Mitigation of checkpoint inhibitor-induced autoimmune hemolytic anemia through modulation of purinergic signaling.

ABSTRACT: Immune checkpoint inhibitors (ICPis) have revolutionized cancer immunotherapy but also can induce autoimmune hemolytic anemia (AIHA), a severe disease with high mortality. However, the cellular and molecular mechanism(s) of AIHA secondary to ICPi therapy (ICPi-AIHA) are unclear, other than being initiated through decreased checkpoint inhibition. Herein, we report ICPi-AIHA in a novel mouse model that shows similar characteristics of known human ICPi-AIHA (eg, autoantibodies, hemolysis, and increased mortality). During ICPi-AIHA, there is the simultaneous reduction of 2 regulatory T-cell populations (FoxP3+ and Tr1 [type 1 regulatory cells]) and an increase in inflammatory T helper cell 17 (TH17). Moreover, a novel CD39+CD73-FoxP3-CD25- CD4+ T-cell subset (ie, CD39 single positive [CD39SP]) emerges, and early increases in CD39SP predict AIHA development; CD39 is an ectonuclease that breaks down adenosine triphosphate (ATP). Additionally, we found that boosting ATPase activity by injecting recombinant apyrase mitigates AIHA development and significant CD39SP reductions, both suggesting a functional role for CD39 and demonstrating a novel therapeutic approach. Importantly, CD39SP are detectable in multiple mouse models developing AIHA and in patients with AIHA, demonstrating applicability to idiopathic and secondary AIHA. Highlighting broader autoimmunity relevance, ICPi-treated NZB mice experienced accelerated onset and severity of lupus, including AIHA. Moreover, ICPi treatment of healthy B6 animals led to detectable CD39SP and development of autoantibodies against multiple autoantigens including those on red blood cells and platelets. Together, our findings provide further insight into the cellular and molecular mechanisms of ICPi-AIHA, leading to novel diagnostic and therapeutic approaches with translational potential for use in humans being treated with ICPi.

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).

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.

Association of ABO mismatch with the outcomes of allogeneic hematopoietic cell transplantation for acute leukemia.

Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative treatment for acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). While many factors influence the outcomes of allo-HCT, the independent impact of donor-recipient ABO mismatching remains unclear. Using the Center for International Blood and Marrow Transplant Research (CIBMTR) database, we identified patients aged ≥18 years with AML or ALL who underwent allo-HCT between 2008 and 2018. Our objectives were to analyze the outcomes of allo-HCT based on the donor-recipient ABO status (match, minor mismatch, major mismatch, bidirectional mismatch). Among 4946 eligible patients, 2741 patients (55.4%) were ABO matched, 1030 patients (20.8%) had a minor ABO mismatch, 899 patients (18.1%) had a major ABO mismatch, and 276 patients (5.6%) had a bidirectional ABO mismatch. In multivariable analyses, compared to ABO matched allo-HCT, the presence of a major ABO mismatch was associated with worse overall survival (HR 1.16, 95% CI 1.05-1.29; p = 0.005), inferior platelet engraftment (HR 0.83, 95% CI 0.77-0.90; p < 0.001), and higher primary graft failure (HR 1.60, 95% CI 1.12-2.30, p = 0.01). Relapse, acute graft versus host disease (GVHD) grades III-IV and chronic GVHD were not significantly associated with ABO status. While donor age was not significantly associated with outcomes, older recipient age was associated with worse survival and non-relapse mortality. Our study demonstrates that donor-recipient ABO status is independently associated with survival and other post-transplantation outcomes in acute leukemia. This underscores the importance of considering the ABO status in donor selection algorithms and its impact in acute leukemia.

Additional efficacy analysis of avatrombopag phase III data for the treatment of adults with immune thrombocytopenia.

Avatrombopag is an oral thrombopoietin receptor agonist (TPO-RA) that was approved in the US in 2019 for treatment of chronic immune thrombocytopenia (ITP). This post hoc analysis of the pivotal phase III study (NCT01438840) of avatrombopag in adult patients with ITP evaluated platelet count response to avatrombopag during the core study in different subgroups, and durability of response data in patients who responded to avatrombopag treatment both during the core phase (total population) and during the core and extension phase (total population and by subgroup). Loss of response (LOR [platelet count <30 × 109/L]) was defined as LOR over two consecutive scheduled visits. The response was generally similar between subgroups though a few differences were observed. The durability of response analysis showed that avatrombopag-treated patients maintained their response for 84.5% of time on treatment during the core phase and 83.3% during the core and extension phase; 55.2% of patients in the core phase and 52.3% in the core and extension phase never experienced LOR. We conclude that the initial response to avatrombopag is both stable and durable.

What is the context? Avatrombopag is a medicine that helps the body produce platelet cells, which are necessary for blood clotting.Avatrombopag is used to treat people with chronic immune thrombocytopenia (ITP); these patients have low numbers of platelet cells in their blood, so their blood may not clot efficiently, putting them at risk of uncontrolled bleeding.A phase III clinical study in patients with chronic ITP showed that platelet counts increased for most patients who were treated with avatrombopag; patients who had a platelet count ≥50 × 10⁹/L were considered to have a response to avatrombopag treatment.The present study analyzes data from a phase III clinical study to determine whether there are any characteristics that make a patient more or less likely to have a loss of response (LOR) while taking avatrombopag, whether the initial response to avatrombopag is stable, and how long the response lasts.For this analysis, LOR is defined as platelet count <30 × 10⁹/L for either four consecutive weeks or for two consecutive office visits while taking avatrombopag.What is new? In general, patient characteristics did not influence the likelihood of LOR.Patients who responded maintained their response for most of the time they were taking avatrombopag.Most patients did not experience LOR.What is the impact? The initial response to avatrombopag is stable and long-lasting in patients with chronic ITP.These findings indicate that patients with a variety of background characteristics can experience a durable platelet response with avatrombopag treatment.

Optimizing haematopoietic stem and progenitor cell apheresis collection from plerixafor-mobilized patients with sickle cell disease.

We adjusted haematopoietic stem and progenitor cell (HSPC) apheresis collection from patients with sickle cell disease (SCD) by targeting deep buffy coat collection using medium or low collection preference (CP), and by increasing anticoagulant-citrate-dextrose-solution A dosage. In 43 HSPC collections from plerixafor-mobilized adult patients with SCD, we increased the collection efficiency to 35.79% using medium CP and 82.23% using low CP. Deep buffy coat collection increased red blood cell contamination of the HSPC product, the product haematocrit was 4.7% with medium CP and 6.4% with low CP. These adjustments were well-tolerated and allowed efficient HSPC collection from SCD patients.

Benralizumab for PDGFRA-Negative Hypereosinophilic Syndrome.

BACKGROUND: Hypereosinophilic syndrome is a group of diseases defined by marked eosinophilia in blood or tissue and eosinophil-related clinical manifestations. Benralizumab is a monoclonal antibody against interleukin-5 receptor α, which is expressed on human eosinophils. METHODS: In this randomized, double-blind, placebo-controlled, phase 2 trial, we administered a series of three monthly subcutaneous injections of either benralizumab (at a dose of 30 mg) or placebo in 20 symptomatic patients who had PDGFRA-negative hypereosinophilic syndrome and an absolute eosinophil count of at least 1000 cells per cubic millimeter; all the patients were receiving stable therapy (drugs or dietary changes) for this disease. This regimen was followed by an open-label phase, during which the patient's background therapy could be tapered as tolerated, and an extension phase. The primary end point of the randomized phase was a reduction of at least 50% in the absolute eosinophil count at week 12. RESULTS: During the randomized phase, the primary end point occurred in more patients in the benralizumab group than in the placebo group (9 of 10 patients [90%] vs. 3 of 10 patients [30%], P = 0.02). During the open-label phase, clinical and hematologic responses were observed in 17 of 19 patients (89%) and were sustained for 48 weeks in 14 of 19 patients (74%); in the latter group, in 9 of 14 patients (64%), background therapies could be tapered. Bone marrow and tissue eosinophilia were also suppressed with benralizumab therapy. The most common drug-related adverse events, headache and an elevated lactate dehydrogenase level, occurred in 32% of the patients after the first dose of benralizumab and resolved within 48 hours in all patients. Other adverse events occurred with similar frequency in the two groups. Of the many potential predictors of response that were examined, only clinical disease subtype appeared to be associated with the initial response or relapse. CONCLUSIONS: In this small phase 2 trial, patients with PDGFRA-negative hypereosinophilic syndrome who received benralizumab for 12 weeks had lower absolute eosinophil counts than those who received placebo. During the open-label phase, clinical and hematologic responses were sustained for 48 weeks in 74% of the patients. Adverse events did not limit treatment. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov numbers, NCT00001406 and NCT02130882.).

The need for uniform and coordinated practices involving centrally manufactured cell therapies.

Cellular therapies have become an important part of clinical care. The treatment of patients with cell therapies often involves the collection of autologous cells at the medical center treating the patient, the shipment of these cells to a centralized manufacturing site, and the return of the cryopreserved clinical cell therapy to the medical center treating the patient for storage until infusion. As this activity grows, cell processing laboratories at many academic medical centers are involved with many different autologous products manufactured by several different centralized laboratories. The handling of these products by medical center-based cell therapy laboratories is complicated and resource-intensive since each centralized manufacturing laboratory has unique methods for labeling, storing, shipping, receiving, thawing, and infusing the cells. The field would benefit from the development of more uniform practices. The development of a coordinating center similar to those established to facilitate the collection, shipping, and transplantation of hematopoietic stem cells from unrelated donors would also be beneficial. In summary, the wide range of practices involved with labeling, shipping, freezing, thawing, and infusing centrally manufactured autologous cellular therapies lack efficiency and consistency and puts patients at risk. More uniform practices are needed.

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.

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.

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.

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.

Establishment of a cell processing laboratory to support hematopoietic stem cell transplantation and chimeric antigen receptor (CAR)-T cell therapy.

Cell processing laboratories are an important part of cancer treatment centers. Cell processing laboratories began by supporting hematopoietic stem cell (HSC) transplantation programs. These laboratories adapted closed bag systems, centrifuges, sterile connecting devices and other equipment used in transfusion services/blood banks to remove red blood cells and plasma from marrow and peripheral blood stem cells products. The success of cellular cancer immunotherapies such as Chimeric Antigen Receptor (CAR) T-cells has increased the importance of cell processing laboratories. Since many of the diseases successfully treated by CAR T-cell therapy are also treated by HSC transplantation and since HSC transplantation teams are well suited to manage patients treated with CAR T-cells, many cell processing laboratories have begun to produce CAR T-cells. The methods that have been used to process HSCs have been modified for T-cell enrichment, culture, stimulation, transduction and expansion for CAR T-cell production. While processing laboratories are well suited to manufacture CAR T-cells and other cellular therapies, producing these therapies is challenging. The manufacture of cellular therapies requires specialized facilities which are costly to build and maintain. The supplies and reagents, especially vectors, can also be expensive. Finally, highly skilled staff are required. The use of automated equipment for cell production may reduce labor requirements and the cost of facilities. The steps used to produce CAR T-cells are reviewed, as well as various strategies for establishing a laboratory to manufacture these cells.

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.

Dexpramipexole as an oral steroid-sparing agent in hypereosinophilic syndromes.

Hypereosinophilic syndromes (HESs) are a heterogeneous group of disorders characterized by peripheral eosinophilia and eosinophil-related end organ damage. Whereas most patients respond to glucocorticoid (GC) therapy, high doses are often necessary, and side effects are common. Dexpramipexole (KNS-760704), an orally bioavailable synthetic aminobenzothiazole, showed an excellent safety profile and was coincidentally noted to significantly decrease absolute eosinophil counts (AECs) in a phase 3 trial for amyotrophic lateral sclerosis. This proof-of-concept study was designed to evaluate dexpramipexole (150 mg orally twice daily) as a GC-sparing agent in HESs. Dual primary end points were (1) the proportion of subjects with ≥50% decrease in the minimum effective GC dose (MED) to maintain AEC <1000/µL and control clinical symptoms, and (2) the MED after 12 weeks of dexpramipexole (MEDD) as a percentage of the MED at week 0. Out of 10 subjects, 40% (95% confidence interval [CI], 12%, 74%) achieved a ≥50% reduction in MED, and the MEDD/MED ratio was significantly <100% (median, 66%; 95% CI, 6%, 98%; P = .03). All adverse events were self-limited, and none led to drug discontinuation. Affected tissue biopsy samples in 2 subjects showed normalization of pathology and depletion of eosinophils on dexpramipexole. Bone marrow biopsy samples after 12 weeks of dexpramipexole showed selective absence of mature eosinophils in responders. Dexpramipexole appears promising as a GC-sparing agent without apparent toxicity in a subset of subjects with GC-responsive HESs. Although the exact mechanism of action is unknown, preliminary data suggest that dexpramipexole may affect eosinophil maturation in the bone marrow. This study was registered at www.clinicaltrials.gov as #NCT02101138.

The impact of aging on primate hematopoiesis as interrogated by clonal tracking.

Age-associated changes in hematopoietic stem and progenitor cells (HSPCs) have been carefully documented in mouse models but poorly characterized in primates and humans. To investigate clinically relevant aspects of hematopoietic aging, we compared the clonal output of thousands of genetically barcoded HSPCs in aged vs young macaques after autologous transplantation. Aged macaques showed delayed emergence of output from multipotent (MP) clones, with persistence of lineage-biased clones for many months after engraftment. In contrast to murine aging models reporting persistence of myeloid-biased HSPCs, aged macaques demonstrated persistent output from both B-cell and myeloid-biased clones. Clonal expansions of MP, myeloid-biased, and B-biased clones occurred in aged macaques, providing a potential model for human clonal hematopoiesis of indeterminate prognosis. These results suggest that long-term MP HSPC output is impaired in aged macaques, resulting in differences in the kinetics and lineage reconstitution patterns between young and aged primates in an autologous transplantation setting.

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.

Prospective Evaluation of a Practical Guideline for Managing Positive Sterility Test Results in Cell Therapy Products.

Product safety assurance is crucial for the clinical use of manufactured cellular therapies. A rational approach for delivering products that fail release criteria (because of potentially false-positive sterility results) is important to avoid unwarranted wastage of highly personalized and costly therapies in critically ill patients where benefits may outweigh risk. Accurate and timely interpretation of microbial sterility assays represents a major challenge in cell therapies. We developed a systematic protocol for the assessment of positive microbial sterility test results using retrospective data from 2007 to 2016. This protocol was validated and applied prospectively between October 2016 and September 2017 to 13 products from which positive sterility results had been reported. Viable and nonviable environmental monitoring (EM) data were collected concurrently as part of a facility control assessment. Three of 13 (23%) positive sterility results were attributable to bone marrow collections that had been contaminated with skin flora during harvest; all were infused without pertinent infectious sequelae. Of the remaining 10, 1 was deemed a true positive and was discarded before infusion, whereas 9 were classified as false positives attributed to laboratory sampling and/or culturing processes. Three products deemed false positive were infused and 6 were withheld because of patient issues unrelated to microbial sterility results. No postinfusion-associated infectious complications were documented. Almost half of the positive EM findings were skin flora. Paired detection of an organism in both product and associated EM was identified in 1 case. Application of our validated protocol to positive product sterility test results allowed for systematic data compilation for regulatory evaluation and provided comprehensive information to clinical investigators to ensure timely and strategic management for product recipients.

How do I structure logistic processes in preparation for outsourcing of cellular therapy manufacturing?

As cell and gene therapies (CGT) assume center stage in early-phase clinical trials for several acute and chronic diseases, there is heightened interest in the standardization and automation of manufacturing processes in preparation for commercialization. Toward this goal, a hybrid and oftentimes geographically separated model comprising regional cell procurement and infusion facilities and a centralized cell manufacturing unit is gaining traction in the field. Although CGT processing facilities in academic institutions are not involved directly in the manufacturing of these therapies, they must be prepared to collaborate with commercial or contract manufacturing organizations (CMOs) and be ready to address several supply-chain challenges that have emerged for autologous and allogeneic CGT. Academic center cell-processing facilities must handle many events up- and downstream of manufacturing such as donor screening, cell collection, product labeling, cryopreservation, transportation, and thaw infusion. These events merit closer evaluation in the context of multifacility manufacturing since standard procedures have yet to be established. Based on our institutional experience, we summarize logistical challenges encountered in the handling and distribution of CGT products in early phase studies, specifically those involving CMO (outsourced) manufacturing. We also make recommendations to standardize processes unique to the CGT supply chain, emphasizing the need to maintain needle-to-needle traceability from product collection to infusion. These guidelines will inform the development of more complex supply-chain models for larger-scale cell and gene therapeutics.