ATA3219: A Potent Next-Generation Allogeneic Off-the-Shelf CD19-CAR T Therapy without the Need for Gene-Editing

INTRODUCTION The development of allogeneic CD19 chimeric antigen receptor (CAR) T cells from healthy donors is a significant focus in cell therapy and is anticipated to overcome the technical and logistical challenges associated with autologous CAR-T cells. Unlike gene-edited approaches, which require inactivation of the endogenous αβ T cell receptor to reduce the risk of Graft-versus-Host Disease (GvHD), allogeneic Epstein-Barr Virus (EBV)-targeted T cells represent a clinically-advanced treatment modality that, to-date, has demonstrated a favorable safety profile with limited risks of GvHD or cytokine release syndrome [Prockop et al. JCI, 2020; Prockop et al. Blood, 2019] . As an allogeneic CAR T cell platform, EBV T cells represent a unique composition that retains critical transducibility and function, and minimizes risks for GvHD and other host interactions, without requiring complex gene editing or other cell engineering approaches to facilitate use in the allogeneic setting. Recent clinical experience with allogeneic CD19 CAR-modified EBV T cells have further supported safe and effective clinical experience in the context of B cell malignancies [Curran KJ et al. TCT 2020]. Recent advances in next-generation stimulatory domains also represent potential for improvement on current CAR-T therapies. Specifically, a modified CD3ζ domain retaining signaling capacity in 1 of 3 immune-receptor-tyrosine-based-activation-motif (ITAM) regions (referred to as 1XX) is designed to extend functional persistence without compromising potency via calibration of antigen induced CAR signaling intensity to more physiologic levels [Feucht et al. Science Trans Med 2018]. Here, we describe the first preclinical evaluation of ATA3219, a next-generation allogeneic CD19 CAR T cell therapy, combining a non-edited allogeneic EBV T cell approach with a CAR signaling domain designed to improve upon the currently clinically validated CD19 targeted CAR therapies. METHODS and RESULTS We generated EBV T cells engineered with a CD19-targeted CAR containing a modified CD3ζ signaling domain, 1XX (CD19-1XX CAR+ EBV T cells). CD19-1XX CAR+ EBV T cells demonstrate high CAR expression, polyfunctionality, expansion and in vitro potency through HLA-independent killing of CD19+ targets. Furthermore, CD19-1XX CAR+ EBV T cells demonstrate highly potent antitumor activity in an established disseminated tumor model of acute lymphoblastic leukemia and is associated with long-term persistence of the product. No treatment-related toxicities were observed in this animal model. CONCLUSIONS This preclinical dataset for CD19-1XX CAR+ EBV T cells demonstrate, persistence, polyfunctional phenotype and efficient targeting of CD19-expressing tumor cells, both in vitro and in vivo, with limited allocytoxicity against antigen-negative, HLA-mismatched targets. These findings support advancing ATA3219 to clinical evaluation. Disclosures Pham: Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Spindler:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Hwang:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Brito:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Bulliard:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company. Aftab:Atara Biotherapeutics: Current Employment, Current equity holder in publicly-traded company.

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98 ATA3271: an armored, next-generation off-the-shelf, allogeneic, mesothelin-CAR T cell therapy for solid tumors

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0098 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A109-A109

Author(s):

Jiangyue Liu ◽

Xianhui Chen ◽

Jason Karlen ◽

Alfonso Brito ◽

Tiffany Jheng ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Next Generation ◽

Phase 3 ◽

T Cell Therapy ◽

Cell Therapies ◽

Car T Cells ◽

Car T

BackgroundMesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein with high expression levels in an array of malignancies including mesothelioma, ovaria, non-small cell lung cancer, and pancreatic cancers and is an attractive target antigen for immune-based therapies. Early clinical evaluation of autologous MSLN-targeted chimeric antigen receptor (CAR)-T cell therapies for malignant pleural mesothelioma has shown promising acceptable safety1 and have recently evolved with incorporation of next-generation CAR co-stimulatory domains and armoring with intrinsic checkpoint inhibition via expression of a PD-1 dominant negative receptor (PD1DNR).2 Despite the promise that MSLN CAR-T therapies hold, manufacturing and commercial challenges using an autologous approach may prove difficult for widespread application. EBV T cells represent a unique, non-gene edited approach toward an off-the-shelf, allogeneic T cell platform. EBV-specific T cells are currently being evaluated in phase 3 trials [NCT03394365] and, to-date, have demonstrated a favorable safety profile including limited risks for GvHD and cytokine release syndrome.3 4 Clinical proof-of-principle studies for CAR transduced allogeneic EBV T cell therapies have also been associated with acceptable safety and durable response in association with CD19 targeting.5 Here we describe the first preclinical evaluation of ATA3271, a next-generation allogeneic CAR EBV T cell therapy targeting MSLN and incorporating PD1DNR, designed for the treatment of solid tumor indications.MethodsWe generated allogeneic MSLN CAR+ EBV T cells (ATA3271) using retroviral transduction of EBV T cells. ATA3271 includes a novel 1XX CAR signaling domain, previously associated with improved signaling and decreased CAR-mediated exhaustion. It is also armored with PD1DNR to provide intrinsic checkpoint blockade and is designed to retain functional persistence.ResultsIn this study, we characterized ATA3271 both in vitro and in vivo. ATA3271 show stable and proportional CAR and PD1DNR expression. Functional studies show potent antitumor activity of ATA3271 against MSLN-expressing cell lines, including PD-L1-high expressors. In an orthotopic mouse model of pleural mesothelioma, ATA3271 demonstrates potent antitumor activity and significant survival benefit (100% survival exceeding 50 days vs. 25 day median for control), without evident toxicities. ATA3271 maintains persistence and retains central memory phenotype in vivo through end-of-study. Additionally, ATA3271 retains endogenous EBV TCR function and reduced allotoxicity in the context of HLA mismatched targets. ConclusionsOverall, ATA3271 shows potent anti-tumor activity without evidence of allotoxicity, both in vitro and in vivo, suggesting that allogeneic MSLN-CAR-engineered EBV T cells are a promising approach for the treatment of MSLN-positive cancers and warrant further clinical investigation.ReferencesAdusumilli PS, Zauderer MG, Rusch VW, et al. Abstract CT036: A phase I clinical trial of malignant pleural disease treated with regionally delivered autologous mesothelin-targeted CAR T cells: Safety and efficacy. Cancer Research 2019;79:CT036-CT036.Kiesgen S, Linot C, Quach HT, et al. Abstract LB-378: Regional delivery of clinical-grade mesothelin-targeted CAR T cells with cell-intrinsic PD-1 checkpoint blockade: Translation to a phase I trial. Cancer Research 2020;80:LB-378-LB-378.Prockop S, Doubrovina E, Suser S, et al. Off-the-shelf EBV-specific T cell immunotherapy for rituximab-refractory EBV-associated lymphoma following transplantation. J Clin Invest 2020;130:733–747.Prockop S, Hiremath M, Ye W, et al. A Multicenter, Open Label, Phase 3 Study of Tabelecleucel for Solid Organ Transplant Subjects with Epstein-Barr Virus-Driven Post-Transplant Lymphoproliferative Disease (EBV+PTLD) after Failure of Rituximab or Rituximab and Chemotherapy. Blood 2019; 134: 5326–5326.Curran KJ, Sauter CS, Kernan NA, et al. Durable remission following ‘Off-the-Shelf’ chimeric antigen receptor (CAR) T-Cells in patients with relapse/refractory (R/R) B-Cell malignancies. Biology of Blood and Marrow Transplantation 2020;26:S89.

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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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CD171- and GD2-specific CAR-T cells potently target retinoblastoma cells in preclinical in vitro testing

BMC Cancer ◽

10.1186/s12885-019-6131-1 ◽

2019 ◽

Vol 19 (1) ◽

Cited By ~ 5

Author(s):

Lena Andersch ◽

Josefine Radke ◽

Anika Klaus ◽

Silke Schwiebert ◽

Annika Winkler ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Cell Lines ◽

T Cell Therapy ◽

Retinoblastoma Cell ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract Background Chimeric antigen receptor (CAR)-based T cell therapy is in early clinical trials to target the neuroectodermal tumor, neuroblastoma. No preclinical or clinical efficacy data are available for retinoblastoma to date. Whereas unilateral intraocular retinoblastoma is cured by enucleation of the eye, infiltration of the optic nerve indicates potential diffuse scattering and tumor spread leading to a major therapeutic challenge. CAR-T cell therapy could improve the currently limited therapeutic strategies for metastasized retinoblastoma by simultaneously killing both primary tumor and metastasizing malignant cells and by reducing chemotherapy-related late effects. Methods CD171 and GD2 expression was flow cytometrically analyzed in 11 retinoblastoma cell lines. CD171 expression and T cell infiltration (CD3+) was immunohistochemically assessed in retrospectively collected primary retinoblastomas. The efficacy of CAR-T cells targeting the CD171 and GD2 tumor-associated antigens was preclinically tested against three antigen-expressing retinoblastoma cell lines. CAR-T cell activation and exhaustion were assessed by cytokine release assays and flow cytometric detection of cell surface markers, and killing ability was assessed in cytotoxic assays. CAR constructs harboring different extracellular spacer lengths (short/long) and intracellular co-stimulatory domains (CD28/4-1BB) were compared to select the most potent constructs. Results All retinoblastoma cell lines investigated expressed CD171 and GD2. CD171 was expressed in 15/30 primary retinoblastomas. Retinoblastoma cell encounter strongly activated both CD171-specific and GD2-specific CAR-T cells. Targeting either CD171 or GD2 effectively killed all retinoblastoma cell lines examined. Similar activation and killing ability for either target was achieved by all CAR constructs irrespective of the length of the extracellular spacers and the co-stimulatory domain. Cell lines differentially lost tumor antigen expression upon CAR-T cell encounter, with CD171 being completely lost by all tested cell lines and GD2 further down-regulated in cell lines expressing low GD2 levels before CAR-T cell challenge. Alternating the CAR-T cell target in sequential challenges enhanced retinoblastoma cell killing. Conclusion Both CD171 and GD2 are effective targets on human retinoblastoma cell lines, and CAR-T cell therapy is highly effective against retinoblastoma in vitro. Targeting of two different antigens by sequential CAR-T cell applications enhanced tumor cell killing and preempted tumor antigen loss in preclinical testing.

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Clinical Development of a Non-Gene-Edited Allogeneic Bcma-Targeting CAR T-Cell Product in Relapsed or Refractory Multiple Myeloma

Blood ◽

10.1182/blood-2020-139516 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 27-28

Author(s):

A. Samer Al-Homsi ◽

Sebastien Anguille ◽

Jason Brayer ◽

Dries Deeren ◽

Nathalie Meuleman ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Gene Editing ◽

Cell Product ◽

Car T Cells ◽

Car T Cell ◽

Car T

Background Autologous CAR T-cell therapy targeting the B-cell maturation antigen (BCMA) has shown impressive objective response rates in patients with advanced multiple myeloma (MM). Clinical grade manufacturing of autologous CAR T-cells has limitations including vein-to-vein delivery time delay and potentially sub-optimal immunological capability of T-cells isolated from patients with advanced disease. Allogeneic CAR T-cell products, whereby cells from healthy third-party donors are used to generate an "off-the-shelf" CAR T-cell product, have the potential to overcome some of these issues. To circumvent the primary potential risk of graft-versus-host disease (GvHD) associated with the use of allogeneic T-cells, abrogation of the T-cell receptor (TCR) expression in the CAR T-cells, via gene editing, is being actively pursued. To avoid the potential safety risks and manufacturing challenges associated with gene editing, the allogeneic CYAD-211 CAR T-cell product exploits short hairpin RNA (shRNA) interference technology to down-regulate TCR expression thus avoiding the risk of life-threatening GvHD. Aim The aim is to generate a BCMA-specific allogeneic CAR T-cell product using a non-gene editing approach and study its activity both in vitro and in vivo. CYAD-211 combines a BCMA-specific CAR with a single optimized shRNA targeting the TCR CD3ζ subunit. Downregulation of CD3ζ impairs the TCR expression on the surface of the donor T-cells, preventing their reactivity with the normal host tissue cells and potential GvHD induction. Maintaining all the elements required for the therapy within a single vector (all-in-one vector) provides some significant manufacturing advantages, as a solitary selection step will isolate cells expressing all the desired traits. Results CYAD-211 cells produce high amounts of interferon-gamma (IFN-γ) during in vitro co-cultures with various BCMA-expressing MM cell lines (i.e., RPMI-8226, OPM-2, U266, and KMS-11). Cytotoxicity experiments confirmed that CYAD-211 efficiently kills MM cell lines in a BCMA-specific manner. The anti-tumor efficacy of CYAD-211 was further confirmed in vivo, in xenograft MM models using the RPMI-8226 and KMS-11 cell lines. Preclinical data also showed no demonstrable evidence of GvHD when CYAD-211 was infused in NSG mice confirming efficient inhibition of TCR-induced activation. Following FDA acceptance of the IND application, IMMUNICY-1, a first-in-human, open-label dose-escalation phase I clinical study evaluating the safety and clinical activity of CYAD-211 for the treatment of relapsed or refractory MM patients to at least two prior MM treatment regimens, is scheduled to begin recruitment. IMMUNICY-1 will evaluate three dose-levels of CYAD-211 (3x107, 1x108 and 3x108 cells/infusion) administered as a single infusion after a non-myeloablative conditioning (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days) according to a classical Fibonacci 3+3 design. Description of the study design and preliminary safety and clinical data from the first cohort will be presented at ASH 2020. Conclusion CYAD-211 is the first generation of non-gene edited allogeneic CAR T-cell product based on shRNA technology. The IMMUNICY-1 clinical study seeks to provide proof of principle that single shRNA-mediated knockdown can generate fully functional allogeneic CAR T-cells in humans without GvHD-inducing potential. We anticipate that subsequent generations of this technology will incorporate multiple shRNA hairpins within a single vector system. This will enable the production of allogeneic CAR T-cells in which multiple genes of interest are modulated simultaneously thereby providing a platform approach that can underpin the future of this therapeutic modality. Figure 1 Disclosures Al-Homsi: Celyad: Membership on an entity's Board of Directors or advisory committees. Brayer:Janssen: Consultancy; Bristol-Myers Squibb, WindMIL Therapeutics: Research Funding; Bristol-Myers Squibb, Janssen, Amgen: Speakers Bureau. Nishihori:Novartis: Other: Research support to institution; Karyopharm: Other: Research support to institution. Sotiropoulou:Celyad Oncology: Current Employment. Twyffels:Celyad Oncology: Current Employment. Bolsee:Celyad Oncology: Current Employment. Braun:Celyad Oncology: Current Employment. Lonez:Celyad Oncology: Current Employment. Gilham:Celyad Oncology: Current Employment. Flament:Celyad Oncology: Current Employment. Lehmann:Celyad Oncology: Current Employment.

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Efficient elimination of primary B-ALL cells in vitro and in vivo using a novel 4-1BB-based CAR targeting a membrane-distal CD22 epitope

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-000896 ◽

2020 ◽

Vol 8 (2) ◽

pp. e000896

Author(s):

Talia Velasco-Hernandez ◽

Samanta Romina Zanetti ◽

Heleia Roca-Ho ◽

Francisco Gutierrez-Aguera ◽

Paolo Petazzi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

T Cell Therapy ◽

High Affinity ◽

Car T Cells ◽

Car T Cell ◽

Car T

BackgroundThere are few therapeutic options available for patients with B-cell acute lymphoblastic leukemia (B-ALL) relapsing as CD19– either after chemotherapy or CD19-targeted immunotherapies. CD22-chimeric antigen receptor (CAR) T cells represent an attractive addition to CD19-CAR T cell therapy because they will target both CD22+CD19– B-ALL relapses and CD19– preleukemic cells. However, the immune escape mechanisms from CD22-CAR T cells, and the potential contribution of the epitope binding of the anti-CD22 single-chain variable fragment (scFv) remain understudied.MethodsHere, we have developed and comprehensively characterized a novel CD22-CAR (clone hCD22.7) targeting a membrane-distal CD22 epitope and tested its cytotoxic effects against B-ALL cells both in in vitro and in vivo assays.ResultsConformational epitope mapping, cross-blocking, and molecular docking assays revealed that the hCD22.7 scFv is a high-affinity binding antibody which specifically binds to the ESTKDGKVP sequence, located in the Ig-like V-type domain, the most distal domain of CD22. We observed efficient killing of B-ALL cells in vitro, although the kinetics were dependent on the level of CD22 expression. Importantly, we show an efficient in vivo control of patients with B-ALL derived xenografts with diverse aggressiveness, coupled to long-term hCD22.7-CAR T cell persistence. Remaining leukemic cells at sacrifice maintained full expression of CD22, ruling out CAR pressure-mediated antigen loss. Finally, the immunogenicity capacity of this hCD22.7-scFv was very similar to that of other CD22 scFv previously used in adoptive T cell therapy.ConclusionsWe report a novel, high-affinity hCD22.7 scFv which targets a membrane-distal epitope of CD22. 4-1BB-based hCD22.7-CAR T cells efficiently eliminate clinically relevant B- CD22high and CD22low ALL primary samples in vitro and in vivo. Our study supports the clinical translation of this hCD22.7-CAR as either single or tandem CD22–CD19-CAR for both naive and anti-CD19-resistant patients with B-ALL.

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Inhibition of PP2A with LB-100 Enhances Efficacy of CAR-T Cell Therapy Against Glioblastoma

Cancers ◽

10.3390/cancers12010139 ◽

2020 ◽

Vol 12 (1) ◽

pp. 139 ◽

Cited By ~ 5

Author(s):

Jing Cui ◽

Herui Wang ◽

Rogelio Medina ◽

Qi Zhang ◽

Chen Xu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Therapeutic Potential ◽

T Cell Therapy ◽

Car T Cells ◽

Car T Cell ◽

Car T Cell Therapy ◽

Car T

Chimeric antigen receptor (CAR)-engineered T cells represent a promising modality for treating glioblastoma. Recently, we demonstrated that CAR-T cells targeting carbonic anhydrase IX (CAIX), a protein involved in HIF-1a hypoxic signaling, is a promising CAR-T cell target in an intracranial murine glioblastoma model. Anti-CAIX CAR-T cell therapy is limited by its suboptimal activation within the tumor microenvironment. LB-100, a small molecular inhibitor of protein phosphatase 2A (PP2A), has been shown to enhance T cell anti-tumor activity through activation of the mTOR signaling pathway. Herein, we investigated if a treatment strategy consisting of a combination of LB-100 and anti-CAIX CAR-T cell therapy produced a synergistic anti-tumor effect. Our studies demonstrate that LB-100 enhanced anti-CAIX CAR-T cell treatment efficacy in vitro and in vivo. Our findings demonstrate the role of LB-100 in augmenting the cytotoxic activity of anti-CAIX CAR-T cells and underscore the synergistic therapeutic potential of applying combination LB-100 and CAR-T Cell therapy to other solid tumors.

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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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Chimeric Antigen Receptor T Cells Targeting Integrin αvβ6 Expressed on Cholangiocarcinoma Cells

Frontiers in Oncology ◽

10.3389/fonc.2021.657868 ◽

2021 ◽

Vol 11 ◽

Author(s):

Nattaporn Phanthaphol ◽

Chalermchai Somboonpatarakun ◽

Kwanpirom Suwanchiwasiri ◽

Thaweesak Chieochansin ◽

Jatuporn Sujjitjoon ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Therapy ◽

Target Antigen ◽

Tissue Samples ◽

T Cell Therapy ◽

Car T Cells ◽

Integrin Αvβ6 ◽

Car T ◽

Signaling Domain

Cholangiocarcinoma (CCA) is a lethal bile duct cancer that responds poorly to current standard treatments. A new therapeutic approach is, therefore, urgently needed. Adoptive T cell transfer using chimeric antigen receptor (CAR) T cells is a new therapeutic modality with demonstrated efficacy in hematologic malignancies. However, its efficacy against solid tumors is modest, and further intensive investigation continues. An important factor that influences the success of CAR T cell therapy is the selection of a target antigen that is highly expressed on cancer cells, but markedly less so in normal cells. Integrin αvβ6 is upregulated in several solid tumors, but is minimally expressed in normal epithelial cells, which suggests integrin αvβ6 as an attractive target antigen for CAR T cell immunotherapy in CCA. We investigated integrin αvβ6 expression in pathological tissue samples from patients with liver fluke-associated CCA. We then created CAR T cells targeting integrin αvβ6 and evaluated their anti-tumor activities against CCA cells. We found overexpression of the integrin αvβ6 protein in 23 of 30 (73.3%) CCA patient tissue samples. Significant association between high integrin αvβ6 expression and short survival time (p = 0.043) was also observed. Lentiviral constructs were engineered to encode CARs containing an integrin αvβ6-binding peptide (A20) derived from foot-and-mouth disease virus fused with a second-generation CD28/CD3ζ signaling domain (A20-2G CAR) or with a fourth-generation CD28/4-1BB/CD27/CD3ζ signaling domain (A20-4G CAR). The A20-2G and A20-4G CARs were highly expressed in primary human T cells transduced with the engineered lentiviruses, and they exhibited high levels of cytotoxicity against integrin αvβ6-positive CCA cells (p < 0.05). Interestingly, the A20-2G and A20-4G CAR T cells displayed anti-tumor function against integrin αvβ6-positive CCA tumor spheroids (p < 0.05). Upon specific antigen recognition, A20-4G CAR T cells produced a slightly lower level of IFN-γ, but exhibited higher proliferation than A20-2G CAR T cells. Thus, the A20-4G CAR T cells with lower level of cytokine production, but with higher proliferation represents a promising potential adoptive T cell therapy for integrin αvβ6-positive CCA.

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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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Engineered Hypoimmune Allogeneic CAR T Cells Exhibit Innate and Adaptive Immune Evasion Even after Sensitization in Humanized Mice and Retain Potent Anti-Tumor Activity

Blood ◽

10.1182/blood-2021-150021 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1690-1690

Author(s):

Xiaomeng Hu ◽

Mo Dao ◽

Kathy White ◽

Corie Gattis ◽

Ryan Clarke ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Activation ◽

Cell Activation ◽

Nk Cell ◽

Publicly Traded ◽

Car T Cells ◽

Car T

Abstract Off-the-shelf CAR T cells may offer advantages over autologous strategies, including ease of manufacturing, improved quality control with avoidance of malignant contamination and T cell dysfunction as well as the ability to generate a final product from healthy T cells. While TCR editing can effectively prevent graft-versus-host reactions, the significant host-versus-graft immune response against histoincompatible T cells prevents the expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. The goal is to achieve improved rates of durable complete remissions by improving allogeneic CD19CAR persistence since it has been shown that autologous CAR T cells have greater durability over years than allogeneic CAR T cells (N Engl J Med. 2021;384(7):673-674). We describe here the engineering of human immune evasive CAR T cells based on our previously described hypoimmune technology (Nat Biotechnol 2019;37(3):252-258 and Proc Natl Acad Sci U S A 2021;118(28):e2022091118). A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced by this strategy. Since CD47 overexpression can block both NK cell and macrophage killing (J Exp Med 2021;218(3):e20200839), we hypothesized that T cells would lose their immunogenicity when human leukocyte antigen (HLA) class I and II genes are inactivated and CD47 is over-expressed. Human T cells from healthy donors were obtained by leukapheresis. To generate hypoimmune CD19CAR T cells, gene editing was used to delete b2m, CIITA, and TCR expression and lentiviral transduction was used to overexpress CD47 and CD19CAR containing a 4-1BB costimulatory domain to generate hypoimmune CAR T cells. Control T cells were unmanipulated except for lentiviral transduction used to overexpress the same CD19CAR and the deletion of the TCR. When transplanted into allogeneic humanized mice, hypoimmune CD19CAR T cells evade immune recognition by T cells even in previously sensitized animals as evidenced by a lack of T cell activation measured using ELISPOT analysis. In contrast, transplantation of non-hypoimmune-edited CD19CAR T cells generated from the same human donor resulted in a significant T cell activation (see figure: mean 59 and 558 spot frequencies for hypoimmune CD19CAR T cells and non-edited CD19CAR T cells, respectively; p<0.0001 unpaired T-test). In addition to evading T cells, immune cell assays show that CD47 overexpression protects hypoimmune CD19CAR T cells from NK cell and macrophage killing in vitro and in vivo. Relative CD47 expression levels were analyzed to understand the relevance of CD47 for protection from macrophage and NK cell killing. A blocking antibody against CD47 made the hypoimmune CAR T cells susceptible to macrophage and NK cell killing in vitro and in vivo, confirming the importance of CD47 overexpression to evade innate immune clearance. The hypoimmune CD19 CAR T cells retained their antitumor activity in both the Daudi and Nalm-6 B cell leukemia models, in vitro and in vivo. This indicated that the hypoimmune technology-i.e. isolated CD47 overexpression, deletion of b2m, CIITA, and TCR- did not show any effect on the cytotoxic potential of CD19 CAR T cells (see figure). These studies demonstrate that in vivo clearance of leukemic cells in NSG mice occurs across a range of tumor cell toCD19 CAR T cell ratios in a manner comparable to control, unedited CD19 CAR T cells (see figure). This result was validated using T cells from 3 different donors These findings show that, in these models, hypoimmune CD19 CAR T cells are functionally immune evasive in allogeneic humanized mouse recipients and have cytotoxic anti-tumor capacity. They suggest that hypoimmune CAR T cells could provide universal CAR T cells that are able to persist without immunosuppression. Furthermore, these data suggest that hypoimmune CD19 CAR T cells can be used in sensitized patients and for re-dosing strategies. Figure 1 Figure 1. Disclosures Hu: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Dao: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. White: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Gattis: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Clarke: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Landry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Basco: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Tham: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Tucker: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Luo: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Bandoro: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Chu: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Young: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Foster: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Dowdle: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Rebar: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Schrepfer: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

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