scholarly journals Apheresis Products from Patients with Multiple Myeloma Treated with G-CSF Are a Suitable Source of T Cells for the Production of BCMA-Targeting CAR-T Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 480-480
Author(s):  
Anthony M Battram ◽  
Aina Oliver-Caldés ◽  
Miquel Bosch i Crespo ◽  
María Suárez-Lledó ◽  
Miquel Lozano ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Progenitor Cells ◽  
Research Funding ◽  
Car T Cells ◽  
Car T

Abstract Background: Autologous chimeric antigen receptor-T (CAR-T) cells that target BCMA (BCMA-CARs) have emerged as a promising treatment for multiple myeloma (MM). Current clinical protocols dictate that BCMA-CAR therapy is only used after patients have repeatedly relapsed. However, at this stage, the immunosuppressive nature of advanced MM and/or side-effects of the previous therapies cause T cell dysfunction and an unfavourable phenotype, such as exhaustion, senescence and loss of early memory cells. An alternative and convenient pool of 'fitter' T cells are apheresis products that are routinely collected to obtain progenitor cells for autologous stem cell transplantation (ASCT), an intervention that is often carried out early in MM treatment. However, to mobilise the progenitor cells, patients are treated with G-CSF, which could have negative effects on T cells such as reduce proliferation, impair CD8 + T cell function and induce regulatory T cell (Treg) expansion. Whether this has an effect on the BCMA-CARs generated from these T cells, however, is unknown. Therefore, we aimed to establish whether G-CSF treatment had detrimental effects on T cell phenotype, and moreover, to ascertain whether BCMA-CARs that are generated from these T cells were impaired compared to those produced from T cells prior to G-CSF infusion. Methods: T cells were isolated from the blood of 9 patients with MM before and after 4 days of subcutaneous G-CSF administration (PRE G-CSF and POST G-CSF, respectively) prior to peripheral blood CD34 + cell harvesting for an ASCT as consolidation after first-line induction treatment. Following stimulation with anti-CD3/anti-CD28 beads and IL-2, T cells were transduced with ARI2h, an anti-BCMA CAR produced at our institution that is currently being explored in a clinical trial for relapsed/refractory MM (NCT04309981). Freshly-isolated T cells or expanded ARI2h cells were analysed by flow cytometry for markers of cell identity, activation, dysfunction and memory, or alternatively, challenged with an MM cell line (ARP-1 or U266) and then tested for cytokine production and cytotoxic ability. In addition, PRE and POST G-CSF ARI2h CARs were injected into ARP-1 tumour-bearing mice to assess their in vivo function. Results: Firstly, the phenotype of PRE G-CSF and POST G-CSF T cells, before CAR production, was analysed to identify effects of G-CSF treatment. Interestingly, there were fewer POST G-CSF CD8 + T cells with a stem cell memory (CCR7 +CD45RA +CD95 +) phenotype, but the proportion of naïve (CCR7 +CD45RA +CD95 -) cells and other memory populations was not significantly different. Moreover, POST G-CSF T cells had a lower CD4:CD8 ratio, but did not contain more senescent-like cells or display evidence of pre-activation or increased expression of exhaustion markers. Due to the known effect of G-CSF on CD4 + Treg expansion, the percentage of Tregs was also compared between the PRE G-CSF and POST G-CSF samples, but no difference was observed. Following T-cell activation and CAR transduction, comparable transduction efficiencies and proliferation rates were obtained. Likewise, the in vitro function of PRE G-CSF and POST G-CSF ARI2h cells, as determined by assessing their cytotoxic response to MM cell lines and ability to produce effector molecules such as granzyme B, was similar. To test the in vivo function of ARI2h CAR-T cells expanded from PRE G-CSF and POST G-CSF samples, they were injected into a murine xenograft model of advanced MM. Mice administered with both PRE and POST G-CSF ARI2h cells survived longer than those given untransduced T cells (p=0.015 and p=0.039, respectively), but there was no difference in the longevity of mice between the PRE G-CSF and POST G-CSF groups (p=0.990) (Figure 1). The similarity of the in vitro and in vivo function of PRE and POST G-CSF ARI2h cells was reflected in the phenotype of the CAR-T cells after ex vivo expansion, with cells from both groups displaying equal levels of activation, exhaustion, and importantly for CAR-T cell activity, memory/effector phenotype. Conclusions: The in vitro and in vivo functions of ARI2h CAR-T cells when generated from either PRE G-CSF or POST G-CSF samples were comparable, despite G-CSF administration decreasing the CD8 + stem cell memory pool. Overall, we conclude that T cells from apheresis products, performed to collect G-CSF-mobilised peripheral blood progenitor cells for ASCT, are suitable for BCMA-CAR manufacture. Figure 1 Figure 1. Disclosures Lozano: Grifols: Honoraria; Terumo BCT: Honoraria, Research Funding; Macopharma: Research Funding. Fernandez de Larrea: BMS: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Takeda: Honoraria, Research Funding; GSK: Honoraria; Sanofi: Consultancy; Janssen: Consultancy, Honoraria, Research Funding.

scholarly journals Targeting the Membrane-Proximal C2-Set Domain of CD33 for Improved CD33-Directed CAR T Cell Therapy

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2776-2776
Author(s):  
Salvatore Fiorenza ◽  
George S. Laszlo ◽  
Tinh-Doan Phi ◽  
Margaret C. Lunn ◽  
Delaney R. Kirchmeier ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Set Domain ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Privately Held

Abstract Background: There is increasing interest in targeting CD33 in malignant and non-malignant disorders, but available drugs are ineffective in many patients. As one limitation, therapeutic CD33 antibodies typically recognize the membrane-distal V-set domain. Likewise, currently tested CD33-directed chimeric antigen receptor (CAR) T cells likewise target the V-set domain and have thus far shown limited clinical activity. We have recently demonstrated that binding closer to the cell membrane enhances the effector functions of CD33 antibodies. We therefore raised antibodies against the membrane-proximal C2-set domain of CD33 and identified antibodies that bound CD33 regardless of the presence/absence of the V-set domain ("CD33 PAN antibodies"). Here, we tested their properties as targeting moiety in CD33 PAN CAR T cell constructs, using a clinically validated lentiviral backbone. Methods: To generate CAR T cells, negatively selected CD8 + T cells were transduced with an epHIV7 lentivirus encoding the scFv from a CD33 PAN antibody (clone 1H7 or 9G2) linked to either a short (IgG 4 hinge only), intermediate (hinge plus IgG 4 CH3 domain), or long (hinge plus IgG 4 CH3 domain plus IgG 4 CH2 domain) spacer, the CD28-transmembrane domain, CD3zeta and 4-1BB intracellular signaling domains, and non-functional truncated CD19 (tCD19) as transduction marker. Similar constructs using scFvs from 2 different V-set domain-targeting CD33 antibodies, including hP67.6 (My96; used in gemtuzumab ozogamicin), were generated for comparison. CAR-T cells were sorted, expanded in IL-7 and IL-15, and used in vitro or in vivo against human AML cell lines endogenously expressing CD33 and cell lines engineered to lack CD33 (via CRISPR/Cas9) with/or without forced expression of different CD33 variants. Results: CD33 V-set-directed CAR T cells exerted significantly more cytolytic activity against AML cells expressing an artificial CD33 variant lacking the C2-set domain (CD33 ΔE3-4) than cells expressing full-length CD33 at similar or higher levels, consistent with the notion that CD33 CAR T cell efficacy is enhanced when targeting an epitope that is located closer to the cell membrane. CD33 PAN CAR T cells were highly potent against human AML cells in a strictly CD33-dependent fashion, with constructs containing the short and intermediate-length spacer demonstrating robust cytokine secretion, cell proliferation, and in vitro cytolytic activity, as determined by 51Cr release cytotoxicity assays. When compared to optimized CD33 V-set CAR T cells, optimized CD33 PAN CAR T cells were significantly more potent in cytotoxicity, proliferation, and cytokine production without appreciably increased acquisition of exhaustion markers. In vivo, CD33 PAN CAR T cells extended survival in immunodeficient NOD.SCID. IL2rg -/- (NSG) mice bearing significant leukemic burdens from various cell line-derived xenografts (HL-60, KG1α and MOLM14) with efficient tumor clearance demonstrated in a dose-dependent fashion. Conclusion: Targeting the membrane proximal domain of CD33 enhances the anti-leukemic potency of CAR T cells. Our data provide the rationale for the further development of CD33 PAN CAR T cells toward clinical testing. Disclosures Fiorenza: Link Immunotherapeutics: Consultancy; Bristol Myers Squibb: Research Funding. Godwin: Pfizer: Research Funding; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Turtle: Allogene: Consultancy; Amgen: Consultancy; Arsenal Bio: Consultancy; Asher bio: Consultancy; Astrazeneca: Consultancy, Research Funding; Caribou Biosciences: Consultancy, Current holder of individual stocks in a privately-held company; Century Therapeutics: Consultancy, Other; Eureka therapeutics: Current holder of individual stocks in a privately-held company, Other; Juno therapeutics/BMS: Patents & Royalties, Research Funding; Myeloid Therapeutics: Current holder of individual stocks in a privately-held company, Other; Nektar therapeutics: Consultancy, Research Funding; PACT Pharma: Consultancy; Precision Biosciences: Current holder of individual stocks in a privately-held company, Other; T-CURX: Other; TCR2 Therapeutics: Research Funding. Walter: Kite: Consultancy; Janssen: Consultancy; Genentech: Consultancy; BMS: Consultancy; Astellas: Consultancy; Agios: Consultancy; Amphivena: Consultancy, Other: ownership interests; Selvita: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Research Funding; Macrogenics: Consultancy, Research Funding; Immunogen: Research Funding; Celgene: Consultancy, Research Funding; Aptevo: Consultancy, Research Funding; Amgen: Research Funding.

A Novel Bcma-Specific, Centyrin-Based CAR-T Product for the Treatment of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2127-2127 ◽  
Author(s):  
David L. Hermanson ◽  
Burton Earle Barnett ◽  
Srinivas Rengarajan ◽  
Rebecca Codde ◽  
Xinxin Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
M Protein ◽  
Manufacturing Processes ◽  
Protein Levels ◽  
Car T

Abstract Chimeric-antigen receptor (CAR)-T cell immunotherapies have been remarkably effective in treating acute lymphoblastic leukemia. However, current strategies generally suffer from difficult, inefficient and costly manufacturing processes, significant patient side effects and poor durability of response in some patients. Here, we report for the first time a CAR-T cell therapeutic comprising a non-immunoglobulin alternative scaffold Centyrin molecule (a "CARTyrin") manufactured with a novel non-viral piggyBacTM (PB) transposon-based system. Our lead candidate, P-BCMA-101, encodes a CARTyrin that targets the B cell maturation antigen (BCMA) for the treatment of multiple myeloma (MM) and has several unique aspects that improve upon earlier CAR-T products. First, P-BCMA-101 is manufactured using only in vitro transcribed mRNA and plasmid DNA without the need for lentivirus or g-retrovirus, resulting in time and cost savings. Importantly, PB is also safer than viral systems due to a less mutagenic insertional profile and is non-oncogenic. Furthermore, PB can efficiently deliver transgenes as large as several hundred kilobases, and, once inserted, transgenes demonstrate more stable, prolonged and higher expression when compared to those delivered by virus. Second, a mutein of the dihydrofolate reductase (DHFR) gene is included in the P-BCMA-101 transgene that can be used in combination with the non-genotoxic drug methotrexate (MTX) to provide a simple and effective method of CARTyrin+ cell enrichment and reduces variability in patient product material. Third, P-BCMA-101 incorporates a safety switch for optional depletion in vivo in case of adverse events. Lastly, the CARTyrin is comprised of a BCMA-specific Centyrin, which are based on a human tenascin fibronectin type III (FN3) consensus sequence. Centyrins have similar binding affinities to the antibody-derived single chain variable fragments (scFv), but are smaller, more thermostable and predicted to be less immunogenic. Importantly, no signs of tonic signaling leading to T cell exhaustion have been observed with CARTyrins unlike scFv-based CAR molecules, which can interact with each other on the surface causing non-specific CAR signaling. The manufacture process of P-BCMA-101 from primary human T cells is straightforward, employs no cytokines, and easily produces enough CARTyrin+ cells to treat patients. Within 18 days of electroporation of purified T cells, we demonstrate > 95% of the cell product is positive for CARTyrin expression and ready to be administered. Notably, our manufacturing process results in > 60% of CARTyrin+ T cells exhibiting a stem-cell memory phenotype (i.e. CD45RA+ CD62L+). P-BCMA-101 cells exhibit specific and robust in vitro activity against BCMA+ tumor targets, ranging from high to very low levels of BCMA, as measured by target-cell killing and CARTyrin-T cell proliferation. Importantly, proliferating P-BCMA-101 cells were highly sensitive in vitro to activation of the safety switch. Finally, we have evaluated the anti-tumor efficacy of P-BCMA-101 in a model of human MM. NSG™ mice were injected IV with 1.5x106 luciferase+ MM.1S cells, an aggressive human MM-derived cell line. After the tumor cells were allowed to grow for 21 days, animals received a single IV administration of 5x106 P-BCMA-101 cells. All untreated control animals demonstrated a marked increase in serum M-protein levels, rapid growth of tumor cells demonstrated by bioluminescent imaging (BLI), and death within four weeks. In stark contrast, 100% of animals that received P-BCMA-101 rapidly eliminated tumors within 7 days as measured by BLI and serum M-protein levels and improved survival out to at least 60 days post-treatment. P-BCMA-101 is the first-in-class of Centyrin-based CAR therapeutics. The CARTyrin, combined with our advanced manufacturing processes, represents a significant improvement over first generation, immunoglobulin-based and virally-transduced CAR-T products. P-BCMA-101 exhibited an advantageous stem-cell memory phenotype and demonstrated specific and potent anti-tumor efficacy against BCMA+ myeloma cells both in vitro and in vivo. Based on these results, we plan to initiate a phase I clinical trial of P-BCMA-101 for the treatment of patients with relapsed and/or refractory MM. Disclosures Hermanson: Poseida Therapeutics: Employment. Barnett:Poseida Therapeutics: Employment. Rengarajan:Poseida Therapeutics: Employment. Codde:Poseida Therapeutics: Employment. Wang:Poseida Therapeutics: Employment. Tan:Poseida Therapeutics: Employment. Martin:Poseida Therapeutics: Employment. Smith:Poseida Therapeutics: Employment. Osertag:Poseida Therapeutics: Employment, Equity Ownership. Shedlock:Poseida Therapeutics: Employment.

scholarly journals Engineering an Optimized Trimeric APRIL-Based CAR to Broaden Targetability of Multiple Myeloma

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2059-2059 ◽  
Author(s):  
Andrea Schmidts ◽  
Maria Ormhoj ◽  
Allison O. Taylor ◽  
Selena J. Lorrey ◽  
Irene Scarfò ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
Research Funding ◽  
Antigen Expression ◽  
Tumor Burden ◽  
Monomeric Form ◽  
Car T Cells ◽  
Car T

Abstract Background: Targeting BCMA (B-cell maturation factor) with chimeric antigen receptor (CAR) T cells has shown great success in the treatment of multiple myeloma (MM), but is limited by heterogeneous antigen expression and imminent antigen escape of tumor cells. Combinatorial antigen targeting may help address these challenges. Taking the naturally occurring receptor-ligand pairs as a model, we designed monomeric and trimeric APRIL- (A proliferation-inducing ligand) based CARs targeting BCMA and TACI (transmembrane activator and CAML interactor) simultaneously. Methods: The following 2nd generation CARs were designed to target BCMA and TACI concurrently: membrane-tethered truncated APRIL monomer ("APRIL-CAR") and three truncated and fused APRIL monomers ("TriPRIL-CAR"). A single chain variable fragment-based anti(α)-BCMA CAR served as control. CAR multimerization and binding affinity to BCMA and TACI were characterized. In vitro effector function was compared by cytotoxic potency, activation (CD69), degranulation (CD107a), cytokine production and proliferation in response to target antigens. In vivo anti-tumor efficiency was assessed in a xenograft mouse model of MM. Results: CAR T cell manufacturing of all three constructs was accomplished successfully (transduction efficiency 46-78%) from three different donors. Western blot analysis of CARs showed multimerized forms of the TriPRIL and α-BCMA CAR, while only the monomeric form of the APRIL CAR was detected. Binding affinity to soluble BCMA and TACI was higher for the TriPRIL CAR compared to the APRIL CAR. Evaluating the cytotoxic potential, activation and degranulation kinetics as well as long-term proliferation against a panel of BCMA and/or TACI positive target cells, the TriPRIL CAR T cells outperformed the APRIL CAR T cells. All three CAR constructs demonstrated robust antigen-specific production of Th1-type cytokines, like Il-2, IFNƔ, GM-CSF and TNFα. Next, we performed an in vivo stress test, engrafting NSG mice with high tumor burden of MM.1s myeloma cells. The TriPRIL and α-BCMA CAR T cells were able to eradicate the tumors while the APRIL CAR T cells only led to a stabilization of tumor burden. In vivo studies with a mixed antigen population aiming at modeling heterogeneous antigen expression and antigen escape are ongoing. Conclusion: Our APRIL-based chimeric antigen receptors were able to redirect T cell cytotoxicity to both BCMA and TACI positive tumor cells. Since both these receptors are consistently up-regulated on malignant plasma cells this is an attractive method to target MM. Furthermore, we found that using a trimeric form of APRIL rather than monomeric form as the CAR binding domain increased recognition of MM antigens in vitro and in vivo. Disclosures Maus: crispr therapeutics: Consultancy, Research Funding; adaptimmune: Consultancy; novartis: Consultancy; kite therapeutics: Consultancy, Research Funding; windmil therapeutics: Consultancy; agentus: Consultancy, Research Funding.

scholarly journals Optimal Dual-Targeted CAR Construct Simultaneously Targeting Bcma and GPRC5D Prevents Bcma-Escape Driven Relapse in Multiple Myeloma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 136-136 ◽  
Author(s):  
Carlos Fernandez de Larrea ◽  
Mette Staehr ◽  
Andrea Lopez ◽  
Yunxin Chen ◽  
Terence J Purdon ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Car T Cells ◽  
Car T Cell ◽  
Dual Targeting ◽  
Car T ◽  
Previously Treated

Multiple myeloma (MM) remains generally incurable, calling for the development of novel treatment strategies such as chimeric antigen receptor (CAR) T cell therapy. Most clinically tested CAR T cell therapies for MM target B cell maturation antigen (BCMA), but despite high response rates, many patients relapse (Raje N. NEJM 2019). BCMA negative-low MM cells are implicated as a reservoir preceding relapse (Brudno J. JCO 2018; Cohen A. JCI 2019). Our aims are to (1) evaluate whether upfront simultaneous targeting of an additional antigen such as G protein-coupled receptor class C group 5 member D (GPRC5D; Smith EL. Sci Trans Med 2019) can mitigate BCMA escape-mediated relapse in MM, and (2) compare dual targeting strategies to identify an optimal approach. Dual targeting for CD19/CD22 malignancies has been investigated, and multiple approaches are feasible; however, approaches have yet to be comprehensively compared head to head. Here, we compare 2 parallel production and 3 single-vector dual targeting strategies (Fig. 1A). To enhance clinical translatability, all strategies are built on the BCMA(125)/4-1BBζ CAR (BCMA scFv 125; Smith EL. Mol Ther 2018), which is currently under multi-center clinical investigation (NCT03430011; Mailankody S. ASH 2018). We confirmed that all dual targeted approaches lyse, proliferate, and secrete polyfunctional cytokines specifically in response to BCMA and GPRC5D mono- and dual-positive cell lines and/or primary patient MM aspirate samples. Activity in vivo was confirmed using the bone marrow-tropic OPM2 MM model (endogenously BCMA+GPRC5D+). In all experiments MM cells (2 x 106) were injected IV into NSG mice and engrafted/expanded for 14 days before treatment. A high dose of all dual targeted CAR T cell approaches (3 x 106 CAR+) induced long-term disease control (median overall survival (mOS) BCMA(125) non-signaling del control 32d vs other groups mOS not reached; p < 0.05). Prevention of latent BCMA escape-mediated relapse was evaluated by re-challenge of previously treated long-surviving mice with 2 x 106 OPM2 BCMA CRISPR KO (OPM2BCMA KO) cells at day 100 without re-treatment. While mice previously treated with BCMA(125)/41BBζ CAR T cells succumbed to OPM2BCMA KO disease, dual targeted approaches prevented OPM2BCMA KO growth (mOS BCMA mono-targeted arm 37d post re-challenge vs other groups mOS not reached; p < 0.05). To better recapitulate human MM and distinguish among dual targeting approaches, we modeled established BCMA heterogeneous disease by spiking 5-10% OPM2BCMA KO into bulk OPM2WT cells for injection. Each OPM2 population was modified to express distinct luciferases for simultaneous in vivo monitoring by bioluminescent imaging (BLI). Treatment with a moderate (5 x 105) dose of CAR T cells eradicated OPM2WT cells in all groups, but anti-GPRC5D CARs with CD28 co-stimulation, whether included within a mixed T cell population or in a bicistronic construct (Fig. 1A ii, iv), failed to control OPM2BCMA KO cells (Fig. 1B). Correspondingly, 4-1BB-only containing CAR T cells had increased in vivo expansion (2.1-4.1-fold increase CAR T cell BLI at day 7 over CD28 containing groups; p < 0.05). As this result is likely from greater activation-induced cell death in the CD28-containing approaches that was not rescued by 4-1BB, we later compared 4-1BB-only containing approaches (Fig. 1A i, iii, v). These 3 dual targeting approaches effectively controlled OPM2WT disease at moderate (1 x 106 CAR+) and low (2.5 x 105 CAR+) doses. However, when using a sub-therapeutic dose (2.5 x 105 CAR+) in the OPM2BCMA KO-spiked model, the tandem scFv-single stalk design was least effective in controlling OPM2BCMA KO disease (Fig 1C). At a dose that is sub-therapeutic to control OPM2WT disease (1 x 105 CAR+), the bicistronic dual 4-1BB design (Fig. 1A iii) was more effective in eradicating tumor compared with the parallel production approach (6-fold difference tumor BLI at day 28; p < 0.05). These results indicate that upfront dual targeting of BCMA/GPRC5D with CAR T cells can mitigate BCMA escape-mediated relapse in a model of MM. While parallel infusion of separate BCMA- and GPRC5D-targeted CAR T cells is effective, a single bicistronic vector encoding two 4-1BB-containing CARs avoids the practical challenges of parallel manufacturing, and uniquely may provide superior anti-MM efficacy. Figure Disclosures Fernandez de Larrea: Takeda: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding. Brentjens:JUNO Therapeutics: Consultancy, Patents & Royalties, Research Funding; Celgene: Consultancy. Smith:Celgene: Consultancy, Patents & Royalties, Research Funding; Fate Therapeutics and Precision Biosciences: Consultancy.

Preclinical Evaluation of Allogeneic Anti-Bcma Chimeric Antigen Receptor T Cells with Safety Switch Domains and Lymphodepletion Resistance for the Treatment of Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 381-381 ◽  
Author(s):  
Bijan Boldajipour ◽  
Roman Galetto ◽  
Cesar Sommer ◽  
Thomas Pertel ◽  
Julien Valton ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
Chimeric Antigen Receptor ◽  
Antigen Receptor ◽  
Preclinical Evaluation ◽  
Car T Cells ◽  
Car T

Abstract Multiple myeloma (MM) is a hematological disease of plasma B cells that remains incurable despite the availability of numerous therapies. The plasma cell-specific expression of the TNF superfamily receptor BCMA may allow targeting of normal and malignant plasma cells. Genetically engineered chimeric antigen-receptor T cells (CAR T) have shown tremendous promise in the treatment of several hematological diseases, including MM. However, conventional autologous CAR T therapies use patient-derived T cells and the logistics of on-demand CAR T manufacture limits their availability to a broad patient pool. Here we describe the preclinical evaluation of an allogeneic CAR T therapy targeting BCMA that has the potential for a readily available, off-the-shelf therapy for MM and other malignancies expressing BCMA. Human T cells were transduced with recombinant lentiviral vectors encoding three BCMA CAR candidates designed with fully human anti-BCMA scFvs, CD8a transmembrane domains and the intracellular signaling domains of 4-1BB and CD3zeta. All CAR T efficiently killed BCMA-expressing multiple myeloma cell lines (KMS12BM, MM1.S, Molp-8 and OPM-2), but not BCMA-negative REH cells in vitro and in vivo. Whereas 2 of the 3 candidates exhibited target-independent cytokine production, accelerated T cell differentiation and reduced target cell-induced expansion in vitro, the third candidate did not exhibit this scFv-induced autoactivation and was chosen as the lead molecule. Due to the allogeneic nature of this T cell therapy, the possibility of graft-versus-host (GvH) reactions can be a safety concern. We applied Cellectis' know-how and TALEN® technology for the gene inactivation of the T cell receptor (TCR) alpha chain to significantly reduce the probability for TCR-mediated GvH reactions and found that TCR knockout did not affect CAR T activity in vitro or in vivo. Furthermore, we incorporated intra-CAR rituximab-recognition domains into the CAR molecule to enable depletion of CAR T cells from patients when necessary. We found that this modified CAR retained anti-BCMA CAR T activity and enabled CAR T depletion by rituximab. Another aspect of allogeneic CAR T therapies is the rejection of the CAR T by host-versus-graft (HvG) reactions. Lymphodepletion prior to CAR T infusion enhances CAR T efficacy in autologous CAR T trials and may also prevent anti-CAR HvG reactions in allogeneic therapy settings. Engineering lymphodepletion resistance into CAR T cells could therefore enable sustained lymphodepletion for enhanced allogeneic CAR T persistence and efficacy. CD52 is expressed on all lymphocytes and administration of the anti-CD52 antibody alemtuzumab for prolonged lymphodepletion is an approved treatment for multiple sclerosis. TALEN®-mediated knockout of CD52 protected BCMA CAR T from alemtuzumab-induced cytotoxicity and did not alter BCMA CAR T anti-tumor activity. Taken together these results support allogeneic BCMA CAR T as an off-the-shelf adoptive immunotherapy for the treatment of multiple myeloma and other BCMA-positive malignancies. Disclosures Boldajipour: Pfizer: Employment. Galetto:Cellectis SA: Employment. Sommer:Pfizer Inc.: Employment. Pertel:Pfizer Inc.: Employment. Valton:Cellectis Inc.: Employment. Park:Pfizer Inc.: Employment. Gariboldi:Cellectis SA: Employment. Chen:Alexo Therapeutics: Employment. Geng:Kodiak Sciences: Employment. Dong:Pfizer Inc.: Employment. Boucher:Pfizer Inc.: Employment. Van Blarcom:Pfizer Inc.: Employment. Chaparro-Riggers:Pfizer Inc.: Employment. Rajpal:Pfizer Inc.: Employment. Smith:Cellectis SA: Employment. Kuo:Pfizer Inc.: Employment. Sasu:Pfizer Inc.: Employment.

scholarly journals Developing a Safer Anti-CD44v6 Chimeric Antigen Receptor T Cell Against Hematological Cancers By Mitigating on-Target Off-Tumor Toxicity

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2796-2796
Author(s):  
Dennis Awuah ◽  
Lawrence Stern ◽  
Ian Schrack ◽  
Tae Yoon Kim ◽  
Joseph Cohen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
T Cells ◽  
T Cell ◽  
T Cell Therapy ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T ◽  
Soluble Scfv

Abstract Adoptively transferred chimeric antigen receptor (CAR) T cells have shown significant promise in targeted immunotherapy against hematological tumors, despite concerns with safety and antigen escape. The CD44 adhesion molecule, which binds components of ECM such as collagen and hyaluronan, has been implicated in growth and survival as well as metastasis of tumor initiating stem cells (TSCs). A splice variant of CD44, CD44v6 is broadly expressed on malignant tumors including AML, CML and multiple myeloma (MM), where it aids tumor migration and predicts poor disease prognosis 1. Moreover, the expression of CD44v6 on healthy tissues including keratinocytes, presents a significant challenge in 'on-target, off-tumor' toxicity 2,3. We have developed a second generation, anti-CD44v6 CAR T cell (CD44v6CAR) that mediates potent cytotoxic and effector function against AML and MM in-vitro. Additionally, we observed lesser potency of our CD44v6CAR T cells against MM in comparison with AML in-vivo. Interestingly, higher levels of soluble CD44v6 antigen were also detected in mice engrafted with MM.1S compared to THP1. Possibly, the presence of soluble CD44v6 in mouse serum culminated in reduced anti-tumor activity against MM in-vivo, most likely due to CAR scFv blockade with antigen. Here, we provide an alternative strategy for improving the CD44v6 CAR T cell therapy and mitigating on-target off-tumor toxicity using CD44v6 CAR T cells that secrete a soluble protease-susceptible version of the CAR-expressed CD44v6 scFv that will block CAR binding in healthy tissue, but will be cleaved by cancer-specific proteases in the tumor site, allowing for CAR T cell binding and activation (Figure 1). We leveraged the presence of matrix metalloproteinase (MMP)-2, which is significantly overexpressed in multiple myeloma tumor microenvironment 4 to develop a modified CAR T construct (sCD44v6CAR) with engineered MMP-2 cut site in the linker between the heavy and light chains of the soluble CD44v6scFv. Kinetic analysis with varying MMP-2 concentration and digestion time showed moderate proteolytic susceptibility of our scFv construct, with digestion efficiency increasing in a dose-dependent manner. Furthermore, our engineered protease-liable scFv demonstrated higher affinity for CD44v6 antigen binding in titration assays compared to CAR scFv suggesting that engineered scFv are able to 1) bind and neutralize soluble antigen and 2) bind antigen on healthy tissues with high affinity to mitigate off-tumor toxicity. As expected, both conventional (CD44v6CAR) and modified sCD44v6 CAR T cells showed effective cytotoxicity against AML in vitro. Interestingly, cytotoxic activity against MM.1S using the modified T cells (sCD44v6CAR) was significantly suppressed, likely resulting from secretion of soluble scFv. The addition of recombinant MMP-2 in co-culture assays cleaved soluble scFvs, rescuing CAR-mediated tumor killing. Taken together, the data confirms our proof-of-concept hypothesis and highlights the protective capacity of engineered sCD44v6CAR T cells, with its ability to potentially neutralize off-target toxicity and improve anti-MM activity in future studies, which has impact on the CAR T cell therapy as a general strategy. Figure 1: Schematic representation of CAR T Cell mitigating off-disease recognition. Presence of protease-susceptible linker in soluble scFv is efficiently cleaved by tumor-specific proteases in tumor microenvironment enabling CAR binding and activation. Lack of specific proteases in healthy tissues leads to high affinity, soluble scFv-target binding and effective blocking. References 1. Heider, K. H., Kuthan, H., Stehle, G. & Munzert, G. CD44v6: a target for antibody-based cancer therapy. Cancer Immunol Immunother 53, 567-579, (2004). 2. Casucci, M. et al. CD44v6-targeted T cells mediate potent antitumor effects against acute myeloid leukemia and multiple myeloma. Blood 122, 3461-3472, (2013). 3. Riechelmann, H. et al. Phase I trial with the CD44v6-targeting immunoconjugate bivatuzumab mertansine in head and neck squamous cell carcinoma. Oral Oncology 44, 823-829, (2008). 4. Shay, G. et al. Selective inhibition of matrix metalloproteinase-2 in the multiple myeloma-bone microenvironment. Oncotarget 8, 41827-41840, (2017). Figure 1 Figure 1. Disclosures Forman: Allogene: Consultancy; Mustang Bio: Consultancy, Current holder of individual stocks in a privately-held company; Lixte Biotechnology: Consultancy, Current holder of individual stocks in a privately-held company. Wang: Pepromene Bio, Inc.: Consultancy.

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

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.

scholarly journals Base-edited CAR T cells for combinational therapy against T cell malignancies

Leukemia ◽  
2021 ◽  
Author(s):  
Christos Georgiadis ◽  
Jane Rasaiyaah ◽  
Soragia Athina Gkazi ◽  
Roland Preece ◽  
Aniekan Etuk ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Specific Binding ◽  
Base Editing ◽  
Car T Cells ◽  
Dna And Rna ◽  
Car T ◽  
T Cell Malignancies

AbstractTargeting T cell malignancies using chimeric antigen receptor (CAR) T cells is hindered by ‘T v T’ fratricide against shared antigens such as CD3 and CD7. Base editing offers the possibility of seamless disruption of gene expression of problematic antigens through creation of stop codons or elimination of splice sites. We describe the generation of fratricide-resistant T cells by orderly removal of TCR/CD3 and CD7 ahead of lentiviral-mediated expression of CARs specific for CD3 or CD7. Molecular interrogation of base-edited cells confirmed elimination of chromosomal translocations detected in conventional Cas9 treated cells. Interestingly, 3CAR/7CAR co-culture resulted in ‘self-enrichment’ yielding populations 99.6% TCR−/CD3−/CD7−. 3CAR or 7CAR cells were able to exert specific cytotoxicity against leukaemia lines with defined CD3 and/or CD7 expression as well as primary T-ALL cells. Co-cultured 3CAR/7CAR cells exhibited highest cytotoxicity against CD3 + CD7 + T-ALL targets in vitro and an in vivo human:murine chimeric model. While APOBEC editors can reportedly exhibit guide-independent deamination of both DNA and RNA, we found no problematic ‘off-target’ activity or promiscuous base conversion affecting CAR antigen-specific binding regions, which may otherwise redirect T cell specificity. Combinational infusion of fratricide-resistant anti-T CAR T cells may enable enhanced molecular remission ahead of allo-HSCT for T cell malignancies.

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

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A121-A121
Author(s):  
Nina Chu ◽  
Michael Overstreet ◽  
Ryan Gilbreth ◽  
Lori Clarke ◽  
Christina Gesse ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Dominant Negative ◽  
Car T Cells ◽  
Car T Cell ◽  
Car T

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

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

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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