Pina M. Cardarelli

Antibody-drug conjugates: current status and future directions.

Antibody-drug conjugates (ADCs) aim to take advantage of the specificity of monoclonal antibodies (mAbs) to deliver potent cytotoxic drugs selectively to antigen-expressing tumor cells. Despite the simple concept, various parameters must be considered when designing optimal ADCs, such as selection of the appropriate antigen target and conjugation method. Each component of the ADC (the antibody, linker and drug) must also be optimized to fully realize the goal of a targeted therapy with improved efficacy and tolerability. Advancements over the past several decades have led to a new generation of ADCs comprising non-immunogenic mAbs, linkers with balanced stability and highly potent cytotoxic agents. Although challenges remain, recent clinical success has generated intense interest in this therapeutic class.

A phase II, randomized, double‐blind, placebo‐controlled study evaluating the efficacy and safety of MDX‐1100, a fully human anti‐CXCL10 monoclonal antibody, in combination with methotrexate in patients with rheumatoid arthritis

OBJECTIVE CXCL10 (also known as interferon-γ-inducible 10-kd protein [IP-10]) is a chemokine that potentially plays a role in the immunopathogenesis of rheumatoid arthritis (RA). We undertook this phase II study to evaluate the efficacy and safety of MDX-1100, a fully human, anti-CXCL10 (anti-IP-10) monoclonal antibody, in RA patients whose disease responded inadequately to methotrexate (MTX). METHODS Patients with active RA receiving stable doses of MTX (10-25 mg weekly) were randomized to receive intravenous doses of 10 mg/kg MDX-1100 (n = 35) or placebo (n = 35) every other week. The primary end point was the proportion of patients meeting the American College of Rheumatology 20% improvement criteria (achieving an ACR20 response) on day 85, and patients were followed up for safety to day 141. RESULTS The ACR20 response rate was significantly higher among MDX-1100-treated patients than among placebo-treated patients (54% versus 17%; P = 0.0024). Statistically significant differences in the ACR20 response rate between treatments were observed starting on day 43 (P < 0.05). The ACR50 and ACR70 response rates on day 85 did not differ between the groups. Overall, 51.4% of MDX-1100-treated patients and 30.3% of placebo-treated patients experienced at least 1 adverse event (AE). No study drug-related serious AEs were reported. CONCLUSION MDX-1100 was well tolerated and demonstrated clinical efficacy in RA patients whose disease responded inadequately to MTX. This is the first study to demonstrate clinical efficacy of a chemokine inhibitor in RA and supports the notion of a potential role of IP-10 in the immunopathogenesis of RA.

BMS-936564/MDX-1338: A Fully Human Anti-CXCR4 Antibody Induces Apoptosis In Vitro and Shows Antitumor Activity In Vivo in Hematologic Malignancies

Purpose: CXCR4 has been identified as a prognostic marker for acute myeloid leukemia (AML) and other malignancies. We describe the development and characterization of a fully human antibody to CXCR4 and its application for therapy of AML, non–Hodgkin lymphoma (NHL), chronic lymphoid leukemia (CLL), and multiple myeloma. Experimental Design: Human transgenic mice were immunized with CXCR4-expressing cells, and antibodies reactive with CXCR4 were analyzed for apoptosis induction and ability to interfere with CXCL12-induced migration and calcium flux. In vivo efficacy was determined in multiple AML, NHL, and multiple myeloma xenograft tumors in severe combined immunodeficient mice. Results: BMS-936564/MDX-1338 is a fully human IgG4 monoclonal antibody that specifically recognizes human CXCR4. In vitro studies show that MDX-1338 binds to CXCR4-expressing cells with low nanomolar affinity, blocks CXCL12 binding to CXCR4-expressing cells, and inhibits CXCL12-induced migration and calcium flux with low nanomolar EC50 values. When given as monotherapy, MDX-1338 exhibits antitumor activity in established tumors including AML, NHL, and multiple myeloma xenograft models. In addition, we show that MDX-1338 induced apoptosis on a panel of cell lines and propose that antibody-induced apoptosis is one of the mechanisms of tumor growth inhibition. Conclusions: BMS-936564/MDX-1338 is a potent CXCR4 antagonist which is efficacious as monotherapy in tumor-bearing mice and is currently in phase I for the treatment of relapsed/refractory AML, NHL, CLL, and multiple myeloma. Clin Cancer Res; 19(2); 357–66. ©2012 AACR.

C1013G/CXCR4 acts as a driver mutation of tumor progression and modulator of drug resistance in lymphoplasmacytic lymphoma

The C-X-C chemokine receptor type 4 (CXCR4) plays a crucial role in modulating cell trafficking in hematopoietic stem cells and clonal B cells. We screened 418 patients with B-cell lymphoproliferative disorders and described the presence of the C1013G/CXCR4 warts, hypogammaglobulinemia, infections, and myelokathexis-associated mutation in 28.2% (37/131) of patients with lymphoplasmacytic lymphoma (Waldenström macroglobulinemia [WM]), being either absent or present in only 7% of other B-cell lymphomas. In vivo functional characterization demonstrates its activating role in WM cells, as demonstrated by significant tumor proliferation and dissemination to extramedullary organs, leading to disease progression and decreased survival. The use of a monoclonal antibody anti-CXCR4 led to significant tumor reduction in a C1013G/CXCR4 WM model, whereas drug resistance was observed in mutated WM cells exposed to Brutons tyrosine kinase, mammalian target of rapamycin, and phosphatidylinositol 3-kinase inhibitors, but not proteasome inhibitors. These findings demonstrate that C1013G/CXCR4 is an activating mutation in WM and support its role as a critical regulator of WM molecular pathogenesis and as an important therapeutic target.

CXCR4 regulates extra-medullary myeloma through epithelial-mesenchymal transition-like transcriptional activation

Extra-medullary disease (EMD) in multiple myeloma (MM) is associated with poor prognosis and resistance to chemotherapy. However, molecular alterations that lead to EMD have not been well defined. We developed bone marrow (BM)- and EMD-prone MM syngeneic cell lines; identified that epithelial-to-mesenchymal transition (EMT) transcriptional patterns were significantly enriched in both clones compared to parental cells, together with higher levels of CXCR4 protein; and demonstrated that CXCR4 enhanced the acquisition of an EMT-like phenotype in MM cells with a phenotypic conversion for invasion, leading to higher bone metastasis and EMD dissemination in vivo. In contrast, CXCR4 silencing led to inhibited tumor growth and reduced survival. Ulocuplumab, a monoclonal anti-CXCR4 antibody, inhibited MM cell dissemination, supported by suppression of the CXCR4-driven EMT-like phenotype. These results suggest that targeting CXCR4 may act as a regulator of EMD through EMT-like transcriptional modulation, thus representing a potential therapeutic strategy to prevent MM disease progression.

Preclinical Development of Ipilimumab and Nivolumab Combination Immunotherapy: Mouse Tumor Models, In Vitro Functional Studies, and Cynomolgus Macaque Toxicology

The monoclonal antibodies ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) have shown remarkable antitumor activity in an increasing number of cancers. When combined, ipilimumab and nivolumab have demonstrated superior activity in patients with metastatic melanoma (CHECKMATE-067). Here we describe the preclinical development strategy that predicted these clinical results. Synergistic antitumor activity in mouse MC38 and CT26 colorectal tumor models was observed with concurrent, but not sequential CTLA-4 and PD-1 blockade. Significant antitumor activity was maintained using a fixed dose of anti-CTLA-4 antibody with decreasing doses of anti-PD-1 antibody in the MC38 model. Immunohistochemical and flow cytometric analyses confirmed that CD3+ T cells accumulated at the tumor margin and infiltrated the tumor mass in response to the combination therapy, resulting in favorable effector and regulatory T-cell ratios, increased pro-inflammatory cytokine secretion, and activation of tumor-specific T cells. Similarly, in vitro studies with combined ipilimumab and nivolumab showed enhanced cytokine secretion in superantigen stimulation of human peripheral blood lymphocytes and in mixed lymphocyte response assays. In a cynomolgus macaque toxicology study, dose-dependent immune-related gastrointestinal inflammation was observed with the combination therapy; this response had not been observed in previous single agent cynomolgus studies. Together, these in vitro assays and in vivo models comprise a preclinical strategy for the identification and development of highly effective antitumor combination immunotherapies.

Pharmacokinetic characterization of BMS‐936561, an anti‐CD70 antibody‐drug conjugate, in preclinical animal species and prediction of its pharmacokinetics in humans

CD70 is a tumor necrosis factor (TNF)‐like type II integral membrane protein that is transiently expressed on activated T‐ and B‐lymphocytes. Aberrant expression of CD70 was identified in both solid tumors and haematologic malignancies. BMS‐936561 (αCD70_MED‐A) is an antibody‐drug conjugate composed of a fully human anti‐CD70 monoclonal antibody (αCD70) conjugated with a duocarmycin derivative, MED‐A, through a maleimide‐containing citrulline‐valine dipeptide linker. MED‐A is a carbamate prodrug that is activated by carboxylesterase to its active form, MED‐B, to exert its DNA alkylation activity. In vitro serum stability studies suggested the efficiencies of hydrolyzing the carbamate‐protecting group in αCD70_MED‐A followed a rank order of mouse > rat > > monkey > dog ~ human. Pharmacokinetics of αCD70_MED‐A was evaluated in mice, monkeys, and dogs after single intravenous doses. In mice, αCD70_MED‐A was cleared rapidly, with no detectable exposures after 15 min following dosing. In contrast, αCD70_MED‐A was much more stable in monkeys and dogs. The clearance of αCD70_MED‐A in monkeys was 58 mL/d/kg, ~2‐fold faster than that in dogs (31 mL/d/kg). The human PK profiles of the total αCD70 and αCD70_MED‐A were predicted using allometrically scaled monkeys PK parameters of αCD70 and the carbamate hydrolysis rate constant estimated in dogs. Comparing the predicted and observed human PK from the phase I study, the dose‐normalized concentration‐time profiles of αCD70_MED‐A and the total αCD70 were largely within the 5th‐95th percentile of the predicted profiles. Copyright

Abstract LB-237: BMS-986012, a fully human anti-fucosyl-GM1 antibody has potent in vitro and in vivo antitumor activity in preclinical models of small cell lung cancer

Fucosyl-GM1, is a sphingolipid monosialoganglioside and tumor-associated antigen with a high prevalence in human small cell lung cancer (SCLC), while its expression is minimal in most normal tissue. Here, we report results of our preclinical studies with BMS-986012, a novel fully human, non-fucosylated, IgG1 monoclonal antibody that specifically binds to FucGM1. The binding affinity of BMS-986012 was evaluated in Biacore® and cell-based assays. BMS-986012 binds with a high affinity to FucGM1 and demonstrates no significant binding to other closely related gangliosides. The antibody was engineered in a non-fucosylated IgG1 format, resulting in a 40-fold increased affinity for FcγRIIIa (CD16). In in vitro assays, BMS-986012 binds to FucGM1 on cancer cells and activates antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-mediated phagocytosis (ADCP). The antitumor activity of BMS-986012 was profiled in a panel of SCLC lung cancer models in immunodeficient mice with varying FucGM1 target expression levels qualified using immunohistochemistry. Dose-dependent antitumor activity was observed in a panel of SCLC xenograft models with tumor regressions occurring at BMS-986012 doses greater than 0.3 mg/kg against DMS79 tumors. BMS-986012 efficacy generally correlated with FucGM1 target expression in tumor models. In mice, the antibody was well tolerated, with no adverse events observed in long term efficacy studies. Preclinical studies also explored combinations of BMS-986012 with standard-of-care (SOC) chemotherapeutics, including cisplatin, etoposide, topotecan and irinotecan. Combination therapy of BMS-986012 with SOC resulted in significantly enhanced antitumor activity compared to monotherapy. Finally, studies conducted with BMS-986012 in combination with anti-CD137 antibody resulted in significant improvement in efficacy. These data provide preclinical support for evaluation of BMS-986012 in SCLC. BMS-986012 is currently in a phase I trial. Citation Format: Bing Chen, Chin Pan, Paul Ponath, Miho Oyasu, Daniel Menezes, Pina Cardarelli. BMS-986012, a fully human anti-fucosyl-GM1 antibody has potent in vitro and in vivo antitumor activity in preclinical models of small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-237.

Abstract LB-252: Enzymology of the mechanism of action for MDX-1203 antibody drug conjugate

MDX-1203 is an antibody drug conjugate under clinical evaluation by Bristol-Myers Squibb. The antibody portion is MDX-1115, a fully human anti-CD70 IgG. The drug portion is MED-2460, a small molecule which itself is composed of the releasable prodrug MED-2284, a cleavable di-peptide linker, and a maleimide reactive group intended to facilitate protein attachment. The mechanism of action of MDX-1203 involves tumor specific delivery and cellular uptake of ADC, followed by the intervention of two different enzymatic pathways. Release of prodrug is accomplished through proteolysis of a citruline-valine di-peptide linker, presumably by Cathepsin B activity in the acidic environment of the lysosome. Activation of released prodrug, MED-2284, occurs in the endoplasmic reticulum via esterase cleavage in the of the piperazine protecting group by the enzyme human Carboxylesterase 2. Michaelis Menten methodologies are used to probe activity of each enzymatic pathway and their relative role in the activity of the drug. Proteolysis is investigated through the use of Human Liver Cathepsin B protease. A kinetic model is constructed for a cysteine modified analogue MED-2460, and then applied to the more complex system involving antibody conjugated drug. Enzymatic activation of released prodrug MED-2284 is investigated with recombinant Human Carboxylesterase 2. For each pathway, the kinetic parameters Km, kcat, and enzymatic efficiency are measured and reported. Parameters are compared in an effort to define the enzymatic rate limiting step in the activation MDX-1203, and define their respective roles in the overall activity of the drug. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-252. doi:1538-7445.AM2012-LB-252

Abstract 2578: Efficacy of anti-CD22 antibody drug-conjugates in Ramos and Raji tumor xenograft models

CD22 is a well-validated target for immunotherapy on B cell tumors. CD22 is a B lymphoid lineage-specific differentiation antigen, and its cell surface expression coincides with IgD expression in human. CD22 expression is upregulated on mature B cells, and is lost upon terminal differentiation to plasma cells. CD22 is also highly expressed on B cell tumors including non-Hodgkin9s lymphoma (NHL) and acute lymphocytic leukemia (ALL). Antibodies to CD22 rapidly internalize, making it an ideal target for an antibody drug conjugate (ADC) therapeutic approach. Human antibodies to CD22 have been generated. The antibodies bind B cells with high affinity and are rapidly internalized upon binding. Anti-CD22 conjugates linked to highly potent synthetic DNA minor-groove binding alkylating (MGBA) agents have been prepared and tested in vivo for the treatment of established Ramos and Raji subcutaneous tumor xenografts. Anti-tumor efficacy of the anti-CD22 conjugate was highly specific in comparison with the isotype control conjugate. The anti-CD22 conjugate at a single dose of 0.3 µmole/kg resulted in regression of established subcutaneous tumors with less than 5% body weight loss and no clinical signs of toxicity. The anti-CD22 conjugate at a single dose of 0.1 µmole/kg was more efficacious than Rituximab treatment on Ramos tumors. The anti-CD22 conjugate also had better tumor growth inhibition than an anti-CD19 conjugate. In summary, anti-CD22-ADCs have been prepared which show superior in vivo efficacy to Rituximab and to anti-CD19-ADCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2578.