Variable heavy chain and variable light chain regions of antibodies to human platelet glycoprotein ib alpha

Abstract

The present invention is directed to a method of selecting a clone that binds to human platelet glycoprotein Ib alpha using a human variable heavy chain and variable light chain immunoglobulin library. The invention is further directed to isolated nucleic acid molecules encoding a variable heavy chain or variable light chain region of an antibody, wherein the antibody binds to human platelet glycoprotein Ib alpha and inhibits aggregation of platelets. Expression vectors and host cells comprising the nucleic acid molecules are also provided, as well as methods for producing the variable heavy chain or the variable light chain region. An isolated variable heavy chain or variable light chain region of an antibody, wherein the antibody binds to human platelet glycoprotein Ib alpha and inhibits aggregation of platelets, is also provided. An antibody comprising the variable heavy chain or variable light chain regions is provided, as is a composition comprising the antibody and a carrier. The subject invention further provides a method of inhibiting aggregation of platelets, as well as a method of binding human platelet glycoprotein Ib alpha. A method of selecting a variable heavy chain or variable light chain region of an antibody is also provided.

1 . A method of selecting a clone that binds to human platelet glycoprotein Ib alpha using a human variable heavy chain and variable light chain immunoglobulin library, the method comprising: 
 incubating a human variable heavy chain and variable light chain immunoglobulin library with cells expressing human platelet glycoprotein Ib, and selecting clones of the library which bind to the cells; and    incubating the selected clones of the library with washed human platelets, and selecting resulting clones which bind to the washed human platelets, wherein the resulting clones bind to human platelet glycoprotein Ib alpha.    
     
     
         2 . The method of  claim 1  wherein the cells are Chinese Hamster Ovary cells.  
     
     
         3 . The method of  claim 1  further comprising incubating the selected resulting clones with further platelets and adding an anti-glycoprotein Ib alpha molecule that may displace clones already bound to the further platelets, and selecting the then-resulting clones that are not bound to the further platelets, the then-resulting clones being capable of binding to human platelet glycoprotein Ib alpha.  
     
     
         4 . The method of  claim 3  wherein the anti-glycoprotein Ib molecule is a murine monoclonal antibody.  
     
     
         5 . The method of  claim 3  wherein the anti-glycoprotein Ib molecule is a peptide.  
     
     
         6 . The method of  claim 5  wherein the peptide has an amino acid sequence as shown in SEQ ID NO:1.  
     
     
         7 . An isolated variable heavy chain or a variable light chain region of an antibody, or a fragment thereof, wherein the antibody binds to human platelet glycoprotein Ib alpha and inhibits aggregation of platelets.  
     
     
         8 . The variable light chain region of an antibody of  claim 7 , wherein the variable light chain region has an amino acid sequence selected from the group consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21.  
     
     
         9 . A fragment of the variable heavy chain region of an antibody of claim  30 .  
     
     
         10 . The fragment of  claim 9  wherein the fragment is a VH3 fragment having an amino acid sequence selected from the group consisting of SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:35 and SEQ ID NO:36.  
     
     
         11 . The fragment of  claim 9  wherein the fragment is a CDR1 fragment having an amino acid sequence selected from the group consisting of SEQ ID NO:29, SEQ ID NO:33 and SEQ ID NO:37.  
     
     
         12 . The fragment of  claim 9  wherein the fragment is a CDR2 fragment having an amino acid sequence selected from the group consisting of SEQ ID NO:30, SEQ ID NO:34 and SEQ ID NO:38.  
     
     
         13 . The fragment of  claim 9  wherein the fragment is a CDR3 fragment having an amino acid sequence selected from the group consisting of SEQ ID NO:39, SEQ ID NO:40 and SEQ ID NO:41.  
     
     
         14 . A fragment of the variable light chain region of an antibody of  claim 7 .  
     
     
         15 . The fragment of  claim 14  wherein the fragment has an amino acid sequence selected from the group consisting of SEQ ID NO:42, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:59, SEQ ID NO:60 and SEQ ID NO:61.  
     
     
         16 . The fragment of  claim 14  wherein the fragment is a CDR1 fragment having an amino acid sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:48, SEQ ID NO:52 and SEQ ID NO:55.  
     
     
         17 . The fragment of  claim 14  wherein the fragment is a CDR2 fragment having an amino acid sequence selected from the group consisting of SEQ ID NO:44, SEQ ID NO:49, SEQ ID NO:53 and SEQ ID NO:56.  
     
     
         18 . The fragment of claim  38  wherein the fragment is a CDR3 fragment having an amino acid sequence selected from the group consisting of SEQ ID NO:45, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:57 and SEQ ID NO:58.  
     
     
         19 . An isolated antibody, or an antigen binding portion thereof, which binds to human platelet glycoprotein Ib alpha and inhibits aggregation of platelets, wherein the isolated antibody comprises a variable heavy chain region and a variable light chain region wherein the variable heavy chain region comprises a CDR3 region comprising the amino acid sequence as shown in SEQ ID NO:39, SEQ ID NO:40 or SEQ ID NO:41.  
     
     
         20 . A pharmaceutical composition comprising the antibody of  claim 20  and a carrier.  
     
     
         21 . A CDR3 peptide of the isolated antibody or isolated antigen binding portion thereof of  claim 19 , wherein the peptide comprises the amino acid sequence as shown in SEQ ID NO:39, SEQ ID NO:40 or SEQ ID NO:41.