Bispecific T-cell Engager Antibody Research Articles

Single-Cell Analyses Identify Brain Mural Cells Expressing CD19 as Potential Off-Tumor Targets for CAR-T Immunotherapies

CD19-directed immunotherapies are clinically effective for treating B cell malignancies but also cause a high incidence of neurotoxicity. A subset of patients treated with chimeric antigen receptor (CAR) Tcells or bispecific Tcell engager (BiTE) antibodies display severe neurotoxicity, including fatal cerebral edema associated with Tcell infiltration into the brain. Here, we report that mural cells, which surround the endothelium and are critical for blood-brain-barrier integrity, express CD19. We identify CD19 expression in brain mural cells using single-cell RNA sequencing data and confirm perivascular staining at the protein level. CD19 expression in the brain begins early in development alongside the emergence of mural cell lineages and persists throughout adulthood across brain regions. Mouse mural cells demonstrate lower levels of Cd19 expression, suggesting limitations in preclinical animal models of neurotoxicity. These data suggest an on-target mechanism for neurotoxicity in CD19-directed therapies and highlight the utility of human single-cell atlases for designing immunotherapies.

Characterization of relapse patterns in patients with acute lymphoblastic leukemia treated with blinatumomab.

Blinatumomab is a CD19/CD3 bispecific T-cell engager (BiTE) antibody that simultaneously binds CD19 on the surface of B-cells and CD3 on the surface of T-cells, resulting in tumor cell lysis. It is approved for the treatment of patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL) and in patients with minimal residual disease after intensive induction chemotherapy. Relapse patterns after treatment with blinatumomab have not been well characterized. We reviewed patients treated with blinatumomab with relapsed, refractory or minimal residual disease-positive B-ALL from 1 December 2014 to 31 December 2018 at a single academic medical center. Patient demographics, blast percentage prior to blinatumomab initiation, prior lines of therapy, blinatumomab treatment duration, sites of relapse, progression free survival, and overall survival were collected. A total of 20 patients were identified. Four (20%) patients developed extramedullary relapse following blinatumomab. The median time from treatment initiation to extramedullary relapse was 179 days (range 47-241). Sites of extramedullary relapse included the pancreas, adrenal gland, kidneys, liver, parotid gland, and brain. Extramedullary relapse occurs frequently following treatment of B-ALL with blinatumomab. Further studies aimed at preventing extramedullary relapse following blinatumomab treatment are warranted.

Frontline Blinatumomab in Older Adults with Philadelphia Chromosome-Negative B-Cell Acute Lymphoblastic Leukemia.

Outcomes of acute lymphoblastic leukemia (ALL) in older adults treated with chemotherapy are poor. The CD19/CD3 bispecific T-cell engager (BiTE) antibody blinatumomab is approved for refractory, relapsed or minimal/measurable residual disease (MRD)-positive B-cell ALL, but there is little experience in the upfront setting, including in older patients. We retrospectively analyzed outcomes of blinatumomab monotherapy in five newly diagnosed Philadelphia chromosome-negative B-cell ALL patients over 70 years. Three had cytokine release syndrome, treated with dexamethasone and/or tocilizumab, and four patients had neurotoxicity, treated with dexamethasone, without blinatumomab interruption. All five achieved complete remission (CR) after cycle one, three with undetectable MRD. All five were alive at 8 to 15 months. Three remained in MRD-negative CR. Two relapsed after cycle 3, one with extramedullary disease. In our small cohort of patients over 70 years, blinatumomab was safe initial therapy and produced a high response rate.

Abstract PR12: Dynamic single-cell imaging of human cancer growth and therapy responses following engraftment into immunodeficient zebrafish

Abstract Cancer xenograft engraftment studies using immune-deficient mice are indispensable for preclinical drug discovery and are required for IND filings that lead to clinical trials. While immune-deficient mice robustly engraft a wide variety of human cancers, high-resolution intravital imaging of transplanted cells is tedious and its high husbandry costs often limit the scale of experiments. In contrast, zebrafish are an ideal cell transplantation model. They are highly fecund; optically clear, permitting dynamic single-cell imaging of fluorescent cancer cells; and are an excellent platform for high-throughput, large-scale studies. We have recently generated two optically clear prkdc-/-, il2rga-/- and rag2-/-, il2rga-/- immune-compromised zebrafish models that lack T-, B-, and NK-cells. These immune-deficient animals can be grown at 37°C and robustly engraft a variety of human cancers including patient-derived xenografts for >30 days. Importantly, tumors grown in immune-deficient zebrafish and mice are indistinguishable in terms of morphology, proliferation rates, apoptosis, and response to therapy. Engraftment of human cells into the superficial periocular muscle also allowed high-resolution, single-cell imaging using conventional confocal microscopy. Using this approach, we performed photoconversion cell lineage tracing of human rhabdomyosarcoma (RMS), a pediatric cancer of the muscle, and identified mutually exclusive migratory and proliferative cell states. We also demonstrated the preclinical efficacy of combination therapy involving olaparib (PARP-inhibitor) and temozolomide (DNA-damaging agent), including the dynamic visualization of therapeutic responses at single-cell resolution through use of the four-color FUCCI cell cycle fluorescent reporter and serial confocal imaging over days. Importantly, this same drug combination also exhibited remarkable efficacy in studies performed in NSG mice and is now moving forward for clinical evaluation in RMS patients. Finally, recent work has focused on assessing the efficacy of immunotherapies in curbing tumor growth, including assessing CAR-T cell and bispecific T-cell engager antibodies (BITES) in vivo. In both of these platforms, dynamic live cell imaging and automated 3D modeling allowed quantification of migratory potential of T cells into the tumor, dynamic remodeling of T-cell morphology changes following interaction with tumor cells, and real-time visualization of T cell-mediated cancer cell killing using fluorescent caspase reporters. In total, our studies have credentialed the immune-deficient zebrafish as a new platform for preclinical drug studies and provides novel technologies that utilize real-time, single-cell imaging for early endpoint analysis. These models are likely to be particularly useful for discovery of immunomodulatory antibodies and immunomedicines in the future. Citation Format: Chuan Yan, Qiqi Yang, Daniel Do, Dalton Brunson, John Iafrate, John Rawls, David M. Langenau. Dynamic single-cell imaging of human cancer growth and therapy responses following engraftment into immunodeficient zebrafish [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR12.

Characterization of clinical pharmacokinetics and exposure-response relationships of AMG 330, a bispecific CD33 T-cell engager antibody construct, in patients with relapsed/refractory AML.

7536 Background: AMG 330 binds both CD33 and CD3 and redirects T cells toward CD33+ cells leading to T-cell‒mediated cytotoxicity against AML blasts. An ongoing open label phase I dose-escalation study (NCT02520427) has shown preliminary activity and acceptable safety in relapsed or refractory (R/R) acute myeloid leukemia (AML) patients (pts) (Ravandi et al. ASH 2018). Pharmacokinetics and exposure-response (E-R) relationships of AMG 330 were characterized in this trial. Methods: A continuous IV infusion of AMG 330 was evaluated at escalating target doses (range from 0.5 to 720 μg/day) using a 3+3 design with pts receiving step dose/s prior to reaching target doses of ≥ 30 μg/day. Population pharmacokinetics (popPK) using non-linear mixed effects modeling and E-R analyses were conducted to characterize relationships between AMG 330 exposure (steady-state concentration [Css]) at target dose, the baseline tumor burden, clinical response per revised IWG criteria and incidence of cytokine release syndrome (CRS). Results: As of Dec 10, 2019, 55 patients (males, 56.4%; median age, 58.0 [18.0–80.0] years) were enrolled in 16 cohorts. AMG 330 PK was best described by a one-compartment linear PK model. Dose dependent increases were observed in AMG 330 Css exposures. Responders typically showed higher AMG 330 Css than non-responders. Preliminary exploratory analysis indicated that higher AMG 330 exposures, lower baseline leukemic burden in bone marrow and CD33+ AML cells in peripheral blood, and higher baseline Effector:Target cell ratio may be associated with clinical response. Additionally, a positive relationship was observed for AMG 330 exposures and baseline leukemic burden (p < 0.05) with probability of CRS occurrence and severity. Based on the model, at a baseline leukemic burden of 20%, a 240 µg/day target dose is predicted to result in a 28% and 4% probability of developing CRS of grade ≥ 2 and ≥ 3, respectively. Conclusions: Clinical pharmacokinetic profile and E-R relationships of AMG 330 were characterized to identify optimal AMG 330 dosing regimens that minimize the risk for CRS in ongoing and planned clinical investigations. Clinical trial information: NCT02520427 .

Characterizing the mechanism of action of a HER2xCD3 bispecific T-cell engager (BiTE) antibody in 2D and 3D immune cell killing assays using a combined live-cell imaging and flow cytometry workflow

Abstract Solid cancers have proved to be a challenge for immunotherapies and targeted approaches are needed. Evaluation of these therapies using relevant models plays a large part in their development. A workflow that combines live-cell imaging and flow cytometry was used to characterize immune cell killing induced by a BiTE antibody. Immune cell co-culture assays were set up in 2D or 3D formats with green nuclear labeled AU565 HER2 positive breast cancer cells and various effector cell types. Immune cell activation was induced using HER2xCD3 BiTE or CD3/CD28 Dynabeads. Images were captured every 4 h with IncuCyte and quantified for numerous cellular parameters. Cytokine analysis of supernatant samples was performed using a multiplex QBead panel alongside assessment of immune cell activation markers using the iQue3. Quantification of PBMC mediated cytotoxicity showed a comparable 70 ± 2% and 62 ± 1% reduction in spheroid growth with BiTE (10 ng mL−1) and Dynabead (1:1 bead-to-cell ratio) activation. In contrast to Dynabeads, BiTE activation did not induce immune cell shape change or upregulation of T cell activation markers; 1 ± 0.2% CD69 expression was observed on CD3+CD8+ cells, compared to 53 ± 0.6%. Further evaluation found BiTE activated isolated CD56+ cells induced less target cell death compared to PBMCs and granzyme B secretion was not detected, suggesting a lack of involvement of NK cells. Release of IL-6 and CCL2 cytokines indicate immune cell mediated cytotoxicity via an inflammatory pathway. These data suggest HER2xCD3 BiTE activity may not be mediated through T cell or NK cell subtypes. At present the mechanism of target cell killing is unclear, however further studies combining imaging and flow cytometry may provide the required insight.

PCN169 COST BY OUTCOMES ANALYSIS OF BLINATUMOMAB AND INOTUZUMAB OZOGAMICIN FOR ACUTE LYMPHOBLASTIC LEUKEMIA FROM THE BRAZILIAN PRIVATE HEALTHCARE PERSPECTIVE

To estimate treatment costs of novel therapies blinatumomab and inotuzumab ozogamicin in the treatment of adult patients with relapsed or refractory B-cell precursor acute lymphoblastic leukemia (ALL) from the Brazilian private healthcare perspective. Treatment costs associated with blinatumomab, a bispecific T-cell engager (BiTE®) antibody construct, and inotuzumab ozogamicin, a CD22 monoclonal antibody were compared. For each therapy, the median number of treatment cycles from its pivotal phase III clinical trial was used as treatment duration. Treatment costs were estimated for the modeled treatment duration considering therapy-specific dosing schedules and mean body surface area for Brazilian adult men/ women. Drug prices were based on December 2019 Brazilian list prices with 18% state tax (PF 18%). No vial sharing was assumed. Total treatment costs, cost per median overall survival (OS) and cost per complete response (CR) were compared. Total treatment costs ranged from 585,601 to 1,054,081 BRL for inotuzumab ozogamicin depending on the mean body surface area (BSA) and treatment response; for blinatumomab the treatment cost presented up to 47% lower than inotuzumab ozogamicin (561,294 BRL) and the treatment cost didn’t vary for blinatumomab once for adult patients with weight greater than or equal to 45kg is considered a fixed-dose. In terms of cost per median OS blinatumomab presented up to 56% lower than inotuzumab ozogamicin (60,354 BRL versus 80,037-136,894 BRL). Similarly, the cost per achieved CR was lower for blinatumomab (1,521,123 BRL) than that for inotuzumab ozogamicin (1,839,660 – 3,146,510 BRL). Results of the present analysis indicate that blinatumomab might be associated with considerably lower treatment costs. When cost analysis included efficacy data, blinatumomab presented lower cost per median OS and cost per CR than inotuzumab ozogamicin from the Brazilian private healthcare perspective.

Bispecific anti-CD22 T-cell engager efficiently promotes cell-mediated cytotoxicity of lymphoma, leukemia and sarcoma targets

Abstract Immunotherapy has revolutionized cancer therapy. Two of the most recently FDA-approved immunotherapies for B-cell malignancies target CD19, in the form of a Bispecific T-Cell Engager (BiTE) antibody construct or CAR-T cells. Blinatumomab, and FDA-approved BiTE, binds to CD19 on B cells and to CD3 on T cells, mediating killer-target cell contact and T cell activation that results in effective elimination of B cells. Although CD19 is expressed by essentially all cases of B cell malignancies at clinical presentation, relapses with loss or reduction of CD19 surface expression are increasingly recognized as a cause of treatment failure. We have developed a novel BiTE consisting of humanized anti-CD22 and anti-CD3 single chain variable fragments, expressed as a single molecule and purified from tissue culture supernatant. Binding of the anti-CD22 and anti-CD3 moieties to B and T cells, respectively, was confirmed by flow cytometry. CD22-BiTE promoted cell-mediated cytotoxicity in a dose and E:T-dependent fashion, with a maximum NHL and ALL-specific killing activity of 90% in vitro. Besides canonical expression in B cells, CD22 has been reported in non-hematologic malignancies. Our analysis of TCGA data suggests CD22 is expressed in a variety of solid tumors, including sarcoma. We verified expression of CD22 message and protein in solid tumor cell lines. Accordingly, our CD22-BiTE mediated killing of HT1080 sarcoma cells in vitro. In conclusion, we report here the development of a new BiTE with killing activity against CD22+ malignancies which could represent a therapeutic option for B-cell malignancies and potentially solid tumors.

Use of real-time live-cell analysis with enhanced data analytics to quantify immune cell tumor killing

Abstract To develop new therapeutics, researchers are exploring the role of the immune system in defending the body against tumors. Modelling induced malignant cell death in vitro is of paramount importance. Tumor and immune cell co-cultures were created in 96 well plates and using live-cell analysis, various parameters of tumor killing were quantified in real-time. Red nuclear labeled target cells and various densities of pre-activated PBMCs (α-CD3/IL2, 4 d) were seeded in combination with IncuCyte Annexin V green apoptosis detection reagent. Images were acquired every 2 h for 3 d using IncuCyte. Analysis of the fluorescence images provides measurement of target cell number and apoptosis. Enhancement of the phase contrast image analytics enabled single cell segmentation, permitting determination of effector cell parameters; cell number, shape and, using fluorescently labeled surface marker antibodies, protein expression levels. In addition, studies into spatial interactions of target and effector cells were conducted. To exemplify how these new analytical features can be used to investigate the biology of tumor cell killing, studies of a α-hCD3xCD19 bi-specific T-cell engager antibody induced cytotoxicity were performed. Further characterization of effects on cell cycle during target cell death and use of more advanced 3D models of immune cell killing were also assessed, demonstrating the flexibility of live-cell analysis as a powerful tool for analyzing immune cell killing. Advances in data analytics has enabled the multiplexing of target cell quantification alongside the interrogation of effector cell properties in live cells. The added insight gained from these approaches will hopefully lead to improved immuno-therapeutics.

Efficacy of B7-H3-Redirected BiTE and CAR-T Immunotherapies Against Extranodal Nasal Natural Killer/T Cell Lymphoma

Extranodal nasal natural killer (NK)/T cell lymphoma (ENKTCL) is a rare but highly aggressive subtype of non-Hodgkin lymphoma (NHL). Nevertheless, despite extensive research, the estimated 5-year overall survival of affected patients remains low. Therefore, new treatment strategies are needed urgently. Recent advances in immunotherapy have the potential to broaden the applications of chimeric antigen receptor-modified T (CAR-T) cells and the bispecific T-cell engaging (BiTE) antibody. Here, we screened a panel of biomarkers including the B7-H3, CD70, TIM-3, VISTA, ICAM-1, and PD-1 in NKTCL cell lines. As a result, we found for the first time that B7-H3 was highly and homogeneously expressed in these cells. Consequently, we constructed a novel anti-B7-H3/CD3 BiTE antibody and B7-H3-redirected CAR-T cells, and evaluated their efficacy against NKTCL cel lines both in vitro and in vivo. Notably, we found that both anti-B7-H3/CD3 BiTE and B7-H3-redirected CAR-T cells effectively targeted and killed NKTCL cells in vitro, and suppressed the growth of NKTCL tumors in NSG mouse models. Thus, B7-H3 might be a promising therapeutic target for treating patients with NKTCL tumors.

Development of mechanism-based targeted therapies for malignant lymphoma

The development of targeted therapies, such as rituximab-an anti-CD20 antibody targeting CD20, has undergone a paradigm shift from conventional chemotherapy for malignant lymphoma (ML). Although a subset of ML patients has been cured, the treatment of refractory and relapsed diseases remains challenging. Fortunately, growing insights on molecular biology for ML have led to the development of a number of innovative agents. Moreover, a plethora of targeted therapies, including novel antibodies targeting surface antigens, and small molecular inhibitors targeting oncogenic signaling pathways, tumor suppressors, and epigenetic regulation are currently under investigation for the improvement of dismal prognosis. In addition, immunotherapies, including immune checkpoint inhibitors, bispecific T-cell engager antibodies, and chimeric antigen receptor T-cells for ML have been rapidly developed to target tumor microenvironment. These promising mechanism-based targeted therapies could lead to a successful ML management.

Novel therapies for pediatric acute myeloid leukemia: building future strategies through incorporation of treatment currently used in adults

In Japan, acute myeloid leukemia (AML) accounts for approximately 25% of all pediatric leukemias, with approximately 150 cases of newly diagnosed AML occurring annually. Approximately 10% of patients have primary induction failure and 30% of patients, who initially achieve remission in primary treatments, subsequently relapse. Novel treatment modalities need to be developed to further improve the prognosis of pediatric AML patients. AML is a heterogeneous genetic disease characterized by changes in the genome of hematopoietic progenitor cells. Recent studies that have made progress in research related to the pathogenesis of AML have suggested that genotype-specific treatment strategies are associated with increased efficacy. Potential new therapeutic alternatives for pediatric AML include: tyrosine kinase inhibitors, monoclonal or bispecific T-cell engager antibodies, chimeric antigen receptor T-cell therapy, and metabolic agents. This review highlights the current landscape of novel therapeutic approaches for childhood AML, including the results of both preclinical and clinical trials, as well as introducing the results of several preceding adult clinical studies, which could potentially be translated into pediatric AML patients.

Combination therapy with B7H3-redirected bispecific antibody and Sorafenib elicits enhanced synergistic antitumor efficacy.

Rationale: Current traditional treatment options are frequently ineffective to fight against ovarian cancer due to late diagnosis and high recurrence. Therefore, there is a vital need for the development of novel therapeutic agents. B7H3, an immune checkpoint protein, is highly expressed in various cancers, representing it a promising target for cancer immunotherapy. Although targeting B7H3 by bispecific T cell-engaging antibodies (BiTE) has achieved successes in hematological malignancies during recent years, attempts to use them for the treatment of solid cancers are less favorable, in part due to the heterogeneity of tumors. Sorafenib is an unselective inhibitor of multiple kinases currently being tested in clinical trials for several tumors, including ovarian cancer which showed limited activity and inevitable side effect for ovarian cancer treatment. However, it is able to enhance antitumor immune response, which indicates sorafenib may improve the efficiency of immunotherapy.Methods: We evaluated the expression of B7H3 in ovarian cancer using online database and validated its expression of tumor tissues by immunohistochemistry staining. Then, B7H3 expression and the effects of sorafenib on ovarian cancer cell lines were determined by flow cytometry. In addition, 2D and 3D ovarian cancer models were established to test the combined therapeutic effect in vitro. Finally, the efficiency of B7H3×CD3 BiTE alone and its combination with sorafenib were evaluated both in vitro and in vivo.Results: Our data showed that B7H3 was highly expressed in ovarian cancer compared with normal samples. Treatment with sorafenib inhibited ovarian cancer cell proliferation and induced a noticeable upregulation of B7H3 expression level. Further study suggested that B7H3×CD3 BiTE was effective in mediating T cell killing to cancer cells. Combined treatment of sorafenib and B7H3×CD3 BiTE had synergistic anti-tumor effects in ovarian cancer models.Conclusions: Overall, our study indicates that combination therapy with sorafenib and B7H3×CD3 BiTE may be a new therapeutic option for the further study of preclinical treatment of OC.

Current Treatment Strategies for Multiple Myeloma.

The treatment of multiple myeloma (MM) continues to evolve with the approval of numerous agents over the past decade. Advances in treatment have led to the incorporation of these newer therapies into the treatment paradigm, with improvements in overall survival and the possibility of deep responses including a minimal residual disease-negative state. The strategy of triplet therapies for patients with newly diagnosed MM, followed by high-dose chemotherapy and autologous stem-cell transplantation for eligible patients, and subsequently consolidation and maintenance therapy, is the current treatment roadmap for patients. However, patients with MM will ultimately develop refractoriness to antimyeloma therapies. In this article, we summarize our current practice of managing patients with MM. We highlight our approach to patients with newly diagnosed MM who are transplantation eligible and ineligible and highlight risk-adapted strategies for these patients. In addition, we discuss our approach to the management of patients with relapsed or refractory MM. Last, we review standard therapies and emerging strategies such as targeted approaches, immune-based therapies, and drugs with novel mechanisms of action. Trials evaluating chimeric antigen receptor T cells targeting B-cell maturation antigen are ongoing and are only one of several novel approaches targeting cell maturation antigen, which include the use of bispecific T-cell engager antibodies and antibody drug conjugates. Emerging therapies offer the promise of more individualized approaches in the management of patients with MM and ultimately may result in the possibility of being one step closer to curing patients with MM.

Abstract LB-B01: Investigation of immuno-oncology drugs with humanized mouse models: a panel of technologies to evaluate different pharmacological approaches

Abstract Immunotherapy is one of the most exciting recent breakthroughs in the field of cancer treatment. Different approaches are developed such as cancer vaccines, adoptive cellular immunotherapy or immune checkpoint blockade, and a number have been regulatory approved or are currently investigated in clinic. Effective immunity against cancer involves complex interaction between the tumor, the host and the environment. The assessment of cancer immunotherapy approaches in preclinical settings requires the use of appropriate animal models that sufficiently reflect the physiological situation in humans and that must be chosen carefully to address specific mechanisms of action. In light of evaluating the therapeutic potential of different immunomodulatory agents in mice xenografted with cancer cell lines or patient-derived xenografts, we developed multiple humanization strategies on different immuno-deficient mouse strains. Either human PBMCs, Hematopoietic Stem Cells (HSCs), or specific human immune cells such as Dendritic Cells (DCs), T cells, subset of T cells (e.g. gamma9 delta2 T cells) and NK cells were used, but also the combinations of immune subpopulations such as the co-transfer of autologous T cells and DCs. We will highlight some results of our therapeutic efficacy investigation and evaluation of novel immuno-oncology therapies eg. adoptive cell therapy, bispecific T-cell engager antibody, and vaccine immunomodulatory agents in such humanized mouse models, assessing survival, tumor growth and effects on tumors and immune cells by flow cytometry and immunohistochemistry analyses. All these different humanized models can permit to evidence significant in vivo antitumor efficacy of immunomodulatory agents and might help to increase the success rate in clinical trials. Citation Format: Caroline Mignard, Olivier Duchamp, Josselin Caradec, Francis Bichat, Damien France, Fabrice Viviani, Jean François Mirjolet. Investigation of immuno-oncology drugs with humanized mouse models: a panel of technologies to evaluate different pharmacological approaches [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B01. doi:10.1158/1535-7163.TARG-19-LB-B01

Blinatumomab for Acute Lymphoblastic Leukemia: The First Bispecific T-Cell Engager Antibody to Be Approved by the EMA for Minimal Residual Disease.

On November 15, 2018, the Committee for Medicinal Products for Human Use (CHMP) recommended the extension of indication for blinatumomab to include the treatment of adults with minimal residual disease (MRD) positive B-cell precursor acute lymphoblastic leukemia (ALL). Blinatumomab was authorized to treat relapsed or refractory B-precursor ALL, and the change concerned an extension of use. On March 29, 2018, the U.S. Food and Drug Administration (FDA) granted accelerated approval to blinatumomab to treat both adults and children with B-cell precursor ALL who are in remission but still have MRD. On July 26, 2018, the CHMP had originally adopted a negative opinion on the extension. The reason for the initial refusal was that although blinatumomab helped to reduce the amount of residual cancer cells in many patients, there was no strong evidence that it led to improved survival. During the re-examination, the CHMP consulted the scientific advisory group. The CHMP agreed with the expert group's conclusion that, although there was no strong evidence of patients living longer, the available data from the main study (MT103-203) indicated a good durable response to blinatumomab, with an overall complete response rate for the primary endpoint full analysis set (defined as all subjects with an Ig or T-cell receptor polymerase chain reaction MRD assay with the minimum required sensitivity of 1 × 10-4 at central lab established at baseline [n = 113]) as 79.6% (90/113; 95% confidence interval, 71.0-86.6), with a median time to complete MRD response of 29.0 days (range, 5-71). Therefore, the CHMP concluded that the benefits of blinatumomab outweigh its risks and recommended granting the change to the marketing authorization. The Committee for Orphan Medicinal Products, following reassessment, considered that significant benefit continued to be met and recommended maintaining the orphan designation and thus 10 years market exclusivity (the Orphan Designation is a legal procedure that allows for the designation of a medicinal substance with therapeutic potential for a rare disease, before its first administration in humans or during its clinical development). The marketing authorization holder for this medicinal product is Amgen Europe B.V. IMPLICATIONS FOR PRACTICE: Immunotherapy with blinatumomab has excellent and sustainable results, offering new hope for patients with minimal residual disease-positive acute lymphoblastic leukemia, a disease with poor prognosis. New recommendations and change of practice for treatment of this patient group are detailed.

Bispecific T-Cell Engager (BiTE) Antibody Based Immunotherapy for Treatment of Relapsed Refractory Multiple Myeloma (RRMM): A Systematic Review of Preclinical and Clinical Trials

Introduction Bispecific T-cell engager (BiTE) antibodies represent a novel therapeutic option for patients with multiple myeloma (MM). BiTE antibodies lack Fc region, and have variable domain only, they can simultaneously bind to two different epitopes i.e. cluster of differentiation 3 (CD3) molecules on tumor-specific T cells, and a specific antigen on myeloma cells, which leads to T-cell dependent destruction of myeloma cells. Currently, blinatumomab, specific for CD3 and CD19 is the only Food and Drug Administration FDA approved BiTE antibody for clinical use in patients with relapsed/refractory (RR) B-cell acute lymphoblastic leukemia, several similar BiTE antibodies are under development. Methods Following PRISMA guidelines, we performed comprehensive literature on 4/15/19 cross-referencing the terms "bispecific antibodies" and "multiple myeloma" using PubMed, Embase, Web of Science, Cochrane Library, Clinicaltrials.gov and review of international medical meeting abstracts. Initially, 256 articles were identified and after detailed scrutiny, one phase 1 clinical trial with prelim results, 4 preclinical and 4 ongoing clinical trials were included. Results Preclinical trials: Anti-BCMA x Anti-CD3 Bispecific Antibody: BiTE antibodies are still in early development in MM, and most of the published data is about the pre-clinical phase. In preclinical trials, Hipp et al. 2017 and Cho et al. 2018 reported that AMG 420 (BI 836909) and AMG 701, which are anti CD3 and B-cell maturation antigen (BCMA), are highly efficacious in vitro in the killing of myeloma cells and potently induces autologous tumor cell lysis in patients with both newly diagnosed and RRMM regardless of their disease status. In mouse xenograft models reconstituted with human T cells, in vivo efficacy of AMG 420 was reported with an overall response in 6 of 10 animals, with all 6 responders became tumor-free at the end of the study. In an orthotopic L-363 xenograft model, treatment with AMG 420 resulted in prolonged median survival of 43-43.5 days. Dilillo et al. 2018 and Ji Li et al. 2017 reported similar in vivo results for REGN5458 and BFCR4350A respectively. Clinical trials: Currently, there are 5 phase 1 ongoing clinical trials (Table 1). Updated results of only first in human phase I AMG 420 are available. Forty-two MM patients with a high tumor burden and four prior lines of therapy were given 2.5 treatment cycles with AMG 420. Overall thirteen (31%) patients responded to AMG 420 therapy, with complete response (CR) in 6 (14.2%) patients, very good partial response (VGPR) in 2 (5%) patients and partial response (PR) in 2(5%) patients. Eleven of these patients responded in the first treatment cycle, with a median response time of 1 month. Twenty-five (57.1%) patients discontinued treatment due to progressive disease. Four deaths were reported; 2 from disease progression and 2 due to adverse events; neither of them was treatment-related. Serious adverse events were reported in twenty-one (50%) patients, the infection was reported in twelve (29%) and polyneuropathy in three (7%), eighteen (43%) required hospitalization. Treatment-related serious adverse events included three (7%) patients with grade 2-3 cytokine release syndrome, three (7%) with polyneuropathy and one (2.3%) with edema. Conclusion After the success of naked antibodies like daratumumab and elotuzumab for MM, there is a need to develop immunotherapy using conjugated antibodies and BiTE antibodies to overcome the challenge of MM resistance and relapse to prior therapies. Preclinical data with BiTE antibodies are promising; AMG 420 anti-CD3/BCMA BiTE has already been granted fast track status by the FDA. We anticipate that drug will enter phase 2 clinical trials for drug development against RRMM Other BiTE antibodies with strong preclinical efficacy are under development and data from larger prospective clinical trials is needed to explore their efficacy in the treatment of multiple myeloma. Table 1 Disclosures Anwer: In-Cyte: Speakers Bureau; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees.

Mesothelin Targeting Bites for Pediatric AML: In Vivo Efficacy and Specificity

Acute myeloid leukemia (AML) remains the type of pediatric leukemia with poorest outcome. Despite maximally intensive therapy, approximately 20% of patients experience recurrent disease. Novel targeted therapies are needed to improve survival. We recently identified that mesothelin, a well-validated target in some cancers, is also highly expressed in a subset of pediatric AML samples (Tarlock et al., Blood, 128:2873, 2016). Considering that it is not expressed in normal tissues in children (Fan et al., Blood, 130:3792, 2017), MSLN is a viable target for immunotherapies such as Bispecific T-cell Engaging antibodies (BiTEs) that combine antibody single chain variable (scFv) regions targeting a cancer antigen and the T-cell co-receptor CD3. We designed and tested the efficacy and specificity of BiTEs targeting MSLN in disseminated xenograft models of pediatric AML. Using scFv sequences derived from Amatuximab, which recognizes the N-terminal domain of the GPI-linked ectodomain of MSLN, targeting region 1 of MSLN, and from Blinatumomab/AMG-330 targeting CD3, we engineered and expressed two kinds of BiTE molecules - a canonical BiTE and an IgG BiTE, a larger molecule with improved serum half life in vivo. To evaluate the specificity and efficacy of canonical BiTEs, MV4;11-MSLN cell line was generated by lentiviral transduction of parental MV4;11 cells which do not constitutively express MSLN (Fig. 1A, B). These two cell lines were injected i.v. into NSG-SGM3 mice. Once engraftment was confirmed, purified human T cells (3 x 106) were injected to act as effector cells. Mice were then treated with the canonical αMSLN-αCD3 BiTE at a dose of 3 mg/kg/day daily for 6 days. A cohort of mice that were untreated or received BiTE or T-cell infusion only served as controls. Mice from both treated and untreated groups had to be euthanized when they presented with distended abdomens due to myeloid sarcomas and no significant differences in survival were observed. Post euthanasia, bone marrows were flushed and evaluated for the percentage of AML cells (human CD45+CD33+) and T cells (human CD45+CD3+). We observed that the αMSLN-αCD3 BiTE was effective in promoting T-cell activation (based on high T-cell counts compared to mice injected with T-cells alone) and greatly reducing leukemic burden in mice injected with MV4;11 cells engineered to express MSLN (Fig. 1C, D). Similar results were obtained using BiTEs targeting a different MSLN epitope. No T-cell expansion and anti-leukemic effect was observed in mice engrafted with parental MV4;11 cells. Although, there were no significant differences between the median survival of untreated and treated miceThese data highlight the specificity and efficacy of the aMSLN-CD3 BiTEs. Among a panel of 8 AML patient-derived xenograft (PDX) lines generated in the laboratory, NTPL-146 bearing MLL-ENL fusion was found to have endogenous MSLN expression (Fig. 1E). We evaluated the efficacy of αMSLN-αCD3 canonical BiTE (3 mg/Kg Qdx6) against NTPL-146 PDX line in NSG-B2m mice by transfusing human CD3+ T-cells to act as effector cells. A Kaplan-Meier survival plot based on the time when each mouse reached experimental end-point (reduced body weight greater than 20%, impaired mobility, hind limb paralysis) showed that the survival benefit for mice receiving BiTE in the presence of human T-cells (4/6 mice survived at the end of experiment) greatly exceeded the efficacy of T-cells alone (22-day improvement in median survival with no surviving mice), or BiTE treatment alone (no improvement in survival) compared to untreated mice (Fig. 1F, P<0.001). These data validate the efficacy of MSLN targeting BiTEs in a PDX model with endogenous MSLN expression. The efficacy of canonical vs IgG BiTEs was evaluated in MV4;11-MSLN xenografted mice. Mice were dosed Qd5x3 for canonical BiTE and Q7dx3 for IgG BiTE as shown (Fig. 1G). IgG BiTE treatment along with T-cell infusion significantly prolonged survival in mice transplanted with MV4;11-MSLN (Fig. 1H), suggesting that IgG BiTE was far more efficacious than canonical BiTEs (P<0.01). Taken together, these data indicate that MSLN-targeting BiTEs could be used as novel immunotherapy for pediatric AML with MSLN expression. Figure 1 Disclosures Kaeding: Celgene: Employment.

PD-L1 Checkpoint Blockade Augments Anti-Tumor Immune Response of GD2 or HER2-Bsab Ex Vivo Armed T-Cells (EVAT) Therapy in Osteosarcoma

Background Ex Vivo Armed T-cells (EVAT) carrying zeptomoles (10-21M) of T-cell engaging GD2-bispecific antibody (GD2-EVAT) or HER2-bispecific antibodies (HER2-EVAT) have potent anti-tumor activity against GD2(+) and/or HER2(+) solid tumors. Strategies to further optimize this approach are highly relevant. PD-1 is a key immune checkpoint receptor expressed mainly by activated T-cells and mediates immune suppression by binding to its ligands PD-L1 or PD-L2. Upregulation of PD-L1 has been found in many cancers including osteosarcoma and associated with aggressive disease and poor outcome. While the use of immune checkpoint inhibitors (ICIs) seems logical, the ideal timing when combined with T-cell engaging bispecific antibody (T-BsAb) or EVAT has yet to be defined. Here, we described the effects of anti-PD-1 or anti-PD-L1 antibodies on GD2-EVAT or HER2-EVAT therapy and explored the impact of its timing in the treatment of osteosarcoma which is GD2(+), HER2(+) and PD-L1(+). Methods GD2-BsAb and HER-BsAb were built using the IgG(L)-scFv format (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from healthy volunteer donors were isolated, and cultured ex vivo in the presence of CD3/CD28 beads plus 30 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T-cells (ATCs) were harvested and armed for 20 minutes at room temperature with GD2-BsAb or HER2-BsAb. In vivo anti-tumor activity against GD2(+), HER2(+), and PD-L1(+) osteosarcoma cell line xenografts was tested in BALB-Rag2-/-IL-2R-γc-KO mice. Anti-human PD-1 antibody (pembrolizumab, anti-PD-1) or anti-human PD-L1 antibody (atezolizumab, anti-PD-L1) were tested for synergy with GD2-EVAT or HER2-EVAT therapy. Results The PD-1 expression increased among T-cells that circulated in the blood, that infiltrated the spleen or the tumor after EVAT therapy. While anti-PD-L1 combination therapy with GD2-EVAT or HER2-EVAT improved anti-tumor response against osteosarcoma (P=0.0123 and P=0.0004), anti-PD-1 did not (all P>0.05). The addition of anti-PD-L1 significantly increased T-cell survival in blood and T-cell infiltration of tumor when compared to GD2-EVAT or HER2-EVAT alone (all P<0.0001). Treatment of GD2-EVAT or anti-PD-L1 plus GD2-EVAT downregulated GD2 expression on tumors, but anti-PD-1 plus GD2-EVAT did not. For the next step we tested the impact of different combination schedules of ICIs on GD2-EVAT therapy. Concurrent anti-PD-1 (6 doses along with GD2-EVAT therapy) interfered with GD2-EVAT, while sequential anti-PD-1 (6 doses after GD2-EVAT) did not make a significant effect (P>0.05). On the other hand, while the concurrent use of anti-PD-L1 did not show benefit on GD2-EVAT, sequentially administered anti-PD-L1 produced a significant improvement in tumor control when compared to anti-PD-L1 or GD2-EVAT alone (P=0.002 and P=0.018). When anti-PD-L1 treatment was extended (12 doses after GD2-EVAT), the anti-tumor effect was most pronounced compared to GD2-EVAT alone (P <0.0001), which translated into improved survival (P=0.0057). These in vivo anti-tumor responses were associated with increased CD8(+) tumor infiltrating lymphocytes (TILs) of tumor. Conclusion In the arming platform, large numbers of target-specific T-cells can be generated, and this EVAT therapy is a highly effective cellular treatment with high potency in preclinical models. In addition, the advantage of ex vivo cytokine release following T-cell arming and activation could reduce or avoid life threatening cytokine storm if such activation was to proceed in vivo. Adoptive T-cell therapy induced immune response upregulates the inhibitory immune checkpoint PD-1/PD-L1 pathway, and combination treatment with anti-PD-L1 antibody, especially when combined as sequential therapy and continuously treated, significantly improved anti-tumor effect of EVAT, partly through increase in CD8(+) TILs infiltration. Disclosures Xu: MSK: Other: co-inventors in patents on GD2 bispecific antibody and HER2 bispecific antibody. Cheung:Ymabs: Patents & Royalties, Research Funding.

Open-Label, Phase 2 Study of Blinatumomab after First-Line Rituximab-Chemotherapy in Adults with Newly Diagnosed, High-Risk Diffuse Large B-Cell Lymphoma

Background: Rituximab combined with chemotherapy (R-chemotherapy) is the standard of care first-line treatment for diffuse large B-cell lymphoma (DLBCL). Despite success with R-chemotherapy, 30% to 50% of patients with high-risk DLBCL will relapse, and outcomes are poor among patients who relapse within one year of diagnosis. Given the challenge of successful salvage, novel first-line therapies are needed. Blinatumomab, a bispecific T-cell engager (BiTE®) antibody construct that directs cytotoxic T cells to lyse CD19-expressing B cells, has shown efficacy as salvage therapy in patients with relapsed or refractory DLBCL. This open-label, multicenter, phase 2 study (ClinicalTrials.gov, NCT03023878) assessed the efficacy and safety of blinatumomab after first-line R-chemotherapy for patients with newly diagnosed, high-risk DLBCL. Methods: Patients (≥18 y) had proven high-risk DLBCL (International Prognostic Index [IPI] 3−5 and double/triple hit or double MYC/BCL2 expressor) and Eastern Cooperative Oncology group performance status ≤2. To be eligible for blinatumomab, patients were required to achieve complete metabolic response (CMR), partial metabolic response (PMR), or stable metabolic response by PET/CT after a run-in period with 6 cycles of R-chemotherapy (R-CHOP, R-DA-EPOCH, or R-CHOEP). Blinatumomab was given by continuous intravenous infusion in a single 84-day cycle 1 (9 μg/day for 7 days, 28 μg/day for 7 days, and 112 μg/day for 42 days, followed by a 28-day treatment-free interval) and an optional 28-day cycle 2 (9 μg/day for 7 days, 28 μg/day for 7 days, and 112 μg/day for 14 days) for patients without progressive metabolic disease (PMD). The primary endpoint was the incidence and severity of adverse events (AEs). Additional endpoints were objective response rate (ORR [CMR + PMR]) per Lugano criteria, minimal residual disease (MRD) by plasma cell−free circulating tumor DNA, overall survival (OS), and pharmacokinetics (PK). Results: Of 47 patients enrolled, 17 (36%) discontinued R-chemotherapy run-in (protocol criteria, n=6; patient request, n=5; disease progression, n=3; ineligibility, n=1; AE, n=1; death, n=1) and 30 (64%) completed the run-in (2 did not proceed to blinatumomab). Of 28 patients who received blinatumomab, 26 (93%) had high or high-intermediate IPI; 8 (29%) were double/triple hit and 10 (36%) were double protein expressors (Table). In total, 26 (93%) patients completed cycle 1; ten of 11 (91%) patients completed optional cycle 2. Blinatumomab PK were consistent with those in previous studies. After the R-chemotherapy run-in before starting blinatumomab, 24 patients had objective metabolic responses and 4 had no metabolic response (NMR). After blinatumomab treatment, the ORR (within 12 weeks of starting blinatumomab) was 89% (25/28 patients; 95% CI, 72−98; Table). The 4 patients with NMR before blinatumomab had objective responses after blinatumomab treatment. Three patients with objective responses before blinatumomab relapsed after blinatumomab. Twenty-six (93%) patients were still alive with a median follow-up time of 8.6 months; 2 died (disease progression; n=1; infection not related to treatment, n=1). Nine of 13 (69%) patients during the R-chemotherapy run-in were MRD positive, all of whom converted to MRD negative after treatment with blinatumomab. After treatment with blinatumomab, 17 of 18 (94%) patients were MRD negative; the MRD positive patient had PMD. During blinatumomab treatment, 11 (39%) patients had grade ≥3 AEs, and 5 (18%) had grade ≥4 AEs. Two (7%) patients discontinued treatment due to AEs (grade 3 neurotoxicity; grade 4 neutropenia). Consistent with previous blinatumomab reports, neurologic events (NEs) were reported in 17 (61%) patients, including 3 (11%) with grade 3 NEs and 1 (4%) with NEs leading to treatment discontinuation. No patients had grade ≥3 cytokine release syndrome. Other grade ≥3 events of interest included neutropenia and febrile neutropenia (n=4; 14%) and infection (n=3; 11%). Conclusions: In patients with newly diagnosed, high-risk DLBCL, blinatumomab monotherapy after first-line R-chemotherapy led to an 89% ORR, and safety was consistent with that in earlier studies in DLBCL. Thus, blinatumomab is a potential treatment option for patients with newly diagnosed disease. Disclosures Katz: Stemline: Speakers Bureau; Dova: Consultancy. Chu:Celgene: Honoraria; Teva: Consultancy; AstraZeneca: Honoraria; Amgen Inc.: Honoraria; Gilead: Honoraria. Thieblemont:Roche: Honoraria, Research Funding; Gilead: Honoraria; Novartis: Honoraria; Kyte: Honoraria; Janssen: Honoraria; Celgene: Honoraria; Cellectis: Membership on an entity's Board of Directors or advisory committees. Morley:Amgen: Membership on an entity's Board of Directors or advisory committees, Other: speaker fees, conference support ; TAKEDA: Other: conference support ; Janssen Pharmaceuticals: Other: speaker fees; ROCHE: Membership on an entity's Board of Directors or advisory committees, Other: conference support; ABBVIE: Other: speaker fees. Chen:Amgen Inc.: Employment, Equity Ownership. Kalabus:Amgen Inc.: Employment, Equity Ownership. Morris:Amgen: Employment, Equity Ownership. Anderson:Amgen Inc.: Employment, Equity Ownership. Avilion:Amgen Inc.: Employment, Equity Ownership. González-Barca:Takeda: Honoraria; Kiowa: Consultancy; Celtrion: Consultancy; Janssen: Consultancy, Honoraria; Celgene: Consultancy; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria.