WO2022056321A1 - Compositions et procédés pour la reprogrammation de tcr au moyen de protéines de fusion spécifiques gpc3 - Google Patents

Compositions et procédés pour la reprogrammation de tcr au moyen de protéines de fusion spécifiques gpc3 Download PDF

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WO2022056321A1
WO2022056321A1 PCT/US2021/049956 US2021049956W WO2022056321A1 WO 2022056321 A1 WO2022056321 A1 WO 2022056321A1 US 2021049956 W US2021049956 W US 2021049956W WO 2022056321 A1 WO2022056321 A1 WO 2022056321A1
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nucleic acid
sequence
acid molecule
domain
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PCT/US2021/049956
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Robert Hofmeister
Dario Gutierrez
Andrew Collard
Robert Tighe
Michael Lofgren
Vania ASHMINOVA
Huan Yang
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TCR2 Therapeutics Inc.
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Publication of WO2022056321A1 publication Critical patent/WO2022056321A1/fr

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    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4632—T-cell receptors [TCR]; antibody T-cell receptor constructs
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    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
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    • C07K2319/00—Fusion polypeptide
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    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention is directed to a novel therapeutics and method for treating GPC3 -related diseases and disorders.
  • Human cancers are by their nature comprised of normal cells that have undergone a genetic or epigenetic conversion to become abnormal cancer cells. In doing so, cancer cells begin to express proteins and other antigens that are distinct from those expressed by normal cells. These aberrant tumor antigens can be used by the body’s innate immune system to specifically target and kill cancer cells. However, cancer cells employ various mechanisms to prevent immune cells, such as T and B lymphocytes, from successfully targeting cancer cells.
  • cancer immunotherapy Most patients with late-stage solid tumors are incurable with standard therapy. In addition, traditional treatment options often have serious side effects. Numerous attempts have been made to engage a patient’s immune system for rejecting cancerous cells, an approach collectively referred to as cancer immunotherapy. However, several obstacles make it rather difficult to achieve clinical effectiveness. Although hundreds of so-called tumor antigens have been identified, these are often derived from self and thus can direct the cancer immunotherapy against healthy tissue, or are poorly immunogenic. Furthermore, cancer cells use multiple mechanisms to render themselves invisible or hostile to the initiation and propagation of an immune attack by cancer immunotherapies.
  • Human T cell therapies rely on enriched or modified human T cells to target and kill cancer cells in a patient.
  • methods have been developed to engineer T cells to express constructs which direct T cells to a particular target cancer cell.
  • CARs Chimeric antigen receptors
  • TCRs engineered T cell receptors
  • T cells Besides the ability of genetically modified T cells expressing a CAR or an engineered TCR to recognize and destroy respective target cells in vitro/ex vivo, successful patient therapy with engineered T cells requires the T cells to be capable of strong activation, expansion, persistence over time, effective tumor targeting, reduced and, in case of relapsing disease, to enable a ‘memory’ response.
  • CAR therapies currently being developed have been associated with the release of high levels of pro-inflammatory cytokines that have been associated with dose-limiting toxicities.
  • TCR subunits including CD3 epsilon, CD3gamma and CD3 delta, and of TCR alpha, TCR beta, TCR gamma, and TCR delta chains with binding domains specific to GPC3 that have the potential to overcome limitations of existing approaches.
  • a recombinant nucleic acid molecule comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP) wherein the TFP comprises: a TCR subunit comprising: at least a portion of a TCR extracellular domain, and a TCR transmembrane domain, a TCR intracellular domain, and an antigen binding domain that specifically binds GPC3; and wherein the TCR subunit and the antigen binding domain are operatively linked.
  • TCR T cell receptor
  • TFP T cell receptor fusion protein
  • the TCR intracellular domain comprises a stimulatory domain from an intracellular signaling domain of CD3 gamma, CD3 delta, or CD3 epsilon.
  • the TFP functionally interacts with an endogenous TCR complex when expressed in a T cell.
  • a T cell expressing the TFP exhibits increased cytotoxicity to a human cell expressing GPC3 compared to a T cell not containing the TFP.
  • the antigen binding domain is connected to the TCR extracellular domain by a linker sequence.
  • the linker is 120 amino acids in length or less.
  • the linker sequence comprises (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 10. In some embodiments, n is an integer from 1 to 4.
  • At least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR alpha. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR beta. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma.
  • At least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta. In some embodiments, at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma. In some embodiments, all three of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit.
  • the antigen binding domain is a camelid antibody or binding fragment thereof. In some embodiments, the antigen binding domain is a murine antibody or binding fragment thereof. In some embodiments, the antigen binding domain is a human or humanized antibody or binding fragment thereof.
  • the antigen binding domain is a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain.
  • the antibody or antibody fragment comprises a heavy chain variable domain comprising a heavy chain complementarity determining region 1 (CDR1), a CDR2, and a CDR3. In some embodiments, the antibody or antibody fragment comprises a light chain variable domain comprising a light chain complementarity determining region 1 (CDR1), a CDR2, and a CDR3. In some embodiments, the antibody or antibody fragment comprises a heavy chain variable domain comprising a heavy chain complementarity determining region 1 (CDR1), a CDR2, and a CDR3 and a light chain variable domain comprising a light chain complementarity determining region 1 (CDR1), a CDR2, and a CDR3.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:6, the HC CDR2 of SEQ ID NO:7, the HC CDR3 of SEQ ID NO:8; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:3, the LC CDR2 of SEQ ID NO:4, the LC CDR3 of SEQ ID NO:5; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:2.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 1.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:22, the HC CDR2 of SEQ ID NO:23, the HC CDR3 of SEQ ID NO:24; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO: 19, the LC CDR2 of SEQ ID NO:20, the LC CDR3 of SEQ ID NO:21; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 18.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 17.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:38, the HC CDR2 of SEQ ID NO:39, the HC CDR3 of SEQ ID NO:40; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:35, the LC CDR2 of SEQ ID NO:36, the LC CDR3 of SEQ ID NO:37; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:34.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:33.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:46, the HC CDR2 of SEQ ID NO:47, the HC CDR3 of SEQ ID NO:48; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:43, the LC CDR2 of SEQ ID NO:44, the LC CDR3 of SEQ ID NO:45; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:42.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:41.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:54, the HC CDR2 of SEQ ID NO:55, the HC CDR3 of SEQ ID NO:56; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:51, the LC CDR2 of SEQ ID NO:52, the LC CDR3 of SEQ ID NO:53; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:50.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:49.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:62, the HC CDR2 of SEQ ID NO:63, the HC CDR3 of SEQ ID NO:64; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:59, the LC CDR2 of SEQ ID NO:60, the LC CDR3 of SEQ ID NO:61; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:58.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:57.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:86, the HC CDR2 of SEQ ID NO:87, the HC CDR3 of SEQ ID NO:88; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:83, the LC CDR2 of SEQ ID NO:84, the LC CDR3 of SEQ ID NO:85; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:82.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:81.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO: 94, the HC CDR2 of SEQ ID NO: 95, the HC CDR3 of SEQ ID NO:96; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:91, the LC CDR2 of SEQ ID NO:92, the LC CDR3 of SEQ ID NO:93; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:90.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:89.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO: 102, the HC CDR2 of SEQ ID NO: 103, the HC CDR3 of SEQ ID NO: 104; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO: 99, the LC CDR2 of SEQ ID NO: 100, the LC CDR3 of SEQ ID NO: 101; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:98.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:97.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:203. In some embodiments, the heavy chain variable domain comprises a sequence of SEQ ID NO:203. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:204. In some embodiments, the light chain variable domain comprises a sequence of SEQ ID NO:204. In some embodiments, the antigen binding domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:202. In some embodiments, the antigen binding domain comprises a sequence of SEQ ID NO:202. In some embodiments, the TFP comprises a sequence having at least about 80% sequence identity to SEQ ID NO:201.
  • the TFP comprises a sequence of SEQ ID NO:201.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:207.
  • the heavy chain variable domain comprises a sequence of SEQ ID NO:207.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:208.
  • the light chain variable domain comprises a sequence of SEQ ID NO:208.
  • the antigen binding domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:206. In some embodiments, the antigen binding domain comprises a sequence of SEQ ID NO:206.
  • the TFP comprises a sequence having at least about 80% sequence identity to SEQ ID NO:205. In some embodiments, the TFP comprises a sequence of SEQ ID NO:205. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:210. In some embodiments, the heavy chain variable domain comprises a sequence of SEQ ID NO:210. In some embodiments, the TFP comprises a sequence having at least about 80% sequence identity to SEQ ID NO:209. In some embodiments, the TFP comprises a sequence of SEQ ID NO:209.
  • a T cell expressing the TFP inhibits tumor growth when expressed in a T cell.
  • a recombinant nucleic acid molecule comprising a nucleic acid encoding an antibody or fragment thereof that specifically binds GPC3.
  • the antibody or antibody fragment is a camelid antibody or binding fragment thereof.
  • the antibody or antibody fragment is a murine, human or humanized antibody or binding fragment thereof.
  • the antibody or antibody fragment is a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain.
  • the antibody or antibody fragment is a single domain antibody (sdAb).
  • the sdAb is a V HH .
  • the antibody or antibody fragment comprises a heavy chain variable domain comprising a heavy chain complementarity determining region 1 (CDR1), a CDR2, and a CDR3. In some embodiments, the antibody or antibody fragment comprises a light chain variable domain comprising a light chain complementarity determining region 1 (CDR1), a CDR2, and a CDR3. In some embodiments, the antibody or antibody fragment comprises a heavy chain variable domain comprising a heavy chain complementarity determining region 1 (CDR1), a CDR2, and a CDR3 and a light chain variable domain comprising a light chain complementarity determining region 1 (CDR1), a CDR2, and a CDR3.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:6, the HC CDR2 of SEQ ID NO:7, the HC CDR3 of SEQ ID NO:8; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:3, the LC CDR2 of SEQ ID NO:4, the LC CDR3 of SEQ ID NO:5; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:2.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 1.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:22, the HC CDR2 of SEQ ID NO:23, the HC CDR3 of SEQ ID NO:24; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO: 19, the LC CDR2 of SEQ ID NO:20, the LC CDR3 of SEQ ID NO:21; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 18.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 17.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:38, the HC CDR2 of SEQ ID NO:39, the HC CDR3 of SEQ ID NO:40; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:35, the LC CDR2 of SEQ ID NO:36, the LC CDR3 of SEQ ID NO:37; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:34.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:33.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:46, the HC CDR2 of SEQ ID NO:47, the HC CDR3 of SEQ ID NO:48; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:43, the LC CDR2 of SEQ ID NO:44, the LC CDR3 of SEQ ID NO:45; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:42.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:41.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:54, the HC CDR2 of SEQ ID NO:55, the HC CDR3 of SEQ ID NO:56; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:51, the LC CDR2 of SEQ ID NO:52, the LC CDR3 of SEQ ID NO:53; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:50.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:49.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:62, the HC CDR2 of SEQ ID NO:63, the HC CDR3 of SEQ ID NO:64; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:59, the LC CDR2 of SEQ ID NO:60, the LC CDR3 of SEQ ID NO:61; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:58.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:57.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:86, the HC CDR2 of SEQ ID NO:87, the HC CDR3 of SEQ ID NO:88; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:83, the LC CDR2 of SEQ ID NO:84, the LC CDR3 of SEQ ID NO:85; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:82.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:81.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO: 94, the HC CDR2 of SEQ ID NO: 95, the HC CDR3 of SEQ ID NO:96; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:91, the LC CDR2 of SEQ ID NO:92, the LC CDR3 of SEQ ID NO:93; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:90.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:89.
  • the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO: 102, the HC CDR2 of SEQ ID NO: 103, the HC CDR3 of SEQ ID NO: 104; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO: 99, the LC CDR2 of SEQ ID NO: 100, the LC CDR3 of SEQ ID NO: 101; or a combination thereof.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:98.
  • the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:97.
  • the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:203. In some embodiments, the heavy chain variable domain comprises a sequence of SEQ ID NO:203. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:204. In some embodiments, the light chain variable domain comprises a sequence of SEQ ID NO:204. In some embodiments, the antigen binding domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:202. In some embodiments, the antigen binding domain comprises a sequence of SEQ ID NO:202. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:207.
  • the heavy chain variable domain comprises a sequence of SEQ ID NO:207. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:208. In some embodiments, the light chain variable domain comprises a sequence of SEQ ID NO:208. In some embodiments, the antigen binding domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:206. In some embodiments, the antigen binding domain comprises a sequence of SEQ ID NO:206. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:210. In some embodiments, the heavy chain variable domain comprises a sequence of SEQ ID NO:210.
  • the recombinant nucleic acid molecule further comprises a sequence encoding a TCR constant domain.
  • the antibody or antibody fragment is operatively linked to the sequence encoding a TCR constant domain, thereby forming a TFP.
  • the TCR constant domain is a TCR alpha constant domain or portion thereof, a TCR beta constant domain or portion thereof, a TCR alpha constant domain or portion thereof and a TCR beta constant domain or portion thereof, a TCR gamma constant domain or portion thereof, a TCR delta constant domain or portion thereof, or a TCR gamma constant domain or portion thereof and a TCR delta constant domain or portion thereof.
  • the recombinant nucleic acid molecule further comprises a leader sequence.
  • the nucleic acid is selected from the group consisting of a DNA and an RNA. In some embodiments, the nucleic acid is a mRNA. In some embodiments, the nucleic acid is a circRNA. In some embodiments, the nucleic acid comprises a nucleotide analog.
  • the nucleotide analog is selected from the group consisting of 2’-O-methyl, 2’-O- methoxyethyl (2’-O-MOE), 2’-O-aminopropyl, 2’-deoxy, 2’-deoxy-2’-fluoro, 2’-O-aminopropyl (2’- O-AP), 2'-O-dimethylaminoethyl (2’-O-DMAOE), 2’-O-dimethylaminopropyl (2’-O-DMAP), 2’-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 morph
  • the recombinant nucleic acid molecule further comprises a promoter.
  • the nucleic acid is an in vitro transcribed nucleic acid.
  • the nucleic acid further comprises a sequence encoding a poly(A) tail.
  • the nucleic acid further comprises a 3’UTR sequence.
  • polypeptide encoded by a recombinant nucleic acid disclosed herein is provided herein.
  • a vector comprising a recombinant nucleic acid molecule encoding a TFP disclosed herein.
  • a vector comprising a recombinant nucleic acid molecule encoding the TFP disclosed herein.
  • the vector further comprises a sequence 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.
  • the vector further comprises a sequence encoding a TCR constant domain.
  • the TCR constant domain is a TCR alpha constant domain or portion thereof, a TCR beta constant domain or portion thereof, a TCR alpha constant domain or portion thereof and a TCR beta constant domain or portion thereof, a TCR gamma constant domain or portion thereof, a TCR delta constant domain or portion thereof, or a TCR gamma constant domain or portion thereof and a TCR delta constant domain or portion thereof.
  • 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.
  • the vector further comprises a promoter.
  • the vector is an in vitro transcribed vector.
  • a nucleic acid sequence in the vector further comprises a poly(A) tail.
  • a nucleic acid sequence in the vector further comprises a 3’UTR.
  • a cell comprising a recombinant nucleic acid molecule disclosed herein, a polypeptide disclosed herein, or a vector disclosed herein.
  • the cell is a T cell.
  • the T cell is a human T cell.
  • the T cell is a CD8+ or CD4+ T cell.
  • the T cell is a human ⁇ T cell.
  • the T cell is a human ⁇ T cell.
  • the cell is a human NKT cell.
  • the cell comprises a functional disruption of an endogenous TCR.
  • the cell is an allogeneic cell.
  • the cell further comprises a heterologous sequence 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.
  • the cell further comprises a heterologous sequence encoding a TCR constant domain.
  • the TCR constant domain is a TCR alpha constant domain or portion thereof, a TCR beta constant domain or portion thereof, a TCR alpha constant domain or portion thereof and a TCR beta constant domain or portion thereof, a TCR gamma constant domain or portion thereof, a TCR delta constant domain or portion thereof, or a TCR gamma constant domain or portion thereof and a TCR delta constant domain or portion thereof.
  • a pharmaceutical composition comprising a cell disclosed herein and a pharmaceutically acceptable carrier.
  • a method of treating a cancer in a subject in need thereof comprising administering to the subj ect an effective amount of a pharmaceutical composition disclosed herein.
  • a method of treating a cancer in a subject in need thereof comprising administering to the subject a pharmaceutical composition comprising (a) a cell disclosed herein; and (b) a pharmaceutically acceptable carrier.
  • the cancer is a cancer associated with elevated expression of GPC3.
  • the disease or the condition is selected from the group consisting of hepatocellular carcinoma (HCC), lung cancer, melanoma, ovarian clear-cell carcinomas, yolk sac tumors, neuroblastoma, hepatoblastoma, Wilms' tumor cells, and gastric cancer.
  • the disease or the condition is selected from the group consisting of kidney cancer, renal cell carcinoma, pancreatic cancer, ovarian cancer, esophageal cancer, nasopharyngeal carcinoma, mesothelioma, glioblastoma, thymic carcinoma, breast cancer, and head and neck cancer.
  • the subject is a human.
  • FIG. 1 is a graph showing binding of the 18 selected clones shown, GC33 and hYP7, to GPC3, a mixture of GPC5 and GPC6 and to a control antigen by ELISA.
  • FIG. 2 is a graph showing binding of the 14 clones shown, GC33, hYP7, a commercial anti- GPC3 antibody, and negative controls to CHO-GPC3 cells and to GPC3-negative CHO cells.
  • FIG. 3 is a schematic diagram showing binning of the binders shown.
  • FIG. 4 is a series of graphs showing expansion of T cells from three donors transduced with mGC33, hYP7, or HN3 TFPs, or untransduced controls.
  • FIGs. 5A-5C are a series of plots showing transduction efficiency and memory phenotype of T cells from three donors transduced with mGC33, hYP7, or HN3 TFPs, or untransduced controls. Each of FIG. 5A, FIG. 5B, and FIG. 5C shows data from an individual donor, respectively.
  • FIG. 6 is a series of plots showing GPC3 expression in HepG2, Hep3B, SNU398, and A549 cell lines.
  • FIG. 7 is a series of graphs showing cytotoxicity of T cells from three donors transduced with mGC33, hYP7, or HN3 TFPs, or non-transduced controls, when co-cultured with HepG2, Hep3B, SNU398, or A549 cell lines at 3: 1 , 1:1 or 1:3 ratios for 24 hours.
  • FIGs. 8A-8D are a series of graphs showing cytokine production when T cells from three donors transduced with mGC33, hYP7, or HN3 TFPs, or non-transduced controls, are co-cultured with HepG2, Hep3B, SNU398, or A549 cell lines at 3:1, 1:1 or 1:3 ratios for 24 hours.
  • Levels of IFN- ⁇ (FIG. 8A), IL-2 (FIG. 8B), TNF- ⁇ (FIG. 8C), and GM-CSF (FIG. 8D) in cell culture supernatants were measured.
  • FIGs. 9A and 9B are a series of graphs showing expansion of T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-18 shown in Table 9, or untransduced controls. Data for T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-8, and untransduced controls is shown in FIG. 9A. Data for T cells transduced with hYP7 TFP, GPC3 scFv constructs 9-18, and untransduced controls is shown in FIG. 9B.
  • FIGs. 10A and 10B are a series of plots showing transduction efficiency and GPC3 binding of T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-18 shown in Table 9, or untransduced controls.
  • Data for T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-8, and untransduced controls is shown in FIG. 10A.
  • Data for T cells transduced with hYP7 TFP, GPC3 scFv constructs 9- 18, and untransduced controls is shown in FIG. 10B.
  • FIGs. 11A-11D are a series of plots showing memory phenotype of T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-12, 15, and 18 shown in Table 9, or untransduced controls.
  • Data for CD4+ T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-8, and untransduced controls is shown in FIG. 11 A.
  • Data for CD4+ T cells transduced with hYP7 TFP, GPC3 scFv constructs 9-12, 15, and 18, and untransduced controls is shown in FIG. 11B.
  • FIG. 11C Data for CD8+ T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-8, and untransduced controls is shown in FIG. 11C.
  • FIG. 11D Data for CD8+ T cells transduced with hYP7 TFP, GPC3 scFv constructs 9-12, 15, and 18, and untransduced controls is shown in FIG. 11D.
  • FIGs. 12A and 12B are a series of plots showing CD69 expression in T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-12, 15, and 18 shown in Table 9, or untransduced controls. Data for T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-8, and untransduced controls is shown in FIG. 12A. Data for T cells transduced with hYP7 TFP, GPC3 scFv constructs 9-12, 15, and 18, and untransduced controls is shown in FIG. 12B.
  • FIGs. 13A and 13B are a series of graphs showing cytotoxicity of T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-12, 15, and 18 shown in Table 9, or untransduced controls, when co- cultured with HepG2, SNU398, or A549 cell lines at 3:1, 1:1 or 1:3 ratios for 24 hours. Cytotoxicity for T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-8, and untransduced controls is shown in FIG. 13A. Cytotoxicity for T cells transduced with hYP7 TFP, GPC3 scFv constructs 9-12, 15, and 18, and untransduced controls is shown in FIG. 13B.
  • FIGs. 14A-14D are a series of graphs showing cytokine production when T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-12, 15, and 18 shown in Table 9, or untransduced controls, are co-cultured with HepG2, SNU398, or A549 cell lines at 3: 1 , 1:1 or 1:3 ratios for 24 hours. Levels of IFN- ⁇ , IL-2, TNF- ⁇ , and GM-CSF were measured. Levels of IFN- ⁇ and IL-2 produced by T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-8, and untransduced controls is shown are FIG. 14A.
  • FIG. 14B Levels of IFN- ⁇ and IL-2 produced by T cells transduced with hYP7 TFP, GPC3 scFv constructs 9-12, 15, and 18, and untransduced controls are shown in FIG. 14B. Levels of TNF- ⁇ and GM-CSF produced by T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-8, and untransduced controls are shown in FIG. 14C. Levels of TNF- ⁇ and GM-CSF produced by T cells transduced with hYP7 TFP, GPC3 scFv constructs 9-12, 15, and 18, and untransduced controls are shown in FIG. 14D.
  • the present disclosure provides a recombinant nucleic acid molecule comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP).
  • TFP can comprise an antigen binding domain that specifically binds GPC3.
  • the TFP can further comprise a TCR subunit comprising: (i) at least a portion of a TCR extracellular domain, (ii) a TCR transmembrane domain, and (iii) a TCR intracellular domain.
  • the TCR subunit and the antigen binding domain that specifically binds GPC3 can be operatively linked.
  • the TFP can functionally interact with an endogenous or exogenous TCR complex when expressed in a T cell.
  • the TCR extracellular domain, the TCR transmembrane domain or the TCR intracellular domain can be derived from a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 gamma chain, a CD3 delta chain, or a CD3 epsilon chain.
  • the TCR extracellular domain, the TCR transmembrane domain or the TCR intracellular domain can be derived from a single TCR chain (e.g., a single TCR alpha chain, a single TCR beta chain, a single TCR gamma chain, a single TCR delta chain, a single CD3 gamma chain, a single CD3 delta chain, or a single CD3 epsilon chain).
  • the TCR intracellular domain can comprise a stimulatory domain from an intracellular signaling domain of CD3 gamma, CD3 delta, or CD3 epsilon.
  • a T cell expressing the TFP can exhibit increased cytotoxicity to a human cell expressing GPC3 compared to a T cell not containing the TFP.
  • the present disclosure provides a TFP molecule or a TCR complex having the TFP molecule incorporated therein.
  • the present disclosure also provides a vector comprising the recombinant nucleic acid molecule encoding the TFP.
  • the present disclosure also provides a cell (e.g., a T cell) comprising the TFP or the recombinant nucleic acid molecule encoding the TFP.
  • Such TFPs when expressed in a cell, can target GPC3 expressing cells (e.g., tumor cells).
  • the present disclosure also provides a pharmaceutical composition comprising a cell comprising the TFP or the recombinant nucleic acid molecule encoding the TFP and a pharmaceutically acceptable carrier.
  • the present disclosure also provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition described herein.
  • the present disclosure provides a method of producing the cell comprising the TFP or the recombinant nucleic acid molecule encoding the TFP.
  • the term “comprise” or variations thereof such as “comprises” or “comprising” are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers.
  • the term “comprising,” is inclusive and does not exclude additional, unrecited integers or method/process steps.
  • compositions and methods comprising or may be replaced with “consisting essentially of’ or “consisting of’.
  • the phrase “consisting essentially of’ is used herein to require the specified integer(s) or steps as well as those which do not materially affect the character or function of the claimed invention.
  • the term “consisting” is used to indicate the presence of the recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) alone.
  • the term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value ⁇ 10%, ⁇ 5%, or ⁇ 1%. In certain embodiments, where applicable, the term “about” indicates the designated value(s) ⁇ one standard deviation of that value(s).
  • antibody refers to a protein, or polypeptide sequences derived from an immunoglobulin molecule, which specifically binds to an antigen.
  • Antibodies can be intact immunoglobulins of polyclonal or monoclonal origin, or fragments thereof and can be derived from natural or from recombinant sources.
  • antigen-binding domain means the portion of an antibody that is capable of specifically binding to an antigen or epitope.
  • an antigen-binding domain is an antigen- binding domain formed by a V H -V L dimer of an antibody.
  • Another example of an antigen-binding domain is an antigen-binding domain formed by diversification of certain loops from the tenth fibronectin type III domain of an Adnectin.
  • antibody fragment refers to at least one portion of an antibody, or recombinant variants thereof, that contains the antigen binding domain, i.e., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen and its defined epitope.
  • antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, single-chain (sc)Fv (“scFv”) antibody fragments, linear antibodies, single domain antibodies (abbreviated “sdAb”) (either V L or V H ), camelid V HH domains, and multi-specific antibodies formed from antibody fragments.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • “Heavy chain variable region” or “V H ” refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.
  • a scFv may have the V L and V H variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise V L -linker-V H or may comprise V H - linker- V L .
  • the portion of the TFP composition of the invention comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb) or heavy chain antibodies HCAb, a single chain antibody (scFv) derived from a murine, humanized or human antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci.
  • sdAb single domain antibody fragment
  • HCAb heavy chain antibodies
  • scFv single chain antibody
  • the antigen binding domain of a TFP composition of the present disclosure comprises an antibody fragment.
  • the TFP comprises an antibody fragment that comprises a scFv or a sdAb.
  • antibody heavy chain refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
  • antibody light chain refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (“k”) and lambda (“ ⁇ ”) light chains refer to the two major antibody light chain isotypes.
  • recombinant antibody refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.
  • antigen or “Ag” refers to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA.
  • any DNA which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full-length nucleotide sequence of a gene.
  • an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample or might be macromolecule besides a polypeptide.
  • a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.
  • GPC3 or “Glypican 3” is a gene is located on human X chromosome and there are three variants, Isoform 1, Isoform 3, and Isoform 4.
  • GPC3 is a member of glypican family, of which members are heparan sulfate proteoglycans that bind to the exocytoplasmic surface of the plasma membrane through a covalent glycosylphosphatidylinositol (GPI) linkage. GPC3 is highly expressed during embryogenesis and organ development. GPC3 and other members of this family regulate the signaling of WNTs, Hedgehogs, fibroblast growth factors, and bone morphogenetic proteins.
  • GPC3 expression level is low and expression is limited to the placenta and endometrium in healthy adult tissues. It is thought that GPC3 suppresses organ growth during organogenesis and loss of function mutation of GPC3 causes an organ overgrowth condition, Simpson-Golabi-Behmel.
  • GPC3 is a known oncofetal antigen with upregulated expression in hepatocellular carcinoma (HCC), lung cancer, melanoma, ovarian clear-cell carcinomas, yolk sac tumors, neuroblastoma, hepatoblastoma, Wilms' tumor cells, gastric cancer, and other cancers. GPC3 may be a prognostic marker for HCC and greater GPC3 expression in tumor cells is associated with worse prognosis.
  • GPC3 knockdown inhibits cell proliferation in HCC cell lines through Y ap and Wnt signaling; on the other hand, overexpression of GPC3 suppresses hepatocyte proliferation and liver regeneration.
  • An exemplary amino acid sequence of GPC3 is listed in SEQ ID NO: 728.
  • the term “anti-tumor effect” refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, decrease in tumor cell proliferation, decrease in tumor cell survival, or amelioration of various physiological symptoms associated with the cancerous condition.
  • An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of tumor in the first place.
  • “Humanized” forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody.
  • a humanized antibody is generally a human antibody (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody).
  • the donor antibody can be any suitable non- human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect.
  • selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody.
  • Humanized antibodies may also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications may be made to further refine antibody function.
  • a “human antibody” is one which possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encoding sequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.
  • affinity refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen or epitope).
  • affinity refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen or epitope).
  • KD dissociation equilibrium constant
  • the kinetic components that contribute to the dissociation equilibrium constant are described in more detail below. Affinity can be measured by common methods known in the art, including those described herein, such as surface plasmon resonance (SPR) technology (e.g., BIACORE®) or biolayer interferometry (e.g., FORTEBIO®).
  • the terms “bind,” “specific binding,” “specifically binds to,” “specific for,” “selectively binds,” and “selective for” a particular antigen (e.g., a polypeptide target) or an epitope on a particular antigen mean binding that is measurably different from a non-specific or non-selective interaction (e.g., with a non-target molecule).
  • Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule.
  • Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. In that case, specific binding is indicated if the binding of the antibody to the target molecule is competitively inhibited by the control molecule.
  • autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • allogeneic refers to any material derived from a different animal of the same species or different patient as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • treating refers to clinical intervention in an attempt to alter the natural course of a disease or condition in a subject in need thereof. Treatment can be performed both for prophylaxis and during the course of clinical pathology. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis.
  • a “therapeutically effective amount” is the amount of a composition or an active component thereof sufficient to provide a beneficial effect or to otherwise reduce a detrimental non- beneficial event to the individual to whom the composition is administered.
  • therapeutically effective dose herein is meant a dose that produces one or more desired or desirable (e.g., beneficial) effects for which it is administered, such administration occurring one or more times over a given period of time. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g. Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).
  • a “T cell receptor (TCR) fusion protein” or “TFP” includes a recombinant polypeptide derived from the various polypeptides comprising the TCR that is generally capable of i) binding to a surface antigen on target cells and ii) interacting with other polypeptide components of the intact TCR complex, typically when co-located in or on the surface of a T cell.
  • a “TFP T cell” is a T cell that has been transduced according to the methods disclosed herein and that expresses a TFP, e.g., incorporated into the natural TCR.
  • the T cell is a CD4+ T cell, a CD8+ T cell, or a CD4+ / CD8+ T cell.
  • the TFP T cell is an NK cell or a regulatory T cell.
  • T cell receptor and “T cell receptor complex” are used interchangeably to refer to a molecule found on the surface of T cells that is, in general, responsible for recognizing antigens.
  • the TCR comprises a heterodimer consisting of a TCR alpha and TCR beta chain in 95% of T cells, whereas 5% of T cells have TCRs consisting of TCR gamma and TCR delta chains.
  • the TCR further comprises one or more of CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ .
  • the TCR comprises CD3 ⁇ .
  • the TCR comprises CD3 ⁇ .
  • the TCR comprises CD3 ⁇ .
  • the TCR comprises CD3 ⁇ .
  • the TCR comprises CD3 ⁇ .
  • the TCR comprises CD3 ⁇ .
  • the constant domain of TCR alpha has a sequence of SEQ ID NO: 711.
  • the constant domain of TCR alpha has an IgC domain having a sequence of SEQ ID NO: 712, a transmembrane domain having a sequence of SEQ ID NO: 713, and an intracellular domain having a sequence of SS.
  • the constant domain of TCR beta has a sequence of SEQ ID NO: 715.
  • the constant domain of TCR beta has an IgC domain having a sequence of SEQ ID NO: 716, a transmembrane domain having a sequence of SEQ ID NO: 717, and an intracellular domain having a sequence of SEQ ID NO: 719.
  • the constant domain of TCR delta has a sequence of SEQ ID NO: 725.
  • the constant domain of TCR delta has an IgC domain having a sequence of SEQ ID NO: 726, a transmembrane domain having a sequence of SEQ ID NO: 727, and an intracellular domain having a sequence of L.
  • the constant domain of TCR gamma has a sequence of SEQ ID NO: 721.
  • the constant domain of TCR gamma has an IgC domain having a sequence of SEQ ID NO: 722, a transmembrane domain having a sequence of SEQ ID NO: 723, and an intracellular domain having a sequence of SEQ ID NO: 724.
  • CD3 epsilon has a sequence of SEQ ID NO: 694.
  • CD3 epsilon has an extracellular domain having a sequence of SEQ ID NO: 696, a transmembrane domain having a sequence of SEQ ID NO: 697, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO: 698.
  • CD3 delta has a sequence of SEQ ID NO: 704.
  • CD3 delta has an extracellular domain having a sequence of SEQ ID NO: 706, a transmembrane domain having a sequence of SEQ ID NO: 707, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO: 708.
  • CD3 gamma has a sequence of SEQ ID NO: 699.
  • CD3 gamma has an extracellular domain having a sequence of SEQ ID NO: 701, a transmembrane domain having a sequence of SEQ ID NO: 702, and an intracellular domain, e.g., an intracellular signaling domain, having a sequence of SEQ ID NO: 703.
  • the term “subject” means a mammalian subject. Exemplary subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, camels, goats, rabbits, and sheep. In certain embodiments, the subject is a human.
  • a “patient” is a subject suffering from or at risk of developing a disease, disorder or condition or otherwise in need of the compositions and methods provided herein. In some embodiments, the subject has cancer, e.g., a cancer described herein.
  • preventing refers to the prevention of the disease or condition, e.g., tumor formation, in the patient. For example, if an individual at risk of developing a tumor or other form of cancer is treated with the methods of the present invention and does not later develop the tumor or other form of cancer, then the disease has been prevented, at least over a period of time, in that individual.
  • the disease or condition e.g., tumor formation
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic or diagnostic products (e.g., kits) that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic or diagnostic products.
  • cytotoxic agent refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction.
  • a “chemotherapeutic agent” refers to a chemical compound useful in the treatment of cancer.
  • Chemotherapeutic agents include “anti-hormonal agents” or “endocrine therapeutics” which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer.
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • cancer cancer
  • tumor is a solid tumor.
  • tumor is a hematologic malignancy.
  • cancer refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.
  • composition refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective in treating a subject, and which contains no additional components which are unacceptably toxic to the subject in the amounts provided in the pharmaceutical composition.
  • modulate and “modulation” refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.
  • increase and activate refer to an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100- fold, or greater in a recited variable.
  • reduce and “inhibit” refer to a decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or greater in a recited variable.
  • agonist refers to the activation of receptor signaling to induce a biological response associated with activation of the receptor.
  • agonist is an entity that binds to and agonizes a receptor.
  • antiagonize refers to the inhibition of receptor signaling to inhibit a biological response associated with activation of the receptor.
  • An “antagonist” is an entity that binds to and antagonizes a receptor.
  • effector T cell includes T helper (i.e., CD4+) cells and cytotoxic (i.e., CD8+) T cells.
  • CD4+ effector T cells contribute to the development of several immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages.
  • CD8+ effector T cells destroy virus-infected cells and tumor cells. See Seder and Ahmed, Nature Immunol., 2003, 4:835-842, incorporated by reference in its entirety, for additional information on effector T cells.
  • regulatory T cell includes cells that regulate immunological tolerance, for example, by suppressing effector T cells.
  • the regulatory T cell has a CD4+CD25+Foxp3+ phenotype.
  • the regulatory T cell has a CD8+CD25+ phenotype.
  • dendritic cell refers to a professional antigen-presenting cell capable of activating a naive T cell and stimulating growth and differentiation of a B cell.
  • the phrase “disease associated with expression of GPC3” includes, but is not limited to, a disease associated with expression of GPC3 or condition associated with cells which express GPC3 including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition.
  • the disease is a cancer.
  • the cancer is hepatocellular carcinoma (HCC), lung cancer, melanoma, ovarian clear-cell carcinomas, yolk sac tumors, neuroblastoma, hepatoblastoma, Wilms' tumor cells, or gastric cancer.
  • the cancer is T cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), an Epstein-Barr virus (EBV) + cancer, or a human papilloma virus (HPV) + cancer.
  • the cancer is kidney cancer, renal cell carcinoma, pancreatic cancer, ovarian cancer, esophageal cancer, nasopharyngeal carcinoma, mesothelioma, glioblastoma, thymic carcinoma, breast cancer, or head and neck cancer.
  • the cancer can be acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia (CLL), chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), glioblastoma, Hodgkin's lymphoma, hypopharynx cancer, kidney cancer, la
  • bladder cancer e.
  • conservative sequence modifications refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing 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 the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR- mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with 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
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • one or more amino acid residues within a TFP of the invention can be replaced with other amino acid residues from the same side chain family and the altered TFP can be tested using the functional assays described herein.
  • 2A refers to a class of peptides, which can induce ribosomal skipping during translation of a protein in a cell. These peptides share a core sequence motif of DxExNPGP, and are found in a wide range of viral families. Exemplary members of 2A include, but are not limited to, P2A, E2A, F2A, and T2A. “T2A” refers to the 2 A derived from thosea asigna virus, and the sequence is EGRGSLLTCGDVEENPGP (SEQ ID NO:742).
  • P2A refers to the 2A derived from porcine teschovirus-1 2A, and the sequence is ATNFSLLKQAGDVEENPGP (SEQ ID NO:743) or GSGATNFSLLKQAGDVEENPG (SEQ ID NO:741).
  • E2A refers to the 2A derived from quine rhinitis A virus, and the sequence is QCTNYALLKLAGDVESNPGP (SEQ ID NO:744).
  • F2A is derived from foot-and-mouth disease virus 18, and the sequence is VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:745).
  • adding the 1 linker “GSG” (Gly-Ser-Gly) on the N-terminal of a 2A peptide helps with efficiency.
  • CD3 or “Cluster of Differentiation 3,” as used herein, refers to a protein complex that is part of the T cell receptor that is involved in activating both the cytotoxic T cell and T helper cells. In some embodiments, it is composed of four distinct chains. For example, in some embodiments, the complex contains a CD3 ⁇ chain, a CD3 ⁇ chain, and two CD3 ⁇ chains in mammals.
  • CD3 ⁇ includes any of the recombinant or naturally-occurring forms of CD3 ⁇ or variants or homologs thereof that have or maintain CD3 ⁇ activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD3 ⁇ .
  • CD3 ⁇ is substantially identical to the protein identified by the UniProt reference number P07766 or a variant or homolog having substantial identity thereto.
  • CD3 ⁇ includes any of the recombinant or naturally-occurring forms of CD3 ⁇ or variants or homologs thereof that have or maintain CD3 ⁇ activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD3 ⁇ .
  • CD3 ⁇ is substantially identical to the protein identified by the UniProt reference number P04234 or a variant or homolog having substantial identity thereto.
  • CD3 ⁇ includes any of the recombinant or naturally-occurring forms of CD3 ⁇ or variants or homologs thereof that have or maintain CD3 ⁇ activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity).
  • the variants or homologs have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across the whole sequence or a portion of the sequence (e.g., a 50, 100, 150 or 200 continuous amino acid portion) compared to a naturally occurring CD3 ⁇ .
  • CD3 ⁇ is substantially identical to the protein identified by the UniProt reference number P09693 or a variant or homolog having substantial identity thereto.
  • stimulation refers to a primary response induced by binding of a stimulatory domain or stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex.
  • a stimulatory domain or stimulatory molecule e.g., a TCR/CD3 complex
  • Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.
  • the term “stimulatory molecule” or “stimulatory domain” refers to a molecule or portion thereof expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway.
  • the primary signal is initiated by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • a primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or “ITAM”.
  • ITAM immunoreceptor tyrosine-based activation motif
  • Examples of an ITAM containing primary cytoplasmic signaling sequence that is of particular use in the invention includes, but is not limited to, those derived from TCRzeta, FcR gamma, FcRbeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”) and CD66d.
  • the term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC’s) on its surface.
  • T cells may recognize these complexes using their T cell receptors (TCRs).
  • TCRs T cell receptors
  • an “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule.
  • the intracellular signaling domain generates a signal that promotes an immune effector function of the TFP containing cell, e.g., a TFP-expressing T cell.
  • immune effector function e.g., in a TFP-expressing T cell
  • examples of immune effector function, e.g., in a TFP-expressing T cell include cytolytic activity and T helper cell activity, including the secretion of cytokines.
  • the intracellular signaling domain can comprise a primary intracellular signaling domain.
  • Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation.
  • the intracellular signaling domain can comprise a costimulatory intracellular domain.
  • Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation.
  • a primary intracellular signaling domain can comprise an ITAM (“immunoreceptor tyrosine- based activation motif’).
  • ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD66d, DAP10 and DAP12.
  • costimulatory molecule refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response.
  • Costimulatory molecules include, but are not limited to, an MHC class 1 molecule, BTLA and a Toll ligand receptor, as well as DAP10, DAP12, CD30, LIGHT, 0X40, CD2, CD27, CD28, CD5, ICAM- 1, LFA-1 (CDl la/CD18), 4-1BB (CD137), IL-15Ra, IL12R, IL18R, IL21R, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • DAP10 DAP12, CD30, LIGHT, 0X40, CD2, CD27, CD28, CD5, ICAM- 1, LFA-1 (CDl la/CD18), 4-1BB (CD137), IL-15Ra, IL12R, IL18R, IL21R,
  • a costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule.
  • a costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, and a ligand that specifically binds with CD83, IL-15Ra, IL12R, IL18R, IL21R, CD27, CD5, ICAM-1, CD7, CD226, Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII, and the like.
  • the intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment thereof.
  • the term "4- 1BB” refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like; and a "4-1BB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or equivalent residues from non- human species, e.g., mouse, rodent, monkey, ape and the like.
  • the term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene, cDNA, or RNA encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • Both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.
  • 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.
  • the phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain one or more introns.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • the term “expression” refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
  • the term “functional disruption” refers to a physical or biochemical change to a specific (e.g., target) nucleic acid (e.g., gene, RNA transcript, of protein encoded thereby) that prevents its normal expression and/or behavior in the cell.
  • a functional disruption refers to a modification of the gene via a gene editing method.
  • a functional disruption prevents expression of a target gene (e.g., an endogenous gene).
  • transfer vector refers to a composition of matter which comprises an isolated nucleic acid and which 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.
  • the term "transfer vector” includes an autonomously replicating plasmid or a virus.
  • the term should also be construed to further include non-plasmid and non- viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like.
  • Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • lentivirus refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided, e.g., in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009).
  • Other examples of lentivirus vectors that may be used in the clinic include but are not limited to, e.g., the LENTIVECTORTM gene delivery technology from Oxford BioMedica, the LENTIMAXTM vector system from Lentigen Technology, and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
  • Circularized RNA refers to a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. CircRNAs are 3-5’ covalently closed RNA rings, and circRNAs do not display Cap or poly(A) tails. CircRNAs lack the free ends necessary for exonuclease-mediated degradation, rendering them resistant to several mechanisms of RNA turnover and granting them extended lifespans as compared to their linear mRNA counterparts. For this reason, circularization may allow for the stabilization of mRNAs that generally suffer from short half-lives and may therefore improve the overall efficacy of mRNA in a variety of applications.
  • CircRNAs are produced by the process of splicing, and circularization occurs using conventional splice sites mostly at annotated exon boundaries (Starke et al., 2015; Szabo et al., 2015).
  • splice sites are used in reverse: downstream splice donors are “backspliced” to upstream splice acceptors (see Jeck and Sharpless, 2014; Barrett and Salzman, 2016; Szabo and Salzman, 2016; Holdt et al., 2018 for review).
  • RNA circularization Three general strategies have been reported so far for RNA circularization: chemical methods using cyanogen bromide or a similar condensing agent, enzymatic methods using RNA or DNA ligases, and ribozymatic methods using self-splicing introns.
  • precursor RNA is synthesized by run-off transcription and then heated in the presence of magnesium ions and GTP to promote circularization. RNA so produced can efficiently transfect different kinds of cells.
  • the template includes sequences for the TFP, CAR, and TCR, or combination thereof.
  • a ribozymatic method utilizing a permuted group I catalytic intron is used. This method is more applicable to long RNA circularization and requires only the addition of GTP and Mg2+ as cofactors.
  • This permuted intron-exon (PIE) splicing strategy consists of fused partial exons flanked by half-intron sequences. In vitro, these constructs undergo the double transesterification reactions characteristic of group I catalytic introns, but because the exons are fused, they are excised as covalently 5’ and 3 ’linked circles.
  • homologous refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules.
  • two nucleic acid molecules such as, two DNA molecules or two RNA molecules
  • polypeptide molecules between two polypeptide molecules.
  • a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position.
  • the homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 10), are matched or homologous, the two sequences are 90% homologous.
  • isolated means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • A refers to adenosine
  • C refers to cytosine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.
  • parenteral administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, intratumoral, or infusion techniques.
  • nucleic acid refers to deoxyribonucleic acids (DNA) or ribonucleic acids (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 as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (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)).
  • peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • a polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.
  • promoter refers to a DNA sequence recognized by the transcription machinery of the cell, or introduced synthetic machinery, that can initiate the specific transcription of a polynucleotide sequence.
  • promoter/regulatory sequence refers to a nucleic acid sequence which can be used for expression of a gene product operably linked to the promoter/regulatory sequence.
  • this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product.
  • the promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.
  • constitutive promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.
  • inducible promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.
  • tissue-specific promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • linker and “flexible polypeptide linker” as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together.
  • the flexible polypeptide linkers include, but are not limited to, (Gly 4 Ser) 4 or (Gly4Ser) 3 .
  • the linkers include multiple repeats of (Gly 2 Ser), (GlySer) or (Gly 3 Ser). Also included within the scope of the invention are linkers described in WO2012/138475 (incorporated herein by reference).
  • a 5’ cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the “front” or 5’ end of a eukaryotic messenger RNA shortly after the start of transcription.
  • the 5’ cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co- transcriptionally, such that each influences the other.
  • RNA polymerase Shortly after the start of transcription, the 5 ’ end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction.
  • the capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.
  • in vitro transcribed RNA refers to RNA, preferably mRNA, which has been synthesized in vitro.
  • the in vitro transcribed RNA is generated from an in vitro transcription vector.
  • the in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.
  • a “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA.
  • the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400.
  • Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.
  • polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
  • mRNA messenger RNA
  • the 3 ’ poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase.
  • the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal.
  • Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm.
  • the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase.
  • the cleavage site is usually characterized by the presence of the base sequence AAUAAA (SEQ ID NO: 689) near the cleavage site.
  • adenosine residues are added to the free 3’ end at the cleavage site.
  • transient refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.
  • signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell.
  • cell surface receptor includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.
  • a “substantially purified” cell refers to a cell that is essentially free of other cell types.
  • a substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state.
  • a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state.
  • the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.
  • terapéutica as used herein means a treatment.
  • a therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.
  • prophylaxis means the prevention of or protective treatment for a disease or disease state.
  • tumor antigen or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” refers to antigens that are common to specific hyperproliferative disorders.
  • the hyperproliferative disorder antigens of the present invention are derived from, cancers including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, gastric cancer, ovarian cancer, NHL, leukemias, uterine cancer, prostate cancer, colon cancer, cervical cancer, bladder cancer, kidney cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, brain cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, endometrial cancer, and stomach cancer.
  • cancers including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, mesothelioma, renal cell carcinoma, stomach cancer, breast cancer, lung cancer, gastric cancer, ovarian cancer, NHL, leukemias, uterine cancer, prostate cancer, colon
  • the disease is a cancer selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia (CLL), chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), glioblastoma, Hodgkin's lymphoma, hypopha
  • the disease is a cancer selected from the group consisting of hepatocellular carcinoma (HCC), lung cancer, melanoma, ovarian clear-cell carcinomas, yolk sac tumors, neuroblastoma, hepatoblastoma, Wilms' tumor cells, and gastric cancer.
  • the disease is a cancer selected from the group consisting of T cell lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), an Epstein-Barr virus (EBV) + cancer, or a human papilloma virus (HPV) + cancer.
  • HCC hepatocellular carcinoma
  • lung cancer melanoma
  • ovarian clear-cell carcinomas yolk sac tumors
  • neuroblastoma hepatoblastoma
  • Wilms' tumor cells and gastric cancer.
  • the disease is a cancer selected from the group consisting of T cell lymph
  • the cancer is kidney cancer, renal cell carcinoma, pancreatic cancer, ovarian cancer, esophageal cancer, nasopharyngeal carcinoma, mesothelioma, glioblastoma, thymic carcinoma, breast cancer, or head and neck cancer.
  • transfected or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cell includes the primary subject cell and its progeny.
  • the term “specifically binds,” refers to an antibody, an antibody fragment or a specific ligand, which recognizes and binds a cognate binding partner (e.g., GPC3) present in a sample, but which does not necessarily and substantially recognize or bind other molecules in the sample.
  • a cognate binding partner e.g., GPC3
  • Ranges throughout this disclosure, various aspects of the present disclosure can 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 present 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, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6.
  • a range such as 95-99% identity includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.
  • T cell receptor (TCR) fusion proteins (TFPs) T cell receptor (TCR) fusion proteins
  • the present disclosure encompasses recombinant nucleic acid constructs encoding TFPs and variants thereof, wherein the TFP comprises a binding domain, e.g., an antigen binding domain, e.g., an antibody or antibody fragment, a ligand, or a ligand binding protein, that binds specifically to GPC3, e.g., human GPC3, wherein the sequence of the binding domain is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • a binding domain e.g., an antigen binding domain, e.g., an antibody or antibody fragment, a ligand, or a ligand binding protein, that binds specifically to GPC3, e.g., human GPC3, wherein the sequence of the binding domain is contiguous with and in the same reading frame as a nucleic acid sequence encoding a TCR subunit or portion thereof.
  • the TFPs provided herein can associate with one or more endogenous (or alternatively, one or more exogenous, or a combination of endogenous and exogenous) TCR subunits in order to form a functional TCR complex.
  • the TFP that specifically binds to GPC3 described herein can be referred to as anti-GPC3 TFP or a GPC3.TFP.
  • the present disclosure also encompasses a binding domain, e.g., an anti-GPC3 antibody or fragment thereof described herein, that is not a component of an anti-GPC3 TFP.
  • the binding domain is comprised solely of an anti-GPC3 antibody described herein and is not fused to any other polypeptide.
  • the anti-GPC3 antibody or fragment thereof described herein is a component of a fusion protein other than a TFP, e.g., a CAR or other fusion protein.
  • the binding domain provided herein can be an antigen binding domain.
  • the antigen binding domain can be an anti-GPC3 binding domain.
  • the binding domain provided herein can be any domain that binds to GPC3 including but not limited to a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single-domain antibody such as a heavy chain variable domain (V H ), a light chain variable domain (V L ) and a variable domain (V HH ) of a camelid derived nanobody, and to an alternative scaffold to function as antigen binding domain, such as a recombinant fibronectin domain, anticalin, DARPIN and the like.
  • antigen binding domain for the TFP can be used as antigen binding domain for the TFP.
  • the antigen binding domain may be derived from the same species in which the TFP will be used in.
  • the antigen binding domain of the TFP can comprise human or humanized residues for the antigen binding domain of an antibody or antibody fragment.
  • the antigen binding domain is a fragment, e.g., a single chain variable fragment (scFv). In some embodiments, the binding domain is any of the scFv binding domains shown in Table 1. In one aspect, the antigen binding domain is a VHH. In one aspect, the antigen binding domain is a Fv, a Fab, a (Fab’)2, or a bi-functional (e.g., bi-specific) hybrid antibody. In one aspect, the antibodies and fragments thereof disclosed herein bind a GPC3 protein with wild-type or enhanced affinity.
  • a humanized antibody or antibody fragment may retain a similar antigenic specificity as the original antibody, e.g., in the present disclosure, the ability to bind human GPC3.
  • a humanized antibody or antibody fragment may have improved affinity and/or specificity of binding to GPC3.
  • the antigen binding domain comprises a humanized or human antibody or an antibody fragment, or a camelid antibody or antibody fragment, or a murine antibody or antibody fragment.
  • the antigen binding domain of the TFP can comprise one or more (e.g., all three) light chain complementary determining region 1 (LC CDR1), light chain complementary determining region 2 (LC CDR2), and light chain complementary determining region 3 (LC CDR3) of a humanized or human anti-GPC3 binding domain described herein, and/or one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-GPC3 binding domain described herein, e.g., a humanized or human anti-GPC3 binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDR
  • the binding domain comprises a humanized or human anti-GPC3 binding domain comprising one or more, e.g., all three, LC CDRs and one or more, e.g., all three, HC CDRs of a scFv binding domain listed in Table 1.
  • the antigen binding domain of the TFP can comprise one or more (e.g., all three) heavy chain complementary determining region 1 (HC CDR1), heavy chain complementary determining region 2 (HC CDR2), and heavy chain complementary determining region 3 (HC CDR3) of a humanized or human anti-GPC3 binding domain described herein.
  • the antigen binding domain of the TFP can comprise one HC CDR1 , HC CDR2, and HC CDR3.
  • the antigen binding domain of the TFP may have two variable heavy chain regions, each comprising a HC CDR1, a HC CDR2 and a HC CDR3 described herein.
  • the antigen binding domain of the TFP can comprise a humanized or human light chain variable region described herein and/or a humanized or human heavy chain variable region described herein. In some embodiments, the binding domain comprises a humanized or human light chain variable region and/or a humanized or human heavy chain variable region of a scFv binding domain listed in Table 1.
  • the antigen binding domain of the TFP can comprise a humanized heavy chain variable region described herein, e.g., at least two humanized or human heavy chain variable regions described herein.
  • the antigen binding domain of the TFP can be a scFv comprising a light chain and a heavy chain.
  • the antigen binding domain of the TFP can be a single domain antibody such as V HH comprising a heavy chain variable region.
  • the antigen binding domain of the TFP is a scFv, and a light chain variable regionis attached to a heavy chain variable region via a linker, e.g., a linker described herein.
  • the antigen binding domain of the TFP includes a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6, preferably 3 or 4.
  • the light chain variable region and heavy chain variable region of a scFv can be, e.g., in any of the following orientations: light chain variable region-linker-heavy chain variable region or heavy chain variable region-linker-light chain variable region.
  • the linker sequence comprises a long linker (LL) sequence.
  • the linker sequence comprises a short linker (SL) sequence.
  • the linker is a Whitlow linker.
  • the antigen binding domain can comprise a LC CDR1 of SEQ ID NO:3, SEQ ID NO: 11, SEQ ID NO: 19, SEQ ID NO:27, SEQ ID NO:35, SEQ ID NO:43, SEQ ID NO:51, SEQ ID NO:59, SEQ ID NO:67, SEQ ID NO:75, SEQ ID NO:83, SEQ ID NO:91, SEQ ID NO:99, SEQ ID NO:107, SEQ ID NO:115, SEQ ID NO:123, SEQ ID NO:131, SEQ ID NO:139, SEQ ID NO:147, SEQ ID NO:155, SEQ ID NO:163, SEQ ID NO:171, SEQ ID NO:179, SEQ ID NO:187, or SEQ ID NO: 195.
  • the antigen binding domain can comprise a LC CDR2 of SEQ ID NO:4, SEQ ID NO: 12, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:36, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:60, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO: 84, SEQ ID NO:92, SEQ ID NO: 100, SEQ ID NO: 108, SEQ ID NO: 116, SEQ ID NO: 124, SEQ ID NO: 132, SEQ ID NO: 140, SEQ ID NO: 148, SEQ ID NO: 156, SEQ ID NO: 164, SEQ ID NO: 172, SEQ ID NO: 180, SEQ ID NO: 188, or SEQ ID NO: 196.
  • the antigen binding domain can comprise a LC CDR3 of SEQ ID NO:5, SEQ ID NO: 13, SEQ ID NO:21, SEQ ID NO:29, SEQ ID NO:37, SEQ ID NO:45, SEQ ID NO:53, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:77, SEQ ID NO:85, SEQ ID NO:93, SEQ ID NO: 101, SEQ ID NO: 109, SEQ ID NO:117, SEQ ID NO:125, SEQ ID NO:133, SEQ ID NO:141, SEQ ID NO:149, SEQ ID NO:157, SEQ ID NO: 165, SEQ ID NO:173, SEQ ID NO:181, SEQ ID NO:189, or SEQ ID NO:197.
  • the antigen binding domain can comprise HC CDR1 of SEQ ID NO:6, SEQ ID NO: 14, SEQ ID NO:22, SEQ ID NO:30, SEQ ID NO:38, SEQ ID NO:46, SEQ ID NO:54, SEQ ID NO:62, SEQ ID NO:70, SEQ ID NO:78, SEQ ID NO:86, SEQ ID NO:94, SEQ ID NO: 102, SEQ ID NO: 110, SEQ ID NO: 118, SEQ ID NO: 126, SEQ ID NO: 134, SEQ ID NO: 142, SEQ ID NO: 150, SEQ ID NO: 158, SEQ ID NO: 166, SEQ ID NO: 174, SEQ ID NO: 182, SEQ ID NO: 190, or SEQ ID NO: 198.
  • the antigen binding domain can comprise HC CDR2 of SEQ ID NO:7, SEQ ID NO: 15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:87, SEQ ID NO:95, SEQ ID NO: 103, SEQ ID NO: 111, SEQ ID NO: 119, SEQ ID NO: 127, SEQ ID NO:135, SEQ ID NO:143, SEQ ID NO:151, SEQ ID NO:159, SEQ ID NO:167, SEQ ID NO: 175, SEQ ID NO: 183, SEQ ID NO: 191, or SEQ ID NO: 199.
  • the antigen binding domain can comprise HC CDR2 of SEQ ID NO:7, SEQ ID NO: 15, SEQ ID NO:23, SEQ ID NO:31, SEQ ID NO:39, SEQ ID NO:47, SEQ ID NO:55, SEQ ID NO:63, SEQ ID NO:71, SEQ ID NO:79, SEQ ID NO:87, SEQ ID NO:95, SEQ ID NO:103, SEQ ID NO:111, SEQ ID NO:119, SEQ ID NO: 127, SEQ ID NO: 135, SEQ ID NO:143, SEQ ID NO:151, SEQ ID NO:159, SEQ ID NO:167, SEQ ID NO:175, SEQ ID NO:183, SEQ ID NO:191, or SEQ ID NO: 199.
  • the antigen binding domain can comprise HC CDR3 of SEQ ID NO: 8, SEQ ID NO: 16, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:40, SEQ ID NO:48, SEQ ID NO:56, SEQ ID NO:64, SEQ ID NO:72, SEQ ID NO:80, SEQ ID NO:88, SEQ ID NO:96, SEQ ID NO: 104, SEQ ID NO: 112, SEQ ID NO: 120, SEQ ID NO: 128, SEQ ID NO: 136, SEQ ID NO: 144, SEQ ID NO: 152, SEQ ID NO: 160, SEQ ID NO: 168, SEQ ID NO: 176, SEQ ID NO: 184, SEQ ID NO: 192, or SEQ ID N0:200.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO:3, a LC CDR2 of SEQ ID NO:4, and a LC CDR3 of SEQ ID NO:5; and/or a HC CDR1 of SEQ ID NO:6, a HC CDR2 of SEQ ID NO:7, and a HC CDR3 of SEQ ID NO:8.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO: 11 , a LC CDR2 of SEQ ID NO: 12, and a LC CDR3 of SEQ ID NO: 13; and/or a HC CDR1 of SEQ ID NO: 14, a HC CDR2 of SEQ ID NO: 15, and a HC CDR3 of SEQ ID NO: 16.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO: 19, a LC CDR2 of SEQ ID NO:20, and a LC CDR3 of SEQ ID NO:21; and/or a HC CDR1 of SEQ ID NO:22, a HC CDR2 of SEQ ID NO:23, and a HC CDR3 of SEQ ID NO:24.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO:27, a LC CDR2 of SEQ ID NO:28, and a LC CDR3 of SEQ ID NO:29; and/or a HC CDR1 of SEQ ID NO:30, aHC CDR2 of SEQ ID NO:31, and a HC CDR3 of SEQ IDNO:32.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO:35, a LC CDR2 of SEQ ID NO:36, and a LC CDR3 of SEQ ID NO:37; and/or a HC CDR1 of SEQ ID NO:38, a HC CDR2 of SEQ ID NO:39, and a HC CDR3 of SEQ ID NO:40.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO:43, a LC CDR2 of SEQ ID NO:44, and a LC CDR3 of SEQ ID NO:45; and/or a HC CDR1 of SEQ ID NO:46, a HC CDR2 of SEQ ID NO:47, and a HC CDR3 of SEQ ID NO:48.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO:51 , a LC CDR2 of SEQ ID NO:52, and a LC CDR3 of SEQ ID NO:53; and/or a HC CDR1 of SEQ ID NO:54, a HC CDR2 of SEQ ID NO:55, and a HC CDR3 of SEQ ID NO:56.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO:59, a LC CDR2 of SEQ ID NO:60, and a LC CDR3 of SEQ ID NO:61; and/or a HC CDR1 of SEQ ID NO:62, a HC CDR2 of SEQ ID NO:63, and a HC CDR3 of SEQ ID NO:64.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO:67, a LC CDR2 of SEQ ID NO:68, and a LC CDR3 of SEQ ID NO:69; and/or a HC CDR1 of SEQ ID NO: 70, a HC CDR2 of SEQ ID NO: 71, and a HC CDR3 of SEQ ID NO: 72.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO:75, a LC CDR2 of SEQ ID NO:76, and a LC CDR3 of SEQ ID NO:77; and/or a HC CDR1 of SEQ ID NO:78, a HC CDR2 of SEQ ID NO:79, and a HC CDR3 of SEQ ID NO:80.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO: 83, a LC CDR2 of SEQ ID NO: 84, and a LC CDR3 of SEQ ID NO: 85; and/or a HC CDR1 of SEQ ID NO:86, a HC CDR2 of SEQ ID NO:87, and a HC CDR3 of SEQ ID NO:88.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO:91, a LC CDR2 of SEQ ID NO:92, and a LC CDR3 of SEQ ID NO:93; and/or a HC CDR1 of SEQ ID NO:94, a HC CDR2 of SEQ ID NO:95, and a HC CDR3 of SEQ ID NO:96.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO:99, a LC CDR2 of SEQ ID NO: 100, and a LC CDR3 of SEQ ID NO : 101 ; and/or a HC CDR 1 of SEQ ID NO : 102, a HC CDR2 of SEQ ID NO : 103 , and a HC CDR3 of SEQ ID NO: 104.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO:107, a LC CDR2 of SEQ ID NO: 108, and a LC CDR3 of SEQ ID NO:109; and/or a HC CDR1 of SEQ ID NO: 110, a HC CDR2 of SEQ ID NO: 111, and a HC CDR3 of SEQ ID NO: 112.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO: 115, a LC CDR2 of SEQ ID NO: 116, and a LC CDR3 of SEQ ID NO: 117; and/or a HC CDR1 of SEQ ID NO:118, a HC CDR2 of SEQ ID NO: 119, and a HC CDR3 of SEQ ID NO: 120.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO: 123, a LC CDR2 of SEQ ID NO: 124, and a LC CDR3 of SEQ ID NO: 125; and/or a HC CDR1 ofSEQ ID NO: 126, a HC CDR2 of SEQ ID NO: 127, and a HC CDR3 of SEQ ID NO: 128.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO:131, a LC CDR2 of SEQ ID NO: 132, and a LC CDR3 of SEQ ID NO:133; and/or a HC CDR1 of SEQ ID NO: 134, a HC CDR2 of SEQ ID NO: 135, and a HC CDR3 of SEQ ID NO: 136.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO: 139, a LC CDR2 of SEQ ID NO: 140, and a LC CDR3 of SEQ ID NO: 141; and/or a HC CDR1 of SEQ ID NO: 142, a HC CDR2 of SEQ ID NO: 143, and a HC CDR3 of SEQ ID NO: 144.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO: 147, a LC CDR2 of SEQ ID NO: 148, and a LC CDR3 of SEQ ID NO: 149; and/or a HC CDR1 of SEQ ID NO: 150, a HC CDR2 of SEQ ID NO: 151 , and a HC CDR3 of SEQ ID NO: 152.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO:155, a LC CDR2 of SEQ ID NO: 156, and a LC CDR3 of SEQ ID NO:157; and/or a HC CDR1 of SEQ ID NO: 158, a HC CDR2 of SEQ ID NO: 159, and a HC CDR3 of SEQ ID NO: 160.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO: 163, a LC CDR2 of SEQ ID NO: 164, and a LC CDR3 of SEQ ID NO: 165; and/or a HC CDR1 of SEQ ID NO: 166, a HC CDR2 of SEQ ID NO: 167, and a HC CDR3 of SEQ ID NO: 168.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO: 171, a LC CDR2 of SEQ ID NO: 172, and a LC CDR3 of SEQ ID NO : 173 ; and/or a HC CDR 1 of SEQ ID NO : 174, a HC CDR2 of SEQ ID NO : 175 , and a HC CDR3 of SEQ ID NO: 176.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO:179, a LC CDR2 of SEQ ID NO: 180, and a LC CDR3 of SEQ ID NO:181; and/or a HC CDR1 of SEQ ID NO: 182, a HC CDR2 of SEQ ID NO: 183, and a HC CDR3 of SEQ ID NO: 184.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO: 187, a LC CDR2 of SEQ ID NO: 188, and a LC CDR3 of SEQ ID NO: 189; and/or a HC CDR1 of SEQ ID NO: 190, a HC CDR2 of SEQ ID NO: 191 , and a HC CDR3 of SEQ ID NO: 192.
  • the antigen binding domain comprises a LC CDR1 of SEQ ID NO: 195, a LC CDR2 of SEQ ID NO: 196, and a LC CDR3 of SEQ ID NO: 197; and/or a HC CDR1 ofSEQ ID NO: 198, a HC CDR2 of SEQ ID NO: 199, and a HC CDR3 of SEQ ID NO:200.
  • the antigen binding domain can comprise a light chain variable region (V L ) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:1, SEQ ID NO:9, SEQ ID NO: 17, SEQ ID NO:25, SEQ ID NO:33, SEQ ID NO:41, SEQ ID NO:49, SEQ ID NO:57, SEQ ID NO:65, SEQ ID NO:73, SEQ ID NO:81, SEQ ID NO:89, SEQ ID NO:97, SEQ ID NO:105, SEQ ID NO:113, SEQ ID NO:121, SEQ ID NO:129, SEQ ID NO:137, SEQ ID NO: 145, SEQ ID NO:153, SEQ ID NO:161, SEQ ID NO:169, SEQ ID NO:177, SEQ ID NO:185, or SEQ ID NO: 193.
  • V L light chain variable region
  • the antigen binding domain can comprise a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:2, SEQ ID NO: 10, SEQ ID NO: 18, SEQ ID NO:26, SEQ ID NO:34, SEQ ID NO:42, SEQ ID NO:50, SEQ ID NO:58, SEQ ID NO:66, SEQ ID NO:74, SEQ ID NO:82, SEQ ID NOVO, SEQ ID NO:98, SEQ ID NO: 106, SEQ ID NO: 114, SEQ ID NO: 122, SEQ ID NO: 130, SEQ ID NO: 138, SEQ ID NO: 146, SEQ ID NO: 154, SEQ ID NO: 162, SEQ ID NO: 170, SEQ ID NO: 178, SEQ ID NO: 186, or SEQ ID NO: 194.
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (V L ) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:1, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:2.
  • V L light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (V L ) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:9, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NOTO.
  • V L light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 17, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 18.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:25, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:26.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:33, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:34.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:41, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:42.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:49, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:50.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:57, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:58.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:65, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:66.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:73, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:74.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:81, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:82.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:89, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NOVO.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:97, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:98.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 105, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 106.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 113, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 114.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 121, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 122.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 129, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 130.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 137, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 138.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 145, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 146.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 153, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 154.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 161, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 162.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 169, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 170.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 177, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 178.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 185, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 186.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a light chain variable region (VL) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 193, and/or a heavy chain variable region (VH) having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 194.
  • VL light chain variable region
  • VH heavy chain variable region
  • the antigen binding domain can comprise a heavy chain variable domain comprising a sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:203.
  • the heavy chain variable domain comprises a sequence of SEQ ID NO:203.
  • the antigen binding domain can comprise the light chain variable domain comprising a sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:204.
  • the light chain variable domain comprises a sequence of SEQ ID NO:204.
  • the antigen binding domain comprises a sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:202. In some cases, the antigen binding domain comprises a sequence of SEQ ID NO:202. In some cases, the TFP comprises a sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:201. In some cases, the TFP comprises a sequence of SEQ ID NO:201.
  • the antigen binding domain can comprise a heavy chain variable domain comprising a sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:207.
  • the heavy chain variable domain comprises a sequence of SEQ ID NO:207.
  • the antigen binding domain can comprise a light chain variable domain comprising a sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:208.
  • the light chain variable domain comprises a sequence of SEQ ID NO:208.
  • the antigen binding domain can comprise a sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:206. In some cases, the antigen binding domain comprises a sequence of SEQ ID NO:206. In some cases, the TFP comprises a sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:205. In some cases, the TFP comprises a sequence of SEQ ID NO:205.
  • the antigen binding domain can comprise a heavy chain variable domain comprising a sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:210.
  • the heavy chain variable domain comprises a sequence of SEQ ID NO:210.
  • the antigen binding domain may be a VHH domain.
  • the TFP can comprise a sequence having at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO:209.
  • the TFP comprises a sequence of SEQ ID NO:209.
  • a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human or fragment thereof.
  • the antigen binding domain is humanized.
  • a humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each ofwhich is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g., European Patent Nos.
  • framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entireties.)
  • a humanized antibody or antibody fragment has one or more amino acid residues remaining in it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain.
  • humanized antibodies or antibody fragments comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions wherein the amino acid residues comprising the framework are derived completely or mostly from human germline.
  • variable domains both light and heavy
  • the choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity.
  • sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences.
  • the human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al. , J. Immunol. , 151 :2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference herein in their entirety).
  • 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 (see, e.g., Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997); Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference herein in their entirety).
  • the framework region e.g., all four framework regions, of the heavy chain variable region are derived from a VH4-4-59 germline sequence.
  • the framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence.
  • the framework region e.g., all four framework regions of the light chain variable region are derived from a VK3-1.25 germline sequence.
  • the framework region can comprise, one, two, three, four or five modifications, e.g., substitutions, e.g., from the amino acid at the corresponding murine sequence.
  • the portion of a TFP composition of the present disclosure that comprises an antibody fragment is humanized with retention of high affinity for the target antigen and other favorable biological properties.
  • humanized antibodies and antibody fragments are prepared by a process of analysis of the 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 permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, e.g., the analysis of residues that influence the ability of the candidate immunoglobulin to bind the target antigen.
  • FR residues can be selected and combined from the recipient and import sequences so that the desired antibody or antibody fragment characteristic, such as increased affinity for the target antigen, is achieved.
  • the CDR residues are directly and most substantially involved in influencing antigen binding.
  • the antigen binding domain (e.g., the anti-GPC3 binding domain) is characterized by particular functional features or properties of an antibody or antibody fragment.
  • the portion of a TFP composition of the present disclosure that comprises an antigen binding domain specifically binds human GPC3.
  • the present disclosure relates to an antigen binding domain comprising an antibody or antibody fragment, wherein the antigen binding domain specifically binds to a GPC3 protein or fragment thereof, wherein the antibody or antibody fragment comprises a variable light chain and/or a variable heavy chain.
  • the antigen binding domain e.g., scFv or a sdAb
  • a target antigen e.g., GPC3
  • V HH domains and scFvs can be prepared according to method known in the art (see, for example, 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 produced by linking V H and V L regions together using flexible polypeptide linkers.
  • the scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The linker length can greatly affect how the variable regions of a scFv fold and interact.
  • the linker sequence comprises a long linker (LL) sequence.
  • the linker sequence comprises a short linker (SL) sequence.
  • a scFv can comprise a linker of about 10, 11, 12, 13, 14, 15 or greater than 15 residues between its V L and V H regions.
  • the linker sequence may comprise any naturally occurring amino acid.
  • the linker sequence comprises amino acids glycine and serine.
  • the linker sequence comprises sets of glycine and serine repeats such as (G 4 S) n , where n is a positive integer equal to or greater than 1.
  • the linker can be (G 4 S) 4 or (G 4 S) 3 . Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.
  • the linker sequence comprises a long linker (LL) sequence.
  • the linker sequence comprises a short linker (SL) sequence.
  • the antigen binding domain described herein can be a camelid antibody or binding fragment thereof.
  • the antigen binding domain can be a murine antibody or binding fragment thereof.
  • the antigen binding domain can be a human or humanized antibody or binding fragment thereof.
  • the antigen binding domain can be a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain.
  • the antigen binding domain is an scFv listed in Table 1.
  • the antigen binding domain can be a single domain antibody (sdAb).
  • the sdAb can be a V HH .
  • an anti-GPC3 binding domain e.g., scFv or sdAb molecules (e.g., soluble scFv or sdAb) can be evaluated in reference to the biophysical properties (e.g., thermal stability) of a conventional control scFv molecule or a full length antibody.
  • the humanized or human scFv has a thermal stability that is greater than about 0.1, about 0.25, about 0.5, about 0.75, about 1, about 1.25, about 1.5, about 1.75, about 2, about 2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10 degrees, about 11 degrees, about 12 degrees, about 13 degrees, about 14 degrees, or about 15 degrees Celsius than a parent scFv in the described assays.
  • the improved thermal stability of the anti-GPC3 binding domain e.g., scFv is subsequently conferred to the entire anti-GPC3 TFP construct, leading to improved therapeutic properties of the anti-GPC3 TFP construct.
  • the thermal stability of the anti-GPC3 binding domain, e.g., scFv can be improved by at least about 2 °C or 3 °C as compared to a conventional antibody.
  • the anti-GPC3 binding domain, e.g., scFv has a 1 °C improved thermal stability as compared to a conventional antibody.
  • the anti-GPC3 binding domain e.g., scFv has a 2 °C improved thermal stability as compared to a conventional antibody.
  • the scFv has a 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 improved thermal stability as compared to a conventional antibody. Comparisons can be made, for example, between the scFv molecules disclosed herein and scFv molecules or Fab fragments of an antibody from which the scFv V H and V L were derived.
  • Thermal stability can be measured using methods known in the art. For example, in one embodiment, TM can be measured. Methods for measuring TM and other methods of determining protein stability are described below.
  • the antigen binding domain such as scFv or sdAb (arising through humanization or mutagenesis of the soluble scFv or sdAb) alter the stability of the antigen binding domain and improve the overall stability of the antigen binding domain and the anti-GPC3 TFP construct. Stability of the humanized antigen binding domain can be compared against the murine antigen binding domain using measurements such as TM, temperature denaturation and temperature aggregation.
  • the antigen binding domain e.g., a scFv or sdAb, can comprise at least one mutation arising from the humanization process such that the mutated antigen binding domain confers improved stability to the anti-GPC3 TFP construct.
  • the anti-GPC3 binding domain e.g., scFv or sdAb, comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mutations arising from the humanization process such that the mutated antigen binding domain confers improved stability to the anti-GPC3 TFP construct.
  • the antigen binding domain of the TFP is engineered by modifying one or more amino acids within one or both variable regions (e.g., V H and/or V L ), for example within one or more CDR regions and/or within one or more framework regions.
  • the TFP composition of the present disclosure comprises an antibody fragment.
  • that antibody fragment comprises a scFv or sdAb.
  • the antibody or antibody fragment of the present disclosure may further be modified such that they vary in amino acid sequence (e.g., from wild-type), but not in desired activity.
  • additional nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues may be made to the protein.
  • a nonessential amino acid residue in a molecule may be replaced with another amino acid residue from the same side chain family.
  • a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members, e.g., a conservative substitution, in which an amino acid residue is replaced with an amino acid residue having a similar side chain, may be made.
  • Families of amino acid residues having similar side chains have been defined in the art, including 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), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid
  • Percent identity in the context of two or more nucleic acids or polypeptide sequences refers to two or more sequences that are the same. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 60% identity, optionally 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%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math.
  • BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent 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, respectively.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • the algorithm parameters for using nucleotide BLAST to determine nucleotide sequence identity may use scoring parameters with a match/mismatch score of 1,-2 and wherein the gap costs are linear.
  • the length of the sequence that initiates an alignment or the word size in a BLAST algorithm may be set to 28 for sequence alignment.
  • the algorithm parameters for using protein BLAST to determine a peptide sequence identity may use scoring parameters with a BLOSUM62 matrix to assign a score for aligning pairs of residues, and determining overall alignment score, wherein the the gap costs may have an existence penalty of 11 and an extension penalty of 1.
  • the matrix adjustment method to compensate for amino acid composition of sequences may be a conditional compositional score matrix adjustment.
  • the length of the sequence that initiates an alignment or the word size in a BLAST algorithm may be set to 6 for sequence alignment.
  • the present disclosure contemplates modifications of a starting antibody or fragment (e.g., scFv or VHH) amino acid sequence that generates functionally equivalent molecules.
  • the V H or V L of a binding domain, e.g., scFv or VHH, comprised in the TFP can be modified to retain at least about 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% identity of the starting V H or V L framework region of the anti-GPC3 binding domain, e.g., scFv or V HH .
  • the present disclosure contemplates modifications of the entire TFP construct, e.g., modifications in one or more amino acid sequences of the various domains of the TFP construct in order to generate functionally equivalent molecules.
  • the TFP construct can be modified to retain at least about 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% identity of the starting TFP construct.
  • the extracellular domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any protein, but in particular a membrane-bound or transmembrane protein. In one aspect the extracellular domain is capable of associating with the transmembrane domain.
  • An extracellular domain of particular use in this present disclosure may include at least the extracellular region(s) of e.g., the alpha, beta, gamma, or delta chain of the T cell receptor, or CD3 epsilon, CD3 gamma, or CD3 delta, or in alternative embodiments, CD28, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD 137, CD 154.
  • the TCR extracellular domain 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 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 amino acid sequences thereof having at least one but not more than 20 modifications.
  • the TCR extracellular domain comprises an extracellular domain or portion thereof of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain.
  • the TCR extracellular domain comprises an IgC domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain.
  • the extracellular domain comprises, or comprises at least 5, 6, 7, 8, 9,
  • the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain.
  • the extracellular domain comprises a sequence encoding the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain having a truncation of 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 acids at the N- or C-terminus or at both the N- and C-terminus.
  • the extracellular domain comprises, or comprises at least 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,
  • the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding an IgC domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma.
  • the extracellular domain comprises a sequence encoding an IgC domain of TCR alpha, TCR beta, TCR delta, or TCR gamma having a truncation of 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 acids at the N- or C-terminus or at both the N- and C-terminus.
  • the extracellular domain comprises, or comprises at least 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,
  • the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular portion of the constant domain TCR alpha, aTCRbeta, aTCR delta, or aTCR gamma.
  • the extracellular domain comprises a sequence encoding the extracellular portion of the constant domain TCR alpha, TCR beta, TCR delta, or TCR gamma having a truncation of 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 acids at the N- or C-terminus or at both the N- and C-terminus.
  • the extracellular domain comprises, or comprises at least 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,
  • the extracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the extracellular domain comprises a sequence encoding the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of 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 acids at the N- or C-terminus or at both the N- and C- terminus.
  • a TFP sequence contains an extracellular domain and a transmembrane domain encoded by a single genomic sequence.
  • a TFP can be designed to comprise a transmembrane domain that is heterologous to the extracellular domain of the TFP.
  • a transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was 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 of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is 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 of the intracellular region).
  • the transmembrane domain can include at least 30, 35, 40, 45, 50, 55, 60 or more amino acids of the extracellular region. In some cases, 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 one that is associated with one of the other domains of the TFP is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex.
  • the transmembrane domain is capable of homodimerization with another TFP on the TFP-T cell surface.
  • the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same TFP.
  • the transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the TFP has bound to a target.
  • the TCR- integrating subunit comprises a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a TCR zeta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, a CD3 delta 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 not more than 20 modifications.
  • the transmembrane domain comprises, or comprises at least 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, or 30 or more consecutive amino acid residues of the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the transmembrane domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the transmembrane domain comprises a sequence encoding the transmembrane domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C-terminus or at both the N- and C-terminus.
  • the transmembrane domain can be attached to the extracellular region of the TFP, e.g., the antigen binding domain of the TFP, via a hinge, e.g., a hinge from a human protein.
  • a hinge e.g., a hinge from a human protein.
  • the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge.
  • a short oligo- or polypeptide linker may form the linkage between the binding element and the TCR extracellular domain of the TFP.
  • a glycine- serine doublet provides a particularly suitable linker.
  • the linker may be at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more in length.
  • the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO: 690) or a sequence (GGGGS)x wherein X is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more.
  • X is 2.
  • X is 4.
  • the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO: 691).
  • the cytoplasmic domain of the TFP can include an intracellular domain.
  • the intracellular domain is from CD3 gamma, CD3 delta, CD3 epsilon, TCR alpha, TCR beta, TCR gamma, or TCR delta.
  • the intracellular domain comprises a signaling domain, if the TFP contains CD3 gamma, delta or epsilon polypeptides; TCR alpha, TCR beta, TCR gamma, and TCR delta subunits generally have short (e.g., 1-19 amino acids in length) intracellular domains and are generally lacking in a signaling domain.
  • An intracellular signaling domain is generally responsible for activation of at least one of the normal effector functions of the immune cell in which the TFP has been introduced. While the intracellular domains of TCR alpha, TCR beta, TCR gamma, and TCR delta do not have signaling domains, the TCR that forms with any TCR fusion protein is able to recruit proteins having a primary intracellular signaling domain described herein, e.g., CD3 zeta, which functions as an intracellular signaling domain.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • intracellular signaling domain refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal.
  • intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.
  • intracellular domains for use in the TFP of the present disclosure include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that are able to act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any recombinant sequence that has the same functional capability.
  • TCR T cell receptor
  • the intracellular domain comprises the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the intracellular domain comprises, or comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 or more consecutive amino acid residues of the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain.
  • the intracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain.
  • the transmembrane domain comprises a sequence encoding the intracellular domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain, or a TCR delta chain having a truncation of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids at the N- or C- terminus or at both the N- and C-terminus.
  • the intracellular domain comprises, or comprises at least 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, or 62 or more consecutive amino acid residues of the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the intracellular domain comprises a sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit.
  • the intracellular domain comprises a sequence encoding the intracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit having a truncation of 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 acids at the N- or C-terminus or at both the N- and C-terminus.
  • naive T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic domain, e.g., a costimulatory domain).
  • a primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way.
  • Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (IT AMs).
  • Examples of IT AMs containing primary intracellular signaling domains that are of particular use in the present disclosure include those of CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
  • a TFP of the present disclosure comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3 epsilon, CD3 delta, or CD3 gamma.
  • a primary signaling domain comprises a modified IT AM domain, e.g., a mutated IT AM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain.
  • a primary signaling domain comprises a modified ITAM-containing primary intracellular signaling domain, e.g., an optimized and/or truncated ITAM-containing primary intracellular signaling domain.
  • a primary signaling domain comprises one, two, three, four or more ITAM motifs.
  • the intracellular signaling domain of the TFP can comprise a CD3 signaling domain, e.g., CD3 epsilon, CD3 delta, CD3 gamma, or CD3 zeta, by itself or it can be combined with any other desired intracellular signaling domain(s) useful in the context of a TFP of the present disclosure.
  • the intracellular signaling domain of the TFP can comprise a CD3 epsilon chain portion and a costimulatory signaling domain.
  • the costimulatory signaling domain refers to a portion of the TFP comprising the intracellular domain of a costimulatory molecule.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands that is required for an efficient response of lymphocytes to an antigen.
  • examples of such molecules include CD27, CD28, 4- IBB (CD 137), 0X40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like.
  • CD27 costimulation has been demonstrated to enhance expansion, effector function, and survival of human TFP-T cells in vitro and augments human T cell persistence and antitumor activity in vivo (Song et al., Blood. 2012; 119(3):696-706).
  • the intracellular signaling sequences within the cytoplasmic portion of the TFP of the present disclosure may be linked to each other in a random or specified order.
  • a short oligo- or polypeptide linker for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences.
  • a glycine-serine doublet can be used as a suitable linker.
  • a single amino acid e.g., an alanine, a glycine, can be used as a suitable linker.
  • the TFP -expressing cell described herein can further comprise a second TFP, e.g., a second TFP that includes a different antigen binding domain, e.g., to the same target (e.g., GPC3) or a different target (e.g., MSLN, CD19, or MUC16).
  • a second TFP e.g., a second TFP that includes a different antigen binding domain, e.g., to the same target (e.g., GPC3) or a different target (e.g., MSLN, CD19, or MUC16).
  • the antigen binding domains of the different TFPs can be such that the antigen binding domains do not interact with one another.
  • a cell expressing a first and second TFP can have an antigen binding domain of the first TFP, e.g., as a fragment, e.g., a scFv, that does not form an association with the antigen binding domain of the second TFP, e.g., the antigen binding domain of the second TFP is a V HH .
  • the TFP-expressing cell described herein can further express another agent, e.g., an agent which enhances the activity of a modified T cell.
  • the agent can be an agent which inhibits an inhibitory molecule.
  • Inhibitory molecules e.g., PD1
  • PD1 can, in some embodiments, decrease the ability of a modified T cell to mount an immune effector response.
  • inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 and TGFR beta.
  • the agent which inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
  • the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD1, LAG3, CTLA4, CD 160, BTLA, LAIR1, TIM3, 2B4 and TIGIT, or a fragment of any of these (e.g., at least a portion of an extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 4-1BB, CD27 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein).
  • an inhibitory molecule such as PD1, LAG3, CTLA4, CD 160, BTLA, LAIR1, TIM3, 2B4 and TIGIT
  • a fragment of any of these e.g., at least a portion of an extracellular domain of any of these
  • a second polypeptide which is an intracellular signal
  • the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g., at least a portion of an extracellular domain of PD1), and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein).
  • PD1 is an inhibitory member of the CD28 family of receptors that 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 Two ligands for PD1, PD-L1 and PD-L2, 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). Immune suppression can be reversed by inhibiting the local interaction of PD 1 with PD-L 1.
  • the agent comprises the extracellular domain (ECD) of an inhibitory molecule, e.g., Programmed Death 1 (PD1) can be fused to a transmembrane domain and optionally an intracellular signaling domain such as 4 IBB and CD3 zeta (also referred to herein as a PD1 TFP).
  • the PD1 TFP when used in combinations with an anti-GPC3 TFP described herein, improves the persistence of the T cell.
  • the TFP is a PD 1 TFP comprising the extracellular domain of PD-1.
  • TFPs containing an antibody or antibody fragment such as a scFv that specifically binds to the Programmed Death-Ligand 1 (PD-L1) or Programmed Death-Ligand 2 (PD-L2).
  • the present disclosure provides a population of TFP-expressing T cells, e.g., TFP-T cells.
  • the population of TFP-expressing T cells comprises a mixture of cells expressing different TFPs.
  • the population of TFP-T cells can include a first cell expressing a TFP having an anti-GPC3 binding domain described herein, and a second cell expressing a TFP having a binding domain specifically targeting a different antigen, e.g., a binding domain described herein that differs from the anti-GPC3 binding domain in the TFP expressed by the first cell.
  • the population of TFP-expressing cells can include a first cell expressing a TFP that includes a first binding domain binding domain, e.g., as described herein, and a second cell expressing a TFP that includes an antigen binding domain to a target other than the binding domain of the first cell (e.g., another tumor-associated antigen).
  • the present disclosure provides a population of cells wherein at least one cell in the population expresses a TFP having a domain described herein, and a second cell expressing another agent, e.g., an agent which enhances the activity of a modified T cell.
  • the agent can be an agent which inhibits an inhibitory molecule.
  • Inhibitory molecules e.g., can, in some embodiments, decrease the ability of a modified T cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, PD-L2, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 and TGFR beta.
  • the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein.
  • the agent is a cytokine.
  • the cytokine is IL- 15.
  • IL- 15 increases the persistence of the T cells described herein.
  • the recombinant nucleic acid further comprises a leader sequence. In some instances, the recombinant nucleic acid further comprises a promoter sequence. In some instances, the recombinant nucleic acid further comprises a sequence encoding a poly(A) tail. In some instances, the recombinant nucleic acid further comprises a 3’UTR sequence. In some instances, the nucleic acid is an isolated nucleic acid or a non-naturally occurring nucleic acid. Non-naturally occurring nucleic acids are well known to those of skill in the art. In some instances, the nucleic acid is an in vitro transcribed nucleic acid.
  • RNA encoding TFPs include methods for producing in vitro transcribed RNA encoding TFPs.
  • the present disclosure also includes a TFP encoding RNA construct that can be directly transfected into a cell.
  • a method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3’ and 5’ untranslated sequence (“UTR”), a 5’ cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length.
  • RNA so produced can efficiently transfect different kinds of cells.
  • the template includes sequences for the TFP.
  • the anti-GPC3 TFP is encoded by a messenger RNA (mRNA).
  • mRNA messenger RNA
  • the mRNA encoding the anti-GPC3 TFP is introduced into a T cell for production of a TFP-T cell.
  • the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection.
  • the RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)- generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase.
  • PCR polymerase chain reaction
  • the source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.
  • the desired template for in vitro transcription is a TFP of the present disclosure.
  • the DNA to be used for PCR contains an open reading frame.
  • the DNA can be from a naturally occurring DNA sequence from the genome of an organism.
  • the nucleic acid can include some or all of the 5’ and/or 3’ untranslated regions (UTRs).
  • the nucleic acid can include exons and introns.
  • the DNA to be used for PCR is a human nucleic acid sequence.
  • the DNA to be used for PCR is a human nucleic acid sequence including the 5’ and 3’ UTRs.
  • the DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism.
  • An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein.
  • the portions of DNA that are ligated together can be from a single organism or from more than one organism.
  • PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection.
  • Methods for performing PCR are well known in the art.
  • Primers for use in PCR are designed to have regions that are substantially complementary to regions of the DNA to be used as a template for the PCR.
  • “Substantially complementary,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementary, or one or more bases are non-complementary, or mismatched. Substantially complementary sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR.
  • the primers can be designed to be substantially complementary to any portion of the DNA template.
  • the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5’ and 3’ UTRs.
  • the primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest.
  • the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5’ and 3’ UTRs.
  • Primers useful for PCR can be generated by synthetic methods that are well known in the art.
  • “Forward primers” are primers that contain a region of nucleotides that are substantially complementary to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified.
  • Upstream is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand.
  • reverse primers are primers that contain a region of nucleotides that are substantially complementary to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified.
  • Downstream is used herein to refer to a location 3’ to the DNA sequence to be amplified relative to the coding strand.
  • Any DNA polymerase useful for PCR can be used in the methods disclosed herein.
  • the reagents and polymerase are commercially available from a number of sources.
  • the RNA preferably has 5’ and 3’ UTRs.
  • the 5’ UTR is between one and 3,000 nucleotides in length.
  • the length of 5’ and 3’ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5’ and 3’ UTR lengths that can be used to achieve optimal translation efficiency following transfection of the transcribed RNA.
  • the 5 ’ and 3 ’ UTRs can be the naturally occurring, endogenous 5 ’ and 3 ’ UTRs for the nucleic acid of interest.
  • UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template.
  • the use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3 ’UTR sequences can decrease the stability of mRNA. Therefore, 3 ’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.
  • the 5’ UTR can contain the Kozak sequence of the endogenous nucleic acid.
  • a consensus Kozak sequence can be redesigned by adding the 5’ UTR sequence.
  • Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation.
  • the 5’ UTR can be 5 ’UTR of an RNA virus whose RNA genome is stable in cells.
  • various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.
  • a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
  • the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
  • the promoter is a T7 polymerase promoter, as described elsewhere herein.
  • Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.
  • the mRNA has both a cap on the 5’ end and a 3’ poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell.
  • RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells.
  • the transcription of plasmid DNA linearized at the end of the 3 ’ UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.
  • phage T7 RNA polymerase can extend the 3’ end of the transcript beyond the last base of the template (Schenbom and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)).
  • the polyA/T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100 T tail (size can be 50-5000 Ts), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination.
  • Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is between 100 and 5000 adenosines.
  • Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP).
  • E-PAP E. coli polyA polymerase
  • increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA.
  • the attachment of different chemical groups to the 3’ end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds.
  • ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.
  • RNAs produced by the methods disclosed herein include a 5’ cap.
  • the 5’ cap is provided 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)).
  • RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence.
  • IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.
  • RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8): 861 -70 (2001)).
  • the recombinant nucleic acid molecule described herein can further comprise a sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain, a TCR alpha constant domain and a TCR beta constant domain, a TCR gamma constant domain, a TCR delta constant domain, or a TCR gamma constant domain and a TCR delta constant domain.
  • the TCR subunit and the antigen binding domain e.g., anti- GPC3 antigen binding domain or antibody domain
  • the TFP can functionally incorporate into a TCR complex (e.g., an endogenous TCR complex) when expressed in a T cell.
  • the sequence encoding the TFP and the sequence encoding the TCR constant domain can be contained within a same nucleic acid molecule.
  • the sequence encoding the TFP and the sequence encoding the TCR constant domain can be contained within different nucleic acid molecules.
  • the sequence can further encode a cleavage site (e.g. , a protease cleavage site) between the encoded TFP and the TCR constant domain.
  • the cleavage site can be a protease cleavage site.
  • the cleavage site can be a self-cleaving peptide such as a T2A, P2A, E2A or F2A cleavage site.
  • the constant domain can comprise a constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant domain can comprise a full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant domain can comprise a fragment (e.g., functional fragment) of the full-length constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant domain can comprise at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of the constant domain of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the sequence encoding the TCR constant domain can further encode the transmembrane domain and/or intracellular region of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the sequence encoding the TCR constant domain can encode a full-length constant region of a TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the constant region of a TCR chain can 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 the intracellular region of the TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain.
  • the TCR alpha chain, a TCR beta chain, a TCR gamma chain or a TCR delta chain described herein can be derived from various species.
  • the TCR chain can be a murine or human TCR chain.
  • the constant domain can comprise a constant domain of a murine or human TCR alpha chain, TCR beta chain, TCR gamma chain or TCR delta chain.
  • the constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.
  • the constant domain can comprise a sequence or fragment thereof of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.
  • the constant domain can 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:711, SEQ ID NO:715, SEQ ID NO :211, SEQ ID NO: 721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:711, SEQ ID NO:715, SEQ ID NO:211, SEQ ID NO:721, SEQ ID NO:725, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, or SEQ ID NO:215.
  • the constant domain described herein can be a human TCR alpha constant domain.
  • the human TCR alpha constant domain can comprise a sequence of SEQ ID NO:711.
  • the human TCR alpha constant domain can comprise truncations, additions, or substitutions of a sequence of SEQ ID NO:711.
  • the human TCR alpha constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:711.
  • the human TCR alpha constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:711.
  • the human TCR alpha constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:711.
  • the human TCR alpha constant domain can comprise a sequence or fragment thereof of SEQ ID NO:711.
  • the constant domain can 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:711.
  • the human TCR alpha constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:711.
  • the human TCR alpha constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:711.
  • the constant domain described herein can be a human TCR beta constant domain.
  • the human TCR beta constant domain can comprise a sequence of SEQ ID NO:715 or SEQ ID NO:211.
  • the human TCR beta constant domain can comprise truncations, additions, or substitutions of a sequence of SEQ ID NO:715 or SEQ ID NO:211.
  • the human TCR beta constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:715 or SEQ ID NO:211.
  • the human TCR beta constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:715 or SEQ ID NO:211.
  • the human TCR beta constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:715 or SEQ ID NO:211.
  • the human TCR beta constant domain can comprise a sequence or fragment thereof of SEQ ID NO:715 or SEQ ID NO:211.
  • the constant domain can 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:715 or SEQ ID NO:211.
  • the human TCR beta constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:715 or SEQ ID NO:211.
  • the human TCR beta constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:715 or SEQ ID NO:211.
  • the constant domain described herein can be a human TCR gamma constant domain.
  • the human TCR gamma constant domain can comprise a sequence of SEQ ID NO:721.
  • the human TCR gamma constant domain can comprise truncations, additions, or substitutions of a sequence of SEQ ID NO:721.
  • the human TCR gamma constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:721.
  • the human TCR gamma constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:721.
  • the human TCR gamma constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:721.
  • the human TCR gamma constant domain can comprise a sequence or fragment thereof of SEQ ID NO:721.
  • the constant domain can 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:721.
  • the human TCR gamma constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:721.
  • the human TCR gamma constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:721.
  • the constant domain described herein can be a human TCR delta constant domain.
  • the human TCR delta constant domain can comprise a sequence of SEQ ID NO:725.
  • the human TCR delta constant domain can comprise truncations, additions, or substitutions of a sequence of SEQ ID NO:725.
  • the human TCR delta constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:725.
  • the human TCR delta constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:725.
  • the human TCR delta constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:725.
  • the human TCR delta constant domain can comprise a sequence or fragment thereof of SEQ ID NO:725.
  • the constant domain can 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:725.
  • the human TCR delta constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:725.
  • the human TCR delta constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:725.
  • the constant domain described herein can be a murine TCR alpha constant domain.
  • the murine TCR alpha constant domain can comprise a sequence of SEQ ID NO:212 or SEQ ID NO:213.
  • the murine TCR alpha constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:212 or SEQ ID NO:213.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:212 or SEQ ID NO:213.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:212 or SEQ ID NO:213.
  • the constant domain can comprise a sequence or fragment thereof of SEQ ID NO:212 or SEQ ID NO:213.
  • the constant domain can 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:212 or SEQ ID NO:213.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:212 or SEQ ID NO:213.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:212 or SEQ ID NO:213.
  • the constant domain described herein can be a murine TCR beta constant domain.
  • the murine TCR beta constant domain can comprise a sequence of SEQ ID NO:214 or SEQ ID NO:215.
  • the murine TCR beta constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein.
  • the constant domain can comprise a truncated version of a constant domain described herein having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid residues of SEQ ID NO:214 or SEQ ID NO:215.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more additional amino acid residues of SEQ ID NO:214 or SEQ ID NO:215.
  • the constant domain can comprise a sequence having at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150 or more amino acid substitutions of SEQ ID NO:214 or SEQ ID NO:215.
  • the constant domain can comprise a sequence or fragment thereof of SEQ ID NO:214 or SEQ ID NO:215.
  • the constant domain can 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:214 or SEQ ID NO:215.
  • the constant domain can comprise at most 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 modification, mutations or deletions of the sequence of SEQ ID NO:214 or SEQ ID NO:215.
  • the constant domain can comprise a sequence having a sequence identity of at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% to the sequence of SEQ ID NO:214 or SEQ ID NO:215.
  • the modified T cells disclosed herein are engineered using a gene editing technique such as clustered regularly interspaced short palindromic repeats (CRISPR®, see, e.g., U.S. Patent No. 8,697,359), transcription activator-like effector (TALE) nucleases (TALENs, see, e.g., U.S. Patent No. 9,393,257), meganucleases (endodeoxyribonucleases having large recognition sites comprising double-stranded DNA sequences of 12 to 40 base pairs), zinc finger nuclease (ZFN, see, e.g., Umov et al., Nat. Rev.
  • CRISPR® clustered regularly interspaced short palindromic repeats
  • TALE transcription activator-like effector
  • TALENs transcription activator-like effector
  • meganucleases endodeoxyribonucleases having large recognition sites comprising double-stranded DNA sequences of 12 to 40 base pairs
  • a chimeric construct may be engineered to combine desirable characteristics of each subunit, such as conformation or signaling capabilities. See also Sander & Joung, Nat. Biotech. (2014) v32, 347-55; and June et al., 2009 Nature Reviews Immunol. 9.10: 704-716, each incorporated herein by reference.
  • one or more of the extracellular domain, the transmembrane domain, or the cytoplasmic domain of a TFP subunit are engineered to have aspects of more than one natural TCR subunit domain (i.e., are chimeric).
  • gene editing techniques are employed to disrupt an endogenous TCR gene.
  • mentioned endogenous TCR gene encodes a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain.
  • mentioned endogenous TCR gene encodes a TCR gamma chain, a TCR delta chain, or a TCR gamma chain and a TCR delta chain.
  • gene editing techniques pave the way for multiplex genomic editing, which allows simultaneous disruption of multiple genomic loci in endogenous TCR gene.
  • multiplex genomic editing techniques are applied to generate gene-disrupted T cells that are deficient in the expression of endogenous TCR, and/or B2M, and/or human leukocyte antigens (HLAs), and/or programmed cell death protein 1 (PD1), and/or other genes.
  • HLAs human leukocyte antigens
  • PD1 programmed cell death protein 1
  • nickase nucleases generate single- stranded DNA breaks (SSB).
  • DSBs may be repaired by single strand DNA incorporation (ssDI) or single strand template repair (ssTR), an event that introduces the homologous sequence from a donor DNA.
  • ssDI single strand DNA incorporation
  • ssTR single strand template repair
  • Genome DNA can be performed using site-specific, rare-cutting endonucleases that are engineered to recognize DNA sequences in the locus of interest.
  • Methods for producing engineered, site-specific endonucleases are known in the art.
  • ZFNs zinc-fmger nucleases
  • ZFNs are chimeric proteins comprising a zinc finger DNA-binding domain fused to the nuclease domain of the Fokl restriction enzyme.
  • the zinc finger domain can be redesigned through rational or experimental means to produce a protein that binds to a pre-determined DNA sequence - 18 basepairs in length.
  • TAL-effector nucleases can be generated to cleave specific sites in genomic DNA.
  • a TALEN comprises an engineered, site-specific DNA-binding domain fused to the Fokl nuclease domain (reviewed in Mak et al. (2013), Curr Opin Struct Biol. 23:93-9).
  • the DNA binding domain comprises a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA basepair.
  • Compact TALENs have an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley et al. (2013), Nat Commun. 4: 1762).
  • a Compact TALEN comprises an engineered, site-specific TAL-effector DNA-binding domain fused to the nuclease domain from the LTevI homing endonuclease. Unlike Fokl, I-TevI does not need to dimerize to produce a double-strand DNA break so a Compact TALEN is functional as a monomer.
  • Engineered endonucleases based on the CRISPR/Cas9 system are also known in the art (Ran et al. (2013), NatProtoc. 8:2281-2308; Mali et al. (2013), Nat Methods 10:957-63).
  • the CRISPR gene- editing technology is composed of an endonuclease protein whose DNA-targeting specificity and cutting activity can be programmed by a short guide RNA or a duplex crRNA/TracrRNA.
  • a CRISPR endonuclease comprises two components: (1) a caspase effector nuclease, typically microbial Cas9; and (2) a short "guide RNA” or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome.
  • a caspase effector nuclease typically microbial Cas9
  • a short "guide RNA” or a RNA duplex comprising a 18 to 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome.
  • CRISPR systems There are two classes of CRISPR systems known in the art (Adli (2016) Nat. Commun. 9:1911), each containing multiple CRISPR types. Class 1 contains type I and type III CRISPR systems that are commonly found in Archaea. And, Class II contains type II, IV, V, and VI CRISPR systems. Although the most widely used CRISPR/Cas system is the type II CRISPR-Cas9 system, CRISPR/Cas systems have been repurposed by researchers for genome editing. More than 10 different CRISPR/Cas proteins have been remodeled within last few years (Adli (2016) Ata. Commun. 9:1911). Among these, such as Casl2a (Cpfl) proteins from Acta- aminococcus sp (AsCpfl) and Lachnospiraceae bacterium (LbCpfl), are particularly interesting.
  • Cpfl Casl2a
  • AsCpfl Acta- aminococcus sp
  • Homing endonucleases are a group of naturally occurring nucleases that recognize 15-40 base-pair cleavage sites commonly found in the genomes of plants and fungi. They are frequently associated with parasitic 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 producing a double -stranded break in the chromosome, which recruits the cellular DNA-repair machinery (Stoddard (2006), Q. Rev. Biophys. 38: 49-95).
  • meganucleases are monomeric proteins with innate nuclease activity that are derived from bacterial homing endonucleases and engineered for a unique target site (Gersbach (2016), Molecular Therapy. 24: 430-446).
  • meganuclease is engineered I-Crel homing endonuclease. In other embodiments, meganuclease is engineered I-Scel homing endonuclease.
  • chimeric proteins comprising fusions of meganucleases, ZFNs, and TALENs have been engineered to generate novel monomeric enzymes that take advantage of the binding affinity of ZFNs and TALENs and the cleavage specificity of meganucleases (Gersbach (2016), Molecular Therapy. 24: 430-446).
  • a megaTAL is a single chimeric protein, which is the combination of the easy-to-tailor DNA binding domains from TALENs with the high cleavage efficiency of meganucleases.
  • nucleases In order to perform the gene editing technique, the nucleases, and in the case of the CRISPR/ Cas9 system, a gRNA, must be efficiently delivered to the cells of interest. Delivery methods such as physical, chemical, and viral methods are also know in the art (Mali (2013). Indian J. Hum. Genet. 19: 3-8.). In some instances, physical delivery methods can be selected from the methods but not limited to electroporation, microinjection, or use of ballistic particles. On the other hand, chemical delivery methods require use of complex molecules such calcium phosphate, lipid, or protein. In some embodiments, viral delivery methods are applied for gene editing techniques using viruses such as but not limited to adenovirus, lentivirus, and retrovirus.
  • viruses such as but not limited to adenovirus, lentivirus, and retrovirus.
  • the instant invention provides vectors comprising the recombinant nucleic acid(s) encoding the TFP and/or additional molecules of interest (e.g., a protein or proteins to be secreted by the TFP T cell).
  • the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, an adeno-associated viral vector (AAV), a Rous sarcoma viral (RSV) vector, or a retrovirus vector.
  • the vector is an AAV6 vector.
  • the vector further comprises a promoter.
  • the vector is an in vitro transcribed vector.
  • nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques.
  • the gene of interest can be produced synthetically, rather than cloned.
  • the present disclosure also provides vectors in which a DNA of the present disclosure is inserted.
  • Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long- term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells.
  • Lentiviral vectors have the added advantage over vectors derived from onco- retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • the vector comprising the nucleic acid encoding the desired TFP of the present disclosure is an adenoviral vector (A5/35).
  • the expression of nucleic acids encoding TFPs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See, e.g., June et al., 2009 Nature Reviews Immunology 9.10: 704-716, which is incorporated herein by reference.
  • the TFP of the present invention may be used in multicistronic vectors or vectors expressing several proteins in the same transcriptional unit.
  • Such vectors may use internal ribosomal entry sites (IRES). Since IRES are not functional in all hosts and do not allow for the stoichiometric expression of multiple protein, self-cleaving peptides may be used instead.
  • IRES internal ribosomal entry sites
  • self-cleaving peptides may be used instead.
  • several viral peptides are cleaved during translation and allow for the expression of multiple proteins form a single transcriptional unit.
  • Such peptides include 2A-peptides, or 2A-like sequences, from members of the Picomaviridae virus family. See for example Szymczak et al., 2004, Nature Biotechnology; 22:589- 594.
  • the recombinant nucleic acid described herein encodes the TFP in frame with the agent, with the two sequences separated by a self-clea
  • the expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art (see, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, each of which is incorporated by reference herein in their entireties).
  • the present disclosure provides a gene therapy vector.
  • the nucleic acid can be cloned into a number of types of vectors.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals.
  • Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • lentivirus vectors are used.
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation.
  • these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well.
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • individual elements can function either cooperatively or independently to activate transcription.
  • a promoter that is capable of expressing a TFP transgene in a mammalian T cell is the EFla promoter.
  • the native EFla promoter drives expression of the alpha subunit of the elongation factor- 1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome.
  • the EFla promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving TFP expression from transgenes cloned into a lentiviral vector (see, e.g., Milone et al., Mol. Ther. 17(8): 1453-1464 (2009)).
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor- la promoter, the hemoglobin promoter, and the creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • inducible promoters are also contemplated as part of the present disclosure.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline-regulated promoter.
  • the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. 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 and the like.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.
  • the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter.
  • Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • a host cell e.g., mammalian, bacterial, yeast, or insect cell
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.
  • Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like (see, e.g., U.S. Pat. Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).
  • Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.
  • an exemplary delivery vehicle is a liposome.
  • lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • Lipids suitable for use can be obtained from commercial sources.
  • DMPC dimyristyl phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 °C.
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine-nucleic acid complexes are also contemplated.
  • assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and western blots) or by assays described herein to identify agents falling within the scope of the present disclosure.
  • moleukin assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and western blots) or by assays described herein to identify agents falling within the scope of the present disclosure.
  • the present disclosure further provides a vector comprising a TFP encoding nucleic acid molecule.
  • a TFP vector can be directly transduced into a cell, e.g., a T cell.
  • the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs.
  • the vector is capable of expressing the TFP construct in mammalian T cells.
  • the mammalian T cell is a human T cell.
  • TFP T cells are transduced with an RNA molecule.
  • the RNA is circular RNA.
  • the circular RNA is exogenous.
  • circular RNA is endogenous.
  • circular RNAs with an internal ribosomal entry site (IRES) can be translated in vitro or in vivo or ex vivo.
  • Circular RNAs are a class of single-stranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Circular RNAs are 3-5’ covalently closed RNA rings, and circular RNAs do not display Cap or poly(A) tails. Since circular RNAs lack the free ends necessary for exonuclease-mediated degradation, rendering them resistant to several mechanisms of RNA turnover and granting them extended lifespans as compared to their linear mRNA counterparts. For this reason, circularization may allow for the stabilization of mRNAs that generally suffer from short half-lives and may therefore improve the overall efficacy of mRNA in a variety of applications.
  • Circular RNAs are produced by the process of splicing, and circularization occurs using conventional splice sites mostly at annotated exon boundaries (Starke et al., 2015; Szabo et al., 2015).
  • splice sites are used in reverse: downstream splice donors are “backspliced” to upstream splice acceptors (see Jeck and Sharpless, 2014; Barrett and Salzman, 2016; Szabo and Salzman, 2016; Holdt et al., 2018 for review).
  • RNA circularization To generate circular RNAs that we could subsequently transfer into cells, in vitro production of circular RNAs with autocatalytic-splicing introns can be programmed.
  • IVT in vitro transcription
  • Three general strategies have been reported so far for RNA circularization: chemical methods using cyanogen bromide or a similar condensing agent, enzymatic methods using RNA or DNA ligases, and ribozymatic methods using self-splicing introns.
  • precursor RNA was synthesized by run-off transcription and then heated in the presence of magnesium ions and GTP to promote circularization. RNA so produced can efficiently transfect different kinds of cells.
  • the template includes sequences for the TFP, CAR, and TCR, or combination thereof.
  • the group I intron of phage T4 thymidylate synthase (td) gene is well characterized to circularize while the exons linearly splice together (Chandry and Bel- fort, 1987; Ford and Ares, 1994; Perriman and Ares, 1998). When the td intron order is permuted flanking any exon sequence, the exon is circularized via two autocatalytic transesterification reactions (Ford and Ares, 1994; Puttaraju and Been, 1995).
  • the group I intron of phage T4 thymidylate synthase (td) gene is used to generate exogenous circular RNA.
  • a ribozymatic method utilizing a permuted group I catalytic intron has been used since it is more applicable to long RNA circularization and requires only the addition of GTP and Mg 2+ as cofactors.
  • This permuted intron-exon (PIE) splicing strategy consists of fused partial exons flanked by half-intron sequences. In vitro, these constructs undergo the double transesterification reactions characteristic of group I catalytic introns, but because the exons are fused, they are excised as covalently 5' to 3' linked circles.
  • a sequence containing a full-length encephalomyocarditis virus such as EMCV) IRES, a gene encoding a TFP, a CAR, a TCR or combination thereof, two short regions corresponding to exon fragments (El and E2), and of the PIE construct between the 3' and 5' introns of the permuted group I catalytic intron in the thymidylate synthase (Td) gene of the T4 phage or the permuted group I catalytic intron in the pre-tRNA gene of Anabaena.
  • EMCV encephalomyocarditis virus
  • the mentioned sequence further comprises complementary ‘homology arms’ placed at the 5' and 3' ends of the precursor RNA with the aim of bringing the 5' and 3' splice sites into proximity of one another.
  • the splicing reaction can be treated with RNase R.
  • the anti-GPC3 TFP is encoded by a circular RNA.
  • the circular RNA encoding the anti-GPC3 TFP is introduced into a T cell for production of a TFP-T cell.
  • the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection.
  • linear precursor RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template as is described herein.
  • PCR polymerase chain reaction
  • modified T cells comprising the nucleic acid encoding the TFP of the nucleic acid disclosed herein or a TFP encoded by the nucleic acid disclosed herein. Further disclosed herein, in some embodiments, are modified allogenic T cells comprising the nucleic acid TFP disclosed herein or a TFP encoded by nucleic acid disclosed herein.
  • the modified T cells comprising the recombinant nucleic acid disclosed herein, or the vectors disclosed herein comprises a functional disruption of an endogenous TCR.
  • modified allogenic T cells comprising the nucleic acid encoding the TFP disclosed herein or a TFP encoded by the nucleic acid disclosed herein.
  • the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR alpha constant domain, a TCR beta constant domain or a TCR alpha constant domain and a TCR beta constant domain.
  • the endogenous TCR that is functionally disrupted is an endogenous TCR alpha chain, an endogenous TCR beta chain, or an endogenous TCR alpha chain and an endogenous TCR beta chain.
  • the T cell further comprises a heterologous sequence encoding a TCR constant domain, wherein the TCR constant domain is a TCR gamma constant domain, a TCR delta constant domain or a TCR gamma constant domain and a TCR delta constant domain.
  • the endogenous TCR that is functionally disrupted is an endogenous TCR gamma chain, an endogenous TCR delta chain, or an endogenous TCR gamma chain and an endogenous TCR delta chain.
  • the endogenous TCR that is functionally disrupted has reduced binding to MHC-peptide complex compared to that of an unmodified control T cell.
  • the functional disruption is a disruption of a gene encoding the endogenous TCR.
  • the disruption of a gene encoding the endogenous TCR is a removal of a sequence of the gene encoding the endogenous TCR from the genome of a T cell.
  • the T cell is a human T cell.
  • the T cell is a CD8+ or CD4+ T cell.
  • the T cell is an allogenic T cell.
  • the T cell is a TCR alpha-beta T cell.
  • the T cell is a TCR gamma-delta T cell.
  • TCR alpha, TCR beta, TCR gamma, and TCR delta have been modified to produce an allogeneic T cell. See, e.g., copending PCT Publication No. WO2019173693, which is herein incorporated by reference.
  • the modified T cells are y5 T cells and do not comprise a functional disruption of an endogenous TCR.
  • the y5 T cells are Vyl+ Vy2- yy T cells.
  • the y5 T cells are Vyl- Vy2+ yy T cells.
  • the y5 T cells are Vyl- Vy2- yy T cells.
  • the modified T cells further comprise a nucleic acid encoding an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain.
  • the inhibitory molecule comprises the first polypeptide comprising at least a portion of PD1 and the second polypeptide comprising a costimulatory domain and primary signaling domain.
  • the modified T cells can further comprise an enhancing agent or a nucleic acid sequence encoding an enhancing agent.
  • the modified T cells can comprise a nucleic acid sequence encoding a TFP described herein and an additional nucleic acid sequence encoding the enhancing agent.
  • the nucleic acid sequence encoding the TFP and the additional nucleic acid sequence encoding the enhancing agent can be on the same nucleic acid molecule or be on different nucleic acid molecules. In some cases, the nucleic acid sequence encoding the TFP and the additional nucleic acid sequence encoding the enhancing agent are operatively linked by a linker.
  • the linker may be a cleavable linker.
  • the linker may comprise a protease cleavage site.
  • the cleavage site can be a self-cleaving peptide, for example, a 2A cleavage site such as a T2A, P2A, E2A or F2A cleavage site.
  • the protease cleavage site is a T2A cleavage site.
  • the enhancing agent can be a TGFBr2 switch polypeptide comprising a transforming growth factor beta receptor II (TGFBr2) extracellular domain or a functional fragment thereof, a PD-1 switch molecule comprising a PD-1 polypeptide and an intracellular domain of a costimulatory polypeptide, an anti-PD-1 antibody, a fusion molecule comprising an anti-PD- 1 antibody or fragment thereof and a transmembrane domain, a IL- 15 polypeptide or a fragment thereof, a IL-15Ra or fragment thereof, a fusion protein comprising a IL- 15 polypeptide or a fragment thereof or a IL-15Ra or fragment thereof, or an exogenous CXCR6 gene or functional fragment thereof.
  • TGFBr2 switch polypeptide comprising a transforming growth factor beta receptor II (TGFBr2) extracellular domain or a functional fragment thereof
  • the modified T cells described herein may comprise a TGFBr2 switch polypeptide or a nucleic acid sequence encoding a TGFBr2 switch polypeptide comprising a transforming growth factor beta receptor II (TGFBr2) extracellular domain or a functional fragment thereof.
  • the nucleic acid sequence can be on the same nucleic acid molecule encoding the TFP described herein or be on a different nucleic acid molecule encoding the TFP described herein.
  • the modified T cells can comprise a first nucleic acid sequence encoding a TFP comprising an anti-GPC3 antigen binding domain and a second nucleic acid sequence encoding a TGFBr2 switch polypeptide.
  • the first nucleic acid sequence and the second nucleic acid sequence can be linked by a linker.
  • the linker comprises a protease cleavage site.
  • the protease cleavage site is a 2A cleavage site.
  • the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.
  • the TGFBr2 extracellular domain or functional fragment thereof comprises a sequence with at least 80% sequence identity to SEQ ID NO:220 or SEQ ID NO:221. In some embodiments, the TGFBr2 extracellular domain or functional fragment thereof comprises the sequence of SEQ ID NO:220 or SEQ ID NO:221.
  • the switch polypeptide further comprises a switch intracellular domain. In some embodiments, the TGFBr2 extracellular domain or functional fragment thereof is operably linked to the switch intracellular domain. In some embodiments, the switch intracellular domain comprises an intracellular domain of a costimulatory polypeptide.
  • the costimulatory polypeptide is selected from the group consisting of CD28, 4-1BB, IL-15Ra, 0X40, CD2, CD27, CDS, ICAM-1, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD 160, CD226, Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • the costimulatory polypeptide is CD28.
  • the costimulatory polypeptide is 4-1BB.
  • the costimulatory polypeptide is IL-15Ra.
  • the switch intracellular domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:223 or SEQ ID NO:224. In some embodiments, the switch intracellular domain comprises the sequence of SEQ ID NO:223 or SEQ ID NO:224. In some embodiments, the switch polypeptide further comprises a switch transmembrane domain. In some embodiments, the TGFBr2 extracellular domain or functional fragment thereof is operably linked to the switch intracellular domain via the switch transmembrane domain. In some embodiments, the switch transmembrane domain is a TGFBr2 transmembrane domain. In some embodiments, the switch transmembrane domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:225.
  • the switch transmembrane domain comprises the sequence of SEQ ID NO:225. In some embodiments, the switch transmembrane domain is a transmembrane domain of the costimulatory polypeptide. In some embodiments, the switch transmembrane domain is a transmembrane domain of CD28. In some embodiments, the switch transmembrane domain is a transmembrane domain of 4- IBB. In some embodiments, the switch transmembrane domain is a transmembrane domain of IL-15Ra. In some embodiments, the switch transmembrane domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:226 or SEQ ID NO:227.
  • the switch transmembrane domain comprises the sequence of SEQ ID NO:226 or SEQ ID NO:227.
  • the switch polypeptide further comprises an additional intracellular domain.
  • the additional intracellular domain is operably linked to the C-terminus of the switch intracellular domain.
  • the additional intracellular domain comprises an intracellular domain of IL-15Ra or signaling domain thereof.
  • the additional intracellular domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:228 or SEQ ID NO:229.
  • the additional intracellular domain comprises the sequence of SEQ ID NO:228 or SEQ ID NO:229.
  • the switch polypeptide comprises a TGFBr2 transmembrane domain and an intracellular signaling domain of 4-1BB. In some embodiments, the switch polypeptide comprises a 4-1BB transmembrane domain and an intracellular signaling domain of 4- IBB. In some embodiments, the switch polypeptide comprises a TGFBr2 transmembrane domain and an intracellular signaling domain of CD28. In some embodiments, the switch polypeptide comprises a CD28 transmembrane domain and an intracellular signaling domain of CD28. In some embodiments, the switch polypeptide comprises a sequence with at least 80% sequence identity to any one selected from SEQ ID NOs: 216, 217, 218, and 219.
  • the switch polypeptide comprises the sequence of SEQ ID NOs: 216, 217, 218, or 219.
  • the third nucleic acid sequence encodes a dominant negative TGFBR2 receptor or a fragment thereof.
  • the dominant negative TGFBR2 receptor or a fragment thereof comprises a sequence with at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO: 230 or SEQ ID NO: 231.
  • the dominant negative TGFBR2 receptor or a fragment thereof comprises the sequence of SEQ ID NO: 230 or SEQ ID NO: 231.
  • the modified cells can comprise an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain.
  • the modified cells can comprise a nucleic acid sequence encoding an inhibitory molecule that comprises a first polypeptide comprising at least a portion of an inhibitory molecule, associated with a second polypeptide comprising a positive signal from an intracellular signaling domain.
  • the inhibitory molecule can be a PD- 1 switch molecule comprising the first polypeptide comprising at least a portion of PD- 1 and the second polypeptide comprising a costimulatory domain and primary signaling domain.
  • a T cell expressing the TFP as described herein, and a PD-1 switch molecule as descried herein can inhibit tumor growth when expressed in a T cell.
  • the PD- 1 switch molecule can enhance the activity of a modified T cell.
  • the nucleic acid sequence can be on the same nucleic acid molecule encoding the TFP described herein or be on a different nucleic acid molecule encoding the TFP described herein.
  • the modified T cells can comprise a first nucleic acid sequence encoding a TFP comprising an anti-GPC3 antigen binding domain and a second nucleic acid sequence encoding a PD-1 switch molecule.
  • the first nucleic acid sequence and the second nucleic acid sequence can be linked by a linker.
  • the linker comprises a protease cleavage site.
  • the protease cleavage site is a 2A cleavage site.
  • the 2A cleavage site is a T2A cleavage site or a P2A cleavage site
  • the PD- 1 switch molecule can comprise a PD- 1 polypeptide.
  • the PD- 1 polypeptide may be operably linked to the N-terminus of an intracellular domain of a costimulatory polypeptide via the C-terminus of the PD-1 polypeptide.
  • the costimulatory polypeptide is selected from the group consisting of 0X40, CD2, CD27, CD5, ICAM-1, ICOS (CD278), 4-1BB (CD137), GITR, CD28, CD30, CD40, IL-15Ra, IL12R, IL18R, IL21R, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, CD226, Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIII.
  • the costimulatory peptide is CD28.
  • an extracellular domain and a transmembrane domain of PD-1 are linked to an intracellular domain of CD28.
  • the nucleic acid sequence encodes a PD-1 switch molecule comprising the extracellular domain and the transmembrane domain of PD- 1 linked to the intracellular domain of CD28.
  • the fusion protein comprises a sequence with at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO: 232 or SEQ ID NO: 233.
  • the fusion protein comprises the sequence of SEQ ID NO: 232 or SEQ ID NO: 233.
  • the nucleic acid sequence encodes a PD-1 switch molecule comprising an extracellular domain and a transmembrane domain of PD-1 linked to an intracellular domain of CD28 linked to IL-15Ra.
  • the fusion protein comprises a sequence with at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO: 234 or SEQ ID NO: 235.
  • the PD-1 switch molecule comprises the sequence of SEQ ID NO: 234 or SEQ ID NO: 235.
  • the enhancing agent described herein can be an anti-PD-1 antibody or fragment thereof (e.g., antigen binding fragment).
  • the modified T cells described herein may comprise a nucleic acid sequence encoding an anti-PD-1 antibody or fragment thereof that specifically binds programmed cell death protein 1 (PD-1).
  • the anti-PD-1 antibody or fragment thereof can inhibit an interaction of PD- 1 with PD-L1 or PD-L2.
  • the anti-PD-1 antibody or fragment thereof can be secreted by the T cell.
  • the modified T cell described herein can comprise a first nucleic acid sequence encoding the TFP described herein and a second nucleic acid sequence encoding the anti-PD-1 antibody or fragment thereof.
  • the anti-PD-1 antibody or fragment thereof comprises a variable domain comprising a complementarity determining region 1 (CDR1), a CDR2, and a CDR3, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:238.
  • the CDR1 comprises the amino acid sequence of SEQ ID NO:236.
  • the CDR2 comprises the amino acid sequence of SEQ ID NO:237.
  • the variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:239.
  • the variable domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:239.
  • variable domain comprises the amino acid sequence of SEQ ID NO:239. In some embodiments, the variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 240-243. In some embodiments, the variable domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 240-243. In some embodiments, the variable domain comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 240-243.
  • the second nucleic acid encodes a fusion protein comprising an anti-PD-1 antibody or fragment thereof that specifically binds PD-1.
  • the anti-PD-1 antibody or antigen binding fragment thereof may be operably linked to the N-terminus of an intracellular domain of a costimulatory polypeptide via the C-terminus of the anti-PD-1 antibody, or antigen binding fragment thereof.
  • the anti-PD-1 antibody is linked to the intracellular domain of the costimulatory polypeptide via the transmembrane domain of PD- 1.
  • the fusion protein further comprises a signal sequence.
  • the signal sequence is a PD-1 signal peptide.
  • the signal sequence comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:244 or SEQ ID NO:245.
  • the signal sequence comprises the amino acid sequence of SEQ ID NO:244 or SEQ ID NO:245.
  • the anti-PD-1 antibody or fragment thereof binds to PD- 1 on the surface of the T cell, PD- 1 on the surface of a bystander T cell, or a combination thereof.
  • the fusion protein further comprises an intracellular domain operatively linked to the transmembrane domain.
  • the fusion protein further comprises a transmembrane domain operatively linked to the intracellular domain and the anti-PD- 1 antibody or fragment thereof.
  • the transmembrane domain of the fusion protein comprises a transmembrane domain of a protein selected from the group consisting of CD28, CD3 ⁇ , CD3 , CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD41, CD64, CD68, CD80, CD86, CD134, CD137, CD154, ICOS, 4-1BB, 0X40, PD-1, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the transmembrane domain of the fusion protein comprises a PD- 1 transmembrane domain. In some embodiments, the transmembrane domain of the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:246. In some embodiments, the transmembrane domain ofthe fusion protein comprises the amino acid sequence of SEQ ID NO:246. In some embodiments, the fusion protein further comprises a PD- 1 stalk domain. In some embodiments, the PD- 1 stalk domain is operatively linked to the transmembrane do-main. In some embodiments, the PD-1 stalk domain is operatively linked to the N-terminus of the transmembrane domain.
  • the PD-1 stalk domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:247. In some embodiments, the PD-1 stalk domain comprises the amino acid sequence of SEQ ID NO:247. In some embodiments, the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:247 operatively linked to an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:246.
  • the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:247 operatively linked to the N terminus of an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:246. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:247 operatively linked to the amino acid sequence of SEQ ID NO:246. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO:247 operatively linked to the N-terminus of the amino acid sequence of SEQ ID NO:246. In some embodiments, the intracellular domain of the fusion protein comprises a co-stimulatory domain.
  • the co-stimulatory domain comprises a co-stimulatory domain of a protein selected from the group consisting of a CD27, CD28, 4- IBB (CD 137), 0X40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, NKG2D, B7-H3, a ligand that specifically binds with CD83, PD-1, CD258, ICAM-1, functional fragments thereof, and amino acid sequences thereof having at least one but not more than 20 modifications.
  • the co-stimulatory domain comprises a 4- IBB (CD 137) co-stimulatory domain.
  • the co-stimulatory domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:224. In some embodiments, the co- stimulatory domain comprises the amino acid sequence of SEQ ID NO:224. In some embodiments, the co-stimulatory domain comprises a CD28 co-stimulatory domain. In some embodiments, the co- stimulatory domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:223. In some embodiments, the co-stimulatory domain comprises the amino acid sequence of SEQ ID NO:223. In some embodiments, the fusion protein comprises two or more anti-PD-1 antibodies or fragments thereof.
  • the two or more anti -PD-1 antibodies or fragments thereof are operatively linked tandemly. In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are identical. In some embodiments, the two or more anti-PD-1 antibodies or fragments thereof are different. In some embodiments, the two or more anti- PD- 1 antibodies or fragments thereof are operatively linked by a linker.
  • the linker comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:248. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO:248.
  • the fusion protein further comprises a PD-1 signal peptide, a PD-1 stalk domain, a PD-1 transmembrane domain, and a CD28 or 4-1BB (CD137) co-stimulatory do-main.
  • the fusion protein comprises, from the N-terminus to the C-terminus, a PD-1 signal peptide operatively linked to the anti-PD-1 antibody or fragment thereof operatively linked to a PD- 1 stalk domain operatively linked to a PD- 1 transmembrane domain operatively linked to a CD28 or 4- IBB (CD 137) co-stimulatory domain.
  • the fusion protein comprises, from the N-terminus to the C-terminus, a PD-1 signal peptide operatively linked to a first anti-PD- 1 antibody or fragment thereof operatively linked to a linker operatively linked to a second anti-PD-1 antibody or fragment thereof operatively linked to a PD-1 stalk domain operatively linked to a PD-1 transmembrane domain operatively linked to a CD28 or 4- IBB (CD 137) co-stimulatory domain.
  • the fusion protein comprises SEQ ID NO:244 or SEQ ID NO:245, SEQ ID NO:239 or SEQ ID NO:241, SEQ ID NO:247, SEQ ID NO:246, and SEQ ID NO:223 or SEQ ID NO:224.
  • the fusion protein comprises, from the N-terminus to the C- terminus, SEQ ID NO:244 or SEQ ID NO:245 operatively linked to SEQ ID NO:239 or SEQ ID NO:241 operatively linked to SEQ ID NO:247 operatively linked to SEQ ID NO:246 operatively linked to SEQ ID NO:223 or SEQ ID NO:224.
  • the fusion protein comprises, from the N-terminus to the C-terminus, SEQ ID NO:244 or SEQ ID NO:245 operatively linked to SEQ ID NO:239 or SEQ ID NO:241 operatively linked to SEQ ID NO:248 operatively linked to SEQ ID NO:239 or SEQ ID NO:241 operatively linked to SEQ ID NO:247 operatively linked to SEQ ID NO:246 operatively linked to SEQ ID NO:223 or SEQ ID NO:224.
  • the fusion protein comprises an amino acid sequence having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to the amino acid sequence of any one of SEQ ID NOs:249-253. In some embodiments, the fusion protein comprises an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs:249-253.
  • the modified T cells described herein may comprise a nucleic acid sequence encoding an IL- 15 polypeptide or a fragment thereof.
  • the modified T cells described herein may comprise a nucleic acid sequence encoding an Interleukin- 15 receptor alpha (IL-15Ra) polypeptide or a fragment thereof.
  • the modified T cells described herein may comprise a nucleic acid sequence encoding a fusion protein comprising an IL- 15 polypeptide or a fragment thereof linked to an IL-15Ra polypeptide or a fragment thereof.
  • the modified T cells described herein may comprise a nucleic acid sequence encoding a fusion protein comprising an IL-15Ra polypeptide or a fragment thereof linked to PD-1 or a fragment thereof and/or CD28 or a fragment thereof.
  • the IL- 15 polypeptide or a fragment thereof may comprise an IL- 15 signal peptide.
  • the IL- 15 polypeptide or a fragment thereof may comprise amino acids 1-29 of IL-15.
  • the IL-15 polypeptide or a fragment thereof may comprise amino acids 1-29 of SEQ ID NO:256.
  • the IL-15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:257.
  • the IL- 15 polypeptide or a fragment thereof may comprise amino acids 30-162 of IL- 15. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise amino acids 30-162 of SEQ ID NO:256. In some embodiments, the IL- 15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:254. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise amino acids 1-162 of SEQ ID NO:256. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:257 and a sequence of SEQ ID NO:254. In some embodiments, IL- 15 polypeptide is secreted when expressed in a cell, such as a T cell.
  • the IL-15Ra polypeptide or a fragment thereof may comprise IL-15Ra signal peptide. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 1-30 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 1-30 of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof does not comprise IL-15Ra signal peptide. In some embodiments, the IL-15Ra polypeptide or a fragment thereof does not comprise amino acids 1-30 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof does not comprise amino acids 1-30 of SEQ ID NO:258.
  • the IL-15Ra polypeptide or a fragment thereof may comprise IL-15Ra Sushi domain. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 31-95 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 31-95 of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:260.
  • the IL-15Ra polypeptide or a fragment thereof may comprise an intracellular domain of IL- 15Ra. In some embodiments, the IL- 15Ra polypeptide or a fragment thereof may comprise amino acids 229-267 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 229-267 of a sequence of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:228.
  • the IL-15Ra polypeptide or a fragment thereof may comprise IL-15Ra
  • the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 31-267 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 31-267 of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:260. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:261. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 96-267 of SEQ ID NO:258. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:260 and a sequence of SEQ ID NO:261.
  • the IL-15Ra polypeptide or a fragment thereof may be a soluble IL- 15Ra (sIL- 15Ra).
  • the IL- 15Ra polypeptide or a fragment thereof may comprise amino acids 21-205 ofIL-15Ra.
  • the IL- 15Ra polypeptide or a fragment thereof may comprise amino acids 21-205 of a sequence of SEQ ID NO:258.
  • the IL- 15Ra polypeptide or a fragment thereof may comprise a sequence of SEQ ID NO:259.
  • the present disclosure encompasses recombinant nucleic acid molecules encoding a fusion protein comprising an IL- 15 polypeptide linked to an IL-15R subunit.
  • IL- 15 and IL-15R subunit are operatively linked by a linker.
  • the IL-15R subunit is IL-15R alpha (IL-15Ra).
  • IL-15 polypeptide may be linked to N-terminus of IL-15Ra subunit.
  • IL- 15 polypeptide may be linked to C-terminus of IL-15Ra subunit.
  • IL- 15 and IL-15Ra are operatively linked by a linker.
  • the linker is not a cleavable linker.
  • the linker may comprise a sequence comprising (G4S)n, wherein G is glycine, S is serine, and n is an integer from 1 to 10. In some embodiments, n is an integer from 1 to 4. In some embodiments, n is 3. In some embodiments, the linker comprises a sequence of SEQ ID NO:255.
  • the fusion protein is expressed on cell surface when expressed in a cell, e.g., a T cell. In some embodiments, the fusion protein is secreted when expressed in a cell, e.g., a T cell.
  • the fusion protein may comprise amino acids 30-162 of IL-15. In some embodiments, the fusion protein may comprise amino acids 30-162 of a sequence of SEQ ID NO:256. In some embodiments, the fusion protein may comprise any one of the sequence listed in Table 7 or a fragment thereof. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:254. In some embodiments, the fusion protein does not comprise IL-15 signal peptide. In some embodiments, the fusion protein does not comprise amino acids 1-29 of IL- 15. In some embodiments, the fusion protein does not comprise amino acids 1-29 of a sequence of SEQ ID NO:256.
  • the fusion protein does not comprise a sequence of SEQ ID NO:257.
  • the fusion protein may comprise a Sushi domain.
  • the fusion protein may comprise amino acids 31-95 of IL-15Ra.
  • the fusion protein may comprise amino acids 31-95 of a sequence of SEQ ID NO:258.
  • the fusion protein may comprise a sequence of SEQ ID NO:260.
  • the fusion protein may comprise the intracellular domain of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 229-267 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 229-267 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:228.
  • the fusion protein may comprise a soluble IL-15Ra (sIL-15Ra). In some embodiments, the fusion protein may comprise amino acids 21-205 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 21-205 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:259.
  • the fusion protein may comprise the transmembrane domain and the intracellular domain of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 96-267 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 96-267 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:261.
  • the fusion protein may comprise the Sushi domain, the transmembrane domain, and the intracellular domain of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 31-267 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 31-267 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:260 and a sequence of SEQ ID NO:261.
  • the fusion protein comprising an IL- 15 polypeptide or a fragment thereof and an IL-15Ra subunit or a fragment thereof comprises a sequence with at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one sequence selected from SEQ ID NO:263.
  • the fusion protein comprising an IL- 15 polypeptide or a fragment thereof and an IL-15Ra subunit or a fragment thereof comprises the sequence of SEQ ID NO:263.
  • the modified T cells described herein may comprise C-X-C chemokine receptor type 6 (CXCR6) or a functional fragment thereof.
  • the modified T cells described herein may comprise a nucleic acid sequence encoding C-X-C chemokine receptor type 6 (CXCR6) or a functional fragment thereof.
  • a modified T cell described herein can comprise a first nucleic acid sequence encoding a TFP described herein (e.g., a TFP comprising an anti-GPC3 antigen binding domain), and a second nucleic acid sequence encoding C-X-C chemokine receptor type 6 (CXCR6) or a functional fragment thereof.
  • the first and the second nucleic acid molecules are expressed in the same operon.
  • the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a sequence encoding a linker.
  • the linker comprises a protease cleavage site.
  • the protease cleavage site is a 2A cleavage site.
  • the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.
  • the first nucleic acid sequence and the second nucleic acid sequence are present on different nucleic acid molecules.
  • the CXCR6 or functional fragment thereof comprises a sequence with at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to any one selected from SEQ ID NOs:264-266.
  • the CXCR6 or functional fragment thereof comprises a sequence of any one selected from SEQ ID NOs:264-266.
  • the sequence of the recombinant nucleic acid is codon optimized.
  • the CXCR6 or functional fragment thereof is encoded by a nucleic acid with at least 60% sequence identity to SEQ ID NO: 285.
  • the CXCR6 or functional fragment thereof is encoded by the nucleic acid of SEQ ID NO: 285. In some embodiments, the CXCR6 or functional fragment thereof comprises at least one, two, three, or four extracellular domains. In some embodiments, the CXCR6 or functional fragment thereof comprises four extracellular domains. In some embodiments, the CXCR6 or functional fragment thereof comprises an N-terminal extracellular region comprising a sequence with at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO: 267.
  • the CXCR6 or functional fragment thereof comprises an N-terminal extracellular region comprising the sequence of SEQ ID NO: 267. In some embodiments, the CXCR6 or functional fragment thereof comprises a CXCL16-b inding domain. In some embodiments, the CXCR6 or functional fragment thereof is associated with the cell membrane when expressed in a T cell. In some embodiments, the CXCR6 or functional fragment thereof comprises a transmembrane region comprising at least one, two, three, four, five, six, or seven transmembrane domains. In some embodiments, the transmembrane region comprises the sequence of any one of SEQ ID NOs:274-280, or any combination thereof.
  • the CXCR6 or functional fragment thereof comprises a transmembrane region comprising seven transmembrane domains. In some embodiments, the CXCR6 or functional fragment thereof further comprises the sequence of SEQ ID NO 270, the sequence of SEQ ID NO 271, the sequence of SEQ ID NO 272, the sequence of SEQ ID NO 281, the sequence of SEQ ID NO 282, the sequence of SEQ ID NO 283, or any combination thereof.
  • the CXCR6 or functional fragment thereof further comprises the sequence of SEQ ID NO 270, the sequence of SEQ ID NO 271, the sequence of SEQ ID NO 272, or a combination thereof; and the sequence of the sequence of SEQ ID NO 281, the sequence of SEQ ID NO 282, the sequence of SEQ ID NO 283, or any combination thereof.
  • the CXCR6 or functional fragment thereof comprises a transmembrane region comprising a sequence with at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO: 273.
  • the CXCR6 or functional fragment thereof comprises a transmembrane region comprising the sequence of SEQ ID NO: 273. In some embodiments, the CXCR6 or functional fragment thereof comprises at least one, two, three, or four cytoplasmic domains. In some embodiments, the CXCR6 or functional fragment thereof comprises four cytoplasmic domains. In some embodiments, the CXCR6 or functional fragment thereof comprises a C-terminal cytoplasmic domain comprising a sequence with at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 95%, 97%, 99.0%, 99.5%, 99.8, or 99.9% sequence identity to SEQ ID NO: 284.
  • the CXCR6 or functional fragment thereof comprises a cytoplasmic domain comprising the sequence of SEQ ID NO: 284.
  • migration of a cell expressing the CXCR6 or functional fragment there -of increases in response to CXCL16.
  • a migration rate of a cell expressing the CXCR6 or functional fragment thereof increases in response to CXCL16
  • the number of cells expressing the CXCR6 or functional fragment thereof that migrate to a tumor site increases in response to CXCL 16, or (iii) a combination thereof.
  • T cells Prior to expansion and genetic modification, a source of T cells is obtained from a subject.
  • the term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • PBMCs peripheral blood mononuclear cells
  • T cells can be obtained from a leukopak.
  • any number of T cell lines available in the art may be used.
  • T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FicollTM separation.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation.
  • a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe® 2991 cell processor, the Baxter OncologyCytoMate, or the Haemonetics® Cell Saver® 5) according to the manufacturer’s instructions.
  • a semi-automated “flow-through” centrifuge for example, the Cobe® 2991 cell processor, the Baxter OncologyCytoMate, or the Haemonetics® Cell Saver® 5
  • the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • buffers such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture media.
  • the T cells are a[:J T cells.
  • the T cells are y5 T cells.
  • y5 T cells are obtained from a bank of umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cells, for example.
  • T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL®gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of T cells such as CD3+, CD28+, CD4+, CD8+, CD45RA+, CD45RO+, alpha-beta, or gamma-delta T cells, can be further isolated by positive or negative selection techniques.
  • CD4+ and CD8+ T cells are isolated with anti-CD4 and anti-CD8 microbeads.
  • T cells are isolated by incubation with anti-CD3/anti-CD28 (e.g., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T or Trans-Act® beads, for a time period sufficient for positive selection of the desired T cells.
  • the time period is about 30 minutes.
  • the time period ranges from 30 minutes to 36 hours or longer and all integer values there between.
  • the time period is at least 1, 2, 3, 4, 5, or 6 hours.
  • the time period is 10 to 24 hours.
  • the incubation time period is 24 hours.
  • TIL tumor infiltrating lymphocytes
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.
  • multiple rounds of selection can also be used in the context of this present disclosure. In certain aspects, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection.
  • Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected.
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8.
  • it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+.
  • T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.
  • a T cell population can be selected that expresses one or more of IFN- ⁇ TNF- ⁇ lpha, IL-17A, IL-2, IL-3, IL -4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g., other cytokines.
  • Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No. WO2013126712, which is herein incorporated by reference.
  • the concentration of cells and surface can be varied.
  • it may be desirable to significantly decrease the volume in which beads and cells are mixed together e.g., increase the concentration of cells, to ensure maximum contact of cells and beads.
  • a concentration of 2 billion cells/mL is used.
  • a concentration of 1 billion cells/mL is used.
  • greater than 100 million cells/mL is used.
  • a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mL is used.
  • concentrations of 125 or 150 million cells/mL can be used.
  • Using high concentrations can result in increased cell yield, cell activation, and cell expansion.
  • use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28- negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain.
  • using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
  • the concentration of cells used is 5xlO 6 /mL. In other aspects, the concentration used can be from about IxlO mL to lxlO 6 /mL, and any integer value in between. In other aspects, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10 °C or at room temperature.
  • T cells for stimulation can also be frozen after a washing step.
  • the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
  • the cells may be suspended in a freezing solution.
  • one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80 °C at a rate of 1 per minute and stored in the vapor phase of a liquid nitrogen storage tank.
  • cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure.
  • a blood sample or an apheresis product is taken from a generally healthy subject.
  • a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use.
  • the T cells may be expanded, frozen, and used at a later time.
  • samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments.
  • the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, and mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, and irradiation.
  • agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, and mycophenolate, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3
  • T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells.
  • the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo.
  • these cells may be in a preferred state for enhanced engraftment and in vivo expansion.
  • mobilization for example, mobilization with GM-CSF
  • conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy.
  • Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.
  • T cells may be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041, and 7,572,631.
  • the T cells of the present disclosure may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells.
  • T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen- binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore.
  • a ligand that binds the accessory molecule is used for co- stimulation of an accessory molecule on the surface of the T cells.
  • a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells.
  • an anti-CD3 antibody and an anti-CD28 antibody include 9.3, B-T3, XR- CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J.
  • T cells are activated by incubation with anti-CD3/anti-CD28-conjugated beads, such as DYNABEADS® or Trans-Act® beads, for a time period sufficient for activation of the T cells.
  • the time period is at least 1, 2, 3, 4, 5, or 6 hours.
  • the time period is 10 to 24 hours, e.g., 24 hours.
  • T cells are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with cytokines that bind the common gamma- chain (e.g., IL -2, IL-7, IL-12, IL-15, IL -21, and others).
  • T cells are activated by stimulation with an anti-CD3 antibody and an anti-CD28 antibody in combination with 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 100 U/mL of IL-2, IL-7, and/or IL-15.
  • the cells are activated for 24 hours.
  • the cells after transduction, are expanded in the presence of anti-CD3 antibody, anti-CD28 antibody in combination with the same cytokines.
  • cells activated in the presence of an anti- CD3 antibody and an anti-CD28 antibody in combination with cytokines that bind the common gamma-chain are expanded in the presence of the same cytokines in the absence of the anti-CD3 antibody and anti-CD28 antibody after transduction.
  • the cells after transduction, the cells are expanded in the presence of anti-CD3 antibody, anti-CD28 antibody in combination with the same cytokines up to a first washing step, when the cells are sub-cultured in media that includes the cytokines but does not include the anti-CD3 antibody and anti-CD28 antibody.
  • the cells are subcultured every 1, 2, 3, 4, 5, or 6 days.
  • cells are expanded for 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, or 30 days.
  • T cells may be stimulated with zoledronic acid (Zometa), alendronic acid (Fosamax) or other related bisphosphonate drugs at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10, or 100 pM in the presence of feeder cells (irradiated cancer cells, PBMCs, artificial antigen presenting cells).
  • Zometa zoledronic acid
  • Fosamax alendronic acid
  • 5.0, 7.5, 10 pM
  • feeder cells irradiated cancer cells, PBMCs, artificial antigen presenting cells.
  • T cells may be stimulated with isopentyl pyrophosphate (IPP), (E)- 4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP or HMB-PP) or other structurutally related compounds at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10, or 100 pM in the presence of feeder cells (irradiated cancer cells, PBMCs, artificial antigen presenting cells).
  • IPP isopentyl pyrophosphate
  • HMBPP or HMB-PP 4-Hydroxy-3-methyl-but-2-enyl pyrophosphate
  • other structurutally related compounds at concentrations of 0.1, 0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.5, 10, or 100 pM in the presence of feeder cells (irradiated cancer cells, PBMCs, artificial antigen presenting cells).
  • the expansion of T cells may be stimulated with synthetic phosphoantigens (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1 PP, or 2-methyl-3-butenyl-l -pyrophosphate in the presence of IL-2 for one-to-two weeks.
  • the expansion of T cells may be stimulated with immobilized anti-TCRyd (e.g., pan TCRY6) in the presence of IL -2, e.g., for approximately 14 days.
  • the expansion of T cells may be stimulated with culture of immobilized anti-CD3 antibodies (e.g., OKT3) in the presence of IL-2.
  • the aforementioned culture is maintained for about seven days prior to subculture in soluble anti-CD3, and IL-2.
  • T cells that have been exposed to varied stimulation times may exhibit different characteristics.
  • typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+).
  • TH, CD4+ helper T cell population
  • TC cytotoxic or suppressor T cell population
  • Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells.
  • infusing a subject with a T cell population comprising predominately of TH cells may be advantageous.
  • an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.
  • TFP TFP
  • various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability of T cells to activate and expand stimulation, and anti-cancer activities in appropriate in vitro and animal models. Assays to evaluate the effects of a TFP are described in further detail below.
  • the TFP T cells provided herein may be useful for the treatment of any disease or condition involving GPC3 (e.g., GPC3-expressing cancers).
  • the disease or condition is a disease or condition that can benefit from treatment with adoptive cell therapy.
  • the disease or condition is a cell proliferative disorder.
  • the disease or condition is a cancer.
  • the disease or condition is a blood cancer.
  • the disease or condition is a tumor.
  • the disease or condition is a viral infection.
  • provided herein is a method of treating a disease or condition in a subject in need thereof by administering an effective amount of a TFP T cell provided herein to the subject.
  • the disease or condition is a cancer.
  • the disease or condition is a viral infection.
  • any suitable cancer may be treated with the TFP T cells provided herein.
  • suitable cancers include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma
  • ALL acute lympho
  • the cancer can be acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, cancer of the anus, anal canal, or anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia (CLL), chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastric cancer, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), glioblastoma, Hodgkin's lymphoma, hypopharynx cancer
  • bladder cancer e.g
  • the cancer can be characterized by the expression of GPC3.
  • the GPC3 can be highly expressed in hepatocellular carcinoma (HCC), lung cancer, melanoma, ovarian clear- cell carcinomas, yolk sac tumors, neuroblastoma, hepatoblastoma, Wilms' tumor cells, and gastric cancer.
  • HCC hepatocellular carcinoma
  • lung cancer melanoma
  • ovarian clear- cell carcinomas ovarian clear- cell carcinomas
  • yolk sac tumors neuroblastoma
  • hepatoblastoma hepatoblastoma
  • Wilms' tumor cells and gastric cancer.
  • the invention provides methods for treating a disease associated with at least one tumor-associated antigen expression.
  • the invention provides methods for treating a disease wherein part of the tumor is negative for the tumor associated antigen and part of the tumor is positive for the tumor associated antigen.
  • the antibody or TFP of the invention is useful for treating subjects that have undergone treatment for a disease associated with elevated expression of said tumor antigen, wherein the subject that has undergone treatment for elevated levels of the tumor associated antigen exhibits a disease associated with elevated levels of the tumor associated antigen.
  • the invention pertains to a vector comprising an anti-tumor-associated antigen antibody or TFP operably linked to promoter for expression in mammalian T cells.
  • the invention provides a recombinant T cell expressing a tumor-associated antigen TFP for use in treating tumor-associated antigen-expressing tumors, wherein the recombinant T cell expressing the tumor- associated antigen TFP is termed a tumor-associated antigen TFP-T.
  • the tumor- associated antigen TFP-T of the invention is capable of contacting a tumor cell with at least one tumor- associated antigen TFP of the invention expressed on its surface such that the TFP-T targets the tumor cell and growth of the tumor is inhibited.
  • the invention pertains to a method of inhibiting growth of a tumor-associated antigen-expressing tumor cell, comprising contacting the tumor cell with a tumor-associated antigen antibody or TFP T cell of the present invention such that the TFP-T is activated in response to the antigen and targets the cancer cell, wherein the growth of the tumor is inhibited.
  • the invention pertains to a method of treating cancer in a subject.
  • the method comprises administering to the subject a tumor-associated antigen antibody, bispecific antibody, or TFP T cell of the present invention such that the cancer is treated in the subject.
  • a cancer that is treatable by the tumor-associated antigen TFP T cell of the invention is a cancer associated with expression of tumor-associated antigen.
  • tumor-associated antigen antibodies or TFP therapy can be used in combination with one or more additional therapies described herein.
  • T cells are genetically modified to express a TFP and the TFP-expressing T cell is infused to a recipient in need thereof.
  • the infused cell is able to kill tumor cells in the recipient.
  • TFP-expressing T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.
  • the T cells administered to the patient, or their progeny persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of the T cell to the patient.
  • T cells are modified, e.g., by in vitro transcribed RNA, to transiently express a TFP and the TFP-expressing T cell is infused to a recipient in need thereof.
  • the infused cell is able to kill tumor cells in the recipient.
  • the T cells administered to the patient is present for less than one month, e.g., three weeks, two weeks, or one week, after administration of the T cell to the patient.
  • the anti-tumor immunity response elicited by the TFP-expressing T cells may be an active or a passive immune response, or alternatively may be due to a direct vs indirect immune response.
  • the TFP transduced T cells exhibit specific proinflammatory cytokine secretion and potent cytolytic activity in response to human cancer cells expressing the tumor-associated antigen, resist soluble tumor-associated antigen inhibition, mediate bystander killing and/or mediate regression of an established human tumor.
  • antigen-less tumor cells within a heterogeneous field of tumor-associated antigen-expressing tumor may be susceptible to indirect destruction by tumor-associated antigen-redirected T cells that has previously reacted against adjacent antigen-positive cancer cells.
  • the human TFP-modified T cells of the invention may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal.
  • the mammal is a human.
  • ex vivo immunization With respect to ex vivo immunization, at least one of the following occurs in vitro prior to administering the cell into a mammal: i) expansion of the cells, ii) introducing a nucleic acid encoding a TFP to the cells or iii) cryopreservation of the cells, as is described herein. [00358] In addition to using a cell-based vaccine in terms of ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.
  • the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised.
  • the TFP-modified T cells of the invention are used in the treatment of diseases, disorders and conditions associated with expression of tumor-associated antigens.
  • the cells of the invention are used in the treatment of patients at risk for developing diseases, disorders and conditions associated with expression of tumor-associated antigens.
  • the present invention provides methods for the treatment or prevention of diseases, disorders and conditions associated with expression of tumor-associated antigens comprising administering to a subject in need thereof, a therapeutically effective amount of the TFP-modified T cells of the invention.
  • the antibodies or TFP-modified T cells of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components as is described in further detail below.
  • the present invention also provides methods for inhibiting the proliferation or reducing a tumor-associated antigen-expressing cell population, the methods comprising contacting a population of cells comprising a tumor-associated antigen-expressing cell with an anti-tumor-associated antigen TFP-T cell of the invention that binds to the tumor-associated antigen-expressing cell.
  • the present invention provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing tumor-associated antigen, the methods comprising contacting the tumor-associated antigen-expressing cancer cell population with an anti-tumor-associated antigen antibody or TFP-T cell of the invention that binds to the tumor-associated antigen-expressing cell.
  • the present invention provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing tumor-associated antigen, the methods comprising contacting the tumor-associated antigen-expressing cancer cell population with an anti-tumor-associated antigen antibody or TFP-T cell of the invention that binds to the tumor-associated antigen-expressing cell.
  • the anti-tumor-associated antigen antibody or TFP-T cell of the invention reduces the quantity, number, amount or percentage of cells and/or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a subject with or animal model for multiple myeloma or another cancer associated with tumor-associated antigen-expressing cells relative to a negative control.
  • the subject is a human.
  • the present invention also provides methods for preventing, treating and/or managing a disease associated with tumor-associated antigen-expressing cells (e.g., a cancer expressing tumor- associated antigen), the methods comprising administering to a subject in need an anti-tumor- associated antigen antibody or TFP-T cell of the invention that binds to the tumor-associated antigen- expressing cell.
  • the subject is a human.
  • disorders associated with tumor-associated antigen-expressing cells include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma) and cancers (such as hematological cancers or atypical cancers expressing tumor-associated antigen).
  • Suitable doses of the TFP-T cells described herein for a therapeutic effect would be at least 10 5 or between about 10 5 and about 10 10 cells per dose, for example, preferably in a series of dosing cycles.
  • An exemplary dosing regimen consists of four one-week dosing cycles of escalating doses, starting at least at about 10 5 cells on Day 0, for example increasing incrementally up to a target dose of about 10 10 cells within several weeks of initiating an intra-patient dose escalation scheme.
  • Suitable modes of administration include intravenous, subcutaneous, intracavitary (for example by reservoir- access device), intraperitoneal, and direct injection into a tumor mass.
  • an effective amount or sufficient number of the isolated, T cells is present in the composition and introduced into the subject such that long-term, specific, anti-cancer and/or anti-tumor responses are established to reduce the size of a tumor or eliminate tumor growth or regrowth than would otherwise result in the absence of such treatment.
  • the amount of T cells introduced into the subject causes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in tumor size when compared to otherwise same conditions wherein the T cells are not present.
  • the amount of T cells administered should take into account the route of administration and should be such that a sufficient number of the T cells will be introduced so as to achieve the desired therapeutic response.
  • the amounts of each active agent included in the compositions described herein e.g., the amount per each cell to be contacted or the amount per certain body weight
  • an antibody or TFP -expressing cell described herein may be used in combination with other known agents and therapies.
  • Administered “in combination”, as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons.
  • the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”.
  • the delivery of one treatment ends before the delivery of the other treatment begins.
  • the treatment is more effective because of combined administration.
  • the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment or the analogous situation is seen with the first treatment.
  • delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other.
  • the effect of the two treatments can be partially additive, wholly additive, or greater than additive.
  • the delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.
  • the “at least one additional therapeutic agent” includes a TFP- expressing cell.
  • T cells that express multiple TFPs, which bind to the same or different target antigens, or same or different epitopes on the same target antigen.
  • populations of T cells in which a first subset of T cells expresses a first TFP and a second subset of T cells expresses a second TFP.
  • a TFP-expressing cell described herein and the at least one additional therapeutic agent can be administered simultaneously, in the same or in separate compositions, or sequentially.
  • the TFP-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.
  • the TFP T cells provided herein are administered with at least one additional therapeutic agent.
  • Any suitable additional therapeutic agent may be administered with a TFP T cell provided herein.
  • the additional therapeutic agent is selected from radiation, a cytotoxic agent, a chemotherapeutic agent, a cytostatic agent, an anti-hormonal agent, an EGFR inhibitor, an immunostimulatory agent, an anti-angiogenic agent, and combinations thereof.
  • a TFP-expressing cell described herein may be used in a treatment regimen in combination with surgery, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and tacrolimus, antibodies, or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies, cyclophosphamide, fludarabine, cyclosporin, tacrolimus, rapamycin, mycophenolic acid, steroids, romidepsin, cytokines, and irradiation, peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg 108:963-971.
  • immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate, and tacrolimus
  • antibodies or other immunoablative agents such as alemtuzumab, anti-CD3 antibodies or other antibody therapies
  • the subject can be administered an agent which reduces or ameliorates a side effect associated with the administration of a TFP-expressing cell.
  • Side effects associated with the administration of a TFP-expressing cell include, but are not limited to, cytokine release syndrome (CRS), and hemophagocytic lymphohistiocytosis (HLH), also termed Macrophage Activation Syndrome (MAS).
  • CRS cytokine release syndrome
  • HHL hemophagocytic lymphohistiocytosis
  • MAS Macrophage Activation Syndrome
  • Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like.
  • the methods described herein can comprise administering a TFP-expressing cell described herein to a subject and further administering an agent to manage elevated levels of a soluble factor resulting from treatment with a TFP-expressing cell.
  • the soluble factor elevated in the subject is one or more of IFN- ⁇ , TNFa, IL-2 and IL-6. Therefore, an agent administered to treat this side effect can be an agent that neutralizes one or more of these soluble factors.
  • agents include, but are not limited to a steroid, an inhibitor of TNFa, and an inhibitor of IL-6.
  • a TNFa inhibitor is etanercept (marketed under the name ENBREL®).
  • An example of an IL-6 inhibitor is tocilizumab (marketed under the name ACTEMRA®).
  • the subject can be administered an agent which enhances the activity of a TFP-expressing cell.
  • the agent can be an agent which inhibits an inhibitory molecule.
  • Inhibitory molecules e.g., Programmed Death 1 (PD1)
  • PD1 can, in some embodiments, decrease the ability of a TFP-expressing cell to mount an immune effector response.
  • Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR beta.
  • Inhibition of an inhibitory molecule e.g., by inhibition at the DNA, RNA or protein level, can optimize a TFP-expressing cell performance.
  • an inhibitory nucleic acid e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA
  • an inhibitory nucleic acid e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA
  • the inhibitor is a shRNA.
  • the inhibitory molecule is inhibited within a TFP-expressing cell.
  • a dsRNA molecule that inhibits expression of the inhibitory molecule is linked to the nucleic acid that encodes a component, e.g., all of the components, of the TFP.
  • the inhibitor of an inhibitory signal can be, e.g., an antibody or antibody fragment that binds to an inhibitory molecule.
  • the agent can be an antibody or antibody fragment that binds to PD1, PD-L1, PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX-101, and marketed as YERVOY®; Bristol-Myers Squibb; tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206)).
  • ipilimumab also referred to as MDX-010 and MDX-101, and marketed as YERVOY®
  • tremelimumab IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206
  • the agent is an antibody or antibody fragment that binds to T cell immunoglobulin and mucin-domain containing-3 (TIM3). In an embodiment, the agent is an antibody or antibody fragment that binds to Lymphocyte-activation gene 3 (LAG3).
  • TIM3 T cell immunoglobulin and mucin-domain containing-3
  • LAG3 Lymphocyte-activation gene 3
  • the agent which enhances the activity of a TFP-expressing cell can be, e.g., a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule, or fragment thereof, and the second domain is a polypeptide that is associated with a positive signal, e.g., a polypeptide comprising an intracellular signaling domain as described herein.
  • the polypeptide that is associated with a positive signal can include a costimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27 and/or ICOS, and/or a primary signaling domain, e.g., of CD3 zeta, e.g., described herein.
  • the fusion protein is expressed by the same cell that expressed the TFP.
  • the fusion protein is expressed by a cell, e.g., a T cell that does not express an anti-tumor- associated antigen TFP.
  • the additional therapeutic agent comprises an immunostimulatory agent.
  • the immunostimulatory agent is an agent that blocks signaling of an inhibitory receptor of an immune cell, or a ligand thereof.
  • the inhibitory receptor or ligand is selected from cytotoxic T-lymphocyte-associated protein 4 (CTLA-4, also known as CD 152), programmed cell death protein 1 (also PD-1 or CD279), programmed death ligand 1 (also PD-L1 or CD274), transforming growth factor beta (TGF ), lymphocyte-activation gene 3 (LAG-3, also CD223), Tim-3 (hepatitis A virus cellular receptor 2 or HAVCR2 or CD366), neuritin, B- and T- lymphocyte attenuator (also BTLA or CD272), killer cell immunoglobulin-like receptors (KIRs), and combinations thereof.
  • the agent is selected from an anti -PD-1 antibody (e.g., pembrolizumab or nivolumab), and anti-PD-Ll antibody (e.g., atezolizumab), an anti-CTLA-4 antibody (e.g., ipilimumab), an anti-TIM3 antibody, carcinoembryonic antigen-related cell adhesion molecule 1 (CECAM-1, also CD66a) and 5 (CEACAM-5, also CD66e), vset immunoregulatory receptor (also VISR or VISTA), leukocyte-associated immunoglobulin-like receptor 1 (also LAIR1 or CD305), CD 160, natural killer cell receptor 2B4 (also CD244 or SLAMF4), and combinations thereof.
  • the agent is pembrolizumab.
  • the agent is nivolumab.
  • the agent is atezolizumab.
  • the additional therapeutic agent is an agent that inhibits the interaction between PD- 1 and PD-L 1.
  • the additional therapeutic agent that inhibits the interaction between PD- 1 and PD-L 1 is selected from an antibody, a peptidomimetic and a small molecule.
  • the additional therapeutic agent that inhibits the interaction between PD- 1 and PD-L 1 is selected from pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), atezolizumab, avelumab, pidilizumab, durvalumab, sulfamonomethoxine 1, and sulfamethizole 2.
  • the additional therapeutic agent that inhibits the interaction between PD- 1 and PD-L 1 is any therapeutic known in the art to have such activity, for example as described in Weinmann et al., Chem Med Chem, 2016, 14: 1576 (DOI: 10.1002/cmdc.201500566), incorporated by reference in its entirety.
  • the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in the same pharmaceutical composition an antibody provided herein.
  • the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in a different pharmaceutical composition from an antibody provided herein.
  • the agent that inhibits the interaction between PD-1 and PD-L1 is administered prior to administration of an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is administered after administration of an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is administered contemporaneously with an antibody provided herein, but the agent and antibody are administered in separate pharmaceutical compositions.
  • the immunostimulatory agent is an agonist of a co-stimulatory receptor of an immune cell.
  • the co-stimulatory receptor is selected from GITR, 0X40, ICOS, LAG-2, CD27, CD28, 4-1BB, CD40, STING, a toll-like receptor, RIG-1, and a NOD-like receptor.
  • the agonist is an antibody.
  • the immunostimulatory agent modulates the activity of arginase, indoleamine-2 3-dioxygenase, or the adenosine A2A receptor.
  • the immunostimulatory agent is a cytokine.
  • the cytokine is selected from IL -2, IL-5, IL-7, IL- 12, IL- 15, IL-21, and combinations thereof.
  • the immunostimulatory agent is an oncolytic virus.
  • the oncolytic virus is selected from a herpes simplex virus, a vesicular stomatitis virus, an adenovirus, a Newcastle disease virus, a vaccinia virus, and a maraba virus.
  • additional therapeutic agents include a taxane (e.g., paclitaxel or docetaxel); a platinum agent (e.g., carboplatin, oxaliplatin, and/or cisplatin); a topoisomerase inhibitor (e.g., irinotecan, topotecan, etoposide, and/or mitoxantrone); folinic acid (e.g., leucovorin); or a nucleoside metabolic inhibitor (e.g., fluorouracil, capecitabine, and/or gemcitabine).
  • the additional therapeutic agent is folinic acid, 5-fluorouracil, and/or oxaliplatin.
  • the additional therapeutic agent is 5-fluorouracil and irinotecan. In some embodiments, the additional therapeutic agent is a taxane and a platinum agent. In some embodiments, the additional therapeutic agent is paclitaxel and carboplatin. In some embodiments, the additional therapeutic agent is pemetrexate. In some embodiments, the additional therapeutic agent is a targeted therapeutic such as an EGFR, RAF or MEK-targeted agent.
  • the additional therapeutic agent may be administered by any suitable means.
  • a medicament provided herein, and the additional therapeutic agent are included in the same pharmaceutical composition.
  • an antibody provided herein, and the additional therapeutic agent are included in different pharmaceutical compositions.
  • administration of the antibody can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about one month of each other.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about one week of each other.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about one day of each other.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about twelve hours of each other.
  • administration of an antibody provided herein, and the additional therapeutic agent occur within about one hour of each other.
  • the additional therapeutic agent is an agent that increases levels of GPC3 in cancer cells associated with elevated expression of GPC3.
  • the agent that increases levels of GPC3 is an agent that inhibits DNA methylation.
  • the agent that increases levels of GPC3 is an agent that inhibits DNA methyltransferease.
  • the agent that increases levels of GPC3 is a hypomethylating agent.
  • the hypomethylating agent includes, but are not limited to 5-azacitidine and decitabine and also includes any hypomethylating agent known in the art.
  • the hypomethylating agent is 5- azacitidine.
  • the hypomethylating agent is decitabine.
  • the hypomethylating agent is a derivative of decitabine or a derivative of 5-azacitidine. In some embodiments, the hypomethylating agent is an esterificated azacytidine, an acetylated azacitidine, an esterificated decitabine, or an acetylated decitabine.
  • an imaging method e.g., an immunopositron emisson tomography (immunoPET) or immunohistochemistry (IHC)
  • IHC immunohistochemistry
  • a liquid chromatography-ion trap-mass spectrometry can be used to measure salivary levels of GPC3.
  • a tumor tissue sample is obtained from a subject and the fraction of cells expressing GPC3 or expression pattern of GPC3 is determined.
  • a blood sample is obtained from a subject and the fraction of cells expressing GPC3 is determined.
  • the relative amount of GPC3 expressed by such cells is determined.
  • the fraction of cells expressing GPC3 and the relative amount of GPC3 expressed by such cells can be determined by any suitable method.
  • flow cytometry is used to make such measurements.
  • fluorescence assisted cell sorting (FACS) is used to make such measurement.
  • the levels of serum GPC3 can be determined.
  • ELISA can be used to determine the serum GPC3 levels.
  • Sandwich ELISA can be used to determine the serum GPC3 levels. See Capurro et al., Li et al., Gastroenterology, 2003, 125 ( 1 ) : 89-97 for methods of evaluating GPC3 expression in peripheral blood.
  • the dual specificity TFP T cells are administered with an additional anti-cancer agent; in some embodiments, the anti- cancer agent is an antibody or fragment thereof, another TFP T cell, a CAR T cell, or a small molecule.
  • tumor-associated antigens include, but are not limited to, oncofetal antigens (e.g., those expressed in fetal tissues and in cancerous somatic cells), oncoviral antigens (e.g., those encoded by tumorigenic transforming viruses), overexpressed/ accumulated antigens (e.g., those expressed by both normal and neoplastic tissue, with the level of expression highly elevated in neoplasia), cancer- testis antigens (e.g., those expressed only by cancer cells and adult reproductive tissues such as testis and placenta), lineage-restricted antigens (e.g., those expressed largely by a single cancer histotype), mutated antigens (e.g., those expressed by cancer as a result of genetic mutation or alteration in transcription), posttranslationally altered antigens (e.g., those tumor-associated alterations in glycosylation, etc.), and idiotypic antigens (e.g., those from highly polymorphic genes where a
  • tumor-associated antigens include, but are not limited to, antigens of alpha-actinin-4, ARTCI, alphafetoprotein (AFP), BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8, beta-catenin, Cdc27, CDK4, CDK12, CDKN2A, CLPP, COA-1, CSNK1A1, CD79, CD79B, dek-can fusion protein, EFTUD2, Elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD, FNDC3B, FN1, GAS7, GPNMB, HAUS3, HSDL1, LDLR-fticosyltransferase AS fusion protein, HLA-A2d, HLA-Al ld, hsp70-2, MART2, MATN, MEI, MUM-lf, MUM-2, MUM-3, neo-PAP, Myosin class I, NFYC
  • compositions of the present invention may comprise a TFP-expressing cell, e.g., a plurality of TFP-expressing cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • Compositions of the present invention are in one aspect formulated for intravenous administration.
  • compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented).
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.
  • the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti- CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.
  • a contaminant e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti- CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.
  • the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A.
  • an immunologically effective amount When “an immunologically effective amount,” “an anti-tumor effective amount,” “a tumor- inhibiting effective amount,” or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 10 4 to 10 9 cells/kg body weight, in some instances 10 5 to 10 6 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).
  • T cells can be activated from blood draws of from 10 cc to 400 cc.
  • T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc.
  • compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection.
  • the T cell compositions of the present invention are administered by i.v. injection.
  • the compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.
  • subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g., T cells.
  • T cell isolates may be expanded by methods known in the art and treated such that one or more TFP constructs of the invention may be introduced, thereby creating a TFP-expressing T cell of the invention.
  • Subjects in need thereof may subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • subjects receive an infusion of the expanded TFP T cells of the present invention.
  • expanded cells are administered before or following surgery.
  • the dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
  • the scaling of dosages for human administration can be performed according to art-accepted practices.
  • the dose for alemtuzumab (CAMPATH®) will generally be in the range 1 to about 100 mg for an adult patient, usually administered daily for a period between 1 and 30 days.
  • the preferred daily dose is 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used (described, e.g., in U.S. Pat. No. 6,120,766).
  • the TFP is introduced into T cells, e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of TFP T cells of the invention, and one or more subsequent administrations of the TFP T cells of the invention, wherein the one or more subsequent administrations are administered less than 15 days, e.g., 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration.
  • more than one administration of the TFP T cells of the invention are administered to the subject (e.g., human) per week, e.g., 2, 3, or 4 administrations of the TFP T cells of the invention are administered per week.
  • the subject receives more than one administration of the TFP T cells per week (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no TFP T cells administrations, and then one or more additional administration of the TFP T cells (e.g., more than one administration of the TFP T cells per week) is administered to the subject.
  • the subject receives more than one cycle of TFP T cells, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days.
  • the TFP T cells are administered every other day for 3 administrations per week.
  • the TFP T cells of the invention are administered for at least two, three, four, five, six, seven, eight or more weeks.
  • tumor-associated antigen TFP T cells are generated using lentiviral viral vectors, such as lentivirus. TFP-T cells generated that way will have stable TFP expression.
  • TFP T cells transiently express TFP vectors for 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days after transduction.
  • Transient expression of TFPs can be effected by RNA TFP vector delivery.
  • the TFP RNA is transduced into the T cell by electroporation.
  • a potential issue that can arise in patients being treated using transiently expressing TFP T cells is anaphylaxis after multiple treatments.
  • anaphylactic response might be caused by a patient developing humoral anti-TFP response, i.e. , anti-TFP antibodies having an anti- IgE isotype. It is thought that a patient’s antibody producing cells undergo a class switch from IgG isotype (that does not cause anaphylaxis) to IgE isotype when there is a ten- to fourteen-day break in exposure to antigen.
  • TFP T cell infusion breaks should not last more than ten to fourteen days.
  • Cytokine release syndrome is a form of systemic inflammatory response syndrome that arises as a complication of some diseases or infections, and is also an adverse effect of some monoclonal antibody drugs, as well as adoptive T cell therapies.
  • TFP T cells can exhibit better killing activity than CAR-T cells.
  • TFP T cells administered to a subject can exhibit better killing activity than CAR-T cells administered to a subject. This can be one of the advantages of TFP T cells over CAR-T cells.
  • TFP T cells can exhibit less cytokine release CAR-T cells.
  • a subject administered TFP T cells can exhibit less cytokine release than a subject administered CAR-T cells. This can be one of the advantages of TFP T cell therapies over CAR-T cell therapies.
  • TFP T cells can exhibit similar or better killing activity than CAR-T cells and the TFP T cells can exhibit less cytokine release than the CAR-T cells.
  • TFP T cells administered to a subject can exhibit similar or better killing activity than CAR-T cells administered to a subject and the subject can exhibit less cytokine release than a subject administered CAR-T cells. This can be one of the advantages of TFP T cell therapies over CAR-T cell therapies.
  • the cytokine release of a treatment with TFP T cells is less than the cytokine release of a treatment with CAR-T cells. In some embodiments, the cytokine release of a treatment with TFP T cells is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% less than the cytokine release of a treatment with CAR-T cells. Various cytokines can be released less in the T cell treatment with TFP T cells than CAR-T cells.
  • the cytokine is IL -2, IFN- ⁇ , IL -4, TNF- ⁇ , IL-6, IL-13, IL-5, IL-10, sCD137, GM- CSF, MIP-la, MIP-ip, or a combination thereof.
  • the treatment with TFP T cells release less perforin, granzyme A, granzyme B, or a combination thereof, than the treatment with CAR-T cells.
  • the perforin, granzyme A, or granzyme B released in a treatment with TFP T cells is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or at least 60% less than a treatment with CAR-T cells.
  • the given cytokine comprises one or more cytokines selected from the group consisting of IL -2, IFN- ⁇ , IL-4, TNF- ⁇ , IL- 6, IL-13, IL-5, IL-10, sCD137, GM-CSF, MIP-la, MIP-1 , and any combination thereof.
  • the TFP T cells may exhibit similar or better activity in killing tumor cells than CAR-T cells.
  • a tumor growth in the mammal is inhibited such that a size of the tumor is at most 10%, at most 20%, at most 30%, at most 40%, at most 50%, or at most 60% of a size of a tumor in a mammal treated with T cells that do not express the TFP after at least 8 days of treatment, wherein the mammal treated with T cells expressing TFP and the mammal treated with T cells that do not express the TFP have the same tumor size before the treatment.
  • the tumor growth in the mammal is completely inhibited.
  • the tumor growth in the mammal is completely inhibited for at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least 100 days, or more.
  • the population of T cells transduced with TFP kill similar amount of tumor cells compared to the CAR-T cells comprising the same binding domain.
  • the TFP T cells can exhibit different gene expression profde than cells that do not express TFP.
  • the TFP T cells may exhibit similar gene expression profdes than CAR-T cells.
  • the TFP T cells may exhibit different gene expression profdes than CAR-T cells.
  • the population of T cells transduced with TFP have a different gene expression profde than the CAR-T cells comprising the same binding domain.
  • an expression level of a gene is different in the T cells transduced with the TFP than an expression level of the gene in the CAR-T cells comprising the same binding domain.
  • the gene has a function in antigen presentation, TCR signaling, homeostasis, metabolism, chemokine signaling, cytokine signaling, toll like receptor signaling, MMP and adhesion molecule signaling, or TNFR related signaling.
  • scFv GPC3 antibodies were generated by phage display. 2 rounds of selection were performed on biotinylated human GPC3 using four Gen3 naive phage libraries. Following the second round of selection, polyclonal phage affinity for GPC3 was confirmed by ELISA and flow cytometry (data not shown).
  • the output of the second round of phage selection was cloned into a yeast display vector for selection by yeast display. Two rounds of selection for binding biotinylated human GPC3 by flow cytometry were performed. Once a population of GPC3 -binding yeast cells was identified, negative selection against GPC 1, 2, 4, 5, and 6 was performed, followed by positive clonal selection for cross- reactivity to mouse or cyno GPC3. NGS and Sanger sequencing were performed on the output of each phage library that had undergone selection for GPC3 binding, selected against GPC 1, 2, 4, 5, and 6 binding and selected for mouse or cyno cross-reactivity in yeast.
  • NGS data identified 1506 unique H3/L3 pairings of triple cross-reactive H3/L3 pairings, comprised of 367 unique HCDR3 sequences and -125 unique LCDR#3 sequences.
  • Sanger sequencing of two 96-well plates of yeast clones identified 99 unique scFv clones with cross-reactivity to human, mouse, and cyno GPC3, and that overlap with NGS data to yield a total of 72 unique HCDR3 sequences from the 4 libraries.
  • Binding affinities (KD) of the 16 scFv-hFc fusions were measured to range from 0.1 - 29.0 nM, as determined by kinetic titration using a Carterra LSA instrument for surface plasmon resonance (SPR) detection of human GPC3 binding to anti-GPC3 scFv-hFc fusions captured by anti-human Fc antibody functionalized to HC30M chips.
  • 3 of 16 scFv-hFc were disqualified due to fast GPC3 off-rates (k o ff> 10’ 2 s’ 1 ).
  • Epitope binning analysis of the remaining 13 clones was performed using SPR detection on an LSA Carterra instrument via a sandwich assay format.
  • Anti-GPC3 TFP constructs were engineered by cloning anti-GPC3 binding domain (V HH domain or scFv domains) DNA fragment linked to a CD3 or TCR DNA fragment by either a DNA sequence encoding a short linker (SL): AAAGGGGSGGGGSGGGGSLE (SEQ ID NO:692) or a long linker (LL): AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE (SEQ ID NO:693) into a lentiviral vector.
  • SL short linker
  • LL long linker
  • AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE
  • the anti-GPC3 binding domain is any of the anti-scFv antibody domains of Table 1 in either the VH-VL or VL-VH orientation. In some embodiments, the anti-GPC3 binding domain is selected from 9F2, GC33, YP7, hYP7, 32A9, HN3, or MDX-1414.
  • Subunits of the human T Cell Receptor (TCR) complex all contain an extracellular domain and a transmembrane domain.
  • the CD3 epsilon, CD3 delta, and CD3 gamma subunits have an intracellular domain.
  • a human TCR complex contains the CD3-epsilon polypeptide, the CD3-gamma poly peptide, the CD3-delta polypeptide, and the TCR alpha chain polypeptide and the TCR beta chain polypeptide or the TCR delta chain polypeptide and the TCR gamma chain polypeptide.
  • TCR alpha, TCR beta, TCR gamma, and TCR delta recruit the CD3 zeta polypeptide.
  • the human CD3 -epsilon polypeptide canonical sequence is Uniprot Accession No. P07766.
  • the human CD3-gamma polypeptide canonical sequence is Uniprot Accession No. P09693.
  • the human CD3-delta polypeptide canonical sequence is Uniprot Accession No. P043234.
  • the human CD3-zeta polypeptide canonical sequence is Uniprot Accession No. P20963.
  • the human TCR alpha chain canonical sequence is Uniprot Accession No. Q6ISU1.
  • the human TCR beta chain C region canonical sequence is Uniprot Accession No. P01850, a human TCR beta chain V region sequence is P04435.
  • the human CD3-epsilon polypeptide canonical sequence is: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQH NDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENC MEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPP VPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 694).
  • the signal peptide of human CD3 ⁇ is:
  • the transmembrane domain of human CD3 ⁇ is:
  • the intracellular domain of human CD3 ⁇ is:
  • the human CD3-gamma polypeptide canonical sequence is: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKM IGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAE IVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO:699).
  • the signal peptide of human CD3 ⁇ is:
  • the transmembrane domain of human CD3 ⁇ is:
  • the intracellular domain of human CD3 ⁇ is:
  • GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO: 703).
  • the human CD3-delta polypeptide canonical sequence is: MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRI LDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFA GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKS (SEQ ID NO:704).
  • the signal peptide of human CD3 ⁇ is:
  • the transmembrane domain of human CD3 ⁇ is: GIIVTDVIATLLLALGVFCFA (SEQ ID NO:707).
  • the intracellular domain of human CD3 ⁇ is:
  • the human CD3-zeta polypeptide canonical sequence is: MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPA YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:709).
  • the human TCR alpha chain canonical sequence is: MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLDSPI WFSAGNGSALDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHSRSTQP MHLSGEASTARTCPQEPLRGTPGGALWLGVLRLLLFKLLLFDLLLTCSCLCDPAGPLPSPAT TTRLRALGSHRLHPATETGGREATS SPRPQPRDRRWGDTPPGRKPGSP VWGEGSYLS SYPTC PAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGAA (SEQ ID NO:710).
  • the human TCR alpha chain C region canonical sequence is: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSA VAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLK VAGFNLLMTLRLWSS (SEQ ID NO:711).
  • the human TCR alpha chain human IgC sequence is:
  • the transmembrane domain of the human TCR alpha chain is:
  • VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO:713).
  • the intracellular domain of the human TCR alpha chain is:
  • the human TCR alpha chain V region CTL-L17 canonical sequence is: MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFL WYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAAKGAGT ASKLTFGTGTRLQVTL (SEQ ID NO:714).
  • the human TCR beta chain C region 1 (hTRBCl) canonical sequence is: EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQ PLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVS AEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO:715).
  • the human TCR beta chain human IgC sequence is:
  • the transmembrane domain of the human TCR beta chain is:
  • the human TCR beta chain V region CTL-L17 canonical sequence is: MGTSLLCWMALCLLGADHADTGVSQNPRHNITKRGQNVTFRCDPISEHNRLYWYRQTLGQ GPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLAGLNQPQ HFGDGTRLSIL (SEQ ID NO:718).
  • the intracellular domain of the human TCR beta chain is:
  • VKRKDF (SEQ ID NO: 719)
  • the human TCR beta chain V region YT35 canonical sequence is: MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMR GLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSFSTCSANYG YTFGSGTRLTVV (SEQ ID NO:720).
  • the human TCR gamma chain C region canonical sequence is:
  • the human TCR gamma human IgC sequence is:
  • transmembrane domain of the human TCR gamma chain is:
  • the intracellular domain of the human TCR gamma chain is:
  • the human TCR delta chain C region canonical sequence is:
  • the human TCR delta human IgC sequence is:
  • the transmembrane domain of the human TCR delta chain is:
  • LGLRMLFAKTVAVNFLLTAKLFF (SEQ ID NO:727).
  • the intracellular domain of the human TCR delta chain is:
  • the murine TCR alpha chain constant (mTRAC) region canonical sequence is:
  • the murine TCR alpha chain (2-137) sequence is:
  • the murine TCR beta chain constant region canonical sequence (murine TCR beta chain constant region 1 canonical sequence) is:
  • the murine TCR beta chain (2-173) sequence is:
  • the transmembrane domain of the murine TCR alpha chain is: MGLRILLLKVAGFNLLMTLRLW (SEQ ID NO:736).
  • the transmembrane domain of the murine TCR beta chain 1 is: ILYEILLGKATLYAVLVS TLWMAMVK (SEQ ID NO:738).
  • the intracellular domain of the murine TCR beta chain 1 is:
  • KRKNS (SEQ ID NO: 739).
  • the murine TCR beta chain constant 2 region canonical sequence is: XDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQ AYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAW GRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO: 740).
  • Expression vectors include: a promoter (eukaryotic elongation factor 1 alpha (EFla promoter), a signal sequence to enable secretion, a polyadenylation signal and transcription terminator (Bovine Growth Hormone (BGH) gene), an element allowing episomal replication and replication in prokaryotes e.g., SV40 origin and ColEl or others known in the art) and elements to allow selection (ampicillin resistance gene and zeocin marker).
  • a promoter eukaryotic elongation factor 1 alpha (EFla promoter)
  • EFla promoter eukaryotic elongation factor 1 alpha
  • BGH Bovine Growth Hormone
  • the TFP-encoding nucleic acid construct was cloned into the lentiviral expression vector as is described above.
  • the anti-GPC3 TFP lentiviral transfer vector was used to produce the genomic material packaged into the VSV-G pseudotyped lentiviral particles.
  • the virus stock preparation is either used for infection immediately or aliquoted and stored at -80°C for future use.
  • TFP T cells expressing GC33, HN3, or hYP7 CD3e TFP T cells were generated for 3 donors.
  • T cells were purified from healthy donor leukopak via positive selection of CD4+ and CD8+ T cells with CD4 and CD8 microbeads from Miltenyi Biotech.
  • T cells freshly isolated or thawed from previously prepared frozen vials, were activated by MACS GMP T cell TransAct (Miltenyi Biotech), in the presence of human IL-7 and IL- 15 (both from Miltenyi Biotech, premium grade).
  • activated T cells were transduced with lentivirus encoding the GPC3 TFP.
  • GPC3 TFPs Following lentiviral transduction, expression of GPC3 TFPs by transduced T cells was confirmed by flow cytometry, using AF647-labeled GPC3, on day 10 of cell expansion. As is shown in FIGs. 5A-5C, GPC3 TPF expression was detected in cells transduced with each of the GPC3 TFP constructs (GC33, HN3, or hYP7 CD3e TFP T cells) in each of the three donors. Transduction efficiency was at least 50% for all constructs in all donors. No TFP expression was detected in untransduced cells.
  • the memory phenotype was also determined for the GPC3 TFP constructs transduced T cells on day 10.
  • the memory status of the T cells was determined by flow cytometry to detect cell surface levels of CD45RA and CCR7 as is shown in FIGs. 5A-5C.
  • the luciferase-based cytotoxicity assay assesses the cytotoxicity of TFP T cells by indirectly measuring the luciferase enzymatic activity in the residual live target cells after co-culture.
  • GPC3- positive and GPC3 negative tumor cell lines (HepG2, Hep3B, SNU398 and A549) were modified to overexpress firefly luciferase via transduction with firefly luciferase gene encoding lentivirus followed with antibiotic selection to generate stable cell lines.
  • GPC3 expression levels of each cell line is shown in FIG. 6.
  • HepG2, Hep3B, and SNU398 expresses high level of GPC3, and A549 expresses very low level (close to negative) of GPC3.
  • the target cells were plated at 10000 cells per well in 96-well plate.
  • the GPC3.TFP transduced or untransduced T cells were added to the target cells at different effector-to-target ratios (3:1, 1:1, or 1:3).
  • the mixture of cells was then cultured for 24 hrs at 37 °C with 5 % CO2 before the luciferase enzymatic activity in the live target cells was measured by the Bright-Glo® Luciferase Assay System (Promega®, Catalogue number E2610). After centrifugation of the 96 well plates, supernatant was taken for the cytokine secretion assay.
  • % Cytotoxicity 100% x [1 - RLU (Tumor cells + T cells) / RLU (Tumor cells)].
  • GPC3 TFP transduced T cells bearing GPC3 binders GC33, HN3, or hYP7 from all three donors demonstrated specific and robust cytotoxicity towards GPC3 high cell lines HepG2, Hep3B, and SNU398, and low to no cytotoxicity against GPC3 low A549 cells.
  • effector T-cell activation and proliferation associated with the recognition of cells bearing cognate antigen is the production of effector cytokines such as interferon-gamma (IFN- y), interleukin 2 (IL-2), GM-CSF, and tumor necrosis factor alpha (TNF- ⁇ ).
  • effector cytokines such as interferon-gamma (IFN- y), interleukin 2 (IL-2), GM-CSF, and tumor necrosis factor alpha (TNF- ⁇ ).
  • Target-specific cytokine production including IFN-y, IL -2, TNF- ⁇ and GM-CSF by TFP T cells was measured from supernatants harvested from the cytotoxicity assay described in Example 4 at 24 hours after the co-culture of T cells with GPC3 high and GPC3 low target cells using the U- PLEX® Biomarker Group I (hu) Assays (Meso Scale Diagnostics®, LLC, catalog number: K15067L- 4).
  • FIGs. 8A-8D consistent with cytotoxicity assay results (FIG. 7), robust production of IFN-y, IL-2, TNF- ⁇ , and GM-CSF was observed for all GPC-3 TFP-cells from all 3 donors, when co-cultured with GPC-3 high HepG2 cells.
  • GPC-3 TFP cells produced less cytokines when co-cultured with Hep3B and SNU398 cells, in comparison to HepG2, which is consistent with the lower expression of GPC3 on the cell lines (FIG. 6).
  • No cytokine production was observed for GPC-3 TFP cells when co-cultured with GPC3 low A549 target cells, or for non-transduced T cells when co-cultured with any tumor cell line.
  • Example 6 Characterization of T cell receptor fusion protein T Cells using novel anti-GPC3 scFv
  • TCR fusion proteins having the following novel anti-GPC3 scFv binders in both the VH-VL and VL-VH fused to CD3 epsilon were generated.
  • T cells from one donor were activated, transduced, and expanded as is described in Example 3.
  • the constructs used are shown in Table 9 below.
  • the hYP7 TFP and untransduced cells were included as controls. Expansion of the cells is shown in FIGs. 9A-9B.
  • GPC3 TFPs Following lentiviral transduction, expression of GPC3 TFPs by transduced T cells was confirmed by flow cytometry, using an anti-Fab antibody, and GPC3 binding was measured, on day 10 of cell expansion. Viral titer in the transduced cells was also measured. As is shown in FIGs. 10A- 10B, GPC3 TPF expression and GPC3 binding was detected in the majority of cells transduced with each of the GPC3 TFPs. However, little GPC3 TFP expression was seen in cells transduced with 1- A08 vHvL TFP or 1-G10 vHvL TFP due to low viral titer. These cells were not studied further. The staining for 2.19 vLvH and 1-H07 vHvL suggested that the samples may contain a mixed population of cells. Therefore, these cells were not characterized for cytotoxicity or cytokine production.
  • the memory phenotype was also determined for the GPC3 TFP constructs transduced T cells on day 10.
  • the memory status of the T cells was determined by flow cytometry to detect cell surface levels of CD45RA and CCR7 in CD4+ T cells and CD8+ T cells as is shown in FIGs. 11A-11D.
  • Basal activation status was also determined by measuring CD69 levels by flow cytometry as is shown in FIGs. 12A-12B.
  • cytotoxicity of the GPC3 TFP T cells towards HepG2, SNU398 and A549 target cells was determined as described in Example 4 using the luciferase-based cytotoxicity assay.
  • GPC3 TFP T cells or untransduced controls were contacted with HepG2, SNU398 or A549 target cells at 3:1, 1:1, or 1:3 ratio for 24 hours and cytotoxicity was determined. The results are shown in FIGs. 13A-13B.
  • TFPs having many of the novel svFv binders showed cytotoxicity that approached that of the hYP7 TPF towards HepG2 and SNU398 cells, particularly at the 3:1 ratio, including at least 1.11 vLvH CD3 ⁇ , 1.12 vLvH CD3 ⁇ , 1-C05 vLvH CD3 ⁇ , 2.6 vLvH CD3 ⁇ , 1.11 vHvL CD3 ⁇ , 1.12 vHvL CD3 ⁇ , 1-C05 vHvL CD3 ⁇ , 2.6 vHvL CD3 ⁇ , 1-H07 vLvH CD3 ⁇ , 2.19 vHvL CD3 ⁇ , 1-A08 vLvH CD3 ⁇ , and 1-G10 vLvH CD3 ⁇ .
  • the GPC3 TFPs showed little to no cytotoxicity against GPC3 low A549 cells.
  • Target-specific cytokine production including IFN- ⁇ , IL-2, TNF- ⁇ and GM-CSF by TFP T cells was measured from supernatants harvested from the cytotoxicity assay described above at 24 hours after the co-culture of T cells with HepG2, SNU398 and A549 target cells using the methods described in Example 5.
  • FIGs. 14A-14D consistent with cytotoxicity assay results (FIGs. 13A-13B), many of the constructs induced cytokine production when contacted with HepG2 target cells, particularly at a 3:1 ratio, though the levels were substantially lower than that induced by the hYP7 TFP.
  • TFP T cells shown to induce cytokine production when contacted with HepG2 target cells include at least those expressing 1.11 vLvH CD3 ⁇ , 1.12 vLvH CD3 ⁇ , 1-C05 vLvH CD3 ⁇ , 2.6 vLvH CD3 ⁇ , 1.11 vHvL CD3 ⁇ , 1.12 vHvL CD3 ⁇ , 1-C05 vHvL CD3 ⁇ , 2.6 vHvL CD3 ⁇ , 1-H07 vLvH CD3 ⁇ , 2.19 vHvL CD3 ⁇ , 1-A08 vLvH CD3 ⁇ , and 1-G10 vLvH CD3 ⁇ .
  • the GPC3 TFPs showed little to no induction of cytokine production when contacted with GPC3 low A549 cells.
  • Table 7 Examples of IL-15 or IL-15R Sequences
  • Table 8 Examples of CXCR6 Sequences

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Abstract

La présente invention concerne des protéines de fusion du récepteur des lymphocytes T (TCR) comprenant des domaines de liaison à GPC3, des lymphocytes T modifiés pour exprimer une ou plusieurs protéines de fusion, ainsi que des procédés d'utilisation associés pour le traitement de maladies, y compris le cancer.
PCT/US2021/049956 2020-09-10 2021-09-10 Compositions et procédés pour la reprogrammation de tcr au moyen de protéines de fusion spécifiques gpc3 WO2022056321A1 (fr)

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WO2023172967A3 (fr) * 2022-03-09 2023-11-02 TCR2 Therapeutics Inc. Compositions et méthodes pour la reprogrammation de tcr au moyen de protéines de fusion spécifiques de gpc3
WO2023249071A1 (fr) * 2022-06-24 2023-12-28 国立研究開発法人国立がん研究センター Récepteur de lymphocytes t
US11965012B2 (en) 2015-05-18 2024-04-23 TCR2 Therapeutics Inc. Compositions and methods for TCR reprogramming using fusion proteins

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WO2019222275A2 (fr) * 2018-05-14 2019-11-21 TCR2 Therapeutics Inc. Compositions et procédés de reprogrammation de tcr utilisant des protéines de fusion inductibles

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Publication number Priority date Publication date Assignee Title
US11965012B2 (en) 2015-05-18 2024-04-23 TCR2 Therapeutics Inc. Compositions and methods for TCR reprogramming using fusion proteins
WO2023172967A3 (fr) * 2022-03-09 2023-11-02 TCR2 Therapeutics Inc. Compositions et méthodes pour la reprogrammation de tcr au moyen de protéines de fusion spécifiques de gpc3
WO2023249071A1 (fr) * 2022-06-24 2023-12-28 国立研究開発法人国立がん研究センター Récepteur de lymphocytes t

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