Engineered Cell Therapies Research Articles

Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-T cell-mediated antitumor activity.

Tumor cell methylation of KRAS G12V epitope in HLA-A2 permits immune evasion, and new TCRs were generated to overcome this with engineered cell therapy.

Scalable intracellular delivery via microfluidic vortex shedding enhances the function of chimeric antigen receptor T-cells.

Adoptive chimeric antigen receptor T-cell (CAR-T) therapy is transformative and approved for hematologic malignancies. It is also being developed for the treatment of solid tumors, autoimmune disorders, heart disease, and aging. Despite unprecedented clinical outcomes, CAR-T and other engineered cell therapies face a variety of manufacturing and safety challenges. Traditional methods, such as lentivirus transduction and electroporation, result in random integration or cause significant cellular damage, which can limit the safety and efficacy of engineered cell therapies. We present hydroporation as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7- to 2-fold higher CAR-T yields compared to electroporation with superior cell viability and recovery. Hydroporated cells exhibited rapid proliferation, robust target cell lysis, and increased pro-inflammatory and regulatory cytokine secretion in addition to improved CAR-T yield by day 5 post-transfection. We demonstrate that scaled-up hydroporation can process 5 × 108 cells in less than 10 s, showcasing the platform as a viable solution for high-yield CAR-T manufacturing with the potential for improved therapeutic outcomes.

Shear-thinning hydrogel for allograft cell transplantation and externally controlled transgene expression

This work establishes the design of a fully synthetic, shear-thinning hydrogel platform that is injectable and can isolate engineered, allogeneic cell therapies from the host. We utilized RAFT to generate a library of linear random copolymers of N,N-dimethylacrylamide (DMA) and 2-vinyl-4,4-dimethyl azlactone (VDMA) with variable mol% VDMA and degree of polymerization. Poly(DMA-co-VDMA) copolymers were subsequently modified with either adamantane (Ad) or β-cyclodextrin (Cd) through amine-reactive VDMA to prepare hydrogel precursor macromers containing complementary guest-host pairing pendant groups that, when mixed, form shear-thinning hydrogels. Rheometric evaluation of the hydrogel library enabled identification of lead macromer structures comprising 15 mol% pendants (Ad or Cd) and a degree of polymerization of 1000; mixing of these Ad and Cd functionalized precursors yielded hydrogels possessing storage modulus above 1000 Pa, tan(δ) values below 1 and high yield strain, which are target characteristics of robust but injectable shear-thinning gels. This modular system proved amenable to nanoparticle integration with surface-modified nanoparticles displaying Ad. The addition of the Ad-functionalized nanoparticles simultaneously improved mechanical properties of the hydrogels and enabled extended hydrogel retention of a model small molecule in vivo. In studies benchmarking against alginate, a material traditionally used for cell encapsulation, the lead hydrogel showed significantly less fibrous encapsulation in a subcutaneous implant site. Finally, this platform was utilized to encapsulate and extend in vivo longevity of inducible transgene-engineered mesenchymal stem cells in an allogeneic transplant model. The hydrogels remained intact and blocked infiltration by host cells, consequently extending the longevity of grafted cell function relative to a benchmark, shear-thinning hyaluronic acid-based gel. In sum, the new synthetic, shear-thinning hydrogel system presented here shows potential for further development as an injectable platform for delivery and in situ drug modulation of allograft and engineered cell therapies.

Assessing a single-cell multi-omic analytic platform to characterize ex vivo-engineered T-cell therapy products.

Genetically engineered CD8+ T cells are being explored for the treatment of various cancers. Analytical characterization represents a major challenge in the development of genetically engineered cell therapies, especially assessing the potential off-target editing and product heterogeneity. As conventional sequencing techniques only provide information at the bulk level, they are unable to detect off-target CRISPR translocation or editing events occurring in minor cell subpopulations. In this study, we report the analytical development of a single-cell multi-omics DNA and protein assay to characterize genetically engineered cell products for safety and genotoxicity assessment. We were able to quantify on-target edits, off-target events, and potential translocations at the targeting loci with per-cell granularity, providing important characterization data of the final cell product. Conclusion: A single-cell multi-omics approach provides the resolution required to understand the composition of cellular products and identify critical quality attributes (CQAs).

Hypoxia-responsive synthetic gene circuits to improve safety and potency of CAR T cell therapy for solid tumors.

37 Background: Engineered cell therapies expressing a chimeric antigen receptor (CAR) in human T cells have demonstrated great promise for the treatment of hematological malignancies. However, unique challenges remain in translating the success of CAR T cell therapies to solid tumors, namely: (1) specificity: there are few truly unique antigens present on solid tumors, leading to off-target effects that damage normal tissue and pose safety risks; and (2) suppression: solid tumors develop microenvironments that are immunosuppressive and decrease T cell survival and tumor-directed cytotoxicity. To address these challenges and improve the safety and potency of solid tumor cell therapy, we engineered cells with synthetic genetic circuits containing a previously validated hypoxia biosensor (HBS) that responds to upregulation of the native HIF1α and HIF2α transcription factors by hypoxia, enabling the cells to sense and respond to the hypoxic tumor microenvironment ubiquitous among solid tumors. Methods: We evaluated CAR expression from circuit architectures containing the HBS promoter driving expression of both CAR and native HIF1α, HIF2α, as well as from circuit architectures containing the HBS promoter and our toolkit of synthetic transcription factors and promoters. Circuits were integrated into HEK293 cells via PiggyBac transposon vector to generate stable cell lines. Cells were subsequently cultured in normoxia (21% O2) and hypoxia (1% O2), and harvested for flow cytometric analysis to evaluate CAR surface expression across a three day time frame. Results: We identified novel circuits providing fast reporter output with minimal background signal under hypoxic conditions, and elucidated design rules that enable or restrict amplification of transgene in native HIF1α and HIF2α-containing circuits. We observed differences in CAR transgene expression in hypoxia between circuits containing HIF1α and HIF2α-mediated feedback, suggesting mechanistic differences in regulation of HIF1α and HIF2α when overexpressed. Additionally, we observed greatly amplified transgene expression in circuit topologies containing feedback mediated by our synthetic transcription factor and promoter toolkit compared to native HIF1α and HIF2α-containing circuits and cells constitutively expressing CAR. Conclusions: We present a new therapeutic technology that senses hypoxic environments and enables high-output sense and respond behaviors with therapeutic outputs. We envision that this technology will enable development of safer, more effective cell therapies for solid tumors, as well as provide new biological and mechanistic insights that can inform the future development of hypoxia-responsive cell therapies.

Scalable intracellular delivery via microfluidic vortex shedding enhances the function of chimeric antigen receptor T-cells.

Adoptive chimeric antigen receptor T-cell (CAR-T) therapy is transformative and approved for hematologic malignancies. It is also being developed for the treatment of solid tumors, autoimmune disorders, heart disease, and aging. Despite unprecedented clinical outcomes, CAR-T and other engineered cell therapies face a variety of manufacturing and safety challenges. Traditional methods, such as lentivirus transduction and electroporation, result in random integration or cause significant cellular damage, which can limit the safety and efficacy of engineered cell therapies. We present hydroporation as a gentle and effective alternative for intracellular delivery. Hydroporation resulted in 1.7- to 2-fold higher CAR-T yields compared to electroporation with superior cell viability and recovery. Hydroporated cells exhibited rapid proliferation, robust target cell lysis, and increased pro-inflammatory and regulatory cytokine secretion in addition to improved CAR-T yield by day 5 post-transfection. We demonstrate that scaled-up hydroporation can process 5 x 108 cells in less than 10 s, showcasing the platform as a viable solution for high-yield CAR-T manufacturing with the potential for improved therapeutic outcomes.

Therapeutic intersections: Expanding benefits of CD19 CAR T cells from cancer to autoimmunity

Anti-CD19 CAR Tcells were among the last decade's scientific breakthroughs, achieving remarkable remissions in patients with B cell leukemias and lymphomas. Now, the engineered cell therapies are traversing disease indications into autoimmunity and resolving disease symptoms in patients with systemic lupus erythematosus (SLE), idiopathic inflammatory myositis, and systemic sclerosis.1.

Building Synthetic Biosensors Using Red Blood Cell Proteins.

As the use of engineered cell therapies expands from pioneering efforts in cancer immunotherapy to other applications, an attractive but less explored approach is the use of engineered red blood cells (RBCs). Compared to other cells, RBCs have a very long circulation time and reside in the blood compartment, so they could be ideally suited for applications as sentinel cells that enable in situ sensing and diagnostics. However, we largely lack tools for converting RBCs into biosensors. A unique challenge is that RBCs remodel their membranes during maturation, shedding many membrane components, suggesting that an RBC-specific approach may be needed. Toward addressing this need, here we develop a biosensing architecture built on RBC membrane proteins that are retained through erythropoiesis. This biosensor employs a mechanism in which extracellular ligand binding is transduced into intracellular reconstitution of a split output protein (including either a fluorophore or an enzyme). By comparatively evaluating a range of biosensor architectures, linker types, scaffold choices, and output signals, we identify biosensor designs and design features that confer substantial ligand-induced signal in vitro. Finally, we demonstrate that erythroid precursor cells engineered with our RBC-protein biosensors function in vivo. This study establishes a foundation for developing RBC-based biosensors that could ultimately address unmet needs including noninvasive monitoring of physiological signals for a range of diagnostic applications.

Abstract 5230: A modified FcγRI expressing T cell (SolidT) exhibits profound anti-tumor activity followed by maturation into effector memory t cells that can be reactivated to kill a secondary tumor

Abstract We previously described a subset of cytotoxic FcγRI (CD64)-expressing T cells in tumors and inflamed tissues (PMID 31449054). We subsequently expressed an engineered modified CD64 into T cells (“SolidT”) and demonstrated that these cells possessed greater specificity for tumor over normal tissues, reduced exhaustion, and attenuated cytokine secretion compared to other engineered cell therapies. SolidT activation correlates with surface tumor antigen density enabling the differentiation between tumor cells and normal cells by use of a specific antibody targeting tumor antigens. We previously showed that SolidT + mAb has anti-tumor activity in vitro and in vivo in several tumor models, including immunodeficient HER2 xenografts and an immunocompetent syngeneic B16-TYRP1 model (PMID 37070661). In this study, we further assessed the effect of SolidT on HER2+ tumors in a rechallenge model and extended the platform to CD20+ lymphoma models by combining SolidT with rituximab. In an in vivo experiment using HER2+, NCI-N87 cells in immunodeficient mice, several mice in the SolidT + trastuzumab group experienced complete tumor clearance. These mice were monitored until day 84, then rechallenged with NCI-N87 cells along with a new control group. After tumor establishment, mice were treated only with trastuzumab weekly for two weeks. While tumors in the control group progressed, the tumors in the rechallenged mice were completely cleared with a trastuzumab boost only, suggesting a SolidT cell memory response. Bone marrow from these rechallenged mice was analyzed and revealed the presence of effector memory SolidT cells. The SolidT model was then studied in a human, CD20+ lymphoma model in vitro and in vivo in immunodeficient mice. SolidT + rituximab was more potent in-vivo against CD20+ lymphoma cells than rituximab. In vivo, mice treated with SolidT + rituximab had significantly greater survival, lower tumor burden, and fewer sites of metastases than mice treated with rituximab alone. Bone marrow from these mice was analyzed and revealed the presence of effector memory SolidT cells in mice treated with SolidT + rituximab. We demonstrated that marrow resident SolidT cells developed an effector memory phenotype that positively correlated with outcome in HER2+ and CD20+ xenograft models, consistent with earlier data from a syngeneic B16 melanoma model. We further demonstrated that SolidT cells can be reactivated in a rechallenge model with only the addition of tumor targeting mAb, resulting in the eradication of the recurring tumors. This data further confirms that the SolidT platform can be targeted against different tumor antigens by using different tumor targeting mAbs. The SolidT platform could represent a paradigm shift in the approach to engineered cellular therapy for hematological malignancies and solid tumors. Citation Format: Shahar Dotan, Noam Pilpel, Peleg Rider, Hana Shpilt, Diana Rasoulouniriana, Yaron Carmi, James E. Wooldridge. A modified FcγRI expressing T cell (SolidT) exhibits profound anti-tumor activity followed by maturation into effector memory t cells that can be reactivated to kill a secondary tumor [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 5230.

IL-15-secreting CAR natural killer cells directed toward the pan-cancer target CD70 eliminate both cancer cells and cancer-associated fibroblasts

BackgroundIt remains challenging to obtain positive outcomes with chimeric antigen receptor (CAR)-engineered cell therapies in solid malignancies, like colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC). A major obstacle is the lack of targetable surface antigens that are not shared by healthy tissues. CD70 emerges as interesting target, due to its stringent expression pattern in healthy tissue and its apparent role in tumor progression in a considerable amount of malignancies. Moreover, CD70 is also expressed on cancer-associated fibroblasts (CAFs), another roadblock for treatment efficacy in CRC and PDAC. We explored the therapeutic potential of CD70 as target for CAR natural killer (NK) cell therapy in CRC, PDAC, focusing on tumor cells and CAFs, and lymphoma.MethodsRNA-seq data and immunohistochemical analysis of patient samples were used to explore CD70 expression in CRC and PDAC patients. In addition, CD70-targeting CAR NK cells were developed to assess cytotoxic activity against CD70+ tumor cells and CAFs, and the effect of cytokine stimulation on their efficacy was evaluated. The in vitro functionality of CD70-CAR NK cells was investigated against a panel of tumor and CAF cell lines with varying CD70 expression. Lymphoma-bearing mice were used to validate in vivo potency of CD70-CAR NK cells. Lastly, to consider patient variability, CD70-CAR NK cells were tested on patient-derived organoids containing CAFs.ResultsIn this study, we identified CD70 as a target for tumor cells and CAFs in CRC and PDAC patients. Functional evaluation of CD70-directed CAR NK cells indicated that IL-15 stimulation is essential to obtain effective elimination of CD70+ tumor cells and CAFs, and to improve tumor burden and survival of mice bearing CD70+ tumors. Mechanistically, IL-15 stimulation resulted in improved potency of CD70-CAR NK cells by upregulating CAR expression and increasing secretion of pro-inflammatory cytokines, in a mainly autocrine or intracellular manner.ConclusionsWe disclose CD70 as an attractive target both in hematological and solid tumors. IL-15 armored CAR NK cells act as potent effectors to eliminate these CD70+ cells. They can target both tumor cells and CAFs in patients with CRC and PDAC, and potentially other desmoplastic solid tumors.

Efficient Generation of Murine Chimeric Antigen Receptor (CAR)-T Cells.

Engineered cell therapies utilizing chimeric antigen receptor (CAR)-T cells have achieved remarkable effectiveness in individuals with hematological malignancies and are presently undergoing development for the treatment of diverse solid tumors. So far, the preliminary evaluation of novel CAR-T cell products has predominantly taken place in xenograft tumor models using immunodeficient mice. This approach is chosen to facilitate the successful engraftment of human CAR-T cells in the experimental setting. However, syngeneic mouse models, in which tumors and CAR-T cells are derived from the same mouse strain, allow evaluation of new CAR technologies in the context of a functional immune system and comprehensive tumor microenvironment (TME). The protocol described here aims to streamline the process of mouse CAR-T cell generation by presenting standardized methods for retroviral transduction and ex vivo T cell culture. The methods described in this protocol can be applied to other CAR constructs beyond the ones used in this study to enable routine evaluation of new CAR technologies in immune-competent systems.

Cellular reprogramming by protein degradation: The next frontier in cancer immunotherapy

In this issue of Cell Chemical Biology, Lane etal.1 introduce a transgene-based system to express fusion proteins that recruit transcription factors to E3 ligases. This approach expands the target repertoire for engineered cell therapies as exemplified by the targeting of SMAD proteins to overcome a TGFβ-mediated suppressive mechanism that weakens anti-tumor responses.

A dual-luciferase bioluminescence system for the assessment of cellular therapies

Bioluminescence imaging is a well-established platform for evaluating engineered cell therapies in preclinical studies. However, despite the discovery of new luciferases and substrates, optimal combinations to simultaneously monitor two cell populations remain limited. This makes the functional assessment of cellular therapies cumbersome and expensive, especially in preclinical invivo models. In this study, we explored the potential of using a green bioluminescence-emitting click beetle luciferase, CBG99, and a red bioluminescence-emitting firefly luciferase mutant, Akaluc, together to simultaneously monitor two cell populations. Using various chimeric antigen receptor Tcells and tumor pairings, we demonstrate that these luciferases are suitable for real-time tracking of two cell types using 2D and 3D cultures invitro and experimental models invivo. Our data show the broad compatibility of this dual-luciferase (duo-luc) system with multiple bioluminescence detection equipment ranging from benchtop spectrophotometers to live animal imaging systems. Although this study focused on investigating complex CAR Tcells and tumor cell interactions, this duo-luc system has potential utility for the simultaneous monitoring of any two cellular components-for example, to unravel the impact of a specific genetic variant on clonal dominance in a mixed population of tumor cells.

Revolutionizing the treatment for nasopharyngeal cancer: the impact, challenges and strategies of stem cell and genetically engineered cell therapies.

Nasopharyngeal carcinoma (NPC) is a distinct malignancy of the nasopharynx and is consistently associated with the Epstein-Barr virus (EBV) infection. Its unique anatomical location and complex aetiology often result in advanced-stage disease at first diagnosis. While radiotherapy (RT) and chemotherapy have been the mainstays of treatment, they often fail to prevent tumour recurrence and metastasis, leading to high rates of treatment failure and mortality. Recent advancement in cell-based therapies, such as chimeric antigen receptor (CAR)-T cell therapy, have shown great promise in hematological malignancies and are now being investigated for NPC. However, challenges such as targeting specific tumour antigens, limited T cell persistence and proliferation, and managing treatment-related toxicities must be addressed. Extensive research is needed to enhance the effectiveness and safety of these therapies, paving the way for their integration into standard clinical practice for better management of NPC and a better quality of life for human health.

Novel, Engineered, Modified Fcγ Receptor I (CD64)-Expressing T Cells (SolidT) Combined with Rituximab Exhibited Profound Anti-Tumor Activity and Memory in a CD20+ Raji Xenograft Model of Lymphoma

Background: CD19 CART cell therapy has been a major advance in the treatment of lymphoma, but is limited due to profound toxicity and relapse, in part due to loss of CD19 expression on malignant cells. Following our discovery of FcγRI (CD64)-expressing T cells in tumors and inflamed tissues (PMID 31449054), we developed novel, engineered cytotoxic T cells (“SolidT”) with increased specificity, reduced exhaustion, and attenuated cytokine secretion compared to other engineered cell therapies (e.g., CAR-T, CAR-NK). SolidT are produced by transducing a retroviral vector encoding an enhanced FcγRI into T cells for use with tumor-targeting monoclonal antibodies (mAbs). SolidT activation correlates with surface tumor antigen density. Unlike CART, where tonic signaling causes premature exhaustion and limited tumor infiltration, SolidT are not exhausted prematurely. They are activated and persist only by mAb bound to tumor-specific antigen at sufficient density, and they take on a T effector memory (T EM) phenotype. We previously showed that SolidT + mAb has anti-tumor activity in vitro and in vivo in several tumor models, including immunodeficient HER2 xenografts and an immunocompetent syngeneic B16-TYRP1 model (PMID 37070661). To further assess the SolidT platform, we examined the effect of SolidT + rituximab (R) in vitro and in vivo in a CD20+ Raji-Luciferase (Luc) xenograft model. Methods: In in vitro Raji-Luc and Daudi-Luc models, SolidT + R was compared to CD19 CART, R + sham transfected T cells (Sham), Sham only, and SolidT only. Cytotoxicity was measured by flow analysis of Annexin V and/or Luc assay. IFNγ and Granzyme B were measured by ELISA. Addition of intravenous immunoglobulin (IVIG; 5mg/ml) to cytotoxicity co-cultures was used to evaluate the effect of non-specific IgGs on the efficacy of the combination treatment. In vitro safety was evaluated in a similar manner using primary cultures of kidney and lung epithelial cells. To assess the anti-tumor activity of SolidT, NSG mice were inoculated intravenously (IV) with 1x10 6 Raji-Luc cells (n=8). SolidT (1x10 7 cells) was given on Day (D) 1. R (250 mg) was given on D1 and then weekly (125 mg). Mice were monitored for weight, clinical signs, and survival until D41 when the experiment was terminated to conduct endpoint analyses including gross pathology, histopathology, and CD3 + immunohistochemistry (IHC). Results: In vitro, SolidT + R showed higher cytotoxicity and attenuated cytokine levels against Raji-Luc compared to CD19 CART cells. Similar results were obtained in a Daudi-Luc model. Addition of IVIG did not activate SolidT cells, and though IFNγ secretion was attenuated, cytotoxicity was not affected. The in vitro co-culture of SolidT cells with primary epithelial cells demonstrated no activation of the effector cells and no death of primary target cells. In vivo, mice treated with SolidT + R had increased survival and reduced tumor burden against Raji-Luc compared to mice treated with R alone (Figure 1). On pathology, 5/8 SolidT + R mice had no tumors detected and 3 had only abdominal tumors. R mice developed tumors in the lungs (2/8), liver (7/8), and abdomen (4/8). Bone marrow immunophenotyping revealed that the presence of T EM phenotype highly correlated to better survival and lower tumor burden. Gross pathology analysis revealed no treatment-related pathology. IHC showed increased inflammation, lymphocyte infiltration, and necrosis in tumors from SolidT + R-treated mice, as well as intense infiltration of CD3 + human cells in the tumors at end point D41. The in vivo Raji-Luc model was performed twice with similar results. Conclusions: Consistent with prior results in solid tumor models targeting HER2 or TYRP1, these data demonstrated that the SolidT platform could be effectively targeted against CD20+ cell lines. A single administration of SolidT cells in combination with R prevented tumor formation and prolonged survival in a CD20+ Raji-Luc model. This experiment demonstrated that marrow-resident SolidT cells developed a T EM phenotype that positively correlated with outcome. These data confirm that the SolidT platform can be targeted against different tumor antigens by using different tumor-targeting mAbs. The SolidT platform could represent a paradigm shift in the approach to engineered cellular therapy for hematological malignancies and solid tumors.

High-Throughput Identification of Graft-Vs-Leukemia TCRs in AML Patient Samples after Allogeneic Stem Cell Transplantation

For patients with intermediate and high-risk AML/MDS, allogeneic stem cell transplantation (aSCT) is a potentially curative therapy. Efficacy is driven by the graft-versus-leukemia (GvL) response, in which donor-derived T cells recognize peptide:MHC antigens presented by leukemia cells, and subsequently kill them. The antigens that drive GvL responses are attractive therapeutic targets for engineered cell therapies and/or biologics. However, only a small number of GvL antigens have been identified, limiting the therapeutic potential of targeted immunotherapies. Deep interrogation of the T cell repertoires of patients who have responded favorably to aSCT presents an opportunity to identify bona fide GvL antigens, but high-throughput methods need to be developed to do this effectively. A popular method to identify antigen-specific T cells is fluorescent peptide:MHC tetramer staining, whereby T cells whose T cell receptors (TCR) bind a particular peptide:MHC antigen become fluorescent and identifiable by flow cytometry. However, a major challenge to identifying GvL T cells using tetramers is that the prediction of immunogenic antigens - that is, those that induce a T cell response in vivo - is poor; studies of solid tumors suggest that only ~1% of predicted MHC-binding neoantigens are immunogenic in patients. In AML/MDS aSCT patients, there are thousands of peptides that are predicted to bind MHCI and represent potential GvL antigens, but due to practical limitations of patient blood and bone marrow sample quantities, only a small subset can be screened by flow cytometry. Other higher throughput methods using heavy-metal tagged peptide:MHC tetramers followed by mass cytometry, or DNA-barcoded tetramers followed by bulk sequencing have been employed to increase antigen screening scale, but these methods are limited in sensitivity and destroy the T cell without providing TCR sequences. To overcome these limitations and rapidly identify GvL T cells in patient samples, we developed a single cell sequencing based method that enables the screening of thousands of peptide:MHC antigens simultaneously, using a single patient sample. We generated peptide: MHCI monomers using UV-mediated peptide exchange and generated large panels of combinatorial DNA-barcoded peptide:MHCI tetramers at microscale. We integrated our methodology into the 10X Genomics Single Cell Immune Profiling workflow to enable simultaneous recovery of TCR antigen-specificity, paired TCR alpha beta sequences, and single cell RNA-Seq data. These methods were used to screen post-transplant AML patient blood and bone marrow, and we identified expanded GvL T cell clonotypes that target novel minor histocompatibility antigens, overexpressed “aberrant self” proteins, and other leukemia associated antigens. We generated primary CD8 T cells that retrovirally express these TCRs and demonstrate that they are potent and have the expected antigen specificities. The methods described are highly sensitive, specific, efficient, and scalable, and enable high confidence identification of antigen specific clonotypes in patient samples. They markedly expand the throughput compared to existing methods, and simultaneously yield paired alpha beta TCR sequences, facilitating a full characterization of the antigen-TCR drivers of GvL. Furthermore, these methods have broad utility beyond SCT, as they are applicable to studying T cell responses to other immune settings, including anti-cancer, autoimmune, and anti-microbial responses.

Safety and Efficacy of Orca-Q with Haploidentical Donors for the Treatment of Advanced Hematologic Malignancies without the Use of Post-Transplant Cyclophosphamide

Srour, Salhotra, Lowsky and Abedi contributed equally as authors BACKGROUND Allogeneic stem cell transplantation (SCT) can be curative for several high-risk hematologic malignancies, but access was previously limited to patients with a fully matched donor. The introduction of post-transplant cyclophosphamide (PTCy) GvHD prophylaxis has increased the use of haploidentical donors (haplo) for patients who lack an HLA-matched donor. However, clinical outcomes associated with PTCy-based regimens remain challenging due to increased relapse rates (particularly in the setting of reduced intensity conditioning) and the adverse event profile including higher incidence of mucositis, cytokine release syndrome (CRS), delayed engraftment & T cell reconstitution, infections, early cardiotoxicity events and death from organ failure (Dulery 2021, Abboud 2021, Nagler 2022, Hoover 2022, Bolaños-Meade 2023). Additionally, the incidence of chronic GvHD after haplo SCT with PTCy is 24 - 33% at 1 year in various historical cohorts. Orca-Q -an investigational precision engineered cell therapy biologic- represents an alternative strategy which may be administered with single agent prophylaxis and does not require PTCy. The cellular composition of Orca-Q includes enriched CD34+ stem cells, combined with specific T-cell subsets, and is intended to reconstitute the blood and immune systems. METHODS Adult patients with high-risk hematologic malignancies eligible for MAC SCT were enrolled on the haplo donor dose expansion arm of a multicenter phase1 study of Orca-Q (NCT03802695). Haplo was defined as ≥ 4/8 but < 7/8 matched related donor at HLA-A, -B, -C, and -DRB1 typed using DNA-based high-resolution. Patients received MAC conditioning and single agent GVHD prophylaxis with tacrolimus (starting on day -1 and taper day +60). Orca-Q was manufactured centrally at Orca Bio Manufacturing Site in Sacramento, CA from G-CSF mobilized peripheral blood apheresis. RESULTS Orca-Q was successfully manufactured and delivered to all subjects with a vein-to-vein time (time between end of donor apheresis to start of recipient's Orca-T infusion) of < 72 hours. A total of 33 patients (21 AML, 10 ALL, 2 CML) were included. Median age was 43 (range, 21 - 63) years, 73% were male. Median follow-up was 375 (range, 73 - 1384) days. Sixteen patients received TBI-based MAC; 17 received busulfan-based MAC (Table). All patients engrafted with median time of 12.0 (range, 8 - 25) and 15.5 (range, 8 - 79) days for neutrophil and platelet engraftment, respectively. Two patients had secondary graft failure. Two patients had Grade 1 CRS; 1 patient had Grade 2 CRS. Grade 2+ aGvHD through Day 180+ occurred in 15% of patients. Grade 3-4 acute GvHD (aGvHD) was rare with only 1 event of grade 3 aGvHD and no grade 4 aGvHD. No patients had moderate-to-severe cGvHD. Estimated incidence of CTCAE grade 2 and > grade 3 infections were 9% and 15% at 1 year, respectively. 5 patients died (2 from relapsed disease) during the study period. NRM was 9% at 1 year. The 1-year relapse-free survival, GvHD-and-relapse free survival (Figure), and overall survival at 1 year was 82%. CONCLUSIONS Our findings reveal promising safety and efficacy outcomes using Orca-Q cell therapy for haplo-SCT despite the use of MAC with only single-agent tacrolimus, and without PTCy. With median follow-up of approximately 1 year, no patients experienced moderate or severe cGvHD, the low AE profile in the haplo setting remains favorable, and 1 year GvHD-free, relapse-free survival of 83% is very encouraging. This phase 1 study continues to enroll patients across the US. REFERENCES Abboud R, Wan F, Mariotti J, et al. Bone Marrow Transplant. 2021;56(11):2763-2770. Bolaños-Meade GM, Hamadani M, Wu J. N Engl J Med. 2023; 388(25):2338-2348. Duléry R, Mohty R, Labopin M, et al. JACC CardioOncol. 2021; 3(2):250-259. Hoover A, O'Leary D, Cao Q, et al. Blood. 2022; 140 (Supplement 1):282-283 Nagler A, Tsirigotis P. Bone Marrow Transplant. 2022; 57(11):1640-1641.

Automated production of gene-modified chimeric antigen receptor T cells using the Cocoon Platform

Autologous cell-based therapeutics have gained increasing attention in recent years because of their efficacy at treating diseases with limited therapeutic options. Chimeric antigen receptor (CAR) T-cell therapy has demonstrated clinical success in hematologic oncology indications, providing critically ill patients with a potentially curative therapy. Although engineered cell therapies such as CAR T cells provide new options for patients with unmet needs, the high cost and complexity of manufacturing may hinder clinical and commercial translation. The Cocoon Platform (Lonza, Basel, Switzerland) addresses many challenges, such as high labor demand, process consistency, contamination risks and scalability, by enabling efficient, functionally closed and automated production, whether at clinical or commercial scale. This platform is customizable and easy to use and requires minimal operator interaction, thereby decreasing process variability. We present two processes that demonstrate the Cocoon Platform's capabilities. We employed different T-cell activation methods—OKT3 and CD3/CD28 Dynabeads (Thermo Fisher Scientific, Waltham, MA, USA)—to generate final cellular products that meet the critical quality attributes of a clinical autologous CAR T-cell product. This study demonstrates a manufacturing solution for addressing challenges with manual methods of production and facilitating the scale-up of autologous cell therapy.

An In Vitro Model to Assess CRS Potential of CAR T Cells Using a Tumor Cell Line and Autologous Monocytes.

Chimeric antigen receptor (CAR) T cell therapy is an engineered cell therapy where T cells are isolated and genetically modified to contain a synthetic CAR with specificity to a tumor cell antigen. Upon antigen binding, the CAR T cell will initiate signaling cascades that result in lysis of the associated tumor cell. Cytokine release syndrome (CRS) is the primary toxicity associated with CAR T cell therapy and remains a prominent safety issue with currently available commercial products. CRS is driven by interaction of the CAR T cells with endogenous monocytes and macrophages, which can lead to immune cell overactivation and an increase in certain cytokines to supraphysiological levels. Identifying the potential of any given CAR construct to drive toxicities in vivo should be assessed in preclinical models prior to human trials. While there are in vivo mouse models available for this purpose, these are often complex xenograft models available in few centers. Thus, there is a need to develop an in vitro assay for measuring the CRS potential of CAR T cells. The assay described here is a preclinical tool for assessing the propensity of any given CAR construct to produce potentially CRS-driving cytokines following tumor cell and monocyte interactions. This article provides a detailed protocol for target cell preparation and isolation of monocytes from peripheral blood mononuclear cells (PBMCs) autologous to the CAR T cells, as well as protocols for seeding the three cell types in a co-culture assay and collecting/analyzing the cytokines produced via an ELISA or multiplex bead array. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of K562 target cells Basic Protocol 2: Isolation of monocytes from autologous PBMCs Basic Protocol 3: Seeding of CAR T cells, monocytes, and K562 cells in 96-well plates Basic Protocol 4: Analysis of co-culture supernatants by single-cytokine ELISA Alternate Protocol: Analysis of co-culture supernatants by multiplex cytokine bead array.

Evolution of molecular switches for regulation of transgene expression by clinically licensed gluconate.

Synthetic biology holds great promise to improve the safety and efficacy of future gene and engineered cell therapies by providing new means of endogenous or exogenous control of the embedded therapeutic programs. Here, we focused on gluconate as a clinically licensed small-molecule inducer and engineered gluconate-sensitive molecular switches to regulate transgene expression in human cell cultures and in mice. Several switch designs were assembled based on the gluconate-responsive transcriptional repressor GntR from Escherichia coli. Initially we assembled OFF- and ON-type switches by rewiring the native gluconate-dependent binding of GntR to target DNA sequences in mammalian cells. Then, we utilized the ability of GntR to dimerize in the presence of gluconate to activate gene expression from a split transcriptional activator. By means of random mutagenesis of GntR combined with phenotypic screening, we identified variants that significantly enhanced the functionality of the genetic devices, enabling the construction of robust two-input logic gates. We also demonstrated the potential utility of the synthetic switch in two in vivo settings, one employing implantation of alginate-encapsulated engineered cells and the other involving modification of host cells by DNA delivery. Then, as proof-of-concept, the gluconate-actuated genetic switch was connected to insulin secretion, and the components encoding gluconate-induced insulin production were introduced intotype-1 diabetic miceas naked DNA via hydrodynamic tail vein injection. Normoglycemia was restored, thereby showcasing the suitability of oral gluconate to regulate in situ production of a therapeutic protein.