Evaluation of ROR1 Targeted Antibody Drug Conjugates in Acute and Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma

Introduction: Receptor tyrosine kinase like orphan receptor 1 (ROR1) is a developmentally restricted receptor tyrosine kinase that is expressed on the surface of multiple hematological and solid malignancies but not on normal adult tissues. Recently, there has been an increased interest in ROR1 as a potential therapeutic target in multiple ROR1 positive leukemia and lymphomas. In this study, we present novel anti-ROR1 monoclonal antibody drug conjugates (ADCs) based on chimeric rabbit/human mAb "XBR1-402" or its humanized version "huXBR1-402", that show both in vitro and in vivo efficacy at eliminating ROR1 positive chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), and pre B cell acute lymphocytic leukemia (pre B cell ALL) leukemic cells.

Methods: From our studies, we saw no difference in activity between XBR1-402-PNU and huXBR1-402-PNU. XBR1-402-PNU was used for our studies in pre B cell ALL and huXBR1-402-PNU was used for our studies in MCL and CLL. For ALL in vitro experiments, we monitored cytotoxicity in Kasumi2 and 697 cell lines. For MCL in vitro experiments we monitored cytotoxicity in Jeko and Mino cell lines. For CLL in vitro experiments, we monitored cytotoxicity in primary CLL samples. For ALL in vivo experiments we monitored overall survival in a murine disseminated 697 model. For CLL in vivo experiments we monitored ROR1+ leukemic cell depletion in human ROR1 transgenic mice engrafted with human ROR1+TCL1 leukemia.

Results: We observed a decrease in viability for all XBR1-402-PNU and huXBR1-402-PNU treated ROR1 positive leukemic cell lines and primary cells in vitro . In the in vitro model of pre B cell ALL we observed a significant decrease in viability of the ROR1 positive pre B cell ALL cell lines 697 and Kasumi-2 when treated with XBR1-402-PNU(EC₅₀ = 77 and 1 ng/mL, respectively) when compared to treatment with the isotype-matched control ADC Trastuzumab-PNU (Tras-PNU) (EC₅₀ = 2987 and 5330 ng/mL, respectively). In the in vitro model of MCL, we observed a decrease in viability for huXBR1-402-PNU treated ROR1 positive Jeko and Mino cell lines but not ROR1 negative Mec-1 and Jurkat cell lines when compared to the unconjugated huXBR1-402 antibody or the control Tras-PNU. Additionally, we tested primary CLL samples confirmed to be positive for ROR1 and found a significant decrease in viability over 96 hours when treated with the antibody-drug-conjugate huXBR1-402 (78.6 + 6.1%)as compared to the unconjugated antibody (100% + 2.4; p=0.022, N=7) and the control Tras-PNU (87.4% +/- 6.1; p=0.038). We have also detected promising in vivo activity with XBR1-402-PNU and huXBR1-402-PNU in ALL and CLL models, respectively. In vivo studies on a murine disseminated 697 ALL model showed that treatment with XBR1-402-PNU increased overall survival when compared to treatment with Tras-PNU control (Median survival of 32 and 23 days after implantation respectively, p=0.021, n=6). In addition, in preliminarily experiments, we have tested depletion of human ROR1+ leukemic cells and normal B cells in vivo using human ROR1 transgenic mice engrafted with human ROR+TCL1 leukemia and observed a preferential depletion of ROR1+TCL1 leukemic cells upon treatment with huXBR1-402-PNU compared to control Tras-PNU.

Conclusion: Our results suggest that anti-ROR1 ADC huXBR1-402-PNU is an effective and promising targeted cytotoxic therapy for ROR1 positive leukemic cells of CLL, MCL, and pre B ALL and warrants further evaluation for clinical consideration in patients with ROR1 positive leukemia and lymphomas. Current long-term studies are ongoing to evaluate the in vivo cytotoxic effects of huXBR1-402-PNU on total tumor burden, disease progression, and overall survival.

(EH is supported by Graduate Pelotonia Fellowship)