Immunotherapy is based on mobilising the patient’s immune system to treat cancer. There has been extensive research into immunotherapy including the development of CAR-T, checkpoint inhibitors and antibody drug-conjugates. However, limitations of these therapies are numerous and include resistance, efficacy in only a subset of patients, and cancer recurrence [1]. In situ vaccination involves injecting immunoadjuvants and immunomodulators intratumorally; these localised immune responses may initiate tumor cell death while also potentiating systemic immune responses [2]. While there have been descriptions of the role of T-cells in in situ vaccines, there has been a lack of research into whether this approach can be used to modify antibody/humoral responses [2, 3].

Humoral immunity in cancer is complex; there is conflicting evidence on the function of B-cells. In some cases, B-cells are thought to have an immunosuppressive, regulatory function [4]. For instance, it has been shown that temporarily depleting B-cells results in an increase in anti-cancer T-cell responses [5]. In contradiction, others found that B-cell depletion resulted in enhanced B16 melanoma growth, metastasis and decreased CD4 and CD8 levels [6]. Studies have also found that total depletion of B cells results in an increase in T-cell activity and a type 1 immune response; however, despite this, the presence of B-cells had a positive role in anti-cancer activity [7]. Therefore, the humoral immune response may be an essential component of an anti-tumor immune response, resulting in the production of antibodies, which can stimulate effector cells and reduce disease progression [8].

Indeed, the use of recombinant, exogenously administered, anti-tumor antibodies is proven to be a successful strategy in cancer therapy. Trastuzumab, a recombinant humanised monoclonal antibody of the IgG1 isotype, which recognises the HER2 receptor overexpressed on some breast cancers, has been shown to improve survival rates in patients with early-stage HER2-positive breast cancer [9]. Trastuzumab induces apoptosis through antibody-dependent cellular cytotoxicity in metastatic breast cancer [10]. A major limitation of exogenously administered antibody drugs is that they only work in a small number of patients and are dependent on antigen expression levels. Inducing the production of endogenous antibodies through in situ vaccination may circumvent this shortcoming, providing a source of patient-specific antibodies. In theory, these in situ generated antibodies may enhance cytotoxic T-cell responses, neutralise circulating tumor cells and prevent dissemination.

However, as with the B-cell responses, the role of endogenous antibodies in cancer is complex. There has been some debate as to whether antibodies have a pro- or anti-tumor effect. Antigen-specific antibodies form immune complexes, which are thought to activate granulocytes and macrophages, resulting in an inflammatory immune response causing enhanced tumor invasion and metastasis [11]. A study found that antibodies can promote neoplastic progression and cause carcinoma development, thought to be due to the activation of Fc receptors on leukocytes in neoplastic tissue [12]. In contrast, Carmi et al. identified allogenic tumor rejection in mice through the production of antibodies. Furthermore, when the antibodies were mixed with dendritic cells and reinjected, mice could resolve metastases and the primary tumor [13]. Clinically, the presence of anti-MUC1 IgG antibodies has correlated with improved disease prognosis in pancreatic cancer [14].

In theory, due to the distribution and function of endogenous antibodies, they may be able to prevent cancer cell dissemination, limiting invasion and secondary metastasis. In agreement, Parratto et al. found, preclinically, that poorly metastatic tumors induce a higher antibody response than non-metastatic variants of the same cell line [15]. Furthermore, clinically, it has been shown that circulating antibodies are higher in patients with non-metastatic melanoma compared to metastasised disease and higher titres are correlated with less severe disease [16]. In agreement, it has been shown that patients with non-metastatic melanoma had a higher antibody titre than those with metastatic disease, suggesting antibody levels are reduced during disease progression [8]. However, it should be noted that these are correlative observations only, and the direction of the correlation or causal link is yet to be confirmed. While antibody titre has been assessed and correlated to non-metastatic disease, the quality of these antibody responses in terms of antibody isotype has not been assessed. This may account for some of the ambiguity in attributing a role for antibodies in the resolution of cancer. In preclinical models, it has been shown that exogenously administered IgG2 antibodies are the most potent in activating effector responses and the only isotype capable of resolving tumor challenge [17].

The current study aims to investigate the following: (1) can in situ vaccination with adjuvants designed to boost antibody responses in infectious disease vaccines enhance endogenous anti-tumor antibody titre, (2) can in situ vaccination of immunoadjuvants skew antibody responses towards a more favourable IgG2 isotype and (3) whether serum from in situ vaccinated mice can neutralise circulating tumor cells in the blood. Adjuvants selected include Addavax (Adda, comparable to MF59), an immunoadjuvant known to increase the antibody titre and induce a mixed IgG1:IgG2 response [18], and Adda + CpG, where CpG is a TLR9 agonist known to skew the antibody response towards the IgG2a isotype (equivalent to IgG2c in C57BL/6 mice) [19]. The use of MF59 and CpG in combination has previously been demonstrated to simultaneously boost antibody titres and increase levels of IgG2c in response to injected tumor antigen [20]. It was hypothesised that in situ vaccination with immunoadjuvants will increase the titre of tumor-specific antibodies, while the inclusion of CpG may skew the antibody response towards IgG2c.