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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • 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
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2799/00—Uses of viruses
  • C12N2799/02—Uses of viruses as vector
  • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
  • C12N2799/023—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a poxvirus

Definitions

• the present invention concerns materials and methods relating to the preparation of immunogenic
• antigens in malignant cells may be used as vaccines to induce tumour-specific cell-mediated immunity (Pardoll, D.W., Nature 369, 357- 358 (1994)) and work has focussed on various sources of antigens, including viral (Papadopoulos, E.B., et al., N.Engl. J. Med. 330, 1185-1191(1994); Feitkamp, M.C.W., et al., Eur. J. Immun. 23, 2242-2249 (1993), fetal and tissue-specific antigens (Pardoll, D.W., 1994 supra.), and point mutations (Peace, D.J., et al., J. Exp. Med. 179, 473-479 (1994).
• antigens may arise by frameshift mutations which give rise to new segments of unique protein.
• Antigens deriving from new segments of unique protein in consequence of a frameshift mutation may be a previously unrecognized source of tumour-specific antigens or antigens specific to any other disease state associated with a frameshift
• fragments can be presented at the surface of somatic cells in association with MHC Class 1 molecules.
• the mechanism is known as antigen presentation. It is thought that viral or host proteins are made on free ribosomes in the cytosol. These proteins are then fragmented by proteolytic enzymes and the peptide fragments transported by a signal-independent mechanism into a compartment of the host cell (probably the endoplasmic reticulum) where they bind to the MHC Class 1 molecules. This complex is transported to the cell surface and the peptide is displayed at the cell surface to circulating cytotoxic lymphocytes.
• the mechanism allows the immune system to detect intracellular viral antigens early in infections. It is also responsible for the rejection of unrelated organs after transplantation. Although the mechanism probably evolved to deal with intracellular infectious agents, it is possible that this mechanism may be active in
• a normal (meaning 'healthy') cell mutates.
• the normal version of the cell will contain a particular gene sequence which encodes the corresponding normal protein.
• the mutation may comprise either the deletion of one or more bases from the normal gene sequence or the addition of one or more bases into the normal gene sequence. This can result in a shift of the reading frame.
• the proteins of the cell are being continually sampled, digested and peptide fragments resulting from the digestion, presented at the cell surface as a complex with MHC molecules for inspection by circulating lymphocytes. Where the proteins are normal for the individual, the
• polypeptide/peptide fragments are recognised by the individual's lymphocytes as being native to the
• lymphocytes are responsible for the following reasons:
• the mutant protein itself and at least some of the peptide/polypeptide fragments deriving therefrom will be new to the individual. In which case when these new fragments are presented in association with MHC to the circulating lymphocytes, they will be seen as foreign and the lymphocytes will mediate an immune response
• translation into the second and third reading frames and peptide/polypeptide fragments deriving from such a mutant protein may be of value as an active ingredient of a pharmaceutical to invoke an immune response against the mutant protein.
• the new polypeptides and peptides may be of value in the formulation of vaccines.
• adenomatous polyposis coli (APC) gene from chromosome 5q21 has been suggested to
• the APC gene has been found to code an unusually large (300 KDa) protein and the gene has been sequenced and a predictive amino acid sequence disclosed (Kinzler et al., Science Vol. 253 p661, (1991)).
• the present inventor has confirmed that the
• a new peptide/ polypeptide sequence is in fact translated between a frameshift mutation and the stop codon and that this new sequence and polypeptide and peptide fragments from it, will be disease (e.g. tumour) specific antigens which when presented at the cell surface in association with MHC will be seen as foreign.
• disease e.g. tumour
• sequences will be unique antigens specific to the disease associated with the frameshift, peptides/ polypeptides comprising part or all of the sequences of these unique antigens may be used to treat individuals in order to induce immune responses to mutant cellular proteins associated with the disease e.g. colon carcinoma.
• the present invention provides proteins and polypeptide and peptide fragments thereof, which proteins have substantially the sequence of corresponding mutant proteins which are produced in consequence of gene mutations associated with a disease state which shift translation into the second or third reading frames downstream of the gene mutation, and which proteins and polypeptide and peptide fragments thereof and mutant proteins are antigenic.
• Such a mutant protein may be encoded by the
• adenomatous polyposis coli (APC) gene of chromosome 5q21 is adenomatous polyposis coli (APC) gene of chromosome 5q21.
• the mutant protein may be encoded by the second reading frame lying between codons 1418 and 1472 of the APC gene.
• the mutant protein may be encoded by the second reading frame lying between codons 1472 and 1506 of the APC gene.
• the peptides may be selected from the sequences
• the present invention also provides nucleotide sequences coding for proteins and polypeptide and peptide fragments thereof and mutant proteins as described above.
• the present invention also provides these nucleotide sequences as incorporated into vectors such as transfer, expression and vaccine vectors.
• the vaccine vector may be based upon vaccinia virus or other useful viral vectors and Ty particles.
• the present invention also provides recombinant host cells which contain the nucleotide sequence or vector.
• the present invention also provides the proteins and polypeptide and peptide fragments in combination with one or more moieties to increase antigenicity.
• combination may be by way of conjugation or admixture.
• compositions which comprise either (i) a protein or polypeptide/peptide fragment thereof as described above; (ii) a nucleotide sequence as described above; or (iii) a vector as described above.
• the pharmaceutical composition may be a therapeutic vaccine comprising a vector based upon virus (such as vaccinia virus) which incorporates a nucleotide sequence as described above.
• virus such as vaccinia virus
• the pharmaceutical composition may be a therapeutic vaccine comprising a vector based on a Ty particle which incorporates a nucleotide sequence as described above.
• the vaccines may also be used prophylactically eg to protect against the development of secondary cancers.
• the present invention also provides treatment methods using these pharmaceuticals/vaccines to treat a patient for a disease condition associated with a
• the present invention also provides a method for identifying a mutant protein and polypeptide and peptide fragments thereof and which are as described above, the method comprising the steps of:
• polypeptide/peptide sequences encoded by the nucleotide sequences selected in step (iv).
• the method may comprise the steps of
• step (iv) on the basis of their encoding amino acid sequences likely to bind MHC molecules and which are in the second or third reading frame may be incorporated into expression vectors and expressed as in step (d).
• the expression product will be tested as in steps (e) and (f).
• proteins/polypeptides/ peptides may be tested in
• any proteins/polypeptides/peptides which as a result of such tests are thought to have therapeutic utility may be both formulated into pharmaceuticals and vaccines and further tested both in vitro and in vivo (animal and human studies) in accordance with techniques and procedures commonly used and well-known in the art.
• the target cells were L cells that had previously been transfected with the HLA gene H-2Db (called LDb). These cells were labelled with Cr51 and exposed to the treatments listed in the figure. They were then mixed with the effector cytotoxic T cells in the ratios shown in the figure (E/T ratio). The amount of killing was measured as % specific lysis as described in Townsend et al Cell Vol. 44, 959- 968 1986.
• Figure lb A similar experiment was done using a colon carcinoma cell line as the target cell.
• the cell line is called LoVo (Brodsky et al Immunol. Rev 47, 3-61, 1979). This cell line lacks expression of beta-2
• an additional vaccinia virus provided expression of beta- 2 microglobulin (/32m) (see Yewdell et al J. Immunol, 152, 1163-1170, 1994).
• the target cells were recognised by the cytotoxic T cells only when either i) the target cells were infected with beta-2 microglobulin vaccinia, and K d vaccinia and the vaccinia encoding APC second reading frame codons 1418-1472, or ii) infected with Beta-2 microglobulin vaccinia and K d vaccinia and then exposed to the peptide KYLKIKHLL.
• FIG. 1 shows the background level of A2 detected in the absence of peptide
• Lane 2 the increase seen when a known binding peptide derived from influenza matrix protein was added to 20 ⁇ m.
• lanes 3-18 a set of peptides derived from the second and third reading frames of APC were added to 20 ⁇ M.
• the sequences QLKPSEKYL (lane 11) and KVLQMDFLV (lane 18) markedly increased the level of A2 detected, indicating binding.
• Lane 1 shows the level of A3 detected by the monoclonal antibody GAPA3 in the absence of peptide.
• Lanes 2 and 3 show a marked increased after addition of the peptide sequence ILYYILPRK to either 50 ⁇ m (lane 2) or 5 ⁇ m (lane 3).
• Lane 4 contained a known HLA A3 binding peptide that is known to be recognised by human A3 restricted cytotoxic T cells specific for HIV nef, as a positive control. Note in Lanes 7 and 8 that the sequence QLKPSEKYL that was shown in Fig. 2 to bind HLA A2, did not bind HLA A3.
• Figure 4 shows a Western Blot.
• Lane 1 a positive control extract from a colon cancer cell line SW480 (described in Smith et al., PNAS 90, 2846-2850, 1993) which expresses a mutant APC protein of approximately 147 kD which is close in size to the expected size of a recombinant form of mutant APC protein deriving from a recombinant vaccinia virus;
• Lanes 2-4 extracts from LK cells (5 x 10 5 cells/lane) infected with (20 p.f.u in 10 s cells; 1.5 hours; 4 hours for recovery) three different subclones (7131c3 VAC, 7131c2 VAC, 7131cl VAC) of a recombinant vaccinia virus encoding a full length mutant APC gene (7131 VAC);
• Lane 5 a positive control extract from a lymphoblastoid cell line derived from a patient with a mutation similar to that carried by the
• Figures 5 show the results for Cr 51 release assays.
• the target cells are all murine L cells transfected with a gene encoding murine K d Class I molecules (LKd cells).
• Lkd Un are Lkd cells not infected with vaccinia virus carrying any of the mutant APC gene.
• Lkd M-53-QVAC are Lkd cells infected with a recombinant vaccinia virus encoding a 53 amino acid fragment containing the KYLKIKHLL epitope.
• VAC are LKd cells infected with a recombinant vaccinia virus encoding the full length mutant APC protein from tumour 7131 and that contains the KYLKIKHLL epitope.
• NP147-55 are LKd cells treated with a peptide representing amino acids 147 to 155 from influenza nucleoprotein as a negative control.
• K-9-L are LKd cells treated with the peptide KYLKIKHLL which is present in the frameshifted APC protein sequence in tumour 7131.
• lytotoxic T eymphocytes were murine and raised to the 9 residue peptide KYLKIKHLL.
• the cytotoxic T lymphocytes were murine and raised to the sequence NP147- 155 of influenza virus.
• the two sets of cytotoxic T lymphocytes were tested against L cells
• codons 1250 and 1550 allow convenient screening for mutations by polymerise chain reaction (PCR) amplification of the APC DNA sequence between these codons, derived from tumour samples.
• PCR polymerise chain reaction
• tumour DNA was extracted from tumour samples in accordance w: th standard techniques (eg as described in Sambrook, Fritsth, Maniatis (1989) Cold Spring Harbour Laboratory Press).
• w th standard techniques (eg as described in Sambrook, Fritsth, Maniatis (1989) Cold Spring Harbour Laboratory Press).
• the nucleotide sequences lying between codons 1418 to 1472 and codons 1472 to 1506 were amplified by PCR in accordance with standard procedures using oligonucleotides A, B, C & D below:
• Primers A and B were used to amplify the nucleotide sequences lying between codons 1418 to 1472 and primers C and D were used to amplify nucleotide sequences lying between codons 1472 to 1506. PCR was performed with Stratagene P.F.U.. The following ingredients were mixed:
• the oligonucleotides were designed to provide an ATG initiation codon contained within an Ncol restriction site at the start of the reading frames and a Bglll restriction site at the end of the reading frames.
• amplified nucleotide sequences were digested with Bglll and Ncol and cloned into the vaccinia
• PSC1130R.2 was derived from the vector pSCll (Chakrabarti, S., et al Molec. Cell
• the oligonucleotide inserted into the Smal site provides a kozac sequence upstream of an ATG initiation site to allow expression of cloned open reading frames.
• nucleotide sequences encoding predictive polypeptide sequences considered as being likely to bind murine K d are sequenced and examined for encoding predictive amino acid sequences that were likely to bind to murine K d Class I molecules on the basis of the nucleotide sequences encoding a Y residue at position 2 and L residue at position 9 (Falk et al Nature 351, 290-296, 1991).
• Class I molecules which were in the second frame (as determined from a comparison with the wild type sequence) were further investigated for their immunogenicity.
• mice were immunised i.v. with 10 7 plaque forming units (p.f.u) of recombinant vaccinia virus having a sequence insertion. At least 10 days later, spleen cells were removed. If a polypeptide is suitably immunogenic and therefore of potential use as a vaccine, it will induce a population of T cells carrying receptors
• T cells having the specific receptor will exist in low concentration in the spleen (about 1 cell in 10,000), the population of specific T cells was expanded by exposure in vitro to the 9 residue peptide KYLKIKHLL (residues 1451-1459), using a method as described by Bastin, J., et al J. Exp. Med. Vol. 165, 1508-1523, 1987. 5-7 days later the growing T cells were either tested in a
• the target cells which release Cr 51 may, for example, be either murine L cells (Townsend et al Cell Vol. 44 959-968, 1986) infected with the recombinant vaccinia virus encoding the appropriate reading frame, or a cell line derived from human colon carcinoma similarly infected.
• murine L cells Townsend et al Cell Vol. 44 959-968, 1986
• vaccinia virus encoding the appropriate reading frame
• cell line derived from human colon carcinoma similarly infected.
• they may be transfected with a gene coding the MHC molecule
• the cytotoxic T cells also recognised both types of target cells coinfected with the vaccinia virus encoding the complete open reading frame (1418-1472) and the vaccinia virus encoding the murine MHC K d molecule. This demonstrates (a) that the 2nd reading frame lying between codons 1418-1472 was immunogenic to Balb/c mice, (b) that the epitope 1451-1459 could be presented from within the longer fragment 1418-1472.
• mice are not immunogenically tolerant to the proteins encoded in the 2nd and 3rd reading frames of human APC.
• the experiments in Part 2 make use of a recombinant vaccinia virus having as a sequence insertion a full length mutant APC gene.
• the first objective was to demonstrate that a full length mutant APC protein would be digested as expected in cells to produce a range of peptide fragments which originate from the polypeptide encoded by the nucleotide sequence downstream of the frameshift mutation. This demonstration required the construction of full length mutant APC coding sequence.
• sequences of the oligonucleotides E and F used to amplify by PCR the fragment comprising mutant sequence from tumour 7131 and the complete sequence of the amplified fragment with the Psp 14061 restriction site shown and the 2 base insertion shown in bold (AA) at codon 1438.
• the principal was to include in the amplified sequence a restriction enzyme site Psp 14061 that is unique to the APC gene. This site can then be used to recombine the normal left hand fragment (5' fragment) of the normal gene with the mutant right hand fragment (3' fragment) amplified from the tumour sample using standard technology as described in Sambrook et al 1989 supra.,.
• the complete mutant cDNA can then be cleaved out with BamHI and Xhol to subclone into pSC65 for making the recombinant vaccinia by standard
• tumour 7131 DNA was extracted from a tumour sample (designated 7131) in accordance with standard techniques (Sambrook et al., 1989 supra).
• the tumour 7131 has a 2 base pair insertion into codon 1438 of the APC gene.
• a short mutant fragment from within exon 15 was cloned as follows.
• the nucleotide sequence comprising the fragment was amplified by PCR in accordance with standard
• Stratagene P.F.U The following ingredients were mixed: (1) template DNA from tumour (titrated); (2) 10 ⁇ ls oligo's E and F from 4 ⁇ molar stock solutions; (3) 10 ⁇ ls x 10 Stratagene P.F.U buffer; (4) 5 ⁇ ls GATC mix from 5 m molar stock solution; (5) H 2 O to 100 ⁇ ls final volume. This mixture was heated to 95°C for 5 mins, then cooled to 80°C. Five units of Stratagene P.F.U were then added. PCR was performed with 35 cycles consisting of 5 sec. at 92°C, 1 min at 58°C and 1 min. at 72°C.
• the amplified fragment was then recombined with a full length wild type APC cDNA followed by cloning into the vaccinia expression vector PSC65 (a gift from Dr. Moss of the National Institute of Health, Washington US: this vector is similar to PSC1130R.2 which could be used as an alternative vector) and sequencing as described in Part 1.
• Recombinant vaccinia encoding the full length mutant APC cDNA were then produced as described in Part 1.
• L cells were then infected (20 p.f.u in 10 6 cells; 1.5 hours; 4 hours for expression) with recombinant vaccinia (subclones 7131c3 VAC, 7131c2 VAC and 7131cl VAC) and the size of proteins expressed determined by Western blot analysis (5 x 10 5 cells/lane) using the antibody FE9 (available from Oncogene Science Inc., 106 Charles Lindbergh Blvd, Uniondale, NY 11553-3649, USA) which reacts with the non-mutant N-terminal region of the protein as described by Smith et al., PNAS 90, 2846-2850 (1995).
• Lane 1 is a positive control extract from a colon cancer cell line SW480 (described in Smith et al., PNAS 1993 supra) which expresses a mutant APC protein of approximately 147 kD which is close in size to the expected size of a recombinant form of mutant APC protein (approximately 165 kD) deriving from the
• Lanes 2 to 4 are extracts from L cells infected with three different subclones (7131c3 VAC, 7131c2 VAC and 7131cl VAC) of vaccinia.
• a major band is seen running just above the SW480 band and close in size to the band in lane 5 from SM501 another positive control derived from a lymphoblastoid cell line from a patient who has inherited a mutation similar to that carried by the recombinant vaccinias.
• Lanes 2 to 4 also show bands representing a series of proteins with mws of less than 147 kD and which also react with the FE9 antibody. These probably result from digestion of the full length mutant APC gene in the L cells.
• cytotoxic T lymphocytes were raised in Balb/c mice that had been immunised as earlier described with a recombinant vaccinia virus having as an insertion the 9 residue peptide KYLKIKHLL (residues 1451-1459) identified in Part 1.
• the 9 residue peptide KYLKIKLL is derived from the commonest frameshift antigen (present in tumour 7131) and is recognised by murine cytotoxic T lymphocytes in association with the K d Class I molecule.
• cytotoxic T lymphocytes were tested in a standard CR 51 release array using as target cells (i) LKd Un which are murine L cells transfected with a gene encoding murine K d Class I molecules but not infected with vaccinia virus carrying any of the mutant APC gene; (ii) Lkd M-53- QVAC which are murine L cells transfected with a gene encoding murine K d class 1 molecules and a recombiant vaccinia virus encoding a 53 amino acid fringement containing the KYLKIKHLL epitope as a positive control; (iii) 7131/3VAC which are murine L cells transfected with a gene encoding murine K d Class I molecules and a
• NP147-155 which are murine L cells transfected with a gene encoding murine K d Class 1 molecules and treated with a peptide representing amino acids 147 to 155 from influenza nucleoprotein as a negative control
• K-9-L which are murine L cells transfected with a gene encoding murine K d Class I molecules and treated with the peptide KYLKIKHLL which is present in the frameshifted APC protein sequence in tumour 7131.
• the figure shows that the cells infected with a recombinant vaccinia virus encoding the full length mutant APC gene from tumour 7131 were recognised by cytotoxic T lymphocytes specific to the 9 residue peptide KYLKIKHLL.
• Fig 5 (b) the same set of target cells were tested with cytotoxic T lymphocytes raised in Balb/c mice that had been immunised with the sequence NP 147-155 of ir-iiuenza virus.
• the results show that cells infected with a recombinant vaccinia virus encoding the full length mutant APC gene from tumour 7131 are recognized only by cytotoxic T lymphocytes specific to the APC frameshift antigens and not by other cytotoxic T
• lymphocytes that recognize other antigens in association with murine K d Class I molecules.
• cytotoxic T lymphocytes were tested against L cells transfected with the histocompatibility molecule Db instead of Kd. The results show that the cytotoxic T lymphocytes will only recognize their antigen when presented in the context of the correct
• the second reading frame sequence lying between codons 1418 and 1472 which is immunogenic to Balb/c mice also contains a sequence motif (Y at position 2 and L at position 9) appropriate for binding the MHC A24 molecule, the commonest allele in Japan where colorectal carcinoma is particularly prevalent.
• volunteers may be immunised with prototype vaccines containing or able to express in vivo the peptides encoded in the 2nd and 3rd reading frames of a gene such as APC.

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