Background: We set out to determine if the administration of subcutaneous (SQ) ALT-803 was non-inferior to standard intravesical BCG treatment in a carcinogen induced mouse (C57BL/6J) bladder cancer model.

Methods: Using this well-established carcinogen induced mouse model, we studied the effects of various dosing schemas of ALT-803 (SQ alone, SQ with intravesical BCG, intravesical alone, intravesical with intravesical BCG) compared to intravesical BCG alone (positive control) and PBS (negative control). The non-inferiority margin for the difference in bladder weight, as a surrogate for tumor mass, was defined as 7%.

Results: All treatment groups (i.e., ALT-803 SQ alone, ALT-803 SQ with intravesical BCG, ALT-803 intravesical alone, ALT-803 intravesical with intravesical BCG and intravesical BCG alone) demonstrated a significant reduction in tumor burden as evident by bladder weights and H&E stain (p < 0.005). Non-inferiority tests between the intravesical BCG alone group and the additional treatment groups showed that SQ ALT-803 alone (p = 0.04) and BCG plus SQ ALT-803 (p = 0.009) were non-inferior to intravesical BCG alone. In this model, we did not see an appreciable infiltration of CD4+ T, CD8+ T or CD161/KLRB1+ natural killer (NK) cells in the bladder/tumor. When assessing peripheral blood mononuclear cells, SQ ALT-803 alone resulted in a robust induction of CD8+ T cells (p < 0.01), NKG2D+ NK cells (p < 0.005) and CD3+/NKG2D+ NKT cells (p < 0.005) compared to other groups, while in splenic tissue, SQ ALT-803 alone resulted in a robust induction of CD3+/NKG2D+ NKT cells (p < 0.005) compared to other groups.

Conclusion: Subcutaneous ALT-803 treatment alone or in combination with intravesical BCG was well tolerated and was not inferior to intravesical BCG alone. CD8+ T, NKG2D+ NK and CD3+/NKG2D+ NKT cell induction along with induction of key cytokines remain steadfast mechanisms behind ALT-803. The enhanced therapeutic index seen with BCG and ALT-803, administered SQ or intravesically, provides a powerful justification for the further development of these regimens.