Antitumor activity of the investigational B7-H3 antibody-drug conjugate, vobramitamab duocarmazine, in preclinical models of neuroblastoma.

INTRODUCTION: B7-H3 is a potential target for pediatric cancers, including neuroblastoma (NB). Vobramitamab duocarmazine (also referred to as MGC018 and herein referred to as vobra duo) is an investigational duocarmycin-based antibody-drug conjugate (ADC) directed against the B7-H3 antigen. It is composed of an anti-B7-H3 humanized IgG1/kappa monoclonal antibody chemically conjugated through a cleavable valine-citrulline linker to a duocarmycin-hydroxybenzamide azaindole (vc-seco-DUBA). Vobra duo has shown preliminary clinical activity in B7-H3-expressing tumors. METHODS: B7-H3 expression was evaluated by flow-cytometry in a panel of human NB cell lines. Cytotoxicity was evaluated in monolayer and in multicellular tumor spheroid (MCTS) models by the water-soluble tetrazolium salt,MTS, proliferation assay and Cell Titer Glo 3D cell viability assay, respectively. Apoptotic cell death was investigated by annexin V staining. Orthotopic, pseudometastatic, and resected mouse NB models were developed to mimic disease conditions related to primary tumor growth, metastases, and circulating tumor cells with minimal residual disease, respectively. RESULTS: All human NB cell lines expressed cell surface B7-H3 in a unimodal fashion. Vobra duo was cytotoxic in a dose-dependent and time-dependent manner against all cell lines (IC50 range 5.1-53.9 ng/mL) and NB MCTS (IC50 range 17.8-364 ng/mL). Vobra duo was inactive against a murine NB cell line (NX-S2) that did not express human B7-H3; however, NX-S2 cells were killed in the presence of vobra duo when co-cultured with human B7-H3-expressing cells, demonstrating bystander activity. In orthotopic and pseudometastatic mouse models, weekly intravenous treatments with 1 mg/kg vobra duo for 3 weeks delayed tumor growth compared with animals treated with an irrelevant (anti-CD20) duocarmycin-ADC. Vobra duo treatment for 4 weeks further increased survival in both orthotopic and resected NB models. Vobra duo compared favorably to TOpotecan-TEMozolomide (TOTEM), the standard-of-care therapy for NB relapsed disease, with tumor relapse delayed or arrested by two or three repeated 4-week vobra duo treatments, respectively. Further increased survival was observed in mice treated with vobra duo in combination with TOTEM. Vobra duo treatment was not associated with body weight loss, hematological toxicity, or clinical chemistry abnormalities. CONCLUSION: Vobra duo exerts relevant antitumor activity in preclinical B7-H3-expressing NB models and represents a potential candidate for clinical translation.

Flotetuzumab and other T-cell immunotherapies upregulate MHC class II expression on acute myeloid leukemia cells.

Acute myeloid leukemia (AML) relapse is one of the most common and significant adverse events following allogeneic hematopoietic cell transplantation (HCT). Downregulation of major histocompatibility class II (MHC-II) surface expression on AML blasts may represent a mechanism of escape from the graft-versus-malignancy effect and facilitate relapse. We hypothesized that T-cell immunotherapies targeting AML antigens would upregulate MHC-II surface expression via localized release of interferon gamma (IFN-γ), a protein known to upregulate MHC-II expression via JAK-STAT signaling. We demonstrate that flotetuzumab (FLZ), a CD123 × CD3 bispecific DART molecule, and chimeric antigen receptor expressing T cells targeting CD123, CD33, or CD371 upregulate MHC-II surface expression in vitro on a THP-1 AML cell line with intermediate MHC-II expression and 4 primary AML samples from patients relapsing after HCT with low MHC-II expression. We additionally show that FLZ upregulates MHC-II expression in a patient-derived xenograft model and in patients with relapsed or refractory AML who were treated with FLZ in a clinical trial. Finally, we report that FLZ-induced MHC-II upregulation is mediated by IFN-γ. In conclusion, we provide evidence that T-cell immunotherapies targeting relapsed AML can kill AML via both MHC-independent mechanisms and by an MHC-dependent mechanism through local release of IFN-γ and subsequent upregulation of MHC-II expression.

Preclinical Evaluation of IMGC936, a Next-Generation Maytansinoid-based Antibody-drug Conjugate Targeting ADAM9-expressing Tumors.

ADAM metallopeptidase domain 9 (ADAM9) is a member of the ADAM family of multifunctional, multidomain type 1 transmembrane proteins. ADAM9 is overexpressed in many cancers, including non-small cell lung, pancreatic, gastric, breast, ovarian, and colorectal cancer, but exhibits limited expression in normal tissues. A target-unbiased discovery platform based on intact tumor and progenitor cell immunizations, followed by an IHC screen, led to the identification of anti-ADAM9 antibodies with selective tumor-versus-normal tissue binding. Subsequent analysis revealed anti-ADAM9 antibodies were efficiently internalized and processed by tumor cells making ADAM9 an attractive target for antibody-drug conjugate (ADC) development. Here, we describe the preclinical evaluation of IMGC936, a novel ADC targeted against ADAM9. IMGC936 is comprised of a high-affinity humanized antibody site-specifically conjugated to DM21-C, a next-generation linker-payload that combines a maytansinoid microtubule-disrupting payload with a stable tripeptide linker, at a drug antibody ratio of approximately 2.0. In addition, the YTE mutation (M252Y/S254T/T256E) was introduced into the CH2 domain of the antibody Fc to maximize in vivo plasma half-life and exposure. IMGC936 exhibited cytotoxicity toward ADAM9-positive human tumor cell lines, as well as bystander killing, potent antitumor activity in human cell line-derived xenograft and patient-derived xenograft tumor models, and an acceptable safety profile in cynomolgus monkeys with favorable pharmacokinetic properties. Our preclinical data provide a strong scientific rationale for the further development of IMGC936 as a therapeutic candidate for the treatment of ADAM9-positive cancers. A first-in-human study of IMGC936 in patients with advanced solid tumors has been initiated (NCT04622774).

Dual checkpoint targeting of B7-H3 and PD-1 with enoblituzumab and pembrolizumab in advanced solid tumors: interim results from a multicenter phase I/II trial.

BACKGROUND: Availability of checkpoint inhibitors has created a paradigm shift in the management of patients with solid tumors. Despite this, most patients do not respond to immunotherapy, and there is considerable interest in developing combination therapies to improve response rates and outcomes. B7-H3 (CD276) is a member of the B7 family of cell surface molecules and provides an alternative immune checkpoint molecule to therapeutically target alone or in combination with programmed cell death-1 (PD-1)-targeted therapies. Enoblituzumab, an investigational anti-B7-H3 humanized monoclonal antibody, incorporates an immunoglobulin G1 fragment crystallizable (Fc) domain that enhances Fcγ receptor-mediated antibody-dependent cellular cytotoxicity. Coordinated engagement of innate and adaptive immunity by targeting distinct members of the B7 family (B7-H3 and PD-1) is hypothesized to provide greater antitumor activity than either agent alone. METHODS: In this phase I/II study, patients received intravenous enoblituzumab (3-15 mg/kg) weekly plus intravenous pembrolizumab (2 mg/kg) every 3 weeks during dose-escalation and cohort expansion. Expansion cohorts included non-small cell lung cancer (NSCLC; checkpoint inhibitor [CPI]-naïve and post-CPI, programmed death-ligand 1 [PD-L1] <1%), head and neck squamous cell carcinoma (HNSCC; CPI-naïve), urothelial cancer (post-CPI), and melanoma (post-CPI). Disease was assessed using Response Evaluation Criteria in Solid Tumors version 1.1 after 6 weeks and every 9 weeks thereafter. Safety and pharmacokinetic data were provided for all enrolled patients; efficacy data focused on HNSCC and NSCLC cohorts. RESULTS: Overall, 133 patients were enrolled and received ≥1 dose of study treatment. The maximum tolerated dose of enoblituzumab with pembrolizumab at 2 mg/kg was not reached. Intravenous enoblituzumab (15 mg/kg) every 3 weeks plus pembrolizumab (2 mg/kg) every 3 weeks was recommended for phase II evaluation. Treatment-related adverse events occurred in 116 patients (87.2%) and were grade ≥3 in 28.6%. One treatment-related death occurred (pneumonitis). Objective responses occurred in 6 of 18 (33.3% [95% CI 13.3 to 59.0]) patients with CPI-naïve HNSCC and in 5 of 14 (35.7% [95% CI 12.8 to 64.9]) patients with CPI-naïve NSCLC. CONCLUSIONS: Checkpoint targeting with enoblituzumab and pembrolizumab demonstrated acceptable safety and antitumor activity in patients with CPI-naïve HNSCC and NSCLC. TRIAL REGISTRATION NUMBER: NCT02475213.

Efficacy of Flotetuzumab in Combination with Cytarabine in Patient-Derived Xenograft Models of Pediatric Acute Myeloid Leukemia.

Children with acute myeloid leukemia (AML) have a poor prognosis despite the intensification of chemotherapy. Future efforts to improve outcomes should focus on more precise targeting of leukemia cells. CD123, or IL3RA, is expressed on the surface of nearly all pediatric AML samples and is a high-priority target for immunotherapy. The efficacy of an investigational dual-affinity retargeting antibody (DART) molecule (CD123 × CD3; MGD006 or flotetuzumab) was assessed in two distinct patient-derived xenograft (PDX) models of pediatric AML. MGD006 simultaneously binds to CD123 on target cells and CD3 on effector T cells, thereby activating T cells and redirecting them to induce cytotoxicity in target cells. The concurrent treatment of cytarabine and MGD006 was performed to determine the effect of cytarabine on T-cell counts and MGD006 activity. Treatment with MGD006 along with an allogeneic human T-cell infusion to act as effector cells induced durable responses in both PDX models, with CD123 positivity. This effect was sustained in mice treated with a combination of MGD006 and cytarabine in the presence of T cells. MGD006 enhanced T-cell proliferation and decreased the burden of AML blasts in the peripheral blood with or without cytarabine treatment. These data demonstrate the efficacy of MGD006 in prolonging survival in pediatric AML PDX models in the presence of effector T cells and show that the inclusion of cytarabine in the treatment regimen does not interfere with MGD006 activity.

A humanized CD3ε-knock-in mouse model for pre-clinical testing of anti-human CD3 therapy.

Pre-clinical murine models are critical for translating drug candidates from the bench to the bedside. There is interest in better understanding how anti-human CD3 therapy works based on recent longitudinal studies of short-term administration. Although several models have been created in this pursuit, each have their own advantages and disadvantages in Type-1 diabetes. In this study, we report a murine genetic knock-in model which expresses both a murine and a humanized-CD3ε-exon, rendering it sensitive to manipulation with anti-human CD3. These huCD3εHET mice are viable and display no gross abnormalities. Specifically, thymocyte development and T cell peripheral homeostasis is unaffected. We tested immune functionality of these mice by immunizing them with T cell-dependent antigens and no differences in antibody titers compared to wild type mice were recorded. Finally, we performed a graft-vs-host disease model that is driven by effector T cell responses and observed a wasting disease upon transfer of huCD3εHET T cells. Our results show a viable humanized CD3 murine model that develops normally, is functionally engaged by anti-human CD3 and can instruct on pre-clinical tests of anti-human CD3 antibodies.

Efficacy of Margetuximab vs Trastuzumab in Patients With Pretreated ERBB2-Positive Advanced Breast Cancer: A Phase 3 Randomized Clinical Trial.

IMPORTANCE: ERRB2 (formerly HER2)-positive advanced breast cancer (ABC) remains typically incurable with optimal treatment undefined in later lines of therapy. The chimeric antibody margetuximab shares ERBB2 specificity with trastuzumab but incorporates an engineered Fc region to increase immune activation. OBJECTIVE: To compare the clinical efficacy of margetuximab vs trastuzumab, each with chemotherapy, in patients with pretreated ERBB2-positive ABC. DESIGN, SETTING, AND PARTICIPANTS: The SOPHIA phase 3 randomized open-label trial of margetuximab plus chemotherapy vs trastuzumab plus chemotherapy enrolled 536 patients from August 26, 2015, to October 10, 2018, at 166 sites in 17 countries. Eligible patients had disease progression on 2 or more prior anti-ERBB2 therapies and 1 to 3 lines of therapy for metastatic disease. Data were analyzed from February 2019 to October 2019. INTERVENTIONS: Investigators selected chemotherapy before 1:1 randomization to margetuximab, 15 mg/kg, or trastuzumab, 6 mg/kg (loading dose, 8 mg/kg), each in 3-week cycles. Stratification factors were metastatic sites (≤2, >2), lines of therapy (≤2, >2), and chemotherapy choice. MAIN OUTCOMES AND MEASURES: Sequential primary end points were progression-free survival (PFS) by central blinded analysis and overall survival (OS). All α was allocated to PFS, followed by OS. Secondary end points were investigator-assessed PFS and objective response rate by central blinded analysis. RESULTS: A total of 536 patients were randomized to receive margetuximab (n = 266) or trastuzumab (n = 270). The median age was 56 (27-86) years; 266 (100%) women were in the margetuximab group, while 267 (98.9%) women were in the trastuzumab group. Groups were balanced. All but 1 patient had received prior pertuzumab, and 489 (91.2%) had received prior ado-trastuzumab emtansine. Margetuximab improved primary PFS over trastuzumab with 24% relative risk reduction (hazard ratio [HR], 0.76; 95% CI, 0.59-0.98; P = .03; median, 5.8 [95% CI, 5.5-7.0] months vs 4.9 [95% CI, 4.2-5.6] months; October 10, 2018). After the second planned interim analysis of 270 deaths, median OS was 21.6 months with margetuximab vs 19.8 months with trastuzumab (HR, 0.89; 95% CI, 0.69-1.13; P = .33; September 10, 2019), and investigator-assessed PFS showed 29% relative risk reduction favoring margetuximab (HR, 0.71; 95% CI, 0.58-0.86; P < .001; median, 5.7 vs 4.4 months; September 10, 2019). Margetuximab improved objective response rate over trastuzumab: 22% vs 16% (P = .06; October 10, 2018), and 25% vs 14% (P < .001; September 10, 2019). Incidence of infusion-related reactions, mostly in cycle 1, was higher with margetuximab (35 [13.3%] vs 9 [3.4%]); otherwise, safety was comparable. CONCLUSIONS AND RELEVANCE: In this phase 3 randomized clinical trial, margetuximab plus chemotherapy had acceptable safety and a statistically significant improvement in PFS compared with trastuzumab plus chemotherapy in ERBB2-positive ABC after progression on 2 or more prior anti-ERBB2 therapies. Final OS analysis is expected in 2021. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02492711.

Flotetuzumab as salvage immunotherapy for refractory acute myeloid leukemia.

Approximately 50% of acute myeloid leukemia (AML) patients do not respond to induction therapy (primary induction failure [PIF]) or relapse after <6 months (early relapse [ER]). We have recently shown an association between an immune-infiltrated tumor microenvironment (TME) and resistance to cytarabine-based chemotherapy but responsiveness to flotetuzumab, a bispecific DART antibody-based molecule to CD3ε and CD123. This paper reports the results of a multicenter, open-label, phase 1/2 study of flotetuzumab in 88 adults with relapsed/refractory AML: 42 in a dose-finding segment and 46 at the recommended phase 2 dose (RP2D) of 500 ng/kg per day. The most frequent adverse events were infusion-related reactions (IRRs)/cytokine release syndrome (CRS), largely grade 1-2. Stepwise dosing during week 1, pretreatment dexamethasone, prompt use of tocilizumab, and temporary dose reductions/interruptions successfully prevented severe IRR/CRS. Clinical benefit accrued to PIF/ER patients showing an immune-infiltrated TME. Among 30 PIF/ER patients treated at the RP2D, the complete remission (CR)/CR with partial hematological recovery (CRh) rate was 26.7%, with an overall response rate (CR/CRh/CR with incomplete hematological recovery) of 30.0%. In PIF/ER patients who achieved CR/CRh, median overall survival was 10.2 months (range, 1.87-27.27), with 6- and 12-month survival rates of 75% (95% confidence interval [CI], 0.450-1.05) and 50% (95% CI, 0.154-0.846). Bone marrow transcriptomic analysis showed that a parsimonious 10-gene signature predicted CRs to flotetuzumab (area under the receiver operating characteristic curve = 0.904 vs 0.672 for the European LeukemiaNet classifier). Flotetuzumab represents an innovative experimental approach associated with acceptable safety and encouraging evidence of activity in PIF/ER patients. This trial was registered at www.clinicaltrials.gov as #NCT02152956.

Development and Preliminary Clinical Activity of PD-1-Guided CTLA-4 Blocking Bispecific DART Molecule.

Combination immunotherapy with antibodies directed against PD-1 and CTLA-4 shows improved clinical benefit across cancer indications compared to single agents, albeit with increased toxicity. Leveraging the observation that PD-1 and CTLA-4 are co-expressed by tumor-infiltrating lymphocytes, an investigational PD-1 x CTLA-4 bispecific DART molecule, MGD019, is engineered to maximize checkpoint blockade in the tumor microenvironment via enhanced CTLA-4 blockade in a PD-1-binding-dependent manner. In vitro, MGD019 mediates the combinatorial blockade of PD-1 and CTLA-4, confirming dual inhibition via a single molecule. MGD019 is well tolerated in non-human primates, with evidence of both PD-1 and CTLA-4 blockade, including increases in Ki67+CD8 and ICOS+CD4 T cells, respectively. In the ongoing MGD019 first-in-human study enrolling patients with advanced solid tumors (NCT03761017), an analysis undertaken following the dose escalation phase revealed acceptable safety, pharmacodynamic evidence of combinatorial blockade, and objective responses in multiple tumor types typically unresponsive to checkpoint inhibitor therapy.

Preclinical Development of MGC018, a Duocarmycin-based Antibody-drug Conjugate Targeting B7-H3 for Solid Cancer.

B7-H3, also referred to as CD276, is a member of the B7 family of immune regulatory proteins. B7-H3 is overexpressed on many solid cancers, including prostate cancer, renal cell carcinoma, melanoma, squamous cell carcinoma of the head and neck, non-small cell lung cancer, and breast cancer. Overexpression of B7-H3 is associated with disease severity, risk of recurrence and reduced survival. In this article, we report the preclinical development of MGC018, an antibody-drug conjugate targeted against B7-H3. MGC018 is comprised of the cleavable linker-duocarmycin payload, valine-citrulline-seco duocarmycin hydroxybenzamide azaindole (vc-seco-DUBA), conjugated to an anti-B7-H3 humanized IgG1/kappa mAb through reduced interchain disulfides, with an average drug-to-antibody ratio of approximately 2.7. MGC018 exhibited cytotoxicity toward B7-H3-positive human tumor cell lines, and exhibited bystander killing of target-negative tumor cells when cocultured with B7-H3-positive tumor cells. MGC018 displayed potent antitumor activity in preclinical tumor models of breast, ovarian, and lung cancer, as well as melanoma. In addition, antitumor activity was observed toward patient-derived xenograft models of breast, prostate, and head and neck cancer displaying heterogeneous expression of B7-H3. Importantly, MGC018 exhibited a favorable pharmacokinetic and safety profile in cynomolgus monkeys following repeat-dose administration. The antitumor activity observed preclinically with MGC018, together with the positive safety profile, provides evidence of a potentially favorable therapeutic index and supports the continued development of MGC018 for the treatment of solid cancers. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/11/2235/F1.large.jpg.

CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors.

PURPOSE: Patients with relapsed pediatric solid tumors and CNS malignancies have few therapeutic options and frequently die of their disease. Chimeric antigen receptor (CAR) T cells have shown tremendous success in treating relapsed pediatric acute lymphoblastic leukemia, but this has not yet translated to treating solid tumors. This is partially due to a paucity of differentially expressed cell surface molecules on solid tumors that can be safely targeted. Here, we present B7-H3 (CD276) as a putative target for CAR T-cell therapy of pediatric solid tumors, including those arising in the central nervous system. EXPERIMENTAL DESIGN: We developed a novel B7-H3 CAR whose binder is derived from a mAb that has been shown to preferentially bind tumor tissues and has been safely used in humans in early-phase clinical trials. We tested B7-H3 CAR T cells in a variety of pediatric cancer models. RESULTS: B7-H3 CAR T cells mediate significant antitumor activity in vivo, causing regression of established solid tumors in xenograft models including osteosarcoma, medulloblastoma, and Ewing sarcoma. We demonstrate that B7-H3 CAR T-cell efficacy is largely dependent upon high surface target antigen density on tumor tissues and that activity is greatly diminished against target cells that express low levels of antigen, thus providing a possible therapeutic window despite low-level normal tissue expression of B7-H3. CONCLUSIONS: B7-H3 CAR T cells could represent an exciting therapeutic option for patients with certain lethal relapsed or refractory pediatric malignancies, and should be tested in carefully designed clinical trials.

Development of MGD007, a gpA33 x CD3-Bispecific DART Protein for T-Cell Immunotherapy of Metastatic Colorectal Cancer.

We have developed MGD007 (anti-glycoprotein A33 x anti-CD3), a DART protein designed to redirect T cells to target gpA33 expressing colon cancer. The gpA33 target was selected on the basis of an antibody-based screen to identify cancer antigens universally expressed in both primary and metastatic colorectal cancer specimens, including putative cancer stem cell populations. MGD007 displays the anticipated-bispecific binding properties and mediates potent lysis of gpA33-positive cancer cell lines, including models of colorectal cancer stem cells, through recruitment of T cells. Xenograft studies showed tumor growth inhibition at doses as low as 4 µg/kg. Both CD8 and CD4 T cells mediated lysis of gpA33-expressing tumor cells, with activity accompanied by increases in granzyme and perforin. Notably, suppressive T-cell populations could also be leveraged to mediate lysis of gpA33-expressing tumor cells. Concomitant with CTL activity, both T-cell activation and expansion are observed in a gpA33-dependent manner. No cytokine activation was observed with human PBMC alone, consistent with the absence of gpA33 expression on peripheral blood cell populations. Following prolonged exposure to MGD007 and gpA33 positive tumor cells, T cells express PD-1 and LAG-3 and acquire a memory phenotype but retain ability to support potent cell killing. In cynomolgus monkeys, 4 weekly doses of 100 µg/kg were well tolerated, with prolonged PK consistent with that of an Fc-containing molecule. Taken together, MGD007 displays potent activity against colorectal cancer cells consistent with a mechanism of action endowed in its design and support further investigation of MGD007 as a potential novel therapeutic treatment for colorectal cancer. Mol Cancer Ther; 17(8); 1761-72. ©2018 AACR.

Tailoring CD19xCD3-DART exposure enhances T-cells to eradication of B-cell neoplasms.

Many patients with B-cell malignancies can be successfully treated, although tumor eradication is rarely achieved. T-cell-directed killing of tumor cells using engineered T-cells or bispecific antibodies is a promising approach for the treatment of hematologic malignancies. We investigated the efficacy of CD19xCD3 DART bispecific antibody in a broad panel of human primary B-cell malignancies. The CD19xCD3 DART identified 2 distinct subsets of patients, in which the neoplastic lymphocytes were eliminated with rapid or slow kinetics. Delayed responses were always overcome by a prolonged or repeated DART exposure. Both CD4 and CD8 effector cytotoxic cells were generated, and DART-mediated killing of CD4+ cells into cytotoxic effectors required the presence of CD8+ cells. Serial exposures to DART led to the exponential expansion of CD4 + and CD8 + cells and to the sequential ablation of neoplastic cells in absence of a PD-L1-mediated exhaustion. Lastly, patient-derived neoplastic B-cells (B-Acute Lymphoblast Leukemia and Diffuse Large B Cell Lymphoma) could be proficiently eradicated in a xenograft mouse model by DART-armed cytokine induced killer (CIK) cells. Collectively, patient tailored DART exposures can result in the effective elimination of CD19 positive leukemia and B-cell lymphoma and the association of bispecific antibodies with unmatched CIK cells represents an effective modality for the treatment of CD19 positive leukemia/lymphoma.

MGD011, A CD19 x CD3 Dual-Affinity Retargeting Bi-specific Molecule Incorporating Extended Circulating Half-life for the Treatment of B-Cell Malignancies.

Purpose: CD19, a B-cell lineage-specific marker, is highly represented in B-cell malignancies and an attractive target for therapeutic interventions. MGD011 is a CD19 x CD3 DART bispecific protein designed to redirect T lymphocytes to eliminate CD19-expressing cells. MGD011 has been engineered with a modified human Fc domain for improved pharmacokinetic (PK) properties and designed to cross-react with the corresponding antigens in cynomolgus monkeys. Here, we report on the preclinical activity, safety and PK properties of MGD011.Experimental Design: The activity of MGD011 was evaluated in several in vitro and in vivo models. PK, safety and pharmacodynamic activity was also assessed in dose-escalation and repeat-dose studies of MGD011 administered once weekly in cynomolgus monkeys.Results: MGD011 mediated killing of human B-cell lymphoma lines by human or cynomolgus monkey PBMCs as well as autologous B-cell depletion in PBMCs from both species. MGD011-mediated killing was accompanied by target-dependent T-cell activation and expansion, cytokine release and upregulation of perforin and granzyme B. MGD011 demonstrated antitumor activity against localized and disseminated lymphoma xenografts reconstituted with human PBMCs. In cynomolgus monkeys, MGD011 displayed a terminal half-life of 6.7 days; once weekly intravenous infusion of MGD011 at doses up to 100 µg/kg, the highest dose tested, was well tolerated and resulted in dose-dependent, durable decreases in circulating B cells accompanied by profound reductions of B lymphocytes in lymphoid organs.Conclusions: The preclinical activity, safety and PK profile support clinical investigation of MGD011 as a therapeutic candidate for the treatment of B-cell malignancies. Clin Cancer Res; 23(6); 1506-18. ©2016 AACR.

Targeting CD123 in acute myeloid leukemia using a T-cell-directed dual-affinity retargeting platform.

T-cell-directed killing of tumor cells using bispecific antibodies is a promising approach for the treatment of hematologic malignancies. Here we describe our preclinical work with a dual-affinity retargeting (DART) molecule generated from antibodies to CD3 and CD123, designed to redirect T cells against acute myeloid leukemia blasts. The CD3×CD123 DART (also referred to as MGD006/S80880) consists of 2 independent polypeptides, each composed of the VH of 1 antibody in tandem with the VL of the other antibody. The target antigen CD123 (interleukin 3RA) is highly and differentially expressed in acute myeloid leukemia (AML) blasts compared with normal hematopoietic stem and progenitor cells. In this study we demonstrate that the CD3×CD123 DART binds to both human CD3 and CD123 to mediate target-effector cell association, T-cell activation, proliferation, and receptor diversification. The CD3×CD123 DART also induces a dose-dependent killing of AML cell lines and primary AML blasts in vitro and in vivo. These results provide the basis for testing the CD3×CD123 DART in the treatment of patients with CD123(+) AML.

Development of PF-06671008, a Highly Potent Anti-P-cadherin/Anti-CD3 Bispecific DART Molecule with Extended Half-Life for the Treatment of Cancer.

Bispecific antibodies offer a promising approach for the treatment of cancer but can be challenging to engineer and manufacture. Here we report the development of PF-06671008, an extended-half-life dual-affinity re-targeting (DART®) bispecific molecule against P-cadherin and CD3 that demonstrates antibody-like properties. Using phage display, we identified anti-P-cadherin single chain Fv (scFv) that were subsequently affinity-optimized to picomolar affinity using stringent phage selection strategies, resulting in low picomolar potency in cytotoxic T lymphocyte (CTL) killing assays in the DART format. The crystal structure of this disulfide-constrained diabody shows that it forms a novel compact structure with the two antigen binding sites separated from each other by approximately 30 Å and facing approximately 90° apart. We show here that introduction of the human Fc domain in PF-06671008 has produced a molecule with an extended half-life (-4.4 days in human FcRn knock-in mice), high stability (Tm1 > 68 °C), high expression (>1 g/L), and robust purification properties (highly pure heterodimer), all with minimal impact on potency. Finally, we demonstrate in vivo anti-tumor efficacy in a human colorectal/human peripheral blood mononuclear cell (PBMC) co-mix xenograft mouse model. These results suggest PF-06671008 is a promising new bispecific for the treatment of patients with solid tumors expressing P-cadherin.

A CD3xCD123 bispecific DART for redirecting host T cells to myelogenous leukemia: preclinical activity and safety in nonhuman primates.

Current therapies for acute myeloid leukemia (AML) are largely ineffective, and AML patients may benefit from targeted immunotherapy approaches. MGD006 is a bispecific CD3xCD123 dual-affinity re-targeting (DART) molecule that binds T lymphocytes and cells expressing CD123, an antigen up-regulated in several hematological malignancies including AML. MGD006 mediates blast killing in AML samples, together with concomitant activation and expansion of residual T cells. MGD006 is designed to be rapidly cleared, and therefore requires continuous delivery. In a mouse model of continuous administration, MGD006 eliminated engrafted KG-1a cells (an AML-M0 line) in human PBMC (peripheral blood mononuclear cell)-reconstituted NSG/ß2m(-/-) mice at doses as low as 0.5 µg/kg per day for ~7 days. MGD006 binds to human and cynomolgus monkey antigens with similar affinities and redirects T cells from either species to kill CD123-expressing target cells. MGD006 was well tolerated in monkeys continuously infused with 0.1 µg/kg per day escalated weekly to up to 1 µg/kg per day during a 4-week period. Depletion of circulating CD123-positive cells was observed as early as 72 hours after treatment initiation and persisted throughout the infusion period. Cytokine release, observed after the first infusion, was reduced after subsequent administrations, even when the dose was escalated. T cells from animals with prolonged in vivo exposure exhibited unperturbed target cell lysis ex vivo, indicating no exhaustion. A transient decrease in red cell mass was observed, with no neutropenia or thrombocytopenia. These studies support clinical testing of MGD006 in hematological malignancies, including AML.

Isolation of cancer stem like cells from human adenosquamous carcinoma of the lung supports a monoclonal origin from a multipotential tissue stem cell.

There is increasing evidence that many solid tumors are hierarchically organized with the bulk tumor cells having limited replication potential, but are sustained by a stem-like cell that perpetuates the tumor. These cancer stem cells have been hypothesized to originate from transformation of adult tissue stem cells, or through re-acquisition of stem-like properties by progenitor cells. Adenosquamous carcinoma (ASC) is an aggressive type of lung cancer that contains a mixture of cells with squamous (cytokeratin 5+) and adenocarcinoma (cytokeratin 7+) phenotypes. The origin of these mixtures is unclear as squamous carcinomas are thought to arise from basal cells in the upper respiratory tract while adenocarcinomas are believed to form from stem cells in the bronchial alveolar junction. We have isolated and characterized cancer stem-like populations from ASC through application of selective defined culture medium initially used to grow human lung stem cells. Homogeneous cells selected from ASC tumor specimens were stably expanded in vitro. Primary xenografts and metastatic lesions derived from these cells in NSG mice fully recapitulate both the adenocarcinoma and squamous features of the patient tumor. Interestingly, while the CSLC all co-expressed cytokeratins 5 and 7, most xenograft cells expressed either one, or neither, with <10% remaining double positive. We also demonstrated the potential of the CSLC to differentiate to multi-lineage structures with branching lung morphology expressing bronchial, alveolar and neuroendocrine markers in vitro. Taken together the properties of these ASC-derived CSLC suggests that ASC may arise from a primitive lung stem cell distinct from the bronchial-alveolar or basal stem cells.

Teplizumab preserves C-peptide in recent-onset type 1 diabetes: two-year results from the randomized, placebo-controlled Protégé trial.

Protégé was a phase 3, randomized, double-blind, parallel, placebo-controlled 2-year study of three intravenous teplizumab dosing regimens, administered daily for 14 days at baseline and again after 26 weeks, in new-onset type 1 diabetes. We sought to determine efficacy and safety of teplizumab immunotherapy at 2 years and to identify characteristics associated with therapeutic response. Of 516 randomized patients, 513 were treated, and 462 completed 2 years of follow-up. Teplizumab (14-day full-dose) reduced the loss of C-peptide mean area under the curve (AUC), a prespecified secondary end point, at 2 years versus placebo. In analyses of prespecified and post hoc subsets at entry, U.S. residents, patients with C-peptide mean AUC >0.2 nmol/L, those randomized ≤6 weeks after diagnosis, HbA1c <7.5% (58 mmol/mol), insulin use <0.4 units/kg/day, and 8-17 years of age each had greater teplizumab-associated C-peptide preservation than their counterparts. Exogenous insulin needs tended to be reduced versus placebo. Antidrug antibodies developed in some patients, without apparent change in drug efficacy. No new safety or tolerability issues were observed during year 2. In summary, anti-CD3 therapy reduced C-peptide loss 2 years after diagnosis using a tolerable dose.

C-reactive protein causes insulin resistance in mice through Fcγ receptor IIB-mediated inhibition of skeletal muscle glucose delivery.

Elevations in C-reactive protein (CRP) are associated with an increased risk of insulin resistance. Whether CRP plays a causal role is unknown. Here we show that CRP transgenic mice and wild-type mice administered recombinant CRP are insulin resistant. Mice lacking the inhibitory Fcγ receptor IIB (FcγRIIB) are protected from CRP-induced insulin resistance, and immunohistochemistry reveals that FcγRIIB is expressed in skeletal muscle microvascular endothelium and is absent in skeletal muscle myocytes, adipocytes, and hepatocytes. The primary mechanism in glucose homeostasis disrupted by CRP is skeletal muscle glucose delivery, and CRP attenuates insulin-induced skeletal muscle blood flow. CRP does not impair skeletal muscle glucose delivery in FcγRIIB(-/-) mice or in endothelial nitric oxide synthase knock-in mice with phosphomimetic modification of Ser1176, which is normally phosphorylated by insulin signaling to stimulate nitric oxide-mediated skeletal muscle blood flow and glucose delivery and is dephosphorylated by CRP/FcγRIIB. Thus, CRP causes insulin resistance in mice through FcγRIIB-mediated inhibition of skeletal muscle glucose delivery.