Trastuzumab-MMAU Antibody-Auristatin Conjugates: Valine-Glucoserine Linker with Stabilized Maleimide Conjugation Improves In Vivo Efficacy and Tolerability.

PURPOSE: Antibody-drug conjugates (ADCs) have shown impressive clinical activity with approval of many agents in hematological and solid tumors. However, challenges remain with both efficacy and safety of ADCs. This study describes novel trastuzumab-auristatin conjugates with the hydrophilic MMAE prodrug MMAU, and optimization of a glycopeptide linker leading to a wider therapeutic window. EXPERIMENTAL DESIGN: Trastuzumab was conjugated with auristatin payloads via a series of linkers using a stabilized maleimide handle. The ADCs were characterized in vitro and their relative in vivo anti-tumor efficacies were assessed in HER2+ xenograft models. Relative linker stabilities and the mechanism of linker cleavage were studied using in vitro assays. Toxicity and toxicokinetics of the best performing ADC were evaluated in cynomolgus monkey (cyno). RESULTS: The trastuzumab-MMAU ADC with stabilized glycopeptide linker showed maleimide stabilization and higher resistance to cleavage by serum and lysosomal enzymes compared to a valine-citrulline conjugated trastuzumab ADC (trastuzumab-vc-MMAE). A single dose of 1 or 2 mg/kg of trastuzumab-MMAU at drug-to-antibody ratios (DAR) of 8 and 4 respectively resulted in xenograft tumor growth inhibition, with superior efficacy to trastuzumab-vc-MMAE. Trastuzumab-MMAU DAR4 was tolerated at doses up to 12 mg/kg in cyno, which represents 2- to 4-fold higher dose than that observed with vedotin ADCs, and had increased terminal half-life and exposure. CONCLUSIONS: The optimized trastuzumab-MMAU ADC showed potent antitumor activity and was well tolerated with excellent pharmacokinetics in non-human primates, leading to a superior preclinical therapeutic window. The data supports potential utility of trastuzumab-MMAU for treatment of HER2+ tumors.

Development of disulfide-stabilized Fabs for targeting of antibody-directed nanotherapeutics.

Antibody-directed nanotherapeutics (ADNs) represent a promising delivery platform for selective delivery of an encapsulated drug payload to the site of disease that improves the therapeutic index. Although both single-chain Fv (scFv) and Fab antibody fragments have been used for targeting, no platform approach applicable to any target has emerged. scFv can suffer from intrinsic instability, and the Fabs are challenging to use due to native disulfide over-reduction and resulting impurities at the end of the conjugation process. This occurs because of the close proximity of the disulfide bond connecting the heavy and light chain to the free cysteine at the C-terminus, which is commonly used as the conjugation site. Here we show that by engineering an alternative heavy chain-light chain disulfide within the Fab, we can maintain efficient conjugation while eliminating the process impurities and retaining stability. We have demonstrated the utility of this technology for efficient ADN delivery and internalization for a series of targets, including EphA2, EGFR, and ErbB2. We expect that this technology will be broadly applicable for targeting of nanoparticle encapsulated payloads, including DNA, mRNA, and small molecules.

Antibody Co-Administration Can Improve Systemic and Local Distribution of Antibody-Drug Conjugates to Increase In Vivo Efficacy.

Several antibody-drug conjugates (ADC) showing strong clinical responses in solid tumors target high expression antigens (HER2, TROP2, Nectin-4, and folate receptor alpha/FRα). Highly expressed tumor antigens often have significant low-level expression in normal tissues, resulting in the potential for target-mediated drug disposition (TMDD) and increased clearance. However, ADCs often do not cross-react with normal tissue in animal models used to test efficacy (typically mice), and the impact of ADC binding to normal tissue antigens on tumor response remains unclear. An antibody that cross-reacts with human and murine FRα was generated and tested in an animal model where the antibody/ADC bind both human tumor FRα and mouse FRα in normal tissue. Previous work has demonstrated that a "carrier" dose of unconjugated antibody can improve the tumor penetration of ADCs with high expression target-antigens. A carrier dose was employed to study the impact on cross-reactive ADC clearance, distribution, and efficacy. Co-administration of unconjugated anti-FRα antibody with the ADC-improved efficacy, even in low expression models where co-administration normally lowers efficacy. By reducing target-antigen-mediated clearance in normal tissue, the co-administered antibody increased systemic exposure, improved tumor tissue penetration, reduced target-antigen-mediated uptake in normal tissue, and increased ADC efficacy. However, payload potency and tumor antigen saturation are also critical to efficacy, as shown with reduced efficacy using too high of a carrier dose. The judicious use of higher antibody doses, either through lower DAR or carrier doses, can improve the therapeutic window by increasing efficacy while lowering target-mediated toxicity in normal tissue.

Peri- and Postoperative Outcomes of Outpatient vs Inpatient Laparoscopic Apical Prolapse Repair.

STUDY OBJECTIVE: To assess the feasibility of outpatient laparoscopic management of apical pelvic organ prolapse along with indicated vaginal repairs and anti-incontinence procedures. DESIGN: Retrospective cohort study. SETTING: Tertiary-care academic center, Boston, MA. PATIENTS: Total of 112 patients seen in the minimally invasive gynecologic surgery and urogynecology clinics with symptomatic pelvic organ prolapse. INTERVENTIONS: Laparoscopic hysterectomy, sacrocervico- or sacrocolpopexy along with vaginal prolapse and anti-incontinence procedures as indicated from 2013 to 2017 at Brigham & Women's Hospital and Brigham & Women's Faulkner Hospital performed by a minimally invasive gynecologic surgery and urogynecology team. MEASUREMENTS AND MAIN RESULTS: Of the 112 patients, 52 were outpatient and 60 were admitted (median stay in admission group = 1 day; range 1-3). Patient baseline characteristics, American Society of Anesthesiologists' class, and pelvic organ prolapse quantification stage were similar between the outpatient and admitted cohorts. Most patients underwent hysterectomy at the time of the sacropexy (65.4% outpatient vs 73.3% admitted, p = .08). Concomitant apical prolapse repair was more common in the outpatient group (98.1% vs 85%, p = .02). The proportion of outpatient procedures increased from 17% in 2013 to a peak of 70% in 2016. Operating room time was shorter for the outpatient cohort (103.9 minutes vs 115.5 minutes, p = .04), but other perioperative outcomes were similar. There were no intraoperative complications. The numbers of postoperative complications, readmission, and reoperations were low and similar between outpatient and admitted cohorts. No factor was predictive of admission on regression analysis. CONCLUSION: Laparoscopic apical prolapse repair with concomitant vaginal repairs can be performed safely as an outpatient procedure. A unique team approach may foster a shorter, more efficient procedure without compromising short-term outcomes.

Antibody-mediated targeting of TNFR2 activates CD8+ T cells in mice and promotes antitumor immunity.

Tumor necrosis factor receptor 2 (TNFR2) is the alternate receptor for TNF and can mediate both pro- and anti-inflammatory activities of T cells. Although TNFR2 has been linked to enhanced suppressive activity of regulatory T cells (Tregs) in autoimmune diseases, the viability of TNFR2 as a target for cancer immunotherapy has been underappreciated. Here, we show that new murine monoclonal anti-TNFR2 antibodies yield robust antitumor activity and durable protective memory in multiple mouse cancer cell line models. The antibodies mediate potent Fc-dependent T cell costimulation and do not result in significant depletion of Tregs Corresponding human agonistic monoclonal anti-TNFR2 antibodies were identified and also had antitumor effects in humanized mouse models. Anti-TNFR2 antibodies could be developed as a novel treatment option for patients with cancer.

Antitumour activity and tolerability of an EphA2-targeted nanotherapeutic in multiple mouse models.

Antibody-mediated tumour targeting and nanoparticle-mediated encapsulation can reduce the toxicity of antitumour drugs and improve their efficacy. Here, we describe the performance of a nanotherapeutic encapsulating a hydrolytically sensitive docetaxel prodrug and conjugated to an antibody specific for EphA2-a receptor overexpressed in many tumours. Administration of the nanotherapeutic in mice led to slow and sustained release of the prodrug, reduced exposure of active docetaxel in the circulation (compared with administration of the free drug) and maintenance of optimal exposure of the drug in tumour tissue. We also show that administration of the nanotherapeutic in rats and dogs resulted in minimal haematological toxicity, as well as the absence of neutropenia and improved overall tolerability in multiple rodent models. Targeting of the nanotherapeutic to EphA2 improved tumour penetration and resulted in markedly enhanced antitumour activity (compared with administration of free docetaxel and non-targeted nanotherapeutic controls) in multiple tumour-xenografted mice. This nanomedicine could become a potent and safe therapeutic alternative for cancer patients undergoing chemotherapy.

MM-131, a bispecific anti-Met/EpCAM mAb, inhibits HGF-dependent and HGF-independent Met signaling through concurrent binding to EpCAM.

Activation of the Met receptor tyrosine kinase, either by its ligand, hepatocyte growth factor (HGF), or via ligand-independent mechanisms, such as MET amplification or receptor overexpression, has been implicated in driving tumor proliferation, metastasis, and resistance to therapy. Clinical development of Met-targeted antibodies has been challenging, however, as bivalent antibodies exhibit agonistic properties, whereas monovalent antibodies lack potency and the capacity to down-regulate Met. Through computational modeling, we found that the potency of a monovalent antibody targeting Met could be dramatically improved by introducing a second binding site that recognizes an unrelated, highly expressed antigen on the tumor cell surface. Guided by this prediction, we engineered MM-131, a bispecific antibody that is monovalent for both Met and epithelial cell adhesion molecule (EpCAM). MM-131 is a purely antagonistic antibody that blocks ligand-dependent and ligand-independent Met signaling by inhibiting HGF binding to Met and inducing receptor down-regulation. Together, these mechanisms lead to inhibition of proliferation in Met-driven cancer cells, inhibition of HGF-mediated cancer cell migration, and inhibition of tumor growth in HGF-dependent and -independent mouse xenograft models. Consistent with its design, MM-131 is more potent in EpCAM-high cells than in EpCAM-low cells, and its potency decreases when EpCAM levels are reduced by RNAi. Evaluation of Met, EpCAM, and HGF levels in human tumor samples reveals that EpCAM is expressed at high levels in a wide range of Met-positive tumor types, suggesting a broad opportunity for clinical development of MM-131.

Efficacy of FemiScan Pelvic Floor Therapy for the Treatment of Anal Incontinence.

OBJECTIVES: Pelvic floor muscle training can be effective in alleviating anal incontinence; however, women need instruction, motivation, and feedback to gain optimal benefit. The FemiScan Pelvic Floor Therapy System is approved in the United States and European Union for the treatment of urinary incontinence. It uses office electromyography and an in-home programmable device. This study was undertaken to document the effect of FemiScan on anal incontinence symptoms of women who completed a physician-supervised program. METHODS: Women referred for treatment of urinary symptoms who also reported anal incontinence symptoms were included in the analysis. We collected patient demographics, electromyographic measurements, and responses to subjective questions about the status of their anal incontinence. RESULTS: Forty eight (55%) of 88 patients who started treatment completed the 8-visit protocol. No adverse events were reported. Mean age was 54.8 ± 12.0 years. There was a statistically significant increase in the mean maximal response comparing the first and final electromyographic measurements obtained during the first and last office visits: left side, 13.7 ± 9.3 µV versus 23.2 ± 13.5 µV, P < 0.001 and right side, 14.6 ± 2.4 µV versus 22.7 ± 10.6 µV, P < 0.001 were analyzed separately. Fifty six percent reported that they were 100% free of symptoms, and 77% considered their symptoms at least 80% improved. Colorectal Anal Distress Inventory results demonstrated a statistically significant improvement when comparing the first and last visit (28.9 ± 17.9 vs 2.1 ± 7.8, P < 0.001). CONCLUSIONS: FemiScan appears to be a safe and effective treatment for anal incontinence with concomitant increased pelvic floor electromyographic activity.

Improving the developability of an anti-EphA2 single-chain variable fragment for nanoparticle targeting.

Antibody-targeted nanoparticles have great promise as anti-cancer drugs; however, substantial developmental challenges of antibody modules prevent many candidates from reaching the clinic. Here, we describe a robust strategy for developing an EphA2-targeting antibody fragment for immunoliposomal drug delivery. A highly bioactive single-chain variable fragment (scFv) was engineered to overcome developmental liabilities, including low thermostability and weak binding to affinity purification resins. Improved thermostability was achieved by modifying the framework of the scFv, and complementarity-determining region (CDR)-H2 was modified to increase binding to protein A resins. The results of our engineering campaigns demonstrate that it is possible, using focused design strategies, to rapidly improve the stability and manufacturing characteristics of an antibody fragment for use as a component of a novel therapeutic construct.

Efficacy of FemiScan Pelvic Floor Therapy for the Treatment of Urinary Incontinence.

OBJECTIVES: Pelvic floor muscle training can be effective in alleviating urinary incontinence; however, women need instruction, motivation, and feedback to gain optimal benefit from pelvic rehabilitation. The Food and Drug Administration-approved FemiScan Pelvic Floor Therapy System uses office electromyography and an in-home programmable device to provide training, motivation, and feedback between office visits. This study was undertaken to document the outcomes of women who completed an MD-supervised program using the FemiScan Pelvic Floor Therapy System. METHODS: In this chart review, women with urinary incontinence, who completed the 8-visit protocol, were included in the analysis. We collected patient demographics, electromyographic measurements, and responses to subjective questions about the status of their urinary incontinence symptoms. RESULTS: Two hundred fifteen (60%) of 361 patients who started treatment with FemiScan completed the 8-visit protocol. No adverse events were reported. The mean age was 54.4 ± 12.7 years. There was a statistically significant increase in the mean maximal response comparing the first and final electromyographic measurements obtained during the first and last electromyography office visits. The left side (15.9 ± 10.2 µV vs 28.0± 15.2 µV, P < 001) and the right side (16.6 µV vs 28.2 µV, P < 0.001) were analyzed separately in peak electromyographic measurements between the first and final visits. Seventy-five percent considered their symptoms at least 80% improved with 45% reporting complete subjective cure. Urinary Distress Inventory 6 results confirmed the subjective report with a statistical significant improvement comparing the first and last visit (9.47 ± 3.66 vs 2.71 ± 3.58, P < 001). CONCLUSIONS: FemiScan appears to be a safe and effective treatment for urinary incontinence with concomitant increased pelvic floor electromyographic activity.

Surgical Outcomes of a Combined Surgical Approach for Apical Prolapse Repair.

Introduction We aimed to evaluate the safety, efficacy and surgical outcomes of combined laparoscopic/vaginal prolapse repair by two surgeons. Material and Methods A retrospective chart review of all patients (n = 135) who underwent apical prolapse repair from February 2009 to December 2012 performed in a collaborative manner by a Minimally Invasive Gynecologic Surgeon and a Urogynecologist. Demographic data (age, body mass index [BMI], race, gravidity, parity) and surgical information (estimated blood loss, operative time, intraoperative complications, readmission and reoperation rates, presence of postoperative infection) were collected. Results The majority of patients were postmenopausal (58.91%), multiparous (mean parity = 2.49) and overweight (mean BMI = 27.71). Nearly 20% had previous prolapse surgery. The most common surgical procedure was laparoscopic supracervical hysterectomy (LSH) with sacrocervicopexy (59.26%), and the most common vaginal repair was of the posterior compartment (78.68%). The median operative time was 149 minutes (82-302), and the estimated blood loss was 100 mL (10-530). Five intra-operative complications, five readmissions and four reoperations were noted. Performance of a concomitant hysterectomy did not affect surgical or anatomical outcomes. Conclusion Combination laparoscopic/vaginal prolapse repair by two separate surgeons seems to be an efficient option for operative management.

Surgical Outcomes of a Combined Surgical Approach for Apical Prolapse Repair / Resultados cirúrgicos de uma abordagem cirúrgica combinada para a correção do prolapso apical

Abstract Introduction We aimed to evaluate the safety, efficacy and surgical outcomes of combined laparoscopic/vaginal prolapse repair by two surgeons. Material and Methods A retrospective chart review of all patients (n =135) who underwent apical prolapse repair from February 2009 to December 2012 performed in a collaborative manner by a Minimally Invasive Gynecologic Surgeon and a Urogynecologist. Demographic data (age, body mass index [BMI], race, gravidity, parity) and surgical information (estimated blood loss, operative time, intraoperative complications, readmission and reoperation rates, presence of postoperative infection) were collected. Results The majority of patients were postmenopausal (58.91%), multiparous (mean parity =2.49) and overweight (mean BMI =27.71). Nearly 20% had previous prolapse surgery. The most common surgical procedure was laparoscopic supracervical hysterectomy (LSH) with sacrocervicopexy (59.26%), and the most common vaginal repair was of the posterior compartment (78.68%). The median operative time was 149 minutes (82-302), and the estimated blood loss was 100 mL (10-530). Five intraoperative complications, five readmissions and four reoperations were noted. Performance of a concomitant hysterectomy did not affect surgical or anatomical outcomes. Conclusion Combination laparoscopic/vaginal prolapse repair by two separate surgeons seems to be an efficient option for operative management.

Resumo Introdução Objetivamos avaliar a segurança, eficácia e desfechos cirúrgicos da via laparoscópica e vaginal combinadas para a correção do prolapso feitos por dois cirurgiões. Métodos Um estudo retrospectivo com análise de prontuário foi realizado em todos os pacientes (n =135) que foram submetidos a correção de prolapso apical de fevereiro de 2009 a dezembro de 2012 de maneira concomitante por um laparoscopista e um uroginecologista. Dados demográficos (idade, índice de massa corporal [IMC], raça, número de gestações e partos) e cirúrgicos (perda sanguínea estimada, tempo operatório, complicações intraoperatórias, taxas de readmissão e reoperação, e presença de infecção pós-operatória) foram analisados. Resultados Operfil da paciente operada era pertencente à pós-menopausa (58,91%), ser multípara (paridade média =2,49) e com sobrepeso (IMC médio =27,71). Aproximadamente 20% havia feito cirurgia prévia para prolapso. O procedimento cirúrgico mais realizado foi a histerectomia supracervical laparoscópica (HSL) com sacrocervicopexia (59,6%); o reparo vaginal mais encontrado foi o para defeito de compartimento posterior (78,68%). O tempo operatório mediano foi de 149 minutos (82-302), e a perda sanguínea estimada foi de 100 ml (10-530). Cinco complicações pós-operatórias, cinco readmissões e quatro reoperações foram encontradas. A realização de uma histerectomia em concomitância aos demais procedimentos não afetou os desfechos cirúrgicos ou anatômicos. Conclusão O reparo combinado do prolapso pela via laparoscópica e vaginal por dois cirurgiões em concomitância aparenta ser uma opção eficiente para o manejo operatório.

Enhanced Targeting of the EGFR Network with MM-151, an Oligoclonal Anti-EGFR Antibody Therapeutic.

Although EGFR is a validated therapeutic target across multiple cancer indications, the often modest clinical responses to current anti-EGFR agents suggest the need for improved therapeutics. Here, we demonstrate that signal amplification driven by high-affinity EGFR ligands limits the capacity of monoclonal anti-EGFR antibodies to block pathway signaling and cell proliferation and that these ligands are commonly coexpressed with low-affinity EGFR ligands in epithelial tumors. To develop an improved antibody therapeutic capable of overcoming high-affinity ligand-mediated signal amplification, we used a network biology approach comprised of signaling studies and computational modeling of receptor-antagonist interactions. Model simulations suggested that an oligoclonal antibody combination may overcome signal amplification within the EGFR:ERK pathway driven by all EGFR ligands. Based on this, we designed MM-151, a combination of three fully human IgG1 monoclonal antibodies that can simultaneously engage distinct, nonoverlapping epitopes on EGFR with subnanomolar affinities. In signaling studies, MM-151 antagonized high-affinity EGFR ligands more effectively than cetuximab, leading to an approximately 65-fold greater decrease in signal amplification to ERK. In cell viability studies, MM-151 demonstrated antiproliferative activity against high-affinity EGFR ligands, either singly or in combination, while cetuximab activity was largely abrogated under these conditions. We confirmed this finding both in vitro and in vivo in a cell line model of autocrine high-affinity ligand expression. Together, these preclinical studies provide rationale for the clinical study of MM-151 and suggest that high-affinity EGFR ligand expression may be a predictive response marker that distinguishes MM-151 from other anti-EGFR therapeutics.

Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle.

Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anti-cancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.

MM-141, an IGF-IR- and ErbB3-directed bispecific antibody, overcomes network adaptations that limit activity of IGF-IR inhibitors.

Although inhibition of the insulin-like growth factor (IGF) signaling pathway was expected to eliminate a key resistance mechanism for EGF receptor (EGFR)-driven cancers, the effectiveness of IGF-I receptor (IGF-IR) inhibitors in clinical trials has been limited. A multiplicity of survival mechanisms are available to cancer cells. Both IGF-IR and the ErbB3 receptor activate the PI3K/AKT/mTOR axis, but ErbB3 has only recently been pursued as a therapeutic target. We show that coactivation of the ErbB3 pathway is prevalent in a majority of cell lines responsive to IGF ligands and antagonizes IGF-IR-mediated growth inhibition. Blockade of the redundant IGF-IR and ErbB3 survival pathways and downstream resistance mechanisms was achieved with MM-141, a tetravalent bispecific antibody antagonist of IGF-IR and ErbB3. MM-141 potency was superior to monospecific and combination antibody therapies and was insensitive to variation in the ratio of IGF-IR and ErbB3 receptors. MM-141 enhanced the biologic impact of receptor inhibition in vivo as a monotherapy and in combination with the mTOR inhibitor everolimus, gemcitabine, or docetaxel, through blockade of IGF-IR and ErbB3 signaling and prevention of PI3K/AKT/mTOR network adaptation.

Rapid optimization and prototyping for therapeutic antibody-like molecules.

Multispecific antibody-like molecules have the potential to advance the standard-of-care in many human diseases. The design of therapeutic molecules in this class, however, has proven to be difficult and, despite significant successes in preclinical research, only one trivalent antibody, catumaxomab, has demonstrated clinical utility. The challenge originates from the complexity of the design space where multiple parameters such as affinity, avidity, effector functions, and pharmaceutical properties need to be engineered in concurrent fashion to achieve the desired therapeutic efficacy. Here, we present a rapid prototyping approach that allows us to successfully optimize these parameters within one campaign cycle that includes modular design, yeast display of structure focused antibody libraries and high throughput biophysical profiling. We delineate this approach by presenting a design case study of MM-141, a tetravalent bispecific antibody targeting two compensatory signaling growth factor receptors: insulin-like growth factor 1 receptor (IGF-1R) and v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ErbB3). A MM-141 proof-of-concept (POC) parent molecule did not meet initial design criteria due to modest bioactivity and poor stability properties. Using a combination of yeast display, structured-guided antibody design and library-scale thermal challenge assay, we discovered a diverse set of stable and active anti-IGF-1R and anti-ErbB3 single-chain variable fragments (scFvs). These optimized modules were reformatted to create a diverse set of full-length tetravalent bispecific antibodies. These re-engineered molecules achieved complete blockade of growth factor induced pro-survival signaling, were stable in serum, and had adequate activity and pharmaceutical properties for clinical development. We believe this approach can be readily applied to the optimization of other classes of bispecific or even multispecific antibody-like molecules.

Case report: a novel method for uterine-sparing hysteropexy.

OBJECTIVE: The objective of this study was to describe a technique for uterine-sparing hysteropexy. CASE REPORT: A 50-year-old multiparous woman with pelvic organ prolapse underwent laparoscopic sacrohysteropexy utilizing polypropylene mesh with good clinical result. CONCLUSIONS: Placement of mesh arms medial to the uterine vessels during a laparoscopic sacrohysteropexy can be facilitated by using blunt needles to introduce the mesh arms.

Antitumor activity of a novel bispecific antibody that targets the ErbB2/ErbB3 oncogenic unit and inhibits heregulin-induced activation of ErbB3.

The prevalence of ErbB2 amplification in breast cancer has resulted in the heavy pursuit of ErbB2 as a therapeutic target. Although both the ErbB2 monoclonal antibody trastuzumab and ErbB1/ErbB2 dual kinase inhibitor lapatinib have met with success in the clinic, many patients fail to benefit. In addition, the majority of patients who initially respond will unfortunately ultimately progress on these therapies. Activation of ErbB3, the preferred dimerization partner of ErbB2, plays a key role in driving ErbB2-amplified tumor growth, but we have found that current ErbB2-directed therapies are poor inhibitors of ligand-induced activation. By simulating ErbB3 inhibition in a computational model of ErbB2/ErbB3 receptor signaling, we predicted that a bispecific antibody that docks onto ErbB2 and subsequently binds to ErbB3 and blocks ligand-induced receptor activation would be highly effective in ErbB2-amplified tumors, with superior activity to a monospecific ErbB3 inhibitor. We have developed a bispecific antibody suitable for both large scale production and systemic therapy by generating a single polypeptide fusion protein of two human scFv antibodies linked to modified human serum albumin. The resulting molecule, MM-111, forms a trimeric complex with ErbB2 and ErbB3, effectively inhibiting ErbB3 signaling and showing antitumor activity in preclinical models that is dependent on ErbB2 overexpression. MM-111 can be rationally combined with trastuzumab or lapatinib for increased antitumor activity and may in the future complement existing ErbB2-directed therapies to treat resistant tumors or deter relapse.

Optimizing properties of antireceptor antibodies using kinetic computational models and experiments.

Monoclonal antibodies are valuable as anticancer therapeutics because of their ability to selectively bind tumor-associated target proteins like receptor tyrosine kinases. Kinetic computational models that capture protein-protein interactions using mass action kinetics are a valuable tool for understanding the binding properties of monoclonal antibodies to their targets. Insights from the models can be used to explore different formats, to set antibody design specifications such as affinity and valence, and to predict potency. Antibody binding to target is driven by both intrinsic monovalent affinity and bivalent avidity. In this chapter, we describe a combined experimental and computational method of assessing the relative importance of these effects on observed drug potency. The method, which we call virtual flow cytometry (VFC), merges experimental measurements of monovalent antibody binding kinetics and affinity curves of antibody-antigen binding into a kinetic computational model of antibody-antigen interaction. The VFC method introduces a parameter χ, the avidity factor, which characterizes the ability of an antibody to cross-link its target through bivalent binding. This simple parameterization of antibody cross-linking allows the model to successfully describe and predict antibody binding curves across a wide variety of experimental conditions, including variations in target expression level and incubation time of antibody with target. We further demonstrate how computational models of antibody binding to cells can be used to predict target inhibition potency. Importantly, we demonstrate computationally that antibodies with high ability to cross-link antigen have significant potency advantages. We also present data suggesting that the parameter χ is a physical, epitope-dependent property of an antibody, and as a result propose that determination of antibody cross-linking and avidity should be incorporated into the screening of antibody panels for therapeutic development. Overall, our results suggest that antibody cross-linking, in addition to monovalent binding affinity, is a key design parameter of antibody performance.