Cell Dose Research Articles

Preclinical Studies and Clinical Trials on Cell-Based Treatments for Meniscus Regeneration.

This study aims at performing a thorough review of cell-based treatment strategies for meniscus regeneration in preclinical and clinical studies. The PubMed, Embase, and Web of Science databases were searched for relevant studies (both preclinical and clinical) published from the time of database construction to December 2022. Data related to cell-based therapies for in situ regeneration of the meniscus were extracted independently by two researchers. Assessment of risk of bias was performed according to the Cochrane Handbook for Systematic Reviews of Interventions. Statistical analyses based on the classification of different treatment strategies were performed. A total of 5730 articles were retrieved, of which 72 preclinical studies and 6 clinical studies were included in this review. Mesenchymal stem cells (MSCs), especially bone marrow MSCs (BMSCs), were the most commonly used cell type. Among preclinical studies, rabbit was the most commonly used animal species, partial meniscectomy was the most commonly adopted injury pattern, and 12 weeks was the most frequently chosen final time point for assessing repair outcomes. A range of natural and synthetic materials were used to aid cell delivery as scaffolds, hydrogels, or other morphologies. In clinical trials, there was large variation in the dose of cells, ranging from 16 × 106 to 150 × 106 cells with an average of 41.52 × 106 cells. The selection of treatment strategy for meniscus repair should be based on the nature of the injury. Cell-based therapies incorporating various "combination" strategies such as co-culture, composite materials, and extra stimulation may offer greater promise than single strategies for effective meniscal tissue regeneration, restoring natural meniscal anisotropy, and eventually achieving clinical translation. Impact Statement This review provides an up-to-date and comprehensive overview of preclinical and clinical studies that tested cell-based treatments for meniscus regeneration. It presents novel perspectives on studies published in the past 30 years, giving consideration to the cell sources and dose selection, delivery methods, extra stimulation, animal models and injury patterns, timing of outcome assessment, and histological and biomechanical outcomes, as well as a summary of findings for individual studies. These unique insights will help to shape future research on the repair of meniscus lesions and inform the clinical translation of new cell-based tissue engineering strategies.

Mesenchymal Stem Cell-Extracellular Vesicle Therapy for Stroke: Scalable Production and Imaging Biomarker Studies.

A major clinical hurdle to translate MSC-derived extracellular vesicles (EVs) is the lack of a method to scale-up the production of EVs with customized therapeutic properties. In this study, we tested whether EV production by a scalable 3D-bioprocessing method is feasible and improves neuroplasticity in animal models of stroke using MRI study. MSCs were cultured in a 3D-spheroid using a micro-patterned well. The EVs were isolated with filter and tangential flow filtration and characterized using electron microscopy, nanoparticle tracking analysis, and small RNA sequencing. Compared to conventional 2D culture, the production-reproduction of EVs (the number/size of particles and EV purity) obtained from 3D platform were more consistent among different lots from the same donor and among different donors. Several microRNAs with molecular functions associated with neurogenesis were upregulated in EVs obtained from 3D platform. EVs induced both neurogenesis and neuritogenesis via microRNAs (especially, miR-27a-3p and miR-132-3p)-mediated actions. EV therapy improved functional recovery on behavioral tests and reduced infarct volume on MRI in stroke models. The dose of MSC-EVs of 1/30 cell dose had similar therapeutic effects. In addition, the EV group had better anatomical and functional connectivity on diffusion tensor imaging and resting-state functional MRI in a mouse stroke model. This study shows that clinical-scale MSC-EV therapeutics are feasible, cost-effective, and improve functional recovery following experimental stroke, with a likely contribution from enhanced neurogenesis and neuroplasticity.

IMMU-21. ELUCIDATING THE CELLULAR MECHANISMS OF THERAPEUTIC BENEFIT UNDERLYING ADOPTIVE CELLULAR THERAPY IN MODELS OF GLIOMA

Abstract Among pediatric cancers, malignancies of the brain carry significant morbidity and mortality, harboring dismal prognosis and poor outcomes. Our group has previously shown that adoptive cellular therapy (ACT) provides therapeutic benefit against central nervous system (CNS) malignancies, including medulloblastoma and high-grade glioma. In this study, we characterized cell cycling differences of immune sub-populations in peripheral lymphoid and tumor tissue compartments in vivo. ACT was conducted in orthotopic KR158 luciferase tumor-bearing mice, a syngeneic murine glioma model. Five days following intracranial implantation of tumor cells, mice were treated with ACT which included the following elements: total body irradiation, hematopoietic stem cell transplantation, one dose of tumor-activated splenocytes and three weekly doses of dendritic cells that had been electroporated with tumor-derived ribonucleic acid. One week after the completion of ACT, bromodeoxyuridine (BRDU) was administered to mice 12-hours prior to tissue collection and flow cytometry was utilized to measure the level of BRDU incorporation. 7-aminoactinomycin-D was used in conjunction with BRDU to provide insight into all phases of the cell cycle. We observed significant differences in cycling populations of myeloid derived suppressor cells (MDSC) and T-cells. In ACT-treated-mice, MDSCs found in secondary lymphoid tissues were significantly more proliferative. In the tumor microenvironment (TME), proliferating MDSCs were significantly reduced compared to mice that did not receive treatment. Frequencies of proliferating CD4+ and CD8+ T-cells were significantly increased in cervical lymph node and TME but reduced in spleen. These findings suggest that ACT alters immune cycling in peripheral lymphoid tissues and TME to potentially promote antitumor T-cell responses and minimize MDSC-mediated immunosuppression. While ACT-mediated alterations to immune cell cycling are likely a major contributor to the therapy’s benefit, it is unlikely to be the sole mechanism. Further exploration into other functional pathways, namely migration, is needed to explain changes to immune populations following ACT.

IMMU-13. PRECLINICAL EFFICACY OF MULTI-ANTIGEN (HER2/EGFR806/B7-H3/IL13RA2) TARGETING QUAD-CAR T CELLS AGAINST DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse midline glioma (DMG), including diffuse intrinsic pontine glioma (DIPG), remains a fatal disease. We have published the preclinical efficacy of HER2, EGFR806, and B7-H3 CAR-T cells and opened 3 trials (BrainChild-01, -02, and -03, respectively) that have delivered >350 intracranial CAR T doses to >70 enrolled children with recurrent/refractory CNS tumors and DIPG/DMG. While these trials have demonstrated the feasibility and tolerability of repeated, locoregional CAR-T cell dosing, responses have not been uniform in part due to tumor heterogeneity. METHODS Seattle Children’s Therapeutics generated multi-antigen (HER2, EGFR806, B7-H3, IL13ra2) quad-CAR T cells and we performed IND-enabling preclinical studies using CNS tumor models including DMG models (PBT-22, PBT-27, and PBT-29). RESULTS Quad-CAR T cells were generated by transducing T cells with a mixture of four vectors. The specificity of these CARs is conferred by scFv binding domains derived from the monoclonal antibodies Herceptin, mAb806, and MGA271 for HER2, EGFR806, and B7-H3, respectively. The IL13-zetakine, aligned with NCT00730613, carries an E13Y mutation in place of an scFv for specificity. In vitro, the quad-CAR T cells demonstrated significant in vitro cytolysis and cytokine (IL-2, IFNy, TNFa) production compared to mock (untransduced) CAR-Ts. In vivo, pilot studies in a GFP:ffLuc+Her2 U87 orthoptic xenograft mouse model showed significant survival (p<0.001). To validate against DMG, we are conducting a larger study of single dose intracranial quad-CAR T cells in an orthotopic xenograft PBT-29 DMG mouse model. While ongoing, at day 19 post-treatment there is already a significant bioluminescence decrease (p<0.001). In vivo studies will be completed and complimented by tumor spatial transcriptomics. CONCLUSION Quad-CAR T cell targeting of DMG is highly effective preclinically. A phase I trial of repeated, locoregional quad-CAR T cells for children with recurrent/refractory CNS tumors and DIPG/DMG is FDA-approved and will open in 2023.

Clinical Trials with Mesenchymal Stem Cell Therapies for Osteoarthritis: Challenges in the Regeneration of Articular Cartilage.

Osteoarthritis (OA) is a whole-joint disease primarily characterized by the deterioration of hyaline cartilage. Current treatments include microfracture and chondrocyte implantation as early surgical strategies that can be combined with scaffolds to repair osteochondral lesions; however, intra-articular (IA) injections or implantations of mesenchymal stem cells (MSCs) are new approaches that have presented encouraging therapeutic results in animal models and humans. We critically reviewed clinical trials with MSC therapies for OA, focusing on their effectiveness, quality, and outcomes in the regeneration of articular cartilage. Several sources of autologous or allogeneic MSCs were used in the clinical trials. Minor adverse events were generally reported, indicating that IA applications of MSCs are potentially safe. The evaluation of articular cartilage regeneration in human clinical trials is challenging, particularly in the inflammatory environment of osteoarthritic joints. Our findings indicate that IA injections of MSCs are efficacious in the treatment of OA and the regeneration of cartilage, but that they may be insufficient for the full repair of articular cartilage defects. The possible interference of clinical and quality variables in the outcomes suggests that robust clinical trials are still necessary for generating reliable evidence with which to support these treatments. We suggest that the administration of just-sufficient doses of viable cells in appropriate regimens is critical to achieve effective and durable effects. In terms of future perspectives, genetic modification, complex products with extracellular vesicles derived from MSCs, cell encapsulation in hydrogels, and 3D bioprinted tissue engineering are promising approaches with which to improve MSC therapies for OA.

Population Pharmacokinetics of Total Rabbit Anti-thymocyte Globulin in Non-obese Adult Patients Undergoing Hematopoietic Cell Transplantation for Hematologic Malignancy.

Rabbit anti-thymocyte globulin (rATG), a therapeutic polyclonal antibody against human T cells, is commonly used in conditioning therapy prior to allogeneic hematopoietic cell transplantation (HCT). Previous studies successfully developed an individualized rATG dosing regimen based on "active" rATG population PK (popPK) analysis, while "total" rATG can be a more logistically favorable alternative for early HCT outcomes. We conducted a novel popPK analysis of total rATG. Total rATG concentration was measured in adult human-leukocyte antigen (HLA) mismatched HCT patients who received a low-dose rATG regimen (total 2.5-3mg/kg) within 3days prior to HCT. PopPK modeling and simulation was performed using nonlinear mixed effect modeling approach. A total of 504 rATG concentrations were available from 105 non-obese patients with hematologic malignancy (median age 47years) treated in Japan. The majority had acute leukemia or malignant lymphoma (94%). Total rATG PK was described by a two-compartment linear model. Influential covariate relations include ideal body weight [positively on both clearance (CL) and central volume of distribution], baseline serum albumin (negatively on CL), CD4+ T cell dose (positively on CL), and baseline serum IgG (positively on CL). Simulated covariate effects predicted that early total rATG exposures were affected by ideal body weight. This novel popPK model described the PK of total rATG in the adult HCT patients who received a low-dose rATG conditioning regimen. This model can be used for model-informed precision dosing in the settings with minimal baseline rATG targets (T cells), and early clinical outcomes are of interest.

CD3+TCRαβ/CD19+-Depleted Mismatched Family or Unrelated Donor Salvage Stem Cell Transplantation for Graft Dysfunction in Inborn Errors of Immunity

A minority of children experience significant graft dysfunction after an allogeneic hematopoietic stem cell transplant (HSCT) for inborn errors of immunity (IEI). The optimal approach to salvage HSCT is unclear with respect to conditioning regimen and stem cell source. This single-centre retrospective case series reports the outcomes of salvage CD3+TCRαβ/CD19 depleted mismatched family or unrelated donor stem cell transplantation (TCRαβ-SCT) between 2013 - 2022 for graft dysfunction in 12 children with IEI. Outcomes of interest were overall survival (OS), event free survival (EFS), graft-versus-host disease (GvHD)-free and event-free survival (GEFS), toxicities, GvHD, viremia and long-term graft function. A retrospective audit of patients who underwent second CD3+TCRαβ/CD19 depleted mismatched donor graft using Treosulfan-based reduced toxicity myeloablative conditioning. Median age at first HSCT was 8.76 months (range, 2.5 months - 6 years) and at second TCRαβ-SCT was 3.6 years (1.2 - 11 years). Median interval between first and second HSCT was 1.7 years (3 months - 9 years). The primary diagnoses were: severe combined immunodeficiency (SCID) (n=5) and non-SCID IEI (n=7). The indications for second HSCT were: primary aplasia (n=1), secondary autologous reconstitution (n=6), refractory aGVHD (n=3) and secondary leukemia (n=1). Donors were either haploidentical parental donors (n=10) or mismatched unrelated donors (n=2). All received TCRαβ/CD19-depleted-PBSC with a median CD34+ cell dose of 9.3 × 106/kg (2.8-32.3 × 106/kg) and a median TCRαβ+ cell dose of 4 × 104/kg (1.3-19.2 × 104/kg). All engrafted with median days to neutrophil and platelet recovery of 15 (12-24) and 12 (9-19). One developed secondary aplasia and one had secondary autologous reconstitution, but both underwent a successful third HSCT. Four (33%) had grade II aGvHD and none had grade III-IV aGvHD. None had cGvHD but one developed extensive cutaneous cGVHD after third HSCT using PBSC and ATG. Nine (75%) were noted to have at least one episode of blood viremia with HHV6 (n=6, 50%), adenovirus (n=6, 50%), EBV (n=3, 25%) or CMV (n=3; 25%). Median duration of follow-up was 2.3 years (range: 0.5 - 10 years) and the 2-year OS, EFS and GEFS were 100% (95% confidence interval, 0-100%), 73% (37-90%) and 73% (37%-90%) respectively. TCRαβ-SCT from mismatched family or unrelated donors, using a chemotherapy only regimen, is a safe alternative donor salvage transplant strategy for second HSCT in patients without a suitably matched donor.

Absolute lymphocyte counts (ALC) recovery and risk of viral reactivation in the setting of haploidentical hematopoietic stem cell transplant.

e19065 Background: The impact of early absolute lymphocyte repopulation after allogeneic hematopoietic stem cell transplant has historically been shown to predict the risk of post-transplant infection, and subsequently, non-relapse mortality (NRM). Previous studies have shown that ALC recovery after allogeneic stem cell transplant is associated with risk of viral reactivation and survival. In this study we aim to investigate the role of ALC recovery in the contemporary era with myeloablative haploidentical hematopoietic stem cell transplant (HI-HSCT). Methods: We retrospectively analyzed adult patients at University of Southern California (USC) Norris Cancer Hospital who received myeloablative conditioning haplo-HSCT (Fludarabine with 12 Gy of radiation) from 2014 to 2020. The primary endpoint was correlation between ALC recovery day 21 and risk of viral reactivation including cytomegalovirus (CMV), Epstein-Barr Virus (EBV) and BK virus. ALC recovery cut off was chosen to be 175/uL on day +21 and 500/uL on day +60 based on prior reports. Results: 58 patients were identified as recipients of MAC haploidentical-HSCT. 17 had ALC recovery at day +21. Mean age was 49 years, 31 (53.4%) were males and 27 (46.6%) were females. 41 (70%) patients were Hispanic, 12 (20%) were non-Hispanic White, and the majority had AML (51%). 76% received > 5*106/kg CD34+ cell dose and 78% received CD3 + cell dose < 3*108/kg. Serum CMV, EBV and urine BK virus were detected in 38 (65.5%), 8 (15.7%) and 11 (32.4%) respectively. Clinically significant CMV reactivation (required treatment with antiviral therapy) was detected in 26 (44.8%) patients. Lack of ALC recovery on day +21 did not show correlation with CMV, EBV and BK virus reactivation. Also, the risk of CMV reactivation was not related to day +60 ALC recovery (p-value = 0.469). On univariate analysis, ALC recovery day +21, day +60, and CMV reactivation did not show correlation with overall survival. In multivariate analysis when adjusted for age and pre-transplant CMV status, higher CD 3+ cell dose of > 3*108/kg was associated with reduced risk of CMV reactivation HR = 0.35 (0.15-0.85) p-value = 0.02. Conclusions: Previous studies have established ALC recovery as a predictor of better outcome. This has not been established in patients undertaking myeloablative HI-HSCT. Our study shows that lack of ALC recovery on day +21 does not affect CMV, EBV and BK virus reactivation and risk of CMV reactivation does not correlate with day +60 ALC recovery. Also, ALC recovery and CMV reactivation does not impact overall survival. The use of Letermovir, effective CMV therapies and close viral surveillance in contemporary practice is likely responsible for mitigating poor outcomes with CMV reactivation. Graft composition needs to be studied prospectively, as higher CD3+ cell dose may present an opportunity to reduce CMV reactivation in HI-HSCT.

A Phase I Study of CD19‐targeted 19(T2)28z1xx CAR T Cells in Adult Patients with Relapsed or Refractory Diffuse Large B‐cell Lymphoma

Background: The redundancy of CD28 and CD3 signaling in a CAR design incorporating all 3 immunoreceptor tyrosine-based activation motifs (ITAMs) might negatively affect T cell differentiation and promote exhaustion. Therefore, we created a novel CD19 CAR construct with calibrated CAR signaling potential by mutating 2 of the 3 ITAMs, termed 1XX. In mouse models, 19-28z1XX CAR successfully induced tumor eradication at low CAR T cell doses with improved survival compared to conventional 19-28z CAR and with enhanced persistence of functional CAR T cells. We report on adult pts with DLBCL treated with 19(T2)28z-1XX CAR T cells (NCT04464200). Methods: This is a single center, phase I, dose-escalation and expansion trial of 19(T2)28z1XX CAR T cells in adult pts with R/R DLBCL. Pts received lymphodepleting chemotherapy followed by escalating doses of CAR T cells: 25–200 × 106. The primary objective was to evaluate safety and tolerability and determine the recommended phase 2 dose. Secondary objectives include overall response rate and duration of response. Results: Sixteen pts were treated on the dose escalation phase of the study. Median age was 62 (range, 50–80), and median number of prior treatments was 2 (range, 1–5). 4, 6, 3 and 3 pts were treated at DL1, DL2, DL3 and DL4 CAR T cells, respectively. 15 pts (94%) experienced cytokine release syndrome (CRS): grade 1 (n = 9), grade 2 (n = 6). No pts experienced ≥ grade 3 CRS. Two pts (13%) experienced neurotoxicity (NTX): 1 pt with grade 1 and 1 pt with grade 3. NTX was transient and reversible in both cases.Ten of 16 pts (63%) achieved a complete response (CR) and 2 pts (13%) achieved a partial response (PR), with overall response rate (ORR) of 75%. One pt who achieved initial PR spontaneously converted to CR without further treatment after month 6, resulting in best CR rate of 69%. Responses were seen across all dose levels. With a median follow-up of 155 days (range, 33–667), no pt in CR has experienced relapse including 7 pts with >6 months of follow-up (Figure 1). Peak CAR T cell expansion occurred at a median of 14 days after CAR T cell infusion (range, 7–83.3 days). CAR T cell detection beyond 350 days has been noted. DL 1 (25 × 10e6 cells) was chosen as the recommended dose for the expansion phase. Updated responses in the dose escalation cohort and the full expansion cohort, as well as PK data, will be presented. Conclusions: Treatment with 19(T2)28z1XX CAR T cells was safe. No severe CRS was observed and only 1 pt experienced transient grade 3 NTX. The overall CR rate was 69% and durable with no relapse observed with a median follow-up of 155 days. CRs were observed at DL1, which was chosen for the expansion phase. These findings corroborate our preclinical data and suggest that the 1XX signaling domain may lead to enhanced efficacy of CD19 CAR and allow infusion of a lower T cell dose with favorable toxicity profile. Keyword: Cellular therapies Conflicts of interests pertinent to the abstract. M. L. Palomba Consultant or advisory role: Synthekine, Cellectar, Kite, Bristol Meyer Squibb I. Riviere Consultant or advisory role: Fate Therapeutics, Akron Biotechnology Stock ownership: Mnemo Therapeutics, J. Li Employment or leadership position: Takeda L. Sellner Employment or leadership position: Takeda M. Sadelain Consultant or advisory role: Atara Research funding: Takeda, Mnemo Therapeutics S. Chen Employment or leadership position: Takeda Y. Zhao Employment or leadership position: Takeda J. H. Park Consultant or advisory role: Novartis, Kite, Kura, Astra-Zeneca, Servier, Curocell, Intella, Autolus, Allogene, Affymmune, Amgen Research funding: Genentech

The ELiPSE-1 study: A phase 1, multicenter, open-label study of CNTY-101 in subjects with relapsed or refractory CD19-positive B-cell malignancies.

TPS7580 Background: Century Therapeutics’ foundational technology is built on induced pluripotent stem cells, differentiated from allogeneic approaches that utilize non-renewable donor-derived cells. This approach allows production of a clonal cell bank of precisely edited cells that can be expanded and differentiated to supply large amounts of homogeneous allogeneic therapeutic products for off-the-shelf use. CNTY-101 is an allogeneic Chimeric Antigen Receptor NK (CAR-NK) cell product with genetic modifications designed to specifically target CD19-expressing B-cell malignancies, reduce allorejection in the recipient allowing for repeat dosing, increase persistence, and enable cell elimination in the event of unexpected adverse events. CNTY-101, as a CAR-NK product, has the potential for improved safety over T-cell based cellular therapies. Methods: This first-in-human study will determine the MTD and recommended Phase 2 regimen (RP2R) of CNTY-101 in combination with subcutaneous (SC) IL-2 in patients with R/R aggressive and indolent CD19-positive B-cell Non-Hodgkin lymphomas including diffuse large B-cell lymphoma, mantle cell lymphoma, primary mediastinal large B-cell lymphoma, follicular lymphoma and marginal zone lymphoma, who are without other treatment options. Part 1 of the study (dose escalation using the BOIN design) will start with a dose of CNTY-101 cells without IL-2 (1 to 3 subjects), followed by dose escalation up to four dose cohorts administering a single dose of CNTY-101 cells in combination with IL-2 daily for 8 days (Sched A). After the single-dose MTD (or the maximal single dose) has been reached, a fractionated schedule of 3 doses per 28-day cycle, administered on Days 1, 8, and 15 (Sched B), will also be explored, starting with approximately one-third the maximum single dose level of CNTY-101, followed by escalation/de-escalation. Subjects in whom CNTY-101 shows signs of clinical benefit (i.e. stable disease or better per Lugano 2014 criteria) may receive additional cycles of CNTY-101 if the Investigator, Sponsor, and appropriate regulatory authorities, as required, concur that the benefit-risk is positive. One overall CNTY-101 RP2R will be declared in Part 1. Part 2, expansion, will further evaluate the safety, pharmacokinetics (PK), and efficacy of the CNTY-101 regimen, treating up to an additional 12 subjects to reach a total of approximately 20 subjects treated at the RP2R. In addition to safety and efficacy endpoints, the trial will evaluate exploratory PK, immunogenicity and pharmacodynamic endpoints including evaluations of tumor cfDNA, iNK tumor trafficking and serum cytokines. Clinical trial information: NCT05336409 .

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Anti-BCMA chimeric antigen receptor (CAR) T-cell therapy showed excellent efficacy in patients with relapsed/refractory multiple myeloma (R/R MM). Point-of-care (POC) CAR manufacturing abrogates the need for cryopreservation and shipment of cells, thus shortening the manufacturing process and reducing the necessity of bridging therapy. We report outcomes of phase 1b/2 single-center clinical trial of autologous POC anti-BCMA CAR T-cell therapy in patients (pts) with R/R MM treated with ≥3 prior therapies (NCT05243212). Methods: Pts underwent a single peripheral blood leukapheresis. Fresh T-cells were transduced with retroviral vector encoding the anti-BCMA CAR (based on 11D5-3 ScFv, CD28 costimulatory domain, and CD3-ζ signaling domain). Cell dose was 6 × 106/kg (dose level I [n = 3]) or 9 × 106/kg (dose level II [n = 29]) CAR T-cells. Response was defined per IMWG criteria. Last follow-up was as of 02/2023. Results: All 32 pts (median age 60, IQR 54–67) enrolled received CAR T-cell infusion in a median of 11 days (IQR 11–11) after leukapheresis. Only 2 pts received bridging chemotherapy. The median number of prior therapies was 4 (IQR 3–5), with 59% and 34% of the pts being penta- and quad-refractory, respectively. 7 (22%) pts had prior exposure to BCMA-targeted therapy (belantamab mafodotin, n = 5 [16%]; talqeutamab, n = 2 [6%]). At enrollment, 10 (34%) pts had high-risk cytogenetics, 7 (22%) had double-hit myeloma, and 17 (57%) had extramedullary involvement. Only 17 (53%) and 2 (16%) pts were eligible to enroll in the KarMMa (NEJM, 2021) and CARTITUDE-1 (Lancet, 2021) studies, respectively. One patient (3%) developed grade 3 cytokine release syndrome. Immune effector cell-associated neurotoxicity syndrome was not noted. Grade 3–4 neutropenia and thrombocytopenia occurred in 31 (97%) and 16 (50%), respectively. Anemia requiring transfusion occurred in 14 (44%) pts. Cellular therapy-related death was not observed. The median follow-up was 3.9 months (IQR 2.6–7.3). Best Overall response rate (PR at least) was 59% (VGPR at least, 40%). Median time to first response was 31 days (95% CI: 26–33). Estimated 6-months OS, PFS and duration of response were 89% (95% CI: 75–100), 48% (95% CI: 33–72), and 63% (95% CI: 41–97), respectively. Patients with prior exposure to BCMA-targeted therapies had an inferior PFS (HR 3.4 [95% CI: 1.2–9.7] p = 0.03; Figure 1). Conclusion: POC anti-BCMA CAR T-cells induced high response rates with an excellent safety profile in high-risk MM patients mostly not eligible to be enrolled in the pivotal trials. The rapid CAR-T production time obviated the need for bridging therapy in most patients. It is noteworthy that prior exposure to BCMA-targeted therapies is associated with dismal PFS, hence those therapies should be carefully considered when CAR T-cell therapy might be intended. Encore Abstract—previously submitted to EHA 2023 Keywords: cellular therapies, multiple myeloma No conflicts of interests pertinent to the abstract. * H. Magen, E. Shkury and A. Avigdor contributed equally to the abstract.

Clinical safety and efficacy observation of ex vivo-expanded peripheral blood-derived allogeneic NK cells in the treatment of refractory or recurrent ovarian cancer.

e17553 Background: Ovarian cancer is the most lethal gynecologic malignancy. Nearly 75% of patients were diagnosed at advanced stages. Despite 60%-80% of patients achieve complete remission after treatment with surgery combined with chemotherapy, about 60% of patients relapse within two years, more than 50% of patients develop chemotherapy tolerance with only 30% five-year survival rate. Platinum resistance-induced recurrence and platinum intolerance result in poor prognosis. Thus, there is an unmet need for innovative therapy of advanced ovarian cancer. We have developed a novel non-genetically modified ex vivo-expanded allogeneic NK cells from peripheral blood (PB). The expanded PB-NK cells have highly cytolytic activity and have the capacity to preferentially reside in ovary. We conducted a phase I clinical trial for evaluation of safety and efficacy of allogeneic NK cells in treatment of patients with refractory or recurrent ovarian cancers. Methods: Patients with advanced ovarian cancer who failed after multiline treatment were enrolled and received the treatment with allogeneic NK cells. The treatment was divided into low, medium and high dose groups with a 28-day cycle. safety and efficacy were evaluated after receiving two cycles of allogeneic NK cell infusion. Results: A total of 4 patients with advanced ovarian cancer who failed multiline therapy completed two courses of cell infusion therapy and evaluation. The median age of patients was 58 years (range 50 to 71 years). The median number of lines of treatment was 6 (range 3-7). One recurrent patient with platinum intolerance received low dose of NK cells and three relapse or refractory patients with platinum resistance received medium dose of allogeneic NK cell therapy. The patient in low-dose group and one patient in medium-dose group were evaluated for stable disease (SD), the other two patients in medium-dose group were disease progression after two courses of treatment. Notable, the patient in low-dose group continued to receive treatment with the same dose and achieved partial response (PR) after three courses of treatment. Upon receiving continuous NK cell therapy, the tumor lesions continued to shrink, with the best result was 60% reduction. It was re-evaluated as progressive disease (PD) 6 months after treatment was discontinued. Adverse events during the follow-up period included grade I fever in 4 (100%), grade I drowsiness in 1 (25%), grade I chills in 1 (25%), and grade I malaise in 1 (25%). No CRS or GVHD occurred. Conclusions: We report the safety and efficacy of allogeneic PB-NK cells in treatment of relapse or refractory ovarian cancer. The results showed that the allogeneic PB-NK cells are safe and feasible for the treatment of ovarian cancer, indicating a promising curative effect. There is no host versus graft reaction upon receiving continuous allogeneic NK cell therapy. Clinical trial information: NCT03619954 .

Phase I clinical trial using a unique immunotherapeutic combination of MUC1-targeted CAR-T cells with PD-1-knockout in the treatment of patients with advanced esophageal cancer.

e16061 Background: Combining CAR-T with programmed cell death-1 (PD-1) inhibition may be a more efficacious approach for treatment of solid tumors. This clinical study was designed to assess the safety and tolerability of PD-1 knockout MUC1-Targeted CAR-T engineered Cells to treat advanced esophageal cancer. Methods: Patients with advanced esophageal cancer were recruited according to the criteria in NCT03706326. PD-1 gene knockout (KO) MUC1-targeted CAR-T cells were prepared by constructing MUC1-specific CARs using the SM3 scFv in patient-derived T-cells, and PD-1 gene KO was achieved in these CAR positive T cells using the CRISPR-Cas9 system. Efficiency and validation were confirmed by sequencing and flow cytometry. MUC1-CAR+/PD-1- KO engineered T cells at a starting dose of 5x107 were infused over 60 min. The dose was escalated to 5x109 cells. Following treatment, the safety and tolerability of dose of the therapeutic cells were assessed using CTCAE v4.0. Patients’ general condition, levels of lymphocytes, IL-6, hs-CRP, PCT, CYFRA21, NSE(E), and SCC were monitored at regular intervals. Circulating CART cells were checked regularly. Tumor size were examined by MRI/CT scans. Efficacy was also assessed. Results: A total of 9 patients (aged 48 to 80) diagnosed with esophageal cancer/esophageal squamous cell carcinoma (IIIb to IV), were included in this study. All participants received at least one cycle of CART cell treatment. Among the 9 treated patients, 4 received 1 cycle, other 5 received 2, 3, 4, 7 and 10 cycles, respectively. The most common adverse events (AE) were acute fever (8 patients, 38.0-40.5 C◦), chills (5 patients, occurred in the subsequent cycle of infusion), skin rash (4 patient). No grade 3-5 AEs were found, and no cytokine release syndrome (CRS) was observed. Of the 9 assessed patients, 6 had stable disease (SD) while 3 had progressive disease. All patients had significant symptom improvements after infusion. Circulating CART cells gradually declined after infusion; the number dropped down to approximately 25% in 4 months after one cycle treatment indicating the necessity of receiving more cycles. Patients who received multiple cycles of infusion showed long-term and steady SD status. Two patients who received a multiple cycle of CAR-T cells had an overall survival (OS) of 24 months. Conclusions: Our data suggests that the treatment of patients with advanced esophageal cancer with PD-1 disrupted MUC1-targeted CAR-T cells is safe and well-tolerated by all patients. No CRS and other serious EAs were observed throughout the study. The efficacy of this unique combined therapy is currently being assessed. Our limited data indicate that the OS of the patients extended significantly in the treated patients. Two out of 6 patients with an OS of greater than 24 months, an outstanding clinical outcome. Clinical trial information: NCT03706326 .

TOP-077 - Safety, tolerability, and preliminary efficacy of ascending doses of Human Allogeneic Liver-derived Progenitor Cells (HepaStem®) in patients with cirrhotic and pre-cirrhotic non-alcoholic steatohepatitis (NASH)

TOP-077 - Safety, tolerability, and preliminary efficacy of ascending doses of Human Allogeneic Liver-derived Progenitor Cells (HepaStem®) in patients with cirrhotic and pre-cirrhotic non-alcoholic steatohepatitis (NASH)

INB-200 phase I study of gene modified autologous gamma-delta (γδ) T cells in patients with newly diagnosed glioblastoma multiforme (GBM) receiving maintenance temozolomide (TMZ).

2007 Background: MHC unrestricted γδ T cells target NKG2D ligands upregulated on tumor cells. IN8bio’s DeltEx chemotherapy resistant cell therapy (CRCT), is a novel γδ T cell genetically engineered to express MGMT to convey temozolomide (TMZ) resistance and allow combination therapy. NCT04165941, a Phase 1 trial assessing the safety of single and multiple infusions of autologous CRCT cells presents updated safety and efficacy data. Methods: Adult newly diagnosed GBM patients with adequate organ function and KPS≥70% are enrolled. Cells engineered from apheresis were infused through a Rickham catheter placed during surgery. Cohorts (C) 1, 2 and 3 each receive 1, 3 and6 doses of cells respectively on day (D) 1 of each 28-day maintenance cycle. Patients receive 1 x 107 γδ T cells intratumorally with 150 mg/m2 of TMZ intravenously on D1 of maintenance with the standard Stupp maintenance regimen on D2-5. Primary endpoint is safety; secondary endpoints include progression free and overall survival. Immunologic and genomic correlative analyses are being conducted. Dose limiting toxicities (DLTs) are defined as treatment related ≥ grade 3 cardiopulmonary or hepatic toxicity, grade 4 toxicity exceeding 72 hours or neurologic deterioration that exceeds 2 weeks. Results: 15 patients (53% male; median age 69 (range: 21-76); 80% IDH-WT, 66.7% MGMT unmethylated) were enrolled with 8 dosed (N=3 in C1, 4 in C2, 1 in C3). No patients experienced DLTs, cytokine release syndrome (CRS), or neurotoxicity (ICANS). The most common adverse events (AEs) were Grade 1/2 events including fever, fatigue, nausea, headache, platelet count decreased, incision site pain attributable to TMZ, radiotherapy or disease. One subject had Grade 3 treatment related AEs of UTI, dehydration, and thrombocytopenia. Three evaluable C1 patients have PFS of 8.3, 11.9, 7.4 months and OS of 15.6, 17.7 and 9.6 months respectively. In C2, four patients have been dosed, with no DLTs in 3 evaluable patients; 2 remain progression free at 18.9 and 14.8 months, while a third died without relapse due to pulmonary embolus at 8.7 months. One C3 patient has completed 5 of 6 planned doses without DLT. Patient recruitment continues with anticipated completion in 2023. Conclusions: Data demonstrates that single, repeat doses of CRCT gamma delta T cells have manageable toxicity with continued encouraging trend in PFS and warrants further assessment. Clinical trial information: NCT04165941 .

A phase 1/2a, multicenter, open-label study of CNA3103 (LGR5-targeted, autologous CAR-T cells) in patients with metastatic colorectal cancer (mCRC).

TPS3632 Background: Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a cancer stem cell (CSC) marker that is abundant in solid tumors, highly expressed in colorectal cancer and involved in tumor initiation, growth, and metastasis. Its expression is linked to poor survival and poor patient response to therapy in mCRC, and presents an attractive opportunity for selectively targeting a highly specific, functional CSC receptor in this disease. CNA3103 is a first-in-class cell product consisting of autologous T cells transduced to express a CAR to target LGR5. It contains the antigen-biding domain of BNC101, a humanized monoclonal LGR5 antibody previously tested in Phase 1 in mCRC subjects and demonstrated to be safe and well tolerated with no off-target binding activity. Methods: This is a first in human, multicenter, open label, Phase 1/2a dose escalation and expansion study to determine the safety of and overall best response to CNA3103 in subjects with mCRC. Up to 44 subjects (24 in Phase 1 and 20 in Phase 2a) will be enrolled. The trial follows a BOIN design with a minimum of 3 consecutively enrolled subjects per cohort, and a 28-day DLT evaluation period. Four cell dose levels (5 × 107, 1.5 × 108, 4.5 × 108, 1.5 × 109) of CNA3103 will be evaluated. A Safety Monitoring Committee (SMC) will oversee the conduct of the study. The pharmacokinetic (PK) profile of CNA3103 in blood will be assessed. Exploratory objectives include evaluation of LGR5+ cells and CNA3103 in tumor biopsies, immune cellular profile, cytokine levels and circulating tumor DNA in blood, and immunogenicity of CNA3103. Main inclusion criteria: mCRC subjects positive for LGR5 expression and failing prior treatment with 5-FU, oxaliplatin and irinotecan-based regimens for metastatic disease; >1 radiologically measurable lesion per RECISTv1.1; >1 biopsiable lesion at baseline; ECOG 0 or 1; serum albumin >3 g/dL; and normal organ and marrow function. Main exclusion criteria: BRAF-mutated disease, clinical ascites/ pleural effusions; active autoimmune/ connective tissue disease; history of inflammatory bowel disease or active peptic ulcer disease. The trial is sponsored by Carina Biotech. Phase 1 enrolment is limited to Australian sites. Phase 2a enrolment will take place in Australia and the US. For information, contact Lina Jablonskis lina@carinabiotech.com or +61 437 891 563.

Intratumoral pan-ErbB targeted CAR-T for head and neck squamous cell carcinoma: interim analysis of the T4 immunotherapy study

BackgroundLocally advanced/recurrent head and neck squamous cell carcinoma (HNSCC) is associated with significant morbidity and mortality. To target upregulated ErbB dimer expression in this cancer, we developed an autologous CD28-based...

Cytokine release syndrome in haploidentical stem cell transplant may impact T-cell recovery and relapse

AbstractCytokine release syndrome (CRS) following haploidentical hematopoietic cell transplantation (HCT) resembles CRS after chimeric antigen receptor-T therapy. We conducted this single-center retrospective study to evaluate the association of posthaploidentical HCT CRS with clinical outcomes and immune reconstitution. One hundred sixty-nine patients who underwent haploidentical HCT between 2011 and 2020 were identified. Of these, 98 patients (58%) developed CRS after HCT. CRS was diagnosed based on the presence of fever within the first 5 days after HCT without evidence of infection or infusion reaction and was graded according to established criteria. The development of posthaploidentical HCT CRS was associated with a lower incidence of disease relapse (P = .024) but with an increased risk of chronic graft-versus-host disease GVHD (P = .01). The association of CRS with a lower incidence of relapse was not confounded by graft source or disease diagnosis. Neither CD34 nor total nucleated cell dose was associated with CRS independently of graft type. In patients developing CRS, CD4+ Treg (P < .0005), CD4+ Tcon (P < .005), and CD8+ T cells (P < .005) increased 1 month after HCT compared with those who did not develop CRS, but not at later time points. The increase in CD4+ regulatory T cells 1 month after HCT was most notable among patients with CRS who received a bone marrow graft (P < .005). The development of posthaploidentical HCT CRS is associated with a reduced incidence of disease relapse and a transient effect on post-HCT immune reconstitution of T cells and their subsets. Therefore, the validation of these observations in a multicenter cohort is required.

Intensity of endogenous thrombocytopenia after autologous stem cell transplantation in patients prophylactically transfused with platelets.

Large clinical trials have demonstrated that some patient groups with hypoproliferative thrombocytopenia benefit from prophylactic platelet transfusions, while in others, a therapeutic transfusion regimen might be sufficient. The remaining capacity to generate endogenous platelets might be helpful to select the platelet transfusion regimen. We assessed whether the recently described method of digital droplet polymerase chain reaction (PCR) can be used to assess the endogenous platelet levels in two groups of patients undergoing high-dose chemotherapy with autologous stem cell transplantation (ASCT). Multiple myeloma (n = 22) patients received high-dose melphalan alone (HDMA); lymphoma patients (n = 15) received BEAM or TEAM (B/TEAM) conditioning. Patients with a total platelet count <10 G/L received prophylactic apheresis platelet concentrates. Daily endogenous platelet counts were measured by digital droplet PCR for at least 10 days post-ASCT. Post-transplantation B/TEAM patients received their first platelet transfusion on average 3 days earlier than HDMA patients (p < 0.001) and required about twofold more platelet concentrates (p < 0.001). The endogenous platelet count fell ≤5 G/L for a median of 115 h (91-159; 95% confidence interval) in B/TEAM-treated patients compared to 12.6 h (0-24) (p < 0.0001) in HDMA-treated patients. Multivariate analysis confirmed this profound effect of the high-dose regimen (p < 0.001). The CD-34+ -cell dose in the graft was inversely correlated with the intensity of endogenous thrombocytopenia in B/TEAM-treated patients. Monitoring endogenous platelet counts detects the direct effects of myelosuppressive chemotherapies on platelet regeneration. This approach may help to develop a platelet transfusion regimen tailored to specific patient groups.

Multiscale Monte Carlo simulations of gold nanoparticle dose-enhanced radiotherapy I: Cellular dose enhancement in microscopic models.

The introduction of Gold NanoParticles (GNPs) in radiotherapy treatments necessitates considerations such as GNP size, location, and quantity, as well as patient geometry and beam quality. Physics considerations span length scales across many orders of magnitude (nanometer-to-centimeter), presenting challenges that often limit the scope of dosimetric studies to either micro- or macroscopic scales. To investigate GNP dose-enhanced radiation Therapy (GNPT) through Monte Carlo (MC) simulations that bridge micro-to-macroscopic scales. The work is presented in two parts, with Part I (this work) investigating accurate and efficient MC modeling at the single cell level to calculate nucleus and cytoplasm Dose Enhancement Factors (n,cDEFs), considering a broad parameter space including GNP concentration, GNP intracellular distribution, cell size, and incident photon energy. Part II then evaluates cell dose enhancement factors across macroscopic (tumor) lengthscales. Different methods of modeling gold within cells are compared, from a contiguous volume of either pure gold or gold-tissue mixture to discrete GNPs in a hexagonal close-packed lattice. MC simulations with EGSnrc are performed to calculate n,cDEF for a cell with radius µm and nucleus µm considering 10to 370keV incident photons, gold concentrations from 4 to 24mgAu /gtissue , and three different GNP configurations within the cell: GNPs distributed around the surface of the nucleus (perinuclear) or GNPs packed into one (or four) endosome(s). Select simulations are extended to cells with different cell (and nucleus) sizes: 5µm (2, 3, and 4µm), 7.35µm (4 and 6µm), and 10µm (7, 8, and 9µm). n,cDEFs are sensitive to the method of modeling gold in the cell, with differences of up to 17% observed; the hexagonal lattice of GNPs is chosen (as the most realistic model) for all subsequent simulations. Across cell/nucleus radii, source energies, and gold concentrations, both nDEF and cDEF are highest for GNPs in the perinuclear configuration, compared with GNPs in one (or four) endosome(s). Across all simulations of the (rcell , rnuc ) = (7.35, 5)µm cell, nDEFs and cDEFs range from unity to 6.83 and 3.87, respectively. Including different cell sizes, nDEFs and cDEFs as high as 21.5 and 5.5, respectively, are observed. Both nDEF and cDEF are maximized at photon energies above the K- or L-edges ofgold by 10 to 20 keV. Considering 5000 unique simulation scenarios, this work comprehensively investigates many physics trends on DEFs at the cellular level, including demonstrating that cellular DEFs are sensitive to gold modeling approach, intracellular GNP configuration, cell/nucleus size, gold concentration, and incident source energy. These data should prove especially useful in research as well as treatment planning, allowing one to optimize or estimate DEF using not only GNP uptake, but also account for average tumor cell size, incident photon energy, and intracellular configuration of GNPs. PartII will expand the investigation, taking the PartI cell model and applying it in cm-scalephantoms.