WO2023172967A2

WO2023172967A2 - Compositions and methods for tcr reprogramming using gpc3 specific fusion proteins

Info

Publication number: WO2023172967A2
Application number: PCT/US2023/063956
Authority: WO
Inventors: Robert Hofmeister; Dario Gutierrez; Jian Ding; Robert Tighe; Michael Lofgren; Vania ASHMINOVA; Huan Yang; Melinda HANES
Original assignee: TCR2 Therapeutics Inc.
Priority date: 2022-03-09
Filing date: 2023-03-08
Publication date: 2023-09-14
Also published as: WO2023172967A3

Abstract

Provided herein are T cell receptor (TCR) fusion proteins (TFPs) comprising GPC3 binding domains, T cells engineered to express one or more TFPs, and methods of use thereof for the treatment of diseases, including cancer.

Description

COMPOSITIONS AND METHODS FOR TCR REPROGRAMMING USING GPC3

SPECIFIC FUSION PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63/318,176, filed March 9, 2022, the entire content of which is incorporated herein by reference in its entirety.

FIELD

[0002] The present invention is directed to a novel therapeutics and method for treating GPC3- related diseases and disorders.

BACKGROUND

[0003] 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.

[0004] 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.

[0005] Human T cell therapies rely on enriched or modified human T cells to target and kill cancer cells in a patient. To increase the ability of T cells to target and kill a particular cancer cell, methods have been developed to engineer T cells to express constructs which direct T cells to a particular target cancer cell. Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs), which comprise binding domains capable of interacting with a particular tumor antigen, allow T cells to target and kill cancer cells that express the particular tumor antigen. [0006] 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. In addition, CAR therapies currently being developed have been associated with the release of high levels of pro-inflammatory cytokines that have been associated with doselimiting toxicities.

SUMMARY

[0007] There is a clear need to develop improved genetically engineered T cells to act against various human malignancies, including GPC3 expressing malignancies. Described herein are novel fusion proteins of 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.

[0008] In an aspect, provided herein is 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.

[0009] In some embodiments, the TCR intracellular domain comprises a stimulatory domain from an intracellular signaling domain of CD3 gamma, CD3 delta, or CD3 epsilon.

[0010] In some embodiments, the TFP functionally interacts with an endogenous TCR complex when expressed in a T cell. In some embodiments, a T cell expressing the TFP exhibits increased cytotoxicity to a human cell expressing GPC3 compared to a T cell not containing the TFP.

[0011] In some embodiments, the antigen binding domain is connected to the TCR extracellular domain by a linker sequence. In some embodiments, the linker is 120 amino acids in length or less. In some embodiments, 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.

[0012] In some embodiments, 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. In some embodiments, 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.

[0013] In some embodiments, 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.

[0014] In some embodiments, the antigen binding domain is a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain.

[0015] 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. 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.

[0016] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:2. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 1.

[0017] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 18. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 17.

[0018] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:34. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:33.

[0019] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:42. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:41.

[0020] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:50. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:49.

[0021] In some embodiments, 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 ID NO:59, the LC CDR2 of SEQ ID NO:60, the LC CDR3 of SEQ ID NO:61; or a combination thereof. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 58. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:57.

[0022] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:82. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:81.

[0023] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:90. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:89.

[0024] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 98. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:97.

[0025] In some embodiments, the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:782, the HC CDR2 of SEQ ID NO:783, the HC CDR3 of SEQ ID NO:784; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:785, the LC CDR2 of SEQ ID NO:786, the LC CDR3 of SEQ ID NO:787; or a combination thereof. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 780. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:781.

[0026] In some embodiments, the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:838, the HC CDR2 of SEQ ID NO:839, the HC CDR3 of SEQ ID NO:840; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO : 841 , the LC CDR2 of SEQ ID NO : 842, the LC CDR3 of SEQ ID NO : 843 ; or a combination thereof. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 836. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:837.

[0027] In some embodiments, 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. In some embodiments, the TFP comprises a sequence of SEQ ID NO:201. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:207. In some embodiments, 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 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:780. In some embodiments, the heavy chain variable domain comprises a sequence of SEQ ID NO:780. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:781. In some embodiments, the light chain variable domain comprises a sequence of SEQ ID NO:781. In some embodiments, the antigen binding domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:854. In some embodiments, the antigen binding domain comprises a sequence of SEQ ID NO:854. In some embodiments, the antigen binding domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:855. In some embodiments, the antigen binding domain comprises a sequence of SEQ ID NO:855. In some embodiments, the TFP comprises a sequence having at least about 80% sequence identity to SEQ ID NO:852. In some embodiments, the TFP comprises a sequence of SEQ ID NO:852. In some embodiments, the TFP comprises a sequence having at least about 80% sequence identity to SEQ ID NO:853. In some embodiments, the TFP comprises a sequence of SEQ ID NO:853. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:836. In some embodiments, the heavy chain variable domain comprises a sequence of SEQ ID NO:836. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:837. In some embodiments, the light chain variable domain comprises a sequence of SEQ ID NO:837. In some embodiments, the antigen binding domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:883. In some embodiments, the antigen binding domain comprises a sequence of SEQ ID NO:883. In some embodiments, the TFP comprises a sequence having at least about 80% sequence identity to SEQ ID NO:881. In some embodiments, the TFP comprises a sequence of SEQ ID NO:881.

[0028] In some embodiments, a T cell expressing the TFP inhibits tumor growth when expressed in a T cell.

[0029] In another aspect, provided herein is a recombinant nucleic acid molecule comprising a nucleic acid encoding an antibody or fragment thereof that specifically binds GPC3. In some embodiments, the antibody or antibody fragment is a camelid antibody or binding fragment thereof. In some embodiments, the antibody or antibody fragment is a murine, human or humanized antibody or binding fragment thereof. In some embodiments, the antibody or antibody fragment is a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain. In some embodiments, the antibody or antibody fragment is a single domain antibody (sdAb). In some embodiments, the sdAb is a VHH. [0030] 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. 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.

[0031] In some embodiments, 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 In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:2. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 1.

[0032] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 18. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 17.

[0033] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:34. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:33.

[0034] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:42. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:41.

[0035] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:50. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:49.

[0036] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 58. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:57.

[0037] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:82. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:81.

[0038] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:90. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:89.

[0039] In some embodiments, 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. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 98. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:97.

[0040] In some embodiments, the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:782, the HC CDR2 of SEQ ID NO:783, the HC CDR3 of SEQ ID NO:784; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:785, the LC CDR2 of SEQ ID NO:786, the LC CDR3 of SEQ ID NO:787; or a combination thereof. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:780. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:781.

[0041] In some embodiments, the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO:838, the HC CDR2 of SEQ ID NO:839, the HC CDR3 of SEQ ID NO:840; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO : 841 , the LC CDR2 of SEQ ID NO : 842, the LC CDR3 of SEQ ID NO : 843 ; or a combination thereof. In some embodiments, the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:836. In some embodiments, the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:837.

[0042] In some embodiments, 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. In some embodiments, 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.

[0043] In some embodiments, the recombinant nucleic acid molecule further comprises a sequence encoding a TCR constant domain. In some embodiments, the antibody or antibody fragment is operatively linked to the sequence encoding a TCR constant domain, thereby forming a TFP. In some embodiments, 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. In some embodiments, the recombinant nucleic acid molecule further comprises a leader sequence.

[0044] In some embodiments, 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. In some embodiments, the nucleotide analog is selected from the group consisting of 2’-O-methyl, 2’-O- methoxyethyl (2’-0-M0E), 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’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2’-fhioro N3-P5’-phosphoramidite.

[0045] In some embodiments, the recombinant nucleic acid molecule further comprises a promoter. In some embodiments, the nucleic acid is an in vitro transcribed nucleic acid. In some embodiments, the nucleic acid further comprises a sequence encoding a poly(A) tail. In some embodiments, the nucleic acid further comprises a 3’UTR sequence. [0046] In another aspect, provided herein is a polypeptide encoded by a recombinant nucleic acid disclosed herein.

[0047] In another aspect, provided herein is a vector comprising a recombinant nucleic acid molecule encoding a TFP disclosed herein.

[0048] In another aspect, provided herein is a vector comprising a recombinant nucleic acid molecule encoding the TFP disclosed herein. In some embodiments, 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. In some embodiments, the vector further comprises a sequence encoding a TCR constant domain. In some embodiments, 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. In some embodiments, 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. In some embodiments, the vector further comprises a promoter. In some embodiments, the vector is an in vitro transcribed vector. In some embodiments, a nucleic acid sequence in the vector further comprises a poly(A) tail. In some embodiments, a nucleic acid sequence in the vector further comprises a 3’UTR.

[0049] In another aspect, provided herein is a cell comprising a recombinant nucleic acid molecule disclosed herein, a polypeptide disclosed herein, or a vector disclosed herein. In some embodiments, the cell is a T cell. In some embodiments, the T cell is a human T cell. In some embodiments, the T cell is a CD8+ or CD4+ T cell. In some embodiments, the T cell is a human aPT cell. In some embodiments, the T cell is a human y6 T cell. In some embodiments, the cell is a human NKT cell. In some embodiments, the cell comprises a functional disruption of an endogenous TCR. In some embodiments, the cell is an allogeneic cell. In some embodiments, 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. In some embodiments, the cell further comprises a heterologous sequence encoding a TCR constant domain. In some embodiments, 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.

[0050] In another aspect, provided herein is a pharmaceutical composition comprising a cell disclosed herein and a pharmaceutically acceptable carrier.

[0051] In another aspect, provided herein is a method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of a pharmaceutical composition disclosed herein.

[0052] In another aspect, provided herein is a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a cell disclosed herein; and (b) a pharmaceutically acceptable carrier. In some embodiments, the cancer is a cancer associated with elevated expression of GPC3. In some embodiments, 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. In some embodiments, 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. In some embodiments, the subject is a human.

INCORPORATION BY REFERENCE

[0053] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0055] 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.

[0056] 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. [0057] FIG. 3 is a schematic diagram showing binning of the binders shown.

[0058] 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.

[0059] 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.

[0060] FIG. 6 is a series of plots showing GPC3 expression in HepG2, Hep3B, SNU398, and A549 cell lines.

[0061] 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.

[0062] 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-y (FIG. 8 A), IL-2 (FIG. 8B), TNF-a (FIG. 8C), and GM-CSF (FIG. 8D) in cell culture supernatants were measured.

[0063] 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.

[0064] 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.

[0065] 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. Data for CD8+ T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-8, and untransduced controls is shown in FIG. 11C. 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.

[0066] 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.

[0067] 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.

[0068] 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-y, IL-2, TNF-a, and GM-CSF were measured. Levels of IFN- y and IL-2 produced by T cells transduced with hYP7 TFP, GPC3 scFv constructs 1-8, and untransduced controls is shown are FIG. 14A. Levels of IFN-y 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-a 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-a 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.

[0069] FIG. 15 shows efficacy of hYP7 TRuC T cells in clearing tumor cells in an in vivo xenograft mouse model.

[0070] FIG. 16 shows exemplary flow cytometry plots demonstrating the surface expression of GPC3 binders in transduced T cells.

[0071] FIG. 17 shows exemplary phenotypic characterization of hYP7 TFP transduced T cells as measured by flow cytometry detection of CD4, CD8, CD69, CD45RA, and CCR7.

[0072] FIG. 18 shows the results of luciferase-based cytotoxicity assay after 24hr co-culture of hYP7 TFP T cells with HepG2, SNU398, or A549 target cells.

[0073] FIG. 19 shows cytokine levels as measured in the supernatant following 24hr co-culture of hYP7 TFP T cells and HepG2, SNU398, or A549 target cells.

[0074] FIG. 20 shows exemplary flow cytometry plots demonstrating the transduction efficiency of GC33 TFP variants.

[0075] FIG. 21 shows exemplary phenotypic characterization of GC33 TFP transduced T cells as measured by flow cytometry detection of CD4, CD8, CD69, CD45RA, and CCR7.

[0076] FIG. 22 shows the results of luciferase-based cytotoxicity assay after 24hr co-culture of GC33 TFP T cells with HepG2, SNU398, or A549 target cells.

[0077] FIG. 23 shows cytokine levels as measured in the supernatant following 24hr co-culture of GC33 TFP cells and HepG2, SNU398, or A549 target cells.

[0078] FIG. 24 shows expression of GPC3 TFPs in cells transduced with the indicated constructs, on Day 10 after transduction. The left panel shows the % frequency of GPC3+ cells and the right panel shows the mean fluorescence intensity (MFI) of TFP+ cells.

[0079] FIG. 25 shows the fold expansion of cells transduced with the indicated constructs, on Day 10 after transduction.

[0080] FIG. 26 provides a flow cytometric analysis of the CD4 and CD8 compartments in cells transduced with the indicated GPC3 TFP constructs, on Day 10 after transduction. The top panel shows the frequency of CD4 and CD8 T cells in each population of cells. The bottom left and bottom right panels show the memory phenotypes of the CD4+ and CD8+ cells, respectively. Memory phenotype was determined by detection of CD45RA and CCR7. Effector memory T cells (TEM) are CD45RA-CCR7-; Central memory T cells (TCM) are CD45RA-CCR7+; Terminally differentiated effector memory cells re-expressing CD45RA (TEMRA) are CD45RA+ CCR7-; and Naive T cells are CD45RA+ CCR7+.

[0081] FIG. 27 shows the cytotoxicity of the indicated transduced cells against HepG2 target cells upon coculture for 24 hours at a 3: 1 or 1 :1 E:T, using a luciferase bioluminescence-based cytotoxicity assay to determine % tumor cell lysis.

[0082] FIG. 28 shows the TCR activation as measured by Caspase 3/7 staining intensity in an Incucyte® assay following incubation of the transduced T cells with HepG2 cells at a 3: 1 or 1 : 1 E:T.

[0083] FIG. 29 shows IL2, IFNy, TNFa, and GMCSF production by transduced T cells after coculture with HepG2 cells for 24 hours at a 1 : 1 E:T.

[0084] FIG. 30 shows IL2, IFNY, TNFa, and GMCSF production by transduced T cells after coculture with HepG2 cells for 24 hours at a 3 : 1 E:T.

[0085] FIG. 31 shows IL2, IFNY, TNFa, and GMCSF production by transduced T cells after coculture with Hep3B cells for 24 hours at a 1 : 1 E:T.

[0086] FIG. 32 shows IL2, IFNY, TNFa, and GMCSF production by transduced T cells after co- culture with Hep3B cells for 24 hours at a 3 : 1 E:T.

[0087] FIG. 33 shows expression of the TFP on transduced CD3+ cells from two different donors on Day 10 using GPC3 protein conjugated to PE and flow cytometry analysis.

[0088] FIG. 34 shows the frequency of CD4+ and CD 8+ cells in transduced cells from two different donors.

[0089] FIG. 35 shows the memory phenotypes (TEMRA, Naive, TCM, and TEM) of the transduced cells from each of the two donors.

[0090] FIG. 36 shows the % tumor lysis from transduced cells from a representative donor (Donor A) upon co-culture with HepG2 (top left panel), Hep3B (bottom left panel), SNU398 (top right panel), or A549 (bottom right panel) at a 9: 1, 3: 1, 1 : 1, or 1 :3 E:T.

[0091] FIG. 37 shows the IL2, IFNy, TNFa, and GMCSF production from T cells from a representative donor (Donor A) following incubation with HepG2 cells at a 9: 1, 3: 1, 1 : 1, or 1:3 E:T.

[0092] FIG. 38 shows the IL2, IFNy, TNFa, and GMCSF production from T cells from a representative donor (Donor A) following incubation with Hep3B cells at a 9: 1, 3: 1, 1 : 1, or 1 :3 E:T.

[0093] FIG. 39 shows the IL2, IFNy, TNFa, and GMCSF production from T cells from a representative donor (Donor A) following incubation with SNU398 cells at a 9: 1, 3: 1, 1 : 1, or 1 :3 E:T.

[0094] FIG. 40 shows the IL2, IFNy, TNFa, and GMCSF production from T cells from a representative donor (Donor A) following incubation with A549 cells at a 9: 1, 3 : 1, 1 : 1, or 1 :3 E:T. [0095] FIG. 41 shows the fold expansion of cells transduced with the indicated constructs (Donor A and Donor B cells combined) in a repeated stimulation assay with HepG2 cells.

DETAILED DESCRIPTION

[0096] The present disclosure provides a recombinant nucleic acid molecule comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP). The 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. As provided herein, 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.

[0097] In another aspect, 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.

[0098] In another aspect, the present disclosure provides a method of producing the cell comprising the TFP or the recombinant nucleic acid molecule encoding the TFP.

Definitions

[0099] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer-defined protocols and conditions unless otherwise noted.

[00100] As used herein, the singular forms “a,” “an,” and “the” include the plural referents unless the context clearly indicates otherwise. The terms “include,” “such as,” and the like are intended to convey inclusion without limitation, unless otherwise specifically indicated.

[00101] As used herein, 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. Thus, as used herein, the term “comprising,” is inclusive and does not exclude additional, unrecited integers or method/process steps.

[00102] In embodiments of any of the compositions and methods provided herein, “comprising” 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. As used herein, 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.

[00103] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[00104] 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).

[00105] The term “antibody,” as used herein, 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.

[00106] The term “antigen-binding domain” means the portion of an antibody that is capable of specifically binding to an antigen or epitope. One example of an antigen-binding domain is an antigen-binding domain formed by a VH-VL dimer of an antibody. Another example of an antigenbinding domain is an antigen-binding domain formed by diversification of certain loops from the tenth fibronectin type III domain of an Adnectin.

[00107] The terms “antibody fragment” or “antibody binding domain” refer 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. Examples of 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 VL or VH), camelid VHH domains, and multi-specific antibodies formed from antibody fragments.

[00108] The term “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.

[00109] “Heavy chain variable region” or “VH” (or, in the case of single domain antibodies, e.g., nanobodies, “VHH”) with regard to an antibody 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.

[00110] Unless specified, as used herein a scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise -linker-Vu or may comprise VH-1 inker- VL.

[00111] 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. Set. USA 85:5879- 5883; Bird et al., 1988, Science 242:423-426). In one aspect, the antigen binding domain of a TFP composition of the present disclosure comprises an antibody fragment. In a further aspect, the TFP comprises an antibody fragment that comprises a scFv or a sdAb.

[00112] The term “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.

[00113] The term “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 (“X”) light chains refer to the two major antibody light chain isotypes.

[00114] The term “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.

[00115] The term “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.

[00116] The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that 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. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that 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. Such 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.

[00117] “ 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. Normally, 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 Yap 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.

[00118] 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.

[00119] “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. In some instances, 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. For further details, see Jones et al., Nature, 1986, 321 :522-525; Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op. Struct. Biol., 1992, 2:593-596, each of which is incorporated by reference in its entirety.

[00120] 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.

[00121] “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). Unless indicated otherwise, as used herein, “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). The affinity of a molecule X for its partner Y can be represented by the dissociation equilibrium constant (KD). 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®).

[00122] With regard to the binding of an antibody or fragment thereof to a target molecule, 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.

[00123] The term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[00124] The term “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.

[00125] The term “xenogeneic” refers to a graft derived from an animal of a different species.

[00126] The term “treating” (and variations thereof such as “treat” or “treatment”) 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.

[00127] As used herein, 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. By “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)).

[00128] As used herein, 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. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, or a CD4+ / CD8+ T cell. In some embodiments, the TFP T cell is an NK cell or a regulatory T cell.

[00129] As is used herein, the terms “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 CD3s, CD3y, and CD38. In some embodiments, the TCR comprises CD3s. In some embodiments, the TCR comprises CD3y. In some embodiments, the TCR comprises CD38. In some embodiments, the TCR comprises CD3(^. Engagement of the TCR with antigen, e.g., with antigen and MHC, results in activation of its T cells through a series of biochemical events mediated by associated enzymes, co-receptors, and specialized accessory molecules. In some embodiments, the constant domain of TCR alpha has a sequence of SEQ ID NO: 711. In some embodiments, 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. In some embodiments, the constant domain of TCR beta has a sequence of SEQ ID NO: 715. In some embodiments, 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. In some embodiments, the constant domain of TCR delta has a sequence of SEQ ID NO: 725. In some embodiments, 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. In some embodiments, the constant domain of TCR gamma has a sequence of SEQ ID NO: 721. In some embodiments, 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. In some embodiments, CD3 epsilon has a sequence of SEQ ID NO: 694. In some embodiments, 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. In some embodiments, CD3 delta has a sequence of SEQ ID NO: 704. In some embodiments, 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. In some embodiments, CD3 gamma has a sequence of SEQ ID NO: 699. In some embodiments, 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.

[00130] As used herein, 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.

[00131] As used 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.

[00132] The term “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.

[00133] The term “cytotoxic agent,” as used herein, refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. [00134] 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.

[00135] The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “cell proliferative disorder,” “proliferative disorder” and “tumor” are not mutually exclusive as referred to herein. The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is a cancer. In some aspects, the tumor is a solid tumor. In some aspects, the tumor is a hematologic malignancy.

[00136] The term “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.

[00137] The term “pharmaceutical 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.

[00138] The terms “modulate” and “modulation” refer to reducing or inhibiting or, alternatively, activating or increasing, a recited variable.

[00139] The terms “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.

[00140] The terms “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.

[00141] The term “agonize” refers to the activation of receptor signaling to induce a biological response associated with activation of the receptor. An “agonist” is an entity that binds to and agonizes a receptor.

[00142] The term “antagonize” 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.

[00143] The term “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.

[00144] The term “regulatory T cell” includes cells that regulate immunological tolerance, for example, by suppressing effector T cells. In some aspects, the regulatory T cell has a CD4+CD25+Foxp3+ phenotype. In some aspects, the regulatory T cell has a CD8+CD25+ phenotype.

[00145] The term “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.

[00146] 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. In one aspect, the disease is a cancer.

[00147] In some cases, the cancer is hepatocellular carcinoma (HCC), lung cancer, melanoma, ovarian clear-cell carcinomas, yolk sac tumors, neuroblastoma, hepatoblastoma, Wilms' tumor cells, or gastric cancer. In some cases, 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. In some cases, 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.

[00148] In some cases, 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, larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung carcinoma), lymphoma, diffuse large-B-cell lymphoma, follicular lymphoma, malignant mesothelioma, mastocytoma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin's lymphoma (NHL), B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, mesentery cancer, pharynx cancer, prostate cancer, RCC, ccRCC, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, or ureter cancer.

[00149] The term “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. These families include 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) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, 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.

[00150] The term “2A,” “2A self-cleaving peptide,” or “2A peptide,” as used herein, 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 2A derived from thosea asigna virus, and the sequence is EGRGSLLTCGDVEENPGP (SEQ ID NO: 742). “P2A” refers to the 2 A 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 2 A 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). In some embodiments, adding the 1 linker “GSG” (Gly-Ser-Gly) on the N-terminal of a 2A peptide helps with efficiency.

[00151] The term “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 CD3y chain, a CD36 chain, and two CD3s chains in mammals.

[00152] “ CD3s,” “CD3s chain,” or “T-cell surface glycoprotein CD3 epsilon chain,” as used herein, includes any of the recombinant or naturally-occurring forms of CD3s or variants or homologs thereof that have or maintain CD3s activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, 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 CD3s. In some embodiments, CD3s is substantially identical to the protein identified by the UniProt reference number P07766 or a variant or homolog having substantial identity thereto.

[00153] “CD36,” “CD36 chain,” or “T-cell surface glycoprotein CD3 delta chain,” as used herein, includes any of the recombinant or naturally-occurring forms of CD36 or variants or homologs thereof that have or maintain CD36 activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, 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 CD36. In some embodiments, CD36 is substantially identical to the protein identified by the UniProt reference number P04234 or a variant or homolog having substantial identity thereto.

[00154] “CD3y,” “CD3y chain,” or “T-cell surface glycoprotein CD3 gamma chain,” as used herein, includes any of the recombinant or naturally-occurring forms of CD3y or variants or homologs thereof that have or maintain CD3y activity (e.g., at least 40% 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity). In some aspects, 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 CD3y. In some embodiments, CD3y is substantially identical to the protein identified by the UniProt reference number P09693 or a variant or homolog having substantial identity thereto.

[00155] The term “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. Stimulation can mediate altered expression of certain molecules, and/or reorganization of cytoskeletal structures, and the like.

[00156] 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. In one aspect, 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 “IT AM”. Examples of an IT AM containing primary cytoplasmic signaling sequence that is of particular use in the invention includes, but is not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as “ICOS”) and CD66d.

[00157] 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). APCs process antigens and present them to T cells.

[00158] 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. 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. In an embodiment, 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. In an embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. [00159] A primary intracellular signaling domain can comprise an ITAM (“immunoreceptor tyrosine-based activation motif’). Examples of 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.

[00160] The term "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 (CD1 la/CD18), 4-1BB (CD137), IL-15Ra, IL12R, IL18R, IL21R, ICOS (CD278), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, CD226, FcyRI, FcyRII, and FcyRIII. 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, CD 160, B7-H3, and a ligand that specifically binds with CD83, IL-15Ra, IL12R, IL18R, IL21R, CD27, CD5, ICAM-1, CD7, CD226, FcyRI, FcyRII, FcyRIII, 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.

[00161] 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. Thus, 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.

[00162] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. 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.

[00163] The term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[00164] The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[00165] The term “expression” refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

[00166] 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. In one embodiment, a functional disruption refers to a modification of the gene via a gene editing method. In one embodiment, a functional disruption prevents expression of a target gene (e.g., an endogenous gene).

[00167] The term "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. Thus, 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.

[00168] The term “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.

[00169] The term “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.

[00170] The term “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 LENTIVECTOR™ gene delivery technology from Oxford BioMedica, the LENTIMAX™ 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.

[00171] The term “circularized RNA” or “circRNA” 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). For circularization, 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).

[00172] 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. In preferred embodiments, 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. In one aspect, the template includes sequences for the TFP, CAR, and TCR, or combination thereof. [00173] In some exemplary embodiments, 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 3Tinked circles.

[00174] The term “homologous” or “identity” 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. When 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.

[00175] The term “isolated” means altered or removed from the natural state. For example, 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.

[00176] In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[00177] The term “operably linked” or “transcriptional control” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, 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. For instance, 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.

[00178] The term “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.

[00179] The term “nucleic acid” or “polynucleotide” 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. Specifically, 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.,Afo/. Cell. Probes 8:91-98 (1994)).

[00180] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer 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. As used herein, 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.

[00181] The term “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.

[00182] The term “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. In some instances, 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. [00183] The term “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.

[00184] The term “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.

[00185] The term “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.

[00186] The terms “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. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1. For example, n=l, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 and n=10. In one embodiment, the flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 or (Gly4Ser)3. In another embodiment, the linkers include multiple repeats of (Gly2Ser), (GlySer) or (GlysSer). Also included within the scope of the invention are linkers described in WO2012/138475 (incorporated herein by reference). In some instances, the linker sequence comprises (G4S)n, wherein n=2 to 4. In some instances, the linker sequence comprises (G4S)_n, wherein n=l to 3.

[00187] As used herein, 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. 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.

[00188] As used herein, “/// vitro transcribed RNA” refers to RNA, preferably mRNA, which has been synthesized in vitro. Generally, 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.

[00189] As used herein, a “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA. In the preferred embodiment of a construct for transient expression, 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.

[00190] As used herein, “polyadenylation” refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3’ end. 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. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. 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. After transcription has been terminated, 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. After the mRNA has been cleaved, adenosine residues are added to the free 3’ end at the cleavage site.

[00191] As used herein, “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.

[00192] The term “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. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.

[00193] The term, 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. In some instances, 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. In some aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

[00194] The term “therapeutic” as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.

[00195] The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state.

[00196] In the context of the present invention, “tumor antigen” or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” refers to antigens that are common to specific hyperproliferative disorders. In certain aspects, 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.

[00197] In some instances, 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, hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung carcinoma), lymphoma, diffuse large-B-cell lymphoma, follicular lymphoma, malignant mesothelioma, mastocytoma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin's lymphoma (NHL), B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, mesentery cancer, pharynx cancer, prostate cancer, RCC, ccRCC, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, or ureter cancer.

[00198] In some cases, 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. In some cases, 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. In some cases, 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.

[00199] The term “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.

[00200] 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.

[00201] 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. As another example, 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)

[00202] 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. 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 term T cell receptor fusion construct or TRuC is also used interchangeably herein with TFP.

[00203] 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. In some embodiments, the binding domain is comprised solely of an anti-GPC3 antibody described herein and is not fused to any other polypeptide. In some embodiments, 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.

[00204] 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 (VH), a light chain variable domain (VL) and a variable domain (VHH) 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. Likewise a natural or synthetic ligand specifically recognizing and binding GPC3 can be used as antigen binding domain for the TFP. In some instances, the antigen binding domain may be derived from the same species in which the TFP will be used in. For example, for use in humans, the antigen binding domain of the TFP can comprise human or humanized residues for the antigen binding domain of an antibody or antibody fragment.

[00205] In one aspect, 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 wildtype or enhanced affinity.

[00206] 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. In some embodiments, a humanized antibody or antibody fragment may have improved affinity and/or specificity of binding to GPC3.

[00207] In an aspect, 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 CDRs. In some embodiments, 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 or Table 14.

[00208] 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. For example, the antigen binding domain of the TFP can comprise one HC CDR1, HC CDR2, and HC CDR3. For another example, 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 or Table 14. 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 VHH comprising a heavy chain variable region. In one embodiment, the antigen binding domain of the TFP is a scFv, and a light chain variable region is attached to a heavy chain variable region via a linker, e.g., a linker described herein. In one embodiment, 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. In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G4S)_n, wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S)_n, wherein n=l to 3. In some embodiments, the linker is a Whitlow linker.

[00209] For example, the antigen binding domain can comprise a LC CDR1 of SEQ ID NO:3, SEQ ID NO: 11, SEQ ID NO: 19, SEQIDNO:27, SEQIDNO:35, SEQIDNO:43, SEQIDNO: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, SEQ ID NO: 195, SEQIDNO:785, SEQIDNO:793, SEQIDNO:801, SEQIDNO:809, SEQ ID NO:817, SEQ ID NO:825, SEQ ID NO:833, SEQ ID NO:841, or SEQ ID NO:849. The antigen binding domain can comprise a LC CDR2 of SEQ ID NON, SEQ ID NO: 12, SEQ IDNO:20, SEQ IDNO:28, SEQIDNO:36, SEQIDNO:44, SEQIDNO:52, SEQIDNO:60, SEQIDNO:68, SEQ IDNO:76, SEQIDNO:84, SEQIDNO: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, SEQ ID NO: 196, SEQIDNO:786, SEQ ID NO: 794, SEQ ID NO: 802, SEQ ID NO: 810, SEQ ID NO: 818, SEQ ID NO: 826, SEQ ID NO:834, SEQ ID NO:842, or SEQ ID NO:850. 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 IDNO:45, SEQIDNO:53, SEQIDNO:61, SEQIDNO:69, SEQIDNO:77, SEQIDNO: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, SEQ ID NO: 197, SEQIDNO:787, SEQIDNO:795, SEQ ID NO:803, SEQIDNO:811, SEQIDNO:819, SEQIDNO:827, SEQIDNO:835, SEQIDNO:843, or SEQ ID NO:851. The antigen binding domain can comprise HC CDR1 of SEQ ID NO:6, SEQ ID NO: 14, SEQIDNO:22, SEQIDNO:30, SEQIDNO:38, SEQIDNO:46, SEQIDNO: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, SEQ ID NO: 198, SEQ ID NO:782, SEQ ID NO:790, SEQ ID NO:798, SEQ ID NO:806, SEQ ID NO:814, SEQ ID NO:822, SEQ ID NO:830, SEQ ID NO:838, or SEQ ID NO:846. The antigen binding domain can comprise HC CDR2 of SEQ ID NO:7, SEQ ID NO: 15, SEQ ID NO:23, SEQ IDNO:31, SEQIDNO:39, SEQIDNO:47, SEQIDNO:55, SEQIDNO:63, SEQIDNO:71, SEQ ID NO:79, SEQ ID NO:87, SEQ ID NO:95, SEQ ID NO: 103, SEQ ID NO: 111, SEQ ID N0: 119, SEQ ID NO: 127, SEQ ID NO: 135, SEQ ID NO: 143, SEQ ID N0: 151, SEQ ID NO: 159, SEQ ID NO: 167, SEQ ID NO: 175, SEQ ID NO: 183, SEQ ID NO: 191, SEQ ID NO: 199, SEQ ID NO:783, SEQ ID NO:791, SEQ ID NO:799, SEQ ID NO:807, SEQ ID NO:815, SEQ ID NO:823, SEQ ID NO:831, SEQ ID NO:839, or SEQ ID NO:847. 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, SEQ ID N0:200, SEQ ID NO:784, SEQ ID NO:792, SEQ ID N0:800, SEQ ID NO:808, SEQ ID NO:816, SEQ ID NO:824, SEQ ID NO:832, SEQ ID NO:840, or SEQ ID NO:848. In some cases, the antigen binding domain comprises a LC CDR1 of SEQ ID NO:3, a LC CDR2 of SEQ ID NON, 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. In some cases, 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. In some cases, 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. In some cases, 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, a HC CDR2 of SEQ ID NON 1, and a HC CDR3 of SEQ ID NO:32. In some cases, 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. In some cases, 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. In some cases, 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, aHC CDR2 of SEQ ID NO:55, and a HC CDR3 of SEQ ID NO:56. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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 CDR1 of SEQ ID NO: 102, a HC CDR2 of SEQ ID NO: 103, and a HC CDR3 of SEQ ID NO: 104. In some cases, 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. In some cases, 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. In some cases, 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 of SEQ ID NO: 126, a HC CDR2 of SEQ ID NO: 127, and a HC CDR3 of SEQ ID NO: 128. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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 CDR1 of SEQ ID NO: 174, a HC CDR2 of SEQ ID NO: 175, and a HC CDR3 of SEQ ID NO: 176. In some cases, 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. In some cases, 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. In some cases, 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 of SEQ ID NO: 198, a HC CDR2 of SEQ ID NO: 199, and a HC CDR3 of SEQ ID NO:200. In some cases, the antigen binding domain comprises a LC CDR1 of SEQ ID NO:785, a LC CDR2 of SEQ ID NO:786, and a LC CDR3 of SEQ ID NO: 787; and/or a HC CDR1 of SEQ ID NO: 782, a HC CDR2 of SEQ ID NO: 783, and a HC CDR3 of SEQ ID NO:784. In some cases, the antigen binding domain comprises a LC CDR1 of SEQ ID NO: 793, a LC CDR2 of SEQ ID NO: 794, and a LC CDR3 of SEQ ID NO: 795; and/or a HC CDR1 of SEQ ID NO: 790, a HC CDR2 of SEQ ID NO: 791, and a HC CDR3 of SEQ ID NO:792. In some cases, the antigen binding domain comprises a LC CDR1 of SEQ ID NO:801, a LC CDR2 of SEQ ID NO: 802, and a LC CDR3 of SEQ ID NO: 803; and/or a HC CDR1 of SEQ ID NO:798, a HC CDR2 of SEQ ID NO:799, and a HC CDR3 of SEQ ID NO:800. In some cases, the antigen binding domain comprises a LC CDR1 of SEQ ID NO:809, a LC CDR2 of SEQ ID NO : 810, and a LC CDR3 of SEQ ID NO : 811 ; and/or a HC CDR 1 of SEQ ID NO : 806, a HC CDR2 of SEQ ID NO:807, and a HC CDR3 of SEQ ID NO:808. In some cases, the antigen binding domain comprises a LC CDR1 of SEQ ID NO:817, a LC CDR2 of SEQ ID NO:818, and a LC CDR3 of SEQ ID NO:819; and/or a HC CDR1 of SEQ ID NO:814, a HC CDR2 of SEQ ID NO:815, and a HC CDR3 of SEQ ID NO:816. In some cases, the antigen binding domain comprises a LC CDR1 of SEQ ID NO:825, a LC CDR2 of SEQ ID NO:826, and a LC CDR3 of SEQ ID NO: 827; and/or a HC CDR1 of SEQ ID NO: 822, a HC CDR2 of SEQ ID NO: 823, and a HC CDR3 of SEQ ID NO:824. In some cases, the antigen binding domain comprises a LC CDR1 of SEQ ID NO:833, a LC CDR2 of SEQ ID NO:834, and a LC CDR3 of SEQ ID NO:835; and/or a HC CDR1 of SEQ ID NO:830, a HC CDR2 of SEQ ID NO:831, and a HC CDR3 of SEQ ID NO:832. In some cases, the antigen binding domain comprises a LC CDR1 of SEQ ID NO:841, a LC CDR2 of SEQ ID NO: 842, and a LC CDR3 of SEQ ID NO: 843; and/or a HC CDR1 of SEQ ID NO:838, a HC CDR2 of SEQ ID NO:839, and a HC CDR3 of SEQ ID NO:840. In some cases, the antigen binding domain comprises a LC CDR1 of SEQ ID NO:849, a LC CDR2 of SEQ ID NO:850, and a LC CDR3 of SEQ ID NO:851; and/or a HC CDR1 of SEQ ID NO:846, a HC CDR2 of SEQ ID NO: 847, and a HC CDR3 of SEQ ID NO: 848. [00210] 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: 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, SEQ ID NO: 193, SEQ ID NO:781, SEQ ID NO:789, SEQ ID NO:797, SEQ ID NO:805, SEQ ID NO:813, SEQ ID NO:821, SEQ ID NO:829, SEQ ID NO:837, or SEQ ID NO:845. 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 NO:90, 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, SEQ ID NO: 194, SEQ ID NO:780, SEQ ID NO:788, SEQ ID NO:796, SEQ ID NO: 804, SEQ ID NO: 812, SEQ ID NO: 820, SEQ ID NO: 828, SEQ ID NO: 836, or SEQ ID NO:844. 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: 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. 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: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 NO: 10. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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 NO:90. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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:781, 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:780. 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:789, 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:788. 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:797, 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:796. 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:805, 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:804. 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: 813, 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:812. 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:821, 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:820. 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:829, 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:828. 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:837, 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:836. 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: 845, 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:844.

[00211] 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, 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. In some cases, the TFP comprises a sequence of SEQ ID NO:209. 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:852. In some cases, the TFP comprises a sequence of SEQ ID NO:852. 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:780. 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:781. 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:853. In some cases, the TFP comprises a sequence of SEQ ID NO:853. 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:780. 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:781. 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:881. In some cases, the TFP comprises a sequence of SEQ ID NO:881. 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:836. 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:837.

[00212] In some aspects, 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. In one aspect, the antigen binding domain is humanized.

[00213] 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 of which is incorporated herein in its entirety by reference), veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91 :969-973, each of which is incorporated herein by its entirety by reference), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein in its entirety by reference), and techniques disclosed in, e.g., U.S. Patent Application Publication No. US2005/0042664, U.S. Patent Application Publication No. US2005/0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169: 1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein in its entirety by reference. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, for example improve, antigen binding. These 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.)

[00214] 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. As provided herein, 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. Multiple techniques for humanization of antibodies or antibody fragments are well-known in the art and can essentially be performed following the method of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference in their entirety). In such humanized antibodies and antibody fragments, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. Humanized antibodies are often human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies and antibody fragments can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., PNAS, 91 :969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the contents of which are incorporated herein by reference in their entirety.

[00215] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity. According to the so-called “best-fit” method, the 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). In some embodiments, the framework region, e.g., all four framework regions, of the heavy chain variable region are derived from a VH4-4-59 germline sequence. In one embodiment, 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. In one embodiment, the framework region, e.g., all four framework regions of the light chain variable region are derived from a VK3-1.25 germline sequence. In one embodiment, 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.

[00216] In some aspects, 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. According to one aspect of the present disclosure, 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. In this way, 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. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[00217] In one aspect, 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. For example, in one aspect, the portion of a TFP composition of the present disclosure that comprises an antigen binding domain specifically binds human GPC3. In one aspect, 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. In certain aspects, the antigen binding domain (e.g., scFv or a sdAb) is contiguous with and in the same reading frame as a leader sequence.

[00218] Also provided herein are methods for obtaining an antibody antigen binding domain specific for a target antigen (e.g., GPC3, or any target antigen described elsewhere herein for targets of fusion moiety binding domains), the method comprising providing by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a VH and/or VL domain a modified VH domain which is an amino acid sequence variant of the VH domain, optionally combining the modified VH domain thus provided with one or more VL domains, and testing the VH domain or VH/VL combination or combinations to identify a specific binding member or an antibody antigen binding domain specific for a target antigen of interest (e.g., GPC3) and optionally with one or more desired properties.

[00219] In some instances, VHH 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 VH and VL 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. In fact, if a short polypeptide linker is employed (e.g., between 5-10 amino acids) intra-chain folding is prevented. Inter-chain folding may also be required to bring the two variable regions together to form a functional epitope binding site. In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G4S)_n, wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S)_n, wherein n=l to 3. For examples of linker orientation and size see, e.g., Hollinger et al. 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent No. 7,695,936, U.S. Patent Application Publication Nos. 20050100543 and 20050175606, and PCT Publication Nos. W02006/020258 and W02007/024715, all of which are incorporated herein by reference.

[00220] A scFv can comprise a linker of about 10, 11, 12, 13, 14, 15 or greater than 15 residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises amino acids glycine and serine. In another embodiment, the linker sequence comprises sets of glycine and serine repeats such as (G4S)_n, where n is a positive integer equal to or greater than 1. In one embodiment, the linker can be (G4S)4 or (G S)?. Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies. In some instances, the linker sequence comprises a long linker (LL) sequence. In some instances, the long linker sequence comprises (G4S)_n, wherein n=2 to 4. In some instances, the linker sequence comprises a short linker (SL) sequence. In some instances, the short linker sequence comprises (G4S)_n, wherein n=l to 3.

[00221] 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. In some embodiments, the antigen binding domain is an scFv listed in Table 1 or Table 14. The antigen binding domain can be a single domain antibody (sdAb). The sdAb can be a VHH.

Stability and Mutations

[00222] The stability of 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. In one embodiment, 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.

[00223] 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. In one embodiment, the anti-GPC3 binding domain, e.g., scFv has a 1 °C improved thermal stability as compared to a conventional antibody. In another embodiment, the anti-GPC3 binding domain, e.g., scFv has a 2 °C improved thermal stability as compared to a conventional antibody. In another embodiment, 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 VH and VL 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.

[00224] Mutations in 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. In one embodiment, 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. In another embodiment, 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.

[00225] In various aspects, the antigen binding domain of the TFP is engineered by modifying one or more amino acids within one or both variable regions (e.g., VH and/or VL), for example within one or more CDR regions and/or within one or more framework regions. In one specific aspect, the TFP composition of the present disclosure comprises an antibody fragment. In a further aspect, that antibody fragment comprises a scFv or sdAb.

[00226] It will be understood by one of ordinary skill in the art that 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. For example, additional nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues may be made to the protein. For example, a nonessential amino acid residue in a molecule may be replaced with another amino acid residue from the same side chain family. In another embodiment, 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.

[00227] 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).

[00228] 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. Optionally, 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.

[00229] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, 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. The 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. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Brent et al., (2003) Current Protocols in Molecular Biology). 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.

[00230] In an aspect, the present disclosure contemplates modifications of a starting antibody or fragment (e.g., scFv or VHH) amino acid sequence that generates functionally equivalent molecules. For example, the VH or VL 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 VH or VL framework region of the anti-GPC3 binding domain, e.g., scFv or VHH. 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.

Extracellular domain

[00231] 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, CD137, CD154. In some instances, 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.

[00232] In some embodiments, 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. In some embodiments, 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.

[00233] In some embodiments, 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, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,

61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,

87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more consecutive amino acid residues of the extracellular domain of a TCR alpha chain, a TCR beta chain, a TCR delta chain, or a TCR gamma chain. In some embodiments, 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. In some embodiments, 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.

[00234] In some embodiments, 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, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,

87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more consecutive amino acid residues of an IgC domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma. In some embodiments, 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. In some embodiments, 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 bland C-terminus.

[00235] In some embodiments, 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,

87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more consecutive amino acid residues of the extracellular portion of the constant domain of TCR alpha, a TCR beta, a TCR delta, or a TCR gamma. In some embodiments, 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, a TCR beta, a TCR delta, or a TCR gamma. In some embodiments, 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 bland C-terminus.

[00236] In some embodiments, 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,

87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more consecutive amino acid residues of the extracellular domain of a CD3 epsilon TCR subunit, a CD3 gamma TCR subunit, or a CD3 delta TCR subunit. In some embodiments, 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. In some embodiments, 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.

Transmembrane Domain

[00237] In general, a TFP sequence contains an extracellular domain and a transmembrane domain encoded by a single genomic sequence. In alternative embodiments, 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). In some cases, 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. In one aspect, the transmembrane domain is capable of homodimerization with another TFP on the TFP-T cell surface. In a different aspect 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.

[00238] 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. In some instances, 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.

[00239] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[00240] In some instances, 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. For example, in one embodiment, the hinge can be a human immunoglobulin (Ig) hinge, e.g., an IgG4 hinge, or a CD8a hinge.

Linkers

[00241] Optionally, a short oligo- or polypeptide linker, between 2 and 10 amino acids in length 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. In some cases, 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. For example, in one aspect, 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. In some embodiments, X is 2. In some embodiments, X is 4. In some embodiments, the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO:

691).

Cytoplasmic Domain

[00242] The cytoplasmic domain of the TFP can include an intracellular domain. In some embodiments, the intracellular domain is from CD3 gamma, CD3 delta, CD3 epsilon, TCR alpha, TCR beta, TCR gamma, or TCR delta. In some embodiments, 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. The term “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. Thus the term “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. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[00243] Examples of 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.

[00244] In some embodiments, 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.

[00245] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[00246] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

[00247] It is known that signals generated through the TCR alone are insufficient for full activation of naive T cells and that a secondary and/or costimulatory signal is required. Thus, 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).

[00248] 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 tyrosinebased activation motifs (ITAMs).

[00249] Examples of ITAMs 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. In one embodiment, 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. In one embodiment, a primary signaling domain comprises a modified ITAM domain, e.g., a mutated ITAM domain which has altered (e.g., increased or decreased) activity as compared to the native ITAM domain. In one embodiment, 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. In an embodiment, a primary signaling domain comprises one, two, three, four or more ITAM motifs.

[00250] 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. For example, 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, 4-1BB (CD137), 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. For example, 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).

[00251] 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. Optionally, 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.

[00252] In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable linker.

[00253] In one aspect, 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). In one embodiment, when the TFP-expressing cell comprises two or more different TFPs, the antigen binding domains of the different TFPs can be such that the antigen binding domains do not interact with one another. For example, 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 VHH.

[00254] In another aspect, the TFP-expressing cell described herein can further express another agent, e.g., an agent which enhances the activity of a modified T cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., PD1, 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, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD 160, 2B4 and TGFR beta. In one embodiment, 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. In one embodiment, the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD1, LAG3, CTLA4, CD160, 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). In one embodiment, 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). 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 ofPDl with PD-Ll.

[00255] In one embodiment, 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 41BB and CD3 zeta (also referred to herein as a PD1 TFP). In one embodiment, the PD1 TFP, when used in combinations with an anti-GPC3 TFP described herein, improves the persistence of the T cell. In one embodiment, the TFP is a PD1 TFP comprising the extracellular domain of PD-1. Alternatively, provided are 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).

[00256] In another aspect, the present disclosure provides a population of TFP-expressing T cells, e.g., TFP-T cells. In some embodiments, the population of TFP-expressing T cells comprises a mixture of cells expressing different TFPs. For example, in one embodiment, the population of TFP-T cells 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. As another example, 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).

[00257] In another aspect, 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. For example, in one embodiment, 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, CD160, 2B4 and TGFR beta. In one embodiment, 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. In some embodiments, the agent is a cytokine. In some embodiments, the cytokine is IL-15. In some embodiments, IL-15 increases the persistence of the T cells described herein.

Recombinant Nucleic Acids Encoding a TFP

[00258] Disclosed herein, in some embodiments, are recombinant nucleic acids encoding the TFPs disclosed herein.

[00259] In some instances, 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.

[00260] Disclosed herein are 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. In one aspect, the template includes sequences for the TFP.

[00261] In one aspect the anti-GPC3 TFP is encoded by a messenger RNA (mRNA). In one aspect the mRNA encoding the anti-GPC3 TFP is introduced into a T cell for production of a TFP-T cell. In one embodiment, 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. 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, the DNA to be used for PCR is a human nucleic acid sequence. In another embodiment, 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.

[00262] 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. For example, 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. In one embodiment, 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.

[00263] 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.

[00264] Chemical structures with the ability to promote stability and/or translation efficiency may also be used. The RNA preferably has 5’ and 3’ UTRs. In one embodiment, 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.

[00265] The 5’ and 3’ UTRs can be the naturally occurring, endogenous 5’ and 3’ UTRs for the nucleic acid of interest. Alternatively, 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.

[00266] In one embodiment, the 5’ UTR can contain the Kozak sequence of the endogenous nucleic acid. Alternatively, when a 5’ UTR that is not endogenous to the nucleic acid of interest is being added by PCR as described above, 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. In other embodiments the 5’ UTR can be 5 ’UTR of an RNA virus whose RNA genome is stable in cells. In other embodiments various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.

[00267] To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5’ end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one preferred embodiment, 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.

[00268] In a preferred embodiment, 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. On a circular DNA template, for instance, plasmid DNA, 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.

[00269] On a linear DNA template, 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)).

[00270] The conventional method of integration of polyA/T stretches into a DNA template is molecular cloning. However, polyA/T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable. That is why a method which allows construction of DNA templates with polyA/T 3’ stretch without cloning highly desirable.

[00271] 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.

[00272] 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). In one embodiment, 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. Additionally, 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. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.

[00273] 5’ caps on also provide stability to RNA molecules. In a preferred embodiment, 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)).

[00274] The RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The 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.

[00275] 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 Then, 12(8):861-70 (2001)).

[00276] For additional information on making and using TFP T cells, see U.S. Patent Nos. 10,442,849, 10,358,473, 10,358,474, and 10,208,285, each of which is herein incorporated by reference.

[00277] In some cases, 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) can be operatively linked. 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.

[00278] 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. For example, 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.

[00279] 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. For example, 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.

[00280] The constant domain can comprise truncations, additions, or substitutions of a sequence of a constant domain described herein. For example, 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. For example, 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. For example, 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.

[00281] The constant domain described herein can be a human TCR alpha constant domain. For example, 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. For example, 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. For example, 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. For example, 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 ormore 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.

[00282] The constant domain described herein can be a human TCR beta constant domain. For example, 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. For example, 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. For example, 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. For example, 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.

[00283] The constant domain described herein can be a human TCR gamma constant domain. For example, 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. For example, 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. For example, 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. For example, 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.

[00284] The constant domain described herein can be a human TCR delta constant domain. For example, 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. For example, 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. For example, 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. For example, 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 ormore 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.

[00285] The constant domain described herein can be a murine TCR alpha constant domain. For example, 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. For example, 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. For example, 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. For example, 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.

[00286] The constant domain described herein can be a murine TCR beta constant domain. For example, 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. For example, 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. For example, 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. For example, 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.

Gene Editing of TCR Complex or Endogenous Protein-coding Genes

[00287] In some embodiments, 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., Urnov et al., Nat. Rev. Genetics (2010) vl 1, 636-646), ormegaTAL nucleases (a fusion protein of a meganuclease to TAL repeats) methods. In this way, 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. In some embodiments, 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).

[00288] Recent developments of technologies to permanently alter the human genome and to introduce site-specific genome modifications in disease relevant genes lay the foundation for therapeutic applications. These technologies are now commonly known as “genome editing.

[00289] In some embodiments, gene editing techniques are employed to disrupt an endogenous TCR gene. In some embodiments, mentioned endogenous TCR gene encodes a TCR alpha chain, a TCR beta chain, or a TCR alpha chain and a TCR beta chain. In some embodiments, mentioned endogenous TCR gene encodes a TCR gamma chain, a TCR delta chain, or a TCR gamma chain and a TCR delta chain. In some embodiments, gene editing techniques pave the way for multiplex genomic editing, which allows simultaneous disruption of multiple genomic loci in endogenous TCR gene. In some embodiments, 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.

[00290] Current gene editing technologies comprise meganucleases, zinc-finger nucleases (ZFN), TAL effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system. These four major classes of gene-editing techniques share a common mode of action in binding a user-defined sequence of DNA and mediating a double-stranded DNA break (DSB). DSB may then be repaired by either non-homologous end joining (NHEJ) or -when donor DNA is present- homologous recombination (HR), an event that introduces the homologous sequence from a donor DNA fragment. Additionally, 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.

[00291] Genetic modification of genomic 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. For example, zinc- finger nucleases (ZFNs) can be engineered to recognize and cut predetermined sites in a genome. 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. By fusing this engineered protein domain to the Fokl nuclease, it is possible to target DNA breaks with genome-level specificity. ZFNs have been used extensively to target gene addition, removal, and substitution in a wide range of eukaryotic organisms (reviewed in Durai et al. (2005) Nucleic Acids Res 33, 5978). Likewise, TAL-effector nucleases (TALENs) can be generated to cleave specific sites in genomic DNA. Like a ZFN, 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). In this case, however, 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 I-TevI 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.

[00292] Engineered endonucleases based on the CRISPR/Cas9 system are also known in the art (Ran et al. (2013), Nat Protoc. 8:2281-2308; Mali et al. (2013), Nat Methods 10:957-63). 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. By expressing multiple guide RNAs in the same cell, each having a different targeting sequence, it is possible to target DNA breaks simultaneously to multiple sites in the genome (multiplex genomic editing).

[00293] There are two classes of CRISPR systems known in the art (Adli (2018) 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 (2018) Nat. Commun. 9: 1911). Among these, such as Casl2a (Cpfl) proteins from Acid- aminococcus sp (AsCpfl) and Lachnospiraceae bacterium (LbCpfl), are particularly interesting.

[00294] 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). Specific amino acid substations could reprogram DNA cleavage specificity of homing nucleases (Niyonzima (2017), Protein Eng Des Sei. 30(7): 503-522). Meganucleases (MN) 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). In some embodiments, meganuclease is engineered I-Crel homing endonuclease. In other embodiments, meganuclease is engineered I-Scel homing endonuclease.

[00295] In addition to mentioned four major gene editing technologies, 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). For example, 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.

[00296] 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.

Vectors

[00297] In some embodiments, 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). In some instances, 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. In some instances, the vector is an AAV6 vector. In some instances, the vector further comprises a promoter. In some instances, the vector is an in vitro transcribed vector.

[00298] The 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. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[00299] 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 longterm 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.

[00300] In another embodiment, the vector comprising the nucleic acid encoding the desired TFP of the present disclosure is an adenoviral vector (A5/35). In another embodiment, 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.

[00301] The TFP of the present invention may be used in multi ci stronic 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. For example, 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 Picornaviridae virus family. See for example Szymczak et al., 2004, Nature Biotechnology; 22: 589-594. In some embodiments, the recombinant nucleic acid described herein encodes the TFP in frame with the agent, with the two sequences separated by a self-cleaving peptide, such as a 2A sequence, or a T2A sequence.

[00302] 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). In another embodiment, the present disclosure provides a gene therapy vector.

[00303] The nucleic acid can be cloned into a number of types of vectors. For example, 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.

[00304] Further, 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. In general, 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). [00305] A number of virally based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged 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. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[00306] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[00307] An example of 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)). Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. However, 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. Further, the present disclosure should not be limited to the use of constitutive promoters. 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. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline-regulated promoter.

[00308] In order to assess the expression of a TFP polypeptide or portions thereof, 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. In other aspects, 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, antibioticresistance genes, such as neo and the like.

[00309] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, 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 Leters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, 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.

[00310] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[00311] 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 [00312] 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.

[00313] 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 submicron sized delivery system.

[00314] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, 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. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, 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.

[00315] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20 °C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “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. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[00316] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present disclosure, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such 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.

[00317] The present disclosure further provides a vector comprising a TFP encoding nucleic acid molecule. In one aspect, a TFP vector can be directly transduced into a cell, e.g., a T cell. In one aspect, 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. In one aspect, the vector is capable of expressing the TFP construct in mammalian T cells. In one aspect, the mammalian T cell is a human T cell.

Circular RNA

[00318] In some embodiments, TFP T cells are transduced with an RNA molecule. In some embodiments, the RNA is circular RNA. In some embodiments, the circular RNA is exogenous. In other embodiments, circular RNA is endogenous. In other embodiments, circular RNAs with an internal ribosomal entry site (IRES) can be translated in vitro or in vivo or ex vivo.

[00319] 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). For circularization, 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).

[00320] To generate circular RNAs that we could subsequently transfer into cells, in vitro production of circular RNAs with autocatalytic-splicing introns can be programmed. A method for generating circular RNA can involve in vitro transcription (IVT) of a precursor linear RNA template with specially designed primers. 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. In preferred embodiments, 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. In one aspect, the template includes sequences for the TFP, CAR, and TCR, or combination thereof.

[00321] 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). In preferred embodiments, the group I intron of phage T4 thymidylate synthase (td) gene is used to generate exogenous circular RNA.

[00322] In some exemplary embodiments, 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 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' to 3' linked circles.

[00323] In one aspect, disclosed herein is 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. In more preferred embodiments, 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. To ensure that the major splicing product was circular, the splicing reaction can be treated with RNase R.

[00324] In one aspect, the anti-GPC3 TFP is encoded by a circular RNA. In one aspect the circular RNA encoding the anti-GPC3 TFP is introduced into a T cell for production of a TFP-T cell. In one embodiment, the in vitro transcribed RNA TFP can be introduced to a cell as a form of transient transfection.

[00325] In some aspects, linear precursor RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template as is described herein.

[00326] For additional information on TFP T cells produced by the methods above, see Provisional Application No. 62/836,977 or International Application No. PCT/US2020/029344, each of which is herein incorporated by reference in its entirety.

Modified T cells

[00327] Disclosed herein are 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.

[00328] In some embodiments, the modified T cells comprising the recombinant nucleic acid disclosed herein, or the vectors disclosed herein comprises a functional disruption of an endogenous TCR. Further disclosed herein, in some embodiments, are modified allogenic T cells comprising the nucleic acid encoding the TFP disclosed herein or a TFP encoded by the nucleic acid disclosed herein.

[00329] In some instances, 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. In some instances, 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. In some instances, 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. In some instances, 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. In some instances, the endogenous TCR that is functionally disrupted has reduced binding to MHC-peptide complex compared to that of an unmodified control T cell. In some instances, the functional disruption is a disruption of a gene encoding the endogenous TCR. In some instances, 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. In some instances, the T cell is a human T cell. In some instances, the T cell is a CD8+ or CD4+ T cell. In some instances, the T cell is an allogenic T cell. In some instances, the T cell is a TCR alpha-beta T cell. In some instances, the T cell is a TCR gamma-delta T cell. In some instances, one or more of 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.

[00330] In some embodiments, the modified T cells are y6 T cells and do not comprise a functional disruption of an endogenous TCR. In some embodiments, the y6 T cells are V<51+ V<52- y<5 T cells. In some embodiments, the y6 T cells are V<51- V<52+ y<5 T cells. In some embodiments, the y6 T cells are V<51- V<52- y<5 T cells.

[00331] In some instances, 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. In some instances, 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.

[00332] The modified T cells can further comprise an enhancing agent or a nucleic acid sequence encoding an enhancing agent. For example, 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. For example, the linker may be a cleavable linker. In some embodiments, 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. In some embodiments, 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 Polypeptides

[00333] 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. For example, 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. In some embodiments, the linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A cleavage site. In some embodiments, the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.

[00334] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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, CD160, CD226, FcyRI, FcyRII, andFcyRIII. In some embodiments, the costimulatory polypeptide is CD28. In some embodiments, the costimulatory polypeptide is 4-1BB. In some embodiments, the costimulatory polypeptide is IL-15Ra. In some embodiments, 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. In some embodiments, 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 4-1BB. 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. In some embodiments, the switch transmembrane domain comprises the sequence of SEQ ID NO:226 or SEQ ID NO:227. In some embodiments, the switch polypeptide further comprises an additional intracellular domain. In some embodiments, the additional intracellular domain is operably linked to the C-terminus of the switch intracellular domain. In some embodiments, the additional intracellular domain comprises an intracellular domain of IL-15Ra or signaling domain thereof. In some embodiments, the additional intracellular domain comprises a sequence with at least 80% sequence identity to SEQ ID NO:228 or SEQ ID NO:229. In some embodiments, the additional intracellular domain comprises the sequence of SEQ ID NO:228 or SEQ ID NO:229. In some embodiments, 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 4-1BB. 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. In some embodiments, the switch polypeptide comprises the sequence of SEQ ID NOs: 216, 217, 218, or 219. In some embodiments, the third nucleic acid sequence encodes a dominant negative TGFBR2 receptor or a fragment thereof. In some embodiments, 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. In some embodiments, the dominant negative TGFBR2 receptor or a fragment thereof comprises the sequence of SEQ ID NO: 230 or SEQ ID NO: 231.

PD-1 Switch Molecules

[00335] 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. In some embodiments, 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. In some embodiments, 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.

[00336] 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. For example, 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. In some embodiments, the linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A cleavage site. In some embodiments, the 2A cleavage site is a T2A cleavage site or a P2A cleavage site.

[00337] The PD-1 switch molecule can comprise a PD-1 polypeptide. In some embodiments, 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. In some embodiments, 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, CD 160, CD226, FcyRI, FcyRII, and FcyRIII. In some embodiments, the costimulatory peptide is CD28. In some embodiments, an extracellular domain and a transmembrane domain of PD-1 are linked to an intracellular domain of CD28.

[00338] In some embodiments, 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. In some embodiments, 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. In some embodiments, the fusion protein comprises the sequence of SEQ ID NO: 232 or SEQ ID NO: 233. In some embodiments, 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. In some embodiments, 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. In some embodiments, the PD-1 switch molecule comprises the sequence of SEQ ID NO: 234 or SEQ ID NO: 235.

Anti-PD-1 Antibodies

[00339] 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. For example, 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.

[00340] In some embodiments, 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. In some embodiments, the CDR1 comprises the amino acid sequence of SEQ ID NO:236. In some embodiments, the CDR2 comprises the amino acid sequence of SEQ ID NO:237. In some embodiments, the variable domain comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:239. In some embodiments, the variable domain comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:239. In some embodiments, the 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.

[00341] In some cases, the second nucleic acid encodes a fusion protein comprising an anti-PD-1 antibody or fragment thereof that specifically binds PD-1. For example, 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. In some embodiments, the anti-PD-1 antibody is linked to the intracellular domain of the costimulatory polypeptide via the transmembrane domain of PD-1.

[00342] In some embodiments, the fusion protein further comprises a signal sequence. In some embodiments, the signal sequence is a PD-1 signal peptide. In some embodiments, 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. In some embodiments, the signal sequence comprises the amino acid sequence of SEQ ID NO:244 or SEQ ID NO:245. In some embodiments, 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. In some embodiments, the fusion protein further comprises an intracellular domain operatively linked to the transmembrane domain. In some embodiments, the fusion protein further comprises a transmembrane domain operatively linked to the intracellular domain and the anti-PD-1 antibody or fragment thereof. In some embodiments, the transmembrane domain of the fusion protein comprises a transmembrane domain of a protein selected from the group consisting of CD28, CD3 E, 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. In some embodiments, 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 of the 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. In some embodiments, 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. 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 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. In some embodiments, the co-stimulatory domain comprises a co-stimulatory domain of a protein selected from the group consisting of a CD27, CD28, 4-1BB (CD137), 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. In some embodiments, the co-stimulatory domain comprises a 4-1BB (CD137) 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: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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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 domain. In some embodiments, 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 4-1BB (CD 137) co-stimulatory domain. In some embodiments, 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 (CD137) co-stimulatory domain. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

IL-15 and/or IL-15Ra

[00343] 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. In some embodiments, 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. In some embodiments, 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.

[00344] In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise an IL- 15 signal peptide. In some embodiments, the IL- 15 polypeptide or a fragment thereof may comprise amino acids 1-29 of IL-15. In some embodiments, the IL-15 polypeptide or a fragment thereof may comprise amino acids 1-29 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. In some embodiments, 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.

[00345] In some embodiments, 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.

[00346] In some embodiments, 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.

[00347] In some embodiments, 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.

[00348] In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise IL- 15Ra Sushi domain, transmembrane domain, and intracellular domain. In some embodiments, 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.

[00349] In some embodiments, the IL-15Ra polypeptide or a fragment thereof may be a soluble IL-15Ra (sIL-15Ra). In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 21-205 of IL-15Ra. In some embodiments, the IL-15Ra polypeptide or a fragment thereof may comprise amino acids 21-205 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:259.

[00350] The present disclosure encompasses recombinant nucleic acid molecules encoding a fusion protein comprising an IL-15 polypeptide linked to an IL-15R subunit. In some embodiments, IL-15 and IL-15R subunit are operatively linked by a linker. In some embodiments, the IL-15R subunit is IL-15R alpha (IL-15Ra). For example, IL- 15 polypeptide may be linked to N-terminus of IL-15Ra subunit. For example, IL-15 polypeptide may be linked to C-terminus of IL-15Ra subunit. In some embodiments, IL- 15 and IL-15Ra are operatively linked by a linker. In some embodiments, the linker is not a cleavable linker. For example, 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. In some embodiments, 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.

[00351] In some embodiments, 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. In some embodiments, the fusion protein does not comprise a sequence of SEQ ID NO:257.

[00352] In some embodiments, the fusion protein may comprise a Sushi domain. In some embodiments, the fusion protein may comprise amino acids 31-95 of IL-15Ra. In some embodiments, the fusion protein may comprise amino acids 31-95 of a sequence of SEQ ID NO:258. In some embodiments, the fusion protein may comprise a sequence of SEQ ID NO:260.

[00353] In some embodiments, 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.

[00354] In some embodiments, 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.

[00355] In some embodiments, 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.

[00356] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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.

CXCR6

[00357] 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. For example, 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.

[00358] In some embodiments, the first and the second nucleic acid molecules are expressed in the same operon. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are operatively linked by a sequence encoding a linker. In some embodiments, the linker comprises a protease cleavage site. In some embodiments, the protease cleavage site is a 2A cleavage site. In some embodiments, the 2A cleavage site is a T2A cleavage site or a P2A cleavage site. In some embodiments, the first nucleic acid sequence and the second nucleic acid sequence are present on different nucleic acid molecules. In some embodiments, 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. In some embodiments, the CXCR6 or functional fragment thereof comprises a sequence of any one selected from SEQ ID NOs:264-266. In some embodiments, the sequence of the recombinant nucleic acid is codon optimized. In some embodiments, the CXCR6 or functional fragment thereof is encoded by a nucleic acid with at least 60% sequence identity to SEQ ID NO: 285. In some embodiments, 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. In some embodiments, 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- binding 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. In some embodiments, 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. 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, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, the CXCR6 or functional fragment thereof comprises a cytoplasmic domain comprising the sequence of SEQ ID NO: 284. In some embodiments, migration of a cell expressing the CXCR6 or functional fragment there-of increases in response to CXCL16. In some embodiments, (i) a migration rate of a cell expressing the CXCR6 or functional fragment thereof increases in response to CXCL16 , (ii) the number of cells expressing the CXCR6 or functional fragment thereof that migrate to a tumor site increases in response to CXCL16, or (iii) a combination thereof.

Sources of T cells

[00359] 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. In some embodiments, T cells can be obtained from a leukopak. In certain aspects of the present disclosure, any number of T cell lines available in the art, may be used. In certain aspects of the present disclosure, 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 Ficoll™ separation. In one preferred aspect, 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. In one aspect, 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. In one aspect of the present disclosure, the cells are washed with phosphate buffered saline (PBS). In an alternative aspect, 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. As those of ordinary skill in the art would readily appreciate 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 Oncol ogyCytoMate, or the Haemonetics® Cell Saver® 5) according to the manufacturer’s instructions. After washing, 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. Alternatively, the undesirable components of the apheresis sample may be removed, and the cells directly resuspended in culture media.

[00360] In embodiments, the T cells are aP T cells. In some embodiments, the T cells are y6 T cells. y6 T cells are obtained from a bank of umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cells, for example.

[00361] In one aspect, 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. For example, in one aspect, CD4+ and CD8+ T cells are isolated with anti-CD4 and anti-CD8 microbeads. In another aspect, 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. In one aspect, the time period is about 30 minutes. In a further aspect, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours. In one aspect, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this 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.

[00362] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In certain aspects, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4+, CD25+, CD62Lhi, GITR+, and FoxP3+. Alternatively, in certain aspects, T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.

[00363] In one embodiment, a T cell population can be selected that expresses one or more of IFN- y TNF-alpha, 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.

[00364] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain aspects, 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. For example, in one aspect, a concentration of 2 billion cells/mL is used. In one aspect, a concentration of 1 billion cells/mL is used. In a further aspect, greater than 100 million cells/mL is used. In a further aspect, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In yet one aspect, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mL is used. In further aspects, 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. Further, 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. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[00365] In a related aspect, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one aspect, the concentration of cells used is 5xlO⁶/mL. In other aspects, the concentration used can be from about lxlO⁵/mL to lxlO⁶/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.

[00366] T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80 °C at a rate of 1 per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20 °C or in liquid nitrogen. In certain aspects, 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.

[00367] Also contemplated in the context of the present disclosure is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one aspect a blood sample or an apheresis is taken from a generally healthy subject. In certain aspects, 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. In certain aspects, the T cells may be expanded, frozen, and used at a later time. In certain aspects, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further aspect, 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.

[00368] In a further aspect of the present disclosure, T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present disclosure to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in certain aspects, mobilization (for example, mobilization with GM-CSF) and 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.

Activation and Expansion of T Cells

[00369] 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.

[00370] Generally, 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. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti- CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells, CD8+ T cells, or CD4+CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc . 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol. Meth. 227(l-2):53-63, 1999). In some embodiments, 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. In one aspect, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred aspect, the time period is 10 to 24 hours, e.g., 24 hours. In some embodiments, 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). In some embodiments, 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. In some embodiments, the cells are activated for 24 hours. In some embodiments, after transduction, the cells are expanded in the presence of anti-CD3 antibody, anti-CD28 antibody in combination with the same cytokines. In some embodiments, 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. In some embodiments, 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. In some embodiments, the cells are subcultured every 1, 2, 3, 4, 5, or 6 days. In some embodiments, 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.

[00371] The expansion of 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). The expansion of 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). In some embodiments, the expansion of T cells may be stimulated with synthetic phosphoantigens (e.g., bromohydrin pyrophosphate; BrHPP), 2M3B1 PP, or 2-methyl-3-butenyl- 1 -pyrophosphate in the presence of IL-2 for one-to-two weeks. In some embodiments, 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. In some embodiments, the expansion of T cells may be stimulated with culture of immobilized anti-CD3 antibodies (e.g., OKT3) in the presence of IL-2. In some embodiments, the aforementioned culture is maintained for about seven days prior to subculture in soluble anti-CD3, and IL-2.

[00372] T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, 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+). 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. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.

[00373] Further, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.

[00374] Once a TFP is constructed, 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.

Therapeutic Applications

[00375] The TFP T cells provided herein may be useful for the treatment of any disease or condition involving GPC3 (e.g., GPC3 -expressing cancers). In some embodiments, the disease or condition is a disease or condition that can benefit from treatment with adoptive cell therapy. In some embodiments, the disease or condition is a cell proliferative disorder. In some embodiments, the disease or condition is a cancer. In some embodiments, the disease or condition is a blood cancer. In some embodiments, the disease or condition is a tumor. In some embodiments, the disease or condition is a viral infection.

[00376] In some embodiments, 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. In some aspects, the disease or condition is a cancer. In some aspects, the disease or condition is a viral infection.

[00377] Any suitable cancer may be treated with the TFP T cells provided herein. Illustrative 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, head and neck cancer, hepatocellular cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cavity cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary, midline tract carcinoma involving NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, my elody splasti c/my el oproliferative neoplasm, nasal cavity and par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, nonsmall cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytomas, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell cancer, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms tumor.

[00378] In some cases, 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, kidney cancer, larynx cancer, leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung carcinoma), lymphoma, diffuse large-B-cell lymphoma, follicular lymphoma, malignant mesothelioma, mastocytoma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin's lymphoma (NHL), B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), Burkitt's lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum, mesentery cancer, pharynx cancer, prostate cancer, RCC, ccRCC, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, stomach cancer, testicular cancer, thyroid cancer, or ureter cancer. The cancer can be characterized by the expression of GPC3. In some cases, 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.

Methods of Treatment

[00379] In one aspect, the invention provides methods for treating a disease associated with at least one tumor-associated antigen expression. In one aspect, 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. For example, 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.

[00380] In one aspect, 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. In one aspect, 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. In one aspect, 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.

[00381] In one aspect, 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.

[00382] In one aspect, 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. An example of 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.

[00383] In some embodiments, tumor-associated antigen antibodies or TFP therapy can be used in combination with one or more additional therapies described herein.

[00384] In one aspect, disclosed herein is a method of cellular therapy wherein 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. Unlike antibody therapies, TFP-expressing T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control. In various aspects, 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.

[00385] In some instances, disclosed herein is a type of cellular therapy where 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. Thus, in various aspects, 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.

[00386] Without wishing to be bound by any particular theory, 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. In one aspect, 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. For example, 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.

[00387] In one aspect, 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. In one aspect, the mammal is a human.

[00388] 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) cry opreservation of the cells, as is described herein.

[00389] 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.

[00390] Generally, 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. In particular, 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. In certain aspects, 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. Thus, 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.

[00391] 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.

[00392] 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 antigenexpressing cell. In a specific aspect, 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. In one aspect, 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. In certain aspects, the antitumor-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 antigenexpressing cells relative to a negative control. In one aspect, the subject is a human.

[00393] 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. In one aspect, the subject is a human. Non-limiting examples of 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).

[00394] Suitable doses of the TFP-T cells described herein for a therapeutic effect would be at least 10⁵ or between about 10⁵ and about 10¹⁰ 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 10⁵ cells on Day 0, for example increasing incrementally up to a target dose of about 10¹⁰ 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.

[00395] 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 antitumor 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. Desirably, 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.

[00396] Accordingly, 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. Furthermore, 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) can vary in different applications. Combination Therapies

[00397] 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. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery”. In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[00398] In some embodiments, the “at least one additional therapeutic agent” includes a TFP- expressing cell. Also provided are 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. Also provided are 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.

[00399] 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. For sequential administration, 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.

[00400] In some embodiments, 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. In some aspects, 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.

I l l [00401] In further aspects, 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.

[00402] In one embodiment, 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). Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like. Accordingly, 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. In one embodiment, the soluble factor elevated in the subject is one or more of IFN-y, 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. Such agents include, but are not limited to a steroid, an inhibitor of TNFa, and an inhibitor of IL-6. An example of a TNFa inhibitor is etanercept (marketed under the name ENBREL®). An example of an IL-6 inhibitor is tocilizumab (marketed under the name ACTEMRA®).

[00403] In one embodiment, the subject can be administered an agent which enhances the activity of a TFP-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Inhibitory molecules, e.g., Programmed Death 1 (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. In embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, can be used to inhibit expression of an inhibitory molecule in the TFP- expressing cell. In an embodiment, the inhibitor is a shRNA. In an embodiment, the inhibitory molecule is inhibited within a TFP-expressing cell. In these embodiments, 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. In one embodiment, the inhibitor of an inhibitory signal can be, e.g., an antibody or antibody fragment that binds to an inhibitory molecule. For example, 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)). In an embodiment, 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 Lymphocyteactivation gene 3 (LAG3).

[00404] In some embodiments, 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. In some embodiments, 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. In one embodiment, the fusion protein is expressed by the same cell that expressed the TFP. In another embodiment, the fusion protein is expressed by a cell, e.g., a T cell that does not express an anti-tumor-associated antigen TFP.

[00405] In some embodiments, the additional therapeutic agent comprises an immunostimulatory agent.

[00406] In some embodiments, the immunostimulatory agent is an agent that blocks signaling of an inhibitory receptor of an immune cell, or a ligand thereof. In some aspects, 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 (TGFP), lymphocyte-activation gene 3 (LAG-3, also CD223), Tim-3 (hepatitis A virus cellular receptor 2 orHAVCR2 or CD366), neuritin, B- and T-lymphocyte attenuator (also BTLA or CD272), killer cell immunoglobulin-like receptors (KIRs), and combinations thereof. In some aspects, 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), CD160, natural killer cell receptor 2B4 (also CD244 or SLAMF4), and combinations thereof. In some aspects, the agent is pembrolizumab. In some aspects, the agent is nivolumab. In some aspects, the agent is atezolizumab.

[00407] In some embodiments, the additional therapeutic agent is an agent that inhibits the interaction between PD-1 and PD-L1. In some aspects, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is selected from an antibody, a peptidomimetic and a small molecule. In some aspects, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-Ll is selected from pembrolizumab (KEYTRUDA), nivolumab (OPDIVO), atezolizumab, avelumab, pidilizumab, durvalumab, sulfamonomethoxine 1, and sulfamethizole 2. In some embodiments, the additional therapeutic agent that inhibits the interaction between PD-1 and PD-L1 is any therapeutic known in the art to have such activity, for example as described in Weinmann et al., ChemMed ('h m, 2016, 14:1576 (DOI: 10.1002/cmdc.201500566), incorporated by reference in its entirety. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in the same pharmaceutical composition an antibody provided herein. In some embodiments, the agent that inhibits the interaction between PD-1 and PD-L1 is formulated in a different pharmaceutical composition from an antibody provided herein. In some embodiments, 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.

[00408] In some embodiments, the immunostimulatory agent is an agonist of a co-stimulatory receptor of an immune cell. In some aspects, 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. In some embodiments, the agonist is an antibody.

[00409] In some embodiments, the immunostimulatory agent modulates the activity of arginase, indoleamine-2 3 -dioxygenase, or the adenosine A2A receptor.

[00410] In some embodiments, the immunostimulatory agent is a cytokine. In some aspects, the cytokine is selected from IL-2, IL-5, IL-7, IL-12, IL-15, IL-21, and combinations thereof.

[00411] In some embodiments, the immunostimulatory agent is an oncolytic virus. In some aspects, 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.

[00412] Further examples of 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). In some embodiments, the additional therapeutic agent is folinic acid, 5 -fluorouracil, and/or oxaliplatin. In some embodiments, 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.

[00413] The additional therapeutic agent may be administered by any suitable means. In some embodiments, a medicament provided herein, and the additional therapeutic agent are included in the same pharmaceutical composition. In some embodiments, an antibody provided herein, and the additional therapeutic agent are included in different pharmaceutical compositions.

[00414] In embodiments where 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. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about one month of each other. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about one week of each other. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about one day of each other. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about twelve hours of each other. In some aspects, administration of an antibody provided herein, and the additional therapeutic agent occur within about one hour of each other.

[00415] In some embodiments, the additional therapeutic agent is an agent that increases levels of GPC3 in cancer cells associated with elevated expression of GPC3. In some embodiments, the agent that increases levels of GPC3 is an agent that inhibits DNA methylation. In some embodiments, the agent that increases levels of GPC3 is an agent that inhibits DNA methyltransferease. In some embodiments, the agent that increases levels of GPC3 is a hypomethylating agent. Examples of the hypomethylating agent includes, but are not limited to 5- azacitidine and decitabine and also includes any hypomethylating agent known in the art. In some embodiments, the hypomethylating agent is 5-azacitidine. In some embodiments, the hypomethylating agent is decitabine. In some embodiments, 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.

Diagnostic Methods

[00416] Also provided are methods for detecting the presence of GPC3 on cells from a subject. Such methods may be used, for example, to predict and evaluate responsiveness to treatment with an antibody provided herein. Any suitable method known to one skilled in the art can be used for diagnosis. In some embodiments, an imaging method, e.g., an immunopositron emisson tomography (immunoPET) or immunohistochemistry (IHC), can be used to assess tumor distribution or GPC3. In some embodiments, a liquid chromatography-ion trap-mass spectrometry can be used to measure salivary levels of GPC3.

[00417] In some embodiments, a tumor tissue sample is obtained from a subject and the fraction of cells expressing GPC3 or expression pattern of GPC3 is determined. In some embodiments, a blood sample is obtained from a subject and the fraction of cells expressing GPC3 is determined. In some aspects, 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. In some embodiments, flow cytometry is used to make such measurements. In some embodiments, fluorescence assisted cell sorting (FACS) is used to make such measurement. In some embodiments, the levels of serum GPC3 can be determined. In some embodiments, ELISA can be used to determine the serum GPC3 levels. In some embodiments, Sandwich ELISA can be used to determine the serum GPC3 levels. See Capurro et al., Li et al., Gastroenterology, 2003, 125(l):89-97 for methods of evaluating GPC3 expression in peripheral blood.

Tumor Antigen Associated Diseases or Disorders

[00418] Many patients treated with cancer therapeutics that are directed to one target on a tumor cell, e.g., BCMA, CD19, CD20, CD22, CD123, MUC16, MSLN, GPC3, etc., become resistant over time as escape mechanisms such as alternate signaling pathways and feedback loops become activated. Dual specificity therapeutics attempt to address this by combining targets that often substitute for each other as escape routes. Therapeutic T cell populations having TCRs specific to more than one tumor-associated antigen are promising combination therapeutics. In some embodiments, 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. Exemplary 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 cell expresses a specific clonotype, e.g., as in B cell, T cell lymphoma/leukemia resulting from clonal aberrancies). Exemplary 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-fucosyltransferase 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, OGT, OS- 9, p53, pml-RARalpha fusion protein, PPP1R3B, PRDX5, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1 or -SSX2 fusion protein, TGF-betaRII, triosephosphate isomerase, BAGE-1, D393-CD20n, Cyclin-Al, GAGE-1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GnTVf, HERV-K-MEL, KK-LC-1, KM-HN-1, LAGE-1, LY6K, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12 m, MAGE-CI, MAGE-C2, mucink, NA88-A, NY-ESO-1 / LAGE-2, SAGE, Spl7, SSX-2, SSX-4, TAG-1, TAG-2, TRAG-3, TRP2-INT2g, XAGE-lb/GAGED2a, Gene / protein, CEA, gplOO / Pmell7, mammaglobin-A, Melan-A / MART-1, NY-BR-1, OA1, PAP, PSA, RAB38 / NY-MEL- 1, TRP-1 / gp75, TRP-2, tyrosinase, adipophilin, AIM-2, ALDH1A1, BCLX (L), BING-4, CALCA, CD45, CD274, CPSF, cyclin DI, DKK1, ENAH (hMena), EpCAM, EphA3, EZH2, FGF5, glypican-3, G250 / MN / CAIX, HER-2 / neu, HLA-DOB, Hepsin, IDO1, IGF2B3, IL13Ralpha2, Intestinal carboxyl esterase, alpha-foetoprotein, Kallikrein 4, KIF20A, Lengsin, M- CSF, MCSP, mdm-2, Meloe, Midkine, MMP-2, MMP-7, MUC1, MUC5AC, p53, PAX5, PBF, PRAME, PSMA, RAGE-1, RGS5, RhoC, RNF43, RU2AS, secernin 1, SOXIO, STEAP1, survivin, Telomerase, TPBG, VEGF, and WT1.

Pharmaceutical Compositions

[00419] Pharmaceutical 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.

[00420] Pharmaceutical 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.

[00421] In one embodiment, 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. In one embodiment, 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.

[00422] 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 10⁴ to 10⁹ cells/kg body weight, in some instances 10⁵ to 10⁶ 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).

[00423] In certain aspects, it may be desired to administer activated T cells to a subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom according to the present invention, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain aspects, T cells can be activated from blood draws of from 10 cc to 400 cc. In certain aspects, 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.

[00424] The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one aspect, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In one aspect, 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.

[00425] In a particular exemplary aspect, 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. These 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. In certain aspects, following or concurrent with the transplant, subjects receive an infusion of the expanded TFP T cells of the present invention. In an additional aspect, expanded cells are administered before or following surgery.

[00426] 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®), for example, 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).

[00427] In one embodiment, 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. In one embodiment, 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. In one embodiment, the subject (e.g., human 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. In another embodiment, the subject (e.g., human 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. In one embodiment, the TFP T cells are administered every other day for 3 administrations per week. In one embodiment, the TFP T cells of the invention are administered for at least two, three, four, five, six, seven, eight or more weeks.

[00428] In one aspect, 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.

[00429] In one aspect, 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. In one aspect, the TFP RNA is transduced into the T cell by electroporation.

[00430] A potential issue that can arise in patients being treated using transiently expressing TFP T cells (particularly with murine scFv bearing TFP T cells) is anaphylaxis after multiple treatments.

[00431] Without being bound by this theory, it is believed that such an 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.

[00432] If a patient is at high risk of generating an anti-TFP antibody response during the course of transient TFP therapy (such as those generated by RNA transductions), TFP T cell infusion breaks should not last more than ten to fourteen days.

Cytokine Release

[00433] 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.

[00434] In some cases, 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. In some embodiments, the cytokine is IL-2, IFN-y, IL-4, TNF-a, IL-6, IL-13, IL-5, IL-10, sCD137, GM-CSF, MIP-la, MIP-ip, or a combination thereof. In some cases, the treatment with TFP T cells release less perforin, granzyme A, granzyme B, or a combination thereof, than the treatment with CAR-T cells. In some embodiments, 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.

[00435] In some embodiments, for a given cytokine, at least 10% less amount of the given cytokine is released following treatment compared to an amount of the given cytokine of a mammal treated with a CAR-T cell comprising the same binding domain. In some embodiments, the given cytokine comprises one or more cytokines selected from the group consisting of IL-2, IFN-y, IL-4, TNF-a, IL-6, IL-13, IL-5, IL-10, sCD137, GM-CSF, MIP-la, MIP-ip, and any combination thereof.

[00436] The TFP T cells may exhibit similar or better activity in killing tumor cells than CAR-T cells. In some embodiments, 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. In some embodiments, the tumor growth in the mammal is completely inhibited. In some embodiments, 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. In some embodiments, 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.

[00437] The TFP T cells can exhibit different gene expression profile than cells that do not express TFP. In some cases, the TFP T cells may exhibit similar gene expression profiles than CAR-T cells. In some other cases, the TFP T cells may exhibit different gene expression profiles than CAR-T cells. In some embodiments, the population of T cells transduced with TFP have a different gene expression profile than the CAR-T cells comprising the same binding domain. In some embodiments, 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. In some embodiments, 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.

EXAMPLES

[00438] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples specifically point out various aspects of the present invention and are not to be construed as limiting in any way the remainder of the disclosure.

[00439] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced, given the general description provided herein.

[00440] The entire disclosures of all patent and non-patent publications cited herein are each incorporated by reference in their entireties for all purposes.

Example 1: Generation of GPC3 antibodies

[00441] 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).

[00442] 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 crossreactivity 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.

[00443] As a pilot assessment, 10 unique yeast clones from each Sanger sequenced 96-well plate were randomly selected and the supernatants were directly tested in an ELISA assay for GPC3 binding along with GC33 and hYP7 GPC3 antibodies. The results are shown in FIG. 1. Binding to GPC5 and GPC6, as well as binding to a negative control antigen were also tested. 16 of the 20 clones showed GPC3 binding by ELISA and did not show binding to GPC5+GPC6 or the negative control antigen, along with GC33 and hYP7. Two of the clones did not show binding and two did not grow. Supernatant from the 18 clones that grew were also tested for the ability to bind GPC3- expressing CHO cells or GPC3-negative CHO control cells by flow cytometry. The results are shown in FIG. 2. 14/18 showed positive and specific binding to GPC3 -expressing cells, along with GC33 and hYP7, and the two clones that did not show ELISA binding did not bind to GPC3- expressing cells.

[00444] Based on this data, 25 Sanger/NGS overlay ed clones with unique HCDR3s, including the 14 clones shown to have positive binding for GPC3 by ELISA and flow cytometry, as described above, were selected for further characterization. The sequences of the 25 anti-GPC scFv antibodies are shown in Table 1. Of the 25 scFv antibodies that were sub-cloned for expression as human Fc (hFc) fusions in a mammalian expression system and purified using Protein A resin, 16 both exhibited cell specific flow cytometric binding to CHO cells expressing GPC3, compared to binding to CHO WT cells, and yielded concentrations sufficient for further biochemical characterization. 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_off> 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. In this assay, individual anti-GPC3 scFv-hFc proteins are immobilized on activated SPR chips, loaded with GPC3 protein, and subsequently challenged in a pairwise format against all other anti-GPC3 scFv-hFc fusions to identify unique and shared GPC3 epitope bins. This analysis identified 5 discrete GPC3-binding epitope bins. The summarized characterization data is shown in Table 2. As is shown, all measurements were not performed for all binders. A schematic illustration of the binning analysis is shown in FIG. 3.

Table 1: Anti-GPC3 scFv Sequences

Table 2: Properties of anti-GPC3 scFvs

Example 2: TFP Constructs

[00445] Anti-GPC3 TFP constructs were engineered by cloning anti-GPC3 binding domain (VHH 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. Various other vectors may be used to generate fusion protein constructs. In some embodiments, the anti-GPC3 binding domain is any of the anti-scFv antibody domains of Table 1 or Table 14 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, MDX-1414, GPC3-001, or GPC3-009.

Source of TCR Subunits

[00446] 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.

[00447] The human CD3-epsilon polypeptide canonical sequence is: MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILW QHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARV CENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQ NKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO:694).

[00448] The signal peptide of human CD3s is:

MQSGTHWRVLGLCLLSVGVWGQ (SEQ ID NO:695).

[00449] The extracellular domain of human CD3s is: DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHL SLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD (SEQ ID NO:696).

[00450] The transmembrane domain of human CD3s is:

VMSVATIVIVDICITGGLLLLVYYWS (SEQ ID NO: 697).

[00451] The intracellular domain of human CD3s is:

KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: 698).

[00452] The human CD3-gamma polypeptide canonical sequence is: MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDG KMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS GFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQ GNQLRRN (SEQ ID NO: 699).

[00453] The signal peptide of human CD3y is:

MEQGKGLAVLILAIILLQGTLA (SEQ ID NO:700).

[00454] The extracellular domain of human CD3y is:

QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAK DPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS (SEQ ID NO:701).

[00455] The transmembrane domain of human CD3y is:

GFLFAEIVSIFVLAVGVYFIA (SEQ ID NO:702).

[00456] The intracellular domain of human CD3y is:

GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN (SEQ ID NO:703).

[00457] The human CD3-delta polypeptide canonical sequence is: MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLG KRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALG VFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKS (SEQ ID NO: 704).

[00458] The signal peptide of human CD36 is:

MEHSTFLSGLVLATLLSQVSP (SEQ ID NO:705).

[00459] The extracellular domain of human CD36 is:

FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKE STVQVHYRMCQSCVELDPATVA (SEQ ID NO:706).

[00460] The transmembrane domain of human CD36 is:

GIIVTDVIATLLLALGVFCFA (SEQ ID NO:707).

[00461] The intracellular domain of human CD36 is:

GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK (SEQ ID NO: 708).

[00462] The human CD3-zeta polypeptide canonical sequence is: MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:709).

[00463] The human TCR alpha chain canonical sequence is: MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLD SPIWFSAGNGSALDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHS RSTQPMHLSGEASTARTCPQEPLRGTPGGALWLGVLRLLLFKLLLFDLLLTCSCLCDPAG

PLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVWGEG SYLSSYPTCPAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGAA (SEQ ID NO:710).

[00464] The human TCR alpha chain C region canonical sequence is: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSN SAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRI LLLKVAGFNLLMTLRLWSS (SEQ ID NO:711).

[00465] The human TCR alpha chain human IgC sequence is:

PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFK SNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS (SEQ ID NO: 712).

[00466] The transmembrane domain of the human TCR alpha chain is:

VIGFRILLLKVAGFNLLMTLRLW (SEQ ID NO:713).

[00467] The intracellular domain of the human TCR alpha chain is:

SS

[00468] The human TCR alpha chain V region CTL-L17 canonical sequence is: MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDY FLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAAK

GAGTASKLTFGTGTRLQVTL (SEQ ID NO:714). [00469] The human TCR beta chain C region 1 (hTRBCl) canonical sequence is: EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK DF (SEQ ID NO:715).

[00470] The human TCR beta chain constant region:

VEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVST DPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKP VTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKR KDF (SEQ ID NO:211).

[00471] The human TCR beta chain human IgC sequence is:

EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYE (SEQ ID NO: 716).

[00472] The transmembrane domain of the human TCR beta chain is:

ILLGKATLYAVLVSALVLMAM (SEQ ID NO:717).

[00473] The human TCR beta chain V region CTL-L17 canonical sequence is: MGTSLLCWMALCLLGADHADTGVSQNPRHNITKRGQNVTFRCDPISEHNRLYWYRQTL GQGPEFLT YFQNEAQLEKSRLLSDRF S AERPKGSF STLEIQRTEQGD S AMYLC AS SL AGL NQPQHFGDGTRLSIL (SEQ ID NO:718).

[00474] The intracellular domain of the human TCR beta chain is:

VKRKDF (SEQ ID NO: 719).

[00475] The human TCR beta chain V region YT35 canonical sequence is: MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTM MRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSFSTCS ANYGYTFGSGTRLTVV (SEQ ID NO:720).

[00476] The human TCR gamma chain C region canonical sequence is:

DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN TMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN CSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS (SEQ ID NO:721). [00477] The human TCR gamma human IgC sequence is:

DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN TMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN CSKDANDTLLLQLTNTSA (SEQ ID NO: 722).

[00478] The transmembrane domain of the human TCR gamma chain is:

YYMYLLLLLKSVVYFAIITCCLL (SEQ ID NO:723).

[00479] The intracellular domain of the human TCR gamma chain is:

RRTAFCCNGEKS (SEQ ID NO: 724).

[00480] The human TCR delta chain C region canonical sequence is:

SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLG KYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTE KVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL (SEQ ID NO: 725).

[00481] The human TCR delta human IgC sequence is:

SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLG KYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTE KVNMMSLTV (SEQ ID NO: 726).

[00482] The transmembrane domain of the human TCR delta chain is:

LGLRMLFAKTVAVNFLLTAKLFF (SEQ ID NO: 727).

[00483] The intracellular domain of the human TCR delta chain is:

L

[00484] The murine TCR alpha chain constant (mTRAC) region canonical sequence is:

XIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSN GAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILL LKVAGFNLLMTLRLWSS (SEQ ID NO:212).

[00485] The murine TCR alpha chain (2-137) sequence is:

IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNG AIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLL KVAGFNLLMTLRLWSS (SEQ ID NO:213).

[00486] The murine TCR beta chain constant region canonical sequence (murine TCR beta chain constant region 1 canonical sequence) is: EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTD PQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNIS AEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO:214).

[00487] The murine TCR beta chain (2-173) sequence is: DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDP QAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISA EAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS (SEQ ID NO:215).

[00488] The transmembrane domain of the murine TCR alpha chain is: MGLRILLLKVAGFNLLMTLRLW (SEQ ID NO: 736).

[00489] The transmembrane domain of the murine TCR beta chain 1 is: ILYEILLGKATLYAVLVS TLVVMAMVK (SEQ ID NO: 738).

[00490] The intracellular domain of the murine TCR beta chain 1 is:

KRKNS (SEQ ID NO: 739).

[00491] The murine TCR beta chain constant 2 region canonical sequence is: XDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVST DPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNI SAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO:740).

TFP Expression Vectors

[00492] Expression vectors are provided that 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).

[00493] 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. Example 3: Generation of T cell receptor fusion protein T Cells

T-cell activation, transduction, and expansion

[00494] 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. On day 0, 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). On day 1, activated T cells were transduced with lentivirus encoding the GPC3 TFP. On day 4, the cells were washed, subcultured in fresh medium with cytokines and then expanded up to day 10 by supplementing fresh medium on day 7. At each day of subculture, cells were harvested, washed, and resuspended with fresh cytokine-containing medium. Expansion of the cells is shown in FIG. 4.

Verification of TFP expression by cell staining

[00495] 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.

[00496] 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.

Example 4: Luciferase-based cytotoxicity assay

[00497] 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 coculture. 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.

[00498] 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. Bright-Glo reagent was then added into each well to lysis cells and detect luciferase enzymatic activity. The percentage of tumor cell killing was then calculated with the following formula: % Cytotoxicity = 100% x [1 - RLU (Tumor cells + T cells) / RLU (Tumor cells)].

[00499] As shown in FIG. 7, 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.

Example 5: Cytokine Secretion measurement by MSP

[00500] A measure of 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-a).

[00501] Target-specific cytokine production including IFN-Y, IL-2, TNF-a 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).

[00502] As shown in FIGs. 8A-8D, consistent with cytotoxicity assay results (FIG. 7), robust production of IFN-Y, IL-2, TNF-a, and GM-CSF was observed for all GPC-3 TFP-cells from all 3 donors, when co-cultured with GPC-3 high HepG2 cells. On the contrary, despite of high level of cytotoxicity, 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

[00503] Constructs encoding 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.

Table 9: GPC3 TFPs with novel scFvs used in this study

Verification of TFP expression by cell staining

[00504] 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.

[00505] 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.

[00506] The 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. As shown, 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 CD3s, 1.12 vLvH CD3s, 1-C05 vLvH CD3s, 2.6 vLvH CD3s, 1.11 vHvL CD3s, 1.12 vHvL CD3s, 1-C05 vHvL CD3s, 2.6 vHvL CD3s, 1-H07 vLvH CD3s, 2.19 vHvL CD3s, 1-A08 vLvH CD3s, and 1-G10 vLvH CD3s. The GPC3 TFPs showed little to no cytotoxicity against GPC3 low A549 cells.

[00507] Target-specific cytokine production including IFN-y, IL-2, TNF-a 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.

[00508] As shown in 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 CD3s, 1.12 vLvH CD3s, 1-C05 vLvH CD3s, 2.6 vLvH CD3s, 1.11 vHvL CD3s, 1.12 vHvL CD3s, 1-C05 vHvL CD3s, 2.6 vHvL CD3s, 1-H07 vLvH CD3s, 2.19 vHvL CD3s, 1-A08 vLvH CD3s, and 1-G10 vLvH CD3s. The GPC3 TFPs showed little to no induction of cytokine production when contacted with GPC3 low A549 cells.

Example 7: Testing of hYP7 TRuC in Hep3B xenograft model

[00509] Based on the findings above, hYP7 CD3e TFP (SEQ ID NO: 205) T cells were further tested for in vivo efficacy in an NSG xenograft mouse model. Mice were subcutaneously inoculated with 5xl0⁶ Hep3B tumor cells two weeks prior to the study start date (i.e., day -14). At the start of the study, on Day 0, 4xl0⁶ hYP7 TRuC+ T cells (4.9xl0⁶ total cells) were intravenously administered to each mouse. Non-transduced and vehicle control groups were also used for comparison. Tumor volume was tracked over time, with data shown in FIG. 15. The hYP7 TRuC T cells proved efficacious in vivo, with successful clearing of the Hep3B tumor cells in this model. Example 8: Engineering of T cell receptor fusion proteins and associated T cells

[00510] To test whether additional engineering of the hYP7 CD3e TFP could further improve the potency and efficacy of the associated hYP7 TRuC T cells, a set of new constructs was designed, prepared and tested. The hYP7 CD3e TFP (SEQ ID NO: 205), having a VH-(G4S)3-VL format (linked via an A3(G4S)3LE linker to CD3e), was used as the baseline comparator construct. In the second construct, the same sequence components were used, but the format of the scFv was inverted, such that a VL-(G4S)3-VH construct was generated (SEQ ID NO: 286). In two other constructs, the (G4S)3 linker was replaced with a Whitlow linker, having a nucleotide sequence given by SEQ ID NO: 396 and an amino acid sequence given by SEQ ID NO: 315, yielding constructs having vH-whitlow-vL (SEQ ID NO: 287) and vL-whitlow-VH (SEQ ID NO: 288) formats for the hYP7 scFv, further linked to CD3e via an A3(G4S)3LE linker.

[00511] Each of these four constructs was packaged into lentiviral vectors and transduced into T cells collected from two donors. After 10-day expansion, the cells were collected and assessed for transduction efficiency based on surface expression of the hYP7 binder as measured by flow cytometry. The data are shown in FIG. 16 and demonstrate that each of the four constructs was able to successfully transduce T cells, with those having the vL-linker-vH format showing somewhat improved transduction efficiency as compared to constructs having the vH-linker-vL hYP7 format.

[00512] The hYP7 TRuC T cells were further characterized by phenotype as determined by CD4, CD8, CD69, CD45Ra, and CCR7 expression by flow cytometry. The four new constructs outlined above were compared to TC-210 (SEQ ID NO: 319) TRuC T cells and non-transduced controls. Exemplary flow cytometry plots from 1 donor are shown in FIG. 17 and showed that the two constructs having the vL-linker-vH hYP7 format yielded an improved phenotype over those having the vH-linker-vL format. The improved phenotype was based on an evident decrease in the activated cell population after 10 days of expansion in T cells transduced with these constructs.

[00513] Cytotoxicity of the four constructs was tested with a luciferase-based assay after 24hr coculture with HepG2, SNU398 and A549 cells. Each of the four constructs demonstrated substantially similar ability to kill cells, indicating that the format of the scFv did not negatively impact the function of these hYP7 TRuC T cells. Data are shown in FIG. 18.

[00514] Cytokine levels (IL-2, TNFa, GM-CSF) in the supernatant after 24hr of co-culture of hYP7 TRuC T cells and HepG2, SNU398 or A549 cells were also measured. Exemplary data for IL-2 from two donors are shown in FIG. 19 but the patterns were consistent for each of the cytokines measured. The vL-linker-vH format hYP7 TRuC T cells demonstrated the ability to induce greater cytokine release in this in vitro assay, suggestive of a preferred phenotype or improved functionality as compared to the vH-linker-vL format hYP7 TRuC T cells. This improvement in induced cytokine release appeared most substantial in co-culture with the GPC- moderate SNU398 cells.

[00515] Taken together, these studies indicated that hYP7 vL-linker-vH TRuC T cells had higher affinity to soluble GPC3, lower basal activation and better memory phenotypes and higher cytokine release than the hYP7 vH-linker-vL TRuC T cells.

Example 9: Generation and characterization of T cell receptor fusion proteins and associated T cells using humanized scFvs

[00516] In an alternative strategy to potentially further improve the potency and efficacy of GPC3 targeting TRuC T cells, humanized variants of mouse antibody GC33 were generated and tested. First, the murine GC33 antibody (mGC33) was humanized using CDR grafting. The resultant hGC33 was incorporated into a vH-(G4S)3-vL-CD3e TFP (SEQ ID NO: 293). After a liability analysis, a mutation was introduced into CDR1 (G34R) and then this mutated humanized hGC33 variant was incorporated into 4 hGC33 TFPs, having formats hGC33Arg vH-(G4S)3-vL (SEQ ID NO: 289), hGC33Arg vL-(G4S)3)-VH (SEQ ID NO: 290), hGC33Arg vH-whitlow-vL (SEQ ID NO: 291) and hGC33Arg vL-whitlow-vH (SEQ ID NO: 292). Each of these hGC33 scFvs were operatively linked to CD3 epsilon via an A3(G4S)3LE linker. Additional constructs used in these studies included, mGC33 vH-(G4S)3-vL CD3e TFP (SEQ ID NO: 201), hYP7 CD3e TFP (SEQ ID NO: 205), and a mGC33 vH-(G4S)3-vL further enhanced with IL21 and IL15 sequences (SEQ ID NO: 316). Constructs were packaged into lentiviral vectors and transduced into T cells collected from healthy donors to generate hGC33 TRuC T cells. Non-transduced (NT) controls were also included.

[00517] T cells transduced with each of the above noted constructs were expanded for 10 days. Expansion was substantially similar across all test groups (data not shown) and two donors, including the non-transduced controls, suggesting that transduction of GC33 TFPs did not negatively impact T cell expansion.

[00518] After expansion, the transduced T cells (TFP T cells or TRuC T cells) were collected and assessed by flow cytometry for surface expression of the GC33 binder. Exemplary flow cytometry plots from two separate donors are shown in FIG. 20. Each of the tested GC33 TFPs showed successful transduction into T cells, however, the humanized variants showed greater transduction efficiency as compared to the murine variant.

[00519] The GC33 TRuC T cells were further characterized by phenotype as determined by CD4, CD8, CD69, CD45Ra, and CCR7 expression by flow cytometry. Exemplary flow cytometry plots from 1 donor are shown in FIG. 21 and showed that the humanized variants had a preferred phenotype, having a smaller activated population after 10 days of expansion, as compared to the TFP T cells expressing a murine GC33 binder.

[00520] The cell killing ability of the GC33 TRuC T cells was tested after 24hrs co-culture with HepG2, SNU398 and A549 cells. All tested GC33 targeting TRuC T cells proved effective at killing tumor cells in vitro with substantially similar efficiency. Representative data are shown in FIG. 22.Cytokine release (IFNg, IL-2, TNFa, GM-CSF) was measured in the supernatant collected after 24hrs of co-culture of GC33 TRuC T cells and HePG2, SNU398 and A549. All tested GC33 TRuC T cells demonstrated the ability to induce substantial cytokine release in co-culture with moderate and high GC33 expressing target cells (SNU398 and HepG2, respectively). TRuC T cells expressing a humanized GC33 binder demonstrated a more powerful ability to induce cytokine release in this in vitro assay, as compared to TRuC T cells expressing a murine version of GC33. Representative data for TNFa levels across 2 donors are shown in FIG. 23.

[00521] Taken together, these studies indicated that TRuC T cells having a humanized GC33 binder showed improved performance in terms of expression, phenotype and cytokine release, when compared to TRuC T cells having the murine GC33 binder.

Example 10: Generation of additional GPC3 antibodies: immunization library

[00522] To identify new GPC3 antibodies for potential use in GPC3 targeting TFPs and TFP T cells, wild type mice will be immunized with KLH conjugated GPC3 peptide (511-560).

Example 11: Generation of additional GPC3 antibodies: re-panning of naive human scFv library

[00523] The library described in Example 1 will be re-screened for additional GPC3 binding variants for testing in TFPs and TFP T cells. This re-screening will focus on C-terminal GPC3 binders. Further, the cross-reactivity threshold set previously will be eased to include additional candidates.

Example 12: Generation of additional GPC3 antibodies: de novo naive human scFv library [00524] A de-novo naive human scFv library will be generated for identification of additional GPC3 binders for potential incorporation into TFPs and TFP T cells.

Example 13. Generation and characterization of additional GPC3 binders and TFPs

[00525] To identify improved GPC3 binders for incorporation into TFPs and TFP T cells, additional constructs from the library described in Example 1 were screened and tested. Rescreening focused on binders that bind to a C-terminal peptide of the human GPC3 protein (amino acids 511-560 of SEQ ID NO: 728). Thirty initial clones were selected and tested. Subsequently, 10 of the 30 clones ("GPC-001" - "GPC3-010") were selected based on the clones' ability to specifically bind to CHO cells expressing GPC3 without binding to wild type CHO cells; the specificity of each of the clones for binding to the GPC3 peptide versus a scrambled 49 amino acid peptide (detected by ELISA and by SPR); and each of the 10 clone having an affinity (KD) for intact GPC3 of below 50nM, as measured by sandwich assay format SPR detection on an LSA Carterra instrument. The affinity, specificity of cell binding, and specificity of peptide binding data are provided below in Table A. The amino acid sequences of the variable heavy and light chains of the selected clones are provided in Table 14.

Table A. Binding and affinity data for GPC3 clones

*Fold over WT cell (Cell Binding) or irrelevant peptide (Peptide Binding) control

[00526] For each of the 10 new selected clones, T cells were transduced with a lentiviral vector comprising a nucleic acid encoding the SCFV-CD3 E TFP, as described in Example 3, to generate TFP T cells. One clone (GPC3-002) did not transduce and was not further studied. Table 15 provides the amino acid sequences of the new GPC3 TFP constructs. For each, constructs were generated with the scFv in both variable chain orientations (VHVL and VLVH).

[00527] To identify functional TFP T cells, transduced cells were assessed for TFP expression, expansion, immunophenotype, and functional properties such as cytotoxicity and cytokine production. GC33Arg(VHVL)CD3 E and hYP7(VLVH)CD3 E TFP transduced cells were used as comparators (SEQ ID NOs: 291 and 288, respectively). FIG. 24 shows the expression of GPC3 TFPs in the transduced T cells as determined by flow cytometry on Day 10 after transduction. Percent frequency of GPC3+ cells is shown in the left panel, and GPC3+ MFI is shown in the right panel. As shown in the figure, TFP expression was detected in cells transduced with each of the GPC3 TFP constructs; transduction efficiency was at least 50% for all constructs except for GPC3- 006VLVH and GPC3-010 VLVH. No TFP expression was detected in control untransduced cells. Fold expansion of the transduced cells at Day 10 is shown in FIG. 25. Each of the new GPC3 scFv constructs exhibited greater fold expansion compared to the GC33 and hYP7 comparators. FIG. 26 (top panel) shows the relative frequency of CD4+ and CD8+ T cells in the transduced cells at Day 10. The memory phenotypes of the T cells was determined by flow cytometry by detection of cell surface levels of CD45RA and CCR7 on CD4+ (FIG. 26, bottom left panel) or CD8+ (FIG. 26, bottom right panel) cells.

[00528] To assess % tumor cell lysis by transduced cells, luciferase bioluminescence-based cytotoxicity assays were performed by incubating the transduced cells with HepG2 cells at a 3: 1 or 1 : 1 E:T (FIG. 27). T cell activation in response to HepG2 cells was also assessed using an Incucyte® Assay. The transduced cells were incubated with HepG2 cells at a 3: 1 or 1 : 1 ET, and activation of T cells was measured using cell imaging and caspase 3/7 staining intensity (Alam et al., J Exp Med 190(12): 1879-90 (1999); McComb et al., PLoS One 5(12): el5328 (2010)) (FIG. 28).

[00529] Production of IL2, IFNy, TNFa, and GMCSF by TFP T cells was measured using an MSD assay as described in Example 5. FIG. 29 shows cytokine production after co-culture of the transduced cells with HepG2 cells for 24 hours at a 1 : 1 E:T. FIG. 30 shows cytokine production after co-culture of the transduced cells with HepG2 cells for 24 hours at a 3 : 1 E:T. FIG. 31 shows cytokine production after co-culture of the transduced cells with Hep3B cells for 24 hours at a 1 : 1 E:T. FIG. 32 shows cytokine production after co-culture of the transduced cells with Hep3B cells for 24 hours at a 3 : 1 E:T.

[00530] Together, the results of these initial assays to screen for improved GPC3 TFP T cells indicated that many of the GPC3 TFP transduced cells, including cells transduced with the GPC3- 001 TFP in either orientation, GPC3-008 VLVH TFP, and GPC3-009 VLVH TFP, exhibited levels of tumor lysis, TCR activation, and/or cytokine production similar to or superior to the comparator cells. Example 14. Further evaluation of lead constructs

[00531] Based on the studies above, four lead constructs were selected as exhibiting superior target cell lysis, T cell activation, and/or cytokine production in the screening assays and were subjected to further tests to confirm the favorable phenotype an activity: GPC3-001 VHVL, GPC3-001 VLVH, GPC3-008 VLVH, and GPC3-009 VLVH. Transduced cells were generated using cells from two additional separate donors, referred to as Donor A and Donor B in the figures of this Example. FIG. 33 shows expression of the TFP on Day 10 after transduction of cells from the additional donors. FIG. 34 shows the CD4 and CD8 distribution, and FIG. 35 shows the memory phenotypes of the transduced cells.

[00532] Tumor cell lysis (using the luciferase-based cytotoxicity assay) and cytokine release (IL2, IFNY, TNFa, and GMCSF by MSD) were assessed. Fourteen days after transduction, transduced cells were normalized for TFP expression, and incubated with HepG2, Hep3B, SNU398, or A549 cells (see FIG. 6 for GPC3 expression in each cell line) at 9: 1, 3: 1, 1 : 1, or 1 :3 E:T. After 24 hours of coculture, tumor lysis and cytokine release were measured. The transduced T cells were each similarly cytotoxic compared to gGC33 and hYP7, as shown in FIG. 36. Cytokine release in the co-cultures with high GPC3 expressing cell lines HepG2 (FIG. 37) and Hep3B (FIG. 38) was also similar or increased compared to the comparators, except for GPC3-008 VLVH which exhibited lower levels of IL2, IFNy, and TNFa. There was less cytokine production in response to SNU398 cells (FIG. 39). None of the cells except GPC3-008 VLVH exhibited cytokine production in response to A549 cells (very low/no GPC3 expression), suggesting off-target, basal activation or non-specific targeting was not present in the GPC3-009 and GPC3-001 cells tested (FIG. 40). Thus, in particular, GPC3-009 VLVH, GPC3-001 VHVL, and GPC3-001 VLVH exhibited favorable lysis and cytokine profiles.

[00533] To assess which of the TFPs provided the most robust responses to HepG2 cells after repeated stimulation, a repeated stimulation assay was conducted using transduced cells from each of Donor A and Donor B. Transduced cells were normalized to 50% transduction, and incubated with HepG2 cells at a 1 :1 ratio. At four subsequent timepoints (4 Days, 8 Days, 12 Days, and 16 Days), supernatants and cells were collected, and HepG2 cells were added again to the culture to provide repeat stimulation. Fold expansion of the transduced cells (combined Donor A and Donor B cells) at Days 4, 8, and 12 is shown in FIG. 41. Compared to the comparator GC33 and hPY7 transduced cells, GPC3-009 and GPC3-001 TFP expressing cells exhibited approximately equivalent (GPC3-009 VLVH) or approximately 35% improved (GPC3- 001 VHVL and GPC3-001 VLVH) enrichment of antigen-specific TFP+ T cells (FIG. 41, left panel); and approximately equivalent (GPC3-009 VLVH) or approximately 46% improved (GPC3-001 VHVL and GPC3-001 VLVH) expansion in response to tumor cells (FIG. 41, right panel).

Example 15. In vivo characterization of lead GPC3 TFP expressing T cells.

[00534] GPC3-009 VLVH, GPC3-001 VHVL, and GPC3-001 VLVH TFP+ T cells are further tested for in vivo efficacy in an NSG xenograft mouse model. Mice are subcutaneously inoculated with Hep2G or Hep3B tumor cells. Approximately 2 weeks later, on Day 0 of the study, TFP+ T cells are intravenously administered to tumor bearing mice. Non-transduced and vehicle control groups are also used for comparison. Tumor volume is tracked over time. The results of the study will show the efficacy of the GPC3-009 VLVH, GPC3-001 VHVL, and GPC3-001 VLVH TFP+ T cells in vivo by demonstrating successful clearing of tumor cells.

Table 3: Additional Anti-GPC3 Binder, TFP Construct and Component Amino Acid

Sequences

Table 4: Examples of TGFBr2 Switch Polypeptide Sequences

Table 5: Examples of PD-1 Switch Molecule Amino Acid Sequences

Table 6: Examples of Anti-PD-1 Antibody Sequences and Fusion Protein Amino Acid

Sequences

Table 7: Examples of IL-15 or IL-15R Construct Amino acid Sequences

Table 8: Examples of CXCR6 Sequences

Table 10: Example Anti-MSLN Binder, TFP Construct and Related Component Amino

Acid Sequences

Table 11: Example Construct Nucleic Acid Sequences

Table 12: Components of Exemplary Constructs - Amino Acid Sequences

Table 13: Components of Exemplary Constructs - Nucleic Acid Sequences

Table 14. Additional GPC3 Binders - Amino acid sequences

Table 15. TFP Constructs with GPC3-001 to GPC3-010 scFvs

OTHER EMBODIMENTS

[00535] The disclosure set forth above may encompass multiple distinct inventions with independent utility. Although each of these inventions has been disclosed in its preferred form(s), the specific embodiments thereof as disclosed and illustrated herein are not to be considered in a limiting sense, because numerous variations are possible. The subject matter of the inventions includes all novel and nonobvious combinations and subcombinations of the various elements, features, functions, and/or properties disclosed herein. The following claims particularly point out certain combinations and subcombinations regarded as novel and nonobvious. Inventions embodied in other combinations and subcombinations of features, functions, elements, and/or properties may be claimed in this application, in applications claiming priority from this application, or in related applications. Such claims, whether directed to a different invention or to the same invention, and whether broader, narrower, equal, or different in scope in comparison to the original claims, also are regarded as included within the subject matter of the inventions of the present disclosure.

APPENDIX: SEQUENCES

SEQ ID NO: 689

AAUAAA

SEQ IDNO: 690

GGGGSGGGGS

SEQ ID NO: 691

GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC

SEQ ID NO: 692

AAAGGGGSGGGGSGGGGSL

SEQ ID NO: 693

AAAIEVMYPPPYLGGGGSGGGGSGGGGSLE

SEQ ID NO: 694

MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILW

QHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARV

CENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQ

NKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

SEQ ID NO: 695

MQ SGTHWRVLGLCLLS VGVWGQ

SEQ ID NO: 696

DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHL

SLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

SEQ ID NO: 697

VMSVATIVIVDICITGGLLLLVYYWS

SEQ ID NO: 698

KNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI

SEQ ID NO: 699

MEQGKGLAVLILAIILLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDG

KMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS

GFLFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQ

GNQLRRN

SEQ ID NO: 700

MEQGKGLAVLILAIILLQGTLA

SEQ ID NO:701

QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAK

DPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATIS

SEQ ID NO: 702

GFLFAEIVSIFVLAVGVYFIA SEQ ID NO: 703

GQDGVRQSRASDKQTLLPNDQLYQPLKDREDDQYSHLQGNQLRRN

SEQ ID NO: 704

MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLG

KRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALG

VFCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNKS

SEQ ID NO: 705

MEHSTFLSGLVLATLLSQVSP

SEQ ID NO: 706

FKIPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKE

STVQVHYRMCQSCVELDPATVA

SEQ ID NO: 707

GIIVTDVIATLLLALGVFCFA

SEQ ID NO: 708

GHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYSHLGGNWARNK

SEQ ID NO: 709

MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADA

PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDK

MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO:710

MAGTWLLLLLALGCPALPTGVGGTPFPSLAPPIMLLVDGKQQMVVVCLVLDVAPPGLD

SPIWFSAGNGSALDAFTYGPSPATDGTWTNLAHLSLPSEELASWEPLVCHTGPGAEGHS

RSTQPMHLSGEASTARTCPQEPLRGTPGGALWLGVLRLLLFKLLLFDLLLTCSCLCDPAG

PLPSPATTTRLRALGSHRLHPATETGGREATSSPRPQPRDRRWGDTPPGRKPGSPVWGEG

SYLSSYPTCPAQAWCSRSALRAPSSSLGAFFAGDLPPPLQAGAA

SEQ ID NO:711

PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFK

SNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIG

FRILLLKVAGFNLLMTLRLWS S

SEQ ID NO: 712

PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFK

SNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLS

SEQ ID NO:713

VIGFRILLLKVAGFNLLMTLRLW

SEQ ID NO:714

MAMLLGASVLILWLQPDWVNSQQKNDDQQVKQNSPSLSVQEGRISILNCDYTNSMFDY

FLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAAK GAGTASKLTFGTGTRLQVTL

SEQ ID NO:715 EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTD PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV TQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRK

DF

SEQ ID NO: 716

EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTD

PQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPV

TQIVSAEAWGRADCGFTSVSYQQGVLSATILYE

SEQ ID NO:717

ILLGKATLYAVLVSALVLMAM

SEQ ID NO:718

MGTSLLCWMALCLLGADHADTGVSQNPRHNITKRGQNVTFRCDPISEHNRLYWYRQTL GQGPEFLT YFQNEAQLEKSRLLSDRF S AERPKGSF STLEIQRTEQGD S AMYLC AS SL AGL NQPQHFGDGTRLSIL

SEQ ID NO: 719

VKRKDF

SEQ ID NO: 720

MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTM

MRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSFSTCS ANYGYTFGSGTRLTVV

SEQ ID NO:721

DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN TMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN CSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFCCNGEKS

SEQ ID NO: 722

DKQLDADVSPKPTIFLPSIAETKLQKAGTYLCLLEKFFPDVIKIHWQEKKSNTILGSQEGN TMKTNDTYMKFSWLTVPEKSLDKEHRCIVRHENNKNGVDQEIIFPPIKTDVITMDPKDN CSKDANDTLLLQLTNTSA

SEQ ID NO: 723

YYMYLLLLLKSVVYFAIITCCLL

SEQ ID NO: 724

RRTAFCCNGEKS

SEQ ID NO: 725

SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLG

KYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTE KVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFFL

SEQ ID NO: 726

SQPHTKPSVFVMKNGTNVACLVKEFYPKDIRINLVSSKKITEFDPAIVISPSGKYNAVKLG

KYEDSNSVTCSVQHDNKTVHSTDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTE KVNMMSLTV SEQ ID NO: 727

LGLRMLFAKTVAVNFLLTAKLFF

SEQ ID NO: 728

MAGTVRTACLVVAMLLSLDFPGQAQPPPPPPDATCHQVRSFFQRLQPGLKWVPETPVPG

SDLQVCLPKGPTCCSRKMEEKYQLTARLNMEQLLQSASMELKFLIIQNAAVFQEAFEIV

VRHAKNYTNAMFKNNYPSLTPQAFEFVGEFFTDVSLYILGSDINVDDMVNELFDSLFPVI

YTQLMNPGLPDSALDINECLRGARRDLKVFGNFPKLIMTQVSKSLQVTRIFLQALNLGIE

VINTTDHLKFSKDCGRMLTRMWYCSYCQGLMMVKPCGGYCNVVMQGCMAGVVEIDK

YWREYILSLEELVNGMYRIYDMENVLLGLFSTIHDSIQYVQKNAGKLTTTIGKLCAHSQ

QRQYRSAYYPEDLFIDKKVLKVAHVEHEETLSSRRRELIQKLKSFISFYSALPGYICSHSP

VAENDTLCWNGQELVERYSQKAARNGMKNQFNLHELKMKGPEPVVSQIIDKLKHINQL

LRTMSMPKGRVLDKNLDEEGFESGDCGDDEDECIGGSGDGMIKVKNQLRFLAELAYDL

DVDDAPGNSQQATPKDNEISTFHNLGNVHSPLKLLTSMAISVVCFFFLVH

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A recombinant nucleic acid molecule comprising a sequence encoding a T cell receptor (TCR) fusion protein (TFP) wherein the TFP comprises:

(a) a TCR subunit comprising:

(i) at least a portion of a TCR extracellular domain, and

(ii) a TCR transmembrane domain,

(iii) a TCR intracellular domain, and

(b) an antigen binding domain that specifically binds GPC3; and wherein the TCR subunit and the antigen binding domain are operatively linked.

2. The recombinant nucleic acid molecule of claim 1, wherein the TFP functionally interacts with an endogenous TCR complex when expressed in a T cell.

3. The recombinant nucleic acid molecule of claim 1 or 2, wherein the TCR intracellular domain comprises a stimulatory domain from an intracellular signaling domain of CD3 gamma, CD3 delta, or CD3 epsilon.

4. The recombinant nucleic acid molecule of any one of claims 1-3, wherein a T cell expressing the TFP exhibits increased cytotoxicity to a human cell expressing GPC3 compared to a T cell not containing the TFP.

5. The recombinant nucleic acid molecule of any one of claims 1-4, wherein the antigen binding domain is connected to the TCR extracellular domain by a linker sequence.

6. The recombinant nucleic acid molecule of claim 5, wherein the linker is 120 amino acids in length or less.

7. The recombinant nucleic acid molecule of claim 5, wherein the linker sequence comprises (G4S)_n, wherein G is glycine, S is serine, and n is an integer from 1 to 10.

8. The recombinant nucleic acid molecule of claim 7, wherein n is an integer from 1 to 4.

9. The recombinant nucleic acid molecule of any one of claims 1-8, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit.

10. The recombinant nucleic acid molecule of claim 9, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR alpha.

11. The recombinant nucleic acid molecule of claim 9, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR beta. The recombinant nucleic acid molecule of claim 9, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR gamma. The recombinant nucleic acid molecule of claim 9, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from TCR delta. The recombinant nucleic acid molecule of claim 9, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 epsilon. The recombinant nucleic acid molecule of claim 9, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 delta. The recombinant nucleic acid molecule of claim 9, wherein at least two of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from CD3 gamma. The recombinant nucleic acid molecule of any one of claims 9-16, wherein all three of the TCR extracellular domain, the TCR transmembrane domain, and the TCR intracellular domain are from the same TCR subunit. The recombinant nucleic acid molecule of any one of claims 1-17, wherein the antigen binding domain is a camelid antibody or binding fragment thereof. The recombinant nucleic acid molecule of any one of claims 1-17, wherein the antigen binding domain is a murine antibody or binding fragment thereof. The recombinant nucleic acid molecule of any one of claims 1-17, wherein the antigen binding domain is a human or humanized antibody or binding fragment thereof. The recombinant nucleic acid molecule of any one of claims 1-20, wherein the antigen binding domain is a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain. The recombinant nucleic acid molecule of any one of claims 1-21, wherein the antigen binding domain comprises a heavy chain variable domain comprising a heavy chain complementarity determining region 1 (HC CDR1), a heavy chain complementarity determining region 2 (HC CDR2), and a heavy chain complementarity determining region 2 (HC CDR3). The recombinant nucleic acid molecule of any one of claims 1-22, wherein the antigen binding domain comprises a light chain variable domain comprising a light chain complementarity determining region 1 (LC CDR1), a light chain complementarity determining region 2 (LC CDR2), and a light chain complementarity determining region 3 (LC CDR3). The recombinant nucleic acid molecule of claim 22 or 23, wherein 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 recombinant nucleic acid molecule of claim 24, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:2. The recombinant nucleic acid molecule of claim 24 or 25, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 1. The recombinant nucleic acid molecule of claim 22 or 23, wherein 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 recombinant nucleic acid molecule of claim 27, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:18. The recombinant nucleic acid molecule of claim 27 or 28, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 17. The recombinant nucleic acid molecule of claim 22 or 23, wherein 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 recombinant nucleic acid molecule of claim 30, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:34. The recombinant nucleic acid molecule of claim 30 or 31, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:33. The recombinant nucleic acid molecule of claim 22 or 23, wherein 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 recombinant nucleic acid molecule of claim 33, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:42. The recombinant nucleic acid molecule of claim 33 or 34, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:41. The recombinant nucleic acid molecule of claim 22 or 23, wherein 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 recombinant nucleic acid molecule of claim 36, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:50. The recombinant nucleic acid molecule of claim 36 or 37, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:49. The recombinant nucleic acid molecule of claim 22 or 23, wherein 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 recombinant nucleic acid molecule of claim 39, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:58. The recombinant nucleic acid molecule of claim 39 or 40, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:57. The recombinant nucleic acid molecule of claim 22 or 23, wherein 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 recombinant nucleic acid molecule of claim 42, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:82. The recombinant nucleic acid molecule of claim 42 or 43, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:81. The recombinant nucleic acid molecule of claim 22 or 23, wherein 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 recombinant nucleic acid molecule of claim 45, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:90. The recombinant nucleic acid molecule of claim 45 or 46, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:89. The recombinant nucleic acid molecule of claim 22 or 23, wherein 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 recombinant nucleic acid molecule of claim 48, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:98. The recombinant nucleic acid molecule of claim 48 or 49, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:97. The recombinant nucleic acid molecule of claim 22 or 23, wherein the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO: 782, the HC CDR2 of SEQ ID NO: 783, the HC CDR3 of SEQ ID NO: 784; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:785, the LC CDR2 of SEQ ID NO:786, the LC CDR3 of SEQ ID NO:787; or a combination thereof The recombinant nucleic acid molecule of claim 51, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 780. The recombinant nucleic acid molecule of claim 51 or 52, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:781. The recombinant nucleic acid molecule of claim 22 or 23, wherein the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO: 838, the HC CDR2 of SEQ ID NO: 839, the HC CDR3 of SEQ ID NO: 840; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:841, the LC CDR2 of SEQ ID NO: 842, the LC CDR3 of SEQ ID NO: 843; or a combination thereof. The recombinant nucleic acid molecule of claim 54, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 836. The recombinant nucleic acid molecule of claim 54 or 55, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:837. The recombinant nucleic acid molecule of claim 22 or 23, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:203. The recombinant nucleic acid molecule of claim 57, wherein the heavy chain variable domain comprises a sequence of SEQ ID NO:203. The recombinant nucleic acid molecule of any one of claims 22, 23, 57 and 58, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:204. The recombinant nucleic acid molecule of claim 59, wherein the light chain variable domain comprises a sequence of SEQ ID NO:204. The recombinant nucleic acid molecule of any one of claims 57-60, wherein the antigen binding domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:202. The recombinant nucleic acid molecule of claim 61, wherein the antigen binding domain comprises a sequence of SEQ ID NO:202. The recombinant nucleic acid molecule of any one of claims 57-62, wherein the TFP comprises a sequence having at least about 80% sequence identity to SEQ ID NO:201. The recombinant nucleic acid molecule of claim 63, wherein the TFP comprises a sequence of SEQ ID NO:201. The recombinant nucleic acid molecule of claim 22 or 23, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:207. The recombinant nucleic acid molecule of claim 65, wherein the heavy chain variable domain comprises a sequence of SEQ ID NO:207. The recombinant nucleic acid molecule of any one of claims 22, 23, 65 and 66, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:208. The recombinant nucleic acid molecule of claim 67, wherein the light chain variable domain comprises a sequence of SEQ ID NO:208. The recombinant nucleic acid molecule of any one of claims 65-68, wherein the antigen binding domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:206. The recombinant nucleic acid molecule of claim 69, wherein the antigen binding domain comprises a sequence of SEQ ID NO:206. The recombinant nucleic acid molecule of any one of claims 65-70, wherein the TFP comprises a sequence having at least about 80% sequence identity to SEQ ID NO:205. The recombinant nucleic acid molecule of claim 71, wherein the TFP comprises a sequence of SEQ ID NO:205. The recombinant nucleic acid molecule of claim 22, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:210. The recombinant nucleic acid molecule of claim 73, wherein the heavy chain variable domain comprises a sequence of SEQ ID NO:210. The recombinant nucleic acid molecule of claim 73 or 74, wherein the TFP comprises a sequence having at least about 80% sequence identity to SEQ ID NO:209. The recombinant nucleic acid molecule of claim 75, wherein the TFP comprises a sequence of SEQ ID NO:209. The recombinant nucleic acid molecule of claim 22 or 23, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:780. The recombinant nucleic acid molecule of claim 77, wherein the heavy chain variable domain comprises a sequence of SEQ ID NO:780. The recombinant nucleic acid molecule of any one of claims 22, 23, 77, and 78, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:781. The recombinant nucleic acid molecule of claim 79, wherein the light chain variable domain comprises a sequence of SEQ ID NO:781. The recombinant nucleic acid molecule of any one of claims 77-80, wherein the antigen binding domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:854. The recombinant nucleic acid molecule of claim 81, wherein the antigen binding domain comprises a sequence of SEQ ID NO:854. The recombinant nucleic acid molecule of any one of claims 77-80, wherein the antigen binding domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:855. The recombinant nucleic acid molecule of claim 83, wherein the antigen binding domain comprises a sequence of SEQ ID NO:855. The recombinant nucleic acid molecule of any one of claims 77-82, wherein the TFP comprises a sequence having at least about 80% sequence identity to SEQ ID NO:852. The recombinant nucleic acid molecule of claim 85, wherein the TFP comprises a sequence of SEQ ID NO:852. The recombinant nucleic acid molecule of any one of claims 77-80, 83, and 84, wherein the TFP comprises a sequence having at least about 80% sequence identity to SEQ ID NO:853. The recombinant nucleic acid molecule of claim 87, wherein the TFP comprises a sequence of SEQ ID NO:853. The recombinant nucleic acid molecule of claim 22 or 23, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:836. The recombinant nucleic acid molecule of claim 89, wherein the heavy chain variable domain comprises a sequence of SEQ ID NO:836. The recombinant nucleic acid molecule of any one of claims 22, 23, 89, and 90, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:837. The recombinant nucleic acid molecule of claim 91, wherein the light chain variable domain comprises a sequence of SEQ ID NO:837. The recombinant nucleic acid molecule of any one of claims 89-92, wherein the antigen binding domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:883. The recombinant nucleic acid molecule of claim 93, wherein the antigen binding domain comprises a sequence of SEQ ID NO:883. The recombinant nucleic acid molecule of any one of claims 89-94, wherein the TFP comprises a sequence having at least about 80% sequence identity to SEQ ID NO:881. The recombinant nucleic acid molecule of claim 95, wherein the TFP comprises a sequence of SEQ ID NO:881. The recombinant nucleic acid molecule of any one of claims 1-76, wherein a T cell expressing the TFP inhibits tumor growth when expressed in a T cell. A recombinant nucleic acid molecule comprising a sequence encoding an antibody or fragment thereof that specifically binds GPC3. The recombinant nucleic acid molecule of claim 98, wherein the antibody or antibody fragment is a camelid antibody or binding fragment thereof. The recombinant nucleic acid molecule of claim 98, wherein the antibody or antibody fragment is a murine, human or humanized antibody or binding fragment thereof. The recombinant nucleic acid molecule of any one of claims 98-100, wherein the antibody or antibody fragment is a single-chain variable fragment (scFv) or a single domain antibody (sdAb) domain. The recombinant nucleic acid molecule of claim 101, wherein the antibody or antibody fragment is a single domain antibody (sdAb). The recombinant nucleic acid molecule of claim 102, wherein the sdAb is a VHH. The recombinant nucleic acid molecule of any one of claims 98-103, wherein 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. The recombinant nucleic acid molecule of any one of claims 98-104, wherein 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. The recombinant nucleic acid molecule of claim 104 or 105, wherein 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 recombinant nucleic acid molecule of claim 106, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:2. The recombinant nucleic acid molecule of claim 106 or 107, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 1. The recombinant nucleic acid molecule of claim 104 or 105, wherein 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 recombinant nucleic acid molecule of claim 109, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:18. The recombinant nucleic acid molecule of claim 109 or 110, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO: 17. The recombinant nucleic acid molecule of claim 104 or 105, wherein 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 recombinant nucleic acid molecule of claim 112, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:34. The recombinant nucleic acid molecule of claim 112 or 113, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:33. The recombinant nucleic acid molecule of claim 104 or 105, wherein 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 recombinant nucleic acid molecule of claim 115, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:42. The recombinant nucleic acid molecule of claim 115 or 116, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:41. The recombinant nucleic acid molecule of claim 104 or 105, wherein 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 recombinant nucleic acid molecule of claim 118, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:50. The recombinant nucleic acid molecule of claim 118 or 119, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:49. The recombinant nucleic acid molecule of claim 104 or 105, wherein 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 recombinant nucleic acid molecule of claim 121, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:58. The recombinant nucleic acid molecule of claim 121 or 122, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:57. The recombinant nucleic acid molecule of claim 104 or 105, wherein 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 recombinant nucleic acid molecule of claim 124, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:82. The recombinant nucleic acid molecule of claim 124 or 125, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:81. The recombinant nucleic acid molecule of claim 104 or 105, wherein 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 recombinant nucleic acid molecule of claim 127, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:90. The recombinant nucleic acid molecule of claim 127 or 128, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:89. The recombinant nucleic acid molecule of claim 104 or 105, wherein 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 recombinant nucleic acid molecule of claim 130, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:98. The recombinant nucleic acid molecule of claim 130 or 131, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:97. The recombinant nucleic acid molecule of claim 104 or 105, wherein the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO: 782, the HC CDR2 of SEQ ID NO: 783, the HC CDR3 of SEQ ID NO: 784; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:785, the LC CDR2 of SEQ ID NO:786, the LC CDR3 of SEQ ID NO:787; or a combination thereof. The recombinant nucleic acid molecule of claim 133, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:780. The recombinant nucleic acid molecule or claim 133 or 134, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:781. The recombinant nucleic acid molecule of claim 104 or 105, wherein the antigen binding domain comprises (i) the heavy chain variable domain comprising the HC CDR1 of SEQ ID NO: 838, the HC CDR2 of SEQ ID NO: 839, the HC CDR3 of SEQ ID NO: 840; (ii) the light chain variable domain comprising the LC CDR1 of SEQ ID NO:841, the LC CDR2 of SEQ ID NO: 842, the LC CDR3 of SEQ ID NO: 843; or a combination thereof. The recombinant nucleic acid molecule of claim 136, wherein the heavy chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:836. The recombinant nucleic acid molecule of claim 136 or 137, wherein the light chain variable domain comprises a sequence having at least about 80% sequence identity to SEQ ID NO:837. The recombinant nucleic acid molecule of any one of claims 98-132, wherein the recombinant nucleic acid molecule further comprises a sequence encoding a TCR constant domain. The recombinant nucleic acid molecule of claim 139, wherein the antibody or antibody fragment is operatively linked to the sequence encoding a TCR constant domain, thereby forming a TFP. The recombinant nucleic acid molecule of claim 139 or 140, wherein 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 of any one of claims 981-141, further comprising a leader sequence. The recombinant nucleic acid molecule of any one of claims 1-142, wherein the nucleic acid is selected from the group consisting of a DNA and an RNA. The recombinant nucleic acid molecule of claim 143, wherein the nucleic acid is a mRNA. The recombinant nucleic acid molecule of claim 143, wherein the nucleic acid is a circRNA. The recombinant nucleic acid molecule of any one of claims 1-145, wherein the nucleic acid comprises a nucleotide analog. The recombinant nucleic acid molecule of claim 146, wherein the nucleotide analog is selected from the group consisting of 2’-O-methyl, 2’-O-methoxy ethyl (2’-0-M0E), 2’-O- aminopropyl, 2’-deoxy, 2’ -deoxy-2’ -fluoro, 2’-O-aminopropyl (2’-O-AP), 2'-O- dimethylaminoethyl (2’-0-DMA0E), 2’-O-dimethylaminopropyl (2’-0-DMAP), 2’-O- dimethylaminoethyloxyethyl (2’-0-DMAE0E), 2’-O-N-methylacetamido (2’-0-NMA) modified, a locked nucleic acid (LNA), an ethylene nucleic acid (ENA), a peptide nucleic acid (PNA), a l’,5’- anhydrohexitol nucleic acid (HNA), a morpholino, a methylphosphonate nucleotide, a thiolphosphonate nucleotide, and a 2’-fluoro N3-P5’- phosphoramidite. The recombinant nucleic acid molecule of any one of claims 1-147, further comprising a promoter. The recombinant nucleic acid molecule of any one of claims 1-148, wherein the nucleic acid is an in vitro transcribed nucleic acid. The recombinant nucleic acid molecule of any one of claims 1-149, wherein the nucleic acid further comprises a sequence encoding a poly(A) tail. The recombinant nucleic acid molecule of any one of claims 1-150, wherein the nucleic acid further comprises a 3’UTR sequence. A polypeptide encoded by the recombinant nucleic acid molecule of any one of claims 1- 151. A vector comprising a recombinant nucleic acid molecule encoding the TFP of any one of claims 1-97, 140, or 141. A vector comprising a recombinant nucleic acid molecule encoding the antibody or antigen binding fragment of claims 98-139. The vector of claim 153, further comprising 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 of claim 153, further comprising a sequence encoding a TCR constant domain. The vector of claim 156, wherein 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 of any one of claims 153-157, wherein the vector is selected from the group consisting of a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, a Rous sarcoma viral (RSV) vector, or a retrovirus vector. The vector of any one of claims 153-158, further comprising a promoter. The vector of any one of claims 153-159, wherein the vector is an in vitro transcribed vector. The vector of any one of claims 153-160, wherein a nucleic acid sequence in the vector further comprises a poly (A) tail. The vector of any one of claims 153-161, wherein a nucleic acid sequence in the vector further comprises a 3’UTR. A cell comprising the recombinant nucleic acid molecule of any one of claims 1-151, the polypeptide of claim 152, or the vector of any one of claims 153-162. The cell of claim 163, wherein the cell is a T cell. The T cell of claim 164, wherein the T cell is a human T cell. The T cell of claim 164 or 165, wherein the T cell is a CD8+ or CD4+ T cell. The T cell of claim 164, wherein the T cell is a human aP T cell. The T cell of claim 164, wherein the T cell is a human y6 T cell. The cell of claim 163, wherein the cell is a human NKT cell. The cell of any one of claims 163-169, wherein the cell comprises a functional disruption of an endogenous TCR. The cell of any one of claims 163-170, wherein the cell is an allogeneic cell. The cell of any one of claims 163-171, wherein 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 of any one of claims 163-172, wherein the cell further comprises a heterologous sequence encoding a TCR constant domain. The cell of claim 173, wherein 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 the cell of any one of claims 163-174 and a pharmaceutically acceptable carrier. A method of treating a cancer in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of claim 175. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) the cell of any one of claims 163-174; and (b) a pharmaceutically acceptable carrier. The method of claim 176 or 177, wherein the cancer is a cancer associated with elevated expression of GPC3. The method of claim 176 or 177, wherein the disease or the condition is selected from the group consisting of hepatocellular carcinoma (HCC), lung cancer, melanoma, ovarian clearcell carcinomas, yolk sac tumors, neuroblastoma, hepatoblastoma, Wilms' tumor cells, and gastric cancer. The method of claim 176 or 177, wherein 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 method of any one of claims 176-180, wherein the subject is a human.