The Anti-B-Cell Maturation Antigen (BCMA) Antibody-α-Amanitin Conjugate Hdp-101 Induces Immunogenic Cell Death and Immunologic Memory in Models of Multiple Myeloma
Authors: Singh Ram Kumar , Jones Richard J. , Shirazi Fazal M. , Zou Jianxuan M. , Wang Hua , Lee Hans C. , Manasanch Elisabet E. , Kuiatse Isere , Pahl Andreas , Orlowski Robert Z. 
Published: February 2021

Background:
Patients with relapsed/refractory myeloma continue to represent an unmet medical need, and this is especially true for those with deletion (del) 17p, who have an inferior prognosis. We previously reported the ability of HDP-101 to induce anti-proliferative and pro-apoptotic effects in myeloma cell lines and primary patient samples. HDP-101 showed enhanced efficacy against del 17p models, which are characterized by concurrent haploinsufficiency of RNA polymerase II subunit A (POLR2A), which is the target of amanitin. This occurred in conjunction with activation of the endoplasmic reticulum stress, and at least two arms of the unfolded protein response. Also, a single dose of HDP-101 was sufficient to induce cures in murine models of myeloma both with and without TP53 and/or POLR2A. Therefore, we sought to better understand the mechanisms of action of HDP-101 and its potential immunologic sequelae.
Methods:
Pre-clinical studies were performed using HDP-101, the unconjugated BCMA antibody, free α-Amanitin, and a non-targeting control Amanitin antibody drug conjugate in myeloma cell line models and in vivo. These included H929, MM1.S, and MOLP-8 TP53 wild-type (WT) lines and isogenic cells in which TP53 had been knocked out (KO) using genome editing techniques. To further model del 17p and POLR2A haploinsufficiency, POLR2A expression was knocked down using sequence-specific shRNAs. Tumor rechallenge experiments were performed after 100 days of tumor free survival in NOD.CB17-Prkdcscid/J mice with longitudinal monitoring of tumor growth kinetics.
Results:
HDP-101 induced immunogenic cell death (ICD) in myeloma cell lines as determined by increased expression of Calreticulin (CRT), Heat shock protein (HSP)-70, and release of High mobility group box 1 (HMGB1) by flow cytometry and Western blotting. Microscopic analysis of ICD upon treatment with HDP-101 in MM1.S cells showed a speckled arrangement of both CRT and HMGB1 on the cell surface. In addition, an increased ADP/ATP ratio was observed across three different cell lines in a TP53-independent manner suggestive of an operational ICD pathway. Using our current knowledge of ER stress activation and induction of ICD, we evaluated the combination of bortezomib and HDP-101 in H929, MM1.S, and MOLP-8 cells and primary samples. A significant loss of cellular viability (p&lt;0.01) was observed upon combination treatment across the cell lines, and primary samples also showed a similar effect but only in CD138+ve populations while sparing the CD138-ve fraction. NOD.CB17-Prkdcscid/J mice injected with MM1.S luciferase-transfected cells uniformly developed systemic disease, and one dose of HDP-101 induced cures with no evidence of relapse out to 100 days. These same mice were then rechallenged with MM1.S cells but none developed recurrent disease, suggesting the generation of immunologic memory. Natural killer (NK) cells isolated from these mice showed ex vivo activity against myeloma cell lines which was greatest against MM1.S cells. Finally, cured mice were treated with the anti-asialo GM1 antibody that depletes NK cells or control serum first, and then rechallenged with MM1.S cells. While MM1.S-based tumors could not become established in control serum treated mice, pre-treatment with the anti-asialo GM1 antibody did allow tumors to develop. Studies to compare the activity of HDP-101 with other antibody drug conjugates are currently ongoing.
Conclusions:
Our results support the statement that HDP-101 is a novel anti-BCMA antibody drug conjugate that shows potent activity against drug-naïve and -resistant models of myeloma, and has the potential to show enhanced anti-tumor activity against del 17 p myeloma. Moreover, its mechanism of action involves both a direct cytotoxic effect, as well as the induction of immunogenic cell death, with the latter potentially leading to immunologic memory. These studies were supported by a Leukemia & Lymphoma Society Specialized Center of Research (SCOR-12206-17).

Disclosures
Jones: Asylia Therapeutics, Inc.: Current equity holder in private company, Patents & Royalties. Lee:Amgen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Genentech: Consultancy; GlaxoSmithKline: Consultancy, Research Funding; Sanofi: Consultancy; Daiichi Sankyo: Research Funding; Regeneron: Research Funding; Genentech: Consultancy. Manasanch:Sanofi: Honoraria, Research Funding; Novartis: Research Funding; JW Pharma: Research Funding; Merck: Research Funding; Quest Diagnostics: Research Funding; Takeda: Honoraria; BMS: Honoraria; Glaxo Smith Kline: Honoraria; Adaptive Biotechnologies: Honoraria. Pahl:Heidelberg Pharma AG: Current Employment. Orlowski:Sanofi-Aventis, Servier, Takeda Pharmaceuticals North America, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen, Inc., AstraZeneca, BMS, Celgene, EcoR1 Capital LLC, Forma Therapeutics, Genzyme, GSK Biologicals, Ionis Pharmaceuticals, Inc., Janssen Biotech, Juno Therapeutics, Kite Pharma, Legend Biotech USA, Molecular Partners, Regeneron Pharmaceuticals, Inc.,: Honoraria, Membership on an entity's Board of Directors or advisory committees; Laboratory research funding from BioTheryX, and clinical research funding from CARsgen Therapeutics, Celgene, Exelixis, Janssen Biotech, Sanofi-Aventis, Takeda Pharmaceuticals North America, Inc.: Research Funding; STATinMED Research: Consultancy; Founder of Asylia Therapeutics, Inc., with associated patents and an equity interest, though this technology does not bear on the current submission.: Current equity holder in private company, Patents & Royalties.