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WO2023211279A1 - Combinaisons d'adjuvants pour vaccins à base de néopeptides - Google Patents

Combinaisons d'adjuvants pour vaccins à base de néopeptides Download PDF

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WO2023211279A1
WO2023211279A1 PCT/NL2023/050230 NL2023050230W WO2023211279A1 WO 2023211279 A1 WO2023211279 A1 WO 2023211279A1 NL 2023050230 W NL2023050230 W NL 2023050230W WO 2023211279 A1 WO2023211279 A1 WO 2023211279A1
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cpg
cancer
tumor
cells
neoantigen
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PCT/NL2023/050230
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Peter D. Katsikis
Ken J. Ishii
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Erasmus University Medical Center Rotterdam
The University Of Tokyo
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    • 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
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention is in the field of cancer treatment, in particular to cancer vaccines, and methods for preparing same.
  • the present invention provides as an anti/cancer vaccine a combination of a neoantigen and an adjuvant.
  • the present invention also provides novel adjuvant combinations.
  • the invention also provides methods of treating disease, including cancer using the vaccine and adjuvant combinations of the invention.
  • peptides that target mutations in the tumor are poor inducers of cytotoxic CD8+ T cell (CTL) responses. This is because externally acquired peptides or proteins need to be cross-presented by professional antigen presenting cells (APC). This has led to the usage in cancer by some investigators of peptide vaccines that employ membranepenetrating peptides or nanoparticles (Ma M, et al. 2020 Scand J Immunol. 91(6):el2875).
  • Nanoparticle delivery of peptide and proteins can increase their immunogenicity and this is believed to be because of enhanced crosspresentation of peptides or proteins (San Roman B, et al. 2014 J Pharm Pharm Sci. 17(4):541-53; Mueller M, et al. 2012 J Control Release
  • the CTL immunity enhancing effect of the combination of K3 CpG and STING agonists is somewhat surprising as STING agonists can inhibit the ability of CpG TLR9 agonists like K3 CpG to induce type I IFN (Deb P, et al. 2020 J Immunol. 205(l):223-236). Such type I IFN production is considered a critical signal 3 for the generation of efficient CTL immunity (Agarwal P, et al. 2009 J Immunol. 183(3): 1695-704; Curtsinger JM, Mescher MF, 2010 Curr Opin Immunol. 22(3):333-40).
  • An adjuvant is an immunopotentiator that is added to enhance the effect of a vaccine.
  • the action mechanism of adjuvants has been gradually elucidated.
  • various immunoregulatory properties of adjuvants are expected to be applied in the prevention or therapy of not only cancer and infections, but also allergies, cancer, and autoimmune diseases.
  • Th2 adjuvants type II adjuvants
  • Thl adjuvants type I adjuvants
  • CpG oligonucleotides While many candidate substances for Thl adjuvants have been reported, CpG oligonucleotides (CpG ODN) are considered the most effective. CpG ODN is demonstrated to be effective as a vaccine adjuvant for cancer or infection.
  • the present inventors have now found a therapeutic anti-cancer vaccine strategy comprising the use of a neopeptide as cancer antigen in a vaccine composition adjuvated with a combination of a CpG oligonucleotide and a STING agonist.
  • the inventors herein provide an adjuvant combination of a CpG oligonucleotide and a STING agonist, wherein the CpG oligonucleotide is K3 CpG and wherein the STING agonist is c-di-AMP.
  • This adjuvant combination was found to induce a strong CTL immunity against neopeptides (free, in solution) without the need to include the peptides in particles (such as nanoparticles, liposomes or virus-like particles), conjugate the peptides to TLR agonists or carrier proteins, or multimerize the peptides (He X, et al. 2021ACS Nano. 15(3):4357-4371; Malonis RJ, et al. 2020 Chem Rev. 120(6):3210-3229; Kuai R, et al. 2017 Nat Mater. 16(4):489-496; Zom GG, et al. 2012 Adv Immunol. 114:177-201; Daftarian P, et al.
  • mice against a further three 20-mer neopeptide antigens designed from mouse tumors B16F10 melanoma, AE17 mesothelioma and 4662 pancreatic adenocarcinoma.
  • Th2 immunity demonstrated by absence of IL-5 producing T cells
  • mice with an established B16F 10 melanoma that express the OVA(257-264) CTL epitope
  • the administration of the OVA(252-27i) antigenbased vaccine composition, comprising the adjuvant combination of the present invention showed to delay tumor growth and lead to increased survival.
  • neopeptides now enable to provision of a personalized cancer vaccine, wherein the neopeptide is a patient-specific tumor peptide.
  • immune checkpoint inhibitors e.g. PD-1 inhibitors
  • the vaccine composition increases the efficacy of checkpoint blockade by anti-PD-1 in B16F10 tumor carrying mice, evidenced by the observation of further delay in tumor growth and increased survival. Therefore the present invention provides the use of the vaccine composition described herein in combination with immune checkpoint inhibitors (ICI) in therapeutic treatment of cancer.
  • ICI immune checkpoint inhibitors
  • the present invention provides an anti-cancer vaccine composition
  • a cancer neoantigen comprising a CTL epitope
  • an adjuvant combination of a CpG oligonucleotide and a STING agonist optionally further comprising a pharmaceutically acceptable vehicle, preferably a non-p articulate aqueous vehicle.
  • the cancer is a (solid) tumor
  • the cancer neoantigen is a tumor neoantigen
  • the CpG oligonucleotide is K3 CpG
  • the STING agonist is c-di-AMP
  • the present invention provides an anti-cancer vaccine composition
  • a cancer neoantigen comprising a CTL epitope
  • an adjuvant combination of a CpG oligonucleotide and a STING agonist as described above, for use in the treatment of cancer.
  • the present invention provides a therapeutic combination, comprising as (sole) active components:
  • an anti-cancer vaccine composition comprising a cancer neoantigen comprising a CTL epitope, and an adjuvant combination of a CpG oligonucleotide and a STING agonist of the present invention
  • the ICI is a cytotoxic T lymphocyte associated protein 4 (CTLA-4) inhibitor, a programmed cell death protein 1 (PD-1) inhibitor, or a PD-L1 inhibitor, more preferably the ICI is selected from pembrolizumab, nivolumab, ipilimumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, and avelumab.
  • CTL-4 cytotoxic T lymphocyte associated protein 4
  • PD-1 programmed cell death protein 1
  • PD-L1 inhibitor more preferably the ICI is selected from pembrolizumab, nivolumab, ipilimumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, and avelumab.
  • the anti-cancer vaccine composition and the immune checkpoint inhibitor are for simultaneous, sequential or separate administration.
  • the cancer is a (solid) tumor
  • the cancer neoantigen is a tumor neoantigen
  • the CpG oligonucleotide is K3 CpG
  • the STING agonist is c-di-AMP
  • the present invention provides a method of treating cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of an anti-cancer vaccine composition of the present invention, or a therapeutically effective amount of a therapeutic combination of the present invention.
  • the cancer is a (solid) tumor
  • the cancer neoantigen is a tumor neoantigen
  • the CpG oligonucleotide is K3 CpG
  • the STING agonist is c-di-AMP
  • the present invention provides an adjuvant combination comprising K3 CpG and c-di-AMP.
  • the present invention provides the anti-cancer vaccine composition of the present invention or the therapeutic combination of the present invention, or the method of treating cancer of the present invention, wherein the neoantigen is an antigenic peptide present in a subject's tumor cell or tissue but not in the subject's corresponding normal cell or tissue, preferably wherein the neoantigen is a peptide having a length of 10-30 consecutive amino acids.
  • FIG. 1 shows the results of Example 1, wherein a potent CD8+ T cell response is induced by K3 CpG and c-di-AMP agonist combination in blood/splenocyte/lymph node cells by A) flow cytometry and staining with OVA(257-264) peptide-loaded MHC-class I tetramers and B) by intracellular cytokine stains and flow cytometry after in vitro stimulation for 6h with OVA(257-264) peptide. IFNy, TNFa, IL-2 cytokines are shown.
  • CD 107a a marker of cellular degranulation and surrogate marker for cytotoxicity, is also shown.
  • Figure 2 shows that the K3 CpG and c-di-AMP adjuvant combination induce potent in vivo T cells responses when used with tumor neoantigen peptide immunization as described in Example 2.
  • Neopeptides (20 a. a. in length) designed from mouse tumor neoantigens (non synonymus mutations, frame shifts or fusions etc.) were combined with K3 CpG and c- di-AMP adjuvant combination. Mice were immunized 3 times with pools of 10 neoantigen peptides each from 3 different mouse tumors: B16F10 melanoma, AE17 mesothelioma and 4662 pancreatic adenocarcinoma. The T cells response against these tumors are shown in panels A, B and C, respectively.
  • Figure 3 shows the results of Example 3.
  • SIINFEKL subcutaneous melanoma B16F10 tumor that expresses the OVA(257-264) CTL epitope
  • Figure 4 shows the results of Example 4, wherein mice carrying an established subcutaneous melanoma B16F 10 tumor that expresses the OVA(257-264) CTL epitope (SIINFEKL) were vaccinated with 20 amino acid OVA(252-271) peptide plus K3 CpG and c-di-AMP adjuvant combination and anti-PD-1. Controls received no anti-PD-1.
  • the combined vaccination and anti-PD-1 treatment delayed tumor growth ( Figure 4, panel B) and increased survival of mice ( Figure 4, panel A).
  • Figure 5. Potent CD8+ T cell response against 20-mer peptide is induced by K3/c-di-AMP combination.
  • mice were immunized once a week for three weeks in the left flank with 100 pl of each vaccine containing 20-mer OVA(252-271) SLP and different adjuvants individually or combined, Addavax or saline as controls.
  • the frequencies of IFN-yand TNF-aproducing CD8+ T cells were analyzed by intracellular staining after restimulation with SIINFEKL peptide (B). Differences between the groups were analyzed by Kurskal- Wallis test followed by Dunn’s post-test after analyzing the data distribution; *p ⁇ 0.05, **p ⁇ 0.01, *** p ⁇ 0.001 and **** p ⁇ 0.0001.
  • TLR9 and STING agonist combination activates DC.
  • BMDC were seeded at 2xl0 5 cells/ml and stimulated ON with 1 jiM of adjuvants, K3 CpG and/or c-di-AMP.
  • LPS (1 pg/ml) was used as a positive control.
  • the expression of CD40, CD80, CD86, MHC-I, and MHC-II was analyzed by flow cytometry. Mean fluorescence intensity (MFI) for each molecule is shown, each dot represents the mean of duplicates in one experiment (six independent experiments) and the bars represent the mean and range.
  • MFI Mean fluorescence intensity
  • Vaccines containing low affinity OVA peptides and K3/c- di-AMP combination induce robust antigen-specific CD8+ T cells.
  • C57BL/6 mice were immunized once a week for three weeks in the left flank with 100 pl of each vaccine containing low affinity 20-mer OVA(252-271) SLP (El or R4) and K3/c-di-AMP or Addavax.
  • mice were harvested and splenocytes were re-stimulated with El or R4 8- mer OVA(257-264) peptides for 6h and cytokine expression was analyzed by intracellular staining.
  • the frequencies of IFN-yand TNF-a expressing CD8+ T cells are shown. Symbols represent individual mice (data from two independent experiments) and the bars represent the mean and range. Differences between the groups were analyzed by Mann-Whitney test after analyzing the data distribution; *p ⁇ 0.05 and **p ⁇ 0.01.
  • FIG. 8 Potent neoantigen-specific T cell responses are induced by vaccines containing neopeptides with K3/c-di-AMP adjuvant.
  • Vaccine containing 20-mer peptide pools of neoantigens from two different tumor models, B16-F10 and AE-17 were used to vaccinate C57BL/6 mice.
  • mice were harvested and the number of IFN-y(left panels) and IL-5 (right panels) producing cells were measured by ELISpot after 24h restimulation of splenocytes with same pool of peptides used for immunization.
  • the number of spot forming cells (SFC) in response to the different pools are shown for B16-F10 (A) and AE-17 (B) neoantigens.
  • SFC spot forming cells
  • FIG. 9 In vitro 20-mer OVA peptide cross-presentation and T cell activation.
  • DC2.4 cells were cultured ON with 20-mer OVA(252-271) SLP (green) or 8-mer OVA(257-264) peptide (blue) or media alone (red), and the loading of SIINFEKL peptide on MHC-I molecules was analyzed by flow cytometry using an antibody that recognizes the H-2Kb/SIINFEKL complex. Data shown as histograms (A).
  • K3/c-di-AMP adjuvant is more potent than poly(I:C) at inducing neoantigen-specific T cell immunity.
  • C57BL/6 mice were immunized 3 times once a week.
  • Vaccines contained a 20-mer neoantigen peptide pool from B16-F10 tumor, in combination with K3/c-di-AMP or poly(I:C) as adjuvants.
  • the number of IFN-y and IL-5 producing T cells were determined by ELISpot after 24h restimulation of splenocytes with the 20-mer neoantigen peptide pool (A).
  • Vaccine containing 20-mer OVA(252-271) SLP and K3/c-di-AMP reduces tumor growth and improves survival of mice with established B16-F10-OVA tumors.
  • K3/c-di-AMP without antigen cannot protect from established B16-F10-OVA tumors.
  • FIG. 13 Vaccines containing 20-mer OVA(252-271) SLP combined with K3/c-di-AMP synergize with anti-PD-1 treatment in mice with established tumors.
  • C57BL/6 mice were injected subcutaneously with 0.5x106 B16-F10-OVA cells and 12 days later mice were vaccinated with vaccines containing 20-mer OVA(252-271) SLP and K3/c-di-AMP or Addavax. Mice were vaccinated once a week for three weeks. Additionally, anti-PD-1 treatment or isotype control was given intraperitoneal twice a week for three weeks. Antibody treatment was started after day 12 posttumor injection (A).
  • FIG. 14 Vaccination with STING agonist c-di-AMP alone induces higher level of IL-5 producing T cells in a non-antigen-specific manner.
  • C57BL/6 mice were immunized once a week for three weeks in the left flank with 100 pl of each vaccine containing different 20-mer peptide pools of neoantigens from two different tumor models, B16-F10 and AE-17, in combination with K3/c-di-AMP, c-di-AMP alone or Addavax.
  • SFC spot forming cells
  • CpG oligonucleotide As used herein, “CpG oligonucleotide”, “CpG oligodeoxynucleotide”, “CpG ODN”, or “simply “CpG” are interchangeably used, and refer to a polynucleotide, preferably an oligonucleotide, comprising at least one non-methylated CG dinucleotide sequence.
  • An oligonucleotide comprising at least one CpG motif may comprise multiple CpG motifs.
  • CpG motif refers to a nonmethylated dinucleotide moiety of an oligonucleotide, comprising a cytosine nucleotide and a subsequent guanosine nucleotide. 5-methylcytosine may also be used instead of cytosine.
  • a CpG oligonucleotide is a short (about 20 base pairs) synthetic single-stranded DNA fragment comprising an immunostimulatory CpG motif.
  • a CpG oligonucleotide is a potent agonist of a toll-like receptor 9 (TLR9), which activates dendritic cells (DCs) and B cells to produce type I interferons (IFNs) and inflammatory cytokines (Hemmi, H., et al. Nature 408, 740-745 (2000); Krieg, A. M. Nature reviews.
  • CTL cytotoxic T-lymphocyte
  • a CpG oligodeoxynucleotide is a synthetic single stranded DNA comprising a non-methylated CpG motif with a immunostimulatory feature due to similarity with a microbial genome, and is recognized by TLR9 in a specific type of natural immune cell [Hartmann et al., J. Immunol. (2000) 164: 944-953; Wagner et al., Trends Immunol. (2004) 25: 1-6], In ligand binding, TLR9 signals through an adapter molecule myD88 to induce the production of IRF7 dependent type I IFN and NF-KB dependent cytokines [Krieg et al., Nat. Rev. Drug Discov.
  • CpG ODN induces a Thl response due to the type of cytokine induced by CpG ODN in ARC in vivo [Krieg et al., Nat. Rev. Drug Discov. (2006) 5: 471-84].
  • type D CpG ODN strongly induces both type I and type II IFN, but cannot induce B cell activation [Krieg et al., Nat. Rev. Drug Discov. (2006) 5: 471-84; Klinman et al., Nat. Rev. Immunol.
  • Type K CpG ODN (K3 CpG) strongly induces B cell activation to induce IL-6 and antibody production, but they only weakly induce type I and type II IFN.
  • type D CpG ODN forms an aggregation, such that only type K CpG can be used for clinical applications [Krieg et al., Nat. Rev. Drug Discov. (2006) 5: 471-84; Klinman et al., Nat. Rev. Immunol. (2004) 4: 1-10],
  • Pathogen derived agents such as LPS or non-methylated CpG DNA (CpG) (CpG ODN) stimulate natural immune cells that produce cytokines such as type I or type II IFN and IL- 12.
  • IL- 12 acts on naive CD4+ T cells to derive the generation of Thl and the production of IFNy [Seder et al., Proc. Natl. Acad. Sci. U.S.A. (1993) 90: 10188-92; Hsieh et al., Science.
  • Thl cells are the main actors in the induction of type 1 immunity, which are distinguished by high phagocytic activity [Spellberg et al., Clin. Infect. Dis. (2001) 90509: 76-102; Mantovani et al., Curr. Opin. Immunol. (2010) 22: 231-237], Furthermore, Thl cells play an important role in the generation of antitumor immunity and are useful in CTL effector functions and suitable activation including IFNy production [Hung et al., J. Exp. Med. (1998) 188: 2357-68; Vesely et al., Annu. Rev. Immunol.
  • agents, CTLs, and NK cells that can induce a strong Thl response may play an important role in the development of a vaccine adjuvant or immunotherapeutic agent that is effective against intracellular pathogens or cancer. Therefore, they are in immediate demand.
  • type D/A induces the production of type I interferon mainly from plasmacytoid dendritic cells (called “plasmacytoid DC” or “pDC”)
  • type K/B induces B cell growth and the production of IgM, IL-6 or the like.
  • Type D/A CpG-DNA strongly induces IFN-a production, but exhibits low pDC maturation inducing activity and no direct immunostimulatory activity to B cells.
  • Type K/B exhibits immunostimulatory activity to B cells, strongly promotes maturation of pDCs, and has high IL- 12 inducing capability, but has low IFN-a inducing capability.
  • type C sequences having repetitive sequences of TCG that are completely thiolated IFN-a production by pDCs or polyclonal B cell activation is induced.
  • Type D/A CpG ODN (also called type A, type D or the like and denoted as CpG-A ODN) is an oligonucleotide characterized by a phoshothioate (PS) bond at the 5' and 3' terminuses and by a poly G motif with a palindrom (palindromic structure) CpG containing sequence of phosphodiester (PO) in the middle. Cell uptake is facilitated due to the presence of phosphorothioate (PS) at the 5' and 3' terminuses.
  • CpG type D/A produces a large quantity of interferon a (IFN-a) in pDCs (different feature from CpG type K/B).
  • IFN-a interferon a
  • ODN Three other types of ODN consist of a PS backbone.
  • Type K/B CpG ODN is also called CpG-type B or CpG-type K. All type K/B CpG ODN with one or more CpG motifs without a poly G motif have a phosphorothioate (PS) backbone. Typically, type K/B CpG ODN contains multiple CpG motifs with a non-palindromic structure. Type K/B CpG has weak IFN-a inducing activity (produces nearly none), but is a very potent Thl adjuvant and a potent B cell response stimulating agent which produces IL-6 and activates and matures pDCs (Verthelyi, D., et al.
  • Type K/B CpGODN has a function of promoting the survival, activating, and maturing both monocyte derived dendritic cells and pDCs.
  • type C and type P CpG ODN comprise one and two palindromic structure CpG sequences, respectively. Both can activate B cells, like type K CpG ODN, and activate pDCs, like type D CpG ODN. Meanwhile, type C CpG ODN more weakly induces IFN-a production relative to type P CpG ODN (Hartmann, G., et al. European journal of immunology 33, 1633-1641 (2003); Marshall, J. D., et al. Journal of leukocyte biology 73, 781-792 (2003).; and Samulowitz, U., et al. Oligonucleotides 20, 93-101 (2010)).
  • Type D/K and type P CpG ODN are shown to form a higher order structure i.e., Hoogsteen base pair forming a four parallel strand structure called G-tetrads and Watson-Crick base pair between a cis palindromic structure site and a trans palindromic structure site, respectively, which are required for potent IFN-a production by pDCs (Samulowitz, U., et al.
  • type C CpG ODN has a complete phosphorothioate (PS) backbone without a poly G motif, but comprises the type A palindromic sequence of CpG in combination with a stimulatory CpG motif. It is reported from an in vivo study that type C CpG ODN is a very potent Thl adjuvant.
  • PS phosphorothioate
  • Type K CpG ODN used in a preferred embodiment in the present invention has a length of 10 nucleotides or longer and comprises the nucleotide sequence set forth in the following formula: 5'-NiN2N 3 T-CpG-WN4N 5 N 6 -3' [Formula 1] wherein the middle CpG motif (described as CpG) is not methylated, W is A or T, and N 1, N2, N3, N4, N5, and N6 may be any nucleotide.
  • type K CpG ODN of the invention has a length of 10 nucleotides or longer and comprises the nucleotide sequence of the above-described formula.
  • the CpG motif of 4 bases in the middle (TCpGW) only needs to be included in the 10 nucleotides.
  • the motif does not necessarily need to be positioned between N3 and N4 in the above-described formula.
  • the N 1, N2, N3, N4, N5, and N6 may be any nucleotide in the above-described formula.
  • Combinations of at least one (preferably one) of N1 and N2, N2 and N3, N3 and N4, N4 and N5, and N5 and N6 may be a two base CpG motif.
  • any two contiguous bases in the middle 4 bases (4th to 7th bases) in the abovedescribed formula may be a CpG motif and the other two bases may be any nucleotide.
  • a part of or the entire phosphodiester bond of an oligodeoxynucleotide may be substituted with a phosphorothioate bond.
  • the entire phosphodiester bond of an oligodeoxynucleotide is substituted with a phosphorothioate bond.
  • Type K CpG ODN suitably used in the present invention contains a non-palindromic structure comprising one or more CpG motifs.
  • Type K CpG ODN more suitably used in the present invention consists of a non- palindromic structure comprising 1 or more CpG motifs.
  • Type K CpG ODN contained in the oligodeoxynucleotide of the invention is preferably humanized. “Humanized” refers to having agonistic activity against human TLR9. Thus, the oligodeoxynucleotide of the invention comprising humanized type K CpG ODN has immunostimulatory activity unique to type K CpG ODN against humans (e.g., activity to activate human B cells to produce IL-6).
  • Humanized type K CpG ODN is generally characterized by a four base CpG motif consisting of TCGA or TCGT. In many cases, a single humanized type K CpG ODN comprises 2 or 3 of the four base CpG motifs.
  • type K CpG ODN contained in the oligodeoxynucleotide of the invention comprises at least 1, more preferably 2 or more, and still more preferably 2 or 3 four base CpG motifs consisting of TCGA or TCGT. When such type K CpG ODN has 2 or 3 four base CpG motifs, these four base CpG motifs may be the same or different. However, this is not particularly limited, as long as there is agonist activity against human TLR9.
  • One preferred type K CpG ODN included in the aspects of the invention comprises the nucleotide sequence set forth in the sequence (atcgactctc gagcgttctc (SEQ ID NO: 1)).
  • CpG ODNs include CpG 1826 (5'- tccatgacgttcctgacgtt-3' (SEQ ID NO: 2)), D35 CpG (5'-ggtgcatcgatgcagggggg- 3' (SEQ ID NO: 3)), and the like.
  • the CpG ODN consists of SEQ ID NO:1 and is herein referred to as K3 CpG.
  • the length of type K CpG ODN is not particularly limited, as long as the oligodeoxynucleotide of the invention activates immunostimulatory activity (e.g., activity to activate B cells (preferably human B cells) to produce IL-6) or has anticancer activity, but the length is preferably 100 nucleotides long or less (e.g., 10 to 75 nucleotides long).
  • the length of type K CpG ODN is more preferably 50 nucleotides long or less (e.g., 10 to 40 nucleotides long).
  • the length of type K CpG ODN is still more preferably 30 nucleotides long or less (e.g., 10 to 25 nucleotides long).
  • the length of type K CpG ODN is most preferably 12 to 25 nucleotides long.
  • STING ((adapter molecule) stimulator of interferon genes) identified as a membrane protein localized in the endoplasmic reticulum plays an important role in the biological defense mechanism against infections of various RNA viruses and DNA viruses. It is also reported that STING plays an important role in inducing natural immune responses against DNA components derived from microbes and viruses, but the molecular mechanism thereof had not been elucidated. STING can form a complex with not only genomic DNA derived from viruses, but also synthetic double stranded DNA of 45 to 90 base pairs called ISD and self-DNA components derived from apoptotic cells. Analysis of DNA interaction region in vitro demonstrated that the C-terminus side region of STING is important.
  • STING Recognition of various DNA components by STING was demonstrated to induce dynamic local change to regions surrounding the nuclear membrane of STING and to induce interferon production via activation of TBK1. It is also suggested that STING is possibly involved in the regulation of chronic inflammatory responses via recognition of not only allo-DNA component from a microorganism, but also auto-DNA component.
  • STING ligand and “STING agonist” are interchangeably used, which is a ligand (agonist) of “STING” ((adapter molecule) stimulator of interferon genes)) inducing type I IFN production and NF-KB mediated cytokine production.
  • STING agonists are considered to be membrane proteins localized in the endoplasmic reticulum.
  • STING agonists in addition to cGAMP, cyclic dinucleotides of microbial origin, c-di- AMP and c-di-GMP, are ligands of adapter molecule stimulators of IFN genes (STING), which signal through the TBK1-IRF3 axis to induce type I IFN production and NF-KB mediated cytokine production [Burdette et al., Nature. (2011) 478: 515-8; Mcwhirter et al., J. Exp. Med. (2009) 206: 1899- 1911], Recent studies report that these cyclic dinucleotides function as a potent vaccine adjuvant due to their ability to enhance antigen-specific T cells and humoral immune responses.
  • STING agonist DMXAA
  • STING-IRF3 STING-IRF3 mediated type I IFN production
  • type 2 immune responses can inhibit a type 1 immune response
  • the clinical usefulness of STING agonists was debatable.
  • the most common adjuvant, aluminum salt (alum) lacks the ability to induce cell-mediated immunity, which is understood to protect against cancer or diseases from intracellular pathogens [Hogenesch et al., Front. Immunol.
  • alums were combined with many different types of adjuvants including monophosphoryl lipid A [Macleod et al., Proc. Natl. Acad. Sci. U.S.A (2011) 108: 7914-7919] and CpG ODN [Weeratna et al., Vaccine. (2000) 18: 1755-1762],
  • monophosphoryl lipid A Macleod et al., Proc. Natl. Acad. Sci. U.S.A (2011) 108: 7914-7919
  • CpG ODN Weeratna et al., Vaccine. (2000) 18: 1755-1762
  • host DNA may also be a sign of danger as in microorganism DNA, which results in interferon and inflammatory cytokine production [Desmet et al., Nat. Rev. Immunol.
  • a recently identified cytosol DNA sensor is a cyclic GMP-AMP synthase (cGAS), which catalyzes the production of nonstandard cyclic dinucleotide cGAMP (2'3'-cGAMP) and contains a nonstandard 2', 5' bond and 3', 5' bond with the purine nucleoside thereof [Sun et al., Science. (2013) 339: 786-91], Standard cGAMP (3'3') is synthesized in a microbe and has more variety of bonds than mammalian 2'3'-cGAMP. GMP and AMP nucleosides bind by a bis-(3',5') bond [Wu et al., Science. (2013) 339: 826-30; Zhang et al., Mol. Cell. (2013) 51: 226-35],
  • examples of STING agonists that can be used in the present invention include cyclic dinucleotides (CDN) such as 2'3'-cGAMP, c-di-AMP, 3'3'-cGAMP, and 3'2'-cGAMP, xanthenone derivatives such as DMXAA, and the like. STING agonists are also explained in WO 2010/017248, whose entire content is incorporated herein by reference.
  • CDN cyclic dinucleotides
  • the STING agonists is c-di-AMP.
  • Cyclic di-adenosine monophosphate c-di-AMP is a second messenger used in signal transduction in bacteria.
  • an “adjuvant” refers to an immunopotentiator that is added to increase the effect of a vaccine, which is an agent that is not a constituent of a specific antigen but increases immune responses to the administered antigen.
  • combination as used herein is to be understood as referring to simultaneous, separate or sequential administration. In one aspect of the invention “combination” refers to simultaneous administration. In another aspect of the invention “combination” refers to separate administration. In a further aspect of the invention “combination” refers to sequential administration. Where the administration is sequential or separate, the delay in administering the second component is preferably such that both agents are present in the body so as to produce the effect of the combination.
  • a combination of two agents may be administered concomitantly, at different times, as part of the same formulation, as a combination of different formulations, in order, or separately.
  • cancer refers to a disease characterized by dysregulated cell proliferation and/or growth.
  • the term comprises benign and malignant cancerous diseases, such as tumors, and may refer to an invasive or non-invasive cancer.
  • the term comprises all types of cancers, including carcinomas, sarcomas, lymphomas, germ cell tumors, and blastomas.
  • the term cancer relates to solid tumor.
  • solid tumor examples include stomach cancer, breast cancer, lung cancer, colorectal cancer, liver cancer, gallbladder cancer, pancreatic cancer, thyroid cancer, prostate cancer, ovarian cancer, uterine cervical cancer, bladder cancer, sarcoma, glioma, mesothelioma, colorectal tumors, hepatic tumors, and head and neck tumors, with preference for breast cancer, lung cancer, colorectal cancer, stomach cancer, prostate cancer, and liver cancer.
  • the term cancer relates to breast cancer.
  • cancer relates to invasive breast cancer.
  • allergen refers to excessive immune responses to a specific antigen. Antigens from the environment causing allergies are especially called allergens.
  • An “allergic disease” refers to a disease induced by an immune response to an exogenous antigen. However, this antigen is often harmless in a quantity that a patient is exposed to in normal life (e.g., pollen during spring time does not have toxicity in and of itself). An immune response resulting in unnecessary discomfort is experienced therewith. This is also called an allergic disease. Examples of typical diseases include atopic dermatitis, allergic rhinitis (hay fever), allergic conjunctivitis, allergic gastroenteritis, bronchial asthma, childhood asthma, food allergy, drug allergy, and hives. Recently, pathological conditions exhibiting a type 1 allergy symptom such as asthma or facial flash only from the scent of citrus or fragrance of gum or the like has drawn attention.
  • autoimmune disease refers is a disease involving an immune response to a constituent substance of the patient's own body as an antigen.
  • Autoimmune diseases may lead to a disorder or inflammation of a specific organ or site or a systemic symptom.
  • Typical examples of such diseases include connective tissue diseases such as rheumatoid arthritis and alopecia areata.
  • Cytotoxic T lymphocytes are key players in the immune control of cancer and (viral or bacterial) infection, as they recognize cancer or pathogen derived peptide epitopes presented by HLA class I molecules on the cancer cell or infected cell surface
  • the anti-cancer vaccine strategy provided by the present invention targets the immune system against specific antigens in cancer. It achieves this by combining 10-30 amino acid long peptides (neopeptides derived from neoantigen protein that are generated from mutations in the tumor) with a CpG oligonucleotide and a STING agonist as adjuvants.
  • the present inventors are the first to show that this adjuvant combination can induce potent CTL responses in vivo.
  • the combination of the adjuvants K3 CpG and c-di-AMP is preferred herein.
  • the adjuvants and antigen may be used in solution, i.e. they do not need to be presented in the form of nanoparticles.
  • this adjuvant combination makes the use of nanoparticles to achieve cross-presentation redundant.
  • the use of the CpG oligonucleotide and a STING agonist combination promotes crosspresentation while stimulating strong CTL immunity. This solves a major hurdle in cancer peptide vaccination as it stimulates very strong CTL responses to peptides that otherwise need cross-presentation.
  • cross-presentation refers to the process by which exogenous antigens captured by phagocytic antigen- presenting cells (APCs) are processed and presented onto MHC-I molecules.
  • APCs phagocytic antigen- presenting cells
  • the vaccine composition provided herein solves the problem of inducing cross-presentation without the need to generate modified peptides or nanoparticles in order to enhance uptake by APCs.
  • modified peptides or nanoparticles would make it much more difficult to produce the vaccine composition under Good Manufacturing Practice (GMP) conditions, due to the chemicals required and complex production process.
  • GMP Good Manufacturing Practice
  • the presently provided vaccine composition requires only 3 components that can be mixed in aqueous solution, such as saline, and is easy to prepare and administer. These 3 components can be lyophilized together greatly facilitating the storage and transport of the vaccine without the need of a cold chain. Such a lyophilized vaccine can be reconstituted with sterile water just before usage.
  • Peptides used in cancer vaccines are derived from the predicted peptide sequence encoded by mutations in the tumor DNA.
  • the use of peptides of around 20 amino acids in length allows the targeting of mutations without any knowledge of the exact 8-10 amino acid sequence that is loaded in the MHC-class I complex for presentation to CTL (theoretically each mutation can yield 8-10 putative peptides for that mutation in different positions).
  • the possibility to use longer peptides makes it much easier to target mutations, but introduces the need for good cross-presentation. This was not possible in prior art methods without the use of nanoparticles.
  • the vaccine compositions of the present invention overcome this problem and allow the use of such relatively long peptides, similarly without exact knowledge of the 8-10 amino acid sequence that is loaded in the MHC- class I complex, whereby efficient cross-presentation of these longer peptides is achieved by using a CpG oligonucleotide and a STING agonist as adjuvants, preferably, the combination of the adjuvants K3 CpG and c-di- AMP.
  • neopeptide with K3 CpG and c-di-AMP can be used as a personalized cancer vaccine alone or in combination with other cancer therapies, preferably in combination with ICI therapy.
  • the neopeptides are preferably non-modified peptides.
  • the safety concerns of non-modified peptides are very low.
  • GMP batches of K3 CpG and c-di-AMP are produced they can be used off-the-shelf with GMP peptides custom-made for each patient.
  • the preparation of the vaccine requires no special technique or equipment and can be done at the bedside.
  • K3 CpG may be substituted with humanized K3 CpG.
  • One highly preferred adjuvant combination of a CpG oligonucleotide and a STING agonist as adjuvants is K3 CpG and c-di-AMP. This adjuvant combination is one aspect of this invention.
  • the adjuvant combination may very suitably be used in combination with cancer neoantigens in (a vaccine for use in) the treatment of cancer.
  • the invention provides the use of a combination of (humanized) K3 CpG oligonucleotide and c-di-AMP as adjuvants together with a short peptide 10-30 amino acid long as antigen. These peptides can be derived from tumor neoantigens.
  • the adjuvant combination may very suitably be used in combination with allergen immunotherapy, also known as desensitization or hypo-sensitization. Allergen-specific Immunotherapy (AIT) is the only available treatment aimed to tackle the underlying causes of allergy.
  • the active components of subcutaneous vaccines traditionally consist of natural or modified allergen extracts which can be combined with adjuvant platforms.
  • the adjuvant platform comprising the combination of K3 CpG and c-di-AMP is provided.
  • the adjuvant combination may very suitably be used in combination with antigenic peptides from pathogens in the preparation of a vaccine that can be used to stimulate immunity against viruses, bacteria etc.
  • T cell targeted immunomodulators blocking the immune checkpoints CTLA-4 and PD1 or PDL1.
  • CTLA4 the first antibody blocking an immune checkpoint
  • monoclonal antibodies targeting PD 1 pembrolizumab and nivolumab
  • PDL1 atezolizumab and durvalumab
  • Anti-PDl/PDLl antibodies have become some of the most widely prescribed anticancer therapies.
  • T-cell-targeted immunomodulators are now used as single agents or in combination with other anti-cancer therapies as first or second lines of treatment for about 50 cancer types.
  • the present invention provides a therapeutic combination of an anti-cancer vaccine, as described herein above, and an immune checkpoint inhibitor (ICI).
  • the ICI may be a cytotoxic T lymphocyte associated protein 4 (CTLA-4) inhibitor, a programmed cell death protein 1 (PD-1) inhibitor, or a PD-L1 inhibitor.
  • the ICI is selected from pembrolizumab, nivolumab, ipilimumab, cemiplimab, atezolizumab, dostarlimab, durvalumab, and avelumab.
  • the combination therapy of ICI/neopeptide vaccination provides for a very effective therapy, wherein the effects of the ICI may be enhanced.
  • Dosing of the ICI may generally be between about 0.1 mg/kg to about 30 mg/kg as body weight (BW)-based dosing regimen, depending on the ICI.
  • Administration of the ICI may suitably be intravenously (IV) into the vein. Dosing may be weekly or biweekly.
  • Tumor neoantigens arise from mutations in a tumor’s DNA and can serve as targets of the immune system.
  • ICI relies on augmenting existing anti-tumor immunity in patients. Therefore, one approach to increase ICI efficacy is to boost anti-tumor immunity by neoantigen vaccination. This has encouraged the pursuit of a personalized cancer vaccine which specifically targets a patient’s tumor neoantigens and could enhance ICI.
  • current neopeptide vaccine strategies do not generate potent T cell immunity. To overcome this obstacle, a strategy was employed that maximizes immunogenicity of neopeptides by enhancing both crosspresentation of peptides and T cell stimulation through a combination of novel adjuvants.
  • the overarching aim of the invention is to develop an efficient personalized cancer vaccine that improves ICI therapy.
  • a sample of a patient’s cancer such as a tumor biopsy
  • the proteome of the cancer is compared to the proteome of the patient’s healthy cells.
  • This may for instance be done by using a genomic approach, wherein tumor DNA/RNA sequencing are combined to identify tumor-restricted (neoantigen) peptides arising from multiple genomic aberrations to generate a highly targetspecific, autologous, personalized therapy based on the use of one of the patient's neopeptides as a neoantigen in a composition of the present invention.
  • the present invention is provided as medicaments (therapeutic agent or prophylactic agent) in various forms described above.
  • Aqueous solutions for injection may be stored, for example, in a vial or a stainless steel container.
  • Aqueous solutions for injections may also be blended with, for example, saline, sugar (e.g., trehalose), NaCl, NaOH, or the like.
  • Therapeutic agents may also be blended, for example, with a buffer (e.g., phosphate buffer), stabilizer, or the like.
  • composition, medicament, therapeutic agent, prophylactic agent, or the like of the present invention comprises a therapeutically effective amount of a therapeutic agent or effective ingredient, and a pharmaceutically acceptable carrier or excipient.
  • pharmaceutically acceptable means that a substance is approved by a government regulatory agency or listed in the pharmacopoeia or other commonly recognized pharmacopoeia for use in animals, more specifically in humans.
  • carrier refers to a diluent, adjuvant, excipient or vehicle administered with a therapeutic agent.
  • Such a carrier can be an aseptic liquid such as water or oil, including, but not limited to, those derived from petroleum, animal, plant or synthesis, as well as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • water is a preferred carrier.
  • saline and aqueous dextrose are preferred carriers.
  • an aqueous saline solution and aqueous dextrose and glycerol solution are used as a liquid carrier of an injectable solution.
  • Suitable excipients include light anhydrous silicic acid, crystalline cellulose, mannitol, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, powdered skim milk, glycerol, propylene, glycol, water, ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl acetal diethylamino acetate, polyvinylpyrrolidone, gelatin, medium -chain fatty acid triglyceride, polyoxyethylene hydrogenated castor oil 60, saccharose, carboxymethylcellulose, corn starch, inorganic salt, and the like.
  • the composition can also contain a small amount of wetting agent, emulsifier, or pH buffer.
  • these compositions can be in a form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained release preparation, or the like.
  • traditional binding agents and carriers such as triglyceride
  • Oral preparation can also comprise a standard carrier such as medicine grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, or magnesium carbonate. Examples of a suitable carrier are described in E. W. Martin, Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S.A.).
  • Such a composition contains a therapeutically effective amount of therapy agent, preferably in a purified form, together with a suitable amount of carrier, such that the composition is provided in a form suitable for administration to a patient.
  • a preparation must be suitable for the administration format.
  • the composition may comprise, for example, a surfactant, excipient, coloring agent, flavoring agent, preservative, stabilizer, buffer, suspension, isotonizing agent, binding agent, disintegrant, lubricant, fluidity improving agent, corrigent, or the like.
  • salts in one embodiment of the present invention include anionic salts formed with any acidic (e.g., carboxyl) group and cationic salts formed with any basic (e.g., amino) group.
  • Salts include inorganic salts and organic salts, as well as salts described in, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19. Examples thereof further include metal salts, ammonium salts, salts with organic base, salts with inorganic acid, salts with organic acid, and the like.
  • “Solvate” in one embodiment of the present invention is a compound formed with a solute or solvent.
  • solvates For example, J Honig et al., The Van Nostrand Chemist's Dictionary P650 (1953) can be referred for solvates.
  • a solvent is water
  • a solvate formed thereof is a hydrate. It is preferable that the solvent does not obstruct the biological activity of the solute.
  • examples of such a preferred solvent include, but not particularly limited to, water and various buffers.
  • Examples of “chemical modification” in one embodiment of the present invention include modifications with PEG or a derivative thereof, fluorescein modification, biotin modification, and the like.
  • various delivery systems which can be used to administer the agent of the invention to a suitable site (e.g., esophagus).
  • a suitable site e.g., esophagus
  • a recombinant cell that can express encapsulated therapeutic agent (e.g., polypeptide) in liposomes, microparticles, and microcapsules; use of endocytosis mediated by a receptor; construction of a therapy nucleic acid as a part of a retrovirus vector or another vector; and the like.
  • the method of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • a medicament can be administered by any suitable route, such as by injection, bolus injection, or by absorption through epithelial or mucocutaneous lining (e.g., oral cavity, rectum, intestinal mucosa, or the like).
  • an inhaler or mistifier using an aerosolizing agent can be used as needed.
  • other biological activating agents can also be administered concomitantly. Administration can be systemic or local.
  • the present invention can be administered by any suitable route such as direct injection into cancer (lesion).
  • a composition can be prepared as a pharmaceutical composition adapted to administration to humans in accordance with a known method. Such a composition can be administered by an injection.
  • a composition for injection is typically a solution in an aseptic isotonic aqueous buffer.
  • a composition can also comprise a local anesthetic such as lidocaine, which alleviates the pain at the site of injection, and a solubilizing agent as needed.
  • ingredients can be supplied individually or by mixing the ingredients together in a unit dosage form; and supplied, for example, in a sealed container such as an ampoule or sachet showing the amount of active agent or as a lyophilized powder or water-free concentrate.
  • composition When a composition is to be administered by injection, the composition can be distributed using an injection bottle containing aseptic agent-grade water or saline.
  • an aseptic water or saline ampoule for injection can also be provided such that the ingredients can be mixed prior to administration.
  • composition, medicament, therapeutic agent, and prophylactic agent of the invention can be prepared as a neutral or base form or other prodrugs (e.g., ester or the like).
  • Pharmaceutically acceptable salts include salts formed with a free carboxyl group, derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, or the like, salts formed with a free amine group, derived from isopropylamine, triethylamine, 2 -ethylaminoethanol, histidine, procaine, or the like; and salts derived from sodium, potassium, ammonium, calcium, ferric hydroxide or the like.
  • the amount of therapeutic agent of the invention that is effective in therapy of a specific disorder or condition may vary depending on the properties of the disorder or condition. However, such an amount can be determined by those skilled in the art with a standard clinical technique based on the descriptions herein. Furthermore, an in vitro assay can be used in some cases to assist the identification of the optimal dosing range.
  • the precise dose to be used for a preparation may also vary depending on the route of administration or the severity of the disease or disorder. Thus, the dose should be determined in accordance with the judgment of the attending physician or the condition of each patient.
  • the dosage is not particularly limited, but may be 0.001, 1, 5, 10, 15, 100, or 1000 mg/kg body weight per dosage or within a range between any two values described above.
  • the dosing interval is not particularly limited, but may be, for example, 1 or 2 doses every 1, 7, 14, 21, or 28 days or 1 or 2 doses in a range of period between any two values described above.
  • the dosage, dosing interval, and dosing method may be appropriately selected depending on the age, weight, symptom, target organ, or the like of the patient.
  • a therapeutic agent contains a therapeutically effective amount of effective ingredients, or an amount of effective ingredients effective for exerting a desired effect.
  • the effective dose can be estimated from a dose-response curves obtained from in vitro or animal model testing systems.
  • each peptide in single dose vaccine injection may be in the range of about lOOjig to 3 mg, preferably 500 pg to 2mg, or more preferably about Img.
  • a single dose vaccine injection may deliver 10, 20 or 30 or more peptides.
  • the amount of N protein per vaccine injection may be in the range of about lOOjig to lOmg, preferably about 500 jig to 5mg, or more preferably about Img to 3mg, or about 2mg.
  • the amount of each adjuvant in the adjuvant combination comprised in a single dose vaccine injection is preferably about or equal in weight.
  • the ratio between the ODN and STING agonist in an adjuvant combination of the invention may be 1:5 or 5:1, preferably the weight ratio is 1:2 to 2:1. Most preferably, the weight ratio is about 1:1.
  • the amount of each adjuvant may be in the range of lOOjig to 3mg.
  • an amount of K3 CpG of lOOjig, 500 jig, Img, 2mg or 3mg may suitably be combined with an amount of c-di-AMP of lOOjig, 500 jig, Img, 2mg or 3mg per vaccine injection.
  • “Patient” or “subject” in one embodiment of the present invention includes humans and mammals excluding humans (e.g., one or more species of mice, guinea pigs, hamsters, rats, rabbits, pigs, sheep, goats, cows, horses, cats, dogs, marmosets, monkeys, and the like).
  • humans e.g., one or more species of mice, guinea pigs, hamsters, rats, rabbits, pigs, sheep, goats, cows, horses, cats, dogs, marmosets, monkeys, and the like).
  • the pharmaceutical composition, therapeutic agent, or prophylactic agent of the invention can be provided as a kit.
  • the present invention provides an agent pack or kit comprising one or more containers filled with one or more ingredients of the composition or medicament of the invention.
  • information indicating approval for manufacture, use, or sale for administration to a human by a government agency regulating the manufacture, use, or sale of medicaments or biological products can be appended to such a container in a stipulated form.
  • the pharmaceutical composition comprising an ingredient of the present invention can be administered via liposomes, microparticles, or microcapsules. In various embodiments of the present invention, it may be useful to use such a composition to achieve sustained release of the ingredient of the present invention.
  • the formulation procedure for the therapeutic agent, prophylactic agent, or the like of the invention as a medicament or the like is known in the art.
  • the procedure is described, for example, in the Japanese Pharmacopoeia, the United States Pharmacopeia, pharmacopeia of other countries, or the like.
  • those skilled in the art can determine the embodiment such as the amount to be used without undue experimentation from the descriptions herein.
  • EXAMPLE 1 Potent CD8+ T cell response is induced by K3 CpG and c-di-AMP agonist combination
  • K3 CpG and c-di-AMP adjuvant combination when used with peptide immunization induce potent cytotoxic CD8+ T cells (CTL) responses.
  • Mice were vaccinated with the 20 amino acid OVA(252-271) peptide of ovalbumin together with K3 CpG and c-di-AMP agonist combination as adjuvants.
  • the OVA(252-271) peptide contains the known in C57BL6 mice 8 amino acid OVA(257-264) CTL epitope (SIINFEKL).
  • mice received OVA(252-271) peptide without adjuvants (saline) or with AddaVax as adjuvant (a squalene based adjuvant).
  • K3 CpG and c-di-AMP combination adjuvant strong CTL immunity is induced against OVA(257-264) peptide. This has been demonstrated (Figure 1) in blood/splenocyte/lymph node cells by A) flow cytometry and staining with OVA(257-264) peptide-loaded MHC-class I tetramers and B) by intracellular cytokine stains and flow cytometry after in vitro stimulation for 6h with OVA(257-264) peptide.
  • IFNy, TNFa, IL-2 cytokines are shown.
  • CD 107a a marker of cellular degranulation and surrogate marker for cytotoxicity.
  • K3 CpG and c-di-AMP adjuvant combination when used with tumor neoantigen peptide immunization induce potent in vivo T cells responses.
  • Twenty amino acid neopeptides designed from mouse tumor neoantigens were combined with K3 CpG and c-di-AMP adjuvant combination.
  • Neoantigen peptide vaccination K3 CpG and c-di-AMP adjuvant combination induce potent Thl T cell immunity in vivo in mice without inducing Th2 immunity.
  • mice were immunized 3 times with pools of 10 neoantigen peptides each from 3 different mouse tumors: B16F10 melanoma, AE17 mesothelioma and 4662 pancreatic adenocarcinoma. The T cells response against these are shown in Figure 2, panels A, B and C, respectively. Mice were vaccinated with pools of 12-14 neoantigen peptides (7pg of each peptide) together with 10 jig of K3 CpG and 10 pg of c-di-AMP. For controls, the same amount of peptides were injected with AddaVax or 10 pg of c-di-AMP alone.
  • mice were vaccinated 3 times with 1-week intervals and the immune response was measured one week after the 3rd vaccination.
  • OVA peptide vaccine reduces B16-F10-OVA tumor growth and improves survival
  • mice that carry an established subcutaneous melanoma B16F10 tumor that expresses the OVA(257-264) CTL epitope (SIINFEKL), results in delayed tumor growth ( Figure 3, panel B) and increased survival of mice ( Figure 3, panel A).
  • EXAMPLE 4 OVA peptide plus K3 CpG + c-di-AMP vaccination synergizes with anti-PD-1 treatment to reduce B16-F10-OVA tumor growth and improve survival
  • Vaccination with 20 amino acid OVA(252-271) peptide plus K3 CpG and c-di-AMP adjuvant combination synergizes with anti-PD-1 treatment to delay tumor growth ( Figure 4, panel B) and increase survival of mice ( Figure 4, panel A).
  • Mice that carry an established subcutaneous melanoma B16F10 tumor that expresses the OVA(257-264) CTL epitope (SIINFEKL) were vaccinated after 12 days of receiving tumor cells. Mice were vaccinated 3 times with 1-week intervals. Animals were vaccinated with lOgg of OVA peptide and 10 pg of K3 CpG and 10 pg of c-di-AMP.
  • mice received peptide in sterile saline or AddaVax.
  • mice received anti-PD-1 (clone RMP1-14, BioLegend, San Diego, USA) or isotype control antibody (100 jig intraperitoneally) every 3-4 days. In this study, vaccination was delayed till day 12 to minimize effects of anti-PD-1 in the absence of vaccination.
  • Each group n 10-12 animals.
  • Ovalbumin (OVA) peptides used for immunizations and in vitro re-stimulations were 20-mer OVA(252-271) peptide (LEQLESIINFEKLTEWTSSN) and 8-mer OVA(257-264) peptide (SIINFEKL) (both purchased from AnaSpec Inc., Fremont, CA, USA).
  • Neoantigens peptides pools, used in vaccines, were purchased from AnaSpec.
  • Full length SARS-CoV-2 nucleocapsid protein N protein; C- terminal His tagged full length protein of YP_009724397.2 was purchased from GeneTex, Inc. Irvine, CA, USA.
  • SARS-CoV-2 nucleoprotein peptide pool JPT PepMix SARS-CoV-2 was purchased from JPT (JPT Peptide Technologies GmbH, Berlin, Germany).
  • Adjuvants used were K3 CpG ((ATCG ACTC TCGA GCGT TCTC); GeneDesign Inc., Osaka, Japan) and cyclic-di-AMP (CAS: 54447-84-6; Yamasa Corporation, Chiba, Japan).
  • AddaVaxTM InVivoGen, San Diego, USA
  • squalene-based commercial adjuvant was included as a control in some immunizations.
  • mice were immunized subcutaneously in the left flank 3 times once a week, with lOOyl volume of each formulation in sterile saline were injected.
  • the OVA vaccination formulations contained lOpg of OVA(252-271) 20-mer peptide, lOpg of K3 CpG and lOpg c-di-AMP adjuvants.
  • the same amount of peptide was injected in in 100 pl of sterile saline or a mix of equal volumes of sterile saline and AddaVaxTM.
  • neoantigen peptide vaccinations pools of 12-14 peptides per vaccine were tested using 7pg per peptide/vaccine, with lOpg of K3 CpG and lOpg c-di-AMP adjuvants.
  • the same amount of peptides were injected in 100 pl sterile saline or 100 pl of a mix of equal volumes of sterile saline and AddaVaxTM.
  • Neoantigen peptides were identified using a proprietary bioinformatics pipeline after whole exome sequencing (WES) of tumor and spleen DNA and RNAseq of RNA from B16- F10 melanoma, AE-17 mesothelioma or 4662 pancreatic adenocarcinoma tumor cell lines.
  • WES whole exome sequencing
  • mice were injected subcutaneously with lOOpl of tumor cell suspension containing 5xl0 5 B16-F10-OVA tumor cells. Once the tumors were established, vaccination and/or ICB treatment was initiated. When vaccination alone was tested, vaccination with 20-mer OVA peptide started on day 7. Vaccinations were given as above in 3 doses once a week using lOgg of OVA(252-271) 20-mer peptide, lOpg of K3 CpG and lOpg c-di-AMP adjuvants per vaccination.
  • Single-cell suspensions of splenocytes and lymph nodes were generated by mechanical disruption of spleens and lymph nodes and then filtering through a 40gm cell strainer (Falcon, San Jose, CA, USA). Cells were washed and counted, 2xl0 6 cells were used for staining. For blood staining 60gl of blood were used. After lysing erythrocytes, cells were washed and stained. In all stains cells were first pre-treated with Fc block for 10 min. For surface stains, cells were stained for 20 min on ice with different panels of antibodies.
  • HBSS Hank's Balanced Salt Solution
  • FBS fetal bovine serum
  • PFA paraformaldehyde
  • cytokine staining cells were stimulated with lOgg/ml OVA(257-264) for 6 hrs at 37°C in 5% CO2 in the presence of GolgiPlugTM (BD Biosciences) and CD107a-APC-Cy7 antibody.
  • Cells were fixed overnight with IC Fixation Buffer at 4°C, washed with a Perm/Wash buffer (both from eBiosciences, Thermo Fisher Scientific, Waltham, MA, USA) and stained for intracellular cytokines for 45 min at 4°C. Fluorochrome conjugated antibodies for CD4 and CD8 were used. Finally, cells were washed twice with Perm/Wash buffer and fixed with 1% PFA. All samples were acquired in a Fortessa Flow Cytometer (BD Biosciences) and analyzed with FlowJo v.9.9.6 software.
  • ELISPOT assay The ELISPOT assays were performed according the manufacturer protocol (ImmunoSpot®, Cellular Technology Ltd (CTL), Shaker Heights, OH, USA). Ninety-six well ELISpot plates pre-coated overnight with anti-murine IFN-y antibody or anti-murine IL-5 antibody according to manufacturer instructions. Plates were washed and IxlO 5 cells for IFN-y plates or 4xl0 5 cells for IL-5 plates were seeded in 200pl 5% FCS DMEM media per well.
  • PMA and lonomycin were used at 20ng/ml and 500ng/ml final concentration respectively.
  • Anti-murine detection antibody was added and incubated at room temperature for 2 hours. After washing 3 times the plates with PBS Tween 0.05%, Streptavidin solution was added and incubated at room temperature for 30min. Plates were then washed two more times with PBS Tween 0.05% and two times with deionized H2O and the developer solution was added and incubated at room temperature for 15min. The reaction was stopped by washing the plates with water, afterwards they were allowed to air-dry for at least 24 hrs before reading. Plates were read and analyzed using a CTL counter with ImmunoSpot Software (CTL, USA).
  • CTL ImmunoSpot Software
  • Immune checkpoint blockade (ICB) therapies have demonstrated a remarkable therapeutic efficacy against a variety of cancers in humans over the past decade. Despite, however, their clinical success, only a subset of patients benefits from ICB therapies and only approximately 30% of different solid tumors respond.
  • One predictor of ICB therapy response in patients is the abundance of tumor mutational load that presumably leads to increased expression of neoantigens, which elicits more antigen-specific T cells that attack tumor cells. Based on the above, amplifying anti-tumor neoantigen-specific T cell responses is expected to improve ICB therapy efficacy and a number of clinical trials have attempted this with neoantigen vaccines.
  • neoantigen vaccine approach is based on neopeptide vaccines that use synthetic peptides in combination with adjuvants to boost anti-tumor immune responses, specifically targeting mutation-derived neoantigen-specific CD4+ and CD8+ T cells with the goal to eliminate tumor cells.
  • neopeptide vaccines have been tested in clinical trials alone or in combination with ICB, clinical benefit has yet to be demonstrated while elicited T cell responses remain low.
  • the most frequently used adjuvant in clinical trials of neopeptide vaccines is the TLR3 agonist poly-ICLC, an attenuated human analogue ofpoly(I:C).
  • Personalized neoantigen vaccines formulating synthetic long peptides (SLPs) with poly-ICLC have been tested in melanoma and glioblastoma patients. These demonstrated the induction of antigen-specific CD4+ and CD8 + T cells but albeit in very low frequencies, especially for the CD8 + T cell compartment, and with no clinical benefit. Therefore, there is still a need for new vaccine strategies that induce better cytotoxic CD8+ T cell responses.
  • neopeptide selection is one aspect of the problem, a more important issue resides in the potency of vaccine adjuvants used.
  • neopeptide vaccines are especially needed in neopeptide vaccines, as neopeptides derived from somatic tumor mutations are often similar to self-peptides and are generally of low affinity while peptides on their own generally lack sufficient immunogenicity. Therefore, employing the appropriate adjuvants is crucial to induce strong CD8 + T cell responses and to produce an effective peptide-based vaccine. For these reasons, in this study the aim was to develop a highly immunogenic vaccine using a novel combination of pattern recognition receptor (PRR) agonists in combination with 20 amino acid long SLPs to induce potent antigen-specific cytotoxic CD8+ T cell responses.
  • PRR pattern recognition receptor
  • SLP are known to be efficiently cross-presented by dendritic cells (DCs) and when combined with appropriate adjuvants they can stimulate T cell immunity.
  • CpG-ODNs CpG oligodeoxynucleotides
  • APCs antigen- presenting cells
  • TLR9 Toll-like receptor 9
  • B K-type of CpG-ODN is capable of inducing both humoral and cellular immune response.
  • stimulator of IFN genes STING
  • cGAMP cyclic guanosine monophosphate-adenosine monophosphate
  • K3 CpG-ODN and STING agonists can act synergistically to induce Thl-type immune responses and suppress tumor growth in mice in vivo.
  • the inventors tested the well-established adjuvants Addavax, a squalene analogue of MF59 and the TLR3 ligand poly(I:C) together with K3 CpG ODN, a TLR9 agonist, and c-di-AMP, a STING agonist, and examined their effect on SLP immunogenicity in vitro and in vivo.
  • the inventors utilized 20-mer synthetic long neopeptides because SLP have been known to induce stronger CD8 + T cell responses in vivo compared to short peptides. Short peptides can induce tolerance, while SLP do not, owing to their efficient and prolonged crosspresentation on MHC-I by professional APCs and their capacity to induce CD4 + T cells that help CD8 + T cell induction.
  • SLP as a vaccine platform for personalized neoantigen vaccines have advantages such as ease of production and cost-effectiveness, a critical component of such peptide based vaccines is the selection of adjuvant that is necessary for potent immunogenicity since peptides alone are generally of low affinity and their immunogenicity relies on adjuvants.
  • One such adjuvant formulation could be the K3 CpG/c-di-AMP combination that was found to be much more immunostimulatory than poly(I:C) and elicited ⁇ 10-fold higher neopeptidespecific T cell immunity. This increased immunostimulatory activity was not accompanied by adverse effects and only rarely was transient skin irritation observed at the injection site which was always less than 1mm in diameter and transient.
  • ICB cancer-infiltrating lymphocytes which presumably are tumor-specific 10 . Therefore, personalized cancer vaccines that elicit neoantigen-specific anti-tumor T cell immunity can be one way to augment and improve the efficacy of ICB. This however has yet to be proven clinically.
  • One phase I clinical trial did utilize a vaccine of neopeptides formulated with poly-ICLC and combined it with anti-PD-1 antibody treatment, however, without an arm with anti-PD-1 antibody treatment alone, it is impossible to confirm added clinical benefit of vaccination.
  • peptide vaccine formulated with the K3/c-di-AMP adjuvant combination conferred antitumor protection to mice and this protection was further increased when vaccine was combined with anti-PD-1 antibody treatment in the ICB resistant B16-F10-OVA tumor. Therefore, the vaccination strategy according to the present invention, using the compositions of the present invention, can convert a non-ICB responsive tumor to ICB responsive. Importantly, the inventors show that vaccine protection when using the K3/c-di-AMP adjuvant combination relied on the presence of antigen and was not due to a systemic non-specific immunostimulatory activity of adjuvants alone.
  • K3/c-di-AMP combination is a more potent adjuvant than c-di-AMP alone.
  • the systemic Th2 priming effect that c-di-AMP induced could impair immunity against tumor.
  • K3 CpG is a Thl inducer in general, it is a weak inducer of IFN-y but a potent IL-6 inducer.
  • adding a STING agonist to K3 CpG strongly boosts Thl and CTL immunity.
  • adding the K3 CpG to the STING agonist inhibited Th2 immunity development by the c-di-AMP.
  • STING agonists can be Th2 inducers and this is IRF3 mediated.
  • the combination of K3 CpG plus STING agonists act synergistically to induce large quantities of IFN-a and IL- 12 by DCs. This high induction of IL- 12 would hinder Th2 development and explain the lack of Th2 immunity with this combination. More recently it has been shown that STING agonists can induce IL-35 by B cells in an IRF-3 dependent manner.
  • This IL-35 in turn can promote tumor growth in mice as it hinders immunity. It would be interesting to investigate whether adding K3 CpG to STING agonists can prevent IL-35 induction and offer an additional explanation for the improved anti-tumor efficacy of the K3/c-di- AMP combination.
  • the present inventors demonstrate that the K3/c-di- AMP adjuvant combination is able to induce potent neopeptide-specific Thl- type and CD8 + T cell immune responses against SLP and neopeptide pools derived from melanoma and mesothelioma tumors.
  • This vaccine formulation did not induce Th2 responses.
  • potent anti-tumor activity in vivo is shown when SLP are combined with the K3/c-di-AMP adjuvant combination and this antitumor activity is synergistically increased by ICB treatment, in the ICB resistant melanoma model B16-F10.
  • the peptide vaccine formulation according to the present invention confers ICB responsiveness to an unresponsive tumor.
  • the present findings indicate that the herein proposed vaccine formulation containing SLPs and K3/c-di-AMP adjuvant combination is a promising candidate for the development of efficient vaccine platforms that can be used in personalized cancer vaccines that potentiate the effect of ICB therapy in cancer patients.
  • the inventors first investigated the in vivo ability of different adjuvants to stimulate T cell immunity against 20-mer OVA(252-27i).
  • the inventors used adjuvants K3 CpG, c-di-AMP and their combination.
  • As control Addavax was used, a squalene-based mimetic of vaccine adjuvant MF59 which is used in approved influenza virus vaccines.
  • both Addavax and MF59 are known to induce robust CD8 + T cell responses against whole proteins.
  • the inventors immunized mice 3 times with different formulations containing 20-mer peptide and adjuvants.
  • the inventors examined whether the combination of K3/c-di-AMP enhances the immunogenicity of low affinity peptides. To test this, the inventors used 20- mer peptides of OVA El or R4 peptides, two variants of SIINFEKL peptide, known for their lower affinity to MHC-I. Mice were immunized with the same schedule as before but with K3/c-di-AMP combination or Addavax containing 20-mer OVA El or R4 peptides. Antigen-specific CD8 + T cell responses against 8-mer OVA El or R4 peptides were determined by measuring intracellular cytokine production upon in vitro restimulation with these peptides.
  • EXAMPLE 6 Potent neoantigen-specific T cell immunity is induced by K3/c-di-AMP adjuvant combination
  • the inventors next investigated the immunogenicity of the K3 CpG plus c-di-AMP vaccine formulations with neopeptides obtained from the 2 different tumor models. They first compared the K3/c-di-AMP combination to c-di-AMP alone and Addavax. The inventors vaccinated mice with neopeptides harboring missense or frame shift mutations from each of melanoma B16-F10 and mesothelioma AE17 tumors. Mice were immunized, as above, with the 20-mer neopeptide pools consisting of 8-14 peptides from each tumor combined with adjuvants. T cell immunity was assessed one week after the last vaccination in spleens.
  • the inventors next compared the K3/c-di-AMP adjuvant combination to poly(I:C), since its attenuated derivative poly-ICLC is currently a favorite in cancer neoantigen vaccines. They immunized mice with a 20-mer neopeptide pool from the B16-F10 tumor 3 times and one week after the last boost analyzed T cell responses.
  • K3/c-di-AMP adjuvant combination induced ⁇ 10-fold higher T cell immunity compared to poly(I:C) ( Figure 10A).
  • K3/c-di-AMP adjuvant combination and poly(I:C) did not differ in IL-5 producing T cells ( Figure 10A).
  • EXAMPLE 7 Formulations containing 20-mer peptide in combination with K3/c-di-AMP adjuvant provide in vivo protection against tumors
  • mice were first inoculated with B16-F10-OVA tumor cells and after seven days, the mice were immunized with 20-mer OVA(252- 271) peptide and K3 CpG and/or c-di-AMP three times as indicated in Figure 11A. It was found that 20-mer OVA(252-27i) peptide combined with K3/c-di- AMP was the most effective formulation at reducing tumor growth and increasing survival of B16-F10-OVA tumor-bearing mice ( Figure 11B, C).
  • the inventors excluded that the protective effect of vaccinating with the K3/c-di-AMP combination was due to a non-specific effect of the adjuvants as no such protection was found when animals were vaccinated with the adjuvants but without OVA(252-27i) peptide, thus confirming that the tumor control was not due to non-specific effects of the adjuvants ( Figures 4 and 12).
  • EXAMPLE 8 Vaccination with 20-mer peptide combined with K3/c- di-AMP adjuvant synergizes in vivo with a-PD-1 treatment to better control an ICB resistant tumor
  • the inventors injected the partially refractory to anti-PD-1 therapy B16-F10- OVA tumor cells and waited for 12 days to establish large enough tumors that do not respond to a-PD-1 antibody therapy. Starting at 12 days, the inventors next vaccinated animals 3 times, as above, with 20-mer OVA ⁇ 252- 271) peptide and K3/c-di-AMP combination as adjuvants. In addition, animals received either a-PD-1 antibody or isotype control antibody twice weekly as indicated in Figure 13A.
  • mice Male and female C57BL/6 mice (8-12 week-old) were used for bone marrow isolation and male and female C57BL/6 Tg (TcraTcrb)1100Mjb/J (OT-I) mice OT-I mice (8-12 week-old) were used for CD8 + T cell isolation (both strains from Charles River France).
  • C57BL/6 female mice were used for immunizations (8-10 week-old) and to test the vaccine effect with or without anti-PD-1 treatment in tumor bearing C57BL/6 female mice (6-8 week-old). Animal studies were carried out in accordance with the recommendations of the local authorities (Instantie voor Dierenwelzijn (IvD) that approved all protocols (license AVD 1010020209604).
  • IvD Instantie voor Dierenwelzijn
  • SPF mice were purchased from Charles Rivers and housed in the Erasmus Medical Center animal facility (Erasmus Dierenexperimenteel Center, EDC) in groups of two to four mice and kept in type IV cages, food and water was administered ad libitum.
  • EDC Erasmus Medical Center animal facility
  • Dendritic cell line DC2.4 cells obtained from Dr. K. Rock (University of Massachusetts Medical School, Worcester, MA, USA) were maintained in RPMI media supplemented with 10% FBS, 100 units/ml Penicillin/Streptomycin and 1% HEPES. Cells were split every 2-3 days.
  • Melanoma cell lines B16-F10-OVA and B16-F10 (kindly provided by Dr. M. Wolkers, San quin, Amsterdam) were cultured in RPMI media supplemented with 10% FBS, 1% L-glutamine, 50 pM B-mercaptoethanol, and 100 units/ml Penicillin/Streptomycin.
  • Mesothelioma cell line AE17 (kind gift from Dr. D.
  • BMDC bone marrow-derived dendritic cells
  • Mouse BMDC were generated in vitro from mouse bone marrow. Briefly, femur and tibia from C57BL/6 mice were collected, cleaned with 70% ethanol and kept in RPMI. Bones were crushed or flushed with a syringe and bone marrow cells were collected and filtered using a 70 gm cell strainer.
  • RPMI media were washed in RPMI media followed by centrifugation at 500 g for 5 min at room temperature (RT) and resuspended in RPMI media supplemented with 10% FBS, 100 units/ml Penicillin/Streptomycin, 50 pM B-mercaptoethanol (complete media) and 20 ng/ml GM-CSF and cultured in non-cell culture-coated 10cm petri dishes. Culture media was refreshed after four days. On day 6, cells were harvested with Ca ++ /Mg ++ free Hanks' Balanced Salt Solution (HBSS)-EDTA, washed with complete media and used for subsequent experiments.
  • HBSS Hanks' Balanced Salt Solution
  • Splenocytes from OT-I mice were harvested and processed to single cell suspension in RPMI media supplemented with 10% FBS, 100 units/ml Penicillin/Streptomycin and 2 mM L-glutamine. Cells were washed with complete media and CD8+ T cells were isolated using EasySepTM Mouse CD8 + T Cell Isolation Kit (Stem Cell Technologies, USA). Briefly, cells were resuspended at IxlO 8 cells/ml, 50 jil/ml of sample of Rat Serum and 50 jil/ml of Isolation Cocktail were added and incubated at RT for 15 min. Rapid Spheres were added at 125 jil/ml of sample, mixed, and incubated at RT for 5 min. Media was added and mix was placed into magnet for 2.5 min at RT. Non-bound CD8 + T cells were poured into a fresh tube and counted for further use.
  • OVA(252-27i) LEQLESIINFEKLTEWTSSN
  • OVA E 1(252-271) LEQLEEIINFEKLTEWTSSN
  • 8-mer OVA El (257-264) EIINFEKL
  • 8-mer OVA R4(257-264) SIIRFEKL (all from GeneScript) and 8-mer OVA(257 -264) peptide SIINFEKL (Anaspec).
  • TLR9 agonist K-type of CpG ODN K3 CpG
  • STING agonist c-di-AMP adjuvants were used in vitro at 1 jiM and in vivo at 10 jig per injection, respectively, in sterile saline.
  • K3 CpG was synthesized by GeneDesign (Japan) and c-di-AMP was kindly provided by Yamasa (Japan).
  • TLR3 agonist polyinosinic-polycy tidy lie acid poly(I:C) HMW VacciGradeTM (InVivoGen, USA) and TLR4 agonist Lipopolysaccharides (LPS, E.coli O111:B4) (Sigma Aldrich) were commercially purchased.
  • AddavaxTM InVivoGen, USA
  • a squalene based adjuvant was used by mixing it with an equal volume of sterile saline.
  • Immunostimulatory capacity of adjuvants was tested in BMDC that were plated at IxlO 5 cells/ml/well in 12-well plates and incubated overnight (ON) at 37°C and 5% CO2. The following day, cells were stimulated ON with 1 jiM K3 CpG and/or c-di-AMP and after harvesting, cells were stained by flow cytometry for co-stimulatory and major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • OT-I CD8+ T cell activation was tested by seeding BMDC at IxlO 5 cells/ml/well in a 12-well plate. Cells were first incubated ON at 37°C and 5% CO2. After that time, OVA peptides were added to the culture and 3h later adjuvants were added and incubated ON at 37°C in 5% CO2. Next day, 2x10 5 cells/ml/well OT-I CD8 + T cells were added to the culture and incubated ON at 37°C in 5% CO2. Cells were then harvested and stained for flow cytometry analysis.
  • mice were subcutaneously immunized with 100 gl of each formulation in sterile saline in the left flank three times, once every week. One week after the last immunization, mice were euthanized and blood, spleen and left axillary and inguinal lymph nodes were collected.
  • OVA peptide immunizations the formulations contained 10 pg of 20-mer OVA(252-27i) peptide and 10 pg of each of the corresponding adjuvants.
  • tumor neoantigen vaccinations a pool of 8-14 20-mer neopeptides were included per vaccine at 7 pg each peptide and 10 pg per adjuvant.
  • Tumor neopeptides for B16-F10 and AE17 tumors were determined by an in-house proprietary bioinformatic neopeptide prediction pipeline using whole exome sequencing (WES) and RNAseq data of the above tumors and C57BL/6 spleens.
  • WES whole exome sequencing
  • mice were injected subcutaneously with 100 pl of tumor cell suspension mixed 1:1 with Matrigel (Corning Life Sciences) containing 0.5xl0 5 cells of B16-F10-OVA cell lines.
  • ICB treatment was initiated.
  • Vaccinations started 7 days post tumor injection and were delivered as above in three doses once a week using 10 jig of 20- mer OVA peptide and 10 pg of individual adjuvants per vaccine. In some experiments animals were vaccinated without peptide.
  • vaccinations were delayed and started 12 days posttumor injection to minimize protective effect of ICB.
  • tissue were mechanically disrupted and filtered through a 40 gm cell strainer (Falcon, San Jose, CA, USA). Cells were washed RPMI and counted, and 2xl0 6 cells were used for staining. For blood staining, 60 gl of blood were used, after lysing erythrocytes, cells were washed and stained. For staining DC2.4 cells and OT-I CD8 + T cells co-cultured with BMDC, the seeded cells were harvested and washed with HBSS containing 3% FBS and 0.02% sodium azide.
  • cells were stimulated with 10 gg/ml of OVA(257-264) for 6h at 37°C in 5% CO2 in the presence of GolgiPlug (BD Bioscience) and anti-CD107a-APC-Cy7 antibody (Table 1) or stimulated with 2 pg/ml of B16-F10 20-mer neopeptide pool ON at 37°C in 5% CO2in the presence of anti-CD107a-APC-Cy7 antibody, and GolgiPlug for the last 6h.
  • GolgiPlug BD Bioscience
  • B16-F10 20-mer neopeptide pool ON at 37°C in 5% CO2in the presence of anti-CD107a-APC-Cy7 antibody, and GolgiPlug for the last 6h.
  • IxlO 5 cells for IFN-y plates and 4xl0 5 cells for IL-5 plates were seeded in 100 pl per well, and 100 pl of media, 8-mer OVA peptide at 10 jig/ml, or neopeptide pools at 2 jig/ml were used to re-stimulate cells.
  • PMA and lonomycin were used at 20 ng/ml and 500 ng/ml final concentration, respectively. Cells were incubated ON at 37°C in 7% CO2.

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Abstract

La présente invention concerne une composition de vaccin anticancéreux comprenant un néo-antigène cancéreux comprenant un épitope de CTL, la composition comprenant en outre en tant qu'adjuvants une combinaison d'un oligonucléotide CpG et d'un agoniste de STING.
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010017248A2 (fr) 2008-08-04 2010-02-11 University Of Miami Sting (stimulateur de gènes d’interféron), un régulateur de réponses immunitaires innées
US11058758B2 (en) * 2014-11-20 2021-07-13 National Institutes Of Biomedical Innovation, Health And Nutrition TH1-inducing adjuvant comprising combination of different nucleic acid adjuvants, and use of same

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Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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US11058758B2 (en) * 2014-11-20 2021-07-13 National Institutes Of Biomedical Innovation, Health And Nutrition TH1-inducing adjuvant comprising combination of different nucleic acid adjuvants, and use of same

Non-Patent Citations (68)

* Cited by examiner, † Cited by third party
Title
AGARWAL P ET AL., J IMMUNOL., vol. 183, no. 3, 2009, pages 1695 - 704
AKONDY RS ET AL., NATURE, vol. 565, no. 7685, 2017, pages 362 - 367
BARBER ET AL., IMMUNOL. REV., vol. 243, 2011, pages 99 - 108
BERGE ET AL., J. PHARM. SCI., vol. 66, 1977, pages 1 - 19
BODE, C. ET AL., EXPERT REVIEW OF VACCINES, vol. 10, 2011, pages 499 - 511
BRAZOLOT MILIAN, C. L ET AL., PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 95, 1998, pages 15553 - 15558
BURCU TEMIZOZ ET AL: "TLR9 and STING agonists synergistically induce innate and adaptive type-II IFN", EUROPEAN JOURNAL OF IMMUNOLOGY, WILEY-VCH, HOBOKEN, USA, vol. 45, no. 4, 5 February 2015 (2015-02-05), pages 1159 - 1169, XP071227632, ISSN: 0014-2980, DOI: 10.1002/EJI.201445132 *
BURDETTE ET AL., NATURE, vol. 478, 2011, pages 515 - 8
CHU, R. S. ET AL., THE JOURNAL OF EXPERIMENTAL MEDICINE, vol. 186, 1997, pages 1623 - 1631
CURTSINGER JMMESCHER MF, CURR OPIN IMMUNOL, vol. 22, no. 3, 2010, pages 333 - 40
DAFTARIAN P ET AL., VACCINE, vol. 23, no. 26, 2005, pages 3453 - 68
DEB P ET AL., J IMMUNOL., vol. 205, no. 1, 2020, pages 223 - 236
DESMET ET AL., NAT. REV. IMMUNOL., vol. 12, 2012, pages 479 - 491
DOROSTKAR FARIBA ET AL: "Co-administration of 2'3'-cGAMP STING activator and CpG-C adjuvants with a mutated form of HPV 16 E7 protein leads to tumor growth inhibition in the mouse model", INFECTIOUS AGENTS AND CANCER, vol. 16, no. 1, 1 December 2021 (2021-12-01), XP093066581, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7836183/pdf/13027_2021_Article_346.pdf> DOI: 10.1186/s13027-021-00346-7 *
GUTJAHR A ET AL., TRENDS IMMUNOL, vol. 37, no. 9, 2016, pages 573 - 587
HARTMANN ET AL., J. IMMUNOL., vol. 164, 2000, pages 944 - 953
HARTMANN, G. ET AL., EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 33, 2003, pages 1633 - 1641
HARTMANN, G.KRIEG, A. M., JOURNAL OF IMMUNOLOGY, vol. 164, 2000, pages 944 - 953
HE X ET AL., ACS NANO, vol. 15, no. 3, 2021, pages 4357 - 4371
HEMMI, H. ET AL., NATURE, vol. 408, 2000, pages 740 - 745
HOGENESCH ET AL., FRONT. IMMUNOL., vol. 3, 2013, pages 1 - 13
HSIEH ET AL., SCIENCE, vol. 260, 1993, pages 547 - 579
HU ZHUTING ET AL: "Personal neoantigen vaccines induce persistent memory T cell responses and epitope spreading in patients with melanoma", NATURE MEDICINE, vol. 27, no. 3, 21 January 2021 (2021-01-21), pages 515 - 525, XP037389701, ISSN: 1078-8956, DOI: 10.1038/S41591-020-01206-4 *
HUNG ET AL., J. EXP. MED., vol. 188, 1998, pages 2357 - 68
J HONIG ET AL.: "The Van Nostrand Chemist's Dictionary P650", 1953
KAWAI ET AL., IMMUNITY, vol. 34, 2011, pages 637 - 650
KERKMANN, M. ET AL., THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 280, 2005, pages 8086 - 8093
KESKIN DB ET AL., NATURE, vol. 565, no. 7738, 2019, pages 234 - 239
KLEIN, D. C.LATZ, E.ESPEVIK, T.STOKKE, B. T., ULTRAMICROSCOPY, vol. 110, 2010, pages 689 - 693
KLINMAN ET AL., NAT. REV. IMMUNOL., vol. 4, 2004, pages 1 - 10
KLINMAN, D. M.: "Immunology", NATURE REVIEWS, vol. 4, 2004, pages 249 - 258
KOCABAS BANU BAYYURT ET AL: "Dual-adjuvant effect of pH-sensitive liposomes loaded with STING and TLR9 agonists regress tumor development by enhancing Th1 immune response", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 328, 22 September 2020 (2020-09-22), pages 587 - 595, XP086411154, ISSN: 0168-3659, [retrieved on 20200922], DOI: 10.1016/J.JCONREL.2020.09.040 *
KRIEG ET AL., NAT. REV. DRUG DISCOV., vol. 5, 2006, pages 471 - 84
KRIEG, A. M.: "Drug discovery", NATURE REVIEWS, vol. 5, 2006, pages 471 - 484
KRUG, A. ET AL., EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 31, 2001, pages 2154 - 2163
KUAI R ET AL., NAT MATER, vol. 16, no. 4, 2017, pages 489 - 496
MA M ET AL., SCAND J IMMUNOL., vol. 91, no. 6, 2020, pages e12875
MACLEOD ET AL., PROC. NATL. ACAD. SCI. U.S.A, vol. 108, 2011, pages 7914 - 7919
MALONIS RJ ET AL., CHEM REV, vol. 120, no. 6, 2020, pages 3210 - 3229
MANTOVANI ET AL., CURR. OPIN. IMMUNOL., vol. 22, 2010, pages 231 - 237
MARSHALL, J. D. ET AL., JOURNAL OF LEUKOCYTE BIOLOGY, vol. 73, 2003, pages 781 - 792
MCHUTCHISON, J. G. ET AL., HEPATOLOGY, vol. 46, 2007, pages 1341 - 1349
MCWHIRTER ET AL., J. EXP. MED., vol. 206, 2009, pages 1899 - 1911
MERIC-BERNSTAM ET AL., CLIN CANCER RES, vol. 28, 2022, pages 677 - 688
MUELLER M ET AL., INT J CANCER, vol. 129, no. 2, 2011, pages 407 - 16
MUELLER M ET AL., J CONTROL RELEASE, vol. 162, no. 1, 2012, pages 159 - 66
OTT PATRICK A ET AL: "An immunogenic personal neoantigen vaccine for patients with melanoma", NATURE, SPRINGER NATURE PUBLISHING AG, LONDON, vol. 547, no. 7662, 13 July 2017 (2017-07-13), pages 217, XP002785348, ISSN: 1476-4687 *
PUIG, M. ET AL., NUCLEIC ACIDS RESEARCH, vol. 34, 2006, pages 6488 - 6495
SAMULOWITZ, U. ET AL., OLIGONUCLEOTIDES, vol. 20, 2010, pages 93 - 101
SAN ROMAN B ET AL., J PHARM PHARM SCI, vol. 17, no. 4, 2014, pages 541 - 53
SCHLOSSER E ET AL., VACCINE, vol. 26, no. 13, 2008, pages 1626 - 37
SEDER ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 90, 1993, pages 10188 - 92
SPELLBERG ET AL., CLIN. INFECT. DIS., vol. 90509, 2001, pages 76 - 102
SUN ET AL., SCIENCE, vol. 339, 2013, pages 826 - 30
TANG ET AL., PLOS ONE, vol. 8, 2013, pages 1 - 6
TEMIZOZ BURCU ET AL: "Anti-tumor immunity by transcriptional synergy between TLR9 and STING activation", IMMUNOLOGY FRONTIER RESEARCH CENTER (IFREC), OSAKA UNIVERSITY , OSAKA , JAPAN, vol. 34, no. 7, 14 April 2022 (2022-04-14), pages 353 - 364, XP093066652, Retrieved from the Internet <URL:https://watermark.silverchair.com/dxac012.pdf?token=AQECAHi208BE49Ooan9kkhW_Ercy7Dm3ZL_9Cf3qfKAc485ysgAAA2gwggNkBgkqhkiG9w0BBwagggNVMIIDUQIBADCCA0oGCSqGSIb3DQEHATAeBglghkgBZQMEAS4wEQQMqfFdE-rEFjumWhHwAgEQgIIDG3zAzbLKnGRF_PuCCP3yT4v_NFvNRR9KzFdEeIsWhb_CIJ3y2Ofh3FFL0ml896j81p2zgIUcq1pn1pBaqGGgonKWEHnb> DOI: 10.1093/intimm/dxac012 *
TRINCHIERI ET AL., IMMUNOL, vol. 7, 2007, pages 179 - 190
VASOU A ET AL., VIRUSES, vol. 9, no. 7, 2017, pages 186
VERTHELYI, D. ET AL., JOURNAL OF IMMUNOLOGY, vol. 166, 2001, pages 2372 - 2377
VESELY ET AL., ANNU. REV. IMMUNOL., vol. 29, 2011, pages 235 - 271
VITALE ET AL., EUR. J. IMMUNOL., vol. 44, 2014, pages 1582 - 1592
VOLLMER, J.KRIEG, A. M., ADVANCED DRUG DELIVERY REVIEWS, vol. 61, 2009, pages 195 - 204
WAGNER ET AL., TRENDS IMMUNOL, vol. 25, 2004, pages 1 - 6
WEERATNA ET AL., VACCINE, vol. 18, 2000, pages 1755 - 1762
YAMAMOTO TORI N ET AL: "Developing neoantigen-targeted T cell-based treatments for solid tumors", NATURE MEDICINE, NATURE PUBLISHING GROUP US, NEW YORK, vol. 25, no. 10, 1 October 2019 (2019-10-01), pages 1488 - 1499, XP036901638, ISSN: 1078-8956, [retrieved on 20191007], DOI: 10.1038/S41591-019-0596-Y *
ZHANG ET AL., MOL. CELL., vol. 51, 2013, pages 226 - 35
ZHAO JING ET AL: "Safety and Efficacy of Therapeutic Cancer Vaccines Alone or in Combination With Immune Checkpoint Inhibitors in Cancer Treatment", FRONTIERS IN PHARMACOLOGY, vol. 10, 11 October 2019 (2019-10-11), pages 1 - 11, XP093052638, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6798079/pdf/fphar-10-01184.pdf> DOI: 10.3389/fphar.2019.01184 *
ZOM GG ET AL., ADV IMMUNOL, vol. 114, 2012, pages 177 - 201

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