Chimeric Antigen Receptor Expression Research Articles

An IMiD-inducible degron provides reversible regulation for chimeric antigen receptor expression and activity

The recent development of successful CAR (chimeric antigen receptor) Tcell therapies has been accompanied by a need to better control potentially fatal toxicities that can arise from adverse immune reactions. Here we present a ligand-controlled CAR system, based on the IKZF3 ZF2 β-hairpin IMiD-inducible degron, which allows for the reversible control of expression levels of type I membrane proteins, including CARs. Testing this system in an established mouse xenotransplantation model for acute lymphoblastic leukemia, we validate the ability of the CAR19-degron to target and kill CD19-positive cells displaying complete control/clearance of the tumor. We also demonstrate that the activity of CAR19-degron can be regulated invivo when dosing a US Food and Drug Administration-approved drug, lenalidomide.

NOTCH and CAR Signaling Control T Cell Lineage Commitment from Pluripotent Stem Cells

Chimeric Antigen Receptor (CAR) T cells are a new treatment paradigm for relapsed/refractory hematopoietic malignancies. However, their autologous nature imposes manufacturing constraints that can delay CAR T cell availability and increase their cost. We previously established proof of principle that αβ T cell-derived induced pluripotent stem cells (TiPSCs) can provide a self-renewing source for in vitro CAR T cell production (Themeli, Nat Biotechnol, 2013). The use of cloned TiPSC further enhances the feasibility of verifying genome integrity of the genetically engineered stem cells and should in principle yield highly homogenous cell products. Using αβ T cell-derived TiPSCs transduced with a well-defined CD19-specific CAR (1928z; Park, NEJM, 2018), we previously demonstrated that TiPSCs can be differentiated into CAR T cells. These T cells retained their endogenous T cell receptor (TCR) and also displayed characteristics of innate lymphoid cells. We have now examined how the timing of CAR expression as well as the CAR signaling strength influence T cell lineage commitment, enabling better control towards αβ T cell lineage commitment. αβ T cell lineage development depends in part on a precisely orchestrated interactions between NOTCH and (pre)TCR signaling, the timing and strength of which are crucial for αβ lineage commitment. Because TiPSCs harbor rearranged TCRα and TCRβ genes, mature TCR expression occurs earlier than if it required VDJ recombination, skewing differentiation towards acquiring innate features including CD4-CD8- double-negative or CD8αα single-positive phenotypes. We show that providing strong NOTCH stimulation counteracts the effects of early antigen receptor expression, facilitating CD4+CD8αβ+ double positive (DP) formation. We hypothesized that CAR signaling in the absence of ligand binding (tonic signaling) may mimic a TCR signal, the strength and timing of which could re-direct lineage commitment. We therefore investigated CARs providing different levels of signaling strength and the impact of delaying the onset of CAR expression. Tonic CAR signaling was measured in peripheral blood T cells expressing 1928z or 1928z-1XX, a construct in which the second and third ITAM in the CD3ζ domain have been mutated to be non-functional (Feucht, Nat Med, 2019), following either retroviral transduction (SFG vector) orTRAC-targeted cDNA integration, placing CAR expression under the transcriptional control of the TCRα promoter (Eyquem, Nature, 2017). CAR signaling in the absence of antigen exposure, measured by phosphorylation of ITAM3, ERK1/2 and ZAP70, was reduced by bothTRAC-targeting and reduction of functional ITAMs, with additive effects when combined inTRAC-1928z-1XX. Three of these engineering strategies (virally expressed 1928z,TRAC-1928z andTRAC-1928z-1XX) were evaluated in the context of TiPSC-derived T cell differentiation. Virally expressed 1928z (resulting in constitutive CAR expression throughout differentiation) resulted in the predominant generation of innate-like CD8αα T cells, associated with the absence of early T cell lineage markers such as CD5, CD2 and CD1a. Delayed expression of 1928z throughTRACtargeting resulted in increased CD5, CD2 and CD1a, but did not yield any more CD4+CD8αβ+ DP cells. In TiPSC expressingTRAClocus-encoded 1928z-1XX, a greater DP population emerged, from which CD8αβ single-positive T cells could be induced. Phenotypic analyses of clonal TRAC-1928z-1XX TiPSC lines further establish the interplay between CAR and NOTCH1 in determining αβ lineage commitment. Together these data show that early TCR and CAR expression skew T cell lineage commitment towards an innate-like T cell fate, which can be overcome by controlling the strength and timing of NOTCH, TCR and CAR signaling. These studies pave the way for the predetermined generation of a variety of CAR T cell types endowed with different functional attributes. Disclosures Whitlock: Fate Therapeutics Inc.:Current Employment, Current equity holder in publicly-traded company.Clarke:Fate Therapeutics Inc.:Current Employment, Current equity holder in publicly-traded company.Valamehr:Fate Therapeutics, Inc:Current Employment, Current equity holder in publicly-traded company.Riviere:Juno Therapeutics:Other: Ownership interest, Research Funding;Takeda:Research Funding;Fate Therapeutics Inc.:Consultancy, Other: Ownership interest , Research Funding;FloDesign Sonics:Consultancy, Other: Ownership interest;Atara:Research Funding.Sadelain:Atara:Patents & Royalties, Research Funding;Fate Therapeutics:Patents & Royalties, Research Funding;Mnemo:Patents & Royalties;Takeda:Patents & Royalties, Research Funding;Minerva:Other: Biotechnologies , Patents & Royalties.

Combinatorial Antigen Targeting Strategy for Acute Myeloid Leukemia

Introduction: Efforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia associated antigen with chimeric antigen receptor T (CAR T) cells have had limited success. We determined whether combinatorial expression of chimeric antigen receptors directed to two different AML associated antigens would augment tumor eradication and prevent relapse in targets with heterogeneous expression of myeloid antigens. Methods: We generated CD123 and CD33 targeting CARs; each containing a 4-1BBz or CD28z endodomain. We analyzed the anti-tumor activity of T cells expressing each CAR alone or in co-transduction with a CLL-1 CAR with CD28z endodomain and CD8 hinge previously optimized for use in our open CAR-T cell trial for AML (NCT04219163). We analyzed CAR-T cell phenotype, expansion and transduction efficacy by flow cytometry and assessed function by in vitro and in vivo activity against AML cell lines expressing high, intermediate or low levels of the target antigens (Molm 13= CD123 high, CD33 high, CLL-1 intermediate, KG1a= CD123 low, CD33 low, CLL-1 low and HL60= CD123 low, CD33 intermediate, CLL-1 intermediate/high) For in vivo studies we used NOD.SCID IL-2Rg-/-3/GM/SF (NSGS) mice with established leukemia, determining antitumor activity by bioluminescence imaging. Results: We obtained high levels of gene transfer and expression with both single (CD33.4-1BBʓ, CD123.4-1BBʓ, CD33.CD28ʓ, CD123.CD28ʓ, CLL-1 CAR) and double transduction CD33/CD123.4-1BBʓ or CD33/CD123.CD28ʓ) although single-transductants had marginally higher total CAR expression of 70%-80% versus 60-70% after co-transduction. Constructs containing CD28 co-stimulatory domain exhibited rapid expansion with elevated peak levels compared to 41BB co-stim domain irrespective of the CAR specificity. (p<0.001) (Fig 1a). In 72h co-culture assays, we found consistently improved anti-tumor activity by CAR Ts expressing CLL-1 in combination either with CD33 or with CD123 compared to T cells expressing CLL-1 CAR alone. The benefit of dual expression was most evident when the target cell line expressed low levels of one or both target antigens (e.g. KG1a) (Fig 1b) (P<0.001). No antigen escape was detected in residual tumor. Mechanistically, dual expression was associated with higher pCD3ʓ levels compared to single CAR T cells on exposure to any given tumor (Fig 1c). Increased pCD3ʓ levels were in turn associated with augmented CAR-T degranulation (assessed by CD107a expression) in both CD4 and CD8 T cell populations and with increased TNFα and IFNɣ production (p<0.001 Fig 1d). In vivo, combinatorial targeting with CD123/CD33.CD28ʓ and CLL-1 CAR T cells improved tumor control and animal survival in lines (KG1a, MOLM13 and HL60) expressing diverse levels of the target antigens (Fig 2). Conclusion: Combinatorial targeting of T cells with CD33 or CD123.CD28z CARs and CLL-1-CAR improves CAR T cell activation associated with superior recruitment/phosphorylation of CD3ʓ, producing enhanced effector function and tumor control. The events that lead to increased pCD3ʓ after antigen engagement in the dual transduced cells may in part be due to an overall increase in CAR expression but may also reflect superior CAR recruitment after antigen engagement. We are now comparing the formation, structure, and stability of immune synapses in single and dual targeting CARs for AML. Disclosures Brenner: Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Founder; Maker Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Memmgen: Membership on an entity's Board of Directors or advisory committees; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Atilla:Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Tumstone: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: founder; Marker Therapeuticsa: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder, Patents & Royalties; Allogene: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Walking Fish: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Memgen: Membership on an entity's Board of Directors or advisory committees; KUUR: Membership on an entity's Board of Directors or advisory committees.

Donor-Derived CAR T Cells Engineered with Sleeping Beauty Achieve Anti-Leukemic Activity without Severe Toxicity

Donor-Derived CAR T Cells Engineered with Sleeping Beauty Achieve Anti-Leukemic Activity without Severe Toxicity

CD33 Targeting Primary CAR-NK Cells Generated By CRISPR Mediated Gene Insertion Show Enhanced Anti-AML Activity

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein complexes (PMID: 29985369 and 32603414). Here we combined this approach with single-stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. We demonstrated that expansion of NK cells on feeder cells (CSTX002) expressing membrane-bound IL21 increases expression of HDR-related genes and provides optimum biological condition for targeted gene insertion. For proof-of-concept, we successfully generated stable mCherry-expressing primary NK cells (up to 89% mCherry+) and determined that sc vectors with 300bp homology arms were optimal. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3ζ and NKG2D/2B4+CD3ζ), which continued to show robust expansion on CSTX002 and stably maintained their CAR expression. This resulted in CAR-NK-cells of high number and purity (mean 68% CAR+) that demonstrated enhanced antileukemic activity against acute myeloid leukemia (AML) cell lines. This efficient method for site-directed insertion of genetic materials into primary NK cells has broad potential for fundamental discovery and therapeutic applications. Keywords: CRISPR, NK, Cas9/RNP, AAV6, CRISPaint, HR, CD33CAR-NK Figure Disclosures Naeimi Kararoudi: Kiadis Pharma Netherlans B.V: Patents & Royalties. Lee:Kiadis Pharma Netherlands B.V: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

A Phase I Study of FT819, a First-of-Kind, Off-the-Shelf, iPSC-Derived TCR-Less CD19 CAR T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Malignancies

A Phase I Study of FT819, a First-of-Kind, Off-the-Shelf, iPSC-Derived TCR-Less CD19 CAR T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Malignancies

Investigation of ALLO-316: A Fratricide-Resistant Allogeneic CAR T Targeting CD70 As a Potential Therapy for the Treatment of AML

CD70, a member of the TNF superfamily, is a type II transmembrane glycoprotein that interacts with its receptor (CD27) to promote survival of primed T cells and leads to formation of effector and memory T cells. Expression of CD70 in normal tissues is restricted to activated T and B lymphocytes and mature dendritic cells. CD70 is also widely expressed in various malignancies, including renal cell carcinoma (RCC) and acute myeloid leukemia (AML). The restricted expression pattern of CD70 in normal tissues makes it an attractive target for cancer therapeutics. Adoptive transfer of T cells expressing chimeric antigen receptors (CARs) is an exciting new therapeutic modality showing great promise in hematologic malignancies. Approval of two CD19-targeting autologous CAR Ts, Kymriah® and Yescarta®, has been followed with promising results from BCMA autologous CAR T clinical trials, showing that activity can extend to other targets. We have previously described the functional screening of a library of anti-CD70 scFv-based CARs and the identification of lead CD70 allogeneic CAR T cells (AlloCAR TTM) with robust activity against RCC cell lines both in vitro and in vivo. Here, we evaluate the anti-tumor activity and safety of a lead CD70 AlloCAR T (ALLO-316) for the treatment of AML. CD70 expression was evaluated and detected on three AML cell lines and in six primary AML patient samples, with 5/6 patient samples showing expression on 24%-99% of cells. CD70 expression will be profiled in a broader subset of AML patients and preliminary data will be presented. Despite the expression of CD70 on activated T cells it was possible to generate CD70 AlloCAR T cells. No CD70 expression was observed on CAR T cells after generation, suggesting either cells are succumbing to fratricide or are being "masked" by the CAR. CD70 was also not detected on Jurkat cells expressing CARs and this data, in combination with results showing CAR expression is protective when overexpressed in RCC cells support the phenomenon of "masking". Cellectis' TALEN® gene-editing was used to inactivate the TRAC and CD52 loci with the intent to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. ALLO-316 cells were highly effective at lysing CD70-expressing target cells and eliminated greater than 99% of cells at the high effector to target (E:T) ratio and were unable to lyse AML cells in which CD70 was knocked out. Moreover, ALLO-316 cells were able to kill primary AML blasts with CD70 expression ex vivo. An orthotopic in vivo model utilizing the AML cell line MV4-11 was developed and anti-tumor activity was observed. In addition to evaluating efficacy against AML cell lines and tumors we also explored potential toxicity liabilities related to ALLO-316 treatment. Previous studies have reported that certain AML tumor antigens can also be expressed on normal hematopoietic progenitors and such expression could potentially lead to toxicity with targeted therapeutics. No detectable CD70 was observed by flow cytometry on purified CD34+ cells from 14 healthy donors. Taken together, our results support clinical development of CD70 AlloCAR T therapy for the treatment of AML. Disclosures Srinivasan: Allogene Therapeutics: Current Employment. Tan:Allogene Therapeutics: Current Employment. Cheng:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang:Allogene Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tacheva-Grigorova:Allogene Therapeutics: Current Employment, Current equity holder in publicly-traded company. Van Blarcom:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Sommer:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Nguyen:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Sasu:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company. Panowski:Allogene Therapeutics, Inc.: Current Employment, Current equity holder in publicly-traded company.

CAR-T Cells with High CAR Expression Intensity Have Improved In Vitro and In Vivo Anti-Lymphoma Effect without Functional Exhaustion

Background While immunotherapy with anti-CD19 chimeric antigen receptor (CAR) T cells has shown significant efficacy in B-cell malignancies, CAR T cells directed against CD30 (CAR30) for the treatment of Hodgkin lymphoma (HL) showed modest antitumor effect, with more than 50% of patients being unresponsive. Several factors related to the infused product and persistence may be relevant to increase clinical efficacy, but further investigation is needed. In this way, CAR expression intensity may play an important role on CAR T cell function, but this has not been systematically explored. Aim We have evaluated the impact of CAR expression intensity on T cell function, cell exhaustion and antitumor efficacy against HL and B cell lymphoma. Methods T cells were generated as previously described (Alvarez-Fernández C et al. 2016) and transduced with third generation lentivirus encoding a 4-1BBz CAR (either CAR30 or CAR19). Two populations of CAR+ T cells were sorted according to mean fluorescence intensity (MFI) of CAR: CARHI (MFI> 5x103) and CARLO (MFI <3x103). Cytotoxicity assays were performed using Raji (CD19+) or L540 (CD30+) tumor cell lines. Multiparametric flow cytometry was used to analyze T-cell inhibition and activation markers. CARHI and CARLOin vivo antitumor effect was tested under stringent therapeutic conditions using 5x106 T cells/mice (iv) in a HL NSG model. Results CAR30+ T cells were sorted into CARLO (MFI: 1064±124.7) and CARHI (MFI: 7068±1377) (p=0.01). TSCM were highly represented in CARLO compared to CARHI (CD4+: 70.14±1.78% vs. 55.61±5.5%, CD8+: 83.78±3.8% vs 72.2±5.47%, respectively) (p<0.01). However, these differences disappear after 24h co-culture with tumor cells due to an increase of TSCM in CARHI (CD4+: 72.52±7.54%, CD8+: 80.26±5.3%). CARHI showed a significantly higher in vitro antitumor effect compared to CARLO (tumor death at 5:1 E:T ratio: 96.6±1.86% vs. 89.1±3.83%; 1.25:1 E:T ratio: 84.61±4.7% vs. 31.15±19.79%; CARHI vs. CARLO, respectively) (p<0.0001). No differences were observed in expression of activation markers (i.e.: CD25, CD69, and HLA-DR) among both populations. Generalizability of this finding was studied using a CAR19. Similarly, CAR19+ T cells were arranged into CARLO (MFI: 1610±187) and CARHI (MFI: 10810±1486) subgroups (p<0.01). TSCM represented the most frequent subtype in both populations (CD4+: CARHI 70,22±9,87%, CARLO 69,22±9,33%; CD8+: CARHI 65,1±10,5%, CARLO 60,9±9,5%) and no differences in T cell subset composition between CARHI and CARLO were found. Again, CARHI exhibited superior antitumor effect compared to CARLO (tumor death at 5:1 E:T ratio:59.9±8.72% vs. 28.8±8.7%; 1.25:1 E:T ratio: 21.6±11.4% vs. 2.9±2.9%, CARHI vs. CARLO, respectively) (p<0.0001). At 24h and 72h of antigen encounter, expression of inhibitory markers was determined in both CAR30+ populations. While CD4+ T cells showed significantly higher PD1 and TIM3 co-expression in CARHI compared to CARLO (p<0.05), CD8+ T cells showed similar co-expression (p=0.4 and p=0.8, at 24h and 72h, respectively). A similar kinetics was observed in CAR19+ T cells, suggesting that it could be related to an inhibitory control of activation, but not cellular exhaustion. To confirm this, functional performance of CAR30HI and CAR30LO T cells was evaluated by continuous tumor exposure. CAR30HI function persisted after sequential re-exposition (n=5) to tumor cells; in contrast, the CAR30LO subpopulation showed progressive loss of cytotoxic activity (i.e., tumor death at ratio E:T 5:1 after 4 expositions: 0% vs. 91.96%, CAR30LO and CAR30HI respectively; representative of 2 independent studies with different donors). To assess if these results were consistent in vivo, the antitumor effect of CAR30HI and CAR30LO were evaluated in a xenograft model of HL. Mice treated with CAR30HI T cells showed reduced tumor growth compared to those treated with CAR30LO T cells, which translated into an improved survival. Conclusion We have shown that high expression of a CAR (either CAR30 or CAR19) confers an enhanced in vitro antitumor effect against HL and B cell lymphoma. This effect is maintained after repetitive exposures to tumor cells and is not associated with T cell exhaustion or differentiation. Notably, this enhanced antitumor effect was also found in vivo. Our data shows that CAR expression intensity should be considered as an additional important factor to improve the efficacy of CAR T cells. Disclosures Sierra: Jazz Pharmaceuticals: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead-Kite: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Generation of Multiplexed Engineered, Off-the-Shelf CAR T Cells Uniformly Carrying Multiple Anti-Tumor Modalities to Prevent Tumor Relapse

The development of chimeric antigen receptor (CAR) T cell therapeutics is widely recognized as a significant advancement for the treatment of cancer. However, several obstacles currently impede the broad use of CAR T cells, including the inherent process variability, cost of manufacturing, the absolute requirement for precise and uniform genetic editing in the allogeneic setting, and the challenge to keep pace with clonal heterogeneity and tumor growth. Utilizing our previously described induced pluripotent stem cell (iPSC)-derived T (iT) cell platform, we illustrate here the unique ability to address these challenges by creating a consistent CAR iT cell product that can be repeatedly manufactured in large quantities from a renewable iPSC master cell bank that has been engineered to mitigate the occurrence of graft versus host disease (GvHD), antigen escape and tumor relapse. Utilizing our proprietary cellular reprogramming and engineering platform and stage-specific T cell differentiation protocol, we demonstrate that iPSC can be engineered at the single cell level to generate a fully characterized clonal iPSC line, which can then be accessed routinely to yield CAR iT cells in a highly scalable manufacturing process (>100,000 fold expansion). Through bi-allelic targeting of a CAR into the T cell receptor alpha constant (TRAC) region, we generated CAR iT cells with uniform CAR expression (99.0 ± 0.5% CAR+) and complete elimination of T cell receptor (TCR) expression to avoid GvHD in the allogeneic setting. We elected to utilize the 1XX-CAR configuration, which has demonstrated superior anti-tumor performance relative to other CAR designs and when introduced into iT cells displayed enhanced antigen specificity (% specific cytotoxicity at E:T=10:1, antigen positive group: 86.4 ± 7.8; antigen null group: 8.9 ± 3.5). To enhance persistence without reliance on exogenous cytokine support, we engineered signaling-fusion complexes, including IL-7 receptor fusion (RF), into iPSC and studied its impact on iT phenotype, persistence, and efficacy. In vitro, IL-7RF clones demonstrated improved anti-tumor activity in a serial antigen dependent tumor challenge assay (Day 10, relative tumor counts, IL-7RF group: 1.95 ± 0.01; control group: 57.56 ± 4.55, P<0.000001). In a preclinical in vivo model of disseminated leukemia, IL-7RF clones demonstrate enhanced tumor growth inhibition (Day 34, Log [BLI], IL-7RF group: 6.68 ± 1.93; control group: 9.99 ± 0.23, P=0.0143). We next investigated a unique strategy to incorporate multi-antigen targeting potential into anti-CD19 1XX CAR iT cells with the addition of a high-affinity non-cleavable CD16 (hnCD16) Fc receptor. The combination of hnCD16 with anti-CD19 1XX CAR culminated in iT cells capable of multi-antigen specificity through combinatorial use with monoclonal antibodies to tackle antigen escape. Utilizing CD19 negative leukemia cells as targets, superior antibody-dependent cellular cytotoxicity (ADCC) was demonstrated by the combination of hnCD16 CAR iT and Rituximab (% specific cytotoxicity at E:T=1:1, hnCD16 group + Rituximab: 75.64 ± 2.12; control group + Rituximab: 16.98 ± 3.87, P<0.001). To address T cell fitness, the role of CD38 knockout (KO) in T cells was investigated, which we have previously shown to mediate NK cell resistance to oxidative stress induced apoptosis. CD38 gene was disrupted at the iPSC stage to generate 1XX-CAR T cells that lack CD38 expression (% CD38+ population, CD38WT group: 69.67 ± 24.34; CD38KO group: 0.12 ± 0.11) and upon antigen mediated stimulation, CD38KO CAR iT cells showed higher percentages of degranulation (2.3-fold increase in CD107a/b), and IFNγ (4.1-fold increase) and TNFα (2.5-fold increase) production. Antigen specific in vitro tumor killing also was enhanced in CD38KO CAR iT cells (EC50, 3.2-fold decrease). Lastly, to avoid the potential host-mediated rejection, the inclusion of allogeneic defense receptor (ADR) which has been shown to significantly reduce host-mediated rejection will be discussed. Collectively, the described studies demonstrate that iPSCs are an ideal cellular source to generate large-quantities of uniformly multi-edited off-the-shelf CAR T cell products that include a best-in-class CAR design, enhanced product modalities, and complete elimination of TCR expression to avoid the potential of GvHD while maintaining high anti-tumor efficacy in allogeneic setting. Disclosures Hsia: Fate Therapeutics Inc.: Current Employment. Clarke:Fate Therapeutics Inc.: Current Employment, Current equity holder in publicly-traded company. Lee:Fate Therapeutics, Inc.: Current Employment. Robbins:Fate Therapeutics, Inc.: Current Employment. Denholtz:Fate Therapeutics, Inc: Current Employment. Hanok:Fate Therapeutics, Inc.: Current Employment. Carron:Fate Therapeutics, Inc.: Current Employment. Navarrete:Fate Therapeutics, Inc.: Current Employment. ORourke:Fate Therapeutics, Inc.: Current Employment. Sung:Fate Therapeutics, Inc.: Current Employment. Gentile:Fate Therapeutics, Inc.: Current Employment. Nguyen:Fate Therapeutics, Inc.: Current Employment. Valamehr:Fate Therapeutics, Inc: Current Employment, Current equity holder in publicly-traded company.

Single-Cell RNA Sequencing Identifies Expression Patterns Associated with Clinical Responses to Dual-Targeted CAR-T Cell Therapy

INTRODUCTION: Chimeric Antigen Receptor (CAR)-T cell therapy is emerging as a powerful treatment for relapsed or refractory B cell lymphomas. However, a variety of escape mechanisms prevent CAR-T cell therapy from being more uniformly effective. To better understand mechanisms of CAR-T failure among patients treated with dual-targeted CAR-T cells, we performed single-cell RNA sequencing of samples from a Phase 1 trial (NCT03019055). The clinical trial used anti-CD20, anti-CD19 CAR-T cells for the treatment of relapsed/refractory B-cell non-Hodgkin Lymphoma. Clinical responses from this study are reported independently (Shah et al. in press in Nat Med). While robust clinical responses occurred, not all patients had similar outcomes. In single-antigen specific CAR-T cells, mechanisms of resistance include antigen down-regulation, phenotype switch, or PD-1 inhibition (Song et al. Int J Mol Sci 2019). However, very little is understood about the mechanisms of failure that are specific to dual-targeted CAR-T cells. Interestingly, loss of CD19 antigen was not observed in treatment failures in the study. METHODS: De-identified patient samples were obtained as peripheral blood mononuclear cells on the day of harvest ("pre" samples), at the peak of in vivo CAR-T cell expansion which varied from day 10 to day 21 after infusion ("peak" samples), and on day 28 post-infusion ("d28" samples). The CAR-T cell infusion product was obtained on day 14 of on-site manufacturing ("product" samples). All samples were cryopreserved and single cell preparation was performed with batched samples using 10X Genomics kits. Subsequent analysis was performed in R studio using the Seurat package (Butler et al. Nat Biotech 2018) with SingleR being used to identify cell types in an unbiased manner (Aran et al. Nat Immunol 2019). RESULTS: We found that distinct T cell clusters were similarly represented in the responder and non-responder samples. The patients' clinical responses did not depend on the level of CAR expression or the percentage of CAR+ cells in the infusion product. At day 28, however, there was a considerable decrease in the percentage of CAR+ cells in the responder samples possibly due to contracture of the CAR+ T cell compartment after successful clearance of antigen-positive cells. In all samples, the CAR-T cell population shifted from a CD4+ to a CD8+ T cell predominant population after infusion. We performed differentially-expressed gene analyses (DEG) of the total and CAR-T cells. In the pre samples, genes associated with T-cell stimulation and cell-mediated cytotoxicity were highly expressed in the responder samples. Since the responders had an effective anti-tumor response, we expected these pathways to also be enriched for in the peak samples; however, this was not the case. We hypothesize that differential expression of the above genes was masked due to homeostatic expansion of the T cells following conditioning chemotherapy. Based on the DEG results, we next interrogated specific genes associated with cytotoxicity, T cell co-stimulation, and checkpoint protein inhibition. Cytotoxicity-associated genes were highly expressed among responder CD8+ T cells in the pre samples, but not in the other samples (Figure 1). Few differences were seen in specific co-stimulatory and checkpoint inhibitor genes at any timepoint in the T cell clusters. We performed gene set enrichment analyses (GSEA). Gene sets representing TCR, IFN-gamma, and PD-1 signaling were significantly increased in the pre samples of the responders but not at later time points or in the infusion products. DISCUSSION: We found a correlation between expression of genes associated with T cell stimulation and cytotoxicity in pre-treatment patient samples and subsequent response to CAR-T cell therapy. This demonstrates that the existing transcriptome of T cells prior to CAR transduction critically shapes anti-tumor responses. Further work will discover biomarkers that can be used to select patients expected to have better clinical outcomes. Figure 1 Disclosures Johnson: Miltenyi Biotec: Research Funding; Cell Vault: Research Funding. Schneider:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties. Dropulic:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Hari:BMS: Consultancy; Amgen: Consultancy; GSK: Consultancy; Janssen: Consultancy; Incyte Corporation: Consultancy; Takeda: Consultancy. Shah:Incyte: Consultancy; Cell Vault: Research Funding; Lily: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria; Verastim: Consultancy; TG Therapeutics: Consultancy; Celgene: Consultancy, Honoraria; Miltenyi Biotec: Honoraria, Research Funding.

Modular Chimeric Antigen Receptor Systems for Universal CAR T Cell Retargeting.

The engineering of T cells through expression of chimeric antigen receptors (CARs) against tumor-associated antigens (TAAs) has shown significant potential for use as an anti-cancer therapeutic. The development of strategies for flexible and modular CAR T systems is accelerating, allowing for multiple antigen targeting, precise programming, and adaptable solutions in the field of cellular immunotherapy. Moving beyond the fixed antigen specificity of traditional CAR T systems, the modular CAR T technology splits the T cell signaling domains and the targeting elements through use of a switch molecule. The activity of CAR T cells depends on the presence of the switch, offering dose-titratable response and precise control over CAR T cells. In this review, we summarize developments in universal or modular CAR T strategies that expand on current CAR T systems and open the door for more customizable T cell activity.

Predicting the Efficacy and Safety of TACTICs (Tumor Angiogenesis-Specific CAR-T Cells Impacting Cancers) Therapy for Soft Tissue Sarcoma Patients.

Simple SummarySarcomas have few effective treatment options due to the rarity and diversity and have a high risk of recurrence and metastasis. Therefore, the development of new therapeutics that can meet their medical needs is required. Our adoptive immunotherapy strategy using T cells to express the chimeric antigen receptor (CAR) against vascular endothelial growth factor receptor 2 (VEGFR2), which is highly expressed on tumor vascular endothelial cells, has the potential to be a novel treatment against diverse sarcomas with abundant vascular invasion. Here, we optimized the manufacturing and transportation of anti-VEGFR2 CAR-mRNA-transfected T cells and collected information that allowed the extrapolation of their efficacy and safety potential for sarcoma patients. Our results support the development of a “first in humans” study to evaluate the potential of our anti-VEGFR2 CAR-T cell therapy as a new treatment option for sarcoma patients.Soft tissue sarcomas (STSs) are heterogeneous and aggressive malignancies with few effective therapies available. We have developed T cells expressing a vascular endothelial growth factor receptor 2 (VEGFR2)-specific chimeric antigen receptor (CAR) to establish a tumor angiogenesis-specific CAR-T cells impacting cancers (TACTICs) therapy. In this study, we optimized the manufacturing and transportation of mRNA-transfected anti-VEGFR2 CAR-T cells and collected information that allowed the extrapolation of the efficacy and safety potential of TACTICs therapy for STS patients. Although 5-methoxyuridines versus uridines did not improve CAR-mRNA stability in T cells, the utilization of CleanCap as a 5′ cap-structure extended the CAR expression level, increasing VEGFR2-specific cytotoxicity. Furthermore, 4 °C preservation conditions did not affect the viability/cytotoxicity of CAR-T cells, contrarily to a freeze-thaw approach. Importantly, immunohistochemistry showed that most of the STS patients’ specimens expressed VEGFR2, suggesting a great potential of our TACTICs approach. However, VEGFR2 expression was also detected in normal tissues, stressing the importance of the application of a strict monitoring schedule to detect (and respond to) the occurrence of adverse effects in clinics. Overall, our results support the development of a “first in humans” study to evaluate the potential of our TACTICs therapy as a new treatment option for STSs.

Abstract 2180: Genetically engineered chimeric antigen receptor (CAR) monocytes demonstrate targeted anti-tumor activity and differentiate into M1-polarized CAR macrophages

Abstract Despite rapid advances in cancer immunotherapy, clinical responses in metastatic solid tumors have been limited. Macrophages are the most abundant immune cell in the solid tumor microenvironment (TME) and are primarily recruited as monocytes by TME-derived chemokines. When not under the control of the TME, macrophages are potent immune effector cells capable of phagocytosis, T cell recruitment, and antigen presentation. We have previously demonstrated that CAR macrophages (CAR-M) have potent anti-tumor activity and overcome several of the barriers to success in solid tumor immunotherapy - trafficking, immunosuppression, and antigen heterogeneity. Currently, CAR-M are generated via ex vivo differentiation of peripheral blood monocytes into macrophages prior to genetic manipulation. To more closely recapitulate normal biologic behavior, we attempted to create CAR monocytes that could traffic and differentiate into CAR macrophages upon tumor penetration. Toward that goal, we genetically engineered CD14+ human monocytes without ex vivo differentiation and with minimal cell culture. Using the chimeric adenoviral vector Ad5f35, we engineered human CAR-monocytes targeted against HER2. CAR expression and viability both exceeded 90%. Ad5f35 transduced CAR monocytes survived and maintained CAR expression ex vivo for at least 21 days. CAR monocytes efficiently differentiated into CAR-expressing macrophages when treated with GM-CSF as determined by FACS-based phenotypic characterization and Wright-Giemsa staining. Anti-HER2 CAR monocytes eradicated HER2 expressing tumor cells in a time and dose-dependent manner, and had comparable potency to anti-HER2 CAR-M. Additionally, the CAR monocyte manufacturing process offered the logistical advantage of a short manufacturing process (approximately two days). We have previously demonstrated that CAR-M are polarized toward a pro-inflammatory M1 phenotype after transduction with Ad5f35. Similarly, CAR monocytes demonstrated elevated expression of M1 markers, and intriguingly after differentiation into CAR-expressing macrophages, HLA-DR, CD80, CD86, and other M1 markers remained elevated - suggesting that transduction prior to differentiation does not impact the pro-inflammatory impact of adenoviral vectors on myeloid cells. Taken together, this abstract describes the successful development of CAR-monocytes with the potential for a rapid manufacturing process. In addition to direct anti-tumor activity while in the monocyte phase, CAR monocytes have the capacity to differentiate into CAR macrophages in situ, which are in turn capable of phagocytosis, T cell recruitment, TME activation, and antigen presentation. Given the previously demonstrated pre-clinical efficacy of CT-0508 (an anti-HER2 CAR macrophage), the CAR monocyte platform described herein offers a shortened manufacturing process and a potential advantage in tumor penetration, which will be directly evaluated in upcoming studies. Citation Format: Konrad Gabrusiewicz, Maggie Schmierer, Andrew Best, Martha Zeeman, Yumi Ohtani, Linara Gabitova, Daniel Cushing, Saar Gill, Michael Klichinsky. Genetically engineered chimeric antigen receptor (CAR) monocytes demonstrate targeted anti-tumor activity and differentiate into M1-polarized CAR macrophages [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2180.

Abstract 3313: iPSC-derived natural killer cells expressing EGFR-CAR against glioblastoma

Abstract Background: Natural Killer (NK) cells are a type of immune cells that belong to the innate immune system and executes a rapid immune reaction against cancer cells and viruses without prior activation. The huge success of chimeric antigen receptor (CAR)-T cells has motivated scientists to genetically modify human NK cells with CARs to further improve their tumor killing capacity. To engineer NK cells with a CAR, NK cells can be isolated from peripheral blood and umbilical cord blood. NK cells can also be derived from induced pluripotent stem cells (iPSCs) to produce a homogenous and standardized group of NK cells for the treatment of cancer. Here, we test the feasibility to engineer our EGFR-CAR construct into iPSC-derived hematopoietic stem cells (iPSC-HSC) to generate EGFR-CAR NK cells that will eradicate glioblastoma (GBM). Material and Methods: iPSCs-derived HSCs were differentiated into NK cells using co-culture with a feeder stroma of OP9-DL1 cells in the presence of human cytokines and NK cells were assessed by flow cytometry. Expanded NK cells (with K562 feeder cells expressing mb-IL-21 and 4-1BB) and HSCs from peripheral blood were efficiently transduced with a retroviral vector, carrying a CAR targeting EGFR. CAR expression was assessed by flow cytometry. After transduction, the CAR-NK cells were collected for cytotoxicity assay to test their efficacy. CAR-HSCs were differentiated into CAR-NK cells utilizing OP9-DL1 stroma cells. Results: Our results demonstrated that iPSCs can be fully differentiated into NK cells expressing typical NK cell surface markers including CD56, NKp46, CD16 and NKG2A, etc. In addition, HSCs can be engineered with EGFR-CAR without affecting their differentiation into CAR-NK cells. Our engineered EGFR-CAR NK cells showed an enhanced antitumor activity against GBM compared to NK cells without the EGFR-CAR. Conclusion: Collectively, our study suggests the feasibility of engineering CAR-EGFR NK cells from iPSCs for effective “off the shelf” immunotherapy. Our approach can be utilized to generate an unlimited number of CAR-NK cells to treat patients with cancer such as GBM in the clinical setting. Citation Format: Melissa Yu, Anthony G. Mansour, Kun-Yu Teng, Guoqiang Sun, Yanhong Shi, Michael A. Caligiuri. iPSC-derived natural killer cells expressing EGFR-CAR against glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3313.

Abstract 4066: A non-integrating, non-viral DNA Nanovector platform for the safe, persistent, and rapid manufacture of recombinant T-cells for Adoptive cell therapy

Abstract The capability to introduce Chimeric Antigen Receptors (CARs) into naïve Human T-Cells represents one of the most promising therapeutic strategies for the treatment of cancer. However, virus mediated adoptive cell therapy (ACT) remain severely limited by two factors: the long lead time and high cost of GMP virus manufacture, and the virus safety profiles. What if the entire ACT process could be sped up, made safer and more cost-effective by at least an order of magnitude? We have invented a novel DNA Vector platform based on scaffold/matrix attachment region (S/MAR) component that provides the opportunity to efficiently generate genetically engineered T-cells. This system is based on a nanovector technology. It contains no immunogenic and comprises only clinically approved sequences. It is easy, simple and cost-efficient to produce. Critically, it does not integrate and replicates autonomously and extrachromosomally in the nuclei of dividing primary human cells, thus avoiding the inherent risk of integrative mutagenesis. Through a process of iterative CpG depletion, selection marker minimalisation, empirical promoter design and elimination of cryptic eukaryotic signals our nano-S/MARt DNA Vector (nS/MARt) can be efficiently transfected into primary human T Cells. nS/MARt vectors are designed to remain stably expressed, and in addition to having the best in class safety profile, they also demonstrate enhanced performance as a biopharmaceutical. Human T-cells engineered to express the CAR receptor against the carcinoembryonic antigen (CEA) using a nS/MARt vector provide more effective killing of human cancer cells in vitro than those engineered with integrative lentivirus. These results hold in vivo, where nS/MARt transfected CAR Tcells outperform the lentivirally transduced cells, attenuating tumour growth and extending mouse survival. Moreover, in pre-clinical studies, the comparison with the FDA approved drug Kymriah®,T cells modified with nS/MARt vectors harbouring the expression of a CD19 CAR are comparable to those engineered with the viral vector. Notably, we have also taken steps to evaluate nS/MARt's scalability and have succeeded in manufacturing a clinically relevant number of CAR-T Cells (2 × 107CAR+ T-cells per kilo, we estimate the production for an individual of 80 Kg). The extension of the results from mice to patients-scale required a 1000x scale up for the processing of T-cell transfection while halving the time for production to hit a meaningful therapeutic window. We have developed a novel manufacturing protocol where nS/MARt vectors can be used "off the shelf" for CAR-T therapy to generate a clinically relevant number of modified cells in just seven days. The delivery of our DNA to CD3+ cells, reaches ~60-70% with cell viability of 60%, that increases in the days that follow the cell electroporation. Thus, the most significant benefit will be for the patients that will be able to access the nS/MARt mediated therapy in 1 week. To translate this technology into a clinical reality a fermentation process that allows the preparation of 2.6 g/L of pure, supercoiled DNA was optimised. There is a pressing need to offer ACT to more oncology patients, and we believe that this novel DNA Vector system provides a unique and innovative approach to this therapeutic strategy for cancer therapy. Citation Format: Matthias Bozza, Alice De Roia, Aileen Berger, Alexandra Tuch, Patrick Schmidt, Richard Harbottle. A non-integrating, non-viral DNA Nanovector platform for the safe, persistent, and rapid manufacture of recombinant T-cells for Adoptive cell therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4066.

Abstract 6598: Eradication of EpCAM expressing solid tumors by low-affinity CAR T cells

Abstract Background: Adoptive cell transfer therapy with genetically engineered chimeric antigen receptor (CAR)-T cells is rapidly revolutionizing treatment against cancers. However, limited success of CAR T cells against large metastatic solid tumors has been made to date. It is largely due to safety concerns regarding on-target off-tumor toxicity to vital healthy tissues, and poor efficacy after adoptive transfer. Epithelial cell adhesion molecule (EpCAM) was found to be frequently over-expressed in a wide variety of carcinomas, including colon, gastric, pancreas, and breast cancers, making EpCAM an attractive molecule for targeted therapeutics. However, clinical trials with anti-EpCAM antibodies (ING-1, 3622W94) and bispecific T cell engager antibodies (Solitomab) have shown significant dose-limiting toxicities, resulting in very limited clinical responses. To selectively target EpCAM high tumors, we are developing affinity tuned EpCAM CAR T cells with varying affinities to EpCAM. Methods: The human glioblastoma cell line U251, breast cancer cell lines MDA-MB-231 and SK-BR-3, gastric cancer cell lines MKN28 and MKN45, pancreatic cancer cell lines SW1990 and Capan2, and colon cancer cell line HT29 were transduced with Luciferase-F2A-GFP (FLuc/GFP) lentivirus. EpCAM expression levels on tumor cell lines were measured by flow cytometry. Primary human T cells were transduced with EpCAM CAR lentivirus twice at 24 and 48 hours after activation with anti-CD3/CD28 Dynabeads. NSG mice bearing intraperitoneal or subcutaneous MKN28 xenografts, systemic metastatic MKN45 xenografts, and orthotopic Capan2 xenografts were treated with EpCAM CAR T cells to test the in vivo efficacy. PET/CT imaging was adopted to monitor T cell dynamics using 18F-NOTA-octreotide which binds specifically to somatostatin receptor 2 (SSTR2) introduced in CAR T cells. Results: Jurkat cells transduced with C215 and UBS54 CARs displayed 3.5 nM and 264.3 nM binding affinities to soluble EpCAM, respectively. EpCAM CAR expression in primary human T cells was higher than 50%. CD8+ to CD4+ T cell ratios were 1:1. Compared to high-affinity C215 CAR T cells, low-affinity UBS54 CAR showed equivalent cytotoxicity against high-density EpCAM-expressing targets (MKN28, MKN45 and HT29), but slower killing of low-density EpCAM-expressing cell line MDA-MB-231. T cell activation as monitored by CD69 upregulation and cytokine secretion (IL2, IFN-γ, TNF-α, GM-CSF and MIP-1β) of C215 and UBS54 CAR T cells were similar, and correlate well with EpCAM density on tumor cell surfaces. In immunodeficient mouse models, low-affinity UBS54 CAR potently eliminated intraperitoneal and subcutaneous gastric tumors (MKN28), systemic metastatic gastric tumor (MKN45), and orthotopic pancreatic tumor (Capan2). Notably, the level of T cell expansion in the course of tumor killing, observed by PET/CT imaging, was significantly lower for UBS54 CAR than high-affinity C215 CAR. Conclusions: Low-affinity EpCAM targeting CAR T cells demonstrated durable antitumor activity against various tumor models in vivo. To enhance safety and therapeutic efficacy, micromolar affinity anti-EpCAM CAR T cells are currently being evaluated to limit cell killing to EpCAM-positive tumor cells while sparing normal cells with basal levels of EpCAM expression. Citation Format: Yanping Yang, Jaclyn E. McCloskey, Huan Yang, Janusz Puc, Angel A. Gomez Gallegos, Yogindra Vedvyas, Irene M. Min, Eric von Hofe, Moonsoo M. Jin. Eradication of EpCAM expressing solid tumors by low-affinity CAR T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6598.

Abstract 888: Vδ1γδT-Cells: A unique allogeneic cell therapy platform for the treatment of a broad range of malignancies

Abstract The treatment of haematologic malignancies with adoptive cell therapy is largely limited to platforms based on patient-derived, autologous αβ T cells. Although successful, this approach comes with challenges including associated toxicities, risk of relapse, high production costs and a requirement to gene edit cells to avoid graft vs host disease (GvHD) risk if the therapy is to be used in an allogeneic setting. In contrast to αβ T cells, human Vδ1 γδ T cells are a subset of T cells defined by expression of heterodimeric T cell receptors (TCRs) composed of a γ chain paired to a Vδ1 chain. Vδ1 γδ T cell function is highly differentiated from αβ T cells, as target cell recognition is not MHC restricted and γδ T cells are not alloreactive. Allogeneic matching of patients is therefore not required for Vδ1 γδ T cell therapeutic approaches. Instead Vδ1 T cells elicit direct anti-tumour responses via activation of diverse receptor repertoires that recognise multiple ligands upregulated on the surface of transformed cells. These key features of Vδ1 T cell biology therefore confer several advantages when generating an allogeneic cell therapy platform. We have developed a good manufacturing practice (GMP) compliant, scalable process to generate αβ T cell depleted, γδ T cell cultures (93.5% ± 3.7 of live), enriched for Vδ1 T cells (67.9% ± 2.2% of live). The cryopreserved therapeutic product shows good recoverability and proliferation after thawing, an activated innate phenotype and produces high levels of IFNγ and chemokines which can stimulate activation of other cells of the immune system. Post-thaw, Vδ1 T cell product has cytotoxic activity against a variety of malignant leukaemia and lymphoma cells in vitro and the cells home to the bone marrow and mediate anti-tumour activity following IV dosing in an in vivo xenograft model. Importantly, Vδ1 T cells have a good safety profile and do not mediate; cytotoxic activity against healthy tissues, mixed lymphocyte reactions or GvHD in in vivo models. Building on our expertise for expanding Vδ1 T cells, we have now developed scalable transient and stable gene engineering platforms for the generation of Vδ1 T cells expressing chimeric antigen receptors (CAR) and other molecules. Genetically engineered cells show good expansion kinetics and recoverability and viability post-cryopreservation. The cells also maintain a favourable activated phenotype post-thaw characterised by high levels of expression of CD27, NCR receptors (DNAM1, NKG2D and NKp30) and stable CAR expression at the cell surface. We have now used the Vδ1 CAR-T platform to develop and screen CAR constructs designed to generate allogeneic CAR-T cell therapies that exploit the unique safety and activity profile of Vδ1 T cells while additionally potentiating on-target tumour cell killing. Using this approach and tool constructs containing CARs targeting CD19, we have demonstrated potent and enhanced killing of a B-cell tumour cell line without eliciting on-target bystander killing of healthy B cells. In addition, the Vδ1 CAR-T anti-tumour activity is not associated with release of cytokines that may potentiate off-target toxicities. These data demonstrate that Vδ1 T cells have the potential to provide a unique CAR-T platform capable of targeting a broader cancer antigen profile than is possible for conventional CAR-T therapies. Citation Format: Istvan Kovacs, Andre Simoes, Tim Recaldin, Elizabeth Reynolds, Katharina Bergerhoff, Mihil Patel, Rebecca Alade, Victoria Hillerdal, Andrew Hutton, Daniel Fowler, Joanna Kawalkowska, Kalle Soderstrom, Valentino Parravicini, Michael Koslowski, Oliver Nussbaumer, Alice Brown. Vδ1γδT-Cells: A unique allogeneic cell therapy platform for the treatment of a broad range of malignancies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 888.

Rapid In-Process Monitoring of Lentiviral Vector Particles by High-Performance Liquid Chromatography

Lentiviral vectors (LVs) are a popular gene delivery tool in cell and gene therapy and they are a primary tool for ex vivo transduction of T cells for expression of chimeric antigen receptor (CAR) in CAR-T cell therapies. Extensive process and product characterization are required in manufacturing virus-based gene vectors to better control batch-to-batch variability. However, it has been an ongoing challenge to make quantitative assessments of LV product because current analytical tools often are low throughput and lack robustness and standardization is still required. This paper presents a high-throughput and robust physico-chemical characterization method that directly assesses total LV particles. With simple sample preparation and fast elution time (6.24 min) of the LV peak in 440 mM NaCl (in 20 mM Tris-HCl [pH 7.5]), this ion exchange high-performance liquid chromatography (IEX-HPLC) method is ideal for routine in-process monitoring to facilitate the development of scalable and robust LV manufacturing processes. Furthermore, this HPLC method is suitable for the analysis of all in-process samples, from crude samples such as LV supernatants to final purified products. The linearity range of the standard curve is 3.13 × 108 to 1.0 × 1010 total particles/mL, and both the intra- and inter-assay variabilities are less than 5%.

Thermal Control of Engineered T-cells.

Genetically engineered T-cells are being developed to perform a variety of therapeutic functions. However, no robust mechanisms exist to externally control the activity of T-cells at specific locations within the body. Such spatiotemporal control could help mitigate potential off-target toxicity due to incomplete molecular specificity in applications such as T-cell immunotherapy against solid tumors. Temperature is a versatile external control signal that can be delivered to target tissues in vivo using techniques such as focused ultrasound and magnetic hyperthermia. Here, we test the ability of heat shock promoters to mediate thermal actuation of genetic circuits in primary human T-cells in the well-tolerated temperature range of 37-42 °C, and introduce genetic architectures enabling the tuning of the amplitude and duration of thermal activation. We demonstrate the use of these circuits to control the expression of chimeric antigen receptors and cytokines, and the killing of target tumor cells. This technology provides a critical tool to direct the activity of T-cells after they are deployed inside the body.

Preclinical evaluation of CD8+ anti-BCMA mRNA CAR T cells for treatment of multiple myeloma.

Chimeric antigen receptor (CAR) T-cell therapy remains limited to select centers that can carefully monitor adverse events. To broaden use of CAR T-cells in community clinics and in a frontline setting, we developed a novel CD8+ CAR T-cell product, Descartes-08, with predictable pharmacokinetics for treatment of multiple myeloma. Descartes-08 is engineered by mRNA transfection to express anti-BCMA CAR for a defined length of time. Descartes-08 express anti-BCMA CAR for 1 week, limiting risk of uncontrolled proliferation; produce inflammatory cytokines in response to myeloma target cells; and are highly cytolytic against myeloma cells regardless of presence of myeloma-protecting bone marrow stromal cells, exogenous a proliferation-inducing ligand, or drug resistance including IMiDs. The magnitude of cytolysis correlates with anti-BCMA CAR expression duration, indicating a temporal limit in activity. In the mouse model of aggressive disseminated human myeloma, Descartes-08 induces BCMA CAR-specific myeloma growth inhibition and significantly prolongs host survival (P<.0001). These preclinical data, coupled with an ongoing clinical trial of Descartes-08 in relapsed/refractory myeloma (NCT03448978) showing preliminary durable responses and a favorable therapeutic index, have provided the framework for a recently initiated trial of an optimized/humanized version of Descartes-08 (i.e., Descartes-11) in newly diagnosed myeloma patients with residual disease after induction therapy.