Single-domain brain-targeting antibody fragments derived from LLAMA antibodies

Abstract

A phage-displayed library of llama single heavy domain antibodies (sdAbs) was enriched for species that selectively bind to and are internalized by human cerebromicrovascular endothelial cells (HCEC). From the enriched library, two sdAbs were selected, sequenced, subcloned, and expressed as fusion proteins with c-myc-His 5 tags. Similarly as phage-displayed sdAbs, these soluble tagged sdAbs were shown to selectively bind to HCEC and to transmigrate across in vitro human blood-brain barrier (BBB) model. In contrast to an unrelated llama sdAb, these sdAbs were also detected in the brain after i.v. injection into mice. These small (˜13 kDa) antibody fragments have essential characteristics of brain-specific delivery vectors and can be used to facilitate drug transport across the BBB.

1 . A therapeutic or diagnostic agent, said agent comprising an antibody fragment derived from a llama antibody, said fragment being adapted to selectively bind to an antigen on the surface of the mammalian cells expressing BBB antigens and transmigrating across the human blood-brain barrier (BBB), said antibody fragment being linked, directly or indirectly, covalently or non-covalently, to a therapeutic or diagnostic entity. 
     
     
         2 . A therapeutic or diagnostic agent according to  claim 1 , wherein the mammalian cells are human cerebromicrovascular endothelial cells (HCEC). 
     
     
         3 . A therapeutic or diagnostic agent according to  claim 2 , wherein the antibody fragment is internalized by the HCEC. 
     
     
         4 . A therapeutic or diagnostic agent according to  claim 3 , wherein the agent is administered by injection. 
     
     
         5 . A therapeutic or diagnostic agent according to  claim 4 , wherein the agent is administered by an intravenous, intraperitoneal, intramuscular, or subcutaneous injection. 
     
     
         6 . A therapeutic or diagnostic agent according to  claim 4 , wherein the agent is localized in brain parenchyma after being administered by injection. 
     
     
         7 . A therapeutic or diagnostic agent according to  claim 3 , wherein the antibody fragment comprises at least a part of the variable heavy domain (V H H or VH) of the llama antibody. 
     
     
         8 . A therapeutic or diagnostic agent according to  claim 3 , wherein the antibody fragment comprises a complete variable heavy domain (V H H or VH) of the llama antibody. 
     
     
         9 . A therapeutic or diagnostic agent according to  claim 8 , wherein the antibody fragment consists essentially of the variable heavy domain (V H H or VH) of the llama antibody. 
     
     
         10 . A therapeutic or diagnostic agent according to  claim 9 , wherein the antibody fragment is selected from the group consisting of polypeptides designated as FC5 [SEQ ID No. 85], FC 44 [SEQ ID No. 86] and FC 7 [SEQ ID No. 87]. 
     
     
         11 . A therapeutic or diagnostic agent according to  claim 1 , wherein the therapeutic or diagnostic entity does not permeate the BBB. 
     
     
         12 . A therapeutic or diagnostic agent according to  claim 11 , wherein the therapeutic or diagnostic entity is a peptide. 
     
     
         13 . A therapeutic or diagnostic agent according to  claim 12 , wherein the peptide is c-myc or His 5  tag. 
     
     
         14 . A therapeutic or diagnostic agent according to  claim 11 , wherein the therapeutic or diagnostic entity is a piece of DNA. 
     
     
         15 . A therapeutic or diagnostic agent according to  claim 11 , wherein the therapeutic or diagnostic entity is a phage. 
     
     
         16 . A therapeutic or diagnostic agent according to  claim 11 , wherein the therapeutic or diagnostic entity is a fluorescent or radioactive label. 
     
     
         17 . A therapeutic or diagnostic agent according to  claim 11 , wherein the antibody fragments are linked to a liposomal or polymeric preparation comprising the therapeutic or diagnostic entity.