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WO1996037234A1 - Immunisation intracellulaire - Google Patents

Immunisation intracellulaire Download PDF

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Publication number
WO1996037234A1
WO1996037234A1 PCT/US1996/007393 US9607393W WO9637234A1 WO 1996037234 A1 WO1996037234 A1 WO 1996037234A1 US 9607393 W US9607393 W US 9607393W WO 9637234 A1 WO9637234 A1 WO 9637234A1
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WO
WIPO (PCT)
Prior art keywords
cells
antibody
hiv
ser
recombinant gene
Prior art date
Application number
PCT/US1996/007393
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English (en)
Inventor
Lingxun Duan
Roger J. Pomerantz
Original Assignee
Thomas Jefferson University
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Publication of WO1996037234A1 publication Critical patent/WO1996037234A1/fr

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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
    • C07K16/1072—Regulatory proteins, e.g. tat, rev, vpt
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/1054—Lentiviridae, e.g. HIV, FIV, SIV gag-pol, e.g. p17, p24
    • 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
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates to an immunological approach using gene therapy to treat infectious disease.
  • HIV human immunodeficiency virus
  • Vaccines contain immunogens that are incapable of producing the disease state, but capable of producing immunity against the pathogen. Vaccines have been very successful in protecting against infection by some pathogens, but ineffective in protecting against infection by others.
  • Another approach is passive immunization, which involves supplying systemically to a host antibodies that can bind the pathogen.
  • the utility of this approach was greatly increased with the development of humanized antibodies and single-chain antibodies, both of which do not provoke an immune response by the host.
  • a current experimental approach for treating infectious disease is to intracellularly express in a host a mutant form of viral protein that can strongly interfere with the replication of the wild-type virus.
  • this strategy has been successfully implemented to produce cell lines with acquired resistance to herpes simplex virus (HSV) and HIV.
  • HSV herpes simplex virus
  • Different approaches to this intracellular binding have been developed for human viral infections, including: (1) transdominant-negative mutant inhibitors; (2) specific target gene ribozymes; (3) anti- sense oligonucleotides; (4) viral receptors and receptor analogs; (5) suicide constructs; (6) virus specific inhibitory molecules; and (7) molecular decoys.
  • the present invention overcomes the limitations of this prior art and vastly expands the therapeutic potential of antibodies.
  • the present invention involves treating diseases by intracellular immunization.
  • Antibody genes are delivered to cells in vectors. They "immunize" the host cells by enabling the intracellular expression of antibodies or modified antibody binding domains which are specific for important disease related antigens. These antibodies bind the antigens, thereby halting, inhibiting or retarding the development or progression of the disease.
  • the invention can provide immunity before or after the development of the disease as well as treatment to control its severity.
  • an improved method for conducting gene therapy is provided. The improvement involves using a recombinant gene encoding an antibody that is selectively specific for an intracellular antigen associated with an intracellular pathogen.
  • the recombinant genes of the invention preferably are prepared so as to be free of a signal sequence.
  • the recombinant genes can be provided with localization sequences, such as a nuclear localization sequence, so that the antibodies can be targeted to desired compartments.
  • the preferred recombinant genes encode single chain antibodies that are selectively specific for intracellular viral antigens and that are part of an infectious agent that is replication-deficient.
  • the recombinant genes encode single or multiple binding domains from one or more antibodies.
  • the antibody gene is under the control of a pathogen promoter such as the HTLV-1 LTR promoter which is expression dependent upon the presence of HIV-1 tat protein, so that intracellular expression of the antibody will not occur until the cell is also infected by a pathogen that can initiate the regulatory effects of that promoter.
  • a pathogen promoter such as the HTLV-1 LTR promoter which is expression dependent upon the presence of HIV-1 tat protein, so that intracellular expression of the antibody will not occur until the cell is also infected by a pathogen that can initiate the regulatory effects of that promoter.
  • a method for treating a subject having a disease caused by an intracellular pathogen is provided.
  • a recombinant gene in an infectious vector is administered to the subject, the gene encoding an antibody that is selectively specific for an intracellular antigen associated with the pathogen.
  • an ex vivo treatment is provided.
  • Cells may be isolated from a subject or derived from another source.
  • a recombinant gene is introduced into the cells, the gene encoding an antibody that is selectively specific for an antigen associated with an intracellular pathogen, to form immunized cells. The immunized cells then are introduced into the subject.
  • Still another aspect of the invention involves a method for inhibiting replication of an intracellular pathogen in a cell by causing to be introduced into the cell a recombinant gene encoding an antibody that is selectively specific for an antigen associated with an intracellular pathogen.
  • the recombinant gene can be as described above.
  • the invention also includes vectors containing the recombinant genes of the invention and cell lines transduced or transfected with such genes.
  • Figure 1 is a diagram of the vector pT7H3-10.
  • Figure 2 is a diagram of the vector p4ZABVKRID0.
  • Figure 3 is a diagram of the vector pSCCribo.
  • Figure 4 is a photograph of a gel depicting ribozyme cleavage of endogenous ABVK RNA.
  • Figure 5 is a diagram of the vector pLXSNCAT.
  • Figure 6 is a diagram of the vector pLX-GAL.
  • Figure 7 is a diagram of the vector pET19vHLc8.
  • Figure 8 is a diagram of the vector p9CESAR.
  • Figure 9 is a graph showing the effect of sFv- anti-rev production on syncytia formation.
  • Figure 10 is a graph depicting the effect of sFv- anti-rev expression on the levels of soluble p24 production.
  • Figure 11 is a graph showing the effect of anti- rev sFv expression on p24 antigen production in different clinically isolated HIV-1 strains.
  • Figure 12 is a bar graph depicting peptide mapping of human anti-tat Fab binding domains.
  • Figure 13 is a bar graph depicting the effect of peptide reduction on binding of human anti-tat Fab to cysteine rich domain peptides 4, 5 and 6.
  • Figure 14 is a bar graph depicting human anti-rev
  • Figure 15 is a diagram of the murine leukemia virus (MLV) -based retroviral vectors used to express the anti-rev sFv and CAT gene products.
  • Figure 16 is a photograph of a CAT assay depicting the patterns of gene expression of retroviral vectors cloned in human T lymphocyte cell lines. Using limiting dilution, forty cellular clones were generated from transduced CEM and Sup-Tl cells which were G418 selected for two weeks prior to being maintained in G418-free media for over two months. A total of 1 X 10 6 cells were collected from the different clonal cell lines in which to assess the amount of CAT expression.
  • MMV murine leukemia virus
  • the figure contains data generated using five representative pLXSN-transduced CEM (lanes 1-5) and Sup-Tl (lanes A-E) cellular clones.
  • - Figure 17, comprising parts A and B, depicts the patterns of gene expression of retroviral vectors, pLXSN and pSLXCMV. in human T lymphocytes.
  • Lane 1 non- transduced Sup-Tl cells
  • Lane 2 non-transduced CEM cells
  • Lane 3 Sup-Tl cells transduced with pLXSN-CAT
  • Lane 4 Sup- Tl cells transduced with pSLXCMV-CAT
  • Lane 5 CEM cells transduced with pLXSN-CAT
  • Lane 6 CEM cells transduced with pSLXCMV-CAT.
  • B CAT expression in peripheral blood mononuclear cells (PBMC) transduced with retroviral vectors. In each case, 2 X 10 6 stimulated PBMC were transduced with CAT-expressing retroviral vectors and CAT activity was measured at 48 hours post-transduction.
  • Lane A human PBMC transduced with pSLXCMV-CAT
  • Lane B human PBMC transduced with pLXSN-CAT.
  • Figure 18 comprising parts A, B and C, depicts, an analysis of sFv gene transduction and sFv protein localization in T lymphocytes.
  • Lanes 1-16 Sixteen representative transduced CEM cellular clones; Lane (-) : non-transduced CEM cells; Lane (+) : pSLXCMV-D8-SFv plasmid control; Lane M: PCR DNA marker. The arrow points to specific amplified bands (356 base pairs) .
  • Figure 19 is a series of graphs depicting HIV replication in mixed CEM and Sup-Tl cell populations transduced with anti-rev sFV.
  • Cells were transduced with CAT- or sFv-expressing retroviral vectors. Untransduced cells served as controls. Cells were then infected with HIV N 4 - 3 or HIV HXB2 at multiplicities of infection (moi) of 0.024and 0.24, respectively. HIV replication was assessed by measuring p24 antigen levels in culture supernatants in an ELISA assay (Dupont) .
  • the data presented are representative of at least two separate sets of experiment ' s.
  • Figure 20 comprises two graphs depicting HIV-1 infection in CEM and Sup-Tl cell clones transduced with anti-rev sFv.
  • Either untransduced or representative CEM and Sup-Tl cell clones transduced with CAT or sFv-expressing retroviral vectors were infected with HIV ⁇ , ⁇ at mois of 0.06 and 0.024.
  • HIV-1 replication was assessed by measuring p24 antigen levels in culture supernatants in an ELISA assay.
  • the data presented are representative of at least two separate sets of experiments.
  • Figure 21 comprising parts A and B, are photomicrographs depicting syncytium formation in sFv- transduced and control cells.
  • Mixed cell populations of anti-rev sFv-transduced (A) and CAT-transduced (B) CEM cells were infected with HIV J - L ⁇ at an moi of 0.12. Syncytium formation was measured and photographed on day 12 post- infection.
  • Figure 22 comprises two graphs depicting HIV-1 replication in anti-rev sFv transduced human PBMC.
  • Stimul-ated human PBMC were transduced with the various retroviral expression vectors as shown in the figure.
  • Mixed populations of PBMC were infected with HIV ⁇ at mois of 0.24 and 0.06.
  • HIV-1 replication was assessed by measuring p24 antigen levels in culture supernatants in an ELISA assay.
  • Figure 23 comprising parts A and B, depicts cell surface CD4 antigen levels and early stages of HIV-1 expression in retroviral vector transduced and in non- transduced T-lymphocytes.
  • (A) CD4 antigen expression CEM and Sup-Tl cells were transduced with retroviral vectors, selected in G418 medium for two weeks and were then incubated in G418-free medium for two months. Cells were stained for CD4 antigen using an FITC-conjugated antibody and were analyzed for CD4 expression via FACS. Non-transduced cells were used as controls in each experiment. The "% positive cells" on the Y-axis represents the percent of cells which were positive for CD4 antigen expression in each cell population.
  • Figure 25 is a diagram of the HIV-1 integrase gene and its protein product depicting the relative positions on the protein to which the monoclonal antibodies bind, which monoclonal antibodies form the basis of the anti-in sFv antibodies of the invention.
  • Figure 26 is a diagram of each of the expression vectors into which the anti-IN sFv gene is inserted.
  • Figure 27 depicts the nucleotide and amino acid sequence of the monoclonal antibody genes directed against HIV-1 invertase.
  • A MAb #21, anti-HIV-1 integrase heavy chain variable domain DNA sequence
  • B MAb #21 heavy chain corresponding amino acid sequence
  • C MAb #21 anti-HIV-1 integrase light chain variable domain DNA sequence
  • D MAb #7 anti-HIV-1 integrase heavy chain variable domain DNA sequence
  • E MAb #7 heavy chain corresponding amino acid sequence
  • F MAb #17 anti-HIV-l integrase heavy chain variable domain DNA sequence
  • G MAb #17 heavy chain corresponding amino acid sequence
  • H MAb #12 anti-HIV-l integrase light chain variable domain DNA sequence
  • I MAb #12 light chain corresponding amino acid sequence
  • J MAb #12 anti-HIV-l integrase heavy chain variable domain DNA sequence
  • K MAb #12 heavy chain
  • the invention provides antibody-based intracellular immunity against intracellular pathogens.
  • Recombinant antibody genes are introduced into cells.
  • the recombinant antibody genes encode antibodies that are selectively specific for antigens associated with the pathogen.
  • the antibodies are expressed intracellularly and the pathogen-associated antigens are present intracellularly.
  • the antibodies bind to the antigens and interfere with the replication of the pathogen, thereby providing the "immunity" or treatment.
  • the invention may be used prophylactically or therapeutically.
  • the invention When used prophylactically, the invention is applied to a subject that is at risk of being infected by an intracellular pathogen.
  • the invention When used therapeutically, the invention is applied to a subject that is known to have or that is suspected of having an infection by an intracellular pathogen.
  • subject means animal.
  • Preferred subjects are mammals, fowl and fish. Most preferred are humans, primates, dogs, cats, horses, cows, sheep, goats, pigs, rodents, chickens and turkeys.
  • Intracellular pathogen means a disease- causing organism which resides, during only part of its life cycle, within a host cell.
  • pathogens include certain viruses, bacteria, fungi and protozoans. Examples include: Human Immunodeficiency Virus including, without limitation, HIV-1 and HIV-2; human T cell leukemia virus (including, without limitation, HTLV-I and HTLV-II) ; herpesvirus including, without limitation, herpes simplex virus type 1 (HSV-1) and type 2, varicella zoster virus; cytomegalovirus (CMV); Epstein-Barr virus (EBV) ; papillomavirus; hepatitis (including, without limitation hepatitis A, B, C, D and E viruses) ; Creutzfeldt-Jacob virus; feline leukemia virus; influenza virus; variola; rubeola; mumps virus; mycobacteria including, without limitation, M.
  • Human Immunodeficiency Virus including,
  • Candida including, without limitation, Candida albicans and Candida tropicalis, mycoplasma, Toxoplasma gondii ; Trypanosoma cruzi ; organisms of the genus Leishmani ; and organisms of the genus Plasmodium.
  • a recombinant gene is an isolated protein-coding sequence operably linked to a promoter, whereby the protein is capable of being produced when the recombinant gene is introduced into a cell " .
  • the coding region can encode a full length gene product or a subfragment thereof, or a novel mutated or fusion sequence as described in greater detail below.
  • the protein coding sequence may be a sequence endogenous to the target cell, although according to the preferred embodiments it typically will not be a sequence endogenous to the target cell.
  • the promoter, with which the coding sequence is operably associated may or may not be one that normally is associated with the coding sequence.
  • the promoters useful in constructing the recombinant genes of the invention may be constitutive or inducible.
  • a constitutive promoter is expressed under all conditions of cell growth.
  • Exemplary constitutive promoters include the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT) , adenosine deaminase, pyruvate kinase, the S-action promoter and others.
  • HPRT hypoxanthine phosphoribosyl transferase
  • adenosine deaminase pyruvate kinase
  • S-action promoter the S-action promoter and others.
  • many viral promoters function constitutively in eukaryotic cells.
  • promoters include: the early and late promoters of SV40; the long terminal repeats (LTRs) of Moloney leukemia virus and other retroviruses, and the thymidine kinase promoter of herpes simplex virus.
  • LTRs long terminal repeats
  • thymidine kinase promoter of herpes simplex virus.
  • Other promoters are known to those of ordinary skill in the art.
  • Inducible promoters are expressed in the presence of an inducing agent.
  • the metallothionein promoter is induced to promote (increase) transcription in the presence of certain metal ions.
  • Other inducible promoters are known to those of ordinary skill in the art.
  • the recombinant genes of the invention are prepared synthetically or, preferably, from isolated nucleic acids. A nucleic acid is "isolated" when purified away from other cellular constituents, i.e., other cellular nucleic acids or proteins, by standard techniques known to those
  • the recombinant genes of the invention can be derived from sequencing information or cell lines publicly available or may be derived from antibody producing cell lines or isolated antibody producing lymphocytes prepared according to a variety of methods.
  • One such method involves the formation of monoclonal antibody producing hybridomas.
  • an animal is immunized with an antigen.
  • a fused cell hybrid then is formed between the antibody-producing cells from the immunized animal and an immortalizing cell line such as a myeloma.
  • cell lines can be produced by directly immortalizing antibody-producing human lymphocytes with Epstein-Barr virus (EBV) .
  • EBV Epstein-Barr virus
  • the recombinant genes of the invention encode antibodies that are selectively specific for intracellular antigens associated with intracellular pathogens.
  • An antibody that is "selectively specific" for an intracellular antigen binds to that antigen, but does not bind to any appreciable degree to native intracellular constituents of the host cell.
  • Antibodies as used herein means any portion of an antibody that retains the variable region binding specificity, including whole antibody, Fab portions, chimeric antibodies or fragments thereof including humanized and human antibodies and single chain antibodies. Single or multiple binding domains from one or more antibodies may be combined to form a chimeric antibody having the specificity of the binding domains of each antibody.
  • the antibodies should be selected such that they interfere with replication of the pathogen upon binding to the antigen.
  • Antibodies that selectively bind to antigens or elements that are conserved which are critical to regulation or which are critical to replication are preferred.
  • the antibodies can be selected to have specificity for important enzymes or regulatory proteins such as HIV-1 integrase, Tat, Rev and RT.
  • HSV antibodies with specificity for HSV-1 IE gene transactivator VP16 and ICP4 can be used.
  • HBV hepatitis B virus
  • HBV polymerase antibodies with specificity for HBV polymerase can be used. It should be understood that the foregoing are merely-' examples of antigens against which antibodies may be directed, and other appropriate antigens well known to or easily identified by those of ordinary skill in the art can be selected depending upon the particular pathogen of interest.
  • Antigens can be derived from virtually any pathogen associated source, including parts, extracts or isolates of pathogens. Recombinant antigens also are useful according to the invention. Many such antigens are available in various forms from commercial sources or from depositories such as the American Type Culture Collection, Rockville, MD.
  • DNase shotgun cleavage is based upon the observation that bovine pancreatic DNA I causes double strand scission of DNA in the presence of Mn ++ . Since cleavage is random and can be controlled by varying the enzyme concentration, temperature and/or incubation time, this method is very useful in the initial step in the generation of representative libraries having virtually any insert size range. Small random-size specific DNAs can be inserted into a vector, such as the pTOPE-T vector (Novagen, Madison, WI; U.S. Patent No. 4,952,496, the entire disclosure of which is incorporated herein by reference) , for expression of fusion proteins. These bacterial expression libraries will represent substantially the epitope domain of the specific antigen.
  • This bacterial expression system is suitable for human antibody epitope screening.
  • the fusion partner can ensure a high level of expression and help protect the target sequence from proteolytic degradation. Desired clones may be identified by direct screening on colony lift filters. Reagents and specific protocols are available in kits, including the Colony FinderTM Immunoscreening Kit sold by Novagen.
  • the recombinant genes of the invention preferably are prepared so as to be free of a signal sequence.
  • Free of a signal sequence means a deletion, mutation or modification of the signal sequence which ordinarily directs antibodies to the secretory compartments.
  • the hydrophobic amino acid core of the signal sequence for secretion can be substituted with hydrophilic residues by site directed mutagenesis. See Biocca, S. et al., "Expression and Targeting of Intracellular Antibodies in Mammalian Cells," European Molecular Biology Organization (EMBO) Journal 1: 101 (1990) .
  • the antibodies also can be targeted to desired compartments.
  • the antibodies can be targeted to the nucleus using the nuclear localization sequence PKKKRKV of the large T antigen of SV40 virus. Id.
  • the preferred recombinant genes encode single chain FV antibodies (sFv) .
  • the sFv antibody is described in U.S. Patent No. 4,946,778 to Genex Corporation, issued August 7, 1990, the entire disclosure of which is incorporated herein by reference.
  • sFv antibodies incorporate the complete antigen-binding Fv domain of an antibody into a single polypeptide by joining the light and heavy variable domains (vL and vH) with a linker peptide.
  • vL and vH light and heavy variable domains
  • sFv antibodies having specificity for haptens, proteins, receptors and tumor antigens have been shown to have binding affinities equivalent to those of the monoclonal antibodies from which they were derived.
  • sFv antibodies are preferred because of their small size and their reported lack of immunogenicity.
  • the recombinant genes of this invention are preferably free of a signal sequence and encode an appropriate targeting sequence as desired.
  • the recombinant genes encoding the sFv antibodies are prepared according to methods well known to those of ordinary skill in the art. See e.g. U.S. Patent No. 4,946,778. Briefly, hybridomas or immortalized B-cells making monoclonal antibodies to the antigens of interest are produced. Heavy and light chain cDNAs then are isolated and characterized, for example, by making DNA libraries from the foregoing immortalized cells and screening these libraries with probes for heavy and light chain clones. The heavy and light chain clones then are studied to determine the sequence of the variable domains. The variable domains of the heavy and light chain are joined by a linker. To design a suitable linker, it is preferred to first define the extent of the variable domains.
  • the sFv antibody may be constructed by joining either vL as the N-terminal domain followed by the linker and vH.
  • a preferred linker for constructing a vH-linker-vL sFv antibody is the single linker designed by Huston et al. , a (gly-gly-gly-ser) 3 linker designed to bridge the 3.5 n gap between C terminals of vH and the N terminals of vL, without exhibiting any propensity for ordered secondary structure (Huston, J.S. et al. , Proc. Natl. Acad. Sci. USA 85 pp 5879-5883, 1988). Minor modifications of this linker design appear to have little effect upon the in vivo performance of an sFv antibody.
  • the sFv gene then can be engineered to encode an identification signal such as the Tat nuclear translocation signal : . Because there exist specific antibodies to this signal, anti-idiotype antibody will not be necessary for immunostaining to determine sFv expression and intracellular location.
  • the sFv recombinant gene may be placed in a cassette that provides for efficient introduction into a cell and subsequent selection, for example, by G418 or gpt selection. After selection, cells can be evaluated for DNA, RNA and protein expression using DNA-PCR, RT-PCR and radioimmune precipitation, as well as immunostaining.
  • the recombinant genes of the invention are introduced into cells using vectors. Almost any delivery vector can be used, although the vector selected will depend upon the particular disease being treated, the particular form of treatment, whether the treated cells are replicating cells and other factors known to those of ordinary skill in the art.
  • Transfection refers to the acquisition by a cell of new genetic material by incorporation of added DNA. Transfection can occur by physical or chemical methods. Many transfection techniques are known to those of ordinary skill in the art including: calcium phosphate DNA co-precipitation; DEAE-dextran DNA transfection; electroporation and cationic liposome-mediated transfection. Transduction refers to the process of transferring nucleic acid into a cell using a DNA or RNA virus. Suitable viral vectors for use as transducing agents include, but are not limited to, retroviral vectors, adeno associated viral vectors and Semliki Forest virus vectors. The treatment of cells may be in vivo or ex vivo .
  • cells are isolated from an animal (preferably a human), transformed (i.e. transduced or transfected in vi tro) with a vector containing a recombinant gene of the invention, and then administered to a recipient.
  • Procedures for removing cells from animals are well known to those of ordinary skill in the art.
  • tissue or the whole or parts of organs may be removed, treated ex vivo and then returned to the patient.
  • cells, tissue or organs may be cultured, bathed, perfused and the like under conditions for introducing the recombinant genes of the invention into the desired cells.
  • the preferred treatment is ex vivo and the preferred cells for ex vivo treatment are stem cells.
  • cells of an animal preferably a mammal and most preferably a human
  • a vector containing a recombinant gene of the invention are transformed in vivo with a vector containing a recombinant gene of the invention.
  • the in vivo treatment may involve systemic treatment with a vector such as intravenously, local internal treatment with a vector such as by perfusion, topical treatment with a vector and the like.
  • the preferred vectors are based on noncytopathic eukaryotic viruses in which nonessential or complementable genes have been replaced with the gene of interest.
  • noncytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA.
  • Retroviruses have recently been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e. capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle) . Such genetically altered retroviral expression vectors have general utility for high-efficiency transduction of genes in vivo . Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M. "Gene Transfer and Expression, a Laboratory Manual", W.H. Freeman Co., New York (1990) and Murry, E.J. e.d. "Methods in Molecular Biology", Vol. 7, Humana Press, Inc., Cliffton, New Jersey (1991).
  • a preferred virus for certain applications is the adeno-associated virus, a double-stranded DNA virus.
  • the adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as: heat and lipid solvent stability, high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Recent reports indicate that the adeno-associated virus can also function in an extrachromosomal fashion.
  • a vector such as dl3-94 can accommodate an insertion of 4.7kb in length.
  • a modified sFv will be approximately 1 to 1.5kb in length, and therefore the adeno- associated virus may be an ideal delivery system.
  • an anti-HIV-l sFv (pAVsFv-Integ) can be constructed by removing all endogenous coding sequences (bases 190-4034) from an infectious molecular clone of an adeno-associated virus (pAVl from ATCC, Rockville, MD.). The RSV long terminal repeat (LTR) driven sFv and the Neo gene under the control of the SV40 early promoter is then inserted into this virus.
  • LTR long terminal repeat
  • Semliki Forest virus vectors which are useful as transducing agents include, but are not limited to, pSFVl and pSFV3 -lacZ (Gibco-BRL) . These vectors contain a polylinker for insertion of foreign genes therein which is followed by a series of stop codons. The gene of choice is inserted into the polylinker region and viruses are generated using the in vi tro packaging helper virus system also provided by Gibco-BRL. Following the directions of the manufacturer and the disclosure contained herein, it is a relatively simple matter for one of skill in the art to generate Semliki Forest virus vectors capable of expressing the sFvs of the invention.
  • Transgenic animals also may be produced according to the invention.
  • a "transgenic animal” is an animal having cells that contain DNA which has been artificially inserted into a cell, which DNA becomes part of the genome of the animal which develops from that cell.
  • Preferred transgenic animals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogs and cats.
  • DNA can be injected into the pronucleus of a fertilized egg before fusion of the male and female pronuclei, or injected into the nucleus of an embryonic cell (e.g., the nucleus of a two-cell embryo) following the initiation of cell division (Brinster et al, Proc. Natl. Acad. Sci. USA 82: 4438-4442, 1985).
  • Embryos can be infected with viruses, especially retroviruses, modified to carry the nucleotide sequences of the invention which encode intracellularly expressed antibodies.
  • Pluripotent stem cells derived from the inner cell mass of the embryo and stabilized in culture can be manipulated in culture to incorporate nucleotide sequences of the invention.
  • a transgenic animal can be produced from such cells through implantation into a blastocyst that is implanted into a foster mother and allowed to come to term. Animals suitable for transgenic experiments can be obtained from standard commercial sources such as Charles River (Wilmington, MA) , Taconic (Germantown, NY) , Harlan Sprague Dawley (Indianapolis, IN), etc.
  • mice are induced to superovulate.
  • Females are placed with males, and the mated females are sacrificed by C0 2 asphyxiation or cervical dislocation and embryos are recovered from excised oviducts. Surrounding cumulus cells are removed. Pronuclear embryos are then washed and stored until the time of injection. Randomly cycling adult female mice are paired with vasectomized males. Recipient females are mated at the same time as donor females. Embryos then are transferred surgically.
  • transgenic rats The procedure for generating transgenic rats is similar to that of mice (see Hammer et al., Cell 63: 1099- 1112, 1990) .
  • a clone containing the sequence(s) of the invention is co- transfected with a gene encoding resistance.
  • the gene encoding neomycin resistance is physically linked to the sequence(s) of the invention.
  • Transfection and isolation of desired clones are carried out by any one of several methods well known to those of ordinary skill in the art (E.J. Robertson, supra) .
  • DNA molecules introduced into ES cells can also be integrated into the chromosome through the process of homologous recombination (Capecchi, Science 244: 1288-1292, 1989) . Methods for positive selection of the recombination event (i.e., neo resistance) and dual positive-negative selection (i.e.
  • neo resistance and gancyclovir resistance have been described by Capecchi, supra and Joyner et al., Nature 338: 153-156 (1989), the teachings of which are incorporated by reference herein.
  • the final phase of the procedure is to inject targeted ES cells into blastocysts and to transfer the blastocysts into pseudopregnant females.
  • the resulting chimeric animals are bred and the offspring are analyzed by Southern blotting to identify individuals that carry the transgene.
  • Intracellular immunization of humans against HIV-l may be accomplished by recovery of cells from the individual followed by ex vivo transduction of these cells with a construct of interest. Cells which are transduced are then returned to the individual by re-infusion.
  • the data presented herein provide a viable approach for the immunization of primary cells using constructs encoding anti-HIV-l sFv.
  • the use of recombinant antibody fragments, expressed intracellularly is especially promising as an approach to inhibit HIV-l replication.
  • the wide diversity and extraordinarily of antibody repertoires allows for timely and efficient targeting of various critical HIV-l specific proteins.
  • HIV-l resistant cells in combination with effective anti-retroviral pharmaceutical agents may arrest this rapid virion turnover (Wei et al. , nature 373:117-122, 1995; Ho et al. , Nature 373:123-126, 1995).
  • the invention should not be construed to be limited to the retroviral vectors disclosed nor be limited to the use of an anti-rev sFv.
  • other transduction vehicles capable of expressing anti-HIV-l sFvs including adeno-associated virus vectors, and Semliki Forest virus vectors may also be used (Muzyczka, Curr. Top.
  • viral vectors comprising sFvs directed against other HIV-l encoded proteins, for example, the HIV-l invertase (in) are also useful for intracellular immunization against HIV-l infection.
  • viral proteins which may be useful as intracellular immunization agents include sFvs directed against any other HIV-l specific function which when expressed in a cell expressing an anti-HIV antibody specific for that gene either diminishes or ablates or otherwise protects the cell and subsequently the human against infection with HIV-l.
  • the following examples are illustrative of the methods and compositions of the invention and are not meant to limit the invention in any way.
  • RNA was prepared from 5 x 10 7 hybridoma cells. The total RNA was used for first strand cDNA synthesis using 17 bp poly-T mixed with either the Vh or VI 3' primer at 42°C for 1 hour in 50 ⁇ l reaction mixture containing 100 ⁇ g of RNA and AMV reverse transcriptase 100 Units, with a standard buffer system. For amplification of vL and vH, 5 ⁇ l of cDNA was subjected to 35 cycles of PCR using reagents, as per the manufacturer's instructions (Gene Amp.
  • vL-5' or vH-5' primer obtained from Novagen, Inc., Madison, WI
  • vL-5' or vH-5' primer obtained from Novagen, Inc., Madison, WI
  • Each PCR cycle consisted of denaturation at 94°C for 1 minute annealing at 50°C for 90 sec, and polymerization at 72°C for 2 minutes, and finally a 10 minute extension.
  • the amplified vL and vH fragments were purified on 1.5% low- melting agarose with the Promega PCR magic purification kit (Madison, WI) .
  • Taq polymerase-amplified PCR products were directly ligated into a modified pT7Blue (R) vector (Novagen) , pT7H3-10, Figure 1, which carries extra T's at the 5' end.
  • R modified pT7Blue
  • pT7H3-10 Figure 1
  • recombinants were selected on X-gal plates.
  • 40 colonies were picked up for mini preparation of plasmid for further enzymatic digestion to check the size of the insert. Plasmids were further prepared for DNA sequencing. All of the plasmids were sequenced on a 373A ABI automatic DNA sequencer (ABI, Foster City, CA) . Finally, the plasmids were confirmed using the USB Sequenase Kit (United States Biochemical, Cleveland, OH) .
  • the original insert DNA sequence was confirmed to be a mutant endogenous K chain by computer homology searching. Because spO/2 myeloma cells have endogenous K chain expression, the Complementarity Determining Region (CDR) sequence specific for endogenous K chains is used for K chain PCR recombinant plasmid screening to eliminate the contamination of this K chain from the recombinant plasmids. Only less than 5% of the plasmid do not contain this K chain and those plasmids are DNA sequenced.
  • CDR Complementarity Determining Region
  • each of the cDNA fragments at least 3 different colonies are sequenced to confirm sequence.
  • Specific primer targeting-CDR derived from those cDNA sequences are designed to repeat RT-PCR for each of the parent hybridomas to confirm the sequence.
  • the first method is to eliminate ABKV RNA background by cleaving ABKV RNA directly with the ribozyme RNA system. This reduces the ABKV RNA RT-PCR background and enhances the specific Ig light chain RNA signal for cloning.
  • a 62 bp ABKV ribozyme DNA fragment was synthesized by PCR and inserted into vector pGEM4Z at the Hindlll-Bamll site to form the plasmid p4ZABVKRIBO ( Figure 2) . After the plasmid is linearized by BamHI digestion, the specific ABKV ribozyme can be synthesized with T7 RNA polymerase in vi tro as follows:
  • Ribozyme RNA can be resuspended in diethylpyrocarbonate (DEPC) treated water and stored at -70°C.
  • DEPC diethylpyrocarbonate
  • Total or polyA RNA which is extracted from the hybridomas and resuspended in 5 ⁇ l DEPC water, is mixed with
  • a second method for eliminating the endogenous ABVK chain is as follows: Manipulation of RNA for extended time periods or in multiple step processes may cause dramatic RNA degradation and affect the efficiency of RT-PCR.
  • This 62 bp ABVK ribozyme fragment into a new plasmid pSCCribo which may be transfected into the packaging cell line PA317 to produce an infectious but replication deficient virus ( Figure 3) .
  • the supernatants containing the virus can be used to introduce the ABVK ribozyme into any hybridoma cell line with high efficiency.
  • the CAT-ABVK ribozyme can specifically target the endogenous ABVK RNA resulting in cleavage. This dramatically reduces the ABVK RNA background and enhances the antigen specific hybridoma Ig light chain for RT-PCR (See Figure 4, Gel) .
  • vL fragments are produced by using commercially available filamentous phage vector systems.
  • the vector systems can concurrently produce free Fab fragments and Fab displayed on the surface of bacteriophage via a vHC H1 -pIII fusion protein. When expressed in a supo
  • the bacteriophage expression is carried out as specified in Barbas and Lerner, Methods: A Companion to
  • RT-PCR DNA encoding the Fd is inserted into a phage vector and transformed into host bacteria.
  • RT-PCR light chain DNA fragments from the same hybridoma are then inserted into the pComb3 vector.
  • the combinatorial libraries are treated to prepare phagemid.
  • Solid phase selection (panning) of the Fab against the antigen of interest proceeds as follows: Microtiter wells were coated with 9.5 ⁇ g of purified E. coli recombinant antigen (such as HIV-l-RT, Tat or Rev) , overnight at 4°C.
  • phage libraries typically >10 1:L colony-forming phage per well
  • the selected phage are then allowed to infect E. coli XL-IBlue cells and used to prepare a new phage stock by infection with the helper phage VCSM 13 (both from Stratagene, La Jolla, CA) .
  • helper phage VCSM 13 both from Stratagene, La Jolla, CA
  • phagemid containing XL-IBlue cells from the last panning against antigen is split in two and one half is packaged with CSM13 helper phage, the other half used to prepare phagemid DNA.
  • Phagemid DNA is digested with restriction enzymes to excise the genelll coding for the phage cap protein allowing the Fabs to be expressed in soluble form.
  • the religated DNA is retransformed into XL- IBlue cells and clone supernatants screened for Fab production by ELISA using microtiter wells coated with 0.1 ⁇ g of antigen, followed by clone supernatant, then goat anti-human F(ab) 2 conjugated to alkaline phosphatase, then alkaline phosphatase substrate.
  • Positive clones are then tested for specificity ' against a umber of different antigens (viral and human) by ELISA and phagemid DNA prepared from each clone.
  • the above methods have been applied to production of human viral neutralizing Fab to HIV-l, respiratory syncytial virus (RSV) , CMV, HSV-I and II (Burton et al.,
  • Ul and ACH2 cells were selected to ' test LXSN expression function.
  • the Ul cell line a U937-derived HIV-l infected clone, has been used as a model for viral latency, and the effects of monocyte-specific cytokines on the induction of
  • ACH2 is derived from an infected T-lymphocyte line which has one copy of provirus integration while Ul cells have two proviral copies. Both cell lines produce very low levels of HIV-l P24 expression and act as an HIV-l latency state. With different stimulation, such as PMA or TNF- ⁇ , both of these cells will increase HIV-l p24 by more than 1000-fold in 48 hours, and will produce infectious functional virus. Those cell lines provide good cell line model systems which not only represent both T-lymphocyte and macrophage type cells but also represent most of the HIV-l infected cellular populations.
  • a 734 bp CAT fragment was inserted into the pLXSN vector (MuLV retrovector) .
  • This pLXSNCAT plasmid ( Figure 5) was transfected into the packaging cell line PA 317 and selected in G418 (1 mg/ml) .
  • 1 x 10 6 PA317 cells were plated in 100mm dishes in 10 ml Dulbecco's Modified Eagles Medium (DMEM) + 10% Fetal Calf Serum (FCS) one day before transfection. Three hours before transfection, the 10 ml of medium was replaced with 10 ml of fresh prewarmed medium. 20 ⁇ g pLXSNCAT was transfected into pA317 cells.
  • DMEM Dulbecco's Modified Eagles Medium
  • FCS Fetal Calf Serum
  • the cultured medium containing the virus was collected and passed through a 0.45 ⁇ m filter to- prepare cell free virus.
  • the medium was mixed with 3 x 10 6 Ul or ACH2 cells with 8 ⁇ g/ml polybrene to help increase efficiency and incubated for 12-16 hours. Cells were then washed twice with serum free medium and resuspended in 10 ml RPMI 1640 with 10% FCS for further culture. From this test, we can detect CAT activity after 48 hours transfection without non-specific stimulation of HIV replication (i.e. maintained at the same level of p24 as prior to superinfection) .
  • a single chain sFv anti-rev antibody was constructed consisting of variable domains of the heavy (vH) and light (vL) chains of a murine monoclonal antibody against HIV-1 IIIB ) rev (the "parent antibody”).
  • Protocols for constructing the vH and vL regions are as follows:
  • the CDR region was compared by computer with the published Ig protein sequences. The full length sequence was then designed. First two synthesized oligonucleotides were used to create a linker DNA fragment with Apal-Bglll sites. This was then cloned into pT7/Blue (R) vector in order to determine the DNA sequence. vH and vL were then reamplified with two new pairs of oligonucleotides with suitable enzyme sites at both ends, cloned into pT7/Blue (R) . After verifying the DNA sequence, the linker DNA, N- GGGGSGGGGSGGGGS-C (Sequence I.D.
  • the DNA sequence of the sFv anti-rev was determined to be as follows (Sequence I.D. Number 1):
  • Rev is one of the essential regulatory proteins of Human Immunodeficiency Virus. It is a 19kD phosphoprotein localized primarily in the nucleolus/nucleus, and acts by binding to Rev Responsive Element (RRE) and promoting the nuclear export, stabilization and utilization of the viral mRNA's containing RRE.
  • RRE Rev Responsive Element
  • the binding affinity of the sFv anti-rev produced in E. coli was then determined by using an ELISA (Enzyme Linked Immunoassay) utilizing recombinant rev conjugated with biotin.
  • the binding affinity was approximately 10 "7 which was comparable to the affinity of the present antibody.
  • the binding efficiency was determined as follows: -" Purified E. coli derived sFv anti-rev was diluted in Phosphate Buffered Saline (PBS) solution at 200 ⁇ g/ml. ELISA plate wells were coated with 200 ⁇ l per well of this solution, overnight at 4°C. The same concentration of BSA/PBS was used for coating control wells. Wells were washed once with PBS and blocked by the addition of 10% BSA/PBS, 200 ⁇ l/well. After blocking for 1 hour at 37°C wells were washed three times with 0.5% Tween 20/PBS.
  • PBS Phosphate Buffered Saline
  • E. coli derived rev has an additional 12 aa leading sequence (Sequence I.D. number 3).
  • the sFv gene was inserted into XhoIBamHI site on the vector. It was driven by the RSV-LTR promoter.
  • the HIV-Tat nuclear translocation signal DNA was cloned by PCR.
  • the HIV Tat cDNA was amplified with two oligo primers and was then ligated into pT7 Blue(R) vector and sequenced.
  • the amino acid sequence of the signal is: N-GRKKRRQRRRAHQN-C (Sequence I.D. number 4) .
  • the corresponding DNA sequence is: 5' GGC AGG AAG AAG CGG AGA CAG CGA CGA AGA GCT CAT CAG AAC AGT CAG ACT 3' (Sequence I.D. number 5).
  • Hela-T4's Hela cells expressing CD4 (Hela-T4's) were then transfected with the pREP 4 -sFv construct which also contained tk driven neomycin resistant gene as a marker. After transfection, the Hela-T4's were incubated with neomycin (G418) to enrich the population of sFv expressing cells. sFv expressing cells and non-transfected Hela-T4's
  • Figure 11 shows that the sFv specifically binds a highly conserved rev domain.
  • the HeLa T4 cells expressed sFv resistance to all of the tested clinically isolated strains of HIV-l.
  • Example 5 Human Lymphocyte RNA Preparation Five milliliters of bone marrow was removed by aspiration from an long term asymptomatic HIV-l positive donor. Immediately, 10 ml of 3M guanidium isothiocyanate containing 71 ⁇ l of 2-mercaptoethanol was added and then RNA was prepared by standard methods.
  • RNA typically 10 ⁇ g was reverse- transcribed as described by Burton, et al. Proc. Natl . Acad. Sci . , U. S.A . , 88, 10134-10137 (1991), incorporated by reference herein in its entirety and ⁇ l (Fd region) and /. ' chains were amplified by PCR.
  • the resulting ⁇ l heavy chain DNA was cut with an excess of the restriction enzymes Xho I and Spe I and typically about 350 ng was ligated with 2 ⁇ g of Xho I/Spe I-linearized pComb3 vector (isolated by agarose gel electrophoresis) in a total volume of 150 ⁇ l with 10 units of ligase (BRL) at 16°C overnight.
  • BRL ligase
  • DNA was precipitated at -20°C for 2 hr by the addition of 2 ⁇ l of 2% (wt/vol) glycogen, 15 ⁇ l of 3 M sodium acetate (pH 5.2), and 330 ⁇ l of ethanol. DNA was pelleted by microcentrifugation at 4°C for 15 minutes.
  • the DNA pellet was washed with cold 70% ethanol and dried under vacuum. The pellet was resuspended in 10 ⁇ l of water and transformed by electroporation into 300 ⁇ l of Escherichia coli XLl-Blue. After transformation, 3 ml of SOC medium (20mM glucose pH 7.0, 2% bacto-tryptone, 0.5% yeast extract, 0.05% NaCl 2 , 2.5mM KC1) was added and the culture was shaken at 220 rpm for 1 hr at 37°C after which 10 ml of SB (super broth; 30 g of tryptone, 20 g of yeast extract, and 10 g of Mops per liter, pH 7) containing carbenicillin (20 ⁇ g/ml) and tetracycline (10 ⁇ g/ml) was added.
  • SOC medium 20mM glucose pH 7.0, 2% bacto-tryptone, 0.5% yeast extract, 0.05% NaCl 2 , 2.5mM KC1
  • SB super broth; 30
  • samples (20, 1, and 0.1 ⁇ l) were withdrawn for plating to determine the library size.
  • the library had about 10 7 members.
  • the culture was grown for an additional hour at 37°C while shaking at 300 rpm. This culture was added to 100 ml of SB containing carbenicillin (50 ⁇ g/ml) and tetracycline (10 ⁇ g/ml) and was grown overnight. Phagemid DNA containing the heavy-chain library was prepared from this overnight culture. To determine the insert frequency of this ligation, 10 colonies from the plates used to titer the library were picked and grown. DNA was prepared and then digested with Xho I and Spe I.
  • phagemid DNA (pcomb3) (10 ⁇ g) was digested as described above except that the restriction enzymes Sac I and Xba I were used.
  • the resulting linearized vector was treated with phosphatase and purified by agarose gel electrophoresis. The desired fragment, 4.7 kilobases long, was excised from the gel. Ligation of this vector with prepared light-chain PCR DNA proceeded as described above for the heavy chain. After transformation, 3 ml of SOC medium was added and the culture was shaken at 220 rpm for 1 hour at 37°C.
  • kanamycin 70 ⁇ g/ml was added and the culture was incubated at 37°C overnight. The supernatant was cleared by centrifugation (400C rpm for 15 minutes in a JA- 10 rotor) at 4°C. Phage were precipitated by addition of 4% (wt/vol) polyethylene glycol 8000 and 3% (wt/vol) NaCl followed by incubation on ice for 30 minutes and centrifugation. Phage pellets were resuspended in 2 ml of phosphate-buffered saline (PBS: 50 mM phosphate, pH 7.2/150 mM NaCl) and microcentrifuged for 3 minutes to pellet debris. Supernatants were transferred to fresh tubes and stored at -20°C.
  • PBS phosphate-buffered saline
  • Example 8 Panning of the Combinatorial Library to Select Antigen Binders
  • Four wells of a microtiter plate (Costar 3690) were coated overnight at 4°C with 25 ⁇ l of recombinantly produced rev or tat protein (40 ⁇ g/ml in 0.1 M bicarbonate., buffer, pH 8.6) .
  • the wells were washed twice with water and blocked by completely filling the well with 1% (wt/vol) bovine serum albumin (BSA) in PBS and incubating the plate at 37°C for 1 hour.
  • Blocking solution was shaken out, 50 ⁇ l of the phage library (typically 10 11 colony-forming units) was added to each well, and the plate was incubated at 37°C for 2 hours.
  • BSA bovine serum albumin
  • elution buffer 0.1 M HCl, adjusted to pH 2.2 with solid glycine and containing 0.1% BSA
  • Phagemid DNA from positive clones was isolated and digested with Spe I and Nhe I. Digestion with these enzymes produces compatible cohesive ends.
  • the 4.7-kilobase D ⁇ A fragment lacking the gene III (cap protein) portion was gel-purified (0.6% agarose) and self-ligated.
  • Transformation of E. coli XLl-Blue afforded the isolation of recombinants lacking the gene III (cap protein) fragment. Clones were examined for removal of the gene III fragment by Xho I/Xba I digestion, which yielded a 1.6- kilobase fragment. Clones were grown in 15 ml of SB containing carbenicillin (50 ⁇ g/ml) and 20 mM MgCl 2 at 37°C until OD 600 of 0.2 was achieved.
  • Isopropyl j ⁇ -D-thiogalactopyranoside (ImM) was added and the culture was incubated overnight at 37°C.
  • Cells were pelleted by centrifugation at 4000 rpm for 15 minutes in a JA-10 rotor (Beckman J2-21) at 4°C.
  • Cells were resuspended in 3 ml of PBS containing 0.2 mM phenylmethylsulfonyl fluoride and lysed by sonication on ice (2-4 min, 50% duty) .
  • the debris was pelleted by centrifugation at 14,000 rpm in a JA-20 rotor at 4°C for 15 minutes. The supernatant was used directly for ELISA analysis and was stored at -20°C.
  • ELISA wells were coated with rev and tat proteins exactly as above, washed five times with water, blocked in 100 ⁇ l of 1% BSA/PBS for 1 hour at 37°C, and then incubated with 25 ⁇ l Fab supernatants for 1 hour at 37°C. After 10 washes with water, 25 ⁇ l of a 1:1000 dilution of alkaline phosphatase-conjugated goat anti-human IgG F(ab') 2 (Pierce) was added and incubated for 1 hour at 37°C. Following 10 washes with water, 50 ⁇ l of p-nitrophenyl phosphate substrate was added and color development was monitored at 405 nm. Positive clones gave A 405 values >1 (mostly >1.5) after 10 minutes, whereas negative clones gave values of 0.1-0.2.
  • VL5-FR1 (VL5-FR1, /GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAXSS
  • ELISA assays were performed as described above using defined peptides of the tat and rev proteins set forth in Tables 2 and 3, respectively.
  • the anti-tat Fab bound to the cysteine rich tat functional domain as shown in Figure 12. Reduction of the antibody reduced binding of the Fab to the functional domain as shown in Figure 13.
  • Binding of anti-rev Fd and Fab is shown in Figure 14.
  • the anti-rev Fd rev9 bound to the sequence immediately adjacent to the basic nucleolar localization domain.
  • Anti- rev Fab revl6 and rev20 were found to be identical and binding was evident to the region immediately adjacent to the activation domain.
  • Examples 13 to 23, provided below, comprise a description of experiments establishing that intracellular immunization of cells can be successfully accomplished using retroviral vectors encoding sFvs directed against essential HIV-l genes.
  • these data provide further support for the feasibility of intracellular immunization as a viable means of treating HIV infection.
  • the data describe the development and characterization of amphotropic murine retroviral shuttle vectors which express intracellularly anti-rev sFv. Using these vectors, human T-lymphotrophic cell lines were transduced with anti-rev sFV and were thereby intracellularly immunized against infection with
  • PBMC peripheral blood mononuclear cells
  • Example 13 Cells and viruses used to generate and test retroviral vectors encoding sFv
  • HIV-l viral strains utilized in the experiments described herein include HIV-l strain NL4-3
  • NL4-3 in which all viral open reading frames are intact and is highly cytopathic for T-lymphocytes
  • HXB2 were produced from transfected Cos cells and propagated in the T-lymphocytic cell-line, CEM.
  • Viral stocks were assayed for their infectious titers on both CEM and H9 cells (Aldovini et al., In: Techniques in HIV Research, Stockton Press, NY, 1990) .
  • HIV-l viral construct containing the CAT gene inserted into the nef open reading frame (Malim et al., J. Exp. Med. 176:1197-1201, 1992), was also used in some experiments as indicated herein.
  • CEM, H9, and Sup-Tl cells are CD4-positive human T-lymphocytic cell lines, which are highly susceptible to infection with HIV-l (Aldovini et al., In: Techniques in HIV Research, Stockton Press, NY, 1990) . These cells were grown in RPMI-1640 medium, supplemented with 10% fetal calf serum (FCS) (Gibco-BRL) . Retroviral packaging cells, PA317 (Miller et al., Biotechniques 7:980-990, 1989), were maintained in Dulbecco's modified Eagle's medium (DMEM) in the presence of 10% FCS.
  • DMEM Dulbecco's modified Eagle's medium
  • Human PBMC were prepared via Ficoll-Hypaque centrifugation of blood from HIV-1- seronegative individuals.
  • the PBMC were stimulated with phytohemagglutinin (PHA) (5 ⁇ g/ml - Sigma) and interleukin- II (IL-2) (50 U/ml - Sigma) for 3 days, following which the PBMC were maintained in RPMI-1640 medium, supplemented with 20% FCS and IL-2 (10 U/ml) .
  • PHA phytohemagglutinin
  • IL-2 interleukin- II
  • LUV murine leukemia virus
  • pLXSN Two murine leukemia virus (MLV) -based retroviral shuttle vectors, pLXSN and PSLXCMV, were used in the experiments described herein.
  • These expression vectors contain the bacterial neomycin-resistance gene (neo) (Miller et al., Biotechniques 7:980-990, 1989) . Genes of interest were inserted in the polylinker regions of these vectors (see Figure 15) .
  • 3-Galactosidase (3-Gal) and CAT-expression retroviral vectors, PLXSP-Gal and PLXSCAT were constructed as described herein.
  • PSLXCMV-CAT was constructed by inserting a 726 bp Hind III (blunt) -Bam HI fragment, containing the CAT gene into pSLXC via Hpa I-Bgl II sites.
  • the anti-rev sFv (D8) moiety used in these studies constructed from the Vj and V h cDNA obtained from a murine hybridoma, is also described herein.
  • anti-rev (D8) sFv expression a 869 bp Xho I-Bam HI sFv-containing fragment was inserted into the Xho I-Bam HI sites of pLSXN, to form pLSXN-D8-SFv.
  • a Xho I (blunt) -Bam HI sFv-containing fragment was ligated into a Hpa I-Bgl II treated pSLXCMV vector, to form pSLXCW-D8-sFv.
  • Helper-free, recombinant MLV viral stocks were produced by transient transfection of retroviral vector plasmids into PA317 cells, which were used in all experiments to produce the various retroviral shuttle vector virions. Briefly, 5 ⁇ g of purified plasmids were transfected into 1 X 10 6 PA317 cells/100 mm dish, using a standard lipofectamine reagent procedure (Gibco-BRL) . Five hours later, 10 ml of pre-warmed DMEM supplemented with 20% FCS was added to the cells. At 48 hours post-transfection, the supernatants were harvested by centrifugation at 1500 g for 5 minutes.
  • transfected PA317 cell supernatants For transduction of CEM and Sup-Tl cells, 5 ml of the transfected PA317 cell supernatants were used to infect 1-2 X 10 6 target cells with polybrene (8 ⁇ g/ml) overnight. Cells were then washed with serum free media and maintained in G418 (400 ⁇ g/ml) for 2 days. Clonal cell lines were isolated by G418 selection and limiting dilution. In addition, mixed cellular populations were isolated by continuous culture in G418 (1 mg/ml) -containing medium for 2 weeks.
  • PBMC For human PBMC, after 3 days of PHA/IL-2 stimulation (PHA: 5 ⁇ g/ml, IL-2: 50 U/ml), 1 X 10 6 cells were cultured with 10 ml of transfected PA317 supernatant and incubated for 3 days with daily replacement of fresh packaging cell line supernatant. The transduced PBMC were then challenged with HIV-l.
  • PHA PHA/IL-2 stimulation
  • IL-2 50 U/ml
  • Example 15 Cat Assays CAT assays were performed as described (Gorman et al., Mol. Cell. Biol. 2:1044-1051, 1982; Ausebel et al. , Current Protocols in Molecular Biology, Volume 1, John Wiley and Sons, NY) . Briefly, 1 X 10 7 cells were transduced using the CAT-expression retroviral vectors, and were harvested by centrifugation. The pelleted cells were lysed in 0.9 ml CAT lysis buffer (Promega, Inc.) and approximately 200 ⁇ l of supernatants, normalized for protein content, were used in standard CAT assays. Percent conversion of chloramphenicol was assessed using a phosphor imager (Molecular Dynamics, Inc.) .
  • PCR cycles were: 94°C for 1 minute and 20 seconds, 50°C for 2 minutes and 72°C for 1 minute and 30 seconds, for 35 cycles with a final extension for 10 minutes at 72°C.
  • PCR amplification products were resolved on 1.5% agarose gels.
  • Intracellular sFv gene expression and protein localization was detected by indirect immunofluorescence assays (Bagasra et al. , Proc. Natl. Acad. Sci. USA 89:6285- 6289, 1992) . Briefly, retroviral vector- ransduced cloned cells were cultured on multichambered glass slides overnight. After removing the culture media, the cells were fixed in 10% methanol and 90% ethanol at -20°C for 2 hours. Cells were then washed twice with PBS and blocked with 5% normal goat serum for 1 hour at 37°C.
  • Cells were further overlayed with 200 ⁇ l of polyclonal rabbit anti-mouse IgG (Fab-specific) (Sigma), for 2 hours at 37°C. After washing five times with PBS, the cells were incubated with fluorescein isothiocyanate (FITC) -conjugated goat anti- rabbit IgG for 1 hour. Following an additional five washes in PBS, the cells were analyzed by epifluorescence microscopy.
  • FITC fluorescein isothiocyanate
  • Example 17 Infection of cells with HIV-l Viral stocks of the HIV-l strains, HXB2 and NL4-3 were used in these studies. G418-selected CEM and Sup-Tl cells were first maintained in G418-free medium for two weeks prior to HIV-l infection. Also prior to infection, cell-surface CD4 antigen expression was analyzed by flow cytometry (see below) . Parental CEM and Sup-Tl cells and CAT-transduced and sFv-transduced CEM and Sup-Tl cells were incubated with infectious HIV-l strains HXB2 and NL4-3 at various input multiplicities of infection for 2 hours following which the cells were washed four times in pre ⁇ warmed, serum-free media.
  • Human PBMC were challenged with HIV-l strain NL4-3 using mois of 0.24 and 0.06.
  • Cells were cultured at an initial concentration of 5 X 10 s cells/ml in RPMI-1640 medium supplemented with 10% FCS for CEM and Sup- Tl cells, and 20% FCS for human PBMC.
  • the cells were split at a ratio of 1:4 to maintain a cell concentration of approximately 5 X 10 5 /ml.
  • the culture supernatants were collected every three days after infection and the level of HIV-l p24 antigen in the supernatants was determined by an HIV-l antigen capture ELISA. Cell viability was monitored by trypan blue exclusion staining.
  • the vector pSLXCMV in which CAT gene expression is driven by an internal cytomegalovirus (CMV) promoter, yielded higher levels of CAT in human PBMC compared with PBMC transduced with pLSXN. These data correlate with those obtained in the HIV-l challenge experiments described below.
  • CMV cytomegalovirus
  • G418-selected, sFv-transduced CEM and Sup-Tl cellular clones were maintained in G418-free media for two months and then assayed for the presence and expression of sFv using immunofluorescence and a DNA- and reverse transcriptase (RT) -PCR assay.
  • RT DNA- and reverse transcriptase
  • Example 21 Inhibition of HIV-l Replication by Anti-Rev SFv in Human PBMC
  • the vector pSLXCMV- D8-sFv may be an even more potent inhibitor of HIV-l at the higher moi in PBMCs compared with the vector pLXSN-D8-sFv.
  • These challenge experiments were performed using fresh PBMC from several different HIV-1-seronegative donors. In each instance, the results obtained were similar.
  • potent inhibition of HIV-l replication by an intracellularly expressed sFv which has as its target a retroviral regulatory protein is now demonstrated in human PBMC.
  • Example 22 Inhibition of HIV-l Replication in sFv-Transduced Cells is Secondary to a Specific Block in Rev Function
  • HIV-l clone HIV-CAT encodes the CAT gene within the nef open reading frame and, thus, CAT is expressed as an early gene product from multiply-spliced viral RNA through a rev- independent mechanism in cells infected with this virus.
  • HIV-CAT expression in different clonal and mixed cell populations clearly establish that the transduced cells are equally sensitive to HIV-l infection at the early stages of virus replication ( Figure 23B) .
  • Example 23 Inhibition of replication of primary isolates of HIV-l in anti-rev transduced human PBMC Human PBMC were stimulated by treatment with 5 ⁇ l/ml of PHA and 50 ⁇ l/ml of IL-2 for three days.
  • Samples of 1 X 10 6 cells so stimulated were cultured in the presence of 10 ml of transfected packaging cell line PA317 supernatant for 3 days with daily replacement of the supernatant.
  • the cells were transduced with the vectors LXSNSF8 . and LXSNCAT as described herein.
  • the transduced PBMC which were cultured in IL-2 alone were then challenged with one of two clinical isolates of HIV-l. These isolates, HIV-l strain NSI #89000641 and HIV-l strain SI #9200611, were isolated from the PBMC of two infected children.
  • strains are resistant to AZT; further, strain NSI #89000641 does not induce syncytium formation whereas strain SI #9200611 induces syncytium formation in infected cells.
  • 100 pg of HIV-l p24 equivalents of virus was mixed with the transduced PBMC for 2 hours at 37°C following which the cells were washed five times in 10 ml of prewarmed serum free medium. The cells were then resuspended in 2 ml of culture medium containing IL-2 following which they were maintained at a density of 0.2 to 0.5 X 10 6 cells/ml by splitting the cells at regular intervals.
  • Example 24 Cloning and characterization of sFvs directed against HIV-l integrase (IN)
  • the HIV-l integrase (IN) gene plays an essential role in the virus replication cycle in that it mediates integration of the viral genome into the host cell genome.
  • the 288. amino acid integrase protein has three domains. The amino terminal domain has a zinc finger-like motif of two histidines followed by two cysteines (HHCC domain) .
  • the central domain comprises the catalytic center containing a highly conserved D,D-35-E motif.
  • the carboxyl terminal domain binds to DNA and is required for efficient 3' processing and DNA strand transfer.
  • anti-IN monoclonal antibodies have been generated which are specific for each of the domains within the IN protein.
  • a diagram of the IN gene is presented in Figure 25.
  • the relative positions of the domains of the protein are also shown as are the relative positions on the molecule to which each monoclonal antibody is directed. Also shown in this figure is the relative binding affinities of each of the monoclonal antibodies to the appropriate domains on IN and the relative ability of each of these antibodies to inhibit IN function is also shown.
  • Vh heavy chain variable genes
  • VI light chain variable genes
  • PCR amplification of cDNA using 3' and 5' Vh primers generated a 450 bp DNA fragment which was then cloned into the T7 Blue (R) vector in order that it be sequenced.
  • VI domain was cloned in a similar manner with the following exceptions: Since the parent hybridoma cell line sp2/0 contains an aberrant variable kappa chain (abVk) , reduction of the level of the abVk mRNA level was accomplished by cocultivation of the hybridoma cell lines with pA317 cells transiently expressing MLV which, as disclosed herein, encodes an abVk specific ribozyme.
  • VI first strand cDNA was synthesized using a 3' VI primer and isolated ribozyme treated hybridoma RNA. Light chain cDNA was amplified by PCR using 3' VI and 5' Vl primers.
  • Progeny clones were screened by PCR for the presence of the abVk sequence using specific abVk primers (200 bp product) . Clones which were negative for abVk were screened by PCR for VI sequences using T7 and U19 pT vector primers (Novagene) (500 bp product) . All of the Vh and VI clones which were obtained were sequenced in both orientations to identify novel anti- IN antibody sequences. Anti-IN sFv molecules were generated by simply replacing rev in the anti-rev sFv constructs disclosed above with the cloned anti-IN sequences. PCR amplification was used to add convenient restriction enzyme sites which are either 5' or 3' to each VI and Vh domain.
  • Each of these anti-IN sFv molecules was sequenced in order to insure that they did not contain additional mutations resulting from the cloning procedure.
  • Each of the anti-IN sFv was cloned into the following expression vectors: pSLX-CMV and pSLXN, retroviral expression vectors; pET-19b, a bacterial expression vector and pSFVl, a high copy expression vector.
  • pSLX-CMV and pSLXN retroviral expression vectors
  • pET-19b a bacterial expression vector
  • pSFVl a high copy expression vector.
  • GGAGGCACCA AGCTGGAAAT CAAACGGGCT GATGGGCCCG GTGGGGGCGG TTCGGGTGGC 480
  • Pro Arg Arg Trp Thr Gin Leu Trp lie Pro Pro Asp Tyr Trp Gly Gin 100 105 110
  • Pro Arg Arg Trp Thr Gin Leu Trp lie Pro Pro Asp Tyr Trp Gly Gin 100 105 110

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Abstract

Méthode d'exécution d'une thérapie génique qui consiste à utiliser un gène de recombinaison qui code un anticorps fixant un antigène associé à une maladie. La méthode selon l'invention est particulièrement utile pour conférer à des cellules une 'immunité' contre des pathogènes intracellulaires. L'invention porte aussi sur des nouveaux vecteurs et lignées cellulaires.
PCT/US1996/007393 1995-05-23 1996-05-23 Immunisation intracellulaire WO1996037234A1 (fr)

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Cited By (7)

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WO2000015259A1 (fr) * 1998-09-10 2000-03-23 The University Of Virginia Patent Foundation Anticorps anti-c3b(i) pouvant diffuser des agents diagnostiques ou therapeutiques dans des cellules cancereuses
WO2001059142A1 (fr) * 2000-02-09 2001-08-16 Medimmune, Inc. Therapie genique a anticorps avec vecteurs viraux adeno-associes
US6572856B1 (en) 1998-09-10 2003-06-03 The University Of Virginia Patent Foundation Methods for the prevention and treatment of cancer using anti-C3b(i) antibodies
WO2003016354A3 (fr) * 2001-08-16 2003-11-06 Morten Steen Hanefeld Dziegiel Anticorps de recombinaison anti-plasmodium falciparum
US7265095B1 (en) * 1999-09-20 2007-09-04 Aberdeen University Monoclonal antibody 3F1H10 neutralising VHSV (viral haemorrhagic septicaemia virus)
US11697801B2 (en) 2017-12-19 2023-07-11 Akouos, Inc. AAV-mediated delivery of therapeutic antibodies to the inner ear
US12365726B2 (en) 2020-12-01 2025-07-22 Akouos, Inc. Anti-VEGF antibody constructs

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CURR. TOP. MICROBIOL. IMMUNOL., 1992, Vol. 158, MUZYCZKA N., "Use of Adeno-Associated Virus as a General Transduction Vector for Mammalian Cells", pages 97-129. *
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PROC. NATL. ACAD. SCI. U.S.A., June 1994, Vol. 91, CHEN S.-Y. et al., "Combined Intra- and Extracellular Immunization Against Human Immunodeficiency Virus Type 1 Infection with a Human Anti-gp120 Antibody", pages 5932-5936. *
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000015259A1 (fr) * 1998-09-10 2000-03-23 The University Of Virginia Patent Foundation Anticorps anti-c3b(i) pouvant diffuser des agents diagnostiques ou therapeutiques dans des cellules cancereuses
US6572856B1 (en) 1998-09-10 2003-06-03 The University Of Virginia Patent Foundation Methods for the prevention and treatment of cancer using anti-C3b(i) antibodies
US7265095B1 (en) * 1999-09-20 2007-09-04 Aberdeen University Monoclonal antibody 3F1H10 neutralising VHSV (viral haemorrhagic septicaemia virus)
WO2001059142A1 (fr) * 2000-02-09 2001-08-16 Medimmune, Inc. Therapie genique a anticorps avec vecteurs viraux adeno-associes
WO2003016354A3 (fr) * 2001-08-16 2003-11-06 Morten Steen Hanefeld Dziegiel Anticorps de recombinaison anti-plasmodium falciparum
US7811569B2 (en) 2001-08-16 2010-10-12 Institut Pasteur Recombinant anti-Plasmodium falciparum antibodies
US11697801B2 (en) 2017-12-19 2023-07-11 Akouos, Inc. AAV-mediated delivery of therapeutic antibodies to the inner ear
US12077783B2 (en) 2017-12-19 2024-09-03 Akouos, Inc. AAV-mediated delivery of antibodies to the inner ear
US12275960B2 (en) 2017-12-19 2025-04-15 Akouos, Inc. AAV-mediated delivery of therapeutic antibodies to the inner ear
US12365726B2 (en) 2020-12-01 2025-07-22 Akouos, Inc. Anti-VEGF antibody constructs

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