Evaluation of a xenogeneic VEGF vaccine in dogs with soft tissue sarcoma

Cancer Immunol Immunother (2007) 56:1299–1309 DOI 10.1007/s00262-007-0282-7

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ORIGINAL ARTICLE

Evaluation of a xenogeneic VEGF vaccine in dogs with soft tissue sarcoma

Debra Kamstock · Robyn Elmslie · Douglas Thamm · Steven Dow

Received: 27 October 2006 / Accepted: 29 December 2006 / Published online: 14 February 2007! Springer-Verlag 2007

Abstract Active immunization against pro-angio-genic growth factors or their receptors is an emergingstrategy for controlling tumor growth and angiogene-sis. Previous studies in rodent tumor models have indi-cated that immunization against xenogeneic growthfactors is more likely to induce eVective anti-tumorresponses than immunization against the autologousgrowth factor. However, the eVectiveness or safety ofthe xenogeneic vaccination approach has not been pre-viously assessed in a clinically relevant outbred, spon-taneous tumor model. Therefore, we investigated thesafety and anti-tumor and anti-angiogenic eVects of axenogeneic vascular endothelial cell growth factor(VEGF) vaccine in pet dogs with spontaneous cancer.Nine dogs with soft tissue sarcoma were immunizedwith a recombinant human VEGF vaccine over a 16-week period. The eVects of immunization on antibod-ies to human and canine VEGF, circulating VEGFconcentrations, tumor microvessel density (MVD), andtumor growth were assessed. The xenogeneic VEGFvaccine was well-tolerated by all dogs and resulted ininduction of humoral responses against both humanand canine VEGF in animals that remained in the

study long enough to receive multiple immunizations.Three of Wve multiply immunized dogs also experi-enced sustained decreases in circulating plasma VEGFconcentrations and two dogs had a signiWcant decreasein tumor MVD. The overall tumor response rate was30% for all treated dogs in the study. We concludetherefore that a xenogeneic VEGF vaccine may be asafe and eVective alternative means of controllingtumor growth and angiogenesis.

Keywords Canine · Cancer · Antibodies · Immunization · Endothelial cell

Introduction

Angiogenesis is critical to the ability of tumors to growprogressively and vascular endothelial cell growth fac-tor (VEGF) is one of the key growth factors regulatingthe process of angiogenesis. A number of previousstudies have demonstrated the importance of VEGF inregulating tumor angiogenesis and growth [1–3]. Forthis reason, strategies to inhibit the biologic eVects ofVEGF have become important approaches to the inhi-bition of tumor angiogenesis [4–9]. A variety of strate-gies to inhibit VEGF have been evaluated, includingthe use of mAbs to neutralize VEGF or block its recep-tors, the use of soluble VEGF receptors, and the use ofsmall molecule inhibitors of VEGF receptor tyrosinekinase activity [7–10]. Notably, the clinical validityof inhibiting tumor angiogenesis by neutralization ofVEGF activity was demonstrated in a pivotal study ofbevacizumab (anti-VEGF mAb) in humans with colo-rectal cancer [11, 12]. This and other studies were theWrst to demonstrate that neutralization of VEGF, in

D. Kamstock · S. Dow (&)Department of Microbiology, Immunology, and Pathology, Colorado State University, Ft Collins, CO 80523, USAe-mail: [email protected]

D. Thamm · S. DowDepartment of Clinical Sciences, Animal Cancer Center, Colorado State University, Ft Collins, CO 80523, USA

R. ElmslieVeterinary Cancer Specialists, Englewood, CO 80110, USA

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combination with chemotherapy, could produce a sig-niWcant survival beneWt in cancer patients [6, 12–14].

Active vaccination against pro-angiogenic factors ortheir receptors is an alternative approach to thera-peutic inhibition of angiogenesis [15–18]. For example,it was found that immunization against xenogeneicVEGF via the use of plasmid DNA vaccination couldelicit cross-reactive antibodies that neutralized endo-genous VEGF and inhibited tumor growth and angio-genesis in a mouse tumor model [19]. Immunizationagainst the VEGF receptor was also found in otherstudies to inhibit tumor angiogenesis in rodent models[20–24]. Vaccination against other pro-angiogenicgrowth factors, such as Wbroblast growth factor (FGF),appears to also inhibit tumor angiogenesis [25]. Analternative method to inhibit tumor angiogenesis is toactively immunize against endothelial cells themselves.For example, immunization with xenogeneic enodothe-lial cells, or more recently with autologous endothelialcells, was shown to elicit therapeutic inhibition ofangiogenesis in several diVerent rodent models [26–28].

One appeal of the immunization approach to angio-genesis inhibition is the ability to achieve sustainedinhibition without the necessity of continuous adminis-tration of angiogenesis inhibiting drugs [17]. However,the vaccine approach may also hold some risk, in asmuch as sustained, eVective neutralization of endoge-nous VEGF activity may elicit deleterious side-eVects,such as coagulopathy and interference with woundhealing [9].

Therefore, to more fully assess the potential eVec-tiveness and safety of VEGF vaccination as a viabletreatment option in humans, we conducted a study ofa xenogeneic VEGF vaccine in pet dogs with cancer.Dogs spontaneously develop a number of cancers thatclosely resemble their related tumors in humans [29,30]. In addition, dogs with cancer have been shown tohave elevated circulating VEGF concentrations [31].Because dogs are a highly outbred species, immunolog-ical studies in dogs are likely to more accurately reXector predict immune responses in humans. For this study,we chose dogs with cutaneous soft tissue sarcoma,which is a relatively slow growing tumor of dogs thatis locally invasive tumor and accessible to repeatedbiopsy [29].

The purpose of the study was to determine whetherimmunization against a xenogeneic VEGF protein(human VEGF165) could elicit therapeutic anti-VEGFresponses. We selected huVEGF as the immunogenbecause the amino acid sequences of human andcanine VEGF (caVEGF) diVer by only 4.8%, indica-tive of a high degree of homology [32]. Moreover, thehomology was close enough that canine VEGF bound

to and activated the human VEGF receptor [32].Unlike most previous studies that relied on DNAvaccination, in this study dogs were immunized withrecombinant human VEGF protein. The vaccine wasadministered using a liposome–DNA adjuvant shownrecently by our laboratory to be very eVective in elicit-ing immune responses against protein antigens [33–35].

The endpoints of the study were induction of anti-VEGF antibody responses, eVects on circulatingVEGF concentrations, eVects on tumor growth andangiogenesis, and safety. Nine dogs were enrolled inthe study and Wve dogs remained in the study longenough to receive at least Wve immunizations. Noneof the dogs enrolled in the study experienced adverseeVects that might be associated with excessive neu-tralization of endogenous VEGF concentrations. Inthe dogs that remained in the study long enough toreceive multiple immunizations, the huVEGF vaccineelicited anti-VEGF antibody responses. Moreover,the induction of VEGF antibody responses was asso-ciated with reduction in circulating VEGF concentra-tions, tumor angiogenesis, and decreased tumorvolume. We concluded from these results that immu-nization against xenogeneic VEGF may be a safe andeVective treatment option for the induction of anti-tumor and anti-angiogenic activity in some patientswith cancer.

Materials and methods

Study design

Client-owned dogs of any age, breed, or gender withspontaneous, initial or recurring, histologically con-Wrmed, cutaneous soft tissue sarcomas measuring<125 cm3 at the time of diagnosis were eligible forentry into the study. Tumor volume was determinedas the product of three tumor measurements (length,width, height). Any dog previously treated with radia-tion therapy or angiogenesis inhibitors and anydog currently receiving steroids or chemotherapy wasexcluded from entry into the study. Dogs with concur-rent disease or evidence of metastatic disease were alsoexcluded from the study. These studies were approvedby the Institutional Animal Care and Use Committeeat Colorado State University. The study was designedto include a series of six immunizations administeredover a course of 16 weeks. Animals were enrolled andtreated at the Colorado State University VeterinaryTeaching Hospital (Ft Collins, CO, USA) or at theVeterinary Referral Center of Colorado (Englewood,CO, USA).

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Preparation of VEGF vaccine

The vaccine was prepared using rhVEGF165, which wasmanufactured by R&D Systems (Minneapolis, MN,USA) and provided by the NCI Reagent Repository.The liposome–DNA complexes used as the vaccine adju-vant were prepared as described previously [33]. BrieXy,liposomes were prepared by dissolving the cationiclipid DOTIM (octadecenolyoxy{ethyl-2-heptadecenyl-3-hydroxyethyl} imidazolinium chloride; Sigma-AldrichChemical Co., St Louis, MO, USA) and cholesterol(Avanti Polar Lipids, Alabaster, AL, USA) in chloro-form and adding equimolar concentrations to roundbottom 15 ml glass tubes. The solution was then driedovernight in a vacuum dessicator to a thin Wlm and thelipids were rehydrated in 5% dextrose in water at 50°Cfor 50 min, followed by extrusion through a series of 1,0.45, and 0.20 !m Wlters to form the Wnal liposomes. Toform the liposome–DNA complexes, non-codingplasmid DNA (prepared by the Valentis Corp, Burlin-game, CA, USA) was added to liposomes in solution(100 !l liposomes per ml D5W) with gentle pipetting ata Wnal concentration of 100 !g DNA per ml of lipo-some solution. The vaccine was then prepared by add-ing rhVEGF directly to the liposome–DNA solution atroom temperature, followed by gentle mixing. Dogswere immunized within 15 min of preparing the VEGFvaccine.

Treatment and monitoring

A total of six immunizations with the VEGF vaccinewere administered intradermally to dogs. Dogs wereinitially immunized once every other week for threeimmunizations, then once every four weeks for threeadditional immunizations. For the Wrst immunization,dogs received 50 !g rhVEGF in 600 !l adjuvant (admin-istered in two diVerent sites over the lateral thorax),while all subsequent immunizations consisted of 25 !grhVEGF in 300 !l adjuvant administered in one siteover the lateral thorax. Dogs enrolled in the study weremonitored for adverse eVects by physical examinationat the time of each immunization. Complete bloodcounts and serum biochemical proWles were evaluatedprior to starting the study and again at week 6 and week16. The vaccine site was evaluated at each repeat immu-nization to assess injection site responses. In addition,dogs were carefully evaluated clinically for evidence ofcoagulopathy (hemorrhage, petechiation) and delayedwound healing at tumor biopsy sites (see below).

Tumor volume was determined using calipers tomeasure tumor dimensions prior to treatment and ateach vaccination boost. Tumor responses were classi-

Wed as partial responses if tumor volume decreased by>50% from pre-treatment volume measurements andcomplete responses if the tumor was no longer detect-able. Stable disease was deWned as tumor volume thatdid not increase or decrease by >50% from starting val-ues, whereas progressive disease was deWned as tumorvolume that increased >50% from starting values. Forany dog where there was documented progressivetumor growth, the dog was removed from the studyand alternative therapy was implemented.

Assessment of anti-huVEGF antibody responses

Plasma and serum samples were obtained from eachdog prior to treatment and immediately prior to vacci-nation for each of the six treatments and stored at¡80°C. Plasma concentrations of anti-huVEGF anti-bodies were determined by enzyme linked immuno-sorbent assay (ELISA). BrieXy, the anti-huVEGFantibody ELISA was prepared by coating Immunolon-3 plates (Dynatech Laboratories, Chantilly, VA, USA)with a solution of 5 !g/ml rhVEGF (R&D Systems) in50 mM carbonate/bicarbonate buVer at a pH of 9.6incubated overnight at 4°C. Wells were blocked witha 5% solution of non-fat dried milk in phosphatebuVered saline (PBS). The plasma samples were pre-diluted 1:1,000 in buVer (PBS with 1% BSA) and incu-bated at room temperature for 90 min. After washing,the plates were incubated with biotinylated-rabbit anti-dog IgG (Jackson ImmunoResearch, West Grove, PA,USA) for 60 min at room temperature. After washing,streptavidin–horseradish peroxidase (HRP) conjugate(Jackson ImmunoResearch) was added for 30 min atroom temperature, then washed again and the sub-strate developed with 3,3!,5,5!-tetramethylbenzadine(Sigma-Aldrich). The colorimetric reaction was stoppedwith 1 M hydrochloric acid and the optical density wasdetermined by ELISA plate reader (Thermo LabSys-tems, Franklin, MA, USA). Plasma concentrations ofanti-caVEGF antibodies were determined followingthe same protocol as described above except that theplates were initially coated with a 5 !g/ml solution ofrecombinant caVEGF. Recombinant caVEGF waspurchased from R&D Systems.

Measurement of plasma VEGF concentrations

Plasma VEGF concentrations in dogs were determinedusing an ELISA kit developed for detection ofhuVEGF (R&D Systems) in accordance with themanufacturer’s directions. Validation of this kit fordetection of caVEGF in plasma has been previouslyreported [31, 36, 37].


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Tumor biopsy and assessment of tumor microvessel density, VEGF expression, and IgG deposition

Tumor biopsies were obtained under local anesthesiaby wedge biopsy immediately prior to the Wrst treat-ment, at week 6, and at week 16. Biopsy specimenswere embedded in Tissue-Tek OCT compound (Ted-Pella Inc., Redding, CA, USA), snap frozen in isopen-tane and dry ice, and stored at ¡80°C. Tissues werecryosectioned to a thickness of 4 !m and adhered topositively charged glass slides. For identiWcation oftumor microvessels, a mAb speciWc for human CD146(clone P1H12, Chemicon, Temecula, CA, USA) whichcross reacts with canine endothelial cells was utilized,as described previously [37]. BrieXy, tissues were Wxedin acetone, then blocked With H2O2 to eliminateendogenous peroxidase activity, then incubated withthe primary antibody (mouse anti-human CD146) for30 min at room temperature, followed by incubationwith donkey anti-mouse IgG antiserum (JacksonImmunoResearch). Slides were then treated withstreptavidin–HRP followed by 3-amino-9-ethylcarbaz-ole (AEC) substrate (Vector Laboratories, Burlin-game, CA, USA), counterstained with hematoxylin, airdried, and cover slipped with aqueous-based crystalmount. Negative controls for IHC included incubationwith irrelevant mAb and omission of the primary anti-bodies. Normal canine spleen was utilized as a positivecontrol tissue for microvessel density (MVD) assess-ment.

To quantitate tumor MVD, four digital photomicro-graphs at 10£ magniWcation were obtained (LeicaMicroscope equipped with digital camera in con-junction with SPOT Advanced Imaging software), asdescribed previously [37]. Adobe Photoshop andReindeer Graphics Quantitative Analysis Plug-ins(Asheville, NC, USA) were utilized to convert thephotomicrographs to binary images. The number ofmicrovessels per section was then calculated as numberof pixels per mm2 tumor tissue. The Wnal MVD valuefor each time tumor section was calculated as the mean(§SEM) number of vessels for the four 10£ Welds.

To assess tumor-associated VEGF expression, cryo-sectioned slides were Wrst Wxed in 10% formalin for5 min rather than Wxation in acetone. Prior to staining,slides were treated in target retrieval solution (Dako,Carpinteria, CA, USA) pH 6.0 at 125°C for 1 min.Staining was then performed utilizing a Dako auto-stainer in conjunction with Dako reagents (LSAB+Link System) according to manufacturer’s directions.Goat anti-caVEGF antibody (R&D Systems) was uti-lized as the primary antibody, followed by incubationwith HRP-conjugated donkey anti-goat IgG (Dako),

followed by incubation with diaminobenzidine as thesubstrate. After staining, slides were dehydratedthrough graded alcohol followed by xylene and covers-lipped with permanent-based mount. All sections frompre- and post-treatment biopsies were evaluatedmicroscopically to assess the percentage of VEGF-pos-itive cells as well as the relative intensity of VEGFstaining.

For assessment of IgG deposition in tumor tissues,cryosectioned biopsy specimens were prepared asdescribed above, except that samples were Wxed usingacetone. Slides were incubated with peroxidase-con-jugated rabbit anti-dog IgG (Jackson ImmunoRe-search) for 30 min at room temperature, followed byincubation with AEC substrate for 15 min at roomtemperature. Incubation with irrelevant primary anti-body (rabbit anti-mouse IgG) and omission of the pri-mary antibody were used as negative controls whilenormal canine splenic tissue was utilized as a positivecontrol. All sections were subjectively evaluatedmicroscopically and assigned a score of 0–3 based onthe overall IgG immunoreactivity and intensity ofstaining.

Statistical analyses

Paired samples were compared statistically using theStudent’s t-test. Comparisons between more thantwo treatment groups or more than two time pointswere done using one-way analysis of variance(ANOVA), followed by Tukey’s multiple meanscomparisons test. Analysis was done using GraphPadPrism statistical software (San Diego, CA, USA).For all analyses, P values <0.05 were considered sta-tistically signiWcant.

Results

EVects of vaccination on induction of anti-huVEGF antibodies

A total of nine dogs with soft tissue sarcoma wereenrolled in the study (Table 1). Of these nine dogs,four dogs remained in the study long enough to receiveWve or more immunizations, while Wve dogs receivedthree or fewer immunizations. The Wve dogs that failedto receive greater than three immunizations wereremoved from the study due to progressive tumorgrowth (Table 1). For the four dogs that received Wveor more immunizations, antibody response generallyincreased over time, with three of the four dogs mount-ing a marked increase in anti-huVEGF antibody


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responses (Fig. 1). None of the Wve dogs receivingthree or fewer vaccinations mounted substantial anti-huVEGF antibody response (data not shown andTable 1). Thus, it appeared that immunization withrhVEGF could elicit anti-VEGF antibody responses,but that multiple immunizations (at least three ormore) were required to elicit signiWcant increases. Posi-tive antibody responses ranged from four- to tenfoldincreases in anti-huVEGF antibody increases comparedto pre-treatment values.

EVects of vaccination on circulating plasma VEGF concentrations

We next assessed the eVects of VEGF immunizationon circulating VEGF concentrations. A decrease inplasma VEGF concentrations was observed at two ormore time points in three of the four dogs that receivedWve or more VEGF immunizations (Fig. 2). In the fourthdog, no changes in circulating VEGF concentrationswere noted despite a large increase in anti-huVEGF

Table 1 Clinical response data for nine dogs with soft tissue sarcomas that were immunized with huVEGF

Nine dogs with soft tissue sarcoma received a series of immunizations with huVEGF. Tumors were classiWed histologically as peripheralnerve sheath tumors (PNST) or undiVerentiated sarcomas (UndiV Src). Tumors were graded on a scale of I to III based on decreasingdegree of diVerentiation, as well as other criteria. Tumor responses were classiWed as partial response (PR; greater than 50% reductionin tumor volume) or progressive disease (PD; greater than 50% increase in tumor size). Anti-huVEGF antibody responses, circulatingVEGF concentrations, and tumor MVD were assessed as described in Sect. “Materials and methods”NC no signiWcant change, NA not evaluated

Dog Histiotype/grade Clinical response

VEGF Ab Plasma VEGF Tumor MVD Number of vaccine

1 PNST/I PR + Decreased NC 62 PNST/I PR + Decreased Decreased 63 PNST/II PR ¡ Decreased NC 64 PNST/I PD + NC Decreased/increased 55 PNST/I PD ¡ Decreased NA 36 PNST/II PD ¡ NC NA 37 PNST/II PD ¡ NC NA 28 UndiV Src/III PD ¡ NC NA 29 PNST/I PD ¡ NC NA 1

Fig. 1 VEGF antibody responses following immunization ofdogs against recombinant huVEGF. Nine dogs with cancer wereenrolled in a study to evaluate the eVects of vaccination againstxenogeneic huVEGF. Plasma samples were collected prior toimmunization (week 0) and at several diVerent time points afterbeginning immunization with rhuVEGF. Plasma samples were

assayed for anti-huVEGF antibody responses, as described inSect. “Materials and methods,” and the mean (§SD) of replicateanti-VEGF antibody determinations was plotted. Anti-huVEGFantibody responses over time were plotted in four dogs (panels a–d and dogs 1–4 in Table 1) that received Wve or more immuniza-tions


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antibodies (see dog 4, Table 1, Figs. 1, 2). In contrast,VEGF concentrations did not decrease in four of theWve dogs that received three or fewer VEGF immuni-zations (Table 1). The one exception was a dog (dog 5,Table 1) that was immunized three times, where adecrease in VEGF concentrations was noted (data notshown). Thus, in some dogs, repeated immunizationwith rhVEGF induced a substantial anti-huVEGF anti-body response that was associated with a large declinein circulating VEGF concentrations (dogs 1 and 2,Table 1). In two other dogs (dogs 3 and 5, Table 1)there was a decrease in circulating VEGF despite thelack of a anti-huVEGF antibody responses. Theseresults suggest that in some dogs, the decrease in circu-lating VEGF concentrations may have been mediatedby mechanisms other than humoral immune responses.

EVects of vaccination on generation of anti-canine VEGF antibody responses

Next, studies were conducted to determine whethervaccination with rhVEGF was capable of elicitingcross-reactive antibodies against endogenous caVEGF.Such a response would be expected if immunizationagainst xenogeneic huVEGF was capable of breakingself-tolerance. Plasma samples from each of the four dogsthat received Wve or more huVEGF immunizationswere assessed by ELISA for the presence of antibod-ies recognizing canine VEGF. We found that anti-caVEGF titers were very modestly increased in threeof the four multiply vaccinated dogs that also mountedstrong anti-huVEGF responses (Fig. 3). However, theantibody responses against caVEGF were very weakcompared to the responses against huVEGF. These

Wndings assume greater importance when one consid-ers the fact that dogs are a highly outbred species witha very diverse MHC repertoire compared to inbredmouse strains [29, 30].

EVects of VEGF vaccination on tumor angiogenesis

Tumor MVD was evaluated in biopsy specimensobtained pre-treatment, on week 6, and on week 16 ofthe study from the four dogs that received multipleVEGF immunizations. Due to the design of the study,post-treatment tumor biopsies could not be obtainedfrom any the Wve dogs that received three or fewerimmunizations as they did not remain in the study longenough to reach the Wrst biopsy time point. Two of thefour multiply vaccinated dogs demonstrated a signiWcantdecrease in tumor MVD at one or more time pointswhen pre- and post-treatment biopsies were compared(Figs. 4, 5). It should be noted however that in one ofthese dogs tumor MVD actually increased at a later timepoint, coincident with progressive growth of the tumor(see dog 4, Table 1, Fig. 4). In the other two dogs, tumorMVD remained relatively constant after VEGF immu-nization was initiated. Thus, it appeared that repeatedVEGF immunization was capable of inhibiting tumorangiogenesis in at least half of the dogs.

EVects of vaccination on intratumoral VEGF expression and immunoglobulin deposition

We also investigated the possible eVects of repeatedVEGF vaccination on intratumoral VEGF concentra-tions. Immunohistochemistry was used to evaluateintracellular expression of VEGF by tumor cells and

Fig. 2 EVects of VEGF vacci-nation on endogenous VEGF concentrations in dogs. Circu-lating VEGF concentrations were measured in dogs prior to vaccination (week 0) and at twice weekly intervals follow-ing vaccination with hu-VEGF. VEGF concentrations were determined by VEGF ELISA, as described in Sect. “Materials and meth-ods” and the mean (§SD) of replicate VEGF determina-tions was plotted. In four dogs (a–d) that received more than Wve immunizations, VEGF concentrations were plotted over time


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inWltrating leukocytes in tumor biopsy specimens fromfour dogs that received more than Wve immunizations.A consistent diVerence in the extent or intensity oftumor VEGF staining was not noted when pre- andpost-vaccination biopsies were compared (data notshown). The deposition of IgG complexes within thetumor was also assessed, as deposition of IgG com-plexes has been noted in tumors of mice immunizedwith xenogeneic VEGF previously [21]. However, wedid not Wnd evidence of a vaccine eVect when pre- andpost-vaccination tumor biopsies were evaluated for thedegree of IgG deposition (data not shown). Theseresults suggested therefore that the major eVects ofimmunization on canine VEGF probably occurred sys-temically, rather than within the tumor itself.

Clinical responses in vaccinated dogs

Tumor responses were also assessed by means oftumor volume measurements in all dogs enrolled in the

study. A partial tumor response (>50% decrease intumor volume) was noted in three of the nine enrolleddogs (Table 1). In the other six dogs, progressive tumorgrowth was observed and all were dropped from thestudy before completing their full series of VEGFimmunizations. The three dogs in which tumor regres-sion was noted were also those that received the fullcourse of six immunizations. For dog 1 (Table 1), thetumor size remained stable over a 1-year period of fol-low-up. In dog 2, progressive tumor growth developedwithin 1 month of completing the vaccine trial. Tumorsize remained stable in dog 3 for 8 months, then pro-gressive tumor growth developed.

One possible explanation for these results is thatreceiving the full course of VEGF immunizations mayhave been associated with tumor regression. For exam-ple, spontaneous tumor regressions are very rare indogs with soft tissue sarcomas. However, selection biasmay have also played a role, inasmuch as dogs withinherently less aggressive tumors were those mostlikely to complete the vaccination regime.

Finally, all treated dogs were also carefully evalu-ated for adverse eVects associated with repeatedVEGF immunization. Vaccine site reactions were notobserved in any of the vaccinated dogs. In addition,evidence of coagulopathies (e.g., excessive bleedingfrom venipuncture or biopsy sites) or changes in

Fig. 3 Antibody responses against canine VEGF in dogs vacci-nated against huVEGF. Dogs were immunized with rhuVEGFand plasma was collected for analysis of anti-caVEGF antibodyresponses by ELISA, as described in Sect. “Materials and meth-ods” and the mean (§SD) of replicate anti-caVEGF antibodydeterminations was plotted. For the four dogs (a–d) that receivedWve or more VEGF vaccines, anti-caVEGF antibody responseswere measured prior to treatment and again at the completion ofthe immunization period (week 12 or week 16)

Fig. 4 EVects of VEGF vaccination on tumor MVD in dogs withsoft tissue sarcomas. Tumor biopsies were obtained from fourdogs (a–d) before treatment, at 6 weeks, and again at 16 weeks af-ter beginning VEGF immunization. Tumor MVD was quanti-tated using CD146 immunohistochemistry, as described inSect. “Materials and methods” and the mean MVD (§SD) foreach tumor was plotted. Pre- and post-treatment tumor MVDswere compared by ANOVA and signiWcant diVerences (P < 0.05)were denoted by asterisk


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complete blood counts or serum biochemistries werenot observed (data not shown). We also did notobserve any interference in wound healing at thetumor biopsy sites in the four dogs that had repeatedtumor biopsies performed. Thus, it appeared based onthis preliminary clinical investigation that dogs immu-nized repeatedly against xenogeneic VEGF did notdevelop serious adverse eVects.

Discussion

In this study we found that repeated immunization ofdogs with spontaneous tumors with xenogeneic VEGFwas associated with induction of anti-VEGF antibodyresponses and decreases in circulating VEGF concen-trations and tumor angiogenesis. Moreover, vaccina-tion was also associated with an overall tumor responserate (partial response) of 30%, in a tumor that is nottypically responsive to chemotherapy or other medicaltreatments. These results, which to our knowledge

represent the Wrst evaluation of xenogeneic VEGF vac-cination in a large animal spontaneous tumor model,indicate that the approach is technically feasible andsafe and is capable of inhibiting tumor angiogenesisand stimulating anti-tumor activity in animals withlarge, spontaneous tumors.

Not surprisingly, the study revealed that the abilityto generate anti-VEGF immune responses was asso-ciated with number of immunizations an animal received.Dogs that received three or fewer immunizationsgenerally failed to produce signiWcant anti-huVEGFantibody responses, whereas signiWcant anti-huVEGFantibody responses were observed in all dogs thatreceived Wve or six immunizations (see Fig. 1). In twoof three dogs with high levels of anti-huVEGF antibod-ies, signiWcant reductions in circulating plasma VEGFconcentrations were also observed (see Table 1). Cir-culating VEGF concentrations were also signiWcantlyreduced in two dogs that did not mount signiWcantanti-huVEGF antibody responses. We postulate thatthe VEGF vaccine was the most likely explanationfor the observed decreases in VEGF concentration.This is based in part on the fact that in a previousstudy we completed of 13 dogs also with soft tissuesarcoma, we did not observe any signiWcant change incirculating VEGF concentration during repeatedsampling over a 12-week period, despite the fact thatsigniWcant tumor responses were observed duringthat time [37].

Evaluation of pre- and post-treatment tumor MVDin the four dogs that received Wve or more VEGFvaccinations revealed a signiWcant decrease in MVDin one dog (dog 2, Table 1), no change in MVD intwo dogs, and a transient decrease in tumor MVD inone dog followed by an increase (dog 4, Fig. 4). Inthe one dog that had a subsequent increase in tumorMVD following the initial decrease, it is possible thatunder the selective pressure of anti-VEGF antibodies,the tumor may have upregulated production of alterna-tive pro-angiogenic growth factors, such as bFGF orIL-8 [25, 38].

We also evaluated tumor responses clinically byserial determinations of tumor volume. From analysisof these data (Table 1), it was apparent that number ofvaccinations correlated closely with clinical tumorresponses. For example, three of four dogs thatreceived Wve or more immunizations had objectiveclinical tumor responses. In two of the dogs with tumorresponses, the responses were durable (8 months and>12 months). In contrast, all Wve dogs that had threeor fewer vaccinations experienced progressive tumorgrowth. It is possible that tumor responses and thenumber of vaccinations each dog received may have

Fig. 5 Determination of tumor MVD by immunohistochemistry.Biopsies were obtained from the tumor of a dog prior to VEGFvaccination and again at week 16 after six VEGF immunizationshad been administered. The tumor MVD was assessed by immu-nostaining for CD146 expression, using a cross-reactive antibodyto human endothelial cells, as described in Sect. “Materials andmethods.” The tumor MVD was markedly decreased in the post-vaccination tumor biopsy, compared to the MVD in the pre-treat-ment tumor biopsy


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also been inXuenced by tumor grade. Dogs with low-grade (grade I or II) soft tissue sarcoma in general tendto have a better outcome (less aggressive tumors, lesslocal recurrence) than dogs with grade III tumors [29].In our study, of the four dogs that received Wve or moreimmunizations, three dogs had grade I tumors and onedog had a grade II tumor. Of the Wve dogs thatreceived fewer than three VEGF vaccinations and hadprogressive tumor growth, there were two dogs withgrade I tumors, two dogs with grade II tumors, and onedog with a grade III tumor.

It is important to note that circulating anti-VEGFantibodies were evaluated in plasma rather than inserum. When blood samples are clotted, the activatedplatelets release large quantities of VEGF into serum[39–41]. This high level of platelet-released VEGFcould then potentially react with any anti-VEGF anti-bodies present in circulation, thereby falsely loweringthe anti-VEGF antibody concentrations detected. Infact, we observed this eVect when we initially com-pared VEGF antibody results from paired serum andplasma samples from some dogs in the study (data notshown). Therefore, we used plasma, which containsrelatively low concentrations of platelet-derivedVEGF, for assessment of both antibody and circulatingVEGF concentration in this study.

One of the premises underlying use of the xeno-geneic VEGF vaccine approach is that this techniquehas the potential to break self-tolerance against endo-genous VEGF. Though some dogs in this study (i.e.,those that received more than Wve immunizations)mounted strong antibody responses against huVEGF,we did not observe substantial antibody responsesagainst caVEGF (see Figs. 1, 3), suggesting thatimmune tolerance was not eVectively broken. How-ever, it is also possible that anti-caVEGF antibodieswere in fact elicited, but were rapidly eliminated due tocomplexing with circulating caVEGF. For example, inthree of the four multiply vaccinated dogs there was asigniWcant reduction in circulating caVEGF concentra-tions (Figs. 1, 2) despite the low levels of anti-caVEGFantibodies elicited (Fig. 3). DiVerences in antibodyaYnity or speciWcity for huVEGF versus caVEGF mayalso help explain the diVerence in levels of anti-huVEGF and anti-caVEGF antibodies noted in thisstudy. For example, antibodies elicited against uniquedeterminants on huVEGF not expressed on caVEGFwould still be detected using the huVEGF ELISA, butwould not be detected using the caVEGF ELISA.Also, if higher aYnity antibodies were elicited againsthuVEGF than against caVEGF, this might be reXectedby detecting higher antibody concentrations againsthuVEGF than against caVEGF.

Our tumor response data suggest that the reductionin circulating VEGF concentrations following vaccina-tion had a functional eVect on tumor growth. Forexample, three of the four dogs that had a reductionin circulating plasma VEGF concentrations also hadobjective tumor responses (Table 1). However, we alsonoted that reductions in circulating VEGF concentra-tions, as well as overt tumor responses, occurred insome dogs before a noticeable increase in anti-VEGFantibody levels. We also observed decreased circulat-ing VEGF concentrations in one dog that did not havedetectable anti-VEGF antibodies (Table 1). TheseWndings suggest that other immunologic mechanisms,such as induction of T cell responses against tumorcells expressing high levels of VEGF, may have alsocontributed to the reduction in circulating VEGF con-centrations as well as anti-tumor activity. One also can-not exclude the possibility that a reduction in tumormass and local production of VEGF was a primarycause of the observed decreases in circulating VEGFconcentrations.

The study was also designed to determine whetherneutralization of VEGF by vaccination was a safeapproach clinically. Since dogs closely resemble humansphysiologically and immunologically, a VEGF vaccinestudy in dogs would be more likely than rodent studiesto provide a realistic assessment of safety in humans.Obvious adverse eVects were not observed in any ofthe dogs treated in this study. In particular, we did notobserve interference with normal angiogenic pro-cesses, such as wound healing. For example, somedogs underwent multiple tumor biopsies over thecourse of the study and did not develop problems withhealing of the tumor biopsy sites. In addition, obviousclotting disturbances were not noted on physicalexamination or following blood collection by veni-puncture. Thus, by clinical criteria vaccination againstxenogeneic VEGF did not appear to inhibit normalangiogenesis or clot formation, though these processeswere not evaluated by laboratory testing. It is notpossible to comment on the possible eVects of VEGFvaccination on fertility, as all of the female dogs in thisstudy were neutered.

One of the major determinants of whether or not atreatment eVect was observed in this study was howmany vaccines could be administered before progres-sive tumor growth developed. The inherent growthrate and/or biological aggressiveness of the tumor mayhave been one of the primary determinants of time totumor progression. To overcome this eVect, it might bepossible in future studies to augment or accelerateimmune responses to the VEGF vaccine. For example,immunization with larger amounts of VEGF antigen or


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by a diVerent route might have elicited more rapidantibody responses of higher magnitude. Of note, priorto this study we completed a smaller pilot study ofxenogeneic VEGF vaccination in four dogs that wereimmunized with rhVEGF165 plasmid DNA, deliveredby intramuscular injection. However, in that study onlyvery weak anti-huVEGF antibody responses wereobserved and no objective tumor responses occurred(S. W. Dow, unpublished data). Therefore, we predictthat use of conventional plasmid DNA immunizationin dogs is unlikely to oVer a signiWcant improvementover the current immunization protocol. We also didnot evaluate cellular immune responses to VEGF andtherefore do not know what role cellular immunitymight play in some of the responses observed. How-ever, we did not observe any consistent increases inmononuclear cell inWltration when pre- and post-vacci-nation tumor biopsy specimens were compared (datanot shown).

In summary, we show here that repeated vaccinationwith xenogeneic VEGF protein is well-tolerated clini-cally in dogs with cancer. Moreover, induction ofstrong anti-huVEGF antibody responses was associ-ated in most animals with a signiWcant decline in circu-lating VEGF concentrations, reduction in tumor MVD,and reduction in tumor volume. These data, generatedin an outbred species, suggest that xenogeneic vaccinationagainst pro-angiogenic growth factors has potential asa safe adjunct to standard therapies for controllingtumor growth and angiogenesis.

Acknowledgments The authors wish to acknowledge the excel-lent technical assistance provided by Julie Willer and JessicaBushanam. We are also grateful to Drs E. J. Ehrhart and ChrisOrton, and Mr Brad Charles for assistance with VEGF immuno-histochemistry. These studies were supported by a grant from theNIH (CA86224-01).

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Evaluation of a xenogeneic VEGF vaccine in dogs with soft tissue sarcoma

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