Compositions and methods for tcr reprogramming using fusion proteins

Abstract

Provided herein are T-cell receptor (TCR) fusion proteins (TFPs), T-cells engineered to express one or more TFPs, and methods of use thereof for the treatment of diseases, including cancer.

What is claimed is: 
     
         1 . An isolated recombinant nucleic acid molecule encoding a T-cell receptor (TCR) fusion protein (TFP) comprising
 (a) a TCR subunit comprising
 (i) at least a portion of a TCR extracellular domain, and 
 (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon; and 
   (b) a human or humanized antibody domain comprising an antigen binding domain that is an anti-mesothelin binding domain;   wherein the TCR subunit and the antibody domain are operatively linked, and   wherein the TFP incorporates into a TCR when expressed in a T-cell.   
     
     
         2 . An isolated recombinant nucleic acid molecule encoding a T-cell receptor (TCR) fusion protein (TFP) comprising
 (a) a TCR subunit comprising
 (i) at least a portion of a TCR extracellular domain, and 
 (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 epsilon; and 
   (b) a human or humanized antibody domain comprising an antigen binding domain that is an anti-mesothelin binding domain;   wherein the TCR subunit and the antibody domain are operatively linked, and   wherein the TFP incorporates into a TCR when expressed in a T-cell.   
     
     
         3 . An isolated recombinant nucleic acid molecule encoding a T-cell receptor (TCR) fusion protein (TFP) comprising
 (a) a TCR subunit comprising
 (i) at least a portion of a TCR extracellular domain, and 
 (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 gamma; and 
 (b) a human or humanized antibody domain comprising an antigen binding domain that is an anti-mesothelin binding domain; 
   wherein the TCR subunit and the antibody domain are operatively linked, and   wherein the TFP incorporates into a TCR when expressed in a T-cell.   
     
     
         4 . An isolated recombinant nucleic acid molecule encoding a T-cell receptor (TCR) fusion protein (TFP) comprising
 (a) a TCR subunit comprising
 (i) at least a portion of a TCR extracellular domain, and 
 (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3 delta; and 
   (b) a human or humanized antibody domain comprising an antigen binding domain that is an anti-mesothelin binding domain;   wherein the TCR subunit and the antibody domain are operatively linked, and   wherein the TFP incorporates into a TCR when expressed in a T-cell.   
     
     
         5 . An isolated recombinant nucleic acid molecule encoding a T-cell receptor (TCR) fusion protein (TFP) comprising
 (a) a TCR subunit comprising
 (i) at least a portion of a TCR extracellular domain, and 
 (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of TCR alpha; and 
   (b) a human or humanized antibody domain comprising an antigen binding domain that is an anti-mesothelin binding domain;   wherein the TCR subunit and the antibody domain are operatively linked, and   wherein the TFP incorporates into a TCR when expressed in a T-cell.   
     
     
         6 . An isolated recombinant nucleic acid molecule encoding a T-cell receptor (TCR) fusion protein (TFP) comprising
 (a) a TCR subunit comprising
 (i) at least a portion of a TCR extracellular domain, and 
 (ii) a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of TCR beta; and 
   (b) a human or humanized antibody domain comprising an antigen binding domain that is an anti-mesothelin binding domain;   wherein the TCR subunit and the antibody domain are operatively linked, and   wherein the TFP incorporates into a TCR when expressed in a T-cell.   
     
     
         7 . An isolated recombinant nucleic acid molecule encoding a T-cell receptor (TCR) fusion protein (TFP) comprising a TCR subunit and a human or humanized antibody domain comprising an antigen binding domain that is an anti-mesothelin binding domain. 
     
     
         8 . The isolated nucleic acid molecule  claim 7 , wherein the TCR subunit and the antibody domain are operatively linked. 
     
     
         9 . The isolated nucleic acid molecule of  claim 7  or  8 , wherein the TFP incorporates into a TCR when expressed in a T-cell. 
     
     
         10 . The isolated nucleic acid molecule of any one of  claims 1 - 9 , wherein the encoded antigen binding domain is connected to the TCR extracellular domain by a linker sequence. 
     
     
         11 . The isolated nucleic acid molecule of  claim 10 , wherein the encoded linker sequence comprises (G 4 S) n  wherein n=1 to 4. 
     
     
         12 . The isolated nucleic acid molecule of any one of  claims 1 - 11 , wherein the TCR subunit comprises a TCR extracellular domain. 
     
     
         13 . The isolated nucleic acid molecule of any one of  claims 1 - 12 , wherein the TCR subunit comprises a TCR transmembrane domain. 
     
     
         14 . The isolated nucleic acid molecule of any one of  claims 1 - 13 , wherein the TCR subunit comprises a TCR intracellular domain. 
     
     
         15 . The isolated nucleic acid molecule of any one of  claims 1 - 14 , wherein the TCR subunit comprises (i) a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain, wherein at least two of (i), (ii), and (iii) are from the same TCR subunit. 
     
     
         16 . The isolated nucleic acid molecule of any one of  claims 1 - 15 , wherein the TCR subunit comprises a TCR intracellular domain comprising a stimulatory domain selected from an intracellular signaling domain of CD3 epsilon, CD3 gamma or CD3 delta, or an amino acid sequence having at least one modification thereto. 
     
     
         17 . The isolated nucleic acid molecule of any one of  claims 1 - 16 , wherein the TCR subunit comprises an intracellular domain comprising a stimulatory domain selected from a functional signaling domain of 4-1BB and/or a functional signaling domain of CD3 zeta, or an amino acid sequence having at least one modification thereto. 
     
     
         18 . The isolated nucleic acid molecule of any one of  claims 1 - 17 , wherein the human or humanized antibody domain comprises an antibody fragment. 
     
     
         19 . The isolated nucleic acid molecule of any one of  claims 1 - 18 , wherein the human or humanized antibody domain comprises a scFv or a V H  domain. 
     
     
         20 . The isolated nucleic acid molecule of any one of  claims 1 - 19 , wherein the human or humanized antibody domain comprises a sdAb or a V HH  domain. 
     
     
         21 . The isolated nucleic acid molecule of any one of  claims 1 - 20 , encoding (i) a light chain (LC) CDR1, LC CDR2 and LC CDR3 of an anti-mesothelin light chain binding domain amino acid sequence with 70-100% sequence identity to a light chain (LC) CDR1, LC CDR2 and LC CDR3 of an anti-mesothelin light chain binding domain provided herein, respectively, and/or (ii) a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of an anti-mesothelin heavy chain binding domain amino acid sequence with 70-100% sequence identity to a heavy chain (HC) CDR1, HC CDR2 and HC CDR3 of an anti-mesothelin heavy chain binding domain provided herein, respectively. 
     
     
         22 . The isolated nucleic acid molecule of any one of  claims 1 - 21 , encoding a light chain variable region, wherein the light chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a light chain variable region amino acid sequence of a light chain variable region provided herein, or a sequence with 95-99% identity to a light chain variable region amino acid sequence of a light chain variable region provided herein. 
     
     
         23 . The isolated nucleic acid molecule of any one of  claims 1 - 22 , encoding a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a heavy chain variable region amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to a heavy chain variable region amino acid sequence of a heavy chain variable region provided herein. 
     
     
         24 . The isolated nucleic acid molecule of any one of  claims 1 - 23 , encoding a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence having at least one but not more than 30 modifications of a heavy chain variable region amino acid sequence of a heavy chain variable region provided herein, or a sequence with 95-99% identity to a single domain antibody amino acid sequence of a V HH  region provided herein. 
     
     
         25 . The isolated nucleic acid molecule of  claim 24 , wherein the sequence of the V HH  region is set forth in a sequence provided herein. 
     
     
         26 . The isolated nucleic acid molecule of any one of  claims 1 - 23 , wherein the TFP includes an extracellular domain of a TCR subunit that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. 
     
     
         27 . The isolated nucleic acid molecule of any one of  claims 1 - 26 , wherein the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. 
     
     
         28 . The isolated nucleic acid molecule of any one of  claims 1 - 27 , wherein the encoded TFP includes a transmembrane domain that comprises a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, CD45, CD2, 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 not more than 20 modifications. 
     
     
         29 . The isolated nucleic acid molecule of any one of  claims 1 - 28 , further comprising a sequence encoding a costimulatory domain. 
     
     
         30 . The isolated nucleic acid molecule of  claim 29 , wherein the costimulatory domain is a functional signaling domain obtained from a protein selected from the group consisting of DAP10, DAP12, CD30, LIGHT, OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), and 4-1BB (CD137), and amino acid sequences thereof having at least one but not more than 20 modifications thereto. 
     
     
         31 . The isolated nucleic acid molecule of any one of  claims 1 - 30 , wherein the at least one but not more than 20 modifications thereto comprise a modification of an amino acid that mediates cell signaling or a modification of an amino acid that is phosphorylated in response to a ligand binding to the TFP. 
     
     
         32 . The isolated nucleic acid molecule of any one of  claims 1 - 31 , wherein the isolated nucleic acid molecule is mRNA. 
     
     
         33 . The isolated nucleic acid molecule of any one of  claims 1 - 32 , wherein the TFP includes an immunoreceptor tyrosine-based activation motif (ITAM) of a TCR subunit that comprises an ITAM or portion thereof of a protein selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, CD3 delta TCR subunit, TCR zeta chain, Fc epsilon receptor 1 chain, Fc epsilon receptor 2 chain, Fc gamma receptor 1 chain, Fc gamma receptor 2a chain, Fc gamma receptor 2b1 chain, Fc gamma receptor 2b2 chain, Fc gamma receptor 3a chain, Fc gamma receptor 3b chain, Fc beta 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 not more than 20 modifications thereto. 
     
     
         34 . The isolated nucleic acid molecule of  claim 33 , wherein the ITAM replaces an ITAM of CD3 gamma, CD3 delta, or CD3 epsilon. 
     
     
         35 . The isolated nucleic acid molecule of  claim 33 , wherein the ITAM is selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit and replaces a different ITAM selected from the group consisting of CD3 zeta TCR subunit, CD3 epsilon TCR subunit, CD3 gamma TCR subunit, and CD3 delta TCR subunit. 
     
     
         36 . The isolated nucleic acid molecule of any one of  claims 1 - 35 , wherein the nucleic acid comprises a nucleotide analog. 
     
     
         37 . The isolated nucleic acid molecule of  claim 36 , wherein the nucleotide analog is selected from the group consisting of 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), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), 2′-O—N-methylacetamido (2′-O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a 1′,5′-anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2′-fluoro N3-P5′-phosphoramidite. 
     
     
         38 . The isolated nucleic acid molecule of any one of  claims 1 - 37 , further comprising a leader sequence 
     
     
         39 . The isolated nucleic acid molecule of any one of  claims 1 - 38 , wherein the human or humanized antibody domain comprising an antigen binding domain that is an anti-mesothelin binding domain encoded by the nucleic acid, or an antibody comprising the anti-mesothelin binding domain, or a cell expressing the anti-mesothelin binding domain encoded by the nucleic acid has an affinity value of at most about 200 nM, 100 nM, 75 nM, a 50 nM, 25 nM, 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM; and/or at least about 100 nM, 75 nM, a 50 nM, 25 nM, 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM; and or about 200 nM, 100 nM, 75 nM, a 50 nM, 25 nM, 20 nM, 15 nM, 14 nM, 13 nM, 12 nM, 11 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM, 0.03 nM, 0.02 nM, or 0.01 nM. 
     
     
         40 . An isolated polypeptide molecule encoded by the nucleic acid molecule of any one of  claims 1 - 39 . 
     
     
         41 . An isolated recombinant TFP molecule comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain. 
     
     
         42 . An isolated recombinant TFP molecule comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide. 
     
     
         43 . An isolated recombinant TFP molecule comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the TFP molecule is capable of functionally integrating into an endogenous TCR complex. 
     
     
         44 . The isolated TFP molecule of  claim 41 , comprising an antibody or antibody fragment comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain. 
     
     
         45 . The isolated TFP molecule of any one of  claims 41 - 44 , wherein the anti-mesothelin binding domain is a scFv or a V H  domain. 
     
     
         46 . The isolated TFP molecule of any one of  claims 41 - 45 , wherein the anti-mesothelin binding domain comprises a heavy chain with 95-100% identity to an amino acid sequence of an anti-mesothelin light chain provided herein, a functional fragment thereof, or an amino acid sequence thereof having at least one but not more than 30 modifications. 
     
     
         47 . The isolated TFP molecule of any one of  claims 41 - 46 , wherein the anti-mesothelin binding domain comprises a light chain with 95-100% identity to an amino acid sequence of an anti-mesothelin heavy chain provided herein, a functional fragment thereof, or an amino acid sequence thereof having at least one but not more than 30 modifications. 
     
     
         48 . The isolated TFP molecule of any one of  claims 41 - 47 , comprising a TCR extracellular domain that comprises an extracellular domain or portion thereof of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta TCR subunit, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications. 
     
     
         49 . The isolated TFP molecule of any one of  claims 41 - 48 , wherein the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of the alpha or beta chain of the T-cell receptor, CD3 delta, CD3 epsilon, or CD3 gamma. 
     
     
         50 . The isolated TFP molecule of any one of  claims 41 - 49 , wherein the anti-mesothelin binding domain is connected to the TCR extracellular domain by a linker sequence. 
     
     
         51 . The isolated TFP molecule of  claim 50 , wherein the linker region comprises (G 4 S) n , wherein n=1 to 4. 
     
     
         52 . The isolated TFP molecule of any one of  claims 41 - 51 , further comprising a sequence encoding a costimulatory domain. 
     
     
         53 . The isolated TFP molecule of any one of  claims 41 - 52 , further comprising a sequence encoding an intracellular signaling domain. 
     
     
         54 . The isolated TFP molecule of any one of  claims 41 - 53 , further comprising a leader sequence. 
     
     
         55 . A nucleic acid comprising a sequence encoding a TFP of any one of  claims 41 - 54 . 
     
     
         56 . The nucleic acid of  claim 55 , wherein the nucleic acid is selected from the group consisting of a DNA and a RNA. 
     
     
         57 . The nucleic acid of  claim 55  or  56 , wherein the nucleic acid is a mRNA. 
     
     
         58 . The nucleic acid of any one of  claims 55 - 57 , wherein the nucleic acid comprises a nucleotide analog. 
     
     
         59 . The nucleic acid of  claim 58 , wherein the nucleotide analog is selected from the group consisting of 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), T-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), 2′-O—N-methylacetamido (2′-O-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2′-fluoro N3-P5′-phosphoramidite. 
     
     
         60 . The nucleic acid of any one of  claims 55 - 59 , further comprising a promoter. 
     
     
         61 . The nucleic acid of any one of  claims 55 - 60 , wherein the nucleic acid is an in vitro transcribed nucleic acid. 
     
     
         62 . The nucleic acid of any one of  claims 55 - 61 , wherein the nucleic acid further comprises a sequence encoding a poly(A) tail. 
     
     
         63 . The nucleic acid of any one of  claims 55 - 62 , wherein the nucleic acid further comprises a 3′UTR sequence. 
     
     
         64 . A vector comprising a nucleic acid molecule encoding a TFP of any one of  claims 41 - 54 . 
     
     
         65 . The vector of  claim 64 , wherein the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector. 
     
     
         66 . The vector of  claim 64  or  65 , further comprising a promoter. 
     
     
         67 . The vector of any one of  claims 64 - 66 , wherein the vector is an in vitro transcribed vector. 
     
     
         68 . The vector of any one of  claims 64 - 67 , wherein a nucleic acid sequence in the vector further comprises a poly(A) tail. 
     
     
         69 . The vector of any one of  claims 64 - 68 , wherein a nucleic acid sequence in the vector further comprises a 3′UTR. 
     
     
         70 . A cell comprising the isolated nucleic acid molecule of any one of  claims 1 - 39 , the polypeptide molecule of  claim 40 , the TFP molecule of any one of  claims 41 - 54 , the nucleic acid of any one of  claims 55 - 63 , the vector of any one of  claims 64 - 69 . 
     
     
         71 . The cell of  claim 70 , wherein the cell is a human T-cell. 
     
     
         72 . The cell of  claim 71 , wherein the T-cell is a CD8+ or CD4+ T-cell. 
     
     
         73 . The cell of  claim 71 , wherein the T-cell is a gamma-delta T cell. 
     
     
         74 . The cell of  claim 71 , wherein the T-cell is an NKT cell. 
     
     
         75 . The cell of any one of  claims 70 - 73 , further comprising a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide that comprises at least a portion of an inhibitory molecule, associated with a second polypeptide that comprises a positive signal from an intracellular signaling domain. 
     
     
         76 . The cell of  claim 75 , wherein the inhibitory molecule comprise first polypeptide that comprises at least a portion of PD1 and a second polypeptide comprising a costimulatory domain and primary signaling domain. 
     
     
         77 . A human CD8+ or CD4+ T-cell comprising at least two TFP molecules, the TFP molecules comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+ or CD4+ T-cell. 
     
     
         78 . A protein complex comprising:
 i) a TFP molecule comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain; and   ii) at least one endogenous TCR subunit or endogenous TCR complex.   
     
     
         79 . The protein complex of  claim 78 , wherein the TCR comprises an extracellular domain or portion thereof of a protein selected from the group consisting of TCR alpha chain, a TCR beta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, and a CD3 delta TCR subunit. 
     
     
         80 . The protein complex of  claim 78  or  79 , wherein the anti-mesothelin binding domain is connected to the TCR extracellular domain by a linker sequence. 
     
     
         81 . The protein complex of  claim 80 , wherein the linker region comprises (G 4 S) n , wherein n=1 to 4. 
     
     
         82 . A protein complex comprising
 (a) a TFP encoded by the isolated nucleic acid molecule of any one of  claims 1 - 39 , and   (b) at least one endogenous TCR subunit or endogenous TCR complex.   
     
     
         83 . A human CD8+ or CD4+ T-cell comprising at least two different TFP proteins per the protein complex of any one of  claims 78 - 82 . 
     
     
         84 . A human CD8+ or CD4+ T-cell comprising at least two different TFP molecules encoded by the isolated nucleic acid molecule of any one of  claims 1 - 39 . 
     
     
         85 . A population of human CD8+ or CD4+ T-cells, wherein the T-cells of the population individually or collectively comprise at least two TFP molecules, the TFP molecules comprising a human or humanized anti-mesothelin binding domain, a TCR extracellular domain, a transmembrane domain, and an intracellular domain, wherein the TFP molecule is capable of functionally interacting with an endogenous TCR complex and/or at least one endogenous TCR polypeptide in, at and/or on the surface of the human CD8+ or CD4+ T-cell. 
     
     
         86 . A population of human CD8+ or CD4+ T-cells, wherein the T-cells of the population individually or collectively comprise at least two TFP molecules encoded by the isolated nucleic acid molecule of any one of  claims 1 - 39 . 
     
     
         87 . A method of making a cell comprising transducing a T-cell with the isolated nucleic acid molecule of any one of  claims 1 - 39 , the nucleic acid of any one of  claims 55 - 63 , or the vector of any one of  claims 64 - 69 . 
     
     
         88 . A method of generating a population of RNA-engineered cells comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell, where the RNA comprises a nucleic acid encoding the TFP molecule of any one of  claims 41 - 54 . 
     
     
         89 . A method of providing an anti-tumor immunity in a mammal comprising administering to the mammal an effective amount of the isolated nucleic acid molecule of any one of  claims 1 - 39 , the polypeptide molecule of  claim 40 , a cell expressing the polypeptide molecule of  claim 40 , the TFP molecule of any one of  claims 41 - 54 , the nucleic acid of any one of  claims 55 - 63 , the vector of any one of  claims 64 - 69 , or the cell of any one of  claims 70 - 77  and  83 - 87 . 
     
     
         90 . The method of  claim 89 , wherein the cell is an autologous T-cell. 
     
     
         91 . The method of  claim 89 , wherein the cell is an allogeneic T-cell. 
     
     
         92 . The method of any one of  claims 89 - 91 , wherein the mammal is a human. 
     
     
         93 . A method of treating a mammal having a disease associated with expression of mesothelin comprising administering to the mammal an effective amount of the isolated nucleic acid molecule of any one of  claims 1 - 39 , the polypeptide molecule of  claim 40 , a cell expressing the polypeptide molecule of  claim 40 , the TFP molecule of any one of  claims 41 - 54 , the nucleic acid of any one of  claims 55 - 63 , the vector of any one of  claims 64 - 69 , or the cell of any one of  claims 70 - 77  and  83 - 86 . 
     
     
         94 . The method of  claim 93 , wherein the disease associated with mesothelin expression is selected from the group consisting of a proliferative disease, a cancer, a malignancy, and a non-cancer related indication associated with expression of mesothelin. 
     
     
         95 . The method of  claim 93 , wherein the disease is a cancer selected from the group consisting of mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, liver cancer, pancreatic cancer, thyroid cancer, bladder cancer, ureter cancer, kidney cancer, endometrial cancer, esophageal cancer, gastric cancer, thymic carcinoma, cholangiocarcinoma and stomach cancer. 
     
     
         96 . The method of  claim 93 , wherein the disease is a cancer selected from the group consisting of mesothelioma, papillary serous ovarian adenocarcinoma, clear cell ovarian carcinoma, mixed Mullerian ovarian carcinoma, endometroid mucinous ovarian carcinoma, pancreatic adenocarcinoma, ductal pancreatic adenocarcinoma, uterine serous carcinoma, lung adenocarcinoma, extrahepatic bile duct carcinoma, gastric adenocarcinoma, esophageal adenocarcinoma, colorectal adenocarcinoma, breast adenocarcinoma, a disease associated with mesothelin expression, and combinations thereof. 
     
     
         97 . The method of  claim 93 , wherein the cells expressing a TFP molecule are administered in combination with an agent that increases the efficacy of a cell expressing a TFP molecule. 
     
     
         98 . The method of any one of  claims 93 - 97 , wherein less cytokines are released in the mammal compared a mammal administered an effective amount of a T-cell expressing an anti-mesothelin chimeric antigen receptor (CAR). 
     
     
         99 . The method of any one of  claims 93 - 98 , wherein the cells expressing a TFP molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a TFP molecule. 
     
     
         100 . The method of any one of  claims 93 - 99 , wherein the cells expressing a TFP molecule are administered in combination with an agent that treats the disease associated with mesothelin. 
     
     
         101 . The isolated nucleic acid molecule of any one of  claims 1 - 39 , the isolated polypeptide molecule of  claim 40 , a cell expressing the polypeptide molecule of  claim 40 , the isolated TFP of any one of  claims 41 - 54 , the nucleic acid of any one of  claims 55 - 63 , the vector of any one  claims 64 - 69 , the complex of any one of  claims 78 - 82 , or the cell of any one of  claims 70 - 77  and  83 - 86 , for use as a medicament. 
     
     
         102 . A method of treating a mammal having a disease associated with expression of mesothelin comprising administering to the mammal an effective amount of the isolated nucleic acid molecule of any one of  claims 1 - 39 , the polypeptide molecule of  claim 40 , a cell expressing the polypeptide molecule of  claim 40 , the TFP molecule of any one of  claims 41 - 54 , the nucleic acid of any one of  claims 55 - 63 , the vector of any one of  claims 64 - 69 , or the cell of any one of  claims 70 - 77  and  83 - 86 , wherein less cytokines are released in the mammal compared a mammal administered an effective amount of a T-cell expressing an anti-mesothelin chimeric antigen receptor (CAR).