Rapid anti-myeloma activity by T cells expressing an anti-BCMA CAR with a human heavy-chain-only antigen-binding domain.

Multiple myeloma (MM) is a rarely curable malignancy of plasma cells. MM expresses B cell maturation antigen (BCMA). We developed a fully human anti-BCMA chimeric antigen receptor (CAR) with a heavy-chain-only antigen-recognition domain, a 4-1BB domain, and a CD3ζ domain. The CAR was designated FHVH33-CD8BBZ. We conducted the first-in-humans clinical trial of T cells expressing FHVH33-CD8BBZ (FHVH-T). Twenty-five patients with relapsed MM were treated. The stringent complete response rate (sCR) was 52%. Median progression-free survival (PFS) was 78 weeks. Of 24 evaluable patients, 6 (25%) had a maximum cytokine-release syndrome (CRS) grade of 3; no patients had CRS of greater than grade 3. Most anti-MM activity occurred within 2-4 weeks of FHVH-T infusion as shown by decreases in the rapidly changing MM markers serum free light chains, urine light chains, and bone marrow plasma cells. Blood CAR+ cell levels peaked during the time that MM elimination was occurring, between 7 and 15 days after FHVH-T infusion. C-C chemokine receptor type 7 (CCR7) expression on infusion CD4+ FHVH-T correlated with peak blood FHVH-T levels. Single-cell RNA sequencing revealed a shift toward more differentiated FHVH-T after infusion. Anti-CAR antibody responses were detected in 4 of 12 patients assessed. FHVH-T has powerful, rapid, and durable anti-MM activity.

Deciphering the importance of culture pH on CD22 CAR T-cells characteristics.

BACKGROUND: Chimeric antigen receptor (CAR) T-cells have demonstrated significant efficacy in targeting hematological malignancies, and their use continues to expand. Despite substantial efforts spent on the optimization of protocols for CAR T-cell manufacturing, critical parameters of cell culture such as pH or oxygenation are rarely actively monitored during cGMP CAR T-cell generation. A comprehensive understanding of the role that these factors play in manufacturing may help in optimizing patient-specific CAR T-cell therapy with maximum benefits and minimal toxicity. METHODS: This retrospective study examined cell culture supernatants from the manufacture of CAR T-cells for 20 patients with B-cell malignancies enrolled in a phase 1/2 clinical trial of anti-CD22 CAR T-cells. MetaFLEX was used to measure supernatant pH, oxygenation, and metabolites, and a Bio-Plex assay was used to assess protein levels. Correlations were assessed between the pH of cell culture media throughout manufacturing and cell proliferation as well as clinical outcomes. Next-generation sequencing was conducted to examine gene expression profiles of the final CAR T-cell products. RESULTS: A pH level at the lower range of normal at the beginning of the manufacturing process significantly correlated with measures of T-cell expansion and metabolism. Stable or rising pH during the manufacturing process was associated with clinical response, whereas a drop in pH was associated with non-response. CONCLUSIONS: pH has potential to serve as an informative factor in predicting CAR T-cell quality and clinical outcomes. Thus, its active monitoring during manufacturing may ensure a more effective CAR T-cell product.

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).

Assessment and comparison of viability assays for cellular products.

BACKGROUND AIMS: Accurate assessment of cell viability is crucial in cellular product manufacturing, yet selecting the appropriate viability assay presents challenges due to various factors. This study compares and evaluates different viability assays on fresh and cryopreserved cellular products, including peripheral blood stem cell (PBSC) and peripheral blood mononuclear cell (PBMC) apheresis products, purified PBMCs and cultured chimeric antigen receptor and T-cell receptor-engineered T-cell products. METHODS: Viability assays, including manual Trypan Blue exclusion, flow cytometry-based assays using 7-aminoactinomycin D (7-AAD) or propidium iodide (PI) direct staining or cell surface marker staining in conjunction with 7-AAD, Cellometer (Nexcelom Bioscience LLC, Lawrence, MA, USA) Acridine Orange/PI staining and Vi-CELL BLU Cell Viability Analyzer (Beckman Coulter, Inc, Brea, CA, USA), were evaluated. A viability standard was established using live and dead cell mixtures to assess the accuracy of these assays. Furthermore, precision assessment was conducted to determine the reproducibility of the viability assays. Additionally, the viability of individual cell populations from cryopreserved PBSC and PBMC apheresis products was examined. RESULTS: All methods provided accurate viability measurements and generated consistent and reproducible viability data. The assessed viability assays were demonstrated to be reliable alternatives when evaluating the viability of fresh cellular products. However, cryopreserved products exhibited variability among the tested assays. Additionally, analyzing the viability of each subset of the cryopreserved PBSC and PBMC apheresis products revealed that T cells and granulocytes were more susceptible to the freeze-thaw process, showing decreased viability. CONCLUSIONS: The study demonstrates the importance of careful assay selection, validation and standardization, particularly for assessing the viability of cryopreserved products. Given the complexity of cellular products, choosing a fit-for-purpose viability assay is essential.

CAR-T cell expansion platforms yield distinct T cell differentiation states.

With investigators looking to expand engineered T cell therapies such as CAR-T to new tumor targets and patient populations, a variety of cell manufacturing platforms have been developed to scale manufacturing capacity using closed and/or automated systems. Such platforms are particularly useful for solid tumor targets, which typically require higher CAR-T cell doses. Although T cell phenotype and function are key attributes that often correlate with therapeutic efficacy, how manufacturing platforms influence the final CAR-T cell product is currently unknown. We compared 4 commonly used T cell manufacturing platforms (CliniMACS Prodigy, Xuri W25 rocking platform, G-Rex gas-permeable bioreactor, static bag culture) using identical media, stimulation, culture length, and donor starting material. Selected CD4+CD8+ cells were transduced with lentiviral vector incorporating a CAR targeting FGFR4, a promising target for pediatric sarcoma. We observed significant differences in overall expansion over the 14-day culture; bag cultures had the highest capacity for expansion while the Prodigy had the lowest (481-fold versus 84-fold, respectively). Strikingly, we also observed considerable differences in the phenotype of the final product, with the Prodigy significantly enriched for CCR7+CD45RA+ naïve/stem central memory (Tn/scm)-like cells at 46% compared to bag and G-Rex with 16% and 13%, respectively. Gene expression analysis also showed that Prodigy CAR-Ts are more naïve, less cytotoxic and less exhausted than bag, G-Rex, and Xuri CAR-Ts, and pointed to differences in cell metabolism that were confirmed via metabolic assays. We hypothesized that dissolved oxygen level, which decreased substantially during the final 3 days of the Prodigy culture, may contribute to the observed differences in T cell phenotype. By culturing bag and G-Rex cultures in 1% O2 from day 5 onward, we could generate >60% Tn/scm-like cells, with longer time in hypoxia correlating with a higher percentage of Tn/scm-like cells. Intriguingly, our results suggest that oxygenation is responsible, at least in part, for observed differences in T cell phenotype among bioreactors and suggest hypoxic culture as a potential strategy prevent T cell differentiation during expansion. Ultimately, our study demonstrates that selection of bioreactor system may have profound effects not only on the capacity for expansion, but also on the differentiation state of the resulting CAR-T cells.

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.

Optimizing a fully automated and closed system process for red blood cell reduction of human bone marrow products.

BACKGROUND AIMS: Hematopoietic stem cell transplantation using bone marrow as the graft source is a common treatment for hematopoietic malignancies and disorders. For allogeneic transplants, processing of bone marrow requires the depletion of ABO-mismatched red blood cells (RBCs) to avoid transfusion reactions. Here the authors tested the use of an automated closed system for depleting RBCs from bone marrow and compared the results to a semi-automated platform that is more commonly used in transplant centers today. The authors found that fully automated processing using the Sepax instrument (Cytiva, Marlborough, MA, USA) resulted in depletion of RBCs and total mononuclear cell recovery that were comparable to that achieved with the COBE 2991 (Terumo BCT, Lakewood, CO, USA) semi-automated process. METHODS: The authors optimized the fully automated and closed Sepax SmartRedux (Cytiva) protocol. Three reduction folds (10×, 12× and 15×) were tested on the Sepax. Each run was compared with the standard processing performed in the authors' center on the COBE 2991. Given that bone marrow is difficult to acquire for these purposes, the authors opted to create a surrogate that is more easily obtainable, which consisted of cryopreserved peripheral blood stem cells that were thawed and mixed with RBCs and supplemented with Plasma-Lyte A (Baxter, Deerfield, IL, USA) and 4% human serum albumin (Baxalta, Westlake Village, CA, USA). This "bone marrow-like" product was split into two starting products of approximately 600 mL, and these were loaded onto the COBE and Sepax for direct comparison testing. Samples were taken from the final products for cell counts and flow cytometry. The authors also tested a 10× Sepax reduction using human bone marrow supplemented with human liquid plasma and RBCs. RESULTS: RBC reduction increased as the Sepax reduction rate increased, with an average of 86.06% (range of 70.85-96.39%) in the 10×, 98.80% (range of 98.1-99.5%) in the 12× and 98.89% (range of 98.80-98.89%) in the 15×. The reduction rate on the COBE ranged an average of 69.0-93.15%. However, white blood cell (WBC) recovery decreased as the Sepax reduction rate increased, with an average of 47.65% (range of 38.9-62.35%) in the 10×, 14.56% (range of 14.34-14.78%) in the 12× and 27.97% (range of 24.7-31.23%) in the 15×. COBE WBC recovery ranged an average of 53.17-76.12%. Testing a supplemented human bone marrow sample using a 10× Sepax reduction resulted in an average RBC reduction of 84.22% (range of 84.0-84.36%) and WBC recovery of 43.37% (range of 37.48-49.26%). Flow cytometry analysis also showed that 10× Sepax reduction resulted in higher purity and better recovery of CD34+, CD3+ and CD19+ cells compared with 12× and 15× reduction. Therefore, a 10× reduction rate was selected for the Sepax process. CONCLUSIONS: The fully automated and closed SmartRedux program on the Sepax was shown to be effective at reducing RBCs from "bone marrow-like" products and a supplemented bone marrow product using a 10× reduction rate.

Reference gene selection for clinical chimeric antigen receptor T-cell product vector copy number assays.

BACKGROUND AIMS: Reference genes are an essential part of clinical assays such as droplet digital polymerase chain reaction (ddPCR), which measure the number of copies of vector integrated into genetically engineered cells and the loss of plasmids in reprogrammed cells used in clinical cell therapies. Care should be taken to select reference genes, because it has been discovered that there may be thousands of variations in copy number from genomic segments among different individuals. In addition, within the same person in the context of cancer and other proliferative disorders, substantial parts of the genome also can differ in copy number between cells from diseased and healthy people. The purpose of this study was to identify reference genes that could be used for copy number variation analysis of transduced chimeric antigen receptor T cells and for plasmid loss analysis in induced pluripotent stem cells using ddPCR. METHODS: We used The Cancer Genome Atlas (TCGA) to evaluate candidate reference genes. If TCGA found a candidate gene to have low copy number variance in cancer, ddPCR was used to measure the copy numbers of the potential reference gene in cells from healthy subjects, cancer cell lines and patients with acute lymphocytic leukemia, lymphoma, multiple myeloma and human papillomavirus-associated cancers. RESULTS: In addition to the rPP30 gene, which we have has been using in our copy number assays, three other candidate reference genes were evaluated using TCGA, and this analysis found that none of the four gene regions (AGO1, AP3B1, MKL2 and rPP30) were amplified or deleted in all of the cancer cell types that are currently being treated with cellular therapies by our facility. The number of copies of the genes AP3B1, AGO1, rPP30 and MKL2 measured by ddPCR was similar among cells from healthy subjects. We found that AGO1 had copy number alteration in some of the clinical samples, and the number of copies of the genes AP3B1, MKL2 and rPP30 measured by ddPCR was similar among cells from patients with the cancer cell types that are currently being treated with genetically engineered T-cell therapies by our facility. CONCLUSIONS: Based on our current results, the three genes, AP3B1, MKL2 and rPP30, are suitable for use as reference genes for assays measuring vector copy number in chimeric antigen receptor T cells produced from patients with acute leukemia, lymphoma, multiple myeloma and human papillomavirus-associated cancers. We will continue to evaluate AGO1 on our future samples.

Point-of-care cell therapy manufacturing; it's not for everyone.

The use of cellular therapies to treat cancer, inherited immune deficiencies, hemoglobinopathies and viral infections is growing rapidly. The increased interest in cellular therapies has led to the development of reagents and closed-system automated instruments for the production of these therapies. For cellular therapy clinical trials involving multiple sites some people are advocating a decentralized model of manufacturing where patients are treated with cells produced using automated instruments at each participating center using a single, centrally held Investigational New Drug Application (IND). Many academic centers are purchasing these automated instruments for point-of-care manufacturing and participation in decentralized multiple center clinical trials. However, multiple site manufacturing requires harmonization of product testing and manufacturing in order to interpret the clinical trial results. Decentralized manufacturing is quite challenging since all centers should use the same manufacturing protocol, the same or comparable in-process and lot release assays and the quality programs from each center must work closely together. Consequently, manufacturing cellular therapies using a decentralized model is in many ways more difficult than manufacturing cells in a single centralized facility. Before an academic center decides to establish a point-of-care cell processing laboratory, they should consider all costs associated with such a program. For many academic cell processing centers, point-of-care manufacturing may not be a good investment.

Genome-wide profiling of retroviral DNA integration and its effect on clinical pre-infusion CAR T-cell products.

BACKGROUND: Clinical CAR T-cell therapy using integrating vector systems represents a promising approach for the treatment of hematological malignancies. Lentiviral and γ-retroviral vectors are the most commonly used vectors in the manufacturing process. However, the integration pattern of these viral vectors and subsequent effect on CAR T-cell products is still unclear. METHODS: We used a modified viral integration sites analysis (VISA) pipeline to evaluate viral integration events around the whole genome in pre-infusion CAR T-cell products. We compared the differences of integration pattern between lentiviral and γ-retroviral products. We also explored whether the integration sites correlated with clinical outcomes. RESULTS: We found that γ-retroviral vectors were more likely to insert than lentiviral vectors into promoter, untranslated, and exon regions, while lentiviral vector integration sites were more likely to occur in intron and intergenic regions. Some integration events affected gene expression at the transcriptional and post-transcriptional level. Moreover, γ-retroviral vectors showed a stronger impact on the host transcriptome. Analysis of individuals with different clinical outcomes revealed genes with differential enrichment of integration events. These genes may affect biological functions by interrupting amino acid sequences and generating abnormal proteins, instead of by affecting mRNA expression. These results suggest that vector integration is associated with CAR T-cell efficacy and clinical responses. CONCLUSION: We found differences in integration patterns, insertion hotspots and effects on gene expression vary between lentiviral and γ-retroviral vectors used in CAR T-cell products and established a foundation upon which we can conduct further analyses.

High efficiency closed-system gene transfer using automated spinoculation.

BACKGROUND: Gene transfer is an important tool for cellular therapies. Lentiviral vectors are most effectively transferred into lymphocytes or hematopoietic progenitor cells using spinoculation. To enable cGMP (current Good Manufacturing Practice)-compliant cell therapy production, we developed and compared a closed-system spinoculation method that uses cell culture bags, and an automated closed system spinoculation method to decrease technician hands on time and reduce the likelihood for microbial contamination. METHODS: Sepax spinoculation, bag spinoculation, and static bag transduction without spinoculation were compared for lentiviral gene transfer in lymphocytes collected by apheresis. The lymphocytes were transduced once and cultured for 9 days. The lentiviral vectors tested encoded a CD19/CD22 Bispecific Chimeric Antigen Receptor (CAR), a FGFR4-CAR, or a CD22-CAR. Sepax spinoculation times were evaluated by testing against bag spinoculation and static transduction to optimize the Sepax spin time. The Sepax spinoculation was then used to test the transduction of different CAR vectors. The performance of the process using healthy donor and a patient sample was evaluated. Functional assessment was performed of the CD19/22 and CD22 CAR T-cells using killing assays against the NALM6 tumor cell line and cytokine secretion analysis. Finally, gene expression of the transduced T-cells was examined to determine if there were any major changes that may have occurred as a result of the spinoculation process. RESULTS: The process of spinoculation lead to significant enhancement in gene transfer. Sepax spinoculation using a 1-h spin time showed comparable transduction efficiency to the bag spinoculation, and much greater than the static bag transduction method (83.4%, 72.8%, 35.7% n = 3). The performance of three different methods were consistent for all lentiviral vectors tested and no significant difference was observed when using starting cells from healthy donor versus a patient sample. Sepax spinoculation does not affect the function of the CAR T-cells against tumor cells, as these cells appeared to kill target cells equally well. Spinoculation also does not appear to affect gene expression patterns that are necessary for imparting function on the cell. CONCLUSIONS: Closed system-bag spinoculation resulted in more efficient lymphocyte gene transfer than standard bag transductions without spinoculation. This method is effective for both retroviral and lentiviral vector gene transfer in lymphocytes and may be a feasible approach for gene transfer into other cell types including hematopoietic and myeloid progenitors. Sepax spinoculation further improved upon the process by offering an automated, closed system approach that significantly decreased hands-on time while also decreasing the risk of culture bag tears and microbial contamination.

Establishment and validation of in-house cryopreserved CAR/TCR-T cell flow cytometry quality control.

BACKGROUND: Chimeric antigen receptor (CAR) or T-cell receptor (TCR) engineered T-cell therapy has recently emerged as a promising adoptive immunotherapy approach for the treatment of hematologic malignancies and solid tumors. Multiparametric flow cytometry-based assays play a critical role in monitoring cellular manufacturing steps. Since manufacturing CAR/TCR T-cell products must be in compliance with current good manufacturing practices (cGMP), a standard or quality control for flow cytometry assays should be used to ensure the accuracy of flow cytometry results, but none is currently commercially available. Therefore, we established a procedure to generate an in-house cryopreserved CAR/TCR T-cell products for use as a flow cytometry quality control and validated their use. METHODS: Two CAR T-cell products: CD19/CD22 bispecific CAR T-cells and FGFR4 CAR T-cells and one TCR-engineered T-cell product: KK-LC-1 TCR T-cells were manufactured in Center for Cellular Engineering (CCE), NIH Clinical Center. The products were divided in aliquots, cryopreserved and stored in the liquid nitrogen. The cryopreserved flow cytometry quality controls were tested in flow cytometry assays which measured post-thaw viability, CD3, CD4 and CD8 frequencies as well as the transduction efficiency and vector identity. The long-term stability and shelf-life of cryopreserved quality control cells were evaluated. In addition, the sensitivity as well as the precision assay were also assessed on the cryopreserved quality control cells. RESULTS: After thawing, the viability of the cryopreserved CAR/TCR T-cell controls was found to be greater than 50%. The expression of transduction efficiency and vector identity markers by the cryopreserved control cells were stable for at least 1 year; with post-thaw values falling within ± 20% range of the values measured at time of cryopreservation. After thawing and storage at room temperature, the stability of these cryopreserved cells lasted at least 6 h. In addition, our cryopreserved CAR/TCR-T cell quality controls showed a strong correlation between transduction efficiency expression and dilution factors. Furthermore, the results of flow cytometric analysis of the cryopreserved cells among different laboratory technicians and different flow cytometry instruments were comparable, highlighting the reproducibility and reliability of these quality control cells. CONCLUSION: We developed and validated a feasible and reliable procedure to establish a bank of cryopreserved CAR/TCR T-cells for use as flow cytometry quality controls, which can serve as a quality control standard for in-process and lot-release testing of CAR/TCR T-cell products.

Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products.

BACKGROUND: Genetically engineered T cells have become an important therapy for B-cell malignancies. Measuring the efficiency of vector integration into the T cell genome is important for assessing the potency and safety of these cancer immunotherapies. METHODS: A digital droplet polymerase chain reaction (ddPCR) assay was developed and evaluated for assessing the average number of lenti- and retroviral vectors integrated into Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR)-engineered T cells. RESULTS: The ddPCR assay consistently measured the concentration of an empty vector in solution and the average number of CAR and TCR vectors integrated into T cell populations. There was a linear relationship between the average vector copy number per cell measured by ddPCR and the proportion of cells transduced as measured by flow cytometry. Similar vector copy number measurements were obtained by different staff using the ddPCR assay, highlighting the assays reproducibility among technicians. Analysis of fresh and cryopreserved CAR T and TCR engineered T cells yielded similar results. CONCLUSIONS: ddPCR is a robust tool for accurate quantitation of average vector copy number in CAR and TCR engineered T cells. The assay is also applicable to other types of genetically engineered cells including Natural Killer cells and hematopoietic stem cells.

Group O plasma as a media supplement for CAR-T cells and other adoptive T-cell therapies.

BACKGROUND: Most chimeric antigen receptor T (CAR-T) cells and other adoptive T-cell therapies (ACTs) are currently manufactured by ex vivo expansion of patient lymphocytes in culture media supplemented with human plasma from group AB donors. As lymphocytes do not express A or B antigens, the isoagglutinins of non-AB plasmas are unlikely to cause deleterious effects on lymphocytes in culture. STUDY DESIGN AND METHODS: Seeding cultures with peripheral blood mononuclear cell (PBMNC) concentrates from group A1 donors and using a CAR-T culture protocol, parallel cultures were performed, each with unique donor plasmas as media supplements (including group O plasmas with high-titer anti-A and group AB plasmas as control). An additional variable, a 3% group A1 red blood cell (RBC) spike, was added to simulate a RBC-contaminated PBMNC collection. Cultures were monitored by cell count, viability, flow cytometric phenotype, gene expression analysis, and supernatant chemokine analysis. RESULTS: There was no difference in lymphocyte expansion or phenotype when cultured with AB plasma or O plasma with high-titer anti-A. Compared to controls, the presence of contaminating RBCs in lymphocyte culture led to poor lymphocyte expansion and a less desirable phenotype-irrespective of the isoagglutinin titer of the plasma supplement used. CONCLUSIONS: This study suggests that ABO incompatible plasma may be used as a media supplement when culturing cell types that do not express ABO antigens-such as lymphocytes for CAR-T or other ACT. The presence of contaminating RBCs in culture was disadvantageous independent of isoagglutinin titer.

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.

Single cell sequencing reveals gene expression signatures associated with bone marrow stromal cell subpopulations and time in culture.

BACKGROUND: Bone marrow stromal cells (BMSCs) are a heterogeneous population that participates in wound healing, immune modulation and tissue regeneration. Next generation sequencing was used to analyze transcripts from single BMSCs in order to better characterize BMSC subpopulations. METHODS: Cryopreserved passage 2 BMSCs from one healthy subject were cultured through passage 10. The transcriptomes of bulk BMSCs from designated passages were analyzed with microarrays and RNA sequencing (RNA-Seq). For some passages, single BMSCs were separated using microfluidics and their transcriptomes were analyzed by RNA-Seq. RESULTS: Transcriptome analysis by microarray and RNA-Seq of unseparated BMSCs from passages 2, 4, 6, 8, 9 and 10 yielded similar results; both data sets grouped passages 4 and 6 and passages 9 and 10 together and genes differentially expressed among these early and late passage BMSCs were similar. 3D Diffusion map visualization of single BMSCs from passages 3, 4, 6, 8 and 9 clustered passages 3 and 9 into two distinct groups, but there was considerable overlap for passages 4, 6 and 8 cells. Markers for early passage, FGFR2, and late passage BMSCs, PLAT, were able to identify three subpopulations within passage 3 BMSCs; one that expressed high levels of FGFR2 and low levels of PLAT; one that expressed low levels of FGFR2 and high levels of PLAT and one that expressed intermediate levels of FGFR2 and low levels of PLAT. CONCLUSIONS: Single BMSCs can be separated by microfluidics and their transcriptome analyzed by next generation sequencing. Single cell analysis of early passage BMSCs identified a subpopulation of cells expressing high levels of FGFR2 that might include skeletal stem cells.

Production of a cellular product consisting of monocytes stimulated with Sylatron® (Peginterferon alfa-2b) and Actimmune® (Interferon gamma-1b) for human use.

BACKGROUND: Monocytes are myeloid cells that reside in the blood and bone marrow and respond to inflammation. At the site of inflammation, monocytes express cytokines and chemokines. Monocytes have been shown to be cytotoxic to tumor cells in the presence of pro-inflammatory cytokines such as Interferon Alpha, Interferon Gamma, and IL-6. We have previously shown that monocytes stimulated with both interferons (IFNs) results in synergistic killing of ovarian cancer cells. We translated these observations to an ongoing clinical trial using adoptive cell transfer of autologous monocytes stimulated ex vivo with IFNs and infused into the peritoneal cavity of patients with advanced, chemotherapy resistant, ovarian cancer. Here we describe the optimization of the monocyte elutriation protocol and a cryopreservation protocol of the monocytes isolated from peripheral blood. METHODS: Counter flow elutriation was performed on healthy donors or women with ovarian cancer. The monocyte-containing, RO-fraction was assessed for total monocyte number, purity, viability, and cytotoxicity with and without a cryopreservation step. All five fractions obtained from the elutriation procedure were also assessed by flow cytometry to measure the percent of immune cell subsets in each fraction. RESULTS: Both iterative monocyte isolation using counter flow elutriation or cryopreservation following counter flow elutriation can yield over 2 billion monocytes for each donor with high purity. We also show that the monocytes are stable, viable, and retain cytotoxic functions when cultured with IFNs. CONCLUSION: Large scale isolation of monocytes from both healthy donors and patients with advanced, chemotherapy resistant ovarian cancer, can be achieved with high total number of monocytes. These monocytes can be cryopreserved and maintain viability and cytotoxic function. All of the elutriated cell fractions contain ample immune cells which could be used for other cell therapy-based applications.

Effects of starting cellular material composition on chimeric antigen receptor T-cell expansion and characteristics.

BACKGROUND: When manufacturing chimeric antigen receptor (CAR) T cells using anti-CD3/anti-CD28 beads, ex vivo T-cell expansion is dependent on the composition of leukocytes used in the manufacturing process. We investigated the effects of leukocyte composition on CAR T-cell expansion and characteristics using an alternative manufacturing method. METHODS: Anti-B-cell maturation antigen and CD19-CAR T cells were manufactured using autologous peripheral blood mononuclear cell (PBMNC) concentrates. The PBMNCs were enriched for lymphocytes using density gradient separation, which were used for CAR T-cell culture initiation. T-cell expansion was stimulated with soluble anti-CD3 and interleukin-2. RESULTS: Fifty-one CAR T-cell products were evaluated; 28 anti-B-cell maturation antigen (BCMA) CAR T cells produced for 24 patients and 27 CD19 CAR T cells produced for 24 patients. CAR T-cell expansion was reduced when greater quantities of monocytes were present in the post-density gradient separation PBMNCs. In addition, the ratio of CD4 to CD8 cells in the CAR T-cell products after 7 days of culture was dependent on the quantity of monocytes, RBCs, and neutrophils in the post-density gradient separation PBMNCs. Greater quantities of monocytes and RBCs were associated with a greater proportion of CD4+ cells and greater quantities of neutrophils were associated with a greater proportion of CD8+ cells. CONCLUSIONS: The composition of leukocytes used to manufacture CAR T cells can affect cell expansion and the composition of CAR T-cell products. More uniform or complete lymphocyte enrichment of PBMNCs improves the consistency of final CAR T-cell products.

Enhanced clinical-scale manufacturing of TCR transduced T-cells using closed culture system modules.

BACKGROUND: Genetic engineering of T-cells to express specific T cell receptors (TCR) has emerged as a novel strategy to treat various malignancies. More widespread utilization of these types of therapies has been somewhat constrained by the lack of closed culture processes capable of expanding sufficient numbers of T-cells for clinical application. Here, we evaluate a process for robust clinical grade manufacturing of TCR gene engineered T-cells. METHODS: TCRs that target human papillomavirus E6 and E7 were independently tested. A 21 day process was divided into a transduction phase (7 days) and a rapid expansion phase (14 days). This process was evaluated using two healthy donor samples and four samples obtained from patients with epithelial cancers. RESULTS: The process resulted in ~ 2000-fold increase in viable nucleated cells and high transduction efficiencies (64-92%). At the end of culture, functional assays demonstrated that these cells were potent and specific in their ability to kill tumor cells bearing target and secrete large quantities of interferon and tumor necrosis factor. Both phases of culture were contained within closed or semi-closed modules, which include automated density gradient separation and cell culture bags for the first phase and closed GREX culture devices and wash/concentrate systems for the second phase. CONCLUSION: Large-scale manufacturing using modular systems and semi-automated devices resulted in highly functional clinical-grade TCR transduced T-cells. This process is now in use in actively accruing clinical trials and the NIH Clinical Center and can be utilized at other cell therapy manufacturing sites that wish to scale-up and optimize their processing using closed systems.

A Phase 1 trial of autologous monocytes stimulated ex vivo with Sylatron® (Peginterferon alfa-2b) and Actimmune® (Interferon gamma-1b) for intra-peritoneal administration in recurrent ovarian cancer.

BACKGROUND: Ovarian cancer has no definitive second line therapeutic options, and largely recurs in the peritoneal cavity. Locoregional immune therapy using both interferons and monocytes can be used as a novel approach. Interferons have both cytostatic and cytotoxic properties, while monocytes stimulated with interferons have potent cytotoxic properties. Due to the highly immune suppressive properties of ovarian cancer, ex vivo stimulation of autologous patient monocytes with interferons and infusion of all three agents intraperitoneally (IP) can provide a strong pro-inflammatory environment at the site of disease to kill malignant cells. METHODS: Patient monocytes are isolated through counterflow elutriation and stimulated ex vivo with interferons and infused IP through a semi-permanent catheter. We have designed a standard 3 + 3 dose escalation study to explore the highest tolerated dose of interferons and monocytes infused IP in patients with chemotherapy resistant ovarian cancer. Secondary outcome measurements of changes in the peripheral blood immune compartment and plasma cytokines will be studied for correlations of response. DISCUSSION: We have developed a novel immunotherapy focused on the innate immune system for the treatment of ovarian cancer. We have combined the use of autologous monocytes and interferons alpha and gamma for local-regional administration directly into the peritoneal cavity. This therapy is highly unique in that it is the first study of its type using only components of the innate immune system for the locoregional delivery consisting of autologous monocytes and dual interferons alpha and gamma. Trial Registration ClinicalTrials.gov Identifier: NCT02948426, registered on October 28, 2016. https://clinicaltrials.gov/ct2/show/NCT02948426.