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Blinatumomab, a CD19/CD3-bispecific T-cell engager (BiTE) immuno-oncology therapy for the treatment of B-cell malignancies, is associated with neurologic adverse events in a subgroup of patients. Here, we provide evidence for a two-step process for the development of neurologic adverse events in response to blinatumomab: (i) blinatumomab induced B-cell–independent redistribution of peripheral T cells, including T-cell adhesion to blood vessel endothelium, endothelial activation, and T-cell transmigration into the perivascular space, where (ii) blinatumomab induced B-cell–dependent T-cell activation and cytokine release to potentially trigger neurologic adverse events. Evidence for this process includes (i) the coincidence of T-cell redistribution and the early occurrence of most neurologic adverse events, (ii) T-cell transmigration through brain microvascular endothelium, (iii) detection of T cells, B cells, and blinatumomab in cerebrospinal fluid, (iv) blinatumomab-induced T-cell rolling and adhesion to vascular endothelial cells in vitro, and (v) the ability of antiadhesive agents to interfere with blinatumomab-induced interactions between T cells and vascular endothelial cells in vitro and in patients. On the basis of these observations, we propose a model that could be the basis of mitigation strategies for neurologic adverse events associated with blinatumomab treatment and other T-cell therapies.Significance:This study proposes T-cell adhesion to endothelial cells as a necessary but insufficient first step for development of blinatumomab-associated neurologic adverse events and suggests interfering with adhesion as a mitigation approach.

Table S1. Designs, primary results, and incidences of neurologic adverse events of the five clinical studies with blinatumomab of which data was used in present analysis; Table S2: Multivariate analyses of blinatumomab-treated patients with r/r NHL or r/r ALL.

Blinatumomab is a first-in-class immunotherapy based on the bispecific T-cell engager (BiTE®) immune-oncology platform, which redirects CD3+ T cells to kill CD19+ target cells. The objective of this analysis was to describe the correlation between B- and T-cell kinetics and response to blinatumomab in patients with relapsed or refractory (r/r) non-Hodgkin lymphoma (NHL). The clinical efficacy of treatment with blinatumomab in patients with r/r NHL was recently investigated in a phase 1 dose-escalation and expansion trial (NCT00274742) wherein 76 patients received blinatumomab by continuous intravenous infusion at various doses (0.5–90 μg/m2/day). B-Cell depletion and expansion of CD3+, CD4+, and CD8+ T cells was analyzed in patients stratified per clinical response (complete response [CR], n = 16; partial response [PR], stable disease [SD], or progressive disease [PD], n = 54) for at least 4 weeks (additional 4 weeks after clinical benefit) from the date of administration of blinatumomab until dose-limiting toxicity or PD. B-cell depletion kinetics were faster in patients who had a CR than in patients who did not have a complete response (PR, SD, or PD). T-cell expansion (T-cell counts exceeding the baseline level on day 22) was more pronounced in patients with CR than in patients without CR. T-cell expansion in patients with CR correlated with increased T-cell counts of both CD4+ and CD8+ T cells compared with patients without CR. Patients with r/r NHL who achieved a CR had faster B-cell depletion and increased expansion of CD3+, CD4+, and CD8+ T cells than patients who did not achieve a CR.

Despite advances in standards of care, the prognosis of relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) remains poor. In these patients, 50–74% fail to respond to next line therapy,...

Immuno-oncology approaches have entered clinical practice, with tremendous progress particularly in the field of T cell-engaging therapies over the past decade. Herein, we provide an overview of the current status of bispecific T cell engager (BiTE) therapy, considering the unprecedented new indication for such therapy in combating minimal (or measurable) residual disease in patients with acute lymphoblastic leukaemia, and the development of novel approaches based on this concept. Key aspects that we discuss include the current clinical data, challenges relating to treatment administration and patient monitoring, toxicities and resistance to treatment, and novel strategies to overcome these hurdles as well as to broaden the indications for BiTE therapy, particularly to common solid cancers. Elucidation of mechanisms of resistance and immune escape and new technologies used in drug development pave the way for new and more-effective therapies and rational combinatorial approaches. In particular, we highlight novel therapeutic agents, such as bifunctional checkpoint-inhibitory T cell engagers (CiTEs), simultaneous multiple interaction T cell engagers (SMITEs), trispecific killer engagers (TriKEs) and BiTE-expressing chimeric antigen receptor (CAR) T cells (CART.BiTE cells), designed to integrate various immune functions into one molecule or a single cellular vector and thereby enhance efficacy without compromising safety. We also discuss the targeting of intracellular tumour-associated epitopes using bispecific constructs with T cell receptor (TCR)-derived, rather than an antibody-based, antigen-recognition domains, termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs), which might broaden the armamentarium of T cell-engaging therapies. The use of bispecific antibodies to engage cells of the immune system that are cytotoxic to cancer cells is a major focus of cancer immunotherapy, with approvals for the treatment of acute lymphoblastic leukaemia. Here, the authors review the clinical results obtained with bispecific antibodies to date. They also discuss the challenges associated with this therapeutic approach and the proposed solutions aimed at preventing or minimizing toxicities, countering immune escape and broadening the indications for these treatments.

Blinatumomab, a CD19/CD3-bispecific T-cell engager (BiTE) immuno-oncology therapy for the treatment of B-cell malignancies, is associated with neurologic adverse events in a subgroup of patients. Here, we provide evidence for a two-step process for the development of neurologic adverse events in response to blinatumomab: (i) blinatumomab induced B-cell–independent redistribution of peripheral T cells, including T-cell adhesion to blood vessel endothelium, endothelial activation, and T-cell transmigration into the perivascular space, where (ii) blinatumomab induced B-cell–dependent T-cell activation and cytokine release to potentially trigger neurologic adverse events. Evidence for this process includes (i) the coincidence of T-cell redistribution and the early occurrence of most neurologic adverse events, (ii) T-cell transmigration through brain microvascular endothelium, (iii) detection of T cells, B cells, and blinatumomab in cerebrospinal fluid, (iv) blinatumomab-induced T-cell rolling and adhesion to vascular endothelial cells in vitro, and (v) the ability of antiadhesive agents to interfere with blinatumomab-induced interactions between T cells and vascular endothelial cells in vitro and in patients. On the basis of these observations, we propose a model that could be the basis of mitigation strategies for neurologic adverse events associated with blinatumomab treatment and other T-cell therapies. Significance: This study proposes T-cell adhesion to endothelial cells as a necessary but insufficient first step for development of blinatumomab-associated neurologic adverse events and suggests interfering with adhesion as a mitigation approach.

T cell-engaging immunotherapies are changing the landscape of current cancer care. However, suitable target antigens are scarce, restricting these strategies to very few tumor types. Here, we report on a T cell-engaging antibody derivative that comes in two complementary halves and addresses antigen combinations instead of single molecules. Each half, now coined hemibody, contains an antigen-specific single-chain variable fragment (scFv) fused to either the variable light (VL) or variable heavy (VH) chain domain of an anti-CD3 antibody. When the two hemibodies simultaneously bind their respective antigens on a single cell, they align and reconstitute the original CD3-binding site to engage T cells. Employing preclinical models for aggressive leukemia and breast cancer, we show that by the combinatorial nature of this approach, T lymphocytes exclusively eliminate dual antigen-positive cells while sparing single positive bystanders. This allows for precision targeting of cancers not amenable to current immunotherapies., The restriction of appropriate tumour-specific antigens is a current limitation for T cell-engaging immunotherapy. Here, the authors have designed a new system constituted by two halve antibodies, which engage T cells once binding to two different antigens, to specifically eliminate double positive cells in preclinical leukemia and breast cancer mouse models.

BackgroundGrowth and differentiation factor 15 (GDF-15) is a TGF-β superfamily member physiologically expressed mainly in placenta and linked to feto-maternal tolerance. Under pathophysiologic conditions, prevention of excessive immune cell infiltration during tissue damage and cachexia induction have been ascribed to GDF-15. Recent research has though indicated a prominant role in modulation of the tumor microenvironment and the immune synapse, too1 2 indicating that GDF-15 may be a major tumor-derived immunosuppressant. Importantly, several cancer entities secrete high levels of GDF-15, correlating with poor prognosis and reduced overall survival [Front Immunol 2020 May 19;11:951]. To block this effect the GDF-15 neutralizing antibody CTL-002 was generated. In preclinical models CTL-002 demonstrated potent effector T cell shifting into tumor tissue by neutralizing GDF-15.MethodsThis is a phase 1, first-in-human (FIH), two-part, open-label clinical trial of intravenous (IV) administration of CTL-002 given as monotherapy and in combination with an anti-PD-1 antibody in subjects with advanced-stage, relapsed/refractory solid tumors who relapsed or were refractory to a prior anti-PD-1/PD-L1 therapy. Eligible subjects have exhausted all available approved standard treatments, including prior anti-PD1/-PD-L1 treatment, and present with a biopsy-accessible tumor for serial biopsy taking. The trial is termed GDFATHER, for ”GDF-15 Antibody-mediaTed Effector cell Relocation”.Main endpoints are safety of CTL-002 monotherapy and CTL-002 combination with an anti-PD-1 antibody, pharmacokinetics, pharmacodynamics (e.g. degree of GDF-15 neutralization achieved and change in immune-cell number and composition in the tumor tissue) as well as preliminary clinical efficacy (tumor mass reduction; anticachexia effect)In part A of the trial (dose escalation) up to 24 subjects will receive escalating doses of CTL-002 IV (0.3 – 20 mg/kg) in a ”mono-followed-by-combination”-design with CTL-002 given as monotherapy and followed by combination with an anti-PD-1 checkpoint inhibitor. In part B (expansion) up to 5 cohorts with up to 25 subjects per cohort with defined tumor entities expected to be GDF-15 dependent will be treated to determine the recommended phase 2 dose (RP2D) and further evaluate safety and preliminary efficacy of CTL-002 monotherapy and the combination.The study was initiated in December 2020 and enrolled the first patient on Dec 09, 2020. Cohort 4 is ongoing at time of submission (07/2021) and so far no DLT has occurred. Updated safety, biomarker and response assessments will be reported at the meeting. The ClinicalTrials.gov Identifier is NCT04725474. For more information please contact info@catalym.com.Trial RegistrationNCT04725474ReferencesWischhusen J, Wistuba-Hamprecht K, Harter PN, Cheng P, Martens A, Gogolla F, Nonomura Y, Romer P, Koch SD, Haake M, Schuberth-Wagner C, Rudiger M, Leo E, Mittelbronn M, Levesque MP, Hackl H, Dummer R, Weide B. Identifying GDF-15 as potential novel immunotherapeutic target linked to immune cell exclusion in tumors and resistance to anti-PD-1 treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27–28 and Jun 22–24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl): Abstract nr 2161.Hurt E, Thomas S, Mulgrew K, Blackmore S, Moynihan J, Cusdin F, Dodd R, Cariuk P, Sigurdardottir A, Brannigan E, Dobson C, Kumar R, Cobbold M. AZD8853: A novel antibody targeting GDF15 for immunotherapy refractory tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10–15 and May 17–21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl): Abstract nr 1828.Ethics ApprovalAll participants gave informed consent prior to participation. EC approval by Gobierno de Navarra, Departamento de Salud, EC_2020/30, Dated: Oct 13, 2020 in Pamplona, Spain. Respective additional national lead EC approvals for Germany (Ethikkommission der Universität Würzburg, 203–20ff of Oct 26, 2020) and Switzerland (Kantonale Ethikkommission Zürich, 2020–02308 of Nov 24, 2020).

Ferroptosis is a form of cell death that has received considerable attention not only as a means to eradicate defined tumour entities but also because it provides unforeseen insights into the metabolic adaptation exploited by tumours to counteract phospholipid oxidation. Here, we identify a pro-ferroptotic activity of 7-dehydrocholesterol reductase (DHCR7) and an unexpected pro-survival function of its substrate, 7-dehydrocholesterol (7-DHC). Although previous studies suggested that high levels of 7-DHC are cytotoxic to developing neurons and favour lipid peroxidation, we now demonstrate that 7-DHC accumulation confers a robust pro-survival function in cancer cells. 7-DHC, due to its far superior reactivity towards peroxyl radicals, is shown here to effectively shield (phospho)lipids from autoxidation and subsequent fragmentation. We further demonstrate in a subset of ferroptosis-sensitive Burkitt lymphomas - where DHCR7 mutations have been reported - that the accumulation of 7-DHC is sufficient to suppress the basal sensitivity of cells toward ferroptosis, thereby translating into an unexpected growth advantage. Conclusively, our findings provide compelling evidence of a yet-unrecognised anti-ferroptotic activity of 7-DHC as a cell-intrinsic mechanism that could be exploited by cancer cells to escape ferroptosis.

TO THE EDITOR: Recently, biallelic (“double hit”) TP53 inactivation, occurring in 2% to 4% of newly diagnosed multiple myeloma (MM) patients, was identified as an ultimate high-risk feature of MM, being associated with median survival of

PurposeCardiotoxic adverse events (AE) are challenging in the field of oncologic therapy. Age-related prevalence of cardiovascular disease and cancer itself may contribute to an increased risk of cardiac morbidity. Cardiac dysfunction impacts cardiac morbidity, survival and quality of life in cancer patients. This study established a cardio-oncological monitoring for the early detection of cardiovascular complications during experimental anti-tumour therapy. MethodsNinety patients referred to experimental anti-tumour therapy underwent prospective monitoring including repeat-electrocardiogram (ECG), echocardiography with speckle tracking imaging (STI), and determination of cardiac biomarkers. Changes in cardiac function were evaluated according to the Common Terminology Criteria of Adverse Events (CTCAE Version 4.03). ResultsConventional ECG, high-sensitive troponin T, echocardiography including STI-analysis, and 24-hour Holter ECG were identified as relevant diagnostic tools. Early signs of cardiotoxicity appeared within the first 3 months after initiating anti-tumour therapy. Most frequent AEs were mitral valve disease (22%), diastolic dysfunction (15.7%), sinus tachycardia (13.8%), and increased hs-TnT (13.8%). ConclusionProspective cardiac monitoring allowed early detection of cardiac dysfunction during experimental anti-tumour therapy. A multi­disciplinary work-up and decision-making during cancer treatment is warranted to foster a deeper understanding of the underlying mechanisms of cardiac AEs and particularly to early detect and counteract cardiotoxicity.

Immuno-oncology approaches have entered clinical practice, with tremendous progress particularly in the field of T cell-engaging therapies over the past decade. Herein, we provide an overview of the current status of bispecific T cell engager (BiTE) therapy, considering the unprecedented new indication for such therapy in combating minimal (or measurable) residual disease in patients with acute lymphoblastic leukaemia, and the development of novel approaches based on this concept. Key aspects that we discuss include the current clinical data, challenges relating to treatment administration and patient monitoring, toxicities and resistance to treatment, and novel strategies to overcome these hurdles as well as to broaden the indications for BiTE therapy, particularly to common solid cancers. Elucidation of mechanisms of resistance and immune escape and new technologies used in drug development pave the way for new and more-effective therapies and rational combinatorial approaches. In particular, we highlight novel therapeutic agents, such as bifunctional checkpoint-inhibitory T cell engagers (CiTEs), simultaneous multiple interaction T cell engagers (SMITEs), trispecific killer engagers (TriKEs) and BiTE-expressing chimeric antigen receptor (CAR) T cells (CART.BiTE cells), designed to integrate various immune functions into one molecule or a single cellular vector and thereby enhance efficacy without compromising safety. We also discuss the targeting of intracellular tumour-associated epitopes using bispecific constructs with T cell receptor (TCR)-derived, rather than an antibody-based, antigen-recognition domains, termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs), which might broaden the armamentarium of T cell-engaging therapies.

Aim: We report results of a first-in-human study of pasotuxizumab, a PSMA bispecific T-cell engager (BiTE®) immune therapy mediating T-cell killing of tumor cells in patients with advanced castration-resistant prostate cancer. Patients & methods: We assessed once-daily subcutaneous (SC) pasotuxizumab. All SC patients developed antidrug antibodies; therefore, continuous intravenous (cIV) infusion was assessed. Results: A total of 47 patients received pasotuxizumab (SC: n = 31, 0.5–172 μg/d; cIV: n = 16, 5–80 μg/d). The SC maximum tolerated dose was 172.0 μg/d. A sponsor change stopped the cIV cohort early; maximum tolerated dose was not determined. PSA responders occurred (>50% PSA decline: SC, n = 9; cIV, n = 3), including two long-term responders. Conclusion: Data support pasotuxizumab safety in advanced castration-resistant prostate cancer and represent evidence of BiTE monotherapy efficacy in solid tumors. Clinical trial registration: NCT01723475 (ClinicalTrials.gov)

Minimal residual disease (MRD) is the strongest predictor of relapse in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In BLAST study (NCT01207388), adults with BCP-ALL in remission with MRD after chemotherapy received blinatumomab, a CD19 BiTE® immuno-oncotherapy, 15 µg/m2/day for up to four 6-week cycles (4 weeks continuous infusion, 2 weeks off). Survival was evaluated for 110 patients, including 74 who received HSCT in continuous complete remission. With a median follow-up of 59·8 months, median survival (months) was 36·5 (95% CI: 22.0–not reached [NR]). Median survival was NR (29.5–NR) for complete MRD responders (n = 84) and 14.4 (3.8–32.3) for MRD non-responders (n = 23; p = 0.002); after blinatumomab and HSCT, median survival was NR (25.7–NR) (n = 61) and 16.5 (1.1–NR) (n = 10; p = 0.065), respectively. This final analysis suggests complete MRD response during blinatumomab treatment is curative. Post-hoc analysis of study data suggests while post blinatumomab HSCT may be beneficial in appropriate patients, long-term survival without HSCT is also possible.

Introduction The CD200/CD200 receptor (CD200R) axis is known to exert immunoregulatory effects in myeloid-derived cells and constitutes a putative immune checkpoint in hematological malignancies, in which CD200 expression is associated with poor prognosis. In multiple myeloma (MM), CD200 is expressed in the majority of patient-derived primary cells. However, its functional importance as well as the related downstream mechanism upon CD200 ligand binding to its CD200R on T cells are not well understood. In this study, we analyze the functional role of CD200 as a potential immune checkpoint in MM and decipher the mechanism of CD200-mediated immune escape. Methods Primary MM cells and MM cell lines were analyzed for CD200 surface expression by flow cytometry. To overexpress CD200 on non-expressing MM cell lines we used a Sleeping Beauty transposon vector system. CD200+/- MM cell lines L363, U266 and MM.1s were co-cultured with CD3/CD28-activated healthy donor T cells. T cell-mediated cytotoxicity in these co-cultures was assessed with flow cytometry and/or luciferase assay. Moreover, to analyze the effects of CD200R activation on downstream signaling pathways, activated T cells were treated with recombinant human CD200 (rhCD200) and/or anti-CD200 blocking antibody and subsequently, Western blotting was performed. Results Of n=120 primary MM samples (n=120) analyzed, CD200 protein expression could be detected in ca. 75 % of the cases. In contrast, all n=9 MM cell lines tested neither displayed surface nor cytoplasmic CD200 expression. Therefore, using a Sleeping Beauty transposon vector system we stably expressed CD200 on MM cell lines for further analyses. In the presence of CD200-expressing MM cells up to 50% decrease in CD3+ T cell-mediated cytotoxicity against MM cells was observed in flow cytometry and luciferase assay. Proliferation rates of MM cell lines remained unchanged regardless of the level of CD200 overexpression as determined by Alamar blue assays. In myeloid-derived cells, CD200R directly interacts with docking protein-2 (DOK2). In activated T cells, we observed DOK2 phosphorylation upon CD200 binding when treated with rhCD200 in a time- and concentration-dependent manner. Applying an anti-CD200 blocking antibody, this effect could be reversed, thus revealing a possible mechanism for the observed attenuation of T cell function. Conclusion Our study shows that anti-MM cytotoxicity from primary healthy donor CD3+ T cells is attenuated by CD200 expression on MM cells. We also demonstrate that this inhibitory mechanism in CD3+ T cells is mediated via DOK2, providing a potential target for immunotherapeutic approaches in MM. Disclosures Einsele: Janssen, Celgene/BMS, Amgen, GSK, Sanofi: Consultancy, Honoraria, Research Funding.

MRD in ALL is defined as the detection of leukemic cells in bone marrow below the microscopic threshold in complete remission (CR). Patients (pts) with molecular failure (MolFail) or molecular relapse (MolRel) after induction/consolidation therapy are at a high risk for hematologic relapse. Targeted therapies should prevent hematologic relapse, reduce MRD load and provide a bridging strategy to allogeneic stem cell transplantation (SCT) and thereby improve overall outcome of these pts. In pts without (wo) SCT option reduction of MRD load is an essential goal as well. Blinatumomab is an antibody construct that redirects CD3+ T cells to CD19+ target cells, resulting in a serial lysis of CD19+ B cells. In a study in pts with MRD ≥10-3, 78% achieved complete MRD response (Gökbuget N et al., Blood 2018). The MolAct1 trial was initiated by the GMALL study group to evaluate the efficacy and tolerability of Blinatumomab in MRD+ ALL including those with MRD below 10-3 and pts with MRD after SCT. Adults (≥18 yrs) with CD19+, Ph-negative BCP ALL in CR after ≥ 3 chemotherapies with MRD ≥10-4 were eligible (NCT03109093). Recruitment of pts with MRD ≥10-3 was stopped after marketing authorization for this entity. After an amendment, which became effective after 44 recruited pts, pts with MRD below 10-4 or non-quantifiable (nq) MRD were eligible. Blinatumomab 28 μg/day was given as 4-wk infusion, followed by a 2-wk break (1 cycle). Responders could receive up to 4 cycles or undergo HSCT after ≥ 1 cycle. MRD after 1 cycle was the primary endpoint. MRD was centrally assessed by allele-specific quantitative real-time PCR of clonal rearrangements of immunoglobulin or T-cell receptor genes. For definition of MRD at inclusion and at response assessment see table 1. 64 pts with a median age of 44 (18-83) yrs were included from 19 centers and 60 were evaluable. 63 pts were treated in first CR (5 after SCT). Overall, 67% achieved MolCR, 10% had MolFail, 23% MolNE. MolNE identifies an intermediate response with different options clarified table 1. 81% of the pts included with MRD ≥10-4 had a molecular response i.e. MolCR or MRD < 10-4. No significant differences in terms of MRD response were observed according to MRD level at inclusion or other patient characteristics (table 1). 60 pts have completed study treatment (40 HSCT, 8 relapses during treatment, 4 completed 4 cycles wo SCT, 2 stopped earlier due to toxicities - 1 with subsequent SCT, 1 due to GvHD, 1 due to physicians' decision and 4 pts returned to standard treatment after 2 cycles). SCT pts had a median age of 42 (18-66) yrs and follow-up is available in 37 / 41 pts (29 CCR, 3 relapse, 5 death in CR). 16 relapses occurred: 8 during treatment (1 after MolCR, 5 MolNE1-3 and 2 with MolFail resp); after SCT in 3/41 pts; 5/11 with CR at end of treatment wo subsequent SCT. The median observation time of surviving pts is 12 (1-38) mo and the median survival is not reached. At 2 yrs the survival probability (OS) was 64%. OS was 70%, 64% and 43% in pts with MRD between 10-4-10-3, 10-3-10-2 and >10-2 at inclusion, resp (p>.05; Fig.1). OS was 71% vs 54% in pts MolFail vs MolRel at inclusion (p>.05). OS was 72%, 40% and 56% in pts with MolCR, MolFail and MolNE after cycle 1 resp (P=0.02; Fig.2). Overall, the results from previous trials were confirmed. In addition, it was demonstrated that pts with MRD between 10-4 and 10-3 had a similar response and a trend towards better outcome compared to pts with higher MRD levels >10-2. So far only 7 pts with MRD below 10-4 were included; more data are needed to evaluate the impact of Blinatumomab in this population; GMALL does currently not recommend SCT for these pts unless there is an indication due to other risk factors. Interestingly, a significant proportion of pts (23%) had an incomplete MRD response and the outcome results indicate that these pts together with those with MolFail may have an inferior survival compared to those with MolCR. The results underline that the clear definition of MRD categories and consideration of level and sensitivity is essential for interpretation, which is possible due to well defined standards for the PCR method used here. 68% of the pts received SCT in CR after Blinatumomab with a so far limited mortality (13%). Further follow-up is certainly required. The GMALL will continue to recruit patients with MRD levels below 10-3 in order to improve their chances for long-term survival This study was supported by Amgen Inc. Disclosures Goekbuget: Servier: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Erytech: Consultancy; Kite: Consultancy; Gilead: Consultancy. Schwartz:Pfizer: Other: personal fees ; AMGEN: Other: personal fees and non-financial support; BTG Intl Inc: Other: personal fees ; Gilead Sciences: Other: personal fees and non-financial support ; MSD Sharp & Dohme: Other: personal fees ; Basilea: Other: non-financial suppor; Novartis: Other: personal fees and non-financial support; Jazz Pharmaceuticals: Other: personal fees and non-financial support. Hüttmann:Lead Discovery Center GmbH: Consultancy; University Hospital Essen, University of Duisburg-Essen, Essen, Germany: Current Employment; Celgene: Honoraria, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Seattle Genetics: Research Funding; Roche: Other: Travel expenses; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company); Gilead: Honoraria. Viardot:Roche: Honoraria, Other: advisory board; Kite/Gilead: Honoraria, Other: advisory board; Novartis: Honoraria, Other: advisory board; Amgen: Honoraria, Other: advisory board. Fiedler:Daiichi Sankyo: Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Jazz Pharmaceuticals: Honoraria, Other: support for meeting attendance; Abbvie: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy, Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria; ARIAD/Incyte: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: support for meeting attendance, Patents & Royalties, Research Funding. Alakel:Pfizer: Consultancy. Stelljes:Pfizer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Speakers Bureau. Subklewe:Seattle Genetics: Research Funding; AMGEN: Consultancy, Honoraria, Research Funding; Morphosys: Research Funding; Gilead Sciences: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria; Janssen: Consultancy; Roche AG: Consultancy, Research Funding. Wäsch:Pfizer: Consultancy; Amgen: Consultancy; Janssen: Consultancy. Vucinic:Celgene: Honoraria. Fransecky:Amgen: Consultancy. Bargou:Amgen: Consultancy, Honoraria, Patents & Royalties; Gemoab: Consultancy, Honoraria; Cellex: Consultancy, Honoraria; Catalym: Consultancy, Honoraria. Topp:Amgen, Boehringer Ingelheim, KITE, Regeneron, Roche: Research Funding; Amgen, KITE, Novartis, Regeneron, Roche: Consultancy. Brüggemann:Regeneron: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy; Incyte: Consultancy; Roche: Consultancy; Affimed: Research Funding; Janssen: Consultancy, Honoraria. OffLabel Disclosure: Blinatumomab in MRD positive disease below 10-3

TPS2658 Background: Growth and differentiation factor 15 (GDF-15) is a TGF-β superfamily member physiologically expressed mainly in placenta and linked to feto-maternal tolerance. Under pathophysiologic conditions, prevention of excessive immune cell infiltration during tissue damage and cachexia induction have been ascribed to GDF-15. A recent study [Haake et al. AACR2020; Abstract #5597] elucidated a mechanism by which GDF-15 inhibits LFA-1 activation on CD8+ T cells, thus interfering with effector T cell recruitment to tissues. Importantly, several cancer entities secrete high levels of GDF-15, correlating with poor prognosis and reduced overall survival [reviewed in Front Immunol 2020 May 19;11:951]. To block this effect the GDF-15 neutralizing antibody CTL-002 was generated. In preclinical models CTL-002 demonstrated potent effector T cell shifting into tumor tissue by neutralizing GDF-15. Methods: This is a phase 1, first-in-human (FIH), two-part, open-label clinical trial of intravenous (IV) administration of CTL-002 given as monotherapy and in combination with an anti-PD-1 antibody in subjects with advanced-stage, relapsed/refractory solid tumors who relapsed post or were refractory to a prior anti-PD-1/PD-L1 therapy. Eligible subjects have exhausted all available approved standard treatments. Further key eligibility criteria include having received at least one prior anti-PD1/-PD-L1 treatment and having relapsed on or after it or having been refractory to it, and presenting with a biopsy-accessible tumor for serial biopsy taking. The trial is termed GDFATHER, for “GDF-15 Antibody-mediaTed Effector cell Relocation”. Main endpoints are safety of CTL-002 monotherapy and CTL-002 combination with an anti-PD-1 antibody, pharmacokinetics, pharmacodynamics (e.g. degree of GDF-15 neutralization achieved and change in immune-cell number and composition in the tumor tissue) as well as preliminary clinical efficacy (tumor mass reduction; anticachexia effect) In part A of the trial (dose escalation) up to 24 subjects will receive escalating doses of CTL-002 IV (0.3 – 20 mg/kg) in a „mono-followed-by-combination“-design with CTL-002 given as monotherapy and followed by combination with an anti-PD-1 checkpoint inhibitor. In part B (expansion) up to 5 cohorts with up to 25 subjects per cohort with defined tumor entities expected to be GDF-15 dependent will be treated to determine the recommended phase 2 dose (RP2D) and further evaluate safety and preliminary efficacy of CTL-002 monotherapy and the combination. The study was initiated in December 2020 and enrolled the first patient on Dec 09, 2020. Cohort 1 has been completed without DLT and enrollment for cohort 2 began in February 2021. Clinical trial information: NCT04725474.

Hintergrund Durch neue Entwicklungen in der Immunonkologie hat die Systemtherapie maligner Erkrankungen in den letzten Jahren erhebliche Fortschritte gemacht.

Lenalidomide, adriamycin, dexamethasone for induction followed by stem-cell transplant in newly diagnosed myeloma

The CD19 antigen is a promising target for immunotherapy of acute lymphoblastic leukemia (ALL), but CD19- relapses remain a major challenge in about 10% to 20% of patients. Here, we analyzed 4 CD19- ALL relapses after treatment with the CD19/CD3 bispecific T-cell engager (BiTE) blinatumomab. Three were on-drug relapses, with the CD19- escape variant first detected after only 2 treatment courses. In 1 patient, the CD19- clone appeared as a late relapse 19 months after completion of blinatumomab treatment. All 4 cases showed a cellular phenotype identical to the primary diagnosis except for CD19 negativity. This argued strongly in favor of an isolated molecular event and against a common lymphoid CD19- progenitor cell or myeloid lineage shift driving resistance. A thorough molecular workup of 1 of the cases with early relapse confirmed this hypothesis by revealing a disrupted CD19 membrane export in the post-endoplasmic reticulum compartment as molecular basis for blinatumomab resistance.

Recent advances in antibody technology to harness T cells for cancer immunotherapy, particularly in the difficult-to-treat setting of relapsed/refractory acute lymphoblastic leukemia (r/r ALL), have led to innovative methods for directing cytotoxic T cells to specific surface antigens on cancer cells. One approach involves administration of soluble bispecific (or dual-affinity) antibody-based constructs that temporarily bridge T cells and cancer cells. Another approach infuses ex vivo-engineered T cells that express a surface plasma membrane-inserted antibody construct called a chimeric antigen receptor (CAR). Both bispecific antibodies and CARs circumvent natural target cell recognition by creating a physical connection between cytotoxic T cells and target cancer cells to activate a cytolysis signaling pathway; this connection allows essentially all cytotoxic T cells in a patient to be engaged because typical tumor cell resistance mechanisms (such as T-cell receptor specificity, antigen processing and presentation, and major histocompatibility complex context) are bypassed. Both the bispecific T-cell engager (BiTE) antibody construct blinatumomab and CD19-CARs are immunotherapies that have yielded encouraging remission rates in CD19-positive r/r ALL, suggesting that they might serve as definitive treatments or bridging therapies to allogeneic hematopoietic cell transplantation. With the introduction of these immunotherapies, new challenges arise related to unique toxicities and distinctive pathways of resistance. An increasing body of knowledge is being accumulated on how to predict, prevent, and manage such toxicities, which will help to better stratify patient risk and tailor treatments to minimize severe adverse events. A deeper understanding of the precise mechanisms of action and immune resistance, interaction with other novel agents in potential combinations, and optimization in the manufacturing process will help to advance immunotherapy outcomes in the r/r ALL setting.

Recent advances in molecular diagnostics have shown that lesions affecting both copies of the gene for tumor suppressor protein 53 (TP53) count among the most powerful predictors for high-risk disease in multiple myeloma (MM). However, the functional relevance and potential therapeutic implications of single hits to TP53 remain less well understood. Here, we have for the first time approximated the different constellations of mono- and bi-allelic TP53 lesions observed in MM patients within the frame of a single MM cell line model and assessed their potential to disrupt p53 system functionality and to impart drug resistance. Both types of common first hit: point mutation with expression of mutant p53 protein or complete loss of contribution from one of two wildtype alleles strongly impaired p53 system functionality and increased resistance to melphalan. Second hits abolished remaining p53 activity and increased resistance to genotoxic drugs even further. These results fit well with the clinical drive to TP53 single- and double-hit disease in MM patients, provide a rationale for the most commonly observed double-hit constellation (del17p+ TP53 point mutation), and underscore the potential increases in MM cell malignancy associated with any type of initial TP53 lesion.

Minimal residual disease (MRD), where leukemic cell levels are lower than the morphologic detection threshold, is the most important prognostic factor for acute lymphoblastic leukemia (ALL) relapse during first-line chemotherapy treatment and is standard of care in treatment monitoring and decision making. Limited data are available on the prognostic value of MRD response after relapse. We evaluated the relationship between MRD response and outcomes in blinatumomab-treated adults with relapsed/refractory (R/R) B-cell precursor ALL. Of 90 patients with complete remission (CR) or CR with partial hematologic recovery (CRh), 64 (71.1%) achieved a complete MRD response (no detectable individual rearrangements of immunoglobulin/T-cell receptor genes by polymerase chain reaction [PCR] at a minimum sensitivity level of 10(−4)). Eleven patients had MRD

Heat shock proteins (HSPs) are molecular chaperones that play a pivotal role in correct folding, stabilization and intracellular transport of many client proteins including those involved in oncogenesis. HSP70, which is frequently overexpressed in prostate cancer (PCa), has been shown to critically contribute to tumor cell survival, and might therefore represent a potential therapeutic target. We treated both the androgen receptor (AR)-positive LNCaP and the AR-negative PC-3 cell lines with the pharmacologic HSP70 inhibitor VER155008. Although we observed antiproliferative effects and induction of apoptosis upon HSP70 inhibition, the apoptotic effect was more pronounced in AR-positive LNCaP cells. In addition, VER155008 treatment induced G1 cell cycle arrest in LNCaP cells and decreased AR expression. Further analysis of the HSP system by Western blot analysis revealed that expression of HSP27, HOP and HSP90β was significantly inhibited by VER155008 treatment, whereas the HSP40, HSP60, and HSP90α expression remained unchanged. Taken together, VER155008 might serve as a novel therapeutic option in PCa patients independent of the AR expression status.

Direct therapeutic targeting of oncogenic RAS is currently still impossible due to lack of suitable pharmacological inhibitors. Because specific blockade of druggable RAS effectors might represent an alternative treatment approach, we evaluated the role of the Raf complex for multiple myeloma (MM) pathobiology. We found frequent overexpression of the Raf isoforms (A-, B- and C-Raf) and downstream activation of MEK1,2/ERK1,2 in MM cells. Concomitant inhibition of all Raf isoforms (pan-Raf inhibition) by RNAi or pharmacological inhibitors was required to strongly induce apoptosis in human MM cell lines (HMCLs), in primary MM cells in vitro, and in a syngeneic MM mouse model in vivo. The anti-MM effect of pan-Raf inhibition did not correlate with the RAS mutation status, and functionally appeared to involve both MEK-dependent and -independent mechanisms. Furthermore, transcriptome analyses revealed that pan-Raf activity affects PI3K-dependent signalling, thus highlighting a functional link between the RAS/Raf and PI3K/mTOR/Akt pro-survival pathways. Accordingly, pharmacological inhibition of PI3K strongly enhanced the anti-MM effect of pan-Raf inhibition in MM cell lines and in primary MM cells in vitro and in vivo. Concomitant pan-Raf/PI3K inhibition was also effective in carfilzomib- and lenalidomide-resistant MM models underscoring that this attractive therapeutic anti-MM strategy is suitable for immediate clinical translation.

BACKGROUND Older adults with relapsed/refractory B-precursor acute lymphoblastic leukemia (r/r ALL) are reported to have a poor prognosis and few therapeutic options. In the current study, the authors evaluated treatment with single-agent blinatumomab in adults aged ≥65 years with r/r ALL. METHODS A total of 261 adults with r/r ALL who were examined across two phase 2 studies received blinatumomab in cycles of 4-week continuous infusion and 2-week treatment-free intervals. The primary endpoint in each study was complete remission (CR) or CR with partial hematologic recovery (CRh) during the first 2 cycles. Data were pooled and analyzed according to patient age at screening (aged ≥65 years vs aged

Blinatumomab is a member of a novel class of T cell-engaging bispecific antibodies, so-called Bispecific T cell Engager (BiTEs). It is directed against the B cell differentiation antigen CD19 and intended for treatment of B cell malignancies. In clinical phase I/II trials, blinatumomab showed remarkable single-agent activity in patients with relapsed and/or refractory (R/R) non-Hodgkin lymphoma and R/R B cell precursor acute lymphoblastic leukemia (B-precursor ALL). Cytokine release syndrome and neurological side effects were dose-limiting. Adverse effects were well manageable and transient in nature. Based on results of an international phase II trial, blinatumomab received FDA approval for the treatment of R/R B-precursor ALL in December 2014. Ongoing and future trials will contribute to further optimization of blinatumomab-based T cell therapy and have to show that integration of blinatumomab in current and innovative treatment protocols improves overall survival and quality of life of patients with B cell malignancies.

Introduction Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by clonal evolution and heterogeneous genetic abnormalities. Deletion of the short arm of chromosome 17 (del17p) harboring TP53 is a high-risk abnormality associated with aggressive disease and therapy resistance. Here, we tested in vitro and ex vivo efficacy of a peptide-drug conjugate melflufen, currently in phase 3 clinical trials for relapsed/refractory multiple myeloma (RRMM), in an isogenic myeloma p53-abrogated cell line model and patient samples. Methods Efficacy of melflufen versus melphalan was tested in the AMO-1 cell line, which displays biallelic wildtype TP53 and retains aspects of a functional p53 system, and AMO-1 clones with either complete loss of p53 or expressing point-mutated p53 protein (R282W hotspot mutation), created using the CRISPR/Cas9 system and modified via Sleeping Beauty vectors. We assessed toxicity and apoptosis using Annexin V and Alamar blue assays. Ex vivo sensitivity to the drugs was examined by flow cytometry in CD138+ plasma cells isolated from MM patients with confirmed del17p or TP53 mutations. We also analyzed response rates in a subpopulation of patients with confirmed del17p from OP-106 HORIZON, an ongoing phase 2 study evaluating the efficacy of melflufen plus dexamethasone in patients with RRMM (NCT02963493). Results Whereas loss of p53 functionality strongly impaired melphalan induced cytotoxicity in the AMO-1 MM cell line model system, treatment with melflufen showed superior efficacy in the parental cells, and was effective independent of the respective TP53 status. Although at low dosages wild-type cells were still more sensitive, in contrast to melphalan this effect was overcome by slight dose increases of melflufen, due to the much steeper dose-effect relationships in the p53 deficient sublines. In contrast to melphalan, the melflufen effect was only partially blocked by combined pre-treatment with inhibitors of apoptosis and necroptosis. Similar results were observed in patient-derived material with melflufen displaying superior activity towards TP53 mutated plasma cells. Analysis of OP-106 HORIZON trial data showed comparable ORR of 20% (CI 95% 4.3-48.1) in the del17p subpopulation (n=15) to 26.2% (CI 95% 18.8-34.6) in the total population (n=136). Conclusions Melflufen is able to trigger myeloma cell death regardless of p53 status, and thus overcome p53-deficiency-mediated melphalan resistance. It demonstrates better ex vivo efficacy in MM patient derived plasma cells harboring del17p or TP53 mutations. Melflufen response rates in the del17p Citation Format: Ana Slipicevic, Umair Munawar, Thorsten Stühmer, Johan Aschan, Fredrik Lehmann, Nina N. Nupponen, Juho Miettinen, Maiju-Emilia Huppunen, Caroline A. Heckman, María-Victoria Mateos, Paula Rodríguez Otero, Paul Richardson, Ralf Bargou. Melflufen efficacy in multiple myeloma with TP53 aberrations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1843.

124 Background: mCRPC has a poor prognosis and immunotherapies are largely ineffective. PSMA is a promising therapeutic target in mCRPC. Pasotuxizumab is a PSMA x CD3 BiTE immune therapy that mediates killing of tumor cells by T cells. Methods: NCT01723475 was a first-in-human, multicenter, dose-escalation study in patients (pts) with mCRPC refractory to standard therapy. Pts received continuous IV infusion of pasotuxizumab in cohorts of 3–4 pts. Dose-escalation followed a continuous reassessment methodology. The primary objective was to determine safety and maximum tolerated dose (MTD); secondary objectives included pharmacokinetics, biomarkers, and tumor response. Results: 16 pts were enrolled into 5 dosing cohorts (5 µg/d, n=3; 10 µg/d, n=4; 20 µg/d, n=3; 40 µg/d, n=4; 80 µg/d, n=2). All pts had ≥1 AE of any grade; most common were fever (94%), chills (69%), and fatigue (50%). 13 pts (81%) had ≥1 AE of grade ≥3; most common were decreased lymphocytes and infections (both 44%). No grade 5 AE occurred. A serious drug-related AE was reported for 1 pt (fatigue, 20 µg/d). No antidrug antibodies were observed. Recruitment was stopped before MTD was reached to allow initiation of a new study sponsored by Amgen. Antitumor activity as indicated by PSA serum level decline was dose dependent, with a mean best PSA change per dosing cohort vs baseline of +0.74% (5 µg/d), −17.9% (10 µg/d), −37.4% (20 µg/d), −42.5% (40 µg/d) and −54.9% (80 µg/d). PSA decreases ≥50% occurred in 3 pts (n=1 each in 20 µg/d, 40 µg/d, 80 µg/d cohorts). One long-term PSA responder was treated for 14 months (40 µg/d) and one for 19.4 months (80 µg/d). The latter pt showed a complete regression of soft-tissue metastases and marked regression of bone metastases by PSMA-PET/CT, >90% reduction in PSA and alkaline phosphatase, and a significant and durable improvement in disease related symptoms. Conclusions: Pasotuxizumab had an acceptable safety profile and dose-dependent clinical activity in mCRPC pts. There were two long term responders in the dose escalation. This is the first clinical study showing that a BiTE immune therapy can be efficacious in solid tumors. Clinical trial information: NCT01723475.

shRNA expression is an established technique to transiently or permanently deplete cells of a particular mRNA/protein. In functional analyses of oncogenic pathways it can thus be used as an alternative to pharmacologic inhibitors, or as a means to address otherwise undruggable targets. Here we describe and functionally validate a simple reiterative cloning system to generate concatenated multi-shRNA expression plasmids. The multi-shRNA expression cassette can eventually be subcloned into any suitably designed vector for the stable transfection of cells, here tested with derivatives of the Sleeping Beauty transposon vector for stable transfection of multiple myeloma cell lines at the lowest biosafety level. We finally test inducible versions of such multi-cassette knockdown vectors and show their efficacy for the induced concerted knockdown of all four components of the MEK/MAPK-module in the Ras/MAPK pathway. The described vector system(s) should be useful for functional knockdown analyses in a wide array of cell line models.

Despite an increasing number of approved therapies, multiple myeloma (MM) remains an incurable disease and only a small number of patients achieve prolonged disease control. Some genes have been linked with response to commonly used anti-MM compounds, including immunomodulators (IMiDs) and proteasome inhibitors (PIs). In this manuscript, we demonstrate an increased incidence of acquired proteasomal subunit mutations in relapsed MM compared to newly diagnosed disease, underpinning a potential role of point mutations in the clonal evolution of MM. Furthermore, we are first to present and functionally characterize four somatic PSMB5 mutations from primary MM cells identified in a patient under prolonged proteasome inhibition, with three of them affecting the PI-binding pocket S1. We confirm resistance induction through missense mutations not only to Bortezomib, but also, in variable extent, to the next-generation PIs Carfilzomib and Ixazomib. In addition, a negative impact on the proteasome activity is assessed, providing a potential explanation for later therapy-induced eradication of the affected tumor subclones in this patient.