Vaccination is generally not potent enough to treat patients with established cancer. A first step towards generating an efficacious vaccine in humans is to develop one that is beneficial in animals bearing established aggressive tumors such as B16 melanoma. A vaccine which is an effective prophylactic generally fails in a tumor bearing host because the tumor itself alters the immunologic milieu, crippling nascent anti-tumor immune responses. We have combined passive administration of mAb TA99 with DNA vaccination against the same antigen, Tyrp1, and found this combination to be highly effective therapy for B16 lung metastasis (). Tyrp1, the target of TA99, is a differentiation antigen without known oncogenic function expressed by human and murine melanomas, and also by normal melanocytes (10
). The therapeutic efficacy of TA99, therefore, unlike that of anti-her2 antibody(9
) or anti-EGF receptor antibody(29
), cannot be attributed to interruption of oncogenic signaling, but rather exclusively to immunologic activity. Thus, we show that mAb against a tumor antigen is an effective vaccine adjuvant in treatment of a poorly immunogenic tumor.
The observed therapeutic interaction between TA99 and vaccine can be explained most simply by a combination of antibody dependent cytotoxicity (ADCC) mediated by the antibody and T cell responses generated by the vaccine.(23
) T cell assays, however, revealed that CD8+ T cell responses in the spleen against Tyrp1 were elevated in the combination therapy group as compared with vaccination alone. This finding is consistent with prior data showing that antibodies can enhance T cell responses to irradiated cellular vaccines (7
). In these studies, the hypothesized mechanism of vaccine enhancement is improved antigen presentation due to direct binding of the antibody to the vaccine cells (8
In the context of DNA vaccination in this study, there are three sources of Tyrp1 antigen to which TA99 presumably binds- the protein product of the DNA vaccine, normal melanocytes, and the B16 tumor. Although Tyrp1 was initially defined as an intracellular antigen, cell surface expression on B16 cells was confirmed by mixed hemadsorption rosetting assay (33
). While we were unable to detect accumulation of TA99 in the skin, perhaps because of lower levels of surface expression, or because of background auto-fluorescence, TA99 was shown to localize rapidly and specifically to B16 lung nodules (). ELISPOT assays, meanwhile, revealed no significant enhancement of the CD8+ T cell response by TA99 in non-tumor bearing animals.
Tumor therefore is required for the immunomodulatory effect of TA99. Baseline responses, however, were higher in non-tumor bearing animals and we cannot exclude the possibility that enhancement did not occur because baseline T cell responses were higher in these non-tumor bearing animals. In this regard, we note that TA99 enhances the immunogenicity of gp100 DNA, a vaccine to which TA99 does not bind (). This strongly suggests that the primary mechanism of immune enhancement is not ligation of the protein product of the DNA vaccine, but rather ligation of the tumor with subsequent cross-presentation of antigen. However, the antibody alone is insufficient to induce T cell responses in the absence of vaccination. Thus, we show requirement for a three-way interaction between antibody, tumor, and vaccine.
This mechanism whereby both tumor and vaccine are required for antibody mediated immunomodulation differs from prior models antibody binds and opsonizes the vaccine itself (9
), and this difference has implications for clinical trial design because it allows antibodies to be combined with diverse vaccination strategies. Intriguingly, in the breast cancer model employed by Kim and colleagues, therapeutic benefit was seen when antibody and tumor were injected synchronously, while the vaccine had been given 14 days prior (8
). Therefore, it is theoretically possible that observed enhanced therapeutic efficacy and CD8+ T cell responses with antibody administration in this other experimental model may also be mediated, in part, by direct binding of the antibody to the tumor.
Antibodies modulate immune responses via Fc domain interactions with cell surface receptors, including complement receptors and/or Fc receptors (6
). When administered synchronously with tumor, TA99 protects mice in a mechanism independent of CD8+ T cells, but dependent on activating Fc receptors, most likely by ligating FcRIV or FcR I on macrophages (25
). Meanwhile, the hypopigmentation produced by the antibody can be mediated by either Fc receptors or the complement system (24
). In this context, we show that the immunomodulatory effect of TA99 on CD8+T cells is FcR dependent. This finding is consistent with the known affinity of IgG2a antibodies for activating Fc receptors (35
), and our results complement data from Kim and colleagues that cleaved antibody is insufficient to mediate enhancement of CD8+T cell responses to a whole cell vaccine (8
). Therapeutic agents modulating Fc receptor signaling would therefore have clinical application in trials combining antibodies with vaccines.
FcRγ−/− mice inoculated with B16 melanoma by tail vein develop B16 lung nodules more rapidly than do wild type animals. Theoretically, this may be due to abrogation of the protection normally provided by naturally occurring antibodies against B16. However, B16 is a very poorly immunogenic tumor and the serum of untreated wild type animals does not contain detectable levels of antibodies against melanoma differentiation antigens(36
) or B16 cell lysates (JD Wolchok, unpublished data). Neither can the enhanced tumor growth in FcRγ−/− mice be attributed to a deficiency in the natural anti-tumor activity of NK cells as this was previously shown to be unimpaired(16
). Nonetheless, the data suggests that activating Fc receptors do play a role in anti-tumor immunosurveillance, possibly by transmitting activating signals induced by tonic binding of non-specific immune complexes.
Furthermore, in the treatment of subcutaneous B16 melanoma, TA99 heightens the therapeutic effect of gp100 DNA vaccination combined with adoptive T cell transfer of a T cell clone specific for g10025–33
. This data shows that TA99 has the potential to enhance T cell immunity in both pulmonary and skin disease in mice. A human anti-Tyrp1 antibody is currently under clinical development (38
), and our data highlights potential advantages of combining this human antibody with T cell based therapies in clinical trials.
It is somewhat surprising that TA99, in combination with gp100 DNA vaccination, yields enhanced CD8+ T cell responses to gp100 and mediates reactivity against Tyrp1. The presence of epitope spreading to Tyrp1455–63 in some animals and not others may reflect differences in the repertoire between genetically identical animals due to thymic selection. In addition, it remains possible that TA99 also mediates spreading to other as of yet unidentified epitopes besides Tyrp1455–63. In any case, as gp100 and Tyrp1 are both localized in the melanosome, TA99 potentially facilitates entry of both proteins into antigen presenting cells. From a clinical perspective, it is noteworthy that antibody TA99 can enhance CD8+T cell responses against two distinct melanosomal antigens. This finding shows that antibodies can be used in conjunction with vaccines targeting tumor antigens to broaden the immune response against cancer.
From an immunologic perspective, the adjuvant potency of TA99 can be explained by cross-presentation of tumor antigen, whereby tumor cells serve as a source of antigen for bone marrow derived cells (39
). These antigen presenting cells express Fc receptors, and are susceptible to modulation by antibody bound tumor antigen. Combination therapy with antibodies and vaccines should be further explored in patients with cancer. Antibodies can be combined with all types of vaccines and should not be restricted to use with whole cell vaccines. Clinical investigators may also consider the option of combining antibodies with vaccines targeting antigens on the same tumor cell, particularly if vaccine and antibody targets are in the same sub-cellular compartment.