A Novel Antibody-Drug Conjugate Directed Towards the Surrogate Light Chain of Malignantly-Transformed B-Cell Precursors Reverses Leukemic Progression in NSG Mice

Field: Pharmacology, Toxicology and Pharmaceutics (Life Sciences)

Background: Antibody-drug conjugates (ADC) utilizing AcBut-linked calicheamicin have improved survival for B- and myeloid lineage hematopoietic malignancies for both adults and children. We recently showed that the VpreB1 component (CD179a) of the B-cell surrogate light chain is ubiquitously expressed on B-lineage lymphoblasts, which may present a suitable target for an immunotherapeutic intervention. Specifically, the VpreB1 component of the surrogate light chain was identifiable in 36 / 36 (100%) of primary patient samples obtained from COG studies AALL0232 and AALL1131 (Blood Adv6(2): 585-589; 2022.) Materials and Methods: Using a human IgG1 subclass monoclonal antibody (mAb) against the VpreB1 component of the surrogate light chain, we created an ADC that is comprised of an AcBut-linker to calicheamicin. Using ex vivo testing conditions, we assessed 50% inhibitory concentrations (IC50s) in the Nalm6, REH and SEM B-ALL cells lines, and in PDXB1 and PDXB2 patient-derived xenographs (PDXs). We tested the VpreB1 ADC to evaluate its potential to rescue NSG mice that were engrafted with Nalm-6 B-lymphoblasts using VpreB1 ADC doses ranging from 0.1 to 3 mg/kg given intraperitonealy three times, four days at apart at leukemic engraftment to assess responses. To assess leukemic burden in the experimental and control conditions, we utilized human FITC-labelled CD45/CD19 antigen expression and Nalm-6-Luciferase imaging to track disease progression in mock, CD179a mAb (alone) and VpreB1 ADC-treated NSG mice. Results: We found a spectrum of IC50s in the B-ALL samples tested under ex vivo conditions, which ranged from 59 ng/ml (Nalm6), 81 ng/ml (REH), 108 ng/ml (PDXB2), 2499 ng/ml (PDXB1) to 5105 ng/ml (SEM), suggesting a range of sensitivities to the mAb. Using flow cytometric analyses of Nalm-6 engraftment, we found that human CD19-expressing cell populations were absent in the VpreB1 ADC treatment group, but present in the mock and CD179a mAb-alone control groups. We employed a series of dose-finding studies and found that a dose of 2 mg/kg (~105 mcg/kg calicheamicin dose) in the experimental group was sufficient to reverse progressive disease. Using Nalm-6-engrafted NSG mice, we found that the VpreB1 ADC (2 mg/kg) treatment cohort had significantly extended lifespans in comparison to those treated with mock or naked mAb control groups (Mantel-Cox log-rank; P<0.0001). Using Nalm-6-luciferase imaging, we determined that the surviving animal population showed no signs of leukemic infiltration in comparison to the negative control groups, and that (4/6; 67%) of the animals in the VpreB1 ADC treatment group survived without evidence for relapsed leukemia for more than 100 days. Two of the animals died for inexplicable reasons, but did not have progressive disease by luciferase imaging. At necropsy, none of the four surviving animals in the experimental treatment group had flow cytometric evidence for leukemia. These findings suggest that the VpreB1 ADC induced "cures” in the surviving Nalm-6-Luciferase treatment group. Conclusions: We have created a novel ADC that neutralizes leukemic progression in our NSG Nalm-6 engrafted murine model. Our preliminary data supports the further evaluation of the ADC in patient-derived xenografts (currently underway). Further studies will be performed in preparation for Phase 1 clinical testing in adults and children with B-lineage acute lymphoblastic leukemia/lymphoma.