Memory Phenotype in Allogeneic Anti-BCMA CAR-T Cell Therapy (P-BCMA-ALLO1) Correlates with In Vivo Tumor Control

Abstract The emergence of CAR-T cell therapy has transformed the treatment of the previously refractory/relapsed multiple myeloma (MM). Yet, autologous CAR-T cells suffer from inconsistent manufacturing, long manufacturing timelines, and high cost, which can limit patient accessibility. To address these issues, we engineered a fully allogeneic anti-BCMA CAR-T cell candidate for MM from healthy donors (P-BCMA-ALLO1). Herein, we demonstrate that P-BCMA-ALLO1 maintains a T stem cell memory phenotype (T SCM) through genetic editing, which correlates with antitumor efficacy. Using Poseida's proprietary non-viral piggyBac® (PB) DNA Delivery System, in combination with the high-fidelity Cas-CLOVER™ (CC) Site-Specific Gene Editing System and a proprietary "booster molecule", we generated P-BCMA-ALLO1 from healthy donor T cells. We used CC to eliminate surface expression of TCR and MHC class I to make fully allogeneic CAR-T cells. In addition to the CAR molecule, PB enables the delivery of a selectable marker allowing the generation of a final cell product that is >95% CAR-positive. The inclusion of the "booster molecule" in the manufacturing process improves the expansion of gene-edited cells without compromising memory phenotype or function. This process can produce up to hundreds of patient doses from a single manufacturing run using one healthy donor, thereby significantly reducing manufacturing cost per dose. We characterized the phenotype and functionality of P-BCMA-ALLO1 using flow cytometry and Nanostring to assess their memory phenotype at both the protein and mRNA levels. Also analyzed was antitumor toxicity and proliferative capacity through multiple rounds of activation using in vitro co-culture assays and serial restimulation, respectively. The relationship of all characterizations with in vivo efficacy was then determined, as defined by control of tumor growth in an immunodeficient RPMI-8226 subcutaneous murine tumor model. We found that P-BCMA-ALLO1 is comprised of a high frequency of T SCM after editing (Fig. 1), and the maintenance of that memory phenotype correlates with antitumor efficacy. In vivo, these CAR-T cells are potent in controlling tumor growth, comparable to or better than autologous anti-BCMA CAR-T cells. Our analysis revealed that the expression of memory markers at the surface protein level (CD27, CD62L, CD127, CCR7) and mRNA level significantly correlate with in vivo tumor control. Conversely, suboptimal research products with worse in vivo outcomes express a more exhausted gene expression profile. We reveal from our analysis that the most effective P-BCMA-ALLO1 in vivo share similar characteristics: (1) these products were a result of efficient manufacturing, with >90% CAR+ and >99% TCR-; (2) they carry a memory phenotype, with 50-70% T scm and high proliferative capacity after multiple rounds of restimulation; (3) they are >90% viable; and (4) they show strong antitumor efficacy both in vitro and in vivo. We demonstrate that Tscm percentage in the final product correlates with antitumor activity. P-BCMA-ALLO1 is advancing rapidly towards the clinic (NCT04960579) to positively impact the outcomes of CAR-T therapy for MM patients. Figure 1: Memory composition of P-BCMA-ALLO1 research products. P-BCMA-ALLO1 consists mostly of stem cell memory (T scm) and central memory (T cm) T cells that are CD62L + as opposed to effector memory (T em) and effector (T eff) T cells. Figure 1 Figure 1. Disclosures Tseng: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Zhang: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Cranert: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Richter: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Marquez: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Qiu: Poseida Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Cho: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tan: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tong: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Domingo: Poseida Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Weiss: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Argus: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Sparks: Poseida Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Ostertag: Poseida: Current Employment, Current equity holder in publicly-traded company. Coronella: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company. Shedlock: Poseida Therapeutics: Current Employment, Current equity holder in publicly-traded company.

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Preservation of T-Cell Stemness with a Novel Expansionless CAR-T Manufacturing Process, Which Reduces Manufacturing Time to Less Than Two Days, Drives Enhanced CAR-T Cell Efficacy

Blood ◽

10.1182/blood-2021-146246 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2848-2848

Author(s):

Boris Engels ◽

Xu Zhu ◽

Jennifer Yang ◽

Andrew Price ◽

Akash Sohoni ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Ex Vivo ◽

Antitumor Efficacy ◽

Publicly Traded ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract Background: Extended T-cell culture periods in vitro deplete the CAR-T final product of naive and stem cell memory T-cell (T scm) subpopulations that are associated with improved antitumor efficacy. YTB323 is an autologous CD19-directed CAR-T cell therapy with dramatically simplified manufacturing, which eliminates complexities such as long culture periods. This improved T-Charge™ process preserves T-cell stemness, an important characteristic closely tied to therapeutic potential, which leads to enhanced expansion ability and greater antitumor activity of CAR-T cells. Methods: The new T-Charge TM manufacturing platform, which reduces ex vivo culture time to about 24 hours and takes <2 days to manufacture the final product, was evaluated in a preclinical setting. T cells were enriched from healthy donor leukapheresis, followed by activation and transduction with a lentiviral vector encoding for the same CAR used for tisagenlecleucel. After ≈24 hours of culture, cells were harvested, washed, and formulated (YTB323). In parallel, CAR-T cells (CTL*019) were generated using a traditional ex vivo expansion CAR-T manufacturing protocol (TM process) from the same healthy donor T cells and identical lentiviral vector. Post manufacturing, CAR-T products were assessed in T-cell functional assays in vitro and in vivo, in immunodeficient NSG mice (NOD-scid IL2Rg-null) inoculated with a pre-B-ALL cell line (NALM6) or a DLBCL cell line (TMD-8) to evaluate antitumor activity and CAR-T expansion. Initial data from the dose escalation portion of the Phase 1 study will be reported separately. Results: YTB323 CAR-T products, generated via this novel expansionless manufacturing process, retained the immunophenotype of the input leukapheresis; specifically, naive/T scm cells (CD45RO -/CCR7 +) were retained as shown by flow cytometry. In contrast, the TM process with ex vivo expansion generated a final product consisting mainly of central memory T cells (T cm) (CD45RO +/CCR7 +) (Fig A). Further evidence to support the preservation of the initial phenotype is illustrated by bulk and single-cell RNA sequencing experiments, comparing leukapheresis and final products from CAR-Ts generated using the T-Charge™ and TM protocols. YTB323 CAR-T cell potency was assessed in vitro using a cytokine secretion assay and a tumor repeat stimulation assay, designed to test the persistence and exhaustion of the cell product. YTB323 T cells exhibited 10- to 17-fold higher levels of IL-2 and IFN-γ secretion upon CD19-specific activation compared with CTL*019. Moreover, YTB323 cells were able to control the tumor at a 30-fold lower Effector:Tumor cell ratio and for a minimum of 7 more stimulations in the repeat stimulation assay. Both assays clearly demonstrated enhanced potency of the YTB323 CAR-T cells in vitro. The ultimate preclinical assessment of the YTB323 cell potency was through comparison with CTL*019 regarding in vivo expansion and antitumor efficacy against B-cell tumors in immunodeficient NSG mouse models at multiple doses. Expansion of CD3+/CAR+ T-cells in blood was analyzed weekly by flow cytometry for up to 4 weeks postinfusion. Dose-dependent expansion (C max and AUC 0-21d) was observed for both YTB323 and CTL*019. C max was ≈40-times higher and AUC 0-21d was ≈33-times higher for YTB323 compared with CTL*019 across multiple doses. Delayed peak expansion (T max) of YTB323 by at least 1 week compared with CTL*019 was observed, supporting that increased expansion was driven by the less differentiated T-cell phenotype of YTB323. YTB323 controlled NALM6 B-ALL tumor growth at a lower dose of 0.1×10 6 CAR+ cells compared to 0.5×10 6 CAR+ cells required for CTL*019 (Fig B). In the DLBCL model TMD-8, only YTB323 was able to control the tumors while CTL*019 led to tumor progression at the respective dose groups. This ability of YTB323 cells to control the tumor at lower doses confirms their robustness and potency. Conclusions: The novel manufacturing platform T-Charge™ used for YTB323 is simplified, shortened, and expansionless. It thereby preserves T-cell stemness, associated with improved in vivo CAR-T expansion and antitumor efficacy. Compared to approved CAR-T therapies, YTB323 has the potential to achieve higher clinical efficacy at its respective lower doses. T-Charge™ is aiming to substantially revolutionize CAR-T manufacturing, with concomitant higher likelihood of long-term deep responses. Figure 1 Figure 1. Disclosures Engels: Novartis: Current Employment, Current equity holder in publicly-traded company. Zhu: Novartis: Current Employment, Current equity holder in publicly-traded company. Yang: Novartis: Current Employment, Patents & Royalties. Price: Novartis: Current Employment. Sohoni: Novartis: Current Employment. Stein: Novartis: Current Employment. Parent: Novartis: Ended employment in the past 24 months; iVexSol, Inc: Current Employment. Greene: iVexSol, Inc: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company. Niederst: Novartis: Current Employment, Current equity holder in publicly-traded company. Whalen: Novartis: Current Employment. Orlando: Novartis: Current Employment. Treanor: Novartis: Current Employment, Current holder of individual stocks in a privately-held company, Divested equity in a private or publicly-traded company in the past 24 months, Patents & Royalties: no royalties as company-held patents. Brogdon: Novartis Institutes for Biomedical Research: Current Employment.

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Novel CAR T Modules (Tmod) to Target HLA-Class I Loss in Lymphoma

Blood ◽

10.1182/blood-2020-134041 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 23-24

Author(s):

Agnes E. Hamburger ◽

Breanna DiAndreth ◽

Mark E. Daris ◽

Melanie L. Munguia ◽

Kiran Deshmukh ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Publicly Traded ◽

Private Company ◽

Car T Cells ◽

The Past ◽

Car T Cell ◽

Car T

Background: Chimeric Antigen Receptor (CAR) T-cell therapy is a proven, powerful clinical modality. However, it is still limited by the fundamental obstacle of cancer therapy: discriminating cancer from normal cells. Current FDA-approved CAR T-cell therapies eliminate normal B cells, leaving patients with B cell aplasia, hypogammaglobulinemia, and susceptible to infection. HLA-Class I loss of heterozygosity (LOH) occurs at an average frequency of ~13% among cancers and specifically ~13% in DLBCL (Broad Institute TCGA database). These losses are irreversible and distinguish the cancer from normal cells. To exploit LOH at the HLA locus, we target the remaining allelic product in tumors with LOH. We evaluated a novel AND NOT Boolean logic gate CAR T module (Tmod) T-cell system to target HLA-A*02 (A2) LOH in lymphoma using both in vitro and in vivo models. Methods: To model tumor cells that have lost A2 via LOH, we used CD19+ Raji lymphoma tumor cells. To model the corresponding "normal" cells, we established CD19+ Raji cells stably expressing A2 (CD19+/A2+ Raji). We then engineered human primary T cells to express a modular signal-integration circuit designed to be activated only by CD19+ lymphoma that do not express A2 (CD19+/A2- Raji). Each primary Tmod CAR T cell expresses both a CD19 activator (A) module using a CD19-targeting 3rd generation CAR, and a separate A2-targeting blocker (B) module using a novel A2-targeting inhibitory receptor. Human primary Tmod CAR T cells were engineered to co-express the A/B modules. First, T cells were stimulated via CD3/CD28 activation, followed by A/B module lentivirus transduction, and enriched for the B module. In vitro Tmod CAR T cells were evaluated for selective killing of CD19+/A2- Raji compared with CD19+/A2+ Raji. For in vivo proof of concept, both CD19+/A2- Raji and CD19+/A2+ Raji cell lines were injected and established into flanks of immunocompromised NGS mice and challenged with adoptive transfer of engineered human primary Tmod CAR T cells. Results: Engineered primary Tmod CAR T cells selectively killed CD19+/A2- Raji and spared CD19+/A2+ Raji (Figure 1). Tmod CAR T cells reversibly cycled from a state of non-killing, "block", to cytotoxicity and back, depending on the CD19+/A2- Raji vs. CD19+/A2+ Raji cells to which they were exposed. Importantly, primary Tmod CAR T cells selectively eliminated only the CD19+/A2- Raji cells in mixed cultures. In vivo, Tmod CAR T cells selectively eradicated CD19+/A2- Raji. More importantly, Tmod CAR T cells did not eradicate CD19+/A2+ Raji in vivo. Conclusions: CD19-targeting Tmod CAR T cells demonstrated robust and selective killing, distinguishing Raji lymphoma lines, one with A2 (CD19+/A2+) and one without (CD19+/A2-), both in vitro and in vivo. A critical requirement for Tmod CAR T-cell therapy is to determine reversibility and lack of anergy in the kill-"block"-kill and "block"-kill-"block" scenarios. This result demonstrates that Tmod CAR T cells do not terminally differentiate into one state (blockade or activation), but rather can switch back and forth as they integrate signals from "normal" and tumor cells. Furthermore, because Tmod CAR T cells can selectively target malignant B cells, it may increase the clinical therapeutic window for CAR T. Tmod CAR T cells may provide a powerful system to address hematologic malignancies and solid tumors with HLA-Class I LOH. Disclosures Hamburger: A2 Biotherapeutics: Current Employment, Current equity holder in private company. DiAndreth:A2 Biotherapeutics: Current Employment. Daris:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Munguia:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Deshmukh:A2 Biotherapeutics: Current Employment. Mock:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Asuelime:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Lim:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Kreke:A2 Biotherapeutics: Current Employment, Current equity holder in private company; Gilead: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Tokatlian:A2 Biotherapeutics: Current Employment, Current equity holder in private company. Maloney:A2 Biotherapeutics: Consultancy, Current equity holder in publicly-traded company, Honoraria; Bioline Rx: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria; Gilead Science: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Juno Therapeutics: Consultancy, Honoraria, Patents & Royalties, Research Funding. Go:A2 Biotherapeutics: Current Employment, Current equity holder in private company; Amgen: Current equity holder in publicly-traded company; Allogene: Divested equity in a private or publicly-traded company in the past 24 months; Gilead: Current equity holder in publicly-traded company; Illumina: Divested equity in a private or publicly-traded company in the past 24 months. Kamb:A2 Biotherapeutics: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

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Orvacabtagene Autoleucel (orva-cel; JCARH125): A Fully Human BCMA-Targeted Second-Generation CAR T Cell Product Characterized By a Predominant Central Memory Phenotype with High in Vitro and In Vivo Proliferative Potential and Sustained In Vivo Persistence

Blood ◽

10.1182/blood-2020-136748 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 11-12

Author(s):

Lucrezia Colonna ◽

Garnet Navarro ◽

Todd Devries ◽

Valeria Beckett ◽

Anthony Amsberry ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Publicly Traded ◽

Memory Phenotype ◽

Drug Products ◽

Car T Cells ◽

Car T Cell ◽

Car T

Background: Orva-cel is an investigational B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T cell product genetically modified with a lentiviral vector to express a CAR construct with a unique fully human single-chain variable fragment, optimized spacer, and 4-1BB costimulatory and CD3ζ activation domains. Orva-cel is currently being evaluated for efficacy and safety in the ongoing phase 1/2 EVOLVE study (NCT03430011) in heavily pretreated patients with relapsed/refractory multiple myeloma. We characterized orva-cel drug products, manufactured using the process in place for the phase 2 portion of the study and intended for commercial manufacturing, for CAR+ T cell purity, phenotype, and function. Methods: Immunophenotyping was performed by flow cytometry of both surface and intracellular markers, including CD3, CD4, CD8, CD45, CCR7, CD45RA, CD28, CD27, and active caspase 3. Cytokine production after challenge with BCMA+ target cells was assessed by intracellular cytokine staining and Luminex multiplex assay of secreted cytokines, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and granzyme B (GrB). CAR-mediated in vitro proliferative capacity was measured after anti-idiotypic antibody stimulation using the IncuCyte Live-Cell Analysis System (Sartorius, Göttingen, Germany). In vivo CAR+ T cell proliferation and persistence were assessed by quantitative polymerase chain reaction (qPCR). Results: The orva-cel manufacturing process was designed to enable consistent production of highly pure CD3+ cell products (median frequency of CD3+ T cells, 99.96%; quartiles 1-3 interquartile range, 99.9%-100.0%; n = 81). Orva-cel drug products were characterized by high frequencies of less-differentiated CAR+ T cells, leading to a dominant central memory-like population (CCR7+CD45RA- CAR+ T cells) and substantial frequencies of naïve-like cells (CCR7+CD45RA+ CAR+ T cells) (Figure). When assayed for in vitro functional activity, orva-cel drug products showed robust antigen-specific cytokine and effector molecule production (IFN-γ, TNF-α, IL-2, and GrB) upon challenge with BCMA+ tumor cells, as well as vigorous proliferation in response to CAR stimulation. Preliminary correlative analysis suggested that the early memory phenotype may be linked to increased CAR+ T cell proliferative capacity, as determined by in vitro experiments and in vivo PK parameters (ie, maximum CAR+ T cell concentration observed in the blood [Cmax], time to Cmax, area under the curve from Day 0 to 28 [AUC0-28], and CAR+ T cell persistence at Month 3 and Month 6). Consistent with the early CAR T cell memory phenotype, qPCR analysis showed robust in vivo proliferation of CAR+ T cells after infusion, with a median Cmax of 1.54 × 105 transgene copies/µg DNA and median AUC0-28 of 1.61 × 106 transgene copies/µg DNA*day, as well as long-term in vivo persistence, with CAR+ T cells detected in 69% of patients at 6 months postinfusion. Conclusions: The orva-cel manufacturing process results in drug products characterized by highly pure T cells, with high frequencies of early memory and polyfunctional CAR+ T cells. Orva-cel drug products showed robust antigen-specific degranulation, production of multiple cytokines, sustained in vitro and in vivo proliferation, and in vivo persistence. Disclosures Colonna: Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Navarro:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Devries:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Beckett:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. Amsberry:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Radhakrishnan:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Piasecki:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Heipel:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Li:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Kavita:Bristol Myers Squibb Company: Current Employment, Current equity holder in publicly-traded company. Works:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. Mujacic:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment.

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Combinatorial Antigen Targeting Strategy for Acute Myeloid Leukemia

Blood ◽

10.1182/blood-2020-141540 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 22-23

Author(s):

Pinar Ataca Atilla ◽

Mary K McKenna ◽

Norihiro Watanabe ◽

Maksim Mamonkin ◽

Malcolm K. Brenner ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Tumor Control ◽

Publicly Traded ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T

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.

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CAR T-Cells Depend on the Coupling of NADH Oxidation with ATP Production

Cells ◽

10.3390/cells10092334 ◽

2021 ◽

Vol 10 (9) ◽

pp. 2334

Author(s):

Juan C. Garcia-Canaveras ◽

David Heo ◽

Sophie Trefely ◽

John Leferovich ◽

Chong Xu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nadh Oxidase ◽

Nadh Oxidation ◽

Antitumor Efficacy ◽

Atp Production ◽

Car T Cells ◽

Car T Cell ◽

Car T

The metabolic milieu of solid tumors provides a barrier to chimeric antigen receptor (CAR) T-cell therapies. Excessive lactate or hypoxia suppresses T-cell growth, through mechanisms including NADH buildup and the depletion of oxidized metabolites. NADH is converted into NAD+ by the enzyme Lactobacillus brevis NADH Oxidase (LbNOX), which mimics the oxidative function of the electron transport chain without generating ATP. Here we determine if LbNOX promotes human CAR T-cell metabolic activity and antitumor efficacy. CAR T-cells expressing LbNOX have enhanced oxygen as well as lactate consumption and increased pyruvate production. LbNOX renders CAR T-cells resilient to lactate dehydrogenase inhibition. But in vivo in a model of mesothelioma, CAR T-cell’s expressing LbNOX showed no increased antitumor efficacy over control CAR T-cells. We hypothesize that T cells in hostile environments face dual metabolic stressors of excessive NADH and insufficient ATP production. Accordingly, futile T-cell NADH oxidation by LbNOX is insufficient to promote tumor clearance.

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147 Memory phenotype in allogeneic anti-BCMA CAR-T cell therapy (P-BCMA-ALLO1) correlates with in vivo tumor control

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.147 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A155-A155

Author(s):

Hubert Tseng ◽

Yan Zhang ◽

Stacey Cranert ◽

Maximilian Richter ◽

Karl Marquez ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

T Cell Therapy ◽

Memory Phenotype ◽

Car T Cells ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T

BackgroundThe emergence of CAR-T cell therapy has transformed the treatment of refractory/relapsed multiple myeloma (MM). Yet, autologous CAR-T cells suffer from many manufacturing challenges including mainly consistency, toxicity, and cost. To address these issues, we engineered a fully allogeneic anti-BCMA CAR-T cell candidate for MM from healthy donors (P-BCMA-ALLO1). Herein, we demonstrate that this therapy maintains a stem cell memory T cell (TSCM) phenotype through editing which correlates with in vivo antitumor efficacy.MethodsUsing Poseida’s non-viral piggyBac® (PB) DNA Delivery System in combination with the high-fidelity Cas-CLOVER™ (CC) Site-Specific Gene Editing System and a proprietary ‘booster molecule’, we generated P-BCMA-ALLO1 from healthy donor T cells. We used CC to eliminate surface expression of both the TCR and MHC class I to make fully allogeneic CAR-T cells. In addition to the CAR molecule, PB enables the delivery of a selectable marker allowing the generation of a final cell product that is >95% CAR-positive. The inclusion of the ‘booster molecule’ in the manufacturing process improves the expansion of gene-edited cells without compromising memory phenotype or function. This process can produce up to hundreds of patient doses from a single manufacturing run which significantly reduces manufacturing cost per dose. We characterized the memory phenotype of P-BCMA-ALLO1 by assessing the mRNA and protein expression profiles of rested and activated CAR-T cells by flow cytometry and Nanostring analysis. We also assessed the antitumor capabilities of these cells using cytotoxicity assays and performed serial in vitro restimulation to assess the ability of P-BCMA-ALLO1 to undergo multiple rounds of activation and expansion. We then evaluated the relationship of these characteristics with in vivo efficacy, as defined by control of tumor in an immunodeficient RPMI-8226 subcutaneous murine tumor model.ResultsP-BCMA-ALLO1 is comprised of a high frequency of TSCM. It has potent in vivo antitumor activity, which is comparable to non-edited autologous anti-BCMA CAR-T cell therapy. Expression of memory markers at both mRNA and protein levels across individual lots significantly correlates with in vivo tumor control. Conversely, suboptimal research products with worse in vivo outcomes expressed an exhausted gene expression profile. Moreover, CAR-T products that are more effective in vivo are also more viable, cytotoxic, and proliferative following multiple rounds of restimulation in vitro.ConclusionsP-BCMA-ALLO1 is a highly potent and safe allogeneic anti-BCMA CAR with a manufacturing process that consistently maintains a TSCM phenotype, which correlates with antitumor efficacy. P-BCMA-ALLO1 is advancing rapidly towards the clinic (NCT04960579).

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4-1BB and optimized CD28 co-stimulation enhances function of human mono-specific and bi-specific third-generation CAR T cells

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-003354 ◽

2021 ◽

Vol 9 (10) ◽

pp. e003354

Author(s):

Emiliano Roselli ◽

Justin C Boucher ◽

Gongbo Li ◽

Hiroshi Kotani ◽

Kristen Spitler ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Function ◽

Third Generation ◽

Memory Phenotype ◽

Car T Cells ◽

Car T Cell ◽

Car T

BackgroundCo-stimulatory signals regulate the expansion, persistence, and function of chimeric antigen receptor (CAR) T cells. Most studies have focused on the co-stimulatory domains CD28 or 4-1BB. CAR T cell persistence is enhanced by 4-1BB co-stimulation leading to nuclear factor kappa B (NF-κB) signaling, while resistance to exhaustion is enhanced by mutations of the CD28 co-stimulatory domain.MethodsWe hypothesized that a third-generation CAR containing 4-1BB and CD28 with only PYAP signaling motif (mut06) would provide beneficial aspects of both. We designed CD19-specific CAR T cells with either 4-1BB or mut06 together with the combination of both and evaluated their immune-phenotype, cytokine secretion, real-time cytotoxic ability and polyfunctionality against CD19-expressing cells. We analyzed lymphocyte-specific protein tyrosine kinase (LCK) recruitment by the different constructs by immunoblotting. We further determined their ability to control growth of Raji cells in NOD scid gamma (NSG) mice. We also engineered bi-specific CARs against CD20/CD19 combining 4-1BB and mut06 and performed repeated in vitro antigenic stimulation experiments to evaluate their expansion, memory phenotype and phenotypic (PD1+CD39+) and functional exhaustion. Bi-specific CAR T cells were transferred into Raji or Nalm6-bearing mice to study their ability to eradicate CD20/CD19-expressing tumors.ResultsCo-stimulatory domains combining 4-1BB and mut06 confers CAR T cells with an increased central memory phenotype, expansion, and LCK recruitment to the CAR. This enhanced function was dependent on the positioning of the two co-stimulatory domains. A bi-specific CAR targeting CD20/CD19, incorporating 4-1BB and mut06 co-stimulation, showed enhanced antigen-dependent in vitro expansion with lower exhaustion-associated markers. Bi-specific CAR T cells exhibited improved in vivo antitumor activity with increased persistence and decreased exhaustion.ConclusionThese results demonstrate that co-stimulation combining 4-1BB with an optimized form of CD28 is a valid approach to optimize CAR T cell function. Cells with both mono-specific and bi-specific versions of this design showed enhanced in vitro and in vivo features such as expansion, persistence and resistance to exhaustion. Our observations validate the approach and justify clinical studies to test the efficacy and safety of this CAR in patients.

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109 Dominant-negative TGFβ receptor 2 enhances GPC3-targeting CAR-T cell efficacy against hepatocellular carcinoma

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0109 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A121-A121

Author(s):

Nina Chu ◽

Michael Overstreet ◽

Ryan Gilbreth ◽

Lori Clarke ◽

Christina Gesse ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Dominant Negative ◽

Car T Cells ◽

Car T Cell ◽

Car T

BackgroundChimeric antigen receptors (CARs) are engineered synthetic receptors that reprogram T cell specificity and function against a given antigen. Autologous CAR-T cell therapy has demonstrated potent efficacy against various hematological malignancies, but has yielded limited success against solid cancers. MEDI7028 is a CAR that targets oncofetal antigen glypican-3 (GPC3), which is expressed in 70–90% of hepatocellular carcinoma (HCC), but not in normal liver tissue. Transforming growth factor β (TGFβ) secretion is increased in advanced HCC, which creates an immunosuppressive milieu and facilitates cancer progression and poor prognosis. We tested whether the anti-tumor efficacy of a GPC3 CAR-T can be enhanced with the co-expression of dominant-negative TGFβRII (TGFβRIIDN).MethodsPrimary human T cells were lentivirally transduced to express GPC3 CAR both with and without TGFβRIIDN. Western blot and flow cytometry were performed on purified CAR-T cells to assess modulation of pathways and immune phenotypes driven by TGFβ in vitro. A xenograft model of human HCC cell line overexpressing TGFβ in immunodeficient mice was used to investigate the in vivo efficacy of TGFβRIIDN armored and unarmored CAR-T. Tumor infiltrating lymphocyte populations were analyzed by flow cytometry while serum cytokine levels were quantified with ELISA.ResultsArmoring GPC3 CAR-T with TGFβRIIDN nearly abolished phospho-SMAD2/3 expression upon exposure to recombinant human TGFβ in vitro, indicating that the TGFβ signaling axis was successfully blocked by expression of the dominant-negative receptor. Additionally, expression of TGFβRIIDN suppressed TGFβ-driven CD103 upregulation, further demonstrating attenuation of the pathway by this armoring strategy. In vivo, the TGFβRIIDN armored CAR-T achieved superior tumor regression and delayed tumor regrowth compared to the unarmored CAR-T. The armored CAR-T cells infiltrated HCC tumors more abundantly than their unarmored counterparts, and were phenotypically less exhausted and less differentiated. In line with these observations, we detected significantly more interferon gamma (IFNγ) at peak response and decreased alpha-fetoprotein in the serum of mice treated with armored cells compared to mice receiving unarmored CAR-T, demonstrating in vivo functional superiority of TGFβRIIDN armored CAR-T therapy.ConclusionsArmoring GPC3 CAR-T with TGFβRIIDN abrogates the signaling of TGFβ in vitro and enhances the anti-tumor efficacy of GPC3 CAR-T against TGFβ-expressing HCC tumors in vivo, proving TGFβRIIDN to be an effective armoring strategy against TGFβ-expressing solid malignancies in preclinical models.Ethics ApprovalThe study was approved by AstraZeneca’s Ethics Board and Institutional Animal Care and Use Committee (IACUC).

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AAV-Mediated In Vivo CAR Gene Therapy for Targeting Human T Cell Leukemia

10.1101/2021.02.15.431201 ◽

2021 ◽

Author(s):

Waqas Nawaz ◽

Bilian Huang ◽

Shijie Xu ◽

Yanlei Li ◽

Linjing Zhu ◽

...

Keyword(s):

Gene Therapy ◽

T Cells ◽

T Cell ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Car T Cells ◽

Car T Cell ◽

Human T Cell ◽

Car T

AbstractChimeric antigen receptor (CAR) T cell therapy is the most active field in immuno-oncology and brings substantial benefit to patients with B cell malignancies. However, the complex procedure for CAR T cell generation hampers its widespread applications. Here, we describe a novel approach in which human CAR T cells can be generated within the host upon injecting an Adeno-associated virus (AAV)vector carrying the CAR gene, which we call AAV delivering CAR gene therapy (ACG). Upon single infusion into a humanized NCG tumor mouse model of human T cell leukemia, AAV generates sufficient numbers of potent in vivo CAR cells, resulting in tumor regression; these in vivo generated CAR cells produce antitumor immunological characteristics. This instantaneous generation of in vivo CAR T cells may bypass the need for patient lymphodepletion, as well as the ex vivo processes of traditional CAR T cell production, which may make CAR therapy simpler and less expensive. It may allow the development of intricate, individualized treatments in the form of on-demand and diverse therapies.Significance StatementAAV can generate enough CAR cells within the host. That act as a living drug, distributed throughout the body, and persist for weeks, with the ability to recognize and destroy tumor cells.

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124 Functionalizing CAR T cells for selective proliferation and dual-targeting using the meditope technology

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.124 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A133-A133

Author(s):

Cheng-Fu Kuo ◽

Yi-Chiu Kuo ◽

Miso Park ◽

Zhen Tong ◽

Brenda Aguilar ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T

BackgroundMeditope is a small cyclic peptide that was identified to bind to cetuximab within the Fab region. The meditope binding site can be grafted onto any Fab framework, creating a platform to uniquely and specifically target monoclonal antibodies. Here we demonstrate that the meditope binding site can be grafted onto chimeric antigen receptors (CARs) and utilized to regulate and extend CAR T cell function. We demonstrate that the platform can be used to overcome key barriers to CAR T cell therapy, including T cell exhaustion and antigen escape.MethodsMeditope-enabled CARs (meCARs) were generated by amino acid substitutions to create binding sites for meditope peptide (meP) within the Fab tumor targeting domain of the CAR. meCAR expression was validated by anti-Fc FITC or meP-Alexa 647 probes. In vitro and in vivo assays were performed and compared to standard scFv CAR T cells. For meCAR T cell proliferation and dual-targeting assays, the meditope peptide (meP) was conjugated to recombinant human IL15 fused to the CD215 sushi domain (meP-IL15:sushi) and anti-CD20 monoclonal antibody rituximab (meP-rituximab).ResultsWe generated meCAR T cells targeting HER2, CD19 and HER1/3 and demonstrate the selective specific binding of the meditope peptide along with potent meCAR T cell effector function. We next demonstrated the utility of a meP-IL15:sushi for enhancing meCAR T cell proliferation in vitro and in vivo. Proliferation and persistence of meCAR T cells was dose dependent, establishing the ability to regulate CAR T cell expansion using the meditope platform. We also demonstrate the ability to redirect meCAR T cells tumor killing using meP-antibody adaptors. As proof-of-concept, meHER2-CAR T cells were redirected to target CD20+ Raji tumors, establishing the potential of the meditope platform to alter the CAR specificity and overcome tumor heterogeneity.ConclusionsOur studies show the utility of the meCAR platform for overcoming key challenges for CAR T cell therapy by specifically regulating CAR T cell functionality. Specifically, the meP-IL15:sushi enhanced meCAR T cell persistence and proliferation following adoptive transfer in vivo and protects against T cell exhaustion. Further, meP-ritiuximab can redirect meCAR T cells to target CD20-tumors, showing the versatility of this platform to address the tumor antigen escape variants. Future studies are focused on conferring additional ‘add-on’ functionalities to meCAR T cells to potentiate the therapeutic effectiveness of CAR T cell therapy.

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