WO2022017895A1 - Treatment of residual disease - Google Patents

Treatment of residual disease Download PDF

Info

Publication number
WO2022017895A1
WO2022017895A1 PCT/EP2021/069697 EP2021069697W WO2022017895A1 WO 2022017895 A1 WO2022017895 A1 WO 2022017895A1 EP 2021069697 W EP2021069697 W EP 2021069697W WO 2022017895 A1 WO2022017895 A1 WO 2022017895A1
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
rhoc
nucleic acid
cells
seq
Prior art date
Application number
PCT/EP2021/069697
Other languages
French (fr)
Inventor
Juliane SCHUHMACHER
Cecile Gouttefangeas
Klaus Brasso
Anders Ljungqvist
Original Assignee
Rhovac Aps
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Rhovac Aps filed Critical Rhovac Aps
Publication of WO2022017895A1 publication Critical patent/WO2022017895A1/en

Links

Classifications

    • 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
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001154—Enzymes
    • A61K39/001164—GTPases, e.g. Ras or Rho
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • 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
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/884—Vaccine for a specifically defined cancer prostate

Definitions

  • the present invention relates generally to the field of treatment of residual disease in cancer patients having undergone initial treatment of a localized cancer by radical surgery and/or radiation therapy.
  • the invention relates to treatment of residual disease in prostate cancer patients having undergone radical prostatectomy and/or radiation therapy.
  • the invention provides RhoC peptides, preferably in the form of vaccine compositions comprising RhoC peptides for use in such treatment.
  • PCa localized prostate cancer
  • RP radical prostatectomy
  • Therapeutic anti-tumor vaccination could provide a safe, cheap, and well-tolerated immunotherapy treatment option for cancer, and many trials are ongoing worldwide, with most recent developments favouring a patient-individual approach.
  • a cancer vaccine should not only induce cytotoxic T lymphocytes (CTL), but also effector CD4 T cells.
  • CD4 T helper cells are important for CD8 T cell activation and expansion, as well as for the generation and maintenance of CD8 T cell memory.
  • CD4 T cells may display a range of anti-tumoral effects, such as secretion of TNF and IFN-gamma, activation of macrophages or natural killer cells, and direct cytotoxicity, which together might be more powerful than the sole tumor killing by CTL (Schuhmacher et al. , submitted).
  • RhoC The Ras homolog gene family member C
  • RhoC is a member of the Rho GTPase family which comprises RhoA, RhoB, and RhoC (85% sequence homology), all involved in the regulation of cytoskeleton organization.
  • RhoA and RhoC being highly homologous there is extensive heterogeneity in the C-terminal of the sequence between RhoA and RhoC.
  • Figure 7 shows an alignment of RhoA; RhoB and RhoC, wherein identical residues (“*”) and residues strong similar properties (“:”) and weak similar properties (“ ”)are marked.
  • RhoC was shown to be an important player in tumor cell motility, invasion, and metastasis formation.
  • RhoC has a limited expression in normal cells, but is highly expressed on advanced cancer cells including metastatic prostate cancer. Thus, expression of RhoC is significantly higher in metastatic tumors compared to primary tumors in different cancers. Furthermore, increased expression of RhoC is positively correlated to poor prognosis (Thomas et al., 2019, Suwa et al., 1998).
  • WO 2009/076966 describes RhoC or peptides derived thereof for treatment of metastatic cancer.
  • RhoC is overexpressed in metastatic cancer, but the present invention surprisingly shows that RhoC also is a suitable target for immunotherapy against non-metastatic cancers, and in particular that RhoC is a suitable target for immunotherapy against residual disease after initial treatment of patients having suffered from localised cancer.
  • the invention provides that RhoC also is a suitable target for immunotherapy against residual disease in patients with no objective signs of distant or systemic metastasis by standard CT scanning with bone scintigraphy or by standard PET-CT scanning.
  • immunotherapy with RhoC peptides inhibits or at least reduces the risk of biochemical recurrence. Furthermore, immunotherapy with RhoC peptides may inhibit cancer progression, even in patients with high risk of biochemical recurrence.
  • PSA is used as a marker for presence of prostate derived cells, and thus elevated serum levels of PSA are found in patients suffering from residual disease.
  • immunotherapy with RhoC peptides interestingly resulted in lower PSA levels than expected, surprisingly demonstrating that immunotherapy with RhoC peptides is effective in treatment of non metastatic residual disease. Even in patients having detectable levels of PSA, which can be considered “high risk” patients, immunotherapy with RhoC peptides resulted in slower increase PSA levels than expected.
  • RhoC peptide or a nucleic acid encoding a RhoC peptide for use in a method of treatment of residual disease in an individual in need thereof, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEC ID NO: 1 , wherein at the most three amino acids of SEC ID NO:1 have been substituted; and wherein the individual has undergone initial treatment of a localized cancer, wherein the initial treatment is selected from the group consisting of cryotherapy, laser therapy, topical therapy, tumour targeting chemo- and immune- therapies, radical surgery and radiation therapy.
  • the invention also provides kit-of-parts comprising a. a RhoC peptide or a nucleic acid encoding a RhoC peptide; and b. a further anti-cancer agent for use in a method of treatment of residual disease in an individual in need thereof, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO:1 have been substituted; and wherein the individual has undergone initial treatment of a localized cancer, wherein the initial treatment is selected from the group consisting of cryotherapy, laser therapy, topical therapy, tumour targeting chemo- and immune-therapies, radical surgery and radiation therapy.
  • RV001 -specific T cells are induced after RhoC vaccination.
  • A Vaccination and monitoring schedule. Patients were vaccinated 11 times. For immunoassays, blood was taken pre-vaccination, three times during the vaccination phase (vaccination) and four times post-vaccination (follow-up 1 and follow-up 2) (blood drops).
  • PBMCs were pre-stimulated with the RV001 and expanded for 12 days before IFN-y ELISpot testing (0.2 x 10 6 cells/well, except for Patient 21 , visits 2-13 and Patient 012, visits 16+17: 0.17 x 10 6 cells/well).
  • B Exemplary result of an ELISpot (Patient 011). ddFLO and PHA were used as negative and positive control, respectively.
  • D; n 7; 31500-2500 spots
  • E weak/non-
  • RV001 -specific mean spot counts per patient and visit normalized to 0.2 x 10 6 cells/well. Light green indicates a statistical significance according to the DFR(2x) permutation test. n 21 patients na: cells not available.
  • RV001 -responding cells are multifunctional. ddhhO stimulated cells harvested from the ELISpot were re-stimulated with the RV001 for 12 h. Expression of CD107a, CD154, IL-2, TNF, and IFN-g was examined by ICS on live CD4 and CD8 lymphocytes.
  • RV001 -specific T cells are effector memory T cells with a stable PD-1 and OX- 40 expression over time.
  • PBMCs from Patient 005 (visit 6 to visit 15) and Patients 009 and 018 (visit 6 to visit 17) were thawed, rested, and stimulated either with RV001 or ddhhO for 12 h.
  • Live RV001 -specific CD4 lymphocytes were identified by TNF expression and were further phenotypically examined for the expression of CD45RA, CCR7, PD-1 , OX- 40, and LAG-3.
  • CD4TNF + cells black are overlayed on the whole CD4 cell population (grey).
  • Numbers indicate CD4TNF + cell count in each quadrant of the dot plot.
  • MFI median fluorescence intensity
  • RV001 sequence comprises several promiscuous HLA-class II epitopes and one epitope presented on the HLA-B*27:05 allele.
  • the expression of CD107a, CD154, IL- 2, TNF, and IFN-y was examined on live CD4 or CD8 cells.
  • (B) LCLs were pre-loaded with RV001, ATR15 or AGL15, and incubated with HLA-matched patient cells in at 1 :2 ratio for 12 h. Shown are the specific percentages of CD4 cells expressing the indicated activation markers.
  • FIG. 5 Gating strategies.
  • A Gating strategy for the intracellular cytokine staining (ICS) analysis to identify RV001 specific T cell populations (Patient 011).
  • CD4 T cells (middle row) and CD8 T cells (lower row) were analyzed for their expression of IL-2, CD154, CD107a, TNF and IFN-g (from left to right).
  • B Gating strategy for the ex vivo flow analysis of RV001 specific CD4 T cells (Patient 018).
  • Gating 1 CD4 cells were examined for CD45RA/CCR7, as well as for TNF expression after RV001 peptide stimulation for 12 hr. The overlay of the TNF+ cells (black) onto the CD45RA/CCR7 dot plot (grey) shows that most of the RV001 -specific CD4 cells were effector memory (CD45RA-CCR7-).
  • Gating 2 The PD-1, OX-40, and LAG-3 expressions were further examined in the TNF+, CD45RA-CCR7- cells.
  • FMOs fluorescence minus one controls
  • Figure 6 Additional ICS results for the identification of RV001-derived HLA-class II epitopes (see also Fig 4).
  • Figure 7 shows an alignment of RhoA; RhoB and RhoC, wherein identical residues (“*”) and residues with conservative (“:”) and semi-conservative (“ ”) substitutions are marked.
  • biochemical recurrence refers to an individual having an increased serum level of PSA after initial treatment of a localized prostate cancer.
  • biochemical recurrence in relation to prostate cancer according to the present invention is defined as follows:
  • initial treatment refers to the first line treatment of a localized cancer.
  • the initial treatment may be selected from the group consisting of cryotherapy, laser therapy, topical therapy, tumour targeting chemo- and immune-therapies, radical surgery and radiation therapy.
  • Initial treatment of localized prostate cancer is either radical prostatectomy or radiation treatment.
  • a patient is considered to suffer from “localized prostate cancer”, when there are no objective signs of distant or systemic metastasis by standard CT scanning with bone scintigraphy or by standard PET-CT scanning.
  • a prostate cancer is considered “localized” even if there is invasion of adjacent organs or tissues.
  • non-metastatic cancer refers to a cancer having no objective signs of distant or systemic metastasis by standard CT scanning with bone scintigraphy or by standard PET-CT scanning.
  • residual disease refers to cancer cells that remain after attempts to remove a cancer have been made. Thus, residual disease may be any cancer cells present or developing after initial treatment. It is noted that the term “residual disease” as used herein always refers to residual disease after initial treatment of cancer.
  • treatment refers to any type of treatment or prevention that imparts a benefit to an individual suffering from or at risk of developing cancer, notably prostate cancer.
  • treatment may e.g. refer to preventive or prophylactic treatment, curative treatment, ameliorating treatment, treatment of symptoms, and/or treatment to delay disease progression.
  • RhoC is overexpressed in metastatic cancer, but the present invention surprisingly shows that RhoC also is a suitable target for immunotherapy against non-metastatic cancers, and in particular RhoC is a suitable target for immunotherapy against residual disease in patients having suffered from localised cancer.
  • RhoC peptide for use in a method of treatment of residual disease in cancer patients having undergone initial treatment.
  • Said RhoC peptide may be provided in the form of a RhoC peptide, or it may be provided in the form of a nucleic acid encoding the RhoC peptide.
  • the RhoC peptide is provided in the form of a RhoC peptide.
  • RhoC refers to a polypeptide of SEQ ID NO 1.
  • a RhoC peptide according to the present invention comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO: 1 , such as at the most two amino acids of SEQ ID NO:1, for example at the most one amino acid of SEQ ID NO:1 has/have been substituted.
  • the RhoC peptide may comprise or consist of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO:1.
  • RhoC peptide may comprise or consist of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO:1 , wherein up to 3 amino acids present in SEQ ID NO:1 has been substituted for another amino acid, for example
  • the RhoC peptides comprises a consecutive sequence of SEQ ID NO:1 comprising amino acids not present in the corresponding sequence of RhoB. In more preferred embodiments the RhoC peptides comprises a consecutive sequence of SEQ ID NO:1 comprising amino acids neither present in the corresponding sequence of RhoA nor RhoB. Accordingly in very preferred embodiments the peptide fragments of the invention contains at least one of amino acid residues I43, Q123, R140, S141, S152, L157, E165, G178, V181, K183, N184, R186, R187, R188,
  • RhoC peptides are peptides comprising amino acid residues, which differ from the sequence of RhoB, but are the same as the sequence of RhoA. Accordingly in less preferred embodiments the peptide fragments of the invention contains at least one of amino acid residues L81 , M82, C83, F84, S85, I86, D87, S88,
  • RhoC mainly differs from RhoA and RhoB in the C-terminal part of the sequence.
  • preferred RhoC peptides are derived from the 120 most C- terminal residues of RhoC, such as the 100 most C-terminal residues of RhoC, for example the 75 most C-terminal residues of RhoC, such as 52 most C-terminal residues of RhoC, for example the 40 most C-terminal residues of RhoC, such as the 30 most C- terminal residues of RhoC, for example the 25 most C-terminal residues of RhoC, such as the 24 most C-terminal residues of RhoC, such as the 23 most C-terminal residues of RhoC, for example the 22 most C-terminal residues of RhoC, such as the 21 most C- terminal residues of RhoC, such as the 20 most C-terminal residues of RhoC of SEQ ID NO 1.
  • Most preferred is a peptide consisting of the 20 most C-terminal
  • the RhoC peptide comprises at least one of the 20 most C-terminal RhoC specific amino acid residues G178, V181, K183, N184,
  • the RhoC peptide comprises or consists of a consecutive sequence of in the range of 8 to 100, such as 8 to 75, for example 8 to 60, for example of 8 to 50, preferably in the range of 9 to 25 consecutive amino acids of said RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO: 1 , such as at the most 2 amino acids of SEQ ID NO: 1 , for example at the most 1 amino acid of SEQ ID NO:1 has been substituted, for example no amino acid is substituted.
  • the RhoC peptide may comprise or consists of a consecutive sequence of in the range of 8 to 60, for example in the range of 8 to 50, such as in the range of 8 to 40, for example in the range of 8 to 10 or in the range of 18 to 60 amino, such as in the range of 18 to 50 amino acids of RhoC of SEQ I D NO: 1 , wherein at the most three, for example at the most two, such as at the most one amino acid(s) of SEQ ID NO:1 have been substituted, for example no amino acid is substituted.
  • the RhoC peptide may comprise (or more preferably consists of) at the most 200, preferably at the most 100, more preferably at the most 60, yet more preferably at the most 25, even more preferably at the most 20, yet even more preferably at the most 15, such as at the most 10, for example in the range of 8 to 10 contiguous amino acids of RhoC of SEQ ID NO:1 or a functional homologue thereof , wherein at the most three amino acids of SEQ ID NO:1 have been substituted.
  • the RhoC peptide may comprise or consist of a consecutive sequence of in the range of 8 to 100, such as 8 to 75, for example 8 to 60, for example of 8 to 50, such as in the range of 9 to 25, for example in the range of 18 to 60, such as in the range of 18 to 50 amino acid of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO: 1 , such as at the most 2 amino acids of SEQ ID NO: 1 , for example at the most one amino acid of SEQ ID NO:1 have been substituted, for example no amino acid is substituted and wherein said sequence comprises at least one of amino acid residues I43, Q123, R140, S141 , S152, L157, E165, G178, V181, K183, N184, R186, R187, R188, P191 or 1192 of RhoC of SEQ ID NO:1.
  • the RhoC peptide comprises or consists of the sequence RXGLQVRKNK, wherein X is selected from the group consisting of alanine and leucine and wherein said RhoC peptide is at the most 200, preferably at the most 100, more preferably at the most 60, yet more preferably at the most 25, even more preferably at the most 20 amino acids long, such as in the range of 10 and 150, preferably between 12 and 120, more preferably between 15 and 75, yet more preferably between 15 and 70, even more preferably between 18 and 60, such as between 18 and 50 amino acids long.
  • said RhoC peptide consists of a consecutive sequence of SEQ ID NO: 1 , wherein at the most three, for example at the most two, such as at the most one amino acid(s) of SEQ ID NO:1 have been substituted, for example no amino acid is substituted.
  • RhoC peptide comprises or consists of RXGLQVRKNK (SEQ ID NO:6), wherein X is selected from the group consisting of alanine and leucine.
  • the RhoC peptide comprises or consists of the sequence ATRAGLQVRKNKRRRGCPIL (SEQ ID NO:2), wherein said RhoC peptide is at the most 200, preferably at the most 100, more preferably at the most 60, yet more preferably at the most 25, even more preferably at the most 20 amino acids long, such as in the range of 20 and 150, preferably between 20 and 120, more preferably between 20 and 75, yet more preferably between 20 and 70, even more preferably between 20 and 60, such as between 18 and 50 amino acids long.
  • said RhoC peptide consists of a consecutive sequence of SEQ ID NO: 1 , wherein at the most three, for example at the most two, such as at the most one amino acid(s) of SEQ ID NO:1 have been substituted, for example no amino acid is substituted.
  • the RhoC peptide comprises or consist of the 20 most C-terminal amino acid residues of RhoC of SEQ ID NO:1. Accordingly in this particular embodiment the RhoC peptide consists of the sequence ATRAGLQVRKNKRRRGCPIL (SEQ ID NO:2).
  • the RhoC peptide comprises or consists of the sequence SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5, wherein said RhoC peptide is at the most 200, preferably at the most 100, more preferably at the most 60, yet more preferably at the most 25, even more preferably at the most 20 amino acids long, such as in the range of 20 and 150, preferably between 20 and 120, more preferably between 20 and 75, yet more preferably between 20 and 70, even more preferably between 20 and 60, such as between 18 and 50 amino acids long.
  • said RhoC peptide consists of a consecutive sequence of SEQ ID NO: 1 , wherein at the most three, for example at the most two, such as at the most one amino acid(s) of SEQ ID NO:1 have been substituted, for example no amino acid is substituted.
  • RhoC peptide may consists of a sequence selected from the group consisting of ATRAGLQVRKNKRRR (SEQ ID NO:3), AGLQVRKNKRRRGCP (SEQ ID NO:4) and LQVRKNKRRRGCPIL (SEQ ID NO:5).
  • the RhoC peptide is relatively long.
  • the longer peptide may be processed into a series of smaller peptides in the cytosol, e.g. by the proteasome or by proteases in the cytosol.
  • An advantage of using a longer peptide that may be processed into a variety of different shorter peptides is that more H LA-class I and II allotypes may be targeted with one peptide than one 8 to 10 amino acid peptide that is restricted to a particular HLA class-l.
  • the immunogenically active peptide fragment of the invention consists of 50 amino acid residues, for example at the most 45 amino acid residues, such as at the most 40 amino acid residues, for example at the most 35 amino acid residues, such as at the most 30 amino acid residues, for example at the most 25 amino acid residues, such as 18 to 25 consecutive amino acids from RhoC of SEQ ID NO:1 or a functional homologue thereof, wherein at the most three amino acids have been substituted and the peptide fragment contains at least one of the RhoC specific amino acid residues I43, Q123, R140, S141, S152, L157, E165, G178, V181, K183, N184, R186, R187, R188, P191 or 1192 of RhoC of SEQ ID NO:1.
  • the immunogenically active peptide fragment of the invention consists of 100 amino acid residues, for example at the most 90 amino acid residues, such as at the most 80 amino acid residues, for example at the most 70 amino acid residues, such as at the most 65 amino acid residues, such as 18 to 60 consecutive amino acids from RhoC of SEQ ID NO:1 or a functional homologue thereof, wherein at the most three amino acids have been substituted and the peptide fragment contains at least one of the RhoC specific amino acid residues I43, Q123, R140, S141, S152, L157, E165, G178, V181, K183, N184, R186, R187, R188, P191 or 1192 of RhoC of SEQ ID NO:1.
  • the RhoC peptide comprises or consists of sequence selected from the group consisting of VYVPTVFENYIADIEVDGKQV (SEQ ID NO:7), ILVGNKKLRQDEHTRRLAK (SEQ ID NO:8) and ELAKMKQEPVRSEEGRDMANR (SEQ ID NO:9), wherein at the most three amino acids have been substituted, such as at the most two amino acids have been substituted, for example at the most one amino acid have been substituted. Preferably no amino acids have been substituted.
  • the RhoC peptide may also comprises (or more preferably consists of) between 8 and 120, preferably between 8 and 100, more preferably between 10 and 75, yet more preferably between 12 and 60, even more preferably between 15 and 40, such as between 18 and 25 contiguous amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids have been substituted, deleted or added.
  • the peptide comprises (or more preferably consists of) between 10 and 150, preferably between 12 and 120, more preferably between 15 and 75, yet more preferably between 20 and 70, even more preferably between 15 and 65, such as between 15 and 60 contiguous amino acids of RhoC of SEQ ID NO:1 or a functional homologue thereof, wherein at the most three amino acids have been substituted, deleted or added.
  • RhoC peptide may also comprise or consist of the following sequences: EEGRDMANRISAFGYKECSAKTKEGVREVFEMATRAGLQVRKNKRRRGCPIL (SEQ ID NO:10) or ATRAGLQVRKNKRRRGCPIL (SEQ ID NO:11) or RAGLQVRKNK (SEQ ID NO:12).
  • the RhoC peptide is RLGLQVRKNK (SEQ ID NO:13) which is an artificial peptide, wherein the alanine of RAGLQVRKNK (SEQ ID NO: 12) has been substituted with leucine.
  • RhoC peptide for use in a method of treatment of residual disease in cancer patients having undergone initial treatment.
  • Said RhoC peptide may be provided in the form of a vaccine, wherein said vaccine comprises the RhoC peptide or a nucleic acid encoding same as well as an adjuvant.
  • the adjuvant may be any substance whose admixture into the vaccine composition increases or otherwise modifies the immune response to the RhoC peptide.
  • Adjuvants may for example be selected from the group consisting of: AIK(SC>4)2, AINa(SC>4)2, AINH4 (SO4), silica, alum, AI(OH)3, Ca3 (PCU kaolin, carbon, aluminum hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(T2'-dipalmitoyl-sn -glycero-3- hydroxphosphoryloxyj-ethylamine (CGP 19835A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in
  • lipid A lipid A
  • FCA Freund's Complete Adjuvant
  • FCA Freund 's Incomplete Adjuvants
  • Merck Adjuvant 65 polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacterium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, Titermax, ISCOMS, Quil A, ALUN (see US 58767 and 5,554,372), Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, Interleukin 1, Interleukin 2, Montanide ISA-51 and QS-21.
  • Preferred adjuvants to be used with the invention include oil/surfactant based adjuvants such as Montanide adjuvants (available from Seppic, Belgium), preferably Montanide ISA-51.
  • Other adjuvants are bacterial DNA based adjuvants, such as adjuvants including CpG oligonucleotide sequences.
  • Yet other adjuvants are viral dsRNA based adjuvants, such as the TLR-ligand poly l:C.
  • Imidazochinilines are yet another example of preferred adjuvants.
  • the most preferred adjuvants are adjuvants suitable for human use. Liposomes are in general not useful adjuvants for the present invention, and preferably the vaccine compositions of the invention therefore do not comprise liposomes.
  • Montanide adjuvants may be selected from the group consisting of Montanide ISA-51, Montanide ISA-50, Montanide ISA-70, Montanide ISA- 206, Montanide ISA-25, Montanide ISA-720, Montanide ISA-708, Montanide ISA-763A, Montanide ISA-207, Montanide ISA-264, Montanide ISA-27, Montanide ISA-35,
  • Montanide ISA 51 F, Montanide ISA 016D and Montanide IMS preferably from the group consisting of Montanide ISA-51, Montanide IMS and Montanide ISA-720, more preferably from the group consisting of Montanide ISA-51 and Montanide ISA-720.
  • Montanide, and in particular Montanide ISA-51 is oil/surfactant based adjuvants in which different surfactants are combined with either a non-metabolizable mineral oil, a metabolizable oil, or a mixture of the two. They are prepared for use as an emulsion with an aqueous solution comprising RhoC peptide.
  • the surfactant is mannide oleate.
  • QS-21 (Antigenics; Aquila Biopharmaceuticals, Framingham, MA) is a highly purified, water- soluble saponin that handles as an aqueous solution.
  • QS-21 and Montanide ISA-51 adjuvants can be provided in sterile, single-use vials.
  • cytokine GM-CSF is another adjuvant and may be GM-CSF as described in WO 97/28816.
  • a vaccine composition according to the present invention may comprise more than one different adjuvant.
  • the vaccine may comprise other useful components in addition to RhoC peptide and adjuvant.
  • the vaccine composition may comprise a carrier.
  • the function of a carrier can for example be to increase the molecular weight of RhoC peptides in order to increase their activity or immunogenicity, to confer stability, to increase the biological activity, or to increase serum half-life.
  • a carrier may aid presenting the protein belonging to the rho gene family or peptide fragments thereof to T-cells.
  • the carrier may be any suitable carrier known to the person skilled in the art, for example a protein or an antigen presenting cell.
  • a carrier protein could be but is not limited to keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.
  • the carrier must be a physiologically acceptable carrier acceptable to humans and safe.
  • tetanus toxoid and/or diptheria toxoid are suitable carriers in one embodiment of the invention.
  • the carrier may be dextrans for example sepharose.
  • the vaccine may also comprise one or more additional pharmaceutically acceptable excipients, for example such excipients described in Remington: The Science and Practice of Pharmacy,
  • the invention relates to a RhoC peptide for use in a method of treatment of residual disease in a cancer patient having undergone initial treatment.
  • “Residual disease” within the meaning of the present invention relates to cancer disease after initial treatment of a localised cancer.
  • a cancer is considered “localised” if no distant or systemic metastases are observed by CT scanning with bone scintigraphy or by standard PET-CT scanning.
  • the present invention has surprisingly found that even though RhoC is mainly associated with metastatic cancer, treatment with RhoC peptide induces a T-cell response, which is useful in treatment of non-metastatic cancer, and notably of residual disease after initial treatment of a localized cancer.
  • the individual to be treated according to the present invention does not have any distant or systemic metastasis as detected by CT scanning or PET-CT scanning.
  • the initial treatment is in general selected according to the type of localised cancer, and will typically be the ordinary initial treatment used for treatment of the particular type of localised cancer.
  • the initial treatment may be selected from the group consisting of cryotherapy, laser therapy, topical therapy, tumour targeting chemo- and immune-therapies, radical surgery and radiation therapy.
  • the initial treatment may for example be radical surgery or radiation therapy.
  • the cancer is prostate cancer.
  • the invention in particular relates to a RhoC peptide for use in a method of treatment of residual disease in an individual having undergone initial treatment for prostate cancer.
  • the invention relates to a method of treating an individual having suffered from prostate cancer, wherein the treatment is administered subsequent to initial treatment of said prostate cancer.
  • the prostate cancer prior to initial treatment may be referred to as the “initial prostate cancer”.
  • the initial prostate cancer is preferably a localized prostate cancer, i.e. a prostate cancer wherein no distant or systemic metastases are detected by CT scanning with bone scintigraphy or by standard PET-CT scanning.
  • Prostate cancer refers to a disease in which cancer develops in the prostate gland of the male reproductive system.
  • Prostate cancer is classified as an adenocarcinoma, or glandular cancer, that begins when normal semen-secreting prostate gland cells mutate into cancer cells.
  • adenocarcinoma or glandular cancer
  • small clumps of cancer cells remain confined to otherwise normal prostate glands, a condition known as carcinoma in situ or prostatic intraepithelial neoplasia (PIN), a prostate precancer. Over time these cancer cells begin to multiply and spread to the surrounding prostate tissue (the stroma), forming a tumor.
  • PIN prostatic intraepithelial neoplasia
  • prostate cancer originates and may remain in the prostate, prostate tumor cells may develop the ability to travel in the bloodstream and lymphatic system and thus be found in other organs or tissues. Prostate cancer most commonly metastasizes to the bones, lymph nodes, rectum, and bladder.
  • One method include classifying a prostate tumor based on the Gleason scoring system.
  • the Gleason scoring system is based on microscopic tumor patterns assessed by a pathologist while interpreting the biopsy specimen.
  • the Gleason score is based upon the degree of loss of the normal glandular tissue architecture (i.e. shape, size and differentiation of the glands).
  • the classic Gleason scoring system has five basic tissue patterns that are technically referred to as tumor "grades.”
  • the microscopic determination of this loss of normal glandular structure caused by the cancer is represented by a grade, a number ranging from 1 to 5, with 5 being the worst grade.
  • Grade 1 is typically where the cancerous prostate closely resembles normal prostate tissue.
  • the glands are small, well-formed, and closely packed. At Grade 2 the tissue still has well-formed glands, but they are larger and have more tissue between them, whereas at Grade 3 the tissue still has recognizable glands, but the cells are darker. At high magnification, some of these cells in a Grade 3 sample have left the glands and are beginning to invade the surrounding tissue. Grade 4 samples have tissue with few recognizable glands and many cells are invading the surrounding tissue. For Grade 5 samples, the tissue does not have recognizable glands, and are often sheets of cells throughout the surrounding tissue. Since prostate cancers are often made up of cancerous cells that have different grades, two grades are assigned for each patient, and the Gleason score is provided as the sum of these two scores. A primary grade may be given to describe the cells that make up the largest area of the tumor and a secondary grade may be given to describe the cells of the next largest area. Unless otherwise indicated the Gleason scores provided herein are provided as the sum of the two scores.
  • the initial, localized prostate cancer of the individual to be treated had a Gleason score in the range of 6 to 10, for example in the range of 6 to 9 prior to initial treatment.
  • the clinical T category is the doctor’s estimate of the disease based on results of physical examination, prostate biopsy and imaging tests.
  • This classification is used to evaluate the primary tumour, and comprises the following clinical T stages (cT): cTO: no evidence of tumor • cT1: tumor present, but not detectable clinically or with imaging o cT1a: tumor was incidentally found in 5% or less of prostate tissue resected (for other reasons) o cT1b: tumor was incidentally found in greater than 5% of prostate tissue resected o cT1c: tumor was found in a needle biopsy performed due to an elevated serum PSA
  • cT2 the tumor can be felt (palpated) on examination, but has not spread outside the prostate o cT2a: the tumor is in half or less than half of one of the prostate gland's two lobes o cT2b: the tumor is in more than half of one lobe, but not both o cT2c: the tumor is in both lobes but within the prostatic capsule
  • cT3 the tumor has spread through the prostatic capsule (if it is only part- way through, it is still T2)
  • o cT3a the tumor has spread through the capsule on one or both sides, e.g. had an extra prostatic extension
  • o cT3b the tumor has invaded one or both seminal vesicles, e.g. had seminal vesicle/bladder neck involvement
  • the initial, localized prostate cancer of the individual to be treated may be classified as cT1, cT2, cT3 or cT4.
  • the initial, localized prostate cancer of the individual to be treated may be classified as a cT1a, cT1b, cT1c, cT2a, cT2b, cT2c, cT3a, cT3b or cT4.
  • the initial, localized prostate cancer of the individual to be treated may be classified as a cT3a, cT3b or cT4.
  • the individual to be treated has in general undergone an initial treatment of said initial localized prostate cancer.
  • Said initial treatment may in particular be either radical prostatectomy and/or radiation therapy, which are typical first line treatment for localized prostate cancer.
  • Radical prostatectomy refers to the surgical removal of the prostate gland.
  • a radical prostatectomy involves the removal of the entire prostate gland, the seminal vesicles and the vas deferens.
  • a radical prostatectomy can be performed, e.g. by open surgery (via a large incision through the lower abdomen), laparoscopically with the help of a robot (a type of minimally invasive surgery), through the urethra or through the perineum.
  • the prostatectomy may be performed in a manner so the blood vessels and nerves that promote penile erections are left behind in the body and not taken out with the prostate.
  • the prostatectomy may be performed with limited pelvic lymph node dissection, so that at least some of the surrounding lymph nodes are removed.
  • lymph nodes may be removed from the area defined by external iliac vein anteriorly, the obturator nerve posteriorly, the origin of the internal iliac artery proximally, Cooper's ligament distally, the bladder medially and the pelvic side wall laterally.
  • the prostatectomy may also be associated with extended pelvic lymph node dissection.
  • lymph nodes further away from the prostate are also removed.
  • the lymph nodes removed during limited pelvic lymph node dissection are also removed, as well as lymph node from the posterior boundary and the floor of the pelvis.
  • the radiation therapy may in particular be either external beam radiation or brachytherapy, which are the most common radiation therapies used in the treatment of localized prostate cancer.
  • EBRT External beam radiation
  • Moderate hypofraction The dose is higher, and there are fewer sessions. 20 sessions over 4 weeks
  • IRT Intensity modulated radiation therapy
  • VMAT Volumetric modulated arc therapy
  • Proton beam radiation therapy delivers beams of protons instead of radiation.
  • Brachytherapy is also known as Internal radiation therapy (IRT), and in general involves placing radioactive pellets inside the body, on the prostate gland. The pellets are in general around the size of a grain of rice.
  • the implant may be temporary or permanent:
  • the individual to be treated may have no detectable serum prostate-specific antigen (PSA).
  • PSA serum prostate-specific antigen
  • the individual suffers from a biochemical recurrence.
  • the individual may have detectable serum PSA, such as a serum level of PSA of >0.05 ng/ml, for example >0.06 ng/ml.
  • the individual has a serum level of PSA of in the range of 0.05 to 20 ng/ml, for example in the range of 0.06 to 20 ng/ml.
  • the individual may have the aforementioned serum level of PSA.
  • the serum level of PSA may be determined by any useful means.
  • PSA may be detected in serum using a PSA specific monoclonal antibody.
  • PSA is detected using electrochemiluminescence immunoassays for measuring either total PSA or free PSA.
  • electrochemiluminescence immunoassays for measuring either total PSA or free PSA.
  • assays can be performed on automated analysers such as Elecsys 1010 and 2010 or COBIS 8000. Many different approved and validated methods are available for determine PSA levels, and any of these may be used. If nothing else is indicated, the level of PSA is the level of total PSA.
  • the invention relates to a RhoC peptide or a nucleic acid encoding the same for use in a method of treatment of residual disease in a cancer patient after initial treatment of localised cancer.
  • the invention relates to a method of treating an individual having suffered from cancer, e.g. a localised cancer, wherein the treatment is administered subsequent to initial treatment of said cancer.
  • the individual to be treated according to the invention is in general a cancer patient, in particular a human cancer patient.
  • Treatment with RhoC peptide may be initiated immediately after termination of the initial treatment. In some cases (in particular if the initial treatment is radiation therapy) the treatment with RhoC peptide may even be initiated during the initial treatment. In other cases the treatment with RhoC peptide may be initiated long after the initial treatment, for example at least 1 week after, e.g. at least 1 month after, such as within 1 to 20 years after completion of the initial treatment. Typically, the RhoC peptide or nucleic acid encoding same is administered to the individual more than once.
  • the RhoC peptide may be administered at least twice, such as at least 5 times, preferably at least 9 times, more preferably at least 10 times, even more preferably at least 11 times, such as in the range of 2 to 50 times, for example in the range of 5 to 40 times, such as in the range of 10 to 30 times, for example in the range of 10 to 20 times, preferably in the range of 11 to 12 times.
  • the RhoC peptide is administered 11 times.
  • the RhoC peptide is administered 12 times.
  • the interval between two administrations of RhoC peptide may vary, but is typically in the range of 2 days to several months, such as in the range of 2 days to 6 months, for example in the range of 1 to 12 weeks, such as in the range of 1 to 8 weeks, for example in the range of 2 to 4 weeks. Within the same treatment regimen, the intervals may also vary. Thus, for example the first administrations may be administered with shorter intervals between two administrations compared to the later administrations.
  • a non limiting example of a useful administration scheme is provided in figure 1A.
  • RhoC peptide may thus be administered over an extended time period, wherein RhoC is administered multiple times with suitable intervals between administrations.
  • the total time period for administration of RhoC may be at least 12 months, such as at least 18 months, for example at least 24 months, such as in the range of 12 to 100 months, for example in the range of 24 to 60 months.
  • RhoC peptide depends on several factors, but may for example be in the range of 0.01 to 10 mg RhoC peptide, such as in the range of 0.05 to 5 mg RhoC peptide, for example in the range of 0.05 to 1 mg RhoC peptide, such as approx. 0.1 mg RhoC peptide. Aforementioned dosages may be useful for treatment of adult human males.
  • the route of administration may be any useful route, for example by injection, such as subcutaneous injection or intradermal injection.
  • the present invention relates to methods of treatment of residual disease in an individual, who has undergone initial treatment of a localised cancer.
  • the methods relate to treatment of non-metastatic, residual disease.
  • the methods relate to treatment of residual disease in an individual having no objective signs of distant or systemic metastasis by standard CT scanning with bone scintigraphy or by standard PET- CT scanning.
  • Vaccination with RhoC peptides has previously been suggested for treatment of metastatic cancer, however, the present invention surprisingly discloses that RhoC peptides or nucleic acids encoding same are useful in treatment of non-metastatic cancer, i.e. of residual disease after initial treatment of a localised cancer.
  • the purpose of the methods is the treatment of the residual, non-metastatic cancer per se.
  • a CD4+ response may be useful in treatment of cancer, because the anti-cancer effect of a CD4+ cell response is not confined to the cytolysis of cells expressing RhoC.
  • a CD4+ cell response also provides a more general anticancer effect, e.g. due to expression of several different cytokines, such as INF-g, TNF and IL-2 with known anti-cancer properties.
  • the RhoC peptide is immunogenic.
  • the RhoC peptide may have at least one of the following characteristics:
  • RhoC peptide upon administration of the RhoC peptide to an individual, said individual produces memory T cells, wherein said T cells - after in vitro expansion with stimulation by RhoC peptide - have the following characteristics:
  • administering preferably leads to one or more of the aforementioned effects in said individual.
  • the treatment with RhoC peptide elicits a clinical response in the individual, wherein the clinical response is significantly reduced risk of biochemical recurrence.
  • the individual to be treated with RhoC peptide is an individual having undergone initial treatment of localized prostate cancer, wherein the individual has a serum level of PSA below the detection limit, then treatment with RhoC peptide results in said individual having reduced risk of developing a serum level of PSA 3 0.2 ng/ml.
  • said individual does not develop a serum level of PSA 3 0.2 ng/ml.
  • the treatment may elicit a clinical response in said individual, wherein the clinical response is a PSA level £ 0.2 ng/ml.
  • said PSA levels are determined within 60 months, such as within 48 months, for example within 36 months from first administration.
  • the individual has a detectable PSA serum level.
  • the treatment with RhoC peptide may elicit a clinical response characterised by an increased PSA serum level doubling time compared to serum PSA level doubling time in said individual prior to treatment with the peptide.
  • the treatment with RhoC peptide may elicit a clinical response in the individual, wherein the clinical response is characterised by at least 1.5-fold increase in the PSA serum level doubling time compared to the PSA serum level doubling time in said individual prior to treatment with the peptide.
  • the treatment with RhoC peptide may also elicit a clinical response in the individual, wherein the clinical response is a decrease in the progression of cancer, e.g. a decrease in progression of prostate cancer compared to the expected progression without treatment.
  • RhoC peptide may also elicit a clinical response in the individual, wherein the clinical response is characterised by a stable disease, a partial response or complete remission.
  • the RhoC peptide or nucleic acid encoding same may also be administered in combination with one or more other anti-cancer agents.
  • the invention also provides a kit-of-parts for use in a method of treating residual disease in a cancer patient having undergone initial treatment, wherein the kit-of-part comprises a. a RhoC peptide or a nucleic acid encoding same, wherein said peptide or nucleic acid optionally is comprised in a vaccine; and b. a further anti-cancer agent.
  • the anti-cancer agent may for example be an anti-cancer agent used for chemotherapy or gene therapy of cancer.
  • the anti-cancer agent may also be immunostimulating substances or antibodies.
  • the immunostimulating substances may for example be cytokines, such as cytokines selected from the group consisting of GM-CSF, type I IFN, interleukin 12 and interleukin 15.
  • the antibody may preferably be an immunostimulating antibody such as anti-CD40, anti-CTLA-4, anti-OX-40 antibodies, anti-PD1 or anti-PD1-L antibodies.
  • the immunostimulatory substance may also be a substance capable of depletion of immune inhibitory cells (e.g. regulatory T-cells) or factors, said substance may for example be E3 ubiquitin ligases.
  • E3 ubiquitin ligases have emerged as key molecular regulators of immune cell function, and each may be involved in the regulation of immune responses during infection by targeting specific inhibitory molecules for proteolytic destruction.
  • HECT and RING E3 proteins have now also been linked to the induction and maintenance of immune self tolerance: c-Cbl, Cbl-b, GRAIL, Itch and Nedd4 each negatively regulate T cell growth factor production and proliferation.
  • anti-cancer agents may also be substances that induce androgen deprivation therapy (ADT) such as LHRH agonists, LHRH antagonists in addition to anti-androgens and CYP17 blockers.
  • ADT androgen deprivation therapy
  • the invention may further be defined by the following items:
  • RhoC peptide or a nucleic acid encoding a RhoC peptide for use in a method of treatment of residual disease in an individual in need thereof, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO:1 have been substituted; and wherein the individual has undergone initial treatment of a localized cancer, wherein the initial treatment is selected from the group consisting of radical surgery, radiation therapy, cryotherapy, laser therapy, topical therapy, tumour targeting chemotherapy and tumour targeting immune therapy.
  • RhoC peptide or a nucleic acid encoding a RhoC peptide for use in a method of treatment of residual disease in an individual in need thereof, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO:1 have been substituted; and wherein the method comprises administering said peptide or nucleic acid to said individual at least 9 times.
  • peptide or nucleic acid for use in the method according to any one of items 3 to 4, wherein said individual has undergone radical prostatectomy for treatment of a localized prostate cancer.
  • peptide or nucleic acid for use in the method according to any one of items 3 to 11 , wherein the individual had a serum level of PSA in the range of 0.1 to 20 ng/ml.
  • peptide or nucleic acid for use in the method according to any one of items 3 to 9, wherein the individual had a serum level of PSA in the range of 0.05 to 20 ng/ml prior to initial treatment.
  • peptide or nucleic acid for use in the method according to any one of items 3 to 13, wherein the localized prostate cancer had an extraprostatic extension and/or was classified as cT3a prostate cancer prior to initial treatment.
  • peptide or nucleic acid for use in the method according to any one of items 3 to 13, wherein the localized prostate cancer had seminal vesicle/bladder neck involvement and/or was classified as cT3b prostate cancer prior to initial treatment.
  • peptide or nucleic acid for use in the method according to any one of items 3 to 13, wherein the localized prostate cancer showed seminal vesicle involvement, tumor fixation, or invasion of adjacent organs and/or was classified as cT4 prostate cancer prior to initial treatment.
  • peptide or nucleic acid for use in the method according to any one of items 3 to 15, wherein the localized prostate cancer had a Gleason score in the range of 6 to 10, for example in the range of 6 to 9 prior to initial treatment.
  • peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the individual does not have distant or systemic metastasis as detected by CT scanning or PET-CT scanning. 19. The peptide or nucleic acid for use in the method according to anyone of the preceding items, wherein said peptide is immunogenic as determined by said peptide having at least one of the following characteristics:
  • peptide or nucleic acid for use in the method according to any one of items 3 to 20, wherein the treatment elicits a clinical response in the individual, wherein the clinical response is characterised by an increased PSA serum level doubling time compared to a PSA serum level doubling time in said individual prior to treatment with the peptide.
  • peptide or nucleic acid for use in the method according to any one of items 3 to 20, wherein the treatment elicits a clinical response in the individual, wherein the clinical response is characterised by at least a 1.5-fold increase in the PSA serum level doubling time compared to a PSA serum level doubling time in said individual prior to treatment with the peptide.
  • peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the treatment elicits a clinical response in the individual, wherein the clinical response is characterised by a stable disease, a partial response or complete remission.
  • RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ I D NO: 1 , wherein at the most two, such as at the most one amino acid(s) of SEQ ID NO:1 have been substituted.
  • RhoC peptide for use in the method according to any one of the preceding items, wherein the RhoC peptide consists of a consecutive sequence of in the range of 8 to 60, for example in the range of 8 to 50, such as in the range of 8 to 40, for example in the range of 8 to 10 or in the range of 18 to 60 amino, such as in the range of 18 to 50 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three, for example at the most two, such as at the most one amino acid(s) of SEQ ID NO:1 have been substituted.
  • RhoC peptide for use in the method according to any one of the preceding items, wherein the RhoC peptide consists of a consecutive sequence of in the range of 8 to 60, for example in the range of 8 to 50, such as in the range of 8 to 40, for example in the range of 8 to 10 or in the range of 18 to 60, such as in the range of 18 to 50 amino acids of RhoC of SEQ ID NO:1.
  • RhoC peptide comprises or consists of a consecutive sequence of amino acids of RhoC of SEQ ID NO:1, wherein said consecutive sequence comprise at least one of amino acid residues I43, Q123, R140, S141, S152, L157, E165, G178, V181, K183, N184, R186, R187, R188, P191 or 1192 of RhoC of SEQ ID NO:1.
  • RhoC peptide for use in the method according to any one of the preceding items, wherein the RhoC peptide consists of in the range of 8 to 60, such as in the range of 8 to 50, for example in the range of 18 to 25 consecutive amino acids from RhoC of SEQ ID NO:1 , wherein at the most two amino acids have been substituted, wherein the peptide fragment contains at least one of amino acid residues I43, Q123, R140, S141, S152, L157, E165, G178, V181,
  • RhoC peptide comprises of consists of the sequence RXGLQVRKNK, wherein X is selected from the group consisting of alanine and leucine and wherein said peptide fragment is at the most 60 amino acids in length.
  • RhoC peptide comprises of consists of the sequence ATRAGLQVRKNKRRRGCPIL.
  • RhoC peptide comprises of consists of a sequence selected from the group consisting of ATRAGLQVRKNKRRR, AGLQVRKNKRRRGCP and LQVRKNKRRRGCPIL.
  • peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the peptide and/or nucleic acid is contained in a vaccine composition comprising a. said peptide and/or nucleic acid; and b. an adjuvant.
  • the adjuvant is a Montanide ISA adjuvant, such as Montanide ISA 51 or Montanide ISA 720.
  • peptide for use in the method according to any one of the preceding items, wherein the peptide is formulated for administration of in the range of 0.01 to 10 mg of said peptide.
  • peptide for use in the method according to any one of the preceding items, wherein the peptide is formulated for administration of in the range of 0.05 to 1 mg of said peptide.
  • peptide or nucleic acid for use in the method according to any one of the preceding items, wherein said method comprises administering said peptide or nucleic acid to said individual at least 9 times, preferably at least 10 times.
  • peptide or nucleic acid for use in the method according to any one of the preceding items, wherein said method comprises administering said peptide or nucleic acid to said individual at least 11 times.
  • peptide or nucleic acid for use in the method according to any one of the preceding items, wherein said method comprises administering said peptide or nucleic acid to said individual in the range 10 to 30 times.
  • peptide or nucleic acid for use in the method according to any one of the preceding items, wherein said method comprises administering said peptide or nucleic acid to said individual in the range of 11 to 12 times.
  • peptide or nucleic acid for use in the method according to any one of the preceding items, wherein said method comprises multiple administrations with a time interval of in the range of 1 to 8 weeks between every administration.
  • peptide or nucleic acid for use in the method according to any one of the preceding items, wherein said method comprises multiple administrations with a time interval of in the range of 2 to 4 weeks between every administration.
  • peptide or nucleic acid for use in the method according to any one of the preceding items, wherein said administration is subcutaneous injection or intradermal injection.
  • a kit-of-parts comprising a. a RhoC peptide or a nucleic acid encoding a RhoC peptide; and b. a further anti-cancer agent for use in a method of treatment of residual disease in an individual in need thereof, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO:1 have been substituted; and wherein the individual has undergone initial treatment of a localized cancer, wherein the initial treatment is selected from the group consisting of radical surgery, radiation therapy, cryotherapy, laser therapy, topical therapy, tumour targeting chemotherapy and tumour targeting immune therapy.
  • kit-of-parts according to item 47, wherein the RhoC peptide is as defined in any one of items 26 to 33 or is comprised within a vaccine composition according to any one of item 34 to 37.
  • kit-of-parts according to any one of items 47 to 48, wherein the method is the method defined in any one of items 1 to 25 or 38 to 46.
  • kit-of-parts according to any one of items 47 to 49, wherein the anti-cancer agent is an antibody.
  • kit-of-parts according to any one of items 47 to 50, wherein the anti-cancer agent is a cytokine.
  • a method of treating residual disease in an individual in need thereof comprising administering to an individual in need thereof an effective amount of a RhoC peptide or a nucleic acid encoding the RhoC peptide, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO:1 have been substituted; and wherein the individual has undergone initial treatment of a localized cancer by radical surgery and/or radiation therapy.
  • RhoC peptide or a nucleic acid encoding a RhoC peptide or a kit-of-parts comprising said RhoC peptide or nucleic acid in the manufacture of a medicament for the treatment of residual disease in an individual in need thereof, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO:1 have been substituted; and wherein the individual has undergone initial treatment of a localized cancer by radical surgery and/or radiation therapy.
  • the method is as defined in any one of items 1 to 46.
  • RhoC also referred to as RV001 herein ATRAGLQVRKNKRRRGCPIL
  • X is selected from the group consisting of alanine and leucine.
  • the example describes a clinical phase I/ll study, showing safety and immunogenicity of a 20mer SLP vaccine specifically targeting the RhoC protein in prostate cancer patients (see also Schuhmacher et al., submitted).
  • BCR biochemical recurrence
  • Table 1a For inclusion and exclusion criteria, see Table 1a.
  • the primary endpoint of the study was the evaluation of the safety and tolerability of the vaccine.
  • Treatment-emergent adverse events (TEAE) were recorded and analyzed in accordance with the common terminology criteria for adverse events (CTCAE), version 4.03.
  • CCAE common terminology criteria for adverse events
  • RV001 refers to a peptide consisting of the 20aa C-terminal sequence of the RhoC protein: ATRAGLQVRKNKRRRGCPIL (SEQ ID NO:2). The 16 last amino-acid sequence of RV001 is found only in RhoC, but not in RhoA/B, and is thus RhoC-specific..
  • Table 1b Main patient ' s characteristics
  • PSA 0.1 pg/L
  • LOQ 0.1 pg/L
  • 3 had received salvage radiation, and one patient refrained from salvage therapy.
  • 2 patients had PSA levels above 0.1 pg/L.
  • One patient was progressing following radical prostatectomy and salvage (Patient 018), the other one following prostatectomy only (Patient 006).
  • Table 3 HLA-typing of individual patients and cell lines
  • MIATA Minimal Information about T cell Assays
  • PBMCs peripheral blood mononuclearcells
  • Blood samples (100 ml, heparinized) were processed within 8 h after blood drawing (DanTrials ApS).
  • the PBMC isolation was performed according to a standard, pre-established protocol, using pre-filled 50 ml LeucosepTM separation tubes (Greiner Bio-One). Cells were counted with trypan blue and Tuerks Solution (both Sigma-Aldrich). Six to 13 x 10 6 cells per cryovial (NuncTM, Sigma-Aldrich) were frozen in 1 ml heat-inactivated (hi) FBS (ThermoFisher) containing 10% dimethylsulfoxide (DMSO, Sigma-Aldrich). Cells were first stored in freezing containers (Nalgene® Mr.
  • PBMCs were thawed in IMDM (Gibco) containing 2.5% hi human serum (HS, Sigma-Aldrich), penicillin 100 units/ml and streptomycin 0.1 mg/ml (P/S, Sigma-Aldrich), and 50 mM b- mercaptoethanol (b-ME, Merck). After one washing step with serum-free medium (SFM, IMDM, P/S, 50 pM b-ME), cells were counted with trypan blue (Merck). The median live cell yield after thawing was 63,5%. No cut-off was applied for further in vitro culture.
  • IMDM Gibco
  • P/S penicillin 100 units/ml and streptomycin 0.1 mg/ml
  • b-ME 50 mM b- mercaptoethanol
  • T cell medium TCM, IMDM with 10% hi HS, 1 x P/S and 50 pM b-ME
  • Cellstar® Greiner bio-one
  • the IFN-g ELISpot protocol has been described by Widenmeyer et al. , 2012. If not otherwise stated, 0.2 x 10 6 cells were cultured per well in the presence of 50 pg/ml RV001 peptide (SEQ ID NO:2) for 26 h at 37°C and 7.5% CO2 (triplicates). ddhhO (6 wells) and phytohemagglutinin- L (PHA-L, 10 pg/ml, Sigma-Aldrich, 1 well) were used as negative and positive control, respectively. Spots were counted with the ImmunoSpot series 6 ultra-V analyzer (CTL Europe GmbH) according to a standard protocol.
  • CTL Europe GmbH ImmunoSpot series 6 ultra-V analyzer
  • RV001 -specific spot counts are defined as the mean spot counts obtained in the RV001 stimulated wells minus the mean spot counts in the ddH 2 0 wells. All spot counts are given in Table 5.
  • PBMCs were analyzed by ICS either ex vivo or after culture. Thawed cells were rested in TCM for 4-6 h prior to the ICS. Pre-cultured cells were directly examined on day 12 (Fig. 2E; Fig. 4), or harvested from the ELISpot plate (Fig. 2A-D, ddH 2 0 cells from the visit with the highest spot count in the ELISpot were used).
  • RV001 SEQ ID NO:2
  • RV001 ATRAGLQVRKNKRRR (ATR 15 )
  • AGLQVRKNKRRRGCP AGL 15
  • LQVRKN KRRRGCPIL LQV 15
  • ddH 2 0 and Staphylococcus enterotoxin B (10 pg/ml, SEB, Sigma- Aldrich) were added as negative and positive control, respectively.
  • the CD107a antibody was added together with the stimulus, the protein transport inhibitors Brefeldin A (10 pg/ml, Sigma- Aldrich) and Golgi Stop (BD) 1 h thereafter. After 12 h at 37°C and 7.5% CO2, cells were stained (Panel 1, Table 4). For ex vivo analysis of PD-1 , OX-40, and LAG-3 expression (Panel 2, Table 4), fluorescence minus one controls were performed.
  • FACS-buffer PBS without Ca/Mg (Lonza), 0,02% NaN 3 , 2 mM EDTA (both Sigma-Aldrich) and 2% hi FBS (Capricorn Scientific) and stained extracellularly for 20 min at 4°C.
  • FACS-buffer PBS without Ca/Mg (Lonza)
  • 2% hi FBS both Sigma-Aldrich
  • permeabilization buffer PBS 1X, 0,02% NaN 3 , 0,5% BSA and 0,1% Saponin (Sigma-Aldrich)
  • Table 5 IFN-g ELISpot raw data spot counts per analysis time and patient
  • 0.2 x 10 6 cells were platted/ well, except for Patient 021 visit 2 to visit 13 and Patient 012 visits 16 and 17, here 0.17 x 10 6 cells were platted/ well.
  • Patient 002 dropped out of the immunological analysis due to inconsistent results (data not shown).
  • Visit 2 for Patient 001 and visit 13 for Patient 015 were not tested as no PBMC samples were available. Spots counts above 2000/well or TNTC (too numerous to count) are set to 2000 and marked in red. For details see section on ELISpot.
  • RV001-derived HLA-class I and -class II potential epitopes were tested by using selected HLA-matched human lymphoblastic cell lines (LCL) as peptide-presenting cells.
  • C1R and its HLA-B*27:05 transfected version were cultured in RPMI 1640 supplemented with 10% hi FBS, P/S, 50 mM b-ME and G418 (Sigma-Aldrich) at 1 mg/ml.
  • MGAR and H0301 were cultured in RPMI 164020% hi FBS, P/S, 50 mM b-ME (for HLA-typing results, see Table 3).
  • LCLs were harvested, counted, and 8 x 10 6 cells per condition were loaded with 50 pg/ml RV001 (SEQ ID NO:2), with 20 pg/ml RV001 -derived single 15mer peptides (ATRi5 (SEQ ID NO:3), AGLI S (SEQ ID NO:4)), or with the respective amount of ddhhO in 1.5 ml of IMDM supplemented with 5% hi HS, P/S and 50 pM b-ME. After 7 h at 37°C, 7.5% CO2 , cells were washed three times to remove unbound peptide.
  • PBMCs stimulated with RV001 SEQ ID NO:2
  • Hiltonol® for 12 days were either incubated with the pre-loaded HLA-matched LCLs at a 2:1 effector to target ratio, with single 50 pg/ml RV001 , or with 10 pg/ml of each single 15mer peptide (ATRis (SEQ ID NO:3), AGLis (SEQ ID NO:4), LQV15 (SEQ ID NO:5)).
  • ddHaO and 10 pg/ml SEB were used as controls. After 12 h, ICS was performed.
  • T cell response was defined by the DFR(2x) permutation resampling method as described by Moodie et al., 2010.
  • T cell reactivity was assessed within CD4 and CD8 T cell subsets.
  • Vaccination against RhoC induces strong and long-lasting RV001 -specific immune responses
  • Fig. 1A An exemplary IFN-y ELISpot result (Patient 011) is shown in Fig. 1 B.
  • strong-responders T-cell frequencies reached a plateau at visit 13 (after 11 vaccinations), which lasted for 13 months post-vaccination.
  • PBMCs from all weak-responders patients show a maximal response mainly at visit 15, 7 months post-vaccination. Specific mean spot counts per patient and visit are displayed in Fig. 1 F. In total, 18/21 (86%) of the patients were responders during vaccination and 19/21 (90%) patients during the follow-up 1/2 (light green).
  • One spontaneous RV001 -specific response was detected in Patient 012 PBMC (visit 2), was boosted by the vaccination (approx. 20 fold,), and lasted until the last follow up visit (Fig. 1F).
  • Vaccine-specific T cells are mainly polyfunctional CD4 T cells
  • Multifunctional flow cytometry analysis was performed to identify RV001 -specific T cells.
  • Cells from one visit during vaccination were re-stimulated with the RV001 peptide (or ddhhO as negative control) for 12 h, and tested in ICS.
  • a representative example for Patient 011 visit 13 is shown in Fig. 5A. Seventeen out of 18 patients (94%) showed a CD4 T cell response against the RV001.
  • boolean gating was performed: 81% of the RV001 -specific CD4 T cells expressed at least two activation markers and of these, almost half (43%) at least three markers simultaneously.
  • especially strong-responders showed a significantly higher frequency of RV001 -specific T cells expressing the 2-3 marker combinations (Figure 2C).
  • RV001 -specific T cells within the CD4 T cell population expressed CD154 and TNF (mean: 2,8%, 95% Cl 1.3 to 4.3), CD154, TNF, and CD107a (mean: 2,5%, 95% Cl 0.8 to 4.3), or CD154, TNF, CD107a, and IFN-y (mean 1,1%, 95% Cl 0.1 to 2.1) ( Figure 2D).
  • 0,49% (95% Cl 0.19 to 0.80) of the RV001 -specific within the CD4 T cell population express all five markers simultaneously.
  • a CD4 T cell response against the RV001 While Patient 007 was still non-responder (not shown).
  • Patient 004 showed at both visits also a CD8 T cell response against the RV001 peptide (Fig. 1 E).
  • the multiparametric analysis shows that RV001 -specific T cells are mainly polyfunctional CD4 effectors, and identified in addition the presence of at least one H LA- class l-presented epitope within the vaccine-peptide sequence.
  • a long-lasting anti-tumor immune response is mediated by T-cell memory formation.
  • RV001 -specific T cells we examined the differentiation status (CD45RA/CCR7), as well as the expression of OX-40 (activation marker), PD-1 (activation/exhaustion marker), and LAG-3 (exhaustion marker) by ex vivo multiparametric flow cytometry.
  • RV001 -specific T cells were mostly effector memory T cells (CD45RAOCR7 ) and detectable already after the 4 th vaccination at visit 6. The response peaked between visit 6 and visit 13 for all patients .
  • PD-1 and to a lesser extent OX-40 were expressed on RV001 -specific cells, however their median fluorescence intensities were similar as for the whole CD4 subset, indicating that vaccine-specific cells did not differentiate towards an exhausted phenotype upon repeated vaccination (Fig. 3B). LAG-3 was not expressed (data not shown).
  • the 20mer RV001 peptide itself might serve as a CD4 T cell epitope itself, but could also contain several shorter CD4 T cell epitopes.
  • Peptide AGL15 was recognized by cells from all three patients, peptide LQVis by cells from Patients 001 and 003, and ATRis by cells from Patient 019 only.
  • HLA-DRB1 *13:02 allele was expressed by Patient 001 , Patient 003, and by H0301, whereas HLA-DRB1*15:01, -DQB1*06:02, -DPB1*04:01 alleles were shared between Patient 019 and MGAR (Table 3).
  • RV001 pre-stimulated PBMCs were cultured with pre-loaded (RV001, ATR 15 or AGL 15 ) LCLS for 12 h in the ICS.
  • the next step was to identify the presenting allele of the RV001 -derived H LA-class I epitope that was recognized by CD8 T cells from Patient 004 (HLA-A*02/-A*30, -B*18/-B*27).
  • PBMCs from Patient 004 visit 15 were pre-stimulated with RV001 , and incubated with either the RV001 or the RV001 pre-loaded C1 R or C1 R-HLA-B*27 LCLs.
  • a CD8 T cell response (CD107a, CD154, TNF, and IL-2 expression) was observed upon stimulation with the RV001 peptide or with the RV001 pre-loaded HLA-B*27 LCL, but not with the non- transfected LCL (Fig. 4C).
  • the RV001 peptide contains at least three different HLA-class II peptides promiscuously presented on various HLA-class II allelic products, as well as one HLA-class I class I peptide presented by the HLA-B*27:05 allele.
  • N 22 patients; */**clinical event, including laboratory test abnormality, with a reasonable time sequence to administration of the vaccine, but which can also be explained by concurrent disease or other drugs or chemicals (*) or which can unlikely be attributed to concurrent disease or other drugs or chemicals (**).
  • Recent cancer vaccine studies have perse been individualized, since they target mutations that mostly occur in individual patients.
  • Other patient-specific approaches that are suitable for tumors with low level of mutational events focus on non-mutated, tumor-specific or tumor-associated antigens that are presented on various malignancies.
  • targeting of antigens associated with metastases could be useful for treatment of metastatic cancer.
  • the vaccination with RV001 against RhoC was safe for all patients over the complete treatment. Side effects related to the treatment were predominantly injection site reactions and fatigue (grade 1/2). These events are most likely related to the adjuvant and carrier Montanide ISA-51 and did not necessitate specific medical intervention.
  • anti-vaccine T cells belonged predominantly to the effector memory CD4 subset and were polyfunctional cells (> 80% expressing at least two of the activation markers tested) that produced TNF, IL-2, and to a lesser extent IFN-g. .
  • the T-cells often expressed the cell-surface degranulation marker CD107a, indicating that at least a fraction of those were cytotoxic effectors.
  • ex vivo phenotyping strongly suggests that RV001 -specific cells did not shift towards an exhausted phenotype after multiple vaccine applications.
  • Tregs CD4 + CD25 + FoxP3 + T regulatory cells
  • a CD8 response restricted by HLA- B*27:05 could be observed in one patient. This response was detected four and seven months post-vaccination. The exact epitope is under characterization and further B27 patients will be assessed.
  • CD154 + CD8 T cells also called CD8 helper cells
  • CD8 helper cells have been shown to support their own expansion and differentiation and to activate dendritic cells to promote anti-tumor immunity.
  • RhoC 20mer contains a nested HLA-A*03 binding peptide, we did not observe any CD8 T cell response in the two patients carrying the HLA-A*03 allele.
  • CD4 T cells are now widely recognized as key players in anti-tumor immunity. Their role in dendritic cell activation (via CD154-CD40 interaction) and CD8 T activation and memory formation (via IL-2 production) are well described. CD4 T cells were also shown to kill HLA- class II positive tumors via granzyme/perforin release or Fas/Fas-L interaction (12,34), and there is also pre-clinical evidence that CD4 T cells can reject tumors better than CD8 T cells. HLA-class II expression has been detected on primary prostate tissues, and on localised prostate cancer (PCa) cell lines, or could be induced by IFN-g.
  • PCa localised prostate cancer
  • Indirect tumor killing by CD4 T cells can also be mediated by IFN-g secretion, as well as by the recruitment of nitric oxide producing macrophages within the tumor.
  • TNF and IL-2 were associated with tumor recognition in vitro. The presented data support that most of the RV001 -specific, polyfunctional CD4 T cells are perfectly equipped for an anti-tumor response as they express exactly these different markers.
  • CD4 T cells Vaccine-based studies in mice and patients have started to unravel the contribution of CD4 T cells in tumor control.
  • Case reports have also documented tumor regressions after adoptive transfer of anti-tumor CD4 cells.
  • CD4 chimeric antigen receptor (CAR) T cells are more potent than CD8 CAR T cells, as they can kill tumors, and in addition, exhibit long-lasting effector function.
  • PSA doubling time is regarded as a surrogate parameter for localised prostate cancer (PCa) progression, and PSA doubling time can predict the risk of mortality in men with localized PCa.
  • PCa localised prostate cancer
  • Three patients progressed biochemically during follow-up (Patients 006, 015, 018). Two of these patients (Patients 006 and 018) had BCR at study entry.
  • Patient 006 had a PSA increase from 0.5 to 1.1 pg/L 29 months following study entry, and Patient 018 from 1.1 to 1.5 pg/ml 24 months following study entry.
  • Patient 015 and 006 had some BCR 13 months post-vaccination (visit 17), however, the PSA for Patient 006 declined the last months on follow-up, and from a clinical perspective, the patient was considered as biochemically stable. No patient developed clinical signs of recurrence.
  • RhoC is a well-tolerated treatment option which induces a strong and long-lasting immune response in the large majority of patients.
  • Vaccine-specific cells are polyfunctional and perfectly equipped for an anti-tumor response.
  • the correlation between the induction of immune responses to RhoC upon vaccination and clinical outcome is examined in a recruiting double-blind, placebo controlled, phase II trial for PCa patients in biochemical relapse (NCT04114825).
  • RhoC vaccination to impair tumor spreading might also synergize with many tumor vaccines (such as those targeting patient-individual, tumor antigens) or other therapies, and extended to other tumor entities.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • Organic Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention provides RhoC peptides, preferably in the form of vaccine compositions comprising RhoC peptides for use in methods of treatment of residual disease in cancer patients having undergone initial treatment, e.g. by radical surgery and/or radiation therapy.

Description

Treatment of residual disease
Technical field
The present invention relates generally to the field of treatment of residual disease in cancer patients having undergone initial treatment of a localized cancer by radical surgery and/or radiation therapy. In particular the invention relates to treatment of residual disease in prostate cancer patients having undergone radical prostatectomy and/or radiation therapy. The invention provides RhoC peptides, preferably in the form of vaccine compositions comprising RhoC peptides for use in such treatment.
Background
The majority of patients with localized prostate cancer (PCa) is treated by radical prostatectomy (RP). Although surgery in most cases is curative, biochemical recurrence (BCR), local tumor recurrence, or distant metastases, as well as cancer-related death occur in up to approximately 30% of the patients within 10 years.
Therapeutic anti-tumor vaccination could provide a safe, cheap, and well-tolerated immunotherapy treatment option for cancer, and many trials are ongoing worldwide, with most recent developments favouring a patient-individual approach. In an ideal scenario, a cancer vaccine should not only induce cytotoxic T lymphocytes (CTL), but also effector CD4 T cells. CD4 T helper cells are important for CD8 T cell activation and expansion, as well as for the generation and maintenance of CD8 T cell memory. In addition, CD4 T cells may display a range of anti-tumoral effects, such as secretion of TNF and IFN-gamma, activation of macrophages or natural killer cells, and direct cytotoxicity, which together might be more powerful than the sole tumor killing by CTL (Schuhmacher et al. , submitted).
The Ras homolog gene family member C (RhoC) is a member of the Rho GTPase family which comprises RhoA, RhoB, and RhoC (85% sequence homology), all involved in the regulation of cytoskeleton organization. Despite RhoA and RhoC being highly homologous there is extensive heterogeneity in the C-terminal of the sequence between RhoA and RhoC. Figure 7 shows an alignment of RhoA; RhoB and RhoC, wherein identical residues (“*”) and residues strong similar properties (“:”) and weak similar properties (“ ”)are marked. RhoC was shown to be an important player in tumor cell motility, invasion, and metastasis formation. RhoC has a limited expression in normal cells, but is highly expressed on advanced cancer cells including metastatic prostate cancer. Thus, expression of RhoC is significantly higher in metastatic tumors compared to primary tumors in different cancers. Furthermore, increased expression of RhoC is positively correlated to poor prognosis (Thomas et al., 2019, Suwa et al., 1998). WO 2009/076966 describes RhoC or peptides derived thereof for treatment of metastatic cancer.
Summary
RhoC is overexpressed in metastatic cancer, but the present invention surprisingly shows that RhoC also is a suitable target for immunotherapy against non-metastatic cancers, and in particular that RhoC is a suitable target for immunotherapy against residual disease after initial treatment of patients having suffered from localised cancer. In other words, the invention provides that RhoC also is a suitable target for immunotherapy against residual disease in patients with no objective signs of distant or systemic metastasis by standard CT scanning with bone scintigraphy or by standard PET-CT scanning.
Thus, immunotherapy with RhoC peptides inhibits or at least reduces the risk of biochemical recurrence. Furthermore, immunotherapy with RhoC peptides may inhibit cancer progression, even in patients with high risk of biochemical recurrence. In prostate cancer, PSA is used as a marker for presence of prostate derived cells, and thus elevated serum levels of PSA are found in patients suffering from residual disease. In treatment of non-metastatic, residual disease in prostate cancers patients, immunotherapy with RhoC peptides interestingly resulted in lower PSA levels than expected, surprisingly demonstrating that immunotherapy with RhoC peptides is effective in treatment of non metastatic residual disease. Even in patients having detectable levels of PSA, which can be considered “high risk” patients, immunotherapy with RhoC peptides resulted in slower increase PSA levels than expected.
Thus it is an aspect of the invention to provide a RhoC peptide or a nucleic acid encoding a RhoC peptide for use in a method of treatment of residual disease in an individual in need thereof, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEC ID NO: 1 , wherein at the most three amino acids of SEC ID NO:1 have been substituted; and wherein the individual has undergone initial treatment of a localized cancer, wherein the initial treatment is selected from the group consisting of cryotherapy, laser therapy, topical therapy, tumour targeting chemo- and immune- therapies, radical surgery and radiation therapy.
The invention also provides kit-of-parts comprising a. a RhoC peptide or a nucleic acid encoding a RhoC peptide; and b. a further anti-cancer agent for use in a method of treatment of residual disease in an individual in need thereof, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO:1 have been substituted; and wherein the individual has undergone initial treatment of a localized cancer, wherein the initial treatment is selected from the group consisting of cryotherapy, laser therapy, topical therapy, tumour targeting chemo- and immune-therapies, radical surgery and radiation therapy.
Description of Drawings
Figure 1: RV001 -specific T cells are induced after RhoC vaccination. (A) Vaccination and monitoring schedule. Patients were vaccinated 11 times. For immunoassays, blood was taken pre-vaccination, three times during the vaccination phase (vaccination) and four times post-vaccination (follow-up 1 and follow-up 2) (blood drops). PBMCs were pre-stimulated with the RV001 and expanded for 12 days before IFN-y ELISpot testing (0.2 x 106 cells/well, except for Patient 21 , visits 2-13 and Patient 012, visits 16+17: 0.17 x 106 cells/well). (B) Exemplary result of an ELISpot (Patient 011). ddFLO and PHA were used as negative and positive control, respectively. (C-E) RV001 -specific mean spot counts per analyzed time and normalized to 0.2 x 106 cells/well. Three independent ELISpot experiments were performed (indicated by the gaps). The sums of RV001 -specific mean spot numbers were calculated for visits 6+8+13 for each patient, and patients were accordingly grouped into strong- (C; n=7; ³2500 spots), intermediate- (D; n=7; ³1500-2500 spots), and weak/non- (E; n=7; 0-1500 spots) responders. (F) RV001 -specific mean spot counts per patient and visit normalized to 0.2 x 106 cells/well. Light green indicates a statistical significance according to the DFR(2x) permutation test. n=21 patients na: cells not available.
Figure 2: RV001 -responding cells are multifunctional. ddhhO stimulated cells harvested from the ELISpot were re-stimulated with the RV001 for 12 h. Expression of CD107a, CD154, IL-2, TNF, and IFN-g was examined by ICS on live CD4 and CD8 lymphocytes. (A) Overview of CD4 T cell responses (all patients during the vaccination phase, n=18). (B) Mean +95% Cl of cumulative marker expression on RV001 -specific CD4 T cells classified per strong- (n=7), intermediate- (n=7) and weak/non-responder (n=4) groups. Kruskal- Wallis test with Dunn's post-test. (C) Min to max percentages of RV001 -specific CD4 T cells expressing one-five markers simultaneously, classified per strong- (n=7), intermediate- (n=7), and weak/non- (n=4) responder groups. Median values are indicated. Two-way ANOVA with Tukey's post-test. (D) Mean +95% Cl of RV001 specific CD4 T cells expressing each of the five activation markers or combinations thereof (n=18). (E) 12 day- cultured PBMCs from Patient 004 at visit 14 were re-stimulated with ddhhO (upper dot plot panel) or the RV001 (lower dot plot panel). The activation marker expression was examined on living CD4 (upper rows) and on CD8 (lower rows) lymphocytes. Percentages of marker+ cells within CD4 or CD8 cells are given. P £ 0,05, **P £ 0,01, ***P £ 0,001. Responder groups are defined based on the ELISpot results.
Figure 3: RV001 -specific T cells are effector memory T cells with a stable PD-1 and OX- 40 expression over time. PBMCs from Patient 005 (visit 6 to visit 15) and Patients 009 and 018 (visit 6 to visit 17) were thawed, rested, and stimulated either with RV001 or ddhhO for 12 h. Live RV001 -specific CD4 lymphocytes were identified by TNF expression and were further phenotypically examined for the expression of CD45RA, CCR7, PD-1 , OX- 40, and LAG-3. (A) Exemplary results (Patient 009): CD4TNF+ cells (black) are overlayed on the whole CD4 cell population (grey). Numbers indicate CD4TNF+ cell count in each quadrant of the dot plot. (B) Expression profile of PD-1 (upper row) and OX-40 (lower row) for n=3 patients at visit 6 and visit 15. Numbers indicate median fluorescence intensity (MFI) ratios between CD4+TNF+CD45RAOCR7- cells (dark grey) and CD4+TNFCD45RA- CCR7 cells (light grey). Histograms show event counts normalized to mode.
Figure 4: The RV001 sequence comprises several promiscuous HLA-class II epitopes and one epitope presented on the HLA-B*27:05 allele. The expression of CD107a, CD154, IL- 2, TNF, and IFN-y was examined on live CD4 or CD8 cells. (A) Cells were re-stimulated with the RV001 or with each RV001 derived 15mer peptide (ATR15, AGL15, LQV15) for 12 h. Shown are the percentage or mean +SD percentage (n=2 repeated measurements) of peptide-specific CD4 cells expressing each activation marker for three patients at visit 16. (B) LCLs were pre-loaded with RV001, ATR15 or AGL15, and incubated with HLA-matched patient cells in at 1 :2 ratio for 12 h. Shown are the specific percentages of CD4 cells expressing the indicated activation markers. (C) Cells were re-stimulated either with the RV001 peptide alone or with RV001 pre-loaded C1 R or C1 R-HLA-B*27:05 cells for 12 h in the ICS. Shown are the percentages of specific marker expression on CD8 cells.
Figure 5: Gating strategies. A: Gating strategy for the intracellular cytokine staining (ICS) analysis to identify RV001 specific T cell populations (Patient 011). Upper row, left to right: sample flow overtime, duplet exclusion (FSC-A/FSC-H), gating on live cells (FSC-A/Zombie Aqua dye), lymphocytes (FSC-A/SSC-A), and CD4/CD8 dot plot. CD4 T cells (middle row) and CD8 T cells (lower row) were analyzed for their expression of IL-2, CD154, CD107a, TNF and IFN-g (from left to right). B: Gating strategy for the ex vivo flow analysis of RV001 specific CD4 T cells (Patient 018). Upper row, left to right: sample flow over time, duplet exclusion (FSC-A/FSC-H), gating on live cells (FSC-A/Zombie Aqua dye), lymphocytes (FSC-A/SSC-A). Gating 1 : CD4 cells were examined for CD45RA/CCR7, as well as for TNF expression after RV001 peptide stimulation for 12 hr. The overlay of the TNF+ cells (black) onto the CD45RA/CCR7 dot plot (grey) shows that most of the RV001 -specific CD4 cells were effector memory (CD45RA-CCR7-). Gating 2: The PD-1, OX-40, and LAG-3 expressions were further examined in the TNF+, CD45RA-CCR7- cells. Lowest 2 rows: As controls for the expression of PD-1 , OX-40, and LAG-3, fluorescence minus one controls (FMOs) were used. FMO gates were set on the whole CD4 T cell population (shown on the dot plots) and were copied to the CD4+CD45RA-CCR7-TNF+ (RV001 -specific) cell population.
Figure 6: Additional ICS results for the identification of RV001-derived HLA-class II epitopes (see also Fig 4). Table: tested patients and visits, as well as the peptides recognized by patients' CD4 T cells (see Examples). Bars indicate percentages of peptide-specific CD4 T cells expressing CD107a, CD154, IL-2, TNF, IFN-y (IFN) when re-stimulated for 12 h with the peptides RV001 (black), ATR15 (middle grey), AGLis (dark grey), LQV15 (light grey). For gating, see Figure 5.
Figure 7 shows an alignment of RhoA; RhoB and RhoC, wherein identical residues (“*”) and residues with conservative (“:”) and semi-conservative (“ ”) substitutions are marked.
Detailed description
Definitions
The term “biochemical recurrence” as used herein in relation to prostate cancer, refers to an individual having an increased serum level of PSA after initial treatment of a localized prostate cancer. Preferably, “biochemical recurrence” in relation to prostate cancer according to the present invention is defined as follows:
• for patients who do not have measurable PSA recorded at onset of treatment, a serum level of PSA ³ 0.2 ng/ml is considered “biochemical recurrence”
• for patients who have measurable PSA recorded at onset of treatment - increase in PSA by 50% or more from level at onset of treatment is considered “Biochemical recurrence”.
The term “initial treatment” as used herein refers to the first line treatment of a localized cancer. Dependent on the localized cancer, the initial treatment may be selected from the group consisting of cryotherapy, laser therapy, topical therapy, tumour targeting chemo- and immune-therapies, radical surgery and radiation therapy. Initial treatment of localized prostate cancer is either radical prostatectomy or radiation treatment.
As used herein, a patient is considered to suffer from “localized prostate cancer”, when there are no objective signs of distant or systemic metastasis by standard CT scanning with bone scintigraphy or by standard PET-CT scanning. Thus, a prostate cancer is considered “localized” even if there is invasion of adjacent organs or tissues.
The term “non-metastatic cancer” as used herein refers to a cancer having no objective signs of distant or systemic metastasis by standard CT scanning with bone scintigraphy or by standard PET-CT scanning.
The term “residual disease” as used herein refers to cancer cells that remain after attempts to remove a cancer have been made. Thus, residual disease may be any cancer cells present or developing after initial treatment. It is noted that the term “residual disease” as used herein always refers to residual disease after initial treatment of cancer.
The term “treatment” as used herein refers to any type of treatment or prevention that imparts a benefit to an individual suffering from or at risk of developing cancer, notably prostate cancer. Thus the term “treatment” may e.g. refer to preventive or prophylactic treatment, curative treatment, ameliorating treatment, treatment of symptoms, and/or treatment to delay disease progression.
RhoC peptide
RhoC is overexpressed in metastatic cancer, but the present invention surprisingly shows that RhoC also is a suitable target for immunotherapy against non-metastatic cancers, and in particular RhoC is a suitable target for immunotherapy against residual disease in patients having suffered from localised cancer.
The invention relates to a RhoC peptide for use in a method of treatment of residual disease in cancer patients having undergone initial treatment. Said RhoC peptide may be provided in the form of a RhoC peptide, or it may be provided in the form of a nucleic acid encoding the RhoC peptide. In preferred embodiments, the RhoC peptide is provided in the form of a RhoC peptide.
If nothing else is specified the term “human RhoC” as used herein refers to a polypeptide of SEQ ID NO 1. A RhoC peptide according to the present invention comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO: 1 , such as at the most two amino acids of SEQ ID NO:1, for example at the most one amino acid of SEQ ID NO:1 has/have been substituted.
Thus, in some embodiments the RhoC peptide may comprise or consist of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO:1.
In other embodiments, RhoC peptide may comprise or consist of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO:1 , wherein up to 3 amino acids present in SEQ ID NO:1 has been substituted for another amino acid, for example
In preferred embodiments of the invention, the RhoC peptides comprises a consecutive sequence of SEQ ID NO:1 comprising amino acids not present in the corresponding sequence of RhoB. In more preferred embodiments the RhoC peptides comprises a consecutive sequence of SEQ ID NO:1 comprising amino acids neither present in the corresponding sequence of RhoA nor RhoB. Accordingly in very preferred embodiments the peptide fragments of the invention contains at least one of amino acid residues I43, Q123, R140, S141, S152, L157, E165, G178, V181, K183, N184, R186, R187, R188,
P191 or 1192 of RhoC of SEQ ID NO:1.
In less preferred embodiments the RhoC peptides are peptides comprising amino acid residues, which differ from the sequence of RhoB, but are the same as the sequence of RhoA. Accordingly in less preferred embodiments the peptide fragments of the invention contains at least one of amino acid residues L81 , M82, C83, F84, S85, I86, D87, S88,
P89, D90, S91, L92, E93, N94, I95, K98, W99, T100, P101, E102, V103, K104, H105, F106, C107, P108, N109, P111 , 1112, 1113, V115, G116, N117, K118, K119, T127, R129, E142, E143, D146, N149, F154, D155, G166, V167, R168, E169, V170, F171 , E172, M173, A174, T175, R176, A177, L179, Q180, R182, K185, G189, C190.
As described herein above, RhoC mainly differs from RhoA and RhoB in the C-terminal part of the sequence. Thus, preferred RhoC peptides are derived from the 120 most C- terminal residues of RhoC, such as the 100 most C-terminal residues of RhoC, for example the 75 most C-terminal residues of RhoC, such as 52 most C-terminal residues of RhoC, for example the 40 most C-terminal residues of RhoC, such as the 30 most C- terminal residues of RhoC, for example the 25 most C-terminal residues of RhoC, such as the 24 most C-terminal residues of RhoC, such as the 23 most C-terminal residues of RhoC, for example the 22 most C-terminal residues of RhoC, such as the 21 most C- terminal residues of RhoC, such as the 20 most C-terminal residues of RhoC of SEQ ID NO 1. Most preferred is a peptide consisting of the 20 most C-terminal residues of RhoC of SEQ ID NO:1.
Accordingly in very preferred embodiments the RhoC peptide comprises at least one of the 20 most C-terminal RhoC specific amino acid residues G178, V181, K183, N184,
R186, R187, R188, P191 or 1192 of RhoC of SEQ ID NO:1.
It is preferred that the RhoC peptide comprises or consists of a consecutive sequence of in the range of 8 to 100, such as 8 to 75, for example 8 to 60, for example of 8 to 50, preferably in the range of 9 to 25 consecutive amino acids of said RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO: 1 , such as at the most 2 amino acids of SEQ ID NO: 1 , for example at the most 1 amino acid of SEQ ID NO:1 has been substituted, for example no amino acid is substituted.
The RhoC peptide may comprise or consists of a consecutive sequence of in the range of 8 to 60, for example in the range of 8 to 50, such as in the range of 8 to 40, for example in the range of 8 to 10 or in the range of 18 to 60 amino, such as in the range of 18 to 50 amino acids of RhoC of SEQ I D NO: 1 , wherein at the most three, for example at the most two, such as at the most one amino acid(s) of SEQ ID NO:1 have been substituted, for example no amino acid is substituted.
The RhoC peptide may comprise (or more preferably consists of) at the most 200, preferably at the most 100, more preferably at the most 60, yet more preferably at the most 25, even more preferably at the most 20, yet even more preferably at the most 15, such as at the most 10, for example in the range of 8 to 10 contiguous amino acids of RhoC of SEQ ID NO:1 or a functional homologue thereof , wherein at the most three amino acids of SEQ ID NO:1 have been substituted.
The RhoC peptide may comprise or consist of a consecutive sequence of in the range of 8 to 100, such as 8 to 75, for example 8 to 60, for example of 8 to 50, such as in the range of 9 to 25, for example in the range of 18 to 60, such as in the range of 18 to 50 amino acid of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO: 1 , such as at the most 2 amino acids of SEQ ID NO: 1 , for example at the most one amino acid of SEQ ID NO:1 have been substituted, for example no amino acid is substituted and wherein said sequence comprises at least one of amino acid residues I43, Q123, R140, S141 , S152, L157, E165, G178, V181, K183, N184, R186, R187, R188, P191 or 1192 of RhoC of SEQ ID NO:1.
In some embodiments the RhoC peptide comprises or consists of the sequence RXGLQVRKNK, wherein X is selected from the group consisting of alanine and leucine and wherein said RhoC peptide is at the most 200, preferably at the most 100, more preferably at the most 60, yet more preferably at the most 25, even more preferably at the most 20 amino acids long, such as in the range of 10 and 150, preferably between 12 and 120, more preferably between 15 and 75, yet more preferably between 15 and 70, even more preferably between 18 and 60, such as between 18 and 50 amino acids long. Preferably, said RhoC peptide consists of a consecutive sequence of SEQ ID NO: 1 , wherein at the most three, for example at the most two, such as at the most one amino acid(s) of SEQ ID NO:1 have been substituted, for example no amino acid is substituted.
In other specific embodiments of the invention the RhoC peptide comprises or consists of RXGLQVRKNK (SEQ ID NO:6), wherein X is selected from the group consisting of alanine and leucine.
In some embodiments the RhoC peptide comprises or consists of the sequence ATRAGLQVRKNKRRRGCPIL (SEQ ID NO:2), wherein said RhoC peptide is at the most 200, preferably at the most 100, more preferably at the most 60, yet more preferably at the most 25, even more preferably at the most 20 amino acids long, such as in the range of 20 and 150, preferably between 20 and 120, more preferably between 20 and 75, yet more preferably between 20 and 70, even more preferably between 20 and 60, such as between 18 and 50 amino acids long. Preferably, said RhoC peptide consists of a consecutive sequence of SEQ ID NO: 1 , wherein at the most three, for example at the most two, such as at the most one amino acid(s) of SEQ ID NO:1 have been substituted, for example no amino acid is substituted.
In a specific embodiment of the invention the RhoC peptide comprises or consist of the 20 most C-terminal amino acid residues of RhoC of SEQ ID NO:1. Accordingly in this particular embodiment the RhoC peptide consists of the sequence ATRAGLQVRKNKRRRGCPIL (SEQ ID NO:2).
In some embodiments the RhoC peptide comprises or consists of the sequence SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5, wherein said RhoC peptide is at the most 200, preferably at the most 100, more preferably at the most 60, yet more preferably at the most 25, even more preferably at the most 20 amino acids long, such as in the range of 20 and 150, preferably between 20 and 120, more preferably between 20 and 75, yet more preferably between 20 and 70, even more preferably between 20 and 60, such as between 18 and 50 amino acids long. Preferably, said RhoC peptide consists of a consecutive sequence of SEQ ID NO: 1 , wherein at the most three, for example at the most two, such as at the most one amino acid(s) of SEQ ID NO:1 have been substituted, for example no amino acid is substituted.
Thus, the RhoC peptide may consists of a sequence selected from the group consisting of ATRAGLQVRKNKRRR (SEQ ID NO:3), AGLQVRKNKRRRGCP (SEQ ID NO:4) and LQVRKNKRRRGCPIL (SEQ ID NO:5).
In some aspects of the present invention, the RhoC peptide is relatively long. When administering a peptide longer than 8 to 10 amino acids the longer peptide may be processed into a series of smaller peptides in the cytosol, e.g. by the proteasome or by proteases in the cytosol. An advantage of using a longer peptide that may be processed into a variety of different shorter peptides is that more H LA-class I and II allotypes may be targeted with one peptide than one 8 to 10 amino acid peptide that is restricted to a particular HLA class-l.
Accordingly in a specific embodiment the immunogenically active peptide fragment of the invention consists of 50 amino acid residues, for example at the most 45 amino acid residues, such as at the most 40 amino acid residues, for example at the most 35 amino acid residues, such as at the most 30 amino acid residues, for example at the most 25 amino acid residues, such as 18 to 25 consecutive amino acids from RhoC of SEQ ID NO:1 or a functional homologue thereof, wherein at the most three amino acids have been substituted and the peptide fragment contains at least one of the RhoC specific amino acid residues I43, Q123, R140, S141, S152, L157, E165, G178, V181, K183, N184, R186, R187, R188, P191 or 1192 of RhoC of SEQ ID NO:1.
Accordingly in another specific embodiment the immunogenically active peptide fragment of the invention consists of 100 amino acid residues, for example at the most 90 amino acid residues, such as at the most 80 amino acid residues, for example at the most 70 amino acid residues, such as at the most 65 amino acid residues, such as 18 to 60 consecutive amino acids from RhoC of SEQ ID NO:1 or a functional homologue thereof, wherein at the most three amino acids have been substituted and the peptide fragment contains at least one of the RhoC specific amino acid residues I43, Q123, R140, S141, S152, L157, E165, G178, V181, K183, N184, R186, R187, R188, P191 or 1192 of RhoC of SEQ ID NO:1.
In a specific embodiment the RhoC peptide comprises or consists of sequence selected from the group consisting of VYVPTVFENYIADIEVDGKQV (SEQ ID NO:7), ILVGNKKLRQDEHTRRLAK (SEQ ID NO:8) and ELAKMKQEPVRSEEGRDMANR (SEQ ID NO:9), wherein at the most three amino acids have been substituted, such as at the most two amino acids have been substituted, for example at the most one amino acid have been substituted. Preferably no amino acids have been substituted.
The RhoC peptide may also comprises (or more preferably consists of) between 8 and 120, preferably between 8 and 100, more preferably between 10 and 75, yet more preferably between 12 and 60, even more preferably between 15 and 40, such as between 18 and 25 contiguous amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids have been substituted, deleted or added.
In a preferred embodiment of the invention the peptide comprises (or more preferably consists of) between 10 and 150, preferably between 12 and 120, more preferably between 15 and 75, yet more preferably between 20 and 70, even more preferably between 15 and 65, such as between 15 and 60 contiguous amino acids of RhoC of SEQ ID NO:1 or a functional homologue thereof, wherein at the most three amino acids have been substituted, deleted or added.
The RhoC peptide may also comprise or consist of the following sequences: EEGRDMANRISAFGYKECSAKTKEGVREVFEMATRAGLQVRKNKRRRGCPIL (SEQ ID NO:10) or ATRAGLQVRKNKRRRGCPIL (SEQ ID NO:11) or RAGLQVRKNK (SEQ ID NO:12). In another preferred embodiment the RhoC peptide is RLGLQVRKNK (SEQ ID NO:13) which is an artificial peptide, wherein the alanine of RAGLQVRKNK (SEQ ID NO: 12) has been substituted with leucine.
Vaccine
The invention relates to a RhoC peptide for use in a method of treatment of residual disease in cancer patients having undergone initial treatment. Said RhoC peptide may be provided in the form of a vaccine, wherein said vaccine comprises the RhoC peptide or a nucleic acid encoding same as well as an adjuvant. The adjuvant may be any substance whose admixture into the vaccine composition increases or otherwise modifies the immune response to the RhoC peptide.
Adjuvants may for example be selected from the group consisting of: AIK(SC>4)2, AINa(SC>4)2, AINH4 (SO4), silica, alum, AI(OH)3, Ca3 (PCU kaolin, carbon, aluminum hydroxide, muramyl dipeptides, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetylmuramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(T2'-dipalmitoyl-sn -glycero-3- hydroxphosphoryloxyj-ethylamine (CGP 19835A, also referred to as MTP-PE), RIBI (MPL+TDM+CWS) in a 2% squalene/Tween-80.RTM. emulsion, lipopolysaccharides and its various derivatives, including lipid A, Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacterium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, Titermax, ISCOMS, Quil A, ALUN (see US 58767 and 5,554,372), Lipid A derivatives, choleratoxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrixes or GMDP, Interleukin 1, Interleukin 2, Montanide ISA-51 and QS-21. Preferred adjuvants to be used with the invention include oil/surfactant based adjuvants such as Montanide adjuvants (available from Seppic, Belgium), preferably Montanide ISA-51. Other adjuvants are bacterial DNA based adjuvants, such as adjuvants including CpG oligonucleotide sequences. Yet other adjuvants are viral dsRNA based adjuvants, such as the TLR-ligand poly l:C. Imidazochinilines are yet another example of preferred adjuvants. The most preferred adjuvants are adjuvants suitable for human use. Liposomes are in general not useful adjuvants for the present invention, and preferably the vaccine compositions of the invention therefore do not comprise liposomes.
Montanide adjuvants (all available from Seppic, Belgium), may be selected from the group consisting of Montanide ISA-51, Montanide ISA-50, Montanide ISA-70, Montanide ISA- 206, Montanide ISA-25, Montanide ISA-720, Montanide ISA-708, Montanide ISA-763A, Montanide ISA-207, Montanide ISA-264, Montanide ISA-27, Montanide ISA-35,
Montanide ISA 51 F, Montanide ISA 016D and Montanide IMS, preferably from the group consisting of Montanide ISA-51, Montanide IMS and Montanide ISA-720, more preferably from the group consisting of Montanide ISA-51 and Montanide ISA-720. Montanide, and in particular Montanide ISA-51 (Seppic, Inc.) is oil/surfactant based adjuvants in which different surfactants are combined with either a non-metabolizable mineral oil, a metabolizable oil, or a mixture of the two. They are prepared for use as an emulsion with an aqueous solution comprising RhoC peptide. The surfactant is mannide oleate. QS-21 (Antigenics; Aquila Biopharmaceuticals, Framingham, MA) is a highly purified, water- soluble saponin that handles as an aqueous solution. QS-21 and Montanide ISA-51 adjuvants can be provided in sterile, single-use vials.
The well-known cytokine GM-CSF is another adjuvant and may be GM-CSF as described in WO 97/28816.
A vaccine composition according to the present invention may comprise more than one different adjuvant.
The vaccine may comprise other useful components in addition to RhoC peptide and adjuvant. For example, the vaccine composition may comprise a carrier. The function of a carrier can for example be to increase the molecular weight of RhoC peptides in order to increase their activity or immunogenicity, to confer stability, to increase the biological activity, or to increase serum half-life. Furthermore, a carrier may aid presenting the protein belonging to the rho gene family or peptide fragments thereof to T-cells. The carrier may be any suitable carrier known to the person skilled in the art, for example a protein or an antigen presenting cell. A carrier protein could be but is not limited to keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid. For immunization of humans, the carrier must be a physiologically acceptable carrier acceptable to humans and safe. However, tetanus toxoid and/or diptheria toxoid are suitable carriers in one embodiment of the invention. Alternatively, the carrier may be dextrans for example sepharose.
In addition to RhoC peptide, nucleic acid encoding same and/or adjuvant, the vaccine may also comprise one or more additional pharmaceutically acceptable excipients, for example such excipients described in Remington: The Science and Practice of Pharmacy,
Lippincott Williams & Wilkins, 21.sup.st ed. (2004), which is herein incorporated by reference in its entirety.
Residual disease
The invention relates to a RhoC peptide for use in a method of treatment of residual disease in a cancer patient having undergone initial treatment. “Residual disease” within the meaning of the present invention relates to cancer disease after initial treatment of a localised cancer. A cancer is considered “localised” if no distant or systemic metastases are observed by CT scanning with bone scintigraphy or by standard PET-CT scanning. The present invention has surprisingly found that even though RhoC is mainly associated with metastatic cancer, treatment with RhoC peptide induces a T-cell response, which is useful in treatment of non-metastatic cancer, and notably of residual disease after initial treatment of a localized cancer. Thus, it is preferred that the individual to be treated according to the present invention does not have any distant or systemic metastasis as detected by CT scanning or PET-CT scanning.
The initial treatment is in general selected according to the type of localised cancer, and will typically be the ordinary initial treatment used for treatment of the particular type of localised cancer. For example the initial treatment may be selected from the group consisting of cryotherapy, laser therapy, topical therapy, tumour targeting chemo- and immune-therapies, radical surgery and radiation therapy.
Thus, the initial treatment may for example be radical surgery or radiation therapy. This is in particular preferred with the cancer is prostate cancer.
Prostate cancer
The invention in particular relates to a RhoC peptide for use in a method of treatment of residual disease in an individual having undergone initial treatment for prostate cancer. In other words, the invention relates to a method of treating an individual having suffered from prostate cancer, wherein the treatment is administered subsequent to initial treatment of said prostate cancer.
Herein the prostate cancer prior to initial treatment may be referred to as the “initial prostate cancer”. The initial prostate cancer is preferably a localized prostate cancer, i.e. a prostate cancer wherein no distant or systemic metastases are detected by CT scanning with bone scintigraphy or by standard PET-CT scanning.
Prostate cancer, as used herein, refers to a disease in which cancer develops in the prostate gland of the male reproductive system. Prostate cancer is classified as an adenocarcinoma, or glandular cancer, that begins when normal semen-secreting prostate gland cells mutate into cancer cells. In the initial stage of prostate cancer, small clumps of cancer cells remain confined to otherwise normal prostate glands, a condition known as carcinoma in situ or prostatic intraepithelial neoplasia (PIN), a prostate precancer. Over time these cancer cells begin to multiply and spread to the surrounding prostate tissue (the stroma), forming a tumor. While prostate cancer originates and may remain in the prostate, prostate tumor cells may develop the ability to travel in the bloodstream and lymphatic system and thus be found in other organs or tissues. Prostate cancer most commonly metastasizes to the bones, lymph nodes, rectum, and bladder.
Different methods are used to classify prostate cancers. One method include classifying a prostate tumor based on the Gleason scoring system. The Gleason scoring system is based on microscopic tumor patterns assessed by a pathologist while interpreting the biopsy specimen. When prostate cancer is present in the biopsy, the Gleason score is based upon the degree of loss of the normal glandular tissue architecture (i.e. shape, size and differentiation of the glands). The classic Gleason scoring system has five basic tissue patterns that are technically referred to as tumor "grades." The microscopic determination of this loss of normal glandular structure caused by the cancer is represented by a grade, a number ranging from 1 to 5, with 5 being the worst grade. Grade 1 is typically where the cancerous prostate closely resembles normal prostate tissue. The glands are small, well-formed, and closely packed. At Grade 2 the tissue still has well-formed glands, but they are larger and have more tissue between them, whereas at Grade 3 the tissue still has recognizable glands, but the cells are darker. At high magnification, some of these cells in a Grade 3 sample have left the glands and are beginning to invade the surrounding tissue. Grade 4 samples have tissue with few recognizable glands and many cells are invading the surrounding tissue. For Grade 5 samples, the tissue does not have recognizable glands, and are often sheets of cells throughout the surrounding tissue. Since prostate cancers are often made up of cancerous cells that have different grades, two grades are assigned for each patient, and the Gleason score is provided as the sum of these two scores. A primary grade may be given to describe the cells that make up the largest area of the tumor and a secondary grade may be given to describe the cells of the next largest area. Unless otherwise indicated the Gleason scores provided herein are provided as the sum of the two scores.
In one embodiment, the initial, localized prostate cancer of the individual to be treated had a Gleason score in the range of 6 to 10, for example in the range of 6 to 9 prior to initial treatment.
Another method for classifying prostate cancer is by determining the extent of the primary tumour. The clinical T category (cT) is the doctor’s estimate of the disease based on results of physical examination, prostate biopsy and imaging tests. This classification is used to evaluate the primary tumour, and comprises the following clinical T stages (cT): cTO: no evidence of tumor • cT1: tumor present, but not detectable clinically or with imaging o cT1a: tumor was incidentally found in 5% or less of prostate tissue resected (for other reasons) o cT1b: tumor was incidentally found in greater than 5% of prostate tissue resected o cT1c: tumor was found in a needle biopsy performed due to an elevated serum PSA
• cT2: the tumor can be felt (palpated) on examination, but has not spread outside the prostate o cT2a: the tumor is in half or less than half of one of the prostate gland's two lobes o cT2b: the tumor is in more than half of one lobe, but not both o cT2c: the tumor is in both lobes but within the prostatic capsule
• cT3: the tumor has spread through the prostatic capsule (if it is only part- way through, it is still T2) o cT3a: the tumor has spread through the capsule on one or both sides, e.g. had an extra prostatic extension o cT3b: the tumor has invaded one or both seminal vesicles, e.g. had seminal vesicle/bladder neck involvement
• cT4: the tumor has invaded other nearby structures
In one embodiment, the initial, localized prostate cancer of the individual to be treated may be classified as cT1, cT2, cT3 or cT4. In particular, the initial, localized prostate cancer of the individual to be treated may be classified as a cT1a, cT1b, cT1c, cT2a, cT2b, cT2c, cT3a, cT3b or cT4. Preferably, the initial, localized prostate cancer of the individual to be treated may be classified as a cT3a, cT3b or cT4.
As described herein above, the individual to be treated has in general undergone an initial treatment of said initial localized prostate cancer. Said initial treatment may in particular be either radical prostatectomy and/or radiation therapy, which are typical first line treatment for localized prostate cancer.
Radical prostatectomy refers to the surgical removal of the prostate gland. In general a radical prostatectomy involves the removal of the entire prostate gland, the seminal vesicles and the vas deferens.
There are multiple ways a radical prostatectomy can be performed, e.g. by open surgery (via a large incision through the lower abdomen), laparoscopically with the help of a robot (a type of minimally invasive surgery), through the urethra or through the perineum.
The prostatectomy may be performed in a manner so the blood vessels and nerves that promote penile erections are left behind in the body and not taken out with the prostate.
The prostatectomy may be performed with limited pelvic lymph node dissection, so that at least some of the surrounding lymph nodes are removed. Typically, lymph nodes may be removed from the area defined by external iliac vein anteriorly, the obturator nerve posteriorly, the origin of the internal iliac artery proximally, Cooper's ligament distally, the bladder medially and the pelvic side wall laterally.
The prostatectomy may also be associated with extended pelvic lymph node dissection. In such cases lymph nodes further away from the prostate are also removed. Typically, the lymph nodes removed during limited pelvic lymph node dissection are also removed, as well as lymph node from the posterior boundary and the floor of the pelvis.
The radiation therapy may in particular be either external beam radiation or brachytherapy, which are the most common radiation therapies used in the treatment of localized prostate cancer.
External beam radiation (EBRT) is in general delivered over several sessions in order to reduce the risk of adverse effects. There are several ways to deliver EBRT including the following:
• Conventional therapy: A person receives 35-75 doses over 7-9 weeks
• Moderate hypofraction: The dose is higher, and there are fewer sessions. 20 sessions over 4 weeks
• Ultra-hypofraction: High doses over five sessions. It is also called stereotactic body radiation therapy (SBRT).
• Three-dimensional conformal radiation therapy (3D-CRT)
• Intensity modulated radiation therapy (IMRT.
• Volumetric modulated arc therapy (VMAT): This version of IMRT delivers treatment rapidly so that each session is shorter.
• Proton beam radiation therapy: Proton therapy delivers beams of protons instead of radiation. Brachytherapy is also known as Internal radiation therapy (IRT), and in general involves placing radioactive pellets inside the body, on the prostate gland. The pellets are in general around the size of a grain of rice. The implant may be temporary or permanent:
In some embodiments, the individual to be treated may have no detectable serum prostate-specific antigen (PSA). However, in other embodiments, the individual suffers from a biochemical recurrence. Thus, the individual may have detectable serum PSA, such as a serum level of PSA of >0.05 ng/ml, for example >0.06 ng/ml. In some embodiments the individual has a serum level of PSA of in the range of 0.05 to 20 ng/ml, for example in the range of 0.06 to 20 ng/ml. At least at the onset of treatment with RhoC peptide, the individual may have the aforementioned serum level of PSA.
The serum level of PSA may be determined by any useful means. For example, PSA may be detected in serum using a PSA specific monoclonal antibody. Typically, PSA is detected using electrochemiluminescence immunoassays for measuring either total PSA or free PSA. Such assays can be performed on automated analysers such as Elecsys 1010 and 2010 or COBIS 8000. Many different approved and validated methods are available for determine PSA levels, and any of these may be used. If nothing else is indicated, the level of PSA is the level of total PSA.
Method of treatment of residual disease
The invention relates to a RhoC peptide or a nucleic acid encoding the same for use in a method of treatment of residual disease in a cancer patient after initial treatment of localised cancer. In other words, the invention relates to a method of treating an individual having suffered from cancer, e.g. a localised cancer, wherein the treatment is administered subsequent to initial treatment of said cancer.
The individual to be treated according to the invention is in general a cancer patient, in particular a human cancer patient.
Treatment with RhoC peptide may be initiated immediately after termination of the initial treatment. In some cases (in particular if the initial treatment is radiation therapy) the treatment with RhoC peptide may even be initiated during the initial treatment. In other cases the treatment with RhoC peptide may be initiated long after the initial treatment, for example at least 1 week after, e.g. at least 1 month after, such as within 1 to 20 years after completion of the initial treatment. Typically, the RhoC peptide or nucleic acid encoding same is administered to the individual more than once. Thus, the RhoC peptide may be administered at least twice, such as at least 5 times, preferably at least 9 times, more preferably at least 10 times, even more preferably at least 11 times, such as in the range of 2 to 50 times, for example in the range of 5 to 40 times, such as in the range of 10 to 30 times, for example in the range of 10 to 20 times, preferably in the range of 11 to 12 times. In one embodiment, the RhoC peptide is administered 11 times. In one embodiment, the RhoC peptide is administered 12 times.
The interval between two administrations of RhoC peptide may vary, but is typically in the range of 2 days to several months, such as in the range of 2 days to 6 months, for example in the range of 1 to 12 weeks, such as in the range of 1 to 8 weeks, for example in the range of 2 to 4 weeks. Within the same treatment regimen, the intervals may also vary. Thus, for example the first administrations may be administered with shorter intervals between two administrations compared to the later administrations. A non limiting example of a useful administration scheme is provided in figure 1A.
RhoC peptide may thus be administered over an extended time period, wherein RhoC is administered multiple times with suitable intervals between administrations. The total time period for administration of RhoC may be at least 12 months, such as at least 18 months, for example at least 24 months, such as in the range of 12 to 100 months, for example in the range of 24 to 60 months.
The dosage of RhoC peptide depends on several factors, but may for example be in the range of 0.01 to 10 mg RhoC peptide, such as in the range of 0.05 to 5 mg RhoC peptide, for example in the range of 0.05 to 1 mg RhoC peptide, such as approx. 0.1 mg RhoC peptide. Aforementioned dosages may be useful for treatment of adult human males.
The route of administration may be any useful route, for example by injection, such as subcutaneous injection or intradermal injection.
The present invention relates to methods of treatment of residual disease in an individual, who has undergone initial treatment of a localised cancer. In particular, the methods relate to treatment of non-metastatic, residual disease. In other words, the methods relate to treatment of residual disease in an individual having no objective signs of distant or systemic metastasis by standard CT scanning with bone scintigraphy or by standard PET- CT scanning. Vaccination with RhoC peptides has previously been suggested for treatment of metastatic cancer, however, the present invention surprisingly discloses that RhoC peptides or nucleic acids encoding same are useful in treatment of non-metastatic cancer, i.e. of residual disease after initial treatment of a localised cancer. Even though effective treatment of residual disease inhibits disease progression, it cannot be regarded solely as a treatment to prevent metastatic cancer. Rather, the purpose of the methods is the treatment of the residual, non-metastatic cancer per se.
Interestingly, the present invention has found that administration of RhoC peptide results in a strong CD4+ cell response in the large majority of the patients. Whereas a general significant immune response does not necessarily correlate with efficacy of cancer treatment with a given peptide, a CD4+ response may be useful in treatment of cancer, because the anti-cancer effect of a CD4+ cell response is not confined to the cytolysis of cells expressing RhoC. Thus, a CD4+ cell response also provides a more general anticancer effect, e.g. due to expression of several different cytokines, such as INF-g, TNF and IL-2 with known anti-cancer properties.
Thus, in one embodiment, the RhoC peptide is immunogenic. In particular, the RhoC peptide may have at least one of the following characteristics:
(i) capable of inducing intracellular IFN-g in CD4+ T cells, and/or
(ii) capable of increasing intracellular TNF in CD4+ T cells, and/or
(iii) capable of increasing intracellular IL2 in CD4+ T cells, and/or
(iv) capable of increasing intracellular CD154+ in CD4+ T cells, and/or
(v) capable of increasing intracellular CD107a in CD4+ T cells and/or CD8+ T cells.
In particular, upon administration of the RhoC peptide to an individual, said individual produces memory T cells, wherein said T cells - after in vitro expansion with stimulation by RhoC peptide - have the following characteristics:
(i) at least 2 fold induction in CD4+T cells expressing INF-g, and/or
(ii) at least 2 fold induction in CD4+ T cells expressing TNF, and/or
(iii) at least 2 fold induction in CD4+ T cells expressing IL2 and/or
(iv) at least 2 fold induction in CD4+ T cells expressing CD154, and/or
(v) at least 2 fold induction in CD4+ T expressing CD107a wherein said induction is the induction compared to the number of CD4+ memory T-cells expressing aforementioned activation markers after in vitro expansion without stimulation with a RhoC peptide.
In thus, administration of the RhoC peptide to an individual preferably leads to one or more of the aforementioned effects in said individual.
In particular, it is preferred that the treatment with RhoC peptide elicits a clinical response in the individual, wherein the clinical response is significantly reduced risk of biochemical recurrence.
In particular, it is preferred that when the individual to be treated with RhoC peptide, is an individual having undergone initial treatment of localized prostate cancer, wherein the individual has a serum level of PSA below the detection limit, then treatment with RhoC peptide results in said individual having reduced risk of developing a serum level of PSA ³ 0.2 ng/ml. Preferably, said individual does not develop a serum level of PSA ³ 0.2 ng/ml. Thus, the treatment may elicit a clinical response in said individual, wherein the clinical response is a PSA level £ 0.2 ng/ml. Preferably, said PSA levels are determined within 60 months, such as within 48 months, for example within 36 months from first administration.
In some embodiments the individual has a detectable PSA serum level. In such embodiments, the treatment with RhoC peptide may elicit a clinical response characterised by an increased PSA serum level doubling time compared to serum PSA level doubling time in said individual prior to treatment with the peptide. Thus, the treatment with RhoC peptide may elicit a clinical response in the individual, wherein the clinical response is characterised by at least 1.5-fold increase in the PSA serum level doubling time compared to the PSA serum level doubling time in said individual prior to treatment with the peptide.
The treatment with RhoC peptide may also elicit a clinical response in the individual, wherein the clinical response is a decrease in the progression of cancer, e.g. a decrease in progression of prostate cancer compared to the expected progression without treatment.
The treatment with RhoC peptide may also elicit a clinical response in the individual, wherein the clinical response is characterised by a stable disease, a partial response or complete remission. Kit-of-parts
The RhoC peptide or nucleic acid encoding same may also be administered in combination with one or more other anti-cancer agents. Thus, the invention also provides a kit-of-parts for use in a method of treating residual disease in a cancer patient having undergone initial treatment, wherein the kit-of-part comprises a. a RhoC peptide or a nucleic acid encoding same, wherein said peptide or nucleic acid optionally is comprised in a vaccine; and b. a further anti-cancer agent.
The anti-cancer agent may for example be an anti-cancer agent used for chemotherapy or gene therapy of cancer. The anti-cancer agent may also be immunostimulating substances or antibodies. The immunostimulating substances may for example be cytokines, such as cytokines selected from the group consisting of GM-CSF, type I IFN, interleukin 12 and interleukin 15. The antibody may preferably be an immunostimulating antibody such as anti-CD40, anti-CTLA-4, anti-OX-40 antibodies, anti-PD1 or anti-PD1-L antibodies. The immunostimulatory substance may also be a substance capable of depletion of immune inhibitory cells (e.g. regulatory T-cells) or factors, said substance may for example be E3 ubiquitin ligases. E3 ubiquitin ligases (the HECT, RING and U-box proteins) have emerged as key molecular regulators of immune cell function, and each may be involved in the regulation of immune responses during infection by targeting specific inhibitory molecules for proteolytic destruction. Several HECT and RING E3 proteins have now also been linked to the induction and maintenance of immune self tolerance: c-Cbl, Cbl-b, GRAIL, Itch and Nedd4 each negatively regulate T cell growth factor production and proliferation.
Other anti-cancer agents may also be substances that induce androgen deprivation therapy (ADT) such as LHRH agonists, LHRH antagonists in addition to anti-androgens and CYP17 blockers.
Items
The invention may further be defined by the following items:
1. A RhoC peptide or a nucleic acid encoding a RhoC peptide for use in a method of treatment of residual disease in an individual in need thereof, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO:1 have been substituted; and wherein the individual has undergone initial treatment of a localized cancer, wherein the initial treatment is selected from the group consisting of radical surgery, radiation therapy, cryotherapy, laser therapy, topical therapy, tumour targeting chemotherapy and tumour targeting immune therapy.
2. A RhoC peptide or a nucleic acid encoding a RhoC peptide for use in a method of treatment of residual disease in an individual in need thereof, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO:1 have been substituted; and wherein the method comprises administering said peptide or nucleic acid to said individual at least 9 times.
3. The peptide or nucleic acid for use in the method according to item 1, wherein the localized cancer is localized prostate cancer.
4. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the initial treatment is radical surgery and/or radiation therapy.
5. The peptide or nucleic acid for use in the method according to any one of items 3 to 4, wherein said individual has undergone radical prostatectomy for treatment of a localized prostate cancer.
6. The peptide or nucleic acid for use in the method according to any one of items 3 to 5, wherein said individual has undergone radiation therapy for treatment of a localized prostate cancer, wherein said radiation therapy is selected from the group consisting of external beam radiation and brachytherapy
7. The peptide or nucleic acid for use in the method according to any one of items 3 to 6, wherein said individual suffers from biochemical recurrence.
8. The peptide or nucleic acid for use in the method according to any one of items 3 to 7, wherein the individual has detectable serum PSA. 9. The peptide or nucleic acid for use in the method according to any one of items 3 to 8, wherein the individual has a serum level of PSA of >0.05 ng/ml.
10. The peptide or nucleic acid for use in the method according to any one of items 3 to 9, wherein the individual has a serum level of PSA >0.06 ng/ml.
11. The peptide or nucleic acid for use in the method according to any one of items 3 to 9, wherein the individual has a serum level of PSA >0.1 ng/ml.
12. The peptide or nucleic acid for use in the method according to any one of items 3 to 11 , wherein the individual had a serum level of PSA in the range of 0.1 to 20 ng/ml.
13. The peptide or nucleic acid for use in the method according to any one of items 3 to 9, wherein the individual had a serum level of PSA in the range of 0.05 to 20 ng/ml prior to initial treatment.
14. The peptide or nucleic acid for use in the method according to any one of items 3 to 13, wherein the localized prostate cancer had an extraprostatic extension and/or was classified as cT3a prostate cancer prior to initial treatment.
15. The peptide or nucleic acid for use in the method according to any one of items 3 to 13, wherein the localized prostate cancer had seminal vesicle/bladder neck involvement and/or was classified as cT3b prostate cancer prior to initial treatment.
16. The peptide or nucleic acid for use in the method according to any one of items 3 to 13, wherein the localized prostate cancer showed seminal vesicle involvement, tumor fixation, or invasion of adjacent organs and/or was classified as cT4 prostate cancer prior to initial treatment.
17. The peptide or nucleic acid for use in the method according to any one of items 3 to 15, wherein the localized prostate cancer had a Gleason score in the range of 6 to 10, for example in the range of 6 to 9 prior to initial treatment.
18. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the individual does not have distant or systemic metastasis as detected by CT scanning or PET-CT scanning. 19. The peptide or nucleic acid for use in the method according to anyone of the preceding items, wherein said peptide is immunogenic as determined by said peptide having at least one of the following characteristics:
(i) capable of inducing intracellular IFN-g in CD4+ T cells, and/or
(ii) capable of increasing intracellular TNF in CD4+ T cells, and/or
(iii) capable of increasing intracellular IL2 in CD4+ T cells, and/or
(iv) capable of increasing intracellular CD154+ in CD4+ T cells, and/or
(v) capable of increasing intracellular CD107a in CD4+ T cells.
20. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the treatment elicits a clinical response in the individual, wherein the clinical response is reduced risk of biochemical recurrence.
21. The peptide or nucleic acid for use in the method according to any one of items 3 to 6, wherein the individual has a PSA level below the detection limit and wherein the treatment elicits a clinical response in the individual, wherein the clinical response is a PSA level £ 0.2 ng/ml.
22. The peptide or nucleic acid for use in the method according to any one of items 3 to 20, wherein the treatment elicits a clinical response in the individual, wherein the clinical response is characterised by an increased PSA serum level doubling time compared to a PSA serum level doubling time in said individual prior to treatment with the peptide.
23. The peptide or nucleic acid for use in the method according to any one of items 3 to 20, wherein the treatment elicits a clinical response in the individual, wherein the clinical response is characterised by at least a 1.5-fold increase in the PSA serum level doubling time compared to a PSA serum level doubling time in said individual prior to treatment with the peptide.
24. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the treatment elicits a clinical response in the individual, wherein the clinical response is a slower prostate cancer progression compared to expected progression without treatment.
25. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the treatment elicits a clinical response in the individual, wherein the clinical response is characterised by a stable disease, a partial response or complete remission.
26. The peptide or nucleic acid for use in the method according to any one of the preceding items wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ I D NO: 1 , wherein at the most two, such as at the most one amino acid(s) of SEQ ID NO:1 have been substituted.
27. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the RhoC peptide consists of a consecutive sequence of in the range of 8 to 60, for example in the range of 8 to 50, such as in the range of 8 to 40, for example in the range of 8 to 10 or in the range of 18 to 60 amino, such as in the range of 18 to 50 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three, for example at the most two, such as at the most one amino acid(s) of SEQ ID NO:1 have been substituted.
28. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the RhoC peptide consists of a consecutive sequence of in the range of 8 to 60, for example in the range of 8 to 50, such as in the range of 8 to 40, for example in the range of 8 to 10 or in the range of 18 to 60, such as in the range of 18 to 50 amino acids of RhoC of SEQ ID NO:1.
29. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the RhoC peptide comprises or consists of a consecutive sequence of amino acids of RhoC of SEQ ID NO:1, wherein said consecutive sequence comprise at least one of amino acid residues I43, Q123, R140, S141, S152, L157, E165, G178, V181, K183, N184, R186, R187, R188, P191 or 1192 of RhoC of SEQ ID NO:1.
30. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the RhoC peptide consists of in the range of 8 to 60, such as in the range of 8 to 50, for example in the range of 18 to 25 consecutive amino acids from RhoC of SEQ ID NO:1 , wherein at the most two amino acids have been substituted, wherein the peptide fragment contains at least one of amino acid residues I43, Q123, R140, S141, S152, L157, E165, G178, V181,
K183, N184, R186, R187, R188, P191 or 1192 of RhoC of SEQ ID NO:1. 31. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the RhoC peptide comprises of consists of the sequence RXGLQVRKNK, wherein X is selected from the group consisting of alanine and leucine and wherein said peptide fragment is at the most 60 amino acids in length.
32. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the RhoC peptide comprises of consists of the sequence ATRAGLQVRKNKRRRGCPIL.
33. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the RhoC peptide comprises of consists of a sequence selected from the group consisting of ATRAGLQVRKNKRRR, AGLQVRKNKRRRGCP and LQVRKNKRRRGCPIL.
34. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein the peptide and/or nucleic acid is contained in a vaccine composition comprising a. said peptide and/or nucleic acid; and b. an adjuvant.
35. The peptide or nucleic acid for use in the method according to item 34, wherein the adjuvant is a water in oil emulsion.
36. The peptide or nucleic acid for use in the method according to any one of items 34 to 35, wherein the adjuvant is a Montanide ISA adjuvant, such as Montanide ISA 51 or Montanide ISA 720.
37. The peptide or nucleic acid for use in the method according to any one of items 34 to 36, wherein the vaccine further comprises a carrier.
38. The peptide for use in the method according to any one of the preceding items, wherein the peptide is formulated for administration of in the range of 0.01 to 10 mg of said peptide.
39. The peptide for use in the method according to any one of the preceding items, wherein the peptide is formulated for administration of in the range of 0.05 to 1 mg of said peptide. 40. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein said method comprises administering said peptide or nucleic acid to said individual at least 9 times, preferably at least 10 times.
41. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein said method comprises administering said peptide or nucleic acid to said individual at least 11 times.
42. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein said method comprises administering said peptide or nucleic acid to said individual in the range 10 to 30 times.
43. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein said method comprises administering said peptide or nucleic acid to said individual in the range of 11 to 12 times.
44. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein said method comprises multiple administrations with a time interval of in the range of 1 to 8 weeks between every administration.
45. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein said method comprises multiple administrations with a time interval of in the range of 2 to 4 weeks between every administration.
46. The peptide or nucleic acid for use in the method according to any one of the preceding items, wherein said administration is subcutaneous injection or intradermal injection.
47. A kit-of-parts comprising a. a RhoC peptide or a nucleic acid encoding a RhoC peptide; and b. a further anti-cancer agent for use in a method of treatment of residual disease in an individual in need thereof, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO:1 have been substituted; and wherein the individual has undergone initial treatment of a localized cancer, wherein the initial treatment is selected from the group consisting of radical surgery, radiation therapy, cryotherapy, laser therapy, topical therapy, tumour targeting chemotherapy and tumour targeting immune therapy.
48. The kit-of-parts according to item 47, wherein the RhoC peptide is as defined in any one of items 26 to 33 or is comprised within a vaccine composition according to any one of item 34 to 37.
49. The kit-of-parts according to any one of items 47 to 48, wherein the method is the method defined in any one of items 1 to 25 or 38 to 46.
50. The kit-of-parts according to any one of items 47 to 49, wherein the anti-cancer agent is an antibody.
51. The kit-of-parts according to any one of items 47 to 50, wherein the anti-cancer agent is a cytokine.
52. A method of treating residual disease in an individual in need thereof, the method comprising administering to an individual in need thereof an effective amount of a RhoC peptide or a nucleic acid encoding the RhoC peptide, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO:1 have been substituted; and wherein the individual has undergone initial treatment of a localized cancer by radical surgery and/or radiation therapy.
53. The method according to item 52, wherein the method is as defined in any one of items 1 to 46.
54. Use of a RhoC peptide or a nucleic acid encoding a RhoC peptide or a kit-of-parts comprising said RhoC peptide or nucleic acid in the manufacture of a medicament for the treatment of residual disease in an individual in need thereof, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO:1 have been substituted; and wherein the individual has undergone initial treatment of a localized cancer by radical surgery and/or radiation therapy. 55. Use according to item 54, wherein the method is as defined in any one of items 1 to 46.
Sequence list
SEQ ID NO:1
Wild-type human RhoC i.e. the naturally occurring non-mutated human RhoC SEQ ID NO:2
20 aa C-terminal fragment of RhoC also referred to as RV001 herein ATRAGLQVRKNKRRRGCPIL
SEQ ID NO:3 ATR15
ATRAGLQVRKNKRRR
SEQ ID NO:4 AGLi5
AGLQVRKNKRRRGCP
SEQ ID NO:5 LQV15
LQVRKNKRRRGCPIL
SEQ ID NO:6 RXGLQVRKNK
X is selected from the group consisting of alanine and leucine.
SEQ ID NO:7
VYVPTVFENYIADIEVDGKQV
SEQ ID NO:8
I LVGN KKLRQDEHTRRLAK
SEQ ID NO:9
ELAKMKQEPVRSEEGRDMANR
SEQ ID NO:10 EEGRDMANRISAFGYKECSAKTKEGVREVFEMATRAGLQVRKNKRRRGCPIL
SEQ ID NO: 11
ATRAGLQVRKN KRRRGCPIL
SEQ ID NO:12 RAGLQVRKNK
SEQ ID NO:13 RLGLQVRKNK
Examples
The invention may further be illustrated by the following examples, which however should not be construed as limiting for the invention.
Example 1
The example describes a clinical phase I/ll study, showing safety and immunogenicity of a 20mer SLP vaccine specifically targeting the RhoC protein in prostate cancer patients (see also Schuhmacher et al., submitted).
Study design and patients
The study was an open-label, phase I/ll trial. Patients having previously been diagnosed with localised prostate cancer and subsequently treated with radical prostatectomy (RP) were identified, informed, and followed at the Department of Urology, Rigshospitalet. Vaccinations were administered at Zelo Phase I Unit, DanTrials ApS, Copenhagen, Denmark. The trial was approved by the local ethical committee (H-1604701) and the European Union regulatory authorities (EuDRACT number: 2016-004189-24), and was conducted according to the Declaration of Helsinki. All patients gave informed consent. Prior to study entry, patients underwent screening procedures including a full physical examination, and in case of biochemical recurrence (BCR), a metastatic workup with computer tomography and bone-scans. For inclusion and exclusion criteria, see Table 1a. The primary endpoint of the study was the evaluation of the safety and tolerability of the vaccine. Treatment-emergent adverse events (TEAE) were recorded and analyzed in accordance with the common terminology criteria for adverse events (CTCAE), version 4.03. The second endpoint was the investigation of the immunological responses against the vaccine. Table 1a: Inclusion and exclusion criteria of the open label clinical phase I/ll study
Figure imgf000033_0001
Figure imgf000034_0001
Altogether, 24 patients were screened and 22 included in the study (Table 1b). Median time for surgery to study entry was 1.2 years (range: 0.3-12.8). Patients were vaccinated subcutaneously alternating between the right and left upper arms with the RV001 vaccine consisting of 0.1 mg/ml RV001 emulsified in Montanide ISA-51 (1 ml). The term “RV001” as used herein refers to a peptide consisting of the 20aa C-terminal sequence of the RhoC protein: ATRAGLQVRKNKRRRGCPIL (SEQ ID NO:2). The 16 last amino-acid sequence of RV001 is found only in RhoC, but not in RhoA/B, and is thus RhoC-specific.. Patients were vaccinated six times every two weeks, then five times every four weeks, resulting in a treatment duration of approximately 30 weeks (11 vaccinations in total). Blood was taken for HLA-class I and II typing (performed by the Department of Clinical Immunology, Tissue Typing Laboratory, Rigshospitalet, Copenhagen) before vaccination (visit 2). For the in vitro immunomonitoring, blood was taken before vaccination (visit 2), and after the 4th, 6th and 11th vaccinations (visits 6, 8 and 13). Follow up samples were obtained every third month up to 13 months post-vaccination (visits 14 to 17). Serum PSA levels were measured by routine clinical testing at every patient visit (median number of PSA measurements from study entry: 15 (range 13-17). The study started in March 2017 and was completed in March 2019, with the final post-vaccination PSA level measurement in December 2019. Median follow-up time for serum PSA was 2.5 years (range 2.4-2.7 years). Of note, Patient 015 withdrew from treatment after seven injections, but completed all visits for blood collection, except visit 13. Study design is shown on Fig. 1 and patient's characteristics, including time between prostatectomy and vaccination, PSA levels, and HLA-typing are shown in Tables 2 and 3.
Table 1b: Main patient's characteristics
Figure imgf000035_0001
T= tumor, N= nodes, R- and R+ = negative and positive margins Table 2: Patient's characteristics
Figure imgf000036_0001
*Patient received only 7-8 vaccinations instead of eleven.
**Assumption 30 days per months
Four patients had experienced biochemical recurrence defined as PSA > 0.1 pg/L (LOQ) prior to study entry. Of these, 3 had received salvage radiation, and one patient refrained from salvage therapy. At study entry, 2 patients had PSA levels above 0.1 pg/L. One patient was progressing following radical prostatectomy and salvage (Patient 018), the other one following prostatectomy only (Patient 006).
Table 3: HLA-typing of individual patients and cell lines
Figure imgf000037_0001
Figure imgf000038_0001
The following sections are prepared in accordance with the Minimal Information about T cell Assays (MIATA) guidelines (Britten et al., 2012 ).
Cell samples
Isolation of peripheral blood mononuclearcells (PBMCs)
Blood samples (100 ml, heparinized) were processed within 8 h after blood drawing (DanTrials ApS). The PBMC isolation was performed according to a standard, pre-established protocol, using pre-filled 50 ml Leucosep™ separation tubes (Greiner Bio-One). Cells were counted with trypan blue and Tuerks Solution (both Sigma-Aldrich). Six to 13 x 106 cells per cryovial (Nunc™, Sigma-Aldrich) were frozen in 1 ml heat-inactivated (hi) FBS (ThermoFisher) containing 10% dimethylsulfoxide (DMSO, Sigma-Aldrich). Cells were first stored in freezing containers (Nalgene® Mr. Frosty, Sigma-Aldrich) at -70°C for 24-72 h and later transferred into liquid nitrogen (-196°C). PBMCs were shipped on temperature-controlled dry ice to the Department of Immunology at the University of Tubingen for immunological analysis. Cells were stored again at -196°C (liquid nitrogen) for approximately 3-12 months before testing. PBMC samples from Patient 001 visit 2 and Patient 015 visit 13 were not available.
Immunological assessments
In vitro stimulation of antigen-specific T cells
PBMCs were thawed in IMDM (Gibco) containing 2.5% hi human serum (HS, Sigma-Aldrich), penicillin 100 units/ml and streptomycin 0.1 mg/ml (P/S, Sigma-Aldrich), and 50 mM b- mercaptoethanol (b-ME, Merck). After one washing step with serum-free medium (SFM, IMDM, P/S, 50 pM b-ME), cells were counted with trypan blue (Merck). The median live cell yield after thawing was 63,5%. No cut-off was applied for further in vitro culture. On day 0, 1.0- 3.5 x 106 PBMCs/well or 3.5-6.5 x 106 cells/well were seeded in T cell medium (TCM, IMDM with 10% hi HS, 1 x P/S and 50 pM b-ME) in a 48-well or 24-well plate, respectively (Cellstar®, Greiner bio-one), and further cultured at 37°C and 7.5% CO2. On day 1, cells were stimulated with 10 pg/ml RV001 (SEC ID NO:2)(Batch AW16147B, purity ³90%, prepared by PolyPeptide Laboratories France SAS) dissolved in 100% deionized water (ddH20; LiChrosolv, Merck) plus 20 pg/ml Poly-ICLC (Hiltonol®, Oncovir). Human interleukin (IL)-2 (R&D Systems) was added to the culture at 2 ng/ml on days 3, 5, 7, and 9. On day 12, cells were harvested, counted (median live cell yield: 76,8%) and tested with enzyme-linked immune spot assay (ELISpot) and intracellular cytokine staining (ICS) assay.
ELISpot The IFN-g ELISpot protocol has been described by Widenmeyer et al. , 2012. If not otherwise stated, 0.2 x 106 cells were cultured per well in the presence of 50 pg/ml RV001 peptide (SEQ ID NO:2) for 26 h at 37°C and 7.5% CO2 (triplicates). ddhhO (6 wells) and phytohemagglutinin- L (PHA-L, 10 pg/ml, Sigma-Aldrich, 1 well) were used as negative and positive control, respectively. Spots were counted with the ImmunoSpot series 6 ultra-V analyzer (CTL Europe GmbH) according to a standard protocol. Altogether, samples from n=21 patients tested by ELISpot were immunologically evaluated (results from Patient 002 were excluded because of inconsistent spot counts). For wells with counts above 2000 spots or stated as TNTC (“too numerous to count”), a count cut-off was set to 2000 spots. RV001 -specific spot counts are defined as the mean spot counts obtained in the RV001 stimulated wells minus the mean spot counts in the ddH20 wells. All spot counts are given in Table 5.
Multiparameter flow cytometry
PBMCs were analyzed by ICS either ex vivo or after culture. Thawed cells were rested in TCM for 4-6 h prior to the ICS. Pre-cultured cells were directly examined on day 12 (Fig. 2E; Fig. 4), or harvested from the ELISpot plate (Fig. 2A-D, ddH20 cells from the visit with the highest spot count in the ELISpot were used). Between 0.5 and 2 x 106 cells per well (96-well plate) were stimulated with 50 pg/ml RV001 (SEQ ID NO:2) or 10 pg/ml of single overlapping 15mers (RV001 -derived: ATRAGLQVRKNKRRR (ATR15) (SEQ ID NO:3), AGLQVRKNKRRRGCP (AGL15) (SEQ ID NO:4), LQVRKN KRRRGCPIL (LQV15) (SEQ ID NO:5), all from JPT Peptide Technologies, ³90% purity). ddH20 and Staphylococcus enterotoxin B (10 pg/ml, SEB, Sigma- Aldrich) were added as negative and positive control, respectively. The CD107a antibody was added together with the stimulus, the protein transport inhibitors Brefeldin A (10 pg/ml, Sigma- Aldrich) and Golgi Stop (BD) 1 h thereafter. After 12 h at 37°C and 7.5% CO2, cells were stained (Panel 1, Table 4). For ex vivo analysis of PD-1 , OX-40, and LAG-3 expression (Panel 2, Table 4), fluorescence minus one controls were performed. For staining, cells were washed in FACS-buffer (PBS without Ca/Mg (Lonza), 0,02% NaN3, 2 mM EDTA (both Sigma-Aldrich) and 2% hi FBS (Capricorn Scientific) and stained extracellularly for 20 min at 4°C. After fixation and permeabilization (Cytofix/Cytoperm, BD), cells were washed with permeabilization buffer (PBS 1X, 0,02% NaN3, 0,5% BSA and 0,1% Saponin (Sigma-Aldrich)) and stained intracellularly for 20 min at 4°C. Cells were washed with permeabilization buffer, resuspended in FACS-buffer, and acquired on the same day on a LSRFortessa™ SORP (BD) equipped with the DIVA Software (Version 6). The analysis was performed with FlowJo (Version 10.6.1), gatings strategies are shown in Fig. 5. All results were audited. Frequencies of RV001 -specific T cells are defined as: % marker positive cells in the RV001 stimulated sample minus % of marker positive cells in the ddH2Q sample. Table 4: Monoclonal antibody panels for flow cytometry analysis
Figure imgf000041_0001
Note: All fluorophore-coupled antibodies were pre-titrated.
Table 5: IFN-g ELISpot raw data spot counts per analysis time and patient
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
R: Rep icate; neg = negative control (ddhhO); na= not available. For all patients and tests, 0.2 x 106 cells were platted/ well, except for Patient 021 visit 2 to visit 13 and Patient 012 visits 16 and 17, here 0.17 x 106 cells were platted/ well. Patient 002 dropped out of the immunological analysis due to inconsistent results (data not shown). Visit 2 for Patient 001 and visit 13 for Patient 015 were not tested as no PBMC samples were available. Spots counts above 2000/well or TNTC (too numerous to count) are set to 2000 and marked in red. For details see section on ELISpot.
Identification of RV001 -presenting allelic products
RV001-derived HLA-class I and -class II potential epitopes were tested by using selected HLA-matched human lymphoblastic cell lines (LCL) as peptide-presenting cells. C1R and its HLA-B*27:05 transfected version (C1R-B*27) were cultured in RPMI 1640 supplemented with 10% hi FBS, P/S, 50 mM b-ME and G418 (Sigma-Aldrich) at 1 mg/ml. MGAR and H0301 were cultured in RPMI 164020% hi FBS, P/S, 50 mM b-ME (for HLA-typing results, see Table 3). LCLs were harvested, counted, and 8 x 106 cells per condition were loaded with 50 pg/ml RV001 (SEQ ID NO:2), with 20 pg/ml RV001 -derived single 15mer peptides (ATRi5 (SEQ ID NO:3), AGLIS (SEQ ID NO:4)), or with the respective amount of ddhhO in 1.5 ml of IMDM supplemented with 5% hi HS, P/S and 50 pM b-ME. After 7 h at 37°C, 7.5% CO2, cells were washed three times to remove unbound peptide. PBMCs stimulated with RV001 (SEQ ID NO:2) plus Hiltonol® for 12 days were either incubated with the pre-loaded HLA-matched LCLs at a 2:1 effector to target ratio, with single 50 pg/ml RV001 , or with 10 pg/ml of each single 15mer peptide (ATRis (SEQ ID NO:3), AGLis (SEQ ID NO:4), LQV15 (SEQ ID NO:5)). ddHaO and 10 pg/ml SEB were used as controls. After 12 h, ICS was performed.
Immunological response definition
Patients were grouped into immunological responder groups (strong, intermediate, low/non) according to the results of the ELISpot assay of PBMCs obtained during vaccination (visits 6, 8, and 13 together). For each visit, a T cell response was defined by the DFR(2x) permutation resampling method as described by Moodie et al., 2010. A patient was defined as an immunological responder if at least two out of three analyzed times were tested positive in the ELIspot. If T cell reactivity against RV001 was already detected at baseline level, the patient was considered as a responder if response was boosted during vaccination (specific spot counts after vaccination ³ 2 x specific spot counts at baseline). If less than three vaccination samples were evaluable (n=1), T cell reactivity detected at one analyzed time was enough to consider the patient as an immunological responder.
For the ICS, T cell reactivity was assessed within CD4 and CD8 T cell subsets. First, each activation marker (n=5) was stated as positive if the percentage of positive cells was ³ three fold for the peptide-stimulated cells compared to the ddH20 stimulated cells. In addition, ³20 marker-positive cells must be counted. Second, at least three out of five markers must be positive. Statistical comparisons
Statistical analyses were performed with GraphPad Prism 6 (version 6.01). The Kolmogorov-Smirnov test with Dallal-Wilkinson-Liliefor corrected P value was used to check for Gaussian distribution. If one comparison was made between groups, a one-way ANOVA was performed, if two, a two-way ANOVA was performed. In all cases, a correction for multiple comparisons was performed. Statistical differences were considered as significant for P £ 0,05 (*), P £ 0,01 (**), and P £ 0,001 (***). The absence of an asterisk indicates that no significant difference was reported. Information on the statistical test and the number of patients included is given for each experiment.
General Lab Operation
All experiments were performed with standard reagents and according to laboratory- standard protocols for culture, assays, and analysis. Protocols for ELISpot and ICS have been validated and the performance of the working group is regularly controlled by participation in external proficiency panels (MHC Multimer & ELISpot, CIP/CIMT, and Immudex).
Results
Vaccination against RhoC induces strong and long-lasting RV001 -specific immune responses
The immunological response against the RV001 vaccine was assessed on PBMC samples obtained during the vaccination phase and at all follow-up times for 21/22 evaluable patients (Fig. 1A). An exemplary IFN-y ELISpot result (Patient 011) is shown in Fig. 1 B. Patients were grouped according to the strength of their T cell response (defined as the sum of vaccine-specific spot counts during the vaccination phase, n=3 timepoints) into strong- (Fig. 2C), intermediate- (Fig. 2D), and weak/non-responders (Fig. 2E). In most cases, vaccine- reactive T cells were detected after 4 vaccinations (visit 6). In strong-responders, T-cell frequencies reached a plateau at visit 13 (after 11 vaccinations), which lasted for 13 months post-vaccination. PBMCs from all weak-responders patients (Patients: 007, 015, 016, 017) show a maximal response mainly at visit 15, 7 months post-vaccination. Specific mean spot counts per patient and visit are displayed in Fig. 1 F. In total, 18/21 (86%) of the patients were responders during vaccination and 19/21 (90%) patients during the follow-up 1/2 (light green). One spontaneous RV001 -specific response was detected in Patient 012 PBMC (visit 2), was boosted by the vaccination (approx. 20 fold,), and lasted until the last follow up visit (Fig. 1F). Interestingly, Patient 010, classified as non-responder during vaccination, developed a statistically significant response against the RV001 peptide at visit 15, which increased further at visit 16 (1.4-fold increase), then at visit 17 (4-fold increase). Only one RV001 -specific response was lost at the last follow-up visit (Patient 007). The high response rate among patients with various MHC allelic products indicates a broad immunogenicity of the vaccine. In addition, T cell responses were mostly detectable for at least 10 months (visit 16) post-vaccination, suggesting the induction of stable T cell memory responses.
Vaccine-specific T cells are mainly polyfunctional CD4 T cells
Multifunctional flow cytometry analysis was performed to identify RV001 -specific T cells. Cells from one visit during vaccination were re-stimulated with the RV001 peptide (or ddhhO as negative control) for 12 h, and tested in ICS. A representative example for Patient 011 visit 13 is shown in Fig. 5A. Seventeen out of 18 patients (94%) showed a CD4 T cell response against the RV001. A response against the RV001 was defined as following: First, each activation marker (n=5) was stated as positive if the percentage of positive cells was ³ three-fold for the peptide-stimulated cells compared to the ddhhO stimulated cells. In addition, ³20 marker-positive cells must be counted. Second, at least three out of five markers must be positive. Cells from Patient 007, classified as a weak-responder by the ELISpot, did not reach the positivity threshold in the ICS (Figure 2A). CD4 T cells of strong- responder patients expressed in mean more activation markers (CD107a, CD154, IL-2, TNF, and/or IFN-g: mean sum 21 ,7%, 95% Cl 8,3 to 35,1) upon RV001 re-stimulation than CD4 T cells of intermediate-responders (mean sum: 9,0%, 95% Cl 3,2 to 14,7) and significantly more as CD4 T cells of weak/non-responders (mean sum: 2,4%, 95% Cl -3,8 to 9,0). (Figure 2B). This supports our previous classification of patients in the three groups according to the ELISpot results. To assess multifunctionality, boolean gating was performed: 81% of the RV001 -specific CD4 T cells expressed at least two activation markers and of these, almost half (43%) at least three markers simultaneously. When comparing the three patient groups, especially strong-responders showed a significantly higher frequency of RV001 -specific T cells expressing the 2-3 marker combinations (Figure 2C). Among all patients, most RV001 -specific T cells within the CD4 T cell population expressed CD154 and TNF (mean: 2,8%, 95% Cl 1.3 to 4.3), CD154, TNF, and CD107a (mean: 2,5%, 95% Cl 0.8 to 4.3), or CD154, TNF, CD107a, and IFN-y (mean 1,1%, 95% Cl 0.1 to 2.1) (Figure 2D). On average, 0,49% (95% Cl 0.19 to 0.80) of the RV001 -specific within the CD4 T cell population express all five markers simultaneously.
PBMCs obtained from n=10 patients (including the non-responder Patient 007) during the follow-up 1 (visit 14 and visit 15) were also examined in ICS to identify RV001 specific cells. Nine out of 10 patients still showed a CD4 T cell response against the RV001, while Patient 007 was still non-responder (not shown). In addition to a CD4 T cell response, Patient 004 showed at both visits also a CD8 T cell response against the RV001 peptide (Fig. 1 E). In summary, the multiparametric analysis shows that RV001 -specific T cells are mainly polyfunctional CD4 effectors, and identified in addition the presence of at least one H LA- class l-presented epitope within the vaccine-peptide sequence.
Vaccination against RhoC induces memory CD4 T cells
A long-lasting anti-tumor immune response is mediated by T-cell memory formation. To address the phenotype of RV001 -specific T cells, we examined the differentiation status (CD45RA/CCR7), as well as the expression of OX-40 (activation marker), PD-1 (activation/exhaustion marker), and LAG-3 (exhaustion marker) by ex vivo multiparametric flow cytometry. A representative gating strategy for Patient 018 is shown in Fig. 5B. Patients (n=3) with a strong or intermediate IFN-y response in the ELISpot were selected for the analysis. RV001 -specific T cells were identified by their TNF expression after stimulation. A representative overlay of CD4TNF+ cells (black) on the CD45RA/CCR7 gated CD4 T cells (grey) is shown for Patient 009 in Fig 3A. RV001 -specific T cells were mostly effector memory T cells (CD45RAOCR7 ) and detectable already after the 4th vaccination at visit 6. The response peaked between visit 6 and visit 13 for all patients . PD-1 and to a lesser extent OX-40 were expressed on RV001 -specific cells, however their median fluorescence intensities were similar as for the whole CD4 subset, indicating that vaccine-specific cells did not differentiate towards an exhausted phenotype upon repeated vaccination (Fig. 3B). LAG-3 was not expressed (data not shown).
Several epitopes are recognized by T cells within the RV001 sequence.
The 20mer RV001 peptide itself might serve as a CD4 T cell epitope itself, but could also contain several shorter CD4 T cell epitopes. To identify such sequences, we expanded RV001 -specific T cells from selected PBMC samples (n=7, two follow up visits per patient) and re-stimulated the cells with RV001, or with single RV001 -derived 15mer peptides (ATRi5, AGLi5, LQV15; 15aa overlap) for 12 h, followed by ICS staining. Representative results for CD4 T cells of n=3 patients from visit 16 samples are shown in Fig. 4A and Fig. 6. We found that all three peptides could be recognized, albeit at various rates. Peptide AGL15 was recognized by cells from all three patients, peptide LQVis by cells from Patients 001 and 003, and ATRis by cells from Patient 019 only.
Next, we used two human LCLs MGAR and H0301 to identify the presenting HLA-class II allele/s for Patients 001 , 003, and 019. According to the four digit HLA-typing, the HLA- DRB1 *13:02 allele was expressed by Patient 001 , Patient 003, and by H0301, whereas HLA-DRB1*15:01, -DQB1*06:02, -DPB1*04:01 alleles were shared between Patient 019 and MGAR (Table 3). RV001 pre-stimulated PBMCs were cultured with pre-loaded (RV001, ATR15 or AGL15) LCLS for 12 h in the ICS. Both 15mer peptides were recognized by CD4 cells of Patients 001 and 003. For Patient 019, only a response against the RV001 pre-loaded onto the LCL was detected. Incubation with the ATRi5 or AG s pre-loaded LCL led to an increase in IFN-g, CD154, and IL-2, but did not reach positivity threshold (Fig. 4B). Based on these findings, we concluded that several epitopes derived from the RV001 sequence are presented by HLA-DRB1*13:02, and possibly by DRB1*15:01 , and/or -DQB1*06:02, and/or -DPB1*04:01, three alleles co expressed in 80% of the HLA-DRB1*15:01 positive patients in the cohort.
The next step was to identify the presenting allele of the RV001 -derived H LA-class I epitope that was recognized by CD8 T cells from Patient 004 (HLA-A*02/-A*30, -B*18/-B*27). PBMCs from Patient 004 visit 15 were pre-stimulated with RV001 , and incubated with either the RV001 or the RV001 pre-loaded C1 R or C1 R-HLA-B*27 LCLs. A CD8 T cell response (CD107a, CD154, TNF, and IL-2 expression) was observed upon stimulation with the RV001 peptide or with the RV001 pre-loaded HLA-B*27 LCL, but not with the non- transfected LCL (Fig. 4C). These findings clearly show that a RV001 -derived sequence is presented to CD8 cells by the HLA-B*27:05 allele.
In summary, the RV001 peptide contains at least three different HLA-class II peptides promiscuously presented on various HLA-class II allelic products, as well as one HLA-class I class I peptide presented by the HLA-B*27:05 allele.
Vaccine safety
No adverse event led to discontinuation of treatment in any patient. Most frequent treatment-related events were fatigue and injection site reactions of grades 1 or 2. All patients experienced at least 1 TEAE of injection site reaction considered related to the RV001 vaccine. Four patients (18%) experienced at least 1 grade 2 TEAE of injection site reaction. One patient experienced a TEAE of fatigue (grade 1) that was probably related to the RV001 vaccine, and one patient a TEAE of hot flush (grade 1), also probably related to the RV001 vaccine. No treatment-related side effects of grade 3 or higher occurred. (Table 6).
Table 6: Treatment-related side effects
Figure imgf000049_0001
Figure imgf000050_0001
N=22 patients; */**clinical event, including laboratory test abnormality, with a reasonable time sequence to administration of the vaccine, but which can also be explained by concurrent disease or other drugs or chemicals (*) or which can unlikely be attributed to concurrent disease or other drugs or chemicals (**). *** indicates the total number of patients reporting at least one event.
Long term study
Approx.. 3 years after last vaccination with RV001 , the patients are analysed for sustainability of the specific RV001 immune response by analysing PBMC samples essentially as described above.
Furthermore, disease progression is evaluated clinically, and PSA levels are measured. Discussion
Recent cancer vaccine studies have perse been individualized, since they target mutations that mostly occur in individual patients. Other patient-specific approaches that are suitable for tumors with low level of mutational events focus on non-mutated, tumor-specific or tumor-associated antigens that are presented on various malignancies. In addition, targeting of antigens associated with metastases, like the RhoC protein, could be useful for treatment of metastatic cancer. The vaccination with RV001 against RhoC was safe for all patients over the complete treatment. Side effects related to the treatment were predominantly injection site reactions and fatigue (grade 1/2). These events are most likely related to the adjuvant and carrier Montanide ISA-51 and did not necessitate specific medical intervention.
Immunogenicity of the vaccine was assessed in n=21 patients. Most of these (94% of the tested patients with available cellular material at pre-vaccination) had developed a T cell response against the RhoC peptide after the 4th vaccination. High IFN-g ELISpot counts (>1000 spots/ 200.000 cells) were frequently observed, indicating a high frequency of in vivo RV001 -specific cells after vaccination and robust proliferation capacity. T cell responses were overall stable in the individual patients and detected almost one year after the last vaccination. This long-lasting functional immune response indicates the establishment of an immunological memory, which is essential for immunosurveillance of recurrent tumors and/or metastases. We found that anti-vaccine T cells belonged predominantly to the effector memory CD4 subset and were polyfunctional cells (> 80% expressing at least two of the activation markers tested) that produced TNF, IL-2, and to a lesser extent IFN-g. . In addition, the T-cells often expressed the cell-surface degranulation marker CD107a, indicating that at least a fraction of those were cytotoxic effectors. Moreover, ex vivo phenotyping strongly suggests that RV001 -specific cells did not shift towards an exhausted phenotype after multiple vaccine applications. We did not directly examine CD4+CD25+FoxP3+ T regulatory cells (Tregs), but the observation that activated Tregs express no or very little CD154, together with the production of IL-2, strongly suggest that essentially T helper cells were induced by the vaccine. Although the vaccine contained a single 20mer peptide, we could identify three HLA-class II 15mer RV001-derived epitopes. The high response rate in our patient cohort indicates that these, and possibly further as yet unidentified class-ll epitopes, can be presented promiscuously on several MHC-class II alleles (including DRB1*13:02). Hence, the RhoC vaccine could be applied broadly, independently of the patient's HLA allotype. In addition, a CD8 response restricted by HLA- B*27:05 could be observed in one patient. This response was detected four and seven months post-vaccination. The exact epitope is under characterization and further B27 patients will be assessed. Interestingly, RV001 -specific CD8 T cells in that patient were polyfunctional, with high level of TNF and/or CD107a, IL-2, CD154. CD154+ CD8 T cells (also called CD8 helper cells) have been shown to support their own expansion and differentiation and to activate dendritic cells to promote anti-tumor immunity. Although the RhoC 20mer contains a nested HLA-A*03 binding peptide, we did not observe any CD8 T cell response in the two patients carrying the HLA-A*03 allele.
CD4 T cells are now widely recognized as key players in anti-tumor immunity. Their role in dendritic cell activation (via CD154-CD40 interaction) and CD8 T activation and memory formation (via IL-2 production) are well described. CD4 T cells were also shown to kill HLA- class II positive tumors via granzyme/perforin release or Fas/Fas-L interaction (12,34), and there is also pre-clinical evidence that CD4 T cells can reject tumors better than CD8 T cells. HLA-class II expression has been detected on primary prostate tissues, and on localised prostate cancer (PCa) cell lines, or could be induced by IFN-g. Indirect tumor killing by CD4 T cells can also be mediated by IFN-g secretion, as well as by the recruitment of nitric oxide producing macrophages within the tumor. In addition, TNF and IL-2 were associated with tumor recognition in vitro. The presented data support that most of the RV001 -specific, polyfunctional CD4 T cells are perfectly equipped for an anti-tumor response as they express exactly these different markers.
Vaccine-based studies in mice and patients have started to unravel the contribution of CD4 T cells in tumor control. High rate of polyfunctional CD4 cells, together with CD8 T cells, were detected in melanoma patients vaccinated with a personalized, neoantigen-based SLP vaccine containing up to 20 long mutated peptides. Four out of six patients had no recurrence 25 months post-vaccination, while the two patients with tumor recurrence achieved tumor regression when treated with an anti-PD1 antibody. Case reports have also documented tumor regressions after adoptive transfer of anti-tumor CD4 cells. In mice models, there is also evidences that CD4 chimeric antigen receptor (CAR) T cells are more potent than CD8 CAR T cells, as they can kill tumors, and in addition, exhibit long-lasting effector function.
Although clinical response was not a primary endpoint of this phase I/ll study, patients were monitored for tumor progression and PSA serum levels. PSA doubling time is regarded as a surrogate parameter for localised prostate cancer (PCa) progression, and PSA doubling time can predict the risk of mortality in men with localized PCa. Three patients progressed biochemically during follow-up (Patients 006, 015, 018). Two of these patients (Patients 006 and 018) had BCR at study entry. Patient 006 had a PSA increase from 0.5 to 1.1 pg/L 29 months following study entry, and Patient 018 from 1.1 to 1.5 pg/ml 24 months following study entry. Patient 015, who received only seven vaccinations, and presented with a pT2c R+ Gleason score 7 (4+3) PCa, developed biochemical progression with a PSA doubling time of 1.2 years and final PSA level of 0.28 pg/L 26 months after end of the vaccination. When comparing the pre-study PSA doubling time to that on study, we observed an increase in PSA doubling time from 1.3 to 2.1 years for Patient 006, and from 1.95 to 3.8 years for Patient 018 indicating a slowdown of disease progression by RV001 vaccination. Patient 015 and 006 had some BCR 13 months post-vaccination (visit 17), however, the PSA for Patient 006 declined the last months on follow-up, and from a clinical perspective, the patient was considered as biochemically stable. No patient developed clinical signs of recurrence.
It is estimated that approximately 1 in 4 patients undergoing RP will, at some point in time, experience BCR. The risk of BCR varies according to preoperative PSA and histopathological findings - specimen Gleason score and margin status. The individual risk of BCR the first year following enrolment in this study ranged from 2 to 19%. Recurrence occurred only in one patient (015), who did not have BCR before enrolment experience recurrence. This suggest that the effect of the vaccine is not limited to metastatic cancers but also is suitable as immunotherapy against non-metastatic cancers and in particular against residual disease after initial treatment of patients having suffered from localised cancer.
In summary, vaccination against RhoC is a well-tolerated treatment option which induces a strong and long-lasting immune response in the large majority of patients. Vaccine-specific cells are polyfunctional and perfectly equipped for an anti-tumor response. The correlation between the induction of immune responses to RhoC upon vaccination and clinical outcome is examined in a recruiting double-blind, placebo controlled, phase II trial for PCa patients in biochemical relapse (NCT04114825). RhoC vaccination to impair tumor spreading might also synergize with many tumor vaccines (such as those targeting patient-individual, tumor antigens) or other therapies, and extended to other tumor entities.
Reference list
Britten CM, Janetzki S, Butterfield LH, Ferrari G, Gouttefangeas C, Huber C, et al. T Cell Assays and MIATA: The Essential Minimum for Maximum Impact. Immunity. 2012. page 1 2
Moodie Z, Price L, Gouttefangeas C, Mander A, Janetzki S, Lower M, et al. Response definition criteria for ELISPOT assays revisited. Cancer Immunol Immunother. 2010;59:1489-501.
Schuhmacher J, Heidu S, Balchen T, Schmeltz C, Sonne J, Schweiker J, Rammensee HG, Straten P T, Roder M A, Brasso K, Gouttefangeas C, Vaccination targeting RhoC induces long-lasting immune responses in prostate cancer patients: results from a phase I/ll clinical trial, Submitted for publication at Clinical Cancer research, 2020)
Suwa H, Ohshio G, Imamura T, Watanabe G, Arii S, Imamura M, et al. Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of the pancreas. Br J Cancer 1998;77(1): 147-52.
Thomas P, Pranatharthi A, Ross C and Srivastava S, RhoC: a fascinating journey from a cytoskeletal organizer to a Cancer stem cell therapeutic target Journal of Experimental & Clinical Cancer Research (2019) 38:328
Widenmeyer M, Griesemann H, Stevanovic S, Feyerabend S, Klein R, Attig S, et al. Promiscuous survivin peptide induces robust CD4 + T-cell responses in the majority of vaccinated cancer patients. Int J Cancer. 2012;131:140-9.

Claims

Claims
1. A RhoC peptide or a nucleic acid encoding a RhoC peptide for use in a method of treatment of residual disease in an individual in need thereof, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO:1 have been substituted; and wherein the individual has undergone initial treatment of a localized cancer, wherein the initial treatment is selected from the group consisting of radical surgery, radiation therapy, cryotherapy, laser therapy, topical therapy, tumour targeting chemotherapy and tumour targeting immune therapy.
2. The peptide or nucleic acid for use in the method according to claim 1, wherein the localized cancer is localized prostate cancer.
3. The peptide or nucleic acid for use in the method according to claim 2, wherein said individual has undergone radical prostatectomy for treatment of a localized prostate cancer.
4. The peptide or nucleic acid for use in the method according to any one of the preceding claims, wherein said individual suffers from biochemical recurrence.
5. The peptide or nucleic acid for use in the method according to any one of claims 2 to 4, wherein the individual has detectable serum PSA.
6. The peptide or nucleic acid for use in the method according to any one of the preceding claims, wherein the individual does not have distant or systemic metastasis as detected by CT scanning or PET-CT scanning.
7. The peptide or nucleic acid for use in the method according to anyone of the preceding claims, wherein said peptide is immunogenic as determined by said peptide having at least one of the following characteristics:
(vi) capable of inducing intracellular IFN-g in CD4+ T cells, and/or
(vii) capable of increasing intracellular TNF in CD4+ T cells, and/or (viii) capable of increasing intracellular IL2 in CD4+ T cells, and/or
(ix) capable of increasing intracellular CD154+ in CD4+ T cells, and/or
(x) capable of increasing intracellular CD107a in CD4+ T cells.
8. The peptide or nucleic acid for use in the method according to any one of claims 2 to 7, wherein the treatment elicits a clinical response in the individual, wherein the clinical response is characterised by an increased PSA serum level doubling time compared to a PSA serum level doubling time in said individual prior to treatment with the peptide.
9. The peptide or nucleic acid for use in the method according to any one of the preceding claims, wherein the RhoC peptide consists of a consecutive sequence of in the range of 8 to 60, for example in the range of 8 to 50, such as in the range of 8 to 40, for example in the range of 8 to 10 or in the range of 18 to 60 amino, such as in the range of 18 to 50 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three, for example at the most two, such as at the most one amino acid(s) of SEQ ID NO:1 have been substituted.
10. The peptide or nucleic acid for use in the method according to any one of the preceding claims, wherein the RhoC peptide consists of in the range of 8 to 60, such as in the range of 8 to 50, for example in the range of 18 to 25 consecutive amino acids from RhoC of SEQ ID NO: 1 , wherein at the most two amino acids have been substituted, wherein the peptide fragment contains at least one of amino acid residues I43, Q123, R140, S141, S152, L157, E165, G178, V181,
K183, N184, R186, R187, R188, P191 or 1192 of RhoC of SEQ ID NO:1.
11. The peptide or nucleic acid for use in the method according to any one of the preceding claims, wherein the RhoC peptide comprises of consists of the sequence selected from the group consisting of RXGLQVRKNK, ATRAGLQVRKNKRRRGCPIL, ATRAGLQVRKNKRRR, AGLQVRKNKRRRGCP and LQVRKNKRRRGCPIL, wherein X is selected from the group consisting of alanine and leucine and wherein said peptide fragment is at the most 60 amino acids in length.
12. The peptide or nucleic acid for use in the method according to any one of the preceding claims, wherein the peptide and/or nucleic acid is contained in a vaccine composition comprising a. said peptide and/or nucleic acid; and b. an adjuvant.
13. The peptide or nucleic acid for use in the method according to claim 12, wherein the adjuvant is a water in oil emulsion.
14. The peptide or nucleic acid for use in the method according to any one of claims 12 to 13, wherein the vaccine further comprises a carrier.
15. The peptide or nucleic acid for use in the method according to any one of the preceding claims, wherein said method comprises administering said peptide or nucleic acid to said individual at least 9 times, preferably at least 10 times.
16. A kit-of-parts comprising a. a RhoC peptide or a nucleic acid encoding a RhoC peptide; and b. a further anti-cancer agent for use in a method of treatment of residual disease in an individual in need thereof, wherein the RhoC peptide comprises or consists of a consecutive sequence of at least 8 amino acids of RhoC of SEQ ID NO: 1 , wherein at the most three amino acids of SEQ ID NO:1 have been substituted; and wherein the individual has undergone initial treatment of a localized cancer, wherein the initial treatment is selected from the group consisting of radical surgery, radiation therapy, cryotherapy, laser therapy, topical therapy, tumour targeting chemotherapy and tumour targeting immune therapy.
PCT/EP2021/069697 2020-07-21 2021-07-15 Treatment of residual disease WO2022017895A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP20186943 2020-07-21
EP20186943.5 2020-07-21

Publications (1)

Publication Number Publication Date
WO2022017895A1 true WO2022017895A1 (en) 2022-01-27

Family

ID=71786732

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2021/069697 WO2022017895A1 (en) 2020-07-21 2021-07-15 Treatment of residual disease

Country Status (1)

Country Link
WO (1) WO2022017895A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997028816A1 (en) 1996-02-09 1997-08-14 Ludwig Institute For Cancer Research Compositions containing immunogenic molecules and granulocyte-macrophage colony stimulating factor, as an adjuvant
WO2009076966A2 (en) 2007-12-19 2009-06-25 Rhovac Aps Rhoc-based immunotherapy

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997028816A1 (en) 1996-02-09 1997-08-14 Ludwig Institute For Cancer Research Compositions containing immunogenic molecules and granulocyte-macrophage colony stimulating factor, as an adjuvant
WO2009076966A2 (en) 2007-12-19 2009-06-25 Rhovac Aps Rhoc-based immunotherapy

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
"Remington: The Science and Practice of Pharmacy", 2004, LIPPINCOTT WILLIAMS & WILKINS
ANDERS MANSSON: "Positive Results from RhoVac's Clinical Phase I/II Study published in Journal of ImmunoTherapy of Cancer", PRESS RELEASE, 1 November 2020 (2020-11-01), XP055754471, Retrieved from the Internet <URL:https://mb.cision.com/Main/13747/3236680/1334767.pdf> [retrieved on 20201126] *
ANDREAS MORITZ ET AL: "High-throughput kinetic screenings of T cell engaging receptors with peptide-receptive HLAA*02:01 molecules", 17TH CIMT ANNUAL MEETING, 23 May 2019 (2019-05-23), pages 22, XP055717784 *
BRITTEN CMJANETZKI SBUTTERFIELD LHFERRARI GGOUTTEFANGEAS CHUBER C ET AL.: "T Cell Assays and MIATA: The Essential Minimum for Maximum Impact", IMMUNITY, 2012, pages 1 - 2
EUROPEAN COMISSSION: "Imminent anti-metastatic vaccine for prostate cancer | Result In Brief | CORDIS | European Commission", 4 March 2019 (2019-03-04), XP055754490, Retrieved from the Internet <URL:https://cordis.europa.eu/article/id/252266-imminent-antimetastatic-vaccine-for-prostate-cancer> [retrieved on 20201126] *
JULIANE SCHUHMACHER ET AL: "Vaccination against RhoC induces long- lasting immune responses in patients with prostate cancer: results from a phase I/II clinical trial", J IMMUNOTHER CANCER, vol. 8, 12 November 2020 (2020-11-12), pages 1157, XP055754684, DOI: 10.1136/jitc-2020-001157 *
MARK C. MARKOWSKI ET AL: "PSA Doubling Time and Absolute PSA Predict Metastasis-free Survival in Men With Biochemically Recurrent Prostate Cancer After Radical Prostatectomy", CLINICAL GENITOURINARY CANCER, vol. 17, no. 6, 1 December 2019 (2019-12-01), US, pages 470 - 475.e1, XP055754679, ISSN: 1558-7673, DOI: 10.1016/j.clgc.2019.08.002 *
MOODIE ZPRICE LGOUTTEFANGEAS CMANDER AJANETZKI SLOWER M ET AL.: "Response definition criteria for ELISPOT assays revisited", CANCER IMMUNOL IMMUNOTHER, vol. 59, 2010, pages 1489 - 501, XP019842218
RHOVAC: "RhoVac receives approval in Sweden to start clinical phase IIb trial", 1 January 2020 (2020-01-01), XP055754475, Retrieved from the Internet <URL:https://mb.cision.com/Main/13747/3139266/1267945.pdf> *
SCHUHMACHER JHEIDU SBALCHEN TSCHMELTZ CSONNE JSCHWEIKER JRAMMENSEE HGSTRATEN P TRODER M ABRASSO K: "Vaccination targeting RhoC induces long-lasting immune responses in prostate cancer patients: results from a phase / clinical trial", CLINICAL CANCER RESEARCH, 2020
SUWA HOHSHIO GIMAMURA TWATANABE GARII SIMAMURA M ET AL.: "Overexpression of the rhoC gene correlates with progression of ductal adenocarcinoma of the pancreas", BR J CANCER., vol. 77, no. 1, 1998, pages 147 - 52
THOMAS PPRANATHARTHI AROSS CSRIVASTAVA S: "RhoC: a fascinating journey from a cytoskeletal organizer to a Cancer stem cell therapeutic target", JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH, vol. 38, 2019, pages 328
WIDENMEYER MGRIESEMANN HSTEVANOVIC SFEYERABEND SKLEIN RATTIG S ET AL.: "Promiscuous survivin peptide induces robust CD4 + T-cell responses in the majority of vaccinated cancer patients", INT J CANCER, vol. 131, 2012, pages 140 - 9, XP003031907, DOI: 10.1002/ijc.26365

Similar Documents

Publication Publication Date Title
JP5502823B2 (en) Proteins belonging to the BCL-2 family and fragments thereof, and their use in cancer patients
EP2547691B1 (en) Composition of tumor-associated peptides and related anti-cancer vaccine for the treatment of gastric cancer and other cancers
Curigliano et al. Breast cancer vaccines: a clinical reality or fairy tale?
EP1879612B1 (en) Use of native peptides and their optimized derivatives for vaccination
Schuhmacher et al. Vaccination against RhoC induces long-lasting immune responses in patients with prostate cancer: results from a phase I/II clinical trial
US6699483B1 (en) Cancer treatments
US20160199469A1 (en) Oncology vaccine
CA3013930A1 (en) Cancer therapy
US20100255020A1 (en) Method for inducing cytotoxic t-cells, cytotoxic t-cell inducers, and pharmaceutical compositions and vaccines employing them
Parizadeh et al. Personalized peptide-based vaccination for treatment of colorectal cancer: rational and progress
US5882654A (en) Polyvalent melanoma vaccine
EP1782827A1 (en) Peptide vaccine for cancer therapy
NO327044B1 (en) Allogeneic immunotherapeutic agent, immunogenic composition, vaccine composition and uses thereof, in the treatment of prostate cancer
WO2022017895A1 (en) Treatment of residual disease
Kouiavskaia et al. Vaccination with agonist peptide PSA: 154-163 (155L) derived from prostate specific antigen induced CD8 T-cell response to the native peptide PSA: 154-163 but failed to induce the reactivity against tumor targets expressing PSA: a phase 2 study in patients with recurrent prostate cancer
US5840317A (en) Composition comprising tumor cell lines containing GD2 ganglioside GM2 ganglioside, M-TAA, and either M-urinary antigen or M-fetal antigen
Schuhmacher Anti-cancer vaccination–Immunomonitoring of a clinical phase I/II study in prostate cancer patients vaccinated with a RhoC-derived synthetic peptide
US20070232558A1 (en) Methods and compositions for treating prostate cancer using dna vaccines directed to cancer testis antigen
Pandha et al. NECTAR
Yamshchikov et al. Sentinel Node Assays
Gendler et al. Immunotherapeutic Strategies in Breast Cancer: Preclinical and Clinical Trials
Lubkov et al. Vaccine-induced memory CD8+ T cells provide clinical benefit in HER2 expressing breast cancer: a mouse to human translational study Erika J. Crosby, William Gwin 2, 3, Kimberly Blackwell 2, Paul K. Marcom 2, Serena Chang 4, Holden T Maecker 4, Gloria Broadwater 5, Terry Hyslop 5, Sungjin Kim 6, Andre Rogatko 6
Marchand " Immunization of melanoma patients with tumor antigens recognized by T lymphocytes, using peptides and a recombinant protein encoded by MAGE-A3

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21745756

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 07/06/2023)

122 Ep: pct application non-entry in european phase

Ref document number: 21745756

Country of ref document: EP

Kind code of ref document: A1