scholarly journals Development of an Allogeneic Anti-Bcma T Cell Therapy Utilizing a Novel Dimeric Antigen Receptor (DAR) Structure

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1942-1942 ◽  
Author(s):  
Bei Bei Ding ◽  
John Dixon Gray ◽  
Nan Zhang ◽  
Yanliang Zhang ◽  
Xia Cao ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Antigen Receptor ◽  
Employment Equity ◽  
T Cell Therapy ◽  
Equity Ownership ◽  
Anti Tumor Activity

Background: Multiple myeloma remains an incurable malignancy of plasma cells. Adoptive transfer of chimeric antigen receptor (CAR)-expressing T cells is a promising new therapy for hematologic malignancies. B-cell maturation antigen (BCMA) is a protein that is selectively expressed by B-lineage cells, including Multiple Myeloma (MM) cells, and represents a suitable target for T cell therapy. We have developed an allogeneic T cell therapy approach utilizing genetic engineering of donor-derived T cells to express an anti-BCMA Dimeric Antigen Receptor (DAR) using a proprietary non-viral vector Knock out/knock in (KOKI) technology. Preclinical data demonstrate potent anti-tumor activity both vitro and in vivo against BCMA-expressing MM cell lines. Methods: Anti-BCMA DAR-T cells were generated through genetic engineering of T cells derived from healthy donors by inserting the anti-BCMA DAR construct into the TRAC gene locus, resulting in loss of endogenous TCR expression while expressing the DAR. Distinct DAR constructs were utilized differing only in their intracellular signaling components, namely combinations of 4-1BB, CD28, and CD3zeta. The anti-BCMA DAR-T cells were expanded and purified for subsequent preclinical studies. Using in vitro assays, the different anti-BCMA DAR-T cells were evaluated against multiple myeloma cell lines for specific cytotoxicity as well as stimulus-induced cytokine secretion and cell expansion. The in vivo anti-tumor activity was assessed using luciferase-expressing RPMI8226 cells in NSG mice in a model of disseminated disease. A single dose of anti-BCMA DAR-T cells or relevant control cells was administered, and tumor burden was assessed weekly using bioluminescence imaging. Results: After purification, the anti-BCMA DAR T cells population contained less than 1% TCR-expressing ab T cells. The DAR-positive T cell population was between 20-50%. All anti-BCMA DAR-T cells exhibited BCMA-specific activation, including cytokine production, proliferation, cytotoxicity, and in vivo tumor eradication. The DAR-T cells using a third generation signaling configuration containing components from 4-1BB, CD28 and CD3zeta signaling domains performed best overall. Conclusions: All tested anti-BCMA DAR-T cells exhibited effective anti-tumor activity. Direct comparison of different cytoplasmic signaling compositions of the DAR allowed for selection of the most potent construct, namely the anti-BCMA DAR utilizing a 3rd generation signaling domain configuration. Based on these data, further development of anti-BCMA DAR-T therapy for hematological malignancies is warranted. These allogeneic abTCR-negative anti-BCMA DAR-T cells have been selected for clinical development. Disclosures Ding: Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Gray:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zhang:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zhang:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Cao:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Krapf:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Deng:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Wei:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Knight:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Guo:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Development of a Genetically-Engineered Allogeneic Anti-CD38 T Cell Therapy Utilizing a Novel Antigen Receptor Structure

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4444-4444
Author(s):  
Bei Bei Ding ◽  
John Dixon Gray ◽  
Irina Krapf ◽  
Yanliang Zhang ◽  
Nan Zhang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Employment Equity ◽  
T Cell Therapy ◽  
Equity Ownership ◽  
Anti Tumor Activity

Background: Autologous Chimeric Antigen Receptor (CAR) T cell therapy has shown great promise as a treatment modality for a variety of hematological malignancies. But autologous cell therapies still face several practical hurdles, including reliance on patient immune cells and manufacturing difficulties. Sorrento has pioneered an allogeneic T cell therapy approach utilizing genetic engineering of donor-derived T cells to express a Dimeric Antigen Receptor (DAR). The first DAR-T cell therapy being developed is targeted against CD38, a clinically-validated antigen in multiple myeloma. Preclinical data demonstrate potent anti-tumor activity in both in vitro assays and in vivo studies against CD38-expressing lymphoma and multiple myeloma (MM) cell lines. Methods: Anti-CD38 DAR-T cells were generated through genetic engineering of T cells derived from healthy donors inserting the anti-CD38 DAR construct into the TRAC gene locus resulting in loss of endogenous TCR expression while expressing the DAR. Three distinct DAR constructs were utilized differing only in the intracellular signaling components, namely CD28/CD3zeta, 4-1BB/CD3zeta and CD28/4-1BB/CD3zeta. The CD38 DAR-T were expanded and purified for subsequent preclinical studies. Using in vitro assays, the 3 different CD38 DAR-T cells were evaluated against multiple myeloma and lymphoma cell lines for specific cytotoxicity as well as stimulus-induced cytokine secretion and cell expansion. The in vivo anti-tumor activity was assessed using luciferase-expressing RPMI8226 cells in NSG mice in a model of disseminated disease. A single dose of anti-CD38 DAR-T cells or relevant control cells was administered and tumor burden was assessed weekly using bioluminescence imaging. Results: An anti-CD38 DAR gene was efficiently integrated into TRAC locus of T cells from healthy donors by one step knock out/knock in (KOKI) methodology with high efficiency (~40-80% CD38 DAR expression and ~90% TCR knock out). Following a CD3-depletion step, the TCR-positive T cells were less 1%. When co-cultured with CD38-positive tumor cells, anti-CD38 DAR T cells killed as effectively as retroviral anti-CD38 CAR-T cells with similar cytokine secretion profiles while no cytotoxicity was observed against CD38-negative cancer cells. Moreover, in vivo DAR-T cells showed better killing activity against multiple myeloma cell lines than CAR-T cell with anti-CD38 4-1BB/CD3zeta DAR demonstrating the best anti-tumor activity in an NSG mouse model. The anti-CD38 DAR-T cells with 41BB/CD3 zeta internal signals have been selected for clinical development. Conclusions: All tested anti-CD38 DAR-T cells exhibited potent in vitro and in vivo anti-tumor activity. Direct comparison of three different cytoplasmic signaling compositions of the DAR allowed for selection of the most potent construct, namely the anti-CD38 DAR utilizing 4-1BB and CD3zeta signaling domains. Based on these data, further development of CD38 DAR-T therapy for hematological malignancies is warranted. GMP manufacturing of the allogeneic anti-CD38 DAR-T cells has been initiated. Disclosures Ding: Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Gray:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Krapf:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zhang:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zhang:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Deng:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Wei:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Knight:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Guo:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

scholarly journals First-in-Human Study of ET019003, a Next Generation Anti-CD19 T-Cell Therapy, in Patients with Relapsed/Refractory Diffuse Large B Cell Lymphoma (r/r DLBCL)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2870-2870 ◽  
Author(s):  
Pengcheng He ◽  
Hong Liu ◽  
Haibo Liu ◽  
Mina Luo ◽  
Hui Feng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Cell Expansion ◽  
T Cell Therapy ◽  
Car T ◽  
Equity Ownership ◽  
T Cell Expansion ◽  
The Absolute

Background : CD19-targeted CAR-T therapies have shown promising efficacy in treating B-cell malignancies. However, treatment-related toxicities, such as cytokine-release syndrome (CRS) and CAR T-cell-related encephalopathy syndrome (CRES), have been one of the major obstacles limiting the use of CAR-T therapies. How to minimize occurrence and severity of toxicity while maintaining efficacy is a major focus for T-cell therapies in development. ET019003 is a next generation CD19-targeted T-cell therapy developed by Eureka Therapeutics, built on the proprietary ARTEMISTM T-cell platform. The ET019003 construct is optimized with the co-expression of an ET190L1 Antibody-TCR (Xu et al, 2018) and novel co-stimulation molecule. We are conducting a First-in-human (FIH) study of ET019003 T cells in CD19+ r/r DLBCL patients. Methods: This FIH study aims to evaluate the safety and efficacy of ET019003 T-cell therapy in CD19+ patients with r/r DLBCL. As of July 2019, six subjects were administered ET019003 T cells. These subjects were pathologically confirmed with DLBCL that is CD19+ (by immunohistochemistry), whose disease have progressed or relapsed after 2-5 lines of prior therapies. All were high-risk patients with rapid tumor progression and heavy tumor burden. Each subject had a Ki67 proliferative index over 60%, 2/6 of the subjects had a Ki67 proliferative index over 90%. Moreover, 5/6 of the subjects had extra-nodal involvement. Following a 3-day preconditioning treatment with Fludarabine (25mg/m2/day)/ Cyclophosphamide (250mg/m2/day), patients received i.v. infusions of ET019003 T cells at an initial dose of 2-3×106 cells/kg. Additional doses at 3×106 cells/kg were administered at 14 to 30-day intervals. Adverse events were monitored and assessed based on CTCAE 5.0. Clinical responses were assessed based on Lugano 2014 criteria. Results: As of July 2019, six subjects have received at least one ET019003 T-cell infusion, and four subjects have received two or more ET019003 T-cell infusions. No Grade 2 or higher CRS was observed in the six subjects. One subject developed convulsions and cognitive disturbance. This subject had lymphoma invasion in the central nervous system before ET019003 T-cell therapy. The subject was treated with glucocorticoid and the symptoms resolved within 24 hours. Other adverse events included fever (6/6, 100%), fatigue (3/6, 50%), thrombocytopenia (3/6, 50%), diarrhea (2/6, 33%), and herpes zoster (1/6, 17%). ET019003 T-cell expansion in vivo (monitored by flow cytometry and qPCR) was observed in all six subjects after first infusion. The absolute peak value of detected ET019003 T cells ranged between 26,000 - 348,240 (median 235,500) per ml of peripheral blood. Tmax (time to reach the absolute peak value) was 6 - 14 days (median 7.5 days). For the four subjects who received multiple ET019003 T-cell infusions, the absolute peak values of detected ET019003 T cells after the second infusion were significantly lower than the absolute peak values achieved after the first infusion. For the two subjects who received three or more infusions of ET019003 T cells, no significant ET019003 T-cell expansion in vivo was observed after the third infusion. All six subjects completed the evaluation of clinical responses at 1 month after ET019003 T-cell therapy. All subjects responded to ET019003 T cells and achieved either a partial remission (PR) or complete response (CR). Conclusions: Preliminary results from six CD19+ r/r DLBCL patients in a FIH study show that ET019003 T-cell therapy is safe with robust in vivo T-cell expansion. The clinical study is on-going and we are monitoring safety as well as duration of response in longer follow-up. Reference: Xu et al. Nature Cell Discovery, 2018 Disclosures Liu: Eureka Therapeutics: Employment, Equity Ownership. Chang:Eureka Therapeutics: Equity Ownership. Liu:Eureka Therapeutics: Employment, Equity Ownership.

scholarly journals GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells

2019 ◽  
Vol 11 (485) ◽  
pp. eaau7746 ◽  
Author(s):  
Eric L. Smith ◽  
Kim Harrington ◽  
Mette Staehr ◽  
Reed Masakayan ◽  
Jon Jones ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
B Cell ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell Therapy ◽  
Car T

Early clinical results of chimeric antigen receptor (CAR) T cell therapy targeting B cell maturation antigen (BCMA) for multiple myeloma (MM) appear promising, but relapses associated with residual low-to-negative BCMA-expressing MM cells have been reported, necessitating identification of additional targets. The orphan G protein–coupled receptor, class C group 5 member D (GPRC5D), normally expressed only in the hair follicle, was previously identified as expressed by mRNA in marrow aspirates from patients with MM, but confirmation of protein expression remained elusive. Using quantitative immunofluorescence, we determined that GPRC5D protein is expressed on CD138+ MM cells from primary marrow samples with a distribution that was similar to, but independent of, BCMA. Panning a human B cell–derived phage display library identified seven GPRC5D-specific single-chain variable fragments (scFvs). Incorporation of these into multiple CAR formats yielded 42 different constructs, which were screened for antigen-specific and antigen-independent (tonic) signaling using a Nur77-based reporter system. Nur77 reporter screen results were confirmed in vivo using a marrow-tropic MM xenograft in mice. CAR T cells incorporating GPRC5D-targeted scFv clone 109 eradicated MM and enabled long-term survival, including in a BCMA antigen escape model. GPRC5D(109) is specific for GPRC5D and resulted in MM cell line and primary MM cytotoxicity, cytokine release, and in vivo activity comparable to anti-BCMA CAR T cells. Murine and cynomolgus cross-reactive CAR T cells did not cause alopecia or other signs of GPRC5D-mediated toxicity in these species. Thus, GPRC5D(109) CAR T cell therapy shows potential for the treatment of advanced MM irrespective of previous BCMA-targeted therapy.

scholarly journals The roles of T cell competition and stochastic extinction events in chimeric antigen receptor T cell therapy

2021 ◽  
Vol 288 (1947) ◽  
Author(s):  
Gregory J. Kimmel ◽  
Frederick L. Locke ◽  
Philipp M. Altrock
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

Chimeric antigen receptor (CAR) T cell therapy is a remarkably effective immunotherapy that relies on in vivo expansion of engineered CAR T cells, after lymphodepletion (LD) by chemotherapy. The quantitative laws underlying this expansion and subsequent tumour eradication remain unknown. We develop a mathematical model of T cell–tumour cell interactions and demonstrate that expansion can be explained by immune reconstitution dynamics after LD and competition among T cells. CAR T cells rapidly grow and engage tumour cells but experience an emerging growth rate disadvantage compared to normal T cells. Since tumour eradication is deterministically unstable in our model, we define cure as a stochastic event, which, even when likely, can occur at variable times. However, we show that variability in timing is largely determined by patient variability. While cure events impacted by these fluctuations occur early and are narrowly distributed, progression events occur late and are more widely distributed in time. We parameterized our model using population-level CAR T cell and tumour data over time and compare our predictions with progression-free survival rates. We find that therapy could be improved by optimizing the tumour-killing rate and the CAR T cells' ability to adapt, as quantified by their carrying capacity. Our tumour extinction model can be leveraged to examine why therapy works in some patients but not others, and to better understand the interplay of deterministic and stochastic effects on outcomes. For example, our model implies that LD before a second CAR T injection is necessary.

scholarly journals Cyclophosphamide and Fludarabine Conditioning Chemotherapy Induces a Key Homeostatic Cytokine Profile in Patients Prior to CAR T Cell Therapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4426-4426 ◽  
Author(s):  
Adrian Bot ◽  
John M. Rossi ◽  
Yizhou Jiang ◽  
Lynn Navale ◽  
Yueh-wei Shen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Therapy ◽  
Fold Increase ◽  
Employment Equity ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Abstract This study is supported in part by funding from the Cooperative Research and Development Agreement (CRADA) between the National Cancer Institute and Kite Pharma Introduction: CAR-engineered autologous T-cell therapy has shown promising activity in relapsed/refractory B-cell malignancies in an ongoing phase 1 study (Kochenderfer et al. J Clin Oncol 2014). Lymphodepleting conditioning chemotherapy is critical for optimal CAR T-cell activity in animal models. We evaluated the effects of conditioning chemotherapy on cytokine and chemokine levels in patients dosed with anti-CD19 CAR T cells. Methods: In this National Cancer Institute clinical trial (NCT00924326), patients with relapsed/refractory B-cell malignancies received conditioning with cyclophosphamide and fludarabine daily for 3 days starting on day -5; followed by anti-CD19 CAR T cells engineered with a CAR comprising CD28 and CD3-zeta signaling domains. Forty one cytokines, chemokines and immune response related markers were measured in the blood of patients pre (day -5) and post conditioning (day 0) by using EMD Millipore Luminex® xMAP® multiplex assays. Data acquisition and analysis were performed using a Luminex 200™ instrument and xPONENT® 3.1 data analysis software. Increases in cytokine and chemokine levels were analyzed pre- and post- conditioning, and the fold-changes in cytokine and chemokine levels were analyzed relative to clinical outcome subsequent to infusion with anti-CD19 CAR T cells. Analyses were performed with the Wilcoxon rank sum test adjusted for multiplicity with a Bonferroni correction, using a nominal level of 0.006 for significance. Results: Samples from 15patients have been evaluated. There were significant increases pre- to post-conditioning in the levels of interleukin 15 (IL-15; p=0.001), interleukin 7 (IL-7; p=0.0002), and monocyte chemoattractant protein-1 (MCP-1; p<0.0025) in blood, five days after the initiation of conditioning chemotherapy. Levels of interferon-gamma induced protein 10 (IP-10) were elevated post-conditioning, but did not meet the threshold for significance (p=0.048). Compared with baseline, levels of IL-15 increased on average 13 fold and levels of IL-7, IP-10 and MCP-1, about 2 fold. Comparison of the fold-increases in IL-15 upon conditioning between responders and non-responders approached significance (p=0.01), but did not meet the threshold after multiplicity adjustment. Larger fold-change increases for responders versus non-responders were also observed with placental growth factor (PLGF) (median fold increase 2.6 v. 1.6, average fold increase 32 v 4.2), C-reactive protein (CRP) (median fold increase 3.5 v 2.4, average fold increase 6.6 v. 2.0), IP-10 (median fold increase 2.1 v. 0.7, average fold increase 2.6 v. 2.8), and interleukin 10 (IL-10) (median fold increase 1.8 v. 0.4, average fold increase 3.1 v. 2.0), but did not meet the threshold for significance. In addition to ongoing analysis of conditioning-mediated cytokine induction and clinical response, we are evaluating the impact of conditioning chemotherapy dose on cytokine levels, as well as the relationship between conditioning-related cytokines and CAR T-cell expansion and persistence. Conclusions: The data obtained to date support the hypothesis that cytokines such as IL-15 play a key role in the clinical outcomes to anti-CD19 CAR T-cell therapy. Our results demonstrate that conditioning chemotherapy significantly increases the levels of homeostatic cytokines known to regulate T-cell expansion, as well as specific pro-inflammatory cytokines and chemokines. Optimization of conditioning chemotherapy is critical to the activity of CAR T-cell therapies. Disclosures Bot: Kite Pharma: Employment, Equity Ownership. Rossi:Amgen: Equity Ownership; Kite Pharma: Employment, Equity Ownership. Jiang:Kite Pharma: Employment, Equity Ownership. Navale:Amgen: Equity Ownership; Kite Pharma: Employment, Equity Ownership. Shen:Kite Pharma: Employment, Equity Ownership. Sherman:Amgen: Equity Ownership; Kite Pharma: Employment, Equity Ownership. Mardiros:Kite Pharma: Employment, Equity Ownership. Yoder:Kite Pharma: Employment, Equity Ownership. Go:Amgen: Equity Ownership; Kite Pharma: Employment, Equity Ownership. Rosenberg:Kite Pharma: Other: CRADA between Surgery Branch-NCI and Kite Pharma. Wiezorek:Kite Pharma: Employment, Equity Ownership, Other: Officer of Kite Pharma. Chang:Kite Pharma: Employment, Equity Ownership, Other: Officer of Kite Pharma. Roberts:Kite Pharma: Employment, Equity Ownership, Other: Officer of Kite Pharma.

scholarly journals Preclinical Characterization of an ANTI-CD38/CD3 T CELL-Redirecting Bispecific Antibody

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4463-4463
Author(s):  
Xiao He ◽  
Yanliang Zhang ◽  
Yun Wei Lai ◽  
Stephanie Baguley ◽  
Yan Li ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cell ◽  
T Cell Activation ◽  
Cytokine Release ◽  
Employment Equity ◽  
Equity Ownership ◽  
Anti Tumor Activity

Introduction: Multiple Myeloma (MM) and Non-Hodgkin Lymphoma (NHL) are hematologic malignancies that remain difficult to treat. While autologous CAR-T cell therapies have shown promise in treating these diseases, these therapies are not without issues, including lack of response in many patients, lengthy time to produce CAR-T cells, occasional production failures, as well as high manufacturing costs. As an alternative approach, protein-based T cell engaging and redirecting bispecific antibodies (BsAbs) have been developed. We have generated anti-CD38/CD3 BsAbs to redirect T cells against CD38, a clinically validated antigen in MM and studied their ability to elicit target-dependent tumor cell lysis. The lead molecule is a humanized, stability-engineered CD3-engaging and CD38 antigen affinity-optimized BsAb with reduced effector function to mitigate antigen-independent T cell toxicity. Preclinical data demonstrate potent anti-tumor activity in vitro assays and in vivo studies against CD38-expressing lymphoma and MM cell lines. Methods: Anti-CD38/CD3 BsAbs were generated by CH3 Fc domain interface engineering for heterodimerization of a CD38-targeting Fab arm and anti-CD3-scFv-Fc fusion chain with hinge mutations for reduced FcR affinities. Novel bispecific molecules that bind to CD38 with various affinities/binding kinetics were evaluated in a series of in vitro and in vivo studies, including target-specific redirected T cell cytotoxicity (RTCC) against cancer cell lines. T cell response profiles, and cytokine release. The lead CD38/CD3 BsAb was selected and further evaluated for its ability to inhibit tumor growth and prolong survival in a disseminated luciferase-expressing Raji xenograft mouse model co-implanted with primary human peripheral blood mononuclear cells (hPBMC). Results: Our lead CD38/CD3 BsAb possesses the desired CD38 and CD3, affinities resulting in effective tumor antigen and T cell engagement for RTCC. The CD38/CD3 BsAb induced potent T cell-dependent lysis of CD38-positive cancer cells in vitro, with the CD38 antigen density positively correlating with the cytotoxicity potency. Antigen dependent and dose-dependent T cell activation and cytokine release were studied in vitro, with the level of T cell activation and cytokine release being indicative of the anti-tumor potency but not necessarily anti-CD3 affinity. In an in vivo study, we evaluated the impact of CD38 affinity of the BsAb on anti-tumor activity of the BsAbs. The data showed that a balanced CD38 vs CD3 affinity was shown to be preferred for T cell stimulation and prolonged anti-tumor activity. In preclinical cytotoxicity assays against a cancer cell line panel using hPBMC from healthy donors, our lead CD38/CD3 BsAb was benchmarked against daratumumab, a marketed anti-CD38 antibody for MM, and demonstrated more potent tumor cell killing. These data suggest a more robust anti-tumor activity exerted by the CD38/CD3 BsAb through RTCC than daratumumab through antibody-dependent cellular cytotoxicity (ADCC). In Raji tumor cell-bearing NSG mice implanted with previously unstimulated hPBMCs, our CD38/CD3 BsAb induced tumor growth inhibition and prolonged survival compared to control BsAb or hPBMCs-only treated animals. Conclusions: Our preclinical data demonstrate that our lead CD38/CD3 BsAb recruits T cells against CD38-positive tumor MM and lymphoma cells in a potent target and dose-dependent manner in preclinical studies. These preclinical characterizations support the rationale for clinical investigation of the lead BsAb in selected CD38-positive malignancies. Disclosures He: Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Zhang:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Lai:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Baguley:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Li:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Cao:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Yan:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Takeshita:Sorrento Therapeutics, Inc.: Employment, Equity Ownership. Zeldis:Sorrento Therapeutics Inc: Employment, Equity Ownership. Ji:Sorrento Therapeutics Inc: Employment, Equity Ownership, Patents & Royalties; Celularity, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Kaufmann:Sorrento Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.

scholarly journals Preclinical Evaluation of ALLO-819, an Allogeneic CAR T Cell Therapy Targeting FLT3 for the Treatment of Acute Myeloid Leukemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3921-3921 ◽  
Author(s):  
Cesar Sommer ◽  
Hsin-Yuan Cheng ◽  
Yik Andy Yeung ◽  
Duy Nguyen ◽  
Janette Sutton ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Target Cells ◽  
Employment Equity ◽  
T Cell Therapy ◽  
Cell Therapies ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Equity Ownership

Autologous chimeric antigen receptor (CAR) T cells have achieved unprecedented clinical responses in patients with B-cell leukemias, lymphomas and multiple myeloma, raising interest in using CAR T cell therapies in AML. These therapies are produced using a patient's own T cells, an approach that has inherent challenges, including requiring significant time for production, complex supply chain logistics, separate GMP manufacturing for each patient, and variability in performance of patient-derived cells. Given the rapid pace of disease progression combined with limitations associated with the autologous approach and treatment-induced lymphopenia, many patients with AML may not receive treatment. Allogeneic CAR T (AlloCAR T) cell therapies, which utilize cells from healthy donors, may provide greater convenience with readily available off-the-shelf CAR T cells on-demand, reliable product consistency, and accessibility at greater scale for more patients. To create an allogeneic product, the TRAC and CD52 genes are inactivated in CAR T cells using Transcription Activator-Like Effector Nuclease (TALEN®) technology. These genetic modifications are intended to minimize the risk of graft-versus-host disease and to confer resistance to ALLO-647, an anti-CD52 antibody that can be used as part of the conditioning regimen to deplete host alloreactive immune cells potentially leading to increased persistence and efficacy of the infused allogeneic cells. We have previously described the functional screening of a library of anti-FLT3 single-chain variable fragments (scFvs) and the identification of a lead FLT3 CAR with optimal activity against AML cells and featuring an off-switch activated by rituximab. Here we characterize ALLO-819, an allogeneic FLT3 CAR T cell product, for its antitumor efficacy and expansion in orthotopic models of human AML, cytotoxicity in the presence of soluble FLT3 (sFLT3), performance compared with previously described anti-FLT3 CARs and potential for off-target binding of the scFv to normal human tissues. To produce ALLO-819, T cells derived from healthy donors were activated and transduced with a lentiviral construct for expression of the lead anti-FLT3 CAR followed by efficient knockout of TRAC and CD52. ALLO-819 manufactured from multiple donors was insensitive to ALLO-647 (100 µg/mL) in in vitro assays, suggesting that it would avoid elimination by the lymphodepletion regimen. In orthotopic models of AML (MV4-11 and EOL-1), ALLO-819 exhibited dose-dependent expansion and cytotoxic activity, with peak CAR T cell levels corresponding to maximal antitumor efficacy. Intriguingly, ALLO-819 showed earlier and more robust peak expansion in mice engrafted with MV4-11 target cells, which express lower levels of the antigen relative to EOL-1 cells (n=2 donors). To further assess the potency of ALLO-819, multiple anti-FLT3 scFvs that had been described in previous reports were cloned into lentiviral constructs that were used to generate CAR T cells following the standard protocol. In these comparative studies, the ALLO-819 CAR displayed high transduction efficiency and superior performance across different donors. Furthermore, the effector function of ALLO-819 was equivalent to that observed in FLT3 CAR T cells with normal expression of TCR and CD52, indicating no effects of TALEN® treatment on CAR T cell activity. Plasma levels of sFLT3 are frequently increased in patients with AML and correlate with tumor burden, raising the possibility that sFLT3 may act as a decoy for FLT3 CAR T cells. To rule out an inhibitory effect of sFLT3 on ALLO-819, effector and target cells were cultured overnight in the presence of increasing concentrations of recombinant sFLT3. We found that ALLO-819 retained its killing properties even in the presence of supraphysiological concentrations of sFLT3 (1 µg/mL). To investigate the potential for off-target binding of the ALLO-819 CAR to human tissues, tissue cross-reactivity studies were conducted using a recombinant protein consisting of the extracellular domain of the CAR fused to human IgG Fc. Consistent with the limited expression pattern of FLT3 and indicative of the high specificity of the lead scFv, no appreciable membrane staining was detected in any of the 36 normal tissues tested (n=3 donors). Taken together, our results support clinical development of ALLO-819 as a novel and effective CAR T cell therapy for the treatment of AML. Disclosures Sommer: Allogene Therapeutics, Inc.: Employment, Equity Ownership. Cheng:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Yeung:Pfizer Inc.: Employment, Equity Ownership. Nguyen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Sutton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Melton:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Valton:Cellectis, Inc.: Employment, Equity Ownership. Poulsen:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Djuretic:Pfizer, Inc.: Employment, Equity Ownership. Van Blarcom:Allogene Therapeutics, Inc.: Employment, Equity Ownership. Chaparro-Riggers:Pfizer, Inc.: Employment, Equity Ownership. Sasu:Allogene Therapeutics, Inc.: Employment, Equity Ownership.

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