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WO2007056304A2 - Formes mutees de ny-eso-1 produites dans de la levure et leurs utilisations - Google Patents

Formes mutees de ny-eso-1 produites dans de la levure et leurs utilisations Download PDF

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WO2007056304A2
WO2007056304A2 PCT/US2006/043226 US2006043226W WO2007056304A2 WO 2007056304 A2 WO2007056304 A2 WO 2007056304A2 US 2006043226 W US2006043226 W US 2006043226W WO 2007056304 A2 WO2007056304 A2 WO 2007056304A2
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eso
mutein
amino acids
seq
cells
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PCT/US2006/043226
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WO2007056304A3 (fr
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K. Dane Wittrup
Shanshan W. Howland
Andrea Piatesi
Gerd Ritter
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Ludwig Institute For Cancer Research
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Publication of WO2007056304A3 publication Critical patent/WO2007056304A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP

Definitions

  • This invention relates to variants of the well known immunologically active molecule known as NY-ESO-I. These variants possess immunological activity that is equal to that of wild type NY-ESO-I, but are more easily produced in usable form by recombinant systems, such as transformed or transfected, prokaryotic and eukaryotic cells.
  • CT antigens are a class of tumor-associated antigens with expression normally restricted to germ cells in the testis, ovaries or trophoblast cells, and not in adult somatic tissues. See, Simpson, et al., Nat. Rev. Cancer, 5(8):615-625 (2005); Scanlan, et al., citiunol. Reviews. 188:22-32 (2002); Scanlan, et al., Cane. Immun.. 4:1-15 (2004).
  • the gene regulation of CTs is disrupted in cancer patients, leading to the aberrant expression of CT antigens in a wide variety of tumors.
  • the first CT antigen, MAGE-I was identified in the early 90's by T-cell epitope cloning.
  • This 180 amino acid long protein was first identified by SEREX in an esophageal squamous cell carcinoma in the late 90' s at the New York Branch of the Ludwig Institute for Cancer Research and represents the most immunogenic CT antigen described to date.
  • NY-ESO-I has been found in a wide variety of tumors, including ovarian cancer, lung cancer, breast cancer, esophageal cancer, bladder cancer and in melanomas. See, Chen, et al, supra; Jungbluth, et al., Int. J. Cane, 92(6):856-860 (2001).
  • HLA Human Leukocyte Antigen
  • the 157-167 NY-ESO-I fragment has dual specificity, with the 157-165 peptide as the natural antigenic tumor epitope and the 159-167 peptide as an immunodominant cryptic epitope that diverts the immune response from tumor recognition. See, Gnjatic, et al., Proc. Natl. Acad. Sci. USA. 99(18):11813-11818 (2002). It therefore appears desirable to sometimes use full-length NY-ESO-I. See, Schnurr, et al., Blood. 105(6):2465-2472 (2005).
  • ISCOMATRIX a saponin-based adjuvant that induces strong humoral and cellular immune responses
  • ISCOMATRIX a saponin-based adjuvant that induces strong humoral and cellular immune responses
  • Davis et al. showed that this formulation induced a broad integrated humoral and CD4+ and CD8+ T-cell response allowing the identification of previously unknown immunogenic NY-ESO-I peptides.
  • the low purity of this recombinant antigen raises concerns over the generation of non-specific immune responses and false positive Delayed Type Hypersensitivity (DTH) skin reactions in response to bacterial contaminants from the same antigen preparation.
  • DTH Delayed Type Hypersensitivity
  • the improved display and secretion achieved by the variant form of NY-ESO-I not only would apply to S. cervisiae but also to other types of yeast, such as Pichia pastoris, and other eukaryotic cells such as, insect cells, mammalian cells, including but not being limited to, Chinese hamster ovary cells (CHO), and mouse myeloma cells such as NSO. It could also be applied to viruses that are intended for delivery of NY-ESO-I to antigen presenting, or dendritic, cells in order to induce an immunogenic reaction in a subject. See Palmowski, et al., J Immunol., 2004 Feb l;172(3):1582-7.
  • viruses examples include; lenti-, fowlpox-, modified vaccinia virus Ankara-, adeno-, herpes simplex-, retro- and vaccinia viruses.
  • delivery vehicles examples include certain bacteria such as those of the Salmonella and Listeria families.
  • This example describes the generation of NY-ESO-I variants.
  • NY-ESO-CA amino acid sequence of NY-ESO-I
  • three variants were prepared: "NY-ESO-CA,” in which cysteine residues at positions 75, 76, and 78 were replaced by Alanine; "NY-ESO-CS,” where these cysteine residues were replaced by serine, and "NY-ESO-T,” where amino acid 1-78 were deleted in their entirety.
  • the rationale for these modifications was based on several factors. First, amino acids 1-80 are glycine rich and unstructured, whereas amino acids 80-180, the C-terminus of the protein is highly immunogenic, and likely to form a helix sheet-helix motif.
  • amino acids 153- 180 are highly hydrophobic, and probably contain a transmembrane domain. See, Chen, et al., Proc. Natl. Acad. Sci. USA, 94(5):1914-1918 (1997).
  • Amino acids 75, 76, and 78 are part of a cysteine cluster from amino acid 75 to amino acid 78. It has been hypothesized that this cluster results, in potential protein misfolding or toxicity effects, which in turn results in almost undetectable protein secretion in S. cervisiae.
  • nucleotides 5-10 constitute an Nhel site
  • nucleotides 5- 10 of SEQ ID NO: 3 form a BamHI site.
  • CA and CS variants were prepared by simultaneous substitution of the three cysteine residues by site directed mutagenesis, using commercially available reagents methods.
  • nucleotides 17-22 were replaced by agcagc, and nucleotides 26-28 by age.
  • Corresponding antisense oligos were also prepared.
  • nucleotides 17-22 and 26-28 were replaced by gccgcc and gcc, respectively.
  • the NY-ESO-CS, NY-ESO-CA and NY-ESO-I ORFs were then used to generate three libraries, referred to herein as libraries “L,” “M” and “H”.
  • the libraries were generated using error prone PCR, using commercially available products and methods, and the libraries had different mutation rates.
  • the error rates were "tuned” by varying the amount of template (NY- ESO-I, ⁇ !CS" or "CA”) used during PCR.
  • For library L this was 600 ng, 60 ng for library M, and 10 ng for library H.
  • Library L was designed to have no more than 2 mutations per gene, library M, 2-4, and library H, more than 4.
  • cagatctcgg agctattaca agtccttctt cagaaataag cttttgttc (SEQ ID NO: 6)
  • each PCR product was independently co- transformed, with a digested and purified PCT vector in EBYlOO, using the electroporation method of Colby, et al., Meth. Enzvmol. 388:348-358 (2004), incorporated by reference. This results in mutant libraries via homologous recombination, with each library containing from 10 to 30 million members.
  • Yeast cell display experiments were then carried out, using S. cervisiae strain EBYlOO, described by Boder, et al., Nature Biotechnology, 15(6):553-557 (1997), incorporated by reference.
  • EBYlOO is an Agal+ strain, prepared from BJ5464, which is an Agal " strain that is available from the American Type Culture Collection. This strain expresses Agalp, which covalently binds to Aga2p, (Boder, et al. Nature Biotechnology. 15(6):553-557 (1997); Boder, et al., Meth.
  • Yeast cells were transformed with the libraries discussed supra, in SD-CAA medium (a selective medium, lacking ura and trp, ph 6.0) 20 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids 5.4 g/L Na 2 HP04, 8.6 g/L NaH 2 PO 4 H 2 O, and 5 g/L cosainino acid, at 30°C, until an OD 6 oo of 2-6 was reached.
  • SD-CAA medium a selective medium, lacking ura and trp, ph 6.0
  • Sorted cells were cultured in SD-CAA at 30°C, in the presence of 25 ⁇ g/ml of Kanamycin, to prevent bacterial contamination, until an OD 600 of about 6 was reached. When it was, an aliquot of enriched EBYlOO population of at least 10Ox excess of sorted cells, was stored as a glycerol stock, at -80°C. Any library produced in this way was then subjected to three, additional induction/labeling/sorting rounds, with increased stringency at each round, to improve display.
  • An additional 6 mutants (one "L”, three "M”, and two "H"), showed about a 10x increase in display.
  • Example 2 refers to a group of 20 mutants. Further studies were carried out on the individual members of this group.
  • PCT shuttle vector for each clone was isolated, using commercially available materials and known techniques. They were then transformed into E. coli XLI blue for plasmid amplification, followed by sequence analysis.
  • Variations in an amino acid sequence can lead to a redirection of an immune response, so there is interest in variants with a few mutations as possible.
  • variations were found with a change at position 156, which is adjacent to the epitope defined by amino acids 157-165, a very thoroughly studied region of NY-ESO-I. See, e.g., Gnjatic, et aU Proc. Natl. Acad. Sd. USA, 99(18): 11813-11818 (2002).
  • One of the variants identified contains AARA at positions 75-78, and a point mutation at position 153, from Leu to His. This variant was selected for further study.
  • Co-transformed yeast were cultured in 5 ml SD-CAA at 30°C, until an OD ⁇ oQ of about 2-4 was reached. Yeast were then pelleted (1000 x g, 10', 2O 0 C), resuspended in SG-CAA containing 0.5 g/L lysozyme, and cultured at 2O 0 C for 72 hours. Samples of supernatant were collected, every 24 hours, and analyzed via SDS-PAGE under reducing conditions, with protein identity confirmed with the E978 mAb (0.5 ⁇ g/ml), and HRP-conjugated goat anti-mouse mAb (20 ng/ml).
  • nucleotides 2-7 are an Nhel site, and 8-31, the HRV 3 C coding sequence, and
  • cagatctcga gctattaatg gtg (SEQ ID NO: 8)
  • nucleotides 14-18 are an Xhol site.
  • PCT product was co-transformed into EBYlOO, with PCT-CON2 that had been digested with Pstl and Xhol. Homologous recombination led to pAH-L5. An additional His 6 tag was then added to the N-terminus of Aga2p, via homologous recombination with three, overlapping DNA fragments . A His 6 tag was added after the Aga2p secretion signal peptide, via PCR, using:
  • His 6 tag is at nucleotide 23-40
  • a second PCR product from pAH-L5 product which started before the EcoRI restriction site and spanned the coding sequence of Aga2p secretion signal peptide, was generated using:
  • the construct maintained a display and secretion level that was essentially identical to the original PCT construct but the fusion protein is significantly smaller, allowing simple purification of L5.
  • EBYlOO yeast cells freshly transformed with pHAH-L5 were cultured in 1 L of SD-CAA in a Tunair flask at 3O 0 C until an OD 6O o of about 4 was reached.
  • the yeast was pelleted (100Ox g, 10', 20°C), resuspended in 4x1 L of SG-CAA and cultured at 20°C for two more days.
  • Induced yeast was harvested by centrifugation (100Ox g, 10', 4°C), washed with 100 ml PBS (pH 7.2) and gently rotated with two pelletO-volumes of PBS (pH 7.2) containing 25 mM TCEP (30', 4°C).
  • the suspension was pelleted and the supernatant was set aside.
  • Surface- displayed Aga2p-Ny-ESO-L5 was extracted twice more by reduction with one pellet-volume of PBS (pH 7.2) containing 25 mM TCEP.
  • the supernatant fractions were combined and dialyzed against PBS (pH 7.2) containing 5 mM ⁇ -mercaptoethanol ( ⁇ -ME).
  • CHAPS was added to a final concentration of 1 mM and the supernatant was combined with 1 mL of a metal affinity resin under gentle shaking (4 hours, 4°C).
  • the resin was subsequently transferred to a column and washed with 12 column volumes of buffer A (PBS, pH 7.2, 5 mM ⁇ -ME, 1 mM CHAPS) followed by 12 column volumes of buffer A containing 10 mM imidazole. Finally, the resin was washed with 2 column volumes of buffer A and incubated overnight at 4°C with 1 column volume of buffer A containing 5 units of GST-tagged HRV 3 C protease. The supernatant was separated from the resin by centrifugation. The maximize protein recovery, the resin was washed four more times with 0.25 mL buffer A.
  • buffer A PBS, pH 7.2, 5 mM ⁇ -ME, 1 mM CHAPS
  • the HRV 3 C protease was removed by incubation with glutathione-agarose and the purified NY-ESO-L5 was dialyzed against PBS (pH 7.2) containing a 5 mM ⁇ -ME. After concentration by ultrafiltration, protein purity and yield were assessed by SDS-PAGE and imaging on a Fluor-S Multilmager. Protein identity was confirmed by Western blotting, as previously described.
  • PBMCs peripheral blood mononuclear cells
  • MoDCs were then generated by culturing MACS purified, CD14 + cells, with GM-CSF and IL-4, for 6 or 7 days.
  • the cell cultures were maintained in RPMI 1640, supplemented with 20 mM HEPES, 60 mg/L penicillin, 12.5 mg/L streptomycin, 2 mM L-glutamine, 1% non-essential amino acids and 10% heat inactivated fetal calf serum.
  • MHC-Class I restricted presentation was assessed by pulsing the MoDc with either the known 157-165 ESO-I peptide, which binds to HLA-A2 + and serves as a positive control, NY-ESO-I protein combined in ICOSOMATRIX, yeast produced L5 or Aga2p-NY- ESO-L5 (all at 10 ⁇ g/ml), and equimolar amounts of empty IMX, which facilitated cytosolic entry.
  • the foregoing disclosure sets forth various features of the invention, which are muteins of the wild type molecule NY-ESO-I, which has the amino acid sequence set forth in SEQ ID NO:1.
  • "Muteins” as referred to herein means proteins which essentially maintain the amino acid sequence of SEQ ID NO:1, except for changes at positions 75, 76, 78 or 153, and are any of the standard amino acids set forth in, e.g., Lehninger, Biochemistry or other standard reference works.
  • the muteins of the invention are preferably at least about 155 amino acids long, and are preferably no more than 180 amino acids long, which is the full length of SEQ ID NO:1.
  • the proteins of the invention contain a further change at amino acid 153, i.e., leucine, in SEQ ID NO:1. This amino acid may be deleted, or be replaced by one other amino acid.
  • the new amino acids may be the same or different, and are preferably the same. Even more preferably, they are all serine or alanine. Deletions at any of these positions are not permitted.
  • amino acid 153 preferably the amino acid is replaced, rather than deleted.
  • the replacement amino acid is one which facilitates the secretion of the protein out of the transformed or transfected cell in which it is produced. Histidine is exemplified, but the ability to prepare point mutations and then to compare the secretion thereof to wild type NY- ESO-I, of SEQ ID NO:1, is an experiment that can easily be carried out, to determine if the mutein does in fact have improved secretion properties. Deletion at position 153 is also permitted.
  • the muteins possess the property of NY-ESO-I which makes it so valuable, i.e., it is processed intracellularly into immunological active peptides which form complexes with MHC molecules, be these of Class I or Class II, leading to a T Cell (CD8+ or CD4+) response.
  • nucleic acid molecules which encode the muteins are also a part of the invention.
  • Exemplary of the host cells which can be employed in this fashion are COS cells, CHO cells, yeast cells, insect cells (e.g., Spodoptera frugiperda), NIH 3T3 cells, and so forth.
  • Prokaryotic cells such as E. coli and other bacteria may also be used.
  • nucleic acid molecules which encode one or more of the muteins may be incorporated into these vectors, which are then the major constituent of nucleic acid based therapies.
  • the invention also involves the use of various materials disclosed herein to "immunize” subjects or as “vaccines".
  • immunize or “vaccination” means increasing or activating an immune response against an antigen. It does not require elimination or eradication of a condition, but rather contemplates the clinically favorable enhancement of an immune response toward an antigen.
  • Generally accepted animal models can be used for testing of immunization against cancer using a cancer associated antigen nucleic acid.
  • human cancer cells can be introduced into a mouse to create a tumor, and one or more cancer associated antigen nucleic acids can be delivered by the methods described herein.
  • the effect on the cancer cells can be assessed as a measure of the effectiveness of the cancer associated antigen nucleic acid immunization.
  • testing of the foregoing animal model using more conventional methods for immunization include the administration of one or more cancer associated antigen polypeptides or peptides derived therefrom, optionally combined with one or more adjuvants and/or cytokines to boost the immune response.
  • Methods for immunization including formulation of a vaccine composition and selection of doses, route of administration and the schedule of administration (e.g. primary and one or more booster doses), are well known in the art.
  • the tests also can be performed in humans, where the end point is to test for the presence of enhanced levels of circulating CTLs against cells bearing the antigen, to test for levels of circulating antibodies against the antigen, to test for the presence of cells expressing the antigen and so forth.
  • one or more of the muteins or stimulatory fragments thereof are administered with one or more adjuvants to induce an immune response or to increase an immune response.
  • An adjuvant is a substance incorporated into or administered with antigen which potentiates the immune response.
  • Adjuvants may enhance the immunological response by providing a reservoir of antigen (extracellularly or within macrophages), activating macrophages and stimulating specific sets of lymphocytes. Adjuvants of many kinds are well known in the art.
  • adjuvants include monophosphoryl lipid A (MPL, SmithKline Beecham), a congener obtained after purification and acid hydrolysis of Salmonella minnesota Re 595 lipopolysaccharide; saponins including QS21 (SmithKline Beecham), a pure QA-21 saponin purified from Quillja saponaria extract; DQS21, described in PCT application WO96/33739 (SmithKline Beecham); QS-7, QS-17, QS- 18, and QS-Ll (So et al., MoI.
  • MPL monophosphoryl lipid A
  • SPL SmithKline Beecham
  • saponins including QS21 (SmithKline Beecham), a pure QA-21 saponin purified from Quillja saponaria extract
  • DQS21 described in PCT application WO96/33739 (SmithKline Beecham)
  • QS-7, QS-17, QS- 18, and QS-Ll So et al., MoI.
  • Immunostimulatory oligonucleotides may also be used as adjuvants. Such oligonucleotides are well known and are described in, for example WO96/02555. Examples of oligonucleotides for use in adjuvants or vaccines of the present invention include CpG containing oligonucleotides, generally containing two or more dinucleotide CpG motifs separated by at least three, more often at least six or more nucleotides. A CpG motif is a cytosine nucleotide followed by a guanine nucleotide. The CpG oligonucleotides are typically deoxynucleotides.
  • the internucleotide in the oligonucleotide is phosphorodithioate, or more preferably a phosphorothioate bond, although phosphodiester and other internucleotide bonds are within the scope of the invention.
  • oligonucleotides with mixed internucleotide linkages are included within the scope of the invention. Methods for producing phosphorothioate oligonucleotides or phosphorodithioate are described in U.S. Patent Nos. 5,666,153 and 5,278,302 and WO95/26204, all of which are incorporated by reference.
  • oligonucleotides include but are not limited to: TCC ATG ACG TTC CTG ACG TT (CpG 1826) (SEQ ID NO: 15) TCT CCC AGC GTG CGC CAT (CpG 1758) (SEQ ID NO: 16) ACC GAT GAC GTC GCC GGT GAC GGC ACC ACG (SEQ ID NO: 17) TCG TCG TTT TGT CGT TTT GTC GTT (CpG 2006) (SEQ ID NO: 18) TCC ATG ACG TTC CTG ATG CT (CpG 1668) (SEQ ID NO: 19) TCG ACG TTT TCG GCG CGC GCC G (CpG 5456) (SEQ ID NO: 20)
  • the sequences may contain phosphorothioate modified internucleotide linkages.
  • Alternative CpG oligonucleotides may comprise one or more sequences above in that they have inconsequential deletions or additions thereto.
  • the CpG oligonucleotides may be synthesized by any method known in the art (for example see EP 468520). Conveniently, such oligonucleotides may be synthesized utilising an automated synthesizer.
  • Adjuvants combinations include 3D-MPL and QS21 (EP 0 671 948 Bl), oil in water emulsions comprising 3D-MPL and QS21 (WO 95/17210, WO 98/56414), or 3D-MPL formulated with other carriers (EP 0 689 454 Bl).
  • Other preferred adjuvant systems comprise a combination of 3D-MPL, QS21 and a CpG oligonucleotide as described in U.S. Patent Nos. 6,558,670 and 6,544,518, both of which are incorporated by reference.
  • the invention also contemplates delivery of nucleic acids, polypeptides or peptides for vaccination. Delivery of polypeptides and peptides can be accomplished according to standard vaccination protocols which are well known in the art. In another embodiment, the delivery of nucleic acid is accomplished by ex vivo methods, i.e. by removing a cell from a subject, genetically engineering the cell to include a cancer associated antigen, and reintroducing the engineered cell into the subject.
  • ex vivo methods i.e. by removing a cell from a subject, genetically engineering the cell to include a cancer associated antigen, and reintroducing the engineered cell into the subject.
  • One example of such a procedure is outlined in U.S. Pat. No. 5,399,346 and in exhibits submitted in the file history of that patent, all of which are publicly available documents.
  • the therapeutic compositions of the present invention can be administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents.
  • the compositions of the invention are administered in effective amounts.
  • An "effective amount" is that amount of a mutein composition that alone, or together with further doses, produces the desired response, e.g. increases an immune response to the mutein.
  • the desired response is inhibiting the progression of the disease.
  • This can be monitored by routine methods or can be monitored according to diagnostic methods of the invention discussed herein.
  • the desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition.
  • the pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of mutein or nucleic acid encoding the mutein for producing the desired response in a unit of weight or volume suitable for administration to a patient.
  • the response can, for example, be measured by determining the immune response following administration of the mutein composition via a reporter system by measuring downstream effects such as gene expression, or by measuring the physiological effects of the mutein composition, such as regression of a tumor or decrease of disease symptoms.
  • Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response.
  • methods for diagnosing or determining the prognosis of a disorder that is characterized by expression of a tumor associated nucleic acid or polypeptide involve contacting a biological sample isolated from a subject with an agent specific for the tumor associated nucleic acid or polypeptide to detect the presence of the tumor associated nucleic acid or polypeptide in the biological sample.
  • a series of tests is carried out over time to determine the subject's prognosis with respect to progression or regression of the disorder.
  • Another aspect of the invention provides methods for diagnosing a disorder characterized by the expression of a tumor rejection antigen derived from a tumor associated polypeptide which forms a complex with HLA molecules.
  • the method involves contacting a biological sample isolated from a subject with an agent that binds the complex and then determining binding between the complex and the agent as a determination of the disorder.
  • fusion proteins where a fusion partner which assists in providing T helper epitopes (immunological fusion partner), such as T helper epitopes recognized by humans, or assists in expressing the protein (expression enhancer) at higher yields than the native recombinant protein, is used in connection with a full length protein.
  • the fusion partner may be both an immunological fusion partner and expression enhancing partner.
  • the immunological fusion partner consists of an amino acid sequence from protein D, a surface protein of the gram-negative bacterium, Haemophilus influenza B (WO91/18926).
  • the protein D derivative comprises approximately the first 1/3 of the protein, in particular approximately the first N-terminal 100-110 amino acids.
  • the protein D derivative is lipidated.
  • the first 109 residues of the Lipoprotein D fusion partner are appended to the N-terminus to provide the vaccine candidate antigen with additional, exogenous T-cell epitopes and increases expression levels, for example when synthesized in E. coli.
  • the lipid tail optimizes presentation of the antigen to antigen presenting cells.
  • fusion partners include, but are not limited to, the non-structural protein from influenzae virus, NSl (hemagglutinin).
  • NSl hemagglutinin
  • N terminal 81 amino acids are utilized, although different fragments may be used, as long as they include T-helper epitopes. These T helper epitopes will be easily recognized to one of skill in the art, and need not be set forth here.
  • the immunological fusion partner is the protein known as LYTA.
  • LYTA the protein known as LYTA.
  • the C terminal portion of the molecule is used.
  • Lyta is a Streptococcus pneumoniae protein. These bacteria synthesize an N-acetyl-L-alanine amidase, amidase LYTA, (coded by the lytA gene (Gene, 43 (1986) page 265-272) an autolysin that specifically degrades certain bonds in peptidoglycan backbones.
  • the C-terminal domain of the LYTA protein is responsible for affinity of the molecule to choline or to choline analogues such as DEAE.

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Abstract

La présente invention concerne une protéine NY-ESO hautement immunogène faiblement produite dans un système eucaryote recombinant. Des techniques d’évolution dirigée et de purification améliorée, ont eu pour résultat une protéine mutée de NY-ESO-I, une culture de cystéine aux positions 75 à 78 (CCRA) étant remplacée par AARA et la position 153 étant mutée de leucine en histidine. La protéine mutée est facilement obtenue à partir de levure et est autant immunogène que NY-ESO-I de type sauvage dans le MHC de classe I, des systèmes HLA-A2+.
PCT/US2006/043226 2005-11-04 2006-11-03 Formes mutees de ny-eso-1 produites dans de la levure et leurs utilisations WO2007056304A2 (fr)

Applications Claiming Priority (2)

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US73343705P 2005-11-04 2005-11-04
US60/733,437 2005-11-04

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WO2007056304A2 true WO2007056304A2 (fr) 2007-05-18
WO2007056304A3 WO2007056304A3 (fr) 2007-07-26

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN Y.T. ET AL.: 'A testicular antigen aberrantly expressed in human cancers detected by autologous antibody screening' P.N.A.S. vol. 94, 1997, pages 1914 - 1918, XP002355506 *
SCHULTZ-THATER E. ET AL.: 'NY-ESO-1 tumour associated antigen is a cytoplasmic protein detectable by specific monoclonal antibodies in cell lines and clinical specimens' BRITISH JOURNAL OF CANCER vol. 83, no. 2, 2000, pages 204 - 208, XP003015668 *

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