Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70503—Immunoglobulin superfamily
  • C07K14/70514—CD4
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• T4 lymphocytes The CD4 (T4) molecule, which is a surface glycoprotein on a subset of T lymphocytes (referred to as T4 lymphocytes) is involved in Class II (Ia) MHC recognition and appears to be the physiological receptor for one or more monomorphi ⁇ regions of class II MHC.
• T4 lymphocytes The CD4 (T4) molecule, which is a surface glycoprotein on a subset of T lymphocytes (referred to as T4 lymphocytes) is involved in Class II (Ia) MHC recognition and appears to be the physiological receptor for one or more monomorphi ⁇ regions of class II MHC.
• Meur S. et al., Proceedings of the National Academy of Sciences, U.S.A., 79:4395-4399 (1982); Biddison, W. et al., J. Exp. Med., 156: 1065-1076 (1982); Gay, D. et al., Nature, 328: 626-629 (1987).
• Human CD4 is also the receptor for the gp120 envelope glycoprotein of the human immunodeficiency virus (HIV) and is essential for virus entry into the host cell, and for membrane fusion, which both contribute to cell-to-cell transmission of the virus and to its cytopathic effects.
• HAV human immunodeficiency virus
• T4 lymphocytes i.e., helper and inducer T lymphocytes
• T4 lymphocytes i.e., helper and inducer T lymphocytes
• the present invention relates to soluble human CD4 (T4) fragments which bind to the HIV gp120 envelope protein (HIV gp120); to soluble human CD4 fragments whose ability to bind to the HIV gp120 envelope protein has been altered; to DNA encoding such types of human CD4 fragments; to methods of using soluble human CD4 fragments in interfering with infection of cells by HIV; to methods of modifying the amino acid sequence of soluble human CD4 fragments; and to methods of modifying or altering the ability of soluble human CD4 fragment to bind HIV gp120.
• T4 and T4 are used herein interchangeably).
• Soluble human CD4 fragments include none of the hydrophobic transmembrane region of CD4 or only a portion (generally six amino acids or less) of the hydrophobic region which does not prevent solubilization of the fragments.
• soluble human CD4 fragments which are capable of binding with HIV gp120 are referred to as biologically active soluble human CD4 fragments.
• biologically active soluble human CD4 fragments can be modified, with the result that the amino acid sequence differs in some way from that of the corresponding portion of naturally-occurring CD4.
• Biologically active soluble human CD4 fragments can be modified in several different ways.
• the amino acid sequence of soluble human CD4 can be:
• Biologically active soluble CD4 fragments of the present invention have the ability to bind to HIV. They will, therefore, also have the capacity to prevent infection of human T-lymphocytes by HIV and to prevent formation of the human T-lymphocyte syncytia which are thought to play a role in transmission of HIV from cell to cell.
• Such biologically active soluble CD4 fragments can be used for diagnostic, therapeutic and preventive purposes. For example, they can be used to determine the presence or absence of HIV gp120 in a biological sample (e.g., blood, urine, saliva, semen) and, thus, to determine whether HIV is present in the sample or not.
• a biological sample e.g., blood, urine, saliva, semen
• they can be used to treat individuals infected with HIV, in vivo (e.g., by administration to infected individuals). They can also be used prophylactically. That is, they can be administered to individuals at risk for HIV infection.
• they can be used to prevent infection by HIV by, for example, being coated onto materials used as barriers against introduction of the virus (e.g., condoms, spermicides, garments, containers for collecting, processing or storing blood, etc.).
• Modified soluble CD4 fragments with altered HIV gp120 binding ability can be used for diagnostic, therapeutic and preventive purposes. They can be used in a similar manner as described above for use of biologically active soluble human CD4 fragments.
• FIG. 1 is the nucleotide sequence of T4 S ⁇ C1 cDNA (referred to as the T4 exl sequence), which encodes 370 amino acids of soluble CD4 protein (referred to as T4 ex1 ). Modifications in cDNA and in the encoded CD4 protein are indicated by the boxed areas; each box represents the nucleotide triplet and encoded amino acid at which the modification is made.
• Figure 2 is a schematic representation of the construction of an expression vector of the present invention.
• Figure 3 is a schematic representation of the method by which the biologically active, modified soluble CD4 fragments of the present invention are produced.
• Figure 4 is a graphic representation of the effects of the soluble CD4 fragments of the invention and the effects of control proteins on viral protein replication.
• Figure 5 is a graph illustrating the lack of inhibition of CTL effector function by the soluble CD4 fragments of the invention.
• Figure 6 is a bar graph illustrating the lack of inhibition of proliferation of normal helper T-lymphocytes by the soluble CD4 fragments of the invention.
• Figure 7 is a schematic representation of the structure of native and recombinant CD4 proteins.
• Figure 7A is a representation of the native CD4 protein structure derived from the cDNA sequence of Figure 1. Numbers in parentheses indicate the four putative extracellular domains; the S at 16, 84, 130, 159, 303 and 345 indicates the position of cysteine residues; Tm: transmembrane; Cty: cytopiasmic region.
• Figure 7B is a schematic representation of the T4 exl protein.
• Figure 7C is the complete amino acid se ⁇ uence of the T4 exl protein.
• Figure 8 is a schematic representation of CD4 protein T4 exl showing the four immunoglobulin-like domains, three disulfide bonds and two potential glycosylation sites. Numbering of amino acids is according to Hussey et al., Nature, 331:78-81 (1988). The positions of 16 mutations (see the Table) are represented below the line. The triangle indicates a stop codon introduced by site directed mutagenesis to create a protein containing only the first 182 amino acids.
• Figure 9 shows results of anti-CD4 immunopre ⁇ ipitation and anti-gp120 co-precipitation of T4 exl and a truncated 182 amino acid version of CD4 from supernatants of 35 S-cysteine labelled Cos-1 cells transfected with the CD4 constructs.
• Lane 1 immunoprecipitation of supernatant from Cos-1 cells transfected with the T4 ex1 containing plasmid and immunoprecipitated with ant ⁇ -T8 (21Thy2D3) (control); lane 2, immunoprecipitation of supernatant from Cos-1 cells transfected with the 182 amino acid truncation using the control anti-T8 antibody; lane 3, immunoprecipitation of T4 ex1 with anti-CD4 antibody (19Thy5D7); lane 4, immunoprecipitation of the 182 amino acid truncation of T4 ex1 with anti-CD4; lane 5, co-precipitation of T4 exl with anti-gp120 (DuPont) in the presence of gp120; lane 6, co-precipitation of T4 exl with anti-gp120 in the absence of gp120; lane 7 , co-precipitation of the 182 amino acid truncation of T4 exl with anti-gt120 in the presence of g
• the molecular weight markers are phosporylase B (97.4KD), bovine serum albumin (69KD), ovaibumin (46KD), carbonic anhydrase (30KD), lactoglobulin A (18.4KD).
• Figure 10 shows results of anti-CD4 immunoprecipitation of 35 S-cynsteine labelled supernatants from Cos- 1 cells transfected with T4 ex 1 ,
• the present invention relates to soluble human CD4 fragments which bind to HIV gp120, as well as to soluble human CD4 fragments having altered gp120 binding ability; to DNA encoding soluble human CD4 fragments; to methods of making soluble human CD4 fragments and to methods of using soluble human CD4 fragments of the present invention to interfere with HIV infection of cells.
• soluble human CD4 fragments in which the amino acid sequence is the same as that of the corresponding region of naturally-occurring human CD4; to soluble human CD4 fragments in which the amino acid sequence has been modified, with the result that their amino acid sequences differ, as described below, from that of the corresponding region of naturally-occurring human CD4; to soluble human CD4 fragments whose binding ability is different from that of naturally-occurring human CD4 or the corresponding human CD4 fragment and to DNA encoding such soluble human CD4 fragments.
• Soluble human CD4 fragments of the present invention include none of tne hydropnobic transmembrane region of naturally-occurring CD4 or contain a portion of the hydrophobic region which is sufficiently short (i.e., generally six amino acids or less) that it does not prevent solubilization of the fragments.
• Soluble human CD4 fragments capable of binding HIV gp120 are referred to herein as biologically active soluble human CD4 fragments.
• Biologically active soluble human CD4 fragments are long enough (e.g., 10 amino acids or longer) that they are able to bind effectively to HIV gp120. Fragments need not exhibit total homology with the amino acid sequence of the corresponding region of human CD4. Rather, they must have sufficient homology to bind to HIV gp120.
• biologically active soluble human CD4 fragments of the present invention are able to exert an anti-HIV effect, as a result of binding HIV gp120, without interfering with the function or proliferation of human T-lymphocytes not infected with HIV. That is, biologically active soluble human CD4 fragments of the present invention have been shown, as described below, to prevent infection of human T-lymphocytes by HIV and to inhibit HIV envelope-induced syncytium formation and HIV replication without inhibiting Class II MHC recognition events (i.e., without inhibiting CTL effector function), even at high concentrations, and without having a discernible effect on Class II-directed physiologic T cell responses.
• soluble human CD4 fragments includes all soluble human CD4 fragments (i.e., those in which the amino acid sequence corresponds to that of naturally-occurring human CD4 and those in which modification of amino acid sequence has been made) capable of bin ⁇ ing HIV gp120.
• Biologically active soluble human CD4 fragments in which the amino acid sequence has been modified are referred to biologically active, modified soluble human CD4 fragments. Fragments whose HIV gp120 binding ability has been changed (with the result that it is different from that of the corresponding or equivalent portion of naturally-occurring CD4) are referred to as modified soluble human CD4 fragments with altered HIV gp120 binding ability.
• Biologically active, modified soluble human CD4 fragments of the present invention differ from that of soluble human CD4 (e.g., from the sequence represented in Figures 1 or 7C) in that the amino acid sequence: 1) is truncated; 2) has been altered as a result of deletion(s) from, substitution(s) in and/or addition(s) to the amino acid sequence of human CD4; or 3 ) it is truncated and the truncated form or portion includes deletion(s) from, substitution s) in and/or add ⁇ tion(s) to the amino acid sequence which occurs in the corresponding portion or segment.
• soluble human CD4 e.g., from the sequence represented in Figures 1 or 7C
• Modified soluble human CD4 fragments having altered HIV gp120 binding ability are modified soluble human CD4 fragments in which the amino acid sequence of soluble human CD4 is altered at a selected site or sites in such a manner that the resulting CD4 fragment has HIV gp120 binding ability or affinity less than that of the corresponding (unaltered) soluble human CD4 fragment, or HIV gp120 binding ability or affinity greater than that of the corresponding (unaltered) human CD4 fragment.
• Such fragments are referred to, respectively, as modified soluble human CD4 fragments with diminished HIV gp120 binding ability and modified soluble human CD4 fragments with enhanced HIV gp120 binding ability.
• CD4 fragments with altered HIV gp120 binding ability differ from soluble human CD4 fragments in that the amino acid sequences of the CD4 fragments with altered gp120 binding ability are different from the amino acid sequence of the soluble CD4 protein at a site or sites which have been found to be critical for gp120 binding.
• critical sites have now been identified by means of oligonucleotide-directed mutagenesis and have been found to occur in domain I ana domain II of human CD4 protein, suggesting that the HIV gp120 binding site is complex and involves both of the NH 2 -terminal domains.
• T4 cDNA Modifications of the T4 cDNA, as it is represented in Figure 1, have been made and the encoded CD4 fragments expressed. Resulting CD4 fragments have been shown to have altered gp120 binding ability in vitro; in these instances, gp120 binding ability has been abrogated. Modifications at these same sites, and at other, as yet unidentified, sites, as described herein, can similarly be made to enhance gp120 binding ability, as well as to reduce or turn down (but not eliminate) gp120 binding ability.
• plasmid vector PAC373/T4 ex which contains the truncated CD4 gene, was constructed from plasmids pAc373 and pSP65-T4.
• a secreted form of the CD4 molecule was produced by releasing the CD4 cDNA insert contained in pSP65-T4.
• the CD4 cDNA insert was digested with Neil to produce a 1.17Kb fragment which lacks the ATG start codon and terminates just before the transmembrane region.
• the 1.17Kb fragment was ligated to a synthetic linker, with the result that either 371 residues (T4 ex1 ) or 370 residues (T4 ex 2) of the mature extracellular segment would be preserved.
• pAc373/T4 ex1 and pAc373/T4 ex2 were characterized in detail.
• the truncated CD4 cDNA constructs were integrated into the Autographa californica nuclear polyhedrosis virus (AcNPV) genome by homologous recombination, using known methods. Smith et al., Proc. Natl. Acad. Sci., U.S.A., 82: 8404-8408 (1985).
• Baculovirus stocks were used to infect Spodoptera frugiperda (SF9) cells, which are publicly available. Subsequently, SF9 cells infected with the recombinant baculovirus containing the T4 ex cDNAs or wild type AcNPV were cultured in 3 5 S-methionine and products were examined by
• the T4 ex1 , polypeptide was shown to be the major secreted product of SF9 cells infected with the T4 ex1 recombinant baculovirus.
• the predominant 35 S labelled protein band in SDS-PAGE analysis of supernatants from SF9 cells obtained 54 hours after
• T4 ex1 recombinant baculoviral infection was a 50KD band under reducing conditions. No CD4 material was precipitated from supernatants of wild type AcNPV-infected cells or detectable in the total supernatant.
• T4 ex2 Each of two representative T4 ex2 preparations yielded a protein that migrated under reducing conditions with a molecular weight of 51KD.
• the different mobility was not unexpected, given that T4 ex2 contains 17 additional carboxy terminal amino acids derived from fusion with the polyhedrin gene.
• T4 ex2 contains 17 additional carboxy terminal amino acids derived from fusion with the polyhedrin gene.
• 50KD T4 ex1 and 51 KD T4 ex2 proteins were the products of the CD4 gene.
• the soluble CD4 fragments were shown to have no discernible effect on Class II-directed physiologic T cell responses; they were shown to have no effect on proliferation of the T4+ tetanus toxoid specific Class II MHC restricted helper T cell clone CTT7 (Example 1).
• T4 ex proteins or their derived peptide fragments were used to further analyze the specific physical interaction between T4 ex proteins or their derived peptide fragments and HIV gp120. This approach is described in detail in Example 2. Briefly, this method made use of size fractionation of SDS-PAGE, followed by electroblotting of the T4 ex2 protein onto polyvinylidine difluoride membranes.
• Enzymic fragmentation of the purified T4 ex protein was also carried out.
• Results of papain digestion showed the presence of a fragment with a mobility of 28KD which binds HIV gp120. It was shown to bind HIV gp120 with the same efficiency as the parent T4 ex1 protein and to be an intact polypeptide chain derived from the amino terminal region of the T4 ex1 protein.
• Similar experiments using trypsin fragmentation of T4 ex2 were also carried out to further define the nature of the HIV gp120 binding fragments, as described in Example 2.
• T4 SEC1 cDNA The cDNA sequence which encodes 270 amino acids of mature CD4 protein (T4 SEC1 cDNA) is represented in Figure 1, as is the deduced amino acid sequence of the encoded CD4 protein. Modifications of the T4 cDNA as represented in Figure 1 have been made and the encoded soluble CD4 fragment expressed.
• CD4 fragments have been shown to bind to HIV gp120 in vitro, as demonstrated by the ability to detect a complex between HIV gp120 and soluble CD4 proteins in solution.
• HIV gp120 and interfere with HIV infection of T cells without interfering with the function or proliferation of human T lymphocytes which are not infected with HIV.
• DNA encoding a soluble CD4 fragment is produced, either by using recombinant DNA techniques, such as excising it from a vector containing cDNA encoding such a fragment (see Example 1) or by synthesizing DNA encoding a soluble CD4 fragment mechanically and/or chemically, using known techniques.
• the DNA obtained encodes a soluble CD4 fragment, capable of binding to the gp120 envelope protein of HIV in vitro, which includes none of the hydrophobic transmembrane region of CD4 or a portion of that region (generally six amino acids or less) small enough that it does not prevent solubilization of the fragment.
• the CD4 fragment is long enough (e.g., 10 amino acids or more) to bind effectively to HIV gp120 envelope protein.
• Templates for subsequent mutagenesis are produced, using the CD4 fragment-encoding cDNA or DNA. As described below, this can be carried out using a single-stranded bacteriophage cloning vehicle, such as M13. This results in production of single-stranded DNA homologous to only one of the two strands of the DNA encoding the soluble CD4 fragment.
• the resulting single-stranded DNA is used as a template for producing the biologically active, modified soluble CD4 fragments, as follows: Oligonucleotides are produced, such that their sequence includes a base change (or changes) which, when incorporated into the nucleotide sequence of DNA subsequently used for the production of soluble CD4 fragments, results in a change in the encoded CD4 protein (i.e., different from that encoded by the nucleotide sequence of Figure 1). Such oligonucleotides are produced using standard methods. Oligonucleotides having a base change or base changes are referred to as mutageinzed or mutant oligonucleotides.
• the mutant oligonucleotide produced in this manner is hybridized to (e.g., by being kinasea) the template produced as described above, to pro ⁇ uce a template-mutant oligonucleotide complex, referred to as a mutant primer/template.
• the mutant primer/template is used for the production or a second strand of DNA, using well- known tecnniques. For example, synthesis of the second DNA strand is carried out by the Klenow fragment of DNA polymerase in the presence of dCTP ⁇ S. Taylor, J.W. et al., Nucleic Acids Research, 13: 8749-8764 (1985); Taylor, J.W.
• the resulting strand of DNA contains a modification (or modifications) in the nucleotide sequence of T4 cDNA (i.e., is different from the nucleotide sequence represented in Figure 1) and is referred to as a mutant strand.
• Unreplicated single-stranded DNA is removed and the double-stranded DNA is nicked with a selected restriction enzyme (e.g., NciI, which does not cut phosphorothioate DNA and, thus, does not cut the new DNA strand containing dCTP ⁇ S or the mutant strand).
• a selected restriction enzyme e.g., NciI, which does not cut phosphorothioate DNA and, thus, does not cut the new DNA strand containing dCTP ⁇ S or the mutant strand.
• Nicked, nonmodified DNA is removed by digestion with another enzyme, such as exonuclease III.
• the resulting gapped DNA is repolymerized and, because the mutant strand serves as the template for repolymerization, the mutation or modification is copied into both strands.
• the double-stranded DNA in which both strands contain the mutation or modification encoding the corresponding modification in the amino acid sequence of the soluble CD4 fragment is introduced into a competent host cell, sucn as a competent bacterial host (e.g., by transformation).
• the resulting plaques are grown and DNA contained in them is isolated, using known techniques, and sequenced to confirm the presence of the mutation.
• the mutated DNA produced in this manner is excised from the M13 vector containing it, introduced into a suitable expression vector, such as CDM8, and transfected into an appropriate host cell, sucn as Cos cells, in which it is expressed.
• a suitable expression vector such as CDM8
• transfected into an appropriate host cell sucn as Cos cells, in which it is expressed.
• CDM8 a suitable expression vector
• mutant CD4 proteins can be assayed, using known techniques.
• the vector-insert ligation mixture is introduced into competent host bacteria, such as the publicly available E. coli MC1061P3, and radiolabelled T4 DNA is used to identify CDM8 containing mutant T4 cDNAs.
• DNA encoding a biologically active, modified soluble CD4 fragment is produced, the encoded CD4 fragment is expressed and its ability to bind the HIV gp120 envelope protein is assessed.
• An alternative approach to producing a biologically active, modified soluble human CD4 fragment of the present invention is to use peptide synthesis to make a peptide or polypeptide having the amino acid sequence of such a fragment.
• the T4 ex cDNA was excised from the plasmid vector pAc373/T4 ex , using the restriction enzyme BamHI. The ends of the fragments were blunted with DNA polymerase I and the fragment was ligated to XbaI linkers. The ligated fragment was cut with XbaI, excess linkers were removed and the linkered fragments were ligated to Xba-cut M13 (replicative form). M13 is a single-stranded bacteriopnage cloning vehicle which has a closed circular DNA genome approximately 6.5Kb in size. Messing, J. and J. Viera, Gene, 19 : 269-276 (1982).
• infected cells release phage particles which contain single-stranded DNA which is homologous to only one of the two strands of cloned DNA and which can be used as a template.
• the resulting ligation mixture was transformed into competent TG1 host bacteria, which were plated out.
• the plaques were screened, using T4 oligonucleotides. Plaques hybridizing to sense oligonucleotides were selected and grown up to produce single-stranded M13 templates for mutagenesis.
• Mutagenesis was carried out by the protocol which is marketed by Amersham and is based on the method of Eckstein (See Example 3).
• Oligonucleotides whose sequence lncluded a base change which, when incorporated, produced an amino acid change in the encoded CD4 protein (different from that encoded by the cDNA protein of Figure 1) were produced, using standard methods. In this case, a truncation of the CD4 molecule was introduced at amino acid #183.
• the normal T4 cDNA sequence is G-AAG-GCC-TCC-AGC-ATA-G (see Figure 1).
• An oligonucleotide having the sequence 5'G-AAG-GCC-TAA-AGC-ATA-G was synthesized. The difference in the two sequences is underlined.
• the modified oligonucleotide was kinased and hybridized to M13 T4 template, which served as a template for synthesis of a second strand of DNA, by the Klenow fragment of DNA polymerase in the presence of dCTP ⁇ S. Taylor, J.W. et al., Nucleic Acids Research, 13:8749-8764 (1985); Taylor, J.W. et al., Nucleic Acids Research, 13:8764-8785 (1985); Nakayame, K. and F. Eckstein, Nucleic Acids Research, 14 :9769-9698 (1986).
• the gapped DNA was repolymerized using DNA polymerase I in the presence of T4 DNA ligase. Because the mutant strand served as the template, the mutation or modification was copied into both strands. The resulting double-stranded DNA was introduced into competent TGI by transformation. Mandel, M. and A. Higa, Journal of Molecular Biology, 53:154 (1970). Derived plaques were grown up and single stranded and replicative form DNAs were isolated. The DNA was sequenced to confirm the presence of the mutation.
• Mutated DNA (DNA including the mutation introduced as a result of the DNA synthesis using the modified oligonucleotide, as described above) was excised from the replicative form of DNA with Xba and ligated to vector CDM8 which had been cut by Xba.
• the CDM8 vector is expressed in Cos cells upon transfection.
• Cos cells are a monkey kidney cell line, which have been transformed by simian virus 40 (SV40) DNA which includes the functional early gene region, and thus constitutively expresses the SV40 large T antigen, but has a defective origin of viral DNA replication.
• SV40 simian virus 40
• CD4 proteins The vector-insert ligation mixture was introduced into competent MC1061F3 nost bacteria and CDM8 containing mutant T4 cDNAs, were identified by hybridization to radiolabelled T4 DNA. Ausubel, F.M. et al. (ed.), Current Protocols in Molecular Biology, Greene Publishing Associates, p. 1.4.9 (1938), Seed, B. and A. Aruffo, Proceedings of the National Academy of Sciences, USA, 84: 3365-3369 (1987). Restriction enzyme analysis of mini-prep DNAs was used to determine the proper orientation of the insert in the CDM8 vector.
• Cos cells transfected with the CDM8 vector containing mutant T4 cDNA were assayed for production of modified soluble CD4 proteins capable of binding HIV, as described briefly in the following sections and in detail in the Exemplification.
• Cos cells transfected with the mutant T4 cDNA-containing CDM8 vector were processed in order to produce dialyzed supernate, which was precleared with control rabbit anti-T cell receptor IgG coupled to a Sepharose support in order to minimize non-specific binding.
• the precleared supernate was immunoprecipitated with a monoclonal anti-CD4 antibody (19Thy5D7) coupled to a Sepharose support.
• 19Thy5D7 is an antibody against a T4 epitopic site which competes with HIV for binding of gp120.
• binding of a component of the supernate to 19Thv5D7 is suggestive of the presence in the supernate of a component capable of binding HIV.
• CD4 fragments produced in this manner to bind to tne HIV exterior gp120 glycoprotein can be directly determined as follows:
• Cos supernates determined to contain optimal levels of recombinant, secreted CD4 protein will be taken for co-precipitation studies. For example, 67 ng of gp120 (1 ul at 67 ug/ml in PBS/0.1% BSA) can be added to 0.5 ml of Cos supernates. As a control, no addition is made to a second 0.5 ml aliquot of supernate. After a 2 hour incubation at 37oC, 500 ng of monoclonal anti-gp120 are added to both supernates, followed by rabbit anti-mouse IgG coupled to Sepharose 4B (10 ul). The samples are then rotated for 2 hours at 4oC.
• T4 ex1 protein protein encoded by the modified T4 DNA
• Rabbit heteroantisera to the CD4 protein is also available for identification of modified CD4 products in which monoclonal CD4 epitopes are no longer present.
• T4 ex1 a modified CD4 protein
• Co-precipitation of gp120 with T4 protein produced in Cos is readily detected in the presence of anti-gp120 antibody plus rabbit anti-mouse Ig.
• the co-precipitated product will be a 50KD band in
• the modified soluble CD4 protein produced as described above includes the amino acid sequence (as shown in Figure 1) of the CD4 protein through amino acid 183.
• this truncated soluble CD4 protein has the further advantage that it lacks the glycosylation sites present in T4 ex1 and should, thus, be less immunogenic.
• the terminal amino acid (histidine) present in the mature CD4 protein encoded by the nucleotide sequence of Figure 1 is absent from the biologically active, modified soluble CD4 fragment of the present invention. It is also absent in the native CD4 molecule. Because the glycosylation sites are not present in the CD4 fragment produced in this manner, fragments of this type can be expressed in a bacterial host.
• Immunoprecipitation of CD4 protein having amino acid residues 1-182 (as represented in Figure 1) identifies a band of approximately 19 kD on SDS-PAGE of 19Thy5D7 immunoprecipitates from transfected Cos supernatants. Co-precipitation studies with gp120 and anti-gp120 antibody identifies the same band.
• truncation at a different amino acid can be carried out.
• truncation of T4 ex1 after amino acid 369 i.e., removal of the carboxy terminal histidine
• a termination codon see Figure 1
• the resulting truncated form will retain the capability of binding HIV.
• such a modified form has the advantage that it lacks the histidine present in the CD4 protein encoded by the cDNA of Figure 1 and not present in the native molecule.
• Another approach is to produce biologically active, modified soluble CD4 fragments in such a manner that fragments which include of one or more domains of the encoded protein are obtained.
• CD4 region of the CD4 molecule that binds gp120 has not yet been defined. Nor is it known whether the same or different segments of CD4 bind to an invariant region of class II MHC molecules(s) which are the presumed pnysiologic CD4 ligand. Meuer, S. et al., Proceedings of the National Academy of Sciences, USA, 79 : 4395 (1982); Biddison, W. et al., Journal of Experimental Medicine, 156:1065 (1982); Gay, D. et al., Nature, 328: 626 (1987). In this regard, sequence analysis of CD4 has suggested an evolutionary origin from a structure with four immunoglobulin-related domains (Figure 7A). Two of these domains (the first two) are involved in HIV gp120 binding. The NH 2 -terminal
• CD4 domain (amino acids 1-92), termed domain 1, bears the most structural homology to Ig light chain variable regions (about 32% at amino acid level).
• the NH 2 -terminal region of CD4, including the immunoglobulin V-like domain, has been snown to be required for gp120 interaction.
• the carboxy terminal half of the molecule containing the two potential N-glycosylation sites does not appear to be necessary.
• T4 ex1 construct A domain 1 T4 ex1 construct and a partial domain 2 mutant will be obtained if a TAG termination codon is inserted after the fourth cysteine, which produces truncation after amino acid 162 ( Figure 1). This will produce a modified soluble CD4 fragment capable of binding HIV and will also make manufacture/production easier because of improved ability to introduce the construct into a cell line.
• the method described herein can be used, with appropriate modification, to convert asparagine and N-linked glycosylation sites at positions 271 and 300 to asparate.
• This can be carried out in the same construct or in two separate constructs (each including one of the two modifications).
• the two codons at the positions indicated will be modified: in the case of the codon for amino acid 271, to GAC and in the case of the codon for amino acid 300, to GAT.
• This modified protein will also bind HIV and has the further advantage that because the glycosylation sites are no longer present, it will be less immunogenic than a fragment which includes such sites.
• Additional modification of the mature CD4 protein can similarly be made, as desired, and subsequently shown to have the capability of binding HIV by the means described herein.
• Expression of additional constructs will be carried out, for example, in baculovirus (e.g., Autographa californica), Chinese hamster ovary (CHO) cells or E. coli.
• Transfer of the T4 ex1 se ⁇ uence from the plasmid vector to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome can be accomplished essentially as described in Smith et al., (1985) Proceedings of the National Academy of Sciences, USA,82:8404-8408.
• SFS Spodoptera frugjperda
• Recombinant AcNPV contains an inactivated polyhedrin gene which no longer forms occlusions in infected cells, thus providing a means by which infected and noninfected cells can be distinguished.
• 2 x 10 6 SF9 cells can be seeded in 100 mM petri dishes 24 hours prior to assay. Ten fold dilutions of viral supernatant are prepared, using final media (Grace's insect medium (Gibco, Grand Island, NY), TC yeastolate 0.33%, lactalbumin hydrosylate 0.33%, 2 mM supplemental glutamine and 50 ug/ml gentamycin containing 10% FCS (Hyclone, Logan, UT).
• Each plate is innoculated with virus (e.g., 1 ml., 10 -3 to 10 -7 dilution) plus 2 ml of final media. After incubation for 2 hours, the innoculum is removed and replaced with 10 ml of 1.5% Sea Plaque agarose (FMC Bioproducts, Rockland, ME) in final media. Plates are transferred to a a humid environment after agarose solidification for 4-6 days at 27oC.
• virus e.g., 1 ml., 10 -3 to 10 -7 dilution
• Plaque assay of the transfection supernatant will demonstrate distinct morphological differences between infected cells; infected cells which are occlusion positive contain wild type AcNPV and infected cells which are occlusion negative contain recombinant CD4 virus. Occlusion-negative plaques are identified, selected, and further plaque purified. DNA from cells infected with putative CD4 recombinant virus will be hybridized with a 32 P labelled CD4 cDNA probe to verify tne presence of the CD4 seguence.
• T4 ex polypeptide Production of the T4 ex polypeptide is carried out as follows: SF9 cells (6 x 10 5 cells per well) are seeded per well in 24 well Nunc plates (Interlab, Thousand Oaks, CA) for 2 hours at 27°C and then adherent SF9 cells are infected with virus at an MOI of 10 in 0.2 ml final media for 2 hours. The innoculum is then removed and cells are cultured in 0.5 ml fresh medium at 27oC for 48 hours. Adherent cells are then washed twice with 0.5 ml Grace's medium lacking serum and methionine. This is followed by incubation in 0.5 ml in the same medium for 1 hour.
• the adherent cells are washed once and then cultured for 6 hours in serum and methionine-free Grace's medium containing 67 uCi 35 S methionine (New England Nuclear, Boston, MA 1134 Ci/nmol). Culture supernatants are harvested, microfuged for 10 minutes, and dialyzed at 4oC against PBS containing 0.5% sodium azide and 10 mM cold methionine.
• Cells are dislodged from the wells, washed twice with Grace's medium at 4oC (by centrifugation in a Sorvall RT6000 for 5 min at 1000 rpm) and finally lysed for 30 min at 4oC by the addition of a RIPA buffer containing 1% Triton X-100, 0.15 M NaCl and a cocktail of protease inhibitors, as described below.
• the lysates are microfuged for 10 min and dialyzed at 4oC using the same procedure as was used for culture supernatants.
• Both lysates and culture supernatants are subjected to immunoprecipitation for 16 h at 4oC with a monoclonal anti-CD4 antibody (19Thy5D7) linked to Affigel-10 beads (5 mg monoclonal antibody/ml gel).
• the beads are washed twice with lysis buffer and bound material is eluted by treatment of the beads with 0.1M glycine-HCl buffer, pH 2.0.
• Eluates and whole samples of lysates or culture supernatants are mixed with SDS sample buffer containing 2-mercaptoethanol, boiled for 5 minutes and electrophoresed in 12.5% mini-slab gels according to Laemmli. Laemmli, Nature, 227: 680-685 (1970). Subsequently, the gels are fixed, dried and autoradiographed using Kodak XAR-5 film.
• High titer viral stocks are generated by infecting SF9 cells at an MOI of 1 and culturing at 1 x 10 6 cells/ml for 4 days in final media. These stocks are used for infecting SF9 cells for production of protein.
• SF9 cells are grown in 2 liter spinner flasks in final media. Cells are harvested and infected with an MOI of 15 (using high titer viral stocks) at a concentration of 10 x 10 6 cells/ml.
• Cells are then pelleted, resuspended in media at 1 x 10 /ml, and cultured for 3 days at 27°C in spinner flasks. At this time, supernatants are collected by centrifuging cultures to remove cells.
• infected SF9 cell culture supernatants are harvested by centrifugation of cells in a Sorvall H-4000 rotor at 800 rpm for 6 minutes at 4°C .
• the culture supernatants are then subjected to protease inhibition by the addition of a cocktail of protease inhibitors made up of leupeptin, antipain, pepstatin, and chymostatin to final concentrations of 0.5 ug/ml; soybean trypsin inhibitor to 0.02 ug/ml; and phenyl metnyl suifonyi fluoride (PMSF) to 1.25 mM, followed by adjustment of the pH to 6.8 by the dropwise addition of 1 M NaOH .
• protease inhibitors made up of leupeptin, antipain, pepstatin, and chymostatin to final concentrations of 0.5 ug/ml
• soybean trypsin inhibitor to 0.02 ug/ml
• the samples are subsequently clarified by centrifugation in a Sorvall GSA rotor at 8000 rpm for 25 minutes at 4°C and pumped at 4°C at a flow-rate of 30 ml/hour through a 2 ml precleared immunoabsorbent column, 21Thy2D3 monoclonal antibody (anti-T8) coupled to Affigel-10 (Biorad), followed in series by a 7 ml column of anti-CD4 monoclonal antibody (19Thy5D7) coupled to Affigel 10 at a concentration of 7.5 mg monoclonal antibody per ml of gel.
• the monoclonal antibodies are made according to conventional methods.
• the anti-CD4 column is then washed with 30 ml of 10 mM Tris-HCl buffer, pH 6.8 followed by 15 ml of 0.1M glycine-HCl, pH 5.0.
• the bound CD4 polypeptides are eluted by pumping 0.1 M glycine-HCl, pH 2.0 through the washed anti-CD4 column and 0.8 ml fractions of eluant are collected into tubes containing 0.15 ml 1 M Tris-HCl, pH 7.5.
• eluate absorption is monitored at 280 nm with a Uvicord 2 (LKB, Gaithersburg, MD) fitted with an event marker.
• Regions or sites on human CD4 critical for HIV gp120 binding were identified, as described below and, based on the identification of critical sites, soluble human CD4 fragments with altered HIV gp120 binding ability were produced, as is described below, particularly in Examples 4 and 5.
• oligonucleotide-directed mutagenesis was used to create each of 16 individual mutant human CD4 molecules containing from 1 to 4 amino acid substitutions.
• Introduction of as few as three amino acids into corresponding positions of human CD4 resulted in CD4 fragments unabl.e to bind gp120.
• These critical residues have been shown to be located in domain I as well as in domain II of CD4 , thus implying that tne gp120 binding site is complex and involves both of the NH 2 -terminaI domains.
• Modelling studies using the 3-dimensional coordinates of the V k Bence-Jones homodimer, REI localize the site of domain I to the C'' ⁇ strand. Thus, domain I is distant from the loops analogous to hypervariable regions.
• Residues of the CD4 structure involved in HIV gp120 binding were characterized through use of a
• T4 ex1 Cos-1 cell expression system and a cDNA encoding the anchor-minus CD4 segment termed T4 ex1 .
• the 370 amino acid T4 ex1 protein (Figure 1) contains 369 of the predicted 372 NH 2 -terminal amino acids of the CD4 extracellular segment and a COOH-terminal histidine. As shown in. Figure 8, this structure is comprised of three intrachain disulfide bonded domains (a domain is defined as residues between and including 20 amino acid residues to either side of the cysteines), and one domain (III) which lacks cysteine residues but, like its counterparts, is immunoglobulin-like. Clark, S., et al., Proc. Natl. Acad. Sci. USA, 84:
• Nanomolar concentrations of T4 ex1 inhibit gp120-transmembrane CD4 interaction, syncytium formation and HIV infection by binding to gp120-expressing cells.
• the T4 ex1 construct was subcloned into the vector CDM8 and transfected into Cos-1 cells.
• Supernatants from metabolically labelled transfected cells were tested by immunoprecipitation with an anti-CD4 monoclonal antibody (19Thy5D7).
• the resulting precipitate was subjected to SDS-PAGE. Results showed the presence of a 50KD CD4-derived molecule in transfected Cos-1 cell supernatants ( Figure 9, lane 3).
• the murine CD4 sequence does not bind gp120, and, thus, it was anticipated that some murine substitutions would abrogate human CD4-gp120 interaction.
• Table 1 15 oligonucleotides were used in a standard site-directed mutagenesis protocol, as described in Example 4, to produce 16 different versions of the human CD4 molecule containing from 104 substitutions each. The positions of these substitutions are listed in Table 1 and diagramatically mapped in Figure 8. All 16 CD4 mutants were assayed after transfeetion into Cos-1 cells by immunoprecipitation with anti-CD4 monoclonal antibody and by gp120 co-precipitation with anti-gp120.
• M1A 223 CAA-TTC-ACC-TCC-AAA-TTC-TCC-CAC-CAG-AGA-AAG 255 aa 30 N to F + +
• M1B 223 CAA-TTC-ACC-TGG-AAA-TTC-TCC-CAC-CAG-AGA-AAC 255 aa 30 N to F + +
• M5 , M10, M7 and M3 react with the anti-CD4 monoclonal antibody 19Thy5D7. As shown in Table 1, 15 of the 16 mutants react with anti-CD4 antibody. Only mutant
• M3 contains three amino acid substitutions in human CD4 domain I at positions 48, 50 and 51. One or more of these changes clearly abrogates the ability of CD4 to bind to HIV gp120.
• M9 contains three amino acid substitutions in domain II of CD4 at positions 121-123. Thus, alteration of a few residues in either CD4 domain I or domain II results in abrogation of HIV gp120 binding.
• M14 demonstrates reduced binding to gp120 (Table 1). M14 also failed to bind to the two anti-CD4 monoclonal antibodies examined.
• CD4 domain 1 to gp120 binding was recognized previously in studies of the T4 6 ⁇ .i. polypeptide produced in a baculovirus system in conjunction with proteoiytic fragmentation analysis, microsequencing and a specific CD4-gp120 binding assay.
• Richardson, N.E., et al., Proc. Natl. Acad. Sci. USA. Richardson and co-workers showed that disruption of the peptide bond at lysine 72 by tryptic cleavage destroyed CD4-gp120 interaction without inducing any detectable alterations in other domains of CD4.
• V k Bence-Jones homodimer REI.
• Use of this model has resulted in accurate prediction of each of three tryptic cleavage sites in domain I to be surface exposed, thus supporting the validity of the CD4 model.
• the region of residues in the alpha carbon skeleton of the REI homodimer corresponding to the mutated CD4 residues which abrogate gp120 binding were determined. This region corresponds to the C'' strand uni ⁇ ue to V domains which connect the two sheets. Williams, A.F., et al., Ann. Rev. Immunol., 6, 381-405 (1988).
• the alignment between REI and CD4 requires a gap in this segment, and, thus., it is not meant to imply that the CD4 alpha carbon skeleton follows an identical course in this region.
• CD4 se ⁇ uence will loop out and be solvent exposed. Furthermore, it should be noted that this site is distinct from the three segments equivalent to the hyper-variable loops of the REI homodimer.
• the region of CD4 domain I implicated as a possible binding site for gp120 is distinct from the loops analagous to hypervariable complementarity determining segments. If those loops form a binding site for class II MHC, the putative natural ligand of CD4 one can speculate that gp120 may be incapable of inhibiting class II recognition events, even after binding to the CD4 structure. Krensky, A.M., et al., Proc. Natl. Acad. Sci. USA, 79: 2365-2369 (1982); Meuer, S.C., et al., Proc. Natl. Acad. Sci. USA, 79: 4395-4399 (1982); Biddison, W., et al., J.
• CD4 mutants described herein should be useful in future analysis of CD 4- class II MHC interactions.
• T4 ex1 M3 fails to bind gp120; M9 has substantially reduced gp120 binding capacity; and M14 demonstrates reduced gp120 binding ability.
• M3 and M9 are recognized by anti-CD4 antibody and M14 is not recognized by either of the two anti-CD4 antibodies used.
• substitutions can be made: 1) at one, two or all three of the critical sites (i.e., at one or more of the three amino acid sites represented by mutants M3 , M9 and M14) and/or 2) of one, two or all three amino acid residues within each site ( i .e., within a critical site, of amino acid residues 1, 2 or 3 individually, 1, 2 ana 3 in any combination of a 2 amino acid residues; or of all three amino acid residues).
• mutant M3 glycine, proline and serine, respectively, replace proline, lysine and leucine, which occur at amino acid positions 48, 50 and 51 of human CD4.
• substitution of one or more of those amino acids by other amino acids of the same type (e..g, at position 48 by another amino acid with a nonpolar R group) as that present at that position in M3 can be made and the effect on gp120 binding ability determined.
• substitutions at these three sites, individually or in combination, of amino acids having characteristics different from those of amino acid whose presence at those sites has been shown to eliminate or reduce gp120 binding ability can also be made and their effect on binding ability assessed using the anti-CD4 immunoprecipitation and anti-gp120 coprecipitation methods described in the Examples.
• substitutions of some or all of the amino acids at one or more of these critical sites which result in modified soluble CD4 fragments with enhanced gp120 binding ability can be made.
• CD4 fragments having enhanced binding ability can be identified.
• modified soluble human CD4 fragments with enhanced gp120 binding ability is as follows: amino acid residues present at the three sites in human CD4 (as represented in Figure 1) and amino acid residues present at the corresponding positions in the three mutant CD4 molecules are excluded from the group of amino acid residues to be assessed for their effects on gp120 binding ability when they are incorporated at these sites. Also excluded are amino acids having similar characteristics (e.g., nonpolar R groups, uncharged polar R groups, etc.). Mutants are then produced to include amino acid residues other than those eliminated from consideration in this manner. Each mutant is then assessed using the anti-CD4 immunoprecipitation and anti-gp120 coprecipitation techniques described.
• modified soluble human CD4 fragments having enhanced gp120 binding ability can be identified. Similar techniques can be used to identify additional critical sites, if such sites exist, and, subsequently, to make substitutions and assess their effects on gp120 binding ability of the resulting modified soluble CD4 fragments.
• Modified soluble CD4 fragments having altered gp120 binding ability are produced using the techniques described in detail in Examples 4 and 5. Briefly, they are produced as follows:
• DNA encoding a desired CD4 fragment is produced, either by using recombinant DNA techniques, such as excising it from a vector containing cDNA encoding such a fragment, or by synthesizing DNA encoding the desired fragment mechanically and/or chemically, using known techniques.
• DNA produced by tnese techniques encodes a soluble CD4 fragment which includes none of the hydrophobic transmembrane region of CD4 or a portion of that region (generally six amino acids or less) small enough that it does not prevent solubilization of the fragment.
• the CD4 fragment is long enough (e.g., 10 amino acids or more) to bind effectively to HIV gp120 envelope protein.
• Templates for subsequent mutagenesis are produced, using the CD4 fragment-encoding cDNA or DNA. As described below, this can be carried out using a single-stranded bacteriophage cloning vehicle, such as M13. This results in production of single-stranded DNA homologous to only one of the two strands of the DNA encoding the desired CD4 fragment. The resulting single-stranded DNA is used as a template for producing the desired modified soluble CD4 fragments, as follows:
• Oligonucleotides are produced, such that their sequence includes a base change or changes which, when incorporated into the nucleotide sequence of DNA subsequently used for the production of CD4 fragments, results in the desired change in the encoded CD4 protein (i.e., different from that encoded by the nucleotide sequence of Figure 1). Such oligonucleotides are produced using standard methods. Oligonucleotides having a base change or base changes are referred to as mutagenized or mutant oligonucleotides.
• the mutant oligonucleotide produced in this manner is hybridized to (e.g., by being kinased) the template produced as described above, to produce a template-mutant oligonucleotide complex, referred to as a mutant primer/template.
• the mutant primer/template is used for the production of a second strand of DNA, using well-known techniques. For example, synthesis of the second DNA strand is carried out by the Klenow fragmnet of DNA polymerase in the presence of dCTP ⁇ S. Taylor, J.W. et al., Nucleic Acids Research, 13: 8749-8764 (1985); Taylor, J.W. et al., Nucleic Acids Research, 13 :8764-8785
• the resulting strand of DNA contains a modification (or modifications) in the nucleotide sequence of T4 cDNA (i.e., is different from the nucleotide se ⁇ uence represented in Figure 1) and is referred to as a mutant strand.
• Unreplicated single-stranded DNA is removed and the double-stranded DNA is nicked with a selected restriction enzyme (e.g., Neil, which does not cut phosphorothioate DNA and, thus, does not cut the new DNA strand containing dCTP S or the mutant strand).
• a selected restriction enzyme e.g., Neil, which does not cut phosphorothioate DNA and, thus, does not cut the new DNA strand containing dCTP S or the mutant strand.
• Nicked, nonmodified DNA is removed by digestion with another enzyme, such as exonuclease III.
• the resulting gapped DNA is repolymerized and, because the mutant strand serves as the template for repolymerization, the mutation or modification is copied into both strands.
• the double-stranded DNA in which both strands contain the mutation or modification encoding the corresponding modification in the amino acid sequence of the desired soluble CD4 fragment is introduced into a competent host cell, such as a competent bacterial host (e.g., by transformations
• a competent host cell such as a competent bacterial host
• the resulting plaques are grown and DNA contained in them is isolated, using known tecnniques, and sequenced to confirm the presence of tne mutation.
• the mutated DNA produced in this manner is excised from the M13 vector containing it, introduced into a suitable expression vector, such as CDM8, and transfected into an appropriate host cell, such as Cos cells, in which it is expressed.
• a suitable expression vector such as CDM8
• mutant CD4 proteins can be assayed, using known techniques.
• the vector-insert ligation mixture is introduced into competent host bacteria, such as the publicly available E. coli MC1061P3, and radiolabelled T4 DNA is used to identify CDM8 containing mutant T4 cDNAs.
• double stranded DNA encoding a modified soluble GD4 fragment having altered gp120 binding ability is produced, the encoded CD4 fragment is expressed and its ability to bind the HIV gp120 envelope protein is assessed.
• modified soluble human CD4 fragment having altered gp120 binding ability is to use peptide synthesis to make a peptide or polypeptide having the amino acid sequence of such a fragment.
• Soluble human CD4 fragments of the present invention have diagnostic, preventative and therapeutic applications.
• biologically active soluble human CD4 fragments can be used for diagnosis, therapy and prevention of infection by
• such fragments can be used therapeutically (in vivo ) to treat individuals infected with HIV.
• Such fragments can be administered by an acceptable route (e.g., intravenously, intramuscularly, intraperitoneally, orally), alone or after combination with an acceptable carrier (e.g., saline buffer). They can be administered to inhibit binding of HIV to T4 lymphocytes and to inhibit HIV transmission from an infected cell to uninfected cells by interfering with syncytium formation.
• the quantity of such CD4 fragments administered will be determined on an individual basis, but will generally range from approximately 10 ug/kg body weight to approximately 500 ug/kg body weight per day (in one or more doses per day).
• Biologically active soluble CD4 fragments of the present invention can also be used for diagnostic purposes. For example, they can be used in known immunoassay procedures for detecting the presence and determining the quantity, if desired, of HIV gp120 envelope protein (and, as a result, of HIV itself) in samples, such as blood, semen and saliva.
• CD4 fragments of the present invention can be, for example, attached or bound by virtue of the CD4 fragment to a solid support, such as latex beads, which are then contacted with a sample to be assayed, in such a manner that if HIV is present in the sample, it will be bound (by virtue of the CD4 frament-gp120 interaction).
• Biologically active soluble CD4 fragments can also be used for the prevention of HIV infection.
• such fragments can be incorporated in or attached to materials which might come in contact with HIV. They can be incorporated into spermicides, incorporated into or attached to surfaces of condoms, materials from which surgical gloves, dressings and other medical equipment are made or attached to the surfaces of containers or other materials (e.g., filters) for receiving, processing and/or storing blood.
• the CD4 fragments of the present invention will bind to HIV gp120 envelope protein (and, thus, to HIV), which will be prevented from further passage (e.g., in the case of spermicides, condoms) or can be removed (e.g., in the case of donated or stored blood).
• modified soluble CD4 fragments of the present invention with altered (i.e., enhanced gp120 binding ability) will be shown to have the same advantage described for soluble human CD4 fragments. That is, it is reasonable to assume such fragments of the present invention have the capacity to bind the KIV gp120 envelope protein and interfere with KIV infection of T cells, but will not interfere with the function or proliferation of human T lymphocytes which are not infected with HIV.
• the capability of fragments to bind gp120 envelope protein and interfere with HIV infection and their lack of interference with uninfected T lymphocytes can be assessed by means described herein.
• Modified soluble human CD4 fragments having altered gp120 binding ability can be used for therapy, diagnosis and prevention of infection by
• fragments having slightly reduced or turned down affinity may improve the effective pharmokinetics of therapy.
• fragments can be used to bind or hold on to gp120 (and, thus, HIV) transiently.
• Such fragments bind the virus long enough to render it ineffective as an infectious agent and to prepare it to bind or accept another therapeutic agent (e.g., one which will destroy the virus).
• the region of the CD4 domain I implicated as a possible binding site for gp120 is distinct from the loops analogous to hypervariable complementarity determining segments. If those loops form a binding site for class II MHC, the putative natural ligand of CD4, one can speculate that gp120 may be incapable of inhibiting class II recognition events, even after it has bound to the CD4 structure. Thus, the CD4 mutant described herein should be useful in future analysis of CD4 class II MHC interactions.
• Fragments of the present invention having enhanced gp120 binding ability can be used therapeutically (in vivo) to treat individuals infected with HIV.
• Such fragments can be administered by an acceptable route (e.g., intravenously, intramuscularly, intraperitoneally, orally), alone or after combination with an acceptable carrier (e.g., saline buffer).
• Modified soluble CD4 fragments with enhanced gp120 binding ability of the present invention can be administered to inhibit binding of HIV to T4 lymphocytes and to inhibit HIV transmission from an infected cell to uninfected cells by interfering with syncytium formation.
• the quantity of such CD4 fragments administered will be determined on an individual basis, but will generally range from approximately 10 ug/kg body weight to approximately 500 ug/kg body weight per day (in one or more doses per day).
• Modified soluble CD4 fragments having enhanced gp120 binding ability can also be used for diagnostic purposes. Because of their enhanced binding ability, they can be used in known immunoassay procedures for detecting the presence and determining the quantity, if desired, of HIV gp120 envelope protein (and, as a result, of HIV itself) in samples, such as blood, semen and saliva.
• CD4 fragments of the present invention can be, for example, attached or bound by virtue of the CD4 fragment to solid support, such as latex beads, which are then contacted with a sample to be assayed, in such a manner that if HIV is present in the sample, it will be bound (by virtue of the CD4 fragment-gp120 interaction). This can be followed by precipitation and/or labelling through contact with an anti-gp120 antibody and detection of the precipitate or labelled product, using known techniques.
• Modified soluble CD4 fragments having enhanced gp120 binding ability can also be used for the prevention of HIV infection.
• such fragments can be incorporated in or attached to materials which might come in contact with HIV. They can be incorporated into spermicides; incorporated into or attached to surfaces of condoms, materials from which surgical gloves, dressings and otner medical equipment are made; or attached to the surfaces of containers or other materials (e.g., filters) for receiving, processing and/or storing blood.
• the CD4 fragments of the present invention will bind to HIV gp120 envelope protein (and, thus, to HIV), which will be prevented from further passage (e.g., in the case of spermicides, condoms, surgical gloves, dressings) or can be removed (e.g., in the case of donated or stored blood).
• cDNA encoding human CD4 was engineered in order to delete the nucleotide sequence encoding the hydrophobic transmembrane region, which ordinarily renders CD4 memDrane bound and insoluble. As a result, cDNA encoding soluble human CD4 fragments was produced.
• Plasmid Construction can best De descriDed with reference to Figure 2.
• plasmid vector pAc373/T4 ex containing the truncated CD4 gene, was constructed from plasmids pAc373 and pSP65-T4.
• the plasmid transfer vector pAc373 contains a single BamHI cloning site 8 base pairs upstream of the polyhedrin ATG start site.
• the plasmid CD4 protein-encoding pSP65-T4 (kindly provided by Dan Littman, Univ.
• CD4 cDNA insert (which can be readily obtained as described in the literature, as in, for example, Madden et al. Cell, 42: 93-104 (1985)).
• the CD4 cDNA insert was subsequently digested with Neil, which cleaves CD4 cDNA at nucleotide positions 83, 1253 and 1604, producing a fragment of 1.17Kb which lacks the ATG start codon and terminates just prior to the transmembrane region.
• oligonucleotides 5'AGGATCCTTAATGAACC3 ' and 5'CGGTTCATTAAGGATCCT3', were synthesized, using standard cyanoethyl phosphoramidite chemistry. They were annealed and kinased to generate a linker molecule which reconstructs the ATG translation initiation codon, includes a stop codon (TAA) for termination of transcription, creates an Neil cohesive end, and adds a BamHI cloning site. Linkers were ligated to the 1.17Kb CD4-encoding fragment and then digested with BamHI to generate BamHI cohesive ends.
• TAA stop codon
• the CD4-encoding fragment was inserted into the BamHI cloning site of the publicly available transfer vector pAc373.
• Recombinant plasmids containing a single copy of the truncated CD4 molecule in the correct orientation were identified by restriction mapping. The constructs were then sequenced by the 35 S-ATP labelled dideoxy method to confirm the expected sequence at the junctions of insertion.
• Recombinant plasmids pAc373/T4 ex1 and pAc373/T4 ex2 were characterized in detail. They contained identical 5' ends. The synthetic linker ligated in the expected orientation in pAc373/T4 ex1 to result in a predicted CD4 protein carboxy-terminus of LPTWSTPVH.
• Transfer of the T4 ex sequence from the plasmid vector to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome was accomplished essentially as described in Smith et al. (1985) P.N.A.S. U.S.A. 82, 8404-8408.
• SF9 S. frugiperda cells
• Recombinant AcNPV contains an inactivated polyhedrin gene which no longer forms occlusions in infected cells.
• 2 x 10 6 SF9 cells were seeded in 100 mM Petri dishes approximately 24 hours prior to assay.
• Ten fold dilutions of viral supernatant were prepared using final media [Grace's insect medium (Gibco, Grand Island, NY), Difco TC yeastolate 0.33%, lactalbumin hydrcsylate 0.231, 2 mM supplemental glutamine and 50 ug/ml gentamycin containing 10% FCS (Hyclone, Loga, UT ) ] .
• Each piate was innoculated with 1 ml of virus (10 -3 to 10 -7 dilution) plus 2 ml of final media. After incubation for 2 hours, the innoculum was removed and replaced with 10 ml of 1.5-5 Sea Plaque agarose (FMC Bioproducts, Rockland, ME) in final media. After agarose solidification, plates were transferred to a humid environment for 4-6 days at 27oC.
• Plaque assay of the transfection supernatant yielded plaques of distinct morphology: either infected cells which are occlusion positive (wild type AcNPV) or occlusion negative (recombinant CD4 virus). Occlusion-negative plaques were identified, selected, and further plaque purified. DNA from cells infected with putative CD4 recombinant virus was hybridized with a 32P labelled CD4 cDNA probe to verify the presence of the CD4 sequence.
• T4 ex polypeptide was carried out as follows: 6 x 10 5 SF9 cells were seede ⁇ per well in 24 well Nunc plates (Interlab, Thousand Oaks, CA) for 2 hours at 27oC and then adherent cells infected with virus at an MOI of 10 in 0.2 ml final media for 2 h. The innoculum was then removed and cells cultured in 0.5 ml fresh medium at 27oC for 48 hours. Adherent cells were then washed twice with 0.5 ml Grace's medium lacking serum and methionine followed by incubation in 0.5 ml in the same medium for 1 hour. The adherent cells were washed once and then cultured for 6 hours in serum and methionine-free Grace's medium containing 67 uCi 3 5 S methionine (New England
• the lysates were microfuged for 10 minutes and dialyzed at 4"C as for culture supernatants. Both lysates and culture supernatants were subjected to immunoprecipitation for 16 hours at 4oC with a monoclonal anti-CD4 antibody (19Thy5D7) linked to Affigel-10 beads (5 mg monoclonal antibody/ml gel). After immunoabsorption, the beads were washed twice with lysis buffer and bound material was eluted by treatment of the beads with 0.1 M glycine-HCl buffer, pH 2.0.
• Cells were harvested and infected with an MOI of 15 (using high titer viral stocks) at a concentration of 10 x 10 6 cells/ml. Cells were then pelleted, resuspended in media at 1 x 10 6 /ml, and cultured for 3 days at 27oC in spinner flasks. At this time, supernatants were collected by centrifuging cultures to remove ceils.
• infected SF9 cell culture supernatants were harvested by centrifugation of cells in a Sorvall H-4000 rotor at 800 rpm tor 6 minutes at 4oC.
• the culture supernatants were then subjected to protease inhibition by the addition of a cocktail of protease inhibitors made up of leupeptin, antipain, pepstatin, and chymostatin to final concentrations of 0.5 ug/ml; soybean trypsin inhibitor to 0.02 ug/ml; and phenyl methyl sulfonyl fluoride
• the anti-CD4 column was then washed with 30 ml of 10 mM Tris-HCl buffer, pH 5.0.
• the bound CD4 polypeptides were eluted by pumping 0.1 M glycine-HCl, pH 2.0, through the washed anti-CD4 column and 0.8 ml fractions of eluant were collected into tubes containing 0.15 ml 1 M Tris-HCl, pH 7.6.
• eluate absorption was monitored at 280 nm with a Uvicord 2 (LKB, Gaithersburg, MD) fitted with an event marker.
• SF9 cells infected with either recombinant baculovirus containing the T4 ex cDNAs or wild type AcNPV virus were cultured in 35 S-methionine and products were examined by SDS-PAGE, followed by autoradiography.
• T4 ex1 polypeptide is the major secreted product of SF9 cells infected with the T4 ex1 recomnbinant nieunlnovirus.
• the predoominant 35 S labelled protein band (45% of total labelled material) in SDS-PAGE analysis of supernatants from
• SF9 cells obtained 54 hours after T4 ex1 recombinant baculoviral infection is a 50KD band under reducing conditions .
• This band co-migrates with material immunoprecipitated by anti-CD4 monoclonal antibody (19Thy5D7) from T4 ex1 baculovirus infected SF9 supernatants or cell lysates.
• the latter shows a strongly labelled band of 52KD which presumably represents the T4 ex1 polypeptide still carrying the uncleaved signal peptide.
• 50KD band is readily detected in the total cell lysate of T4 ex1 virus infected cells even in the absence of immunoprecipitation with anti-CD4 monoclonal antibody, it is a minor component of a complex mixture of labelled intracellular polypeptides. As expected, no CD4 material was precipitated from supernatants of wild type AcNPV-infected cells or detectable in the total supernatant.
• T4 ex2 preparations yielded a protein that migrated under reducing conditions with a molecular weight of 5 1KD (and is glycosylated as indicated by endoglycosidase F experiments), whereas the T4 ex1 protein migrated slightly faster with a molecular weight of 50KD.
• T4 ex1 and T4 ex2 proteins are not unexpected, since T4 ex2 contains 17 additional carboxy terminal amino acids derived from fusion with the polyhedrin gene.
• T4 ex1 protein is faster than under reducing conditions, consistent with previous predictions that there are intrachain disulfide bonds in the CD4 external segment, and also showing the absence of covalent disulfide linked polymers of T4 ex1 protein.
• the protein production strategy described above routinely yields 1-2 mg of secreted T4 ex1 or T4 ex2 proteins per liter of SF9 cells (1-2 x 10 9 cells) over a 72 hour culture period.
• the codon for the amino terminal residue was determined to be AAG, rather than AAC, as given in the original CD4 cDNA cloning paper (Madden et al., id) without other differences noted. Whether this single nucleotide discrepancy represents a mutation resulting from cloning into pAc373 is not known, but appears unlikely in view of the lysine residue found at the N-terminus of the homologous rat CD4 sequence. From these data, it was concluded that the amino terminus of mature human CD4 begins with two lysine residues, followed by two valine residues and that T4 ex1 and T4 ex2 are CD4 derived polypeptides.
• this data shows that the baculovirus expression system has the capacity to enzymatically cleave the signal peptide from the T4 ex polypeptide precursor, allowing it to be secreted.
• the hydrophobic transmembrane portion of the CD4 protein which ordinarily causes the protein to be insoluble, is deleted, as are the first three or four external amino acids adjacent the transmembrane portion.
• the truncated soluble CD4 polypeptides have 371 amino acid residues (T4 ex1 ) or 370 amino acid residues (T4 ex2 ), compared to the 374 amino acid mature extracellular segment.
• HIV gp120 exterior glycoprotein the following two reciprocal coprecipitation experiments were carried out.
• metabolically labelled gp120 protein derived from HIV virions was incubated with unlabelled purified T4 ex2 either in the absence or presence of monoclonal antibodies directed against distinct epitopes of the CD4 protein (OKT4 and OKT4A).
• the OKT4A antibody (like 19Thy5D7), but not the OKT4 monoclonal antibody, is known to inhibit the binding of gp120 to the CD4 molecule.
• Culture supernatants were collected from 1 x 10 7 Molt-3lymphocytes stably infected with the HIV strain IIIB that were metabolically labelled overnight with 100 uCi/ml. 35S-cysteine in total volume of 1.5 ml.
• NP-40 was added to a final concentration of 0.5% and the supernatants were incubated with 10 ug of T4 ex2 for 1 hour at 37oC, with or without preincubation of the soluble T4 with 5 ug/ml of OKT4A (Ortho Pharmaceutical, Raritan, NJ). The samples were then immunoprecipitated with the monoclonal antibody OKT4 and run on SDS polyacrylamide gels as described in Kowalski et al., Science, 237: 1351-1355 (1987).
• the mixture was then immunoprecipitated with a goat antiserum raised against purified HIV gp120 protein.
• the iodinated T4 ex2 protein was coprecipitated by the anti-gp120 serum only when HIV virions were present, indicating that the T4 2x2 protein was capable of binding to an HIV virion component.
• T4 ex1 protein could inhibit the binding of gp120 protein to CD4+ lymphocytes
• metabolically labelled gp120 protein from the supernatants of virus-infected cells was preincubated with T4 ex1 protein or a control protein made in the baculovirus system (an extracellular T11 segment), as follows. 1 x 10 7 H9 lymphocytes stably infected with the
• HIV strain IIIB were metabolically labelled with S-cysteine overnight in a total volume of 1.5 ml.
• the supernatants containing labelled HIV proteins were incubated for 1 hour at 37oC with either: phosphate buffer saline (PBS), 2.5 ug/ml OKT4A; 30 ug/ml T4 ex1 ; or 30 ug/ml T11.
• PBS phosphate buffer saline
• the SupTl cells were centrifuged, washed once with PBS, lysed with 0.75 ml of lysis buffer and the gp120 bound to the SupT1 cells was immunoprecipitated as described in Kowalski et al., id.
• the OKT4 and OKT4A monoclonal antibodies were added to the SupT1 cells prior to the addition of the labelled protein to control for specificity of the binding.
• the OKT4A but not the OKT4 monoclonal antibody was found to inhibit the binding of the labelled gp120 protein to the SupT1 cells.
• the T4 ex1 protein significantly inhibited the binding of labelled gp120 to the surface of SupT1 cells. No inhibition was observed using up to a 30 ug/ml concentration of the control protein, whereas inhibition of gp120 binding was seen at 0.5 ug/ml concentration of T4 ex1 protein.
• T4 ex1 and T4 ex2 and anti-T4 (19Thy5D7) exhibited significant inhibition of viral protein expression and virus production.
• the T4 ex1 and T4 ex2 proteins were able to decrease HIV p24 protein expression at concentrations of 0.2 ug/ml.
• the induction of syncytia by the HIV envelope depends upon binding of the gp120 exterior glycoprotein to the CD4 molecule, followed by events involved in membrane fusion.
• T4 ex2 protein could inhibit the formation of syncytia by the HIV envelope, cells chronically infected with
• HIV were cocultivated with CD4+ SupT1 lymphocytes in the presence or absence of the T4 ex2 protein.
• Table 3 shows that the pretreatment of the envelope-expressing cells (H9/HTLVTIIB) was as effective at syncytia inhibition as was pretreatment of both envelope-expressing and CD4+ (SupTl) cells.
• incubation of the "target" SupTl cells with the T4 ex2 protein exhibited only slight effects on syncytium formation.
• the soluble CD4 fragments appears to exert their syncytium-inhibiting effect through its interaction with the envelope-expressing cells.
• H9/KTLYIIIB lymphocytes or 4 x 10 6 SupT1 lymphocytes were incubated ia medium with either ovalbumin orT4 ex 2 protein at a concentration of 20 ug/ml at 37 C for 30 min.
• the cells were then centrifuged and washed with phospnate suffered saline, centrifuged and resuspended in medium.
• the treated H9/KTLVIIIB were chen mixed with the treated SupT1 cells in 24 well dishes and returned to a 37°C, 5%. CO 2 incubator for 5 h, when total syncytia per well were counted.
• T4 ex2 like the control protein BSA and the anti-T8 monoclonal antibody, failed to inhibit CTL effector function even at concentrations as high as 100 ug/ml. In contrast, as little as 1-3 ug/ml of specific anti-T4 (19Thy5D7) monoclonal antibody reduced cytolysis by less than 50%. T4 ex1 also was without effect on cell lysis. Lack of Effect on T-Cell Proliferation bv Soluble CD4 Fragments
• the CTT7 clone is activated to undergo proliferation only in the presence of tetanus toxoid and the autologous antigen presenting cell.
• the anti-CD4 (19Thy5D7) monoclonal antibody inhibited 3 H-TdR incorporation by 80%, consistent with the important role of CD4 in helper T cell response.
• equivalent amounts of T4 ex1 , ovalbumin, or anti-T8 monoclonal antibody have no effects.
• T4 ex protein binds HIV gp120 and thereby inhibits binding of gp120 to its receptor, HIV envelope-induced syncytium formation and HIV replication, it has no discernable effect on class II directed physiologic T cell response at identical concentrations under these experimental conditions. The basis for this difference remains to be resolved.
• One possibility is that the affinity of CD4 for gp120 is substantially higher than CD4 for its native ligand (presumably class II MHC).
• CD4 is only one of several elements (others including LFA-1, T11, etc.) that facilitate cell-cell interactions between CTL and targets or inducer T cells and antigen presenting cells, partial abrogation of the CD4 function with T4 ex1 protein may still leave the T cell activation process uninhibited.
• soluble CD4 fragments in the picomolar range inhibit syncytium formation and HIV infection.
• anti-T4 the effects of soluble CD4 protein are exerted ar the level of gp120 expressing cells.
• class II specific T cell interactions are functionally unimpeded by soluble CD4 protein, whereas they are virtually abrogated by equivalent amounts of anti-T4 antibody under the same experimental conditions. Whether this selective effect is a consequence of substantial differences in CD4 affinity for gp120 compared to antibody remains to be determined.
• the extracellular segment of the CD4 protein or peptide fragments derived from it can be useful in competitively inhibiting the interaction between the native transmembrane CD4 structure on T lymphocytes and the viral gp120 protein.
• these soluble CD4 proteins should allow the establishment of assays designed to detect drugs which might interfere with gp120-CD4 interactions.
• T4 ex proteins themselves or fragments derived from T4 ex may have clinical utility in inhibiting gp120 binding to membrane bound CD4 on T lymphocytes, monocytes, or brain cells without interfering with the normal physiological role of surface CD4 on healthy cells.
• T4 ex2 75 ug in neutralized immunoaffinity eluate
• TPCK trypsin (Worthington) was added to an enzyme:protein ratio of 1:50 (w/w) and digestion was carried out at 37oC. 25 ug aliquots were removed at 10, 20 and 45 minutes.
• Digestion was stopped by the addition of a non-reduced SDS sample buffer and heating to 100 oC for 5 minutes. Aliquots were electrophoresed on 12.5% mini slab gel under non-reduced conditions. Gels were subsequently electroblotted, using the method of Matsudaira, onto poiyvinylidene diflouride memebrane (Millipore; 0.45 urn pore size). Mastsudaira, P., J. Biol. Chem., 262: 10035 (1987). Duplicate tracks were either stained with Coomassie blue or blocked with a 5% dried milk solution in PBS/azide for 2 hours at room temp.
• Electroblots after blocking were assembled in a miniblotter apparatus (Immunetics) and slots overlying the appropriate tracks filled with 50 ul of purified, native HIV gp120 at 20ug/ml. in 1% dried milk in PBS for testing for HIV gp120 reactivity. Incubation with shaking was carried our overnight at 4oC. Following rhree 5 minute washes with PBS/0.05% Triton X-100, the blors were incubared with radioiodinated mouse monoclonal IgG, anti-HTLVIII gp120 2.6 ug (specific activity,
• T4 ex2 The single band of T4 ex2 at 50KD MW when electrophoresed unreduced was shown to be capable of binding HIV gp120 strongly. In contrast, identical amounts of T4 ex2 , either reduced or reduced and amidomethylated, did not bind gp120 when similarly examined. The lack of gp120 binding to reduced and alkvlated T4 ex2 is not due to the modification during alkylation of the cysteine residues themselves, as shown by the concurrent lack of gp120 binding to reduced T4 ex2 . The fact that the electrophoretic mobility of the T4 ex2 protein after reduction is slower than when not reduced is consistent with rhe prediction that there are intrachain disulfide bonds in the external segment of human T4.
• the migration of the non-reduced T4 ex2 protein as a single moiety of 50KD shows that the purified protein does not contain disulfide-linked polvmers of T4 ex2 protein.
• Identical results to the above were obtained when using T4 ex1 proteins. Taken together, these results demonstrated that under these conditions, it is likely that the binding of gp120 to T4 ex proteins is dependent on the presence of intact disulfide bridges within the T4 ex protein, which are presumably stabilizing the tertiary structure of their binding region.
• Enzymic fragmentations on the purified T4 ex protein carried out as described above produced a wide range of fragments.
• HIV gp120 binding analysis of blotted material from a 45 minute papain digest demonstrates that, in addition to the expected binding by the 50KD residual T4 ex1 protein, a fragment is present with a mobility of 28KD which binds gp120.
• 40-fold more T4 ex1 protein was digested with papain and separated by preparative SDS-PAGE.
• a portion of the blot was subjected to analysis for gp120 binding, and comparison of densitometric scans of the stained blot and the autoradiograph showed that the 28 kD material boudn a relative amount of gp120 similar to that bound by the residual T4 ex1 protein in the same track.
• the 28KD fragment bound HIV gp 120 with the same efficiency as the parent T4 ex2 protein.
• the 28 KD band was excised from the Coomassie blue stained portion of this same blot and subjected to amino terminal microsequencing. The first 11 cycles yielded a single unambiguous sequence of KKWLGKKGDT, showing that the 28KD fragment is an intact polypeptide chain derived from the amino terminal region of the T4 ex1 protein.
• the papain cleavage of T4 ex1 yielding the 28KD fragment can be located as being C-terminal to the cysteine residue at position 159 and with domain 3 proximal to the oligosaccharide addition sites at positions 256 and 300 ( Figure 7C).
• the binding of HIV gp120 to T4ex1 does not involve the C-terminal stretch of amino acids in domain 3 containing both N-linked glycosylation sites of the T4 structure, or domain 4. This result defines the gp120 binding portion of T4 as being in the N-terminal region of, the protein.
• This cluster may be involved in the binding of HIV gp120 to the CD4 modecule.
• the above results indicate that a linear stretch of amino acids is unlikely to be an effective, high affinity inhibitor of CD4-HIV gp120 interaction; the results show that disulfide bridging of T4 € ⁇ . protein plays a key role in such interaction.
• the finding that cleavage at lysine residue 72 (i.e., between the cvsteine residues in the V-like domain) is sufficient to destabilize both the gp120 and 19Thy5D7 binding region is consistent with this view.
• T4 cDNA Modified Soluble Human CD4 Fragments and Assessment of Their Activity Modifications of the T4 cDNA were produced using an H13 T4 template.
• the T4 ex cDNA fragment was excised from plasmid vector pAc373/T4 using BamHI.
• the plasmid vector pAc373/T4ex is described in
• the ends of the fragment were blunted with DNA polymerase I and the fragment ligated to XbaI linkers (New England Biolabs).
• the linkered fragment was digested with XbaI, gel purified to remove excess linkers and ligated to Xba cut M13 replicative form.
• the ligation mixture was transformed into competent TGI host bacteria, plated out and the resulting plaques were screened by hybridization to T4 oligonucleotides. Plaques hybridizing to sense oligonucleotides were grown up to produce single-stranded M13 templates for mutagenesis.
• the mutagenesis protocol is that marketed by Amersham and is based on the method of Eckstein (Taylor, et al., Nucleic Acids Research, 13:8749 (1985); Taylor, et al., Nucleic Acids Research, 13:8764 (1985); Nakayama and Eckstein, Nucleic Acids Research, 14:9679 (1986)). Oligonucleotides were produced containing in their se ⁇ uence a base change which, when incorporated, produced a stop codon, resulting in a truncated T4 protein. This resulred in truncation of the T4 molecules at amino acid #183.
• An oligonucleotide comprising the sequence 5' G-AAG-GCC-TAA-AGC-ATA-G was synthesized.
• the normal T4 sequence is G-AAG-GCC-TCC-AGC-ATA-G.
• the mutant oligonucleotide was kinased and hybridized to 10 ug of the M13 T4 template.
• a second strand of DNA was synthesized, using the M13 T4 template and oligonucleotide primer, by the Klenow fragment of DNA polymerase in the presence of the thionucleotide dCTP ⁇ S.
• Any unreplicated single stranded DNA was removed by filtration through a nirro-cellulose filrer and rhe purified double-stranded DNA was nicked with the restriction enzyme Neil. Neil will not cut phosphorothioate DNA. Thus, the new strand containing dCTB ⁇ S and the mutation were not nicked.
• the nicked DNA was digested with exonuclease III, which digested away the nicked, non-mutant DNA strand.
• the gapped DNA was repolymerized by DNA polymerasel, in the presence of T4 DNA ligase. In this step, the mutant strand served as the template so the mutation was copied into both srrands.
• the resulting DNA was transformed into competent TGI and derived plaques were grown up. Single stranded and replicative form DNAs were isolared and the DNA was sequenced to confirm the presence of the mutation. Mutated DNA was excised from the replicative form of DNA with Xba and ligated to Xba cut vector CDM8. This vector was developed and provided by Dr. Brian Seed (Massachusetts General Hospital). CDM8 is expressed in Cos cells upon transfection. Thus, the mutant T4 proteins could be assayed after transfection into Cos cells. CDM8 containing mutant T4 cDNAs were identified by hybridization to radiolabelled T4 DNA after transformation of the vector-insert ligation into competent MC1061P3 host bacteria. The proper orientation of insert in vector was determined by restriction enzyme analysis of mini-prep DNAs . Large scale plasmid preparations were used for transfection.
• Cos cells were plated in 100 cm dishes in RPMI-10% FCS-T% glutamine-1% pen-strep-10 ug/ml gentamycin. 12-24 hours later, the cells were washed with RPMI and incubated for 2-2.5 hours in the presence of 4 ml DME containing 400 ug/ml DEAE-dextran and 45 ug plasmid DNA. The cells were washed with RPMI and incubated in 10 ml DME-2% FCS-1% glutamine-15 pen-strep-10 ug/ml gentamycin-120 uM chloroquine for 3 hours. The cells are washed with RPMI and incubated for 2 days in the original media.
• the 1.17Kb T4 ex1 fragment was excised using BamHI from pAC 373/T4 ex1 , blunted using the Klenow fragment of DNA polymerase I, ligated to XhoI linkers (New England Biolabs) and subcloned in the XhoI site of the vector CDM8.
• CDM8 constructs into Cos cells 2-3 X 10 6 cells are plated in 100 X 15 cm dishes in RPMI 1640 (Gibco) containing 10% fetal bovine serum (FBS). Twelve to twenty-four hours later, 45 ug of plasmid DNA are added to 2.5 ml RPMI and mixed with 2.5 ml RPMI-800 ug/ml DEAE dextran, then added to the washed Cos cells.
• RPMI 1640 Gibco
• FBS fetal bovine serum
• the cells are washed and then incubated in RPMI containing 2% FCS, 1% glutamine, 1% penicillinstreptomycin, 10 ug/ml gentamycin and 150 uM chloroquine for 3 hours.
• the cells were incubated at 37o C for 2 days in RPMI 10% FCS.
• the transfected Cos cells (2 days after transfection) and incubated for 1 hour in 5 ml RPMI minus cysteine containing 10% FCS.
• the media is removed and the cells are incubated in RPMI minus cysteine containing 10% dialyzed FCS and 100 uCi/ml of 3 5 S-cysteine for 5-6 hours at 37o C.
• the supernatants are removed, centrifuged at 200 g for 10 minutes and dialyzed vs. PBS/0.025% azide/lOmM cold cysteine overnight at 4oC.
• 5 ml of the dialyzed 35 S-cysteine labelled supernatant is precleared by a 45 minute incubation at 4o C with 20 ul anti-T8 antibody (21Thy2D3) on Affigel- 10 (Biorad) beads (about 5 mg antibody per ml beads).
• the precleared supernatant is then incubated with 20 ul anti-CD4 (19Thy5D7) on Affigel-10 beads for 3 hours at 4o C.
• the beads are washed once in 10 ml 10 mM Tris, pH 6.8/0.1% Triton X-100/0.1% SDS/0.5% DOC, once in ⁇ 1 ml of the same buffer and once in 1ml 0.1 M glvcine, pH 5/0.1% Triton X-100 and then eluted with 35 ul 0.1 M glycine, pH 2/0.1% Triton X0100 and neutralized with 6 ul 1 M Tris , pH 7 . 6 .
• the sample is run on a 0.75 or 1.5 mm 12.5% mini-polyacrylamide-SDS gel under non-reducing conditions. The gel is fixed, dried and autoradiographed at about 70° C from 1-7 days.
• Immunoprecipitation with anti-CD8 was carried out as above except that 20 ul anti-CD8 on Affigel-10 beads is used for immunoprecipitation.
• gp120 kind gift of Dr. Bolognesi, Duke University
• 0.5 ml of labelled supernatant is incubated with 67 ng native gp120 for 2 hours at 37o C.
• Five hundred ng anti-gp120 (Dupont) and 10 ul rabbit anti-mouse IgG Sepnarose 4B beads are added and rotated for 2 hours at 4° C. The beads are washed once in 10 ml and once in 1 ml cold PBS, eluted and the sample run in SDS-PAGE as above.
• the CD4 protein (182 amino acids long) was created using the thionucle ⁇ tide method of oligonucleotide site directed mutagenesis. Taylor, J.W. et al., Nucl. Acids Res., 13:8749-8765 (1985); Taylor, J.W. et al., Nucl. Acids Res. 13:8765-8785 (1985); Nakayame et al., Nucl. Acids Res. 14:9679-9698 (1986) The XhoI insert of T4 ex1 was excised from CDM8, blunted with the Klenow fragment of DNA polymerase I ligated to XbaI linkers (New England Biolabs) and subcloned into M13mp18.
• Single stranded DNA was prepared as a template and mutagenesis was carried out according to the manufacturer's recommendations (Amersham).
• the oligonucleotide 5' GAAGGCCTAAAGCATAG 3' was synthesized using standard cyanoethyl phosphoramodite chemistry.
• the termination codon which converts the serine (TCC) at amino acid 183 to a stop codon is underlined.
• the presence of the mutation was confirmed by sequencing the M13mp18-T4 construct and mini preps of the replicative form of the mutation-containing DNA were prepared.
• the mutated insert was excised with XbaI and ligated into the XbaI site of CDM8.
• Example 4 As shown in Table 1, 15 oligonucleotides were used, in a standard site-directed mutagenesis protocol (Example 4), to produce 16 different version of the human CD4 molecule, each containing from 1 to
• M3 failed to bind gp120
• M9 has substantially reduced gp120 binding capacity
• M14 also demonstrates reduced gp120 binding capacity.
• the amino acid substitutions made in each are as follows:
• proline S serine lysine
• V valine leucine
• D aspartic acid
• F phenylalanine threonine
• N asparagine

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