Brentuximab Vedotin-Driven Microtubule Disruption Results in Endoplasmic Reticulum Stress Leading to Immunogenic Cell Death and Antitumor Immunity.

Brentuximab vedotin, a CD30-directed antibody-drug conjugate (ADC), is approved for clinical use in multiple CD30-expressing lymphomas. The cytotoxic payload component of brentuximab vedotin is monomethyl auristatin E (MMAE), a highly potent microtubule-disrupting agent. Preclinical results provided here demonstrate that treatment of cancer cells with brentuximab vedotin or free MMAE leads to a catastrophic disruption of the microtubule network eliciting a robust endoplasmic reticulum (ER) stress response that culminates in the induction of the classic hallmarks of immunogenic cell death (ICD). In accordance with the induction of ICD, brentuximab vedotin-killed lymphoma cells drove innate immune cell activation in vitro and in vivo. In the "gold-standard" test of ICD, vaccination of mice with brentuximab vedotin or free MMAE-killed tumor cells protected animals from tumor rechallenge; in addition, T cells transferred from previously vaccinated animals slowed tumor growth in immunodeficient mice. Immunity acquired from killed tumor cell vaccination was further amplified by the addition of PD-1 blockade. In a humanized model of CD30+ B-cell tumors, treatment with brentuximab vedotin drove the expansion and recruitment of autologous Epstein-Barr virus-reactive CD8+ T cells potentiating the activity of anti-PD-1 therapy. Together, these data support the ability of brentuximab vedotin and MMAE to drive ICD in tumor cells resulting in the activation of antigen-presenting cells and augmented T-cell immunity. These data provide a strong rationale for the clinical combination of brentuximab vedotin and other MMAE-based ADCs with checkpoint inhibitors.

SGN-B6A: A New Vedotin Antibody-Drug Conjugate Directed to Integrin Beta-6 for Multiple Carcinoma Indications.

Integrin beta-6, a component of the heterodimeric adhesion receptor alpha-v/beta-6, is overexpressed in numerous solid tumors. Its expression has been shown by multiple investigators to be a negative prognostic indicator in diverse cancers including colorectal, non-small cell lung, gastric, and cervical. We developed SGN-B6A as an antibody-drug conjugate (ADC) directed to integrin beta-6 to deliver the clinically validated payload monomethyl auristatin E (MMAE) to cancer cells. The antibody component of SGN-B6A is specific for integrin beta-6 and does not bind other alpha-v family members. In preclinical studies, this ADC has demonstrated activity in vivo in models derived from non-small cell lung, pancreatic, pharyngeal, and bladder carcinomas spanning a range of antigen expression levels. In nonclinical toxicology studies in cynomolgus monkeys, doses of up to 5 mg/kg weekly for four doses or 6 mg/kg every 3 weeks for two doses were tolerated. Hematologic toxicities typical of MMAE ADCs were dose limiting, and no significant target-mediated toxicity was observed. A phase I first-in-human study is in progress to evaluate the safety and antitumor activity of SGN-B6A in a variety of solid tumors known to express integrin beta-6 (NCT04389632).

An in Vitro Assay Using Cultured Kupffer Cells Can Predict the Impact of Drug Conjugation on in Vivo Antibody Pharmacokinetics.

While antibody-drug conjugates (ADCs) are advancing through clinical testing and receiving new marketing approvals, improvements to the technology continue to be developed in both academic and industrial laboratories. Among the key ADC attributes that can be improved upon with new technology are their biodistribution and pharmacokinetic properties. During the course of ADC development, it has become apparent that conjugation of drugs to the surface of a monoclonal antibody can alter its physicochemical characteristics in a manner that results in increased nonspecific interactions and more rapid elimination from plasma. Researchers in the field have typically relied upon in vivo studies in preclinical models to understand how a particular ADC chemistry will impact these biological characteristics. In previous work, we described how animal studies have revealed a relationship between ADC hydrophobicity, pharmacokinetics, and nonspecific hepatic clearance, particularly by sinusoidal endothelium and Kupffer cells. Here, we describe a fluorescence-based assay using cultured Kupffer cells to recapitulate the nonspecific interactions that lead to ADC clearance in an in vitro setting with the aim of developing a tool for predicting the pharmacokinetics of novel ADC designs. Output from this assay has demonstrated an excellent correlation with plasma clearance for a series of closely related ADCs bearing discrete PEG chains of varying length and has proven useful in interrogating the mechanism of the interactions between ADCs and Kupffer cells.

Characterization of SGN-CD123A, A Potent CD123-Directed Antibody-Drug Conjugate for Acute Myeloid Leukemia.

Treatment choices for acute myelogenous leukemia (AML) patients resistant to conventional chemotherapies are limited and novel therapeutic agents are needed. IL3 receptor alpha (IL3Rα, or CD123) is expressed on the majority of AML blasts, and there is evidence that its expression is increased on leukemic relative to normal hematopoietic stem cells, which makes it an attractive target for antibody-based therapy. Here, we report the generation and preclinical characterization of SGN-CD123A, an antibody-drug conjugate using the pyrrolobenzodiazepine dimer (PBD) linker and a humanized CD123 antibody with engineered cysteines for site-specific conjugation. Mechanistically, SGN-CD123A induces activation of DNA damage response pathways, cell-cycle changes, and apoptosis in AML cells. In vitro, SGN-CD123A-mediated potent cytotoxicity of 11/12 CD123+ AML cell lines and 20/23 primary samples from AML patients, including those with unfavorable cytogenetic profiles or FLT3 mutations. In vivo, SGN-CD123A treatment led to AML eradication in a disseminated disease model, remission in a subcutaneous xenograft model, and significant growth delay in a multidrug resistance xenograft model. Moreover, SGN-CD123A also resulted in durable complete remission of a patient-derived xenograft AML model. When combined with a FLT3 inhibitor quizartinib, SGN-CD123A enhanced the activity of quizartinib against two FLT3-mutated xenograft models. Overall, these data demonstrate that SGN-CD123A is a potent antileukemic agent, supporting an ongoing trial to evaluate its safety and efficacy in AML patients (NCT02848248). Mol Cancer Ther; 17(2); 554-64. ©2017 AACR.

Tumor-Associated Macrophages Can Contribute to Antitumor Activity through FcγR-Mediated Processing of Antibody-Drug Conjugates.

The primary mechanism of antibody-drug conjugates (ADC) is targeted delivery of a cytotoxic payload to tumor cells via cancer-associated membrane receptors. However, the tumor microenvironment likely plays a role in ADC penetration, distribution, and processing and thus impacts the overall antitumor activity. Here, we report on the potential contribution of Fc-FcγR interactions between ADCs and tumor-associated macrophages (TAM) to the preclinical antitumor activities of ADCs. In the CD30+ L-428 Hodgkin lymphoma model, anti-CD30-vcMMAE and a non-binding control (hIgG-vcMMAE) demonstrated similar antitumor activity as well as similar payload release in the tumors. IHC analysis revealed L-428 tumors contained highly abundant TAMs, which were confirmed to bind ADCs by IHC and flow cytometry. The infiltration of TAMs was further found to correlate with the antitumor activity of the non-binding hIgG-vcMMAE in five additional xenograft models. hIgG1V1-vcMMAE, bearing a mutation in the Fc region which ablates Fc gamma receptor (FcγR) binding, lost antitumor activity in three TAM-high xenograft models, suggesting Fc-FcγR interactions modulate the TAM-ADC interaction. Our results suggest that TAMs can contribute to ADC processing through FcγR interaction in preclinical tumor models and may represent an important additional mechanism for drug release from ADCs. Correlative studies in clinical trials will further shed light on whether TAMs play a role in patients' response to ADC therapies. Mol Cancer Ther; 16(7); 1347-54. ©2017 AACR.

Intracellular Released Payload Influences Potency and Bystander-Killing Effects of Antibody-Drug Conjugates in Preclinical Models.

Antibody-drug conjugates (ADC) comprise targeting antibodies armed with potent small-molecule payloads. ADCs demonstrate specific cell killing in clinic, but the basis of their antitumor activity is not fully understood. In this study, we investigated the degree to which payload release predicts ADC activity in vitro and in vivo ADCs were generated to target different receptors on the anaplastic large cell lymphoma line L-82, but delivered the same cytotoxic payload (monomethyl auristatin E, MMAE), and we found that the intracellular concentration of released MMAE correlated with in vitro ADC-mediated cytotoxicity independent of target expression or drug:antibody ratios. Intratumoral MMAE concentrations consistently correlated with the extent of tumor growth inhibition in tumor xenograft models. In addition, we developed a robust admixed tumor model consisting of CD30(+) and CD30(-) cancer cells to study how heterogeneity of target antigen expression, a phenomenon often observed in cancer specimens, affects the treatment response. CD30-targeting ADC delivering membrane permeable MMAE or pyrrolobenzodiazepine dimers demonstrated potent bystander killing of neighboring CD30(-) cells. In contrast, a less membrane permeable payload, MMAF, failed to mediate bystander killing in vivo, suggesting local diffusion and distribution of released payloads represents a potential mechanism of ADC-mediated bystander killing. Collectively, our findings establish that the biophysical properties and amount of released payloads are chief factors determining the overall ADC potency and bystander killing. Cancer Res; 76(9); 2710-9. ©2016 AACR.

Reducing hydrophobicity of homogeneous antibody-drug conjugates improves pharmacokinetics and therapeutic index.

The in vitro potency of antibody-drug conjugates (ADCs) increases with the drug-to-antibody ratio (DAR); however, ADC plasma clearance also increases with DAR, reducing exposure and in vivo efficacy. Here we show that accelerated clearance arises from ADC hydrophobicity, which can be modulated through drug-linker design. We exemplify this using hydrophilic auristatin drug linkers and PEGylated ADCs that yield uniform, high-DAR ADCs with superior in vivo performance.

Anti-leukemic activity of lintuzumab (SGN-33) in preclinical models of acute myeloid leukemia.

Despite therapeutic advances, the long-term survival rates for acute myeloid leukemia (AML) are estimated to be 10% or less, pointing to the need for better treatment options. AML cells express the myeloid marker CD33, making it amenable to CD33-targeted therapy. Thus, the in vitro and in vivo anti-tumor activities of lintuzumab (SGN-33), a humanized monoclonal anti-CD33 antibody undergoing clinical evaluation, were investigated. In vitro assays were used to assess the ability of lintuzumab to mediate effector functions and to decrease the production of growth factors from AML cells. SCID mice models of disseminated AML with the multi-drug resistance (MDR)-negative HL60 and the MDR(+), HEL9217 and TF1-alpha, cell lines were developed and applied to examine the in vivo antitumor activity. In vitro, lintuzumab significantly reduced the production of TNFalpha-induced pro-inflammatory cytokines and chemokines by AML cells. Lintuzumab promoted tumor cell killing through antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities against MDR(-) and MDR(+) AML cell lines and primary AML patient samples. At doses from 3 to 30 mg/kg, lintuzumab significantly enhanced survival and reduced tumor burden in vivo, regardless of MDR status. Survival of the mice was dependent upon the activity of resident macrophages and neutrophils. The results suggest that lintuzumab may exert its therapeutic effects by modulating the cytokine milieu in the tumor microenvironment and through effector mediated cell killing. Given that lintuzumab induced meaningful responses in a phase 1 clinical trial, the preclinical antitumor activities defined in this study may underlie its observed therapeutic efficacy in AML patients.

A novel mutation in CD83 results in the development of a unique population of CD4+ T cells.

Using a mouse mutagenesis screen, we have identified CD83 as being critical for the development of CD4(+) T cells and for their function postactivation. CD11c(+) dendritic cells develop and function normally in mice with a mutated CD83 gene but CD4(+) T cell development is substantially reduced. Additionally, we now show that those CD4(+) cells that develop in a CD83 mutant animal fail to respond normally following allogeneic stimulation. This is at least in part due to an altered cytokine expression pattern characterized by an increased production of IL-4 and IL-10 and diminished IL-2 production. Thus, in addition to its role in selection of CD4(+) T cells, absence of CD83 results in the generation of cells with an altered activation and cytokine profile.

Broad-spectrum caspase inhibition paradoxically augments cell death in TNF-alpha -stimulated neutrophils.

It is increasingly clear that there are caspase-dependent and -independent mechanisms for the execution of cell death and that the utilization of these mechanisms is stimulus- and cell type-dependent. Intriguingly, broad-spectrum caspase inhibition enhances death receptor agonist-induced cell death in a few transformed cell lines. Endogenously produced oxidants are causally linked to necroticlike cell death in these instances. We report here that broad-spectrum caspase inhibitors effectively attenuated apoptosis induced in human neutrophils by incubation with agonistic anti-Fas antibody or by coincubation with tumor necrosis factor-alpha (TNF-alpha) and cycloheximide ex vivo. In contrast, the same caspase inhibitors could augment cell death upon stimulation by TNF-alpha alone during the 6-hour time course examined. Caspase inhibitor-sensitized, TNF-alpha-stimulated, dying neutrophils exhibit apoptoticlike and necroticlike features. This occurred without apparent alteration in nuclear factor-kappaB (NF-kappaB) activation. Nevertheless, intracellular oxidant production was enhanced and sustained in caspase inhibitor-sensitized, TNF-alpha-stimulated neutrophils obtained from healthy subjects. However, despite reduced or absent intracellular oxidant production following TNF-alpha stimulation, cell death was also augmented in neutrophils isolated from patients with chronic granulomatous disease incubated with a caspase inhibitor and TNF-alpha. These results demonstrate that, in human neutrophils, TNF-alpha induces a caspase-independent but protein synthesis-dependent cell death signal. Furthermore, they suggest that TNF-alpha activates a caspase-dependent pathway that negatively regulates reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity.

A role for cysteinyl leukotrienes in airway remodeling in a mouse asthma model.

Airway inflammation and remodeling in chronic asthma are characterized by airway eosinophilia, hyperplasia of goblet cells and smooth muscle, and subepithelial fibrosis. We examined the role of leukotrienes in a mouse model of allergen-induced chronic lung inflammation and fibrosis. BALB/c mice, after intraperitoneal ovalbumin (OVA) sensitization on Days 0 and 14, received intranasal OVA periodically Days 14-75. The OVA-treated mice developed an extensive eosinophil and mononuclear cell inflammatory response, goblet cell hyperplasia, and mucus occlusion of the airways. A striking feature of this inflammatory response was the widespread deposition of collagen beneath the airway epithelial cell layer and also in the lung interstitium in the sites of leukocytic infiltration that was not observed in the saline-treated controls. The cysteinyl leukotriene(1) (CysLT(1)) receptor antagonist montelukast significantly reduced the airway eosinophil infiltration, mucus plugging, smooth muscle hyperplasia, and subepithelial fibrosis in the OVA-sensitized/challenged mice. The presence of Charcot-Leyden-like crystals in airway macrophages and the increased interleukin (IL)-4 and IL-13 mRNA expression in lung tissue and protein in BAL fluid seen in OVA-treated mice were also inhibited by CysLT(1) receptor blockade. These data suggest an important role for cysteinyl leukotrienes in the pathogenesis of chronic allergic airway inflammation with fibrosis.