A 3D matrix platform for the rapid generation of therapeutic anti-human carcinoma monoclonal antibodies

Abstract

Efforts to develop unbiased screens for identifying novel functionblocking monoclonal antibodies (mAbs) in human carcinomatous states have been hampered by the limited ability to design in vitro models that recapitulate tumor cell behavior in vivo. Given that only invasive carcinoma cells gain permanent access to type I collagen-rich interstitial tissues, an experimental platform was established in which human breast cancer cells were embedded in 3D aldimine cross-linked collagen matrices and used as an immunogen to generate mAb libraries. In turn, cancer-cell-reactive antibodies were screened for their ability to block carcinoma cell proliferation within collagen hydrogels that mimic the in vivo environment. As a proof of principle, a single function-blocking mAb out of 15 identified was selected for further analysis and found to be capable of halting carcinoma cell proliferation, inducing apoptosis, and exerting global changes in gene expression in vitro. The ability of this mAb to block carcinoma cell proliferation and metastatic activity was confirmed in vivo, and the target antigen was identified by mass spectroscopy as the α 2 subunit of the α 2 β 1 integrin, one of the major type I collagen-binding receptors in mammalian cells. Validating the ability of the in vitro model to predict patterns of antigen expression in the disease setting, immunohistochemical analyses of tissues from patients with breast cancer verified markedly increased expression of the α 2 subunit in vivo. These results not only highlight the utility of this discovery platform for rapidly selecting and characterizing function-blocking, anticancer mAbs in an unbiased fashion, but also identify α 2 β 1 as a potential target in human carcinomatous states.n mammalian systems, a specialized form of extracellular matrix (ECM), termed the basement membrane, normally separates epithelial cells from the underlying type I collagen-rich interstitial matrix (1, 2). Thus, in mature animals and under physiologic conditions, the epithelium does not establish stable physical contacts with interstitial tissues (1, 2). In contrast, in neoplastic states, transformed epithelial cells (i.e., carcinomas) dissolve the intervening basement membrane barrier and establish adhesive interactions with the newly exposed type I collagen fibrillar network (1-5). As carcinoma cells begin to infiltrate the interstitial matrix, they rapidly adapt themselves to their 3D environment and initiate the proliferative phenotypes that define tumor progression at both primary and metastatic sites (2, 6, 7). Indeed, emphasizing the importance of the tumor-ECM interface, carcinoma cells do not simply use the surrounding interstitial matrix as a passive substrate, but actively promote increased type I collagen deposition within the peritumoral microenvironment as a means of further enhancing invasive activity, local growth, and cancer stem cell formation (7-12).Despite the importance of the carcinoma cell-type I collagen interface in vivo, therapeutic interventions that directly inte...

Tailoring nanoparticle designs to target cancer based on tumor pathophysiology

“…Self-assembled hydrogels composed of either 2.5 or 4.0 mg/mL of type I collagen were used to mimic stromal changes caused by tumor growth. Type I collagen was selected as a stromal phantom because type I collagen is the primary component of the tumor-blood vessel interface (30,31). Entry of AuNPs from a fluid reservoir into the hydrogel was kinetically monitored by AuNP fluorescence using scanning confocal microscopy at different time points over 900 min.…”

Integrin α2β1 inhibits MST1 kinase phosphorylation and activates Yes-associated protein oncogenic signaling in hepatocellular carcinoma

“…α2β1 integrin of the β1-integrin family is an extracellular matrix receptor for collagen and laminin [26]. In addition to its physiologic function, α2β1 integrin contributes to the tumor-stromal interaction that is important to cancer cell survival and metastasis [27-29]. Herein, the present study reports α2β1 integrin as a potential upstream negative regulator of the Hippo pathway in HCC, which upon activation through binding to collagen, directly inhibits MST1 kinase and activates the YAP-driven transcriptional activities.…”

Phenotypic screening: the future of antibody discovery

“…As early as 1995, de Kruif et al [11] screened for target cell specificity and more recent examples have followed [12][13][14]. Functional screens have also been performed that have identified antibodies that induce apoptosis [15], inhibit cell proliferation [16], or internalise [17,18].…”

“…To date, most antibodies identified by phenotypic screens have been found using a two-step process. First, antibody populations have been enriched against a target cell or tissue type before being screened for function [11][12][13][14][15][16][17][18]. A two-step process is dependent on the efficiency of both steps; however, the functional screen poses the greatest challenge and is often the bottleneck in the process.…”

“…3D phenotypic screens are now being successfully applied to antibody discovery. Examples have highlighted the benefits of this approach and have successfully identified tumour-relevant targets, such as α2β1 integrin [16] and CDCP1 [54], whose relevance could be translated in subsequent disease models in vivo. 3D screening is a clear improvement to the phenotypic screening paradigm, provided that challenges around assay robustness and reproducibility can be overcome.…”