CD70-Targeted Allogeneic CAR T-Cell Therapy for Advanced Clear Cell Renal Cell Carcinoma.

Therapeutic approaches for clear cell renal cell carcinoma (ccRCC) remain limited; however, chimeric antigen receptor (CAR) T-cell therapies may offer novel treatment options. CTX130, an allogeneic CD70-targeting CAR T-cell product, was developed for the treatment of advanced or refractory ccRCC. We report that CTX130 showed favorable preclinical proliferation and cytotoxicity profiles and completely regressed RCC xenograft tumors. We also report results from 16 patients with relapsed/refractory ccRCC who received CTX130 in a phase I, multicenter, first-in-human clinical trial. No patients encountered dose-limiting toxicity, and disease control was achieved in 81.3% of patients. One patient remains in a durable complete response at 3 years. Finally, we report on a next-generation CAR T construct, CTX131, in which synergistic potency edits to CTX130 confer improved expansion and efficacy in preclinical studies. These data represent a proof of concept for the treatment of ccRCC and other CD70+ malignancies with CD70-targeted allogeneic CAR T cells. SIGNIFICANCE: Although the role of CAR T cells is well established in hematologic malignancies, the clinical experience in solid tumors has been disappointing. This clinical trial demonstrates the first complete response in a patient with RCC, reinforcing the potential benefit of CAR T cells in the treatment of solid tumors.

Tumor-Infiltrating Lymphocyte and Other Cell Therapies for Metastatic Melanoma.

ABSTRACT: Major progress in prolonging survival of patients with advanced melanoma has been made in the past decade because of the development and approval of immune checkpoint inhibitor and targeted therapies. However, for nonresponding or relapsing patients, their prognosis is still dismal. Based on clinical trial data, treatment with adoptive cell therapies holds great promise. In patients with metastatic melanoma progressing on or nonresponsive to single-agent anti-programmed cell death 1, infusion of tumor-infiltrating lymphocytes can produce responses in up to half of patients, with durable complete responses in up to 20%. Genetic modification of peripheral blood T cells with T-cell receptors derived from tumor-specific T cells, or with chimeric antigen receptors, has the potential to further improve treatment outcomes in this refractory population. In this review, we will discuss the historical development, current status, and future perspectives of adoptive T-cell therapies in melanoma.

Tumour-infiltrating lymphocyte therapy for patients with advanced-stage melanoma.

Immunotherapy with immune-checkpoint inhibitors (ICIs) and targeted therapy with BRAF and MEK inhibitors have revolutionized the treatment of melanoma over the past decade. Despite these breakthroughs, the 5-year survival rate of patients with advanced-stage melanoma is at most 50%, emphasizing the need for additional therapeutic strategies. Adoptive cell therapy with tumour-infiltrating lymphocytes (TILs) is a therapeutic modality that has, in the past few years, demonstrated long-term clinical benefit in phase II/III trials involving patients with advanced-stage melanoma, including those with disease progression on ICIs and/or BRAF/MEK inhibitors. In this Review, we summarize the current status of TIL therapies for patients with advanced-stage melanoma, including potential upcoming marketing authorization, the characteristics of TIL therapy products, as well as future strategies that are expected to increase the efficacy of this promising cellular immunotherapy.

CLDN6-specific CAR-T cells plus amplifying RNA vaccine in relapsed or refractory solid tumors: the phase 1 BNT211-01 trial.

The oncofetal antigen Claudin 6 (CLDN6) is highly and specifically expressed in many solid tumors, and could be a promising treatment target. We report dose escalation results from the ongoing phase 1/2 BNT211-01 trial evaluating the safety and feasibility of chimeric antigen receptor (CAR) T cells targeting the CLDN6 with or without a CAR-T cell-amplifying RNA vaccine (CARVac) at two dose levels (DLs) in relapsed/refractory CLDN6-positive solid tumors. The primary endpoints were safety and tolerability, maximum tolerated dose and recommended phase 2 dose (RP2D). Secondary endpoints included objective response rate (ORR) and disease control rate. We observed manageable toxicity, with 10 out of 22 patients (46%) experiencing cytokine release syndrome including one grade 3 event and 1 out of 22 (5%) with grade 1 immune effector cell-associated neurotoxicity syndrome. Dose-limiting toxicities occurred in two patients at the higher DL, resolving without sequelae. CAR-T cell engraftment was robust, and the addition of CARVac was well tolerated. The unconfirmed ORR in 21 evaluable patients was 33% (7 of 21), including one complete response. The disease control rate was 67% (14 of 21), with stable disease in seven patients. Patients with germ cell tumors treated at the higher DL exhibited the highest response rate (ORR 57% (4 of 7)). The maximum tolerated dose and RP2D were not established as the trial has been amended to utilize an automated manufacturing process. A repeat of the dose escalation is ongoing and will identify a RP2D for pivotal trials. ClinicalTrials.gov Identifier: NCT04503278 .

The emerging role for CAR T cells in solid tumor oncology.

In recent years, treatment with chimeric antigen receptor (CAR) T-cells has revolutionized the outcomes of patients with relapsed or refractory hematological malignancies with long-term remissions in >30% of patients. Similarly, the introduction of immune checkpoint inhibitor therapy changed the therapeutic landscape for several solid malignancies also leading to impressive long-term remission in patients. However, so far CAR T-cell therapy in solid tumors has shown low response rates and especially a lack of long-term remissions. This review focuses on the latest clinical advances and discusses promising results seen with CAR T-cells exploring new target antigens. We then review relevant challenges limiting long-term responses with CAR T-cell therapy in solid tumors like CAR T-cell persistence and target antigen expression. In addition, there is an increasing understanding on T-cell function and dysfunction within the immunosuppressive tumor microenvironment. This comprises of inhibitory cytokines and checkpoint molecules limiting the killing capacity of CAR T-cells. Finally, we will discuss how this deeper knowledge can be used to develop CAR T-cell therapies overcoming these inhibitory factors and results in CAR T-cell products with higher efficacy and safety. These technological developments will hopefully lead to enhanced clinical activity and improved solid tumor patient outcomes in the near future.

Human T cells recognize HLA-DP-bound peptides in two orientations.

Human leukocyte antigen (HLA) molecules present small peptide antigens to T cells, thereby allowing them to recognize pathogen-infected and cancer cells. A central dogma over the last 50+ y is that peptide binding to HLA molecules is mediated by the docking of side chains of particular amino acids in the peptide into pockets in the HLA molecules in a conserved N- to C-terminal orientation. Whether peptides can be presented in a reversed C- to N-terminal orientation remains unclear. Here, we performed large-scale identification of peptides bound to HLA-DP molecules and observed that in addition to peptide binding in an N- to C-terminal orientation, in 9 out of 14 HLA-DP allotypes, reverse motifs are found, compatible with C- to N-terminal peptide binding. Moreover, we isolated high-avidity human cytomegalovirus (CMV)-specific HLA-DP-restricted CD4+ T cells from the memory repertoire of healthy donors and demonstrate that such T cells recognized CMV-derived peptides bound to HLA-DPB1*01:01 or *05:01 in a reverse C- to N-terminal manner. Finally, we obtained a high-resolution HLA-DPB1*01:01-CMVpp65(142-158) peptide crystal structure, which is the molecular basis for C- to N-terminal peptide binding to HLA-DP. Our results point to unique features of HLA-DP molecules that substantially broaden the HLA class II bound peptide repertoire to combat pathogens and eliminate cancer cells.

LDHB Overexpression Can Partially Overcome T Cell Inhibition by Lactic Acid.

Accelerated glycolysis leads to secretion and accumulation of lactate and protons in the tumor environment and determines the efficacy of adoptive T cell and checkpoint inhibition therapy. Here, we analyzed effects of lactic acid on different human CD4 T cell subsets and aimed to increase CD4 T cell resistance towards lactic acid. In all CD4 T cell subsets analyzed, lactic acid inhibited metabolic activity (glycolysis and respiration), cytokine secretion, and cell proliferation. Overexpression of the lactate-metabolizing isoenzyme LDHB increased cell respiration and mitigated lactic acid effects on intracellular cytokine production. Strikingly, LDHB-overexpressing cells preferentially migrated into HCT116 tumor spheroids and displayed higher expression of cytotoxic effector molecules. We conclude, that LDHB overexpression might be a promising strategy to increase the efficacy of adoptive T cell transfer therapy.

Promiscuity of Peptides Presented in HLA-DP Molecules from Different Immunogenicity Groups Is Associated With T-Cell Cross-Reactivity.

In the context of HLA-DP-mismatched allogeneic stem cell transplantation, mismatched HLA-DP alleles can provoke profound allo-HLA-DP-specific immune responses from the donor T-cell repertoire leading to graft-versus-leukemia effect and/or graft-versus-host disease in the patient. The magnitude of allo-HLA-DP-specific immune responses has been shown to depend on the specific HLA-DP disparity between donor and patient and the immunogenicity of the mismatched HLA-DP allele(s). HLA-DP peptidome clustering (DPC) was developed to classify the HLA-DP molecules based on similarities and differences in their peptide-binding motifs. To investigate a possible categorization of HLA-DP molecules based on overlap of presented peptides, we identified and compared the peptidomes of the thirteen most frequently expressed HLA-DP molecules. Our categorization based on shared peptides was in line with the DPC classification. We found that the HLA-DP molecules within the previously defined groups DPC-1 or DPC-3 shared the largest numbers of presented peptides. However, the HLA-DP molecules in DPC-2 segregated into two subgroups based on the overlap in presented peptides. Besides overlap in presented peptides within the DPC groups, a substantial number of peptides was also found to be shared between HLA-DP molecules from different DPC groups, especially for groups DPC-1 and -2. The functional relevance of these findings was illustrated by demonstration of cross-reactivity of allo-HLA-DP-reactive T-cell clones not only against HLA-DP molecules within one DPC group, but also across different DPC groups. The promiscuity of peptides presented in various HLA-DP molecules and the cross-reactivity against different HLA-DP molecules demonstrate that these molecules cannot be strictly categorized in immunogenicity groups.

A Randomized Phase II Trial Comparing the Efficacy and Safety of Pioglitazone, Clarithromycin and Metronomic Low-Dose Chemotherapy with Single-Agent Nivolumab Therapy in Patients with Advanced Non-small Cell Lung Cancer Treated in Second or Further Line (ModuLung).

Background: Most non-small cell lung cancers occur in elderly and frequently comorbid patients. Therefore, it is necessary to evaluate the efficacy of biomodulatory active therapy regimen, concertedly interfering with tumor-associated homeostatic pathways to achieve tumor control paralleled by modest toxicity profiles. Patients and Methods: The ModuLung trial is a national, multicentre, prospective, open-label, randomized phase II trial in patients with histologically confirmed stage IIIB/IV squamous (n = 11) and non-squamous non-small cell (n = 26) lung cancer who failed first-line platinum-based chemotherapy. Patients were randomly assigned on a 1:1 ratio to the biomodulatory or control group, treated with nivolumab. Patients randomized to the biomodulatory group received an all-oral therapy consisting of treosulfan 250 mg twice daily, pioglitazone 45 mg once daily, clarithromycin 250 mg twice daily, until disease progression or unacceptable toxicity. Results: The study had to be closed pre-maturely due to approval of immune checkpoint inhibitors (ICi) in first-line treatment. Thirty-seven patients, available for analysis, were treated in second to forth-line. Progression-free survival (PFS) was significantly inferior for biomodulation (N = 20) vs. nivolumab (N = 17) with a median PFS (95% confidence interval) of 1.4 (1.2-2.0) months vs. 1.6 (1.4-6.2), respectively; with a hazard ratio (95% confidence interval) of 1.908 [0.962; 3.788]; p = 0.0483. Objective response rate was 11.8% with nivolumab vs. 5% with biomodulation, median follow-up 8.25 months. The frequency of grade 3-5 treatment related adverse events was 29% with nivolumab and 10% with biomodulation. Overall survival (OS), the secondary endpoint, was comparable in both treatment arms; biomodulation with a median OS (95% confidence interval) of 9.4 (6.0-33.0) months vs. nivolumab 6.9 (4.6-24.0), respectively; hazard ratio (95% confidence interval) of 0.733 [0.334; 1.610]; p = 0.4368. Seventy-five percent of patients in the biomodulation arm received rescue therapy with checkpoint inhibitors. Conclusions: This trial shows that the biomodulatory therapy was inferior to nivolumab on PFS. However, the fact that OS was similar between groups gives rise to the hypothesis that the well-tolerable biomodulatory therapy may prime tumor tissues for efficacious checkpoint inhibitor therapy, even in very advanced treatment lines where poor response to ICi might be expected with increasing line of therapy.

HLA-DPB1 Reactive T Cell Receptors for Adoptive Immunotherapy in Allogeneic Stem Cell Transplantation.

HLA-DPB1 antigens are mismatched in about 80% of allogeneic hematopoietic stem cell transplantations from HLA 10/10 matched unrelated donors and were shown to be associated with a decreased risk of leukemia relapse. We recently developed a reliable in vitro method to generate HLA-DPB1 mismatch-reactive CD4 T-cell clones from allogeneic donors. Here, we isolated HLA-DPB1 specific T cell receptors (TCR DP) and used them either as wild-type or genetically optimized receptors to analyze in detail the reactivity of transduced CD4 and CD8 T cells toward primary AML blasts. While both CD4 and CD8 T cells showed strong AML reactivity in vitro, only CD4 T cells were able to effectively eliminate leukemia blasts in AML engrafted NOD/SCID/IL2Rγc-/- (NSG) mice. Further analysis showed that optimized TCR DP and under some conditions wild-type TCR DP also mediated reactivity to non-hematopoietic cells like fibroblasts or tumor cell lines after HLA-DP upregulation. In conclusion, T cells engineered with selected allo-HLA-DPB1 specific TCRs might be powerful off-the-shelf reagents in allogeneic T-cell therapy of leukemia. However, because of frequent (common) cross-reactivity to non-hematopoietic cells with optimized TCR DP T cells, safety mechanisms are mandatory.

IL6-receptor antibody tocilizumab as salvage therapy in severe chronic graft-versus-host disease after allogeneic hematopoietic stem cell transplantation: a retrospective analysis.

Chronic graft-versus-host disease (cGvHD) remains the most relevant factor affecting survival after allogeneic hematopoietic stem cell transplantation (alloHSCT). Besides corticosteroids (and ibrutinib in the USA), there is no established therapy for cGvHD. Tocilizumab, a humanized IgG1 IL6-receptor antibody, has shown efficacy in acute GvHD and cGvHD. We retrospectively analyzed the efficacy and safety of tocilizumab for the treatment of advanced cGvHD. Eleven patients with severe steroid refractory cGvHD (median age 49; range 21-62 years) that received at least two prior lines of therapy for cGvHD (range 2-8 regimens) were treated with tocilizumab (q4w, dosage 8 mg/kg IV) with a median number of 15 cycles (range 2-31). NIH consensus criteria grading for cGvHD were recorded prior to tocilizumab administration and after 3, 6, and 12 months of therapy. All patients received additional concomitant immunosuppression (IS) but no new IS within the last 4 weeks before start of tocilizumab and response assessment was terminated before start of any new IS. The median number of days between alloHSCT and initiation of tocilizumab therapy was 1033 days. Organs involved at initiation of tocilizumab therapy were skin (100%, all grade 3), eyes (82%), fascia (82%), mouth (64%), lungs (55%), and genitals (18%). Overall, 7/10 patients (70%) showed partial remission, 2/10 patients (20%) showed progressive cGvHD, 1/10 patient (10%) showed mixed response, and 1 patient died due to sepsis before first response assessment 1.5 months after initiation of treatment. Four patients required subsequent new immunosuppressive treatment. Two patients developed bacterial sepsis, one of whom died. The overall survival and relapse-free survival were 82% with an average follow-up of 22 months (range 1.5-52 months). Tocilizumab seems a promising treatment option in advanced cGvHD but further evaluation within a phase II trial is required.

Long-term follow-up of rituximab in treatment of chronic graft-versus-host disease-single center experience.

Rituximab was recently described also as first-line therapy of chronic graft-versus-host disease (cGvHD). We retrospectively analyzed the efficacy and safety of all patients receiving rituximab for treatment of cGvHD between 2005 and 2016 at the Regensburg University transplant center with a median follow-up after rituximab therapy of 2.8 years. Responses of 29 allogeneic stem cell-transplanted patients (median age 49) with previous failure of response to steroids including one patient after donor lymphocyte infusion were assessed. Three months after rituximab application, the overall response rate was 31% (7% complete (n = 2) and 24% partial remission (n = 7)). At 12 months, overall survival was 72% (n = 21) and failure-free survival was 24% (n = 7). We further analyzed associations of rituximab response with clinical characteristics showing a higher response rate in steroid-dependent cGvHD patients (89% of 9 responding compared to steroid refractory patients (11% responding)). However, this difference was not statistically significant. Seven patients (24%) (including four lethal infectious complications) developed serious infections requiring hospitalization within 1-9 months after rituximab therapy exclusively in patients failing to respond to rituximab. In conclusion, rituximab appears to be an effective treatment of cGvHD especially in steroid dependent patients, but identification of biomarker predicting response will be crucial to avoid long-term infectious morbidity and mortality in non-responders.

The role of checkpoint blockade after allogeneic stem cell transplantation in diseases other than Hodgkin's Lymphoma.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment option for many malignant high-risk hematological diseases. The Graft-vs.-Tumor (GvT) effect is the major hallmark of this treatment approach. However, disease relapse remains a major limitation. Boosting the GvT effect by checkpoint inhibitors (CI) is an attractive option in this desperate situation although potentially triggering Graft-vs.-Host Disease (GvHD). Early reports in patients with Hodgkin's lymphoma support the idea that CI therapy after HSCT is feasible and effective. We have retrospectively analyzed CI therapy for treatment of disease recurrence after allo-HSCT other than Hodgkin's lymphoma including 21 patients from eight German transplant centers. The median follow-up was 59 days. The overall response rate (ORR) was 43%. Patients receiving donor lymphocyte infusion (DLI) in combination with CI had superior response (ORR 80%). Severe acute GvHD grade III-IV and moderate to severe chronic GvHD were observed in 29% of all patients. Taken together, CI therapy in relapsed patients after HSCT, especially in combination with DLI, is effective but induces severe GvHD in a considerable proportion of patients. Thus, prospective trials or EBMT registry-based validation of different dosing and application schedules including immunosuppressive regimens in those patients are urgently needed.

Anakoinosis: Correcting Aberrant Homeostasis of Cancer Tissue-Going Beyond Apoptosis Induction.

The current approach to systemic therapy for metastatic cancer is aimed predominantly at inducing apoptosis of cancer cells by blocking tumor-promoting signaling pathways or by eradicating cell compartments within the tumor. In contrast, a systems view of therapy primarily considers the communication protocols that exist at multiple levels within the tumor complex, and the role of key regulators of such systems. Such regulators may have far-reaching influence on tumor response to therapy and therefore patient survival. This implies that neoplasia may be considered as a cell non-autonomous disease. The multi-scale activity ranges from intra-tumor cell compartments, to the tumor, to the tumor-harboring organ to the organism. In contrast to molecularly targeted therapies, a systems approach that identifies the complex communications networks driving tumor growth offers the prospect of disrupting or "normalizing" such aberrant communicative behaviors and therefore attenuating tumor growth. Communicative reprogramming, a treatment strategy referred to as anakoinosis, requires novel therapeutic instruments, so-called master modifiers to deliver concerted tumor growth-attenuating action. The diversity of biological outcomes following pro-anakoinotic tumor therapy, such as differentiation, trans-differentiation, control of tumor-associated inflammation, etc. demonstrates that long-term tumor control may occur in multiple forms, inducing even continuous complete remission. Accordingly, pro-anakoinotic therapies dramatically extend the repertoire for achieving tumor control and may activate apoptosis pathways for controlling resistant metastatic tumor disease and hematologic neoplasia.

Clinical Efficacy of a Novel Therapeutic Principle, Anakoinosis.

Classic tumor therapy, consisting of cytotoxic agents and/or targeted therapy, has not overcome therapeutic limitations like poor risk genetic parameters, genetic heterogeneity at different metastatic sites or the problem of undruggable targets. Here we summarize data and trials principally following a completely different treatment concept tackling systems biologic processes: the principle of communicative reprogramming of tumor tissues, i.e., anakoinosis (ancient greek for communication), aims at establishing novel communicative behavior of tumor tissue, the hosting organ and organism via re-modeling gene expression, thus recovering differentiation, and apoptosis competence leading to cancer control - in contrast to an immediate, "poisoning" with maximal tolerable doses of targeted or cytotoxic therapies. Therefore, we introduce the term "Master modulators" for drugs or drug combinations promoting evolutionary processes or regulating homeostatic pathways. These "master modulators" comprise a broad diversity of drugs, characterized by the capacity for reprogramming tumor tissues, i.e., transcriptional modulators, metronomic low-dose chemotherapy, epigenetically modifying agents, protein binding pro-anakoinotic drugs, such as COX-2 inhibitors, IMiDs etc., or for example differentiation inducing therapies. Data on 97 anakoinosis inducing schedules indicate a favorable toxicity profile: The combined administration of master modulators, frequently (with poor or no monoactivity) may even induce continuous complete remission in refractory metastatic neoplasia, irrespectively of the tumor type. That means recessive components of the tumor, successively developing during tumor ontogenesis, are accessible by regulatory active drug combinations in a therapeutically meaningful way. Drug selection is now dependent on situative systems characteristics, to less extent histology dependent. To sum up, anakoinosis represents a new substantive therapy principle besides novel targeted therapies.

Biomodulatory Treatment With Azacitidine, All-trans Retinoic Acid and Pioglitazone Induces Differentiation of Primary AML Blasts Into Neutrophil Like Cells Capable of ROS Production and Phagocytosis.

Effective and tolerable salvage therapies for elderly patients with chemorefractory acute myeloid leukemia (AML) are limited and usually do not change the poor clinical outcome. We recently described in several chemorefractory elderly AML patients that a novel biomodulatory treatment regimen consisting of low-dose azacitidine (AZA) in combination with PPARγ agonist pioglitazone (PGZ) and all-trans retinoic acid (ATRA) induced complete remission of leukemia and also triggered myeloid differentiation with rapid increase of peripheral blood neutrophils. Herein, we further investigated our observations and comprehensively analyzed cell differentiation in primary AML blasts after treatment with ATRA, AZA, and PGZ ex vivo. The drug combination was found to significantly inhibit cell growth as well as to induce cell differentiation in about half of primary AML blasts samples independent of leukemia subtype. Notably and in comparison to ATRA/AZA/PGZ triple-treatment, effects on cell growth and myeloid differentiation with ATRA monotherapy was much less efficient. Morphological signs of myeloid cell differentiation were further confirmed on a functional basis by demonstrating increased production of reactive oxygen species as well as enhanced phagocytic activity in AML blasts treated with ATRA/AZA/PGZ. In conclusion, we show that biomodulatory treatment with ATRA/AZA/PGZ can induce phenotypical and functional differentiation of primary AML blasts into neutrophil like cells, which aside from its antileukemic activity may lower neutropenia associated infection rates in elderly AML patients in vivo. Clinical impact of the ATRA/AZA/PGZ treatment regimen is currently further investigated in a randomized clinical trial in chemorefractory AML patients (NCT02942758).

Cutaneous Leukemic Infiltrates Successfully Treated With Biomodulatory Therapy in a Rare Case of Therapy-Related High Risk MDS/AML.

Cutaneous manifestations in hematologic malignancies, especially in leukemia, are not common and may be very variable. Here we report a very unusual case of a patient (female, 70 years old) who was admitted to the hospital in 2016 because of skin lesions on the face, the trunk of the body and the extremities. She had a history of breast cancer in the year 2004 (pT1b, pN0, cM0, L0, V0, R0) which had been resected and treated with adjuvant radiation and chemotherapy (cyclophosphamide, methotrexate, 5-fluorouracile) as well as psoriasis treated with methotrexate and cyclosporine. Because of mild cytopenia a bone marrow aspirate/biopsy was performed showing myelodysplastic syndrome (MDS) with multilineage dysplasia. Cytogenetic review revealed a complex aberrant karyotype denoting adverse outcome. Simultaneously, a skin biopsy could confirm leukemic skin infiltration. Consequently, a therapy with azacitidine was started. After the first cycle the patient developed severe pancytopenia with a percentage of 13% peripheral blasts (previously 0-2%) as well as fever without evidence for infection which was interpreted as progressive disease. Therefore, the therapeutic regimen was changed to a biomodulatory therapy consisting of low-dose azacitidine 75 mg/day (given sc d1-7 of 28), pioglitazone 45 mg/day per os, and all-trans-retinoic acid (ATRA) 45 mg/m2/day per os. After cycle 1 of this combined biomodulatory therapy the patient showed hematologic recovery; besides a mild anemia (hemoglobin 11.1 g/dl) she developed a normal blood count. Moreover, the cutaneous leukemic infiltrates which had been unaffected by the azacitidine ameliorated tremendously after 2 cycles resulting in a complete remission of the skin lesions after cycle 6. In conclusion, we report a very unusual case with cutaneous infiltrates being the first clinical manifestation of hematologic disease, preceding the development of acute myeloid leukemia. While azacitidine alone was ineffective, a combined biomodulatory approach resulted in a complete remission of the cutaneous manifestation.

Combined Metabolic Targeting With Metformin and the NSAIDs Diflunisal and Diclofenac Induces Apoptosis in Acute Myeloid Leukemia Cells.

The accelerated metabolism of tumor cells, inevitable for maintaining high proliferation rates, is an emerging target for tumor therapy. Increased glucose and lipid metabolism as well as mitochondrial activity have been shown in solid tumors but also in leukemic cells. As tumor cells are able to escape the blockade of one metabolic pathway by a compensatory increase in other pathways, treatment strategies simultaneously targeting metabolism at different sites are currently developed. However, the number of clinically applicable anti-metabolic drugs is still limited. Here, we analyzed the impact of the anti-diabetic drug metformin alone or in combination with two non-steroidal anti-inflammatory drugs (NSAIDs) diclofenac and diflunisal on acute myeloid leukemia (AML) cell lines and primary patient blasts. Diclofenac but not diflunisal reduced lactate secretion in different AML cell lines (THP-1, U937, and KG-1) and both drugs increased respiration at low concentrations. Despite these metabolic effects, both NSAIDs showed a limited effect on tumor cell proliferation and viability up to a concentration of 0.2 mM. In higher concentrations of 0.4-0.8 mM diflunisal alone exerted a clear effect on proliferation of AML cell lines and blocked respiration. Single treatment with the anti-diabetic drug metformin blocked mitochondrial respiration, but proliferation and viability were not affected. However, combining all three drugs exerted a strong cytostatic and cytotoxic effect on THP-1 cells. Comparable to the results obtained with THP-1 cells, the combination of all three drugs significantly reduced proliferation of primary leukemic blasts and induced apoptosis. Furthermore, NSAIDs supported the effect of low dose chemotherapy with cytarabine and reduced proliferation of primary AML blasts. Taken together we show that low concentrations of metformin and the two NSAIDs diclofenac and diflunisal exert a synergistic inhibitory effect on AML proliferation and induce apoptosis most likely by blocking tumor cell metabolism. Our results underline the feasibility of applying anti-metabolic drugs for AML therapy.

In-vitro blockade of the CD4 receptor co-signal in antigen-specific T-cell stimulation cultures induces the outgrowth of potent CD4 independent T-cell effectors.

T-cell receptor (TCR) redirected T cells are promising tools for adoptive cancer immunotherapy. Since not only CD8 but also CD4 T cells are key players for efficient antitumor responses, the targeted redirection of both subsets with the same antigen-specific TCR comes more and more into focus. Although rapidly evolving technologies enable the reliable genetic re-programming of T cells, the limited availability of TCRs that induce T-cell activation in both T-cell subsets without CD4/CD8 co-receptor contribution hampers the broad application of this approach. We developed a novel stimulation approach, which drives the activation and proliferation of CD4 T-cell populations capable of inducing effector functions in a CD4-independent manner. Naive-enriched CD4 T cells were stimulated against dendritic cells (DC) expressing allogeneic HLA-DP antigens upon RNA transfection and CD4/HLA interactions were blocked by the addition of CD4 binding antibody. Evolving CD4 T-cell populations were specifically activated independent of the CD4 co-signal and induced strong TCR-mediated IFN-γ secretion as well as cytolysis upon recognition of leukemia cells expressing HLA-DP antigen. Our novel stimulation approach may facilitate the generation of CD4 T cells as source for co-receptor independent TCRs for future immunotherapies.

Potential Role of the PD-1/PD-L1 Axis in the Immune Regulation of Chronic GVHD.

BACKGROUND: Antibodies blocking the PD-1/PD-L1 axis have been shown to have substantial antitumor effects also in the treatment of Hodgkin's lymphoma (HL) relapsing after conventional chemotherapy or even autologous hematopoietic stem cell transplantation (autoHSCT). In the case of allogeneic HSCT (alloHSCT), this treatment bears the risk of inducing graft-versus-host disease (GVHD). So far, only a small number of patients who developed acute GVHD after PD-1 antibody administration are described in the literature. CASE REPORTS: We herein report the cases of 2 HL patients after alloHSCT who both responded well to the therapy; however, 1 patient developed chronic GVHD (cGVHD) within 3 days of administration of nivolumab. This patient already had a history of cGVHD and interestingly showed manifestations at the very same sites. The other patient never showed any signs of cGVHD, even with the administration of 13 cycles of anti-PD-1 therapy and large doses of donor lymphocytes. CONCLUSION: The rapid reappearance of cGVHD after blockade of PD-1 implies an important role of PD-1/PD-L1 in peripheral immune tolerance in cGVHD after alloHSCT and warrants further investigation.