WO1993015747A1 - Procede d'utilisation des molecules contenant le recepteur cellulaire cd4 pour reduire considerablement le risque de transmission du vih dans le milieu professionnel - Google Patents
Procede d'utilisation des molecules contenant le recepteur cellulaire cd4 pour reduire considerablement le risque de transmission du vih dans le milieu professionnel Download PDFInfo
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- WO1993015747A1 WO1993015747A1 PCT/US1993/001193 US9301193W WO9315747A1 WO 1993015747 A1 WO1993015747 A1 WO 1993015747A1 US 9301193 W US9301193 W US 9301193W WO 9315747 A1 WO9315747 A1 WO 9315747A1
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- hiv
- patient
- infected
- chimeric
- containing molecule
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Classifications
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- 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
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- C—CHEMISTRY; METALLURGY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
- C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- 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"
Definitions
- HIV-Seroprevalence Several important factors affect the risk of occupational HIV transmission. These include i) the HIV seroprevalence of the patient population; ii) the risk of occupational blood exposure; and iii) the risk of seroconversion after blood exposure. i) HIV-Seroprevalence
- the HIV seroprevalence is significant. For example, at a trauma center in Hannover, Germany, 0.1% of surgical patients were HIV-seropositive over an 18-month period (5) . In contrast, 13.6% of all patients admitted with penetrating traumas at the Johns Hopkins Hospital in Baltimore, Maryland were HIV- seropositive (6) . In the Johns Hopkins study, the HIV- seroprevalence of 2,302 adult patients screened in the emergency room was 5.2%. Furthermore, 29% of all surgical patients at San Francisco General Hospital had risk factors for HIV infection (7).
- the cumulative lifetime risk of HIV infection to a surgeon can be estimated by probability models. Using two recent models, it has been estimated that the cumulative lifetime risk of seroconversion for the average surgeon is between 1% and 20% (20, 21) .
- the San Francisco General Hospital study estimates that every eight years, one of their staff surgeons will become infected with HIV as a result of occupational transmission (7) .
- HIV infects primarily helper T lymphocytes and monocytes/macrophages—cells that express surface CD4— leading to a gradual loss of immune function which results in the development of the human acquired immune deficiency syndrome (AIDS) .
- the initial phase of the HIV replicative cycle involves the high affinity interaction between the HIV exterior envelope glycoprotein gpl20 and the cellular receptor CD4 (25) .
- viral and target cell membranes fuse, resulting in the introduction of the viral genome into the cytoplasm.
- Several lines of evidence demonstrate the requirement of this interaction for viral infectivity.
- the introduction of a functional cDNA encoding CD4 into human cells which do not express CD4 is sufficient to render otherwise resistant cells susceptible to HIV infection (26) .
- CD4 is a nonpolymorphic, lineage-restricted cell surface glycoprotein that is a member of the immunoglobulin gene superfa ily. High-level expression of both full-length and truncated, soluble versions of CD4 (sCD4) have been described in stable expression systems. The availability of large quantities of purified sCD4 has permitted a detailed understanding of the structure of this complex glycoprotein.
- Mature CD4 has a relative molecular mass (Mr) of 55 kilodaltons and consists of an amino-terminal 372 amino acid extracellular domain containing four tandem im unoglobulin-like regions denoted V1-V4, followed by a 23 amino acid transmembrane domain and a 38 amino acid cytoplasmic segment.
- Mr relative molecular mass
- VI has defined a discrete gpl20 binding site (residues 38-52 of the mature CD4 protein) that comprises a region structurally homologous to the second complementarity-determining region (CDR2) of immunoglobulins (29).
- CDR2 complementarity-determining region
- CD4 Since the discovery of CD4's role as the cellular receptor for HIV, researchers have explored its potential uses as a therapeutic agent to treat patients with HIV disease. A water-soluble version of the entire extracellular segment of CD4 (V1-V4, termed sCD4) has been described (30).
- sCD4 acts as a "molecular decoy" by binding to HIV gpl20 and competitively inhibiting viral attachment to and subsequent infection of human cells; 2) sCD4 "strips" the viral envelope glycoprotein gpl20 from the viral surface, thereby inactivating HIV particles directly; and 3) sCD4 blocks the intercellular spread of virus from HIV-infected cells to uninfected cells by inhibiting virus-mediated cell fusion (25, 31).
- pretreatment of chimpanzees with a CD4-based molecule prior to challenge with HIV protects them from infection (32) .
- the subject invention provides a method of substantially reducing the risk of occupational transmission of HIV comprising administering to a patient during a suitable time period an amount of a CD4-containing molecule effective to substantially reduce the likelihood of a non-infected medical practitioner becoming infected with HIV by virtue of contact with the patient's bodily fluid during a bodily fluid-exposing medical procedure.
- the method of the subject invention does not involve therapeutically treating an HIV-infected individual. Accordingly, the potential problems of short serum half-life and limited in vivo therapeutic efficacy during chronic use of certain CD4-containing molecules, discussed supra, are not a barrier to practicing this invention.
- This invention provides a method of substantially reducing the likelihood of a non-infected medical practitioner becoming infected with HIV during a blood-exposing medical procedure involving a patient which comprises administering to the patient during a suitable time period an amount of a CD4-containing molecule effective to substantially reduce the likelihood of the non-infected medical practitioner becoming infected with HIV by virtue of contact with the patient's blood during the medical procedure.
- This invention also provides a method of substantially reducing the likelihood of a non-infected medical practitioner becoming infected with HIV during a blood-exposing medical procedure involving a patient which further comprises administering to the patient during a suitable time period a suitable dose of a mixture comprising an HIV-neutralizing antibody and a CD4-containing molecule, such mixture being effective to substantially reduce the likelihood of the non- infected medical practitioner becoming infected with HIV by virtue of contact with the patient's blood during the medical procedure.
- this invention provides a method of substantially reducing the likelihood of a non- infected medical practitioner becoming infected with HIV during a bodily fluid-exposing medical procedure involving a patient which comprises administering to the patient during a suitable time period an amount of a CD4-containing molecule effective to substantially reduce the likelihood of the non-infected medical practitioner becoming infected with
- the plasmid ⁇ CD4-IgG 1 HC-pRcCMV, CD4-kLC-pRcCMV, CD4IgG 2 - pcDNAl and CD4-IgG 2 HC-pRcCMV were deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession Nos. 75192, 75194, 40952 and 75193, respectively.
- ATCC American Type Culture Collection
- this invention provides a method of substantially reducing the likelihood of a non-infected medical practitioner becoming infected with HIV during a blood-exposing medical procedure involving a patient which comprises administering to the patient during a suitable time period an amount of a CD4-containing molecule effective to substantially reduce the likelihood of the non-infected medical practitioner becoming infected with HIV by virtue of contact with the patient's blood during the medical procedure.
- the patient is HIV-infected, i.e., possesses HIV particles in his bloodstream.
- substantially reducing the likelihood of a non-infected medical practitioner becoming infected with HIV means reducing the likelihood by at least two-fold. For example, if the likelihood of infection were 1 infection occurring for every 1,000,000 blood-exposing medical procedures performed, a two-fold reduction in the likelihood of infection would result in a likelihood of 1 infection occurring for every 2,000,000 blood-exposing medical procedures performed. In a preferred embodiment of this invention, substantially reducing the likelihood of a non-infected medical practitioner becoming infected with HIV means reducing the likelihood by at least ten-fold.
- a blood-exposing medical procedure is any procedure which places the medical practitioner at risk of contacting the patient's blood in a manner sufficient to cause HIV in the patient's blood to be transmitted to the medical practitioner's body, and thereby infect the medical practitioner with HIV.
- the medical procedure may comprise, but is not limited to, a dental procedure such as oral surgery or dental cleaning, a surgical procedure such as an emergency care surgical procedure or a surgical transplant procedure, a dialysis procedure, a catheterization procedure, an obstetric procedure or a gynecological procedure.
- the administering of the CD4-containing molecule may be effected or performed using any of the various methods known to those skilled in the art.
- the administering comprises administering intravenously.
- the administering comprises administering intramuscularly.
- the administering comprises administering subcutaneously.
- the suitable time period during which a CD4-containing molecule is administered can be any time period such that, when the CD4-containing molecule is administered within this time period, an optimal prophylactic concentration of CD4-containing molecule is maintained in the patient's bloodstream throughout the medical procedure.
- the suitable time period comprises a time period sufficiently prior to the medical procedure. An example of administration within this time period would be a single injection of a CD4-containing molecule prior to a medical procedure.
- the suitable time period comprises a continuous time period spanning the duration of the procedure.
- An example of administration within this time period would be a continual IV infusion of a CD4-containing molecule throughout the duration of a medical procedure.
- the suitable time period comprises intermittent time periods spanning the duration of the procedure.
- An example of administration within this time period would be an intermittent series of injections of a CD4-containing molecule throughout the duration of the medical procedure.
- the amount of the CD4-containing molecule is sufficient to maintain the concentration of circulating CD4-containing molecule in the patient's blood during the medical procedure above the minimum amount necessary to substantially reduce the likelihood of the non- infected medical practitioner becoming infected with HIV by virtue of contact with the patient's blood during the medical procedure.
- the amount of the CD4-containing molecule sufficient to maintain the necessary concentration of circulating CD4-containing molecule depends on the molecule's weight and the number of gpl20-binding sites it possesses. For example, assume that it requires amount X of sCD4 , having a molecular weight of A and 1 gpl20-binding site, to achieve a sufficient serum concentration of CD4- containing molecule. For a CD4-containing molecule of molecular weight 4A having 1 gpl20-binding site, it would require 4X of this molecule to achieve the same concentration.
- a CD4-containing molecule may be any molecule comprising a portion of the CD4 protein capable of forming a complex with the HIV-1 gpl20 envelope glycoprotein.
- Examples of CD4-containing molecules include, but are in no way limited to, the CD4-containing molecules discussed infra.
- the CD4-containing molecule may be soluble CD4 (sCD4) , which comprises the extracellular domain of CD4.
- the amount is greater than l ⁇ g of sCD4 per ml of serum.
- the amount is greater than lO ⁇ g of sCD4 per ml of serum.
- the amount is greater than lOO ⁇ g of sCD4 per ml of serum.
- the CD4-containing molecule may comprise a gpl20-binding fragment of soluble CD4 (sCD4) .
- the CD4-containing molecule may also be a CD4-immunoconjugate.
- the CD4-immunoconjugate may be a CD4-gammal chimeric heavy chain homodimer.
- the CD4-immunoconjugate may also be a heterotetramer comprising two heavy chains and two light chains, both heavy chains being either a) IgGl heavy chains or b) chimeric CD4- IgGl heavy chains, and both light chains being either a) kappa light chains or b) chimeric CD4-kappa light chains, with the proviso that either both heavy chains or both light chains or all four chains are CD4 chimeras.
- the heterotetramer may be a heterotetramer wherein the chimeric CD4-IgGl heavy chains are encoded by the expression vector designated CD4-IgGlHC-pRcCMV (ATCC No. 75192) , and the chimeric CD4-kappa light chains are encoded by the expression vector designated CD4-kLC-pRcCMV (ATCC No. 75194) .
- the CD4-immunoconjugate may be a CD4-gamma2 chimeric heavy chain homodimer.
- the CD4-gamma2 chimeric heavy chain homodimer is the CD4-gamma2 chimeric heavy chain homodimer whose chains are encoded by the expression vector designated CD4IgG 2 -pcDNAl (ATCC No. 40952) .
- the CD4-immunoconjugate may also be a heterotetramer comprising two heavy chains and two light chains, both heavy chains being either a) IgG2 heavy chains or b) chimeric CD4- IgG2 heavy chains, and both light chains being either a) kappa light chains or b) chimeric CD4-kappa light chains, with the proviso that either both heavy chains or both light chains or all four chains are CD4 chimeras.
- the heterotetramer may be a heterotetramer wherein the chimeric CD4-IgG2 heavy chains are encoded by the expression vector designated CD4-IgG2HC-pRcCMV (ATCC No. 75193) , and the chimeric CD4-kappa light chains are encoded by the expression vector designated CD4-kLC-pRcCMV . (ATCC No. 75194) .
- This invention also provides a method of substantially reducing the likelihood of a non-infected medical practitioner becoming infected with HIV during a blood- exposing medical procedure involving a patient which further comprises administering to the patient during a suitable time period a suitable dose of a mixture comprising an HIV- neutralizing antibody and a CD4-containing molecule, such mixture being effective to substantially reduce the likelihood of the non-infected medical practitioner becoming infected with HIV by virtue of contact with the patient's blood during the medical procedure.
- the humoral immune response to HIV is characterized by neutralizing antibodies.
- Neutralizing antibodies directly neutralize HIV particles by preventing the initiation of infection and are believed to be one of the most important component of the immune response against HIV and many other viruses.
- HIV neutralizing antibodies are directed against the envelope glycoprotein (gpl20/gp41) of the virus. There are at least two major classifications of HIV neutralizing antibodies. These two classifications are the type-specific HIV neutralizing antibodies and the group-common HIV neutralizing antibodies. Type-specific HIV neutralizing antibodies primarily recognize the third variable domain (V3) of gpl20, a highly variable loop structure termed the principal neutralizing determinant. Because of the extreme variability in amino acid sequence, type-specific HIV neutralizing antibodies generally neutralize only a particular isolate or closely related strains. Type- specific HIV neutralizing antibodies generally act by inhibiting HIV membrane fusion with the target cell. In contrast, group-common HIV neutralizing antibodies possess the ability to neutralize a broader set of isolates.
- group-common neutralizing antibodies recognize a site on gpl20 at or near the CD4-binding site, although antibodies directed against other regions of gpl20 may have broadly neutralizing activity as well. Consequently, many group- common HIV-neutralizing antibodies inhibit viral attachment to the target cell by blocking the interaction between gpl20 and surface CD4. Both type-specific and group-common neutralizing activity have been detected in HIV antibody- positive human sera. In addition, both type-specific and group-common HIV monoclonal neutralizing antibodies have been generated in several species.
- a third kind of HIV-neutralizing antibody is directed toward the gp41 component of the HIV envelope glycoprotein.
- Anti- gp41 neutralizing antibodies recognize an extracellular component of this trans embrane glycoprotein.
- Anti-gp41 antibodies have been generated which possess either strain- specific reactivity (like the type-specific neutralizing antibody) or more broad reactivity (like the group-common neutralizing antibody) .
- the neutralizing antibody may be a group- common neutralizing antibody.
- the neutralizing antibody may also be a type-specific neutralizing antibody.
- the type-specific neutralizing antibody is an anti-V3 loop antibody.
- the neutralizing antibody may also be an anti-gp41 antibody.
- this invention provides a method of substantially reducing the likelihood of a non-infected medical practitioner becoming infected with HIV during a bodily fluid-exposing medical procedure involving a patient which comprises administering to the patient during a suitable time period an amount of a CD4-containing molecule effective to substantially reduce the likelihood of the non-infected medical practitioner becoming infected with HIV by virtue of contact with the patient's bodily fluid during the medical procedure.
- a bodily fluid is any fluid which is present in the human body and is capable of containing infectious HIV in an HIV-infected patient.
- Bodily fluids include, but are not limited to, saliva, cerebrospinal fluid, tears, vaginal secretions, urine, alveolar fluid, synovial fluid and pleural fluid.
- a bodily fluid-exposing medical procedure is any procedure which places the medical practitioner at risk of contacting the patient's bodily fluid in a manner sufficient to cause HIV in the patient's bodily fluid to be transmitted to the medical practitioner's -body, and thereby infect the medical practitioner with .HIV.
- Sera were obtained from HIV-seropositive men who were asymptomatic or had chronic lymphadenopathy. Serum from one patient with clinical AIDS was also tested. Sera were stored at -70°C and were heat inactivated at 56°C for 30 minutes before use. The IgG fraction was purified from some sera by ammonium sulfate precipitation and DEAE chro atography as previously described (36) .
- the lymphadenopathy virus (LAV-i) prototype strain of HIV-i was used in the infectivity assays.
- clinical isolates or first passage isolates were obtained by coculture of patient lymphocytes with phytohemagglutinin (PHA)-stimulated normal peripheral blood lymphocytes.
- PHA phytohemagglutinin
- Soluble CD4 (a genetically-engineered, water-soluble extracellular fragment of human CD4) is disclosed, for example, in Patent Cooperation Treaty International Publication No. WO 88/01304. Soluble CD4 is also commercially available.
- Soluble CD4 also designated sCD4 or sCD4 fragments, may be produced by truncating pT4B (ATCC No. 68389) after the V4J4 domain. Such DNA fragments terminate before the transmembrane segment, which begins at approximately nucleotide position 1264.
- soluble CD4 fragments Purification and characterization of soluble CD4 fragments is greatly enhanced by constructing a cell line (preferably mammalian) which overexpresses the secreted protein fragment.
- Strategies which allow the overexpression of proteins may be employed in bacteria, yeast, insect and mammalian systems. Inducible expression systems may also be employed in bacteria and yeast to overproduce proteins which may be toxic if constitutively expressed.
- Overexpression of soluble CD4 fragments may be accomplished by amplifying a soluble CD4 expression vector, resulting in constitutive overexpression. The amplification of dihydrofolate reductase (dhfr) genes by growth in progressively increased concentrations of the drug methotrexate, an antagonist of dhfr, is widely employed.
- dhfr dihydrofolate reductase
- dhfr may be used as a selectable marker and as a means of coamplifying newly introduced sequences. This strategy may be successfully employed to increase the expression of several different genes cotransformed with dhfr plasmids.
- a vector expressing a secreted, soluble, extracellular fragment of CD4 encoded by the human cDNA clone pT4B may be generated.
- Base pairs 1- 1252 of pT4B encode the leader peptide of CD4 needed for the synthesis of secreted protein, as well as the extracellular portion of CD4 encompassing the four VJ-like domains (V1J1- V4J4) , but not the transmembrane and cytoplasmic regions which anchor the protein in the membrane.
- This vector contains sequences encoding the extracellular portion of the CD4 protein which contains the HIV binding domain. These sequences are placed downstream from the SV40 early region promoter.
- a TAA termination codon followed by the polyadenylation region of the bovine growth hormone gene is placed downstream from the truncated CD4 cDNA to provide the signals necessary for the termination of protein synthesis, transcription termination, and polyadenylation of the RNA transcript.
- the resulting soluble CD4 minigene is then ligated to the mouse dihydrofolate reductase (dhfr) gene to generate a plasmid capable of being amplified after introduction into dhfr-deficient (dhfr-) Chinese hamster ovary (CHO) cells.
- the 1.8 kb EcoRI-BamHI fragment of pT4B which contains the entire CD4 coding sequence, is inserted between the StuI and Bell sites of the mammalian expression vector DSP modified to contain the SV-40 early promoter and the bovine growth hormone polyadenylation sequence.
- the Haell (bp 124) - Hpall (bp 1252) fragment of pT4B is inserted between the Kpnl and Xbal sites of the plasmid pUCl ⁇ .
- a soluble CD4 expression vector is created by ligating:
- DXB-11 a clone of Chinese hamster ovary cells deficient in dhfr, is transfected with the soluble CD4 expression plasmid.
- the DXB-11 transformants are then grown in F12 medium, without hypoxanthine or thymidine, containing 10% dialyzed fetal bovine serum.
- Clones are selected and subjected to stepwise increasing concentrations of methotrexate (mtx) , an antagonist of dhfr, to select for stable transformants which have amplified the newly introduced dhfr gene and adjacent soluble CD4 sequences.
- mtx methotrexate
- CosM5 cells grown in DMEM containing 10% fetal calf serum were split to 75% confluence. On the following day, the cells were transfected for 16-20 hours with 10 micrograms of a CsCl-purified plasmid encoding a chain of a CD4-IgGl heavy chain homodimer by the standard CaPO(4) precipitation technique. After transfection, fresh medium was added to the cells.
- Dhfr- Chinese hamster ovary cells were transfected with 20 microgra s of CsCl-purified DNA in a 1000:1 molar ratio of CD4IgGl-pcDNAl:p410 (p410 is an expression plasmid containing the dhfr gene) , although other ratios may also be used. Approximately 3-5 days post-transfection, cells were placed in selective medium (nucleoside-free alpha MEM containing 10% dialyzed fetal calf serum) .
- CD4-gammal chimeric heavy chain homodimer Approximately 10-15 days post-selection, individual cell clones were picked and analyzed for stable expression of CD4-gammal chimeric heavy chain homodimer by several screening techniques, such as ELISA and precipitation with Protein A- sepharose beads followed by SDS-PAGE under reducing and non- reducing conditions. Clones expressing the highest levels were subjected to successive rounds of amplification of the newly introduced DNA sequences in increasing concentrations of methotrexate. Stable CHO cell lines were thus generated which secrete between 10-100 micrograms/ illiliter of CD4- gammal chimeric heavy chain homodimer.
- CD4-gammal chimeric heavy chain homodimer was purified by column chromatography. CHO cells secreting CD4-gammal chimeric heavy chain homodimer were grown to high density in roller bottles in medium containing alpha MEM with 10% IgG- free fetal calf serum. Conditioned media was collected, clarified by centrifugation, and diluted 1:1 with PBS either with or without detergent (i.e. Tween) in this and subsequent buffers. The diluted media was then applied to a 5ml column of Protein A-Sepharose fast flow previously equilibrated with PBS, at a flow rate of 60ml/hour.
- detergent i.e. Tween
- the pooled fractions were then applied to a 10 ml column of S-sepharose fast flow previously equilibrated with 50mM BES pH 7.0 at a flow rate of 120ml/hr.
- a step elution gradient (consisting of the following 4 steps: 5 column volumes of 50mM BES pH 7.0, 4 column volumes of 50mM BES pH 7.0, lOOmM NaCl, 6 column volumes of 50mM BES pH 7.0 225mM NaCl, followed by 8 column volumes of 50mM BES pH 7.0, 500mM NaCl) was employed for specific elution of the CD4-gammal chimeric heavy chain homodimer.
- the CD4-gammal chimeric heavy chain homodimer was eluted from the column in 50mM BES pH 7.0, 500mM NaCl. The peak fractions were then pooled and concentrated to yield a final protein concentration of at least lmg/ml.
- CosM5 cells grown in DMEM containing 10% fetal calf serum are split to 75% confluence. On the following day, the cells are transfected for 16-20 hours with 5 micrograms of
- CsCl-purified CD4-IgGlHC-pRcCMV DNA and 5 micrograms of CsCl-purified CD4-kLC-pRcCMV plasmid DNA by the standard CaPO(4) precipitation technique. After transfection, fresh medium is added to the cells. Analysis of the products synthesized 48-72 hours post-transfection is performed by radiolabelling of transfectants with 35 S-methionine for 12-18 hours followed by precipitation of media and cell lysates using anti-CD4 antibodies or by incubation with Protein A-sepharose beads alone followed by SDS-PAGE under reducing or non-reducing conditions. In addition, analysis of media and cell lysates is performed 48-72 hours post- transfection by standard Western blotting procedures.
- Stable expression Dhfr- Chinese hamster ovary cells are transfected with 20 micrograms of CsCl purified DNA in a ratio of 1000:1000:1 CD4-IgGlHC-pRcCMV:CD4-kLC-pRcCMV:p410 (p410 is an expression plasmid containing the dhfr gene) , although other ratios may also be used.
- p410 is an expression plasmid containing the dhfr gene
- cells are placed in selective medium (nucleoside-free alpha MEM containing 10% dialyzed fetal calf serum) .
- individual cell clones are picked. The clones are then analyzed for stable expression of CD4-IgGl chimeric heterotetramers by several screening techniques, such as ELISA and precipitation with
- CD4-IgGl chimeric heterotetramers are purified using Protein A-Sepharose column chromatography. CHO cells secreting CD4- IgGl chimeric heterotetramers are grown to high density in roller bottles in medium containing alpha MEM with 10% IgG- free fetal calf serum. Conditioned media is collected, clarified by centrifugation, and diluted 1:1 with PBS either with or without detergent (i.e. Tween) in this and subsequent buffer ⁇ . The diluted media is then applied to a 5ml column of Protein A-Sepharose fast flow previously equilibrated with PBS, at a flow rate of 60ml/hour.
- CosM5 cells grown in DMEM containing 10% fetal calf serum were split to 75% confluence.
- the cells were transfected for 16-20 hours with 10 micrograms of CsCl-purified plasmid CD4IgG2-pcDNAl DNA by the standard CaPO(4) precipitation technique. After transfection, fresh medium was added to the cells.
- Analysis of the products synthesized 48-72 hours post-transfection was performed by radiolabelling of transfectants with 35 S-methionine for 12-18 hours followed by precipitation of media and cell lysates using anti-CD4 antibodies or by incubation with Protein A-sepharose beads alone followed by SDS-PAGE under reducing or non-reducing conditions.
- analysis of media and cell lysates was performed 48-72 hours post- transfection by standard Western blotting procedures.
- Dhfr- Chinese hamster ovary cells were transfected with 20 micrograms of CsCl-purified DNA in a 1000:1 molar ratio of CD4IgG2-pcDNAl:p410 (p410 is an expression plasmid containing the dhfr gene) , although other ratios may also be used. Approximately 3-5 days post-transfection, cells were placed in selective medium (nucleoside-free alpha MEM containing 10% dialyzed fetal calf serum) .
- CD4-gamma2 chimeric heavy chain homodimer Approximately 10-15 days post-selection, individual cell clones were picked and analyzed for stable expression of CD4-gamma2 chimeric heavy chain homodimer by several screening techniques, such as ELISA and precipitation with Protein A- sepharose beads followed by SDS-PAGE under reducing and non- reducing conditions. Clones expressing the highest levels were subjected to successive rounds of amplification of the newly introduced DNA sequences in increasing concentrations of methotrexate. Stable CHO cell lines were thus generated which secrete between 10-100 micrograms/milliliter of CD4- gamma2 chimeric heavy chain homodimer.
- CD4-gamma2 chimeric heavy chain homodimer was purified by column chromatography. CHO cells secreting CD4-gamma2 chimeric heavy chain homodimer were grown to high density in roller bottles in medium containing alpha MEM with 10% IgG- free fetal calf serum. Conditioned media was collected, clarified by centrifugation, and diluted 1:1 with PBS either with or without detergent (i.e. Tween) in this and subsequent buffers. The diluted media was then applied to a 5ml column of Protein A-Sepharose fast flow previously equilibrated with PBS, at a flow rate of 60ml/hour.
- detergent i.e. Tween
- a step elution gradient (consisting of the following 4 steps: 5 column volumes of 50mM BES pH 7.0, 4 column volumes of 50mM BES pH 7.0, lOOmM NaCl, 6 column volumes of 50mM BES pH 7.0 225mM NaCl, followed by 8 column volumes of 50mM BES pH 7.0, 500mM NaCl) was employed for specific elution of the CD4-gamma2 chimeric heavy chain homodimer.
- the CD4-gamma2 chimeric heavy chain homodimer was eluted from the column in 50mM BES pH 7.0, 500mM NaCl.
- the peak fractions were then pooled and concentrated to yield a final protein concentration of at least 1 mg/ml.
- the pooled and concentrated fractions were then applied to a 120 ml column of Sephacryl S-300HR previously equilibrated with PBS, at a flow rate of 8ml/hr.
- the CD4-gamma2 chimeric heavy chain homodimer fraction was specifically eluted in PBS, and concentrated to at least lmg/ml.
- CosM5 cells grown in DMEM containing 10% fetal calf serum are split to 75% confluence.
- the cells are transfected for 16-20 hours with 5 micrograms of CsCl-purified CD4-IgG2HC-pRcCMV DNA and 5 micrograms of CsCl-purified CD4-kLC-pRcCMV plasmid DNA by the standard CaPO(4) precipitation technique. After transfection, fresh medium is added to the cells.
- Analysis of the products synthesized 48-72 hours post-transfection is performed by radiolabelling of transfectant ⁇ with 35 S-methionine for 12-18 hours followed by precipitation of media and cell lysates using anti-CD4 antibodies or by incubation with Protein A-sepharose beads alone followed by SDS-PAGE under reducing or non-reducing conditions.
- analysis of media and cell lysates is performed 48-72 hours post- transfection by standard Western blotting procedures.
- Dhfr- Chinese hamster ovary cells are transfected with 20 micrograms of CsCl-purified DNA in a ratio of 1000:1000:1 CD4-IgG2HC-pRcCMV:CD4-kLC-pRcCMV:p 10 (p410 is an expression plasmid containing the dhfr gene) , although other ratios may also be used.
- p410 is an expression plasmid containing the dhfr gene
- cells are placed in selective medium (nucleoside-free alpha MEM containing 10% dialyzed fetal calf serum) .
- selective medium nucleoside-free alpha MEM containing 10% dialyzed fetal calf serum
- the clones are then analyzed for stable expression of CD4-IgG2 chimeric heterotetramers by several screening techniques, such as ELISA and precipitation with Protein A-sepharose beads followed by SDS-PAGE under reducing or non-reducing conditions. Clones expressing the highest levels are subjected to successive rounds of amplification of the newly introduced DNA sequences in increasing concentrations of methotrexate. Stable CHO cell lines are thus generated which secrete high levels of CD4- IgG2 chimeric heterotetramer.
- CD4-IgG2 chimeric heterotetramers are purified using Protein A-Sepharose column chro atography.
- CHO cells secreting CD4- IgG2 chimeric heterotetramers are grown to high density in roller bottles in medium containing alpha MEM with 10% IgG- free fetal calf serum.
- Conditioned media is collected, clarified by centrifugation, and diluted 1:1 with PBS either with or without detergent (i.e. Tween) in this and subsequent buffer ⁇ .
- the diluted media is then applied to a 5ml column of Protein A-Sepharose fast flow previou ⁇ ly equilibrated with PBS, at a flow rate of 60ml/hour.
- Group-common and type-specific neutralizing antibodies are commercially available. Specifically, anti-gpl20 antibodies, anti-V3 loop antibodies and anti-gp41 antibodies are commercially available.
- PBMCs Peripheral blood mononuclear cells
- EBV Epstein Barr Virus
- B95-8 cell supernatants Epstein Barr Virus
- Colonies of EBV-immortalized B lymphocytes grow out and those colonies producing anti-gp41 antibodies are identified by immunoprecipitation of gp41 from metabolically radiolabelled cells expre ⁇ ing gpl20/gp41, or by western blotting.
- Hybrids are selected by culture in selective medium in the presence of feeder cells, and stable antibody- secreting hybrids are cloned and expanded.
- the microculture as ⁇ ay for productive viral replication was used (36). Briefly, sCD4, antibody, or a mixture was preincubated for 30 minutes at room temperature with an HIV-1 inoculum (100 TCID-50) . The mixtures were added to PHA-stimulated lymphocytes and incubated at 37°C overnight. The cells were washed by centrifugation, plated in microculture (1 x 10 4 cells/culture; 10 cultures/dilution) , and monitored for reproductive viral replication by detection of HIV antigen in culture supernates 8 and 12 days later. In this assay, antibody and sCD4 are washed away after initial exposure of virus to cells and before plating in microculture. They are not replaced in the plating and feeding media. Residual antibody or sCD4 does not interfere with the detection of supernate viral antigen as determined by testing mixture ⁇ of ⁇ upernate ⁇ from cultures incubated with sCD4/antibody alone and from cultures with HIV-1 alone.
- HIV preparation was combined with sCD4, anti-HIV serum, or a mixture of the two in a 100 microliter volume. Final concentrations were 50 micrograms/milliliter for sCD4 and a 1:4 dilution for serum. The preparations were incubated for
- sCD4 In order to determine whether sCD4 would neutralize HIV in the presence of HIV-1 antibody-positive human sera, the following assay was performed. Purified IgG or anti-HIV human sera and sCD4, alone and in combination, were tested at multiple dilutions in a microculture assay for neutralization of productive HIV infection. A representative plot of fraction inhibited (% inhibition/100) versus dose of antibody/sCD4 is shown in Figure 1. The results demonstrate that sCD4 or HIV antibody neutralize HIV infection, and inhibition by sCD4 is more potent than inhibition by HIV antibody. Moreover, the reduction in HIV infectivity was greatest with mixtures of sCD4 and HIV antibody.
- results demonstrate a general additive effect or slight synergism between HIV antibody and sCD4, In other words, the reduction in HIV infectivity mediated by mixtures of sCD4 and antibody was always equal to or greater than the arithmetic sum of the reductions by either agent alone. Similar additive effects were obtained with a fresh clinical HIV isolate that requires higher concentrations of sCD4 for inhibition in the neutralization assay.
- sCD4 causes shedding of gpl20 from virions resulting in irreversible inactivation of HIV infectivity (30, 40) .
- a model sy ⁇ tem wa ⁇ designed to measure this effect in the presence of HIV antibody-positive human sera.
- An HIV inoculum was mixed with ⁇ CD4, sera, or a mixture of the two, and incubated for 2 hours at 37°C.
- Serial 10-fold dilutions were made, added to cells, and viral replication was monitored.
- HIV, sCD4, and sera were preincubated separately at 37°C, dilutions were made, and the dilutions were combined just before addition to cells.
- the inactivation and control culture ⁇ are identical with re ⁇ pect to HIV, ⁇ CD4, and ⁇ era concentration ⁇ , but differ in the opportunity for interaction at 37°C before addition to cell ⁇ . It should be noted that with dilution, the concentration of sCD4/ ⁇ erum in the culture ⁇ is orders of magnitude lower than that required for inhibition in the neutralization assay where graded doses of sCD4/serum are mixed with a constant amount of HIV just before addition to cells (see supra .
- HIV was expo ⁇ ed to anti-HIV ⁇ erum (final concentration 1:4) and/or sCD4 (50 microgram ⁇ /milliliter in Expo ⁇ ures 1 and 3; 25 micrograms/milliliter in Exposure 2) in a 100 microliter volume for 2 hours at 37°C before dilution and microculture infectivity titration.
- Titer reduction is the difference between thi ⁇ titration and the re ⁇ pective control titration.
- titer reduction is the difference between the experimental titration and the titration of HIV in the absence of sCD4 or ⁇ erum.
- a CD4-containing molecule In order to substantially reduce the risk of occupational HIV transmission in this high risk setting, a CD4-containing molecule would be administered to trauma patients either before or during treatment in the emergency room and operating room.
- the CD4-containing molecule might be administered by IV (intravenous) bolus or continual IV infusion, IM (intramuscular) injection, or SC (subcutaneous) injection.
- IV intravenous
- IM intramuscular
- SC subcutaneous
- the dose of the CD4-containing molecule administered would depend on the serum concentration required to substantially reduce the infectivity of HIV in the patient's blood. It is presumed that the above options will vary depending on the type and pharmacokinetics of the CD4-containing molecule administered.
- a patient with penetrating trauma upon arrival at an emergency room, a patient with penetrating trauma would receive an IV bolus of a CD4-immunoglobulin chimeric molecule in order to reach a serum concentration of greater than 10 micrograms/milliliter. IV administration is preferred in order to achieve peak concentrations rapidly. For a CD4-immunoglobulin chimeric molecule, this might require a dose equal to or greater than 1.0 milligram/kilogram of body weight. Since the serum half-life of CD4-immunoglobulin chimeric molecules is on the order of several days, additional administration of the CD4-containing molecule might not be required during the initial treatment.
- Periodic blood sample ⁇ would be assayed for the serum concentrations of the CD4-containing molecule in order to ascertain whether the desired blood levels have been achieved. Additional dose ⁇ of the CD4-containing molecule might be admini ⁇ tered during the cour ⁇ e of hospitalization or additional operative procedures.
- a patient would receive a CD4-containing molecule prior to an operative procedure.
- Such procedures include, but are not limited to, ob ⁇ tetric and gynecologic procedures, general ⁇ urgical procedure ⁇ , cardiac and va ⁇ cular procedure ⁇ , orthopedic procedures, and urological procedures.
- the CD -containing molecule might be administered by IV bolus or continual IV infusion, IM injection or SC injection. Depending on the route of admini ⁇ tration and the nature of the treatment, the CD4-containing molecule might be given continuously or intermittently.
- the dose of the CD4-containing molecule administered would depend on the serum concentration required to substantially reduce the infectivity of HIV in the patient's blood. It is presumed that the above options will vary depending on the type and phar acokinetics of the CD4-containing molecule administered.
- a CD4-immunoglobulin chimeric molecule might be delivered by IV bolus in order to rapidly achieve serum concentrations of greater than 10 micrograms/milliliter. For a CD4-immunoglobulin chimeric molecule, this might require a dose equal to or greater than 1.0 milligram/kilogram of body weight. Since the serum half-life of CD4-immunoglobulin molecules is several days, additional administration of the CD4-containing molecule might not be required during the initial procedure. Periodic blood samples would be assayed for the serum concentrations of the CD4-containing molecule in order to ascertain whether the desired blood levels have been achieved. Additional dose ⁇ of the CD4-containing molecule might be administered during the course of hospitalization or additional operative procedures.
- HIV-seroprevalence of hospitalized patients i ⁇ ⁇ ignificant.
- other health care worker ⁇ such as physicians, dentists, nurses, medical students, and phlebotomist ⁇ perform procedure ⁇ which put them at ri ⁇ k of expo ⁇ ure to patient ⁇ ' blood.
- patients who are known to be HIV seropositive or who have risk factors for HIV infection would receive a CD4-containing molecule.
- a patient would receive a CD4-containing molecule during the course of hospitalization.
- the CD4- containing molecule might be admini ⁇ tered by IV bolu ⁇ or continuou ⁇ IV infu ⁇ ion, intramu ⁇ cular injection, or ⁇ ubcutaneous injection. Depending on the route of administration and the nature of the treatment, the CD4- containing molecule might be given continuously or intermittently.
- the dose of the CD4-containing molecule administered would depend on the serum concentration required to ⁇ ub ⁇ tantially reduce the infectivity of HIV in the patient' ⁇ blood. It i ⁇ pre ⁇ umed that the above options will vary depending on the type and pharmacokinetic ⁇ of the CD4-containing molecule administered.
- a CD4- immunoconjugate might be delivered by IV bolus in order to rapidly achieve ⁇ erum concentration ⁇ of greater than 10 micrograms/milliliter.
- a CD4-immunoconjugate this might require a dose equal to or greater than 1.0 milligram/kilogram of body weight.
- additional administration of the CD4- containing molecule might not be required during the hospitalization.
- Periodic blood ⁇ ample ⁇ would be assayed for the ⁇ erum concentration ⁇ of the CD4-containing molecule in order to a ⁇ certain whether the desired blood level ⁇ have been achieved. Additional dose ⁇ of the CD4-containing molecule might be administered during the course of hospitalization.
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Abstract
L'invention concerne un procédé pour réduire considérablement les risques, chez un praticien non-infecté, d'être infecté par le VIH s'il se trouve exposé au sang ou lors d'une intervention chirurgicale l'exposant au liquide biologique du patient. Ce procédé consiste à administrer au patient sur une durée appropriée une quantité d'une molécule contenant le CD4 ou un mélange comportant un anticorps neutralisant le VIH, ainsi qu'une molécule contenant le CD4 efficace pour réduire considérablement chez un praticien non injecté, les risques d'être infecté par le VIH par contact virtuel avec le sang du patient lors d'une intervention chirurgicale; ou à administrer une quantité d'une molécule contenant le CD4, efficace pour réduire considérablement les risques, chez un praticien non-infecté, d'être infecté par le VIH par contact virtuel avec le liquide biologique d'un patient lors d'une intervention chirurgicale.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US83335892A | 1992-02-10 | 1992-02-10 | |
US07/833,358 | 1992-02-10 |
Publications (1)
Publication Number | Publication Date |
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WO1993015747A1 true WO1993015747A1 (fr) | 1993-08-19 |
Family
ID=25264205
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1993/001193 WO1993015747A1 (fr) | 1992-02-10 | 1993-02-10 | Procede d'utilisation des molecules contenant le recepteur cellulaire cd4 pour reduire considerablement le risque de transmission du vih dans le milieu professionnel |
Country Status (2)
Country | Link |
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AU (1) | AU3662293A (fr) |
WO (1) | WO1993015747A1 (fr) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0687183A4 (fr) * | 1993-02-24 | 1997-06-25 | Progenics Pharm Inc | Composition synergique d'une proteine a base de cd4 et d'un anticorps dirige contre le vih-1, et procedes d'utilisation |
WO2006044410A3 (fr) * | 2004-10-14 | 2006-07-06 | Us Gov Health & Human Serv | Proteines hybrides comprenant l'anticorps monoclonal a32, utiles comme inhibiteurs du vih et vaccins anti-vih |
US7378093B2 (en) | 2001-10-16 | 2008-05-27 | The United States Of America As Represented By The Department Of Health And Human Services | Broadly cross-reactive neutralizing antibodies against Human Immunodeficiency Virus selected by Env-CD4-co-receptor complexes |
US7566451B2 (en) | 2002-05-06 | 2009-07-28 | The United States Of America As Represented By The Department Of Health And Human Services | Human immunodeficiency virus-neutralizing human antibodies with improved breadth and potency |
US7803913B2 (en) | 2002-05-06 | 2010-09-28 | The United States Of America As Represented By The Department Of Health And Human Services | Identification of novel broadly cross-reactive neutralizing human monoclonal antibodies using sequential antigen panning of phage display libraries |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1988001304A1 (fr) * | 1986-08-21 | 1988-02-25 | The Trustees Of Columbia University In The City Of | Adn de codage de la proteine t4 de la surface des cellules t et utilisation de fragments de t4 pour le traitement du sida |
-
1993
- 1993-02-10 AU AU36622/93A patent/AU3662293A/en not_active Abandoned
- 1993-02-10 WO PCT/US1993/001193 patent/WO1993015747A1/fr active Application Filing
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
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WO1988001304A1 (fr) * | 1986-08-21 | 1988-02-25 | The Trustees Of Columbia University In The City Of | Adn de codage de la proteine t4 de la surface des cellules t et utilisation de fragments de t4 pour le traitement du sida |
Non-Patent Citations (8)
Title |
---|
ANNALS OF INTERNAL MEDICINE, Volume 112, No. 4, issued 15 February 1990, R.T. SCHOOLEY et al., "Recombinant Soluble CD4 Therapy in Patients with the Acquired Immunodeficiency Syndrome (AIDS) and AIDS-Related Complex", pages 247-253. * |
CELL, Volume 57, issued 05 May 1989, J. ARTHOS et al., "Identification of the Residues in Human CD4 Critical for the Binding of HIV", pages 469-481. * |
MEDICAL MICROBIOLOGY AND IMMUNOLOGY, Volume 180, issued 1991, I.H. CHOWDHURY et al., "Evaluation of Anti-Human Immunodeficiency Virus Effect of Recombinant CD4-Immunoglobulin In Vitro: A Good Candidate for AIDS Treatment", pages 183-192. * |
NATURE, Volume 339, issued 04 May 1989, A. TRAUNECKER et al., "Highly Efficient Neutralization of HIV with Recombinant CD4-Immunoglobulin Molecules", pages 68-70. * |
NATURE, Volume 344, issued 12 April 1990, R.A. BYRN et al., "Biological Properties of a CD4 Immunoadhesin", pages 667-670. * |
NATURE, Volume 352, issued 01 August 1991, R.H.R. WARD et al., "Prevention of HIV-1 IIIB Infection in Chimpanzees by CD4 Immunoadhesin", pages 434-436. * |
SCIENCE, Volume 250, issued 23 November 1990, J.P. MOORE et al., "Dissociation of gp120 from HIV-1 Virions Induced by Soluble CD4", pages 1139-1142. * |
THE LANCET, issued 23 December 1989, R.J. GERETY et al., "Human Recombinant Soluble CD4 Therapy", pages 1521. * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0687183A4 (fr) * | 1993-02-24 | 1997-06-25 | Progenics Pharm Inc | Composition synergique d'une proteine a base de cd4 et d'un anticorps dirige contre le vih-1, et procedes d'utilisation |
US7378093B2 (en) | 2001-10-16 | 2008-05-27 | The United States Of America As Represented By The Department Of Health And Human Services | Broadly cross-reactive neutralizing antibodies against Human Immunodeficiency Virus selected by Env-CD4-co-receptor complexes |
US7566451B2 (en) | 2002-05-06 | 2009-07-28 | The United States Of America As Represented By The Department Of Health And Human Services | Human immunodeficiency virus-neutralizing human antibodies with improved breadth and potency |
US7803913B2 (en) | 2002-05-06 | 2010-09-28 | The United States Of America As Represented By The Department Of Health And Human Services | Identification of novel broadly cross-reactive neutralizing human monoclonal antibodies using sequential antigen panning of phage display libraries |
US8110192B2 (en) | 2002-05-06 | 2012-02-07 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Human immunodeficiency virus type 1 (HIV-1)-neutralizing human single-chain antibodies with improved breadth and potency |
WO2006044410A3 (fr) * | 2004-10-14 | 2006-07-06 | Us Gov Health & Human Serv | Proteines hybrides comprenant l'anticorps monoclonal a32, utiles comme inhibiteurs du vih et vaccins anti-vih |
Also Published As
Publication number | Publication date |
---|---|
AU3662293A (en) | 1993-09-03 |
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