WO2008029138A2 - Peptides et procédés - Google Patents

Peptides et procédés Download PDF

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Publication number
WO2008029138A2
WO2008029138A2 PCT/GB2007/003355 GB2007003355W WO2008029138A2 WO 2008029138 A2 WO2008029138 A2 WO 2008029138A2 GB 2007003355 W GB2007003355 W GB 2007003355W WO 2008029138 A2 WO2008029138 A2 WO 2008029138A2
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Prior art keywords
seq
polypeptide
afp
treatment
cells
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PCT/GB2007/003355
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English (en)
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WO2008029138A3 (fr
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Shahriar Behboudi
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Ucl Business Plc
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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
    • C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
    • 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
    • A61K39/00118—Cancer antigens from embryonic or fetal origin
    • A61K39/001181—Alpha-feto protein

Definitions

  • the invention relates to novel peptides and to their use in preparation of medicaments for, and in the treatment of, cancer.
  • the cancer is preferably one which expresses alpha fetoprotein, and is preferably hepatocellular carcinoma (HCC).
  • HCC hepatocellular carcinoma
  • Hepatocellular carcinoma has a dismal prognosis.
  • the lack of effective therapies gives rise to an overall 6% probability of being alive at a time point five years from diagnosis.
  • 94% of patients diagnosed with HCC will have died within 5 years. This is the lowest survival rate for any type of cancer listed by the surveillance epidemiology and end results of the National Cancer Institute.
  • novel therapies for this devastating disease are needed.
  • Butterfield et al disclose detailed responses to HLA-A* 0201 immunodominant peptides derived from ⁇ - Fetoprotein in patients with hepatocellular cancer.
  • This document presents details of various ⁇ -Fetoprotein peptides, and discusses CD8+ responses against those peptides.
  • Liu et al disclose the hierarchy of ⁇ -Fetoprotein specific T-cell responses in subjects with AFP positive hepatocellular cancer.
  • This publication describes the ordering of responses to various AFP derived peptides into immunodominant and subdominant hierarchies. Again, this document focuses on CD8+ T cell responses to AFP peptides.
  • Alisa et al disclose an analysis of CD4+ T-cell responses to a novel ⁇ -Fetoprotein derived epitope in hepatocellular carcinoma patients. Specifically, a promiscuous and immunodominant AFP derived HLA-DR restricted peptide was identified. CD4 T cell response to this epitope was analyzed in patients with HCC and found to be detected in high frequency in large number of HCC patients with early stage of disease. This publication observes a CD4+ T-cell response to AFP.
  • WO 01/58922 discloses a method and compositions for treating hepatocellular cancer. This document is principally concerned with the generation of AFP peptide specific T- lymphocytes which are cytotoxic T-lymphocytes. Various different peptides are disclosed for various different applications.
  • WO 98/35981 discloses prevention and treatment of hepatocellular cancer. This document focuses on attempts to generate immune responses against parts of the AFP molecule. Various peptides are presented as apparently conforming to the HLA- A* 0201 class I binding motif. A very small proportion of these disclosed peptides are shown to have promise in the context of cytotoxic T-cell responses.
  • Peptide SB32 (PLFQVPEPV - AFP137-145) is disclosed in Butterfield et al (2003 Clinical Cancer Research vol 9 pp5902-5908) at page 5903 r-col.
  • the peptide is disclosed as one of several peptides which may elicit CD8+ T cell responses.
  • Peptide SB45 (AVIADFSGL - AFP570-578) is disclosed in WO98/35981 at page 7 (Table I - 'AFP24'). The peptide is disclosed as one of 72 theoretical peptides which may conform to the HLA A2.1 class I binding motif.
  • Peptide SB66 (QLAVSVILRV - AFP364-373) is disclosed in Alisa et al (2005 Clinical Cancer Research vol 11 pp6686-6694) at page 6687 first paragraph. This peptide is disclosed as playing a role in the induction of CD4+ T-cell responses to AFP.
  • the present invention seeks to overcome problem(s) associated with the prior art.
  • CD4 epitopes derived from ⁇ -Fetoprotein can generate clinically useful anti-tumour responses.
  • the prior art contains examples of CD8 responses against ⁇ -Fetoprotein, there has been no previous disclosure of the clinical value of manipulating CD4 responses to ⁇ -Fetoprotein.
  • the inventors have derived revealing insights into the nature of the immune response against tumours following such treatments. Furthermore, the inventors have discovered how to manipulate and enhance these immune responses to provide novel, clinical benefits such as delaying or preventing reoccurrence of the tumour following necrotising treatment, and extension of life expectancy.
  • the invention is based upon these surprising findings. Specifically, the invention involves the use of CD4 AFP epitopes in stimulating immune responses following necrotising treatment. Furthermore, a very precise window of opportunity is defined during which administration of these epitopes is especially beneficial to HCC patients. These two novel aspects combine synergistically in order to enhance life expectancy and to inhibit or prevent tumour recurrence following necrotising treatment. These are key technical benefits of the present invention.
  • the invention provides a recombinant or isolated polypeptide, wherein said polypeptide is or comprises a CD4+ T cell epitope; wherein said polypeptide is no more than 14 amino acids in length; and wherein said polypeptide comprises amino acid sequence selected from the group consisting of
  • said polypeptide is no more than 12 amino acids in length.
  • said polypeptide is no more than 10 amino acids in length.
  • the invention provides a recombinant or isolated polypeptide, wherein said polypeptide is or comprises a CD4+ T cell epitope; wherein said polypeptide is no more than 14 amino acids in length; and wherein said polypeptide comprises amino acid sequence selected from the group consisting of
  • said polypeptide is no more than 12 amino acids in length.
  • said polypeptide is no more than 10 amino acids in length.
  • polypeptide comprises amino acid sequence selected from the group consisting of preferably refers to a situation 'wherein said polypeptide comprises contiguous amino acid sequence selected from the group consisting of, i.e. preferably the polypeptide being defined by reference to the recited amino acid sequences and variants thereof must comprise a whole, uninterrupted amino acid sequence or variant referred to.
  • peptides in this group share the unexpected technical benefits and applications disclosed herein, particularly in connection with HCC.
  • the therapeutic significance of these peptides in the context of HCC is disclosed herein for the first time.
  • polypeptide comprises amino acid sequence of a fragment of human alpha fetoprotein.
  • the invention provides a polypeptide or epitope string comprising amino acid sequence of two or more amino acid sequences selected from the group consisting of
  • the two or more amino acid sequences may be consecutive i.e. contiguous or adjacent to one another in the polypeptide/epitope string, or may be separated by intervening sequence such as spacer sequence, linker sequence, kinker sequence, scaffold sequence or other such polypeptide element(s).
  • the two or more amino acid sequences are adjacent or contiguous in the polypeptides/epitope strings of the invention.
  • the invention provides a polypeptide or an epitope string as described above for use as a medicament.
  • the invention provides use of a recombinant or isolated polypeptide, wherein said polypeptide is or comprises a CD4+ T cell epitope; wherein said polypeptide is no more than 14 amino acids in length; and wherein said polypeptide comprises amino acid sequence selected from the group consisting of
  • said polypeptide is no more than 12 amino acids in length.
  • Preferably said polypeptide is no more than 10 amino acids in length.
  • the invention provides a polypeptide as described above for use in the treatment of cancer, wherein said cancer expresses alpha fetoprotein.
  • the invention provides use of a polypeptide as described above in the induction of a CD4+ T cell response against AFP, wherein said peptide comprises amino acid sequence selected from the group consisting of
  • said uses are in vitro uses. Such uses advantageously avoid manipulation of the human or animal body. In some embodiments, preferably said uses are in vivo uses. Such uses may advantageously be carried out on the human or animal body.
  • the invention provides a polypeptide or use as described above wherein said treatment of cancer comprises
  • the invention provides a method of treating cancer in a subject, wherein said cancer expresses alpha fetoprotein, said method comprising
  • said polypeptide is no more than 12 amino acids in length.
  • said polypeptide is no more than 10 amino acids in length.
  • the invention provides a method or a polypeptide or use as described above wherein the polypeptide or medicament is administered after the necrotising treatment.
  • polypeptide in an further aspect, there is provided a polypeptide, a method, a use, a kit or a composition substantially as described herein and with reference to any one of the Examples or Figures.
  • polypeptide or medicament is administered approximately 2, 3, 4 or 12 weeks after necrotising treatment.
  • polypeptide or medicament is administered approximately 2, 3, 4 and 12 weeks after necrotising treatment.
  • polypeptide or medicament is administered approximately 14-21 days after the necrotising treatment.
  • polypeptide or medicament is administered 14 days after the necrotising treatment.
  • polypeptide or medicament is administered 21 days after the necrotising treatment.
  • said cancer is hepatocellular carcinoma (HCC).
  • HCC hepatocellular carcinoma
  • the invention provides a method for stimulating expansion of AFP- peptide reactive T-cells comprising contacting said T-cells with a polypeptide as described above.
  • the invention provides a method for activating AFP-peptide reactive T-cells comprising contacting said T-cells with a polypeptide as described above.
  • said polypeptide is no more than 12 amino acids in length.
  • Preferably said polypeptide is no more than 10 amino acids in length.
  • the invention provides a method for activating AFP-peptide reactive
  • CD4+ T-cells comprising contacting said T-cells with a polypeptide, wherein said polypeptide is or comprises a CD4+ T cell epitope; wherein said polypeptide is no more than 14 amino acids in length; and wherein said polypeptide comprises amino acid sequence selected from the group consisting of
  • said polypeptide is no more than 12 amino acids in length.
  • said polypeptide is no more than 10 amino acids in length.
  • said contacting is performed in vitro, advantageously avoiding manipulation of the human or animal body.
  • the invention provides a composition comprising a polypeptide, wherein said polypeptide is or comprises a CD4+ T cell epitope; wherein said polypeptide is no more than 14 amino acids in length; and wherein said polypeptide comprises amino acid sequence selected from the group consisting of
  • composition is for stimulating or enhancing an immune response against said polypeptide.
  • polypeptide is no more than 12 amino acids in length.
  • polypeptide is no more than 10 amino acids in length.
  • composition is for immunising a subject against said polypeptide.
  • composition is a vaccine composition.
  • the invention provides a kit comprising
  • polypeptide (ii) a polypeptide, wherein said polypeptide is or comprises a CD4+ T cell epitope; wherein said polypeptide is no more than 14 amino acids in length; and wherein said polypeptide comprises amino acid sequence selected from the group consisting of
  • said polypeptide is no more than 12 amino acids in length.
  • said polypeptide is no more than 10 amino acids in length.
  • the kit is supplied together with instructions for administration of the polypeptide to a subject after administration of the necrotising agent.
  • kit further comprises adjuvant.
  • kit further comprises apparatus for administration of the polypeptide and/or necrotising agent.
  • the invention provides a method for identifying peptides useful in the methods and uses disclosed herein, said method comprising
  • binding of peptides to a HLA of interest is predicted using a suite of in silico prediction tools
  • the peptides are pooled into groupings of approximately 5-10 peptides per pool. 6) The peptides are tested using IOday PBMC cultures from patients and successful peptides are selected.
  • the invention also relates to a peptide obtainable by this method.
  • said peptides are obtained by this method. More detailed methods are presented in the examples section.
  • tumour-specific T cell immune responses Despite the spontaneous presence and induction by vaccination of tumour-specific T cell immune responses in humans, clearance of established tumours by endogenous immune mechanisms is rare. It is increasingly recognised that tumours create an immunosuppressive network that promotes tumour growth and attenuates immunotherapeutic efficiency. In-situ induction of necrosis of tumour cells may provide a means of subverting these tolerizing conditions, for example, by producing local inflammation and presenting tumour rejection antigens in an immunostimulatory context. In-vitro and clinical vaccination studies have shown that necrotic tumour cells release danger signals which can prime antigen-specific immune responses. However, whether a necrosis-inducing cancer treatment can activate tumour rejection antigen- specific immunity in humans is not known from the prior art. According to the present invention, by breaking tolerance in this manner, the combination of in vivo tumour destruction and immune potentiating strategies provides a relatively simple way of inducing effective in situ antitumour immune responses.
  • TACE/TAE induced necrosis could subvert the regulatory effects of advanced HCC and unmask AFP-specific T cell responses thereby providing an attractive window for immunotherapy.
  • 'peptide' has its ordinary meaning in the art and refers to a polypeptide.
  • 'Peptide sequence' refers to the amino acid sequence of a given polypeptide.
  • An 'epitope string' is a polypeptide comprising a number of epitopes, and which may comprise a number of 'peptide sequences' as presented herein.
  • Alpha-fetoprotein AFP
  • Alpha-fetoprotein is an oncofetal antigen which is re-expressed in the majority of patients with HCC and used in clinical practice as a diagnostic and prognostic serum marker. It also serves as a tumour rejection antigen and therefore is an attractive target for immunotherapy. It has been suggested that the rationale behind an effective epitope-based therapeutic vaccine is to enhance both tumour-specific CD8 + and CD4 + T cell responses, thus controlling tumour growth.
  • AFP-specific cytotoxic lymphocyte responses exist which can be generated by epitope-based vaccination in patients with hepatocellular carcinoma.
  • CD4 + T cells which recognised an immunodominant AFP-derived epitope in HCC patients, predominantly in early stage disease.
  • the present invention teaches new peptides and new therapeutic applications, particularly in the field of HCC.
  • Peptides of the invention are preferably derived from human alphafetoprotein (AFP).
  • AFP human alphafetoprotein
  • References to human alphafetoprotein preferably refer to the amino acid sequence as disclosed in Swiss-Prot/TrEMBL accession number P02771. For ease of reference, the sequence is presented herein (http://ca.expasy.org/uniprot/P02771): AFP sequence:
  • the peptide sequences shown in Table A are CD4 T cell epitopes.
  • the peptides used may be variants of the peptides shown in Table A.
  • variants of the peptides include longer peptides which comprise a core sequence shown in Table A, but which are not full length recombinant AFP.
  • Preferred variants of the peptides of Table A have the amino acid sequences shown in Table A, but shortened by one or two amino acids.
  • the sequence may be as shown in Table A ('parent peptide') but with a one or two amino acid N-terminal truncation, or a one or two amino acid C-terminal truncation, or a one amino acid N- terminal truncation and a one amino acid C-terminal truncation.
  • VNFTEIQKL laa N-terminal truncation - SEQ ID NO:9
  • NFTEIQKL 2aa N-terminal truncation - SEQ ID NO: 10
  • KVNFTEIQK laa C-terminal truncation - SEQ ED NO: 11
  • KVNFTEIQ laa C-terminal truncation - SEQ ED NO: 12
  • VNFTEIQK laa N-terminal truncation and laa C-terminal truncation - SEQ ID NO: 13
  • the corresponding variants of the other peptides shown in table A are preferred variant peptides according to the present invention (see table B):
  • variant peptides according to the present invention retain the CD4 epitope of the parent peptide on which they are based.
  • polypeptides of the invention are not peptides as disclosed in WO98/35981, WO01/58922, Alisa et al (2005 Clin Cancer Res vol 11 p6686), Liu et al (2006 J.Immunol, vol 177 p712), or Butterfield et al (2003 Clin Cancer Res vol 9 p5902).
  • peptides according to the present invention comprise one or more of the amino acid sequences presented in table A.
  • peptides according to the present invention consist of one or more of the amino acid sequences presented in table A.
  • peptides according to the present invention are not so long as to represent recombinant AFP.
  • peptides according to the present invention are 100 amino acids or less in length, preferably 90 amino acids or less in length, preferably 80 amino acids or less in length, preferably 70 amino acids or less in length, preferably 60 amino acids or less in length, preferably 50 amino acids or less in length, preferably 40 amino acids or less in length, preferably 30 amino acids or less in length, preferably 25 amino acids or less in length, preferably 23 amino acids or less in length, preferably 21 amino acids or less in length, preferably 19 amino acids or less in length, preferably 18 amino acids or less in length, preferably 17 amino acids or less in length, preferably 16 amino acids or less in length, preferably 15 amino acids or less in length, preferably 14 amino acids or less in length, preferably 13 amino acids or less in length, preferably 12 amino acids or less in length, preferably 11 amino acids or less in length, preferably 10 amino acids or less in length, preferably 9 amino acids or less
  • peptides of the invention are 14, 12 or 10 amino acids in length. These lengths have the advantage of being tractable and easily handled. Furthermore, their relatively short length enables them to be easily manufactured, and promotes specificity of presentation of the epitope of interest.
  • peptides of the invention are 14 amino acids in length. This length has the advantage of being a most commonly used length and therefore makes standard protocols easier to follow and makes it easier to validly compare results with other peptides of this standard length.
  • the peptide(s) of the invention retain at least one CD4+ epitope, and must therefore comprise sufficient amino acids to permit this.
  • a suitable test should be conducted such as one or more of the following techniques to detect CD4 T cell responses to an epitope:
  • CD4 T cells recognise a peptide epitope and produce cytokines (such as IFN- gamma).
  • cytokines such as IFN- gamma
  • the production of cytokines can be detected using an intracellular cytokine assay, ELISPOT assay or ELISA assay.
  • cytokine assay e.g. IL-12 assay
  • ELISPOT assay e.g., ELISPOT assay
  • ELISA assay e-e- Instead of (or as well as) detecting the ability of cells to produce cytokines upon peptide recognition, phenotypic characterisations of cytokine producing CD4 T cells by intracellular cytokine assay could be utilised.
  • CD4 T cells recognise a peptide epitope and proliferate. This proliferation can be detected using a standard proliferation assay (eg. Alisa et al. 2005 ibid).
  • peptides according to the present invention comprise one or more amino acid sequences selected from the group consisting of
  • peptides according to the present invention comprise one or more amino acid sequences selected from the group consisting of
  • the peptide is SB94 (KVNFTEIQKL (AFP249-258)).
  • This peptide has the advantage of acting in a necrosis-specific manner. In other words, this peptide is selectively active in subjects having had necrotising treatment.
  • the functional significance of these peptides is to prevent and/or delay recurrence of the tumour, particularly to prevent and/or delay recurrence of AFP expressing HCC tumours previously subjected to a necrotising treatment.
  • Peptides may be used in combination.
  • peptides When used in combination, peptides may be concatenated (i.e. fused into a single polypeptide comprising amino acid sequences of a number of peptides disclosed herein) or may be used as a cocktail (i.e. prepared as separate entities but administered as a combination of said separate entities). Use without concatenation (e.g. as a cocktail) is preferred.
  • An advantage to using peptides in combination is that different patients can respond to different peptides. Thus, by using them in combination the effective coverage for a particular preparation is advantageously increased. Furthermore, if a subject responds to more than one of the peptides, then giving more than one of those peptides correspondingly increases the effectiveness of the treatment.
  • one administration event comprises multiple injections.
  • Each peptide may be administered as a separate injection or the peptides may be mixed and administered in one or more combined injections.
  • the administration will comprise separate injections of the different peptides in a single administration event.
  • Preferred combinations of peptides include SB66 (QLAVSVILRV (AFP 364-373)) together with any other peptide or variant disclosed herein. The advantage of this particular combination is that at least 25-30% of patients respond to this.
  • SB94 KVNFTEIQKL (AFP249-258)
  • SB66 and SB94 optionally with third or further peptide(s).
  • the technical advantage of this combination is the excellent coverage provided by SB66 coupled with the necrosis-specific effectiveness of SB94.
  • the peptides may be combined by concatenation or fusion into larger polypeptides comprising the sequences of the individual peptides. These fused polypeptides are known as epitope strings. Such epitope strings may be made by fusion/concatenation of any two or more peptides from table A or table B, preferably three or more, preferably 4 or more, preferably 5 or more, preferably 6 or more, preferably 7 or more, preferably eight peptides are joined to form an epitope string. Preferably all eight peptides from Table A are joined to make an epitope string. Preferably said peptides are joined in order SEQ ED NO: 1 to SEQ ID NO:8.
  • a preferred epitope string is QLAVSVILRVPLFQVPEPVLQTMKQEFLIKVNFTEIQKLEMTPVNPGVCLENQL PAFLAVIADFSGLSSGEKNIFL,or KVNFTEIQKLEMTPVNPGVCLENQLPAFLAVIADFSGLSSGEKNIFL 5 Or KVNFTEIQKLEMTPVNPGVCLENQLPAFLSSGEKNIFL, most preferably QLAVSVILRVPLFQVPEPVLQTMKQEFLIKVNFTEIQKLEMTPVNPGVCLENQL PAFLAVIADFSGLSSGEKNIFL.
  • the peptides are preferably supplied and/or administered together with an appropriate adjuvant.
  • said adjuvant is approved for human use.
  • said adjuvant is or comprises mannide oleate.
  • said adjuvant is or comprises mannide oleate, Montamide ISA-51 (Seppic Inc., USA), Freund's incomplete adjuvant or aluminium hydroxide.
  • the peptides may be provided in any suitable form. Preferably the peptides are provided as free peptides in solution.
  • the invention also relates to provision of the peptides as nucleic acid such as in the form of a DNA encoding the peptide(s) of interest.
  • the invention also relates to the provision of the peptides in the form of nucleic acid encoding them in a viral vector such as an adenoviral vector, a vaccinia viral vector such as modified vaccinia Ankara (MVA) or other suitable viral vector.
  • Preferred vectors are poxvirus vectors such as vaccinia virus, fowl pox virus and adenovirus based vectors which are known in the art.
  • the viral vectors are those which have been used in humans.
  • the peptides may be provided in the form of a cell expressing same such as an engineered dendritic cell or similar cell.
  • the peptides may be provided on the cell surface of a cell which does not express them but onto which they have been loaded.
  • An example of such a form would be the provision of a T2 cell loaded with peptide(s) of the invention.
  • the (immune) treatment of the invention is administered to a subject in combination with a necrotising treatment.
  • a necrotising treatment means that the treatment of the invention (the immune treatment) is administered together with or after the necrotising treatment.
  • the treatment of the invention is not given before necrotising treatment.
  • the treatment of the invention is administered after the necrotising treatment. This has the advantage of optimising the efficacy of the treatment of the invention. Preferably the treatment of the invention is administered at least 2-3 weeks after the necrotising treatment.
  • the timing of the treatment is a key teaching of the invention. It is disclosed herein for the first time that immune treatment should be conducted after the necrotising treatment. More in particular, the immune treatment should be administered before recurrence of the tumour following necrotising treatment. Specifically, after the last necrotising treatment (e.g. embolism), there is a window before recurrence of the tumour. This window may vary between subjects, but recurrence is not earlier than 4 weeks after the last necrotising treatment, and the time to recurrence has a median value of about 3 months. Thus, the window for optimum treatment is typically 2-3 weeks. According to the present invention, this window is the time at which significant clinical benefits can be achieved by the immune treatment of the invention. Thus, preferably the immune treatment of the invention is administered 2-3 weeks after the last necrotising treatment.
  • the last necrotising treatment e.g. embolism
  • the peak of response to the necrotising treatment is typically about day 7 (day 0 being the dayof the last treatment such as TACE/TAE).
  • the immune treatment of the invention is administered 1-2 weeks after the peak of immune response associated with the last necrotising treatment.
  • Figure 6 shows a preferred scheme of immune treatment following necrotising treatment. As noted above, this peak of immune response usually occurs approximately 1 week after the last necrotising treatment.
  • distinct clinical benefits may be achieved.
  • an extra 2 months can be added to the life expectancy of the subject, preferably an extra 3 months can be added, preferably an extra 3-6 months can be added. Since patients currently have a 94% likelihood of death 5 years from diagnosis, extending life expectancy by these amounts is clearly a valuable technical effect of the present invention.
  • use of AFP peptide vaccination as disclosed herein improves the 5 -year survival rate to approximately 60-70%.
  • the immune treatment is given on day 14, day O being the day of the last necrotising treatment.
  • one or two boosts are given, preferably at one or more of days 21 and 28.
  • two boosts are given, one at day 21 and one at day 28.
  • the necrotising treatment may be any treatment suitable for causing necrosis of the tumour, and may comprise embolism, i.e. blocking or restricting one or more blood vessels supplying the tumour, or chemotherapeutically induced necrosis.
  • embolism i.e. blocking or restricting one or more blood vessels supplying the tumour, or chemotherapeutically induced necrosis.
  • Necrosis by embolism may be accomplished by use of an air bubble, such as a balloon or inflatable stent, or other foreign object, or a chemical block in the appropriate blood vessel.
  • Trans-arterial chemoembolisation (TACE) or embolisation (TAE) are the most preferred necrotising treatments for hepatocellular carcinoma (HCC), especially unresectable HCC.
  • TACE or TAE involves the injection of coils or particles into the hepatic artery with or without a chemotherapeutic agent, leading to obstruction of the hepatic artery. This produces necrosis of the well-vascularised tumour and a reduction in tumour burden.
  • the necrotising treatment comprises TACE or TAE.
  • embolism in the context of HCC is conducted by passing an instrument into the subject's circulatory system, entering in the leg. This instrument is then manoeuvred up the leg, into the abdomen and thereby into the liver. At the appropriate location with respect to the tumour to be addressed, blocker injection is effected in order to create the embolism and deprive or starve the tumour of blood.
  • the necrotising treatment may be given in multiple administrations.
  • the times 'after the necrotising treatment' are preferably measured from the last necrotising treatment.
  • this may not be the very last necrotising treatment in the lifetime of the subject being treated, but rather 'last necrotising treatment' refers to the last treatment in a given series, course or regime of necrotising treatments.
  • the treatment of the invention may be interleaved into the necrotising treatment.
  • it may be given in between individual administrations of necrotising agent.
  • Administration may be interleaved into the necrotising treatment.
  • it may be given in between individual administrations of necrotising agent.
  • the peptides are for administration intradermally.
  • administration is biweekly.
  • peptides are administered at least once, preferably at least twice, preferably at least three times.
  • An administration is a single clinical event, for example taking place in a single 24 hour period.
  • An administration is preferably a single operation such as a single injection.
  • an administration may comprise multiple operations such as multiple injections over a single event e.g. 24 hour period.
  • Benefits of this approach include for example splitting the dose up or spreading the administration of the dose to different sites, or avoiding or reducing local acute reactions or to provide other benefits such as lower volumes per injection or other similar effect.
  • Individual administration events typically comprise a total dose of 1 to 1000 ⁇ g peptide, preferably 10 to 1000 ⁇ g peptide, preferably 10 to 900 ⁇ g peptide, preferably 50 to 900 ⁇ g peptide, preferably 50 to 800 ⁇ g peptide, preferably 100 to 800 ⁇ g peptide, preferably 100 to 700 ⁇ g peptide, preferably 100 to 600 ⁇ g peptide, preferably 100 to 500 ⁇ g peptide, preferably lOO ⁇ g peptide, preferably 200 ⁇ g peptide, preferably 300 ⁇ g peptide, preferably 400 ⁇ g peptide, preferably 500 ⁇ g peptide.
  • Doses are given for a typical adult human subject. Doses for other subjects may be calculated on the basis of the guidance provided herein, adjusting for height, weight and other relevant characteristics known to the skilled operator. Immune Responses
  • CD8+ T cell responses An observation in connection with CD8+ T cell responses is no indication with respect to likely CD4+ T cell responses (if any).
  • teachings provided herein with respect to CD4+ responses represent a part of the contribution to the art.
  • the evidence and experimental material provided herein show for the first time the memory response, whereas prior work has focussed on effector responses, which represent a different population of cells.
  • the present inventors undertook a study of the AFP protein using sequence analysis to help to select epitopes capable of HLA-A2 binding. 94 predicted sequences were selected. These were tested for their effects in a CD8 (memory) setting. Cells were co-stained for IFNgamma and CD8. The expectation was to see a IFNgamma and CD8 cell population. However, to the inventors' astonishment, the IFNgamma positive cells were negative for CD8. In the course of subsequent investigations to try to understand what might be happening, it was surprisingly found that the IFNgainma positive cells were in fact CD4 positive. This is an unexpected finding which underlies the present invention.
  • CD4 inducing elements could have significant therapeutic benefits at set out herein. This is because the focus in the art has been on CD8 responses. CD4 responses are classically regarded as antibody related responses, and therefore not thought to be of significant use for tumour protection. It is thus a further surprise that the beneficial antitumour effects of the present invention could be produced using CD4 epitopes.
  • TACE/TAE necrosis inducing cancer treatment
  • HCC advanced malignancy
  • an effective peptide-based tumour vaccine for HCC may be most effective if both CD8 + and CD4 + T cell responses against tumour-associated antigens are induced.
  • a prior art study of patients with advanced HCC has demonstrated that AFP-specific CD8 + T cell responses can be generated by vaccination with class I epitopes but the lack of tumour responses highlights the need to improve the effectiveness of this strategy.
  • the generation of an effective anti-tumour CD4 + T cell response according to the present invention may improve efficacy. Indeed, studies of patients with advanced breast or ovarian cancer have shown that when T helper epitopes are combined with CTL epitopes they provide a more powerful longer lasting immunity than CTL epitopes alone. Whilst there have been single reports of CD4 + responses to both AFP and cancer-testis antigens in HCC patients, there is a need to characterise the epitopes recognised by T helper cells for future use in peptide vaccination studies.
  • AFP 24 9- 2 58 specific CD4 + T cells This indicates that processing and presentation of tumour-derived AFP antigen by antigen-presenting cells through the exogenous pathway was the mechanism through which spontaneous responses occurred in-vivo.
  • AFP specific CD4 + T cells were generally detectable in patients at an early stage of disease (low tumour burden) and low or moderately elevated serum AFP levels but not in control patients.
  • the reason for the lack of anti-AFP CD4 + responses to these peptides in advanced disease is unknown but may be due to immunoregulatory effects of AFP, or regulatory cytokines released by tumour.
  • a recent study has demonstrated higher frequencies of effector AFP-specific CD8 + T cells in HCC patients at a more advanced stage of disease. Without wishing to be bound by theory, our contrasting results may be explained by the different cell populations (short-term T cell lines vs ex-vivo) or cell types (CD4 + vs CD8 + T cells) being analysed in the respective studies. Nevertheless, the effectiveness of our approaches is demonstrated herein.
  • AFP 137-I4S has been described previously as an A2 restricted epitope and here we show that this peptide is capable of stimulating both CD4 + and CD8 + T cells.
  • Vaccination with dual specific epitopes may be more efficacious than a mixture of CTL and T helper cell epitopes as they could allow CD4 + and CD8 + cells to interact with the same antigen presenting cell thereby improving communication between cells.
  • tumour-specific T cell responses As our inter and intra-assay variation was low and AFP 249-258 specific responses only detected in treated patients, the changes in the levels of AFP specific CD4 + T cell responses can be attributed to the effects of TACE/TAE.
  • the frequency of AFP specific CD4 + T cell responses after treatment was significantly higher in clinical responders than non responders. As patients with clinical responses had larger volumes of necrosis than clinical non responders this indicates a 'dose- response' relationship between necrosis and the stimulation of AFP specific CD4 + T cells.
  • CT evidence of a response correlates well with a longer time to progression of disease and an improved survival, it appears that these tumour-specific T cell responses advantageously play a role in protection against progression or recurrence of malignancy.
  • TACE/TAE can be viewed as a preferred form of in-situ vaccination (e.g. a 'prime', with administration of polypeptides of the invention being a 'boost') leading to presentation of AFP-derived epitopes in an immunostimulatory context.
  • a 'prime' with administration of polypeptides of the invention being a 'boost'
  • necrotic cells release danger signals such as heatshock proteins and uric acid which activate dendritic cells, a prerequisite for priming naive T cells.
  • the identification of a population of AFP specific CD4 + T cells during therapy that are not detectable in untreated patients could be explained by work showing that necrotic cells induce a different set of class II peptides than cells dying via apoptosis.
  • tumour burden/regulatory factors by resection or embolisation may explain in part the observed concomitant expansion of pre-existing tumour immunity.
  • necrosis of tumour cells may be essential for their induction, and is thus a preferred first step in a two-step treatment of the invention, the second step being boosting or stimulation of the immune response by administration of appropriate peptide epitope(s).
  • necrosis produced by TACE/TAE unmasks tumour rejection antigen- specific T cell responses. It represents a relatively simple method of in-situ immune response induction which can advantageously be combined with immunotherapy to control tumour growth and improve survival.
  • the methods and materials of the present invention are preferably for treatment of cancer, preferably human cancer. They also find application in the generation, boosting or maintenance of anti-AFP immune responses, preferably CD4+ anti-AFP responses.
  • cancer is testicular carcinoma or hepatocellular carcinoma (HCC), preferably HCC.
  • the 'prime' in a prime-boost regime is a necrotising treatment.
  • tumour necrosis unmasks alpha-fetoprotein-specific CD4+ T cell responses in hepatocellular carcinoma. We further describe necrosis induced AFP-specific T cell responses.
  • each human subject has two HLA-DR haplotypes (this could be two similar or different haplotypes).
  • a cancer patient may have HLA- DR3 and HLA-DR4. If there is a HLA-DR restricted CD4 T cell response to an epitope in this patient, it will be restricted to DR3 and/or DR4 in this patient.
  • Finding a CD4 T cell response to the same epitope in a different patient means that this epitope is restricted to HLA-DR13 or DRl 5 in this patient.
  • finding this response in patients with different HLA-DR haplotypes demonstrates that this epitope can be incorporated into a composition such as a vaccine composition (or a composition for inducing an immune response) for inducing CD4 T cell responses to this epitope in patients with different HLA-DR haplotypes (ie.
  • the invention can be universally applied).
  • the invention finds application in mammalian subjects regardless of their HLA-DR haplotype.
  • the invention is applied to subjects having one or more HLA-DR haplotypes selected from the group consisting of DR3, DR4, DR 13 and DR15.
  • polypeptides may have deletions, insertions or substitutions of amino acid residues, which produce a silent change and result in a functionally equivalent polypeptide.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the functionally of the polypeptide is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • Homologous substitution substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue
  • substitution and replacement may occur i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc.
  • Non-homologous substitution may also occur i.e. from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.
  • Z ornithine
  • B diaminobutyric acid ornithine
  • O norleucine ornithine
  • pyriylalanine pyriylalanine
  • thienylalanine
  • Replacements or additions may also be made by unnatural amino acids which include; alpha* and alpha-disubstituted* amino acids, N-alkyl amino acids*, lactic acid*, halide derivatives of natural amino acids such as trifluorotyrosine*, p-Cl-phenylalanine*, p- Br-phenylalanine*, p-I-phenylalanine*, L-allyl-glycine*, ⁇ -alanine*, L- ⁇ -amino butyric acid*, L- ⁇ -amino butyric acid*, L- ⁇ -amino isoburyric acid*, L- ⁇ -amino caproic acid*, 7-amino heptanoic acid*, L-methionine sulfone* * , L-norleucine*, L-norvaline*, p-nitro-L-phenylalanine*, L-hydroxyproline # , L-thioproline*,
  • Polypeptides may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or ⁇ -alanine residues.
  • alkyl groups such as methyl, ethyl or propyl groups
  • amino acid spacers such as glycine or ⁇ -alanine residues.
  • the presence of one or more amino acid residues in peptoid form is also contemplated and will be well understood by those skilled in the art.
  • the peptoid form is used to refer to amino acid residues wherein the ⁇ -carbon substituent group is on the residue's nitrogen atom rather than the ⁇ -carbon.
  • Figure 1 shows naturally occurring AFPn 7-I4S -SPeCiCc T cells in patients with HCC.
  • AFPi 37 ,i 45 -specific CD4 + T cells recognised AFPi 37-H s (Ia) and purified AFP (Ib) but not an irrelevant peptide or control protein (BSA) and produced ThI type cytokines (Ia, Ib, Ic).
  • AFP ⁇ . ⁇ s-specific CD4 + T cells recognised AFP 137-J4S in a HLA-DR restricted manner (Id).
  • AFPi 37-I45 was also recognised by CD8 + T cells isolated from HCC patients (Ie). Columns, each experiment were performed in duplicate and the results are representative of two different experiments on different days.
  • Figure 2 shows AFP 249 - 2 s 8- specific CD4 + T cells generated after embolisation in a patient with HCC.
  • AFP 249-2 s 8 -specific CD4 + T cells recognised (2a) and purified AFP (2b) but not an irrelevant peptide or control protein (BSA) and produced ThI type cytokines (2c). was recognised by CD4 + T cells in an HLA-DR restricted manner (2d).
  • BSA irrelevant peptide or control protein
  • CD4 + T cells in an HLA-DR restricted manner
  • FIG. 3 shows transarterial chemoembolisation (TACE) or embolisation (TAE)- induced tumour necrosis unmasks AFP-specific CD4 + T cells.
  • TACE transarterial chemoembolisation
  • TAE embolisation
  • FIG. 4 shows TACE/TAE induced necrosis significantly increases AFP-specif ⁇ c CD4 + T cell responses which are associated with clinical outcome 4a) each dot represents average percentage of AFP137-145, AFP364-373 or AFP 24 9 -258 -specific CD4 + T cells before, during and one month after the last treatment. 4b). Columns, average percentage of CD4 + T cells recognising AFP 137- I 4 S, AFP364-373 and AFP 2 49-25S epitopes are shown before and one month after TACE/TAE in clinical responders and non- responders.
  • Figure 5 shows pre-existing anti-AFP CD4 + T cell responses were expanded in one of three patients with HCC undergoing curative tumour resection.
  • Short term T cell lines were generated from PBMCs isolated from the three HCC patients before resection and four weeks after surgery. The samples were analysed in duplicate (average) and the results are representative of two different experiments on different days.
  • Figure 6 shows a preferred immune treatment regime according to the present invention.
  • PBMCs Peripheral blood mononuclear cells
  • AFP derived peptides were synthesised by Mimotopes Pty Ltd. (Clayton Victoria, Australia). These peptide epitopes were: a HLA-DR restricted T cell epitope QLAVSVILRV (AFP 364-373 ), three previously reported HLA-A2 restricted epitopes: PLFQVPEPV (AFP 137-145 ), GLSPNLNRFL (AFP 325-334 ), GVALQTMKQ (AFP 542-550 ), and a preferred AFP-derived peptide epitope KVNFTEIQKL (AFP 249-258 ). Generation of T cell lines and intracellular cytokine assay
  • PBMCs were separated by Ficoll centrifugation. T cell lines were generated as described previously (Alisa A, Ives A, Pathan AA, Navarrete CV, Williams R, Bertoletti A, Behboudi S. Analysis of CD4+ T-CeIl responses to a novel alpha- fetoprotein-derived epitope in hepatocellular carcinoma patients. Clin Cancer Res 2005;l 1:6686-94). In brief, PBMCs were re-suspended in AIM-V medium (Gibco) and cultured in duplicate with individual peptides (1 ⁇ M).
  • AIM-V medium Gibco
  • Recombinant IL-2 (25 IU/ ml) was added on day 2-3 of culture and cells were analysed after a total of 10-12 days of culture.
  • AFP-specif ⁇ c T cell lines were incubated for 5 hours at 37 0 C with the AFP- derived peptides (1 ⁇ M) and Brefeldin A. Cells were surface stained with Cy-chrome- conjugated anti-CD4 + or anti-CD8 + antibodies. The cells were then permeabilised and fixed using Cytof ⁇ x/CytopermTM (BD PharMingen, Cowley, UK).
  • the cells were stained for intracellular cytokines with FITC-conjugated anti IFN- ⁇ , FITC- conjugated anti-TNF- ⁇ , PE-conjugated anti-IL-5 or isotype controls (R & D systems, Abingdon, UK), washed twice and the frequency of AFP-specific T cell responses quantified by flow cytometry.
  • An immunological response/responder was defined as a two fold increase in frequency of cytokine-producing cells above control peptides or proteins.
  • An immunological responder to TACE/TAE or surgery was defined as a two fold increase in an immunological response during or after treatment compared with pre-treatment.
  • the murine mAbs HL-39 and SPVL-3 (which block peptide presentation to CD4 + T cells by HLA-DR and HLA-DQ, respectively) were added (5 ⁇ g/ml) 90 minutes before peptide stimulation. All samples were tested in duplicate and peptide-specific intracellular IFN- ⁇ -production was analysed using flow cytometry.
  • the murine monoclonal Ab w6/32 (which blocks peptide presentation by HLA class I to CD8 + T cells) was used as described above.
  • the chi-square, Mann Whitney and unpaired t tests were used to test for a difference in variables in treatment naive patients between immunological responders and non- responders.
  • the changes in frequency of AFP-specific CD4 + T cells during and after treatment with TACE/TAE were compared in a pair-wise fashion using the Wilcoxon rank sum test.
  • the Mann Whitney test was used to determine if there was a difference in the frequency of AFP-specific T cells after treatment between clinical responders and non responders.
  • AU tests were two-tailed and p values ⁇ 0.05 were considered as being statistically significant.
  • Example 1 Method for obtaining peptides of the invention.
  • binding of AFP peptides to a HLA of interest is predicted using a suite of in silico prediction tools
  • the software tools are used to generate a roster of candidate peptide sequences which were predicted to bind to the HLA of interest.
  • the HLA is HLA- A2.
  • the SYFPEITHI, MIMAS, MHCPred and ProPred-1 programs were used to predict HLA-A2 binding ability of AFP-derived peptides.
  • the candidate peptides are ordered with respect to their predicted binding affinities
  • the highest affinity binders are selected. 3) The solubilities of the peptides are predicted.
  • the peptides are pooled into groupings of approximately 5-10 peptides per pool.
  • AFP alpha-fetoprotein
  • Methods In the peripheral blood of 42 treatment naive HCC patients and 24 controls, AFP-specific T cell responses to AFP-derived peptides were analysed using intracellular cytokine and MHC blocking assays. AFP-specific T cell responses were studied longitudinally for six months in ten patients undergoing chemoembolisation/ embolisation.
  • AFP-specific T cells (AFPi 37- i 45 -specific, both HLA-A2 and DR restricted) & (AFP 249 - 2S8 -specific, HLA-DR restricted) which were tumour-specific and together with AFP3 64 - 3 73-specific CD4 + T cells significantly more likely to be detectable in patients with a low serum AFP (p ⁇ 0.05) and at an early stage of disease (pO.Ol).
  • AFP-specific CD4 + T cells were significantly expanded or induced in-vivo during (p ⁇ 0.0001) or after embolisation (p ⁇ 0.002). The development of higher frequencies of AFP-specific CD4 + T cells after treatment were significantly associated with the induction of >50% necrosis of tumour and an improved clinical outcome (p ⁇ 0.007).
  • Example 3 T cell responses to AFP-derived epitopes in treatment naive HCC patients and control groups
  • AFP-derived CD8 + T cells have been identified and we have reported the presence of AFP36 4 -373 specific CD4 + T cells in treatment naive and treated patients with HCC. To date, there has not been a systematic analysis of naturally occurring immune responses to these epitopes in treatment naive HCC patients. In the present study, we analyzed T cell responses to AF?36 4 -373 and three class I restricted peptide epitopes (AFPB 7- HS, AFP325-334 and AFP5 42-5 5o) in treatment naive patients. These AFP-derived CD4 + and CD8 + T cell epitopes are presented by HLA-DR and HLA-A2 molecules, respectively.
  • a treatment naive HLA-A2+ HCC patient was recruited and short term T cell lines generated individually in the presence of AFP364-373, AFPB 7 -HS, AFP325-334 and AFP 542- 550 .
  • Ten days later, the presence of peptide-specific T cells were analyzed using an intracellular cytokine assay for EFN- ⁇ , TNF- ⁇ and IL-5.
  • S S0 was recognised by T cell lines and stimulated significantly higher levels of ThI type cytokines by CD4 + cells as compared with samples with irrelevant peptides ( Figure Ia, Ib and Ic).
  • AFPi3 7 - H 5-specific short term T cell lines were incubated with purified AFP (13 ⁇ g/ml) and the production of antigen-specific IFN- ⁇ production assessed using an intracellular cytokine assay.
  • AFPn 7-145 -specific CD4 + cells recognised purified AFP but not control protein ( Figure Ib).
  • T cell lines were generated from PBMCs and hilar lymph node derived mononuclear cells of two HLA-A2+ patients with HCC.
  • both CD4 + and CD8 + T cells recognised AFP] 37- 14 5 and produced peptide-specific IFN- ⁇ (data not shown).
  • AFPi 37- i 45 was only recognised by CD8 + T cells ( Figure Ie).
  • AFP 137 . 145 - pulsed T2 cells HLA- A2+ TAP-2-deficient cell line
  • H s-specif ⁇ c CD4 + cells were identified in two treatment naive patients, one of whom also had AFP36 4 -373 specific CD4 + T cells. No CD4 + or CD8 + T cell responses to AFP 32 5-33 4 and AFPs 42 .55o were detected in treatment naive patients.
  • CD4 + or CD8 + T cell responses to any of the epitopes were detected in control patients.
  • AFP specific CD4 + T cells were significantly more likely to be detectable in patients with a lower serum AFP (p ⁇ 0.05) and at an early stage of disease
  • AFPi 37-14 5-specific CD8 + T cells were detected in two treatment-naive patients out of 42 HCC patients. There were no CD8 + T cell responses to AFP3 25 -3 34 , AFp 364 - 37> or AFPs4 2 -550 in treatment na ⁇ ve HCC patients or control groups.
  • Example 4 Recognition of a novel AFP-derived epitope by CD4 + T cells in a patient treated with transarterial embolisation
  • tumour necrosis produced by embolisation may induce tumour- specific T cell responses that are not present in treatment na ⁇ ve patients.
  • PBMCs from an A2 positive patient with HCC before and after TAE.
  • Short term T cell lines were generated in the presence of AFP 249-2S g (a previously uncharacterised AFP derived epitope) AFPn 7-H s 1 AFP 32 5- 334 or AFPs 42-SS o
  • CD4 + T cells recognised AFP 2 49- 2 s8 and produced IFN- ⁇ and TNF- ⁇ ( Figure 2a, 2b and 2c).
  • the average inter and intra-assay variation was ⁇ 5% as calculated from the coefficients of variation of five treated patients (20 x duplicates) repeated on different days.
  • AFP 36 4 .373 specific CD4 + T cells were detected in six out of the ten patients whilst only one patient had a detectable AFP 137-14 S CD4 + T cell response. No T cell response was detected to AFP 24 9 -2 s8 before treatment.
  • Pre-existing CD4 + T cell responses were expanded in six patients and responses to at least one peptide epitope were induced in all ten ( Figure 3).
  • T cell lines stimulated with PMA/ionomycin and analysed with an intracellular cytokine assay for IFN- ⁇ were used to detect changes in the non-specific T cell response during and after treatment.

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Abstract

La présente invention concerne un polypeptide recombinant ou isolé, lequel polypeptide est ou comprend un épitope T CD4+, la longueur de ce polypeptide n'excédant pas 14 acides aminés, ledit polypeptide comprenant une séquence d'acides aminés sélectionnée dans le groupe comprenant KVNFTEIQKL (AFP249-258 = SB94 = SEQ ID NO:4); LQTMKQEFLI (AFP545-554 = SB85 = SEQ ID NO:3); EMTPVNPGV (AFP489-497 = SB44 = SEQ ID NO:5); CLENQLPAFL (AFP86-95 = SB93 = SEQ ID NO:6); SSGEKNIFL (AFP343-351 = SB48 = SEQ ID NO: 8); et des variants de celui-ci. Cette invention concerne également d'autres polypeptides tels que définis dans les revendications, ainsi que leurs utilisations dans la fabrication de médicaments servant au traitement du cancer, de préférence dans le cas où le cancer exprime une alpha-foetoprotéine, idéalement dans le cas où le cancer est un carcinome hépatocellulaire (CHC).
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Publication number Priority date Publication date Assignee Title
CN104371017A (zh) * 2014-07-04 2015-02-25 嘉兴博泰生物科技发展有限公司 一种高效表达与制备外分泌型人源afp的方法
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WO2018194203A1 (fr) * 2017-04-20 2018-10-25 한국기초과학지원연구원 PROCÉDÉ DE DIAGNOSTIC DE CANCER DU FOIE PAR SPECTROMÉTRIE DE MASSE DE GLYCOPEPTIDES DÉRIVÉS DE α-FÉTOPROTÉINE

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