CTLA-4 blockade is currently being evaluated clinically in many different solid and hematologic malignancies. Treatment with anti-CTLA-4 antibodies presumably potentiates immunosurveillance to endogenous tumor antigens by relieving a crucial immune checkpoint. However, the specific endogenous antigen response has been difficult to define, particularly in the absence of a co-administered vaccine where the vaccine antigens may be known. Nevertheless CTLA-4 blockade can induce clinical responses in the absence of a vaccine. None of our patients received an administered tumor vaccine as part of their treatment, so their clinical effects must be dependent on endogenous antigens. By using protein microarrays representing approximately one third of the human proteome, we were able to profile the antibody responses induced with treatment to a broad spectrum of autoantigens. Moreover, a significant proportion of patients clinically responded to our treatment allowing us to examine whether antigen-specific responses could distinguish the clinical responders from non-responders.
Based upon these antibody responses, clinical responders developed a broader immune response as seen by the induced antibodies to a greater number of endogenous antigens compared to the non-responders. This difference was observed at four fold but not at two fold up-modulation of antibodies indicating that the induced responses were also at higher intensities for the responders compared to the non-responders. The modulated antibody responses were quite diverse, and there was very little overlap between the antigens identified in responders versus the non-responders. These results show that patients who can clinically respond to treatment may also be immunologically distinct from non-responders based on their autoantibody profiles. These differences could reflect the capacity of tumors in different patients to avoid immunosurveillance. Alternatively, the clinical responders may have tumors that are inherently more immunogenic or may have differing levels of tumor associated immunosuppression. The majority of the antigens are unique for each patient, which could also reflect the diversity of their T and B cell repertoires and/or the heterogeneity of antigens expressed in prostate tumors. Although common pathways could be affected in cancer, different genetic alterations are observed in cancer patients (24
), which can give rise to individualized antigenic milieu. Therefore, by modulating the immune system to recognize patient specific endogenous antigens, CTLA-4 blockade could represent a form of personalized immunotherapy.
Another unresolved question regarding the treatment’s mechanism of action is whether CTLA-4 blockade enhances pre-existing immune responses, or whether the treatment potentiates de novo antigen-specific responses. In all of our patients, we did not see any modulation of antibody responses to the control antigen Influenza A H3N2, supporting the notion that the treatment-induced modulation of antibodies reflects the antigen milieu in the host. Antibody responses were in fact induced to antigens both with and without detectable preexisting antibodies prior to treatment. This is exemplified with Pak6, to which patient 19 had low levels of antibody prior to treatment while patient 20 had undetectable levels prior to treatment. Interestingly, clinical responders were more likely than non-responders to generate antibodies against antigens to which preexisting antibodies could be detected. These results suggest that induction of preexisting rather than de novo (non-preexisting) immune responses may be important in generating antitumor activity in CTLA-4 blockade therapy. With one of the clinical responders (patient 19), detectable preexisting IgG to Pak6 could be detected, but no T cell response to Pak6 could be detected at baseline. Following treatment, a CD4 T cell response to Pak6 was induced coinciding with an enhancement of IgG responses. These results would indicate that an immune response to Pak6 was generated spontaneously in the patient, but was subsequently dampened perhaps by tumor-induced immunosuppression. Nevertheless, relieving a crucial immunologic checkpoint with CTLA-4 blockade may be sufficient to recover immune responses to such tumor associated antigens.
Antibodies that were down-modulated were also detected in CTLA-4 blockade therapy. However, the number of down-modulated antibodies between the responders and non-responders were not significantly different. Total levels of IgG were not significantly changed with treatment, so these changes could not be due to dilution. However, the mechanism for down-modulation of antibodies by CTLA-4 blockade is unclear at present.
Most of the antigens with induced autoantibodies following treatment were intracellular proteins. Presumably, immune responses could have been initiated to these antigens as they are released from dying cells, especially since tumor cells have a propensity for increased cell turnover as well as for apoptosis and necrosis (26
). All of our patients received and clinically progressed on androgen deprivation therapy, which would have also induced cancer cell death and release of antigens. However, there may be insufficient danger signals to drive an effective immune response in the absence of CTLA-4 blockade. The antigen that we focused upon, Pak6, could be considered a novel tumor-associated antigen. Pak6 is expressed in prostate cancer and is known to co-translocate into the nucleus with androgen receptor (AR) in response to androgen and inhibits the transcriptional activity of AR (18
). Alterations in Pak6 itself or in the regulation and expression of Pak6 could render Pak6 more immunogenic. Indeed, missense mutations have already been detected in Pak6 in the prostate cancer cell lines PC3 and LAPC9 as well as in primary prostate cancer (MSKCC Prostate Oncogenome Project, http://cbio.mskcc.org/prostate-portal/
). Pak6 has to be recognized by T cells to mediate anti-tumor effects. As we have demonstrated, Pak6-specific T cell responses are induced in the immunized mice, and Pak6-specific T cell responses could be detected directly in the post-treatment blood of the clinical responders who have induced antibody responses to this antigen. The patients’ reactive T cells produced IFN-γ to Pak6 that was consistent with a Th1 response and were of a magnitude beyond what has been spontaneously detected with previously described prostate-associated antigens (27
Whether these autoantigens represent immune targets that can mediate antitumor immunity or represent bystander antigens resulting from tumor cell death in human remains a critical question. Nevertheless, we found that Pak6 immunization can lead to tumor protection in both the Myc-Cap and Tramp transplantable models of prostate cancer, indicating that inducing immunity to such a self-protein can in fact lead to antitumor responses. However, the protection afforded by Pak6 immunization was not complete, suggesting that immune recognition of other antigens can also contribute to anti-tumor responses. Knockdown of Pak6 with siRNA has been shown to inhibit prostate cancer growth in nude mice (28
) and increase radiosensitivity of prostate cancer cell lines (29
), further supporting Pak6 as a viable target for cancer therapy.
While our patient cohort is relatively small, the number of clinical responses we observed provided a unique opportunity to characterize how the breadth of the antigen immune response induced by treatment is associated with clinical outcome. Our results with Pak6 represent only one of the antigens that we have identified with our approach. Nevertheless, immune responses to more than one cancer antigen will likely be required for maximal efficacy. As more treated patients are analyzed with this antibody profiling, other novel antigens will undoubtedly be identified including additional shared targets. Moreover, this approach may provide us with an immunologic perspective not only into molecular aberrations in these tumors, but also the heterogeneity of these alterations between patients. Alternatively, patients who developed treatment-induced immune-related adverse events may also provide unique opportunities to perhaps identify relevant autoantigens that might mediate these side effects. We did not see specific toxicities (e.g. only three patients had diarrhea) at sufficient frequency to assess for these associations. Nevertheless, defining an immune profile that is associated with specific side effects could also allow for improved patient selection for these immune therapies, especially as ipilimumab is more widely used. Finally, understanding the nature and targets of the adaptive immune response elicited by immune checkpoint blockade could result in the development of improved multi-targeted vaccines, which could direct the immune response more specifically to the tumor, thus increasing the therapeutic efficacy and perhaps reducing the frequency of immune mediated side effects seen with immunotherapy.