Antibody Constructs Research Articles

IMMU-05. A PHASE I STUDY IN PROGRESS OF HEGFRVIII-CD3 BI-SCFV (BRITE) IN PATIENTS WITH WHO GRADE IV MALIGNANT GLIOMA

Abstract INTRODUCTION Approximately 30% of glioblastomas harbor the tumor specific EGFRvIII mutation. hEGFRvIII-CD3 bi-scFv (Brain Bi-Specific T Cell Engager - BRiTE) is a novel bispecific antibody construct which can redirect a patient’s entire repertoire of T cells (TCR-agnostic) to specifically lyse EGFRvIII-positive tumor cells. Compared to CAR-T therapy, it offers a highly potent yet off-the-shelf approach for glioblastoma. BRiTE is now entering Phase I clinical trials (NCT04903795) and will be trialed as a monotherapy or with autologous T cell infusion. Migration of T cell binding macromolecules across the blood-brain barrier may be facilitated by activated T cells which adhere to the microvascular endothelium and enter the brain parenchyma. Concurrent administration of activated T cells could therefore enhance trafficking of BRiTE and other antibody-based macromolecules into the intracerebral compartment. HYPOTHESIS We hypothesize that treatment of EGFRvIII-positive WHO grade IV malignant glioma with BRiTE alone or with peripheral T-cell infusion is safe and can induce objective tumor shrinkage at tolerable doses. TRIAL DESIGN/OBJECTIVES A maximum of 18 patients with newly diagnosed or recurrent WHO grade IV malignant EGFRvIII+ glioma will be enrolled after undergoing standard of care treatment. The primary objective will be evaluating the safety of BRiTE alone and with autologous T cell infusion. Patients will receive a single BRiTE infusion, followed 14 days later by an infusion of activated autologous T cells and a second bolus BRiTE infusion. Dose escalation and de-escalation will be managed using a Bayesian optimal interval design. Secondary objectives will be to describe clinical benefit as determined by objective response rate per mRANO criteria, and to evaluate BRiTE pharmacokinetics in serum. CONCLUSION We describe a first-in-human trial of bispecific T cell engager therapy for glioblastoma. We also describe our novel approach for enhancing intracranial penetration of BRiTE using autologous T cell infusion.

Optimal Timing of Blinatumomab for the Treatment of B-Lymphoblastic Leukemia

Introduction Blinatumomab is a CD19-targeting bi-specific T-cell engager antibody construct developed for the treatment of CD19-expressing B-cell malignancies. After early impressive in vitro and in vivo pre-clinical results,1–3 blinatumomab was quickly incorporated into clinical investigations. After a phase I non-Hodgkin lymphoma (NHL) trial established a safe and effective dose, delivered via continuous intravenous (IV) infusion,4 trials exploring its use in B-lymphoblastic leukemia (B-ALL) soon followed. The results of these trials have been largely positive, though the extent of response and benefit appears to vary based on disease status and burden at the time of treatment. Thus, the optimal incorporation of blinatumomab into B-ALL therapy is still being refined.

Targeting BCMA in Multiple Myeloma.

Despite considerable advances in the treatment of multiple myeloma (MM) in the last decade, a significant number of patients still progress on current available therapies. Here, we review treatment modalities used to target BCMA in the treatment of MM, specifically antibody-drug conjugates (ADC), bispecific antibody constructs, and chimeric antibody receptor (CAR) modified T-cell therapies. We will provide an overview of therapies from these classes that have presented or published clinical data, as well as data on mechanisms of resistance to these novel agents. Clinical trials exploring different BCMA-targeting modalities to treat multiple myeloma are underway and demonstrate promising results. In relapsed/refractory multiple myeloma, anti-BCMA ADCs and bispecific antibody constructs are showing impressive efficacy with manageable side effect profiles. In parallel, adoptive cellular therapy has induced dramatic durable responses in multiply relapsed and refractory myeloma patients. Therapeutic approaches targeting BCMA hold significant potential in the management of multiple myeloma and will soon be incorporated in combination with current standard therapies to improve outcomes for patients with multiple myeloma. In addition, novel approaches are being evaluated to overcome resistance mechanisms to anti-BCMA therapies.

Increasing the efficacy and safety of a human complement inhibitor for treating post-transplant cardiac ischemia reperfusion injury by targeting to a graft-specific neoepitope

Post-transplant ischemia reperfusion injury (IRI) is a recognized risk factor for subsequent organ dysfunction, alloresponsiveness, and rejection. The complement system is known to play a role in IRI and represents a therapeutic target. Complement is activated in transplanted grafts when circulating IgM antibodies bind to exposed ischemia-induced neoepitopes upon reperfusion, and we investigated the targeting of a human complement inhibitor, CR1, to a post-transplant ischemia-induced neoepitope. A fragment of human CR1 was linked to a single chain antibody construct (C2 scFv) recognizing an injury-specific neoepitope to yield C2-CR1. This construct, along with a soluble untargeted counterpart, was characterized in a cardiac allograft transplantation model of IRI in terms of efficacy and safety. CR1 was similarly effective against mouse and human complement. C2-CR1 provided effective protection against cardiac IRI at a lower dose than untargeted CR1. The increased efficacy of C2-CR1 relative to CR1 correlated with decreased C3 deposition, and C2-CR1, but not CR1, targeted to cardiac allografts. At a dose necessary to reduce IRI, C2-CR1 had minimal impact on serum complement activity, in contrast to CR1 which resulted in a high level of systemic inhibition. The circulatory half-life of CR1 was markedly longer than that of C2-CR1, and whereas a minimum therapeutic dose of CR1 severely impaired host susceptibility to infection, C2-CR1 had no impact. We show the translational potential of a human complement inhibitor targeted to a universal ischemia-induced graft-specific epitope, and demonstrate advantages compared to an untargeted counterpart in terms of efficacy and safety.

Abstract 1737: Understanding the geometry and valency of bispecific antibodies in the optimization of tumor-dependent activation of 4-1BB

Abstract 4-1BB is a TNF family receptor expressed on the surface of tumor-infiltrating T cells. Activation of 4-1BB enhances the activation, metabolism and function of tumor-infiltrating T cells and promotes tumor regression. Several anti-4-1BB antibodies have entered the clinic and have suffered from liver toxicity or lack of activity. To address the clinical liabilities with 4-1BB targeting, we designed bispecific 4-1BB x tumor associated antigen (TAA) antibodies to selectively activate T cells within the tumor microenvironment. To understand the impact of antibody format on potency of T cell activation, we generated a panel of 4-1BBxTAA bispecific antibodies in different formats with the AzymetricTM and EFECTTM platforms. We compared potency and maximal activity of these constructs in co-culture assays with tumor cells and T or Jurkat reporter cells, in comparison to urelumab as a clinical benchmark. The AzymetricTM platform allowed the production and evaluation of unique antibody formats from which an extensive structure activity relationship analysis was performed. We also examined the accessible epitope space by sampling the possible paratope binding conformations of the molecule. From this we determined the geometric limits of antigen engagement. Bispecific antibody constructs utilizing monovalent 4-1BB targeting showed inferior activity than urelumab in co-culture assays with TAAhi tumor cells. Activity greater than urelumab was seen with constructs containing two 4-1BB arms with one or two TAA binding scFv on the C-terminus of the Fc. Bivalent 4-1BB antibodies with an anti-TAA scFv fused to the N-terminus of the 4-1BB Fab showed decreased activity, potentially due to reduced ability to engage 4-1BB in a bivalent manner. Critically, the activity seen was transferable across multiple TAAs. The activity of the 4-1BB bispecific constructs was dependent on the expression level of the TAA. Without the presence of tumor cells, or with constructs where the TAA scFv was replaced by an irrelevant scFv, minimal activity was seen. The modelling of the space accessible to the 4-1BB paratopes showed that the constructs with which we saw the most activity had a distance between 4-1BB and TAA binding paratopes similar to that seen between T and APC membranes as part of an immune synapse, suggesting that this may be a key characteristic for the development of conditional T cell agonist bispecifics. This work represents an investigation of 4-1BBxTAA formats which would be difficult to make without an efficient bispecific technology. We identified a series of 4-1BBxTAA bispecific agonist antibody formats which were transferable between multiple TAAs. These formats also allowed optimization of activity and selectivity to promote maximal therapeutic index and efficacy, key factors which are potentially able to contribute to improved clinical outcomes. Citation Format: Anna von Rossum, Harsh Pratap, Lisa Newhook, Elizabeth C. Halvorsen, Lee Freiburger, Renee Duan, Charles Stevens, Dimitri Tcaciuc, Gesa Volkers, Duncan Browman, Brandon Clavette, David Mills, Thomas Spreter, Daniel T. Patton. Understanding the geometry and valency of bispecific antibodies in the optimization of tumor-dependent activation of 4-1BB [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 1737.

CD33/CD3-Bispecific T-Cell Engaging (BiTE®) Antibody Constructs Efficiently Target Monocytic CD14+ hla-DRlow IDO+ aml-MDSCs

Acute Myeloid Leukemia (AML) is the most common acute leukemia amongst adults with a 5-year overall survival lower than 25%. Emerging evidence suggest that immune alterations favor leukemogenesis and/or AML relapse thereby negatively impacting disease outcome. Over the last years myeloid derived suppressor cells (MDSCs) have been gaining momentum in the field of cancer research. MDSCs are a heterogeneous cell population morphologically resembling either monocytes or granulocytes and sharing some key features including myeloid origin, aberrant (immature) phenotype, and immunosuppressive activity. Increasing evidence suggests that accumulating MDSCs are involved in hampering anti-tumor immune responses and immune-based therapies. In this study we sought to investigate whether the CD33/CD3-bispecific BiTE® antibody construct (AMG 330) with documented pre-clinical activity against AML blasts can also target CD33+ AML-MDSCs by redirecting and activating T-cells and thereby enhance AMG 330 mediated anti-leukemic activity.First, we assessed the presence of circulating CD14+ cells that co-express CD33 but lack HLA-DR expression (HLA-DRlo) in patients with newly diagnosed AML. These monocytic cells represent one of the best-defined human MDSC subsets. Frequency of CD14+HLA-DRlo cells was significantly increased in untreated AML patients as compared to healthy controls (28,98±4,19%, n=13 versus 3,28±0,75%, n=37) but did not correlate with the number of AML blasts. Purified CD14+HLA-DRlo cells suppressed in vitro T-cell proliferation in a concentration-dependent manner allowing us their denomination as MDSCs.Interestingly, we also observed that primary AML blasts and human AML cell lines (THP, OCI-AML, and HL-60) induced HLA-DRlo cells from healthy donor-derived monocytes that were T-cell suppressive and expressed the immune regulatory indoleamine-2,3-dioxygenase (IDO). Functional and phenotypic reprogramming of monocytes was cell-contact independent.Using ex vivo co-culturing models of primary AML blasts/AML cell lines (=target cells) and T-cells (=effector cells) we observed AMG 330-triggered antibody construct-dependent cell-mediated cytotoxicity/ADCC. Short-term (three to six days) treatment of AML patient-derived peripheral blood mononuclear cells (PBMCs) with 10 or 100 pM AMG 330 led to T-cell activation based on proliferation and on increased expression/production of CD25, CD69, CD137, CD154, IL-2, and IFN-g. The initial T-cell frequency ranging from 0,16 to 14,30% had no impact on their responsiveness. Moreover, we observed a bystander activation of NK-cells. The addition of AML-MDSCs into these co-cultures did not negatively impact ADCC activity. On the contrary, T-cells formed immunological synapses with AML-MDSCs and triggered their elimination (also in presence of AML blasts) overcoming their T-cell suppressive effect. MDSC levels in AML-PBMCs had no effect on AMG 330-mediated T-cell (and bystander cell) activation, which was further corroborated in experiments where the total CD14+ cell compartment (comprising AML-MDSCs) was removed prior AMG 330 application.However, we did observe an impact of MDSCs on BiTE® mediated T-cell proliferation, which has been shown as highly relevant for clinical activity of BiTE® antibody constructs (Zugmaier et al. Blood 2015). Here we found a 1,68-fold enhanced (p=0.001) T-cell expansion in AML-PBMCs treated with 100 pM AMG 330 following CD14+ cell depletion. Given the importance of T-cell proliferation for clinical activity this finding warrants further exploration.Taken together, our results suggest that anti-CD33/CD3 bispecific BiTE® antibody constructs may achieve anti-leukemic efficacy not only through direct T cell mediated cytotoxicity against AML blasts but also through circumventing immune evasion via MDSCs targeting. Although therapeutic targeting of MDSCs in patients has not yet been successfully accomplished, bystander killing of CD33+ MDSCs via anti-CD33/CD3-bispecific BiTE® antibody constructs could represent a very promising approach to increase the anti-leukemic T-cell response in AML patients and to reverse immune evasion. DisclosuresMougiakakos:AMGEN: Research Funding. Kischel:AMGEN: Employment. Michael:AMGEN: Employment. Dos Santos:Amgen Inc.: Employment. Jitschin:AMGEN: Research Funding. Mackensen:AMGEN: Research Funding.

Generation of Half-Life Extended Anti-BCMA Bite® Antibody Construct Compatible with Once-Weekly Dosing for Treatment of Multiple Myeloma (MM)

Multiple myeloma (MM) is a hematologic malignancy characterized by accumulation of abnormal plasma cells in the bone marrow. Although several treatment options are available, patients frequently develop relapsed/refractory disease leading to significant morbidity and mortality. More effective induction and elimination of residual disease are required for curative treatments. One potential strategy for improving patient outcomes is the use of T cell engaging agents such as BiTE® antibody constructs and CAR-T cells.The bispecific T cell engager (BiTE®) antibody construct blinatumomab, which targets CD19-positive cells, has demonstrated efficacy in treating certain CD19-positive hematological malignancies. Blinatumomab comprises a single chain variable fragment (scFv) that binds CD19 on B cells and a scFv that targets the T cell CD3 protein. The short serum half-life of blinatumomab necessitates administration by continuous IV infusion to maintain therapeutic serum concentrations. To enable once-weekly dosing, next generation BiTE® antibody constructs have been modified by fusion to an Fc domain to extend half-life.BCMA, a member of the TNF receptor super family, supports plasma cell survival, and is widely expressed on MM and normal plasma cells. A half-life extended (HLE) anti-BCMA BiTE® was generated and evaluated in vitro, in mouse xenograft models and in non-human primates (NHP). In vitro assays evaluated 1) binding to both human and NHP BCMA and CD3 proteins, and 2) cytotoxicity using human and NHP target and effector cells. The anti-BCMA BiTE® demonstrated picomolar binding affinity and efficacy in cytotoxicity assays. In a mouse xenograft model in which BCMA-positive NCI-H929 cells and human PBMC were co-administered SC, the anti-BCMA HLE BiTE® completely inhibited tumor formation in contrast to vehicle control-treated groups with or without PBMC. BCMA cell surface protein expression was assessed by flow cytometry and observed on NHP bone marrow plasma cells but not on blood or bone marrow B cells. The serum half-life in NHP was 112 hours following a single 15 µg/kg dose. The PK/PD relationship in NHP was subsequently evaluated in a repeat-dose study. To assess elimination of BCMA-positive plasma cells in blood and bone marrow, a sensitive and specific ddPCR-based method was developed. Robust depletion of BCMA mRNA was observed in NHP following anti-BCMA BiTE® treatment, indicating effective elimination of BCMA-positive plasma cells from blood and bone marrow. These results demonstrate that HLE anti-BCMA BiTE® is effective in vitro and in vivo, and may be suitable for once-weekly dosing in MM patients. DisclosuresGoyos:Amgen: Employment. Li:Amgen, Inc.: Employment. Deegen:Amgen: Employment. Bogner:Amgen: Employment. Thomas:Amgen Research (Munich): Employment. Matthias:Amgen: Employment. Wahl:Amgen Research (Munich): Employment. Goldstein:Amgen: Employment. Coxon:Amgen, Inc.: Employment. Balazs:Amgen Inc.: Employment, Equity Ownership. Chapman-Arvedson:Amgen, Inc.: Employment, Equity Ownership.

Generation of a Half-Life Extended Anti-CD19 BiTE® Antibody Construct Compatible with Once-Weekly Dosing for Treatment of CD19-Positive Malignancies

CD19 is an immunoglobulin family member expressed ubiquitously on B cell lineage cells, from the pre-B cell to the mature B cell stage, until downregulation during terminal differentiation into plasma cells. CD19 is important for B cell activation, maturation and proliferation, and is widely expressed on the majority of B cell neoplasms.The T cell engaging bispecific antibody construct (BiTE®) blinatumomab, which targets CD19, is approved for the treatment of relapsed or refractory B cell precursor acute lymphoblastic leukemia in adults and children in the US and has shown great promise for treating other CD19-positive hematological malignancies. The short serum half-life of blinatumomab necessitates administration by continuous IV infusion to maintain therapeutic serum concentrations. To extend the half-life and allow for a more convenient administration, a next generation BiTE® antibody construct designated CD19 HLE BiTE® has been generated. The CD19 HLE BiTE® comprises a new CD19- and CD3-targeting tandem single-chain Fv antibody fused to an Fc domain.The biological properties of CD19 HLE BiTE® were evaluated in vitro and in vivo . CD19 HLE BiTE® binds with high affinity to both human and non-human primate (NHP) CD19 as well as CD3. In vitro experiments demonstrated that CD19 HLE BiTE® can recruit and activate human and NHP T cells, mediating redirected lysis of CD19-expressing cells at half-maximal effective concentrations in the low picomolar range. The fusion to an Fc domain resulted in a serum half-life of 210 hours following a single intravenous administration of a 5 µg/kg dose in NHP. In a repeat-dose study CD19 HLE BiTE® caused robust depletion of circulating B cells, B lineage bone marrow cells, and peripheral lymphoid organ B cells in NHPs, whilst no overt signs of toxicity were observed in clinical and laboratory examinations of the animals.These results demonstrate that CD19 HLE BiTE® effectively eliminated CD19-positive cells in vitro and in vivo, and may be suitable for once weekly dosing in patients with CD19-positive malignancies. DisclosuresLorenczewski:Amgen: Employment, Equity Ownership. Friedrich:Amgen Research (Munich) GmbH: Employment, Equity Ownership. Kischel:AMGEN: Employment. Dahlhoff:Amgen Research (Munich): Employment, Equity Ownership. Anlahr:Amgen: Employment, Equity Ownership. Balazs:Amgen Inc.: Employment, Equity Ownership. Rock:Amgen, Inc.: Employment, Equity Ownership. Boyle:Amgen: Employment. Goldstein:Amgen: Employment. Coxon:Amgen, Inc.: Employment. Chapman-Arvedson:Amgen, Inc.: Employment, Equity Ownership.

Midostaurin Reduces Bite® Mediated Cytotoxicity Against Acute Myeloid Leukemia

T-cell recruiting antibody constructs represent a promising approach to recruit T-cells to tumor cells independent of TCR-specificity. So far, CD33 has been the most commonly targeted antigen in AML. However, its ubiquitous expression pattern within the healthy myeloid hematopoietic system remains a major challenge. To reduce on-target off-leukemia toxicity, alternative AML-associated targets with a more restricted expression pattern are sought after. We evaluated CD135 (FLT3 ; FSM-like tyrosine kinase 3) as a target antigen for T-cell recruiting antibody based immunotherapy in AML. In 159/192 of AML patient samples (83%; MFI ratio of >1.5) a significant expression level was detected by flow cytometry with no differences in expression level at time of initial diagnosis versus relapse (Initial diagnosis: n=161, median MFI ratio 3.0 vs relapse: n=18, median MFI ratio 3.5). Expression level could neither be correlated to FLT3 mutational status (median MFI ratio FLT3-ITD vs FLT3 wild type (WT): 3.7 vs 4.0, n=52 and 173 respectively) nor to FLT3 allelic ratio (median MFI ratio allelic ratio high vs low: 4.1 vs 4.5, n=13 and 19 respectively). Importantly, CD34+/CD38- leukemia initiating cells (LICs) also expressed CD135 at a similar level compared to AML bulk cells (Median MFI ratio LIC: 2.4 vs bulk: 2.6, p=0.1, n=81). Bone marrow progenitor cells (CD45DIM/SSCLOW) cells and CD34+/CD38- expressing hematopoietic precursors from healthy bone marrow expressed CD135 to a significantly lower level compared to AML bulk cells and LICs, respectively (median MFI Ratio: AML bulk vs healthy progenitor cells: 3.2 vs 2.1, p<0.0001; LICs vs HSCs: 3.1 vs 0.7, p=0.0003). Next, we evaluated a CD135xCD3 BiTE® antibody construct (FLT3 BiTE®) for its ability to mediate cytotoxicity against AML cell lines and primary AML cells. Target antigen expression level was relevant for FLT3 BiTE® mediated cytotoxicity as demonstrated by preferential lysis of CD135bright (MM6, MFI ratio 15) vs CD135dim (OCI-AML3, MFI ratio 1.5) AML cell lines. Cytotoxicity against primary AML cells was tested in our long-term culture system using either allogeneic or autologous T cells (Krupka et al, Blood 2014). In the allogeneic set-up, with a fixed E:T ratio of 1:5, the FLT3 BiTE® antibody construct mediated efficient elimination of primary AML cells within 9 days of culture (median % specific lysis 75.9, n=6). This was accompanied by a strong T-cell proliferation (fold change CD2+ T cells: 7.9, n=6). In the autologous system, the E:T ratio was not fixed and influenced by the number of residual T-cells in the respective AML sample. Despite low E:T ratios (1:12-1:74) the FLT3 BiTE® induced high cytotoxicity and T-cell proliferation against primary AML cells within 14 days of culture (median specific lysis: 34.4%; fold change CD2 T cells: 6.6, n=7). We further investigated the influence of Midostaurin on FLT3 BiTE® mediated cytotoxicity against AML cells. Midostaurin (1 µM) drastically decreased FLT3 BiTE® mediated cytotoxicity in 7/8 patient samples (median % cytotoxicity FLT3 BiTE® vs +Midostaurin: 59.0 vs 39.9, p=0.02). This corresponded to a significant decrease in BiTE®- as well as CD3/CD28-mediated T-cell proliferation (median % CD3/CD28 mediated T-cell proliferation FLT3 BiTE® vs +Midostaurin: 56.4 vs 1.46, n=5). It has been reported that Midostaurin inhibits Akt phosphorylation (Kawai, J Bio Sci 2015), a major factor in T-cell proliferation, which might explain the decrease in T-cell proliferation and cytotoxicity. In summary, CD135 was shown to be expressed in the majority of AML patients on AML bulk cells as well as LICs with a restricted expression pattern in the healthy hematopoietic compartment. The FLT3 BiTE® antibody construct mediated strong cytotoxicity against primary AML cells. This correlated to profound T-cell activation and proliferation. The combinatorial approach with Midostaurin abrogated BiTE® mediated cytotoxicity due to a decrease in T-cell proliferation. As T-cell recruiting antibody construct mediated cytotoxicity relies on T-cell function, our data do not support the combinatorial approach with the FLT3 -TKI Midostaurin for targeted therapy of AML. DisclosuresLindl:AMGEN Inc: Research Funding. Krupka:Amgen, Inc: Research Funding; Amgen Research (Munich): Research Funding. Chapman-Arvedson:Amgen, Inc.: Employment, Equity Ownership. Kischel:AMGEN: Employment. Kufer:AMGEN Research Munich: Employment. Subklewe:Amgen, Inc.: Research Funding; Amgen Research (Munich): Research Funding.

Evaluation of a FLT3 Bite® for Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a disease with high unmet medical need. Standard induction chemotherapy can lead to complete remission in patients with a favorable genetic risk profile; however, about half of patients are ineligible for this intensive regimen. In those patients with a non-favorable genetic risk profile, the preferred treatment strategy is allogeneic stem cell transfer, but the majority of patients are ineligible due to age and other comorbidities.FLT3 is a receptor tyrosine kinase that induces cell growth in myeloid progenitor cells. Activating mutations in FLT3 have been identified in approximately 30% of AML patients. Expression of mutant FLT3 and overexpression of wild type FLT3 are associated with poor prognosis in AML, leading to clinical investigation of targeted FLT3 inhibitors, including midostaurin which was recently approved for patients with newly diagnosed FLT3-mutant AML. These inhibitors are likely to be effective only in certain patient subpopulations and may ultimately give rise to acquired resistance. To drive cures in AML, effective therapies for broad patient populations that can eliminate residual disease are needed.Bispecific T cell engaging antibody constructs (BiTE®) are a class of immunotherapy agents which redirect T lymphocytes to a specific antigen expressed on tumor cells. Blinatumomab, a BiTE® containing a single chain variable fragment (scFv) that binds CD19 on B lymphocytes and an scFv that binds CD3 on T lymphocytes, has shown clinical efficacy for treating CD19-positive hematological malignancies such as ALL. To extend the serum half-life, next generation BiTE® antibody constructs have been modified to contain Fc-moieties, allowing for weekly dosing.To evaluate FLT3 as a BiTE® target in AML, we assessed expression of FLT3 on primary AML patient blasts using flow cytometry. Over 83% of AML patients (n = 192) were FLT3-positive (MFI ratio > 1.5) with similar expression in bulk and leukemic stem cells. A half-life extended FLT3 BiTE® (FLT3 HLE BITE®) was generated and evaluated in vitro as well as in vivo in a mouse xenograft tumor model and non-human primates (NHP). FLT3 HLE BiTE® had subnanomolar binding affinity for human FLT3 and nanomolar binding affinity for human CD3 and NHP FLT3 and CD3. In cytotoxic assays targeting human AML cell lines, FLT3 HLE BiTE® exhibited picomolar efficacy using as effector cells human pan T cells in a 10:1 E:T ratio (range: 2 to 10 pM) or NHP PBMCs in a 5:1 E:T ratio (range: 30 to 80 pM). In mice injected with FLT3-positive MOLM13 cells and human CD3-positive T cells, FLT3 HLE BiTE® induced robust anti-tumor activity and extended survival ≥ 2-fold at all doses (n = 10 per group, p ≤ 0.0015). Following a single 5 µg/kg dose in NHP (n=2), FLT3 HLE BiTE® exhibited a reproducible pharmacokinetic profile with a 66 hour half-life. In this study, we observed hallmarks of BiTE® activation such as induction of IL-6 and MCP-1 and indication of a pharmacodynamic effect through increased soluble FLT3 ligand. Following repeat-dosing with FLT3 HLE BiTE®, digital droplet PCR was used to measure FLT3 transcript in NHP blood and bone marrow. Robust depletion of FLT3+ cells was observed in both NHP blood (≥ 95% reduction) and bone marrow (≥ 68% reduction) at all doses (5 µg/kg, 25 µg/kg, 75 µg/kg, n = 3/group). Taken together, FLT3 HLE BiTE® has potent in vitro and in vivo activity and may provide therapeutic benefit to AML patients. DisclosuresGoldstein:Amgen: Employment. Henn:Amgen Research (Munich) GmbH: Employment, Equity Ownership. Archibeque:Amgen: Employment. Frank:Amgen Inc.: Employment, Equity Ownership. Balazs:Amgen Inc.: Employment, Equity Ownership. Dahlhoff:Amgen Research (Munich): Employment, Equity Ownership. Raum:Amgen Research (Munich): Employment. Li:Amgen, Inc.: Employment. Wahl:Amgen Research (Munich): Employment. Rock:Amgen, Inc.: Employment, Equity Ownership. Thomas:Amgen Research (Munich): Employment. Karbowski:Amgen, Inc.: Employment. Krupka:Amgen Research (Munich): Research Funding; Amgen, Inc: Research Funding. Subklewe:Amgen Research (Munich): Research Funding; Amgen, Inc.: Research Funding. Coxon:Amgen, Inc.: Employment. Chapman-Arvedson:Amgen, Inc.: Employment, Equity Ownership.

The Efficacy and Toxicity of Blinatumomab in Patients with Relapsed/Refractory Acute Lymphoblastic Leukemia: Russian Multicenter Experience

Introduction. The prognosis of patients (pts) with relapsed/refractory acute lymphoblastic leukemia (R/R ALL), especially after allogeneic hematopoietic stem cell transplantation (allo-HSCT), is very poor. Therapeutic options for these pts are limited. Blinatumomab is a bispecific T-cell engager (BiTE®) antibody construct with dual specificity for CD19 and CD3. BiTE® therapy may help to overcome the resistance to chemotherapy (CT) with minimal toxicity, and may be a bridge to allo-HSCT.Patients and Methods. We analyzed data of 52 pts from 7 hematologic centers in Russian Federation with CD19 positive R/R ALL, who received BiTE®. The median age was 21 (range 1-53) years, 18 (35%) pts <18 yrs, 34 (65%) pts ≥18 yrs. The diagnosis was ALL B-I (EGIL) subtype in 15 pts, B-II - in 29, B-III - in 5 pts, B-IV - in 2 pts, and 1 patient had mixed phenotype leukemia (M5 (FAB) and B-ALL). Five (10%) pts had Philadelphia positive (Ph+) ALL. Primary refractory in 1 pts. In 19 pts it was the 1st relapse of ALL, in 17 - 2nd, in 15 - 3rd. In 47 pts it was isolated bone marrow relapse, in 5 pts - combined relapse (bone marrow and extramedullary sites). The bone marrow blast infiltration was <50% in 27 pts, >50% in 25 pts. Disease relapse was revealed after CT in 31 pts. Allo-HSCT after BiTE® therapy was performed 12 (39%) pts (4 - from related, 5 - from unrelated, 3 - from haploidentical donors). Twenty one pts received BiTE® due to relapse after allo-HSCT (6 - from related, 12 - from unrelated, 3 - from haploidentical donors). Six pts received 2nd allo-HSCT after BiTE® therapy. In 10 pts with posttransplant relapse donor lymphocyte infusion (DLI) was used in combination with BiTE®. Every patient received from 1 to 7 cycles (median 2) of BiTE®.Results. Complete remission (CR) was achieved in 31/52 (60%) pts: in 18/31 (58%) pts with ALL relapse after CT, in 13/21 (62%) pts - after allo-HSCT. In 22/31 (71%) pts with CR after BiTE® therapy minimal residual disease was negative. Pts with less than 50% blasts in bone-marrow at baseline experienced substantially higher response rates compared with patients with 50% blasts or higher (70% (19/27) vs 48% (12/25)). Response rates were similar although the number of relapse - 58% (11/19) in 1st relapse, 59% (10/17) in 2ndrelapse and 60% (9/15) in 3drelapse. Pts with posttransplant ALL relapse who received BiTE® in combination with DLI had higher response rate than pts, who received BiTE® as monotherapy: 70% (7/10) vs 54% (6/11) respectively. One-year OS was 50% (95% CI 25-75%). One-year DFS was 42% (95% CI 18-66%). Grade ≥3 hematological toxicity (neutropenia, thrombocytopenia) was observed in 14 (27%) pts, grade ≥3 liver toxicity - in 6 (12%) pts. Ten pts (19%) developed toxic neuropathy during the therapy. Cytokine release syndrome occurred in 5 (9%) pts. One pts after allo-HSCT (but not after DLI) developed grade I aGVHD. One pts after allo-HSCT developed grade II aGVHD after BiTE® in combination with DLI. There were no fatal treatment related toxicity. Nine (29%) pts who responded to BiTE® had relapse, 6 of them bone marrow and 3 extramedullary sites. Nine (29%) pts died: 5 pts - due to R/R ALL, 2 pts due to infection, 1 case of transplant related mortality and 1 pts due to second solid tumor (synovial sarcoma).Conclusion. The treatment of R/R ALL with BiTE® is effective and has acceptable toxicity. We demonstrated high efficacy in therapy of posttransplant ALL relapses, especially when BiTE® was combined with DLI, perhaps, due to additional immunological effect of the transplant. DisclosuresNo relevant conflicts of interest to declare.

Baseline Biomarkers Influencing Clinical Outcomes in Adults with Relapsed/Refractory B-Precursor Acute Lymphoblastic Leukemia (R/R ALL) Treated with Blinatumomab Versus Standard-of-Care Chemotherapy (SOC) from a Randomized Phase 3 Study

Introduction: Blinatumomab, an anti-CD3/CD19 bispecific T-cell engager antibody construct, showed antileukemic activity and significantly longer overall survival (OS) vs SOC in adults with advanced Philadelphia chromosome-negative R/R ALL (median OS 7.7 vs 4.0 months; P=0.01) (N Engl J Med 2017;376:836−847). Here we aimed to identify baseline biomarkers that were prognostic (disease-related) or predictive (treatment-related) of clinical outcomes.Methods: Adults with R/R ALL (refractory to primary induction therapy or salvage therapy, first relapse <1 year, second or later relapse, or relapse after allogeneic hematopoietic cell transplantation [alloHCT]) were randomized to receive either blinatumomab or SOC. Blinatumomab was given by continuous infusion (4 wks on/2 wks off) for up to 5 cycles (9 μg/d on d1‒7 of cycle 1 and 28 μg/d thereafter). Up to 4 additional maintenance cycles (4 wks on/8 wks off) were allowed for up to 1 year. Evaluated biomarkers included leukocytes, monocytes, lymphocytes, granulocytes, platelets, neutrophils, CD45+ CD3+ CD4+ T cells, CD45+ CD3+ CD8+ T cells, CD45+ CD3- CD19+ B cells, CD3- CD16+ CD56+ cells, bone marrow (BM) blasts, CD3+ T cells, CD19+ cells, and ratios of CD3+/CD19+ and CD3+%/BM blast%. A multivariate model was used to select potential biomarkers associated with treatment outcomes. Effect on outcome was determined if either the main effect or interaction term between the biomarker and treatment had a P value ≤0.3. Prognostic and predictive associations of baseline biomarkers with OS, hematologic response (complete remission [CR] or CR with full, partial, or incomplete hematologic recovery [CR/CRh/CRi]) within the first 12 wks, and minimal residual disease (MRD) response were assessed by Cox regression for OS and multivariate logistic regression for hematologic response and MRD response. Predictive biomarkers were identified using interaction tests with treatment group.Results: A total of 405 patients received blinatumomab (n=271) or SOC (n=134). Baseline characteristics including age, prior alloHCT, salvage status, granulocytes, platelets, and lymphocyte subpopulations (CD45+ CD3+ CD8+, CD45+ CD3- CD19+, and CD3+) were generally balanced between treatment arms. Associations of baseline biomarkers with clinical outcomes are presented in the table. Higher platelet counts were associated with decreased risk of death (HR 0.43; 95% CI 0.29-0.65) and increased CR (odds ratio [OR] 4.12; 95% CI 2.04-8.33) and CR/CRh/CRi (HR 1.64; 95% CI 0.90-2.99). Higher granulocyte counts were associated with decreased risk of death (HR 0.75; 95% CI 0.57-0.99), whereas total leukocyte counts were not (HR 1.04; 95% CI 0.70-1.54). Lower BM blast percentage was associated with increased CR (OR 0.90; 95% CI 0.82-0.97) and CR/CRh/CRi (OR 0.87; 95% CI 0.80-0.95) but not OS (HR 1.04; 95% CI 0.98-1.09). Higher CD3+ T cells were associated with increased MRD response (OR 1.75; 95% CI 0.99-3.08). Higher counts of CD45+ CD3- CD19+ cells increased the risk of death for blinatumomab patients (HR 1.19; 95% CI 1.09-1.29) but also was not associated with a survival advantage for SOC patients (HR 1.03; 95% CI 0.93-1.15). Higher CD45+ CD3+ CD8+ T cells were associated with increased CR (OR 1.39; 95% CI 1.18-1.65) and CR/CRh/CRi (OR 1.44; 95% CI 1.22-1.70) in patients treated with blinatumomab but did not correlate with increased hematologic response in SOC patients. Multiple logistic regression modeling for adverse events (AEs) of interest showed that BM blasts and lymphocyte markers (CD3+ and CD45+ CD3+ CD8+) were not associated with grade ≥3 neurologic events, infections, or cytokine release syndrome.Conclusions: Higher platelet and granulocyte counts, lower BM blasts, and higher numbers of CD3+ T cells were prognostic for improved OS, hematologic response, and MRD response, respectively, in patients with R/R ALL who received blinatumomab vs SOC. A higher frequency of CD45+ CD3+ CD8+ cells was predictive of increased hematologic response to blinatumomab, whereas a higher frequency of CD45+ CD3- CD19+ cells was predictive of decreased OS. BM blast percentage and lymphocyte markers were not associated with AEs of interest. Overall, these data demonstrate that responses to blinatumomab can be seen across a variety of subgroups, including those expressing specific baseline biomarkers. Further study is needed to identify subgroups most likely to benefit from blinatumomab. [Display omitted] DisclosuresWei:AbbVie, Celgene, Servier: Research Funding; AbbVie, Celgene, Novartis, Amgen, Servier: Membership on an entity's Board of Directors or advisory committees; AbbVie, Celgene, Novartis, Amgen, Servier: Honoraria. Ribera:Janssen: Honoraria; Gilead: Honoraria; Celgene: Honoraria; Pfizer: Research Funding, Speakers Bureau; Amgen Inc.: Research Funding, Speakers Bureau; Incyte: Research Funding, Speakers Bureau; ARIAD: Research Funding, Speakers Bureau; Roche: Honoraria. Larson:Daiichi Sankyo: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen Inc.: Research Funding. Ritchie:Amgen Inc.: Honoraria. Chen:Amgen Inc.: Employment, Other: Stock holder. Dos Santos:Amgen Inc.: Employment. Zimmerman:Amgen Inc.: Employment, Equity Ownership. Kantarjian:Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding; Amgen: Research Funding; Pfizer: Research Funding; Delta-Fly Pharma: Research Funding; ARIAD: Research Funding.

Changes in Leukemic Blasts CD19 Expression in Children with Relapsed/Refractory B-Cell Precursor ALL Treated with Blinatumomab

Blinatumomab is a bispecific T-cell engager (BiTE) antibody construct targeting CD3 and CD19, resulting in T-cell-mediated lysis of malignant B cells. Currently the main application of blinatumomab is the treatment of relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Although promising results were obtained therapy with blinatumomab fails in 30-60% of patients. CD19 downexpression is shown to be one of the possible mechanisms of tumor cells adaptation and subsequent escape from treatment. Moreover, loss of CD19 is a serious obstacle for flow cytometric minimal residual disease (MRD) assessment, necessary for control the leukemia response to therapy. The aim of the present study was to evaluate changes of CD19 expression in children with BCP-ALL treated with blinatumomab and possible monitoring of MRD using additional markers.Patients and methods. From December 2015 to July 201739 (18 F/21M) patients with median age of 8 years (8 mo-20 y) with relapsed/refractory (R/R) BCP-ALL completed at least one course of blinatumomab. 5 patients (13%) carried various types of KMT2A-gene rearrangements and in 3 (7,5%) children ETV6-RUNX1 fusion transcript was detected. MRD monitoring was performed by 8-color flow cytometry. After CD19-directed treatment CD22 and CD24 were added to conventional antibodies' combinations for MRD detection (as suggested by Cherian S. et al, Cytometry B. 2016) to avoid negative influence of potential CD19 loss on cytometric data analysis and interpretation. MRD-negativity was defined as undetectable at the sensitivity level of 0.01%.Results. 20 out of 39(51%) patients continue in complete continuous remission(CCR) after one or two blinatumomab courses, followed by alloHSCT.In 8 (40%) patients in CCR, MRD was never detected since completion of first blinatumomab course, and they were excluded from further analysis. In other 12 pts were found to be MRD-positive at least once after blinatumomab administration. Among them in 5 patients leukemic blasts were still CD19-positive and in 7 patients residual leukemic cells were CD19-negative.Among 19 out of 39 patients (49%) who were resistant to(n=7) or developed bone marrow relapse after blinatumomab (n=12), in 12 (63%) cases tumor cells at relapse were CD19-positive, whereas 5 children experienced CD19-negative relapse. Among them only CD19-positive MRD was detected in one case, only CD19-negative residual blasts - in two cases and in other two children both types of CD19 expression were found atdifferent time-points.Two additional patients, who had CD19-negative MRD, then developed relapse as a lineage “switch” from BCP-ALL to acute myeloid leukemia (AML). In these patients myeloid switch developed in a different ways: one patient carried KMT2A-AF4 fusion gene which is more or less typical for switching AL, the second patient with germline KMT2A hadvery small AML population at the time of relapse diagnosis. Thus in this case positive selection of myeloid subclone during the blinatumomab treatment could be the main pathway of AML development.Conclusions. In our group the remission rate after blinatumomab treatment was about 50%. In those children, who didn't respond and/or developed subsequent relapse, CD19 downexpression seems to be one of the significant pathways for tumor escape from blinatumomab - 26% of relapses were CD19-negative, and in 42% of all relapsed/resistant casesCD19-negative blasts were detected at least on MRD level. Rather frequent loss of CD19 could be a serious obstacle for flow cytometric MRD monitoring, but application of expanded antibodies panel and use of 10-12-color investigation allows residual blasts detection in nearly all cases. Other mechanisms of leukemia resistance including myeloid switch (even in KMT2A-germline patients) can cause blinatumomab failure. DisclosuresNo relevant conflicts of interest to declare.

Blinatumomab Treatment in Korean Adult Patients with Relapse/Refractory Acute Lymphoblastic Leukemia

Blinatumomab is a bispecific T- cell engaging antibody construct targeting CD19 on B-cell lymphoblast and has demonstrated the efficacy for relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL). In this study, we evaluated the efficacy and toxicity of blinatumomab monotherapy in Korean patients with relapse/refractory ALL. A total of 40 patients received blinatumomab monotherapy between July 2016 and July 2017 in Korea. Median time to blinatumomab treatment from initial diagnosis was 13.6 months (range 1.3 - 104.7). Twenty-four patients completed induction therapy (2 cycles of blinatumomab), 15 ended in cycle 1 due to disease progression or toxicity, and 1 died in 1 cycle due to infection. Among 39 evaluable patients, 17 patients (42.5%) achieved complete response (CR) or CR with incomplete blood count recovery, and 10 responders underwent subsequent allogenic stem cell transplantation. Nine patients had extramedullary disease at relapse and 3 (33.3%) achieved CR. Median progression free survival (PFS) and overall survival (OS) were 2.8 months and 5.7 months, respectively. Patients responded to blinatumomab had significantly better PFS and OS compare to those who did not. In multivariate analysis, patients with 50% or more of bone marrow blast at relapse and relapse within 12 months after initial diagnosis were significantly associated with poor response to blinatumomab. The most common non-hematologic toxicity was neurologic toxicity. Thirteen patients (32.5%) had neurologic events and severe neurologic events (grade 3-4) occurred in two patients in 2nd cycle of blinatumomab. Severe cytokine release syndrome (grade 3) occurred in 2 patients (5.0%) despite pretreatment with dexamethasone. In conclusion, blinatumomab monotherapy was effective and tolerable in Korean adult patients with relapse/refractory ALL. DisclosuresNo relevant conflicts of interest to declare.

A Phase 1 Study Investigating the Combination of AFM13 and the Monoclonal Anti-PD-1 Antibody Pembrolizumab in Patients with Relapsed/Refractory Hodgkin Lymphoma after Brentuximab Vedotin Failure: Data from the Dose Escalation Part of the Study

BackgroundAFM13 is a bispecific, tetravalent NK cell-engaging antibody construct binding to CD30 on Hodgkin Lymphoma (HL) cells and CD16A on NK cells. By engaging CD16A-positive NK cells, AFM13 leads to NK-cell mediated killing of CD30-positive lymphoma cells (Reusch et al., 2014). Pembrolizumab is a PD-1 blocking antibody that prevents tumor immune evasion and has shown to induce high single-agent response rates in patients (pts) with relapsed or refractory (R/R) HL (Armand et al., 2016). AFM13 has shown first signs of clinical activity in R/R HL as single agent in a preceding Phase 1 study (Rothe et al., 2015). Preclinical in vivo data of the combination of AFM13 with PD-1 inhibition suggest synergistic activity and the potential for induction of cross-talk between innate and adaptive immunity (Zhao et al., 2016). The combination of the two agents could improve outcomes in pts with R/R HL.MethodsA Phase 1b study is ongoing to evaluate the safety and tolerability of the combination of AFM13 with pembrolizumab (Keytruda) as salvage therapy after failure of standard therapies including brentuximab vedotin in HL (NCT02665650). Pts receive escalating doses of AFM13 (Table 1) in combination with pembrolizumab at a flat dose of 200 mg administered every 3 weeks following the classical 3+3 design. Upon completion, recruitment continues into an extension cohort. Response assessment is performed every 12 weeks by PET/CT according to the Lugano Classification Revised Staging System (Cheson 2014).ResultsAs of July 24th 2017, 12 pts have been enrolled into the dose escalation part of the study. All pts have relapsed disease and have failed standard treatments including brentuximab vedotin. 66 % (8/12) have failed prior autologous stem cell transplantation.At the time of the data extract, all 12 pts had completed the 6-week dose-limiting toxicity (DLT) observation period. 3 pts were enrolled into cohorts 1 and 2 each and 6 patients were enrolled into cohort 3. While no grade 3 or 4 adverse events related to the study treatment were observed, one DLT per protocol definition was observed in cohort 3, which was a repeated grade 2 infusion-related reaction (IRR), leading to discontinuation of AFM13 treatment. No further DLTs occurred. The most frequent adverse events (AEs) were IRRs (9 pts), headache (4 pts), rash (4 pts), nausea (4 pts) and cough (4 pts). All these events were of grade 1 or 2. All observed IRRs were related to the AFM13 administration. Premedication with antihistamine and steroid was not mandatory during dose escalation. Only one grade 3 AE, a duodenal ulcer, was observed, which was assessed as not related to the study treatment. Thus the extension cohort will continue with the highest dose explored during dose escalation.3-month response data from the 12 patients enrolled into cohorts 1, 2 and 3 will be reported at the ASH 2017 annual meeting.ConclusionsEarly data suggest that the combination of AFM13 and pembrolizumab is a well-tolerated salvage therapy in pts with R/R HL. While IRRs were observed frequently, all of these events were of mild or moderate severity and only one event led to discontinuation of treatment in one patient. Preliminary antitumor activity will be further analyzed in the currently recruiting extension cohort. [Display omitted] DisclosuresAnsell:Merck: Research Funding; Bristol-Myers Squibb: Research Funding; Celldex: Research Funding; Seattle Genetics: Research Funding; Affimed: Research Funding. Chen:Merck: Consultancy, Speakers Bureau; Genentech: Speakers Bureau; Seattle Genetics: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Affimed: Research Funding; Pharmacyclics: Consultancy, Research Funding. Forero-Torres:Juno: Research Funding; 47 Pharmaceutical: Research Funding; Affimed: Research Funding; Pfizer: Research Funding; Seattle Genetics: Consultancy, Research Funding, Speakers Bureau; Genentech/Roche: Research Funding; GSK: Research Funding; Novartis: Research Funding; Incyte: Research Funding. Armand:Tensha: Research Funding; Affimed: Research Funding; Roche: Research Funding; Otsuka: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Infinity: Consultancy; Merck & Co., Inc.: Consultancy, Research Funding; Genmab: Consultancy; Sequenta/Adaptive: Research Funding; Sigma Tau: Research Funding. Lossos:Affimed: Research Funding. Reeder:Novartis: Research Funding; Celgene: Research Funding; Bristol-Myers Squibb: Research Funding; Millennium: Research Funding; Affimed: Research Funding. Strassz:Affimed: Employment. Kerber:Affimed: Employment. Bartlett:Celgene: Research Funding; Seattle Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck & Co: Research Funding; Bristol-Meyers Squibb: Research Funding; Immune Design: Research Funding; Forty Seven: Research Funding; Affimed: Research Funding; Janssen: Research Funding; Pharmacyclics: Research Funding; Millenium: Research Funding; Astra Zeneca: Research Funding; ImaginAB: Research Funding; Novartis: Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Blinatumomab in B-Cell Acute Lymphoblastic Leukemia with Positive Minimal Residual Disease: Initial Results of a Phase II Trial

Blinatumomab in B-Cell Acute Lymphoblastic Leukemia with Positive Minimal Residual Disease: Initial Results of a Phase II Trial

Maintenance Therapy with Blinatumomab in Adults with Relapsed/Refractory B-Precursor Acute Lymphoblastic Leukemia (ALL): Overall Survival in Adults Enrolled in a Phase 3 Open-Label Trial

IntroductionHematopoietic stem cell transplant (HSCT) after induction/consolidation therapy with standard chemotherapy or targeted therapies, such as blinatumomab or inotuzumab (Kantarjian H, et al. N Engl J Med . 2016;375:740-53), is considered the only treatment option with curative intent for patients with relapsed/refractory Philadelphia chromosome-negative B-cell precursor acute lymphoblastic leukemia (r/r Ph- B-precursor ALL). A recent randomized phase 3 study of blinatumomab, a bispecific T-cell engager antibody construct, in patients with r/r Ph- B-precursor ALL, included a maintenance treatment phase to allow patients in hematologic remission to continue blinatumomab (Kantarjian H, et al. N Engl J Med . 2017;376:836-847). In this exploratory analysis, we examined overall survival (OS) for patients who received blinatumomab maintenance therapy and compared outcomes with data pooled from patients who did not receive maintenance therapy in the phase 3 and phase 2 (Topp MS, et al. Lancet Oncol . 2015;16:57-66) blinatumomab trials.MethodsAdult patients (≥18 years) in this phase 3 trial were randomized to either blinatumomab or standard of care (SOC) chemotherapy. Patients randomized to blinatumomab received this treatment by continuous intravenous infusion (4 weeks on, 2 weeks off; 9 μg/day on days 1-7 of Cycle 1 and 28 μg/day thereafter) during induction and consolidation cycles. Patients could receive HSCT at any time after cycle 1. Patients who received blinatumomab, and had bone marrow blasts <5% after two induction and three consolidation cycles of blinatumomab, were eligible for blinatumomab maintenance, administered for up to an additional 12 months (4 weeks on therapy, 8 weeks off). Maintenance therapy was discontinued in case of transition to HSCT, investigator discretion, toxicity, relapse, or use of protocol-excluded medications. The cumulative probability of OS over time was estimated using the Simon-Makuch method and the effect of maintenance was analyzed using the Mantel-Byar method. OS was measured from the start of maintenance therapy for patients who were in remission until the start of Cycle 6 (for those who received maintenance) or at Day 211 (landmark date for those who did not receive maintenance). Data from the phase 3 study and an earlier phase 2 study in which maintenance was not offered were pooled to increase the sample size for this OS analysis.ResultsAs of the primary analysis cutoff, 119 of 271 (43.9%) patients receiving blinatumomab achieved a best response of CR/CRh/CRi within two treatment cycles, and 27 patients continued to the blinatumomab maintenance phase. Three patients completed the maintenance phase, 4 transitioned to HSCT, and one discontinued due to adverse events (AE). Twenty-one (77.8%) patients had a best response of CR during maintenance, 1 (3.7%) patient each had CRh or blast free hypoplastic or aplastic bone marrow, and 2 (7.4%) patients each had hematologic relapse or were not evaluable. The Mantel-Byar analysis demonstrated a relative OR for OS of 0.59 (95% CI, 0.20 - 1.73) P = 0.33 comparing maintenance vs. no maintenance (Figure), representing a 41% reduction in the risk of death associated with maintenance therapy, though not statistically significant. Among patients receiving blinatumomab who entered the maintenance phase, the incidence of adverse events of interest generally was less compared with the incidence during the induction or consolidation phases (Table).ConclusionsIn r/r ALL patients who achieved a hematologic response following blinatumomab induction/consolidation, high response rates with blinatumomab maintenance therapy (beyond cycle 5) were observed, with longer OS compared with patients receiving no maintenance. During maintenance with blinatumomab, no new safety concerns were identified. A lower incidence of adverse events of interest was seen during the maintenance phase, compared with events recorded during the induction/consolidation phases of this, and prior blinatumomab studies.Research supported by Amgen Inc. [Display omitted] DisclosuresRambaldi:Novartis, Roche/Genentech, Amgen, Italfarmaco: Consultancy; Novartis, Amgen, Celgene, Sanofi: Other: Travel, Accomodations, Expenses. Rigal-Huguet:Bristol Myers-Squibb, Novartis, Pfizer, Amgen, Incyte, Jazz Pharma: Consultancy; Bristol Myers-Squibb, Novartis, Pfizer, Amgen, Incyte, Jazz Pharma: Honoraria. Nie:Amgen: Employment, Equity Ownership. Zimmerman:Amgen Inc.: Employment, Equity Ownership. Topp:Amgen: Consultancy, Honoraria, Other: Travel, Research Funding; Regeneron: Consultancy, Honoraria, Research Funding; Celgene: Other: Travel; Roche: Consultancy, Research Funding; Macrogenics: Consultancy, Research Funding.

Novel Bispecific Antibody Targeting CD45 and 90Y-DOTA Is Effective Therapy for Acute Myeloid Leukemia in Preclinical Murine Models

Background : Acute myeloid leukemia (AML) is a heterogeneous disease with a variety of chromosomal and molecular aberrations, rendering AML a challenging disease to cure with targeted agents. Despite the genetic variability, AML uniformly responds to radiation therapy. We have previously shown therapeutic efficacy targeting CD45+ AML using the beta-emitter 90Y and a streptavidin-biotin (SA) pretargeted radioimmunotherapy (PRIT) system. Theoretical concerns, such as endogenous biotin in humans, may limit translation of this approach to the clinic. To circumvent such hurdles, we developed bispecific antibody constructs targeting both CD45 and 90Y-labeled-DOTA-biotin, and evaluated this approach in murine models of AML.Methods : We constructed a pair of plasmids using the pfuse system that code for heavy and light chains for antibody bispecific to murine CD45 (30F11) and Y-DOTA (C825). Plasmid pairs were co-transfected into HEK 293T cells for production and purificaiton of the bispecific antibody. Binding to CD45 and 90Y-labeled-DOTA-biotin was confirmed, after which biodistribution studies with 30F11-Fc-C825 were performed in a disseminated murine leukemia model. B6SJLF1 mice (n=5 per group) were injected intravenously with 105 SJL murine leukemia cells, followed 2 days later with 0.67 nmol 30F11-Fc-C825, then 5 μg DOTA-Y-dextran clearing agent 23 hr later to clear any unbound bispecific construct from circulation. Mice were then injected with 50 µCi 90Y-DOTA-biotin IP 24 hr after the bispecific antibody infusion. Tissues were harvested at 6 and 24 hr after radiolabeled DOTA-biotin, and then analyzed for radioactivity to calculate the percent injected dose per gram of tissue (% ID/g).Results : Target organs (spleen and bone marrow) from leukemia bearing mice treated with 30F11-Fc-C825 had 9.0 ± 1.5 and 8.1 ± 1.2% ID/g, respectively, 24 hr post injection. Non-target organs such as kidneys and lungs each had <0.5% ID/g at this time point. Targeting translated into a therapeutic benefit, as mice bearing SJL leukemia treated with 30F11-Fc-C825 and 800 µCi of 90Y-DOTA-biotin had median overall survival of 43 days, compared to leukemic control mice (30 days, p<0.0001).To investigate this approach in a clinically relevant human leukemia model, an anti-human CD45 (BC8) scFv fusion protein was similarly engineered onto a high-affinity anti-Y-DOTA (C825) scFv fusion protein. This bispecific construct (BC8-Fc-C825) was expressed and purified from CHO cells. Binding to human CD45 and 90Y-labeled-DOTA-biotin was confirmed by flow cytometry. In biodistribution studies, female athymic mice (n=5 per group) were injected subcutaneously with 107 human AML cells (HEL). Mice were then injected intravenously with 1.4 nmol BC8-Fc-C825, BC8-SA, or non-targeting CC49-Fc-C825 as the first step before receiving clearing agent. Radiolabeled 90Y-DOTA-biotin (50 µCi) was injected IP 24 hr after bispecific delivery. HEL tumors from mice treated with BC8-Fc-C825 showed peak uptake at 24 hr post injection with 7.75 ± 0.02% ID/g, with minimal uptake in non-target organs [e.g., 0.5 ± 0.3% ID/g in kidneys]. This compared favorably with HEL tumors from mice treated with BC8-SA as the first step, which had peak uptake of 6.7 ± 0.01% ID/g 24 hr post-injection. Therapy studies took athymic mice (n=10 per group) that had been given subcutaneous HEL xenografts, before receiving BC8-Fc-C825, BC8-SA, or non-binding control CC49-Fc-C825. Treated mice were given clearing agent, followed by 800 to 1500 µCi of 90Y-labeled DOTA-biotin. Mice bearing HEL tumors treated with BC8-Fc-C825 followed by 1400 µCi of 90Y-DOTA-biotin, resulted in 6 of 10 mice surviving 170 days post-injection, whereas untreated controls and HEL-bearing mice treated with non-targeting negative control CC49-Fc-C825 and 1400 µCi of 90Y-DOTA-biotin required euthanasia due to tumor size by day 26 and 32, respectively.Conclusion : Our bispecific constructs both showed selectivity for CD45+ targets and 90Y-DOTA. In two leukemia models, targeting of 90Y-DOTA to target hematopoietic tissues translated into survival benefits. Given the therapeutic efficacy, bispecific constructs with the capacity to pretarget CD45 and capture 90Y-DOTA should be added to the armamentarium of PRIT approaches. [Display omitted] DisclosuresOrozco:Actinium Pharmaceuticals: Other: Research Funding to Institution for sponsored Clinical Trials. Till:Mustang Bio: Patents & Royalties, Research Funding; Genentech: Research Funding. Gopal:Seattle Genetics: Consultancy, Research Funding. Orcutt:inviCRO: Employment, Equity Ownership. Pagel:Actinium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Press:Roche: Honoraria, Research Funding; BMS: Honoraria; Bayer: Consultancy.

Single-chain variable fragment antibody constructs neutralize measles virus infection in vitro and in vivo.

Single-chain variable fragment antibody constructs neutralize measles virus infection in vitro and in vivo.

Improving the yield of recalcitrant Nanobodies® by simple modifications to the standard protocol

Nanobodies are single-domain antibody constructs derived from the variable regions of heavy chain only (VHH) camelid IgGs. Their small size and single gene format make them amenable to various molecular biology applications that require a protein affinity-based approach. These features, in addition to their high solubility, allows their periplasmic expression, extraction and purification in E. coli systems with relative ease, using standardized protocols. However, some Nanobodies are recalcitrant to periplasmic expression, extraction and purification within E. coli systems. To improve their expression would require either a change in the expression host, vector or an increased scale of expression, all of which entail an increase in the complexity of their expression, and production cost. However, as shown here, specific changes in the existing standard E. coli culture protocol, aimed at reducing breakdown of selective antibiotic pressure, increasing the initial culture inoculum and improving transport to the periplasmic space, rescued the expression of several such refractory Nanobodies. The periplasmic extraction protocol was also changed to ensure efficient osmolysis, prevent both protein degradation and prevent downstream chelation of Ni2+ ions during IMAC purification. Adoption of this protocol will lead to an improvement of the expression of Nanobodies in general, and specifically, those that are recalcitrant.