Purpose: The oncofetal antigen, human chorionic gonadotropin β subunit (hCGβ), is expressed by a number of carcinomas and is a prognostic indicator in renal, colorectal, bladder, and pancreatic cancers. We describe the development of a novel antibody-based dendritic cell (DC)-targeted cancer vaccine capable of eliciting cellular immune responses directed against hCGβ.

Experimental Design: The tumor-associated antigen hCGβ was coupled genetically to a human anti-DC antibody (B11). The resulting fusion protein (B11-hCGβ) was evaluated for its ability to promote tumor antigen-specific cellular immune responses in a human in vitro model. Monocyte-derived human DCs from normal donors were exposed to purified B11-hCGβ, activated with CD40 ligand, mixed with autologous lymphocytes, and tested for their ability to promote hCGβ-specific proliferative and cytotoxic T-lymphocyte responses.

Results: B11-hCGβ was found to be a soluble, well-defined, and readily purified product that specifically recognized the human mannose receptor via the B11 antibody portion of the fusion protein. B11-hCGβ functionally promoted the uptake and processing of tumor antigen by DCs, which led to the generation of tumor-specific HLA class I and class II-restricted T-cell responses, including CTLs capable of killing human cancer cell lines expressing hCGβ.

Conclusions: Although other hCG vaccines have been shown to be capable of eliciting antibody responses to hCGβ, this is the first time that cellular immune responses to hCGβ have been induced by a vaccine in a human system. This DC-targeted hCGβ vaccine holds promise for the management of a number of cancers and merits additional clinical development.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone secreted by trophoblastic cells during normal gestation that is composed of noncovalently associated α (hCGα) and β subunits (hCGβ). Importantly, hCGβ also is an established tumor-associated antigen that is overexpressed in a variety of common cancers, including those of the colon, lung, pancreas, esophagus, breast, bladder, cervix, stomach, and prostate (1). In patients with trophoblastic and testicular neoplasms, serum hCG levels have proved to be a useful diagnostic tool and a convenient means for monitoring tumor burden and for evaluating the effectiveness of therapeutic intervention (2, 3). Elevated hCGβ serum levels and/or tissue expression also have been shown to be an independent predictor of an unfavorable disease outcome and are associated with a more aggressive disease course in renal, colorectal, bladder, and pancreatic cancers (4, 5, 6, 7). It has been proposed mechanistically that hCG may act at several different levels to facilitate cancer progression, as a transforming growth factor, an immunosuppressive agent, an inducer of metastasis, and/or as an angiogenic factor (1).

The realization that this oncofetal antigen exhibits a fairly selective expression profile and may play an active role to promote oncogenesis has spurred interest in the development of hCGβ as a target for active immunotherapy. Clinical studies investigating the safety and efficacy of hCG vaccines as contraceptive agents provided an important proof-of-principle finding by demonstrating that tolerance to this self-antigen could be broken. Immunization of subjects with various forms of hCG/hCGβ conjugated to an immunogenic toxoid often induced antibody responses to native hCG of a sufficient magnitude as to neutralize the biological effects of this hormone and prevent gestation (1). One of these contraceptive vaccines, originally described by Triozzi et al. (8), also is being developed currently as a cancer therapeutic agent based on its acceptable safety profile and ability to stimulate an anti-hCG antibody response. This vaccine comprises the COOH-terminal 37 amino acid peptide of hCGβ coupled to diphtheria toxoid (CTP37-DT) and has been reported to show efficacy for the management of pancreatic and colorectal cancers in Phase II clinical studies (9). Although hCGβ has been shown to contain several antigenic determinants recognized by in vitro-induced CD8+ and CD4+ T cells, none of the current hCG vaccines appear capable of generating hCG-specific cellular responses in humans (10).

Because CTLs are recognized to play a major role in the protection against tumors and in the establishment of antitumor immunity, developing a vaccine capable of inducing cellular and humoral responses to hCGβ would represent a significant advancement. In this study, we describe a novel approach to elicit MHC class I- and class II-restricted cellular immune responses to soluble hCGβ. By coupling hCGβ to a fully human antibody that recognizes the mannose receptor present on antigenpresenting cells (APCs), we are able to efficiently direct full-length hCGβ to dendritic cells (DCs) for processing and demonstrate induction of CTL responses specific for this tumor-associated antigen.

Derivation and Characterization of B11 Antibody.

A fully human IgG antimannose receptor antibody, B11, was generated. Briefly, transgenic mice containing unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences were immunized with human monocyte-derived DCs. Hybridomas were produced using standard methods and screened for selective binding to human monocyte-derived DCs. The specificity of B11 antibody for the mannose receptor was demonstrated by N-terminal sequencing of protein immunoprecipitated from DC lysates. All of the animal studies were conducted in accordance with Institutional Review Board-approved protocols.

Generation of the pB11-hCGβ Construct.

A cDNA fragment encompassing the entire coding region of the mature hCGβ subunit was amplified by reverse transcription-PCR using RNA prepared from the human BeWo choriocarcinoma cell line (American Type Culture Collection, Manassas, VA). The hCGβ fragment was sequenced and inserted in-frame at the 3′-end of the B11 antibody heavy chain gene present in the pMMV4 mammalian expression vector. In addition to the B11 heavy chain-hCGβ fusion protein gene, the resulting pB11-hCGβ vector contains a B11 κ chain gene, a dihydrofolate reductase gene, and the ampicillin and neomycin resistance genes. The same hCGβ fragment also was inserted in-frame at the 3′-end of a recombinant B11 single chain variable region fusion gene to generate the plasmid pB11ScFv-hCGβ.

Production and Purification of B11-hCGβ Vaccine.

Chinese hamster ovary cells were transfected with linearized pB11-hCGβ plasmid DNA using SuperFect reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Transfectants were selected in αMEM medium supplemented with 10% dialyzed fetal bovine serum and 500 μg/ml of G418 (Calbiochem-Novabiochem, San Diego, CA). To induce amplification of the transfected genes, G418-resistant cells also were subjected to methotrexate up to a final concentration of 320 nm. Cells were cloned by limiting dilution, and the highest producing clonal line was selected for all of the subsequent studies. HEK-293 cells (American Type Culture Collection) were maintained in DMEM supplemented with 10% fetal bovine serum and transiently transfected with pB11ScFv-hCGβ using Superfect reagent as per manufacturer’s suggestions (Qiagen). Clarified supernatants were purified by standard protein A or protein L affinity chromatography.

SDS-PAGE and Immunoblot Analysis.

Purified B11-hCGβ fusion protein was subjected to SDS-PAGE on 4–20% gradient gels (Bio-Rad, Hercules, CA) under reducing or nonreducing conditions, and protein bands were identified by direct staining (Coomassie or silver stain) or immunoblotting. For immunoblot analysis, proteins were transferred from polyacrylamide gels to nitrocellulose membranes, blocked, and incubated with the indicated primary antibody [either goat antihuman IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) or mouse monoclonal antihuman hCGβ antibody (Wako Chemicals USA, Richmond, VA)] and secondary antibody (APconjugated antigoat or antimouse IgG; Sigma, St. Louis, MO). CDP-Star chemiluminescence substrate (Roche Molecular Biochemicals, Mannheim, Germany) was used for detection.

Preparation of Monocyte-Derived DCs.

Peripheral blood mononuclear cells were suspended in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 40 μg/ml gentamicin, and 50 μm β-mercaptoethanol. Cells were allowed to adhere to flasks for 90 min at 37°C. Nonadherent cells were removed gently, washed, and cryopreserved. Immature DCs were generated by culturing adherent cells in the above growth medium supplemented with 25 ng/ml granulocyte macrophage colony-stimulating factor and 100 ng/ml interleukin 4 (IL-4; R&D Systems, Minneapolis, MN). DCs were harvested on days 5–6 and either were used as stimulators or cryopreserved for later use.

Binding Assays.

A sandwich ELISA assay was used to examine the binding of B11-hCGβ to a soluble 6XHis-tagged fragment of the human mannose receptor (sMR). Briefly, recombinantly expressed sMR was purified by nickel-NTA chromatography and added to 96-well EIA plates that had been coated with the murine anti-MR monoclonal antibody (mAb) clone 19.2 (Research Diagnostics, Inc., Flanders, NJ). Samples (i.e., B11 mAb, B11-hCGβ, or isotype control mAb) subsequently were added to the wells, incubated, washed, and detected with alkaline phosphatase-conjugated goat anti-Fc Fab (human specific; Jackson ImmunoResearch Laboratories) using pNPP (Pierce Biotechnology, Rockford, IL) as a substrate. Plates were read at 405 nm using an ELISA plate reader (Molecular Devices, Sunnyvale, CA).

Flow cytometry analysis was used to examine the binding of B11-hCGβ to immature monocyte-derived DCs. Briefly, purified proteins (i.e., B11-hCGβ, B11 mAb, and an irrelevant human IgG1) were labeled directly with FITC according to manufacturer’s instructions (Molecular Probes, Eugene, OR). A total of 2 × 105 DCs were incubated for 30 min at 4°C with 2 μg/ml of FITC-conjugated protein, washed, and analyzed using an LSR flow cytometer (BD, Franklin Lakes, NJ). Blocking experiments were performed as described previously except that DCs were preincubated at 4°C for 30 min with 10 μg/ml of either unlabeled B11 mAb or unlabeled isotype control antibody before incubation with FITC-conjugated B11-hCGβ.

Immunohistochemical Analysis.

Snap frozen unfixed tissues were obtained from the Cooperative Human Tissue Network (Philadelphia, PA) and the National Disease Research Institute (Philadelphia, PA). Tissues were sectioned and then fixed with acetone or methanol for 10 min at room temperature. After blocking endogenous peroxidase and nonspecific binding, sections were incubated for 1 h with 0.2 μg/ml of primary antibody: FITC-B11-hCGβ, FITC-B11, or FITC-IgG1 (isotype control). This was followed by 30-min incubation with murine anti-FITC secondary antibody (Sigma). Using an EnVision kit (DAKO, Carpinteria, CA,), sections were stained using an immunoperoxidase method as per manufacturer’s instructions and were counterstained with Mayer’s hematoxylin.

Internalization Assay and Confocal Microscopy.

Purified hCGβ (US Biological, Swampscott, MA) was FITC-labeled as described previously. Immature DCs were incubated for 1 h on ice with 3 μg/ml human γ globulin (Sigma) in PBS to block nonspecific binding or with 10 μg/ml free B11 antibody to block specific binding. Cells then were incubated for 30 min on ice with FITC-B11-hCGβ or FITC-hCGβ (2 μg/ml final concentration in blocking buffer) and then allowed to internalize at 37°C for various times between 0 and 90 min. Following internalization, cells were washed and fixed overnight in ice-cold 1% methanol-free formaldehyde (Polysciences, Warrington, PA). Fixed cells were washed, centrifuged onto microscope slides, and imaged using a Bio-Rad MRC-1024 confocal scanning laser microscope system and LaserSharp version 3.2 software (Bio-Rad). Images were captured as a single section from the center plane of cells and are representative of 30 fields captured per slide. The percentage of DCs internalizing antigen was quantitated by counting the number of FITC-positive cells among all of the cells in 30 fields (∼15 total cells per field).

T-Cell Stimulation and Expansion.

Nonadherent peripheral blood mononuclear cells obtained from frozen stocks were used as a source of T cells and stimulated every week for 4–5 weeks with autologous DCs that had been exposed to B11-hCGβ. Briefly, 1.2 × 106 immature DCs were exposed to 20 μg of B11-hCGβ in 1.0 ml of RPMI 1640 containing 20 mm HEPES for 45 min and allowed to mature for 24 h in the presence of 20 ng/ml CD40L (Peprotech, Rocky Hill, NJ) in complete medium (RPMI 1640, 10% fetal bovine serum, 2 mm glutamine, 20 mm HEPES, and 40 μg/ml gentamicin). T cells were cocultured with B11-hCGβ-treated DCs in 24-well culture plates, and the T cell:DC ratio was 20. IL-7 (10 ng/ml) was added to cocultures on day 0, followed by IL-10 (10 ng/ml) on day 1, and IL-2 (20 units/ml) on day 2. The cultures were restimulated on days 8, 16, and 24, and the T cell:DC ratio was maintained at 20 throughout the course of stimulations by weekly pooling and counting all of the T cells and adding the appropriate number of treated DCs. IL-2 was added every 3–4 days. T cells were maintained as bulk cultures (containing CD4+ and CD8+ T cells). Effector T cells were expanded on allogeneic mitomycin C-treated peripheral blood mononuclear cell feeder layers (pooled from three donors) in the presence of anti-CD3 antibody and IL-2, and maintained by refeeding with fresh medium containing IL-2. T-cell cultures were harvested and assayed between days 10–12 or cryopreserved for later use. Purified CD8+ and CD4+ cells were isolated from bulk T-cell cultures on day 6 or 7 following the third round of stimulation using antibody-coated magnetic microbeads (11).

Proliferation Assay.

T cells that had been subjected to multiple rounds of stimulation with B11-hCGβ-treated DCs were cocultured with antigen-naive DCs or DCs that had been exposed to B11-hCGβ, hCGβ, or B11 antibody in a final volume of 0.2 ml of RP-10 medium. Alternatively, T cells were cocultured with an autologous B lymphoblastoid cell line (B-LCLs) that was loaded exogenously with hCGβ38–58 peptide, the hCGβ COOH-terminal peptide (hCGβ123–145), or a B11-derived peptide. On day 4, cultures were pulsed with [3H]-thymidine (1 μCi/well) for 18 h. Cells then were harvested onto filters with a cell harvester (Wallac Instruments, Shelton, CT), and filter-bound radioactivity was measured using a β-scintillation counter (1450 MicroBeta Jet; Perkin-Elmer Life Sciences, Boston, MA). Results are expressed as cpm. In experiments designed to examine the relative requirements for the presence of MHC class I or class II molecules, B11-hCGβ-stimulated DCs were preincubated for 30 min at room temperature with 20 μg/ml of an anti-pan HLA class I-specific mAb (W6/32), an anti-pan HLA-DR antibody (L243), or appropriate isotype-matched controls. All of the assays were performed in triplicate with error bars indicating experimental SD.

Cytolytic Assay.

T cells (bulk or purified) that had been stimulated with B11-hCGβ-treated DCs were tested for cytolytic activity in a standard 4-h 51Cr-release assay. Targets cells included autologous DCs exposed to targeted antigen (B11-hCGβ fusion protein), untargeted antigen (hCGβ), vehicle (B11 antibody), or no antigen/vehicle; 174xCEM.T2 (T2) cells loaded with peptides; EBV-immortalized autologous B-LCLs; and cancer cell lines obtained from the American Type Culture Collection (e.g., ScaBer, BeWo, Colo 205, SK-MEL19, A375, SKRC-4, SKOV3.A2, SK-Br-3, HT29, MCF-7, T-24, LNCaP, PC-3, Daudi, HUT 78, and CCRF-CEM) and elsewhere (e.g., INT.OV2; Ref. 11). The T2 cell line is a human T-B lymphoblast hybrid that is defective in transporter of antigenic peptides-mediated antigen processing and expresses HLA-A2.1 but no HLA class-II antigens (American Type Culture Collection). Cancer cell lines were treated with 100 units/ml of IFN-γ 48 h before the assay. In some experiments, target cells were preincubated for 30 min at room temperature with either 20 μg/ml of an anti-pan HLA class I-specific mAb (W6/32) or isotype-matched control antibody. Radioactivity was measured using a gamma counter (Wizard 1470; Wallac Instruments). All of the assays were performed in triplicate, and error bars indicate experimental SD.

Development of B11-hCGβ Vaccine.

We have developed a mannose receptor-specific human mAb, B11, which is rapidly internalized by myeloid DCs. We have exploited here the B11 antibody as a vehicle to deliver hCGβ to DCs for processing and presentation by creating a genetic fusion protein between these two molecules. A mammalian expression vector was created specifically that contained the B11 κ gene in addition to the sequence encoding the entire mature hCGβ subunit genetically fused in-frame to the COOH-terminus of the B11 heavy chain. Selected Chinese hamster ovary cell lines stably transfected with this plasmid were identified that produced >100 mg/l of fusion protein in their culture supernatants. B11-hCGβ protein purified by routine chromatography steps developed for the purification of standard mAbs was characterized by SDS-PAGE and immunoblotting. Under reducing conditions (Fig. 1,A), the B11 light chain (∼25 kD) and the B11 heavy chain-hCGβ fusion (∼80 kD) were observed to be in agreement with their predicted sizes. Detection of the B11-hCGβ heavy chain fusion with an antibody that binds the COOH-terminal region of hCGβ also suggests that the fusion protein is full length and not prematurely truncated. The fusion of hCGβ to the B11 heavy chain did not adversely effect its association with the B11 light chain or the formation of properly assembled antibody molecules as evidenced by the band of ∼210 kD observed under nonreducing conditions [predicted size = (2 × 80 kD) + (2 × 25 kD)] (Fig. 1 B).

B11-hCGβ Efficiently Binds and Is Internalized by DCs.

The B11-hCGβ fusion product retained its ability to bind recombinant human mannose receptor (data not shown) and monocyte-derived DCs expressing this receptor. Immunohistochemical analysis of sections from 12 different normal human tissues (skin, lymph node, spleen, tonsil, liver, lung, heart, kidney, ovary, testis, cerebrum, and cerebellum) revealed an identical staining pattern for B11 mAb and B11-hCGβ. Dermal DC and macrophages of the skin and interstitial DCs (all of the tissues), Kupffer cells (liver), alveolar macrophages (lung), and endothelial cells (spleen and liver) were recognized by B11-hCGβ and B11 (Fig. 2 A and data not shown). Importantly, B11-hCGβ was not observed to bind cells known to expresses the LH/hCG receptor (e.g., testicular Leydig cells), suggesting that this molecule will not possess hCG agonist properties. These findings are consistent with the studies of Narayan et al. (12), who demonstrated that individual α and β subunits of hCG cannot act independently as functional agonists.

In agreement with our in situ results, no differences between B11-hCGβ and B11 were observed with respect to their ability to bind in vitro cultured monocyte-derived DCs either by fluorescence-activated cell sorter or confocal microscopy (data not shown). In addition, pretreatment of DCs with B11 antibody was able to block the binding of FITC-B11-hCGβ. However, confocal microscopy analysis did reveal dramatic differences between targeted (FITC-labeled B11-hCGβ) and nontargeted antigen (FITC-labeled hCGβ) with respect to their binding to and internalization by monocyte-derived DCs (Fig. 2 B). The nontargeted hCGβ antigen was not observed to bind DCs at 4°C. However, following incubation at 37°C some cells (∼20%) were able to internalize nontargeted antigen, presumably by a nonspecific macropinocytotic mechanism. In contrast, FITC-labeled B11-hCGβ readily bound to DCs, and >95% of the cells contained intracellular label at the conclusion of the experiment. Such differences could not be attributed to the relative strength of FITC signals because the labeling efficiency was approximately threefold greater for the nontargeted hCGβ compared with its B11-hCGβ counterpart. These experiments suggest that coupling hCGβ to the antimannose receptor-specific B11 human antibody is an efficient way to direct this tumor-associated antigen into myeloid DCs.

DC Uptake of B11-hCGβ Induces T-Helper Responses and HLA Class I-Restricted Cytolytic Effectors.

The functional consequences of the directed delivery of hCGβ to DCs via B11-hCGβ were investigated using an in vitro culture system comprising autologous DCs and T cells obtained from four sources: three from different normal donors and the fourth from a patient with metastatic colon cancer. After several rounds of stimulation with autologous DCs exposed to sensitizing antigen (B11-hCGβ), T cells were analyzed for their proliferative and cytolytic capacities. Although the magnitude of individual responses varied, the pattern of responses illustrated in Fig. 3 and Fig. 4 is representative of those observed for all of the donors. Proliferative responses were observed when T cells were restimulated with DCs that had been pulsed with B11-hCGβ. Similarly, restimulation of T cells with B-LCL pulsed with the predicted HLA class II hCGβ38–58 peptide also promoted proliferation, indicating the response was hCGβ-specific (Fig. 3). Moreover, the response was reduced significantly in the presence of an anti-class II antibody, suggesting that a major T-helper component was responsible for the observed effects. The fact that an anti-class I antibody also was able to partially abrogate proliferative responses suggests that ongoing stimulation of CD8 T cells by CD4 helper cells in the bulk culture may be occurring. No significant enhancement of proliferative responses resulted from restimulation of T-cell cultures with untreated DCs or DCs that had been exposed to the antigen (hCGβ) or vehicle alone (B11 antibody), indicating that the response depended on the presence of mannose receptor-targeted hCGβ antigen.

The cytolytic effector response of B11-hCGβ-stimulated T cells was investigated initially using a standard chromium release assay with radiolabeled autologous DCs as targets. DCs that are able to internalize and process soluble exogenous antigen and subsequently present this processed antigen in the form of a peptide-class I complex, we reasoned, also would be recognized as targets by appropriately primed autologous CTLs. We observed that B11-hCGβ-stimulated bulk cultures efficiently lysed autologous DCs that had been exposed to the mannose receptor-targeted tumor antigen (B11-hCGβ; Fig. 4,A) and T2 cells exogenously loaded with hCGβ-derived MHC class I peptides (Fig. 4,B). In contrast, no CTL activity was observed when the targets were (a) antigen-naive DCs, (b) DCs exposed to either nontargeted antigen (hCGβ) or vehicle alone (B11 antibody), or (c) T2 cells that were loaded with irrelevant peptides. CTL activity was restricted to the purified CD8+ T-cell population (Fig. 4 C), and no lytic activity could be detected in the CD4+ T-cell fraction (data not shown). Qualitatively, the specificity of the lytic activity mediated by isolated CD8+ effector T cells appeared to be similar to that observed for bulk T-cell cultures. In addition, this CTL activity was abolished in the presence of an anti-pan HLA class I-specific antibody (W6/32), indicating that HLA class I molecules are required for a lytic response to hCGβ.

In addition to C-type lectins, Fc receptors present on APCs also are capable of capturing exogenous antigen in the form of immune complexes and delivering it to the MHC class I processing pathway (13, 14). To investigate the contribution of the Fc region of B11-hCGβ to the observed immunologic responses, we created a fusion protein that lacked the Fc region by genetically linking hCGβ to a single chain fragment comprising the B11 VL and VH domains (B11ScFv-hCGβ). DCs pulsed with this fusion protein were suitable targets for lysis by hCGβ-specific CTLs, indicating that the Fc region is not required for mannose receptor-directed uptake and processing of B11-hCGβ (data not shown).

CTL Raised to B11-hCGβ–Treated DCs Lyse hCGβ-Expressing Cancer Cell Lines.

Seventeen cancer cell lines were examined for their susceptibility to lysis by B11-hCGβ–sensitized T cells. In addition, K562 cells were included as a control for natural killer-mediated killing. Only cancer cell lines, such as Colo205 or Ov2, that expressed hCGβ and shared at least a partial HLA match with the effector cells were recognized as targets for cell-mediated lysis (Fig. 5). Significantly, this is the first demonstration that hCGβ expressed by human tumor cells can be processed and presented in the context of HLA class I molecules, making these cells targets for cytotoxic T-cell-mediated lysis. In addition, these results confirm that the CTL responses we have observed contain an hCGβ-specific component because B11 is not associated with tumor cell lines. This result also is supported by the finding that T2 cells (Fig. 4 B) or autologous B-LCL (data not shown) pulsed with peptides corresponding to predicted hCGβ T-cell epitopes are rendered targets for lysis by hCGβ-specific CTLs.

Free hCGβ has proved to be poorly immunogenic in humans. Although human immune responses to hCGβ are enhanced dramatically by coupling this antigen to an immunogenic carrier, these responses appear to be restricted to the induction of humoral immunity (1). Using autologous DCs pulsed with hCGβ-specific peptides as stimulators, Dangles et al. (10) were the first to demonstrate that CD4+ and CD8+ human T-cell responses to hCGβ could be induced. Our study extends these observations by demonstrating that hCGβ-specific CTLs recognize and lyse relevant human tumor cell lines. In addition, because the B11-hCGβ vaccine described here delivers full-length hCGβ into the appropriate class I and class II processing pathways, all of the possible T-helper and CTL epitopes are made available for presentation by DCs. Therefore, such a vaccine approach circumvents individual HLA restrictions associated with traditional peptide vaccines and has appeal in the universality of its application. Furthermore, this vaccine offers the potential to eliminate tumor cells expressing hCGβ by inducing hCGβ-specific CTLs and by promoting hCGβ-neutralizing antibody responses.

The mannose receptor and other C-type lectins present on APCs play a pivotal role in host defense and immunomodulation by providing a critical link between innate and adaptive immunity (15, 16). These pattern recognition receptors facilitate the capture, endocytosis, processing, and clearance of foreign substances/organisms and endogenous glycoconjugates by recognizing the presence of distinctive carbohydrate signatures. We have sought to exploit this recognition system to promote DC-mediated hCGβ antigen uptake and presentation by genetically linking this tumor-associated antigen to a fully human antimannose receptor antibody. This strategy not only enhances the efficiency of antigen delivery and promotes antigen-specific immune responses but also results in a readily purified, uniform vaccine product.

Whether using antibodies as a vehicle to deliver antigen to APCs either directly (13, 17, 18) or in the form of an immune complex (19), it appears that the activation/maturation state of these cells will influence significantly the outcome of the ensuing immune response. Here we have used CD40 ligand to stimulate DC activation/maturation in vitro. Engagement of CD40 on DCs has been demonstrated to induce the expression of DC maturation markers and the production of IL-12 and promote antitumor immunity (20, 21, 22). One of the major challenges of translating this approach into an effective in vivo therapy will be not only to delineate the factors required to promote efficient DC differentiation in vivo but also to determine the sequence in which they must be applied. Underscoring this point, targeting antigen to immature DCs via an antibody to the mannose receptor family member DEC-205 has been demonstrated to promote antigen-specific tolerization rather than immune enhancement in the absence of an activating agent (23). To elucidate a pathway for in vivo DC maturation and facilitate the clinical development of B11-hCG, we are developing transgenic mice that express the human mannose receptor and second-generation surrogate antibodies that recognize mannose receptors from other species.3

The entire coding region of the mature hCGβ was incorporated into the B11-hCGβ construct to maximize the number of hCGβ epitopes available for presentation. Although such an approach offers the potential to generate a more efficacious antitumor response, it also holds the potential to generate immune responses against hormones that have a high degree of homology with hCGβ (e.g., luteinizing hormone, follicle-stimulating hormone) and the cells that produce them. Even though generation of cross-reactive antibody responses to human luteinizing hormone in Phase I/II clinical trials of hCGβ vaccines has not been associated with any serious side effects or alterations in ovulation or menstrual cycle, it remains to be determined what, if any, side effects would result from a strong cellular immune response to hCGβ (1).

By genetically linking tumor antigen to a fully human antibody that targets APCs, we have generated a well-defined, uniform, soluble, readily purified, protein vaccine that is capable of inducing cellular immune responses against hCGβ. Such a vaccine holds promise for the management of a number of cancers and merits additional clinical development.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Notes: Dr. Connolly is currently at the Baylor Institute for Immunology Research, Dallas, TX and Dr. Wallace is currently at the Roswell Park Cancer Institute, Buffalo, NY.

Requests for reprints: Michael J. Endres, Medarex, Inc., 519 Route 173 West, Bloomsbury, NJ 08804. Phone: 908-479-2427; Fax: 908-479-2402; E-mail: mendres@medarex.com

3

Unpublished observations.

Fig. 1.

Purified human anti-dendritic cell antibody (B11)-human chorionic gonadotropin β subunit (hCGβ) fusion protein. A, purified B11-hCGβ fusion protein and control B11 antibody (200 ng/lane) were separated by SDS-PAGE under reducing conditions. Immunoblotting was performed with primary antibodies as indicated. Fusion, B11 heavy chain-hCGβ fusion; H. C., heavy chain of B11; L. C., light chain of B11-hCGβ or B11 antibody. B, purified B11-hCGβ fusion protein and control B11 antibody (2 μg/lane) were separated by SDS-PAGE under nonreducing conditions and stained with Coomassie Blue.

Fig. 1.

Purified human anti-dendritic cell antibody (B11)-human chorionic gonadotropin β subunit (hCGβ) fusion protein. A, purified B11-hCGβ fusion protein and control B11 antibody (200 ng/lane) were separated by SDS-PAGE under reducing conditions. Immunoblotting was performed with primary antibodies as indicated. Fusion, B11 heavy chain-hCGβ fusion; H. C., heavy chain of B11; L. C., light chain of B11-hCGβ or B11 antibody. B, purified B11-hCGβ fusion protein and control B11 antibody (2 μg/lane) were separated by SDS-PAGE under nonreducing conditions and stained with Coomassie Blue.

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Fig. 2.

A, immunohistochemical labeling of human skin with human anti-dendritic cell (DC) antibody (B11)-human chorionic gonadotropin β subunit (hCGβ). Normal human skin sections were incubated with FITC-B11-hCGβ, FITC-B11, or FITC-human IgG1 (control) and detected using an immunoperoxidase method as described in “Materials and Methods.” Positive labeling (brown) for B11-hCGβ and B11 was associated with dermal DC and macrophages (arrowheads). The inserts represent higher power magnification. B, binding and internalization of B11-hCGβ by DCs. Monocyte-derived DCs were pulsed with FITC-B11-hCGβ or FITC-hCGβ for 30 min on ice. Cells then were incubated at 37°C for the indicated periods to allow for internalization. Images were taken by confocal microscopy, and green staining indicates the presence of FITC-labeled molecules.

Fig. 2.

A, immunohistochemical labeling of human skin with human anti-dendritic cell (DC) antibody (B11)-human chorionic gonadotropin β subunit (hCGβ). Normal human skin sections were incubated with FITC-B11-hCGβ, FITC-B11, or FITC-human IgG1 (control) and detected using an immunoperoxidase method as described in “Materials and Methods.” Positive labeling (brown) for B11-hCGβ and B11 was associated with dermal DC and macrophages (arrowheads). The inserts represent higher power magnification. B, binding and internalization of B11-hCGβ by DCs. Monocyte-derived DCs were pulsed with FITC-B11-hCGβ or FITC-hCGβ for 30 min on ice. Cells then were incubated at 37°C for the indicated periods to allow for internalization. Images were taken by confocal microscopy, and green staining indicates the presence of FITC-labeled molecules.

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Fig. 3.

Human anti-dendritic cell (DC) antibody (B11)-human chorionic gonadotropin β subunit (hCGβ)-treated DCs induce T-cell proliferation. Bulk T cells raised from B11-hCGβ-treated DCs were pulsed with [3H]thymidine and subjected to a final round of stimulation with DCs exposed to various molecules as indicated. Preincubation of DCs with anti-HLA class I or class II antibody, but not their isotype-matched control IgG, partially blocked the T-cell proliferation response. Other specificity controls included a final round of stimulation with autologous B lymphoblastoid cell line (B-LCL) that had been loaded with the predicted HLA class II peptide hCGβ38–58, the non-class II epitope hCGβ123–145, or a B11-derived class II peptide. The experiment was done in triplicate, and error bars indicate experimental SDs. Representative results from one of four different donors are shown.

Fig. 3.

Human anti-dendritic cell (DC) antibody (B11)-human chorionic gonadotropin β subunit (hCGβ)-treated DCs induce T-cell proliferation. Bulk T cells raised from B11-hCGβ-treated DCs were pulsed with [3H]thymidine and subjected to a final round of stimulation with DCs exposed to various molecules as indicated. Preincubation of DCs with anti-HLA class I or class II antibody, but not their isotype-matched control IgG, partially blocked the T-cell proliferation response. Other specificity controls included a final round of stimulation with autologous B lymphoblastoid cell line (B-LCL) that had been loaded with the predicted HLA class II peptide hCGβ38–58, the non-class II epitope hCGβ123–145, or a B11-derived class II peptide. The experiment was done in triplicate, and error bars indicate experimental SDs. Representative results from one of four different donors are shown.

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Fig. 4.

Human anti-dendritic cell (DC) antibody (B11)-human chorionic gonadotropin β subunit (hCGβ)-treated DCs induce hCGβ-specific CTL responses. A, bulk T cells stimulated with B11-hCGβ-treated DCs were used as effector cells, and autologous DCs or autologous DCs loaded with B11-hCGβ, hCGβ, or B11 monoclonal antibody were used as target cells. Triplicate samples were evaluated using a 4-h 51Cr release assay at the effector-to-target (E:T) ratios indicated, and error bars indicate experimental SDs. B, bulk T cells that had been stimulated with B11-hCGβ-treated DCs were used as effector cells, and T2 cells loaded with the indicated peptides were used as target cells. The 4-h 51Cr release assay was done in triplicate. C, CD8+ cells isolated from bulk T-cell cultures that had been stimulated with B11-hCGβ-treated DCs were used as effector cells. Autologous DCs pulsed with B11-hCGβ and preincubated with or without anti-HLA class I antibody or its isotype-matched IgG were used as targets. Antigen-naive autologous DCs and DCs pulsed with B11 antibody were used as target controls. A 4-h 51Cr release assay was performed in triplicate, and error bars indicate experimental SD. Illustrated are results for two of four donors with different types of HLA class I genotypes (donor 1, A2, 31, B13, and 35; donor 2, A1, 30, B7, and 13) at an E:T ratio of 40.

Fig. 4.

Human anti-dendritic cell (DC) antibody (B11)-human chorionic gonadotropin β subunit (hCGβ)-treated DCs induce hCGβ-specific CTL responses. A, bulk T cells stimulated with B11-hCGβ-treated DCs were used as effector cells, and autologous DCs or autologous DCs loaded with B11-hCGβ, hCGβ, or B11 monoclonal antibody were used as target cells. Triplicate samples were evaluated using a 4-h 51Cr release assay at the effector-to-target (E:T) ratios indicated, and error bars indicate experimental SDs. B, bulk T cells that had been stimulated with B11-hCGβ-treated DCs were used as effector cells, and T2 cells loaded with the indicated peptides were used as target cells. The 4-h 51Cr release assay was done in triplicate. C, CD8+ cells isolated from bulk T-cell cultures that had been stimulated with B11-hCGβ-treated DCs were used as effector cells. Autologous DCs pulsed with B11-hCGβ and preincubated with or without anti-HLA class I antibody or its isotype-matched IgG were used as targets. Antigen-naive autologous DCs and DCs pulsed with B11 antibody were used as target controls. A 4-h 51Cr release assay was performed in triplicate, and error bars indicate experimental SD. Illustrated are results for two of four donors with different types of HLA class I genotypes (donor 1, A2, 31, B13, and 35; donor 2, A1, 30, B7, and 13) at an E:T ratio of 40.

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Fig. 5.

CTL responses directed at cancer cell lines. A, T cells stimulated with human anti-dendritic cell (DC) antibody (B11)-human chorionic gonadotropin β subunit (hCGβ)-treated DCs from donor 1 (HLA-A2, 31, B13, and 35) were used as effector cells. Various cancer cell lines were used as targets. Expression of hCGβ in cancer cell lines was examined at the protein and RNA levels by immunocytochemistry and reverse transcription-PCR, respectively. Autologous B lymphoblastoid cell line (B-LCL) was used as an HLA-matched control. The 4-h 51Cr release assay was performed in triplicate, and error bars indicate experimental deviation. B, T cells stimulated with B11-hCGβ-treated DCs from donor 1 (HLA-A2, 31, B13, and 35) were used as effector cells. The hCGβ-positive HLA-partially matched cancer cell lines Colo205 (HLA-A1, 2, B7, and 8) and OV2 (HLA-A1, 2, B35, and 49) were incubated with or without anti-HLA class I antibody or its isotype-matched IgG and then were used as targets. The 4-h 51Cr release assay was performed in triplicate, and error bars indicate experimental SD.

Fig. 5.

CTL responses directed at cancer cell lines. A, T cells stimulated with human anti-dendritic cell (DC) antibody (B11)-human chorionic gonadotropin β subunit (hCGβ)-treated DCs from donor 1 (HLA-A2, 31, B13, and 35) were used as effector cells. Various cancer cell lines were used as targets. Expression of hCGβ in cancer cell lines was examined at the protein and RNA levels by immunocytochemistry and reverse transcription-PCR, respectively. Autologous B lymphoblastoid cell line (B-LCL) was used as an HLA-matched control. The 4-h 51Cr release assay was performed in triplicate, and error bars indicate experimental deviation. B, T cells stimulated with B11-hCGβ-treated DCs from donor 1 (HLA-A2, 31, B13, and 35) were used as effector cells. The hCGβ-positive HLA-partially matched cancer cell lines Colo205 (HLA-A1, 2, B7, and 8) and OV2 (HLA-A1, 2, B35, and 49) were incubated with or without anti-HLA class I antibody or its isotype-matched IgG and then were used as targets. The 4-h 51Cr release assay was performed in triplicate, and error bars indicate experimental SD.

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We thank Drs. Robert Graziano, Dan Rohrer, Michael Yellin, Gisela Ferreira, and Michael Fanger for helpful discussions and critical reading of the manuscript, Dr. G. Parmiani (Istituto Nazionale Tumori, Milan, Italy) for the gift of the OV2 cell line, Lori Fritz and Victoria Shi for DNA sequencing, and Kim Wunder and Beth Jacobs for excellent administrative support.

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