qPCR assay for detection of Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Elements from CAR-T and TCR-T cells in fresh and formalin-fixed tissue.

As adoptive cellular therapies become more commonplace in cancer care, there is a growing need to monitor site-specific localization of engineered cells-such as chimeric antigen receptor T (CAR-T) cells and T-cell receptor T (TCR-T) cells-in patients' tissues to understand treatment effectiveness as well as associated adverse events. Manufacturing CAR-T and TCR-T cells involves transduction with viral vectors commonly containing the WPRE gene sequence to enhance gene expression, providing a viable assay target unique to these engineered cells. Quantitative PCR (qPCR) is currently used clinically in fresh patient tissue samples and blood with target sequences specific to each immunotherapy product. Herein, we developed a WPRE-targeted qPCR assay that is broadly applicable for detection of engineered cell products in both fresh and archival formalin-fixed paraffin embedded (FFPE) tissues. Using both traditional PCR and SYBR Green PCR protocols, we demonstrate the use of this WPRE-targeted assay to successfully detect two CAR-T cell and two TCR-T cell products in FFPE tissue. Standard curve analysis reported a reproducible limit of detection at 100 WPRE copies per 20µL PCR reaction. This novel and inexpensive technique could provide better understanding of tissue abundance of engineered therapeutic T cells in both tumor and second-site toxicity tissues and provide quantitative assessment of immune effector cell trafficking in archival tissue.

Lisocabtagene Maraleucel in Relapsed/Refractory Mantle Cell Lymphoma: Primary Analysis of the Mantle Cell Lymphoma Cohort From TRANSCEND NHL 001, a Phase I Multicenter Seamless Design Study.

PURPOSE: To report the primary analysis results from the mantle cell lymphoma (MCL) cohort of the phase I seamless design TRANSCEND NHL 001 (ClinicalTrials.gov identifier: NCT02631044) study. METHODS: Patients with relapsed/refractory (R/R) MCL after ≥two lines of previous therapy, including a Bruton tyrosine kinase inhibitor (BTKi), an alkylating agent, and a CD20-targeted agent, received lisocabtagene maraleucel (liso-cel) at a target dose level (DL) of 50 × 106 (DL1) or 100 × 106 (DL2) chimeric antigen receptor-positive T cells. Primary end points were adverse events (AEs), dose-limiting toxicities, and objective response rate (ORR) by independent review committee per Lugano criteria. RESULTS: Of 104 leukapheresed patients, liso-cel was infused into 88. Median (range) number of previous lines of therapy was three (1-11) with 30% receiving ≥five previous lines of therapy, 73% of patients were age 65 years and older, 69% had refractory disease, 53% had BTKi refractory disease, 23% had TP53 mutation, and 8% had secondary CNS lymphoma. Median (range) on-study follow-up was 16.1 months (0.4-60.5). In the efficacy set (n = 83; DL1 + DL2), ORR was 83.1% (95% CI, 73.3 to 90.5) and complete response (CR) rate was 72.3% (95% CI, 61.4 to 81.6). Median duration of response was 15.7 months (95% CI, 6.2 to 24.0) and progression-free survival was 15.3 months (95% CI, 6.6 to 24.9). Most common grade ≥3 treatment-emergent AEs were neutropenia (56%), anemia (37.5%), and thrombocytopenia (25%). Cytokine release syndrome (CRS) was reported in 61% of patients (grade 3/4, 1%; grade 5, 0), neurologic events (NEs) in 31% (grade 3/4, 9%; grade 5, 0), grade ≥3 infections in 15%, and prolonged cytopenia in 40%. CONCLUSION: Liso-cel demonstrated high CR rate and deep, durable responses with low incidence of grade ≥3 CRS, NE, and infections in patients with heavily pretreated R/R MCL, including those with high-risk, aggressive disease.

Timing of anti-PD-L1 antibody initiation affects efficacy/toxicity of CD19 CAR T-cell therapy for large B-cell lymphoma.

ABSTRACT: More than half of the patients treated with CD19-targeted chimeric antigen receptor (CAR) T-cell immunotherapy for large B-cell lymphoma (LBCL) do not achieve durable remission, which may be partly due to PD-1/PD-L1-associated CAR T-cell dysfunction. We report data from a phase 1 clinical trial (NCT02706405), in which adults with LBCL were treated with autologous CD19 CAR T cells (JCAR014) combined with escalating doses of the anti-PD-L1 monoclonal antibody, durvalumab, starting either before or after CAR T-cell infusion. The addition of durvalumab to JCAR014 was safe and not associated with increased autoimmune or immune effector cell-associated toxicities. Patients who started durvalumab before JCAR014 infusion had later onset and shorter duration of cytokine release syndrome and inferior efficacy, which was associated with slower accumulation of CAR T cells and lower concentrations of inflammatory cytokines in the blood. Initiation of durvalumab before JCAR014 infusion resulted in an early increase in soluble PD-L1 (sPD-L1) levels that coincided with the timing of maximal CAR T-cell accumulation in the blood. In vitro, sPD-L1 induced dose-dependent suppression of CAR T-cell effector function, which could contribute to inferior efficacy observed in patients who received durvalumab before JCAR014. Despite the lack of efficacy improvement and similar CAR T-cell kinetics early after infusion, ongoing durvalumab therapy after JCAR014 was associated with re-expansion of CAR T cells in the blood, late regression of CD19+ and CD19- tumors, and enhanced duration of response. Our results indicate that the timing of initiation of PD-L1 blockade is a key variable that affects outcomes after CD19 CAR T-cell immunotherapy for adults with LBCL.

Abnormal bone marrow findings in patients following treatment with chimeric antigen receptor-T cell therapy.

BACKGROUND: Bone marrow (BM) assessment after CAR-T cell immunotherapy infusion is not routinely performed to monitor adverse events such as cytopenias, hemophagocytic lymphohistiocytosis, or infections. Our institution has performed BM biopsies as part of CAR-T cell treatment protocols, encompassing pre- and post-treatment time points and during long-term follow-up. METHODS: We conducted a systematic retrospective review of BM abnormalities observed in samples from 259 patients following CAR-T cell immunotherapy. We correlated BM pathology findings with mortality, relapse/residual disease, and laboratory values. RESULTS: At a median of 35.5 days post-CAR-T infusion, 25.5% showed severe marrow hypocellularity, and 6.2% showed serous atrophy, and peripheral blood cytopenias corroborated these observations. Marrow features associated with reduced disease burden post-CAR-T infusion include increased lymphocytes seen in 16 patients and an increase of macrophages or granulomatous response seen in 25 patients. However, a 100-day landmark analysis also showed increased marrow histiocytes were associated with lower survival (median OS 6.0 vs. 21.4 months, p = .026), as was grade 2-3 marrow reticulin (18 patients) (median OS 12.5 vs. 24.2 months, p = .034). CONCLUSIONS: These data represent the first systematic observations of BM changes in patients receiving CAR-T cell immunotherapy.

Factors associated with long-term outcomes of CD19 CAR T-cell therapy for relapsed/refractory CLL.

High response rates have been reported after CD19-targeted chimeric antigen receptor-modified (CD19 CAR) T-cell therapy for relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL), yet the factors associated with duration of response in this setting are poorly characterized. We analyzed long-term outcomes in 47 patients with R/R CLL and/or Richter transformation treated on our phase 1/2 clinical trial of CD19 CAR T-cell therapy with an updated median follow-up of 79.6 months. Median progression-free survival (PFS) was 8.9 months, and the 6-year PFS was 17.8%. Maximum standardized uptake value (hazard ratio [HR], 1.15; 95% confidence interval [CI], 1.07-1.23; P < .001) and bulky disease (≥5 cm; HR, 2.12; 95% CI, 1.06-4.26; P = .034) before lymphodepletion were associated with shorter PFS. Day +28 complete response by positron emission tomography-computed tomography (HR, 0.13; 95% CI, 0.04-0.40; P < .001), day +28 measurable residual disease (MRD) negativity by multiparameter flow cytometry (HR, 0.08; 95% CI, 0.03-0.22; P < .001), day +28 MRD negativity by next-generation sequencing (HR, 0.21; 95% CI, 0.08-0.51; P < .001), higher peak CD8+ CAR T-cell expansion (HR, 0.49; 95% CI; 0.36-0.68; P < .001), higher peak CD4+ CAR T-cell expansion (HR, 0.47; 95% CI; 0.33-0.69; P < .001), and longer CAR T-cell persistence (HR, 0.56; 95% CI, 0.44-0.72; P < .001) were associated with longer PFS. The 6-year duration of response and overall survival were 26.4% and 31.2%, respectively. CD19 CAR T-cell therapy achieved durable responses with curative potential in a subset of patients with R/R CLL. This trial was registered at www.clinicaltrials.gov as #NCT01865617.

Anakinra for Refractory Cytokine Release Syndrome or Immune Effector Cell-Associated Neurotoxicity Syndrome after Chimeric Antigen Receptor T Cell Therapy.

Chimeric antigen receptor-engineered (CAR)-T cell therapy remains limited by significant toxicities, including cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). The optimal management of severe and/or refractory CRS/ICANS remains ill-defined. Anakinra has emerged as a promising agent based on preclinical data, but its safety and efficacy in CAR-T therapy recipients are unknown. The primary objective of this study was to evaluate the safety of anakinra to treat refractory CRS and ICANS after CAR-T therapy. The secondary objective was to evaluate the impact of key treatment-, patient-, and disease-related variables on the time to CRS/ICANS resolution and treatment-related mortality (TRM). We retrospectively analyzed the outcomes of 43 patients with B cell or plasma cell malignancies treated with anakinra for refractory CRS or ICANS at 9 institutions in the United States and Spain between 2019 and 2022. Cause-specific Cox regression was used to account for competing risks. Multivariable cause-specific Cox regression was used to estimate the effect of anakinra dose on outcomes while minimizing treatment allocation bias by including age, CAR-T product, prelymphodepletion (pre-LD) ferritin, and performance status. Indications for anakinra treatment were grade ≥2 ICANS with worsening or lack of symptom improvement despite treatment with high-dose corticosteroids (n = 40) and grade ≥2 CRS with worsening symptoms despite treatment with tocilizumab (n = 3). Anakinra treatment was feasible and safe; discontinuation of therapy because of anakinra-related side effects was reported in only 3 patients (7%). The overall response rate (ORR) to CAR-T therapy was 77%. The cumulative incidence of TRM in the whole cohort was 7% (95% confidence interval [CI], 2% to 17%) at 28 days and 23% (95% CI, 11% to 38%) at 60 days after CAR-T infusion. The cumulative incidence of TRM at day 28 after initiation of anakinra therapy was 0% in the high-dose (>200 mg/day i.v.) recipient group and 47% (95% CI, 20% to 70%) in the low-dose (100 to 200 mg/day s.c. or i.v.) recipient group. The median cumulative incidence of CRS/ICANS resolution from the time of anakinra initiation was 7 days in the high-dose group and was not reached in the low-dose group, owing to the high TRM in this group. Univariate Cox modeling suggested a shorter time to CRS/ICANS resolution in the high-dose recipients (hazard ratio [HR], 2.19; 95% CI, .94 to 5.12; P = .069). In a multivariable Cox model for TRM including age, CAR-T product, pre-LD ferritin level, and pre-LD Karnofsky Performance Status (KPS), higher anakinra dose remained associated with lower TRM (HR, .41 per 1 mg/kg/day increase; 95% CI, .17 to .96; P = .039. The sole factor independently associated with time to CRS/ICANS resolution in a multivariable Cox model including age, CAR-T product, pre-LD ferritin and anakinra dose was higher pre-LD KPS (HR, 1.05 per 10% increase; 95% CI, 1.01 to 1.09; P = .02). Anakinra treatment for refractory CRS or ICANS was safe at doses up to 12 mg/kg/day i.v. We observed an ORR of 77% after CAR-T therapy despite anakinra treatment, suggesting a limited impact of anakinra on CAR-T efficacy. Higher anakinra dose may be associated with faster CRS/ICANS resolution and was independently associated with lower TRM. Prospective comparative studies are needed to confirm our findings.

CD19 CAR-T therapy in solid organ transplant recipients: case report and systematic review.

Post-transplant lymphoproliferative disorder (PTLD) is a leading cause of cancer death in solid organ transplant recipients (SOTRs). Relapsed or refractory (R/R) PTLD portends a high risk of death and effective management is not well established. CD19-targeted CAR-T cell therapy has been utilized, but the risks and benefits are unknown. We report the first case of diffuse large B-cell lymphoma (DLBCL) PTLD treated with lisocabtagene maraleucel and present a systematic literature review of SOTRs with PTLD treated with CD19 CAR-T therapy. Our patient achieved a complete response (CR) with limited toxicity but experienced a CD19+ relapse 8 months after infusion despite CAR-T persistence. Literature review revealed 14 DLBCL and 2 Burkitt lymphoma PTLD cases treated with CD19 CAR-T cells. Kidney (n = 12), liver (n = 2), heart (n = 2), and pancreas after kidney (n = 1) transplant recipients were analyzed. The objective response rate (ORR) was 82.4% (14/17), with 58.5% (10/17) CRs and a 6.5-month median duration of response. Among kidney transplant recipients, the ORR was 91.7% (11/12). Allograft rejection occurred in 23.5% (4/17). No graft failure occurred. Our analysis suggests that CD19 CAR-T therapy offers short-term effectiveness and manageable toxicity in SOTRs with R/R PTLD. Further investigation through larger datasets and prospective study is needed.

A novel polymer-conjugated human IL-15 improves efficacy of CD19-targeted CAR T-cell immunotherapy.

Chimeric antigen receptor (CAR)-modified T-cell therapies targeting CD19 represent a new treatment option for patients with relapsed/refractory (R/R) B-cell malignancies. However, CAR T-cell therapy fails to elicit durable responses in a significant fraction of patients. Limited in vivo proliferation and survival of infused CAR T cells are key causes of failure. In a phase 1/2 clinical trial of CD19 CAR T cells for B-cell malignancies (#NCT01865617), low serum interleukin 15 (IL-15) concentration after CAR T-cell infusion was associated with inferior CAR T-cell kinetics. IL-15 supports T-cell proliferation and survival, and therefore, supplementation with IL-15 may enhance CAR T-cell therapy. However, the clinical use of native IL-15 is challenging because of its unfavorable pharmacokinetic (PK) and toxicity. NKTR-255 is a polymer-conjugated IL-15 that engages the entire IL-15 receptor complex (IL-15Rα/IL-2Rßγ) and exhibits reduced clearance, providing sustained pharmacodynamic (PD) responses. We investigated the PK and immune cell PDs in nonhuman primates treated with NKTR-255 and found that NKTR-255 enhanced the in vivo proliferation of T cells and natural killer cells. In vitro, NKTR-255 induced dose-dependent proliferation and accumulation of human CD19 CAR T cells, especially at low target cell abundance. In vivo studies in lymphoma-bearing immunodeficient mice demonstrated enhanced antitumor efficacy of human CD19 CAR T cells. In contrast to mice treated with CAR T cells alone, those that received CAR T cells and NKTR-255 had markedly higher CAR T-cell counts in the blood and marrow that were sustained after tumor clearance, without evidence of persistent proliferation or ongoing activation/exhaustion as assessed by Ki-67 and inhibitory receptor coexpression. These data support an ongoing phase 1 clinical trial of combined therapy with CD19 CAR T cells and NKTR-255 for R/R B-cell malignancies.

Impact of CD19 CAR T-cell product type on outcomes in relapsed or refractory aggressive B-NHL.

CD19-targeted chimeric antigen receptor-engineered (CD19 CAR) T cells are novel therapies showing great promise for patients with relapsed or refractory (R/R) aggressive B-cell non-Hodgkin lymphoma (B-NHL). Single-arm studies showed significant variations in outcomes across distinct CD19 CAR T-cell products. To estimate the independent impact of the CAR T-cell product type on outcomes, we retrospectively analyzed data from 129 patients with R/R aggressive B-NHL treated with cyclophosphamide and fludarabine lymphodepletion followed by either a commercially available CD19 CAR T-cell therapy (axicabtagene ciloleucel [axicel] or tisagenlecleucel [tisacel]), or the investigational product JCAR014 on a phase 1/2 clinical trial (NCT01865617). After adjustment for age, hematopoietic cell transplantation-specific comorbidity index, lactate dehydrogenase (LDH), largest lesion diameter, and absolute lymphocyte count (ALC), CAR T-cell product type remained associated with outcomes in multivariable models. JCAR014 was independently associated with lower cytokine release syndrome (CRS) severity compared with axicel (adjusted odds ratio [aOR], 0.19; 95% confidence interval [CI]; 0.08-0.46), with a trend toward lower CRS severity with tisacel compared with axicel (aOR, 0.47; 95% CI, 0.21-1.06; P = .07). Tisacel (aOR, 0.17; 95% CI, 0.06-0.48) and JCAR014 (aOR, 0.17; 95% CI, 0.06-0.47) were both associated with lower immune effector cell-associated neurotoxicity syndrome severity compared with axicel. Lower odds of complete response (CR) were predicted with tisacel and JCAR014 compared with axicel. Although sensitivity analyses using either positron emission tomography- or computed tomography-based response criteria also suggested higher efficacy of axicel over JCAR014, the impact of tisacel vs axicel became undetermined. Higher preleukapheresis LDH, largest lesion diameter, and lower ALC were independently associated with lower odds of CR. We conclude that CD19 CAR T-cell product type independently impacts toxicity and efficacy in R/R aggressive B-NHL patients.

Severe cytokine release syndrome is associated with hematologic toxicity following CD19 CAR T-cell therapy.

CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy in patients with relapsed/refractory B-cell malignancies; however, it is associated with toxicities including cytokine release syndrome (CRS), neurotoxicity, and impaired hematopoietic recovery. The latter is associated with high-grade cytopenias requiring extended growth factor or transfusional support, potentially leading to additional complications such as infection or hemorrhage. To date, the factors independently associated with hematologic toxicity have not been well characterized. To address this deficit, we retrospectively analyzed 173 patients who received defined-composition CD19 CAR T-cell therapy in a phase 1/2 clinical trial (https://clinicaltrials.gov; NCT01865617), with primary end points of absolute neutrophil count and platelet count at day-28 after CAR T-cell infusion. We observed cumulative incidences of neutrophil and platelet recovery of 81% and 75%, respectively, at 28 days after infusion. Hematologic toxicity was noted in a significant subset of patients, with persistent neutropenia in 9% and thrombocytopenia in 14% at last follow-up. Using debiased least absolute shrinkage selector and operator regression analysis for high-dimensional modeling and considering patient-, disease-, and treatment-related variables, we identified increased CRS severity as an independent predictor for decreased platelet count and lower prelymphodepletion platelet count as an independent predictor of both decreased neutrophil and platelet counts after CD19 CAR T-cell infusion. Furthermore, multivariable models including CRS-related cytokines identified associations between higher peak serum concentrations of interleukin-6 and lower day-28 cell counts; in contrast, higher serum concentrations of transforming growth factor-ß1 were associated with higher counts. Our findings suggest that patient selection and improved CRS management may improve hematopoietic recovery after CD19 CAR T-cell therapy.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. II: CAR-T cell therapy for patients with CD19+ acute lymphoblastic leukemia.

Chimeric antigen receptor T (CAR-T) cell therapy is a novel therapeutic modality for acute lymphoblastic leukemia (ALL) with robust outcomes in patients with refractory or relapsed disease. At the same time, CAR-T cell therapy is associated with unique and potentially fatal toxicities, such as cytokine release syndrome (CRS) and neurological toxicities (ICANS). This manuscript aims to provide a consensus of specialists in the fields of Hematology Oncology and Cellular Therapy to make recommendations on the current scenario of the use of CAR-T cells in patients with ALL.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. III: anti-CD19 CAR-T cell therapy for patients with non-Hodgkin lymphoma.

The treatment and evolution of B-cell non-Hodgkin lymphoma (B-NHL) has undergone important changes in the last years with the emergence of targeted therapies, such as monoclonal antibodies, small molecules, antibody-drug conjugates, and bispecific antibodies. Nevertheless, a significant portion of patients remains refractory or relapsed (R/R) to the new therapeutic modalities, representing thus an unmet medical need. The use of CAR-T cells for the treatment of B-NHL patients has shown to be a promising therapy with impressive results in patients with R/R disease. The expectations are as high as the imminent approval of CAR-T cell therapy in Brazil, which it is expected to impact the prognosis of R/R B-NHL. The aim of this manuscript is to offer a consensus of specialists in the field of onco-hematology and cellular therapy, working in Brazil and United States, in order to discuss and offer recommendations in the present setting of the use of CAR-T cells for patients with B-NHL.

Associação Brasileira de Hematologia, Hemoterapia e Terapia Celular Consensus on genetically modified cells. I: Structuring centers for the multidisciplinary clinical administration and management of CAR-T cell therapy patients.

Chimeric antigen receptor T-cells (CAR-T cells) are a new modality of oncological treatment which has demonstrated impressive response in refractory or relapsed diseases, such as acute lymphoblastic leukemia (ALL), lymphomas, and myeloma but is also associated with unique and potentially life-threatening toxicities. The most common adverse events (AEs) include cytokine release syndrome (CRS), neurological toxicities, such as the immune effector cell-associated neurotoxicity syndrome (ICANS), cytopenias, infections, and hypogammaglobulinemia. These may be severe and require admission of the patient to an intensive care unit. However, these AEs are manageable when recognized early and treated by a duly trained team. The objective of this article is to report a consensus compiled by specialists in the fields of oncohematology, bone marrow transplantation, and cellular therapy describing recommendations on the Clinical Centers preparation, training of teams that will use CAR-T cells, and leading clinical questions as to their use and the management of potential complications.

Antibodies against vaccine-preventable infections after CAR-T cell therapy for B cell malignancies.

BACKGROUNDLittle is known about pathogen-specific humoral immunity after chimeric antigen receptor-modified T (CAR-T) cell therapy for B cell malignancies.METHODSWe conducted a prospective cross-sectional study of CD19-targeted or B cell maturation antigen-targeted (BCMA-targeted) CAR-T cell therapy recipients at least 6 months posttreatment and in remission. We measured pathogen-specific IgG against 12 vaccine-preventable infections and the number of viral and bacterial epitopes to which IgG was detected ("epitope hits") using a serological profiling assay. The primary outcome was the proportion of participants with IgG levels above a threshold correlated with seroprotection for vaccine-preventable infections.RESULTSWe enrolled 65 children and adults a median of 20 months after CD19- (n = 54) or BCMA- (n = 11) CAR-T cell therapy. Among 30 adults without IgG replacement therapy (IGRT) in the prior 16 weeks, 27 (90%) had hypogammaglobulinemia. These individuals had seroprotection to a median of 67% (IQR, 59%-73%) of tested infections. Proportions of participants with seroprotection per pathogen were comparable to population-based studies, but most individuals lacked seroprotection to specific pathogens. Compared with CD19-CAR-T cell recipients, BCMA-CAR-T cell recipients were half as likely to have seroprotection (prevalence ratio, 0.47; 95% CI, 0.18-1.25) and had fewer pathogen-specific epitope hits (mean difference, -90 epitope hits; 95% CI, -157 to -22).CONCLUSIONSeroprotection for vaccine-preventable infections in adult CD19-CAR-T cell recipients was comparable to the general population. BCMA-CAR-T cell recipients had fewer pathogen-specific antibodies. Deficits in both groups support the need for vaccine and immunoglobulin replacement therapy studies.FUNDINGSwiss National Science Foundation (Early Postdoc Mobility grant P2BSP3_188162), NIH/National Cancer Institute (NIH/NCI) (U01CA247548 and P01CA018029), NIH/NCI Cancer Center Support Grants (P30CA0087-48 and P30CA015704-44), American Society for Transplantation and Cellular Therapy, and Juno Therapeutics/BMS.

Detection of engineered T cells in FFPE tissue by multiplex in situ hybridization and immunohistochemistry.

Identifying engineered T cells in situ is important to understand the location, persistence, and phenotype of these cells in patients after adoptive T cell therapy. While engineered cells are routinely characterized in fresh tissue or blood from patients by flow cytometry, it is difficult to distinguish them from endogenous cells in formalin-fixed, paraffin-embedded (FFPE) tissue biopsies. To overcome this limitation, we have developed a method for characterizing engineered T cells in fixed tissue using in situ hybridization (ISH) to the woodchuck hepatitis post-transcriptional regulatory element (WPRE) common in many lentiviral vectors used to transduce chimeric antigen receptor T (CAR-T) and T cell receptor T (TCR-T) cells, coupled with alternative permeabilization conditions that allows subsequent multiplex immunohistochemical (mIHC) staining within the same image. This new method provides the ability to mark the cells by ISH, and simultaneously stain for cell-associated proteins to immunophenotype CAR/TCR modified T cells within tumors, as well as assess potential roles of these cells in on-target/off-tumor toxicity in other tissue.

Factors associated with outcomes after a second CD19-targeted CAR T-cell infusion for refractory B-cell malignancies.

CD19-targeted chimeric antigen receptor-engineered (CD19 CAR) T-cell therapy has shown significant efficacy for relapsed or refractory (R/R) B-cell malignancies. Yet, CD19 CAR T cells fail to induce durable responses in most patients. Second infusions of CD19 CAR T cells (CART2) have been considered as a possible approach to improve outcomes. We analyzed data from 44 patients with R/R B-cell malignancies (acute lymphoblastic leukemia [ALL], n = 14; chronic lymphocytic leukemia [CLL], n = 9; non-Hodgkin lymphoma [NHL], n = 21) who received CART2 on a phase 1/2 trial (NCT01865617) at our institution. Despite a CART2 dose increase in 82% of patients, we observed a low incidence of severe toxicity after CART2 (grade ≥3 cytokine release syndrome, 9%; grade ≥3 neurotoxicity, 11%). After CART2, complete response (CR) was achieved in 22% of CLL, 19% of NHL, and 21% of ALL patients. The median durations of response after CART2 in CLL, NHL, and ALL patients were 33, 6, and 4 months, respectively. Addition of fludarabine to cyclophosphamide-based lymphodepletion before the first CAR T-cell infusion (CART1) and an increase in the CART2 dose compared with CART1 were independently associated with higher overall response rates and longer progression-free survival after CART2. We observed durable CAR T-cell persistence after CART2 in patients who received cyclophosphamide and fludarabine (Cy-Flu) lymphodepletion before CART1 and a higher CART2 compared with CART1 cell dose. The identification of 2 modifiable pretreatment factors independently associated with better outcomes after CART2 suggests strategies to improve in vivo CAR T-cell kinetics and responses after repeat CAR T-cell infusions, and has implications for the design of trials of novel CAR T-cell products after failure of prior CAR T-cell immunotherapies.

Cytokine elevation in severe and critical COVID-19: a rapid systematic review, meta-analysis, and comparison with other inflammatory syndromes.

The description of a so-called cytokine storm in patients with COVID-19 has prompted consideration of anti-cytokine therapies, particularly interleukin-6 antagonists. However, direct systematic comparisons of COVID-19 with other critical illnesses associated with elevated cytokine concentrations have not been reported. In this Rapid Review, we report the results of a systematic review and meta-analysis of COVID-19 studies published or posted as preprints between Nov 1, 2019, and April 14, 2020, in which interleukin-6 concentrations in patients with severe or critical disease were recorded. 25 COVID-19 studies (n=1245 patients) were ultimately included. Comparator groups included four trials each in sepsis (n=5320), cytokine release syndrome (n=72), and acute respiratory distress syndrome unrelated to COVID-19 (n=2767). In patients with severe or critical COVID-19, the pooled mean serum interleukin-6 concentration was 36·7 pg/mL (95% CI 21·6-62·3 pg/mL; I2=57·7%). Mean interleukin-6 concentrations were nearly 100 times higher in patients with cytokine release syndrome (3110·5 pg/mL, 632·3-15 302·9 pg/mL; p<0·0001), 27 times higher in patients with sepsis (983·6 pg/mL, 550·1-1758·4 pg/mL; p<0·0001), and 12 times higher in patients with acute respiratory distress syndrome unrelated to COVID-19 (460 pg/mL, 216·3-978·7 pg/mL; p<0·0001). Our findings question the role of a cytokine storm in COVID-19-induced organ dysfunction. Many questions remain about the immune features of COVID-19 and the potential role of anti-cytokine and immune-modulating treatments in patients with the disease.