Combining lapatinib (GW572016), a small molecule inhibitor of ErbB1 and ErbB2 tyrosine kinases, with therapeutic anti-ErbB2 antibodies enhances apoptosis of ErbB2-overexpressing breast cancer cells.

Antibodies and small molecule tyrosine kinase inhibitors targeting ErbB2 exhibit distinct, noncross resistant mechanisms of action. Here, apoptosis of ErbB2-overexpressing breast cancer cells was enhanced by combining lapatinib, an inhibitor of ErbB1 and ErbB2 tyrosine kinases, with anti-ErbB2 antibodies, including (i) trastuzumab, a humanized monoclonal antibody, and (ii) pAb, rabbit polyclonal antisera generated by vaccination with a human ErbB2 fusion protein. Treating ErbB2-overexpressing breast cancer cell lines with a relatively low concentration of lapatinib alone resulted in a minimal increase in tumor cell apoptosis with an associated decrease in steady-state protein levels of p-ErbB2, p-Akt, p-Erk1/2, and notably survivin, compared to baseline. Exposure to pAb alone reduced total ErbB2 protein, disrupting ErbB3 transactivation, leading to a marked inhibition of p-Akt; however, survivin protein levels remained unchanged and apoptosis only increased slightly. Treatment with trastuzumab alone had relatively little effect on survivin and apoptosis was unaffected. Combining lapatinib with either pAb or trastuzumab markedly downregulated survivin protein and enhanced tumor cell apoptosis. The association between the inhibition of survivin and enhanced apoptosis following the combination of ErbB2-targeted therapies provides a biological effect in order to identify therapeutic strategies that promote tumor cell apoptosis and might improve clinical response.

A Comprehensive Preclinical Model Evaluating the Recombinant PRAME Antigen Combined With the AS15 Immunostimulant to Fight Against PRAME-expressing Tumors.

The PRAME tumor antigen is a potential target for immunotherapy. We assessed the immunogenicity, the antitumor activity, and the safety and the tolerability of a recombinant PRAME protein (recPRAME) combined with the AS15 immunostimulant (recPRAME+ AS15) in preclinical studies in mice and Cynomolgus monkeys. Four groups of 12 CB6F1 mice received 4 injections of phosphate-buffered saline (PBS), recPRAME, AS15, or recPRAME+AS15. Immunized mice were injected with tumor cells expressing PRAME (CT26-PRAME) 2 weeks or 2 months after the last injection. The mean tumor surface was measured twice a week. Two groups of 10 monkeys received 7 injections of saline or recPRAME+ AS15. T-cell responses were measured by flow cytometry using intracellular cytokine staining (ICS). In CB6F1 mice, repeated injections of recPRAME+ AS15 induced high PRAME-specific antibody titers and mostly CD4+ T cells producing cytokines. This immune response was long-lasting in these animals and was associated with protection against a challenge with PRAME-expressing tumor cells (CT26-PRAME) applied either 2 weeks or 2 months after the last injection; these data indicate the induction of an immune memory. In HLA-A02.01/HLA-DR1 transgenic mice, recPRAME+ AS15 induced both CD4+ and CD8+ T-cell responses, indicating that this antigen can be processed by the human leukocyte antigen and is potentially immunogenic in humans. In addition, a repeated-dose toxicity study in monkeys showed that 7 biweekly injections of recPRAME+ AS15 were well tolerated, and induced PRAME-specific antibodies and T cells. In conclusion, these preclinical data indicate that repeated injections of the PRAME cancer immunotherapeutic are immunogenic and have an acceptable safety profile.

Tumor mouse model confirms MAGE-A3 cancer immunotherapeutic as an efficient inducer of long-lasting anti-tumoral responses.

PURPOSE: MAGE-A3 is a potential target for immunotherapy due to its tumor-specific nature and expression in several tumor types. Clinical data on MAGE-A3 immunotherapy have raised many questions that can only be addressed by using animal models. In the present study, different aspects of the murine anti-tumor immune responses induced by a recombinant MAGE-A3 protein (recMAGE-A3) in combination with different immunostimulants (AS01, AS02, CpG7909 or AS15) were investigated. EXPERIMENTAL DESIGN AND RESULTS: Based on cytokine profile analyses and protection against challenge with MAGE-A3-expressing tumor, the combination recMAGE-A3+AS15 was selected for further experimental work, in particular to study the mechanisms of anti-tumor responses. By using MHC class I-, MHC class II-, perforin-, B-cell- and IFN-γ- knock-out mice and CD4+ T cell-, CD8+ T cell- and NK cell- depleted mice, we demonstrated that CD4+ T cells and NK cells are the main anti-tumor effectors, and that IFN-γ is a major effector molecule. This mouse tumor model also established the need to repeat recMAGE-A3+AS15 injections to sustain efficient anti-tumor responses. Furthermore, our results indicated that the efficacy of tumor rejection by the elicited anti-MAGE-A3 responses depends on the proportion of tumor cells expressing MAGE-A3. CONCLUSIONS: The recMAGE-A3+AS15 cancer immunotherapy efficiently induced an antigen-specific, functional and long-lasting immune response able to recognize and eliminate MAGE-A3-expressing tumor cells up to several months after the last immunization in mice. The data highlighted the importance of the immunostimulant to induce a Th1-type immune response, as well as the key role played by IFN-γ, CD4+ T cells and NK cells in the anti-tumoral effect.