Abstract 2551: B cells induced during CD4+ T-cell mediated destruction of primary tumors in neuN mice produce mAbs that are capable of inhibiting spontaneous tumor growth
Authors: Wang Li-Xin , Patel Mona , Berk Michael , Plautz Gregory 
Published: May 2015

Abstract
We previously identified an induced serologic response in FVB-neuN mice bearing primary breast tumors following adoptive therapy with CD4+ T cells. Because the CD4+ T cells were initially primed with breast tumor progenitor cells, we tested whether the B cells induced in treated mice also recognized antigens expressed specifically by breast tumor progenitor cells. We generated hybridomas and then screened supernatant for binding to target cells by FACS analysis. We isolated three clones that produced IgG (mAb1-2, mAb2, and mAb3) which recognized cell surface molecules on tumorsphere cultures from a NeuN breast tumor line but not the parental adherent culture from which they were derived. We used a Her-2/neu antigen loss variant to show that Her-2/neu was not itself the antigen. Interestingly, the three mAbs also recognized 4T1tumorspheres, an independently derived breast tumor from another mouse strain, but not adherent 4T1 cultures. Moreover, the mAbs were not reactive with normal spleen, liver, or kidney cells. To investigate whether any of these mAbs had anti-tumor function we used Her-2/neuN transgenic mice which develop mammary adenocarcinoma starting at 5 months of age. We selected mice with a single spontaneous tumor in situ when tumors became palpable and visible. The mice received mAb1-2, mAb2, mAb3 (150 μg/dose) or PBS on day 0, day 1 then every other day until day 23. There was no toxicity from the treatment and mice treated with PBS or mAb3 all showed progressive tumor growth. However, administration of mAb1-2 inhibited primary tumor progression in 5/8 mice and mAb2 inhibited tumor progression in 9/13 mice (p&lt;0.05). Some mice with response to mAb had ongoing attenuation of tumor growth even after completion of mAb treatments. These data suggest that mice responding to CD4 T cell immunotherapy were able to stimulate endogenous B cells with reactivity against auto-antigens expressed by tumor progenitor cells. Our approach restricted our screen to cell surface antigens because we used FACS to identify hybridoma supernatant which specifically bound to tumor progenitor cells. This allowed us to focus on mAb that might have some direct therapeutic effect and two of the antibodies did inhibit tumor growth. At this point we do not know whether mAb1-2 or mAb2 inhibit tumor growth through blockade of signaling or through cytotoxic mechanisms but the antigen seems to be expressed by the majority of spontaneous breast tumors arising in FVB-neuN mice. Our current studies are attempting to identify the antigen recognized by each mAb.
Citation Format: Li-Xin Wang, Mona Patel, Michael Berk, Gregory Plautz. B cells induced during CD4+ T-cell mediated destruction of primary tumors in neuN mice produce mAbs that are capable of inhibiting spontaneous tumor growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2551. doi:10.1158/1538-7445.AM2014-2551