Murine allogeneic CAR T cells integrated before or early after posttransplant cyclophosphamide exert antitumor effects.

Relapse limits the therapeutic efficacy both of chimeric antigen receptor (CAR) T cells and allogeneic hematopoietic cell transplantation (allo-HCT). Patients may undergo these therapies sequentially to prevent or treat relapsed malignancy. However, direct integration of the 2 therapies has been avoided over concerns for potential induction of graft-versus-host disease (GVHD) by allogeneic CAR T cells. We have shown in murine T-cell-replete MHC-haploidentical allo-HCT that suppressive mechanisms induced immediately after posttransplant cyclophosphamide (PTCy), given on days +3/+4, prevent GVHD induction by alloreactive T cells infused as early as day +5. Therefore, we hypothesized that allogeneic CAR T cells given in a similarly integrated manner in our murine MHC-haploidentical allo-HCT model may safely exert antitumor effects. Indeed, allogeneic anti-CD19 CAR T cells given early after (day +5) PTCy or even prior to (day 0) PTCy cleared leukemia without exacerbating the cytokine release syndrome occurring from the MHC-haploidentical allo-HCT or interfering with PTCy-mediated GVHD prevention. Meanwhile, CAR T-cell treatment on day +9 or day +14 was safe but less effective, suggesting a limited therapeutic window. CAR T cells infused before PTCy were not eliminated, but surviving CAR T cells continued to proliferate highly and expand despite PTCy. In comparison with infusion on day +5, CAR T-cell infusion on day 0 demonstrated superior clinical efficacy associated with earlier CAR T-cell expansion, higher phenotypic CAR T-cell activation, less CD4+CD25+Foxp3+ CAR T-cell recovery, and transcriptional changes suggesting increased activation of CD4+ CAR T cells and more cytotoxic CD8+ CAR T cells. This study provides mechanistic insight into PTCy's impact on graft-versus-tumor immunity and describes novel approaches to integrate CAR T cells and allo-HCT that may compensate for deficiencies of each individual approach.

CD19/22 CAR T cells in children and young adults with B-ALL: phase 1 results and development of a novel bicistronic CAR.

Remission durability following single-antigen targeted chimeric antigen receptor (CAR) T-cells is limited by antigen modulation, which may be overcome with combinatorial targeting. Building upon our experiences targeting CD19 and CD22 in B-cell acute lymphoblastic leukemia (B-ALL), we report on our phase 1 dose-escalation study of a novel murine stem cell virus (MSCV)-CD19/CD22-4-1BB bivalent CAR T-cell (CD19.22.BBζ) for children and young adults (CAYA) with B-cell malignancies. Primary objectives included toxicity and dose finding. Secondary objectives included response rates and relapse-free survival (RFS). Biologic correlatives included laboratory investigations, CAR T-cell expansion and cytokine profiling. Twenty patients, ages 5.4 to 34.6 years, with B-ALL received CD19.22.BBζ. The complete response (CR) rate was 60% (12 of 20) in the full cohort and 71.4% (10 of 14) in CAR-naïve patients. Ten (50%) developed cytokine release syndrome (CRS), with 3 (15%) having ≥ grade 3 CRS and only 1 experiencing neurotoxicity (grade 3). The 6- and 12-month RFS in those achieving CR was 80.8% (95% confidence interval [CI]: 42.4%-94.9%) and 57.7% (95% CI: 22.1%-81.9%), respectively. Limited CAR T-cell expansion and persistence of MSCV-CD19.22.BBζ compared with EF1α-CD22.BBζ prompted laboratory investigations comparing EF1α vs MSCV promoters, which did not reveal major differences. Limited CD22 targeting with CD19.22.BBζ, as evaluated by ex vivo cytokine secretion and leukemia eradication in humanized mice, led to development of a novel bicistronic CD19.28ζ/CD22.BBζ construct with enhanced cytokine production against CD22. With demonstrated safety and efficacy of CD19.22.BBζ in a heavily pretreated CAYA B-ALL cohort, further optimization of combinatorial antigen targeting serves to overcome identified limitations (www.clinicaltrials.gov #NCT03448393).

Characterization of HLH-like manifestations as a CRS variant in patients receiving CD22 CAR T cells.

Chimeric antigen receptor (CAR) T-cell toxicities resembling hemophagocytic lymphohistiocytosis (HLH) occur in a subset of patients with cytokine release syndrome (CRS). As a variant of conventional CRS, a comprehensive characterization of CAR T-cell-associated HLH (carHLH) and investigations into associated risk factors are lacking. In the context of 59 patients infused with CD22 CAR T cells where a substantial proportion developed carHLH, we comprehensively describe the manifestations and timing of carHLH as a CRS variant and explore factors associated with this clinical profile. Among 52 subjects with CRS, 21 (40.4%) developed carHLH. Clinical features of carHLH included hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, coagulopathy, hepatic transaminitis, hyperbilirubinemia, severe neutropenia, elevated lactate dehydrogenase, and occasionally hemophagocytosis. Development of carHLH was associated with preinfusion natural killer(NK) cell lymphopenia and higher bone marrow T-cell:NK cell ratio, which was further amplified with CAR T-cell expansion. Following CRS, more robust CAR T-cell and CD8 T-cell expansion in concert with pronounced NK cell lymphopenia amplified preinfusion differences in those with carHLH without evidence for defects in NK cell mediated cytotoxicity. CarHLH was further characterized by persistent elevation of HLH-associated inflammatory cytokines, which contrasted with declining levels in those without carHLH. In the setting of CAR T-cell mediated expansion, clinical manifestations and immunophenotypic profiling in those with carHLH overlap with features of secondary HLH, prompting consideration of an alternative framework for identification and management of this toxicity profile to optimize outcomes following CAR T-cell infusion.

Potent preclinical activity of FLT3-directed chimeric antigen receptor T-cell immunotherapy against FLT3- mutant acute myeloid leukemia and KMT2A-rearranged acute lymphoblastic leukemia.

Chimeric antigen receptor (CAR) T-cell immunotherapies targeting CD19 or CD22 induce remissions in the majority of patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL), although relapse due to target antigen loss or downregulation has emerged as a major clinical dilemma. Accordingly, great interest exists in developing CAR T cells directed against alternative leukemia cell surface antigens that may help to overcome immunotherapeutic resistance. The fms-like tyrosine kinase 3 receptor (FLT3) is constitutively activated via FLT3 mutation in acute myeloid leukemia (AML) or wild-type FLT3 overexpression in KMT2A (lysine-specific methyltransferase 2A)-rearranged ALL, which are associated with poor clinical outcomes in children and adults. We developed monovalent FLT3-targeted CAR T cells (FLT3CART) and bispecific CD19xFLT3CART and assessed their anti-leukemia activity in preclinical models of FLT3-mutant AML and KMT2A-rearranged infant ALL. We report robust in vitro FLT3CART-induced cytokine production and cytotoxicity against AML and ALL cell lines with minimal cross-reactivity against normal hematopoietic and non-hematopoietic tissues. We also observed potent in vivo inhibition of leukemia proliferation in xenograft models of both FLT3-mutant AML and KMT2A-rearranged ALL, including a post-tisagenlecleucel ALL-to-AML lineage switch patient-derived xenograft model pairing. We further demonstrate significant in vitro and in vivo activity of bispecific CD19xFLT3CART against KMT2Arearranged ALL and posit that this additional approach might also diminish potential antigen escape in these high-risk leukemias. Our preclinical data credential FLT3CART as a highly effective immunotherapeutic strategy for both FLT3- mutant AML and KMT2A-rearranged ALL which is poised for further investigation and clinical translation.

Digital polymerase chain reaction strategies for accurate and precise detection of vector copy number in chimeric antigen receptor T-cell products.

BACKGROUND AIMS: Vector copy number (VCN), an average quantification of transgene copies unique to a chimeric antigen receptor (CAR) T-cell product, is a characteristic that must be reported prior to patient administration, as high VCN increases the risk of insertional mutagenesis. Historically, VCN assessment in CAR T-cell products has been performed via quantitative polymerase chain reaction (qPCR). qPCR is reliable along a broad range of concentrations, but quantification requires use of a standard curve and precision is limited. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared with qPCR include simplicity, reproducibility, sensitivity and lack of dependency on a standard curve for definitive quantification. In the present study, the authors describe a dPCR assay developed for analysis of the novel bicistronic CD19 × CD22 CAR T-cell construct. METHODS: The authors compared the performance of the dPCR assay with qPCR on both the QX200 droplet dPCR (ddPCR) system (Bio-Rad Laboratories, Inc, Hercules, CA, USA) and the QIAcuity nanoplate-based dPCR (ndPCR) system (QIAGEN Sciences, Inc, Germantown, MD, USA). The primer-probe assay was validated with qPCR, ndPCR and ddPCR using patient samples from pre-clinical CAR T-cell manufacturing production runs as well as Jurkat cell subclones, which stably express this bicistronic CAR construct. RESULTS: ddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product. Additionally, the authors' assay gave accurate, precise and reproducible CAR T-cell VCN measurements across qPCR, ndPCR and ddPCR modalities. CONCLUSIONS: The authors demonstrate that dPCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, with improved test-retest reliability, and that specific assays can be developed for detection of unique constructs.

Children's Oncology Group's 2023 blueprint for research: Cellular therapy and stem cell transplantation.

Since the publication of the last Cellular Therapy and Stem Cell Transplant blueprint in 2013, Children's Oncology Group cellular therapy-based trials advanced the field and created new standards of care across a wide spectrum of pediatric cancer diagnoses. Key findings include that tandem autologous transplant improved survival for patients with neuroblastoma and atypical teratoid/rhabdoid brain tumors, one umbilical cord blood (UCB) donor was safer than two UCB donors, killer immunoglobulin receptor (KIR) mismatched donors did not improve survival for pediatric acute myeloid leukemia when in vivo T-cell depletion is used, and the depth of remission as measured by next-generation sequencing-based minimal residual disease assessment pretransplant was the best predictor of relapse for acute lymphoblastic leukemia. Plans for the next decade include optimizing donor selection for transplants for acute leukemia/myelodysplastic syndrome, using novel engineered cellular therapies to target a wide array of malignancies, and developing better treatments for cellular therapy toxicities such as viral infections and graft-vs-host disease.

Murine pre-B-cell ALL induces T-cell dysfunction not fully reversed by introduction of a chimeric antigen receptor.

Adoptive transfer of patient-derived T cells modified to express chimeric antigen receptors (CARTs) has demonstrated dramatic success in relapsed/refractory pre-B-cell acute lymphoblastic leukemia (ALL), but response and durability of remission requires exponential CART expansion and persistence. Tumors are known to affect T-cell function, but this has not been well studied in ALL and in the context of chimeric antigen receptor (CAR) expression. Using TCF3/PBX1 and MLL-AF4-driven murine ALL models, we assessed the impact of progressive ALL on T-cell function in vivo. Vaccines protect against TCF3/PBX1.3 but were ineffective when administered after leukemia injection, suggesting immunosuppression induced early during ALL progression. T cells from leukemia-bearing mice exhibited increased expression of inhibitory receptors, including PD1, Tim3, and LAG3, and were dysfunctional following adoptive transfer in a model of T-cell receptor (TCR)-dependent leukemia clearance. Although expression of inhibitory receptors has been linked to TCR signaling, pre-B-cell ALL induced inhibitory receptor expression, at least in part, in a TCR-independent manner. Finally, introduction of a CAR into T cells generated from leukemia-bearing mice failed to fully reverse poor in vivo function.

Disease detection methodologies in relapsed B-cell acute lymphoblastic leukemia: Opportunities for improvement.

BACKGROUND: Accurate disease detection is integral to risk stratification in B-cell acute lymphoblastic leukemia (ALL). The gold standard used to evaluate response in the United States includes morphologic evaluation and minimal residual disease (MRD) testing of aspirated bone marrow (BM) by flow cytometry (FC). This MRD assessment is usually made on a single aspirate sample that is subject to variability in collection techniques and sampling error. Additionally, central nervous system (CNS) assessments for ALL include evaluations of cytopathology and cell counts, which can miss subclinical involvement. PROCEDURE: We retrospectively compared BM biopsy, aspirate, and FC samples obtained from children and young adults with relapsed/refractory ALL to identify the frequency and degree of disease discrepancies in this population. We also compared CNS FC and cytopathology techniques. RESULTS: Sixty of 410 (14.6%) BM samples had discrepant results, 41 (10%) of which were clinically relevant as they resulted in a change in the assignment of marrow status. Discrepant BM results were found in 28 of 89 (31.5%) patients evaluated. Additionally, cerebrospinal fluid (CSF) FC identified disease in 9.7% of cases where cytopathology was negative. CONCLUSIONS: These results support further investigation of the role of concurrent BM biopsy, with aspirate and FC evaluations, and the addition of FC to CSF evaluations, to fully assess disease status and response, particularly in patients with relapsed/refractory ALL. Prospective studies incorporating more comprehensive analysis to evaluate the impact on clinical outcomes are warranted.

Chimeric Antigen Receptors Incorporating D Domains Targeting CD123 Direct Potent Mono- and Bi-specific Antitumor Activity of T Cells.

Chimeric antigen receptor (CAR) T cell therapies have demonstrated impressive initial response rates in hematologic malignancies. However, relapse rates are significant, and robust efficacies in other indications, such as solid tumors, will likely require novel therapeutic strategies and CAR designs. To that end, we sought to develop simple, highly selective targeting domains (D domains) that could be incorporated into complex, multifunctional therapeutics. Herein, we describe the identification and characterization of D domains specific for CD123, a therapeutic target for hematologic malignancies, including acute myelogenous leukemia (AML). CARs comprised of these D domains mediate potent T cell activation and cytolysis of CD123-expressing target cells and induce complete durable remission in two AML xenograft models. We describe a strategy of engineering less immunogenic D domains through the identification and removal of putative T cell epitopes and investigate the binding kinetics and affinity requirements of the resultant D domain CARs. Finally, we extended the utility of D domains by generating functional, bi-specific CARs comprised of a CD123-specific D domain and a CD19-specific scFv. The properties of D domains suggest that this class of targeting domain may facilitate the development of multi-functional CARs where conventional, scFv-based designs may be suboptimal.

Effect of Cryopreservation on Autologous Chimeric Antigen Receptor T Cell Characteristics.

As clinical applications for chimeric antigen receptor T cell (CART) therapy extend beyond early phase trials, commercial manufacture incorporating cryopreservation steps becomes a logistical necessity. The effect of cryopreservation on CART characteristics is unclear. We retrospectively evaluated the effect of cryopreservation on product release criteria and in vivo characteristics in 158 autologous CART products from 6 single-center clinical trials. Further, from 3 healthy donor manufacturing runs, we prospectively identified differentially expressed cell surface markers and gene signatures among fresh versus cryopreserved CARTs. Within 2 days of culture initiation, cell viability of the starting fraction (peripheral blood mononuclear cells [PBMNCs]) decreased significantly in the cryo-thawed arm compared to the fresh arm. Despite this, PBMNC cryopreservation did not affect final CART fold expansion, transduction efficiency, CD3%, or CD4:CD8 ratios. In vivo CART persistence and clinical responses did not differ among fresh and cryopreserved final products. In healthy donors, compared to fresh CARTs, early apoptotic cell-surface markers were significantly elevated in cryo-thawed CARTs. Cryo-thawed CARTs also demonstrated significantly elevated expression of mitochondrial dysfunction, apoptosis signaling, and cell cycle damage pathways. Cryopreservation during CART manufacture is a viable strategy, based on standard product release parameters. The clinical impact of cryopreservation-related subtle micro-cellular damage needs further study.

Cytomegalovirus Infection Incidence and Risk Factors Across Diverse Hematopoietic Cell Transplantation Platforms Using a Standardized Monitoring and Treatment Approach: A Comprehensive Evaluation from a Single Institution.

Human cytomegalovirus (CMV) infection and disease remains a significant cause of morbidity and mortality for hematopoietic cell transplantation (HCT) recipients. Disruption of or weak reconstitution of virus-specific cellular immune function, such as with certain HCT approaches, poses significant risk for CMV-related complications. The incidence of and risk factors for CMV infection and the nature of CMV disease were evaluated retrospectively among 356 consecutive HCT recipients transplanted at the National Institutes of Health using all graft sources, including bone marrow, peripheral blood stem cell (PBSC), and umbilical cord blood (UCB), and a range of in vivo and ex vivo approaches for graft-versus-host disease (GVHD) prophylaxis. The cumulative incidence of CMV infection was higher for CMV-seropositive recipients at 33%, regardless of donor CMV serostatus. Patients transplanted with CMV-seropositive donors had a significantly shorter duration of antiviral therapy. Among graft sources UCB was associated with the highest cumulative incidence of CMV infection at 65% and significantly longer treatment duration at a median of 36days, whereas PBSC HCT was associated with the lowest incidence at 26% and the shortest CMV treatment duration at a median of 21days. There were significant differences in the cumulative incidence of CMV infection by T cell manipulation strategy when systemic steroids were included as a risk-modifying event. Over one-third of CMV infections occurred in the setting of systemic steroid administration. CMV disease occurred in 5% of HCT recipients, with 70% of cases in the setting of treatment for GVHD. Although factors related to serostatus, graft source, and GVHD prophylaxis were associated with varied CMV infection incidence, unplanned post-HCT corticosteroid therapy contributed greatly to the incidence of both CMV infection and disease across HCT approaches, highlighting this post-HCT intervention as a key time to potentially tailor the approach to monitoring, preemptive therapy, and even prophylaxis.

Outcomes of Measurable Residual Disease in Pediatric Acute Myeloid Leukemia before and after Hematopoietic Stem Cell Transplant: Validation of Difference from Normal Flow Cytometry with Chimerism Studies and Wilms Tumor 1 Gene Expression.

We enrolled 150 patients in a prospective multicenter study of children with acute myeloid leukemia undergoing hematopoietic stem cell transplantation (HSCT) to compare the detection of measurable residual disease (MRD) by a "difference from normal" flow cytometry (ΔN) approach with assessment of Wilms tumor 1 (WT1) gene expression without access to the diagnostic specimen. Prospective analysis of the specimens using this approach showed that 23% of patients screened for HSCT had detectable residual disease by ΔN (.04% to 53%). Of those patients who proceeded to transplant as being in morphologic remission, 10 had detectable disease (.04% to 14%) by ΔN. The disease-free survival of this group was 10% (0 to 35%) compared with 55% (46% to 64%, P < .001) for those without disease. The ΔN assay was validated using the post-HSCT specimen by sorting abnormal or suspicious cells to confirm recipient or donor origin by chimerism studies. All 15 patients who had confirmation of tumor detection relapsed, whereas the 2 patients with suspicious phenotype cells lacking this confirmation did not. The phenotype of the relapse specimen was then used retrospectively to assess the pre-HSCT specimen, allowing identification of additional samples with low levels of MRD involvement that were previously undetected. Quantitative assessment of WT1 gene expression was not predictive of relapse or other outcomes in either pre- or post-transplant specimens. MRD detected by ΔN was highly specific, but did not identify most relapsing patients. The application of the assay was limited by poor quality among one-third of the specimens and lack of a diagnostic phenotype for comparison.

Murine allogeneic CD19 CAR T cells harbor potent antileukemic activity but have the potential to mediate lethal GVHD.

Acute lymphoblastic leukemia (ALL) persisting or relapsing following bone marrow transplantation (BMT) has a dismal prognosis. Success with chimeric antigen receptor (CAR) T cells offers an opportunity to treat these patients with leukemia-redirected donor-derived T cells, which may be more functional than T cells derived from patients with leukemia but have the potential to mediate graft-versus-host disease (GVHD). We, together with others, have previously demonstrated tumor-specific T-cell dysfunction in the allogeneic environment. Here, we studied CAR T-cell function following BMT using an immunocompetent murine model of minor mismatched allogeneic transplantation followed by donor-derived CD19-CAR T cells. Allogeneic donor-derived CD19-CAR T cells eliminated residual ALL with equal potency to those administered after syngeneic BMT. Surprisingly, allogeneic CAR T cells mediated lethal acute GVHD with early mortality, which is atypical for this minor mismatch model. We demonstrated that both allogeneic and syngeneic CAR T cells show initial expansion as effector T cells, with a higher peak but rapid deletion of allogeneic CAR T cells. Interestingly, CAR-mediated acute GVHD was only seen in the presence of leukemia, suggesting CAR-target interactions induced GVHD. Indeed, serum interleukin (IL)-6 was elevated only in the presence of both leukemia and CAR T cells, and IL-6 neutralization ameliorated the severity of GVHD in a delayed donor lymphocyte infusion model. Finally, allogeneic CD4(+) CAR T cells were responsible for GVHD, which correlated with their ability to produce IL-6 upon CAR stimulation. Altogether, we demonstrate that donor-derived allogeneic CAR T cells are active but have the capacity to drive GVHD.

Generation of clinical-grade CD19-specific CAR-modified CD8+ memory stem cells for the treatment of human B-cell malignancies.

Long-lived, self-renewing, multipotent T memory stem cells (TSCM) can trigger profound and sustained tumor regression but their rareness poses a major hurdle to their clinical application. Presently, clinically compliant procedures to generate relevant numbers of this T-cell population are undefined. Here, we provide a strategy for deriving large numbers of clinical-grade tumor-redirected TSCM starting from naive precursors. CD8(+)CD62L(+)CD45RA(+) naive T cells enriched by streptamer-based serial-positive selection were activated by CD3/CD28 engagement in the presence of interleukin-7 (IL-7), IL-21, and the glycogen synthase-3ß inhibitor TWS119, and genetically engineered to express a CD19-specific chimeric antigen receptor (CD19-CAR). These conditions enabled the generation of CD19-CAR-modified CD8(+) TSCM that were phenotypically, functionally, and transcriptomically equivalent to their naturally occurring counterpart. Compared with CD8(+) T cells generated with clinical protocols currently under investigation, CD19-CAR-modified CD8(+) TSCM exhibited enhanced metabolic fitness and mediated robust, long-lasting antitumor responses against systemic acute lymphoblastic leukemia xenografts. This clinical-grade platform provides the basis for a phase 1 trial evaluating the activity of CD19-CAR-modified CD8(+) TSCM in patients with B-cell malignancies refractory to prior allogeneic hematopoietic stem cell transplantation.

Chimeric Antigen Receptor T-Cell Therapy.

Patients with relapsed or refractory (R/R) cancers have a poor prognosis and limited treatment options. The recent approval of 2 chimeric antigen receptor (CAR) autologous T-cell products for R/R B-cell acute lymphoblastic leukemia and non-Hodgkin's lymphoma treatment is setting the stage for what is possible in other diseases. However, there are important factors that must be considered, including patient selection, toxicity management, and costs associated with CAR T-cell therapy. To begin to address these issues, NCCN organized a task force consisting of a multidisciplinary panel of experts in oncology, cancer center administration, and health policy, which met for the first time in March 2018. This report describes the current state of CAR T-cell therapy and future strategies that should be considered as the application of this novel immunotherapy expands and evolves.

Tumor Antigen and Receptor Densities Regulate Efficacy of a Chimeric Antigen Receptor Targeting Anaplastic Lymphoma Kinase.

We explored the utility of targeting anaplastic lymphoma kinase (ALK), a cell surface receptor overexpressed on pediatric solid tumors, using chimeric antigen receptor (CAR)-based immunotherapy. T cells expressing a CAR incorporating the single-chain variable fragment sequence of the ALK48 mAb linked to a 4-1BB-CD3ζ signaling domain lysed ALK-expressing tumor lines and produced interferon-gamma upon antigen stimulation but had limited anti-tumor efficacy in two xenograft models of human neuroblastoma. Further exploration demonstrated that cytokine production was highly dependent upon ALK target density and that target density of ALK on neuroblastoma cell lines was insufficient for maximal activation of CAR T cells. In addition, ALK CAR T cells demonstrated rapid and complete antigen-induced loss of receptor from the T cell surface via internalization. Using a model that simultaneously modulated antigen density and CAR expression, we demonstrated that CAR functionality is regulated by target antigen and CAR density and that low expression of either contributes to limited anti-tumor efficacy of the ALK CAR. These data suggest that stoichiometric relationships between CAR receptors and target antigens may significantly impact the anti-tumor efficacy of CAR T cells and that manipulation of these parameters could allow precise tuning of CAR T cell activity.

Elutriated lymphocytes for manufacturing chimeric antigen receptor T cells.

BACKGROUND: Clinical trials of Chimeric Antigen Receptor (CAR) T cells manufactured from autologous peripheral blood mononuclear cell (PBMC) concentrates for the treatment of hematologic malignancies have been promising, but CAR T cell yields have been variable. This variability is due in part to the contamination of the PBMC concentrates with monocytes and granulocytes. METHODS: Counter-flow elutriation allows for the closed system separation of lymphocytes from monocytes and granulocytes. We investigated the use of PBMC concentrates enriched for lymphocytes using elutriation for manufacturing 8 CD19- and 5 GD2-CAR T cell products. RESULTS: When compared to PBMC concentrates, lymphocyte-enriched elutriation fractions contained greater proportions of CD3+ and CD56+ cells and reduced proportions of CD14+ and CD15+ cells. All 13 CAR T cell products manufactured using the elutriated lymphocytes yielded sufficient quantities of transduced CAR T cells to meet clinical dose criteria. The GD2-CAR T cell products contained significantly more T cells and transduced T cells than the CD19-CAR T cell products. A comparison of the yields of CAR T cells produced from elutriated lymphocytes with the yields of CAR T cells previous produced from cells isolated from PBMC concentrates by anti-CD3/CD28 bead selection or by anti-CD3/CD28 bead selection plus plastic adherence found that greater quantities of GD2-CAR T cells were produced from elutriated lymphocytes, but not CD19-CAR T cells. CONCLUSIONS: Enrichment of PBMC concentrates for lymphocytes using elutriation increased the quantity of GD2-CAR T cells produced. These results provide further evidence that CAR T cell expansion is inhibited by monocytes and granulocytes.