hybridoma , huvec and b16f10 melanoma cells were cultured as described previously.8 antibody generation was obtained by hybridoma technology6 and vegfbinding antibody detection by elisa.9 detailed procedures are described in data s1 . to generate an antibevacizumab idiotype antibody , four balb / c mice were immunized with klhconjugated bevacizumab . screening was based on the inhibition of biotinylated bevacizumab binding to hvegf in the presence of immune serum . after limiting dilution cloning , stable antibevacizumab idiotypesecreting hybridoma clone 10.d7 was selected and the secreted antibody was determined to be an igg1 isotype . we used the b1610 tumor model to examine the therapeutic potential of our proposed antiid mab vaccination . mice were challenged with b16f10 cells on the 10th day after boost immunization , according to the experimental schedule shown in figure  1a . vaccination with 10.d7 antiid mab resulted in significantly reduced tumor growth , compared to the adjuvant and isotype immunized control groups ( p  <  0.05 ; oneway anova ) . mice were immunized with 10.d7 antiid monoclonal antibody ( mab ) ( or controls ) . ( c ) elisa detection of vegfbinding antibodies in 1:100diluted serum samples from mice 10  days after immunization boost . adjuvant control , n  =  6 ; isotype control mabimmunized , n  =  8 ; 10.d7 antiid mabimmunized group , n  =  8 . * p  <  0.05 ; oneway anova / bonferroni 's posttest . the presence of detectable levels of hvegfbinding antibodies in serum samples from 10.d7 antiid mabimmunized mice immediately before b16f10 cell injection was verified by elisa ( fig . it is important to observe here that such immune sera recognize not only hvegf but also the murine form of this factor . bevacizumab , in comparison , could bind only to hvegf , as expected ( fig . serum from 10.d7 antiidiotype ( antiid ) antibodyimmunized mice recognizes murine and human vascular endothelial growth factor ( vegf ) . histological analyses of hestained sections revealed an increase of necrotic area in tumors from mice immunized with 10.d7 antiid mab : the mean percentage of tumor necrotic area was approximately fivefold higher than that observed in the control groups ( p  <  0.05 ; oneway anova ) . this result was accompanied by a decreased cd31positive vascular network in subcutaneous tumors excised from the 10.d7 antiid mabimmunized group , compared to controls ( p  <  0.05 ; oneway anova ; fig . in addition , a reduced number of cd68positive cells were observed in tumors from 10.d7 mabimmunized mice ( p  <  0.05 , oneway anova ) ( fig . 10.d7 antiidiotype ( antiid ) monoclonal antibody ( mab ) vaccination increases necrosis and reduces cd31positive vessel area and cd68positive cells in a murine tumor model . ( a ) representative hestained b16f10 tumor sections from the group immunized with 10.d7 antiid mab ( or controls ) . adjuvant control , n  =  6 ; isotypic control mabimmunized , n  =  7 ; 10.d7 antiid mabimmunized group , n  =  5 . adjuvant control , n  =  5 ; isotypic control mabimmunized , n  =  7 ; 10.d7 antiid mabimmunized group , n  =  7 . adjuvant control , n  =  5 ; isotypic control mabimmunized , n  =  7 ; 10.d7 antiid mabimmunized group , n  =  7 . * p  <  0.05 ; oneway anova / bonferroni 's posttest , related to controls . furthermore , such angiogenic inhibition was also observed by in  vitro tubulogenesis assay . as shown in figure  4 , huvec incubation with sera obtained after 10.d7 antiid mab immunization gave a reduced and poorly organized capillarylike plexus , compared with the serum controls ( p  <  0.05 ; oneway anova ) . representative images obtained after huvec 6h incubation with 1:100diluted pool sera from 10.d7 antiid mabimmunized mice ( or controls ) . to generate an antibevacizumab idiotype antibody , four balb / c mice were immunized with klhconjugated bevacizumab . screening was based on the inhibition of biotinylated bevacizumab binding to hvegf in the presence of immune serum . after limiting dilution cloning , stable antibevacizumab idiotypesecreting hybridoma clone 10.d7 was selected and the secreted antibody was determined to be an igg1 isotype . we used the b1610 tumor model to examine the therapeutic potential of our proposed antiid mab vaccination . mice were challenged with b16f10 cells on the 10th day after boost immunization , according to the experimental schedule shown in figure  1a . vaccination with 10.d7 antiid mab resulted in significantly reduced tumor growth , compared to the adjuvant and isotype immunized control groups ( p  <  0.05 ; oneway anova ) . mice were immunized with 10.d7 antiid monoclonal antibody ( mab ) ( or controls ) . ( c ) elisa detection of vegfbinding antibodies in 1:100diluted serum samples from mice 10  days after immunization boost . ; isotype control mabimmunized , n  =  8 ; 10.d7 antiid mabimmunized group , n  =  8 . * p  <  0.05 ; oneway anova / bonferroni 's posttest . the presence of detectable levels of hvegfbinding antibodies in serum samples from 10.d7 antiid mabimmunized mice immediately before b16f10 cell injection was verified by elisa ( fig . it is important to observe here that such immune sera recognize not only hvegf but also the murine form of this factor . bevacizumab , in comparison , could bind only to hvegf , as expected ( fig . serum from 10.d7 antiidiotype ( antiid ) antibodyimmunized mice recognizes murine and human vascular endothelial growth factor ( vegf ) . histological analyses of hestained sections revealed an increase of necrotic area in tumors from mice immunized with 10.d7 antiid mab : the mean percentage of tumor necrotic area was approximately fivefold higher than that observed in the control groups ( p  <  0.05 ; oneway anova ) . this result was accompanied by a decreased cd31positive vascular network in subcutaneous tumors excised from the 10.d7 antiid mabimmunized group , compared to controls ( p  <  0.05 ; oneway anova ; fig . in addition , a reduced number of cd68positive cells were observed in tumors from 10.d7 mabimmunized mice ( p  <  0.05 , oneway anova ) ( fig . 10.d7 antiidiotype ( antiid ) monoclonal antibody ( mab ) vaccination increases necrosis and reduces cd31positive vessel area and cd68positive cells in a murine tumor model . ( a ) representative hestained b16f10 tumor sections from the group immunized with 10.d7 antiid mab ( or controls ) . adjuvant control , n  =  6 ; isotypic control mabimmunized , n  =  7 ; 10.d7 antiid mabimmunized group , n  =  5 . adjuvant control , n  =  5 ; isotypic control mabimmunized , n  =  7 ; 10.d7 antiid mabimmunized group , n  =  7 . adjuvant control , n  =  5 ; isotypic control mabimmunized , n  =  7 ; 10.d7 antiid mabimmunized group , n  =  7 . * p  <  0.05 ; oneway anova / bonferroni 's posttest , related to controls . furthermore , such angiogenic inhibition was also observed by in  vitro tubulogenesis assay . as shown in figure  4 , huvec incubation with sera obtained after 10.d7 antiid mab immunization gave a reduced and poorly organized capillarylike plexus , compared with the serum controls ( p  <  0.05 ; oneway anova ) . representative images obtained after huvec 6h incubation with 1:100diluted pool sera from 10.d7 antiid mabimmunized mice ( or controls ) . the use of antiid antibodies acting as an internal image of tumorexpressing antigens on vaccination settings offers the opportunity to overcome tolerance to autoantigens10 and to control tumor progression,11 , 12 with some studies already in clinical development ( table  s1 ) . herein , we show the effectiveness of an antibevacizumab idiotype antibody in generating an active vegfbinding immune response . as far as we know , this is the first time that an antiid approach has been used to inhibit angiogenesis . the b16f10 tumor model was chosen to investigate the therapeutic viability of our antibevacizumab idiotype mab vaccination approach . despite clinical studies pointing to the nonresponsiveness of melanomas to vegftargeting therapies,13 , 14 the previous reported impairments of b16f10 solid tumor growth and vascular density by antivegf antibodies15 the effectiveness of the studied antiid mab vaccination was revealed by the detection of serum vegfbinding antibodies , in b16f10challenged animals , leading to tumors with reduced size and higher necrotic areas , compared to those obtained in the control groups . the antiangiogenic activity obtained by antiid vaccination was evidenced by the significantly lower cd31positive tumor vascular area , as well as by the hampered in  vitro formation of tubelike structures of huvec , known to secrete vegf.17 these results are in agreement with previous reports stating that vegf inhibition , mainly in the early stages of tumor growth , impairs tumor angiogenesis and its further progression.16 , 18 
  it is already known that even though bevacizumab seems to interact with degraded products of murine vegf,19 it fails in inhibiting mvegfinduced activity and in displaying any effect on murine tumor growth.20 despite this , sera from mice immunized with 10.d7 antibevacizumab idiotype antibody recognize both murine and human vegf , which makes it reasonable to assume that the in  vivo data presented here might be , at least in part , the result of mvegf immunoneutralization by vegfbinding antibody serum content . given that vegf acts in several processes in the tumor microenvironment , mechanisms other than impairing tumor angiogenesis might be behind our therapeutic findings . of note , it is known that vegf blocking strategies led to reduced recruitment and retention of circulating myeloid cells , like monocytes , to the perivascular area,21 as well as to enhanced activation of dendritic cells.22 in fact , analyses of tumors from 10.d7 mabimmunized mice pointed in the same direction . in addition , fcmediated mechanisms , such as complement or antibody dependent cellmediated cytotoxicities , might contribute to the reduced tumor growth observed . overall , mice immunization with an antibevacizumab idiotype antibody was effective in eliciting vegfbinding humoral igg antibodies capable of impairing vascular density and tumor growth . the proposed vaccination should be considered as an antiangiogenic therapeutic strategy for controlling the progression of vegfdependent tumors , including postsurgical resection relapse scenarios , where vegf plays an important role.23 , 24 status of the clinical development of antiidiotype antibodies for cancer therapy . click here for additional data file .
tumors require blood supply and , to overcome this restriction , induce angiogenesis . 
 vascular endothelial growth factor ( vegf ) plays an important role in this process , which explains the great number of antiangiogenic therapies targeting vegf . 
 the research and development of targeted therapy has led to the approval of bevacizumab , a humanized antivegf monoclonal antibody ( mab ) , in clinical settings . however , side effects have been reported , usually as a consequence of bolusdose administration of the antibody . 
 this limitation could be circumvented through the use of antiidiotype ( i d ) antibodies . in the present study 
 , we evaluated the efficacy of an active vegfbinding immune response generated by an antibevacizumab idiotype mab , 10.d7 . 
 the 10.d7 antiid mab vaccination led to detectable levels of vegfbinding antiantiid antibodies . in order to examine whether this humoral immune response could have implications for tumor development , 
 10.d7immunized mice were challenged with b16f10 tumor cells . 
 mice immunized with 10.d7 antiid mab revealed reduced tumor growth when compared to control groups . 
 histological analyses of tumor sections from 10.d7immunized mice showed increased necrotic areas , decreased cd31positive vascular density and reduced cd68positive cell infiltration . 
 our results encourage further therapeutic studies , particularly if one considers that the antiid therapeutic vaccination maintains stable levels of vegfbinding antibodies , which might be useful in the control of tumor relapse .