EP1100544A1 - Allogene, zelluläre immunogene, die als krebsimpfstoff einsetzbar sind - Google Patents

Allogene, zelluläre immunogene, die als krebsimpfstoff einsetzbar sind

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Publication number
EP1100544A1
EP1100544A1 EP99933855A EP99933855A EP1100544A1 EP 1100544 A1 EP1100544 A1 EP 1100544A1 EP 99933855 A EP99933855 A EP 99933855A EP 99933855 A EP99933855 A EP 99933855A EP 1100544 A1 EP1100544 A1 EP 1100544A1
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EP
European Patent Office
Prior art keywords
oncogene
transgene
proto
cells
host
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EP99933855A
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English (en)
French (fr)
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EP1100544A4 (de
Inventor
Michael S. Halpern
James M. England
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Alleghney University of Health Sciences
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Alleghney University of Health Sciences
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Publication of EP1100544A1 publication Critical patent/EP1100544A1/de
Publication of EP1100544A4 publication Critical patent/EP1100544A4/de
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001103—Receptors for growth factors
    • A61K39/001106—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001152—Transcription factors, e.g. SOX or c-MYC
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A61K39/001164—GTPases, e.g. Ras or Rho
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination

Definitions

  • oncogenes Genes that confer the ability to convert cells to a tumo ⁇ gemc state aie known as oncogenes
  • the transforming ability of a number of retroviiuses has been localized in individual viral oncogenes (generally v-onc)
  • Cellulai oncogenes generally c-onc
  • retroviral oncogenes may represent escaped and or partially metamorphosed cellular genes that are incorporated into the genomes of transmissible, infectious agents, the retroviruses
  • Some c-onc genes mt ⁇ nsicalh lack oncogenic properties, but be converted by mutation into oncogenes whose transforming activity reflects the acquisition of new properties, or loss of old properties
  • Amino acid substitution can convert a cellular proto-oncogene into an oncogene
  • Gene amplification provides another mechanism by which oncogene expression may be increased
  • Many tumor cell lines have visible regions of chromosomal amplification
  • a 20-fold c-m ⁇ c amplification has been observed in certain human leukemia and lung carcinoma lines
  • the related oncogene N-myc is five to one thousand fold amplified m human neuroblastoma and retinoblastoma
  • the proto-oncogene c-myb is amplified five to ten fold
  • established cell lines are prone to amplify genes, the presence of known oncogenes in the amplified regions, and the consistent amplification of particular oncogenes in many independent tumors of the same type, strengthens the correlation between increased expression and tumor growth
  • Immunity has been successfully induced against tumor formation by inoculation with DNA constructs containing v-onc genes, or by inoculation with v-onc proteins or peptides
  • a series of reports describe a form of "homologous" challenge m which an animal test subject is inoculated with either v-src oncoprotein or DNA constructs containing the v-src gene.
  • Protective immunity was induced against tumor formation by subsequent challenge with v- src DNA or v-src-induced tumor cells. See, Kuzumaki et al , JNCI (1988), 80:959-962; Wisner et al. , J. Virol. (1991), 65:7020-7024; Halpern et al.
  • a challenge is said to be “homologous” where reactivity to the product of a targeted gene is induced by immunization with the same gene, the corresponding gene product thereof, or fragment of the gene product.
  • a challenge is "heterologous” where reactivity to the product of a targeted gene is induced by immunization with a different gene, gene product or fragment thereof.
  • WO 92/14756 (1992) describes synthetic peptides and oncoprotein fragments which are capable of eliciting T cellular immunity, for use in cancer vaccines.
  • the peptides and fragments have a point mutation or translocation as compared to the corresponding fragment of the proto-oncogene.
  • the aim is to induce immunoreactivity against the mutated proto-oncogene, not the wild-type proto-oncogene.
  • WO 92/14756 thus relates to a form of homologous challenge.
  • EP 119.702 (1984) describes synthetic peptides having an amino acid sequence corresponding to a determinant of an oncoprotein encoded by an oncogemc virus, which determinant is vicinal to an active site of the oncoprotein.
  • the active site is a region of the oncoprotein required for oncoprotein function. e.g.. catalysis of phosphorylation.
  • the peptides may be used to immunize hosts to elicit antibodies to the oncoprotein active site.
  • EP 119.702 is thus directed to a form of homologous challenge.
  • the protein product encoded by a proto-oncogene constitutes a self antigen and.
  • the immunogen selected to induce immunity comprised a purified peptide of the pl85 HER_2 " e " protein, and not a cellular immunogen.
  • cytotoxic T cells elicited in the latter report were not, however, shown to recognize tumor cells, but only targets that bound the synthesized peptides.
  • Other work (Dahl et al.. J. Immunol. (1996), 157:239- 246) has demonstrated that cytotoxic cells may recognize targets that bind peptide but fail to recognize targets that endogenously synthesize peptide. It is thus unclear whether the cytotoxic cells elicited by Disis et al. would be capable of recognizing rumor cells. In any event, no protection against tumor growth was demonstrated by Disis et al. Peoples et al , Proc Natl Acad Sci USA (1995), 92 432-436.
  • Non-mutant, peptide antigen-presenting cells have their HLA class I molecules already loaded with endogenous peptides.
  • mutant lines lack the TAP genes (encoding the transporters associated with antigen presentation) Class I binding of mternally-de ⁇ ved peptides is significantly lowered, and empty class I molecules are present on the cell surface and available for binding of exogenously added peptides This availability of peptide binding sites on membrane-bound class I allows examination of whether a given peptide will (l) even bind to class I, and (n) function as a target m cytotoxic T cell assay s
  • the need for a mutant cell line for deduction of candidate immunizing peptide sequences limits the usefulness of peptide-based immunization schemes Fendly et al. , J.
  • Biol Response Modifiers (1990), 9:449-455 ' present an account of a polypeptide-based immunotherapy.
  • Purified polypeptide corresponding to the extracellular domain of the pl85 HER"2 " e " protein was obtained from a transfected cell line.
  • the purified peptide was employed in the immunization of guinea pigs.
  • the immunized animals developed a cellular immune response, as monitored by delayed-type hypersensitivity .
  • Antisera derived from immunized animals specifically inhibited the in vitro growth of human breast tumor cells overexpressing pl85 HER 2/ "TM.
  • peptides for immunization are of necessity limited to immunization with a single haplotype. There are approximately thirty HLA types in man. In each case of peptide immunization, one must be careful to select peptides which match the host HLA type. The selected peptide must be immunogenic in the host and be capable of presentation to host immune system cells.
  • a method of vaccinating a host against disease associated with the overexpression of a target proto-oncogene comprises
  • transfecting allogeneic donor cells with at least one transgene construct comprising at least one transgene cognate to the target proto-oncogene and a strong promoter to drive the expression of the transgene in the transfected cells, the transgene encoding a gene product which induces host immunoreactivity to host self-determinants of the product of the target proto-oncogene gene,
  • the transgene comprises wild-type or mutant retroviral oncogene DNA
  • the transgene comprises wild- type or mutant proto-oncogene DNA of a species different from the host species
  • the mutant DNA is preferably nontrans forming.
  • the mutant DNA preferably comprises a deletion mutation in a region of the DNA which is essential for transformation.
  • the donor cells are transfected with a plurality, most preferably at least five, different transgene constructs, each construct encoding a different deletion mutation.
  • the mutant DNA has at least about 75 % homology, more preferably at least about 80% homology, most preferably at least about 90% homology, with the corresponding wild-type oncogene or proto-oncogene DNA.
  • the invention is further directed to a cellular immunogen for immunizing a host against the effects of the product of a target proto-oncogene. the overexpression of which is associated with a cancer.
  • the cellular immunogen comprises the allogeneic donor cells which have been transfected with at least one transgene construct, as described above.
  • the invention is also directed to a method of preparing the cellular immunogen, by (a) providing allogeneic donor cells, and (b) transfecting the cells with at least one transgene construct, as described above.
  • the cells transfected with the transgene are preferably rendered non-dividing prior to insertion into the body of the host.
  • allogeneic as used herein to describe the genetic relationship between organisms has its ordinary meaning in biology, that is. organisms are allogeneic if they are genetically dissimilar (i.e. , non-identical) members of the same species.
  • Cells comprising an "allogeneic" graft are derived from organisms which are the same species as the host, but are not genetically identical .
  • a polynucleotide sequence is homologous (i.e.. is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence. or that a polypeptide sequence is identical to a reference polypeptide sequence.
  • the term "cognate” as used herein refers to a gene sequence that is evolutionarily and functionally related between species
  • the human c-myc gene is the cognate gene to the mouse c-myc gene, since the sequences and structures of these two genes indicate that they are highly homologous and both genes encode proteins which are functionally equivalent
  • donor cell is meant a cell which is used herein for immunization of a host
  • the expression “donor cell” is meant to include not only cells freshly harvested from donor individuals, but also cultured cells which may have been obtained from a cell or tissue bank or other biological repository
  • allogeneic donor cell is meant a cell which is allogeneic with respect to the host, l e . the donor cell, upon transplantation into the host, would compromise an allogeneic graft
  • homology is meant the degree of sequence similarity between two different amino acid sequences, as that degree of sequence similarity is derived by the FASTA program of Pearson and Lipman, Proc Natl Acad Sci USA (1988). 85 2444-2448, the entire disclosure of which is incorporated herein by reference
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship
  • a nucleic acid is "operably linked' when it is placed into a functional relationship with another nucleic acid sequence
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence
  • Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein coding regions, contiguous and m reading frame
  • transfection is meant to have its ordinary meaning that is. the introduction of foreign DNA into eukaryotic cells
  • transgene is meant a foreign gene that is introduced into one or more donor cells
  • transgene construct is meant DNA containing a transgene and • additional regulatory DNA, such as promoter elements, necessary for the expression of the transgene in the donor cells used to innoculate the host.
  • Figs. 1A and IB are plots of the mean tumor diameter over time following subcutaneous wing web inoculation of 1-day-old line TK (Fig. 1A) and line SC (Fig. IB) chickens with 100 ug ⁇ of tumorigenic plasmids pcsrc527 (—* • —), pVSRC-Cl (— •— ) or pMvsrc ( — ⁇ — ).
  • the mean tumor diameter (mm) at a particular time point and for any one group of TK or SC line chickens inoculated was computed as the sum of the diameters of the primary tumors divided by the number of chickens surviving to that point.
  • the ratios at each time point show, for a particular group, the number of chickens bearing palpable tumors to the total number of survivors to that point (standard typeface for pcsrc527, italics for pVSRC-Cl , bold typeface for pMVsrc). Error bars (unless obscured by the symbol) indicate standard error.
  • Figs. 2A and 2B are plots of the growth of challenge (wing web) tumors in test and control line TK chickens under conditions of (i) priming and homologous challenge with plasmid p ⁇ src527 (Fig. 2A: — ⁇ — . test; — * — . control), or (ii) priming and homologous challenge with plasmid pVSRC-Cl (Fig. 2B: — O — , test; — • — , control). Test chickens were primed at 1 day posthatch with 100 ⁇ g of construct; test and control chickens were challenged at five weeks posthatch with 200 ⁇ g of construct. The mean challenge diameter was computed as in Figs. 1A and IB.
  • the ratio of chickens bearing palpable challenge tumors to total number of survivors to that point is indicated (standard typeface for control group . bold typeface for test group) .
  • the statistical comparison between the mean challenge tumor diameters of the test versus the control group at a particular time point was made using a two-tailed student's t test, *(p ⁇ 0.05). **(p ⁇ 0.01). ***(p ⁇ 0.001).
  • the statistical comparison between the ratios of chickens bearing palpable challenge tumors to total number of survivors of the test versus the control group at a particular time • point was made using a chi-squared test; the paired ratios are underlined for only those time points where p ⁇ 0.05. Error bars indicate standard error.
  • Figs. 3 A and 3B are plots of the growth of challenge (wing web) tumors in TK chickens under conditions of (i) priming with plasmid pVSRC-C 1 and heterologous challenge with plasmid pcsrc527 (Fig. 3 A: — ⁇ — , test; — * — . control) or (ii) priming with p ⁇ src527 and heterologous challenge with pVSRC-Cl (Fig. 3B: — O — , test; — • — . control). Test chickens were primed at 1 day posthatch with 100 ⁇ g of construct; test and control chickens were challenged at five weeks posthatch with 200 ⁇ g of construct.
  • the mean challenge tumor diameter was computed as in Figs. 1A and IB. At each time point the ratio of chickens bearing palpable challenge tumors to total number of survivors to that point is indicated (standard typeface for control group, bold typeface for test group). Statistical comparisons were made between test and control groups at a particular time point as described for Figs. 2 A and 2B. [*(p ⁇ 0.05). **(p ⁇ 0.01). ***(p ⁇ 0.001), for the student's t test] , and the paired ratios are underlined for only those time points where, in the chi-squared test. p ⁇ 0.05. Error bars indicate standard error.
  • a vaccination strategy is provided to prevent development of cancers.
  • the vaccination method may be carried out on a subject at risk for a particular cancer, but before the development of the cancer.
  • the practice of the invention may serve for the immunoprevention of prevalent human cancers, such as colon carcinoma, breast carcinoma, and various lymphomas whose progress is accompanied by the overexpression of a cellular proto-oncogene.
  • the vaccination strategy of the present invention relies on the induction of an immune response that targets tumor cells by virtue of the recognition of the proto-oncogene-specific antigenicity .
  • the aim of the vaccine protocol is to induce reactivity to self-determinants of an overexpressed proto- oncogene product.
  • the strategy exploits the structural relatedness between the product of the cellular proto-oncogene and that of the product of genes cognate to the target proto-oncogene
  • the cognate gene may comprise a wild-type or mutant cognate retroviral oncogene or a wild-type or mutant proto-oncogene of a species different from the host species
  • the starting point of the vaccine strategy is the high degree of primary sequence homology that exists between the protein product of a targeted proto-oncogene and that of its cognate retroviral oncogene. or between the proto-oncogene product and the product of a cognate proto-oncogene from a different species
  • the present invention is not based on the immune recognition of a
  • this sequence homology permits application of the following strategy, which can be employed either prophylactically or therapeutically under conditions of cell- surface expression, oi other forms of adjuvamcity , as chosen to enhance immunogenicity (a) transfection of allogeneic donor cells with a DNA construct comprising a transgene cognate to the target proto-oncogene, which transgene encodes a gene product which induces host lmmunoreactivity to host self- determmants of the product of the target proto-oncogene, (b) transplantation of the transfected cells into the body of the host to obtain expression of the transgene in the host, and thus immunity against the pioto-oncogene product
  • the invention relies on the targeting of a self-determinant found on an overexpressed or overabundant proto-oncogene-encoded product
  • the foreign peptide elements of the immunizing oncogene product will trigger peripheral lymphocytes exhibiting a weak cross reactivity for
  • the immune strategy exploits the antigemcity of two alternative types of determinants (1) tumor-associated antigemc determmant(s) induced as a consequence of the activity of the oncogene product, e.g. , an enzymatic • modification of a cellular protein effected by the oncogene product, or (2) tumor associated antigenic determinant(s) intrinsic to the oncogene-encoded product itself.
  • the difficulty in exploiting the first alternative by traditional means, i.e. , antigen purification is that at present little or no systematic information exists bearing on the properties of an antigen that, though oncogene-induced, is not oncogene-encoded. This situation makes purification of any such antigen problematic. However, this problem is obviated from the outset by the present invention which utilizes alogeneic donor cells which, as transfected in culture by the cognate retroviral oncogene, would express the relevant antigenicity .
  • the protocol of immunization according to the present invention primes the host to determinants of the oncogene product itself.
  • a consequence of this immunization is induction of T-cell reactivity to the divergent, i.e foreign, peptide determinants of the retroviral oncogene product, i.e.. those peptide determinants that show sequence differences with the positionally homologous determinants of the cellular proto-oncogene product.
  • the present vaccination method does not have as its object the deliberate targeting of non-self determinants generated by proto-oncogene mutations. Unlike prior • vaccination methods designed to target such mutation-driven non-self determinants, it is the aim of the present invention to induce reactivity for self- determinants in the overexpressed product of tumor associated and overexpressed proto-oncogenes.
  • the value of a non-self, but closely related to self, peptide is that it may more readily activate those T cells that have both a weak cross reactivity for the cognate self peptide and an activation threshold (determined by the tightness of binding to the T cell receptor) too high to be triggered by the self peptide.
  • cognate non-self is inductive of a good immune response, simply because it does in fact constitute nonself.
  • the non-self immune response is expected to predispose the induction of the inevitably weaker response to the self determinants on the same protein product, since the resultant cytokine release provides local help to initiate the weaker anti-self response.
  • immunization with cells transfected with a transgene construct expressing the v-src oncogene product induces reactivity to the product of the c- src proto-oncogene, thereby conferring protection against the growth of tumors displaying overexpression of the c-src proto-oncogene.
  • the methodology described therein utilizes excised host cells for preparing the cellular immunogen.
  • the cellular immunogen is prepared from cells obtained from a donor other than the patient, and other than an identical twin of the patient.
  • PCT/US97/00582 describes an inoculation constituting a syngeneic transfer
  • the present invention relies upon an allogeneic transfer. Given the outbredness of the human population, there will inevitably be allelic differences between the donor and the patient. These differences do not impede the induction of immunity to the cognate proto-oncogene encoded product overexpressed in the allogeneic transfectants used as immunogen.
  • This pathway of antigen transfer from the donor cells to the bone marrow-derived antigen presenting cells of the recipient exemplifies an in vivo process designated the "exogenous pathway , to distinguish it from the "classical pathway”: the latter as based on processing of antigen-presenting cells themselves.
  • exogenous pathway permits recipient priming to a cellular antigen introduced in the context of a non-recipient class I allele. thereby permitting the use of allogeneic donor cells, mismatched with reference to the recipient at none or at all of the diploid 6 human class I loci, for priming to an overexpressed cognate proto-oncogene.
  • patients with a family history of a cancer characterized by the overexpression of a particular proto-oncogene are selected for immunization.
  • patients whose tumors can be shown to overexpress the proto-oncogene are selected.
  • Overexpression of a proto-oncogene may derive from an increase over a basal level of transcription.
  • Overexpression may also derive from gene amplification, that is, an increase in gene copy number, coupled with a basal or elevated level of transcription.
  • Proto- oncogene overexpression may be assayed by conventional probing techniques. such as described in Molecular Cloning: A Laboratory Manual J. Sambrook et al . eds.. Cold Spring Harbor Laboratory Press, 2nd ed. 1989.
  • the level of target proto-oncogene expression may be determined by probing total cellular RNA from patient cells with a complementary probe for the relevant mRNA.
  • Total RNA from the patient cells is fractionated in a glyoxal/agarose gel, trans- ferred to nylon and hybridized to an appropriately labelled nucleic acid probe for the target mRNA.
  • the number of relevant mRNA transcripts found in the patient cells is compared to that found in cells taken from the same tissue of a normal control subject.
  • the expression level of a target proto-oncogene may be assessed by assaying the amount of encoded protein which is formed.
  • Western blotting is a standard protocol in routine use for the determination of protein levels. See Molecular Cloning, supra. Chapter 18, incorporated herein by reference. Accordingly, a cell lysate or other cell fraction containing protein is electrophoresed on a polyacrylamide gel. followed by protein transfer to nitrocellulose, and probing of the gel with an antibody specific for the protein in question. The probe step permits resolution of the desired protein from all other proteins in the starting mixture.
  • the bound antibody may be prelabeled, e.g. , by a radioisotope such as 125 I, so as to permit its detection on the gel.
  • a secondary reagent (usually an anti- immunoglobin or protein A) may be radiolabeled or covalently coupled to an enzyme such as horseradish peroxidase or alkaline phosphatase
  • an enzyme such as horseradish peroxidase or alkaline phosphatase
  • the strength of the signal is proportional to the amount of the target protein
  • the strength of the signal is compared with the signal from a sample analyzed in the same manner but taken from normal as opposed to tumor tissue
  • Table 1 includes a partial list of representative proto-oncogenes. the overexpression of which has been associated with one or more malignancies Each listed proto-oncogene is a target proto-oncogene according to the present invention The corresponding oncogene, of which the target proto-oncogene is the normal cellular homolog, is also identified This list of target proto- oncogenes is intended to be representative, and not a complete list
  • AKT-2 ovarian v-Akt is the oncogene of the AKT8 virus, which induces lymphomas in mice
  • AKT-2 pancreatic Cheng et al (1996) Proc. Natl. Acad. Sci. USA 93(8):3636-41 : Amplification of AKT-2 has been detected in 10% of pancreatic carcinomas.
  • c-crbB-2 bladder c-Erb -2 is also known as ⁇ ER2/neu.
  • V-erbB is the oncogene of the avian erythroblastosis virus.
  • MDM-2 leukemia MDM-2 is the mu ⁇ ne double m ⁇ nute-2 oncogene
  • c-m ⁇ b colon V- vb is the oncogene of the avian myeloblastoma virus 1 Ramsay et al , (1992) Cell Growth and Diff 3(10) 723-30 c-myb levels were alway s higher in colon cancer samples than normal tissue
  • c-m ⁇ c breast V-myc is the oncogene of the avian myelocytoma virus 1 Lonn et al , (1995) Cancer 75(11) 2681- 7 Amplification of c-myb occurs in 16% of patients with breast cancer. 2. Hehir et al , ⁇ (1993) J of Surg. Oncology 54(4): 207-9: c-m c overexpression was found in 60% of breast carcinoma samples. 3. Kreipe et al , (1993) Cancer Research 53(8): 1956-61 : Amplification of c-myc was found in 52.6% of samples that displayed a Ki-Sl labelling index exceeding 30% . 4. Watson et al , (1993) J. Nat. Cancer Inst. 85(l l):902-7: Amplification of c-myc occurs in up to 20 - 30% of breast cancers. 5. Berns et al .
  • c-ras lung Ras oncogenes were first recognized as the transforming genes of Harvey and Kirsten murine sarcoma viruses. Lorenz et al , (1994) Clin. Invest. 72(2): 156-63: a 13-fold increase in overexpression of c-Ki-ras was observed . 18 % of tumors displayed strong overexpression of c-Ki- ras.
  • c-src breast V-src is the oncogene of the Rous sarcoma virus. which induces sarcomas in chickens . Muthuswamy et al , (1994) Mol. and Cell. Biol 14(1) 735-43 c-erbB-2-mduced mammary tumors possessed 6-8-fold higher c-src kinase activity than adjacent epithelium
  • c- ⁇ es colon V-ves is the oncogene of two avian sarcoma viruses. Esh sarcoma virus and Y73 1 Pena et al , (1995) Gastroent 108(1) 117-24 Twelve to fourteen-fold higher expression of c-ves was found m colomc transforming oncogene adenomas compared to normal mucosa Activity of c-yes was elevated in adenomas that are at greatest risk foi developing cancer 2 Park et al . (1993) Oncogene 8(10):2627-35: A ten to 20-fold higher ⁇ than normal activity of c-yes was observed in 3 out of 5 colon carcinoma cell lines. A 5-fold higher than normal activity was found in 10 out of 21 primary colon cancers, compared to normal colonic cells.
  • a transgene construct is engineered comprising a transgene which is cognate to the target proto-oncogene (hereinafter "cognate transgene” or “CTG”).
  • CCG target proto-oncogene
  • the transgene is selected such that it encodes a gene product which induces host immunoreactivity to host self- determinants of the product of the target proto-oncogene.
  • the transgene should be expressed to very high levels in the transfectants.
  • the construct should contain a strong promoter.
  • the product encoded by the cognate gene must have a high degree of sequence homology with the product of the target proto-oncogene. but also must display some amino acid differences with the target proto-oncogene product.
  • xenogenic proto-oncogene is intended to have its normal biological meaning, that is. a property or characteristic referring or relating to a different species.
  • a xenogenic proto-oncogene is meant to include the a homologous proto-oncogene of a species other than the host organism species. It may be appreciated that in the case of a target proto- oncogene. e.g.
  • a xenogenic homologue is advantageously utilized as the source of the DNA for the cognate transgene.
  • a more effective immunogenic stimulus would depend ⁇ on the particular sequence, and not on the distinction between a retroviral oncogene and a xenogenic proto-oncogene in terms of their relative transforming capacity.
  • a retroviral oncogene may be better at providing a tolerance-breaking immunogenic stimulus, and in other cases, a xenogenic proto-oncogene may be more effective.
  • the retroviral oncogene or xenogenic proto-oncogene DNA forming the CTG may comprise the wild type oncogene or proto-oncogene DNA. More preferably, a mutant DNA is utilized, which is engineered so as to be non- transforming in the host.
  • the DNA is mutated to include one or more nucleotide insertions, deletions or substitutions which will encode an oncogene product which is nontransforming in the host, but retains the requisite degree of sequence homology with respect to the target proto-oncogene.
  • a cognate transgene deletion mutant hereinafter "dCTG" is preferred.
  • a protein sequence is generally considered " cognate " with respect to the target proto-oncogene-encoded protein if it is evolutionarily and functionally related between species.
  • a more precise view of cognation is based upon the following sequence comparison carried out utilizing the FASTA program of Pearson and Lipman, Proc. Natl. Acad. Sci. USA (1988), 85:2444- 2448. the entire disclosure of which is incorporated herein by reference. Cognation is attained upon satisfying two criteria imposed by FASTA; (i) alignment of segments corresponding to at least 75 % of the target proto- oncogene 's encoded amino acid sequence; (ii) at least 80% amino acid identity within the aligned sequences.
  • the segments of the target proto-oncogene protein sequence and protein test sequence satisfying the two criteria are referred to as "homology regions" . Accordingly, at least 75 % of the target proto-oncogene protein sequence is alignable with the test sequence.
  • the alignable segments or homology regions may. however, represent less than 75 % of the total test polypeptide chain for the case of test sequences that may significantly exceed the target proto-oncogene protein in length.
  • One skilled in the art armed with the FASTA program, may ⁇ survey existing sequence data bases (either protein sequences or DNA sequences, insofar as the amino acid sequence is determined by FASTA for all reading frames) for test sequences which are cognate with respect to the target proto- oncogene.
  • cognate test sequences e.g. feline MDM-2, as likely to be cognate to human MDM-2
  • FASTA FASTA to verify the presumed cognation, according to the criteria set forth above.
  • an immunogenic stimulus is provided that (i) is directed against the foreign protein and (ii) with a lower probability, induce an anti-self response.
  • the CTG is selected such that the gene product will yield the greatest immunogenic stimulus to induce anti-self reactivity.
  • overall sequence homology preferably greater than about 75 %) is maintained, the presence of scattered amino acid differences is desired, since any one residue would likely have a relatively low probability of inducing self- reactivity. Moreover, the greatest number of residue differences would be advantageous, consistent with maintaining the requisite degree of general sequence homology.
  • cells are biopsied from a normal volunteer of particular haplotype
  • the cells are transfected with a CTG construct, preferably a dCTG construct, satisfying the criteria set for cognition More preferably, the cells are transfected with multiple dCTGs preferably at least five dCTGs, satisfying the criteria for cognition
  • the at least five dCTGs are selected to display ammo acid differences that essentially extend throughout the polypeptide chains of the encoded sequences
  • the transfected cells are then used to immunize the volunteer in accordance with the immunization method of the present invention After immunization, the human subject is tested m a standard delayed hypersensitivity (DH) reaction with 10 4 -10 6 irradiated, autologous fibroblasts as transfected with the same dCTG (or series of dCTGs
  • DH delayed hypersensitivity
  • any given amino acid difference between the CTG-encoded product and the proto-oncogene-encoded product has a low probability of being a "tolerance-breaker" .
  • the number of different dCTGs is preferably five or more.
  • the multiple dCTGs show amino acid differences that essentially extend throughout the polypeptide chains of the encoded sequences.
  • the dCTGs would be selected to maximize amino acid differences and, at the same time, make sure that differences are found all along the polypeptide chain. It would thus not be preferable to select a battery of deletions all from within the same domain of the polypeptide chain.
  • Non-transforming cognate transgene variants are most advantageously derived via deletion of a sequence essential for transformation. Unlike point mutations which are potentially reversible due to back mutations, deletion mutations are irreversible. Furthermore, deletion mutations do not possess the inherent disadvantage attaching to point mutations, namely, even though the requirement for generation of an acceptable cognate transgene is for a qualitative difference with the wild type, i.e. , non-transforming versus transforming, any given point mutation may be neutral or else quantitative in its effect, that is.
  • the mutation may reduce but not totally eliminate transformability
  • a deletion is created in a region of the cognate transgene which encodes an amino acid sequence required for transformation Consonant with non-transformability, the smallest deletion possible so as to leave intact the bulk of the antigenicity of the transgene product is selected
  • the deletion mutant is engineered to include at least a part of the region identified as critical for transformation In those cases where essential amino acids have been identified, the deletion will span these residues
  • PCR polymerase chain reaction
  • Each CTG or dCTG cell culture is inoculated into nude mice in the absence of any treatment to render the cells non-dividing Those dCTGs which do not ⁇ leld tumors in the mice even after prolonged observation are then utilized as transgenes for the biopsied human cells which, upon transfection with the transgene.
  • the dCTGs are selected with the smallest deletion mutant consonant with non-transformability Some CTGs representing xenogenic proto-oncogenes may not be tumo ⁇ genic in the 3T3/nude mouse assay For any such non-transforming CTG.
  • the deletion would be engineered so as to remove the homologous region to that deleted in the particular dCTG that corresponds to the deletion in the corresponding retroviral oncogene dCTG
  • transgene construct may comprise mutant oncogene or proto-oncogene DNA which is nontransforming
  • the cells are irradiated with a radiation dosage sufficient to render them non-dividmg
  • an oncogemcity testing regimen may take the form of three separate assays (I) dCTG transfection of NIH 3T3 cells, followed by inoculation into nude mice, (n) dCTG transfection of human fibroblasts, followed by inoculation into nude mice, and (in) dCTG transfection of human fibroblasts, followed by an in vitro test of anchorage-dependent growth In principle, all three should be negative to ⁇ ahdate the use of any given dCTG in the vaccination method of the present m ⁇ ention According to the oncogemcity assay (1), after stable transfection of NIH 3T3 cells with the test dCTG, the transfectants are inoculated into nude mice Tumo ⁇ genicity of the transfectants in the mice is
  • Anchorage independence as defined by the ability of cells to grow when suspended in semisohd medium, is a common phenotype acquired by human tumor cells particularly those tumor cells of mesenchymal ongm, such as fibrosarcomas While assay (in) has no ⁇ n o readout, it offers an independent test of the critical issue of dCTG oncogemcity m human cells
  • each dish is trypsinized and reseeded into a 175-c ⁇ r ⁇ flask.
  • cultures are selected in G418 (400 ⁇ g/ml), and the flasks are then trypsinized and cells are replated in the same flask to disperse the G418-resistant colonies into a diffuse lawn of cells.
  • the cells are harvested and washed with serum-free medium prior to injection.
  • One injection of 5 X 10 6 cells into the right flank and one injection of 1 X 10 7 cells into the left flank, each in a volume of 200 ⁇ l, are done on each nude mouse. Injection sites are monitored at 3- or 4-day intervals for 100 days.
  • Assay (iii) involves a test of the in vitro anchorage-dependent growth of dCTG-transfected human fibroblasts. The assay is carried out as described in Stevens et al. , J. CancerRes. and Clin. Oncol. 1989, 115: 118-128. 1 x 10 s cells are seeded per 60-mm dish into 0.33 % Noble agar over a 6-ml 0.5 % agar base layer in Hams F10 supplemented with 6% fetal bovine serum. A portion of the agar suspension is diluted with Hams F10 plus 6% fetal calf serum to 200 cells '5 ml to determine the cloning efficiency of these cells when seeded into plastic 60-mm dishes.
  • Agar dishes are fed with 1 ml Hams F10 supplemented with 6% fetal bovine serum on the 1st and 15th day after seeding.
  • all agar colonies > 75 ⁇ m in diameter are counted and the colony counts are normalized to the plating efficiencies which aliquots of the initially seeded cells showed on plastic.
  • This comparison, or normalization, of the agar colony counts to the plastic dish colony counts is useful in identifying and correcting for any mechanical artifacts which might result from the seeding into agar of dead cells that had persisted from the initial transfection treatment or from heat-induced cell death, which might have occurred while suspending cells in molten agar during the process of seeding the agar dishes.
  • the following is a partial list of various deletions which, based upon published accounts of experiments with human or animal cells, are believed to render the identified CTG non-tumorigenic.
  • a dCTG The engineering of vectors for expression of a particular CTG. preferably a dCTG. is based on standard methods of recombinant DNA technology, i.e. insertion of the dCTG via the polylinker of standard or commercially available expression vectors.
  • the dCTG is operably linked to a strong promoter.
  • a strong promoter is a promoter which achieves constitutively high expression of the dCTG in the transfected cells.
  • Each promoter should include all of the signals necessary for initiating transcription of the relevant downstream sequence.
  • the allogeneic donor cells which may be transfected to derive the cellular immunogens of the present invention must express class I MHC and be susceptible to isolation and culture.
  • Fibroblasts express class I MHC and may be cultured.
  • Other preferred donor cells are bone marrow-derived antigen- • presenting cells such as macrophages, follicular dendritic cells, and Langerhans cells, for example.
  • the cells may comprise primary explants or established cell lines.
  • Primary skin fibroblasts may be obtained as follows. Punch biopsies can be performed by a competent physician as a standard clinical procedure. Each biopsy yields a starting population of 1-2 X 10 7 cells that would proliferate in culture. Methods for the preparation of tissue cultures of human fibroblasts are well developed and widely used.
  • the expression vector carrying the dCTG is used to transfect allogeneic donor cells according to conventional transfection methods.
  • One method of transfection involves the addition of DEAE-dextran to increase the uptake of the naked DNA molecules by a recipient cell. See McCutchin and Pagano, J. Natl. Cancer Inst. (1968) 41 :351-7.
  • Another method of transfection is the calcium phosphate precipitation technique which depends upon the addition of Ca" to a phosphate-containing DNA solution. The resulting precipitate apparently includes DNA in association with calcium phosphate crystals. These crystals settle onto a cell monolayer; the resulting apposition of crystals and cell surface appears to lead to uptake of the DNA. A small proportion of the DNA taken up becomes expressed in a transfectant.
  • transfection is carried out by cationic phospholipid- • mediated delivery.
  • polycationic liposomes can be formed from N- [l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) or related liposome-forming materials.
  • DOTMA N- [l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOTMA N- [l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • RNA-transfection RNA-transfection
  • LipofectAMINETM Reagent Cat. No. 18324-012, Life Technologies, Inc. , Gaithersburg, MD
  • DOSP A 3-dioleyloxy-N-[2(sperminecarboxamido)ethyl-N,N-dimethyl- 1 -propanaminium trifluoroacetate
  • DOSP A Cosmetic Abstracts Registry name : N-[2-( ⁇ 2.5-bis[(3-aminopropyl)amino]-l-oxypentyl ⁇ amino)ethyl]-N,N-dimethyl- 2,3-bis(9-octadecenyloxy)-l-propanaminium trifluoroacetate)
  • DOPE neutral lipid dioleoyl phosphatidylethanolamine
  • transient expression is its rapidity, i.e. there is no requirement for cellular proliferation to select for stable integration events . This rapidity could conceivably be of major clinical importance, in cases of an already metastatic tumor burden, wherein the weeks required for selection of stable transfectants may simply not be available to the clinician.
  • transient transfection There are, nonetheless, two general disadvantages to the use of transient transfection.
  • the first is that expression usually peters out after a few days, in contrast to the continual expression in the case of stable transfection. This is not particularly crippling in terms of our immunization protocol.
  • the inoculated, irradiated cells used for immunization would likely not survive in vivo for more than 4 or 5 days, in any case. (For the same reason, immunological rejection of the allogeneic donor cells by the host is not a concern).
  • the nominal advantage accruing to stable transfection that of a long-duration expression by the progeny of the parental inoculated cell, is not of particular relevance in the case of the immunizing regime described herein, which is based • on the use of non-dividing, probably short-lived cells.
  • transient transfection resides in the fact that it yields a cell population, only a subset of which has actually been transfected and thus expresses the protein encoded by the transgene.
  • This problem is obviated in the case of stable transfection, wherein over time one can develop a pure population of transfectants via selection for a resistance marker, such as neo, under conditions of clonal proliferation of the initial stable transfectants. i. e. daughter cells of transiently transfected cells lack the transgene. in contrast to the case with stable transfectants.
  • the percentage of cells exhibiting dCTG expression may be determined by an immunohistology assay.
  • an immunohistology assay In this procedure, a small number of cells (-500) from the harvested pellet following centrifugation of transfected cells are deposited on a cover slip and fixed with cold acetone. At this point, a standard immunohistological assay is carried out with the cells on the cover slip. i.e. addition of a primary monoclonal antibody reactive to the dCTG-encoded protein, followed by the addition of a developing antibody, e.g. a fluorescent tagged antibody reactive to the primary monoclonal antibody.
  • Measurement of the percentage of cells scoring as dCTG-positive in the fluorescent assay allows a determination of the number of positive transfectants in the starting culture, and thus the number of total cells to be used for immunization to arrive at the desired number of dCTG-positive cells to be inoculated in the patient. If, as would be almost certain, the percentage of cells scoring as • dCTG-positive is less than one hundred percent, one can simply increase the number of cells to be used for immunization, so as to include the desired number of transfectants. The non-transfected cells in the immunizing population would simply represent x-irradiated, autologous fibroblasts that would constitute no danger to the patient.
  • the transfected cells Prior to innoculation into the host, the transfected cells are preferably irradiated.
  • the transfectants are irradiated with a radiation dose sufficient to render them non-dividing, such as a dose of 25 By or 2500R.
  • the cells are then counted by trypan blue exclusion, and about 2 X 10 7 irradiated transfectants are resuspended in a volume of 0.2-0.4 ml of Hanks Balanced Salt Solution.
  • the transfected cells are inserted into the host to achieve vaccination.
  • Intramuscular, intraperitoneal or subcutaneous inoculation will suffice to yield a systemic response.
  • patients are vaccinated with the transfected cells accordingly.
  • s-crc overexpression associated with colon carcinoma partial venous inoculation is preferred, as the liver is a frequent site of metastases.
  • systemic immunization is preferred.
  • the efficacy of the • inoculation schedule may be monitored by a delayed hypersensitivity reaction administered to the patient. A course of about up to 10 inoculations, at 2-3 week intervals, may be utilized. It may be appreciated that the inoculation schedule - may be modified in view of the immunologic response of the individual patient, as determined with resort to the delayed-type hypersensitivity (DTH) reaction.
  • DTH delayed-type hypersensitivity
  • HBSS Hanks buffered saline solution
  • One advantage to the DTH assay is that it can independently assess the induction of T cell reactivity to (i) the transfectants used for immunization (i.e. the set of 5 or more dCTGs chosen for immunization purposes, each containing non-self determinants) and (ii) transfectants, as transfected with the human dCTG itself containing only self determinants.
  • the induction of reactivity to the transfectants used for immunization establishes that the immunizing transfectants are in fact immunogenic, that is, the patient has not exhibiting a much weakened capacity for immune response.
  • the oncogene c-src(527) is an activated form of chicken c-src. Its protein product pp60 c srL(5:7) differs from the protein product of c-src, pp60 c src , by only a single amino acid substitution, phenylalanine for tyrosine at residue 527 (Kmiecik and Shalloway , ( 1987) Cell 49 , 65-73) . This substitution eliminates the negative regulatory influence exerted on pp60 c src phosphokinase activity by the enzymatic phosphorylation of the position 527 tyrosine.
  • the protein product of v-src, pp60' "src , shows a number of sequence differences with pp60 c src (Takeya and Hanafusa, (1983) Cell 32, 881-890), including scattered single amino acid substitutions within the first 514 residues and a novel C terminus of 12 amino acids (residues 515-526). in place of the nineteen C terminal amino acids of pp60 c src (residues 515-533). Both the v-src-positive plasmid, pMvsrc, and the c-src(527)-positive plasmid, pcsrc527.
  • the pVSRC-Cl plasmid was prepared as described by Halpern et al. , (1991) Virology 180, 857-86. Essentially, the plasmid was derived from the pRU -src plasmid (Halpern et al. , (1990) Virology 175, 328-331) by subcloning the v-src( + ) Xhol-EcoRl fragment of the latter into the multiple cloning sequence of pSP65 (Melton et al. , (1984) Nucleic Acids Res.
  • This insert included the v-src oncogene of the subgroup A strain of Prague RSV, as flanked downstream by a portion of the long terminal repeat (LTR) of RSV (from the 5' start of the LTR, to the single EcoRI site).
  • LTR long terminal repeat
  • the pM vsrc plasmid was generously provided by Dr. David Shalloway. Cornell University, Ithaca, NY.
  • the plasmid is prepared according to Johnson et al , (1985) Mol. Cell. Biol. 5. 1073-1083. Briefly, the 3.1- b BamHl-Bg/ll Schmidt Ruppin A v-src fragment from plasmid pN4 (Iba et al. , (1984) Proc. Nat. Acad. Sci. USA 81. 4424-4428) is inserted into the p ⁇ VX plasmid (Kriegler et al.
  • This fragment contains 276 bp of pBR322 DNA from the pBR322 BamHI to Sail sites followed by 2.8 kb of Rous sarcoma virus (RSV) DNA from the Sail site that is about 750 bp upstream of the env termination codon down to the Nrul site that is about 90 bp downstream of the v-src termination codon.
  • RSV Rous sarcoma virus
  • the pM src plasmid was restricted with Nhel, so as to liberate a tumorigenic fragment.
  • the fragment included the v-src oncogene of the subgroup A strain of Schmidt-Ruppin RSV, as flanked upstream by most of the Moloney murine leukemia virus (MoMLV) LTR (from the Nhel site near the 5 ' start of the LTR, to the 3' end of this LTR) and downstream by a small portion of the MoMLV LTR (from the 5' start to the Nhel site).
  • MoMLV Moloney murine leukemia virus
  • pcsrc527 The pcsrc527 plasmid is prepared according to Kmiecik and
  • a plasmid is constructed by cleaving expression vector pEVX (Kriegler et al , (1984) Cell 38,483-491 at its unique 5gIII site lying between two MoMLV LTRs and inserting the 3.2 kilobase (kb) pair 5 ⁇ 3mHI-5gIII hybrid src fragment from plasmid pHB5 in the proper orientation.
  • This fragment contains sequences from pBR322, the SRA env 3 ' region, SRA v-src, src from recovered ASV, and chicken c-src.
  • the Bglll site is generated by insertion of a linker at the Sacl site about 20 bp downstream from the c-src termination codon.
  • the restriction map of pMHB5 contains the MoMLV splice donor about 60 bp downstream from the 3 'end of the upstream LTR and the v-src splice acceptor about 75 bp upstream from the src ATG.
  • Plasmid pMHB5527 is constructed by inserting the synthetic double-stranded D ⁇ A oligomer
  • Equimolar amounts of the double- stranded oligomer and three gel-purified tandem restriction fragments from pMHB5 are ligated in one reaction, which contains the following the ohgomei with Banll and Bglll complementary ends, the 3 kb Bglll-Bgll (Bgll in the pEVX ampicilhn resistance gene) partial digest fragment, the adjacent 6 1 kb Bgll-Bgll (downstream Bg/7 m c-s/c) fragment, and the 0 38 kb Bgll-Baril (Banll at c-src codon 524) fragment
  • Plasmid pcsrc527 is constructed by replacing the 2 kb Sail (in env)-Mlul (in c-src) fragment in plasmid pMHB5527, with the homologous fragment from plasmid p5H
  • This fragment contains the coding sequence for the c-s/ c amino region (codons 1 to 257) that have been isolated by molecular cloning of a c-src provirus and previously shown by sequencing to contain authentic c-src sequence without the mutation at codon 63 (Levy et al , (1986) Proc Natl Acad Sci USA 83, 4228-4232)
  • Equimolar amounts of complementary gel-purified Sall-Mlul fragments from p5H and the other plasmids are ligated
  • the pcsrc527 plasmid was restricted with Nhel, so as to liberate a tumo ⁇ genic fragment
  • TK chickens Growth of challenge (wing web) tumors in test and control line TK chickens was determined under conditions of (i) priming and homologous challenge with pcsrc527, or (ii) priming and homologous challenge with pVSRC- Cl.
  • Test chickens were primed at 1 day posthatch with 100 ⁇ g of construct; test and control chickens were challenged at five weeks posthatch with 200 ⁇ g of construct.
  • the mean challenge tumor diameter was computed as described in the preceding section. At each time point the ratio of chickens bearing palpable challenge mmors to total number of survivors to that point is indicated for • priming and homologous challenge with pcsrc527 (Fig. 2A) and priming and homologous challenge with pVSRC-Cl (Fig.
  • TK chickens under Conditions of Priming with pVSRC-Cl and Heterologous Challenge with pcsrc527, or Priming with pcsrc527 and Heterologous Challenge with pVSRC-Cl
  • a punch biopsy of skin is obtained from a donor (other than the patient-to-be-treated or identical twin thereof) by a trained physician following standard medical practice.
  • the skin obtained by punch biopsy is put in a tube with 10 ml of the following wash medium: Dulbecco's Modified Eagle Medium (DMEM), containing sodium bicarbonate (30 ml/liter of a 5.6% solution) and penicillin/streptomycin (2 ml/liter of a pen-strep stock solution containing 5000 units penicillin and 5000 g of streptomycin/ml, pH 7.2-7.4.).
  • DMEM Dulbecco's Modified Eagle Medium
  • penicillin/streptomycin 2 ml/liter of a pen-strep stock solution containing 5000 units penicillin and 5000 g of streptomycin/ml, pH 7.2-7.4.
  • the biopsy is then finely minced with two scalpels, and 2-4 pieces ( ⁇ 1 mm 3 ) of the minced biopsied are placed in the middle part of one or more T25 flasks.
  • the flask is placed in a tissue culture incubator at 37 °C for one half hour with the cap firmly ⁇ closed, then opened for 10 minutes.
  • the following culture medium is prepared: DMEM containing sodium bicarbonate; antibiotics; and 10% fetal calf serum containing 2.5 ⁇ g/ml fungizone, 40 ⁇ g/ml gentamicin, and 1 % glutamine( 3 % W/V).
  • trypsin solution 0.05/0.02 % trypsin in PBS. without Ca+ + or Mg+ +
  • 2 ml of culmre fluid is added to stop the action of the trypsin.
  • the cells are then transferred to a larger flask (T75) and incubated at 37°C in 15 ml of culture fluid, which is changed every 2 days.
  • the fibroblasts (2 X 10' cells) are washed twice in DMEM without serum or antibiotics.
  • a LipofectAMINETM-DNA solution is prepared by mixing in tube #1 mix 400 ⁇ l DMEM and 10 ⁇ of dCTG vector DNA (l ⁇ g/ul). In tube
  • Transfectants are irradiated to a dose of 25 By or 2500R. the cells are then counted by trypan blue exclusion. 2 X 10 7 irradiated transfectants are resuspended in a volume of 0.2-0.4 ml of Hanks Balanced Salt Solution.
  • Patients are vaccinated by subcutaneous inoculation of 2 X 10 irradiated cells at 2-3 week intervals. A shorter or longer regimen is used, depending upon the results of delayed type hypersensitivity (DTH) reaction monitoring (described below).
  • DTH delayed type hypersensitivity
  • the v-myc retroviral oncogene of avian myelocytomatosis virus MC29 was obtained from the American Type Culmre Collection, Rockville. MD. 20852. as the pSVv-myc vector (ATCC No. 45014).
  • the v-myc-positive EcoRl-Kpnl fragment of pSVv-myc was ligated into the polylinker sites of the pBK-CMV plasmid (Stratagene Cloning Systems. La Jolla, CA).
  • Stable transfection using the pBK-CMV-v-myc vector was carried out on a line of A31 fibroblasts (Balb/c origin), obtained from the ATCC.
  • 2 X lO "1 cells were seeded in a 100 mm/dish and allowed to grow for 18-20 h (RPMI 1640 medium and 10% fetal bovine serum), at which time the cells reached 50- 70% confluence. The cells were then washed twice in Dulbecco's Modified Eagles Medium (without serum or antibiotics).
  • a LipofectAMINETM-DNA solution was prepared according to Example 2.C. , with the pBK-CMV-v-myc vector DNA.
  • the cells were then incubated for 6 hours at 37 °C, washed once with Hank's Balanced Salt Solution, and then refed with the growth medium and incubated for an additional 24 hour at 37 °C. Thereafter, the cells were fed once every two days with growth medium containing 250 ⁇ g/ml geneticin (G418; Gibco BRL cat. no. 11811) as the selective marker. Within two weeks, colonies were picked and expanded into permanent cell lines. The cells were then washed and collected by centrifugation.
  • G418 Gibco BRL cat. no. 11811

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