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

Significance To select for novel monoclonal antibodies (mAbs) with anticancer activity, an experimental platform was established wherein human breast cancer cells were embedded in 3D collagen matrices and used as an immunogen to generate mAb libraries. Fifteen mAbs capable of inhibiting carcinoma cell growth in vitro were generated. A single function-blocking mAb was selected for further analysis and then validated as a potent inhibitor of carcinoma cell behavior in vivo. The target antigen was identified as the α ₂ subunit of the α ₂β ₁ integrin, a major type I collagen-binding receptor whose expression was confirmed in tissues of patients with breast cancer. These findings describe a new discovery platform that allows for the rapid selection of function-blocking antibodies and identify α ₂β ₁ as a potential target in carcinomatous states. Efforts to develop unbiased screens for identifying novel function-blocking 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 ₂β 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, anticance