(530f) Annexin A5-DM1 Protein Drug Conjugate Combined with Immune Stimulation and mTOR Inhibitor Treatment of Triple-Negative Breast Cancer

While major advancements in biomarker discoveries have enhanced chemotherapy delivery to the tumor microenvironment, triple-negative breast cancer (TNBC) remains one of the few cancers that does not have an FDA approved targeted treatment. TNBC cells lack all three hormonal receptors (estrogen, progesterone, and HER2) found on other breast cancer subtypes, and are highly aggressive. Once the tumor has metastasized, there is approximately a 12% 5-year survival rate. The need to target both the primary TNBC tumor and metastatic lesions has never been greater. Here we report a novel protein drug conjugate that targets and exploits cancer’s upregulation of phosphatidylserine (PS).

We have developed a novel protein drug conjugate consisting of annexin A5 (ANXA5) and emtansine (DM1). ANXA5 (~36 kDa) is a naturally occurring human protein that binds with high affinity (Kd ~ 1 nM) and specificity to PS in the presence of calcium. PS is the most abundant anionic phospholipid in the lipid bilayer; its asymmetric expression is tightly regulated. In healthy cells, PS is almost exclusively found on the cytosolic leaflet and regulates phagocytotic cell death when it is extracellularly expressed. The tumor, metastatic lesions, and tumor vasculature all have an upregulated and ubiquitous expression of PS, making it a useful biomarker for cancer therapy.

We have successfully conjugated DM1 to ANXA5 (ANXA5-DM1). DM1 is a microtubule inhibitor and causes mitotic arrest leading to apoptosis. While DM1 was considered to be a promising anticancer drug because of its high cytotoxicity, it induced widespread systemic toxicity, limiting its therapeutic potential. By conjugating DM1 to ANXA5, we have harnessed DM1’s therapeutic potential while decreasing systemic side effects. On top of DM1 being an attractive cytotoxic agent, it also induces immunogenic cell death, which leads to the activation of professional antigen-presenting cells and T-cells causing antitumoral immunity. The immunogenic cell death caused by ANXA5-DM1 increases the therapeutic potential for checkpoint inhibitors such as anti-PD-1. Additionally, ANXA5-DM1 also targets tumor vasculature cutting off the tumor’s nutrient supply, inducing growth in the presence of hypoxia as a result of the activation of hypoxia-inducible factor-1 (HIF-1). Mammalian target of rapamycin (mTOR) inhibitors (such as rapamycin, everolimus, and temsirolimus) are thought to reduce angiogenesis by indirectly reducing the synthesis of HIF-1 subunits. By combining ANXA5-DM1, anti-PD1 antibody and rapamycin, a wholistic treatment regime is established.

DM-1 is linked to ANXA5 in a two-step reaction. First, the lysine residues on ANXA5 are activated with sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxyalte (Sulfo-SMCC). Second, DM1 is introduced and the malemide on ANXA5-sulfo-SMCC reacts with the lone sulfhydryl group on DM1. Six DM1 molecules were successfully loaded per ANXA5.

The conjugate has been tested both in vitro and in vivo. When compared to the free DM1, the ANXA5-DM1 conjugate was 130 times more effective against EMT6 mouse breast cancer cells, 377 times more effective against 4T1 mouse breast cancer cells, and 910 times more effective against MCF-7 human breast cancer cells when comparing the IC50 values (concentration where the cell viability is reduced by 50%). An in vivo dosing regime was also optimized to find the lowest dose that is effective. Immune-competent female mice were inoculated orthotopically with 4T1 cancer cells. Once the tumor reached ~5 mm in diameter, the mice were treated with the ANXA5-DM1 conjugate at doses in the range of 0.0025 to 0.25 mg/kg (weight of DM1/weight of mouse) once every 7 days. This optimization study found that the dose of 0.025 mg/kg gave the lowest tumor volume of the doses tested. Furthermore, a combination therapy approach was also explored against 4T1 cancer. After 31 days since tumor inoculation (6 days after treatment ended), the combination of ANXA5-DM1, rapamycin, and anti-PD-1 resulted in the tumor volume being smaller by 55% compared to the phosphate buffered saline (PBS) control, a statistically significant difference (p =0.0002). Validation of the treatment is currently ongoing with EMT6 mouse breast cancer tumors, and further studies exploring immune activation after treatment are ongoing.