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

Abstract

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.

Biology and therapeutic targeting of molecular mechanisms in MPNs

“…Because the mutant CALR-MPL complex traffics to the cell surface, interest in therapeutically targeting mutant CALR using a mutant CALR blocking antibody ( Figure 1 C) has grown. Three recent studies reported the development and testing of therapeutic monoclonal antibodies targeting the C-terminal mutant CALR sequence in preclinical models, 136 , 137 , 138 with 1 of the studies also testing the antibody (4D7) against primary MPN cells in vitro and demonstrating reduced megakaryocyte proliferation, specifically in CALR -mutant (but not JAK2- mutant) cells. 136 Similar findings in primary MPN cells were recently reported for a fully human immunoglobulin G1–mutant CALR antibody (INCA033989) as a plenary abstract at the 2022 American Society of Hematology meeting.…”

Antibody targeting of mutant calreticulin in myeloproliferative neoplasms

“…Recent research efforts have therefore centered on immunotherapeutic approaches with the goal of targeting the mutant calreticulin neoepitope while sparing normal haematopoiesis. In 2020, Kihara et al reported (in abstract form) the generation of the mouse chimeric monoclonal antibody B3, specifically targeting mutant calreticulin 27 . In a CALR del52 ET mouse model, treatment with B3 reduced platelets in the peripheral blood and numbers of megakaryocytes in the bone marrow of the mice.…”

“…In 2020, Kihara et al reported (in abstract form) the generation of the mouse chimeric monoclonal antibody B3, specifically targeting mutant calreticulin. 27 In a CALR del52 ET mouse model, treatment with B3 reduced platelets in the peripheral blood and numbers of megakaryocytes in the bone marrow of the mice. Soon after, Achyutuni and colleagues generated a murine IgG2a raised against the human calreticulin neoantigen and treated homozygous CALR del52 transgenic mice.…”

Recent Advances in Molecular Diagnostics and Targeted Therapy of Myeloproliferative Neoplasms

“…Investigative efforts are ongoing, as, e.g., for JAK inhibitors with a type II mode of binding similar to BCR-ABL inhibitors [86,87]. In addition, mutant calreticulin, which is exposed at the cell surface in association with MPL, could be addressed as an therapeutic target, directly relating to JAK2-STAT signaling in CALR mutant patients [38,88,89].…”