Cancer Immunol Immunother (2011) 60:87–97

DOI 10.1007/s00262-010-0921-2

ORIGINAL ARTICLE

An autologous dendritic cell canine mammary tumor hybrid-cell fusion vaccine

R. Curtis Bird · Patricia DeInnocentes · Allison E. Church Bird · Frederik W. van Ginkel · Joni Lindquist · Bruce F. Smith

Received: 23 June 2010 / Accepted: 13 September 2010 / Published online: 11 November 2010© Springer-Verlag 2010

Abstract Mammary cancer is among the most prevalentcanine tumors and frequently resulting in death due to met-astatic disease that is highly homologous to human breastcancer. Most canine tumors fail to raise eVective immunereactions yet, some spontaneous remissions do occur.Hybrid canine dendritic cell–tumor cell fusion vaccineswere designed to enhance antigen presentation and tumorimmune recognition. Peripheral blood-derived autologousdendritic cell enriched populations were isolated from dogsbased on CD11c+ expression and fused with canine mam-mary tumor (CMT) cells for vaccination of laboratory Bea-gles. These hybrid cells were injected into popliteal lymphnodes of normal dogs, guided by ultrasound, and includedCpG-oligonucleotide adjuvants. Three rounds of vaccina-tion were delivered. SigniWcant IgG responses wereobserved in all vaccinated dogs compared to vehicle-injected controls. Canine IgG antibodies recognized sharedCMT antigens as was demonstrated by IgG-recognition ofthree unrelated/independently derived CMT cell lines, andrecognition of freshly isolated, unrelated, primary biopsy-derived CMT cells. A bias toward an IgG2 isotype responsewas observed after two vaccinations in most dogs. Neither

signiWcant cytotoxic T cell responses were detected, noradverse or side-eVects due to vaccination or due to theinduced immune responses noted. These data provideproof-of-principle for this cancer vaccine strategy and dem-onstrate the presence of shared CMT antigens that promoteimmune recognition of mammary cancer.

Keywords Autologous · Hybrid-cell fusion · Vaccine · Canine mammary cancer · Dendritic cells

Introduction

Spontaneous mammary cancer is among the most commonmalignancies aZicting unspayed female dogs with mostcases resulting in death from widespread metastatic disease[1]. Canine mammary tumors (CMTs) have been character-ized extensively for both genetic defects and phenotypes,and share many characteristics with human breast cancerincluding hormone dependence [2–6], frequent oncogeneHer-2/neu activation [7] and defects in expression of cyclindependent kinases (CDKs) and their inhibitors (CKIs) suchas p16/INK4A and p21/Cip1 [8–10]. CMTs represent oneof the best homologous models of human breast cancerknown consisting of the same malignant tissue types,highly similar pathology and natural history, and involveno viral etiology [2, 3, 11].

Tumors express speciWc antigens that are recognized bythe immune system [12, 13] and in some cases promote nat-ural remission [14–16]. A therapeutic strategy that couldreliably stimulate immune recognition could promote bettermanagement strategies in treatment of neoplastic diseaseparticularly in metastatic cases. However, naturally occur-ring immune management is, at best, unreliable and, atworst, ineVective in eliminating tumor cells.

Electronic supplementary material The online version of this article (doi:10.1007/s00262-010-0921-2) contains supplementary material, which is available to authorized users.

R. C. Bird (&) · P. DeInnocentes · A. E. Church Bird · F. W. van Ginkel · J. Lindquist · B. F. SmithDepartment of Pathobiology, Auburn University, Auburn, AL 36849-5519, USAe-mail: birdric@vetmed.auburn.edu

F. W. van Ginkel · B. F. SmithScott-Ritchey Research Center, College of Veterinary Medicine Auburn University, Auburn, AL 36849, USA

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One approach, designed to improve immune recognition,is based on creation of hybrid-cell vaccines through fusionof antigen presenting cells (APCs) with tumor cells [17–20]. Hybrid-cell fusion constructs express both tumor-spe-ciWc antigens and necessary machinery for antigen presen-tation and, if they are autologous (MHC I matched), T cellactivation is possible. Among APCs, dendritic cells (DCs)are considered most eVective in antigen presentation. Theyexpress MHC class I and II molecules as well as costimula-tory and adhesion molecules [17–20]. Because DCs cantransport presented antigens to activate eVector T cells inlymph nodes they have been suggested as promising candi-dates for the APC component of hybrid-cell vaccines [19].

Our work and others, have demonstrated that immunerecognition can be enhanced by vaccination with cellularconstructs composed of allogeneic hybrid-cell fusions ofAPCs and tumor cells [17, 19–23]. Uptake, processing andpresentation of tumor cell antigens by APCs, includingantigen loading of DCs, is thought to be promoted by cellfusion stimulating antigen recognition. Such hybrid-cellfusions express both tumor-speciWc antigens and the neces-sary machinery needed for antigen presentation but havebeen thought to require MHC matching for T cell activa-tion. We have previously demonstrated that MHC-match-ing was not required and that measurable cytotoxic T cellresponses could be induced using only allogeneic cell linesthat could be explained by cross-presentation between allo-geneic and autologous DCs [21, 24]. Although some suc-cess has been reported using cellular cancer vaccines,suggesting that these strategies possess the potential toimprove immune recognition of tumors, and they remainexperimental [12, 25–28].

Canine mammary tumor cell lines have been developedand represent stable lines derived from malignant caninemammary cancers with well characterized morphology andgenetic changes [2, 3, 7, 9–11, 21, 29]. We have previouslypublished a strategy for creating allogeneic (unmatched)canine hybrid-cell fusion vaccines against canine mammarycancer utilizing CMT cell lines and their fusion to a DC-like cell line [21]. The resulting fusions were of very highquality typically fusing at rates of 40–70% of all cells andvaccination demonstrated a clear, statistically signiWcantenhancement of cytotoxic T lymphocyte (CTL) activity innormal healthy dogs. However, because no component cellin the allogeneic hybrid-cell fusions was MHC matched,there was uncertainty as to the mechanism of immune pre-sentation. We have now developed the technology for high-speed sorting of relatively rare canine DCs isolated fromperipheral blood to provide a population of APCs suitablefor making hybrid-DC fusions that are autologous (MHCmatched) to each dog vaccinated. We now report the fusionof these well described CMT cell models and primary DC-enriched populations sorted from fresh canine peripheral

blood to create autologous DC hybrid-cell fusions for useas cancer vaccines.

Materials and methods

Cell culture

CMT12, CMT27, and CMT28 canine mammary tumor-derived cells were grown under standard conditions inLeibovitz’s L-15-medium, as previously described, in prep-aration for hybrid-cell fusion [8, 10, 21]. Primary CMTcells were librated from fresh canine mammary tumorbiopsy materials as previously described [2].

Primary autologous dendritic cell sorting

Flow cytometry and high-speed cell sorting were used toisolate DC populations labeled with speciWc antibodies toCD antigens. Peripheral blood mononuclear cell (PBMC)populations were prepared from whole canine blood (40–60 ml) collected from normal outbred Beagles into EDTAtubes (Becton, Dickinson), gently mixed and centrifuged30 min at 1,000g at room temperature. The buVy coat con-taining the PBMCs was isolated in 1–2 ml and combinedwith 8 ml HBS (HEPES-buVered saline, pH 7.2, no Mg/Ca,Gibco). The cell suspension was layered over 5 ml Hist-opaque 1077 (Sigma-Aldrich) and centrifuged at 1,000g atroom temperature for 30 min. The PBMC containing bandwas extracted in 1–2 ml and diluted with 2 ml HBS, mixed,and centrifuged. The supernatant was removed and cellsgently resuspend in 5 ml HBS. Cells were centrifuged onceand resuspended in 4 ml Xow wash buVer (FWB: HBS,10% fetal bovine serum) and incubated at room tempera-ture for 40 min.

PBMC populations (1–3 £ 109 cells in 1 ml) werelabeled with antibodies against canine CD4 (FITC-conju-gated polyclonal rat anti-canine, Serotec), CD8 (RPE-con-jugated polyclonal rat anti-canine, Serotec), and CD11c(monoclonal mouse anti-canine, Serotec) as previouslydescribed [21] in combinations of up to three antibodies(100 �l of each antibody solution per 1 ml reaction vol-ume). Anti-CD11c antibodies were labeled with secondaryanti-mouse monoclonal antibody conjugated to Alexa-Fluor 660 (70 �l/reaction) as previously described [21].Cells were analyzed and sorted in a MoFlo Xow cytometerwith 488 and 635 nm lasers (Beckman). Dot-plots and his-tograms were analyzed using Summit 4.3 (Beckman). Theentire cell suspension was sorted for each experiment andsorted cells were collected under sterile conditions intotubes containing 1 ml of fetal bovine serum. Both sampleand sorted cell populations were maintained at 4°C.Following sorting, cells were gently mixed, collected by

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centrifugation, as described above, and cultured in RPMI-1640 (Gibco/BRL) containing 10% fetal bovine serum(Hyclone) and penicillin/streptomycin/fungizone (Gibco/BRL) as described above.

Hybrid-DC fusion and vaccine preparation

Populations of CMT28 and autologous DCs were fusedby incubation with sterile solutions of 50% w/v PEG(»3,350 MW) in Improved MEM as previously described[21]. Parallel fusions of stained and unstained cells pro-vided cells to be analyzed by Xow cytometry and unstainedcells for injection as previously described except CellTrac-ker Orange CMTMR (Invitrogen) was used to stain DCpopulations (1:2 dilution of CellTracker Orange CMTMRstock to sterile clear 1£ Hanks and then 1 �l of the diluteddye added per 1 £ 106 cells for 30 min at 37°C) [21]. Cellsuspensions were collected by centrifugation, resuspendedin Improved MEM and incubated for 15 min followed bycentrifugation and washed three times with Hanks and5 £ 104 cells analyzed by Xow cytometry on a MoFlo Xowcytometer (green 530 § 20 nm and orange 580 § 15 nm) todetermine percentages of single or dual-stained populationsfollowing fusion. Populations of CMT28/DC fusions(5 £ 106 DCs fused to 1 £ 107 CMT28 cells, ratio of 1:2 in0.5 ml PBS containing CpG oligonucleotides, adjusted forDC yield) were prepared as previously described [21, 30].

Polyclonal canine IgG assay

Polyclonal antibody-containing sera from vaccinated dogswere assayed for CMT-speciWc cIgG (canine IgG) by col-lecting clotted blood samples at 37°C from each dog, rim-ming each clot followed by clot contraction at 4°C for 1 hand then centrifugation and extraction of the serum andstorage in aliquots at ¡85°C. For the standard assay, serawere thawed on ice and aliquots (100 �l) of live 5 £ 105

CMT28 cells in growth medium were reacted with 5 �l ofpolyclonal dog serum (or HBS for no-primary antibodycontrols) for 60 min at room temperature, washed threetimes in 1 ml HBS and resuspended in 100 �l FWB. Sec-ondary antibody (4 �l/assay, sheep pan-anti-dog IgG-FITC,Serotec) was added and incubated 60 min at room tempera-ture in the dark, washed two times in 1 ml HBS, resus-pended in 700 �l HBS with 1% w/v BSA (fraction V), andWltered to 53 �m [21]. Live labeled cells were analyzed byXow cytometry on a MoFlo Xow cytometer (Beckman).Volumes of primary and secondary antibodies were alteredin those experiments designed to optimize the standardassay. In some experiments FITC-conjugated cIgG class 1(goat anti-dog IgG1, Serotec) or cIgG class 2 (sheep anti-dog IgG1, Serotec) antibodies were substituted or an anti-

MHC I antibody known to react with canine MHC I (mousemonoclonal anti-MHC I/poly-mammalian, H58A, VMRD,Inc.) was reacted with target cells prior to assay. This is theonly type of commercially anti-MHC I antibody availablethat has been proven to bind canine MHC I. In other casesthe order of antibody addition was reversed by applyingpolyclonal cIgG Wrst and then the anti-MHC class Iantibody followed by secondary Alexa660-conjugatedanti-mouse IgG monoclonal antibody (Invitrogen). All cytom-etry analysis ensured collection of at least 10,000 events inthe positive gates.

MHC I typing of dogs and canine cell lines

Each vaccinated dog and the CMT27 cell line were par-tially typed by direct DNA sequencing of exon 2 of themajor MHC I locus DLA88 in the canine genome as previ-ously described [21].

Vaccine injection and blood collection

All experiments were conducted under oversight of theAuburn University IACUC committee in AAALACapproved animal care facilities. Healthy adult, reproduc-tively intact (unspayed) female Beagles (1–2 years old)were acclimated for 2 weeks and randomly assigned (threedogs/group: vaccinated and vehicle injected control). Vac-cines were prepared and injected into popliteal lymphnodes, guided by ultrasound, of mildly sedated dogs givenlocal anesthesia at the site of injection as previouslydescribed at weeks 1, 5, and 9 [21]. Blood was collectedprior to, and one week after, each vaccination from eachanimal by venipuncture. Both sera and PBMC populationswere isolated and frozen for storage at each time point.Each dog was examined daily for physical signs of tumorgrowth at the injection site and any signs of physical dis-tress. Regular examinations for acute cytotoxicity and long-term eVects were also conducted [21].

Cell-mediated immune assays

Cell-mediated immune (CMI) assays were assessed by51Cr-release using vaccine strain CMT28 cells as targets aswell as other unmatched CMT cell lines and primary CMTcells freshly isolated from tumor biopsy specimens as pre-viously described [21]. Target cells were incubated withdilutions (25:1 or 50:1) of eVector lymphocytes:targetsfrom vaccinated or unvaccinated dogs, or medium aloneand supernatants counted for released radioactivity by liq-uid scintillation counting in triplicate. Corrected percentlysis was calculated to determine relative CTL activity aspreviously described [21].

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Results

Construction of autologous DC hybrid-cell fusion vaccines

We previously published a strategy for creating allogeneiccanine hybrid-DC fusion vaccines against canine cancer[21]. Although fusions were of high quality (typically fus-ing at rates of 40–70%) and elicited an enhancement ofcytotoxic T lymphocyte (CTL) activity in normal healthydogs no component of this vaccine was MHC matched asdetermined by individual DLA88 (HLA equivalent) genesequencing. To provide MHC-matched DCs, we havedeveloped technology to isolate relatively rare peripheralblood subsets of autologous canine DCs from individualdogs based on combinations of CD4/CD8/CD11c stainingof PBMC populations and sorting the CD11c+ cells underlive and aseptic conditions. Canine PBMCs labeled forCD4, CD8, and CD11c were analyzed by Xow cytometryand sorted into CD11c+/CD4¡/CD8+ (blue), CD11c+/CD4+/CD8¡ (green), CD11c+/CD4¡/CD8¡ (red), and CD11c++/CD4 +/CD8 + (light blue) antigen expressing populationsrepresenting DC-enriched populations from peripheralblood (Fig. 1). We routinely sort 1–3 £ 109 PBMCs recov-ering approximately 1–5 £ 106 CD11c+ cells suYcient toprepare a single hybrid-DC fusion vaccine or approxi-mately 0.1–0.5% of total PBMCs.

Blood was collected from six normal healthy femaleBeagles and DC-enriched populations were sorted fromthree of them for preparation of autologous DC hybrid-cellfusion vaccines. Fusions were injected three times at 4-weekintervals into popliteal lymph nodes in formulations

composed of cell fusions in pyrogen-free PBS with CpG-oligonucleotide adjuvant (Fig. 2) as previously described[21]. Three control dogs were bled identically but injectedwith vehicle with CpG alone. No acute or longer-termeVects of vaccination were detected and blood was col-lected on 1 and 2 weeks following each vaccination forimmune assessment (Fig. 2).

Autologous DC hybrid-cell fusion vaccination induces a robust IgG response

To overcome barriers with Wxed CMT cell targets in ELISAfor cIgGs [21], a live CMT cell target assay was developedusing Xow cytometry to provide greater sensitivity andquantitation by single cell Xuorescence. Amount of primary

Fig. 1 Cytometry and cell sorting of primary canine DC populationsfrom PBMCs. Canine PBMCs (60 ml fresh EDTA-treated blood) werelabeled for CD4, CD8, and CD11c, analyzed by Xow cytometry andsorted into CD11c+/CD4¡/CD8+ (b lower right gate), CD11c+/CD4+/CD8¡ (a lower right gate), CD11c+/CD4¡/CD8¡ (b lower left gateexcluding CD11c+/CD4+/CD8¡ cells in the a lower right gate), andCD11c++/CD4+/CD8+ (b upper left gate) populations enriched in

peripheral blood-derived DCs. DC-enriched/labeled populations areindicated by color back-gates. Sorted populations were gated Wrst onwhole cells in forward/side scatter dot plots and then analyzed for Xuo-rescence. Typical sorts involved labeling approximately 1–3 £ 109

PBMCs recovering 1–5 £ 106 CD11c+ cells suYcient to prepare asingle hybrid-DC fusion vaccine

Fig. 2 Autologous DC hybrid-cell fusion vaccine and blood collec-tion strategy. Six normal healthy reproductively intact female adultlaboratory Beagles were randomly divided among two groups: vacci-nated (animals Di, M, and L) and control vehicle injected (animals Da,B, G). Vaccines were composed of LPS-free PBS, CpG oligonucleo-tides, and hybrid-DC fusions while controls were injected with theidentical formulation minus the cell fusions both delivered into thepopliteal lymph node. Autologous DC hybrid-cell fusion vaccines(V£) were administered at weeks 1, 5, and 9 and blood (B) was col-lected (40 ml/dog) 1 and 2 weeks after each vaccination. Acute eVectswere monitored daily for 1 week and then twice weekly for 12 weeks

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cIgG-containing sera from dogs and secondary anti-cIgGantibody was varied to ensure that primary antibodies wereapplied at less than saturating levels and secondary antibod-ies were applied at saturating levels to allow quantitation ofamount of the CMT-speciWc cIgG present in each canineserum sample (Fig. 3). Levels of 5 �l of primary serum and4 �l of secondary serum (for 100 �l reactions containing5 £ 105 CMT targets) were optimal providing suYcientdynamic range for assay of varying levels of CMT-speciWccIgG.

A time course of cIgG activities binding CMT28 cellsfor each dog was prepared from sera collected on 1 and2 weeks following each of three vaccinations (Fig. 4a).Activities were expressed relative to an average for cellslabeled with secondary antibody only (no primary anti-body). Control injected animals ranged near the mean with-out signiWcant response while hybrid-DC fusion vaccinateddogs reacted strongly but to varying degrees. All vaccinated

dogs demonstrated a statistically signiWcant anamnesticresponse to subsequent vaccinations after week 3 with vac-cine matched CMT28 cell targets.

Antibody immunity in sera, derived 1 week after vacci-nation 3 against vaccine matched CMT28 cells, was com-pared to reactions using MHC-unmatched CMT12 andCMT27 cells. In vaccinated dogs, antibody recognition ofshared CMT cell antigens was evident in all CMT cell lines(Fig. 4b). Reactions in all CMT cell targets (matched andunmatched) suggested expression of shared antigensalthough reactions to CMT12 were approximately half thatobserved for CMT27 and CMT28 cells. No antibody recog-nition was detected with any cell targets reacted with serafrom control animals. DiVerences in reactions to CMT28,CMT27, and CMT12 cells were all statistically signiWcantfrom control sera.

To ensure that such immune reactions were not recog-nizing common antigens expressed as a consequence of

Fig. 3 Anti-CMT cIgG assay optimization. Flow cytometry assays of cIgG recognizing CMT antigens were developed such that primary cIgG was delivered below saturation of live CMT28 cell targets and sec-ondary sheep anti-cIgG-FITC was applied at saturating con-centrations so that mean channel Xuorescence (MCF) was propor-tional to strength of the primary cIgG reaction to CMT28 anti-gens. Histograms show Xuores-cence (FITC-channel) for cells treated with serum containing cIgG from naïve control dogs (Control), for cells reacted with primary cIgG containing serum from vehicle-injected dogs (Vehicle), and for cells reacted with cIgG containing serum from vaccinated dogs (V£). Primary antibodies (1oAb) were applied at 5 or 10 �l serum per 100 �l cells (5 £ 105) and sec-ondary antibodies (2oAb) were applied at 2 or 4 �l serum per 100 �l cells (5 £ 105) as noted. Optimal Xuorescence protocol (arrow) was judged as lowest level of primary serum used and maximum level of secondary antibody used without back-ground staining. Data shown is the last summary experiment of several trials adjusting serum levels for optimization assay conditions as 10 �l primary anti-body, 4 �l secondary antibody to 5 £ 105 cells in 100 �l reaction volume

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culture in vitro, primary CMT cells freshly recovered froma canine mammary tumor biopsy were also evaluated as tar-gets of cIgG binding. Although limited numbers of cellsreduced the number of assays possible and the binding lev-els were lower, there was a clear statistically signiWcantbinding of primary CMT cells by the vaccinated dog sera

compared to control sera (Fig. 4c). This data reinforce theconclusion that the sera from vaccinated dogs recognizedshared antigens among distinct canine mammary tumor-derived cell lines that were also shared by primary tumors.No signiWcant reaction of these antisera with unrelatedtumor-derived cell lines from canine melanoma or lym-phoma was detected (data not shown).

Each of the dogs and cell lines express unique MHC Inucleotide sequences (data not shown) and, with one excep-tion (CMT27 cells and Dog Di/6), also express uniqueMHC I alleles as demonstrated by direct DNA sequencingof exon 2 of the canine DLA88 locus (Fig. S1) encodingmost of the canine DLA88 locus variable region of MHC I[21]. In an attempt to assess the contribution of MHC classI mismatch to cIgG recognition assays, CMT cells werepre-reacted with anti-MHC class I antibody, known to react

Fig. 4 Anti-CMT cIgG assay time course and cross-reactivity. Flowcytometry assays of cIgG from vaccinated dogs recognizing CMT anti-gens were delivered at below saturation of live CMT28 cell targets andusing secondary sheep anti-cIgG-FITC was applied at saturating con-centrations were performed. Mean channel Xuorescence (MCF) wasproportional to strength of the primary cIgG reaction to CMT28 anti-gens for each dog. Titers were calculated as fold diVerence relative toan average for cells labeled with secondary antibody only. Relativelevels from 2 naïve (never injected) Beagles are shown for comparison.a Time course of autologous DC vaccinated (V £ 1, 2, and 3) and con-trol (Control 1, 2, and 3) dog antibody titers against CMT28 target cellsfor each of six blood collections (1 and 2 weeks after each of three vac-cinations weeks 2, 3, 6, 7, 10, and 11 for each dog). Inset: examples ofpolyclonal CMT-speciWc cIgG binding from naïve dog (black), DogV £ 3/L 1 week post vaccination 1 (light gray) and 1 week post vacci-nation 3 (medium gray). Statistical analysis employed a mixed modelfor repeated measures analysis of variance. Statistical signiWcance(asterisk) was achieved by the Wrst blood collection after the secondvaccination (week 6) for all vaccinated animals (week 6 p = 0.0274,week 7 p = 0.013, week 10 p = 0.0004, week 11 p = 0.0029).b Autologous DC vaccinated (Vaccine) and control (Control) dog IgGtiters (1 week following vaccination 3) against vaccine matchedCMT28 target cells and allogeneic target CMT27 and CMT12 cells.For each cell target, sera from 3 control (left) and 3 vaccinated (right)dogs from 1 week post vaccine 3 are shown. Relative MCF levels werecalculated as described above. Inset: examples of polyclonal CMT-speciWc cIgG binding allogeneic CMT12 (black) and CMT27 (lightgray) cells as well as vaccine-matched CMT28 (medium gray) cell tar-gets. Histograms are plotted as frequency/cell number against log Xuo-rescence. Statistical signiWcance (asterisk) was achieved for all serafrom vaccinated animals against all three cell lines (unpaired t test:CMT12 p = 0.0105, CMT27 p = 0.0068, CMT28 p = 0.0134). c Anti-CMT cIgG assay cross-reactivity against primary CMT cells. Flowcytometry assays of cIgG recognizing CMT antigens in primary biopsy-derived canine CMT cells were performed to evaluate autologous DCvaccinated (Vaccine) and control (Control) dog IgG titers (1 week fol-lowing vaccination 3). Left: Percent-labeled population (hatched) wascalculated compared to controls (gray) analyzed without primary anti-body (representative example shown). Right: Percent-labeled popula-tions compared for 2 control (Da and B) and 2 vaccinated (M and L)dog sera. SuYcient primary cells were recovered to evaluate sera from2 control and 2 vaccinated dogs. T test (1 tailed) analysis revealed sta-tistical signiWcance between vaccinated and unvaccinated sera againstprimary biopsy-derived CMT cells at p = 0.0866

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with canine MHC I on CMT cells [21], prior to addition ofvaccinated dog sera (Fig. S2). Pre-reaction to block MHCclass I neither results in diminished reactions againstCMT28 vaccine cells nor did such attempts to block reac-tion aVect binding to unmatched CMT12 or CMT27 cellsemploying sera from any of the vaccinated dogs (Di, M, L).This data could suggest that MHC I mismatch made littlecontribution to cIgG reactions although, because the anti-cMHC I antibody reacts to an epitope conserved in multiplespecies, it is also possible that this antibody does not pro-vide suYcient steric hindrance to interfere with polyclonalcIgG binding to MHC I determinants on target cells. Theseresults were conWrmed by reversing the order of reactionapplying the cIgG Wrst and then the anti-MHC class I anti-body followed by Alexa 660-conjugated anti-mouse IgGantibody. This experiment produced essentially the sameresults where cIgG pre-reaction was unable to block anti-

bodies directed against MHC I (Fig. S3). Because the onlycommercially available anti-MHC I antibodies proven tobind canine MHC I react with multiple species no furtherconclusions were possible.

To determine if IgG class switching occurred duringvaccination, IgG class 1- and 2-speciWc anti-cIgG antibod-ies were substituted as secondary antibodies stainingmatched CMT28 and unmatched CMT27 targets assayingsera from vaccinated dogs 1 week following vaccination 1and 3 (Fig. 5). In all vaccinated dogs there were strongbalanced IgG1/2 reactions to matched CMT28 cells aftervaccination 1. However, statistically signiWcant bias towardstronger IgG2 reactions to CMT28 cells following the thirdvaccination was detected. Although not signiWcant, this wasalso observed with unmatched CMT27 targets except indog Di where a weaker near background level IgG2 reac-tion was observed.

Fig. 5 Anti-CMT cIgG assay of isotype switching in vaccinated dogs.Flow cytometry analysis of cIgG isotype (1 or 2) binding to matchedCMT28 and unmatched CMT27 cell targets was performed. Sera fromthe three vaccinated dogs (Di, M, and L) from 1 week followingvaccination 1 and 3 (V£ 1 and 3 for each isotype and each dog) werereacted with target cells as noted (Target). FITC-conjugated secondaryantibody speciWc for canine IgG class 1 or class 2 (IgG1 or IgG2) wereused for secondary labeling. Negative controls included primary seraomitted (N1, N2) and no antibody (N) for both target cells. Inset:

Representative examples of polyclonal CMT cIgG-speciWc histogramsreacted against CMT28 cells for IgG1 (gray) and IgG2 (hatched) iso-types for dogs Di and L at 1 week following vaccination 3. StatisticalsigniWcance (asterisk) was achieved for all vaccinated animals forCMT28 targets detecting cIgG2 after vaccination 3 (paired t test:p = 0.0363). Mean channel Xuorescence (MCF) was expressed as aproportion of the cIgG reaction (in fold) to CMT28 antigens asdescribed above

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Autologous DC hybrid-cell fusion vaccination results in a weak CTL response

CMT-speciWc CTL assays were performed by 51Cr-releasefrom vaccine matched CMT28 or unmatched CMT12 celltargets by control and hybrid-DC fusion injected dogPBMCs at dilution ratios of 50:1 and 25:1 (PBMCs to 51Cr-loaded CMT targets in triplicate) and percent-speciWc lysiscalculated as a percentage of total releasable isotope avail-able [21]. Some signiWcant increase in CTL activity wasdetected at ratios of 25:1 against CMT28 targets but noconsistent pattern of CTL activity was evident suggestingthat, despite the presence of CpG oligonucleotides, immunereactions were biased toward an IgG response (Fig. 6).

Discussion

Mammary neoplasms express surface antigens that are rec-ognized by the patient’s immune system but fail to promotean eVective killing response [13]. We have previouslydemonstrated the potential of inducing such immuneresponses to mammary cancer in vaccine developmentthrough fusion of CMT cells with DC-like cells utilizing

all-allogeneic cell line sources [21]. This strategy appearsto potentiate uptake and promote processing and presenta-tion of CMT antigens by DCs enhancing antigen recogni-tion [13, 21, 24]. The success of CMT cell-DC fusions asvaccines suggests that they can overcome the rate limitingstep, which is thought to be the transfer of antigens to theDC [13]. Cell fusion strategies for DC loading also avoidintroduction of antigen selection bias although a theoreticalrisk of induction of autoimmunity has been postulated.There appears to be no evidence of this potential [13, 21–24]. We have previously reported only mild and rapidlyresolving side-eVects from lymph node vaccination ofthese hybrid-DC fusions and our current data also supportsprevious reports suggesting that injection of live tumorcells does not result in establishment of cell colonies [21,31]. To date we have vaccinated approximately 30 dogs inwhich more than 100 hybrid-cell fusion injections havebeen made revealing no long-term adverse eVects [21].Adverse aVects were conWned to mild and rapidly resolv-ing lameness (within 2 days) in the injected leg and tran-sient and mild elevation of body temperature. Previousinvestigations also revealed a signiWcant CTL response invaccinated dogs as a consequence of vaccination with anallogeneic cell construct [21].

Fig. 6 Cell-mediated immunity of hybrid-DC fusion immunized dogsagainst matched and unmatched CMT cells. CMT-speciWc cytotoxicT cell (CTL) assays were performed by standard 51Cr-release assaysfrom vaccine matched CMT28 or unmatched CMT27 cell targets in thepresence of PBMCs recovered from immunized or control vehicleinjected animals. CTL activity in PBMC populations was measuredindependently in triplicate and relative CTL response calculated as per-cent-speciWc lysis (% CMI) for each of 2 PBMC:CMT target cell ratios(50:1—50£ or 25:1—25£) calculated as the percentage of total

releasable isotope available due to speciWc lysis [21]. PBMCs wereobtained from dogs 2 weeks after vaccine 1 (1.2 samples) and 1 weekafter vaccine 3 (3.1 samples). CMI assays for hybrid-DC fusion vac-cine and control groups were performed on labeled vaccine-matchedCMT28 cell targets (right) and unmatched CMT12 target cells (left).The entire experiment was repeated twice and representative data isshown. Some statistical signiWcance was detected after vaccine 1 at aratio of 25:1 but no consistent pattern of CTL responses was evident(mixed model for repeated measures analysis of variance)

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We have further developed this strategy to incorporatean autologous DC population isolated directly from labora-tory Beagles to provide an individualized MHC-matchedcomponent to these vaccine constructs. Preparative isola-tion of primary canine DCs from individual dogs was possi-ble due to their size and blood volume which allowedrecovery of suYcient PBMCs to isolate approximately1–5 £ 106 DCs from each animal for each vaccine con-struct. Such volumes bode well for the translation of suchstrategies to humans from this intermediate canine model asbody size and blood volume are comparable. Previouslypublished procedures for cell fusion were also successfullyadapted to autologous DC hybrid-cell fusions [21].

Immune responses to antigens common to several CMTcell lines were assessed by comparing IgG reactions toCMT cells matched or unmatched for MHC to the vaccineconstruct based on CMT28 cells [21]. The assay measuredvariation in level of CMT-speciWc cIgGs present in serumable to stably bind CMT cells from unrelated dogs. Thedata demonstrate that a speciWc antibody response could bereliably detected and recognized native CMT antigens onlive cells when healthy dogs were vaccinated with autolo-gous DC hybrid-cell fusions composed of autologous DCsand CMT28 cells. Further, the response was also detectedin unmatched CMT12 and CMT27 cells and even freshlyisolated primary CMT cells from a tumor biopsy. Suchcomparisons strongly suggested the presence and detectionof shared CMT cell antigens common to these CMT cellssince the CMT cell lines used here have been previouslyshown to encode unique canine MHC class I DLA88(HLA-equivalent) alleles [21]. Thus, a positive immunereaction with any of the CMT target cells, unmatched forMHC I, would strongly suggest that shared CMT antigenswere recognized. This was true even though the reactionwas less intense than that detected against vaccine-matchedCMT28 cells. The data also suggest that the antigens recog-nized were not the result of culture in vitro as primary CMTcells were also recognized by these polyclonal sera. Wehave presumed that these antigens are located on the cellsurface, as antibodies eVectively labeled live cells, althoughat present we do not have any direct indication as to theidentity of these apparently shared antigens. We have evi-dence that the important breast cancer antigen mucin 1(MUC1) is expressed in CMT cells although not at theunusually high levels observed in human breast cancer lines(Fig. S4) [32]. Canine mammary tumors have previouslybeen shown to express MUC1 protein by immunohisto-chemistry and this protein could account for some of thisshared antigen [33].

There was also a distinct class shift detected in the cIgGresponse following the third vaccination from IgG1 to astronger IgG2 response. This was true for all vaccinated ani-mals when vaccine-matched CMT28 cell targets were used

and in two of three vaccinated animals when non-matchedCMT27 cells were employed as targets. A clear class shiftbiased toward the IgG2 from the IgG1 isotype was detected.Such observations in canine systems cannot be directly asso-ciated with speciWc T-helper type responses [34] although ithas been suggested that a class shift to an IgG2-biased pro-Wle can be associated with a Th1-type response with a cyto-kine proWle characterized as similar to human immuneresponses [35–38]. The enhanced cIgG2/Th1 induction,with characteristic cytokine expression, is suppressed inmany cancers and is reduced in dogs with spontaneous meta-static disease [39]. The shift to a cIgG2 immune responsemay indicate potential for a more eVective anti-tumorresponse even though direct correlation between cIgG iso-type and Th1/Th2 responses is not yet possible in caninemodels. That recognition of MHC I miss-match likelyaccounted for part of the IgG response was suggested by thelower reaction with unmatched CMT cell lines. Limitationsin the speciWcity of available antibodies recognizing canineMHC I precluded further conclusions regarding dependenceof this reaction on MHC I recognition.

Subsequent assessment of CTL responses were surpris-ingly low in vaccinated dogs in these experiments comparedto allogeneic hybrid-DC fusion vaccinated dogs [21]. Thiscould be due to a diVerence in the autologous nature of theDCs employed and the mechanisms of antigen presentationnow available. It is also possible that regulatory T cells (Treg

or suppressor T cells) were induced by this improved vaccinestrategy thus suppressing a CTL response. Further investiga-tion will be needed to explore these responses but evidenceconcerning cIgG responses clearly demonstrated that vacci-nation stimulated immune recognition of canine mammarytumor antigens and that reaction was biased toward an anti-body response that in most cases peaked after two vaccina-tions. We have previously determined that enhanced immunerecognition of shared CMT cell antigens using allogeneicvaccines were the result of enhanced CTL recognition mostlikely of shared CMT antigens. Development of the autolo-gous DC vaccine component has caused most of the reactionto be directed through an antibody response. The possibleconsequences of an antibody bias and any enhanced inXam-matory response remain to be elucidated.

We have established that vaccination with autologousDC hybrid-cell fusions constructed using freshly isolatedautologous DC-enriched cell populations can be accom-plished and such cellular vaccines have the potential todevelop enhanced individualized immune responses indogs. This demonstrates the potential of the hybrid-DCfusion strategy and the practicality of this technology forpromoting immune recognition of mammary tumors. Thisstrategy will serve as a platform for development of animmunotherapeutic vaccine treatment of canine mammarycancer.

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Acknowledgments The authors thank Drs. S. Ewald and E. Thackerfor valuable consultations, Mr. Stephen Waters for expert animal man-agement, Dr. L. G. Wolfe for original cell cultures, and Dr. J. Wrightfor statistical analysis. Supported by Scott-Ritchey Research Center.

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