Tumor-associated embryonic antigen-expressing vaccines that target CCR6 elicit potent CD8+ T cell-mediated protective and therapeutic antitumor immunity

Abstract

Despite its potency, the wider use of immunotherapy for B cell malignancies is hampered by the lack of well-defined tumor-specific Ags. In this study, we demonstrate that an evolutionarily conserved 37-kDa immature laminin receptor protein (OFA-iLRP), a nonimmunogenic embryonic Ag expressed by a variety of tumors, is rendered immunogenic if targeted to the APCs using the CCR6 ligands MIP3alpha/CCL20 and mDF2beta. The CCR6 targeting facilitated efficient Ag cross-presentation and induction of tumor-neutralizing CTLs. Although the Ag targeting alone, without activation of dendritic cells (DCs), is proposed to induce tolerance, and MIP3alpha does not directly activate DCs, the MIP3alpha-based vaccine efficiently induced protective and therapeutic antitumor responses. The responses were as strong as those elicited by the OFA-iLRP fusions with moieties that activated DCs and Th1-type cytokine responses, mDF2beta, or mycobacterial Hsp70 Ag. Although the same cDNA encodes the dimerized high-affinity mature 67-kDa mLRP that is expressed in normal tissues to stabilize the binding of laminin to cell surface integrins, the vaccines expressing OFA-iLRP elicited long-term protective CD8(+) T cell-mediated memory responses against syngeneic B cell lymphoma, indicating the potential application of these simple vaccines as preventive and therapeutic formulations for human use.

FIGURE 1

Genetic immunizations with constructs expressing mDF2β fusions with nonimmunogenic TAAs induce protective antilymphoma response. A, BALB/c mice (10 per group), immunized with pmDF2β-OFA (#x25CF) or pmDF2β-sFv20 (▽), were challenged i.p. with 2.5 × 10⁵ A20 lymphoma cells. A separate group of mice was injected with a mixture of pmDF2β-sFv20 and pMIP3β-OFA (■) or mock with PBS (◇). The logrank p value is for comparison between pmDF2β-OFA or pmDF2β-sFv20 and PBS. Data are representative of at least three independent experiments, all yielding similar results. B, Mice immunized with pmDF2β-OFA generate significant OFA-specific IgG1 (▽) and IgG2a (▲). Shown is a representative plot of experiments of mixed sera from five mice per group. No OFA-specific Ab was detected in the sera of mock immunized mice (IgG1, ○ and IgG2a, #x25CF). Titrated amounts of immune or naive mouse sera were incubated for 1 h on the same plate coated with 3 μg/ml recombinant TARC-OFA, and the Ig isotypes were determined using goat antimouse IgG1-or IgG2a-HRP Abs (Caltag Laboratories).

FIGURE 2

Vaccine induces T cell responses. A, Splenocytes from mice immunized with pMIP3α-OFA or with the iLR_58-66 peptide/IFA specifically lyse A20 lymphoma cells (pMIP3α-OFA/A20 and OFA peptide/A20), but not HLA-matched (H-2K^d) OFA^- MOPC315 (pMIP3α-OFA/MOPC315) and OFA peptide/MOPC315, or mismatched (H-2^b) EL-4 (pMIP3α-OFA and OFA peptide/EL4) tumor cells. Control splenocytes from mice injected with PBS or immunized with OFA fusions with a mutant MIP3α (pMIP3α-D-OFA), which could not bind CCR6, failed to lyse either of the cells. Percentage of cytotoxicity (Y-axis) of two representative and independent experiments with similar results, performed in triplicate, are shown. X-axis is the E:T ratio of cells used. B, Tumor protection requires the presence of OFA-specific effector CD8⁺ T cells. Mice were immunized with a pMIP3α-OFA plasmid as above and randomly allocated (ten per group) to treatment with anti-CD8 mAb GK2.43, anti-CD4 mAb GK1.5, or normal rat IgG. The p values refer to comparison between anti-CD8 mAb and IgG injected groups. Flow cytometry analysis of splenocytes from normal mice treated with these mAb in parallel 1 and 2 wk after treatment confirmed a >90% depletion of the appropriate subset with normal levels of the other subset (data not shown).

FIGURE 3

A, Chemoattractants facilitate the CCR6-mediated uptake, processing, and presentation of OFA to MHC-I molecules. Naive BALB/C mouse iDCs (target cells) were incubated overnight with 100 ng/ml MIP3α-OFA or mDF2β-OFA. Then, after extensive washings and irradiation, they were cocultured with immune effector splenocytes from BALB/C mice (immunized with the iLR_58-66 peptide/IFA) and IFN-γ release was measured after overnight incubation. Effector cell specificity was validated using splenocytes pulsed with 1 μg/ml of the iLR_58-66 (OFA peptide) or MOPC315 peptides (irrelevant peptide); or incubating with OFA⁺ A20 lymphoma or OFA^- MOPC315 tumor cells. Control DCs treated with MIP3α fused with an irrelevant tumor Ag or MC148-D-mOFA (data not shown) or mixture of untreated effector cells with splenocytes (E + T) failed to stimulate T cells. Some iDC were also treated in presence of 0.4 M sucrose, pertussis toxin, chloroquine, brefeldin A, or lactacystin. The p values refer to comparisons after treatment with chloroquine. B, Colocalization study. To enable internalization, cells were prechilled on ice and placed at 37°C for the time indicated by the column headings. Green, MIP3α-fusions stained with anti-myc mAb 1.9 μg/ml and goat anti-mouse Alexa 488 2 μg/ml. Red, Alexa 568 conjugated to goat antirabbit IgG, specific for either clathrin (top), LAMP (middle), or proteasomes (bottom). Merged signal is yellow. Transmission light image is of the 0 min time cell. Scale bar, 5 μm(□). C, Processed OFA is presented on MHC-I molecules. iDCs were incubated with mDF2β-OFA or MIP3α-OFA in the presence of neutralizing anti-MHC-I (H-2^d)or iso-type-matched control Abs. The same treatment was performed for control iDCs incubated with 1 μg/ml the OFA or MOPC315 peptides. The p values refer to comparisons with control Abs. Shown, representative data of at least two (C) and three (A and B) independent experiments yielding similar results.

FIGURE 4

Treatment with pMIP3α-OFA eradicates established A20 lymphoma. BALB/c mice (10 mice/group) bearing A20 lymphoma were treated with pMIP3α-OFA or pHsp70-OFA. Control mice were mock treated with PBS or electroporated with pMIP3α-D-OFA. Tumor-free survival was followed for 100 days after tumor challenge. The data shown is representative of four independent experiments, which yielded similar results. The p value refers to comparison with pMIP3α-D-OFA.

FIGURE 5

Eradication of A20 lymphoma promote long-term T cell-mediated memory that protects mice from rechallenge with A20 lymphoma. A, Sixteen mice that were free of tumors for ∼9 mo (○) and control ten age-matched naive BALB/C mice (#x25CF) were challenged with A20 lymphoma cells. The p value refers to comparison with control mice. B, In parallel, splenocytes of long-term survivor mice (E, effector cells) were in vitro stimulated for 1 wk on DCs pulsed with OFA-peptide and tested against target cells (T), such as A20, 4T1, and B16 tumors, at the indicated ratio (T:E). The percentage of cytotoxicity (Y-axis) of a representative experiment performed in triplicate is shown. C, OFA-iLRP is expressed on the surface of A20 lymphoma and B16 melanoma cells, but not 4T1 tumor cells. OFA expression was determined with Alexa 488-conjugated anti-OFA mAb (bold lines) vs control Alexa 488-conjugated isotype-matched Ab. D, Mice that survived A20 tumor challenge (A20-survivor + 4T1 (see also A) or control BALB/c mice (HBs + 4T1) immunized with control constructs expressing HBsAg were rechallenged with 4T1 tumors cells.