a TCR subunit comprising an intracellular domain, or an intracellular domain comprising a stimulatory domain from the intracellular signaling domain of CD3ε, CD3γ, or CD3δ; and (ii) binds to a receptor or polypeptide expressed on the surface of the cell a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising a ligand or an antigen binding domain comprising a fragment thereof; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain comprises A modification comprising a sequence that is a TCRα constant domain or a TCRβ constant domain, or a TCRα constant domain and a TCRβ constant domain, wherein the TCR subunit and the antigen binding domain are functionally linked, and which comprises functional disruption of the endogenous TCR A recombinant nucleic acid is provided that, when expressed in a T cell, functionally integrates TFP into the TCR complex.

[0375] In some embodiments, the TCR constant domain is a mouse TCRγ constant domain, eg, a mouse TCRα constant domain or a mouse TCRβ constant domain, or a mouse TCRα constant domain and a mouse TCRβ constant domain. In some embodiments, the extracellular domain comprises at least a portion of a TCRα extracellular domain or a TCRβ extracellular domain. In some embodiments, the TCRα or TCRβ extracellular domain is a mouse TCRα or mouse TCRβ extracellular domain. In some embodiments, the extracellular domain comprises at least part of a TCRα or TCRβ constant domain. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a TCRα transmembrane domain and a sequence encoding a TCRβ transmembrane domain. In some embodiments, the recombinant nucleic acid further comprises at least a subsequence encoding a TCRγ constant domain, a subsequence encoding a TCRδ constant domain, or a subsequence of both the TCRγ and TCRδ constant domains. In some embodiments the antigen binding domain comprises a ligand. In some embodiments, the ligand binds to a receptor on the cell. In some embodiments, the ligand binds to a polypeptide expressed on the surface of the cell. In some embodiments, the receptor or polypeptide expressed on the surface of the cell comprises a stress response receptor or polypeptide. In some embodiments, the receptor or polypeptide expressed on the surface of the cell is an MHC class I-associated glycoprotein. In some embodiments, the MHC class I-associated glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4, and combinations thereof. In some embodiments, the antigen binding domain is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. Contains mers. In some embodiments, the antigen binding domain comprises a monomer or dimer of the ligand or fragment thereof.

[0376] In some embodiments, the ligand or fragment thereof is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonameric isomers, or decamers. In some embodiments, the ligand or fragment thereof is monomeric or dimeric. In some embodiments the antigen binding domain does not comprise an antibody or fragment thereof. In some embodiments, the antigen binding domain does not contain a variable region. In some embodiments, the antigen binding domain does not contain CDRs. In some embodiments, the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or fragment thereof. In some embodiments, the TCR constant domain is incorporated into a functional TCR complex upon expression in T cells. In some embodiments, the TCR constant domain is incorporated into the same functional TCR complex that incorporates TFP when expressed in T cells. In some embodiments, the TFP-encoding sequence and the TCR constant domain-encoding sequence are contained within the same nucleic acid molecule. In some embodiments, the TFP-encoding sequence and the TCR constant domain-encoding sequence are contained within different nucleic acid molecules. In some embodiments, the TCR subunit and antibody domain, antigen binding domain, or binding ligand, or fragment thereof are operably linked by a linker sequence. In some embodiments, the linker sequence comprises (G ₄ S) _n , where n=1-4. In some embodiments, the transmembrane domain is a TCR transmembrane domain from CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRδ, or TCRγ. In some embodiments, the encoded intracellular domain is derived from CD3ε only, CD3γ only, CD3δ only, TCRα only, TCRβ, TCRγ, or TCRδ. In some embodiments, the encoded TCR subunit comprises (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain, (i) At least two of (ii) and (iii) are from the same TCR subunit. In some embodiments, the TCR
The extracellular domain comprises a TCRα chain, a TCRβ chain, a TCRγ chain, a TCRδ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. the extracellular domain of a protein or a portion thereof selected from the group consisting of In some embodiments, the TCR subunit is TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, TCRζ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16 , CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. contains a transmembrane domain containing the transmembrane domain of In some embodiments, the TCR subunit comprises a stimulatory domain of a protein selected from the intracellular signaling domains of CD3ε, CD3γ, or CD3δ, or an amino acid sequence having at least one modification thereto. Including internal domains. In some embodiments, the TCR subunit has a stimulatory domain of a protein selected from the functional signaling domain of 4-1BB and/or the functional signaling domain of CD3ζ, or at least one modification thereto. further includes an intracellular domain comprising an amino acid sequence having

[0377] In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a costimulatory domain. In some embodiments, the co-stimulatory domain is OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137), and at least A functional signaling domain of a protein selected from the group consisting of amino acid sequences thereof having one but no more than 20 modifications. In some embodiments, the TCR subunit is CD3ζ TCR subunit, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, TCRζ chain, Fcε receptor 1 chain, Fcε receptor 2 chain, Fcγ receptor 1 chain, Fcγ Receptor 2a chain, Fcγ receptor 2b1 chain, Fcγ receptor 2b2 chain, Fcγ receptor 3a chain, Fcγ receptor 3b chain, Fcβ receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, Immunoreceptor tyrosine activation of a protein selected from the group consisting of CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. ITAMs of TCR subunits containing motifs (ITAMs) or portions thereof. In some embodiments, the ITAM replaces the ITAM of CD3γ, CD3δ, or CD3ε. In some embodiments, the ITAM is selected from the group consisting of a CD3ζ TCR subunit, a CD3ε TCR subunit, a CD3γ TCR subunit, and a CD3δ TCR subunit, wherein: Replaces another ITAM selected from the group consisting of: In some embodiments, TFP, TCRα constant domain, TCRβ domain, and any combination thereof can functionally interact with an endogenous TCR complex and/or at least one endogenous TCR polypeptide is.

[0378] In some embodiments, the TCR constant domain is a TCRα constant domain, and TFP is functionally integrated into a TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or combinations thereof or the TCR constant domain is a TCRβ constant domain and the TFP is functionally integrated into a TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or combinations thereof, or the TCR constant domain is a TCRα constant domain domain and the TCRβ constant domain, which functionally integrates TFP into the TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or combinations thereof.

[0379] In another aspect, the present disclosure provides (a)(i) (1) at least a portion of the extracellular domain of mouse TCRα or mouse TCRβ, (2) the transmembrane domain of mouse TCRα or mouse TCRβ, and ( 3) a sequence encoding a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising the intracellular domain of mouse TCRα or mouse TCRβ and (ii) an antibody or fragment thereof comprising an antigen binding domain and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a mouse TCRα constant domain or a mouse TCRβ constant domain, or a mouse TCRα constant domain and a mouse TCRβ constant domain, wherein the TCR sub The unit and antibody are operably linked and provide a recombinant nucleic acid that, when expressed in a modified T cell containing functional disruption of the endogenous TCR, functionally integrates TFP into the TCR complex.

[0380] In another aspect, the present disclosure provides (a)(i) (1) at least a portion of the extracellular domain of mouse TCRα or mouse TCRβ, (2) the transmembrane domain of mouse TCRα or mouse TCRβ, and ( 3) a T cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit comprising the intracellular domain of mouse TCRα or mouse TCRβ and (ii) a binding ligand or fragment thereof capable of binding to an antibody or fragment thereof; (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a mouse TCRα constant domain or a mouse TCRβ constant domain, or a mouse TCRα constant domain and a mouse TCRβ constant domain , a TCR subunit and a binding ligand or fragment thereof are operably linked, and TFP is operably integrated into the TCR complex when expressed in a modified T cell comprising a functional disruption of the endogenous TCR. I will provide a.

[0381] In some embodiments, the antigen- or ligand-binding domain encoding sequence is operably linked to the α constant domain encoding sequence. In some embodiments, the intracellular domain is that of TCRβ. In some embodiments, the antigen- or ligand-binding domain-encoding sequence is operably linked to the beta constant domain-encoding sequence. In some embodiments, the intracellular domain is that of TCRα. In some embodiments, the sequences are, from 5′ to 3′, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene. Contains arrays. In some embodiments, the sequence encodes a polypeptide set forth in SEQ ID NO:10. In some embodiments, the recombinant nucleic acid further comprises at least one leader sequence and at least one linker. In some embodiments, a binding ligand can bind to the Fc domain of an antibody. In some embodiments, the binding ligand can selectively bind to an IgG1 antibody. In some embodiments, the binding ligand can specifically bind to an IgG4 antibody. In some embodiments, the antibody or fragment thereof binds to a cell surface antigen. In some embodiments, the antibody or fragment thereof is murine, human, or humanized. In some embodiments, the antibody or fragment thereof binds to a cell surface antigen on the surface of tumor cells. In some embodiments, the binding ligand is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. Including body. In some embodiments, binding ligands do not include antibodies or fragments thereof. In some embodiments, the binding ligand comprises a CD16 polypeptide or fragment thereof. In some embodiments, the binding ligand comprises a CD16 binding polypeptide. In some embodiments, the binding ligand is human or humanized. In some embodiments, the recombinant nucleic acid further comprises a nucleic acid sequence encoding an antibody or fragment thereof that can be bound by a binding ligand. In some embodiments, the antibody or fragment thereof is secretable from the cell.

[0382] In another aspect, the present disclosure provides (a)(i) (1) at least a portion of the extracellular domain of mouse TCRα or mouse TCRβ, (2) the transmembrane domain of mouse TCRα or mouse TCRβ, and ( 3) T cells comprising a TCR subunit comprising the intracellular domain of mouse TCRα or mouse TCRβ, and (ii) an antigen-binding domain comprising a ligand or fragment thereof that binds to a receptor or polypeptide expressed on the surface of the cell. a sequence encoding a receptor (TCR) fusion protein (TFP); and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a mouse TCRα constant domain or a mouse TCRβ constant domain, or a mouse TCRα constant domain. and a sequence that is a murine TCRβ constant domain, wherein the TCR subunit and antigen binding domain are operably linked, and TFP joins the TCR complex when expressed in a modified T cell containing a functional disruption of the endogenous TCR. Functionally integrated recombinant nucleic acids are provided.

[0383] In some embodiments, the extracellular domain comprises at least a portion of a TCRα or TCRβ constant domain. In some embodiments, the recombinant nucleic acid further comprises at least a subsequence encoding a TCRγ constant domain, a subsequence encoding a TCRδ constant domain, or a subsequence of both the TCRγ and TCRδ constant domains. In some embodiments the antigen binding domain comprises a ligand. In some embodiments, the ligand binds to a receptor on the cell. In some embodiments, the ligand binds to a polypeptide expressed on the surface of the cell. In some embodiments, the receptor or polypeptide expressed on the surface of the cell comprises a stress response receptor or polypeptide. In some embodiments, the receptor or polypeptide expressed on the surface of the cell is an MHC class I-associated glycoprotein. In some embodiments, the MHC class I-associated glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4, and combinations thereof. In some embodiments, the antigen binding domain is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. Contains mers. In some embodiments, the antigen binding domain comprises a monomer or dimer of the ligand or fragment thereof. In some embodiments, the ligand or fragment thereof is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or It is decamer. In some embodiments, the ligand or fragment thereof is monomeric or dimeric. In some embodiments the antigen binding domain does not comprise an antibody or fragment thereof. In some embodiments, the antigen binding domain does not contain a variable region. In some embodiments, the antigen binding domain does not contain CDRs. In some embodiments, the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or fragment thereof. In some embodiments, the TCR constant domain is incorporated into a functional TCR complex upon expression in T cells. In some embodiments, the TCR constant domain is incorporated into the same functional TCR complex that incorporates TFP when expressed in T cells. In some embodiments, the TFP-encoding sequence and the TCR constant domain-encoding sequence are contained within the same nucleic acid molecule. In some embodiments, the TFP-encoding sequence and the TCR constant domain-encoding sequence are contained within different nucleic acid molecules. In some embodiments, the TCR subunit and antibody domain, antigen binding domain, or binding ligand, or fragment thereof, are operably linked by a linker sequence. In some embodiments, the linker sequence comprises (G4S) _n , where n=1-4. In some embodiments, the transmembrane domain is a TCRα or TCRβ, eg, a TCR transmembrane domain from mouse TCRα or TCRβ.

[0384] In some embodiments, the encoded TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, ( At least two of i), (ii) and (iii) are from the same TCR subunit.

[0385] In some embodiments, the TCR extracellular domain is a TCRα or TCRβ chain, such as a murine TCRα or TCRβ chain extracellular domain or a portion thereof, a functional fragment thereof, and at least one but 20 Including that amino acid sequence with the following modifications. In some embodiments, the TCR subunit is a TCR α or TCR β chain, such as the transmembrane domain of a murine TCR α or TCR β chain, a functional fragment thereof, and an amino acid sequence thereof having at least one but no more than 20 modifications. containing a transmembrane domain. In some embodiments, the TCR subunit comprises a TCR α or TCR β chain, eg, the TCR intracellular domain of a murine TCR α or TCR β chain, or an amino acid sequence having at least one modification thereto. In some embodiments, the TCR subunit has a stimulatory domain of a protein selected from the functional signaling domain of 4-1BB and/or the functional signaling domain of CD3ζ, or at least one modification thereto. further includes an intracellular domain comprising an amino acid sequence having

[0386] In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a costimulatory domain. In some embodiments, the co-stimulatory domain is OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137), and at least A functional signaling domain of a protein selected from the group consisting of amino acid sequences thereof having one but no more than 20 modifications. In some embodiments, the TCR subunit is CD3ζ TCR subunit, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, TCRζ chain, Fcε receptor 1 chain, Fcε receptor 2 chain, Fcγ receptor 1 chain, Fcγ Receptor 2a chain, Fcγ receptor 2b1 chain, Fcγ receptor 2b2 chain, Fcγ receptor 3a chain, Fcγ receptor 3b chain, Fcβ receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, Immunoreceptor tyrosine activation of a protein selected from the group consisting of CD32, CD64, CD79a, CD79b, CD89, CD278, CD66d, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. Includes ITAMs of TCR subunits containing motifs (ITAMs) or portions thereof. In some embodiments, the ITAM replaces the ITAM of CD3γ, CD3δ, or CD3ε. In some embodiments, the ITAM is selected from the group consisting of a CD3ζ TCR subunit, a CD3ε TCR subunit, a CD3γ TCR subunit, and a CD3δ TCR subunit, wherein: Replaces another ITAM selected from the group consisting of: In some embodiments, TFP, TCRα constant domain, TCRβ domain, and any combination thereof can functionally interact with an endogenous TCR complex and/or at least one endogenous TCR polypeptide is. In some embodiments, the TCR constant domain is a TCRα constant domain, and TFP is functionally integrated into a TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or a combination thereof; domain is a TCRβ constant domain and TFP is functionally integrated into a TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or combinations thereof; domain that functionally integrates TFP into the TCR complex containing endogenous subunits of CD3ε, CD3γ, CD3δ, or combinations thereof.

[0387] In some embodiments, the nucleic acid is selected from the group consisting of DNA and RNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is circRNA. In some embodiments, a recombinant nucleic acid comprises a nucleic acid analogue that is absent from the coding sequence of the recombinant nucleic acid. In some embodiments, the nucleic acid analogue is 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T- deoxy-2′-fluoro, 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2 '-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA) modification, locked nucleic acid (LNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1′,5′-anhydrohexitol nucleic acid (HNA), morpholino, methylphosphonate nucleotide, thiolphosphonate nucleotide, and 2′-fluoro N3-P5′-phospho selected from the group consisting of roamidites; In some embodiments, the recombinant nucleic acid further comprises a leader sequence. In some embodiments, the recombinant nucleic acid further comprises a promoter sequence. In some embodiments, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some embodiments, the recombinant nucleic acid further comprises a 3'UTR sequence. In some embodiments, the nucleic acid is an isolated or non-naturally occurring nucleic acid. In some embodiments, the nucleic acid is an in vitro transcribed nucleic acid.

[0388] In another aspect, the disclosure provides vectors comprising the recombinant nucleic acids described herein.
[0389] In some embodiments, the vector is the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, adeno-associated viral vector (AAV), Rous sarcoma virus (RSV) vector, or retroviral vector. is selected from In some embodiments the vector is an AAV6 vector. In some embodiments the vector further comprises a promoter. In some embodiments, the vector is an in vitro transcribed vector.

[0390] In another aspect, the disclosure provides a modified T cell comprising a recombinant nucleic acid as described herein, or a vector as described herein, wherein the modified T cell is a functional Including destruction.

[0391] In another aspect, the present disclosure provides a modified T cell comprising a sequence encoding a TFP of a nucleic acid described herein or a TFP encoded by a sequence of a nucleic acid described herein encoding a TFP. and the modified T cells comprise a functional disruption of the endogenous TCR.

[0392] In another aspect, the present disclosure provides a modified allogeneic T cell comprising a TFP encoded by a sequence encoding a TFP described herein, or a sequence of a nucleic acid described herein encoding a TFP. offer.

[0393] In some embodiments, the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein said TCR constant domain is a TCRγ constant domain, a TCRδ constant domain, or a TCRγ constant domain and a TCRδ constant domain. In some embodiments, the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein said TCR constant domain is a TCRα constant domain, a TCRβ constant domain, or a TCRα constant domain and a TCRβ constant domain. In some embodiments, the TCR constant domain, e.g., TCRα constant domain, TCRβ constant domain, or TCRα and TCRβ constant domains, is a mouse TCRγ constant domain, e.g., mouse TCRα constant domain, mouse TCRβ constant domain, or mouse The TCRα constant domain and the mouse TCRβ constant domain. In some embodiments, the endogenous TCR that is functionally disrupted is an endogenous TCRα chain, an endogenous TCRβ chain, or an endogenous TCRα chain and an endogenous TCRβ chain. In some embodiments, endogenous TCRs that are functionally disrupted have reduced binding to MHC-peptide complexes compared to those of unmodified control T cells. In some embodiments, the functional disruption is disruption of the gene encoding the endogenous TCR. In some embodiments, disruption of the gene encoding the endogenous TCR is removal of the sequence of the gene encoding the endogenous TCR from the genome of the T cell. In some embodiments, the T cells are CD4 cells, CD8 cells, naive T cells, memory stem T cells, central memory T cells, double negative T cells, effector memory T cells, effector T cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, α/β T cells, γ/δ T cells, natural killer (NK) cells, natural killer T (NKT) cells, hematopoietic stem cells, and many Human T cells selected from potential stem cells. In some embodiments, the T cells are CD8 ⁺ T cells or CD4 ⁺ T cells. In some embodiments, the T cells are allogeneic T cells. In some embodiments, the engineered T cell has an inhibitory molecule comprising a first polypeptide comprising at least a portion of the inhibitory molecule associated with a second polypeptide comprising a positive signal from an intracellular signaling domain. It contains the encoding nucleic acid. In some embodiments, the inhibitory molecule comprises a first polypeptide comprising at least a portion of PD1 and a second polypeptide comprising a co-stimulatory domain and a primary signaling domain.

[0394] In another aspect, the present disclosure provides pharmaceutical compositions comprising the engineered T cells described herein and a pharmaceutically acceptable carrier.
[0395] In another aspect, the present disclosure provides a method of making a modified T cell described herein, wherein the method comprises endogenous cells encoding a TCRα chain, a TCRβ chain, or a TCRα chain and a TCRβ chain. disrupting the TCR gene, thereby producing a T cell comprising a functional disruption of the endogenous TCR gene; and transducing the nucleic acid or vector described herein.

[0396] In some embodiments, the disruption is a nuclease protein that targets an endogenous gene encoding a TCRα chain, a TCRβ chain, or a TCRα chain and a TCRβ chain, or a nucleic acid sequence encoding a nuclease protein, to a T cell. Including transducing.

[0397] In another aspect, the present disclosure provides a method of making a modified T cell as described herein, wherein the T cell comprising a functional disruption of an endogenous TCR gene is transformed into a T cell described herein. transducing a recombinant nucleic acid as described herein or a vector as described herein.

[0398] In some embodiments, the T cell comprising a functional disruption of an endogenous TCR gene comprises a functional disruption of an endogenous TCR gene encoding a TCRα chain, a TCRβ chain, or a TCRα chain and a TCRβ chain. T cells contained. In some embodiments, the T cells are human T cells. In some embodiments, T cells comprising a functional disruption of an endogenous TCR gene have reduced binding to MHC-peptide complexes compared to those of unmodified control T cells. In some embodiments, the nuclease is a meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR/Cas nuclease, or megaTAL nuclease. In some embodiments, the sequence contained in the recombinant nucleic acid or vector is inserted into the endogenous TCR subunit gene at the cleavage site, and the insertion of the sequence into the endogenous TCR subunit gene results in an endogenous TCR subunit. functionally destroyed. In some embodiments the nuclease is a meganuclease. In some embodiments, the meganuclease comprises a first subunit and a second subunit, the first subunit binding to the first recognition half-site of the recognition sequence and the second subunit comprising Binds to the second recognition half-site of the recognition sequence. In some embodiments, the meganuclease is a single-stranded meganuclease comprising a linker, which covalently joins the first subunit and the second subunit.

[0399] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. Including.

[0400] In another aspect, the disclosure provides a method of treating cancer in a subject in need thereof, comprising: (a) a modified T cell produced according to the methods described herein; and (b) administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0401] In another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising (a) a modified T cell produced according to the methods described herein; and (b) administering to the subject a pharmaceutical composition comprising a pharmaceutically acceptable carrier.

[0402] In some embodiments, the engineered T cells are allogeneic T cells. In some embodiments, less cytokines are released in the subject compared to subjects administered an effective amount of unmodified control T cells. In some embodiments, less cytokines are released in a subject compared to a subject administered an effective amount of modified T cells comprising a recombinant nucleic acid described herein or a vector described herein. In some embodiments, the method comprises administering the pharmaceutical composition in combination with an agent that increases the effectiveness of the pharmaceutical composition. In some embodiments, the method comprises administering the pharmaceutical composition in combination with an agent that ameliorates one or more side effects associated with the pharmaceutical composition. In some embodiments, the cancer is solid tumor, lymphoma, or leukemia. In some embodiments, the cancer is from the group consisting of renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney cancer, and gastric cancer selected. In some embodiments, less cytokines are released in a subject compared to a subject administered an effective amount of autologous T cells expressing TFP as described herein. In some embodiments, the method does not induce graft-versus-host disease. In some embodiments, the subject has a reduced risk of developing graft-versus-host disease compared to a subject administered an effective amount of autologous T cells expressing TFP described herein.

[0403] In another aspect, the present disclosure provides a recombinant nucleic acid as described herein, a vector as described herein, a modified T as described herein, for use as a medicament or in the preparation of a medicament. Cells or pharmaceutical compositions described herein are provided.

[0404] In some embodiments, disclosed herein are (a)(i) (1) at least a portion of the TCR extracellular domain, (2) the transmembrane domain, and (3) CD3ε , CD3γ, CD3δ, TCRγ, TCRδ, TCRα, or TCRβ intracellular signaling domains, and (ii) a human or humanized antibody comprising an antigen binding domain. and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRγ constant domain, a TCRδ constant domain, or a TCRγ A recombinant nucleic acid comprising a constant domain and a sequence that is a TCRdelta constant domain, wherein the TCR subunit and antibody are operably linked, and when expressed in a T cell, TFP is operably integrated into the TCR complex .

[0405] In some embodiments, disclosed herein are (a)(i) (1) at least a portion of the TCR extracellular domain, (2) the transmembrane domain, and (3) CD3ε (ii) a binding ligand or fragment thereof capable of binding to an antibody or fragment thereof; and (b) a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCRγ constant domain, a TCRδ constant domain, or The TCR subunits and binding ligands or fragments thereof are operably linked, and TFP contains a functional disruption of the endogenous TCR when expressed in T cells. A recombinant nucleic acid that is functionally incorporated into the complex.

[0406] In one embodiment, the sequence encoding the antigen- or ligand-binding domain is operably linked to the sequence encoding the delta constant domain. In another embodiment, the antigen- or ligand-binding domain encoding sequence is operably linked to both the TCRδ constant domain or fragment thereof encoding sequence and the TCRγ constant domain or fragment thereof encoding sequence. there is In another embodiment, the intracellular signaling domain is CD3ε. In another embodiment, the intracellular signaling domain is CD3γ. In another embodiment, the recombinant nucleic acid further comprises at least one leader sequence and at least one linker. In another embodiment, the recombinant nucleic acid further comprises a portion of the TCRα constant domain, a portion of the TCRβ domain, or both.

[0407] In another embodiment, the recombinant nucleic acid sequence comprises, from 5' to 3', a first leader sequence, an antigen binding domain sequence, a linker, a TRDC gene sequence, a cleavable linker sequence, a second leader sequence. sequence, and TRGC gene sequence.

[0408] In another embodiment, the recombinant nucleic acid sequence comprises, in the 5'-3' direction, a first leader sequence, a TRDC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, It contains a linker sequence and a TRGC gene sequence.

[0409] In another embodiment, the recombinant nucleic acid sequence comprises, in the 5'-3' direction, a first leader sequence, an antigen binding domain sequence, a first linker sequence, a TRDC gene sequence, a cleavable linker, a 2 leader sequences, a second antigen binding domain sequence, a second linker sequence, and a TRGC gene sequence.

[0410] In another embodiment, the recombinant nucleic acid sequence comprises, in the 5'-3' direction, a first leader sequence, a TRDC gene sequence, a first cleavable linker sequence, a second leader sequence, a TRGC gene a sequence, a second cleavable linker sequence, a third leader sequence, an antigen binding domain sequence, a linker sequence, and a CD3ε gene sequence.

[0411] In another embodiment, the recombinant nucleic acid sequence comprises, in the 5'-3' direction, a first leader sequence, a first antigen binding domain sequence, a first linker sequence, a TRDC gene sequence or a fragment thereof, A TRAC gene sequence or fragment thereof, a cleavable linker sequence, a second leader sequence, a second antigen binding domain sequence, a second linker sequence, a TRGC gene sequence or fragment thereof, and a TRBC gene sequence or fragment thereof.

[0412] In one embodiment, the sequence encodes the polypeptide set forth in SEQ ID NO:1.
In another embodiment, the sequence encodes the polypeptide set forth in SEQ ID NO:2. In another embodiment, the sequence encodes the polypeptide set forth in SEQ ID NO:3. In another embodiment, the sequence encodes a polypeptide set forth in SEQ ID NO:4. In another embodiment, the sequence encodes a polypeptide set forth in SEQ ID NO:5. In one embodiment, the recombinant nucleic acid sequence further comprises at least a subsequence encoding a TCRα constant domain, a subsequence encoding a TCRβ constant domain, or a subsequence of both the TCRα and TCRβ constant domains.

[0413] Optionally, the binding ligand can bind to the Fc domain of the antibody. In some cases, the binding ligand can selectively bind the IgG1 antibody. In some cases, the binding ligand can specifically bind the IgG1 antibody. Optionally, the antibody or fragment thereof binds to a cell surface antigen. Optionally, the antibody or fragment thereof binds to a cell surface antigen on the surface of tumor cells. Optionally, the binding ligand comprises a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer . Optionally, the binding ligand does not include an antibody or fragment thereof. Optionally, the binding ligand comprises a CD16 polypeptide or fragment thereof. Optionally, the binding ligand comprises a CD16 binding polypeptide. Optionally, the binding ligand is human or humanized. Optionally, the recombinant nucleic acid further comprises a nucleic acid sequence encoding an antibody or fragment thereof that can be bound by a binding ligand. In some cases, the antibody or fragment thereof can be secreted from the cell.

[0414] In some embodiments, disclosed herein are (a)(i) (1) at least a portion of the TCR extracellular domain, (2) the transmembrane domain, and (3) CD3ε , CD3γ, CD3δ, TCRα, TCRβ, TCRγ, or a TCR subunit comprising an intracellular domain comprising a stimulatory domain from the intracellular signaling domain of TCRδ; (b) a sequence encoding a TCR constant domain, the TCR constant domain comprising TCRγ constant domain, TCRδ constant domain, or a sequence that is a TCRγ constant domain and a TCRδ constant domain, wherein the TCR subunit and antigen domain are operably linked, and the TFP comprises functional disruption of the endogenous TCR A recombinant nucleic acid that is functionally integrated into the TCR complex when expressed in a T cell. Optionally, the antigen domain includes a ligand. In some cases, the ligand binds to a receptor on the cell. In some cases, the ligand binds to a polypeptide expressed on the surface of the cell. Optionally, the receptor or polypeptide expressed on the surface of the cell comprises a stress response receptor or polypeptide. In some cases, the receptor or polypeptide expressed on the surface of the cell is an MHC class I-associated glycoprotein. Optionally, the MHC class I-associated glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4, and combinations thereof. Optionally, the antigen domain comprises a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. . Optionally, the antigen domain comprises a monomer or dimer of the ligand or fragment thereof. Optionally, the ligand or fragment thereof is a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. including. Optionally, the ligand or fragment thereof is monomeric or dimeric. Optionally, the antigen domain does not include an antibody or fragment thereof. In some cases, the antigen domain does not include variable regions. Optionally, the antigen domain does not contain CDRs. Optionally, the ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or fragment thereof.

[0415] In some embodiments, with respect to the recombinant nucleic acids disclosed above, the TCR constant domain is incorporated into a functional TCR complex upon expression in a T cell. In some cases, the TCR constant domain is incorporated into the same functional TCR complex that incorporates TFP when expressed in T cells. In some instances, the TFP-encoding sequence and the TCR constant domain-encoding sequence are contained within the same nucleic acid molecule. In some cases, T.
The FP-encoding sequence and the TCR constant domain-encoding sequence are contained within different nucleic acid molecules. Optionally, the TCR subunit and antibody domain, antigen domain, or binding ligand, or fragment thereof are operably linked by a linker sequence. Optionally, the linker sequence comprises (G ₄ S) _n , where n=1-4. Optionally, the transmembrane domain is a TCR transmembrane domain from CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRγ, or TCRδ. Optionally, the intracellular domain is derived from CD3ε only, CD3γ only, CD3δ only, TCRα only, TCRβ only, TCRγ only, or TCRδ only. Optionally, the TCR subunit comprises (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain, wherein (i), (ii), and ( At least two of iii) are from the same TCR subunit. Optionally, the TCR extracellular domain comprises a TCRα chain, a TCRβ chain, a TCRγ chain, a TCRδ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, functional fragments thereof, and at least one but no more than 20 modifications. the extracellular domain of a protein or a portion thereof selected from the group consisting of amino acid sequences thereof having Optionally, the TCR subunit is a TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ chain, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, A transmembrane protein selected from the group consisting of CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications. Contains a transmembrane domain containing domain. Optionally, the TCR subunit comprises a stimulatory domain of a protein selected from the intracellular signaling domains of CD3ε, CD3γ, or CD3δ, or a TCR intracellular domain comprising an amino acid sequence having at least one modification thereto. include. Optionally, the TCR subunit is a stimulatory domain of a protein selected from the functional signaling domain of 4-1BB and/or the functional signaling domain of CD3ζ, or an amino acid sequence having at least one modification thereto. containing an intracellular domain containing Optionally, the recombinant nucleic acid further comprises a sequence encoding a co-stimulatory domain. Optionally, the co-stimulatory domain is OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137), and at least one but Include a functional signaling domain of a protein selected from the group consisting of amino acid sequences thereof having 20 or fewer modifications. Optionally, the TCR subunit is CD3ζ TCR subunit, CD3ε TCR subunit, CD3γ TCR subunit, CD3δ TCR subunit, Fcε receptor chain 1, Fcε receptor 2 chain, Fcγ receptor 1 chain, Fcγ receptor 2a chain, Fcγ Receptor 2b1 chain, Fcγ receptor 2b2 chain, Fcγ receptor 3a chain, Fcγ receptor 3b chain, Fcβ receptor 1 chain, TYROBP (DAP12), CD5, CD16a, CD16b, CD22, CD23, CD32, CD64, CD79a, A protein immunoreceptor tyrosine-activating motif (ITAM) or its ITAM of the TCR subunit, including part. In some cases, the ITAM replaces the ITAM of CD3γ, CD3δ, or CD3ε. Optionally, the ITAM is selected from the group consisting of a CD3ζ TCR subunit, a CD3ε TCR subunit, a CD3γ TCR subunit, and a CD3δ TCR subunit; Replaces another ITAM that is selected. In some cases, TFP, the TCRγ constant domain, the TCRδ constant domain, and any combination thereof are capable of functionally interacting with the endogenous TCR complex and/or at least one endogenous TCR polypeptide. Optionally, (a) the TCR constant domain is a TCRγ constant domain and the TFP is functionally integrated into a TCR complex comprising endogenous subunits of TCRδ, CD3ε, CD3γ, CD3δ, or a combination thereof; (b) the TCR constant domain is a TCRδ constant domain, and the TFP is functionally integrated into a TCR complex comprising endogenous subunits of TCRγ, CD3ε, CD3γ, CD3δ, or a combination thereof; or (c) The TCR constant domains are the TCRγ constant domain and the TCRδ constant domain, and TFP is functionally integrated into the TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or combinations thereof. Optionally, at least one but no more than 20 modifications thereto include modifications of amino acids that mediate cell signaling or modifications of amino acids that are phosphorylated in response to ligand binding to TFP. Optionally, the antibody is an antibody fragment. Optionally, the antibody fragment is a scFv, single domain antibody domain, _VH domain, or _VL domain. Optionally, the human or humanized antibody comprising the antigen binding domain is an anti-CD19 binding domain, an anti-B cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-MUC16 binding domain, an anti-IL13Rα2 binding domain , anti-CD22 binding domain, anti-PD-1 binding domain, anti-PD-L1 binding domain, anti-BAFF or BAFF receptor binding domain, and anti-ROR-1 binding domain. Optionally, the nucleic acid is selected from the group consisting of DNA and RNA. In some cases, the nucleic acid is mRNA. Optionally, a recombinant nucleic acid comprises a nucleic acid analogue, which is not in the coding sequence of the recombinant nucleic acid. In some cases, the nucleic acid analogue is 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2 '-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O -DMAP), TO-dimethylaminoethyloxyethyl (2′-O-DMAEOE), 2′-ON-methylacetamide (2′-O-NMA) modification, locked nucleic acid (LNA), ethylene nucleic acid ( ENA), peptide nucleic acids (PNA), 1′,5′-anhydrohexitol nucleic acids (HNA), morpholinos, methylphosphonate nucleotides, thiolphosphonate nucleotides, and 2′-fluoro N3-P5′-phosphoramidites. selected from the group consisting of Optionally, the recombinant nucleic acid further comprises a leader sequence. Optionally, the recombinant nucleic acid further comprises a promoter sequence. Optionally, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. Optionally, the recombinant nucleic acid further comprises a 3'UTR sequence. Optionally, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. Optionally, the nucleic acid is an in vitro transcribed nucleic acid. Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRα transmembrane domain. Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRβ transmembrane domain. Optionally, the recombinant nucleic acid further comprises a sequence encoding a TCRα transmembrane domain and a sequence encoding a TCRβ transmembrane domain.

[0416] In some embodiments, disclosed herein are vectors comprising the recombinant nucleic acids disclosed herein. Optionally, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors (AAV), Rous sarcoma virus (RSV) vectors, or retroviral vectors. Optionally, the vector is an AAV6 vector. Optionally, the vector further comprises a promoter. In some cases, the vector is in
It is an in vitro transcribed vector.

[0417] In some embodiments, disclosed herein is a modified T cell comprising a recombinant nucleic acid disclosed above or a vector disclosed above, wherein the modified T cell is endogenous Including functional disruption of the TCR. In some embodiments, further disclosed herein is a TFP encoded by a sequence encoding a TFP of a nucleic acid disclosed above, or a sequence of a nucleic acid disclosed above encoding a TFP. A modified T cell comprising a functional disruption of an endogenous TCR. Also disclosed herein are engineered allogeneic T cells comprising a TFP encoded by a sequence encoding a TFP disclosed above or a sequence of a nucleic acid encoding a TFP disclosed above. Optionally, the T cell further comprises a heterologous sequence encoding a TCR constant domain, which TCR constant domain is a TCRγ constant domain, a TCRδ constant domain, or a TCRγ and a TCRδ constant domain. Optionally, the endogenous TCR that is functionally disrupted is an endogenous TCRα chain, an endogenous TCRβ chain, or an endogenous TCRα chain and an endogenous TCRβ chain. In some cases, endogenous TCRs that are functionally disrupted have reduced binding to MHC-peptide complexes compared to those of unmodified control T cells. In some cases, the functional disruption is disruption of the gene encoding the endogenous TCR. In some cases, disruption of the endogenous TCR-encoding gene is removal of sequences of the endogenous TCR-encoding gene from the genome of the T cell. Optionally, the T cells are human T cells. Optionally, the T cells are CD8 ⁺ T cells, CD4 ⁺ T cells, naive T cells, memory stem T cells, central memory T cells, double negative T cells, effector memory T cells, effector T cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, γδT cells, natural killer (NK) cells, natural killer T (NKT) cells, hematopoietic stem cells, or pluripotent stem cells. Optionally, the T cells are CD8 ⁺ T cells or CD4 ⁺
T cells. In some embodiments, the T cells are CD4 ⁺ CD8 ⁺ T cells. Optionally, the T cells are allogeneic T cells. Optionally, the modified T cell is a nucleic acid encoding an inhibitory molecule comprising a first polypeptide comprising at least a portion of the inhibitory molecule associated with a second polypeptide comprising a positive signal from an intracellular signaling domain further includes Optionally, the inhibitory molecule comprises a first polypeptide comprising at least a portion of PD1 and a second polypeptide comprising a co-stimulatory domain and a primary signaling domain.

[0418] In some embodiments, disclosed herein are pharmaceutical compositions comprising (a) the engineered T cells of the present disclosure and (b) a pharmaceutically acceptable carrier.
[0419] In some embodiments, disclosed herein is a method of making a modified T cell of the present disclosure, comprising: (a) a TCRα chain, a TCRβ chain, or a TCRα chain; disrupting the endogenous TCR gene encoding the TCR beta chain, thereby generating a T cell comprising a functional disruption of the endogenous TCR gene; and (b) a T cell comprising a functional disruption of the endogenous TCR gene. and transducing the recombinant nucleic acid or vector disclosed herein. Optionally, the disruption comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding the nuclease protein that targets the endogenous genes encoding the TCRα chain, the TCRβ chain, or the TCRα and TCRβ chains. . In some embodiments, further disclosed herein are methods of making the modified T cells of the present disclosure, wherein T cells comprising a functional disruption of an endogenous TCR gene including transducing the recombinant nucleic acid or vector disclosed herein. Optionally, the T cell with a functional disruption of an endogenous TCR gene is a T cell with a functional disruption of an endogenous TCR gene encoding a TCR α chain, a TCR β chain, or a TCR α chain and a TCR β chain. Optionally, the T cells are human T cells. In some cases, T cells containing a functional disruption of the endogenous TCR gene have reduced binding to MHC-peptide complexes compared to those of unmodified control T cells. Optionally, the nuclease is a meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), CRISPR/Cas nuclease, or megaTAL nuclease. Optionally, the sequence contained in the recombinant nucleic acid or vector is inserted into the endogenous TCR subunit gene at the cleavage site, and insertion of the sequence into the endogenous TCR subunit gene renders the endogenous TCR subunit functional. be destroyed. Optionally the nuclease is a meganuclease. Optionally, the meganuclease comprises a first subunit and a second subunit, the first subunit binding to the first recognition half-site of the recognition sequence and the second subunit binding to the recognition sequence. Binds to the second recognition half-site. Optionally, the meganuclease is a single-stranded meganuclease comprising a linker, the linker covalently joining the first subunit and the second subunit.

[0420] In some embodiments, disclosed herein is a method of treating cancer in a subject in need thereof, the method comprising a therapeutically effective amount of to the subject. In some embodiments, also disclosed herein is a method of treating cancer in a subject in need thereof, the method comprising: (a) made according to the methods disclosed herein; and (b) a pharmaceutically acceptable carrier. Optionally, the modified T cells are allogeneic T cells. In some cases, less cytokines are released in the subject compared to subjects receiving an effective amount of unmodified control T cells. In some cases, less cytokines are released in a subject compared to a subject administered an effective amount of a modified T cell comprising a recombinant nucleic acid disclosed herein or a vector disclosed herein. Optionally, the method includes administering the pharmaceutical composition in combination with an agent that increases the effectiveness of the pharmaceutical composition. Optionally, the method includes administering the pharmaceutical composition in combination with an agent that ameliorates one or more side effects associated with the pharmaceutical composition. In some cases, the cancer is solid tumor, lymphoma, or leukemia. Optionally, the cancer is selected from the group consisting of renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney cancer, and gastric cancer .

[0421] In some embodiments, disclosed herein are recombinant nucleic acids, vectors, modified T cells, or A pharmaceutical composition.

engineered T cells
[0422] In some embodiments, disclosed herein is a modified T cell comprising a recombinant nucleic acid disclosed herein or a vector disclosed herein, wherein the modified T cell Cells contain a functional disruption of the endogenous TCR. In some embodiments, disclosed herein is also a TFP-encoding sequence of a nucleic acid disclosed herein, or a TFP encoded by a sequence of a nucleic acid disclosed herein. A modified T cell comprising a functional disruption of an endogenous TCR. In some embodiments, further disclosed herein are modifications comprising a TFP encoded by a sequence encoding a TFP disclosed herein, or a sequence of a nucleic acid disclosed herein Allogeneic T cells.

[0423] Optionally, the T cell further comprises a heterologous sequence encoding a TCR constant domain, the TCR constant domains being a TCRα constant domain, a TCRβ constant domain, a TCRα constant domain and a TCRβ constant domain, a TCRγ constant domain, a TCRδ constant domain. domains, or the TCRγ constant domain and the TCRδ constant domain. Optionally, the endogenous TCR that is functionally disrupted is an endogenous TCRα chain, an endogenous TCRβ constant domain, or an endogenous TCRα constant domain and an endogenous TCRβ constant domain, an endogenous TCRγ chain, an endogenous TCRδ chain, or endogenous TCRγ chain and endogenous TCRδ chain. In some cases, endogenous TCRs that are functionally disrupted have reduced binding to MHC-peptide complexes compared to those of unmodified control T cells. In some cases, the functional disruption is disruption of the gene encoding the endogenous TCR. In some cases, disruption of the endogenous TCR-encoding gene is removal of sequences of the endogenous TCR-encoding gene from the genome of the T cell. Optionally, the T cells are human T cells. Optionally, the T cells are CD8 ⁺ T cells or CD4 ⁺ T cells. Optionally, the T cells are allogeneic T cells. Optionally, the modified T cell associates a nucleic acid encoding an inhibitory molecule comprising a first polypeptide comprising at least a portion of the inhibitory molecule with a second polypeptide comprising a positive signal from an intracellular signaling domain. and further includes Optionally, the inhibitory molecule comprises a first polypeptide comprising at least a portion of PD1 and a second polypeptide comprising a co-stimulatory domain and a primary signaling domain.

source of T cells
[0424] Prior to expansion and genetic modification, a source of T cells is obtained from the subject. The term "subject" is intended to include organisms (eg, mammals) capable of eliciting an immune response. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, splenic tissue, and tumors. Certain aspects of the present disclosure may use any number of T cell lines available in the art. In certain aspects of the present disclosure, T cells can be obtained from blood units taken from a subject using any number of techniques known to those of skill in the art, such as Ficoll™ separation. In one preferred aspect, cells from the blood stream of an individual are obtained by apheresis. Apheresis products usually contain T cells, monocytes, granulocytes, lymphocytes including B cells, other nucleated leukocytes, red blood cells, and platelets. In one aspect, cells harvested by apheresis may be washed to remove the plasma fraction and placed in appropriate buffers or media in preparation for subsequent processing steps. In one aspect of the disclosure, cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the wash solution may be free of calcium, free of magnesium, or free of many, if not all, divalent cations. Activation can be increased by the absence of calcium in the initial activation step. As will be readily appreciated by those skilled in the art, washing steps may be performed using methods known to those skilled in the art, such as semi-automatic "flow-through" centrifuges (e.g., Cobe® 2991 cell processor, Baxter Oncology CytoMate). ®), or Haemonetics® Cell Saver® 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in various biocompatible buffers such as Ca-free PBS, Mg-free PBS, PlasmaLyte A, or other saline with or without buffers. . Alternatively, unwanted components of the apheresis sample may be removed and the cells directly resuspended in culture medium.

[0425] In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, e.g., by centrifugation on a PERCOLL® gradient, or countercurrent centrifugal elution. . Specific T cell subpopulations, 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, incubating with anti-CD3/anti-CD28 (eg, 3×28) conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a period of time sufficient for positive selection of the desired T cells. T cells are isolated by. 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 therebetween. In a further aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10-24 hours. In one aspect, the incubation period is 24 hours. Longer incubations for T cell isolation may be required in any situation where T cells are scarce compared to other cell types, such as when isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals. Time may be used. Furthermore, the capture efficiency of CD8 ⁺ T cells can be increased by using longer incubation times. Thus, simply shortening or lengthening the time allows T cells to bind to the CD3/CD28 beads and/or increases or decreases the bead to T cell ratio (as described further herein). This allows preferential selection of subpopulations of T cells at the beginning of the culture or at other points in the process, depending on availability. In addition, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on beads or other surfaces, preferential selection of subpopulations of T cells may or may not be made at culture initiation or other desired time points. can do. Those skilled in the art will recognize that multiple selections can also be used in the context of this disclosure. In certain aspects, it may be desirable to perform a selection procedure and use "non-selected" cells in the activation and expansion process. "Unselected" cells may also be subjected to several more rounds of selection.

[0426] Enrichment of the T cell population by negative selection can be performed using a combination of antibodies against surface markers specific to the negatively selected cells. One method is cell sorting and/or sorting by negative magnetic immunoadherence or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on negatively selected cells. For example, to enrich for CD4 ⁺ cells by negative selection, a monoclonal antibody cocktail is usually used for C
Includes antibodies against D14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain aspects, it may be desirable to enrich for or positively select regulatory T cells that normally express CD4 ⁺ , CD25 ⁺ , CD62Lhi, GITR ⁺ , and FoxP3 ⁺ . Alternatively, in certain embodiments, regulatory T cells are depleted by anti-C25 conjugated beads or other similar selection methods.

[0427] In one embodiment, IFN-γ, TNF-α, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, Or T cell populations can be selected that express one or more other suitable molecules, such as other cytokines. Cellular expression screening methods can be determined, for example, as described in PCT Publication No. WO2013/126712.

[0428] When isolating the desired cell population by positive or negative selection, the concentration of cells and surfaces (eg, particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the amount of bead-cell mixing (eg, increase the cell concentration) to maximize cell-bead contact. For example, in one aspect a concentration of 2 billion cells/mL is used. In one aspect, a concentration of 1 billion cells/mL is used. In a further aspect, greater than 100 million cells/mL is used. In further aspects, cell concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells/mL are used. In yet another aspect, cell concentrations of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells/mL are used. In further aspects, concentrations of 125 million or 150 million cells/mL can be used. Using high concentrations can result in increased cell yield, cell activation, and cell proliferation. Furthermore, the use of high cell concentrations allows cells that may weakly express the target antigen of interest, such as CD28-negative T cells, or from samples rich in tumor cells (e.g., leukemic blood, tumor tissue, etc.). cells can be captured more efficiently. Such cell populations may have therapeutic value and would be desirable to obtain. For example, the use of high concentrations of cells allows efficient selection of CD8 ⁺ T cells that normally have weak CD28 expression.

[0429] In related embodiments, it may be desirable to use low concentrations of cells. By greatly diluting the mixture of T cells and surfaces (eg, particles such as beads), interactions between particles and cells are minimized. This selects cells that express large amounts of the desired antigen to bind to the particles. For example, CD4 ⁺ T cells express higher levels of CD28 and are trapped more efficiently than CD8 ⁺ T cells at dilute concentrations. In one aspect, the concentration of cells used is 5×10 ⁶ /mL. In other aspects, the concentration used can be from about 1×10 ⁵ /mL to 1×10 ⁶ /mL, and all integer values therebetween. In other embodiments, the cells can be incubated on a rotator at various speeds at 2-10° C. or room temperature for various lengths of time.

[0430] T cells for stimulation can also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing step removes granulocytes and to some extent monocytes from the cell population, resulting in a more uniform product. After washing steps to remove plasma and platelets, the cells may be suspended in a freezing solution. Many freezing solutions and parameters are known in the art and useful in this context, but one method is PBS containing 20% DMSO and 8% human serum albumin, or 10% dextran 40 and 5%. Culture medium containing 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% Use culture medium containing dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, then freeze the cells at -80°C each time. Freezing at a rate of 1 minute and storing in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods may be used as well as uncontrolled rapid freezing in -20°C or liquid nitrogen. In certain aspects, cryopreserved cells are thawed as described herein, washed, left at room temperature for 1 hour, and then activated using the methods of the present disclosure.

[0431] Also contemplated, in the context of the present disclosure, is taking a blood sample or apheresis product from a subject at a time before expansion of the cells described herein may be required. Thus, sources of cells to expand can be harvested at any time as needed and T cells directed to any number of diseases or conditions for which T cell therapy as described herein is beneficial. It can be isolated and frozen for later use in therapy. In one aspect, the blood sample or apheresis is taken from a generally healthy subject. In certain embodiments, the blood sample or apheresis is taken from a generally healthy subject who is at risk of developing disease but has not yet developed disease, and the subject's cells are isolated for later use. Frozen. In certain aspects, T cells may be expanded, frozen, and later used. In certain aspects, a sample is taken from a patient shortly after being diagnosed with a particular disease described herein, but prior to any treatment. In a further aspect, the cells are isolated from a blood sample or apheresis from the subject prior to any number of relevant therapeutic modalities. Such treatments include drugs such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, and mycophenolate, antibodies, or alemtuzumab, anti-CD3 antibodies, cytoxan. , fludarabine, cyclosporine, tacrolimus, rapamycin, mycophenolic acid, steroids, other immunodepleting drugs such as romidepsin, and treatment with irradiation.

[0432] In a further aspect of the present disclosure, T cells are obtained from a patient directly after treatment that leaves the subject with functional T cells. In this regard, after certain cancer treatments, particularly with drugs that damage the immune system, the quality of the T cells obtained during the immediate posttreatment period, when the patient is normally recovering from the treatment, is It has been observed that ex vivo growth may be optimal or enhanced. Similarly, after ex vivo manipulation using the methods described herein, these cells may be in a favorable state for engraftment and enhanced in vivo proliferation. Therefore, it is contemplated within the context of the present disclosure to harvest blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage during this recovery period. Furthermore, in certain aspects, mobilization (e.g., mobilization with GM-CSF) and conditional regimens are used to repopulate, recirculate, repopulate, recirculate, and repopulate specific cell types, particularly for a defined timeframe following therapy. Conditions can be created in a subject that favor regeneration and/or proliferation. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

T cell activation and proliferation
[0433] T cells are generally described, for example, in US Pat. 858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, Nos. 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867 , 041 and 7,572,631 can be used to activate and propagate.

[0434] In general, the T cells of the present disclosure are contacted with a surface bound thereto with an agent that stimulates a CD3/TCR complex-associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cell. can be enhanced by In particular, the T-cell population may be stimulated by contact with a surface-immobilized anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody, or protein kinase C activity with a calcium ionophore, as described herein. can be stimulated, such as by contact with a stimulating factor such as bryostatin. Costimulation of accessory molecules on the surface of T cells uses ligands that bind to the accessory molecules. For example, a population of T cells can be contacted with anti-CD3 and anti-CD28 antibodies under conditions suitable to stimulate proliferation of the T cells. Anti-CD3 and anti-CD28 antibodies for stimulating proliferation of either CD4 ⁺ T cells or CD8 ⁺ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and can be used as well as other methods commonly known in the art ( Ber
g et al. , Transplant Proc. 30(8):3975-3977, 1998, Haanen et al. , J. Exp. Med. 190(9): 13191328, 1999, Garland et al. , J. Immunol Meth. 227(1-2):53-63, 1999). T cells can be further activated and proliferated in the presence of cytokines, with or without anti-CD3 and/or CD28 antibodies. Exemplary cytokines include IL-2, IL-7, IL-15, and IL-21.

[0435] T cells exposed to stimuli for varying times may exhibit different characteristics. For example, normal blood or apheresis peripheral blood mononuclear cell products have a greater helper T cell population (TH, CD4 ⁺ ) than a cytotoxic or suppressor T cell population (TC, CD8 ⁺ ). Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors produces a T cell population consisting primarily of TH cells before about days 8-9, but after about days 8-9. , the population of T cells becomes increasingly rich in populations of TC cells. Therefore, depending on the purpose of treatment, it may be advantageous to infuse a subject with a T cell population comprising primarily TH cells. Similarly, where an antigen-specific subset of TC cells has been isolated, it may be beneficial to expand this subset to a higher degree.

[0436] Furthermore, in addition to the CD4 and CD8 markers, other phenotypic markers also vary significantly, but mostly reproducibly, during the course of cell proliferation. Such reproducibility therefore allows tailoring of the activated T cell product for specific purposes.

[0437] Anti-CD19, anti-BCMA, anti-CD22, anti-ROR1, anti-PD-1, or anti-BAFF, anti-MUC16, anti-mesothelin, anti-HER2, anti-PMSA, anti-CD20, anti-CD70, anti-GPC3, anti-nectin-4, After constructing an anti-Trop2 or anti-CD79b TFP, various assays can be used to assess the activity of the molecule, including the ability to proliferate T cells after antigen stimulation, the absence of restimulation. and anti-cancer activity in suitable in vitro and animal models. anti-CD19, anti-BCMA, anti-GPC3, anti-Nectin-4, anti-Trop2, anti-CD22, anti-MSLN, anti-CD79B, anti-ROR1, anti-PD-1, anti-IL13Ra2, anti-PD-L1, anti-CD20, anti-CD70, or Assays for evaluating the effects of anti-BAFF or BAFFR TFPs are described in further detail below.

[0438] Western blot analysis of TFP expression in primary T cells can be used to detect the presence of monomers and dimers (see, eg, Milone et al., Molecular Therapy 17(8):1453-1464). (2009)). Very briefly, TFP-expressing T cells (a 1:1 mixture of CD4 ⁺ and CD8 ⁺ T cells) were grown in vitro for more than 10 days before being lysed and exposed to reducing conditions. Apply to SDS-PAGE with . TFP is detected by Western blotting using an antibody against the TCR chain. The same subset of T cells is used for SDS-PAGE analysis under non-reducing conditions to allow assessment of covalent dimer production.

[0439] In vitro proliferation of TFP ⁺ T cells after antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4 ⁺ and CD8 ⁺ T cells are stimulated with αCD3/αCD28 and APC, then transduced with a lentiviral vector expressing GFP under the control of a promoter, and analyzed. Exemplary promoters include the CMV IE gene, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK) promoters. On day 6 of culture with CD4 ⁺ and/or CD8 ⁺ T cell subsets, GFP fluorescence is assessed by flow cytometry (eg, Milone et al., Molecular Therapy 17(8):1453-1464 (2009)). ). Alternatively, a mixture of CD4 ⁺ and CD8 ⁺ T cells was stimulated on day 0 with αCD3/αCD28-coated magnetic beads and on day 1 a bicistronic lentiviral vector expressing TFP was injected into the 2A ribosomes. Used with eGFP with a skipping sequence to transduce TFP. After washing, TAA ⁺ K562 cells (K562-TAA), wild-type K562 cells (K562 wild-type), or K562 cells expressing hCD32 and 4-1BBL in the presence of anti-CD3 and anti-CD28 antibodies (K562-BBL-3 /28). Exogenous IL-2 is added to the cultures at 100 IU/mL every other day. GFP ⁺ T cells are counted by flow cytometry using bead-based counting (see, eg, Milone et al., Molecular Therapy 17(8):1453-1464 (2009)).

[0440] Sustained TFP ⁺ T cell proliferation without restimulation can also be measured (see, eg, Milone et al., Molecular Therapy 17(8):1453-1464 (2009)). Briefly, after stimulation with αCD3/αCD28-coated magnetic beads on day 0 and transduction with the indicated TFPs on day 1, mean T cell volumes (fl) on day 8 of culture were measured by Coulter Measured using a Multisizer III particle counter.

[0441] Animal models can also be used to measure TFP-T activity. For example, xenograft models using, for example, human CD19-specific TFP ⁺ T cells can be used to treat primary human pre-B ALL in immunodeficient mice (see, eg, Milone et al., Molecular Therapy 17 ( 8): 1453-1464 (2009)). After establishment of ALL, mice are randomized to treatment groups. Different numbers of engineered T cells are co-injected at a 1:1 ratio into NOD/SCID/γ ^{−/− mice} with B-ALL. The copy number of each vector in splenic DNA from mice is assessed at various time points after T cell infusion. Animals are evaluated for leukemia at weekly intervals. Peripheral blood CD19 ⁺ B-ALL blast counts are determined in mice injected with αCD19-ζ TFP ⁺ T cells, or mock-transduced T cells. Survival curves for each group are compared using the log-rank test. In addition, absolute numbers of peripheral blood CD4 ⁺ and CD8 ⁺ T cells 4 weeks after T cell infusion in NOD/SCID/γ ^{−/− mice} can also be analyzed. Mice are injected with leukemic cells and three weeks later are injected with T cells engineered to express TFP with a bicistronic lentiviral vector encoding TFP linked to eGFP. 45-50% input GFP ⁺ by mixing with mock-transduced cells prior to injection.
T cells are normalized to T cells and confirmed by flow cytometry. Animals are evaluated for leukemia at weekly intervals. Survival curves of TFP ⁺ T cell populations are compared using the log-rank test.

[0442] Dose-dependent TFP treatment response can be assessed (e.g., Milone
et al. , Molecular Therapy 17(8):1453-1464 (2009)). For example, on day 21, in mice injected with TFP T cells, mice injected with the same number of mock-transduced T cells, or mice without T cells, peripheral blood transfusions were observed 35-70 days after leukemia establishment. is collected. On days 35 and 49, mice in each group are randomly bled, peripheral blood CD19 ⁺ ALL blast counts are measured, and then sacrificed. The remaining animals are evaluated on days 57 and 70.

[0443] For evaluation of cell proliferation and cytokine production, see, eg, Milone et al. , Molecular Therapy 17(8):1453-1464 (2009). Briefly, TFP-mediated proliferation was assessed by quantifying washed T cells into tumor-associated antigens (TAAs such as CD19), K562 cells expressing CD19 (K19), or K562 cells expressing CD32 and CD137 (KT32-BBL). ) with a final T cell:K562 cell ratio of 2:1 and performed in microtiter plates. K562 cells are irradiated with gamma rays before use. Anti-CD3 (clone OKT3) and anti-CD28 (clone 9.3) monoclonal antibodies are added to cultures containing KT32-BBL cells to serve as positive controls to stimulate T cell proliferation. This is because this signal supports long-term ex vivo expansion of CD8 ⁺ T cells. T cells are counted in culture using CountBright™ fluorescent beads (Invitrogen) and flow cytometry according to the manufacturer's instructions. TFP ⁺ T cells are identified by expression of GFP using T cells engineered with a TFP-expressing lentiviral vector linked to eGFP-2A. TFP ⁺ not expressing GFP
For T cells, TFP ⁺ T cells are detected with biotinylated recombinant CD19 protein and a secondary avidin-PE conjugate. Expression of CD4 ⁺ and CD8 ⁺ on T cells is also detected simultaneously by specific monoclonal antibodies (BD Biosciences). Cytokine measurements are performed on supernatants harvested 24 hours after restimulation using the Human TH1/TH2 Cytokine Cytometry Bead Array Kit (BD Biosciences) according to the manufacturer's instructions. Fluorescence is assessed using a FACScalibur™ flow cytometer (BD Biosciences) and data are analyzed according to the manufacturer's instructions.

[0444] Cytotoxicity can be assessed by a standard ⁵¹ Cr release assay (see, eg, Milone et al., Molecular Therapy 17(8):1453-1464 (2009)). Target cells (strain K562 and primary pro-B-ALL cells) were loaded with ⁵¹ Cr (NaCrO ₄ , New England Nuclear) for 2 hours at 37° C. with frequent agitation, which was then mixed with complete RPMI for 2 hours. Wash twice and seed in microtiter plates. Effector T cells are mixed with target cells in wells of complete RPMI at various effector cell:target cell (E:T) ratios. In addition, wells containing medium alone (spontaneous release, SR) or a 1% solution of the detergent Triton-X 100 (total release, TR) are prepared. Supernatant is harvested from each well after 4 hours of incubation at 37°C. The released ⁵¹ Cr is then measured using a gamma particle counter (Packard Instrument Co., Waltham, Mass.). Each condition was performed in at least triplicate and the percent cell lysis was calculated using the formula: percent cell lysis = (ER-SR)/(TR-SR), where ER is the release in each experimental condition. represents the average of ⁵¹ Cr obtained).

[0445] Imaging techniques can be used to assess specific trafficking and proliferation of TFP in tumor-bearing animal models. Such assays are described, for example, in Barrett et al.
al. , Human Gene Therapy 22:1575-1586 (2011). T cells 4 hours after electroporation of TFP constructs 7 days after intravenous injection of Nalm-6 cells (ATCC® CRL-3273™) in NOD/SCID/γc ^−/− (NSG) mice administered intravenously. T cells are stably transfected with a lentiviral construct expressing firefly luciferase and the mice are imaged for bioluminescence. Alternatively, the therapeutic efficacy and specificity of a single injection of TFP ⁺ T cells in the Nalm-6 xenograft model can be determined as follows. NSG mice are injected with Nalm-6 transduced to stably express firefly luciferase, followed 7 days later by a single tail vein injection of TAA TFP-electroporated T cells. Animals are imaged at various time points after injection. For example, photon density heatmaps of firefly luciferase-positive leukemia in representative mice on day 5 (2 days before treatment) and day 8 (24 hours after TFP ⁺ PBL) can be generated.

[0446] Other assays disclosed herein, including those described in the Examples section of this specification, as well as those known in the art, may also be used for anti-CD19, anti-BCMA, anti-CD22, anti-CD22, MSLN, anti-CD79B, anti-GPC3, anti-nectin-4, anti-Trop2, anti-IL13Ra2, anti-PD-1, anti-ROR1, anti-PD-L1, or anti-BAFF or BAFFR
It can be used to evaluate TFP constructs.

Pharmaceutical composition
[0447] In some embodiments, disclosed herein are pharmaceutical compositions comprising (a) the engineered T cells of the present disclosure, and (b) a pharmaceutically acceptable carrier. Such compositions may include buffers such as neutral buffered saline, phosphate buffered saline, glucose, mannose, sucrose or dextran, carbohydrates such as mannitol, proteins, polypeptides or amino acids such as glycine, Oxidizing agents, chelating agents such as EDTA or glutathione, adjuvants (eg aluminum hydroxide), and preservatives may also be included. Compositions of the disclosure are, in one aspect, formulated for intravenous administration.

[0448] The pharmaceutical compositions of this disclosure can be administered in a manner appropriate for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient's disease, but an appropriate dose may be determined by clinical trials.

[0449] In one embodiment, the pharmaceutical composition comprises, for example, endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibody , pooled human serum, bovine serum albumin, bovine serum, culture medium components, vector packaging cell or plasmid components, bacteria, and fungi, substantially free of, e.g. detectable, contaminants. no significant levels of contaminants.一実施形態では、細菌は、Ａｌｃａｌｉｇｅｎｅｓ ｆａｅｃａｌｉｓ、Ｃａｎｄｉｄａ ａｌｂｉｃａｎｓ、Ｅｓｃｈｅｒｉｃｈｉａ ｃｏｌｉ、Ｈａｅｍｏｐｈｉｌｕｓ ｉｎｆｌｕｅｎｚａ、Ｎｅｉｓｓｅｒｉａ ｍｅｎｉｎｇｉｔｉｄｅｓ、Ｐｓｅｕｄｏｍｏｎａｓ ａｅｒｕｇｉｎｏｓａ、Ｓｔａｐｈｙｌｏｃｏｃｃｕｓ ａｕｒｅｕｓ、Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｐｎｅｕｍｏｎｉａ、及びＳｔｒｅｐｔｏｃｏｃｃｕｓ ｐｙｏｇｅｎｅｓ Ａ群からなる群から選択される少なくとも１種である。

[0450] When referring to an "immunologically effective amount,""antitumor effective amount,""tumor inhibiting effective amount," or "therapeutic amount," the precise amount of the composition of this disclosure to be administered is , age, weight, tumor size, degree of infection or metastasis, and individual differences in patient (subject) condition. Generally, pharmaceutical compositions comprising the T cells described herein will be administered at a concentration of 10 ⁴ to 10 ⁹ cells/kg body weight, optionally 10 ⁵ to 10 ⁶ cells/kg body weight, including all integer values within the range. It can be said that it can be administered in doses. Also, the T cell composition may be administered multiple times at these dosages. Cells can be administered using injection techniques commonly known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).

[0451] In certain embodiments, the activated T cells are administered to a subject, followed by subsequent rebleeding (or apheresis), activation of the T cells therefrom according to the present disclosure, and activation and proliferation of the T cells derived therefrom. It may be desirable to reinfuse the patient with the obtained T cells. This process can be performed multiple times every few weeks. In certain aspects, T cells can be activated from 10cc to 400cc blood draws. In certain aspects, T cells can be activated from 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc blood draws.

[0452] Administration of the compositions may be by any convenient method, including by aerosol inhalation, injection, oral ingestion, infusion, infusion, or implantation. The compositions described herein can be administered to patients by intraarterial, subcutaneous, intradermal, intratumoral, intralymphatic, intramedullary, intramuscular, or intravenous (i.v.) injection. administration, or intraperitoneal administration. In one aspect, the T cell compositions of this disclosure are administered to a patient by intradermal or subcutaneous injection. In one aspect, the T cell composition of the present disclosure comprises i. v. Administered by injection. T cell compositions may be injected directly into a tumor, lymph node, or site of infection.

[0453] In certain exemplary embodiments, the subject may undergo leukapheresis. This is ex
Leukocyte collection, enrichment, or depletion in vivo to select and/or isolate cells of interest, such as T cells. Such T cell isolates are grown by methods known in the art and treated to introduce one or more TFP constructs of the present disclosure to generate the modified TT cells of the present disclosure. be able to. Subjects in need thereof may subsequently receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain aspects, a subject receives an infusion of expanded engineered T cells of the present disclosure after or concurrently with transplantation. In additional aspects, the proliferating cells are administered preoperatively or postoperatively.

[0454] The dosage of the above treatments to be administered to a patient will vary depending on the condition being treated and the exact nature of the recipient of the treatment. Dosage weighing for human administration can be performed according to art-accepted practices. For example, doses of alemtuzumab generally range from 1 to about 100 mg for adult patients, usually administered daily for a period of 1 to 30 days. A preferred daily dose is 1-10 mg/day, although higher doses up to 40 mg/day may be used in some cases (as described in US Pat. No. 6,120,766).

[0455] In one embodiment, TFP is introduced into T cells, eg, using in vitro transcription, followed by an initial administration of TFP T cells of the disclosure, followed by one or more TFPs of the disclosure.
Administration of T cells is to a subject (e.g., a human), wherein one or more subsequent administrations are administered less than 15 days after the previous administration, e.g., 14, 13, 12, 11, 10, 9, 8, It is administered after 7, 6, 5, 4, 3, or 2 days. In one embodiment, multiple administrations of TFP T cells of the present disclosure are administered to a subject (e.g., human) on a weekly basis, e.g., 2, 3, or 4 administrations of TFP T cells of the present disclosure are Conduct weekly. In one embodiment, administration of TFP T cells multiple times per week (e.g., administration 2, 3, or 4 times per week) (also referred to herein as cycles) is administered to a subject (e.g., a human subject) Subjects undergo no TFP T cell administration for 1 week, followed by one or more additional TFP T cell administrations (e.g., multiple TFP T cell administrations per week) . In another embodiment, a subject (eg, a human subject) receives multiple cycles of TFP T cells, and each cycle is separated by less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, TFP T cells are administered every other day for 3 administrations per week. In one embodiment, the TFP T cells of this disclosure are administered for at least 2, 3, 4, 5, 6, 7, 8 weeks, or longer.

[0456] In one aspect, CD19 TFP T cells are produced using a lentiviral-type viral vector, such as a lentivirus. TFP-T cells produced in that manner stably express TFP.

[0457] In one aspect, the TFP T cells transiently express the TFP vector 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction. . Transient expression of TFP can be achieved by delivery of RNA TFP vectors. In one aspect, TFP RNA is transduced into T cells by electroporation.

[0458] A potential problem that may arise in patients treated using transiently expressing TFP T cells (particularly mouse scFv-bearing TFP T cells) is anaphylaxis after combination therapy.

[0459] Without wishing to be bound by theory, it is believed that such anaphylactic reactions may be induced by patients developing a humoral anti-TFP reaction, i.e., anti-TFP antibodies with the anti-IgE isotype. be Patients' antibody-producing cells are thought to class switch from the IgG isotype (which does not cause anaphylaxis) to the IgE isotype when antigen exposure is discontinued for 10-14 days.

[0460] If the patient is at high risk of developing an anti-TFP antibody response during transient TFP therapy (such as that produced by RNA transduction), discontinuation of TFP T cell infusion should be more than 10-14 days. should not continue for long.

Method for producing modified T cells
[0461] In some embodiments, disclosed herein is a method of making a modified T cell of the present disclosure, comprising: (a) TCRα chain, TCRβ chain, TCRγ chain, TCRδ disrupting an endogenous TCR gene encoding a chain, or a combination thereof, thereby generating a T cell comprising a functional disruption of the endogenous TCR gene; and (b) functional disruption of the endogenous TCR gene. transducing the containing T cells with a recombinant nucleic acid of the disclosure or a vector disclosed herein. Optionally, the disruption comprises transducing the T cell with a nuclease protein or a nucleic acid sequence encoding the nuclease protein that targets the endogenous genes encoding the TCRα chain, the TCRβ chain, or the TCRα and TCRβ chains. .

[0462] In some embodiments, further disclosed herein are methods of making the modified T cells of the present disclosure, wherein the methods comprise functional disruption of an endogenous TCR gene. Including transducing a cell with a recombinant nucleic acid disclosed herein or a vector disclosed herein. Optionally, the T cell with a functional disruption of an endogenous TCR gene is a T cell with a functional disruption of an endogenous TCR gene encoding a TCR α chain, a TCR β chain, or a TCR α chain and a TCR β chain.

[0463] Optionally, the T cell is a human T cell. In some cases, T cells containing a functional disruption of the endogenous TCR gene have reduced binding to MHC-peptide complexes compared to those of unmodified control T cells.

[0464] Optionally, the nuclease is a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, a CRISPR/Cas nickase, or a megaTAL nuclease. Optionally, the sequence contained in the recombinant nucleic acid or vector is inserted into the endogenous TCR subunit gene at the cleavage site, and insertion of the sequence into the endogenous TCR subunit gene renders the endogenous TCR subunit functional. be destroyed. Optionally the nuclease is a meganuclease. Optionally, the meganuclease comprises a first subunit and a second subunit, the first subunit binding to the first recognition half-site of the recognition sequence and the second subunit binding to the recognition sequence. Binds to the second recognition half-site. Optionally, the meganuclease is a single-stranded meganuclease comprising a linker, the linker covalently joining the first subunit and the second subunit.

gene editing technology
[0465] In some embodiments, the engineered T cells disclosed herein are characterized by clustered regularly interspaced short palindromic repeats (CRISPR®, see, e.g., US Pat. No. 8,697,359), transcription Activator-like effector (TALE) nucleases (TALENs, see, eg, US Pat. No. 9,393,257), meganucleases (endodeoxyribonucleases with large recognition sites containing double-stranded DNA sequences of 12-40 base pairs) ), zinc finger nuclease (ZFN, see for example Urnov et al., Nat. Rev. Genetics (2010) vl1, 636-646), or megaTAL nuclease (a fusion protein between a meganuclease and a TAL repeat) method. Manipulated using editing techniques. In this way, chimeric constructs can be engineered to combine desirable properties of each subunit, such as conformation or signaling ability. Sander & Joung, Nat. Biotech. (2014) v32, 347-55, and June et al.
al. , 2009 Nature Reviews Immunol. 9.10:704-716. In some embodiments, one or more extracellular, transmembrane, or cytoplasmic domains of a TFP subunit are engineered to have aspects of multiple naturally occurring TCR subunit domains (i.e., are chimeric). be.

[0466] In recent years, techniques have been developed to permanently alter the human genome and introduce site-specific genomic modifications into disease-associated genes, which are the cornerstone of therapeutic applications. These techniques are now commonly known as "genome editing".

[0467] Endogenous TCR genes encoding TCRα chains, TCRβ chains, or TCRα and TCRβ chains can be inactivated in the engineered cells (eg, engineered T cells) described herein. Inactivation can include disruption of genomic loci, gene silencing, inhibition or reduction of transcription, or inhibition or reduction of translation. Endogenous TCR genes can be silenced by inhibitory nucleic acids such as, for example, siRNAs and shRNAs. Translation of endogenous TCR genes can be inhibited by inhibitory nucleic acids such as microRNAs. In some embodiments, gene editing techniques are used to disrupt the endogenous TCR gene. In some embodiments, the endogenous TCR gene of interest encodes a TCRα chain, a TCRβ chain, or a TCRα chain and a TCRβ chain. In some embodiments, gene editing technology facilitates multiplex genome editing that allows simultaneous disruption of multiple genomic loci in the endogenous TCR gene. In some embodiments, gene editing techniques are used to disrupt the endogenous TCR gene. In some embodiments, the endogenous TCR gene of interest encodes a TCRα chain, a TCRβ chain, or a TCRα chain and a TCRβ chain. In some embodiments, gene editing technology facilitates multiplex genome editing that allows simultaneous disruption of multiple genomic loci in the endogenous TCR gene. In some embodiments, multiplex genome editing techniques are applied to express endogenous TCRs, and/or human leukocyte antigens (HLA), and/or programmed cell death protein 1 (PD1), and/or other genes. produces gene-disrupted T cells that are deficient in

[0468] Current gene editing technologies include meganucleases, zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems. These four major classes of gene-editing techniques share a common mechanism of action: binding of user-defined sequences of DNA and mediation of double-stranded DNA breaks (DSBs). Additionally, DSBs can be repaired by either non-homologous end joining (NHEJ) or (if donor DNA is present) homologous recombination (HR), an event that introduces homologous sequences from a donor DNA fragment. In addition, nickase nucleases produce single-strand DNA breaks (SSBs). DSBs can be repaired by single-strand DNA integration (ssDI) or single-strand template repair (ssTR), events that introduce homologous sequences from the donor DNA.

[0469] Genetic modification of genomic DNA can be performed using site-specific rare-cutting endonucleases engineered to recognize the DNA sequence of the locus of interest. Methods of making engineered site-specific endonucleases are known in the art. For example, zinc finger nucleases (ZFNs) can be engineered to recognize and cleave predetermined sites within the genome. ZFNs are chimeric proteins comprising a zinc finger DNA binding domain fused to the nuclease domain of the Fok1 restriction enzyme. A zinc finger domain can be redesigned by rational or experimental means to produce a protein that binds to a given DNA sequence that is 18 base pairs in length. By fusing this engineered protein domain to the Fok1 nuclease, it is possible to target DNA cleavage with genomic-level specificity. ZFNs are widely used for targeting gene additions, deletions, and replacements in a wide range of eukaryotes (Durai et al.
al. (2005), Nucleic Acids Res 33, 5978). Similarly, TAL effector nucleases (TALENs) can be produced that cleave specific sites in genomic DNA. Similar to ZFNs, TALENs contain an engineered site-specific DNA-binding domain fused to the Fok1 nuclease domain (Mak et al.
al. (2013), Curr Opin Struct Biol. 23:93-9). However, for TALENs, the DNA binding domain comprises a tandem array of TAL effector domains, each of which specifically recognizes a single DNA base pair. Small TALENs have an alternative endonuclease structure that does not require dimerization (Beurdeley et al. (2013), Nat Commun. 4:1762). Small TALENs have engineered site-specific TAL effector DNA-binding domains. fused to the nuclease domain of the I-TevI homing endonuclease. Unlike Fok1, I-TevI does not require dimerization to generate double-stranded DNA breaks, so the small TALENs function as monomers.

[0470] Engineered endonucleases based on the CRISPR/Cas9 system are also known in the art (Ran et al. (2013), Nat Protoc. 8:2281-2308, Mali et al. (2013) , Nat Methods 10:957-63). CRISPR gene editing technology consists of endonuclease proteins whose DNA targeting specificity and cleavage activity are programmable by short guide RNAs or duplex crRNA/TracrRNAs. A CRISPR endonuclease is a short "guide RNA" containing (1) a caspase effector nuclease, usually microbial Cas9, and (2) a targeting sequence of 18-20 nucleotides that directs the nuclease to a location of interest within the genome. or RNA duplex. By expressing multiple guide RNAs in the same cell, each with a different targeting sequence, it is possible to simultaneously target DNA cleavage to multiple sites within the genome (
multiplex genome editing).

[0471] There are two classes of CRISPR systems known in the art (Adli (2018) Nat. Commun. 9:1911), each containing multiple CRISPR types. Class 1 includes Type I and Type III CRISPR systems commonly found in archaea. Class II also includes Types II, IV, V, and VI CRISPR systems. The most widely used CRISPR/Cas system is the type II CRISPR-Cas9 system, although CRISPR/Cas systems have been repurposed by genome editing researchers. Within the past few years, over 10 different CRISPR/Cas proteins have been engineered (Adli (2018) Nat. Commun. 9:1911). Of particular interest are Cas12a (Cpf1) proteins derived from Acid-aminococcus sp (AsCpf1) and Lachnospiraceae bacterium (LbCpf1).

[0472] Homing endonucleases are a group of naturally occurring nucleases that recognize 15-40 base pair cleavage sites commonly found in plant and fungal genomes. They frequently associate with parasite DNA elements such as group 1 self-splicing introns and inteins. They naturally promote homologous recombination or gene insertion at specific locations in the host genome by generating double-strand breaks in chromosomes and recruiting cellular DNA repair machinery (Stoddard (2006), Q. Rev. Biophys. 38:49-95). Substitution of specific amino acids can reprogram the DNA-cleaving specificity of homing nucleases (Niyonzima (2017), Protein Eng Des Sei. 30(7):503-522). Meganucleases (MNs) are monomeric proteins with innate nuclease activity, derived from bacterial homing endonucleases and engineered for unique target sites (Gersbach (2016), Molecular Therapy. 24 : 430-446). In some embodiments, the meganuclease is an engineered I-CreI homing endonuclease. In other embodiments, the meganuclease is an engineered I-SceI homing endonuclease.

[0473] In addition to the four major gene-editing techniques mentioned, chimeric proteins containing fusions of meganucleases, ZFNs, and TALENs have been engineered to enhance the binding affinity of ZFNs and TALENs and the cleavage specificity of meganucleases. A novel monomeric enzyme has been produced that utilizes (Gersbach (2016), Molecular Therapy. 24:430-446). For example, megaTAL is a single chimeric protein that combines the ease of tuning of the TALEN-derived DNA-binding domain with the high cleavage efficiency of meganucleases.

[0474] Gene-editing techniques may require that the nuclease, and in the case of the CRISPR/Cas9 system, the gRNA, be efficiently delivered to the cells of interest. Delivery methods such as physical, chemical and viral methods are also known in the art (Mali (2013). Indian J. Hum. Genet. 19:3-8.). In some cases, the physical delivery method can be selected from, but not limited to, electroporation, microinjection, or the use of microprojectile bombardment. Chemical delivery methods, on the other hand, require the use of complex molecules such as calcium phosphate, lipids, or proteins. In some embodiments, viral delivery methods are applied to gene editing techniques using viruses such as, but not limited to, adenoviruses, lentiviruses, and retroviruses.

[0475] As an example, endogenous TCR genes (e.g., TRAC loci or TRBC loci) encoding TCRα chains, TCRβ chains, or TCRα and TCRβ chains can be inactivated by the CRISPR/Cas9 system. . A gRNA used to inactivate (eg, disrupt) the TRAC locus may comprise the sequence of SEQ ID NO:196. A gRNA used to disrupt the TRBC locus may comprise the sequence of SEQ ID NO:197.

[0476] CTCGACCAGCTTTGACATCAC (SEQ ID NO: 196).
[0477] ACACTGGTGTGCCTGGCCAC (SEQ ID NO: 197).
Method of treatment
[0478] In some embodiments, disclosed herein is a method of treating cancer in a subject in need thereof, wherein the method comprises a therapeutically effective amount of to the subject. In some embodiments, further disclosed herein is a method of treating cancer in a subject in need of treatment, the method comprising: (a) made according to the methods disclosed herein; and (b) a pharmaceutically acceptable carrier.

[0479] Optionally, the modified T cell is an allogeneic T cell. In some cases, less cytokines are released in the subject compared to subjects receiving an effective amount of unmodified control T cells. In some cases, less cytokines are released in a subject compared to a subject administered an effective amount of a modified T cell comprising a recombinant nucleic acid disclosed herein or a vector disclosed herein.

[0480] Optionally, the method comprises administering the pharmaceutical composition in combination with an agent that increases the effectiveness of the pharmaceutical composition. Optionally, the method includes administering the pharmaceutical composition in combination with an agent that ameliorates one or more side effects associated with the pharmaceutical composition.

[0481] Optionally, the cancer is a solid tumor, lymphoma, or leukemia. Optionally, the cancer is selected from the group consisting of renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney cancer, and gastric cancer .

[0482] The present disclosure includes a type of cell therapy in which T cells are genetically modified to express TFP and TCRγ and/or δ constant domains and the modified T cells are infused into a recipient in need thereof. Injected cells can kill the recipient's tumor cells. Unlike antibody therapy, these modified T cells are able to replicate in vivo and thus can survive long-term and provide long-lasting tumor control. In various embodiments, the T cells administered to the patient, or passages thereof, are administered for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months after administration of the T cells to the patient. 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months , 22 months, 23 months, 2 years, 3 years, 4 years, or 5 years.

[0483] The present disclosure also includes modifying T cells to transiently express TFP and TCR gamma and/or delta constant domains, e.g., by in vitro transcribed RNA, requiring modified T cells It also includes the type of cell therapy that is injected into the recipient. Injected cells can kill the recipient's tumor cells. Thus, in various aspects, the T cells administered to the patient are present for less than one month, such as three weeks, two weeks, or one week after administration of the T cells to the patient.

[0484] Without being bound by any particular theory, the anti-tumor immune response elicited by the engineered T cells may be an active immune response or a passive immune response, Alternatively, it may result from a direct or indirect immune response.

[0485] In one aspect, the human engineered T cells of this disclosure can be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one aspect, the mammal is a human.

[0486] For ex vivo immunization, prior to administering the cells to a mammal, i) proliferation of the cells, ii) introduction of nucleic acids encoding TFP and TCRγ and/or δ constant domains into the cells, or iii) the cells. At least one of the cryopreservation of is performed in vitro.

[0487] Ex vivo procedures are well known in the art and are discussed exhaustively below. Briefly, cells are isolated from a mammal (eg, human) and genetically modified (ie, transduced or transfected in vitro) with the vectors disclosed herein. Engineered T cells can be administered to a mammalian recipient to provide therapeutic benefit. The mammalian recipient may be human and the modified cells may be autologous to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic to the recipient.

[0488] Procedures for ex vivo expansion of hematopoietic stem and progenitor cells are described in US Pat. No. 5,199,942, incorporated herein by reference, and can be applied to the cells of the present disclosure. can. This disclosure is not limited to any particular method for ex vivo expansion of cells, as other suitable methods are known in the art. Briefly, ex vivo culture and expansion of T cells involves (1) recovery of mammalian-derived CD34 ⁺ hematopoietic stem and progenitor cells from peripheral blood draws or bone marrow grafts, and (2) ex vivo Including proliferation. In addition to the cell growth factors described in US Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligand are used in cell culture and growth can do.

[0489] With respect to ex vivo immunization, in addition to the use of cell-based vaccines, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response to a patient's antigen.

[0490] In general, cells activated and expanded as described herein can be used to treat and prevent diseases that occur in immunocompromised individuals.
[0491] The modified T cells of this disclosure can be administered alone or as pharmaceutical compositions in combination with diluents and/or other components such as IL-2 or other cytokines or cell populations. good.

combination therapy
[0492] The engineered T cells described herein can be used in combination with other known agents and therapies. As used herein, administering "in combination" means that two (or more) different treatments are delivered to a subject for the duration that the subject is afflicted with the disease, e.g. and before the disorder heals or disappears or is discontinued for other reasons, two or more treatments are delivered. In some embodiments, there is an overlap in dosing as delivery of the first therapy continues at the beginning of delivery of the second. This is sometimes referred to herein as "simultaneous" or "simultaneous delivery." In other embodiments, delivery of the first therapy ends before delivery of the other therapy begins. In some embodiments of either case, the combination of administrations enhances the effectiveness of the treatment. e.g., the second treatment is more effective than what is observed when the second treatment is administered without the first treatment, or similar conditions are observed with the first treatment, e.g. , the second treatment is equally effective, or the symptoms are greatly reduced with the second treatment. In some embodiments, the delivery is such that the relief of symptoms, or other parameter associated with the disease, is greater than would be observed with one treatment delivered without the other treatment. delivery. The effect of the two treatments may be partially additive, fully additive, or greater than additive. Delivery can be such that the effect of a first delivered therapy remains detectable when a second therapy is delivered.

[0493] In some embodiments, the "at least one additional therapeutic agent" comprises engineered T cells. Also provided are T cells expressing multiple TFPs that bind to the same or different target antigens, or to the same or different epitopes on the same target antigen. a first subset of T cells expressing a first TFP and a TCR gamma and/or delta constant domain and a second subset of T cells expressing a second TFP and a TCR gamma and/or delta constant domain Groups are also offered.

[0494] The engineered T cells described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or separate compositions, or sequentially. For sequential administration, the modified T cells described herein may be administered first and the additional agent second, or the order of administration may be reversed.

[0495] In a further aspect, the modified T cells described herein are treated with surgery, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and tacrolimus, antibodies, or other drugs such as alemtuzumab. immunodepletion drugs, anti-CD3 antibodies, or other antibody therapies, cytoxan, fludarabine, cyclosporine, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, cytokines, and irradiation, Izumoto et al. 2008 JNeurosurg 108:963-971 in combination with peptide vaccines in therapeutic regimens.

[0496] In one embodiment, a subject can be administered an agent that reduces or ameliorates side effects associated with administration of engineered T cells. Side effects associated with administration of engineered T cells include, but are not limited to, cytokine release syndrome (CRS) and hemophagocytic lymphohistiocytosis (HLH), also known as macrophage activation syndrome (MAS). Symptoms of CRS include high fever, nausea, transient hypotension, and hypoxia. Accordingly, the methods disclosed herein include administering to a subject the modified T cells described herein, and further administering an agent that manages elevated levels of soluble factors resulting from treatment with the modified T cells. can include doing In one embodiment, the soluble factor elevated in the subject is one or more of IFN-γ, TNFα, IL-2, and IL-6. Therefore, agents administered to combat this side effect may be agents that neutralize one or more of these soluble factors. Such agents include, but are not limited to, steroids, inhibitors of TNFα, and inhibitors of IL-6. An example of a TNFα inhibitor is etanercept. An example of an IL-6 inhibitor is tocilizumab (toc).

[0497] In one embodiment, the subject can be administered an agent that enhances the activity of engineered T cells. For example, in one embodiment, an agent can be an agent that inhibits inhibitory molecules. Suppressive molecules such as programmed death 1 (PD1) can, in some embodiments, reduce the ability of engineered T cells to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFRβ. For example, inhibition of suppressor molecules by inhibition at the DNA, RNA, or protein level can optimize performance of engineered T cells. In each embodiment, an inhibitory nucleic acid, eg, an inhibitory nucleic acid, eg, a dsRNA, eg, siRNA or shRNA, can be used to inhibit expression of a suppressor molecule in a TFP-expressing cell. In one embodiment, the inhibitor is an shRNA. In certain embodiments, the inhibitory molecule is inhibited in engineered T cells. In these embodiments, a dsRNA molecule that inhibits expression of a suppressor molecule is linked to nucleic acids encoding all of the components of the TFP, eg, the components. In one embodiment, an inhibitor of inhibitory signals can be, for example, an antibody or antibody fragment that binds to the inhibitory molecule. For example, the agent can be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2, or CTLA4 (eg, ipilimumab (also known as MDX-010 and MDX-101, marketed as Yervoy™, Bristol- Myers Squibb, Tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly Ticilimumab, CP-675,206) In certain embodiments, the agent is an antibody or antibody fragment that binds to TIM3. is an antibody or antibody fragment that binds to LAG3.

[0498] In some embodiments, an agent that enhances the activity of an engineered T cell can be, for example, a fusion protein comprising a first domain and a second domain, wherein the first domain is A molecule or fragment thereof, wherein the second domain is a positive signal-associated polypeptide, eg, a polypeptide comprising an intracellular signaling domain as described herein. In some embodiments, the positive signal-associated polypeptide is CD28, CD27, the co-stimulatory domain of ICOS, such as CD28, CD27, and/or the intracellular signaling domain of ICOS, and/or the primary signal, such as CD3ζ. It may include a transduction domain, such as those described herein. In one embodiment, the fusion protein is expressed by the same cells that express TFP. In another embodiment, the fusion protein is expressed by cells that do not express anti-TAA TFPs, such as T cells.

[0499] The present invention is further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, this invention should not be construed in any way as limited to the following examples, but rather as encompassing any and all modifications that become apparent as a result of the teachings presented herein. should. Without further elaboration, it is believed that one skilled in the art, using the preceding description and the following illustrative examples, can make and use the compounds of the present invention and practice the claimed methods. . The following examples illustrate various aspects of the present invention and should not be construed as limiting the remainder of the disclosure in any way.

Background of Examples
[0500] The T cell receptor (TCR) is formed by a complex of dimeric TCRα/β, CD3γ/ε, CD3δ/ε, and homodimeric CD3ζ/ζ. Some specific T cells express TCRγ/δ instead of TCRα/β to form a functional TCR. TCRα/β/γ/δ have constant domains common to all T cells and antigen-specific variable domains. The TRAC, TRBC, TRGC, and TRDC genes encode constant C-terminal regions of TCRα, TCRβ, TCRγ, and TCRδ, respectively. Despite the high structural homology between these molecules, TCRα only pairs with TCRβ and TCRγ only with TCRδ. Thus, the TCR complex is formed by TCRα/β in α/β T cells or by TCRγ/δ in γ/δ T cells.

[0501] Disruption of the TCR α/β/γ/δ constant region(s) blocks translocation of TCR protein(s) to the cell surface, thereby inhibiting assembly of the TCR receptor complex. Reducing translocation of TCRα or TCRβ is sufficient to inhibit overall TCR receptor assembly in TCRα/β T cells. Similarly, reducing translocation of TCRγ or TCRδ is sufficient to inhibit assembly of the entire TCR receptor in TCRγ/δ T cells. Thus, inactivation of the TCR complex can be achieved using gene editing methods using the clustered regularly interspaced short palindromic repeat (CRISPR) method, transcription activator-like effector nucleases (TALENs), zinc finger nucleases, or meganucleases. This can be done by targeting the TRAC or TRBC genes. However, TFP T cells based on CD3ε or CD3γ or CD3δ fusion proteins require surface expression of TCRα/β or TCRγ/δ to integrate into functional TCR complexes.

[0502] Activation of the TCR complex on the surface of alloreactive donor T cells by HLA-mismatched molecules or alloantigens (i.e., recognition of antigen presented by the major histocompatibility complex (MHC) of antigen-presenting cells) , can cause undesirable effects such as graft-versus-host disease (GvHD) and cytokine release syndrome (CRS). Thus, the following examples are truncated TCRα (mouse) or TCRβ (mouse) with binding agents to one or both, or CD3 TFP, or one or both of the truncated TCRs, or binding agents to CD3 TFP. Methods are described to introduce transgenes encoding truncated or full-length TCRγ or TCRδ, plus the fusion protein itself separated by a self-cleavage signal (eg, T2A), into TCRα or TCRβ knockout cells. In one embodiment, a truncated form of TCRγ or TCRδ comprises the transmembrane domain and binding peptide domain (CP) and constant domain of TCRγ or TCRδ. In another embodiment, a truncated form of TCRγ or TCRδ comprises the transmembrane domain and binding peptide domain (CP) and constant domain of TCRα or TCRβ. In another embodiment, the antigen-binding domain of TFP is fused to the N-terminus of one or both of truncated TCRγ and/or TCRδ.

Example 1: Design of crRNA (CRISPR RNA)
[0503] crRNAs that inactivate TRA were designed using the "Dunne 2017" algorithm published on the DeskGen™ CRISPR library website (www.deskgen.com). Any crRNA that binds to the TRA locus can efficiently generate a double-strand break in the TRA gene. CRISP
To minimize off-target activity of the R endonuclease, the crRNAs used had an off-target score >90% and were matched to the closest homologous sequences within the Genome Reference Consortium Human Genome Build 38 (GRCh38/hg38) genome. Contains at least 3 mismatches. In a preferred embodiment, one mismatch is located 8 bp upstream of the protospacer adjacent motif (PAM). Tables 1-2 show exemplary crRNA sequences selected to inactivate the TRA gene (Table 1) and predicted off-target activity (Table 2).

[0504] A crRNA that inactivates the TRBC gene was designed using the Dunne 2017 algorithm described above. Since the constant region of TCRβ is encoded by two genes, TRBC1 and TRBC2, the crRNA is directed to the same sequence in both TRBC1 and TRBC2. As a result, off-target scores generated by DeskGen™ are less than 94%. However, except for the targeting of TRBC1 and TRBC2, other homologous sequences between crRNA and GRCh38/hg38 genome have at least 3 mismatches. In preferred embodiments, one of these mismatches is located 8 bp upstream of the protospacer adjacent motif (PAM). Tables 3-4 show exemplary crRNA sequences selected to inactivate the TRB gene (Table 3) and predicted off-target activity (Table 4).

Example 2: Editing of Endogenous TCRα or β in Jurkat Cells
[0505] Inactivation of the TRAC or TRBC genes in Jurkat cells was performed by electroporation of SpCas9 ribonucleoprotein (RNP) against the TRAC or TRBC genes. Cells were maintained at 0.2×10 ⁶ cells per mL in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 300 mg/L L-glutamine until electroporation. SpCas9 ribonucleoprotein targeting the TRA or TRB gene by annealing crRNA targeting either TRAC (TRAC2-4598) or TRBC (TRBC-44345) and tracrRNA at a molecular ratio of 1:1 prepared. Annealed duplexes were mixed with SpCas9 protein at a molar ratio of 1.5:1. 0.61 μM RNP was mixed with 2.5×10 ⁶ T cells and electroporated according to the manufacturer's protocol for the Neon Transfection System (Thermo Fisher Scientific). Electroporation was set at 1600 V, 10 ms, 3 pulses. After pulsing, cells were immediately transferred to warm medium and incubated at 37° C. for 3 days.

[0506] The editing effect was evaluated by observing the decrease in surface expression of TCRαβ and CD3ε by flow cytometry. Results are shown in Figure 1 for TRA-edited cells (left panel) and TRB-edited cells (right panel). Edited Jurkat cells were purified by magnetic cell separation (MACS, Miltenyi Biotec) cell separation system. Edited Jurkat cells were negatively selected against anti-TCRαβ (clone IP27) (eBioscience #17-9986-42) and anti-CD3ε (clone SK7) antibodies (eBioscience #25-0036-42). Cells expressing TCRαβ or CD3ε on their surface were fixed to MACS MS (Cat #130-041-301) or LS (Cat #130-041-306) columns and negative for both TCRαβ and CD3ε. , edited Jurkat cells were harvested by column flow-through and maintained in culture at 0.4×10 ⁶ cells/mL in the medium specified above. TCRα and TCRβ knockout cells are referred to herein as TRA ^−/− or TRB ^−/− Jurkat cells.

Example 3: Generation of Allogeneic T Cell Receptor Fusion Protein T Cells
Transduction of Jurkat cells
[0507] The TFP transgene was introduced into Jurkat cells using lentivirus, eg, as described in co-pending US Patent Publication No. 2017-0166622. Jurkat cells were incubated with virus at a multiplicity of infection (MOI) of 5. After 24 hours of incubation, the medium was changed. Transduction efficacy and TFP expression were assessed by flow cytometry using ligands specific for the TFP-binding factor of interest and/or surface expression of TCRαβ and CD3ε. TRAC ^-/- and TRBC ^-/- Jurkat cells were transduced with TCRγδ TFP and showed restoration of surface TCR by highly positive CD3ε staining (Fig. 2). TRBC ^-/- Jurkat cells were also transduced with anti-CD19 TCRβ-TFP or anti-CD19 TCRβ-TFP with only the constant domain of TCRβ (Fig. 12B). The results show that TFPs with the entire human TCRβ are expressed on the cell surface, whereas TFPs with only the constant domain of human TCRβ are not expressed on the cell surface (FIG. 13). This is because the constant domain of TCRβ cannot localize to the cell surface, although the constant domains of mouse TCRβ and mouse-human chimeric TCRβ are known to be able to localize to the cell surface.

Transduction of T cells
[0508] The TFP transgene was introduced into T cells using lentivirus, eg, as described in co-pending US Patent Publication No. 2017-0166622. T cells were mixed with virus at a multiplicity of infection (MOI) of 5 plus 100 ng/mL LentiBOOST® (Sirion Biotech). Transduction efficacy and TFP expression were assessed by flow cytometry using ligands specific for the TFP-binding factor of interest and/or surface expression of TCRαβ and CD3ε.

Description of the transgene
[0509] In α/β T cells, inactivation of TRAC or TRBC blocks translocation of all TCR subunits to the cell surface. TCRα or TCRβ cannot pair with TCRγ or TCRδ. As a result, exogenous TRGC and TRDC transgenes or TRAC and TRBC transgenes are expressed in TRAC ^−/− or TRBC ^−/− cells to restore functional TFP T cells.

Expression of human TCRγ/δ TFP
[0510] TCRα-negative cells still express TCRβ, and conversely TCRα is expressed in TCRβ-negative cells. However, TCRα or TCRβ cannot pair with TCRγ or TCRδ. Therefore, TCRγ TFP and TCRδ TFP were co-expressed in TRAC ^−/− or TRB ^−/− cells. Multiple forms of TCRγ/δ TFPs were tested in TCR-negative cells to determine the optimal conformation to restore translocation of the entire TCR complex (see Figure 3 schematic). In one embodiment, TCRγ/δ TFPs were generated by assembling TCRγ or/and TCRδ constant domains with an antigen binding agent (eg, scFv or sdAb). In another embodiment, the TCRγ/δ constant domain is co-expressed with CD3ε TFP (Figure 4). TRGC1 and TRDC residues are listed according to the sequences provided herein and according to the international ImMunoGeneTics information system (IMGT).

TCR subunit source
[0511] The TCR complex comprises a CD3-ε polypeptide, a CD3-γ polypeptide, a CD3-δ polypeptide, and a TCR α chain polypeptide and a TCR β chain polypeptide or a TCR δ chain polypeptide and a TCR γ chain polypeptide . TCRα, TCRβ, TCRγ, and TCRδ recruit CD3ζ polypeptides. The canonical sequence for the human CD3-ε polypeptide is Uniprot accession number P07766. The canonical sequence for human CD3-γ polypeptide is Uniprot accession number P09693. The canonical sequence for human CD3-delta polypeptide is Uniprot accession number P043234. The canonical sequence for the human CD3-zeta polypeptide is Uniprot accession number P20963. The canonical sequence for the human TCRα chain is Uniprot accession number Q6ISU1. The canonical sequence for the mouse TCRα chain is Uniprot accession number A0A075B662. The canonical sequence for the constant region of the human TCR beta chain is Uniprot accession number P01850. The canonical sequence of the constant region of the murine TCR beta chain is:
Uniprot accession number P01852.

[0512] The canonical sequence of the human CD3-ε polypeptide is as follows:
ＭＱＳＧＴＨＷＲＶＬＧＬＣＬＬＳＶＧＶＷＧＱＤＧＮＥＥＭＧＧＩＴＱＴＰＹＫＶＳＩＳＧＴＴＶＩＬＴＣＰＱＹＰＧＳＥＩＬＷＱＨＮＤＫＮＩＧＧＤＥＤＤＫＮＩＧＳＤＥＤＨＬＳＬＫＥＦＳＥＬＥＱＳＧＹＹＶＣＹＰＲＧＳＫＰＥＤＡＮＦＹＬＹＬＲＡＲＶＣＥＮＣＭＥＭＤＶＭＳＶＡＴＩＶＩＶＤＩＣＩＴＧＧＬＬＬＬＶＹＹＷＳＫＮＲＫＡＫＡＫＰＶＴＲＧＡＧＡＧＧＲＱＲＧＱＮＫＥＲＰＰＰＶＰＮＰＤＹＥＰＩＲＫＧＱＲＤＬＹＳＧＬＮＱＲＲＩ（配列番号１２４）。

[0513] The mature human CD3-ε polypeptide sequence is as follows:
ＤＧＮＥＥＭＧＧＩＴＱＴＰＹＫＶＳＩＳＧＴＴＶＩＬＴＣＰＱＹＰＧＳＥＩＬＷＱＨＮＤＫＮＩＧＧＤＥＤＤＫＮＩＧＳＤＥＤＨＬＳＬＫＥＦＳＥＬＥＱＳＧＹＹＶＣＹＰＲＧＳＫＰＥＤＡＮＦＹＬＹＬＲＡＲＶＣＥＮＣＭＥＭＤＶＭＳＶＡＴＩＶＩＶＤＩＣＩＴＧＧＬＬＬＬＶＹＹＷＳＫＮＲＫＡＫＡＫＰＶＴＲＧＡＧＡＧＧＲＱＲＧＱＮＫＥＲＰＰＰＶＰＮＰＤＹＥＰＩＲＫＧＱＲＤＬＹＳＧＬＮＱＲＲＩ（配列番号２５８）。

[0514] The signal peptide for human CD3ε is as follows:
MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO: 125).
[0515] The extracellular domain of human CD3ε is as follows:
DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO: 126).

[0516] The transmembrane domain of human CD3ε is as follows:
VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID NO: 127).
[0517] The intracellular domain of human CD3ε is as follows:
KNRKAKAKPVTRGAGAGGRQRGQNKERPPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 128).

[0518] A canonical sequence for a human CD3-γ polypeptide is as follows:
ＭＥＱＧＫＧＬＡＶＬＩＬＡＩＩＬＬＱＧＴＬＡＱＳＩＫＧＮＨＬＶＫＶＹＤＹＱＥＤＧＳＶＬＬＴＣＤＡＥＡＫＮＩＴＷＦＫＤＧＫＭＩＧＦＬＴＥＤＫＫＫＷＮＬＧＳＮＡＫＤＰＲＧＭＹＱＣＫＧＳＱＮＫＳＫＰＬＱＶＹＹＲＭＣＱＮＣＩＥＬＮＡＡＴＩＳＧＦＬＦＡＥＩＶＳＩＦＶＬＡＶＧＶＹＦＩＡＧＱＤＧＶＲＱＳＲＡＳＤＫＱＴＬＬＰＮＤＱＬＹＱＰＬＫＤＲＥＤＤＱＹＳＨＬＱＧＮＱＬＲＲＮ（配列番号１２９）。

[0519] The mature human CD3-γ polypeptide sequence is as follows:
QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFFLFAEIIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLSLKDLDQ0 (sequence number).

[0520] The signal peptide for human CD3γ is as follows:
MEQGKGLAVLILAIILLQGTLA (SEQ ID NO: 131).
[0521] The extracellular domain of human CD3γ is as follows:
QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS (SEQ ID NO: 132).

[0522] The transmembrane domain of human CD3γ is as follows:
GFLFAEIVSIFVLAVGVYFIA (SEQ ID NO: 133).
[0523] The intracellular domain of human CD3γ is as follows:
GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 134).

[0524] A canonical sequence for a human CD3-delta polypeptide is as follows:
MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTS
ITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKS (SEQ ID NO: 135).

[0525] The mature human CD3-delta polypeptide sequence is as follows:
FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKS (SEQ ID NO: 13).

[0526] The signal peptide for human CD3delta is as follows:
MEHSTFLSGLVLATLLSQVSP (SEQ ID NO: 137).
[0527] The extracellular domain of human CD3delta is as follows:
FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVA (SEQ ID NO: 138).

[0528] The transmembrane domain of human CD3delta is as follows:
GIIVTDVIATLLLLALGVFCFA (SEQ ID NO: 139).
[0529] The intracellular domain of human CD3delta is as follows:
GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO: 140).

[0530] ヒトＣＤ３－ζポリペプチドの標準配列は、以下の通りである：ＭＫＷＫＡＬＦＴＡＡＩＬＱＡＱＬＰＩＴＥＡＱＳＦＧＬＬＤＰＫＬＣＹＬＬＤＧＩＬＦＩＹＧＶＩＬＴＡＬＦＬＲＶＫＦＳＲＳＡＤＡＰＡＹＱＱＧＱＮＱＬＹＮＥＬＮＬＧＲＲＥＥＹＤＶＬＤＫＲＲＧＲＤＰＥＭＧＧＫＰＱＲＲＫＮＰＱＥＧＬＹＮＥＬＱＫＤＫＭＡＥＡＹＳＥＩＧＭＫＧＥＲＲＲＧＫＧＨＤＧＬＹＱＧＬＳＴＡＴＫＤＴＹＤＡＬＨＭＱＡＬＰＰＲ（配列番号１４１）。

[0531] The canonical sequence of the constant region of the human TCRα chain is as follows: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLLK4 (number) sequence.

[0532] The human IgC sequence for the human TCRα chain is as follows:
IQNPDPAVYQLRDSKSSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSSMTFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS (SEQ ID NO: 143).

[0533] The transmembrane domain of the human TCRα chain is as follows:
VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO: 144).
[0534] The intracellular domain of the human TCRα chain is as follows: SS (SEQ ID NO: 145).

[0535] The canonical sequence of the constant (mTRAC) region of the mouse TCRα chain is as follows:
XIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 146).

[0536] The transmembrane domain of the mouse TCRα chain is as follows:
MGLRILLLKVAGFNLLMTLRLW (SEQ ID NO: 147).
[0537] The intracellular domain of the mouse TCRα chain is as follows: SS.

[0538] ヒトＴＣＲβ鎖の定常領域（ｍＴＲＢＣ）の標準配列は、以下の通りである：ＥＤＬＮＫＶＦＰＰＥＶＡＶＦＥＰＳＥＡＥＩＳＨＴＱＫＡＴＬＶＣＬＡＴＧＦＦＰＤＨＶＥＬＳＷＷＶＮＧＫＥＶＨＳＧＶＳＴＤＰＱＰＬＫＥＱＰＡＬＮＤＳＲＹＣＬＳＳＲＬＲＶＳＡＴＦＷＱＮＰＲＮＨＦＲＣＱＶＱＦＹＧＬＳＥＮＤＥＷＴＱＤＲＡＫＰＶＴＱＩＶＳＡＥＡＷＧＲＡＤＣＧＦＴＳＶＳＹＱＱＧＶＬＳＡＴＩＬＹＥＩＬＬＧＫＡＴＬＹＡＶＬＶＳＡＬＶＬＭＡＭＶＫＲＫＤＦ（配列番号１４８）。

[0539] The human IgC sequence for the human TCR beta chain is as follows:
EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQGVLSATQGVLSATQ49E (SEQ ID NO: 1Y49)
[0540] The transmembrane domain of the human TCR beta chain is as follows:
ILLGKATLYAVLVSALVLMAM (SEQ ID NO: 150).

[0541] The intracellular domain of the human TCR beta chain is as follows:
VKRKDF (SEQ ID NO: 151).
[0542] The canonical sequence of the constant region of the mouse TCR beta chain is as follows:
ＥＤＬＲＮＶＴＰＰＫＶＳＬＦＥＰＳＫＡＥＩＡＮＫＱＫＡＴＬＶＣＬＡＲＧＦＦＰＤＨＶＥＬＳＷＷＶＮＧＫＥＶＨＳＧＶＳＴＤＰＱＡＹＫＥＳＮＹＳＹＣＬＳＳＲＬＲＶＳＡＴＦＷＨＮＰＲＮＨＦＲＣＱＶＱＦＨＧＬＳＥＥＤＫＷＰＥＧＳＰＫＰＶＴＱＮＩＳＡＥＡＷＧＲＡＤＣＧＩＴＳＡＳＹＱＱＧＶＬＳＡＴＩＬＹＥＩＬＬＧＫＡＴＬＹＡＶＬＶＳＴＬＶＶＭＡＭＶＫＲＫＮＳ（配列番号１５２）。

[0543] The transmembrane domain of the mouse TCR beta chain is:
ILYEILLGKATLYAVLVS TLVVMAMVK (SEQ ID NO: 153).
[0544] The intracellular domain of the mouse TCR beta chain is as follows:
KRKNS (SEQ ID NO: 154).

[0545] The canonical sequence of the constant region of the human TCRγ chain is as follows:
ＤＫＱＬＤＡＤＶＳＰＫＰＴＩＦＬＰＳＩＡＥＴＫＬＱＫＡＧＴＹＬＣＬＬＥＫＦＦＰＤＶＩＫＩＨＷＱＥＫＫＳＮＴＩＬＧＳＱＥＧＮＴＭＫＴＮＤＴＹＭＫＦＳＷＬＴＶＰＥＫＳＬＤＫＥＨＲＣＩＶＲＨＥＮＮＫＮＧＶＤＱＥＩＩＦＰＰＩＫＴＤＶＩＴＭＤＰＫＤＮＣＳＫＤＡＮＤＴＬＬＬＱＬＴＮＴＳＡＹＹＭＹＬＬＬＬＬＫＳＶＶＹＦＡＩＩＴＣＣＬＬＲＲＴＡＦＣＣＮＧＥＫＳ（配列番号２１）。

[0546] The human IgC sequence for human TCRγ is as follows:
DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGNTMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDNCSKDANDTLLLQLTNTSA (SEQ ID NO: 155)
[0547] The transmembrane domain of the human TCRγ chain is as follows:
YYMYLLLLLKSVVYFAIITCCLL (SEQ ID NO: 156).

[0548] The intracellular domain of the human TCRγ chain is as follows:
RRTAFCCNGEKS (SEQ ID NO: 157)
[0549] The canonical sequence of the C region of the human TCR delta chain is as follows:
SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVLFAVNFLL3 (SEQ ID NO: 2).

[0550] The human IgC sequence of human TCRδ is as follows:
SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLGKYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTE
KVNMMSLTV (SEQ ID NO: 265).

[0551] The transmembrane domain of the human TCRδ chain is as follows:
LGLRMLFAKTVAVNFLLTAKLFF (SEQ ID NO: 158).
[0552] The intracellular domain of the human TCR delta chain is as follows: L.

TCRγ/δ FMC63 TFP expressed in TRA ^−/− or TRB ^−/− cells
Nt-FMC63-TRDC(1-153)-T2A-TRGC1(1-173)-Ct (according to IMGT numbering, Nt-FMC63-TRDC(-6,189)-2A-TRGC1(1 .8,189)-Ct):
ＭＬＬＬＶＴＳＬＬＬＣＥＬＰＨＰＡＦＬＬＩＰＤＩＱＭＴＱＴＴＳＳＬＳＡＳＬＧＤＲＶＴＩＳＣＲＡＳＱＤＩＳＫＹＬＮＷＹＱＱＫＰＤＧＴＶＫＬＬＩＹＨＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＳＬＴＩＳＮＬＥＱＥＤＩＡＴＹＦＣＱＱＧＮＴＬＰＹＴＦＧＧＧＴＫＬＥＩＴＧＳＴＳＧＳＧＫＰＧＳＧＥＧＳＴＫＧＥＶＫＬＱＥＳＧＰＧＬＶＡＰＳＱＳＬＳＶＴＣＴＶＳＧＶＳＬＰＤＹＧＶＳＷＩＲＱＰＰＲＫＧＬＥＷＬＧＶＩＷＧＳＥＴＴＹＹＮＳＡＬＫＳＲＬＴＩＩＫＤＮＳＫＳＱＶＦＬＫＭＮＳＬＱＴＤＤＴＡＩＹＹＣＡＫＨＹＹＹＧＧＳＹＡＭＤＹＷＧＱＧＴＳＶＴＶＳＳＡＡＡＧＧＧＧＳＧＧＧＧＳＧＧＧＧＳＬＥＳＱＰＨＴＫＰＳＶＦＶＭＫＮＧＴＮＶＡＣＬＶＫＥＦＹＰＫＤＩＲＩＮＬＶＳＳＫＫＩＴＥＦＤＰＡＩＶＩＳＰＳＧＫＹＮＡＶＫＬＧＫＹＥＤＳＮＳＶＴＣＳＶＱＨＤＮＫＴＶＨＳＴＤＦＥＶＫＴＤＳＴＤＨＶＫＰＫＥＴＥＮＴＫＱＰＳＫＳＣＨＫＰＫＡＩＶＨＴＥＫＶＮＭＭＳＬＴＶＬＧＬＲＭＬＦＡＫＴＶＡＶＮＦＬＬＴＡＫＬＦＦＬＧＳＧＥＧＲＧＳＬＬＴＣＧＤＶＥＥＮＰＧＰＭＬＬＬＶＴＳＬＬＬＣＥＬＰＨＰＡＦＬＬＩＰＤＫＱＬＤＡＤＶＳＰＫＰＴＩＦＬＰＳＩＡＥＴＫＬＱＫＡＧＴＹＬＣＬＬＥＫＦＦＰＤＶＩＫＩＨＷＱＥＫＫＳＮＴＩＬＧＳＱＥＧＮＴＭＫＴＮＤＴＹＭＫＦＳＷＬＴＶＰＥＫＳＬＤＫＥＨＲＣＩＶＲＨＥＮＮＫＮＧＶＤＱＥＩＩＦＰＰＩＫＴＤＶＩＴＭＤＰＫＤＮＣＳＫＤＡＮＤＴＬＬＬＱＬＴＮＴＳＡＹＹＭＹＬＬＬＬＬＫＳＶＶＹＦＡＩＩＴＣＣＬＬＲＲＴＡＦＣＣＮＧＥＫＳ（配列番号１）。

Nt-FMC63-TRDC(1-153)-T2A-FMC63-TRGC1(1-173)-Ct (According to IMGT numbering, Nt-FMC63-TRDC(-6,189)-2A-FMC63-TRGC1:
ＭＬＬＬＶＴＳＬＬＬＣＥＬＰＨＰＡＦＬＬＩＰＤＩＱＭＴＱＴＴＳＳＬＳＡＳＬＧＤＲＶＴＩＳＣＲＡＳＱＤＩＳＫＹＬＮＷＹＱＱＫＰＤＧＴＶＫＬＬＩＹＨＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＳＬＴＩＳＮＬＥＱＥＤＩＡＴＹＦＣＱＱＧＮＴＬＰＹＴＦＧＧＧＴＫＬＥＩＴＧＳＴＳＧＳＧＫＰＧＳＧＥＧＳＴＫＧＥＶＫＬＱＥＳＧＰＧＬＶＡＰＳＱＳＬＳＶＴＣＴＶＳＧＶＳＬＰＤＹＧＶＳＷＩＲＱＰＰＲＫＧＬＥＷＬＧＶＩＷＧＳＥＴＴＹＹＮＳＡＬＫＳＲＬＴＩＩＫＤＮＳＫＳＱＶＦＬＫＭＮＳＬＱＴＤＤＴＡＩＹＹＣＡＫＨＹＹＹＧＧＳＹＡＭＤＹＷＧＱＧＴＳＶＴＶＳＳＡＡＡＧＧＧＧＳＧＧＧＧＳＧＧＧＧＳＬＥＳＱＰＨＴＫＰＳＶＦＶＭＫＮＧＴＮＶＡＣＬＶＫＥＦＹＰＫＤＩＲＩＮＬＶＳＳＫＫＩＴＥＦＤＰＡＩＶＩＳＰＳＧＫＹＮＡＶＫＬＧＫＹＥＤＳＮＳＶＴＣＳＶＱＨＤＮＫＴＶＨＳＴＤＦＥＶＫＴＤＳＴＤＨＶＫＰＫＥＴＥＮＴＫＱＰＳＫＳＣＨＫＰＫＡＩＶＨＴＥＫＶＮＭＭＳＬＴＶＬＧＬＲＭＬＦＡＫＴＶＡＶＮＦＬＬＴＡＫＬＦＦＬＧＳＧＥＧＲＧＳＬＬＴＣＧＤＶＥＥＮＰＧＰＭＬＬＬＶＴＳＬＬＬＣＥＬＰＨＰＡＦＬＬＩＰＤＩＱＭＴＱＴＴＳＳＬＳＡＳＬＧＤＲＶＴＩＳＣＲＡＳＱＤＩＳＫＹＬＮＷＹＱＱＫＰＤＧＴＶＫＬＬＩＹＨＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＳＬＴＩＳＮＬＥＱＥＤＩＡＴＹＦＣＱＱＧＮＴＬＰＹＴＦＧＧＧＴＫＬＥＩＴＧＳＴＳＧＳＧＫＰＧＳＧＥＧＳＴＫＧＥＶＫＬＱＥＳＧＰＧＬＶＡＰＳＱＳＬＳＶＴＣＴＶＳＧＶＳＬＰＤＹＧＶＳＷＩＲＱＰＰＲＫＧＬＥＷＬＧＶＩＷＧＳＥＴＴＹＹＮＳＡＬＫＳＲＬＴＩＩＫＤＮＳＫＳＱＶＦＬＫＭＮＳＬＱＴＤＤＴＡＩＹＹＣＡＫＨＹＹＹＧＧＳＹＡＭＤＹＷＧＱＧＴＳＶＴＶＳＳＡＡＡＧＧＧＧＳＧＧＧＧＳＧＧＧＧＳＬＥＤＫＱＬＤＡＤＶＳＰＫＰＴＩＦＬＰＳＩＡＥＴＫＬＱＫＡＧＴＹＬＣＬＬＥＫＦＦＰＤＶＩＫＩＨＷＱＥＫＫＳＮＴＩＬＧＳＱＥＧＮＴＭＫＴＮＤＴＹＭＫＦＳＷＬＴＶＰＥＫＳＬＤＫＥＨＲＣＩＶＲＨＥＮＮＫＮＧＶＤＱＥＩＩＦＰＰＩＫＴＤＶＩＴＭＤＰＫＤＮＣＳＫＤＡＮＤＴＬＬＬＱ
LTNTSAYYMYLLLLLKSVVYFAIITCCLLRTAFCCNGEKS (SEQ ID NO: 2).

Nt-TRDC(1-153)-T2A-TRGC1(1-173)-T2A-FMC63-CD3ε(1-185)-Ct (According to IMGT numbering, Nt-TRDC(-6,189)-2A- TRGC1(1.8,189)-2A-FMC63-CD3ε(1,186)-Ct):
ＭＬＬＬＶＴＳＬＬＬＣＥＬＰＨＰＡＦＬＬＩＰＳＱＰＨＴＫＰＳＶＦＶＭＫＮＧＴＮＶＡＣＬＶＫＥＦＹＰＫＤＩＲＩＮＬＶＳＳＫＫＩＴＥＦＤＰＡＩＶＩＳＰＳＧＫＹＮＡＶＫＬＧＫＹＥＤＳＮＳＶＴＣＳＶＱＨＤＮＫＴＶＨＳＴＤＦＥＶＫＴＤＳＴＤＨＶＫＰＫＥＴＥＮＴＫＱＰＳＫＳＣＨＫＰＫＡＩＶＨＴＥＫＶＮＭＭＳＬＴＶＬＧＬＲＭＬＦＡＫＴＶＡＶＮＦＬＬＴＡＫＬＦＦＬＧＳＧＥＧＲＧＳＬＬＴＣＧＤＶＥＥＮＰＧＰＭＬＬＬＶＴＳＬＬＬＣＥＬＰＨＰＡＦＬＬＩＰＤＫＱＬＤＡＤＶＳＰＫＰＴＩＦＬＰＳＩＡＥＴＫＬＱＫＡＧＴＹＬＣＬＬＥＫＦＦＰＤＶＩＫＩＨＷＱＥＫＫＳＮＴＩＬＧＳＱＥＧＮＴＭＫＴＮＤＴＹＭＫＦＳＷＬＴＶＰＥＫＳＬＤＫＥＨＲＣＩＶＲＨＥＮＮＫＮＧＶＤＱＥＩＩＦＰＰＩＫＴＤＶＩＴＭＤＰＫＤＮＣＳＫＤＡＮＤＴＬＬＬＱＬＴＮＴＳＡＹＹＭＹＬＬＬＬＬＫＳＶＶＹＦＡＩＩＴＣＣＬＬＲＲＴＡＦＣＣＮＧＥＫＳＧＳＧＥＧＲＧＳＬＬＴＣＧＤＶＥＥＮＰＧＰＭＬＬＬＶＴＳＬＬＬＣＥＬＰＨＰＡＦＬＬＩＰＤＩＱＭＴＱＴＴＳＳＬＳＡＳＬＧＤＲＶＴＩＳＣＲＡＳＱＤＩＳＫＹＬＮＷＹＱＱＫＰＤＧＴＶＫＬＬＩＹＨＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＳＬＴＩＳＮＬＥＱＥＤＩＡＴＹＦＣＱＱＧＮＴＬＰＹＴＦＧＧＧＴＫＬＥＩＴＧＳＴＳＧＳＧＫＰＧＳＧＥＧＳＴＫＧＥＶＫＬＱＥＳＧＰＧＬＶＡＰＳＱＳＬＳＶＴＣＴＶＳＧＶＳＬＰＤＹＧＶＳＷＩＲＱＰＰＲＫＧＬＥＷＬＧＶＩＷＧＳＥＴＴＹＹＮＳＡＬＫＳＲＬＴＩＩＫＤＮＳＫＳＱＶＦＬＫＭＮＳＬＱＴＤＤＴＡＩＹＹＣＡＫＨＹＹＹＧＧＳＹＡＭＤＹＷＧＱＧＴＳＶＴＶＳＳＡＡＡＧＧＧＧＳＧＧＧＧＳＧＧＧＧＳＬＥＤＧＮＥＥＭＧＧＩＴＱＴＰＹＫＶＳＩＳＧＴＴＶＩＬＴＣＰＱＹＰＧＳＥＩＬＷＱＨＮＤＫＮＩＧＧＤＥＤＤＫＮＩＧＳＤＥＤＨＬＳＬＫＥＦＳＥＬＥＱＳＧＹＹＶＣＹＰＲＧＳＫＰＥＤＡＮＦＹＬＹＬＲＡＲＶＣＥＮＣＭＥＭＤＶＭＳＶＡＴＩＶＩＶＤＩＣＩＴＧＧＬＬＬＬＶＹＹＷＳＫＮＲＫＡＫＡＫＰＶＴＲＧＡＧＡＧＧＲＱＲＧＱＮＫＥＲＰＰＰＶＰＮＰＤＹＥＰＩＲＫＧＱＲＤＬＹＳＧＬＮＱＲＲＩ（配列番号３）。

Nt-TRDC(1-153)-T2A-FMC63-TRGC1(1-173)-Ct-(According to IMGT numbering, Nt-TRDC(-6,189)-2A-FMC63-TRGC1(1.8, 189)-Ct) (including signal peptide):
ＭＬＬＬＶＴＳＬＬＬＣＥＬＰＨＰＡＦＬＬＩＰＳＱＰＨＴＫＰＳＶＦＶＭＫＮＧＴＮＶＡＣＬＶＫＥＦＹＰＫＤＩＲＩＮＬＶＳＳＫＫＩＴＥＦＤＰＡＩＶＩＳＰＳＧＫＹＮＡＶＫＬＧＫＹＥＤＳＮＳＶＴＣＳＶＱＨＤＮＫＴＶＨＳＴＤＦＥＶＫＴＤＳＴＤＨＶＫＰＫＥＴＥＮＴＫＱＰＳＫＳＣＨＫＰＫＡＩＶＨＴＥＫＶＮＭＭＳＬＴＶＬＧＬＲＭＬＦＡＫＴＶＡＶＮＦＬＬＴＡＫＬＦＦＬＧＳＧＥＧＲＧＳＬＬＴＣＧＤＶＥＥＮＰＧＰＭＬＬＬＶＴＳＬＬＬＣＥＬＰＨＰＡＦＬＬＩＰＤＩＱＭＴＱＴＴＳＳＬＳＡＳＬＧＤＲＶＴＩＳＣＲＡＳＱＤＩＳＫＹＬＮＷＹＱＱＫＰＤＧＴＶＫＬＬＩＹＨＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＳＬＴＩＳＮＬＥＱＥＤＩＡＴＹＦＣＱＱＧＮＴＬＰＹＴＦＧＧＧＴＫＬＥＩＴＧＳＴＳＧＳＧＫＰＧＳＧＥＧＳＴＫＧＥＶＫＬＱＥＳＧＰＧＬＶＡＰＳＱＳＬＳＶＴＣＴＶＳＧＶＳＬＰＤＹＧＶＳＷＩＲＱＰＰＲＫＧＬＥＷＬＧＶＩＷＧＳＥＴＴＹＹＮＳＡＬＫＳＲＬＴＩＩＫＤＮＳＫＳＱＶＦＬＫＭＮＳＬＱＴＤＤＴＡＩＹＹＣＡＫＨＹＹＹＧＧＳＹＡＭＤＹＷＧＱＧＴＳＶＴＶＳＳＡＡＡＧＧＧＧＳＧＧＧＧＳＧＧＧＧＳＬＥＤＫＱＬＤＡＤＶＳＰＫＰＴＩＦＬＰＳＩＡＥＴＫＬＱＫＡＧＴＹＬＣＬＬＥＫＦＦＰＤＶＩＫＩＨＷＱＥＫＫＳＮＴＩＬＧＳＱＥＧＮＴＭＫＴＮＤＴＹＭＫＦＳＷＬＴＶＰＥＫＳＬＤＫＥＨＲＣＩＶＲＨＥＮＮＫＮＧＶＤＱＥＩＩＦＰＰＩＫＴＤＶＩＴＭＤＰＫＤＮＣＳＫＤＡＮＤＴＬＬＬＱＬＴＮＴＳＡＹＹＭＹＬＬＬＬＬＫＳＶＶＹＦＡＩＩＴＣＣＬＬＲＲＴＡＦＣＣＮＧＥＫＳ（配列番号４）。

Nt-FMC63-TRDC(1-129)-TRAC(116-140)-T2A-
FMC63-TRGC1(1-106)-TRBC1(145-177)-Ct (according to IMGT numbering Nt-FMC63-TRDC(-6,165)-TRAC(150,174)-2A-FMC63-TRGC1( 1.8,189)-TRBC1(142,173)-Ct):
ＭＬＬＬＶＴＳＬＬＬＣＥＬＰＨＰＡＦＬＬＩＰＤＩＱＭＴＱＴＴＳＳＬＳＡＳＬＧＤＲＶＴＩＳＣＲＡＳＱＤＩＳＫＹＬＮＷＹＱＱＫＰＤＧＴＶＫＬＬＩＹＨＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＳＬＴＩＳＮＬＥＱＥＤＩＡＴＹＦＣＱＱＧＮＴＬＰＹＴＦＧＧＧＴＫＬＥＩＴＧＳＴＳＧＳＧＫＰＧＳＧＥＧＳＴＫＧＥＶＫＬＱＥＳＧＰＧＬＶＡＰＳＱＳＬＳＶＴＣＴＶＳＧＶＳＬＰＤＹＧＶＳＷＩＲＱＰＰＲＫＧＬＥＷＬＧＶＩＷＧＳＥＴＴＹＹＮＳＡＬＫＳＲＬＴＩＩＫＤＮＳＫＳＱＶＦＬＫＭＮＳＬＱＴＤＤＴＡＩＹＹＣＡＫＨＹＹＹＧＧＳＹＡＭＤＹＷＧＱＧＴＳＶＴＶＳＳＡＡＡＧＧＧＧＳＧＧＧＧＳＧＧＧＧＳＬＥＳＱＰＨＴＫＰＳＶＦＶＭＫＮＧＴＮＶＡＣＬＶＫＥＦＹＰＫＤＩＲＩＮＬＶＳＳＫＫＩＴＥＦＤＰＡＩＶＩＳＰＳＧＫＹＮＡＶＫＬＧＫＹＥＤＳＮＳＶＴＣＳＶＱＨＤＮＫＴＶＨＳＴＤＦＥＶＫＴＤＳＴＤＨＶＫＰＫＥＴＥＮＴＫＱＰＳＫＳＣＨＫＰＫＡＩＶＨＴＥＫＶＮＭＭＳＬＴＶＶＩＧＦＲＩＬＬＬＫＶＡＧＦＮＬＬＭＴＬＲＬＷＳＳＧＳＧＥＧＲＧＳＬＬＴＣＧＤＶＥＥＮＰＧＰＭＬＬＬＶＴＳＬＬＬＣＥＬＰＨＰＡＦＬＬＩＰＤＩＱＭＴＱＴＴＳＳＬＳＡＳＬＧＤＲＶＴＩＳＣＲＡＳＱＤＩＳＫＹＬＮＷＹＱＱＫＰＤＧＴＶＫＬＬＩＹＨＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＳＬＴＩＳＮＬＥＱＥＤＩＡＴＹＦＣＱＱＧＮＴＬＰＹＴＦＧＧＧＴＫＬＥＩＴＧＳＴＳＧＳＧＫＰＧＳＧＥＧＳＴＫＧＥＶＫＬＱＥＳＧＰＧＬＶＡＰＳＱＳＬＳＶＴＣＴＶＳＧＶＳＬＰＤＹＧＶＳＷＩＲＱＰＰＲＫＧＬＥＷＬＧＶＩＷＧＳＥＴＴＹＹＮＳＡＬＫＳＲＬＴＩＩＫＤＮＳＫＳＱＶＦＬＫＭＮＳＬＱＴＤＤＴＡＩＹＹＣＡＫＨＹＹＹＧＧＳＹＡＭＤＹＷＧＱＧＴＳＶＴＶＳＳＡＡＡＧＧＧＧＳＧＧＧＧＳＧＧＧＧＳＬＥＤＫＱＬＤＡＤＶＳＰＫＰＴＩＦＬＰＳＩＡＥＴＫＬＱＫＡＧＴＹＬＣＬＬＥＫＦＦＰＤＶＩＫＩＨＷＱＥＫＫＳＮＴＩＬＧＳＱＥＧＮＴＭＫＴＮＤＴＹＭＫＦＳＷＬＴＶＰＥＫＳＬＤＫＥＨＲＣＩＶＲＨＥＮＮＫＮＧＶＤＱＥＩＩＦＰＴＩＬＹＥＩＬＬＧＫＡＴＬＹＡＶＬＶＳＡＬＶＬＭＡＭＶＫＲＫＤＦ（配列番号５）。

Nt-FMC63-TRDC(1-153)-T2A-TRGC1(1-173)-Ct (amino acid sequence) without signal peptide (according to IMGT numbering, Nt-FMC63-TRDC(-6,189)- 2A-TRGC1(1.8,189)-Ct):
ＤＩＱＭＴＱＴＴＳＳＬＳＡＳＬＧＤＲＶＴＩＳＣＲＡＳＱＤＩＳＫＹＬＮＷＹＱＱＫＰＤＧＴＶＫＬＬＩＹＨＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＳＬＴＩＳＮＬＥＱＥＤＩＡＴＹＦＣＱＱＧＮＴＬＰＹＴＦＧＧＧＴＫＬＥＩＴＧＳＴＳＧＳＧＫＰＧＳＧＥＧＳＴＫＧＥＶＫＬＱＥＳＧＰＧＬＶＡＰＳＱＳＬＳＶＴＣＴＶＳＧＶＳＬＰＤＹＧＶＳＷＩＲＱＰＰＲＫＧＬＥＷＬＧＶＩＷＧＳＥＴＴＹＹＮＳＡＬＫＳＲＬＴＩＩＫＤＮＳＫＳＱＶＦＬＫＭＮＳＬＱＴＤＤＴＡＩＹＹＣＡＫＨＹＹＹＧＧＳＹＡＭＤＹＷＧＱＧＴＳＶＴＶＳＳＡＡＡＧＧＧＧＳＧＧＧＧＳＧＧＧＧＳＬＥＳＱＰＨＴＫＰＳＶＦＶＭＫＮＧＴＮＶＡＣＬＶＫＥＦＹＰＫＤＩＲＩＮＬＶＳＳＫＫＩＴＥＦＤＰＡＩＶＩＳＰＳＧＫＹＮＡＶＫＬＧＫＹＥＤＳＮＳＶＴＣＳＶＱＨＤＮＫＴＶＨＳＴＤＦＥＶＫＴＤＳＴＤＨＶＫＰＫＥＴＥＮＴＫＱＰＳＫＳＣＨＫＰＫＡＩＶＨＴＥＫＶＮＭＭＳＬＴＶＬＧＬＲＭＬＦＡＫＴＶＡＶＮＦＬＬＴＡＫＬＦＦＬＧＳＧＥＧＲＧＳＬＬＴＣＧＤＶＥＥＮＰＧＰＭＬＬＬＶＴＳＬＬＬＣＥＬＰＨＰＡＦＬＬＩＰＤＫＱＬＤＡＤＶＳＰＫＰＴＩＦＬＰＳＩＡＥＴＫＬＱＫＡＧＴＹＬＣＬＬＥＫＦＦＰＤＶＩＫＩＨＷＱＥＫＫＳＮＴＩＬＧＳＱＥＧＮＴＭＫＴＮＤＴＹＭＫＦＳＷＬＴＶＰＥＫＳＬＤＫＥＨＲＣＩＶＲＨＥＮＮＫＮＧＶＤＱＥＩＩＦＰＰＩＫＴＤＶＩＴＭＤＰＫＤＮＣＳＫＤＡＮＤＴＬＬＬＱＬＴＮＴＳＡＹＹＭＹＬＬＬＬＬＫＳＶＶＹＦＡＩＩＴＣＣＬＬＲＲＴＡＦＣＣＮＧＥＫＳ（配列番号６）。

Nt-FMC63-TRDC(1-153)-T2A-TRGC1(1-173)-Ct (nucleic acid sequence) without signal peptide (according to IMGT numbering, Nt-FMC63-TRDC(-6,189)- 2A-TRGC1(1.8,189)-Ct):
ＧＡＣＡＴＣＣＡＧＡＴＧＡＣＡＣＡＧＡＣＴＡＣＡＴＣＣＴＣＣＣＴＧＴＣＴＧＣＣＴＣＴＣＴＧＧＧＡＧＡＣＡＧＡＧＴＣＡＣＣＡＴＣＡＧＴＴＧＣＡＧＧＧＣＡＡＧＴＣＡＧＧＡＣＡＴＴＡＧＴＡＡＡＴＡＴＴＴＡＡＡＴＴＧＧＴＡＴＣＡＧＣＡＧＡＡＡＣＣＡＧＡＴＧＧＡＡＣＴＧＴＴＡＡＡＣＴＣＣＴＧＡＴＣＴＡＣＣＡＴＡＣＡＴＣＡＡＧＡＴＴＡＣＡＣＴＣＡＧＧＡＧＴＣＣＣＡＴＣＡＡＧＧＴＴＣＡＧＴＧＧＣＡＧＴＧＧＧＴＣＴＧＧＡＡＣＡＧＡＴＴＡＴＴＣＴＣＴＣＡＣＣＡＴＴＡＧＣＡＡＣＣＴＧＧＡＧＣＡＡＧＡＡＧＡＴＡＴＴＧＣＣＡＣＴＴＡＣＴＴＴＴＧＣＣＡＡＣＡＧＧＧＴＡＡＴＡＣＧＣＴＴＣＣＧＴＡＣＡＣＧＴＴＣＧＧＡＧＧＧＧＧＧＡＣＴＡＡＧＴＴＧＧＡＡＡＴＡＡＣＡＧＧＣＴＣＣＡＣＣＴＣＴＧＧＡＴＣＣＧＧＣＡＡＧＣＣＣＧＧＡＴＣＴＧＧＣＧＡＧＧＧＡＴＣＣＡＣＣＡＡＧＧＧＣＧＡＧＧＴＧＡＡＡＣＴＧＣＡＧＧＡＧＴＣＡＧＧＡＣＣＴＧＧＣＣＴＧＧＴＧＧＣＧＣＣＣＴＣＡＣＡＧＡＧＣＣＴＧＴＣＣＧＴＣＡＣＡＴＧＣＡＣＴＧＴＣＴＣＡＧＧＧＧＴＣＴＣＡＴＴＡＣＣＣＧＡＣＴＡＴＧＧＴＧＴＡＡＧＣＴＧＧＡＴＴＣＧＣＣＡＧＣＣＴＣＣＡＣＧＡＡＡＧＧＧＴＣＴＧＧＡＧＴＧＧＣＴＧＧＧＡＧＴＡＡＴＡＴＧＧＧＧＴＡＧＴＧＡＡＡＣＣＡＣＡＴＡＣＴＡＴＡＡＴＴＣＡＧＣＴＣＴＣＡＡＡＴＣＣＡＧＡＣＴＧＡＣＣＡＴＣＡＴＣＡＡＧＧＡＣＡＡＣＴＣＣＡＡＧＡＧＣＣＡＡＧＴＴＴＴＣＴＴＡＡＡＡＡＴＧＡＡＣＡＧＴＣＴＧＣＡＡＡＣＴＧＡＴＧＡＣＡＣＡＧＣＣＡＴＴＴＡＣＴＡＣＴＧＴＧＣＣＡＡＡＣＡＴＴＡＴＴＡＣＴＡＣＧＧＴＧＧＴＡＧＣＴＡＴＧＣＴＡＴＧＧＡＣＴＡＣＴＧＧＧＧＴＣＡＡＧＧＡＡＣＣＴＣＡＧＴＣＡＣＣＧＴＣＴＣＣＴＣＡＧＣＧＧＣＣＧＣＡＧＧＴＧＧＡＧＧＡＧＧＴＴＣＴＧＧＡＧＧＴＧＧＴＧＧＡＴＣＡＧＧＴＧＧＴＧＧＡＧＧＡＴＣＴＴＴＡＧＡＡＡＧＴＣＡＧＣＣＴＣＡＴＡＣＣＡＡＡＣＣＡＴＣＣＧＴＴＴＴＴＧＴＣＡＴＧＡＡＡＡＡＴＧＧＡＡＣＡＡＡＴＧＴＣＧＣＴＴＧＴＣＴＧＧＴＧＡＡＧＧＡＡＴＴＣＴＡＣＣＣＣＡＡＧＧＡＴＡＴＡＡＧＡＡＴＡＡＡＴＣＴＣＧＴＧＴＣＡＴＣＣＡＡＧＡＡＧＡＴＡＡＣＡＧＡＧＴＴＴＧＡＴＣＣＴＧＣＴＡＴＴＧＴＣＡＴＣＴＣＴＣＣＣＡＧＴＧＧＧＡＡＧＴＡＣＡＡＴＧＣＴＧＴＣＡＡＧＣＴＴＧＧＴＡＡＡＴＡＴＧＡＡＧＡＴＴＣＡＡＡＴＴＣＡＧ ＴＧＡＣＡＴＧＴＴＣＡＧＴＴＣＡＡＣＡＣＧＡＣＡＡＴＡＡＡＡＣＴＧＴＧＣＡＣＴＣＣＡＣＴＧＡＣＴＴＴＧＡＡＧＴＧＡＡＧＡＣＡＧＡＴＴＣＴＡＣＡＧＡＴＣＡＣＧＴＡＡＡＡＣＣＡＡＡＧＧＡＡＡＣＴＧＡＡＡＡＣＡＣＡＡＡＧＣＡＡＣＣＴＴＣＡＡＡＧＡＧＣＴＧＣＣＡＴＡＡＡＣＣＣＡＡＡＧＣＣＡＴＡＧＴＴＣＡＴＡＣＣＧＡＧＡＡＧＧＴＧＡＡＣＡＴＧＡＴＧＴＣＣＣＴＣＡＣＡＧＴＧＣＴＴＧＧＧＣＴＡＣＧＡＡＴＧＣＴＧＴＴＴＧＣＡＡＡＧＡＣＴＧＴＴＧＣＣＧＴＣＡＡＴＴＴＴＣＴＣＴＴＧＡＣＴＧＣＣＡＡＧＴＴＡＴＴＴＴＴＣＴＴＧＧＧＧＴＣＡＧＧＣＧＡＧＧＧＣＡＧＡＧＧＡＡＧＴＣＴＧＣＴＡＡＣＡＴＧＣＧＧＴＧＡＣＧＴＣＧＡＧＧＡＧＡＡＴＣＣＴＧＧＡＣＣＴＡＴＧＣＴＡＣＴＡＣＴＴＧＴＧＡＣＣＴＣＡＣＴＡＴＴＧＴＴＡＴＧＣＧＡＡＣＴＣＣＣＴＣＡＴＣＣＣＧＣＡＴＴＣＴＴＧＣＴＧＡＴＴＣＣＡＧＡＴＡＡＡＣＡＡＣＴＴＧＡＴＧＣＡＧＡＴＧＴＴＴＣＣＣＣＣＡＡＧＣＣＣＡＣＴＡＴＴＴＴＴＣＴＴＣＣＴＴＣＡＡＴＴＧＣＴＧＡＡＡＣＡＡＡＧＣＴＣＣＡＧＡＡＧＧＣＴＧＧＡＡＣＡＴＡＣＣＴＴＴＧＴＣＴＴＣＴＴＧＡＧＡＡＡＴＴＴＴＴＣＣＣＴＧＡＴＧＴＴＡＴＴＡＡＧＡＴＡＣＡＴＴＧＧＣＡＡＧＡＡＡＡＧＡＡＧＡＧＣＡＡＣＡＣＧＡＴＴＣＴＧＧＧＡＴＣＣＣＡＧＧＡＧＧＧＧＡＡＣＡＣＣＡＴＧＡＡＧＡＣＴＡＡＣＧＡＣＡＣＡＴＡＣＡＴＧＡＡＡＴＴＴＡＧＣＴＧＧＴＴＡＡＣＧＧＴＧＣＣＡＧＡＡＡＡＧＴＣＡＣＴＧＧＡＣＡＡＡＧＡＡＣＡＣＡＧＡＴＧＴＡＴＣＧＴＣＡＧＡＣＡＴＧＡＧＡＡＴＡＡＴＡＡＡＡＡＣＧＧＡＧＴＴＧＡＴＣＡＡＧＡＡＡＴＴＡＴＣＴＴＴＣＣＴＣＣＡＡＴＡＡＡＧＡＣＡＧＡＴＧＴＣＡＴＣＡＣＡＡＴＧＧＡＴＣＣＣＡＡＡＧＡＣＡＡＴＴＧＴＴＣＡＡＡＡＧＡＴＧＣＡＡＡＴＧＡＴＡＣＡＣＴＡＣＴＧＣＴＧＣＡＧＣＴＣＡＣＡＡＡＣＡＣＣＴＣＴＧＣＡＴＡＴＴＡＣＡＴＧＴＡＣＣＴＣＣＴＣＣＴＧＣＴＣＣＴＣＡＡＧＡＧＴＧＴＧＧＴＣＴＡＴＴＴＴＧＣＣＡＴＣＡＴＣＡＣＣＴＧＣＴＧＴＣＴＧＣＴＴＡＧＡＡＧＡＡＣＧＧＣＴＴＴＣＴＧＣＴＧＣＡＡＴＧＧＡＧＡＧＡＡＡＴＣＡ（配列番号７）。

Nt-TRDC(1-153)-T2A-FMC63-TRGC1(1-173)-Ct (no signal peptide; amino acid sequence) (according to IMGT numbering, Nt-TRDC
(−6,189)-2A-FMC63-TRGC1(1.8,189)-Ct):
ＳＱＰＨＴＫＰＳＶＦＶＭＫＮＧＴＮＶＡＣＬＶＫＥＦＹＰＫＤＩＲＩＮＬＶＳＳＫＫＩＴＥＦＤＰＡＩＶＩＳＰＳＧＫＹＮＡＶＫＬＧＫＹＥＤＳＮＳＶＴＣＳＶＱＨＤＮＫＴＶＨＳＴＤＦＥＶＫＴＤＳＴＤＨＶＫＰＫＥＴＥＮＴＫＱＰＳＫＳＣＨＫＰＫＡＩＶＨＴＥＫＶＮＭＭＳＬＴＶＬＧＬＲＭＬＦＡＫＴＶＡＶＮＦＬＬＴＡＫＬＦＦＬＧＳＧＥＧＲＧＳＬＬＴＣＧＤＶＥＥＮＰＧＰＭＬＬＬＶＴＳＬＬＬＣＥＬＰＨＰＡＦＬＬＩＰＤＩＱＭＴＱＴＴＳＳＬＳＡＳＬＧＤＲＶＴＩＳＣＲＡＳＱＤＩＳＫＹＬＮＷＹＱＱＫＰＤＧＴＶＫＬＬＩＹＨＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＳＬＴＩＳＮＬＥＱＥＤＩＡＴＹＦＣＱＱＧＮＴＬＰＹＴＦＧＧＧＴＫＬＥＩＴＧＳＴＳＧＳＧＫＰＧＳＧＥＧＳＴＫＧＥＶＫＬＱＥＳＧＰＧＬＶＡＰＳＱＳＬＳＶＴＣＴＶＳＧＶＳＬＰＤＹＧＶＳＷＩＲＱＰＰＲＫＧＬＥＷＬＧＶＩＷＧＳＥＴＴＹＹＮＳＡＬＫＳＲＬＴＩＩＫＤＮＳＫＳＱＶＦＬＫＭＮＳＬＱＴＤＤＴＡＩＹＹＣＡＫＨＹＹＹＧＧＳＹＡＭＤＹＷＧＱＧＴＳＶＴＶＳＳＡＡＡＧＧＧＧＳＧＧＧＧＳＧＧＧＧＳＬＥＤＫＱＬＤＡＤＶＳＰＫＰＴＩＦＬＰＳＩＡＥＴＫＬＱＫＡＧＴＹＬＣＬＬＥＫＦＦＰＤＶＩＫＩＨＷＱＥＫＫＳＮＴＩＬＧＳＱＥＧＮＴＭＫＴＮＤＴＹＭＫＦＳＷＬＴＶＰＥＫＳＬＤＫＥＨＲＣＩＶＲＨＥＮＮＫＮＧＶＤＱＥＩＩＦＰＰＩＫＴＤＶＩＴＭＤＰＫＤＮＣＳＫＤＡＮＤＴＬＬＬＱＬＴＮＴＳＡＹＹＭＹＬＬＬＬＬＫＳＶＶＹＦＡＩＩＴＣＣＬＬＲＲＴＡＦＣＣＮＧＥＫＳ（配列番号８）。

Nt-TRDC(1-153)-T2A-FMC63-TRGC1(1-173)-Ct (with signal peptide; nucleic acid sequence) (according to IMGT numbering, Nt-TRDC(-6,189)-2A-FMC63 -TRGC1(1.8,189)-Ct):
ＡＧＴＣＡＧＣＣＴＣＡＴＡＣＣＡＡＡＣＣＡＴＣＣＧＴＴＴＴＴＧＴＣＡＴＧＡＡＡＡＡＴＧＧＡＡＣＡＡＡＴＧＴＣＧＣＴＴＧＴＣＴＧＧＴＧＡＡＧＧＡＡＴＴＣＴＡＣＣＣＣＡＡＧＧＡＴＡＴＡＡＧＡＡＴＡＡＡＴＣＴＣＧＴＧＴＣＡＴＣＣＡＡＧＡＡＧＡＴＡＡＣＡＧＡＧＴＴＴＧＡＴＣＣＴＧＣＴＡＴＴＧＴＣＡＴＣＴＣＴＣＣＣＡＧＴＧＧＧＡＡＧＴＡＣＡＡＴＧＣＴＧＴＣＡＡＧＣＴＴＧＧＴＡＡＡＴＡＴＧＡＡＧＡＴＴＣＡＡＡＴＴＣＡＧＴＧＡＣＡＴＧＴＴＣＡＧＴＴＣＡＡＣＡＣＧＡＣＡＡＴＡＡＡＡＣＴＧＴＧＣＡＣＴＣＣＡＣＴＧＡＣＴＴＴＧＡＡＧＴＧＡＡＧＡＣＡＧＡＴＴＣＴＡＣＡＧＡＴＣＡＣＧＴＡＡＡＡＣＣＡＡＡＧＧＡＡＡＣＴＧＡＡＡＡＣＡＣＡＡＡＧＣＡＡＣＣＴＴＣＡＡＡＧＡＧＣＴＧＣＣＡＴＡＡＡＣＣＣＡＡＡＧＣＣＡＴＡＧＴＴＣＡＴＡＣＣＧＡＧＡＡＧＧＴＧＡＡＣＡＴＧＡＴＧＴＣＣＣＴＣＡＣＡＧＴＧＣＴＴＧＧＧＣＴＡＣＧＡＡＴＧＣＴＧＴＴＴＧＣＡＡＡＧＡＣＴＧＴＴＧＣＣＧＴＣＡＡＴＴＴＴＣＴＣＴＴＧＡＣＴＧＣＣＡＡＧＴＴＡＴＴＴＴＴＣＴＴＧＧＧＧＴＣＡＧＧＣＧＡＧＧＧＣＡＧＡＧＧＡＡＧＴＣＴＧＣＴＡＡＣＡＴＧＣＧＧＴＧＡＣＧＴＣＧＡＧＧＡＧＡＡＴＣＣＴＧＧＡＣＣＴＡＴＧＣＴＡＣＴＡＣＴＴＧＴＧＡＣＣＴＣＡＣＴＡＴＴＧＴＴＡＴＧＣＧＡＡＣＴＣＣＣＴＣＡＴＣＣＣＧＣＡＴＴＣＴＴＧＣＴＧＡＴＴＣＣＡＧＡＣＡＴＴＣＡＧＡＴＧＡＣＴＣＡＡＡＣＡＡＣＴＴＣＣＡＧＣＣＴＣＴＣＣＧＣＣＴＣＡＣＴＣＧＧＣＧＡＣＣＧＣＧＴＡＡＣＡＡＴＡＡＧＣＴＧＴＣＧＧＧＣＣＴＣＧＣＡＡＧＡＴＡＴＴＡＧＴＡＡＧＴＡＣＣＴＧＡＡＴＴＧＧＴＡＴＣＡＧＣＡＡＡＡＡＣＣＣＧＡＴＧＧＴＡＣＡＧＴＣＡＡＧＣＴＴＣＴＧＡＴＣＴＡＣＣＡＴＡＣＣＡＧＴＣＧＴＣＴＧＣＡＣＡＧＣＧＧＴＧＴＣＣＣＣＡＧＣＡＧＧＴＴＣＡＧＣＧＧＣＴＣＡＧＧＡＴＣＴＧＧＴＡＣＣＧＡＴＴＡＴＴＣＡＣＴＧＡＣＧＡＴＴＴＣＣＡＡＣＣＴＴＧＡＧＣＡＧＧＡＧＧＡＣＡＴＣＧＣＣＡＣＣＴＡＣＴＴＣＴＧＣＣＡＧＣＡＧＧＧＴＡＡＴＡＣＴＣＴＧＣＣＧＴＡＣＡＣＡＴＴＣＧＧＧＧＧＣＧＧＴＡＣＣＡＡＧＣＴＣＧＡＧＡＴＣＡＣＧＧＧＴＴＣＡＡＣＡＡＧＣＧＧＴＴＣＴＧＧＣＡＡＧＣＣＡＧＧＣＡＧＣＧＧＣＧＡＧＧＧＧＡＧＴＡＣＡＡＡＧＧＧＧＧＡＧＧＴＧＡＡＧＴＴＧＣＡＧＧＡＡＡＧＴＧＧＣＣＣＴＧＧＡＴＴＧＧＴＧＧ CCCCGAGCCAGAGTCTGTCTGTCACCTGCACAGTTTCCGGAGTAAGTCTGCCTGATTACGGAGTGTCCTGGATCAGACAGCCACCTCGAAAGGGCTTGGAGTGGCTTGGGGTCATTTGGGGCAGTGAAACCATACTACAACAGCGCTCTTAAGTCCAG
ＧＣＴＣＡＣＴＡＴＣＡＴＣＡＡＧＧＡＣＡＡＴＴＣＡＡＡＧＡＧＣＣＡＡＧＴＡＴＴＣＴＴＧＡＡＡＡＴＧＡＡＴＴＣＣＣＴＧＣＡＧＡＣＴＧＡＴＧＡＣＡＣＣＧＣＴＡＴＴＴＡＴＴＡＴＴＧＣＧＣＴＡＡＡＣＡＴＴＡＴＴＡＣＴＡＴＧＧＡＧＧＴＴＣＴＴＡＴＧＣＣＡＴＧＧＡＣＴＡＣＴＧＧＧＧＧＣＡＧＧＧＴＡＣＣＴＣＴＧＴＧＡＣＡＧＴＧＡＧＴＴＣＡＧＣＴＧＣＡＧＣＴＧＧＡＧＧＴＧＧＡＧＧＴＡＧＣＧＧＡＧＧＣＧＧＴＧＧＴＡＧＴＧＧＡＧＧＧＧＧＴＧＧＴＴＣＴＣＴＧＧＡＡＧＡＴＡＡＡＣＡＡＣＴＴＧＡＴＧＣＡＧＡＴＧＴＴＴＣＣＣＣＣＡＡＧＣＣＣＡＣＴＡＴＴＴＴＴＣＴＴＣＣＴＴＣＡＡＴＴＧＣＴＧＡＡＡＣＡＡＡＧＣＴＣＣＡＧＡＡＧＧＣＴＧＧＡＡＣＡＴＡＣＣＴＴＴＧＴＣＴＴＣＴＴＧＡＧＡＡＡＴＴＴＴＴＣＣＣＴＧＡＴＧＴＴＡＴＴＡＡＧＡＴＡＣＡＴＴＧＧＣＡＡＧＡＡＡＡＧＡＡＧＡＧＣＡＡＣＡＣＧＡＴＴＣＴＧＧＧＡＴＣＣＣＡＧＧＡＧＧＧＧＡＡＣＡＣＣＡＴＧＡＡＧＡＣＴＡＡＣＧＡＣＡＣＡＴＡＣＡＴＧＡＡＡＴＴＴＡＧＣＴＧＧＴＴＡＡＣＧＧＴＧＣＣＡＧＡＡＡＡＧＴＣＡＣＴＧＧＡＣＡＡＡＧＡＡＣＡＣＡＧＡＴＧＴＡＴＣＧＴＣＡＧＡＣＡＴＧＡＧＡＡＴＡＡＴＡＡＡＡＡＣＧＧＡＧＴＴＧＡＴＣＡＡＧＡＡＡＴＴＡＴＣＴＴＴＣＣＴＣＣＡＡＴＡＡＡＧＡＣＡＧＡＴＧＴＣＡＴＣＡＣＡＡＴＧＧＡＴＣＣＣＡＡＡＧＡＣＡＡＴＴＧＴＴＣＡＡＡＡＧＡＴＧＣＡＡＡＴＧＡＴＡＣＡＣＴＡＣＴＧＣＴＧＣＡＧＣＴＣＡＣＡＡＡＣＡＣＣＴＣＴＧＣＡＴＡＴＴＡＣＡＴＧＴＡＣＣＴＣＣＴＣＣＴＧＣＴＣＣＴＣＡＡＧＡＧＴＧＴＧＧＴＣＴＡＴＴＴＴＧＣＣＡＴＣＡＴＣＡＣＣＴＧＣＴＧＴＣＴＧＣＴＴＡＧＡＡＧＡＡＣＧＧＣＴＴＴＣＴＧＣＴＧＣＡＡＴＧＧＡＧＡＧＡＡＡＴＣＡ（配列番号９）。

Expression of mouse TCRβ/α TFP
[0533] TCRα-negative cells still express TCRβ, and conversely TCRα is expressed in TCRβ-negative cells. However, TCRα or TCRβ TFPs generated by assembling TCRα and/or TCRβ constant domains and antigen binding agents (e.g., scFvs or sdAbs) can be paired with TCRα or TCRβ constant domains. However, the constant domains of human TCRα and TCRβ are unable to translocate to the cell surface. Mouse or human-mouse chimeric TCRα and TCRβ constant domains can be used. FIG. 12A is a schematic showing which TFPs can and cannot reconstitute at the cell surface. A mouse anti-CD19 TCRα TFP comprising the constant, intracellular and transmembrane domains of mouse TCRα and a protein comprising the constant, intracellular and transmembrane domains of TCRβ were transfected into TRA ^−/− cells or TRB ^{−/ -} co-expressed in cells. In one embodiment, TCRα TFPs were generated by assembling the constant domain of TCRα and an antigen binding agent (eg, scFv or sdAb).

TCRγ/δ FMC63 TFP expressed in TRA ^−/− or TRB ^−/− cells
Nt-pLRPO FMC63-mTRAC(82-137)T2A mTRBC(123-173)-Ct (according to IMGT numbering Nt-pLRPO FMC63-mTRAC(197-252)T2A mTRBC(233-283)-Ct) ( amino acid sequence):
ＤＩＱＭＴＱＴＴＳＳＬＳＡＳＬＧＤＲＶＴＩＳＣＲＡＳＱＤＩＳＫＹＬＮＷＹＱＱＫＰＤＧＴＶＫＬＬＩＹＨＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＳＬＴＩＳＮＬＥＱＥＤＩＡＴＹＦＣＱＱＧＮＴＬＰＹＴＦＧＧＧＴＫＬＥＩＴＧＧＧＧＳＧＧＧＧＳＧＧＧＧＳＥＶＫＬＱＥＳＧＰＧＬＶＡＰＳＱＳＬＳＶＴＣＴＶＳＧＶＳＬＰＤＹＧＶＳＷＩＲＱＰＰＲＫＧＬＥＷＬＧＶＩＷＧＳＥＴＴＹＹＮＳＡＬＫＳＲＬＴＩＩＫＤＮＳＫＳＱＶＦＬＫＭＮＳＬＱＴＤＤＴＡＩＹＹＣＡＫＨＹＹＹＧＧＳＹＡＭＤＹＷＧＱＧＴＳＶＴＶＳＳＧＡＴＹＰＳＳＤＶＰＣＤＡＴＬＴＥＫＳＦＥＴＤＭＮＬＮＦＱＮＬＳＶＭＧＬＲＩＬＬＬＫＶＡＧＦＮＬＬＭＴＬＲＬＷＳＳＧＳＧＥＧＲＧＳＬＬＴＣＧＤＶＥＥＮＰＧＰＧＲＡＤＣＧＩＴＳＡＳＹＱＱＧＶＬＳＡＴＩＬＹＥＩＬＬＧＫＡＴＬＹＡＶＬＶＳＴＬＶＶＭＡＭＶＫＲＫＮＳ（配列番号１０）。

Nt-pLRPO FMC63-mTRAC(82-137)T2A mTRBC(123-173)-Ct (according to IMGT numbering Nt-pLRPO FMC63-mTRAC(197-252)T2A mTRBC(233-283)-Ct) ( nucleic acid sequence)
:
ＧＡＣＡＴＣＣＡＧＡＴＧＡＣＡＣＡＧＡＣＴＡＣＡＴＣＣＴＣＣＣＴＧＴＣＴＧＣＣＴＣＴＣＴＧＧＧＡＧＡＣＡＧＡＧＴＣＡＣＣＡＴＣＡＧＴＴＧＣＡＧＧＧＣＡＡＧＴＣＡＧＧＡＣＡＴＴＡＧＴＡＡＡＴＡＴＴＴＡＡＡＴＴＧＧＴＡＴＣＡＧＣＡＧＡＡＡＣＣＡＧＡＴＧＧＡＡＣＴＧＴＴＡＡＡＣＴＣＣＴＧＡＴＣＴＡＣＣＡＴＡＣＡＴＣＡＡＧＡＴＴＡＣＡＣＴＣＡＧＧＡＧＴＣＣＣＡＴＣＡＡＧＧＴＴＣＡＧＴＧＧＣＡＧＴＧＧＧＴＣＴＧＧＡＡＣＡＧＡＴＴＡＴＴＣＴＣＴＣＡＣＣＡＴＴＡＧＣＡＡＣＣＴＧＧＡＧＣＡＡＧＡＡＧＡＴＡＴＴＧＣＣＡＣＴＴＡＣＴＴＴＴＧＣＣＡＡＣＡＧＧＧＴＡＡＴＡＣＧＣＴＴＣＣＧＴＡＣＡＣＧＴＴＣＧＧＡＧＧＧＧＧＧＡＣＴＡＡＧＴＴＧＧＡＡＡＴＡＡＣＡＧＧＡＧＧＴＧＧＡＧＧＴＴＣＴＧＧＴＧＧＡＧＧＡＧＧＴＴＣＡＧＧＡＧＧＴＧＧＴＧＧＡＡＧＴＧＡＧＧＴＧＡＡＡＣＴＧＣＡＧＧＡＧＴＣＡＧＧＡＣＣＴＧＧＣＣＴＧＧＴＧＧＣＧＣＣＣＴＣＡＣＡＧＡＧＣＣＴＧＴＣＣＧＴＣＡＣＡＴＧＣＡＣＴＧＴＣＴＣＡＧＧＧＧＴＣＴＣＡＴＴＡＣＣＣＧＡＣＴＡＴＧＧＴＧＴＡＡＧＣＴＧＧＡＴＴＣＧＣＣＡＧＣＣＴＣＣＡＣＧＡＡＡＧＧＧＴＣＴＧＧＡＧＴＧＧＣＴＧＧＧＡＧＴＡＡＴＡＴＧＧＧＧＴＡＧＴＧＡＡＡＣＣＡＣＡＴＡＣＴＡＴＡＡＴＴＣＡＧＣＴＣＴＣＡＡＡＴＣＣＡＧＡＣＴＧＡＣＣＡＴＣＡＴＣＡＡＧＧＡＣＡＡＣＴＣＣＡＡＧＡＧＣＣＡＡＧＴＴＴＴＣＴＴＡＡＡＡＡＴＧＡＡＣＡＧＴＣＴＧＣＡＡＡＣＴＧＡＴＧＡＣＡＣＡＧＣＣＡＴＴＴＡＣＴＡＣＴＧＴＧＣＣＡＡＡＣＡＴＴＡＴＴＡＣＴＡＣＧＧＴＧＧＴＡＧＣＴＡＴＧＣＴＡＴＧＧＡＣＴＡＣＴＧＧＧＧＴＣＡＡＧＧＡＡＣＣＴＣＡＧＴＣＡＣＣＧＴＣＴＣＣＴＣＡＧＧＡＧＣＣＡＣＣＴＡＣＣＣＣＡＧＴＴＣＡＧＡＣＧＴＴＣＣＣＴＧＴＧＡＴＧＣＣＡＣＧＴＴＧＡＣＴＧＡＧＡＡＡＡＧＣＴＴＴＧＡＡＡＣＡＧＡＴＡＴＧＡＡＣＣＴＡＡＡＣＴＴＴＣＡＡＡＡＣＣＴＧＴＣＡＧＴＴＡＴＧＧＧＡＣＴＣＣＧＡＡＴＣＣＴＣＣＴＧＣＴＧＡＡＡＧＴＡＧＣＣＧＧＡＴＴＴＡＡＣＣＴＧＣＴＣＡＴＧＡＣＧＣＴＧＡＧＧＣＴＧＴＧＧＴＣＣＡＧＴＧＧＣＡＧＣＧＧＣＧＡＧＧＧＣＡＧＡＧＧＡＡＧＴＣＴＧＣＴＡＡＣＡＴＧＣＧＧＴＧＡＣＧＴＣＧＡＧＧＡＧＡＡＴＣＣＴＧＧＡＣＣＴＧＧＴＣＧＡＧＣＡＧＡＣＴＧＴＧＧＴＡＴＴＡＣＣＴＣＡＧＣＡＴＣＣＴＡＴＣＡＡＣ AAGGAGTCTTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAAGCCACCCTGTATGCTGTGCTTGTCAGTACACTGGTGGTGATGGCTATGGTCAAAAGAAAGAATTCA (SEQ ID NO: 11).

pLRPO FMC63-TCRβ1 (amino acid sequence)
ＤＩＱＭＴＱＴＴＳＳＬＳＡＳＬＧＤＲＶＴＩＳＣＲＡＳＱＤＩＳＫＹＬＮＷＹＱＱＫＰＤＧＴＶＫＬＬＩＹＨＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＳＬＴＩＳＮＬＥＱＥＤＩＡＴＹＦＣＱＱＧＮＴＬＰＹＴＦＧＧＧＴＫＬＥＩＴＧＳＴＳＧＳＧＫＰＧＳＧＥＧＳＴＫＧＥＶＫＬＱＥＳＧＰＧＬＶＡＰＳＱＳＬＳＶＴＣＴＶＳＧＶＳＬＰＤＹＧＶＳＷＩＲＱＰＰＲＫＧＬＥＷＬＧＶＩＷＧＳＥＴＴＹＹＮＳＡＬＫＳＲＬＴＩＩＫＤＮＳＫＳＱＶＦＬＫＭＮＳＬＱＴＤＤＴＡＩＹＹＣＡＫＨＹＹＹＧＧＳＹＡＭＤＹＷＧＱＧＴＳＶＴＶＳＳＡＡＡＧＧＧＧＳＧＧＧＧＳＧＧＧＧＳＬＥＬＧＡＧＰＶＤＳＧＶＴＱＴＰＫＨＬＩＴＡＴＧＱＲＶＴＬＲＣＳＰＲＳＧＤＬＳＶＳＷＹＱＱＳＬＤＱＧＬＱＦＬＩＱＹＹＮＧＥＥＲＡＫＧＮＩＬＥＲＦＳＡＱＱＦＰＤＬＨＳＥＬＮＬＳＳＬＥＬＧＤＳＡＬＹＦＣＡＳＳＰＲＴＧＬＮＴＥＡＦＦＧＱＧＴＲＬＴＶＶＥＤＬＮＫＶＦＰＰＥＶＡＶＦＥＰＳＥＡＥＩＳＨＴＱＫＡＴＬＶＣＬＡＴＧＦＦＰＤＨＶＥＬＳＷＷＶＮＧＫＥＶＨＳＧＶＳＴＤＰＱＰＬＫＥＱＰＡＬＮＤＳＲＹＣＬＳＳＲＬＲＶＳＡＴＦＷＱＮＰＲＮＨＦＲＣＱＶＱＦＹＧＬＳＥＮＤＥＷＴＱＤＲＡＫＰＶＴＱＩＶＳＡＥＡＷＧＲＡＤＣＧＦＴＳＶＳＹＱＱＧＶＬＳＡＴＩＬＹＥＩＬＬＧＫＡＴＬＹＡＶＬＶＳＡＬＶＬＭＡＭＶＫＲＫＤＦ（配列番号１２）。

pLRPO FMC63-TCRβ1 (nucleic acid sequence)
GACATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAGTCACCATCAGTTGCAGGGCAAGTCAGGACATTAGTAAAATTTAAATTGGTATCAGCAGAAACCA
ＧＡＴＧＧＡＡＣＴＧＴＴＡＡＡＣＴＣＣＴＧＡＴＣＴＡＣＣＡＴＡＣＡＴＣＡＡＧＡＴＴＡＣＡＣＴＣＡＧＧＡＧＴＣＣＣＡＴＣＡＡＧＧＴＴＣＡＧＴＧＧＣＡＧＴＧＧＧＴＣＴＧＧＡＡＣＡＧＡＴＴＡＴＴＣＴＣＴＣＡＣＣＡＴＴＡＧＣＡＡＣＣＴＧＧＡＧＣＡＡＧＡＡＧＡＴＡＴＴＧＣＣＡＣＴＴＡＣＴＴＴＴＧＣＣＡＡＣＡＧＧＧＴＡＡＴＡＣＧＣＴＴＣＣＧＴＡＣＡＣＧＴＴＣＧＧＡＧＧＧＧＧＧＡＣＴＡＡＧＴＴＧＧＡＡＡＴＡＡＣＡＧＧＣＴＣＣＡＣＣＴＣＴＧＧＡＴＣＣＧＧＣＡＡＧＣＣＣＧＧＡＴＣＴＧＧＣＧＡＧＧＧＡＴＣＣＡＣＣＡＡＧＧＧＣＧＡＧＧＴＧＡＡＡＣＴＧＣＡＧＧＡＧＴＣＡＧＧＡＣＣＴＧＧＣＣＴＧＧＴＧＧＣＧＣＣＣＴＣＡＣＡＧＡＧＣＣＴＧＴＣＣＧＴＣＡＣＡＴＧＣＡＣＴＧＴＣＴＣＡＧＧＧＧＴＣＴＣＡＴＴＡＣＣＣＧＡＣＴＡＴＧＧＴＧＴＡＡＧＣＴＧＧＡＴＴＣＧＣＣＡＧＣＣＴＣＣＡＣＧＡＡＡＧＧＧＴＣＴＧＧＡＧＴＧＧＣＴＧＧＧＡＧＴＡＡＴＡＴＧＧＧＧＴＡＧＴＧＡＡＡＣＣＡＣＡＴＡＣＴＡＴＡＡＴＴＣＡＧＣＴＣＴＣＡＡＡＴＣＣＡＧＡＣＴＧＡＣＣＡＴＣＡＴＣＡＡＧＧＡＣＡＡＣＴＣＣＡＡＧＡＧＣＣＡＡＧＴＴＴＴＣＴＴＡＡＡＡＡＴＧＡＡＣＡＧＴＣＴＧＣＡＡＡＣＴＧＡＴＧＡＣＡＣＡＧＣＣＡＴＴＴＡＣＴＡＣＴＧＴＧＣＣＡＡＡＣＡＴＴＡＴＴＡＣＴＡＣＧＧＴＧＧＴＡＧＣＴＡＴＧＣＴＡＴＧＧＡＣＴＡＣＴＧＧＧＧＴＣＡＡＧＧＡＡＣＣＴＣＡＧＴＣＡＣＣＧＴＣＴＣＣＴＣＡＧＣＧＧＣＣＧＣＡＧＧＴＧＧＡＧＧＡＧＧＴＴＣＴＧＧＡＧＧＴＧＧＡＧＧＴＴＣＡＧＧＴＧＧＡＧＧＴＧＧＴＴＣＡＣＴＣＧＡＧＣＴＧＧＧＡＧＣＡＧＧＣＣＣＡＧＴＧＧＡＴＴＣＴＧＧＡＧＴＣＡＣＡＣＡＡＡＣＣＣＣＡＡＡＧＣＡＣＣＴＧＡＴＣＡＣＡＧＣＡＡＣＴＧＧＡＣＡＧＣＧＡＧＴＧＡＣＧＣＴＧＡＧＡＴＧＣＴＣＣＣＣＴＡＧＧＴＣＴＧＧＡＧＡＣＣＴＣＴＣＴＧＴＧＴＣＡＴＧＧＴＡＣＣＡＡＣＡＧＡＧＣＣＴＧＧＡＣＣＡＧＧＧＣＣＴＣＣＡＧＴＴＣＣＴＣＡＴＴＣＡＧＴＡＴＴＡＴＡＡＴＧＧＡＧＡＡＧＡＧＡＧＡＧＣＡＡＡＡＧＧＡＡＡＣＡＴＴＣＴＴＧＡＡＣＧＡＴＴＣＴＣＣＧＣＡＣＡＡＣＡＧＴＴＣＣＣＴＧＡＣＴＴＧＣＡＣＴＣＴＧＡＡＣＴＡＡＡＣＣＴＧＡＧＣＴＣＴＣＴＧＧＡＧＣＴＧＧＧＧＧＡＣＴＣＡＧＣＴＴＴＧＴＡＴＴＴＣＴＧＴＧＣＣＡＧＣＡＧＣＣＣＣＣＧＧＡＣＡＧＧＣＣＴＧＡＡＣＡＣＴＧＡＡＧ ＣＴＴＴＣＴＴＴＧＧＡＣＡＡＧＧＣＡＣＣＡＧＡＣＴＣＡＣＡＧＴＴＧＴＡＧＡＧＧＡＣＣＴＧＡＡＣＡＡＧＧＴＧＴＴＣＣＣＡＣＣＣＧＡＧＧＴＣＧＣＴＧＴＧＴＴＴＧＡＧＣＣＡＴＣＡＧＡＡＧＣＡＧＡＧＡＴＣＴＣＣＣＡＣＡＣＣＣＡＡＡＡＧＧＣＣＡＣＡＣＴＧＧＴＧＴＧＣＣＴＧＧＣＣＡＣＡＧＧＣＴＴＣＴＴＣＣＣＣＧＡＣＣＡＣＧＴＧＧＡＧＣＴＧＡＧＣＴＧＧＴＧＧＧＴＧＡＡＴＧＧＧＡＡＧＧＡＧＧＴＧＣＡＣＡＧＴＧＧＧＧＴＣＡＧＣＡＣＧＧＡＣＣＣＧＣＡＧＣＣＣＣＴＣＡＡＧＧＡＧＣＡＧＣＣＣＧＣＣＣＴＣＡＡＴＧＡＣＴＣＣＡＧＡＴＡＣＴＧＣＣＴＧＡＧＣＡＧＣＣＧＣＣＴＧＡＧＧＧＴＣＴＣＧＧＣＣＡＣＣＴＴＣＴＧＧＣＡＧＡＡＣＣＣＣＣＧＣＡＡＣＣＡＣＴＴＣＣＧＣＴＧＴＣＡＡＧＴＣＣＡＧＴＴＣＴＡＣＧＧＧＣＴＣＴＣＧＧＡＧＡＡＴＧＡＣＧＡＧＴＧＧＡＣＣＣＡＧＧＡＴＡＧＧＧＣＣＡＡＡＣＣＣＧＴＣＡＣＣＣＡＧＡＴＣＧＴＣＡＧＣＧＣＣＧＡＧＧＣＣＴＧＧＧＧＴＡＧＡＧＣＡＧＡＣＴＧＴＧＧＣＴＴＴＡＣＣＴＣＧＧＴＧＴＣＣＴＡＣＣＡＧＣＡＡＧＧＧＧＴＣＣＴＧＴＣＴＧＣＣＡＣＣＡＴＣＣＴＣＴＡＴＧＡＧＡＴＣＣＴＧＣＴＡＧＧＧＡＡＧＧＣＣＡＣＣＣＴＧＴＡＴＧＣＴＧＴＧＣＴＧＧＴＣＡＧＣＧＣＣＣＴＴＧＴＧＴＴＧＡＴＧＧＣＣＡＴＧＧＴＣＡＡＧＡＧＡＡＡＧＧＡＴＴＴＣ（配列番号１３）。

pLRPO FMC63 endoL TRBC1 (126-177) (according to IMGT numbering pLRPO FMC63 endoL TRBC1 (132-309)) (amino acid sequence)
ＤＩＱＭＴＱＴＴＳＳＬＳＡＳＬＧＤＲＶＴＩＳＣＲＡＳＱＤＩＳＫＹＬＮＷＹＱＱＫＰＤＧＴＶＫＬＬＩＹＨＴＳＲＬＨＳＧＶＰＳＲＦＳＧＳＧＳＧＴＤＹＳＬＴＩＳＮＬＥＱＥＤＩＡＴＹＦＣＱＱＧＮＴＬＰＹＴＦＧＧＧＴＫＬＥＩＴＧＳＴＳＧＳＧＫＰＧＳＧＥＧＳＴＫＧＥＶＫＬＱＥＳＧＰＧＬＶＡＰＳＱＳＬＳＶＴＣＴＶＳＧＶＳＬＰＤＹＧＶＳＷＩＲＱＰＰＲＫＧＬＥＷＬＧＶＩＷＧＳＥＴＴＹＹＮＳＡＬＫＳＲＬＴＩＩＫＤＮＳＫＳＱＶＦＬＫＭＮＳＬＱＴＤＤＴＡＩＹＹＣＡＫＨＹＹＹＧＧＳＹＡＭＤＹＷＧＱＧＴＳＶＴＶＳＳＧＶＥＤＬＮＫＶＦＰＰＥＶＡＶＦＥＰＳＥＡＥＩＳＨＴＱＫＡＴＬＶＣＬＡ
TGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF).

pLRPO FMC63 endoL TRBC1 (126-177) (according to IMGT numbering pLRPO FMC63 endoL TRBC1 (132-309)) (nucleic acid sequence)
ＧＡＣＡＴＣＣＡＧＡＴＧＡＣＡＣＡＧＡＣＴＡＣＡＴＣＣＴＣＣＣＴＧＴＣＴＧＣＣＴＣＴＣＴＧＧＧＡＧＡＣＡＧＡＧＴＣＡＣＣＡＴＣＡＧＴＴＧＣＡＧＧＧＣＡＡＧＴＣＡＧＧＡＣＡＴＴＡＧＴＡＡＡＴＡＴＴＴＡＡＡＴＴＧＧＴＡＴＣＡＧＣＡＧＡＡＡＣＣＡＧＡＴＧＧＡＡＣＴＧＴＴＡＡＡＣＴＣＣＴＧＡＴＣＴＡＣＣＡＴＡＣＡＴＣＡＡＧＡＴＴＡＣＡＣＴＣＡＧＧＡＧＴＣＣＣＡＴＣＡＡＧＧＴＴＣＡＧＴＧＧＣＡＧＴＧＧＧＴＣＴＧＧＡＡＣＡＧＡＴＴＡＴＴＣＴＣＴＣＡＣＣＡＴＴＡＧＣＡＡＣＣＴＧＧＡＧＣＡＡＧＡＡＧＡＴＡＴＴＧＣＣＡＣＴＴＡＣＴＴＴＴＧＣＣＡＡＣＡＧＧＧＴＡＡＴＡＣＧＣＴＴＣＣＧＴＡＣＡＣＧＴＴＣＧＧＡＧＧＧＧＧＧＡＣＴＡＡＧＴＴＧＧＡＡＡＴＡＡＣＡＧＧＣＴＣＣＡＣＣＴＣＴＧＧＡＴＣＣＧＧＣＡＡＧＣＣＣＧＧＡＴＣＴＧＧＣＧＡＧＧＧＡＴＣＣＡＣＣＡＡＧＧＧＣＧＡＧＧＴＧＡＡＡＣＴＧＣＡＧＧＡＧＴＣＡＧＧＡＣＣＴＧＧＣＣＴＧＧＴＧＧＣＧＣＣＣＴＣＡＣＡＧＡＧＣＣＴＧＴＣＣＧＴＣＡＣＡＴＧＣＡＣＴＧＴＣＴＣＡＧＧＧＧＴＣＴＣＡＴＴＡＣＣＣＧＡＣＴＡＴＧＧＴＧＴＡＡＧＣＴＧＧＡＴＴＣＧＣＣＡＧＣＣＴＣＣＡＣＧＡＡＡＧＧＧＴＣＴＧＧＡＧＴＧＧＣＴＧＧＧＡＧＴＡＡＴＡＴＧＧＧＧＴＡＧＴＧＡＡＡＣＣＡＣＡＴＡＣＴＡＴＡＡＴＴＣＡＧＣＴＣＴＣＡＡＡＴＣＣＡＧＡＣＴＧＡＣＣＡＴＣＡＴＣＡＡＧＧＡＣＡＡＣＴＣＣＡＡＧＡＧＣＣＡＡＧＴＴＴＴＣＴＴＡＡＡＡＡＴＧＡＡＣＡＧＴＣＴＧＣＡＡＡＣＴＧＡＴＧＡＣＡＣＡＧＣＣＡＴＴＴＡＣＴＡＣＴＧＴＧＣＣＡＡＡＣＡＴＴＡＴＴＡＣＴＡＣＧＧＴＧＧＴＡＧＣＴＡＴＧＣＴＡＴＧＧＡＣＴＡＣＴＧＧＧＧＴＣＡＡＧＧＡＡＣＣＴＣＡＧＴＣＡＣＣＧＴＣＴＣＣＴＣＡＧＧＡＧＴＡＧＡＧＧＡＣＣＴＧＡＡＣＡＡＧＧＴＧＴＴＣＣＣＡＣＣＣＧＡＧＧＴＣＧＣＴＧＴＧＴＴＴＧＡＧＣＣＡＴＣＡＧＡＡＧＣＡＧＡＧＡＴＣＴＣＣＣＡＣＡＣＣＣＡＡＡＡＧＧＣＣＡＣＴＣＴＡＧＴＡＴＧＴＣＴＡＧＣＴＡＣＡＧＧＣＴＴＣＴＴＣＣＣＴＧＡＣＣＡＣＧＴＧＧＡＧＣＴＧＡＧＣＴＧＧＴＧＧＧＴＧＡＡＴＧＧＧＡＡＧＧＡＧＧＴＧＣＡＣＡＧＴＧＧＧＧＴＣＡＧＣＡＣＧＧＡＣＣＣＧＣＡＧＣＣＡＣＴＴＡＡＡＧＡＡＣＡＧＣＣＡＧＣＴＣＴＣＡＡＴＧＡＣＴＣＣＡＧＡＴＡＣＴＧＴＣＴＡＡＧＣＡＧＴＣＧＡＣＴＴＡＧＡＧＴＣＴＣＧＧＣＴＡＣＡＴＴＴＴ ＧＧＣＡＡＡＡＴＣＣＴＣＧＡＡＡＣＣＡＣＴＴＣＣＧＣＴＧＴＣＡＡＧＴＣＣＡＧＴＴＣＴＡＣＧＧＧＣＴＣＴＣＧＧＡＧＡＡＴＧＡＣＧＡＧＴＧＧＡＣＣＣＡＧＧＡＴＡＧＧＧＣＣＡＡＡＣＣＣＧＴＣＡＣＣＣＡＧＡＴＣＧＴＣＡＧＣＧＣＣＧＡＧＧＣＣＴＧＧＧＧＴＡＧＡＧＣＡＧＡＣＴＧＴＧＧＣＴＴＴＡＣＣＴＣＧＧＴＧＴＣＣＴＡＣＣＡＧＣＡＡＧＧＧＧＴＣＣＴＧＴＣＴＧＣＣＡＣＣＡＴＣＣＴＣＴＡＴＧＡＧＡＴＣＣＴＧＣＴＡＧＧＧＡＡＧＧＣＣＡＣＣＣＴＧＴＡＴＧＣＴＧＴＧＣＴＧＧＴＣＡＧＣＧＣＣＣＴＴＧＴＧＴＴＧＡＴＧＧＣＣＡＴＧＧＴＣＡＡＧＡＧＡＡＡＧＧＡＴＴＴＣ（配列番号１５）。

Editing of primary human T cells
[0554] Next, the TRA or TRB gene is inactivated in primary T cells from human donors. Four days before electroporation, T cells were activated with TransAct® (Milltenyi) supplemented with IL7/IL15 in TexMac medium containing 3% human serum. Annealing SpCas9 ribonucleoprotein (RNP) targeting the TRA or TRB gene with crRNA and tracrRNA targeting either TRAC (TRAC2-4598) or TRBC (TRBC-44345) at a molecular ratio of 1:1 Prepared by Annealed duplexes were mixed with SpCas9 protein at a molar ratio of 1.5:1. 0.61 μM RNP was mixed with 2.5×10 ⁶ T cells and electroporated according to the manufacturer's protocol for the Neon Transfection System. Electroporation was set at 1600 V, 10 ms, 3 pulses. Cells were immediately transferred to warm medium and incubated at 37° C. to allow the edited T cells to expand with a doubling time of approximately 3-5 days. Editing effects were evaluated by measuring the reduction in surface expression of TCRαβ and CD3ε by flow cytometry. Edited T cells were purified 10 days after activation using a magnetic cell sorting (MACS®, Miltenyi Biotec) cell separation system according to the manufacturer and treated with anti-TCRαβ (clone IP27) antibody ( eBioscience #14-9986-82) and anti-CD3ε (clone SK7) antibody (eBioscience #16-0036-81). Cells expressing TCRαβ or CD3ε on their surface were subjected to MACS
Edited T cells, fixed to MS (Cat #130-041-301) or LS (Cat #130-041-306) columns and negative for both TCRαβ and CD3ε, were collected in the column flow-through. , were maintained in culture at 10 ⁶ cells/mL in the medium specified above.

Phenotypic characterization of allogeneic TFP T cells
[0555] Allogeneic TFP T cells were treated with human TCRαβ (anti-human TCR, Miltenyi Bio, using clone BW242/412), human CD3ε (anti-human CD3ε BioLegend, using clone UCHT1), human CD4 (anti-human CD4, BioLegend, CD19 binding factor by TFP (using clone RPA-T4), human CD8 (using anti-human CD8, BioLegend, clone SK-1), human CD45RA and human CCR7, and TFP (Biotinylated CD19 (Cat# CD9-H8259, AcroBio)). using detection of FMC63). Wild-type T cells from the same donor as comparisons and transduced TFPs (no editing) containing control TCRβ full-length (FL) constructs were examined in the same panel.

[0556] The results are shown in Figures 5-9. After TFP transduction and TRAC editing, T cells remain double positive for surface expression of human TCRαβ and CD3ε (upper right panel). In contrast, T cells edited to remove the TRAC gene show two cell populations (bottom right panel). Most cells lack surface expression of TCRαβ and CD3ε. In contrast, TCRγδ TFP-transduced T cells (cells transduced with vectors expressing SEQ ID NOs: 1, 2, or 4) show three distinct populations (left panel). Although most cells lack surface expression of TCRαβ, here there was a new subset of cells expressing CD3ε without expression of TCRαβ, suggesting successful reassembly of the entire TCR complex. ing. This population corresponds to cells that have been edited to remove TCRαβ and transduced to express TCRγδ. FIG. 6 shows cells after MACS purification to remove residual cells that expressed TCRαβ. T cells transduced with TCRγδ TFP now have two cell populations. Both are negative for TCRαβ and one population expresses high levels of CD3ε. FIG. 7 shows TFP expression of TCRγδ-transduced TFPs compared to TCRβ full-length (FL) control TFPs and non-transduced control T cells. Expression of human CD4 and CD8 is not significantly different between TCRβFL TFP control and TCRγδ TFP T cells. There is also no difference in memory status as defined by CD45RA and CCR7 expression (Figures 8 and 9, respectively).

[0557] Figure 14 shows the α-CD19 scFv TFP constructs: αCD19-CD3ε, αCD19-TCRγ (constant) -2A-TCRδ (constant) (according to IMGT numbering, FMC63-TRDC(1-153)-T2A- TRGC1 (1-173) or FMC63-TRDC (−6,189)-2A-TRGC1 (1.8,189)), TCRγ (constant)-2A-αCD19-TCRδ (constant) (according to IMGT numbering, TRDC(1-153)-T2A-FMC63-TRGC1(1-173) or TRDC(-6,189)-2A-FMC63-TRGC1(1.8,189)), or αCD19-TCRα (mouse constant)-2A - shows primary T cells transduced 24 hours after lentiviral activation containing mouse TCRβ (constant) (pLRPC FMC63mTRAC_T2A_mTRBC). Endogenous TCR was inactivated with CRISPR/Cas9 72 hours after activation by targeting the TCRα constant region locus in all TFP-expressing cells except αCD19-CD3ε-expressing cells. T cells were purified to remove residual T cells still expressing endogenous TCR by negative selection. The panel of flow cytometry shown was performed on days 9 and 10 to assess transduction, editing efficiency, and T cell phenotype. The results demonstrate successful engineering of allogeneic TFP-expressing cells by replacing the endogenous T-cell receptor α and β subunits with TCR transgenes containing the constant regions of mouse TCRα and TCRβ or human TCRγ and TCRδ. It shows that

MLR of human TCR-negative T cells expressing TFP
[0558] The mixed lymphocyte reaction (MLR) assay was used to assess the homogeneity of TFP-expressing human TCR-negative T cells. Mismatched PBMC donor cells are first B cell depleted by magnetic cell separation of CD19 negative cells. X-irradiated PBMCs (Astarte Biologics) are labeled with the cell-labeling dye CellTrace® (Thermo Fisher Scientific). At the same time, different colored CellTrace dyes are incorporated into target T cells. Human TCR-negative T cells expressing TFP are then co-cultured with wild-type T cells from the same donor in a 1:1 ratio (PBMC to T cells), or T cells alone are cultured. Donor T-cell proliferation is monitored by following the labeling dye over time points of 6-12 days. Since the dye is diluted in half during cell division, the amount of proliferation occurring in T cells is assessed and compared to wild-type controls.

Example 4: Cytotoxicity and Cytokine Production of TFP-Expressing Allogeneic TCR T Cells
[0559] The luciferase-based cytotoxicity assay ("Luc-Cyto" assay) assesses TFP T cell cytotoxicity by indirectly measuring luciferase enzymatic activity in surviving target cells after co-culture. It is a thing.

Production of firefly luciferase (Luc)-expressing tumor cells
[0560] The target cells used for the Luc-Cyto assay were stably transduced Nalm6 (DSMZ Cat # ACC128) and K562 (ATCC™ Cat # CCL-243™) cells expressing firefly luciferase. Nalm6-Luc (CD19 positive) and K562-Luc (CD19 negative) produced by DNA encoding firefly luciferase was obtained from GeneArt® (Thermo
Fisher Scientific) and inserted into the multiple cloning site of the single-promoter lentiviral vector pCDH527A-1 (System Biosciences). Lentivirus was encapsulated according to the manufacturer's instructions. Tumor cells were then transduced with lentivirus for 24 hours and selected with puromycin (5 μg/mL). Successful production of Nalm6-Luc and K562-Luc cells was confirmed by measuring intracellular luciferase enzymatic activity with the Bright-Glo™ Luciferase Assay System (Promega).

Luc-Cyto Assay to Assess Allogeneic T-Cell Cytotoxicity and Cytokine Production
[0561] The Luc-Cyto assay was set up by mixing T cells with tumor cells at various effector (T cell) to target (tumor cell) (E to T) ratios. Target cells (Nalm6-Luc or K562-Luc) were seeded at 10,000 cells per well in 96-well plates in RPMI-1640 medium supplemented with 10% heat-inactivated (HI) FBS. Allogeneic TFP T cells were added to tumor cells at 30,000, 10,000, 3333, or 1111 cells per well such that the E to T ratio was 3:1, 1:1, or 1:3, or 1:9. added. The cell mixture was incubated for 24 hours at 37°C with 5% _CO2 . Luciferase enzymatic activity was measured using the Bright-Glo™ Luciferase Assay System (Promega), which measures activity from surviving target cells in co-cultures of T cells and tumor cells.

[0562] The results are shown in FIG. Allogeneic TCRγδ-TFP T cells showed robust and specific lysis against CD19-positive tumor cells Nalm6-Luc, but not CD19-negative tumor cells K562-Luc.

[0563] Supernatants were harvested from the same co-culture assay after 24 hours and assessed for allogeneic T cell production of the following cytokines: GM-CSF, IFNγ, IL2, and TNFα. Cytokine production was analyzed using the U-PLEX Biomarker Group I (hu) Assay (Cat#: K15067L-4) using MesoScale Discovery Technology (MesoScale Diagnostics, LLC). Robust cytokine secretion was observed in a dose-dependent manner from all constructs (Fig. 11).

[0564] Results from additional experiments for the same constructs as in Figure 14 are shown in Figures 15 and 16 . Allogeneic TCRγδ TFP T cells and murine TCRαβ T cells exhibited robust and specific lysis against CD19-positive tumor cells Nalm6-Luc, but not CD19-negative tumor cells K562-Luc (Fig. 15). ). Robust cytokine secretion was observed in a dose-dependent manner from all constructs (Figure 16). To account for differences in TFP transduction efficiencies between constructs, the percentage of TFP ⁺ population is graphically displayed. Allogeneic TFP cells containing αCD19 scFv efficiently lysed CD19 ⁺ tumor cells in vitro and produced cytokines at levels similar to non-edited αCD19 TFP cells.

[0565] Results from additional experiments are shown in Figures 20-23.同種異系マウスＴＣＲα（ＦＭＣ６３ｍＴＲＡＣ＿Ｐ２Ａ＿ｍＴＲＢＣ Ｕ５；ＦＭＣ６３ＳＬｍＴＲＡＣ＿Ｐ２Ａ＿ｍＴＲＢＣ Ｕ５；ＦＭＣ６３ｍＴＲＡＣ＿Ｔ２Ａ＿ｍＴＲＢＣ；ＦＭＣ６３ｍＴＲＡＣ＿Ｔ２Ａ＿ｍＴＲＢＣ Ｕ５）及びＴＣＲαβ（ＦＭＣ６３ＳＬｍＴＲＡＣ＿Ｐ２Ａ＿ＦＭＣ６３ＳＬｍＴＲＢＣ Ｕ５）ＴＦＰ Ｔ細胞は、ＣＤ１９陽性腫瘍細胞Ｎａｌｍ６－Ｌｕｃに対して頑強かつ特異的な溶解を示したが、ＣＤ１９ It did not show negative tumor cells K562-Luc (Fig. 20). To account for differences in TFP transduction efficiencies between constructs, the percentage of TFP ⁺ population is graphically displayed. Robust cytokine secretion was observed in a dose-dependent manner from all constructs (Figure 21). Allogeneic TCRδ (FMC63TRDC T2A TRGC1), TCRγδ (FMC63TRDC_T2A_FM63opTRGC1), and TCRε (FMC63TCR γ9G115 T2A δ2cl5 P2A FMC63ε) TFP T cells showed robust and specific lysis against CD19-positive tumor cells Nalm6-Luc. , but not in the CD19-negative tumor cell K562-Luc (Fig. 22). To account for differences in TFP transduction efficiencies between constructs, the percentage of TFP ⁺ population is graphically displayed. Robust cytokine secretion was observed in a dose-dependent manner from all constructs (Figure 23). In Figures 22 and 23, TFP T cells were added to target cells at 3:1, 1:1, and 0.1:1 ratios. Figures 21 and 23 show cytokine production normalized to cytokine production in non-edited T cells with anti-CD19-CD3ε TFPs.

MLR of TFP-expressing human TCR-negative T cells relative to cytokine expression
[0566] The mixed lymphocyte reaction (MLR) assay was used to assess TFP-expressing human TCR-negative or TCR-positive T cells for homogeneity. HLA-mismatched, in vitro-derived dendritic cells were co-cultured with TCR-negative or TCR-positive TFP-expressing cells at a 1:3 (T:DC) ratio for 72 hours. Secreted cytokines in the supernatant were measured by the MSD assay as described above. TCR-negative TFP-expressing T cells secreted significantly less cytokines than non-editing TFP-expressing T cells and were not alloreactive in a mixed lymphocyte reaction with HLA-mismatched dendritic cells. (Fig. 17).

Example 5: In vivo efficacy of TFP-expressing allogeneic TCR T cells
[0567] The Nalm6-luc tumor mouse model was used to assess the in vivo efficacy of TFP-expressing allogeneic TCR T cells. Tumor cells were injected into NSG mice on day -10. Mice were injected once with 2.2×10 ⁶ non-edited or allogeneic TFP cells on day 0 after tumor establishment. Mice were imaged every 3 days after TFP cell injection to assess tumor burden. In vivo, non-editing TFP-expressing cells exhibited full anti-tumor activity, whereas allogeneic TFP-expressing cells exhibited tumor regression (FIG. 18).

[0568] Fifty days after injection of TFP-expressing T cells, livers were harvested from non-tumor bearing mice,
Graft versus host disease (GvHD) was assessed by tissue infiltration of human cells. Flow cytometric analysis of human CD7 ⁺ cells in mouse liver revealed infiltration of human cells in animals treated with non-edited T cells expressing αCD19-CD3ε (left panel). No invasion was observed with TFP-expressing allogeneic cells, and mice treated with allogeneic cells showed no signs of GvHD. Invasion analysis was confirmed by IHC analysis of CD7 ⁺ cell staining (n=2; right panel).

Array of vectors:
pLRPO V5-mTRAC _(82-137) T2A mTRBC _(123-173) (SEQ ID NO: 159)

pLRPO FMC63-mTRAC _(82-137) T2A mTRBC _(123-173 (SEQ ID NO: 160)

pLRPO FMC63mTRAC_P2A_Mtrbc (SEQ ID NO: 161)

pLRPO Jet_FMC63mTRAC_T2A_mTRBC (SEQ ID NO: 162)

pLRPC FMC63SLmTRAC _(82-137) T2A optiFMC63SLmTRBC _(123-173) (SEQ ID NO: 163)

pLRPC FMC63mTRAC_T2A_mTRBC (SEQ ID NO: 164)

pLRPC FMC63mTRAC_T2A_mTRBC U5 (SEQ ID NO: 165)

pLCUS FMC63mTRAC_P2A_mTRBC (SEQ ID NO: 166)

pLCUS FMC63SLmTRAC_P2A_mTRBC (SEQ ID NO: 167)

pLCUS FMC63SLmTRAC_P2A_FMC63SLmTRBC (SEQ ID NO: 168)

pLKaUS FMC63mTRAC_P2A_FMC63mTRBC (SEQ ID NO: 169)

pLRPO FMC63 endoL TRBC1(126-177) (SEQ ID NO: 170)

pLRPO FMC63 SL TRBC1(131-177) (SEQ ID NO: 171)

pLRPO FMC63 endoL TRBC1 (SEQ ID NO: 172)

pLRPO V5-TRAC(94-140) T2A PuroR (SEQ ID NO: 173)

pLRPO V5-TRAC(7-140) T2A PuroR (SEQ ID NO: 174)

pLRPO FMC63 endoL TRBC1(126-177) T2AW trEGFR (SEQ ID NO: 175)

pLRPO FMC63-TCRbeta1 (37GRK) (SEQ ID NO: 176)

pLRPO FMC63-HAP17(1-258) (SEQ ID NO: 177)

pLRPO FMC63-HAP17(35-258) (SEQ ID NO: 178)

pLRPO FMC63-HAP17(79-258) (SEQ ID NO: 179)

pLRPC FMC63-TCRβ1 (CRISPR resistant) (SEQ ID NO: 180)

pLRPO FMC63 TRDC T2AW FMC63op TRGC1 (SEQ ID NO: 181)

pLKaUS FMC63 TRDC P2AW FMC63op TRGC1 (SEQ ID NO: 182)

pLRPC TRDC T2AW FMC63op TRGC1 (SEQ ID NO: 183)

pLRPC FMC63 TRDC T2AW TRGC1 (SEQ ID NO: 184)

pLRPCU TRDC T2AW FMC63op TRGC1 (SEQ ID NO: 185)

pLRPC_TRDC P2AW FMC63op TRGC1 (SEQ ID NO: 186)

pLRPCU TRDC P2AW FMC63op TRGC1 (SEQ ID NO: 187)

pLCUS FMC63 TRDC P2AW TRGC1 (SEQ ID NO: 188)

pLCUS FMC63 TRDC P2AW FMC63op TRGC1 (SEQ ID NO: 189)

pLRPC m971 TRDC T2AW m971op TRGC1 (SEQ ID NO: 190)

pLRPS TCR γ9G115 T2A δ2cl5 (SEQ ID NO: 191)

pLRPS TCR γ9G115 T2A δ2cl5 P2A FMC63ε (SEQ ID NO: 192)

pLKaUS mTRAC(82-137) T2A mTRBC(123-173) P2A MH1CD3ε (SEQ ID NO: 193)

pLKaUS MH1mTRAC(2-137) P2A MH1mTRBC(2-173) (SEQ ID NO: 194)

pLKaUS mTRAC(2-137) T2A mTRBC(2-173) P2A
MH1CD3ε (SEQ ID NO: 195)

anti-MSLN-CD3ε (SEQ ID NO: 198)

anti-CD19-CD3ε (SEQ ID NO: 199)

Claims (290)

(a) a sequence encoding a T-cell receptor (TCR) fusion protein (TFP),
(i) below:
(1) at least a portion of a TCR extracellular domain; and (2) a TCR subunit comprising a transmembrane domain; and (ii) an antibody comprising an antigen binding domain;
(b) a sequence encoding a TCR constant domain, wherein said TCR constant domain is a TCRγ constant domain or a TCRδ constant domain, or a sequence encoding a TCRγ constant domain and a TCRδ constant domain;
wherein said TCR subunit and said antibody are operably linked;
and a recombinant nucleic acid that, when expressed in a modified T cell containing a functional disruption of an endogenous TCR, functionally integrates said TFP into the TCR complex. (a) a sequence encoding a T-cell receptor (TCR) fusion protein (TFP),
(i) below:
(1) at least a portion of the TCR extracellular domain;
(2) a TCR subunit comprising a transmembrane domain; and (ii) a binding ligand or fragment thereof capable of binding to the antibody or fragment thereof;
(b) a sequence encoding a TCR constant domain, wherein said TCR constant domain is a TCRγ constant domain or a TCRδ constant domain, or a sequence encoding a TCRγ constant domain and a TCRδ constant domain;
said TCR subunit and binding ligand or fragment thereof are operably linked, and said TFP is functionally integrated into the TCR complex when expressed in a modified T cell comprising a functional disruption of the endogenous TCR. nucleic acid. 10. Claim 1 or claim, wherein said TCR subunit further comprises an intracellular domain comprising a stimulatory domain from the intracellular domain of TCRα, TCRβ, TCRγ, or TCRδ, or the intracellular signaling domain of CD3ε, CD3γ, or CD3δ. Item 3. The recombinant nucleic acid of item 2. The recombinant nucleic acid of any one of claims 1-3, wherein said TCR constant domain is the TCRδ constant domain. 5. The TCRdelta constant domain comprises SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 243, or SEQ ID NO: 265, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications. A recombinant nucleic acid as described. The recombinant nucleic acid of any one of claims 3-5, wherein said intracellular domain is the intracellular domain of TCRγ. 6. Any of claims 3-5, wherein said sequence encoding said TCRδ constant domain further encodes a second antigen or ligand binding domain operably linked to said sequence encoding said TCRδ constant domain. or the recombinant nucleic acid according to claim 1. 8. The recombinant nucleic acid of claim 7, wherein said second antigen binding domain or said ligand binding domain is the same as or different from said antigen binding domain or said ligand binding domain of said TFP. 8. The recombinant nucleic acid of claim 7, wherein said second antigen binding domain or said ligand binding domain is operably linked via a linker to said sequence encoding said TCR[delta] constant domain. The recombinant nucleic acid of any one of claims 1-3, wherein said TCR constant domain is the TCRγ constant domain. 11. The recombinant nucleic acid of claim 10, wherein said TCRγ constant domain comprises SEQ ID NO:21 or SEQ ID NO:155, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications. 12. The recombinant nucleic acid of claim 10 or 11, wherein said intracellular domain is the intracellular domain of TCR[delta]. 13. Any of claims 8 to 12, wherein said sequence encoding said TCRγ constant domain further encodes a second antigen or ligand binding domain operably linked to said sequence encoding said TCRγ constant domain. or the recombinant nucleic acid according to claim 1. 14. The recombinant nucleic acid of claim 13, wherein said second antigen binding domain or said ligand binding domain is the same as or different from said antigen binding domain or said ligand binding domain of said TFP. 14. The recombinant nucleic acid of claim 13, wherein said second antigen binding domain or said ligand binding domain is operably linked via a linker to said sequence encoding said TCR[gamma] constant domain. The recombinant nucleic acid of any one of claims 1-3, wherein the recombinant nucleic acid comprises sequences encoding a TCRγ constant domain and a TCRδ constant domain. 17. The recombinant nucleic acid of claim 16, wherein said TCRγ constant domain comprises SEQ ID NO:21 or SEQ ID NO:155, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications. 18. A recombinant nucleic acid according to claim 16 or 17, wherein said sequence encoding said TCR[gamma] constant domain further encodes a TCR[gamma] variable domain and thus encodes the entire TCR[gamma] domain. 19. The recombinant nucleic acid of claim 18, wherein said entire TCRγ domain is γ9 or γ4. 20. The recombinant nucleic acid of claim 18 or 19, wherein the entire TCRγ domain comprises SEQ ID NO:255, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications. 16-, wherein said TCRδ constant domain comprises SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 243, or SEQ ID NO: 265, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications 21. The recombinant nucleic acid of any one of 20. A recombinant nucleic acid according to any one of claims 16 to 21, wherein said sequence encoding said TCRδ constant domain further encodes a TCRδ variable domain and thus encodes the entire TCRδ domain. 23. The recombinant nucleic acid of claim 22, wherein the entire TCR[delta] domain is [delta]2 or [delta]l. 24. The recombinant nucleic acid of claim 22 or 23, wherein the entire TCRdelta constant domain comprises SEQ ID NO:256, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications. 25. The recombinant nucleic acid of any one of claims 16-24, wherein the intracellular signaling domain is CD3ε, CD3γ, or CD3δ. The recombinant nucleic acid of any one of claims 16-25, wherein said intracellular signaling domain is CD3ε. 27. The recombinant nucleic acid of any one of claims 1-26, further comprising at least one leader sequence and at least one linker. 28. The recombinant nucleic acid of claim 27, further comprising a portion of the TCRα constant domain, a portion of the TCRβ domain, or both. 4. The claim, wherein said sequence comprises, from 5' to 3', a first leader sequence, an antigen binding domain sequence, a linker, a TRDC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRGC gene sequence. 1. The recombinant nucleic acid according to 1. wherein said sequence comprises, in 5′-3′ direction, a first leader sequence, a TRDC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker sequence and a TRGC gene sequence. Item 1. The recombinant nucleic acid of Item 1. The sequences are, in the 5′-3′ direction, a first leader sequence, an antigen binding domain sequence, a first linker sequence, a TRDC gene sequence, a cleavable linker, a second leader sequence, a second antigen binding domain. 2. The recombinant nucleic acid of claim 1, comprising a sequence, a second linker sequence, and a TRGC gene sequence. the sequences are, in the 5′-3′ direction, a first leader sequence, a TRDC gene sequence, a first cleavable linker sequence, a second leader sequence, a TRGC gene sequence, a second cleavable linker sequence; 2. The recombinant nucleic acid of Claim 1, comprising a third leader sequence, an antigen binding domain sequence, a linker sequence, and a CD3 epsilon gene sequence. The sequences are, in the 5′-3′ direction, a first leader sequence, a first antigen binding domain sequence, a first linker sequence, a TRDC gene sequence or a fragment thereof, a TRAC gene sequence or a fragment thereof, a cleavable linker. 2. The recombinant nucleic acid of claim 1, comprising a sequence, a second leader sequence, a second antigen binding domain sequence, a second linker sequence, a TRGC gene sequence or fragment thereof, and a TRBC gene sequence or fragment thereof. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the polypeptide shown in SEQ ID NO:1. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the polypeptide shown in SEQ ID NO:2. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the polypeptide shown in SEQ ID NO:3. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the polypeptide shown in SEQ ID NO:4. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the polypeptide shown in SEQ ID NO:5. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the sequence of SEQ ID NO:242. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the sequence of SEQ ID NO:244. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the sequence of SEQ ID NO:245. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the sequence of SEQ ID NO:246. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the sequence of SEQ ID NO:248. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the sequence of SEQ ID NO:250. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the sequence of SEQ ID NO:252. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the sequence of SEQ ID NO:257. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the sequence of SEQ ID NO:263. 34. The recombinant nucleic acid of any one of claims 1-33, wherein said sequence encodes the sequence of SEQ ID NO:264. 2. The recombinant nucleic acid of claim 1, wherein said binding ligand is capable of binding the Fc domain of said antibody. 2. The recombinant nucleic acid of claim 1, wherein said binding ligand is capable of selectively binding an IgGl antibody. 2. The recombinant nucleic acid of claim 1, wherein said binding ligand is capable of specifically binding an IgG4 antibody. 2. The recombinant nucleic acid of claim 1, wherein said antibody or fragment thereof binds to a cell surface antigen. 2. The recombinant nucleic acid of claim 1, wherein said antibody or fragment thereof is murine, human, or humanized. 2. The recombinant nucleic acid of claim 1, wherein said antibody or fragment thereof binds to a cell surface antigen on the surface of tumor cells. 4. The binding ligand comprises a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. 1. The recombinant nucleic acid according to 1. 2. The recombinant nucleic acid of Claim 1, wherein said binding ligand does not comprise an antibody or fragment thereof. 57. The recombinant nucleic acid of claim 56, wherein said binding ligand comprises a CD16 binding polypeptide or fragment thereof. 57. The recombinant nucleic acid of claim 56, wherein said binding ligand comprises a CD16 binding polypeptide. 2. The recombinant nucleic acid of claim 1, wherein said binding ligand is human or humanized. 2. The recombinant nucleic acid of claim 1, further comprising a nucleic acid sequence encoding an antibody or fragment thereof to which said binding ligand can bind. 61. The recombinant nucleic acid of claim 60, wherein said antibody or fragment thereof is secretable from the cell. (a) a sequence encoding a T-cell receptor (TCR) fusion protein (TFP),
(i) below:
(1) at least a portion of a TCR extracellular domain, and (2) a TCR subunit comprising a transmembrane domain and (ii) a ligand or fragment thereof that binds to a receptor or polypeptide expressed on the surface of a cell. the sequence comprising domains and
(b) a sequence encoding a TCR constant domain, wherein said TCR constant domain is a TCRγ constant domain or a TCRδ constant domain, or a sequence encoding a TCRγ constant domain and a TCRδ constant domain;
said TCR subunit and said antigen binding domain are operably linked;
and a recombinant nucleic acid that, when expressed in a modified T cell containing a functional disruption of an endogenous TCR, functionally integrates said TFP into the TCR complex. 63. The method of claim 62, wherein said TCR subunit further comprises an intracellular domain comprising a stimulatory domain from the intracellular domain of TCRα, TCRβ, TCRγ, or TCRδ, or the intracellular signaling domain of CD3ε, CD3γ, or CD3δ. of recombinant nucleic acids. 64. The recombination of any one of claims 1-63, further comprising at least a subsequence encoding a TCRα constant domain, a subsequence encoding a TCRβ constant domain, or a subsequence of both the TCRα and TCRβ constant domains. nucleic acid. 63. The recombinant nucleic acid of claim 62, wherein said antigen binding domain comprises a ligand. 63. The recombinant nucleic acid of claim 62, wherein said ligand binds to a cellular receptor. 63. The recombinant nucleic acid of claim 62, wherein said ligand binds to said polypeptide expressed on the surface of a cell. 63. The recombinant nucleic acid of claim 62, wherein said receptor or polypeptide expressed on the surface of a cell comprises a stress response receptor or polypeptide. 63. The recombinant nucleic acid of claim 62, wherein said receptor or polypeptide expressed on the surface of a cell is an MHC class I-associated glycoprotein. 70. The recombinant nucleic acid of claim 69, wherein said MHC class I-associated glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4, and combinations thereof. wherein said antigen binding domain comprises a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer 63. The recombinant nucleic acid of Paragraph 62. 72. The recombinant nucleic acid of claim 71, wherein said antigen binding domain comprises a monomer or dimer of said ligand or fragment thereof. said ligand or fragment thereof comprises a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer; 63. The recombinant nucleic acid of claim 62. 74. The recombinant nucleic acid of claim 73, wherein said ligand or fragment thereof is monomeric or dimeric. 63. The recombinant nucleic acid of claim 62, wherein said antigen binding domain does not comprise an antibody or fragment thereof. 63. The recombinant nucleic acid of claim 62, wherein said antigen binding domain does not contain a variable region. 63. The recombinant nucleic acid of claim 62, wherein said antigen binding domain does not contain CDRs. 63. The recombinant nucleic acid of claim 62, wherein said ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or fragment thereof. 79. The recombinant nucleic acid of any one of claims 1-78, wherein said TCR constant domain is integrated into a functional TCR complex when expressed in a T cell. 80. The recombinant nucleic acid of any one of claims 1-79, wherein said TCR constant domain integrates into the same functional TCR complex as said functional TCR complex that incorporates said TFP when expressed in a T cell. 81. The recombinant nucleic acid of any one of claims 1-80, wherein the TFP-encoding sequence and the TCR constant domain(s)-encoding sequence are contained within the same nucleic acid molecule. 82. The recombinant nucleic acid of any one of claims 1-81, wherein said encoded TFP and said encoded TCR constant domain are operably linked by a first linker sequence. 83. The recombinant nucleic acid of claim 82, wherein said first linker comprises a protease cleavage site. 84. The recombinant nucleic acid of claim 83, wherein said protease cleavage site is a 2A, such as a T2A or P2A cleavage site. 85. The recombinant nucleic acid of any one of claims 1-84, wherein the TFP-encoding sequence and the TCR constant domain-encoding sequence are contained within different nucleic acid molecules. Claims 1-85, wherein said TCR subunit of said TFP and said antibody domain, said antigen binding domain, or said binding ligand, or fragments thereof, are operably linked by a second linker sequence. A recombinant nucleic acid according to . 87. The recombinant nucleic acid of claim 86, wherein said second linker sequence comprises (G4S) _n , where n= _1-4 . 88. The recombinant nucleic acid of any one of claims 1-87, wherein the transmembrane domain is a TCR transmembrane domain from CD3ε, CD3γ, CD3δ, TCRα, TCRβ, TCRδ, or TCRγ. The recombinant nucleic acid of any one of claims 3-61 and 63-88, wherein said intracellular domain is derived from CD3ε only, CD3γ only, CD3δ only, TCRα only, TCRβ, TCRγ, or TCRδ. said TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein (i), (ii) and (iii) 89. The recombinant nucleic acid of any one of claims 1-89, at least two of which are derived from the same TCR subunit. said TCR extracellular domain is selected from the group consisting of a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, a functional fragment thereof, or an amino acid sequence thereof having at least one but no more than 20 modifications; 91. The recombinant nucleic acid of any one of claims 1-90, comprising the extracellular domain of a protein, or a portion thereof, of a 92. The TCR subunit of claim 91, wherein the TCR subunit comprises (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain of a TCR gamma or TCR delta chain. recombinant nucleic acid. 93. The recombination of claim 92, wherein said TCR extracellular domain comprises the extracellular portion of a constant domain of a TCRγ or TCRδ chain, a functional fragment thereof, or an amino acid sequence thereof having at least one but no more than 20 modifications. nucleic acid. said TCR subunit comprises (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain, and is a delta constant domain or a fragment thereof, or 94. The recombinant nucleic acid of claim 92 or 93, comprising: 95. The delta constant domain has SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 243, or SEQ ID NO: 265, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications. A recombinant nucleic acid as described. wherein said TCR subunit comprising (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain is or comprises a gamma constant domain. 94. The recombinant nucleic acid of paragraph 92 or 93. 97. The recombinant nucleic acid of claim 96, wherein the gamma constant domain has the sequence of SEQ ID NO:21 or SEQ ID NO:155, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications. 98. The recombinant nucleic acid of any one of claims 94-97, wherein the extracellular domain of TFP does not comprise a gamma or delta chain variable domain. wherein said TCR subunit comprises a stimulatory domain of a protein selected from the intracellular signaling domains of CD3ε, CD3γ, or CD3δ, or a TCR intracellular domain comprising an amino acid sequence having at least one modification thereto; The recombinant nucleic acid of any one of paragraphs 3-61 and 63-88. 100. The recombinant nucleic acid of claim 99, wherein said TFP TCR subunit comprises the extracellular, transmembrane and intracellular domains of CD3[epsilon]. 101. The recombinant nucleic acid of claim 100, wherein the TCR subunit of CD3ε comprises SEQ ID NO: 258, the sequence of a functional fragment thereof, or the amino acid sequence thereof with at least one but no more than 20 modifications. Claims 1-101, wherein said TFP, said TCRγ constant domain, said TCRδ constant domain and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide Recombinant nucleic acid according to any one of (a) said TCR constant domain is a TCRγ constant domain and said TFP is functionally integrated into a TCR complex comprising endogenous subunits of TCRδ, CD3ε, CD3γ, CD3δ, or combinations thereof;
(b) said TCR constant domain is a TCRδ constant domain, and said TFP is functionally integrated into a TCR complex comprising endogenous subunits of TCRγ, CD3ε, CD3γ, CD3δ, or combinations thereof; c) said TCR constant domains are a TCRγ constant domain and a TCRδ constant domain, and said TFP is functionally integrated into a TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or combinations thereof. 103. The recombinant nucleic acid of any one of 1-102. said at least one but no more than 20 modifications comprise modifications of amino acids that mediate cell signaling or modifications of amino acids that are phosphorylated in response to ligand binding to said TFP;
The recombinant nucleic acid of any one of claims 1-103. The recombinant nucleic acid of any one of claims 1, 3-61, and 85-104, wherein said antibody is an antibody fragment. 106. The recombinant nucleic acid of claim 105, wherein said antibody fragment is a scFv, single domain antibody domain, _VH domain, or _VL domain. The antigen binding domain comprises an anti-CD19 binding domain, an anti-B cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, an anti-79b binding domain, an anti-HER2 binding domain, an anti- PMSA binding domain, anti-MUC16 binding domain, anti-CD22 binding domain, anti-PD-L1 binding domain, anti-BAFF or BAFF receptor binding domain, anti-nectin-4 binding domain, anti-TROP-2 binding domain, anti-GPC3 binding domain, and 107. The recombinant nucleic acid of any one of claims 1, 3-61, and 85-106, selected from the group consisting of anti-ROR-1 binding domains. 108. The recombinant nucleic acid of claim 107, wherein said anti-MSLN binding domain comprises CDR1 of SEQ ID NO:60, CDR2 of SEQ ID NO:61, and CDR3 of SEQ ID NO:62. 108. The recombinant nucleic acid of claim 107, wherein said anti-MSLN binding domain comprises CDR1 of SEQ ID NO:63, CDR2 of SEQ ID NO:64, and CDR3 of SEQ ID NO:65. 108. The recombinant nucleic acid of claim 107, wherein said anti-MSLN binding domain comprises CDR1 of SEQ ID NO:66, CDR2 of SEQ ID NO:67, and CDR3 of SEQ ID NO:68. 108. The recombinant nucleic acid of claim 107, wherein said anti-MSLN binding domain comprises a sequence having at least about 80% sequence identity to the sequence of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71. 108. The recombinant nucleic acid of claim 107, wherein said anti-CD19 binding domain comprises light chain CDR1 of SEQ ID NO:73, CDR2 of SEQ ID NO:75, and CDR3 of SEQ ID NO:77. 108. The recombinant nucleic acid of claim 107, wherein said anti-CD19 binding domain comprises heavy chain CDR1 of SEQ ID NO:79, CDR2 of SEQ ID NO:81, and CDR3 of SEQ ID NO:83. wherein said anti-CD19 binding domain has at least about 80% sequence identity to the sequence of SEQ ID NO:85 and/or at least about 80% sequence identity to the sequence of SEQ ID NO:87. 108. The recombinant nucleic acid of claim 107, comprising a heavy chain variable region having a 115. The recombinant nucleic acid of any one of claims 1-114, further comprising a sequence encoding a TCRα transmembrane domain. 115. The recombinant nucleic acid of any one of claims 1-114, further comprising a sequence encoding a TCRβ transmembrane domain. 115. The recombinant nucleic acid of any one of claims 1-114, further comprising a sequence encoding a TCRα transmembrane domain and a sequence encoding a TCRβ transmembrane domain. (a) a sequence encoding a T-cell receptor (TCR) fusion protein (TFP),
(i) below:
(1) at least a portion of the extracellular domain of mouse TCRα or mouse TCRβ; and (2) a TCR subunit comprising the transmembrane domain of mouse TCRα or mouse TCRβ; and (ii) an antibody or fragment thereof comprising an antigen binding domain. said sequence comprising
(b) a sequence encoding a TCR constant domain(s), wherein said TCR constant domain is a mouse TCRα constant domain or a mouse TCRβ constant domain, or a mouse TCRα constant domain and a mouse TCRβ constant domain; an encoding sequence and
said TCR subunit and said antibody are operably linked;
and a recombinant nucleic acid that, when expressed in a modified T cell containing a functional disruption of an endogenous TCR, functionally integrates said TFP into the TCR complex. (a) a sequence encoding a T-cell receptor (TCR) fusion protein (TFP),
(i) below:
(1) at least a portion of the extracellular domain of mouse TCRα or mouse TCRβ; and (2) a TCR subunit comprising the transmembrane domain of mouse TCRα or mouse TCRβ; or a fragment thereof;
(b) a sequence encoding a TCR constant domain(s), wherein said TCR constant domain is a mouse TCRα constant domain or a mouse TCRβ constant domain, or a mouse TCRα constant domain and a mouse TCRβ constant domain; an encoding sequence and
said TCR subunit and binding ligand or fragment thereof are operably linked;
and a recombinant nucleic acid that, when expressed in a modified T cell containing a functional disruption of an endogenous TCR, functionally integrates said TFP into the TCR complex. 120. The recombinant nucleic acid of claim 118 or 119, wherein said TCR subunit comprises the intracellular domain of mouse TCR[alpha] or mouse TCR[beta]. 121. The recombinant nucleic acid of any one of claims 118 or 120, wherein said TCR constant domain is the TCR[alpha] constant domain. said TCRα constant domain comprises SEQ ID NO: 17, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO: 207, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications; 122. The recombinant nucleic acid of claim 121. 122. The recombinant nucleic acid of claim 121, wherein said TCRα constant domain comprises a mouse TCRα constant domain. 124. The recombinant nucleic acid of claim 123, wherein said mouse TCRα constant domain comprises amino acids 2-137 of mouse TCRα constant domain. 125. The recombinant nucleic acid of claim 124, wherein said mouse TCRα constant domain comprises amino acids 2-137 of SEQ ID NO:146. 125. The recombinant nucleic acid of claim 124, wherein said mouse TCRα constant domain comprises the sequence of SEQ ID NO:207. 124. The recombinant nucleic acid of claim 123, wherein said mouse TCRα constant domain comprises amino acids 82-137 of SEQ ID NO:146. 128. The recombinant nucleic acid of claim 127, wherein said mouse TCRα constant domain comprises the sequence of SEQ ID NO:17. 129. The recombinant nucleic acid of any one of claims 122-128, wherein said intracellular domain is the intracellular domain of TCRβ. 130. Any of claims 121 to 129, wherein said sequence encoding said TCRα constant domain further encodes a second antigen or ligand binding domain operably linked to said sequence encoding said TCRα constant domain. or the recombinant nucleic acid according to claim 1. 131. The recombinant nucleic acid of claim 130, wherein said second antigen binding domain or said ligand binding domain is the same as or different from said antigen binding domain or said ligand binding domain of said TFP. 131. The recombinant nucleic acid of claim 130, wherein said second antigen binding domain or said ligand binding domain is operably linked via a linker to said sequence encoding said TCRα constant domain. The recombinant nucleic acid of any one of claims 118-120, wherein said TCR constant domain is the TCRβ constant domain. said TCRβ constant domain comprises SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO: 209, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications; 134. The recombinant nucleic acid of claim 133. 135. The recombinant nucleic acid of claim 134, wherein said TCR[beta] constant domain comprises a mouse TCR[beta] constant domain. 135. The recombinant nucleic acid of claim 134, wherein said mouse TCRβ constant domain comprises amino acids 2-173 of said mouse TCRβ constant domain. 135. The recombinant nucleic acid of claim 134, wherein said mouse TCRβ constant domain comprises amino acids 2-173 of SEQ ID NO:152. 135. The recombinant nucleic acid of claim 134, wherein said mouse TCRβ constant domain comprises SEQ ID NO:209. 135. The recombinant nucleic acid of claim 134, wherein said TCRβ constant domain comprises amino acids 123-173 of SEQ ID NO:152. 135. The recombinant nucleic acid of claim 134, wherein said TCRβ constant domain comprises SEQ ID NO:18. 141. The recombinant nucleic acid of any one of claims 133-140, wherein said intracellular domain is the intracellular domain of TCRα. 142. Any of claims 118 to 141, wherein said sequence encoding said TCRβ constant domain further encodes a second antigen or ligand binding domain operably linked to said sequence encoding said TCRβ constant domain. or the recombinant nucleic acid according to claim 1. 143. The recombinant nucleic acid of claim 142, wherein said second antigen binding domain or said ligand binding domain is the same as or different from said antigen binding domain or said ligand binding domain of said TFP. 143. The recombinant nucleic acid of claim 142, wherein said second antigen binding domain or said ligand binding domain is operably linked via a linker to said sequence encoding said TCR[beta] constant domain. The recombinant nucleic acid of any one of claims 118-120, wherein said recombinant nucleic acid comprises sequences encoding a TCRα constant domain and a TCRβ constant domain. said TCRα constant domain comprises SEQ ID NO: 17, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO: 207, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications; 146. The recombinant nucleic acid of claim 145. said TCRβ constant domain comprises SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO: 209, a functional fragment thereof, or an amino acid sequence thereof with at least one but no more than 20 modifications; 147. The recombinant nucleic acid of claim 145 or 146. 148. The recombinant nucleic acid of any one of claims 118-147, wherein said intracellular signaling domain is CD3ε, CD3γ, or CD3δ. The recombinant nucleic acid of any one of claims 118-147, wherein said intracellular signaling domain is CD3ε. 4. The claim, wherein said sequence comprises, from 5' to 3', a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene sequence. 145. The recombinant nucleic acid according to 145. 3. The claim, wherein said sequence comprises, from 5' to 3', a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, and a TRBC gene sequence. 145. The recombinant nucleic acid according to 145. 4. The claim, wherein said sequence comprises, from 5' to 3', a first leader sequence, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker, and a TRBC gene sequence. 145. The recombinant nucleic acid according to 145. The sequences are, from 5′ to 3′, a first leader sequence, an antigen binding domain sequence, a linker, a TRAC gene sequence, a cleavable linker sequence, a second leader sequence, an antigen binding domain sequence, a linker, and a TRBC. 146. The recombinant nucleic acid of claim 145, comprising a gene sequence. the sequences are, in the 5′-3′ direction, a first leader sequence, a TRAC gene sequence, a first cleavable linker sequence, a second leader sequence, a TRBC gene sequence, a second cleavable linker sequence; 146. The recombinant nucleic acid of claim 145, comprising a third leader sequence, an antigen binding domain sequence, a linker sequence, and a CD3 epsilon gene sequence. 155. The recombinant nucleic acid of any one of claims 115-154, wherein said sequence encodes the polypeptide set forth in SEQ ID NO:10. 155. The recombinant nucleic acid of any one of claims 115-154, wherein said sequence encodes the polypeptide set forth in SEQ ID NO:204. 155. The recombinant nucleic acid of any one of claims 115-154, wherein said sequence encodes the polypeptide set forth in SEQ ID NO:206. 155. The recombinant nucleic acid of any one of claims 115-154, wherein said sequence encodes the polypeptide shown in SEQ ID NO:210. 155. The recombinant nucleic acid of any one of claims 115-154, wherein said sequence encodes the polypeptide shown in SEQ ID NO:211. 155. The recombinant nucleic acid of any one of claims 115-154, wherein said sequence encodes the polypeptide shown in SEQ ID NO:217. 155. The recombinant nucleic acid of any one of claims 115-154, wherein said sequence encodes the polypeptide shown in SEQ ID NO:218. 155. The recombinant nucleic acid of any one of claims 115-154, wherein said sequence encodes a polypeptide shown in SEQ ID NO:219. 155. The recombinant nucleic acid of any one of claims 115-154, wherein said sequence encodes the polypeptide shown in SEQ ID NO:220. 155. The recombinant nucleic acid of any one of claims 115-154, wherein said sequence encodes a polypeptide set forth in SEQ ID NO:259. 155. The recombinant nucleic acid of any one of claims 115-154, wherein said sequence encodes the polypeptide shown in SEQ ID NO:261. 155. The recombinant nucleic acid of any one of claims 115-154, wherein said sequence encodes the polypeptide set forth in SEQ ID NO:262. 167. The recombinant nucleic acid of any one of claims 115-166, further comprising at least one leader sequence and at least one linker. The recombinant nucleic acid of any one of claims 119-167, wherein said binding ligand is capable of binding the Fc domain of said antibody. 168. The recombinant nucleic acid of any one of claims 119-167, wherein said binding ligand is capable of selectively binding an IgGl antibody. 168. The recombinant nucleic acid of any one of claims 119-167, wherein said binding ligand is capable of specifically binding an IgG4 antibody. The recombinant nucleic acid of any one of claims 118 and 120-170, wherein said antibody or fragment thereof binds to a cell surface antigen. The recombinant nucleic acid of any one of claims 118 and 120-170, wherein said antibody or fragment thereof is murine, human, or humanized. The recombinant nucleic acid of any one of claims 118 and 120-170, wherein said antibody or fragment thereof binds to a cell surface antigen on the surface of tumor cells. 4. The binding ligand comprises a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer. 119-167. 168. The recombinant nucleic acid of any one of claims 119-167, wherein said binding ligand does not comprise an antibody or fragment thereof. 176. The recombinant nucleic acid of claim 175, wherein said binding ligand comprises a CD16 binding polypeptide or fragment thereof. 176. The recombinant nucleic acid of claim 175, wherein said binding ligand comprises a CD16 binding polypeptide. 168. The recombinant nucleic acid of any one of claims 119-167, wherein said binding ligand is human or humanized. 168. The recombinant nucleic acid of any one of claims 119-167, further comprising a nucleic acid sequence encoding an antibody or fragment thereof to which said binding ligand can bind. The recombinant nucleic acid of any one of claims 118 and 120-170, wherein said antibody or fragment thereof is secretable from the cell. (a) a sequence encoding a T-cell receptor (TCR) fusion protein (TFP),
(i) below:
(1) at least a portion of the extracellular domain of mouse TCRα or mouse TCRβ; and (2) a TCR subunit comprising the transmembrane domain of mouse TCRα or mouse TCRβ; an antigen binding domain comprising a ligand or fragment thereof that binds to
(b) a sequence encoding a TCR constant domain, wherein said TCR constant domain is a mouse TCRα constant domain or a mouse TCRβ constant domain, or a sequence encoding a mouse TCRα constant domain and a mouse TCRβ constant domain; including
said TCR subunit and said antigen binding domain are operably linked;
and a recombinant nucleic acid that, when expressed in a modified T cell containing a functional disruption of an endogenous TCR, functionally integrates said TFP into the TCR complex. 182. The recombinant nucleic acid of claim 181, wherein said TCR subunit comprises the intracellular domain of mouse TCRα or mouse TCRβ. 183. The recombinant nucleic acid of any one of claims 118-182, wherein said extracellular domain comprises an extracellular portion of a TCRα constant domain or a TCRβ constant domain, or a fragment thereof. 184. The recombination of any one of claims 118-183, further comprising at least a subsequence encoding a TCRγ constant domain, a subsequence encoding a TCRδ constant domain, or a subsequence of both the TCRγ and TCRδ constant domains. nucleic acid. 185. The recombinant nucleic acid of any one of claims 181-184, wherein said antigen binding domain comprises a ligand. 186. The recombinant nucleic acid of claim 185, wherein said ligand binds to a cellular receptor. 186. The recombinant nucleic acid of claim 185, wherein said ligand binds to said polypeptide expressed on the surface of a cell. 186. The recombinant nucleic acid of claim 185, wherein said receptor or polypeptide expressed on the surface of a cell comprises a stress response receptor or polypeptide. 186. The recombinant nucleic acid of claim 185, wherein said receptor or polypeptide expressed on the surface of a cell is an MHC class I-associated glycoprotein. 190. The recombinant nucleic acid of claim 189, wherein said MHC class I-associated glycoprotein is selected from the group consisting of MICA, MICB, RAET1E, RAET1G, ULBP1, ULBP2, ULBP3, ULBP4, and combinations thereof. wherein said antigen binding domain comprises a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer 186. The recombinant nucleic acid of Paragraph 185. 192. The recombinant nucleic acid of claim 191, wherein said antigen binding domain comprises a monomer or dimer of said ligand or fragment thereof. said ligand or fragment thereof comprises a monomer, dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, or decamer; 186. The recombinant nucleic acid of claim 185. 194. The recombinant nucleic acid of claim 193, wherein said ligand or fragment thereof is monomeric or dimeric. 186. The recombinant nucleic acid of claim 185, wherein said antigen binding domain does not comprise an antibody or fragment thereof. 186. The recombinant nucleic acid of claim 185, wherein said antigen binding domain does not contain a variable region. 186. The recombinant nucleic acid of claim 185, wherein said antigen binding domain does not contain CDRs. 186. The recombinant nucleic acid of claim 185, wherein said ligand or fragment thereof is a Natural Killer Group 2D (NKG2D) ligand or fragment thereof. when expressed in T cells, said TCR constant domain is incorporated into a functional TCR complex;
The recombinant nucleic acid of any one of claims 118-198. 200. The recombinant nucleic acid of any one of claims 199, wherein said TCR constant domain integrates into the same functional TCR complex as said functional TCR complex that incorporates said TFP when expressed in a T cell. The recombinant nucleic acid of any one of claims 118-200, wherein the TFP-encoding sequence and the TCR constant domain(s)-encoding sequence are contained within the same nucleic acid molecule. The recombinant nucleic acid of any one of claims 118-200, wherein said TFP and said TCR constant domain are operably linked by a first linker sequence. 203. The recombinant nucleic acid molecule of claim 202, wherein said first linker comprises a protease cleavage site. 204. The recombinant nucleic acid molecule of claim 203, wherein said protease cleavage site is a 2A, such as a T2A or P2A cleavage site. 201. The recombinant nucleic acid of any one of claims 118-200, wherein the TFP-encoding sequence and the TCR constant domain(s)-encoding sequence are contained within different nucleic acid molecules. Claims 118-205, wherein said TCR subunit of said TFP and said antibody domain, said antigen binding domain, or said binding ligand, or fragments thereof, are operably linked by a second linker sequence Recombinant nucleic acid according to any one of The recombinant nucleic acid of paragraph 206, wherein said second linker sequence comprises (G ₄ S) _n , where n=1-4. 208. The recombinant nucleic acid of any one of claims 118-207, wherein said transmembrane domain is TCRα or TCRβ, eg, the TCR transmembrane domain from mouse TCRα or TCRβ. 208. Any one of claims 118-207, wherein said TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain of TCRα or TCRβ. The recombinant nucleic acid according to the paragraph. said TCR extracellular domain comprises a TCRα or TCRβ chain, such as the extracellular portion of the constant domain of the murine TCRα or TCRβ chain, a functional fragment thereof, or an amino acid sequence thereof having at least one but no more than 20 modifications The recombinant nucleic acid of any one of claims 118-207. wherein said TCR subunit comprises a transmembrane domain of a TCRα or TCRβ chain, such as a murine TCRα or TCRβ chain, a functional fragment thereof, or an amino acid sequence thereof having at least one but no more than 20 modifications. 208. The recombinant nucleic acid of any one of claims 118-207, comprising a 208. Any one of claims 118-207, wherein said TCR subunit comprises a TCR alpha or TCR beta chain, such as the TCR intracellular domain of a murine TCR alpha or TCR beta chain, or an amino acid sequence having at least one modification thereto. The recombinant nucleic acid according to the paragraph. wherein said TCR subunit comprises (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain and is or comprises an alpha constant domain. 208. The recombinant nucleic acid of any one of paragraphs 118-207. said alpha constant domain is the sequence of SEQ ID NO: 17, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 146, or SEQ ID NO: 207, a functional fragment thereof, or an amino acid sequence thereof having at least one but no more than 20 modifications; 214. The recombinant nucleic acid of claim 213, comprising amino acids 2-137 of SEQ ID NO:146, for example. wherein the encoded TCR comprising (i) at least a portion of the TCR extracellular domain, (ii) the TCR transmembrane domain, and (iii) the TCR intracellular domain is or comprises a beta constant domain. 208. The recombinant nucleic acid of any one of paragraphs 118-207. said beta constant domain is the sequence of SEQ ID NO: 18, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 152, or SEQ ID NO: 209, a functional fragment thereof, or an amino acid sequence thereof having at least one but no more than 20 modifications; 216. The recombinant nucleic acid of claim 215, comprising, for example, amino acids 2-173 of SEQ ID NO:152. 217. The recombinant nucleic acid of claim 215 or 216, wherein said extracellular domain of said TCR subunit does not comprise an alpha or beta chain variable domain. wherein said TCR subunit comprises a stimulatory domain of a protein selected from the intracellular signaling domains of CD3ε, CD3γ, or CD3δ, or a TCR intracellular domain comprising an amino acid sequence having at least one modification thereto; 218. The recombinant nucleic acid of any one of paragraphs 1-217. 219. The recombinant nucleic acid of claim 218, wherein said TFP TCR subunit comprises the extracellular, transmembrane, and intracellular domains of CD3[epsilon]. 220. The recombinant nucleic acid of claim 218 or 219, wherein the TCR subunit of CD3ε comprises SEQ ID NO: 258, the sequence of a functional fragment thereof, or the amino acid sequence thereof with at least one but no more than 20 modifications. of claims 1-220, wherein said TFP, said TCRα constant domain, said TCRβ domain, and any combination thereof is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide A recombinant nucleic acid according to any one of claims 1 to 3. (a) said TCR constant domain is a TCRα constant domain and said TFP is functionally integrated into a TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or a combination thereof;
(b) said TCR constant domain is a TCRβ constant domain and said TFP is functionally integrated into a TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or a combination thereof; or (c) 2. The TCR constant domains are a TCRα constant domain and a TCRβ constant domain, and the TFP is functionally integrated into a TCR complex comprising endogenous subunits of CD3ε, CD3γ, CD3δ, or combinations thereof. 221. The recombinant nucleic acid of any one of paragraphs 1-221. 223. Any of claims 220-222, wherein said at least one but no more than 20 modifications comprise modifications of amino acids that mediate cell signaling or modifications of amino acids that are phosphorylated in response to ligand binding to said TFP. 2. Recombinant nucleic acid according to item 1. The recombinant nucleic acid of any one of claims 118 and 120-180, wherein said antibody is an antibody fragment. 225. The recombinant nucleic acid of claim 224, wherein said antibody fragment is a scFv, single domain antibody domain, _VH domain, or _VL domain. The antigen binding domain comprises an anti-CD19 binding domain, an anti-B cell maturation antigen (BCMA) binding domain, an anti-mesothelin (MSLN) binding domain, an anti-CD20 binding domain, an anti-CD70 binding domain, an anti-79b binding domain, an anti-HER2 binding domain, an anti- PMSA binding domain, anti-MUC16 binding domain, anti-CD22 binding domain, anti-PD-L1 binding domain, anti-BAFF or BAFF receptor binding domain, anti-nectin-4 binding domain, anti-TROP-2 binding domain, anti-GPC3 binding domain, and 181. The recombinant nucleic acid of any one of claims 118 and 120-180, selected from the group consisting of anti-ROR-1 binding domains. 227. The recombinant nucleic acid of claim 226, wherein said anti-MSLN binding domain comprises CDR1 of SEQ ID NO:60, CDR2 of SEQ ID NO:61, and CDR3 of SEQ ID NO:62. 227. The recombinant nucleic acid of claim 226, wherein said anti-MSLN binding domain comprises CDR1 of SEQ ID NO:63, CDR2 of SEQ ID NO:64, and CDR3 of SEQ ID NO:65. 227. The recombinant nucleic acid of claim 226, wherein said anti-MSLN binding domain comprises CDR1 of SEQ ID NO:66, CDR2 of SEQ ID NO:67, and CDR3 of SEQ ID NO:68. 227. The recombinant nucleic acid of claim 226, wherein said anti-MSLN binding domain comprises a sequence having at least about 80% sequence identity to the sequence of SEQ ID NO:69, SEQ ID NO:70, or SEQ ID NO:71. 227. The recombinant nucleic acid of claim 226, wherein said anti-CD19 binding domain comprises light chain CDR1 of SEQ ID NO:73, CDR2 of SEQ ID NO:75, and CDR3 of SEQ ID NO:77. 227. The recombinant nucleic acid of claim 226, wherein said anti-CD19 binding domain comprises heavy chain CDR1 of SEQ ID NO:79, CDR2 of SEQ ID NO:81, and CDR3 of SEQ ID NO:83. wherein said anti-CD19 binding domain has at least about 80% sequence identity to the sequence of SEQ ID NO:85 and/or at least about 80% sequence identity to the sequence of SEQ ID NO:87. 227. The recombinant nucleic acid of claim 226, comprising a heavy chain variable region having a 4. The recombinant nucleic acid of any one of the preceding claims, wherein said nucleic acid is selected from the group consisting of DNA and RNA. 4. The recombinant nucleic acid of any one of the preceding claims, wherein said nucleic acid is mRNA. 4. The recombinant nucleic acid of any one of the preceding claims, wherein said nucleic acid is circRNA. 4. The recombinant nucleic acid of any one of the preceding claims, wherein said recombinant nucleic acid comprises a nucleic acid analogue, said nucleic acid analogue being absent from the coding sequence of said recombinant nucleic acid. The nucleic acid analog is 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, T-deoxy-2′-fluoro , 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP) , TO-dimethylaminoethyloxyethyl (2′-O-DMAEOE), 2′-ON-methylacetamide (2′-O-NMA) modification, locked nucleic acid (LNA), ethylene nucleic acid (ENA), from the group consisting of peptide nucleic acids (PNA), 1′,5′-anhydrohexitol nucleic acids (HNA), morpholinos, methylphosphonate nucleotides, thiolphosphonate nucleotides, and 2′-fluoro N3-P5′-phosphoramidites 238. The recombinant nucleic acid of claim 237, selected. 12. The recombinant nucleic acid of any one of the preceding claims, further comprising a leader sequence. 12. The recombinant nucleic acid of any one of the preceding claims, further comprising a promoter sequence. 4. The recombinant nucleic acid of any one of the preceding claims, further comprising a sequence encoding a poly(A) tail. 12. The recombinant nucleic acid of any one of the preceding claims, further comprising a 3'UTR sequence. 4. The recombinant nucleic acid of any one of the preceding claims, wherein said nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. 4. The recombinant nucleic acid molecule of any one of the preceding claims, wherein said nucleic acid is an in vitro transcribed nucleic acid. A vector comprising a recombinant nucleic acid according to any one of the preceding claims. 246. The vector of claim 245, wherein said vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, adeno-associated viral vector (AAV), Rous sarcoma virus (RSV) vector, or retroviral vector. Vector described. 247. The vector of claim 245 or 246, wherein said vector is an AAV6 vector. The vector of any one of claims 245-247, further comprising a promoter. The vector of any one of claims 245-248, wherein said vector is an in vitro transcribed vector. A modified T cell comprising the recombinant nucleic acid of any one of claims 1-244 or the vector of any one of claims 245-249, wherein said modified T cell is capable of endogenous TCR function Said modified T cell comprising targeted destruction. Said sequence encoding said TFP of the nucleic acid of any one of claims 1-244, or encoded by said sequence of said nucleic acid of any one of claims 1-244 encoding TFP A modified T cell comprising said TFP, said modified T cell comprising a functional disruption of an endogenous TCR. said sequence encoding a TFP according to any one of claims 1-244 or said TFP encoded by said sequence of said nucleic acid according to any one of claims 1-244 encoding a TFP Engineered allogeneic T cells, comprising: 253. Any one of claims 250-252, wherein said T cell further comprises a heterologous sequence encoding a TCR constant domain, said TCR constant domain being a TCRγ constant domain, a TCRδ constant domain, or a TCRγ and TCRδ constant domain. The modified T cell according to the paragraph. 254. Any one of claims 250-253, wherein said T cell further comprises a heterologous sequence encoding a TCR constant domain, said TCR constant domain being a TCRα constant domain, a TCRβ constant domain, or a TCRα and TCRβ constant domain. The modified T cell according to the paragraph. Said TCR constant domain, e.g., said TCRα constant domain, said TCRβ constant domain, or said TCRα constant domain and said TCRβ constant domain is a mouse TCRγ constant domain, e.g., mouse TCRα constant domain, mouse TCRβ constant domain, or mouse TCRα constant 255. The engineered T cell of claim 254, which is a domain and a mouse TCRβ constant domain. 256. The modified T of any one of claims 250-255, wherein the endogenous TCR that is functionally disrupted is an endogenous TCRα chain, an endogenous TCRβ chain, or an endogenous TCRα chain and an endogenous TCRβ chain. cell. 257. The modification of any one of claims 250-256, wherein said functionally disrupted endogenous TCR has reduced binding to MHC-peptide complexes compared to that of unmodified control T cells. T cells. The engineered T cell of any one of claims 250-257, wherein said functional disruption is disruption of the gene encoding said endogenous TCR. 259. The modified T cell of claim 258, wherein said disruption of said gene encoding said endogenous TCR is removal of a sequence of said gene encoding said endogenous TCR from the genome of said T cell. The T cells are CD4 cells, CD8 cells, naive T cells, memory stem T cells, central memory T cells, double negative T cells, effector memory T cells, effector T cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, α/β T cells, γ/δ T cells, natural killer (NK) cells, natural killer T (NKT) cells, hematopoietic stem cells, and pluripotent stem cells 260. The modified T cell of any one of claims 250-259, which is a human T cell that is a human T cell. The engineered T cell of any one of claims 250-260, wherein said T cell is a CD8 ⁺ T cell or a CD4 ⁺ T cell. The modified T cell of any one of claims 250-261, wherein said T cell is an allogeneic T cell. Claims 250-262, further comprising a nucleic acid encoding an inhibitory molecule comprising a first polypeptide comprising at least a portion of the inhibitory molecule associated with a second polypeptide comprising a positive signal from an intracellular signaling domain. The modified T cell according to any one of Claims 1 to 3. 264. The engineered T cell of claim 263, wherein said inhibitory molecule comprises a first polypeptide comprising at least a portion of PDl and a second polypeptide comprising a costimulatory domain and a primary signaling domain. (a) the modified T cell of any one of claims 250-264;
(b) a pharmaceutically acceptable carrier; and a pharmaceutical composition. A method of making the modified T cell of any one of claims 250-264, comprising:
(a) disrupting an endogenous TCR gene encoding a TCRα chain, a TCRβ chain, or a TCRα chain and a TCRβ chain, thereby producing a T cell containing a functional disruption of the endogenous TCR gene;
(b) a T cell comprising a functional disruption of an endogenous TCR gene with the recombinant nucleic acid of any one of claims 1-244 or the vector of any one of claims 245-249; The above method, comprising transducing. 12. The method of claim 1, wherein said disruption comprises transducing T cells with a nuclease protein or a nucleic acid sequence encoding a nuclease protein that targets endogenous genes encoding the TCRα chain, the TCRβ chain, or the TCRα and TCRβ chains. 266. 264. A method of making the modified T cell of any one of claims 250-264, wherein a T cell comprising a functional disruption of an endogenous TCR gene is added to the T cell of any one of claims 1-244. Said method comprising transducing a recombinant nucleic acid according to any one of claims 245-249 or a vector according to any one of claims 245-249. 268, wherein said T cell comprising a functional disruption of an endogenous TCR gene is a T cell comprising a functional disruption of an endogenous TCR gene encoding a TCRα chain, a TCRβ chain, or a TCRα chain and a TCRβ chain The method described in . The method of any one of claims 266-269, wherein said T cells are human T cells. 271. Any one of claims 266-270, wherein said T cells comprising a functional disruption of an endogenous TCR gene have reduced binding to MHC-peptide complexes compared to unmodified control T cells. The method described in section. 272. The method of any one of claims 267-271, wherein said nuclease is a meganuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, or a megaTAL nuclease. . said sequence contained in said recombinant nucleic acid or said vector is inserted into said endogenous TCR subunit gene at said cleavage site, said insertion of said sequence into said endogenous TCR subunit gene resulting in said endogenous TCR subunit 273. The method of any one of claims 266-272, wherein is functionally disrupted. 272. The method of any one of claims 267-271, wherein said nuclease is a meganuclease. said meganuclease comprising a first subunit and a second subunit;
275. The method of claim 274, wherein the subunit of binds to a first recognition half-site of said recognition sequence and said second subunit binds to a second recognition half-site of said recognition sequence. 276. The method of claim 275, wherein said meganuclease is a single-stranded meganuclease comprising a linker, said linker covalently joining said first subunit and said second subunit. 266. A method of treating cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of the pharmaceutical composition of claim 265. 277. A method of treating cancer in a subject in need thereof, comprising: (a) an engineered T cell produced according to the method of any one of claims 266-276; and (b) a pharmaceutically acceptable said method comprising administering to said subject a pharmaceutical composition comprising said carrier. 277. A method of treating cancer in a subject in need thereof, comprising: (a) an engineered T cell produced according to the method of any one of claims 266-276; and (b) a pharmaceutically acceptable said method comprising administering to said subject a pharmaceutical composition comprising said carrier. The method of any one of claims 277-279, wherein said modified T cells are allogeneic T cells. 281. The method of any one of claims 277-280, wherein less cytokines are released in said subject compared to a subject administered an effective amount of unmodified control T cells. compared to a subject administered an effective amount of modified T cells comprising the recombinant nucleic acid of any one of claims 1-244 or the vector of any one of claims 245-249, said subject 282. The method of any one of claims 277-281, wherein less cytokines are released in. 283. The method of any one of claims 277-282, wherein said method comprises administering said pharmaceutical composition in combination with an agent that increases the effectiveness of said pharmaceutical composition. 284. The method of any one of claims 277-283, wherein said method comprises administering said pharmaceutical composition in combination with an agent that ameliorates one or more side effects associated with said pharmaceutical composition. 285. The method of any one of claims 277-284, wherein the cancer is solid tumor, lymphoma, or leukemia. 4. The cancer is selected from the group consisting of renal cell carcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, kidney cancer, and gastric cancer. 277-285. Claims 277-286, wherein less cytokines are released in said subject compared to a subject administered an effective amount of autologous T cells expressing the TFP of claims 1-244. Method. The method of any one of claims 277-287, wherein said method does not induce graft-versus-host disease. of claims 277-288, wherein the risk of said subject developing graft-versus-host disease is reduced compared to a subject administered an effective amount of autologous T cells expressing the TFP of claims 1-244 A method according to any one of paragraphs. A recombinant nucleic acid according to any one of claims 1 to 244, a vector according to any one of claims 245 to 249, a vector according to any one of claims 250 to 264, for use as a medicament or for the preparation of a medicament. 266. The engineered T cell of any one or the pharmaceutical composition of claim 265.

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