[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

WO1991009625A1 - Anticorps monoclonaux qui neutralisent l'infection par hiv-1 et leurs anti-idiotypes - Google Patents

Anticorps monoclonaux qui neutralisent l'infection par hiv-1 et leurs anti-idiotypes Download PDF

Info

Publication number
WO1991009625A1
WO1991009625A1 PCT/US1990/007535 US9007535W WO9109625A1 WO 1991009625 A1 WO1991009625 A1 WO 1991009625A1 US 9007535 W US9007535 W US 9007535W WO 9109625 A1 WO9109625 A1 WO 9109625A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
hiv
cells
specific
idiotypic
Prior art date
Application number
PCT/US1990/007535
Other languages
English (en)
Inventor
Tse Wen Chang
Michael S. C. Fung
Cecily R. Y. Sun
Bill N. C. Sun
Nancy T. Chang
Original Assignee
Tanox Biosystems, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tanox Biosystems, Inc. filed Critical Tanox Biosystems, Inc.
Publication of WO1991009625A1 publication Critical patent/WO1991009625A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • AIDS Neutralizing and Anti-CD4 Binding Site Antibodies
  • AIDS is probably the most serious health threat confronting society. It could reach epidemic proportions in the general population before the end of this century. The disease runs a painful and debilitating course and results in the death of its victim. In fact, from diagnosis onward, the average life span of an AIDS victim is only a few years.
  • AIDS is caused by a virus which has at various times been called human T-cell lymphotropic virus type III (HTLV-III), or lymphoadenopathy-associated virus (LAV).
  • HTLV-III human T-cell lymphotropic virus type III
  • LAV lymphoadenopathy-associated virus
  • the virus is currently known as human immunodeficiency virus I (HIV-1).
  • HIV-1 also causes a somewhat less serious immunodeficiency syndrome known as AIDS related complex (ARC).
  • ARC will often precede the onset of AIDS.
  • T cells T helper/inducer lymphocytes
  • B cells T helper/inducer lymphocytes
  • cytotoxic T lymphocytes killer T cells
  • macrophages macrophages
  • natural killer cells T helper/inducer lymphocytes
  • numerous other regulator and effector functions of the immune system HIV-1 infection severely compromises the immune response, leaving the victim unable to defend against secondary opportunistic infections. It is often the secondary infections which debilitate the victim and cause death.
  • AIDS victims In addition to their susceptibility to secondary infections, AIDS victims frequently develop otherwise rare conditions. A large number develop a rare form of skin cancer known as Kaposi's sarcoma.
  • Infection of a T cell with HTV-1 follows from interaction between an epitope borne by HIV-1 and a receptor site which is located on the T cell surface, known as the CD4 antigen.
  • the epitope on HIV-1 is borne by the envelope glycoprotein gpl20
  • glycoprotein gpl20 (molecular weight 120,000 daltons).
  • the glycoprotein gpl20 is produced when a precursor glycoprotein gpl60 is cleaved apart into gp41 (molecular weight 41,000 daltons) and gpl20.
  • HIV-1 is a retrovirus.
  • a viral enzyme called reverse transcriptase transcribes the viral genomic RNA into DNA in the host cell nucleus.
  • the newly synthesized DNA is incorporated into the host cell genome under a variety of activation conditions, and the infected T cell begins to transcribe the new DNA to make copies of messenger RNA and genomic RNA.
  • the viral genomic RNA's are packed with core proteins, reverse transcriptase, and certain other proteins. They are then enveloped by parts of the cellular membrane and budded off from the cell as newly synthesized virions. These new virions can enter and infect other T cells.
  • HIV-1 HIV-1 is transmitted to other T cells.
  • Direct, cell-to-cell transmission occurs when an infected cell, which expresses the viral gp 120 on its surface, binds with the CD4 antigen of an uninfected cell or cells. As a result, the cells fuse and virions can pass to the uninfected cell(s).
  • Direct cell-to-cell contact and the resulting fusion are a significant source of cellular infection, and may be a major mechanism of T cell destruction in HIV-1 infected individuals.
  • syncytia multi-nucleated aggregates known as syncytia.
  • the cell fusion causes the death of cells in the syncytia. See Lifson et al.
  • HIV-1 titers of neutralizing antibodies in the serum of infected individuals is usually so low as to be insufficient to neutralize the HIV-1 infection.
  • monoclonal antibodies which neutralize HIV-1 would be particularly useful for treatment.
  • Monoclonal antibodies are produced by hybridoma cells.
  • Hybridomas are cells which have all been cloned from a single fused cell. All the clones are identical to the parent. Accordingly, all the hybridomas of the same clone produce antibodies of the same idiotype which bind to the same epitope of the antigen.
  • a host animal usually a mouse, is immunized with an antigen and then sacrificed. Lymphocytes containing B-cells are then removed, usually from the spleen or other lymphoid tissues.
  • the removed lymphocytes are fused with myeloma cells to form hybridomas.
  • the hybridomas which produce antibody against the designated epitopes of the immunizing antigen are cloned and screened. These hybridomas are then used to manufacture the desired monoclonal antibodies.
  • a monoclonal antibody that inhibits infection of susceptible cells by many strains of HIV-1, either by preventing attachment of free virions or by inhibiting direct cell-to-cell transmission of virus through syncytium formation, has great potential therapeutic value.
  • Such an antibody could be useful in treating patients with AIDS or ARC, or could be used to prevent AIDS in asymptomatic healthy HIV-1 infected individuals, or in individuals in high-risk groups for AIDS exposure and infection.
  • Such an antibody could target the CD4 binding site of the virus or another neutralization site on the virus.
  • a vaccine derived from such monoclonal antibodies could be used as an alternative to administering neutralizing monoclonal antibodies.
  • the Ab2 are, therefore, useful as vaccines, because they induce production of endogenous Ab3. If the Abl originally administered were specific to the CD4 binding site of HIV-1, or were Abl which otherwise neutralized HIV-1, then the resulting Ab3
  • the monoclonal antibodies (mAbs) of the invention bind to the CD4 binding region of HIV-1 or to a neutralizing epitope in the principal neutralizing determinant region on gpl20. They inhibit HIV-1 infection of T cells by free virions, and they also inhibit syncytium formation.
  • the monoclonal antibodies of the invention are group specific and can neutralize and cross-protect
  • the mAbs of the invention can be used for treatment of
  • the antibodies can be used as
  • Polyclonal or monoclonal anti-idiotype antibodies against the paratope of the antibodies of the invention can be used to stimulate a neutralizing immune response against HIV-1.
  • the mAbs and anti-idiotypes of this invention can be used in vivo as antibodies derived wholly from mice or other animals.
  • the mAbs and anti- idiotypes can be made in the form of whole human antibodies, animal/human chimeric antibodies, single chain antibodies, or antobody fragments.
  • the constant region is human-derived
  • the variable region is animal-derived.
  • the mAbs and anti-idiotypes of this invention are produced by continuous, stable antibody-producing cell lines. These cell lines can be produced by hybridoma techniques and by genetic engineering techniques.
  • This invention also pertains to peptides which correspond to epitopic segments of gpl20 recognized by the antibodies of this invention.
  • the peptides can be used in vaccine compositions for generating a cross-protective, neutralizing immune response against HIV-1. They can also be used to detect neutralizing antibodies against HIV-1 in a biological fluid.
  • Figure 1 is a plot showing the relative effectiveness of four of the mAbs of the invention (BAT085, BAT123, BAT267,
  • BAT509 in neutralizing HIV-1 infection of H9 cells, as compared with another anti-gpl20 mAb (BAT496) and an irrelevant murine mAb to human chorionic gonadotropin ( ⁇ -HcG). The percentage of infected cells was determined nine days after infection.
  • Figure 2 is a plot showing the relative effectiveness of the four of the mAbs of the invention in neutralizing HIV-1 infection of H9 cells, as compared with the irrelevant mAb ( ⁇ -HcG). The percentage of infected cells was determined thirteen days after infection.
  • Figure 3 is a plot of purification of the immunoconjugate.
  • Immunoconjugate was eluted with a NaCl gradient ( — ) and absorbance at 280 nm was recorded ( ). The immunoconjugate elutes as a single peak at 110 mM NaCl.
  • Figure 4 is a flow cytometric analysis plot showing the relative binding activities of the immunoconjugates BAT123-PAP-S and G3.519-PAP-S to HTLV-III B infected H9 cells. Infected H9 cells were treated without immunoconjugates (a), with
  • Figure 5 is a plot showing the cytotoxic effects of
  • Figure 6 is a plot showing the cytotoxic effects of
  • BAT123-PAP-S when presented to H9 cells uninfected (filled circle) or infected with HTLV-III B (open circle), HTLV-III MN (open triangle), or HTLV-ITV (open square). BAT123-PAP-S was most effective at killing H9 cells infected with HTLV-III B , moderately effective at killing H9 cells infected with HTLV-III MN , and relatively ineffective at killing H9 cells infected with HTLV-IIV
  • Figure 7 is a chart showing the specificity of the cytotoxic effects of immunoconjugates.
  • BAT123-PAP-S is the immunoconjugate in Panel A, and
  • G3.519-PAP-S is the immunoconjugate in Panel B, both represented by the open columns.
  • An irrelevant monoclonal antibody, which does not inhibit the cytotoxicity of the immunoconjugate, is represented by the hatched column.
  • Figure 8A shows the results of competition assays of the binding between BAT123 and AB19-4 by synthetic peptides corresponding to the BAT123 binding regions in HTLV-III B ,
  • HTLV-III MN and HTLV-III RJ are irrelevant peptide used as control.
  • Figure 8B shows the results of competition assays of the binding between BAT123 and AB 19-31 by synthetic peptides corresponding to the BAT123 binding regions in HTLV-III B ,
  • HTLV-III MN and HTLV-III RF (T64-63-6 is an irrelevant peptide used as control).
  • Figure 9 shows specific gpl20 binding of Ab3s generated in rabbits immunized respectively with AB19-4 and AB19-31.
  • Figure 10 shows the reactivity of AB19-4-HRP conjugate with solid-phase antibodies in ELISA.
  • Wells of Immunlon 2 plates were coated with 100 ⁇ l of BAT123 (filled circle), CAGl-51-4
  • FIG. 11 shows the inhibition of the binding between
  • Microtest plates were coated with 100 ⁇ l of BAT123 (10 ⁇ g/ml) for ELISA as described in Fung et al., J. Immunol. 145:2199-2206
  • Figure 12 shows inhibition of the binding between AB19-4- HRP conjugate and solid-phase BAT123 by the synthetic epitope peptides.
  • the amino acid sequences of R15K (open circle), R15N (filled circle), and S15Q (open triangle), defining the corresponding peptidic segments in the gpl20 of HTLV-III B , HTLV-HI MN , and HTLV-IH RP respectively were shown in Table I, Fung et al., J. Immunol. 145:2199-2206 (1990).
  • Peptide T19V defining a distinct segment in the C2 region (amino acid residue #254-275) of HTLV-III B gpl20 was used as control (filled triangle).
  • Figures 13A and 13B respectively, show reactivity of goat
  • Figure 14 shows reactivity of gpl20-affinity purified Ab3
  • Figures 15A and 15B show flow cytometric analysis of the binding of gpl20-affinity purified Ab3 (Fig. 16A) and of BAT123
  • HTLV-III B filled circle, open circle
  • HTLV-III MN filled triangle, open triangle
  • gpl20-affi ⁇ ity purified Ab3 filled symbols
  • sham purified pre-immune rabbit serum substances open symbols
  • Vn is the mean number of syncytia in triplicate test wells
  • Vo the mean number of syncytia in the triplicate control wells.
  • the vertical bars represent SD.
  • Figure 17 shows neutralization of HIV-1 by BAT123.
  • Figure 18 shows the binding of AB20-4 to solid-phase
  • Figure 19 shows the inhibition of binding of G3.519-HRP solid-phase HIV-1 gpl20 by AB20-4 (filled circles), G3.519 (open circles) and an irrelevant murine IgGl (open triangles).
  • Figure 20 shows the inhibition of binding of AB20-4-HRP to solid-phase G3.519 by peptide T35S (having the sequence of the
  • the monoclonal antibodies of the invention bind to the viral envelope glycoprotein gpl20.
  • gp41 is a transmembrane protein and is largely not exposed.
  • gpl20 is an external envelope protein which is extracellular.
  • the gpl20 protein offers binding epitopes for the monoclonal antibodies of the invention.
  • the mAbs of the invention include mAbs
  • the monoclonal antibodies of the invention were found to be effective in inhibiting infectivity and in inhibiting syncytium formation. This indicates that they will likely be very effective for in vivo
  • the antibodies can neutralize different strains and different isolates of HIV-1 (i.e. the antibodies are group specific).
  • the neutralizing antibodies also inhibit syncytium formation by various strains of HIV-1 which have a substantial degree of heterogeneity in the amino acid sequence of gpl20.
  • the neutralizing antibodies of this invention can have high potency in neutralizing infectivity.
  • the mAbs against the principal neutralizing determinant (“PND”) can inhibit, with an
  • the PND is the peptide segment on gpl20 from amino acid residue numbers 296 to 331, as determined from the gpl20 sequence of the HTLV-III B , or sequences of the corresponding regions from other HIV-1 strains. See Devash, Y., Proc. Nat'l Acad. Sci. USA 87:3445-3449 (1990).
  • the PND peptide segment is in the relatively variable region, V3, of gpl20.
  • V3 variable region
  • the amino acid sequences of PND segments in field HIV-1 isolates from patients are closely related. See LaRosa, G.J. et. al, Science 249:932-935 (1990). Antibodies which target the
  • PND are generally effective in neutralizing HIV-1 infection.
  • Abl which target the PND and neutralize HIV-1 include BAT123 and BAT267.
  • Suitable monoclonal Abl including BAT123 and BAT267,
  • a suitable antigen which in this case is inactivated HIV-1.
  • the antigen can be in whole form, e.g., whole HIV-1 virions, or cells infected with a virus and expressing the virus or its immunogenic domains can also be used. Specific viral proteins, such as the envelope glycoproteins, may be purified from the lysates of infected cells or viruses.
  • the immunogenic domains of HIV-1 on gpl20, or synthetic or recombinant peptides which have the same or an immunologically equivalent sequence to these immunogenic domains, can also be used. These synthetic or recombinant peptides for use in immunization can be synthesized by conventional techniques, such as with the RaMPS system (DuPont).
  • recombinant peptides containing these peptides may be biosynthesized by expressing in is. coli or eukaryotic cells the gene segments containing the appropriate coding sequences.
  • a synthetic peptide segment as an immunogen, it is usually more effective to conjugate it to a protein carrier, for example, HBsAg, hepatitis B virus core antigen, ovalbumin, bovine serum albumin, or preferably keyhole lympethemocyanin ("KLH").
  • the peptidic segment lacks a lysine residue or if the lysine residue is in the middle part of the segment, it is desirable to add a lysine residue at the C-terminal end. Because the N-terminus already has an ⁇ -amino group, the modified synthetic peptide will have two available amino groups for linking.
  • peptides can be conjugated to each molecule of the carrier to make the immunogen.
  • KLH a preferred molar ratio for peptide/KLH is 10.
  • the conjugation can be done with well established methods using glutaraldehyde or bis
  • One preferred immunization protocol for preparing the Abl monoclonal antibodies is to inject into each mouse 50 ⁇ g of the conjugate of KLH and the aforementioned recombinant or synthetic peptides in Freund's complete adjuvant. Two and four weeks later, the same amount of antigen is given subcutaneously in Freund's incomplete adjuvant. After about six weeks, the fourth antigen injection is given intraperitoneally in saline. Mice are sacrificed 4 days after the last injection and the spleens (or sometimes the lymph nodes) are removed for preparing single cell suspensions for fusion with myeloma cells. Lymphocytes from the spleens (or lymph nodes) which have been removed from the mice can be fused with myeloma cells to prepare hybridomas secreting the Abl monoclonal antibodies.
  • the fusion procedure with polyethylene glycol and other various procedures concerning the cloning and the culturing of hybridomas have been well established.
  • One preferred protocol is the well- known one described by Hudson, L. and Hay, F.C. (Practical Immunology, 2nd edition, pp. 303-313, 1980, Blackwell Publishing Co., Boston), in which the lymphocytes are fused with non- secreting mouse myeloma cells, such as NS-1 or Sp2/0 cells, using polyethylene glycol.
  • the fusion reagent used to make BAT123 was polyethylene glycol mixed with dimethyl sulfoxide (DMSO) in calcium magnesium-free phosphate buffered saline (PBS).
  • DMSO dimethyl sulfoxide
  • PBS calcium magnesium-free phosphate buffered saline
  • Reagents other than those discussed can be used for the chemical fusion.
  • Another alternative is to use electrical fusion rather than chemical fusion to form hybridomas. This technique is well-established.
  • electrical fusion one can also transform a B-cell to make it immortal using, for example, an Epstein Barr Virus or a tranforming gene. (For a method of transforming a B-cell, See “Continuously Proliferating Human Cell Lines Syn ⁇ thesizing Antibody of Predetermined Specificity," Zurawski, V.R.
  • the screening of hybridomas for monoclonal antibodies reactive with the immunogen can be performed with an enzyme
  • a synthetic or recombinant peptide having the same sequence as a portion of the immunogen is used as the solid-phase antigen.
  • a preferred solid phase antigen is the conjugate of such a synthetic or recombinant peptide with a carrier protein different from that used with the immunogen.
  • An appropriate carrier protein can be bovine serum albumin or ovalbumin, provided they were not used as carriers in the immunization.
  • Clones of hybridomas which showed highest reactivities with the PND of gpl20 were selected for further screening by an immunofluorescence assay.
  • the immunofluorescence assay was run to determine which of the ELISA positive monoclonal antibodies would bind specifically to intact, live infected T cells, but not to uninfected T cells. This was determined using immunofluorescence flow cytometric analysis of staining of HTLV- III B -infected H9 cells.
  • the clones which showed the highest reactivities with the CD4 region of gpl20 were screened using a p24 assay of HTLV-III B -infected H9 cells.
  • G3.519 is to first conjugate Abl to KLH using glutaraldehyde as described by Maloney et al, Hybridoma 4:191 (1985). Mice are then immunized intraperitoneally with 100 ⁇ g of the Abl-KLH conjugate at one month intervals for three months. Three days after the final immunization, the mice are killed, and the spleen cells are isolated and fused with Sp2/0 myeloma cells to create the
  • the second functional test of neutralization is by syncytium inhibition.
  • infected T cells were added to a well seeded with HeLa cells which had been artifically transfected with CD4 genes and expressed the CD4 antigen on their surface.
  • the CD4 antigen on the cell surface fuses with infected T cells to form multi-nucleated giant cells. It was determined which concentrations of mAbs to the PND, and which mAbs to the anti-CD4 binding region (International Application No. PCT/US90/02261), would inhibit giant cell formation.
  • the antibodies are tested in these assays to determine their ability to neutralize different viral strains and isolates.
  • the prophylactic and therapeutic uses for the monoclonal antibodies of the invention include both jn vivo immunotherapy
  • Direct in vivo treatment with the monoclonal antibodies of the invention involves administering them internally, preferably via intravenous injection. They can be administered subcutaneously or intramuscularly.
  • the monoclonal antibody may be coupled to an agent, such as certain lipophilic substances, which allows it to pass through the blood-brain barrier.
  • agent such as certain lipophilic substances
  • the antibodies of this invention can neutralize different strains and isolates of HIV-1 in the patient population.
  • blood leukocyctes are removed from the patient and treated with neutralizing antibody.
  • the monoclonal antibody is then added to the leukocytes.
  • the leukocytes can also be stimulated with immunopotentiating drugs, for example interleukin-2.
  • the leukocytes are then returned to the patient.
  • the mouse-derived monoclonal antibodies of the invention can be used for both direct in vivo and extracorporeal
  • the preferred antibodies of the invention have human constant regions. These preferred antibodies include whole human antibodies, chimeric antibodies wherein the variable region is of murine origin and the constant region is of human origin, and antibodies wherein only the complementarity determining regions
  • CDR CDR
  • V H , V L , F- * Fd, Fab and F(ab') 2 are of murine origin and the remainder of the variable regions, and the entire constant regions, are of human origin.
  • antibody fragements such as V H , V L , F- * Fd, Fab and F(ab') 2 , none of which have complete constant regions, can be used.
  • Chimeric antibodies can be produced by transfecting non-producing mouse myeloma cells with the hybrid genomic DNA, or cDNA. See V.T. Oi et al., Bio Techniques 4(4 ⁇ :214-221 (1986); L.K. Sun et al.,"Chimeric Antibodies with 17-1A-Derived Variable and Human Constant Regions". Hybridoma 5 (1986).
  • the hybrid genomic DNA or cDNA will contain the human constant regions and the mouse variable region. If one is making an antibody in which only the CDRs are of mouse origin, the hybrid genomic
  • DNA or cDNA will contain human constant regions, mouse CDR regions, and the remainder of the variable regions will be human.
  • Human antibodies can be produced by using human expression libraries (e.g., Stratagene Corp., La Jolla, California) to produce fragments of human antibodies (V H , V L , F v , Fd, Fab, or
  • F(ab') 2 One can use the fragments to construct whole human antibodies using techniques similar to those for producing chimeric antibodies. One can also create single peptide chain antibodies. In such antibodies, the heavy and light chain F v regions are connected. See Huston, J.S. et al., Proc. Natl. Acad. Sci. USA
  • Another alternative form of monoclonal antibody suitable for use in therapy are derivative antibodies which draw cytotoxic cells such as macrophages or cytotoxic T cells toward the targeted
  • bi-specific antibodies having a specificity for a receptor of a cytotoxic cell and a specificity for the infected cells.
  • Such hybrid bi-specific antibodies can include two different Fab moieties, one Fab moiety having antigen specificity for the targeted infected cells, and the other Fab moiety having antigen specificity for a surface antigen of a cytotoxic cell, such as CD3 or CD8.
  • the bi-specific antibodies of the invention can be a single antibody having two specificities, or a heteroaggregate of two or more antibodies or antibody fragments. See. e.g. ⁇ C. Reading, U.S. Patent Nos. 4,474,893 and 4,714,681; Segal et al, U.S. Patent No. 4,676,980.
  • GM-CSF granulocyte monocyte-colony stimulation factor
  • M-CSF monocyte-colony stimulation factor
  • CSF have been shown to augment the ADCC activity on tumor cells mediated by monoclonal antibodies specific for surface antigens expressed on the tumor cells.
  • the therapeutic effect of specific monoclonal antibodies of the invention, conjugates, or polyclonal antibodies in suppressing the immune response could perhaps be enhanced by combining them with factors that augment ADCC activities.
  • Immunotherapy for patients with AIDS or ARC is appropriate with the mAbs and related products of the invention.
  • a variant of immunotherapy is protection through passive immunization.
  • the antibodies of this invention are particularly
  • the targets include fetuses born in or babies born to HIV-1-carrier mothers and health professionals working with AIDS patients, or blood products.
  • the agent for treatment again, can be the mAbs of the invention, or the human or humanized antibodies, or the fragments, discussed above.
  • Much attention in the effort to stop AIDS has focused on the search for a vaccine.
  • the immunizing agent is a portion of HIV-1 which itself is non-infective but which nonetheless induces antibody production.
  • Monoclonal antibodies which neutralize HIV-1 can help in the search for such a vaccine. They can be used to help locate, identify, and study the "neutralizing" epitopes on HIV-1 which bind the monoclonal antibodies. These epitopes are likely to be the non-infective but nonetheless immunogenic portion of the molecule. Study of these epitopes allows synthesis of a non-pathogenic immunogen with a structure which is the same or
  • the immunogen can be a peptide which comprises an amino acid sequence that is the same or similar to the epitope bound by an anti-HIV-1 antibody which neutralizes HIV-1.
  • This segment represents a 25 amino acid residue long segment of gpl20 (residue # 294 to residue # 318).
  • One antibody (BAT267) reacts with a peptide having the sequence RPNNNTRKRIRIQRG (peptide a) and the other antibody (BAT123) reacts with a peptide having the sequence RIQRGPGRAFVTIGK (peptide b).
  • Other strains of HIV have regions corresponding to this segment.
  • peptide "a” represents the segment of residue #294 to residue #308 and peptide "b” of residue #304 to #318.
  • BAT267 reacts with peptide "a” and not peptide "b”, which shares five amino acids RIQRG, or another 15 amino acid long peptide, which represents a segment of gpl20 (residues #284 to #298) adjacent to peptide "a” and shares five amino acids RPNNN.
  • BAT267 recogmzes an epitope either borne entirely by all or a
  • PGRAF or formed by the combination of all of a part of PGRAF with some of the flanking amino acid residues.
  • the CD4 receptor binding region on gpl20 includes a s e g m e n t h aving t h e ami n o a c i d s e qu e n c e
  • the peptidic immunogens of this invention can comprise the above-identified amino acid sequences or immunochemical and immunological equivalents thereof. These equivalents include, for example, any of the actual epitope portions of any of these sequences, corresponding peptidic regions from various HIV-1 strains and peptides generated by various changes such as insertions, deletions and substitutions of amino acids.
  • the peptides of this invention can be coupled together to form larger, multivalent oligopeptides.
  • the peptides may be prepared by chemical synthesis. Alternatively, they may be prepared by recombinant DNA technology where DNA sequences
  • encoding the peptides are synthesized or isolated from HIV-1 DNA and expressed in an appropriate expression system.
  • the peptides may be used in immunoassays to identify neutralizing antibody or to screen for the presence of neutralizing antibody in serum.
  • the peptides may also be used individually or in combination to elicit a immune response against HIV-1.
  • the peptides may be formulated in vaccine compositions, generally for administration at concentrations in the range of 1 ⁇ g to 20 mg/kg of host.
  • Physiologically acceptable vehicles such as water, saline, or phosphate buffered saline can be used in the formulations.
  • Adjuvants such as aluminum hydroxide gel, can also be employed.
  • the route of administration can be intramuscular, intraperitoneal, subcutaneous, or intravenous.
  • the compositions can be given one time or mutiple times, usually at one to four week intervals.
  • the peptides are coupled to a carrier protein such as a foreign keyhole limpet hemocyanin. This can enhance the immunogenicity of the haptenic peptides.
  • a carrier protein such as a foreign keyhole limpet hemocyanin.
  • Another type of vaccine employs anti-idiotype antibodies
  • parotope-specific anti-idiotypic antibodies with partially the same structure as the PND on HIV-1 can be made by immunizing an animal with the monoclonal antibody to the PND of HIV-1.
  • HIV-1 can be made by immunizing an animal with the monoclonal antibody to the CD4 binding region of HIV-1.
  • anti-idiotype antibodies consist of protein and do not carry any viral nucleic acid, they would be of much less concern for pathogenicity than the killed or inactivated virus.
  • derivative antibodies and fragments which are less immunogenic than murine mAbs, e., human, chimeric mouse/human, single chain, and the human antibody fragments V H , V L , F ⁇ Fd, Fab and F(ab') 2 , are preferable for the anti-idiotype antibodies of the invention.
  • single chain polypeptides containing the antigen combining region (paratope) of the anti-idiotypic antibody can be used.
  • Such polypeptides can be produced by genetic
  • the anti-idiotypes of the invention can be used for active immunization, and are preferably administered together with appropriate adjuvants, such as threonyl muramyl dipeptide.
  • the anti-idiotypes can also be used as boosters, to enhance the immunogenicity of another type of vaccine.
  • the other vaccine could be a protein subunit vaccine, such as the peptidic immunogens of the invention described above, or killed or inactivated HIV-1.
  • the anti-idiotypes of the invention would enhance the anti-HIV-1 immune response, and thus enhance the immonogenicity of the other vaccine.
  • the other vaccine would be admimstered simultaneously or shortly after administering the anti- idiotype.
  • the mAbs of the invention can also be used to aid in the delivery of cytotoxic or antiviral agents, by incorporating them into, for example, microcarriers or liposomes.
  • cytotoxic agents include pokeweed antiviral protein from seeds (PAP-S), cytotoxic steriods, gelonin, abrin, ricin and phospholipases.
  • antiviral agents are interferon, azidothymidine and ribovirin.
  • Example I Preparation of the Hybridomas and Monoclonal Antibodies a) Preparation of Virus for mAbs against the PND
  • a virus stock was prepared as follows.
  • the H9 clones of the human T cell line (which is described by M. Robert-Guroff et al. in Nature 316:72-74, supra) were maintained in culture. These H9 cells were infected with HIV-1 (HTLV ⁇ i B ), which was a gift from Dr. R.
  • the H9 cells were cultured in a growth medium of 20%
  • Purified HIV-1 was obtained by first centrifuging the cell culture at 1000 g for ten minutes to remove the cells and debris. The supernatant was then centrifuged at 90,000 g for one hour. The virus pellet was resuspended in minimal volume of phosphate buffered saline pH 7.4 and loaded into a centrifuge tube with a preformed sucrose gradient (20%-60%). The sample was then centrifuged at 100,000 g for sixteen hours. The virus was collected at the gradient of 38%. The virus was then aliquoted and frozen at -80°C after the protein content was measured. b ⁇ Immunization Procedure for mAbs against the PND
  • mice Male Balb/c mice were used for the immunization. Each mouse received 100 micrograms of inactivated HIV-1. The inactivation of the virus was performed according to FDA approved protocol, by UV irradiation and addition of a detergent,
  • Nonidet P-40 (0.1%). A volume of suspension containing 100 micrograms of virus per mouse was suspended in 200 microliters phosphate buffered saline (PBS), and emulsified with equal volumes of Freund's complete adjuvant.
  • PBS phosphate buffered saline
  • mice were immunized subcutaneously with 100 micrograms of the emulsified virus.
  • the mice were injected at sites with high concentrations of lymph nodes, for example, the underside of the intersection of the limbs and the trunk.
  • the boosters were prepared essentially in the same manner as was the first injection, except that for the boosters the emulsification was done in Freund's incomplete adjuvant.
  • each mouse was reimmunized subcutaneously with 100 micrograms of virus suspended in PBS.
  • mice were injected subcutaneously at the intersection of each limb with the trunk, and intraperitoneally. Three days after the last injection, the mice were sacrificed and their spleens were removed. The spleen cells were then fused with myeloma cells by the following procedure. c) Fusion
  • Suspensions containing a five-to-one ratio of spleen cells to myeloma cells were prepared.
  • the myeloma cells chosen were NS-1.
  • the NS-1 cells were conditioned to have a doubling time about every seventeen hours. They were used for fusion when in the log phase.
  • the NS-1 cells were subcultured in bacteriological plates (100 mm) at a concentration of 6 x 10 4 cells/ml in 10 ml of Dulbecco's Modified Eagle's Medium (DMEM) containing 5% Fetal Bovine Serum (FBS), 100 units/ml of penicillin and 100 micrograms/ml of streptomycin. The medium was changed every three days. Alternatively, the cells were subcultured at 1.54 x 10 5 cells/ml in 10 ml of the same medium, and the medium was changed every two days.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS Fetal Bovine Serum
  • the spleen cells were prepared by placing the spleen on a bacteriological plate (100 mm) and injecting 20 ml of calcium magnesium free PBS (CMF-PBS) into both ends of the spleen to flush out the spleen cells. The flushed spleen cells were then transferred to a 50 ml centrifuge tube.
  • CMF-PBS calcium magnesium free PBS
  • the spleen cells were centrifuged at 400 g for five minutes, and then suspended in 5 ml of 0.83% NH 4 C1 (0.155 M) for ten minutes at room temperature to lyse the erythrocytes. 5 ml of
  • CMF-PBS was added to the tube to stop the lysis.
  • the cells were then pelleted, and resuspended in 10 ml of CMF-PBS.
  • the concentration of lymphocytes was determined by adding 40 microliters of cell suspension to 10 ml of saline together
  • TM with 3 drops of Zapoglobin .
  • the number of lymphocytes was counted with a hemacytometer and from this value the concentration of cells was determined. The concentration was then multiplied by the dilution factor of 250 to yield the actual concentration of cells in the suspension.
  • the Sp2/0 cells were transferred from five of the bacteriological plates (100 mm) to a 50 ml centrifuge tube. The cell concentration was determined using the counting technique described above. 5 x 10 7 of the Sp2/0 cells were then suspended in 10 ml of CMF-PBS and mixed with 2.5 X 10 8 spleen cells in a 50 ml centrifuge tube. The cells were spun down and washed once with 10 ml of
  • a fusion mixture Prior to preparing the cells, a fusion mixture had been prepared as follows. 5 g of polyethylene glycol 1450 (purchased from Kodak) had been mixed with 5 ml of CMF-PBS and 0.5 ml
  • Millipore filter in order to sterilize it. 1.0 ml aliquots had been added to Cryotubes, and these had been stored at -70°C.
  • the 1.0 ml aliquot of polyethylene glycol fusion mixture was added to the cell suspension and the suspension was mixed well. Forty-five seconds after the polyethylene glcyol fusion mixture had been added, 2.0 ml of the pre-heated DMEM (without serum) was added dropwise with mixing. The remaining 8 ml of the pre-heated DMEM (without serum) was then added. The cells were left at room temperature for 10 minutes. 2.0 ml of FBS was added to the suspension and the suspensions were mixed well. The combination of the FBS and the DMEM can help prevent adherence of cells to the test tube walls. The suspensions were then centrifuged at 400 g for four minutes. After having been spun down, the cells were suspended in
  • the concentration of the cell suspension was adjusted to be ⁇ [0,1] ⁇ [0,1] ⁇ [0,1] ⁇ [0,1] ⁇ [0,1] ⁇ [0,1] ⁇ [0,1] ⁇ [0,1] ⁇ [0,1] ⁇ [0,1] ⁇ [0,1] ⁇ [0,1] ⁇ [0,1] ⁇ [0,1] ⁇ [0,5-phosphatethyl)
  • the envelope glycoprotein, gpl20 was used in immunizing to make the mAbs against the CD4-binding domain.
  • the gpl20 was prepared from H9/HTLV-IIIB cell extracts. The immunization, virus preparation, fusion and screening for preparation of these mABs is described in published International Application PCT/US90/02261. e) ELISA Procedure for Preparing the mAbs Against the PND
  • ELISA immunosorbent assay
  • Purified gpl20 protein was prepared as described in W.G. Robey, "Prospect for Prevention of Human Immunodeficienty Virus Infection: Purified 120-kD Envelope Glycoprotein Induces Neutralizing Antibody", Proc. Natl. Acad. Sci. USA J&7023-27
  • the cell fusion supernatant will contain the antibody which
  • the antibody which is specific to gpl20 will bind thereto. Inasmuch as the gpl20 is bound to the Immunlon I plate, the antibody specific to gpl20 will also become bound to the plate.
  • the next stage is to add the marker which will indicate the amount of bound antibody in each well.
  • the marker chosen was horseradish peroxidase. This marker was conjugated with goat anti-mouse IgG to yield peroxidase-conjugated goat anti-mouse
  • the goat anti-mouse IgG will bind to any mouse monoclonal antibody which is bound to the palte.
  • the peroxidase marker can then be activated to indicate the quantity of bound antibody by an exzyme reaction.
  • the next step is to activate the peroxidase marker which is conjugated to the goat anti-mouse IgG. This is done by adding
  • the color reaction is stopped by adding 50 microliters of 2.0 M H 2 S0 4 .
  • the intensity of color was determined with an ELISA reader at 450 nm.
  • the amount of antibody specific to gpl20 is proportional to the intensity of the color.
  • An immunofluorescence assay was performed to determine whether any of the antibodies which were reactive with gpl20 in the ELISA would bind specifically to live HIV-1 infected H9 cells.
  • the H9 cell line is permissive to persistent infection by HIV-1. This cell line was obtained from the American Type Culture
  • infected cells but not uninfected cells, is probably selective to a domain of the HIV-1 envelope protein on the extracellular side of the cell membrane.
  • the immunofluorescence assay helps to select those gpl20 reactive antibodies which have a high potential to recognize the neutralization epitopes on the HIV-1 virion, and to inhibit syncytium formation by infected T-cells. Cultures of infected H9 cells were maintained as described above under the heading (a), "Preparation of Virus". The procedure by which the assay was performed is described below.
  • the cells were then resuspended in PBS, placed onto individual slides and cover-slipped. The cells were viewed with a fluorescence microscope.
  • Cell suspensions from each of the thirty-nine ELISA positive wells were expanded in the wells of a twenty-four well plate. After five days of growth in the twenty-four well plate, the cell suspension from the seven wells tested immunoreactive to infected H9 cells which were diluted to thirty, fifty and one hundred cells per ml. 0.1 ml of the diluted cell suspensions
  • gpl20 are the ones which are desired.
  • the procedure is described below. 30 micrograms of HIV-1 was solubilized by heating it in a sample buffer (which contained 2% SDS and 5% beta-mercaptoethanol) at 100°C for five minutes. It was then loaded onto a 12% slab poly-acrylamide gels 1.5 mm thick. The gel was run at constant voltage of 35 mV for 8 hours at room temperature. The procedure was described in "Procedure for
  • Blotto buffer consists of 50 g of non-fat dry milk, 1.0 g of antifoam A
  • nitrocellulose sheets where then rinsed in PBS/0.05% Tween 20 and dried on a paper towel between weighted plexiglass plates. The nitrocellulose sheets were then cut into strips 0.5 cm
  • the strips can either be used immediately or stored dry and in the dark for up to one month.
  • the strips which carry the gpl20 band were to be used in the next stage.
  • the gpl20 nitrocellulose strips were then prepared to allow
  • the positive control was made of 2.0 ml of Blotto buffer/4% goat serum (which is made by mixing 100 ml of Blotto buffer and 4 ml of heat inactivated normal goat serum) added to one strip after which 10 microliters of heat inactivated AIDS patient serum was added to the well. 2.0 ml of supernatant was withdrawn from each of the thirty-nine wells in the microtiter plates which contained ELISA positive clones. Mixtures were made which consisted of 2.0 ml of supernatant, 5% non-fat dry milk, 50 microliters of 1 M HEPES (pH 8.0), and merthiolate.
  • the strips were then reacted with the staining reagents, which permit visualization of specific antibody binding to gpl20.
  • the reagent chosen was horseradish-peroxidase. This reagent exhibits color when contacted by a working substrate which consists of 10 ml of PBS, pH 7.4, 2.0 ml of substrate stock, and 4.0
  • Substrate stock is made by dissolving 0.3 g of 4-chloro-l-napthol in 100 ml of anhydrous methanol.
  • the seven immunofluorescence positive clones which have situated in the wells in the second twenty-four well plate, were grown up in a 100 mm tissue culture plate. The expanded culture of the selected seven single-cell clones were then separately injected into the peritoneal cavity of pristane treated mice, using five million cells per mouse. After seven days the ascites fluid of each mouse was collected and frozen.
  • the monoclonal antibodies in the ascites fluid were purified as follows.
  • the frozen ascites fluid was thawed and filtered through a nylon cloth to remove viscous material.
  • Sufficient phenylmethyl sulfonyl fluoride was added to the ascites fluid so that there was a final concentration of 0.1 mM.
  • 0.05 ml of 1.2M acetate buffer (pH 4.0) was added for every milliliter of ascites fluid.
  • the final concentration of the acetate buffer was 60 mM.
  • the pH was adjusted to 4.5.
  • caprylic acid For every milliliter of treated ascites fluid, 25 microliters of caprylic acid (MW of 144.21, density of 0.91) was added dropwise with vigorous stirring. The suspension was kept at room tempera- ure and stirred continuously for 30 more minutes. The suspension was then centrifued at 15,000 g for ten minutes in order to remove the precipitate. The supernatant, which contains IgG, was neutralized by adding a volume of 1 M HEPES buffer (pH 8.0) equal to one-tenth the volume of the supernatant. The IgG was then precipitated with 50% (NH 4 ) 2 S0 4 .
  • the precipitate was then dissolved in HEPES saline buffer.
  • HEPES buffer saline contains purified dissolved IgG. The purified
  • H9 cells were selected for the neutralization assay. i) Preparing the Virus. Antibody and Cells H9 cells were prepared by washing a cell culture with H9 growth medium. The H9 growth medium contained 20% FBS
  • the cells were then resuspended to a final concentration of 2 x 10 6 cells/ml.
  • the suspension was then incubated with 2 micrograms/ml of polybrene in a water bath at 37°C for twenty minutes.
  • the cells were spun down at 700 g for seven minutes. The supernatant was then discarded, and the cells were resuspended in H9 growth medium and washed again to remove the polybrene. The cells were then resuspended to 2 x 10 6 cells/ml in growth medium.
  • Virus at 20 TCID 50 was used in the infection of H9 cells.
  • the TCID 50 value of the virus preparation was determined in previous infectivity assays under the same experimental conditions. It is defined as the virus titer at which 50% of the experimental wells are infected. 20 TCID TM was equivalent to roughly a 4.72 x 10 5 dilution of the viral stock.
  • 30 microliters of virus suspension, and 30 microliters of each of the antibody solutions were mixed in the wells of a microtiter plate at 4°C for one hour. Each well was done in duplicate. The plate was then warmed in an incubator at 37° C and 5% C0 2 for thirty minutes. 30 microliters of the polybrene treated H9 cell suspensions was then added to each well.
  • microtiter plates were then incubated for one hour at
  • the plates were incubated for three days, and new growth medium was replaced every three days. Cells were collected on the third, sixth, ninth and thirteenth day.
  • This suspensions were air dried and then fixed with 1:1 acetone/methanol for ten minutes, air dried and stored at -20°C before assay.
  • the fixed cells were rehydrated in PBS for twenty minutes and then incubated with 5% normal goat serum in
  • Fig. 1 the percentage of immunofluorescence cells is plotted against the concentration of antibody in suspension.
  • Fig. 2 the results in Fig. 1 are from cells collected on day 9.
  • Fig. 2 the cells were collected on day 13.
  • FIGs. 1 and 2 it can be seen that four of the six antibodies tested (designated as BAT123, 267, 509, and 085) were effective in inhibiting infection.
  • BAT123 showed almost complete inhibition of infection on day 9.
  • This results is to be contrasted with the negative control anti-HcG antibody, which exhibited virtually no inhibition.
  • Nearly 100% of the cells treated with anti-HcG were immunofluorescent, irrespective of the concentration of antibody. The similar result was obtained with
  • BAT496 which is reactive with gpl20 but shows no neutralization activity. For this reason, BAT496 was not assayed on day 13 and does not appear in Fig. 2.
  • FIGs. 1 and 2 because it was found less effective in syncytium formation inhibition.
  • a comparison of Figs. 1 and 2 shows that as time goes on, more of the cells in the suspension become infected. This result is expected.
  • the amount of antibody in suspension available to neutralize the virus is decreasing due to change in medium and probably degradation or internalization.
  • the infected H9 cells continually produce more virus, and this virus eventually infects all the cells.
  • Another test for the monoclonal antibodies of the invention was to determine whether they inhibited syncytium formation.
  • the syncytium assay was based on the assumption that the exterior envelope protein of the virus in infected H9 cells binds to the CD4 antigen which is carried by T cells.
  • infected H9 cells are added to a well containing CD4 DNA transfected HeLa cells.
  • HeLa cells are used because they adhere, in a monolayer, to the bottom of the well.
  • These transfected HeLa cells express abundantly CD4 antigen on their cell surface. Thus, they have the ability to fuse with infected H9 cells. Therefore, if syncytium formation occurs, aggregates of HeLa and H9 cells will be bound to the well. These multi-nucleated giant cells can readily be observed and counted.
  • HeLa-CD4 + cells which express the CD4 antigen on the surface
  • HeLa-T4 growth medium which
  • FBS heat inactivated
  • the cell suspension was first washed twice with H9 growth medium (20% FBS in RPMI 1640, 5 mM of
  • HeLa-T 4 at a concentration of 0.4 million/ml.
  • the antibodies were prepared by first performing a sterile filtration on the seven antibody solutions which had been used in the neutralization assay. Six of these solutions contained antibodies of the invention, and the seventh contained the anti-HcG. Each solution was then diluted to make two final
  • the microtiter plate wells had previously been coated with the HeLa-CD4 + cells.
  • infected H9 cell suspension was added without the addition of antibody. This well was to serve as a positive control.
  • uninfected H9 cell suspension was added. This well was to serve as a negative control.
  • the experiments were done in triplicate. The plates were then incubated for eighteen hours at 37° C and 5% C0 2 . The plates were washed gently twice with DMEM in order to remove unattached H9 cells.
  • the DMEM was removed and the cells were fixed by adding 200 microliters of methanol per well for seven minutes. After removing the methanol, the cells were air dried, and then stained with 100 microliters of 1.4% methylene blue for ten minutes. The cells were rinsed with distilled water three times.
  • BAT496 was almost ineffective in both applications as was, of course, the negative control anti-HcG. Although BAT085 was effective in neutralization, it was not among the most effective in syncytium inhibition.
  • BAT401 was not very effective at syncytium inhibition, although it was effective in the neutralization assay. This result indicates that antibodies which are effective in inhibiting HIV-1 infection are not necessarily effective in inhibiting syncytia formation. Accordingly, the hybridoma producing BAT123, which was most effective at inhibiting both infectivity by the HIV-1 virions and syncytium formation, was deposited at the ATCC in Rockville, Maryland, under Accession number HB 10315. The Table II results demonstrate that, similar to neutralization as shown in Table I, syncytium inhibition is also dosage-dependent. The solutions with 10 microgram/ml of antibody were generally more effective in inhibition than the 1 microgram/ml solutions.
  • Example III Neutralization of Different Strains and Isolates of
  • HIV-1 bv the anti-PND mAbs
  • HIV-1 with a substantial degree of heterogeneity in the amino acid sequence of gpl20.
  • the strains selected for the study were the RF, AL, MN, Z84 and Z34 strains. See Starcich £t .al., supra.
  • the neutralization antibody BAT123 was chosen for use in the study because it was shown to elicit highest potency in the neutralization of the virus.
  • blood specimens were randomly collected from infected individuals in different parts of the United States (Houston, Texas; Los Angeles, California; Boston,
  • the procedure used is similar to that described earlier. 30 ml of heparinized blood from each patient was freshly collected and processed for mononuclear leukocytes by density-gradient centrifugation. Briefly, the whole blood was diluted with equal volume of phosphate-buffered saline (PBS). 25 ml of the diluted blood was laid over 10 ml of Ficoll-Paque (Pharmacia) and centrifuged at 1500 x g for 30 minutes. After centrifugation, the interphase containing mononuclear leukocytes was removed and washed twice in PBS. The mononuclear leukocytes were then
  • PHA phytohaemagglutinin
  • HIV-1 strains HIV-1 B , HIV-IRF, HIV-1 ⁇ , HIV-1 MN , HIV-l ⁇ ,
  • HlV-l ⁇ and HIV-l ⁇ -o ⁇ by approximately 50%, and HIV-l ⁇ by approximately 50%, and HIV-l ⁇ by approximately 50%
  • the co-culture experiments used lymphocytes isolated from the peripheral blood of patient clinically diagnosed as positive but in an asymptomatic state for AIDS or ARC. Out of 32 patient blood specimen tested, the virus had been isolated from 18 samples as measured for reverse transcriptase activities.
  • Example IV Determining The Peptidic Segments of gpl20 Reactive With anti-PND Monoclonal Antibodies
  • the synthetic peptides on the strips are 8-20 amino acid residue long. These peptides represent overlapping peptidic segments across the entire length of gpl20 of HTV-1B strain. Several tens of peptide solutions had been adsorbed on individual strips in equally spaced regions. The strips were provided in a dry form.
  • the immunoblotting procedure using the nitrocellulose strips is the same as the Western blot procedure used to determine whether the monoclonal antibodies react with gpl20 described in the preceding section.
  • BAT123 overlap by 5 amino acids. However, the antibodies react with just one of them and do not react with the other to any measurable extent. The antibodies do not react with peptides overlapping at the other ends either, i.e. BAT267 does not react with LNQSVRINCTRPNNN and BAT123 does not react with
  • Murine monoclonal antibody G3.519 (IgGl) binds to a conserved region in or near the CD4 receptor binding site on gpl20 and is known to have neutralizing activities against diverse strains of HTV-1. See Published International Application
  • BAT123 (IgGl) recognizes a relatively variable peptidic segment in gpl20 and exhibits effective neutralizing activity against HTLV-III B strain. The details of the neutralizing activity of BAT123 is described above. BAT123 and G3.519 were used in the immunoconjugate study.
  • PAP-S was purified from seeds of Phytolacca americana (pokeweed) using a method of Barbieri et al. (L. Barbieri, et al..
  • N-succinimidyl-3-(2-pyridyldithio) propionate (Pharmacia) at 1:3 molar ratio as described by Carlsson et al. (J. Carlsson, et al..
  • H9 cells either uninfected or chronically infected by HIV-1 strains (HTLV-ffl B , HTLV-HI RF , and HTLV-IH MN ) were maintained in log phase in RPMI1640 supplemented with 15% heat-inactivated fetal bovine serum, 100 U/ml of penicillin and 100 ⁇ g/ml of streptomycin.
  • HTLV-ffl B uninfected or chronically infected by HIV-1 strains
  • HTLV-HI RF HTLV-HI RF
  • HTLV-IH MN HTLV-IH MN
  • the controls were an irrelevant immunoconjugate of PAP-S or a mixture of the unconjugated antibody and PAP-S at equivalent concentrations under the identical conditions.
  • the cell cultures were kept at 37°C for 24
  • H9 cells infected by HTLV-IIIB were washed twice in PBS containing 1% bovine serum albumin at 4°C. The cells were resuspended at 1 x 10 7 /ml in the same buffer. Fifty ⁇ l of the cell suspension were incubated with 50 ⁇ l of diluted immunoconjugates (10-0.1 ⁇ g/ml) at 4°C for 30 minutes. The cells were then washed with 3 ml of the buffer.
  • Immunoconjugate was isolated from uncoupled monoclonal antibody using a cation exchange column (Mono S). Since the isoelectric points of both mAbs BAT123 and G3.519 are approximately 6.8, unmodified mAb did not bind to the column during the sample loading in 5 mM phosphate buffer, pH 6.0.
  • the immunoconjugate was eluted from the Mono S column as a single peak at 110 mM NaCl concentration (Fig.3). However, this peak, when analyzed by 7.5% SDS-PAGE under the non-reducing condition, resolved into two protein bands. The higher molecular weight band represented the conjugate containing two molecules of PAP-S per molecule of antibody and the lower band a conjugate with one molecule of PAP-S per antibody molecule.
  • SUBSTITUTE SHEET G3.519 are specific to gpl20 but recognize distinct epitopes with different binding constants (2.9 x 10 10 M "1 and 6.9 x 10 8 M '1 respectively). As shown in Figure 4, BAT123-PAP-S bound more
  • G3.519-PAP-S binds to the CD4-binding region of gpl20 in which the amino acid sequence is conserved. H9 cells infected separately with three diverse strains of HIV-1 were all sensitive to
  • the cytotoxic activity of the BAT123-PAP-S immunoconjugate was evaluated using H9 cells infected with the same diverse strains of HIV tested with G3.519-PAP-S. Incubation of H9 cells infected by these diverse strains of HIV-1 with
  • BAT123-PAP-S showed various degrees of specific killing (Figure 6). H9 cells infected with HTLV-III B were most susceptible to BAT123-PAP-S treatment with an IC 5Q of 5.2 x 10 "11 M. At 2.6 x 10 "9 M BAT123-PAP-S inhibited DNA synthesis by more than
  • BAT123-PAP-S also killed H9 cells infected with
  • BAT123-PAP-S was relatively ineffective in killing H9 cells infected with the
  • Irrelevant monoclonal antibody did not inhibit the cytotoxicity of immunoconjugates even at 200 fold excess of the free antibody.
  • BAT123-PAP-S and G3.519-PAP-S showed specific cytotoxicity against human T cells infected with HIV-1.
  • Epitope mapping studies using synthetic polypeptides revealed that mAb BAT123 recongizes the relatively variable region (amino acid 308-322) of gpl20 and mAb G3.519 recognizes a relatively conserved region
  • BAT123 is directed against the variable region in gpl20 of HTLV- ⁇ i B , BAT123-PAP-S still was effective in killing H9 infected with HTLV-III MN while H9 cells infected with HTLV-III RP were not effectively killed. However, in neutralization studies, BAT123 showed a similar degree of ineffectiveness against both HTLV-III RP and HTLV-i ⁇ MN . The recent data with
  • BAT123-PAP-S showing its ability to kill H9 cells infected with both HTLV-III B and HTLV-III MN suggests a broader application of this antibody for use as an immunoconjugate.
  • Epitope mapping of G3.519 revealed that the binding site of the antibody resides on the CD4 binding region of gpl20 which is conserved among diverse strains of HIV.
  • G3.519-PAP-S specifically killed H9 cells infected with three diverse strains. It is interesting to note that even though mAb G3.519 was generated against gpl20 of the HTLV-III B strain, H9 cells infected with
  • HTLV- ⁇ i MN strain were more sensitive to G3.519-PAP-S than H9
  • Antibodies can be induced by the individually unique idiotypic determinants (idiotopes) of the first antibodies (Ab-1) that are induced by specific antigens. A subset of these anti-idiotypic antibodies or anti-id (Ab-2) recognizes the antigen-combining site (paratope) of Ab-1 and thus bear the internal image of the
  • Ab-2 ⁇ antigen recognizing other idiotypic determinants are classified as Ab2 ⁇ and Ab-2 ⁇ : those whose binding to Ab-1 can be inhibited by the antigen are Ab-2 ⁇ and those whose binding to Ab-1 cannot be inhibited by the antigen are Ab2 ⁇ (Jerne, N.K. _ ⁇ i ⁇ l. EMBO J.
  • BAT123 was shown to neutralize the infectivity of HTLV-IIIB virions and to block syncytium formation between HTLV-IIIB-infected T cells and uninfected CD4 + HeLa cells (see above). In immunofluorescene flow cytometric analysis, BAT123 was shown to bind specifically to HTLV-III B and HTLV-III MN -infected T cell line (H9). Recent studies suggest that HTLV-i ⁇ B and HIV-III MN may be among the prevalent HTV-1 strains in the HIV-infected populations in the U.S.A. and in
  • the anti-CD4 binding region mAb G3.519 was also selected to generate anti-ids. G3.519 exhibited significant inhibition of binding to HTLV-III B and HTLV-III RP to CD4+ C8166 cells.
  • G3.519-PAP-S immunoconjugate was cytotoxic to H9 cells infected with HIV-1 strains HTLV-III B , HTLV-III MN , and HTLV-III RF .
  • mice were immunized i.p. with 100 ⁇ g of BAT123-KLH conjugate in CFA at 1-month intervals for 3 months.
  • mice were sacrificed, and spleen cells isolated and fused with Sp2/0 myeloma cells as described by Fung, M.C. et al, Bio /Technology 5:940 (1987). After selection using
  • HAT medium culture supernatants were tested for reactivity with BAT123 by ELISA, in which anti-BAT123 antibodies were bound by solid-phase BAT123, and detected by BAT123-HRP conjugate, which was prepared by the method of Wilson and Nakane, "Recent developments in the periodate method of conjugating horseradish peroxidase (HRPO) to antibodies.” Immunofluorescence and Related Staining Techniques. W. Knapp ed. Elsevier/North- Holland, Amsterdam and New York, p. 215. (1978). Briefly, wells of 96-well ELISA plates (Immunlon 2,
  • Reactive culture supernatants with OD greater than 0.2 were further tested for the ability to inhibit the binding of 125 I- labeled gpl20 to immobilized BAT123.
  • the gpl20 was purified from HTLV-i ⁇ B -infected H9 cell lysates as described by Sun et al, /. Virol 63:3579 (1989), and radioiodinated by the method described by Bolton, A. E. et al Biochem. J. 133:529 (1973).
  • IgGl, K was selected for further characterization.
  • the mAb was Ab2 ⁇ or Ab2 ⁇ . To confirm the anti-Id nature of this mAb, its identical binding to BAT123 and its chimeric form
  • CAGl-51-4 (the V regions of CAGI-51-4 are identical to those of
  • BAT123 was used as a control for reactivity with C rather than with V regions.
  • the AB19-4-HRP conjugate bound specifically to BAT123 but not to murine G3.519. Also, AB19-4 reacted with CAGI-51-4 and with BAT123 (Fig. 11). To aid in characterizing the binding region of AB19-4 and
  • HTLV-III MN and HTLV-III RP were run. The results are shown in Figures 8A and 8B. T64-63-6 is an irrelevant peptide used as control.
  • oligopeptides were synthesized using a DuPont RaMPS peptide synthesis system (Wilmington, DE).
  • a DuPont RaMPS peptide synthesis system WiPont RaMPS peptide synthesis system (Wilmington, DE).
  • wells of Immunlon 2 plate were coated with 100 ⁇ l of BAT123 (10 ⁇ g/ml).
  • HTLV-III B -gpl20 inhibited the binding between AB19-4-
  • AB19-4 is an Ab2 ⁇ , it should be only the intact gpl20 but not the peptides that can inhibit its binding to BAT123 via steric
  • AB19-4 is an Ab2 ⁇
  • the peptides should also inhibit the binding because they bind to the CDR.
  • Figures 13A and 13B show that both the goat and sheep antisera bound to solid-phase AB19-4 but not to normal mouse
  • Ab3 in antisera showing anti-gpl20 reactivity was affinity- purified by AffiGel-10 (BioRad, Richmond, CA) coupled with purified HTLV-III B gp 120. Five ml of diluted antiserum (1:1 with
  • the protein in the eluate was quantitated by the BCA protein assay (Pierce Chemical Co., Rockford, IL) and its purity examined by SDS-PAGE under reducing and non-reducing conditions. Pre- immune sera were sham purified by the same procedure and the eluate used as control.
  • HRP-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG are examples of HRP-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG (Fisher Scientific, Springfield, NJ), respectively.
  • the sham purified pre-immune serum substances as well as the irrelevant mAb anti-hCG did not bind to the uninfected or HIV-1- infected H9 cells.
  • Vn is the SFUs in the test wells and Vo the SFUs in the control without test antibodies.
  • Sham purified pre-immune rabbit serum substances and a non-HIV-neutralizing mAb BAT496 were used as controls.
  • the Ab3 was found to neutralize HTLV-III B and HTLV-III MN with ID ⁇ 0.7 and 0.33 ⁇ g/ml respectively (Fig. 18), whereas BAT123 neutralized only HTLV-IH B with ID 50 0.11 ⁇ g/ml, but not HTLV-III MN (Fig. 19). Both the Ab3 and BAT123 did not neutralize HTLV-III RP at concentrations as high as 2 ⁇ g/ml (data not shown). The sham purified pre-immune serum substances as well as the control mAb BAT496 showed no effect on the infectivity of the viruses in the assays.
  • Ab-2 ⁇ anti-id
  • Ab-1 anti-id
  • Ab-2 ⁇ anti-id of BAT123
  • the Ab-3 produced in Ab-2-immunized rabbits elicited an Ab-1 like Ab-3 response which was specific to gpl20 and to its specific epitope peptide and conferred HIV-neutralizing activities. Further, there was broadening of neutralizing activities of Ab-3 for HIV strains including both MN and IIIB as compared to Ab-1.
  • the finding that Ab-3 (AB19-4) exhibit broader reactivity to HIV is important, because it shows that via some as yet undefined mechanisms of jn vivo immunomodulation, a type-specific anti-HIV humoral immunity can be transformed, into a broader anti-HIV reactivity.
  • the paratope-specific anti-id's generated from broadly reacting HIV-neutralizing antibodies may have wider application.
  • Anti-idiotypes to mAb G3.519 were generated and screened
  • anti-idiotype AB19-4 was made by essentially the same process as the anti-idiotype AB19-4 was made.
  • the resulting anti-idiotypes were designated the AB20 series, and AB20-4 was deemed the one with the most suitable properties for further studies.
  • the peptide T35S has the same sequence as the gpl20 CD4-binding site of HTLV-IH B (amino acid residue numbers 413- 447, amino acid sequence TTTLPCRIKQIINMWQKVGKAMYAPPISGQIRCSS).
  • an assay was run with G3.519 as the solid-phase antigen, and AB20-4-HRP as the marker, where T35S was used as an inhibitor. The results are shown in Fig. 20, where it can be seen that T35S does inhibit binding between G3.519 and AB20-4-HRP,
  • AB20-4 is a suitable anti-idiotype for use as a vaccine against HIV-1.
  • the hybridoma cell line producing AB20-4 was placed on deposit at the American Type

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Oncology (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • AIDS & HIV (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

On décrit des anticorps monoclonaux qui se lient à la protéine gp120 sur l'enveloppe du HIV-1, dans la zone du PND aussi bien que dans la zone de liaison du CD4. Lesdits anticorps neutralisent l'HIV-1. ILs provoquent l'inhibition de l'infection des cellules en T et inhibent également la formation de syncytium. De plus, les anticorps sont spécifiques à des groupes et neutralisent différentes souches et isolats du HIV-1. Ces anticorps ont divers emplois, y compris le traitement et la prévention du Sida et du complexe lié à celui-ci (ARC). On utilise les anticorps dans des immunotoxines qui sont toxiques de façon spécifique pour les cellules en T infectées par le HIV-1. Par ailleurs, on peut utiliser les anticorps anti-idiotypiques envers ces anticorps neturalisants du HIV, ou des produits de dérivés tels que des anticorps chimériques et des fragments d'anticorps humains afin d'immuniser contre le HIV-1.
PCT/US1990/007535 1989-12-21 1990-12-19 Anticorps monoclonaux qui neutralisent l'infection par hiv-1 et leurs anti-idiotypes WO1991009625A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US45416189A 1989-12-21 1989-12-21
US454,161 1989-12-21
US53178990A 1990-06-12 1990-06-12
US531,789 1990-06-12

Publications (1)

Publication Number Publication Date
WO1991009625A1 true WO1991009625A1 (fr) 1991-07-11

Family

ID=27037357

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1990/007535 WO1991009625A1 (fr) 1989-12-21 1990-12-19 Anticorps monoclonaux qui neutralisent l'infection par hiv-1 et leurs anti-idiotypes

Country Status (2)

Country Link
AU (1) AU7160691A (fr)
WO (1) WO1991009625A1 (fr)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5558865A (en) * 1991-08-22 1996-09-24 Nissin Shokuhin Kabushiki Kaisha HIV immunotherapeutics
US5618922A (en) * 1994-07-25 1997-04-08 Nissin Shokuhin Kabushiki Kaisha NM03 antibody materials and methods
US5922325A (en) * 1990-10-26 1999-07-13 Public Health Research Institute Of The City Of New York, Inc. Synergistic neutralization of HIV-1 by human monoclonal antibodies and other antibodies directed against the v3 loop and the CD-4 binding site of GP-120,and the use for immunotherapy of HIV-1 infection
US6193982B1 (en) 1995-04-27 2001-02-27 The United States Of America As Represented By The Department Of Health & Human Services Anti-cyanovirin antibody with an internal image of gp120, a method of use thereof, and a method of using a cyanovirin to induce an immune response to gp120
US6420336B1 (en) 1995-04-27 2002-07-16 The United States Of America As Represented By The Department Of Health And Human Services Methods of using cyanovirins topically to inhibit viral infection
US6428790B1 (en) 1995-04-27 2002-08-06 The United States Of America As Represented By The Secretary Department Of Health And Human Services Cyanovirin conjugates and matrix-anchored cyanovirin and related compositions and methods of use
US6780847B2 (en) 1995-04-27 2004-08-24 The United States Of America As Represented By The Department Of Health And Human Services Glycosylation-resistant cyanovirins and related conjugates, compositions, nucleic acids, vectors, host cells, methods of production and methods of using nonglycosylated cyanovirins
US7048935B2 (en) 1995-04-27 2006-05-23 The United States Of America As Represented By The Department Of Health And Human Services Cyanovirin conjugates and matrix-anchored cyanovirin and related compositions and methods of use
US7339037B2 (en) 2001-03-22 2008-03-04 The United States Of America As Represented By The Department Of Health And Human Services Glycosylation-resistant cyanovirins and related conjugates, compositions, nucleic acids, vectors, host cells, methods of production and methods of using nonglycosylated cyanovirins
US7754420B2 (en) 1995-04-27 2010-07-13 The United States Of America As Represented By The Department Of Health And Human Services Methods of using cyanovirins to inhibit viral infection
US9534203B2 (en) 2007-06-08 2017-01-03 Wake Forest University Health Sciences Selective cell therapy for the treatment of renal failure
US9580688B2 (en) 2007-06-08 2017-02-28 Wake Forest University Health Sciences Kidney structures and methods of forming the same
US10590391B2 (en) 2007-06-08 2020-03-17 Wake Forest University Health Sciences Selective cell therapy for the treatment of renal failure

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4834976A (en) * 1986-07-03 1989-05-30 Genetic Systems Corporation Monoclonal antibodies to pseudomonas aeruginosa flagella
US4908203A (en) * 1987-09-09 1990-03-13 Johnson & Johnson Method for inducing HIV neutralizing antibodies using an internal image idiotope

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4834976A (en) * 1986-07-03 1989-05-30 Genetic Systems Corporation Monoclonal antibodies to pseudomonas aeruginosa flagella
US4908203A (en) * 1987-09-09 1990-03-13 Johnson & Johnson Method for inducing HIV neutralizing antibodies using an internal image idiotope

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIO/TECHNOLOGY, Vol. 5, issued September 1987, M.S.C. FUNG et al., "Monoclonal Antibodies that Neutralize HIV-1 Virions and Inhibit Syncytium Formation by Infected Cells", pages 940-946. *
JOURNAL OF IMMUNOLOGY, Vol.145, No. 7, issued 01 October 1990, M.S.C. FUNG et al., "Monoclonal Anti-Idiotypic Antibody Mimicking the Principal Neutralization Site in HIV-1 gp120 Induces HIV-1 Neutralizing Antibodies in Rabbits", pages 2199-2206. *
JOURNAL OF VIROLOGY, Vol. 63(9), issued September 1989, SUN et al., "Generation and Characterization of Monoclonal Antibodies to the Putative CD-4 Binding Domain of Human Immunodeficiency Virus Type 1 hp120", pages 3579-3585. *
SCIENCE, Vol. 226, issued 14 December 1984, M.K. MCNAMARA et al., "Monoclonal Idiotope Vaccine Against Streptococcus Pneumoniae Infection", pages 1325-1326. *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5922325A (en) * 1990-10-26 1999-07-13 Public Health Research Institute Of The City Of New York, Inc. Synergistic neutralization of HIV-1 by human monoclonal antibodies and other antibodies directed against the v3 loop and the CD-4 binding site of GP-120,and the use for immunotherapy of HIV-1 infection
US5558865A (en) * 1991-08-22 1996-09-24 Nissin Shokuhin Kabushiki Kaisha HIV immunotherapeutics
US5618922A (en) * 1994-07-25 1997-04-08 Nissin Shokuhin Kabushiki Kaisha NM03 antibody materials and methods
EP0848013A1 (fr) 1994-07-25 1998-06-17 Nissin Shokuhin Kabushiki Kaisha NM03, un anticorps monoclonal dirige contre la VIH-1 gp120 protéine
US6780847B2 (en) 1995-04-27 2004-08-24 The United States Of America As Represented By The Department Of Health And Human Services Glycosylation-resistant cyanovirins and related conjugates, compositions, nucleic acids, vectors, host cells, methods of production and methods of using nonglycosylated cyanovirins
US6420336B1 (en) 1995-04-27 2002-07-16 The United States Of America As Represented By The Department Of Health And Human Services Methods of using cyanovirins topically to inhibit viral infection
US6428790B1 (en) 1995-04-27 2002-08-06 The United States Of America As Represented By The Secretary Department Of Health And Human Services Cyanovirin conjugates and matrix-anchored cyanovirin and related compositions and methods of use
US6743577B2 (en) 1995-04-27 2004-06-01 The United States Of America As Represented By The Department Of Health And Human Services Methods of using cyanovirins to inhibit viral infection
US6193982B1 (en) 1995-04-27 2001-02-27 The United States Of America As Represented By The Department Of Health & Human Services Anti-cyanovirin antibody with an internal image of gp120, a method of use thereof, and a method of using a cyanovirin to induce an immune response to gp120
US7048935B2 (en) 1995-04-27 2006-05-23 The United States Of America As Represented By The Department Of Health And Human Services Cyanovirin conjugates and matrix-anchored cyanovirin and related compositions and methods of use
US7105169B2 (en) 1995-04-27 2006-09-12 The United States Of America As Represented By The Department Of Health And Human Services Cyanovirin conjugates and matrix-anchored cyanovirin and related compositions and methods of use
US7754420B2 (en) 1995-04-27 2010-07-13 The United States Of America As Represented By The Department Of Health And Human Services Methods of using cyanovirins to inhibit viral infection
US7339037B2 (en) 2001-03-22 2008-03-04 The United States Of America As Represented By The Department Of Health And Human Services Glycosylation-resistant cyanovirins and related conjugates, compositions, nucleic acids, vectors, host cells, methods of production and methods of using nonglycosylated cyanovirins
US9534203B2 (en) 2007-06-08 2017-01-03 Wake Forest University Health Sciences Selective cell therapy for the treatment of renal failure
US9580688B2 (en) 2007-06-08 2017-02-28 Wake Forest University Health Sciences Kidney structures and methods of forming the same
US10590391B2 (en) 2007-06-08 2020-03-17 Wake Forest University Health Sciences Selective cell therapy for the treatment of renal failure

Also Published As

Publication number Publication date
AU7160691A (en) 1991-07-24

Similar Documents

Publication Publication Date Title
EP0366718B1 (fr) Anticorps monoclonaux neutralisant le hiv-1
US5834599A (en) Immunoconjugates which neutralize HIV-1 infection
US5245015A (en) Monoclonal antibodies which neutralize HIV-1 through reaction with a conformational epitope in vitro
US5922325A (en) Synergistic neutralization of HIV-1 by human monoclonal antibodies and other antibodies directed against the v3 loop and the CD-4 binding site of GP-120,and the use for immunotherapy of HIV-1 infection
US8110203B2 (en) Adjuvant comprising non-toxic cross-linked muramyl dipeptide (MDP) microparticles derived from Propionibacterium acnes
US5166050A (en) Monoclonal antibodies and peptides useful in treating and diagnosing HIV infections
US5854400A (en) Monoclonal antibodies which neutralize HIV-1 infection
KR920008744B1 (ko) Hiv 감염의 치료 및 진단용 모노클로날항체 및 펩티드
AU633570B2 (en) Antibodies specific for cd4-binding domain of hiv
EP0492560A2 (fr) Anticorps monoclonaux humains contre la glycoprotéine transmembranaire (gp41) de HIV-1, et peptides associés
WO1991009625A1 (fr) Anticorps monoclonaux qui neutralisent l'infection par hiv-1 et leurs anti-idiotypes
US6309880B1 (en) Antibodies specific for CD4-binding domain of HIV-1
US5266478A (en) Antibodies which target a neutralization site within the second variable region of human immunodeficiency virus type 1 gp120
US5981278A (en) Chimeric monoclonal antibodies which neutralize HIV-1 infection and their applications in therapy and prevention for AIDS
WO1993004693A1 (fr) Inhibition synergique du vih-1
Dickey et al. Murine monoclonal antibodies biologically active against the amino region of HIV-1 gp120: isolation and characterization
Viveros et al. Characterization of a novel human immunodeficiency virus type 1 neutralizable epitope within the immunodominant region of gp41
WO1990000617A1 (fr) Anticorps specifiques envers gp 48
PL161520B1 (pl) Sposób wytwarzania nowego peptydu PL
AU2006200455A1 (en) Compositions and methods for treating viral infections
MXPA99003380A (en) Compositions and methods for treating viral infections

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BB BG BR CA DK ES FI HU JP KP KR LK MC MG MW NO RO SD SU US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE BF BJ CF CG CH CM DE DK ES FR GA GB GR IT LU ML MR NL SE SN TD TG

NENP Non-entry into the national phase

Ref country code: CA