Compositions and Methods for the Treatment of Cancer Using CD8-Modified T Cell Therapy

Abstract

Compositions and methods for the treatment of cancer using neoTCR-based cell therapy with CD8-expressing constructs. In certain embodiments, the presently disclosed subject matter provides cells comprising an exogenous T cell receptor (TCR) and exogenous CD8. In certain embodiments, the exogenous CD8 comprises at least one monomer. In certain embodiments, the 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.

Description

Technical Field

[0001] [Cross - Reference to Related Applications] This application claims priority based on 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 contents of which are incorporated herein by reference in their entirety.

[0002] [Sequence Listing] This application is electronically filed in ASCII format and includes a sequence listing that is incorporated herein by reference in its entirety. The ASCII copy created on April 30, 2020, is named 087520_0145_SL.txt and is 120,294 bytes in size.

Background Art

[0003] Activation of T cells requires signal transduction via the T cell receptor (TCR) and its coreceptor molecules. CD4 and CD8 are membrane proteins expressed on T helper cells and cytotoxic T lymphocytes that function as coreceptors to enhance TCR signal transduction by stabilizing the interaction between the peptide-major histocompatibility antigen (pMHC) ligand and the TCR (Li QJ et al. (2004) CD4 enhances T cell sensitivity to antigen by coordinating Lck accumulation at the immunological synapse. Nat Immunol 5:791-799; Holler PD, Kranz DM (2003) Quantitative analysis of the contribution of TCR / pepMHC affinity and CD8 to T cell activation. Immunity 18:255-264). Specifically, the CD4 and CD8 coreceptors are essential for the initiation of signal transduction as they facilitate the recruitment of kinases to the TCR-pMHC complex. Furthermore, studies have shown that both the CD4 and CD8 coreceptors enhance the sensitivity of T cells to their ligands, but only CD8 plays a role in stabilizing the TCR-pMHC interaction.

[0004] Furthermore, naturally occurring MHC-I TCRs are presumed to require the concurrent CD8 coreceptor to assist in stabilizing peptide-MHC binding, while high-affinity TCRs drive CD8-independent target binding and T cell activation. Thus, CD4 T cells, when modified with high-affinity NeoTCRs, can recognize peptide-MHC-I targets and trigger effector T cell function. However, low-affinity TCRs are dependent on the CD8 coreceptor to trigger T cell activation.

[0005] Thus, NeoTCR cell therapies modified to express the CD8 coreceptor can stabilize the TCR-pMHC interaction and enhance the efficacy of NeoTCR cell therapies that include low-affinity TCRs that are dependent on such CD8 coreceptors.

SUMMARY OF THE INVENTION

[0006] In certain embodiments, the subject matter of the present disclosure provides cells comprising an exogenous T cell receptor (TCR) and an exogenous CD8. In certain embodiments, the exogenous CD8 comprises at least one monomer. In certain embodiments, at least one monomer of the exogenous CD8 comprises an extracellular domain, a transmembrane domain, an intracellular domain, fragments thereof, or combinations thereof. In certain embodiments, the extracellular domain comprises a CD8α extracellular domain or a CD8β extracellular domain. In certain embodiments, the transmembrane is a CD8α transmembrane domain or a CD8β transmembrane domain. In certain embodiments, the intracellular domain comprises a CD8α intracellular domain or a CD8β intracellular domain. In certain embodiments, the intracellular domain comprises a CD4 intracellular domain.

[0007] In certain embodiments, at least one monomer comprises a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8α intracellular domain. In certain embodiments, at least one monomer comprises a CD8β extracellular domain, a CD8β transmembrane domain, and a CD8β intracellular domain. In certain embodiments, at least one monomer comprises a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8β intracellular domain. In certain embodiments, at least one monomer comprises a CD8α extracellular domain, a CD8α 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.

[0008] 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. 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.

[0009] In certain embodiments, the exogenous TCR is a patient-derived TCR. In certain embodiments, the exogenous TCR comprises a signal sequence, a first 2A sequence 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. 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 naive 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+.

[0010] In certain embodiments, the cells further comprise genetic modifications to enhance cell persistence and / or enhance memory cell differentiation. In certain embodiments, the killing activity of the cells increases between about 10% and about 500% compared to the killing activity of cells without exogenous CD8. In certain embodiments, the proliferation of the cells upon binding of the TCR to the antigen increases between about 10% and about 500% compared to the proliferation of cells without exogenous CD8. In certain embodiments, the secretion of pro-inflammatory cytokines upon binding of the TCR to the antigen by the cells increases between about 10% and about 500% compared to the secretion by cells without exogenous CD8. In certain embodiments, the LCK affinity of the cells increases between about 10% and about 500% compared to the LCK affinity of cells without exogenous CD8. In certain embodiments, the persistence of the cells increases between about 10% and about 500% compared to the persistence of cells without exogenous CD8. In certain embodiments, the tumor infiltration ability of the cells increases between about 10% and about 500% compared to the tumor infiltration ability of cells without exogenous CD8.

[0011] In certain embodiments, the exogenous TCR is a CD8-dependent TCR. In certain embodiments, the exogenous TCR is a CD8-independent TCR. In certain embodiments, the exogenous CD8 is encoded by CD8 construct 1, CD8 construct 2, CD8 construct 3, or CD8 construct 4. In certain embodiments, 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. 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), and a CD8β intracellular domain (SEQ ID NO: 147); or 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 CD4 intracellular domain (SEQ ID NO: 148).

[0012] In certain embodiments, the subject matter of the present disclosure provides a method of modifying a cell, the method comprising introducing into the cell a homologous recombination (HR) template nucleic acid sequence, wherein the HR template comprises a first homology arm and a second homology arm homologous to a first target nucleic acid sequence and a second target nucleic acid sequence, a TCR gene sequence located between the first homology arm and the second homology arm, and a CD8 gene sequence located between the first homology arm and the second homology arm; and recombining the HR template nucleic acid into an 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 have different codons from each other. In certain embodiments, the HR template comprises a sequence encoding the amino acid sequence Gly Ser Gly located immediately upstream of the first, second, and / or third 2A coding sequence. In certain embodiments, the HR template further 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.

[0013] In certain embodiments, the HR template includes a second TCR sequence located between a third 2A coding sequence and a second homology arm. In certain embodiments, the HR template includes 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 includes 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 CD8 gene sequence and the second CD8 gene sequence. In certain embodiments, the sequence encoding the amino acid sequence Gly Ser Gly is located between the first CD8 gene sequence and the second CD8 gene sequence. In certain embodiments, the sequence encoding a furin cleavage site is located between the first CD8 gene sequence and the second CD8 gene sequence.

[0014] In certain embodiments, the CD8 gene sequence includes a sequence encoding an extracellular domain, a sequence encoding an intracellular domain, a sequence encoding an intracellular domain, fragments thereof, or combinations thereof. In certain embodiments, the sequence encoding the extracellular domain includes a sequence encoding a CD8α extracellular domain or a CD8β extracellular domain. In certain embodiments, the sequence encoding the transmembrane domain includes a sequence encoding a CD8α transmembrane domain or a CD8β transmembrane domain. In certain embodiments, the sequence encoding the intracellular domain includes a sequence encoding a CD8α intracellular domain or a CD8β intracellular domain. In certain embodiments, the sequence encoding the intracellular domain includes a sequence encoding a CD4 intracellular domain.

[0015] In certain embodiments, the CD8 gene sequence includes sequences encoding the CD8α extracellular domain, the CD8α transmembrane domain, and the CD8α intracellular domain. In certain embodiments, the CD8 gene sequence includes sequences encoding the CD8β extracellular domain, the CD8β transmembrane domain, and the CD8β intracellular domain. In certain embodiments, the CD8 gene sequence includes sequences encoding the CD8α extracellular domain, the CD8α transmembrane domain, and the CD8β intracellular domain. In certain embodiments, the CD8 gene sequence includes sequences encoding the CD8α extracellular domain, the CD8α transmembrane domain, and the CD4 intracellular domain.

[0016] In certain embodiments, the HR template includes 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 homology arm and the second homology arm of the HR template are each about 300 bases to about 2000 bases in length. In certain embodiments, the first homology arm and the second homology arm of the HR template are each about 600 bases to about 2000 bases in length.

[0017] In certain embodiments, the exogenous TCR is a patient-derived TCR. In certain embodiments, the exogenous TCR includes a signal sequence, a first 2A sequence 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. In certain embodiments, the HR template is non-viral. In certain embodiments, the HR template is circular DNA. In certain embodiments, the HR template is linear DNA. In certain embodiments, the introduction occurs via electroporation.

[0018] In certain embodiments, recombination involves cleavage of an endogenous locus by a nuclease and recombination of an 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 Repeats (CRISPR) family nuclease, or a derivative thereof. In certain embodiments, the nuclease further comprises a gRNA.

[0019] In certain embodiments, the method further comprises culturing the cells. In certain embodiments, the culturing is performed in the presence of at least one cytokine. In certain embodiments, the culturing is performed in the presence of IL2, IL7, IL15, or any combination thereof. In certain embodiments, the culturing is performed in the presence of IL7 and IL15. In certain embodiments, the method comprises genetic modifications that enhance cell persistence and / or enhance memory cell differentiation.

[0020] In certain embodiments, the cells are primary cells. In certain embodiments, the cells are patient-derived cells. In certain embodiments, the cells are lymphocytes. In certain embodiments, the cells are T cells. In certain embodiments, the cells are naive T cells. In certain embodiments, the cells are CD45RA+, CD62L+, CD28+, CD95-, CCR7+, and CD27+. In certain embodiments, the cells are CD45RA+, CD62L+, CD28+, CD95+, CD27+, CCR7+. In certain embodiments, the cells are CD45RO+, CD62L+, CD28+, CD95+, CCR7+, CD27+, CD127+.

[0021] In certain embodiments, the killing activity of the cells increases between about 10% and about 500% compared to the killing activity of cells that do not have the CD8 gene sequence. In certain embodiments, the proliferation of the cells upon binding of the TCR to the antigen increases between about 10% and about 500% compared to the proliferation of cells that do not have the CD8 gene sequence. In certain embodiments, the secretion of pro-inflammatory cytokines upon binding of the TCR to the antigen by the cells increases between about 10% and about 500% compared to the secretion by cells that do not have the CD8 gene sequence. In certain embodiments, the LCK affinity of the cells increases between about 10% and about 500% compared to the LCK affinity of cells that do not have the CD8 gene sequence. The method of any one of claims 43-95, wherein the persistence of the cells increases between about 10% and about 500% compared to the persistence of cells that do not have the CD8 gene sequence. In certain embodiments, the tumor infiltration ability of the cells increases between about 10% and about 500% compared to the tumor infiltration ability of cells that do not have the CD8 gene sequence.

[0022] In certain embodiments, the TCR gene encodes a CD8-dependent TCR. In certain embodiments, the TCR gene encodes a 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, 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, and a CD8β intracellular domain; or a CD8α extracellular domain, a CD8α transmembrane domain, and a 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), and a CD8β intracellular domain (SEQ ID NO: 147); or 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 CD4 intracellular domain (SEQ ID NO: 148).

[0023] In certain embodiments, the subject matter of the present disclosure provides a cell modified by any of the methods disclosed herein. In certain embodiments, the subject matter of the present disclosure provides a composition comprising an effective amount of the cells disclosed herein. In certain embodiments, the composition is a pharmaceutical composition further comprising pharmaceutically acceptable additives. In certain embodiments, the composition is administered to a patient in need of treatment for the treatment of 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.

[0024] In certain embodiments, the subject matter of the present disclosure provides a method of treating cancer in a subject in need of treatment, the method comprising administering a therapeutically effective amount of the cells or composition disclosed herein. In certain embodiments, a non-myeloablative lymphocyte depletion regimen is administered to the subject prior to administering a therapeutically effective amount of the cells. 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, thoracic 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 cancer, cholangiocarcinoma, renal cell cancer, 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.

[0025] In certain embodiments, the subject matter of the present disclosure provides a kit comprising the cells disclosed herein, a reagent for performing the methods disclosed herein, or the composition disclosed herein. In certain embodiments, the kit further comprises written instructions for treating cancer.

[0026] In certain embodiments, the subject matter of the present disclosure provides a cell comprising an exogenous T cell receptor (TCR); and an exogenous CD8 comprising 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.

[0027] In certain embodiments, the subject matter of the present disclosure provides a cell comprising an exogenous T cell receptor (TCR); and an 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), and a CD8β intracellular domain (SEQ ID NO: 147); or 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 CD4 intracellular domain (SEQ ID NO: 148).

[0028] In certain embodiments, the subject matter of the present disclosure provides a method of modifying a cell, the method comprising introducing into the cell a homologous recombination (HR) template nucleic acid sequence, wherein the HR template comprises a first homology arm and a second homology arm homologous to a first target nucleic acid sequence and a second target nucleic acid sequence; a TCR gene sequence located between the first homology arm and the second homology arm; a CD8 gene sequence located between the first homology arm and the second homology arm; and 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; or a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain.

[0029] In certain embodiments, the subject matter of the present disclosure provides a method of modifying a cell, the method comprising introducing into the cell a homologous recombination (HR) template nucleic acid sequence, wherein the HR template comprises a first homology arm and a second homology arm homologous to a first target nucleic acid sequence and a second target nucleic acid sequence, respectively; a TCR gene sequence positioned between the first homology arm and the second homology arm; and a CD8 gene sequence positioned between the first homology arm and the second homology arm, and 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), 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), and a CD8β intracellular domain (SEQ ID NO: 147); or 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 CD4 intracellular domain (SEQ ID NO: 148).

[0030] In certain embodiments, the subject matter of the present disclosure provides a composition comprising a cell, wherein the cell comprises an exogenous T cell receptor (TCR) and an 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.

[0031] In certain embodiments, the subject matter of the present disclosure provides a composition comprising a cell, wherein the cell comprises an exogenous T cell receptor (TCR) and an 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), and a CD8β intracellular domain (SEQ ID NO: 147); or 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 CD4 intracellular domain (SEQ ID NO: 148).

[0032] In certain embodiments, the subject matter of the present disclosure 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 an 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.

[0033] In certain embodiments, the subject matter of the present disclosure 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 an 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), and a CD8β intracellular domain (SEQ ID NO: 147); or 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 CD4 intracellular domain (SEQ ID NO: 148). BRIEF DESCRIPTION OF THE DRAWINGS

[0034]

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Mode for Carrying Out the Invention

[0035] [Definition] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art. The following references provide one of ordinary skill in the art with many general definitions of terms used in the subject matter of this disclosure: 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 and Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meaning ascribed to them below unless specifically stated otherwise.

[0036] It is understood that the aspects and embodiments of the invention described herein include aspects and embodiments of "comprising", "consisting of", and "consisting essentially of". The terms "comprises" and "comprising" are intended to have the broad meaning ascribed to them in the United States Patent Laws and may mean "includes", "including", etc.

[0037] As used herein, the term "about" or "approximately" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art, and depends in part on how the value is measured or determined, i.e., on the limitations of the measurement system. For example, "about" can mean within three or more standard deviations according to the practice in the art. Alternatively, "about" can mean within a range of up to 20% of a given value, e.g., up to 10%, up to 5%, or up to 1%. Alternatively, especially with respect to biological systems or processes, this term can mean within one order of magnitude of the value, e.g., within five or two times the value.

[0038] As used herein, the term "antibody" is used in the broadest sense and includes, without limitation, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and trispecific antibodies), and antibody fragments (e.g., bis-Fab), provided that they exhibit the desired antigen-binding activity, encompassing various antibody structures. As used herein, "antibody fragment" refers to a molecule other than an intact antibody that includes a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, without limitation, bis-Fab; Fv; Fab; Fab, Fab'-SH; F(ab')2; diabody; linear antibody; single-chain antibody molecule (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0039] The terms "cancer" and "tumor" are used interchangeably herein. As used herein, the terms "cancer" or "tumor" refer to all neoplastic cell growth and proliferation, and all pre-cancerous and cancerous cells and tissues, whether malignant or benign. The term is further used to refer to or describe a physiological state in a mammal that is typically characterized by uncontrolled cell growth / proliferation. Cancer can affect various cell types, tissues, or organs, including, without limitation, those selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, 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 their tissue or cell types. Cancer includes cancers such as sarcoma, carcinoma, or plasmacytoma (malignant tumor of plasma cells). Examples of cancer include, without limitation, those described herein. The terms "cancer" or "tumor" and "proliferative disease" are not mutually exclusive as used herein.

[0040] "CD8" is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediate efficient cell-cell interactions within the immune system. The CD8 antigen acts as a co-receptor together with the T cell receptor on T lymphocytes, and recognizes antigens displayed by antigen-presenting cells in the context of class I MHC molecules. As used herein, "CD8 cells" means one or more cells that have been engineered to express one or more NeoTCRs and CD8 constructs. As used herein, "CD8 construct" means any one of CD8 construct 1, CD8 construct 2, CD8 construct 3, or CD8 construct 4. As used herein, "CD8 product" means a product that includes CD8 cells.

[0041] "CD8 construct 1" and "CD8 product 1" refer to a construct that includes a NeoTCR and CD8α (CD8α extracellular domain, CD8α transmembrane domain, and CD8α intracellular domain), and the resulting product that includes the expressed NeoTCR and CD8α. Non-limiting examples of CD8 product 1 are provided in FIGS. 2A, 3A, and 6. Non-limiting examples of CD8 product 1 are provided in FIG. 4A. "CD8 construct 2" and "CD8 product 2" refer to a construct that includes a 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 that includes the expressed NeoTCR, CD8α, and CD8β. Non-limiting examples of CD8 product 2 are provided in FIGS. 2B, 3B, and 7. Non-limiting examples of CD8 product 1 are provided in FIG. 4A.

[0042] "CD8 construct 3" and "CD8 product 3" refer to a construct comprising a NeoTCR, CD8α extracellular domain, CD8α transmembrane domain, and CD8β intracellular domain, and a resulting product comprising the expressed NeoTCR, CD8α extracellular domain, CD8α transmembrane domain, and CD8β intracellular domain. Non-limiting examples of CD8 product 3 are provided in FIGS. 2C, 3C, and 8. Non-limiting examples of CD8 product 1 are provided in FIG. 4B. "CD8 construct 4" and "CD8 product 4" refer to a construct comprising a NeoTCR, CD8α extracellular domain, CD8α transmembrane domain, and CD4 intracellular domain, and a resulting product comprising the expressed NeoTCR, CD8α extracellular domain, CD8α transmembrane domain, and CD4 intracellular domain. Non-limiting examples of CD8 product 4 are provided in FIGS. 2D, 3D, and 9. Non-limiting examples of CD8 product 1 are provided in FIG. 4B.

[0043] "Conservative substitution" or "conservative amino acid" refers to a substitution by an amino acid that is chemically or functionally similar to the amino acid. Tables of conservative substitutions that provide similar amino acids are well known in the art. In certain embodiments, acidic amino acids D and E are conservative substitutions of each other; basic amino acids K, R, and H are conservative substitutions of each other; hydrophilic uncharged amino acids S, T, N, and Q are conservative substitutions of each other; aliphatic uncharged amino acids G, A, V, L, and I are conservative substitutions of each other; nonpolar uncharged amino acids C, M, and P are conservative substitutions of each other; 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; and C and M are conservative substitutions of each other. Additional conservative substitutions can be found, for example, in Creighton, Proteins: Structures and Molecular Properties 2nd Edition (1993) W.H. Freeman & Co., New York, NY.

[0044] "Treat", "Treatment", and "treating" are used interchangeably and, as used herein, mean obtaining a beneficial or desirable result including a clinical outcome. Desirable effects of treatment include, but are not limited to, prevention of the onset or recurrence of a disease, alleviation of symptoms, reduction of any direct or indirect pathological consequence of the disease, prevention of metastasis, reduction of the rate of disease progression, regression or alleviation of the medical condition, and improvement of remission or prognosis. In some embodiments, the NeoTCR products of the invention are used to delay the onset of a proliferative disease (e.g., cancer) or to slow the progression of such a disease.

[0045] As used herein, "dextramer" means a multimerized neoepitope-HLA complex that specifically binds to its cognate NeoTCR. As used herein, the terms "neoantigen", "neoepitope", or "neoE" refer to newly formed antigenic determinants that arise, for example, from somatic mutations and are recognized as "non-self". Mutations that give rise to "neoantigen", "neoepitope", or "neoE" can include frameshift or non-frameshift indels, missense or nonsense substitutions, splice site changes (e.g., alternatively spliced transcripts), genomic rearrangements or gene fusions, any genomic or expression changes, or any post-translational modifications.

[0046] As used herein, "NeoTCR", "NeoE TCR", and "exogenous TCR" mean neoepitope-specific T cell receptors that are introduced into T cells, for example, by gene editing methods. As used herein, the term "TCR gene sequence" refers to the NeoTCR gene sequence. As used herein, "NeoTCR cells" means one or more cells that have been 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 of the patient to whom the NeoTCR product is administered. The terms "NeoTCR cells" and "NeoTCR-P1 T cells" and "NeoTCR-P1 cells" are used interchangeably herein.

[0047] As used herein, "NeoTCR product" means a pharmaceutical formulation comprising one or more NeoTCR cells. The NeoTCR product consists of autologous, precisely genome-edited CD8+ and CD4+ T cells. Using a non-viral precise genome engineering approach via a target DNA, the expression of the endogenous TCR is eliminated and replaced by a patient-specific NeoTCR isolated from peripheral CD8+ T cells targeting tumor-exclusive neoepitopes. In certain embodiments, the resulting modified CD8+ or CD4+ T cells express the NeoTCR on their surface with natural sequences, at natural expression levels, and with natural TCR function. The sequences of the NeoTCR extracellular binding domain and the cytoplasmic signaling domain are unmodified from the TCR isolated from natural CD8+ T cells. Regulation of NeoTCR gene expression is driven by the native endogenous TCR promoter located upstream of the location where the NeoTCR gene cassette is integrated into the genome. By this approach, the natural level of NeoTCR expression is observed in the unstimulated antigen-activated T cell state.

[0048] The NeoTCR product manufactured for each patient represents a defined dose of autologous CD8+ and / or CD4+ T cells that have been precisely genome-edited to express a single neoE-specific TCR cloned from neoE-specific CD8+ T cells isolated individually from the same patient's peripheral blood. As used herein, "NeoTCR viral product" has the same definition as NeoTCR product, except that the genome modification is performed using a virus-mediated method.

[0049] "Pharmaceutical formulation" refers to a formulation in a form that enables the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation can be administered. For clarity, the amount of DMSO used in the NeoTCR product is not considered to be unacceptably toxic. As used herein, the terms "subject", "patient", or "individual" for purposes of treatment refer to any animal classified as a mammal, including humans, farm animals, zoo animals, sports, or pet animals such as dogs, horses, cats, cows, etc. Preferably, the mammal is a human.

[0050] As used herein, "TCR" means T cell receptor. As used herein, the term "tumor antigen" refers to an antigen (e.g., a polypeptide) that is uniquely or differentially expressed on tumor cells as compared to normal or non-tumor cells. In certain embodiments, tumor antigens include any polypeptide expressed by a tumor that can activate or induce an immune response via an antigen recognition receptor or can suppress an immune response via receptor-ligand binding. "2A" and "2A peptide" are used interchangeably herein and refer to a class of 18 - 22 amino acid long viral self-cleaving peptides that can mediate peptide cleavage during translation in eukaryotic cells.

[0051] 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 porcine teschovirus-1 2A. The E2A peptide was first identified in equine rhinitis A virus. The F2A peptide was first identified in foot-and-mouth disease virus. The self-cleavage mechanism of the 2A peptide is the result of the ribosome skipping the formation of the glycyl-prolyl peptide bond at the C-terminus of 2A. Specifically, the 2A peptide has a C-terminal conserved sequence necessary for creating steric hindrance and ribosome skipping. Ribosome skipping can result in one of three options: 1) Skipping is successful, translation resumes, and two cleaved proteins are produced (upstream of the 2A protein that binds to the complete 2A peptide excluding the C-terminal proline and downstream of the 2A protein that binds to one proline at the N-terminus); 2) Skipping is successful, but translation is interrupted by ribosome dropout, and only the protein upstream of 2A is produced; or 3) Skipping fails and translation continues (i.e., a fusion protein).

[0052] As used herein, the term "endogenous" typically refers to a nucleic acid molecule or polypeptide that is expressed in a cell or tissue. As used herein, the term "exogenous" refers to a nucleic acid molecule or polypeptide that is not endogenously present within a cell. Thus, the term "exogenous" would encompass any recombinant nucleic acid molecule or polypeptide expressed within a cell, such as foreign, heterologous, and overexpressed nucleic acid molecules and polypeptides. An "exogenous" nucleic acid means a nucleic acid that is not present in a natural wild-type cell; for example, an exogenous nucleic acid can differ from its endogenous counterpart by sequence, position / location, or both. For clarity, an exogenous nucleic acid can have the same or a different sequence compared to its natural endogenous counterpart; it can be introduced into the cell itself or its progenitor cells by genetic engineering and optionally ligated to alternative regulatory sequences such as a non-natural promoter or secretion sequence.

[0053] When related to T cells, "naive" or "more naive" or "naive T cells" refer to memory stem cells (T MSC ) and central memory cells (T CMThis means. These cells show T cell proliferation upon specific activation and are competent for multiple cell divisions. They also engraft after reinfusion and rapidly differentiate into effector T cells upon exposure to their cognate antigen, having the ability not only to target and kill tumor cells but also to persist for the surveillance and control of ongoing cancer.

[0054] [NeoTCR product] In some embodiments, using gene editing techniques and neoTCR isolation techniques described in PCT / US2020 / 017887 and PCT / US2019 / 025415, which are hereby incorporated by reference in their entirety, the NeoTCR is cloned into autologous CD8+ and CD4+ T cells from the same cancer patient that have been precisely genome edited to express the neoTCR (using the DNA-mediated (non-viral) method described in FIGS. 1A - 1C). In other words, the NeoTCR that is tumor-specific is identified in a cancer patient, such NeoTCR is then cloned, and the cloned NeoTCR is then inserted into the T cells of the cancer patient. Next, the T cells expressing the NeoTCR are expanded in a form that maintains the "naive" T cell phenotype, resulting in a NeoTCR-P1 product (i.e., the NeoTCR product) in which the majority of the T cells exhibit the T memory stem cell and T central memory phenotypes.

[0055] These "naive" or "more naive" or less differentiated T cell phenotypes are described as conferring improved engraftment ability and long-term persistence after infusion. Thus, administration of the NeoTCR product significantly composed of the "naive" T cell phenotype may bring benefits to cancer patients in eradicating tumor cells throughout the body through improved engraftment potential, extended persistence after infusion, and rapid differentiation into effector T cells. Mechanistic studies ex vivo have also been performed with NeoTCR products manufactured from T cells of cancer patients. Equivalent gene editing efficiency and functional activity, measured by antigen specificity of T cell killing activity, proliferation, and cytokine production, were observed, demonstrating that the manufacturing method described herein was successful in producing a product using T cells from cancer patients as starting materials.

[0056] In certain embodiments, the method of manufacturing NeoTCR products involves electroporation of a dual ribonucleoprotein species of CRISPR-Cas9 nuclease bound to a guide RNA sequence, where each species targets the genomic TCRα and genomic TCRβ loci. The specificity of targeting Cas9 nuclease to each genomic locus has been previously described in the literature as highly specific. Comprehensive testing of NeoTCR products was performed with in vitro and in silico analyses using COSMID and GUIDE-seq, respectively, to investigate potential off-target genomic cleavage sites. Multiple NeoTCR products or equivalent cell products from healthy donors were evaluated by deep sequencing for cleavage at candidate off-target sites, corroborating published evidence that the selected nuclease is highly specific.

[0057] Further aspects of the precision genome engineering method have been evaluated for safety. Evidence of genomic instability after precision genome modification was not found in the evaluation of multiple NeoTCR products by target locus amplification (TLA) or standard FISH cytogenetics. Off-target integration of the NeoTCR sequence into the genome was not detected. Evidence of Cas9 remaining in the cell products was not found. Comprehensive evaluation of NeoTCR products and the precision genome engineering method has shown that NeoTCR products are well tolerated after injection into patients.

[0058] The genome modification approach described herein enables the highly efficient generation of custom NeoTCR T cells (i.e., NeoTCR products) for personalized adoptive cell therapies for patients with solid and liquid tumors. Furthermore, the modification approach is not limited to use in T cells and has been successfully applied to other primary cell types, including natural killer cells and hematopoietic stem cells.

[0059] [CD8 product] Co-expression of MHC class I-restricted neoTCR and ectopic CD8 receptor in precision genome-edited CD4 T cells significantly enhances antigen-specific effector function.

[0060] Neoepitopes from tumor-exclusive mutations represent a compelling target for individualized neoE-specific autologous TCR-T cell therapy for solid tumor patients. The imPACT isolation technology described in PCT / US2020 / 17887, which is hereby incorporated by reference in its entirety, is a highly sensitive and high-throughput method for capturing neoE-specific CD8 T cells from the blood of solid cancer patients. Using this technology, neoepitope-specific MHC class I-restricted TCRs ("MHC-I neoTCRs") were cloned from individually captured CD8 T cells. Fresh CD8 and CD4 T cells from the same cancer patients were modified to express MHC-I neoTCRs (simultaneously with removal of the endogenous TCR) using DNA-mediated (non-viral) gene editing as described in Example 1.

[0061] It was hypothesized that naturally occurring MHC-I TCRs require the simultaneous assistance of the CD8 coreceptor to stabilize peptide-MHC binding, but high-affinity TCRs were able to promote CD8-independent target binding and T cell activation. Thus, CD4 T cells, when modified with high-affinity neoTCRs, were able to recognize peptide-MHC-I targets and trigger effector T cell function. However, low-affinity TCRs were dependent on the CD8 coreceptor to trigger T cell activation. By precisely genome-editing the CD8 coreceptor gene into CD4 T cells together with neoTCRs, MHC-I neoTCRs were enabled to have the ability to trigger antigen-specific effector T cell function.

[0062] CD8 stabilizes TCR-pMHC interactions and synergizes with the TCR with respect to binding activity. CD8 also functions to enhance the binding activity from low-affinity TCRs, although the intracellular domain of CD8α is important for enhancing T cell activation. CD8 expression can enhance CD4 T cell responses that may not respond to physiological concentrations of pMHC. Disruption of CD8 binding to MHC can convert catch bond TCR-pMHC to slip bond and emphasizes the importance of the CD8-MHC interaction even for TCRs that bind pMHC independent of CD8.

[0063] CD8α has a lower affinity for LCK than CD8β, suggesting that either co-expression of both CD8α and CD8β or generation of chimeric CD8α-CD8β molecules containing the extracellular CD8α and the intracellular domain of CD8β can improve efficacy. The CD8 constructs described herein are as follows: 1. CD8α homodimer (CD8 construct 1) 2. CD8α-P2A-CD8β (CD8 construct 2) 3. CD8α containing the CD8β intracellular domain (CD8 construct 3) 4. CD8α homodimer containing the CD4 intracellular domain (CD8 construct 4)

[0064] In some embodiments, the NeoTCR products described above include additional modifications including the expression of CD8 construct 1, CD8 construct 2, CD8 construct 3, or CD8 construct 4 (each a CD8 product). Specifically, using the gene editing techniques and neoTCR isolation techniques described in PCT / US2020 / 17887 and PCT / US2019 / 025415, which are hereby incorporated by reference in their entirety, the NeoTCR is precisely genome-edited to express the neoTCR (using the DNA-mediated (non-viral) method described in FIGS. 1A-1C) and cloned into autologous CD8+ and CD4+ T cells of the same cancer patient.

[0065] Each of the CD8 constructs, when expressed, results in a CD8 product. Table 1 provides descriptions of each construct and product. JPEG0007717619000001.jpg99169

[0066] In certain embodiments, CD8 product 1 comprises a NeoTCR and a CD8 homodimer. In certain embodiments, CD8 product 1 comprises the expression of a NeoTCR, a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8α intracellular domain. In certain embodiments, CD8 product 1 further comprises the expression of a CD8α signal peptide. In certain embodiments, CD8 product 1 comprises the translated elements presented in FIG. 3A. In a non-limiting exemplary embodiment, CD8 product 1 comprises a NeoTCR, 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). In certain embodiments, sequence modifications to the CD8α signal peptide, CD8α extracellular domain, CD8α transmembrane domain, and CD8α intracellular domain can be made to preserve or substantially preserve the function of each element. In certain embodiments, such sequence modifications are conservative substitutions of amino acids.

[0067] In a non-limiting embodiment, the CD8 product 1 is produced from the CD8 construct 1 provided in FIG. 6. In a given embodiment, the sequence of the CD8 construct 1 provided in FIG. 6 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 leaves the function of each element preserved. In a given embodiment, the order of each element of the CD8 construct 1 in FIG. 6 remains the same, but the sequence of each individual element can be varied as long as the amino acids encoded by the nucleic acid remain the same or include only conservative substitutions. In a given embodiment, the order of each element of the CD8 construct 1 in FIG. 6 remains the same, but the sequence of each individual element can be varied as long as the function of the encoded protein does not change substantially.

[0068] In certain embodiments, CD8 product 2 comprises a NeoTCR, CD8α, and CD8β. In certain embodiments, CD8α and CD8β are separated for expression by a protease cleavage site and a 2A peptide in the CD8 product 2 construct. In certain embodiments, CD8 product 2 comprises the expression of a NeoTCR, CD8α extracellular domain, CD8α transmembrane domain, CD8α intracellular domain, CD8β extracellular domain, CD8β transmembrane domain, and CD8β intracellular domain. In certain embodiments, 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, CD8 product 2 comprises the translated elements presented in Figure 3B. In a non-limiting exemplary embodiment, CD8 product 2 comprises a 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, sequence modifications of the CD8α signal peptide, CD8α extracellular domain, CD8α transmembrane domain, CD8α intracellular domain, CD8β extracellular domain, CD8β transmembrane domain, and CD8β intracellular domain can be made to preserve or substantially preserve the function of each element. In certain embodiments, such sequence modifications are conservative substitutions of amino acids.

[0069] In a non-limiting embodiment, the CD8 product 2 is produced from the CD8 construct 2 provided in FIG. 7. In certain embodiments, the sequence of the CD8 construct 2 provided in FIG. 7 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 leaves the function of each element preserved. In certain embodiments, the order of each element of the CD8 construct 2 of FIG. 7 remains the same, but the sequence of each individual element can be changed as long as the amino acids encoded by the nucleic acid remain the same or include only conservative substitutions. In certain embodiments, the order of each element of the CD8 construct 2 of FIG. 7 remains the same, but the sequence of each individual element can be changed as long as the function of the encoded protein does not substantially change.

[0070] In certain embodiments, the CD8 product 3 includes CD8α along with the NeoTCR and the CD8β intracellular domain. In certain embodiments, the CD8 product 3 includes the expression of the NeoTCR, CD8α extracellular domain, CD8α transmembrane domain, and CD8β intracellular domain. In certain embodiments, the CD8 product 3 further includes the expression of the CD8α signal peptide. In certain embodiments, the CD8 product 3 includes the translated elements presented in FIG. 3C. In a non-limiting exemplary embodiment, the CD8 product 3 includes the 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, sequence modifications of the CD8α signal peptide, CD8α extracellular domain, CD8α transmembrane domain, and CD8β intracellular domain can be made to preserve or substantially preserve the function of each element. In certain embodiments, such sequence modifications are conservative substitutions of amino acids.

[0071] In non-limiting embodiments, the CD8 product 3 is produced from the CD8 construct 3 provided in FIG. 8. In certain embodiments, the sequence of the CD8 construct 3 provided in FIG. 8 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 leaves the function of each element preserved. In certain embodiments, the order of each element of the CD8 construct 3 of FIG. 8 remains the same, but the sequence of each individual element can be varied as long as the amino acids encoded by the nucleic acid remain the same or include only conservative substitutions. In certain embodiments, the order of each element of the CD8 construct 3 of FIG. 8 remains the same, but the sequence of each individual element can be varied as long as the function of the encoded protein does not substantially change.

[0072] In certain embodiments, the CD8 product 4 comprises a CD8α homodimer together with a NeoTCR and a CD4 intracellular domain. In certain embodiments, the CD8 product 4 comprises the expression of a NeoTCR, a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain. In certain embodiments, the CD8 product 4 further comprises the expression of a CD8α signal peptide. In certain embodiments, the CD8 product 4 comprises the translated elements presented in FIG. 3D. In a non-limiting, exemplary embodiment, the CD8 product 4 comprises a NeoTCR, 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 CD4 intracellular domain (SEQ ID NO: 148). In certain embodiments, sequence modifications of the CD8α signal peptide, CD8α extracellular domain, CD8α transmembrane domain, and CD4 intracellular domain can be made to preserve or substantially preserve the function of each element. In certain embodiments, such sequence modifications are conservative substitutions of amino acids.

[0073] In a non-limiting embodiment, the CD8 product 4 is produced from the CD8 construct 4 provided in FIG. 9. In a given embodiment, the sequence of the CD8 construct 4 provided in FIG. 9 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 leaves the function of each element preserved. In a given embodiment, the order of each element of the CD8 construct 4 in FIG. 9 remains the same, but the sequence of each individual element can be varied as long as the amino acids encoded by the nucleic acid remain the same or contain only conservative substitutions. In a given embodiment, the order of each element of the CD8 construct 4 in FIG. 9 remains the same, but the sequence of each individual element can be varied as long as the function of the encoded protein does not substantially change.

[0074] In a given embodiment, the CD8 product includes a TET2 knockout or TET2 knockdown. In a given embodiment, the cells of the CD8 product are further modified to knockout the TET2 gene using a non-viral method. In a given embodiment, the cells of the CD8 product are further modified to knockout the TET2 gene using a viral method. In a given embodiment, the cells of the CD8 product are further modified to increase T cell persistence by using a non-viral method to knockout, knockdown, or modify the function of a gene associated with T cell persistence. In a given embodiment, the cells of the CD8 product are further modified to increase T cell persistence by using a viral method to knockout, knockdown, or modify the function of a gene associated with T cell persistence.

[0075] In some embodiments, the CD8 product comprises cells modified to express the NeoTCR and the CD8 construct using a viral method (i.e., the CD8 viral product). In certain embodiments, the cells of the CD8 viral product are further modified to knockout the TET2 gene using a non-viral method. In certain embodiments, the cells of the CD8 viral product are further modified to knockout the TET2 gene using a viral method. In certain embodiments, the cells of the CD8 viral product are further modified to increase T cell persistence by using a non-viral method to knockout, knockdown, or modify the function of a gene associated with T cell persistence. In certain embodiments, the cells of the CD8 viral product are further modified to increase T cell persistence by using a viral method to knockout, knockdown, or modify the function of a gene associated with T cell persistence.

[0076] In certain embodiments, the T cell persistence gene having a function that is knocked out, knocked down, or modified is a gene that results in downregulation of T cell activity. In certain embodiments, the gene that is knocked out, knocked down, or modified is a gene that downregulates T cell memory function. In certain embodiments, the gene that is knocked out, knocked down, or modified is a gene that decreases the function, proliferation, and / or survival of T cells.

[0077] In certain embodiments, additional modifications to the CD8 product and its CD8 cells include modifications that increase the resilience of the tumor microenvironment, increase T cell activity, increase homing / retention in the tumor microenvironment, increase T cell persistence, and increase ectopic effector function of T cells. In certain embodiments, tumor microenvironment resilience includes, but is not limited to, converting / antagonizing negative environmental signals. In certain embodiments, TCR-mediated signal enhancement includes, but is not limited to, an increase in T cell activity. In certain embodiments, homing / retention in the tumor microenvironment includes, but is not limited to, enhanced tumor infiltration. In certain embodiments, functional T cell persistence includes, but is not limited to, metabolic and transcriptional regulation. In certain embodiments, ectopic effector function includes, but is not limited to, TCR-induced antibody, cytokine, or peptide secretion.

[0078] In certain embodiments, functional T cell persistence can be achieved by genetic engineering as described herein, by co-administration of an agent that improves the functional T cell persistence of modified T cells (modified to incorporate at least a neoTCR as described herein), and by the manufacturing and culture conditions of modified T cells (modified to incorporate at least a neoTCR as described herein). In certain embodiments, the TCR-induced antibody used to improve ectopic effector function is any one of the antibodies or functional fragments thereof described herein. 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 cytokines, or any combination thereof. In certain embodiments, the TCR-induced cytokine is not a cytokine, but rather another cofactor or expression element that induces endogenous cytokine production.

[0079] In certain embodiments, the CD8 product and additional modifications to the CD8 cells include modifications to knock in one or more additional genes and / or functional proteins. In certain embodiments, the genes and / or functional proteins to be knocked in 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 a gene is an antibody or a functional fragment thereof that is an antagonist of the gene. For example, dominant negative PD-1 can be an anti-PD-1 antibody or a functional fragment thereof. In certain embodiments, any of the knock-in genes and / or functional proteins are functional fragments of the genes and / or proteins (including, but not limited to, truncations).

[0080] In certain embodiments, the CD8 product and additional modifications to the CD8 cells include modifications to knock out or knockdown one or more additional genes and / or functional proteins. In certain embodiments, the genes and / or functional proteins to be knocked out or knockdown include, but are not limited to, TET2, IFNGR1, RICTOR, NR4A1, DNMT3A, SUV39H1, PPP2RD, adenosine 2A receptor, PP2A3, and PP2A4. In certain embodiments, instead of knocking in a gene for the expression of a functional protein, a different gene that would otherwise be knocked in can be regulated by the genetic engineering described herein to upregulate the expression of the gene. In certain embodiments, instead of knocking out or knocking down a gene, a different gene that would otherwise be knocked out can be regulated by the genetic engineering described herein to downregulate the expression of the gene.

[0081] The method for producing CD8 products involves electroporation of a double ribonucleoprotein species of CRISPR-Cas9 nuclease bound to a guide RNA sequence, and the various species target the genomic TCRα and genomic TCRβ loci. The specificity of targeting Cas9 nuclease to each genomic locus has been described in the literature as being highly specific previously. An exhaustive test of the CD8 products was performed with in vitro and in silico analyses using COSMID and GUIDE-seq respectively to examine potential off-target genomic cleavage sites. Multiple CD8 products or equivalent cell products from healthy donors were evaluated for cleavage of candidate off-target sites by deep sequencing, corroborating the published evidence that the selected nuclease is highly specific.

[0082] 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. In certain embodiments, the CD8 cells are expanded to maintain a "naive" T cell phenotype, resulting in a CD8 product in which the majority of the T cells exhibit a T memory stem cell and T central memory phenotype. These "naive" or "more naive" or less differentiated T cell phenotypes are described as conferring improved engraftment ability and long-term persistence after infusion. Thus, administration of a CD8 product significantly composed of the "naive" T cell phenotype may provide cancer patients with the benefit of eradicating tumor cells throughout the body through improved engraftment potential, extended persistence after infusion, and rapid differentiation into effector T cells.

[0083] In certain embodiments, the CD8 cells of the CD8 product mainly comprise memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 25% of the CD8 cells of the CD8 product comprise memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 30% of the CD8 cells of the CD8 product comprise memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 35% of the CD8 cells of the CD8 product comprise memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 40% of the CD8 cells of the CD8 product comprise memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 45% of the CD8 cells of the CD8 product comprise memory stem cells (Tmsc) and / or central memory cells (Tcm). In certain embodiments, at least 50% of the CD8 cells of the CD8 product comprise 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 is characterized as cells that are CD45RA+CD62L+, CD28+CD95+, and CCR7+CD27+.Tcm is characterized as cells that are CD45RO+CD62L+, CD28+CD95+, and CCR7+CD27+CD127+. Both Tmsc and Tcm are characterized by weak effector T cell function, strong proliferative ability, strong engraftment ability, and long telomeres.

[0084] In certain embodiments, the CD8 cells disclosed herein exhibit improved properties (e.g., cytotoxic activity, cell proliferative ability, cytokine secretion ability, LCK affinity, persistence, tumor infiltration ability) of 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% compared to cells that do not have the CD8 construct.

[0085] [Method for Producing a CD8 Product with a Naive Phenotype] In certain embodiments, the present disclosure relates, in part, to the production of modified "naive" T cells. In certain embodiments, the present disclosure includes methods for producing antigen-specific cells, such as T cells, ex vivo, including activating, engineering, and expanding antigen-specific cells originally obtained from a subject or isolated from such a sample. In certain embodiments, the method for activating the cells includes the step of activating the TCR / CD3 complex. For example, without limitation, the T cells can be incubated and / or cultured with a CD3 agonist, a CD28 agonist, or a combination thereof.

[0086] In certain embodiments, modified activated antigen-specific cells, such as modified activated T cells, can be expanded by culturing the modified activated antigen-specific cells, such as T cells, together with cytokines, chemokines, soluble peptides, or combinations thereof. In certain embodiments, modified activated antigen-specific cells, such as modified activated T cells, can be cultured with one or more cytokines. In certain embodiments, the cytokine can be IL2, IL7, IL15, or combinations thereof. For example, modified activated antigen-specific cells, such as modified activated T cells, can be cultured with IL7 and IL15. In certain embodiments, the cytokines used in connection with the culture of modified activated antigen-specific cells, such as modified activated T cells, can be present at concentrations from about 1 pg / ml to about 1 g / ml, from about 1 ng / ml to about 1 g / ml, from about 1 μg / ml to about 1 g / ml, or from about 1 mg / ml to about 1 g / ml, and any value therebetween.

[0087] [Pharmaceutical formulation] A pharmaceutical formulation of the CD8 product is prepared by combining CD8 cells in a solution that can preserve the "naive" phenotype of the cells in a cryopreserved state. Table 1 provides an example of such a pharmaceutical formulation. Alternatively, a pharmaceutical formulation of the CD8 product can be prepared by combining CD8 cells in a solution that can preserve the "naive" phenotype of the cells without the need to freeze or cryopreserve the product (i.e., the CD8 product is maintained in an aqueous solution or as a non-frozen / cryopreserved cell pellet). Additional pharmaceutically acceptable carriers, buffers, stabilizers, and / or preservatives can also be added to the cryopreservation solution or aqueous storage solution (if the CD8 product is not cryopreserved). The CD8 product can be cryopreserved using any cryopreservative and / or medium, including but not limited to CryoStor, CryoStor CS5, CELLBANKER, and custom cryopreservation media that may contain DMSO in some cases.

[0088] [Gene editing method] In certain embodiments, the present disclosure includes, in part, methods of modifying human cells, such as modified T cells or modified human stem cells. In certain embodiments, the present disclosure includes, in part, methods of modifying human cells, such as NK cells, NKT cells, macrophages, hematopoietic stem cells (HSCs), cells derived from HSCs, or dendritic / antigen presenting cells. In certain embodiments, such modifications include genome editing. For example, but not limited to, such genome editing can be achieved using nucleases that target one or more endogenous loci, such as the TCR alpha (TCRα) locus and the TCR beta (TCRβ) locus. In certain embodiments, the nuclease can generate a single-stranded DNA nick or a double-stranded DNA break in the endogenous target sequence. In certain embodiments, the nuclease can target the coding or non-coding portion of the genome, such as exons, introns. In certain embodiments, nucleases contemplated herein include homing endonucleases, meganucleases, megaTAL nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / Cas nucleases. In certain embodiments, the nuclease can itself be modified, such as by introduction of amino acid substitutions and / or deletions, to enhance the efficiency of its cleavage activity.

[0089] In certain embodiments, the CRISPR / Cas nuclease system is used to modify human cells. In certain embodiments, the CRISPR / Cas nuclease system comprises a Cas nuclease and one or more RNAs that direct the Cas nuclease to an endogenous target sequence, such as a single guide RNA. In certain embodiments, the Cas nuclease and the RNA are introduced into the cell separately using different vectors or compositions, or 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 bacterial species including, but not limited to, Streptococcus pyogenes or Neisseria meningitidis. Additional 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, 1 1911 (2018), which is hereby incorporated by reference in its entirety for all that it teaches.

[0090] In certain embodiments, genome editing is performed at one or more genomic loci that regulate the immune response. In certain embodiments, the loci include, but are not limited to, the TCR alpha (TCRα) locus, the TCR beta (TCRβ) locus, the TCR gamma (TCRγ), and the TCR delta (TCRδ). In certain embodiments, the locus for inserting the CD8 construct is somewhere 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 the two TRBC loci. In certain embodiments, the locus for inserting the CD8 construct is a locus other than the TRAC locus or the TRAB locus. In certain embodiments, the locus for inserting the CD8 construct is inserted into a locus at which such a 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 into the TET2 locus or the AAVS1 locus. In certain embodiments, the insertion of the CD8 construct is concurrent with the NeoTCR insertion. In certain embodiments, the insertion of the CD8 construct is at a locus separate from the NeoTCR insertion.

[0091] In certain embodiments, genome editing is performed using a non-viral delivery system. For example, administration of nucleic acids in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 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), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or microinjection under surgical conditions (Wolff et al., Science 247:1465, 1990) can introduce nucleic acid molecules into cells. Other non-viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be potentially useful for delivery of DNA to cells. Transplantation of a normal gene into the affected tissue of a subject can also be achieved by introducing the normal nucleic acid into a cell type that can be cultured ex vivo (e.g., autologous or heterologous primary cells or their progeny), and then the cells (or their progeny) are injected into the target tissue or systemically injected.

[0092] In certain embodiments, genome editing is performed 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 lentiviral approaches). In certain embodiments, viral delivery will be achieved without integration of nucleases. In such embodiments, the viral delivery system can be Lentiflash or another similar delivery system.

[0093] [Homologous recombination template] In certain embodiments, the present disclosure provides genomic editing of cells by introducing and recombining a homologous recombination (HR) template nucleic acid sequence into an endogenous locus of the cell. 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 minicircle, or a nanoplasmid. In certain embodiments, the HR template nucleic acid sequence comprises a first homology arm and a second homology arm. In certain embodiments, the homology arms can be from about 300 bases to about 2000 bases. For example, each homology arm can be 1000 bases. In certain embodiments, the homology arms can be homologous to a first endogenous sequence and a second endogenous sequence of the cell. In certain embodiments, the endogenous locus is a TCR locus. For example, the first endogenous sequence and the second endogenous sequence are within the TCR alpha locus or the TCR beta 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 non-limiting embodiments, the TCR gene sequence can be identified and obtained using the methods described in PCT / US2020 / 017887, the contents of which are hereby incorporated by reference. In certain embodiments, the HR template comprises a TCR alpha gene sequence and a TCR beta gene sequence.

[0094] 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) within the sequence. In certain embodiments, the HR template comprises a 2A peptide (e.g., P2A, T2A, E2A, and F2A). Additional information regarding the HR template nucleic acid and methods of modifying cells therewith can be found in International Patent Application PCT / US2018 / 058230, the contents of which are hereby incorporated by reference.

[0095] [Treatment Method] The subject matter of the present disclosure provides methods 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 extend the survival of a subject afflicted 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 an effective amount of the CD8 product, or a composition comprising the same (e.g., a pharmaceutical composition), to achieve the desired effect, whether for alleviation of an existing condition or prevention of recurrence. In treatment, the amount administered is an amount effective to produce the desired effect. The effective amount can be provided in a single or series of administrations. The effective amount can be provided by bolus or continuous perfusion.

[0096] In certain embodiments, the CD8 product can be used to treat viral or bacterial diseases. In certain embodiments, the CD8 product can be used to treat autoimmune diseases. In certain embodiments, an effective amount of the CD8 product is delivered by IV administration. In certain embodiments, the CD8 product is delivered by IV administration as a single administration. In certain embodiments, the CD8 product is delivered by IV administration in multiple administrations. In certain embodiments, the CD8 product is delivered by IV administration in two or more administrations. In certain embodiments, the CD8 product is delivered by IV administration in two administrations. In certain embodiments, the CD8 product is delivered by IV administration in three administrations. The subject matter of the present disclosure provides a method for treating and / or preventing cancer in a subject. In certain embodiments, the method comprises administering an effective amount of the CD8 product to a subject suffering from cancer.

[0097] Non-limiting examples of cancer include blood cancers (e.g., leukemia, lymphoma, and myeloma), 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, pharyngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate cancer and small cell lung cancer). Suitable carcinomas further include, but are not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, epithelioma, medulloblastoma, primitive neuroectodermal tumor (PNET), chondrosarcoma, osteosarcoma, pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchioloalveolar carcinoma, epithelial adenocarcinoma, and their liver metastases, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatocellular tumor, cholangiocarcinoma, synovioma, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon cancer, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchiogenic lung carcinoma, renal cell carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, epithelioma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenström macroglobulinemia, and heavy chain disease, breast tumors such as ductal and lobular adenocarcinoma, squamous and adenocarcinoma of the uterine cervix, uterine and ovarian epithelial cancer, prostate adenocarcinoma, transitional squamous cell carcinoma of the bladder, B and T cell lymphomas (nodular and diffuse) plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma, and any other known in the field of oncology. In certain embodiments, the cancer is selected from the group consisting of blood cancers (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 pharyngeal cancer. In certain embodiments, the disclosed naive T cells and compositions containing them can be used to treat and / or prevent blood cancers (e.g., leukemia, lymphoma, and myeloma) or ovarian cancer for which conventional therapeutic interventions are not acceptable.

[0098] In certain embodiments, the cancer is a solid cancer or a 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 adenocarcinoma, rectal adenocarcinoma, colon adenocarcinoma, esophageal cancer, endometrial cancer of the corpus uteri, breast cancer, cutaneous melanoma, lung adenocarcinoma, gastric adenocarcinoma, cervical and uterine cervical cancer, renal clear cell carcinoma, testicular germ cell tumor, and aggressive B cell lymphoma.

[0099] The subject may have a progressive disease, in which case the treatment objective may include alleviation or reversal of disease progression and / or improvement of side effects. The subject may have a history of being already treated, in which case the treatment objective will typically include reduction or delay of the risk of recurrence.

[0100] Human subjects suitable for treatment typically include two treatment groups distinguishable by clinical criteria. Subjects with "advanced disease" or "high tumor burden" are subjects with clinically measurable tumors. Clinically measurable tumors are tumors detectable based on tumor mass (e.g., by palpation, CAT scan, ultrasound, mammogram, or x-ray; positive biochemical or histopathological markers alone are insufficient to identify this population). The pharmaceutical composition is administered to these subjects for the purpose of alleviating their condition to induce an anti-tumor response. Ideally, this results in a decrease in tumor mass, but any clinical improvement constitutes an advantage. Clinical improvements include a decrease in the risk or rate of progression or a decrease in the pathological outcome of the tumor.

[0101] [Manufactured product] CD8 products can be used in combination with a manufactured product. Such a manufactured product can be useful for the prevention or treatment of proliferative diseases (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 accompanying documents on or associated with the containers. The container can be made of any material that is acceptable for the storage and preservation of CD8 cells within the CD8 product. In certain embodiments, the container can be an intravenous solution bag or a vial with a stopper penetrable by a hypodermic needle. For example, the container can be a CryoMACS freezing bag. The label or accompanying document indicates that the CD8 product is to be used to treat a selected condition and patient population. Since the CD8 product is made from autologous cells and is designed as a patient-specific individualized therapeutic agent, the patient is identified on the container of the CD8 product.

[0102] The manufactured product can include 1) a first container that contains the CD8 product therein. The manufactured product can include 1) a first container that contains the CD8 product therein; and 2) a second container that contains the same CD8 product as in the first container. Optionally, additional containers containing the same CD8 product as the first and second containers can be provided and manufactured. Optionally, additional containers containing compositions that include different cytotoxic or other therapeutic agents can also be combined with the above-described containers. The manufactured product can include 1) a first container that contains the CD8 product therein; and 2) a second container that contains a composition therein, the composition including a further cytotoxic or other therapeutic agent.

[0103] The manufactured product can include 1) a first container that contains two CD8 products therein; and 2) a second container that contains a composition therein, the composition including a further cytotoxic or other therapeutic agent. The manufactured product 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 that includes an additional cytotoxic or other therapeutic agent. In certain embodiments, the first CD8 product and the second CD8 product are different CD8 products. In certain embodiments, the first CD8 product and the second CD8 product are the same CD8 product.

[0104] The manufactured product may include: 1) a first container containing three CD8 products; and 2) optionally, a second container containing a composition that includes an additional cytotoxic or other therapeutic agent. 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 that includes an additional cytotoxic 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 product. In certain embodiments, two of the first, second, and third CD8 products are the same CD8 product.

[0105] The manufactured product may include: 1) a first container containing four CD8 products; and 2) optionally, a second container containing a composition that includes an additional cytotoxic or other therapeutic agent. 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 contains additional cytotoxic or other therapeutic agents. 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 CD8 product. In certain embodiments, two of the first, second, third, and fourth CD8 products are the same CD8 product. In certain embodiments, three of the first, second, third, and fourth CD8 products are the same CD8 product.

[0106] The manufactured product may include: 1) a first container containing five or more CD8 products; and 2) optionally, a second container containing a composition, wherein the composition contains additional cytotoxic or other therapeutic agents. 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, additional containers numbered six or above, each containing a CD8 product numbered six or above; and 7) optionally, additional containers containing a composition, wherein the composition contains additional cytotoxic or other therapeutic agents. In certain embodiments, all of the containers of CD8 products contain different CD8 products. In certain embodiments, all of the containers of CD8 products contain the same CD8 product. In certain embodiments, based on the availability of detectable CD8 in the patient's tumor sample, the need and / or desire for multiple CD8 products for the patient, and the availability of any one CD8 product for which one or more containers are needed or would benefit therefrom, any combination of the same or different CD8 products may be present in five or more containers.

[0107] The manufactured product may include: 1) a first container containing a CD8 product therein; 2) a second container containing a second CD8 product therein; and 3) a third container containing a third CD8 product therein. The manufactured product may include: 1) a first container containing a CD8 product therein; 2) a second container containing a second CD8 product therein; 3) a third container containing a third CD8 product therein; and 4) optionally, a fourth container containing a fourth CD8 product therein. The manufactured product may include: 1) a first container containing a CD8 product therein; 2) a second container containing a second CD8 product therein; 3) a third container containing a third CD8 product therein; 4) a fourth container containing a fourth CD8 product therein; and 5) optionally, a fifth container containing a fourth CD8 product therein.

[0108] The manufactured product may include a container containing one type of CD8 product therein. The manufactured product may include a container containing two types of CD8 products therein. The manufactured product may include a container containing three types of CD8 products therein. The manufactured product may include a container containing four types of CD8 products therein. The manufactured product may include a container containing five types of CD8 products therein. The manufactured product may include: 1) a first container containing one type of CD8 product therein, and 2) a second container containing two types of CD8 products therein. The manufactured product may include: 1) a first container containing two types of CD8 products therein, and 2) a second container containing one type of CD8 product therein. 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.

[0109] Furthermore, any container of the CD8 products described herein can be divided into two, three, or four separate containers for multiple dosing times and / or based on the appropriate dosage for the patient. In certain embodiments, the CD8 product is provided in a kit. The kit can include, by way of non-limiting example, package inserts, labels, instructions for use of the CD8 product, syringes, disposal instructions, administration instructions, tubes, needles, and other items that a clinician may need to properly administer the CD8 product.

[0110] [Therapeutic Composition and Method of Manufacture] As described herein, a plasmid DNA-mediated precision genome engineering method for the Good Manufacturing Practice (GMP) manufacture of the CD8 product was developed. Targeted integration of patient-specific neoTCRs was achieved by electroporating CRISPR endonuclease ribonucleoprotein (RNP) together with an individualized neoTCR gene cassette encoded by plasmid DNA. In addition to neoTCRs, CD8 constructs were inserted by introducing them into the neoTCR vector and then electroporating with the above-described CRISPR endonuclease ribonucleoprotein (RNP).

[0111] The CD8 product can be formulated into a pharmaceutical using a clinical manufacturing method. In this method, the CD8 product is cryopreserved in CryoMACS freezing bags. 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 autologous CD8 and CD4 T cells that are precisely genome-modified to express one or more autologous neoTCRs that target neoepitopes that complex with one of the endogenous HLA receptors that are uniquely present on the surface of the patient's tumor cells.

[0112] The final product will contain 5% dimethyl sulfoxide (DMSO), human serum albumin, and Plasma-Lyte. The final cell product will contain the components of the list provided in Table 2. JPEG0007717619000002.jpg54169

[0113] [Composition and Vector] The subject matter of the present disclosure provides compositions comprising the cells disclosed herein (e.g., immunoreactive cells). In certain embodiments, the subject matter of the present disclosure provides nucleic acid compositions comprising polynucleotides encoding the NeoTCRs disclosed herein. In certain embodiments, the nucleic acid compositions disclosed herein comprise polynucleotides encoding the CD8 constructs disclosed herein. Also provided are cells comprising such nucleic acid compositions.

[0114] In certain embodiments, the nucleic acid composition further comprises a promoter operably linked to the NeoTCR disclosed herein. In certain embodiments, the nucleic acid composition further comprises a promoter operably linked to the CD8 construct disclosed herein. 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 transcriptional response element (TRE) promoter, CD69 promoter, CD25 promoter, IL-2 promoter, IL-12 promoter, p40 promoter, and Bcl-xL promoter.

[0115] The compositions and nucleic acid compositions can be administered to a subject and / or delivered to cells 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 homogeneous cell composition with a recombinant DNA construct. In certain embodiments, a retroviral vector (either a gamma-retroviral vector or a lentiviral vector) is used to introduce the DNA construct into the cells. Non-viral vectors can also be used.

[0116] Possible transfection methods include, for example, direct co-culture of cells with producer cells by the method of Bregni et al. (1992) Blood 80:1418-1422, or, for example, culture using only virus supernatant or concentrated vector stocks with or without appropriate growth factors and polycations by the methods of Xu et al. (1994) Exp. Hemat. 22:223-230; and Hughes et al. (1992) J. Clin. Invest. 89:1817.

[0117] Cells can be modified using other transduction viral vectors. In certain embodiments, the selectable vectors exhibit 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. U.S.A. 94:10319, 1997). Other viral vectors that can be used include, for example, adenovirus, lentivirus, and adeno-associated viral vectors, vaccinia virus, bovine papillomavirus, or herpes virus, such as Epstein-Barr virus (see, e.g., 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 have been particularly well developed and are used in the clinical setting (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Patent No. 5,399,346).

[0118] Non-viral approaches can also be used for genetic modification of cells. For example, nucleic acid molecules can be introduced into cells by administering the nucleic acid molecules in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 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), in the presence of asialorosomucoid-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). Other non-viral means for gene transfer include transfection in vitro using calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes may also be potentially useful for the delivery of DNA into cells. Transplantation of normal genes into diseased tissues of a subject can also be achieved by introducing the normal nucleic acid into a cell type that can be cultured ex vivo (e.g., autologous or heterologous primary cells or their progeny), and then the cells (or their progeny) are injected into the target tissue or injected systemically.

[0119] Polynucleotide therapies can be directed from any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoter) and can be regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron construct). For example, if desired, the expression of the nucleic acid can be directed using an enhancer known to preferentially direct gene expression in a particular cell type. Enhancers that can be used include, but are not limited to, those characterized as tissue-specific or cell-specific enhancers. Alternatively, when a genomic clone is used as the therapeutic construct, regulation can be mediated by cognate regulatory sequences or, if desired, by regulatory sequences from a heterologous source that includes any of the above promoters or regulatory elements.

[0120] The resulting cells can be grown under conditions similar to those of unmodified cells, thereby allowing the modified cells to be expanded and used for various purposes.

[0121] [Kit] The subject matter of the present disclosure provides kits for inducing, and / or enhancing, and / or treating, and / or preventing an immune response, cancer, or pathogen infection in a subject. In certain embodiments, the kit includes an effective amount of a cell of the present disclosure or a pharmaceutical composition comprising the same. In certain embodiments, the kit includes a sterile container; such container can be in the form of a box, ampule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container form known in the art. Such container can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding a medicament. In certain non-limiting embodiments, the kit includes an isolated nucleic acid molecule encoding the HR template of the present disclosure.

[0122] If desired, the cells and / or nucleic acid molecules are provided with instructions for administering the cells or nucleic acid molecules to a subject having or at risk of developing cancer or a pathogen or immunodeficiency. The instructions generally include information regarding the use of the composition for the treatment and / or prevention of cancer or pathogen infection. In certain embodiments, the instructions include at least one of the following: a description of the therapeutic agent; an administration schedule and administration for the treatment or prevention of neoplasms, pathogen infections, or immunodeficiency or their symptoms; cautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical trials; and / or references. The instructions may be printed directly on the container, if present, or as a label affixed to the container, or printed as a separate sheet, pamphlet, card, or folder provided within or with the container. The resulting cells can be grown under conditions similar to unmodified cells, thereby allowing the modified cells to be grown and used for various purposes.

[0123] [Exemplary Embodiments] A. In certain non-limiting embodiments, the subject matter of the present disclosure provides cells comprising an exogenous T cell receptor (TCR) and an exogenous CD8. A1. The cells of A above, wherein the exogenous CD8 comprises at least one monomer. A2. The cells of A1 above, wherein at least one monomer of the exogenous CD8 comprises an extracellular domain, a transmembrane domain, an intracellular domain, fragments thereof, or combinations thereof. A3. The cells of A2 above, wherein the extracellular domain comprises a CD8α extracellular domain or a CD8β extracellular domain. A4. The cells of A2 or A3 above, wherein the transmembrane is a CD8α transmembrane domain or a CD8β transmembrane domain. A5. The cells of A2 - A4 above, wherein the intracellular domain comprises a CD8α intracellular domain or a CD8β intracellular domain. A6. The cells of A2 - A4 above, wherein the intracellular domain comprises a CD4 intracellular domain. A7. The aforementioned cells of A1 - A5, wherein at least one monomer comprises a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8α intracellular domain. A8. The aforementioned cells of A1 - A5, wherein at least one monomer comprises a CD8β extracellular domain, a CD8β transmembrane domain, and a CD8β intracellular domain. A9. The aforementioned cells of A1 - A5, wherein at least one monomer comprises a CD8α extracellular domain, a CD8α transmembrane domain, and a CD8β intracellular domain. A10. The aforementioned cells of A1 - A6, wherein at least one monomer comprises a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain. A11. The aforementioned cells of A1 - A10, wherein at least one monomer comprises a signal peptide. A12. The aforementioned cells of A11, wherein the signal peptide is a CD8 signal peptide. A13. The aforementioned cells of A2 - A12, wherein the extracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 140 or SEQ ID NO: 145. A14. The aforementioned cells of A2 - A13, wherein the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 141 or SEQ ID NO: 146. A15. The aforementioned cells of A2 - A14, wherein the intracellular domain comprises the amino acid sequence set forth in SEQ ID NO: 142, SEQ ID NO: 147, or SEQ ID NO: 148. A16. The aforementioned cells of A11 - A15, wherein the signal peptide comprises the amino acid sequence set forth in SEQ ID NO: 139 or SEQ ID NO: 144. A17. The aforementioned cells of A - A16, wherein the exogenous CD8 comprises a 2A sequence. A18. The aforementioned cells of A - A17, wherein the exogenous CD8 comprises a linker. A19. The aforementioned cells of A18, wherein the linker comprises the amino acid sequence set forth in SEQ ID NO: 137. A20. The aforementioned cells of A - A19, wherein the exogenous CD8 comprises a protease cleavage site. A21. The aforementioned cells of A - A20, wherein the protease cleavage site is a furin cleavage site. The cell of A-A21, wherein the exogenous TCR is a patient-derived TCR. The cell of A-A22, wherein the exogenous TCR comprises a signal sequence, a first 2A sequence and a second 2A sequence, and a TCR polypeptide sequence. The cell of A-A23, wherein the exogenous TCR recognizes a cancer antigen. The cell of A24, wherein the cancer antigen is a neoantigen. The cell of A24, wherein the cancer antigen is a patient-specific antigen. The cell of A-A26, wherein the cell is a primary cell. The cell of A-A26, wherein the cell is a patient-derived cell. The cell of A-A26, wherein the cell is a lymphocyte. The cell of A-A26, wherein the cell is a T cell. The cell of A-A26, wherein the cell is a naive T cell. The cell of A31, wherein the cell is CD45RA+, CD62L+, CD28+, CD95-, CCR7+, and CD27+. The cell of A31, wherein the cell is CD45RA+, CD62L+, CD28+, CD95+, CD27+, CCR7+. The cell of A31, wherein the cell is CD45RO+, CD62L+, CD28+, CD95+, CCR7+, CD27+, CD127+. The cell of A-A34, further comprising a genetic modification for enhancing cell persistence and / or enhancing memory cell differentiation. The cell of A-A35, wherein the killing activity of the cell is increased by about 10% to about 500% compared to the killing activity of a cell without exogenous CD8. The cell of A-A36, wherein the proliferation of the cell upon binding of the TCR to the antigen is increased by about 10% to about 500% compared to the proliferation of a cell without exogenous CD8. The cell of A-A37, wherein the secretion of inflammatory cytokines upon binding of TCR to an antigen by the cell is increased between about 10% and about 500% as compared to the secretion by cells without exogenous CD8. The cell of A-A38, wherein the LCK affinity of the cell is increased between about 10% and about 500% as compared to the LCK affinity of cells without exogenous CD8. The cell of A-A39, wherein the persistence of the cell is increased between about 10% and about 500% as compared to the persistence of cells without exogenous CD8. The cell of A-A40, wherein the tumor infiltration ability of the cell is increased between about 10% and about 500% as compared to the tumor infiltration ability of cells without exogenous CD8. The cell of A-A41, wherein the exogenous TCR is a CD8-dependent TCR. The cell of A-A41, wherein the exogenous TCR is a CD8-independent TCR. The cell of A-A43, wherein the exogenous CD8 is encoded by CD8 construct 1, CD8 construct 2, CD8 construct 3, or CD8 construct 4. The cell of A-A43, 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. A46. The exogenous CD8 consists of 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), and a CD8β intracellular domain (SEQ ID NO: 147); or 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 CD4 intracellular domain (SEQ ID NO: 148), and the aforementioned cells of A - A43.

[0124] B. In certain non - limiting embodiments, the subject matter of the present disclosure provides a method of 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 homology arm and a second homology arm homologous to a first target nucleic acid sequence and a second target nucleic acid sequence, a TCR gene sequence located between the first homology arm and the second homology arm, and a CD8 gene sequence located between the first homology arm and the second homology arm, and further comprising the step of recombining the HR template nucleic acid into an endogenous locus of the cell. B1. 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 different codons from each other, and the aforementioned method of B. The method according to B or B1, wherein the HR template comprises a sequence encoding the amino acid sequence Gly Ser Gly located immediately upstream of the first, second, and / or third 2A coding sequences. The method according to B1 or B2, wherein the HR template further comprises a sequence encoding a furin cleavage site located upstream of the first, second, and / or third 2A coding sequences. The method according to B - B3, 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. The method according to B - B4, wherein the HR template comprises a second TCR sequence located between the third 2A coding sequence and the second homology arm. The method according to B5, wherein 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. The method according to B - B6, wherein the HR template comprises a second CD8 gene sequence located between the first CD8 gene sequence and the second 2A coding sequence. The method according to B7, wherein the 2A coding sequence is located between the first CD8 gene sequence and the second CD8 gene sequence. The method according to B7 or B8, wherein the sequence encoding the amino acid sequence Gly Ser Gly is located between the first CD8 gene sequence and the second CD8 gene sequence. The method according to B7 - B9, wherein the sequence encoding the furin cleavage site is located between the first CD8 gene sequence and the second CD8 gene sequence. The method according to B - B10, wherein the CD8 gene sequence comprises a sequence encoding an extracellular domain, a sequence encoding an intracellular domain, a sequence encoding an intracellular domain, fragments thereof, or combinations thereof. The method according to B11, wherein the sequence encoding the extracellular domain comprises a sequence encoding a CD8α extracellular domain or a CD8β extracellular domain. The method of B11 or B12, wherein the sequence encoding the transmembrane domain comprises a sequence encoding the CD8α transmembrane domain or the CD8β transmembrane domain. The method of B11 - B13, wherein the sequence encoding the intracellular domain comprises a sequence encoding the CD8α intracellular domain or the CD8β intracellular domain. The method of B11 - B14, wherein the sequence encoding the intracellular domain comprises a sequence encoding the CD4 intracellular domain. The method of B11 - B15, wherein the CD8 gene sequence comprises a sequence encoding the CD8α extracellular domain, the CD8α transmembrane domain, and the CD8α intracellular domain. The method of B11 - B15, wherein the CD8 gene sequence comprises a sequence encoding the CD8β extracellular domain, the CD8β transmembrane domain, and the CD8β intracellular domain. The method of B11 - B15, wherein the CD8 gene sequence comprises a sequence encoding the CD8α extracellular domain, the CD8α transmembrane domain, and the CD8β intracellular domain. The method of B11 - B15, wherein the CD8 gene sequence comprises a sequence encoding the CD8α extracellular domain, the CD8α transmembrane domain, and the CD4 intracellular domain. The method of B7 - B19, wherein 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. The method of B4 - B20, wherein the signal sequence is a CD8 signal sequence, a human growth hormone signal sequence, a fragment thereof, or a combination thereof. The method of B - B21, wherein the first homology arm and the second homology arm of the HR template are each about 300 bases to about 2000 bases in length. The method of B - B22, wherein the first homology arm and the second homology arm of the HR template are each about 600 bases to about 2000 bases in length. The method of B-B23, wherein the exogenous TCR is a patient-derived TCR. The method of B-B24, wherein the exogenous TCR comprises a signal sequence, a first 2A sequence and a second 2A sequence, and a TCR polypeptide sequence. The method of B-B25, wherein the exogenous TCR recognizes a cancer antigen. The method of B26, wherein the cancer antigen is a neoantigen. The method of B26, wherein the cancer antigen is a patient-specific antigen. The method of B-B28, wherein the HR template is non-viral. The method of B-B29, wherein the HR template is circular DNA. The method of B-B29, wherein the HR template is linear DNA. The method of B-B31, wherein the introduction occurs via electroporation. The method of B-B32, wherein the recombination comprises cleavage of an endogenous locus by a nuclease; and recombination of the HR template nucleic acid sequence into the endogenous locus by homology-directed repair. The method of B33, wherein the nuclease is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) family nuclease, or a derivative thereof. The method of B34, further comprising a gRNA. The method of B-B35, further comprising culturing the cells. The method of B36, wherein the culturing is performed in the presence of at least one cytokine. The method of B36 or B37, wherein the culturing is performed in the presence of IL2, IL7, IL15, or any combination thereof. The method of B36 or B37, wherein the culturing is performed in the presence of IL7 and IL15. The method of B-B39, further comprising a genetic modification that enhances cell persistence and / or enhances memory cell differentiation. The method of B-B40, wherein the cell is a primary cell. The method of B-B40, wherein the cell is a patient-derived cell. The method of B-B40, wherein the cell is a lymphocyte. The method of B-B40, wherein the cell is a T cell. The method of B-B40, wherein the cell is a naive T cell. The method of B45, wherein the cell is CD45RA+, CD62L+, CD28+, CD95-, CCR7+, and CD27+. The method of B45, wherein the cell is CD45RA+, CD62L+, CD28+, CD95+, CD27+, CCR7+. The method of B45, wherein the cell is CD45RO+, CD62L+, CD28+, CD95+, CCR7+, CD27+, CD127+. The method of B-B48, wherein the killing activity of the cell is increased by about 10% to about 500% compared to the killing activity of a cell not having a CD8 gene sequence. The method of B-B49, wherein the proliferation of the cell upon binding of the TCR to the antigen is increased by about 10% to about 500% compared to the proliferation of a cell not having a CD8 gene sequence. The method of B-B50, wherein the secretion of pro-inflammatory cytokines upon binding of the TCR to the antigen by the cell is increased by about 10% to about 500% compared to the secretion by a cell not having a CD8 gene sequence. The method of B-B51, wherein the LCK affinity of the cell is increased by about 10% to about 500% compared to the LCK affinity of a cell not having a CD8 gene sequence. The method of B-B52, wherein the persistence of the cell is increased by about 10% to about 500% compared to the persistence of a cell not having a CD8 gene sequence. The method of B-B53, wherein the tumor infiltration ability of the cell is increased by about 10% to about 500% compared to the tumor infiltration ability of a cell not having a CD8 gene sequence. B55. The method of B-B54, wherein the TCR gene encodes a CD8-dependent TCR. B56. The method of B-B54, wherein the TCR gene encodes a CD8-independent TCR. B57. The method of B-B56, wherein the CD8 gene sequence is encoded by CD8 construct 1, CD8 construct 2, CD8 construct 3, or CD8 construct 4. B58. The method of B-B56, 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, and a CD8β intracellular domain; or a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain. B59. The method of B-B56, 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), 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), and a CD8β intracellular domain (SEQ ID NO: 147); or 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 CD4 intracellular domain (SEQ ID NO: 148).

[0125] C. In certain non-limiting embodiments, the subject matter of the present disclosure provides cells modified by the method of B-B57.

[0126] D. In certain non-limiting embodiments, the subject matter of the present disclosure provides a composition comprising an effective amount of the cells of A-A46 or the cells of C. D1. The aforementioned composition of D, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable additive. D2. The aforementioned composition of D or D1, wherein the composition is administered to a patient in need of treatment for the treatment of cancer. D3. The aforementioned composition of D-D2, wherein the composition comprises a cryopreservative. D4. The aforementioned composition of D-D3, wherein the composition comprises serum albumin. D5. The aforementioned composition of D-D4, wherein the composition comprises Plasma-Lyte A, HSA, and CryoStor CS10.

[0127] E. In certain non-limiting embodiments, the subject matter of the present disclosure provides a method of treating cancer in a subject in need of treatment, the method comprising administering a therapeutically effective amount of the cells of A-A46, or the cells of claim C, or the composition of D-D5. E1. The aforementioned method of E, wherein a non-myeloablative lymphocyte depletion regimen is administered to the subject prior to administering the therapeutically effective amount of cells. E2. The aforementioned method of E or E1, wherein the cancer is a solid tumor. E3. The aforementioned method of E or E1, wherein the cancer is a liquid tumor. E4. The aforementioned method of E2, wherein the solid tumor is selected from the group consisting of melanoma, thoracic 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 cancer, cholangiocarcinoma, renal cell cancer, testicular cancer, sarcoma, and colorectal cancer. E5. The aforementioned method of E3, wherein the liquid tumor is selected from the group consisting of follicular lymphoma, leukemia, and multiple myeloma.

[0128] F. In certain non-limiting embodiments, the subject matter of the present disclosure provides a kit comprising cells of A-A46, reagents for practicing the method of B-B57, cells of C, or compositions of D-D5. F1. The aforementioned kit of F, wherein the kit further comprises written instructions for treating cancer.

[0129] G. In certain non-limiting embodiments, the subject matter of the present disclosure provides cells comprising an exogenous T cell receptor (TCR); and an exogenous CD8 comprising 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.

[0130] H. In certain non-limiting embodiments, the subject matter of the present disclosure provides cells comprising an exogenous T cell receptor (TCR); and an 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), and a CD8β intracellular domain (SEQ ID NO: 147); or 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 CD4 intracellular domain (SEQ ID NO: 148).

[0131] I. In certain non-limiting embodiments, the subject matter of the present disclosure is a method of 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 homology arm and a second homology arm that are homologous to a first target nucleic acid sequence and a second target nucleic acid sequence, respectively; a TCR gene sequence located between the first homology arm and the second homology arm; a CD8 gene sequence located between the first homology arm and the second homology arm; and 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; or a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain.

[0132] J. In certain non-limiting embodiments, the subject matter of the present disclosure is a method of modifying a cell, the method comprising introducing into the cell a homologous recombination (HR) template nucleic acid sequence, wherein the HR template comprises a first homology arm and a second homology arm that are homologous to a first target nucleic acid sequence and a second target nucleic acid sequence, respectively; a TCR gene sequence located between the first homology arm and the second homology arm; and a CD8 gene sequence located between the first homology arm and the second homology arm; and 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), 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), and a CD8β intracellular domain (SEQ ID NO: 147); or 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 CD4 intracellular domain (SEQ ID NO: 148).

[0133] K. In certain non-limiting embodiments, the subject matter of the present disclosure is a composition comprising a cell, wherein the cell comprises an exogenous T cell receptor (TCR) and an 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.

[0134] I. In certain non-limiting embodiments, the subject matter of the present disclosure is a composition comprising a cell, wherein the cell comprises an exogenous T cell receptor (TCR) and an 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), and a CD8β intracellular domain (SEQ ID NO: 147); or 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 CD4 intracellular domain (SEQ ID NO: 148).

[0135] M. In certain non-limiting embodiments, the subject matter of the present disclosure is a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of cells, where the cells comprise an exogenous T cell receptor (TCR) and an exogenous CD8, and where 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, provides a method.

[0136] N. A method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of cells, where the cells comprise an exogenous T cell receptor (TCR) and an exogenous CD8, and where 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), and a CD8β intracellular domain (SEQ ID NO: 147); or 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 CD4 intracellular domain (SEQ ID NO: 148), provides a method.

Example

[0137] Next are examples of the methods and compositions of the present invention. It is understood that various other embodiments can be implemented, given the general description provided above.

[0138] Example 1 Generation of NeoTCR Products Using neoepitope-specific TCRs identified by the imPACT isolation technique described in PCT / US2020 / 17887 (incorporated herein by reference in its entirety), homologous recombination (HR) DNA templates were generated. These HR templates were transfected into primary human T cells in tandem with site-specific nucleases (see FIGS. 1A - 1C). By one-step non-viral precision genome modification, the endogenous TCR was seamlessly replaced with the patient's neoepitope-specific TCR expressed by the endogenous promoter. The TCR expressed on the surface has a completely natural sequence. The accuracy of neoTCR-T cell genome modification was evaluated by target locus amplification (TLA) for off-target integration hotspots or translocations, and also by next-generation sequencing-based off-target cleavage assays, and no evidence of unintended results was found.

[0139] As shown in FIGS. 1A-1C, a construct containing the gene of interest was inserted into the endogenous locus. This was achieved using a homologous repair template containing the coding sequence of the gene of interest flanked by left and right HR arms. In addition to the HR arms, the gene of interest was flanked between a 2A peptide, a protease cleavage site upstream of the 2A peptide for removing the 2A peptide from the translated gene upstream of the gene of interest, and a signal sequence (FIG. 1B). When integrated into the genome, the gene of the expression gene cassette of interest was transcribed as a single messenger RNA. During translation of this gene of interest in the messenger RNA, the adjacent region was cleaved from the gene of interest by the self-cleaving 2A peptide, and the protease cleavage site was cleaved for removal of the 2A peptide upstream of the translated gene of interest (FIG. 1C). In addition to the 2A peptide and protease cleavage site, a gly-ser-gly (GSG) linker was inserted in front of each 2A peptide to further facilitate separation of the gene of interest from other elements within the expression cassette.

[0140] The P2A peptide was determined to be superior to other 2A peptides of cell products for its efficient cleavage. Thus, two (2) P2A peptides and codon branching were used to express the gene of interest without introducing any exogenous epitopes from the remaining amino acids at either end of the gene of interest from the P2A peptide. The advantage of gene-edited cells without exogenous epitopes (i.e., no P2A peptide amino acids flanking both sides of the gene of interest) is that the immunogenicity is strongly reduced, and the likelihood that a cell product containing gene-edited cells that elicit an immune response against the gene-edited cells will be injected into a patient is low.

[0141] As described in PCT / US / 2018 / 058230, NeoTCR was integrated into the TCRα locus of T cells. Specifically, a homologous repair template containing the NeoTCR coding sequence flanked by left and right HR arms was used. In addition, the endogenous TCRβ locus was disrupted to express only the TCR sequence encoded by the NeoTCR construct. A general strategy was applied using circular HR templates as well as linear templates.

[0142] The target TCRα locus (Cα) is shown together with the plasmid HR template, and the resulting edited sequences and downstream mRNA / protein products are shown in FIGS. 1B and 1C. The target TCRα locus (endogenous TRAC) and its CRISPR Cas9 target sites (cleavage sites indicated by horizontal stripes and arrows) are shown (FIGS. 1A-1C). A circular plasmid HR template containing a polynucleotide encoding NeoTCR is located between the left and right homology arms (referred to as "LHA" and "RHA", respectively). The region of TRAC introduced by the codon-optimized HR template is shown (vertical stripes). The TCRβ constant domain was derived from TRBC2, which has been shown to be functionally equivalent to TRBC1. Other elements in the NeoTCR cassette include: 2A = 2A ribosome skipping element (in a non-limiting example, the 2A peptides used in the cassette are both P2A sequences, used in combination with codon branching to eliminate any non-endogenous epitopes that occur in other forms in the translation product); P = protease cleavage site upstream of 2A that removes the 2A tag from the upstream TCRβ protein (in a non-limiting example, the protease cleavage site can be a furin protease cleavage site); SS = signal sequence (in 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 contains two adjacent homology arms that direct the insertion of the CRISPR Cas9 nuclease RNP complexed with TCRα guide RNA into the TCRα genomic locus. These homology arms (LHA and RHA) are adjacent to the neoE-specific TCR sequence of the NeoTCR expression gene cassette. The protease cleavage site used in this example was a furin protease cleavage site, but any suitable protease cleavage site known to those skilled in the art can be used. Similarly, HGH was the signal sequence selected for this example, but any signal sequence known to those skilled in the art can be selected and used based on the desired transport.

[0143] Once integrated into the genome (Figure 1C), the NeoTCR expression gene cassette is transcribed as a single messenger RNA from the endogenous TCRα promoter, still containing a portion of the endogenous TCRα polypeptide from its individual T cells (Figure 1C). During ribosomal polypeptide translation of this single NeoTCR messenger RNA, the NeoTCR sequence is separated from the endogenously CRISPR-disrupted TCRα polypeptide by self-cleavage at the P2A peptide (Figure 1C). The encoded NeoTCRα and NeoTCRβ polypeptides are also separated from each other by cleavage by the endogenous cellular human furin protease and through a second self-cleaving P2A sequence motif contained within the NeoTCR expression gene cassette (Figure 1C). The NeoTCRα and NeoTCRβ polypeptides are separately targeted to the endoplasmic reticulum by signal leader sequences (derived from human growth hormone, HGH) for multimer construction and transport of the NeoTCR protein complex to the T cell surface. Inclusion of the furin protease cleavage site promotes removal of the 2A sequence from the upstream TCRβ chain, reducing potential interference with TCRβ function. Inclusion of a gly-ser-gly linker in front of each 2A (not shown) further promotes separation of the three polypeptides.

[0144] In addition, three repeat protein sequences are codon-branched within the HR template to promote genome stability. Two P2As are codon-branched within the TCR gene cassette to promote stability of the NeoTCR cassette sequence introduced into the genome of ex vivo modified T cells, and the two HGH signal sequences are likewise codon-branched with each other. Similarly, the re-introduced 5’ end (vertical stripe) of the TRAC exon 1 reduces the likelihood that the entire cassette will be lost over time by removing the intervening sequence of two direct repeats. In addition to the NeoTCR product, this method can be used for any CD8 product.

[0145] In-Out PCR was used to confirm the accurate targeting integration of the NeoE TCR cassette. The agarose gel shows the results of PCR using primers specific to the integrated cassette, producing products of the expected size only for cells treated with both the nuclease and the DNA templates (KOKI and KOKIKO), demonstrating site-specific and accurate integration.

[0146] Furthermore, target locus amplification (TLA) was used to confirm the specificity of the target integration. By cross-linking, ligation, and using primers specific to the NeoTCR insert fragment, the sequences around the integration site were obtained. Reads mapped to the genome were binned at 10 kb intervals. Significant read depths were obtained only around the target site of the integration site on chromosome 14, indicating no evidence of common off-target insertion sites.

[0147] Antibody staining of endogenous TCR and peptide-HLA staining of neoTCR revealed that the modification led to a high frequency of knock-in of neoTCR, with some TCR cells and a small number of WT T cells remaining. The knock-in was demonstrated by the expression of neoTCR in the absence of an exogenous promoter. Similar results were obtained when the modification was performed multiple times using the same neoTCR. Thus, efficient and consistent expression of neoTCR and knockout of endogenous TCR in the modified T cells were achieved.

[0148] Example 2 Preparation of CD8 Product 1 [T cell isolation and editing] CD4 and CD8 T cells were isolated from healthy donor PBMCs using Miltenyi Prodigy or Miltenyi MACS separation columns according to the manufacturer's instructions. CD4 and CD8 T cells selected as positive (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 with TexMACS (Miltenyi) + 10% human AB serum (Valley Biomedical), and seeded at a density of 2 × 106 cells per mL in TexMACS + 3% human AB serum (medium). One day after thawing, or immediately if used fresh, the cells were washed and reseeded at a density of 1.46 × 106 cells per mL in medium + 12.5 ng / mL IL7 + 12.5 ng / mL IL15 + TransACT T cell activation reagent at a ratio of 1:17.5 by volume (all reagents from Miltenyi). Two days after activation, the T cells were electroporated with i) a plasmid for the production of NeoTCR products (see, e.g., FIG. 1B) or ii) CD8 construct 1 (consisting of the coding sequence of CD8α adjacent to the P2A site upstream of the neoTCR beta and alpha sequences and gRNA-Cas9 RNP targeting the TCR alpha and beta loci; see, e.g., FIG. 2A). An exemplary expression construct of CD8 construct 1 is shown in FIG. 11A. The T cells were electroporated using a Lonza X unit and program EO-115 in a 100 μL cuvette. The T cells were grown in medium supplemented with 12.5 ng / mL of IL7 + 12.5 ng / mL of IL15. The supplemented medium was changed every 2-3 days until the end of the study 13 days after activation.

[0149] [Preparation of comPACT and comPACT-dextramers] Neoantigen-specific peptide-HLA complex polypeptides (each "comPACT") were prepared according to the method described in PCT / US2019 / 025415, which is hereby incorporated by reference in its entirety. ComPACT-dextramer complexes were generated for labeling of neoTCR-expressing T cells. Biotinylated comPACT protein was incubated with streptavidin-conjugated fluorophore for 10 minutes at room temperature (RT). Biotin-40-dextran (NANOCS) was added to the mixture and incubated for an additional 10 minutes at RT. ComPACT-dextramers were stored at 4°C.

[0150] [Confirmation of comPACT binding to neoTCR-edited T cells] T cells were stained for flow cytometry. Cells were first stained with a viability dye for 20 minutes at 4°C, then washed and stained with comPACT-dextramer for 10 minutes at 4°C. Surface antibodies (anti-CD8α, anti-CD8β, anti-CD4) were added to the cell and comPACT-dextramer suspension, and the cells were incubated for an additional 20 minutes at 4°C. Next, the cells were washed and fixed with an intracellular fixation buffer (BD Biosciences). All cells were acquired on an Attune NxT flow cytometer (ThermoFisher Scientific), and the data were analyzed with either FCS Express or FlowJo.

[0151] [Cytometry Bead Array (CBA)] Streptavidin-coated plates (Eagle Biosciences) were washed three times with wash buffer (PBS supplemented with 1% BSA and 0.05% Tween 20), and then coated with comPACT at various concentrations ranging from 100 - 0.01 ng / well. Wells without comPACT and wells coated with mismatched comPACT 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 Tween 20. T cells were washed twice with TexMACS supplemented with 3% human AB serum and resuspended in TexMACS supplemented with 3% human AB serum and 1X penicillin-streptomycin solution at 1 million cells / mL. T cells were seeded onto comPACT-coated plates at 100 μL / well and incubated at 37°C, 5% CO2. After 24 hours, the supernatants were collected and cytokine concentrations were analyzed using the BD Cytometry Bead Array (CBA) Human Th1 / Th2 Cytokine Kit II (Catalog No. 551809) according to the manufacturer's protocol. Capture beads were mixed with the culture supernatants, protected from light with the detection reagent, incubated at RT for 3 hours, washed, and resuspended in wash buffer. Samples were assayed on an Attune NxT flow cytometer and the data were analyzed with FlowJo. The EC50 represents the concentration of homologous comPACT that induces 50% of the maximal response and is calculated using least-squares fitting of IFNγ secretion across a range of comPACT concentrations.

[0152] [Intracellular Staining] T cells were stained for flow cytometry on the indicated day. T cells were first stained with a viability dye for 20 minutes at 4°C, then washed and incubated with surface antibodies (anti-CD8α, anti-CD8β, anti-CD4) for an additional 20 minutes at 4°C. Next, the T cells were washed and permeabilized for intracellular staining. The T cells were stained with anti-2A peptide, or anti-IFNγ, anti-TNF, or anti-IL2 in permeabilization buffer for 20 minutes at 4°C. The T cells were fixed with intracellular fixation buffer (BD Biosciences). Samples were assayed on an Attune NxT flow cytometer (ThermoFisher Scientific) and the data were analyzed with FCS Express or FlowJo.

[0153] [T Cell Proliferation Assay] Edited CD4 and CD8 T cells were labeled with e450 proliferation dye (eBioscience) according to the manufacturer's instructions. The labeled cells were stimulated on comPACT-coated plates at the above concentration ranges. T cells were collected over 48 - 96 hours and proliferation was analyzed by measuring the dilution of the e450 dye.

[0154] [T Cell Killing Assay] HLA-matched cell lines were pulsed with cognate neoantigen peptides or mismatched peptides at 37°C in 5% CO2 for 1 hour. After the cells were washed three times with medium to remove unbound peptides, they were co-cultured with edited CD4 and CD8 T cells labeled with the above e450 proliferation dye. The co-cultures were incubated at 37°C in 5% CO2 for 48 hours before collection. The cells were washed and stained with a fixable viability dye to determine the killing efficiency. The e450 proliferation dye was used to distinguish between edited T cells and target cells.

[0155] [Generation and Verification of CD8 Product 1] Synthesize an expression construct consisting of the coding sequence (CDS) of CD8α adjacent to the P2A site upstream of the neoTCR beta and alpha sequences. Briefly, the CDS of human CD8α is synthesized with a GSG linker and a P2A site adjacent to the upstream of the restriction site. Incubate the synthesized CD8α construct with the target neoTCR expression vector and restriction enzymes, and ligate them together to generate the final HDR construct. CD8 construct 1 was electroporated with gRNA-Cas9RNP targeting the TCRα and β loci. Using a model neoTCR (e.g., TCR097) known to bind dextramers among CD8 T cells but not among CD4 T cells, it was demonstrated that the expression of the CD8α transgene enabled these TCRs to bind dextramers among CD4 T cells.

[0156] To test the efficiency of gene transfer and expression of CD8α on the surface of CD4 T cells, the modified CD4 T cells were stained with anti-CD8α antibody, and the surface expression of the transgene was confirmed by flow cytometry as described above. Using a NeoTCR (e.g., TCR097) known to bind dextramers only among CD8 T cells, it was demonstrated that the expression of the CD8α transgene enabled these TCRs to bind dextramers among CD4 T cells. See FIGS. 13A, 13B, 14, 15A, and 15B. Furthermore, the CD8α expression in wild-type and genetically engineered CD8α T cells was evaluated to determine whether the addition of the CD8α transgene increased the surface level of CD8α on CD8 T cells. CD4 T cells modified to express the CD8α transgene were double-positive for CD4 and CD8α.

[0157] CD8 T cells were also modified to express the CD8α transgene and characterized as described above. FIGS. 15A and 15B. The relative CD8α gene expression was also quantified by RT-qPCR and compared to control unmodified CD8 T cells. CD8 T cells expressing the CD8α transgene had higher levels than the endogenous levels of CD8α expression.

[0158] [Effect of CD8 expression on T cell proliferation upon encounter with cognate antigen] The edited T cells were stained with the proliferation dye as described above. After staining, the T cells were stimulated with cognate comPACT protein at a range of concentrations. After 48 - 72 hours, the T cells were collected and stained with anti - CD4, anti - CD8, and anti - 2A peptide as described above. The edited T cells were identified by 2A expression. Proliferation of CD4 and CD8 T cells was determined by quantifying the dilution of the 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 than CD4 T cells lacking CD8α expression.

[0159] [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 product and CD8 product 1 were stimulated with cognate comPACT at various concentrations in the presence of brefeldin A for 5 hours. After stimulation, the T cells were stained with anti - CD4 and anti - CD8α. The 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 than neoTCR CD4 T cells lacking CD8α expression.

[0160] [Effect of CD8α expression on killing activity upon encounter with cognate antigen] To evaluate effector function, the edited CD4 and CD8 T cells were cultured with HLA - compatible target cells pulsed with cognate peptide as described above. CD4 and CD8 T cells were edited separately to evaluate the ability of CD4 T cells expressing CD8α to kill target cells. NeoTCR CD4 T cells expressing the CD8α transgene killed a greater percentage of target cells presenting cognate peptide than neoTCR CD4 T cells lacking CD8α expression.

[0161] Example 3 Preparation of CD8 Product 2 CD4 T cells modified to express CD8α lack the expression of CD8β. 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 along with CD8α (i.e., CD8 Product 2). An additional construct was made that includes CD8β adjacent to the P2A site, CD8α adjacent to the P2A site, followed by the previously described TRB and TRA alleles. CD4 and CD8 T cells expressing CD8α and CD8β were evaluated using the same assay as described above. An exemplary expression construct containing CD8α and CD8β sequences is shown in Figure 11B.

[0162] The expression of CD8β was also evaluated in the edited CD4 T cells. NeoTCR CD4 T cells expressing CD8α and CD8β transgenes proliferated in response to a lower concentration of cognate comPACT than CD4 T cells expressing only the CD8α transgene. NeoTCR CD4 T cells expressing CD8α and CD8β transgenes also produced effector cytokines in response to a lower concentration of cognate comPACT than neoTCR CD4 T cells expressing only the CD8α transgene. Also, neoTCR CD4 T cells expressing CD8α and CD8β transgenes killed a greater percentage of target cells presenting cognate peptides than neoTCR CD4 T cells expressing only the CD8α transgene.

[0163] Example 4 Preparation of Chimeric CD8α and CD8β Constructs and CD8 Products 3 and 4 To ensure efficient editing of T cells and expression of neoTCR, a chimeric protein was created consisting of the coding sequence of the extracellular domain and transmembrane domain of CD8α linked to the intracellular domain of CD8β. An exemplary expression construct having the extracellular domain and transmembrane domain of CD8α linked to the intracellular domain of the CD8β sequence (i.e., CD8 product 3) is shown in Figure 11C.

[0164] Furthermore, the intracellular domain of CD4 has a higher affinity for LCK than CD8 (Irie et al., 1998). Therefore, a second chimeric protein was created that includes the coding sequence of the extracellular domain and transmembrane domain of CD8α linked to the intracellular domain of CD4. An exemplary expression construct having the extracellular domain and transmembrane domain of CD8α linked to the intracellular domain of the CD4 sequence (i.e., CD8 product 4) is shown in Figure 11D. CD4 and CD8 T cells expressing CD8 products 3 and 4 were evaluated using the same assay as described above.

[0165] NeoTCR CD4 T cells expressing the CD8α-CD8β-ID transgene proliferated in response to a lower concentration of cognate comPACT than CD4 T cells expressing only the CD8α transgene. NeoTCR CD4 T cells expressing the CD8α-CD8β-ID transgene produced effector cytokines in response to a lower concentration of cognate comPACT than neoTCR CD4 T cells expressing only the CD8α transgene. NeoTCR CD4 T cells expressing the CD8α-CD8β-ID transgene killed a greater percentage of target cells presenting the cognate peptide than neoTCR CD4 T cells expressing the CD8α-CD8β-ID transgene.

[0166] NeoTCR CD4 T cells expressing the CD8α-CD4-ID transgene proliferated in response to lower concentrations of cognate comPACT than 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 than neoTCR CD4 T cells expressing the CD8α-CD8β-ID transgene.

[0167] NeoTCR CD4 T cells expressing the CD8α-CD4-ID transgene killed a greater percentage of 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-ID transgene killed a greater percentage of target cells presenting cognate peptides than neoTCR CD8 T cells lacking the transgene.

[0168] Example 5 CD8 products have increased sensitivity to neoE-HLA target recognition and trigger pro-inflammatory and cytotoxic functions 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 genome-edited to express the cloned MHC-I neoTCR alone or to include the modification of a heterologous CD8 co-receptor in gene-edited T cells. Flow cytometry analysis was used to evaluate the surface expression of each of the neoTCR and the heterologous CD8 co-receptor (i.e., the CD8 and CD4 components of CD8 constructs 1-4). Rescue of neoTCR binding between CD4 T cells to lower affinity CD8-dependent neoTCRs was observed. Importantly, in response to stimulation with cognate antigen, CD107a and intracellular IFNγ staining revealed a 10- to 100-fold increase in the sensitivity of MHC-I neoTCR-induced effector functions by CD4 T cells without affecting specificity. No changes in functionality or sensitivity were seen in CD8 T cells with the expression of additional CD8 co-receptors.

[0169] These results demonstrate that by simultaneously and precisely genome-editing CD8 co-receptors together with CD8-dependent MHC-I neoTCRs in CD4 T cells (i.e., CD8 products 1-4), their sensitivity to neoE-HLA target recognition is significantly enhanced, triggering pro-inflammatory and cytotoxic functions without compromising antigen specificity.

[0170] Example 6 Generation and Design of CD8 Products with Various LCK Affinities As described herein, four classes of CD8 products were generated: 1. CD8α homodimer (CD8 construct 1) 2. CD8α-P2A-CD8β (CD8 construct 2) 3. CD8α containing the CD8β intracellular domain (CD8 construct 3) 4. CD8α homodimer containing the CD4 intracellular domain (CD8 construct 4) As shown in Figure 10, these CD8 constructs and the resulting CD8 products were designed to allow for various degrees of LCK affinity. As predicted, CD8 product 1 had the lowest LCK affinity, followed by CD8 product 2, CD8 product 3, and CD8 product 4 in that order, with CD8 product 4 shown to have the highest LCK affinity. Based on the high affinity of CD8 product 4, this product was used in a cytotoxicity assay to demonstrate an increase in the cytotoxic ability of CD8 products 1 - 4 compared to the NeoTCR product.

[0171] CD4+ T cells were modified as described herein to express CD8 product 4 (the product containing TCR097 as NeoTCR) described in Figures 2D and 3D. The SW620 cell line was modified to heterologously express the R20Q mutation (the cognate antigen of TCR097). CD8 product 4 expressing NeoTCR097 was combined with SW620 heterologous cells. As shown in Figures 13A and 13B, CD8 product 4 resulted in substantially better killing of the cognate antigen expressed by SW620 cells than the NeoTCR product also expressing TCR097. The experiment shown in Figure 13A was performed at an E:T ratio of 1:1, and as shown in the graph, the NeoTCR product expressing TCR097 showed no effect in killing the cognate antigen expressed by SW620 cells. The experiment shown in Figure 13B was performed at an E:T ratio of 2:1, and the NeoTCR product expressing TCR097 showed some ability to kill the cognate antigen expressed by SW620 cells, but it was clear from the experiment that CD8 product 4 expressing TCR097 had an excellent effect.

[0172] Using the data provided in FIGS. 13A and 13B, the same modified CD4+ cells used in the above experiments were also tested in the SW620 cell line modified to homozygously express the R20Q mutation. In this experiment, high expression of the cognate antigen was able to complement the low-affinity NeoTCR097, and both the NeoTCR product expressing TCR097 and the CD8 product 4 expressing TCR097 showed an effect on the killing of SW620 cells. However, high expression of the cognate antigen in homozygous SW620 cells is not physiologically relevant, and this experiment serves to highlight the ability to rescue NeoTCR products with low-affinity TCRs that cannot effectively participate in and kill tumor cells by modifying them to also contain a CD8α homodimer with a CD4 intracellular domain (i.e., CD8 product 4).

[0173] As a final control and proof of efficacy of the CD8 product, CD8 T cells were also transfected to express a CD8α homodimer with NeoTCR097 and a CD4 intracellular domain (i.e., CD8 product 4 using CD8 T cells instead of CD4 T cells). As shown in the upper graphs of FIGS. 15A and 15B, CD8 product 4 resulted in substantially better killing of SW620 cells than the NeoTCR product when the product was made from CD4 T cells. However, when CD8 product 4 and the NeoTCR product were made from CD8 T cells (lower graphs of FIGS. 15A and 15B), the ability of the NeoTCR product was rescued due to endogenous CD8 expression in CD8 T cells. Although an overlay of the graphs is not shown, the CD8 product 4 in CD4 T cells shown in the upper graphs of FIGS. 15A and 15B appears to have a superior effect compared to the CD8 product 4 of CD8 T cells in the lower two graphs; note also that this suggests the superior ability of the CD8 products described herein to bind to the cognate antigen of tumor cells to cause tumor death and provide an effective treatment for cancer patients in need of treatment.

[0174] Example 6 CD8 Product Increased the Sensitivity of CD4 T Cells While Maintaining NeoTCR Sensitivity To confirm that the CD8 constructs were properly expressed, CD8 products 1, 2, 3, and 4 were tested to determine surface expression of CD8α. It was shown that each of CD8 products 1, 2, 3, and 4 exhibited normal CD8α surface expression. Representative data for CD8 product 4 are shown in Figure 16.

[0175] It was also important to determine how the CD8 constructs affected the sensitivity of CD4 T cells and the specificity of the NeoTCR expressed in the CD8 products for cognate antigens. Sensitivity experiments were performed and it was shown that CD8 products 1-4 exhibited an increase in CD4 T cell sensitivity (Figure 17A). Specificity experiments were also performed with CD8 products 1-4. CD8 products 1, 2, 3, and 4 were made using NeoTCR097. Experiments were performed to test CD8 products 1, 2, 3, and 4 (expressing NeoTCR097) to evaluate the specificity of these products for the cognate antigen of NeoTCR097. As shown in Figure 17B, CD8 products 1, 2, 3, and 4 (expressing NeoTCR097) were specific for the cognate antigen of NeoTCR097 and showed no activity when exposed to mismatched antigens. Specificity was determined by INFγ and CD107 production, which are evidence of T cell activation. Thus, the CD8 products described herein have increased sensitivity to CD4 T cells and maintain their specificity for the cognate antigens of the expressed NeoTCR compared to NeoTCR products expressing the same NeoTCR.

[0176] Finally, experiments were conducted to examine the effect of the CD8 constructs on CD8-dependent and CD8-independent NeoTCRs. Since CD8 cells are modified to express CD8α, it was predicted that CD8-dependent NeoTCRs would show an increase in sensitivity to cognate antigen, while CD8-independent NeoTCRs would not show an increase in sensitivity due to NeoTCR independence. However, it was shown that NeoTCR products 1, 2, 3, and 4 significantly increased the sensitivity of both CD8-dependent (e.g., NeoTCR097) and CD8-independent (e.g., NeoTCR089) NeoTCRs. Thus, it was shown that CD8 constructs 1, 2, 3, and 4 can improve NeoTCR binding and T cell killing of tumor cells by cognate NeoTCR antigens for all NeoTCRs, regardless of whether they are CD8-dependent or CD8-independent.

[0177] The present invention has been described in some detail and with some particularity with respect to some of the described embodiments, but is not intended to be limited to any such details or embodiments or any particular embodiment, and in light of the prior art, provides the broadest possible interpretation of such claims, and thus should be construed with reference to the appended claims so as to effectively encompass the intended scope of the present invention.

[0178] All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the section headings, materials, methods, and examples are illustrative only and not intended to be limiting.

Claims

1. a. An exogenous T cell receptor (TCR); and b. An exogenous CD8 comprising a T cell, wherein the exogenous CD8 comprises a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain.

2. The T cell according to claim 1, wherein the exogenous CD8 comprises a signal peptide, a 2A sequence, a linker, a protease cleavage site, a furin cleavage site, or a combination thereof, optionally, the signal peptide is a CD8 signal peptide, and optionally, the linker comprises the amino acid sequence set forth in SEQ ID NO:

137.

3. The exogenous TCR is: (a) a TCR derived from a patient; (b) comprises a signal sequence, a first 2A sequence and a second 2A sequence, and a TCR polypeptide sequence; or (c) recognizes a cancer antigen, optionally, the cancer antigen is a neoantigen or a patient-specific antigen, The T cell according to claim 1 or 2.

4. The T cell is a primary cell, a patient-derived cell, or a naive T cell, optionally, the T cell is (a) CD45RA+, CD62L+, CD28+, CD95−, CCR7+, and CD27+; (b) CD45RA+, CD62L+, CD28+, CD95+, CD27+, CCR7+; or (c) CD45RO+, CD62L+, CD28+, CD95+, CCR7+, CD27+, CD127+ The T cell according to any one of claims 1 to 3.

5. Further comprising a genetic modification for enhancing cell persistence and / or enhancing memory cell differentiation, optionally, (a) the killing activity of the T cell is increased between about 10% and about 500% compared to the killing activity of cells without exogenous CD8; (b) the proliferation of the T cell upon binding of the TCR to the antigen is increased between about 10% and about 500% compared to the proliferation of cells without exogenous CD8; (c) the secretion of pro-inflammatory cytokines upon binding of the TCR to the antigen by the cell is increased between about 10% and about 500% compared to the secretion by cells without exogenous CD8; (d) the LCK affinity of the T cell is increased between about 10% and about 500% compared to the LCK affinity of cells without exogenous CD8; (e) the persistence of the T cell is increased between about 10% and about 500% compared to the persistence of cells without exogenous CD8; and / or ​ (f)The tumor infiltration ability of the T cells is increased between about 10% and about 500% as compared to the tumor infiltration ability of cells without exogenous CD8. The T cells according to any one of claims 1 to 4. **Claim 6** The T cells according to any one of claims 1 to 5, wherein the exogenous TCR is a CD8-dependent TCR or a CD8-independent TCR. **Claim 7** The T cells according to any one of claims 1 to 6, wherein the exogenous CD8 comprises a CD8α signal peptide having the amino acid sequence of SEQ ID NO: 139, a CD8α extracellular domain having the amino acid sequence of SEQ ID NO: 140, a CD8α transmembrane domain having the amino acid sequence of SEQ ID NO: 141, and a CD4 intracellular domain having the amino acid sequence of SEQ ID NO:

148. **Claim 8** In a method for modifying T cells, (a) A step of introducing a homologous recombination (HR) template nucleic acid sequence into the T cells, wherein the HR template comprises (i) a first homology arm and a second homology arm homologous to a first target nucleic acid sequence and a second target nucleic acid sequence, optionally each about 300 bases to about 2000 bases in length, and optionally each about 600 bases to about 2000 bases in length, the first homology arm and the second homology arm; (ii) a TCR gene sequence located between the first homology arm and the second homology arm; and (iii) a CD8 gene sequence located between the first homology arm and the second homology arm, comprising a CD8α extracellular domain, a CD8α transmembrane domain, and a CD4 intracellular domain, and optionally, the CD8α extracellular domain comprises the amino acid sequence of SEQ ID NO: 140, the CD8α transmembrane domain comprises the amino acid sequence of SEQ ID NO: 141, and the CD4 intracellular domain comprises the amino acid sequence of SEQ ID NO: 148, the CD8 gene sequence comprising the steps; and (b) A step of recombining the HR template nucleic acid into the endogenous locus of the cell comprising the method. **Claim 9** The HR template comprises (a) 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 different codons from each other, and optionally, a sequence encoding the amino acid sequence Gly Ser Gly is located immediately upstream of the first, second, and / or third 2A coding sequences, and optionally, a sequence encoding a furin cleavage site is located immediately upstream of the first, second, and / or third 2A coding sequences; (b) A sequence encoding a signal sequence located immediately upstream of the TCR gene sequence and / or the CD8 gene sequence, wherein optionally, the signal sequence is a CD8 signal sequence, a human growth hormone signal sequence, a fragment thereof, or a combination thereof; and / or (c) A second TCR gene sequence located between the third 2A coding sequence and the second homology arm, wherein optionally, a sequence encoding a second signal sequence is located immediately upstream of the second TCR gene sequence, optionally, a 2A coding sequence is located between the first TCR gene sequence and the second TCR gene sequence, optionally, a sequence encoding the amino acid sequence Gly Ser Gly is located between the first TCR gene sequence and the second TCR gene sequence, and optionally, a sequence encoding a furin cleavage site is located between the first TCR gene sequence and the second TCR gene sequence; The method according to claim 8, comprising the above.

10. The method according to claim 8 or 9, wherein the TCR gene sequence encodes a TCR derived from a patient and / or a TCR that recognizes a cancer antigen, optionally, the cancer antigen is a neoantigen or a patient-specific antigen, and optionally, the TCR is a CD8-dependent TCR or a CD8-independent TCR.

11. (a) The HR template is non-viral, and optionally, the HR template is circular DNA or linear DNA; (b) The introduction occurs via electroporation; (c) the recombination involves cleavage of an endogenous locus by a nuclease and recombination of an HR template nucleic acid sequence into the endogenous locus by homology-directed repair, optionally, the nuclease is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) family nuclease, or a derivative thereof, optionally, the nuclease further comprises a gRNA; and / or (d) the method further comprises culturing the cells in the presence of at least one cytokine, optionally, the culturing is performed in the presence of IL2, IL7, IL15, or any combination thereof, The method according to any one of claims 8 to 10.

12. The T cells are (a) CD45RA+, CD62L+, CD28+, CD95-, CCR7+, and CD27+; (b) CD45RA+, CD62L+, CD28+, CD95+, CD27+, CCR7+; or (c) CD45RO+, CD62L+, CD28+, CD95+, CCR7+, CD27+, CD127+ The method according to any one of claims 8 to 11, which is a primary cell, a patient-derived cell, or a naive T cell.

13. A method for producing a cell, comprising modifying a cell by the method according to any one of claims 8 to 12.

14. A composition comprising an effective amount of the cell according to any one of claims 1 to 7, optionally, a pharmaceutical composition further comprising a pharmaceutically acceptable additive, optionally, a cryopreservative and / or serum albumin.

15. A cell according to any one of claims 1 to 7 or a composition according to claim 14 for use in a method of treating cancer in a subject in need of treatment, optionally, the subject has been subjected to a non-myeloablative lymphodepletion regimen.

16. The cancer is (a) a solid tumor, optionally a solid tumor selected from the group consisting of melanoma, thoracic 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 cancer, cholangiocarcinoma, renal cell cancer, testicular cancer, sarcoma, and colorectal cancer; or (b)A liquid tumor, optionally a liquid tumor selected from the group consisting of follicular lymphoma, leukemia, and multiple myeloma The cell or composition according to claim 15, which is such. **Claim 17** A kit for treating cancer, comprising the cell according to any one of claims 1 to 7 or the composition according to claim 14.

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