A Cell Surface Heat Shock Protein 70 (csHSP70)×CD3 Bispecific T-Cell Engaging Antibody Exhibits Anti-Tumor Efficacy in Pre-Clinical T-Cell Lymphoma and Multiple Myeloma Models
Authors: Ray Upasana , Jones Richard J. , Orlowski Robert Z. 
Published: December 2024

Background:
Neoplasms of T-cell or natural killer (NK)/T-cell origin account for 10-15% of all non-Hodgkin lymphomas (NHLs) in the United States, and up to 30% or more of NHLs in African and Asian countries. Tumors from post-thymic or peripheral T-cells, referred to collectively as peripheral T-cell lymphomas (PTCLs), are heterogeneous malignancies diagnosed by their clinical, morphologic, and immunophenotypic features, along with selected molecular assays. Despite recent advances, PTCLs still have a poorer prognosis than most B-cell NHLs, and &gt;75% of patients (excluding those with Anaplastic lymphoma kinase (ALK)-rearranged anaplastic large cell lymphoma) fail to respond to, or relapse within 2 years after current regimens. We previously identified externalization of csHSP70 on PTCL cell lines and developed a csHSP70-targeted monoclonal antibody that showed anti-tumor efficacy as an antibody drug conjugate in both in vitro and in vivo models, and next sought to convert this into a bispecific antibody format.
Methods:
We developed both small molecule (bispecific T-cell engager (BiTE), dual-affinity re-targeting protein, diabody) and large molecule (knob-in-hole, duobody) engager formats based on the single chain variable fragment sequences of our csHSP70-targeted antibody. These were linked with sequences derived from the murine SP34 or UCHT1 antibodies recognizing human CD3. Binding assays were performed across different T-cell NHL and myeloma cell lines. Activation of the T-cell effector (E) population isolated from healthy donor blood was performed in a co-culture model with target (T) csHSP70-positive cancer cells (E:T 10:1) and examined by flow for surface markers (CD3, CD69, CD71). Cytotoxicity assays were performed using a similar co-culture model (E:T 10:1) in the presence of engagers verses parental antibody controls for 48 hours, and cells were stained with Annexin V/PI, and the percent apoptotic events were analyzed by flow cytometry.
Results:
Binding of histidine-tagged bispecific constructs to csHSP70-positive lymphoma and myeloma cells, and to CD3 expressed on PBMC-derived T-cells, was confirmed by flow cytometry using and anti-histidine-PE tagged secondary antibody. Activation of effector T-cells was demonstrated by appearance of CD69- and CD71-positivity in the CD3-positive T-cell population using a co-culture system with PBMCs and calcein AM-stained csHSP70-positive cancer cells at nanomolar bispecific concentrations over 48 hours. As binding was optimal for the BiTE-UCHT1 construct, we further tested its efficacy in inducing apoptosis in target cancer cell lines. Both early (Annexin+/PI-) and late (Annexin+/PI+) apoptosis was enhanced by the BiTE-UCHT1 in a dose-dependent manner over a range of concentrations (25-100 nM) compared to UCHT1 and/or csHSP70 antibody controls. Notably, addition of excess intact csHSP70 antibody impaired the pro-apoptotic effects of BiTE-UCHT1, further validating its specificity. To study possible combination regimens of interest, target cell death was studied in co-culture and analyzed by staining with the Zombie near Infrared (ZNIR) dye that detects dead cells. The proteasome inhibitor (PI) bortezomib or the deacetylase inhibitor (HDACi) vorinostat enhanced csHSP70 and, in combination with BiTE-UCHT1, enhanced the anti-tumor efficacy in both T-cell NHL and myeloma cell line models in a synergistic manner. Cell line-derived xenograft testing is underway, as is pre-clinical testing with NK-cell engagers, and will be available at the time of the Annual Meeting.
Conclusions:
Bispecific T-cell engagers recognizing csHSP70 demonstrate robust pre-clinical activity against tumor models that externalize enhanced levels of csHSP70, including PTCL and myeloma cell lines, at physiologically relevant concentrations. Combination strategies with agents already used in these tumors that act in part through immunogenic cell death, including PIs and HDACis, show enhanced, synergistic activity. Taken together, the data support further pre-clinical development of csHSP70-targeting bispecific immune effector cell engager therapeutics, and a rationale to consider their translation to the clinic.