Stem Cell Memory Research Articles

Humoral and cellular immune response in patients of liver cirrhosis and immunocompetent recipient of ChAdOx1nCoV-19 Vaccine (Covishield)

Despite the effectiveness of COVID-19 vaccination in reducing the severity of the disease, the demand for booster is increasing in vulnerable populations like elderly and immunocompromised individuals especially with each new wave of COVID-19 in different countries. There is limited data on the sustained immunity against COVID-19 in patients with liver cirrhosis. The study was aimed to compare the T cell and humoral immune response after 1 year of ChAdOx1nCoV-19 Vaccine in patients with liver cirrhosis and healthy health care workers (HCW). This was a prospective observational study including 36 HCW, 19 liver cirrhosis patients and 10 unvaccinated individuals. Anti-SARS-CoV-2S antibody, neutralizing antibody and memory T cell subsets were evaluated by ELISA and flow cytometry, respectively, in all three groups after 1 year of initial vaccination. Compared to HCW and unvaccinated individuals, liver cirrhosis patients had significantly depleted T cells, although CD4:CD8 + T cell ratio was normal. Both cirrhotic patients and HCW developed memory T cell subset [effector memory RA (P = 0.141, P < 0.001), effector memory (P < 0.001, P < 0.001), central memory (P < 0.001, P < 0.01), stem cell memory (P = 0.009, P = 0.08) and naïve (P < 0.001, P = 0.02)] compared to unvaccinated unexposed individuals of CD4 + T and CD8 + T, respectively. However, among HCW and cirrhotic group no difference was noted on central memory and stem cell memory cells on T cells. Patients with liver cirrhosis developed comparable memory T cells after vaccination which can evoke sustainable immune response on reinfection. Therefore, additional vaccine doses may not be necessary for cirrhosis patients.

Kinetics of Recovery of Naïve and Memory T Cells in Acute Leukemia Patients after Allogeneic Stem Cell Transplantation Depending on Different GVHD Prophylaxis Regimens.

Background: Memory T cells are a heterogeneous population of immune cells that provide adaptive immunity. Its full recovery seems essential for graft-versus-tumor reactions that provide an opportunity for biological cure in patients with acute leukemia. The use of mismatched or haploidentical donors has increased, which has become possible because of modifications in graft versus host disease (GVHD) prophylaxis. Materials and Methods: Sixty-five leukemia patients (acute myeloid leukemia - 40, acute lymphoblastic leukemia - 25), median age 33 (17-61) years, underwent allo-HSCT from 2016 to 2019 in the National Research Centre for Hematology. Patients were divided into three groups based on the impact of GVHD prophylaxis on T cell recovery: horse antithymocyte globulin (ATG)-based regimen (n=32), horse ATG combined with posttransplant cyclophosphamide (PT-Cy) (n=18), and ex vivo T cell depletion (n=15). Results: The early period after transplantation (before day +100) was characterized by significantly lower absolute numbers of T naïve, memory stem and T central memory cells in peripheral blood in patients after ATG+PT-Cy-regimen or ex vivo T cell depletion than after ATG-based prophylaxis (p<0.05). Moreover, strong depletion of naïve T and memory stem cells prevents the development of GVHD, and determining the absolute number of CD8+ naïve T and memory stem cells with a cutoff of 1.31 cells per microliter seems to be a perspective in assessing the risks of developing acute GVHD (p=0.008). The dynamics of T cell recovery showed the involvement of either circulating or bone marrow resident T effector cells shortly after allogeneic transplantation in all patients, but the use of manipulated grafts with ex vivo T cell depletion requires the involvement of naïve and memory stem cells. There was no significant effect of T cell recovery on leukemia relapse after allogeneic transplantation. Conclusion: These experimental outcomes contribute to providing the best understanding of immunological events that occur early after transplantation and help in the rational choice of GVHD prophylaxis in patients who will undergo allogeneic transplantation. Our study demonstrated the comparable immunological effects of posttransplant cyclophosphamide and ex vivo T cell depletion and immunological inefficiency of horse ATG for GVHD prevention.

A novel strategy of co-expressing CXCR5 and IL-7 enhances CAR-T cell effectiveness in osteosarcoma.

Solid tumors are characterized by a low blood supply, complex stromal architecture, and immunosuppressive milieu, which inhibit CAR-T cell entry and survival. CXCR5 has previously been employed to increase CAR-T cell infiltration into CXCL13+ cancers. On the other hand, IL-7 improves the survival and persistence of T cells inside a solid tumor milieu. We constructed a novel NKG2D-based CAR (C5/IL7-CAR) that co-expressed CXCR5 and IL-7. The human osteosarcoma cell lines U-2 OS, 143B, and Mg63 highly expressed MICA/B and CXCL13, thus presenting a perfect avenue for the present study. Novel CAR-T cells are superior in their activation, degranulation, and cytokine release competence, hence lysing more target cells than conventional CAR. Furthermore, CXCR5 and IL-7 co-expression decreased the expression of PD-1, TIM-3, and TIGIT and increased Bcl-2 expression. Novel CAR-T cells show enhanced proliferation and differentiation towards the stem cell memory T cell phenotype. C5/IL7-CAR-T cells outperformed conventional CAR-T in eradicating osteosarcoma in mouse models and displayed better survival. Additionally, CXCR5 and IL-7 co-expression enhanced CAR-T cell numbers, cytokine release, and survival in implanted tumor tissues compared to conventional CAR-T cells. Mechanistically, C5/IL7-CAR-T cells displayed enhanced STAT5 signaling. These findings highlight the potential of CXCR5 and IL-7 co-expression to improve CAR-T cell therapy efficacy against osteosarcoma.

Diversity of Memory CD8+ T Cells in Tumor-Draining Lymph Nodes from Patients with Bladder Cancer.

The role of memory T cells in orchestrating memory responses to previously known tumor antigens is well documented. The aim of this study was to assess the frequency of different memory T cell subsets in tumor-draining lymph nodes of patients with bladder cancer (BC) and their prognostic significance. Mononuclear cells were isolated from 50 tumor-draining lymph nodes of untreated patients with BC and stained with antibodies against the markers CD8, CD95, CD45RO and CCR7. Data were collected using the FACSCalibur flow cytometer and analyzed using FlowJo software. Among the CD8+ cytotoxic lymphocytes, the frequency of different subsets was determined including total memory cells (CD8+CD45RO+CD95+), T central memory (TCM: CD8+CCR7+CD45RO+CD95+), T effector memory (TEM: CD8+CCR7-CD45RO+CD95+), T stem cell memory (TSCM: CD8+CCR7+CD45RO-CD95+) and naïve T cells (CD8+CCR7+CD45RO-CD95-). The analysis revealed that on average 49.32±20.15 (between 1.62% and 87.20%) percent of CD8+ lymphocytes in draining lymph nodes of BC had a memory phenotype. TCM cells showed the highest frequency (34.71±17.04), while TSCM cells (7.51±8.53) demonstrated the lowest. The total frequency of memory cells tended to be higher in patients with tumor invasion to muscle layer (P=0.052) and stage III (P=0.042) than in patients without invasion and stage I. The TCM subset was more frequent in patients with necrotic tumors than in patients without necrosis (P=0.048). TSCM significantly increased in patients with N2 compared to N0 (P=0.042). Conversely, the ratio of TSCM cells to total memory cells was higher in lower tumor stages (P=0.059), tumors without muscle invasion (P=0.026) and low T grouping (P=0.043). Overall the data indicated an increase in the frequency of memory T cells and their TSCM and TCM cells with tumor progression. In contrast, the ratio of TSCM to total memory cells was higher in less advanced tumors. These results suggest that the immune system is frequently exposed to tumor antigens and strives to create a memory T cell reservoir, but this is suppressed by inhibitory factors provided by the tumor. These findings emphasize the importance of understanding the dynamic interplay between memory T cell subsets and BC progression.

Expansion and Precise CRISPR-Cas9 Gene Repair of Autologous T-Memory Stem Cells from Patients with T-Cell Immunodeficiencies.

The adoptive transfer of autologous, long-lived, gene-repaired T cells is a promising way to treat inherited T-cell immunodeficiencies. However, adoptive T-cell therapies require a large number of T cells to be manipulated and infused back into the patient. This poses a challenge in primary immunodeficiencies that manifest early in childhood and where only small volumes of blood samples may be available. Our protocol describes the ex vivo expansion of potentially long-lived human T memory stem cells (TSCM), starting from a limited number of peripheral blood mononuclear cells (PBMCs). Using the perforin gene as an example, we provide detailed instructions for precise gene repair of human T cells and the expansion of TSCM. The efficiency of precise gene repair can be increased by suppressing unintended non-homologous end-joining (NHEJ) events. Our protocol yields edited T-cell populations that are ready for phenotyping, genome-wide off-target analysis, and functional characterization. Key features • Expansion and enrichment of TSCM from PBMCs using IL-7 and IL-15. • Phenotyping of TSCM. • Design of "off-the-shelf" gene-repair strategies based on knock-in of a single exon or complete cDNA and design of effective guide RNAs and DNA donor templates. • High-efficiency gene targeting using CRISPR-Cas9, recombinant adeno-associated virus serotype 6 (rAAV6), and a selective small molecule inhibitor of DNA-dependent protein kinase (DNA-PK).

Insights into the Role of Histone Methylation in Brain Aging and Potential Therapeutic Interventions.

Epigenetic mechanisms play a primary role in the cellular damage associated with brain aging. Histone posttranslational modifications represent intrinsic molecular alterations essential for proper physiological functioning, while divergent expression and activity have been detected in several aspects of brain aging. Aberrant histone methylation has been involved in neural stem cell (NSC) quiescence, microglial deficits, inflammatory processes, memory impairment, cognitive decline, neurodegenerative diseases, and schizophrenia. Herein, we provide an overview of recent studies on epigenetic regulation of brain tissue aging, mainly focusing on the role of histone methylation in different cellular and functional aspects of the aging process. Emerging targeting strategies of histone methylation are further explored, including neuroprotective drugs, natural compounds, and lifestyle modifications with therapeutic potential towards the aging process of the brain.

Harnessing T cell exhaustion and trogocytosis to isolate patient-derived tumor-specific TCR.

To study and then harness the tumor-specific T cell dynamics after allogeneic hematopoietic stem cell transplant, we typed the frequency, phenotype, and function of lymphocytes directed against tumor-associated antigens (TAAs) in 39 consecutive transplanted patients, for 1 year after transplant. We showed that TAA-specific T cells circulated in 90% of patients but display a limited effector function associated to an exhaustion phenotype, particularly in the subgroup of patients deemed to relapse, where exhausted stem cell memory T cells accumulated. Accordingly, cancer-specific cytolytic functions were relevant only when the TAA-specific T cell receptors (TCRs) were transferred into healthy, genome-edited T cells. We then exploited trogocytosis and ligandome-on-chip technology to unveil the specificities of tumor-specific TCRs retrieved from the exhausted T cell pool. Overall, we showed that harnessing circulating TAA-specific and exhausted T cells allow to isolate TCRs against TAAs and previously not described acute myeloid leukemia antigens, potentially relevant for T cell-based cancer immunotherapy.

CRISPR Activation Screen to Optimize Chimeric Antigen Receptor (CAR) T Cell Immunophenotype

Background Adoptive cellular therapies (ACT) offer potential cures for a wide range of human diseases. Immune cells genetically engineered with chimeric antigen receptors (CAR) have a clear role in B cell malignancies, and broader applications to other malignancies are under investigation. Significant obstacles to the long-term durability and potency of these therapies remain. As CAR T-cells remain persistently activated and exposed to the tumor microenvironment, they may become functionally impaired. This exhaustion can result in reduced production of cytokines, cytotoxic molecules, and other factors necessary for effective tumor cell killing. While CAR T-cell exhaustion remains a challenge, ongoing research and clinical trials aim to further understand the underlying mechanisms and develop strategies to overcome this limitation. Clinical studies have demonstrated that sustained in vivo persistence and function of CAR T-cells is correlated with infusion product phenotype. Persistence is associated with a less differentiated, memory-like phenotype. Naive (T N), Central Memory (T CM) and Stem Cell Memory (T SCM) are related to good proliferative potential and durability. CRISPR technologies offer powerful, functional genomic screens that permit the inference and modification of gene regulatory networks to optimize CAR T-cell persistence and function. Genome-wide CRISPR knock out screens have identified the CD2-CD58 axis as critical to T-cell potency and the knock-out of the mediator kinase module as beneficial to potency. Here we report on in vitro CRISPR activation screening of human CAR T-cells for gene expression that enhances T cell phenotypes associated with durable function. Method We designed 9,983 single guide RNA (sgRNA) to target the transcription start sites (TSS) of all known human transcription factors as well as a select set of genes with critical functions in T-cells. The vast majority of genes was targeted with 5 different sgRNA. To this we added a pool of 1,000 non-targeting sgRNA. We lentivirally transduced the sgRNA in tandem to a second-generation anti-CD20 CAR into primary T cells from healthy human donors. In a second lentiviral transduction we integrated catalytically dead Cas9 (dCas9) fused to the strong transcription activator, VP64 and in tandem to a puromycin resistance gene. After puromycin selection, we cultured the CRISPR activated CAR T-cells either alone or together with Raji or K562 cells for nine days before immunophenotypically sorting the CAR T-cells into CD8 + Naive, CD8 + Transitional Memory, CD8 + Central Memory, CD8 + Stem Cell Memory, CD8 + Transitional Effector, CD8 + Effector Memory, CD4 + Naive and CD4 + Memory T-cell populations. We extracted DNA from the sorted cells and prepared sequencing libraries targeting the sgRNA. We carried out two-way ANOVA analysis on the experimental conditions (trivial, Raji or K562 co-culture versus addition of CD2/CD28 activation beads) and T-cell subset to determine which genes targeted by CRISPRa resulted in favorable phenotypes. Result Across the different culture conditions, sgRNA activating IL23A and KLF4 were enriched in Transitional Memory versus Stem Cell Memory T-cells. Across the different CAR T-cell immunophenotypes, sgRNA targeting the TGFß (including KLF10), TNFα (including NFATC4) and FOXO signaling pathways were enriched in CAR T-cells cultured alone compared to CAR T-cells activated with CD2/CD28. Conclusion In this study we have investigated genes whose activation results in CAR T-cell immunophenotypes that are associated with more potent and durable CAR T-cell therapies. Our experiment suggests that the activation of genes, such as IL23 and KLF4, enriches for a Transitional Memory over Stem Cell Memory phenotype in a manner that is largely independent of environment (culture conditions). The activation of other genes, such as KLF10 and NFATC4, results in enrichment of CAR T-cells across all T-cell subsets, but in a manner that is dependent on environment. Gene expression requires coordinated control of gene-proximal and -distal cis-regulatory elements (CREs), whose state varies as cells differentiate or respond to environmental signals. Abrogating or adding CRE may result in more durable and potent adoptive cellular therapies independent of environment.

Role of Cell Therapy Product Characteristics in Patient Response to TAK-940 (CD19(T2)28z1xx Chimeric Antigen Receptor (CAR) T): Biomarker Analyses from a Phase I Study in Adult Patients with Relapsed or Refractory B-Cell Malignancies

TAK-940 (CD19(T2)28z1XX Chimeric Antigen Receptor (CAR) T) is an autologous CD19 CAR T cell therapy utilizing an optimized 1XX signaling domain substituting for the natural CD3ζ immunoreceptor tyrosine-based activation motifs. This 1XX design extends the functional persistence of CAR T cells with increased potency in mice (Feucht J et al. Nat Med 2019). TAK-940 is being evaluated in a Ph1 trial in patients with Relapsed or Refractory B-cell malignancies (NCT04464200) with demonstrated early signals of clinical efficacy (Park J et al. ASH 2022). The present study aims to provide a better understanding of the clinical performance of CD19(T2)28z1XX CAR T cells by in depth characterization of the cellular products and patient samples from this Ph1 trial. A total of 28 subjects were enrolled and treated in the dose escalation and expansion cohorts of the Ph1 trial. Responses were observed across all dose levels (25x10 6 to 200x10 6 CAR T cells), achieving ORR 82%, CR 71%. Immunophenotyping by flow cytometry was conducted on patient apheresis material, un-transduced cells, transduced cellular drug product and peripheral blood samples from infused subjects. Circulating cytokine profiling was performed on patient serum samples collected pre and post treatment with TAK-940. In the transduced cellular drug products, CAR+ and CAR- CD8+ central memory and transitional memory T cells were more abundant than naïve, stem cell memory or terminal effector populations. This elevation in central and transitional memory T cells seen in the drug product was not observed in the patient apheresis material, indicating that this effect is likely driven by the production process. CD57 and TIM3 expression in both CD4+ and CD8+ CAR+ T cells in the drug product showed a positive association with clinical response, while CTLA4 or LAG3 expression independently was negatively associated with response. The drug product also contained low numbers of dysregulated/exhausted T cells, with no observable difference between responders and non-responders. Interestingly, in the starting apheresis material, increased expression of TIM3 as well as decreased expression of either LAG3 or KLRG1 in CD8 T cells, independently associated with responses. Current analysis is based on associations with complete response. Additional analyses to be reported will include associations with ORR and durability of responses. These findings suggest potential utility of these T cell markers in predicting patient response and warrant evaluation in a larger population. Although CD57 expression has been associated with a senescent phenotype, we hypothesize that the observed increase in CD57 expression in CD4+ and CD8+ CAR+ T cells in the product may indicate higher cytotoxic potential of those cells. Increase in CD57+ CAR+ T cell subsets has been previously associated with durable CRs in large B cell lymphoma patients treated with axi-cel (Good Z et al. Nat Med 2022). Additional analyses to further characterize the CD57+ cell populations and establish their role in clinical responses to CAR T cell therapy with the 1XX CAR design are in progress. The increased expression of CD57 and the activation marker TIM3, and decreased expression of inhibitory receptors CTLA4 and LAG3 in the drug product from responders, together with the robust clinical responses observed with doses as low as 25x10 6 CAR T cells, suggest that the product consists of highly potent T cells, in accordance with the preclinical results obtained with the 1XX CAR. Low levels of circulating cytokines were observed following treatment with TAK-940, including IFNg, IL-6, IL-10, indicating a safe and tolerable CAR T product consistent with the observed clinical safety profile of TAK-940. Correlative analysis of T cell immunophenotypes observed in the product to clinical parameters including durability of responses, metabolic tumor volume/tumor burden, and induction of cytokines and chemokines is ongoing and will be reported. This will further contribute to our understanding of CD19 CAR T cells endowed with a 1XX signaling domain and potentially support the identification of product and/or apheresis biomarkers that may predict patient response.

Mitochondrial Isocitrate Dehydrogenase Inhibition Enhances CAR T-Cell Function By Restraining Antioxidant Metabolism and Histone Acetylation

Introduction: The efficacy of chimeric antigen receptor (CAR) T-cell therapy is hampered by relapse in hematologic malignancies and by hyporesponsiveness in solid tumors. Mitochondria are vital for the regulation of memory T-cell formation or exhaustion. A mitochondria-related compound screening was performed and we found that the FDA-approved isocitrate dehydrogenase 2 (IDH2) inhibitor enasidenib enhances long-lived memory CAR T-cell formation, and improves tumor clearance in vivo. IDH2 as the key component of the tricarboxylic acid (TCA) cycle, we hypothesized that IDH2 inhibition might promote CAR T-cell persistence by reprogramming the metabolism. The study aims to systematically evaluate the role and the mechanism of IDH2 inhibition on CAR T cells, and explore the application of enasidenib in CAR T-cell therapy. Methods and Results: To identify mitochondrial components that affect the long-term efficacy of CAR T-cells, we performed an in vitro screening using a mitochondria-related compound library based on the enrichment of the CD62L + CAR T-cell subset, which mainly contains T memory stem cells (T SCM) and central memory T cells (T CM). Among several candidate compounds that promoted memory CAR T-cell formation, enasidenib (ENA), an inhibitor of both wild-type and mutant IDH2 enzymes, was the most effective one. The proportion of CD62L has increased by over 20%. To determine the effect of ENA on exhaustion induced by tonic CAR signaling and tumor antigen stimulation, we measured the expression of inhibitory receptors PD-1, TIM-3, and LAG-3 and the level of apoptosis in freshly expanded CAR T cells and B-ALL encountered CAR T cells. CAR T cells treated with ENA exhibited reduced surface levels of those inhibitory receptors. We measured the production of granzyme B and IFNγ, and tumor rechallenge assay in CAR T cells after ENA treatment to assess the effector function, ENA treated CAR T cells exerted enhanced and sustained cytotoxicity in a tumor rechallenge assay at an extreme E:T ratio of 1:10. These results were observed not only in CD19-41BBζ CAR T cells, but also in CD19-28ζ and GD2-28ζ CAR T cells. CAR T cells expanded in the presence of ENA significantly prolonged survival of recipients after Nalm-6 infusion (p &amp;lt; 0.0001), extended in vivo ENA treatment further enhanced CAR T-cell expansion and tumor suppression. The median survival was extended from 45 to 67.5 days. Consistent with the effect of ENA treatment, IDH2 knockdown increased the proportions of the T scm/T n and T cm subsets, and alleviated CAR T-cell exhaustion. IDH2-knockdown CAR T cells had sustained cytotoxicity and higher CD62L expression after rounds of killing B-ALL cells in vitro. To systematically explore the effects of IDH2 inhibition on the metabolism of CAR T cells, relative metabolite amounts was measured using mass spectrometry (MS). IDH2-inhibited CAR T cells exhibited a substantial reduction of metabolites in the TCA cycle, including succinate, fumarate, and malate, and in the glycolysis pathway, including lactate, phosphoenolpyruvate. Mechanistically, IDH2 inhibition reprogram central carbon metabolism of CAR T cells by redirects glucose carbon utilization from glycolysis to the pentose phosphate pathway. In addition, IDH2 limits cytosolic acetyl-CoA level to prevent histone acetylation that promotes memory cell formation. Significance: Our study indicates that metabolic intervention in CAR T cells with the FDA-approved IDH2 inhibitor enasidenib can advance current treatment, with better tumor eradication and CAR T-cell persistence.

Development of a Same-Day Closed CAR-T Cell Manufacturing Process to Generate Lentivector Loaded Lymphocytes Ready for Subcutaneous Injection

Background: Adoptive T-cell therapies with CD19 chimeric antigen receptors (CAR) are now the standard of care for refractory/recurrent hematologic B-cell malignancies. However, challenges remain in delivering a more fit cell product that can be manufactured in a more timely and cost-effective manner. To address these challenges, a rapid same-day fully closed manufacturing process was developed to generate a more fit product for subcutaneous administration and to enable an efficient transition from early-phase clinical trials to commercial-scale production. Methods: A lentivector (LV) was designed with the capability of directly targeting and activating T cells simultaneously to enable manufacturing without prior T-cell selection. Experiments were performed to understand the kinetics of direct lentivector-induced T-cell activation, internalization, reverse transcription, and vector genome integration. After establishing critical process parameters for efficient loading of T cells with LVs, a closed manufacturing process was developed from peripheral blood mononuclear cells (PBMC) starting material derived from whole blood or apheresis. PBMCs were isolated in an automated closed system and exposed to LV for a four-hour incubation period. Cells were washed in an automated closed system to remove residual LV, formulated, and vialed with release testing for identity, potency, purity, and safety. Three full-scale engineering lots were manufactured from donor-derived apheresis in addition to three small-scale analytical lots with patient-derived PBMC. Results: For targeted gene delivery into T cells within PBMC, the CD19 CAR LV was designed to contain a CD3 targeting motif in its envelope to enable T-cell binding, activation, and viral entry into CD3+ T cells. The CD3-directed LV encoding a CD19-CAR and a novel synthetic driver co-stimulatory domain was manufactured in a chemically defined 25L clinical scale suspension-based process and vialed to a target unit potency for ease of cell loading. The purified CD3-directed LV is capable of inducing T-cell receptor internalization and activation through specific bioassays. In surrogate analytical cultures, following LV exposure and automated washing, viral entry into T cells was complete. Analytical cultures demonstrate the ability to successfully generate CAR+ cells with a unique CD3+CD56+CD45RA+CCR7+ T memory-stem cell with NK-like features and a vector copy number of less than 5 vector copies/transduced cell (1.95 +/- 0.58). In preparation for clinical investigation, full-scale runs were performed in GMP facilities with qualified methods for release testing against identity, potency, purity, and safety. Small-scale analytical processes utilizing B-cell lymphoma patient-derived PBMC from whole blood were evaluated for differences relative to donor-derived material. Conclusions: To address current challenges in the manufacturing of autologous cell products, a same-day closed manufacturing process was developed to produce cells with enhanced fitness. Qualification of this novel design included 1) completion of the production of the CD3-directed LV encoding a CD19 CAR with a novel synthetic co-stimulatory driver element at the 25L scale; 2) completion of three full-scale closed engineering runs along with the qualification of QC release testing for identity, potency, purity, and safety and 3) comparability studies utilizing patient-derived PBMC.

New Optimized Academic Anti-BCMA CAR (CARTemis-1): How Does the Manufacturing Process Impact CAR-T Cell Features?

Introduction CAR therapy targeting BCMA (B-cell maturation antigen) is under research as treatment for multiple myeloma (MM). However, given the lack of a plateau in most studies, it becomes imperative to pursue more effective alternatives. BCMA downmodulation, the presence of soluble BCMA or the immune dysfunction observed in MM are some limitations that may affect efficacy. Next-generation CAR-T cells are being designed exploring new approaches to overcome restrictions to CAR-T efficacy in MM. We present a novel optimized anti-BCMA CAR (CARTemis-1) and its translation to the clinic. We have specifically focused on the study of the dynamics of the immunophenotype of CARTemis-1 along the manufacturing process, analyzing how these variables might impact the quality of the final product and influence the release decision. Materials and methods CARTemis-1 was evaluated in preclinical in vitro and in vivo models. CARTemis-1 generation from three healthy donors was validated under GMP-conditions in CliniMACS Prodigy with IL-7/IL-15. Flow cytometry was performed to characterize the immunophenotype along the manufacturing process. Quality controls of the final CARTemis-1 product were performed according to the criteria of the Spanish Medicine Agency. Stability of the final cryopreserved CARTemis-1 cell product was also analyzed. Results CARTemis-1 is a second generation anti-BCMA CAR that co-expresses the truncated epidermal growth factor receptor (EGFRt) as a transduction marker and suicide gene. Two different versions of CARTemis-1 were synthesized with different spacer regions (short vs long) with the long version showing significantly greater anti-myeloma function in vitro compared to the short version (p&amp;lt;0.001) (Figure 1A). During the preclinical validation, CARTemis-1 demonstrated potent anti-tumor capacity in vitro and in vivo, showing no susceptibility to the presence of soluble BCMA. Expansion with IL-2 or IL-7/IL-15 was compared (n=8), obtaining increased proliferation (mean 1.59x10 7 vs 3.07x10 7, respectively, p=0.002), increased stem cell memory and naïve population (mean 37.48% vs 43.95%, respectively, p=0.0052), and less PD1+LAG3+ levels (1.38% vs 0.55%, respectively, p=0.0338) with IL-7/IL-15. The generation of CARTemis-1 was validated under GMP-conditions closely monitoring the immunophenotype dynamics from leukapheresis to the final product. Through comprehensive evaluations, the optimal time-point for product release to achieve the best-fitness product was &amp;gt;6 (specifically, day 8-10) with reduced levels of exhaustion markers (Figure 1B) maintaining hallmarks of T cell activation and anti-tumor potency. In vivo biodistribution of CARTemis-1 cells was studied in mice models without myeloma cells, finding preserved tissue structure without relevant morphological alterations in brain, cerebellum, intestine, lung, endometrium, ovary, testicle, spleen, and liver. We performed graft-versus-host disease (GVHD) in vivo mice models and found no increase in GVHD incidence following CARTemis-1 inoculation. Additionally, we assessed the cryopreserved product stability, and it met all specifications and retained its functionality for up to 12 months after cryopreservation. With this positive outcome and having obtained the approval of the Spanish Medicine Agency, we plan to proceed with a phase I/II clinical trial using CARTemis-1 for patients with multiple myeloma who have experienced relapse following allogeneic transplantation (EudraCT 2021-001955-15). Conclusions CARTemis-1 has been rationally designed to increase anti-tumor efficacy and overcomes sBCMA inhibition. To our knowledge, this is the first study analyzing the impact of the manufacturing process in the dynamics of the CAR-T immunophenotype highlighting the importance of the immunophenotypic characterization of CAR-T cells throughout the manufacturing process to define the optimal cell culture protocol and expansion time to increase CAR-T cell product fitness.

Restoration of LAT Signaling in CD33-Directed CAR T Cells Improves Potency and Persistence Against Acute Myeloid Leukemia

Despite the development of novel therapies, the majority of patients with acute myeloid leukemia (AML) will not achieve long-term remissions. Patients with relapsed/refractory (r/r) AML have particularly poor outcomes, as conventional chemotherapy regimens are often unable to elicit long-term remissions. Allogeneic hematopoietic stem cell transplant (HSCT) is potentially curative in eligible patients, however, bridging therapies which can induce deep remissions prior to HSCT are needed. Second generation chimeric antigen receptor (CAR) T cells targeting lineage-restricted antigens, such as CD19, have induced remissions in up to 80 - 90% of pediatric patients with r/r B-lineage acute lymphoblastic leukemia (ALL), however, post-CAR relapses are common and only ~50% of treated patients achieve durable remissions. Similar strategies targeting myeloid-restricted antigens with 2 nd generation CAR T cells in AML have not replicated the success seen in ALL. As clinical experience has highlighted the impact of CAR T cell potency, persistence, and antigen-sensitivity to long-term efficacy in ALL, strategies to boost these properties in myeloid-directed CAR T cells are necessary to extend the clinical benefit of this therapy to patients with AML. Utilizing a global phosphoproteomics screen, we recently identified that inefficient phosphorylation of the Linker for activation of T cells (LAT) and the subsequent decrease in the downstream signaling pathways results in suboptimal CAR T cell activation. To overcome inefficient LAT activation, we developed a novel bicistronic CAR platform, consisting of a 2 nd generation CAR along with an Adjunctive LAT-Activating CAR (ALA-CART) which improved sensitivity to low-antigen leukemia cells, increased in vivo potency, and enhanced persistence in xenograft models of ALL. We hypothesized that the restoration of LAT signaling via the ALA-CART platform would also improve potency and persistence in pre-clinical models of AML. We found that while CD33 ALA-CART cells responded to CD33+ AML cells in vitro, they were less cytotoxic and produced lower levels of cytokines than CD33-28z or CD33-BBz 2 ndgeneration CAR T cells. However, CD33 ALA-CART cells demonstrated increased potency in xenograft models, eradicating AML cells at much lower doses than CD33-28z and CD33-BBz CAR T cells (Fig. 1A, p&amp;lt;0.0001). This in vivo potency correlated with an enrichment of T stem cell memory cells in the ALA-CART product, resulting in increased persistence of CD33 ALA-CART cells after leukemia clearance (Fig. 1B, p=0.0139). The ALA-CART platform was associated with increased mitochondrial mass and function, suggesting that the increased signaling through LAT results in an enhanced capacity for oxidative phosphorylation, potentially contributing to the increased persistence of CD33 ALA-CART cells in vivo. Thus, the ALA-CART platform, designed to restore LAT signaling in CAR T cells, demonstrates that CAR molecules can be rationally designed to enhance both in vivo potency and persistence, and represents a promising strategy for the development for more effective AML-directed CAR T cells.

Immune Graft Composition Is Important for Deepening Response and Outcome after Autologous Stem Cell Transplantation in Patients with Multiple Myeloma Who Do Not Receive Maintenance Therapy

Maintenance therapy with Lenalidomide or proteasome inhibitors after autologous stem cell transplantation (ASCT) in multiple myeloma (MM) is an important component of first line treatment, resulting in deepening response (DR), converting PR to VGPR/CR or VGPR to CR in most patients and, in significantly longer PFS and OS. Of note, randomised clinical trials and real-world data confirm that DR also occurs in about 30% of patients receiving placebo or no maintenance at all. We analysed 90 patients with MM treated with first line ASCT in Hammersmith Hospital between 2017 - 2020, who did not receive maintenance therapy. We observed DR in 20 patients (22%) in the first two years. Patients achieving a DR had significantly higher PFS at 36 months post ASCT (89.4% v 41.1%, p=0.001 - Figure 1A). Baseline and ASCT characteristics, including high-risk cytogenetics, ISS, induction therapy and conditioning were comparable in DR and non-DR patients, however the use of the CXCR4 antagonist Plerixafor plus G-CSF vs G-CSF alone in the mobilisation regimen was associated with a higher probability for DR in the first 24 months post ASCT (58.3% v 23.2%, p=0.011 - Figure 1B). We suggest that improvement in depth of response after ASCT without pharmacological intervention indicates an immune anti-MM effect, underpinned by the immune composition of autologous grafts. We used 14-colour flow cytometry to analyse the immune cell frequencies in graft in 59 of the above patients, including 17 patients mobilised with Plerixafor plus G-CSF and 42 with G-CSF alone. The cell dose of each cell population was calculated from the relative frequency of CD34 cells in the graft and the CD34 cell dose infused in ASCT. In line with previous reports, the use of Plerixafor results in higher frequency and cell dose (in x10^6 cells/kg) of T cells in the graft (70.6% v 61.3%, p=0.015 and 306.2 v 161.4, p=0.007), driven mostly by a higher frequency (67.8% v 48.7%, p= 0.001) and dose (224 v 78.2, p=0.001) of central memory (CM) CD4 T cells. On the contrary, frequency and cell dose of effector memory (EM) CD4 T cells in Plerixafor plus G-CSF mobilised grafts were lower (6.7% v 21.5%, p=0.006 and 15.5 v 34.7, p=0.063). Apart from mobilisation regimen, univariate analysis showed a higher probability of achieving DR in grafts with high frequency of CD4 CM T cells (p= 0.012), NK cells (p=0.018 , Figure 1C), CD8 CM cells (p=0.06), higher dose of CD8 memory stem cells (p=0.033), lower frequency and dose of CD4 EM (p= 0.017 and p=0.007) and a lower dose of CD4 terminal effectors (TE) (p=0.039). In multivariate analysis, only a high NK cell frequency (p=0.011) and low CD4 TE cell dose (p=0.01), driven by their PD1 expression, predicted a higher probability for DR. Tregs frequency and cell dose were not affected from mobilisation regimen. However, Tregs dose was the stronger predictor of PFS at 36 months (75% with dose &amp;lt;5.06 x10^6/kg v 34.5% with dose &amp;gt;5.06 x10^6/kg, p= 0.001). Similarly, lower cell doses of CD4 EM (p=0.012) and frequency of CD4 TE (p=0.034) predicted a higher probability of PFS, but only Tregs cell dose retained significance in multivariate analysis (Figure 1D, p=0.001). In conclusion, spontaneous DR occurs in the first 2 years post ASCT in the absence of maintenance therapy and is associated with better PFS. CD4 memory subsets and NK cell graft composition appears significant for a potential immunological effect in DR. Additionally, a lower cell dose of immunosuppressive Tregs appears more important for sustaining long-term remission post ASCT.

Comprehensive immune profiling of SARS-CoV-2 infected kidney transplant patients.

The immune responses of kidney transplant recipients against SARS-CoV-2 remains under studied. In this prospective pilot study, we performed comprehensive immune profiling using cellular, proteomic, and serologic assays on a cohort of 9 kidney transplant recipients and 12 non-transplant individuals diagnosed with COVID-19. Our data show that in addition to having reduced SARS-CoV-2 specific antibody levels, kidney transplant recipients exhibited significant cellular differences including a decrease in naïve-but increase in effector T cells, a high number of CD28+ CD4 effector memory T cells, and increased CD8 T memory stem cells compared with non-transplant patients. Furthermore, transplant patients had lower concentrations of serum cytokine MIP-1β as well as a less diverse T cell receptor repertoire. Overall, our results show that compared to non-transplant patients, kidney transplant recipients with SARS-CoV-2 infection exhibit an immunophenotype that is reminiscent of the immune signature observed in patients with severe COVID-19.

Immune Control in Repeated Babesia microti Infection in a Patient With B-Cell Deficiency.

The immunology of human babesiosis is poorly investigated. We present a comprehensive investigation of a 75-year-old man with B-cell deficiency who experienced 3 episodes of babesiosis over a 6-year period. Slowly evolving clinical immunity was observed, as evidenced by milder clinical symptoms and lower peak parasite burden after each subsequent babesiosis episode. The patient exhibited several striking immunologic findings. First, the patient had exceptionally high Babesia microti-specific antibodies despite very few circulating B cells, which predominantly coexpressed CD27 (memory marker) and CD95 (death receptor). Second, we demonstrated the presence of long-lasting NK cells and expansion of T memory stem cells. Third, levels of the IP-10 cytokine directly correlated with parasite burden. These results raise fundamental questions on the priming, maintenance, and location of a B-cell population that produces high antibody levels in the face of severe B-cell deficiency. Our results should invoke interest among researchers to study the immunology and pathogenesis of human babesiosis.

Memory T Cells Discrepancies in COVID-19 Patients.

The immune response implicated in Coronavirus disease 2019 (COVID-19) pathogenesis remains to be fully understood. The present study aimed to clarify the alterations in CD4+ and CD8+ memory T cells' compartments in SARS-CoV-2-infected patients, with an emphasis on various comorbidities affecting COVID-19 patients. Peripheral blood samples were collected from 35 COVID-19 patients, 16 recovered individuals, and 25 healthy controls, and analyzed using flow cytometry. Significant alterations were detected in the percentage of CD8+ T cells and effector memory-expressing CD45RA CD8+ T cells (TEMRA) in COVID-19 patients compared to healthy controls. Interestingly, altered percentages of CD4+ T cells, CD8+ T cells, T effector (TEff), T naïve cells (TNs), T central memory (TCM), T effector memory (TEM), T stem cell memory (TSCM), and TEMRA T cells were significantly associated with the disease severity. Male patients had more CD8+ TSCMs and CD4+ TNs cells, while female patients had a significantly higher percentage of effector CD8+CD45RA+ T cells. Moreover, altered percentages of CD8+ TNs and memory CD8+CD45RO+ T cells were detected in diabetic and non-diabetic COVID-19 patients, respectively. In summary, this study identified alterations in memory T cells among COVID-19 patients, revealing a sex bias in the percentage of memory T cells. Moreover, COVID-19 severity and comorbidities have been linked to specific subsets of T memory cells which could be used as therapeutic, diagnostic, and protective targets for severe COVID-19.

Mitochondrial Reprogramming By Bcl-2 Inhibitor Venetoclax Enhances αCD19 CAR-T Cell Fitness and Anti-Tumor Efficacy

Introduction Patients undergoing anti-CD19 chimeric antigen receptor T cell (CAR T) therapy for the treatment of aggressive B-cell non-Hodgkin lymphomas (B-NHL) can expect a complete response rate of merely 43-68%. Insubstantial clinical responses to CAR T therapy and a small window of survival for patients facing relapsed/refractory (R/R) disease highlights the critical need for improvement of CAR T therapy in aggressive B-NHL cases. Recent findings suggest that enriching for a CD8 + T Stem Cell Memory (T SCM) population within adoptive cell therapies translates to enhanced antitumor function and persistence in vivo, and thus produces favorable clinical outcomes. T SCM cells display heightened self-renewal, low mitochondrial membrane potential (∆Ψm), and are metabolically dependent upon oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO). Selection of CD8 + T SCM subsets is therefore necessary to ensure a robust antitumor response. Previously, we revealed synergy between the Bcl-2 inhibitor, Venetoclax, and CAR T cells in the context of B-NHL. Venetoclax exposure increased CD8 + T SCM cell frequency, decreased ∆Ψm, reduced T Regulatory (T REG) cell frequency, and bolstered effector cytokine production. Though the role of Bcl-2 inhibition within B-NHLs is well characterized (FDA-approved for frontline and relapsed/refractory chronic lymphocytic leukemia [CLL] treatment), further investigation regarding the impact of Venetoclax on the patient T cell compartment is needed. In this study, we demonstrate a role for Venetoclax as a qualitative enhancer of bulk CAR T cell product using both healthy-donor and B-NHL patient-derived T cells, and implicate modulation of mitochondrial pathways as a likely mechanism of action. Methods Whole peripheral blood mononuclear cells were collected from consenting healthy human donors or CLL patients undergoing ramp-up with Venetoclax (BDR 164122) at Roswell Park Comprehensive Cancer Center, and activated with αCD3/αCD28. CLL samples were collected at baseline, 1 day, 7 days, and 1-month timepoints. Transduction was performed with lentiviral vectors encoding CD28 or 4-1BB-based α-CD19 CAR constructs 24 hours post-activation. Transduced cells were cultured in RPMI with hIL-2 and hIL-7. In vivo studies were performed on 7-week-old SCID mice using Raji-Luc, Rec-1, and U2932. Venetoclax was given daily [100mg/kg] via oral gavage. CAR T cells were delivered by single infusion via tail-vein injection at a dose of either 1x10 6 or 3x10 6 cells total.Mice were imaged via IVIS spectrum. Western blot was performed on stimulated untransduced T cells or CAR T cells exposed to Venetoclax. Total protein was quantified, and membranes were probed for apoptotic and mitochondrial protein expression. Patient sample immunophenotyping was performed on CLL patient samples using panels interrogating memory differentiation, exhaustion, and T REG populations. Results Consistent with our hypothesis, a significant increase in mitochondrial proteins associated with FAO and OXPHOS - ACC, PDH, and AMPKα - was observed in exposed CAR T cells, as well increased expression of transcription factor FOXO1 - an essential driver of CD8 + memory differentiation. Voltage-dependent anion channel (VDAC) expression increased, suggesting heightened efflux of respiratory substrates into the tricarboxylic acid cycle. Hexokinase-I, a critical glycolytic enzyme, was significantly downregulated. We next investigated our combination therapy in vivo and observed extended survival outcome by 15 days. Further in vivo studies investigating preclinical models of mantle cell lymphoma and activated B-cell diffuse large B-cell lymphoma are currently ongoing. Within our CLL patient samples, 24-hour and 7-day timepoints reflected a favorable population increase in CD8 + T SCM cells as compared to baseline samples, and marginally restored CD8 + T cell frequencies. Conclusion Metabolic properties guide T cell memory differentiation and fitness. There exists potential to manipulate this mechanism and select the most metabolically fit T cell population for adoptive cell therapies. In addition to answering fundamental questions of CAR T cell biology, the combination of Venetoclax and CAR T cell therapy may provide a solution to the observed clinical gap, in which CAR T therapy as a single agent does not afford favorable response rates in the context of aggressive and R/R B-NHLs.

An Assessment of Marrow-Infiltrating T Cells in Early Relapsed Hematologic Cancer Patients Post Allogeneic Hematopoietic Stem Cell Transplantation

Background: Relapse is the major treatment failure of allogeneic hematopoietic stem cell transplantation (HSCT) in patients with hematologic malignancies. Especially, early relapse had seriously bad prognosis. And tumor-infiltrating T cells play an important role in disease progression and recurrence. However, there is lack of data about the marrow-infiltrating T cell of early relapsed patients compared to those with complete remission (CR) at an early timepoint after HSCT. Therefore, we analyzed the differentiation stage and expression of inhibitory receptors of the marrow-infiltrating T cell in the early stages of allogeneic HSCT. Method: A total of 40 samples from patients treated at Chungnam National University Hospital were collected between April 2021 and October 2022. We used flow cytometry to classify T cells into five subtypes using CD45RA, CD95, and CCR7: naïve, stem cell memory (SCM), central memory (CM), effector memory (EM) and effector memory with positive CD45RA (EMRA). Otherwise, we analyzed regulatory T cell (Treg) by defining as CD25 highCD127 low and divided them into three categories using CD25 and CD45RA: naïve, active and non-suppressive Treg. We defined early relapse (ER) as relapse within 6 months of allogeneic HSCT. High-throughput single-cell RNAseq assays from BM lymphocytes isolated by density gradients were performed to determine the expression and cell composition of previously irradiated markers. Results: With this approach, marrow-infiltrating T cells were analyzed in 40 patients (10 patients with early relapse) with a median age of 57 years (ranged from 30 to 71). AML is the most common disease at 60%, followed by MDS (20%), ALL (15%) and PMF (2.5%). Stem cell sources were 32.5% from matched sibling donor, 37.5% from haplo-identical donor and 27.5% from matched unrelated donor. All patients received mobilized peripheral blood stem cell transplantation. Marrow-infiltrating CD3+T cell counts were lower in patients with allogeneic HSCT compared to normal subjects (CD3+T cells: 13.89% in HSCT patient and 9.116% in ER patients vs. 34.19% in normal subjects, p&amp;lt; 0.0001). In addition, CD3+CD8+T cells were higher in CR than ER patients (CD3+CD8+T cells: 50.39% in CR vs. 30.41% in ER patients, p&amp;lt; 0.0033). In HSCT patients, naïve was higher, and EM and EMRA were lower than those in normal subjects among CD4+T cells (naïve; 8.629% in ER patients and 5.119% in CR patient's vs 45.76% in normal subjects, p&amp;lt; 0.0001/EM; 54.04% in ER patients and 55.93% in CR patients vs 18.61 in normal subject, p= 0.0003 and p&amp;lt; 0.0001 respectively). Of CD3+T cells, the proportion and expression level of TIM-3 was higher ER patients than in CR patients (56.33% in ER patients vs 39.20% in CR patients, p= 0.0185/MFI; 1873 in ER patients vs. 826.7 in CR patients, p= 0.0331). Of CD4+T cells, the percentage of TIM-3 was lower in normal subjects than in allogeneic HSCT patients (7.794% in normal vs 30.11% in CR patients and 26.04% in ER patients, p= 0.0002 and p= 0.0102 respectively). In patients with ER, naïve and EM was higher than those in CR patients among CD8+T cells (naïve; 8.680% in ER patients vs 4.066% in CR patients, p= 0.4001 /EM; 54.44% in ER patients vs 44.33% in CR patients, p= 0.3039). Of CD8+ EMRA T cell, the expression of TIM-3 was higher in ER patients than in CR patients (MFI; 1441 in HSCT patients vs 421.7 in normal, p=0.1038). Otherwise, of CD3+CD4-CD8-, The percentage of TIM3 was higher in ER patients than CR patients (80.33 % in ER patients vs 62.49% in CR patients, p= 0.0333). Treg levels were no significant difference between CR and ER. We have obtained single-cell sequencing data from two CR (complete response) patients and two ER (early relapse) patients, representing each group. Since the classified groups exhibited ambiguous phenotypes, they were reclassified based on specific markers of cell groups. The reclassified groups showed a higher proportion of HCS (Hematopoietic Stem Cell)-like cells in relapsed patients (+10.14%, P=0.368) and a lower proportion of CD8+ T cells (-13.40%, p=0.068). The HSC-like cells in relapsed patients exhibited higher expression of TIM3 ligand than in CR patients Conclusion: Among the IRs, TIM3 had the highest expression of all cell groups (CD4 T cells, CD8 T cells and Treg) compared with other IRs at the early stage of allogeneic HSCT. So, TIM3 might be a potential target for preventing relapse in hematologic cancer patients after allogeneic HSCT in the future.

Early Safety Results of P-BCMA-ALLO1, a Fully Allogeneic Chimeric Antigen Receptor T-Cell (CAR-T), in Patients with Relapsed / Refractory Multiple Myeloma (RRMM)

P-BCMA-ALLO1 is an allogeneic CAR-T that targets B-cell Maturation Antigen (BCMA) and is currently being investigated for the treatment of RRMM. P-BCMA-ALLO1 cells are manufactured from healthy donor T-cells using non-viral transposon-based integration (piggyBac® DNA Delivery System) that introduces a human anti-BCMA V H-based CAR and an iCas9 safety switch. The piggyBac® DNA delivery system produces a highly enriched T stem cell memory product. The Cas-CLOVER™ Site-Specific Gene Editing System eliminates endogenous T cell receptor (TCR) expression via knockout of the TCR beta chain 1 gene to prevent graft-vs-host disease (GvHD), and the beta-2 microglobulin gene to reduce Major Histocompatibility Complex (MHC) class I expression to eliminate host-vs-graft responses. P-BCMA-ALLO1 demonstrated compelling activity in MM xenografts, providing rationale for this first-in-human phase I study. The primary objective is to assess the safety and maximum tolerated dose (MTD) of P-BCMA-ALLO1 based on dose limiting toxicity (DLT) in RRMM patients with measurable disease who have received a proteasome inhibitor (PI), immunomodulatory agent (IMiD) and anti-CD38 monoclonal antibody therapy. Key secondary objective will assess the anti-myeloma effect of P-BCMA-ALLO1. Exploratory objectives will assess CAR-T related cytokines, serum BCMA levels and cellular kinetics. The trial utilizes a standard 3 + 3 dose escalation design to test seven planned dose levels of intravenous P-BCMA-ALLO1 ranging from 0.0625 × 10 6 to 15 × 10 6 cells/kg (NCT04960579). Multiple lymphodepletion (LD) strategies and cyclic administration of P-BCMA-ALLO1 are being investigated. Repeat P-BCMA-ALLO1 administration is allowed for patients who do not achieve at least a partial response with the first dose after 4 weeks of follow up. We are currently dosing patients in arm S (Cyclophosphamide (Cy) 300 mg/m 2 + Fludarabine (Flu) 30 mg/m 2 X 3 days), arm P1 (Cy 500 mg/m 2 + Flu 30 mg/m 2 X 3 days), arm P2 (Cy 1,000 mg/m 2 + Flu 30 mg/m 2 X 3 days) and arm C (multiple P-BCMA-ALLO1 doses following Cy 300 mg/m 2 + Flu 30 mg/m 2 X 3 days). The median patient age was 73 (33, 85) years, 61% were female, and median time since MM diagnosis was 6.27 (1.48,18.95) years. Eight (35%) patients had high risk disease by cytogenetic criteria. The enrolled patients were heavily pretreated, having received median 7 (2,16) prior lines of therapy, with 30% having undergone prior BCMA targeting therapy. As of 10 Jul 2023, 24 patients received P-BCMA-ALLO1 at 4 dose levels. Twenty-two were treated in arm S, 1 each in arms P1 and P2. One patient received 0.25 X 10 6 cells/kg (cohort -1, arm S), 7 received 0.75 X 10 6 cells/kg (cohort 1, arm S), 12 received 2 X 10 6 cells/kg (cohort 2, 10 in arm S, 1 in arm P1 and 1 in arm P2), and 4 received 6 X 10 6 cells/kg (cohort 3). One of the cohort 2 patients received a second P-BCMA-ALLO1 infusion at the cohort 2 dose level following disease progression. The median time from enrollment to start of LD chemotherapy was 2 (1-8) days and from enrollment to CAR-T infusion was 7 (6-13) days. No patient needed bridging therapy between enrollment and the start of LD. Twenty-two (all in arm S) of the 24 treated patients completed DLT evaluation by data cutoff. None of the patients treated thus far had DLTs. Most common treatment emergent adverse events (TEAEs) were anemia (36%), neutropenia (36%), constipation (36%) and leukopenia (32%). Most common ≥ grade (G) 3 TEAEs were neutropenia (36%), leukopenia (32%) and anemia (23%). Most of the AEs were considered unrelated to P-BCMA-ALLO1. Three (14%) patients developed grade 1 Cytokine Release Syndrome (CRS). One (4%) patient experienced grade 1 Immune Effector Cell Associated Neurotoxicity Syndrome (ICANS), which resolved with one dose of corticosteroids. None of the patients experienced GvHD by the last follow up. P-BCMA-ALLO1 is a non-viral transposon-generated allogeneic “off-the-shelf” CAR-T that is rapidly available for administration and well tolerated in a heavily pretreated RRMM patient population with minimal CRS/ICANS risk. Updated results including safety, preliminary efficacy, and selected biomarkers will be presented at the American Society of Hematology 2023 meeting.