Chimeric Antigen Receptor mRNA Research Articles

Abstract 3615: Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells

Abstract Background: Neuroblastoma (NB) is a common malignant extracranial solid tumor in children, responsible for 11% of cancer deaths in pediatric patients under 15 (Smith et al, Cancer, 2014). High-risk neuroblastoma is a metastatic disease in children aged 18 months or older with L2, M, or MS disease with amplification of the MYCN oncogene (Chuang Pediatr Blood Cancer 2021). Recent analyses by COG show that HR Neuroblastoma still has a 5-year event free survival of 51% despite an extensive treatment regimen (Steven et al, ASCO, 2022). To address improved tumor targeting and overcome antigen escape, chimeric antigen receptor (CAR) technology to Natural Killer (NK) cell therapy has been adapted (Chu/Cairo, et al, Frontiers in Oncology, 2019). TGFβ is a potent mechanism of the NK cell anti-tumor resistance and is present at elevated levels in the NB tumor microenvironment (TME) (Foltz, Cancers, 2018). Studies demonstrated that blockade of TGF-β signaling in NK cells caused the accumulation of NK cells that produce IFN-γ and neutralization of TGF-β prevented NKG2D downregulation and also restored NK cell anti-tumor reactivity (Nayyar/Chu/Cairo, et al Frontierls in Oncology, 2018). ROR1, a transmembrane protein is highly expressed on the majority of NB (Rebagay/Yan Front Oncol, 2012). Our group has successfully expanded functional and active peripheral blood NK cells (exPBNK) with irradiated feeder cells and electroporated CAR mRNA to exPBNK (Chu/Cairo, et al Cancer Immunol Res, 2015). Objective: To determine in vitro cytotoxicity against neuroblastoma cells by ex vivo expanded TGFβ imprinting (TGFβi) NK cells. Methods: TGFβi NK cells are generated by culturing expanded NK cells with irradiated K562-41BBL-mbIL21 in 50 IU/mL IL-2 plus 10 ng/mL TGFβ every 2-3 days for two weeks. NK and TGFβi NK cells specific in-vitro cytotoxicity were compared via luciferase reporter assay against ROR1+ NB cell lines as we previously described (Chu/Cairo, et al, JITC, 2022). Cytokine/chemokine levels secreted by NK and TGFβi NK cells were compared using ELISA assays (Chu/Cairo, et al, JITC, 2022). Results: We have successfully expanded TGFβi NK cells. These TGFβi NK cells showed significantly enhanced in vitro cytotoxicity against NB cells compared to NK cells under TGFβ environment (20+2.4% vs 0%; p<0.001) at E:T=3:1 and consistently, TGFβ-imprinting NK secreted significantly high level of TNF-alpha (393.7pg/ml+16.77pg/ml) against NB cells (p<0.001) compared to NK cells (79.7pg/ml+14.7pg/ml) under TGFβ environment. Conclusion: Our data indicate TGFβi NK cells may be a promising novel therapy to overcome the immunosuppression of TGFβ in the tumor microenvironment for pediatric NB tumors. We will further engineer these TGFβi NK cells to express anti-ROR1 CAR to enhance the targeting specificity against NB tumors and investigate the anti-tumor effects of anti-ROR1 CAR TGFβi NK cells against NB tumors in-vitro and in-vivo (Funded by U54 CA232561). Citation Format: Jessica A. Ayala-Cuesta, Yaya Chu, Keira Foley, Meijuan Tian, Kayleigh Klose, Alyssa S. Mendelowitz, Timothy P. Cripe, Dean A. Lee, Mitchell S. Cairo. Targeting high-risk neuroblastoma by TGFβ imprinted natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3615.

Abstract P20: Anti-CD123 CAR NK cells and cam161533 Trispecific Killer Engager have synergistic activity against Acute Myeloid Leukemia

Abstract P20: Anti-CD123 CAR NK cells and cam161533 Trispecific Killer Engager have synergistic activity against Acute Myeloid Leukemia

In Vivo mRNA CAR T Cell Engineering via Targeted Ionizable Lipid Nanoparticles with Extrahepatic Tropism.

With six therapies approved by the Food and Drug Association, chimeric antigen receptor (CAR) T cells have reshaped cancer immunotherapy. However, these therapies rely on ex vivo viral transduction to induce permanent CAR expression in T cells, which contributes to high production costs and long-term side effects. Thus, this work aims to develop an in vivo CAR T cell engineering platform to streamline production while using mRNA to induce transient, tunable CAR expression. Specifically, an ionizable lipid nanoparticle (LNP) is utilized as these platforms have demonstrated clinical success in nucleic acid delivery. Though LNPs often accumulate in the liver, the LNP platform used here achieves extrahepatic transfection with enhanced delivery to the spleen, and it is further modified via antibody conjugation (Ab-LNPs) to target pan-T cell markers. The in vivo evaluation of these Ab-LNPs confirms that targeting is necessary for potent T cell transfection. When using these Ab-LNPs for the delivery of CAR mRNA, antibody and dose-dependent CAR expression and cytokine release are observed along with B cell depletion of up to 90%. In all, this work conjugates antibodies to LNPs with extrahepatic tropism, evaluates pan-T cell markers, and develops Ab-LNPs capable of generating functional CAR T cells in vivo.

Deploying an RNA-Based Gene Writer System and Lipid Nanoparticle (LNP) Delivery to Generate Functional Chimeric Antigen Receptor (CAR) T Cells with in Vitro and In Vivo Anti-Tumor Activity

The FDA approval of autologous Chimeric Antigen Receptor (CAR) T-cell therapies have added a powerful tool in the armamentarium to treat relapsed and refractory leukemias and lymphomas. However, significant challenges limit these therapies from accommodating current patient demand including supply chain limitations and wait times associated with availability of GMP quality viral vectors and long needle-to-needle time from initiation of treatment to receiving the final drug product. As of yet other competitive therapies including bi-specific antibodies and allogeneic CAR-T have not achieved the same level of therapeutic efficacy as conventional autologous T cell therapies highlighting the critical unmet need for same day treatment options for autologous CAR-T products. RNA Gene Writers leverage target-primed reverse transcription (TPRT) biochemistry evolved from non-LTR retrotransposon mobile genetic elements to modify the genetic information in cells using RNA templates and without the need to introduce DNA breaks. Moreover, RNA Gene Writers can be engineered to catalyze a variety of editing reactions, such as the introduction of gene-length DNA sequences, substitutions, insertions, and deletions. These edits can be achieved with all-RNA delivery in primary cells and in vivo, eliminating the necessity for viral vectors and DNA template-based gene editing. LNP-RNA delivery of RNA Gene Writers resulted in the integration and expression of transgenes in >60% of primary human T cells in vitro. Furthermore, our LNPs deliver RNA to T cells in vivo, with 80% reporter expression in a humanized mouse model and 45% in non-human primates (NHP). We have demonstrated LNP delivery of RNA Writers into primary human T cells with an RNA template encoding a CAR cassette can achieve >20% CAR+ T cells without detriments to cell viability or proliferative capacity and have demonstrated ability to mediate tumor cell killing in vitro. Moreover, RNA Gene Writer derived CAR-T cells can be introduced into mouse xenograft models to clear antigen specific tumors in vivo. CAR mRNA can also be packaged into proprietary LNP formulations and delivered to primary human T cells in a mixture of lymphocytes commonly found in patient leukapheresis to generate CAR-T cells in vitro. This opens the possibility of using RNA Gene Writers to develop a same-day CAR-T treatment. In addition, the modularity of our RNA Gene Writing technology allows multiplex editing to co-introduce multiple genetic changes including generation of universal and more potent CAR-T cells through the knock-out of B2M and TRAC at comparable levels (>80% double knockout) to Cas9 nucleases. Further, we show that we can achieve both edits simultaneously in 80% of T cells that have achieved CAR transgene insertion via another RNA Gene Writer enzyme. Importantly, we have demonstrated successful B2M and TRAC double knock-out without inducing translocations (undetectable vs 8% translocations with Cas9). RNA Gene Writing technology is uniquely positioned for same-day delivery of all-RNA components toenable a wide range of editing applications, from gene knock-out to gene integration. The ability to package the RNA Gene Writer system into LNPs unlocks the potential for the genetic engineering of autologous T cell therapies to enable same-day delivery a current limitation of conventional lentiviral-based CAR-T therapies.

IMMU-26. MRNA CAR T CELL TRAFFICKING FOLLOWING INTRA-VENTRICULAR ADMINISTRATION IN PRECLINICAL MODELS OF DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND GD2-directed chimeric antigen receptor (CAR) T cells have shown promise as a potential therapeutic for diffuse midline glioma. We have previously shown intra-tumoral infusions of mRNA CAR T cells result in tumor regression with improved safety and toxicity profile in murine models. For human trials, the optimal route of CAR T cell administration into the central nervous system should maximize in vivo potency while minimizing procedural morbidity. Prior mRNA CAR T literature has reported decreased solid tumor infiltration with systemic delivery, so we sought to determine mRNA CAR T cell trafficking into pontine tumors from the cerebral spinal fluid to inform clinical translation of this therapy. METHODS Mice engrafted with SU-DIPG13P* tumor cells in the pons were treated with 5x106 mRNA CAR T cells into the lateral ventricle (LV) and brains were harvested 24 hours later. Tumor and T cells were analyzed using confocal microscopy to determine the migration of CAR T cells. RESULTS LV injection of GD2-directed CAR T cells resulted in T cell trafficking to the subarachnoid spaces, tumor site, and surrounding parenchyma. The highest concentration of T cells was in the subarachnoid space directly adjacent to the tumor (p< 0.0001) and decreased on a gradient to subcortical tumor. Even at 24 hours after injection, T cells were identified within the center of the tumor mass. CD19-directed control CAR T cells showed similar distribution within the subarachnoid with significantly decreased T cells at the tumor site. CONCLUSION Ventricular infusions of mRNA CAR T cells showed effective migration to pontine tumors within 24 hours. While CAR T cells did not fully penetrate the parenchyma within that timeframe, GD2-directed mRNA CAR T cells were detected within the center of the tumor mass, indicating ventricular administration may be sufficient for clinical delivery. Additional routes are being explored in ongoing work.

Ionizable Lipid Nanoparticles with Integrated Immune Checkpoint Inhibition for mRNA CAR T Cell Engineering.

The programmed cell death protein 1 (PD-1) signaling pathway is a major source of dampened T cell activity in the tumor microenvironment. While clinical approaches to inhibiting the PD-1 pathway using antibody blockade have been broadly successful, these approaches lead to widespread PD-1 suppression, increasing the risk of autoimmune reactions. This study reports the development of an ionizable lipid nanoparticle (LNP) platform for simultaneous therapeutic gene expression and RNA interference (RNAi)-mediated transient gene knockdown in T cells. In developing this platform, interesting interactions are observed between the two RNA cargoes when co-encapsulated, leading to improved expression and knockdown characteristics compared to delivering either cargo alone. This messenger RNA (mRNA)/small interfering RNA (siRNA) co-delivery platform is adopted to deliver chimeric antigen receptor (CAR) mRNA and siRNA targeting PD-1 to primary human T cells ex vivo and strong CAR expression and PD-1 knockdown are observed without apparent changes to overall T cell activation state. This delivery platform shows great promise for transient immune gene modulation for a number of immunoengineering applications, including the development of improved cancerimmunotherapies.

Enhancing the effectiveness of γδ T cells by mRNA transfection of chimeric antigen receptors or bispecific T cell engagers

Adoptive cell therapy (ACT) utilizing γδ Tcells is becoming a promising option for the treatment of cancer, because it offers an off-the-shelf allogeneic product that is safe, potent, and clinically effective. Approaches to engineer or enhance immune-competent cells for ACT, like expression of chimeric antigen receptors (CARs) or combination treatments with bispecific Tcell engagers, have improved the specificity and cytotoxic potential of ACTs and have shown great promise in preclinical and clinical settings. Here, we test whether electroporation of γδ Tcells with CAR or secreted bispecific Tcell engager (sBite) mRNA is an effective approach to improve the cytotoxicity of γδ Tcells. Using a CD19-specific CAR, approximately 60% of γδ Tcells are modified after mRNA electroporation and these cells show potent anticancer activity invitro and invivo against two CD19-positive cancer cell lines. In addition, expression and secretion of a CD19 sBite enhances γδ Tcell cytotoxicity, both invitro and invivo, and promotes killing of target cells by modified and unmodified γδ Tcells. Taken together, we show that transient transfection of γδ Tcells with CAR or sBite mRNA by electroporation can be an effective treatment platform as a cancer therapeutic.

Abstract 1785: Multi-step engineering of gene-edited CAR T cells using RNA lipid nanoparticles

Abstract Autologous chimeric antigen receptor (CAR) T therapies utilize patient cells and can be limited by cell quality, and the high manufacturing burden of viral vectors. As such, there is a need for allogeneic, “off-the-shelf” CAR T cells to make these transformative treatments widely available. However, allogeneic therapies require multiple genetic engineering steps to express CAR and to delete proteins responsible for graft-versus-host disease. Messenger RNA (mRNA) is a promising approach for expression of therapeutic proteins and gene editing nucleases. In this work, we demonstrate a new method for multi-step engineering of gene-edited CAR T cells using RNA lipid nanoparticles (LNPs). LNPs encapsulating Spy-Cas9 mRNA, TCR and CD52 guide RNA (sgRNA), and CAR mRNA were produced using microfluidics. The CAR construct contained an anti-CD19 scFv binding domain and CD3ζ/4-1BB co-stimulatory domains. Microgram quantities of RNA LNPs were produced to optimize LNP packaging, cargo ratios, and sgRNA combinations. Lead candidates were scaled to milligrams. Purified human primary T cells were cultured, activated, and expanded in serum-free media in plates, flasks and bioreactors. CAR+, TCR− or CD52− cells were generated by addition of the corresponding LNP to activated cells. Cytotoxic killing was determined by co-culture assays with leukemia cells. Gene knockout, CAR expression, viability and cell killing were measured using flow-cytometry. CD19 CAR was selected as a relevant protein for expression, with TCR and CD52 proteins as gene knockout targets. Single-step addition of CAR LNPs to T cells resulted in transfection efficiencies of 95.0 ± 2.1% and high protein expression. Upon TCR or CD52 LNP addition to T cells, the onset of gene editing was within 48 hours, reaching single target knockout efficiencies of 92.3 ± 3.0% (TCR−), and double knockouts (TCR−/CD52−) of 74.5 ± 6.1%. Similar results were obtained when comparing different LNP batch sizes (microgram to milligram RNA) and cell culture vessels (125,000 to 45 million cells), demonstrating scalability of both the LNP production and cell treatment. Cell viabilities above 90% were maintained at all steps and for all RNA LNPs. Finally, as proof-of-concept for multi-step engineering, sequential addition of TCR LNPs and CAR LNPs resulted in simultaneous CAR expression and TCR gene knockout. These “off-the-shelf” gene-edited CAR T cells were functionally equivalent to non-edited cells in a B cell killing assay, efficiently clearing over 80% of leukemia target cells at a 1:1 ratio. Our findings demonstrate the advantages of LNPs for RNA delivery to T cells. The simple and gentle nature of LNP cell treatment allows for multiple genetic engineering steps for simultaneous expression and deletion of proteins. Furthermore, LNPs can be easily manufactured using microfluidics, enabling small-scale screening of RNA libraries and rapid scale-up of lead candidates for clinical translation. Citation Format: Samuel Clarke, R Geczy, A Balgi, S Park, R Zhao, M Swaminathan, R Tieu, N Hoang, C Webb, E Watt, M Wong, M Fujisawa, N Jain, Angela Zhang, Anitha Thomas. Multi-step engineering of gene-edited CAR T cells using RNA lipid nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1785.

Nanotechnology-based chimeric antigen receptor T-cell therapy in treating solid tumor

Chimeric Antigen Receptor (CAR) T cells have changed the therapeutic landscape of hematological malignancies with overwhelming success. The clinical success of CAR T-cell therapy in hematologic malignancies has fueled interest in exploring the technology in solid tumors. However, the treatment of solid tumors presents a unique set of challenges compared to hematological tumors. The biggest impediments to the success of CAR T cell treatment are the paucity of tumor-specific antigens that are produced selectively and uniformly and the immunosuppressive tumor microenvironment. To overcome these significant challenges, nanotechnology has been involved to improve the efficacy of CAR-T cells. In this review, we systematically introduced the components of different generations of CARs and summarized recent innovations in nano-based CAR-T cell therapy to conquer therapeutically resistant non-hematologic malignancies, including mRNA and hydrogel-based CAR T cells delivery, photothermal-remodeling, and tumor microenvironment-based CAR T cell therapy. These nanotechnologies remarkably facilitate in vivo generation of CAR T cells and hold promise as a therapeutic platform to treat solid tumors and even other diseases.

Development of GPC2-directed chimeric antigen receptors using mRNA for pediatric brain tumors

BackgroundPediatric brain tumors are the leading cause of cancer death in children with an urgent need for innovative therapies. Glypican 2 (GPC2) is a cell surface oncoprotein expressed in neuroblastoma...

Research advance in lipid nanoparticle-mRNA delivery system and its application in CAR-T cell therapy.

Chimeric antigen receptor (CAR) T cell therapy has shown significant efficacy for hematological malignancies, however, it needs to be further optimized. Recently, the lipid nanoparticle (LNP)-mRNA delivery system as a nonviral gene transfer vector has gained rapid progress in CAR-T cell therapy. The claudin-6 (CLDN6) mRNA is delivered to antigen presenting cells (APCs) through LNP system, thereby enhancing the function of CLDN6 CAR-T cells for the clearance of solid tumor cells. For treatment of acute cardiac injury, the fibroblast activation protein (FAP) CAR mRNA can be delivered to T cells through LNP system for the in vivo production of FAP CAR-T cells, thereby blocking the process of myocardial fibrosis. The LNP-mRNA delivery system has advantages including having no integration in host genome, inexpensiveness, low toxicity and modifiability; on the other hand, it has certain disadvantages such as limited cell persistence caused by transient protein expression and limitations in preparation techniques. This article reviews the research advance in LNP-mRNA in vivo delivery system and its application in CAR-T cell therapy.

In Vitro Engineering Chimeric Antigen Receptor Macrophages and T Cells by Lipid Nanoparticle-Mediated mRNA Delivery.

Chimeric antigen receptor (CAR)-engineered adoptive cell therapy marks a revolution in cancer treatment based on the highly successful responses to CAR T cell therapy in the treatment of blood cancers. Due to the versatile structure of CARs, this technology can be easily adapted to other immune cell types, including macrophages and NKs, and applied in the treatment of many other cancers. However, high costs and fatal adverse effects represent significant concerns for future development. In vitro transcribed (IVT) mRNA therapeutics, which possess a high safety profile and straightforward production methods, could provide a useful alternative for CAR cell construction. However, the low stability and transfection efficiency of IVT-mRNA in immune cells limit further applications. In this work, we successfully engineered CAR macrophages (CAR-Ms) and CAR T cells with CAR mRNA using lipid nanoparticles (LNPs). Both the LNP formulations and mRNA modifications were optimized for in vitro mRNA transfection. More importantly, the CAR macrophages and CAR T cells both demonstrated significant cytotoxic effects on B lymphoma in vitro, underscoring the great potential of mRNA-engineered adoptive cell therapy.

Anti-CD117 CAR T Cells Incorporating a Safety Switch Eradicate Acute Myeloid Leukemia and Deplete Human Hematopoietic Stem Cells

Anti-CD117 CAR T Cells Incorporating a Safety Switch Eradicate Acute Myeloid Leukemia and Deplete Human Hematopoietic Stem Cells

Orthogonal Design of Experiments for Optimization of Lipid Nanoparticles for mRNA Engineering of CAR T Cells.

Viral engineered chimeric antigen receptor (CAR) T cell therapies are potent, targeted cancer immunotherapies, but their permanent CAR expression can lead to severe adverse effects. Nonviral messenger RNA (mRNA) CAR T cells are being explored to overcome these drawbacks, but electroporation, the most common T cell transfection method, is limited by cytotoxicity. As a potentially safer nonviral delivery strategy, here, sequential libraries of ionizable lipid nanoparticle (LNP) formulations with varied excipient compositions were screened in comparison to a standard formulation for improved mRNA delivery to T cells with low cytotoxicity, revealing B10 as the top formulation with a 3-fold increase in mRNA delivery. When compared to electroporation in primary human T cells, B10 LNPs induced comparable CAR expression with reduced cytotoxicity while demonstrating potent cancer cell killing. These results demonstrate the impact of excipient optimization on LNP performance and support B10 LNPs as a potent mRNA delivery platform for T cell engineering.

CD8+ Anti-BCMA mRNA CAR T-Cells Effectively Kill Human Multiple Myeloma Cells In Vitro and In Vivo

Potent anti-multiple myeloma (MM) responses have been observed in early-stage clinical trials with virally-transduced chimeric antigen receptor (CAR) T-cells redirected to B-cell maturation antigen (BCMA), a membrane protein with extremely high sensitivity and specificity for MM and plasma cells. Despite significant potency, CAR T-cells engineered by virally-mediated gene transfer present a poor risk profile. Permanent modification enhances CAR persistence but also increases the risk of severe cytokine release syndrome (CRS), neurotoxicity, and prolonged plasma cell aplasia due to uncontrolled in vivo expansion and long-term persistence of the permanently-modified CAR T-cells. In an effort to address these shortcomings while preserving efficacy, we have developed CD8+ anti-BCMA CAR T-cells modified transiently by mRNA transfection (Descartes-08). Descartes-08 manufactured at clinical scale showed high (>90%) purity, post-cryopreservation viability, and transfection efficiency. Descartes-08 demonstrated robust dose- and time- dependent killing of BCMA+ MM cell lines, including those with acquired resistance to immunomodulatory drugs (IMiDs), as well as primary MM cells co-cultured with autologous bone marrow stromal cells. Compared with transfected pan- (CD3+) anti-BCMA CAR T-cells, Descartes-08 (CD8+) demonstrated enhanced transfection and killing but reduced secretion of IFN-g, an inflammatory cytokine highly correlated with CRS. Descartes-08 also demonstrated robust dose-dependent efficacy in a disseminated human MM model (i.v. MM1SLuc cells in NSG mice), and could be safely administered repetitively to deepen responses. These results advocate for rapid clinical translation of Descartes-08, which may improve the benefit: risk profile of CAR T-cells in MM and enable their use in patients with earlier-stage disease. DisclosuresKurtoglu:2Cartesian Therapeutics, Inc: Employment, Equity Ownership. Holderness:Cartesian Therapeutics, Inc: Employment, Equity Ownership. Kalayoglu:Cartesian Therapeutics, Inc: Employment, Equity Ownership. Richardson:Celgene: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees. Anderson:Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; Oncopep: Other: scientific founder; C4 Therapeutics: Other: scientific founder.

In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo

Engineering chimeric antigen receptors (CAR) or T cell receptors (TCR) helps create disease-specific T cells for targeted therapy, but the cost and rigor associated with manufacturing engineered T cells ex vivo can be prohibitive, so programing T cells in vivo may be a viable alternative. Here we report an injectable nanocarrier that delivers in vitro-transcribed (IVT) CAR or TCR mRNA for transiently reprograming of circulating T cells to recognize disease-relevant antigens. In mouse models of human leukemia, prostate cancer and hepatitis B-induced hepatocellular carcinoma, repeated infusions of these polymer nanocarriers induce sufficient host T cells expressing tumor-specific CARs or virus-specific TCRs to cause disease regression at levels similar to bolus infusions of ex vivo engineered lymphocytes. Given their ease of manufacturing, distribution and administration, these nanocarriers, and the associated platforms, could become a therapeutic for a wide range of diseases.

Abstract 6594: Targeting Ewing sarcoma and osteosarcoma with anti-MCAM chimeric antigen receptor modified NK cells

Abstract Ewing sarcoma and osteosarcoma are the two most common pediatric malignant bone tumors. Patients who present with metastatic disease or relapse have a dismal average 5-year survival of only 20%. Novel therapeutic approaches are desperately needed. The melanoma cell adhesion molecule (MCAM/CD146/MUC18) is highly expressed in sarcoma and metastatic pediatric tumors. MCAM depletion inhibits Ewing sarcoma cell migration in vitro and tumor metastasis in vivo. High MCAM levels in patient tumors are associated with poor survival in Ewing sarcoma. MCAM therefore constitutes a unique and novel target for immunotherapy against MCAM+ sarcomas. We previously demonstrated that natural killer (NK) cell engineered to express chimeric antigen receptor (CAR) against CD20 significantly reduced tumor burden, prevented tumor dissemination, and extended survival of treated mice compared to mock NK treatment in humanized Burkitt lymphoma xenograft mouse model. We hypothesize that anti-MCAM CAR NK cell will enhance the inherent NK-sarcoma cytotoxicity by better targeting sarcoma cells. Here we develop an anti-MCAM CAR NK cell and investigate its efficacy in promoting NK cell cytotoxicity against Ewing sarcoma and osteosarcoma. MCAM expression in sarcoma cell lines and tumor samples were validated by flow cytometry and western blotting. Peripheral blood mononuclear cells (PBMCs) are expanded into NK cells ex vivo using K562-mbIL21-41BBL artificial antigen presenting cells (aAPC). Anti-MCAM CAR NK cells are generated by non-viral electroporation of anti-MCAM CAR mRNA into expanded NK cells. Anti-MCAM CAR NK cytotoxicity is evaluated in vitro by luciferase based cytotoxicity assay. We detected various levels of MCAM expression in Ewing sarcoma and osteosarcoma cell lines and tumors. In addition, we noticed an interesting patchy expression pattern of MCAM in the Ewing sarcoma tumor sample. Importantly, we found a significantly increased cytotoxicity of anti-MCAM CAR NK cells compared to mock NK cells (59.9±10 vs 13.3±3.7 at E:T ratio of 5:1, p=0.006; and 65.6±7 vs 13.7±3.4 at 10:1, p=0.004) against Ewing sarcoma TC71 cells after co-culturing for 4 hours. A similar result was observed at the time point of 48 hours (91.5±8 vs 59±0.5 at E:T of 5:1, p=0.018). Anti-MCAM CAR NK cells also showed significantly enhanced cytotoxicity against U2OS osteosarcoma cells compared to mock NK cells (32.6±23.2 vs 0.5±19.5 at E:T of 0.5:1, p=0.004; 47.1±27.4 vs 10.2±24 at E:T of 1:1, p=0.001) at the time point of 48 hours. In summary, these findings demonstrated enhanced efficacy of anti-MCAM CAR NK cells compared to mock NK cells against Ewing sarcoma and osteosarcoma cells and support a further preclinical investigation whether targeting MCAM by CAR NK will limit sarcoma growth and/or metastasis and increase survival in vivo. Citation Format: Aliza Gardenswartz, Wen Luo, Jeremy M. Rosenblum, Janet Ayello, Mitchell S. Cairo. Targeting Ewing sarcoma and osteosarcoma with anti-MCAM chimeric antigen receptor modified NK cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6594.

Preclinical evaluation of CD8+ anti-BCMA mRNA CAR T cells for treatment of multiple myeloma.

Chimeric antigen receptor (CAR) T-cell therapy remains limited to select centers that can carefully monitor adverse events. To broaden use of CAR T-cells in community clinics and in a frontline setting, we developed a novel CD8+ CAR T-cell product, Descartes-08, with predictable pharmacokinetics for treatment of multiple myeloma. Descartes-08 is engineered by mRNA transfection to express anti-BCMA CAR for a defined length of time. Descartes-08 express anti-BCMA CAR for 1 week, limiting risk of uncontrolled proliferation; produce inflammatory cytokines in response to myeloma target cells; and are highly cytolytic against myeloma cells regardless of presence of myeloma-protecting bone marrow stromal cells, exogenous a proliferation-inducing ligand, or drug resistance including IMiDs. The magnitude of cytolysis correlates with anti-BCMA CAR expression duration, indicating a temporal limit in activity. In the mouse model of aggressive disseminated human myeloma, Descartes-08 induces BCMA CAR-specific myeloma growth inhibition and significantly prolongs host survival (P<.0001). These preclinical data, coupled with an ongoing clinical trial of Descartes-08 in relapsed/refractory myeloma (NCT03448978) showing preliminary durable responses and a favorable therapeutic index, have provided the framework for a recently initiated trial of an optimized/humanized version of Descartes-08 (i.e., Descartes-11) in newly diagnosed myeloma patients with residual disease after induction therapy.

Feasibility and preliminary safety and efficacy of first-in-human intraperitoneal delivery of MCY-M11, anti-human-mesothelin CAR mRNA transfected into peripheral blood mononuclear cells, for ovarian cancer and malignant peritoneal mesothelioma.

3014 Background: MCY-M11 is a mesothelin-targeting chimeric antigen receptor (CAR) therapy made by a non-viral, mRNA-based platform, for rapid ( &lt; 1 day) CAR manufacturing. We are conducting a phase I dose escalation trial in ovarian cancer and malignant peritoneal mesothelioma (MPM) (NCT03608618). Methods: MCY-M11 are fresh, non-expanded, autologous peripheral blood mononuclear cells (PBMCs) transfected by flow electroporation with mRNA encoding a human anti-mesothelin CAR. Following a 3+3 design, patients are treated in dose level (DL) escalating cohorts (DL1 1.0 x 107, DL2 5.0 x 107, DL3 1.0 x 108, DL4 5.0 x 108 cells/dose), in one cycle of weekly x 3 doses, intraperitoneal (ip) without preconditioning chemotherapy. Results: By January 2020, CP-M11-101 study successfully completed DL1 and DL2 without safety concerns. Based on 11 patients treated in DL1, DL2 and DL3, ip infusion of MCY-M11 is safe and well tolerated. No infusion-related adverse events and no dose limiting toxicities (DLTs) have occurred. No neurotoxicity has been observed. Most reported treatment-related adverse events have been Grades 1-2 per NCI CTCAE. One patient in DL3 presented with G2 pericarditis, fever and transient neutropenia clinically assessed as related SAEs, that resolved without further complications. These events were assessed as on-target off-tumor effects and possibly G1 cytokine release syndrome (CRS). Two unrelated SAEs (G2 confusion in a patient in DL2; G3 enterocutaneous fistula in a patient in DL3) were reported. These 2 patients have been replaced as they did not complete the evaluation period (3 weekly infusions and the DLT 43 day follow up). There have been no treatment-related discontinuations or deaths. Three patients in DL2 showed stable disease (SD) by RECIST 1.1 at the end of the DLT period. Of them, 1 completed the study and did not participate in additional follow up, 1 remained in SD 6 months, and 1 remained in SD 2 months. In DL3, 1 patient remains in SD at 2 months, and evaluation is pending for the other 2 patients. Enrollment is ongoing. Conclusions: Feasibility of 1-day manufacturing of MCY-M11 for ip delivery is demonstrated. Treatment has been safe. Initial SD observed in DL2 and DL3 with one-cycle infusions is encouraging and supports exploration of additional strategies such as the addition of preconditioning chemotherapy and multiple cycles to increase efficacy. Clinical trial information: NCT03608618 .

Human CAR-T Cells engineered using the Solupore Ex Vivo Cell Engineering Platform are highly cytotoxic and specific against CD19+ cells in vitro

Background & Aim Solupore® is a non-viral ex vivo cell engineering platform that enables development and manufacture of cell therapies, using reversible permeabilization to achieve rapid intracellular delivery of cargos. The technology achieves intracellular delivery and subsequent reversal of cell permeabilization by precisely controlling the contact of the target cells with a solution containing the cargo. We have termed the method ‘soluporation’. Non-viral methods, such as soluporation, that enable intracellular delivery of various cargo types for clinical applications are attractive candidates for next-generation delivery modalities because of potential benefits for manufacture, safety and regulation compared with viral vectors. While electroporation is currently the most widely-used non-viral method used for cell engineering, it can be damaging to cells. In contrast, we have previously demonstrated that soluporation causes minimal perturbation of human T cells. Methods, Results & Conclusion Here we have further investigated the impact of soluporation and nucleofection on T cells by carrying out gene expression profiling in transfected cells. In nucleofected cells, 263 out of 574 immune related genes were misregulated at 6 h post-transfection. These genes clustered to several immune-related pathways including T cell activation, metabolism and exhaustion. In contrast, 8 genes were misregulated in soluporated cells. This indicates that soluporation maintains cellular function similar to untreated control cells, unlike nucleofection. We also assessed whether Solupore® could be used to engineer T cells to express a chimeric antigen receptor (CAR) and if these cells could effectively kill cancer cells in vitro. CD19 CAR mRNA was delivered to primary human T cells with on average 60% CD19 CAR expression detected at 24 h post-transfection. These CD19-CAR T cells were able to kill target cancer cells in a density-dependent manner. These studies demonstrate that the Solupore® technology is a gentle modality for efficient engineering of immune cells. The technology is highly reproducible, automated, and scalable and has the potential to enable a broad range of T cell engineering applications.