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WO1994006813A1 - Molecules de systeme majeur d'histocompatibilite et leurs modifications - Google Patents

Molecules de systeme majeur d'histocompatibilite et leurs modifications Download PDF

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Publication number
WO1994006813A1
WO1994006813A1 PCT/US1993/008715 US9308715W WO9406813A1 WO 1994006813 A1 WO1994006813 A1 WO 1994006813A1 US 9308715 W US9308715 W US 9308715W WO 9406813 A1 WO9406813 A1 WO 9406813A1
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Prior art keywords
molecule
modified
fragment
major histocompatibility
histocompatibility complex
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PCT/US1993/008715
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English (en)
Inventor
Jan Geliebter
Krishna V. Kesari
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The Rockefeller University
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Priority to AU49235/93A priority Critical patent/AU4923593A/en
Publication of WO1994006813A1 publication Critical patent/WO1994006813A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • 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

  • the present invention relates to modifications of MHC to modulate epitope recognition by the immune system. This may take into account both endogenous and foreign molecules, and may involve altering the immunogenicity thereof or the immune recognition and response thereto.
  • antigens are "presented" to the immune system by antigen presenting cells (APCs) , including, for instance, macrophages and B-cells in the context of major histocompatibility complex molecules (MHC) which are present on the APC surface.
  • APCs antigen presenting cells
  • MHC major histocompatibility complex molecules
  • Modification of MHC molecules and modulation of the normal recognition thereof changes the recognition and reactivity of otherwise non-recognized or weakly recognized epitopes.
  • tumor-specific antigens are, for example, known to elicit little or no antibody, cytotoxic or other immune response, or may elicit only a weak response. It has therefore been difficult or impossible, for instance, to raise antibodies or cytotoxic T-cells against these antigens, and the treatment of tumors immunologically has met with little success.
  • the present invention involves modifications of MHC molecules to increase or decrease the immune system recognition of antigens which are presented in the context of MHC. By altering the MHC molecu-te of interest, one may raise or suppress an immune response to molecules which may be present or newly introduced in non-modified form. Altered MHC molecules may effect the presentation or recognition of different molecules to which the immune system is ordinarily unresponsive or ineffective. The epitope may be essentially unrecognized or inactive. Similarly, such unmodified molecules may be weakly recognized or responded to by the immune system under normal conditions.
  • One object of the present invention is thus to modulate the immune system recognition of undesirable components, e.g., tumor tissue or cells.
  • Another object of the present invention is to differentiate immunologically between tumor and non-tumor cells.
  • Another object of the present invention is to utilize allogenic MHC molecules which can be expressed on tumor cells by introducing the gene for allogenic MHC into the tumor by any of various methods, e.g. , transfection, electroporation and lipofection.
  • Another object of the present invention is to provide an immunologically based treatment modality for undesirable components, e.g., tumors.
  • a modified MHC molecule which is modified by including at least one pair of crosslinkable residues in a domain thereof, which pair of crosslinkable residues crosslink, or by crosslinking residues capable of being crosslinked in a domain thereof.
  • nucleotide sequence which codes on expression for the MHC molecule modified to include at least one pair of crosslinkable residues, along with a plasmid containing the nucleotide sequence.
  • fragments of the MHC polypeptide molecule which react with antigenic molecules are coded by the nucleotide sequences noted above, fragments of the gene sequence which code MHC in modified form, ribonucleotide containing derivatives of the gene coding modified MHC, sense and anti-sense molecules which correspond to the DNA and RNA molecules and fragments, and various methods of preparation and use.
  • fragments of the MHC contain the pair of crosslinkable residues in crosslinked form.
  • a method of treating a tumor or infection in a mammal comprising administering the modified MHC molecules described above to said mammal, as well as various methods of analysis, diagnosis, detection and prevention, and compositions and kits for performing such methods.
  • FIGURE 1 is a diagram of the structure of the MHC class I polypeptide in relation to a cell membrane
  • FIGURE 2 is depiction of an MHC molecule with the alpha-1 (al) and alpha (a2) helices and the beta pleated sheet configuration. Two alpha helices are seen atop seven antiparallel beta strands. The position of the natural a2 disulfide bond is shown between residues 101 and 164. The site directed mutagenized cysteine residues 11 and 74 are shown in a proposed disulfide bond.
  • FIGURE 3 is a map of the exon structure of the Kb gene with the Xma 1 restriction sites designated. Eight exons of the Kb gene are indicated with numbers on top. Exon 1 encodes the leader peptide, exon 2 encodes the al domain, etc. The Xma 1 sites that excise exon two are indicated with arrows.
  • FIGURE 4 is a depiction of the oligonucleotide sequences used, with additional Xma I site situated approximately 60 basepairs 3* to the Xmal site shown in the intron between exons 2 and 3, for site directed mutagenesis.
  • FIGURE 5 is an autoradiogram of the mobility of 35 S- synthetically labelled KDC and Kb under reducing and non- reducing conditions using a 14% SDS-PAGE gel. Lanes 1 and 2 were run under reducing conditions. Lanes 3 and 4 were run under non-reducing conditions. Lanes 1 and 3 contain Kb and lanes 2 and 4 contain DC.
  • FIGURE 6 is a FACS analysis of P815, P815-DC and P815-Kb.
  • A-I Eight monoclonal antibodies specific for the Kb molecule and one irrelevant (141-30) monoclonal antibody were incubated with the various cells for thirty minutes on ice, and then washed two times. Cells were then incubated with FITC conjugated Goat anti-mouse Ig for thirty minutes on ice, washed three times and analyzed on a Becton-Dickenson FACSCAN.
  • FIGURE 7 is a graph of cell lysis, Measured by 51- Chromium release over 5 hours as a result of polyclonal activity exhibited by cytotoxic T-lymphocytes (CTL) with both P815-Kb and P815-DC. DBA/2 anti C57BL/6 (B6) activity was assessed. H2 d anti H-2 b bulk CTL were generated by standard MLC conditions, and assayed for cytotoxic reactivity on the indicated 51 Cr labeled target cells at various effector: target ratios.
  • CTL cytotoxic T-lymphocytes
  • FIGURE 8 is a bar graph of cell lysis as measured by Chromium release over 5 hours by primary CTLs restimulated in a secondary MLC against P815-DC. Effector cells from the MLC of Figure 7 were incubated for 5 days with X-irradiated P815-DC cells at a 1:1 ratio and assayed for reactivity on the various targets at a 20:1 effector:target ratio. Secondary DBA/2 anti P815-DC CTL activity is the same on P815-DC and P815-Kb.
  • FIGURE 9 is a graph of tumor progression in DBA/2 mice injected with P815 tumor cells as compared to those injected with p815 tumor cell line expressing the Kb-DC gene. Mice were inoculated with one million tumor cells subcutaneously in the right flank. Mice that were killed when the tumor load was too large were still counted as tumor bearing after their death. Six mice were in each group.
  • FIGURE 10 contains a pair of graphs of monoclonal antibody (Y-3 and FITC) reactivity with MHC I-DC and Kb expressed by the RMA cell line. FACS analysis of RMA, RMA-S, RMA-S-DC and RMA-S-Kb cells.
  • Y-3 is a Kb specific monoclonal antibody.
  • FITC is a non-specific (control) antibody.
  • FIGURE 11 is a graph of tumor progression in C57BL/6 mice treated with RMA, RMA-S, RMA-S + Kb and RMA-S + DC.
  • B6 mice were injected subcutaneously with one million of the indicated cells in the right flank. Mice that were killed when their tumor load was too large were included in the tumor bearing group after their death. The numbers of mice in each group were: RMA, 10; RMA-S, 9; RMA-S + Kb, 9, and RMA-S-DC, 10.
  • FIGURE 12 is a graph of tumor resistance in non-immunized and immunized mice and tumor resistance, showing rejection of RMA-S-Kb tumors by C57BL/6 mice after immunization with RMA-S-DC.
  • B6 were injected with one million RMA-S-DC cells subcutaneously in the right flank.
  • mice Twenty one days after immunization, immune and control mice were challenged with one million RMA-S-Kb cells in the left flank. Eight mice were in the control group and six mice were in the immune group.
  • Expression control sequence a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence.
  • a DNA sequence is operatively linked to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term "operatively linked” includes having an appropriate start signal (e.g. , ATG) upstream (in front of) the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • start signal e.g. , ATG
  • Standard hybridization conditions salt and temperature conditions substantially equivalent to 5 x SSC and 65 ⁇ C for both hybridization and wash.
  • DNA sequence polynucleotide sequences purified, prepared or isolated using recombinant DNA techniques. These include cDNA sequences, DNA sequences isolated from their native genome and synthetic DNA sequences. The term as used in the claims is not intended to include naturally occurring DNA sequences as they exist in nature.
  • Receptor and receptor complex includes both the singular and plural, and contemplates the existence of one or more structures comprised of the protein(s) which make up the ligand recognition site.
  • one or more proteins may be involved, as well as one or more compounds not directly involved in ligand recognition, which even though not included in the recognition reaction, are required, preferred or are typically present when the recognition reaction occurs. All such compounds are included, taken individually as well as in combinations.
  • Binding of the receptor or the antigen means reaction and association between the molecules involved, but need not, and generally does not, include the formation of covalent bonds.
  • Expression of recombinant molecules as used herein may involve the post-translational modification of a resultant polypeptide coded by the sequence present in the host cell.
  • expression might include, among other things, the production of an mRNA molecule or a polypeptide, glycosylation, lipidation or phosphorylation of the polypeptide, or cleavage of a signal sequence to produce a "mature" protein.
  • polypeptide encompasses full-length polypeptides, fragments of mature proteins and modifications or derivatives thereof, such as glycosylated versions of such polypeptides, polypeptides retaining a signal peptide, truncated polypeptides having comparable biological activity and the like.
  • Allogen refers to an antigenic substance which is recognized as foreign, taking into account the modified MHC described herein. This may take into account different members of the same species, members from different species (xenogens) and the recognition of self as antigenic.
  • the term “universal allogen” refers to a substance, like the modified MHC described herein, which can be used to render any cell which contains it alloantigenic.
  • the universal allogen can thus be used to modulate an immune response between genetically different individuals within the same specie, or between genetically identical members of the specie. This is particularly important in the treatment of tumors which have down-regulated their expression of Class I MHC, thus essentially avoiding recognition by the immune system.
  • expression of a "derivative" of the sequence may involve the production of an intermediate molecule, which is generated during the expression of the protein. Typically, this involves the expression of mRNA which likewise codes for the particular polypeptide to be ultimately expressed. In this instance, the mRNA molecule is deemed to be a derivative of the DNA coding sequence contained in the particular expression vector.
  • DNA and RNA molecules are both forms of nucleic acid molecules.
  • Derivatives of a polypeptide thus may include fragments and modified polypeptides, e.g., glycosylated, phosphorylated polypeptides, etc. as noted above. It may also include the gene sequence deduced from the peptide.
  • derivatives can refer to other polynucleotides, sense as well as antisense molecules, ribozymes, vectors, unicellular hosts and other species which embody the polynucleotide in coding sequence directly or complementarily.
  • Signal and signal transduction refer to changes which occur in response to binding of a receptor by reaction with a ligand. Examples of such changes include initiation of a cascade of enzyme reactions, rapid increases in calcium flux, increase in phosphoinositol (IP3) turnover and secretion of lymphokines from lymphocytes.
  • MHC Major Histocompatibility Complex
  • MHC is known to exist in a number of different forms, which have been classified based upon the structure of the molecule, the reactivity with different antigenic substances and the function which the particular MHC molecule appears to serve.
  • the preferred molecular substrate for the modifications described herein is an MHC Class I molecule, based upon its involvement in recognition by cytotoxic T- lymphocytes, preferably the al domain of the MHC class I molecule.
  • the Class I histocompatibility molecule from human cell membranes has two structural motifs.
  • the membrane-proximal end of the glycoprotein contains two domains with immunoglobulin folds that are paired.
  • the region distal to the membrane is in the form of a platform with eight antiparallel beta strands topped by alpha helices.
  • MHC Class I molecules are also known as HLA-A, B and C in humans and H-2K, D and L in mice.
  • MHC Class I (MHC I or Class I) molecules contain a heavy peptide chain having a molecular weight of about 43-44 kD, noncovalently linked to a smaller peptide, about 11 kD, which is termed B 2 - microglobulin (B2-m) .
  • B2-m microglobulin
  • the largest part of the heavy chain is organized into three globular domains, (al, a2 and a3) . These domains protrude from the cell surface.
  • a hydrophobic section of MHC I anchors the molecule in the membrane and a short hydrophilic sequence carries the C-terminus into the cytoplasm.
  • the heavy chain spans the membrane bilayer, with the light chain B2-m noncovalently bound to the heavy chain.
  • the three globular domains of the extracellular portion of the heavy chain are each about 90 amino acids long, and encoded on separate exons.
  • the a3 domain and B2-m are relatively conserved and show amino acid sequence ho ology to immunoglobulin constant or variable domains.
  • MHC class II molecules are heterodimers composed of ⁇ and ⁇ chains. Each chain contains two globular domains, ⁇ l and 2, and ⁇ l and ⁇ 2. The ⁇ l and ⁇ l domains correspond to the al and a2 domains of class I molecules. Like al of the class I molecule, the ⁇ l domain of the class II molecule does not contain an intradomain disulfide bridge, but ⁇ l does.
  • the structure of both class I and class II MHC molecules are well known in the art (see, e.g., reference 4; Paul, Fundamental Immunology, 2nd Edition; and Hood et al., Immunology, Second Edition).
  • This invention takes advantage of the presentation of antigen in the context of modified MHC and/or the recognition of MHC by the immune system. This is important for epitope recognition by receptors and related molecules on the surface of cells which are involved in immune recognition, in particular T- lyraphocytes, natural killer (NK) cells and lymphokine activated killer (LAK) cells. It also takes advantage of the DNA molecules, fragments of such molecules and derivatives thereof, e.g., mRNA, which code on expression for modified MHC. Sense and anti-sense RNA molecules which likewise code for such molecules or for other molecules are also included.
  • the invention is drawn to modified MHC molecules that render cells carrying the modified MHC on the membrane thereof substantially recognizable by the immune system. This is accomplished by introducing a crosslink into such molecules.
  • the crosslink can be introduced by substituting a pair of crosslinkable residues in the sequence of the MHC molecule, or by introducing one crosslinkable residue that can form a crosslink with a crosslinkable residue already present in the MHC molecule.
  • a more stable and immunogenic class I molecule has been engineered by the introduction of two cysteine residues into the al domain of MHC I.
  • This double cysteine molecule (DC) is universally allogenic (“universal allogen") . It is used to more strongly immunize the host.
  • the basis of the stronger immunogenicity may be the formation of a stabilizing disulfide bond and/or the presentation of novel peptides or stable "empty" molecules to the immune system.
  • the expression of the extracellular domains on transplanted cells or tissues is recognized by the immune system of the host and vigorously rejected as being foreign or "alloantigenic", i.e., recognized as antigenic by the members of a particular specie.
  • X-ray crystallographic analysis of human MHC class I molecules indicates that the al and a2 domains form one large superdomain on a base which is formed by the a3 domain and beta-2 microglobulin.
  • This superdomain contains a beta-pleated sheet platform upon which two alpha helical coils form a binding pocket that can accommodate cellular peptides (Fig. 2) .
  • This trimolecular complex of MHC class I, beta-2 microglobulin and peptide is required for stable cell surface expression of the MHC class I molecule.
  • the multimeric complex of class I and peptide (cellular, viral, tumor) is recognized by the T cell receptor, and leads to T cell activation and immune effector function.
  • MHC class I molecules in vertebrate species contain cysteine residues that form disulfide bridges in both the a2 and a3 domains (Fig. 1) .
  • class I molecules do not contain disulfide bonds in the al domain.
  • no cysteine residue has been found in the al domain of any Class I mammalian molecule. It is possible that the lack of a disulfide bridge in the al domain reflects the need for additional flexibility in one domain of the peptide binding portion of Class I.
  • the al domain is chemically or genetically modified so that at least one pair of crosslinkable residues are present in this particular fragment.
  • crosslinkable residues are inserted into domains a2 or a3, then at least one crosslink would be present in addition to the cysteine residues already present. In this instance, the conformation may be changed based upon the formation of additional crosslinks.
  • MHC II molecules which also lack a disulfide in the ⁇ l domain. Insertion or addition of crosslinkable groups is irrespective of the presence of disulfide bridges already present in the molecule or fragment thereof.
  • Class II MHC can likewise be included in the group of molecules which can be modified to increase or decrease antigen recognition by the immune system.
  • the molecule is modified as described above, or the nucleotide sequence coding for the polypeptide is modified to express the MHC II molecule with at least one crosslinkable group contained in the polypeptide.
  • the most preferred crosslinkable group which can be included in MHC I, domain al, as well as in the remainder of the molecule and in MHC II, is a pair of cysteine residues.
  • a cysteine residue may be introduced to a domain of the class I or class II molecule in order to render the molecule allogeneic.
  • MHC I it is most preferred to use a pair of cysteine residues, which can form a disulfide bridge within the al domain.
  • This modification is most preferably made by modifying the gene which codes MHC I, domain al, inserting the gene into an expression vector with its own regulatory sequences or others, if appropriate, and expressing the protein or protein fragment in modified form.
  • transfecting an appropriate cell line with an expression vector containing the coding sequence for the desired polypeptide in modified form the location of the cysteine residue(s) in the al domain can be readily controlled.
  • the most preferred crosslinkable groups which can be included in the MHC molecules described herein are multiple cysteine groups, which can be expressed in the appropriate alpha helical domains of the MHC molecule.
  • the most preferred cysteine groups are positioned to form disulfide bridges.
  • the most preferred moiety for use in connection herein is a pair of cysteine residues, which can form the disulfide bridge across MHC I in the al domain.
  • cysteine groups can be inserted into the MHC II molecule at the appropriate domain to form disulfide bridges in the molecule.
  • a cystine residue is introduced into the al domain of MHC class I or the ⁇ l domain of MHC class II.
  • the positioning of the crosslinkable groups can be determined by predicting the structure of the MHC molecule of known sequence. Structural prediction has been greatly facilitated by the work of Bjorkman et al. (4, supra) .
  • crosslinkable groups can be used without rendering the MHC molecule ineffective in reacting or combining with antigens.
  • a pair of crosslinkable residues can be glutamic acid and lysine, in which the ⁇ -carboxylic acid of glutamic acid forms an amide bond with the e-amino group of lysine. Such bonds can be formed enzymatically or chemically.
  • chemical treatment of an expressed MHC molecule can effectuate crosslinking between the desired peptides, utilizing conventional hydroxyl, amine and carboxyl reactive compounds.
  • MHC I and II The modification of MHC I and II is not limited to human or murine MHC; it encompasses MHC molecules derived from other organisms as well.
  • Another preferred embodiment of the invention relates to DNA constructs containing the gene which codes for such modified MHC or particular domains of MHC, with at least one added crosslinkable group contained therein, as well as the plasmids and vectors incorporating such constructs.
  • oligonucleotide sequences used for site-directed mutagenesis of MHC al appear in Figure 4. These sequences can be incorporated into the desired MHC I gene and used to facilitate expression of the proteins of interest. If appropriate, various promoter and enhancer sequences can be included, if necessary to facilitate the expression of the proteins of interest. Such combinations are within the scope of this invention.
  • nucleic acid molecules encoding such modified MHC molecules can be introduced into cells in vivo as well as in vitro.
  • the DNA sequences of the invention may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host.
  • Such operative linking of a DNA sequence of the invention to an expression control sequence includes if not already part of the DNA, the provision of an initiation codon, e.g., ATG, in the correct reading frame upstream of the DNA sequence.
  • a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of the invention.
  • Useful expression vectors for example, may consist of segments of chromosomal, nonchromosomal or synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.
  • coli plasmids col El, pCRl, pBR322, pMB9 and their derivatives; plasmids such as RP4; phage BNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989 and other phage DNA, e.g., M13, and Filamentous single stranded phage DNA; yeast plasmids such as the 2 mu plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs such as plasmids that have been modified to employ phage DNA or other expression control sequences, and the like.
  • plasmids such as RP4
  • phage BNAs e.g., the numerous derivatives of phage lambda, e.g., NM989 and other phage DNA, e
  • the gene encoding modified MHC is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV) , papillomavirus, Epstein Barr virus (EBV) , adenovirus, adeno-associated virus (AAV) , and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are preferred. Defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells.
  • the viral vector is a retroviral vector, in particular a recombinant Maloney virus vector. Retroviral vectors are preferred since they will be active in replicating cells, such as cancer cells.
  • the vector can be introduced in vivo by lipofection.
  • liposomes for encapsulation and transfection of nucleic acids .in vitro.
  • Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of the modified MHC gene (Feigner, et. al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:7413-7417; see Mackey, et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031)).
  • cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes (Feigner and Ringold, 1989, Science 337:387-388).
  • Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to limited types would be particularly advantageous to target the specific cells desired for destruction.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting (see Mackey, et al., 1988, supra) .
  • Targeted peptides e.g., hormones or cytokines, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • the vector containing the gene encoding modified MHC can be introduced via a DNA vector transporter (see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990).
  • a DNA vector transporter see, e.g., Wu et al., 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263:14621-14624; Hartmut et al., Canadian Patent Application No. 2,012,311, filed March 15, 1990.
  • Vectors are introduced into the desired host cells in vitro by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion) , use of a gene gun, using a viral vector, with a DNA vector transporter, and the like.
  • Promoters which may be used to control expression of the modified MHC gene include, but are not limited to, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3 1 long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A.
  • elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol.
  • mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485- 495)
  • albumin gene control region which is active in liver
  • alpha-fetoprotein gene control region which is active in liver
  • alpha 1- antitrypsin gene control region which is active in the liver
  • beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234:1372-1378).
  • a promoter is chosen to provide high level expression of the modified MHC gene is a transfected cell. If the promoter is a tissue specific promoter, preferably it is chosen to ensure greater expression of the modified MHC in the cell in which expression is desired.
  • a wide variety of unicellular host cells is also useful in expressing the DNA sequences of the invention.
  • These hosts may include well known eukary ⁇ tic and prokaryotic hosts, such as strains of E. coli f Pseudomonas. Bacillus. fungi such as yeasts, and animal cells, such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells, e.g., COS 1, COS 7, BSC1, BSC40 and BMT10, insect cells, e.g., Sf9, and human and plant cells in culture.
  • eukary ⁇ tic and prokaryotic hosts such as strains of E. coli f Pseudomonas.
  • Bacillus. fungi such as yeasts
  • animal cells such as CHO, Rl.l, B-W and L-M cells, African Green Monkey kidney cells, e.g., COS 1, COS 7, BSC1, BSC40 and BMT10, insect cells,
  • Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed and the ease of purification of the expression products.
  • DNA sequences of this invention can be synthesized using polymerase chain reaction technology.
  • the extent of incorporation of the oligonucleotide- modified gene by the expression vector can be selected readily by including a gene for antibiotic resistance, e.g., neomycin, ampicillin etc; and by growing the organisms in the presence of the antibiotic.
  • the effectiveness of the site directed mutagenesis can thereafter be evaluated by hybridization or polymerase chain reaction.
  • sequences can be obtained by screening a genomic library with an appropriate DNA probe. In this method, the genome is isolated, fragmented and the fragments inserted into appropriate vectors. The sequence of interest can then be screened for directly.
  • mRNA can be isolated from cells transfected with the sequences, and cDNA made from it. The cDNA can then be inserted into an appropriate expression vector to express relatively large quantities of the desired protein or polypeptide fragment.
  • Another preferred embodiment of the invention relates to a unicellular host transfected with the plasmids described herein.
  • the present invention provides methods for the treatment of cancer, tumors, infections, and the like in any subject. Accordingly, the therapeutic methods of the invention can be practiced on a mammalian subject in need of such therapy.
  • the subject is a human, although the methods of the invention are contemplated for use in other mammalian species, including but not limited to domesticated animals (canine and feline) ; farm animals (bovine, ovine, equine, caprine, porcine, and the like); rodents; and undomesticated animals.
  • polypeptide and DNA sequences mentioned above can be used in many different ways within the context of the present invention.
  • the protein or protein fragment and the DNA coding the protein may be used directly by administering either of these components to an organism. More particularly, the modified MHC proteins described herein can be used in whole or in part to modify immune system recognition of epitopes.
  • modified genes or polypeptides in the form of a composition which can be introduced into cells and administered to a host organism therapeutically or as an immunogen to elicit a cellular immune response, preferably a CTL response, to the cells.
  • the polypeptide is water soluble, e.g., lacks the transmembrane domain
  • the polypeptide can be linked to a water insoluble protein, e.g., an integral membrane protein, present in the cell membrane of an invasive organism, tumor or other cancer cell.
  • a water insoluble protein e.g., an integral membrane protein
  • the expression of the modified, allogeneic MHC class I and class II molecules on transplantable tumor cells leads to rejection of the tumor cells by the host.
  • the tumor is recognized and treated essentially as a "allograft.” Furthermore, the rejection can extend to non "alloantigenic" tumor cells.
  • allogeneic class I or class II molecules or both are expressed on tumor cells by introducing the allogeneic genes into the genome of the cells by various methods (transfection, electroporation, lipofection) .
  • mice that reject tumor cells expressing allogeneic MHC molecules can survive a challenge by the parental transplantable tumor. It is thought that the vigorous anti-allograft reaction during the primary immunization provides all the necessary lymphokines in the microenvironment of the tumor allograft to allow the host to mount a successful immune response against the previously weak immunogenic parental tumor.
  • the use of the universal allogen for tumor immunization or immunotherapy provides a reagent to be used in situations regardless of the MHC type of the host.
  • class I molecule may have applications for the immunotherapy of tumors that have down-regulated their expression of class I as a means of evading the immune system.
  • the MHC molecules described herein can be used in a number of different treatment modalities to render otherwise unrecognized antigens immune reactive. These MHC molecules can be used alone as a single therapeutic agent or in combination with other agents.
  • One of the preferred treatment methods described herein includes the treatment of cancer or tumors.
  • a nucleic acid vector encoding the modified MHC molecule can be introduced to the cancer or tumor cells -in vivo or -in vitro. These cells can then express the modified MHC in the body of the patient, and elicit an immune response against the cells.
  • the modified MHC molecule is targeted to such cancer or tumor cells, and associates with the cells, thus eliciting an immune response against the cells.
  • the treatment of cancer or tumors may involve the administration of cells modified by transfection with the gene which codes modified MHC or the administration of the gene which codes MHC in modified form.
  • the modified MHC can be associated with the cells, either by covalent attachment to the cell surface, or introduction into the cell membrane.
  • the cells may be recognized as allogenic, allowing an antibody or cellular immune response to occur. More importantly, as demonstrated in the examples herein, the immune response can extend to reject non-allogeneic cells.
  • Tumor or cancer cells for transfection with a nucleic acid of the invention may be taken from the patient directly, such as during a tumor resection, or may be derived from a genetically identical individual from the same species.
  • the gene which codes modified MHC is administered to the patient, the gene may be combined with the tumor or malignant cells, whereupon these cells will become allogenic.
  • the effectiveness of the present invention in eliciting tumor-specific immunity can be shown in numerous model systems.
  • Various tumor cell lines available commercially or from depositories such as, but not limited to, the American Tissue-type Culture Collection (ATCC) ,
  • Rockville, Maryland can be transfected .in vitro or in vivo with vectors of the invention, and the resistance of animals to transplanted tumors, the ability of such transfected tumors to induce resistance to transplanted native (untransfected, non-allogenic) tumors, and the ability of such transfected tumors to cause regression of native tumors, can be evaluated.
  • Such tumor cell lines include, but are not limited to, the T cell lymphoma tumors S49.1 (ATCC accession # TIB 28), EL-4 (available in many laboratories or ATCC accession # TIB 39) , and BW5147.3 (ATCC accession # TIB 47); melanoma cell lines B16 (ATCC accession #s CRL 6322 and CRL 6323) and S91 (ATCC accession # CRL 53.1); the fibrosarcoma cell line WEHI 1.64 (ATCC accession # CRL 1751); and Lewis lung carcinoma (ATCC accession # CRL 1642) .
  • a human tumor cell line is used.
  • Transfections can be performed by preparing the cDNA corresponding to the mutated MHC class I molecule, such as the molecule described in the Examples infra (termed DC) , and incorporating the cDNA in a mammalian expression vector.
  • expression vectors include, but are not limited to, pRC/CMV and pRC/RSV, which are available from Invitrogen.
  • retroviral vectors such as a Maloney virus vector, can be used. Maloney viral vectors can be used in gene therapy for humans.
  • the modified MHC, the transfected cells or the gene itself can also preferably be used to treat chronic infection.
  • infection will preferably be such that the infectious organism is intracellularly located, and thus evades immune surveillance.
  • the modified MHC molecules or fragments thereof, the transfected cells or the gene or gene fragment for modified MHC may be administered to the patient and allowed to combine with cells harboring the pathogenic organism, and render such cells, and thus such organisms, recognizable by the immune system.
  • Representative types of infection which can be treated in accordance with the teachings herein include bacterial, viral, retroviral and mycobacterial infections of the tissue, blood or other body fluid.
  • the modified MHC molecules are administered to the mammalian host in an amount effective to treat the infection, which in general is the amount which is necessary to combine with the cells containing the invasive organism and render it recognizable by the immune system.
  • a preferred method of treatment relates to the treatment of persistent or chronic infection, particularly by organisms for which conventional therapy has been found to be inadequate.
  • modified MHC is believed to be useful in the treatment of HIV viruses.
  • modified MHC, organisms or cells which express MHC in modified form or the gene which codes MHC in modified form it may be possible to use modified MHC, organisms or cells which express MHC in modified form or the gene which codes MHC in modified form as a sole therapeutic agent.
  • the modified MHC, organism or cell which codes MHC in modified form or gene which codes modified MHC is administered to the patient in an amount effective to treat the infection.
  • this may constitute a single therapeutic agent useful in treating the infection, it is more likely to be used in combination with antiviral compounds, immunoreplacement therapy, immune system stimulation, such as via the interferons, interleukins , colony stimulating factors and other agents.
  • Another preferred method of treatment involves combining modified MHC molecules, a derivative or fragment thereof with a tumor cell or infectious organism or a fragment or derivative of said tumor cell or organism.
  • the combination is thereafter administered to, the patient in need of such treatment, to render the epitope(s) present on the tumor cell, organism, fragment or derivative thereof recognizable by the cellular immune system.
  • the modified MHC molecules derivatives or fragments taken alone or in combination with some antigen, are preferably administered to the patient in combination with other therapeutic agents, e.g., lymphokines, factors, hormones, anticancer or anti-infective drugs, e.g., antibiotics and the like.
  • other therapeutic agents e.g., lymphokines, factors, hormones, anticancer or anti-infective drugs, e.g., antibiotics and the like.
  • replacement therapy may be added.
  • therapeutic doses of antibodies, T- lymphocytes, naturally occurring MHC I or II or other immune system components may be administered.
  • compositions for the prevention or treatment of disease are also included. These therapeutic compositions can be topical, transder al, oral or injectable compositions containing the therapeutic compound or specie(s) in combination with a pharmaceutically acceptable carrier.
  • the therapeutic compositions of the invention will be viral vectors, in vivo transfection compositions, or cells that include the modified MHC molecules.
  • such therapeutic compositions will be administered parenterally, e.g., via intravenous, intraarteoral, intraperitoneal, intramuscular, etc. injection.
  • the invention contemplates using any route of administration known to the skilled physician. According to the present invention, therapy of any cancer or tumor can be effected or enhanced.
  • dysproliferative changes are treated or prevented in epithelial tissues such as those in the cervix, esophagus, and lung.
  • epithelial tissues such as those in the cervix, esophagus, and lung.
  • the present invention provides for treatment of conditions known or suspected of preceding progression to neoplasia or cancer, in particular, where non-neoplastic cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology. 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79) .
  • Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or function.
  • endometrial hyperplasia often precedes endometrial cancer.
  • Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells.
  • Atypical metaplasia involves a somewhat disorderly metaplastic epithelium.
  • Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non- neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells.
  • Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism.
  • Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
  • Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.
  • the cancer can be a leukemia or lymphoma, e.g., multiple myeloma, T cell-ALL, hairy cell leukemia, Burkitts lymphoma, and the like.
  • a leukemia or lymphoma e.g., multiple myeloma, T cell-ALL, hairy cell leukemia, Burkitts lymphoma, and the like.
  • the therapy is of prostate cancer.
  • a vector encoding modified MHC class I is introduced in vivo to prostate cancer cells.
  • prostate cancer cells obtained by biopsy are transfected with an expression vector of the invention, and the transfected cells are introduced back into the subject.
  • the carrier may be comprised of one or more agents which are typically found in injectable dosage forms, so long as the carrier is compatible with the pharmacologically active agent.
  • intravenous compositions may contain water for injection, saline, buffering agents, tonicity adjusting agents, preservatives and the like.
  • compositions noted above also include biologicals, such as vaccines and antisera which contain the modified MHC molecule or a fragment thereof, alone or in combination with the molecule to be recognized, antibodies which recognize and bind to the causative agent, alone or in conjunction with the modified MHC molecules described herein. Other agents may also be included. Likewise, a method of making the vaccine and antiserum are included.
  • RMA is a cell line of virally induced lymphoma raised in C57BL/6 mice. Via -in vitro mutagenesis and selection, a mutant cell line, designated RMA-S, was derived which lacks the normal cell surface expression of Class I. RMA-S was found to be deficient in a peptide transport molecule which moves peptides from the cytosol to the endoplasmic reticulum (ER) . As a result, the MHC class I molecules of RMA-S (Kb and Db) do not come into contact with peptides as they transit through the ER and golgi and therefore reach the surface as unstable molecules. These "empty" molecules react poorly with monoclonal antibodies that react with "normal” Kb and Db. In addition, "empty " class I molecules are poorly immunogenic, confirming that the more stable trimolecular complex is necessary for eliciting an immune response.
  • T helper T helper, T cytotoxic cells, antigen presenting cells such as dendritic cells or macrophages, B cells
  • B cells B cells
  • T helper T cytotoxic cells
  • antigen presenting cells such as dendritic cells or macrophages, B cells
  • MHC class II molecules that stimulate T helper cells, that then produce lymphokines, a required second signal, that activates cytotoxic T cells which respond against peptides presented by class I molecules.
  • the lack of an efficient host immune response against tumors may reflect a lack of stimulation of some of these cell types, and the resultant absence of necessary soluble factors.
  • a tumor that expresses tumor specific antigens in the context of Class I, but does not express Class II molecules may not be able to provide the signal to T-helper cells that leads to activation of cytotoxic T lymphocytes. Indeed, it has been suggested that the stimulation of potentially cytotoxic cells without the required helper signal results in "anergy", the lack of an immune response.
  • helper lymphokines may be circumvented, in vivo and .in vitro, by providing exogenous lymphokines such as various interleukins as has been mentioned above.
  • Another aspect of the invention relates to the detection or characterization of the molecule with which such modified MHC compounds bind, i.e., the antigen presented by the MHC molecule. This may involve the post- translational modification of the molecules of interest, or of the binding partners of such molecules.
  • cysteine was engineered into codons 11 and 74 of the Kb gene (the K allele of the C57BL/6 mouse) (Fig. 2) .
  • Residue 11 is on the beta-pleated sheet base and residue 74 is on the bottom of the alpha helical coil of Kb. It was assumed that these two residues are close enough to form a disulfide bond.
  • the Kb gene was obtained.
  • Exon 2 which codes for the al domain is excised from the clone by digestion with the restriction enzyme Xma 1 (Fig. 3) .
  • This 300bp fragment is then subcloned into M13 and site-directed mutagenized by two complete rounds of mutagenesis using oligonucleotides containing codons for cysteine and the Amersham site-directed mutagenesis kit (Fig. 4) .
  • the codon 11 construct was made, and then this clone was then mutagenized at position 74. Successful mutagenesis was confirmed by sequencing this 300bp subclone by dideoxynucleotide sequencing techniques and cloned back into the Kb gene.
  • P815-DC and P815-Kb P815 cell populations.
  • P815 is a DBA/2 mastocytoma, transplantable tumor cell line, that expresses Kd, Dd, and Ld MHC class I molecules.
  • B6-DC DC transgenic mice
  • Spleen cells from B6-DC mice were used as stimulators for B6 responder spleen cells.
  • the effector cells lysed P815 cells that expressed DC, but not several other H-2b haplotype tumor cell lines, nor B6 spleen cells (data not shown) .
  • P815-DC or P815 cells were injected subcutaneously into DBA/2 mice and monitored for tumor formation. Five of six mice injected with P815 developed progressive tumors by day 10 (Fig 9) . The data presented in Fig. 9 indicate that DC functions as a strong alloantigen and prevents tumor formation in 5 of 6 mice until 33 days after injection. Four of six mice remained tumor-free throughout the experiment. One P815-DC injection produced a tumor after 33 days. Another P815- DC injection produced a very slowly growing tumor which had lost expression of DC. Analysis of the tumor cells indicated that the cells had lost the expression of DC. Four of the six mice injected with P815-Kb developed tumors.
  • RMA-S-DC and RMA-S-Kb were transfected into RMA-S cells.
  • RMA and RMA-S already contain endogenous Kb genes-.
  • RMA-S cells express 5% - 15% of the serologically detectable level of class I as compared to parental RMA
  • transfectants RMA-S-DC and RMA-S-Kb expressed near parental RMA levels of MHC class I using the Y-3 MAB (Fig. 10) . Similar results were observed with eight additional mABs specific for the Kb molecule.
  • RMA-S cells and the transfectants RMA-S-DC and RMA-S-Kb are not efficiently lysed by d anti-b CTL.
  • mice were inoculated subcutaneously with one million RMA, RMA-S, RMA-S-DC or RMA-S-Kb. All cell lines except RMA-S-DC rapidly formed progressively growing tumors (Fig 11) . The compilation involving 70 mice indicates that C57BL/6 mice reject a challenge of one million RMA-S-DC 66% of the time.
  • mice immunized (s.c.) with one million RMA-S-DC were challenged with one million RMA- S-Kb subcutaneously on the opposite flank. As indicated in Figure 12, 5 of 6 immunized mice were still tumor free after more than 3 months.
  • the immunotherapy of cancer requires the management of established tumors.
  • Three DBA/2 mice were inoculated s.c with 0.1 ml P815 ascites cells.
  • established tumors of approximately 4 mm diameter were apparent.
  • Two mice received intratumor injections of one million P815-DC cells.
  • the third mouse did not receive an immunotherapeutic inoculation and served as control.

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Abstract

L'invention se rapporte à des molécules du système majeur d'histocompatibilité (MHC) modifiées, facilitant la reconnaissance des antigènes par des anticorps et des constituants cellulaires du système immunitaire. Ceci permet, en particulier, d'augmenter la capacité de reconnaissance des tumeurs du système immunitaire. Les molécules de MHC modifiées peuvent être considérées comme un allogène universel dans la mesure où elles peuvent rendre antigénique toute cellule les contenant. Des gènes codant l'expression des molécules de MHC modifiées, des cellules les contenant, des anticorps et des constituants cellulaires reconnaissant les espèces transformées sont également décrits.
PCT/US1993/008715 1992-09-15 1993-09-15 Molecules de systeme majeur d'histocompatibilite et leurs modifications WO1994006813A1 (fr)

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WO1999054496A3 (fr) * 1998-04-20 2000-03-02 Innogenetics Nv Methode de groupage d'alleles de hla
WO2000015665A3 (fr) * 1998-09-14 2000-08-17 Lars Oestergaard Pedersen Procede de production d'une proteine fonctionnelle de la superfamille des immunoglobulines

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998007441A1 (fr) * 1996-08-23 1998-02-26 Massachusetts Institute Of Technology Complexes d'histocompatibilite allogenes mediateurs de destruction cellulaire
WO1999054496A3 (fr) * 1998-04-20 2000-03-02 Innogenetics Nv Methode de groupage d'alleles de hla
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WO2000015665A3 (fr) * 1998-09-14 2000-08-17 Lars Oestergaard Pedersen Procede de production d'une proteine fonctionnelle de la superfamille des immunoglobulines

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