AB-3A4 for the treatment of invasive carcinomas
Alethia is developing a therapeutic monoclonal antibody for the treatment of ovarian cancer and other invasive carcinomas such as triple-negative breast tumors and renal cancer. AB-3A4 is an IgG1 monoclonal antibody against KAAG-1, a cell-surface protein involved in tumor invasion.
Background and rationale
Ovarian cancer is a terrible disease that affects greater than 1 in 70 women in North America. The 5-year survival rate for all stages is only 35% to 38% clearly illustrating the urgent needs in this disease. One of the ways to progress towards fulfilling these needs, is to identify new and more specific molecular targets. Alethia has directed its ovarian cancer program to address these therapeutic requirements.
KAAG-1 – A Cell Surface Antigen that is Expressed in Greater than 90% of Ovarian Tumors
Using Alethia’s innovative discovery platform, analysis of subtracted cDNA libraries from five ovarian tumors resulted in the identification of hundreds of genes exhibiting increased expression compared to benign tumors. The most promising target to emerge from this discovery effort was kidney-associated antigen-1 (KAAG-1), an antigen that is expressed in ovarian tumors in greater than 90% of patients. Moreover, KAAG-1 is expressed in few normal tissues underscoring its high degree of ovarian cancer specificity.
Starting from a library of recombinant mouse antibodies, a promising candidate antibody termed AB-3A4 was selected based on its high affinity, its ability to detect KAAG-1 on the surface of ovarian cancer cells and its ability to prevent the formation of spheroids in ovarian cancer and melanoma cell lines.
Antibodies against for KAAG-1 as ovarian tumor specific ADCs
Given the high degree of specificity of KAAG1 and the ability of AB-3A4 to detect the protein on the surface of cancer cells, the potential for developing an antibody-drug conjugate (ADC) was explored. We discovered that KAAG1-positive ovarian cancer cells internalized the KAAG1/AB3A4 complex from the surface of the cells. Indeed, using flow cytometry, the fluorescence signal that was observed on the surface of the cells at time 0 was markedly decreased over a period of 30 – 45 minutes. In parallel experiments, a similar decrease in fluorescence signal was observed in other KAAG1-positive cancer cells. To illustrate this internalization, additional studies were performed using SKOV-3 ovarian cancer cells to determine if the internalized KAAG1/AB-3A4 complex was followed known internalization pathways. As shown below, AB-3A4 was detected inside the cells after the incubation period (see red staining, left panel) in a pattern that was consistent late endosomal/lysosomal structures located in the peri-nuclear area of the inside of the cells (arrows, red fluorescence). Furthermore, the cells were stained with an anti-LAMP1 antibody, a marker for lysosomes. When the images were merged, a co-localization of the signals was observed suggesting that the internalization of the AB-3A4/KAAG-1 complex followed the endosomal/lysosomal pathway. This data is in complete agreement with the FCM data and confirmed that KAAG-1 bound to its specific antibody on the surface of cells was internalized.
Taken together, these studies suggest that AB-3A4, which binds to KAAG-1 has tremendous potential for an ADC to specifically target ovarian tumors. We have also found that KAAG-1 is highly expressed in breast cancer, including triple-negative breast cancer, for which there is currently no available targeted therapies.
Recently, ADCs have re-emerge as promising therapeutic strategies with the improvements that were made to linkers which conjugate the mAb to the toxin. Thus, the high level of ovarian cancer specificity of KAAG-1 coupled with the capacity of this target to be internalized in cells would support the development of an ADC.