121 ICAM-1-specific affinity tuned CAR T cells expressing SSTR2 for real-time imaging

BackgroundThe use of CAR T cells for solid tumors has a number of challenges, such as lack of tumor-specific targets, CAR T cell exhaustion, and the immunosuppressive tumor microenvironment. To address these challenges, AffyImmune has developed technologies to affinity tune and track CAR T cells in patients. The targeting moiety is affinity tuned to preferentially bind to tumor cells overexpressing the target while leaving normal cells with low basal levels untouched, thereby increasing the therapeutic window and allowing for more physiological T cell killing. The CAR T cells are designed to express SSTR2 (somatostatin receptor 2), which allows for the tracking of CAR T cells in vivo via PET/CT scan using FDA-approved DOTATATE.MethodsAIC100 was generated by affinity tuning the I-domain of LFA-1, the physiological ligand to ICAM-1. Various mutants with 106-fold difference in affinity were evaluated for affinity. This allowed structure activity relationships to be conducted using CAR T cells expressing the various affinity mutants against targets with varying antigen densities. The variant with micromolar affinity was clearly the most effective in non-clinical animal models. AIC100 is currently being evaluated to assess safety, CAR T expansion, tumor localization, and preliminary activity in patients with advanced thyroid cancer in a phase I study (NCT04420754). Our study uses a modified toxicity probability interval design with three dosage groups of 10 x 106, 100 x 106, and 500 x 106 cells.ResultsPreclinical studies demonstrated greater in vivo anti-tumor activity and safety with lower affinity CAR T cells. A single dose of AIC100 resulted in tumor elimination and significantly improved survival of animals. AIC100 activity was confirmed in other high ICAM-1 tumor models including breast, gastric, and multiple myeloma. In a Phase I patient given 10-million CAR T cells, near synchronous imaging of FDG and DOTATATE revealed preliminary evidence of transient CAR T expansion and tumor reduction at multiple tumor lesions, with the peak of CAR T density coinciding with the spike in CAR T numbers in blood.ConclusionsWe have developed affinity tuned CAR T cells designed to selectively target ICAM-1 overexpressing tumor cells and to spatiotemporally image CAR T cells. Near-synchronous FDG and DOTATATE scans will enhance patient safety by early detection of off-tumor CAR T activity and validation of tumor response. We anticipate that our ‘tune and track’ technology will be widely applicable to developing potent yet safe CAR T cells against hard-to-treat solid cancers.Trial RegistrationNCT04420754Ethics ApprovalIRB number19-12021154IACUC (animal welfare): All animal experiments were performed in accordance with the National Institute of Health’s Guide for the Care and Use of Laboratory Animals. Animal handling protocols were approved by the Institutional Laboratory Animal Use and Care Committee of Weill Cornell Medicine (Permit Number: 2012–0063).

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116 Multi-antigen targeting of heterogenous solid tumors using CAR T cells secreting bi-specific T-cell engagers

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0116 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A128-A128

Author(s):

Martin Hosking ◽

Bishwas Shrestha ◽

Megan Boyett ◽

Soheila Shirinbak ◽

Angela Gentile ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Solid Tumors ◽

Tumor Cells ◽

Cytolytic Activity ◽

Laboratory Animals ◽

Car T Cells ◽

Car T

BackgroundAlthough CAR T cells have been shown to be effective and potent in treating several hematologic malignancies, engineered T-cell therapies have had limited success in addressing solid tumors. Unlike liquid tumors where uniformly expressed antigens are accessible and can be effectively targeted, tumor access and antigen heterogeneity are a significant barrier to the successful development of CAR-T cells in solid tumors.MethodsHere we demonstrate that the combination of a bi-specific T-cell engager (BiTE) targeting EpCAM with a CAR T cell targeting HER2 enhances the in vitro and in vivo anti-tumor activity against heterogenous solid tumors.ResultsWe observed a dose-dependent enhancement of cytolytic activity when EpCAM-specific BiTEs were titrated alongside 4D5-based HER2-specific CAR T cells against HER2low tumors, enhancing maximal cytolysis by two-fold compared to CAR T cells alone (figure 1). Moreover, the escape of HER2low tumor cells in mixed heterogenous culture systems was circumvented by the combination of HER2-specific CAR T cells and EpCAM-specific BiTEs. The enhancement of efficacy was further demonstrated in an established HER2low MDA-MB-231 xenografts. HER2-specific CAR T cells were unable to contain Her2low tumors, whereas tumor growth was effectively controlled in mice receiving both EpCAM-specific BiTEs and HER2-specific CAR T cells.Abstract 116 Figure 1EpCAM specific BiTEs supplement CAR-T efficacy in vitro (A) HER2 and EpCAM expression of SKOV3, MDA-MB-231, and K562 tumor cells was assessed by flow cytometry. (B) HER2 specific CAR-T rapidly targeted and lysed HER2High SKOV3 tumor cells as measured via xCelligence RTCA assay. (C) SKOV3 were co-cultured with untransduced CD8+ T cells and the indicated concentrations of EpCAM BiTE and specific cytolysis was assessed. (D) MDA-MB-231 (HER2low) tumor cells were co-cultured with HER2 CAR-T ± EpCAM BiTE and specific cytolysis was determinedConclusionsCollectively, these data demonstrate that multi-antigen targeting mediated by BiTEs and CARs extends overall anti-tumor efficacy in preclinical models of heterogenous solid tumors. Fate Therapeutics is currently using its proprietary induced pluripotent stem cell (iPSC) product platform to generate iPSC-derived CAR T cells and iPSC-derived CAR NK cells that secrete BiTEs for the treatment of solid tumors.Ethics ApprovalThese studies were approved by Fate Therapeutics Institutional Animal Care and Use Committee and were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals.

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CD19-Targeted Donor T Cells Exert Potent Graft Versus Lymphoma Activity and Attenuated Gvhd

Blood ◽

10.1182/blood.v120.21.451.451 ◽

2012 ◽

Vol 120 (21) ◽

pp. 451-451 ◽

Cited By ~ 2

Author(s):

Arnab Ghosh ◽

Marco L. Davila ◽

Lauren F. Young ◽

Christopher Kloss ◽

Gertrude Gunset ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Tumor Cells ◽

Acute Gvhd ◽

Tcr Signaling ◽

Car T Cells ◽

Donor T Cells ◽

Car T

Abstract Abstract 451 Chimeric antigen receptors (CAR) represent a potent strategy to target T cells against selected tumor antigens. Ongoing clinical trials indicate that autologous T cells expressing CARs targeting CD19, a B cell-associated antigen, can induce complete remission and B cell aplasia in patients with B cell malignancies. Donor CD19-CAR+ T cells could potentially be used to treat recipients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the risk of alloreactivity mediated by endogenous T cell receptors (TCR) triggering an acute GVHD is not known. This is partly due to the absence of in vivo models to study the relative effects of CAR and endogenous TCR signaling. For the first time, we have evaluated the relative effects of CD19-targeted donor T cells on the elimination of CD19+ B cells and endogenous TCR-mediated alloreactivity in mouse models of allo-HSCT. We generated a panel of retroviral vectors encoding mouse CD19-specific CARs: as a control, CD19-delta, a tail-less CAR lacking the CD3ζ signaling domain; CD19z1, which signals through its CD3ζ endodomain; and CD19-28z, which signals through CD28 and CD3ζ (Figure 1A). CD19z1+ and CD19-28z+ T cells mediated specific lysis of CD19-expressing tumors in vitro, while CD19-delta+ T cells did not. In order to assess the anti-tumor capacity of CD19-CAR+ T cells in vivo, we transferred the transduced B6 donor T cells into lethally irradiated BALB/c recipients that were administered T cell-depleted allografts and CD19+ lymphoma A20-TGL (B6–> BALB/c+A20-TGL). CD19-CAR+ T cells (CD19z1 and CD19-28z) mediated clearance of A20 tumor cells visualized by in vivo imaging of luciferase-expressing tumor cells (Figure 1B and data not shown) and significantly improved tumor free survival. CD19-CAR+ B6 T cells could sustain prolonged B cell hypoplasia when adoptively transferred into lethally irradiated haploidentical CBF1 recipients of T cell-depleted allografts (B6–> CBF1, Figure 1C). These data indicate that under alloreactive conditions, donor CD19-CAR+ T cell signaled through the CAR leading to specific elimination of CD19+ tumors and B lineage cells. In order to determine the risk of GVHD, we transferred the donor CD19-CAR+ T cells into haploidentical HSCT recipients. Interestingly, CD19-CAR+ T cells mediated significantly less acute GVHD, resulting in improved survival and lower GVHD scores (Figure 1D). Donor CD19-delta+ T cells however mediated lethal GVHD, indicating that the endogenous TCR mediated strong alloreactivity in the absence of CAR signaling. Similar results were obtained from experiments using MHC-mismatched (B6–> BALB/c) models. It is known that signaling through endogenous TCR is accompanied by down-regulation of surface TCR expression. We found significant decreases in surface CD3ϵ, TCRβ and CD90 expressions in donor CD19-delta+ T cells under alloreactive conditions. In contrast, donor CD1928z+ T cells failed to down-regulate surface TCR expression under similar conditions, suggesting that endogenous TCR function was altered in CAR-activated T cells. In the context of allo-HSCT, preferential CAR signaling at the expense of alloreactive endogenous TCR signaling may thus lead to reduced alloreactivity and attenuation of GVHD. These results provide the first pre-clinical evidence suggesting that CAR-modified, unselected donor T cells may be safely applied in an allogeneic context. Disclosures: No relevant conflicts of interest to declare.

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Piggybac Transposon Mediated CD19 CAR-T Cells Derived from CD45RA-Positive PBMCs Possess Potent and Sustained Antileukemic Function

Blood ◽

10.1182/blood-2021-145863 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2807-2807

Author(s):

Masaya Suematsu ◽

Shigeki Yagyu ◽

Nobuyoshi Nagao ◽

Susumu Kubota ◽

Yuto Shimizu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Research Funding ◽

Stress Test ◽

Transduction Efficiency ◽

Antigen Stimulation ◽

Car T Cells ◽

Car T

Abstract Background: The quality of chimeric antigen receptor (CAR)-T cell products, including the expression of memory and exhaustion markers, has been shown to influence their long-term functionality. We previously demonstrated that piggyBac (PB) transposon-mediated CD19 CAR-T cells exhibit memory-rich phenotype that is characterized by a high proportion of CD45RA+/CCR7+ T cell fraction. To further investigate the favorable phenotype of PB-CD19 CAR-T cells, we generated PB-CD19 CAR-T cells from CD45RA+ and CD45RA− peripheral blood mononuclear cells (PBMCs) (RA+ CAR and RA− CAR, respectively), and compared their phenotype and antitumor function. Methods: CD45RA+ and CD45RA− PBMCs were isolated by magnetic selection from whole PBMCs, then the CD19-CAR transgene was transduced into these cells using the PB transposon system, as described previously. Transduction efficiency of CD19 CAR transgene was determined 24 hours by flow cytometry after transduction. The phenotype of CD19 CAR-T was evaluated by flow cytometry on day 14. High throughput RNA sequencing was performed to see the T cell activation/exhaustion profile upon antigen stimulation. Sequential killing assays were performed by adding fresh tumor cells into CAR-T cells co-cultured with tumor cells every three days by restoring an effector target ratio of 1:1. To see the durable antitumor efficacy in vivo, we performed in vivo stress test, in which CAR T-cells dosage was lowered to the functional limits, so that these CAR-T cells should be maintained and expanded in vivo, to achieve the antitumor efficacy. We injected 5 x 10 5 of firefly luciferase-labeled CD19+ tumor cells (REH) into NSG mice via tail vein, then these mice were treated with 1 x 10 5 of CD19 RA+ CAR-T, RA− CAR-T, or control CAR-T cells, respectively, at day 6 after the tumor injection. Results: RA+ CAR T cells demonstrated better transient transduction efficiency 24 h after transduction (RA+ CAR-T: 77.5 ± 9.8% vs RA− CAR-T: 39.7 ± 3.8%), and superior expansion capacity after 14 days of culture than RA− CAR-T cells (RA+ CAR-T: 32.5 ± 9.3-fold vs RA− CAR-T: 11.1 ± 5.4-fold). RA+ CAR-T cells exhibited dominant CD8 expression (RA+ CAR-T: 84.0 ± 3.4% vs RA− CAR-T: 34.1 ± 10.6%), less expression of exhaustion marker PD-1 (RA+ CAR-T: 3.1 ± 2.5% vs RA− CAR-T: 19.2 ± 6.4%) and T cell senescence marker CD57 (RA+ CAR-T: 6.8 ± 3.6% vs RA− CAR-T: 20.2 ± 6.9%), and enrichment of naïve/stem cell memory fraction (CAR+/CD45RA+CCR7+ fraction; RA+ CAR-T: 71.9 ± 9.7% vs RA− CAR-T: 8.0 ± 5.3%), which were associated with longevity of CAR-T cells. Transcriptome analysis revealed that RA+ CAR-T cells exhibited the enrichment of naïve/memory phenotype and less expression of canonical exhaustion markers, and these exhaustion profiles even maintained after the antigen stimulation. RA+ CAR-T cells demonstrated sustained killing activity even after multiple tumor rechallenges in vitro, without inducing exhaustion marker expression of PD-1. Although antigen stimulation could increase CAR expression, leading to tonic CAR signaling and exhaustion, in our study, the expression of CAR molecule on the cell surface following antigen stimulation in RA+ CAR was controlled at a relatively lower level that in RA− CAR-T cells. RA+ CAR-T cells achieved prolonged tumor control with expansion of CAR-T cells than RA− CAR-T cells in in vivo stress test (Fig.1A-C). On day15, bone marrow studies in RA+ CAR group exhibited abundant human CD3 positive T cells with less expression of PD-1, and relatively smaller amount of REH cells than RA− CAR group (Fig.1D). Furthermore, in two of long-lived mice in RA+ CAR group, human CD3 positive T cells were expanded even day 50 after treatment as confirmed by sequential bone marrow studies (Fig.1E), which indicated the antigen-induced proliferation and long-term functionality of RA+ CAR-T cells in vivo. Conclusion: Our results suggest that PB-mediated RA+ CAR-T cells exhibit memory-rich phenotype and superior antitumor function, thereby indicating the usefulness of CD45RA+ PBMC as a starting material of PB-CAR-T cells. Figure 1 Figure 1. Disclosures Yagyu: AGC Inc.: Research Funding. Nagao: AGC Inc.: Current Employment. Kubota: AGC Inc.: Current Employment. Shimizu: AGC Inc.: Current Employment. Nakazawa: AGC Inc.: Research Funding; Toshiba Corporation: Research Funding.

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IL-18 Secreting CAR T Cells Enhance Cell Persistence, Induce Prolonged B Cell Aplasia and Eradicate CD19+ Tumor Cells without Need for Prior Conditioning

Blood ◽

10.1182/blood.v128.22.816.816 ◽

2016 ◽

Vol 128 (22) ◽

pp. 816-816 ◽

Cited By ~ 10

Author(s):

Mauro P. Avanzi ◽

Dayenne G. van Leeuwen ◽

Xinghuo Li ◽

Kenneth Cheung ◽

Hyebin Park ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Tumor Cells ◽

Car T Cells ◽

Car T Cell ◽

Cytokine Analysis ◽

Car T ◽

Ifn Γ

Abstract Chimeric antigen receptor (CAR) T cell therapy has consistently shown significant results against acute lymphoblastic leukemia (ALL) in clinical trials1. However, results with other hematological or solid malignancies have been far more modest2. These disparate outcomes could be partially due to an inhibitory tumor microenvironment that suppresses CAR T cell function3. Thus, in order to expand the anti-tumor CAR T cell applications, a novel strategy in which these cells are capable of overcoming the hostile tumor microenvironment is needed. The cytokine interleukin-18 (IL-18) induces IFN-γ secretion, enhances the Th1 immune response and activates natural killer and cytotoxic T cells4. Early phase clinical trials that utilized systemic administration of recombinant IL-18 for the treatment of both solid and hematological malignancies have demonstrated the safety of this therapy5. We hypothesize that CAR T cells that constitutively secrete IL-18 could enhance CAR T cell survival and anti-tumor activity, and also activate cells from the endogenous immune system. To generate CAR T cells that constitutively secrete IL-18, we modified SFG-1928z and SFG-19m28mz CAR T cell constructs and engineered bicistronic human and murine vectors with a P2A element to actively secrete the IL-18 protein (1928z-P2A-hIL18 and 19m28mz-P2A-mIL18, respectively). Human and mouse T cells were transduced with these constructs and in vitro CAR T cell function was validated by coculturing the CAR T cells with CD19+ tumor cells and collecting supernatant for cytokine analysis. Both human and mouse CAR T cells secreted increased levels of IL-18, IFN-γ and IL-2. Proliferation and anti-tumor cytotoxic experiments were conducted with human T cells by coculturing CAR T cells with hCD19+ expressing tumor cells. 1928z-P2A-hIL18 CAR T cells had enhanced proliferation over 7 days and enhanced anti-tumor cytotoxicity over 72 hours when compared to 1928z CAR T cells (p=0.03 and 0.01, respectively) Next, the in vivo anti-tumor efficacy of the IL-18 secreting CAR T cell was tested in xenograft and syngeneic mouse models. Experiments were conducted without any prior lympho-depleting regimen. In the human CAR T cell experiments, Scid-Beige mice were injected with 1x106 NALM-6 tumor cells on day 0 and 5x106 CAR T cells on day 1. Survival curves showed a significant improvement in mouse survival with the 1928z-P2A-hIL18 CAR T cell treatment when compared to 1928z CAR T cell (p=0.006). Subsequently, to determine if IL-18 secreting CAR T cells could also improve anti-tumor efficacy in immunocompetent mice, we tested the murine 19m28mz-P2A-mIL18 CAR T cells in a syngeneic mouse model. The C57BL/6 hCD19+/- mCD19+/- mouse model was utilized and injected with 1x106 EL4 hCD19+ tumor cells on day 0 and 2.5 x106 CAR T cells on day 1. Mice treated with 19m28mz-P2A-mIL18 CAR T cells had 100% long-term survival, when compared to 19m28mz (p<0.0001). 19m28mz-P2A-mIL18 CAR T cells were detected in peripheral blood for up to 30 days after injection, whereas the 19m28mz CAR T cells were not detectable at any time point. In addition, 19m28mz-P2A-mIL18 CAR T cells were capable of inducing B cell aplasia for greater than 70 days, whereas 19m28mz treatment was not capable of inducing B cell aplasia. In vivo serum cytokine analysis demonstrated that 19m28mz-P2A-mIL18 CAR T cells, as compared to 19m28mz, significantly increased the levels of IFN-γ and TNF-α in the peripheral blood for up to 14 days after injection (p<0.0001 and 0.01, respectively). Despite the increase in IFN-γ and TNF-α cytokines, there was no increase in IL-6 levels. Our findings demonstrate that anti-CD19 CAR T cells that constitutively secrete IL-18 significantly increase serum cytokine secretion, enhance CAR T cell persistence, induce long-term B cell aplasia and improve mouse survival, even without any prior preconditioning. To our knowledge, this is the first description of an anti-CD19 CAR T cell that constitutively secretes IL-18 and that induces such high levels of T cell proliferation, persistence and anti-tumor cytotoxicity. We are currently investigating other mechanisms by which this novel CAR T cell functions, its interactions with the endogenous immune system, as well as testing its applicability in other tumor types. We anticipate that the advances presented by this new technology will expand the applicability of CAR T cells to a wider array of malignancies. Disclosures Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

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EXTH-20. SYNGENEIC B7-H3-SPECIFIC CAR T-CELLS HAVE POTENT ANTI-BRAIN TUMOR ACTIVITY VIA LOCAL OR SYSTEMIC DELIVERY

Neuro-Oncology ◽

10.1093/neuonc/noaa215.374 ◽

2020 ◽

Vol 22 (Supplement_2) ◽

pp. ii91-ii91

Author(s):

Dalia Haydar ◽

Zhongzhen Yi ◽

Haley Houke ◽

Martine F Roussel ◽

Chris DeRenzo ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Brain Tumors ◽

Tumor Cells ◽

Systemic Delivery ◽

Car T Cells ◽

Car T ◽

Anti Tumor Activity ◽

Gl261 Glioma

Abstract BACKGROUND We and others have identified B7-H3 (CD276) as a promising target for CAR T-cell-based immunotherapies for pediatric brain tumors. So far, B7-H3-CAR T cells have only been studied in xenograft models for brain tumors, which do not recapitulate the immunosuppressive tumor microenvironment (TME). To overcome this obstacle, we decided to adapt the immune competent GL261 murine glioma model which mimics human disease and host immune barriers. METHODS To evaluate their safety and efficacy, murine B7-H3-CAR T-cells were generated using retroviral particles encoding a 2nd generation B7-H3-CAR with a CD28.z signaling domain. Expansion, persistence, and anti-tumor activity were evaluated in vitro and in vivo. Components of the brain TME were then evaluated using flow cytometry and immunostaining. RESULTS B7-H3-CAR T cells only killed B7-H3+ tumor cells, secreted significant levels of IFNγ and IL-2 in an antigen-dependent manner and expanded an average of 85-fold in repeat stimulation assay with B7-H3+ tumor cells in contrast to control CAR T-cells. In vivo, intratumoral (2x106) or systemic (3x106) injection of syngeneic B7-H3-CAR T-cells into mice with orthotopic GL261 glioma induced complete regression in 60% of treated mice resulting in a significant survival advantage. Mice showed no evidence of acute or long-term toxicities related to CAR T-cell infusions. We confirmed this encouraging safety profile by systemic administration of a high dose (1x107) B7-H3-CAR T-cells and performing histological analyses of all major organs on day 14 post T-cell injection, which showed no notable signs of injury or on-target/off-tumor toxicities. CONCLUSIONS We successfully generated syngeneic B7-H3-CAR T-cells and have demonstrated that these cells have potent anti-tumor activity in the immune competent GL261 glioma model via local or systemic delivery without apparent toxicities. Our study paves the way for future testing of B7-H3-CAR T-cells in early phase clinical studies.

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CD4-Targeted Fusosomes Are Capable of Transducing Resting T Helper Cells to Generate Highly Potent CAR-T Cells

Blood ◽

10.1182/blood-2021-148469 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2942-2942

Author(s):

Christie Ciarlo ◽

Zach Frye ◽

Andre DeGroot ◽

Walter Flores ◽

Kutlu Elpek ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Cd4 T Cells ◽

Transduction Efficiency ◽

Publicly Traded ◽

Car T Cells ◽

Car T ◽

Integrating Vector

Abstract Introduction: Chimeric antigen receptor T cell therapy (CAR T) is a successful treatment for B cell malignancies; however, the time, complexity and cost of manufacturing autologous CAR T products limits the availability of these therapies to patients. Furthermore, ex vivo manipulation of T cells is likely to have a negative impact on quality. In vivo gene delivery of CAR T transgenes by systemic infusion of standard lentiviral vectors may increase therapeutic accessibility but is limited by off-target transduction and the requirement for T cell activation. Here, we demonstrate that a paramyxovirus-based integrating vector (fusosome) engineered with a CD4 re-targeted envelop (CD4 fusogen) can efficiently and specifically transduce resting and activated CD4+ T cells to generate functional CD4+ CD19-specific CAR T cells capable of eliminating CD19+ lymphoma cells. Methods: Anti-CD4 single chain variable fragments () and single variable domain (VHHs) were screened for CD4 binding, specificity, and NHP cross-reactivity and inserted into receptor binding paramyxovirus fusogen. CD4-targeted fusosomes expressing GFP were screened for high on-target titer against the CD4+ SupT1 cell line and low off-target transduction on non-CD4 expressing cells. Subsequently, a CD19-specific CAR encoding 4-1BB and the CD3z endo-domains (CD19 CAR) was generated to examine CD4+ CAR T transduction efficiency and functionality. PBMCs were thawed and activated with anti-CD3/anti-CD28 beads and exposed to GFP, CD4-targeted fusosomes and specificity of targeting CD4+ T cells was measured by flow cytometry. Subsequently, CD19 CAR fusosomes targeting CD4 were used to test transduction efficiency against activated (CD3/CD28 or IL-7 treated) or resting T cells, and to measure T cell function against CD19+ and CD19 knockout (CRISPR/Cas9-edited) Nalm-6 lymphoma cells (e.g., tumor co-culture and rechallenge assays and cytokine production) in vitro. Vector copy number (VCN) was determined by a multiplex ddPCR assay and reported as copies per diploid genome (c/dg). Results: To target CD4+ T cells, we generated fusogens encoding scFvs and VHHs specific to the CD4 T cell co-receptor (n = 399). Using fusosomes carrying the GFP transgene, NHP cross-reactive CD4-targeted fusogens that efficiently transduced CD4+ SupT1 cells were selected (n = 12 with crude SupT1 titers >1E6). Activated PBMCs transduced with a CD4-targeted fusosomes exhibited specific CD4 T cell transduction whereas VSV-G pseudotyped vectors showed broad transduction including CD4+ and CD8+ T cells. CD4-targeted CD19 CAR fusosomes could efficiently transduce both activated (34% ± 1.5% CD4+CAR+; 0.54 ± 0.18 c/dg) and resting T cells, albeit at a lower expression and integration rate (20% ± 0.5% CD4+CAR+; 0.28 ± 0.14 c/dg). Resting CD4-transduced CAR T cells demonstrated specific cytotoxicity and cytokine production (GM-CSF, IFN-g, TNF-a, IL-2, IL-6, and IL-10) against CD19+ Nalm-6 but did not recognize CD19 knockout tumor cells. In long-term co-culture assays with repetitive stimulation with fresh tumor cells, resting CD4+ CD19 CAR T cells continued to show potent tumor cell killing. Future experiments will evaluate the efficacy of CD4 fusosomes against CD19+ tumors in vivo. Summary: CD4-specific fusosomes can efficiently deliver an integrating CAR payload to resting and activated CD4+ T cells. Modified CD4+ CAR T cells demonstrate potent anti-tumor activity against CD19+ tumor cells. These data suggest that targeting the CD4 co-receptor through in vivo delivery using a novel pseudotyped integrating vector can produce functional CAR T cells to target cancer. Disclosures Ciarlo: Sana Biotechnology: Current Employment. Frye: Sana Biotechnology: Current Employment. DeGroot: Sana Biotechnology: Current Employment. Flores: Sana Biotechnology: Current Employment. Elpek: Sana Biotechnology: Current Employment. Pepper: Sana Biotechnology: Current Employment. Johnson: Sana Biotechnology: Current Employment. Shah: Sana Biotechnology: Current Employment. Foster: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company. Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

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Repurposing Bi-Specific Chimeric Antigen Receptor (CAR) Approach to Enhance CAR T Cell Activity Against Low Antigen Density Tumors

Blood ◽

10.1182/blood-2021-146995 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1727-1727

Author(s):

Sherly Mardiana ◽

Olga Shestova ◽

Stephan A. Grupp ◽

Marco Ruella ◽

David M. Barrett ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Line ◽

Tumor Cells ◽

Therapeutic Efficacy ◽

Research Funding ◽

Car T Cells ◽

Car T Cell ◽

Car T

Abstract BACKGROUND Chimeric antigen receptor (CAR) T cell therapy has revolutionized the treatment of relapsed/refractory B-cell malignancies, as highlighted by high complete remission rates and FDA approval of CD19-specific CAR T cell products. However, depth and duration of remission are limited by antigen loss/downregulation on tumors, as observed in clinical trials using CAR T cells targeting the CD19 or CD22 in leukemia and lymphoma, BCMA in multiple myeloma, and EGFRvIII in glioblastoma. This observation forms the basis of current efforts to develop multi-targeting CAR T cells to prevent antigen-negative escape. Antigen density is an important factor modulating CAR T cell response, since antigen expression below a certain threshold fails to trigger the full range of T cell functions. Given that signal strength induced upon antigen encounter determines CAR T cell activity, we hypothesized that simultaneous targeting of two dimly-expressed antigens will result in enhanced CAR T cell signaling and anti-tumor function, approaching that seen in response to one highly-expressed antigen. This is important given the heterogeneity of antigen expression in various cancers. Therefore, the bi-specific CAR T cells currently being developed to prevent antigen-negative escape could also be used to enhance efficacy against low antigen density (LAD) tumors. Results from this study will provide a novel rationale for using multi-specific CAR T cells and illuminate the mechanisms of successful CAR T cell therapy. METHODS Lentivirus transduction was performed to generate CAR T cells from healthy human T cells, using second generation 4-1BBz CARs specific for either human CD19 or CD22, or both in cis, herein referred to as CAR19, CAR22, or CAR19/22, respectively (Figure 1A). For in vitro functional characterization, we performed co-culture assay of T cells and B cell leukemia cell line NALM6, which is known to express high levels of both CD19 and CD22. To assess T cell function against LAD tumor cells, primary patients' B-ALL samples expressing low antigen density in comparison to the NALM6 cell line were used (Figure 1B). CAR T cell anti-tumor potency was determined by assessing CAR T cell cytotoxicity and cytokine production. For in vivo therapeutic study, primary patients' B-ALL samples with dimly expressed CD19 and CD22 were used to evaluate and compare the therapeutic efficacy of mono- versus bi-specific CAR T cells. Additionally, we generated a LAD tumor model by deleting the highly expressed CD19 and CD22 from the ALL cell line NALM6 using CRISPR/Cas9, transducing the now antigen-negative cell line with CD19 and CD22, followed by single cell cloning to generate a cell line expressing low antigen density for both the CD19 and CD22. We engrafted tumor cells in NSG mice, followed by administration of CAR19, CAR22, CAR19/22 or untransduced T cells. Therapeutic efficacy was assessed by measuring tumor burden using either flow cytometry or bioluminescent imaging. RESULTS Cytotoxicity assay revealed that the bi-specific CAR19/22 T cells killed tumor cells more rapidly than CAR19 or CAR22 T cells. Further, compared to mono-specific CAR T cells, the bi-specific CAR19/22 T cells produced significantly more pro-inflammatory cytokines including IL-2 and IFNg, in response to stimulation with LAD primary samples or NALM6 cells. This increased cytokine-producing capacity compared to mono-specific CAR T cells was maintained following repeated antigen stimulation when in vitro exhaustion assay was performed. In vivo, enhanced tumor elimination was observed in mice receiving bi-specific CAR19/22 T cells compared to either of the mono-specific CAR T cells, in both low antigen density primary ALL and NALM6 tumor models. This translated to increased survival rates seen in mice treated with the bi-specific CAR19/22 T cells (Figure 1C-D). CONCLUSIONS Here we showed that bi-specific CAR19/22 T cells are superior to mono-specific CAR19 or CAR22 T cells, not only against LAD tumors but also tumor cells expressing high antigen density, NALM6. This was demonstrated by their enhanced cytokine-producing function, cytotoxic capacity, and therapeutic efficacy in vivo. Results from this study provide a novel rationale for repurposing multi-specific CAR T cells as a strategy to improve efficacy against LAD tumors, in addition to the recognized benefit of reducing antigen-negative escape. Figure 1 Figure 1. Disclosures Shestova: Hemogenyx Pharmaceuticals LLC: Research Funding. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Kite, Vertex, and Servier: Research Funding; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Ruella: viTToria biotherapeutics: Research Funding; Novartis: Patents & Royalties; BMS, BAYER, GSK: Consultancy; AbClon: Consultancy, Research Funding; Tmunity: Patents & Royalties. Gill: Novartis: Other: licensed intellectual property, Research Funding; Interius Biotherapeutics: Current holder of stock options in a privately-held company, Research Funding; Carisma Therapeutics: Current holder of stock options in a privately-held company, Research Funding.

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High-Affinity Chimeric Antigen Receptor With Cross-Reactive scFv to Clinically Relevant EGFR Oncogenic Isoforms

Frontiers in Oncology ◽

10.3389/fonc.2021.664236 ◽

2021 ◽

Vol 11 ◽

Author(s):

Radhika Thokala ◽

Zev A. Binder ◽

Yibo Yin ◽

Logan Zhang ◽

Jiasi Vicky Zhang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Chimeric Antigen Receptor ◽

Antigen Receptor ◽

Tumor Escape ◽

Car T Cells ◽

Car T Cell ◽

Car T

Tumor heterogeneity is a key reason for therapeutic failure and tumor recurrence in glioblastoma (GBM). Our chimeric antigen receptor (CAR) T cell (2173 CAR T cells) clinical trial (NCT02209376) against epidermal growth factor receptor (EGFR) variant III (EGFRvIII) demonstrated successful trafficking of T cells across the blood–brain barrier into GBM active tumor sites. However, CAR T cell infiltration was associated only with a selective loss of EGFRvIII+ tumor, demonstrating little to no effect on EGFRvIII- tumor cells. Post-CAR T-treated tumor specimens showed continued presence of EGFR amplification and oncogenic EGFR extracellular domain (ECD) missense mutations, despite loss of EGFRvIII. To address tumor escape, we generated an EGFR-specific CAR by fusing monoclonal antibody (mAb) 806 to a 4-1BB co-stimulatory domain. The resulting construct was compared to 2173 CAR T cells in GBM, using in vitro and in vivo models. 806 CAR T cells specifically lysed tumor cells and secreted cytokines in response to amplified EGFR, EGFRvIII, and EGFR-ECD mutations in U87MG cells, GBM neurosphere-derived cell lines, and patient-derived GBM organoids. 806 CAR T cells did not lyse fetal brain astrocytes or primary keratinocytes to a significant degree. They also exhibited superior antitumor activity in vivo when compared to 2173 CAR T cells. The broad specificity of 806 CAR T cells to EGFR alterations gives us the potential to target multiple clones within a tumor and reduce opportunities for tumor escape via antigen loss.

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CAR-T cells to deliver engineered peptide antigens and reprogram antigen specific T cell responses against solid tumors.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.2530 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 2530-2530

Author(s):

Daniel Lee ◽

Andy J Minn ◽

Lexus R Johnson

Keyword(s):

T Cells ◽

T Cell ◽

Solid Tumors ◽

Tumor Cells ◽

In Vivo Studies ◽

Peptide Antigens ◽

Car T Cells ◽

Car T

2530 Background: Neoantigen depleted malignancies such as colorectal cancer demonstrate primary resistance to immune checkpoint blockade, and solid tumors in general have shown resistance to chimeric antigen receptor (CAR) T cell therapy. However, CAR-T cells have been shown to be capable of delivering various therapeutic molecules in a targeted fashion to the tumor microenvironment, in some cases through extracellular vesicles (EVs). In vivo studies have shown that the presentation of foreign viral peptides by solid tumors can reprogram bystander virus-specific cytotoxic T cells (CTLs) against tumor cells. In this study, we demonstrate that CAR-T cells can deliver engineered peptide antigens to solid tumors, leading to presentation on tumor cells and anti-tumor response. Methods: Second generation CAR-T cells (41BB endodomain) targeting human CD19 (19BBz) or human mesothelin (M5BBz) were generated via retroviral and lentiviral transduction respectively. CAR-T cells were engineered to co-express peptides such as SIINFEKL of ovalbumin and NLVPMVATV of CMV pp65 among others. Peptides were isolated from EVs via ultracentrifugation. For in vivo studies, C57BL/6 or NSG mice were injected on the flank with relevant tumors and treated with peptide-CAR-T cells. In vitro studies utilized flow cytometry and xCELLigence killing assays. Results: Murine 19BBz CAR-T cells expressing the SIINFEKL peptide of ovalbumin (ova-19BBz) were found to transfer SIINFEKL peptide to tumor cells via EVs in vitro and in vivo, leading to peptide presentation on MHC-I of tumor cells. This resulted in significantly delayed tumor growth in tumor bearing mice transfused with OT-I T cells to mimic an existing antigen specific T cell pool. We expanded on these findings by isolating EVs from human M5BBz CAR-T cells expressing CMV viral peptides. Peptide-CAR-T EVs were co-cultured with human ovarian cancer cells to assess presentation to Jurkat T cells. Finally, we utilized primary human T cells from CMV+ healthy donors to assess the clinical feasibility of our peptide delivery approach. Conclusions: CAR-T cells can be engineered to deliver peptides to tumor cells for presentation and subsequent targeting by antigen specific CTLs. This represents a novel strategy for the treatment of non-immunogenic tumors.

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First Results from the Dose Escalation Segment of the Phase I Clinical Study Evaluating Cyad-02, an Optimized Non Gene-Edited Engineered NKG2D CAR T-Cell Product, in Relapsed or Refractory Acute Myeloid Leukemia and Myelodysplastic Syndrome Patients

Blood ◽

10.1182/blood-2020-139667 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 36-36 ◽

Cited By ~ 1

Author(s):

Dries Deeren ◽

Johan A. Maertens ◽

Tara Lin ◽

Yves Beguin ◽

Benjamin Demoulin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Phase I ◽

Research Funding ◽

Next Generation ◽

Cell Product ◽

Car T Cells ◽

Car T Cell ◽

Car T

Background T-cells engineered to express a chimeric antigen receptor (CAR) based on the NKG2D receptor (NKG2D CAR) targeting the 8 NKG2D ligands (MICA/B, ULBP1-6) over-expressed by a large variety of malignancies have been developped to treat patients, including patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Previously, CYAD-01, the first generation of NKG2D CAR T-cell products, was evaluated in several Phase I clinical trials and showed initial signals of objective clinical responses in patients with r/r AML and MDS, albeit with short durability. Preclinical data have shown that NKG2D ligands MICA and MICB are transiently upregulated on activated CAR T-cells, and target-dependent killing of CAR T-cells post-infusion can potentially occur, leading to short in vivo persistence. In an effort to increase the persistence and potency of the NKG2D CAR T-cells, CYAD-02 was developed as a next-generation product using a non-gene editing approach to silence the expression of MICA and MICB. Aim MICA and MICB were down-regulated by inserting a single optimized short hairpin RNA (shRNA) targeting both MICA and MICB within the NKG2D CAR construct. This next-generation NKG2D CAR T-cell product is manufactured with the OptimAb process, resulting in CAR T-cells with a higher frequency of early memory T-cells secreting high levels of cytokines upon activation, and is referred to as CYAD-02. Results As compared to CYAD-01, CYAD-02 cell expansion in vitro was 3-fold increased. In an in vivo AML model, CYAD-02 showed 10-fold higher engraftment 1 week after injection and improved anti-tumor activity as compared to CYAD-01 manufactured with the initial mAb process. This led to a 2.6-fold increase of mouse survival as compared to CYAD-01 in a stress-test aggressive AML model where the dose of CYAD-01 was titrated down for minimal activity (figure). The first-in-human study evaluating CYAD-02, the CYCLE-1 study (NCT04167696), has been initiated in early 2020 in patients with r/r AML/MDS. The study evaluates three dose-levels of CYAD-02 (1x108, 3x108 and 1x109 cells/infusion), administered as a single infusion after non-myeloablative preconditioning chemotherapy (cyclophosphamide 300 mg/m²/day and fludarabine 30 mg/m²/day, daily for 3 days, CyFlu) according to a classical Fibonacci 3+3 design. As of August 2020, 6 patients have been treated with CYAD-02 at the dose of 1x108 or 3x108 cells/infusion. To date, the results demonstrate the safety and tolerability for CYAD-02 in patients with r/r AML and MDS with no dose-limiting toxicity observed. The study is currently enrolling at 1x109 cells/infusion. The CYAD-02 safety profile and preliminary clinical activity data together with the pharmacokinetics evaluation from the complete dose escalation segment will be provided at the time of presentation. Conclusion/summary The CYAD-02 is the first autologous CAR T-cell product based on the non-gene edited shRNA technology used to treat patients. This next generation NKG2D CAR T-cell product is currently investigated in the CYCLE-1 Phase I study in r/r AML/MDS patient population, a difficult to target disease due in part to the absence of truly AML-specific surface antigens, its rapid clinical progression and the absence of disease control by the CyFlu preconditioning. Both the anti-MICA and MICB shRNA hairpin and the OptimAb manufacturing process for CYAD-02 aim to improve CAR T-cell persistence and clinical responses. Figure Disclosures Lin: Mateon Therapeutics: Research Funding; Aptevo: Research Funding; Abbvie: Research Funding; Ono Pharmaceutical: Research Funding; Incyte: Research Funding; Gilead Sciences: Research Funding; Jazz: Research Funding; Astellas Pharma: Research Funding; Bio-Path Holdings: Research Funding; Celgene: Research Funding; Celyad: Research Funding; Genetech-Roche: Research Funding; Seattle Genetics: Research Funding; Tolero Pharmaceuticals: Research Funding; Trovagene: Research Funding; Prescient Therapeutics: Research Funding; Pfizer: Research Funding. Demoulin:Celyad Oncology: Current Employment. Fontaine:Celyad Oncology: Current Employment. Sotiropoulou:Celyad Oncology: Current Employment. Alcantar-Orozco:Celyad Oncology: Current Employment. Breman:Celyad Oncology: Current Employment. Dheur: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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