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Immunotherapies, such as chimeric antigen receptor (CAR) modified T cells and antibody‐drug conjugates (ADCs), have revolutionized the treatment of cancer, especially of lymphoid malignancies. The application of targeted immunotherapy to patients with acute myeloid leukemia (AML) has been limited in particular by the lack of a tumor‐specific target antigen. Gemtuzumab ozogamicin (GO), an ADC targeting CD33, is the only approved immunotherapeutic agent in AML. In our study, we introduce a CD33‐directed third‐generation CAR T‐cell product (3G.CAR33‐T) for the treatment of patients with AML. 3G.CAR33‐T cells could be expanded up to the end‐of‐culture, that is, 17 days after transduction, and displayed significant cytokine secretion and robust cytotoxic activity when incubated with CD33‐positive cells including cell lines, drug‐resistant cells, primary blasts as well as normal hematopoietic stem and progenitor cells (HSPCs). When compared to second‐generation CAR33‐T cells, 3G.CAR33‐T cells exhibited higher viability, increased proliferation and stronger cytotoxicity. Also, GO exerted strong antileukemia activity against CD33‐positive AML cells. Upon genomic deletion of CD33 in HSPCs, 3G.CAR33‐T cells and GO preferentially killed wildtype leukemia cells, while sparing CD33‐deficient HSPCs. Our data provide evidence for the applicability of CD33‐targeted immunotherapies in AML and its potential implementation in CD33 genome‐edited stem cell transplantation approaches. What's new? In the development of immunotherapy for acute myeloid leukemia (AML), a target of interest is CD33, which is expressed on blast cells in more than 90 percent of AML patients. CD33 is also expressed on healthy myeloid and progenitor cells, however, raising the risk for off‐target effects with CD33 therapies. Here, the authors introduce a CD33‐directed third‐generation chimeric antigen receptor (CAR) T‐cell product (3G.CAR33‐T). 3G.CAR33‐T cells were effective against CD33‐positive cells, including AML blasts, and successfully overcame AML drug resistance. Genomic deletion of CD33 in hematopoietic stem and progenitor cells resulted in preferential killing of leukemia cells by 3G.CAR33‐T cells. [ABSTRACT FROM AUTHOR]

Cellular immunotherapy with chimeric antigen receptor (CAR)-T cells (CARTs) represents a breakthrough in the treatment of hematologic malignancies. CARTs are genetically engineered hybrid receptors that combine antigen-specificity of monoclonal antibodies with T cell function to direct patient-derived T cells to kill malignant cells expressing the target (tumor) antigen. CARTs have been introduced into clinical medicine as CD19-targeted CARTs for refractory and relapsed B cell malignancies. Despite high initial response rates, current CART therapies are limited by a long-term loss of antitumor efficacy, the occurrence of toxicities, and the lack of biomarkers for predicting therapy and toxicity outcomes. In the past decade, the gut microbiome of mammals has been extensively studied and evidence is accumulating that human health, apart from our own genome, largely depends on microbes that are living in and on the human body. The microbiome encompasses more than 1000 bacterial species who collectively encode a metagenome that guides multifaceted, bidirectional host-microbiome interactions, primarily through the action of microbial metabolites. Increasing knowledge has been accumulated on the role of the gut microbiome in T cell-driven anticancer immunotherapy. It has been shown that antibiotics, dietary components and gut microbes reciprocally affect the efficacy and toxicity of allogeneic hematopoietic cell transplantation (allo HCT) as the prototype of T cell-based immunotherapy for hematologic malignancies, and that microbiome diversity metrics can predict clinical outcomes of allo HCTs. In this review, we will provide a comprehensive overview of the principles of CD19-CART immunotherapy and major aspects of the gut microbiome and its modulators that impact antitumor T cell transfer therapies. We will outline i) the extrinsic and intrinsic variables that can contribute to the complex interaction of the gut microbiome and host in CART immunotherapy, including ii) antibiotic administration affecting loss of colonization resistance, expansion of pathobionts and disturbed mucosal and immunological homeostasis, and ii) the role of specific gut commensals and their microbial virulence factors in host immunity and inflammation. Although the role of the gut microbiome in CART immunotherapy has only been marginally explored so far, this review may open a new chapter and views on putative connections and mechanisms. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor T (CAR-T) cells targeting CD19 came into clinical practice for the treatment of B cell lymphoma in 2018. However, patients being treated for B cell lymphoma often suffer from comorbidities such as chronic pain, cardiovascular diseases and arthritis. Thus, these patients frequently receive concomitant medications that include nonsteroidal anti-inflammatory drugs (NSAIDs) like cyclooxygenase (COX) inhibitors. Celecoxib, a selective COX-2 inhibitor, and aspirin, a non-selective COX-1 and COX-2 inhibitor, are being used as anti-inflammatory, analgesic and anti-pyretic drugs. In addition, several studies have also focused on the anti-neoplastic properties of COX-inhibitors. As the influence of COX-inhibitors on CD19.CAR-T cells is still unknown, we investigated the effect of celecoxib and aspirin on the quantity and quality of CD19.CAR-T cells at different concentrations with special regard to cytotoxicity, activation, cytokine release, proliferation and exhaustion. A significant effect on CAR-T cells could be observed for 0.1 mmol/L of celecoxib and for 4 mmol/L of aspirin. At these concentrations, we found that both COX-inhibitors could induce intrinsic apoptosis of CD19.CAR-T cells showing a significant reduction in the ratio of JC-10 red to JC-10 green CAR-T cells from 6.46 ± 7.03 (mean ± SD) to 1.76 ± 0.67 by celecoxib and to 4.41 ± 0.32 by aspirin, respectively. Additionally, the ratios of JC-10 red to JC-10 green Daudi cells were also decreased from 3.41 ± 0.30 to 0.77 ± 0.06 by celecoxib and to 1.26 ± 0.04 by aspirin, respectively. Although the cytokine release by CD19.CAR-T cells upon activation was not hampered by both COX-inhibitors, activation and proliferation of CAR-T cells were significantly inhibited via diminishing the NF-ĸB signaling pathway by a significant down-regulation of expression of CD27 on CD4+ and CD8+ CAR-T cells, followed by a clear decrease of phosphorylated NF-ĸB p65 in both CD4+ and CD8+ CAR-T cells by a factor of 1.8. Of note, COX-inhibitors hampered expansion and induced exhaustion of CAR-T cells in an antigen stress assay. Collectively, our findings indicate that the use of COX-inhibitors is a double-edged sword that not only induces apoptosis in tumor cells but also impairs the quantity and quality of CAR-T cells. Therefore, COX-inhibitors should be used with caution in patients with B cell lymphoma under CAR-T cell therapy. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor T (CART) cells targeting CD19 have shown promising results in the treatment of chronic lymphocytic leukemia (CLL). However, efficacy seems to be inferior compared to diffuse large B‐cell lymphoma or acute lymphoblastic leukemia. Impaired T‐cell fitness of CLL patients may be involved in treatment failure. Less‐differentiated naïve‐like T cells play an important role in CART expansion and long‐term persistence in vivo. These cells are sparse in CLL patients. Therefore, optimization of CART cell production protocols enriching less differentiated T cell subsets may overcome treatment resistance. The B‐cell receptor inhibitor ibrutinib targeting Bruton's tyrosine kinase (BTK) is approved for the treatment of CLL. Besides BTK, ibrutinib additionally inhibits interleukin‐2‐inducible T‐cell kinase (ITK) which is involved in T‐cell differentiation. To evaluate the effect of ibrutinib on CART cell production, peripheral blood mononuclear cells from nine healthy donors and eight CLL patients were used to generate CART cells. T‐cell expansion and phenotype, expression of homing and exhaustion makers as well as functionality of CART cells were evaluated. CART cell generation in the presence of ibrutinib resulted in increased cell viability and expansion of CLL patient‐derived CART cells. Furthermore, ibrutinib enriched CART cells with less‐differentiated naïve‐like phenotype and decreased expression of exhaustion markers including PD‐1, TIM‐3 and LAG‐3. In addition, ibrutinib increased the cytokine release capacity of CLL patient‐derived CART cells. In summary, BTK/ITK inhibition with ibrutinib during CART cell culture can improve yield and function of CLL patient‐derived CART cell products. What's new? Chimeric antigen receptor T (CART) cells targeting CD19 have shown promising results in chronic lymphocytic leukemia (CLL). However, naïve‐like T cells play an important role in CART expansion and long‐term persistence in vivo, and these cells are sparse in CLL patients. Here, the authors show that BTK/ITK inhibition with Ibrutinib during CART cell generation may improve CLL patient‐derived CART cell products and enhance CART cell function. Supplementing CART cell production with ibrutinib increases CART cell yields and enriches CART cells with less‐differentiated phenotypes and lower expression of exhaustion markers, representing a potential avenue to improve the clinical outcome of patients. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cell therapy has shown promising responses in patients with refractory or relapsed aggressive B-cell malignancies that are resistant to conventional chemotherapy or stem cell transplantation. A potentially combinatorial therapeutic strategy may be the inhibition of anti-apoptotic Bcl-2 family proteins, overexpressed in most cancer cells. In this study we investigated the combination of 3rd-generation CD19.CAR-T cells and the BH3 mimetics venetoclax, a Bcl-2 inhibitor, or S63845, a Mcl-1 inhibitor, under three different treatment conditions: pre-sensitization of cancer cells with BH3 mimetics followed by CAR-T cell treatment, simultaneous combination therapy, and the administration of BH3 mimetics after CAR-T cell treatment. Our results showed that administration of CAR-T cells and BH3 mimetics had a significant effect on the quantity and quality of CD19.CAR-T cells. The administration of BH3 mimetics prior to CAR-T cell therapy exerted an enhanced cytotoxic efficacy by upregulating the CD19 expression and pro-apoptotic proteins in highly sensitive tumor cells, and thereby improving both CD19.CAR-T cell cytotoxicity and persistence. In simultaneous and post-treatment approaches, however, the quantity of CAR-T cells was adversely affected. Our findings indicate pre-sensitization of highly sensitive tumor cells with BH3 mimetics could enhance the cytotoxic efficacy of CAR-T cell treatment. [ABSTRACT FROM AUTHOR]

Despite major advances in the treatment of multiple myeloma (MM), it remains a largely incurable disease with long‐term control often dependent on continuous therapy. More effective, better tolerated treatments are therefore required to achieve durable remissions and to improve the quality of life of MM patients. Adoptive immunotherapy employing T cells expressing chimeric antigen receptors (CAR) is currently among the most promising treatment approaches in cancer. Within the target portfolio for MM immunotherapy, B‐cell maturation antigen (BCMA) is among the most widely studied target antigens. BCMA is consistently expressed on MM cells and, importantly, is not expressed in critical healthy tissue. For this reason, it is an ideal target for MM immunotherapy. Several clinical trials evaluating different BCMA‐targeting CAR constructs have been initiated and early results are very promising. However, in this rapidly developing clinical landscape, the ultimate role of BCMA‐specific CAR‐T cell therapy remains unclear. In this review, we will summarize currently available clinical data on BCMA‐directed CAR‐T cells and discuss potential future perspective for this promising treatment approach in MM. [ABSTRACT FROM AUTHOR]

Despite encouraging results with chimeric antigen receptor T (CART) cells, outcome can still be improved by optimization of the CART cell generation process. The proportion of less‐differentiated T cells within the transfused product is linked to enhanced in vivo CART cell expansion and long‐term persistence. The clinically approved PI3Kδ inhibitor idelalisib is well established in the treatment of B cell malignancies. Besides B cell receptor pathway inhibition, idelalisib can modulate T cell differentiation and function. Here, detailed longitudinal analysis of idelalisib‐induced effects on T cell phenotype and function was performed during CART cell production. A third generation CD19.CAR.CD28.CD137zeta CAR vector system was used. CART cells were generated from peripheral blood mononuclear cells of healthy donors (HDs) and chronic lymphocytic leukemia (CLL) patients. Idelalisib‐based CART cell generation resulted in an enrichment of less‐differentiated naïve‐like T cells (CD45RA+CCR7+), decreased expression of the exhaustion markers PD‐1 and Tim‐3, as well as upregulation of the lymph node homing marker CD62L. Idelalisib increased transduction efficiency, but did not impair viability and cell expansion. Strikingly, CD4:CD8 ratios that were altered in CART cells from CLL patients were approximated to ratios in HDs by idelalisib. Furthermore, in vivo efficacy of idelalisib‐treated CART cells was validated in a xenograft mouse model. Intracellular TNF‐α and IFN‐γ production decreased in presence of idelalisib. This effect was reversible after resting CART cells without idelalisib. In summary, PI3Kδ inhibition with idelalisib can improve CART cell products, particularly when derived from CLL patients. Further studies with idelalisib‐based CART cell generation protocols are warranted. What's new? Despite encouraging results with anti‐CD19 chimeric antigen receptor T (CART) cell therapy for B cell malignancies, clinical outcome could still be improved by optimization of the CART cell generation process. Here, the authors show that ex vivo PI3Kδ inhibition by the B cell malignancy drug idelalisib during CART cell generation can increase the proportion of less‐differentiated T cells and lead to a less exhausted T cell phenotype. Idelalisib optimized the phenotype of CART cells derived from chronic lymphocytic leukemia (CLL) patients. This study thus provides important novel and potentially practice‐changing findings for CART cell generation and expansion, especially for CLL patients. [ABSTRACT FROM AUTHOR]

Introduction Chimeric antigen receptor (CAR) T cells spark hope for patients with CD19+ B cell neoplasia, including relapsed or refractory (r/r) acute lymphoblastic leukaemia (ALL) or r/r non-Hodgkin's lymphoma (NHL). Published studies have mostly used second-generation CARs with 4-1BB or CD28 as costimulatory domains. Preclinical results of third-generation CARs incorporating both elements have shown superiority concerning longevity and proliferation. The University Hospital of Heidelberg is the first institution to run an investigatorinitiated trial (IIT) CAR T cell trial (Heidelberg Chimeric Antigen Receptor T cell Trial number 1 [HD-CAR-1]) in Germany with third-generation CD19-directed CAR T cells. Methods and analysis Adult patients with r/r ALL (stratum I), r/r NHL including chronic lymphocytic leukaemia, diffuse large B-cell lymphoma, follicular lymphoma or mantle cell lymphoma (stratum II) as well as paediatric patients with r/r ALL (stratum III) will be treated with autologous T-lymphocytes transduced by third-generation RV-SFG.CD19.CD28.4-1BB zeta retroviral vector (CD19.CAR T cells). The main purpose of this study is to evaluate safety and feasibility of escalating CD19.CAR T cell doses (1-20×106 transduced cells/m²) after lymphodepletion with fludarabine (flu) and cyclophosphamide (cyc). Patients will be monitored for cytokine release syndrome (CRS), neurotoxicity, i.e. CART- cell-related encephalopathy syndrome (CRES) and/or other toxicities (primary objectives). Secondary objectives include evaluation of in vivo function and survival of CD19. CAR T cells and assessment of CD19.CAR T cell antitumour efficacy. HD-CAR-1 as a prospective, monocentric trial aims to make CAR T cell therapy accessible to patients in Europe. Currently, HD-CAR-1 is the first and only CAR T cell IIT in Germany. A third-generation Good Manufacturing Practice (GMP) grade retroviral vector, a broad spectrum of NHL, treatment of paediatric and adult ALL patients and inclusion of patients even after allogeneic stem cell transplantation (alloSCT) make this trial unique. Ethics and dissemination Ethical approval and approvals from the local and federal competent authorities were granted. Trial results will be reported via peer-reviewed journals and presented at conferences and scientific meetings. [ABSTRACT FROM AUTHOR]

CD56bri natural killer (NK) cells play an important role in the pathogenesis of graft-vs. -host disease (GVHD) and immune defense in the early period after allogeneic hematopoietic stem cell transplantation. Extracorporeal photopheresis (ECP) as an immunomodulating therapy has been widely used for GVHD treatment. However, the mechanism of action of ECP still remains to be elucidated, particularly the influence of ECP on NK cells. Thirty-four patients with steroid-refractory/resistant acute GVHD (aGVHD) ≥ °II and moderate to severe chronic GVHD (cGVHD) received ECP therapy. Patient samples obtained during intensive and long-term treatment were analyzed. Immunomonitoring with respect to cell phenotype and function was performed on rested peripheral blood mononuclear cells (PBMCs) using multiparametric flow cytometry. NK activity in terms of cytokine release was analyzed by intracellular cytokine staining after co-culture with K562 cells. Moreover, the proliferative capacity of NK cells, CD4+, and CD8+ T cells was determined by carboxyfluorescein succinimidyl ester (CFSE) staining. Clinically, 75% of aGVHD and 78% of cGVHD patients responded to ECP therapy. Moreover, our data show that aGVHD, cGVHD patients and healthy donors (HDs) present distinct NK patterns: aGVHD patients have a higher frequency of CD56bri NK subsets with stronger NKG2D and CD62L expression, while CD56−CD16+ NK cells with higher expression of CD57 and CD11b stand out as a signature population for cGVHD. ECP therapy could significantly decrease CD56briCD16− NK cells with shifting the quality from a cytotoxic to a regulatory pattern and additionally mature CD56dim NK cells via upregulation of CD57 in complete responding aGVHD patients. Moreover, ECP could keep the anti-viral and anti-leukemic effects intact via maintaining specialized anti-viral/leukemic CD57+NKG2C+CD56dim NK cells as well as remaining the quality and quantity of cytokine release by NK cells. The proliferative capacity of effector cells remained constant over ECP therapy. In conclusion, ECP represents an attractive option to treat GVHD without compromising anti-viral/leukemic effects. Shaping of CD56bri NK cell compartment by downregulating the cytotoxic subset while upregulating the regulatory subset contributes to the mechanisms of ECP therapy in aGVHD. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor (CAR) T cell therapy has recently achieved impressive clinical outcome in patients with CD19‐positive hematologic malignancies. Extrapolation of CAR T cell treatment to solid tumors, however, has not yet yielded similar results. This might be due to intrinsic causes, e.g. insufficient CAR T cell activation or CAR toxicity as well as extrinsic factors displaying an unfavorable tumor environment for CAR T cells by raising physical and chemical barriers. In this review, we discuss the advantages as well as major obstacles of CAR T cell therapy, particularly in the context of solid tumors, and focus on efforts and novel strategies in CAR T cell development. [ABSTRACT FROM AUTHOR]

Novel therapies with chimeric antigen receptor (CAR)-transduced T cells (TCs) sparked new hope for patients with relapsed or refractory CD19-positive leukemia or lymphoma even after stem cell therapies. This review focuses on CARs recognizing the B cell antigen CD19. Both retroviral and lentiviral vectors are used, encoding various anti-CD19 CAR constructs comprising costimulatory molecules such as CD28, CD137/4-1BB, and OX40 either alone (second-generation CARs) or in combination (third-generation CARs). Current, up-to-date published studies on anti-CD19 CAR therapy for acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and non-Hodgkin lymphoma (NHL) with observed side effects are discussed and an outlook on 58 ongoing trials is given. Clinical responses were achieved in up to 81% of ALL, 50% of CLL, and 40% of NHL patients. Factors with potential influence on the clinical outcome might be the design of the vector, the preconditioning regimen, and the number and quality of transfused CAR TCs. The applicability of clinical CAR TC therapy might include relapse after allogeneic stem cell transplantation (alloSCT), and ineligibility for or 'bridging' until alloSCT. In summary, CAR therapy represents a highly promising treatment option even in heavily pretreated patients. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic threatens patients with a compromised immune and endothelial system, including patients who underwent allogeneic stem cell transplantation (alloSCT). Thus, there is an unmet need for optimizing vaccination management in this high-risk cohort. Here, we monitored antibodies against SARS-CoV-2 spike protein (anti-S1) in 167 vaccinated alloSCT patients. Humoral immune responses were detectable in 81% of patients after two vaccinations with either mRNA-, vector-based, or heterologous regimens. Age, B-cell counts, time interval from vaccination, and the type of vaccine determined antibody titres in patients without systemic immunosuppression (sIS). Similar to a healthy control cohort, mRNA vaccine-based regimens induced higher titres than vector-based vaccines. Patients on two or more immunosuppressants rarely developed immunity. In contrast, 62% and 45% of patients without or on only one immunosuppressant, respectively, showed a strong humoral vaccination response (titre > 100). Exacerbation of cGVHD upon vaccination was observed in 6% of all patients and in 22% of patients receiving immunosuppression for cGVHD. cGVHD exacerbation and low antibody titres were both associated with higher angiopoietin-2 (ANG2) serum levels. In conclusion, mRNA-based vaccines elicit strong humoral responses in alloSCT patients in the absence of double sIS. Biomarkers such as ANG2 might help with weighing cGVHD risk versus beneficial responses. [ABSTRACT FROM AUTHOR]

Adoptive cell therapy with NY-ESO-1-specific T cells is a promising option for the treatment of soft tissue sarcoma (STS) but achieves only transient tumor control in the majority of cases. A strategy to optimize this cell therapeutic approach might be the modulation of the expression of the cancer-testis antigen NY-ESO-1 using histone deacetylase inhibitors (HDACis). In this study, the ex vivo effect of combining NY-ESO-1-specific T cells with the clinically approved pan HDACis panobinostat or vorionstat was investigated. Our data demonstrated that STS cells were sensitive to HDACis. Administration of HDACi prior to NY-ESO-1-specific T cells exerted enhanced lysis against the NY-ESO-1+ STS cell line SW982. This correlated with an increase in the NY-ESO-1 and HLA-ABC expression of SW982 cells, as well as increased CD25 expression on NY-ESO-1-specific T cells. Furthermore, the immune reactivity of NY-ESO-1-specific CD8+ T cells in terms of cytokine release was enhanced by HDACis. In summary, pretreatment with HDACis represents a potential means of enhancing the cytotoxic efficacy of NY-ESO-1-specific T cells against NY-ESO-1-positive STS. [ABSTRACT FROM AUTHOR]

Chimeric-antigen-receptor-T (CAR-T) cells are currently revolutionizing the field of cancer immunotherapy. Therefore, there is an urgent need for CAR-T cell monitoring by clinicians to assess cell expansion and persistence in patients. CAR-T cell manufacturers and researchers need to evaluate transduction efficiency and vector copy number for quality control. Here, CAR expression was analyzed in peripheral blood samples from patients and healthy donors by flow cytometry with four commercially available detection reagents and on the gene level by quantitative polymerase chain reaction (qPCR). Flow cytometric analysis of CAR expression showed higher mean CAR expression values for CD19 CAR detection reagent and the F(ab')2 antibody than Protein L and CD19 Protein. In addition, the CD19 CAR detection reagent showed a significantly lower median background staining of 0.02% (range 0.007–0.06%) when compared to the F(ab')2 antibody, CD19 protein and Protein L with 0.80% (range 0.47–1.58%), 0.65% (range 0.25–1.35%) and 0.73% (range 0.44–1.23%). Furthermore, flow cytometry-based CAR-T cell frequencies by CD19 CAR detection reagent showed a good correlation with qPCR results. In conclusion, quality control of CAR-T cell products can be performed by FACS and qPCR. For the monitoring of CAR-T cell frequencies by FACS in patients, CAR detection reagents with a low background staining are preferable. [ABSTRACT FROM AUTHOR]

Simple Summary: Chimeric antigen receptor T (CAR-T) cells have become clinical practice for the treatment of acute lymphoblastic leukemia and non-Hodgkin lymphoma. The aim of this retrospective study was to assess infection complications after lymphodepletion and CAR-T cell therapy. Infections were commonly detected, but manageable in most cases. Fast and appropriate identification as well as treatment were critical, especially in this very vulnerable patient group. Effective strategies to prevent infections as well as adequate medical management also include standardized prophylaxis and additional supportive therapy. Chimeric antigen receptor T (CAR-T) cell therapy has proven to be very effective in patients with relapsed/refractory acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). However, infections—related either due to lymphodepletion or the CAR-T cell therapy itself—can result in severe and potentially life-threatening complications, while side effects such as cytokine release syndrome (CRS) might complicate differential diagnosis. Sixty-seven dosings of CAR-T cells in sixty adult patients with NHL (85%) and ALL (15%) receiving CAR-T cell therapy were assessed for infectious complications. Almost two-thirds of patients (61%) developed fever following lymphodepletion and CAR-T cell dosing. Microbiological or radiological findings were observed in 25% of all cases (bacterial 12%, viral 5%, fungal 8%). Inpatient infections were associated with more lines of therapy and more severe CRS. However, overall serious complications were rare after CAR-T therapy, with one patient dying of infection. Pathogen detection after inpatient stay was infrequent and mostly occurred in the first 90 days after dosing. Infections in CAR-T cell treated patents are common. Fast and suitable identification and treatment are crucial in these heavily pretreated and immunocompromised patients. In most cases infectious complications are manageable. Nonetheless, standardized anti-infective prophylaxis and supportive therapy are mandatory to reduce morbidity and mortality in CAR-T cell therapy. [ABSTRACT FROM AUTHOR]

NY-ESO-1-specific T cells have shown promising activity in the treatment of soft tissue sarcoma (STS). However, standardized protocols for their generation are limited. Particularly, cost-effectiveness considerations of cell production protocols are of importance for conducting clinical studies. In this study, two different NY-ESO-1-specific T cell production protocols were compared. Major differences between protocols 1 and 2 include culture medium, interleukin-2 and retronectin concentrations, T cell activation strategy, and the transduction process. NY-ESO-1-specific T cells generated according to the two protocols were investigated for differences in cell viability, transduction efficiency, T cell expansion, immunophenotype as well as functionality. NY-ESO-1-specific T cells showed similar viability and transduction efficiency between both protocols. Protocol 1 generated higher absolute numbers of NY-ESO-1-specific T cells. However, there was no difference in absolute numbers of NY-ESO-1-specific T cell subsets with less-differentiated phenotypes accounting for efficient in vivo expansion and engraftment. Furthermore, cells generated according to protocol 1 displayed higher capacity of TNF-α generation, but lower cytotoxic capacities. Overall, both protocols provided functional NY-ESO-1-specific T cells. However, compared to protocol 1, protocol 2 is advantageous in terms of cost-effectiveness. Cell production protocols should be designed diligently to achieve a cost-effective cellular product for further clinical evaluation. [ABSTRACT FROM AUTHOR]

Simple Summary: To monitor patients after CAR T cell treatment, measuring frequencies of chimeric antigen receptor (CAR) T cells is crucial. However, experimental assays to quantify CAR T cells are lacking. Here, we describe a quantitative single copy gene-based PCR approach to measure frequencies of CAR T cells based on the FMC63 single chain variable fragment (scFv) including commercially available CAR T cell products. Besides enabling to monitor development of CAR T cells after treatment and guide further therapeutic decisions, this quantification assay proved highly useful for diagnosis of CAR T cell associated neurotoxic side effects. Overall, this quantification approach contributes significantly to the better monitoring and safety of treatment of patients with CAR T cells. Chimeric antigen receptor (CAR) T cell (CART) therapy has been established as a treatment option for patients with CD19-positive lymphoid malignancies in both the refractory and the relapsed setting. Displaying significant responses in clinical trials, two second-generation CART products directed against CD19, axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel), have been approved and integrated into the clinical routine. However, experimental assay for quantitative monitoring of both of these CART products in treated patients in the open domain are lacking. To address this issue, we established and validated a quantitative single copy gene (SCG)-based duplex (DP)-PCR assay (SCG-DP-PCR) to quantify CARTs based on the FMC63 single chain variable fragment (scFv), i.e., axi-cel and tisa-cel. This quantitative PCR (qPCR) approach operates without standard curves or calibrator samples, offers a tool to assess cellular kinetics of FMC63 CARTs and allows direct comparison of CART-copies in axi-cel versus tisa-cel patient samples. For treating physicians, SCG-DP-PCR is an important tool to monitor CARTs and guide clinical decisions regarding CART effects in respective patients. [ABSTRACT FROM AUTHOR]

Background: T lymphocyte collection through leukapheresis is an essential step for chimeric antigen receptor T (CAR-T) cell therapy. Timing of apheresis is challenging in heavily pretreated patients who suffer from rapid progressive disease and receive T cell impairing medication. Methods: A total of 75 unstimulated leukaphereses were analyzed including 45 aphereses in patients and 30 in healthy donors. Thereof, 41 adult patients with Non-Hodgkin's lymphoma (85%) or acute lymphoblastic leukemia (15%) underwent leukapheresis for CAR-T cell production. Results: Sufficient lymphocytes were harvested from all patients even from those with low peripheral lymphocyte counts of 0.18/nL. Only four patients required a second leukapheresis session. Leukapheresis products contained a median of 98 × 108 (9 - 341 × 108) total nucleated cells (TNC) with 38 × 108 (4 - 232 × 108) CD3+ T cells. Leukapheresis products from healthy donors as well as from patients in complete remission were characterized by high TNC and CD3+ T lymphocyte counts. CAR-T cell products could be manufactured for all but one patient. Conclusions: Sufficient yield of lymphocytes for CAR-T cell production is feasible also for patients with low peripheral blood counts. Up to 12–15 L blood volume should be processed in patients with absolute lymphocyte counts ≤ 1.0/nL. [ABSTRACT FROM AUTHOR]

Chimeric antigen receptor T cell (CART) therapy is currently one of the most promising treatment approaches in cancer immunotherapy. However, the immunosuppressive nature of the tumor microenvironment, in particular increased reactive oxygen species (ROS) levels, provides considerable limitations. In this study, we aimed to exploit increased ROS levels in the tumor microenvironment with prodrugs of ROS accelerators, which are specifically activated in cancer cells. Upon activation, ROS accelerators induce further generation of ROS. This leads to an accumulation of ROS in tumor cells. We hypothesized that the latter cells will be more susceptible to CARTs. CD19-specific CARTs were generated with a CD19.CAR.CD28.CD137zeta third-generation retroviral vector. Cytotoxicity was determined by chromium-51 release assay. Influence of the ROS accelerators on viability and phenotype of CARTs was determined by flow cytometry. The combination of CARTs with the ROS accelerator PipFcB significantly increased their cytotoxicity in the Burkitt lymphoma cell lines Raji and Daudi, as well as primary chronic lymphocytic leukemia cells. Exposure of CARTs to PipFcB for 48 h did not influence T cell exhaustion, viability, or T cell subpopulations. In summary, the combination of CARTs with ROS accelerators may improve adoptive immunotherapy and help to overcome tumor microenvironment-mediated treatment resistance. [ABSTRACT FROM AUTHOR]

Despite high response rates after initial chemotherapy in patients with acute myeloid leukemia (AML), relapses occur frequently, resulting in a five-year-survival by <30% of the patients. Hitherto, allogeneic hemotopoietic stem cell transplantation (allo-HSCT) is the best curative treatment option in intermediate and high risk AML. It is the proof-of-concept for T cell-based immunotherapies in AML based on the graft-versus-leukemia (GvL)-effect, but it also bears the risk of graft-versus-host disease. CD19-targeting therapies employing chimeric antigen receptor (CAR) T cells are a breakthrough in cancer therapy. A similar approach for myeloid malignancies is highly desirable. This article gives an overview on the state-of-the art of preclinical and clinical studies on suitable target antigens for CAR T cell therapy in AML patients. [ABSTRACT FROM AUTHOR]

• ECP was able to increase Tfh cells to ameliorate GvHD. • Upregulation of Th22 cells was observed in aGvHD patients with CR. • Tim-3 expression was downregulated on effector T cells by ECP. • ECP shows immunomodulatory effects in GvHD setting. Extracorporeal photopheresis (ECP), a personalized cellular immunotherapy, constitutes a promising treatment for steroid-refractory/-resistant graft-versus-host disease (SR-GvHD), with encouraging clinical response rates. To further investigate its mechanism of action, ECP's effects on T helper (Th) cells as well as on expression of immune checkpoint (PD-1 and Tim-3) and apoptotic (Fas receptor [FasR]) molecules were investigated in 27 patients with SR-GvHD. Our data show that GvHD patients had significantly higher levels of Th2, Th17, Th22 and granulocyte-macrophage colony-stimulating factor (GM-CSF)-positive Th (ThG) cells and clearly lower levels of T follicular helper (Tfh) cells, including Th1- and Th2-like cells, compared with healthy donors. ECP therapy for GvHD was effective through the modulation of different Th subsets: increases of Th22 (1.52-fold) and Tfh cells (1.48-fold) in acute GvHD (aGvHD) and increases of Th2-like Tfh cells (1.74-fold) in chronic GvHD (cGvHD) patients were associated with clinical response. Expression of FasR was further upregulated in CD4+CD8+ T cells. Additionally, Tim-3–expressing effector T cells associated with the severity of GvHD were reduced. Taken together, these data show that ECP therapy exerts immunomodulatory effects by promoting a balanced immune reconstitution and inducing immune tolerance. Therefore it represents an attractive option for the treatment of GvHD. [ABSTRACT FROM AUTHOR]

Introduction: Donor-derived modified immune cells (MIC) induced long-term specific immunosuppression against the allogeneic donor in preclinical models of transplantation. In a phase I clinical trial (TOL-1 Study), MIC treatment resulted in a cellular phenotype that was directly and indirectly suppressive to the recipient's immune system allowing for reduction of conventional immunosuppressive therapy. Here, we describe a protocol for a randomised controlled, multicentre phase-IIb clinical trial of individualised immunosuppression with intravenously administered donor MIC compared with standard-of-care (SoC) in living donor kidney transplantation (TOL-2 Study)., Methods and Analysis: Sixty-three living donor kidney transplant recipients from six German transplant centres are randomised 2:1 to treatment with MIC (MIC group, N=42) or no treatment with MIC (control arm, N=21). MIC are manufactured from donor peripheral blood mononuclear cells under Good Manufacturing Practice conditions. The primary objective of this trial is to determine the efficacy of MIC treatment together with reduced conventional immunosuppressive therapy in terms of achieving an operational tolerance-like phenotype compared with SoC 12 months after MIC administration. Key secondary endpoints are the number of patient-relevant infections as well as a composite of biopsy-proven acute rejection, graft loss, graft dysfunction or death. Immunosuppressive therapy of MIC-treated patients is reduced during follow-up under an extended immunological monitoring including human leucocyte antigen-antibody testing, and determination of lymphocyte subsets, for example, regulatory B lymphocytes (Breg) and antidonor T cell response. A Data Safety Monitoring Board has been established to allow an independent assessment of safety and efficacy., Ethics and Dissemination: Ethical approval has been provided by the Ethics Committee of the Medical Faculty of the University of Heidelberg, Heidelberg, Germany (AFmu-580/2021, 17 March 2022) and from the Federal Institute for Vaccines and Biomedicines, Paul-Ehrlich-Institute, Langen, Germany (Vorlage-Nr. 4586/02, 21 March 2022). Written informed consent will be obtained from all patients and respective donors prior to enrolment in the study. The results from the TOL-2 Study will be published in peer-reviewed medical journals and will be presented at symposia and scientific meetings., Trial Registration Number: NCT05365672., Competing Interests: Competing interests: CM, ASchmitt, MS, CK, GO, PT, MZ and MSchaier together with the University of Heidelberg, are cofounders of TolerogenixX GmbH, Heidelberg, Germany, a biotechnology company that holds licenses for MIC treatment. CK, GO, and PT hold a patent for MIC treatment. CM, ASchmitt, MSchmitt, CK, GO, CSüsal, PT, MZ, VD and MSchaier together with the University of Heidelberg and TolerogenixX GmbH filed a patent application for MIC treatment. JR is cofounder and shareholder of Trisaq, a biopharmaceutical company that develops novel therapy for kidney diseases., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Chimeric antigen receptor T (CAR-T) cells targeting CD19 came into clinical practice for the treatment of B cell lymphoma in 2018. However, patients being treated for B cell lymphoma often suffer from comorbidities such as chronic pain, cardiovascular diseases and arthritis. Thus, these patients frequently receive concomitant medications that include nonsteroidal anti-inflammatory drugs (NSAIDs) like cyclooxygenase (COX) inhibitors. Celecoxib, a selective COX-2 inhibitor, and aspirin, a non-selective COX-1 and COX-2 inhibitor, are being used as anti-inflammatory, analgesic and anti-pyretic drugs. In addition, several studies have also focused on the anti-neoplastic properties of COX-inhibitors. As the influence of COX-inhibitors on CD19.CAR-T cells is still unknown, we investigated the effect of celecoxib and aspirin on the quantity and quality of CD19.CAR-T cells at different concentrations with special regard to cytotoxicity, activation, cytokine release, proliferation and exhaustion. A significant effect on CAR-T cells could be observed for 0.1 mmol/L of celecoxib and for 4 mmol/L of aspirin. At these concentrations, we found that both COX-inhibitors could induce intrinsic apoptosis of CD19.CAR-T cells showing a significant reduction in the ratio of JC-10 red to JC-10 green CAR-T cells from 6.46 ± 7.03 (mean ± SD) to 1.76 ± 0.67 by celecoxib and to 4.41 ± 0.32 by aspirin, respectively. Additionally, the ratios of JC-10 red to JC-10 green Daudi cells were also decreased from 3.41 ± 0.30 to 0.77 ± 0.06 by celecoxib and to 1.26 ± 0.04 by aspirin, respectively. Although the cytokine release by CD19.CAR-T cells upon activation was not hampered by both COX-inhibitors, activation and proliferation of CAR-T cells were significantly inhibited via diminishing the NF-ĸB signaling pathway by a significant down-regulation of expression of CD27 on CD4 + and CD8 + CAR-T cells, followed by a clear decrease of phosphorylated NF-ĸB p65 in both CD4 + and CD8 + CAR-T cells by a factor of 1.8. Of note, COX-inhibitors hampered expansion and induced exhaustion of CAR-T cells in an antigen stress assay. Collectively, our findings indicate that the use of COX-inhibitors is a double-edged sword that not only induces apoptosis in tumor cells but also impairs the quantity and quality of CAR-T cells. Therefore, COX-inhibitors should be used with caution in patients with B cell lymphoma under CAR-T cell therapy., Competing Interests: MS received funding for collaborative research from Apogenix, Hexal and Novartis, travel grants from Hexal and Kite, he received financial support for educational activities and conferences from bluebird bio, Kite and Novartis, he is a board member for MSD and (co-)PI of clinical trials of MSD, GSK, Kite and BMS, as well as co-founder and shareholder of TolerogenixX Ltd. AS received travel grants from Hexal and Jazz Pharmaceuticals, research grant from Therakos/Mallinckrodt and is co-founder of TolerogenixX Ltd. AS and LW are part- or full-time employers of TolerogenixX Ltd. LS was employed by Takeda Pharma Vertrieb GmbH & Co. KG. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yang, Wang, Ni, Neuber, Wang, Gong, Sauer, Schubert, Hückelhoven-Krauss, Xia, Ge, Kleist, Eckstein, Sellner, Müller-Tidow, Dreger, Schmitt and Schmitt.)

Chimeric antigen receptor (CAR) T cells are considered genetically modified organisms (GMOs) and constitute gene therapy medicinal products. Thus, CAR T cell manufacturing for clinical application is strictly regulated. Appropriate methods to assess vector copy numbers (VCNs) in CAR T cell products and monitoring of CAR T cell frequencies in patients are required. Quantitative polymerase chain reaction (qPCR) is the preferred method for VCN assessment. However, no standardized procedure with high reproducibility has been described yet. Here, we report on a single copy gene (SCG)-based duplex (DP)-qPCR assay (SCG-DP-PCR) to determine VCN in CAR T cell products. SCG-DP-PCR was validated and compared to the absolute standard curve method (ACM) within the framework of a clinical trial treating patients with good manufacturing practice (GMP)-grade CAR T cells at the University Hospital Heidelberg. Methodologically, SCG-DP-PCR displayed technical advantages over ACM and minimized mathematical analysis. SCG-DP-PCR, as a highly reproducible approach, can be used for clinical follow-up of patients treated with CAR T cells or other GMOs and might replace established methods for VCN quantification. This work will enable clinicians to assess VCN, as well as CAR T cell frequencies, in patients as a basis for decisions on subsequent therapies, including repeated CAR T cell administration., (© 2020 The Author(s).)

Introduction: Chimeric antigen receptor (CAR) T cells spark hope for patients with CD19+ B cell neoplasia, including relapsed or refractory (r/r) acute lymphoblastic leukaemia (ALL) or r/r non-Hodgkin's lymphoma (NHL). Published studies have mostly used second-generation CARs with 4-1BB or CD28 as costimulatory domains. Preclinical results of third-generation CARs incorporating both elements have shown superiority concerning longevity and proliferation. The University Hospital of Heidelberg is the first institution to run an investigator-initiated trial (IIT) CAR T cell trial (Heidelberg Chimeric Antigen Receptor T cell Trial number 1 [HD-CAR-1]) in Germany with third-generation CD19-directed CAR T cells., Methods and Analysis: Adult patients with r/r ALL (stratum I), r/r NHL including chronic lymphocytic leukaemia, diffuse large B-cell lymphoma, follicular lymphoma or mantle cell lymphoma (stratum II) as well as paediatric patients with r/r ALL (stratum III) will be treated with autologous T-lymphocytes transduced by third-generation RV-SFG.CD19.CD28.4-1BB zeta retroviral vector (CD19.CAR T cells). The main purpose of this study is to evaluate safety and feasibility of escalating CD19.CAR T cell doses (1-20×10 6 transduced cells/m 2 ) after lymphodepletion with fludarabine (flu) and cyclophosphamide (cyc). Patients will be monitored for cytokine release syndrome (CRS), neurotoxicity, i.e. CAR-T-cell-related encephalopathy syndrome (CRES) and/or other toxicities (primary objectives). Secondary objectives include evaluation of in vivo function and survival of CD19.CAR T cells and assessment of CD19.CAR T cell antitumour efficacy.HD-CAR-1 as a prospective, monocentric trial aims to make CAR T cell therapy accessible to patients in Europe. Currently, HD-CAR-1 is the first and only CAR T cell IIT in Germany. A third-generation Good Manufacturing Practice (GMP) grade retroviral vector, a broad spectrum of NHL, treatment of paediatric and adult ALL patients and inclusion of patients even after allogeneic stem cell transplantation (alloSCT) make this trial unique., Ethics and Dissemination: Ethical approval and approvals from the local and federal competent authorities were granted. Trial results will be reported via peer-reviewed journals and presented at conferences and scientific meetings., Trial Registration Number: Eudra CT 2016-004808-60; NCT03676504; Pre-results., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)