Patent 6,737,268

Dominant negative alleles of human mismatch repair genes can be used to generate hypermutable cells and organisms. By introducing these genes into cells and transgenic animals, new cell lines and animal varieties with novel and useful properties can be prepared more efficiently than by relying on the natural rate of mutation. These methods are useful for generating genetic diversity within genes encoding for therapeutic antigens to produce altered polypeptides with enhanced antigenic and immunogenic activity. Moreover, these methods are useful for generating effective vaccines.

SUMMARY OF THE INVENTION

The invention described herein is directed to the use of random genetic mutation of a polypeptide in vivo by blocking the endogenous mismatch repair (MMR) activity of a host cell yielding structurally altered antigens that can be screened for antigenicity and immunogenicity in comparison to the wild type molecule. The use of mammalian cell-based high throughput screens as taught by this application will facilitate identification of randomly altered antigens that may serve as effective vaccines. Moreover, the invention describes methods for repeated in vivo genetic alterations and selection for antigens with enhanced immunogenicity and pharmacokinetic profiles.

The ability to develop and screen genetically altered mammalian cells that secrete structurally altered polypeptides in a high throughput manner provides a valuable method for creating vaccines for therapeutic development. A potential problem in generating potent vaccine antigens against endogenous to the mammalian host is the source of antigen production. In many instances recombinant polypeptides that are naturally produced by mammalian cells are generated recombinantly using insect, yeast or bacterial expression systems. These sources typically produce large amounts of proteins that are distinct from the mammalian-produced polypeptides, and may differ from the natural protein due to altered folding or altered post-translational modifications such as hyperglycosylation. The invention described herein is directed to the creation of genetically altered mammalian cell hosts that produce structurally altered polypeptides as vaccine agents via the blockade of MMR.

The present invention facilitates the generation of highly antigenic polypeptides as vaccines. The advantages of the present invention are further described in the examples and figures described herein.

The present invention provides methods for generating genetically altered antigens in vivo, whereby the antigen possesses desired biochemical property(s), such as, but not limited to, increased antigenicity and immunogenicity. One method for identifying antigens with increased antigenicity is through the screening of mismatch repair ("MMR") defective cell clones that produce desired antigens.

The invention also provides methods for rendering cells expressing a target antigen hypermutable. The cells include, but are not limited to rodent, primate, human, plant, yeast or bacterial cells. The antigens can be generated from endogenous genes or from introduced transgenes.

The invention also provides methods for generating genetically altered cell lines that express antigenic polypeptides.

In some embodiments, the invention provides methods for generating genetically altered cell lines that produce immunogenic polypeptides.

In other embodiments, the invention provides methods for producing an antigen expression cassette for high throughput screening of altered polypeptides in vivo.

In other embodiments, the invention provides methods of mutating a gene of interest in a mismatch repair defective cell.

In some embodiments, the invention provides methods of creating genetically altered antigens in vivo by blocking the MMR activity of the cell host.

Still other embodiments of the invention provide methods of creating genetically altered polypeptides in vivo by transfecting genes encoding for an antigen in a MMR defective cell host.

The invention also embraces methods of creating antigens with increased immunogencity due to genetic alterations within the antigen-encoding gene by blocking endogenous MMR of the cell host.

In some embodiments, the invention provides methods of creating a library of randomly altered antigens from mammalian cells by blockade of MMR of the cell host.

In other embodiments, the invention provides methods of creating antigens with enhanced pharmacokinetic profiles due to genetic changes within the encoding gene by blocking endogenous MMR of the cell host.

The invention also provides methods of creating genetically altered antigens in MMR defective cells as vaccine agents.

In some embodiments, the invention provides methods for high throughput screening of antigens produced by MMR defective cells.

These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention, a method for making MMR defective cell lines expressing a target antigen will be provided. A polynucleotide encoding a dominant negative allele of an MMR gene is introduced into a target antigen-producing cell. The cell becomes hypermutable as a result of the introduction of the gene.

In another embodiment of the invention, an isolated hypermutable cell producing antigenic peptides is provided. The cell is defective for mismatch repair and exhibits an enhanced rate of hypermutation. The cell produces a polypeptide from a mutated gene encoding for the polypeptide.

In another embodiment of the invention, a method is provided for introducing a mutation into an endogenous gene encoding for a target polypeptide. A polynucleotide encoding a dominant negative allele of a MMR gene is introduced into a cell. The cell becomes hypermutable as a result of the introduction and expression of the MMR gene allele. The cell further comprises a gene of interest. The cell is grown and tested to determine whether the gene encoding for a polypeptide of interest harbors a mutation.

In another embodiment of the invention, a method is provided for producing a cell-based screening assay to identify antigenic proteins as vaccines. A polynucleotide encoding a dominant negative allele of a MMR gene is introduced into a cell expressing a secreted antigen. The cell becomes hypermutable as a result of the introduction of the gene. The cell is grown and conditioned medium from the cell is tested for the expression of antigenic polypeptides.

In another embodiment of the invention, a gene, or set of genes encoding for polypeptides or a combination therein, are introduced into a mammalian cell host that is defective in MMR. The cell is grown and clones are analyzed for antigens with enhanced antigenicity.

In another embodiment of the invention, a method is provided for producing a cell-based screening assay to identify antigenic proteins as vaccines. A polynucleotide encoding a secreted antigen is introduced into a naturally MMR defective cell. The gene is hypermutable as a result of the introduction of MMR deficiency. The cell is grown and conditioned medium from the cell is tested for the expression of antigenic polypeptides.

In another embodiment of the invention, a method will be provided for restoring genetic stability in a cell containing a polynucleotide encoding for a dominant negative allele of a MMR gene. The expression of the dominant negative MMR gene is suppressed and the cell restores its genetic stability including but not limited to genetic stability within the antigen-encoding genes.

In another embodiment of the invention, a method will be provided for restoring genetic stability in a cell containing a polynucleotide encoding a dominant negative allele of an MMR gene and a newly selected phenotype. The expression of the dominant negative mismatch repair gene is suppressed and the cell restores its genetic stability and the new phenotype is stable.

These and other embodiments of the invention provide the art with methods that can generate enhanced mutability in cells and animals as well as providing cells and animals harboring potentially useful mutations for the large-scale production of highly antigenic polypeptides as potent vaccines.

Detailed Description of the Invention

The inventors have discovered a method for developing hypermutable cells producing therapeutic antigens by taking advantage of the conserved mismatch repair (MMR) process of host cells. Dominant negative alleles of such genes, when introduced into cells or transgenic animals, increase the rate of spontaneous mutations by reducing the effectiveness of DNA repair and thereby render the cells or animals hypermutable. Hypermutable cells or animals can then be utilized to develop new mutations in a gene or genes of interest. Blocking MMR in cells producing antigens (including, but not limited to, mammalian cells, plant cells, yeast cells, and prokaryotic cells) can enhance the rate of mutation within the gene encoding for the antigen that can be screened to identify clones producing structurally altered polypeptides with enhanced antigenicitiy and immunogenicity.

In one aspect of the invention, the methods are useful for the production of antigens that have increased antigenicity and/or immunogenicity. Such antigens may be used as immunogens to elicit immune responses in animals against these antigens.

The antigens may be derived from, for example, pathogenic organisms or cancer cells such that an immune response is directed against the pathogenic organism or cancer cell and exerts an effect on the organism or cancer cell. The effect may be, for example, to prevent, inhibit or terminate the growth of the pathogenic organism or cancer cell when an immunogenic amount of the antigen is administered to an animal.

The pathogenic organisms from which antigens may be derived include bacteria, fungi, parasitic protozoa, helminths, and viruses. Non-limiting examples include species of the following genera: Staphylococcus, Streptococcus, Bacillus, Bordetella, Clostridium, Escherichia, Haemophilus, Helicobacter, Klebsiella, Listeria, Salmonella, Vibrio, Yersinia, Neisseria, Treponema, Borrelia, Corynebacterium, Mycobacterium, Mycoplasma, Chlamydia, Acremonium, Aspergillus, Blastomyces, Candida, Acanthamoeba, Ascaris, Babesia, Cryptosporidium, Echinococcus, Entamoeba, Giardia, Necator, Ancylostoma, Unicinaria, Leishmania, Onchocerca, Plasmodium, Schistosoma, Strongyloides, Taenia, Toxoplasma, Trichinella, Trichomonas, Trichuris, Trypanosoma, Dirofilaria, Brugia, Wuchereria, and Eimeria. Non-limiting examples of viruses include adenovirus, arborviruses, coronavirus, cytomegalovirus, enteroviruses, Epstein-Barr virus, hepatitis viruses, herpes viruses, immunodeficiency viruses (e.g., HIV, FIV SIV), papilloma viruses, T-cell leukemia viruses, influenza viruses, mumps viruses, parainfluenzae viruses, parvoviruses, poxviruses, Rabies virus, respiratory syncytial virus, rhinoviruses, rotaviruses, Rubella viruses, and varicella-zoster viruses.

The antigens derived from the pathogenic organisms, for example, may be antigens known to elicit an immune response, for which an enhanced immune response is desired, or the antigen may be one that is known to generate a weak response for which an enhanced response is desired. It is also possible that some antigens that did not previously elicit an immune response will become antigenic as a result of the methods of the invention and the phenomenon of hypermutability of cells which contain dominant negative alleles of mismatch repair genes.

The antigens produced by the method of the invention are novel immunogens that may be administered in an appropriate pharmaceutical carrier, such as an adjuvant, for administration to animals as a vaccine. The antigens of the invention may be administered to animals in immunogenic amounts such that an antibody and/or a cell-mediated immune response is elicited. The administration of the antigens of the invention may be administered as a single dose, or, preferably as a plurality of doses to effect a boosted immune response. The route of administration may be any accepted route of immunization including, for example, oral, intrmuscular, intrperitoneal, subcutaneous, intradermal, intranasal, or transdermal.

Doses for humans can readily be extrapolated from animal studies as taught by Katocs et al., Chapter 27 of REMINGTON'S PHARMACEUTICAL SCIENCES, 18^th Edition, Gennaro (Ed.) Mack Publishing Co., Easton, Pa., 1990. Immunogenic dosages can be adjusted by one skilled in the art, and may vary depending on several factors, including the age, health, physical condition, weight, type and extent of the disease or disorder of the recipient, frequency of treatment, the nature of concurrent therapy, if required, and the nature and scope of the desired effect(s) (Nies et al., Chapter 3, GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 9th Ed., Hardman et al., Eds., McGraw-Hill, New York, N.Y., 1996). Typically, an immunogenic amount of the antigens of the invention will be in the range of about 5 to about 100 g.

The antigens of the present invention may be administered as single antigens or may be administered as combinations of antigens. As a non-limiting example, the antigen combinations may be antigens of the same pathogenic organism, or may be antigens of different pathogenic organisms, such that immune responses are elicited to more than one pathogenic organism.

The antigens of the present invention are hypermutated by the methods of the invention which take advantage of the mismatch repair system. The process of MMR, also called mismatch proofreading, is carried out by protein complexes in cells ranging from bacteria to mammalian cells. A MMR gene is a gene that encodes for one of the proteins of such a mismatch repair complex. Although not wanting to be bound by any particular theory of mechanism of action, a MMR complex is believed to detect distortions of the DNA helix resulting from non-complementary pairing of nucleotide bases. The non-complementary base on the newer DNA strand is excised, and the excised base is replaced with the appropriate base, which is complementary to the older DNA strand. In this way, cells eliminate many mutations that occur as a result of mistakes in DNA replication.

Dominant negative alleies cause a MMR defective phenotype even in the presence of a wild-type allele in the same cell. An example of a dominant negative allele of a MMR gene is the human gene hPMS2-134, which carries a truncating mutation at codon 134. The mutation causes the product of this gene to abnormally terminate at the position of the 134th amino acid, resulting in a shortened polypeptide containing the N-terminal 133 amino acids. Such a mutation causes an increase in the rate of mutations, which accumulate in cells after DNA replication. Expression of a dominant negative allele of a mismatch repair gene results in impairment of mismatch repair activity, even in the presence of the wild-type allele. Any allele that produces such effect can be used in this invention.

Dominant negative alleles of a MMR gene can be obtained from the cells of humans, animals, yeast, bacteria, or other organisms (Prolla T. A. et al. (1994) Science 264:1091-1093; Strand M. et al. (1993) Nature 365:274-276; Su, S. S. et al. (1988) J. Biol. Chem. 263:6829-6835). Such alleles can be identified by screening cells for defective MMR activity. Cells from animals or humans with cancer can be screened for defective mismatch repair. Cells from colon cancer patients may be particularly useful. Genomic DNA, cDNA, or mRNA from any cell encoding a MMR protein can be analyzed for variations from the wild type sequence. Dominant negative alleles of a MMR gene can also be created artificially, for example, by producing variants of the hPMS2-134 allele or other MMR genes. Various techniques of site-directed mutagenesis can be used. The suitability of such alleles, whether natural or artificial, for use in generating hypermutable cells or animals can be evaluated by testing the mismatch repair activity caused by the allele in the presence of one or more wild-type alleles, to determine if it is a dominant negative allele.

A cell or an animal into which a dominant negative allele of a mismatch repair gene has been introduced will become hypermutable. This means that the spontaneous mutation rate of such cells or animals is elevated compared to cells or animals without such alleles. The degree of elevation of the spontaneous mutation rate can be at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, or 1000-fold that of the normal cell or animal. The use of chemical mutagens such as but limited to methane sulfonate, dimethyl sulfonate, O6-methyl benzadine, MNU, ENU, etc. can be used in MMR defective cells to increase the rates an additional 10 to 100 fold that of the MMR deficiency itself.

According to one aspect of the invention, a polynucleotide encoding for a dominant negative form of a MMR protein is introduced into a cell. The gene can be any dominant negative allele encoding a protein, which is part of a MMR complex, for example, PMS2, PMS1 , MLH1, or MSH2. The dominant negative allele can be naturally occurring or made in the laboratory. The polynucleotide can be in the form of genomic DNA, cDNA, RNA, or a chemically synthesized polynucleotide.

The polynucleotide can be cloned into an expression vector containing a constitutively active promoter segment [such as but not limited to CMV, SV40, Elongation Factor (EF) or LTR sequences] or to inducible promoter sequences such as the steroid inducible pIND vector (InVitrogen), tetracycline, or MMTV, where the expression of the dominant negative MMR gene can be regulated. The polynucleotide can be introduced into the cell by transfection.

According to another aspect of the invention, a gene, a set of genes or a chimeric gene encoding for whole or parts of a therapeutic antigen can be transfected into MMR deficient cell hosts, the cell is grown and screened for clones containing genetically altered genes encoding for antigens with new biochemical features including but not limited to increased antigenicity. MMR defective cells may be of human, primates, mammals, rodent, plant, yeast or of the prokaryotic kingdom.

Transfection is any process whereby a polynucleotide is introduced into a cell. The process of transfection can be carried out in a living animal, e.g., using a vector for gene therapy, or it can be carried out in vitro, e.g., using a suspension of one or more isolated cells in culture. The cell can be any type of eukaryotic cell, including, for example, cells isolated from humans or other primates, mammals or other vertebrates, invertebrates, and single celled organisms such as protozoa, yeast, or bacteria.

In general, transfection will be carried out using a suspension of cells, or a single cell, but other methods can also be applied as long as a sufficient fraction of the treated cells or tissue incorporates the polynucleotide so as to allow transfected cells to be grown and utilized. The protein product of the polynucleotide may be transiently or stably expressed in the cell. Techniques for transfection are well known. Available techniques for introducing polynucleotides include but are not limited to electroporation, transduction, cell fusion, the use of calcium chloride, and packaging of the polynucleotide together with lipid for fusion with the cells of interest. Once a cell has been transfected with the dominant negative MMR gene, the cell can be grown and reproduced in culture. If the transfection is stable, such that the gene is expressed at a consistent level for many cell generations, then a cell line results.

An isolated cell is a cell obtained from a tissue of humans or animals by mechanically separating out individual cells and transferring them to a suitable cell culture medium, either with or without pretreatment of the tissue with enzymes, e.g., collagenase or trypsin. Such isolated cells are typically cultured in the absence of other types of cells. Cells selected for the introduction of a dominant negative allele of a mismatch repair gene may be derived from a eukaryotic organism in the form of a primary cell culture or an immortalized cell line, or may be derived from suspensions of single-celled organisms.

A polynucleotide encoding for a dominant negative form of a MMR protein can be introduced into the genome of an animal by producing a transgenic animal. The animal can be any species for which suitable techniques are available to produce transgenic animals. For example, transgenic animals can be prepared from domestic livestock, e.g., bovine, swine, sheep, goats, horses, etc.; from animals used for the production of recombinant proteins, e.g., bovine, swine, or goats that express a recombinant polypeptide in their milk; or experimental animals for research or product testing, e.g., mice, rats, guinea pigs, hamsters, rabbits, etc. Cell lines that are determined to be MMR defective can then be used as a source for producing genetically altered genes encoding for therapeutic antigens in vitro by introducing whole, intact genes and/or chimeric genes encoding for a therapeutic antigen(s) into MMR defective cells from any tissue of the MMR defective animal.

Once a transfected cell line or a colony of transgenic animals has been produced, it can be used to generate new mutations in one or more gene(s) of interest. A gene of interest can be any gene naturally possessed by the cell line or transgenic animal or introduced into the cell line or transgenic animal. An advantage of using such cells or animals to induce mutations is that the cell or animal need not be exposed to mutagenic chemicals or radiation, which may have secondary harmful effects, both on the object of the exposure and on the workers. However, chemical mutagens may be used in combination with MMR deficiency, which renders such mutagens less toxic due to an undetermined mechanism. Hypermutable animals can then be bred and selected for those producing genetically variable cells that may be isolated and cloned to identify new cell lines that are useful for producing structurally altered polypeptides. Once an altered polypeptide is identified, the dominant negative MMR gene allele can be removed by directly knocking out the allele by technologies used by those skilled in the art or by breeding to mates lacking the dominant negative allele to select for offspring with a desired trait and a stable genome. Another alternative is to use a CRE-LOX expression system, whereby the dominant negative allele is spliced from the animal genome once an animal containing a genetically diverse protein profile has been established. Yet another alternative is the use of inducible vectors such as the steroid induced pIND (InVitrogen) or pMAM (Clonetech) vectors which express exogenous genes in the presence of corticosteroids.

Mutations can be detected by analyzing for alterations in the genotype of the cells or animals, for example by examining the sequence of genomic DNA, cDNA, messenger RNA, or amino acids associated with the gene of interest. Mutations can also be detected by screening for the production of antigenicity. A mutant polypeptide can be detected by identifying alterations in electrophoretic mobility, spectroscopic properties, or other physical or structural characteristics of a protein encoded by a mutant gene. One can also screen for altered function of the protein in situ, in isolated form, or in model systems. One can screen for alteration of any property of the cell or animal associated with the function of the gene of interest, such as but not limited to antigenicity.

According to another aspect of the invention, a high throughput mammalian cell-based assay is presented. A MMR defective cell line is transfected with a secretion cassette containing a leader sequence for secretion at the N-terminus fused to the target antigen. Cells are grown and clones are plated by limiting dilution into microtitre plates and conditioned medium are screened for antigenic peptides. The advantage of such an approach is that the antigen is more similar to the natural polypeptide than it would be if produced by bacterial, yeast or baculovirus systems which tend to cause misfolding and/or distorted post-translational modifications.

Claim 1 of 10 Claims

We claim:

1. A method for making a therapeutically hypermutated immunogen, comprising introducing into a cell that expresses a gene encoding a preselected immunogen in vitro a polynucleotide comprising a dominant negative allele of a mismatch repair gene, wherein said dominant negative allele is a truncation mutant of a PMS2, and selecting cells that comprise a mutation in said gene encoding said preselected immunogen.

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