Compositions and methods for treating cancer using CD8 engineered T cell therapy

Abstract

Compositions of neoTCR-based cell therapies with CD8 expression constructs and methods for treating cancer. In certain embodiments, the currently disclosed subject matter provides cells comprising exogenous T cell receptors (TCRs) and exogenous CD8. In certain embodiments, exogenous CD8 comprises at least one monomer. In certain embodiments, at least one monomer of exogenous CD8 comprises an extracellular domain, a transmembrane domain, an intracellular domain, a fragment thereof, or a combination thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 841,748, filed on May 1, 2019, and U.S. Provisional Application No. 62 / 841,753, filed on May 1, 2019, the entire contents of which are incorporated herein and claim priority.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on April 30, 2020, is named 087520_0145_SL.txt, and is 120,294 bytes in size. Background Art

[0005] The activation of T cells requires signaling through the T cell receptor (TCR) and its co-receptor molecules. CD4 and CD8 are membrane proteins expressed on T helper cells and cytotoxic T lymphocytes. They act as co-receptors to enhance TCR signaling by stabilizing the interaction between peptide major histocompatibility (pMHC) ligands and TCR (Li QJ, et al. (2004) CD4 enhances T cell sensitivity to antigen by coordinating Lckaccumulation at the immunological synapse. Nat Immunol 5: 791–799; Holler PD, Kranz DM (2003) Quantitative analysis of the contribution of TCR / pepMHCaffinity and CD8 to T cell activation. Immunity 18: 255–264). Specifically, CD4 and CD8 co-receptors are essential for initiating signaling because they help recruit kinases to the TCR-pMHC complex. Furthermore, studies have shown that, while both CD4 and CD8 coreceptors enhance the sensitivity of T cells to their ligands, only CD8 plays a role in stabilizing TCR-pMHC interactions.

[0006] Furthermore, while naturally occurring MHC-I TCRs presumably require the co-present CD8 coreceptor to help stabilize peptide-MHC binding, higher affinity TCRs drive CD8-independent target engagement and T cell activation. CD4 T cells, when engineered with high-affinity NeoTCRs, are thus able to recognize peptide-MHC-I targets and trigger effector T cell function. However, lower affinity TCRs rely on the CD8 coreceptor to trigger T cell activation.

[0007] Therefore, NeoTCR cell therapies engineered to express CD8 coreceptors can stabilize TCR-pMHC interactions and improve the efficacy of NeoTCR cell therapies containing low-affinity TCRs that rely on such CD8 coreceptors. Summary of the invention

[0008] In certain embodiments, the subject matter disclosed herein provides a cell comprising an exogenous T cell receptor (TCR) and exogenous CD8. In certain embodiments, exogenous CD8 comprises at least one monomer. In certain embodiments, at least one monomer of exogenous CD8 comprises an extracellular domain, a transmembrane domain, an intracellular domain, a fragment thereof, or a combination thereof. In certain embodiments, the extracellular domain comprises a CD8 α extracellular domain or a CD8 β extracellular domain. In certain embodiments, a transmembrane CD8 α transmembrane domain or a CD8 β transmembrane domain. In certain embodiments, the intracellular domain comprises a CD8 α intracellular domain or a CD8 β intracellular domain. In certain embodiments, the intracellular domain comprises a CD4 intracellular domain.

[0009] In certain embodiments, at least one monomer comprises a CD8 α extracellular domain, a CD8 α transmembrane domain, and a CD8 α intracellular domain. In certain embodiments, at least one monomer comprises a CD8 β extracellular domain, a CD8 β transmembrane domain, and a CD8 β intracellular domain. In certain embodiments, at least one monomer comprises a CD8 α extracellular domain, a CD8 α transmembrane domain, and a CD8 β intracellular domain. In certain embodiments, at least one monomer comprises a CD8 α extracellular domain, a CD8 α transmembrane domain, and a CD8 β intracellular domain. In certain embodiments, at least one monomer comprises a CD8 α extracellular domain, a CD8 α transmembrane domain, and a CD4 intracellular domain. In certain embodiments, at least one monomer comprises a signal peptide. In certain embodiments, the signal peptide is a CD8 signal peptide.

[0010] In certain embodiments, the extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 140 or SEQ ID NO: 145. In certain embodiments, the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 141 or SEQ ID NO: 146. In certain embodiments, the intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 142, SEQ ID NO: 147, or SEQ ID NO: 148. In certain embodiments, the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 139 or SEQ ID NO: 144.

[0011] In certain embodiments, the exogenous CD8 comprises a 2A sequence. In certain embodiments, the exogenous CD8 comprises a linker. In certain embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO: 137. In certain embodiments, the exogenous CD8 comprises a protease cleavage site. In certain embodiments, the protease cleavage site is a furin cleavage site.

[0012] In certain embodiments, the exogenous TCR is a TCR derived from a patient. In certain embodiments, the exogenous TCR comprises a signal sequence, a first and a second 2A sequence, and a TCR polypeptide sequence. In certain embodiments, the exogenous TCR recognizes a cancer antigen. In certain embodiments, the cancer antigen is a neoantigen. In certain embodiments, the cancer antigen is a patient-specific antigen.

[0013] In certain embodiments, the cell is a primary cell. In certain embodiments, the cell is a patient-derived cell. In certain embodiments, the cell is a lymphocyte. In certain embodiments, the cell is a T cell. In certain embodiments, the cell is a young T cell. In certain embodiments, the cell is CD45RA+, CD62L+, CD28+, CD95-, CCR7+ and CD27+. In certain embodiments, the cell is CD45RA+, CD62L+, CD28+, CD95+, CD27+, CCR7+. In certain embodiments, the cell is CD45RO+, CD62L+, CD28+, CD95+, CCR7+, CD27+, CD127+.

[0014] In certain embodiments, the cell further comprises genetic modification to enhance cell persistence and / or enhance memory cell differentiation. In certain embodiments, compared with the killing activity of cells without exogenous CD8, the killing activity of the cell increases by about 10% to about 500%. In certain embodiments, compared with the proliferation of cells without exogenous CD8, the proliferation of cells after TCR binds to antigen increases by about 10% to about 500%. In certain embodiments, compared with the secretion of cells without exogenous CD8, when TCR binds to antigen, the secretion of proinflammatory cytokines of cells increases by about 10% to about 500%. In certain embodiments, compared with the LCK affinity of cells without exogenous CD8, the LCK affinity of cells increases by about 10% to about 500%. In certain embodiments, compared with the persistence of cells without exogenous CD8, the persistence of cells increases by about 10% to about 500%. In certain embodiments, compared with the tumor infiltration ability of cells without exogenous CD8, the tumor infiltration ability of cells increases by about 10% to about 500%.

[0015] In certain embodiments, wherein exogenous TCR is CD8 dependent TCR. In certain embodiments, exogenous TCR is CD8 independent TCR. In certain embodiments, exogenous CD8 is encoded by CD8 construct 1, CD8 construct 2, CD8 construct 3 or CD8 construct 4. In certain embodiments, exogenous CD8 includes: CD8 α extracellular domain, CD8 α transmembrane domain and CD8 α intracellular domain; CD8 α extracellular domain, CD8 α transmembrane domain, CD8 α intracellular domain, CD8 β extracellular domain, CD8 β transmembrane domain and CD8 β intracellular domain; CD8 α extracellular domain, CD8 α transmembrane domain and CD8 β intracellular domain; or CD8 α extracellular domain, CD8 α transmembrane domain and CD4 intracellular domain. In certain embodiments, the exogenous CD8 comprises: a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), and a CD8α intracellular domain (SEQ ID NO: 142); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147). NO: 141) and CD8β intracellular domain (SEQ ID NO: 147); or CD8α signal peptide (SEQ ID NO: 139), CD8α extracellular domain (SEQ ID NO: 140), CD8α transmembrane domain (SEQ ID NO: 141) and CD4 intracellular domain (SEQ ID NO: 148).

[0016] In certain embodiments, the currently disclosed subject matter provides a method for modifying a cell, the method comprising introducing a homologous recombination (HR) template nucleic acid sequence into a cell, wherein the HR template comprises a first and a second homology arm homologous to the first and second target nucleic acid sequences, a TCR gene sequence between the first and second homology arms, a CD8 gene sequence between the first and second homology arms, and the HR template nucleic acid is recombined into the endogenous locus of the cell. In certain embodiments, the HR template comprises a first 2A coding sequence located upstream of the CD8 gene sequence, a second 2A coding sequence located downstream of the CD8 gene sequence and upstream of the TCR gene sequence, and a third 2A coding sequence located downstream of the TCR gene sequence; wherein the first, second and third 2A coding sequences encode the same amino acid sequence and codon diverge relative to each other. In certain embodiments, the HR template comprises a sequence encoding an amino acid sequence Gly Ser Gly immediately upstream of the first, second and / or third 2A coding sequence. In certain embodiments, the HR template also comprises a sequence encoding a furin cleavage site located upstream of the first, second and / or third 2A coding sequence. In certain embodiments, the HR template further comprises a sequence encoding a signal sequence located immediately upstream of the TCR gene sequence and / or the CD8 gene sequence.

[0017] In certain embodiments, the HR template comprises a second TCR sequence between the third 2A coding sequence and the second homology arm. In certain embodiments, the HR template comprises a sequence encoding a first signal sequence located immediately upstream of the first TCR gene sequence and a sequence encoding a second signal sequence located immediately upstream of the second TCR gene sequence. In certain embodiments, the HR template comprises a second CD8 gene sequence located between the first CD8 gene sequence and the second 2A coding sequence. In certain embodiments, the 2A coding sequence is located between the first and second CD8 gene sequences. In certain embodiments, the sequence encoding the amino acid sequence Gly Ser Gly is located between the first and second CD8 gene sequences. In certain embodiments, the sequence encoding the furin cleavage site is located between the first and second CD8 gene sequences.

[0018] In certain embodiments, the CD8 gene sequence comprises a sequence encoding an extracellular domain, a sequence encoding an intracellular domain, a sequence encoding an intracellular domain, a fragment thereof, or a combination thereof. In certain embodiments, the sequence encoding the extracellular domain comprises a sequence encoding a CD8 α extracellular domain or a CD8 β extracellular domain. In certain embodiments, the sequence encoding the transmembrane domain comprises a sequence encoding a CD8 α transmembrane domain or a CD8 β transmembrane domain. In certain embodiments, the sequence encoding the intracellular domain comprises a sequence encoding a CD8 α intracellular domain or a CD8 β intracellular domain. In certain embodiments, the sequence encoding the intracellular domain comprises a sequence encoding a CD4 intracellular domain.

[0019] In certain embodiments, the CD8 gene sequence comprises a sequence encoding a CD8 α extracellular domain, a CD8 α transmembrane domain, and a CD8 α intracellular domain. In certain embodiments, the CD8 gene sequence comprises a sequence encoding a CD8 β extracellular domain, a CD8 β transmembrane domain, and a CD8 β intracellular domain. In certain embodiments, the CD8 gene sequence comprises a sequence encoding a CD8 α extracellular domain, a CD8 α transmembrane domain, and a CD8 β intracellular domain. In certain embodiments, the CD8 gene sequence comprises a sequence encoding a CD8 α extracellular domain, a CD8 α transmembrane domain, and a CD4 intracellular domain.

[0020] In certain embodiments, the HR template comprises a sequence encoding a first signal sequence located immediately upstream of the first CD8 gene sequence and a sequence encoding a second signal sequence located immediately upstream of the second CD8 gene sequence. In certain embodiments, the signal sequence is a CD8 signal sequence, a human growth hormone signal sequence, a fragment thereof, or a combination thereof. In certain embodiments, the first and second homology arms of the HR template each have a length of about 300 bases to about 2,000 bases. In certain embodiments, the first and second homology arms of the HR template each have a length of about 600 bases to about 2,000 bases.

[0021] In certain embodiments, the exogenous TCR is a TCR derived from a patient. In certain embodiments, the exogenous TCR comprises a signal sequence, a first and a second 2A sequence, and a TCR polypeptide sequence. In certain embodiments, the exogenous TCR recognizes a cancer antigen. In certain embodiments, the cancer antigen is a new antigen. In certain embodiments, the cancer antigen is a patient-specific antigen. In certain embodiments, the HR template is non-viral. In certain embodiments, the HR template is a circular DNA. In certain embodiments, the HR template is a linear DNA. In certain embodiments, the introduction occurs by electroporation.

[0022] In certain embodiments, the recombination comprises cleavage of the endogenous locus by a nuclease, and recombination of the HR template nucleic acid sequence into the endogenous locus by homology-directed repair. In certain embodiments, the nuclease is a clustered regularly interspaced short palindromic repeat (CRISPR) family nuclease or a derivative thereof. In certain embodiments, the nuclease further comprises a gRNA.

[0023] In certain embodiments, the method further comprises culturing cells. In certain embodiments, the culturing is carried out in the presence of at least one cytokine. In certain embodiments, the culturing is carried out in the presence of IL2, IL7, IL15 or any combination thereof. In certain embodiments, the culturing is carried out in the presence of IL7 and IL15. In certain embodiments, the method comprises genetic modification to enhance cell persistence and / or enhance memory cell differentiation.

[0024] In certain embodiments, the cell is a primary cell. In certain embodiments, the cell is a patient-derived cell. In certain embodiments, the cell is a lymphocyte. In certain embodiments, the cell is a T cell. In certain embodiments, the cell is a young T cell. In certain embodiments, the cell is CD45RA+, CD62L+, CD28+, CD95-, CCR7+ and CD27+. In certain embodiments, the cell is CD45RA+, CD62L+, CD28+, CD95+, CD27+, CCR7+. In certain embodiments, the cell is CD45RO+, CD62L+, CD28+, CD95+, CCR7+, CD27+, CD127+.

[0025] In certain embodiments, the killing activity of the cell is increased by about 10% to about 500% compared to the killing activity of cells without the CD8 gene sequence. In certain embodiments, the proliferation of the cell after TCR binds to the antigen increases by about 10% to about 500% compared to the proliferation of cells without the CD8 gene sequence. In certain embodiments, the secretion of pro-inflammatory cytokines of the cell after TCR binds to the antigen increases by about 10% to about 500% compared to the secretion of cells without the CD8 gene sequence. In certain embodiments, the LCK affinity of the cell increases by about 10% to about 500% compared to the LCK affinity of cells without the CD8 gene sequence. The method of any one of claims 43-95, wherein the persistence of the cell increases by about 10% to about 500% compared to the persistence of cells without the CD8 gene sequence. In certain embodiments, the tumor infiltration ability of the cell increases by about 10% to about 500% compared to the tumor infiltration ability of cells without the CD8 gene sequence.

[0026] In certain embodiments, the TCR gene encodes CD8 dependent TCR. In certain embodiments, the TCR gene encodes CD8 independent TCR. In certain embodiments, the CD8 gene sequence is encoded by CD8 construct 1, CD8 construct 2, CD8 construct 3 or CD8 construct 4. In certain embodiments, wherein the CD8 gene sequence comprises: CD8α extracellular domain, CD8α transmembrane domain and CD8α intracellular domain; CD8α extracellular domain, CD8α transmembrane domain, CD8α intracellular domain, CD8β extracellular domain, CD8β transmembrane domain and CD8β intracellular domain; CD8α extracellular domain, CD8α transmembrane domain and CD8β intracellular domain; or CD8α extracellular domain, CD8α transmembrane domain, CD4 intracellular domain. In certain embodiments, the CD8 gene sequence comprises: a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), and a CD8α intracellular domain (SEQ ID NO: 142); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147). NO: 141) and CD8β intracellular domain (SEQ ID NO: 147); or CD8α signal peptide (SEQ ID NO: 139), CD8α extracellular domain (SEQ ID NO: 140), CD8α transmembrane domain (SEQ ID NO: 141) and CD4 intracellular domain (SEQ ID NO: 148).

[0027] In certain embodiments, the presently disclosed subject matter provides cells modified by any of the methods disclosed herein.

[0028] In certain embodiments, the presently disclosed subject matter provides a composition comprising an effective amount of cells disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient. In certain embodiments, the composition is administered to a patient in need to treat cancer. In certain embodiments, the composition comprises a cryopreservative. In certain embodiments, the composition comprises serum albumin. In certain embodiments, the composition comprises Plasma-Lyte A, HSA, and CryoStor CS10.

[0029] In certain embodiments, the presently disclosed subject matter provides methods of treating cancer in a subject in need thereof, the methods comprising administering a therapeutically effective amount of a cell or composition disclosed herein.

[0030] In certain embodiments, a non-myeloablative lymphodepletion regimen is administered to a subject prior to administering a therapeutically effective amount of cells disclosed herein. In certain embodiments, the cancer is a solid tumor. In certain embodiments, the cancer is a liquid tumor. In certain embodiments, the solid tumor is selected from the group consisting of melanoma, breast cancer, lung cancer, ovarian cancer, breast cancer, pancreatic cancer, head and neck cancer, prostate cancer, gynecological cancer, central nervous system cancer, skin cancer, HPV+ cancer, esophageal cancer, thyroid cancer, gastric cancer, hepatocellular carcinoma, bile duct cancer, renal cell carcinoma, testicular cancer, sarcoma and colorectal cancer. In certain embodiments, the liquid tumor is selected from the group consisting of follicular lymphoma, leukemia and multiple myeloma.

[0031] In certain embodiments, the presently disclosed subject matter provides a kit comprising a cell disclosed herein, an agent for performing a method disclosed herein, or a composition disclosed herein. In certain embodiments, the kit further comprises written instructions for treating cancer.

[0032] In certain embodiments, the presently disclosed subject matter provides a cell comprising: an exogenous T cell receptor (TCR); exogenous CD8, wherein the exogenous CD8 comprises: a CD8α extracellular domain, a CD8α transmembrane domain, a CD8α intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, a CD8α intracellular domain, a CD8β extracellular domain, a CD8β transmembrane domain, and a CD8β intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8β intracellular domain; or a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain.

[0033] In certain embodiments, the presently disclosed subject matter provides a cell comprising: an exogenous T cell receptor (TCR); and exogenous CD8, the exogenous CD8 comprising: a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), and a CD8α intracellular domain (SEQ ID NO: 142); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147); NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD8β intracellular domain (SEQ ID NO:147); or CD8α signal peptide (SEQ ID NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD4 intracellular domain (SEQ ID NO:148).

[0034] In certain embodiments, the presently disclosed subject matter provides a method for modifying a cell, the method comprising: introducing a homologous recombination (HR) template nucleic acid sequence into the cell, wherein the HR template comprises: first and second homology arms homologous to the first and second target nucleic acid sequences; a TCR gene sequence located between the first and second homology arms; a CD8 gene sequence located between the first and second homology arms; and recombination of the HR template nucleic acid into an endogenous locus of the cell, wherein the CD8 gene sequence comprises: a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8α intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, a CD8α intracellular domain, a CD8β extracellular domain, a CD8β transmembrane domain, and a CD8β intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, a CD8β intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, a CD8β intracellular domain; or a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain.

[0035] In certain embodiments, the presently disclosed subject matter provides a method for modifying a cell, the method comprising: introducing a homologous recombination (HR) template nucleic acid sequence into the cell, wherein the HR template comprises: first and second homology arms homologous to first and second target nucleic acid sequences; a TCR gene sequence located between the first and second homology arms; a CD8 gene sequence located between the first and second homology arms; and recombination of the HR template nucleic acid into an endogenous locus of the cell, wherein the CD8 gene sequence comprises: a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), and a CD8α intracellular domain (SEQ ID NO: 142); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145). NO:145), CD8β transmembrane domain (SEQ ID NO:146) and CD8β intracellular domain (SEQ ID NO:147); CD8α signal peptide (SEQ ID NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD8β intracellular domain (SEQ ID NO:147); or CD8α signal peptide (SEQ ID NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD4 intracellular domain (SEQ ID NO:148).

[0036] In certain embodiments, the presently disclosed subject matter provides a composition comprising a cell, wherein the cell comprises an exogenous T cell receptor (TCR) and exogenous CD8, wherein the exogenous CD8 comprises: a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8α intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, a CD8α intracellular domain, a CD8β extracellular domain, a CD8β transmembrane domain, and a CD8β intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8β intracellular domain; or a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain.

[0037] In certain embodiments, the presently disclosed subject matter provides a composition comprising a cell, wherein the cell comprises an exogenous T cell receptor (TCR) and exogenous CD8, wherein the exogenous CD8 comprises: a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), and a CD8α intracellular domain (SEQ ID NO: 142); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147); NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD8β intracellular domain (SEQ ID NO:147); or CD8α signal peptide (SEQ ID NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD4 intracellular domain (SEQ ID NO:148).

[0038] In certain embodiments, the presently disclosed subject matter provides a method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of cells, wherein the cells comprise an exogenous T cell receptor (TCR) and exogenous CD8, wherein the exogenous CD8 comprises: a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8α intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, a CD8α intracellular domain, a CD8β extracellular domain, a CD8β transmembrane domain, and a CD8β intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8β intracellular domain; or a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain.

[0039] In certain embodiments, the presently disclosed subject matter provides a method of treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of cells, wherein the cells comprise an exogenous T cell receptor (TCR) and exogenous CD8, wherein the exogenous CD8 comprises: a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), and a CD8α intracellular domain (SEQ ID NO: 142); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147); NO:147); CD8α signal peptide (SEQ ID NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD8β intracellular domain (SEQ ID NO:147); or CD8α signal peptide (SEQ ID NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD4 intracellular domain (SEQ ID NO:148). BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figures 1A-1C . Figures 1A-1C Examples of NeoE TCR cassettes and gene editing methods that can be used to make NeoTCR products are shown. Figure 1A Shown is a schematic representation of the general targeting strategy for integrating neoantigen-specific TCR constructs (neoTCR) into the TCRα locus. Figure 1B and 1C A neoantigen-specific TCR construct design for integration of the NeoTCR into the TCRα locus is shown, with the cassette shown having a signal sequence ("SS"), a protease cleavage site ("P"), and a 2A peptide ("2A"). Figure 1B Shown are the target TCRα locus (endogenous TRAC, top panel) and its CRISPR Cas9 target site (horizontal stripes, cleavage site indicated by arrows), as well as a circular plasmid HR template (bottom panel) carrying the polynucleotide encoding the neoTCR, which is located between the left and right homology arms (“LHA” and “RHA”, respectively) before integration. Figure 1C Shown are the integrated neoTCR in the TCRα locus (upper panel), the transcribed and spliced ​​neoTCR mRNA (middle panel), and the translation and processing of the expressed neoTCR (lower panel).

[0041] Figures 2A-2D . Figures 2A-2D Circular plasmids encoding CD8 constructs 1, 2, 3 and 4 are shown. Figure 2A CD8 construct 1 used to produce CD8 product 1 is shown. Figure 2B CD8 construct 2 used to produce CD8 product 2 is shown. Figure 2C CD8 construct 3 used to produce CD8 product 3 is shown. Figure 2D The CD8 construct 4 used to produce the CD8 product 4 is shown. Figures 2A-2D As shown in Figure 1, SS stands for signal sequence. Figure 6 , 7 As described in , 8 and 9, SS can be HGH; however, other signal sequences can be used as needed for proper trafficking. Figures 2A-2D As shown in Figure 1, P represents a protease cleavage site. Figure 6 , 7 As described in , 8 and 9, P can be furin; however, other protease cleavage sites can be used as appropriate to provide the cleavage effects described herein. Figures 2A-2D As shown, 2A represents 2A peptide. Figure 6 , 7 As described in , 8 and 9, 2A can be a P2A peptide; however, other 2A peptides can also be used.

[0042] Figure 3. Figures 3A-3D The transcription / splicing and translation processing of each CD8 construct 1, 2, 3 and 4 to produce CD8 products 1, 2, 3 and 4 are shown. Figures 3A-3D As shown in FIG, SS stands for signal sequence. Figure 6 , 7 As described in , 8 and 9, SS can be HGH; however, other signal sequences can be used as needed for proper trafficking. Figures 3A-3D As shown in Figure 1, P represents a protease cleavage site. Figure 6 , 7 As described in , 8 and 9, P can be furin; however, other protease cleavage sites can be used as appropriate to provide the cleavage effects described herein. Figures 3A-3D As shown, 2A represents 2A peptide. Figure 6 , 7 As described in , 8 and 9, 2A can be a P2A peptide; however, other 2A peptides can also be used.

[0043] Figure 4A and 4B . Figure 4A The translation products of CD8 product 1 and CD8 product 2 are shown. Figure 4B The translation products of CD8 product 3 and CD8 product 4 are shown.

[0044] Figure 5 . Figure 5 Provided Figures 1A-1C The exemplary DNA sequences of the NeoTCR constructs described in . Conservative substitutions of nucleic acids can be used throughout to produce the same translation product. In addition, when amino acids can be substituted without changing the function, Figure 5 Substitution of the nucleic acids provided in the can also be used to achieve that the substituted amino acid confers substantially similar or identical function to the translated protein.

[0045] Figure 6 . Figure 6 Provided Figure 2A , 3A and 4A, an exemplary DNA sequence of CD8 construct 1 (and translated CD8 product 1). Conservative substitutions of nucleic acids can be used throughout to produce the same translation product. In addition, when amino acids can be substituted without changing function, Figure 6 Substitution of the nucleic acids provided in the can also be used to achieve that the substituted amino acid confers substantially similar or identical function to the translated protein.

[0046] Figure 7 . Figure 7 Provided Figure 2B , 3B and 4A, an exemplary DNA sequence of CD8 construct 2 (and translated CD8 product 2). Conservative substitutions of nucleic acids can be used throughout to produce the same translation product. In addition, when amino acids can be substituted without changing function, Figure 7 Substitution of the nucleic acids provided in the can also be used to achieve that the substituted amino acid confers substantially similar or identical function to the translated protein.

[0047] Figure 8 . Figure 8 Provided Figure 2C , 3C and 4B are exemplary DNA sequences of CD8 construct 3 (and translated CD8 product 3). Conservative substitutions of nucleic acids can be used throughout to produce the same translation product. In addition, when amino acids can be substituted without changing function, Figure 8 Substitution of the nucleic acids provided in the can also be used to achieve that the substituted amino acid confers substantially similar or identical function to the translated protein.

[0048] Fig. 9 . Fig. 9 Provided Figure 2D , 3D and 4B are exemplary DNA sequences of CD8 construct 4 (and translated CD8 product 4). Conservative substitutions of nucleic acids can be used throughout to produce the same translation product. In addition, when amino acids can be substituted without changing function, Fig. 9 Substitution of the nucleic acids provided in the can also be used to achieve that the substituted amino acid confers substantially similar or identical function to the translated protein.

[0049] Fig.10 . Fig.10 CD8 products 1, 2, 3 and 4 and a visual depiction of the predicted LCK activity of each CD8 product 1, 2, 3 and 4 are shown.

[0050] Figures 11A-11D . Fig.11A Exemplary expression constructs for CD8 Product 1 are provided. Fig. 11B Exemplary expression constructs for CD8 Product 2 are provided. Fig. 11C Exemplary expression constructs for CD8 Product 3 are provided. Fig.11D An exemplary expression construct for CD8 Product 4 is provided.

[0051] Fig.12 . Fig.12 Schematic representation of the design of the Incucyte experiment to demonstrate the killing ability of the CD8 product.

[0052] Fig.13A and 13B . Fig.13A and 13B The results show that in a CD4 T cell population, the tumor killing ability of CD8 product 4 is increased compared to a NeoTCR product having the same NeoTCR as CD8 product 4. Specifically, both CD8 product 4 and NeoTCR product express TCR097. However, CD8 product 4 also expresses CD8 construct 4, which contains the extracellular domain of CD8α and the intracellular domain of CD4. The effector: target cell ratio (E:T ratio) is 1:1 ( Fig.13A ) or 2:1( Fig. 13B ), and each was normalized for gene editing in each cell line. The SW620 COX6C R20Q heterozygous tumor cells used in this experiment are a cell line expressing the cognate antigen of TCR097 (CD8-dependent TCR). This shows that the low affinity binding of TCR097 can be rescued by co-expressing the extracellular domain of CD8α and the intracellular domain of CD4.

[0053] Fig.14 . Fig.14 It is shown that in a CD4 T cell population, the tumor killing ability of CD8 product 4 is increased compared to a NeoTCR product with the same NeoTCR as CD8 product 4. Specifically, both CD8 product 4 and NeoTCR product express TCR097. However, CD8 product 4 also expresses CD8 construct 4, which contains the extracellular domain of CD8a and the intracellular domain of CD4. The effector: target cell ratio (E:T ratio) was 1:1, 1:2, or 1:4, and each was normalized for gene editing of each cell line. The SW620 COX6C R20Q homozygous tumor cells used in this experiment are a cell line expressing the TCR097 cognate antigen. Fig.13A and 13B Increased killing can be seen in this experiment compared to that shown because the cell line is homozygous for cognate antigen expression; thus, even though TCR97 is a low affinity TCR, the increased amount of cognate antigen overcomes the limitation of low affinity.

[0054] Fig.15A and 15B . Fig.15A and 15B An exemplary control experiment is provided showing that the CD4 T cell population ( Fig.15A and 15B in the upper panel) or CD8 T cell population ( Fig.15A and 15B In the lower panel of Figure 3 , the tumor killing capacity of CD8 Product 4 is increased compared to the NeoTCR product with the same NeoTCR as CD8 Product 4, and CD8 Product 4 kills tumors more effectively than simply expressing the NeoTCR construct and CD8 Product 4 in CD8 T cells. Fig.15A The E:T ratio is 2:1. Fig. 15B The E:T ratio is shown to be 1:1.

[0055] Fig.16 . Fig.16 Surface expression of CD8 construct 4 is shown. Peak #1 is the NeoTCR product in CD4+ / CD8- T cells (expressing TCR089). Peak #2 is the CD8 product 4 in CD8+ / CD4+ T cells. Peak #3 is the NeoTCR product in CD8+ / CD4- T cells. As shown, CD8 construct 4 exhibits appropriate and comparable surface expression to the NeoTCR product. Similar results were obtained for other CD8 constructs (data not shown).

[0056] Fig.17A and 17B . Fig.17A and 17BCD8α expression was shown to enhance CD4 T cell sensitivity while maintaining specificity. Fig.17A It was shown that expression of CD8 constructs 1-4 increased the sensitivity of T cells and did not alter the specificity of the CD8 product for NeoTCR. As shown, co-expression of the CD8 constructs reduced the EC50, indicating increased sensitivity to NeoTCR. Fig. 17B It was shown that CD8 products 1-4 were specific for the cognate antigen of NeoTCR 097, because CD8 cells with CD8 products 1-4 only produced INFγ in the presence of cognate antigen (i.e., CD8 cells with CD8 products 1-4 did not produce INFγ in the presence of mismatched antigen). Fig.17A and 17B The same experiments shown in were performed with CD107a instead of INFγ and showed the same increased sensitivity and maintained specificity for NeoTCR (data not shown) confirming the results using INFγ.

[0057] Fig.18 . Fig.18 It is shown that CD8α expression increases the sensitivity of CD4 T cells in CD8-independent NeoTCRs. The figure shows data from NeoTCR089, which is independent of CD8. This shows that even though the NeoTCR is independent of CD8, the sensitivity of CD8 product 4 is increased. This is an unexpected result: it was found that sensitivity can be increased even for TCRs that are independent of CD8. Similar results were obtained with CD8 products 1-3 (data not shown). Therefore, compared to NeoTCR products expressing the same NeoTCR, CD8-dependent (such as NeoTCR097) and CD8-independent (such as NeoTCR089) show increased sensitivity.

[0058] Detailed description

[0059] definition

[0060] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. The following references provide general definitions of many terms used in the disclosed subject matter for technicians: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, unless otherwise indicated, the following terms have the following meanings assigned to them.

[0061] It should be understood that aspects and embodiments of the invention described herein include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments. The terms "comprises" and "comprising" are intended to have the broad meaning given to them under U.S. patent law and may mean "includes," "including," and the like.

[0062] As used herein, the term "about" or "approximately" means within an acceptable error range for a particular value as determined by one skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 3 or more than 3 standard deviations. Alternatively, "about" can mean a range of up to 20%, such as up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, such as within 5-fold or 2-fold.

[0063] As used herein, the term "antibody" is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and trispecific antibodies) and antibody fragments (e.g., bis-Fab), as long as they exhibit the desired antigen binding activity. As used herein, "antibody fragment" refers to a molecule other than an intact antibody, which contains a portion of an intact antibody and binds to the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to bis-Fab; Fv; Fab'; Fab, Fab'-SH; F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed by antibody fragments.

[0064] The terms "cancer" and "tumor" are used interchangeably herein. As used herein, the term "cancer" or "tumor" refers to all tumor cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. These terms are also used to refer to or describe the physiological conditions of mammals, which are generally characterized by unregulated cell growth / proliferation. Cancer can affect a variety of cell types, tissues or organs, including but not limited to organs selected from the group consisting of bladder, bone, brain, breast, cartilage, glial, esophagus, fallopian tube, gallbladder, heart, intestine, kidney, liver, lung, lymph node, nervous tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus and vagina, or its tissue or cell type. Cancer includes cancer, such as sarcoma, carcinoma or plasmacytoma (malignant tumor of plasma cells). Examples of cancer include but are not limited to those described herein. As used herein, the terms "cancer" or "tumor" and "proliferative disorders" are not mutually exclusive.

[0065] "CD8" is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates potent cell-cell interactions within the immune system. The CD8 antigen acts as a coreceptor, together with the T cell receptor on T lymphocytes, to recognize antigens displayed by antigen presenting cells in the context of class I MHC molecules.

[0066] As used herein, "CD8 cell" refers to one or more cells precisely engineered to express one or more NeoTCR and CD8 constructs.

[0067] As used herein, "CD8 construct" refers to any one of CD8 construct 1, CD8 construct 2, CD8 construct 3 or CD8 construct 4.

[0068] As used herein, "CD8 product" refers to a product comprising CD8 cells.

[0069] "CD8 construct 1" and "CD8 product 1" refer to a construct comprising NeoTCR and CD8α (CD8α extracellular domain, CD8α transmembrane domain, and CD8α intracellular domain), and the resulting product comprising expressed NeoTCR and CD8α. Figure 2A , 3A and Figure 6 A non-limiting example of a CD8 product 1 is provided in . Figure 4A A non-limiting example of a CD8 product 1 is provided in .

[0070] "CD8 Construct 2" and "CD8 Product 2" refer to a construct comprising NeoTCR, CD8α (CD8α extracellular domain, CD8α transmembrane domain, and CD8α intracellular domain) and CD8β (CD8β extracellular domain, CD8β transmembrane domain, and CD8β intracellular domain), and the resulting product comprising expressed NeoTCR, CD8α, and CD8β. Figure 2B , Figure 3B and Figure 7 A non-limiting example of a CD8 product 2 is provided in . Figure 4A A non-limiting example of a CD8 product 1 is provided in .

[0071] "CD8 construct 3" and "CD8 product 3" refer to a construct comprising NeoTCR, CD8α extracellular domain, CD8α transmembrane domain and CD8β intracellular domain, and the resulting product comprising expressed NeoTCR, CD8α extracellular domain, CD8α transmembrane domain and CD8β intracellular domain. Figure 2C , Figure 3C and Figure 8 A non-limiting example of a CD8 product 3 is provided in . Figure 4B A non-limiting example of a CD8 product 1 is provided in .

[0072] "CD8 construct 4" and "CD8 product 4" refer to a construct comprising a NeoTCR, a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain, and the resulting product comprising an expressed NeoTCR, a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain. Figure 2D , Figure 3D and Fig. 9 A non-limiting example of a CD8 product 4 is provided in . Figure 4B A non-limiting example of a CD8 product 1 is provided in .

[0073] "Conservative substitution" or "conservative amino acid" refers to the substitution of an amino acid with a chemically or functionally similar amino acid. Conservative substitution tables providing similar amino acids are well known in the art.

[0074] In certain embodiments, the acidic amino acids D and E are conservative substitutions of each other; the basic amino acids K, R and H are conservative substitutions of each other; the hydrophilic uncharged amino acids S, T, N and Q are conservative substitutions of each other; the aliphatic uncharged amino acids G, A, V, L and I are conservative substitutions of each other; the non-polar uncharged amino acids C, M and P are conservative substitutions of each other; the aromatic amino acids F, Y and W are conservative substitutions of each other; A, S and T are conservative substitutions of each other; D and E are conservative substitutions of each other; N and Q are conservative substitutions of each other; R and K are conservative substitutions of each other; I, L and M are conservative substitutions of each other; F, Y and W are conservative substitutions of each other; A and G are conservative substitutions of each other; D and E are conservative substitutions of each other; N and Q are conservative substitutions of each other; R, K and H are conservative substitutions of each other; I, L, M and V are conservative substitutions of each other; F, Y and W are conservative substitutions of each other; S and T are conservative substitutions of each other; C and M are conservative substitutions of each other.

[0075] Other conservative substitutions can be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd ed. (1993) WH Freeman & Co., New York, NY.

[0076] "Treat", "treatment" and "treating" are used interchangeably and as used herein refer to obtaining beneficial or desired results, including clinical results. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, alleviating or improving prognosis. In some embodiments, the NeoTCR products of the present invention are used to delay the development of proliferative disorders (e.g., cancer) or slow the progression of such diseases.

[0077] As used herein, "dextramer" refers to a multimeric neo-epitope-HLA complex that specifically binds to its cognate NeoTCR.

[0078] As used herein, the term "neoantigen", "neo-epitope" or "neoE" refers to a newly formed antigenic determinant that results from, for example, somatic mutation and is recognized as "non-self". Mutations that generate "neoantigens", "neo-epitopes" or "neoE" may include frameshift or non-frameshift insertions and deletions, missense or nonsense substitutions, splice site changes (e.g., alternative splicing transcripts), genomic rearrangements or gene fusions, any genomic or expression changes, or any post-translational modifications.

[0079] As used herein, "NeoTCR", "NeoE TCR" and "exogenous TCR" refer to a new epitope-specific T cell receptor introduced into a T cell, for example, by a gene editing method. As used herein, the term "TCR gene sequence" refers to a NeoTCR gene sequence.

[0080] As used herein, "NeoTCR cells" refers to one or more cells precisely engineered to express one or more NeoTCRs. In certain embodiments, the cells are T cells. In certain embodiments, the T cells are CD8+ and / or CD4+ T cells. In certain embodiments, the CD8+ and / or CD4+ T cells are autologous cells from a patient to whom the NeoTCR product will be administered. The terms "NeoTCR cells" and "NeoTCR-P1 T cells" and "NeoTCR-P1 cells" are used interchangeably herein.

[0081] As used herein, "NeoTCR product" refers to a pharmaceutical preparation comprising one or more NeoTCR cells. NeoTCR products are composed of CD8+ and CD4+T cells of autologous precise genome modification. Using a non-viral precise genome modification method mediated by targeted DNA, the expression of endogenous TCR is eliminated and replaced by a patient-specific NeoTCR isolated from peripheral CD8+T cells, which targets a new epitope unique to tumors. In certain embodiments, the resulting modified CD8+ or CD4+T cells express NeoTCR on their native sequence surface, natural expression level, and natural TCR function. The sequences of NeoTCR external binding domains and cytoplasmic signaling domains are not modified from TCRs isolated from natural CD8+T cells. The regulation of NeoTCR gene expression is driven by a natural endogenous TCR promoter located upstream of the position where the NeoTCR gene cassette is integrated into the genome. By this method, the natural level of NeoTCR expression is observed in the unstimulated and antigen-activated T cell state.

[0082] The NeoTCR product produced for each patient represents a dose of autologous CD8+ and / or CD4+ T cells that have been precisely genomically engineered to express a single NeoE-specific TCR cloned from NeoE-specific CD8+ T cells that were individually isolated from the peripheral blood of the same patient.

[0083] As used herein, "NeoTCR virus product" has the same definition as NeoTCR product, except that the genome modification is performed using a virus-mediated approach.

[0084] A "pharmaceutical formulation" is a formulation that is in a form that permits the biological activity of the active ingredient contained therein to be effective and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered. For clarity, the amount of DMSO used in the NeoTCR product is not considered to be unacceptably toxic.

[0085] "Subject," "patient," or "individual" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sport or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is a human.

[0086] As used herein, "TCR" refers to T cell receptor.

[0087] As used herein, the term "tumor antigen" refers to an antigen (e.g., polypeptide) that is uniquely or differentially expressed on tumor cells compared to normal or non-tumor cells. In certain embodiments, tumor antigens include any polypeptide expressed by a tumor that is capable of activating or inducing an immune response through an antigen recognition receptor or capable of inhibiting an immune response through receptor-ligand binding.

[0088] As used herein, "2A" and "2A peptide" are used interchangeably to refer to a class of viral self-cleaving peptides of 18-22 amino acids in length that are capable of mediating peptide cleavage during translation in eukaryotic cells.

[0089] Four well-known members of the 2A peptide class are T2A, P2A, E2A, and F2A. The T2A peptide was first identified in Thosea asigna virus 2A. The P2A peptide was first identified in teschovirus-1 2A. The E2A peptide was first discovered in equine rhinitis A virus. The F2A peptide was first identified in foot-and-mouth disease virus.

[0090] The self-cleavage mechanism of the 2A peptide is the result of ribosome jumping to form a glycyl-prolyl peptide bond at the C-terminus of 2A. Specifically, the 2A peptide has a C-terminal conserved sequence that is necessary to generate steric hindrance and ribosome jumping. Ribosome jumping may lead to one of the following three options: 1) successful jumping and restarting translation, resulting in two cleaved proteins (in addition to the C-terminal proline, the upstream of the 2A protein connected to the complete 2A peptide, and the downstream of the 2A protein connected to a proline at the N-terminus; 2) successful jumping but ribosome shedding leads to interrupted translation, with only the protein upstream of 2A; or 3) unsuccessful jumping and continued translation (i.e., fusion protein).

[0091] As used herein, the term "endogenous" refers to a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue.

[0092] As used herein, the term "exogenous" refers to a nucleic acid molecule or polypeptide that is not endogenously present in a cell. Thus, the term "exogenous" will include any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and overexpressed nucleic acid molecules and polypeptides. An "exogenous" nucleic acid refers to a nucleic acid that is not present in a naturally occurring wild-type cell; for example, an exogenous nucleic acid may differ from an endogenous counterpart in sequence, position / localization, or both. For clarity, an exogenous nucleic acid may have the same or different sequence relative to its naturally occurring endogenous counterpart; it may be introduced into the cell itself or its progenitors by genetic engineering, and may optionally be linked to an alternative control sequence, such as a non-native promoter or secretion sequence.

[0093] “Young” or “younger” or “young T cells” in relation to T cells refers to memory stem cells (T MSC ) and central memory cells (T CM ). These cells have T-cell proliferation upon specific activation and are capable of multiple cell divisions. They also have the ability to engraft after re-infusion, rapidly differentiate into effector T cells upon exposure to their cognate antigens and target and kill tumor cells, as well as ongoing cancer monitoring and control.

[0094] NeoTCR Products

[0095] In some embodiments, the NeoTCR is cloned into autologous CD8+ and CD4+ T cells from the same patient with cancer (using the gene editing technology and neoTCR isolation technology described in PCT / US2020 / 17887 and PCT / US2019 / 025415 (which are incorporated herein in their entirety) by precise genome engineering. Figures 1A-1C In other words, a tumor-specific NeoTCR is identified in a cancer patient, such NeoTCR is then cloned, and the cloned NeoTCR is then inserted into the cancer patient's T cells. The NeoTCR-expressing T cells are then expanded in a manner that retains the "young" T cell phenotype, resulting in a NeoTCR-P1 product (i.e., a NeoTCR product), most of which exhibits T memory stem cell and T central memory phenotypes.

[0096] These "younger" or "younger" or less differentiated T cell phenotypes are described as conferring improved engraftment potential and prolonged persistence after infusion. Therefore, administration of NeoTCR products (composed primarily of "young" T cell phenotypes) has the potential to benefit cancer patients by improving engraftment potential, prolonged persistence after infusion, and rapid differentiation into effector T cells to eradicate tumor cells throughout the body.

[0097] In vitro mechanism of action studies were also performed on NeoTCR products made with T cells from cancer patients. Comparable gene editing efficiency and functional activity, as measured by antigen specificity of T cell killing activity, proliferation, and cytokine production, were observed, demonstrating that the manufacturing process described herein successfully produced products using T cells from cancer patients as starting materials.

[0098] In certain embodiments, the NeoTCR product manufacturing process involves electroporation of a binuclear ribonucleoprotein type of CRISPR-Cas9 nuclease in conjunction with a guide RNA sequence, each type targeting a genomic TCRα and a genomic TCRβ locus. The specificity of the Cas9 nuclease targeting each genomic locus has been previously described in the literature as being highly specific. The NeoTCR products were thoroughly tested in vitro and in silico using COSMID and GUIDE-seq, respectively, to investigate possible off-target genomic cleavage sites. Multiple NeoTCR products from healthy donors or equivalent cell products were evaluated for cleavage at candidate off-target sites by deep sequencing, supporting published evidence that the selected nucleases are highly specific.

[0099] Other aspects of the precision genome engineering process have been evaluated for safety. No evidence of genomic instability following precision genome engineering was found when multiple NeoTCR products were evaluated by targeted locus amplification (TLA) or standard FISH cytogenetics. No off-target integration of the NeoTCR sequence anywhere in the genome was detected. No evidence of residual Cas9 was found in the cell products.

[0100] Comprehensive evaluation of the NeoTCR product and the precise genomic engineering process demonstrated that the NeoTCR product would be well tolerated after infusion back into the patient.

[0101] The genome engineering method described in this article can efficiently generate customized NeoTCR T cells (i.e., NeoTCR products) for personalized adoptive cell therapy for patients with solid and liquid tumors. In addition, the engineering method is not limited to use in T cells, but has also been successfully applied to other primary cell types, including natural killer cells and hematopoietic stem cells.

[0102] CD8 products

[0103] Co-expression of an MHC class I-restricted neoTCR and an ectopic CD8 receptor in precisely genomically engineered CD4 T cells significantly enhances antigen-specific effector functions.

[0104] Neo-epitopes derived from tumor-exclusive mutations represent attractive targets for personalized neoE-specific autologous TCR-T cell therapy for patients with solid tumors. The imPACT separation technology described in PCT / US2020 / 17887, which is incorporated herein by reference in its entirety, is an ultrasensitive and high-throughput process for capturing neoE-specific CD8 T cells from the blood of patients with solid cancers. Using this technology, neo-epitope-specific MHC class I-restricted TCRs ("MHC-I neoTCRs") are cloned from individually captured CD8 T cells. Using DNA-mediated (non-viral) gene editing as described in Example 1, fresh CD8 and CD4 T cells from the same cancer patient are engineered to express the MHC-I neoTCR (while eliminating endogenous TCRs).

[0105] While naturally occurring MHC-I TCRs presumably require the co-present CD8 coreceptor to help stabilize peptide-MHC binding, higher affinity TCRs are able to drive CD8-independent target engagement and T cell activation. CD4 T cells, when engineered with high affinity neoTCRs, are thus able to recognize peptide-MHC-I targets and trigger effector T cell function. However, lower affinity TCRs are dependent on the CD8 coreceptor to trigger T cell activation. By precisely genomic engineering the CD8 coreceptor gene along with the neoTCR into CD4 T cells, the MHC-I neoTCR was enabled to trigger antigen-specific effector T cell function.

[0106] CD8 stabilizes TCR-pMHC interactions and cooperates with TCRs to gain avidity. CD8 also serves to enhance the avidity of low-affinity TCRs, and the intracellular domain of CD8α is essential for enhancing T cell activation. Expression of CD8 may enhance CD4 T cell responses, which may not respond to physiological concentrations of pMHC. Disruption of CD8 binding to MHC can convert a capture-bond TCR-pMHC into a slip-bond, highlighting the importance of CD8-MHC interactions even for TCRs that do not rely on CD8 to bind pMHC.

[0107] CD8α has a lower affinity for LCK than CD8β, suggesting that co-expression of CD8α and CD8β or generation of chimeric CD8α-CD8β molecules comprising the extracellular CD8α and the intracellular domain of CD8β may improve effectiveness. The CD8 constructs described herein are:

[0108] 1. CD8α homodimer (CD8 construct 1)

[0109] 2.CD8α-P2A-CD8β (CD8 construct 2)

[0110] 3. CD8α and CD8β intracellular domains (CD8 construct 3)

[0111] 4.CD8α homodimer with CD4 intracellular domain (CD8 construct 4)

[0112] In some embodiments, the above-mentioned NeoTCR products include additional modifications to include expression of CD8 construct 1, CD8 construct 2, CD8 construct 3, or CD8 construct 4 (each a CD8 product). Specifically, using the gene editing technology and neoTCR isolation technology described in PCT / US2020 / 17887 and PCT / US2019 / 025415 (all of which are incorporated herein), NeoTCRs are cloned into autologous CD8+ and CD4+ T cells from the same patient with cancer (using Figures 1A-1C The DNA-mediated (non-viral) method described in ) was used to express the neoTCR.

[0113] Each CD8 construct will produce a CD8 product when expressed. Table 1 provides a description of each construct and product.

[0114] Table 1 CD8 products and CD8 constructs

[0115]

[0116] In certain embodiments, the CD8 product 1 comprises a NeoTCR and a CD8 homodimer. In certain embodiments, the CD8 product 1 comprises expression of a NeoTCR, a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8α intracellular domain. In certain embodiments, the CD8 product 1 further comprises expression of a CD8α signal peptide. In certain embodiments, the CD8 product 1 comprises Figure 3A In a non-limiting exemplary embodiment, CD8 product 1 comprises NeoTCR, CD8α signal peptide (SEQ ID NO: 139), CD8α extracellular domain (SEQ ID NO: 140), CD8α transmembrane domain (SEQ ID NO: 141) and CD8α intracellular domain (SEQ ID NO: 142). In certain embodiments, CD8α signal peptide, CD8α extracellular domain, CD8α transmembrane domain and CD8α intracellular domain can be sequence modified to retain or substantially retain the function of each element. In certain embodiments, such sequence modifications are conservative substitutions of amino acids.

[0117] In a non-limiting embodiment, CD8 product 1 is Figure 6 In certain embodiments, Figure 6 The sequence of CD8 construct 1 provided in can be modified in any number of ways, as long as the translation of the CD8α signal peptide, CD8α extracellular domain, CD8α transmembrane domain, and CD8α intracellular domain maintains the conserved function of each element. In certain embodiments, Figure 6 The order of each element of CD8 construct 1 remains the same, but the sequence of each individual element can be changed as long as the amino acid encoded by the nucleic acid remains the same or contains only conservative substitutions. In certain embodiments, Figure 6 The order of each element in CD8 construct 1 remains the same, but the sequence of each individual element can be varied as long as the function of the encoded protein remains essentially unchanged.

[0118] In certain embodiments, the CD8 product 2 comprises NeoTCR, CD8α, and aCD8β. In certain embodiments, CD8α and CD8β are separated by a protease cleavage site and a 2A peptide in the CD8 product 2 construct for expression. In certain embodiments, the CD8 product 2 comprises the expression of NeoTCR, CD8α extracellular domain, CD8α transmembrane domain, CD8α intracellular domain, CD8β extracellular domain, CD8β transmembrane domain, and CD8β intracellular domain. In certain embodiments, the CD8 product 2 further comprises the expression of a CD8α signal peptide. In certain embodiments, the CD8 product further comprises the expression of a CD8β signal peptide. In certain embodiments, the CD8 product further comprises the expression of a CD8α signal peptide and a CD8β signal peptide. In certain embodiments, the CD8 product 2 comprises Figure 3B In a non-limiting exemplary embodiment, CD8 product 2 comprises NeoTCR, CD8α signal peptide (SEQ ID NO: 139), CD8α extracellular domain (SEQ ID NO: 140), CD8α transmembrane domain (SEQ ID NO: 141), CD8α intracellular domain (SEQ ID NO: 142), CD8β signal peptide (SEQ ID NO: 144), CD8β extracellular domain (SEQ ID NO: 145), CD8β transmembrane domain (SEQ ID NO: 146) and CD8β intracellular domain (SEQ ID NO: 147). In certain embodiments, CD8α signal peptide, CD8α extracellular domain, CD8α transmembrane domain, CD8α intracellular domain, CD8β extracellular domain, CD8β transmembrane domain and CD8β intracellular domain can be sequence modified to retain or substantially retain the function of each element. In certain embodiments, such sequence modifications are conservative substitutions of amino acids.

[0119] In a non-limiting embodiment, CD8 Product 2 is Figure 7 In certain embodiments, Figure 7 The sequence of CD8 construct 2 provided in can be modified in any number of ways, as long as the translation of the CD8α signal peptide, CD8α extracellular domain, CD8α transmembrane domain, CD8α intracellular domain, CD8β extracellular domain, CD8β transmembrane domain, and CD8β intracellular domain is such that each element retains a conserved function. In certain embodiments, Figure 7 The order of each element of CD8 construct 2 remains the same, but the sequence of each individual element can be changed as long as the amino acid encoded by the nucleic acid remains the same or contains only conservative substitutions. In certain embodiments, Figure 7 The order of each element in CD8 Construct 2 remains the same, but the sequence of each individual element can be varied as long as the function of the encoded protein remains essentially unchanged.

[0120] In certain embodiments, the CD8 product 3 comprises NeoTCR and CD8α and CD8β intracellular domains. In certain embodiments, the CD8 product 3 comprises expression of NeoTCR, CD8α extracellular domain, CD8α transmembrane domain, and CD8β intracellular domain. In certain embodiments, the CD8 product 3 further comprises expression of CD8α signal peptide. In certain embodiments, the CD8 product 3 comprises Figure 3C In a non-limiting exemplary embodiment, CD8 product 3 comprises NeoTCR, CD8α signal peptide (SEQ ID NO: 139), CD8α extracellular domain (SEQ ID NO: 140), CD8α transmembrane domain (SEQ ID NO: 141) and CD8β intracellular domain (SEQ ID NO: 147). In certain embodiments, CD8α signal peptide, CD8α extracellular domain, CD8α transmembrane domain and CD8β intracellular domain can be sequence modified to retain or substantially retain the function of each element. In certain embodiments, such sequence modifications are conservative substitutions of amino acids.

[0121] In a non-limiting embodiment, CD8 product 3 is Figure 8 In certain embodiments, Figure 8 The sequence of CD8 construct 3 provided in can be modified in any number of ways, as long as the translation of the CD8α signal peptide, CD8α extracellular domain, CD8α transmembrane domain, and CD8β intracellular domain maintains the conserved function of each element. In certain embodiments, Figure 8The order of each element of CD8 construct 3 remains the same, but the sequence of each individual element can be changed as long as the amino acid encoded by the nucleic acid remains the same or contains only conservative substitutions. In certain embodiments, Figure 8 The order of each element in CD8 construct 3 remains the same, but the sequence of each individual element can be varied as long as the function of the encoded protein remains essentially unchanged.

[0122] In certain embodiments, CD8 product 4 comprises a NeoTCR and CD8α homodimer and a CD4 intracellular domain. In certain embodiments, CD8 product 4 comprises expression of a NeoTCR, a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain. In certain embodiments, CD8 product 4 further comprises expression of a CD8α signal peptide. In certain embodiments, CD8 product 4 comprises Figure 3D In a non-limiting exemplary embodiment, CD8 product 4 comprises NeoTCR, CD8α signal peptide (SEQ ID NO: 139), CD8α extracellular domain (SEQ ID NO: 140), CD8α transmembrane domain (SEQ ID NO: 141) and CD4 intracellular domain (SEQ ID NO: 148). In certain embodiments, CD8α signal peptide, CD8α extracellular domain, CD8α transmembrane domain and CD4 intracellular domain can be sequence modified to retain or substantially retain the function of each element. In certain embodiments, such sequence modifications are conservative substitutions of amino acids.

[0123] In a non-limiting embodiment, the CD8 product 4 is Fig. 9 In certain embodiments, Fig. 9 The sequence of CD8 construct 4 provided in can be modified in any number of ways, as long as the translation of the CD8α signal peptide, CD8α extracellular domain, CD8α transmembrane domain, and CD4 intracellular domain maintains the conserved function of each element. In certain embodiments, Fig. 9 The order of each element of CD8 construct 4 remains the same, but the sequence of each individual element can be changed as long as the amino acid encoded by the nucleic acid remains the same or contains only conservative substitutions. Fig. 9 The order of each element in CD8 construct 4 remains the same, but the sequence of each individual element can be varied as long as the function of the encoded protein remains essentially unchanged.

[0124] In certain embodiments, the CD8 product comprises TET2 knockout or TET2 knockdown. In certain embodiments, the cells of the CD8 product are further modified to use a non-viral method to knock out the TET2 gene. In certain embodiments, the cells of the CD8 product are further modified to use a viral method to knock out the TET2 gene. In certain embodiments, the cells of the CD8 product are further modified to increase T cell persistence by using a non-viral method to knock out, knock down or modify the function of a gene associated with T cell persistence. In certain embodiments, the cells of the CD8 product are further modified to increase T cell persistence by using a viral method to knock out, knock down or modify the function of a gene associated with T cell persistence.

[0125] In some embodiments, the CD8 product comprises cells (i.e., CD8 viral products) transformed to express NeoTCR and CD8 constructs using a viral method. In certain embodiments, the cells of the CD8 viral product are further transformed to knock out the TET2 gene using a non-viral method. In certain embodiments, the cells of the CD8 viral product are further transformed to knock out the TET2 gene using a viral method. In certain embodiments, the cells of the CD8 viral product are further transformed to increase T cell persistence by knocking out, knocking down or modifying the function of a gene associated with T cell persistence using a non-viral method. In certain embodiments, the cells of the CD8 viral product are further transformed to increase T cell persistence by knocking out, knocking down or modifying the function of a gene associated with T cell persistence using a viral method.

[0126] In certain embodiments, the T cell persistence gene that is knocked out, knocked down or has a modified function is a gene that gives T cell activity down regulation. In certain embodiments, the gene that is knocked out, knocked down or modified is a gene that down regulates T cell memory function. In certain embodiments, the gene that is knocked out, knocked down or modified is a gene that reduces T cell function, proliferation and / or viability.

[0127] In certain embodiments, additional modifications to CD8 products and CD8 cells thereof include modifications that increase tumor microenvironment resilience, increase T cell activity, increase tumor microenvironment homing / retention, increase T cell persistence, and increase ectopic effector functions of T cells. In certain embodiments, tumor microenvironment resilience includes but is not limited to conversion / offsetting negative environmental signals. In certain embodiments, TCR-mediated signal enhancement includes but is not limited to increasing T cell activity. In certain embodiments, tumor microenvironment homing / retention includes but is not limited to enhancing tumor infiltration. In certain embodiments, functional T cell persistence includes but is not limited to metabolic and transcriptional regulation. In certain embodiments, ectopic effector functions include but are not limited to TCR-induced antibody, cytokine or peptide secretion.

[0128] In certain embodiments, functional T cell persistence can be achieved by genetic engineering as described herein, by co-administration of an agent that improves functional T cell persistence of modified T cells (modified to incorporate at least a neoTCR as described herein), and by manufacturing and culture conditions of modified T cells (modified to incorporate at least a neoTCR as described herein).

[0129] In certain embodiments, the TCR-induced antibody used to improve ectopic effector function is any of the antibodies described herein or their functional fragments. In certain embodiments, the TCR-induced cytokine used to improve ectopic effector function is a naturally occurring cytokine, a modified cytokine, a fusion protein of a cytokine, or any combination thereof. In certain embodiments, the TCR-induced cytokine is not a cytokine, but other auxiliary factors or expression elements that induce the production of endogenous cytokines.

[0130] In certain embodiments, additional modifications to CD8 products and CD8 cells thereof include modifications of one or more additional genes and / or functional proteins. In certain embodiments, the knocked-in genes and / or functional proteins include but are not limited to c-Myb, dominant negative FAS, FAS truncation, FBXW7, CTP1A, OPA1, GLUT1, CA-STAT5A, dominant negative TGFβR, DNMT3a, dominant negative PD-1R, dominant negative PD-1 or PD-L1, or PD-L2, dominant negative SHIP-1 protein, integrin, chemokine receptor, cytokine and interleukin. In certain embodiments, the dominant negative form of the gene is an antibody or a functional fragment thereof as a gene antagonist. For example, dominant negative PD-1 can be an anti-PD-1 antibody or a functional fragment thereof. In certain embodiments, any knock-in in a gene and / or functional protein is a functional fragment of a gene and / or protein (including but not limited to truncation).

[0131] In certain embodiments, additional modifications to CD8 products and CD8 cells thereof include knocking out or reducing modifications of one or more additional genes and / or functional proteins. In certain embodiments, the knocked out or reduced genes and / or functional proteins include but are not limited to TET2, IFNGR1, RICTOR, NR4A1, DNMT3A, SUV39H1, PPP2RD, adenosine 2A receptor, PP2A3 and PP2A4.

[0132] In certain embodiments, instead of knocking in a gene for expression of a functional protein, a different gene can be regulated by genetic engineering as described herein, which upregulates the expression of the gene that would otherwise be knocked in.

[0133] In certain embodiments, instead of knocking out or knocking out a gene, a different gene can be regulated by genetic engineering as described herein, which downregulates the expression of the gene that would otherwise be knocked out.

[0134] The CD8 product manufacturing process involves electroporation of a binuclear ribonucleoprotein type of CRISPR-Cas9 nuclease bound to a guide RNA sequence, each targeting the genomic TCRα and genomic TCRβ loci. The specificity of targeting the Cas9 nuclease to each genomic locus has been previously described in the literature as highly specific. The CD8 products were thoroughly tested in vitro and in silico using COSMID and GUIDE-seq, respectively, to investigate possible off-target genomic cleavage sites. Multiple CD8 products from healthy donors or equivalent cell products were evaluated for cleavage at candidate off-target sites by deep sequencing, supporting published evidence that the selected nucleases are highly specific.

[0135] In certain embodiments, the CD8 products described herein can be T cells, NK cells, NKT cells, macrophages, hematopoietic stem cells (HSCs), cells derived from HSCs, or dendritic / antigen presenting cells.

[0136] In certain embodiments, CD8 cells are expanded in a manner that retains a "young" T cell phenotype, producing a CD8 product in which a majority of T cells exhibit T memory stem cell and T central memory phenotypes.

[0137] These "younger" or "younger" or less differentiated T cell phenotypes are described as conferring improved engraftment potential and prolonged persistence after infusion. Therefore, administration of CD8 products (consisting primarily of "young" T cell phenotypes) has the potential to benefit cancer patients by improving engraftment potential, prolonged persistence after infusion, and rapid differentiation into effector T cells to eradicate tumor cells throughout the body.

[0138] In certain embodiments, the CD8 cells of the CD8 product mainly include memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 25% of the CD8 cells of the CD8 product include memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 30% of the CD8 cells of the CD8 product include memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 35% of the CD8 cells of the CD8 product include memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 40% of the CD8 cells of the CD8 product include memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 45% of the CD8 cells of the CD8 product include memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 50% of the CD8 cells of the CD8 product include memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 55% of the CD8 cells of the CD8 product comprise memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 60% of the CD8 cells of the CD8 product comprise memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 65% of the CD8 cells of the CD8 product comprise memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 70% of the CD8 cells of the CD8 product comprise memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 75% of the CD8 cells of the CD8 product comprise memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, more than 75% of the CD8 cells of the CD8 product comprise memory stem cells (Tmsc) and / or central memory cells (Tcm). Tmsc are characterized as cells of CD45RA+CD62L+, CD28+CD95+ and CCR7+CD27+. Tcm are characterized as cells that are CD45RO+CD62L+, CD28+CD95+, and CCR7+CD27+CD127+. Both Tmsc and Tcm are characterized as having weak effector T cell function, strong proliferation, strong engraftment, and long telomeres.

[0139] In certain embodiments, the CD8 cells disclosed herein exhibit about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 400%, about 450%, or about 500% improved properties (e.g., cytotoxic activity, cell proliferation, cytokine secretion, LCK affinity, persistence, tumor infiltration ability) compared to cells without a CD8 construct.

[0140] Methods for producing CD8 products with a youthful phenotype

[0141] In certain embodiments, the disclosure relates in part to the production of engineered "young" T cells. In certain embodiments, the disclosure includes methods for ex vivo production of antigen-specific cells, such as T cells, comprising activation, engineering, and expansion of antigen-specific cells initially obtained from a subject or isolated from such a sample.

[0142] In certain embodiments, the method of activating cells includes the step of activating the TCR / CD3 complex. For example, but not limited to, T cells can be incubated and / or cultured with a CD3 agonist, a CD28 agonist, or a combination thereof.

[0143] In certain embodiments, the activated antigen-specific cells (e.g., T cells) of the transformation can be expanded by cultivating the activated antigen-specific cells (e.g., T cells) of the transformation with cytokines, chemokines, soluble peptides, or a combination thereof. In certain embodiments, the activated antigen-specific cells of the transformation, such as the activated T cells of the transformation, can be cultivated together with one or more cytokines. In certain embodiments, the cytokine can be IL2, IL7, IL15, or a combination thereof. For example, the activated antigen-specific cells of the transformation, such as the activated T cells of the transformation, can be cultivated together with IL7 and IL15. In certain embodiments, the cytokine used in combination with the activated antigen-specific cells (e.g., activated T cells of the transformation) cultures of the transformation can be present with a concentration of about 1pg / ml to about 1g / ml, about 1ng / ml to about 1g / ml, about 1μg / ml to about 1g / ml, or about 1mg / ml to about 1g / ml, and any value therebetween.

[0144] Pharmaceutical preparations

[0145] The pharmaceutical preparation of the CD8 product is prepared by mixing the CD8 cells in a solution that preserves the "young" phenotype of the cells in a cryopreserved state. An example of such a pharmaceutical preparation is provided in Table 1. Alternatively, the pharmaceutical preparation of the CD8 product can be prepared by mixing the CD8 cells in a solution that preserves the "young" phenotype of the cells without freezing or cryopreserving the product (i.e., the CD8 product is stored in an aqueous solution or as a non-frozen / cryopreserved cell pellet).

[0146] Additional pharmaceutically acceptable carriers, buffers, stabilizers and / or preservatives may also be added to the cryopreservation solution or aqueous storage solution (if the CD8 product is not cryopreserved). Any cryopreservative and / or culture medium may be used to cryopreserve the CD8 product, including but not limited to CryoStor, CryoStor CS5, CELLBANKER, and customized cryopreservation medium (optionally containing DMSO).

[0147] Gene Editing Methods

[0148] In certain embodiments, the disclosure is partially related to methods for transforming human cells such as transformed T cells or transformed human stem cells. In certain embodiments, the disclosure is partially related to methods for transforming human cells, such as NK cells, NKT cells, macrophages, hematopoietic stem cells (HSC), cells derived from HSC, or dendritic / antigen presenting cells. In certain embodiments, such transformations are related to genome editing. For example, but not limited to, this genome editing can be completed with nucleases targeting one or more endogenous loci, such as TCRα (TCRα) loci and TCRβ (TCRβ) loci. In certain embodiments, nucleases can produce single-stranded DNA nicks or double-stranded DNA breaks in endogenous target sequences. In certain embodiments, nucleases can target coding or non-coding parts of the genome, such as exons, introns. In certain embodiments, the nucleases contemplated herein include homing endonucleases, large-range nucleases, megaTAL nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and clustered regularly spaced short palindromic repeats (CRISPR) / Cas nucleases. In certain embodiments, the nuclease itself can be engineered, for example by introducing amino acid substitutions and / or deletions, to increase the efficiency of the cleavage activity.

[0149] In certain embodiments, the CRISPR / Cas nuclease system is used to transform human cells. In certain embodiments, the CRISPR / Cas nuclease system comprises a Cas nuclease and one or more RNAs that recruit the Cas nuclease to an endogenous target sequence such as a single guide RNA. In certain embodiments, the Cas nuclease and RNA are introduced into cells separately, for example, using different vectors or compositions, or used together, for example, in a polycistronic construct or a single protein-RNA complex. In certain embodiments, the Cas nuclease is Cas9 or Cas12a. In certain embodiments, the Cas9 polypeptide is obtained from a bacterial species, including but not limited to Streptococcus pyogenes or Neisseria meningitidis. Other examples of CRISPR / Cas systems are known in the art. See Adli, Mazhar. "The CRISPR tool kit for genome editing and beyond." Nature communications vol. 9, 11911 (2018), all contents of which are taught are incorporated herein by reference.

[0150] In certain embodiments, genome editing occurs in one or more genomic loci regulating immune responses. In certain embodiments, loci include but are not limited to TCRα (TCRα) loci, TCRβ (TCRβ) loci, TCRγ (TCRγ) and TCRδ (TCRδ). In certain embodiments, the locus for inserting the CD8 construct is located at any position in the genome. In certain embodiments, the locus for inserting the CD8 construct is the TRAC locus. In certain embodiments, the locus for inserting the CD8 construct is one of two TRBC loci. In certain embodiments, the locus for inserting the CD8 construct is a locus different from the TRAC locus or the TRAB locus. In certain embodiments, the locus for inserting the CD8 construct is inserted into a gene locus, wherein the gene is knocked out. As a non-limiting example, if the desired phenotype of the CD8 product is the expression of NeoTCR, the expression of the CD8 construct, and the knockout of the TET2 gene or the AAVS1 gene, the CD8 construct can be inserted in the TET2 locus or the AAVS1 locus. In certain embodiments, the insertion of the CD8 construct is in tandem with the NeoTCR insertion. In certain embodiments, the insertion of the CD8 construct is a different locus than the NeoTCR insertion.

[0151] In certain embodiments, genome editing is performed by using a non-viral delivery system. For example, nucleic acids can be administered in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialo-seromucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Science 247:1465,1990) to introduce nucleic acid molecules into cells. Other non-viral methods for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation and protoplast fusion. Liposomes may also be beneficial for delivering DNA into cells. Transplanting normal genes into affected tissues of subjects can also be achieved by transferring normal nucleic acids into ex vivo culturable cell types (e.g., autologous or heterologous primary cells or their progeny), which are then injected into target tissues or systemically injected.

[0152] In certain embodiments, genome editing is performed by using a viral delivery system. In certain embodiments, viral methods include targeted integration (including but not limited to AAV) and random integration (including but not limited to slow virus methods). In certain embodiments, viral delivery will be completed without integrating nucleases. In such embodiments, the viral delivery system can be Lentiflash or another similar delivery system.

[0153] Homologous recombination template

[0154] In certain embodiments, the present disclosure provides genome editing of cells, which is performed by introducing and recombining homologous recombination (HR) template nucleic acid sequences into the endogenous loci of cells. In certain embodiments, the HR template nucleic acid sequence is linear. In certain embodiments, the HR template nucleic acid sequence is circular. In certain embodiments, the circular HR template can be a plasmid, a mini-circle or a nanoplasmid. In certain embodiments, the HR template nucleic acid sequence comprises a first and a second homology arm. In certain embodiments, the homology arm can be about 300 bases to about 2,000 bases. For example, each homology arm can be 1,000 bases. In certain embodiments, the homology arm can be homologous to the first and second endogenous sequences of the cell. In certain embodiments, the endogenous locus is a TCR locus. For example, the first and second endogenous sequences are within the TCR α locus or the TCR β locus. In certain embodiments, the HR template comprises a TCR gene sequence. In non-limiting embodiments, the TCR gene sequence is a patient-specific TCR gene sequence. In non-limiting embodiments, the TCR gene sequence is tumor-specific. In a non-limiting embodiment, the TCR gene sequence can be identified and obtained using the method described in PCT / US2020 / 017887, and the contents of PCT / US2020 / 017887 are incorporated herein by reference. In certain embodiments, the HR template comprises a TCRα gene sequence and a TCRβ gene sequence.

[0155] In certain embodiments, the HR template is a polycistronic polynucleotide. In certain embodiments, the HR template comprises a sequence encoding a flexible polypeptide sequence (e.g., a Gly-Ser-Gly sequence). In certain embodiments, the HR template comprises a sequence encoding an internal ribosome entry site (IRES). In certain embodiments, the HR template comprises a 2A peptide (e.g., P2A, T2A, E2A, and F2A). Additional information about HR template nucleic acids and methods for modifying cells thereof can be found in International Patent Application No. PCT / US2018 / 058230, the contents of which are incorporated herein by reference.

[0156] Treatment

[0157] The currently disclosed subject matter provides a method for inducing and / or increasing an immune response in a subject in need thereof. The CD8 product can be used to treat and / or prevent cancer in a subject. The CD8 product can be used to prolong the survival of a subject with cancer. The CD8 product can also be used to treat and / or prevent cancer in a subject. The CD8 product can also be used to reduce the tumor burden of a subject. Such methods include administering a CD8 product or a composition (e.g., a pharmaceutical composition) comprising the same in an effective amount to achieve the desired effect, whether to alleviate an existing condition or to prevent recurrence. For treatment, the dosage is an effective amount that produces the desired effect. The effective amount can be provided in one or a series of administrations. The effective amount can be provided by bolus or by continuous infusion.

[0158] In certain embodiments, CD8 products can be used to treat viral or bacterial diseases. In certain embodiments, CD8 products can be used to treat autoimmune diseases.

[0159] In certain embodiments, an effective amount of a CD8 product is delivered by IV administration. In certain embodiments, a CD8 product is delivered by IV administration in a single administration. In certain embodiments, a CD8 product is delivered by IV administration in multiple administrations. In certain embodiments, a CD8 product is delivered by IV administration in two or more administrations. In certain embodiments, a CD8 product is delivered by IV administration in two administrations. In certain embodiments, a CD8 product is delivered by IV administration in three administrations.

[0160] The presently disclosed subject matter provides methods for treating and / or preventing cancer in a subject. In certain embodiments, the method comprises administering an effective amount of a CD8 product to a subject having cancer.

[0161] Non-limiting examples of cancer include blood cancers (e.g., leukemias, lymphomas, and myelomas), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcomas, and various cancers (including prostate cancer and small cell lung cancer). Suitable cancers also include any known in the art of oncology, including but not limited to astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and its liver metastasis, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, bile duct carcinoma, synovioma, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, ramus Tracheal cancer, renal cell carcinoma, bile duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumors, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia and heavy chain disease, breast tumors such as ductal and lobular adenocarcinoma, squamous and adenocarcinoma of the cervix, epithelial carcinoma of the uterus and ovary, prostate adenocarcinoma, bladder transitional squamous cell carcinoma, B and T cell lymphoma (nodular and diffuse) plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma and leiomyosarcoma. In certain embodiments, the neoplasia is selected from the group consisting of blood cancer (e.g., leukemia, lymphoma, and myeloma), ovarian cancer, prostate cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, and laryngeal cancer. In certain embodiments, the currently disclosed young T cells and compositions comprising the same can be used to treat and / or prevent blood cancer (e.g., leukemia, lymphoma, and myeloma) or ovarian cancer that are refractory to conventional therapeutic intervention.

[0162] In certain embodiments, the neoplasia is a solid cancer or solid tumor. In certain embodiments, the solid tumor or solid cancer is selected from the group consisting of glioblastoma, prostate adenocarcinoma, renal papillary cell carcinoma, sarcoma, ovarian cancer, pancreatic cancer, rectal adenocarcinoma, colon adenocarcinoma, esophageal cancer, endometrioid carcinoma, breast cancer, skin melanoma, lung adenocarcinoma, gastric adenocarcinoma, cervical cancer and uterine cervical cancer, renal clear cell carcinoma, testicular germ cell tumor and aggressive B-cell lymphoma.

[0163] Subjects may have advanced disease, in which case treatment goals may include slowing or reversing disease progression, and / or improving side effects. Subjects may have a history of disease for which they have already been treated, in which case treatment goals often include reducing or delaying the risk of relapse.

[0164] Suitable human subjects for treatment generally include two treatment groups that can be distinguished by clinical criteria. Subjects with "advanced disease" or "high tumor burden" are those carrying clinically measurable tumors. Clinically measurable tumors are tumors that can be detected based on tumor mass (e.g., by palpation, CAT scan, ultrasonography, mammography, or X-ray; positive biochemical or histopathological markers alone are insufficient to identify this population). Pharmaceutical compositions are administered to these subjects to elicit an anti-tumor response with the goal of alleviating their condition. Ideally, the result is a reduction in tumor mass, but any clinical improvement constitutes a benefit. Clinical improvement includes reducing the risk or rate of progression or alleviating the pathological consequences of the tumor.

[0165] manufactures

[0166] The CD8 product can be used in combination with a manufactured product. Such manufactured products can be used to prevent or treat proliferative disorders (e.g., cancer). Examples of manufactured products include, but are not limited to, containers (e.g., infusion bags, bottles, storage containers, flasks, vials, syringes, tubes, and IV solution bags) and labels or package inserts on or associated with the containers. The container can be made of any material acceptable for storing and preserving CD8 cells in a CD8 product. In certain embodiments, the container can be an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle. For example, the container can be a CryoMACS freezing bag. The label or package insert indicates that the CD8 product is used to treat selected disorders and original patients. Since the CD8 product is made of autologous cells and is transformed into patient-specific and personalized treatments, the patient is identified on the container of the CD8 product.

[0167] The article of manufacture may include: 1) a first container containing a CD8 product.

[0168] The article of manufacture may include: 1) a first container containing a CD8 product; 2) a second container having the same CD8 product as the first container. Optionally, additional containers having the same CD8 product as the first and second containers may be prepared and manufactured. Optionally, additional containers containing compositions including different cytotoxic agents or other therapeutic agents may also be combined with the above containers.

[0169] The article of manufacture may include: 1) a first container containing a CD8 product; and 2) a second container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent.

[0170] The article of manufacture may include: 1) a first container containing two CD8 products; and 2) a second container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent.

[0171] The manufactured article may include: 1) a first container containing a CD8 product; 2) a second container containing a second CD8 product; and 3) optionally a third container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. In certain embodiments, the first and second CD8 products are different CD8 products. In certain embodiments, the first and second CD8 products are the same CD8 product.

[0172] The article of manufacture may include: 1) a first container containing three CD8 products; and 2) optionally, a second container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent.

[0173] The manufactured product may include: 1) a first container containing a CD8 product; 2) a second container containing a second CD8 product; 3) a third container containing a third CD8 product; and 4) optionally a fourth container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. In certain embodiments, the first, second, and third CD8 products are different CD8 products. In certain embodiments, the first, second, and third CD8 products are the same CD8 products. In certain embodiments, two of the first, second, and third CD8 products are the same CD8 products.

[0174] The article of manufacture may include: 1) a first container containing four CD8 products; and 2) optionally, a second container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent.

[0175] The manufactured product may include: 1) a first container containing a CD8 product; 2) a second container containing a second CD8 product; 3) a third container containing a third CD8 product; 4) a fourth container containing a fourth CD8 product; and 5) optionally a fifth container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. In certain embodiments, the first, second, third, and fourth CD8 products are different CD8 products. In certain embodiments, the first, second, third, and fourth CD8 products are the same NeoTCR products. In certain embodiments, two of the first, second, third, and fourth CD8 products are the same NeoTCR products. In certain embodiments, three of the first, second, third, and fourth CD8 products are the same CD8 products.

[0176] The article of manufacture may include: 1) a first container containing five or more CD8 products; and 2) optionally, a second container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent.

[0177] The manufactured product may include: 1) a first container containing a CD8 product; 2) a second container containing a second CD8 product; 3) a third container containing a third CD8 product; 4) a fourth container containing a fourth CD8 product; 5) a fifth container containing a fifth CD8 product; 6) optionally, a sixth or more additional container containing a sixth or more CD8 products; and 7) optionally, an additional container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. In certain embodiments, all containers of CD8 products are different CD8 products. In certain embodiments, all containers of CD8 products are the same CD8 product. In certain embodiments, based on the availability of detectable CD8 in patient tumor samples, the need and / or desire to have a variety of CD8 products for patients, and the availability of any one CD8 product that may need or benefit from one or more containers, there may be any combination of the same or different CD8 products in five or more containers.

[0178] The article of manufacture may include: 1) a first container containing a CD8 product; 2) a second container containing a second CD8 product; and 3) a third container containing a third CD8 product.

[0179] The article of manufacture may include: 1) a first container containing a CD8 product; 2) a second container containing a second CD8 product; 3) a third container containing a third CD8 product; and 4) optionally, a fourth container containing a fourth CD8 product.

[0180] The manufactured product may include: 1) a first container containing a CD8 product; 2) a second container containing a second CD8 product; 3) a third container containing a third CD8 product; 4) a fourth container containing a fourth CD8 product; and 5) optionally a fifth container containing a fourth CD8 product.

[0181] The manufactured product may include a container containing one CD8 product. The manufactured product may include a container containing two CD8 products. The manufactured product may include a container containing three CD8 products. The manufactured product may include a container containing four CD8 products. The manufactured product may include a container containing five CD8 products.

[0182] A manufactured product may include 1) a first container containing one CD8 product, and 2) a second container containing two CD8 products. A manufactured product may include 1) a first container containing two CD8 products, and 2) a second container containing one CD8 product. In the above examples, a third and / or fourth container containing one or more additional CD8 products may be included in the manufactured product. Additionally, a fifth container containing one or more additional CD8 products may be included in the manufactured product.

[0183] Furthermore, any CD8 product container described herein can be divided into two, three, or four separate containers for multiple dosing time points and / or appropriate dosing based on the patient.

[0184] In certain embodiments, the CD8 product is provided in a kit. By way of non-limiting example, the kit may include a package insert, a label, instructions for using the CD8 product, a syringe, handling instructions, dosing instructions, tubes, needles, and any other items a clinician needs in order to properly administer the CD8 product.

[0185] Therapeutic compositions and methods of manufacture

[0186] As described herein, a plasmid DNA-mediated precise genome modification method for good manufacturing practices (GMP) of CD8 products was developed. Targeted integration of patient-specific neoTCRs was achieved by electroporation of CRISPR endonuclease ribonucleoproteins (RNPs) and personalized neoTCR gene cassettes encoded by plasmid DNA. In addition to neoTCRs, CD8 constructs were inserted by integrating them into neoTCR vectors and then electroporating with CRISPR endonuclease ribonucleoproteins (RNPs) as described above.

[0187] The CD8 product can be formulated into a drug product using clinical manufacturing methods. During this process, the CD8 product is cryopreserved in CryoMACS cryobags. One or more bags can be shipped to the site for each patient, depending on the patient's needs. The product consists of apheresis-derived patient's autologous CD8 and CD4 T cells that have been precisely genomically engineered to express one or more autologous neoTCRs targeting a neo-epitope complexed with an endogenous HLA receptor that is only present on the surface of the patient's tumor cells.

[0188] The final product will contain 5% dimethyl sulfoxide (DMSO), human serum albumin, and Plasma-Lyte. The final cell product will contain the list of components provided in Table 2.

[0189] Table 2 Composition of CD8 products

[0190]

[0191]

[0192] Composition and carrier

[0193] The presently disclosed subject matter provides compositions comprising the cells (eg, immunoreactive cells) disclosed herein.

[0194] In certain embodiments, the presently disclosed subject matter provides a nucleic acid composition comprising a polynucleotide encoding a NeoTCR disclosed herein. In certain embodiments, the nucleic acid composition disclosed herein comprises a polynucleotide encoding a CD8 construct disclosed herein. Cells comprising such nucleic acid compositions are also provided.

[0195] In certain embodiments, the nucleic acid composition further comprises a promoter operably linked to a NeoTCR disclosed herein. In certain embodiments, the nucleic acid composition further comprises a promoter operably linked to a CD8 construct disclosed herein.

[0196] In certain embodiments, the promoter is endogenous or exogenous. In certain embodiments, the exogenous promoter is selected from the group consisting of elongation factor (EF) -1 promoter, CMV promoter, SV40 promoter, PGK promoter, long terminal repeat (LTR) promoter and metallothionein promoter. In certain embodiments, the promoter is an inducible promoter. In certain embodiments, the inducible promoter is selected from the group consisting of NFAT transcription response element (TRE) promoter, CD69 promoter, CD25 promoter, IL-2 promoter, IL-12 promoter, p40 promoter and Bcl-xL promoter.

[0197] Compositions and nucleic acid compositions can be applied to a subject or and / or delivered to a cell by methods known in the art or as described herein. Genetic modification of cells (e.g., T cells) can be achieved by transducing a substantially uniform cell composition with a recombinant DNA construct. In certain embodiments, a retroviral vector (gamma retroviral vector or lentiviral vector) is used to introduce the DNA construct into the cell. Non-viral vectors can also be used.

[0198] Possible transduction methods also include direct co-cultivation of cells with producer cells, for example by the method of Bregni, et al. (1992) Blood 80:1418-1422, or culturing with viral supernatant alone or concentrated vector stock with or without appropriate growth factors and polycations, for example by the method of Xu, et al. (1994) Exp. Hemat. 22:223-230; and Hughes, et al. (1992) J. Clin. Invest. 89:1817.

[0199] Other transduction virus vectors can be used to modify cells. In certain embodiments, the selected vector exhibits high infection efficiency and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. USA 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus and adeno-associated virus vectors, vaccinia virus, bovine papilloma virus or herpes virus, such as Epstein-Barr virus (see also, for example, Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., US Pat. No. 5,399,346).

[0200] Non-viral methods can also be used for genetic modification of cells. For example, nucleic acids can be administered in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialo-seromucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by microinjection under surgical conditions (Wolff et al., Journal of Biological Chemistry 265:16985, 1989). al., Science 247: 1465, 1990) to introduce nucleic acid molecules into cells. Other non-viral methods for gene transfer include in vitro transfection using calcium phosphate, DEAE dextran, electroporation and protoplast fusion. Liposomes may also be beneficial for delivering DNA into cells. Transplanting normal genes into affected tissues of subjects can also be achieved by transferring normal nucleic acids into ex vivo culturable cell types (e.g., autologous or heterologous primary cells or their progeny), and then injecting the cells (or their progeny) into target tissues or systemic injections.

[0201] Polynucleotide therapeutic approaches can be initiated directly from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters) and regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, if desired, enhancers known to preferentially direct gene expression in a particular cell type can be used to direct expression of the nucleic acid. Enhancers used include, but are not limited to, those characterized as tissue or cell specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by cognate regulatory sequences or, if desired, by regulatory sequences derived from heterologous sources, including any of the promoters or regulatory elements described above.

[0202] The resulting cells can be grown under conditions similar to unmodified cells, allowing the modified cells to be expanded and used for a variety of purposes.

[0203] Reagent test kit

[0204] The presently disclosed subject matter provides kits for inducing and / or enhancing an immune response and / or treating and / or preventing cancer or pathogen infection in a subject. In certain embodiments, the kit comprises an effective amount of the presently disclosed cells or a pharmaceutical composition comprising the same. In certain embodiments, the kit comprises a sterile container; such a container may be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a blister pack, or other suitable container forms known in the art. Such a container may be made of plastic, glass, laminated paper, metal foil, or other material suitable for holding a drug. In certain non-limiting embodiments, the kit comprises an isolated nucleic acid molecule encoding the presently disclosed HR template.

[0205] If necessary, the cells and / or nucleic acid molecules are provided together with instructions for administering the cells or nucleic acid molecules to subjects suffering from or at risk of cancer or pathogens or immune disorders. The instructions typically include information about using the composition to treat and / or prevent cancer or pathogen infection. In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosage regimen and administration for treating or preventing neoplasia, pathogen infection, or immune disorders or their symptoms; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions can be printed directly on the container (if any), or as a label applied to the container, or as a separate paper, brochure, card, or folder provided in the container or provided with the container. The resulting cells can be grown under conditions similar to unmodified cells, so that the modified cells can be amplified and used for various purposes.

[0206] Exemplary embodiments

[0207] A. In certain non-limiting embodiments, the presently disclosed subject matter provides cells comprising an exogenous T cell receptor (TCR) and exogenous CD8.

[0208] A1. The aforementioned A, wherein the exogenous CD8 comprises at least one monomer.

[0209] A2. The cell of the aforementioned A1, wherein at least one monomer of the exogenous CD8 comprises an extracellular domain, a transmembrane domain, an intracellular domain, a fragment thereof, or a combination thereof.

[0210] A3. The cell of A2 above, wherein the extracellular domain comprises a CD8α extracellular domain or a CD8β extracellular domain.

[0211] A4. The cell of A2 or A3 described above, wherein the transmembrane domain is a CD8α transmembrane domain or a CD8β transmembrane domain.

[0212] A5. The cell of A2-A4 above, wherein the intracellular domain comprises the CD8α intracellular domain or the CD8β intracellular domain.

[0213] A6. The cell of A2-A4 described above, wherein the intracellular domain comprises a CD4 intracellular domain.

[0214] A7. The cell of A1-A5 described above, wherein the at least one monomer comprises a CD8α extracellular domain, a CD8α transmembrane domain and a CD8α intracellular domain.

[0215] A8. The cell of A1-A5 described above, wherein the at least one monomer comprises a CD8β extracellular domain, a CD8β transmembrane domain and a CD8β intracellular domain.

[0216] A9. The cell of A1-A5 described above, wherein the at least one monomer comprises a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8β intracellular domain.

[0217] A10. The cell of the aforementioned A1-A6, wherein the at least one monomer comprises a CD8α extracellular domain, a CD8α transmembrane domain and a CD4 intracellular domain.

[0218] A11. The cell of the aforementioned A1-A10, wherein the at least one monomer comprises a signal peptide.

[0219] A12. The cell of the aforementioned A11, wherein the signal peptide is a CD8 signal peptide.

[0220] A13. The cell of A2-A12 described above, wherein the extracellular domain comprises the amino acid sequence listed in SEQ ID NO:140 or SEQ ID NO:145.

[0221] A14. The cell of A2-A13 described above, wherein the transmembrane domain comprises the amino acid sequence listed in SEQ ID NO:141 or SEQ ID NO:146.

[0222] A15. The cell of A2-A14 described above, wherein the intracellular domain comprises the amino acid sequence listed in SEQ ID NO:142, SEQ ID NO:147 or SEQ ID NO:148.

[0223] A16. The cell of the aforementioned A11-A15, wherein the signal peptide comprises the amino acid sequence listed in SEQ ID NO:139 or SEQ ID NO:144.

[0224] A17. The cell of A-A16 described above, wherein the exogenous CD8 comprises a 2A sequence.

[0225] A18. The cell of aforementioned A-A17, wherein the exogenous CD8 comprises a linker.

[0226] A19. The cell of A18 described above, wherein the linker comprises the amino acid sequence listed in SEQ ID NO:137.

[0227] A20. The cell of A-A19 described above, wherein the exogenous CD8 comprises a protease cleavage site.

[0228] A21. The cell of A-A20 described above, wherein the protease cleavage site is a furin cleavage site.

[0229] A22. The cell of A-A21 above, wherein the exogenous TCR is a patient-derived TCR.

[0230] A23. The cell of A-A22 above, wherein the exogenous TCR comprises a signal sequence, a first and a second 2A sequence, and a TCR polypeptide sequence.

[0231] A24. The cell of A-A23 described above, wherein the exogenous TCR recognizes a cancer antigen.

[0232] A25. The cell of the aforementioned A24, wherein the cancer antigen is a neoantigen.

[0233] A26. The cell of the aforementioned A24, wherein the cancer antigen is a patient-specific antigen.

[0234] A27. The cell of any of A-A26 above, wherein the cell is a primary cell.

[0235] A28. The cell of A-A26 above, wherein the cell is a patient-derived cell.

[0236] A29. The cell of any of A-A26 above, wherein the cell is a lymphocyte.

[0237] A30. The cell of A-A26 described above, wherein the cell is a T cell.

[0238] A31. The cell of A-A26 described above, wherein the cell is a young T cell.

[0239] A32. The cell of the aforementioned A31, wherein the cell is CD45RA+, CD62L+, CD28+, CD95-, CCR7+ and CD27+.

[0240] A33. The cell of the aforementioned A31, wherein the cell is CD45RA+, CD62L+, CD28+, CD95+, CD27+, CCR7+.

[0241] A34. The cells of the aforementioned A31, wherein the cells are CD45RO+, CD62L+, CD28+, CD95+, CCR7+, CD27+, CD127+.

[0242] A35. The cell of A-A34 described above, further comprising a genetic modification to enhance cell persistence and / or enhance memory cell differentiation.

[0243] A36. The cell of the aforementioned A-A35, wherein the killing activity of the cell is increased by about 10% to about 500% compared to the killing activity of cells without exogenous CD8.

[0244] A37. The cell of A-A36 above, wherein proliferation of the cell after TCR binding to the antigen increases by about 10% to about 500% compared to proliferation of cells without exogenous CD8.

[0245] A38. The cell of A-A37 above, wherein secretion of pro-inflammatory cytokines by the cell is increased by about 10% to about 500% when TCR binds to an antigen, compared to secretion by a cell without exogenous CD8.

[0246] A39. The cell of A-A38 above, wherein the LCK affinity of the cell is increased by about 10% to about 500% compared to the LCK affinity of the cell without exogenous CD8.

[0247] A40. The cell of A-A39 above, wherein the persistence of the cell is increased by about 10% to about 500% compared to the persistence of the cell without exogenous CD8.

[0248] A41. The cell of A-A40 above, wherein the tumor infiltrating ability of the cell is increased by about 10% to about 500% compared to the tumor infiltrating ability of the cell without exogenous CD8.

[0249] A42. The cell of A-A41 above, wherein the exogenous TCR is a CD8-dependent TCR.

[0250] A43. The cell of A-A41 above, wherein the exogenous TCR is a CD8-independent TCR.

[0251] A44. The cell of any of A-A43 above, wherein the exogenous CD8 is encoded by CD8 construct 1, CD8 construct 2, CD8 construct 3 or CD8 construct 4.

[0252] A45. The cells of A-A43 described above, wherein the exogenous CD8 includes: CD8α extracellular domain, CD8α transmembrane domain and CD8α intracellular domain; CD8α extracellular domain, CD8α transmembrane domain, CD8α intracellular domain, CD8β extracellular domain, CD8β transmembrane domain and CD8β intracellular domain; CD8α extracellular domain, CD8α transmembrane domain and CD8β intracellular domain; or CD8α extracellular domain, CD8α transmembrane domain and CD4 intracellular domain.

[0253] A46. The cell of A-A43 described above, wherein the exogenous CD8 comprises: a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), and a CD8α intracellular domain (SEQ ID NO: 142); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147). NO: 140), CD8α transmembrane domain (SEQ ID NO: 141), and CD8β intracellular domain (SEQ ID NO: 147); or CD8α signal peptide (SEQ ID NO: 139), CD8α extracellular domain (SEQ ID NO: 140), CD8α transmembrane domain (SEQ ID NO: 141), and CD4 intracellular domain (SEQ ID NO: 148).

[0254] B. In certain non-limiting embodiments, the presently disclosed subject matter provides a method for modifying a cell, the method comprising introducing a homologous recombination (HR) template nucleic acid sequence into the cell, wherein the HR template comprises first and second homology arms homologous to the first and second target nucleic acid sequences, a TCR gene sequence located between the first and second homology arms, a CD8 gene sequence located between the first and second homology arms, and recombining the HR template nucleic acid into an endogenous locus of the cell.

[0255] B1. The method of B described above, wherein the HR template comprises a first 2A coding sequence located upstream of the CD8 gene sequence, a second 2A coding sequence located downstream of the CD8 gene sequence and upstream of the TCR gene sequence, and a third 2A coding sequence located downstream of the TCR gene sequence; wherein the first, second and third 2A coding sequences encode the same amino acid sequence and have codon divergences relative to each other.

[0256] B2. The method of B or B1 above, wherein the HR template comprises a sequence encoding the amino acid sequence Gly Ser Gly immediately upstream of the first, second and / or third 2A coding sequence.

[0257] B3. The method of B1 or B2 above, wherein the HR template further comprises a sequence encoding a furin cleavage site located upstream of the first, second and / or third 2A coding sequence.

[0258] B4. The method of B-B3 above, wherein the HR template further comprises a sequence encoding a signal sequence located immediately upstream of the TCR gene sequence and / or the CD8 gene sequence.

[0259] B5. The method of B-B4 above, wherein the HR template comprises a second TCR sequence located between the third 2A coding sequence and the second homology arm.

[0260] B6. The method of B5 above, wherein the HR template comprises a sequence encoding a first signal sequence located immediately upstream of a first TCR gene sequence; and a sequence encoding a second signal sequence located immediately upstream of a second TCR gene sequence.

[0261] B7. The method of B-B6 above, wherein the HR template comprises a second CD8 gene sequence located between the first CD8 gene sequence and the second 2A coding sequence.

[0262] B8. The method of B7 described above, wherein the 2A coding sequence is located between the first and second CD8 gene sequences.

[0263] B9. The method of B7 or B8 described above, wherein the sequence encoding the amino acid sequence Gly Ser Gly is located between the first and second CD8 gene sequences.

[0264] B10. The method of B7-B9 described above, wherein the sequence encoding the furin cleavage site is located between the first and second CD8 gene sequences.

[0265] B11. The method of B-B10 described above, wherein the CD8 gene sequence comprises a sequence encoding an extracellular domain, a sequence encoding an intracellular domain, a sequence encoding an intracellular domain, a fragment thereof, or a combination thereof.

[0266] B12. The method of the aforementioned B11, wherein the sequence encoding the extracellular domain comprises a sequence encoding a CD8α extracellular domain or a CD8β extracellular domain.

[0267] B13. The method of the aforementioned B11 or B12, wherein the sequence encoding the transmembrane domain comprises a sequence encoding the CD8α transmembrane domain or the CD8β transmembrane domain.

[0268] B14. The method of aforementioned B11-B13, wherein the sequence encoding the intracellular domain comprises a sequence encoding the CD8α intracellular domain or the CD8β intracellular domain.

[0269] B15. The method of aforementioned B11-B14, wherein the sequence encoding the intracellular domain comprises a sequence encoding the CD4 intracellular domain.

[0270] B16. The method of B11-B15 above, wherein the CD8 gene sequence comprises a sequence encoding a CD8α extracellular domain, a CD8α transmembrane domain and a CD8α intracellular domain.

[0271] B17. The method of B11-B15 described above, wherein the CD8 gene sequence comprises a sequence encoding a CD8β extracellular domain, a CD8β transmembrane domain and a CD8β intracellular domain.

[0272] B18. The method of B11-B15 described above, wherein the CD8 gene sequence comprises a sequence encoding a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8β intracellular domain.

[0273] B19. The method of aforementioned B11-B15, wherein the CD8 gene sequence comprises a sequence encoding a CD8α extracellular domain, a CD8α transmembrane domain and a CD4 intracellular domain.

[0274] B20. The method of B7-B19 described above, wherein the HR template comprises a sequence encoding a first signal sequence located immediately upstream of a first CD8 gene sequence and a sequence encoding a second signal sequence located immediately upstream of a second CD8 gene sequence.

[0275] B21. The method of B4-B20 described above, wherein the signal sequence is a CD8 signal sequence, a human growth hormone signal sequence, a fragment thereof, or a combination thereof.

[0276] B22. The method of B-B21 above, wherein the first and second homology arms of the HR template each have a length of about 300 bases to about 2,000 bases.

[0277] B23. The method of B-B22 above, wherein the first and second homology arms of the HR template each have a length of about 600 bases to about 2,000 bases.

[0278] B24. The method of B-B23 described above, wherein the exogenous TCR is a patient-derived TCR.

[0279] B25. The method of B-B24 above, wherein the exogenous TCR comprises a signal sequence, a first and a second 2A sequence, and a TCR polypeptide sequence.

[0280] B26. The method of B-B25 described above, wherein the exogenous TCR recognizes a cancer antigen.

[0281] B27. The method of the aforementioned B26, wherein the cancer antigen is a neoantigen.

[0282] B28. The method of the aforementioned B26, wherein the cancer antigen is a patient-specific antigen.

[0283] B29. The method of B-B28 above, wherein the HR template is non-viral.

[0284] B30. The method of B-B29 described above, wherein the HR template is circular DNA.

[0285] B31. The method of B-B29 described above, wherein the HR template is linear DNA.

[0286] B32. The method of B-B31 above, wherein the introduction is performed by electroporation.

[0287] B33. The method of B-B32 above, wherein the recombination comprises cutting the endogenous locus by a nuclease; and recombining the HR template nucleic acid sequence into the endogenous locus by homology-directed repair.

[0288] B34. The method of B33 above, wherein the nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR) family nuclease or a derivative thereof.

[0289] B35. The method of B34 described above also includes gRNA.

[0290] B36. The method of B-B35 described above, wherein the method further comprises culturing cells.

[0291] B37. The method of B36 described above, wherein the culturing is carried out in the presence of at least one cytokine.

[0292] B38. The method of B36 or B37 described above, wherein the culture is carried out in the presence of IL2, IL7, IL15 or any combination thereof.

[0293] B39. The method of B36 or B37 described above, wherein the culture is carried out in the presence of IL2 and IL15.

[0294] B40. The method of B-B39 described above, further comprising genetic modification to enhance cell persistence and / or enhance memory cell differentiation.

[0295] B41. The method of B-B40 described above, wherein the cells are primary cells.

[0296] B42. The method of B-B40 described above, wherein the cells are patient-derived cells.

[0297] B43. The method of B-B40 described above, wherein the cell is a lymphocyte.

[0298] B44. The method of B-B40 described above, wherein the cell is a T cell.

[0299] B45. The method of B-B40 described above, wherein the cells are young T cells.

[0300] B46. The method of the aforementioned B45, wherein the cells are CD45RA+, CD62L+, CD28+, CD95-, CCR7+ and CD27+.

[0301] B47. The method of B45 described above, wherein the cells are CD45RA+, CD62L+, CD28+, CD95+, CD27+, CCR7+.

[0302] B48. The method of B45 described above, wherein the cells are CD45RO+, CD62L+, CD28+, CD95+, CCR7+, CD27+, CD127+.

[0303] B49. The method of the aforementioned B-B48, wherein the killing activity of the cells is increased by about 10% to about 500% compared to the killing activity of cells not having the CD8 gene sequence.

[0304] B50. The method of B-B49 described above, wherein the proliferation of cells after TCR binding to the antigen increases by about 10% to about 500% compared to the proliferation of cells not having the CD8 gene sequence.

[0305] B51. The method of B-B50 described above, wherein the secretion of pro-inflammatory cytokines by the cells increases by about 10% to about 500% when the TCR binds to the antigen, compared to the secretion by cells that do not have the CD8 gene sequence.

[0306] B52. The method of B-B51 above, wherein the LCK affinity of the cells is increased by about 10% to about 500% compared to the LCK affinity of cells not having the CD8 gene sequence.

[0307] B53. The method of B-B52 above, wherein the persistence of the cells is increased by about 10% to about 500% compared to the persistence of cells without the CD8 gene sequence.

[0308] B54. The method of the aforementioned B-B53, wherein the tumor infiltration ability of the cells is increased by about 10% to about 500% compared to the tumor infiltration ability of cells not having the CD8 gene sequence.

[0309] B55. The method of aforementioned B-B54, wherein the TCR gene encodes a CD8-dependent TCR.

[0310] B56. The method of B-B54 described above, wherein the TCR gene encodes a TCR that is independent of CD8.

[0311] B57. The method of B-B56 described above, wherein the CD8 gene sequence is encoded by CD8 construct 1, CD8 construct 2, CD8 construct 3 or CD8 construct 4.

[0312] B58. The method of the aforementioned B-B56, wherein the CD8 gene sequence comprises: CD8α extracellular domain, CD8α transmembrane domain and CD8α intracellular domain; CD8α extracellular domain, CD8α transmembrane domain, CD8α intracellular domain, CD8β extracellular domain, CD8β transmembrane domain and CD8β intracellular domain; CD8α extracellular domain, CD8α transmembrane domain and CD8β intracellular domain; or CD8α extracellular domain, CD8α transmembrane domain, CD4 intracellular domain.

[0313] B59. The method of the aforementioned B-B56, wherein the CD8 gene sequence includes: CD8α signal peptide (SEQ ID NO: 139), CD8α extracellular domain (SEQ ID NO: 140), CD8α transmembrane domain (SEQ ID NO: 141) and CD8α intracellular domain (SEQ ID NO: 142); CD8α signal peptide (SEQ ID NO: 139), CD8α extracellular domain (SEQ ID NO: 140), CD8α transmembrane domain (SEQ ID NO: 141), CD8α intracellular domain (SEQ ID NO: 142), CD8β signal peptide (SEQ ID NO: 144), CD8β extracellular domain (SEQ ID NO: 145), CD8β transmembrane domain (SEQ ID NO: 146) and CD8β intracellular domain (SEQ ID NO: 147); CD8α signal peptide (SEQ ID NO: 139), CD8α extracellular domain (SEQ ID NO: 140), CD8α transmembrane domain (SEQ ID NO: 141), CD8α intracellular domain (SEQ ID NO: 142), CD8β signal peptide (SEQ ID NO: 144), CD8β extracellular domain (SEQ ID NO: 145), CD8β transmembrane domain (SEQ ID NO: 146) and CD8β intracellular domain (SEQ ID NO: 147). NO: 140), CD8α transmembrane domain (SEQ ID NO: 141), and CD8β intracellular domain (SEQ ID NO: 147); or CD8α signal peptide (SEQ ID NO: 139), CD8α extracellular domain (SEQ ID NO: 140), CD8α transmembrane domain (SEQ ID NO: 141), and CD4 intracellular domain (SEQ ID NO: 148).

[0314] C. In certain non-limiting embodiments, the presently disclosed subject matter provides cells modified by the method of B-B57.

[0315] D. In certain non-limiting embodiments, the presently disclosed subject matter provides a composition comprising an effective amount of A-A46 cells or C cells.

[0316] D1. The composition of the aforementioned D, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0317] D2. The composition of D or D1 above, wherein the composition is administered to a patient in need thereof to treat cancer.

[0318] D3. The composition of the aforementioned D-D2, wherein the composition comprises a cryopreservative.

[0319] D4. The composition of the aforementioned D-D3, wherein the composition comprises serum albumin.

[0320] D5. The composition of the aforementioned D-D4, wherein the composition comprises Plasma-Lyte A, HSA and CryoStorCS10.

[0321] E. In certain non-limiting embodiments, the presently disclosed subject matter provides methods of treating cancer in a subject in need thereof, the methods comprising administering a therapeutically effective amount of a cell of A-A46, a cell of claim C, or a composition of D-D5.

[0322] E1. The method of E above, wherein prior to administering the therapeutically effective amount of cells, a non-myeloablative lymphodepletion regimen is administered to the subject.

[0323] E2. The method of E or E1 above, wherein the cancer is a solid tumor.

[0324] E3. The method of E or E1 above, wherein the cancer is a liquid tumor.

[0325] E4. The method of E2 above, wherein the solid tumor is selected from the group consisting of melanoma, breast cancer, lung cancer, ovarian cancer, breast cancer, pancreatic cancer, head and neck cancer, prostate cancer, gynecological cancer, central nervous system cancer, skin cancer, HPV+ cancer, esophageal cancer, thyroid cancer, gastric cancer, hepatocellular carcinoma, bile duct cancer, renal cell carcinoma, testicular cancer, sarcoma and colorectal cancer.

[0326] E5. The method of the aforementioned E3, wherein the liquid tumor is selected from the group consisting of follicular lymphoma, leukemia and multiple myeloma.

[0327] F. In certain non-limiting embodiments, the presently disclosed subject matter provides a kit comprising the cells of A-A46, reagents for performing the methods of B-B57, the cells of C, or the compositions of D-D5.

[0328] F1. The kit of F above, wherein the kit further comprises written instructions for treating cancer.

[0329] G. In certain non-limiting embodiments, the presently disclosed subject matter provides a cell comprising: an exogenous T cell receptor (TCR); and exogenous CD8, wherein the exogenous CD8 includes: a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8α intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, a CD8α intracellular domain, a CD8β extracellular domain, a CD8β transmembrane domain, and a CD8β intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8β intracellular domain; or a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain.

[0330] H. In certain non-limiting embodiments, the presently disclosed subject matter provides a cell comprising: an exogenous T cell receptor (TCR); and exogenous CD8, the exogenous CD8 comprising: a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), and a CD8α intracellular domain (SEQ ID NO: 142); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147); NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD8β intracellular domain (SEQ ID NO:147); or CD8α signal peptide (SEQ ID NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD4 intracellular domain (SEQ ID NO:148).

[0331] I. In certain non-limiting embodiments, the presently disclosed subject matter provides a method for modifying a cell, the method comprising: introducing a homologous recombination (HR) template nucleic acid sequence into the cell, wherein the HR template comprises: a first and a second homology arm homologous to a first and a second target nucleic acid sequence; a TCR gene sequence located between the first and the second homology arms; a CD8 gene sequence located between the first and the second homology arms; recombining the HR template nucleic acid into an endogenous locus of the cell, wherein the CD8 gene sequence comprises: a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8α intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, a CD8α intracellular domain, a CD8β extracellular domain, a CD8β transmembrane domain, and a CD8β intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, a CD8β intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, a CD8β intracellular domain; or a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain.

[0332] J. In certain non-limiting embodiments, the presently disclosed subject matter provides a method for modifying a cell, the method comprising: introducing a homologous recombination (HR) template nucleic acid sequence into the cell, wherein the HR template comprises: first and second homology arms homologous to first and second target nucleic acid sequences; a TCR gene sequence located between the first and second homology arms; a CD8 gene sequence located between the first and second homology arms; recombining the HR template nucleic acid into an endogenous locus of the cell, wherein the CD8 gene sequence comprises: a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), and a CD8α intracellular domain (SEQ ID NO: 142); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145); NO:145), CD8β transmembrane domain (SEQ ID NO:146) and CD8β intracellular domain (SEQ ID NO:147); CD8α signal peptide (SEQ ID NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD8β intracellular domain (SEQ ID NO:147); or CD8α signal peptide (SEQ ID NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD4 intracellular domain (SEQ ID NO:148).

[0333] K. In certain non-limiting embodiments, when the disclosed subject matter provides a composition comprising a cell, wherein the cell comprises an exogenous T cell receptor (TCR) and exogenous CD8, wherein the exogenous CD8 comprises: a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8α intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, a CD8α intracellular domain, a CD8β extracellular domain, a CD8β transmembrane domain, and a CD8β intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8β intracellular domain; or a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain.

[0334] L. In certain non-limiting embodiments, the presently disclosed subject matter provides a composition comprising a cell, wherein the cell comprises an exogenous T cell receptor (TCR) and exogenous CD8, wherein the exogenous CD8 comprises: a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), and a CD8α intracellular domain (SEQ ID NO: 142); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147); NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD8β intracellular domain (SEQ ID NO:147); or CD8α signal peptide (SEQ ID NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD4 intracellular domain (SEQ ID NO:148).

[0335] M. In certain non-limiting embodiments, the presently disclosed subject matter provides a method for treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of cells, wherein the cells comprise an exogenous T cell receptor (TCR) and exogenous CD8, wherein the exogenous CD8 comprises: a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8α intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, a CD8α intracellular domain, a CD8β extracellular domain, a CD8β transmembrane domain, and a CD8β intracellular domain; a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8β intracellular domain; or a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain.

[0336] N. A method for treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of cells, wherein the cells comprise an exogenous T cell receptor (TCR) and exogenous CD8, wherein the exogenous CD8 comprises: a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), and a CD8α intracellular domain (SEQ ID NO: 142); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147); a CD8α signal peptide (SEQ ID NO: 139), a CD8α extracellular domain (SEQ ID NO: 140), a CD8α transmembrane domain (SEQ ID NO: 141), a CD8α intracellular domain (SEQ ID NO: 142), a CD8β signal peptide (SEQ ID NO: 144), a CD8β extracellular domain (SEQ ID NO: 145), a CD8β transmembrane domain (SEQ ID NO: 146), and a CD8β intracellular domain (SEQ ID NO: 147); NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD8β intracellular domain (SEQ ID NO:147); or CD8α signal peptide (SEQ ID NO:139), CD8α extracellular domain (SEQ ID NO:140), CD8α transmembrane domain (SEQ ID NO:141) and CD4 intracellular domain (SEQ ID NO:148). Example

[0337] The following are examples of methods and compositions of the present invention.It is understood that various other embodiments may be practiced, given the general description provided above.

[0338] Example 1 Generation of NeoTCR Products

[0339] Neo-epitope-specific TCRs identified by the imPACT isolation technique described in PCT / US2020 / 17887 (incorporated herein by reference in its entirety) were used to generate homologous recombination (HR) DNA templates. These HR templates were transfected into primary human T cells in tandem with site-specific nucleases (see Figures 1A-1C ). Single-step non-viral precise genome engineering results in seamless replacement of endogenous TCR with the patient’s own neo-epitope-specific TCR, expressed from an endogenous promoter. The surface expressed TCR is completely native in sequence.

[0340] The precision of neoTCR-T cell genome engineering was assessed by targeted locus amplification (TLA) to identify off-target integration hotspots or translocations and by next-generation sequencing-based off-target cleavage assays, which revealed a lack of evidence for unexpected outcomes.

[0341] like Figures 1A-1C As shown, a construct containing the target gene is inserted into the endogenous locus. This is accomplished by using a homology repair template that contains the coding sequence of the target gene flanked by left and right HR arms. In addition to the HR arms, the target gene is sandwiched between a 2A peptide, a protease cleavage site, which is upstream of the 2A peptide to remove the 2A peptide from the upstream translated target gene, and a signal sequence ( Figure 1B Once integrated into the genome, the gene of the target expression gene cassette is transcribed into a single messenger RNA. During the translation of the target gene in the messenger RNA, the flanking region is disconnected from the target gene by the self-cleaving 2A peptide, and the protease cleavage site is cleaved to remove the 2A peptide upstream of the target translated gene ( Figure 1C In addition to the 2A peptide and the protease cleavage site, a glycine-serine-glycine (GSG) linker was inserted before each 2A peptide to further enhance the separation of the target gene from other elements in the expression cassette.

[0342] It has been determined that the P2A peptide is superior to other 2A peptides in terms of cell products because of its efficient cleavage ability. Therefore, two (2) P2A peptides and codon divergence are used to express the target gene without introducing any exogenous epitopes from the remaining amino acids at either end of the target gene of the P2A peptide. The benefit of gene-edited cells being free of exogenous epitopes (i.e., no flanking P2A peptide amino acids on either side of the target gene) is that immunogenicity is significantly reduced, and patients who are infused with cell products containing the gene-edited cells are less likely to develop an immune response to the gene-edited cells.

[0343] NeoTCR was integrated into the TCRα locus of T cells as described in PCT / US / 2018 / 058230. Specifically, a homology repair template was used that contained the NeoTCR coding sequence flanked by left and right HR arms. In addition, the endogenous TCRβ locus was disrupted, resulting in the expression of only the TCR sequence encoded by the NeoTCR construct. The general strategy was applied using circular HR templates as well as linear templates.

[0344] The target TCRα locus (Cα) is shown together with the plasmid HR template and the resulting edited sequence and downstream mRNA / protein products. Figure 1B and 1CThe target TCRα locus (endogenous TRAC) and its CRISPR Cas9 target site (horizontal stripes, arrows indicate the cleavage site) are shown ( Figures 1A-1C ). A circular plasmid HR template with a polynucleotide encoding the NeoTCR is located between the left and right homology arms ("LHA" and "RHA", respectively). The TRAC region introduced by the codon-optimized HR template is shown (vertical stripes). The TCRβ constant domain is derived from TRBC2, which is shown to be functionally equivalent to TRBC1. Other elements in the NeoTCR cassette include: 2A = 2A ribosomal skipping element (as a non-limiting example, the 2A peptides used in the cassette are all P2A sequences, which are used in conjunction with codon divergence to eliminate any non-endogenous epitopes that would otherwise appear in the translation product); P = a protease cleavage site upstream of 2A, which removes the 2A tag from the upstream TCRβ protein (as a non-limiting example, the protease cleavage site can be a furin cleavage site); SS = signal sequence (as a non-limiting example, the protease cleavage site can be a human growth hormone signal sequence). The HR template of the NeoTCR expression gene cassette includes two flanking homology arms for direct insertion of the TCRα genomic locus targeted by the CRISPR Cas9 nuclease RNP and the TCRα guide RNA. These homology arms (LHA and RHA) flank the neoE-specific TCR sequence of the NeoTCR expression gene cassette. Although the protease cleavage site used in this example is a furin cleavage site, any suitable protease cleavage site known to those skilled in the art may be used. Similarly, although HGH is the signal sequence selected for this example, any signal sequence known to those skilled in the art may be selected and used based on the desired transport.

[0345] Once integrated into the genome ( Figure 1C ), the NeoTCR expression gene cassette is transcribed into a single messenger RNA from the endogenous TCRα promoter, which still includes a portion of the endogenous TCRα polypeptide from the individual's T cells ( Figure 1C During ribosomal polypeptide translation of this single NeoTCR messenger RNA, the NeoTCR sequence is disconnected from the endogenous, CRISPR-disrupted TCRα polypeptide by self-cleavage at the P2A peptide ( Figure 1C The encoded NeoTCRα and NeoTCRβ polypeptides are also disconnected from each other by cleavage by endogenous cellular human furin and a second self-cleaving P2A sequence motif contained in the NeoTCR expression gene cassette ( Figure 1C). NeoTCRα and NeoTCRβ polypeptides are each targeted to the endoplasmic reticulum by a signal leader sequence (derived from human growth hormone, HGH) for multimer assembly and transport of the NeoTCR protein complex to the T cell surface. The inclusion of a furin cleavage site facilitates the removal of the 2A sequence from the upstream TCRβ chain to reduce potential interference with TCRβ function. The inclusion of a gly-ser-gly linker before each 2A (not shown) further enhances the separation of the three polypeptides.

[0346] In addition, three repeated protein sequences are codon-diverged within the HR template to promote genome stability. The two P2A codons within the TCR gene box diverge relative to each other and the two HGH signal sequences diverge relative to each other to promote the stability of the introduced NeoTCR box sequence in the ex vivo engineered T cell genome. Similarly, the 5' end of the reintroduced TRAC exon 1 (vertical stripe) reduces the possibility of the entire box being lost over time by removing the intervening sequences of the two direct repeats.

[0347] In addition to NeoTCR products, this method can be used for any CD8 product.

[0348] In-Out PCR was used to confirm precise target integration of the NeoE TCR cassette. Agarose gel shows the results of PCR using primers specific for the integration cassette and site-generated products of the expected size for cells treated with nuclease and DNA template (KOKI and KOKIKO), demonstrating site-specific and precise integration.

[0349] In addition, targeted locus amplification (TLA) was used to confirm the specificity of targeted integration. Sequences surrounding the integration site were obtained by crosslinking, ligation, and using primers specific for the NeoTCR insert sequence. Reads mapped to the genome were binned at 10 kb intervals. Significant read depth was obtained only around the expected site (i.e., the integration site on chromosome 14), with no evidence of common off-target insertion sites.

[0350] Antibody staining of endogenous TCRs and peptide-HLA staining of neoTCRs showed that the modification resulted in high-frequency knock-in of NeoTCRs, with some TCR- cells and a small number of WT T cells remaining. In the absence of an exogenous promoter, neoTCR expression demonstrated knock-in. The modification was performed multiple times using the same neoTCR with similar results. Thus, efficient and consistent expression of NeoTCRs and knockout of endogenous TCRs were achieved in engineered T cells.

[0351] Example 2 Production of CD8 Product 1

[0352] T cell isolation and editing. CD4 and CD8 T cells were isolated from healthy donor PBMC using Miltenyi Prodigy or MiltenyiMACS separation columns according to the manufacturer's instructions. Positively selected CD4 and CD8 T cells (using Miltenyi antibodies and separation columns) were used fresh or cryopreserved in 1% human serum albumin (Gemini), 49% plasmalyte (Baxter), and 50% CS10 (Sigma). Cryopreserved cells were thawed, washed in TexMACS (Miltenyi) + 10% human AB serum (Valley Biomedical), and 2x10 per ml. 6 Cells were plated at a density of 10 cells / mL in TexMACS + 3% human AB serum (medium). One day after thawing, or immediately for fresh use, cells were washed and plated at 1.46 x 10 cells / mL. 6 The cells were re-seeded at a density of 10 cells / mL in culture medium + 12.5 ng / mL IL7 + 12.5 ng / mL IL15 + 1:17.5 by volume TransACT T cell activation reagent (all reagents from Miltenyi). After two days of activation, the cells were re-seeded with i) a plasmid for production of NeoTCR products (see, e.g., Figure 1B ) or ii) CD8 construct 1 (e.g., consisting of a CD8α coding sequence flanked by a P2A site upstream of the neoTCRβ and α sequences and a gRNA-Cas9 RNP targeting the TCRα and β sites; see, e.g., Figure 2A ) T cells were electroporated. An exemplary expression construct of CD8 construct 1 is shown in Fig.11A T cells were electroporated using a Lonza X-unit in a 100 μL cuvette and program EO-115. T cells were expanded in medium supplemented with 12.5 ng / mL IL7 + 12.5 ng / mL IL15. The supplemented medium was changed every 2-3 days until the end of the study 13 days after activation.

[0353] comPACT and compPACT-Dextramer preparation. Neoantigen-specific peptide-HLA complex polypeptides (each "comPACT") were prepared according to the methods described in PCT / US2019 / 025415 (incorporated herein by reference in its entirety). The compPACT-dextramer complex was prepared for labeling T cells expressing neoTCR. The biotinylated compPACT protein was incubated with a streptavidin-coupled fluorophore at room temperature (RT) for 10 minutes. Biotin-40-dextran (NANOCS) was added to the mixture and incubated for another 10 minutes at room temperature. The comPACT-Dextramer was stored at 4°C.

[0354] Confirmation of compPACT binding to neoTCR-edited T cells. T cells were stained for flow cytometry. Cells were first stained with viability dye for 20 min at 4°C, then washed and stained with compPACT-dextramer for 10 min at 4°C. Surface antibodies (anti-CD8α, anti-CD8β, anti-CD4) were added to the suspension of cells and compPACT-dextramer, and the cells were incubated for an additional 20 min at 4°C. The cells were then washed and fixed in intracellular fixation buffer (BD Biosciences). All cells were acquired on an Attune NxT flow cytometer (ThermoFisher Scientific), and data were analyzed using FCSExpress or FlowJo.

[0355] Cytometric bead array (CBA). Streptavidin-coated plates (Eagle Biosciences) were washed three times with wash buffer (PBS supplemented with 1% BSA and 0.05% tween20) and then coated with compPACT at different concentrations from 100 to 0.01 ng / well. Wells without compPACT and wells coated with mismatched compPACT were used as controls. The plates were incubated at room temperature for 2 hours, washed three times with wash buffer, and then washed three times with TexMACS supplemented with 3% human AB serum to remove tween20. T cells were washed twice with TexMACS supplemented with 3% human AB serum and resuspended at 1 million cells / mL in TexMACS supplemented with 3% human AB serum and 1× penicillin-streptomycin solution. T cells were seeded onto compACT-coated plates at 100 μL / well and incubated at 37°C, 5% CO 2 . Supernatants were collected after 24 hours and analyzed for cytokine concentrations using the BD Cytometric Bead Array (CBA) Human Th1 / Th2 Cytokine Kit II (Cat. No. 551809) following the manufacturer's protocol. Capture beads were mixed with culture supernatants, incubated with detection reagents for 3 hours at room temperature in the dark, washed, and resuspended in wash buffer. Samples were analyzed on an Attune NxT flow cytometer and data were analyzed using FlowJo. EC50 represents the concentration of the cognate comPACT that elicits 50% of the maximal response and was calculated using a least squares fit of IFNγ secretion over a range of comPACT concentrations.

[0356] Intracellular staining. T cells were stained for flow cytometry on the specified days. T cells were first stained with viability dye for 20 minutes at 4°C, then washed and incubated with surface antibodies (anti-CD8α, anti-CD8β, anti-CD4) for another 20 minutes at 4°C. T cells were then washed and permeabilized for intracellular staining. T cells were stained with anti-2A peptide or anti-IFNγ, anti-TNF or anti-IL2 in permeabilization buffer at 4°C for 20 minutes. T cells were fixed in intracellular fixation buffer (BD Biosciences). Samples were analyzed on an Attune NxT flow cytometer (ThermoFisher Scientific) and data were analyzed using FCSExpress or FlowJo.

[0357] T cell proliferation assay. Edited CD4 and CD8 T cells were labeled with e450 proliferation dye (eBioscience) according to the manufacturer's instructions. Labeled cells were stimulated on compACT-coated plates at a concentration range as described above. T cells were harvested within 48-96 hours and proliferation was analyzed by diluting the e450 dye.

[0358] T cell killing assay. HLA-matched cell lines were incubated with cognate neoantigen peptides or mismatched peptides at 37°C and 5% CO 2 The cells were then pulsed for 1 hour. The cells were washed 3 times with culture medium to remove any unbound peptide and then co-cultured with edited CD4 and CD8 T cells labeled with the e450 proliferation dye described above. The co-cultures were incubated at 37°C, 5% CO before harvesting. 2 Incubate for 48 hours at 4 °C. Cells were washed and stained with a fixable viability dye to determine killing efficiency. E450 proliferation dye was used to distinguish edited T cells from target cells.

[0359] Generation and validation of CD8 product 1. An expression construct consisting of the coding sequence (CDS) of CD8α flanked by P2A sites upstream of the neoTCRβ and α sequences was synthesized. Briefly, the CDS of human CD8α was synthesized with a GSG linker and the upstream P2A site flanked by restriction sites. The target neoTCR expression vector and the synthetic CD8α construct were incubated with restriction endonucleases and ligated together to produce the final HDR construct. CD8 construct 1 was electroporated with gRNA-Cas9 RNPs targeting the TCRα and β loci. Model neoTCRs known to bind dextramers in CD8 T cells but not in CD4 T cells (e.g., TCR097) were used to demonstrate that expression of the CD8α transgene enabled these TCRs to bind dextramers in CD4 T cells.

[0360] To test the efficiency of CD8α gene transfer and expression on the CD4 T cell surface, engineered CD4 T cells were stained with anti-CD8α antibodies and surface expression of the transgene was confirmed by flow cytometry as described above. NeoTCRs (e.g., TCR 097) known to bind dextramers only in CD8 T cells were used to demonstrate that expression of the CD8 transgene enabled these TCRs to bind dextramers in CD4 T cells. Fig.13A , 13B , 14, 15A, and 15B. In addition, CD8α expression on wild-type and genetically engineered CD8α T cells was assessed to determine whether the addition of the CD8α transgene increased the surface level of CD8α on CD8 T cells. CD4 T cells engineered to express the CD8α transgene were double positive for CD4 and CD8α.

[0361] CD8 T cells were also engineered to express the CD8α transgene and characterized as described above. Fig.15A and 15B Relative CD8α gene expression was also quantified by RT-qPCR and compared to control non-engineered CD8 T cells. CD8 T cells expressing the CD8α transgene expressed CD8α at levels above endogenous levels.

[0362] Effect of CD8 expression on T cell proliferation after encountering cognate antigen. Edited T cells were stained with proliferation dye as described above. After staining, T cells were stimulated with a series of concentrations of cognate comPACT protein. After 48-72 hours, T cells were harvested and stained with anti-CD4, anti-CD8, and anti-2A peptide as described above. Edited T cells were identified by 2A expression. Proliferation of CD4 and CD8 T cells was determined by quantifying the dilution of proliferation dye. NeoTCR-expressing T cells lacking the CD8α transgene were used as a negative control. NeoTCR CD4 T cells expressing the CD8α transgene proliferated in response to lower concentrations of cognate comPACT compared to CD4 T cells lacking CD8α expression.

[0363] Effect of CD8α expression on cytokine production upon encounter with cognate antigen. In addition to proliferation, effector cytokine production was measured by intracellular cytokine staining. In this assay, NeoTCR products and CD8 product 1 were stimulated with varying concentrations of cognate comPACT in the presence of brefeldin A for 5 h. After stimulation, T cells were stained with anti-CD4 and anti-CD8α. Cells were permeabilized and stained with anti-P2A peptide, anti-IFNγ, anti-TNF, and anti-IL2. NeoTCR CD4 T cells expressing the CD8α transgene produced effector cytokines in response to lower concentrations of cognate comPACT compared to NeoTCR CD4 T cells lacking CD8α expression.

[0364] Effect of CD8α expression on killing activity when encountering cognate antigens. To assess effector function, edited CD4 and CD8 T cells were cultured with HLA-matched target cells pulsed with the above cognate peptides. CD4 and CD8 T cells were edited separately to assess the ability of CD8α-expressing CD4 T cells to kill target cells. NeoTCR CD4 T cells expressing the CD8α transgene killed more target cells presenting cognate peptides than neoTCR CD4 T cells lacking CD8α expression.

[0365] Example 3 Production of CD8 Product 2

[0366] CD4 T cells engineered to express CD8α lack CD8β expression. However, CD8β has a higher affinity for LCK (Irie et al., 1998, J. Immunol, 161(1), 183–191). Therefore, T cells were edited to co-express CD8β and CD8α (i.e., CD8 product 2). Additional constructs were generated containing CD8β flanked by P2A sites, CD8α flanked by P2A sites, and the TRB and TRA alleles described above. CD4 and CD8 T cells expressing CD8α and CD8β were evaluated using the same assay described above. Fig. 11B Exemplary expression constructs with sequences for CD8α and CD8β are shown.

[0367] CD8β expression was also assessed in edited CD4 T cells. NeoTCR CD4 T cells expressing both CD8α and CD8β transgenes proliferated in response to lower concentrations of cognate comPACT compared with CD4 T cells expressing only the CD8α transgene. NeoTCR CD4 T cells expressing both CD8α and CD8β transgenes also produced effector cytokines in response to lower concentrations of cognate comPACT compared with NeoTCR CD4 T cells expressing only the CD8α transgene. NeoTCR CD4 T cells expressing both CD8α and CD8β transgenes killed more target cells presenting cognate peptides compared with neoTCRCD4 T cells expressing only the CD8α transgene.

[0368] Example 4 Generation of Chimeric CD8α and CD8β Constructs and CD8 Products 3 and 4

[0369] To ensure efficient editing of T cells and expression of the neoTCR, a chimeric protein consisting of the coding sequence of the extracellular and transmembrane domains of CD8α linked to the intracellular domain of CD8β was generated. An exemplary expression construct with the extracellular and transmembrane domains of CD8α linked to the intracellular domain of CD8β sequence (i.e., CD8 product 3) is shown in Fig. 11C Shown in.

[0370] Furthermore, the intracellular domain of CD4 has an even higher affinity for LCK than CD8 (Irie et al., 1998). Therefore, a second chimeric protein was generated that contained the coding sequence for the CD8α extracellular and transmembrane domains linked to the CD4 intracellular domain. Fig.11D An exemplary expression construct is shown in which the extracellular and transmembrane domains of CD8α are linked to the intracellular domain of the CD4 sequence (ie, CD8 product 4).

[0371] CD4 and CD8 T cells expressing CD8 products 3 and 4 were assessed using the same assay described above.

[0372] NeoTCR CD4 T cells expressing the CD8α-CD8β-ID transgene proliferated in response to lower concentrations of cognate comPACT compared with CD4 T cells expressing the CD8α transgene alone. NeoTCR CD4 T cells expressing the CD8α-CD8β-ID transgene produced effector cytokines in response to lower concentrations of cognate comPACT compared with NeoTCR CD4 T cells expressing the CD8α transgene alone. NeoTCR CD4 T cells expressing the CD8α-CD8β-ID transgene killed more target cells presenting the cognate peptide than neoTCR CD4 T cells expressing the CD8α-CD8β-ID transgene.

[0373] NeoTCR CD4 T cells expressing the CD8α-CD4-ID transgene proliferated in response to lower concentrations of cognate comPACT compared with neoTCR CD4 T cells expressing the CD8α-CD8β-ID transgene. NeoTCR CD4 T cells expressing the CD8α-CD4 transgene produced effector cytokines in response to lower concentrations of cognate comPACT compared with NeoTCR CD4 T cells expressing the CD8α-CD8β-ID transgene.

[0374] NeoTCR CD4 T cells expressing the CD8α-CD4-ID transgene killed more target cells presenting cognate peptides than neoTCR CD4 T cells expressing the CD8α-CD8β-ID transgene. NeoTCR CD8 T cells expressing the CD8α-CD4-ID transgene proliferated in response to lower concentrations of cognate comPACT than neoTCR CD8 T cells lacking the transgene. NeoTCR CD8 T cells expressing the CD8α-CD4 transgene produced effector cytokines in response to lower concentrations of cognate comPACT than neoTCR CD8 T cells lacking the transgene. NeoTCR CD8 T cells expressing the CD8α-CD4 transgene killed more target cells presenting cognate peptides than neoTCR CD8 T cells lacking the transgene.

[0375] Example 5 CD8 production increases sensitivity to neoE-HLA target recognition and triggers pro-inflammatory and cytotoxic functions

[0376] The MHC-I neoTCR was cloned from neoE-specific T cells captured from the blood of colorectal cancer patients. Healthy donor CD8 and CD4 T cells were precisely genomically engineered to express the cloned MHC-I neoTCR alone or to include an ectopic CD8 coreceptor in the gene-edited T cells. Flow cytometric analysis was used to assess surface expression of the neoTCR and ectopic CD8 coreceptors (i.e., the CD8 and CD4 components of CD8 constructs 1-4), respectively. Rescuing the low affinity of NeoTCR binding in CD4 T cells, a CD8-dependent NeoTCR was observed. Importantly, in response to stimulation with cognate antigens, CD107a and intracellular IFNγ staining revealed a 10- to 100-fold increase in the sensitivity of CD4 T cells to MHC-I neoTCR-induced effector function without affecting specificity. No changes in function or sensitivity were observed on CD8 T cells by expressing additional CD8 coreceptors.

[0377] These results demonstrate that precise genomic engineering of both the CD8 coreceptor and the CD8-dependent MHC-1neoTCR into CD4 T cells (i.e., CD8 products 1–4) significantly increases their sensitivity to neoE-HLA target recognition and triggering of proinflammatory and cytotoxic functions without compromising antigen specificity.

[0378] Example 6 Generation and design of CD8 products with different LCK affinities

[0379] As described herein, four classes of CD8 products were generated:

[0380] 1. CD8α homodimer (CD8 construct 1)

[0381] 2.CD8α-P2A-CD8β (CD8 construct 2)

[0382] 3. CD8α and CD8β intracellular domains (CD8 construct 3)

[0383] 4.CD8α homodimer with CD4 intracellular domain (CD8 construct 4)

[0384] like Fig.10 As shown, these CD8 constructs and the resulting CD8 products were designed to allow for different degrees of LCK affinity. As predicted, CD8 product 1 was shown to have the lowest LCK affinity, followed by CD8 product 2, CD8 product 3, and CD8 product 4 (in this order, CD8 product 4 had the highest LCK affinity).

[0385] Based on the high affinity of CD8 product 4, this product was used in a cell killing assay to demonstrate that CD8 products 1-4 have higher cell killing ability compared to NeoTCR products.

[0386] CD4+ T cells were engineered as described herein to express Figure 2D and 3D The CD8 product 4 described in (where TCR097 is used as the NeoTCR in the product). The SW620 cell line was modified to heterologously express the R20Q mutation (the cognate antigen of TCR097). The CD8 product 4 expressing NeoTCR097 was combined with SW620 heterologous cells. Fig.13A and 13B As shown, CD8 product 4 provided substantially better killing of SW620 cells expressing cognate antigens compared to the NeoTCR product that also expressed TCR097. Fig.13A The experiment shown in was performed at an E:T ratio of 1:1, and as shown, the NeoTCR product expressing TCR097 showed no efficacy in killing SW620 cells expressing the cognate antigen. Fig. 13B The experiment shown in was performed at an E:T ratio of 2:1, and while the NeoTCR product expressing TCR097 showed some ability to kill SW620 cells expressing cognate antigens, it is clear from the experiment that the CD8 product 4 expressing TCR097 has superior efficacy.

[0387] Use with Fig.13A and 13B The same engineered CD4+ cells used in the above experiments with the data presented in were also tested on a SW620 cell line engineered to homozygously express the R20Q mutation. In this experiment, high expression of cognate antigens was able to compensate for the low affinity NeoTCR097, and both the NeoTCR product expressing TCR097 and the CD8 product 4 expressing TCR097 showed efficacy in killing SW620 cells. However, high expression of cognate antigens in homozygous SW620 cells is physiologically irrelevant, and this experiment was designed to highlight the ability to rescue NeoTCR products with low affinity TCRs that are unable to effectively engage with tumor cells and kill them by further engineering them to also contain CD8α homodimers and the CD4 intracellular domain (i.e., CD8 product 4).

[0388] As a final control and demonstration of the efficacy of the CD8 product, CD8 T cells were also transfected to express NeoTCR097 and the CD8α homodimer and CD4 intracellular domain (i.e., CD8 T cells were used instead of CD4 T cells for the CD8 product 4). Fig.15A and 15B As shown in the above graph, CD8 product 4 provided significantly better killing of SW620 cells than the NeoTCR product when the products were made from CD4 T cells. However, when CD8 product 4 and the NeoTCR product were made from CD8 T cells ( Fig.15A and 15B The lower panel in Figure 2 shows that the ability of NeoTCR production is rescued by endogenous CD8 expression in CD8 T cells. Although the overlap is not shown, it should also be noted that Fig.15A and 15B The CD8 product 4 in CD4 T cells shown in the upper middle panel appears to have better efficacy than the CD8 product 4 in CD8 T cells in the lower two panels; this suggests that the CD8 products described herein have a superior ability to engage with cognate antigens of tumor cells, thereby leading to tumor death and effectively treating cancer patients in need of treatment.

[0389] Example 6 CD8 product increases the sensitivity of CD4 T cells while maintaining NeoTCR sensitivity

[0390] To confirm that the CD8 constructs were expressed correctly, CD8 products 1, 2, 3, and 4 were tested to determine the surface expression of CD8α. The results showed that CD8 products 1, 2, 3, and 4 all showed normal CD8α surface expression. Representative data for CD8 product 4 are shown in Figure 2. Fig.16 shown.

[0391] It will also be important to determine how the CD8 constructs affect the sensitivity and specificity of CD4 T cells to the NeoTCR cognate antigen expressed in the CD8 product. Sensitivity experiments were performed and the results showed that CD8 products 1-4 exhibited increased CD4 T cell sensitivity ( Fig.17A ). Specificity experiments were also performed on CD8 products 1-4. CD8 products 1, 2, 3 and 4 were made by NeoTCR097. Experiments were performed to test CD8 products 1, 2, 3 and 4 (expressing NeoTCR097) to evaluate the specificity of these products for NeoTCR097 cognate antigens. Fig. 17B As shown, CD8 products 1, 2, 3 and 4 (expressing NeoTCR097) are specific for the cognate antigen of NeoTCR097 and do not show activity when exposed to mismatched antigens. Specificity is determined by the production of INFγ and CD107, which are evidence of T cell activation. Therefore, compared to NeoTCR products expressing the same NeoTCR, the CD8 products described herein have increased sensitivity to CD4 T cells and maintain their specificity for the cognate antigen of the expressed NeoTCR.

[0392] Finally, experiments were conducted to study the effects of CD8 constructs on CD8-dependent and CD8-independent NeoTCRs. It is expected that CD8-dependent NeoTCRs will show increased sensitivity to cognate antigens because CD8 cells will be transformed to express CD8α, while CD8-independent NeoTCRs will not show increased sensitivity because of the independent nature of NeoTCRs. However, the results showed that NeoTCR products 1, 2, 3, and 4 surprisingly increased the sensitivity of CD8-dependent (e.g., NeoTCR097) and CD8-independent (e.g., NeoTCR089) NeoTCRs. Therefore, the results show that CD8 constructs 1, 2, 3, and 4 can improve the engagement of NeoTCRs and for all NeoTCRs, improve T cell killing of tumor cells with cognate NeoTCR antigens, whether they are CD8-dependent or CD8-independent.

[0393] While the present invention has been described at some length and with some specificity with respect to several described embodiments, it is not intended to be limited to any such details or embodiments or to any particular embodiment, but rather should be construed by reference to the appended claims so as to provide the broadest possible interpretation of such claims in light of the prior art and thereby effectively encompass the intended scope of the invention.

[0394] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail. In addition, section headings, materials, methods, and examples are illustrative only and not restrictive.

Claims

1. Peripheral T cells, comprising: a) exogenous T cell receptors (TCRs) that recognize neoantigens; and b) Exogenous CD8, which comprises the CD8α extracellular domain, the CD8α transmembrane domain and the CD4 intracellular domain.

2. The cell of claim 1, wherein the exogenous CD8 comprises at least one monomer.

3. The cell of claim 2, wherein the at least one monomer comprises a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain.

4. The cell of any one of claims 2-3, wherein the at least one monomer comprises a signal peptide.

5. The cell of claim 4, wherein the signal peptide is a CD8 signal peptide.

6. The cell of any one of claims 1-3, wherein the amino acid sequence of the CD8α extracellular domain is shown in SEQ ID NO:

140.

7. The cell of any one of claims 1-3, wherein the amino acid sequence of the CD8α transmembrane domain is shown as SEQ ID NO:

141.

8. The cell of any one of claims 1-3, wherein the amino acid sequence of the CD4 intracellular domain is shown as SEQ ID NO:

148.

9. The cell of claim 4, wherein the amino acid sequence of the signal peptide is shown in SEQ ID NO:

139.

10. The cell of any one of claims 1-3, wherein the exogenous CD8 comprises a 2A sequence.

11. The cell of any one of claims 1-3, wherein the exogenous CD8 comprises a linker.

12. The cell of claim 11, wherein the amino acid sequence of the linker is shown in SEQ ID NO:

137.

13. The cell of any one of claims 1-3, wherein the exogenous CD8 comprises a protease cleavage site.

14. The cell of claim 13, wherein the protease cleavage site is a furin cleavage site.

15. The cell of any one of claims 1-3, wherein the exogenous TCR is a patient-derived TCR.

16. The cell of any one of claims 1-3, wherein the exogenous TCR comprises a signal sequence, first and second 2A sequences, and a TCR polypeptide sequence.

17. The cell of any one of claims 1-3, wherein the cell is a primary cell.

18. The cell of any one of claims 1-3, wherein the cell is a patient-derived cell.

19. The cell of any one of claims 1-3, wherein the cell is a young T cell.

20. The cell of claim 19, wherein the cell is CD45RA+, CD62L+, CD28+, CD95-, CCR7+, and CD27+.

21. The cell of claim 19, wherein the cell is CD45RA+, CD62L+, CD28+, CD95+, CD27+, CCR7+.

22. The cell of claim 19, wherein the cell is CD45RO+, CD62L+, CD28+, CD95+, CCR7+, CD27+, CD127+.

23. The cell of any one of claims 1-3, further comprising a genetic modification to enhance cell persistence and / or enhance memory cell differentiation.

24. The cell of any one of claims 1-3, wherein the killing activity of the cell is increased by 10% to 500% compared to the killing activity of a cell without exogenous CD8.

25. The cell of any one of claims 1-3, wherein proliferation of the cell is increased by 10% to 500% following TCR binding to an antigen compared to proliferation of a cell without exogenous CD8.

26. The cell of any one of claims 1-3, wherein secretion of pro-inflammatory cytokines by the cell is increased by 10% to 500% upon TCR binding to an antigen compared to secretion by a cell without exogenous CD8.

27. The cell of any one of claims 1-3, wherein the LCK affinity of the cell is increased by 10% to 500% compared to the LCK affinity of a cell without exogenous CD8.

28. The cell of any one of claims 1-3, wherein the persistence of the cell is increased by 10% to 500% compared to the persistence of a cell without exogenous CD8.

29. The cell of any one of claims 1-3, wherein the tumor infiltrating ability of the cell is increased by 10% to 500% compared to the tumor infiltrating ability of a cell without exogenous CD8.

30. The cell of any one of claims 1-3, wherein the exogenous TCR is a CD8-dependent TCR.

31. The cell of any one of claims 1-3, wherein the exogenous TCR is a CD8-independent TCR.

32. The cell of any one of claims 1-3, wherein the exogenous CD8 is encoded by CD8 construct 4 set forth in SEQ ID NO:

136.

33. The cell of claim 1, wherein the exogenous CD8 comprises a CD8α signal peptide, a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain.

34. The cell of claim 1, wherein the exogenous CD8 comprises a CD8α signal peptide as shown in SEQ ID NO: 139, a CD8α extracellular domain as shown in SEQ ID NO: 140, a CD8α transmembrane domain as shown in SEQ ID NO: 141, and a CD4 intracellular domain as shown in SEQ ID NO:

148.

35. A method for modifying peripheral T cells, the method comprising: a) introducing a homologous recombination (HR) template nucleic acid sequence into a cell, wherein the HR template comprises: i) first and second homology arms homologous to the first and second target nucleic acid sequences; ii) a TCR gene sequence encoding an exogenous TCR that recognizes a new antigen, located between the first and second homology arms; iii) a CD8 gene sequence located between the first and second homology arms; wherein the CD8 gene sequence comprises a CD8α extracellular domain, a CD8α transmembrane domain and a CD4 intracellular domain; and b) Recombination of the HR template nucleic acid into the endogenous locus of the cell.

36. The method of claim 35, wherein the HR template comprises a first 2A coding sequence located upstream of a CD8 gene sequence, a second 2A coding sequence located downstream of the CD8 gene sequence and upstream of a TCR gene sequence, and a third 2A coding sequence located downstream of the TCR gene sequence; wherein the first, second, and third 2A coding sequences encode the same amino acid sequence and have codon divergences relative to each other.

37. The method of claim 35 or 36, wherein the HR template comprises a sequence encoding the amino acid sequence Gly Ser Gly immediately upstream of the first, second and / or third 2A coding sequence.

38. The method of claim 36, wherein the HR template further comprises a sequence encoding a furin cleavage site upstream of the first, second and / or third 2A coding sequence.

39. The method of any one of claims 35-36, wherein the HR template further comprises a sequence encoding a signal sequence located immediately upstream of the TCR gene sequence and / or the CD8 gene sequence.

40. The method of any one of claims 35-36, wherein the HR template comprises a second TCR sequence positioned between a third 2A coding sequence and a second homology arm.

41. The method of claim 40, wherein the HR template comprises: a. a sequence encoding a first signal sequence located immediately upstream of the first TCR gene sequence; and b. A sequence encoding a second signal sequence located immediately upstream of the second TCR gene sequence.

42. The method of any one of claims 35-36, wherein the HR template comprises a second CD8 gene sequence located between the first CD8 gene sequence and the second 2A coding sequence.

43. The method of claim 42, wherein the 2A coding sequence is located between the first and second CD8 gene sequences.

44. The method of claim 42, wherein the sequence encoding the amino acid sequence Gly Ser Gly is located between the first and second CD8 gene sequences.

45. The method of claim 42, wherein the sequence encoding the furin cleavage site is located between the first and second CD8 gene sequences.

46. ​​The method of claim 42, wherein the HR template comprises: a. a sequence encoding a first signal sequence located immediately upstream of the first CD8 gene sequence; and b. A sequence encoding a second signal sequence located immediately upstream of the second CD8 gene sequence.

47. The method of claim 46, wherein the signal sequence is a CD8 signal sequence, a human growth hormone signal sequence, a fragment thereof, or a combination thereof.

48. The method of any one of claims 35-36, wherein the first and second homology arms of the HR template are each 300 bases to 2,000 bases in length.

49. The method of any one of claims 35-36, wherein the first and second homology arms of the HR template are each 600 bases to 2,000 bases in length.

50. The method of any one of claims 35-36, wherein the exogenous TCR is a patient-derived TCR.

51. The method of any of claims 35-36, wherein the exogenous TCR comprises a signal sequence, a first and a second 2A sequence, and a TCR polypeptide sequence.

52. The method of any one of claims 35-36, wherein the HR template is non-viral.

53. The method of any one of claims 35-36, wherein the HR template is circular DNA.

54. The method of any one of claims 35-36, wherein the HR template is linear DNA.

55. The method of any one of claims 35-36, wherein introducing is performed by electroporation. E 56. The method of any one of E 35-36, wherein said recombination comprises: a. cleavage of the endogenous locus by nucleases; and b. Recombining the HR template nucleic acid sequence into the endogenous locus by homology directed repair.

57. The method of claim 56, wherein the nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR) family nuclease or a derivative thereof.

58. The method of claim 57, further comprising a gRNA.

59. The method of any one of claims 35-36, wherein the method further comprises culturing the cells.

60. The method of claim 59, wherein the culturing is performed in the presence of at least one cytokine.

61. The method of claim 59, wherein the culturing is performed in the presence of IL2, IL7, IL15, or any combination thereof.

62. The method of claim 59, wherein the culturing is performed in the presence of IL7 and IL15.

63. The method of any one of claims 35-36, further comprising genetic modification to enhance cell persistence and / or enhance memory cell differentiation.

64. The method of any one of claims 35-36, wherein the cell is a primary cell.

65. The method of any one of claims 35-36, wherein the cell is a patient-derived cell.

66. The method of any one of claims 35-36, wherein the cells are young T cells.

67. The cell of claim 66, wherein the cell is CD45RA+, CD62L+, CD28+, CD95-, CCR7+, and CD27+.

68. The cell of claim 66, wherein the cell is CD45RA+, CD62L+, CD28+, CD95+, CD27+, CCR7+.

69. The cell of claim 66, wherein the cell is CD45RO+, CD62L+, CD28+, CD95+, CCR7+, CD27+, CD127+.

70. The method of any one of claims 35-36, wherein the killing activity of the cells is increased by 10% to 500% compared to the killing activity of cells not having the CD8 gene sequence.

71. The method of any one of claims 35-36, wherein proliferation of the cells upon TCR binding to the antigen is increased by 10% to 500% compared to proliferation of cells without the CD8 gene sequence.

72. The method of any one of claims 35-36, wherein secretion of pro-inflammatory cytokines by the cell is increased by 10% to 500% when the TCR is bound to the antigen, compared to secretion by cells that do not have the CD8 gene sequence.

73. The method of any one of claims 35-36, wherein the LCK affinity of the cell is increased by 10% to 500% compared to the LCK affinity of a cell that does not have the CD8 gene sequence.

74. The method of any one of claims 35-36, wherein the persistence of the cells is increased by 10% to 500% compared to the persistence of cells without the CD8 gene sequence.

75. The method of any one of claims 35-36, wherein the tumor infiltrating ability of the cells is increased by 10% to 500% compared to the tumor infiltrating ability of cells not having the CD8 gene sequence.

76. The method of any one of claims 35-36, wherein the TCR gene encodes a CD8-dependent TCR.

77. The method of any one of claims 35-36, wherein the TCR gene encodes a CD8-independent TCR.

78. The method of any one of claims 35-36, wherein the CD8 gene sequence is encoded by CD8 construct 4 shown in SEQ ID NO:

136.

79. The method of claim 35, wherein the CD8 gene sequence encodes exogenous CD8 comprising the following: CD8α signal peptide, CD8α extracellular domain, CD8α transmembrane domain and CD4 intracellular domain.

80. The method of claim 38, wherein the CD8 gene sequence encodes an exogenous CD8 comprising the following: a CD8α signal peptide as shown in SEQ ID NO: 139, a CD8α extracellular domain as shown in SEQ ID NO: 140, a CD8α transmembrane domain as shown in SEQ ID NO: 141, and a CD4 intracellular domain as shown in SEQ ID NO:

148.

81. A cell modified by the method of any one of claims 35-80.

82. A composition comprising an effective amount of the cell of any one of claims 1-34 or the cell of claim 81.

83. The composition of claim 82, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

84. The composition of claim 82 or 83, wherein the composition is administered to a patient in need thereof to treat cancer.

85. The composition of any one of claims 82-83, wherein the composition comprises a cryopreservative.

86. The composition of any one of claims 82-83, wherein the composition comprises serum albumin.

87. The composition of any one of claims 82-83, wherein the composition comprises Plasma-Lyte A, HSA, and CryoStor CS10.

88. Use of the cell of any one of claims 1-34, the cell of claim 81, or the composition of any one of claims 82-87 in the preparation of a medicament for treating cancer in a subject in need thereof.

89. The use of claim 88, wherein the cancer is a solid tumor.

90. The use of claim 88, wherein the cancer is a liquid tumor.

91. The use of claim 89, wherein the solid tumor is selected from the group consisting of melanoma, breast cancer, lung cancer, ovarian cancer, breast cancer, pancreatic cancer, head and neck cancer, prostate cancer, gynecological cancer, central nervous system cancer, skin cancer, HPV+ cancer, esophageal cancer, thyroid cancer, gastric cancer, hepatocellular carcinoma, bile duct cancer, renal cell carcinoma, testicular cancer, sarcoma and colorectal cancer.

92. The use of claim 90, wherein the liquid tumor is selected from the group consisting of follicular lymphoma, leukemia, and multiple myeloma.

93. A kit comprising a cell of any one of claims 1-34, reagents for performing the method of any one of claims 35-80, a cell of claim 81, or a composition of any one of claims 82-87.

94. The kit of claim 93, wherein the kit further comprises written instructions for treating cancer.

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