US2003049603A1PendingUtilityA1

Methods for construction and screening of libraries of chemokine variants

43
Priority: Sep 5, 2001Filed: Sep 5, 2001Published: Mar 13, 2003
Est. expirySep 5, 2021(expired)· nominal 20-yr term from priority
Inventors:Guy GorochovOliver HartleyKarim DorghamPatrice DebreRobin Offord
A61P 31/18C12N 15/1037A61P 29/00C40B 40/02C07K 14/521C07K 14/523A61K 38/04
43
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Claims

Abstract

The present invention provides a method for the design and/or the selection of agonist or antagonist chemokine variants combining a phage display technology and a screening on living cells expressing the receptor of the corresponding native chemokine. It also provides RANTES variants having agonist properties towards said receptor, and methods for preventing and/or curing viral deseases.

Claims

exact text as granted — not AI-modified
1 . A method for the design and/or the selection of chemokines variants having agonist or antagonist property towards a ligand of GPCR of animal cells comprising the following steps: 
 A) obtaining a phage displayed library expressing on their surface said chemokine variants mutated within the domain responsible for their effector function,    B) having a culture of animal cells expressing on their membranes the GPCR,    C) Incubating the cell culture with the phage library obtained In A),    D) harvesting the cells after removal of non specifically bond and surface receptor bound phages,    E) Releasing the phages internalised in step C) by lysis of cells obtained in D)    F) Infecting an  E. coli  culture with the released phages obtained in E) and amplifying the clones previously internalised,    G) Obtaining a phage library enriched in internalising chemokines ligands,    H) Assaying the agonist or antagonist property of the chemokine variants versus the native one.    
     
     
         2 . The method according to  claim 1  wherein the chemokine is RANTES.  
     
     
         3 . The method according to  claim 1  wherein the GPCR expressed within the membrane of animal cells is CCR5.  
     
     
         4 . The method according to  claim 1  wherein the animal cells are human cells.  
     
     
         5 . The method according to  claim 2  wherein the phage library of RANTES variants is obtained using a method comprising the following steps: 
 Obtaining a DNA sequence coding for human RANTES resulting from the ampification of cDNA prepared from activated PBMCs,  
 Performing a PCR mutagenesis of the 5′portion of the DNA sequence of RANTES using a specific downstream primer and a degenerate upstream primer containing recognition sites for restriction enzymes in order to insert the PCR amplification products into the phage display vector,  
 Inserting the purified PCR products into a phage display vector,  
 Production of the phage library by introducing the vector containing the purified PCR products into an  E. coli  culture.  
 
     
     
         6 . The method according to  claim 2  wherein anti-HIV activity is assayed.  
     
     
         7 . A method for the design and/or the selection of chemokines having agonist or antagonist property towards a GPCR of animal cells comprising the following steps: 
 A) obtaining a phage displayed library expressing on their surface said chemokine mutated within the domain responsible for their effector function,    B) having a culture of animal cells expressing on their membranes the GPCR,    C) Incubating the cell culture with the phage library obtained in A),    D) Eliminating the non specifically bond phages from the cells, by a process keeping the specifically bound phages on the said receptor    E) Incubating the cells obtained in D) with an  E. coli  culture and amplifying the clones being infected by the phages bound to the said receptor on animal cells,    F) Obtaining a phage library enriched in externally bound phages,    G) Assaying the agonist or antagonist property of the chemokine variants versus the native chemokine.    
     
     
         8 . The method according to  claim 7  wherein the chemokine is RANTES.  
     
     
         9 . The method according to  claim 7  wherein the GPCR expressed within the membrane of animal cells is CCR5.  
     
     
         10 . The method according to  claim 7  wherein the animal cells are human cells.  
     
     
         11 . The method according to  claim 8  wherein the phage library of RANTES variants is obtained using a method comprising the following steps: 
 Obtaining a DNA sequence coding for human RANTES resulting from the ampification of cDNA prepared from activated PBMCs,  
 Performing a PCR mutagenesis of the 5′portion of the DNA sequence of RANTES using a specific downstream primer and a degenerate upstream primer containing recognition sites for restriction enzymes in order to insert the PCR amplification products into the phage display vector,  
 Inserting the purified PCR products into a phage display vector,  
 Production of the phage library by introducing the vector containing the purified PCR products into an  E; coli  culture.  
 
     
     
         12 . The method according to  claim 8  wherein anti-HIV activity is assayed.  
     
     
         13 . A compound having antagonist properties to RANTES or to MIP-1α the following formula: *S&#SSQ&&&-RANTES(10-68), in which 
 * is L or an aromatic residue,  
 # is L, M ouV  
 & is S, P, T or A.  
 
     
     
         14 . The compound according to  claim 13  having one of the following formulae: 
 LSPVSSQSSA (P 1 )  
 FSPLSSQSSA (P 2 )  
 LSPMSSQSPA  
 WSPLSSQSPA  
 WSPLSSQSSP  
 LSPQSSLSSS  
 ASSGSSQSTS  
 ISAGSSQSTS  
 RSPMSSQSSP  
 YSPSSSLAPA  
 MSPLSSQASA  
 ASPMSSQSSS  
 QSPLSSQAST  
 QSPLSSTASS  
 LSPLSSQSAA  
 GSSSSSQTPA  
 YSPLSSQSSP  
 FSSVSSQSSS,  
 
     
     
         15 . The compound according to  claim 14)  having the formula: FSPLSSQSSA-RANTES(10-68).  
     
     
         16 . The compound according to  claim 14)  having the formula: LSPVSSQSSA-RANTES (10-68).  
     
     
         17 . A pharmaceutical composition which comprises of a compound having the formula *S&#SSQ&&&-RANTES(10-68), in which 
 * is L or an aromatic residue,    # is L, M ouV    & is S, P, T or A,    or a pharmaceutical salt thereof, in a mixture with one or more pharmaceutically acceptable excipient.    
     
     
         18 . The composition of  claim 17)  in which the compound have the formula: LSPVSSQSSA-RANTES(10-68).  
     
     
         19 . The composition of  claim 17)  in which the compound have the formula: FSPLSSQSSA-RANTES(10-68).  
     
     
         20 . A method for preventing and/or inhibiting HIV infection in humans comprising a step of treatment with a composition of  claim 18) .  
     
     
         21 . A method for preventing and/or inhibiting HIV infection in humans comprising a step of treatment with a composition of claim  19 ).

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