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Efficient derivation of chimeric-antigen receptor-modified TSCM cells

Chimeric-antigen receptor (CAR) T-cell immunotherapy employs autologous-T cells modified with an antigen-specific CAR. Current CAR-T manufacturing processes tend to yield products dominated by effector T cells and relatively small proportions of long-lived memory T cells. Those few cells are a so-called stem cell memory T (TSCM) subset, which express naïve T-cell markers and are capable of self-renewal and oligopotent differentiation into effector phenotypes. Increasing the proportion of this subset may lead to more effective therapies by improving CAR-T persistence; however, there is currently no standardized protocol for the effective generation of CAR-TSCM cells. Here we present a simplified protocol enabling efficient derivation of gene-modified TSCM cells: Stimulation of naïve CD8+ T cells with only soluble anti-CD3 antibody and culture with IL-7 and IL-15 was sufficient for derivation of CD8+ T cells harboring TSCM phenotypes and oligopotent capabilities. These in-vitro expanded TSCM cells were engineered with CARs targeting the HIV-1 envelope protein as well as the CD19 molecule and demonstrated effector activity both in vitro and in a xenograft mouse model. This simple protocol for the derivation of CAR-TSCM cells may facilitate improved adoptive immunotherapy.


Introduction
Gene-engineered T cells with desired antigen-specific receptors such as chimeric-antigen receptors (CARs), aim to confer directed and enhanced cytotoxic T lymphocyte responses (1)(2)(3)(4). CARs contain an antigen-binding domain specific for targets and an internal-signaling domain derived from CD3z chain as well as 41BB, CD28, or other co-stimulatory molecules (5,6). When a CAR encounters its target ligand, it signals the cell in a T cell receptor-like, but human-leukocyte antigen (HLA)independent manner, thus allowing this approach to be used in any patient. In human clinical trials for B-cell malignancies, CAR-modified autologous T cells targeting CD19 have shown robust effector responses (5)(6)(7)(8)(9)(10). For HIV-1 infection, CD4z CAR has been widely and longitudinally tested in patients. Treatment was safe and well-tolerated for over a decade, but antiviral effects were limited due to low levels of gene-modified cell persistence (11)(12)(13)(14).
The novel memory T cell subset known as stem cell-memory T (T SCM ) harbors self-renewing and oligopotency capabilities. The T SCM subset has been identified in mice (15), non-human primates (16,17), and humans (18,19). In humans, approximately 2-4% of peripheral blood T cells consist of T SCM cells (20). The T SCM cells are phenotypically defined as naïve T (T N ) cells by the expression of T N cell markers, such as CD45RA, CD62L and CCR7, but are distinguishable from T N cells by two memory T cell markers: CD95 and CD122. Thus, they exhibit a gene profile between T N and central-memory T (T CM ) cells. Of note, the T SCM cellsunlike other memory T cellscan be expanded ex vivo while maintaining their stemness, allowing enrichment of the gene-modified population prior to transplant.
Increasing evidence indicates that the T SCM cells exhibit a lesser extent of proliferation and effector activities compared to other memory T cells. Upon antigen stimulation, the T SCM cells have the ability to differentiate into T CM (which are thought to be a premature memory subset that differentiates into the effector subset upon antigen re-encounter), followed by effector-memory T (T EM ) cells (which are considered to be committed progenitor cells that undergo terminal differentiation after a limited number of cell divisions) (18, [21][22][23][24]. The frequency and activity of T SCM cells from clinical samples also supports their prolonged precursor potential in humans (25,26). Importantly, individual gene-modified T SCM cell clones did not show the emergence of clonal dominance for over a decade after infusion, indicating that gene engineering of T SCM cells does not bring in them a tumorigenic change. These evidence well support that T SCM cells would be an ideal host cell for CAR engineering.
We here developed a simple condition for the derivation and expansion of gene-modified-T cells harboring T SCM -surface phenotype and validated its applicability for gene engineering of T cells using both anti-HIV-1 and anti-CD19 CARs. Our results further demonstrate that these cells can effectively differentiate to functional-T cells conferring CAR-dependent effector activity against target cells in vitro as well as in a xenograft NSG mouse model.

Cells
Peripheral blood mononuclear cells (PBMCs) from healthy human donors were obtained from the CFAR Virology Core at UCLA without personal identifying information. To minimize a potential induction of T-cell stimulation by events such as crosslinking of cell-surface molecules, T N -cell population was negatively selected in one step using an EasySep ™ Human Naive CD8+ T Cell Enrichment Kit (StemCell Technologies, Inc., Vancouver, Canada), which consistently provides >95% purity of CD8+ T N cells (Supplementary Figure 1). Cells were maintained in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 1% GlutaMAX supplement and Antibiotic-Antimycotic (Life Technologies, Grand Island, NY), 20% FCS (SH30070.03E; GE Healthcare Life Sciences, South Logan, UT), and 0.1 mM 2mercaptoethanol (Sigma-Aldrich St. Louis, MO) (T-cell medium) as reported previously (27). Prior to lentiviral vector transduction, CD8+ T N cells were incubated with various concentrations of anti-CD3 antibody (Hit3a; BioLegend, San Diego, CA) with or without 2 µg/mL of anti-CD28 antibodies (CD28.2; BioLegend) for 2 days in T-cell medium supplemented with 5 ng/mL IL-7 and IL-15 (R&D systems, Minneapolis, MN) as summarized in Figure 1A. All cells were incubated at 37°C in 5% CO 2 .
For in vitro cytotoxicity assay, 5 x 10 4 CAR-or EGFPmodified CD8+ T cells as a negative control were plated using 100µL of T-cell medium in a 96-well round bottom plate. The same numbers of genetically-labeled DKS and HXBC2 cells for CD4z or BCBL-1 and Ramos cells for FMC63-IgG4z were added to each well and co-incubated for 4 or 16 hours [Effector: Target (E:T) ratio = 1:1]. Antigen specificity of both CARs was also validated with HXBC2 cells culturing with doxycycline to suppress HIV-1 HXBC2 envelope protein expression for CD4z CAR which cells cannot be a target for CD4z CAR, as well as human CD19 overexpressing HXBC2 cells culturing with doxycycline for FMC63-IgG4z CAR (data not shown). Total numbers of each cell were determined by MACSQuant (Milteny Biotech, Germany) and relative cytotoxicity of target cells relative to non-target cells was determined by the following formula: Relative cytotoxicity (%) =100 x (1target cell number/non-target cell number).

Viruses
Lentiviral vectors were generated and transduced as described elsewhere (27,34,35). p24 Gag ELISA assays were performed by the CFAR Virology Core at UCLA. Lentiviral vector information used in this research will be provided upon request.

Flow cytometry
The following antibodies were used in flow cytometry:

Statistical analyses
Results are expressed as mean ± standard deviations (SDs). Errors depict SD. Comparisons between two groups were performed using an unpaired two-tailed t-test with Welch's correction. A p-value less than 0.05 was considered statistically significant.

Stimulation of CD8+ T N cells with soluble anti-CD3 antibody enables efficient gene-modification by lentiviral vector while minimizing T-cell differentiation
Two independent protocols for the derivation of T SCM have been described by Gattinoni et al. (18) and Cieri et al. (19). The major differences between those two protocols are that the former used an inhibitor of glycogen synthase kinase 3b (GSK-3b), TWS119 (15, 36), and the latter used a low concentration of two cytokines, IL-7 and IL-15 with no chemical inhibitor. Our preliminary experiments for a side-by-side comparison of those two protocols showed that there was minimal benefit of the use of TWS119 for the derivation of T SCM cells due to a poor cell expansion by the former protocol. Whereas, the latter protocol was able to yield a nearly 5-fold higher number of cells harboring surface phenotypes corresponding to T SCM (0.41 x 10 6 vs 1.88 x 10 6 , Supplementary Table 1). However, a considerable number of T EM cells were derivated under both conditions, presumably due to the potent stimulation condition of using anti-CD3/CD28 antibody coated beads. We thus assessed alternative stimulation conditions for their ability to derive large numbers of T SCM while minimizing T-cell differentiation and allowing for gene modification via lentiviral vectors. Negatively selected CD8+ T N cells were incubated with different amounts of soluble anti-CD3 antibody with or without anti-CD28 antibody. The cells were then transduced with a lentiviral vector encoding EGFP as a marker gene and cultured for an additional 12 days with IL-7 and IL-15 followed with Cieri's protocol ( Figure 1A). In the no antibody condition, approximately 20% of CD8+ T N cells were successfully gene-modified, indicating that a small portion of CD8+ T cells are transducable without active stimulation and that culturing in the presence of IL-7 and IL-15 allows maintenance of the CD8+ T N cell phenotype for at least 14 days ( Figure 1B). However, the no antibody condition resulted in extremely poor expansion of the cells (<2-fold, Figure 1C). The inclusion of increasing concentrations of anti-CD3 antibody and/or the presence of anti-CD28 antibody increased the levels of gene marking and Tcell expansion ( Figure 1C), but also increased the proportion of differentiated cells as defined by the loss of CD45RA or CD62L expression (37-39) (Figures 1D, 2A). The differences shown above became more obvious after an additional 14 days of culture (total 28 days of culture) ( Figure 2B, Day 28), indicating that cells harboring T SCM -surface phenotype slowly but continuously expanded in the presence of IL-7 and IL-15. The average numbers from each population in EGFP-positive cells after a total of 28 days of culture in Figure 2B were summarized in Supplementary Table 2: a higher recovery of the EGFP-marked T SCM cells was observed with 0.5 µg (7.35 x 10 6 ) or 1.0 µg/mL anti-CD3 (7.84 x 10 6 ) stimulation rather than the condition with 0.5 µg/mL anti-CD3 + 2.0 µg of anti-CD28 antibody (5.61 x 10 6 ), which generated the highest gene marking as in Figure 1C. Importantly, EGFP-marked T SCM cells were expanded nearly 2fold following a total of 28 days of culture, whereas the number of T SCM cells in other populations decreased over the course of the culture (Supplementary Figure 3). These results indicate that stimulation of CD8+ T N cells with only soluble anti-CD3 antibody (0.5-1.0 µg/mL) and prolonged-expansion (28 days) in the presence of IL-7 and IL-15 allowed for the most efficient derivation of gene-marked CD8+ T cells harboring T SCM -surface phenotype with a minimum level of T-cell differentiation.
A key feature of the T SCM subset is its oligopotency allowing differentiation into more terminal subsets upon a n t i g e n s t i m u l a t i o n ( 1 8 , 1 9 ) . W e c o n fi r m e d t h e differentiation ability of these in-vitro generated T SCM cells via anti-CD3/CD28 co-stimulation. The status of T-cell differentiation was evaluated by surface phenotypes at 14days after the 2 nd stimulation ( Figure 2C, 2 nd stimulation). The number and prevalence of CD8+ T cells harboring the T SCM and T N phenotype was substantially reduced after the 2 nd stimulation. Concomitantly, the proportion of differentiated cells ─ especially the T EM population ─ increased under this condition. Thus, these in-vitro generated T SCM cells retain their oligopotency.
Derivation of CAR-modified CD8+ T cells with T SCM -surface phenotype As above, we were able to validate the oligopotency of the invitro generated gene-engineered cells with T SCM -surface phenotypes. We next applied this method for the generation of anti-HIV-1 CAR-T cells. We utilized the Triple-CD4z CAR that targets HIV-1 gp120 on the infected cell surface (27) with coexpression of two anti-HIV-1 shRNAs: sh1005 which suppresses surface expression of the key HIV-1 co-receptor CCR5 (40) and sh516 which prevents HIV-1 infection to both CD4+ T and CD8+ T cells mediated via CD4z expression (34). Thus, the Triple-CD4z CAR can exert potent anti-HIV-1-effector activity while protecting its transduced cells from HIV-1 infection. We previously demonstrated its potent anti-HIV-1 effects in vivo using an HIV-1 challenged-humanized mouse model (41,41). Although the condition established above allowed successful modification of CD8+ T N cells with the Triple-CD4z CAR while maintaining a population with the T SCM -surface phenotype, the proportion of transduced cells was relatively lower compared to that of control EGFP vector (42.9% vs 8.8%, Supplementary Figure 4). This is potentially due to a larger size of the transgene (42). Nevertheless, these gene-marked cells were successfully differentiated to cells harboring surface phenotypes of T CM and T EM cells upon anti-CD3/CD28 stimulation ( Figure 3A).

FIGURE 2
Prolonged culture increases % population of CD8+ T cells harboring T SCM -surface phenotype with maintaining oligopotency Freshly isolated CD8+ T N cells were transduced with lentiviral vector encoding EGFP following stimulation with various antibody conditions shown in Figure 1A We next assessed the effector function of Triple-CD4zmodified CD8+ T cells by a flow-cytometry based cytotoxicity assay. The cells modified with Triple-CD4z cells were incubated with their target Jurkat cells expressing HIV-1 HXBC2 envelope protein (HXBC2) upon doxycycline removal from culture medium (28). To validate the level of non-specific target cell killing, control Jurkat cells which do not encode HIV-1-envelope protein (DKS, non-target cell) were included (28). The former is labeled by mCherry and the latter by Tag-BFP, thus both cells can be distinguished by flow cytometry. The numbers of remaining mCherry+ and TagBFP+ cells following incubation with Triple-CD4z-modified CD8+ T cells correspond to levels of anti-HIV-1 CAR-dependent and independent cytotoxicity, respectively. Following a co-culture with the above three cells for 4 and 16 hours, the total numbers of mCherry+ and TagBFP+ cells were analyzed by MACSQuant, and relative cytotoxicity against target cells compared to that against non-target cells was determined ( Figure 3B). The pre-differentiated cells shown in Figure 3A were used as a positive control for the assay. There were minimal levels of change in the cell surface phenotypes of Triple-CD4z following B A FIGURE 3 Induced differentiation of Triple-CD4z modified CD8+ T cells harboring T SCM -surface phenotype with anti-CD3 and CD28 co-stimulation.  Figure 5). A 4-hour incubation was sufficient for the induction of cytotoxic activity by the pre-differentiated cells transduced with Triple-CD4z, whereas a 16-hour incubation was required to achieve a similar level of cytotoxicity without predifferentiation, indicating that longer incubation was required to exert an effector activity for undifferentiated Triple-CD4zmodified CD8+ T cells.

4-hour and 16-hour incubations with the target cells (Supplementary
Additionally, we generated anti-CD19 (FMC63-IgG4z) modified CD8+ T cells using the same conditions as above (Supplementary Figure 6). The level of gene marking by FMC63-IgG4z was similarly lower than control vector as seen with Triple-CD4z. FMC63-IgG4z-modified cells also maintained high levels of expression for T SCM cell markers, and a large portion of the cells harbored T N -and T SCM -surface phenotypes. Similar to Triple-CD4z modified-CD8+ T cells, FMC63-IgG4zmodified cells also exerted a potent antigen dependent cytotoxicity following 16, but not 4, hours of incubation.
Triple-CD4z CAR-modified CD8+ T cells generated with sole anti-CD3 antibody stimulation exert antigen-specific effector functions in a xenograft NOD-SCID mouse model As above, CD8+ CAR-T cells carrying the T SCM -surface phenotype were generated in vitro with an antigen-specific effector activity. We next tested their effector activity in vivo using a xenograft NOD-SCID mouse model. NOD-SCID mice were engrafted with two different lymphoma cell lines: TF228.1.16a derivative of BJAB (29) or HXBC2a derivative of Jurkat (28). These lines express envelope protein from either the HIV-1 BH10 or HIV-1 HXBC2 strains, respectively. In vitro, the TF228.1.16 cells were killed by Triple-CD4zmodified CD8+ T cells like the HXBC2 cells, but relatively weakly by FMC63-IgG4z-modified CD8+ T cells. Although TF228.1.16 is CD19+ and can be a target for FMC63-IgG4zmodified CD8+ T cells in vitro (Supplementary Figure 7), our FMC63-IgG4z construct contains an IgG hinge-CH2-CH3 domain, which makes ineffective this CAR in vivo due to an Fcg receptor 1 mediated CAR-T cell elimination (43)(44)(45)(46). Thus, the FMC63-IgG4z serves as negative control. Those two tumor cells were genetically marked by mStrawberry (TF228.1.16) or mCherry (HXBC2), and subcutaneously engrafted into the left and right hind limbs of NOD-SCID mice, respectively. We confirmed that the expression levels of HIV-1 envelope proteins in tumor tissues developed in xenograft mice were similar to those in cells maintained in vitro by western blotting using anti-GP120 (2G12) and anti-GP41 (2F5) antibodies (data not shown).
At 14 days post-engraftment, we infused CD8+ T cells engineered with either Triple-CD4z CAR or FMC63-IgG4z CAR derived as above. We analyzed these CAR-dependent effector activities by bioimaging using a Maestro 2 multispectral imaging system ( Figure 4). As expected, we observed poor in vivo effector activity of the cells modified by FMC63-IgG4z CAR. On the other hand, the cells modified by Triple-CD4z CAR showed stronger anti-tumor effector activity against both tumors, with tumor burdens decreasing approximately 1.9-fold for TF228.1.16 tumors (blue bars, Figure 4B) and 5.5-fold for HXBC2 tumors (orange bars, Figure 4B). Compared to HXBC2 tumor, the effector activity on TF228.1.16 tumor was weaker with no statistical significance; this may be due to the faster growth rate of TF228.1.16 cells. These results provide additional evidence that our simplified protocol allows for the derivation of CAR-modified CD8+ T cells harboring T SCM -surface phenotype while maintaining antigenspecific effector activity in vivo.

Discussion
Though there is currently no standardized protocol for CAR-T SCM cell manufacturing, the use of CAR-T SCM products represents a promising approach for improving the outcome of CAR-T based therapies. Here, we refined the protocol for the derivation of CAR-modified CD8+ T cells harboring T SCMsurface phenotype and T SCM -like oligopotency.
The first key to generating CAR-T SCM -like cells is the purification of CD8+ T N cells as a starting population. Flowcytometry based cell sorting is commonly used to isolate this population, but has 2 major drawbacks: decreased cell viability (47,48), and the potential for sorting antibodies to induce unwanted activation via binding to T N cell surface markers. By this reasoning, negative selection may be more effective for isolating T N populations because these processes leave the cells 'untouched' and also without any antibodies remaining bound to the cells in the final product. By using an EasySep ™ Human Naïve CD8+ T Cell Enrichment Kit which enables negative selection of CD8+ T N cells, we consistently obtained CD8+ T N cells with >95% purity (Supplementary Figure 1).
Next, we evaluated the utility of different stimulation conditions for generating gene-modified T SCM -like CD8+ T cells and found that stimulation with only soluble anti-CD3 antibody in the presence of IL-7 and IL-15 was the most efficient. Since this condition does not require the use of novel agents, it is readily translatable for clinical applications. Cieri et al. also tested a stimulation condition with only anti-CD3 antibody for expansion of cells with a CD8+/CD45RA+/CD62L+/CD95phenotype, but this attempt was unsuccessful (19). There were several differences compared to our protocol; they used a different anti-CD3 antibody clone -OKT3at >16times lower concentration (30 ng/mL) in a plate-bound format. With this condition, they observed a poor expansion of CD8+ T N cells compared to the cells co-stimulated with anti-CD3/ CD28 antibody coated beads. Surprisingly, most of the cells lost expression of CD45RA and CD62L after 16 days of culture with their experimental conditions (19). We did not observe this by the use of soluble form of anti-CD3 antibody, suggesting that the precoated anti-CD3 antibodies probably induced a stronger stimulation via TCR crosslinking, resulting in Tcell differentiation.
Whereas both CD4+ and CD8+ T cells are needed for an efficient CAR-T cell therapy (49)(50)(51); the former mainly serve as CAR-dependent helper cells and the latter as CAR-dependent effector cells (52). We have already adopted the derivation of CAR-modified CD4+ T SCM cells by the use of EasySep Human Naive CD4+ T cell Isolation kit, which enables negative selection of CD4+ T N cells (Stemcell technologies, #19555). Unlike CD8+ T N cells, CD4+ T N cells required co-stimulation by soluble forms of anti-CD3 (1.0 mg/mL) and anti-CD28 (0.1 mg/mL) antibodies for an efficient expansion of cells with T SCM -surface phenotype (data not shown). The level of gene marking as well as oligopotential activity of CAR-engineered CD4+ T SCM cells were similarly reproduced as those confirmed in CAR-engineered CD8+ T SCM cells. We are currently validating the effector activity of CAR-engineered CD4+ T cells as well as seeking to derivate CAR-engineered T SCM cells from negatively selected naive Pan-T cells using EasySep Human Naive Pan T cell Isolation kit (Stemcell technology, #17961) without preseparating CD4+ or CD8+ T N cells. We believe that these approaches will enable more efficient and reproducible procedures for the manufacturing of highly effective CAR-T cells.
CAR-modified CD8+ T cells harboring T SCM -surface phenotype required longer incubation to exert CAR-dependent effector activity compared to the pre-differentiated cells (16 hours vs 4 hours, Figures 3B and Supplementary Figure 6B). The pre-differentiated cells consisted of more differentiated cells, which have potent cytotoxicity than cells harboring T SCM cell phenotype. We also confirmed that 16 hours of incubation was not sufficient to fully induce differentiation of the cells with T SCM -surface phenotype to cells with differentiated phenotypes, such as T CM or T EM (Supplementary Figure 5). We thus expected the cells derived with our protocol to have less effector activity B A FIGURE 4 Triple-CD4z CAR-modified CD8+ T cells harboring T SCM -surface phenotype eliminate tumor cells expressing HIV-1 envelope proteins in a xenograft mouse model. Two million mStrawberry-labeled CD19+ TF228.1.16 cells expressing envelope protein from HIV-1 BH10 or mCherrylabeled Jurkat cells expressing envelope protein from HIV-1 HXBC2 (HXBC2), were mixed with Matrigel at a 1:1 ratio and subcutaneously engrafted to the left or right hind limbs of NOD-SCID mice from ventral side, respectively (n = 4). Freshly isolated CD8+ T N cells were stimulated for 2 days with 0.5 µg/mL of anti-CD3 antibody in T-cell medium, followed by transduction with a lentiviral vector encoding either Triple-CD4z or FMC63-IgG4z. than those with differentiated phenotypes, and to require longer incubation before acquiring cytotoxicity. These possibilities are currently under investigation.
In recent years, immunotherapy utilizing CAR-engineered T cells has become a highly promising approach, especially for the treatment of blood cancers. T SCM cells have the capacity for both self-renewal and oligopotent differentiation into effector cells upon encounter with antigens; thus, T SCM cells re-directed against their targets could be more effective than mature T cells employed in current clinical trials. Due to the low frequency of T SCM cells in peripheral blood, establishing techniques for efficient expansion with high levels of gene modification will be important for translation to clinical purposes. Our protocol described here is applicable for a prompt implementation of T SCM cell-based immunotherapies against not only cancer but also infectious diseases such as AIDS.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The animal study was reviewed and approved by Guido Eibl, UCLA.