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Six-Transmembrane Epithelial Antigen of the Prostate 1–4 (STEAP1–4) compose a family of metalloproteinases involved in iron and copper homeostasis and other cellular processes. Thus far, five homologs are known: STEAP1, STEAP1B, STEAP2, STEAP3, and STEAP4. In prostate cancer, STEAP1, STEAP2, and STEAP4 are overexpressed, while STEAP3 expression is downregulated. Although the metalloreductase activities of STEAP1–4 are well documented, their other biological functions are not. Furthermore, the properties and expression levels of STEAP heterotrimers, homotrimers, heterodimers, and homodimers are not well understood. Nevertheless, studies over the last few decades have provided sufficient impetus to investigate STEAP1–4 as potential biomarkers and therapeutic targets for prostate cancer. In particular, STEAP1 is the target of many emerging immunotherapies. Herein, we give an overview of the structure, physiology, and pathophysiology of STEAP1–4 to provide context for past and current efforts to translate STEAP1–4 into the clinic.

The B cell surface antigen CD19 is a target for treating B cell malignancies, such as B cell precursor acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma. The BiTE® immuno-oncology platform includes blinatumomab, which is approved for relapsed/refractory B cell precursor acute lymphoblastic leukemia and B cell precursor acute lymphoblastic leukemia with minimal residual disease. Blinatumomab is also being evaluated in combination with other agents (tyrosine kinase inhibitors, checkpoint inhibitors, and chemotherapy) in various treatment settings, including frontline protocols. An extended half-life BiTE molecule is also under investigation. Patients receiving blinatumomab may experience cytokine release syndrome and neurotoxicity; however, these events may be less frequent and severe than in patients receiving other CD19-targeted immunotherapies, such as chimeric antigen receptor T cell therapy. We review BiTE technology for treating malignancies that express CD19, analyzing the benefits and limitations of this bispecific T cell engager platform from clinical experience with blinatumomab.

TPS5101 Background: Six-transmembrane epithelial antigen of prostate 1 (STEAP1) is overexpressed on the surface of prostate cancer cells with low or no expression on normal tissue. AMG 509 is a bispecific XmAb 2+1 T-cell engager that simultaneously binds to STEAP1 on tumor cells and the CD3 complex on T cells resulting in T-cell mediated lysis of STEAP1-expressing cells. AMG 509 demonstrated significant antitumor activity in preclinical prostate cancer models. Methods: This 4-part, first-in-human study will evaluate AMG 509 in pts with mCRPC previously treated with novel hormonal therapy (NHT) and will assess the safety, tolerability, pharmacokinetics (PK), and efficacy of AMG 509 to establish the maximum tolerated dose (MTD) and/or the recommended phase 2 dose (RP2D). Part 1 is currently enrolling pts previously treated with NHT and up to 2 prior taxanes. As of 31 January 2022, 60 of up to 110 pts have been enrolled for treatment with intravenous (IV) AMG 509 with initial inpatient dosing followed by outpatient treatment. Part 2 will evaluate subcutaneous dosing of AMG 509 in the same patient population as in Part 1. Part 3 will explore AMG 509 IV dosing in chemotherapy-naïve pts who have been treated previously with one NHT and will provide additional data on the safety, PK, efficacy, pharmacodynamics, and correlative biomarkers at the MTD or RP2D determined in Part 1. Part 4 will evaluate the combination of AMG 509 with abiraterone (Part 4A) or enzalutamide (Part 4B) in pts previously treated with one NHT and up to 1 prior taxane for castration-sensitive disease. Primary outcome measures include dose-limiting toxicities, treatment-emergent and treatment-related adverse events, and change in clinical and laboratory parameters. Key secondary outcome measures include PK, objective response per RECIST 1.1, prostate-specific antigen response, progression-free survival, and overall survival. Key inclusion criteria are men ≥18 years of age with pathologically confirmed mCRPC, refractoriness to NHT, evidence of progressive disease, and ECOG performance status of 0–1. Key exclusion criteria are pure small cell or neuroendocrine carcinoma of the prostate, untreated CNS metastases or leptomeningeal disease, recent anticancer therapy or immunotherapy within 4 weeks of start of first dose not including luteinizing hormone-releasing hormone/gonadotropin-releasing hormone analogue (agonist/antagonist) therapy, radiation therapy, and a history of or current autoimmune disease or any disease requiring chronic immunosuppressive therapy. The trial is being carried out in investigative sites in North America, Australia, Asia, and Europe. The study opened in January 2020 and is recruiting pts for the dose exploration phase of Part 1; parts 2 and 4 are open for enrollment. Part 3 will be initiated once the MTD and/or RP2D have been determined in Part 1. Clinical trial information: NCT04221542.

TPS183 Background: Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is a cell surface antigen that is highly expressed in prostate cancer. AMG 509 is a potent bispecific XmAb 2+1 immune therapy designed to direct T effector cells to STEAP1-expressing cells. AMG 509 contains two identical humanized anti-STEAP1 Fab domains that bind STEAP1-expressing cells, an anti-CD3 scFv domain that binds T cells, and an Fc domain, engineered to lack effector function, that extends serum half-life. In preclinical studies, AMG 509 induced potent and specific T-cell-mediated lysis of STEAP1-expressing cancer models. Methods: This open-label, phase 1, first-in-human study will evaluate the safety, tolerability, pharmacokinetics (PK), and efficacy of AMG 509 in patients with relapsed/refractory mCRPC. The dose exploration phase (n=40) will estimate the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D) using a Bayesian logistic regression model. The dose expansion phase (n=30) will confirm safety, PK, and pharmacodynamics at the MTD or RP2D and collect further safety, efficacy, and biomarker data. Efficacy will be assessed by prostate-specific antigen response, circulating tumor cell response, and objective tumor response per RECIST 1.1 with Prostate Cancer Working Group 3 modifications. Key inclusion criteria: men ≥18 years with histologically/cytologically confirmed mCRPC who are refractory to novel hormonal therapy (e.g., abiraterone and/or enzalutamide) and have failed 1–2 taxane regimens or are medically unsuitable for or have refused taxanes; ongoing castration with total serum testosterone ≤50 ng/dL; evidence of progressive disease; ECOG performance status 0–1; life expectancy ≥3 months; and adequate hematologic, renal, hepatic, and cardiac function. In the dose exploration phase, novel antiandrogen therapy must have been given in the metastatic setting. Key exclusion criteria: pure small cell or neuroendocrine carcinoma of the prostate; untreated CNS metastases or leptomeningeal disease; any anticancer therapy or immunotherapy, radiation therapy, or major surgery

Adults with relapsed/refractory acute lymphoblastic leukemia have an unfavourable prognosis, which is influenced by disease and patient characteristics. To further evaluate these characteristics, a retrospective analysis of 1,706 adult patients with Ph-negative relapsed/refractory B-precursor acute lymphoblastic leukemia diagnosed between 1990-2013 was conducted using data reflecting the standard of care from 11 study groups and large centers in Europe and the United States. Outcomes included complete remission, overall survival, and realization of stem cell transplantation after salvage treatment. The overall complete remission rate after first salvage was 40%, ranging from 35%-41% across disease status categories (primary refractory, relapsed with or without prior transplant), and was lower after second (21%) and third or greater (11%) salvage. The overall complete remission rate was higher for patients diagnosed from 2005 onward (45%, 95% CI: 39%-50%). One- and three-year survival rates after first, second, and third or greater salvage were 26% and 11%, 18% and 6%, and 15% and 4%, respectively, and rates were 2%-5% higher among patients diagnosed from 2005. Prognostic factors included younger age, longer duration of first remission, and lower white blood cell counts at primary diagnosis. This large dataset can provide detailed reference outcomes for patients with relapsed/refractory Ph-negative B-precursor acute lymphoblastic leukemia. clinicaltrials.gov identifier: 02003612.

TPS5589 Background: Six transmembrane epithelial antigen of the prostate 1 (STEAP1) is a cell surface antigen that is highly expressed in prostate cancer. AMG 509 is a potent bispecific XmAb 2+1 immune therapy designed to direct T-effector cells to STEAP1-expressing cells. AMG 509 contains two identical humanized anti-STEAP1 Fab domains that bind STEAP1-expressing cells, an anti-CD3 scFv domain that binds T cells, and an Fc domain, engineered to lack effector function, that extends serum half-life. In preclinical studies, AMG 509 induced potent and specific T-cell-mediated lysis of STEAP1-expressing cancer models. Methods: This open-label, phase I, first-in-human study will evaluate the safety, tolerability, pharmacokinetics (PK), and efficacy of AMG 509 in patients with relapsed/refractory mCRPC. The dose exploration phase (n=40) will estimate the maximum tolerated dose (MTD) or recommended phase II dose (RP2D) using a Bayesian logistic regression model. The dose expansion phase (n=30) will confirm safety, PK, and pharmacodynamics at the MTD or RP2D and collect further safety, efficacy, and biomarker data. Efficacy will be assessed by prostate-specific antigen response, circulating tumor cell response, and objective tumor response per RECIST 1.1 with Prostate Cancer Working Group 3 modifications. Key inclusion criteria: men ≥18 years with histologically/cytologically confirmed mCRPC who are refractory to novel hormonal therapy (e.g., abiraterone and/or enzalutamide) and have failed 1–2 taxane regimens or are medically unsuitable for or have refused taxanes; ongoing castration with total serum testosterone ≤50 ng/dL; evidence of progressive disease; ECOG performance status 0–1; life expectancy ≥3 months; and adequate hematologic, renal, hepatic, and cardiac function. In the dose exploration phase, novel antiandrogen therapy must have been given in the metastatic setting. Key exclusion criteria: pure small-cell or neuroendocrine carcinoma of the prostate; untreated CNS metastases or leptomeningeal disease; any anticancer therapy or immunotherapy, radiation therapy, or major surgery

Various approaches of T-cell-based cancer immunotherapy are currently under investigation, among these are BiTE® (bispecific T-cell engager) antibody constructs, which have a unique design and mechanism of action. They are constructed by genetically linking onto a single polypeptide chain the minimal binding domains of monoclonal antibodies for tumor-associated surface antigens and for the T-cell receptor-associated molecule CD3. Concurrent engagement of the target cell antigen and CD3 leads to activation of polyclonal cytotoxic T-cells, resulting in target cell lysis. Blinatumomab, a BiTE targeting CD19, is being investigated in a broad range of B-cell malignancies and has recently been approved in the USA by the US FDA for Philadelphia chromosome-negative relapsed/refractory B-acute lymphoblastic leukemia under the trade name BLINCYTO™. The BiTE platform is one of the clinically most advanced T-cell immunotherapy options.

Approximately 30-50% of adults with acute lymphoblastic leukemia (ALL) in hematologic complete remission following multiagent therapy exhibit minimal residual disease (MRD) by reverse transcriptase-polymerase chain reaction or flow cytometry. MRD is the strongest predictor of relapse in ALL. In this open-label, single-arm study, adults with B-cell precursor ALL in hematologic complete remission with MRD (≥10-3) received blinatumomab 15 µg/m2/day by continuous intravenous infusion for up to four cycles. Patients could undergo allogeneic hematopoietic stem cell transplantation any time after cycle 1. The primary endpoint was complete MRD response status after one cycle of blinatumomab. One hundred sixteen patients received blinatumomab. Eighty-eight of 113 evaluable patients (78%) achieved a complete MRD response. In the subgroup of 110 patients with Philadelphia-chromosome negative ALL in hematologic remission, the Kaplan-Meier estimate of relapse-free survival (RFS) at 18 months was 53%. Median overall survival was 36.5 months. In a landmark analysis, complete MRD responders had longer relapse-free survival (23.6 vs 5.7 months; P=0.002) and overall survival (OS) (38.9 vs 12.5 months; P=0.002) compared with MRD nonresponders. Adverse events were consistent with previous studies of blinatumomab. Twelve (10%) patients and three (3%) patients had grade 3 or 4 neurologic events, respectively. Four (3%) patients had cytokine release syndrome (two grade 1 and two grade 3), all during cycle 1. Following treatment with blinatumomab in a population of patients with MRD-positive B-cell precursor ALL, the majority achieved a complete MRD response, which was associated with significantly longer RFS and OS compared with MRD nonresponders. The study is registered to https://clinicaltrials.gov as NCT01207388.

A novel bispecific antibody-derived recombinant protein targeting leukemias and lymphomas was designed, a single-chain Fv triple body (sctb) consisting of 1 polypeptide chain with 3 scFvs connected in tandem. The distal scFvs were specific for the tumor antigen CD19, and the central scFv for the trigger molecule CD16 (FcgammaRIII) on natural killer (NK) cells and macrophages. We had previously built a disulphide stabilized (ds) bsscFv [19 x 16] with monovalent binding for CD19 from ds components. The sctb ds[19 x 16 x 19] also used ds components and displayed 3-fold greater avidity for CD19 than the bsscFv (KD = 13 vs. 42 nM), whereas both had equal affinity for CD16 (KD = 58 nM). Plasma half-lives in mice were 4 and 2 hours for the sctb and the bsscFv, respectively. In antibody-dependent cellular cytotoxicity reactions with human mononuclear cells as effectors, the sctb promoted equal lysis of leukemic cell lines and primary cells from leukemia and lymphoma patients at 10-fold to 40-fold lower concentrations than the bsscFv. This new format may also be applicable to a variety of other tumor antigens and effector molecules. With half-maximum effective concentrations (EC50) in the low picomolar range, the sctb ds[19 x 16 x 19] is an attractive candidate for further preclinical evaluation.

The clinical efficacy of the CD20-specific chimeric monoclonal antibody rituximab is significantly hampered by intrinsic or acquired resistance to therapy. Rituximab activates antibody-dependent cellular cytotoxicity/complement-dependent cytotoxicity–dependent lysis but also induces apoptosis by cross-linking of its target antigen CD20. Recent reports indicate that this apoptotic activity of rituximab can be synergized by cotreatment with Fas agonists. Here, we report on a strategy designed to exploit and optimize the synergy between rituximab and Fas signaling by genetically fusing a rituximab-derived antibody fragment to soluble Fas ligand (sFasL). The resultant fusion protein, designated scFvRit:sFasL, potently induced CD20-restricted apoptosis in a panel of malignant B-cell lines (10 of 11) and primary patient-derived malignant B cells (two of two non–Hodgkin lymphoma and five of six B cell chronic lymphocytic leukemia). ScFvRit:sFasL efficiently activated CD20 and Fas apoptotic signaling, resulting in a far superior proapoptotic activity compared with cotreatment with rituximab and Fas agonists. ScFvRit:sFasL lacked activity toward normal human B cells and also lacked systemic toxicity in nude mice with no elevation of aspartate aminotransferase and alanine aminotransferase levels or liver caspase-3 activity. In conclusion, scFvRit:sFasL efficiently activates CD20 and Fas-apoptotic signaling and may be useful for the elimination of malignant B cells. [Cancer Res 2008;68(2):597–604]

Although the treatment outcome of lymphoid malignancies has improved in recent years by the introduction of transplantation and antibody-based therapeutics, relapse remains a major problem. Therefore, new therapeutic options are urgently needed. One promising approach is the selective activation of apoptosis in tumor cells by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). This study investigated the pro-apoptotic potential of a novel TRAIL fusion protein designated scFvCD19:sTRAIL, consisting of a CD19-specific single-chain Fv antibody fragment (scFv) fused to the soluble extracellular domain of TRAIL (sTRAIL). Potent apoptosis was induced by scFvCD19:sTRAIL in several CD19-positive tumor cell lines, whereas normal blood cells remained unaffected. In mixed culture experiments, selective binding of scFvCD19:sTRAIL to CD19-positive cells resulted in strong induction of apoptosis in CD19-negative bystander tumor cells. Simultaneous treatment of CD19-positive cell lines with scFvCD19:sTRAIL and valproic acid (VPA) or Cyclosporin A induced strongly synergistic apoptosis. Treatment of patient-derived acute B-lymphoblastic leukemia (B-ALL) and chronic B-lymphocytic leukemia (B-CLL) cells resulted in strong tumoricidal activity that was further enhanced by combination with VPA. In addition, scFvCD19:sTRAIL prevented engraftment of human Nalm-6 cells in xenotransplanted NOD/Scid mice. The pre-clinical data presented here warrant further investigation of scFvCD19:sTRAIL as a potential new therapeutic agent for CD19-positive B-lineage malignancies.

CD19 is a B-lineage-specific transmembrane signaling protein participating in the control of proliferation and differentiation. It is present at high surface density on chronic B-lymphocytic leukemia (B-CLL) cells and cells of other B-cell malignancies, and is a prime target for therapy with antibody-derived agents. Many attempts have been made to target malignant cells via CD19, but to date none of these agents have received drug approval. Here we report the design of a monovalent immunotoxin consisting of a CD19-specific single-chain Fv antibody fragment fused to a derivative of Pseudomonas Exotoxin A. This fusion protein induced efficient antigen-restricted apoptosis of several human leukemia- and lymphoma-derived cell lines including Nalm-6, which it eliminated at an effective concentration (EC(50)) of 2.5 nM. The agent displayed synergistic toxicity when used in combination with valproic acid and cyclosporin A in cell-culture assays. It induced apoptosis of primary malignant cells in 12/12 samples from B-CLL patients, including patients responding poorly to fludarabine, and of cells from one pediatric acute lymphoblastic leukemia patient. In NOD/SCID mice transplanted with Nalm-6 cells, the toxin prevented engraftment and significantly prolonged survival of treated mice. Owing to its efficient antigen-restricted antileukemic activity, the agent deserves further development towards clinical testing.

Few patients with relapsed/refractory diffuse large B-cell lymphoma (DLBCL) achieve prolonged disease-free survival. Blinatumomab, a bispecific T-cell engaging antibody construct, transiently links CD3-positive T cells to CD19-positive B cells. This phase 2 study evaluated stepwise (9-28-112 μg/d with weekly dose increases; n = 23) or flat (112 μg/d; n = 2) dosing of blinatumomab by continuous infusion, with dexamethasone prophylaxis, in patients with relapsed/refractory DLBCL. Patients received a median of 3 prior lines of therapy. Median time since last regimen was 1.5 months. Seventeen patients ended treatment in cycle 1 (induction), 7 in cycle 2 (consolidation), and 1 in retreatment. Among 21 evaluable patients, the overall response rate after 1 blinatumomab cycle was 43%, including complete responses (CRs) in 19%. Three patients had late CR in follow-up without other treatment. The most common adverse events with stepwise dosing were tremor (48%), pyrexia (44%), fatigue (26%), and edema (26%). Grade 3 neurologic events with stepwise dosing were encephalopathy and aphasia (each 9%) and tremor, speech disorder, dizziness, somnolence, and disorientation (each 4%). Of 5 (22%) patients who discontinued stepwise dosing because of adverse events, 4 (17%) had neurologic events. Most neurologic events resolved. The flat-dose cohort was stopped because of grade 3 neurologic events in both patients. Blinatumomab monotherapy appears effective in patients with relapsed/refractory DLBCL, a heavily pretreated patient population with a high unmet medical need. Further studies need to define the optimal approach to achieve the target dose without early dropout. The study was registered at www.clinicaltrials.gov as #NCT01741792.

Introduction. MRD in ALL is defined as detection of leukemic cells in bone marrow by polymerase chain reaction (PCR) or flow cytometry with hematologic complete remission (CR). Pts with persistent/recurrent MRD after first-line induction therapy have a higher risk of relapse than those with complete MRD response (no detectable MRD with minimum sensitivity 0.01%). Interventions, including hematopoietic stem cell transplantation (HSCT), are used to improve the outcome of these pts. Blinatumomab, a bispecific T cell engager (BiTE®) antibody construct, redirects CD3+ T cells to CD19+ target cells, resulting in serial lysis of CD19+ B cells. In a multicenter, international phase 2 study in MRD+ ALL (Goekbuget N et al. Blood 2014;124:379), blinatumomab resulted in complete MRD response in cycle 1 in 78% of pts including multiple subgroups such as pts in second-line treatment, those with high MRD burden, and older pts. No subgroups with higher MRD complete response rates were identified. This analysis evaluated long-term outcomes, including overall survival (OS), relapse-free survival (RFS), and duration of remission (DOR). Methods. Adults (≥18 years) with B-cell precursor ALL with hematologic CR ( Results. 116 pts enrolled and received blinatumomab. Median age was 45 years (range 18-76); 15 (13%) pts were age ≥65 years. 90 (78%) pts received HSCT after blinatumomab. 62 (53%) pts were still being followed. 35 pts relapsed and 26 pts died in CR (23 of them after subsequent HSCT). Median OS, with median follow-up of 29.5 mo, was 36.5 mo (95% CI, 19.1 mo to not reached [n.r.]): 40.4 vs 12.0 (P =.001) in pts with (n=88) or without (n=24) MRD complete response in cycle 1. 110 pts were evaluable (CR at study entry, Ph-) for RFS and DOR. Median RFS was 18.9 mo (95% CI, 12.3 to 35.2 mo): 24.6 vs 11.0 (P =0.005) in pts treated in first (n=75) vs later (n=35) remission; and 35.2 vs 7.1 (P =0.002) in pts alive and relapse-free after 45 days with (n=85) or without (n=15) MRD complete response in cycle 1 (Figure). Median DOR was n.r. (95% CI, 24.6 mo to n.r.): n.r. vs 15.0 mo (P =0.002) in pts treated in first vs later remission; and n.r. vs 15.0 mo (P =0.015) in pts with DOR ≥ 45 days with (n=85) or without (n=13) MRD complete response in cycle 1. In time-dependent Cox model analyses, HSCT vs no HSCT were not different for OS (hazard ratio [HR], 1.39; 95% CI, 0.68 to 2.82; P =0.368) or RFS (HR, 0.89; 95% CI, 0.47 to 1.69; P =0.730); DOR (treating death as a competing risk) was longer for HSCT vs no HSCT (HR, 0.36; 95% CI, 0.17 to 0.77; P =0.008). All pts experienced at least one AE. The most clinically relevant were neurologic events, including tremor (30%), aphasia (13%) dizziness (8%), ataxia and paraesthesia (6% each), and encephalopathy (5%). Rates decreased over time (cycles 1, 2, 3, and 4) for any neurologic event (47%, 24%, 15%, and 15%) and any grade ≥3 neurologic event (10%, 4%, 0%, and 0%). 12 (10%) pts interrupted treatment due to grade ≥3 neurologic events: 5 resumed without another interruption and 2 resumed then stopped treatment for another neurologic event. Investigators reported 4 deaths as fatal AEs during follow-up (brain injury, disease progression, gastrointestinal hemorrhage, and multiorgan failure); all 4 pts received HSCT after blinatumomab. Conclusion. In this long-term follow-up analysis of the first large prospective trial with an experimental compound in MRD+ ALL, MRD complete response induced by single-agent blinatumomab treatment was associated with longer OS, RFS, and DOR compared with not achieving an MRD complete response after blinatumomab treatment. This strengthens the current strategy of MRD-based treatment in ALL before occurrence of clinical relapse. Disclosures Gökbuget: Sanofi: Equity Ownership; Pfizer: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Honoraria, Research Funding; Erytech: Consultancy; Eusapharma/Jazz: Consultancy, Honoraria, Research Funding; Gilead Sciences: Consultancy; Kite: Consultancy; Medac: Consultancy, Honoraria, Research Funding; Mundipharma: Consultancy, Honoraria, Research Funding; Bayer: Equity Ownership; SigmaTau: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria. Off Label Use: Blinatumomab (BLINCYTO®) is approved for the treatment of Philadelphia chromosome-negative relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL). It has not been approved for use in patients in hematologic remission, but with presence of minimal residual disease (MRD), from ALL.. Dombret:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Bonifacio:Amgen: Consultancy; Ariad Pharmaceuticals: Consultancy; Pfizer: Consultancy; Novartis Farma: Research Funding. Reichle:University Hospital Regensburg: Employment. Graux:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Faul:Amgen: Honoraria. Topp:Astra: Consultancy; Regeneron: Consultancy; Affimed: Consultancy, Research Funding; Roche: Consultancy, Other: Travel Support; Jazz: Consultancy; Pfizer: Consultancy; Amgen: Consultancy, Honoraria, Other: Travel Support. Brüggemann:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria. Horst:Pfizer: Research Funding; MSD: Research Funding; Gilead: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Boehringer Ingleheim: Research Funding. Stieglmaier:Amgen Research (Munich) GmbH: Employment; Amgen Inc: Equity Ownership. Wessels:Amgen: Employment. Haddad:Amgen Ltd.: Employment; Amgen Inc.: Equity Ownership. Zugmaier:Amgen Res. Munich: Employment. Nagorsen:Amgen: Employment, Equity Ownership, Patents & Royalties: Inventor on blinatumomab-related patent. Bargou:University of Wuerzburg, Germany: Employment; Novartis: Consultancy, Honoraria; GEMoaB GmbH: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Patents & Royalties: Patent for blinatumomab.

To investigate the influence of N-linked oligosaccharides at asparagines-297 on the cytolytic potential of chimeric CD19 antibodies, three distinct variants were generated by production in different expression systems. The same chimeric CD19 antibody was produced in Sf21 insect cells, human 293 T cells, and 293 T cells expressing a co-transfected beta1,4-N-acetylglucosaminyltransferase III (GnTIII). The N-glycan structures and the cytolytic potential of the antibodies produced in these three systems were directly compared. After expression in insect cells, the antibody carried paucimannosidic N-linked oligosaccharides, distinct from the complex biantennary carbohydrate moieties attached to the product from human cells. After co-expression with GnTIII in human cells, the antibody carried an eightfold greater percentage of oligosaccharides with a bisecting N-acetylglucosamine (78.7% versus 9.6%) and a 30-fold increased proportion of bisecting, defucosylated oligosaccharides (15.9% versus 0.5%). The insect cell product triggered stronger antibody-dependent cellular cytotoxicity (ADCC) of a human leukemia-derived cell line than the product from non-re-engineered 293 T cells and was equally effective at 50- to 100-fold lower concentrations. The antibody from glyco-engineered 293 T cells had comparable lytic activity as the insect cell product. Both mediated significant ADCC at lower effector-to-target cell ratios than the antibody from non-re-engineered 293 T cells, and both were highly effective against primary blasts from pediatric leukemia patients. The data demonstrate the influence of the N-glycosylation pattern on the ADCC activity of chimeric CD19 antibodies and point to the importance of suitable expression systems for the production of highly active therapeutic antibodies.

Introduction: Treatment of relapsed or refractory DLBCL can be challenging and little progress has been made in recent years. Blinatumomab, a bispecific T-cell engager (BiTE®) antibody construct, engages CD3+ cytotoxic T cells, resulting in T-cell expansion and lysis of CD19+ B cells. In a prior phase 1 study, blinatumomab treatment resulted in an overall response rate (ORR) of 55% in a subset of patients with diffuse large B-cell lymphoma (DLBCL). In the present phase 2 study, we compared stepwise versus flat dosing of blinatumomab, and evaluated its efficacy in patients with relapsed/refractory (r/r) DLBCL. Methods: Eligible patients were ≥18 years of age, had an Eastern Cooperative Oncology Group performance status ≤2 and had DLBCL; patients were refractory to treatment, had relapsed following autologous HSCT, or had relapsed and were ineligible for autologous hematopoietic stem cell transplantation (HSCT). Blinatumomab was administered over 8 weeks by continuous intravenous infusion. In stage 1, stepwise dosing (cohort I: 9, 28, and 112 μg/day after weeks 1, 2, respectively) was compared to constant dosing of 112 μg/day (cohort II). Based on the benefit/risk assessment from stage 1, stepwise dosing (9, 28, and 112 μg/day) was chosen for cohort III in stage 2. Patients achieving response after 8 weeks of treatment could receive a 4-week consolidation cycle after a 4-week treatment-free period. All patients received prophylactic dexamethasone (2 × 20 mg before infusion start and at infusion start; 3 × 8 mg/day for the first 2 days after infusion start and at dose step). The primary endpoint was ORR by Cheson revised response criteria for malignant lymphomas. Response was evaluated by independent radiologic assessment. Results: As of the primary analysis, 25 patients have been enrolled and treated: 9, 2, and 14 in cohorts I, II, and III, respectively. Fifty-six percent of patients were men, and the median age was 66 years (range, 34–85). Seven (28%) patients had received prior autologous HSCT. Blinatumomab was received as a fourth-line systemic therapy following a median (range) of 3 (1-7) prior treatments. Median (interquartile range) duration of exposure for stepwise dosing (cohorts I and III) was 46.8 (22.1−76.9) days. Twenty-one patients were evaluable for response (cohort I, n=7; cohort II, n=1; cohort III, n=13). Four patients were not evaluable for ORR per protocol definition due to early treatment discontinuation (1 patient were disorientation, encephalopathy, aphasia, and epilepsy [n=2 each]). There were no grade 4 or 5 neurologic events. Conclusions: In this phase 2 study, a stepwise dosing regimen (9, 28, and 112 μg/day) was established as the preferred dosing for blinatumomab in DLBCL. Treatment with blinatumomab showed an acceptable safety profile and resulted in objective and durable responses in heavily pretreated patients with r/r DLBCL. Disclosures Viardot: Amgen Inc.: Membership on an entity's Board of Directors or advisory committees, Travel support Other; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Travel support, Travel support Other; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Travel support Other. Off Label Use: This presentation will discuss the off-label use of blinatumomab, as this agent is not approved for use by the FDA, EMA or any other regulatory authorities.. Libicher:Amgen Inc.: Consultancy. Degenhard:Amgen Inc.: Equity Ownership; Amgen Research (Munich) GmbH: Employment. Stieglmaier:Amgen Inc.: Equity Ownership; Amgen Research (Munich) GmbH: Employment. Zhang:Amgen Inc.: Employment. Nagorsen:Amgen Inc.: Blinatumomab-related Patents & Royalties, Employment, Equity Ownership. Bargou:Amgen Inc.: Consultancy, Honoraria.

Introduction: In ALL, minimal residual disease (MRD) is defined as the detection of leukemic cells in bone marrow by PCR or flow cytometry in the presence of hematological complete remission (CR). Patients with persistent/recurrent MRD after induction therapy have a higher risk of relapse than those with no detectable MRD. Effective treatment of patients with MRD aims to avoid hematologic relapse, reduce MRD load, and provide a bridge to subsequent HSCT. Blinatumomab, an investigational BiTE® antibody construct, redirects CD3+ T cells to CD19+ target cells, resulting in serial lysis of CD19+ B cells. In a phase 2 study of blinatumomab in 21 patients with MRD+ ALL in first-line treatment, 80% of evaluable patients achieved a complete MRD response. BLAST, a confirmatory single-arm, phase 2 study evaluated efficacy, safety, and tolerability of blinatumomab in patients with MRD+ ALL in a larger population. Methods: Adults (≥18 years) with B-precursor ALL in hematologic CR ( Results: 116 patients enrolled and received blinatumomab. Median (range) age was 45 (18–76) years; 15 (13%) patients were ≥65 years of age. 35% of the patients were treated in second or later remission (Table). As of February 2014, 106 patients had ended treatment: 74 had completed treatment (4 cycles or 1 cycle followed by HSCT) and 32 had discontinued treatment due to AEs, disease relapse, or investigators' decision; 79 patients were still alive and being followed. Three patients were excluded from the efficacy analysis: one had no central lab assay and two had assays with a sensitivity of 5×10-4. Eighty-eight patients (78% [95% CI, 69%–85%]) had a complete MRD response after 1 cycle of treatment. The lower CI bound exceeded 44% (the null hypothesis response rate), confirming that the study met its primary objective. Two additional patients had a complete MRD response after >1 cycle of blinatumomab; across all cycles the complete MRD response rate was 80%. The rate of complete MRD response did not differ significantly across baseline age, sex, line of treatment, and MRD burden categories (Table). All patients experienced ≥1 AE. AEs occurring in ≥20% of patients included pyrexia (88%), headache (38%), tremor (29%), chills (25%), fatigue (24%), nausea (22%) and vomiting (22%). Serious AEs (SAEs) occurred in 60% of patients; 59% and 27% of patients had grade ≥3 and grade ≥4 AEs, respectively. SAEs occurring in ≥5% of patients were pyrexia (15%), tremor (7%), aphasia (5%), encephalopathy (5%) and overdose (5%). Two fatal AEs occurred on treatment: subdural hemorrhage and atypical pneumonia (the latter was deemed treatment-related). Conclusion: This is the largest prospective trial with an experimental compound in MRD+ ALL.Blinatumomab treatment resulted in complete MRD response across multiple patient demographics including patients in second-line treatment and those with high MRD burden. With a complete MRD response rate of 78%, the study met its primary objective. Among patients with a complete MRD response, 98% had a response within the first treatment cycle. In patients with MRD+ ALL following intensive therapy, rapid MRD response induced by blinatumomab has the potential to improve patient outcomes. Figure 1 Figure 1. Disclosures Goekbuget: Amgen Inc.: Consultancy, Honoraria, Research Funding. Off Label Use: This presentation will discuss the off-label use of blinatumomab, as this agent is not approved for use by the FDA, EMA or any other regulatory authorities.. Dombret:Amgen Inc.: Honoraria, Research Funding. Bonifacio:Amgen Inc.: Consultancy. Graux:Amgen Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees. Buss:Amgen Inc.: Reimbursement for participation in clinical studies, Reimbursement for participation in clinical studies Other; BMS: Travel support, Travel support Other; Novartis: Travel support Other; Pfizer: Reimbursements for participation in clinical studies, Reimbursements for participation in clinical studies Other. Bruggemann:Amgen Inc.: Consultancy, Research Funding. Stieglmaier:Amgen Inc.: Equity Ownership; Amgen Research (Munich) GmbH: Employment. Wessels:Amgen Inc.: Equity Ownership; Amgen Research (Munich) GmbH: Employment. Haddad:Amgen Ltd.: Employment. Zugmaier:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Nagorsen:Amgen Inc.: Employment, Equity Ownership, Related to blinatumomab Patents & Royalties. Bargou:Amgen Inc.: Consultancy, Honoraria.

Introduction Blinatumomab, a bispecific T-cell engager (BiTE®) that redirects cytotoxic T cells to CD19+ B-lineage cells, has shown anticancer activity and acceptable toxicity in a phase 1 study in patients with relapsed/refractory non-Hodgkin lymphoma, including diffuse large B-cell lymphoma (DLBCL). Using a stepwise dose escalation treatment, the overall response rate (ORR) in patients with DLBCL was 55% (Goebeler et al. Hematol Oncol 2013;31[suppl 1]:197). This open-label phase 2 study has been initiated to investigate the efficacy and tolerability of blinatumomab in patients with relapsed/refractory DLBCL, comparing stepwise dose escalation with constant target dosing. Methods In this ongoing study, eligible patients must be ≥18 years of age, must have relapsed/refractory DLBCL and Eastern Cooperative Oncology Group performance status ≤2. Blinatumomab is administered by continuous intravenous infusion over 8 weeks. In part 1 of the study, two cohorts were evaluated using a double-step or flat dose escalation regimen, respectively, in order to achieve the target dose of 112 μg/d. Part 2 will investigate the selected treatment schedule from part 1. Data from part 1 (cohorts I and II) are presented herein. Patients in cohort I received stepwise blinatumomab dosing of 9, 28, and 112 μg/d during weeks 1, 2, and thereafter, respectively; patients in cohort II received blinatumomab at 112 μg/d throughout. After a 4-week treatment-free period, patients achieving an objective response were permitted to receive a 4-week consolidation cycle. All patients received prophylactic dexamethasone. The primary endpoint is ORR by Cheson (2007) revised response criteria for malignant lymphomas. Results To date, 11 patients have been enrolled and treated in part 1 of the study: nine in cohort I and two in cohort II. The median age was 73 years (range, 55–85); 64% of patients were women. Six (55%) patients had received ≥3 lines of previous systemic antitumor therapy; study treatment was given as fourth-line (median) systemic treatment. Three patients had received autologous hematopoietic stem cell transplantation. At the time of this analysis, seven patients were evaluable for response (cohort I, n=6; cohort II, n=1). The ORR based on independent radiological assessment was 57% (cohort I: complete response, n=1; partial response, n=2; cohort II: partial response, n=1). Three patients had progressive disease (all in cohort I). Four patients were not evaluable for ORR per protocol definition: early treatment discontinuation after Conclusions In this ongoing phase 2 study, blinatumomab was tolerable and showed antitumor activity in adult, heavily pretreated patients with relapsed/refractory DLBCL. Part 1 of the study established a recommended blinatumomab dose for this patient population. The study continues to enroll patients in part 2 (cohort III). Disclosures: Libicher: Amgen Inc.: Consultancy. Degenhard:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Stieglmaier:Amgen Research (Munich) GmbH: Employment; Amgen Inc.: Equity Ownership. Zhang:Amgen Inc.: Employment; Amgen Inc.: Equity Ownership. Nagorsen:Amgen Inc.: Employment; Amgen Inc.: Equity Ownership. Bargou:University of Würzburg: Consultancy; University of Würzburg: Honoraria; Amgen Inc.: patent, patent Other.