Strong Expansion of Human Regulatory T Cells for Adoptive Cell Therapy Results in Epigenetic Changes Which May Impact Their Survival and Function

Adoptive transfer of regulatory T cells (Treg) is a promising new therapeutic option to treat detrimental inflammatory conditions after transplantation and during autoimmune disease. To reach sufficient cell yield for treatment, ex vivo isolated autologous or allogenic Tregs need to be expanded extensively in vitro during manufacturing of the Treg product. However, repetitive cycles of restimulation and prolonged culture have been shown to impact T cell phenotypes, functionality and fitness. It is therefore critical to scrutinize the molecular changes which occur during T cell product generation, and reexamine current manufacturing practices. We performed genome-wide DNA methylation profiling of cells throughout the manufacturing process of a polyclonal Treg product that has proven safety and hints of therapeutic efficacy in kidney transplant patients. We found progressive DNA methylation changes over the duration of culture, which were donor-independent and reproducible between manufacturing runs. Differentially methylated regions (DMRs) in the final products were significantly enriched at promoters and enhancers of genes implicated in T cell activation. Additionally, significant hypomethylation did also occur in promoters of genes implicated in functional exhaustion in conventional T cells, some of which, however, have been reported to strengthen immunosuppressive effector function in Tregs. At the same time, a set of reported Treg-specific demethylated regions increased methylation levels with culture, indicating a possible destabilization of Treg identity during manufacturing, which was independent of the purity of the starting material. Together, our results indicate that the repetitive TCR-mediated stimulation lead to epigenetic changes that might impact functionality of Treg products in multiple ways, by possibly shifting to an effector Treg phenotype with enhanced functional activity or by risking destabilization of Treg identity and impaired TCR activation. Our analyses also illustrate the value of epigenetic profiling for the evaluation of T cell product manufacturing pipelines, which might open new avenues for the improvement of current adoptive Treg therapies with relevance for conventional effector T cell products.

Detection of Vector Copy Number in Bicistronic CD19xCD22 CAR T Cell Products with Digital PCR

Chimeric antigen receptor (CAR) T cell therapy is a rapidly evolving immunotherapeutic treatment modality for adult and pediatric patients with a variety of cancers, which has been most extensively investigated in B-cell malignancies. Given that CAR T cell immunotherapy involves changing the genetic composition of a patient's T cells, this living drug presents unique safety and quality control challenges. Vector copy number (VCN), a measurement of transgene copies within a CAR T cell product, is a product-specific characteristic that must be quantified prior to patient administration as high VCN increases the risk of insertional mutagenesis. Historically, VCN assessment in CAR T cell products has been performed via qPCR. qPCR is reliable along a broad range of concentrations but has inherent limitations in its lower limit of detection and limit of quantification. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared to qPCR include simplicity, reproducibility, lower limit of detection, and definitive quantification. In this present study, we developed an assay for analysis of the novel bicistronic UCD19x22 CAR T cell construct, which was developed in the laboratory of Dr. Terry Fry at the University of Colorado and will be moving in to clinical trials later this year. Custom primer-probe assays were designed using Primer Express v3.0.1 and the ThermoFisher Custom TaqMan Assay Design Tool. As an internal control, forward and reverse primers as well as a VIC-labeled probe specific to human albumin (NCBI gene 213, HGNC:399) were designed. Primers and a FAM-labeled probe assay, specific for the bicistronic CD19x22 CAR T cell product, were designed at the junction site between the two distinct CARs. This study compares two different digital PCR modalities: (1) droplet digital PCR (ddPCR) via the BioRad QX200 system which utilizes water-in-oil droplet partitions and (2) the QIAcuity digital PCR system utilizing a nanoplate-based partitioning platform. While dPCR is a newer methodology compared to ddPCR, the two apply parallel procedures, data generation, and analyses. The primer/probe assay was validated with qPCR, dPCR and ddPCR using patient samples from preclinical CAR T cell manufacturing production runs, as well as Jurkat cell subclones which stably express this bicistronic CAR T product. We successfully developed an assay to specifically detect and quantify our bicistronic CD19xCD22 CAR transgene. ddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product without any signal detected in samples containing untransduced T cells or T cells transduced with CD19 only CARs. Additionally, our assay gives accurate, precise, and reproducible CAR T cell VCN measurements across qPCR, dPCR, and ddPCR modalities. We demonstrate that digital PCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, and that specific assays can be developed for detection of unique constructs. Future studies will evaluate the utility of this assay with digital PCR modalities in measuring CAR T cell persistence in clinical trial patient samples after receiving this novel CAR T cell product. Figure 1 Figure 1. Disclosures Fry: Sana Biotechnology: Current Employment, Current equity holder in publicly-traded company.

Cryopreserved CAR-like NK Cells Pre-Complexed with the CD30/CD16A Bispecific Innate Cell Engager (ICE®) AFM13 for the Treatment of CD30 + Malignancies

AFM13 is a bispecific tetravalent innate cell engager (ICE®) with two binding sites for CD16A and CD30 on immune cells and tumor cells, respectively. AFM13 is designed to potently activate anti-tumoral responses of natural killer (NK) cells, through antibody-dependent cellular cytotoxicity (ADCC) and macrophages via antibody-dependent cellular phagocytosis (ADCP) towards CD30 + tumor cells. It is widely known that the number of NK cells and their anti-tumor activity are often compromised in patients with cancer, including CD30 + malignancies such as Hodgkin lymphoma (HL) (Reiners, K.S. et al., Mol Ther 2013;21:895-903). Accumulating evidence suggests that the selective, high affinity binding of ICE® to CD16A enables stable pre-complexing with NK cells to enhance ADCC. Early clinical studies with AFM13 have demonstrated safety and efficacy, both as mono- and combination therapy with an anti-PD-1 checkpoint inhibitor (pembrolizumab) (Bartlett, N.L. et al., Blood 2020;136(21):2401-2409), in patients with relapsed or refractory (R/R) HL or peripheral T cell lymphoma (PTCL). Currently, AFM13 monotherapy is being evaluated in a registration-directed Phase 2 study to treat patients with PTCL (NCT04101331). Additionally, a Phase 1b/2a study in patients with PTCL (NCT03192202) previously demonstrated that NK cell numbers pre- and post-dosing of AFM13 may positively correlate with anti-tumoral response (Sawas, A. et al., Blood 2020;136(1):25-26). Therefore, the safety and efficacy of freshly prepared cord blood-derived allogeneic NK cells pre-complexed with AFM13, followed by three weekly infusions of AFM13 monotherapy, is currently being evaluated in patients with R/R CD30 + lymphomas in a Phase 1 dose escalation study (NCT04074746). The first four enrolled patients, all with HL, have shown a response rate of 100% without any safety concerns (Rezvani, K. et al., presentation AACR 2021). To further develop this CAR-like NK cell immunotherapeutic towards an off-the-shelf product, we evaluated the feasibility of cryopreservation of pre-complexed NK cells and assessed the biological activity of such cryopreserved and pre-complexed NK cells after thawing. In this study, the cell surface retention, suitability for NK cell preloading, and cytotoxic activity of AFM13 and other CD30-specific antibody formats was assessed. Furthermore, the compatibility of these formats with cryopreservation of the resulting CAR-like NK cell product was investigated. Cell surface retention assays on primary NK cells confirm substantially slower dissociation kinetics of the tetravalent bispecific ICE® compared to corresponding IgG1 or Fc-enhanced IgG1, enabling efficient and durable pre-complexing of NK cells with ICE®. Surface plasmon resonance analyses confirm long retention of ICE® independent of the CD16A polymorphism. More importantly, these assays demonstrate that the high ADCC potency and efficacy of NK cells, pre-complexed with CD16A-specific tetravalent ICE®, is maintained after one freeze-thaw cycle. These data suggest that high-affinity pre-complexing of adoptive NK cells with bispecific, CD16A-selective ICE® could be a novel cryopreserved off-the-shelf NK cell product for the effective depletion of tumor cells without the limitations and potential risks associated with the CAR-NK cell technology. Disclosures Reusch: Affimed: Current Employment, Current holder of stock options in a privately-held company. Ellwanger: Affimed: Current Employment, Current holder of stock options in a privately-held company. Fucek: Affimed: Current Employment, Current holder of stock options in a privately-held company. Müller: Affimed GmbH: Current Employment, Current holder of stock options in a privately-held company. Schniegler-Mattox: Affimed: Current Employment, Current holder of stock options in a privately-held company. Pahl: Affimed: Current Employment, Current holder of stock options in a privately-held company. Tesar: Affimed: Current Employment, Current holder of stock options in a privately-held company. Koch: Affimed: Current Employment, Current holder of stock options in a privately-held company.

Phase 1/2 Study of Nexi-002 Autologous Multi-Antigen-Specific CD8+ T Cells for the Treatment of Relapsed or Refractory Multiple Myeloma

The NEXI-002 study is a prospective, multicenter, open-label phase 1/2 trial designed to characterize the safety, immunologic, and preliminary anti-myeloma activity of the NEXI-002 antigen specific CD8+ T cell product. Multiple myeloma (MM) is an incurable malignancy that occurs predominantly in older patients and is characterized by the growth of malignant plasma cells in the bone marrow. Despite substantial advances in therapy, virtually all patients relapse after treatment, emphasizing the unmet medical need for additional effective treatments. The NEXI-002 product is an autologous non-genetically engineered therapy of CD8+ T cells that recognize HLA 02.01-restricted peptides from the WT1, CD138, CS1, and NY-ESO-1 antigens. This T-cell product includes key memory phenotypes such as stem-like memory, central memory, and effector memory cells. Eligible patients have relapsed or refractory multiple myeloma (RRMM) who have received at least three prior lines of treatment that included at least an immunomodulatory agent, a proteasome inhibitor, and an anti-CD38 agent. Three patients were enrolled into the Safety Evaluation phase and received a single infusion of 80 million (M) to 100M cells of NEXI-002 product. In this phase of the study the primary endpoint is safety and secondary endpoints include expansion, persistence, and trafficking of the NEXI-002 cells. Bridging anti-MM treatment was permitted during the manufacture of the cellular product with a wash-out period of at least 14 days prior to lymphodepletion (LD) chemotherapy (intravenous fludarabine 30 mg/m 2 and cyclophosphamide 300 mg/m 2), which was administered on Days -5, -4, and -3 prior to the infusion of the NEXI-002 product up to 72 hours later (Day1). Treatment-related adverse events, including infusion reactions, events that prolong hospitalization post infusion, CRS, and neurotoxicity (ICANS) have not developed in these patients who received the NEXI-002 product. Lymphocyte recovery to baseline levels occurred within a few days after the infusion of the NEXI-002 product, demonstrating robust CD4 and CD8 T cell reconstitution following LD chemotherapy. NEXI-002 antigen specific T cells were detected in peripheral blood (PB) by multimer staining and proliferated over time and trafficked to the bone marrow (BM). The phenotype composition of detectable antigen specific T cells at both sites maintained that of the infused product. These NEXI-002 T cells persisted in PB and BM during follow-up. T-cell receptor (TCR) sequencing assays revealed T cell clones in the NEXI-002 product that were not detected in PB of patients tested at baseline. These clones subsequently expanded and persisted over time in the PB and BM. In conclusion, these results show that infusion of the NEXI-002 product is safe, well tolerated, and capable of generating a cell-mediated immune response that may lead to clinical activity. RNA Seq transcriptional profiling of the CD8+ T cells is planned. Additional patients have recently received NEXI-002 infusions and the trial will continue to be expanded to gain additional safety, immunologic, and clinical activity experience. Disclosures Knight: Neximmune, Inc: Current Employment. Varela: Nexlmmune: Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Kite: Speakers Bureau. Edavana: Neximmune, Inc: Current Employment. Lu: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Suarez: Neximmune, Inc: Current Employment. Oelke: Neximmune, Inc: Current Employment. Bednarik: Neximmune, Inc: Current Employment.

951 A phase 1 first in human study of adenovirally transduced anti-HER2 CAR macrophages in subjects with HER2 overexpressing solid tumors: preliminary safety, pharmacokinetics, and TME reprogramming data

BackgroundCT-0508 is an autologous monocyte-derived pro-inflammatory macrophage cell product engineered with Ad5f35 to express an anti-HER2 CAR. In pre-clinical studies CT-0508 was safe and effective. This abstract contains preliminary results from the first-in-human experience with CAR macrophages (CAR-M).MethodsThis First-In-Human Phase 1, multi-center, open-label study is evaluating the safety, tolerability, manufacturing feasibility, pharmacokinetics and mechanism of action of CT-0508 in 18 subjects with advanced solid tumors overexpressing HER2 who have progressed on prior therapies, including HER2 targeted therapies if indicated.Patients receive four doses of filgrastim for monocyte mobilization prior to apheresis. CT-0508 CAR-M is manufactured from autologous apheresis products and delivered as a cryopreserved cell product. Group 1 subjects enter an intra-patient fractionated dose escalation regimen, receiving CT-0508 on D1, D3 and D5, followed by Group 2 subjects who receive CT-0508 on D1. There is no preparative chemotherapy prior to CT-0508 infusion.Pre and post treatment biopsies and blood samples are collected to investigate correlates of safety, serum cytokines and chemokines, pharmacokinetics, TME modulation, and induction of an adaptive anti-tumor immune response.ResultsTo date, two subjects have been treated with CT-0508 (esophageal adenocarcinoma and extrahepatic cholangiocarcinoma). Patient product was successfully manufactured, CT-0508 treatment was well tolerated, with no dose limiting and no major organ toxicities observed.One subject experienced Grade 2 CRS on Day 3 which resolved on the same day.Grade 3 AEs included anemia (present at baseline for both subjects) and lymphopenia (present at baseline in one subject). One subject experienced one SAE of Grade 4 tumor bleeding which was unrelated to CT-0508, 88 days after the last infusion.CAR-M were transiently detected in the peripheral blood following each infusion, demonstrating rapid egress from the periphery into tissues within hours. Transient cytokine/chemokine elevations were observed (peak: 2 hours, back to baseline at 48 hours). Single cell RNAseq analysis of dissociated tumor tissue samples (pre-treatment, day 8 and week 4) demonstrated dynamic TME reprogramming, with recruitment of inflammatory innate immune cells and naïve T cells at day 8, and significant CD8+ T cell infiltration, activation, and proliferation at week 4.ConclusionsCT-0508 has been administered to two subjects thus far, exhibiting safety, good tolerability, T cell repertoire modulation, and reprogramming of the TME consistent with the induction of anti-tumor immunity. The study continues to recruit patients and updated data will be presented.Trial RegistrationNCT04660929ReferenceKlichinsky M, Ruella M, Shestova O, et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol 2020;38(8):947–953.Ethics ApprovalEthics approvals have been obtained from the clinical sites enrolling patients: the University of Pennsylvania (844106/IORG0000029), the University of North Carolina and City of Hope Comprehensive Cancer Center (20201732/IORG0000432).

391 Biomarker correlates of response in patients with advanced myxoid/round cell liposarcoma (MRCLS) treated with NY-ESO-1 TCR T cells (Letetresgene autoleucel)

BackgroundThis is an open label pilot study (NCT02992743) on letetresgene autoleucel (lete-cel; GSK3377794), an NY-ESO-1-specific autologous CD4+ and CD8+ T cells expressing a high affinity T-cell receptor which recognizes the NY-ESO-1 antigen epitope in complex with specific HLA- alleles A*02, which exhibited anti-tumor activity and manageable safety profiles in patients with advanced MRCLS based on interim analysis (IA) data.1 Lymphodepletion has been shown to enhance the expansion, persistence, and homing of therapeutically infused T-cells, thereby potentiating therapeutic efficacy against malignant diseases.2 Initial T-cell kinetics data from this study demonstrated that lymphodepletion regimen (LDR)-B robustly depleted lymphocytes at infusion and was trended with higher peak cell expansion (Cmax) vs. LDR-A. The peak expansion was significantly associated with weight-normalized transduced cell dose and trended with response.1 Here, we will be discussing additional cell kinetics data and other exploratory biomarker correlates of response.MethodsPatients with advanced MRCLS were enrolled to 2 cohorts and received either planned A (N=10) or B (N=10) LDRs prior to lete-cel infusion (table 1). Response was assessed per RECIST v1.1. Transduced cell kinetics were measured by quantitative PCR of transgene vector copies in DNA from peripheral blood mononuclear cells (PBMCs). Serum cytokines (Meso Scale Discovery immunoassay) as pharmacodynamic (PD) markers of response and their association with T cell kinetics will be discussed. Phenotypic characterization of the cell product (pre- and post- infusion) via Flow cytometry using Cytek Aurora (23 color panel), to help understand correlation of response with engineered cell product attributes, will be presented. Potential biomarker correlates of clinical response were tested using generalized linear models.ResultsFive out of 6 responders with available lab data exhibited robust lymphocyte depletion at infusion (0–25 cell/µL) and high Cmax (>50,000 vector copies/µg gDNA) with LDR. Only 6/14 non-responders exhibited low lymphocytes counts at infusion and high Cmax. LDR-B also induced strong depletion of monocytes at infusion (p=0.03) vs. LDR-A, but depletion of monocytes did not show association with response. Higher Cmax was correlated with exposure (AUC0–28d) (Adj. R2=0.606). AUC0–28d was a better predictor of response in patients receiving LDR-B (p=0.0182), with AUC0–28d trending towards predicting response in the LDR-A cohort. AUC0–28d was associated with tumor volume reduction (p=0.0569).Abstract 391 Table 1ConclusionsExposure–response analysis of this study reveals that efficacy appears to be driven by weight-normalized transduced cell dose and LDR via AUC0–28d. Higher AUC0–28d was correlated with Cmax and maximum tumor volume reduction.AcknowledgementsThis study (208469; NCT02992743) was funded by GlaxoSmithKline.Trial RegistrationNCT02992743ReferencesD’Angelo SP, et al. J Clin Oncol 2021;39:15_suppl:11521.Bechman, Maher. Expert Opin Biol Ther 2021;21(5):627–637.Ethics ApprovalThis study was approved by institutional review boards (IRB) at the six participating sites.