Development of an Antibody-Drug Conjugate Targeting CADM1 in Adult T-Cell Leukemia/Lymphoma

Background: Adult T-cell leukemia/lymphoma (ATL) is a T-cell malignancy with poor prognosis caused by HTLV-1 infection. Effective therapeutic approaches to improve the prognosis of HTLV-1-related diseases including ATL has been long explored. Recently, a remarkable efficacy of Antibody-drug conjugate (ADC), brentuximab vedotin (BV) has been reported in CD30-positive T-cell lymphoma (ECHELON-2). Although the number of ATL cases analyzed in ECHELON-2 is small and validation by other prospective clinical trials with a sufficient number of ATL patients has not been achieved yet, ADC is attracting attention as a new therapeutic agent for refractory ATL. However, low to moderate expression and heterogenicity of CD30 among ATL cells limit the therapeutic efficacy of BV, and better selection of target antigens may provide more clinical benefit. CADM1 is a cell adhesion molecule overexpressed in ATL cells and HTLV-1 infected T cells of almost all ATL patients and HTLV-1 carriers. It is reported that expression pattern of CADM1 versus CD7 analyzed by flow cytometry reflects disease progression from HTLV-1 carrier status to ATL. Thus, CADM1 is considered as a promising target, and aim of this study is to develop ADC targeting CADM1.

Methods: As an anti-CADM1 monoclonal antibody for ADC, YTH-W-2C2 was developed by immunizing rat with mixture of CADM1 isoforms as a monomer and a dimer form. Obtained YTH-W-2C2 rat IgG was further engineered to rat-human chimeric antibody (cIgG). Physicochemical properties of cIgG were examined by surface plasmon resonance (SPR) and differential scanning calorimetry (DSC). Binding and internalization to CADM1 positive cells were analyzed by flow cytometry and fluorescence microscopy. Cytotoxic function against CADM1 positive cell was evaluated by ADCC assay. The fluorescent-labeled cIgG and CADM1 expressing cell was injected to immunodeficient mice (NOJ) and co-localization were subsequently analyzed by in vivo imaging. Chimeric IgG was modified with VcMMAE. Finally, CADM1 specific cell growth inhibition and cytotoxic effect of cIgG-VcMMAE were evaluated in cell lines, PBMCs derived from indolent ATL patients, and a NOJ mouse model-grafted ATL cell line. Additionally, cross-reactivity to murine CADM1 and adverse effect to normal tissue was analyzed in mouse model by histopathological analysis. Furthermore, investigation of a more ATL specific isoform and unique epitope to reduce side effect was performed by RNA-seq data analysis of normal tissue derived from public database.

Results: SPR analysis revealed that cIgG showed high affinity to CADM1, and thermal stability analysis using DSC showed that rat IgG was successfully converted to a chimeric IgG without compromising the thermal stability. Specific binding to CADM1(+) ATL and HTLV-1 infected T-cell lines were confirmed, and IgG internalized immediately followed by subcellular localization in lysosomes. Furthermore, cIgG displayed ADCC activity to target CADM1(+) T-cell line. In vivo tracking of the cIgG in NOJ mice grafted with CADM1(+) T-cell lines demonstrated the co-localization of antibody and target cells, confirming the specific accumulation of cIgG to target cell. Generated cIgG-VcMMAE suppressed cell growth of CADM1(+) T-cell lines significantly and specifically. Ex vivo viability assays in primary PBMCs derived from indolent ATL showed that the ADC reduced the size of ATL and HTLV-1 infected cell populations, with minimal effects on the uninfected CD4⁺ T-cell. Finally, the ADC treatments in TL-Om1-grafted NOJ mice showed significant reduction of the tumor volume compared with PBS control treatment. Additionally, Histopathological evaluation to evaluate the adverse effect to normal tissue and RNA-seq data analysis derived from a public database to compare the proportion of CADM1 isoforms in normal tissues and searching ATL cell-specific epitopes are underway.

Conclusion: The CADM1-ADC showed specific and effective elimination of CADM1-expressing T cells, providing evidence that the CADM1-targeting ADC has promising potential as a new therapeutic agent for ATL. Since it is necessary to minimize undesirable side effects to normal tissue as well as effective cytotoxicity to ATL cells, the adverse effects are currently investigated in a mouse model. Furthermore, discovery of more specific CADM1 isoforms among ATL cells as a target of antibody is our next target point to refine the safety of ADC.