Cirmtuzumab Vedotin (UC-961ADC3), An Anti-ROR1-Monomethyl Auristatin E Antibody-Drug Conjugate, Is a Potential Treatment For ROR1-Positive Leukemia and Solid Tumors

Abstract ROR1 is an onco-embryonic antigen that is expressed on the neoplastic cells of patients with chronic lymphocytic leukemia (CLL), other B-cell lymphomas, acute leukemias, or many different solid-tumors, but not on non-neoplastic post-partum tissues, except for the uncommon precursor B cells known as hematogones. Because of its restricted expression on human malignancies, we have generated a series of monoclonal antibodies (mAb) against the extracellular domain of human ROR1 and are advancing our lead candidate mAb UC-961 (cirmtuzumab) into human clinical trials. Along with the anti-leukemia activity of this mAb, we have also shown that the UC-961 anti-ROR1 antibody is internalized by CLL B cells, and by several B cell leukemia and lymphoma cell lines, at a greater rate and degree than other anti-ROR1 mAb that bind other epitopes of the extracellular domain of ROR1. As such, we speculated that cirmtuzumab may have an enhanced specific cytotoxic activity when employed as an antibody-drug-conjugate (ADC). To test this hypothesis, we generated a series of cirmtuzumab ADCs using proprietary (Concortis Biosystems, San Diego) linkers and toxins. We examined the cytotoxic activity of each of the various cirmtuzumab-ADC against ROR1 expressing cells (e.g. CLL cells, JeKo-1 cells, or solid tumor cells) or ROR1-negative cells (e.g. Ramos and Jurkat cells). Cirmtuzumab stably bound to a modified monomethyl auristatin E (MMAE) through a light chain, constant region, lysine-linker with an antibody-drug ratio (ADR) of 2.5 (designated UC-961ADC3) showed the highest specific activity for killing ROR1+ cells without generating any toxic activities on ROR1-Neg cells in vitro. For example, UC-961ADC3 kills ROR1+ JeKo-1 (LD 50=1.4 μg/ml) or primary CLL cells (LD 50=39.9 μg/ml) in a dose dependent manner, but had no or low cytotoxicity for ROR1-Neg cell lines, Ramos (LD 50>200 μg/ml) or Jurkat (LD50> 95 μg/ml). To expand these findings, we examined the specific activity of UC-961ADC3 in clearing ROR1+ JeKo-1 cells engrafted in immune deficient Rag-2-/-/γc-/- mice. In these studies, groups of animals were given intravenous injections of 1 x 105 JeKo-1 cells. Three weeks after transfer, each mouse cohort was treated with weekly injections of 10 mg/kg UC-961ADC3, or control IgG antibody. Six weeks after adoptive transfer, the animals were sacrificed and spleens analyzed for the presence of the leukemic cells. Animals treated with UC-961ADC3 had a 5-fold lower median number of leukemia JeKo-1 cells in the spleen than animals treated with control IgG (p<0.001). We also tested the activity of UC-961ADC3 in a murine in vivo human primary CLL xenograph, niche-dependent model. In a representative experiment, control and test mouse cohorts were intraperitoneal implanted on day 0 with 2 x 107 primary human CLL cells. On day 1 the animals were treated with a single 1mg/kg i.p. injection of either UC-961ADC3 or control antibody. One week after implantation the animals were sacrificed and the residual CLL cells determined. In mice treated with UC-961ADC3 only an average of 3 ± 2 x 103 CLL cells were harvested. This number of cells was significantly less than the average number of CLL cells harvested from control IgG treated mice (9.3 ± 3.3 x 105, p <0.01). Finally, we have also demonstrated that UC-961ADC3 also could induce specific killing of ROR1-expressing adenocarcinomas of the breast or pancreas. These studies indicate that a cirmtuzumab-ADC can directly target and kill ROR1-expressing cancer cells in vitro and in vivo without detectable cytotoxicity for ROR1 negative cells. Because of the restricted expression of ROR1 on human malignancies, we propose that UC-961ADC3 might have potential utility in the treatment of patients with ROR1 positive leukemias and solid tumors with an enhanced therapeutic index relative to other therapies. Disclosures: No relevant conflicts of interest to declare.