Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; October 19-20, 2020 The concept that the B-cell Receptor Complex (BCRC) initiates a driver pathway in leukemia-lymphoma has been validated by clinical data. Previously, we reported the generation of a first-in-class antibody, mAb4, to a neo-epitope specific to the BCRC’s membrane IgM (mIgM) subunit, which inhibits cell growth and induces apoptosis (Welt, et al. 2016; US Patent Nos. 9,926,381 and 10,227,419). mAb4 epitope mapping demonstrates that it does not bind to the complementarity-determining regions (CDRs), nor to the micro-clustering site of mIgM that is believed to play a role in BCRC signal amplification. To characterize the mechanism of action of this antibody, we are investigating the inhibition of tyrosine kinase activity of several BCRC downstream signaling pathways. We hypothesize that mAb4 inhibits signal transduction of multiple BCRC-dependent downstream pathways. We are currently evaluating kinase activity across three sites on BTK (Ser180, Tyr223, and Tyr551). Phosphorylation of other common clinical targets, including Syk, Lyn, BCL2, and PI3K, will also be studied. To do this, we are using antibody reagents against phosphorylation sites to assess activation or inhibition of these kinases by mAb4 compared to control mAb-treated IgM-expressing B-cells, and, where available, cells treated with drugs against these targets. In preliminary ELISA analyses we find that mAb4 and a BTK inhibitor drug, inhibit BTK phosphorylation at Tyr223 and Tyr551, but not Ser180, across all anti-phospho-tyrosine/serine antibody reagents tested (Ser180: clones 3i5 and 3D3; Tyr223: clones A16128B and 720101; and Tyr 551: clones A16064A, 797837, and M4G3LN). In vitro, mAb4-treated leukemia and lymphoma cells undergo growth inhibition and apoptosis while the BTK inhibitor drug mediates only cell growth inhibition, as reported by others. Here we show that mAb4 inhibits phosphorylation of BTK tyrosine upon binding to a determinant that spans the extracellular proximal domain and the constant domain 4 (μC4) regions of mIgM. While BTK inhibitor drugs induce a cell growth inhibitory effect, we find that mAb4 mediates BCRC internalization, and in low-density cultures, cell growth inhibition, anti-clonogenic activity, and apoptosis, and may therefore represent a next generation of BTK inhibitor. Though mAb4 does not interact with the mIgM’s CDRs nor the micro-clustering site on μC4, it modulates transmembrane signal transduction. We predict that continued analyses of additional downstream kinases (Syk, Lyn, BCL2, and PI3K) will similarly show an effect following BCRC inhibition by mAb4. These finding indicate that mAb4 may target mIgM at a critical structural site, which interrupts specific ligand binding signal transduction. Furthermore, mAb4 is highly specific to IgM-expressing B-cells, thus this novel approach to modulating B-cell signaling to induce a cytotoxic response may represent an exciting new direction in clinical development. Citation Format: Rachel S. Welt, Jonathan A. Welt, Virginia Raymond, David Kostyal, Sydney Welt. Anti-membrane-IgM monoclonal antibody, mAb4, inhibits the BCRC, modulating downstream signaling pathways [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO020.

中文翻译：

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摘要PO020：抗膜IgM单克隆抗体mAb4抑制BCRC，调节下游信号通路

摘要：AACR虚拟特别会议：肿瘤免疫学和免疫治疗；2020年10月19日至20日，B细胞受体复合物（BCRC）启动白血病-淋巴瘤中的驱动器途径的概念已通过临床数据验证。以前，我们报道了针对BCRC膜IgM（mIgM）亚基特异的新表位的一流抗体mAb4的产生，该表位抑制细胞生长并诱导凋亡（Welt等人，2016年;美国专利9,926,381和10,227,419）。mAb4表位作图表明，它不与互补决定区（CDR）结合，也不与mIgM的微簇位点结合，后者被认为在BCRC信号放大中起作用。为了表征该抗体的作用机理，我们正在研究几种BCRC下游信号通路对酪氨酸激酶活性的抑制作用。我们假设mAb4抑制多个BCRC依赖下游通路的信号转导。我们目前正在评估BTK的三个位点（Ser180，Tyr223和Tyr551）的激酶活性。其他常见的临床靶标，包括Syk，Lyn，BCL2和PI3K的磷酸化也将进行研究。为此，我们使用抗磷酸化位点的抗体试剂来评估mAb4与对照mAb处理的表达IgM的B细胞以及（如果有的话）针对这些靶点的药物处理过的细胞相比，mAb4对这些激酶的激活或抑制作用。在ELISA的初步分析中，我们发现mAb4和BTK抑制剂药物可抑制Tyr223和Tyr551的BTK磷酸化，但不能抑制Ser180，（Ser180：克隆3i5和3D3； Tyr223：克隆A16128B和720101； Tyr 551：克隆A16064A，797837和M4G3LN）中检测到的所有抗磷酸酪氨酸/丝氨酸抗体试剂。在体外，mAb4处理的白血病和淋巴瘤细胞会受到生长抑制和凋亡的影响，而BTK抑制剂药物仅介导细胞生长的抑制作用，这已被其他人报道。在这里，我们显示，mAb4结合跨越mIgM的细胞外近端域和恒定域4（μC4）区域的决定簇后，抑制BTK酪氨酸的磷酸化。虽然BTK抑制剂药物可诱导细胞生长抑制作用，但我们发现mAb4介导BCRC内在化，并且在低密度培养物中可抑制细胞生长，抗克隆活性和凋亡，因此可能代表了下一代BTK抑制剂。尽管mAb4既不与mIgM的CDR相互作用，也不与μC4上的微簇位点相互作用，但它可调节跨膜信号转导。我们预测，继续分析其他下游激酶（Syk，Lyn，BCL2和PI3K）将类似地显示出mAb4抑制BCRC后的作用。这些发现表明，mAb4可以在关键结构位点靶向mIgM，这会中断特异性配体结合信号的转导。此外，mAb4对表达IgM的B细胞具有高度特异性，因此这种调节B细胞信号传导以诱导细胞毒性反应的新方法可能代表了临床开发中令人兴奋的新方向。引文格式：雷切尔·S·威尔特，乔纳森·A·威尔特，弗吉尼亚·雷蒙德，大卫·科斯蒂尔，悉尼·威尔特。抗膜IgM单克隆抗体mAb4抑制BCRC，调节下游信号通路[摘要]。在：摘要：AACR虚拟特别会议：肿瘤免疫学和免疫疗法；2020年10月19日至20日。费城（PA）：AACR；Cancer Immunol Res 2021; 9（2增刊）：摘要编号PO020。