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.
We generated over 70 hybridomas, each producing a monoclonal-antibody (mAb) specific for the extracellular domain of ROR1 and found only one (D10) that had anti-leukemia activity in a niche-dependent assay, despite having a relatively low ROR1-binding affinity (Kd 40 nM). We generated high-affinity mAbs specific for the epitope recognized by D10 using recombinant phage-display libraries and found one (designated 961) that bound ROR1 with high affinity (Kd 800 pM) and had similar anti-leukemia activity as D10. To mitigate immunogenicity, we identified both light and heavy chain complementary determining regions (CDR) and framework junctions in the 961 mAb. Using conservative CDR and parallel framework substitutions (BioAtla-San Diego) we generated a panel of 21 humanized 961 variants. We selected one (cirmtuzumab or UC-961) that had high specificity and affinity for ROR1 (Kd 4nM). Moreover, UC-961 does not react with normal adult tissues, as assessed by immunohistochemical studies on FDA normal tissue arrays or fresh-frozen adult tissues, but reacted strongly with ROR1+ neoplastic cells (e.g. CLL cells or solid-tumor tissues).
We inserted the optimized genetic sequence of the UC-961 mAb into selectable expression cassettes and used these to transfect Chinese hamster ovary cells (CHO-Selexis, Switzerland). Upon successive rounds of single cell cloning, we identified a single clone (Acp7) that stably expressed greater that 2 g/L of UC-961. In pilot studies, we have expanded this clone to over 50 population doublings without change in production quantity or quality of UC-961 during prolonged expansion.
Acp7 has been banked, tested, released and used to produce pilot-scale material for process validation, GLP tissue cross and pre-clinical pharmacology/ toxicology testing. We have developed a scalable, 5-step process that generates a purified antibody that results in the removal of >12 logs of test viruses in clearance studies and have transitioned Acp7 into GMP manufacturing (Pacific GMP-San Diego), using a Wave Reactor (GE). From this, we generated approximately 1 g/L of the UC-961 mAb with an overall yield of >75% for the final formulated product.
The UC-961 mAb has similar, if not greater, anti-leukemia activity as D10 in our niche-dependent assays. For this we assessed whether the UC-961 anti-human ROR1 mAb could effect clearance of human-ROR1 expressing murine leukemia cells engrafted in immunodeficient recipient mice. Groups of eight RAG2-/-γc-/- mice were each injected intravenously with 0, 3, 10, or 30 mg/kg of UC-961 and then given an intravenous injection of 1x104 CD5+B220lo human ROR1+ murine leukemia cells derived from a ROR1xTCL1 transgenic mouse. Treatment with UC-961 mAb resulted in a 95% clearance of leukemic cells in the spleen at all doses tested, compared to control animals (p <0.01). We also tested UC-961 mAb for its capacity to induce clearance of human ROR1+ CLL cells engrafted into the peritoneal cavity of Rag-2-/-/γc-/- immune deficient mice. For this, mice received a single dose of UC-961 (30, 10, 3, 1, and 0.3 mg/kg) or control vehicle one day after engraftment. Seven days later, the CLL cells were harvested peritoneal lavage, counted, and analyzed by flow cytometry. In a representative experiment, the UC-961 significantly reduced the average number of harvested CLL cells in the peritoneal lavage in a dose dependent manner compared to controls (92% ± 4%, 84% ± 5%, 71% ± 8%, 69% ±14% and 60% ± 10% reduction, respectively, p < 0.001, n = 6 per group), demonstrating the in vivo anti-leukemic activity of the ROR1 targeted mAb. Pharmacokinetic and toxicology studies in Wistar rats have yet to demonstrate dose-limiting toxicity. A one compartment PK description of average data reveals that t1/2 = 11.4 days V= 1.18 mL (47 mL/kg) and CL = 0.072 mL/day (0.12 mL/hr/kg). Collectively, these studies indicate that UC-961 may be suitable for clinical studies in patients with CLL or other ROR1-expressing cancers.
Disclosures:
No relevant conflicts of interest to declare.
Non-malignant B cells and chronic lymphocytic leukemia cells induce a pro-survival phenotype in CD14+ cells from peripheral blood