Integrin ␣v␤3 targeted therapy for Kaposi’s sarcoma
with an in vitro evolved antibody1
CHRISTOPH RADER,2 MIKHAIL POPKOV, JOHN A. NEVES, AND CARLOS F. BARBAS III2
Department of Molecular Biology and The Skaggs Institute for Chemical Biology,
The Scripps Research Institute, La Jolla, California, USA
SPECIFIC AIMS
The goal of this study was to evaluate integrin ␣v␤3 as a
molecular target for antibody therapy for Kaposi’s
sarcoma (KS). We used in vitro evolution based on
phage display to generate a humanized and affinitymatured antibody directed to integrin ␣v␤3 to evaluate
tumor targeting and tumor growth inhibition in a
mouse model of KS.
PRINCIPAL FINDINGS
1. In vitro evolution of antibody JC-7U directed to
integrin ␣v␤3
Using a phage display strategy that preserves only the
original CDR3 sequences of light and heavy chain while
subjecting the remaining sequence to selection from
naive human V gene libraries, we previously reported
the full humanization of mouse monoclonal antibody
(mAb) LM609 directed to human integrin ␣v␤3. Here
we further improved the affinity of humanized LM609
using a phage display strategy for sequential and parallel optimization of three complementarity determining
regions (LCDR1, HCDR1, and HCDR3) of the antibody
molecule. The evolved Fab, designated JC-7U, had an
affinity of 150 pM and was converted into IgG1 using a
new mammalian expression vector (Fig. 1).
2. Tumor targeting of antibody JC-7U in a mouse
model of Kaposi’s sarcoma
A previously described nude mouse model of KS based
on human KS cell line SLK was used for our studies. As
an isotype control for JC-7U IgG1, we used human b12
IgG1, which binds to gp120 of HIV-1. Flow cytometry
revealed strong binding of JC-7U IgG1 to SLK cells,
whereas no binding was detected for b12 IgG1. For in
vivo studies, tumor induction was performed by subcutaneous injection of 5 ⫻ 106 SLK cells in the right flank
of nude mice. We first analyzed tumor targeting by
injecting 100 ␮g of JC-7U IgG1 or b12 IgG1 into the tail
vein of mice with an established SLK xenograft. After
24 h, tumor, lung, liver, and spleen were removed and
processed for the detection of bound human IgG1 by
immunohistochemistry. As shown in Fig. 2, JC-7U IgG1
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was found to have intensely penetrated the tumor tissue
whereas control antibody b12 IgG1 revealed only slight
nonspecific uptake. By contrast, neither JC-7U IgG1
nor b12 IgG1 were found in normal tissue.
3. Antibody JC-7U inhibits tumor growth in a mouse
model of Kaposi’s sarcoma
With the selective tumor targeting of JC-7U IgG1 established, we next analyzed whether JC-7U IgG1 can
inhibit tumor growth. Three different groups of five
animals each were treated on days 1, 4, 7, 10, and 13
after tumor induction. Each treatment involved a 200
␮l tail vein injection of 1) 0.5 mg/mL JC-7U IgG1 in
PBS, 2) 0.5 mg/mL b12 IgG1 in PBS, or 3) PBS alone.
Thus, animals in the antibody groups received five
doses of 100 ␮g or 5 mg/kg. For comparison, clinical
studies with Vitaxin, another humanized derivative of
LM609, have used doses up to 4 mg/kg with little or no
toxicity, which suggests that our dosing regimen is in a
clinically relevant range. Study of animals treated with
JC-7U IgG1 revealed no obvious signs of toxicity for this
protein, as indicated by no weight loss or behavioral
change during the course of therapy. Tumor volumes
of treated animals were measured every third day
starting on day 9 and ending on day 54 (Fig. 3). Tumor
growth in animals treated with PBS or b12 IgG1 revealed no difference compared with tumor growth in
untreated animals, resulting in an average tumor volume of 650 and 685 mm3, respectively, tumor growth in
animals treated with JC-7U IgG1 was notably slower
(Fig. 3). The difference in tumor growth was statistically
significant (P⬍0.05) from day 27 until the end of the
experiment 27 days later. On day 54, average tumor
volume in the animals treated with JC-7U IgG1 was 292
mm3, which is ⬃45% of the average tumor volume
observed in the control groups. As an additional measurement, all tumors were surgically removed on day 54
and weighed. The average tumor weight in the three
treatment groups confirmed the results obtained for
1
To read the full text of this article, go to http://www.
fasebj.org/cgi/doi/10.1096/fj.02– 0281fje; to cite this article,
use FASEB J. (October 18, 2002) 10.1096/fj.02– 0281fje
2
Correspondence: Department of Molecular Biology,
BCC-526, The Scripps Research Institute, 10550 North
Torrey Pines Road, La Jolla, CA 92037, USA. E-mail:
crader@scripps.edu and carlos@scripps.edu
0892-6638/02/0016-2000 © FASEB
Figure 1. Tumor growth inhibition in a nude mouse model of
KS in the presence of JC-7U IgG1. JC-7U IgG1 (black) or
control antibody b12 IgG (white circles) was given in five
doses of 5 mg/kg every third day starting on day 1 after tumor
induction. Statistically significant differences in tumor volume were observed 14 days after the last treatment until the
end of the experiment. Shown are average tumor volumes ⫾
sd (n⫽5 for both groups).
the measurement of the tumor volume. We did not
observe growth inhibition in vitro at or beyond the
administered in vivo concentration of JC-7U IgG1,
Figure 3. Tumor targeting of JC-7U IgG1. JC-7U IgG1 (A, E)
and control antibody b12 IgG1 (B, F ) were injected into the
tail vein of nude mice with an established human KS tumor.
After 24 h, tumor (A, B) and lung tissue (E, F ) were removed
and processed for the detection of bound human IgG using
goat anti-human IgG secondary antibodies. C, D) Mouse
endothelial cells that infiltrate the human KS tumor were
detected with a rat anti-mouse CD31 mAb, followed by rabbit
anti-rat IgG secondary antibodies. Note that the sites of CD31
expression and JC-7U IgG1 tumor penetration overlap. Scale
bar ⫽ 50 ␮m for panels A–F.
suggesting that antibody-dependent cellular cytotoxicity (ADCC) is the main mechanism of tumor growth
inhibition of human JC-7U IgG1 in our nude mouse
model of KS. Like its parental antibody, mouse mAb
LM609, JC-7U binds to human but not mouse integrin
␣v␤3. In the SLK xenograft, JC-7U recognizes human
integrin ␣v␤3 expressed by tumor cells but not mouse
integrin ␣v␤3 expressed by angiogenic endothelial cells.
Thus, the reduction of KS tumor growth in the presence of JC-7U is not an antiangiogenic effect but can be
attributed to a specific, direct response against the
tumor, which allows us to dissect out the antitumor
effect of this antibody therapy.
4. Antibody JC-7U partially blocks the binding of
HIV-1 Tat protein to integrin ␣v␤3
Figure 2. Schematic diagram illustrating the in vitro evolution
of antibody JC-7U. Because of its high affinity and its high
degree of humanization, JC-7U IgG1 is an excellent drug
candidate for therapeutic applications that involve integrin
␣v␤3 as the molecular target. Of particular interest is therapy
for KS, breast cancer, melanoma, and other cancers in which
integrin ␣v␤3 is expressed on both angiogenic endothelial
cells and tumor cells, a dual antiangiogenic and antitumor
strike with a single drug. Mouse sequences are shown in
yellow, human sequences in blue, optimized sequences in
red.
INTEGRIN ␣V␤3 TARGETED THERAPY OF KAPOSI’S SARCOMA
Based on published evidence that the HIV-1 Tat protein can act as a growth factor for KS by binding to RGD
receptors, including integrin ␣v␤3, that are expressed
on the surface of KS cells, we investigated whether
JC-7U can interfere with this interaction. We established a binding assay in which a mixture of human
integrin ␣v␤3 and synthetic HIV-1 Tat-biotin was incubated in solution in the presence or absence of JC-7U
Fab. Complexes of integrin ␣v␤3 and Tat-biotin were
then captured with immobilized streptavidin and were
detected by using the anti-␤3 mouse mAb AP-3, which
does not interfere with JC-7U binding, followed by goat
anti-mouse secondary antibodies. Background signals
were determined by running the same assay in the
absence of Tat-biotin. In the absence of JC-7U, a strong
signal above background was observed, which revealed
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an interaction of integrin ␣v␤3 and Tat-biotin. In the
presence of a threefold molar excess of JC-7U Fab over
Tat-biotin or at equimolar concentrations, the signal
was reduced to ⬃45%. The same reduction was observed in the presence of JC-7U IgG1. Thus, JC-7U
partially blocks the binding of HIV-1 Tat protein to
human integrin ␣v␤3.
CONCLUSIONS AND SIGNIFICANCE
In view of the fact that KS tumors are of endothelial
origin and characterized by intense angiogenesis, it is
logical that many of the recently developed antiangiogenic drugs are being studied for the treatment of KS.
With the emerging picture of KS pathogenesis and
angiogenesis, molecular targets are being revealed that
may provide effective targeting of drugs to both the KS
vasculature and the KS tumor itself. We hypothesized
that integrin ␣v␤3 might fulfill these criteria. Integrin
␣v␤3 is up-regulated on endothelial cells in response to
angiogenic growth factors and has been established as a
target for antiangiogenic therapy. Soluble antagonists
of integrin ␣v␤3 such as mAb LM609, RGD peptides, or
peptidomimetics, initiate endothelial cell apoptosis and
thereby inhibit angiogenesis.
In addition to its expression on the surface of angiogenic endothelial cells, integrin ␣v␤3 is expressed on
the surface of tumor cells in a variety of cancers. In
2002
Vol. 16
December 2002
melanoma and breast cancer, for example, tumor progression and metastasis correlate with integrin ␣v␤3
expression. Thus, independent of its role in tumor
angiogenesis, integrin ␣v␤3 is likely to be functionally
implicated in the pathogenesis of a variety of cancers.
This is particularly apparent for KS, where a central
position of integrin ␣v␤3 within angiogenesis and KS
pathogenesis has emerged, suggesting that integrin
␣v␤3 may become a prime molecular target for KS
therapy. The hypervascular morphology of KS makes it
highly accessible to therapeutic macromolecules, such
as antibodies, whose general applicability for cancer
therapy is limited by poor tumor penetration.
With JC-7U, we developed a highly advanced drug
candidate for therapeutic applications that involve integrin ␣v␤3 as the molecular target. JC-7U IgG1 was
found to selectively target tumors in a mouse model of
KS and inhibit tumor growth at a therapeutically relevant dose. We anticipate that the tumor growth-inhibiting activity of JC-7U will be even more effective in KS
patients as compared with mouse models of KS. First, in
the presence of human angiogenic endothelial cells,
JC-7U is expected to exert an antiangiogenic effect
based on initiation of endothelial cell apoptosis and
ADCC in addition to an antitumor effect. Second, in
AIDS-associated KS, JC-7U will partially block the binding of HIV-1 Tat protein to human integrin ␣v␤3 and,
thus, potentially diminish its tumor growth-promoting
activity.
The FASEB Journal
RADER ET AL.
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            
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
            



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
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              



 

            



              
           
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           



                


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               
             



                


             

              
            

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
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
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
                 




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
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

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
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
           
             







   

            
            
          






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
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           
            
               


                   
                  
                  




          


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
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               
             
             









    

              
          











     





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


      
             
             
              

           




          

              

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
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           

           


            
              

             


            

         

         
           
             

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
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              

             
        
         
           



        

           
             


 
             
            
        
          
              

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



              


        

           


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
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

             
         
            
          

          
             
            
           

            
           
             

             
           


           
              

             
               




            


              
            

            
            

            








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 
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
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
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
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
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
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
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             
            

            
           




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
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             

              


                  

           

                
          

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
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

             
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           
             

        


           

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
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


                 
          
          

              

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
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
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











             

            


               




   
              
             
           


            
            



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


          
                

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
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
                


                
            

          

               


                
               
              
                

            

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

                  
           
               
             

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
               


                  

                 

               



               
             

              

              
               

                 

              

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

              


            

           
                
            
                  
              


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
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               
             
              



             

           




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              


              


                


                
              
            

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             

               
             

             
              


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
              

            
                
                 

             
         

              

             

               


          

               
           
          

 
            

             
                




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             


              

            





               

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               
            

             
 
             

             

             
                
            
            

             

               

              

           

             



             
               
            

              



                
             
                 
              
              

             
              



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
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

                 


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
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
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               

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

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

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
         

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               
             

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
         

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

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

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


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
               

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


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
            

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               

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               


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
   
                 
           

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                 
            

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
            

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

             

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
                



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
             


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               

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                


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

         

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


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
               

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
             

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
            

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
             

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             


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
          

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            


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

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              

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
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
                 

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              


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               


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

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

          

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


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

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

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

            

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Table 1
Binding parameters of Fab directed to human integrin αvβ3
Fab
Mouse LM609 b
kon/104
(M−1 s−1)
8.6
a
koff/10−4
(s−1)
Kd
(nM)
8.6
10
Humanized LM609-7
23 (18 b)
5.9 (5.4 b)
2.6 (3.0 b)
JC-7U
24
0.35
0.15
a
Binding kinetics were determined by using surface plasmon resonance. Human integrin αvβ3 was immobilized
on the sensor chip. Kd was calculated from koff/kon. All Fab fragments were produced by using E. coli
expression. bData from Rader et al. (10).
Fig. 1
Figure 1. Affinity maturation of humanized LM609 by CDR walking. CDR walking involves the sequential or
parallel optimization of CDRs by focused mutagenesis and subsequent selection by phage display. Shown is a combination
of sequential and parallel CDR optimization used for the affinity maturation of humanized LM609. Randomized
sequences are hatched; optimized sequences are black. CDR3 of the light chain was optimized first, followed by a parallel
optimization of CDR1 and CDR3 of the heavy chain. Optimized CDR1 and CDR3 of the heavy chain were combined in
the last step. This focused mutagenesis strategy involved the randomization of four hypervariable codons in each targeted
CDR encoding sequence. LC, light chain; HC, heavy chain.
Fig. 2
Figure 2. Randomized and selected sequences of LCDR3, HCDR1, and HCDR3 for one of the selected clones,
designated JC-7U. The top and middle panels show the four codons of each CDR that were subjected to randomization
by NNK (nnk in the figure) doping (n = a, c, g, or t; k = g or t). The bottom panel shows the selected NNK codons and
their corresponding amino acids (single-letter amino acid code). As shown in bold type, 10 of 12 codons but only 5 of 12
amino acids differed between the original and the selected sequence. No amino acid mutations in nonrandomized CDR
and framework regions were found.
Fig. 3
Figure 3. Overlayed Biacore 1000 sensorgrams obtained from the binding of humanized LM609-7 Fab and JC-7U
Fab to immobilized human integrin αvβ3. For association, Fab fragments were injected at 80 nM between t = 125 s and
t = 370 s, with a flow rate of 10 µl/min. For dissociation, the flow rate was increased to 50 µl/min. RU, resonance units.
Fig. 4
Figure 4. Flow cytometry histograms demonstrating the binding of JC-7U Fab and humanized LM609-7 Fab to
the human KS cell line SLK. For indirect immunofluorescence staining, cells were incubated with 2 µg/ml JC-7U Fab
(bold line) or 2 µg/ml humanized LM609-7 (fine line) or buffer alone (dotted line), followed by FITC-conjugated
secondary antibodies. The y axis gives the number of events in linear scale, the x axis the fluorescence intensity in
logarithmic scale. Note that JC-7U Fab revealed a slightly stronger binding than did humanized LM609-7. This difference
was confirmed at Fab concentrations of 200 and 20 ng/ml and was also seen when human melanoma M21 cells were used
instead of SLK cells.
Fig. 5
Figure 5. Vector PIGG designed for antibody expression in mammalian cells. The 9-kb vector contained both
heavy- and light-chain expression cassettes driven by a bidirectional CMV promoter construct. Cloning sites for the
variable domain encoding sequences were designed for compatibility with pComb3 phage display vectors. By using
PIGG, Fab selected by phage display can be readily converted into IgG.
Fig. 6
Figure 6. JC-7U IgG1 did not inhibit the growth of human KS cell line SLK in vitro. Shown is a proliferation assay
based on [3H]thymidine incorporation during DNA synthesis. None of the tested concentrations in a range of 0.1 nM to 1
µM revealed a significant difference between JC-7U IgG1 and the control antibody b12 IgG1. Background signals were
determined by running the same assay in the absence of antibody and were defined as 0% inhibition (n = 3).
Fig. 7
Figure 7. Tumor targeting of JC-7U IgG1. JC-7U IgG1 (A and E) and control antibody b12 IgG1 (B and F) were
injected into the tail vein of nude mice with an established human KS tumor. After 24 h, tumor (A and B) and lung tissue
(E and F) were removed and processed for the detection of bound human IgG by using goat anti-human IgG secondary
antibodies. C, D) Mouse endothelial cells that infiltrate the human KS tumor were detected with a rat anti-mouse CD31
mAb followed by rabbit anti-rat IgG secondary antibodies. Note that the sites of CD31 expression and JC-7U IgG1 tumor
penetration overlap. Scale bar = 50 µm for A-F.
Fig. 8
Figure 8. Tumor growth inhibition in a nude mouse model of KS in the presence of JC-7U IgG1. JC-7U IgG1
(black circles) or control antibody b12 IgG1 (white circles) was given in five doses of 5 mg/kg every third day starting on
day 1 after tumor induction. Statistically significant differences in tumor volume were observed 14 days after the last
treatment until the end of the experiment. Shown are average tumor volumes ± SD (n = 5 for both groups).
Fig. 9
Figure 9. Tumor growth inhibition in a nude mouse model of KS in the presence of JC-7U IgG1. Shown are tumor
weights after treatment with JC-7U IgG1, b12 IgG1, or PBS. Tumors were removed surgically on day 54 after tumor
initiation and weighed. Tumor weights are averages ± SD (n = 5 for JC-7U and b12; n = 4 for PBS [1 animal in this PBS
group died before day 54]). The differences between treatment with JC-7U IgG1 and b12 IgG1 (P=0.0019) or PBS
(P=0.0004) were statistically significant.
Fig. 10
Figure 10. JC-7U Fab partially blocked the binding of HIV-1 Tat protein to human integrin αvβ3. A mixture of
human integrin αvβ3 and synthetic HIV-1 Tat-biotin was incubated in solution in the presence or absence of JC-7U Fab.
Complexes of integrin αvβ3 and Tat-biotin were then captured with immobilized streptavidin and were detected by using
an independent mouse mAb directed to integrin subunit β3 followed by goat anti-mouse secondary antibodies.
Background signals were determined by running the same assay in the absence of Tat-biotin. Background-depleted
signals are shown (n = 3; P=0.0058).