Purpose: Blockade of immune checkpoint proteins such as programmed death (PD1) and programmed death ligand-1 (PDL1) has emerged as a groundbreaking strategy for anticancer therapy. However, transient expression of PD1/PDL1 proteins and abundance of dense dysplastic stroma (DDS) leads to immune evasion in the tumor environment. To overcome these limitations, we developed novel antibody drug conjugates (ADCs) by conjugating PD1 and PDL1 antibody inhibitors with chemotherapeutic drugs, such as Gemcitabine (GEM) and Doxorubicin (Dox) respectively. The PD1 and PDL1 ADCs are connected through the tumor milieu responsive linkers such as hydrazine and dithiol, which allow the ADC to deliver the GEM/Dox in extracellular DDS followed by release, thus improving penetration of PD1/PDL1 antibody into the core of the tumor. The purpose of developing PD1-/PDL1-ADC is to induce synergistic anti-tumor effect through multimodal effect of tumor cell killing, disruption of tumor stroma and resurrection of tumor killing immune cells.
Methods: In this work we developed two ADCs, namely PD1-GEM and PDL1-Dox. The PD1 antibody was conjugated with GEM using mild TCEP reduction of antibody followed by conjugation of GEM through dithiol (S-S) bond formation to obtain PD1-GEM ADC. The PD1-GEM ADC was characterized by gel electrophoresis and MALDI analysis, and the average drug to antibody ratio (DAR) for PD1-GEM was found to be 3. For synthesis of PDL1-Dox, we used clinically used PDL1 antibody to couple with Dox via acidic pH responsive hydrazone linker.
Results: The drug kinetics of PDL-Dox revealed 90% and 30% of Dox release when subjected to pH 5.5 and 7.5 conditions at 50 h respectively. The sustained and acidic pH stimuli-responsive release of Dox from PDL1-Dox supports our hypothesis. Cell cytotoxicity analysis of PDL1-Dox demonstrated significant killing in triple negative breast cancer cells, MDA-MB-231 cells after 72 h of treatment compared to Dox treatment. The IC50 of Dox and PDL1-Dox in MDA-MB-231 was 4 µM and 1.25 µM respectively. The cell killing effect of PDL1-Dox treatment in MDA-MB-231 can be associated with induction of early stage apoptosis with 2-fold higher than control cells. Up-modulation of pro-inflammatory cytokine, IFN-γ in PDL1-Dox treated macrophage and MDA-MB-231 co-culture demonstrated the PDL1-Dox mediated inhibition of PD1 and PDL1 interaction. The significant tumor growth inhibition of highly aggressive pancreatic cancer (PDAC) tumor in treatment of PD1 antibody combined with chemotherapy supports the rational of using PD1-GEM ADC for synergistic therapeutic benefits in deadliest PDAC tumor. The combination of PD1 antibody treatment demonstrated activation of tumor killing M1-macrophages and upmodulating of cytotoxic CD8+ T cell in tumor mass as analyzed by multi-color flow cytometry.
Conclusion: The immune check point ADC opens up a new avenue to achieve the durable anticancer response in a range of solid tumors. Our data provide insights into activation of tumor killing immune cell in synergy with apoptosis medicated cancer cell killing.
Samaresh Sau– Detroit, Michigan
Samaresh Sau– Detroit, Michigan
Rami Alzhrani– Detroit, Michigan
Ketki Bhise– Detroit, Michigan
Alex Petrovici– Detroit, Michigan
Hashem Alsaab– Dearborn, Michigan
Arun Iyer– Detroit, Michigan