Abstract 5036: Single dose treatment with a novel Yttrium-90-labeled high affinity anti-B7-H3 antibody selective for the 4Ig-B7-H3 isoform provides long term survivors for established radioresistant colorectal carcinoma
Authors: Glazer Sarah , Sutton Margie , Yang Ping , Pisaneschi Federica , Gammon Seth , Piwnica-Worms David 
Published: April 2023

Abstract
The immune checkpoint antigen B7-H3 (CD276), expressed on cell surfaces of a variety of epithelial solid tumors, is a type 1 transmembrane protein consisting of an extracellular domain of repeating immunoglobulin constant and immunoglobulin variable domains (IgV, IgC, IgV, IgC), known as the 4Ig-B7-H3 isoform. 4Ig-B7-H3 is the dominant isoform in human cancers and is expressed at much lower levels on normal tissues. Humans also express a sheddable 2Ig-B7-H3 isoform containing only one immunoglobulin variable and constant extracellular domain (IgV, IgC) that is found in circulation (soluble 2Ig-B7-H3) and presents a challenge for systemic targeting of solid tumors. To develop an antibody suitable for fulfilling the promise of B7-H3 as a systemic therapeutic target, herein we report a murine antibody (MIL33B) that demonstrates mid-picomolar affinity to human 4Ig-B7-H3 (Kd, 72 pM) with 8-fold selectivity over 2Ig-B7-H3 (Kd, 580 pM) extracellular domains. Furthermore, MIL33B maintains high affinity to porcine 4Ig-B7-H3 (Kd, 102 pM), selectivity over murine 2Ig-B7-H3 (Kd, 41 nM), facilitating pre-clinical testing, and low cross-reactivity to other B7 family members (Kd, &gt;1 μM). MIL33B-AF495 conjugates showed selective binding by live cell fluorescence microscopy to HeLa and HCT116 cells with high endogenous 4Ig-B7-H3 expression and 4T1, B16F10 and CT26 cells induced to express human 4Ig-B7-H3 compared to HeLa 4Ig-B7-H3 KO cells or murine cell lines with endogenous 2Ig-B7-H3 expression. Compared to uptake in respective KO or vector control tumors, quantitative analysis in vivo with 89Zr-DFO-MIL33B and PET imaging of tumor models demonstrated significantly higher normalized tumor-specific uptake in tumors with high endogenous expression of 4Ig-B7-H3 (HeLa) (3.25 ± 0.65 vs. 0.79 ± 0.13 SUV ratio, * p = 0.02) or those induced to express human 4Ig-B7-H3 (B16F10 and CT26) (3.16 ± 0.30 vs. 1.20 ± 0.69 SUV ratio, ** p = 0.0032; and 3.62 ± 1.62 vs. 1.89 ± 0.85 SUV ratio, *** p &lt; 0.0001, respectively). As a first radio-theranostic treatment application, MIL33B labeled with yttrium-90 (90Y-DOTA-MIL33B), a therapeutic beta-emitter, administered as a single dose I.V. (100 mCi, 3700 kBq), induced complete tumor regression and long-term survival of &gt; 50% of mice harboring established syngeneic radioresistant colorectal CT26 tumors expressing human 4Ig-B7-H3 compared to vector control tumors (* p = 0.0376, log-rank test) 90Y-DOTA-MIL33B-responsive mice developed immunologic memory and depletion assays in vivo demonstrated that CD8b+ cells contributed to the therapeutic efficacy of 90Y-DOTA-MIL33B. These results point to the promise of 90Y-DOTA-MIL33B as a selectively-targeted immune priming agent for radioligand therapy of 4Ig-B7-H3-expressing solid tumors.
Citation Format: Sarah Glazer, Margie Sutton, Ping Yang, Federica Pisaneschi, Seth Gammon, David Piwnica-Worms. Single dose treatment with a novel Yttrium-90-labeled high affinity anti-B7-H3 antibody selective for the 4Ig-B7-H3 isoform provides long term survivors for established radioresistant colorectal carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5036.