Eradication of the CLECL1+ CLL Subset That Is Prone to Carrying Novel Genomic Mutations and Promoting a Pro-Tumor Microenvironment By a Series of New Monoclonal Antibodies Reactive with Distinct Target Epitopes
Authors: Chen Shih-Shih , Buhl Susan , Ng Anita Kar Yun , Schipper Laila , Cinquemani Bianca , Trepeta Nikhil , Cardillo Martina , Blanco Gonzalo , Wong Kelly , Chen Yu-Ju , Yan Xiao J. , Scharff Matthew , Chiorazzi Nicholas 
Published: December 2024

Despite the often long clinical course and the frequent, but not requisite presence of a genetic lesion that causes over production of anti-apoptotic proteins, it is the birth rate, not the death rate, of chronic lymphocytic leukemia (CLL) clones that associates with clinical course. We documented this in patients who drank “heavy” water (2H2O), which labeled dividing CLL cells and allowed birth and death rate measurements. We next determined that the CXCR4DimCD5Bright “proliferative fraction”, PF, is markedly enriched in recently born cells compared to the CXCR4BrightCD5Dim “resting fraction”, RF. Then, by comparing the transcriptomes of the PF and RF, we discovered that adding expression of CLECL1 to the PF phenotype (CLECL1+ PF), enriched for even more recently born cells, thereby allowing studying the biology of these cells in a more precise manner. The CLECL1+ PF is especially dangerous for patients as it can be the source of subclones with new genetic abnormalities since it underwent DNA replication in the presence of DNA mutators AID and high levels of ROS. Since previous studies revealed that recombinant CLECL1 (rCLECL1) elicited IL-4 producing T cells that promote an immunosuppressive tumor microenvironment (TME), we documented this in CLL, also finding that CLECL1+ PF cells were more responsive to IL-4 derived survival signals than CLECL1- PF cells.

To further decipher the heterogeneity of the PF, we performed single-cell RNA sequencing (scRNA) on PBMCs from 9 CLL patients. This revealed that CLECL1 expression associates with an intra-PF cluster that is superior at interacting with T cells and monocytes in the TME, as determined by CellPhoneDB cell-cell interaction analyses. To assess whether interactions between CLECL1+ PF and accessory cells led to similar biological effects in vivo, we evaluated the capacity of CLECL1+ PF to generate Th2 cells in xenografts. CLL fractions sorted from 3 CLL patients were transferred into NSG mice along with resting autologous CD4+ T cells. This recapitulated the in vitro findings: only PF and CLECL1+ PF, but not CLECL1- PF cells activated T cells and induced IL-4 producing Th2 T cells. The CLECL1-stimulated Th2 cells further supported monocytes to differentiate into CD206+CD80- M2 macrophages. Importantly, consistent with CellPhoneDB results, we found that rCLECL1 and CLECL1+ but not CLECL1- cells directly induced M2 macrophages, independent of T cells, although the level of M2 induction was greater with T cell help.

As CLECL1+ PF cells are recent progeny of CLL cells born in solid tissues and are a critical CLL subset promoting a pro-tumor TME, we developed 3 new therapeutic monoclonal antibodies (mAbs) against human CLECL1, by fusing spleen cells from mice immunized with the 12-mer C-terminal peptide (IQDITAMVRFNI) of the CLECL1 external domain conjugated to KLH. Each mAb reacted with the immunizing peptide and with the external domain of CLECL1 in ELISA. Notably, one mAb (221) reacted with the immunizing peptide in its linear state, whereas the two other mAbs (17 and 243) reacted with the peptide in a conformational state. The latter was documented by loss of binding after rCLECL1 was pretreated with 2-mercaptoethanol + iodoacetamide to cleave disulfide bonds. Moreover, each mAb enumerates CLECL1+ CLL cells by flow cytometry at levels like those defined by a standard, available IgM anti-CLECL1 mAb. Functionally, each anti-CLECL1 mAb induced significant antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and antibody-dependent cellular cytotoxicity (ADCC) on primary CLL cells in vitro. Finally, each mAb blocked conversion to/expansion of Th2 cells in vitro, and the growth of MEC1 cells in NSG mice. The latter is especially striking since activity was found using 5-fold less of each mAb (2 mg/kg/bw) than optimal.

Overall, our studies further characterize the dangerous CLECL1+ PF subpopulation of CLL cells, which directly influences disease progression and indirectly promotes Th2 cells and M2 macrophages. The anti-CLECL1 IgG mAbs have striking efficacy in ADCP, ADCC and CDC, and eliminating xenografts, suggesting that they will have clinical applicability. This would be especially beneficial in the setting of BTKi resistance in CLL and other B-cell leukemias/ lymphomas. Since CLECL1+ PF cells are recent descendants of cell division, which BTKi normally eliminates, the mAbs will do the same, albeit after birth.