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Nature Medicine 8, 1369 - 1375 (2002)
Published online: 4 November 2002; | doi:10.1038/nm1202-794
A DNA vaccine against VEGF receptor 2 prevents effective angiogenesis and inhibits tumor growth
Andreas G. Niethammer1, Rong Xiang1, Jürgen C. Becker3, Harald Wodrich2, Ursula Pertl1, Gabriele Karsten1, Brian P. Eliceiri4 & Ralph A. Reisfeld1
1 Department of Immunology, Scripps Research Institute, La Jolla, California, USA

2 Department of Cell Biology, Scripps Research Institute, La Jolla, California, USA

3 Universitaets Hautklinik, Wuerzburg, Germany

4 La Jolla Institute for Molecular Medicine, San Diego, California, USA

Correspondence should be addressed to Ralph A. Reisfeld reisfeld@scripps.edu

Tumor cells are elusive targets for immunotherapy due to their heterogeneity and genetic instability. Here we describe a novel, oral DNA vaccine that targets stable, proliferating endothelial cells in the tumor vasculature rather than tumor cells. Targeting occurs through upregulated vascular-endothelial growth factor receptor 2 (FLK-1) of proliferating endothelial cells in the tumor vasculature. This vaccine effectively protected mice from lethal challenges with melanoma, colon carcinoma and lung carcinoma cells and reduced growth of established metastases in a therapeutic setting. CTL-mediated killing of endothelial cells indicated breaking of peripheral immune tolerance against this self antigen, resulting in markedly reduced dissemination of spontaneous and experimental pulmonary metastases. Angiogenesis in the tumor vasculature was suppressed without impairment of fertility, neuromuscular performance or hematopoiesis, albeit with a slight delay in wound healing. Our strategy circumvents problems in targeting of genetically unstable tumor cells. This approach may provide a new strategy for the rational design of cancer therapies.

The inhibition of tumor growth by attacking the tumor's vascular supply offers a primary target for anti-angiogenic intervention. This approach, pioneered by Folkman and colleagues1, 2, 3, 4, 5, is attractive for several reasons. First, the inhibition of tumor-associated angiogenesis is a physiological host mechanism and should not lead to the development of resistance. Second, each tumor capillary has the potential to supply hundreds of tumor cells, so that targeting the tumor vasculature actually potentiates the antitumor effect. Third, direct contact of the vasculature with the circulation leads to efficient access of therapeutic agents6.

Extensive studies by many investigators established that angiogenesis has a central role in the invasion, growth and metastasis of solid tumors2, 7, 8, 9. In fact, angiogenesis is a rate-limiting step in the development of tumors since tumor growth is generally limited to 1−2 mm3 in the absence of a blood supply6, 10. Beyond this minimum size, tumors often become necrotic and apoptotic under such circumstances11.

Because tumor cells frequently mutate in response to therapy and also downregulate major histocompatibility (MHC) antigens required for T cell−mediated antitumor responses12, 13, efforts have been made to eradicate tumors by therapies directed against the tumor microenvironment. One such report links calreticulin with a model viral tumor antigen, thus combining antitumor therapy with anti-angiogenesis14. Yet another approach is the administration of xenogeneic endothelial cells as a vaccine that yielded anti-angiogenic effects15. This approach differs from those of other investigators applying specific chemical or biological inhibitors of angiogenesis, which often require their constant administration at relatively high dose levels16.

A more molecularly-defined alternative to xenoimmunization is offered by receptor tyrosine kinases (RTKs) and their growth-factor ligands required for tumor growth. Among these receptors, the vascular endothelial growth factor receptor 2 (VEGFR2, also known as FLK-1) that binds the five isomers of murine VEGF has a more restricted expression on endothelial cells and is upregulated once these cells proliferate during angiogenesis in the tumor vasculature. FLK-1 is strongly implicated as a therapeutic target, as it is necessary for tumor angiogenesis and has an important role in tumor growth, invasion and metastasis7, 8, 17, 18, 19, 20, 21, 22, 23, 24. In fact, several approaches have been used to block FLK-1, including dominant-negative receptor mutants, germ-line disruption of VEGFR genes, monoclonal antibodies against VEGF and a series of synthetic RTK inhibitors24, 25.

Here we describe a novel strategy for achieving an antitumor immune response with a FLK-1-based DNA vaccine. Our vaccine causes the collapse of tumor vessels by evoking a T cell−mediated immune response against proliferating endothelial cells overexpressing this growth-factor receptor in the tumor vasculature.

A FLK-1 based DNA vaccine inhibits tumor growth
We tested our hypothesis by demonstrating that an effective antitumor immune response was induced against subcutaneous tumors by an orally administered DNA vaccine encoding murine FLK-1 carried by attenuated Salmonella typhimurium. To this end, we constructed the vector pcDNA3.1-FLK1 (Fig. 1a). Protein expression of FLK-1 was demonstrated by western blotting of transfected COS-7 cells (Fig. 1b). We established the efficacy of gene transfer from attenuated S. typhimurium into Peyer's patches by GFP expression in the cells derived from Peyer's patches at different time points after oral administration of mice (data not shown).

Figure 1. Construction and functionality of expression vector.

a, The DNA encoding the entire murine Flk-1 gene was inserted into the pcDNA3.1 vector between the restriction sites KpnI (5') and XbaI (3') b, This construct was verified by nucleotide sequencing and protein expression by western blots after transient transfection into COS-7 cells. The protein appears in the lysate in its glycosylated form at 220 kD and to a lesser extent in its unglycosylated form at approximately 150 kD.

Full Figure and legend (53K)

Marked inhibition of subcutaneous (s.c.) tumor growth was observed in C57BL/6J mice challenged two weeks after the third vaccination with pcDNA3.1-FLK1 by s.c. injection of either B16G3.26 murine melanoma cells or D121 non−small cell Lewis lung carcinoma cells (Figs. 2a and b). In contrast, animals vaccinated with only the empty vector pcDNA3.1, carried by the attenuated bacteria, revealed uniformly rapid s.c. tumor growth.

Figure 2. Effect of the FLK-1 based DNA vaccine on tumor growth.

a, 2 wk after the last vaccination, mice were challenged (c). with a lethal dose of B16 melanoma cells. The average tumor growth of 8 mice is depicted. , immunization () with the vector encoding FLK-1; , control vector; , PBS. P 50% = 3. We injected CT-26 murine colon carcinoma cells (5 104) i.v. into BALB/c mice inducing experimental pulmonary metastases 2 wk after the last immunization. We tested our treatment in a therapeutic setting by vaccinating animals 10 d after i.v. injection of CT-26 cells.

Activation of CD8+ T cells.
We created the B16G3.26-FLK-1 melanoma and CT-26-FLK-1 colon carcinoma cell lines by retroviral transduction with FLK-1. One week after immunization, splenocytes were collected from C57BL/6J mice (n = 4), vaccinated with pcDNA3.1-FLK1 or the empty control vector. Cells were cocultured overnight with B16G3.26-FLK-1 or B16G3.26 tumor cells. Flow-cytometric analyses were performed using FITC-conjugated antibody to CD8 (#01044) in combination with PE-conjugated anti-mouse monoclonal antibodies to CD2 (#01175), CD25 (#01105A) or CD69 (#01505B) (BD-Pharmingen, La Jolla, California) as described24. We also used splenocytes in a standard 4-h 51Cr-release assay to asses cytotoxicity against CT-26-FLK-1 and CT-26 target cells.

Evaluation of anti-angiogenic effects.
C57BL/6J mice (n = 8) were injected into the sternal region with 250 l growth factor−reduced Matrigel (#354230, BD Biosciences, Bedford, Massachusetts) containing 400 ng/ml murine VEGF (#450-32, PeproTech, Rocky Hill, New Jersey) or bFGF (#100-18B). Endothelium was stained 6 d later by i.v. injection of 200 l (0.1 mg/ml) Bandiera simplofica lectin I, Isolectin B4 conjugated with fluorescein (Vector Labaratories, Burlingame, California). 30 min later, mice were killed and lectin-FITC was extracted from 100 g per plug in 500 l RIPA lysis buffer, centrifuged and its content in the supernatant quantified by fluorimetry (490 nm).

In vivo depletion of CD8+ T cells.
We depleted CD8+ T cells by weekly i.p. injections of 500 g rat anti-mouse monoclonal antibody to CD8 (RH.495) as described32. Controls included non-depleted animals either vaccinated with pcDNA3.1-FLK1 or pcDNA3.1.

Immunohistochemistry.
We stained cryosections (10 m) fixed in paraformaldehyde. Antibody to CD31 (Pharmingen, San Diego, California) was incubated with rhodamine-conjugated secondary antibody, blocked with rat serum, followed by immunostaining with a FITC−conjugated antibody to CD8. Photomicrographs were captured with a laser scanning confocal microscope (Biorad, Hercules, California). Frozen tissue sections were stained with the Techmate Automate (Dako, Hamburg, Germany). Single stained serial sections were incubated for 30 min with biotinylated antibodies, followed by the streptavidin-peroxidase complex (DAKO) and the chromogen AEC (DAKO). Double stainings were performed as described33. Density of antigen-expressing cells was determined by counting of high-power fields.

Evaluation of possible side effects.
To test wound healing, wounding was performed as described34, 35. We inflicted 4 circular wounds of 3-mm diameter each on the upper back of C57BL/6J mice (n = 6), 2 wk after immunization with pcDNA3.1-FLK1 or the empty vector. Time until wound closure was noted. To evaluate fertility, 2 wk after the third immunization with either pcDNA3.1-FLK1 or with empty vector, female C57BL/6J mice (n = 9) were allowed to cohabitate with 3 males. The days until parturition and number of pups were noted. To test neuromuscular performance, we evaluated vaccinated and control mice by both the wire hang test and the footprint test36, 37 as well as by overall behavior and determination of body weight. To test hematopoiesis, animals were subjected to complete peripheral blood counts and differentials up to 10 mo after immunization.

Statistical analysis.
The statistical significance of differential findings between experimental groups and controls was determined by Student's t-test and considered significant if two-tailed P values were <0.05.

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Received 13 August 2002; Accepted 8 October 2002; Published online: 4 November 2002.

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Michael S. O'Reilly's comments - ll_626 (8266 bytes) 2006-4-19, 07:29:54
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