WO2022066100A1

WO2022066100A1 - Methods for improved t cell manufacturing

Info

Publication number: WO2022066100A1
Application number: PCT/SG2021/050574
Authority: WO
Inventors: Tongjin WU; Lih Feng Cheow
Original assignee: National University Of Singapore
Priority date: 2020-09-22
Filing date: 2021-09-22
Publication date: 2022-03-31

Abstract

The invention relates generally to the field of immunotherapy. The invention is directed to a method of preparing a T cell therapeutic as well as methods of treating a subject. In one embodiment, there is provided a method of preparing a cell therapeutic, the method comprising the step of a) culturing a population of lymphocytes comprising CD8+ T cells and CD4+ T cells; wherein the method comprises selecting for a population of lymphocytes prior to or following step a) based on side scatter.

Description

METHODS FOR IMPROVED T CELL MANUFACTURING

Field of Invention

The invention relates generally to the field of immunotherapy. In particular, the invention is directed to a method of preparing a T cell therapeutic as well as methods of treating a subject.

Background

Adoptive immunotherapy has shown great potential in cancer treatment. Nevertheless, the varying efficiencies and some undesirable side effects call for development of better and more innovative cell manufacturing approaches. Insight into attributes of therapeutic potency based on redirected T cells such as chimeric antigen receptor (CAR) T cells and TCR-T cells is a central goal of T cell immunotherapy. Quality of T cell products generated ex vivo has been recognized as a critical factor affecting immunotherapy efficacy. Unfractionated peripheral blood mononuclear cell (PBMC) concentrates are commonly used for T cell production, leading to uncontrollable batch- to-batch variation. Pre-selection techniques of T-cell subsets is available. However, such techniques involve using multiple rounds of cell separation to improve product consistency.

Accordingly, it is generally desirable to overcome or ameliorate one or more of the above mentioned difficulties.

Summary

Disclosed herein is a method of preparing a cell therapeutic, the method comprising the step of a) culturing a population of lymphocytes comprising CD8⁺ T cells and CD4⁺ T cells; wherein the method comprises selecting for a population of lymphocytes prior to or following step a) based on side scatter. Disclosed herein is a method of preparing a cell therapeutic, the method comprising the step of a) culturing a population of lymphocytes comprising CD8⁺ T cells and CD4⁺ T cells; wherein the population of lymphocytes comprises CD8⁺ T cells and CD4⁺ T cells at a pre-determined ratio

Disclosed herein is a method of improving the quality and/or efficacy of a cell therapeutic, the method comprising performing a method as defined herein.

In one embodiment, there is provided a cell therapeutic obtained according a method as defined herein.

In one embodiment, there is provided a cell therapeutic derived from a population of lymphocyte with different side-scattering intensity.

Disclosed herein is a method of treating a subject in need, the method comprising administering to the subject a cell therapeutic that is prepared according to a method as defined herein.

Brief Description of Drawings

Embodiments of the present invention are hereafter described, by way of non-limiting example only, with reference to the accompanying drawings in which:

Figure 1: Experimental design for co-culture of purified lymphocytes and data collection (A) Representative plots of cell purity validation post sorting before reconstituted for co-culture assay. (B) Percentage of major lymphocyte populations from each donor (left) and relative composition analysis as normalized to CD3+ CD4+ T cells (right). (C) Experimental design to study the mutual effects of cell-cell communication during T cell expansion ex vivo. The purified CD4+ T and CD8+ T cells were reconstituted into each other as indicated with or without B/NK cells, and the initial number of cells in each well was fixed to 0.06 M while 0.15 M for B/NK-spiked in group. Figure 2. CD4⁺ T-CD8⁺ T crosstalk improves the yields of T cell product with flexible ratio of CD4/CD8 T cells (A)-(D) Counts of total nucleated cells (A), total CD3+ cells (B), and fold increase of CD3⁺ CD8⁺ T cells (C) and CD3⁺ CD4⁺ T cells (D) along the time course of ex vivo expansion. (E) Percentage change of CD3⁺ CD4⁺ T and CD3⁺ CD8⁺ T cells during expansion procedure. (F) Ratio change of CD4/CD8 T cells post expansion derived from different culture conditions.

Figure 3. CD4⁺ T-CD8⁺ T crosstalk synergistically contributes to TCM pool maintenance (A) Gating strategy to identify TN/TCM/TEM/TEFF subtypes within CD3⁺ CD4⁺ T or CD3⁺ CD8⁺ T cells. (B)-(D). Percentages of differentiated T cell lineages among CD3⁺ CD8⁺ T cells (B) and corresponding expression level of CCR7 (C) and CD45RA (D) on TCM or TEM subsets. (E)-(F). Percentages of differentiated T cell lineages among CD3⁺ CD4⁺ T cells (E) and corresponding expression level of CCR7 (F) and CD45RA (G) on TCM or TEM subsets. The TN and TEFF were omitted here due to only minor proportion in some groups for in parallel comparison. Only TCM were considered for CCR7 MFI analysis given that TEM is characterized as CCR7 negative.

Figure 4. CD4⁺ T-CD8⁺ T crosstalk contributes to production of naive-like CD8⁺ TCM precursors (CD8⁺ TCMRA) (A) In-depth gating strategy to analyze the dynamic changes of TCM with varied CCR7/CD45RA expression, and TEM as well with different CD45RA level (day 7). (B)-(C) The histogram graph summarizes percentage change of subtle effector/memory T subsets among CD3⁺ CD8⁺ T (B) or CD3⁺ CD4⁺ T cells (C) along the expansion course.

Figure 5. CD4⁺ T-CD8⁺ T crosstalk synergistically contributes to optimal T cell functionality of final products (A)-(C) Expression of granzyme B at day 7 of expanded T cells as shown by histogram plot (A) and summary of the granzyme B percentage and MFI in CD3⁺ CD8⁺ T cells (B) and CD3⁺ CD4⁺ T cells (C). The cells at day 0 before expansion were set as un-stimulated control to indicate the background of granzyme B expression. (D)-(E) Percentage and MFI of total cytokine expression at day 7 among CD3⁺ CD8⁺ T cells (D) and CD3⁺ CD4⁺ T cells (E). Multi-functional cytokine expression profile at day 7 of expanded T cells as shown by pie charts (F). Figure 6. Side scatter-based segregation of major lymphocyte subtypes from human PBMC. (A) FSC/SSC plots of T cell populations from each donor (left panel) and compositional analysis among SSC^hl and SSC^low cell clusters, correspondingly (right panel). The cell samples were stained and fixed overnight and the gating is based on the cluster of total lymphocytes which has been hidden. (B) FSC/SSC plots of B/NK cells from each donor (left panel) and corresponding proportion among SSC^hl and SSC^low cell clusters (right panel). The cell samples were stained and fixed overnight and the gating is based on the total lymphocytes which has been hidden. (C) SSC^hl and SSC^low cells were directly sorted by FACS before lymphocyte subtypes determination. Results are shown as three independent experiments for each donor.

Figure 7. Distinct naive/memory cell composition of lymphocytes characterized by side-scattering intensity (A) Representative gating approach for naive/memory T cell characterization and shown is the side-scattering distribution of each naive/memory T cell subset among CD4⁺ T or CD8⁺ T cells (bottom panel). The cell samples were stained and fixed overnight and the gating is based on the amount of total lymphocytes which has been hidden. (B) Compositional analysis of naive/memory T cell subsets in manually gated SSC^hl and SSC^low cell populations as depicted in (A). (C) Compositional analysis of naive/memory T cell subsets in pre-sorted SSC^hl and SSC^low cell populations. The results are represented as three independent experiments of each donor.

Figure 8. Discernable functionality profile of lymphocytes with different side scattering intensity (A)-(B) Abundance of CCR7 (A) and CD45RA (B) expressed on CD3⁺ CD4⁺ T or CD3⁺ CD8⁺ T cells among sorted SSC^hl and SSC^low cell populations. (C) Representative gating strategy for multifunctional cytokine-expressing analysis of lymphocytes with different SSC intensity. The percentage of cytokine-expressing cells was further normalized to its source of cell subsets. (D) - (F) Representative cytokine expression profile of sorted lymphocytes based on SSC intensity before further stimulated with PMA plus ionomycin for 4 hrs.

Figure 9. Fixation/permeabilization and staining cause no observed effects on the tendency of side-scattering distribution of CD4/CD8 T cells or T cell naive/memory subsets (A) Representative FSC/SSC pattern of PBMC (donor 1) with or without subjected to surface marker staining procedure (B) T cell side scattering distribution (donor 1) in situation where only single fluorescence was used to exclude fluorescence compensation-induced interferences (C) Effects of fixation/permeabilization on CD4/CD8 T cells scattering distribution (donor 1 and 3) (D) Effects of fixation/permeabilization on naive/memory T cells scattering distribution (donor 1 and 3).

Figure 10. Forward-scatter intensity enables partial segregation of B/NK cells but not CD4/CD8 T cells or naive/memory T cells from resting PBMCs (A) - (C). FSC/SSC plots of T cell populations (A), B/NK cells (B), and naive/memory T cell subsets (C) from each donor and compositional analysis among FSC^hl and FSC^low cell clusters, correspondingly. The cell samples were stained and fixed overnight and the gating is based on the total lymphocytes which are hidden here.

Figure 11. Comparable auto-fluorescence (Ex/Em: 488 nm/525 nm) distribution of lymphocyte populations in resting PBMCs (A) Shown are the general autofluorescence distribution of lymphocyte populations in resting PBMCs (donor 1). (B) Representative gating plot by FSC/auto-fluorescence for cell sorting. (C) Compositional analysis of T cell and NK cell among sorted cell populations with different autofluorescence intensity (D) Histogram summary of naive/memory T cell subsets in sorted cell populations with different auto-fluorescence intensity.

Figure 12. Double side-scattering by SSC-A (UV405)/SSC-A (B488) for segregation of lymphocyte populations (A) Representative gating strategy (donor 2) for cell sorting. (B) CD4⁺ T and CD8⁺ T lymphocyte composition among sorted SSC^hl and SSC^low cell populations (C) B and NK proportion among sorted SSC^hl and SSC^low cell populations (D) Histogram summary of naive/memory T cell subsets in sorted SSC^hl and SSC^low cell populations

Figure 13. Infeasibility for T cell subsets re-segregation from rapidly expanding PBMCs by either pure auto-fluorescence or light scattering CD3/CD28 beads- activated PBMCs at day 7 were sorted into SSC^hl and SSC^low populations by side-scatter (A), or FSC^hl and FSC^low populations by forward-scatter (B), or FITC^hl, FITC^med and FITC^low populations by auto-fluorescence (FITC channel). The percentage of CD3⁺ CD8" T cells and CD3⁺ CD8⁺ T cells and also naive/memory composition, correspondingly, within the sorted cell clusters were further analyzed by immunostaining and flow cytometry analysis.

Figure 14. Pre-separated T cell populations simply by side-scatter improves the quantity of T cell products with flexible ratio of CD4/CD8 T cells (A) Expansion efficiency of TNC starting from SSC^hlgh, SSC^low, or reconstituted SSC^hlgh+low cells at ratio of 1:1. (B) CD4⁺ T or CD8⁺ T cells expansion potency corresponding to (A). (C) Ratio change of CD4/CD8 T cells along the time course of rapid expansion procedure.

Figure 15. Pre-separated TN from TM favors selection of phenotypically optimal T cell products post expansion (A) Gating strategy to show the dynamic changes of TCM and TEM with varied CCR7/CD45RA expression along the time course of expansion procedure. (B)-(C) The histogram graphs summarize percentage change of effector/memory subsets in CD4⁺ T (B) or CD8⁺ T cells (C) based on the gating strategy in (A). (D) Correlation analysis of starting TN with the percentage of generated TCMRA- hi among cell products of each time point along the expansion procedure. Data of the same day from all of the three donors (B&C) were combined together. Each dot represents the percentage of generated cell subset against the indicated cell component of its starting cell material. The solid line in black indicates combined linear regression using all of the dots in that plot.

Figure 16. Pre-separated TN from TM favors the selection of functionally less- differentiated T cell products (A)-(C) Representative histogram plot of IFN-y/IL- 2/TNF-a expression of cell product (day 11) derived from cell groups with different side-scatter intensity (A) and summary of data from two donors at day 7 (B) and three donors at day 11 (C). (D) - (E) Multifunctional cytokine expression of total CD3⁺ T cells at day 7 (D) and day 11 (E). F-K. Multifunctional cytokine analysis of CD3⁺ CD8" T cells (F) or CD3⁺ CD8⁺ T cells (I) at day 7 and day 1. Shown are results of PCA plots either using cytokine profile only (G and J) or combining both cytokine expression and phenotypic profile (H and K). For PCA plotting, the same cell subsets with percentage lower that 4% in all groups were omitted. Figure 17. Increased TCMRA-W derived from SSC^low cell cluster resembles TscM-uke central memory precursors (A-D) Longitudinal assessment of phenotypic change of TcMRA-hi compared to other T cell subsets. Average expression and abundance per cell of selected phenotypic markers within CD4⁺ T (A-B) or CD8⁺ T subsets (C-D) (n = 3). The relative fluorescence intensity (RFI) was reported after normalization to that of TcMRA-hi of the same time points respectively (B and D). The cross label indicates omitted data due to low cell percentages for marker expression identification or insufficient counts for MFI calculation. (E-H) Correlation analysis of starting effector/memory composition, including TN (E), TCMO (F), TCMRA-IOW (G), and total TEMRA (H), with the percentage of generated TCMRA-H; after expansion. Data from the same day were combined (n = 3). Each dot represents the percentage of produced TCMRA- hi versus that of initial cellular composition (day 0). The solid black line indicates combined linear regression. Unpaired two-tailed t-test.

Figure 18. SSClow-derived CART19 possesses favorable phenotypes and functionality in vitro (A-B) Lentiviral transduction efficiency (A) and transgenic stability as represented by CAR abundance per cell (B) of T cells initially with different SSC intensity. Data are mean ± S.E.M (n = 4). *P < .05, **P < .01, paired two-tailed t- test. (C-D) Longitudinal assessment of CART19 subset composition (C) and absolute numbers (D) accumulated over time. Average data of four donor samples was represented. (E) Dynamic expression of CD27 and CD28 in generated CART19 cells. The relative MFI was shown after normalization to marker expression of SSChi group. (F-H) Representative gating to identify the cell states in in vitro killing assay (F). For the measurement of short-term killing potency, an E:T ratio of 4:1 was used, effector/target percentage (G) and live target numbers as well (H) were shown after 4 hr co-culture. (I) A low E:T ratio at 1:4 and 1:10 was adopted to assess the capability of CART19 in responding to recursive stimulation by Raji cells. Shown was duplicate experiments for each donor sample.

Detailed Description The present invention teaches a method of preparing a cell therapeutic. The present specification teaches a method of preparing a cell therapeutic, the method comprising the step of a) culturing a population of lymphocytes comprising CD8⁺ T cells and CD4⁺ T cells.

Disclosed herein is a method of preparing a cell therapeutic, the method comprising the step of a) culturing a population of lymphocytes comprising CD8⁺ T cells and CD4⁺ T cells; wherein the method comprises selecting for a population of lymphocytes prior to or following step a) based on side scatter.

Without being bound by theory, the inventors have also found that the side-scatter (SSC) can be used to segregate lymphocytes into subpopulations which contain various composition of CD4⁺ T/CD8⁺ T/NK/B cells. The cell cluster of low SSC (SSC^low) has a significantly higher number of CD4⁺ T cells and B cells but less NK cells. In contrast, the cell cluster of high SSC (SSC^hlgh) consists of more CD8⁺ T cells and NK cells. SSC is efficient to separate less-differentiated T cell subsets (e.g. the naive T cells (TN) and naive-like central memory precursors with high CD45RA expression (TCMRA-M) from the more-differentiated T cells.

Disclosed herein is a method of preparing a cell therapeutic, the method comprising the step of a) culturing a population of lymphocytes comprising CD8⁺ T cells and CD4⁺ T cells; wherein the population of lymphocytes comprises CD8⁺ T cells and CD4⁺ T cells at a pre-determined ratio.

The inventors also found that the expansion potency of CD8⁺ T cells can be regulated by the co-culture of CD4⁺ T cells. A higher ratio of CD4⁺ T/CD8⁺ T cells rather than CD8⁺ T cells culture alone is critical for optimal CD8⁺ T cell expansion while the CD4⁺ T cells proliferation is nearly unaffected. Co-culture with CD4⁺ T cells reduces the formation of more-differentiated T cell subtypes.

Manufactured cell therapeutics contemplated herein may be useful in the treatment or prevention of numerous conditions including, but not limited to cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency. In one embodiment, the method comprises harvesting cells from a subject and isolating a population of cells using a closed system process. In some embodiments, cells may be isolated from any suitable fresh or frozen sources. In some embodiments, the isolated population of cells comprises peripheral blood mononuclear cells (PBMCs). The isolated population of cells may be seeded to initiate cultures and T cells are activated and stimulated by contacting the cells with primary and costimulatory ligands. In some embodiments, populations of cells comprising activated T cells are transduced with a viral vector in order to redirect the transduced cells to a particular target antigen. In some embodiments, the cells are transduced with a viral vector encoding a CAR or engineered TCR. Transduced cells or non-transduced cells may then be cultured in growth medium for expansion. Manufactured immune effector cell compositions may then be used to treat subjects in need thereof or frozen for later use.

As used herein, the phrase “preparing a cell therapeutic” or “manufacturing a cell therapeutic” may refer to the process of producing a therapeutic composition of T cells, which manufacturing methods may comprise one or more of, or all of the following steps performed one or more times on a population of cells comprising T cells or a population of purified T cells: harvesting, isolating, washing, stimulating, activating, modifying, expanding, cryopreserving, and thawing, or any suitable combination thereof.

The terms “T cell” or “T lymphocyte” are art-recognized and are intended to include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. Illustrative populations of T cells suitable for use in particular embodiments include but are not limited to helper T cells (CD4⁺ T cell), a cytotoxic T cell (CD8⁺ T cell), CD4⁺CD8⁺ T cell, CD4 CD8" T cell, naive T cells “TN”, stem-cell memory T cells (“TSCM”), central memory T cell (“TCM”), effector memory T cell (“TEM”), terminal effector T cell (“TEMRA”) or any other subset of T cells. Other illustrative populations of T cells suitable for use in particular embodiments include, but are not limited to, T cells expressing one or more of the following markers: CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD 127, CD 197, and HLA-DR and if desired, can be further isolated by positive or negative selection techniques.

A peripheral blood mononuclear cell (PBMC) is defined as any blood cell with a round nucleus (i.e., a lymphocyte or a monocyte). These blood cells are a critical component in the immune system to fight infection and adapt to intruders. The lymphocyte population may comprise CD4⁺ and CD8⁺ T cells, B cells and Natural Killer cells. These cells are often separated from whole blood or from leukopacks using FICOLL™, a hydrophilic polysaccharide that separates layers of blood, with monocytes and lymphocytes forming a huffy coat under a layer of plasma. In one embodiment, “PBMCs” refers to a population of cells comprising at least T cells, B cells, NK cells, monocytes, and granulocytes.

In one embodiment, the method comprises selecting for a population of lymphocytes from a sample obtained from a subject.

In some embodiments, T cells can be obtained from a number of sources including, but not limited to, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, a population of cells comprising T cells, e.g., PBMCs, is used in the manufacturing methods contemplated herein. In other embodiments, an isolated or purified population of T cells is used in the manufacturing methods contemplated herein.

The T cells as referred to herein may be autologous/autogeneic (“self’) or non- autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic). In some embodiments, cell populations comprising T cells are obtained from an individual and subjected to a method as contemplated herein. In one embodiment, cells from the circulating blood of an individual are obtained by a method of apheresis, e.g., leukapheresis. The apheresis product may contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets or may be a leukapheresis product comprising lymphocytes, including T cells, monocytes, granulocytes, B cells, and other nucleated white blood cells. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing. The cells can be washed with PBS or with another suitable solution that lacks calcium, magnesium, and most, if not all other, divalent cations.

As used herein, a “subject” is a mammal, such as a human or other animal (such as a primate), and typically is human. The subject may be one who exhibits a symptom of a cancer, infectious disease, immunodeficiency, inflammatory disease or auto-immune disorder that can be treated with a T cell therapeutic that is prepared according to a method as defined herein.

In one embodiment, the present invention is directed to a method of preparing a modified T cell therapeutic (such as a CART cell therapeutic).

“Modified T cells” refer to T cells that have been modified by the introduction of a polynucleotide encoding an engineered TCR or CAR contemplated herein. Modified T cells include both genetic and non-genetic modifications (e.g., episomal or extrachromosomal) .

As used herein, the term “genetically engineered” or “genetically modified” refers to the addition of extra genetic material in the form of DNA or RNA into the total genetic material in a cell.

The terms, “genetically modified cells” “modified cells” and, “redirected cells” are used interchangeably.

As used herein, the term “gene therapy” refers to the introduction of extra genetic material in the form of DNA or RNA into the total genetic material in a cell that restores, corrects, or modifies expression of a gene, or for the purpose of expressing a therapeutic polypeptide, e.g., a TCR or CAR and/or one or more cytokines. In particular embodiments, T cells are modified to express an engineered TCR or CAR without modifying the genome of the cells, e.g., by introducing an episomal vector that expresses the TCR or CAR into the cell. In one embodiment, the method comprises a step of modifying the population of lymphocytes to generate a modified T cell therapeutic.

In one embodiment is a method of preparing a modified T cell therapeutic, the method comprising the step of a) culturing a population of lymphocytes comprising CD8+ modified T cells and CD4+ modified T cells.

In some embodiments, after isolation of PBMC, both cytotoxic and/or helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after activation, expansion, and/or genetic modification.

In one embodiment, the method comprises selecting for a population of lymphocytes prior to or following step a). The method may, for example, selecting for a population of lymphocytes from PBMCs that has been isolated from a subject. The method may also comprise selecting a population of lymphocytes from a population of T cells such that the population lymphocytes comprises a population of CD8⁺ T cells and a population of CD4⁺ T cells. The method may also comprise selecting a population of lymphocytes from a population of modified T cells, wherein the population of modified T cells can include a population of modified CD8⁺ T cells and a population of modified CD4⁺ T cells.

The method may comprise selecting for a population of lymphocytes using light scattering. The term “light scattering” is well-known in the art and is understood to refer to either “forward scattering” which measures cell size or “side scattering” which measures cellular complexity (e.g. granularity).

Herein, the term “forward scattering” or “forward scatter signal” refers to the light scatter signal measured less than 10° from the incident light. The term of “side scattering” or “side scatter signal” refers to the light scatter signal at about 90° or at the right angle from the incident light, generated by a cell passing through an aperture of a flow cell. The term of “side scatter measurement” or “forward scatter measurement” refers to the measurement of the side scatter signals by an optical detector. Most commercially available flow cytometers are equipped with a detection system which enables measurement of the forward scatter and side scatter signals.

An initial sample (such as an initial PBMCs sample) may appear as 2 or 3 distinct populations on a flow cytometer based on forward scatter and side scatter. For example, the lymphocytes would generally appear within one distinct population of cells while monocytes and/or granulocytes would appear as one or more separate population of cells. The method as defined herein may comprise gating the population of cells that comprises lymphocytes. The population of cells that comprises lymphocytes may further comprise B cells and NK cells. The method may comprise separating the population of cells that comprise lymphocytes into two distinct populations that has low and high side scatter. The two distinct populations may be non-overlapping.

In one embodiment, the method comprises selecting a population of lymphocytes that has a low side scatter as compared to a reference. The low side scatter may be associated with improved quality and/or efficacy of a cell therapeutic. The population of lymphocytes with a low side scatter may comprise an enriched population of less differentiated T cells. The population of lymphocytes with a low side scatter may comprise an enriched population of CD4⁺ T cells and B cells.

In one embodiment, the population of lymphocytes has a high side scatter as compared to a reference The population of lymphocytes with a high side scatter may comprise an enriched population of NK. This allows NK cells to be further separated (such as using immunomagnetic selection) and allows NK cells to be manufactured (e.g., CAR-NK).

The term “low side scatter” may, for example, refer to an intensity that is lower than a reference, such as an intensity that is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more below a reference.

The term “high side scatter” may, for example, refer to an intensity that is higher than a reference, such as an intensity that is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more above a reference. The reference may be an average intensity of a population of lymphocytes.

Alternatively, the reference may be the median intensity of a population of lymphocytes.

A population of lymphocytes may, for example, be separate into 2 distinct groups of lymphocytes, one with a “high side scatter” and one with a “lower side scatter”, wherein the group of lymphocytes with a “high side scatter” has an average side scatter that is higher than the average or median side scatter of the overall population of lymphocytes, whereas the group with a “low side scatter” has an average side scatter that is lower than the average or median side scatter of the overall population of lymphocytes. In some embodiments, a population of lymphocytes with “low side scatter” has an average side scatter that is no more than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10% of the average or median side scatter of the overall population of lymphocytes. In some embodiments, a population of lymphocytes with “high side scatter” has an average side scatter that is more than 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 350%, 400%, 450% or 500% of the average or median side scatter of the overall population of lymphocytes.

Alternatively, each lymphocyte within the “high side scatter” population can have an individual side scatter that is higher than the average side scatter of the overall population of lymphocytes while each lymphocyte within the “low side scatter” population can have an individual side scatter than is lower than the average side scatter of the overall population of lymphocytes.

The use of side scatter may allow for selection of a population of cells with an optimal ratio of CD4⁺ T cells and CD8⁺ T cells. The ratio of CD4⁺ T cells and CD8⁺ T cells in the selected population of cells may be higher than the ratio of CD4⁺ T cells and CD8⁺ T cells in a reference population of cells. The reference population of cells may be a population of cells that has not been selected or gated for any side scatter (e.g. low or high side scatter). The optimal ratio of CD4⁺ T cells and CD8⁺ T cells may be 5: 1, 4.9: 1, 4.8:1, 4.7:1, 4.6:1, 4.5:1, 4.4:1, 4.3:1, 4.2:1, 4.1:1, 4.0:1, 3.9:1, 3.8:1, 3.7:1, 3.6:1, 3.5: 1, 3.4:1, 3.3:1, 3.2:1, 3.1:1, 3.0:1, 2.9:1, 2.8:1, 2.7:1, 2.6:1, 2.5:1, 2.4:1, 2.3:1, 2.2:1, 2.1: 1, 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1: 1, 1:1, 0.9:1, 0.8:1, 0.7: 1,

0.6:1 or 0.5:1.

The use of side scatter may allow for selection of a population of cells with an optimal ratio of less-differentiated (younger) T-cell subsets and more-differentiated T-cell subsets. The less-differentiated T cells may include TN and early TCM or TCM with high CD45RA expression (TCMRA-H;) while the more-differentiated T cells may include late TCM, TEM, and TEMRA- The ratio of less-differentiated T cells to more-differentiated T cells may be higher than the ratio of less-differentiated T cells to more-differentiated T cells in a reference population of cells. The reference population of cells may be a population of cells that has not been selected or gated for any side scatter (e.g. low or high side scatter). The optimal ratio of less-differentiated T cells and more-differentiated T cells may be 10:1, 9.5:1, 9:1, 8.5:1, 8:1, 7.5:1, 7:1, 6.5: 1, 6:1, 5.5:1, 5:1, 4.5:1, 4: 1, 3.5:1, 3:1, 2.5:1 or 2: 1.

The selection technique using light scattering may allow the population of lymphocytes to be selected without any label. The method may comprise selecting for a population of lymphocytes using a cell sorter. The selection of a population of lymphocytes with low side scatter may allow an optimum ratio of CD4⁺ T cells and CD8⁺ T cells to be isolated from a sample (such as from PBMCs). The selection of a population of lymphocytes with low side scatter may allow an optimum ratio of less differentiated T cells and more differentiated T cells to be isolated from a sample (such as from PBMCs).

The method may comprise culturing the population of lymphocytes comprising CD4⁺ T cells and CD8⁺ T cells at a pre-determined ratio. In one embodiment, the ratio of CD4⁺ T cells and CD8⁺ T cells is 5: 1, 4.9:1, 4.8:1, 4.7:1, 4.6:1, 4.5: 1, 4.4:1, 4.3:1, 4.2:1, 4.1: 1, 4.0:1, 3.9:1, 3.8:1, 3.7:1, 3.6:1, 3.5:1, 3.4:1, 3.3:1, 3.2:1, 3.1:1, 3.0:1, 2.9:1, 2.8:1, 2.7: 1, 2.6:1, 2.5:1, 2.4:1, 2.3:1, 2.2:1, 2.1:1, 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3: 1, 1.2:1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1 or 0.5:1.

The method may comprise culturing a population of lymphocytes comprising CD4⁺ T cells, CD8⁺ T cells, B cells and NK cells. In one embodiment, the ratio of CD4⁺ T to CD8⁺ T cells may be 5:1, 4.9:1, 4.8:1, 4.7:1, 4.6:1, 4.5:1, 4.4:1, 4.3:1, 4.2:1, 4.1:1, 4.0: 1, 3.9:1, 3.8:1, 3.7:1, 3.6:1, 3.5:1, 3.4:1, 3.3:1, 3.2:1, 3.1:1, 3.0:1, 2.9:1, 2.8:1, 2.7:1, 2.6: 1, 2.5:1, 2.4:1, 2.3:1, 2.2:1, 2.1:1, 2.0:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2: 1, 1.1:1, 1:1, 0.9:1, 0.8:1, 0.7:1, 0.6:1 or 0.5:1. The ratio of younger T-cell subsets to late- differentiated counterpart may be 10:1, 9.5:1, 9:1, 8.5:1, 8:1, 7.5:1, 7:1, 6.5:1, 6:1, 5.5: 1, 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5: 1 or 2:1.

In order to achieve sufficient therapeutic doses of T cell therapeutic, existing methods for manufacturing T cells are often subject to one or more rounds of stimulation, activation and/or expansion. In one embodiment, culture initiation and activation comprises seeding cell populations in a cell culture vessel, e.g., cell culture bag, GREX bioreactor, WAVE bioreactor, etc. and activating T cells through primary and costimulatory T cell signaling pathways. The cellular compositions may further be cultured in the presence of one or more additional growth factors or cytokines, e.g., IL- 2, IL-7, and/or IL- 15, or any suitable combination thereof.

In some embodiments, culture initiation comprises seeding a population of cells comprising T cells, e.g., PBMCs, in a cell culture vessel, at a desired density, e.g., 1-5 x 10⁶ cells/mL in a suitable cell culture medium containing one or more cytokines, primary stimulatory ligands, and co-stimulatory ligands. In another embodiment, the cytokines, stimulatory, and co-stimulatory ligands may be subsequently added to the PBMCs in the cell culture medium.

In some embodiments, the cells are seeded in a cell culture vessel comprising a suitable cell culture medium. Illustrative examples of suitable cell culture media include, but are not limited to T cell growth medium (TCGM; X-VIV0™15 supplemented with 2mM GlutaMAX™-!, lOrnM HEPES, and 5% human AB serum), CTS™ OpTmizer™ T Cell Expansion SFM (Life Technologies), CTS™ AIM V® Medium (Life Technologies), RPMI 1640, Clicks, DMEM, MEM, a-MEM, F-12, X-Vivo 15 (Lonza), CellGro® Serum-Free Medium (CellGenix), and X-Vivo 20 (Lonza) with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Cell culture media contemplated herein may further comprise one or more factors including, but not limited to serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, IL-21, GM-CSF, IL- 10, IL- 12, IL-15, TGF(3, and TNF-a.

In one embodiment, the cell culture medium may comprise one or more cytokines, e.g., such as IL-2, IL-7, and/or IL- 15, or any suitable combination thereof.

In one embodiment, the method comprises activating the population of lymphocytes. The activation of the population of lymphocytes may comprise contacting the population of lymphocytes with an anti-CD3 antibody or an antigen-binding fragment thereof and an anti-CD28 antibody or an antigen-binding fragment thereof. Alternatively, the activation may comprise contacting the population of lymphocytes with phorbol 12-myristate 13-acetate (PMA) and ionomycin.

The term, “activation” refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. In particular embodiments, activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are proliferating. Signals generated through the TCR alone are insufficient for full activation of the T cell and one or more secondary or costimulatory signals are also required. Thus, T cell activation comprises a primary stimulation signal through the TCR/CD3 complex and one or more secondary costimulatory signals. Costimulation can be evidenced by proliferation and/or cytokine production by T cells that have received a primary activation signal, such as stimulation through the CD3/TCR complex or through CD2.

As used herein, the term “proliferation” refers to an increase in cell division, either symmetric or asymmetric division of cells. In particular embodiments, “proliferation” refers to the symmetric or asymmetric division of T cells. “Increased proliferation” occurs when there is an increase in the number of cells in a treated sample compared to cells in a non-treated sample.

The T cell manufacturing methods contemplated herein comprise modifying immune effector cells and/or T cells to express an engineered T cell receptor (TCR) or chimeric antigen receptor (CAR). In particular embodiments, a population of cells comprising T cells is activated, modified to express an engineered TCR or CAR, and then cultured for expansion. The steps may take place in the same cell culture vessel or in different vessels. “Transduction” refers to the delivery of a gene(s) or other polynucleotide sequence, e.g., an engineered TCR or CAR to immune effector cells, including T cells, using a retroviral or lentiviral vector by means of viral infection. In one embodiment, retroviral vectors are transduced into a cell through infection and provirus integration. In certain embodiments, a target cell, e.g., a PBMC or a T cell, is “transduced” if it comprises a gene or other polynucleotide sequence delivered to the cell by infection using a viral or retroviral vector. In particular embodiments, a transduced cell comprises one or more genes or other polynucleotide sequences delivered by a retroviral or lentiviral vector in its cellular genome.

In one embodiment, the method comprises transducing the population of activated cells with a viral vector.

In some embodiments, the present invention provides T cells genetically engineered with vectors designed to express CARs that redirect cytotoxicity toward tumor cells. CARs are molecules that combine antibody-based specificity for a target antigen (e.g., tumor antigen) with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific anti-tumor cellular immune activity. As used herein, the term, “chimeric,” describes being composed of parts of different proteins or DNAs from different origins.

The CARs contemplated herein comprise an extracellular domain that binds to a specific target antigen (also referred to as a binding domain or antigen-specific binding domain), a transmembrane domain and an intracellular signaling domain. The main characteristic of CARs are their ability to redirect immune effector cell specificity, thereby triggering proliferation, cytokine production, phagocytosis or production of molecules that can mediate cell death of the target antigen expressing cell in a major histocompatibility (MHC) independent manner, exploiting the cell specific targeting abilities of monoclonal antibodies, soluble ligands or cell specific coreceptors.

In particular embodiments, a CAR comprises an extracellular binding domain including but not limited to an antibody or antigen binding fragment thereof, a tethered ligand, or the extracellular domain of a coreceptor, that specifically binds a target antigen that is a tumor- associated antigen (TAA) or a tumor-specific antigen (TSA). In certain embodiments, the TAA or TSA is expressed on a blood cancer cell. In another embodiment, the TAA or TSA is expressed on a cell of a solid tumor. In particular embodiments, the solid tumor is a glioblastoma, a non-small cell lung cancer, a lung cancer other than a non-small cell lung cancer, breast cancer, prostate cancer, pancreatic cancer, liver cancer, colon cancer, stomach cancer, a cancer of the spleen, skin cancer, a brain cancer other than a glioblastoma, a kidney cancer, a thyroid cancer, or the like.

In particular embodiments, the TAA or TSA is selected from the group consisting of alpha folate receptor, 5T4, a_v 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY- ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-llRcx, IL-13Ra2, Lambda, Lewis-Y, Kappa, Mesothelin, Mucl, Mucl6, NCAM, NKG2D Ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, and VEGFR2.

The T cell manufacturing platforms contemplated herein may comprise one or more rounds of T cell expansion. In some embodiments, populations of cells comprising T cells that have been activated and/or transduced are seeded into suitable cell culture vessels comprising cell culture medium suitable for expansion. In some embodiments, cells are seeded into a bioreactor and are periodically harvested without reseeding. In certain embodiments, cells are reseeded at a density to maintain log-phase growth of the cells.

In one embodiment, the method comprises expanding the population of lymphocytes.

Disclosed herein is a T cell therapeutic that has been obtained according to a method as defined herein.

In one embodiment, there is provided a T cell therapeutic comprising a population of lymphocytes comprising CD8⁺ T cells and CD4⁺ T cells. In one embodiment, there is provided a T cell composition comprising a population of lymphocytes comprising CD8⁺ T cells and CD4⁺ T cells.

Disclosed herein is a method of manufacturing a T cell therapeutic, the method comprising performing a method as defined herein.

In one embodiment, there is provided a method of manufacturing a cell therapeutic, the method comprising performing a method as defined herein.

Disclosed herein is a method of improving the quality and/or efficacy of a T cell composition, the method comprising performing a method as defined herein.

In one embodiment, there is provided a method of improving the quality and/or efficacy of a T cell therapeutic, the method comprising performing a method as defined herein.

Disclosed herein is a method of treating a subject in need, the method comprising administering to the subject a T cell therapeutic that is prepared according to a method as defined herein

In one embodiment, there is provided a method of treating a subject in need, the method comprising administering to the subject a T cell therapeutic that is prepared according to a method as defined herein

As used herein “treatment” or “treating,” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., cancer. Treatment can involve optionally either amelioration of, or complete reduction of, one or more symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. A “therapeutically effective amount” of a genetically modified therapeutic cell may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the T cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transduced therapeutic cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).

As used herein, “prevent,” and similar words such as “prevented,” “preventing” etc., indicate an approach for preventing, inhibiting, or reducing the likelihood of the occurrence or recurrence of, a disease or condition, e.g., cancer. It also refers to delaying the onset or recurrence of a disease or condition or delaying the occurrence or recurrence of the symptoms of a disease or condition. As used herein, “prevention” and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to onset or recurrence of the disease or condition.

A “prophylactically effective amount” refers to an amount of a genetically modified therapeutic cell effective to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is less than the therapeutically effective amount.

The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized in part by unregulated cell growth. As used herein, the term “cancer” refers to non-metastatic and metastatic cancers, including early stage and late stage cancers. The term “precancerous” refers to a condition or a growth that typically precedes or develops into a cancer. By “non-metastatic” is meant a cancer that is benign or that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site. Generally, a non-metastatic cancer is any cancer that is a Stage 0, 1, or II cancer, and occasionally a Stage III cancer. By “early stage cancer” is meant a cancer that is not invasive or metastatic or is classified as a Stage 0, I, or II cancer. The term “late stage cancer” generally refers to a Stage III or Stage IV cancer, but can also refer to a Stage II cancer or a substage of a Stage II cancer. One skilled in the art will appreciate that the classification of a Stage II cancer as either an early stage cancer or a late stage cancer depends on the particular type of cancer. Illustrative examples of cancer include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, colorectal cancer, lung cancer, hepatocellular cancer, gastric cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, brain cancer, non-small cell lung cancer, squamous cell cancer of the head and neck, endometrial cancer, multiple myeloma, rectal cancer, and esophageal cancer.

An “infectious disease” refers to a disease that can be transmitted from person to person or from organism to organism, and is caused by a microbial agent (e.g., common cold). Infectious diseases are known in the art and include, for example, hepatitis, sexually transmitted diseases (e.g., Chlamydia, gonorrhea), tuberculosis, HIV/AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.

An “autoimmune disease” refers to a disease in which the body produces an immunogenic (i.e., immune system) response to some constituent of its own tissue. In other words the immune system loses its ability to recognize some tissue or system within the body as “self’ and targets and attacks it as if it were foreign. Autoimmune diseases can be classified into those in which predominantly one organ is affected (e.g., hemolytic anemia and anti-immune thyroiditis), and those in which the autoimmune disease process is diffused through many tissues (e.g., systemic lupus erytnematosus). For example, multiple sclerosis is thought to be caused by T cells attacking the sheaths that surround the nerve fibers of the brain and spinal cord. This results in loss of coordination, weakness, and blurred vision. Autoimmune diseases are known in the art and include, for instance, Hashimoto's thyroiditis, Grave's disease, lupus, multiple sclerosis, rheumatic arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like. As used herein, the term “inflammatory disease” refers to either an acute or chronic inflammatory condition, which can result from infections or non-infectious causes. Various infectious causes include meningitis, encephalitis, uveitis, colitis, tuberculosis, dermatitis, and adult respiratory distress syndrome. Non-infectious causes include trauma (burns, cuts, contusions, crush injuries), autoimmune diseases, and organ rejection episodes.

An “immunodeficiency” means the state of a patient whose immune system has been compromised by disease or by administration of chemicals. This condition makes the system deficient in the number and type of blood cells needed to defend against a foreign substance. Immunodeficiency conditions or diseases are known in the art and include, for example, AIDS (acquired immunodeficiency syndrome), SCID (severe combined immunodeficiency disease), selective IgA deficiency, common variable immunodeficiency, X-linked agammaglobulinemia, chronic granulomatous disease, hyper-IgM syndrome, and diabetes.

By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, position or length that varies by as much 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, position or length.

As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

As used in this application, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes a plurality of agents, including mixtures thereof.

Throughout this specification and the statements which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.

Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

EXAMPLES

EXAMPLE 1

Key Reagents

The apheresis blood from healthy donors were a gift from the Health Sciences Authority (HSA), Singapore. Ficoll® Paque Plus (GE Healthcare, 17-1440-02) was used for peripheral blood mononuclear cells (PBMC) enrichment. The cell culture medium RPMI1640 (ThermoFisher, A1049101) and fetal bovine serum (FBS) (ThermoFisher, 10270106) were used for primary cell culture. In some experiments, human recombinant IL-2 (ThermoFisher, PHC0021) and anti-CD3/CD28 Dynabeads™ (ThermoFisher, 1116 ID) were supplemented for T cell expansion. Phorbol ester (PMA) (Sigma- Aldrich, P8139) plus ionomycin (Sigma- Aldrich, 19657) were used for cell stimulation to produce cytokines. All of the fluorescent antibodies were purchased from BD Biosciences or Biolegend except that the cytokine-detecting antibodies were from Miltenyi Biotec. Cytofix/Cytoperm™ reagent kit (BD Biosciences, 554714) was used for cell fixation and permeabilization before intracellular staining.

Peripheral blood mononuclear cells (PBMC) isolation and usage

The PBMC were isolated from apheresis blood of healthy donors by density gradient centrifugation. The enriched PBMC were counted and cryopreserved in medium (90% FBS and 10% DMSO) at 10 million cells/vial and stored in liquid nitrogen. For each experiment, the cells were recovered overnight in RPMI1640 supplemented with 10% FBS.

Cell sorting

PBMC were pre-stained with fluorescent anti-CD4 and anti-CD8 before sorted by Moflo Astrios (Fig. la). Of note, only the cells with the high expression level of CD8 marker were sorted as CD3⁺ CD8⁺ T cells because some NK cells also express diminished CD8. Also, anti-CD3 was not used for cell sorting experiments where the sorted cells would be subject to anti-CD3/CD28 engaged stimulation. The sorted cells were validated to have enough purity before reconstituted for co-culture assay (Fig. la).

Co-expansion assay

As briefly depicted in Fig. 1c, the sorted CD4⁺ T cells and CD8⁺ T cells were reconstituted at indicated ratios with final total cell numbers of 0.06 million per group. For the B/NK cells-spiked in group, instead additional 0.09 million B/NK cells were added into cell group with 1:1 of CD4⁺ T: CD8⁺ T. All of the cell groups were then cultured in 96- well plate with the ratio of anti-CD3/CD28 microbeads to cells at 3:1 and IL-2 concentration at 5 ng/ml. The culture medium would be half changed with fresh medium supplemented with 10 ng/ml IL-2 at day 3 and day 5. During expansion, the cell numbers in each group was counted to make sure that total cell numbers were below 0.3 million/well, otherwise split into more wells with 0.1 million/well. Data collection includes total nucleated cells (TNC), CD4/CD8 ratio, effector/memory composition, granzyme B and cytokine expression at indicated time points post activation.

Cell effector/memory phenotypes identification

There are two approaches to identify the T cell differentiated lineages. Roughly, as illustrated in Fig. 3a, they were classified as naive (TN, CD45RA^brigbtCCR7^brigbt), central memory (TCM, CD45RA^dim/_CCR7^dim), effector memory (TEM, CD45RA^dimA CCR7 ) and terminally differentiated effector (TEFF or TEMRA, CD45RA^bngbt CCR7 ). For further analysis, an in-depth gating strategy (Fig. 4a) was adapted wherein the TCM was dissected into CD45RA- TCM (TCMO) and naive phenotype-closest CD45RA⁺ TCM (TCMRA) while TEM with CD45RA expression as CD45RA^low TEM (TEMRA-1OW) and CD45RA^blgb TEM (TEMRA-M).

Cell functionality characterization

To determine the immune response of expanded T cells, cell of final products were removed of residual anti-CD3/CD28 microbeads and re-stimulated with or without 50 ng/ml PMA plus 1 pg/ml ionomycin for 2 hours. Intracellular staining was carried out for the detection of both granzyme B abundance and cytokine level in cells. Notably, the CD4 marker has a substantial downregulation post PMA stimulation, thus the CD3⁺ CD8 T cells were gated as CD3⁺ CD4⁺ T cells for data presentation.

Results

CD4⁺ T help is essential for optimal CD8⁺ T cells proliferation To study the role of heterotypic T-T interaction in T cell proliferation by CD3/CD28 engagement plus IL-2, CD4⁺ T cells and CD8⁺ T cells were purified while the remained population mainly consisting of B/NK cells (hereafter named as B/NK cells) was also collected (Fig. la, b) before co-culture at designed combination (Fig. 1c). Generally, CD4⁺ T cells help is necessary for optimal total T cells expansion (Fig. 2a, b). However, the mutual effects of CD4⁺ T, CD8⁺ T and B/NK cells on cellular proliferation show some distinctive characteristics. Help from CD4⁺ T cells is required for CD8⁺ T cells maximal proliferation (6-fold increase at day 7) in a ratio-dependent pattern while presence of B/NK cells significantly counteracts this ascent (Fig. 2c). In turn, CD8⁺ T cells moderately reduce CD4⁺ T cells expansion with negligible effect, which however is further inhibited by B/NK cells (Fig. 2d). The ratio of CD4/CD8 T cells gradually decreases during T cell expansion course (Fig. 2e), consistent with previous reports where anti-CD3/CD28 microbeads was used for T cell stimulation. The orchestrated growing dynamics within cell types results in final T cell products of not only different absolute cell numbers (Fig. 2a, b) but also discernable CD4/CD8 T cell composition (Fig. 2e). Of note, the changing rate of CD4/CD8 ratio during expansion positively correlated with its initial numbers of CD4⁺ T cells (Fig. 2f).

CD4⁺ T-CD8⁺ T crosstalk synergistically contributes to TCM pool maintenance

Further analyses of T cell differentiation lineages identify a distinctive phenotypic profile of cell products derived from different co-culture conditions or varied proliferation stages. Herein, according to the expression level of CD45RA/CCR7 during T cell lineage differentiation, in this work the T cells are classified as naive (TN, CD45RA^brigbt CCR7^brigbt), central memory (TCM, CD45RA^dim/_ CCR7^dim), effector memory (TEM, CD45RA^dimA CCR7 ) and terminally differentiated effector (TEFF or TEMRA, CD45RA^brigbt CCR7 ) (Fig. 3a). Obviously, help from CD4⁺ T cells enables the maximal maintenance of CD8⁺ TCM cells and less CD8 TEM (Fig. 3b). During the early activation/expansion stages (day 0 - day 7), when compared to CD8⁺ T cells culture alone, CD4⁺ T help regardless of the ratio of CD4/CD8 T cells contribute significantly to the generation of CD8⁺ TCM with a higher abundance of CCR7/CD45RA (Fig. 3c, d) and CD8⁺ TEM with increased expression level of CD45RA as well (Fig. 3d), suggesting production of more naive-like CD8⁺ TCM and also survival of terminally effector-like CD8⁺ TEM- B/NK cells, instead, tend to inhibit CD8⁺ T cells expansion (Fig. 3b) and moderately promote CD8⁺ T cells differentiation as indicated by a reduction of CCR7/CD45RA expression during the early expansion phase (Fig. 3c, d). In turn, CD8⁺ T cell co-existence does not affect the general effector/memory pool of CD4⁺ T cells (Fig. 3e). Notably, compared to CD4⁺ T cells culture alone, the abundance of CCR7 in CD4⁺ TCM is increased (Fig. 3f) and the level of CD45RA for both CD4⁺ TCM and CD4⁺ TEM are generally lower in the presence of CD8⁺ T cells along the entire expansion course (Fig. 3g), indicating generation of more memory-like CD4⁺ TCM and terminally effector-like CD4⁺ TEM- B/NK cells slightly increase CD4⁺ T cells differentiation with delayed recovery of CCR7 but increased CD45RA expression in CD4⁺ T cells (Fig. 3f, g)-

CD4⁺ T-CD8⁺ T crosstalk contributes to production of naive-like CD8⁺ TCM precursors

To get a clear insight into effector/memory subsets switch in different co-culture systems, TCM is further dissected into CD45RA- TCM (TCMO) and naive phenotype- closest CD45RA⁺ TCM (TCMRA) while TEM with CD45RA expression as CD45RA^low TEM (TEMRA-IOW) and CD45RA^hlgh TEM (TEMRA-H;) (Fig. 4a). As can be inferred, CD4⁺ T help contributes mainly to the maintenance of TCMRA and effect of which is CD4⁺ T abundance less related (Fig. 4b). Instead, CD8⁺ T cells favor the production of CD4⁺ TCMO in the period of initial activation/expansion phases (Fig. 4c).

CD4⁺ T-CD8⁺ T crosstalk synergistically contributes to optimal T cell functionality of final products

To determine the immune responding capabilities of expanded T cells, expression level of granzyme B (Gran B), IFN-y, IL-2 and TNF-a were determined by intracellular staining. The functionality of CD8⁺ T cells ex vitro can be regulated by the co-culture of CD4⁺ T cells. A higher ratio of CD4⁺ T/CD8⁺ T cells rather than CD8⁺ T cells culture alone have increased granzyme B expression in CD8⁺ T cells (Fig. 5a, b) while in turn CD8⁺ T cells also favor the granzyme B expression in CD4⁺ T cells in a ratio-dependent manner (Fig. 5c). In addition, CD4⁺ T help is critical for the enhanced total IL-2 secretion but reduced total IFN-y/TNF-a secretion of CD8⁺ T cells (Fig. 5d) and vice versa in spite of a less extent (Fig. 5e). A closer look at the cytokine-releasing multifunctionality of final T cell products confirms these findings (Fig. 5f), especially that CD8+ T cells in the presence of CD4+ T cells co-culture have more IL-2 expression but less IFN-y-related polyfunctionality response (e.g. IFN-y, IFN-y/TNF-a, and IFN-y/IL- 2/TNF-a). The remained non-T cell proportion in peripheral blood mononuclear cells (PBMC), mainly NK and B cells, instead drives T cell differentiation which functionally manifests more IFN-y but less IL-2/TNF-a secretion, as well as decreased granzyme B expression (Fig. 5b-f). Taken together, these results indicate that co-culture of CD4⁺ T and CD8⁺ T cells synergistically contributes to the formation of cell pools with naive- like cytokine -releasing potency while maintaining highly effector granzyme B expression.

Discussion

In summary, the expansion potency of CD8⁺ T cells ex vitro can be regulated by the coculture of CD4⁺ T cells. A higher ratio of CD4⁺ T/CD8⁺ T cells rather than CD8⁺ T cells culture alone is critical for optimal CD8⁺ T cell expansion (> 6-fold higher at day 7) while the CD4⁺ T cells proliferation is nearly unaffected.

Compared to CD8⁺ T cells culture alone, help from CD4⁺ T cells contributes to the maintenance of less-differentiated central memory CD8⁺ T cells (CD8⁺ TCM) with high expression of both CCR7 and CD45RA which is phenotypically more naive-like. In turn, CD8⁺ T cells show no apparent effects on the percentages of CD4⁺ T effector/memory components during co-culture. However, the CD4⁺ TCM in the presence of CD8⁺ T cells, however, has a slightly higher expression of CCR7 but a significantly lower expression of CD45RA, suggesting cell pool of more memory-like CD4⁺ TCM-

Compared to CD8⁺ T cells culture alone, help from CD4⁺ T cells reduces the formation of more-differentiated cell subsets such as effector memory (CD8⁺ TEM) and effector memory with low CD45RA expression (CD8⁺ TEMRA-1OW). The functionality of CD8⁺ T cells ex vitro can be regulated by the co-culture of CD4⁺ T cells. A higher ratio of CD4⁺ T/CD8⁺ T cells rather than CD8⁺ T cells culture alone is critical for the generation of functionally less-differentiated CD8⁺ T cells which express less IFN-y/TNF-a but more IL-2, as well as increased granzyme B expression. Similarly, CD4⁺ T cells in the presence of CD8⁺ T cells also tend to secret less IFN-y/TNF-a but enhanced granzyme B expression. These findings indicate co-culture of CD4⁺ T and CD8⁺ T cells synergistically contributes to the formation of cell pools with naive-like cytokinereleasing potency while maintaining highly effector granzyme B expression.

The remaining non-T cell proportion in peripheral blood mononuclear cells (PBMC), mainly NK and B cells, instead counteracts the expansion of both CD4⁺ T and CD8⁺ T cells, and drives T cell differentiation as well which functionally manifests more IFN-y but less IL-2/TNF-a secretion, as well as decreased granzyme B expression. The cell products derived from a high ratio of CD4/CD8 T cells can still maintain relative high number of CD4⁺ T cells. This is important for future adoptive transfer considering that help from CD4+ T cells in vivo is also required for improved therapeutic efficacy.

EXAMPLE 2

Key Reagents

The apheresis blood from healthy donors were a gift from the Health Sciences Authority (HSA), Singapore. Ficoll® Paque Plus (GE Healthcare, 17-1440-02) was used for peripheral blood mononuclear cells (PBMC) enrichment. The cell culture medium RPMI1640 (ThermoFisher, A 1049101) with fetal bovine serum (FBS) (ThermoFisher, 10270106) were used for the culture of primary cells and Raji (human B lymphoblastoid cell line). In some experiments, human recombinant IL-2 (ThermoFisher, PHC0021) and anti-CD3/CD28 Dynabeads™ (ThermoFisher, 11161D) were supplemented for T cell expansion. Phorbol ester (PMA) (Sigma- Aldrich, P8139) plus ionomycin (Sigma- Aldrich, 19657) were used for cell stimulation to produce cytokines. All of the fluorescent antibodies were purchased from BD Biosciences or Biolegend except that the cytokine-detecting antibodies were from Miltenyi Biotec. Cytofix/Cytoperm™ reagent kit (BD Biosciences, 554714) was used for cell fixation and permeabilization before intracellular staining. Peripheral blood mononuclear cells (PBMC) isolation and usage

The PBMC were isolated from apheresis blood of healthy donors by density gradient centrifugation. The enriched PBMC were counted and cryopreserved in medium (90% FBS and 10% DMSO) at 10 million cells/vial and stored in liquid nitrogen. For each experiment, the cells were recovered overnight in RPMI1640 supplemented with 10% FBS before downstream usage.

Label-free cell sorting

For label-free cell sorting, overnight-recovered PBMC were briefly washed and resuspended in in cold phosphate buffer (PBS) before equally sorted into SSC^hlgh and SSC^low populations on Moflo Astrios (Beckman Coulter). The sorted cells were stained to characterize the composition of CD3⁺ CD4⁺ T/CD3⁺ CD8⁺ T, NK/B cell, or TN/TCM/TEM/TEMRA- The excitation wavelength for generating SSC (488/6-nm filter) and FSC (488/6-nm filter) was 488 nm. Where indicated, a combined excitation of 488 nm (488/6-nm filter) and 405 nm (405/6-nm filter) was adopted to sort lymphocytes of different SSC or FSC.

T cell activation and expansion in vitro

Sorted cells were rested in culture medium for 4 hours before activation. Cells were seeded at 0.06-0.08 million in 96-well plate and activated by anti-CD3/CD28 Dynabeads™ (3:1 bead-to-cell ratio) (Gibco) with 5 ng/ml IL-2 (Gibco) supplemented. In some experiments, the expanded T cells were re-stimulated by anti-CD3/CD28 microbeads at 1: 1 bead-to-cell ratio under 5 ng/ml IL-2, or 5ng/ml each of IL-7 and IL- 15 (PeproTech). Half of the culture medium was replaced with fresh medium containing IL-2 (5 ng/ml) every two days during initial activation/expansion stages (day 3 to day 7) and daily during late expansion phases (day 7 to day 11 or more where indicated). Cell numbers were counted by trypan blue exclusion and split into approximately 0.1 million per well. Cell phenotypes and functionality were determined at indicated time points post activation. Cell effector/memory lineages identification

In this study, two approaches are shown to identify the T cell differentiated lineages. For resting PBMC, as illustrated in Fig. 7a, they can be roughly classified as naive (TN, CD45RA^brigbt CCR7^brigbt), central memory (TCM, CD45RA^dim/_ CCR7^dim), effector memory (TEM, CD45RA^dimA CCR7 ) and terminally differentiated effector (TEFF or TEMRA, CD45RA^brigbt CCR7 ). For activated PBMC, instead an in-depth gating strategy (Fig. 16a) was adapted wherein the TCM was dissected into CD45RA- TCM (TCMO) and naive phenotype-closest CD45RA^blgb TCM (TCMRA-M) and memory phenotype-closest CD45RA^low TCM (TCMRA-1OW) while TEM with CD45RA expression as CD45RA^low TEM (TEMRA-IOW) and CD45RA^blgb TEM (TEMRA-H;)-

Cell functionality characterization

To determine the immune response of expanded T cells, except specially indicated, cells of final products were removed of residual anti-CD3/CD28 microbeads and restimulated with or without 50 ng/ml PMA plus 1 pg/ml ionomycin for 2 hours. Intracellular staining was carried out for the detection of both granzyme B abundance and cytokine level in cells. Notably, the CD4 marker has a substantial downregulation post PMA stimulation, thus the CD3⁺ CD8 T cells were gated as CD3⁺ CD4⁺ T cells for data presentation. Cytokine-expression multifunctionality was analyzed as demonstrated in Fig. 9c.

In vitro migration assay

CCL19 and CCL21 (300 ng/ml each) (PeproTech) in 100 pl culture medium were added to the lower chamber of 96-well transwell plate (5 pm porosity) (Corning). Equal numbers of expanded T cells (0.1 million) as indicated were seeded into the upper chamber in 80 pl culture medium. After five hours, the numbers of cells migrating to the lower chamber were counted by trypan blue exclusion.

CART19 generation Label-free sorted cells were rested for 4-12 hours before activated by anti-CD3/CD28 microbeads (2:1 bead-to-cell ratio) for additional 24 hours in 24-well plates in AIM-V medium (2% human AB serum). Stimulated cells were then transduced with lentiviral vector encoding green fluorescent protein (GFP)-fused CD19-directed CAR construct (CAR.19-4-lBBz-GFP) at a multiplicity of infection (MOI) of 5.0. RetroNectin-coated 24-well plate and spinoculation at 1000 g for 60 min at 25°C were used to enhance transduction. The viruses were removed after 3 days post transduction and the cells were expanded for 9-10 more days in G-Rex 24-well plates in AIM-V medium supplemented with 5 ng/ml IL-2. Half of the culture medium was changed with fresh medium containing 5 ng/ml IL-2 at an interval of 3-4 days. The expanded CART 19 cells were harvested and cryopreserved for future experiments.

Statistical methods

All values with error bars are reported as mean ± SEM. Statistical analysis was performed using GraphPad Prism software. Unpaired two-tailed /-test was used to evaluate statistical significances between two groups. Where indicated, paired two- tailed /-test was used instead.

Results and Analysis

Side-scatter (SSC) based selection of lymphocyte populations and naive/memory subpopulations

Firstly, it was shown that the side-scattering distribution of CD4⁺ T/CD8⁺ T/NK/B cells within the total lymphocyte population, despite normally presenting to be very homogenous (Fig 6. a, b, left panel), can still be discernable post further dissection. Generally, in the pre-stained and fixed PBMCs (Fig. 6a, b), CD4⁺ T cells and B cells tend to locate within the SSC^low cell cluster while CD8⁺ T cells and NK cells instead tend to be in the SSC^hl group (Fig. 6a, b). The inventors therefore directly sorted the lymphocytes merely based on their side-scattering intensity into SSC^hl and SSC^low populations and further determined the cell composition by immunostaining. As depicted in Fig. 6c, a similar side-scattering pattern was obtained for both CD4⁺ T/CD8⁺ T/NK cells (top panel) and NK/B cells (bottom panel). Of note, the B cells proportion within sorted SSC^hl and SSC^low cell groups is more comparable despite a trend of more B cells in SSC^low group (Fig.6a, c), indicating absence of fixation may slightly reduce the scattering resolution of B cells against other cell types.

Unexpectedly, the naive/memory T cells also found to manifest a very distinctive sidescattering profile (Fig. 7a). Either for pre-stained and fixed PBMCs (Fig. 7b) or presorted cell samples before staining (Fig. 7c), also regardless of CD4⁺ or CD8⁺ T cells, the SSC^low cell cluster contains more naive T cells (TN) while the SSC^hl group consists of mainly differentiated cell subsets such as TCM, TEM and TEFF-

Discernable functionality of lymphocytes with different side-scattering intensity

Of note, unequal expression level of CCR7/CD45RA was observed among T cells with different side-scattering intensity (Fig. 8a, b). Especially, TCM with low side-scatter has a higher abundance of both CCR7 (Fig. 8a) and CD45RA (Fig. 8b) when compared to that with higher side-scatter, indicating cell pool of more naive-like central memory T cells in the SSC^low cluster.

Naive T cells show more inert in immediate cytokine-secreting polyfunctionality compared to memory T cells. Thus, it should be anticipated that the cytokine-expressing profile of cells with low side-scatter which consists of more TN and naive-like TCM could be less polyfunctional in responding to external stimulation. To verify this, the lymphocytes were pre-sorted by side-scattering intensity into two populations and further stimulated with PMA plus ionomycin for 4 hours before immunostaining for polyfunctional analysis (Fig. 8c). Obviously, the T cells among SSC^low cell cluster response generally slower in polyfunctional cytokines expression but with stronger IL- 2 expression (Fig. 8d, e), indicating superior proliferative capacity. The large amount of IFN-y expression in non-T cell of SSC^hlgh group resulting from a high proportion of NK cells (Fig. 8f)

Side-scatter is a robust biophysical property for lymphocytes segregation The SSC is measured at a wider angle from the light beam propagation axis and intensity of which generally reflects the object granularity. Thus, it was sought to understand possible factors that may affect the stability of this method. Firstly, it was shown that the findings aforementioned is not artificially introduced by either staining procedures (Fig. 9a) or immunofluorescence compensation wherein only single fluorescence- labeled antibody was used (Fig. 9b). Also, the general tendency of side-scattering distribution for both CD4⁺ T/CD8⁺ T cells (Fig. 9c) and TN/TCM/TEM/TEFF (Fig. 9d) remain unaltered with fixation/permeabilization treatment despite that slight disturbance does occur for the side-scattering profile of CD4⁺ T and CD8⁺ T cells post fixation/permeabilization (Fig. 9c).

Attempts using other light-scattering parameters have also been provided here for label- free lymphocytes enrichment. First of all, it was shown that, for resting PBMC, forwardscatter only is inefficient to segregate either CD4⁺ T or CD8⁺ T cells (Fig. 10a) or their naive/memory counterparts (Fig. 10c) despite that it does work for NK and B cells (Fig. 10b). Also, auto-fluorescence, being equal to FITC channel with excitation/emission wavelength as 488 nm/ 526 nm for cell sorting (Fig. Ila, b), is inefficient to segregate CD8⁺ T cells (Fig. 11c) and incapable of discriminating naive/memory T cell lineages (Fig. 1 Id). In addition, considering that the smaller wavelength should return a more detailed scattering pattern and provide more information about the morphometric structure of the cells, UV laser with smaller wavelength (405 nm) is speculated to have a better scattering resolution than blue laser (488 nm). However, as depicted in Fig. 7, compared with the side-scattering profile of blue laser-excited lymphocytes (Fig. 6c and Fig. 7c), double side-scatter (UV405/B488) (Fig. 12a) based cell sorting strategy does not improve the general segregation efficiency of CD4⁺ T/CD8⁺ T cells (Fig. 12b), NK/B cells (Fig. 12c) and naive/memory T cells (Fig. 12d) as well. Of note, neither side-scatter (Fig. 13a) or forward-scatter (Fig. 13b ) nor auto-fluorescence (Fig. 13c) can be applicable for the segregation of fast expanding T cells that were pre-activated by anti-CD3/CD28 engagement, cells of which manifest a large size compared to that of resting condition. These findings suggest that side-scattering property can be an intrinsic and robust biophysical characteristic to reflect the granularity and contents differences of different lymphocyte populations or subpopulations. Nevertheless, size of over-enlarged lymphocytes could be a confounding factor that might mitigate the detailed information of scattering pattern intrinsically manifested by different lymphocyte populations among resting/quiescent status.

Re-constituted lymphocytes by side-scatter improves the yields of T cell products with selectable CD4/CD8 T cell ratio

As demonstrated previously, both proliferative capacity and functional acquisition of T cells are affected by not only T-T communication but also T-B/NK interaction, it is interesting to investigate how compositional difference of starting T cell materials as redistributed solely by side-scatter may affect the diversity of their final T cell products.

Basically, in responding to anti-CD3/CD28 stimulation, SSC^low cells consisting of more TN cells and naive-like TCM cells (Fig. 7c) proliferate dramatically faster (an average of 2.4-fold at day 7 and 4-fold at day 11) than those from SSC^hlgh population which instead has more already differentiated cell composition (e.g. TCM/TEM/TEFF) (Fig. 14a). This is more evident by looking at the expansion dynamics of CD4⁺ T or CD8⁺ T cells independently with more that 3-fold increase for CD4⁺ T and 5-fold increase for CD8⁺ T at day 11 (Fig. 14b). The relatively higher percentage of B/NK cells in the SSC^hlgh population would further counteract the expanding potency of T cells of this group. (Fig. 7a). Unexpectedly, despite being with a lower proportion of B/NK cells and differentiated T cell subpopulations of the reconstituted SSC^hlgh+low cell group, its expansion potency appears to be equivalent to that of SSC^hlgh population (Fig. 14a), possibly due to the generally reduced cell proliferative capacity caused by differentiation-promoting effects of existing effector/memory cells on naive cells. Of note, CD8⁺ T cells grow faster than CD4⁺ T cells during co-expansion as indicated by a gradual decrease of CD4/CD8 T cells, a shaper change of which was observed for cells of lower side-scatter initially with a higher ratio of CD4/CD8 T cells which however continues to maintain a relatively higher ratio of CD4/CD8 T compared to the SSC^hlgh or SSC^hlgh+low group (Fig. 14c). This phenomenon supports the idea that CD4⁺ T help is critical for maximal CD8⁺ T cells expansion. Pre-separated TN from TM favors the selection of phenotypically optimal T cell products post expansion

It was next sought to unravel phenotypic traits of the cell products generated from cell materials of different side-scattering intensity. Herein, similar to what has been depicted in Fig. 6a, the TCM is further divided into CD45RA- TCM (TCMO) and naive phenotype- closest CD45RA^low TCM ( CMRA-1OW) and CD45RA^hlgh TCM (TCMRA-H;) while TEM with CD45RA-regaining as CD45RA^low TEM (TEMRA-1OW) and CD45RA^hlgh TEM (TEMRA-M) (Fig. 15a).

It was demonstrated that the SSC^low and SSC^hl cells show not only distinctive phenotypic composition before expansion but also maintained component differences along the whole course of expansion procedure regardless of CD4⁺ or CD8⁺ T cells. However, the reconstituted SSC^hlgh+low cells are generally less discernable from the SSC^low group during the late expansion stages (Fig. 15b, c). The effector/memory composition that enables segregation of SSC^low- and SSC^hl-derived cell products changes proceeding with cell activation/proliferation. At day 0 before activation, both CD4⁺ T and CD8⁺ T cells from the SSC^low group have a significantly higher percentage of TN but less differentiated TCMO for CD4⁺ T cells (Fig. 15b) or FEMRA-M for CD8⁺ T cells (Fig. 15c). Also, CD4⁺ T cells from SSC^low group have a relatively higher number of TcMRA-hi despite which only accounts for a minor portion in the starting materials (Fig. 15b). Post initial activation at day 2, a higher proportion of TN and TCMRA-1U can be positively assigned to the SSC^low group for both CD4⁺ T and CD8⁺ T cells. Of note, the TEMRA-hi subset occurs within SSC^low group post activation and tends to gradually accumulate in the SSC^low group till end of cell expansion. In contrast, TEMRA-M in both SSC^hlgh and SSC^hlgh+low groups have a apparent reduction post activation. At day 7 and day 11, a combination of reduced TEMRA-IOW but increased TCMRA-1U collectively characterizes the phenotypic traits of SSC^low cells-derived CD4⁺ T descendants. For CD8⁺ T cells of the SSC^low group from day 7 to day 11, instead, a lower proportion of TCMRA-1OW and TEMRA-1OW together with increased TcMRA-hi and TEMRA-M could be discriminated from that of SSC^hlgh cluster. Importantly, it was shown that the burst increase of FCMRA-M at the beginning of T cell activation/expansion (e.g. day 0-2) is well correlated with the initial proportion of TN, and tendency of which maintains during the following expansion procedures (e.g. day 7 and day 11) (Fig. 15d) in spite of a general decrease of this cell subsets in all cell groups post extensive expansion (Fig. 15b, c).

Taken together, these findings suggest that, although similar effector/memory composition of final T cell products is normally observed post extensive expansion in vitro, pre-enriched less-differentiated cells simply by side-scatter enables the maintenance of phenotypically more naive-like TCM (TCMRA-M) and paradoxically a gradual accumulation of terminally effector-like TEM (TEMRA-H), suggesting a favorable environment for the survival of not only memory but also terminally differentiated cell progenies.

Pre-separated TN from TM favors the selection of functionally less-differentiated T cell products post expansion

Of great importance are, in the context of T cell fast expansion, whether the cytokinereleasing functionality of a given T cell product could be correlated to its phenotypic traits wherein common antigens CCR7/CD45RA are used for phenotyping, and how the expansion pattern aforementioned may affect cellular functionality.

Generally, the SSC^low cells-derived products response faster to short period of PMA stimulation in secreting more IL-2 and TNF-a but less IFN-y (Fig. 16a-c). Regarding of polyfunctionality, SSC^low cells-derived descendants also produce the highest amount of early effector cytokines (e.g. IL-2⁺ and IL-2⁺ TNF-a⁺) while SSC^hlgh cells-derived products incline to generate later effector cytokines (e.g. IFN-y⁺, IFN-y⁺ IL-2⁺ and IFN- y⁺ TNF-a⁺) (Fig. 16d, e). Of note, the composition of total triple-cytokines positive (IFN-y⁺ IL-2⁺ TNF-a⁺) cells is more fluctuating among different cell groups due to a mixture response of total CD4⁺ T and CD8⁺ T cells. This tendency of global cytokineexpressing profile is applicable for CD4⁺ T cells from all of the tested cell groups at both day 7 and day 11 (Fig. 16f). While cytokine-expressing functionality shows to be adequate to discriminate the CD4⁺ T cells products descended from SSC^low and SSC^hlgh cell clusters (Fig. 16g), inclusion of phenotypic characteristics further improves the segregating resolution for some samples (e.g. SSC^hlgh from donor 1) (Fig. 16h). Obviously, the cytokine-secreting profile of CD4⁺ T cells is relatively consistent with their subtle formation of effector/memory pools (Fig. 15b, c), especially the expressing magnitude of early effector factors (e.g. IL-2⁺/IL-2⁺ TNF-a⁺) is better associated with the increased abundance of early memory CD4⁺ T precursors (e.g. TCMRA-M) versus those more-differentiated ones (e.g. TCMO/TEM and TEMRA-1OW) (Fig. 16h).

As for CD8⁺ T cells, they present a more diverse cytokine profile (Fig. 16i). CD8⁺ T cells within the SSC^low-derived products at day 7 do lean towards producing more IL- 2/TNF-a but less IFN-y (Fig. 16i, left panel) despite contrast of which is less apparent than CD4⁺ T cells (Fig. 16f). However, at day 11 when most of the cells have experienced extensive proliferation, CD8⁺ T cells derived from the SSC^low group from one of the donors (donor 3) manifest a trait of comparable or even higher expression of IFN-y-included polyfunctional cytokines compared to that of SSC^hlgh group (Fig. 16i, right panel), resulting in a less-discernable clusters of these cell groups (Fig. 16j , bottom panel) but can still be distinctive when phenotypic properties were considered together (Fig. 16k), for example, the increased ratio of TCMRA-M to TCMRA-IOW can be meaningful to discriminate SSC^low- and SSC^hlgh-derived CD8⁺ T cells (Fig. 16k, bottom panel). This unexpected discrepancy is more likely related to a higher expansion magnitude of CD8⁺ T cells and dramatic reduction of CD4/CD8 ratio in SSC^low population from donor 3 (Fig. 14b, c), leading to substantially accumulated terminal effector-like CD8⁺ TEMRA-M cells in the SSC^low group (Fig. 15c). However, global help from CD4⁺ T cells which continuously possess less-differentiated cytokine-expressing functionality among the SSC^low group could potentially harmonize the immune response of final T cell products to be more naive- or memory-like even in the scenario of CD8⁺ T cells over- activation/differentiation post extensive expansion (Fig. 16d, e), which partially involving the feedback regulation of the growing numbers of CD8⁺ T cell in promoting formation of less-differentiated CD4⁺ T cell pool. Also, these findings suggest that combinatory analysis of phenotypic profile and cytokine-expressing capacities could be more informative in understanding therapeutic potential of T cell products.

Increased TCMRA-W derived from the SSC^low cluster resembles TscM-like TCM precursors It was next sought to define the natural characteristics of TCMRA-H; subset, significantly accumulated in cells derived from SSC^low group, that falls in the conventional central memory gate of the cell products (Fig. 15a). Firstly, TcMRA-hihad a higher expression of canonical memory marker CD45RO than TN but lower than TCMRA-IOW and TCMO (Fig. 17a-d). The expression of CD95 (a marker used to distinguish TSCM from TN) was also higher in TCMRA-M compared to TN before activation but became similar post expansion. In addition, there was an enhanced and more comparable expression of persistence- associated marker CD27 in both TCMRA-M and TN, especially for CD8⁺ T subtype (Fig. 17c-d), Finally, with regard to inhibitory or senescent factors, the expression of CD57 and LAG-3 was similar or more comparable between CD8⁺ TCMRA-H; and CD8⁺ TN but less than other CD8⁺ T subsets at resting state (Fig. 17c). These findings suggest that TcMRA-hi is phenotypically distinct from TN and other TCM subsets, and resembles the central memory precursor with Tscwlike phenotypic traits (CCR7⁺ CD45RA^hlgh CD95⁺ CD28⁺ CD27⁺), which could be taken into consideration in the evaluation of therapeutic T-cell products.

It was further shown that the abundance of TCMRA-M throughout the expansion course was well correlated with initial proportion of TN (Fig. 17e). In contrast, the presence of more-differentiated CD4⁺ TCMO (Fig. 17f) and CD4⁺ TCMRA-IOW (Fig. 17g) in starting cell materials tended to counteract the generation of CD4⁺ TCMRA-H;- Likewise, a higher number of CD8⁺ TCMRA-IOW (Fig. 17g) and CD8⁺ TEMRA (Fig. 17h) were more likely to reduce the production of CD8⁺ TCMRA-H;- Taken together, the pre-enrichment of TN and early TCM cells simply by SSC enables a favorable generation of TscM-like TCMRA-M.

SSC^low-derived CART19 possesses superior phenotypes and functionality in vitro

Next, it was tested how this SSC-derived younger T-cell subsets enrichment can be adopted for the production of CART 19 cells. It was found that cells derived from SSC^low cluster had a higher lentiviral transduction efficiency (Fig. 18a) but also gradually enhanced transgenic stability over the two- weeks’ expansion course in vitro (Fig. 18a, b). In addition, there was a favorable CART19 composition produced from the SSC^low group, characterized by a relatively higher proportion of less-differentiated CART subsets (e.g., TN, TOMRA-M and/or TCMRA-IOW) but less late-differentiated ones (e.g., T_EMRA-I_OW and/or TCMO) (Fig. 18c), also reflected on the increased cell numbers of TN, cMRA-hi, CMRA-1OW as well as TEMRA-M (Fig. 18d). In fact, compared to total unsorted lymphocytes, the sorted SSC^low cells that is equal to half of the unsorted one had already achieved a higher number of TN and TCMRA-M for some donor samples, or to some less extent in part of the tested donor samples in which a comparable amount of these CART19 subsets could be observed when combined with SSC^hlgh group. Moreover, SSC^low-derived CART 19 possessed higher co-stimulatory receptors like CD27 and CD28 (Fig. 18e). In line with this, these cells were endowed with not only superior immediately target-killing potency (Fig. 18f-h) but also stronger proliferative capability in response to multiple stimulation by Raji targets (Fig. 18i), together with a balanced CD4/CD8 ratio. These results imply the potential utility of using SSC-preselected T- cell subsets for long-term control of tumor progress in vivo.

Discussion

In summary, the side-scatter (SSC) can be used to segregate lymphocytes into subpopulations which contain various composition of CD4⁺ T/CD8⁺ T/NK/B cells with the cell cluster of low SSC (SSC^low) has a significantly higher number of CD4⁺ T cells and B cells but less NK cells. In contrast, the cell cluster of high SSC (SSC^hlgh) consists of more CD8⁺ T cells and NK cells.

SSC is efficient to separate less-differentiated T cell subsets especially naive T cells (TN) and naive-like central memory precursors with high CD45RA expression (TCMRA- hi) from those more-differentiated cells including central memory T cells with lower or negative CD45RA expression (TCMRA-IOW or TCMO), effector memory T cells (TEM) and terminal effector T cells (TEMRA).

This has been successfully applied to T cell manufacturing for the purpose of improved immunotherapy. Briefly, the whole lymphocytes were pre-segregated simply by SSC intensity into two populations, the SSC^low cluster consists of more TN and TCMRA-M and higher CD4/CD8 ratio while SSC^hlgh group has more differentiated T cell subsets (e.g. TCMRA-IOW, TEM and TEMRA) with lower ratio of CD4/CD8 but more NK cells. SSC^low cells show not only increased product yields (> 3-fold increase for CD4⁺ T and > 5-fold increase for CD8⁺ T at day 11) but also improved quality of final T cell product which possesses a higher proportion of central memory cells and functionally manifests memory- or naive-like cytokine-expressing capability even post extensive expansion ex vivo. Adoptive transfer of less-differentiated T cells are gradually recognized to provide better protective potential than their more-differentiated counterparts. Therefore, the invention described here resorting to side-scatter is a feasible and cost-effective approach to achieving this goal without the need of immunomagnetic pre-selection. Meanwhile, this strategy also enables production of two batches of cell products (SSC^low- or SSC^hlgh-derived) with distinctive cellular quality simply from one packet of apheresis product.

Finally, the concept can be useful for immunotherapies using allogeneic NK cells as effector source, a rising approach for cancer therapy. The majority of NK cells can be assigned to the cell group of high side-scatter, which would be valuable if antibody-free untouched methods are preferred for NK cells enrichment to reduce cost and improve cell transformation efficiency.

Ex vivo stimulated immune cells either with specific antigens or regulatory factors (e.g. cytokines or pharmaceutical compounds) could be an idea source for cancer therapy or alternative regimen for vaccination. Thus, the method described herein enables separation of TN/TCMRA-M from those more differentiated counterparts, and usage of these sorted cells for direct ex vivo stimulation before reinfusion could be an interest area to explore.

T cells characteristic of more memory-like phenotypes (e.g, CD27⁺ CD45RO- CD8⁺ T) before CAR T engineering were associated with better outcome in leukemia patients. CART19 cells derived from SSC^low group were manifested with these favourable attributes. One is the significantly elevated CAR TCWRA-H! subpopulation (CCR7⁺ CD45RA^hlgh CD95⁺ CD28⁺ CD27⁺), which resembles Tscw-iike cells reported to be a critical cell subpopulation in persistent immune memory. The other is the relatively higher CD4/CD8 CAR T-cell ratio before infusion. CD4⁺ T help is critical for optimal CD8⁺ T expansion and early TCM pool maintenance even in the absence of specific cognate antigens. CD4⁺ CAR T was reported to be better than CD8⁺ CAR T with regard to its long-lasting activity in inhibiting solid tumors. Use of defined ratio of CD4/CD8 CAR T products has also seen preliminary success in both manufacturing and antileukemic studies. Hence, it would be of great interest to study whether pre-designed CD4/CD8 T-cell ratio before CAR T manufacture may further potentiate therapeutic efficacy when combined with the defined ratio of CD4/CD8 CAR T cells for infusion. This could be more clinically meaningful considering that patients with some tumors or chronic diseases tend to manifest a decreased ratio of CD4/CD8 T cells.

Much effort has been devoted to improving T cell manufacturing processes for better immunotherapy. For example, shortening the time frame of T cell expansion from around two weeks to a few days or even without expansion in so called FasT CAR-T technology to prevent CAR T exhaustion due to overexpansion, using homeostatic cytokine cocktails (e.g., IL-7 and IL- 15) to maintain pools of less-differentiated T cells. Definitely, the method can be flexibly adapted into these manufacturing workflows.

In conclusion, this study represents a significant advance in the development of alternative CAR T-manufacturing protocols for improved therapeutic index. The results strongly suggest that, the generation of a favorable CAR T product with regard to its persistent activity in vivo results from not only a high proportion of TN and early TCM but also a relatively high ratio of CD4/CD8 T cells before CAR T engineering, which can be simply achieved via SSC-relied T cell pre-segregation. This work could also have opened a new window for the development of integrated microfluidic systems aimed to improve the characterization of T-cell subsets, and label-free biomedical diagnosis based on the abnormal change of T-cell differentiation state.

Claims

1. A method of preparing a cell therapeutic, the method comprising the step of a) culturing a population of lymphocytes comprising CD8⁺ T cells and CD4⁺ T cells; wherein the method comprises selecting for a population of lymphocytes prior to or following step a) based on side scatter.

2. The method of claim 1, wherein the population of lymphocytes have a side scatter associated with improved quality and/or efficacy when used as a T cell therapeutic.

3. The method of claim 1 or 2, wherein the population of lymphocytes has a low side scatter as compared to a reference.

4. The method of any one of claims 1 -3, wherein the population of lymphocytes with a low side scatter comprises an enriched population of less differentiated T cells.

5. The method of any one of claims 1 -4, wherein the population of lymphocytes with a low side scatter comprises an enriched population of CD4⁺ T cells and B cells.

6. The method of claim 1 or 2, wherein the population of lymphocytes has a high side scatter as compared to a reference.

7. The method of claim 6, wherein the population of lymphocytes with a high side scatter comprises an enriched population of NK cells.

8. The method of any one of claims 1-7, wherein the method comprises selecting for a population of lymphocytes from a sample obtained from a subject.

44 The method of any one of claims 1-8, wherein the method comprises activating the population of lymphocytes. The method of any one of claims 1-9, wherein the method comprises transducing the activated population with a viral vector. The method of any one of claims 1-10, wherein the method comprises expanding the population of lymphocytes. A method of preparing a cell therapeutic, the method comprising the step of a) culturing a population of lymphocytes comprising CD8⁺ T cells and CD4⁺ T cells; wherein the population of lymphocytes comprises CD8⁺ T cells and CD4⁺ T cells at a pre-determined ratio. A method of improving the quality and/or efficacy of a cell therapeutic, the method comprising performing a method according to any one of claims 1 to 12. A cell therapeutic composition that is prepared according to a method of any one of claims 1-12. A method of treating a subject in need, the method comprising administering to the subject a cell therapeutic that is prepared according to a method of any one of 1 to 12.

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