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The Journal of Immunology, 2007, 179: 1381-1388.
Copyright © 2007 by The American Association of Immunologists, Inc.
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Tumor-Associated Embryonic Antigen-Expressing Vaccines that Target CCR6 Elicit Potent CD8+ T Cell-Mediated Protective and Therapeutic Antitumor Immunity1

Arya Biragyn2,*, Roberta Schiavo3,*, Purevdorj Olkhanud*, Kenya Sumitomo*, Alan King{ddagger}, Megan McCain{dagger}, Fred E. Indig{dagger}, Giovanni Almanzar* and Dolgor Baatar*

* Laboratory of Immunology and {dagger} Research Resources Branch, Gerontology Research Center, National Institute on Aging, Baltimore, MD 21224; and {ddagger} Cyto Pulse Sciences, Glen Burnie, MD 21061


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 MIP3{alpha}/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 MIP3{alpha} does not directly activate DCs, the MIP3{alpha}-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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Induction of tumor-specific immunity by immunizing patients with the antigenic components of their tumors, tumor-associated Ags (TAAs)4, is thought to be an effective way of eliminating residual malignant cells and preventing disease relapse. However, TAAs are often poorly immunogenic, and their repertoire for immunotherapeutic use is quite limited. Unlike solid tumors, immunotherapy for B cell malignancies is further hampered by a lack of well-defined TAAs, except for the patient’s unique idiotypic Ab (Id). Although efficacy of the Id vaccines, both in preclinical studies and phase I-II clinical tests is demonstrably potent (3), a broader application of the vaccines may not be feasible, due to the unpredictability of their T cell epitopes (4) needed for T cell responses, and the suppressive nature of tumor-derived Id in the absence of continuing T cell help (5). In addition, Id vaccines have to be custom tailored and individually produced for each patient. Recently, the oncofetal Ag (OFA)-immature laminin receptor 37-kDa protein (OFA-iLRP) was reported to be specifically expressed in different human tumors, such as breast, renal, lung, and ovarian cancers, and in hematological malignancies (1). Although the 37-kDa OFA-iLRP is not expressed by adult differentiated tissues (6), the same cDNA encodes the dimerized high-affinity mature 67-kDa laminin receptor protein (mLRP) that acts as a cofactor to stabilize the binding of laminin to cell surface integrins (2). The 67-kDa mLRP precursor is expressed on the surface of normal and cancer cells and shown to participate in metastasis (7, 8). It appears that there is a strong selective evolutionary pressure to maintain OFA/mLPR unchanged, as murine and human sequences are almost identical. The immunotherapeutic potential of OFA-iLRP has been recently proposed, as HLA-A2-specific CD8+ cells, generated from the peripheral blood of healthy donors or cancer patients, were able to lyse OFA-iLRP+ acute myeloid leukemia and chronic lymphocytic leukemia cells (9, 10). Interestingly, patients immunized with renal tumor RNA-transfected dendritic cells (DC) also generated T cell responses to OFA (11). Moreover, it has been reported that the survival of mice challenged with syngeneic tumor could be augmented in mice immunized with DCs transfected with the mRNA of OFA-iLRP (9). However, OFA-iLRP appears to be a poorly immunogenic Ag, as our initial attempts to elicit anti-OFA responses in mice immunized with DNA expressing OFA-iLRP failed (data not shown).

We have recently demonstrated that effective immunity against poorly immunogenic lymphoma Id can be enhanced by targeting their uptake by APCs through their cell surface chemokine receptors (12, 13). Mice immunized with chemokine fused to Id elicited potent protective and therapeutic antitumor responses when challenged with a lethal dose of syngeneic tumor cells. The vaccine required that Id was physically fused with a functionally active chemokine to be efficiently taken up, processed, and presented by APCs both in an MHC class I (MHC-I)- and class II-restricted fashion (14, 15). Therefore, we wanted to test whether the same simple vaccine strategy could also induce antitumor immunity against murine B cell lymphomas when OFA-iLRP would be used. To test this, mice were immunized with DNA constructs expressing OFA-iLRP fused with MIP3{alpha}/CCL20 or beta-defensin mDF2beta, chemoattractant ligands of CCR6 (pMIP3{alpha}-OFA and pmDF2beta-OFA, respectively). CCR6 is differentially expressed on immature DCs (iDC) and Langerhans cells that are recruited to the periphery during inflammation and infection (16, 17). We demonstrate that immunizations with pMIP3{alpha}-OFA and pmDF2beta-OFA elicit both protective and therapeutic antitumor responses in mice challenged with syngeneic A20 B cell tumor cells. Of note, the A20 tumor model is considered to be the most challenging (18), as most cancer vaccines, including prototype human idiotypic vaccine (Id-KLH) that showed promises both in mice and in human tests, failed to protect against it (12). However, it appears that chemoattractant-based vaccines are able to induce long-term tumor-neutralizing memory responses, because the majority of mice that survived the first tumor challenge remained tumor free, even after the second injection with A20 tumors after 9 mo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Fusion gene cloning and protein production

Generation of DNA vaccine constructs expressing murine MIP3{alpha}/CCL20, murine beta-defensin 2 (mDF2beta), and Hsp70 fused with tumor Ags (OFA-iLRP or sFv20) was previously described (14). Mycobacterium Hsp70 cDNA was a gift from Dr. T. Lehner (Guy’s Hospital, London, U.K.). Murine OFA-iLRP, (OFA, GenBank number AF140348) was cloned from murine B cell A20 lymphoma (American Type Culture Collection, (ATCC) Manassas, VA). All constructs were verified by DNA sequencing (Keck DNA Sequencing Laboratory, New Haven, CT). To generate the DNA vaccine, the chemokine-OFA was cloned in pVAX1 plasmid (Invitrogen Life Technologies). Chemoattractant-OFA proteins were produced from isopropyl beta-D-thiogalacto-induced BL21(DE3) cells (Stratagene) using bacterial expression vector pET11d (Stratagene), and purified (>90% purity) from inclusion bodies as described previously (12, 19). The peptides iLR58–66 (LLLAARAIV) (9) and MOPC-315 Ig 91–101 (ALWFRNHFVFGGGTK) (20) were all synthesized by Peptide Technology to a purity >99% by HPLC.

Cell lines

The A20 B cell lymphoma (H-2d, OFA-iLRPhigh), MOPC315 plasmacytoma (H-2d, OFA-iLRP–/low), and 4T1 (H-2d, OFA-iLRPlow) and EL-4 thymoma (H-2b, OFA-iLRPHigh) cell lines were purchased from ATCC. The B6/129 macrophage cell line (H-2d, CCR6+) was a gift from Dr. Howard Young (National Cancer Institute, Frederick, MD). Murine bone marrow-derived DC preparation was previously described (21). Cells used on day 4–5 of cultivation usually yield iDCs (14, 15). Surface expression of OFA-iLRP was evaluated using anti-OFA-iLRP mAb 43515 (gift from Drs. A. Barsoum and J. Coggin, Mobile, AL).

Immunizations of mice

All animals were housed at the National Institute on Aging animal facility (Baltimore, MD). Animal care was provided in accordance with the procedures outlined in a Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 86-23, 1985). For tumor protection study, 6- to 8-wk-old female BALB/C mice (10/group) were immunized three times every 2 wk by electroporating 25 µg DNA in 50 µl endo-free water intradermally in the base of the tail using a parallel row needle array (with two rows of four needles per row, a 1-mm gap between needles within a row, a 4-mm gap between rows, and a needle length of 3 mm) connected to a PA-4000 electroporation system (Cyto Pulse Sciences). A Pulse AgileR electroporation protocol was used, consisting of two pulses of 450 V (1125 V/cm), 0.125 seconds pulse interval and 0.05 mS pulse duration followed by eight pulses of 110 V (275 V/cm) 0.125 seconds pulse interval and 10 mS pulse duration. Control mice were immunized with pmDF2beta-sFv20 that is shown to induce protective antitumor responses in mice (14). Two weeks after the last immunization, mice were challenged i.p. with 2 x 105 A20 lymphoma cells, and mice were followed for tumor survival. For therapy studies, mice were challenged i.p. with 2 x 105 A20 lymphoma cells at day 0, and then immunized with DNA constructs at days 3, 8, and 18. For rechallenge experiments, the surviving mice were pooled together and i.p. injected with 2 x 105 A20 lymphoma cells. The mice used were tumor free ≥9 mo after the first A20 tumor challenge. Subsequently, the surviving and tumor-free mice were i.p. injected with 3000 4T1 or 105 SP2.0 cells. Differences in survival between groups were determined by a nonparametric logrank test (BMDP Statistical Software).

Preparation of immune effector cells and cytolytic assay for immune splenocytes

Mice were electroporated with plasmid constructs as described above or s.c. immunized twice at 3-wk intervals with 10 µg human iLR58–66 peptide emulsified in 100 µl IFA. Splenocytes were cultured with 20 IU/ml rhIL-2 and 1 µg/ml of corresponding peptide (irrelevant MOPC-315 Ig 91–101 or iLR58–66, respectively) and used on days 5–7 after the initiation of the culture. For CTL, the CD8-enriched cells were cocultured with irradiated tumor cells and autologous CD4+ lymphocytes and DCs in 48-well plates with IL-2 (added at 25 U/ml after 2 days of culture). After 7 days, cells were restimulated with irradiated tumor cells and autologous splenocytes. The cytotoxicity as lactate dehydrogenase release in the cell supernatants was measured using the Cytotoxicity Detection Kit (Roche) following the manufacturer’s instructions, with the absorbance measured at 570 nm with a 630-nm reference filter on a plate reader 680XR (Bio-Rad). The average values for wells performed in triplicate were used for calculations after the medium controls were subtracted. The percent-specific cytotoxicity was calculated as follows: percent cytotoxicity = (experimental – effector alone) – target spontaneous/target maximum – target spontaneous.

In vivo T cell subset depletions

In vivo effector T cell depletions started 2 wk after vaccination by i.p injecting 400 µg anti-CD8 mAb GK 2.43 or anti-CD4 mAb GK1.5 (National Cancer Institutes-Frederick Cancer Research and Development Center), or normal rat IgG (Sigma-Aldrich) three times every other day 2 wk after the last immunization and before tumor challenge. Depletion of lymphocyte subsets was assessed 1 wk after final treatment by flow cytometry analysis of splenocytes from normal mice treated with these mAb in parallel (12).

Intracellular Ag processing

iDC from naive BALB/c mice were incubated overnight with 0.01–1 µg/ml fusion protein. Then, cells were irradiated (2000 Rad), washed twice with PBS, and cocultured for 24–48 h with T cells from the iLR58–66 (or irrelevant MOPC-315 Ig91–101) peptide-immunized mice. Some DCs were also treated in the presence of various inhibitors as follows: pertussis toxin (2.5 ng/ml), sucrose (0.4 M), brefeldin A (500 µM), chloroquine (50, 10, and 1 µM), and lactacystin (50, 10, and 1 µM) (purchased from Sigma-Aldrich).

Confocal microscopy

B6/129 cells were cultured overnight in covered glass-bottom dishes (MatTek Corporation) as described elsewhere (22). The slides were incubated on ice with 25 µg/ml MIP3{alpha}-fusion proteins in 10% FBS/RPMI 1640. After two washes in ice-cold PBS, warm 10% FBS/RPMI 1640 (37°C) was added, and slides were incubated at 37°C for 0, 10, 30, and 60 min before fixation with 3.7% formaldehyde and permeabilization with 0.2% Triton X-100. The following primary Abs were used: anti-myc mAb (clone 9E10; Sigma-Aldrich), rabbit anti-LAMP-1 Ab (H-228) or rabbit anti-Clathrin HC (H-300; both from Santa Cruz Biotechnology), or rabbit anti-proteasome 20S subunit {alpha}-5 (Affinity BioReagents). The secondary Abs, goat anti-mouse or goat anti-rabbit IgG, were conjugated to Alexa Fluor 488 or Alexa Fluor 568 (Molecular Probes). Images were acquired with a x63 objective on a Zeiss LSM 410 confocal system and processed using Adobe Photoshop.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
DNA vaccines expressing OFA fused to chemoattractants elicit potent anti-A20 lymphoma protection

Embryonic Ag OFA-iLRP is an attractive target for cancer immunotherapy, as it is abundantly expressed in various malignancies, including murine A20 lymphoma, but is not found in normal adult tissues (1). Our initial attempts to induce anti-A20 lymphoma responses in naive BALB/c mice immunized with plasmid DNA-expressing OFA failed, presumably due to poor immunogenicity of the Ag. Therefore, to render OFA immunogenic through the CCR6-mediated targeting of iDCs, we generated constructs that expressed OFA fusions with mDF2beta (pmDF2beta-OFA) or MIP3{alpha}/CCL20 (pMIP3{alpha}-OFA). Ten naive BALB/c mice per group were immunized with either pmDF2beta-OFA or with pmDF2beta-sFv20, a positive control construct that encoded mDF2beta fusion to an A20-specific Ig fragment (single chain Fv) shown to be immunogenic (13). Two weeks after the last immunization, mice were challenged with a lethal dose of A20 lymphoma cells. Almost all mice mock immunized with PBS succumbed to cancer (Fig. 1a). In contrast, mice immunized with pmDF2beta-OFA or pmDF2beta-sFv20 acquired significant protection against A20 lymphoma (p < 0.05, as compared with PBS-treated mice; Fig. 1a). The response required targeting of CCR6, as control vaccines that expressed OFA fused to mutant MIP3{alpha} that did not bind CCR6 due to a single point mutation (15) and failed to protect (pMIP3{alpha}-D-OFA; see Fig. 4). Thus, both pmDF2beta-OFA and pmDF2beta-sFv20 induce comparable protective anti-B cell lymphoma responses. However, unlike Id, OFA-based vaccines would not require individual formulations for each patient; by contrast, they would allow treatment of any OFA-expressing cancers.


Figure 1
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  		FIGURE 1. Genetic immunizations with constructs expressing mDF2beta fusions with nonimmunogenic TAAs induce protective antilymphoma response. A, BALB/c mice (10 per group), immunized with pmDF2beta-OFA (•) or pmDF2beta-sFv20 ({triangledown}), were challenged i.p. with 2.5 x 105 A20 lymphoma cells. A separate group of mice was injected with a mixture of pmDF2beta-sFv20 and pMIP3{alpha}-OFA ({blacksquare}) or mock with PBS ({diamond}). The logrank p value is for comparison between pmDF2beta-OFA or pmDF2beta-sFv20 and PBS. Data are representative of at least three independent experiments, all yielding similar results. B, Mice immunized with pmDF2beta-OFA generate significant OFA-specific IgG1 ({triangledown}) and IgG2a ({blacktriangleup}). 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, {circ} and IgG2a, •). 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 anti-mouse IgG1- or IgG2a-HRP Abs (Caltag Laboratories).

 

Figure 4
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  		FIGURE 4. Treatment with pMIP3{alpha}-OFA eradicates established A20 lymphoma. BALB/c mice (10 mice/group) bearing A20 lymphoma were treated with pMIP3{alpha}-OFA or pHsp70-OFA. Control mice were mock treated with PBS or electroporated with pMIP3{alpha}-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 pMIP3{alpha}-D-OFA.

 
Tumor protection is not improved by the use of multiple TAA-encoding vaccines

Because either of the vaccines that expressed different tumor Ags, pmDF2beta-sFv20 or pMIP3{alpha}-OFA, elicited comparable responses, we tested whether they would also act additively when used together (pmDF2beta-sFv20 plus pMIP3{alpha}-OFA). As shown in Fig. 1a, mice were protected against A20 lymphoma at similar levels, regardless of whether they were immunized with the vaccine mixture or with a single Ag-expressing vaccine (see pmDF2beta-sFv20 plus pMIP3{alpha}-OFA vs pmDF2beta-OFA or pmDF2beta-sFv20; Fig. 1a). Thus, immune responses elicited against a single TAA are sufficient to protect against tumors, and the use of additional Ags may not be necessary or beneficial.

Chemoattractant vaccines elicit CTLs that recognize H-2Kd-restricted epitopes

Mice immunized with pmDF2beta-OFA or pMIP3{alpha}-OFA (data not shown) generated not only OFA-specific IgG1 Abs ({triangleup}; Fig. 1b), but also significant levels of IgG2a Ab ({blacktriangleup}; Fig. 1b), indicating that they may produce Th1 responses (23). Moreover, mice immunized with the vaccines generated CTLs capable of specifically killing A20 tumor cells in vitro (Fig. 2a). The CTLs were specific to OFA, as they did not lyse irrelevant HLA-matched MOPC315 cells, which did not express OFA (Fig. 2a). The response was dependent on the ability of the vaccine to target CCR6, because splenocytes from mice immunized with the construct-expressing OFA fused to a mutant MIP3{alpha} (pMIP3{alpha}-D-OFA; Fig. 2a) did not kill A20 lymphoma cells.


Figure 2
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  		FIGURE 2. Vaccine induces T cell responses. A, Splenocytes from mice immunized with pMIP3{alpha}-OFA or with the iLR58–66 peptide/IFA specifically lyse A20 lymphoma cells (pMIP3{alpha}-OFA/A20 and OFA peptide/A20), but not HLA-matched (H-2Kd) OFA– MOPC315 (pMIP3{alpha}-OFA/MOPC315) and OFA peptide/MOPC315, or mismatched (H-2b) EL-4 (pMIP3{alpha}-OFA and OFA peptide/EL4) tumor cells. Control splenocytes from mice injected with PBS or immunized with OFA fusions with a mutant MIP3{alpha} (pMIP3{alpha}-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 pMIP3{alpha}-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).

 
To determine the T cell epitopes responsible for CD8+ CTLs, we predicted an 8-mer (underlined) or 9-mer peptide (LLLAARAIV) that contained a comparably high Parker binding score for H-2Kd (96 and 24, respectively; www-bimas.cit.nih.gov). The sequence of the peptide closely matched to OFA-iLRP (LLLAARAIV), which was originally described by others as the iLR58–66 peptide recognized by HLA-A02 (9). Presumably, the iLR58–66 epitope is expressed by A20 lymphoma cells, as immune CD8+ T cells from mice immunized with the iLR58–66 peptide/IFA specifically lysed A20 lymphoma, but not the HLA-matched and OFA-iLRP– MOPC315 cells or the HLA-mismatched but OFA-iLRP+ EL4 cells in vitro (see OFA peptide groups; Fig. 2). Control CD8+ T cells from irrelevant peptide-immunized mice failed to lyse either of the cells (data not shown). The chemoattractant-based vaccines appear to also present the iLR58–66 epitope, because splenocytes from mice immunized with pMIP3{alpha}-OFA or pmDF2beta-OFA were stimulated in vitro by the iLR58–66 peptide-pulsed APCs (data not shown); as well as splenocytes from mice immunized with the peptide were readily stimulated by iDCs incubated with MIP3{alpha}- or mDF2beta-fused OFA-iLRP proteins (via Ag cross-presentation, will be discussed below, Fig. 3a).


Figure 3
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  		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 MIP3{alpha}-OFA or mDF2beta-OFA. Then, after extensive washings and irradiation, they were cocultured with immune effector splenocytes from BALB/C mice (immunized with the iLR58–66 peptide/IFA) and IFN-{gamma} release was measured after overnight incubation. Effector cell specificity was validated using splenocytes pulsed with 1 µg/ml of the iLR58–66 (OFA peptide) or MOPC315 peptides (irrelevant peptide); or incubating with OFA+ A20 lymphoma or OFA– MOPC315 tumor cells. Control DCs treated with MIP3{alpha} 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, MIP3{alpha}-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 anti-rabbit 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 ({square}). C, Processed OFA is presented on MHC-I molecules. iDCs were incubated with mDF2beta-OFA or MIP3{alpha}-OFA in the presence of neutralizing anti-MHC-I (H-2d) or isotype-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.

 
Tumor protection elicited by chemoattractant vaccines is mediated by effector CD8+ CTLs

Because mice immunized with the iLR58–66 peptide generated CD8+ T cells capable of specifically killing A20 lymphoma cells in vitro, we tested whether these CTLs would also protect mice from challenge with a lethal dose of A20 lymphoma cells. Surprisingly, although the chemoattractant-based vaccines elicited significant protection (Fig. 1a and Fig. 2b), we failed to detect any protection in mice immunized with the iLR58–66 peptide/IFA (data not shown). Despite these results, induction of CTLs might be important for protection against the A20 lymphoma, as control mice immunized with pMIP3{alpha}-D-OFA, a mutant MIP3{alpha} fusion with OFA-iLRP, did not induce any CTLs and tumor protection both in vitro (Fig. 2a) and in vivo (see Fig. 4). To study this possibility, before the challenge with A20 lymphoma cells, we depleted effector CD8+ or CD4+ cells by injecting specific Abs 2 wk after the last immunization with pMIP3{alpha}-OFA. Analysis of the spleens from the mice (two per group) injected in parallel with the Abs indicated that the depletion of the appropriate subset with normal levels of the other subsets was >90% (data not shown). Injections of isotype-matched irrelevant IgG (pMIP3{alpha}-OFA plus IgG; Fig. 2b), or the depletion of effector CD4+ T cells (pMIP3{alpha}-OFA plus {alpha}CD4 Ab; Fig. 2b) did not have any effect on survival, and mice immunized with pMIP3{alpha}-OFA remained protected. In contrast, the protection was completely abolished in mice that were depleted of effector CD8+ T cells (pMIP3{alpha}-OFA plus {alpha}CD8 Ab; Fig. 2b). Taken together, these data clearly indicate, as we proposed for Id-mediated anticancer protection (13), that the biological activity of the CCR6-targeting chemoattractant-based OFA-iLRP vaccines is primarily dependent on the activation of cellular immunity, particularly in their ability to elicit tumor specific CD8+ T cell responses.

The CCR6-targeted OFA is efficiently taken up and cross-presented to MHC-I molecules

These data indicate that targeting of CCR6 with MIP3{alpha}- or mDF2b-fused OFA-iLRP promotes the induction of tumor-specific CD8+ CTL responses. Because CCR6 is known to internalize upon binding with its ligands MIP3{alpha} and mDF2beta (13), and no tumor protection was elicited by immunizations with OFA alone or fusions with mutant MIP3{alpha}, we hypothesized that the CCR6-mediated internalization facilitated an efficient cross-presentation of MIP3{alpha}-OFA and mDF2beta-OFA to MHC-I molecules. To test this notion, naive bone marrow iDCs from BALB/c mice were incubated overnight with nanomol concentrations of purified recombinant MIP3{alpha}-OFA or mDF2beta-OFA proteins. Then, after the extensive washing and irradiation steps, the cells were mixed with immune splenocytes from syngeneic mice immunized with the peptide OFA-iLRP58–66 in IFA, which elicited CTLs capable of the specific killing of A20 lymphoma cells in vitro, but not control HLA-matched MOPC315 cells that did not express OFA (Fig. 2a). The assumption was that if CCR6 mediated cross-presentation, APCs incubated with MIP3{alpha}-OFA or mDF2beta-OFA, but not free OFA, would stimulate the OFA-iLRP58–66 peptide-specific T cells. As shown in Fig. 3a, only iDCs incubated with as little as 100 ng/ml MIP3{alpha}- or mDF2beta-OFA fusion proteins induced significant IFN-{gamma} secretion from the OFA peptide-specific T cells, suggesting that chemoattractant-fused OFA-iLRP was processed and presented to MHC-I molecules. Control DCs incubated with OFA alone, or MIP3{alpha}-sFv20 or mDF2betasFv20 (irrelevant tumor Ag fusions, data not shown), did not stimulate the splenocytes, ruling out nonspecific effects from the chemoattractants used. Thus, these data indicate that OFA-iLRP fusions with MIP3{alpha} and mDF2beta were efficiently cross-presented. This was an active receptor-mediated process, as pertussis toxin (which abrogates Gi{alpha}-coupled receptor signaling; Fig. 3a) or high hypertonic sucrose solution (which inhibits clathrin-coated pit-dependent endocytosis, data not shown) completely abolished the ability of APCs to stimulate T cells. Similarly, chloroquine, the serine and cysteine protease inhibitor of lysosomal protein degradation, or brefeldin A, a fungal metabolite that inhibits vesicle transport of newly synthesized MHC molecules between the endoplasmic reticulum and Golgi (24), completely abrogated the response (Fig. 3a), indicating the importance of lysosomal activity in the chemoattractant-induced MHC-I presentation of OFA-iLRP. Proteins were shown to be processed directly within endosomal/lysosomal compartments and loaded to MHC-I molecules, which resided in classical MHC-II compartments, using TAP-independent and NH4Cl-sensitive cross-presentation pathways (25, 26). However, the CCR6-targeted OFA used classical cross-presentation pathway in the cytosol, since lactacystin, a specific inhibitor of proteasomal protein degradation, completely abrogated the response (Fig. 3a). The pharmacological inhibitors used in this experiment did not cause nonspecific suppression, because they did not affect the stimulation of T cells induced by iDCs that were directly pulsed with the OFA-iLRP58–66 peptide (that did not require internalization and processing; Fig. 3a). These findings are supported by confocal microscopy studies, demonstrating that the MIP3{alpha}-fusions, before internalization, were colocalized with clathrin vesicles on the cell surface (0 min; Fig. 3b). However, within 10 min after the internalization of MIP3{alpha}-fusions, they were found in lysosomes or colocalized with proteasomes in the cytosol (Fig. 3b). The processed MIP3{alpha}-fusions were presumably degraded within 1 h after the internalization by lysosomal enzymes and proteasomes (because the colocalized signal disappeared by 60 min of incubation; Fig. 3b). Perhaps, 60 min is sufficient to present processed peptides to MHC molecules, because iDCs incubated with MIP3{alpha}-OFA for as little as 1 h were capable of stimulating immune T cells (although at much lower levels, data not shown). The processed peptides were presented onto H-2d molecules, as the blocking Ab, but not control isotype-matched Ab, completely abolished the ability of iDCs incubated with MIP3{alpha}-OFA or mDF2beta-OFA to stimulate immune T cells (Fig. 3c). Taken together, these data clearly demonstrate that the potency of MIP3{alpha}-OFA or mDF2beta-OFA is in their ability to use the CCR6-mediated uptake, processing, and cross-presentation pathways.

The vaccine also elicits therapeutic antitumor immunity in tumor-bearing mice

Next, we tested the therapeutic use of the DNA vaccines by immunizing BALB/c mice with established A20 lymphoma. As shown in Fig. 4, tumor-bearing mice treated with pMIP3{alpha}-OFA, but not control mutant MIP3{alpha}-fusion (pMIP3{alpha}-D-OFA; Fig. 4), elicited significant therapeutic antitumor responses. As a result, a significant proportion of the mice (up to 30–60% in three independent experiments with 10 mice per group each) were tumor free when observed for 100 days. However, the vaccine did not protect all immunized mice, although a separate experiment indicated that pMIP3{alpha}-OFA could induce A20 lymphoma-specific CTLs in every immunized mouse (data not shown; see also Fig. 2a). The importance of DC activation or inflammation in the induction of potent T cell immune responses has been widely emphasized. MIP3{alpha} is not known to directly activate DCs, although it could induce inflammation via recruitment of proinflammatory cells at the vaccine site. To study this, A20 lymphoma-bearing mice were treated with two different vaccines, which expressed OFA-iLRP fused to moieties shown to directly activate DCs, such as mDF2beta (pmDF2beta-OFA) (27) or a C-terminal fragment of Hsp70 Ag from Mycobacterium tuberculosis (pHsp70-OFA) (28). As shown in Fig. 4, survival of mice treated with pmDF2beta-OFA or Hsp70-OFA was similar to that of mice immunized with pMIP3{alpha}-OFA. Taken together, the MIP3{alpha}-based vaccine is potent, and the inability to elicit 100% protection is presumably not due to the lack of sufficient activation of APCs.

The vaccine-induced protection is long lasting

OFA-iLRP shares the same protein backbone with the 67 kDa LRP that is expressed in normal tissues. Despite this, chemoattractant-OFA vaccines were able to induce tumor-neutralizing CD8+ T cell-mediated responses without the apparent harmful side effects in immunized and protected mice even after one-and-a-half years of observations. To assess the long-term memory anti-OFA CTL responses, protected mice that were tumor free for almost 9 mo postchallenge were rechallenged with the lethal dose of A20 tumor cells. In parallel, some mice were culled and tested for the presence of CTL memory responses by stimulating against A20 tumor cells or DCs pulsed with the iLR58–66 peptide. As shown in Fig. 5a, the majority of rechallenged mice (14 of 16 mice) were protected, while almost all age-matched control BALB/c mice succumbed to tumor. The protection correlated with the presence of CTLs against OFA-iLRP, as splenocytes from the long-term survivor mice stimulated against A20 tumor cells (data not shown) or DCs pulsed with the iLR58–66 peptide (Fig. 5b) induced significant lysis of A20 tumor cells, but not the OFA-iLRP-negative 4T1 tumor cells (H-2d, OFA-iLRPlow; see profile, Fig. 5c) and OFA-iLRP-positive but HLA-mismatched melanoma B16 cells (see profile, Fig. 5c). In concordance, no protection was detected when the survivors from A20 tumor challenge were injected with 4T1 tumor cells (Fig. 5d). Taken together, these data suggest that chemoattractant-OFA vaccine induced potent protective and long-lasting cellular memory antitumor responses.


Figure 5
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  		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 ({circ}) and control ten age-matched naive BALB/C mice (•) 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.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
We recently reported that fusions with chemokines enabled extrinsic Ags to be taken up for both MHC-I and -II processing and presentation pathways (14, 15). We speculated that this would presumably activate both CD4+ Th and cytolytic CD8+ T cell responses to the weakly immunogenic tumor-associated embryonic Ag, OFA-iLRP. Indeed, we demonstrate that chemoattractant-OFA constructs that target CCR6, a receptor expressed on iDCs and Langerhans cells, can induce OFA-specific CD8+ T cell-mediated cytolytic responses. As a result, constructs expressing OFA-iLRP fused with mMIP3{alpha} or mDF2beta elicit significant protective and therapeutic immunity against A20 lymphoma, a representative model for human B cell lymphomas known for its resistance to immunotherapy (18, 29). In contrast, no protection was observed in mice immunized with the OFA-iLRP58–66 peptide/IFA, despite the fact that they generated CTLs capable of killing of A20 tumor cells in vitro (Fig. 2a). Thus, in concordance with our earlier report that chemokine fusions enhance MHC-II presentation of extrinsic Ags (14), it is tempting to speculate that this may be due to the ability of chemoattractant-OFA constructs to also induce a necessary CD4+ T help at the priming stage of CD8+ T cells, which would be absent in mice immunized with the OFA-iLRP58–66 peptide/IFA. In concordance, CD8+ T cells primed in the absence of CD4+ T cell help are shown to undergo TRAIL-dependent death upon Ag restimulation, despite their ability to mediate effector functions, such as cytotoxicity (30). However, we cannot exclude the possibility that CD8+ T cell responses induced by the chemoattractant-OFA vaccines recognized additional epitopes with a higher therapeutic value than the OFA-iLRP58–66 epitope. Recently, while our manuscript was in preparation, a set of H-2Kd-restricted mouse CD8+ CTL and T-inhibitory nanopeptides on OFA-iLRP was described by Rohrer et al. (31). Among which, the M3 CTL peptide (RTWEKLLLAARA) has overlapping sequence (underlined) with our H-2Kd-restricted epitope (LLLAARAIV), which was originally described as the HLA-A02-restricted iLR58–66 peptide by Siegel and colleagues (9).

Optimal adaptive immune responses particularly to self-tumor Ags are thought to require "proper" activation of APCs, specifically DCs. It was proposed that strategies that only target or deliver Ags to APCs would not be effective, as agonistic anti-DEC205 mAb-fused Ags induced tolerance, unless DCs were first activated by CD40 engagement, despite their efficient Ag uptake and processing (35). In this respect, it is quite striking that the MIP3{alpha}-based vaccine, despite its inability to directly activate APCs in vitro (28), rendered nonimmunogenic self-tumor Ags immunogenic, eliciting both protective responses in syngeneic mice similar to those elicited by pmDF2beta-OFA (cf, Fig. 2b with Fig. 1a), which was shown to induce the maturation of iDCs via TLR-4 (28), or that were induced by the OFA fusion with immunostimulatory Hsp70 Ag from Mycobacterium tuberculosis (pHsp70-OFA), which was shown to induce Th1-polarized cytokine responses and activate DCs via binding with CD40 (27, 36). Thus, the MIP3{alpha}-based vaccine is potent, and can presumably indirectly activate APC needed for the generation of potent T cell immune responses against self-tumor Ags (perhaps via promoting cross-talk between various inflammatory cells recruited at the vaccine site). It should be noted that besides the recruitment or activation of APCs, the vaccine requires a physical linkage/fusion with a functionally active chemokine moiety, because, as we reported for other Ags (13, 37), no protection was detected in mice immunized with mutant chemokine fusions, which could not induce chemotaxis or bind CCR6 (pMIP3{alpha}-D-OFA; Fig. 4).

Idiotypic vaccines for some B cell malignancies are shown to be effective both in animal models (13, 38, 39, 40) and in phase I-III clinical trials (3). However, a major limitation of the vaccine is that the T cell epitopes essential for the protection may not always be expressed on Id, and the vaccine has to be individually produced for each patient (see review in Refs. 41 , 42). In contrast, OFA-iLRP may have a higher therapeutic and practical value, as it is an evolutionarily conserved Ag that is expressed in a number of human cancers, including in B cell lymphomas (1). Despite the fact that OFA-iLRP shares the same sequence with a normal 67-kDa OFA-mLRP cell surface Ag (6), mice immunized with plasmid DNA expressing chemoattractant-OFA are able to mount significant and long-lasting CD8+ cytolytic T cell responses and protective and therapeutic antitumor responses against OFA-iLRP-expressing tumors without any apparent autoimmune side effects. It appears that patients with B cell malignancies and renal cell carcinoma also contain circulating precursor T cells, as they could be readily expanded to generate cytolytic CD8+ T cell specific to OFA-iLRP-expressing tumors (10, 11). Moreover, our data that long-lasting memory and protective CTLs can be detected in mice even after almost year suggest that the OFA-based vaccines may also be used as a preventive formulation for the induction of protective memory responses in healthy people at a high risk for cancer.


   Acknowledgments
 
We are grateful to Dr. Howard Young (National Cancer Institutes/National Institutes of Health) for the gift of the B6/129 macrophage cell line; Drs. Adel Barsoum and Joseph Coggin (Mobile, AL) for the gift of recombinant OFA-iLRP and anti-OFA Abs; Thomas Lehner (Guy’s Hospital, London, U.K.) for the gift of cDNA for mycoHsp70; Drs. Dan Longo and Ashani Weeraratna (National Institute on Aging/National Institutes of Health) for helpful comments and suggestions; and Ana Lustig (National Institute on Aging/National Institutes of Health) for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute on Aging. Back

2 Address correspondence and reprint requests to Dr. Arya Biragyn, Laboratory of Immunology, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Box 21, Baltimore, MD 21224. E-mail address: biragyna{at}mail.nih.gov Back

3 Current address: Falck Division of Medical Oncology, Ospedale Niguarda Ca’ Granda, Milan, Italy. Back

4 Abbreviations used in this paper: TAA, tumor-associated Ag; Id, idiotypic Ab; OFA, oncofetal Ag; OFA-iLRP, OFA-immature laminin receptor 37-kDa protein; DC, dendritic cells; MHC-I, MHC class I; iDC, immature DC; mDF2beta, murine beta-defensin 2. Back

Received for publication August 30, 2006. Accepted for publication May 1, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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