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WO1990001035A1 - Agent cytotoxique pour le traitement d'infections virales specifiques - Google Patents

Agent cytotoxique pour le traitement d'infections virales specifiques Download PDF

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Publication number
WO1990001035A1
WO1990001035A1 PCT/US1989/003267 US8903267W WO9001035A1 WO 1990001035 A1 WO1990001035 A1 WO 1990001035A1 US 8903267 W US8903267 W US 8903267W WO 9001035 A1 WO9001035 A1 WO 9001035A1
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Prior art keywords
hiv
cells
protein
human
gpl20
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PCT/US1989/003267
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English (en)
Inventor
Edward A. Berger
Bernard Moss
Thomas R. Fuerst
Tamio Mizukami
Ira H. Pastan
David J. P. Fitzgerald
Vijay K. Chaudhary
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The United States Of America, Represented By The Secretary, United States Department Of Commerce
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Priority claimed from US07/223,270 external-priority patent/US5206353A/en
Application filed by The United States Of America, Represented By The Secretary, United States Department Of Commerce filed Critical The United States Of America, Represented By The Secretary, United States Department Of Commerce
Priority to AU40690/89A priority Critical patent/AU623924B2/en
Publication of WO1990001035A1 publication Critical patent/WO1990001035A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • the present invention is related generally to the control of viral infection. More particularly, the present invention is related to the construction of* a chi eric gene expressing a recombinant fusion protein which selectively kills specific virus-infected cells; to a recombinant soluble truncated form of CD4 containing the active binding site for human immunodeficiency virus; and to a multivalent product ' having substantially long half-life, bonding avidity and the capacity to direct components of the native immune system to kill HIV infected cells or HIV virions vivo relative to mono- meric forms.
  • a hybrid fusion protein having selective cytotoxicity against HIV infected cells has been made.
  • HIV human immunodeficiency virus
  • AIDS acquired immune deficiency syndrome
  • Anti-viral agents, immunomodulators and inhibi ⁇ tors of specific HIV functions are being tested as poten ⁇ tial treatments to alleviate the high morbidity and mortality related to AIDS.
  • a potent cytotoxic agent targeted to selectively kill HIV-infected cells has not heretofore been developed.
  • CD4 derivatives are uniquely suitable for AIDS treatment, since divergent strains of HIV-1 and HIV-2 all infect human T lymphocytes by binding to surface CD4.
  • a recent report describes recombinant proteins containing soluble CD4 linked to human immunoglobulin heavy constant region sequences (Capon et al. 1989. Nature (London) 337, 525-531). However, the molecules did not bind com ⁇ plement component Cg , and no activity against HIV- infected cells or virions was presented.
  • the hybrid proteins of the present invention have distinctive pro-, perties not heretofore known or described.
  • CD4 is an integral membrane glycoprotein of human helper T lymphocytes that serves as an essential component of the receptor for the human immunodeficiency virus (HIV) (Popovic et al. 1984. Clin. Res. 33, 560A abstr.; and Maddon et al. 1986. Cell 47, 333-348), the causative agent of acquired immunodeficiency syndrome. HIV binding and fusion with the cell are mediated by specific interaction between the external subunit of the viral envelope glycoprotein (gpl20) and CD4 on the target cell surface [McDougal et al. 1986, Science 231, 382-385; Sodroski et al. 1986. Nature (London) 322,470-474; and Lundin et al. 1987. J. Immunol. Methods 97, 93-100].
  • HIV human immunodeficiency virus
  • CD4 gene (Littman, D.R. 1987. Annu. Rev. Immunol. 5, 561- 584) supports this notion of structural and possibly functional domains. Of particular interest is the exist ⁇ ence of conserved pairs of cysteine residues that prob-, ably form intradomain disulfide bonds within the first, second, and fourth external domains (Classon et al. 1986. Proc. Natl. Acad. Sci. USA 83, 4499-4503). These struc ⁇ tural features deduced from cDNA sequencing have been complemented by epitope analyses using panels of anti-CD4 monoclonal antibodies (mAbs).
  • mAbs monoclonal antibodies
  • an object of the present, invention to provide a chimeric gene which directs the synthesis, in a suitable expression vector, of a hybrid protein comprising a virus binding region from a cellular receptor sequence linked to a protein toxin sequence containing a region essential for cell toxicity.
  • It is another object of the present invention to provide an anti-HIV composition comprising a polypep- tide molecule made of about 180 amino acid residues representing the first two immunoglobulin-like domain of CD4 and having immunological and functional properties of an active HIV-binding site.
  • an object of the present inven ⁇ tion to provide a method of inhibiting HIV infection, comprising administering to an HIV-infected host, an effective amount of the truncated CD4 molecule of the present invention to inhibit the infection of host cells by HIV.
  • Figure 1 is a schematic representation of the plasmid used for expressing CD4( ⁇ -78)-PE40.
  • Figure 2 shows the re'sults of column chro a- tography (2A) and gel electrophoresis (2B) of the CD4(178)-PE40;
  • Figure 3 demonstrates the binding of CD4(178)- PE40 to HIV envelope protein gpl20 by (3A) coprecipita-, tion technique and (3B, 3C, 3D and 3E) by immunofluores- cence microscopy; and 5
  • Figure 4 shows the selective cytotoxic effect of CD4(178)-PE40 on cells expressing HIV-1 envelope glycoprotein: 4A. Cells expressing the HIV-1 envelope glycoprotein encoded by a recombinant vaccinia virus.
  • Closed symbols represent cells infected with vPE-16, a 10. vaccinia ' recombinant containing the HIV-1 gpl60 gene linked to the vaccinia 7.5 K promoter, inserted within the thymidine kinase locus.
  • Open symbols represent cells infected with a control vaccinia recombinant, vTF7-3, which contains the bacteriophage T7 RNA polymerase gene 5 also linked to the vaccinia 7.5 K promoter and inserted within the thymidine kinase locus.
  • the toxin prepara ⁇ tions used were: ⁇ , A PE, 0, • PE40, Q , ⁇ CD4(178)- PE40. 4B.
  • FIG. 5 shows schematic construction of plas id pCD4f.
  • Plasmid pTK7-5 contains the bacteriophage T7 gene 10 promoter (P- j -y) and the T7 terminator ( my) separated by a unique BamHI site. This region is flanked 0 t,y vaccinia virus thymidine kinase left (TKT ) and right (TK R ) sequences.
  • An adapter made by using two partially complementary synthetic oligonucleotides (GATCGAATTCAGGC- CTAATTAATTAAGTCGAC and GATCGTCGACTTAATTAATTAGGCCTGAATTC) was ligated into the BamHI site of pTF7-5 by using the 5 BamHI 5' overhangs of the adapter.
  • the BamHI site is destroyed in the desired recombinants.
  • the reac ⁇ tion mixture was digested with BamHI to linearize recir- cularized plasmids lacking the insert, and ampicillin- resistant transformants were screened by restrictio ⁇ mapping to identify those containing the insert in the desired orientation.
  • Plasmid pEB-2 contains unique sites for EcoRI and Stu I, followed by a universal termination sequence (UTS) providing termination codons in all three reading frames, followed by a unique Sal I site.
  • the desired CD4 DNA fragment was obtained by digesting pCD4- GEM4 with Nhe I (which cleaves the plasmid at a unique site at nucleotide 678 of the CD4 cDNA sequence), filling in the staggered end with the Klenow fragment of DN polymerase I and dNTPs, and then digesting with EcoRI ⁇
  • the resulting 0.68-kilobase EcoRI-Nhe I fragment, whie-h contains the ATG initiation codon of CD4, was force- " cloned into pEB-2, which had been digested with " EcoRI and Stu I.
  • FIG. 6 shows the results of analysis of the metabolically labeled transient expression products.
  • Transient metabolic labeling reactions were performed in cells infected with vTF7-3 and transfected with either plasmid pEB-2 or pCD4f, and the media were collected.
  • reaction mixes were prepared containing 1.1 ml of transfection medium, 0.99 ml of protease inhibitor buffer, and 0.11 ml of 20% (vol/vol) Nonidet P-40. The samples were cleared by incubation with 0.13 ml of a 20% (vol/vol) suspension of protein A- agarose for 1 hr at 4°C; this was followed by centrifuga- tion.
  • Figure 7 shows the results of epitope analysis of the CD4 fragment.
  • Cells were metabolically labeled with 35 s _ C y Ste j_ ne a fter infection with vTF7-3 and trans- 25 fection with pCD4f.
  • the medium was collected.
  • a mixture was prepared containing 0.55 ml of this transfection medium, 0.55 ml of protease inhibitor buffer, 0.07 ml of 20% (vol/vol) Nonidet P-40, and 1.58 ml of phosphate- buffered saline containing 0.02% (wt/vol) sodium azide. 0
  • the mixture was cleared with 0.28 ml of a 20% suspension of protein A-agarose as described for Fig.
  • Lane 1 total transfection medium (0.05 ml); lane 2, immunoprecipitate obtained with a control mAb (2E12.1). Immunoprecipitates were obtained with a battery of anti-CD4 mAbs: lane 3, MT151; lane 4, Leu3A; lane 5, 0KT4; lane 6, 0KT4A; lane 7, 0KT4B; lane 8, 0KT4C; lane 9, 0KT4D; lane 10, 0KT4E; lane 11, 0KT4F.
  • Molecular weight markers are shown on the left (expressed as M f x 10" 3 ) .
  • Figure 8 demonstrates the interaction of the CD4 fragment with gpl20.
  • Medium from cells metabolically labeled after infection with vTF7-3 and transfection with pCD4 was used as the source of the CD4 fragment.
  • Media from unlabeled or metabolically labeled cells doubly infected with vTF7-3 plus vPE-6 served as the source of the gpl20.
  • Molecular weight markers are shown on the left (expressed as Mf x 10 ⁇ 3 ).
  • Lane 1 total reaction mixture from the incuba- tion containing the transfection medium and the vTF7-3 plus vPE-6 double infection medium.
  • the immune precipi-? tates were obtained from reactions containing the follow ⁇ ing additions: lane 2, transfection medium, vTF7-3 plus vPE-G double infection medium, no antibody; lane 3, normal medium containing 2.5% fetal bovine serum, VTF7-3 plus vPE-6 double infection medium, anti-gpl20; lane 4, transfection medium, normal medium containing 2.5% fetal bovine serum, anti-gpl20; lane 5, transfection medium, vTP7-3 single infection medium, anti-gpl20; lane 6, transfection medium, vTF7-3 plus vPE-6 double infection medium, anti-gpl20; lane 7, transfection medium, normal medium containing 2.5% fetal bovine serum, anti-CD4 ' .
  • Immune complexes were collected and processed as described in the legend to Fig. 6 and elec- trophoresed on 12% gels.
  • the supplementary media added during the initial incubation were as follows: lane 8, normal medium containing 2.5% fetal bovine serum; lane 9,
  • VTF7-3 single infection medium lane 10, vTF7-3 plus vPE- 6 double infection medium.
  • Figure 9 is a schematic representation of CD4- i munoglobulin hybrid proteins which were made in this invention.
  • Figure 10 shows the construction scheme of the intermediate plasmid, pCD4CHl which was used for the construction of the following plasmids for the expression of the hybrid proteins.
  • Figure 11 shows the construction scheme of pCD4ITM10 and pCD4ITM10G which express CD4(109)CH.
  • Figure 12 shows the construction scheme of pCD4ITM20 and pCD4ITM20G which express CD4(178)CH.
  • Figure 13 shows the construction scheme of pCD4ITM30 and pCD4ITM30G which express CD4(372)CH.
  • Figure 14 shows the construction scheme of pCD4ITM40G which expresses CD4(181)CL.
  • FIG. 15 shows the expression of CD4(109)CH
  • Figure 16 shows the pattern of CD4(109)CH
  • Lanes 1, 2, 3, 4 and 5 represent the analysis of the extracts of cells obtained by transfec ⁇ tion of CV-1 cells by pEB2, pCD4LTMl, pCD4ITM10,
  • Lanes 6, 7, 8, 9 and 10 represent the analysis of the culture media obtained by transfection of CV-1 cells by pEB2, pCD4LTMl, PCD4ITM10, pCD4lTM20, and pCD4lTM30, respectively.
  • FIG. 17 shows the coexpression of CD4(178)CH
  • Lanes 1 and 5 represent the analysis of the culture media of pTM3-transfected cells, lanes 2 and*, 6 for the culture media of pCD4ITM20G- transfected cells, lanes 3 and 7 for the culture media of pCD4ITM40G-transfected cells, and lanes 4 and 8 for the culture media of pCD4lTM20G and pCD48ITM40G doubly- transfected cells.
  • Figure 18 shows the schematic model structure of CD4(176)CH and CD4(181)CL tetrameric complex.
  • Figure 19 shows the expression of CD4(109)CH,
  • CD4(178)CH, and CD4(372)CH in RPMI8226 cells.
  • the immunocomplexes were analzyed by SDS-polyacrylamide gel (7.5%) electrophoresis in reducing conditions (lanes 1-8) or non-reducing conditions (lanes 9-16).
  • Lanes 1, 9 and 2, 10 represent the analysis of the culture media and the extracts, respectively, of non-transfected cells, lanes 3, 11 and 4, 12 for the culture media and the extracts, respectively, of pCD4lTM10-transfected cells, lanes 5, 13 and 6, 14 for the culture media and the extracts, respec- tively, of pCD4ITM20 transfected cells, and lanes 7, 15 and 8, 16 for the culture media and the extracts of pCD4ITM30-transfected cells.
  • Figure 20 shows the analysis of the binding properties of CD4(178)CH and soluble CD4(372) which were secreted from the CV-1-transfected cells.
  • Lanes 1-6 represent the analysis of soluble CD4, and lanes 7-12 represent the analysis of CD4(178)CH.
  • the culture media were incubated with the following antibodies and ligands, and analyzed on each lane.
  • solCD4 and 178H denote soluble CD4(372) and CD4(178)CH, respectivel .
  • Figure 21 shows the analysis of the binding property of CD4(178)CH which was secreted from the RPMl8226-transfected cells.
  • the culture media were incu ⁇ bated with the following antibodies and ligands, and analyzed on each lane.
  • Lane 1 (0KT4), 2 (OKT4A) , 3 (gpl20), 4 [anti-human IgG (Fc)], 5 (protein A-agarose)
  • Lane 1 (0KT4), 2 (OKT4A) , 3 (gpl20), 4 [anti-human IgG (Fc)], 5 (protein A-agarose)
  • FIG. 6 shows the analysis of the binding properties of CD4(178)CH and CD4(181)CL coexpressed in CV-1 cells.
  • the culture media of pCD4ITM20G and pCD4ITM40G doubly-transfected cells were incubated with the following antibodies and ligands, and analyzed on each lane. Lane 1 (no addition), 2 (0KT4), 3 (0KT4A) , 4 (gpl20), 5 [anti-human IgG (Fc)], 6 (protein A-agarose),
  • 178H and 161L denote CD4(178)Ch and CD4(181)CL, respectively.
  • a chimeric gene which encodes a recombinant fusion protein having selective toxicity against specific virus-infected cells.
  • the principal aspect of the present invention is that a toxin, or a cytotoxic part thereof, could be genetically attached to a receptor protein (or a fragment thereof) so that the fusion product binds to cells infected with a virus, since all viruses depend on a cellular receptor for entry.
  • CD4 is one such receptor required by HIVs of different types.
  • such a chimeric gene encodes recombi ⁇ nant hybrid proteins comprising the HIV-gpl20 binding regions of the human CD4 molecule and constant regions of the human immunoglobulin heavy and/or light chain mole- cules.
  • this invention is demonstrated by a par ⁇ tial but essential CD4 linked fusion cytotoxic product. The same principle can be applied for other viruses.
  • substantially pure as used herein meai ⁇ s a product which is at least 80% pure monomeric hybrid protein.
  • selective as used herein means that the fusion protein of the present inveniton preferential ⁇ ly attacks cells such as HIV-infected cells without sig ⁇ nificantly affecting the activity of other cells.
  • Part I of the detailed description of the invention below relates to the construction of a chimeric gene expressing a recombinant fusion protein which selec ⁇ tively kills specific virus-infected cells.
  • Part II of the detailed description of the invention below relates to a recombinant soluble trun ⁇ cated form of CD4 containing the active binding site for human immunodeficiency virus.
  • Part III of the detailed description of the invention below relates to a multivalent product housing substantially long half-life, bonding avidity and the capacity to direct components of the native immune system to kill HIV infected cells or HIV virions _in_ vivo rela ⁇ tive to monomeric forms.
  • the plasmid pVC403 was constructed as described below.
  • pVC403 carries a fusion gene encoding the first 178 amino acids of mature CD4 [referred to as CD4(178)] based on amino acid sequence data and amino acids 1-3 and 253-613 of PE (referred to as PE40).
  • the fusion gene is under control of a T7 late promoter.
  • E. coli strain BL21 ( DE3) carrying pVC403 was used to express the fusion protein upon IPTG induction.
  • the direction of transcrip-r tion from the T7 promoter and for the B-lactamase gene is show by solid arrows in Figure 1.
  • the circled- numbers are the amino acids of CD4 and the boxed numbers are the amino acids of PE.
  • pVC403 Plasmid pVC4 which carries a full length PE gene attached to a T7 promoter was cut with Ndel and Asp718 and ligated to a 52 bp linker containing codons for the first 16 amino acids of mature CD4 and Ndel and Asp718 cohesive ends.
  • This intermediate plamid (pVC401) has 3 Rsal sites, one between the first 16 codons of mature CD4 and the remainder of PE gene. pVC401 was partially cut with Rsal, then with Xhol, and a 2.8 Kb fragment was isolated.
  • Plasmic pCD4SPE40TMl which carries a fusion gene between the first 178 amino acids of CD4 [CD4(178)] and PE40 under a T7 promoter was restricted with Rsal and Xhol, and a 1.3 Kb fragment was isolated. Construction of pCD4SPE40TMl is described below. The 1.3 Kb fragment, from pCD4SPE40TMl was ligated to a 2.8 Kb fragment from pVC401 to produce plasmid pVC403. This plasmid has a Nde I site at the junction of the CD4(178) and PE40 genes that can be used to introduce various other PE genes. Construction of pCD4SPE40TMl:
  • an intermediate plasmid, pCD4PE40TMl was constructed as follows.
  • a 1.23-kb frag ⁇ ment containing PE40 was excised from pVC8 by digesting it with EcoRI, filling in the cohesive end with DNA poly ⁇ merase I Klenow fragment, and digesting it with Xbal.
  • the fragment was ligated with a 5.21-kb fragment of pCD4LTMl (an expression plasmid for 372 amino-acid CD4); the fragment was obtained by digesting the plasmid with Sail, filling in the cohesive end with DNA polymerase I Klenow fragment, and digesting with Nhel, yielding pCD4PE40TMl.
  • pCD4SPE40TMl a 546 amino-acid fusion proteing consisting of the first 178 amino acids of DC4 at the amino terminus, followed by histidine and methionine residues derived from the Ndel site used for joining the two molecules, followed by the carboxy terminal PE40 sequence.
  • a deposit of the plasmid pVC 403 has been made at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A., on June 29, 1988 under the accession number 67739.
  • the deposit shall be viably maintained, replacing if it becomes non-viable, for a period of 30 years from the date of the deposit, or for 5 years..from the last date of request for a sample of the deposit, whichever is longer, and made available to the public without restriction in accordance with the provisions of the law.
  • the Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.
  • BL21 ( DE3) carrying plasmid pVC403 was grown in LB medium at 37°C with ampicillin (100 ug/ l), induced at OD s50nm ⁇ ' ⁇ with InM isopropyl D-thiogalactosid ⁇ (IPTG) and the incubation continued for 90 minutes at 37°C.
  • the cells were fractionated into periplasm and spheroplasts.
  • the spheroplasts were suspended in TE (50 M Tris pH 8.0, I M EDTA) , sonicated three times at 100 watts for 30 seconds each and spun at 100,000 x g for 60 minutes to isolate the supernatant (cytoplasm) and pellet (containing inclusion bodies).
  • the pellet was suspended in TE and the various fractions were analyzed by ADP-ribosylation assays and by SDS-PAGE, using Coomassie Blue staining and immunoblot- ting with rabbit antibodies to PE.
  • a denaturation/renaturation procedure was employed for partial purificaton of CD4(178)-PE40.
  • a 500 ml culture of BL21 (J ⁇ DE3) containing pVC403 was induced and the inclusion body pellet fraction prepared as described above.
  • the pellet was suspended in 6.5 ml extraction buffer (guanidine HC1 7M, Tris HC1 0.1M pH 8.0, EDTA ImM and DTT ImM) and sonicated for 20 seconds three times.
  • the suspension was stirred for 1 hour in the cold and centrifuged at 100,000 x g for 15 minutes, and the supernatant saved.
  • the supernatant (6.5 ml) was added dropwise to 500 ml cold phosphate buffered saline with rapid stirring.
  • the second method involved immunofluro- escence.
  • Confluent monolayers of CV-1 cells in 35 mm wells of 6-2311 plates (Costar) were infected with a recombinant vaccinia virus encoding gpl60 (HIV-1, IIIB isolate) under control of the vaccinia 7.5K promoter. The multiplicity of infection was 1.5.
  • As a control cells were infected with vTF7-3 (see above).
  • CD4(178)-PE40 fraction 19 of the mono Q column, see Fig. 2 was added to a final * toxin concentra ⁇ tion of 50 ⁇ _g/ml in PBS plus 0.2%-(w/v) bovine serum albumin.
  • the test systems were employed. The first involved cells expressing the HIV envelope glycoprotein encoded by a recombinant vaccinia virus. Duplicate essays were performed in 16 mm wells of 24-well plates (Costar) . CV-1 cells were grown to 90% confluence (2 x 10 5 cells per well). The indicated recombinant vaccinia viruses were added to the wells at a multiplicity of infection of 20 in 0.25 ml of Dulbecco's MEM supplemented with 2.5% fetal bovine serum. After 90 in with occasional rocking, the medium was removed and replaced with 1 ml of the same medium containing 10% of the normal methionine concentration.
  • the second test system employed cells chron ⁇ ically infected with HIV.
  • 8E5 is a human T-cell line which contains a single integrated copy of the HIV-1 (LAV) genome.
  • the virions produced are non-infectious due to a premature chain termination mutation in the reverse transcriptase gene.
  • the parental non-infected A3.01 cell line was used.
  • Assays were per- formed in duplicate in 24-well plates. Individual wells were seeded with 7 x 10 5 cells of the indicated cell line in 0.9 ml of medium containing 1 part RPMI supplemented with 10% fetal bovine serum plus 8 parts of the same medium lacking methionine.
  • toxin prepara ⁇ - tions were added in 0.02 ml Dulbecco's PBS to give 111% of the final concentration shown. After 17.5 hr, lO ⁇ Ci of 3 -S-methionine in 0.1 ml of complete medium was added to each well, and the incubations continued for 1 hr.
  • RESULTS As shown in Figure 1, a chimeric gene encoding the first 178 amino acids of CD4 and amino acids 1 to 3 and 253 to 613 of PE was constructed ( Figure 1).
  • This segment of PE (designated PE40) lacks domain I but retains domains II and III which are responsible for translocation and ADP-ribosylation, respectively.
  • the plasmid, pVC403, also contained a bacteriophage T7 late promoter and the Shine-Delgarno ribosome binding sequence for high expression in Escherichia coli BL21 ( ⁇ DE3).
  • the chimeric protein designated CD4(178)-PE40, was synthe ⁇ sized in large amounts, remained intracellular and appeared to be primarily associated with inclusion bodies in the 100,000xg pellet of sonicated spheroplasts.
  • the denatured protein had the expected Mr of approximately 60,000 and reacted with polyclonal antibodies to PE by immunoblot analysis (see below).
  • CD4(178)-PE40 To determine the ability of CD4(178)-PE40 to bind gpl20, two types of assays were performed ( Figure 3). First, CD4(178)-PE40 was mixed with soluble [ S]methionine-labeled gpl20, and the immune complexes obtained with anti-PE serum were bound to protein A agarose and resolved by SDS polyacrylamide gel electro ⁇ phoresis. As shown in Figure 3A, labeled gpl20 was spe ⁇ cifically coprecipitated along with CD4(178)-PE40. Second, the binding of CD4(178)-PE40 to cell-associated gpl20 was established by immunofluorescence microscopy.
  • the HIV envelope glycoprotein was produced in CV-1 cells using a vaccinia based expression system. Both the external gpl20 and transmembrane gp41 subunits are present on the surface of cells infected with a recombi ⁇ nant vaccinia virus encoding gp 160; furthermore, such cells form extensive syncytia when mixed with CD4-bearing human cells.
  • Figure 3B shows that CD4(178)-PE40 bound to
  • Figure 4A shows that protein synthesis in CV-1 cells infected with a recombinant vaccinia virus encoding
  • an uninfected human lymphocyte cell line (A3.01) and a daughter cell line (8E5) that is chronically infected with HIV were tested as targets.
  • the 8E5 cells are especially suitable for experimental studies since they contain a single inte ⁇ grated viral genome, constitutively synthesize HIV pro ⁇ teins including gpl20, form syncytia when mixed with CD4- bearing cells, and produce budding particles.
  • CD4 major histocompat- ibility
  • B-lymphocytes and macrophages might be affected , by the chimeric toxin.
  • MHC major histocompat- ibility
  • CD4(178)-PE40 concentrations of purified renatured CD4(178)-PE40 required for -50% inhibition of protein synthesis in three experiments ranged from 27 to 100 ng per ml. Based on data with other PE fusion proteins, it is not difficult to attain • such levels in animals without significant non-specific toxicity. Furthermore, CD4(178)-PE40 could be useful against cells infected with diverse strains of* HIV-1 as well as HIV-2, since the envelope proteins of all- these viruses retain binding specificity for CD4 despite extensive antigenic variation.
  • a thera ⁇ Treatmentic composition in accordance with the present inven ⁇ tion comprises an effective amount of the recombinant toxin to kill HIV-infected cells in a pharmaceutically acceptable vehicle, if necessary, such as physiological saline, buffered solutions and the like.
  • the toxin may be administered by any suitable route, systemically or locally as deemed more effective.
  • the method of con ⁇ trolling or treating AIDS comprises contacting HIV- infected cells with the effective amount of the recombi- nant toxin [CD4(178)-PE40 fusion protein] to kill HIV- infected cells or inhibit fusion and syncytia formation resulting subsequent to HIV-infection.
  • CD4(178)-PE40 fusion protein of the present invention possesses numerous advantages over this immunotoxin: (a) In the case of the immunotoxin the antibody used is type specific, and does not bind to gpl20 from diverse isolates of HIV-1. In contrast, CD4(178)-PE40 may be used against divergent strains of HIV-1 as well as against HIV-2, since all these viruses use CD4 as the receptor.
  • CD4 portion This can be achieved, for example, by differences in length of the CD4 sequencer. Shorter or longer versions of the CD4 sequence can be found which can also be attached to toxins to achieve selective killing of HIV-infected cells.
  • the length of the CD4 sequence can have important consequences for the affinity for gpl20, for e relative affinities for gpl20 vs. class II antigens, for the phys ⁇ ical accessibility to different regions within the body, and for the immunogenicity.
  • site-specific- ' mutagenesis can be used to decrease the affinity of CD4 for normal cellular antigens, and/or increase the affinity for gpl20. Such mutations would widen the window between effective therapeutic dosages and unwanted toxic side effects.
  • C Expression systems Bacterial. By employing, for example, certain _E_.
  • coli expression system secreted forms of the hybrid toxin can be made obviating the need for denaturation/re- naturation.
  • Enzymes Restriction endonucleases were obtained from New England Biolabs or Bethesda Research Laboratories. The Klenow fragment of DNA polymerase I and T4 DNA ligase were from New England Biolabs. Antibodies. Murine anti-CD4 mAbs were obtained from the following sources: MT151, Boehringer Mannheim; Leu3A, Becton Dickinson; 0KT4, 0KT4A, 0KT4B, 0KT4C, 0KT4D, 0KT4E, and 0KT4F, M. Talle, Ortho Diagnostics.
  • Two murine anti-gpl20 mAbs were employed: 2E12.1 (Epitope, Beaverton, OR) and a tissue culture supernatant from hybridoma 902 (B. Chesebro, National Institute of Allergy and Infectious Diseases, Hamilton, MT) .
  • Rabbit antiserum to mouse IgG was purchased from ICN. Plasmids.
  • the CD4 cDNA was donated by D.
  • Plasmid pCD4-GEM4 (obtained from A. Rabson, National Institute of Allergy and Infectious Diseases, Bethesda, MD) contains a full-length copy of the CD4 cDNA with 5'EcoRI and 3' BamHi linkers (Maddon et al, supra) cloned into the EcoRi-BamHI site of pGEM4 (Promega Biotec, Madison, WI).
  • pTF7-5 contains a bacteriophage T7 pro ⁇ moter and terminator separated by a unique BamHI site and flanked by the left and right vaccinia thymidine kinase gene sequences (see Fig. 1).
  • Vaccinia virus recombinant vTF7-3 contains the bacteriophage T7 gene 1 (encoding the T7 RNA polymerase) under control of the vaccinia P7.5 promoter (Fuerst et al. 1986. Proc. Natl. Acad. Sci. USA 83, . 8122-8126).
  • vPE6 is a vaccinia recombinant derived from pTF7-5 containing a bacteriophage T7 promoter linked to the HIV-1 envelope gene (IIIB isolate, clone BH8) with a termination codon inserted by _in_ vitro mutagenesis immediately preceding the sequence encoding the consensus retroviral envelope cleavage site Arg-Glu-Lys-Arg. This virus directs high-level expression of a secreted form of gpl20 in cells doubly infected with vTF7-3.
  • CV-1 monkey kidney cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Expression Conditions. Transfection exper ⁇ iments for transient expression were performed by using conditions similar to those described in Fuerst et al, supra.
  • CV-1 cells were grown to 90-95% confluence in 25- cm 2 flasks ( 2.5 x 10 5 cells) and infected with vTF7-3 at a multiplicity of 30 plaque-forming units (pfu) per cell in medium with 2.5% fetal bovine serum.
  • the virus was allowed to adsorb for 30 min at 37°C with occasional rocking of the flask, whereupon the inoculum was removed and replaced with 1 ml of transfection buffer containing 5 ug of calcium phosphate-precipitated plasmid DNA. After incubation for 30 min at 37°C with occasional rock ⁇ ing, 5 ml of medium containing 2.5% fetal bovine serum was added. The medium was removed after 4 hr at 37°C, and the cells were incubated with 2-4 ml of cysteine-free medium containing 2.5% dialyzed fetal bovine serum.' for 15-60 min.
  • double virus infections were performed by using protocols similar to those reported (Fuerst et al. (1987. Mol. Cell. Biol. 7, 2538-2544).
  • CV-1 cells grown in flasks as described above were infected with vTF7-3 and vPE6 (15 pfu each per cell) in 1 ml of medium containing 2.5% fetal bovine serum. After 90 min at 37°C, the virus inoculum was removed and replaced with fresh medium containing 2.5% fetal bovine serum. In the case of unlabeled infection, the incubation was continued at 37°C for 23 hr, after which the medium was collected.
  • the incubation was continued for 10.5 hr, at which time the medium was removed and the cells were incubated for 15 min at room temperature in cysteine-free medium with 2,5% dialyzed fetal bovine serum. This medium was then replaced with 2.25 ml of the same medium supplemented with 0.1 mCi of [ 35 S]cysteine per ml. After 5 hr of labeling at 37 C°, 0.25 ml of complete medium containing 2.5% fetal bovine serum was added. The incubation was continued for an additional 7 hr and the medium was then collected. Media from these infections were centrifuged as described above for the transfection experiments.
  • Control infections were performed identically, except that the virus inoculum contained vTF7-3 only (30 pfu per cell) .
  • Protease inhibitor buffer contained 0.1 mM N ⁇ ***--.-(p-tosyl)-lysine chloromethyl ketone, 0.1 mM L-l-tosylamido-2-phenylethyl chloromethyl ketone, 50 mM iodoacetimide, 0.01 mM leupeptin, and 70 Kallikrein units of aprotinin per ml in phosphate- buffered saline with 0.02% (wt/vol) sodium azide.
  • RESULTS Expression of the Soluble CD4 Fragment The expression system employed in the present study is based • on that of Fuerst et al, supra.
  • Mammalian cells are • infected with a recombinant vaccinia virus (vTF7-3) con ⁇ taining the bacteriophage T7 RNA polymerase gene linked to a vaccinia promoter and then transfected with a plasmid vector containing the target gene of interest flanked by bacteriophage T7 promoter and transcriptional terminator regions.
  • the T7 RNA polymerase mediates high- level transcription of the target gene in the cytoplasm of the transfected cells.
  • a plasmid vector (pEB-2) was designed that contains, between the T7 promoter and transcriptional terminator, two new unique restriction sites (EcoRI and Stu I) directly followed by a universal translational termination sequence. After cleaving this vector wit ⁇ i EcoRI and Stul, any DNA fragment containing an EcoRI site and a 3' blunt end can be force-cloned in the proper orientation. If the DNA insert contains the translation initiation codon but only a portion of the adjacent cod ⁇ ing region for a particular gene, a truncated polypeptide is expressed. Depending on which synthetic termination codon is in frame, the shortened polypeptide may also contain up to three additional C-terminal amino acids encoded by the vector.
  • pCD4 the EcoRI-Nhe I DNA fragment of the CD4 cDNA was inserted into pEB-2 to obtain another plasmid, designated pCD4 (Fig. 5). Based on the reported cDNA sequence (Maddon et al, supra) , pCD4 would be expected to encode a truncated variant of CD4 with a normal N terminus; cleavage of the signal sequence would result in a polypeptide containing the first 177 amino acid residues of mature CD4 (the first two immunoglobulin-like domains) and containing no con ⁇ sensus N-linked glycosylation sites. This fragment might therefore be expected to be secreted into the medium. DNA sequence analysis of pCD4£ indicated that the frag ⁇ ment also contains C-terminal proline and arginine resi- dues derived from the UTS sequence of the vector.
  • Figures 6A and 6B show the results of a transient metabolic labeling experiment with cells infected with vTF7-3 and transfected wiht different plasmids.
  • NaDodSO ⁇ /polyacrylamide gel electrophoretic analysis revealed that the medium of cells transfected with control plasmid pEB-2 contained a complex pattern of polypeptide bands (lane 3); medium from cells transfected with pCD4f contained the same complex pattern of poly- peptides as well as an additional faint band at the posi- tion expected for the truncated CD4 polypeptide encoded by this plasmid (lane 1) .
  • CD4f encodes the expected fragment representing ' the N-terminal 177 amino acid residues of the extracellular region of the CD4 molecule (plus two residues • from the vector), that this fragment is secreted in soluble. form into, the medium, and that it displays reactivity with anti-CD4 mAbs.
  • mAb 0KT4 failed to immuno- precipitate the fragment, consistent with its reported inability to block CD4 interaction with membrane- associated or soluble gpl20.
  • mAb 0KT4C displayed barely detectable reactivity with the fragment in keeping witlj its reported failure to block interaction of intact HIV with CD4 (McDougal et al, supra; Sattenatu et al, supra) and its relatively weak capacity to inhibit binding of soluble gpl20 to CD4 (Lundin et al, supra) .
  • the epitopes detected by the HIV-blocking anti-CD4 mAbs are contained within the N-terminal 177 amino acid residues of the extracellular region of CD4 and that functional epitopes for these mAbs can be produced when less than half of the full-length CD4 molecule is synthe ⁇ sized.
  • CD4 fragment (lane 1), the anti-gpl20 mAb specifically precipitated two proteins: gpl20 and the CD4 fragment i (lane 6) .
  • the identity of the CD4 fragment band was confirmed by its absence when the fragment-containing medium was omitted from the reaction (only the gpl20 band was observed) (lane 3) and by its comigration with the single band observed when the fragment-containing medium was immunoprecipitated with an anti-CD4 mAB (lane 7) .
  • I ⁇ virus expressing T7 RNA polymerase (lane 5) was used in place of the gpl20-containing medium. Analysis of the s ⁇ pernatants remaining after immunoprecipitation indi ⁇ cated that under the conditions of this experiment, : nearly all of the CD4 fragment was complexed to gpl20,
  • unlabeled gpl20 could compete for the immunoprecipitation of the CD4 fragment by an anti-CD4 mAb.
  • unlabeled medium containing gpl20 strongly inhibited the immunoprecipitation of the CD4 fragment by the 0KT4A mAb (compare lanes 1 and 3).
  • medium lacking gpl20 strongly inhibited the immunoprecipitation of the CD4 fragment by the 0KT4A mAb (compare lanes 1 and 3).
  • the present invention now makes it possible to prepare a composition for the prevention of HIV infec-
  • Such a composition comprises an effective amount of the recombinant, soluble, truncated form of the CD4 containing the first two immunoglobulin-type domains within the first 177 amino acid residues from the N- terminal half of the CD4 molecule, to inhibit binding of the HIV to the host cells.
  • a method for inhibiting HIV infection of the host cells comprises providing an effec- tive amount of the recombinant, soluble truncated form of CD4 molecule of the present invention, to bind HIV.
  • the invention relies on basic host immuno- logical defense mechanisms which normally involve humoral antibody.
  • the resultant molecules acquire high affinity for all HIV envelope variants, plus selected effector function pro ⁇ vided by the particular heavy chain constant regions employed. This overcomes the problems associated with the failure of HIV-infected individuals to raise high affinity antibodies against conserved determinants of the HIV envelope glycoprotein.
  • hybrid molecules of the present inven ⁇ tion contain only human sequences, thereby minimizing problems arising from host immune responses to foreign proteins.
  • the present invention does not involve the use of molecules which are inherently cytotoxic. Thus, problems related to non-specific toxicity are greatly reduced compared to therapies involving CD4-toxins and immunotoxins.
  • the CD4-immunoglobulin hybrid proteins of the present invention are disulfide-bonded multimers (dimers or tetramers, depending on the particular con- structs). Multivalency greatly enhances the avidity of CD4 for gpl20 on both the cell surface and the virion, compared to the monomeric CD4 derivatives. This also enhances direct neutralization of viral infectivity or inhibition of viral spread by cell-to-cell fusion.
  • the immunoglobulin region also prolongs the survival of the CD4 derivatives in the circulation, thereby enhancing beneficial effects mediated by both direct neutralization and effector-mediated mechanisms.
  • CD4-immunoglobulin hybrid proteins may lyse intact virions as well as HIV-infected cells, in contrast to CD4 toxins which act only against cells. Indeed, there is support for complement-mediated lysis of certain retroviruses in vitro (Cooper et al, 1979. Springer Semin. Immunopathol, 2, 285-320).
  • CD4 In order to produce the CD -immunoglobulin hybrid proteins of the present invention (shown schemati- cally in Fig. 9), a number of factors must be consid ⁇ ered. The first involves the region of CD4 to be employed. Most critically, it must contain the high affinity binding site for gpl20. Experiments with trun ⁇ cated soluble derivatives of CD4 (Traunecker et al. 1988, Nature (London) 331, 84-86; Berger et al, supra) indicate that the amino terminal half of the extracellular region (approximately 180 amino acid residues, representing the first two immunoglobulin-like domains) contains the gpl20 binding site. Site directed mutagenesis studies [Clayton et al.
  • CD4 sequence Other factors to consider in choosing the CD4 sequence include the ease of expression of the corre ⁇ sponding immunoglobulin hybrid proteins in secreted form, their stability in the circulation, their accessibility to different sites in the body, and the possibility that they may also contain the determinants for binding MHC Class II antigens (the surface molecules on antigen- presenting cells with which cellular CD4 is believed to interact). Such binding could potentially impair the _ activity of CD4- cells in normal function, though experi ⁇ ments with soluble CD4 [Hussey et al.
  • IgG segment contains the CHI, hinge, CH2 and CH3 regions.
  • the resultant hybrid pro ⁇ teins are then secreted as disulfide-bonded homodimers which specifically bind gpl20, antibodies to CD4 and to human IgG, Protein A, complement (specifically Cg), and FC receptors on appropriate cells of the immune system (e.g. , macrophages) .
  • a third factor to consider is co-expression of the CD4-IgG heavy chain proteins along with human light chains. Under these conditions, disulfide-bonded hetero- tetramers analogous to normal human IgG are produced thereby enhancing expression and secretion of the CD4- heavy chain hybrid molecules. Furthermore, the binding of complement seems dependent on factors other than simply the presence of relevant sequences on the heavy chain constant region, since different subclasses of human IgG which contain the known complement binding sequence differ widely in their complement binding capacity. Thus, higher order structural features appear to be important, and it is likely that these are depen-, dent on the heterotetrameric structure with the light chains.
  • a particular embodiment of this approach involves co-expression of the CD4-heavy chain molecules, not with normal human light chains but instead with recombinant proteins containing CD4 sequences linked to the constant regions of human light chains.
  • the result- ing heterotetramers each contain four copies of the CD4 sequence, and this multivalency leads to extremely high
  • the following antibodies were obtained , from the indicated sources: 0KT4 and 0KT4A (Ortho Pharmaceuti ⁇ cals); anti-Leu-3A (Becton Dickinson); a murine anti- human kappa light chain mAb (Boehringer Mannheim); a murine anti-gpl20 monoclonal antibody (mAb) from hybridoma 902 (National Institute of Allergy and Infec ⁇ tious Diseases, Hamilton, MT); biotinylated goat anti- mouse IgG and biotinylated goat anti-human IgG (Fc) anti ⁇ bodies (Bethesda Research Laboratories); biotinylated goat anti-human lambda light chain antibody (Amersham) .
  • Protein A-agarose and streptavidin agarose were purchased from Bethesda Research Laboratories.
  • the intermediate plasmid, pCD4CHl was constructed as follows ( Figure 10). A 1.7- kilobase (kb) EcoRl-BamHl fragment containing a whole human CD4 cDNA was isolated from pDE4GEM4 (obtained from
  • TM 44 (GACACCCACCTGCTTGCCTCCACCAAGGGCC) and TM 45 (CTTGGTGGAGGCAAGCAGGTGGGTGTC) .
  • TM 44 (GACACCCACCTGCTTGCCTCCACCAAGGGCC)
  • TM 45 (CTTGGTGGAGGCAAGCAGGTGGGTGTC) .
  • the resulting plasmid, pCD4ITM10 is capable of expressing CD4(109)CH under control of the bacteriophage T7 promoter.
  • pCD4lTM10G was constructed by ligating a 2.2-kb Xbal-Sall fragment containing the cod- ing sequences for CD4(109)CH from pCD4ITM10 with a 6.1-kb Xbal-Sall fragment from pTM3 which contains an Eco-gpt transcription unit as a selective marker.
  • CD4(178)CH Figure 9 which comprises the amino-terminal two Ig-like domains (1-178) of CD4 and three constant domains of the human IgGl heavy chain molecule, pCD4ITM20 and pCD4ITM20G were constructed as follows ( Figure 12).
  • An adapter consisting of two synthetic oligonucleotides, TM46 (CTAGCCGCCTCCACCAAGGGCC) , and TM47 (CTTGGTGGAGGCGG), was ligated into the Apal and Nhel sites of pCD4CHl in which the Nhel site exists in the carboxyl-terminal region of the second domain of CD4.
  • pCD4ITM20 is capable of expressing CD4(178)CH under control of the bacteriophage T7 promoter
  • pCD4ITM20G was constructed by ligating a 2.4-kb Xbal-Sall fragment containing the coding sequences Q for CD4(178)CH from pCD4ITM20 with a 6.1-kb Xbal-Sall fragment from pTM3 which contains an Eco-gpt transcrip ⁇ tion unit as a selective marker.
  • pCD4ITM30 and pCD4ITM30G To express the hybrid protein, CD4(372)CH ( Figure 9) which 5 consists of the amino-terminal four domains (1-372) of CD4 and three constant domains of the human IgGl heavy chain molecule, pCD4ITM30 and pCD4ITM30G were constructed as follows ( Figure 13). A 0.65-kb Sacl-Hpall fragment corresponding to nucleotides 598-1252 of the cDNA 0 sequence reported by Maddon et al (1985, Cell 42, 93-104) was isolated from pCD4GEM4. The fragment, together with an adapter consisting of TM48 (CGGTGCAGCCAATGGCCTCCACCAA- GGGCC).
  • pCD4lTM30 is cap ⁇ able of expressing CD4(372)CH under control of the bacteriophage T7 promoter.
  • pCD4ITM30G was constructed by ligating a 3.0-kb Xbal-Sall fragment containing the cod- 0 ing sequences for CD4(372)CH from pCD4lTM30 with a 6.1-kb Xbal-Sall fragment from pTM3 which contains an Eco-gpt transcription unit as a selective marker.
  • CD4(181)CL Figure 9 which comprises the 5 amino-terminal 181 amino acids of CD4, one amino acid (Leu) artificially created by introduction of a Hindlll restriction site, three amino acids (Gin-Met-Lys) of the joining region of the human Ig kappa light chain, and the whole constant region of the human Ig kappa light chain, pCD4ITM40G was constructed as follows ( Figure 14).
  • Ig kappa light chain was isolated from pING1480 in which the Hindlll site was introduced into the joining region and the Xhol site was introduced into the 3 noncoding region of the human Ig kappa light chain cDNA.
  • PCD4LTM1G a derivative of pCD4LTMl (Mizukami et al, supra) , which contains an Eco-gpt transcription unit as a selective marker.
  • 0.23 ml of the processed culture media or 0.115 ml of the cell extracts were added with 0.5 to 1.0 micro- gram of 0KT4A and anti-Leu-3A, respectively, and incu ⁇ bated for 8 hr at 4°C.
  • the immune complexes were collected by adding 0.1 ml of 20% (vol/vol) suspension of protein A-agarose and washed as described by Berger et al
  • 10 processed culture media were used for the binding exper ⁇ iments.
  • the media were added with 0.5 microgram of the mAbs, incubated for 8 hr at 4°C, then added with 10 microgram of biotinylated goat anti-moust.
  • hybrid proteins [CD4(109)CH, CD4(178)CH and CD4(372)CH] with different lengths (1-109, 1-178, and 1-372 of the human CD4 extracellular region linked to human IgGl heavy chain constant region, and one hybrid protein [CD4(181)CL] comprising the amino-terminal 181 amino acid residues of CD4 and the human Ig light chain constant region were designed ( Figure 9).
  • a vaccinia virus-based expression system was used, and the plasmids for the expression of these hybrid proteins were constructed ( Figures 10-14). In those plasmids the coding sequences for the hybrid proteins are placed under the T7 promoter and can be expressed upon coexpression of T7 RNA poly ⁇ merase.
  • CD4(178)CH, and CD4(372)CH in CV-1 cells was investigated ( Figure 15).
  • 38-kilodalton (kd) 38-kilodalton (kd), 65-kd, and 88-kd proteins were detected by transfection of pCD4lTM10, pCD4ITM20, and pCD4ITM30, respectively, after immunoprecipitation with anti-CD4 mAbs followed by trapping with protein A- agarose.
  • the calculated total amino acid residue numbers of CD4(109)CH, CD4(178)CH, and CD4(372)CH are 439, 508, and 702, respectively.
  • the observed molecular weights of the expressed proteins are slightly heavier than those estimated by the residue numbers.
  • the differences pre ⁇ sumably come from glycosylation of the proteins; the CH2 domain of the Ig heavy chain and the third and fourth Ig- like domains of CD4 each have one asparagine-linked glycosylation site.
  • a considerable portion of the syn ⁇ thesized proteins were secreted into the culture media even in the absence of Ig light chain expression; this was unexpected, since it has been shown that Ig heavy chain secretion is poor in the absence of light chain, expression (Pepe et al, 1986, J. Immunol. 137:2367-2372).
  • the subunit structure of the hybrid proteins which were expressed in CV-1 cells was then analyzed by SDS-polyacrylamide-gel electrophoresis in non-reducing conditions (Figure 16).
  • CD4(109)CH, CD4(178)CH, and CD4(372)CH moved to the high molecular weight positions, presumably in dimer positions; by contrast a soluble form of CD4 consisting of 1-372 amino acid residues of CD4 migrated at the expected monomer position.
  • CD4(109)CH, CD4(178)CH, and CD4(372)CH exist as disulphide-linked dimers both in the culture media and inside of the cells.
  • CD4(181)CL the other hybrid pro ⁇ tein, CD4(181)CL
  • pCD4lTM40G the expression of the other hybrid pro ⁇ tein, CD4(181)CL.
  • CV-1 cells expressed a 34-38 kd protein which is most likely CD4(181)CL.
  • the major portion of this protein migrated in the monomer position also in the absence of reducing agent, indicating that CD4(181)CL exists as monomers.
  • CD4(178)CH migrated in the dimer position in a non-reducing condition, indicat- ing that CD4(178)CL exists as disulphide-linked dimers.
  • CD4(109)CH, CD4(178)CH, and CD4(372)CH with the normal human Ig lambda light chain was examined, by transfecting pCD4ITM10, pCD4lTM20, and pCD4ITM30 into RPMI 8226, a human myeloma cell line (ATCC CCL 155) which secretes human Ig lambda light chains ( Figure 19). All of those three hybrid proteins were synthesized and secreted effi ⁇ ciently into the culture media, although much of the expressed proteins accumulated in the inside of the cells.
  • the esti ⁇ mated molecular weights of major complexes in the culture media of the cells transfected with pCD4lTM10, ⁇ CD4lTM20, and pCD4lTM30 are 180 kd, 210 kd, and >220 kd, respec ⁇ tively. This result indicates that these complexes represent tetrameric structures composed of two subunits of each hybrid protein and two subunits of the human Ig lambda light chain from the host cell. These molecules may, therefore, have structures analogous to natural Ig molecules.
  • CD4(178)CH which was expressed and secreted from the CV-1- transfected cells, was first analyzed ( Figure 20). A soluble form of CD4 containing full extracellular four
  • Ig-like domains was also expressed and analyzed as a control.
  • CD4(178)CH bound to the HIV gpl20, 0KT4A, anti- human IgG (Fc) antibody, and protein A-agarose, but did not bind to OKT4.
  • the soluble CD4 bound to the HIV gpl20, 0KT4, and 0KT4A. However, it did not bind to anti-human IgG (Fc) antibody and protein A-agarose.
  • CD4(178)CH The binding regions for gpl20 and 0KT4A have been identified in the first domain of CD4 (Clayton et al, supra; Landau et al, supra; Peterson et al, supra; Mizukami et al, supra) , whereas the binding region for 0KT4 is believed to exist in the third or fourth domains of CD4. These results are consistent with the binding properties of .
  • CD4(178)CH which was coexpressed with the host- derived human Ig lambda chain by the transfected-RPMI8226 cells was then examined for the binding property to various ligands and antibodies (Figure 21).
  • CD4(178-)CH was also coimmunoprecipitated with it, demonstrating that CD4(178)CH makes a complex wiht the host-derived lambda chain.
  • CD4(178)CH and CD4(181)CL which were coex ⁇ pressed in CV-1 cells were also analyzed for the binding properties to various ligands and antibodies ( Figure 22). Although 0KT4 did not immunoprecipitate CD4(178)CH or CD4(181)CL, OKT4A could immunoprecipitate both these hybrid proteins. The gpl20 could also bind to both pro ⁇ teins as well. Anti-human IgG (Fc) antibody and protein A-agarose could immunoprecipitate CD4(178)CH, and also coprecipitate CD4(181)CL with it. Anti-human Ig " kappa mAb could immunoprecipitate CD4(181)CL, . and also copre ⁇ cipitate CD4(178)CH with it.
  • this part of the present invention teaches the construction of multimeric recombinant pro ⁇ teins comprising the gpl20 binding region of human CD4 linked to constant regions of heavy and light chain com ⁇ ponents of human IgG, the resulting recombinant proteins at least possessing the property of inhibiting HIV proliferation by either neutralizing HIV activity, by killing HIV-infected cells, or by lysing HIV virions.
  • a method of treating or inhibiting HIV infection comprises administering an effective amount of the above mentioned composition to a host in need of protection or treatment against HIV to kill or inhibit HIV infection.
  • a deposit of plasmids pCD4lTM10G, pCD4ITM20G, pCD4lTM30G and pCD4ITM40G for the production of recombi- nant hybrid proteins in accordance with the. present invention have been made at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852, U.S.A., on April 27, 1989 under accession numbers 67940, 67941, 67942 and 67943, respectively.
  • the deposit shall be viably maintained, replacing if it becomes non-viable during the life of the patent, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer, and made available to the public without restriction in accordance with the provisions of the law.
  • the Commissioner of Patents and Trademarks, upon request shall have access to the deposit.

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Abstract

Un gène chimérique dirige la synthèse d'une protéine hybride recombinante obtenue par fusion dans un vecteur approprié d'expression. La protéine obtenue par fusion présente une cytotoxicité spécifique à l'égarol de cellules infectées par des virus spécifiques. Une protéine hybride obtenue par fusion, CD4(178)-PE40, tue sélectivement des cellules infectées par VIH. Une forme recombinante, soluble et tronquée de CD4 contient le site actif de liaison du virus d'immunodéficience humaine. De nouvelles protéines hybrides qui contiennent des séquences de CD4 humaine liées à des zones constantes de l'immunoglobuline humaine inhibent l'infection par des VIH.
PCT/US1989/003267 1988-07-22 1989-07-24 Agent cytotoxique pour le traitement d'infections virales specifiques WO1990001035A1 (fr)

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WO1992013947A1 (fr) * 1991-02-08 1992-08-20 Progenics Pharmaceuticals, Inc. CHIMERES DE CD4-GAMMA2 ET DE CD4-IgG2
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EP0561953A1 (fr) * 1990-12-03 1993-09-29 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Immunotoxine recombinee composee d'un anticorps monocatenaire reagissant avec le recepteur de la transferrine humaine et la toxine diphterique
WO1994004689A1 (fr) * 1992-08-14 1994-03-03 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Toxine recombinee a demi-vie prolongee
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US7070991B2 (en) 1991-02-08 2006-07-04 Progenics Pharmaceuticals, Inc. Cells expressing a CD4-IgG2 chimeric heterotetramer

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Biotechniques, Volume 4, Number 3, published 1986, V. OI et al., "Chimeric antibodies", pp. 214-220, see entire article. *
Cell, Volume 42, published August 1985, P.J. MADDON et al., "The isolation and nucleotide sequence of a cDNA encoding the T cell surface protein T4; a new member of the immunoglobulin gege family", pp. 93-104, see entire article. *
Proc. Natl. Acad. Sci. USA, Volume 81, published November 1984, S. MORRISON et al., "Chimeric human antibody molecules: mouse antigen-binding domains with human constant region domains", pp. 6851-6855, see entire article. *
Proc. Natl. Acad. Sci. USA, Volume 84, published July 1987, V.K. CHAUDHARY et al., "Activity of a recombinant fusion protein between transforming growth factor type alpha and Pseudomonas toxin", pp. 4538-4542, see entire article. *
Science, Volume 231, published 24 January 1986, J.S. McDOUGEL et al., "Binding of HTLV-III/LAV to T4+ cells by a complex of the 110K viral protein and the T4 molecule", pp. 382-385, see entire article. *
See also references of EP0428603A4 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5498538A (en) * 1990-02-15 1996-03-12 The University Of North Carolina At Chapel Hill Totally synthetic affinity reagents
US5852167A (en) * 1990-02-15 1998-12-22 The University Of North Carolina At Chapel Hill Totally synthetic affinity reagents
US5844076A (en) * 1990-02-15 1998-12-01 The University Of North Carolina At Chapel Hill Totally synthetic affinity reagents
US5625033A (en) * 1990-02-15 1997-04-29 The University Of North Carolina At Chapel Hill Totally synthetic affinity reagents
US5510256A (en) * 1990-08-06 1996-04-23 The Upjohn Company Eliminating internal initiation of soluble CD4 gene
WO1992002626A1 (fr) * 1990-08-06 1992-02-20 The Upjohn Company Elimination de l'initiation interne de gene de cd4 soluble
US5759852A (en) * 1990-08-06 1998-06-02 Pharmacia & Upjohn Company Expression vector containing PL6M promoter and TAT32 ribosome binding site and host cells transformed therewith
EP0561953A4 (en) * 1990-12-03 1994-08-10 Us Commerce Recombinant immunotoxin composed of a single chain antibody reacting with the human transferrin receptor and diphtheria toxin
EP0561953A1 (fr) * 1990-12-03 1993-09-29 THE UNITED STATES OF AMERICA as represented by the Secretary United States Department of Commerce Immunotoxine recombinee composee d'un anticorps monocatenaire reagissant avec le recepteur de la transferrine humaine et la toxine diphterique
WO1992013947A1 (fr) * 1991-02-08 1992-08-20 Progenics Pharmaceuticals, Inc. CHIMERES DE CD4-GAMMA2 ET DE CD4-IgG2
US6187748B1 (en) 1991-02-08 2001-02-13 Progenics Pharmaceuticals, Inc. Uses of CD4-gamma2 and CD4-IgG2 chimeras
US6451313B1 (en) 1991-02-08 2002-09-17 Progenics Pharmaceuticals, Inc. CD4-gamma2 and CD4-IGG2 chimeras
US7070991B2 (en) 1991-02-08 2006-07-04 Progenics Pharmaceuticals, Inc. Cells expressing a CD4-IgG2 chimeric heterotetramer
US5234905A (en) * 1991-02-22 1993-08-10 University Of Colorado Foundation, Inc. Soluble CD4 molecules modified to prolong circulating half-life
WO1994004191A1 (fr) * 1992-08-13 1994-03-03 Antisoma Limited Traitement medical
WO1994004689A1 (fr) * 1992-08-14 1994-03-03 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Toxine recombinee a demi-vie prolongee

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IL91070A (en) 1995-03-30
EP0428603A1 (fr) 1991-05-29
IL91070A0 (en) 1990-02-09
EP0428603A4 (en) 1991-11-13

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