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- Material and Methods
- Supporting Information
Metastatic forms of cancers remain the main cause of death in cancer patients. In this study, we demonstrate that directing a sustained antibody response towards the homotypic binding function of CEA interferes with the implantation and development of tumor foci in CEA-expressing transgenic (CEA.Tg) mice. Specifically, vaccinating CEA.Tg mice with a recombinant, altered self-form of the CEA Ig V-like N domain led to the production of circulating IgG1 and IgG2a antibodies that inhibited CEA-mediated adhesion of murine carcinoma expressing CEA (MC38.CEA) and mediated antibody-dependent lysis of tumor cells. Moreover, vaccinated CEA.Tg mice were resistant to the development of tumor nodules in the lungs and the peritoneal cavity, suggesting that mounting a focused antibody response to the CEA N domain may represent a simple therapeutic strategy to control the establishment of metastatic foci in cancer patients.
There remains a strong need to develop therapies aimed at blocking or preventing the formation of metastatic tumor foci in cancer patients, in light of the fact that most cancer deaths are accounted for by patients with metastatic disease. Aberrantly expressed surface antigens, involved in intercellular adhesion, represent suitable targets for developing antiadhesive or antiaggregative therapies. One such surface marker is the carcinoembryonic antigen (CEA, CEACAM5 and CD66e), a GPI-linked glycoprotein linked to cell transformation and metastasis. CEA is frequently over-expressed on epithelial carcinomas of the intestinal and respiratory tracts, as well as cancers of the breast, pancreas, stomach and ovary.1–5 From a clinical perspective, high preoperative serum concentrations of CEA correlate with metastasis, treatment failure and poor overall prognosis.6–10 Specifically, a recent prospective study of CEA levels in the serum of 2,062 breast cancer patients has revealed that a CEA level >7.5 μg/L is associated with a high probability of subclinical metastases and a significant reduction in disease free and overall survival rates.11
The association of CEA with cancer progression has led to its use as an immunogen in designing anticancer vaccines.2, 5 Mechanistically, the intercellular homophilic binding property of CEA correlates with cancer invasion and metastasis.5, 12–14 CEA is composed of seven extracellular Ig-like domains (N, A1, B1, A2, B2, A3 and B3) where the binding of N and A3B3 Ig-like modules on distinct tumor cells promotes cell aggregation (defined as homotypic binding and homophilic cellular interactions).13–15 Experimentally, monoclonal antibodies (mAbs) directed at epitopes found in the N domain of CEA,16, 17 or cyclic peptides derived from sequences within the N domain of CEA15 have been shown to inhibit CEA-specific cellular adhesion in vitro. Similarly, the administration of a mAb or (Fab')2, recognizing epitopes located between the N and A1 domains of CEA, has been shown to increase the survival of nude mice harboring CEA-expressing lung micrometastases.16, 17 These findings suggest that an immune response specifically focused at blocking interactions involving the N domain of CEA may halt or limit the formation of tumor metastases in patients.
Previous attempts at developing CEA-based antitumor vaccines have centered on mounting cell-mediated immune responses using vaccine formulations based either on dendritic cells preloaded with predicted T-cell epitopes or recombinant viruses delivering the full length molecule.5, 18–22 The majority of putative T-cell epitopes have been short sequences located in the central region of this molecule.23–25 Unfortunately, the lack of immunogenicity of these epitopes coupled with the presence of immuno-suppressive regulatory T (Treg) cells to this self-antigen in tumor microenvironments have been shown to compromise the efficacy of CEA-based antitumor vaccines.20, 26 These limitations have been partly circumvented either through the depletion of Treg cells20, 26 or by co-administering CEA in combination with costimulatory molecules.21, 22 A vaccine aimed at blocking CEA-dependent adhesion events and the establishment of tumor foci may represent a more appropriate and achievable objective. Importantly, the role of CEA in metastasis is linked to its over-expression and self-association which correlates with the early inactivation of caspase-9, the activation of the PI3-K/Akt survival pathway as well as the inactivation of caspase-827 presumably by directly binding TRAIL-R2 (DR5) through its PELPK motif (residues 108–112 of the N domain of CEA).28 This peptide motif is responsible for mediating the lodging of metastasizing cells to the hepatic parenchyma leading to the development of metastatic foci through homophilic interactions involving the IgV-like N- and IgC-like A3 domains.5, 15, 29, 30
In view of the importance of the CEA N domain in tumor metastasis, we generated a folded recombinant form of the molecule (rCEA N) and used it as an immunogen as part of a simple vaccination procedure aimed at eliciting immune responses capable of simultaneously blocking CEA-mediated cellular interactions as well as killing CEA-expressing tumor cells both in vitro and in vivo.
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- Material and Methods
- Supporting Information
CEA is a useful clinical biomarker for monitoring recurrence and the management of metastatic cancers and has been investigated as a candidate cancer vaccine Ag5–7, 18 in light of its association with tumor progression from neogenesis to metastasis.5, 15, 27, 28 One known biological function of CEA is its role in both homotypic and heterotypic interactions13, 15, 41 which strongly correlates with the establishment and growth of tumor metastases in distal sites such as the liver, lung and the peritoneal cavity.5, 13, 28, 29, 38 Structurally, the IgV-like N domain of CEA strongly interacts with the IgC-like A3B3 domain, allowing adjacent CEA molecules to homotypically adhere to each other. Such homotypic adhesion events on CEA-expressing cells yield networks of homophilic intercellular interactions that further contribute to lodging additional cells within the context of expanding nascent metastatic foci.12, 13, 15, 28, 29, 42
Previous attempts at developing CEA-based cancer vaccines have traditionally revolved around the idea of mounting cellular (TH1) immune responses towards the full-length molecule via the injection of either recombinant viruses and/or Ag-pulsed dendritic cells with the intention of eradicating primary tumor masses.5, 18–22, 43, 44 The success of these vaccination strategies has been hampered by central and peripheral tolerance to CEA.20, 26 We hypothesized that mounting a polyclonal antibody response focused on the CEA N domain would yield antibodies capable of blocking homophilic cell adhesion events between CEA-positive cells (that can prevent tumor implantation and growth) as well as antibodies capable of destroying CEA-bearing tumor cells through ADCC and CDC. To that effect, a recombinant CEA N domain was used to vaccinate CEA.Tg mice. Unlike the full length molecule, the IgV-like rCEA N represents an altered form of a self-antigen since it lacks naturally occurring N-linked glycans and displays an unnatural C-terminus; features which we hypothesize would contribute to overcoming immunological tolerance to CEA.45 Moreover, the use of a single domain of CEA involved in homotypic interactions to serve as the immunogen narrows the immune response to a focused and distinct set of CEA determinants. This CEA module was mixed with Poly I:C and administered to CEA.Tg mice with the view of mounting a protective immune response. Poly I:C was chosen as the adjuvant in view of its capacity to stimulate both Type 1 responses through TLR-3/7 signalling46 as well as B cell activation,47 a combination of immune responses shown to positively influence the development of protective antitumor immune responses in both mice and patients.46 Additionally, this formulation was administered i.p since this route of immunization represents an established route of immunization successfully used to mount anti-CEA antibodies.48
MC38.CEA cells were implanted into CEA.Tg mice using three distinct approaches. As a first implantation model, MC38.CEA cells were introduced subcutaneously into the hind leg of CEA-expressing transgenic mice; an approach that led to a rapid establishment and growth of localized tumor masses. Vaccination of animals following tumor establishment provided a significant delay in tumor growth (Fig. 1), while displaying no signs of autoimmunity (data not shown). However, we projected that a mounted immune response to the rCEA N domain would serve a more appropriate role in blocking the establishment of new tumor foci or the expansion of micrometastases, rather than in arresting the uncontrolled localized growth of a solid tumor mass. Vaccinated CEA.Tg mice were thus challenged with either i.v. or i.p. injected MC38.CEA tumor cells. In both instances, only vaccinated animals were protected from developing tumor nodules, whereas all non-immunized and adjuvant-treated mice displayed numerous tumor nodules in the lungs or viscera (Figs. 2 and 3), suggesting that vaccination prevented the lodging and establishment of tumor foci.
The present vaccination strategy is appealing over previously published vaccine protocols, since the engendered response targets a narrower range of potentially relevant epitopes, bypassing antigenic competition from irrelevant epitopes19, 22–25 present in full length CEA. One report describes the use of CEA-based subunit vaccine, where a recombinant CEA A3B3 domain was mixed with CpG oligonucleotides and subcutaneously injected into C57BL/6 mice.40 The authors reported that this strategy produced a weak CEA-specific immune response that failed to protect C57BL/6 mice against a lethal tumor implant (when compared with a TAT-fused construct).40 In contrast, the present study uses the native CEA N domain sequence, mixed with poly I:C to produce an effective CEA-specific immune responses in CEA.Tg mice capable of blocking tumor implantation to the lungs and peritoneal cavity (Figs. 1, 2, 3, 4, 5, 6, 1–6).
The engendered CEA-specific immune response in CEA.Tg mice is dominated by the production of IgG1 and IgG2a antibodies directed at the N domain of CEA (Fig. 4). The overall response to the CEA N domain is thus distinct from most cancer vaccine strategies aimed at producing a CEA-specific cellular immune response5, 43, 44 and beneficial in modulating or blocking the growth of implanted tumors (Fig. 2, 3, 4, 5, 6, 2–6), by virtue of inducing Ab-dependent tumor lysis (by both ADCC and CDC) and in interfering with CEA-mediated cellular adhesion (Fig. 5). The importance of B lymphocyte populations in vaccinated mice was further confirmed by adoptively transferring CEA-specific B cells or sera from vaccinated animals into naïve CEA.Tg mice and rescuing them from developing peritoneal tumor nodules (Fig. 6). These observations are supported by Park et al.49 who demonstrated the value of mounting a Her-2/neu-specific polyclonal antibody response in curing mice from pulmonary as well as large established subcutaneous ErbB-2–expressing tumors.49 Finally, the recent demonstration that a monoclonal antibody directed at the N domain of CEA can suppress the growth of a colorectal tumor xenograft in nude mice further supports the value of mounting a selective antibody response to the N domain.17
In summary, the simple (i.p.) injection of an altered-self form of the CEA N domain elicited an antibody response in CEA.Tg mice that prevented tumor colonization and the development of tumor nodules. This antibody-dominated response led to the specific killing of CEA-expressing cells by ADCC and CDC in addition to impeding CEA-dependent intercellular adhesion. Since high circulating levels of CEA in the serum of cancer patients frequently correlate with a higher incidence of metastatic relapse, a vaccine formulation using this rCEA N domain as an immunogen may represent a safe and simple adjuvant therapy delaying or preventing tumor metastasis in cancer patients displaying elevated serum CEA levels prior to surgery.