Adheson variants

Abstract

Novel derivatives of cell surface proteins which are homologous to the immunoglobulin superfamily (adhesons) are provided. Amino acid sequence variations are introduced into the adheson, the most noteworthy of which are those in which the transmembrane and, preferably, cytoplasmic domains are rendered functionally inactive, and in which adheson extracellular domains replace an immunoglobulin variable region. These variants are useful in therapy or diagnostics, in particular, CD4 variants are therapeutically useful in the treatment of HIV infections.

1 . Nucleic acid encoding an amino acid sequence variant of an adheson.  
     
     
         2 . The nucleic acid of  claim 1  wherein the adheson is a CD4 polypeptide.  
     
     
         3 . The nucleic acid of  claim 2  wherein the variant is a CD4 polypeptide in which nucleic acid encoding the transmembrane domain has been modified whereby the CD4 polypeptide encoded thereby contains an inactivated transmembrane domain.  
     
     
         4 . The nucleic acid of  claim 3  wherein the transmembrane domain has been inactivated by its deletion or by substituting for the transmembrane domain an amino acid sequence having a substantially hydrophilic hydropathy profile.  
     
     
         5 . The nucleic acid of  claim 2  wherein the variant comprises a fusion of (a) a polypeptide different from the CD4 and (b) a CD4 polypeptide.  
     
     
         6 . The nucleic acid of  claim 5  wherein the polypeptide different from the CD4 bears a non-CD4 immune epitope.  
     
     
         7 . The nucleic acid of  claim 6  wherein the polypeptide different from CD4 is fused to the amino or carboxyl terminus of mature CD4 and the transmembrane domain of CD4 has been inactivated.  
     
     
         8 . The nucleic acid of  claim 5  wherein the different polypeptide comprises a signal sequence.  
     
     
         9 . The nucleic acid of  claim 5  wherein the different polypeptide contains about from 5 to 1000 residues.  
     
     
         10 . The nucleic acid of  claim 9  wherein the different polypeptide is capable of eliciting a humoral immune response in an animal.  
     
     
         11 . The nucleic acid of  claim 10  wherein the different polypeptide is a viral polypeptide or an allergen.  
     
     
         12 . The nucleic acid of  claim 5  wherein the different polypeptide is a human plasma protein having a plasma half life greater than from which the transmembrane domain has been deleted.  
     
     
         13 . The nucleic acid of  claim 12  wherein the variant is a fusion of a polypeptide comprising at least one V-like domain of CD4 fused with a polypeptide comprising an immunoglobulin constant domain.  
     
     
         14 . The nucleic acid of  claim 1  wherein the adheson is CD4, CD8 or the high affinity IgE receptor.  
     
     
         15 . The nucleic acid of  claim 2  wherein the variant consists essentially of the V 1  through V 4  or V 1  through V 2  regions of the CD4 antigen.  
     
     
         16 . The nucleic acid of  claim 2  which consists essentially of the CD4 insert of pCD4DNla.  
     
     
         17 . The nucleic acid of  claim 12  wherein the different polypeptide is albumin, apolipoprotein or transferrin.  
     
     
         18 . The nucleic acid of  claim 8  wherein the signal sequence is a bacterial signal sequence.  
     
     
         19 . The nucleic acid of  claim 15  wherein the variant consists essentially of CD4 residues 1-368.  
     
     
         20 . The nucleic acid of  claim 15  wherein the variant consists essentially of CD4 residues 1-180.  
     
     
         21 . The nucleic acid of  claim 13  wherein the immunoglobulin constant domain is the constant domain of an IgG heavy chain.  
     
     
         22 . The nucleic acid of  claim 5  wherein the different polypeptide is a cytotoxic polypeptide.  
     
     
         23 . The nucleic acid of  claim 5  wherein the cytotoxic polypeptide is the diptheria toxin A.  
     
     
         24 . A composition comprising an adheson amino acid sequence variant which is incapable of cell membrane anchorage.  
     
     
         25 . The composition of  claim 24  wherein the adheson variant comprises a CD4 amino acid sequence capable of binding gp120.  
     
     
         26 . The composition of  claim 25  further comprising an agent for inhibiting the aggregation of the variant selected from the group of a predetermined protein and a surfactant.  
     
     
         27 . The composition of  claim 26  wherein the agent is a surfactant.  
     
     
         28 . The composition of  claim 27  wherein the surfactant is Tween 80 or Tween 20.  
     
     
         29 . The composition of  claim 25  wherein the CD4 transmembrane domain has been deleted or has been substituted for by an amino acid sequence having a substantially hydrophilic hydropathy profile.  
     
     
         30 . The composition of  claim 29  which is sterile and which further comprises a physiologically acceptable carrier.  
     
     
         31 . The composition of  claim 25  wherein the variant comprises an immunoglobulin amino acid sequence.  
     
     
         32 . The composition of  claim 31  wherein the immunoglobulin sequence comprises a constant domain sequence of an immunoglobulin heavy chain.  
     
     
         33 . The composition of  claim 32  wherein the constant domain is linked at its N-terminus to the C-terminus of a transmembrane-deleted CD4 polypeptide.  
     
     
         34 . The composition of  claim 33  wherein the CD4 polypeptide contains V 1 V 2 .  
     
     
         35 . The composition of  claim 33  wherein the CD4 polypeptide contains V 1 V 2 V 3 V 4 .  
     
     
         36 . The composition of  claim 31  wherein the the variant is in the form of a dimer.  
     
     
         37 . The composition of  claim 36  wherein the composition comprises a fusion of a CD4 V-like domain to an immunoglobulin heavy chain constant domain.  
     
     
         38 . The composition of  claim 31  wherein the variant is selected from the group consisting of 
 (a) AC L ;  
 (b) AC L -AC L ;  
 (c) AC H -[AC H , AC L -AC H , AC L -V H C H , V L C L -AC H , or V L C L -V H C H ];  
 (d) AC L -AC H -[AC H , AC L -AC H , AC L -V H C H , V L C L -AC H , or V L C L -V H C H ];  
 (e) AC L -V H C H -[AC H , AC L -AC H , AC L -V H C H , V L C L -AC H , or V L C L -V H C H ];  
 (f) V L C L -AC H -[AC H , AC L -AC H , AC L -V H C H , V L C L -AC H , or V L C L -V H C H ]; or  
 (g) [A-Y] n -[V L C L -V H C H ] 2    
 wherein A is a CD4 polypeptide containing a CD4 variable region-like domain; V L , V H , C L  and C H  represent light or heavy chain variable or constant domains of an immunoglobulin; n is an integer; and Y designates the residue of a covalent cross-linking agent.  
 
     
     
         39 . The composition of  claim 38  wherein the V L  and V H  domains are capable of binding a predetermined antigen.  
     
     
         40 . The composition of  claim 31  wherein the immunoglobulin sequence is obtained from IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgD or IgM.  
     
     
         41 . The composition of  claim 25  wherein the variant comprises a polypeptide different from CD4 which is nonimmunogenic in humans.  
     
     
         42 . The composition of  claim 41  wherein the variant comprises a polypeptide which is immunogenic in humans.  
     
     
         43 . The composition of  claim 41  wherein the variant comprises a polypeptide having a human plasma half life which is greater than about 20 hours.  
     
     
         44 . The composition of  claim 41  wherein the variant comprises a human transferrin, apolipoprotein or albumin polypeptide.  
     
     
         45 . The composition of  claim 25  wherein the variant comprises a cytotoxic polypeptide.  
     
     
         46 . The composition of  claim 45  wherein the cytotoxic polypeptide is ricin A chain or diptheria toxin A.  
     
     
         47 . A polypeptide comprising a CD4 amino acid sequence capable of binding gp120 which is cross-linked to (a) polypeptide having a plasma half life of greater than about 20 hours or (b) a cytotoxic polypeptide.  
     
     
         48 . The polypeptide of  claim 47  wherein the polypeptide of (a) is transferrin, an apolipoprotein or albumin.  
     
     
         49 . The polypeptide of  claim 47  wherein the cytotoxic polypeptide is cross-linked to the CD4 variable-like domain by a bifunctional cross-linking agent.  
     
     
         50 . A method for preparing an adheson variant comprising transfecting a host cell with the nucleic acid of  claim 1 .  
     
     
         51 . A method for preparing an adheson variant comprising recovering the variant from the culture of a host cell transfected with the nucleic acid of  claim 1 .  
     
     
         52 . The method of  claim 51  wherein the adheson is CD4 and the variant is recovered from the culture medium of the host cell or from the cell itself.  
     
     
         53 . The method of  claim 52  wherein the variant is recovered by adsorption onto a cation exchange resin.  
     
     
         54 . The method of  claim 53  wherein the variant is recovered by adsorption of contaminants onto an anion exchange resin.  
     
     
         55 . The method of  claim 52  wherein the variant lacks a functional transmembrane domain.  
     
     
         56 . The method of  claim 52  wherein wherein a salt is added to the culture medium to occupy charged domains of the variant, the resulting solution is contacted with a hydrophobic affinity chromatography resin to adsorb the variant, and the variant eluted from the resin by washing the resin with a declining. gradient of salt.  
     
     
         57 . The method of  claim 52  wherein the variant is recovered by immunoaffinity chromatography.  
     
     
         58 . The method of  claim 57  wherein the immunoaffinity chromatography is directed against a polypeptide different from CD4 which is fused to CD4.  
     
     
         59 . A method for the treatment of an HIV infection comprising administering to a patient infected with HIV a therapeutically effective dose of an amino acid sequence variant of CD4.  
     
     
         60 . A replicable vector comprising the nucleic acid of  claim 1.