WO1995031212A1 - Compositions a base de cd7 humaine et leurs modes d'utilisation - Google Patents

Compositions a base de cd7 humaine et leurs modes d'utilisation Download PDF

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Publication number
WO1995031212A1
WO1995031212A1 PCT/US1995/005936 US9505936W WO9531212A1 WO 1995031212 A1 WO1995031212 A1 WO 1995031212A1 US 9505936 W US9505936 W US 9505936W WO 9531212 A1 WO9531212 A1 WO 9531212A1
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Prior art keywords
human
cells
hiv
protein
antibodies
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PCT/US1995/005936
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English (en)
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David B. Weiner
Williams V. Williams
Alice I. Sato
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The Trustees Of The University Of Pennsylvania
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Publication of WO1995031212A1 publication Critical patent/WO1995031212A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to human CD7 and fragments thereof; antibodies, antibody fragments and peptides that comprise portions of antibodies which each bind to human CD7; and anti-CD7 antisense compounds.
  • the present invention relates to pharmaceutical compositions to treat individuals infected with human immunodeficiency virus (HIV) which comprise human CD7 and fragments thereof; antibodies, antibody fragments and peptides that comprise portions of antibodies which each bind to human CD7; and anti-CD7 antisense compounds, and to methods of using the same.
  • HIV human immunodeficiency virus
  • the present invention also relates to transgenic animals that comprise human CD7.
  • the HIV envelope glycoproteins alone are capable of mediating the cell surface membrane fusion required for infection by cell-free virions and in syncytium formation (Kowalski, M. , et al . 1987 Science 237:1351) .
  • the requirements for the target cell membrane in this fusion process appear to include molecules other than the CD4 receptor, such as cell-surface proteases and LFA-1 as well as several as of yet unidentified proteins (Qureshi, N.M. , et al. 1990 AIDS 4:553, Ehenbichler, C.F., et al . 1993 AIDS 7:489, Hildreth, J.E.K., et al.
  • anti-HIV therapeutics for starting materials to make anti-HIV therapeutics, for assays useful to identify anti-HIV therapeutics, for methods of treating individuals infected with HIV and for transgenic animals useful as HIV models and/or models to identify and evaluate anti-HIV therapeutics.
  • the present invention relates to essentially pure soluble human CD7 and fragments thereof.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising soluble human CD7 or fragments thereof and a pharmaceutically acceptable carrier.
  • the present invention relates to antibodies, antibody fragments and peptides comprising portions of antibodies which bind to human CD7.
  • the present invention relates to a hybridoma cell line which produced monoclonal antibodies that bind to human CD7.
  • the present invention relates to a monoclonal antibody that binds to human CD7.
  • the present invention relates to a pharmaceutical composition comprising antibodies, antibody fragments and peptides comprising portions of antibodies which bind to human CD7, and a pharmaceutically acceptable carrier.
  • the present invention relates to an isolated nucleic acid molecule that comprises a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of human CD7.
  • the present invention relates to a pharmaceutical composition comprising an isolated nucleic acid molecule that comprises a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of human CD7 and a pharmaceutically acceptable carrier.
  • the present invention relates to a method of treating an individual suspected of being infected by human immunodeficiency virus comprising the steps of administering a pharmaceutical composition that comprises a pharmaceutically acceptable carrier and an active ingredient selected from the group consisting of: soluble human CD7 protein, fragments thereof, antibodies that bind to human CD7, antibody fragments that bind to human CD7, peptides comprising portions of antibodies which bind to human CD7 and isolated nucleic acid molecules that comprise a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of human CD7.
  • a pharmaceutical composition that comprises a pharmaceutically acceptable carrier and an active ingredient selected from the group consisting of: soluble human CD7 protein, fragments thereof, antibodies that bind to human CD7, antibody fragments that bind to human CD7, peptides comprising portions of antibodies which bind to human CD7 and isolated nucleic acid molecules that comprise a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of
  • the present invention relates to a non-human transgenic mammal, essentially all of the somatic cells of which comprise a nucleotide sequence that encodes human CD7 operably linked to regulatory sequences required for expression of said nucleotide sequence in said cells, wherein said cells produce human CD7 protein.
  • Figure 1 has two graphs showing the effect of CD7-6B7 on HIV-l mediated syncytium formation.
  • Figure 1 has two panels: panel A and panel B. Representative data of anti-CD7 antibody CD7-6B7 (closed box) inhibition cell-to-cell fusion mediated by HIV-1 isolate III B (panel A) or MN (panel B) as compared to positive (anti-CD4 antibody Leu3a, closed circle) and negative (mouse serum IgG, open box) controls are shown. The total number of syncytia per well as determined by visual inspection is plotted against the concentration of antibody used.
  • Figure 2 is a bar graph showing results from experiments performed for epitope mapping by competition binding assay. SupT 1 cells were pretreated with each test antibody prior to binding of 3A1-FITC monoclonal antibody. All anti-CD7 antibodies except CD7-6B7 compete with 3A1 for binding. Control anti-beta-2-macroglobulin antibody BBMI does not affect 3A1-FITC binding.
  • Figure 3 is a bar graph showing FACS Analysis of CD7- cell line. A CD7- variant of SupTl, line F8.E5 (white bars), was selected as described in the Materials and Methods and tested in flow cytometric analysis in comparison to SupTl
  • CD4 Leu3a
  • CD7 3A1, CD7-6B7, B-F12
  • Figure 5 is two graphs showing Anti-I-FA- 1 inhibition of HIV-mediated syncytium formation in CD7- cells.
  • Figure 5 has two panels: panel A and panel B.
  • Syncytium inhibition assays were performed with SupTl (panel A) and F8.E5 cells
  • Figure 6 shows results from experiments performed to study cell-free infection of CD7+ and CD7- cell lines.
  • Culture supernatants taken at day 10 following infection were analyzed for evidence of reverse transcriptase activity. Background activity (Lanes 1 and 2) for SupTl cells (row A) and F8.E5 cells was minimal.
  • Evidence of productive infection in SupTl cells (row A, lanes 3 and 4) but not F8.E5 cells (row B, lanes 3 and 4) exposed to 100 TCID 50 of cell-free HIV-1 III B demonstrates significantly reduced susceptibility of the CD7- cells to infection.
  • the CD7 antigen is a T cell surface antigen with a poorly understood immunological function.
  • CD7 is expressed on a wide number of cells in the immune system, including approximately 80% of peripheral T cells (Reiter, C. 1989, Cluster report:CD7. In Leukocyte Typing IV, ed. by W. Kapp et al . , Oxford Univ. Press, Oxford 341) .
  • the level of cell surface CD7 is directly related to the activation state of the T cell.
  • CD7 is rapidly induced in vi tro through PHA or anti- CD3 treatment (Lazrovits, A.I., et al . 1987, Modulation of CD7 is associated with T lymphocyte function.
  • Leukocyte Typing III ed. by A.
  • CD7 acts as an accessory molecule in HIV-1 infection.
  • the presence of CD7 in addition to CD4 renders cells infectable by cell free virions. Infected cells which contain both CD7 and CD4 can form syncytium.
  • CD7 binds to HIV proteins, or interact with proteins which may bind to HIV proteins.
  • the human CD7 antigen fulfills several criteria necessary for an accessory molecule for HIV fusion.
  • Antibodies to the CD7 molecule are capable of limiting the scope of infection and in vi tro cytopathic effects 5 of HIV, the infectious agent for acquired immunodeficiency syndrome (AIDS) which selectively impairs the ability of T lymphocytes to respond to antigenic challenge. Furthermore, the dependence of SupTl cells on the presence of CD7 for infection by cell-free virions suggests that the role of CD7 in
  • HIV-mediated fusion is more profound than simply providing additional adhesive contacts as LFA-1 is thought to do in syncytium formation.
  • CD7 may be specifically downmodulated by viral infection as CD4 is suggestive of a direct interaction between it and viral components.
  • 25 protein has been identified by two other groups using crosslinking of T cell lysates to gp41-derived peptides (Qureshi, N.M. , et al . 1990 AIDS 4:553, and Chen, Y.H. , et al. 1992 AIDS 6:533) or bacterially produced soluble external gp41 (Ehenbichler, C.F., et al . 1993 AIDS 7:489) .
  • CD7 serve as a receptor for a component of the virion, possibly as a co-receptor for the viral envelope proteins which in this system is important for virus entry.
  • CD7 serve as a receptor for a component of the virion, possibly as a co-receptor for the viral envelope proteins which in this system is important for virus entry.
  • the role of CD7 in syncytia formation may be substituted by another unknown molecule which
  • 35 may be involved in cell adhesion.
  • soluble human CD7 protein is meant to refer to human CD7 free from cells and fragments of human CD7 which retain there ability to bind to HIV proteins and/or inhibit HIV infection.
  • Soluble CD7 protein is preferably a truncated form of the naturally occurring CD7 in which all or part of the transmembrane region has been deleted.
  • HIV-binding fragment is meant to refer to a fragment of human CD7 which includes the region of CD7 which binds to HIV proteins and which retains its ability to bind to HIV proteins and/or inhibit HIV infection. Fragments of CD7 comprise a portion having at least about 5 amino acids derived from CD7 and may further comprise non-CD7 amino acid sequences.
  • CD7 portion The portion of an HIV-binding fragment that is derived from CD7, which is referred to herein as a "CD7 portion" directly interacts with HIV proteins and/or inhibits HIV infection. Truncated versions of CD7 may be prepared and tested using routine methods and readily available starting material. One having ordinary skill in the art can readily determine whether a protein or peptide is an HIV-binding fragment of CD7 by: 1) comparing the sequence of the peptide or protein with the sequence of CD7 to identify a CD7 portion, 2) testing the peptide or protein to determine whether it has the ability to bind to HIV proteins and/or inhibit HIV infection and 3) determining whether the CD7 portion is involved in binding of the peptide or protein to HIV proteins and/or inhibiting HIV infection.
  • One having ordinary skill in the art can readily determine whether a protein or peptide is an HIV-binding fragment of CD7 without undue experimentation. Sequence analysis can be performed routinely. Similarly, binding studies to determine affinity of a peptide or protein to an HIV protein and infection assays to determine whether a peptide or protein inhibits HIV infection can be performed routinely. Using antibody mapping techniques, the portion of a peptide or protein that binds to another peptide or protein can be determined without undue experimentation.
  • Contemplated equivalents of soluble human CD7 protein including HIV-binding fragments thereof include peptides, polypeptides, molecules comprising amino acids linked by non- peptidal bonds, or proteins which comprise an amino acid sequence that is identical or substantially homologous to at least a portion of the CD7 protein amino acid sequence and which are capable of binding to HIV proteins and/or inhibit HIV infection.
  • substantially homologous refers to an amino acid sequence that has conservative substitutions.
  • One aspect of the present invention provides essentially pure, isolated soluble human CD7 protein.
  • the isolated soluble human CD7 protein is provided essentially free from other cellular proteins and materials.
  • Soluble human CD7 protein may be used in diagnostic assays and kits to identify HIV in a sample.
  • an assay is performed wherein soluble human CD7 protein is combined with a sample and complexes that are formed which include soluble human CD7 protein bound to HIV particles which can then be detected.
  • the presence of the complex is detected by migrating the assay products through an electrophoresis gel and comparing the distance travelled by proteins in the assay product with a control which include uncomplexed soluble human CD7 protein and/or a control that includes complexed soluble human CD7 protein/HIV particle or a molecule that migrates with the same apparent molecule weight of the complex.
  • the soluble human CD7 protein may be fixed to a solid phase and contacted with a sample. Complexes formed between the immobilized soluble human CD7 protein and HIV particles can be detected indicating the presence of HIV particles in the sample. Kits are designed to provide each of the various reagents in containers. Those having ordinary skill in the art can readily use soluble human CD7 protein and well known techniques and starting materials to practice these or other assays to identify the presence of HIV particles in a sample. By obtaining samples from individuals and performing such assays, individuals can be identified as being infected with HIV.
  • the soluble human CD7 protein may also be used in HIV purification assays and kits. For example, columns can be loaded or charged with soluble human CD7 protein to produce columns with immobilized soluble human CD7 protein and samples which contain HIV are passed through the column. HIV in the sample binds to the immobilized soluble human CD7 protein and the column is washed of other components form the sample. The conditions in the column are then changed to bring about the release of the HIV from the immobilized soluble human CD7 protein thereby allowing the pure HIV to be collected.
  • Those having ordinary skill in the art can readily apply standard techniques to purify HIV using soluble human CD7 protein.
  • the pure, isolated soluble human CD7 protein is useful as an anti-HIV agent.
  • Anti-HIV therapeutics which comprise soluble human CD7 protein bind to HIV proteins when contacted with cell-free HIV virions or cells that are infected with HIV and which display HIV proteins on their cell surfaces. By binding to the HIV protein, the soluble human CD7 protein prevents the HIV protein from interacting with CD7 on the cell surface and thus interferes with HIV infection and cell to cell spread of HIV.
  • one aspect of the present invention is a pharmaceutical composition comprising soluble human CD7 protein in combination with a pharmaceutically acceptable carrier.
  • Soluble human CD7 protein is combined with a pharmaceutically acceptable carrier or diluent to prepare a pharmaceutical compositions which can be administered in therapeutically effective amounts to persons diagnosed as or suspected of being infected with HIV in a method of treating such persons. Such persons are readily identifiable by those having ordinary skill in the art.
  • Another aspect of the present invention is a method of treating an individual suspected of being infected with HIV which comprises the step of administering to said individual a therapeutically effective amount of a soluble human CD7 protein.
  • the term "effective amount of a soluble human CD7 protein" is meant to refer to the amount of protein necessary to inhibit HIV infection of cells in the individual and/or the amount of anti-viral material necessary to inhibit cell-cell spread of viral infection by inhibiting syncytium formation between infected and uninfected cells. Effective amounts include both the amount effective to eliminate the progress of infection as well as the amount effective to slow the progress of infection relative to the rate of progress that would be observed in the absence of the soluble human CD7 protein.
  • Soluble CD7 may be produced by routine means using readily available starting materials as described above.
  • the nucleic acid sequence encoding soluble CD7 as well as the amino acid sequence of the protein are well known. Complete nucleotide and amino acid sequences are reported in Schanberg, L.E. et al . (1991) Proc . Na tl . Acad. Sci . USA 88:603-607 and Aruffo, A and B. Seed (1987) EMBO ⁇ 7.6 (11) .3313-3316, each of which is incorporated herein by reference.
  • DNA molecules that encode CD7 may be isolated by those having ordinary skill in the art from readily available starting material routine techniques. Isolation of a DNA sequence encoding human CD7 permits the production of soluble human CD7 protein using recombinant techniques now known in the art as well as the design and production of truncated forms of human CD7 which are also soluble human CD7 proteins.
  • Nucleic acid molecules that comprise nucleotide sequences that encode human CD7 can be obtained from human genetic material or can be prepared chemically using nucleotide sequence synthesizer or other standard techniques. When the coding DNA is prepared synthetically, advantage can be taken of known codon preferences of the intended host where the DNA is to be expressed.
  • One having ordinary skill in the art can, using well known techniques, obtain a DNA molecule that comprises a nucleotide sequence that encodes a soluble human CD7 protein and insert that DNA molecule into a commercially available expression vector for use in well known expression systems.
  • the commercially available plasmid pSE420 (Invitrogen, San Diego, CA) may be used for production in E. coli .
  • the commercially available plasmid pYES2 (Invitrogen, San Diego, CA) may be used for production in S. cerevisiae strains of yeast.
  • the commercially available MaxBacTM Invitrogen, San Diego, CA
  • complete baculovirus expression system may be used for production in insect cells.
  • the commercially available plasmid pcDNA I (Invitrogen, San Diego, CA) may be used for production in mammalian cells such as Chinese Hamster Ovary cells.
  • subtili ⁇ and Pseudomonas are also useful.
  • Suitable control sequences for prokaryotic systems include both constitutive and inducible promoters including the lac promoter, the trp promoter, hybrid promoters such as tac promoter, the lambda phage PI promoter.
  • foreign proteins may be produced in these hosts either as fusion or mature proteins.
  • the sequence produced may be preceded by a methionine which is not necessarily efficiently removed. Accordingly, the peptides and proteins claimed herein may be preceded by an N-terminal Met when produced in bacteria.
  • constructs may be made wherein the coding sequence for the peptide is preceded by an operable signal peptide which results in the secretion of the protein.
  • the signal sequence is removed upon secretion.
  • eukaryotic hosts are also now available for production of recombinant foreign proteins. As in bacteria, eukaryotic hosts may be transformed with expression systems which produce the desired protein directly, but more commonly signal sequences are provided to effect the secretion of the protein. Eukaryotic systems have the additional advantage that they are able to process introns which may occur in the genomic sequences encoding proteins of higher organisms. Eukaryotic systems also provide a variety of processing mechanisms which result in, for example, glycosylation, carboxy-terminal amidation, oxidation or derivatization of certain amino acid residues, conformational control, and so forth.
  • eukaryotic systems include, but is not limited to, yeast, fungal cells, insect cells, mammalian cells, avian cells, and cells of higher plants.
  • Suitable promoters are available which are compatible and operable for use in each of these host types as well as are termination sequences and enhancers, as e.g. the baculovirus polyhedron promoter.
  • promoters can be either constitutive or inducible.
  • the mouse metallothionene promoter can be induced by the addition of heavy metal ions.
  • the DNA encoding it is suitably ligated into the expression vector of choice and then used to transform the compatible host which is then cultured and maintained under conditions wherein expression of the foreign gene takes place.
  • the protein of the present invention thus produced is recovered from the culture, either by lysing the cells or from the culture medium as appropriate and known to those in the art.
  • One having ordinary skill in the art can, using well known techniques, isolate and purify the soluble human CD7 protein produced using such expression systems.
  • soluble human CD7 protein In addition to producing these proteins by recombinant techniques, automated amino acid synthesizers may also be employed to produce soluble human CD7 protein. If soluble human CD7 proteins are made synthetically, substitution by amino acids which are not encoded by a gene may also be made. Alternative residues include, for example, the ⁇ amino acids of the formula H 2 N(CH 2 ) n C00H wherein n is 2-6. These are neutral, nonpolar amino acids, as are sarcosine (Sar) , t-butylalanine
  • Phenylglycine for example, can be substituted for Trp, Tyr or Phe, an aromatic neutral amino acid; citrulline (Cit) and methionine sulfoxide (MSO) are polar but neutral, cyclohexyl alanine (Cha) is neutral and nonpolar, cysteic acid (Cya) is acidic, and ornithine (Orn) is basic.
  • the conformation conferring properties of the proline residues may be obtained if one or more of these is substituted by hydroxyproline (Hyp) .
  • the soluble human CD7 proteins can be formulated as pharmaceutical compositions which can then be administered to individuals infected with or suspected of being infected with HIV.
  • Another aspect of the present invention provides antibodies, antibody fragments which specifically bind to human CD7 and peptides that specifically bind to human CD7 which include portions of antibodies that specifically bind to human CD7.
  • fragment of antibodies is meant to refer to Fab fragments, (Fab) 2 fragments, and any other truncated form of an antibody molecule which retains affinity to CD7 including peptides and proteins which comprise a portion of such antibodies.
  • anti-CD7 antibodies is meant to refer to: rodent-derived antibodies which specifically bind to human CD7 including murine antibodies which specifically bind to human CD7 and rat antibodies which specifically bind to human CD7; fragments of rodent-derived antibodies which specifically bind to human CD7 including fragments of murine antibodies which specifically bind to human CD7 and fragments of rat antibodies which.specifically bind to human CD7; human antibodies that specifically bind to human CD7; fragments of human antibodies which specifically bind to human CD7; humanized antibodies that specifically bind to human CD7; fragments of humanized antibodies which specifically bind to human CD7; chimeric antibodies that specifically bind to human CD7 and fragments of chimeric antibodies which specifically bind to human CD7.
  • One aspect of the invention is anti-CD7 antibodies.
  • a preferred embodiments is monoclonal antibodies that specifically bind to human CD7.
  • One aspect of the invention is hybridomas which generate monoclonal antibodies that specifically bind to human CD7.
  • Anti-CD7 antibodies may be used in soluble human CD7 protein purification assays and kits. For example, columns can be loaded or charged with anti-CD7 antibodies to produce column with immobilized anti-CD7 antibodies and samples which contain human CD7 protein such as cell extracts are passed through the column. Soluble human CD7 protein in the sample binds to the immobilized anti-CD7 antibodies and the column is washed of other components from the sample. The conditions in the column are then changed to bring about the release of the soluble human CD7 protein from the immobilized anti-CD7 antibody thereby allowing the pure soluble human CD7 protein to be collected. Those having ordinary skill in the art can readily apply standard techniques to purify soluble human CD7 protein using anti-CD7 antibodies.
  • anti-CD7 antibodies When contacted cells that have CD7, anti-CD7 antibodies are useful as anti-HIV agents.
  • Anti-HIV therapeutics which comprise anti-CD7 antibodies bind to membrane-bound CD7 on human cells which display CD7 and thereby prevent HIV particles from binding to the CD7. By preventing HIV from interacting with CD7 on the cell surface, HIV infection and cell to cell spread of HIV by HIV infected cells is inhibited.
  • one aspect of the present invention is a pharmaceutical composition
  • a pharmaceutical composition comprising anti-CD7 antibodies in combination with a pharmaceutically acceptable carrier.
  • Anti- CD7 antibodies are combined with a pharmaceutically acceptable carrier or diluent to prepare a pharmaceutical compositions which can be administered in therapeutically effective amounts to persons diagnosed as or suspected of being infected with HIV in a method of treating such persons. Such persons are readily identifiable by those having ordinary skill in the art.
  • Another aspect of the present invention is a method of treating an individual suspected of being infected with HIV which comprises the step of administering to said individual a therapeutically effective amount of anti-CD7 antibodies.
  • the term "effective amount of anti-CD7 antibodies" is meant to refer to the amount of anti-CD7 antibody necessary to inhibit HIV infection of cells in the individual and/or the amount of anti-viral material necessary to inhibit cell-cell spread of viral infection by inhibiting syncytium formation between infected and uninfected cells. Effective amounts include both the amount effective to eliminate the progress of infection as well as the amount effective to slow the progress of infection relative to the rate of progress that would be observed in the absence of the anti-CD7 antibodies.
  • Those having ordinary skill in the art can produce monoclonal antibodies which specifically bind to CD7 and are useful as anti-HIV therapeutics using standard techniques and readily available starting materials.
  • the techniques for producing monoclonal antibodies are outlined in Harlow, E. and D. Lane, (1988) ANTIBODIES: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor NY, which is incorporated herein by reference, provide detailed guidance for the production of hybridomas which produce monoclonal antibodies which specifically bind to target proteins and the production of the monoclonal antibodies themselves. Briefly for example, human CD7 is injected into mice. The spleen of the mouse is removed, the spleen cells are isolated and fused with immortalized mouse cells.
  • hybrid cells or hybridomas, are cultured and those cells which secrete antibodies are selected.
  • the antibodies are analyzed and, if found to specifically bind to the protein of interest, the hybridoma which produces them is cultured to produce a continuous supply of antigen specific antibodies.
  • nucleic acid molecule that comprises a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of human CD7 and pharmaceutical compositions which contain such nucleic acid molecules.
  • Nucleic acid molecules that comprises a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of human CD7 may be used in methods for modulating the activity of RNA that encodes human CD7.
  • nucleic acid molecules that comprises a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of human CD7 relate to the field of "antisense" compounds, compounds which are capable of specific hybridization with a nucleotide sequence of an RNA molecule.
  • anti-CD7 antisense compound is meant to refer to nucleic acid molecules that comprises a nucleotide sequence that is complementary to a nucleotide sequence that encodes a portion of human CD7.
  • Anti-CD7 antisense are capable of specific hybridization with a nucleotide sequence of an RNA molecule and thereby block translation of mRNA that encodes CD7, thus inhibiting production of the protein.
  • the anti-CD7 antisense compounds of the present invention are useful to inhibit production of human CD7 in cells that would otherwise produce the protein.
  • Anti-CD7 antisense compounds are useful in methods of inhibiting production of human CD7 and thereby making cells less infectable by HIV and less capable of forming syncytia with HIV infected cells.
  • Anti-CD7 antisense compounds are useful to regulate gene expression, assaying for RNA and for RNA products through the employment of antisense interactions with such RNA.
  • Anti-CD7 antisense compounds of the present invention inhibit the production of human CD7 by interactions with molecules that direct their synthesis, intracellular RNA. It is the general object of such therapeutic approaches to interfere with or otherwise modulate gene expression leading to CD7 production.
  • Antisense compositions according to the present invention comprise oligonucleotide molecules which are complementary to the nucleotide sequence of the DNA molecule that encodes human CD7.
  • the oligonucleotides in accordance with this aspect of the invention preferably comprise from about 5 to about 200 nucleotides.
  • the oligonucleotides in accordance with this aspect of the invention more preferably comprise from about 5 to about 50 nucleotides. It is more preferred that such oligonucleotides comprise from about 8 to 25 nucleotides, and still more preferred to have from about 12 to 25 nucleotides.
  • oligonucleotides used in accordance with this aspect of the invention may be conveniently and routinely made through the well-known technique of solid phase synthesis using the information provided in Aruffo, A and B. Seed (1987) EMBO J. 6 (11) :3313-3316, which has been incorporated herein by reference, between nucleotide 50 and nucleotide 1480.
  • Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed, however the actual synthesis of the oligonucleotides are well within the talents of the one having ordinary skill in the art. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives.
  • messenger RNA includes not only the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5' -untranslated region, the 3' -untranslated region, the 5' cap region and intron/exon junction ribonucleotides.
  • oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides.
  • the oligonucleotide is specifically hybridizable with a transcription initiation site, a translation initiation site, an intron/exon junction or sequences in the 5'- or 3'- untranslated region or 5' cap region.
  • Oligonucleotides useful in the invention are complementary to the DNA or to the corresponding messenger RNA (mRNA) or pre-messenger RNA.
  • mRNA messenger RNA
  • the oligonucleotides in accordance with the invention preferably have one of the foregoing sequences or an effective portion thereof.
  • compositions according to the invention comprise a pharmaceutically acceptable carrier in combination with an active ingredient selected from the group consisting of soluble human CD7 protein, anti-CD7 antibodies and anti-CD7 antisense molecules.
  • the pharmaceutical compositions of the invention may be formulated by one having ordinary skill in the art with compositions selected depending upon the chosen mode of administration. Suitable pharmaceutical carriers are described in the most recent edition of Remington 's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.
  • the active ingredient is formulated based upon the nature of the ingredient and how it is to be administered.
  • the active ingredient can be, for example, formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle.
  • parenteral vehicle examples include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used.
  • the vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives) .
  • the formulation is sterilized by commonly used techniques.
  • a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution.
  • the pharmaceutical compositions according to the present invention may be administered as a single doses or in multiple doses.
  • the pharmaceutical compositions of the present invention may be administered either as individual therapeutic agents or in combination with other therapeutic agents.
  • soluble human CD7 protein may be administered in conjunction with soluble CD4 and/or AZT.
  • the treatments of the present invention may be combined with conventional therapies, which may be administered sequentially or simultaneously.
  • compositions may be administered by any means that enables the active agent to reach the agent's site of action in the body. Because proteins are subject to being digested when administered orally, parenteral administration, i.e., intravenous, subcutaneous, intramuscular, would ordinarily be used to optimize absorption. Intravenous is the preferred route of administration.
  • a daily dosage of soluble human CD7 protein can be about 1 ⁇ g to 100 milligrams per kilogram of body weight. Ordinarily 0.5 mg to 50 mg, and preferably 1 mg to 10 mg per kilogram per day given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.
  • a daily dosage of anti-CD7 antibody can be about 5 ⁇ g to 5000 mg of antibody. In some preferred embodiments, 50 ⁇ g to 500 mg of antibody may be administered. In other preferred embodiments, 500 ⁇ g to 50 mg of antibody may be administered. In a preferred embodiment, 5 mg of antibody is administered.
  • the pharmaceutical compositions may be administered in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.
  • transgenic animals particularly transgenic mice
  • the transgenic animals according to the invention contain a nucleic acid molecule which encodes human CD7.
  • Such transgenic mice may be used as animal models for studying HIV infection and for use in drug evaluation and discovery efforts to find compounds effective to prevent or impede HIV infection mediated by CD7.
  • Transgenic animals according to the present invention may further comprise a nucleic acid molecule that encodes human CD4.
  • One having ordinary skill in the art using standard techniques, such as those taught in U.S. Patent No. 4,873,191 issued October 10, 1989 Wagner and U.S. Patent No. 4,736,866 issued April 12, 1988 to Leder, both of which are incorporated herein by reference, can produce transgenic animals which produce the human CD7 and use the animals in drug evaluation and discovery projects.
  • a major cytopathic effect seen upon in vi tro infection of CD4+ human T cells by the human immunodeficiency virus (HIV) is cell-to-cell fusion which results in giant cell (or syncytium) formation.
  • Membrane fusion is required for infection by cell-free virions and in syncytium formation.
  • vi tro syncytium formation is considered to be a model for cell-cell transmission of infection.
  • the human T cell surface molecule, CD7 has been discovered to be important for fusion process.
  • CD7 is a roughly 40 kDa glycoprotein member of the immunoglobulin supergene family that is expressed early in the ontogeny of thymocytes and on the majority of peripheral blood T cells, as well as on natural killer (NK) cells and a small subpopulation of B cells.
  • Anti-CD7 monoclonal antibodies inhibited HIV-1 induced cell-cell fusion in several CD4+ T cell lines tested, (SupT 1, HuT-7 8, and CEM-SS) .
  • the anti-syncytial activity of the CD7 antibodies is not due to crossreactivity with CD4 or with viral proteins.
  • Epitope mapping revealed at least two regions of the molecule which are important for this effect.
  • Cells rendered CD7- are poorly infectable by cell-free virus. Additionally, cells rendered CD7- are more easily inhibited from fusing in syncytium formation assays. The collective results support a central role for human CD7 in the process of HIV infection.
  • the major lymphocyte receptor for gpl20 utilized in cell-to-cell infection resulting in syncytium formation is CD4. Binding of gp 120 depends primarily upon a short (approximately 20 amino acid) region of the first domain of CD4 which is structurally similar to an immunoglobulin kappa light chain CDR2 loop. A second region of CD4 is also suggested to play a role in gpl20 binding CD4 not only serves as a receptor but appears to induce a conformational shift in gp 120 which increases the antibody accessibility of the V3 loop of gp 120 and of certain gp4l epitopes.
  • the N-terminal region of the transmembrane gp4l includes a hydrophobic 'spike' . Following gpl20-CD4 binding, this 'spike' is believed to protrude into the target membrane and facilitate membrane fusion by bringing the two membranes into close apposition. A separate receptor molecule for gp41 has been hypothesized to be important for fusion to occur.
  • LFA-1 leukocyte adhesion molecule
  • Syncytium formation can be prevented by treatment of target PHA blasts with several anti-LFA-1 antibodies.
  • Anti-LFA-1 antibody inhibition of syncytium formation in T cell lines CEM and Jurkat has been reported, but inhibition of fusion by the same antibodies has not been observed in other T cell lines, including SupTl.
  • the inability of anti-LFA-1 antibodies to inhibit syncytium formation of certain T cell lines may relate to the function of LFA-1 as an adhesion molecule. It is likely that cell to cell adhesion is a prerequisite for syncytia formation.
  • T cell lines or even different subclones of the same line may demonstrate differences in their ability to form cell-cell conjugates and that only certain cell lines can prevented frog forming fusion permissive contact through antibodies directed against LFA-1 alone.
  • metabolic inhibitors such as cycloheximide and aphidicolin to block cell-cell fusion following viral infection suggests that cell surface adhesion itself and not an associated signaling function is necessary for syncytium formation.
  • Antiserum directed against the T cell surface and recognizing several membrane associated proteins has been reported to be able inhibit cell fusion driven by the HIV envelope glycoproteins. This antiserum did not react with the human CD4 antigen.
  • a multimolecular complex upon immunoprecipitation of human T cell lysates with a soluble form of the external domain of HIV-1 gp41 or HIV-2 gp36 has been identified.
  • the present invention is based upon the discovery that CD7, a 40 kDa immunoglobulin supergene family member expressed early in the ontogeny of thymocytes and on the majority of peripheral blood T cells is an accessory molecule for HIV entry.
  • H9/III B and H9/MN were obtained from the AIDS Reference Reagent Repository (Frederick MD) .
  • Sup Tl, CEM-SS, SP2/0 and H9 cells were obtained from the American Type Culture Collection (ATCC; Rockville MD) .
  • Cell lines were maintained at 37oc and 5% C0 2 in RPMI 1640 or Kennett's HY media (JRH Biosciences; Lenexa KS) supplemented with 10% fetal bovine serum (FBS; HyClone, Logan UT) and L- glutamine.
  • Transfected cells including the SP2-CD4 cell line
  • TCID50 values were determined by titration upon MT-2 target cells as described in Wang, B., et al . (1993) Proc . Natl . Acad. Sci . USA 90:4156, which is incorporated herein by reference.
  • Anti-LFA-1 antibodies H52 and MHM.24 were the kind gift of Dr. J. Hildreth (The Johns Hopkins Medical Institute) .
  • Purified anti-CD7 antibody 3A1, 3A1-FITC conjugated antibody, and control mouse IgG were purchased from Sigma Chemicals.
  • Syncytia Inhibition Assay To analyze the effect of the various antibodies on HIV-1 fusion, CD4+ T cell lines SupTl, Hut-78, or CEM-SS were used as target cells for infection. Two-fold dilutions of antibodies were made in 96 well plates in RPMI 1640 media containing 10% fetal calf serum
  • HIV-1 infected cells MN or III B isolate
  • SupTl target cells were added (5 X 10 4 well) and syncytium formation was
  • Hybridoma 10:673 which is incorporated herein by reference. Briefly, for surface marker analysis, cells were washed with FACS buffer (1% bovine serum albumin, 0.1% sodium azide in phosphate buffered saline) and resuspended in 100 ⁇ l total volume. Primary antibody was added to each sample and allowed
  • FITC-conjugated goat anti-mouse whole immunoglobulin antiserum (Fisher Scientific; Pittsburgh PA) was added at 1:200 dilution in FACS buffer and incubated again for 30 minutes on ice. Cells were then washed twice with FACS buffer and then resuspended in PBS containing 1% paraformaldehyde and fixed. The cells were washed and resuspended in FACS buffer prior to flow cytometric analysis.
  • Soluble CD4 ELISA Assay Dynatech ELISA 96 well plates were precoated with 50 ⁇ l of a 1 ⁇ g/ml solution of either BSA or soluble CD4 (Repligen, Cambridge MA) in carbonate-bicarbonate buffer (pH 9.6) at 4 ⁇ c overnight. Plates were washed 5 times with 0.05% Tween 20 in PBS to remove free protein and blocked against non-specific binding by incubation of 200 ⁇ l FACS buffer without azide for 1 hour at 37oc Ten- fold dilutions of primary antibody in PBS were allowed to bind for 1 hour at 37oc before washing and addition of 100 ⁇ l 1:1000 diluted goat anti-mouse IgG-HRPO (Fisher Scientific; Pittsburgh PA) . Color development was performed using o-phenylenediamine dihydrochloride (OPD) substrate and optical densities of the wells at 492 nm were determined on a Dynatech MR5000 ELISA plate reader.
  • OPD o-pheny
  • CD7 Expressing Cells The plasmid pCDM7-CD7-28 (Aruffo, A., and B. Seed (1987) EMBO J. 6:3313, which is incorporated herein by reference) which contains a cDNA clone of CD7, and pCDM8 (a vector similar to the pCDM7 vector except that it lacks the polyoma ori) , were kindly provided by Dr. B. Seed (Massachusetts General Hospital) .
  • One ⁇ g of column purified plasmid DNA (Qiagen Inc., Chatsworth CA) was used to transfect C0S7 cells by standard DEAE dextran/chloroquine methods (Kriegler, M. (1990) Gene transfer and expression : a laboratory manual . Stockton Press, New York, which is incorporated herein by reference. Cells were washed extensively following transfection and grown for 48 hours at 37oC before analysis.
  • CD7-Cells Cells were rendered cell surface antigen-negative by the method of Hillman et al . (1990) J. Immun . 6:2131, which is incorporated herein by reference. Briefly, SupT 1 cells at a density of 2 x 10-cells/mi were grown in 25 ml of a 1:5000 dilution of ethyl-methanesulfonate (EMS) in RPMI/10% FBS medium for 18 hours. After washing three times in DMEM/2% FBS, cells were resuspended in RPMI/10% FBS and allowed to recover for 48 hours prior to selection.
  • EMS ethyl-methanesulfonate
  • HIV-1 cytopathicity It is possible to measure one aspect of HIV-1 cytopathicity by assaying the ability of the viral envelope proteins, gpl20 and gp41, to mediate cell membrane fusion resulting in syncytium formation.
  • Syncytium Inhibitory activity for HIV-1 isolate III B was therefore assessed using SupTl target cells.
  • published reports of inhibitory antibodies against non-CD4 molecules have implicated cell surface proteases and LFA-1 as important for virally mediated fusion.
  • the broadly reactive polyclonal anti-human cell membrane antisera could block HIV-driven syncytium formation has been reported (Weiner, D.B., et al . (1989) Vaccines Cold Spring Harbor Press:115, which is incorporated herein by reference) .
  • Anti-CD7 antibody CD7-6B7 had significant anti-syncytial activity, inhibiting syncytium formation by 50% at a concentration of 8 ⁇ g/ml.
  • Figure 1, panel A Characterization of Anti-CD7 Antibody Syncytium Inhibition.
  • SupTl cells in the presence of virus infected cells form large syncytia distributed throughout the tissue culture plate within 24-48 hours of coculture.
  • HuT-78 normally grow as small clumps and upon addition of H9/III B cells will form more numerous medium-sized syncytia with similar kinetics as seen in SupTl cells.
  • CEM-SS cells grow as single cells and form fewer, very small syncytia at a slower rate, (48-72 hours) . All three cell lines show strong reactivity on FACS analysis with Leu3a and 3A1.
  • CD7 Reagents Do Not Crossreact with CD4 CD7 is a member of the immunoglobulin supergene family, with a gene structure most similar to Thy-1. Structural analysis suggests that while the CD7 protein most closely resembles kappa light chain structures, it possesses both structural as well as limited amino acid homology to CD4. Experiments were designed to rule out the possibility that the anti-syncytial activity of the effective anti-CD7 antibodies was due to crossreactivity of the antibodies for CD4 and therefore an artefact of the concentrations of antibody used in the system.
  • Solid-phase enzyme-linked immunosorbant assay against a recombinant soluble CD4 protein (Repligen; Cambridge MA) demonstrated no significant crossreactivity of the antibodies for CD4.
  • Antibodies were also tested for their ability to bind to CD4 in flow cytometric assays.
  • Antibody reactivity was compared with a mouse ly phoma cell line, SP2/0, and the stably transfected SP2-CD4 cell line (30) which expresses high levels of human CD4. No significant binding to the CD4+ cells was noted for any of the CD7 antibodies tested (inhibitory or non-inhibitory) whereas strong reactivity was seen for these cells with Leu3a.
  • CD7+CD4-cells were used to verify that our antibody preparations recognized CD7 and that a CD4-like epitope (that recognized by Leu3a) with which the anti-CD7 antibodies might bind by crossreactivity was not present on CD7.
  • a pCDM7-CD7 plasmid construct was transfected into COST cells by standard DEAE-dextran techniques. Strong reactivity with anti-CD7 antibodies 3A1 and 142-9 but not Leu3a was detected in transfected cells when compared to mock or vector (pCDM8) transfected control cells.
  • CD7 Reagents Do Not Appear to Directly Bind Virion Components It was possible that the anti-CD7 antibodies were not binding the target cells in the fusion assay but were binding instead to either CD7 molecules expressed on the surface of the infected cells (H9/III B or H9/MN cells) or to crossreactive epitopes located on the viral proteins. Comparison of the HIV- 1 III B cells with the SupT 1 target cells (Table III) strongly suggests that the anti-syncytial activity of the anti-CD7 antibodies is due to interaction with CD7 on the target cells. Similarly, the antibodies did not show any reactivity with viral protein lysates on Western blot analysis.
  • CD7-cell lines were constructed by EMS mutagenesis of SupTl cells, followed by antibody selection with antibody 3A1 and limiting dilution cloning.
  • the CD7-status of the derived cell lines was confirmed by FACS analysis with anti-CD7 antibodies 3A1, B-F 12 and CD7-6B7 ( Figure 3) . Further FACS analysis was used to examine the cell surface phenotype of the CD7-cell clones.
  • CD7-clone F8.E5 clearly remained CD4+ CDlla+ CD18+ as determined by reactivity with monoclonal antibodies Leu3a, TS1/22, and TSl/18, respectively ( Figure 3) .
  • the morphology and rate of proliferation ( Figure 4) of the CD7-cell line was indistinguishable from those of the parental SupT 1 cell line ( Figure 4) .
  • CD7-SupT 1 cells Using this clonal cell line, the ability of CD7-SupT 1 cells to fuse with HIV-1 infected cell lines was assessed. Both CD7- and CD7+ cell lines fused with H9 cells chronically infected with isolates III B , RF, and MN. The size and number of syncytia formed by the CD7+ and CD7-cells were comparable. The ability of anti-CD4 and anti-LFA-1 antibodies to inhibit syncytium formation in F8.E5 cells was assayed next.
  • CD7+ and CD7- cells Direct comparison of the abilities of CD7+ and CD7- cells to be infected by cell-free virus revealed striking differences in infectivity between the two lines.
  • the CD7- SupTl cells demonstrated no evidence of infection using as much as 100 TCID50's of HIV-1 III B or of HIV-lr ⁇ , at up to 15 days following addition of the virus.
  • the CD7+SupTl cell line was infectable with as little as 12.5 TCID50's with evidence of infection observed by 5 days. This dramatic change in the susceptibility of the T cell line to undergo cell-free infection by HIV suggests that CD7 is important direct infection of cells by cell-free virus whereas its role in syncytium formation is more complex.
  • CD7 CD7-6B7 0.008 CD7 B-F12 0.500 CD7 124-1D1 0.125 CD7 142-9 0.125 CD7 142-24 0.125
  • Table II Comparison of anti-CD7 antibody syncytium inhibition on different target cell lines. The lowest concentration (in mg/ml) at which > 50% inhibition of syncytium formation relative to mouse serum IgG control is observed in the CD4+ CD7+ human T cell lines SupTl, HuT-78, and CEM-SS are given for three anti-CD7 antibodies tested.
  • Table III FACS staining of target and infected cell lines. 2 X 10 s cells per sample were preincubated with antibody as listed above prior to treatment with goat anti-mouse IgG-FITC secondary antibody. After washing and paraformaldehyde fixation, cells were analyzed on a FACScan machine (Becton- Dickinson) . Mean channel number for each sample is shown.

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Abstract

L'invention concerne des protéines CD7 humaines pures solubles, des anticorps anti-CD7, des composés anti-sens anti-CD7. L'invention concerne également des compositions pharmaceutiques contenant des ingrédients actifs choisis dans le groupe constitué d'une protéine CD7 humaine soluble, d'anticorps anti-CD7 ainsi que de composés anti-sens anti-CD7. De plus, l'invention concerne des animaux transgéniques comprenant la CD7 humaine, des procédés de purification contre une infection à virus d'immunodéficience humaine, ainsi que des procédés de diagnostic et de traitement d'individus infectés par le virus de l'immunodéficience humaine.
PCT/US1995/005936 1994-05-13 1995-05-12 Compositions a base de cd7 humaine et leurs modes d'utilisation WO1995031212A1 (fr)

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WO2020212710A1 (fr) * 2019-04-18 2020-10-22 Kymab Limited Anticorps anti-cd7 antagonistes

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US5126433A (en) * 1986-08-21 1992-06-30 The Trustees Of Columbia University In The City Of New York Soluble forms of the t cell surface protein cd4

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IMMUNOGENETICS, Vol. 33, issued 1991, YOSHIKAWA et al., "Molecular Cloning of the Gene Coding for the Human T Cell Differentiation Antigen CD7", pages 352-360. *
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Publication number Priority date Publication date Assignee Title
WO2020212710A1 (fr) * 2019-04-18 2020-10-22 Kymab Limited Anticorps anti-cd7 antagonistes
CN114007699A (zh) * 2019-04-18 2022-02-01 科马布有限公司 拮抗剂抗cd7抗体

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