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.

Expanding the reach of commercial cell therapies requires changes at medical centers.

The clinical application of cell therapies is becoming increasingly important for the treatment of cancer, congenital immune deficiencies, and hemoglobinopathies. These therapies have been primarily manufactured and used at academic medical centers. However, cell therapies are now increasingly being produced in centralized manufacturing facilities and shipped to medical centers for administration. Typically, these cell therapies are produced from a patient's own cells, which are the critical starting material. For these therapies to achieve their full potential, more medical centers must develop the infrastructure to collect, label, cryopreserve, test, and ship these cells to the centralized laboratories where these cell therapies are manufactured. Medical centers must also develop systems to receive, store, and infuse the finished cell therapy products. Since most cell therapies are cryopreserved for shipment and storage, medical centers using these therapies will require access to liquid nitrogen product storage tanks and develop procedures to thaw cell therapies. These services could be provided by the hospital pharmacy or transfusion service, but the latter is likely most appropriate. Another barrier to implementing these services is the variability among providers of these cell therapies in the processes related to handling cell therapies. The provision of these services by medical centers would be facilitated by establishing a national coordinating center and a network of apheresis centers to collect and cryopreserve the cells needed to begin the manufacturing process and cell therapy laboratories to store and issue the cells. In addition to organizing cell collections, the coordinating center could establish uniform practices for collecting, labeling, shipping, receiving, thawing, and infusing the cell therapy.

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.

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.

Healthcare center-based cell therapy laboratories supporting off-site manufactured cell therapies: The experiences of a single academic cell therapy laboratory.

BACKGROUND: Healthcare center-based cell therapy laboratories (HC CTLs) evolved from solely processing hematopoietic stem cells for transplantation to manufacturing various advanced cellular therapies. With increasing interest in cellular therapy applications, off-site manufactured products are becoming more common. HC CTLs play a critical role in supporting these products by shipping out cellular starting material (CSM) for further manufacturing and/or receiving, storing, and distributing final products. The experiences and challenges encountered by a single academic HC CTL in supporting these products are presented. METHODS: All off-site manufacturing protocols supported before 2023 were reviewed. Collected data included protocol characteristics (treatment indication, product type), process logistics (shipping, receiving, storage, thawing, distribution, documentation), and product handling volumes (CSM shipping and final product infusions). RESULTS: Between 2012 and 2022, 15 off-site manufactured cellular therapy early-phase, single- and multicenter clinical trials were supported. Trials were sponsored by academic/research and commercial entities. The number of protocols supported annually increased each year, with few ending. Products included cancer immunotherapies and gene therapies. Autologous CSM was collected and shipped, while autologous and allogeneic final products were received, stored, thawed, and distributed. Process differences among protocols included CSM shipping conditions, laboratory analyses, final product thaw conditions and procedures, number of treatments, and documentation. DISCUSSION: HC CTLs must contend with several challenges in supporting off-site manufacturing protocols. As demand for cellular therapies increases, stakeholders should collaborate from the early phases of clinical trials to streamline processes and standardize procedures to increase value, improve safety, and reduce the burden on HC CTLs.

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.

Effects of extended transport on cryopreserved allogeneic hematopoietic progenitor cell (HPC) product quality and optimal methods to assess HPC stability.

BACKGROUND: Since the beginning of the COVID-19 pandemic, cryopreservation of hematopoietic progenitor cell (HPC) products has been increasingly used to ensure allogeneic donor graft availability prior to recipient conditioning for transplantation. However, in addition to variables such as graft transport duration and storage conditions, the cryopreservation process itself may adversely affect graft quality. Furthermore, the optimal methods to assess graft quality have not yet been determined. STUDY DESIGN AND METHODS: A retrospective review was performed on all cryopreserved HPCs processed and thawed at our facility from 2007 to 2020, including both those collected onsite and by the National Marrow Donor Program (NMDP). HPC viability studies were also performed on fresh products, retention vials, and corresponding final thawed products by staining for 7-AAD (flow cytometry), AO/PI (Cellometer), and trypan blue (manual microscopy). Comparisons were made using the Mann-Whitney test. RESULTS: For HPC products collected by apheresis (HPC(A)), pre-cryopreservation and post-thaw viabilities, as well as total nucleated cell recoveries were lower for products collected by the NMDP compared to those collected onsite. However, there were no differences seen in CD34+ cell recoveries. Greater variation in viability testing was observed using image-based assays compared to flow-based assays, and on cryo-thawed versus fresh samples. No significant differences were observed between viability measurements obtained on retention vials versus corresponding final thawed product bags. DISCUSSION: Our studies suggest extended transport may contribute to lower post-thaw viabilities, but without affecting CD34+ cell recoveries. To assess HPC viability prior to thaw, testing of retention vials offers predictive utility, particularly when automated analyzers are used.

The Potential Use of THP-1, a Monocytic Leukemia Cell Line, to Predict Immune-Suppressive Potency of Human Bone-Marrow Stromal Cells (BMSCs) In Vitro: A Pilot Study.

Adoptive transfer of cultured BMSCs was shown to be immune-suppressive in various inflammatory settings. Many factors play a role in the process, but no master regulator of BMSC-driven immunomodulation was identified. Consequently, an assay that might predict BMSC product efficacy is still unavailable. Below, we show that BMSC donor variability can be monitored by IL-10 production of monocytes/macrophages using THP-1 cells (immortalized monocytic leukemia cells) co-cultured with BMSCs. Using a mixed lymphocyte reaction (MLR) assay, we also compared the ability of the different donor BMSCs to suppress T-cell proliferation, another measure of their immune-suppressive ability. We found that the BMSCs from a donor that induced the most IL-10 production were also the most efficient in suppressing T-cell proliferation. Transcriptome studies showed that the most potent BMSC batch also had higher expression of several known key immunomodulatory molecules such as hepatocyte growth factor (HGF), PDL1, and numerous members of the PGE2 pathway, including PTGS1 and TLR4. Multiplex ELISA experiments revealed higher expression of HGF and IL6 by the most potent BMSC donor. Based on these findings, we propose that THP-1 cells may be used to assess BMSC immunosuppressive activity as a product characterization assay.

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.

Identification of genomic determinants contributing to cytokine release in immunotherapies and human diseases.

BACKGROUND: Cytokine release syndrome (CRS) is a strong immune system response that can occur as a result of the reaction of a cellular immunotherapy with malignant cells. While the frequency and management of CRS in CAR T-cell therapy has been well documented, there is emerging interest in pre-emptive treatment to reduce CRS severity and improve overall outcomes. Accordingly, identification of genomic determinants that contribute to cytokine release may lead to the development of targeted therapies to prevent or abrogate the severity of CRS. METHODS: Forty three clinical CD22 CAR T-cell products were collected for RNA extraction. 100 ng of mRNA was used for Nanostring assay analysis which is based on the nCounter platform. Several public datasets were used for validation purposes. RESULTS: We found the expression of the PFKFB4 gene and glycolytic pathway activity were upregulated in CD22 CAR T-cells given to patients who developed CRS compared to those who did not experience CRS. Moreover, these results were further validated in cohorts with COVID-19, influenza infections and autoimmune diseases, and in tumor tissues. The findings were similar, except that glycolytic pathway activity was not increased in patients with influenza infections and systemic lupus erythematosus (SLE). CONCLUSION: Our data strongly suggests that PFKFB4 acts as a driving factor in mediating cytokine release in vivo by regulating glycolytic activity. Our results suggest that it would beneficial to develop drugs targeting PFKFB4 and the glycolytic pathway for the treatment of CRS.

Longitudinal transcriptional analysis of peripheral blood leukocytes in COVID-19 convalescent donors.

BACKGROUND: SARS-CoV2 can induce a strong host immune response. Many studies have evaluated antibody response following SARS-CoV2 infections. This study investigated the immune response and T cell receptor diversity in people who had recovered from SARS-CoV2 infection (COVID-19). METHODS: Using the nCounter platform, we compared transcriptomic profiles of 162 COVID-19 convalescent donors (CCD) and 40 healthy donors (HD). 69 of the 162 CCDs had two or more time points sampled. RESULTS: After eliminating the effects of demographic factors, we found extensive differential gene expression up to 241 days into the convalescent period. The differentially expressed genes were involved in several pathways, including virus-host interaction, interleukin and JAK-STAT signaling, T-cell co-stimulation, and immune exhaustion. A subset of 21 CCD samples was found to be highly "perturbed," characterized by overexpression of PLAU, IL1B, NFKB1, PLEK, LCP2, IRF3, MTOR, IL18BP, RACK1, TGFB1, and others. In addition, one of the clusters, P1 (n = 8) CCD samples, showed enhanced TCR diversity in 7 VJ pairs (TRAV9.1_TCRVA_014.1, TRBV6.8_TCRVB_016.1, TRAV7_TCRVA_008.1, TRGV9_ENST00000444775.1, TRAV18_TCRVA_026.1, TRGV4_ENST00000390345.1, TRAV11_TCRVA_017.1). Multiplexed cytokine analysis revealed anomalies in SCF, SCGF-b, and MCP-1 expression in this subset. CONCLUSIONS: Persistent alterations in inflammatory pathways and T-cell activation/exhaustion markers for months after active infection may help shed light on the pathophysiology of a prolonged post-viral syndrome observed following recovery from COVID-19 infection. Future studies may inform the ability to identify druggable targets involving these pathways to mitigate the long-term effects of COVID-19 infection. TRIAL REGISTRATION: https://clinicaltrials.gov/ct2/show/NCT04360278 Registered April 24, 2020.

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.

NAFLD causes selective CD4(+) T lymphocyte loss and promotes hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer-related death. Non-alcoholic fatty liver disease (NAFLD) affects a large proportion of the US population and is considered to be a metabolic predisposition to liver cancer. However, the role of adaptive immune responses in NAFLD-promoted HCC is largely unknown. Here we show, in mouse models and human samples, that dysregulation of lipid metabolism in NAFLD causes a selective loss of intrahepatic CD4(+) but not CD8(+) T lymphocytes, leading to accelerated hepatocarcinogenesis. We also demonstrate that CD4(+) T lymphocytes have greater mitochondrial mass than CD8(+) T lymphocytes and generate higher levels of mitochondrially derived reactive oxygen species (ROS). Disruption of mitochondrial function by linoleic acid, a fatty acid accumulated in NAFLD, causes more oxidative damage than other free fatty acids such as palmitic acid, and mediates selective loss of intrahepatic CD4(+) T lymphocytes. In vivo blockade of ROS reversed NAFLD-induced hepatic CD4(+) T lymphocyte decrease and delayed NAFLD-promoted HCC. Our results provide an unexpected link between lipid dysregulation and impaired anti-tumour surveillance.

Combined haploidentical and cord blood transplantation for refractory severe aplastic anaemia and hypoplastic myelodysplastic syndrome.

Umbilical cord blood (UCB) transplantation is a potentially curative treatment for patients with refractory severe aplastic anaemia (SAA), but has historically been associated with delayed engraftment and high graft failure and mortality rates. We conducted a prospective phase 2 trial to assess outcome of an allogeneic transplant regimen that co-infused a single UCB unit with CD34+ -selected cells from a haploidentical relative. Among 29 SAA patients [including 10 evolved to myelodysplastic syndrome (MDS)] who underwent the haplo cord transplantation (median age 20 years), 97% had neutrophil recovery (median 10 days), and 93% had platelet recovery (median 32 days). Early myeloid engraftment was from the haplo donor and was gradually replaced by durable engraftment from UCB in most patients. The cumulative incidences of grade II-IV acute and chronic graft-versus-host disease (GVHD) were 21% and 41%, respectively. With a median follow-up of 7·5 years, overall survival was 83% and GVHD/relapse-free survival was 69%. Patient- and transplant-related factors had no impact on engraftment and survival although transplants with haplo-versus-cord killer-cell immunoglobulin-like receptor (KIR) ligand incompatibility had delayed cord engraftment. Our study shows haplo cord transplantation is associated with excellent engraftment and long-term outcome, providing an alternative option for patients with refractory SAA and hypoplastic MDS who lack human leucocyte antigen (HLA)-matched donors.

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.