Background Chimeric antigen receptor (CAR) T cell therapy has demonstrated transformative outcomes in hematologic malignancies; however, many challenges remain, such as manufacturing complexity, high cost, requirements for lymphodepletion, and poor product persistence. To overcome these challenges, Umoja has developed VivoVec, a scalable, off-the-shelf lentiviral vector platform designed to drive efficient and targeted in vivo T cell transduction following direct administration to patients. VivoVec particles are surface-engineered with the cocal fusion glycoprotein and incorporate an anti-CD3 single chain variable fragment (scFv) and T cell costimulatory ligands on the particle surface to promote T cell binding, activation, and transduction. Here, we present preclinical studies demonstrating the biodistribution and preliminary safety of the VivoVec platform when delivered via either intranodal (IN) or intravenous (IV) routes of administration (ROAs). Methods VivoVec particle selectivity for T cell binding and transduction was assessed in vitro using PBMCs. Toxicology and biodistribution studies were conducted in canines and humanized mice. A surrogate ROA was used (intraperitoneal) in mice as IN is not feasible. Results VivoVec particles cultured with unprimed human PBMCs in vitro selectively bind, activate, and transduce CD3+ T cells with no detectable transduction of other immune cell subtypes. VivoVec particles displayed high selectivity and avidity for T cell binding within 5 minutes of incubation. In vivo toxicology studies demonstrate a favorable safety and biodistribution profile. VivoVec administered to canines was well tolerated and resulted in transduction that was largely restricted to the injected lymph nodes when delivered IN Evaluation of VivoVec safety and biodistribution in the presence of human CD3+ T cells was performed in CD34-humanized NSG mice. IP or IV administration of up to 4 ×107 VivoVec transducing units was well tolerated; efficacy in humanized mouse models (B cell aplasia and tumor clearance) is observed at these doses. Comprehensive tissue analysis via qPCR at 1–13 weeks post treatment primarily detected vector copies in the liver, spleen and injection site, and RNA in situ hybridization analysis demonstrated that the predominant cell types expressing the viral payload were human T cells and murine macrophages. Conclusions Nonclinical studies in two species demonstrate a favorable VivoVec safety and biodistribution profile following multiple ROAs. These findings support the potential of the VivoVec platform to generate safe and effective CAR T cells in vivo, which could expand patient access to CAR T technology in both hematologic and solid tumors without the need for ex vivo cell therapy manufacturing or lymphodepletion.

Background Chimeric antigen receptor (CAR) T cells are potent cancer-killing drugs that have revolutionized the treatment of hematologic malignancies, with potential application as a pan-cancer therapy. To realize this potential, Umoja's platform is designed to address several challenges facing autologous CAR T cell therapies. Methods VivoVec products will be off-the-shelf lentiviral vectors that engineer T cells in vivo, forgoing costly cell therapy manufacturing and toxic lymphodepletion associated with ex vivo approaches. The resulting in vivo engineered T cells will express an anti-fluorescein CAR (TagCAR) that facilitates tumor targeting using fluorescein-conjugated small molecule ligands (TumorTags). This approach enables us to target multiple tumor and stromal antigens with our single TagCAR, potentially allowing us to overcome tumor heterogeneity, immunosuppressive tumor microenvironments, and antigen loss that undermine traditional fixed-specificity CAR therapies. Results UB-VV200 is a VivoVec drug product candidate that generates TagCAR T cells in vivo. UB-VV200 particles are surface-engineered to express anti-CD3 single chain variable fragment and T cell costimulatory ligands in a multidomain fusion protein format. UB-VV200 particles displayed dose-dependent and selective binding, transduction, and activation of T cells following culture with PBMCs. To direct TagCAR T cells to their tumor targets, we developed TumorTags, which are tumor antigen ligands conjugated to fluorescein. Folate receptors alpha (FRa) and beta (FRb) and prostate-specific membrane antigen (PSMA) are well-established tumor-specific targets. UB-TT440 binds PSMA, which is expressed on prostate tumors, as well as most tumor-driven neovasculature. UB-TT170 targets tumors and their microenvironment by binding to FRa and FRb, which are expressed on tumor cells and associated macrophages, respectively. To characterize the bispecific nature of our TumorTags, we determined their on-cell binding affinity to TagCAR T cells and antigen-expressing tumor cells. Both TumorTags displayed dose-dependent and antigen-dependent binding, with Kd values in the picomolar to nanomolar range. Importantly, TagCAR T cells mediated antigen-specific and dose-dependent cytolytic activity, cytokine release, and proliferation in response to TumorTag treated tumor cells in vitro. Finally, Umoja's integrated platform was efficacious against solid tumors in vivo. UB-VV200 particles generated TagCAR T cells in non-activated PBMC-humanized NSG MHC-I/II double knockout mice bearing PSMA+ FRa+ MDA-MB-231 tumors without any evidence of acute toxicities. In response to either PSMA (UB-TT440) or FRa (UB-TT170) targeting TumorTags, TagCAR T cells proliferated and inhibited tumor growth. Conclusions These data demonstrate that, in comparison to autologous CAR T cells, UB-VV200 together with TumorTags have the potential to be a more accessible and more affordable off-the-shelf pan-cancer therapy.

Background Induced pluripotent stem cells (iPSCs) are a renewable, modifiable, and scalable starting material for manufacturing cell-based therapies. However, current approaches for differentiating iPSCs into therapeutic immune effector cells, such as natural killer (NK) cells, require complex growth factors and feeder cells to achieve sufficient yields. Here, we present Synthetic Receptor Enabled Differentiation (ShRED), a directed differentiation and expansion process controlled by the Rapamycin-Activated Cytokine Receptor (RACRTM). RACR is activated via the addition of its synthetic ligand rapamycin, which induces a JAK/STAT signal that drives differentiation and expansion of cells into hematopoietic progenitors (HPs) and then into immune effector cells, termed RACR-induced Cytotoxic Innate Lymphocytes (RACR-iCILs). Furthermore, because rapamycin is a safe, effective, and approved therapeutic for immune suppression, we beleive RACR can also be engaged in vivo through rapamycin dosing to increase the persistence of RACR-iCILs, while simultaneously protecting these cells from allogeneic rejection. Methods RACR was introduced into iPSCs through CRISPR/Cas9 editing, enabling consistent expression of RACR throughout differentiation and expansion. Next, RACR was activated at the start of the manufacturing process, and the resulting differentiated cells were phenotyped by flow cytometry and assessed functionally in cytotoxicity assays. Results Activating RACR in our engineered iPSCs resulted in enhanced HP differentiation, consistently producing a 300x yield of HPs (CD43+/CD45+/CD34+ cells) from iPSCs, two orders of magnitude greater than conventional protocols (figure 1). Continued engagement of RACR during differentiation resulted in >300,000x yield of RACR-iCILs (CD45+/CD56+/LFA1+ cells) from iPSCs in a completely feeder-free process (figure 1). These cells were highly pure and expressed receptors associated with NK cytotoxicity (figure 2A). Functionally, RACR-iCILs demonstrated potent cytotoxicity against solid tumors, and activation of RACR enhanced the cells' ability to clear tumor cells in a manner similar to potency-enhancing cytokines (figure 2B). Lastly, in a preclinical tumor xenograft model, RACR-NK cells demonstrated robust rapamycin-mediated expansion and clearance of breast adenocarcinoma tumors, highlighting RACR's ability to replace cytokine support and potentially increase cell persistence in vivo. Conclusions These data demonstrate the potential of RACR to solve both the manufacturing and in vivo persistence challenges of iPSC-derived cell therapies. ShRED improves the manufacturing of iPSC-derived cells, producing unprecedented yields of immune effector cells (RACR-iCILs) and eliminating the need for complex cytokines and feeder expansions. In vivo, RACR activation increases the proliferation of RACR-expressing NK cells, potentially enhancing the persistence of cell immunotherapies and eliminating the need for cytokine dosing and lymphodepletion.

Off-the-shelf immune cell products derived from induced pluripotent stem cells (iPSC) have the potential to address many of the current limitations of engineered autologous and allogeneic blood-derived cell therapies. IPSC-based platforms offer a scalable and renewable source of allogeneic cells that can be engineered to augment cell phenotype and function. Current approaches to generating iPSC-derived immune effector cells have focused on recapitulating natural differentiation pathways to defined effector cell types (e.g., CD8 T cells or NK cells) utilizing feeder cells and exogenous cytokines. By engineering cells to express an artificial cytokine receptor, termed rapamycin activated cytokine receptor (RACR), we can control immune effector cell differentiation and expansion through synthetic signals independent of endogenous receptor expression, reducing the complexity and variability of cell differentiation while deriving cells with unique phenotypic and functional features not found in conventionally defined native immune cells. Here we demonstrate the ability to differentiate and expand synthetic cytotoxic innate lymphoid cells through engineering progenitor cells to express an artificial cytokine receptor, the RACR system, that is activated by a non-native small molecule ligand, rapamycin. This approach involves a period of feeder cell free hematopoietic and lymphoid progenitor specification followed by rapamycin-mediated activation of the artificial cytokine receptor (RACR), inducing an IL-2/IL-15-like signal to drive cytotoxic innate lymphocyte differentiation and feeder cell free expansion resulting in approximately 3000-fold expansion from the iPSC starting material. The protocol has demonstrated robustness in the small-scale setting with multiple research-grade iPSC lines and enables extensive effector cell expansion at low cost, enabled by the stability of the rapamycin ligand and elimination of complex raw materials such as exogenous cytokines. The resulting RACR-induced cytotoxic innate lymphocytes (RACR-iCILs) exhibit potent polyfunctional anti-tumor activity driven by the synergistic activity of innate immune receptors and the engineered expression of a chimeric antigen receptor (CAR), including cytolytic activity and the secretion of IFNγ and TNFα. The artificial cytokine receptor system can be used ex vivo to generate cells but also has the potential to enable rapamycin-controlled and selective expansion and survival of the engineered cells in vivo. Taken together, our data demonstrate the potential for de novo engineering of novel synthetic cytotoxic effector cells that show significant potential as “off the shelf” cancer therapeutics. We are currently moving these novel cells into humanized mouse models to further evaluate their unique properties and in vivo anti-tumor activity. Citation Format: Samantha O'Hara, Teisha Rowland, Ryan Koning, David Vereide, Michele Hoffmann, Ashley Yingst, Chris Nicolai, Mark Pankau, Kristen Mittelsteadt, Kathryn Michels, Seungjin Shin, Laurie Beitz, Byoung Ryu, Ryan Crisman, Andrew Scharenberg, Chris Garbe, Ryan Larson. Generation of synthetic cytokine receptor-induced cytotoxic innate lymphocytes (iCILs) from iPSCs as off-the-shelf cancer therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 547.

Background Chimeric antigen receptor (CAR) T cell therapies have demonstrated limited efficacy against solid tumors, in part due to challenges overcoming solid tumor heterogeneity and CAR T cell exhaustion associated with the immunosuppressive tumor microenvironment (TME). Our integrated platform aims to overcome these roadblocks by engineering T cells in vivo to express a universal TagCAR which binds to a common tag on bispecific adaptor TumorTags, bridging TagCAR T cells to TumorTag-bound tumor- and TME-associated antigens, including folate receptor (FR) which is upregulated on many tumor types as well as immunosuppressive tumor-associated macrophages. Additionally, our TagCAR T cells are engineered to express a rapamycin-activated cytokine receptor (RACR) which selectively provides survival signals to TagCAR T cells in the presence of rapamycin. Here, we identify a universal TagCAR that demonstrates potent in vitro and in vivo anti-tumor polyfunctionality against FR+ target cells with a folate receptor-targeting TumorTag (UB-TT170). Methods PBMCs from healthy donors were transduced in vitro with surface-engineered lentiviral vectors with TagCAR/RACR payloads. Resultant TagCAR T cell anti-tumor activity and persistence was assessed using a co-culture approach with FR-expressing tumor cells and titrated doses of UB-TT170. To assess in vivo anti-tumor activity, lentiviral particles containing TagCAR/RACR payloads were administered to PBMC-humanized NSG mice with established FR+ xenograft solid tumors to generate TagCAR T cells in vivo. Mice were treated with UB-TT170 and efficacy was determined by assessing tumor regression and UB-TT170-mediated TagCAR T cell expansion. Results TagCAR T cells containing a CD8α hinge/transmembrane domain and 41bbζ endodomain were superior to other construct candidates in eliminating FR+ target cells in the presence of UB-TT170 in vitro. These TagCAR T cells demonstrated UB-TT170-mediated expansion and proinflammatory cytokine production in the presence of FR+ target cells, and repeated elimination of target cells and enhanced persistence properties with serial antigen-exposure. Cells transduced with this vector exhibited RACR-mediated expansion and improved function in the presence of rapamycin. Administration of TagCAR/RACR payload-containing lentiviral particles to PBMC-humanized NSG mice resulted in generation of TagCAR T cells in vivo, which expanded and mediated clearance of FR+ solid tumors with UB-TT170 Conclusions We have identified a universal TagCAR that displays robust anti-tumor activity and persistence qualities against FR+ target cells in vitro and in vivo with UB-TT170. These data support development of this platform as a new cellular therapy approach against solid tumors, using combinatorial targeting of tumor- and TME-associated antigens with an in vivo-generated universal TagCAR and multiple TumorTags.

Background Autologous chimeric antigen receptor (CAR) T cell therapies have revolutionized the treatment of B cell malignancies, leading to long-term remission in 30-40% of certain patient populations. Despite the promising clinical efficacy of CAR T cells in hematologic malignancies, major limitations hinder their widespread application, including challenges for patient access, complex manufacturing, and high cost. Methods To overcome these challenges, we have developed VivoVec, a surface-engineered lentiviral vector-based platform harboring a CAR transgene that is being developed for off-the-shelf use for the generation of CAR T cells in vivo. To achieve specific and efficient in vivo T cell transduction, VivoVec particles are pseudotyped with the Cocal fusion glycoprotein and an anti-CD3 single chain variable fragment (scFv), and we have previously shown that these first-generation particles generate CAR T cells in vivo that mediate antitumor activity. Results We have advanced the VivoVec platform through incorporating costimulatory molecules into the particle surface, in addition to the anti-CD3 scFv and Cocal fusion glycoprotein. These second-generation VivoVec particles exhibit enhanced T cell binding and activation, resulting in increased transduction and greater numbers of CAR+ T cells in vitro. In addition, CAR T cells generated with second-generation VivoVec particles exhibited a less-differentiated, central memory-like phenotype and enhanced CAR-antigen-specific polyfunctionality, including cytokine production, proliferation, and tumor cell killing. Finally, in a humanized NSG mouse model of B cell malignancy we observed that second-generation VivoVec particles generated greater numbers of CAR T cells in the blood, resulting in enhanced antitumor activity at lower doses compared to first-generation particles. Our results indicate that incorporation of costimulatory molecules onto the surface of VivoVec particles increases both the overall number and functionality of the resulting CAR T cells, greatly augmenting VivoVec mediated CAR T cell generation and antitumor activity in vivo. Conclusions Overall, these data demonstrate that second-generation VivoVec particles efficiently generate large numbers of highly functional CAR T cells able to mediate durable tumor control in a preclinical model of B cell malignancy. VivoVec particles have the potential to overcome many of the limitations associated with the current class of CAR T cell therapies.

With an increasing number of clinical developments and commercial launches of autologous chimeric antigen receptor T-cell therapies, appropriate specification strategies throughout the development lifecycle are critical to ensuring product safety, quality, and patient access on commercialization. A unique aspect of autologous chimeric antigen receptor T-cell therapies is the individualized nature of this class of drug. The heterogeneity in patient leukapheresis material and its impact on drug product quality must be considered in developing a risk-based product specification. This review describes a framework for establishing a patient-centric specification based on correlative analysis linking product attributes with clinical outcomes, variance component analysis deciphering the sources of variability, and appropriate statistical analysis of clinical lot release data.

Background TAK-573, a humanized, anti-CD38, IgG4, monoclonal antibody genetically fused to two attenuated IFNα2b molecules, was designed for targeted delivery of attenuated IFNα2b to CD38 expressing (CD38+) cells, utilizing a unique epitope of CD38 that does not compete with current anti-CD38 therapies. Preclinical evaluation of TAK-573 confirmed activation of type I IFN signaling in CD38+ cells inducing direct anti-proliferative effects on multiple myeloma (MM) cells and direct and indirect immune cell activation. Here we provide the preliminary analyses of the pharmacodynamic data currently available from the ongoing Ph I/II TAK-573-1501 clinical study in patients with relapsed/refractory MM (NCT03215030). Methods Peripheral blood (PB) and bone marrow (BM) aspirates were collected from patients at pre- and post-dose time points for exploratory biomarker analyses. CD38 receptor occupancy (RO) and receptor density (RD) were determined using a 9-color flow cytometry assay. Whole transcriptome sequencing of bulk RNA was performed and analyzed to assess the type I IFN gene signature. Serum samples were analyzed using Olink’s Proximity Extension Assay Immuno-Oncology panel to measure changes in cytokine levels. Mass cytometry-based immunophenotyping was utilized to characterize changes in immune cell prevalence and activation status of cryopreserved cells. Results Administration of TAK-573 resulted in a dose dependent increase in CD38 RO of PB-derived immune cells with saturation detected 4 hours after the end of infusion (EOI) at doses ≥ 0.2 mg/kg. The duration of saturation was dose dependent with doses ≥ 0.75 mg/kg saturating CD38 RO through 24 hours. All dose levels tested resulted in increases in the type I IFN gene signature at 24 hours. Consistent with CD38 being an IFN stimulated gene, TAK-573 treatment resulted in CD38 RD increases most notably on NK cells, but also on other CD38+ cells including MM cells. Circulating levels of IFN-associated cytokines were also elevated, with maximal induction 4 hours after the EOI. CD8+ T-cells in BM showed increased CD69 expression in 7 of 9 patients analyzed, 3 of whom also showed increases in both IFNγ and granzyme B positivity suggesting TAK-573 treatment results in increased BM cytolytic CD8+ T-cells, in a subset of patients. Conclusions These preliminary biomarker data indicate that TAK-573 is a pharmacologically active molecule that mediates its effect through IFNAR pathway modulation. Additional data are being collected to further refine the mechanism of action (Image 1), which will inform the recommended phase 2 dose and optimal schedule of administration for the development of TAK-573. Trial Registration ClinicalTrials. gov: NCT03215030 Ethics Approval The TAK-573-1501 study is approved by WIRB-Copernicus Group, University of Nebraska Medical Center, Dana Farber Cancer Institute and Advarra IRBs.