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Abstract Background Relapsed and refractory acute myeloid leukemia (AML) carries a dismal prognosis. CAR T cells have shown limited efficacy in AML, partially due to dysfunctional autologous T cells and the extended time for generation of patient specific CAR T cells. Allogeneic NK cell therapy is a promising alternative, but strategies to enhance efficacy and persistence may be necessary. Proteasome inhibitors (PI) induce changes in the surface proteome which may render malignant cells more vulnerable to NK mediated cytotoxicity. Here, we investigated the potential benefit of combining PIs with CAR-expressing allogeneic NK cells against AML. Methods We established the IC50 concentrations for Bortezomib and Carfilzomib against several AML cell lines. Surface expression of class-I HLA molecules and stress-associated proteins upon treatment with proteasome inhibitors was determined by multiparameter flow cytometry. Using functional in vitro assays, we explored the therapeutic synergy between pre-treatment with PIs and the anti-leukemic efficacy of NK cells with or without expression of AML-specific CAR constructs against AML cell lines and primary patient samples. Also, we investigated the tolerability and efficacy of a single PI application strategy followed by (CAR-) NK cell infusion in two different murine xenograft models of AML. Results AML cell lines and primary AML patient samples were susceptible to Bortezomib and Carfilzomib mediated cytotoxicity. Conditioned resistance to Azacitidine/Venetoclax did not confer primary resistance to PIs. Treating AML cells with PIs reduced the surface expression of class-I HLA molecules on AML cells in a time-and-dose dependent manner. Stress-associated proteins were upregulated on the transcriptional level and on the cell surface. NK cell mediated killing of AML cells was enhanced in a synergistic manner. PI pre-treatment increased effector-target cell conjugate formation and Interferon-γ secretion, resulting in enhanced NK cell activity against AML cell lines and primary samples in vitro. Expression of CD33- and CD70-specific CARs further improved the antileukemic efficacy. In vivo, Bortezomib pre-treatment followed by CAR-NK cell infusion reduced AML growth, leading to prolonged overall survival. Conclusions PIs enhance the anti-leukemic efficacy of CAR-expressing allogeneic NK cells against AML in vitro and in vivo, warranting further exploration of this combinatorial treatment within early phase clinical trials.

IntroductionCAR T-cell therapy is highly effective, but also associated with unique toxicities. Because of the origin of T cells in patients who previously underwent allogeneic hematopoietic cell transplantation (alloHCT), graft-versus-host disease (GVHD) in the post-CAR T-cell setting poses a relevant concern but is only scarcely studied. Potential risk factors and mitigation strategies (from CAR T-cell modifications to clinical management) are yet to be determined.MethodsSharing our retrospective experience and a mini-review of the literature, our aim is to better understand the frequency and risk of the potential occurrence of GVHD after CAR T cells, which are most likely underestimated. ResultsHere, we present a cohort of 11 patients with symptoms suggestive of GVHD out of 25 allografted patients treated with CAR T cells, of whom 3 patients (12%) had GVHD most likely triggered by the preceding CAR T-cell treatment. Severe chronic pulmonary GVHD occurred in a patient after CD19-directed CAR T-cell therapy. Extracorporeal photopheresis (ECP) mediated successful long-term control of GVHD without causing relapse of the underlying disease. Discussion/ConclusionIn conclusion, CD19-directed CAR T-cell therapy seems to be feasible in patients after alloHCT but might comprise the potential risk of triggering GVHD, most likely depending on the T-cell source, donor compatibility, and the specific CAR construct used.

Background Chimeric antigen receptor (CAR) T-cell therapy has demonstrated significant benefits in the treatment of relapsed/refractory multiple myeloma (RRMM). However, these outcomes can be compromised by severe complications, including cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS) and immune effector cell-associated hematotoxicity (ICAHT), predisposing for life-threatening infections.Methods This retrospective observational study examined a total of 129 patients with RRMM who had received idecabtagene vicleucel (ide-cel) at two major myeloma centers in Germany and one center in the USA to assess the Endothelial Activation and Stress Index (EASIX) as a risk marker for an unfavorable clinical course and outcome after CAR T-cell therapy. EASIX is calculated by lactate dehydrogenase (U/L) × creatinine (mg/dL) / platelets (109 cells/L) and was determined before lymphodepletion (baseline) and at the day of CAR T-cell infusion (day 0). The analysis was extended to EASIX derivatives and the CAR-HEMATOTOX score.Results An elevated baseline EASIX (>median) was identified as a risk marker for severe late ICAHT, manifesting with an impaired hematopoietic reconstitution and pronounced cytopenias during the late post-CAR-T period. Patients with high EASIX levels (>upper quartile) were particularly at risk, as evidenced by an increased rate of an aplastic phenotype of neutrophil recovery, severe late-onset infections and ICANS. Finally, we found associations between baseline EASIX and an inferior progression-free and overall survival. Moreover, the EASIX at day 0 also demonstrated potential to serve as a risk marker for post-CAR-T complications and adverse outcomes.Conclusions In conclusion, EASIX aids in risk stratification at clinically relevant time points prior to CAR T-cell therapy with ide-cel. Increased EASIX levels might help clinicians to identify vulnerable patients to adapt peri-CAR-T management at an early stage.

Abstract Background While quadruplet induction therapies deepen responses in newly diagnosed multiple myeloma patients, their impact on peripheral blood stem cell (PBSC) collection remains incompletely understood. This analysis aims to evaluate the effects of prolonged lenalidomide induction and isatuximab- or elotuzumab-containing quadruplet induction therapies on PBSC mobilization and collection. Methods A total of 179 transplant-eligible patients with newly diagnosed MM treated at a single academic center were included. The patients were evaluated based on PBSC mobilization and collection parameters, including overall collection results, CD34+ cell levels in peripheral blood, leukapheresis (LP) delays, overall number of LP sessions, and the rate of rescue mobilization with plerixafor. The patients underwent four different induction regimens: Lenalidomide, bortezomib, and dexamethasone (RVd, six 21-day cycles, n = 44), isatuximab-RVd (six 21-day cycles, n = 35), RVd (four 21-day cycles, n = 51), or elotuzumab-RVd (four 21-day cycles, n = 49). Results The patients' characteristics were well balanced across the different groups. Collection failures, defined as the inability to collect three sufficient PBSC transplants, were rare (n = 3, 2%), with no occurrences in the isatuximab-RVd and elotuzumab-RVd groups. Intensified induction with six 21-day cycles of RVd did not negatively impact the overall number of collected PBSCs (9.7 × 106/kg bw versus 10.5 × 106/kg bw, p = 0.331) compared to four 21-day cycles of RVd. Plerixafor usage was more common after six cycles of RVd compared to four cycles (16% versus 8%). Addition of elotuzumab to RVd did not adversely affect overall PBSC collection (10.9 × 106/kg bw versus 10.5 × 106/kg bw, p = 0.915). Patients treated with isatuximab-RVd (six cycles) had lower numbers of collected stem cells compared to those receiving RVd (six cycles) induction (8.8 × 106/kg bw versus 9.7 × 106/kg bw, p = 0.801), without experiencing significant delays in LP or increased numbers of LP sessions in a multivariable logistic regression analysis. Plerixafor usage was more common after isatuximab plus RVd compared to RVd alone (34% versus 16%). Conclusions This study demonstrates that stem cell collection is feasible after prolonged induction with isatuximab-RVd without collection failures and might be further explored as induction therapy. Trial registration Patients were treated within the randomized phase III clinical trials GMMG-HD6 (NCT02495922, 24/06/2015) and GMMG-HD7 (NCT03617731, 24/07/2018). However, during stem cell mobilization and -collection, no study-specific therapeutic intervention was performed.

Abstract Light chain amyloidosis (AL) is a rare disease caused by the generalized deposition of misfolded free light chains. Patients with immunoglobulin M gammopathy (IgM) and indolent B-cell lymphoma such as marginal zone lymphoma (MZL) may in some instances develop AL amyloidosis. So far, CAR T cells for AL amyloidosis have only been reported utilizing the B cell maturation antigen as target, while CD19 has so far not been used in AL amyloidosis. We report the case of a 71-year-old male, diagnosed with systemic AL kappa amyloidosis and MZL, receiving third-generation CAR T cell therapy targeting CD19. Prior treatment included bendamustine/rituximab and cyclophosphamide/ dexamethasone with subsequent autologous stem cell transplantation. CAR T application was well tolerated despite heart and kidney amyloid manifestations, and only early low-grade procedure-specific toxicities were observed. A continuous decrease in IgM, kappa light chains and kappa-to-lambda light chain difference was observed in the patient from day + 30 on, resulting in a deep hematological response six months after treatment. In summary, we present a novel case of CAR T cell treatment with third generation CD19 directed infusion for AL amyloidosis with an underlying secretory active B cell lymphoma, showing that this is an effective treatment modality and can be applied to patients with subsequent AL amyloidosis.

Abstract Background Third-generation chimeric antigen receptor (CAR)-engineered T cells (CARTs) might improve clinical outcome of patients with B cell malignancies. This is the first report on a third-generation CART dose-escalating, phase-1/2 investigator-initiated trial treating adult patients with refractory and/or relapsed (r/r) acute lymphoblastic leukemia (ALL). Methods Thirteen patients were treated with escalating doses of CD19-directed CARTs between 1 × 106 and 50 × 106 CARTs/m2. Leukapheresis, manufacturing and administration of CARTs were performed in-house. Results For all patients, CART manufacturing was feasible. None of the patients developed any grade of Immune effector cell-associated neurotoxicity syndrome (ICANS) or a higher-grade (≥ grade III) catokine release syndrome (CRS). CART expansion and long-term CART persistence were evident in the peripheral blood (PB) of evaluable patients. At end of study on day 90 after CARTs, ten patients were evaluable for response: Eight patients (80%) achieved a complete remission (CR), including five patients (50%) with minimal residual disease (MRD)-negative CR. Response and outcome were associated with the administered CART dose. At 1-year follow-up, median overall survival was not reached and progression-free survival (PFS) was 38%. Median PFS was reached on day 120. Lack of CD39-expression on memory-like T cells was more frequent in CART products of responders when compared to CART products of non-responders. After CART administration, higher CD8 + and γδ-T cell frequencies, a physiological pattern of immune cells and lower monocyte counts in the PB were associated with response. Conclusion In conclusion, third-generation CARTs were associated with promising clinical efficacy and remarkably low procedure-specific toxicity, thereby opening new therapeutic perspectives for patients with r/r ALL. Trial registration This trial was registered at www.clinicaltrials.gov as NCT03676504.

Summary: Background: Assessment of cardiovascular risk is critical for patients with cancer. Previous retrospective studies suggest potential cardiotoxicity of CAR T cell therapies. We aimed to prospectively assess cardiotoxicity and the predictive value of cardiac biomarkers and classical risk factors (age, cardiac function, diabetes, arterial hypertension, smoking) for cardiac events and all-cause mortality (ACM). Methods: In this prospective cohort study, all patients treated with CAR T cell constructs (axi-cel, tisa-cel, brexu-cel, ide-cel, or the 3rd generation CAR HD-CAR-1) from Oct 1, 2018, to Sept 30, 2022 at the University Hospital Heidelberg were included. Surveillance included cardiac assessment with biomarkers (high-sensitive Troponin T (hs-cTnT), N-terminal brain natriuretic peptide (NT-proBNP)), 12-lead-ECG, and 2D echocardiography. ACM was defined as the primary study endpoint, while cardiotoxicity, defined by clinical syndromes of heart failure or decline in ejection fraction, served as a secondary endpoint. Findings: Overall, 137 patients (median age 60, range 20–83, IQR 16), were included in the study. 46 patients died during the follow up period (median 0.75 years, range 0.02–4.33, IQR 0.89) 57 month, with a median survival of 0.57 years (range 0.03–2.38 years, IQR 0.79). A septal wall thickness above 11 mm (HR 2.48, 95%-CI = 1.10–5.67, p = 0.029) was associated with an increased risk of ACM, with a trend seen for reduced left ventricular ejection fraction prior to therapy (LVEF

Background Relapse and graft-versus-host disease (GVHD) are the main causes of death after allogeneic hematopoietic cell transplantation (HCT). Preclinical murine models and clinical data suggest that invariant natural killer T (iNKT) cells prevent acute and chronic GVHD. In addition, iNKT cells are crucial for efficient immune responses against malignancies and contribute to reduced relapse rates after transplantation. Chimeric antigen receptors (CAR) redirect effector cells to cell surface antigens and enhance killing of target cells. With this study, we aimed to combine enhanced cytotoxicity of CD19-CAR-iNKT cells against lymphoma cells with their tolerogenic properties.Methods iNKT cells were isolated from peripheral blood mononuclear cells and transduced with an anti-CD19-CAR retrovirus. After in vitro expansion, the functionality of CD19-CAR-iNKT cells was assessed by flow cytometry, image stream analysis and multiplex analysis in single-stimulation or repeated-stimulation assays. Moreover, the immunoregulatory properties of CD19-CAR-iNKT cells were analyzed in apoptosis assays and in mixed lymphocyte reactions. The effect of checkpoint inhibition through nivolumab was analyzed in these settings.Results In this study, we could show that the cytotoxicity of CD19-CAR-iNKT cells was mediated either through engagement of their CAR or their invariant T-cell receptor, which may circumvent loss of response through antigen escape. However, encounter of CD19-CAR-iNKT cells with their target induced a phenotype of exhaustion. Consequently, checkpoint inhibition increased cytokine release, cytotoxicity and survival of CD19-CAR-iNKT cells. Additionally, they showed robust suppression of alloreactive immune responses.Conclusion In this work, we demonstrate that CAR-iNKT cells are a powerful cytotherapeutic option to prevent or treat relapse while potentially reducing the risk of GVHD after allogeneic HCT.

BackgroundThe administration of modified immune cells (MIC) before kidney transplantation led to specific immunosuppression against the allogeneic donor and a significant increase in regulatory B lymphocytes. We wondered how this approach affected the continued clinical course of these patients.MethodsTen patients from a phase I clinical trial who had received MIC infusions prior to kidney transplantation were retrospectively compared to 15 matched standard-risk recipients. Follow-up was until year five after surgery.ResultsThe 10 MIC patients had an excellent clinical course with stable kidney graft function, no donor-specific human leukocyte antigen antibodies (DSA) or acute rejections, and no opportunistic infections. In comparison, a retrospectively matched control group receiving standard immunosuppressive therapy had a higher frequency of DSA (log rank P = 0.046) and more opportunistic infections (log rank P = 0.033). Importantly, MIC patients, and in particular the four patients who had received the highest cell number 7 days before surgery and received low immunosuppression during follow-up, continued to show a lack of anti-donor T lymphocyte reactivity in vitro and high CD19+CD24hiCD38hi transitional and CD19+CD24hiCD27+ memory B lymphocytes until year five after surgery.ConclusionsMIC infusions together with reduced conventional immunosuppression were associated with good graft function during five years of follow-up, no de novo DSA development and no opportunistic infections. In the future, MIC infusions might contribute to graft protection while reducing the side effects of immunosuppressive therapy. However, this approach needs further validation in direct comparison with prospective controls.Trial registrationhttps://clinicaltrials.gov/, identifier NCT02560220 (for the TOL-1 Study). EudraCT Number: 2014-002086-30.

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.

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.

Abstract Background High-dose (HD) chemotherapy followed by autologous blood stem-cell transplantation (ASCT) is the standard treatment for multiple myeloma (MM) patients. However, the collection of sufficient peripheral blood stem cell (PBSC) grafts can be challenging, and the question arises whether reinfusion of low-dose grafts will lead to a hematopoietic recovery. Methods The hematopoietic recovery of 148 MM patients who underwent HD melphalan chemotherapy and received PBSC transplants with varying CD34+ cells doses (3–4 × 106 [n = 86], 2–2.5 × 106 [n = 53],

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.

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.

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.

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.

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.

Introduction: Cytomegalovirus (CMV) reactivation occurs in seronegative patients after solid organ transplantation (SOT) particularly from seropositive donors and can be lethal. Generation of CMV-specific T cells helps to prevent CMV reactivation. Therefore, we initiated a clinical phase I CMVpp65 peptide vaccination trial for seronegative end-stage renal disease patients waiting for kidney transplantation. Methods: The highly immunogenic nonamer peptide NLVPMVATV derived from CMV phosphoprotein 65(CMVpp65) in a water-in-oil emulsion (Montanide™) plus imiquimod (Aldara™) as an adjuvant was administered subcutaneously four times biweekly. Clinical course as well as immunological responses were monitored using IFN-γ ELISpot assays and flow cytometry for CMV-specific CD8+ T cells. Results: Peptide vaccination was well tolerated, and no drug-related serious adverse events were detected except for Grade I–II local skin reactions. Five of the 10 patients (50%) mounted any immune response (responders) and 40% of the patients presented CMV-specific CD8+ T cell responses elicited by these prophylactic vaccinations. No responders experienced CMV reactivation in the 18 months post-transplantation, while all non-responders reactivated. Conclusion: CMVpp65 peptide vaccination was safe, well tolerated, and clinically encouraging in seronegative end-stage renal disease patients waiting for kidney transplantation. Further studies with larger patient cohorts are planned.

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.

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.

Graft-vs.-host disease (GvHD), a severe complication of allogeneic hematopoietic stem cell transplantation, significantly affects the post-transplant morbidity and mortality. Systemic steroids remain the gold standard for the initial management of GvHD. However, up to 60% of patients will not sufficiently respond to steroids. Extracorporeal photopheresis (ECP), a cell-based immunotherapy, has shown good clinical results in such steroid-refractory/resistant GvHD patients. Given its immunomodulatory, but not global immunosuppressive and steroid-sparing capacity, ECP constitutes an attractive option. In the case of GvHD, the balance of immune cells is destroyed: effector cells are not any longer efficiently controlled by regulatory cells. ECP therapy may restore this balance. However, the precise mechanism and the impact of ECP on anti-viral/anti-leukemic function remain unclear. In this study, 839 ECP treatments were performed on patients with acute GvHD (aGvHD) and chronic GvHD (cGvHD). A comprehensive analysis of effector and regulatory cells in patients under ECP therapy included multi-parametric flow cytometry and tetramer staining, LuminexTM-based cytokine, interferon-γ enzyme-linked immunospot, and chromium-51 release assays. Gene profiling of myeloid-derived suppressor cells (MDSCs) was performed by microarray analysis. Immunologically, modulations of effector and regulatory cells as well as proinflammatory cytokines were observed under ECP treatment: (1) GvHD-relevant cell subsets like CD62L+ NK cells and newly defined CD19hiCD20hi B cells were modulated, but (2) quantity and quality of anti-viral/anti-leukemic effector cells were preserved. (3) The development of MDSCs was promoted and switched from an inactivated subset (CD33−CD11b+) to an activated subset (CD33+CD11b+). (4) The frequency of Foxp3+CD4+ regulatory T cells (Tregs) and CD24+CD38hi regulatory B cells was considerably increased in aGvHD patients, and Foxp3+CD8+ Tregs in cGvHD patients. (5) Proinflammatory cytokines like IL-1β, IL-6, IL-8, and TNF-α were significantly reduced. In summary, ECP constitutes an effective immunomodulatory therapy for patients with steroid-refractory/resistant GvHD without impairment of anti-viral/leukemia effects.

IntroductionTherapy with chimeric antigen receptor T (CART) cells for hematological malignancies has shown promising results. Effectiveness of CART cells may depend on the ratio of naive (TN) vs. effector (TE) T cells, TN cells being responsible for an enduring antitumor activity through maturation. Therefore, we investigated factors influencing the TN/TE ratio of CART cells.Materials and methodsCART cells were generated upon transduction of peripheral blood mononuclear cells with a CD19.CAR-CD28-CD137zeta third generation retroviral vector under two different stimulating culture conditions: anti-CD3/anti-CD28 antibodies adding either interleukin (IL)-7/IL-15 or IL-2. CART cells were maintained in culture for 20 days. We evaluated 24 healthy donors (HDs) and 11 patients with chronic lymphocytic leukemia (CLL) for the composition of cell subsets and produced CART cells. Phenotype and functionality were tested using flow cytometry and chromium release assays.ResultsIL-7/IL-15 preferentially induced differentiation into TN, stem cell memory (TSCM: naive CD27+ CD95+), CD4+ and CXCR3+ CART cells, while IL-2 increased effector memory (TEM), CD56+ and CD4+ T regulatory (TReg) CART cells. The net amplification of different CART subpopulations derived from HDs and untreated CLL patients was compared. Particularly the expansion of CD4+ CARTN cells differed significantly between the two groups. For HDs, this subtype expanded >60-fold, whereas CD4+ CARTN cells of untreated CLL patients expanded less than 10-fold. Expression of exhaustion marker programmed cell death 1 on CARTN cells on day 10 of culture was significantly higher in patient samples compared to HD samples. As the percentage of malignant B cells was expectedly higher within patient samples, an excessive amount of B cells during culture could account for the reduced expansion potential of CARTN cells in untreated CLL patients. Final TN/TE ratio stayed 2 in samples from HDs stimulated with IL-7/IL-15, thus demonstrating efficient CARTN expansion.ConclusionUntreated CLL patients might constitute a challenge for long-lasting CART effects in vivo since only a low number of TN among the CART product could be generated. Depletion of malignant B cells before starting CART production might be considered to increase the TN/TE ratio within the CART product.

Cytomegalovirus (CMV) disease constitutes a serious complication after stem cell transplantation and has been treated by adoptive transfer of donor-derived CMV-specific CD8+ T cells. CMV-specific CD8+ T cells were selected by multimers, and the technologies may alter the function of these T cells. Therefore, here we evaluated the impact of multimer reagents on the function of CD8+ T lymphocytes. Methods: CMV-specific CD8+ T cells were purified from the peripheral blood of donors using tetra- and streptamer technologies. The functional status of purified CMV-specific CD8+ T cells was assessed by multiparametric immunophenotyping and carboxyfluorescein succinimidyl ester proliferation assays as well as by enzyme-linked immunospot assays. Results: A similar percentage of CMV-specific CD8+ T cells could be purified by both tetra- (90%) and streptamer (92%) technologies. That constitutes a 30- to 50-fold concentration of CMV-specific CD8+CD45RA+CCR7−effector T cells. Selected cells secreted interferon-gamma and granzyme B upon stimulation with CMVpp65 peptide, thus demonstrating their functionality. Conclusion: Our study demonstrated that both tetra- and streptamer technologies can be used to purify CMV-specific cytotoxic CD8+ T cells for adoptive T-cell transfer. Both multimer technologies did not have any negative influence on the proliferation of selected T cells. Importantly, streptamer technology is available at good manufacturing practice level.

While most secreted proteins contain defined signal peptides that direct their extracellular transport through the ER-Golgi pathway, nonclassical transport of leaderless peptides/proteins was first described 20 years ago and the mechanisms responsible for unconventional export of such proteins have been thoroughly reviewed. In addition to directed nonclassical secretion, a number of leaderless secreted proteins have been classified as damage-associated molecular-pattern (DAMP) molecules, which are nuclear or cytoplasmic proteins that, under necrotic or apoptotic conditions, are released outside the cell and function as proinflammatory signals. A strong association between persistent release of DAMPs, chronic inflammation, and the hypoxic tumor microenvironment has been proposed. Thus, protein localization and function can change fundamentally from intracellular to extracellular compartments, often under conditions of inflammation, cancer, and arthritis. If we are truly to understand, model, and treat such biological states, it will be important to investigate these multifunctional proteins and their contribution to degenerative diseases. Here, we will focus our discussion on intracellular proteins, both cytoplasmic and nuclear, that play critical extracellular roles. In particular, the multifunctional nature of HMMR/RHAMM and survivin will be highlighted and compared, as these molecules are the subject of extensive biological and therapeutic investigations within hematology and oncology fields. For these and other genes/proteins, we will highlight points of structural and functional intersection during cellular division and differentiation, as well as states associated with cancer, such as tumor-initiation and epithelial-to-mesenchymal transition (EMT). Finally, we will discuss the potential targeting of these proteins for improved therapeutic outcomes within these degenerative disorders. Our goal is to highlight a number of commonalities among these multifunctional proteins for better understanding of their putative roles in tumor initiation, inflammation, arthritis, and cancer.

Background Recently, we demonstrated immunological and clinical responses to a RHAMM-R3 peptide vaccine in patients with acute myeloid leukemia, myelodysplastic syndrome and multiple myeloma. To improve the outcome of the vaccine, a second cohort was vaccinated with a higher dose of 1,000 μg peptide.Design and Methods Nine patients received four vaccinations subcutaneously at a biweekly interval. Immunomonitoring of cytotoxic CD8+ as well as regulatory CD4+ T cells was performed by flow cytometry as well as by enzyme-linked immunospot (ELISpot) assays. Parameters of clinical response were assessed.Results In 4 of 9 patients (44%) we detected positive immunological responses. These patients showed an increase of CD8+RHAMM-R3_tetramer+/CD45RA+/CCR7−/CD27−/CD28− effector T cells and an increase of R3-specific CD8+ T cells. Two of these patients showed a significant decrease of regulatory T cells (Tregs). In one patient without response Tregs frequency increased from 5 to 16%. Three patients showed clinical effects: one patient with myelodysplastic syndrome RAEB-1 showed a reduction of leukemic blasts in the bone marrow, another myelodysplastic syndrome patient an improvement of peripheral blood counts and one patient with multiple myeloma a reduction of free light chains. Clinical and immunological reactions were lower in this cohort than in the 300 μg cohort.Conclusions High-dose RHAMM-R3 peptide vaccination induced immunological responses and positive clinical effects. Therefore, RHAMM constitutes a promising structure for further targeted immunotherapies in patients with different hematologic malignancies. However, higher doses of peptide did not improve the frequency and intensity of immune responses in this trial. (This study is registered at http://ISRCTN.org as ISRCTN32763606)