CBX250 Is a Novel Cathepsin G Peptide-HLA-Targeting T Cell Engager That Exhibits High Tumor Antigen Selectivity and Potent Antileukemic Activity In Vivo
Authors: Lee Benjamin H. , Paulus Geraldine , Helble Jennifer , Sankaran Preethi , Rahman Tanzila , O'Connor Delainey , Bikowitz Melissa , Jaffe Sarah , Kulesha Alona , Ban Bhupal , Mak Nga Sze Amanda , Princiotta Michael , Shi Chunhua , Yan Jun , He Hong , Feng Ningping , Molldrem Jeffrey J. , Heffernan Timothy , Alatrash Gheath , Wiederschain Dmitri 
Published: December 2024

Cathepsin G (CG) is an intracellular serine protease whose endogenous expression is primarily restricted to cells of myeloid lineage where it is normally stored in azurophilic granules. Compared to normal hematopoietic progenitors, CG is highly expressed and ubiquitinated in AML blasts and leukemic stem cells, where it is aberrantly localized and processed for antigen presentation. CG1 is an HLA-A*02:01 restricted peptide (FLLPTGAEA) derived from the CG protein leader sequence and is abundantly presented by leukemic compared to normal myeloid cells1,2. Moreover, the detection of CG1-specific cytotoxic T lymphocytes (CTLs) in AML patients following allogeneic stem cell transplantation underscores its attractive potential as a novel immunotherapeutic target in AML1,2.

CBX-250 has been developed as a TCR-mimetic (TCRm) bispecific T Cell Engager (TCE) antibody (Ab) that binds to the CG1/HLA-A2*02:01 pMHC complex with high affinity. CBX-250 induces potent T cell mediated killing in vitro of leukemia cell lines with varying levels of target expression at sub-nanomolar EC50 levels and exhibits highly effective tumor control in vivo against multiple AML cell line (CDX) murine models at doses as low as 0.005 mg/kg. Importantly, CBX-250 also demonstrates potent efficacy, reduced tumor burden, and a clear survival benefit vs. a control arm in an aggressive primary AML patient derived (PDX) tumor mouse model that harbors FLT3-ITD, DNMT3a, and NPM1 mutations. Consistent with these results, CBX-250 was able to induce significant levels of IFNg release from several primary AML patient samples co-cultured with donor effector T cells vs. a negative control.

Successful immunotherapeutic approaches for the treatment of AML have been limited due to a lack of tumor specific surface targets (e.g., CD33, CD123) that avoid undesirable toxicities due to their expression on normal hematopoietic cells. Significantly, CBX-250 does not induce T cell activation or IFNg production when co-cultured with HLA-A2 positive healthy donor (HD) neutrophils and displays no toxicity towards normal hematopoietic stem cells (HSC) and progenitor cells in a colony forming unit (CFU) assay, in contrast to a cytarabine-treated positive control. In keeping with these findings, CBX-250 does not induce IFNg or T cell activation when co-cultured with HD CD34+ HSCs and PBMCs, whereas a significant increase in IFNg levels is observed when these stem cells are co-cultured with a CD123 TCE bispecific Ab. Moreover, when HD PBMCs are incubated with increasing concentrations of CBX-250 in the absence of tumor cells for 3 days, no changes are observed in any immune cell subpopulation (T, B cells, monocytes). By comparison, a CD123 TCE Ab induces a dose-dependent reduction in monocytes (CD123+, CD14+) and a concomitant increase in T cells under similar conditions, reflecting on-target T cell-mediated killing by the CD123 TCE Ab that is not observed with CBX-250.

Bystander killing has been recognized as a feature of TCE bispecific Abs that may help to overcome potential resistance mechanisms which may arise due to heterogeneous tumor antigen expression3-6. The ability to kill both CG1/pMHC-positive tumor cells and proximal CG1/pMHC-negative cells (bystanders) would be desirable to prevent escape and outgrowth of target-negative tumor cells during treatment. To investigate CBX-250-mediated bystander killing, we measured cytotoxicity of CG1/pMHC-negative AML cells in vitro when co-cultured with CG1/pMHC-positive tumor cells, effector T cells, and CBX-250 at varying concentrations. Our results demonstrated that CBX-250 was able to induce dose-dependent bystander killing of neighboring CG1/pMHC target-negative leukemia cells that was observed only in the presence of CG1/pMHC positive cells, highlighting the beneficial potency effects of CBX-250 in a tumor microenvironment with heterogenous target antigen expression.

Taken together, these data provide strong preclinical evidence of the potency, specificity, and safety of CBX-250 against a novel tumor antigen that may provide a wider therapeutic index relative to other well characterized AML targets. These findings support further development in the clinic and IND-enabling studies for CBX-250 are currently ongoing. 1.Zhang et al CCR 20132.Alatrash et al Leukemia 20173.Ross et al PLOS One 20174.Upadhyay et al Can Disc 20215.Teijeira et al Theranostics 20226.Gaspar et al JITC 2023