Active Immunization Against the Vascular Endothelial Growth Factor Receptor flk1 Inhibits Tumor Angiogenesis and Metastasis

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Active Immunization Against the Vascular Endothelial Growth Factor Receptor flk1 Inhibits Tumor Angiogenesis and Metastasis
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    C   orrection   •  The Journal of Experimental Medicine   Li et al. Vol. 195, No. 12, June 17, 2002. Pages 1575–1584.The authors regret that the grant number and telephone number of the corresponding address were incorrect. The corrected acknowledge-ments and corresponding address appear below.This work was supported in part by Small Business Innovation Research (SBIR) grant CA86649-01 from the National Institutes of Health to Y. Li. Address correspondence to Dr. Yiwen Li, Department of Immunology, ImClone Systems Incorporated, 180 Varick St., New York, NY10014. Phone: 646-638-5173; Fax: 212-645-2054; E-mail:   on S  e p t   em b  er 1 4  ,2  0 1 4  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published June 10, 2002   on S  e p t   em b  er 1 4  ,2  0 1 4  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published June 10, 2002   on S  e p t   em b  er 1 4  ,2  0 1 4  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published June 10, 2002   on S  e p t   em b  er 1 4  ,2  0 1 4  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published June 10, 2002   on S  e p t   em b  er 1 4  ,2  0 1 4  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published June 10, 2002   on S  e p t   em b  er 1 4  ,2  0 1 4  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published June 10, 2002   on S  e p t   em b  er 1 4  ,2  0 1 4  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published June 10, 2002   on S  e p t   em b  er 1 4  ,2  0 1 4  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published June 10, 2002   on S  e p t   em b  er 1 4  ,2  0 1 4  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published June 10, 2002   on S  e p t   em b  er 1 4  ,2  0 1 4  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published June 10, 2002   on S  e p t   em b  er 1 4  ,2  0 1 4  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published June 10, 2002   on S  e p t   em b  er 1 4  ,2  0 1 4  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published June 10, 2002    on S  e p t   em b  er 1 4  ,2  0 1 4  j   em.r  u pr  e s  s . or  gD  ownl   o a d  e d f  r  om  Published June 10, 2002    J. Exp. Med. ©  The Rockefeller University Press • 0022-1007/2002/06/1575/10 $5.00Volume 195, Number 12,June 17, 20021575–1584  1575  Active Immunization Against the Vascular Endothelial Growth Factor Receptor flk1 Inhibits Tumor Angiogenesis and Metastasis   Yiwen Li, Mei-Nai Wang, Hongli Li, Karen D. King, Rajiv Bassi, Haijun Sun, Angel Santiago, Andrea T. Hooper, Peter Bohlen, and Daniel J. Hicklin   ImClone Systems Incorporated, New York, NY 10014  Abstract   The vascular endothelial growth factor (VEGF) receptor fetal liver kinase 1 (flk1; VEGFR-2,KDR) is an endothelial cell–specific receptor tyrosine kinase that mediates physiological andpathological angiogenesis. We hypothesized that an active immunotherapy approach targetingflk1 may inhibit tumor angiogenesis and metastasis. To test this hypothesis, we first evaluatedwhether immune responses to flk1 could be elicited in mice by immunization with dendriticcells pulsed with a soluble flk1 protein (DC-flk1). This immunization generated flk1-specificneutralizing antibody and CD8      cytotoxic T cell responses, breaking tolerance to self-flk1 anti-gen. Tumor-induced angiogenesis was suppressed in immunized mice as measured in an alginatebead assay. Development of pulmonary metastases was strongly inhibited in DC-flk1–immu-nized mice challenged with B16 melanoma or Lewis lung carcinoma cells. DC-flk1 immuniza-tion also significantly prolonged the survival of mice challenged with Lewis lung tumors. Thus,an active immunization strategy that targets an angiogenesis-related antigen on endothelium caninhibit angiogenesis and may be a useful approach for treating angiogenesis-related diseases.Key words:angiogenesis • antibody • cytotoxic T lymphocytes • cancer vaccine • tumor antigen  Introduction   Tumor metastasis is the main cause for failure of conven-tional cancer therapy, necessitating the reevaluation of current cancer therapy strategies. Conventional approachesfor cancer immunotherapy usually target antigens expressedby tumor cells and are aimed at eradicating tumor cells bydirect or indirect immunological attack (1–3). Despite theidentification of tumor-associated antigens and the demon-strated effectiveness of immunotherapy in many experimen-tal animal tumor models, immunotherapy currently has lim-ited clinical utility for human cancers. The limitations of cancer immunotherapy are thought to be due in part topoor immunogenicity, immune tolerance, and escape of tu-mor cells from immune surveillance through antigen modu-lation, decreased MHC expression by tumor cells, lack of    costimulatory molecules, or secretion of immunosuppressivemolecules by tumor cells (4, 5). One possible alternative toovercome these obstacles of conventional immunotherapy isto target the blood vessels nourishing the growing tumor cells rather than the tumor cells themselves.Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is a tightly regulated process importantin fetal development and wound healing and in pathologi-cal conditions such as tumor growth and metastasis (6, 7).   Vascular endothelial growth factor (VEGF)  *   and its recep-tors fetal liver kinase 1 (flk1; murine) and KDR (human)play a critical role in regulating the process of normal andpathological angiogenesis. Targeted inactivation of the genefor VEGF or flk1 in mice results in embryonic lethality atday 7.5 due to severely impaired vascular development (8,9). The importance of VEGF and flk1/KDR in tumor an-giogenesis is exemplified in studies using a dominant-nega-tive flk1 receptor (10), neutralization of VEGF by mono-clonal antibodies (11), neutralizing flk1/KDR mAb (12,13) or flk1/KDR kinase inhibitors (14), all of which wereshown to inhibit angiogenesis and tumor growth. More-over, overexpression of VEGF and KDR is strongly associ-ated with invasion and metastasis in human malignant dis-ease (15, 16). Therefore, overexpressed flk1/KDR could bea potential target for tumor immunotherapy.   Address correspondence to Dr. Yiwen Li, Department of Immunology,ImClone Systems Incorporated, 180 Varick St., New York, NY 10014.Phone: 212-638-5173; Fax: 212-645-2054; E-mail:   *    Abbreviations used in this paper:   AP, alkaline phosphatase; DC, dendriticcell; flk1, fetal liver kinase 1; VEGF, vascular endothelial growth factor.  1576  Inhibition of Tumor Angiogenesis and Metastasis by Vaccination Against flk1   We have hypothesized that a therapeutically effectiveimmune response can be elicited that targets flk1 expressedon tumor blood vessels and thus, inhibit tumor angiogene-sis and growth. To test this hypothesis, we used a dendriticcell (DC) immunization strategy, which has been success-fully used in mice to overcome immune tolerance to other antigens (17). In this report, we demonstrate that immuni-zation with DCs pulsed with soluble flk1 induces neutral-izing antibody and CD8      cytotoxic T cell responses,suppresses tumor angiogenesis, and strongly inhibits the de-velopment of metastasis in two mouse models.  Materials and Methods   Tumor Cell Lines.   Lewis lung carcinoma cell line D122–96(H-2  b   ) was provided by Dr. L. Eisenbach (Weizman Institute of Science, Rehovot, Israel). H5V endothelial cell line (H-2  b   ),which expresses both flk1 and MHC class I, was provided by Dr.A. Vecchi (Istituto Mario Negri, Milan, Italy; reference 18).Mouse endothelial cell line bEND.3 (H-2  d   ), which expresses flk1(19), was obtained from Dr. T. Sato (University of Texas South-western Medical Center, Dallas, TX). Melanoma cell line B16(H-2  b   ), EL4 lymphoma cell line (H-2  b   ), and the NK-sensitive YAC-1 cell line were all purchased from American Type CultureCollection. The cell lines were maintained in DMEM media (In-vitrogen) containing 10% FCS (HyClone Laboratories).    Animals.   Female C57BL/6 mice, 6–8 wk of age, were pur-chased from Harlan Sprague Dawley, Inc. and housed under pathogen-free conditions.   Soluble flk1-AP Protein.   Construction of the expression plas-mid vector Aptag-flk1, which contains the cDNA correspondingto the extracellular domain of flk1 fused to alkaline phosphatase(AP), was described previously (20). Soluble flk1-AP protein waspurified using an anti-AP affinity chromatography, followed by asize exclusion chromatography. It was then subjected to SDS-PAGE, and shown as a single band with molecular weight of    180KD (      95% purity). The purified protein could be recognized by aflk1-specific monoclonal antibody, DC101 in Western blot. Thebinding of flk1-AP to VEGF was examined by an ELISA, and theprotein was found to be active. The purified protein was tested for endotoxin using the Pyrogent  ®   plus Limulus Amebocyte Lysate as-say kit (BioWhittaker). All protein preparations used in animalstudies contained    1.25 EU/ml of endotoxin.   Recombinant flk1-His Protein.   The flk1 insert from the Aptag-flk1 vector was subcloned into the plasmid pET28a vector with apolyhistidine-encoding sequence at the amino-terminal (BD Bio-sciences/CLONTECH Laboratories, Inc.). The construct wasverified by automatic sequence analysis. The recombinant flk1-His protein was expressed in Escherichia coli    , purified from inclu-sion bodies by preparative electrophoresis, and assessed for purityby SDS-PAGE and binding to VEGF by ELISA.   DC Generation.   DCs were generated from the bone marrowas described with modifications (21). Briefly, C57BL/6 micewere killed and bone marrow harvested from tibia and femurs.Bone marrow cells were depleted of existing T cells, B cells, mac-rophages, and granulocytes by incubation with a cocktail of anti-bodies including anti-CD4 (GK1.5), anti-CD8 (2.43), anti-Ia(B21–2), anti-B220 (RA3–3A1/6.1), and anti-Gr-1 (RB6–8C5/1;all from BD PharMingen), for 30 min at 4      C and then with rab-bit complement (Accurate Chemical) for additional 30 min at37      C. The remaining cells were cultured in 10% FCS supple-mented RPMI 1640 in the presence of GM-CSF (20 ng/ml) and   IL-4 (50 ng/ml; PeproTech) at 37      C, 5% CO  2   , for 9 d. Nonad-herent cells were then harvested and confirmed to be mature DCsby their morphology and phenotypic profile (CD40     , CD81     ,CD86     , Ia     , and CD14     ) on flow cytometric analysis.    Antigen Pulsing of DCs and Immunization Protocol.   DCs werepulsed with antigen, as described previously (22). Briefly, DCswere washed twice in the serum-free medium AIM V (Invitro-gen) and incubated with soluble flk1-AP protein or human AP(50    g/ml) in AIM V for 16 h. The cells were then washed twicein AIM V before use for vaccination. For immunization, micewere injected intravenously with 5    10  4   flk1-AP-pulsed DCs,AP-pulsed DCs, or PBS (200    l) per mouse at 8–10 d intervals.   CTL Culture and Assay.   Mice were immunized three timeswith DCs pulsed with flk1-AP (DC-flk1), DCs pulsed with AP(DC-AP), or PBS as described above. CTL response was assessedas described previously (23). Briefly, spleen cells were preparedfrom immunized mice (two mice per group) and restimulatedwith DCs pulsed with flk1-AP (at 100:1 effector/stimulator ratio)in a 24-well plate for 5 d in RPMI 1640 (Invitrogen) with 10%FCS. The CTL activity was tested in a 4-h 51   Cr release assayagainst a panel of target cells including flk1     H5V endothelialcells, flk1     D122–96 tumor cells, flk1-AP-pulsed DCs, AP-pulsedDCs, and NK-sensitive YAC-1 cells. The percentage cytotoxicitywas calculated using the formula: (experimental release    sponta-neous release)/(maximum release    spontaneous release)    100.   Detection of Antibody Response.   Mice were immunized threetimes with flk1-AP-pulsed DCs, AP-pulsed DCs, or PBS as de-scribed above. Blood samples were collected from mice beforeand 7 d after vaccinations. Anti-flk1 antibody in the sera was de-tected by ELISA. Briefly, a 96-well plate was incubated with 200ng/well of flk1-His protein overnight at 4      C. After three washeswith 0.1% Tween in PBS, 2% BSA was added to the plate andincubated at room temperature for 1 h. Diluted sera were thenadded to wells and incubated for 1 h. Wells were washed threetimes and then incubated with 100    l goat anti–mouse peroxi-dase for 1 h. Wells were washed three times and then incubatedwith 50    l of 3,3      , 5,5      -tetra-methylbenzidine (TMB) substrate(Kirkegaard and Perry Lab, Inc.) for 15 min. The reaction wasstopped by adding 50    l of 1 M phosphoric acid and wells read at450 nm on a microtiter plate reader. For flk1-VEGF blocking as-says, wells were coated with 100 ng of recombinant humanVEGF  165   (provided by Dr. P. Kussie, ImClone) overnight at 4      C.Wells are blocked as described above and then incubated for 1 hat room temperature with 100 ng of flk-AP that had been prein-cubated for 2 h with various concentrations of sera from immu-nized mice. Wells were washed and incubated with p-nitrophe-nyl phosphate (PNPP; Sigma-Aldrich). Color was developed for 30 min at room temperature and was then read at 405 nm on amicrotiter plate reader.   Cell-based Ligand-binding Competition Experiments.   Cell-basedligand-binding assays were performed as described with modifica-tions (24). Flk1-expressing bEND.3 cells (10  5   per well) weregrown in 24-well plates in DMEM 10% FCS for 48 h. Cells werewashed three times with binding buffer (DMEM with 0.1%BSA). Pooled immune sera from DC-flk1–immunized mice or from DC-AP–immunized mice and unlabeled recombinant hu-man VEGF  165   were serially diluted in binding buffer as indicated.In a total volume of 400    l/well, cells were incubated with thesamples for 4 h at 4      C. Cells were then washed with bindingbuffer and incubated with 10 nCi [  125   I]VEGF (Amersham Phar-macia Biotech) in 400    l binding buffer per well for 1 h. After incubation, cells were washed using cold PBS with 0.1% BSA.Cells were harvested by adding 200    l of 0.5 M NaOH. The ra-  1577  Li et al.  dioactivity bound to the cells was determined in a Wizard 1470gamma counter (PerkinElmer).    Alginate In Vivo Angiogenesis Assay.   An alginate bead assaywas designed to measure in vivo angiogenesis induced by tumor cells (25). Lewis lung tumor cells were suspended in a 1.5% solu-tion of sodium alginate and added drop by drop into a swirling37      C solution of 250 mM calcium chloride. Alginate beads wereformed containing    5    10  4   tumor cells per bead. Mice wereanesthetized and four beads implanted subcutaneously through anincision made on the dorsal side. Incisions were closed with sur-gical clips. After 12 d, mice were injected intravenously with 100      l of FITC-dextran solution (20 mg/ml). Animals were killed af-ter 20 min, beads removed, and incubated overnight at roomtemperature in 1 ml of buffer (1 mM Tris-HCl, pH 8). The beadswere ground briefly with a hand-held mixer and an additional 1ml of buffer was added. Samples were then vortexed and centri-fuged at 1,500 rpm for 5 min. Fluorescence of the sample super-natants was quantitated against a standard curve of FITC-dextran.   B16 Metastasis Model.   Mice were immunized three timeswith flk1-AP–pulsed DCs, AP-pulsed DCs, or PBS as describedabove. 10 d after the last immunization, mice were injected intra-venously with 10  6   B16 cells. Mice were killed based on the met-astatic death in the control groups. Tumor load was assessed bycounting the tumor nodules on the lung surface.   Lewis Lung Metastasis Model.   Mice were immunized threetimes with flk1-AP–pulsed DCs, AP-pulsed DCs, or PBS as de-scribed above. 10 d after the last immunization, mice were chal-lenged with an intrafootpad injection with 2    10  5   of D122–96tumor cells. When tumors reached    5 mm in diameter, the tu-mor-bearing leg was surgically removed. Mice were killed basedon the metastatic death in the control groups. Tumor load wasassessed by counting the tumor nodules on the lung surface. Inseparate experiments, mice were monitored daily for survival.   In Vivo T Cell Depletion Experiment.   1 d before DC-flk1 im-munization, mice received intraperitoneal injection of 0.5 mg of either anti-CD4 (GK1.5), or anti-CD8 (clone 116), or control ratIgG (Jackson ImmunoResearch Laboratories). To ensure com-plete depletion of respective T cell population, one mouse fromeach group was killed the next day and splenocytes were analyzedby FACS  ®   after staining with FITC-conjugated anti-CD4 (L3T4)and PE-conjugated anti-CD8 (Ly2; BD PharMingen). The samedepletion procedure was repeated every 2 wk to prevent recoveryof depleted T cell populations. The mice were immunized threetimes with DCs pulsed with flk1-AP and then challenged withLewis lung tumor as described in the Lewis lung metastasis model.Tumor load in the lungs were compared among the groups.   Mouse Pregnancy Experiment.   To test whether flk1 immuniza-tion could affect mouse pregnancy by interfering with prenatalangiogenic process, female mice were immunized with DCspulsed with flk1-AP or PBS as described above. In an additionalcontrol, another group of mice were treated with intraperitonealinjections of the anti-flk1 antibody DC101 (800    g per injection,twice weekly for 30 d; reference 12). Mice were then mated withmales and monitored daily for signs of pregnancy. Number of pups of each delivery was recorded. The pups were also carefullyexamined for signs of sickness and abnormality.   Wound Healing Experiment.   10 d after the last immunizationwith flk1-AP–pulsed DCs, a full thickness wound, including thepanniculus carnous, was excised from the dorsum of each mouse.A 1.60-cm  2   circular defect was outlined 2.0 cm from the nape of the animal’s neck using a fine-tipped marking pen. The defectwas created by elevating the skin and panniculus carnosus in thecenter of the outlined defect using forceps, followed by excisionof the outlined area using microdissecting scissors. Wound areawas measured twice weekly. 15 d after this excision, mice werekilled, and scar tissues were removed for histological examination.   Histology.   Lung samples and scar tissues were fixed overnightin 10% zinc formalin at 4      C, embedded in paraffin, and sectionedat 5    m onto saline-coated slides. Hematoxylin and eosin stain- Figure 1. Flk1-specific neutralizing antibody induced by vaccinationwith DCs pulsed with soluble flk1 protein. (A) Mice were immunizedthree times with flk1-AP-pulsed DCs (DC-flk1), AP-pulsed DCs (DC-AP),or PBS. Postimmunization sera (1:100 dilution) were analyzed for anti-flk1 antibody using ELISA in plates coated with flk1-His protein. Miceimmunized with DC-flk1 exhibited significantly higher levels of anti-flk1antibody compared with control groups. (B) Serially diluted sera fromimmunized mice were coincubated with soluble flk1-AP protein and thentested for binding to VEGF by ELISA. Results indicated that sera fromDC-flk1–immunized mice blocked binding of VEGF to soluble flk1 re-ceptor. (C) Serially diluted sera from immunized mice were incubatedwith flk1-expressing bEND.3 cells at 4  C for 4 h in a total volume of 0.4ml. In parallel, the cells were incubated with a decreasing amount of unla-beled VEGF (serially diluted from a 400 ng/ml solution). After incubation,cells were washed and incubated with 10 nCi [ 125 I]VEGF for 1 h at roomtemperature. The cells were washed and counted in a   counter. Resultssuggested that sera from DC-flk1–immunized mice blocked binding of VEGF to flk1 expressed at the surface of endothelial cells.
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