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716 Therapeutic Potential of an Antibody Targeting the Cleaved Form of Mutant Calreticulin in Myeloproliferative Neoplasms

Program: Oral and Poster Abstracts
Type: Oral
Session: 635. Myeloproliferative Syndromes: Basic Science
Hematology Disease Topics & Pathways:
Biological, Diseases, Therapies, MPN, Biological Processes, immunotherapy, Myeloid Malignancies, molecular interactions
Monday, December 7, 2020: 2:15 PM

Yoshihiko Kihara1,2*, Marito Araki, PhD3*, Misa Imai, PhD1,2*, Yosuke Mori, MD2*, Mei Horino2*, Satoko Ogata, PhD2*, Syunpei Yoshikawa2,4*, Teppei Taguchi2*, Nami Masubuchi, PhD5*, Yo Mabuchi, PhD6*, Yinjie Yang, PhD2*, Yasutaka Fukuda, MD, PhD2*, Soji Morishita, PhD3*, Takehiro Suzuki, PhD7*, Naoshi Domae, PhD7*, Motoyuki Shimonaka, PhD4*, Chihiro Akazawa, MD, PhD8*, Akimichi Ohsaka, MD, PhD3* and Norio Komatsu, MD, PhD2

1Leading Center for the Development and Research of Cancer Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan
2Department of Hematology, Juntendo University Graduate School of Medicine, Tokyo, Japan
3Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University Graduate School of Medicine, Tokyo, Japan
4Faculty of Science, Tokyo University of Science, Tokyo, Japan
5Department of Hematology, Juntendo University Graduate School of Medicine, Bunkyo-Ku, TKY, Japan
6Department of Biochemistry and Biophysics, Tokyo Medical and Dental University Graduate School of Medical and Dental Sciences, Tokyo, Japan
7Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Saitama, Japan
8Intractable Disease Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan

Mutant calreticulin (CALR) has been shown to play a causal role in the development of essential thrombocythemia (ET) and primary myelofibrosis (PMF) via activation of the thrombopoietin receptor MPL. The oncogenic property of mutant CALR originates from a +1 frameshift mutation in its carboxyl-terminal domain, which is found in approximately 30% of patients with ET and PMF. Because the domain is uniquely found in mutant CALR, it has been recognized as a neoantigen and can therefore be used to target CALR-mutant cells using immunotherapy. In the present study, we found that a large portion of the domain generated by the frameshift in the mutant CALR was cleaved by an endoprotease belonging to the subtilisin family in multiple cell lines and primary cells. The cleaved form of mutant CALR was detected in the cell lysate; however, it was more abundant in the culture supernatant, implying that the cleavage occurred on the cell surface and/or outside the cells. Using mass spectrometric analysis, we determined the cleavage site of mutant CALR. To examine whether the cleavage was required for the oncogenic properties of mutant CALR, we introduced point mutations at the cleavage site. The mutant CALR construct that was resistant to protease cleavage exhibited full oncogenic capacity when expressed in UT-7/TPO cells. Consistent with this observation, chemical inhibition of the protease, which blocked the cleavage of mutant CALR, did not interfere with mutant CALR-dependent cell growth in UT-7/TPO cells. Next, we generated B3, a rat monoclonal antibody that recognized the mutant-specific sequence, even after cleavage. B3 recognized both the uncleaved and cleaved forms of mutant CALR by immunoblot in cell lysates prepared from the platelets and peripheral blood cells of CALR-mutant ET and PMF patients. B3 also recognized mutant CALR expressed on the cell surfaces of monocytes and granulocytes from CALR-mutant ET and PMF patients. Based on these results, we developed B3-chimera, a mouse chimeric antibody of B3, and evaluated its therapeutic potential for ET in vivo. We used an ET mouse model created by transplantation of LSK (lin-sca1+c-kit+) cells transduced with CALR del52 into the bone marrow. Intravenous injection of B3-chimera markedly suppressed the thrombocytosis induced by CALR del52, which was associated with a significant reduction in the megakaryocyte count in the bone marrow (Figure 1). In conclusion, we demonstrated that targeting the cleaved form of mutant CALR on the cell surface was a promising strategy for the treatment of CALR-mutant myeloproliferative neoplasms.

Disclosures: Akazawa: ITOCHU CHEMICAL FRONTIER Corporation: Membership on an entity's Board of Directors or advisory committees. Komatsu: Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; PPMX: Consultancy, Research Funding; Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding.

*signifies non-member of ASH