AU2021288213A1

AU2021288213A1 - Compositions and methods of manufacturing autologous T cell therapies

Info

Publication number: AU2021288213A1
Application number: AU2021288213A
Authority: AU
Inventors: Martin Giedlin; Kyle JACOBY; Stefanie MANDL-CASHMAN; Ines MENDE; Ramya H. TUNUGUNTLA; Nathaniel R. TZIZIK-SWANSON; Gregory S. YEDINAK
Original assignee: Adoc Ssf LLC
Current assignee: Adoc Ssf LLC
Priority date: 2020-06-12
Filing date: 2021-06-11
Publication date: 2023-02-02
Also published as: CA3181117A1; WO2021252898A1; EP4165190A1

Abstract

The present disclosure relates to methods, cells, and compositions for preparing cells and compositions for genetic engineering and cell therapy. Provided in certain embodiments are streamlined cell preparation methods, e.g., for isolation, processing, incubation, and genetic engineering of cells and populations of cells. Also provided are cells and compositions produced by the methods and methods of their use.

Description

COMPOSITIONS AND METHODS OF MANUFACTURING AUTOLOGOUS T CELL

THERAPIES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial No. 63/038,516, file on June 12, 2020, and to U.S. Provisional Patent Application Serial No. 63/161,283, file on March 15, 2021, the content of each of which is incorporated by reference in its entirety, and to each of which priority is claimed.

BACKGROUND OF THE INVENTION

Various methods are available for preparing cells for therapeutic use. For example, methods are available for isolating, processing, and engineering cells, including T cells and other immune cells. Methods are available to isolate such cells and to express genetically engineered antigen receptors, such as high affinity T cell receptors (TCRs) and chimeric antigen receptors (CARs). Methods are available to adoptively transfer such cells into subjects.

Presently, standardized methods for cell therapy manufacturing are still missing, with the overall processes being extremely complex, as comprising multiple handling steps, each one capable of causing operator errors, compromising the overall reproducibility. Thus, improved and standardized methods are needed for the preparation (e.g., isolation, processing, culturing, and engineering) of cells for use in cell therapy. In particular, methods are needed for the preparation and engineering of cells, e.g., a plurality of isolated cell types or sub-types, with improved efficiency, safety, variability, and conservation of resources. The present disclosure provides methods, cells, compositions, kits, and systems that meet such needs.

SUMMARY OF THE INVENTION

The present disclosure provides for compositions and methods for manufacturing autologous cell therapies. In certain embodiments, the presently disclosed subject matter provides a method of producing a therapeutic population of T cells comprising: a) obtaining a sample comprising T cells from a subject; b) isolating a first population of T cells; c) activating the first population of T cells, wherein the activation is performed in a closed container providing a gas-permeable surface area; d) transfecting the first population of T cells to express an exogenous nucleic acid; e) expanding the first population of T cells to obtain a second population of T cells, wherein the expansion is performed in a closed container providing a gas-permeable surface area; f) harvesting the second population of T cells, and g) transferring the harvested second population of T cells in an infusion bag, whereby the second population of T cells is the therapeutic population of T cells. In certain embodiments, the method is performed within a closed system. In certain embodiments, the closed system is a sterile environment.

In certain embodiments, the expansion is performed by culturing the T cells to produce a population of young T cells. In certain embodiments, the expansion is performed by culturing the first population of T cells in the presence of IL2, IL7, IL15, IL21, or any combination thereof. In certain embodiments, the expansion is performed by culturing the first population of T cells in the presence of IL7 and IL15. In certain embodiments, the population of young T cells comprises cells that are CD45RA+, CD62L+, CD28+, CD95-, CCR7+, and CD27+.

In certain embodiments, the population of young T cells comprises cells that are CD45RA+, CD62L+, CD28+, CD95+, CD27+, CCR7+. In certain embodiments, the population of young T cells comprises cells that are CD45RO+, CD62L+, CD28+, CD95+, CCR7+, CD27+, CD127+. In certain embodiments, the expansion is performed by culturing the first population of T cells in the presence of fibronectin, insulin, transferrin, or any combination thereof. In certain embodiments, the expansion is performed by culturing the first population of T cells at a glucose concentration of at least about 3.7 g/L.

In certain embodiments, the activation is performed by using non-magnetic beads. In certain embodiments, the first population of T cells comprises CD4 T cells. In certain embodiments, the first population of T cells comprises CD8 T cells.

In certain embodiments, the method further comprises cry opreserving the second population of T cells. In certain embodiments, the cryopreservation is performed by adding CS10 media to the infusion bag. In certain embodiments, the CS10 media has a final concentration of about 50%. In certain embodiments, the cryopreservation is performed by adding human serum albumin to the infusion bag. In certain embodiments, the human serum albumin has a final concentration of about 1%. In certain embodiments, the cryopreservation is performed by adding a crystalloid solution to the infusion bag. In certain embodiments, the crystalloid solution has a final concentration of about 46%.

In certain embodiments, the first population of T cells is concentrated by centrifugation.

In certain embodiments, the centrifugation occurs between the activating and the transfecting. In certain embodiments, the second population of T cells is concentrated by centrifugation. In certain embodiments, the centrifugation occurs during the harvesting. In certain embodiments, the centrifugation is a counter-flow centrifugation. In certain embodiments, the transfer of cells occurs by using a peristaltic pump.

In certain embodiments, the transfecting occurs via viral vector. In certain embodiments, transfecting occurs via electroporation. In certain embodiments, the transfecting comprises introducing into a T cell a polynucleotide, comprising: first and second homology arms homologous to first and second target nucleic acid sequences and oriented to facilitate homologous recombination; a nucleotide sequence encoding a TCR polypeptide sequence positioned between the first and second homology arms; and a first nucleotide sequence encoding a P2A ribosome skipping element positioned upstream of the nucleotide sequence encoding the TCR polypeptide and a second nucleotide sequence encoding a P2A ribosome skipping element positioned downstream of the nucleotide sequence encoding the TCR polypeptide, wherein the first and second nucleotide sequences encoding the P2A ribosome skipping elements are codon- diverged relative to each other.

In certain embodiments, the first and second homology arms of the polynucleotide are each from about 300 bases to about 2,000 bases in length. In certain embodiments, the polynucleotide further comprises: a nucleotide sequence encoding for the amino acid sequence Gly Ser Gly positioned immediately upstream of the nucleotide sequences encoding the 2A ribosome skipping elements; a nucleotide sequence encoding for a Furin cleavage site upstream of the second nucleotide sequence encoding a 2A ribosome skipping element; and a nucleotide sequence encoding for a human growth hormone signal peptide positioned upstream of the nucleotide sequence encoding the TCR. In certain embodiments, the polynucleotide further comprises a second nucleotide sequence encoding a TCR polypeptide sequence between the second nucleotide sequence encoding a P2A ribosome skipping element and the second homology arm. In certain embodiments, the polynucleotide further comprises a second nucleotide sequence encoding for a human growth hormone signal peptide positioned upstream of the second nucleotide sequence encoding the TCR polypeptide. In certain embodiments, the polynucleotide is a circular DNA.

In certain embodiments, the second population of T cells expresses an exogenous TCR gene sequence encoding for a TCR that recognizes a tumor antigen. In certain embodiments, the tumor antigen is a neoantigen. In certain embodiments, the tumor antigen is a patient specific neoantigen. In certain embodiments, the exogenous TCR gene sequence is a patient specific TCR gene sequence. In certain embodiments, the transfecting comprises cleavage of an endogenous locus by a nuclease. In certain embodiments, the nuclease is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) family nuclease or derivative thereof. In certain embodiments, the nuclease further comprises an sgRNA.

In certain embodiments, the activating is performed within a period of about 2 days, or about 3 days. In certain embodiments, the expanding is performed within a period of about 10 days, about 11 days, or about 12 days. In certain embodiments, the method is performed within a period of about 13 days, about 14 days, about 15 days. In certain embodiments, the second population of T cells comprises at least 20% Tmsc and Tcm collectively, at least 25% Tmsc and Tcm collectively, at least 30% Tmsc and Tcm collectively, at least 35% Tmsc and Tcm collectively, at least 40% Tmsc and Tcm collectively, at least 45% Tmsc and Tcm collectively, at least 50% Tmsc and Tcm collectively, at least 55% Tmsc and Tcm collectively, at least 60% Tmsc and Tcm collectively or more than 61% Tmsc and Tcm collectively. In certain embodiments, the therapeutic population of T cells is infused into the subject.

In certain embodiments, the presently disclosed subject matter provides a composition comprising the therapeutic population of T cells produced by any one of the method disclosed herein. In certain embodiments, the composition comprises a pharmaceutical excipient. In certain embodiments, the composition comprises a therapeutically effective amount. In certain embodiments, the therapeutic population of T cells comprises at least 1 x 10⁶ cells/ml. In certain embodiments, the therapeutic population of T cells comprises at least 10 x 10⁶ cells/ml. In certain embodiments, the therapeutic population of T cells comprises at least 100 x 10⁶ cells/ml.

In certain embodiments, the presently disclosed subject matter provides a method of treating cancer comprising administering the therapeutic population of T cells produced by any one of the methods disclosed herein to a subject in need thereof. In certain embodiments, the presently disclosed subject matter provides a method of treating cancer comprising administering the composition disclosed herein to a subject in need thereof.

In certain embodiments, the presently disclosed subject matter provides a method comprising: contacting a cell with an activation reagent; editing the cell to express an exogenous nucleic acid; culturing the edited cell in a cell culture medium; harvesting the edited cell; and transferring the edited cell in a container; wherein the method is performed within a closed system. In certain embodiments, the presently disclosed subject matter provides a method comprising: contacting a cell with an activation reagent; editing the cell to express an exogenous nucleic acid; culturing the cell in a cell culture medium to obtain a population of cells; harvesting the population of cells; and transferring the population of cells in a container; wherein the method is performed within a closed system. In certain embodiments, the method further comprises a counter-flow centrifugation. In certain embodiments, the method occurs within a period of about 13 days, about 14 days, about 15 days.

In certain embodiments, the editing comprises introducing into the cell a polynucleotide. In certain embodiments, the polynucleotide comprises: first and second homology arms homologous to first and second target nucleic acid sequences and oriented to facilitate homologous recombination; a nucleotide sequence encoding a TCR polypeptide sequence positioned between the first and second homology arms; and a first nucleotide sequence encoding a P2A ribosome skipping element positioned upstream of the nucleotide sequence encoding the TCR polypeptide and a second nucleotide sequence encoding a P2A ribosome skipping element positioned downstream of the nucleotide sequence encoding the TCR polypeptide, wherein the first and second nucleotide sequences encoding the P2A ribosome skipping elements are codon- diverged relative to each other. In certain embodiments, the first and second homology arms of the polynucleotide are each from about 300 bases to about 2,000 bases in length. In certain embodiments, the polynucleotide further comprises: a nucleotide sequence encoding for the amino acid sequence Gly Ser Gly positioned immediately upstream of the nucleotide sequences encoding the 2A ribosome skipping elements; a nucleotide sequence encoding for a Furin cleavage site upstream of the second nucleotide sequence encoding a 2A ribosome skipping element; and a nucleotide sequence encoding for a human growth hormone signal peptide positioned upstream of the nucleotide sequence encoding the TCR. In certain embodiments, the polynucleotide further comprises a second nucleotide sequence encoding a TCR polypeptide sequence between the second nucleotide sequence encoding a P2A ribosome skipping element and the second homology arm. In certain embodiments, the polynucleotide further comprises a second nucleotide sequence encoding for a human growth hormone signal peptide positioned upstream of the second nucleotide sequence encoding the TCR polypeptide. In certain embodiments, the polynucleotide is a circular DNA.

In certain embodiments, the edited cell expresses an exogenous TCR gene sequence encoding for a TCR that recognizes a tumor antigen. In certain embodiments, the population of cells comprises a cell expressing an exogenous TCR gene sequence encoding for a TCR that recognizes a tumor antigen. In certain embodiments, the tumor antigen is a neoantigen. In certain embodiments, the tumor antigen is a patient specific neoantigen. In certain embodiments, the exogenous TCR gene sequence is a patient specific TCR gene sequence.

In certain embodiments, the transfecting comprises cleavage of an endogenous locus by a nuclease. In certain embodiments, the nuclease is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) family nuclease or derivative thereof. In certain embodiments, thenuclease further comprises an sgRNA. In certain embodiments, the editing comprises a viral infection. In certain embodiments, the editing comprises electroporation.

In certain embodiments, the cell is a primary cell, a lymphocyte, or a T cell. In certain embodiments, the population of cells comprises a primary cell, a lymphocyte, a T cell, or a combination thereof. In certain embodiments, the T cell is a CD8 or a CD4 T cell. In certain embodiments, the T cell is a young T cell. In certain embodiments, the young T cell is CD45RA⁺, CD62L⁺, CD28⁺, CD95\ CCR7⁺, and CD27⁺. In certain embodiments, the young T cell is CD45RA⁺, CD62L⁺, CD28⁺, CD95⁺, CCR7⁺, CD27⁺. In certain embodiments, the young T cell is CD45RO⁺, CD62L⁺, CD28⁺, CD95⁺, CCR7⁺, CD27⁺, CD127⁺. In certain embodiments, the young T cell is a memory stem cell (TMSC). In certain embodiments, the young T cell is a central memory cells (TCM).

In certain embodiments, the population of cells comprises at least about 20% of TMSC and TCM collectively, at least about 25% TMSC and TCM collectively, at least about 30% TMSC and TCM collectively, at least about 35% TMSC and TCM collectively, at least about 40% TMSC and TCM collectively, at least about 45% Tmsc and Tcm collectively, at least about 50% TMSC and TCM collectively, at least about 55% TMSC and TCM collectively, at least about 60% TMSC and TCM collectively or more than about 61% TMSC and TCM collectively.

In certain embodiments, the cell is obtained from a subject. In certain embodiments, the cell is obtained by leukapheresis. In certain embodiments, the cell is obtained by a tissue sample. In certain embodiments, the tissue sample is a tumor sample. In certain embodiments, the cell is cryopreserved.

In certain embodiments, the cell culture medium comprises interleukin 2 (IL2), interleukin 7 (IL7), interleukin 15 (IL15), interleukin (IL21), or any combination thereof. In certain embodiments, the cell culture medium comprises IL7 and IL15. In certain embodiments, the cell culture medium comprises fibronectin, insulin, transferrin, or any combination thereof.

In certain embodiments, the cell culture medium comprises glucose concentration of at least about 3.7 g/L. In certain embodiments, the culturing is performed within a period of about 10 days, about 11 days, or about 12 days.

In certain embodiments, the activation reagent comprises an anti-CD3 antibody, an anti- CD2 antibody, an anti-CD28 antibody, or a combination thereof. In certain embodiments, the activation reagent comprises non-magnetic beads or magnetic beads. In certain embodiments, the activation reagent comprises an artificial APCs. In certain embodiments, the contacting is performed within a period of about 2 days, or about 3 days.

In certain embodiments, the method further comprises cry opreserving the edited cell. In certain embodiments, the method further comprises cry opreserving the population of cells.

In certain embodiments, the edited cell or population of cells is cryopreserved in a pharmaceutical formulation. In certain embodiments, the pharmaceutical formulation comprises a cryopreservation medium, a serum albumin, a crystalloid solution, or a combination thereof. In certain embodiments, the cryopreservation medium is at a final concentration of about 50% v/v. In certain embodiments, the serum albumin is at a final concentration of about 1% w/v. In certain embodiments, the crystalloid solution is at a final concentration of about 46% v/v. In certain embodiments, the pharmaceutical formulation comprises CryoStor® CS10, human serum albumin, Plasma-Lyte A, or a combination thereof.

In certain embodiments, the closed system comprises a peristaltic pump. In certain embodiments, the edited cell is infused into a subject. In certain embodiments, the population of cells is infused into the subject.

In certain embodiments, the presently disclosed subject matter also provides a composition comprising the edited cells obtained by the methods disclosed herein. In certain embodiments, the presently disclosed subject matter provides a composition comprising the population of cells obtained by the methods disclosed herein. In certain embodiments, the composition further comprises a pharmaceutical excipient. In certain embodiments, the composition comprises a therapeutically effective amount of cells. In certain embodiments, the composition comprises at least 1 x 10⁶ cells/ml. In certain embodiments, the composition comprises at least 10 x 10⁶ cells/ml. In certain embodiments, the composition comprises at least 100 x 10⁶ cells/ml. In certain embodiments, the composition comprises at least about 4.0 x 10⁸ gene-edited cells. In certain embodiments, the composition comprises at least about 1.3 x 10⁹ gene-edited cells. In certain embodiments, the composition comprises at least about 4.0 x 10⁹ gene-edited cells. In certain embodiments, the composition comprises at least about 1.3 x 10¹⁰ gene-edited cells. In certain embodiments, the composition comprises at least about 4.0 x 10¹⁰ gene-edited cells.

In certain embodiments, the presently disclosed subject matter further provides methods of treating a cancer comprising administering the edited cell obtained by the methods disclosed herein, the population of cells obtained by the methods disclosed herein, or the compositions disclosed herein to a subject in need thereof.

In certain embodiments, the presently disclosed subject matter provides the edited cell obtained by the methods disclosed herein, the population of cells obtained by the methods disclosed herein, or the compositions disclosed herein for use in the treatment of a cancer. In certain embodiments, the presently disclosed subject matter provides the use of the edited cell obtained by the methods disclosed herein, the population of cells obtained by the methods disclosed herein, or the compositions disclosed herein for the manufacture of a medicament for the treatment of cancer.

In certain embodiments, the presently disclosed subject matter provides a method of manufacturing NeoTCR Cells using Process 1, Process 2, Process 3, or Process 4 described herein. In certain embodiments, the presently disclosed subject matter also provides a composition comprising NeoCells manufactured using Process 1, Process 2, Process 3, or Process 4 described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1C. Figures 1A-1C show an example of a NeoE TCR cassette and gene editing methods that can be used to make NeoTCR Products. Figure 1A shows a schematic representing the general targeting strategy used for integrating neoantigen-specific TCR constructs (NeoTCRs) into the TCRa locus. Figures IB and 1C show a neoantigen-specific TCR construct design used for integrating a NeoTCR into the TCRa locus wherein the cassette is shown with signal sequences (“SS”), protease cleavage sites (“P”), and 2A peptides (“2A”). Figure IB shows a target TCRa locus (endogenous TRAC, top panel) and its CRISPR Cas9 target site (horizontal stripes, cleavage site designated by the arrow), and the circular plasmid HR template (bottom panel) with the polynucleotide encoding the NeoTCR, which is located between the left and right homology arms (“LHA” and “RHA” respectively) prior to integration. Figure 1C shows the integrated NeoTCR in the TCRa locus (top panel), the transcribed and spliced NeoTCR mRNA (middle panel), and translation and processing of the expressed NeoTCR (bottom panel).

Figure 2. Figure 2 shows a schematic for Process 1 as described in the Examples.

Figure 3. Figure 3 shows a schematic for Process 2 as described in the Examples.

Figure 4. Figure 4 shows a schematic for Process 2 as described in the Examples as it pertains to a 3 NeoTCR Product. Specifically, the middle column shows the process steps for each of the three different sublots for a 3 NeoTCR Product, while the left column lists the processing equipment utilized at each step. The right column illustrates QC sampling during the process.

Figures 5A and 5B. Figures 5A and 5B show the results of unit-operations-based optimizations, T cell activation, and gene-editing efficiencies. Figure 5A shows CD25 expression as measured by flow cytometry following two days of activation of CD4/CD8 cells in either Prodigy or G-Rex 100M flasks using the same ratio of TransAct. Figure 5B shows T cell activation in G-Rex flasks results in similar gene-editing rates (% NeoTCR+) as compared to T cells activated in Prodigy at end of culture (day 13). WT= wild-type cells (non-edited cells expressing endogenous T cell receptor), KO: knock-out, i.e. cells in which endogenous TCR was knocked out, but no NeoTCR knock-in occurred, NeoTCR: cells expressing only the neoantigen- specific TCR.

Figures 6A and 6B. Figures 6A and 6B show the results of unit-operations-based optimizations and T cell expansion and phenotype. For evaluation of feasibility of cell expansion in G-Rex flasks, cells were activated in G-Rex 100M flask and following day 2 electroporation cells were transferred to a G-Rex 100M flask for further cell expansion. On day 9, cells were transferred to G-Rex 500M (1:5 split) until end of culture on day 13. Cells from the same donor (split-run) were activated on the Prodigy, nucleofected on day 2, and expanded in Prodigy CentriCult chamber until day 13. Figure 6A shows that cells expanded in G-Rex flasks have enhanced cell expansion as compared to cells expanded within Prodigy CentriCult unit. Figure 6B shows that cells expanded in G-Rex show similar T cell phenotype at the end of culture (day 13) as compared to cells expanded in the Prodigy. Tn: naive T cells, Tmsc+Tcm: memory stem cell T cells and central memory T cells, Ttm +Tem: transitional memory T cells and effector memory T cells, T eff: effector T cells.

Figure 7. Figure 7 shows the post-rebuffering cell recovery in the Rotea v. the Prodigy. Results show day 2 percent post rebuffering cell recovery using either current Prodigy CAP rebuffering process (Process 1) (n=7, 5 donors) or optimized process using Rotea (Process 2) (n=12, 6 donors). % Cell recovery was calculated based on total viable cell counts pre- and post- rebuffering into electroporation buffer. Cells used for Rotea rebuffering process were activated for two days in G-Rex 100M flasks, while cells rebuffered using Prodigy were activated in Prodigy.

Figure 8. Figure 8 shows the percent recovery after final formulation using the Rotea v. the Prodigy. Results show a comparison of % cell recovery post formulation into Plasmalyte + 2% HSA using either current Prodigy harvest procedure (Process 1) (n=7, 4 different donors) or Rotea formulation procedure (Process 2) (n=9, 6 donors). Cells used for Rotea final formulation process were activated and expanded in G-Rex flasks (Process 2), while cells for Prodigy control were activated and expanded in the Prodigy (Process 1). % Cell recovery was calculated based on total viable cell counts pre- and post-formulation into Plasmalyte + 2% HSA.

Figure 9. Figure 9 shows post-thaw viability following cryopreservation. Post-thaw viability was measured for the NeoTCR Product formulated at a target cell concentration of 10 million cells/mL cryopreserved in either CryoMACS 250 bags with a fill volume of 35 mL or a CryoMACS 500 bag with 70mL fill volume. Cells used for final formulation were manufactured using either the current Prodigy manufacturing process (Process 1) or optimized manufacturing process (Process 2).

Figure 10. Figure 10 shows post-thaw cell concentration by final formulation method. Post-thaw cell concentration was measured for the NeoTCR Product formulated at a target cell concentration of 10 million cells/mL cryopreserved in CryoMACS 250 bags with a fill volume of 35 mL and CryoMACS 500 bags with 70mL fill volume. Cells used for final formulation were manufactured using either the current Prodigy manufacturing process (Process 1) or optimized manufacturing process (Process 2).

Figure 11. Figure 11 shows the percent NeoTCR+ cells (i.e., NeoTCR Cells) by final formulation method. The percentage of NeoTCR Cells was measured for the NeoTCR Product formulated at a target cell concentration of 10 million cells/mL cryopreserved in CryoMACS 250 bags (35 mL fill volume), CryoMACS 500 bag (70mL fill volume), and QC sample vial. Cells used for final formulation were manufactured using either the current Prodigy manufacturing process (Process 1) or optimized manufacturing process (Process 2).

Figure 12. Figure 12 shows T cell phenotype by cryopreservation method. T cell phenotype was assessed for NeoTCR Product formulated at a target cell concentration of 10 million cells/mL cryopreserved in CryoMACS 250 bags (35 mL fill volume), CryoMACS 500 bag (70mL fill volume), and QC sample vial. Cells used for final formulation were manufactured using either the current Prodigy manufacturing process (Process 1) or optimized manufacturing process (Process 2). Tnaive: naive T cells, Tcm+Tmsc: central memory T cells and memory stem cell T cells, Tem+Ttm: effector memory T cells and transitional memory T cells, T eff: effector T cells.

Figure 13. Figure 13 shows cell expansion for Process 2 as compared to Process 1.

Data shown are the results from five direct split comparison runs (same donor) with fold expansion (day 2 to end of study) shown in blue for Process 1 and red (optimized Process 2). Historical cell expansion data from original engineering and clinical readiness runs at Miltenyi (vl .0) are shown for comparison in black.

Figure 14. Figure 14 shows percentage of NeoTCR+ cells post gene-editing for optimized process (Process 2) as compared to Process 1. Data shown are the results from five direct split comparison runs (same donor) with the % NeoTCR+ of final product shown in blue for Process 1 and red for the optimized Process 2. Historical percentage of NeoTCR+ expression levels from original engineering and clinical readiness runs at Miltenyi are shown in black for comparison.

Figure 15. Figure 15 shows NeoTCR+ cell yield for the optimized process (Process 2) as compared to Process 1. Data shown are the results from five direct split comparison runs (same donor) with total NeoTCR+ cell yield at end of study shown in full circles for Process 1 and full diamonds for Process 2. Historical NeoTCR+ cell yield from original engineering and clinical readiness runs at Miltenyi are shown in empty circles for comparison.

Figure 16. Figure 16 shows percent viability of NeoTCR Cells for optimized process (Process 2) as compared to Process 1. Data shown are the results from five direct split comparison nans (same donor) with % viability (NeoTCR Product, pre-cry opreservation) shown in blue for Process 1 and red for Process 2. Historical % Viability (NeoTCR Product QC release data) from original engineering and clinical readiness runs at Miltenyi are shown for comparison.

Figure 17. Figure 17 shows IFN-gamma production of NeoTCR Cells for the optimized process (Process 2) as compared to Process 1. Data shown are the results for NeoTCR Product IFN-gamma production from four direct split comparison runs (same donor) shown in blue for Process 1 and red for Process 2. Samples for IFN-gamma analysis were taken pre-harvest as per quality control (QC) sampling plan for the current NeoTCR Product. Historical IFN-gamma production (NeoTCR Product QC release data) from original engineering and clinical readiness runs at Miltenyi are shown in black for comparison.

Figures 18A and 18B. Figures 18A and 18B show NeoTCR Cells generated using either the optimized process (Process 2) or Process 1 induced highly specific anti-tumor cytotoxicity. Data shown are results from IncuCyte® killing assays, in which NeoTCR Cells from Process 2, Process 1 or media only as negative control were added to target cells expressing the NeoTCR target (Figure 18A) or wild-type cell line (Figure 18B) at a 5: 1 ratio. These cell lines only differ in the expression of one amino acid which is altered in the neoE sequence. The reduction in % nRFP is a measure of target cell death.

Figure 19 Figure 19 shows a high level diagram of Process 2 and Process 3.

Figures 20A and 20. Figures 20A and 20B show a comparison of activation markers and proliferation data for TexMACS media supplemented with 3% human serum compared to Prime- XV media supplemented with 2% Physiologix and shows that Prime XV media can support activation and proliferation in a huAB serum-free environment. Figure 20A shows cell surface markers CD25 and CD69 (associated with T cell activation) and KI-67 (an intracellular marker of proliferation) which were assessed by flow cytometry on day 2 from all the experimental arms.

As shown, there was greater than 10% increases in all three markers for Prime-XV media versus the TexMACs media. Figure 20B shows the increased T cell growth kinetics for the Prime-XV media (correlating to the increase in activation markers shown in Figure 20A).

Figures 21A and 21B. Figures 21A and 21B show that in addition to the overall support of Prime XV medium +2% Physiologix for T cell activation and proliferation, a 5-fold increase in NeoTCR Cells over that seen with TexMACS medium with 3 % huAB serum was also observed (Figure 21A), supported by the relative increase in Dex+ IP26+ T cells. Figure 21B shows that there was a slight decrease in the CD4/CD8 ratio. Based on this, enhanced T cell proliferation also supported expansion of the NeoTCR Cells. Figure 21A is represented as follows: Yellow Diamond: total viable NeoTCR Cells ( calculated as % NeoTCR+ (Dex+/IP26+) x total viable cells based on NC200; Green bar: % NeoTCR+ cells (Dex+/IP26+); Black Bar: WT cells not knockout cells, as they are still expressing TCR; Grey bar: % knockout cells, which neither express NeoTCR nor WT TCR. Overall, the use of Prime-XV supports improved gene editing (partially due to better survival of gene-edited cells post electroporation and less huAB residuals). As better editing in CD8 T cells in large scale was shown, skewing the CD4/CD8 ratio towards CD8 will increase the total percent of NeoTCR Cells.

Figure 22. Figure 22 shows that improved expansion observed in PrimeXV media did not result in loss of CD62L expression (i.e., the increased proliferation and better support of the NeoTCR Cells did not promote further differentiation of the T cells).

Figures 23A and 23B. Figure 23A shows a 2 fold increase in gene editing efficiency without serum/serum replacement and 4 fold increase in total edited cells on Day 13. Figure 23B shows a 3 fold increase in cell expansion with Prime-XV supplemented with 2%

Physiologix XF compared to TexMACs and improved cell expansion observed even in the absence of serum/serum replacement.

Figures 24 A and 24B. Figure 24A shows the gene-editing efficiency at Day 8 with different media using large-scale manufacturing and electroporation methods. Figure 24B shows the cell growth kinetics for the cells cultured under conditions presented in Figure 24A. See Figure 21A for a description of the bars and diamonds in Figure 24A.

Figure 25. Figure 25 shows a diagram of the custom-designed Gravity Drain to harvest cells. It was used on Day 2 of Process 2 to transfer activated cells from G-Rex to a transfer pack that was subsequently welded on the Rotea; used on Day 13 of Process 2 to split culture dependent on cell density; used on Day 13 of Process 2 to harvest cells from the G-Rex into a transfer pack that was welded on the Rotea. The same design was also used for Process 3; however, the timing of when the Gravity Drain was used (i.e., Days 2 and 13) varied based on the other manufacturing optionalities described for Process 3.

Figure 26. Figure 26 shows a diagram of the custom media removal setup that was used on Day 8 of Process 2 to reduce culture volume to - 100m L for media exchange/culture split and on Day 13 of Process 2 to reduce culture volume for cell substance harvest and formulation. The same design was also used for Process 3; however, the timing of when the custom media removal setup was used (i.e., Days 8 and 13) varied based on the other manufacturing optionalities described for Process 3.

Figure 27. Figure 27 shows a diagram for the custom-designed post-electroporation cell transfer that was used on Day 2 after electroporation in Process 2. The cells were transferred into a G-Rex containing pre-warmed media. The same design was also used for Process 3; however, the timing of when the custom post-electroporation cell transfer was used (i.e., Day2) varied based on the type of activation agent used in the process.

Figure 28. Figure 28 shows the use of the WMFG 530S enables seamless closed transfer with accurate volume delivery (WMFG 530S is an example of the closed transfer machine diagramed in Figure 19). The WMFG 530S was used in Process 2 for the following steps: Activation setup, Expansion setup, Day 8 Culture Split, Harvest, and CS10 Addition.

Figure 29. Figure 29 shows that 40% of cells were washed out during the processing of cells using standard centrifugation as described in Example 5. In contrast, the use of counterflow centrifugation (using the Rotea) resulted in minimal cell loss and resulting in minimal cell loss.

Figure 30. Figure 30 shows glucose measurements obtained from cultures after the rebuffering procedures using standard centrifugation and washes/buffer exchanges (the Prodigy) compared to counterflow centrifugation (G-Rex, using the Rotea). This shows that counterflow centrifugation is able to significantly increase media removal compared to standard centrifugation methods.

Figure 31. Figure 31 shows two variations of counterflow centrifugation methods: Version 1 (VI) and Version 2 (V2). VI was set at a centrifugal force of 2500g with a fluid flow rate of 30mL/min and V2 was set at a centrifugal force of 2700g with a fluid flow rate of lOmL/min. As shown, V2 with an increased g force and slower fluid flow rate resulted in a significant increase in the rate of cell recovery.

Figure 32 Figure 32 shows variable T cell expansion in ENG runs. The observed variability prompted Medium A investigation. Medium A is TexMACs.

Figure 33. Figure 33 shows the study design for evaluation of incoming Medium A lots. Medium A is TexMACs.

Figure 34. Figure 34 shows activation markers of T cells cultured with different cell media. No significant differences were observed across media lots. Medium A is TexMACs. Medium B is PrimeXV.

Figure 35. Figure 35 shows cell expansion and viability on day 8. Cell expansion and viability of T cells had comparable results with ENG runs on day 8. Medium A is TexMACs. Medium B is PrimeXV.

Figure 36. Figure 36 shows gene editing efficiencies on day 8. Substantial differences in gene editing were observed on day 8 across medium A lots. Medium A is TexMACs.

Medium B is PrimeXV. Figure 37. Figure 37 shows cell expansion and viability on day 13. The differences in cell expansion and viability were sustained at harvest on day 13. Medium A is TexMACs. Medium B is PrimeXV.

Figure 38. Figure 38 shows gene editing efficiencies on day 13. The differences in gene editing were maintained at harvest on day 13. Medium A is TexMACs. Medium B is PrimeXV.

Figure 39. Figure 39 shows a summary of the data and results disclosed in Figures 32- 38. Medium A is TexMACs. Medium B is PrimeXV.

Figure 40. Figure 40 shows capillary electrophoresis - mass spectrometry (CE-MS) analysis of T cell media. Significant variability was observed across medium A lots identified through extensive screening. CE-MS analysis revealed absence of glutamine in low-performance lots. Medium A is TexMACs. Medium B is PrimeXV.

Figure 41. Figure 41 shows that Medium B improves post EP recovery and gene editing efficiency. Significant improvement in gene editing efficiency was observed in medium B (m=24.5% vs. 14.6%). Medium B attenuated EP induced lag phase between day 2-8 and improved overall expansion (approximately 2-fold). This evidence shows that even greater expansion can be achieved with additional media exchanges to mitigate lactate accumulation. Medium A is TexMACs. Medium B is PrimeXV.

Figure 42 Figure 42 shows increased NeoTCR+ cell yield with Medium B CDM at clinical MFG scale. Medium A is TexMACs. Medium B is PrimeXV.

Figure 43. Figure 43 shows T cell immunophenotype characterization and comparable subset. Medium A is TexMACs. Medium B is PrimeXV

Figure 44. Figure 44 shows cell product characterization (P<Ng secretion and cytotoxicity). Medium A is TexMACs. Medium B is PrimeXV.

Figure 45. Figure 45 shows a summary of the medium B CDM implementation.

DETAILED DESCRIPTION

The present disclosure is directed to compositions and methods for the manufacture of cell therapeutics, e g., autologous cell therapeutics such as NeoTCR Cells. Non-limiting embodiments of the compositions and methods for the manufacture of cell therapeutics are described by the present description and examples. For purposes of clarity of disclosure and not by way of limitation, the detailed description is divided into the following subsections:

1. Definitions

2. Adoptive Cell Therapies 3. Therapeutic Compositions and Methods of Manufacturing

4. Arti cl e of Manufacture

5. Methods of Treatment

6. Kits

L _ Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art. The following references provide one of skill with a general definition of many of the terms used in the present 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 & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting," and "consisting essentially of' aspects and embodiments. The terms “comprises” and “comprising” are intended to have the broad meaning ascribed to them in U.S. Patent Law and can mean “includes”, “including” and the like.

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

As used herein, the term “cell culture medium” refers to a composition, e.g., liquid or gel, designed to support the growth, maintenance, and/or differentiation of cells. The composition can include sources of energy and additional compounds regulating the cell functions. In certain non-limiting embodiments, the cell culture medium includes essential or non-essential amino acids (e.g., cysteine and glutamic acid), vitamins, inorganic salts, glucose, serum, hormones (e.g., insulin), lipids, chemokines, and cytokines. In certain non-limiting embodiments, the cell culture medium is serum-free. As used herein, the term “cryopreservation medium” refers to a composition comprising a cryopreservative, e.g., DMSO. Non-limiting examples of cryopreservation medium include CryoStor® CS10, PSC Cryopreservation Medium, CryoDefend® Cell Lines Media, and HypoThermosol® FRS.

As used herein, the term “culturing” refers to contacting a cell with a cell culture medium under conditions suitable to the growth, maintenance, and/or differentiation of the cell.

As used herein, “Medium A” refers to TexMACs. As used herein, “Medium B” refers to PrimeXV.

As used herein, the terms “closed system” or “closed-system” refer to processes that have no exposure to the surrounding environment. Closed systems prevent the ingress of microbes from the environment and incorporates single-use disposable components for all materials that come into contact with the product, e.g., NeoTCR Cells. In certain embodiments, all components and reagents are presterilized by terminal or filter sterilization. In certain embodiments, closed systems enable the processing of multiple patient batches in parallel in the same area. In certain embodiments, closed systems eliminate the need for the processing of products in a biosafety cabinet (BSC) within a high-grade cleanroom (Grade B/Class 10,000).

“Dextramer” as used herein means a multimerized neoepitope-HLA complex that specifically binds to its cognate NeoTCR.

The term “tumor antigen” as used herein refers to an antigen (e.g., a polypeptide) that is uniquely or differentially expressed on a tumor cell compared to a normal or non-neoplastic cell. In certain embodiments, a tumor antigen includes any polypeptide expressed by a tumor that is capable of activating or inducing an immune response via an antigen-recognizing receptor or capable of suppressing an immune response via receptor-ligand binding.

As used herein, the terms "neoantigen", “neoepitope” or “neoE” refer to a newly formed antigenic determinant that arises, e.g., from a somatic mutation(s) and is recognized as "non self.” A mutation giving rise to a "neoantigen", “neoepitope” or “neoE” can include a frameshift or non-frameshift indel, missense or nonsense substitution, splice site alteration (e.g., alternatively spliced transcripts), genomic rearrangement or gene fusion, any genomic or expression alterations, or any post-translational modifications.

“TCR” as used herein means T cell receptor.

“NeoTCR” and “NeoE TCR” and “exogenous TCR” as used herein mean a neoepitope- specific T cell receptor that is introduced into a T cell, e.g., by gene-editing methods. As used herein, the term “TCR gene sequence” refers to a NeoTCR gene sequence. “NeoTCR cells” as used herein means one or more cells precision-engineered to express one or more NeoTCRs. In certain embodiments, the cells are T cells. In certain embodiments, the T cells are CD8+ and/or CD4+ T cells. In certain embodiments, the CD8+ and/or CD4+ T cells are autologous cells from the patient for whom a NeoTCR Product will be administered.

The terms “NeoTCR cells” and “NeoTCR+ cells” and “NeoTCR-Pl T cells” and “NeoTCR-Pl cells” are used interchangeably herein.

“Cell Product” as used herein means a gene-edited cell therapy wherein one or more 2A peptides are used in the gene-editing process. In certain embodiments, the Cell Product is made through the insertion of DNA wherein the gene of interest is inserted between two 2A sequences (see, e.g., Figure 2A). In certain embodiments, the DNA is linear or circular (e g., plasmid DNA). In certain embodiments, the Cell Product is made through the insertion of DNA wherein the gene of interest is flanked on one side by a 2A peptide. In certain embodiments, when there is more than one 2 A peptide sequence, such sequences are the same 2 A peptides (e.g., two P2A sequences, two T2A sequences, two E2A sequences, or 2 F2A sequences). In certain embodiments, when there is more than one 2A peptide sequence, such sequences are different 2A peptides (e.g., but not limited to, one T2A and one P2A). In certain embodiments, Cell Products are made using viral gene-editing methods. In certain embodiments, Cell Products are made using targeted or non-targeted viral methods. In certain embodiments, Cell Products are made using non-viral gene-editing methods. Cell Products include but are not limited to T cell products, NK cell products HSCs, TILs, and cell products derived from HSCs. Cell Products can also include any other naturally occurring cell that can be edited using a 2A peptide as part of the gene-editing process. Cell Products can be used, for example, for the treatment of autoimmune diseases, neurological diseases and injuries (including but not limited to Alzheimer’s disease, Parkinson’s disease, spinal cord, and nerve injuries and/or damage), cancer, infectious diseases, joint disease (including but not limited to rebuilding damaged cartilage in joints), improving the immune system, cardiovascular disease and abnormalities, aging, immune deficiencies (including but not limited to multiple sclerosis and amyotrophic lateral sclerosis), allergies, and genetic disorders. Cell Products include NeoTCR Products and NeoTCR Viral Products.

“NeoTCR Product” as used herein means a pharmaceutical formulation comprising one or more NeoTCR cells. NeoTCR Product consists of autologous precision genome-engineered CD8+ and CD4+ T cells. Using a targeted DNA-mediated non-viral precision genome engineering approach, expression of the endogenous TCR is eliminated and replaced by a patient-specific NeoTCR isolated from peripheral CD8+ T cells targeting the tumor-exclusive neoepitope. In certain embodiments, the resulting engineered CD8+ or CD4+ T cells express NeoTCRs on their surface of native sequence, native expression levels, and native TCR function. The sequences of the NeoTCR external binding domain and cytoplasmic signaling domains are, in certain embodiments, unmodified from the TCR isolated from native CD8+ T cells.

Regulation of the NeoTCR gene expression is, in certain embodiments, driven by the native endogenous TCR promoter positioned upstream of where the NeoTCR gene cassette is integrated into the genome. Through such approaches, native levels of NeoTCR expression are observed in unstimulated and antigen-activated T cell states.

The NeoTCR Product manufactured for each patient represents a defined dose of autologous CD8+ and/or CD4+ T cells that are precision genome engineered to express a single neoE-specific TCR cloned from neoE-specific CD8+ T cells individually isolated from the peripheral blood of that same patient.

As used herein, the term “CD8 Product” refers to a pharmaceutical composition comprising one or more NeoTCR cells expressing an exogenous CD8. Additional information on the CD8 Products can be found in U.S. Patent Publication No. 2021/0085721, the content of which is incorporated by reference in its entirety.

“NeoTCR Viral Product” as used herein has the same definition of NeoTCR Product except that the genome engineering is performed using viral-mediated methods.

"Pharmaceutical Formulation" refers to a preparation that is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. For clarity, quantities of DMSO used in a Cell Product or a NeoTCR Product are not considered unacceptably toxic.

“Treat,” “Treatment,” and “treating” are used interchangeably and as used herein mean obtaining beneficial or desired results including clinical results. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, the NeoTCR Product of the present disclosure is used to delay the development of a proliferative disorder (e.g., cancer) or to slow the progression of such disease.

A "subject," "patient," or an "individual" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human. 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, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms are further used to refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. Cancer can affect a variety of cell types, tissues, or organs, including but not limited to an organ selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestines, kidney, liver, lung, lymph node, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and vagina, or a tissue or cell type thereof. Cancer includes cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumor of the plasma cells). Examples of cancer include, but are not limited to, those described herein. The terms “Cancer” or “Tumor” and “Proliferative Disorder” are not mutually exclusive as used herein.

“2A” and “2A peptide” are used interchangeably herein and mean a class of 18-22 amino acid long, viral, self-cleaving peptides that are able to mediate cleavage of peptides during translation in eukaryotic cells. Four well-known members of the 2A peptide class are T2A, P2A, E2A, and F2A. The T2A peptide was first identified in the Thosea asigna virus 2A. The P2A peptide was first identified in the porcine teschovirus-1 2A. The E2A peptide was first identified in the equine rhinitis A virus. The F2A peptide was first identified in the foot-and-mouth disease virus. The self-cleaving mechanism of the 2A peptides is a result of ribosome skipping the formation of a glycyl-prolyl peptide bond at the C-terminus of the 2A Specifically, the 2A peptides have a C-terminal conserved sequence that is necessary for the creation of steric hindrance and ribosome skipping. The ribosome skipping can result in one of three options: 1) successful skipping and recommencement of translation resulting in two cleaved proteins (the upstream of the 2A protein which is attached to the complete 2A peptide except for the C- terminal proline and the downstream of the 2A protein which is attached to one proline at the N- terminal; 2) successful skipping but ribosome fall-off that results in discontinued translation and only the protein upstream of the 2A; or 3) unsuccessful skipping and continued translation (i.e., a fusion protein). The term “endogenous” as used herein refers to a nucleic acid molecule or polypeptide that is normally expressed in a cell or tissue.

The term “exogenous” as used herein refers to a nucleic acid molecule or polypeptide that is not endogenously present in a cell. The term “exogenous” would therefore encompass any recombinant nucleic acid molecule or polypeptide expressed in a cell, such as foreign, heterologous, and over-expressed nucleic acid molecules and polypeptides. By “exogenous” nucleic acid is meant a nucleic acid not present in a native wild-type cell; for example, an exogenous nucleic acid may vary from an endogenous counterpart by sequence, by position/location, or both. For clarity, an exogenous nucleic acid may have the same or different sequence relative to its native endogenous counterpart; it may be introduced by genetic engineering into the cell itself or a progenitor thereof, and may optionally be linked to alternative control sequences, such as a non-native promoter or secretory sequence.

As used herein, the term “population of cells” refers to a group of two or more cells. For example, and without any limitation, a population of cells can include different cell types, e.g., T cells and K cells, or different clones, e.g., NeoTCR cells expressing different NeoTCRs. In certain non-limiting embodiments, the population of cells includes at least about 2, at least about 10, at least about 100, at least about 200, at least 500, at least 1000 cells, at least about 1 x 10⁴ cells, at least about 1 x 10⁵ cells, at least about 1 xlO⁶ cells, at least about 1 x 10⁷ cells, or at least about 1 x 10⁸ cells.

“Young” or “Younger” or “Young T cell” as it relates to T cells means memory stem cells (TMSC) and central memory cells (TCM). These cells have T cell proliferation upon specific activation and are competent for multiple cell divisions. They also can engraft after re-infusion, to rapidly differentiate into effector T cells upon exposure to their cognate antigen and target and kill tumor cells, as well as to persist for ongoing cancer surveillance and control.

2. Adoptive Cell Therapies

In one aspect, the present disclosure is directed to methods and compositions relating to the development and improvement of cell-based therapies, referred herein as adoptive cell therapies. As described in detail herein, adoptive cell therapies involve the use of cells to target and facilitate the elimination of diseased cells, e.g., cancer cells. In certain non-limiting embodiments, the present disclosure is directed to methods useful for the manufacturing of adoptive cell therapies. For example, without any limitation, the methods disclosed herein allow for the manufacturing of adoptive cell therapies in closed systems to minimize the risk of contamination.

As used herein, “adoptive cell therapy” refers to a therapy in which cells from the immune system are infused into a subject to help the body fight diseases. In certain embodiments, the adoptive cell therapy involves the use of cells taken from a subject’s own immune system, expanded ex vivo , and then infused into the subject to help the subject’s immune system fight a disease. In certain embodiments, the adoptive cell therapy relates to the infusion of cells that are engineered to improve their ability to target a cancer cell. In certain embodiments, the adoptive cell therapy relates to the infusion of cells of the immune system or hematopoietic stem cells. For example, without any limitation, adoptive cell therapies can involve the infusion of CD8+ T cells, CD4+ T cells, NK-cells, delta-gamma T-cells, regulatory T-cells, or tumor-infiltrating lymphocytes. In certain embodiments, the adoptive cell therapy involves the infusion of T cells. In certain embodiments, the adoptive cell therapy involves the infusion of NK cells. In certain embodiments, the adoptive cell therapy is directed to the use of Cell Products, as described in Section 2.1. In certain embodiments, the adoptive cell therapy is directed to the use of NeoTCR Products, described in Section 2.2. In certain embodiments, the adoptive cell therapy includes NeoTCR Viral Products, as described below.

2.1. Cell Products

Cell Products comprise cell therapies derived from cells that are gene-edited using constructs containing one or more 2A peptides. Such cell products can be made using non-viral or viral methods wherein a gene of interest is inserted into a genome using 2A peptides in the constructs. The use of the 2A peptide sequences enables the expression of multiple proteins within a single open reading frame through co-translational cleavage events and can overcome the problem of uneven expression of different proteins which overcomes a major hurdle in gene editing.

Cell Products include gene-edited cells that retain all or a portion of a 2A peptide sequence in the translated product such that the gene or genes of interest inserted into a genome retain all or a portion of the 2A peptide on one or both of the flanking ends of the gene(s).

Cell Products also include gene-edited cells that fully cleave off the 2A peptide from the gene of interest during translation such that the gene or genes of interest inserted into a genome do not have all or a portion of the 2A peptide on either of the flanking ends of the gene(s). In this scenario, the inserted gene of interest does not contain any non-native epitopes caused by one or more amino acids of the 2A peptide including on either of the flanking ends of the gene(s).

Cell Products include gene-edited cells that are edited using viral and/or non-viral methods.

2.2. NeoTCR Products

In certain embodiments, using the gene-editing technology and NeoTCR isolation technology described in PCT/US2020/17887 and PCT/US2019/025415, which are incorporated herein in their entireties, NeoTCRs are cloned in autologous CD8+ and CD4+ T cells from the same patient with cancer by precision genome engineered (using a DNA-mediated (non-viral) method as described in Figures 1A-1C) to express the NeoTCR. In other words, the NeoTCRs that are tumor-specific are identified in cancer patients, such NeoTCRs are then cloned, and then the cloned NeoTCRs are inserted into the cancer patient’s own T cells. NeoTCR expressing T cells are then expanded in a manner that preserves “young” T cell phenotypes, resulting in a NeoTCR Product in which the majority of the T cells exhibit T memory stem cell and T central memory phenotypes.

These ‘young’ or ‘younger’ or less-differentiated T cell phenotypes are described to confer improved engraftment potential and prolonged persistence post-infusion. Thus, the administration of NeoTCR Product, consisting significantly of ‘young’ T cell phenotypes, has the potential to benefit patients with cancer, through improved engraftment potential, prolonged persistence post-infusion, and rapid differentiation into effector T cells to eradicate tumor cells throughout the body.

Ex vivo mechanism-of-action studies were also performed with NeoTCR Product manufactured with T cells from patients with cancer. Comparable gene editing efficiencies and functional activities, as measured by antigen-specificity of T cell killing activity, proliferation, and cytokine production, were observed demonstrating that the manufacturing process described herein is successful in generating product with T cells from patients with cancer as starting material.

In certain embodiments, the NeoTCR Product manufacturing process involves electroporation of dual ribonucleoprotein species of CRISPR-Cas9 nucleases bound to guide RNA sequences, with each species targeting the genomic TCRa and the genomic TCRp loci. The specificity of targeting Cas9 nucleases to each genomic locus has been previously described in the literature as being highly specific. Comprehensive testing of the NeoTCR Product was performed in vitro and in silico analyses to survey possible off-target genomic cleavage sites, using COSMID and GUIDE-seq, respectively. Multiple NeoTCR Products or comparable cell products from healthy donors were assessed for cleavage of the candidate off-target sites by deep sequencing, supporting the published evidence that the selected nucleases are highly specific.

Further aspects of the precision genome engineering process have been assessed for safety. No evidence of genomic instability following precision genome engineering was found in assessing multiple NeoTCR Products by targeted locus amplification (TLA) or standard FISH cytogenetics. No off-target integration anywhere into the genome of the NeoTCR sequence was detected. No evidence of residual Cas9 was found in the cell product.

The comprehensive assessment of the NeoTCR Product and precision genome engineering process indicates that the NeoTCR Product will be well tolerated following infusion back to the patient.

The genome engineering approach described herein enables highly efficient generation of bespoke NeoTCR T cells (i.e., NeoTCR Products) for personalized adoptive cell therapy for patients with solid and liquid tumors. Furthermore, the engineering method is not restricted to the use in T cells and has also been applied successfully to other primary cell types, including natural killer and hematopoietic stem cells.

2.3. Pharmaceutical Formulations

Pharmaceutical formulations of the Cell Products are prepared by combining the NeoTCR cells in a solution that can preserve the ‘young’ phenotype of the cells in a cryopreserved state.

Additional pharmaceutically acceptable carriers, buffers, stabilizers, and/or preservatives can also be added to the cryopreservation solution or the aqueous storage solution. Any cryopreservation agent and/or media can be used to cryopreserve the Cell Product, including but not limited to CryoStor, CryoStor CS5, CELLBANKER, and custom cryopreservation media that optionally include DMSO.

In certain embodiments, the Cell Products are NeoTCR Products or NeoTCR Viral Products. In certain embodiments, the Cell Products are NeoTCR Viral Products. In certain embodiments, the Cell Products are NeoTCR Products.

2.4. Gene-Editing Methods

In certain embodiments, the present disclosure involves, in part, methods of engineering human cells, e.g., engineered T cells or engineered human stem cells. In certain embodiments, the present disclosure involves, in part, methods of engineering human cells, e g., NK cells, NKT cells, macrophages, hematopoietic stem cells (HSCs), cells derived from HSCs, or dendritic/antigen-presenting cells. In certain embodiments, such engineering involves genome editing. For example, but not by way of limitation, such genome editing can be accomplished with nucleases targeting one or more endogenous loci, e.g., TCR alpha (TCRa) locus and TCR beta (TCRb) locus. In certain embodiments, the nucleases can generate single-stranded DNA nicks or double-stranded DNA breaks in an endogenous target sequence. In certain embodiments, the nuclease can target coding or non-coding portions of the genome, e.g., exons, introns. In certain embodiments, the nucleases contemplated herein comprise homing endonuclease, meganuclease, megaTAL nuclease, transcription activator-like effector nuclease (TALEN), zinc-finger nuclease (ZFN), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas nuclease. In certain embodiments, the nucleases can themselves be engineered, e.g., via the introduction of amino acid substitutions and/or deletions, to increase the efficiency of the cutting activity.

In certain embodiments, a CRISPR Cas nuclease system is used to engineer human cells. In certain embodiments, the CRISPR/Cas nuclease system comprises a Cas nuclease and one or more RNAs that recruit the Cas nuclease to the endogenous target sequence, e.g ., single guide RNA. In certain embodiments, the Cas nuclease and the RNA are introduced in the cell separately, e.g. using different vectors or compositions, or together, e.g., in a polycistronic construct or a single protein-RNA complex. In certain embodiments, the Cas nuclease is Cas9 or Casl2a. In certain embodiments, the Cas9 polypeptide is obtained from a bacterial species including, without limitation, Streptococcus pyogenes or Neisseria menengitidis. 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), herein incorporated by reference for all that it teaches.

In certain embodiments, genome editing occurs at one or more genome loci that regulate immunological responses. In certain embodiments, the loci include, without limitation, TCR alpha (TCRa) locus, TCR beta (TCRP) locus, TCR gamma (TCRy), and TCR delta (TCR5).

In certain embodiments, genome editing is performed by using non-viral delivery systems. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid 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. I. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal ofBiological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection 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 can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a subject can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue or are injected systemically.

In certain embodiments, genome editing is performed by using viral delivery systems. In certain embodiments, the viral methods include targeted integration (including but not limited to AAV) and random integration (including but not limited to lentiviral approaches). In certain embodiments, the viral delivery would be accomplished without integration of the nuclease. In such embodiments, the viral delivery system can be Lentiflash or another similar delivery system.

In certain embodiments, the gene-editing methods comprise insertion (i.e., knock-in) of a NeoTCR into a cell in combination with the knockout of the endogenous TCR. In certain embodiments, the knock-in of a NeoTCR and knockout of the endogenous TCR gene-edited cell further comprises a knock-in of an additional expression element. In certain embodiments, the knock-in of a NeoTCR and knockout of the endogenous TCR gene-edited cell further comprises a knockout of an additional endogenous element. Additional information regarding the gene editing can be found in International Patent Application Nos. PCTVUS20/031007, PCT/US20/030818, and PCT/US20/030704.

2.5. Homolosv Recombination Templates

In certain embodiments, the present disclosure provides genome editing of a cell by introducing and recombining homologous recombination (HR) template nucleic acid sequence into an endogenous locus of a 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 first and second homology arms. In certain embodiments, the homology arms can be of about 300 bases to about 2,000 bases. For example, each homology arm can be 1,000 bases. In certain embodiments, the homology arms can be homologous to first and second endogenous sequences of the cell. In certain embodiments, the endogenous locus is a TCR locus. For example, the first and second endogenous sequences are within a TCR alpha locus or a 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 content of which is herein incorporated by reference. In certain embodiments, the HR template comprises a TCR alpha gene sequence and a TCR beta gene sequence.

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

2.6. Nucleic Acid Compositions and Vectors

The present disclosure provides compositions comprising cells (e.g., T cells) disclosed herein. In certain embodiments, the present disclosure provides nucleic acid compositions comprising a polynucleotide encoding aNeoTCR. In certain embodiments, the nucleic acid compositions disclosed herein comprise a polynucleotide encoding a NeoTCR and one or more additional expression elements. In certain embodiments, the nucleic acid compositions disclosed herein comprise a polynucleotide encoding a NeoTCR and one or more additional elements to knockdown or knockout the expression of an endogenous protein. Also provided are cells comprising such nucleic acid compositions.

In certain embodiments, the nucleic acid composition further comprises a promoter that is operably linked to the NeoTCR disclosed herein. In certain embodiments, the nucleic acid composition further comprises a promoter that is operably linked to one or more additional expression elements disclosed herein.

In certain embodiments, the promoter is endogenous or exogenous. In certain embodiments, the exogenous promoter is selected from the group consisting of an elongation factor (EF)-l promoter, a CMV promoter, a SV40 promoter, a PGK promoter, a long terminal repeat (LTR) promoter, and a metallothionein promoter. In certain embodiments, the promoter is an inducible promoter. In certain embodiments, the inducible promoter is selected from the group consisting of an NFAT transcriptional response element (TRE) promoter, a CD69 promoter, a CD25 promoter, an IL2 promoter, an IL12 promoter, a p40 promoter, and a Bcl-xL promoter.

The compositions and nucleic acid compositions can be administered to subjects or and/delivered into cells by suitable art-known methods or as described herein. Genetic modification of a cell (e.g., a T cell) can be accomplished 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 employed for the introduction of the DNA construct into the cell. Non-viral vectors may be used as well.

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

Other transducing viral vectors can be used to modify a cell. In certain embodiments, the chosen vector exhibits high efficiency of infection 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, adenoviral, lentiviral, and adena- associated viral vectors, vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of 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; Cometta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; LeGal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S- 83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346).

Non-viral approaches can also be employed for genetic modification of a cell. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid 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 ofBiological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection 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 can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a subject can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue or are injected systemically.

Polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element or intron (e.g. the elongation factor la enhancer/promoter/intron structure). For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

The resulting cells can be grown under conditions similar to those for unmodified cells, whereby the modified cells can be expanded and used for a variety of purposes.

3. _ Therapeutic Compositions and Methods of Manufacturing

In another aspect, the present disclosure is directed to methods of manufacturing the adoptive cell therapeutic compositions described herein. Manufacture of adoptive cell therapeutic compositions (each being an adoptive cell therapy) is a highly complex multi-step process involving the isolation, activation, expansion, and formulation of cells in preparation for their subsequent infusion into patients. The present disclosure provides methods for reducing not only the complexity of the manufacturing process but also the risks for contamination, while simultaneously improving the quality and scalability of the manufacturing processes.

3.1. Cell Collection and Isolation

In certain non-limiting embodiments, the present disclosure provides methods for obtaining a sample comprising cells, e.g., immune cells, for use in the context of adoptive cell therapy from a subject. In certain embodiments, the cells, e.g., immune cells, can be obtained by using suitable methods known in the art. For example, but without any limitation, immune cells can be obtained by leukapheresis. Leukapheresis refers to a specific apheresis strategy where blood is removed from a subject and white blood cells, i.e., leukocytes, are separated and collected and the remaining blood is returned to the subject. In certain embodiments, the leukapheresis can be autologous (i.e., the cells collected will be subsequently infused into the donor of the cells) or allogeneic (i.e., the cells collected will be subsequently infused into a patient that was not the donor).

In certain embodiments, leukapheresis is employed to produce a leukopak. A leukopak can comprise any suitable container, typically a flexible bag, for the collection and storage of leukocytes. In certain embodiments the leukopak while be configured to contain a target volume of about 100 mL up to about 400 mL (e.g., following addition of autologous plasma).

In certain embodiments, the cells, e.g., immune cells, can be obtained from a tumor sample. For example, but without any limitation, immune cells can be tumor-infiltrating lymphocytes (TILs). In certain embodiments, the TILs cell and can be isolated after dissection, fragmentation, and isolation from a solid tumor sample.

In certain embodiments, the cells obtained from a subject can be stored at a temperature between about 2°C and about 8°C. In certain embodiments, the cells are processed within 24 hours from collection. In certain embodiments, the cells can be optionally frozen after collection (e.g., cryopreserved) and stored before further processing.

Following collection, e.g., by leukapheresis, the cells can be isolated and enriched for their phenotype. In certain embodiments, but without any limitation, the cells can be isolated by their positivity for a suitable cell marker. In certain non-limiting embodiments, the marker can be CD3, CD4, CD8, CD45, or any combination thereof. In certain embodiments, the isolation occurs by suitable methods known in the art. In certain embodiments, the cells can be isolated by using chromatography, magnetic beads, or fluorescence-activated cell sorting (FACS), centrifugation, filtration, and other methods known in the art. In certain embodiments, the isolation is performed by FACS using antibodies reacting against CD3, CD8, CD4, CD45, or any combination thereof. In certain embodiments, the isolation comprises centrifugation. In certain embodiments, the isolation comprises counterflow centrifugation. In certain embodiments, the isolation comprises counterflow centrifugation elutriation.

In certain embodiments, following initial sampling for cell count/viability, including but not limited to cell count/viability and flow cytometry (for cell characterization) a CliniMACS Prodigy or other suitable system can be employed to facilitate the enrichment process. For example, but not by way of limitation, a leukopak can be loaded onto a CliniMACS Prodigy instrument, or other suitable system, e.g., by sterile welding to the sterile single use disposable Prodigy TS520 kit. In certain embodiments, CD4+ and CD8+ T cells can be positively enriched for further processing using the Prodigy’s “T Cell Transduction Process Program” or other suitable enrichment program In certain embodiments, such enrichment will involve discarding other cell types/impurities. In certain embodiments, cells can again be sampled for cell count/viability and flow cytometry (cell characterization assays).

In certain embodiments, the target cell count after enrichment is < about 5 x 10⁹ cells. In certain embodiments, the target cell count after enrichment is from about 1 x 10⁷ cells to about 5 x 10⁹ cells. In certain embodiments, the target cell count after enrichment is from about 1 x 10⁸ cells to about 5 x 10⁹ cells. In certain embodiments, the target cell count after enrichment is from about 1 x 10⁹ cells to about 5 x 10⁹ cells.

3.2. Cell Activation

In certain non-limiting embodiments, the present disclosure provides methods including activation of the cells, e.g., T cells. In certain embodiments, the methods disclosed herein include contacting the cells with an activation reagent. As used herein, an “activation reagent” refers to a composition capable of activating certain biological processes. When related to T cells, an “activation reagent” refers to a composition capable of activating T cells. In certain embodiments, the activation reagent includes an antigen-presenting cell (APC). In certain embodiments, the APC is a dendritic cell. In certain embodiments, the APC is a B cell. In certain embodiments, the APC presents an antigen in the major histone compatibility complex (MHC). In certain embodiments, the activation reagent includes a second cell providing a costimulatory signal. For example, but without any limitation, the second cell can be a monocyte expressing aB7 molecule.

In certain embodiments, the activation reagent includes an artificial antigen-presenting cell (aAPC). In certain embodiments, the aAPC comprises artificial lipids and one or more costimulatory molecules. Additional information on aAPC can be found in Latouche and Sadelain, Nat Biotechnol. 2000 Apr; 18(4):405-9; or Perica et al., Biochim Biophys Acta.

2015; 1853(4):781-790.

In certain embodiments, the activation reagent includes a costimulatory ligand. A costimulatory ligand can be soluble or provided on a cell surface. Non-limiting examples of costimulatory ligand include CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL,

OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor,

3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3.

In certain embodiments, the activation reagent includes one or more antibodies. In certain embodiments, the antibody is selected from the group consisting of anti-CD2, anti-CD3, anti-CD27, anti-CD28, anti-4-lBB, anti-OX40, anti-CD30, anti-CD40, anti-PD-1, anti-ICOS, anti -lymphocyte function-associated antigen-1 (LFA-1), anti-CD7, anti-LIGHT, anti-NKG2C, anti-B7-H3, anti-CD83, or a combination thereof. In certain embodiments, the activation reagent includes an anti-CD3 antibody. Non-limiting examples of anti-CD3 antibodies include OKT-3, T3, CD3s, otelixizumab, teplizumab, and visilizumab. In certain embodiments, the activation reagent includes an anti-CD28 antibody. In certain embodiments, the activation reagent includes an anti-CD2 antibody. In certain embodiments, the activation reagent includes an anti-CD2, an anti-CD3, and an anti-CD28 antibody. In certain embodiments, the activation reagent includes an anti-CD3 and an anti-CD28 antibody.

In certain embodiments, the antibody is soluble. In certain embodiments, the antibody is bound to a surface. For example, without any limitation, the antibody is bound to a polymeric surface, a magnetic bead, a non-magnetic bead, or an agarose bead. Non-limiting examples of antibodies used in the methods disclosed herein include T Cell TransAct™, Dynabeads™ Human T -Activator CD3/CD28, and ImmunoCult™ Human CD3/CD28 T Cell Activator. In certain embodiments, the activation reagent includes a CD3 and a CD28 agonist.

In certain embodiments, the activation reagent includes growth factors and/or cytokines. In certain embodiments, the activation reagent includes a cytokine selected from the group consisting of IL2, IL7, ILIO, IL12, ILLS, IL21, or a combination thereof. In certain embodiments, the cytokine is IL2. In certain embodiments, the cytokine is IL7. In certain embodiments, the cytokine is IL15.

In certain embodiments, the enriched cells are transferred to a G-Rex lOOM-CS flask, or other suitable container, for activation. In certain embodiment, a non-bead-based activation strategy (TransAct, Miltenyi) will be employed. For example, but not by limitation, T cells can be activated by incubation with TransAct (aCD3/CD28 reagent). In certain embodiments, the aCD3/CD28 reagent will be employed at a ratio of 1 : 17.5, In certain embodiments, the aCD3/CD28 reagent will be contacted with the cells in a suitable medium, e.g., TexMACS medium. In certain embodiments, the medium will comprise one or more suitable supplements. For example, but not by way of limitation, the media can be supplemented with 3% human AB serum. In certain embodiments the media will be supplemented with additional activation reagents, e g., IL7 and/or IL15. In certain embodiments, the media will be supplemented with about 12.5 ng/mL IL7 and / about 12.5 ng/mL IL15. In certain embodiments, the cells can be cultured in the activation medium for about 48 to about 72 hours In certain embodiments, the activation will take place in an incubator at about 37°C and about 5% CO2

In certain embodiments, the activation reagent is removed and/or separated from the cells. For example, but without any limitation, the activation reagent is removed by exchanging cell culture media, by affinity chromatography, or by counterflow centrifugation. In certain embodiments, the cell activation occurs in a closed system.

3.3. Cell Editing and Transfection

In certain non-limiting embodiments, the present disclosure comprises methods including editing and/or transfection of a cell, e.g., T cell, to express an exogenous nucleic acid and/or protein, e.g., a NeoTCR. Various transfection methods can be used including, but without any limitation, viral infection, electroporation, membrane disruption, and combination thereof.

In certain embodiments, the transfection can occur with a viral vector. In certain embodiments, the viral vector can be a retrovirus. In certain embodiments, the viral vector can be a lentivirus. In certain embodiments, the viral vector can be an adena-associated virus (AAV). Additional information on the viral vectors contemplated by the present disclosure and used by the methods disclosed herein can be found in Section 2.6. In certain embodiments, the transfection can occur with non-viral vectors and/or non-viral methods. For example, without limitation, the cells can be transfected by electroporation. In certain embodiments, the electroporation is a large-scale electroporation. As used herein, the term “large-scale” refers to experimental and/or manufacturing conditions using an amount of cells of at least about 100 x 10⁶ cells, at least about 1 x 10⁷ cells, at least about 10 x 10⁷ cells, at least about 100 x 10⁷ cells, at least about 1 x 10⁸ cells, at least about 10 x 10⁸ cells, at least about 100 x 10⁸ cells, at least about 1 x 10⁹ cells, at least about 10 x 10⁹ cells, at least about 100 x 10⁹ cells, or at least about 1 x 10¹⁰ cells. In certain embodiments, the large-scale electroporation produces at least about 100 x 10⁶ edited cells, at least about 1 x 10⁷ edited cells, at least about 10 x 10⁷ edited cells, at least about 100 x 10⁷ edited cells, at least about 1 x 10⁸ edited cells, at least about 10 x 10⁸ edited cells, at least about 100 x 10⁸ edited cells, at least about 1 x 10⁹ edited cells, at least about 10 x 10⁹ edited cells, at least about 100 x 10⁹ edited cells, or at least about 1 x 10¹⁰ edited cells. Additional information on the non-viral vectors and non-viral methods contemplated by the present disclosure can be found in Sections 2.4-2.6 above and in International Patent Application No. PCT/US2018/058230, the content of which is incorporated herein in its entirety.

In certain embodiments, after activation, the cell is collected using a counterflow centrifugation system and mixed with the transfection reagents, e g., DNA plasmid. Collection of the cell and concentration in a small volume facilitates and increases the transfection efficiency. In certain embodiments, after transfection, the cell is transferred in a gas-permeable flask. In certain embodiments, the cell transfection occurs in a closed system.

In certain embodiments, the transfected cell can be selected. For example, but without any limitation, the transfected cell can be selected for the expression of the NeoTCR using peptide-major histocompatibility complex (pMHC) multimers bound to dextramer. Additional information regarding selection of transfected cells can be found in International Patent Publication Nos. W02019195310A1, W02020056173A1, and WO2020167918A1, the content of each of which is incorporated herein in its entirety. In certain embodiments, the transfected cell is not selected.

3.4. Cell Expansion

In certain non-limiting embodiments, the present disclosure provides methods including culturing the cell, e.g., T cell. In certain embodiments, the cell is edited. In certain embodiments, the cell is cultured to obtain a population of cells. In certain embodiments, the cell is cultured in a cell culture medium. In certain embodiments, the cells are transferred to a cell culture chamber for cell proliferation and expansion. In certain embodiments, the cell culture is designed to promote the cells to maintain, develop, and/or retain a stem-like state (i.e., T cells that have a memory stem cell or stem cell (Tmsc or Tcm) phenotype).

For example, in certain experiments, the cells can be cultured in cell culture chambers in an incubator (5% CO2, 37°C) in culture medium. In certain experiments, the cell culture chambers is a G-Rex (Wilson Wolf) cell culture chambers, or other suitable chamber. In certain embodiments, alternative static gas exchange cell culture chambers can be used based on such static gas exchange cell culture chamber’s ability to allow for sufficient cell proliferation of gene edited cells that possess a memory stem cell or stem cell (Tmsc or Tcm) phenotype).

In certain experiments, the media used to culture the cells following electroporation is a chemically-defined, animal component-free medium shown to promote T cell expansion while maintaining T cell functionality and potency. In certain experiments, the media can be PRIME- XV T Cell CDM (Irvine Scientific CDM), ImmunoCult XF (Stemcell), ExCellerate (R&D Systems), LumphoOne (Takara Bio), GT-T551 (TakaraBio), X-VIVO 15, AIM V, CTS OpTmizer (Gibco), and any other suitable medias with similar physiological attributes as those described herein. Suitable medias are known to those of skill in the art that are animal component-free, that enable efficient T cell expansion without the addition of serum or plasma, and promote expansion and growth of T cells with a naive phenotype (e.g., Tmsc and Tcm) can be used in the medias and methods described herein.

Serum free substitute additives can also be used in the medias described herein. For example, but not by way of limitation, Physiol ogix (Nucleus Biologies), human platelet lysate (a growth factor-rich cell culture supplement derived from healthy donor human platelets, Stem Cell), CTS Immune Cell Serum Replacement (Gibco) are suitable supplements that can be used in the context of the methods of the present disclosure. Additional suitable serum free substitutes are known to those of skill in the art to enable efficient cell, e.g., T cell, expansion without the addition of serum or plasma, and promote expansion and growth of cells, e.g., T cells, with a naive phenotype (e.g., Tmsc and Tcm) and thus can be used in the medias and methods described herein.

In certain embodiments, the addition of cytokines can also be used in the context of the medias and methods described herein. In certain experiments, the media can be supplemented with or otherwise contain IL2. In certain experiments, the media can be supplemented with or otherwise contains IL7. In certain experiments, the media can be supplemented with or otherwise contains IL15. In certain experiments, the media can be supplemented with or otherwise contains IL21. In certain experiments, the media does not contain or is not supplemented with IL2. In addition to supplementation with IL2, IL7, IL15, and/or IL21 described above as single agents or combinations thereof for the supplementation of media, IL12, alpha interferon, or beta interferon can be used alone or in combination with each other or with the IL2, IL7, IL15, and/or IL21. Furthermore, any cytokine or chemokine that is involved in lymphocyte proliferation and differentiation can be added to any single IL2, IL7, IL12, IL15, IL21, alpha interferon or beta interferon, or any combination thereof. The concentration and ratios of each of the cytokines and/or chemokines can be adjusted based on the single agent use or combination use and titrated based on the lymphocyte proliferation and differentiation desired.

In certain embodiments, the media does not contain IL2. In certain embodiments, the media contains one or more cytokines but does not contain IL2. In certain embodiments, the media contains IL7 but does not contain IL2. In certain embodiments, the media contains IL15 but does not contain IL2. In certain embodiments, the media contains IL7 and IL15 but does not contain IL2.

In certain embodiments, the cytokine is present in the cell culture medium at a concentration between about 0.05 ng/ml to about 100 ng/ml. For example, but without any limitation, the cytokine is present in the cell culture medium at a concentration of about 0.05 ng/ml, about 0.1 ng/ml, about 0.5 ng/ml, about 1 ng/ml, about 2 ng/ml, about 5 ng/ml, about 8 ng/ml, about 10 ng/ml, about 20 ng/ml, about 30 ng/ml, about 50 ng/ml, about 60 ng/ml, about 80 ng/ml, or about 100 ng/ml.

In addition to the addition of serum free substitute additives and/or chemokines and/or cytokines as described herein, the addition of fatty acids can be beneficial in achieving the desired proliferation and differentiation.

In certain embodiments, fibronectin, insulin, and/or transferrin can be included in the media. In certain embodiments, the transferrin used is recombinant transferrin. In certain embodiments, the transferrin used is non-recombinant transferrin. In certain embodiments, it can be useful to increase the concentration of transferrin when recombinant transferrin is used compared to non-recombinant transferrin in order to achieve the same benefits of lymphocyte proliferation and differentiation to achieve T cells in culture with a naive phenotype (e.g., Tmsc and Tcm).

In certain embodiments, different concentrations of glucose in the cell medias can be used. For example, but not by way of limitations, the glucose concentration can be less than about 3.7 g/L glucose. In certain embodiments, the glucose concentration is between about 3.7 about 4.0 g/L glucose. In certain embodiments, the glucose concentration is between about 4.0 - about 4.2 g/L glucose. In certain embodiments, the glucose concentration is between about 4.2 - about 4.5 g/L glucose. In certain embodiments, the glucose concentration is between about 4.3 - about 4.4 g/L glucose. In certain embodiments, the glucose concentration is between about 4.4 - about 4.5 g/L glucose. In certain embodiments, the glucose concentration is greater than about 4.5 g/L glucose. As cell density in culture increases, so can the concentration of glucose. For example, for a high density cell culture the glucose concentration can be increased up to 100 g/L.

In certain embodiments, antioxidants can be added to the media to promote lymphocyte proliferation and differentiation to achieve T cells in culture with a naive phenotype (e.g., Tmsc and Tcm).

In certain embodiments, reducing agents can be added to the media to promote lymphocyte proliferation and differentiation to achieve T cells in culture with a naive phenotype (e.g., Tmsc and Tcm).

In order to promote automation of the manufacturing processes, e.g., NeoTCR Product manufacture, stir bioreactors can be used to culture the cells instead of a static gas exchange cell culture chamber. Such stir bioreactors allow for real-time analytics and reaction to changes in conditions. For example, a stir bioreactor can be designed to have in line bioanalytics to measure cell mass, lactate, etc., in a closed system without manual sampling.

Alternatively, in order to promote automation of the manufacturing processes, e.g., NeoTCR Product manufacture, shaking/rotating bioreactors can be used to culture the cells instead of a static gas exchange cell culture chamber. Such shaking/rotating bioreactors allow for real-time analytics and reaction to changes in conditions. For example, a shaking/rotating bioreactor can be designed to have in line bioanalytics to measure cell mass, lactate, etc., in a closed system without manual sampling.

Furthermore, bioreactors (e.g., stir, shanking, rotating, etc.) can be designed and programmed to automatically add media supplements to the culture in order to increase or decrease the concentration of certain components in the media. For example, the bioreactor can be designed and programmed to detect lactate levels in the cell culture and add in glucose in order to keep the glucose: lactate levels optimal for lymphocyte proliferation and differentiation to achieve cell, e.g., T cells, in culture with a naive phenotype (e.g., Tmsc and Tcm). In other examples, the bioreactors can be designed and programmed to remove lactate during the culture process in order to promote lymphocyte proliferation and differentiation to achieve cells, e.g., T cells, in culture with a naive phenotype (e.g., Tmsc and Tcm). Another example of the use of a bioreactor is to design and program the bioreactor to detect dissolved oxygen as a negative indicator of a desired cell environment. In certain experiments, cell counts can be taken throughout the culture period. In certain experiments, the cells are taken from the static gas exchange culture chambers (e.g., a G-Rex flask) at the half-way point of cell culture (i.e., the halfway point between the time of electroporation and the time when the cells are cryopreserved, e.g., as a NeoTCR Product) and split into two new static gas exchange culture chambers with fresh media.

In certain embodiments, the cell culture medium includes fibronectin. In certain embodiments, the cell culture medium includes insulin. In certain embodiments, the cell culture medium includes transferrin.

In certain non-limiting embodiments, the cell culture medium induces controlled growth and proliferation of the cells in order to increase the total cell number. In certain non-limiting embodiments, the total cell number is at least about 1 ^x 10⁶ cells, at least about 3 ^x 10⁶ cells, at least about 5 ^x 10⁶ cells, at least about 7 ^x 10⁶ cells, at least about 9 ^x 10⁶ cells, at least about 1 ^x 10⁷ cells, at least about 5 ^x 10⁷ cells, at least about 1 ^x 10⁸ cells, at least about 5 ^x 10⁸ cells, at least about 1 ^x 10⁹ cells, at least about 3 ^x 10⁹ cells, or at least about 5 ^x 10⁹ cells.

In certain embodiments, the culturing occurs in a total volume of from about 0.1 L to about 5 L, from about 0.1 L to about 2 L, or from about 0.2 L to about 2 L. In certain embodiments, the total volume is of about 0.1 L, about 0.2 L, about 0.3 L, about 0.4 L, about 0.5 L, about 0.6 L, about 0.7 L, about 0.8 L, about 0.9 L or about 1.0 L. In certain non-limiting embodiments, as illustrated in the Example section, the total volume can vary based on the total number of cells.

In certain embodiments, the culturing includes obtaining a young T cell. In certain embodiments, the young T cell is CD45RA⁺, CD62L⁺, CD28⁺, CD95 , CCR7⁺, and CD27⁺. In certain embodiments, the young T cell is CD45RA⁺, CD62L⁺, CD28⁺, CD95⁺, CD27⁺, CCR7⁺.

In certain embodiments, the young T cells is CD45RO⁺, CD62L⁺, CD28⁺, CD95⁺, CCR7⁺, CD27⁺, CD127⁺. In certain embodiments, the young T cell is a T memory stem T cells (T_MSC).

In certain embodiments, the young T cell is a T central memory T cells (T_CM). Additional information regarding young T cells and methods for producing, obtaining, and/or culturing them can be found in International Patent Application No. PCT/US2020/025758, the content of which is incorporated herein in its entirety.

In certain embodiments, the culturing includes obtaining a population of cells. In certain embodiments, the population of cells comprises TMSC and TCM. In certain embodiments, the population of cells comprises at least about 20% of TMSC and TCM collectively, at least about 25% TMSC and TCM collectively, at least about 30% TMSC and TCM collectively, at least about 35%

TMSC and TCM collectively, at least about 40% TMSC and TCM collectively, at least about 45% TMSC and TCM collectively, at least about 50% TMSC and TCM collectively, at least about 55%

TMSC and TCM collectively, at least about 60% TMSC and TCM collectively or more than about 61% TMSC and TCM collectively. In certain embodiments, the population of cells comprises at least about 65% TMSC and TCM collectively, at least about 70% TMSC and TCM collectively, at least about 75% TMSC and TCM collectively, at least about 80% TMSC and TCM collectively, at least about 85% TMSC and TCM collectively, at least about 90% TMSC and TCM collectively, or at least about 95% TMSC and TCM collectively.

In certain embodiments, the culturing occurs in a closed system. In certain embodiments, the culturing occurs in a gas permeable system. Non-limiting examples of gas permeable system include G-rex®, Cell Factory, MACS GMP Cell Expansion Bag, VueLife, and Evolve. In certain embodiments, the culturing includes counterflow centrifugation.

In certain embodiments, the cells are cultured for a period of time sufficient to obtain a particular total cell number. In certain embodiments, the cells are expanded for about 10 days.

In certain embodiments, the cells are expanded for about 11 days. In certain embodiments, the cells are expanded for about 12 days.

In certain embodiments, after transfection, the cells are cultured in flasks at about 37°C and about 5% CO2 to facilitate expansion. In certain embodiments, the culture can make use of any suitable media. In certain embodiments, the suitable media is TexMACS GMP medium. In certain embodiments the suitable media is Prime XY media. In certain embodiments the suitable media is Prime XV media supplemented with Physiologix™ XF SR. In certain embodiments, the Physiologix™ XF SR is supplemented at a concentration of 2% or approximately 2%. In certain embodiments, the suitable media is a media with substantially the same components as Prime XV media. In certain embodiments, the suitable media is a media with equivalent components as Prime XV media. In certain embodiments, the suitable media is a media with substantially the same or equivalent components as Prime XV media and is supplemented with Physiologix™ XF SR. In certain embodiments, the suitable media is a media with substantially the same or equivalent components as Prime XV media and is supplemented with a serum free additive that is substantially the same or equivalent to Physiologix™ XF SR.

In certain embodiments, the media will comprise supplements. For example, but not by way of limitation, the media can be supplemented with about 3% human AB serum or other suitable serum. For example, but not by way of limitation, the media can be supplemented with a serum free additive. In certain embodiments, the serum free additive is Physiologix™ XF SR or a supplement that is substantially similar or equivalent to Physiologix™ XF SR. In certain embodiments, the media can comprise one or more cytokine supplements. For example, but no by way of limitation, the media can comprise IL7 (at about 12.5ng/mL) and/or IL15 (at about 12.5 ng/mL).

In certain embodiments, the cell density of the culture will be monitored to ensure appropriate expansion. For example, but not limitation, on a suitable day post-transfection, e.g., on day 8, a cell count can be performed. Based on the cell number obtained, the cells can be split into one or more flasks to allow further expansion.

3.5. Harvesting

In certain non-limiting embodiments, the present disclosure provides methods including harvesting the cell, e g., T cell. In certain embodiments, the cell is edited. In certain embodiments, the present disclosure provides methods including harvesting the population of cells.

In certain embodiments, the cells can be harvested once a particular total cell number has been achieved. In certain embodiments, the population cells can be harvested once a particular total cell number has been achieved. In certain non-limiting embodiments, the total cell number is at least about 1 ^x 10⁶ cells, at least about 3 ^x 10⁶ cells, at least about 5 ^x 10⁶ cells, at least about 7 ^x 10⁶ cells, at least about 9 ^x 10⁶ cells, at least about 1 ^x 10⁷ cells, at least about 5 ^x 10⁷ cells, at least about 1 ^x 10⁸ cells, at least about 5 ^x 10⁸ cells, at least about 1 ^x 10⁹ cells, at least about 3 ^x 10⁹ cells, or at least about 5 ^x 10⁹ cells.

In certain embodiments, harvesting can include one or more of centrifugation, filtration, e.g., TFDF, acoustic wave separation, flocculation, and cell removal technologies. In certain embodiments, harvesting includes counterflow centrifugation. In certain embodiments, harvesting occurs in a closed system.

3.6. Transferrins in Infusion Bass and Final Formulation

In certain non-limiting embodiments, the present disclosure provides methods including transferring the cell, e.g., harvested cell, to a container for use in administration to a patient (e.g., “infusion bag"). In certain embodiments, the present disclosure provides methods including transferring the population of cells to a container for use in administration to a patient.

In certain embodiments, the cell is transferred to the container in a closed system. For example, but without any limitation, the cell can be transferred using a peristaltic pump. In certain embodiments, the cell is collected before transferring via centrifugation. In certain embodiments, the centrifugation is a counterflow centrifugation. In certain embodiments, the cell is prepared for being cryopreserved. In certain embodiments, the cell is prepared for administration to a patient. In certain embodiments, the population of cells is transferred to the container in a closed system. For example, but without any limitation, the population of cells can be transferred using a peristaltic pump. In certain embodiments, the population of cells is collected before transferring via centrifugation. In certain embodiments, the centrifugation is a counterflow centrifugation. In certain embodiments, the population of cells is prepared for being cryopreserved. In certain embodiments, the population of cells is prepared for administration to a patient.

In certain non-limiting embodiments, the container comprises a formulation. In certain embodiments, the formulation is a pharmaceutical formulation. In certain embodiments, the formulation is suitable for administration to a patient.

In certain embodiments, the pharmaceutical formulation comprises at least about 1 ^x 10⁶ gene-edited cells, at least about 3 ^x 10⁶ gene-edited cells, at least about 5 ^x 10⁶ gene-edited cells, at least about 7 ^x 10⁶ gene-edited cells, at least about 9 ^x 10⁶ gene-edited cells, at least about 1 ^x 10⁷ gene-edited cells, at least about 5 ^x 10⁷ gene-edited cells, at least about 1 ^x 10⁸ gene-edited cells, at least about 5 ^x 10⁸ gene-edited cells, or at least about 1 ^x 10⁹ gene-edited cells. In certain embodiments, the pharmaceutical formulation comprises about 4.0 ^x 10⁸ gene-edited cells, about 1.3 ^x 10⁹ gene-edited cells, about 4.0 ^x 10⁹ gene-edited cells, approxim about ately 1.3 ^x 10¹⁰ gene-edited cells, or about 4.0 ^x 10¹⁰ gene-edited cells. In certain embodiments, the pharmaceutical formulation comprises about 4.0 ^x 10⁸ gene-edited cells. In certain embodiments, the pharmaceutical formulation comprises about 1.3 ^x 10⁹ gene-edited cells. In certain embodiments, the pharmaceutical formulation comprises about 4.0 ^x 10⁹ gene-edited cells In certain embodiments, the pharmaceutical formulation comprises about 1.3 ^x 10¹⁰ gene-edited cells. In certain embodiments, the pharmaceutical formulation comprises about 4.0 ^x 10¹⁰ gene- edited cells.

In certain embodiments, the pharmaceutical formulation comprises a crystalloid solution. As used herein, the term “crystalloid solution” refers to an intravenous solution useful in clinical setting. In certain non-limiting embodiments, crystalloid solutions can be used for intravenous medication delivery. Non-limiting examples of crystalloid solution include 0.9% sodium chloride (NaCl), Hartmann’s (or Ringer’s lactate or compound sodium lactate) solution, or PlasmaLyte. In certain embodiments, the crystalloid solution is PlasmaLyte. In certain embodiments, the crystalloid solution is present in the formulation at a final concentration from about 30% v/v to about 60 % v/v, from about 35% v/v to about 60% v/v, from about 40% v/v to about 60 % v/v, from about 45% v/v to about 60 % v/v, from about 50% v/v to about 60 % v/v, from about 40% v/v to about 50 % v/v, or from about 45% v/v to about 50 % v/v. In certain embodiments, the crystalloid solution is present in the formulation at a final concentration of about 46% v/v.

In certain embodiments, the pharmaceutical formulation comprises a serum albumin. In certain embodiments, the serum albumin is human. In certain embodiments, the serum albumin is present in the formulation at a final concentration from about 0.1% w/v to about 5% w/v, from about 0.2% w/v to about 5% w/v, from about 0.3% w/v to about 5% w/v, from about 0.5% w/v to about 5% w/v, from about 0.7% w/v to about 5% w/v, from about 0.8% w/v to about 5% w/v, from about 0.9% w/v to about 5% w/v, from about 1.0% w/v to about 5% w/v, from about 1.5% w/v to about 5% w/v, from about 2% w/v to about 5%, from about 3% w/v to about 5% w/v, or from about 4% w/v to about 5% w/v. In certain embodiments, the serum albumin is present in the formulation at a final concentration of about 1% w/v.

In certain embodiments, the pharmaceutical formulation comprises a cryopreservation medium. In certain embodiments, the cryopreservation medium is CryoStor CS10. In certain embodiments, the cryopreservation medium is present in the formulation at a final concentration from about 10% v/v to about 70 % v/v, from about 20% v/v to about 70% v/v, from about 30% v/v to about 70 % v/v, from about 40% v/v to about 70 % v/v, from about 50% v/v to about 70 % v/v, from about 60% v/v to about 70 % v/v, or from about 45% v/v to about 55 % v/v. In certain embodiments, the cryopreservation medium is present in the formulation at a final concentration of about 50% v/v. In certain embodiments, the final pharmaceutical formulation contains 5% dimethyl sulfoxide (DMSO), human serum albumin, and Plasma-Lyte. In certain embodiments, the final pharmaceutical formulation contains the list of components provided in Table 1.

Table 1. Composition of Exemplary Pharmaceutical Formulations

3. 7. Quality Control and Additional Features

In certain embodiments, the methods of the present disclosure include the monitoring of parameters on-line (e.g., by direct connection to an analyzer) or off-line (e.g., by user intervention). In certain embodiments, the monitored parameters include temperature, pH, glucose, lactate, glucose/lactate ratio, oxygen, carbon dioxide, cell count, viability, gene and/or protein expression, complete blood count, potency, mycoplasma, functional features (e.g., cytokine secretion), sterility, endotoxins, and/or cell characterization.

In certain embodiments, the methods disclosed herein include adjustment of the parameters. For example, but without any limitation, if the monitored oxygen level is too low to achieve the cell expansion, the oxygen level is increased by introducing an oxygenated cell culture medium or by replacing the cell culture medium with an oxygenated cell culture medium. In certain embodiments, the adjustment is performed manually. In certain embodiments, the adjustment is performed automatically.

In certain embodiments, the methods disclosed herein further include characterization of the edited cell or the population of cells. In certain embodiments, the characterization includes determining the expression levels of an exogenous nucleic acid. For example, but without any limitation, the expression levels of a NeoTCR are determined by gene expression analysis or by FACS analysis. In certain embodiments, the characterization includes determining the gene knockout levels in the adoptive cell therapy. For example, but without any limitation, the expression levels of an endogenous gene, e.g., endogenous TCR, can be determined. In certain embodiments, the characterization includes genetic tests and sequencing of the cell’s genome. Non-limiting examples of genetic tests include Targeted Locus Amplification (TLA), Next Generation Sequencing (NGS), deep sequencing, targeted deep sequences, and Fluorescence In Situ Hybridization (FISH).

In certain embodiments, the characterization includes determining the cell subtypes in the adoptive cell therapy. For example, but without any limitation, the percentage of young T cells is determined.

In certain non-limiting embodiments, the present disclosure comprises the use of flasks and vessels for cell culturing. In certain embodiments, the flasks and vessels are gas permeable. In certain embodiments, the cell activation can be performed in flasks or gas-permeable bags. In certain embodiments, the cell activation can be performed in gas-permeable flasks. In certain embodiments, the cell expansion can be performed in flasks or gas-permeable bags. In certain embodiments, the cell expansion can be performed in gas-permeable flasks. In certain non limiting embodiments, the present disclosure comprises methods comprising the use of a process and/or apparatus for aseptically concentrating and washing T cells. In certain embodiments, the method comprises centrifugation. In certain embodiments, the method comprises counterflow centrifugation separation technology. Non-limiting examples of counterflow centrifugation include Gibco® Rotea and Ksep® System.

In certain non-limiting embodiments, the present disclosure comprises methods using an instrument or device that can pump a solution comprising cells in a sterile and/or closed system environment, allowing for continuous flow and cell processing. In certain embodiments, the instrument or device can perform cell separation, washing, fluid exchange, concentration, and/or other cell processing steps in a closed, sterile system.

4. Articles of Manufacture

In certain embodiments, the present disclosure provides articles of manufacture comprising the adoptive cell therapeutics disclosed herein. In certain embodiments, the articles of manufacture comprising adoptive cell therapeutics are obtained by the methods disclosed herein. In certain embodiments, the adoptive cell therapeutic is a Cell Product. In certain embodiments, the adoptive cell therapeutic is aNeoTCR Product.

The Cell Products can be used in combination with articles of manufacture. Such articles of manufacture can be useful for the prevention or treatment of proliferative disorders (e.g., cancer). Examples of articles of manufacture include but are not limited to containers (e.g., infusion bags, bottles, storage containers, flasks, vials, syringes, tubes, and IV solution bags) and a label or package insert on or associated with the container The containers may be made of any material that is acceptable for the storage and preservation of the NeoTCR cells within the Cell Products. In certain embodiments, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle. For example, the container may be a CryoMACS freezing bag. The label or package insert indicates that the Cell Products are used for treating the condition of choice and the patient of origin. The patient is identified on the container of the Cell Product because the Cell Product is made from autologous cells and engineered as a patient-specific and individualized treatment.

The article of manufacture may comprise: 1) a first container with a Cell Product contained therein.

The article of manufacture may comprise: 1) a first container with a Cell Product contained therein; and 2) a second container with the same Cell Product as the first container contained therein. Optionally, additional containers with the same Cell Product as the first and second containers may be prepared and made. Optionally, additional containers containing a composition comprising a different cytotoxic or otherwise therapeutic agent may also be combined with the containers described above. The article of manufacture may comprise: 1) a first container with a Cell Product contained therein; and 2) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.

The article of manufacture may comprise: 1) a first container with two Cell Products contained therein; and 2) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.

The article of manufacture may comprise: 1) a first container with a Cell Product contained therein; 2) a second container with a second Cell Product contained therein; and 3) optionally a third container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. In certain embodiments, the first and second Cell Products are different Cell Products. In certain embodiments, the first and second Cell Products are the same Cell Products.

The article of manufacture may comprise: 1) a first container with three Cell Products contained therein; and 2) optionally a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.

The article of manufacture may comprise: 1) a first container with a Cell Product contained therein; 2) a second container with a second Cell Product contained therein; 3) a third container with a third Cell Product contained therein; and 4) optionally a fourth container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. In certain embodiments, the first, second, and third Cell Products are different Cell Products. In certain embodiments, the first, second, and third Cell Products are the same Cell Products. In certain embodiments, two of the first, second, and third Cell Products are the same Cell Products.

The article of manufacture may comprise: 1) a first container with four Cell Products contained therein; and 2) optionally a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.

The article of manufacture may comprise: 1) a first container with a Cell Product contained therein; 2) a second container with a second Cell Product contained therein; 3) a third container with a third Cell Product contained therein; 4) a fourth container with a fourth Cell Product contained therein; and 5) optionally a fifth container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. In certain embodiments, the first, second, third, and fourth Cell Products are different Cell Products. In certain embodiments, the first, second, third, and fourth Cell Products are the same NeoTCR Products. In certain embodiments, two of the first, second, third, and fourth Cell Products are the same NeoTCR Products. In certain embodiments, three of the first, second, third, and fourth Cell Products are the same Cell Products.

The article of manufacture may comprise: 1) a first container with five or more Cell Products contained therein; and 2) optionally a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent.

The article of manufacture may comprise: 1) a first container with a Cell Product contained therein; 2) a second container with a second Cell Product contained therein; 3) a third container with a third Cell Product contained therein; 4) a fourth container with a fourth Cell Product contained therein; 5) a fifth container with a fifth Cell Product contained therein; 6) optionally a sixth or more additional containers with a sixth or more Cell Product contained therein; and 7) optionally an additional container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. In certain embodiments, all of the containers of Cell Products are different Cell Products. In certain embodiments, all of the containers of Cell Products are the same Cell Products. In certain embodiments, there can be any combination of same or different Cell Products in the five or more containers based on the availability of detectable Cells in a patient’s tumor sample(s), the need and/or desire to have multiple Cell Products for the patient, and the availability of any one Cell Product that may require or benefit from one or more container.

The article of manufacture may comprise: 1) a first container with a Cell Product contained therein; 2) a second container with a second Cell Product contained therein; 3) a third container with a third Cell Product contained therein.

The article of manufacture may comprise: 1) a first container with a Cell Product contained therein; 2) a second container with a second Cell Product contained therein; 3) a third container with a third Cell Product contained therein; 4) optionally a fourth container with a fourth Cell Product contained therein.

The article of manufacture may comprise: 1) a first container with a Cell Product contained therein; 2) a second container with a second Cell Product contained therein; 3) a third container with a third Cell Product contained therein; 4) a fourth container with a fourth Cell Product contained therein; 5) optionally a fifth container with a fourth Cell Product contained therein.

The article of manufacture may comprise a container with one Cell Product contained therein. The article of manufacture may comprise a container with two Cell Products contained therein. The article of manufacture may comprise a container with three Cell Products contained therein. The article of manufacture may comprise a container with four Cell Products contained therein. The article of manufacture may comprise a container with five Cell Products contained therein.

The article of manufacture may comprise 1) a first container with one Cell Product contained therein, and 2) a second container with two Cell Products contained therein. The article of manufacture may comprise 1) a first container with two Cell Products contained therein, and 2) a second container with one Cell Product contained therein. In the examples above, a third and/or fourth container comprising one or more additional Cell Products may be included in the article of manufacture. Additionally, a fifth container comprising one or more additional Cell Products may be included in the article of manufacture.

Furthermore, any container of Cell Product described herein can be split into two, three, or four separate containers for multiple time points of administration and/or based on the appropriate dose for the patient.

In certain embodiments, the Cell Products are provided in a kit. The kit can, by means of non-limiting examples, contain package insert(s), labels, instructions for using the Cell Product(s), syringes, disposal instructions, administration instructions, tubing, needles, and anything else a clinician would need in order to properly administer the Cell Product(s).

In certain embodiments, the Cell Products used in the methods of manufacture disclosed herein are NeoTCR Products or NeoTCR Viral Products. In certain embodiments, the Cell Products used in the methods of manufacture disclosed herein are NeoTCR Products. In certain embodiments, the Cell Products used in the methods of manufacture disclosed herein are NeoTCR Viral Products.

5. Methods of Treatment

The present disclosure provides methods for inducing and/or increasing an immune response in a subject in need thereof. In certain embodiments, the methods include administering the adoptive cell therapies disclosed herein. In certain embodiments, the methods include administering the adoptive cell therapies obtained by the methods disclosed herein. In certain embodiments, the adoptive cell therapy comprises a Cell Product. In certain embodiments, the adoptive cell therapy comprises a NeoTCR Product.

The Cell Products can be used for treating and/or preventing a cancer in a subject. The Cell Products can be used for prolonging the survival of a subject suffering from a cancer. The Cell Products can also be used for treating and/or preventing a cancer in a subject. The Cell Products can also be used for reducing tumor burden in a subject. Such methods comprise administering the Cell Products in an amount effective or a composition ( e.g ., a pharmaceutical composition) comprising thereof to achieve the desired effect, be it palliation of an existing condition or prevention of recurrence. For treatment, the amount administered is an amount effective in producing the desired effect. An effective amount can be provided in one or a series of administrations. An effective amount can be provided in a bolus or by continuous perfusion.

In certain embodiments, the Cell Products can be used for treating viral or bacterial diseases. In certain embodiments, the Cell Products can be used for treating autoimmune diseases.

In certain embodiments, an effective amount of the Cell Products are delivered through IV administration. In certain embodiments, the Cell Products are delivered through IV administration in a single administration. In certain embodiments, the Cell Products are delivered through IV administration in multiple administrations. In certain embodiments, the Cell Products are delivered through IV administration in two or more administrations. In certain embodiments, the Cell Products are delivered through IV administration in two administrations. In certain embodiments, the Cell Products are delivered through IV administration in three administrations.

The present disclosure provides methods for treating and/or preventing cancer in a subject. In certain embodiments, the method comprises administering an effective amount of the Cell Products to a subject having cancer.

Non-limiting examples of cancer include blood cancers (e g. leukemias, lymphomas, and myelomas), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, glioblastoma, throat cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate and small cell lung cancer). Suitable carcinomas further include any known in the field of oncology, including, but not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neural ectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small and large cell lung adenocarcinomas, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, bronchoalveolarcarcinoma, epithelial adenocarcinoma, and liver metastases thereof, lymphangiosarcoma, lymphangioendotheliosarcoma, hepatoma, cholangiocarcinoma, synovioma, mesothelioma, Ewing’ s tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms’ tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom’s macroglobulinemia, and heavy chain disease, breast tumors such as ductal and lobular adenocarcinoma, squamous and adenocarcinomas of the uterine cervix, uterine and ovarian epithelial carcinomas, prostatic adenocarcinomas, transitional squamous cell carcinoma of the bladder, B and T cell lymphomas (nodular and diffuse) plasmacytoma, acute and chronic leukemias, malignant melanoma, soft tissue sarcomas and leiomyosarcomas. In certain embodiments, the neoplasia is selected from the group consisting of blood cancers (e.g. leukemias, lymphomas, and myelomas), 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 throat cancer. In certain embodiments, the presently disclosed young T cells and compositions comprising thereof can be used for treating and/or preventing blood cancers (e.g., leukemias, lymphomas, and myelomas) or ovarian cancer, which are not amenable to conventional therapeutic interventions.

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, kidney papillary cell carcinoma, sarcoma, ovarian cancer, pancreatic adenocarcinoma, rectum adenocarcinoma, colon adenocarcinoma, esophageal carcinoma, uterine corpus endometrioid carcinoma, breast cancer, skin cutaneous melanoma, lung adenocarcinoma, stomach adenocarcinoma, cervical and endocervical cancer, kidney clear cell carcinoma, testicular germ cell tumors, and aggressive B-cell lymphomas

The subjects can have an advanced form of disease, in which case the treatment objective can include mitigation or reversal of disease progression, and/or amelioration of side effects. The subjects can have a history of the condition, for which they have already been treated, in which case the therapeutic objective will typically include a decrease or delay in the risk of recurrence.

Suitable human subjects for therapy typically comprise two treatment groups that can be distinguished by clinical criteria. Subjects with “advanced disease” or “high tumor burden” are those who bear a clinically measurable tumor. A clinically measurable tumor is one that can be detected on the basis of tumor mass (e.g., by palpation, CAT scan, sonogram, mammogram, or X-ray; positive biochemical or histopathologic markers on their own are insufficient to identify this population). A pharmaceutical composition is administered to these subjects to elicit an anti tumor response, with the objective of palliating their condition. Ideally, reduction in tumor mass occurs as a result, but any clinical improvement constitutes a benefit. Clinical improvement includes decreased risk or rate of progression or reduction in pathological consequences of the tumor.

In certain embodiments, the Cell Products used in the methods of treatment disclosed herein are NeoTCR Products or NeoTCR Viral Products. In certain embodiments, the Cell Products used in the methods of treatment disclosed herein are NeoTCR Products. In certain embodiments, the Cell Products used in the methods of treatment disclosed herein are NeoTCR Viral Products.

6. _ Kits

In certain embodiments, the present disclosure provides kits for obtaining adoptive cell therapies disclosed herein.

The present disclosure provides kits for inducing and/or enhancing immune response and/or treating and/or preventing cancer or a pathogen infection in a subject. In certain embodiments, the kit comprises an effective amount of presently disclosed cells or a pharmaceutical composition comprising thereof. In certain embodiments, the kit comprises a sterile container; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

In certain non-limiting embodiments, the kit includes an isolated nucleic acid molecule encoding a presently disclosed HR template.

If desired, the cells and/or nucleic acid molecules are provided together with instructions for administering the cells or nucleic acid molecules to a subject having or at risk of developing cancer or pathogen, or immune disorder. The instructions generally include information about 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: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of a neoplasia, pathogen infection, or immune disorder or symptoms thereof; precautions; warnings; indications; counter-indications; over-dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. The resulting cells can be grown under conditions similar to those for unmodified cells, whereby the modified cells can be expanded and used for a variety of purposes.

In certain embodiments, the present disclosure provides kits for performing the methods disclosed herein. In certain embodiments, the kits include reagents (e g., activation reagent, DNA plasmid), materials (e.g., electroporation cells, gas-permeable flasks, infusion bags), and instructions for carrying out the methods disclosed herein.

EXAMPLES

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

Examyle 1. Generation of NeoTCR Products

Neoepitope-specific TCRs identified by the imPACT Isolation Technology described in PCT/US2020/17887 (which is herein incorporated by reference in its entirety) were used to generate homologous recombination (HR) DNA templates. These HR templates were transfected into primary human T cells in tandem with site-specific nucleases ( see Figures 1A-1C). The single-step non-viral precision genome engineering resulted in the seamless replacement of the endogenous TCR with the patient’s neoepitope-specific TCR, expressed by the endogenous promoter. The TCR expressed on the surface is entirely native in sequence.

The precision of NeoTCR-T cell genome engineering was evaluated by Targeted Locus Amplification (TLA) for off-target integration hot spots or translocations, and by next generation sequencing based off-target cleavage assays and found to lack evidence of unintended outcomes.

As shown in Figures 1A-1C, constructs containing genes of interest were inserted into endogenous loci. This was accomplished with the use of homologous repair templates 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 sandwiched between 2A peptides, a protease cleavage site that is upstream of the 2A peptide to remove the 2A peptide from the upstream translated gene of interest, and signal sequences (Figure IB). Once integrated into the genome, the gene of interested expression gene cassette was transcribed as single messenger RNA. During the translation of this gene of interest in messenger RNA, the flanking regions were unlinked from the gene of interest by the self-cleaving 2A peptide and the protease cleavage site was cleaved for the removal of the 2A peptide upstream from the translated gene of interest (Figure 1C). In addition to the 2A peptide and protease cleavage site, a gly-ser-gly (GSG) linker was inserted before each 2A peptide to further enhance the separation of the gene of interest from the other elements in the expression cassette.

It was determined that P2A peptides were superior to other 2A peptides for Cell Products because of its efficient cleavage. Accordingly, two (2) P2A peptides and codon divergence were used to express the gene of interest without introducing any exogenous epitopes from remaining amino acids on either end of the gene of interest from the P2A peptide. The benefit of the gene edited cell having no exogenous epitopes (i.e., no flanking P2A peptide amino acids on either side of the gene of interest) is that immunogenicity is drastically decreased and there is less likelihood of a patient infused with a Cell Product containing the gene edited cell to have an immune reaction against the gene edited cell.

As described in PCT/US/2018/058230, NeoTCRs were integrated into the TCRa locus of T cells. Specifically, a homologous repair template containing aNeoTCR coding sequence flanked by left and right HR Arms was used. In addition, the endogenous TCRP locus was disrupted leading to the expression of only TCR sequences encoded by the NeoTCR construct. The general strategy was applied using circular HR templates as well as with linear templates.

The target TCRa locus (Ca) is shown along with the plasmid HR template, and the resulting edited sequence and downstream mRNA/protein products in Figures IB and 1C. The target TCRa locus (endogenous TRAC) and its CRISPR Cas9 target site (horizontal stripe, cleavage site designated by arrow) are shown (Figures 1A-1C). The circular plasmid HR template with the polynucleotide encoding the NeoTCR is located between left and right homology arms (“LHA” and “RHA” respectively). The region of the TRAC introduced by the HR template that was codon optimized is shown (vertical stripe). The TCR constant domain was derived from TRBC2, which is indicated as being functionally equivalent to TRBCl. Other elements in the NeoTCR cassette include: 2A = 2A ribosome skipping element (by way of non limiting example, the 2A peptides used in the cassette are both P2A sequences that are used in combination with codon divergence to eliminate any otherwise occurring non-endogenous epitopes in the translated product); P = protease cleavage site upstream of 2A that removes the 2A tag from the upstream TCRfl protein (by way of non-limiting example the protease cleavage site can be a furin protease cleavage site); SS = signal sequences (by way of non-limited example the protease cleavage site can be a human growth hormone signal sequence). The HR template of the NeoTCR expression gene cassette includes two flanking homology arms to direct insertion into the TCRa genomic locus targeted by the CRISPR Cas9 nuclease RNP with the TCRa guide RNA. These homology arms (LHA and RHA) flank the neoE-specific TCR sequences of the NeoTCR expression gene cassette. While the protease cleavage site used in this example was a furin protease cleavage site, any appropriate protease cleavage site known to one of skill in the art could be used. Similarly, while HGH was the signal sequence chosen for this example, any signal sequence known to one of skill in the art could be selected based on the desired trafficking and used. Once integrated into the genome (Figure 1C), the NeoTCR expression gene cassette is transcribed as a single messenger RNA from the endogenous TCRa promoter, which still includes a portion of the endogenous TCRa polypeptide from that individual T cell (Figure 1C). During ribosomal polypeptide translation of this single NeoTCR messenger RNA, the NeoTCR sequences are unlinked from the endogenous, CRISPR-disrupted TCRa polypeptide by self- cleavage at a P2A peptide (Figure 1C). The encoded NeoTCRa and NeoTCRp polypeptides are also unlinked from each other through cleavage by the endogenous cellular human furin protease and a second self-cleaving P2A sequence motifs included in the NeoTCR expression gene cassette (Figure 1C). The NeoTCRa and NeoTCRp polypeptides are separately targeted by signal leader sequences (derived from the human growth hormone, HGH) to the endoplasmic reticulum for multimer assembly and trafficking of the NeoTCR protein complexes to the T cell surface. The inclusion of the furin protease cleavage site facilitates the removal of the 2A sequence from the upstream TCRp chain to reduce potential interference with TCRp function. Inclusion of a gly-ser-gly linker before each 2A (not shown) further enhances the separation of the three polypeptides.

Additionally, three repeated protein sequences are codon diverged within the HR template to promote genomic stability. The two P2A are codon diverged relative to each other, as well as the two HGH signal sequences relative to each other, within the TCR gene cassette to promote stability of the introduced NeoTCR cassette sequences within the genome of the ex vivo engineered T cells. Similarly, the re-introduced 5’ end of TRAC exon 1 (vertical stripe) reduces the likelihood of the entire cassette being lost over time through the removal of intervening sequence of two direct repeats.

In-Out PCR was used to confirm the precise target integration of the NeoE TCR cassette. Agarose gels show the results of a PCR using primers specific to the integration cassette and site generate products of the expected size only for cells treated with both nuclease and DNA template (KOKI and KOKIKO), demonstrating site-specific and precise integration.

Furthermore, Targeted Locus Amplification (TLA) was used to confirm the specificity of targeted integration. Crosslinking, ligation, and use of primers specific to the NeoTCR insert were used to obtain sequences around the site(s) of integration. The reads mapped to the genome are binned in 10 kb intervals. Significant read depths were obtained only around the intended site the integration site on chromosome 14, showing no evidence of common off-target insertion sites.

Antibody staining for endogenous TCR and peptide-HLA staining for NeoTCR revealed that the engineering results in high frequency knock-in of the NeoTCR, with some TCR- cells and few WT T cells remaining. Knock-in is evidenced by NeoTCR expression in the absence of an exogenous promoter. Engineering was carried out multiple times using the same NeoTCR with similar results. Therefore, efficient and consistent expression of the NeoTCR and knockout of the endogenous TCR in engineered T cells was achieved.

Example 2. Example of Process 1

The NeoTCR Product was manufactured with Process 1 using a fully enclosed and programmable manufacturing process on the CliniMACS Prodigy instrument (Miltenyi). Open manipulations were only required for media, buffer, and reagent preparation, as well as during final formulation of NeoTCR Product, which were performed in an ISO 5 biosafety cabinet. Changeover procedures and utilization of disposable materials were in place to avoid cross contamination.

A schematic of the complete manufacturing process, from patient leukopak through NeoTCR Product, is shown in Figure 2. The leukopak was collected at the clinical site and shipped to manufacturing facility overnight at 2-8°C. Depending on whether the NeoTCR Product was manufactured to comprise one or more NeoTCRs, the initial leukopak was split into one or more bags (i.e., one bag per NeoTCR) with each bag loaded onto a separate CliniMACS Prodigy unit. Using the Prodigy’s “T Cell Transduction Process Program” CD4 and CD8 T cells were positively enriched for further processing, and other cell types/impurities were discarded. After CD8 and CD4 T cell enrichment, the designated number of cells were activated in the CentriCult chamber of the Prodigy using non-bead-based activation (TransAct, Miltenyi) and cultured for 48 hours. On day 2, the cells were precision-genome engineered to express the NeoTCR by electroporation using Lonza 4D-Nucleofector™ LY. For this purpose, cells were concentrated by centrifugation within the CentriCult of the Prodigy, resuspended in electroporation buffer and pumped to a custom-made reservoir using a specifically developed Prodigy Custom Application Program (CAP). The reservoir was connected by sterile welding to the electroporation system. Reagents (DNA plasmid and RNPs) were transferred to a sterile single use pouch within a biosafety cabinet (BSC), which was connected to the tubing set of the electroporator cuvette. Cells were mixed together with subject-specific plasmid DNA and ribonucleoproteins (RNPs) reagents within the nucleocuvette immediately prior to electroporation for precision genome engineering to knock out the endogenous TCR and replace with the NeoTCR. Following electroporation, cells are pumped back from the output reservoir to the Prodigy CentriCult chamber using a Prodigy Custom Application Program.

Using the Prodigy’s “T Cell Transduction Process Program”, the cells were cultured in the Prodigy’s CentriCult chamber in TexMACS GMP medium supplemented with 3% human AB serum, IL7 (12.5ng/mL) and IL15 (12.5 ng/mL) for the remainder of the manufacturing period until harvest on day 13. Starting from process day 4 (2 days post electroporation), daily media changes were performed to maintain optimal growth conditions until harvest on day 13. During the harvest, the cells were washed in 2% HSA (w/v) in Plasma-Lyte A, concentrated and eluted into a target cell bag (part of Miltenyi Prodigy TS520 tubing set) in approximately 100 mL total volume of 2% HSA in Plasma-Lyte A. Following harvest, cells were formulated into two CroMACS250 bags filled with 35mL of cells in final formulation medium (46% Plasma-Lyte A, 1% HSA (w/v), 50%CryoStor CS10). A separate set of CroMACS250 bag was used for each NeoTCR — depending on how many NeoTCRs were used for that product. For example, if there is one NeoTCR per NeoTCR Product, two CroMACS250 bags would be used; splitting the total cells into two bags. Alternatively, if there are three NeoTCRs per NeoTCR Product, six CroMACS250 bags would be used. Cells were then cryopreserved in controlled rate freezer and stored in vapor phase liquid nitrogen until shipment to clinical site for infusion.

Material information for the final formulated and filled NeoTCR Product is provided in Table 2.

Table 2. Patient-specific NeoTCR Product Information

Leukopak collection. The leukopak was collected at the clinical site according to medically acceptable protocols and shipped to the manufacturing facility overnight using a qualified shipper with temperature monitoring to maintain temperature between 2-8°C during transport. Upon arrival at the manufacturing site, the leukopak was inspected following standard operating procedures to ensure the leukopak met quality requirements and matched study participant identification requirements prior to transport into controlled environment room for initiation of the manufacturing process. The leukopak details are provided in Table 3. Table 3. Starting Material Parameters

CD4/CD8 Enrichment. Following initial sampling for cell count/viability, including but not limited to cell count/viability and flow cytometry (for cell characterization), the leukopak was removed from biosafety cabinet and then loaded onto the CliniMACS Prodigy instrument by sterile welding to the sterile single use disposable Prodigy TS520 kit. Using the Prodigy’s “T Cell Transduction Process Program” CD4 and CD8 T cells were positively enriched for further processing, and other cell types/impurities were discarded. Following enrichment, cells were again sampled for cell count/viability and flow cytometry (cell characterization assays). The CD4/CD8 election parameters are provided in Table 4.

Table 4. CD4/CD8 Enrichment Parameters T cell activation. After CD8 and CD4 T cell enrichment, the designated number of cells were activated in the CentriCult chamber of the Prodigy using non-bead-based activation (TransAct, Miltenyi). Specifically, T cells were activated by incubation with TransAct (CD3/CD28 reagent at a ratio of 1:17.5) in TexMACS medium supplemented with 3% human AB serum, 12.5 ng/mL IL7 and 12.5 ng/mL IL15. Culture occurred for 48 hours in the Prodigy CentriCult chamber at 37°C and 5% C02 (Programmed Prodigy setting of 39°C translates to actual desired temperature of 37°C).

Electroporation. On day 2, the cells were precision-genome engineered to express the NeoTCR. For this purpose, cells were concentrated by centrifugation within the CentriCult of the Prodigy, resuspended in electroporation buffer and pumped to a custom-made reservoir (Saint Gobain) using a product specific Prodigy Custom Application Program (CAP). The reservoir was connected to the Prodigy by aseptic tube welding to maintain a closed single-patient system (see, e.g., Figure 25). Following rebuffering the cells in the rebuffering solution were detached from the Prodigy using a heat sealer and connected to the cell input (upper line) of the LV Nucleocuvette cartridge tubing set of the electroporation system (Lonza 4D-Nucleofector™ LV). Reagents (DNA plasmid and RNPs) were aseptically transferred to a sterile single use pouch within a biosafety cabinet (BSC), which was then connected to the reagent input (lower line) of the electroporator cuvette tubing set. Upon start of the Lonza electroporation program, the cells were mixed together with subject-specific plasmid DNA and ribonucleoproteins (RNPs) reagents (i.e. GMP Cas9, sgRNA TRACI and sgRNA TRBC2) by the electroporator immediately prior to electroporation within the nucleocuvette. This process allowed precision genome engineering to knock out the endogenous TCR and replace with the NeoTCR. Following electroporation, the electroporated cells were pumped into the output reservoir. The output reservoir with the cells was then detached from the LV Nucleocuvette cartridge tubing set and welded back onto the Prodigy kit. Cells were then pumped back from the output reservoir to the Prodigy CentriCult chamber using Prodigy CAP for cell expansion.

Table 5. Electroporation Conditions

T cell expansion. Using the Prodigy’s “T Cell Transduction Process Program”, the cells were cultured in the Prodigy’s CentriCult chamber in TexMACS GMP medium supplemented with 3% human AB serum, IL7 (12.5ng/mL) and IL15 (12.5 ng/mL) for the remainder of the manufacturing period until harvest on day 13. Starting from process day 4 (2 days post electroporation), daily media changes were performed to maintain optimal growth conditions until harvest on day 13. Cell growth was monitored by sampling for cell count and viability at regular intervals (day 3, day 6, day 8, daylO). Table 6. T Cell Culture Parameters

Harvest and Final Formulation. Prior to harvest, the cell culture was sampled to collect cells for potency, a final product release test, in process cell count and viability, as well as for characterization tests. Cells were washed with 2% HSA in Plasma-Lyte, then harvested into target cell bag of Prodigy TS520 tubing system in a total volume of approximately 100 mL in 2% HSA in Plasma-Lyte. Cells in the target cell bag (cell substance) was sealed off from the Prodigy kit and transported into the BSC in preparation for final formulation and cryopreservation of final cell product (drug product manufacturing).

Within the BSC, cells were transferred to sterile conical tubes, centrifuged and the supernatant was discarded. Cells were resuspended in Plasmalyte/2%HSA and transferred to a CryoMACS250 bag. For final formulation, the cells in Plasmalyte/2%HSA were mixed with an equal volume of cold Cryostor CS10 and distributed into two CryoMACS bags (35 ml total volume per bag post QC sampling). Table 7. Harvest and Final Parameters

Cryopreservation Storage. The final formulated cell product (i.e., the NeoTCR Product) in the cryopreservation bags was placed into metal storage cassettes and frozen in the controlled rate freezer using an optimized freezing program. The sample temperature profile in the controlled rate freezer was monitored and data filed with the batch records. Once cryopreserved, cell product was stored in vapor phase liquid nitrogen at below -120°C until shipment to clinical site. Furthermore, the NeoTCR Product remained below -120 °C during transport to and storage at the clinical site.

Hold Times. The processing time limits and in-process material hold times were provided to ensure consistent manufacturing operations and performance (see Table 8).

Table 8. Hold Times

Example 3. Example of Process 2

The cell manufacturing Process 2 encompasses the process from receipt of patient leukapheresis in the GMP manufacturing facility, enrichment and activation of CD4/CD8 T cells from leukapheresis, followed by gene-editing using a NeoTCR plasmid, expansion of NeoTCR Cells, harvest, final formulation and cryopreservation, as well as QC release of final NeoTCR Product.

The NeoTCR Product was manufactured under Process 2 using a fully enclosed and programmable manufacturing process.

A schematic of the complete manufacturing process, from patient leukopak through the final cell product, is shown in Figures 2 and 3. The leukopak was collected at the clinical site and shipped to manufacturing facility overnight using a qualified shipper at 2-8°C. Following inspection, the leukopak was loaded onto the Prodigy. Using the Prodigy’s “T Cell Transduction Process Program” CD4 and CD8 T cells were positively enriched for further processing, and other cell types/impurities are discarded (specifically aiming to remove the monocytes and NK cells from the CD4+ and CD8+ T cells, along with other non- CD4+ and CD8+ cells). After CD8 and CD4 T cell enrichment, the designated number of cells was activated in a G-Rex lOOM-CS flask per NeoTCR-sublot using non-bead-based activation (in this example, TransAct™, Miltenyi) and cultured for 48 hours. On day 2, the cells were precision-genome engineered according to the process described in Example 1, to express the NeoTCR by electroporation using Lonza 4D-Nucleofector™ LV. For this purpose, cells were harvested from the G-REX 100-CS flask using peristaltic pump and then rebuffered in electroporation buffer and pumped to a custom-designed reservoir using the Rotea Counterflow Centrifugation System (Thermofisher) (Figure 25). The reservoir was connected by sterile welding to the electroporation system. Reagents (DNA plasmid and RNPs) were transferred to a sterile single use pouch within a biosafety cabinet (BSC), which was then connected to the tubing set of the electroporator cuvette. Cells were then mixed together with subject-specific plasmid DNA and ribonucleoproteins (RNPs) reagents within the nucleocuvette immediately prior to electroporation for precision genome engineering to knock out the endogenous TCR and replace with the NeoTCR. Following electroporation, the cells were pumped from the output reservoir to a new G-REX-100M-CS flask for further expansion (Figure 27).

The cells were then cultured in the G-Rex in an incubator (5% CO2, 37°C) in culture medium (TexMACS GMP medium supplemented with 3% human AB serum, IL7 (12.5ng/mL) and IL15 (12.5 ng/mL)). On day 8, a cell count was performed, and cells were split into an appropriate number of G-Rex flasks (G-Rex lOOM-CS or G-REX 500M-CS) using fresh culture medium and cultured until day 13 (Figure 26). On day 13, the cells were collected from the G- Rex flasks using a peristaltic pump into a collection bag, which was then loaded onto the Rotea for further processing (Figure 26). Using the Rotea “final formulation program”, the cells were washed in 2% HSA (w/v) in Plasma-Lyte A, concentrated and eluted into one CryoMACS500 bag in ½ of target final formulation volume (Figure 25). Following closed system addition of an equal volume cold CryoStor CS10 using the peristaltic pump, approximately ½ of final formulated cell suspension was transferred into a second CryoMACS500 bag. CryoMACS 500 bags filled with 70mL of cells in final formulation medium (46% Plasma-Lyte A, 1% HSA (w/v), 50% CryoStor CS10) were then cryopreserved in controlled rate freezer and stored in vapor phase liquid nitrogen until shipment to clinical site for infusion (see Table 9).

Table 9. Patient-specific NeoTCR Product Information

The leukopak was collected at the clinical site and shipped to the manufacturing facility overnight using a qualified shipper with temperature monitoring to maintain temperature between 2-8°C during transport. Leukopak collection. The leukopak was collected as described in Example 2 except the collection volume of blood was 200mL target volume up to 400mL (following addition of autologous plasma).

CD4/CD8 Enrichment. The CD4/CD8 enrichment was performed as described in Example 2 except the number of target cells for enrichment was < 5 x 10⁹. T Cell Activation. After CD8 and CD4 T cell enrichment, the designated number of cells were transferred to a G-Rex 100M-CS flask using non-bead-based activation (TransAct, Miltenyi). Specifically, T cells were activated by incubation with TransAct (aCD3/CD28 reagent at a ratio of 1 : 17.5) in TexMACS medium supplemented with 3% human AB serum, 12.5 ng/mL IL7 and 12.5 ng/mL IL15. The cells were culture in the activation medium for 48 hours in an incubator at 37°C and 5% CO2 (Table 10). Table 10. T Cell Activation Specifications

Electroporation. On day 2, the cells were precision-genome engineered to express the NeoTCR ( see Table 11). For this purpose, cells were harvested from the G-Rex lOOM-CS flask using a peristaltic pump into a bag, which was then connected to a Rotea single-use disposable tubing set (1 set per each NeoTCR) by sterile tube welding to maintain closed system. Using the Rotea’ s automated rebuffering program, the cells were concentrated by counter-flow centrifugation, resuspended in electroporation buffer and pumped to a custom-made reservoir (Saint Gobain). Following rebuffering, the cells in the rebuffering solution were detached from the Rotea kit using a heat sealer and connected to the cell input (upper line) of the LV Nucleocuvette cartridge tubing set of the electroporation system (Lonza 4D-Nucleofector™ LV). Reagents (DNA plasmid and RNPs) were aseptically transferred to a sterile single use pouch, which was then connected to the reagent input (lower line) of the electroporator cuvette tubing set. Upon start of the Lonza electroporation program, the cells were mixed together with subject- specific plasmid DNA and ribonucleoproteins (RNPs) reagents (i.e. GMP Cas9, sgRNA TRACI and sgRNA TRBC2) by the electroporator immediately prior to electroporation within the

Nucleocuvette. This process allowed precision genome engineering to knock out the endogenous TCR and replace with the NeoTCR. Following electroporation, the electroporated cells were pumped into the output reservoir. The output reservoir with the cells was then detached from the LV Nucleocuvette cartridge tubing set and welded onto a custom tube adapter, which was connected to the tubing of a new G-Rex 100M-CS flask and culture media bag. Cells were then diluted with culture media and pumped back from the output reservoir to the G-Rex lOOM-CS filled with culture medium. In the case of a 2-NeoTCR or 3-NeoTCR process (or any NeoTCR Product with greater than 3 NeoTCRs), this process was repeated for each TCR-sublot. While the same Rotea kit may be used for all three sublots, separate reagents and materials are required for electroporation and transfer to G-Rex 100M flasks.

Table 11. T Cell Nucleofection Parameters

T Cell Expansion. The cells were cultured in G-Rex-lOOM-CS flasks in tissue culture incubators at 37°C and 5% CO2 using 1L TexMACS GMP medium supplemented with 3% human AB serum, IL7 (12.5ng/mL) and IL15 (12.5 ng/mL). On day 8, a cell count was performed. Depending on cell numbers, cells were split into one or more G-Rex culture vessels to maintain optimal growth conditions. Glucose/Lactate was monitored by sampling at regular intervals (day 3, day 6, day 8, day 10). See Table 12.

Table 12. T Cell Culture Parameters

Harvest and Final Formulation. Cells from the G-Rex flasks were combined into a single collection bag per TCR sublot using a peristaltic pump after reducing the volume to approximately 100 mL. The combined culture was then sampled to collect cells for potency, a final product release test, in process cell count and viability, as well as for characterization tests. The bag with the cell suspension was then connected to a Rotea tubing set for further processing. Using Rotea’ s “final formulation program”, cells were washed with 2% HSA in Plasma-Lyte, then harvested into one CryoMACS 500 bag in a total volume of approximately 75 mL in 2% HSA in Plasma-Lyte. Cells in the CryoMACS 500 bag (cell substance) was sealed off from the Rotea kit and connected to custom single use disposable adapter and bag with cryopreservation medium (CryoStor CS10) by sterile welding. The peristaltic pump was then used to dispense an equal amount of cold CryoStor CS 10 to the cell suspension (final formulation 46%Plasmalyte A+ 1% HSA (w/v) + 50% CryoStor CS10; the NeoTCR Product). Approximately ½ of the cell suspension was transferred to second CryoMACS 500 bag.

See Table 13 for the harvest and final formulation parameters.

Table 13. Harvest and Final Formulation Example 4. Process 2 Has Superior Properties to Process 1

These experiments demonstrate that changes from manufacturing Process 1 to Process 2 allows for improved yields and efficiency without changing the qualitative nature of the product. The changes to the manufacturing Process 2 compared to Process 1 enhanced the viable cell yield and increase manufacturing efficiency necessary to meet the manufacturing demands required for the NeoTCR Product. The experiments in Example 4 demonstrates that the NeoTCR Product manufactured using Process 2 allows for increased NeoTCR cell yields whilst being of the same quality (identity, safety and potency) as cell product produced by Process 1.

NeoTCR Process 2 was developed in order to overcome limitations of the current clinical manufacturing process with regard to NeoTCR cell yield and improve scalability and manufacturing capacity. The current clinical manufacturing process utilizes the closed, automated CliniMACS Prodigy for T cell selection, T cell activation, T cell rebuffering prior to day 2 electroporation on the Lonza nucleofector and T cell expansion (day 2 to day 13). Final formulation was performed manually within an ISO 5 BSC. Overview of current 3 NeoTCR manufacturing process is shown in Figure 4.

The limited capacity of the CentriCult chamber of the Prodigy is the primary limitation of obtaining higher cell yields. Therefore, the optimized process replaced the CentriCult chamber with a closed system that is easily scalable, namely sterile single-use G-Rex flasks, either G-Rex lOOM-CS or G-Rex 500M-CS as the culture vessel during T cell activation and T cell expansion. Another benefit of using the G-Rex system is that G-Rex flasks are placed into standard tissue culture incubators, which can easily be monitored and maintained and are generally less susceptible to failure than more complex instrumentation. Furthermore, gas exchange is facilitated through a membrane at the bottom of the device allowing the cells to be undisturbed for longer amounts of time (rather than shaking and constant media exchanges necessary in the CentriCult). Importantly, no changes were made with respect to T cell activation reagent (including same ratio of cells to reagent and volume) or the culture medium to ensure similar T cell activation levels as compared to manufacturing Process 1.

Due to the change in culture vessel, the closed system transfer method was changed from Prodigy to the Rotea Counterflow Centrifugation System (Thermofisher) for initial seeding of G- Red flasks for T cell activation as well as for rebuffering of the cells into electroporation buffer for nucleofection. The Rotea is a counterflow centrifugation device that allows gentle concentration of the cells into small volumes with minimal cell loss. The Rotea utilizes GMP sterile single-use disposable kits. Due to the higher accuracy of the Rotea at small volumes (approximately +/- 1ml as opposed to +/- 5ml of Prodigy), this change resulted in improved consistency in cell suspension volume and cell concentration for the nucleofection process. No changes were introduced to the gene-editing process using the Lonza XL-LV nucleofector.

As an additional safety improvement, a closed system harvest and final formulation process was developed to replace the current manual open final formulation within BSC. For this purpose, the cells were harvested from the single use disposable sterile G-Rex culture vessels using a peristaltic pump then formulated into defined volume of PlasmaLyte +2% HSA using the Rotea. Closed system addition of CryoStor CS10 was performed using a peristaltic pump. Due to increased cell yield using the optimized process, the final product container was changed from CryoMACS 250 bag with a fill volume of 35 ml to two CryoMACS 500 with a final fill volume of 70ml to keep same number of bags and same cell concentration as produced in the manufacturing Process 1. An overview of optimized manufacturing Process 2 is shown in Figure 4, while Table 14 summarizes changes between NeoTCR Product manufacturing versions. Table 14. Comparison of Process 1 and Process 2

As part of the process evaluation, the individual unit operations were initially evaluated separately and compared to Process 1.

To evaluate the feasibility of T cell activation in G-Rex flasks the same number of CD4/CD8 enriched cells were activated in the CentriCult unit of the Prodigy or in a G-Rex 100M flask using same ratio of TransAct for two days followed by nucleofection and small-scale expansion (6M G-Rex). Cells from both conditions showed similar expression of activation markers (Figure 5A), as well as similar levels of % NeoTCR+ gene-editing efficiency (Figure 5B).

In parallel, to evaluate T cell activation in G-Rex flasks, experiments to compare T cell expansion in the Prodigy CentriClut or G-Rex flasks were performed. These experiments showed that cell expansion in G-Rex flasks resulted in higher-fold expansion (Figure 6A), but similar T cell phenotype (Figure 6B) as compared to cell expansion in the Prodigy CentriCult unit. Subsequently the G-Rex seeding density and appropriate time for cell split was evaluated for robust expansion across multiple donors. Based on these results, a day 8 split with a target seeding density between 5xl0⁶to lxlO⁷ cells/cm² was selected.

As a result of moving the T cell activation into the G-Rex, a new closed system rebuffering process was developed to concentrate and resuspend cells into electroporation buffer prior to nucleofection. The Rotea Counterflow Centrifugation System was used for this purpose. While cell recovery was variable, average cell recovery was generally higher as compared to the Prodigy CAP rebuffering process (Figure 7).

The performance of the Rotea during final formulation of the NeoTCR Cells into formulation buffer (Plasmalyte A+ 2% human serum albumin (HSA)) was compared to formulation using the Prodigy. Rotea formulation resulted in a higher average % of cell recovery as compared to the Prodigy with an average cell recovery of 91% Rotea vs 81% cell recovery in the Prodigy (Figure 8).

Due to the overall higher T cell yield using the optimized Process 2 (Figures 6A and 6B and Figure 15), the final formulation container was switched from a CryoMACS 250 bag with a 35 mL fill volume to a CryoMACS 500 bag with a 70 mL fill volume. No changes were implemented with regard to final formulation or controlled rate freezer program.

In order to assess whether this change would impact the NeoTCR Product quality, post thaw QC release attributes of NeoTCR Cells using Process 1 cryopreserved in CyroMACS250 bags (35 mL fill volume), NeoTCR Cells using Process 2 formulated in CyroMACS250 bags (35 mL fill volume) and NeoTCR Cells using Process 2 formulated using Rotea and cryopreserved in CyroMACS 500 bags (70 mL fill volume) were compared. Bags were filled with a target cell concentration of 10 Million cells/mL. All conditions showed similar post thaw viability (Figure 9) and post thaw cell concentration (Figure 10) suggesting that change in final product container does not negatively impact NeoTCR Product quality. In addition, similar post thaw % NeoTCR+ expression was observed in cells cryopreserved in QC (quality control) vials, CryoMACS 250 bags (35 mL fill volume) or CryoMACS 500 bags (70mL fill volume). In this particular run, a NeoTCR Product obtained using Process 2 had slightly lower %NeoTCR+ as compared to a NeoTCR Product obtained using Process 1 due to normal variability of the Lonza nucleofector electroporation method, but no impact on %NeoTCR+ was noticed as a result of formulation or cryopreservation method (Figure 11).

No significant changes to T cell phenotype were observed as a result of the cryopreservation method, as cells manufactured using optimized Process 2 showed similar T cell phenotype regardless of whether cells were cryopreserved in QC vials, CryoMACS 250 bags (35 mL fill volume) or CryoMACS 500 bags (70mL fill volume) (Figure 12). Similarly, no changes in phenotype were noted for cells generated using Process 1 and cryopreserved in QC vials or CryoMACS 250 bag with 35 ml fill volume (Figure 12).

Following evaluation of the individual unit operations, side-by side feasibility studies from the same donor were performed to evaluate Process 2 at scale in comparison to manufacturing Process 1 using upgraded Prodigy software with TCT version 2.0. Data comparisons were performed to historical data from engineering runs and clinical readiness runs. A total of five comparison runs were completed with last two runs utilizing a fully closed system. Earlier runs were performed using few open steps while all major unit operations (T cell selection, T cell activation, Rotea rebuffering, expansion in G-Rex 100M) were in place.

Data from the studies show that using G-Rex as the culture vessel successfully increased overall T cell expansion as compared to Process 1 in the Prodigy (Figure 13).

As the gene-editing procedure remained unchanged between with both processes utilizing the Lonza LV-XL nucleofector and same reagents, percentages of NeoTCR Cells in the final product of Process 2 remained within normal variability of manufacturing Process 1 with the average percentage of NeoTCR Cells trending slightly higher (Figure 14). The positive trend towards improved gene-editing is due both to the higher cell concentration and reduced residual media post rebuffering using the Rotea as well as increased survival of electroporated cells in the static culture conditions of the G-Rex.

Due to the improved cell expansion, overall yield of NeoTCR Cells using Process 2 was significantly increased (Figure 15) as compared to manufacturing Process 1 (average number of NeoTCR Cells of 1.4 xlO⁹ cells for a single TCR). The increase allows higher dose levels (1.4 xlO⁹ NeoTCR Cells per TCR would meet dose the required number of cells for a 1 NeoTCR, 2 NeoTCR, or 3 NeoTCR Product (including the ability to expand to have 4 or more NeoTCR Products)).

Whilst the NeoTCR Cell yield was significantly increased for Process 2, % viability of the NeoTCR Product (based on post-harvest, pre-cry opreservation sample) is similar between the two processes (Figure 16) with average % viability above 90% for either process.

In addition, the NeoTCR Cells from Process 2 retain the ability to secrete IFN-gamma at a similar level to manufacturing Process 1, confirming that the cells are functional (Figure 17). Levels of IFN-gamma secreted for Process 2 trend slightly higher in a direct comparison using the same donor.

Cytotoxic activity of the NeoTCR Product from a split comparison run was analyzed using an IncuCyte® killing assay as an additional comparability measure. The NeoTCR Product was generated using a NeoTCR for which a matching tumor cell line expressing cognate neoantigen and a WT control cell line are available. The cells were then used as target cells for the killing assay, while wild-type tumor cells lacking respective neoantigen expression served as assay control. Based on IncuCyte® killing assay data, the NeoTCR Product from Process 2 and Process 1 induced specific cytotoxicity in cell line expressing the NeoTCR target at similar levels. No killing activity was observed in target cells not expressing the cognate neoE target (Figures 18A and 18B)

Example 5. Process 3

Leukopak. For Process 3, the leukopak was gathered pursuant to standard acceptable medical procedures and it was shipped to the manufacturing site no later than overnight from the date of acquisition.

Enclosed manufacturing process . Process 3 comprises programable manufacturing process that is either substantially or fully enclosed. Open manipulations are only permitted for media, buffer, and reagent preparation, as well as during final formulation of NeoTCR Product. Open manipulations, if any, were performed in an ISO 5 biosafety cabinet or substantially similar sterile environment.

A schematic of the complete manufacturing process, starting from the loading of the leukopack onto a device to select and isolate the CD4+ and CD8+ cells from the leukopak through the cryopreservation of the finished NeoTCR Product, is provided in Figure 19. As shown, following CD8+ and CD4+ T cell selection and isolation, the designated number of cells are transferred to and activated in a cell culture chamber that is capable of expanding T cells.

Ideal cell culture chambers provide a gas permeable membrane. In certain instances, the gas permeable membrane used in Process 3 was a static gas exchange cell culture chamber. Such ideal cell culture chambers allow for cell expansion with minimal disturbance of the T cells. In certain instances, the cell culture chambers provide a gas permeable membrane and require nor more than two (2) media changes per week. In certain instances, the cell culture chambers provide a gas permeable membrane and requires one (1) media change during the manufacturing process. In certain instances, the cell culture chambers provide a gas permeable membrane and require no media changes during the manufacturing process. An example of a cell culture chamber used in Process 3 is the G-Rex (Wilson Wolf) which, as described as an element above, allows for cell expansion with minimal disturbance of the T cells in culture.

The activation of the CD4+ and CD8+ T cells occurred using non-magnetic beads.

One (1) to three (3) days following activation, the T cells were precision-genome engineered to express a NeoTCR using the methods described herein. In certain experiments, the T cells were precision genome engineered approximately two (2) days following activation. In certain experiments the T cells were precision genome engineered two (2) days following activation. Given the large volume of activated cells needed for electroporation in order to produce a clinical or commercial product, large scale electroporation devices were used. Certain devices that were used include chamber-based electroporation systems (e.g., chambers that hold approximately 0.5mL - 1.5mL cell suspension). In certain experiments, chamber-based electroporation systems that hold approximately lmL of cell suspension were used. In other experiments, certain devices that were used include flow-through electroporation devices (wherein suspended cells are passed through a chamber or device using a pump-based or microfluidic device system).

The cells were transferred from the cell culture chambers to the electroporation device using peristaltic pump. Prior to the transfer of the cells from the cell culture chambers to the electroporation device, the cells were rebuffered in electroporation buffer and pumped to a custom-designed reservoir using a centrifugation system. In certain experiments, the centrifugation was a performed using a traditional centrifugation system. In certain experiments, the centrifugation was performed using a counter flow centrifugation system. It was found that the counter flow centrifugation system provided less agitation to the cells and resulted in a more complete replacement of the activation media with the electroporation media (Figure 30). Such replacement of media was found to be an important factor in the electroporation efficiency of the cells. The custom-designed reservoir was connected by sterile welding to the electroporation system. Furthermore, another benefit that was discovered with counter flow centrifugation was the ability to declump the cultured T cells without additional force and agitation. Such force and agitation which was previously used to declump the cells prior to electroporation was shown to adversely affect the health and stability of the cells which in turn resulting in lower cell editing rate and lower cell survival rate following the electroporation.

It was shown that the counterflow centrifugation provided increased cell recovery efficiencies over traditional centrifugation (Figure 29). As shown, the recover was not insubstantial; rather it was shown cells could be washed and concentrated with minimal cell loss compared to traditional centrifugation which resulted in up to 40% loss of cells. Furthermore, additional experiments using counterflow centrifugation were performed in order to determine the optimal centrifuge speeds and times. Experiments were performed using centrifuge speed from ranging from 2500g(Vl) to 2700g(V2) during cells washing and pump speeds ranging from lOmL/min to 30mL/min. In a specific experiment (data shown in Figure 31), two versions of a counterflow centrifugation procedure were performed: Version 1 (VI) and Version 2 (V2). VI was set at a centrifugal force of 2500g with a fluid flow rate of 30mL/min and V2 was set at a centrifugal force of 2700g with a fluid flow rate of lOmL/min.

In prior experiments, VI had shown improvement over the former cell wash and harvest program used on the Prodigy. The particles comprising the fluidized bed within the counterflow centrifugation chamber experience two forces, the centripetal force generated by rotation around the chamber’s axis, and the fluidic drag force generated by the fluid flow. These two forces are at a balance if the particle is stabilized within the fluidized bed. Therefore, there was a question of whether compacting the fluidized bed by increasing the centripetal force via increased centrifuge speed would yield better cell recoveries during processing. Furthermore, there was a question of whether reducing the fluidic drag force would also compound to cell retention within the CFC chamber. Accordingly, VI and V2 were run in parallel to determine it would be possible to reduce cell loss from centrifugation. In fact, it was shown that V2 resulted in reduced cell loss by over 30%.

Furthermore, the traditional centrifugation procedures that used a double centrifugation step to first rid the culture of residual spent media, then performed a wash using a buffered solution, and finally harvested the cells into a reservoir for processing. After such processing the cell viability was shown to be reduced simply due to handling. Thus, selecting counterflow centrifugation was key to being able to effectively create a manufacturing process that is clinically and commercially relevant in order to produce sufficient number of gene edited cells and harvest such cells at a high efficiency without substantial cell loss and without damaging the cells.

As shown in Figure 19, following electroporation the cells were pumped from the output reservoir to a new cell culture chamber (as described above) for further expansion. Following the electroporation, the cells were transferred to cell culture chambers for cell proliferation and expansion that promoted the cells to maintain, develop, and/or retain a stem-like state (i.e., T cells that have a memory stem cell or stem cell (Tmsc or Tcm) phenotype).

In certain experiments, the cells were cultured in cell culture chambers in an incubator (5% CO2, 37°C) in culture medium. In certain experiments, the cell culture chambers that were used were the G-Rex (Wilson Wolf) cell culture chambers. Alternatively, another static gas exchange cell culture chamber could be used based on such static gas exchange cell culture chamber’s ability to allow for sufficient cell proliferation of gene edited cells that possess a memory stem cell or stem cell (Tmsc or Tcm) phenotype).

The preferred culture media allows for cell expansion and for cells to maintain, develop, and/or retain a stem-like state (i.e., T cells that have a memory stem cell or stem cell (Tmsc or Tcm) phenotype). In certain experiments, the media that was used to culture the cells following electroporation was the TexMACS GMP medium supplemented with 3% human AB serum, IL7 (12.5ng/mL) and IL15 (12.5 ng/mL)). In certain experiments, the media that was used to culture the cells following electroporation was a chemically-defined, animal component-free medium that was shown to promote T cell expansion while maintaining T cell functionality and potency. An example of the increased benefit of using a chemically-defined, animal component-free medium that was shown to promote T cell expansion while maintaining T cell functionality and potency is provided in Figures 20A-24B. In certain experiments, the media that was used to culture the cells following electroporation was PRIME-XV T Cell CDM (Irvine Scientific CDM). In certain experiments, the media that was used to culture the cells following electroporation was ImmunoCult XF (Stemcell). In certain experiments, the media that was used to culture the cells following electroporation was ExCellerate (R&D Systems). Additional non limiting examples of T cell medias that can be used to promote T cell expansion while maintaining T cell functionality and potency include LumphoOne (Takara Bio), GT-T551 (TakaraBio), X-VIVO 15, AIM V, CTS OpTmizer (Gibco), and all other medias with similar physiological attributes as those described herein. Additional medias known to one of skill in the art that are animal component-free, that enable efficient T cell expansion without the addition of serum or plasma, and promote expansion and growth of T cells with a naive phenotype (e.g., Tmsc and Tcm) can be used in the medias and methods described herein.

Serum free substitute additives were also used in the medias and experiments described herein. In certain experiments, Physiologix (Nucleus Biologies) was a media supplement used in the media. In certain experiments, human platelet lysate (a growth factor-rich cell culture supplement derived from healthy donor human platelets; Stem Cell) can be used as a media supplement for the serum-free medias. In certain experiments, CTS Immune Cell Serum Replacement (Gibco) was a media supplement used in the media. Additional serum free substitutes known to one of skill in the art that enable efficient T cell expansion without the addition of serum or plasma, and promote expansion and growth of T cells with a naive phenotype (e.g., Tmsc and Tcm) can be used in the medias and methods described herein.

In addition to the medias and serum free substitutes, the addition of cytokines can also be used in the medias and methods described herein. In certain experiments, the media was supplemented or contained IL2. In certain experiments, the media was supplemented or contained IL7. In certain experiments, the media was supplemented or contained IL15. In certain experiments, the media was supplemented or contained IL21. In certain experiments, the media did not contain or was not supplemented with IL2. In certain experiments, the media did not contain or was not supplemented with IL2 and did contain or was supplemented with IL7 and IL15. In certain experiments, the media did not contain or was not supplemented with IL2 and did contain or was supplemented with IL7, IL15, and/or IL21. In certain experiments, the media was supplemented or contained IL2, IL7, IL15, and IL21. In certain experiments, the media was supplemented or contained IL2, IL7, and IL15. In certain experiments, the media was supplemented or contained IL7, IL15, and IL21.

In addition to the IL2, IL7, IL15, and IL21 described above as single agents or combinations thereof for the supplementation of media, IL12, alpha interferon, or beta interferon can be used alone or in combination with each other or with the IL2, IL7, IL15, and IL21. Furthermore, any cytokine or chemokine that is involved in lymphocyte proliferation and differentiation can be added to any single IL2, IL7, IL12, IL15, IL21, alpha interferon or beta interferon, or any combination thereof. The concentration and ratios of each of the cytokines and/or chemokines should be adjusted based on the single agent use or combination use and titrated based on optimizing lymphocyte proliferation and differentiation.

In addition to the addition of serum free substitute additives and/or chemokines and/or cytokines as described herein, in certain experiments the addition of fatty acids was shown to be beneficial for the optimization of proliferation and differentiation.

In certain experiments, fibronectin, insulin, and/or transferrin were included in the media. In certain experiments, the transferrin used was recombinant transferrin. In certain experiments, the transferrin used was non-recombinant transferrin. In certain experiments it was determined that it was beneficial to increase the concentration of transferrin when recombinant transferrin was used compared to non-recombinant transferrin in order to achieve the same benefits of lymphocyte proliferation and differentiation to achieve T cells in culture with a naive phenotype (e.g., Tmsc and Tcm).

In certain experiments, different concentrations of glucose in the cell medias were tested. It was determined that increased glucose concentrations resulted in improved T cell activation and gene editing efficiency. In certain experiments, the increased glucose concentration was less than 3.7 g/L glucose. In certain experiments, the increased glucose concentration was between 3.7 - 4.0 g/L glucose. In certain experiments, the increased glucose concentration was between 4.0 - 4.2 g/L glucose. In certain experiments, the increased glucose concentration was between 4.2-4.5 g/L glucose. In certain experiments, the increased glucose concentration was between 4.3 - 4.4 g/L glucose. In certain experiments, the increased glucose concentration was between 4.4 - 4.5 g/L glucose. In certain experiments, the increased glucose concentration was greater than 4.5 g/L glucose. As cell density in culture increases, so can the concentration of glucose. For example, for a high density cell culture the glucose concentration can be increased up to 100 g/L.

In certain experiments, antioxidants were added to the media to promote lymphocyte proliferation and differentiation to achieve T cells in culture with a naive phenotype (e.g., Tmsc and Tcm).

In certain experiments, reducing agents were added to the media to promote lymphocyte proliferation and differentiation to achieve T cells in culture with a naive phenotype (e.g., Tmsc and Tcm). In certain experiments, reducing agents were not added to the media.

In order to promote automation of the NeoTCR Product manufacture, stir bioreactors can be used to culture the cells instead of a static gas exchange cell culture chamber Such stir bioreactors allow for real-time analytics and reaction to changes in conditions. For example, a stir bioreactor can be designed to have in line bioanalytics to measure cell mass, lactate, etc., in a closed system without manual sampling.

Alternatively, in order to promote automation of the NeoTCR Product manufacture, shaking/rotating bioreactors can be used to culture the cells instead of a static gas exchange cell culture chamber. Such shaking/rotating bioreactors allow for real-time analytics and reaction to changes in conditions. For example, a shaking/rotating bioreactor can be designed to have in line bioanalytics to measure cell mass, lactate, etc., in a closed system without manual sampling.

Furthermore, bioreactors (e.g., stir, shanking, rotating, etc.) can be designed and programmed to automatically add media supplements to the culture in order to increase or decrease the concentration of certain components in the media. For example, the bioreactor can be designed and programmed to detect lactate levels in the cell culture and add in glucose in order to keep the glucose: lactate levels optimal for lymphocyte proliferation and differentiation to achieve T cells in culture with a naive phenotype (e.g., Tmsc and Tcm). In other examples, the bioreactors can be designed and programmed to remove lactate during the culture process in order to promote lymphocyte proliferation and differentiation to achieve T cells in culture with a naive phenotype (e.g., Tmsc and Tcm). Another example of the use of a bioreactor is to design and program the bioreactor to detect dissolved oxygen as a negative indicator of an optimal cell environment.

In certain experiments, cell counts are taken throughout the culture period following electroporation. In certain experiments, the cells are taken from the static gas exchange culture chambers (e.g., a G-Rex flask) at the half-way point of post-electroporation culture (i.e., the halfway point between the time of electroporation and the time when the cells are cryopreserved as a NeoTCR Product) and split into two new static gas exchange culture chambers with fresh media.

At the end of the post-electroporation cell culture, the cells were collected from the static gas exchange chambers using peristaltic pump into a collection bag, which is then loaded onto a counterflow centrifugation system and the cells were washed in 2% HSA (w/v) in Plasma-Lyte A, and concentrated and eluted into two freezing bags (equal volume of cells in each bag) that have EVA tubing, leur connectors, roller clamps and an injection port(s). An equal volume of a cryopreservation media (e.g., CryoStor CS10) was added to the freezing bags (the final volume of the freezing bag is ½ cell suspension and ½ cryopreservation media). The final bags of cells were then cryopreserved in controlled rate freezer and stored in vapor phase liquid nitrogen until shipment to clinical site for infusion.

CD4/CD8 Enrichment. The CD4/CD8 enrichment was performed using a matrix to positively select for CD4+ and CD8+ cells in order to minimize the number of other blood cells prior to activation and electroporation. Specifically, the CD4+ and CD8+ selection was performed to remove NK cells and B cells.

T Cell Activation. After CD8 and CD4 T cell enrichment, the designated number of cells are transferred to a static gas exchange culture chamber and activated using non-magnetic beads. Magnetic beads (and any metal-based matrix) was avoided because metal has a sturdy architecture that was shown to stress and/or harm the cells. Furthermore, electroporation efficiency and gene edited cell expansion was shown to be dependent on the health of the cells at the time of electroporation. Accordingly, activation reagents and/or media that allow for easy detachment from the T cells were preferred and utilized. In certain experiments, TransAct (aCD3/CD28 reagent at a ratio of 1 : 17.5) was used as an activation reagent. Alternatively, agonists to CD3, CD2, and/or CD28 can be used to activate the T cells. Examples of such reagents include but are not limited to ImmunoCult Human CD3/CD28 T Cell Activator (Stem Cell), Cloudz Cell Activation (R&D Systems), and any other CD3, CD2, and/or CD28 reagent that is gentle on T cells and that does not interfere with electroporation efficiencies. Table 10.

Electroporation. Following the activation, the cells were precision-genome engineered to express the NeoTCR. For this, the cells are harvested from cell culture chamber where the activation occurred, washed to remove all activation agents and cell media, concentrated using a counter-flow centrifugation, and resuspended in electroporation buffer. The cells were then added to a custom-made reservoir (Saint Gobain). The cells were then transferred into an electroporation chamber. Given the large volume of activated cells needed for electroporation in order to produce a clinical or commercial product, large scale electroporation devices were used. Certain devices that were used include chamber-based electroporation systems (e.g., chambers that hold approximately 0.5mL - 1.5mL cell suspension). In certain experiments, chamber-based electroporation systems that hold approximately lmL of cell suspension were used. In other experiments, certain devices that were used include flow-through electroporation devices (wherein suspended cells are passed through a chamber or device using a pump-based or microfluidic device system). In certain experiments, the chamber-based system was a cuvette- style vessel.

For the electroporation, the cells were mixed together with subject-specific plasmid DNA and ribonucleoproteins (RNPs) reagents (i.e. GMP Cas9, sgRNA TRACI and sgRNA TRBC2) by the electroporator immediately prior to electroporation within the electroporation chamber. This process allowed precision genome engineering to knock out the endogenous TCR and replace with the neoE targeted TCR as described in Example 1. Following electroporation, the electroporated cells were pumped into the output reservoir. The output reservoir and then transferred to a new static gas exchange culture chamber.

T Cell Expansion. The cells are cultured as described above in this example.

Harvest and Final Formulation. The harvest and final formulation was performed as described above in this example.

Example 6. Cell Products

The methods and processes described in Examples 2-5 can be modified to be applicable to all Cell Products. Specifically, while Examples 2-5 describe gene editing that is accomplished using non-viral methods to yield a NeoTCR Product, the electroporation step can be substituted with a viral transduction step. Specifically, as it applies to Examples 3-5, Process 2 and Process 3 can be modified for a viral transduction step instead of a non-viral electroporation step while maintaining the cell culture and manufacturing steps following the electroporation described therein.

Leukopak collection. The leukopak is collected as described in Examples 2-5. The collection volume of blood is at least lOOmL.

CD4/CD8 Enrichment. The CD4/CD8 enrichment is performed as described in Example 2 with target cells for enrichment of less than 5 x 10⁹.

T Cell Activation. After CD8 and CD4 T cell enrichment, the cells are transferred to a flask as described in Examples 2-5. In particular, the T cells are activated by incubation with TransAct (aCD3/CD28 reagent at a ratio of 1:17.5) in TexMACS medium supplemented with 3% human AB serum, 12.5 ng/mL IL7 and 12.5 ng/mL IL15. The cells are cultured in the activation medium for 48 hours in an incubator at 37°C and 5% CO2 (Table 10).

Transfection. On day 2, the cells are engineered to express the NeoTCR. Briefly, the cells are harvested from the flask using a peristaltic pump into a bag, which is connected to a Rotea single-use disposable tubing set (1 set per each NeoTCR) by sterile tube welding to maintain a closed system. The cells are concentrated by counter-flow centrifugation and incubated with retrovirus comprising the NeoTCR construct. After incubation with the viral construct, the cells are concentrated by counter-flow centrifugation and diluted with culture media and pumped back to the flask. All the reagents and materials used during the transfection are sterile.

T Cell Expansion. After transfection, the cells are cultured in flasks at 37°C and 5% C02 using TexMACS GMP medium supplemented with 3% human AB serum, IL7 (12.5ng/mL) and IL15 (12.5 ng/mL). On day 8, a cell count is performed. Based on the cell number, the cells are split into one or more flasks to allow further expansion.

Harvest and Final Formulation. The cells from the flasks are combined into a single collection bag per TCR sublot. The cells can be transferred using a peristaltic pump. The cells are tested for quality control analysis such as determination of potency, viral contamination, viability, cell counts, as well as other characterization tests. The cell suspension is centrifuged and washed with 2% HSA in Plasma-Lyte, then harvested into one CryoMACS 500 bag in a Plasma-Lyte solution comprising 2% HAS. Preferably, the cell suspension is centrifuged using a counter-flow centrifugation system, e.g., Rotea. The cell suspension is further diluted for cryopreservation by adding an equal amount of cold CryoStor CS10 (final formulation 46% Plasmalyte A+ 1% HSA (w/v) + 50% CryoStor CS10; the NeoTCR Product). The final formulation is described in Examples 2-5. Example 7. Process 4

Introduction. Effective personalized autologous cell therapy is dependent on optimal cell culture methods that enable ex vivo activation and expansion. Furthermore, the NeoTCR Products described herein are also dependent on delivery of a patient-specific NeoTCR plasmid via electroporation with RNPs. Various commercial cell culture media formulations, including serum-free media, have been developed to improve the expansion of human T cells building on common formulations such as RPMI 1640, Isocove’s modified Dulbecco’s medium (IMDM), DMEM, and F 12. An often-overlooked aspect of cell manufacturing is understanding how the composition of the growth media medium impacts the functionality and efficacy of a cell product.

The area of immunometabolism is a critical aspect of adoptive cell therapy and significant progress has been made on understanding the unique metabolic requirements of human T cells. Upon activation through co-stimulatory signaling via CD3/CD28 domains, T cells rewire their metabolism from primarily oxidative phosphorylation toward aerobic glycolysis in order to satisfy increased cellular demands. Required for de novo synthesis of macromolecules including proteins, lipids, and nucleic acids, T cells are reliant on an exogenous source of nutrients and metabolites. For example, glucose, glutamine, and serine are instrumental in promoting this metabolic adaptation and are essential for T cell function and proliferation (van der Wint et al 2012; Olenchock et al. 2017; O’Sullivan et al. 2019). As exogeneous nutrients in the microenvironment impact activation, proliferation, phenotype, and propensity towards homology directed repair via electroporation with plasmid and RNPs, the overall outcome of ex vivo processing of T cells is highly dependent on the properties of the growth medium used.

Manufacturing of NeoTCR Products using TexMACS media as described herein for T cell activation and expansion requires supplementation with 3% human AB serum. Although TexMACS is labeled as a serum-free media by the vendor, it is evident that omission of serum from the formulation results in significant cell losses following electroporation and poor cell expansion of NeoTCR Cells. The use of huAB in clinical manufacturing is less than desirable as it suffers from supply concerns around rapidly increased global demand and lot-to-lot variability. Furthermore, significant variability across TexMACS lots was observed. In light of these limitations, there was an identified need to develop a manufacturing process and determine optimal media and growth conditions for NeoTCR Cells to generate NeoTCR Products.

Prime-XV T cell medium (Irvine Scientific / FujiFilm) is a chemically defined, cGMP- grade, animal component-free medium for T cell culture that has been optimized for consistent expansion of human T cells while maintaining functionality and potency. Supplementation of Prime-XV with human AB serum is not required for cell expansion. However, the addition of 2% (v/v) Physiologix XF serum replacement (Nucleus Biologies) following electroporation was found to improve cell recovery and expansion. In addition to the removal of human AB serum from the media as described in Processes 1-3 above, Process 4 features an additional media exchange which was discovered to be needed for optimal cell growth and expansion to make a NeoTCR Product.

Summary. On average, Prime-XV showed 35-fold expansion from day 2 to day 13 as compared to 22-fold expansion observed in cultures expanded with TexMACS in large-scale split comparison runs (n=5). With the additional media exchange on day 6, an average of 47-fold expansion was achieved (n=3). Comparable cell viability (>95%) was maintained in cell substance following expansion in Prime-XV CDM. Significant improvement in % of NeoTCR+ expression was measured on day 13 in cells activated and expanded in Prime-XV T cell CDM relative to TexMACS, 26.4% and 20.2% respectively (n=5). Increasing the final fill volume by switching from the CryoMACS250 bags with 35 mL to CryoMACS500 with a 70 mL final fill volume did not have a significant impact on post-thaw viability, cell concentration, % of NeoTCR expression by dextramer staining, and T cell phenotype. A 2-fold increase in total NeoTCR+ cell yield was obtained with Prime-XV relative to TexMACS (4.6 x 10⁹ and 2.3 x 10⁹ NeoTCR+ cells respectively) (n=5). Additional media exchange on day 6 resulted in a further increase in NeoTCR+ cell yield with an average of 6.2 x 10⁹ (n=3). Cells expanded in Prime-XV maintained comparable T-cell phenotype and functional activity in comparison with TexMACS.

Materials and Methods. All split comparison runs described in this study were executed according to their respective research procedures. For these runs, leukapheresis was collected from healthy donors (HemaCare, Cat # PBOOlF-1), one donor per comparability run or intermediate-scale development experiment, and shipped to manufacturing facility via controlled shipper at 2-8 °C.

The CliniMACS Prodigy® was used to enrich healthy donor Leukopaks for CD4+ and CD8+ T cells using the TS520 tubing set, standard TCT protocol, and the Miltenyi MACS separation columns according to the Manufacturer’s instructions. The enriched target cells were eluted in culture media (TexMACS™ with 3% human AB serum [Valley Medical] and EL-7 and IL-15 [12.5 ng/mL each]). For all conditions cultured in Prime-XV, 715 x 10⁶or 71.5 xlO⁶ cells, for large-scale and intermediate-scale experiments respectively, were dispensed into 50 mL conical tubes, centrifuged at 300 x g for 10 minutes, and resuspended in Prime-XV CDM with IL-7 and IL-15 [12.5 ng/mL each] prior to activation. Following enrichment, TransACT™ was added to the cell suspension at (1 : 17.5) in either 25 or 250 mL of culture medium with a G- Rex6w plate or G-RexlOOM-CS. Forty-eight hours after activation, cells were collected from the G-Rex and either manually buffer exchanged and concentrated via centrifugation at 300 x g for 10 minutes or in the context of large-scale experiment, buffer exchanged and concentrated using the CTS Rotea.

Electroporation was performed on a Lonza 4D Nucleofector™ device either in LV XL cartridges for large-scale processing, as detailed in RP031 : Lonza LV XL Setup or RP030: Lonza HR Nucleofection using pre-complexed RNPs. Cells were electroporated with RNPs and plasmid DNA that carries the Neol2 or PACT035 TCR cassettes in a WT TCR backbone. sgRNA (TRAC-1 and TRBC-2) and Cas9 Nuclease protein were previously complexed into RNPs. Cells were electroporated using the EO-115 pulse code on the Lonza nucleofector unit for all the samples.

After a 10-minute rest period, cells were either manually transferred out of the Lonza LV lmL cartridge via pipette into 100 mL of pre-warmed culture medium plated in a G-Rex6M plate or via peristaltic pump into a G-RexlOOM-CS with a total volume of 1L for large-scale split runs. For all Prime-XV conditions the culture medium following electroporation contains 2% Physiologix XF SR (Nucleus Biologies), IL-7, and IL-15 [12.5 ng/mL] The cells were cultured in the G-REX100M-CS until Day 8 at which the cells were split if the total viable cell count exceeded lxlO⁹ cells to maintain an optimal surface density between 5-10x10^® cells/cm². Cells were harvested on Day 13, washed in 2% HSA (w/v) in Plasma-Lyte A and concentrated in 70 mL using the CTS Rotea. Following formulation of the cell substance in PlasmaLyte A supplemented with 2% HSA, an equal volume of CryoStor CS10 is added to the CryoMACS500 bag and the contents are split via the peristaltic pump into a secondary bag with a final fill volume of 70 mL in each bag resulting in a final viable cell concentration between lOxlO⁶- lOOxlO⁶ cells/mL. The final product bags were placed in storage cassette and frozen in a controlled rate freezer using the optimized freezing program.

Results from Research Scale Manufacturing . These studies suggest that expansion in Prime-XV results in improved cell proliferation following electroporation relative to TexMACS, regardless of whether 2% Physiologix XF SR was added to the culture. Prime-XV showed greater than 30-fold expansion as compared to only 6.7-fold expansion with TexMACS. However, addition of 2% Physiologix XF SR at 2% throughout the culture (37.2-fold expansion) or only during T cell expansion post electroporation (41.6-fold expansion) further improved cell expansion as compared to Prime-XV without any serum supplements except IL-7 and IL-15 (30.6-fold expansion). While presence of 2% Physiologix XF SR during the T cell expansion phase post electroporation improved overall cell expansion, optimal gene-editing rates were observed if Prime-XV without Physiologix XF SR was used during T cell activation phase from Day 0 to Day 2 (26.5% and 17.5% respectively compared to 12.8% using TexMACS). Omission of 2% Physiologix XF SR altogether resulted in a decrease in NeoTCR+ expression (21.1%) relative to the condition where 2% Phx was added post electroporation. Based on these experiments, it was determined that Physiologix XF SR plays an important role in attenuating an electroporation induced lag phase and thus recovery of NeoTCR Cells. With regard to addition of Physiologix XF SR during the activation phase, the observed decrease in NeoTCR Cell expression was due to presence of residuals that alter gene-editing outcomes.

Among the tested Prime-XV supplementation strategies, Prime-XV without Physiologix XF SR during T cell activation, followed by addition of 2% Physiologix XF SR to the media during the expansion phase, resulted in a significant increase in NeoTCR+ cell yield, as compared to the control.

As cell expansion of cells from cancer patients generally trends lower as compared to cells from healthy donors with an average 8-fold expansion in patients (n=17 sublots, 7 different patients) as compared to an average 12-fold expansion for healthy donors (n=15 sublots, 8 different donors), experiments were designed and performed to interrogate the ability of Prime- XV to expand cells from cancer patients. Interestingly and unexpectedly, the results showed that the use of Prime-XV T cell media to expand cells from cancer patients significantly increased cell expansion with an average of 18-fold expansion between day 2 and day 13 as compared to the control (with a mean 8-fold expansion). Including an additional media exchange on Day 6 with Prime-XV medium resulted in a slight further increase in cell expansion with an average 20- fold expansion between day 2 and day 13.

Results from Large/Clinical Scale Manufacturing. Following initial research experiments aimed at optimizing the media formulation and feeding schedule, top conditions including Prime- XV with 2% Phx added following electroporation and the day 6 medium exchange were evaluated in several large-scale split runs. Extracellular glucose and lactate concentrations were monitored at various time points of the culture to confirm the optimum feeding schedule identified using the scale-down model. Aside from evaluation of parameters directly contributing to NeoTCR Cell yield, including %NeoTCR+, cell viability, and cell expansion, NeoTCR Product from split large-scale development runs were further characterized to assess potential changes in T cell phenotype and function. Furthermore, NeoTCR Products generated using Prime-XV were assessed for functionality via an IFN-g release assay as well as using IncuCyte® killing assay to determine cytotoxicity.

NeoTCR Cells expanded in Prime-XV showed significantly increased cell expansion with an average 35-fold expansion from day 2 to day 13 as compared to the 22-fold expansion observed in TexMACS cultures (p=0.0048). Metabolite analysis using the Cedex Bioanalyzer throughout the culture duration revealed that the relatively fast growth kinetics obtained using Prime-XV resulted in increased lactate concentrations in Prime-XV cultures. This increase in lactate production due to metabolic remodeling toward anabolic growth and biomass accumulation was mediated by ex vivo activation via binding of costimulatory aCD3/CD28 antibodies. Upon stimulation, T cells rely primarily rely on aerobic glycolysis in which glucose is converted into lactate and in turn generates various metabolic intermediates that are essential for cell proliferation. Although this glycolytic phenotype is a hallmark of T cell activation, lactate accumulation has been shown to significantly inhibit proliferation of T-cells. In addition to acidification of the culture medium, lactic acid represses cytokine production, particularly with respect to IL-2 and IFN-g expression. Furthermore, production of these cytokines is entirely ablated in the presence of 20 mM lactic acid.

As high lactate concentrations are known to negatively impact cell expansion and functionality of T cells, it was determined through experimentation that an additional media exchange on day6 to minimize lactate build-up and further improve cell expansion was beneficial. Indeed, the average cell expansion for this condition was highest with an average fold expansion of 47-fold. Differences in cell expansion as compared to TexMACS were statistically significant (paired Student’s t-test (n=3 runs) p=0.0080 and unpaired Student’s t-test, p=0.0040 (n=5 TexMACS, n=3 Prime-XV with additional media exchange)). Percent viability of resulting NeoTCR Product at end of culture was comparable regardless of expansion medium used.

Comparison of percent NeoTCR+ expression via dextramer binding between NeoTCR Cells generated using TexMACS, Prime-XV and Prime-XV with additional media exchange on day 6 showed improved gene-editing with an average % NeoTCR+ of 20.2 for TexMACS, 26.4 for Prime-XV and 26.2 for Prime-XV with day 6 feed. Differences were statistically significant for TexMACS and Prime-XV based on paired Student’s t-test with a p-value of 0.0004 (n= 5 runs), but not for TexMACS and Prime-XV with day 6 likely as a result of having a smaller sample size (n=3 runs) with a p-value of 0.0538 (paired Student’s t-test).

Comparison of overall NeoTCR Cell yield showed that the highest average number of NeoTCR+ cells was obtained through cultivation in Prime-XV medium with a day 6 feed yielding an average of 6.2 x 10⁹ NeoTCR+ cells as compared to 4.6 x 10⁹ NeoTCR+ cells using Prime-XV without the additional media exchange and 2.3 x 10⁹ NeoTCR+ cells using TexMACS. This significant improvement in T cell expansion, particularly of theNeoTCR+ population, supports the use of Prime-XV to achieve at least 4xl0⁹ gene-edited cells per manufacturing run. Differences were statistically significant for TexMACS and Prime-XV (p=0.0319, paired Student’s t-test, n=5 runs), but not for TexMACS and Prime-XV with day 6 feed (p=0.0839, paired Student’s t-test, n=3 runs) likely a result of having a smaller sample size. Example 8. Process 4 Improvements to Electroporation Efficiencies

Introduction. In order to further improve NeoTCR Cell yield, different electroporation devices and conditions were tested. It was determined that a large-scale cuvette electroporation device was able to improve NeoTCR Cell yield.

Pulse Protocols. Given that there was no data on gene editing efficiency of primary T cells using RNPs and plasmid DNA, multiple electroporation protocols were tested. Two of the top protocols tested were as follows: (1) Voltage (V)=2500, Pulse Width (ms)=3, # Pulses=4, and Energy Density=30000; and (2) Voltage (V)=2500, Pulse Width (ms)=4, # Pulses=4, and Energy Density=40000. It was determined that both (1) and (2) described above increased the total edited cells and that (1) was the optimal pulse protocol. These protocols using a large-scale cuvette-based electroporation device yielded an increased efficiency of gene editing compared to the gene-editing efficiencies of Processes 1-4 described above when they used the Lonza electroporation units. It was surprising to see intermediate scale runs (runs smaller than the ones used to generate NeoTCR Products for infusion into patients) consistently yielding approximately 30% gene-edited cells (2500V, 4 pulses, 500ms rest interval) with 4.24-7.35 x 10⁹ gene-edited cells. Furthermore, the large-scale cuvette-based electroporation device was shown to consistently produce 1.3xl0¹⁰ gene-edited cells per electroporation run which is more than twice the amount of total edited cells produced using the Lonza unit.

Conclusion. Based on the data generated it was determined that large-scale cuvette-based electroporation devices are capable of producing high numbers of gene-edited primary T cells that are sufficient for treating patients with NeoTCR Products.

While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention.

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

Claims

WHAT IS CLAIMED IS:

1. A method comprising: a) contacting a cell with an activation reagent; b) editing the cell to express an exogenous nucleic acid; c) culturing the edited cell in a cell culture medium; d) harvesting the edited cell; and e) transferring the edited cell in a container; wherein the method is performed within a closed system.

2. A method comprising: a) contacting a cell with an activation reagent; b) editing the cell to express an exogenous nucleic acid; c) culturing the cell in a cell culture medium to obtain a population of cells; d) harvesting the population of cells; and e) transferring the population of cells in a container; wherein the method is performed within a closed system.

3. The method of claim 1 or 2, further comprising a counter-flow centrifugation.

4. The method of any one of claims 1-3, wherein the method occurs within a period of about 13 days, about 14 days, about 15 days.

5. The method of any one of claims 1-4, wherein the editing comprises introducing into the cell a polynucleotide, comprising: a) first and second homology arms homologous to first and second target nucleic acid sequences and oriented to facilitate homologous recombination; b) a nucleotide sequence encoding a TCR polypeptide sequence positioned between the first and second homology arms; and c) a first nucleotide sequence encoding a P2A ribosome skipping element positioned upstream of the nucleotide sequence encoding the TCR polypeptide and a second nucleotide sequence encoding a P2A ribosome skipping element positioned downstream of the nucleotide sequence encoding the TCR polypeptide, wherein the first and second nucleotide sequences encoding the P2A ribosome skipping elements are codon-diverged relative to each other.

6. The method of claim 5, wherein the first and second homology arms of the polynucleotide are each from about 300 bases to about 2,000 bases in length.

7. The method of claim 5 or 6, wherein the polynucleotide further comprises: a) a nucleotide sequence encoding for the amino acid sequence Gly Ser Gly positioned immediately upstream of the nucleotide sequences encoding the 2A ribosome skipping elements; b) a nucleotide sequence encoding for a Furin cleavage site upstream of the second nucleotide sequence encoding a 2A ribosome skipping element; and c) a nucleotide sequence encoding for a human growth hormone signal peptide positioned upstream of the nucleotide sequence encoding the TCR.

8. The method of any one of claim 5-7, wherein the polynucleotide further comprises a second nucleotide sequence encoding a TCR polypeptide sequence between the second nucleotide sequence encoding a P2A ribosome skipping element and the second homology arm.

9. The method of claim 8, wherein the polynucleotide further comprises a second nucleotide sequence encoding for a human growth hormone signal peptide positioned upstream of the second nucleotide sequence encoding the TCR polypeptide.

10. The method of any one of claims 5-10, wherein the polynucleotide is a circular DNA.

11. The method of any one of claims 1-10, wherein the edited cell expresses an exogenous TCR gene sequence encoding for a TCR that recognizes a tumor antigen.

12. The method of any one of claims 1-35, wherein the population of cells comprises a cell expressing an exogenous TCR gene sequence encoding for a TCR that recognizes a tumor antigen.

13. The method of claim 11 or 12, wherein the tumor antigen is a neoantigen.

14. The method of claim 11 or 12, wherein the tumor antigen is a patient specific neoantigen.

15. The method of any one of claims 5-14, wherein the exogenous TCR gene sequence is a patient specific TCR gene sequence.

16. The method of any one of claims 1-15, wherein the transfecting comprises cleavage of an endogenous locus by a nuclease.

17. The method of claim 16, wherein the nuclease is a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) family nuclease or derivative thereof.

18. The method of claim 17, further comprising an sgRNA.

19. The method of any one of claims 1-18, wherein the editing comprises a viral infection.

20. The method of any one of claims 1-18, wherein the editing comprises electroporation.

21. The method of any one of claims 1-20, wherein the cell is a primary cell, a lymphocyte, or a T cell.

22. The method of any one of claims 1-21, wherein the population of cells comprises a primary cell, a lymphocyte, a T cell, or a combination thereof.

23. The method of claim 21 or 22, wherein the T cell is a CD8 or a CD4 T cell.

24. The method of claim 21 or 22, wherein the T cell is a young T cell.

25. The method of claim 24, wherein the young T cell is CD45RA⁺, CD62L⁺, CD28⁺, CD95 , CCR7⁺, and CD27⁺.

26. The method of claim 24, wherein the young T cell is CD45RA⁺, CD62L⁺, CD28⁺, CD95⁺, CCR7⁺, CD27⁺.

27. The method of claim 24, wherein the young T cell is CD45RO⁺, CD62L⁺, CD28⁺, CD95⁺, CCR7⁺, CD27⁺, CD127⁺.

28. The method of claim 24, wherein the young T cell is a memory stem cell (TMSC).

29. The method of claim 24, wherein the young T cell is a central memory cells (T_CM).

30. The method of any one of claims 22-29, wherein the population of cells comprises at least about 20% of TMSC and TCM collectively, at least about 25% TMSC and TCM collectively, at least about 30% TMSC and TCM collectively, at least about 35% TMSC and TCM collectively, at least about 40% TMSC and TCM collectively, at least about 45% Tmsc and Tcm collectively, at least about 50% TMSC and TCM collectively, at least about 55% TMSC and TCM collectively, at least about 60% TMSC and TCM collectively or more than about 61% TMSC and TCM collectively.

31. The method of any one of claims 1-30, wherein the cell is obtained from a subject.

32. The method of claim 31, wherein the cell is obtained by leukapheresis.

33. The method of claim 31, wherein the cell is obtained by a tissue sample.

34. The method of claim 33, wherein the tissue sample is a tumor sample.

35. The method of any one of claims 1-34, wherein the cell is cryopreserved.

36. The method of any one of claims 1-35, wherein the cell culture medium comprises interleukin 2 (IL2), interleukin 7 (IL7), interleukin 15 (TLl 5), interleukin (TL21), or any combination thereof.

37. The method of claim 36, wherein the cell culture medium comprises IL7 and IL15.

38. The method of any one of claims 1-37, wherein the cell culture medium comprises fibronectin, insulin, transferrin, or any combination thereof.

39. The method of any one of claims 1-38, wherein the cell culture medium comprises glucose concentration of at least about 3.7 g/L.

40. The method of any one of claims 1-39, wherein the culturing is performed within a period of about 10 days, about 11 days, or about 12 days.

41. The method of any one of claims 1-40, wherein the activation reagent comprises an anti- CD3 antibody, an anti-CD2 antibody, an anti-CD28 antibody, or a combination thereof.

42. The method of claim 41, wherein the activation reagent comprises non-magnetic beads or magnetic beads.

43. The method of claim 41, wherein the activation reagent comprises an artificial APCs.

44. The method of any one of claims 1-43, wherein the contacting is performed within a period of about 2 days, or about 3 days.

45. The method of any one of claims 1-44, further comprising cry opreserving the edited cell.

46. The method of any one of claims 2-44, further comprising cryopreserving the population of cells.

47. The method of claim 45 or 46, wherein the edited cell or population of cells is cryopreserved in a pharmaceutical formulation.

48. The method of any one of claims 1-47, wherein the pharmaceutical formulation comprises a cryopreservation medium, a serum albumin, a crystalloid solution, or a combination thereof.

49. The method of claim 48, wherein the cryopreservation medium is at a final concentration of about 50% v/v.

50. The method of claim 48 or 49, wherein the serum albumin is at a final concentration of about 1% w/v.

51. The method of any one of claims 48-50, wherein the crystalloid solution is at a final concentration of about 46% v/v.

52. The method of any one of claims 48-51, wherein the pharmaceutical formulation comprises CryoStor® CS10, human serum albumin, Plasma-Lyte A, or a combination thereof.

53. The method of any one of claims 1-52, wherein the closed system comprises a peristaltic pump.

54. The method of any one of claims 1-53, wherein the edited cell is infused into a subject.

55. The method of any one of claims 1-53, wherein the population of cells is infused into the subject.

56. A composition comprising the edited cells obtained by the method of any one of claims 1- 55.

57. A composition comprising the population of cells obtained by the method of any one of claims 2-55

58. The composition of claim 56 or 57, further comprising a pharmaceutical excipient.

59. The composition of any one of claims 56-58, comprising a therapeutically effective amount of cells.

60. The composition of any one of claims 56-59, comprising at least about 1 x 10⁶ cells/ml.

61. The composition of any one of claims 56-59, comprising at least about 10 x 10⁶ cells/ml.

62. The composition of any one of claims 56-59, comprising at least about 100 x 10⁶ cells/ml.

63. The composition of any one of claims 56-59, comprising at least about 4.0 x 10⁸ gene- edited cells.

64. The composition of any one of claims 56-59, comprising at least about 1.3 x 10⁹ gene- edited cells.

65. The composition of any one of claims 56-59, comprising at least about 4.0 x 10⁹ gene- edited cells.

66. The composition of any one of claims 56-59, comprising at least about 1.3 x 10¹⁰ gene- edited cells.

67. The composition of any one of claims 56-59, comprising at least about 4.0 x 10¹⁰ gene- edited cells.

68. A method of treating a cancer comprising administering the edited cell obtained by the method of any one of claims 1-55, the population of cells obtained by the method of any one of claims 2-55, or the composition of any one of claims 56-67 to a subject in need thereof.

69. The edited cell obtained by the method of any one of claims 1-55, the population of cells obtained by the method of any one of claims 2-55, or the composition of any one of claims 56-672 for use in the treatment of a cancer.

70. Use of the edited cell obtained by the method of any one of claims 1-55, the population of cells obtained by the method of any one of claims 2-55, or the composition of any one of claims 56-67 for the manufacture of a medicament for the treatment of cancer.

71. A method of manufacturing NeoTCR Cells using Process 1, Process 2, Process 3, or Process 4 described herein.

72. A composition comprising the NeoTCR Cells of Claim 71.