WO2024089258A1 - Albumin conjugated to cpg oligodeoxynucleotides as super-boosters of immune response - Google Patents

Albumin conjugated to cpg oligodeoxynucleotides as super-boosters of immune response Download PDF

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WO2024089258A1
WO2024089258A1 PCT/EP2023/080098 EP2023080098W WO2024089258A1 WO 2024089258 A1 WO2024089258 A1 WO 2024089258A1 EP 2023080098 W EP2023080098 W EP 2023080098W WO 2024089258 A1 WO2024089258 A1 WO 2024089258A1
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albumin
cpg
antigen
cells
compound according
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PCT/EP2023/080098
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French (fr)
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Kenneth Alan Howard
Diego PILATI
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Aarhus Universitet
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    • 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
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6875—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin
    • A61K47/6879—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody being a hybrid immunoglobulin the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
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    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • A61K2039/55516—Proteins; Peptides
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the present invention relates to vaccines and compounds for treating cancerous disease.
  • the present invention relates to albumin conjugated CpG oligonucleotides to one or more partner molecules, such as immune checkpoint inhibitors and multispecific antigen-targeting moieties, these compounds are in particular useful for treating cancerous disease.
  • CpG ODN Oligodeoxynucleotides containing unmethylated CG dinucleotides
  • TLR9 Toll-like receptor 9
  • Human serum albumin is a natural transport protein with binding sites for both endogenous and exogenous ligands, a free cysteine at position 34, and has a long circulatory half-life of ⁇ 19 days. This long in vivo half-life is the result of the engagement of HSA with FcRn, a receptor located in the endosome that rescues bound proteins from lysosomal degradation.
  • FcRn FcRn
  • albumin variants with altered binding affinity to FcRn has been described in WO 2011 /051489, WO 2011/124718, WO 2012/059486, WO 2012/150319, WO 2011/103076, WO 2012/112188 WO 2013/075066, WO 2014/072481, WO 2014/125082 and WO 2015/63611.
  • WO 2013/135896 discloses albumin variants having one or more (e.g. several) alterations in Domain I and one or more (e.g. several) alterations in Domain III.
  • WO 2015/036579 discloses albumin variants having one or more (e.g. several) alterations in Domain II. In sum, several albumin variants are known to the skilled person.
  • Liu et al. (Structure-based programming of lymph node targeting in molecular vaccines. 2014
  • amphiphiles (amph- vaccines) comprising an antigen or adjuvant cargo (such as CpG-DNA) linked to a lipophilic albumin-binding tail by a solubility-promoting polar polymer chain.
  • Vaccines require adjuvants
  • cancer cells increase immune checkpoint receptors to lower the immune response and cancer treatments, in general, benefit from an increased immune response.
  • combination therapies combining an agent for stimulating the immune system with a vaccine/ cancer treatment, result in a spatial displacement of activation of immune response and treatment/vaccination.
  • the present invention overcomes these unmet challenges through the conjugation of CpG ODN to albumin variants or albumin fusions engineered for tuned optimal binding to the neonatal Fc receptor (FcRn) that facilitates extended half-life, lymph node accumulation and greater endosomal accumulation rich in toll-like receptors (figure 1).
  • the albumin fusions being applicable for use in vaccines and cancer treatments.
  • Zhu et al. and Liu et al. employs endogenous albumin, and is silent in respect of using albumin variants and fusing albumin to other proteins. Furthermore, Liu et al. is silent in respect of coupling via Cys34, and the use of albumin variants.
  • an aspect of the present invention is an albumin compound comprising an albumin variant and one or more ODNs covalently conjugated to albumin.
  • the inventors of the present invention found that when fusing an immune checkpoint inhibitor with an albumin molecule, conjugated to CpG ODN, a surprisingly high stimulation of the immune system was observed (example 3). This effect was further confirmed when albumin was fused to a bispecific T-cell engager (CpG-Albu-LiTE), as shown in example 4.
  • CpG-rHA-anti-PD-Ll showed a high degree of endosomal-driven recycling and retained function of CpG, anti-PD-Ll nanobody and albumin. Both CpG-rHA-anti- PD-L1 and CpG-Albu-LiTE exhibited a surprisingly high degree of synergistic effect (example 3 and 4).
  • Examples 5-6 further shows how vaccines can be generated (cat allergen Fel dl and the corona virus derived RBD (receptor binding domain) protein), that may either induce tolerance or increase the immune response against the parts included in the vaccine.
  • vaccines can be generated (cat allergen Fel dl and the corona virus derived RBD (receptor binding domain) protein), that may either induce tolerance or increase the immune response against the parts included in the vaccine.
  • Example 7 shows how the presentation to the immune system, more specifically in lymph nodes, can be tuned via the use of high binding albumin.
  • one aspect of the invention relates to a compound comprising: a. a protein conjugate comprising an albumin, preferably a highbinder albumin, and one or more proteinaceous parts; b. one or more CpG oligonucleotides (ODN) covalently conjugated to the albumin, preferably via thiols such as Cys34 of albumin; and c. optionally, a linker connecting the albumin and the one or more proteinaceous parts; wherein the one or more proteinaceous parts are selected from the group consisting of an antigen-targeting moiety or fragment thereof, an immunogenic protein or a fragment thereof, and a tolerogenic protein or fragment thereof.
  • ODN CpG oligonucleotides
  • Another aspect of the present invention relates to a composition, comprising the compound according to the first aspect of the invention.
  • the invention relates to these compounds and compositions as for use as medicaments and/or vaccines.
  • an aspect relates to a method of treatment, comprising administering a therapeutic amount of the compound according to the first aspect, or the composition the second aspect to a subject in need thereof.
  • an aspect of the invention is also a vaccine comprising the compound or the composition as defined herein.
  • Yet another aspect of the present invention is to provide a method for producing the compound or the composition according to the previous aspects, the method comprising: a. providing one or more nucleic acid(s) encoding the protein conjugate or its individual parts according to the previous aspects, or one or more expression vector(s) comprising said nucleic acid(s); b. introducing the nucleic acid(s) or the vector(s) into one or more host cell(s), such as host cells selected from the group consisting of: bacteria and eukaryotes; c. growing the host cell(s) under conditions that allow for expression of the protein conjugate from the nucleic acid(s) or the vector(s); d. purifying the protein conjugate, or its individual parts; e.
  • CpG oligonucleotides providing CpG oligonucleotides; and f. conjugating the CpG oligonucleotides to the purified protein conjugate or its individual parts, thereby obtaining the compound or the composition, optionally, wherein the CpG oligonucleotides are Maleimide modified oligonucleotides, such as monobromide maleimide (MBM), lysine-modified oligonucleotides, preferably Maleimide modified oligonucleotides, and optionally purifying the compound obtained in step f, optionally, bringing the protein conjugate's individual parts in contact with each other to allow them anneal.
  • MBM monobromide maleimide
  • Still another aspect of the present invention is to provide a kit comprising: a. the protein conjugate or its individual parts according to the previous aspects; b. a CpG oligonucleotide, such as a CpG-NH2, such as a Maleimide modified oligonucleotide; c. reagents for conjugating the CpG oligonucleotide to the protein conjugate.
  • a CpG oligonucleotide such as a CpG-NH2
  • Maleimide modified oligonucleotide such as a Maleimide modified oligonucleotide
  • Figure 1 shows 1) CpG-conjugated albumin fusion enters the endosome of a dendritic cell. 2) The acidification of the endosome generates conformational changes in albumin and FcRn that results in albumin-FcRn binding. This FcRn- driven endosomal compartmentalisation facilitates CpG co-localisation with TLR9 found in the endosome and the activation of TLR9 by CpG (conjugated to rHA). Higher FcRn binding of albumin variants potentiates this effect. 3) The activated receptors mediate the production and secretion of IFNs and proinflammatory cytokines in DCs. 4) FcRn and albumin are recycled outside the cell and evade lysosomal degradation.
  • Figure 2 shows a SYBR Gold and Coomassie Blue stained Native-PAGE showing purified rHA-anti-PD-Ll WT (rHA-@PD-Ll in figures), CpG-rHA WT and CpG-rHA- anti-PD-Ll WT (CpG-@rHA PD-L1 in figures).
  • the bands of CpG-rHA in the SYBR Gold stain show a high degree of CpG conjugation and purity. The presence of multiple bands indicates the formation of dimers.
  • FIG. 3 shows secretion of TNFo.
  • Figure 5 shows coomassie stained SDS-PAGE showing purified rHA-anti-PD-Ll, WT and HB variants. rHA included as control
  • Figure 6 shows FcRn binding of rHA-anti-PD-Ll.
  • the sensorgrams show binding to FcRn (solid black line) and 1: 1 binding model fit (dashed grey lines).
  • BLI sensorgrams showing association (90-210 s) and dissociation (210-510 s) to immobilised human FcRn of a) rHA WT, b) rHA-anti-PD-Ll WT, c) rHA HB and d) rHA-anti-PD-Ll HB.
  • the analytes contain a five step two-fold dilution series from 3 pM.
  • N l.
  • Figure 8 shows PD-1/PD-L1 Blockade Bioassay.
  • the graphs show the fold induction of luminescence as a function of the loglO of the concentration of i) anti-PD-Ll antibody, ii) anti-PD-Ll nanobody, iii) rHA-anti-PD-Ll and iv) CpG- rHA-anti-PD-Ll.
  • N 3.
  • FIG. 11 shows schematic representations of embodiments described herein.
  • A) shows an embodiment where albumin is conjugated to a CpG oligonucleotide (ODN).
  • ODN CpG oligonucleotide
  • B) and C) show embodiments where albumin is conjugated to an ODN and one or more proteinaceous part through linkers.
  • D) shows an embodiment where albumin and a proteinaceous part are themselves conjugated to ODNs, the ODNs being complementary to each other and form a double stranded ODN, thereby linking the albumin to the proteinaceous part.
  • E) shows an embodiment which is a combination of B) and D).
  • F) shows an embodiment which is a combination of C) and D).
  • G shows an embodiment where albumin is conjugated to multiple ODNs.
  • Figure 13 shows a) Chromatogram of IEX HPLC purification of CpG-RBD-QMP. b) SYBR Gold and Coomassie stained Native-PAGE showing purified CpG-RBD-QMP. CpG 1826 and RBD-QMP included as controls
  • Figure 14 shows in vitro validation of CpG-RBD-QMP.
  • b) fusions binding to human FcRn binding measured as binding to FcRn at pH 5.5 in ELISA,
  • Figure 15 shows intranasal vaccination with CpG-RBD-QMP induces antibodies toward the subunit vaccine antigen a) Percentage antigen-specific B-cells in mediastinal lymph nodes, b) RBD-specific IgG and IgA in nose at endpoint after intranasal vaccination of Tg32-hFc mice.
  • Figure 16 shows in vivo accumulation of albumin WT and HB in lymph nodes and serum.
  • protein conjugate refers to a compound comprising at least one protein, i.e. albumin, conjugated to one or more proteinaceous parts. Since the “protein conjugate” is comprised of separable constituents, in aspects of the invention is referred to the "protein conjugate's" or its individual parts thereby meaning the separable constituents of the protein conjugate, such as albumin and a proteinaceous part.
  • proteinaceous part is understood as a constituent of the protein conjugate, which is primarily comprised of amino acids.
  • a “proteinaceous part” is thus also understood as a constituent only comprised of amino acids.
  • albumin refers to serum albumin and fragments or variants thereof.
  • the albumin may be from a human or non-human species including primates, and laboratory test animals e.g. mice, rats, rabbits, guinea pigs, hamsters.
  • One such serum albumin is human serum albumin according to SEQ ID NO: 1 or the secreted version according to SEQ ID NO: 2.
  • albumin highbinder or highbinder albumin refers to a variant of albumin having one or more improved pharmacokinetic properties when compared with wild type albumin such as providing a long lasting effect, having an increased binding affinity towards the FcRn receptor, having a longer half-life, having a higher binding affinity to FcRn, and/or improved endosomal- mediated cellular recycling.
  • highbinders are: a. an albumin having one or more of the mutations E505Q, T527M, and/or K573P, such as the QMB highbinder according to SEQ ID NO: 6, in preferred embodiments referred to as the QMP highbinder, or simply just QMP; b. highbinder I (HBI) according to SEQ ID NO: 4; and c. highbinder II (HBII) according to SEQ ID NO: 5.
  • linker refers to a molecule that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., a nucleic acid molecule that hybridises to one complementary sequence at the 5' end and to another complementary sequence at the 3' end, thus joining two non-complementary sequences.
  • immune checkpoint inhibitor refers to the class of immunotherapy drugs called immune checkpoint inhibitors which asserts their effect by blocking checkpoint proteins from binding with their partner proteins, thus allowing the immune system to be more active.
  • oligonucleotide refers to a sequence of DNA or RNA nucleotide residues that form a nucleic acid molecule. Oligonucleotides can bind their complementary sequences to form duplexes (double-stranded fragments) or even fragments of a higher order.
  • CpG oligonucleotide or "ODN” is understood as a nucleic acid comprising at least one CpG motif.
  • a CpG motif is a short synthetic single-stranded DNA molecule comprising unmethylated CpG dinucleotides.
  • an "immune response” is understood as either a response triggered by the immune system's recognition of ODNs (1) via an innate response, e.g. TLRs, or the immune system's recognition of "non-self proteins” via MHC presentation (2) via an adaptive immune response.
  • the "immune response” is thus an organism's reaction against foreign antigens.
  • the first contact that an organism has with a particular antigen will result in the production of effector T and B cells which are activated cells that defend against the antigen.
  • the production of these effector cells as a result of the first-time exposure is called a primary immune response.
  • Memory T and memory B cells are also produced in the case that the same antigen enters the organism again. If the organism does happen to become reexposed to the same antigen, a secondary immune response will kick in and the immune system will be able to respond in both a fast and strong manner because of antibodies and the memory cells from the first exposure.
  • an antigen-targeting moiety may be understood as a polypeptide that binds to an antigen present on a cell in need of immunestimulation or in a tissue in need of immunestimulation.
  • an "immunogenic polypeptide” is understood as a polypeptide that induces an immune response.
  • a "tolerogenic polypeptide” is understood as a polypeptide for which it is intended to lower the future immune response against.
  • TLRs Toll-like receptors
  • TLRs are understood as a class of proteins that play a key role in the innate immune system. They are usually expressed on cells that recognise structurally conserved molecules derived from microbes, such as B-cells, macrophages and dendritic cells. Once these microbes have reached physical barriers such as the skin or intestinal tract mucosa, they are recognised by TLRs, which activate immune cell responses.
  • the TLRs include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. TLR3, TLR7, TLR8, and TLR9 are located in intracellular vesicles and are specific against nucleic acids.
  • FcRn receptor refers to the neonatal Fc receptor. FcRn extends the half-life of IgG and serum albumin by reducing lysosomal degradation of these proteins. Following entry into cells, the two most abundant serum proteins, IgG and serum albumin, are bound by FcRn at the slightly acidic pH ( ⁇ 6.5) within early (sorting) endosomes, sorted and recycled to the cell surface where they are released at the neutral pH (>7.0) of the extracellular environment.
  • sequence identity indicates a quantitative measure of the degree of homology between two amino acid sequences of substantially equal length or between two nucleic acid sequences of substantially equal length.
  • the two sequences to be compared must be aligned to best possible fit with the insertion of gaps or alternatively, truncation at the ends of the protein (N re f-N dlf )100 sequences.
  • the sequence identity can be calculated as ref , wherein
  • Ndif is the total number of non-identical residues in the two sequences when aligned and wherein N re f is the number of residues in one of the sequences.
  • Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program (W.R Pearson and D . Lipman (1988)).
  • alignment is performed with the sequence alignment method ClustalW with default parameters as described by J.D. Thompson et al (1994), available at http://www2.ebi.ac.uk/clustalw/.
  • sequence alignment method ClustalW with default parameters as described by J.D. Thompson et al (1994), available at http://www2.ebi.ac.uk/clustalw/.
  • sequence identity is computed as described above.
  • a preferred minimum percentage of sequence identity is at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.
  • the inventing team has designed the ingenious novel compounds comprising albumin, ODNs and proteinaceous parts, where the proteinaceous parts are chosen with the intended use in mind.
  • Professional antigen presenting cells APCs
  • albumin is engulfed and thus taken up by the cell.
  • Some of the albumin is salvaged through structural changes occuring upon acidification of the endosomes, the structural changes allows for albumin to bind to FcRn receptors. Since the endosomes are rich in TLRs, the present invention ingeniously exploits this, and stimulates the APC via the ODN.
  • the present disclosure discloses a compound comprising: a. a protein conjugate comprising an albumin, preferably a highbinder albumin, and one or more proteinaceous parts; b. one or more CpG oligonucleotides (ODN) covalently conjugated to the albumin, preferably via thiols such as Cys34 of albumin; and c. optionally, a linker connecting the albumin and the one or more proteinaceous parts; wherein the one or more proteinaceous parts are selected from the group consisting of an antigen-targeting moiety or fragment thereof, an immunogenic protein or a fragment thereof, and a tolerogenic protein or fragment thereof.
  • ODN CpG oligonucleotides
  • the proteinaceous parts are chosen such that the compound exert the intended effect, be it as a vaccine or a cancer treatment.
  • example 3 and 4 are disclosed such compounds for cancer treatment comprising either an immune checkpoint inhibitor against PD-L1 or a bispecific anti-EGFR/anti-CD3.
  • immune checkpoint inhibitor and the bispecific antibody in the same compound, as well as to exchange the compounds for other compounds, with similar effect.
  • Further examples are for instance the immunogenic protein from the spike protein of the SARS COV2 virus used in example 6 (RBD domain), or the cat allergen as shown in example 5 (Fel dl), showing that the compound finds use in vaccines as well.
  • the inventors of the present disclosure have found that it can be advantageous to connect the ODN, such as via thioether bonds, since it is then possible to specifically design where the ODN is positioned and design the specific stoichiometry.
  • the ODN is conjugated to the albumin via a thioether bond, such as to Cys34 of albumin.
  • the ODN is conjugated to the albumin amide bonds, such as via one or more lysines on the albumin.
  • the ODN is conjugated to the albumin at the 5'end or the 3'end of the ODN, preferably the 5' end.
  • TLR9 agonists Three classes of TLR9 agonists exist, CpG ODN Class A, B, and C, each pose a distinct biologic activity, due to their unique structures and different cell activations, to produce varying innate immune signaling cascades.
  • Class A CpG ODNs are single CpG motifs of partially phosphorothioated (PS- modified) and phosphodiester backbone bases in a palindromic sequence that induce the production of IFNo by peripheral dendritic cells (pDCs) and indirectly activate NK cells.
  • the ODN comprises single CpG motifs of partially phosphorothioated (PS-modified) and phosphodiester backbone bases in a palindromic sequence
  • Class B CpG ODNs are multiple CpG motifs composed of fully PS-modified nucleotides. Unlike Class A CpG ODNs, Class B contain B-cell activators and stimulate pDC maturation. In one embodiment of the present disclosure, the ODN comprises multiple CpG motifs composed of fully PS-modified nucleotides.
  • the Class C CpG ODNs which combine characteristics of the A and B classes, are generally fully PS-modified, double-stranded palindromic motifs that induce strong IFNo production, pDC maturation, and efficient B cell activation.
  • the ODN comprises fully PS-modified, double-stranded palindromic motifs, preferably inducing strong IFNo production, pDC maturation, and efficient B cell activation.
  • the ODN comprises a class A ODN, class B ODN, or a class C ODN, preferably class C ODN, such as CpG 2395 according to SEQ ID NO: 13.
  • the ODN is selected from the group consisting of CpG 2395 according to SEQ ID NO: 13, and CpG 2006 according to SEQ ID NO: 12.
  • a class B ODN is preferred, such as SEQ ID NO: 12 or SEQ ID NO: 14.
  • the ODN can be of different sizes.
  • CpG 2395 is 21 nucleotides long, and CpG 2006 is 24 nucleotides long, however the ODN can have any length between 5-100 nucleotides.
  • the ODN has a length in the range 5-100 nucleotides, preferably 10-50 nucleotides, or 15- 30 nucleotides, such as being 21 or 24 nucleotides long.
  • Examples 2-4 of the present disclosure provide specific embodiments of using the CpG 2395 ODN.
  • Examples 5-6 of the present disclosure provide specific embodiments of using the CpG 1826 ODN and CpG 2006 ODN.
  • the ODN can be modified in many ways to achieve different results. Many such alterations have the purpose of prolonging half-life in circulation and decrease degradation. Most of such modifications will not have an impact on the effect achieved on cells by the compounds of the present disclosure thus it is not the object of the present disclosure to limit the ODNs used to a specific set of modifications.
  • One example being the use of phosphorothiorates in the backbone of ODNs, which will decrease the degradation.
  • the ODN comprises modified nucleotides, such as comprising one or more artificial nucleotides, such as one or more locked nucleic acids (LNAs), such as comprising or consisting of one or more phosphorothioate bonds between individual nucleotides in the backbone of the ODN.
  • LNAs locked nucleic acids
  • the ODN activates an immune response, such as via activation of TLR preferably via the TLR9 receptor.
  • the ODN activates one or more immune cells, such as dendritic cells, B cells, macrophages and/or NK cells, preferably antigen-presenting cells.
  • the binding of the ODN to the TLR receptor stimulates secretion of one or more cytokines, such as TNFo, TNFg, IL-12, IL-6, IL-10, IFN-y, and/or IFN-o.
  • cytokines such as TNFo, TNFg, IL-12, IL-6, IL-10, IFN-y, and/or IFN-o.
  • the compounds and compositions disclosed herein preferably comprise albumin and further proteinaceous parts, to achieve a specific effect.
  • the proteinaceous parts can be selected among several different types, depending on the effect to be achieved, such as for treating a cancer cell or presentation on antigen presenting cells.
  • the one or more proteinaceous parts is an antigen-targeting moiety or a fragment thereof such as an antibody, single domain antibody, diabody, a single-chain variable fragment, affibody or a DARPin.
  • an immune checkpoint inhibitor targeting PD-L1 used in example 3 anti-PD-Ll single domain antibody
  • a bispecific antibody a T-cell engager, targeting both CD3 and EGFR
  • an immune checkpoint inhibitor targeting PD-L1 used in example 3 anti-PD-Ll single domain antibody
  • a bispecific antibody a T-cell engager
  • T-cell engager targeting both CD3 and EGFR
  • one or more proteinaceous parts can both be understood as a single protein part being the single domain antibody, the bispecific antibody (figure 11 C) or several proteinaceous parts being the CD3 targeting moiety and the EGFR targeting moiety (also figure 11 C).
  • the one or more proteinaceous parts can also be positioned at different sites at the albumin.
  • one proteinaceous part could be connected via the ODN, and one proteinaceous part via second linker (figure 11 E-F).
  • the skilled person will be able to select proteinaceous parts, depending on the effect to be achieved, and design experiments to assemble the constructs by the methods disclosed further herein.
  • the antigen-targeting moiety is a multispecific antigen-targeting moiety, such as a bispecific antigen-targeting moiety.
  • Checkpoint inhibitor therapy is a form of cancer immunotherapy.
  • the therapy targets immune checkpoints, key regulators of the immune system that when stimulated can dampen the immune response to an immunologic stimulus. Some cancers can protect themselves from attack by stimulating immune checkpoint targets.
  • Checkpoint therapy can block inhibitory checkpoints, restoring immune system function.
  • the first anti-cancer drug targeting an immune checkpoint was ipilimumab, a CTLA4 blocker approved in the United States in 2011.
  • PD-1 is the transmembrane programmed cell death 1 protein (also called PDCD1 and CD279), which interacts with PD-L1 (PD-1 ligand 1, or CD274).
  • PD-L1 on the cell surface binds to PD-1 on an immune cell surface, which inhibits immune cell activity.
  • PD-L1 functions is a key regulatory role on T cell activities. It appears that (cancer-mediated) upregulation of PD-L1 on the cell surface may inhibit T cells that might otherwise attack. Antibodies that bind to either PD-1 or PD-L1 and, therefore, block the interaction may allow the T-cells to attack the tumor.
  • the proteinaceous parts can thus be an inhibitor of immune checkpoints.
  • the antigen-targeting moiety is an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor inhibits an immune checkpoint selected from the group consisting of PD-1, PD-L1, CTLA-4, VISTA, TIM-3, LAG-3, IDO, KIR2D, A2AR, B7- 1, B7-H3, TIGIT and BTLA.
  • the immune checkpoint inhibitor is selected from the group consisting of PD-1 antagonists, PD-L1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-1 antagonists, B7-H3 antagonists, B7-H4 antagonists, and BTLA antagonists.
  • the immune checkpoint inhibitor is capable of blocking the signal of the immune checkpoint.
  • the antigen-targeting moiety is an immune checkpoint costimulatory agonist, such as 4-1BB, CD28 or CD16A.
  • a compound comprising the PD-L1 antagonist according to amino acids 1-121 of SEQ ID NO: 8, is presented in example 3 of the present disclosure.
  • the proteinaceous part is a PD-L1 antagonist, such as the PD-L1 antagonist according to amino acids 1-121 of SEQ ID NO: 8 or a variant thereof having at least 80% sequence identity to amino acids 1-121 of SEQ ID NO: 8.
  • the PD-L1 antagonist has at least 80% sequence identity to the amino acids 1-121 of SEQ ID NO: 8.
  • the PD-L1 antagonist has at least 85% sequence identity to the amino acids 1-121 of SEQ ID NO: 8, such as at least 90%, at least 95%, at least 99% sequence identity to the amino acids 1-121 of SEQ ID NO: 8.
  • the PD-L1 antagonist is the amino acids 1-121 of SEQ ID NO: 8. To maintain the binding affinity of the PD-L1 antagonist, it is preferred that the sequence variance is outside of the CDR regions.
  • the multispecific antigen-targeting moiety targets at least one antigen located on a cancer cell or an immune cell. In other embodiments, the multispecific antigen-targeting moiety targets at least one antigen on both a cancer cell and an immune cell.
  • the at least one antigen located on a cancer cell is selected from the group consisting of EGFR, PSMA, folate receptor, RGD peptide, transferrin, FcRn, and HEU2. In one embodiment of the present disclosure, the at least one antigen located on a cancer cell is selected from the group consisting of EGFR, PSMA, folate receptor, RGD peptide, transferrin, FcRn, and HER2.
  • the at least one antigen located on a cancer cell is selected from the group consisting of BTLA, 0X40, LAG3, NRP1, VEGF, HER2, CEA, CD19, CD20, Amyloid beta, HER3, IGF-1R, MUC1, EpCAM, CD22, VEGFR-2, PSMA, GM-CSF, CXCR4, CD30, CD70, FGFR2, BCMA, CD44, ICAM-1, Notchl, MHC, CD28, IL-1R1, TCR, Notch3, FGFR3, TGF-g, TGFBR1, TGFBR2, CD109, GITR, CD47, Alpha-synuclein, CD26, LRP1, CD52, IL-4R0, VAP-1, EPO Receptor, Integrin ov, TIM-3, Grp78, LIGHT, TLR2, TLR3, PAR-2, NRP2, GLP- 1 receptor, Hedgehog, and Syndecan 1.
  • the at least one antigen located on an immune cell is selected from the group consisting of CD3, CD16, and CD16A.
  • a compound comprising the bispecific T-cell engager targeting EGFR and CD3 is presented in example 4 of the present disclosure.
  • the proteinaceous part is a bispecific antigentargeting moiety against EGFR and CD3, such as the bispecific antibody according to the amino acids 1-385 of SEQ ID NO: 10 or a variant thereof having at least 80% sequence identity to the amino acids 1-385 of SEQ ID NO: 10.
  • the bispecific antibody has at least 80% sequence identity to the sequence according to the amino acids 1-385 of SEQ ID NO: 10.
  • the bispecific antibody has at least 85% sequence identity to the sequence according to the amino acids 1- 385 of SEQ ID NO: 10, such as at least 90%, at least 95%, at least 99% sequence identity to the sequence according to SEQ ID NO: 10.
  • the bispecific antibody is the sequence according to the amino acids 1-385 of SEQ ID NO: 10. To maintain the binding affinity of the bispecific antibody, it is preferred that the sequence variance is outside of the CDR regions of the bispecific antibody.
  • HLA is a major histocompatibility complex (MHC) in humans.
  • MHC major histocompatibility complex
  • MHC class I and MHC II are two primary classes of MHC molecules.
  • MHC class I and MHC class II are interchangeably used herein with HLA class I and HLA class II.
  • Another aspect of the present disclosure is to design the proteinaceous parts such that an immune response against the proteinaceous parts is moderated.
  • Such an immune response can for example be moderated by degrading the proteinaceous part and presenting it via MHC molecules.
  • Another example can be a whole B-cell antigen, that will trigger an immune response without being degraded.
  • a compound can be used as a vaccine.
  • the compounds can be designed for both creating an immunogenic response such that the immune system activates a response toward a given antigen in the future, and also for creating a tolerogenic response such that the immune system does not react toward a given antigen in the future.
  • the one or more proteinaceous parts is an immunogenic- or tolerogenic protein or a fragment thereof.
  • the immunogenic- or tolerogenic protein or a fragment thereof is capable of binding to a B-cell or an MHC-I or MHC-II molecule on an antigen-presenting cell.
  • the proteinaceous part is selected from non-human proteins, and/or proteins comprising non-human sequences. In another embodiment, the proteinaceous part is selected from non-murine proteins, and/or proteins comprising non-human sequences. In another embodiment, the proteinaceous part is selected from a sequence not present in the species of which the proteinaceous part is to be injected into, i.e. non-species specific proteins, and/or proteins comprising nonspecies specific sequences.
  • the proteinaceous part comprises at least one epitope capable of binding to a MHC molecule, such as an immunogenic epitope.
  • the proteinaceous part comprises at least one epitope of a length between 5 and 20 amino acids. In some embodiments, the epitope has a length of from 7 to 11 amino acids for MHC class I presentation. In other embodiments, the epitope has a length of 15 amino acids for MHC class II presentation. In some embodiments, the epitope has a length suitable for presentation by MHC (major histocompatibility complex). In one embodiment of the present disclosure, the proteinaceous part comprises at least one immunogenic epitope having a predicted ability to bind to MHC class I/II alleles.
  • the binding of the epitopes to HLA I and HLA II alleles can for instance be predicted using NetMHCpan 4.0 and NetMHCIIpan 3.2 (https://services.hea lthtech.dtu.dk/service.php7NetMHCIIpan-3.2).
  • the compounds of the present disclosure comprises albumin or a variant thereof.
  • Albumin is the most abundant protein in human blood, and is thus a well known target for increasing the half-life of compounds.
  • the skilled person knows that a wide range of artificial albumins exist, which are engineered for different technical purposes.
  • the albumin or a variant thereof has at least 80% sequence identity to the sequence according to SEQ ID NO: 2.
  • the albumin or a variant thereof has at least 81% sequence identity to the sequence according to SEQ ID NO: 2, such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% sequence identity to the sequence according to SEQ ID NO: 2.
  • the albumin is the sequence according to SEQ ID NO: 2.
  • the albumin is capable of binding to the FcRn receptor.
  • the albumin comprises one or more additional artificial cysteines, such as the cysteine introducing mutations K93C, or E294C of albumin.
  • additional artificial cysteines such as the cysteine introducing mutations K93C, or E294C of albumin.
  • the skilled person selects one of the mutations K93C and E294C, the compound will be able to bind two ODNs, with the proviso that Cys34 is not alterated.
  • the albumin or the variant thereof is an albumin or a variant thereof as described above, with the proviso that one or more cysteines selected from the list Cys34, Cys93, and Cys 294, are present.
  • albumin highbinders are engineered such that they bind more strongly (compared to wildtype albumin) to the FcRn receptor, when the pH lowers inside the endosomes.
  • the albumin or a variant thereof is a highbinder albumin, such as having one or more improved pharmacokinetic properties when compared with wild type albumin such as providing a long lasting effect, having an increased binding affinity towards the FcRn receptor, having a longer half-life, having a higher binding affinity to FcRn, and/or endosomal-mediated cellular recycling, preferably wherein the highbinder is selected from the group consisting of: a.
  • the albumin as described herein having one or more of the mutations E505Q, T527M, and/or K573P such as the QMB highbinder preferably referred to as the QMP highbinder according to SEQ ID NO: 6; b. highbinder I (HBI) according to SEQ ID NO: 4; and c. highbinder II (HBII) according to SEQ ID NO: 5.
  • Linker preferably referred to as the QMP highbinder according to SEQ ID NO: 6; b. highbinder I (HBI) according to SEQ ID NO: 4; and c. highbinder II (HBII) according to SEQ ID NO: 5.
  • linkers may consist of peptides or nucleic acids.
  • the albumin and the one or more proteinaceous parts is connected via a linker, such as a flexible- or a rigid linker, preferably a peptide linker, such as a GS linker.
  • linker used in example 3 and 4 is the GS linker known as GGGGS (SEQ ID NO: 15), however the skilled person knows that several other linkers can be selected.
  • the albumin and the one or more proteinaceous parts is a genetic fusion, e.g. they are translated from a single transcript, such as the albumin and the one or more proteinaceous parts is consisting of one continuous polypeptide chain.
  • the albumin is positioned at or near the N-terminal- or the C-terminal end of the protein conjugate, in relation to the one or more proteinaceous parts.
  • N-terminal- or the C-terminal means that the albumin have at most 50 additional amino acids between albumin and the N-terminal- or the C-terminal, such as at most 40, at most 30, at most 20, at most 15, at most 10, at most 5, at most 4, at most 3, at most 2, such as 1 additional amino acid between albumin and the N-terminal- or the C-terminal end of the protein conjugate.
  • the ODN as a linker, and thus connect two different protein entities via the ODN.
  • the albumin and the one or more proteinaceous parts is connected via the ODN.
  • the compounds of the present disclosure are modular compounds, since the proteinaceous parts can easily be exchanged via typical molecular biological techniques, e.g. cloning.
  • a particularly modular compound of the present disclosure is a compound where the proteinaceous parts are connected via the ODN, since such compounds can be assembled even without typical molecular biological techniques, e.g. when an albumin conjugated to an ODN is brought into contact with a proteinaceous part conjugated to a complementary ODN, the albumin and the proteinaceous part will spontaneously assemble.
  • several separated proteinaceous parts are connected at different sites to the albumin.
  • At least one of the one or more proteinaceous parts is connected via a peptide linker and at least one of the one or more proteinaceous parts is connected via the ODN.
  • the compound can comprise several linkers, such as a first linker, a second linker, a third linker, and a fourth linker. The individual linkers being selected from the linkers described herein.
  • the connection via the ODN is through a double stranded ODN, wherein the albumin is conjugated to a first ODN and the one or more proteinaceous parts is conjugated to a second ODN, the first- and second ODN being complementary to each other thereby capable of forming the double stranded ODN upon contact.
  • composition composition, medical use and methods of treatment
  • a second aspect of the present disclosure relates to a composition, comprising the compound as described herein.
  • the composition comprises a pharmaceutically acceptable carrier.
  • a composition can also be referred to as a pharmaceutical composition.
  • the compound or the composition described herein is for use as a medicament.
  • the compound or the composition described herein is for use as a vaccine.
  • the compound or the composition described herein is for use in the treatment of cancer, such as a solid cancer, such as a solid cancer selected from the group consisting of breast cancer, colorectal cancer, lung cancers, pancreatic cancers, liver cancers, intestinal cancers, prostate cancers, bladder cancers, kidney cancers, such as renal clear cell carcinoma, ovarian cancers, cervical cancers, adenocarcinomas, squamous cell carcinomas, head and neck cancer, ear or nose or throat cancers, skin cancers, such as basal cell carcinomas, melanomas, non-melanoma skin cancers, squamous cell carcinomas of the skin, such as a liquid cancer such as leukemia, such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML).
  • ALL acute lymphocytic leukemia
  • AML acute myelogenous leukemia
  • the compounds and compositions can also be used in methods of treatment, thus, in another aspect, the disclosure relates to a method of treatment, the method comprising administering a therapeutic amount of the compound as described herein, or the composition as described herein to a subject in need thereof.
  • the disclosure relates to a method for producing the compound or the composition described herein, the method comprising: a. Providing one or more nucleic acid(s) encoding the protein conjugate or its individual parts as described herein, or one or more expression vector(s) comprising said nucleic acid(s); b. introducing the nucleic acid(s) or the vector(s) into one or more host cell(s), such as host cells selected from the group consisting of: bacteria and eukaryotes; c. growing the host cell(s) under conditions that allow for expression of the protein conjugate from the nucleic acid(s) or the vector(s); d. purifying the protein conjugate, or its individual parts; e.
  • CpG oligonucleotides providing CpG oligonucleotides; and f. conjugating the CpG oligonucleotides to the purified protein conjugate or its individual parts, thereby obtaining the compound or the composition, optionally, wherein the CpG oligonucleotides are Maleimide modified oligonucleotides, such as monobromide maleimide (MBM), lysine-modified oligonucleotides, preferably Maleimide modified oligonucleotides, and optionally purifying the compound obtained in step f, optionally, bringing the protein conjugate's individual parts in contact with each other to allow them anneal.
  • MBM monobromide maleimide
  • the Maleimide modified oligonucleotides are prepared by combining a CpG-NHz oligonucleotide with Maleimide, such as Maleimide-(PEG) n -NHS-ester, wherein n is a number between 1-10, or succinimidyl 4-(N- maleimidomethyl)cyclohexane-l-carboxylate (SMCC), and optionally purifying the Maleimide modified oligonucleotides.
  • Maleimide such as Maleimide-(PEG) n -NHS-ester, wherein n is a number between 1-10, or succinimidyl 4-(N- maleimidomethyl)cyclohexane-l-carboxylate (SMCC)
  • the Maleimide modified oligonucleotides is a monobromidemaleimide and prepared by conjugating a Tetrazine-(PEG)n-NHS-ester with a CpG-NHz, wherein n is a number between 1- 10, and conjugating a monobromomaleimide-bicyclo[6.1.0]nonyne (BCN) to cysteines on albumin. Tetrazine is then conjugated to BCN and thereby generating an MBM (Dinesen A et al.
  • a Maleimide-(PEG)8-NHS-ester is used in examples 2-4 to illustrate an embodiment of the invention.
  • the nucleic acids encoding the protein conjugate is operably linked to a promotor and optionally, additional regulatory sequences that regulate expression of said nucleic acid.
  • the compounds of the present disclosure comprises separable constituents, and thus can also be delivered by its individual parts, and assembled later on.
  • a kit comprising: a. the protein conjugate, or its individual parts, as described herein; b. a CpG oligonucleotide, such as a CpG-NHz, such as a Maleimide modified oligonucleotide as described herein; c. reagents for conjugating the CpG oligonucleotide to the protein conjugate.
  • the vaccines can be generic vaccines, but can also be individualised to an individual by selecting epitopes that are particularly displayed by that individuals MHC molecules. As presented earlier, the individualised vaccines can in particular benefit from the modular design employing double stranded ODN presented herein.
  • the vaccines can thus be prefabricated and combined with the proteinaceous parts suitable for a particular individual later on. The vaccines will thus alter the future immune response against, the antigen comprised therein, i.e. the immunogenic- or tolerogenic protein or fragments thereof.
  • the vaccines can be both immune stimulating, hence resulting in activation of an immune response on later encounters with the antigen, and the vaccines can induce tolerance (also known as an allergy-vaccine), hence resulting in a lower or no immune response on later encounters with the antigen.
  • tolerance also known as an allergy-vaccine
  • fragments of the immunogenic- or tolerogenic proteins are used as proteinaceous parts, it is desirable to use fragments that will bind more strongly to MHC molecules, and thus increase the likelihood of presentation from antigen presenting cells.
  • an immunogenic polypeptide may be inserted into the construct.
  • the immunogenic polypeptide is selected from an infectious agent known to cause disease in subjects, preferably human subjects.
  • that immunogenic polypeptide is an immunogenic fragment of the infectious agent, preferably the fragment is capable of binding to and being presented on MHC molecules as described previously.
  • the infectious agent may be any type of infectious agent, in preferred embodiments, the infectious agent is derived from a virus, a bacteria, or a fungus.
  • a tolerogenic polypeptide may be inserted into the construct.
  • the tolerogenic peptide is selected from an allergy antigen known to cause allergies in subjects, preferably human subjects.
  • the tolerogenic polypeptide is a fragment of the allergy antigen, preferably the fragment is capable of binding to and being presented on MHC molecules as described previously.
  • example 6 is shown a construct according the present invention, wherein an RBD domain from the spike protein of the SARS COV2 virus is inserted. Given the large degree of increase in B-cells, and induction of RBD-specific antibodies, this example clearly shows how the constructs of the invention can be used as vaccines.
  • example 5 provides an example showing that the cat allergen Fel dl can be inserted into the construct and result in a high stimulus of cells.
  • constructs can both function as vaccines capable of increasing immune responses, as well as vaccines capable of decreasing immune responses, i.e. inducing tolerance.
  • an aspect relates to the compound or composition according to the invention for use as a vaccine.
  • the disclosure also provides albumin compounds comprising ODNs.
  • albumin compounds comprising ODNs.
  • the skilled person will understand that the albumin compounds disclosed below will also find use in the compositions, vaccines, use as medicament, use in treating cancers, methods of treatment, etc. disclosed above.
  • an albumin compound comprising a highbinder albumin and one or more CpG oligonucleotides (ODN) covalently conjugated to albumin, preferably via Cys34, wherein the highbinder is an albumin, such as having one or more improved pharmacokinetic properties when compared with wild type albumin such as providing a long lasting effect, having an increased binding affinity towards the FcRn receptor, having a longer half-life, and/or having a higher binding affinity to FcRn.
  • ODN CpG oligonucleotides
  • an albumin compound comprising an albumin and one or more ODNs covalently conjugated to albumin as described above.
  • the one or more ODNs can be conjugated via Cys34, additional artificial cysteines engineered in the albumin, or lysines on the albumin.
  • the one or more ODNs is one ODN, are two ODNs, or even three ODNs covalently conjugated to albumin comprises.
  • the disclosure relates to a method for producing the albumin compound described herein, the method comprising: a. Providing a nucleic acid encoding an albumin as described herein, or an expression vector comprising said nucleic acid; b. introducing the nucleic acid or the vector into a host cell, such as host cells selected from the group consisting of: bacteria and eukaryotes; c. growing the host cell under conditions that allow for expression of albumin from the nucleic acid or the vector; d. purifying the albumin; e. providing CpG oligonucleotides; and f.
  • a host cell such as host cells selected from the group consisting of: bacteria and eukaryotes
  • c. growing the host cell under conditions that allow for expression of albumin from the nucleic acid or the vector d. purifying the albumin; e. providing CpG oligonucleotides; and f.
  • the CpG oligonucleotides conjugating the CpG oligonucleotides to the purified albumin, thereby obtaining the albumin compound, optionally, wherein the CpG oligonucleotides are Maleimide modified oligonucleotides, such as monobromide maleimide (MBM) lysine-modified oligonucleotides, preferably Maleimide modified oligonucleotides, and optionally purifying the albumin compound obtained in step f.
  • MBM monobromide maleimide
  • Albumin WT was purchased from Sigma-Aldrich (A6608), rHA NB or highbinder (HB).
  • HBII (SEQ ID NO: 5) is used in all instances where albumin is used alone or conjugated with CpG ODN, unless otherwise indicated.
  • QMP (SEQ ID NO: 6) is the HB variant used in fusion proteins (such as rHA-anti- PD-L1 or Albu-LiTE).
  • the CpG-ODN (2395, 2006 and 1826) and control ODN were provided by Integrated DNA Technologies.
  • HEK293E Human embryonic kidney 293E
  • HEK293E ATCC, CRL-10852
  • Jurkat cells included in the PD-1/PD-L1 Blockade Bioassay (Promega, cat# J 1252) and PBMC cells were cultured in RPMI medium (Gibco, cat# 61870-010) supplemented with 10% fetal bovine serum (FBS, Gibco, cat# 10500-064) and 1% penicillin/streptomycin (P/S) (Gibco, cat# 14140-122) at 37°C, 5% CO2.
  • RPMI medium Gibco, cat# 61870-010
  • FBS fetal bovine serum
  • P/S penicillin/streptomycin
  • RAW264.7 cells ATCC, CRL-1420
  • DMEM Dulbecco's modified eagle medium
  • the amino acid sequence of the anti-PD-Ll nanobody was obtained from the patent WO 2019/166622A1 and used to generate a human codon optimised DNA sequence for expression in human cell line with the software CLC Main Workbench (version 21.0.4). This was subcloned by GenScript into pcDNA3.1 plasmids as a fusion to NB, WT, and HB albumin variants. Between the nanobody C-terminal and albumin N-terminal, a GGTGGCGGGGGAAGC (SEQ ID NO: 14) sequence encoding a GGGGS (SEQ ID NO: 15) flexible peptide linker was added.
  • the Albu-LiTE plasmid was generated in a similar manner as described for rHA- anti-PD-Ll.
  • the LiTE sequence formed by EgAl (aEGFR) nanobody and the OKT3 (aCD3) scFv connected by a GGGGS (SEQ ID NO: 15) flexible peptide linker, were subcloned by GenScript into pcDNA3.1 plasmids as a fusion to NB, WT, and HB albumin variants.
  • Plasmids were amplified by transformation through heat shock (40 min on ice, 2 min at 42°C and 5 min on ice) into TOPIO chemically competent E. coli and plated on agar plates (1% peptone (Merck, cat# 82303), 0.5% yeast extract (Fisher Scientific, cat# BP9727), 0.8% NaCI (Acros Organics, cat# 207790010), 1.5% agar (Sigma, cat# 05040), and 0.1 mg/mL ampicillin (Fisher Scientific, cat# BP1760)).
  • the amino acid sequence of Fel dl chains 1 + 2 was obtained from the protein database registry (PDB code: 1ZKR) and used to generate a human codon optimised DNA sequence using the CLC Main Workbench software (version 21.0.4). This sequence was subcloned by GenScript into pcDNA3.1 plasmids as a fusion with human albumin carrying the flexible GGGGS (SEQ ID NO: 15)peptide linker.
  • Plasmids were amplified by transformation through heat shock (40 min on ice, 2 min at 42°C and 5 min on ice) into TOPIO chemically competent E. coli and plated on agar plates (1% peptone (Merck, cat# 82303), 0.5% yeast extract (Fisher Scientific, cat# BP9727), 0.8% NaCI (Acros Organics, cat# 207790010), 1.5% agar (Sigma, cat# 05040), and 0.1 mg/mL ampicillin (Fisher Scientific, cat# BP1760)).
  • PEI Polyethylenimine
  • plasmid DNA in a 4: 1 ratio were diluted separately in Optimem (Gibco, cat# 11058-021), subsequently mixed and incubated at RT for 15 min before mixing with Freestyle 293 expression medium (Gibco, cat# 12338-018) and added to 55-65% confluent HEK293E cells in 5-layer bottles (Corning, cat# 353144).
  • the rHA-fusion protein was purified on a CaptureSelect anti-HSA affinity column (Matrix: Sigma, cat# 19129701L, packed by Repligen to a column volume (CV) of 5 mL) on an Akta Start system (Cytiva).
  • the program with a flow rate of 1 mL/min, was as follows: tube flushing with 5 CV phosphate buffered saline (PBS, Sigma, cat# D8537) and 0.05% sodium azide (NaN3, ReagentPlus, cat# S200-2), sample addition, wash with 6 CV PBS/NaN3, elution with 10 CV 2 M MgCI2 (Sigma, cat# M2670) with 20 mM Tris (Sigma, cat# T5941) pH 7.4, wash with 10 CV PBS/ NaN3, stripping with 0.1 M glycine (Sigma, G8898) pH 3, and column neutralisation with 10 CV PBS/ NaN3.
  • PBS phosphate buffered saline
  • NaN3, ReagentPlus sodium azide
  • sample addition wash with 6 CV PBS/NaN3, elution with 10 CV 2 M MgCI2 (Sigma, cat# M2670) with 20 mM Tris (Sigma, cat# T5941) pH 7.4,
  • the sequences of the ODNs used for conjugation are shown in the sequence listing below.
  • Amine-modified ODNs were conjugated to the NHS-ester-modified linkers by mixing DMSO (Sigma, cat# 34869), ImM ODN, 0.1 M (4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES, Sigma, cat# H4034), and 100 mM Maleimide-(PEG)8-NHS-ester (SM(PEG)8) linker (Sigma, cat# 746207)) at a volume ratio of 3:2: 10: 1, respectively. After incubation overnight at room temperature (RT) with 650 rpm orbital shaking, excess linker was removed by ethanol precipitation (see below).
  • ODN-linkers were then conjugated to rHA or rHA-anti-PD-Ll.
  • ODN-maleimide was mixed with a 2-fold molar excess of rHA in 0.1 M HEPES pH 7 and incubated as previously described.
  • rHA-anti-PD-Ll conjugation the ODN-maleimide was mixed in a 1.3-fold molar excess compared to rHA-anti-PD-Ll.
  • the conjugates were purified with HPLC using an IEX column (see below).
  • the setup utilised in the experimental work includes an Ultimate 3000 automated fraction collector connected to an LPG-3400RS pump and a VWD-3400RS detector (AFC, all from Thermo Fisher Scientific).
  • the samples were filtered with a 0.2 pm polypropylene (PP) filter (Kinesis, cat# ESF-PP-04-022) before injection on the HPLC with a 100 pL Hamilton syringe (Sigma-Aldrich).
  • PP polypropylene
  • IEX chromatography was performed with a Mono Q 5/50 GL anion exchange column (Cytiva) running the following program at 0.3 mL/min: 5 min buffer A (20 mM Tris and 10 mM NaCI at pH 7.6), a 70 min gradient to buffer B (20 mM Tris and 3 M NaCI at pH 7.6), 5 min of buffer B, a 5 min gradient to buffer A and 5 min of buffer A. Absorbance was measured at 224 and 260 nm. The collected fractions were desalted and concentrated by centrifugation through 10 kDa cut-off membrane filters.
  • SDS sodium dodecyl sulphate
  • PAGE polyacrylamide gel electrophoresis
  • the mixture was poured into 1 mm cassettes (Invitrogen, cat# NC2010) and let to polymerise for 45 min with a 10-well comb (NC3010).
  • Samples were mixed with 20x reducing agent (Invitrogen, cat# NP0009) and 4x loading buffer (Invitrogen, cat# NP0007), heated at 95°C for 5 minutes and run in 3-(N-morpholino)pro- panesulfonic acid (MOPS, Invitrogen, cat# NP0001) buffer.
  • MOPS 3-(N-morpholino)pro- panesulfonic acid
  • a benchmark (Invitrogen, cat# 10747012) protein ladder was run in a well for comparison.
  • BCA Bicinchoninic acid
  • BCA Protein Assay Kit (Thermo Fisher, cat# 23227) was used following the manufacturer's instructions. rHA was used to create a standard curve for the BCA assay. A working reagent was prepared by mixing BCA Reagent A and Reagent B in a 50: 1 ratio. Then, 10 pL of the rHA standard and samples were added in triplicate to a 96 well plate. 200 pL working reagent were added to each well and were incubated for 45 min at 37 °C. The plate was then let to cool to room temperature and the absorbance at 562 nm was measured with a Clariostar plate reader (BMG Labtech).
  • a BLItz system (Sartorius) was used for BLI investigation rHA/FcRn binding kinetics. Streptavidin coated biosensors (Sartorius, cat# 18-5019) were hydrated for 10 minutes in 0.02% PBST, followed by immobilisation of 70 nM biotinylated FcRn protein (Immunitrack, cat# ITF01) in 0.01% PBST and then washed in new 0.01% PBST.
  • the association buffer was 0.01% PBST at pH 5.5 with 25 mM C2H3NaO2, 25 mM NaH2PO4, 150 mM NaCI.
  • HMEC-1 cells engineered to overexpress human FcRn were used to quantify the cellular-mediated recycling of samples. After coating a 48 well plate with 100 pL of GelTrexTM (Gibco, cat# A1413202) diluted 1: 50 with ice-cold PBS for 1 h at 37° C, the wells were aspirated, and 1 x 105 cells were seeded in MCDB141 complete medium and incubated overnight.
  • a Maxisorp 96-well plate (Invitrogen, cat# 44-2404-21) was coated with 1: 1000 polyclonal goat anti-human albumin (Sigma-Aldrich, cat# A-7544) in PBS, 100 pL/well and incubated overnight at 4°C. The plate was then blocked for 2 hours at room temperature with 2 % Skim Milk (Merk, cat# 70166-500G) in PBS. After 3 washings with PBST 0.05 %, dilution series of albumin and constructs, prepared in complete media without FBS, and the conditioned media from the recycling experiment were loaded onto the and incubated for 2 hours at room temperature.
  • the assay (Promega, cat# J1252) was performed according to the manufacturer protocol. 4 x 105 PD-L1 aAPC/CHO-Kl cells per well were seeded in 96-well white-bottom plates (Falcon, cat# 353377) and incubated at 37°C, 5% CO2, overnight. The day of the assay, a 8 step 2.5-fold dilution series of the reference anti-PD-1 (Biolegend, cat# 329902) and test antibodies, starting at 25 pg/mL was prepared with RPMI supplemented with 10% FBS. The medium of the CHO-K1 cells was then discarded and 40 pL of the reference and test antibodies were added to the wells.
  • PBMCs peripheral blood mononuclear cells
  • the PBMCs were washed with PBS and centrifuged at 400 g and 4°C for 15 min, then the PBS was removed, and the cells treated with ACK lysing buffer (Gibco, cat# A1049201) to remove remaining red blood cells. The PBMCs were subsequently washed again in PBS and resuspended in 37°C culture medium.
  • ACK lysing buffer Gibco, cat# A1049201
  • RAW 264.7 murine macrophages were seeded in 48 well plates (3.5 x 105 cells per well) or 96 well plates (2 x 104 cells per well) and let to incubate at 37°C and 5% CO2 for 24 h.
  • the medium was then aspirated from the 48 well plate and substituted with 500 pL (or 125 pL for the 96 well plate) of CpG-rHA 0.25 pM in culture medium at 37°C.
  • Human PBMC were instead directly seeded as 1 x 106 cells per well in 500 pL of culture medium containing 0.25 pM CpG-rHA.
  • the cells and the samples were incubated for 24 h at 37°C and 5% CO2, the conditioned medium was harvested, centrifuged at 1000 g for 5 min and the supernatant collected for TNFa detection.
  • the assay kits for mouse TNFa (Invitrogen, cat# 88- 7324-88) and for human TNFa (Invitrogen, cat# 88-7346-86) were used, following the manufacturer instructions.
  • 96-well plates (Corning, cat# CLS9018) were coated with 100 pL of capture antibody diluted 1:250 in PBS and incubated at 4°C overnight. The wells were aspirated, washed three times with 0.05% PBST and blocked for 1-2 h at RT with the kit ELISA diluent.
  • the human or mouse TNFa was resuspended with NFW and used to prepare a standard dilution series.
  • the wells were washed once and 100 pL of the TNFa standard and cell supernatant harvested from TNFa secretion assay were added into appropriate wells and left to incubate at 4°C overnight.
  • the wells were washed 3-5 times, and 100 pL of detection antibody diluted 1:250 in ELISA diluent were added and incubated at RT for 1-2 h.
  • 100 pL of Streptavidin-HRP diluted 1: 100 in ELISA diluent were added to the wells and incubated for 30 min.
  • the wells were washed again 5-7 times and TMB was added.
  • the reaction was stopped after 5 min with 0.2 M H2SO4, and the absorbance at 450 nm was measured with a Clariostar plate reader (BMG Labtech) with background subtraction of the absorbance at 570 nm.
  • THP-1 cells (WT, MyD88 KO, STING KO) were seeded (3.5 x 105 cells per well) in 24 well plate with 20 pM phorbol 12-myristate-13-acetate (PMA, Sigma-Aldrich, cat# P1585) in RPMI 10 % FBS and incubated at 37°C and 5% CO2 overnight. The cells were then washed with PBS and fresh medium without PMA was added and incubated overnight. Cells were then stimulated with CpG-rHA 0.250 pM in 500 pL RPMI 10 % for 6 h. The cells were collected and lysed for RNA purification using High Pure RNA Isolation Kit (Roche, cat# 11828665001).
  • RNA per sample 2 pL were reverse transcribed and analysed by real-time qPCR with TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems, cat# 4392938) using either TaqMan (Applied Biosystems) gene expression assays for human g-actin (hs00357333_gl), IFNg (hs01077958_sl) or TNFo (Hs00174128_ml).
  • the experiment was conducted with the Real-Time PCR system QuantStudio 5 (Applied Biosystem).
  • RBD-albumin vector and protein production cDNA encoding RBD was synthesized and cloned (GenScript).
  • GenScript human serum albumin
  • HSA human serum albumin
  • the vectors were transiently transfected into Expi293F cells (Gibco) using the ExpiFectamine 293 Transfection Kit (Gibco) according to the manufacturer's instructions.
  • RBD-HSA fusions were purified using columns packed with CaptureSelect Human Albumin Affinity Matrix (Thermo Fisher Scientific) by Repligen and were preequilibrated and equilibrated with lxPBS before elution with buffer consisting of 2 M MgC (Sigma-Aldrich) and 20 mM Tris (Sigma-Aldrich) (pH 7.0). Eluted fractions were collected, up-concentrated and buffer-changed to lxPBS (Sigma- Aldrich) using Amicon Ultra Centrifugal Filter Units (Merck Millipore) with appropriate cutoff (10-100K) and volume (0.5 mL, 4 mL or 15 mL). Protein concentrations were determined using a DS- 11+ (M/C) Spectrophotometer (DeNovix) programmed with the protein's extinction coefficients and molecular weights,
  • CpG-linkers were made.
  • 5' amine-modified CpG 1826 (IDT) was conjugated to an NHS-ester-modified linker by mixing DMSO (Sigma-Aldrich), ImM ODN, 0.1 M 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich), and 100 mM Maleimide-(PEG)8-NHS-ester (SM(PEG)8) linker (Sigma-Aldrich) at a volume ratio of 3:2: 10: 1, respectively.
  • RBD-QMP was conjugated to RBD-QMP.
  • RBD-QMP was mixed with a 1.3 molar excess of CpG-maleimide in 0.1 M HEPES (pH 7) and incubated overnight at RT with 650 rpm orbital shaking.
  • the CpG-RBD-QMP was purified as described above.
  • Protein purity after direct conjugation was assessed using native PAGE as described above.
  • mice B6.Cg-Tg(FCGRT)32Dcr Fcgrt tmlDcr Zg/7gl em2 ( IGHG1 ) Mvw /MvwJ; The Jackson Laboratory) (female and male, 6-13 or 31 weeks; 5-6 mice per group.
  • the mice were anesthetized by intraperitoneally administering 5 mL/kg of ZRF cocktail containing 250 mg/mL of Zoletil Forte, 20 mg/mL of Rompun, and 50 pg/mL of Fentanyl in saline solution (Section for Comparative Medicine, Rikshospitalet, Oslo University Hospital).
  • nasal flushes were collected at endpoint for selected experiments. Nostrils were flushed with sterile PBS, and samples were directly transferred to protease inhibitor to a final concentration of xl-x2. The nose was flushed with 15 pL of PBS in each nostril. Samples with protease inhibitors were centrifuged at 17 000 x g for 10-25 minutes at 4°C to remove debris. All samples were stored at -20°C until analyses of antigen-specific immune responses, using FCBA as described below. At endpoint mediastinal lymph nodes (also inguinal for PBS groups) were harvested for flow cytometry analyses. The animal experiments were approved by the Norwegian Food Safety Authority and carried out in accordance with the national guidelines and regulations at the Section for Comparative Medicine, Oslo University Hospital (Rikshospitalet).
  • HRP horseradish peroxidase
  • Assays using alkaline phosphatase (ALP)-conjugated antibodies were developed by adding 100 pL/well p- nitropenylphospate substrate tablets (Sigma-Aldrich) dissolved in diethanolamine buffer (pH 9.8) (Sigma-Aldrich) to a final concentration of 1 mg/mL (pNpp).
  • Absorbance measurements were performed using a Sunrise spectrophotometer (TECAN) at 405 nm or 450 nm for pNpp or TMB substrates, respectively, with a reference wavelength of 620 nm.
  • RBD-albumin fusions were tested by adding a 4-fold serial dilution from 4 pg/mL to plates coated with 1 pg/mL of the monoclonal SARS-CoV-2 antibodies sotrovimab (GSK), cilgavimab or tixogevimab (AstraZeneca), before detection using, anti-HSA-ALP as above.
  • GSK monoclonal SARS-CoV-2 antibodies sotrovimab
  • cilgavimab or tixogevimab AstraZeneca
  • a bead-based flow cytometric assay was adapted for the detection of antibodies against RBD from ancestral SARS-CoV-2 (Wuhan) as well as SARS- CoV-2 variants.
  • Bead-based arrays with content of virus proteins were incubated for 1 hour or overnight at RT, for measurements of IgGs and IgA, respectively, in sera, or overnight for measurements in other mucosal flushes, diluted in an assay buffer consisting of PBS supplemented with 1% Tween 20 (PBT) (Sigma-Aldrich), 1% Bovine serum albumin (BSA) (Sigma-Aldrich), 10 pg/mL neutravidin (NA) (Thermo Fischer Scientific), 10 pg/mL D-biotin (Sigma-Aldrich) and 0.1% sodium azide.
  • PBT Tween 20
  • BSA Bovine serum albumin
  • NA neutravidin
  • D-biotin D-biotin
  • the dilutions were as follows: serum - 1: 100, or 1: 300 for IgG in Tg32-hFc from intranasal vaccination, and 1:6 for nasal flushes.
  • the beads were then washed three times with PBT to remove unbound immunoglobulins and labeled for 30 minutes at RT with 10 pL of goat anti-hlgG (Jackson Immunoresearch), or digoxigenin (DIG)-conjugated rat anti-mlgA (Mabtech).
  • Stocks were diluted 1:200, 1:200, 1: 100 and 1: 100, respectively, in PBT containing 1% BSA and 0.1% sodium azide.
  • the anti-mlgA-DIG conjugate was prepared by first changing the buffer of the antibody to PBS without sodium azide using 100K cutoff Amicon Ultra Centrifugal Filter Units (Merck Millipore). Then, 1 mg/mL anti-mlgA was mixed with 200 pg/mL DIG-N-hydroxysuccinimidyl-ester (DIG-NHS) (Sigma), and incubated overnight. Excess DIG-NHS was removed by centrifugation through Amicon Ultra Centrifugal Filter Units (Merck Millipore) with 100K cutoff twice, before the solution was passed over a G50-Sephadex column equilibrated with PBS. Anti-DIG was conjugated to PE using standard thiol- maleimide chemistry. Flow cytometry on mediastinal lymph nodes
  • mice Collected mediastinal lymph nodes from mice were pooled according to treatment group and kept on ice or at 4°C continuously from harvest to analysis and protected from light after addition of viability dye. Between steps, cells were centrifuged at 400 x g for 7 minutes at 4°C before transfer to a 96-well plate, and 500 x g for 5 minutes at 4°C after plating. Lymph nodes were mashed through a 70 pm cell strainer (VWR) to a single-cell suspension, before treatment with 3 mL RBC lysis buffer for 5 minutes. Cells were then washed with FACS buffer (2% FBS in PBS), and subsequently divided into triplicates of approximately 2x 106 cells. Next, the cells were stained with ghostDyeV510 (Tonbo Bioscience) (1:400) for 20 minutes followed by blocking with 100 pL 25% normal rat serum (produced inhouse) in PBS for another 20 minutes.
  • VWR cell strainer
  • TCR0-BB7OO (BD Bioscience) (1:200), CD19-APC/Cy7 (BD Bioscience) (1:200), CD38-BUV395 (BD Bioscience) (1:200), CD45R(B220)-BUV805 (BD Bioscience) (1:200), GL7-Alexa Fluor 488 (Biolegend) (1:200) and PE-Klickmer-coupled RBD.
  • the staining buffer also included BD Horizon Brilliant Stain Buffer (BD Bioscience) (1: 50).
  • PE-Klickmer (Immudex) was used per sample, and it was coupled prior to use in a 1: 15 molar ratio to biotinylated 6xHis- and Avi-tagged Wuhan RBD 69 according to the manufacturer's protocol, yielding approximately 3.5 pL total volume per sample.
  • the PE-Klickmer-coupled RBD was added to the cells first along with 5 pL FACS buffer and was allowed to incubate for 10 minutes before addition of 45 pL of the premixed surface antibody staining solution, followed by incubation for 30 minutes.
  • the cells were fixated with 100 pL 4% paraformaldehyde (Sigma) for 10 minutes, washed, and finally resuspended in 350 pL FACS buffer before analysis on a FACSymphony A5 (BD Bioscience) running FACSDiva (BD Bioscience).
  • the Albumus (hFcRn+/+, hAlb+/+ mice) were injected with Alexa 680-conjugated albumin variants wild type (WT) and high-binder (HB) (both variants manufactured by Albumedix, no ref #).
  • the albumin variants diluted in PBS (#cat: 7778-77-0) were injected subcoutaneously at the tail base, at a dose of 3 nmol/mouse and max volume of 100 uL/20 g mouse.
  • Blood samples of 20 uL were drawn from clipped tail tip 24 h after the injection. After 24 h mice were euthanized by cervical dislocation under isoflurane (#cat: 26675-46-7) anesthesia and organs were be harvested from the mice for subsequent analysis with an Amersham Typhoon 5 (Cytiva).
  • the CpG-ODN used in the design belongs to class C, and it can stimulate both murine and human cells.
  • TLR9 and FcRn are crucial aspects of this project.
  • the use of albumin variants with high FcRn affinity not only enables the extension of the in vivo circulatory half-life and accumulation in tumours, but also enhances the effect of on immune cells.
  • the CpG was conjugated to rHA through an SM(PEG)8 crosslinker, followed by purification with IEX HPLC.
  • the purified construct CpG-rHA WT was run on a Native-PAGE, with unconjugated rHA as a control, to evaluate the purity of the constructs ( Figure 2).
  • nucleic acids were stained with SYBR Gold, and after SYBR staining the gels were stained with Coomassie Blue to detect proteins.
  • the native-PAGE stained with SYBR Gold shows two bands of CpG (lane 1).
  • the appearance of two CpG bands (lane 1) is most likely due to the presence of a 3' palindromic sequence, that could result in dimerisation or formation of a 3' hairpin.
  • the band shift between lane 1 and 3 clearly shows successful conjugation of CpG to albumin.
  • TNFo The secretion of TNFo was evaluated in murine RAW 264.7 and human PBMC cells (Figure 3). Both cell lines were incubated for 24 h with the samples before collection of the supernatant. The presence of secreted TNFo, either murine or human, in the supernatant was then detected with an ELISA kit.
  • the amount of TNFa secretion from RAW 264.7 cells ( Figure 3a) in the untreated (UT) control (vehicle control) is equal to that of ODN and rHA WT, while CpG induces a low, but detectable signal.
  • the amount of TNFa secretion stimulated by the three CpG-rHA variants (around 2000 pg/mL) is ⁇ 9-10 times higher than the amount induced by free CpG. Showing that the constructs works as intended.
  • the equal stimulation achieved by the three CpG-rHA variants could indicate that a 24 h incubation is too short to detect differences between variants. LPS was added as a positive control.
  • TNFa secretion from human PBMCs Figure 3b.
  • UT control, ODN and rHA WT have equal amount of secreted TNFa.
  • the secretion induced by CpG is higher of that induced by the ODN control, however, the PBMCs show a lower secretion compared to that detected in RAW 264.7 cells.
  • the three CpG-rHA variants induce different amounts of TNFa (CpG- rHA NB, WT and HB induce 700 pg/mL, 1100 pg/mL and 800 pg/mL, respectively), but all higher than the free CpG.
  • TNFa secretion induced by CpG-rHA variants compared to free CpG in both human and murine cells could be the result of the rHA endosomal trafficking facilitating the accumulation of CpG in the endosomes, where TLR9 is localised.
  • ODN-rHA WT also promotes secretion of TNFa, an amount 3.6 times lower compared to CpG-rHA WT. The experiment showed a strong effect of CpG-rHA on both murine RAW 264.7 and human PBMC TNFa secretion.
  • TNFa The measurement of the TNFa was performed in collaboration with Professor Soren Riis Paludan (Department of Biomedicine, Aarhus University), with the qPCR experiment being performed by Marie Beck Iversen.
  • TLRs and STING can recognise DNA in the endosomes and the cytoplasm, respectively.
  • PRR pattern recognition receptors
  • THP-1 is a human monocytic cell line; the THP-1 STING KO line does not express the cytosolic DNA sensor STING, while knockout of MyD88 inhibits TLR signaling in THP-1 MyD88 KO.
  • the cells were differentiated in macrophages with PMA for 24 h, then allowed to stabilise in PMA-free medium for 24 h.
  • CpG-rHA variants and controls were incubated with the cells for 6 h, and the amount of TNFo and IFNP mRNA in the cells, compared to 0-actin, was quantified through qPCR ( Figure 4).
  • the stimulation of wild-type THP-1 ( Figure 4a) with the three variants of CpG-rHA resulted in a strong response, with ⁇ 30-36 times more TNFo expression compared to the UT cells.
  • the type of rHA variant conjugated to CpG appears to have only a small effect on TNFo expression after 6 h of incubation.
  • the control ODN-rHA WT also stimulated TNFo expression, but only 10 times more than the UT cells.
  • the TNFo expression of rHA WT was equal to that of the UT control, indicating that rHA has no immunostimulatory effect.
  • the lack of TNFo expression in the rHA WT control support the hypothesis that the high response seen with CpG-rHA is caused by CpG-mediated TLR signalling.
  • rHA could improve CpG internalisation in the endosomal compartment, where TLR9 is localised, thus improving CpG stimulation.
  • the coadministration of CpG and rHA WT did not influence TNFo expression, demonstrating that the improvement in CpG efficacy is dependent on the conjugation to rHA.
  • the free CpG control also did not have an effect on TNFo expression. This is probably due to the low concentration, or the short 6 hours incubation times, used in the experiment.
  • IFNP expression was affected only by the presence of cGAMP, which was added only in wild-type THP-1 cells ( Figure 4a) as a positive control.
  • the rest of the samples had no effect on IFNP expression in either of the cells lines used in the experiment ( Figure 4a, b and c). This could be explained by IFN[3 being mainly a marker of STING activation, suggesting CpG-rHA is not entering the cells cytoplasm; for detection of TLR9 activation, IFNo expression has been canonically used.
  • TNFo expression level of the UT control, and other controls, in THP-1 STING KO is higher than that in the wild-type THP-1 cells ( Figure 4b). It has been previously shown that mouse STING-deficient macrophages were hyperresponsive to TLR ligands and produced "abnormally high levels of proinflammatory cytokines". The knockout of STING could therefore cause the increased TNFo expression in THP-1 STING KO cells.
  • the high levels of cytokines could also be related to the differentiation of THP-1 cells with PMA; a longer "rest period" in PMA free medium could help to restore the expression of TNFo to the levels detected with the wild-type THP-1 cells ( Figure 4a).
  • TNFo expression by the CpG conjugated are similar to the wild-type THP-1 cells (figure 4b).
  • the TNFo induction in cells not expressing STING supports the hypothesis that CpG-rHA is not activating the cytosolic STING pathway, but that the response is caused by CpG-mediated activation of TLRs in the endosomes.
  • the hypothesis is reinforced by the complete absence of TNFo induction, compared to the UT control, in the THP-1 MyD88 KO cells ( Figure 4c), in which TLR signaling is inhibited.
  • a partner molecule comprising an anti-PD-Ll nanobody (patent WO 2019/166622A1) fused to albumin variants, to which a single CpG-ODN is conjugated on albumin cysteine 34, synergistically combines the effect of checkpoint inhibition with TLR9 stimulation.
  • albumin as a scaffold for the combination of checkpoint inhibitor and CpG ODN in one single construct would ensure spatiotemporal control on the effect of the drug, synergistic effect of the two drugs to boost both the innate and adaptive immune systems, and long lasting effects. Furthermore, the combination would reduce the number of injections required per cycle of treatment, improving patient compliance.
  • the proteins were designed using the software CLC Main Workbench (version 21.0.4).
  • the secreted version, without propeptide, of the rHA sequence (UniProtKB - Q56G89), modified to obtain NB and HB (QMP) variants, were fused at the N-terminal to the anti-PD-Ll nanobody (patent WO 2019/166622A1) C- terminal with a flexible GGGGS (SEQ ID NO: 15) peptide linker and inserted into pcDNA3.1 plasmids.
  • the plasmids encoding the three rHA-anti-PD-Ll variants (NB, WT and HB) were amplified in E.
  • rHA-anti-PD-Ll was purified from the supernatant through affinity chromatography with an anti-HSA CaptureSelect® column. The albumin-containing fractions were pooled, concentrated and the buffer exchanged to PBS with 30 MWCO spin filters.
  • the purified proteins were run on an SDS-PAGE and stained with Coomassie brilliant blue ( Figure 5) Coomassie stained SDS-PAGE showing purified rHA-anti- PD-Ll NB, WT and HB variants. rHA included as control.
  • the thick bands of the three rHA-anti-PD-Ll variants are aligned with the protein ladder 80 kDa band (predicted MW of rHA-anti-PD-Ll of 80.11 kDa). This result confirms the integrity and purity of the proteins.
  • the CpG was conjugated to rHA-anti-PD-Ll through an SM(PEG)8 crosslinker, followed by purification with IEX HPLC.
  • the purified construct CpG-rHA-anti-PD-Ll was run on a Native-PAGE, with unconjugated rHA, rHA-anti-PD-Ll and CpG-rHA WT as controls, to evaluate the purity of the constructs ( Figure 2).
  • nucleic acids were stained with SYBR Gold, and after SYBR staining the gels were stained with Coomassie Blue to detect proteins.
  • the bands of CpG-rHA-anti- PD-L1 in the SYBR Gold stain show high degree of CpG conjugation and purity. The presence of multiple bands indicates the formation of dimers. Affinity studies towards FcRn
  • the HB variants of rHA ( Figure 6c) and rHA-anti-PD-Ll ( Figure 6d) showed an affinity of 30 nM and 43 nM respectively, ⁇ 93 and ⁇ 83 times higher than the WT counterparts.
  • the steric hindrance of fused moieties at the N-terminal of albumin can lower the affinity of rHA for FcRn, however, the difference in the KD between rHA and corresponding rHA-anti-PD-Ll variants detected with BLI are not likely to produce appreciable differences physiologically.
  • the cellular FcRn-mediated rescue of albumin-based designs from lysosomal degradation was measured with a recycling assay.
  • the ability of rHA-anti-PD-Ll and CpG-rHA-anti-PD-Ll to be internalised by cells, interact with FcRn in the endosome and be directed and released in the supernatant was compared to the rHA variants ( Figure 7). While the rHA NB (non-binder) was not rescued, the rHA WT and HB variants show recycling, with 2.7-fold more of the rHA HB released back into the medium compared with the WT.
  • rHA-anti-PD-Ll and CpG-rHA- anti-PD-Ll the recycled amount was respectively 2.4-fold and 11.6-fold higher than the rHA WT.
  • the high recycling detected for CpG-rHA-anti-PD-Ll is likely the result of the conjugation of the fusion protein with the phosphorothioate backbone oligo, that interact with other receptor on the cell surface and allow internalisation.
  • the constructs mediated inhibition of the PD-1/PD-L1 axis was measured with a PD- 1/PD-L1 Blockade Bioassay (Promega ⁇ ).
  • the assay includes two engineered cells lines: Jurkat T cells expressing PD-1 and, upon activation of TCR signaling, a luciferase reporter; and CHO-K1 cells expressing human PD-L1 and an engineered surface protein that activates TCR in an antigen-independent manner.
  • the PD-1/PD-L1 interaction inhibits the TCR induced production of luciferase in Jurkat cells. Disruption of the checkpoint molecules interaction results in the production of luciferase, that can in turn be used to generate a measurable signal upon the addition of luciferin.
  • the EC50 of the anti-PD-Ll antibody positive is an order of magnitude lower of those of the other constructs, possibly due to antibodies possessing two antigen-binding sites, while the rHA-anti-PD-Ll has only one.
  • the human PBMCs are exposed to CpG-rHA-anti-PD-Ll, following the protocol used in disclosed above (Secretion of TNFo).
  • the design follows the same rationale as CpG-rHA-anti-PD-Ll, using albumin as a scaffold to combine LiTE (see below) and CpG ODN in order to boost the innate and adaptive immune systems, and long lasting effects.
  • Albu-LiTE comprises a bispecific "light" T-cell engager (LiTE) antibody, formed by an anti-EGFR nanobody fused with an anti-CD3 scFv, fused to albumin variants.
  • a single CpG-ODN is conjugated on albumin cysteine 34, to combine the T-cell redirection with TLR9 stimulation.
  • the secretion of TNFo induced by CpG-Albu-LiTE was also evaluated in murine RAW 264.7 ( Figure 10), following the same protocol as for CpG-rHA-anti-PD-Ll.
  • Albu-LiTE induces a TNFo secretion of 3579 pg/mL, 2.39 times higher compared to CpG-rHA WT (1428 pg/mL).
  • the conjugation of CpG and Albu-LiTE on CpG- Albu-LiTE increases the amount of secreted TNFo to 11670 pg/mL, showing the same synergistic effect measured with CpG-rHA-anti-PD-Ll.
  • the conjugate CpG-AlbuFel dl elicited a surprisingly high level of TNFa secretion, with 977 pg/mL.
  • the RPMI control showed no TNFa secretion (below detection of the ELISA kit).
  • ODN-rHA WT also promotes secretion of TNFa, an amount 3.6 times lower compared to CpG-rHA WT.
  • the experiment showed a strong effect of CpG- AlbuFel dl on human PBMC TNFa secretion.
  • CpG-AlbuFel dl exploits albumin for the co-delivery of a CpG ODN class B (2006, human specific) and the cat allergen Fel dl.
  • the conjugation of CpG to the protein fusion yielded higher TNFa secretion from PBMCs in vitro, an unexpected synergistic secretion of TNFa, as seen in the previous examples.
  • the study further tests, whether engagement to FcRn and ACE2 is retained within the construct, and retained availability of epitopes on the RBD antigen. Lastly, the study further shows improved intranasal vaccination in vivo, measurement of antigen-positive B-cells in lymph nodes and antibody titer. Results
  • the vaccine CpG-RBD-QMP joins a CpG class B (1826, mouse specific) and an antigen, on an albumin scaffold with high-FcRn affinity (QMP).
  • the CpG-RBD-QMP conjugate retained the affinity for FcRn, ACE2 and the accessibility to the epitopes on RBD.
  • CpG-RBD-QMP yielded higher amount of antigen-specific B-cells in LN and antibody titer in the nose mucosa compared to RBD-QMP mixed with CpG non-covalently.
  • Example 7 wt and HB albumin accumulation in lymph nodes
  • the Alexa680-albumin variants WT and HB were injected subcutaneously at the tail base, and after 24 h a blood sample and the inguinal, axillary and brachial lymph nodes were collected (Figure 16 a).
  • the background signal from PBS control was subtracted from the lymph nodes (LN) signal and the resulting signal normalised to WT albumin.
  • the average signal from all the LN of each mouse shows a ⁇ 2 fold increase of HB accumulation ( Figure 16 b).
  • the inguinal LN show a ⁇ 2 fold increase from WT to HB (Figure 16 ci), the axillary a ⁇ 3 fold increase ( Figure 16 cii) and the brachial a ⁇ 2 fold increase ( Figure 16 ciii).
  • the similar concentration of WT and HB variants in the inguinal LN could be due to the proximity to the injection site, while more distant LN show a different trend, with higher HB accumulation.
  • the concentration of albumin WT and HB in the serum were significantly different at 24 h, with a 1.5 fold increase for the HB variant ( Figure 16 d).
  • Albumin variants accumulate in the lymph nodes, verifying the rationale for conjugating to albumin, to increase the immune system presentation.
  • albumin WT and HB resulted in different pharmacokinetic profiles of the two variants.
  • Albumin HB concentration in the serum and LN, 24 h after the injection was higher than the WT variant. This result shows that in some instances, albumin variants, with high FcRn affinity, may preferably be used, for example to further increase lymph node accumulation and increase immune system presentation.
  • rHA-anti-PD-Ll fusions retained the affinity of rHA variants for FcRn and of the nanobody for PD-L1, as demonstrated by Bio-Layer Interferometry, cellular recycling and PD-1/PD-L1 Blockade Bioassay, respectively.
  • CpG-rHA induced higher TNFo secretion, compared to free CpG, in murine RAW 264.7 and human PBMC cells.
  • CpG-rHA induces TNFo expression through activation of TLR, and not STING, in THP-1 cells.
  • CpG-rHA-anti-PD-Ll showed a high degree of endosomal-recycling and retained function of CpG, anti-PD-Ll nanobody and albumin.
  • Both CpG-rHA-anti-PD-Ll and CpG-Albu-LiTE exhibited a surprisingly high degree of synergistic effect, compared to the previous constructs, as shown in the stimulation of TNFo secretion from RAW 264.7 murine cells.
  • CpG-AlbuFel dl exploits albumin for the co-delivery of a CpG ODN class B (2006, human specific) and the cat allergen Fel dl.
  • the conjugation of CpG to the protein fusion yielded higher TNFo secretion from PBMCs in vitro.
  • the vaccine CpG-RBD-QMP joins a CpG class B (1826, mouse specific) and an antigen, on an albumin scaffold with high-FcRn affinity (QMP).
  • the CpG-RBD-QMP conjugate retained the affinity for FcRn, ACE2 and the accessibility to the epitopes on RBD.
  • CpG-RBD-QMP yielded higher amount of antigen-specific B-cells in LN and antibody titer in the nasal mucosa compared to RBD-QMP mixed with CpG non-covalently.
  • Albumin variants accumulate in the lymph nodes, verifying the rationale for conjugating to albumin, to increase the immune system presentation.
  • albumin WT and HB resulted in different pharmacokinetic profiles of the two variants.
  • Albumin HB concentration in the serum and LN, 24 h after the injection was higher than the WT variant. This result shows that in some instances, albumin variants, with high FcRn affinity, may preferably be used, for example to further increase lymph node accumulation and increase immune system presentation.
  • a compound comprising: a. a protein conjugate comprising an albumin, preferably a highbinder albumin, and one or more proteinaceous parts; b. one or more CpG oligonucleotides (ODN) covalently conjugated to the albumin, preferably via thiols such as Cys34 of albumin; and c. optionally, a linker connecting the albumin and the one or more proteinaceous parts; wherein the one or more proteinaceous parts are selected from the group consisting of an antigen-targeting moiety or fragment thereof, an immunogenic protein or a fragment thereof, and a tolerogenic protein or fragment thereof.
  • ODN CpG oligonucleotides
  • ODN comprises a class A ODN, class B ODN, such as CpG 2006 according to SEQ ID NO: 12, or a class C ODN, preferably class C ODN, such as CpG 2395 according to SEQ ID NO: 13.
  • ODN is selected from the group consisting of CpG 2395 according to SEQ ID NO: 13, and CpG 2006 according to SEQ ID NO: 12.
  • the ODN comprises modified nucleotides, such as comprising one or more artificial nucleotides, such as one or more locked nucleic acids (LNAs), such as comprising or consisting of one or more phosphorothioate bonds between individual nucleotides in the backbone of the ODN.
  • LNAs locked nucleic acids
  • the ODN activates an immune response, such as via activation of TLR preferably via the TLR9 receptor.
  • the ODN activates one or more immune cells, such as dendritic cells, B cells, macrophages and/or NK cells, preferably antigen-presenting cells.
  • immune cells such as dendritic cells, B cells, macrophages and/or NK cells, preferably antigen-presenting cells.
  • the one or more proteinaceous parts is an antigen-targeting moiety or a fragment thereof such as an antibody, single domain antibody, diabody, a singlechain variable fragment, affibody or a DARPin.
  • antigen-targeting moiety is a multispecific antigen-targeting moiety, such as a bispecific antigen-targeting moiety.
  • an immune checkpoint selected from the group consisting of PD-1, PD-L1, CTLA-4, VISTA, TIM-3, LAG-3, IDO, KIR2D, A2AR, B7-1, B7-H3, TIGIT and BTLA.
  • the immune checkpoint inhibitor is selected from the group consisting of PD-1 antagonists, PD-L1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-1 antagonists, B7- H3 antagonists, B7-H4 antagonists, and BTLA antagonists.
  • the immune checkpoint inhibitor is capable of blocking the signal of the immune checkpoint.
  • the antigen-targeting moiety is an immune checkpoint costimulatory agonist, such as 4-1BB, CD28 or CD16A.
  • the immune checkpoint inhibitor is a PD-L1 antagonist, such as the PD-L1 antagonist according to amino acids 1-121 of SEQ ID NO: 8.
  • the multispecific antigen-targeting moiety targets at least one antigen located on a cancer cell or an immune cell.
  • the compound according to item 18, wherein the at least one antigen located on a cancer cell is selected from the group consisting of EGFR, PSMA, folate receptor, RGD peptide, transferrin, FcRn, and HEU2.
  • the compound according to item 18, wherein the at least one antigen located on an immune cell is selected from the group consisting of CD3, CD16, and CD16A.
  • the compound according to any of items 18-20, wherein the multispecific antigen-targeting moiety is a bispecific antigen-targeting moiety targeting EGFR and CD3, such as the bispecific antibody according to SEQ ID NO: 10.
  • the one or more proteinaceous parts is an immunogenic- or tolerogenic protein or a fragment thereof.
  • the compound according to item 22, wherein the immunogenic- or tolerogenic protein or a fragment thereof is capable of binding to a B-cell or an MHC-I or MHC-II molecule on an antigen-presenting cell.
  • the albumin has at least 80% sequence identity to the sequence according to SEQ ID NO: 2.
  • the compound according to any of the preceding items, wherein the albumin is capable of binding to the FcRn receptor.
  • the albumin comprises one or more additional artificial cysteines, such as the cysteine introducing mutations K93C, or E294C of albumin.
  • albumin is a highbinder albumin, such as having one or more improved pharmacokinetic properties when compared with wild type albumin such as providing a long lasting effect, having an increased binding affinity towards the FcRn receptor, having a longer half-life, having a higher binding affinity to FcRn, and/or endosomal-mediated cellular recycling, preferably wherein the highbinder is selected from the group consisting of: a. the albumin according to any of the preceding items having one or more of the mutations E505Q, T527M, and/or K573P, such as the QMB highbinder according to SEQ ID NO: 6; b.
  • HBI highbinder I
  • HBII highbinder II
  • connection via the ODN is through a double stranded ODN
  • albumin is conjugated to a first ODN
  • the one or more proteinaceous parts is conjugated to a second ODN
  • the first- and second ODN being complementary to each other thereby capable of forming the double stranded ODN upon contact.
  • albumin is positioned at or near the N-terminal- or the C-terminal end of the protein conjugate, in relation to the one or more proteinaceous parts, such as at most 50 additional amino acids between albumin and the N- terminal- or the C-terminal end of the protein conjugate.
  • composition comprising the compound according to any of the preceding items.
  • composition according to item 34 wherein the composition comprises a pharmaceutically acceptable carrier.
  • a solid cancer such as a solid cancer selected from the group consisting of breast cancer, colorectal cancer, lung cancers, pancreatic cancers, liver cancers, intestinal cancers, prostate cancers, bladder cancers, kidney cancers, such as renal clear cell carcinoma, ovarian cancers, cervical cancers, adenocarcinomas, squamous cell carcinomas, head and neck cancer, ear or nose or throat cancers, skin cancers, such as basal cell carcinomas, melanomas, non-melanoma skin cancers, squamous cell carcinomas of the skin, such as a liquid cancer such as leukemia, such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML).
  • ALL acute lymphocytic leukemia
  • AML acute myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelog
  • a method of treatment comprising administering a therapeutic amount of the compound according to any of items 1-33, or the composition according to any of items 34-35 to a subject in need thereof.
  • a method for producing the compound or the composition according to any of items 1-35 comprising: a. providing one or more nucleic acid(s) encoding the protein conjugate or its individual parts according to any of items 1-33, or one or more expression vector(s) comprising said nucleic acid(s); b. introducing the nucleic acid(s) or the vector(s) into one or more host cell(s), such as host cells selected from the group consisting of: bacteria and eukaryotes; c.
  • the host cell(s) under conditions that allow for expression of the protein conjugate from the nucleic acid(s) or the vector(s); d. purifying the protein conjugate, or its individual parts; e. providing CpG oligonucleotides; and f. conjugating the CpG oligonucleotides to the purified protein conjugate or its individual parts, thereby obtaining the compound or the composition.
  • the CpG oligonucleotides are Maleimide modified oligonucleotides, such as monobromide maleimide (MBM), lysine-modified oligonucleotides, preferably Maleimide modified oligonucleotides.
  • step 40 or item 41 wherein the compound obtained in step f is purified.
  • step f The method according to any of items 40-42, wherein the protein conjugate's individual parts of step f are brought in contact with each other to allow them anneal.
  • step f The method according to any of items 40-43, wherein Maleimide modified oligonucleotides are prepared by combining a CpG-NH2 oligonucleotide with Maleimide, such as Maleimide-(PEG) n -NHS-ester, wherein n is a number between 1-10, succinimidyl 4-(N-maleimidomethyl)cyclohexane- 1-carboxylate (SMCC), and optionally purifying the Maleimide modified oligonucleotides.
  • Maleimide modified oligonucleotides are prepared by combining a CpG-NH2 oligonucleotide with Maleimide, such as Maleimide-(PEG) n -
  • kits comprising: a. the protein conjugate or its individual parts according to any of the items items 1-33; b. a CpG oligonucleotide, such as a CpG-NH2, such as a Maleimide modified oligonucleotide; c. reagents for conjugating the CpG oligonucleotide to the protein conjugate.
  • a vaccine comprising the compound or the composition as defined in any of items 1-9 and 22-33.
  • a compound comprising: a. a protein conjugate comprising an albumin, preferably a highbinder albumin, and one or more proteinaceous parts; b. one or more CpG oligonucleotides (ODN) covalently conjugated to the albumin, preferably via thiols such as Cys34 of albumin; and c. optionally, a linker connecting the albumin and the one or more proteinaceous parts; wherein the one or more proteinaceous parts are selected from the group consisting of an antigen-targeting moiety or fragment thereof, an immunogenic protein or a fragment thereof, and a tolerogenic protein or fragment thereof.
  • ODN CpG oligonucleotides
  • TLR9 activates one or more immune cells via TLR9, such as dendritic cells, B cells, macrophages and/or NK cells, preferably antigen-presenting cells.
  • the one or more proteinaceous parts is an antigen-targeting moiety or a fragment thereof such as a multispecific antigen-targeting moiety, such as an antibody, single domain antibody, diabody, a single-chain variable fragment, affibody or a DARPin.
  • the antigen-targeting moiety is an immune checkpoint inhibitor selected from the group consisting of PD-L1 antagonists, such as SEQ ID NO: 8, PD-1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-1 antagonists, B7-H3 antagonists, B7-H4 antagonists, and BTLA antagonists.
  • PD-L1 antagonists such as SEQ ID NO: 8
  • PD-1 antagonists such as SEQ ID NO: 8
  • PD-L2 antagonists such as SEQ ID NO: 8
  • CTLA-4 antagonists such as PD-1 antagonists
  • VISTA antagonists such as PD-1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-1 antagonist
  • the multispecific antigen-targeting moiety is a bispecific antigen-targeting moiety targeting EGFR and CD3, such as the bispecific antibody according to SEQ ID NO: 10.
  • the compound according to any of embodiments 1-2, wherein the one or more proteinaceous parts is an immunogenic protein or a fragment thereof or a tolerogenic protein or a fragment thereof, capable of binding to a B-cell or an MHC-I or MHC-II molecule on an antigen-presenting cell.
  • albumin is a highbinder albumin, such as having one or more improved pharmacokinetic properties when compared with wild type albumin such as providing a long lasting effect, having an increased binding affinity towards the FcRn receptor, having a longer half-life, having a higher binding affinity to FcRn, and/or endosomal-mediated cellular recycling, preferably wherein the highbinder is selected from the group consisting of: a. the albumin according to any of the preceding embodiments having one or more of the mutations E505Q, T527M, and/or K573P, such as the QMB highbinder according to SEQ ID NO: 6; b.
  • HBI highbinder I
  • HBII highbinder II
  • SEQ ID NO: 5 The compound according to any of the preceding embodiments, wherein the albumin and the one or more proteinaceous parts are connected via the ODN.
  • a composition comprising the compound according to any of the preceding embodiments.
  • a solid cancer such as a solid cancer selected from the group consisting of breast cancer, colorectal cancer, lung cancers, pancreatic cancers, liver cancers, intestinal cancers, prostate cancers, bladder cancers, kidney cancers, such as renal clear cell carcinoma, ovarian cancers, cervical cancers, adenocarcinomas, squamous cell carcinomas, head and neck cancer, ear or nose or throat cancers, skin cancers, such as basal cell carcinomas, melanomas, non-melanoma skin cancers, squamous cell carcinomas of the skin, and b.
  • breast cancer colorectal cancer
  • lung cancers pancreatic cancers
  • liver cancers intestinal cancers
  • prostate cancers bladder cancers
  • kidney cancers such as renal clear cell carcinoma, ovarian cancers, cervical cancers, adenocarcinomas, squamous cell carcinomas, head and neck cancer, ear or nose or throat cancers
  • skin cancers such as basal cell carcinomas, melanomas, non-
  • a liquid cancer such as leukemia, such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML).
  • ALL acute lymphocytic leukemia
  • AML acute myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • nucleic acid(s) or the vector(s) into one or more host cell(s), such as host cells selected from the group consisting of: bacteria and eukaryotes; c. growing the host cell(s) under conditions that allow for expression of the protein conjugate from the nucleic acid(s) or the vector(s); d. purifying the protein conjugate, or its individual parts; e. providing CpG oligonucleotides, such as Maleimide modified oligonucleotides; and f. conjugating the CpG oligonucleotides to the purified protein conjugate or its individual parts, thereby obtaining the compound or the composition. vaccine comprising the compound as defined in any of embodiments 1- or 8-10.

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Abstract

The present invention relates to albumin-oligodeoxynucleotides conjugates, such as compounds, and compositions comprising a protein conjugate, the conjugate comprising an albumin, one or more proteinaceous parts, and one or more CpG oligonucleotides (ODN). These compounds and compositions finds use as medicaments and/or vaccines, and in methods of treatment. Also disclosed are methods for producing the compound or the composition and kits comprising the necessary tools.

Description

Albumin conjugated to CpG oligodeoxynucleotides as super-boosters of immune response
Technical field of the invention
The present invention relates to vaccines and compounds for treating cancerous disease. In particular, the present invention relates to albumin conjugated CpG oligonucleotides to one or more partner molecules, such as immune checkpoint inhibitors and multispecific antigen-targeting moieties, these compounds are in particular useful for treating cancerous disease.
Background of the invention
Oligodeoxynucleotides containing unmethylated CG dinucleotides (CpG ODN) adjuvants have potent immunostimulatory effects and can enhance the anticancer activity of a variety of therapeutics. CpG ODN interact with Toll-like receptor 9 (TLR9) in the endosomal compartment of dendritic cells, macrophages and B-cells to induce differentiation and cytokine production. The short in vivo half-life of CpG ODN, however, together with the negatively charged DNA backbone repelled by cell membranes, make it difficult to attain an appropriate cell accumulation, endosomal localisation, and immune stimulation. Furthermore, inadequate accumulation of adjuvants in lymphoid organs rich in immunocompetent cells reduces the efficacy.
Human serum albumin (HSA) is a natural transport protein with binding sites for both endogenous and exogenous ligands, a free cysteine at position 34, and has a long circulatory half-life of ~19 days. This long in vivo half-life is the result of the engagement of HSA with FcRn, a receptor located in the endosome that rescues bound proteins from lysosomal degradation. A number of natural albumin variants have been described. Otagiri et ai, 2009, Biol. Pharm. Bull. 32(4), 527-534, discloses 77 known albumin variants. Furthermore, human made albumin variants with altered binding affinity to FcRn has been described in WO 2011 /051489, WO 2011/124718, WO 2012/059486, WO 2012/150319, WO 2011/103076, WO 2012/112188 WO 2013/075066, WO 2014/072481, WO 2014/125082 and WO 2015/63611. WO 2013/135896 discloses albumin variants having one or more (e.g. several) alterations in Domain I and one or more (e.g. several) alterations in Domain III. WO 2015/036579 discloses albumin variants having one or more (e.g. several) alterations in Domain II. In sum, several albumin variants are known to the skilled person.
Zhu et al. Albumin/vaccine nanocomplexes that assemble in vivo for combination cancer immunotherapy. Nature Communications volume 8, Article number: 1954 (2017)) discloses conjugating molecular vaccines with Evans blue (EB) into albumin-binding vaccines.
Liu et al. (Structure-based programming of lymph node targeting in molecular vaccines. 2014 | Vol 507 | NATURE | 519) discloses 'albumin hitchhiking' approach to molecular vaccines, through the synthesis of amphiphiles (amph- vaccines) comprising an antigen or adjuvant cargo (such as CpG-DNA) linked to a lipophilic albumin-binding tail by a solubility-promoting polar polymer chain.
Induction of the immune system is sought after for many different applications. Vaccines require adjuvants, cancer cells increase immune checkpoint receptors to lower the immune response and cancer treatments, in general, benefit from an increased immune response. However, combination therapies, combining an agent for stimulating the immune system with a vaccine/ cancer treatment, result in a spatial displacement of activation of immune response and treatment/vaccination.
Hence, it would be advantageous to increase the half-life and access to CpG oligonucleotides, for immune system stimulation, and in particular to spatially combine this increase in immune stimulation with known therapies.
Summary of the invention
The present invention overcomes these unmet challenges through the conjugation of CpG ODN to albumin variants or albumin fusions engineered for tuned optimal binding to the neonatal Fc receptor (FcRn) that facilitates extended half-life, lymph node accumulation and greater endosomal accumulation rich in toll-like receptors (figure 1). The albumin fusions being applicable for use in vaccines and cancer treatments. Zhu et al. and Liu et al. employs endogenous albumin, and is silent in respect of using albumin variants and fusing albumin to other proteins. Furthermore, Liu et al. is silent in respect of coupling via Cys34, and the use of albumin variants.
The albumin-ODN conjugates have a surprisingly high activation on the immune system on their own - CpG-rHA induced higher TNFo secretion, compared to free CpG, in murine RAW 264.7 and human PBMC cells (example 2). Thus, an aspect of the present invention is an albumin compound comprising an albumin variant and one or more ODNs covalently conjugated to albumin.
Surprisingly, the inventors of the present invention found that when fusing an immune checkpoint inhibitor with an albumin molecule, conjugated to CpG ODN, a surprisingly high stimulation of the immune system was observed (example 3). This effect was further confirmed when albumin was fused to a bispecific T-cell engager (CpG-Albu-LiTE), as shown in example 4.
CpG-rHA-anti-PD-Ll showed a high degree of endosomal-driven recycling and retained function of CpG, anti-PD-Ll nanobody and albumin. Both CpG-rHA-anti- PD-L1 and CpG-Albu-LiTE exhibited a surprisingly high degree of synergistic effect (example 3 and 4).
Examples 5-6 further shows how vaccines can be generated (cat allergen Fel dl and the corona virus derived RBD (receptor binding domain) protein), that may either induce tolerance or increase the immune response against the parts included in the vaccine.
Example 7 shows how the presentation to the immune system, more specifically in lymph nodes, can be tuned via the use of high binding albumin.
These examples, thus, effectively show how coupling CpG and proteinaceous parts to endosomal recycling, via albumin or variants thereof, result in highly effective molecules.
Thus, one aspect of the invention relates to a compound comprising: a. a protein conjugate comprising an albumin, preferably a highbinder albumin, and one or more proteinaceous parts; b. one or more CpG oligonucleotides (ODN) covalently conjugated to the albumin, preferably via thiols such as Cys34 of albumin; and c. optionally, a linker connecting the albumin and the one or more proteinaceous parts; wherein the one or more proteinaceous parts are selected from the group consisting of an antigen-targeting moiety or fragment thereof, an immunogenic protein or a fragment thereof, and a tolerogenic protein or fragment thereof.
Another aspect of the present invention relates to a composition, comprising the compound according to the first aspect of the invention.
In a further aspect, the invention relates to these compounds and compositions as for use as medicaments and/or vaccines.
Thus, an aspect relates to a method of treatment, comprising administering a therapeutic amount of the compound according to the first aspect, or the composition the second aspect to a subject in need thereof.
As such an aspect of the invention is also a vaccine comprising the compound or the composition as defined herein.
Yet another aspect of the present invention is to provide a method for producing the compound or the composition according to the previous aspects, the method comprising: a. providing one or more nucleic acid(s) encoding the protein conjugate or its individual parts according to the previous aspects, or one or more expression vector(s) comprising said nucleic acid(s); b. introducing the nucleic acid(s) or the vector(s) into one or more host cell(s), such as host cells selected from the group consisting of: bacteria and eukaryotes; c. growing the host cell(s) under conditions that allow for expression of the protein conjugate from the nucleic acid(s) or the vector(s); d. purifying the protein conjugate, or its individual parts; e. providing CpG oligonucleotides; and f. conjugating the CpG oligonucleotides to the purified protein conjugate or its individual parts, thereby obtaining the compound or the composition, optionally, wherein the CpG oligonucleotides are Maleimide modified oligonucleotides, such as monobromide maleimide (MBM), lysine-modified oligonucleotides, preferably Maleimide modified oligonucleotides, and optionally purifying the compound obtained in step f, optionally, bringing the protein conjugate's individual parts in contact with each other to allow them anneal.
Still another aspect of the present invention is to provide a kit comprising: a. the protein conjugate or its individual parts according to the previous aspects; b. a CpG oligonucleotide, such as a CpG-NH2, such as a Maleimide modified oligonucleotide; c. reagents for conjugating the CpG oligonucleotide to the protein conjugate.
Brief description of the figures
Figure 1 shows 1) CpG-conjugated albumin fusion enters the endosome of a dendritic cell. 2) The acidification of the endosome generates conformational changes in albumin and FcRn that results in albumin-FcRn binding. This FcRn- driven endosomal compartmentalisation facilitates CpG co-localisation with TLR9 found in the endosome and the activation of TLR9 by CpG (conjugated to rHA). Higher FcRn binding of albumin variants potentiates this effect. 3) The activated receptors mediate the production and secretion of IFNs and proinflammatory cytokines in DCs. 4) FcRn and albumin are recycled outside the cell and evade lysosomal degradation.
Figure 2 shows a SYBR Gold and Coomassie Blue stained Native-PAGE showing purified rHA-anti-PD-Ll WT (rHA-@PD-Ll in figures), CpG-rHA WT and CpG-rHA- anti-PD-Ll WT (CpG-@rHA PD-L1 in figures). CpG and rHA included as control. The bands of CpG-rHA in the SYBR Gold stain show a high degree of CpG conjugation and purity. The presence of multiple bands indicates the formation of dimers.
Figure 3 shows secretion of TNFo. Secretion of TNFo in 48 well plate induced by CpG-rHA variants and controls (ODN, CpG, LPS, rHA and vehicle control) from a) murine RAW 264.7 cells b) human PBMC, as detected by ELISA. (N = l for CpG- rHA NB; N = 2 for CpG-rHA highbinder (HB), ODN-rHA WT, LPS and rHA; N = 3 for ODN, CpG, CpG-rHA WT and vehicle control)
Figure 4 shows expression of TNFo. Expression fold change of TNFo and IFN[3, compared to p-actin, induced by CpG-rHA variants and control (untreated, cGAMP, rHA WT, CpG and CpG + rHA WT) in THP-1 a) WT, b) STING KO and c) MyD88 KO cells as detected by qPCR. N = 1
Figure 5 shows coomassie stained SDS-PAGE showing purified rHA-anti-PD-Ll, WT and HB variants. rHA included as control
Figure 6 shows FcRn binding of rHA-anti-PD-Ll. The sensorgrams show binding to FcRn (solid black line) and 1: 1 binding model fit (dashed grey lines). BLI sensorgrams showing association (90-210 s) and dissociation (210-510 s) to immobilised human FcRn of a) rHA WT, b) rHA-anti-PD-Ll WT, c) rHA HB and d) rHA-anti-PD-Ll HB. The analytes contain a five step two-fold dilution series from 3 pM. N = l.
Figure 7 shows FcRn-mediated cellular recycling. Detection of recombinant albumin in supernatant from human FcRn expressing endothelial cells. N = 2 (rHA HB N = l).
Figure 8 shows PD-1/PD-L1 Blockade Bioassay. The graphs show the fold induction of luminescence as a function of the loglO of the concentration of i) anti-PD-Ll antibody, ii) anti-PD-Ll nanobody, iii) rHA-anti-PD-Ll and iv) CpG- rHA-anti-PD-Ll. N = 3.
Figure 9 shows secretion of TNFo. Secretion of TNFo in 96 well plate induced by CpG-rHA and CpG-rHA-anti-PD-Ll and controls (ODN, CpG, and vehicle control) from murine RAW 264.7 cells, as detected by ELISA. N = 2.
Figure 10 shows secretion of TNFo. Secretion of TNFo in 96 well plate induced by CpG-rHA and CpG-AlbuLITE and controls (ODN, CpG, AlbuLITE and vehicle control) from murine RAW 264.7 cells, as detected by ELISA. N = 2.
Figure 11 shows schematic representations of embodiments described herein.
A) shows an embodiment where albumin is conjugated to a CpG oligonucleotide (ODN). B) and C) show embodiments where albumin is conjugated to an ODN and one or more proteinaceous part through linkers.
D) shows an embodiment where albumin and a proteinaceous part are themselves conjugated to ODNs, the ODNs being complementary to each other and form a double stranded ODN, thereby linking the albumin to the proteinaceous part.
E) shows an embodiment which is a combination of B) and D).
F) shows an embodiment which is a combination of C) and D).
G) shows an embodiment where albumin is conjugated to multiple ODNs.
Figure 12 shows secretion of TNFo. Secretion of TNFo in 96 well plate induced by CpG-AlbuFel dl and controls (AlbuFel dl, CpG, AlbuFel dl + CpG, and vehicle control) from human PBMC, as detected by ELISA. (N = 1)
Figure 13 shows a) Chromatogram of IEX HPLC purification of CpG-RBD-QMP. b) SYBR Gold and Coomassie stained Native-PAGE showing purified CpG-RBD-QMP. CpG 1826 and RBD-QMP included as controls
Figure 14 shows in vitro validation of CpG-RBD-QMP. a) fusions binding to human ACE2. b) fusions binding to human FcRn binding, measured as binding to FcRn at pH 5.5 in ELISA, c) Determination of RBD epitope availability, using the commercial monoclonal antibodies i) cilgavimab, ii) tixagevimab, and iii) sotrovimab, performed in ELISA.
Figure 15 shows intranasal vaccination with CpG-RBD-QMP induces antibodies toward the subunit vaccine antigen a) Percentage antigen-specific B-cells in mediastinal lymph nodes, b) RBD-specific IgG and IgA in nose at endpoint after intranasal vaccination of Tg32-hFc mice.
Figure 16 shows in vivo accumulation of albumin WT and HB in lymph nodes and serum. A) schematic of in vivo study, b) accumulation in all LN. c) accumulation in i) inguinal, ii) axillary, and iii) brachial LN. d) serum accumulation of albumin WT and HB
The present invention will now be described in more detail in the following. Detailed description of the invention
Definitions
Prior to discussing the present invention in further details, the following terms and conventions will first be defined:
Protein conjugate
In the present context, the term "protein conjugate" refers to a compound comprising at least one protein, i.e. albumin, conjugated to one or more proteinaceous parts. Since the "protein conjugate" is comprised of separable constituents, in aspects of the invention is referred to the "protein conjugate's" or its individual parts thereby meaning the separable constituents of the protein conjugate, such as albumin and a proteinaceous part.
Proteinaceous part
In the present context, the term "proteinaceous part" is understood as a constituent of the protein conjugate, which is primarily comprised of amino acids. A "proteinaceous part" is thus also understood as a constituent only comprised of amino acids.
Albumin
In the present context, the term "albumin" refers to serum albumin and fragments or variants thereof. The albumin may be from a human or non-human species including primates, and laboratory test animals e.g. mice, rats, rabbits, guinea pigs, hamsters. One such serum albumin is human serum albumin according to SEQ ID NO: 1 or the secreted version according to SEQ ID NO: 2.
Albumin highbinder
In the present context, the term albumin highbinder or highbinder albumin refers to a variant of albumin having one or more improved pharmacokinetic properties when compared with wild type albumin such as providing a long lasting effect, having an increased binding affinity towards the FcRn receptor, having a longer half-life, having a higher binding affinity to FcRn, and/or improved endosomal- mediated cellular recycling. Examples of highbinders are: a. an albumin having one or more of the mutations E505Q, T527M, and/or K573P, such as the QMB highbinder according to SEQ ID NO: 6, in preferred embodiments referred to as the QMP highbinder, or simply just QMP; b. highbinder I (HBI) according to SEQ ID NO: 4; and c. highbinder II (HBII) according to SEQ ID NO: 5.
Linker
In the present context, the term "linker" refers to a molecule that joins two other molecules, either covalently, or through ionic, van der Waals or hydrogen bonds, e.g., a nucleic acid molecule that hybridises to one complementary sequence at the 5' end and to another complementary sequence at the 3' end, thus joining two non-complementary sequences.
Immune checkpoint inhibitor
In the present context, the term "immune checkpoint inhibitor" refers to the class of immunotherapy drugs called immune checkpoint inhibitors which asserts their effect by blocking checkpoint proteins from binding with their partner proteins, thus allowing the immune system to be more active.
Oligonucleotide
In the present context, the term "oligonucleotide" refers to a sequence of DNA or RNA nucleotide residues that form a nucleic acid molecule. Oligonucleotides can bind their complementary sequences to form duplexes (double-stranded fragments) or even fragments of a higher order.
CpG oligonucleotide (ODN)
In the present context, the term a "CpG oligonucleotide" or "ODN" is understood as a nucleic acid comprising at least one CpG motif. A CpG motif is a short synthetic single-stranded DNA molecule comprising unmethylated CpG dinucleotides. Immune response
In the present context, the term an "immune response" is understood as either a response triggered by the immune system's recognition of ODNs (1) via an innate response, e.g. TLRs, or the immune system's recognition of "non-self proteins" via MHC presentation (2) via an adaptive immune response. The "immune response" is thus an organism's reaction against foreign antigens.
In the adaptive immune response, the first contact that an organism has with a particular antigen will result in the production of effector T and B cells which are activated cells that defend against the antigen. The production of these effector cells as a result of the first-time exposure is called a primary immune response. Memory T and memory B cells are also produced in the case that the same antigen enters the organism again. If the organism does happen to become reexposed to the same antigen, a secondary immune response will kick in and the immune system will be able to respond in both a fast and strong manner because of antibodies and the memory cells from the first exposure.
Antigen-targeting moiety
In the present context, the term an antigen-targeting moiety may be understood as a polypeptide that binds to an antigen present on a cell in need of immunestimulation or in a tissue in need of immunestimulation.
Immunogenic polypeptide
In the present context, the term an "immunogenic polypeptide" is understood as a polypeptide that induces an immune response.
Tolerogenic polypeptide
In the present context, the term a "tolerogenic polypeptide" is understood as a polypeptide for which it is intended to lower the future immune response against.
TLR receptors
In the present context, the term "Toll-like receptors" or "TLRs" are understood as a class of proteins that play a key role in the innate immune system. They are usually expressed on cells that recognise structurally conserved molecules derived from microbes, such as B-cells, macrophages and dendritic cells. Once these microbes have reached physical barriers such as the skin or intestinal tract mucosa, they are recognised by TLRs, which activate immune cell responses. The TLRs include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and TLR13. TLR3, TLR7, TLR8, and TLR9 are located in intracellular vesicles and are specific against nucleic acids.
FcRn receptor
In the present context, the term "FcRn receptor" refers to the neonatal Fc receptor. FcRn extends the half-life of IgG and serum albumin by reducing lysosomal degradation of these proteins. Following entry into cells, the two most abundant serum proteins, IgG and serum albumin, are bound by FcRn at the slightly acidic pH (<6.5) within early (sorting) endosomes, sorted and recycled to the cell surface where they are released at the neutral pH (>7.0) of the extracellular environment.
Sequence identity
In the present context, the term "sequence identity" indicates a quantitative measure of the degree of homology between two amino acid sequences of substantially equal length or between two nucleic acid sequences of substantially equal length. The two sequences to be compared must be aligned to best possible fit with the insertion of gaps or alternatively, truncation at the ends of the protein (Nref-Ndlf)100 sequences. The sequence identity can be calculated as ref , wherein
Ndif is the total number of non-identical residues in the two sequences when aligned and wherein Nref is the number of residues in one of the sequences. Hence, the DNA sequence AGTCAGTC will have a sequence identity of 75% with the sequence AATCAATC (Ndif=2 and Nref=8). A gap is counted as non-identity of the specific residue(s), i.e. the DNA sequence AGTGTC will have a sequence identity of 75% with the DNA sequence AGTCAGTC (Ndif=2 and Nref=8). Sequence identity can alternatively be calculated by the BLAST program e.g. the BLASTP program (W.R Pearson and D . Lipman (1988)). In one embodiment of the invention, alignment is performed with the sequence alignment method ClustalW with default parameters as described by J.D. Thompson et al (1994), available at http://www2.ebi.ac.uk/clustalw/. For calculations of sequence identity when comparing polypeptide fragments with longer amino acid sequences, the polypeptide fragment is aligned with a segment of the longer amino acid sequence. The polypeptide fragment and the segment of the longer amino acid sequence may be of substantially equal length. Thus, the polypeptide fragment and the segment of the longer amino acid sequence may be of equal length. After alignment of the polypeptide fragment with the segment of the longer amino acid sequence, the sequence identity is computed as described above.
A preferred minimum percentage of sequence identity is at least 80%, such as at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.
Compound
As described above, and outlined in the example section, the inventing team has designed the ingenious novel compounds comprising albumin, ODNs and proteinaceous parts, where the proteinaceous parts are chosen with the intended use in mind. Professional antigen presenting cells (APCs) scavenge their surroundings for potential foreign material. In this process, albumin is engulfed and thus taken up by the cell. Some of the albumin is salvaged through structural changes occuring upon acidification of the endosomes, the structural changes allows for albumin to bind to FcRn receptors. Since the endosomes are rich in TLRs, the present invention ingeniously exploits this, and stimulates the APC via the ODN.
More specifically, the present disclosure discloses a compound comprising: a. a protein conjugate comprising an albumin, preferably a highbinder albumin, and one or more proteinaceous parts; b. one or more CpG oligonucleotides (ODN) covalently conjugated to the albumin, preferably via thiols such as Cys34 of albumin; and c. optionally, a linker connecting the albumin and the one or more proteinaceous parts; wherein the one or more proteinaceous parts are selected from the group consisting of an antigen-targeting moiety or fragment thereof, an immunogenic protein or a fragment thereof, and a tolerogenic protein or fragment thereof. As is detailed below, the proteinaceous parts are chosen such that the compound exert the intended effect, be it as a vaccine or a cancer treatment. In example 3 and 4 are disclosed such compounds for cancer treatment comprising either an immune checkpoint inhibitor against PD-L1 or a bispecific anti-EGFR/anti-CD3. However, as will be present from the below disclosure it is possible to combine immune checkpoint inhibitor and the bispecific antibody in the same compound, as well as to exchange the compounds for other compounds, with similar effect. Further examples are for instance the immunogenic protein from the spike protein of the SARS COV2 virus used in example 6 (RBD domain), or the cat allergen as shown in example 5 (Fel dl), showing that the compound finds use in vaccines as well.
CpG oligonucleotide
The inventors of the present disclosure have found that it can be advantageous to connect the ODN, such as via thioether bonds, since it is then possible to specifically design where the ODN is positioned and design the specific stoichiometry. In one embodiment of the present disclosure, the ODN is conjugated to the albumin via a thioether bond, such as to Cys34 of albumin.
In another embodiment, the ODN is conjugated to the albumin amide bonds, such as via one or more lysines on the albumin.
The skilled person knows that ODNs will generally be conjugated either at the 3'- or 5' ends, however previous attempts at conjugating ODNs to albumin have only been done at the 3' end. Thus, to much of a surprise, the inventors found that they were able to conjugate an ODN the 5' end. In one embodiment of the present disclosure, the ODN is conjugated to the albumin at the 5'end or the 3'end of the ODN, preferably the 5' end.
Three classes of TLR9 agonists exist, CpG ODN Class A, B, and C, each pose a distinct biologic activity, due to their unique structures and different cell activations, to produce varying innate immune signaling cascades.
Class A CpG ODNs are single CpG motifs of partially phosphorothioated (PS- modified) and phosphodiester backbone bases in a palindromic sequence that induce the production of IFNo by peripheral dendritic cells (pDCs) and indirectly activate NK cells. In one embodiment of the present disclosure, the ODN comprises single CpG motifs of partially phosphorothioated (PS-modified) and phosphodiester backbone bases in a palindromic sequence
Class B CpG ODNs are multiple CpG motifs composed of fully PS-modified nucleotides. Unlike Class A CpG ODNs, Class B contain B-cell activators and stimulate pDC maturation. In one embodiment of the present disclosure, the ODN comprises multiple CpG motifs composed of fully PS-modified nucleotides.
The Class C CpG ODNs, which combine characteristics of the A and B classes, are generally fully PS-modified, double-stranded palindromic motifs that induce strong IFNo production, pDC maturation, and efficient B cell activation. In one embodiment of the present disclosure, the ODN comprises fully PS-modified, double-stranded palindromic motifs, preferably inducing strong IFNo production, pDC maturation, and efficient B cell activation. In one embodiment of the present disclosure, the ODN comprises a class A ODN, class B ODN, or a class C ODN, preferably class C ODN, such as CpG 2395 according to SEQ ID NO: 13. In one embodiment of the present disclosure, the ODN is selected from the group consisting of CpG 2395 according to SEQ ID NO: 13, and CpG 2006 according to SEQ ID NO: 12. In a vaccine approach, where B-cells are specifically targeted, it may be preferred to use class B ODN, thus in another embodiment, a class B ODN is preferred, such as SEQ ID NO: 12 or SEQ ID NO: 14.
It is thus advantageous for the skilled person to choose a suitable amount of CpGs and position CpGs with different ordering- and clustering patterns, depending on the response to be achieved, and whether or not the ODN shall be complementary to another ODN. Further to this, the ODN can be of different sizes. CpG 2395 is 21 nucleotides long, and CpG 2006 is 24 nucleotides long, however the ODN can have any length between 5-100 nucleotides. Thus, in an embodiment, the ODN has a length in the range 5-100 nucleotides, preferably 10-50 nucleotides, or 15- 30 nucleotides, such as being 21 or 24 nucleotides long.
Examples 2-4 of the present disclosure provide specific embodiments of using the CpG 2395 ODN.
Examples 5-6 of the present disclosure provide specific embodiments of using the CpG 1826 ODN and CpG 2006 ODN.
ODNs can be modified in many ways to achieve different results. Many such alterations have the purpose of prolonging half-life in circulation and decrease degradation. Most of such modifications will not have an impact on the effect achieved on cells by the compounds of the present disclosure thus it is not the object of the present disclosure to limit the ODNs used to a specific set of modifications. One example being the use of phosphorothiorates in the backbone of ODNs, which will decrease the degradation. In one embodiment of the present disclosure, the ODN comprises modified nucleotides, such as comprising one or more artificial nucleotides, such as one or more locked nucleic acids (LNAs), such as comprising or consisting of one or more phosphorothioate bonds between individual nucleotides in the backbone of the ODN.
As detailed above, it is an object of the present disclosure to provide compounds capable of activating the immune system. In one embodiment of the present disclosure, the ODN activates an immune response, such as via activation of TLR preferably via the TLR9 receptor. In one embodiment of the present disclosure, the ODN activates one or more immune cells, such as dendritic cells, B cells, macrophages and/or NK cells, preferably antigen-presenting cells.
In one embodiment of the present disclosure, the binding of the ODN to the TLR receptor, such as the TLR9 receptor, stimulates secretion of one or more cytokines, such as TNFo, TNFg, IL-12, IL-6, IL-10, IFN-y, and/or IFN-o.
Proteinacous
The compounds and compositions disclosed herein preferably comprise albumin and further proteinaceous parts, to achieve a specific effect. The proteinaceous parts can be selected among several different types, depending on the effect to be achieved, such as for treating a cancer cell or presentation on antigen presenting cells. In one embodiment of the present disclosure, the one or more proteinaceous parts is an antigen-targeting moiety or a fragment thereof such as an antibody, single domain antibody, diabody, a single-chain variable fragment, affibody or a DARPin.
In the examples provided in the present disclosure are provided different proteinaceous parts to exemplify how the compounds can be generated, such as for example an immune checkpoint inhibitor targeting PD-L1 used in example 3 (anti-PD-Ll single domain antibody) and a bispecific antibody, a T-cell engager, targeting both CD3 and EGFR (example 4). Further examples are for instance the immunogenic protein from the spike protein of the SARS COV2 virus used in example 6 (RBD domain), or the cat allergen as shown in example 5 (Fel dl). As apparent from the examples (and figure 11) one or more proteinaceous parts can both be understood as a single protein part being the single domain antibody, the bispecific antibody (figure 11 C) or several proteinaceous parts being the CD3 targeting moiety and the EGFR targeting moiety (also figure 11 C). However, the one or more proteinaceous parts can also be positioned at different sites at the albumin. As such one proteinaceous part could be connected via the ODN, and one proteinaceous part via second linker (figure 11 E-F). The skilled person will be able to select proteinaceous parts, depending on the effect to be achieved, and design experiments to assemble the constructs by the methods disclosed further herein.
In one embodiment of the present disclosure, the antigen-targeting moiety is a multispecific antigen-targeting moiety, such as a bispecific antigen-targeting moiety.
Checkpoint inhibitor therapy is a form of cancer immunotherapy. The therapy targets immune checkpoints, key regulators of the immune system that when stimulated can dampen the immune response to an immunologic stimulus. Some cancers can protect themselves from attack by stimulating immune checkpoint targets. Checkpoint therapy can block inhibitory checkpoints, restoring immune system function. The first anti-cancer drug targeting an immune checkpoint was ipilimumab, a CTLA4 blocker approved in the United States in 2011.
Currently approved checkpoint inhibitors target the molecules CTI_A4, PD-1, and PD-L1. PD-1 is the transmembrane programmed cell death 1 protein (also called PDCD1 and CD279), which interacts with PD-L1 (PD-1 ligand 1, or CD274). PD-L1 on the cell surface binds to PD-1 on an immune cell surface, which inhibits immune cell activity. Among PD-L1 functions is a key regulatory role on T cell activities. It appears that (cancer-mediated) upregulation of PD-L1 on the cell surface may inhibit T cells that might otherwise attack. Antibodies that bind to either PD-1 or PD-L1 and, therefore, block the interaction may allow the T-cells to attack the tumor.
The proteinaceous parts can thus be an inhibitor of immune checkpoints. In one embodiment of the present disclosure, the antigen-targeting moiety is an immune checkpoint inhibitor. In one embodiment of the present disclosure, the immune checkpoint inhibitor inhibits an immune checkpoint selected from the group consisting of PD-1, PD-L1, CTLA-4, VISTA, TIM-3, LAG-3, IDO, KIR2D, A2AR, B7- 1, B7-H3, TIGIT and BTLA. In one embodiment of the present disclosure, the immune checkpoint inhibitor is selected from the group consisting of PD-1 antagonists, PD-L1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-1 antagonists, B7-H3 antagonists, B7-H4 antagonists, and BTLA antagonists. In one embodiment of the present disclosure, the immune checkpoint inhibitor is capable of blocking the signal of the immune checkpoint. In other embodiments of the present disclosure, the antigen-targeting moiety is an immune checkpoint costimulatory agonist, such as 4-1BB, CD28 or CD16A.
A compound comprising the PD-L1 antagonist according to amino acids 1-121 of SEQ ID NO: 8, is presented in example 3 of the present disclosure.
In a preferred embodiment, the proteinaceous part is a PD-L1 antagonist, such as the PD-L1 antagonist according to amino acids 1-121 of SEQ ID NO: 8 or a variant thereof having at least 80% sequence identity to amino acids 1-121 of SEQ ID NO: 8. In one embodiment of the present disclosure, the PD-L1 antagonist has at least 80% sequence identity to the amino acids 1-121 of SEQ ID NO: 8. In one embodiment of the present disclosure, the PD-L1 antagonist has at least 85% sequence identity to the amino acids 1-121 of SEQ ID NO: 8, such as at least 90%, at least 95%, at least 99% sequence identity to the amino acids 1-121 of SEQ ID NO: 8. In one embodiment of the present disclosure, the PD-L1 antagonist is the amino acids 1-121 of SEQ ID NO: 8. To maintain the binding affinity of the PD-L1 antagonist, it is preferred that the sequence variance is outside of the CDR regions.
In one embodiment of the present disclosure, the multispecific antigen-targeting moiety targets at least one antigen located on a cancer cell or an immune cell. In other embodiments, the multispecific antigen-targeting moiety targets at least one antigen on both a cancer cell and an immune cell. In one embodiment of the present disclosure, the at least one antigen located on a cancer cell is selected from the group consisting of EGFR, PSMA, folate receptor, RGD peptide, transferrin, FcRn, and HEU2. In one embodiment of the present disclosure, the at least one antigen located on a cancer cell is selected from the group consisting of EGFR, PSMA, folate receptor, RGD peptide, transferrin, FcRn, and HER2.
In another embodiment of the present disclosure, the at least one antigen located on a cancer cell is selected from the group consisting of BTLA, 0X40, LAG3, NRP1, VEGF, HER2, CEA, CD19, CD20, Amyloid beta, HER3, IGF-1R, MUC1, EpCAM, CD22, VEGFR-2, PSMA, GM-CSF, CXCR4, CD30, CD70, FGFR2, BCMA, CD44, ICAM-1, Notchl, MHC, CD28, IL-1R1, TCR, Notch3, FGFR3, TGF-g, TGFBR1, TGFBR2, CD109, GITR, CD47, Alpha-synuclein, CD26, LRP1, CD52, IL-4R0, VAP-1, EPO Receptor, Integrin ov, TIM-3, Grp78, LIGHT, TLR2, TLR3, PAR-2, NRP2, GLP- 1 receptor, Hedgehog, and Syndecan 1.
In one embodiment of the present disclosure, the at least one antigen located on an immune cell is selected from the group consisting of CD3, CD16, and CD16A. A compound comprising the bispecific T-cell engager targeting EGFR and CD3 is presented in example 4 of the present disclosure.
In a preferred embodiment, the proteinaceous part is a bispecific antigentargeting moiety against EGFR and CD3, such as the bispecific antibody according to the amino acids 1-385 of SEQ ID NO: 10 or a variant thereof having at least 80% sequence identity to the amino acids 1-385 of SEQ ID NO: 10. In one embodiment of the present disclosure, the bispecific antibody has at least 80% sequence identity to the sequence according to the amino acids 1-385 of SEQ ID NO: 10. In one embodiment of the present disclosure, the bispecific antibody has at least 85% sequence identity to the sequence according to the amino acids 1- 385 of SEQ ID NO: 10, such as at least 90%, at least 95%, at least 99% sequence identity to the sequence according to SEQ ID NO: 10. In one embodiment of the present disclosure, the bispecific antibody is the sequence according to the amino acids 1-385 of SEQ ID NO: 10. To maintain the binding affinity of the bispecific antibody, it is preferred that the sequence variance is outside of the CDR regions of the bispecific antibody.
HLA is a major histocompatibility complex (MHC) in humans. There are two primary classes of MHC molecules, MHC class I and MHC II. The terms MHC class I and MHC class II are interchangeably used herein with HLA class I and HLA class II.
Another aspect of the present disclosure is to design the proteinaceous parts such that an immune response against the proteinaceous parts is moderated. Such an immune response can for example be moderated by degrading the proteinaceous part and presenting it via MHC molecules. Another example can be a whole B-cell antigen, that will trigger an immune response without being degraded. In many instances such a compound can be used as a vaccine. The compounds can be designed for both creating an immunogenic response such that the immune system activates a response toward a given antigen in the future, and also for creating a tolerogenic response such that the immune system does not react toward a given antigen in the future. In one embodiment of the present disclosure, the one or more proteinaceous parts is an immunogenic- or tolerogenic protein or a fragment thereof. In one embodiment of the present disclosure, the immunogenic- or tolerogenic protein or a fragment thereof is capable of binding to a B-cell or an MHC-I or MHC-II molecule on an antigen-presenting cell.
Since such compounds can particularly benefit from a more modular design (see further below) than compounds comprising antigen targeting moieties, it can be advantageous to link the proteinaceous parts via complementary double stranded ODNs (as described further below). If the proteinaceous parts are individually designed for the binding to a specific person's MHC molecules, the compounds can thus be prefabricated and combined with proteinaceous parts later on.
FcRn is involved in antigen presentation and cross-presentation, therefore, albumin-based delivery and consequent antigen FcRn-engagement in an environment rich in FcRn may lead to increased antigen presentation and associated induction of immune responses. In another embodiment, the proteinaceous part is selected from non-human proteins, and/or proteins comprising non-human sequences. In another embodiment, the proteinaceous part is selected from non-murine proteins, and/or proteins comprising non-human sequences. In another embodiment, the proteinaceous part is selected from a sequence not present in the species of which the proteinaceous part is to be injected into, i.e. non-species specific proteins, and/or proteins comprising nonspecies specific sequences.
As present from the examples, single domain antibodies, RBD, and Fel dl all result in a synergistic effect. A common feature among these exemplified proteinaceous parts is that they comprise non-human epitopes and/or non-murine epitopes, that have a varying degree of immunogenic response, when injected into the test subjects, such as a mouse or cells derived from humans or mice. Thus, when they are injected as part of a compound as disclosed herein, they are capable of activating the immune system in varying degrees. Thus, in one embodiment of the present disclosure, the proteinaceous part comprises at least one epitope capable of binding to a MHC molecule, such as an immunogenic epitope. In one embodiment of the present disclosure, the proteinaceous part comprises at least one epitope of a length between 5 and 20 amino acids. In some embodiments, the epitope has a length of from 7 to 11 amino acids for MHC class I presentation. In other embodiments, the epitope has a length of 15 amino acids for MHC class II presentation. In some embodiments, the epitope has a length suitable for presentation by MHC (major histocompatibility complex). In one embodiment of the present disclosure, the proteinaceous part comprises at least one immunogenic epitope having a predicted ability to bind to MHC class I/II alleles. The binding of the epitopes to HLA I and HLA II alleles can for instance be predicted using NetMHCpan 4.0 and NetMHCIIpan 3.2 (https://services.hea lthtech.dtu.dk/service.php7NetMHCIIpan-3.2).
Albumin
As detailed previously, the compounds of the present disclosure comprises albumin or a variant thereof. Albumin is the most abundant protein in human blood, and is thus a well known target for increasing the half-life of compounds. Thus the skilled person knows that a wide range of artificial albumins exist, which are engineered for different technical purposes. In one embodiment of the present disclosure, the albumin or a variant thereof has at least 80% sequence identity to the sequence according to SEQ ID NO: 2. In one embodiment of the present disclosure, the albumin or a variant thereof has at least 81% sequence identity to the sequence according to SEQ ID NO: 2, such as at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% sequence identity to the sequence according to SEQ ID NO: 2. In one embodiment of the present disclosure, the albumin is the sequence according to SEQ ID NO: 2.
As it is an object of the present disclosure to provide compounds exploiting the endosomal recycling of albumins (figure 1), it is desirable that this function is retained in such variants. In one embodiment of the present disclosure, the albumin is capable of binding to the FcRn receptor.
The skilled person is also able to design the albumin, such that several ODNs can be conjugated to the albumin via additional thiols. In one embodiment of the present disclosure, the albumin comprises one or more additional artificial cysteines, such as the cysteine introducing mutations K93C, or E294C of albumin. In a compound comprising Cys34, and the mutations K93C and E294C, three available cysteines will allow the skilled person to bind three ODNs to the albumin. If the skilled person selects one of the mutations K93C and E294C, the compound will be able to bind two ODNs, with the proviso that Cys34 is not alterated. Thus, in one embodiment of the present disclosure, the albumin or the variant thereof is an albumin or a variant thereof as described above, with the proviso that one or more cysteines selected from the list Cys34, Cys93, and Cys 294, are present.
Another tool the skilled person have available, is the use of albumin highbinders. Such highbinders are engineered such that they bind more strongly (compared to wildtype albumin) to the FcRn receptor, when the pH lowers inside the endosomes. In one embodiment of the present disclosure, the albumin or a variant thereof is a highbinder albumin, such as having one or more improved pharmacokinetic properties when compared with wild type albumin such as providing a long lasting effect, having an increased binding affinity towards the FcRn receptor, having a longer half-life, having a higher binding affinity to FcRn, and/or endosomal-mediated cellular recycling, preferably wherein the highbinder is selected from the group consisting of: a. the albumin as described herein having one or more of the mutations E505Q, T527M, and/or K573P, such as the QMB highbinder preferably referred to as the QMP highbinder according to SEQ ID NO: 6; b. highbinder I (HBI) according to SEQ ID NO: 4; and c. highbinder II (HBII) according to SEQ ID NO: 5. Linker
To connect the albumin to the one or more proteinaceous parts, it can be advantageous to use a linker, linkers may consist of peptides or nucleic acids. In one embodiment of the present disclosure, the albumin and the one or more proteinaceous parts is connected via a linker, such as a flexible- or a rigid linker, preferably a peptide linker, such as a GS linker.
The linker used in example 3 and 4 is the GS linker known as GGGGS (SEQ ID NO: 15), however the skilled person knows that several other linkers can be selected.
In one embodiment of the present disclosure, the albumin and the one or more proteinaceous parts is a genetic fusion, e.g. they are translated from a single transcript, such as the albumin and the one or more proteinaceous parts is consisting of one continuous polypeptide chain. In one embodiment of the present disclosure, the albumin is positioned at or near the N-terminal- or the C-terminal end of the protein conjugate, in relation to the one or more proteinaceous parts. Near the N-terminal- or the C-terminal means that the albumin have at most 50 additional amino acids between albumin and the N-terminal- or the C-terminal, such as at most 40, at most 30, at most 20, at most 15, at most 10, at most 5, at most 4, at most 3, at most 2, such as 1 additional amino acid between albumin and the N-terminal- or the C-terminal end of the protein conjugate.
It is also possible for the skilled person to use the ODN as a linker, and thus connect two different protein entities via the ODN. In one embodiment of the present disclosure, the albumin and the one or more proteinaceous parts is connected via the ODN.
The compounds of the present disclosure are modular compounds, since the proteinaceous parts can easily be exchanged via typical molecular biological techniques, e.g. cloning. A particularly modular compound of the present disclosure, is a compound where the proteinaceous parts are connected via the ODN, since such compounds can be assembled even without typical molecular biological techniques, e.g. when an albumin conjugated to an ODN is brought into contact with a proteinaceous part conjugated to a complementary ODN, the albumin and the proteinaceous part will spontaneously assemble. In one embodiment of the present disclosure, several separated proteinaceous parts are connected at different sites to the albumin. In one embodiment of the present disclosure, at least one of the one or more proteinaceous parts is connected via a peptide linker and at least one of the one or more proteinaceous parts is connected via the ODN. Some of these embodiments are presented in figure 11. For simplicity, the compound can comprise several linkers, such as a first linker, a second linker, a third linker, and a fourth linker. The individual linkers being selected from the linkers described herein.
Thus, it is possible to design a construct comprising several ODNs, the individual ODNs being used for connecting the albumin with the proteinaceous parts. In one embodiment of the present disclosure, the connection via the ODN is through a double stranded ODN, wherein the albumin is conjugated to a first ODN and the one or more proteinaceous parts is conjugated to a second ODN, the first- and second ODN being complementary to each other thereby capable of forming the double stranded ODN upon contact.
Composition, medical use and methods of treatment
A second aspect of the present disclosure relates to a composition, comprising the compound as described herein.
In one embodiment of the present disclosure, the composition comprises a pharmaceutically acceptable carrier. Such a composition can also be referred to as a pharmaceutical composition.
Further aspects of the present disclosure relates to the use of the compounds and compositions. Thus, in one embodiment of the present disclosure, the compound or the composition described herein is for use as a medicament. In one embodiment of the present disclosure, the compound or the composition described herein is for use as a vaccine.
Some of the compounds described herein are preferably for use in treating cancers, thus in one aspect of the present disclosure, the compound or the composition described herein is for use in the treatment of cancer, such as a solid cancer, such as a solid cancer selected from the group consisting of breast cancer, colorectal cancer, lung cancers, pancreatic cancers, liver cancers, intestinal cancers, prostate cancers, bladder cancers, kidney cancers, such as renal clear cell carcinoma, ovarian cancers, cervical cancers, adenocarcinomas, squamous cell carcinomas, head and neck cancer, ear or nose or throat cancers, skin cancers, such as basal cell carcinomas, melanomas, non-melanoma skin cancers, squamous cell carcinomas of the skin, such as a liquid cancer such as leukemia, such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML).
The compounds and compositions can also be used in methods of treatment, thus, in another aspect, the disclosure relates to a method of treatment, the method comprising administering a therapeutic amount of the compound as described herein, or the composition as described herein to a subject in need thereof.
Method of production
Since the compounds of the present disclosure are tunable to the intended use, it is desirable to provide how to generate the compounds of the disclosure.
Thus, in an aspect, the disclosure relates to a method for producing the compound or the composition described herein, the method comprising: a. Providing one or more nucleic acid(s) encoding the protein conjugate or its individual parts as described herein, or one or more expression vector(s) comprising said nucleic acid(s); b. introducing the nucleic acid(s) or the vector(s) into one or more host cell(s), such as host cells selected from the group consisting of: bacteria and eukaryotes; c. growing the host cell(s) under conditions that allow for expression of the protein conjugate from the nucleic acid(s) or the vector(s); d. purifying the protein conjugate, or its individual parts; e. providing CpG oligonucleotides; and f. conjugating the CpG oligonucleotides to the purified protein conjugate or its individual parts, thereby obtaining the compound or the composition, optionally, wherein the CpG oligonucleotides are Maleimide modified oligonucleotides, such as monobromide maleimide (MBM), lysine-modified oligonucleotides, preferably Maleimide modified oligonucleotides, and optionally purifying the compound obtained in step f, optionally, bringing the protein conjugate's individual parts in contact with each other to allow them anneal.
The skilled person know several ways to conjugate an ODN to a protein, without the choice of chemistry used for conjugating is impacting the effect exerted by the compound or composition described herein. In one embodiment of the present disclosure, the Maleimide modified oligonucleotides are prepared by combining a CpG-NHz oligonucleotide with Maleimide, such as Maleimide-(PEG)n-NHS-ester, wherein n is a number between 1-10, or succinimidyl 4-(N- maleimidomethyl)cyclohexane-l-carboxylate (SMCC), and optionally purifying the Maleimide modified oligonucleotides. Preferably a Maleimide-(PEG)8-NHS-ester is used. In another embodiment of the present disclosure, the Maleimide modified oligonucleotides is a monobromidemaleimide and prepared by conjugating a Tetrazine-(PEG)n-NHS-ester with a CpG-NHz, wherein n is a number between 1- 10, and conjugating a monobromomaleimide-bicyclo[6.1.0]nonyne (BCN) to cysteines on albumin. Tetrazine is then conjugated to BCN and thereby generating an MBM (Dinesen A et al. Albumin Biomolecular Drug Designs Stabilized through Improved Thiol Conjugation and a Modular Locked Nucleic Acid Functionalized Assembly. Bioconjug Chem. 2022 Feb 16;33(2): 333-342. PMID: 35129956).
When preparing the Maleimide modified oligonucleotides, it is preferred to prepare an MBM.
A Maleimide-(PEG)8-NHS-ester is used in examples 2-4 to illustrate an embodiment of the invention.
In one embodiment of the present disclosure, the nucleic acids encoding the protein conjugate is operably linked to a promotor and optionally, additional regulatory sequences that regulate expression of said nucleic acid.
Kit
The compounds of the present disclosure comprises separable constituents, and thus can also be delivered by its individual parts, and assembled later on. Thus it is also an object of the present disclosure to provide a kit comprising: a. the protein conjugate, or its individual parts, as described herein; b. a CpG oligonucleotide, such as a CpG-NHz, such as a Maleimide modified oligonucleotide as described herein; c. reagents for conjugating the CpG oligonucleotide to the protein conjugate.
Vaccine
Since the compounds of the present disclosure can be designed with the intention of moderating the immune response against the proteinaceous parts, such compounds can also be understood as vaccines. The vaccines can be generic vaccines, but can also be individualised to an individual by selecting epitopes that are particularly displayed by that individuals MHC molecules. As presented earlier, the individualised vaccines can in particular benefit from the modular design employing double stranded ODN presented herein. The vaccines can thus be prefabricated and combined with the proteinaceous parts suitable for a particular individual later on. The vaccines will thus alter the future immune response against, the antigen comprised therein, i.e. the immunogenic- or tolerogenic protein or fragments thereof. As such, the vaccines can be both immune stimulating, hence resulting in activation of an immune response on later encounters with the antigen, and the vaccines can induce tolerance (also known as an allergy-vaccine), hence resulting in a lower or no immune response on later encounters with the antigen. When fragments of the immunogenic- or tolerogenic proteins are used as proteinaceous parts, it is desirable to use fragments that will bind more strongly to MHC molecules, and thus increase the likelihood of presentation from antigen presenting cells.
As such in one embodiment of the present disclosure, an immunogenic polypeptide may be inserted into the construct. In some embodiments of the present disclosure, the immunogenic polypeptide is selected from an infectious agent known to cause disease in subjects, preferably human subjects. In some embodiments, that immunogenic polypeptide is an immunogenic fragment of the infectious agent, preferably the fragment is capable of binding to and being presented on MHC molecules as described previously. The infectious agent may be any type of infectious agent, in preferred embodiments, the infectious agent is derived from a virus, a bacteria, or a fungus.
Further, in one embodiment of the present disclosure, a tolerogenic polypeptide may be inserted into the construct. In some embodiments of the present disclosure, the tolerogenic peptide is selected from an allergy antigen known to cause allergies in subjects, preferably human subjects. In some embodiments, the tolerogenic polypeptide is a fragment of the allergy antigen, preferably the fragment is capable of binding to and being presented on MHC molecules as described previously.
As presented by the examples 5-6, two specific types of vaccines are shown with the present application. In example 6 is shown a construct according the present invention, wherein an RBD domain from the spike protein of the SARS COV2 virus is inserted. Given the large degree of increase in B-cells, and induction of RBD-specific antibodies, this example clearly shows how the constructs of the invention can be used as vaccines.
To further show that also tolerogenic proteins can be inserted, example 5 provides an example showing that the cat allergen Fel dl can be inserted into the construct and result in a high stimulus of cells.
In all, these examples clearly shows that the constructs can both function as vaccines capable of increasing immune responses, as well as vaccines capable of decreasing immune responses, i.e. inducing tolerance.
It is thus an object of the present disclosure to provide a vaccine comprising the compound as described herein. Thus, an aspect relates to the compound or composition according to the invention for use as a vaccine.
Albumin conjugates
The ingenious use of coupling ODNs to albumin by the inventors of the present disclosure is also provided. Thus, in several aspects the disclosure also provides albumin compounds comprising ODNs. The skilled person will understand that the albumin compounds disclosed below will also find use in the compositions, vaccines, use as medicament, use in treating cancers, methods of treatment, etc. disclosed above.
One such aspect is an albumin compound comprising a highbinder albumin and one or more CpG oligonucleotides (ODN) covalently conjugated to albumin, preferably via Cys34, wherein the highbinder is an albumin, such as having one or more improved pharmacokinetic properties when compared with wild type albumin such as providing a long lasting effect, having an increased binding affinity towards the FcRn receptor, having a longer half-life, and/or having a higher binding affinity to FcRn.
Another aspect being an albumin compound comprising an albumin and one or more ODNs covalently conjugated to albumin as described above. As detailed above, the one or more ODNs can be conjugated via Cys34, additional artificial cysteines engineered in the albumin, or lysines on the albumin. In one embodiment of the present disclosure, the one or more ODNs is one ODN, are two ODNs, or even three ODNs covalently conjugated to albumin comprises.
The production of such albumin compounds will be by a more simplified version than the method disclosed above thus, in an aspect, the disclosure relates to a method for producing the albumin compound described herein, the method comprising: a. Providing a nucleic acid encoding an albumin as described herein, or an expression vector comprising said nucleic acid; b. introducing the nucleic acid or the vector into a host cell, such as host cells selected from the group consisting of: bacteria and eukaryotes; c. growing the host cell under conditions that allow for expression of albumin from the nucleic acid or the vector; d. purifying the albumin; e. providing CpG oligonucleotides; and f. conjugating the CpG oligonucleotides to the purified albumin, thereby obtaining the albumin compound, optionally, wherein the CpG oligonucleotides are Maleimide modified oligonucleotides, such as monobromide maleimide (MBM) lysine-modified oligonucleotides, preferably Maleimide modified oligonucleotides, and optionally purifying the albumin compound obtained in step f.
It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.
All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.
The invention will now be described in further details in the following non-limiting examples. Examples
Example 1 - Materials and methods
Albumin variants
Albumin WT was purchased from Sigma-Aldrich (A6608), rHA NB or highbinder (HB).
HBII (SEQ ID NO: 5) is used in all instances where albumin is used alone or conjugated with CpG ODN, unless otherwise indicated.
QMP (SEQ ID NO: 6) is the HB variant used in fusion proteins (such as rHA-anti- PD-L1 or Albu-LiTE).
ODN
The CpG-ODN (2395, 2006 and 1826) and control ODN were provided by Integrated DNA Technologies.
Cell culture
Human embryonic kidney 293E (HEK293E, ATCC, CRL-10852), Jurkat cells included in the PD-1/PD-L1 Blockade Bioassay (Promega, cat# J 1252) and PBMC cells were cultured in RPMI medium (Gibco, cat# 61870-010) supplemented with 10% fetal bovine serum (FBS, Gibco, cat# 10500-064) and 1% penicillin/streptomycin (P/S) (Gibco, cat# 14140-122) at 37°C, 5% CO2.
RAW264.7 cells (ATCC, CRL-1420) and Chinese hamster ovary (CHO) KI cells included in the PD-1/PD-L1 Blockade Bioassay (Promega, cat# J 1252) cells were cultured in Dulbecco's modified eagle medium (DMEM, Gibco cat# 41965-039) with 10% FBS and 1% P/S. The cells were discarded after passage 28. rHA-anti-PD-Ll plasmid design, amplification, and purification
The amino acid sequence of the anti-PD-Ll nanobody was obtained from the patent WO 2019/166622A1 and used to generate a human codon optimised DNA sequence for expression in human cell line with the software CLC Main Workbench (version 21.0.4). This was subcloned by GenScript into pcDNA3.1 plasmids as a fusion to NB, WT, and HB albumin variants. Between the nanobody C-terminal and albumin N-terminal, a GGTGGCGGGGGAAGC (SEQ ID NO: 14) sequence encoding a GGGGS (SEQ ID NO: 15) flexible peptide linker was added. Albu-LiTE plasmid design
The Albu-LiTE plasmid was generated in a similar manner as described for rHA- anti-PD-Ll. The LiTE sequence, formed by EgAl (aEGFR) nanobody and the OKT3 (aCD3) scFv connected by a GGGGS (SEQ ID NO: 15) flexible peptide linker, were subcloned by GenScript into pcDNA3.1 plasmids as a fusion to NB, WT, and HB albumin variants.
Plasmid amplification and purification
Plasmids were amplified by transformation through heat shock (40 min on ice, 2 min at 42°C and 5 min on ice) into TOPIO chemically competent E. coli and plated on agar plates (1% peptone (Merck, cat# 82303), 0.5% yeast extract (Fisher Scientific, cat# BP9727), 0.8% NaCI (Acros Organics, cat# 207790010), 1.5% agar (Sigma, cat# 05040), and 0.1 mg/mL ampicillin (Fisher Scientific, cat# BP1760)). After 24 h incubation, a single colony was transferred and grown overnight in peptone yeast extract (3.2% peptone, 2% yeast extract, and 0.5% NaCI), and 0.1 mg/mL ampicillin selection. The bacteria cells were then pelleted by centrifugation, and the plasmids were purified with the NucleoBond Xtra Maxi kit (Macherey-Nagel cat# 740414), an anion-exchange column for plasmid purification, following to the manufacturer's protocol.
AlbuFel dl plasmid design, amplification, and purification
The amino acid sequence of Fel dl chains 1 + 2 was obtained from the protein database registry (PDB code: 1ZKR) and used to generate a human codon optimised DNA sequence using the CLC Main Workbench software (version 21.0.4). This sequence was subcloned by GenScript into pcDNA3.1 plasmids as a fusion with human albumin carrying the flexible GGGGS (SEQ ID NO: 15)peptide linker.
Plasmids were amplified by transformation through heat shock (40 min on ice, 2 min at 42°C and 5 min on ice) into TOPIO chemically competent E. coli and plated on agar plates (1% peptone (Merck, cat# 82303), 0.5% yeast extract (Fisher Scientific, cat# BP9727), 0.8% NaCI (Acros Organics, cat# 207790010), 1.5% agar (Sigma, cat# 05040), and 0.1 mg/mL ampicillin (Fisher Scientific, cat# BP1760)). After 24 h incubation, a single colony was transferred and grown overnight in peptone yeast extract (3.2% peptone, 2% yeast extract, and 0.5% NaCI), and 0.1 mg/mL ampicillin selection. The bacteria cells were then pelleted by centrifugation, and the plasmids were purified with the NucleoBond Xtra Maxi kit (Macherey-Nagel cat# 740414), an anion-exchange column for plasmid purification, following to the manufacturer's protocol.
Protein production
Polyethylenimine (PEI, PolySciences, cat# 24765-1) and plasmid DNA in a 4: 1 ratio were diluted separately in Optimem (Gibco, cat# 11058-021), subsequently mixed and incubated at RT for 15 min before mixing with Freestyle 293 expression medium (Gibco, cat# 12338-018) and added to 55-65% confluent HEK293E cells in 5-layer bottles (Corning, cat# 353144). After 7 days of incubation at 37°C and 5% CO2, complete protease inhibitor (Roche, cat# 11873580001) was added to the conditioned medium and centrifuged (300g, 10 min) to remove cells before sterile filtering (Corning, cat# 431098). The filtered medium was stored at 4°C.
Protein purification
The rHA-fusion protein was purified on a CaptureSelect anti-HSA affinity column (Matrix: Sigma, cat# 19129701L, packed by Repligen to a column volume (CV) of 5 mL) on an Akta Start system (Cytiva). The program, with a flow rate of 1 mL/min, was as follows: tube flushing with 5 CV phosphate buffered saline (PBS, Sigma, cat# D8537) and 0.05% sodium azide (NaN3, ReagentPlus, cat# S200-2), sample addition, wash with 6 CV PBS/NaN3, elution with 10 CV 2 M MgCI2 (Sigma, cat# M2670) with 20 mM Tris (Sigma, cat# T5941) pH 7.4, wash with 10 CV PBS/ NaN3, stripping with 0.1 M glycine (Sigma, G8898) pH 3, and column neutralisation with 10 CV PBS/ NaN3.
An ELISA was performed on the eluted fractions to assess the presence of the protein of interest, and the chosen fractions were centrifuged through 30 kDa cutoff membrane filters (Sartorius, cat# VS0221) for desalting and buffer exchange to PBS. The concentration of the fusion protein was determined by measuring the absorbance at 280 nm with a NanoDrop (Thermo Fisher Scientific).
Conjugation of rHA to oligonucleotides
The sequences of the ODNs used for conjugation are shown in the sequence listing below. Amine-modified ODNs were conjugated to the NHS-ester-modified linkers by mixing DMSO (Sigma, cat# 34869), ImM ODN, 0.1 M (4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid (HEPES, Sigma, cat# H4034), and 100 mM Maleimide-(PEG)8-NHS-ester (SM(PEG)8) linker (Sigma, cat# 746207)) at a volume ratio of 3:2: 10: 1, respectively. After incubation overnight at room temperature (RT) with 650 rpm orbital shaking, excess linker was removed by ethanol precipitation (see below). ODN-linkers were then conjugated to rHA or rHA-anti-PD-Ll. For rHA conjugation, ODN-maleimide was mixed with a 2-fold molar excess of rHA in 0.1 M HEPES pH 7 and incubated as previously described. For rHA-anti-PD-Ll conjugation, the ODN-maleimide was mixed in a 1.3-fold molar excess compared to rHA-anti-PD-Ll. The conjugates were purified with HPLC using an IEX column (see below).
For AlbuFel dl Tetrazine-(PEG)5-NHS-ester linker (BroadPharm, cat# BP-22681) (250 nmol, 50 equiv) in DMSO (2.5 pL) was conjugated to CpG oligonucleotide (5' Amine modified ODN 2006 purchased from IDT, Sequence: 5' /5AmMC6/T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T-3') (SEQ ID NO: 12) (5 nmol, 1.0 equiv) in NFW (10 pL) with 7.5 pL of DMSO and 15 pL of 0.1 M HEPES pH 8.0 incubating overnight at RT with 650 rpm orbital shaking. Excess linker was removed by ethanol precipitation (Section 2.2.7) MBM-BCN linker (50nmol 5.0 equiv) in DMSO (10 pL) was conjugated to AlbuFel dl (lOnmol, 1.0 equiv) and diluted with 0.1 M HEPES pH 7.0 by incubating for 15 min at RT with 650 rpm orbital shaking. Excess linker was removed by spin filtration through a 10 kDa molecular weight cutoff membrane filter (Merck, cat# UFC501096) The BCN-modified AlbuFel dl (~10 nmol, 1.0 equiv) in 0.1 M HEPES pH 7.0 (~35 pL) was conjugated to 5 nmol (~0.5 equiv) tetrazine-modified CpG oligonucleotide. The oligonucleotide conjugate was purified with high-performance liquid chromatography (HPLC) using an ion-exchange (IEX) column with a Mono Q 5/50 GL anion exchange column (Cytiva) (Section 2.2.8).
Ethanol precipitation
ODN, absolute ethanol (EtOH, VWR, cat# 20821.310), and 3 M sodium acetate (NaOAc, Sigma, cat# S2889) were mixed in a 5:31:4 volume ratio and incubated at -18°C for 3-4 hrs followed by centrifugation at 17,000 g for 45 min. The pellet was then washed with EtOH and centrifuged again 17,000 g for 30 min. The supernatant was aspirated and pellet was dried for 10 min and subsequently dissolved in nuclease-free water (NFW, Invitrogen, cat# AM9937).
HPLC IEX protein purification
The setup utilised in the experimental work includes an Ultimate 3000 automated fraction collector connected to an LPG-3400RS pump and a VWD-3400RS detector (AFC, all from Thermo Fisher Scientific). The samples were filtered with a 0.2 pm polypropylene (PP) filter (Kinesis, cat# ESF-PP-04-022) before injection on the HPLC with a 100 pL Hamilton syringe (Sigma-Aldrich). IEX chromatography was performed with a Mono Q 5/50 GL anion exchange column (Cytiva) running the following program at 0.3 mL/min: 5 min buffer A (20 mM Tris and 10 mM NaCI at pH 7.6), a 70 min gradient to buffer B (20 mM Tris and 3 M NaCI at pH 7.6), 5 min of buffer B, a 5 min gradient to buffer A and 5 min of buffer A. Absorbance was measured at 224 and 260 nm. The collected fractions were desalted and concentrated by centrifugation through 10 kDa cut-off membrane filters.
Gel electrophoresis
10% sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) gels: 3.4 mL ProtoGel (National Diagnostics, cat# EC-890), 4 mL MilliQ water, 2.8 mL 1.5 M Tris pH 8.7, 138 pL 10% SDS (Sigma, cat# L4509), 77.5 pL 10% ammonium persulfate (APS) (Sigma, cat# A3678), and 6.9 pL N,N,N',N'- tetramethylethylenediamine (TEMED, Sigma, cat# T9281) were mixed. The mixture was poured into 1 mm cassettes (Invitrogen, cat# NC2010) and let to polymerise for 45 min with a 10-well comb (NC3010). Samples were mixed with 20x reducing agent (Invitrogen, cat# NP0009) and 4x loading buffer (Invitrogen, cat# NP0007), heated at 95°C for 5 minutes and run in 3-(N-morpholino)pro- panesulfonic acid (MOPS, Invitrogen, cat# NP0001) buffer. A benchmark (Invitrogen, cat# 10747012) protein ladder was run in a well for comparison.
15% native PAGE gel: 5 mL ProtoGel, 4 mL MilliQ water, 1 mL 10X tris, borate, ethylenediamine tetraacetic (EDTA) (TBE, Thermo Fisher Scientific, cat# 15581- 044), 100 pL 10% APS, and 10 pL TEMED were mixed. The mixture was poured into 1 mm cassettes and let to polymerise for 45 min with a 12-well comb (NC3012). Samples were mixed with loading buffer to a final concentration of 10% glycerol (VWR, cat# 24388.295) and 1 g/L Orange G (Sigma, cat# 03756) and loaded in a gel, run with TBE buffer. Gels were run at 150 V for ~75 min at RT with an EPS 601 electrophoresis power supply (Amersham Biosciences). For the Coomassie staining, the gel was submerged in 0.3 g/L Coomassie Brilliant Blue (Sigma, cat# B7920), 4.5% methanol (Sigma, cat# 34860) and 1% acetic acid (Merck, cat# 101830) for 20 min with orbital shaking. The gel destaining was performed in 10% methanol and 7.5% acetic acid overnight. SYBR Gold (Invitrogen, cat# S11494) staining was performed by submerging the gel in IX SYBR Gold in MilliQ for 15 min. Imaging of Coomassie stained gels were performed with a Gel Doc EZ Imager (BioRad), while SYBR Gold and SYBR Green were imaged on an Amersham Typhoon 5 (Cytiva).
Bicinchoninic acid (BCA) assay
BCA Protein Assay Kit (Thermo Fisher, cat# 23227) was used following the manufacturer's instructions. rHA was used to create a standard curve for the BCA assay. A working reagent was prepared by mixing BCA Reagent A and Reagent B in a 50: 1 ratio. Then, 10 pL of the rHA standard and samples were added in triplicate to a 96 well plate. 200 pL working reagent were added to each well and were incubated for 45 min at 37 °C. The plate was then let to cool to room temperature and the absorbance at 562 nm was measured with a Clariostar plate reader (BMG Labtech).
Bio-Layer Interferometry (BLI)
A BLItz system (Sartorius) was used for BLI investigation rHA/FcRn binding kinetics. Streptavidin coated biosensors (Sartorius, cat# 18-5019) were hydrated for 10 minutes in 0.02% PBST, followed by immobilisation of 70 nM biotinylated FcRn protein (Immunitrack, cat# ITF01) in 0.01% PBST and then washed in new 0.01% PBST. The association buffer was 0.01% PBST at pH 5.5 with 25 mM C2H3NaO2, 25 mM NaH2PO4, 150 mM NaCI. Association was performed for 120 s and dissociation for 300 s in association buffer; regeneration was performed for 30 s in 0.01% PBST. Binding kinetics analysis was performed in the BLItz Pro software (version 1.2.0.49) using a 1: 1 binding model to determine kinetic parameters. FcRn-mediated Recycling
HMEC-1 cells engineered to overexpress human FcRn (HMEC-l-FcRn) were used to quantify the cellular-mediated recycling of samples. After coating a 48 well plate with 100 pL of GelTrexTM (Gibco, cat# A1413202) diluted 1: 50 with ice-cold PBS for 1 h at 37° C, the wells were aspirated, and 1 x 105 cells were seeded in MCDB141 complete medium and incubated overnight. Cells were then washed twice in pre-warmed PBS and exposed to 0.15 pM constructs or albumin controls in pre-warmed Hank's Buffered Saline Solution (HBSS, Lonza, cat# BE10-547F) mixed with MES (Sigma-Aldrich, cat# M1317), pH 6.0. The well plate was incubated at 37°C, 5 % CO2 for 1 hour, and the cells were then washed 5 times in ice cold PBS. After the washes, 160 pL of complete media without FBS at 37°C were added to the wells and let to incubate at 37°C, 5 % CO2 for 1 hour. The conditioned media was then harvested, and the amount of construct released from the cells was determined using ELISA.
Recycling ELISA
A Maxisorp 96-well plate (Invitrogen, cat# 44-2404-21) was coated with 1: 1000 polyclonal goat anti-human albumin (Sigma-Aldrich, cat# A-7544) in PBS, 100 pL/well and incubated overnight at 4°C. The plate was then blocked for 2 hours at room temperature with 2 % Skim Milk (Merk, cat# 70166-500G) in PBS. After 3 washings with PBST 0.05 %, dilution series of albumin and constructs, prepared in complete media without FBS, and the conditioned media from the recycling experiment were loaded onto the and incubated for 2 hours at room temperature. The washing steps were repeated and a 1: 10.000 polyclonal HRP-conjugated goat anti-human albumin antibody (Abeam, #abl9183) diluted in 2 % mPBS was added and let to incubate at RT for 2 h. The washing steps were repeated and the plate was let to develope with 100 pL TMB, the reaction was then stopped with 100 pL of 0.2M H2SO4. Absorbance was quantified using a CLARIOstar (BMG LABTECH).
PD-1/PD-L1 Blockade Bioassay
The assay (Promega, cat# J1252) was performed according to the manufacturer protocol. 4 x 105 PD-L1 aAPC/CHO-Kl cells per well were seeded in 96-well white-bottom plates (Falcon, cat# 353377) and incubated at 37°C, 5% CO2, overnight. The day of the assay, a 8 step 2.5-fold dilution series of the reference anti-PD-1 (Biolegend, cat# 329902) and test antibodies, starting at 25 pg/mL was prepared with RPMI supplemented with 10% FBS. The medium of the CHO-K1 cells was then discarded and 40 pL of the reference and test antibodies were added to the wells. Immediately after, 40 pL of medium containing 5 x 105 PD-1 Effector Cells was added to the wells and the plate was left to incubate at 37°C, 5% CO2 for 6 h. The plate was then left at RT for 10 min and subsequently 80 pL of Bio-Gio™ Reagent at RT were added to the wells. After 15 min incubation at RT, the luminescence was measured with the LUMIstar OPTIMA luminometer (BMG Labtech). The Bio-Gio™ Reagent background relative light units (RLU) was subtracted from the test and reference antibody signal and the fold induction = RLU (induced) /RLU (no antibody control) data was fitted with the GraphPad Prism® software (version 8.4.3).
PBMC isolation
Human blood was collected from a healthy donor in EDTA-coated Vacutainer tubes (Becton Dickson, cat# 367525). Blood sample was diluted 1: 1 with PBS, and the mixture was carefully layered over Ficoll-Paque PLUS solution (GE Healthcare, cat# 17144002) in a 50 mL Falcon tube. The tubes were centrifuged at 400 g and 20°C for 30 min, with both acceleration and brakes turned off. After the top layer containing plasma and platelets was discarded, the buffy coat containing the peripheral blood mononuclear cells (PBMCs) was carefully isolated using a pipette. The PBMCs were washed with PBS and centrifuged at 400 g and 4°C for 15 min, then the PBS was removed, and the cells treated with ACK lysing buffer (Gibco, cat# A1049201) to remove remaining red blood cells. The PBMCs were subsequently washed again in PBS and resuspended in 37°C culture medium.
Secretion of TNFo
For the secretion of TNFo, RAW 264.7 murine macrophages were seeded in 48 well plates (3.5 x 105 cells per well) or 96 well plates (2 x 104 cells per well) and let to incubate at 37°C and 5% CO2 for 24 h. The medium was then aspirated from the 48 well plate and substituted with 500 pL (or 125 pL for the 96 well plate) of CpG-rHA 0.25 pM in culture medium at 37°C. Human PBMC were instead directly seeded as 1 x 106 cells per well in 500 pL of culture medium containing 0.25 pM CpG-rHA. The cells and the samples were incubated for 24 h at 37°C and 5% CO2, the conditioned medium was harvested, centrifuged at 1000 g for 5 min and the supernatant collected for TNFa detection.
Sandwich ELISA for TNFa detection
For the detection of TNFa, the assay kits for mouse TNFa (Invitrogen, cat# 88- 7324-88) and for human TNFa (Invitrogen, cat# 88-7346-86) were used, following the manufacturer instructions. 96-well plates (Corning, cat# CLS9018) were coated with 100 pL of capture antibody diluted 1:250 in PBS and incubated at 4°C overnight. The wells were aspirated, washed three times with 0.05% PBST and blocked for 1-2 h at RT with the kit ELISA diluent. The human or mouse TNFa was resuspended with NFW and used to prepare a standard dilution series. The wells were washed once and 100 pL of the TNFa standard and cell supernatant harvested from TNFa secretion assay were added into appropriate wells and left to incubate at 4°C overnight. The wells were washed 3-5 times, and 100 pL of detection antibody diluted 1:250 in ELISA diluent were added and incubated at RT for 1-2 h. After 3-5 washing steps, 100 pL of Streptavidin-HRP diluted 1: 100 in ELISA diluent were added to the wells and incubated for 30 min. The wells were washed again 5-7 times and TMB was added. The reaction was stopped after 5 min with 0.2 M H2SO4, and the absorbance at 450 nm was measured with a Clariostar plate reader (BMG Labtech) with background subtraction of the absorbance at 570 nm.
Real-Time Quantitative Polymerase Chain Reaction (qPCR)
THP-1 cells (WT, MyD88 KO, STING KO) were seeded (3.5 x 105 cells per well) in 24 well plate with 20 pM phorbol 12-myristate-13-acetate (PMA, Sigma-Aldrich, cat# P1585) in RPMI 10 % FBS and incubated at 37°C and 5% CO2 overnight. The cells were then washed with PBS and fresh medium without PMA was added and incubated overnight. Cells were then stimulated with CpG-rHA 0.250 pM in 500 pL RPMI 10 % for 6 h. The cells were collected and lysed for RNA purification using High Pure RNA Isolation Kit (Roche, cat# 11828665001). 2 pL of RNA per sample were reverse transcribed and analysed by real-time qPCR with TaqMan RNA-to-CT 1-Step Kit (Applied Biosystems, cat# 4392938) using either TaqMan (Applied Biosystems) gene expression assays for human g-actin (hs00357333_gl), IFNg (hs01077958_sl) or TNFo (Hs00174128_ml). The experiment was conducted with the Real-Time PCR system QuantStudio 5 (Applied Biosystem).
METHODS for CpG-RBD-QMP
RBD-albumin vector and protein production cDNA encoding RBD was synthesized and cloned (GenScript). To generate RBD- albumin fusions, the RBD (Wuhan) was N-terminally fused to human serum albumin (HSA) (WT or E505Q/T527M/K573P (QMP)). The vectors were transiently transfected into Expi293F cells (Gibco) using the ExpiFectamine 293 Transfection Kit (Gibco) according to the manufacturer's instructions.
Protein purification and validation
RBD-HSA fusions were purified using columns packed with CaptureSelect Human Albumin Affinity Matrix (Thermo Fisher Scientific) by Repligen and were preequilibrated and equilibrated with lxPBS before elution with buffer consisting of 2 M MgC (Sigma-Aldrich) and 20 mM Tris (Sigma-Aldrich) (pH 7.0). Eluted fractions were collected, up-concentrated and buffer-changed to lxPBS (Sigma- Aldrich) using Amicon Ultra Centrifugal Filter Units (Merck Millipore) with appropriate cutoff (10-100K) and volume (0.5 mL, 4 mL or 15 mL). Protein concentrations were determined using a DS- 11+ (M/C) Spectrophotometer (DeNovix) programmed with the protein's extinction coefficients and molecular weights,
Direct conjugation of adjuvant to albumin fusion
P purified fraction of RBD-QMP was directly conjugated to the adjuvant. First, CpG-linkers were made. In brief, 5' amine-modified CpG 1826 (IDT) was conjugated to an NHS-ester-modified linker by mixing DMSO (Sigma-Aldrich), ImM ODN, 0.1 M 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich), and 100 mM Maleimide-(PEG)8-NHS-ester (SM(PEG)8) linker (Sigma-Aldrich) at a volume ratio of 3:2: 10: 1, respectively. After overnight incubation at room temperature (RT) with 650 rpm orbital shaking, excess linker was removed by ethanol precipitation, which entailed mixing CpG, absolute ethanol (EtOH) (VWR), and 3 M sodium acetate (Sigma-Aldrich) in a 5: 31:4 volume ratio and incubate at -18°C for 3-4 hours followed by centrifugation at 17 000 x g for 45 min. The pellet was then washed with EtOH and centrifuged again at 17 000 x g for 30 minutes. The supernatant was aspirated, and the pellet was dried for 10 minutes and subsequently dissolved in nuclease-free water (Invitrogen).
Subsequently, the CpG-linkers were conjugated to RBD-QMP. RBD-QMP was mixed with a 1.3 molar excess of CpG-maleimide in 0.1 M HEPES (pH 7) and incubated overnight at RT with 650 rpm orbital shaking. The CpG-RBD-QMP was purified as described above.
Native PAGE
Protein purity after direct conjugation was assessed using native PAGE as described above.
In vivo vaccine studies Tg32-hFc mice (B6.Cg-Tg(FCGRT)32Dcr FcgrttmlDcr Zg/7glem2(IGHG1)Mvw/MvwJ; The Jackson Laboratory) (female and male, 6-13 or 31 weeks; 5-6 mice per group. The mice were anesthetized by intraperitoneally administering 5 mL/kg of ZRF cocktail containing 250 mg/mL of Zoletil Forte, 20 mg/mL of Rompun, and 50 pg/mL of Fentanyl in saline solution (Section for Comparative Medicine, Rikshospitalet, Oslo University Hospital). After sedation, 20 pL of vaccination solutions containing 0.22 nM RBD-QMP fusion, or RBD-QMP mixed with 20 pg CpG ODN 1826 VacciGrade (InvivoGen), or CpG-RBD-QMP, or lxPBS or saline (NaCI) alone were intranasally administered to each mouse (10 pL/nostril). A subsequent dose with 10% of the RBD-QMP mixed with 20 pg CpG or CpG-RBD-QMP was given 3-4 weeks after the initial dose. Blood samples were collected from the saphenous vein at weeks 1, 2, 4 and/or 5 after vaccination. Sera were isolated by centrifugation at 17 000 x g for 10-25 minutes at 4°C. In addition, nasal flushes were collected at endpoint for selected experiments. Nostrils were flushed with sterile PBS, and samples were directly transferred to protease inhibitor to a final concentration of xl-x2. The nose was flushed with 15 pL of PBS in each nostril. Samples with protease inhibitors were centrifuged at 17 000 x g for 10-25 minutes at 4°C to remove debris. All samples were stored at -20°C until analyses of antigen-specific immune responses, using FCBA as described below. At endpoint mediastinal lymph nodes (also inguinal for PBS groups) were harvested for flow cytometry analyses. The animal experiments were approved by the Norwegian Food Safety Authority and carried out in accordance with the national guidelines and regulations at the Section for Comparative Medicine, Oslo University Hospital (Rikshospitalet).
ELISA
All consecutive ELISAs were performed according to the following protocol, unless otherwise stated. Corning 96-well EIA/RIA Polystyrene High Bind Microplates (Corning) were coated with 100 pL/well of the desired protein diluted in lxPBS overnight at 4°C or 2 hours at RT. Wells were blocked with 200 pL/well of PBS containing 4% (w/v) skimmed milk powder (S) (Sigma-Aldrich) (PBS/S) for 1 hour at RT before being washed with 250 pL/well of PBS supplemented with 0.05% (v/v) Tween 20 (Sigma-Aldrich) (PBS/T), three times consecutively. Next, 100 pL/well of samples or proteins (50 pL/well for sera or BALF) diluted in PBS/T supplemented with 4% (w/v) S (PBS/T/S) were added, incubated for 1-2 hours at RT, and washed as above and 100 pL/well of a secondary antibody was added for 1 hour at RT, followed by another wash cycle. Development of horseradish peroxidase (HRP)-conjugated antibodies was done by adding 100 pL/well 3,3',5,5'-Tetramethylbenzidine (TMB) solution (Calbiochem), and the reaction was stopped by adding 100 pL/well of IM HCI. Assays using alkaline phosphatase (ALP)-conjugated antibodies were developed by adding 100 pL/well p- nitropenylphospate substrate tablets (Sigma-Aldrich) dissolved in diethanolamine buffer (pH 9.8) (Sigma-Aldrich) to a final concentration of 1 mg/mL (pNpp). Absorbance measurements were performed using a Sunrise spectrophotometer (TECAN) at 405 nm or 450 nm for pNpp or TMB substrates, respectively, with a reference wavelength of 620 nm.
To determine binding between FcRn and albumin fusions, 10 pg/mL of His-tagged mouse FcRn or human FcRn diluted in PBS/S/T (pH 5.5) was added to plates coated with 8 pg/mL of a human IgGl mutant (M252Y/S254T/T256E/H433K/N434F) with specificity for 4-hydroxy-3-iodo-5- nitrophenylactic acid, followed by addition of serial dilutions of RBD-albumin fusions (225.6 nM-0.004 nM) in PBS/S/T (pH 5.5). RBD-albumin fusions were detected with anti-HSA-ALP as above.
To investigate binding between ACE2 and RBD-albumin fusions, plates were coated with 2 pg/mL of His-tagged recombinant human ACE2 (Sino Biological) before serial dilutions of RBD-HSA fusions (150 nM-0.023 nM) were added, and detected with anti-HSA-ALP. Binding to monoclonal antibodies with known epitope specificity was used as a measure for epitope availability on the fusions. In short, RBD-albumin fusions were tested by adding a 4-fold serial dilution from 4 pg/mL to plates coated with 1 pg/mL of the monoclonal SARS-CoV-2 antibodies sotrovimab (GSK), cilgavimab or tixogevimab (AstraZeneca), before detection using, anti-HSA-ALP as above.
Quantification of RBD-specific antibodies in FCBA
Previously, a bead-based flow cytometric assay was adapted for the detection of antibodies against RBD from ancestral SARS-CoV-2 (Wuhan) as well as SARS- CoV-2 variants. Bead-based arrays with content of virus proteins were incubated for 1 hour or overnight at RT, for measurements of IgGs and IgA, respectively, in sera, or overnight for measurements in other mucosal flushes, diluted in an assay buffer consisting of PBS supplemented with 1% Tween 20 (PBT) (Sigma-Aldrich), 1% Bovine serum albumin (BSA) (Sigma-Aldrich), 10 pg/mL neutravidin (NA) (Thermo Fischer Scientific), 10 pg/mL D-biotin (Sigma-Aldrich) and 0.1% sodium azide. The dilutions were as follows: serum - 1: 100, or 1: 300 for IgG in Tg32-hFc from intranasal vaccination, and 1:6 for nasal flushes. The beads were then washed three times with PBT to remove unbound immunoglobulins and labeled for 30 minutes at RT with 10 pL of goat anti-hlgG (Jackson Immunoresearch), or digoxigenin (DIG)-conjugated rat anti-mlgA (Mabtech). Stocks were diluted 1:200, 1:200, 1: 100 and 1: 100, respectively, in PBT containing 1% BSA and 0.1% sodium azide. After incubation with anti-mlgA-DIG, beads were washed three times before labeling for 30 minutes with 30 pL mouse anti-DIG-PE (Jackson Immunoresearch) (0.5 pg/mL). Following a final wash, the beads were resuspended in PBT containing 0.1% BSA, and run on Attune NxT Flow Cytometer (Thermo Fisher Scientific). Specific antibody binding was measured as the ratio of PE median fluorescence intensity (MFI) of antigen-coupled beads to beads coupled with NA only, referred to as relative MFI (rMFI). The anti-mlgA-DIG conjugate was prepared by first changing the buffer of the antibody to PBS without sodium azide using 100K cutoff Amicon Ultra Centrifugal Filter Units (Merck Millipore). Then, 1 mg/mL anti-mlgA was mixed with 200 pg/mL DIG-N-hydroxysuccinimidyl-ester (DIG-NHS) (Sigma), and incubated overnight. Excess DIG-NHS was removed by centrifugation through Amicon Ultra Centrifugal Filter Units (Merck Millipore) with 100K cutoff twice, before the solution was passed over a G50-Sephadex column equilibrated with PBS. Anti-DIG was conjugated to PE using standard thiol- maleimide chemistry. Flow cytometry on mediastinal lymph nodes
Collected mediastinal lymph nodes from mice were pooled according to treatment group and kept on ice or at 4°C continuously from harvest to analysis and protected from light after addition of viability dye. Between steps, cells were centrifuged at 400 x g for 7 minutes at 4°C before transfer to a 96-well plate, and 500 x g for 5 minutes at 4°C after plating. Lymph nodes were mashed through a 70 pm cell strainer (VWR) to a single-cell suspension, before treatment with 3 mL RBC lysis buffer for 5 minutes. Cells were then washed with FACS buffer (2% FBS in PBS), and subsequently divided into triplicates of approximately 2x 106 cells. Next, the cells were stained with GhostDyeV510 (Tonbo Bioscience) (1:400) for 20 minutes followed by blocking with 100 pL 25% normal rat serum (produced inhouse) in PBS for another 20 minutes.
The following surface markers were used : TCR0-BB7OO (BD Bioscience) (1:200), CD19-APC/Cy7 (BD Bioscience) (1:200), CD38-BUV395 (BD Bioscience) (1:200), CD45R(B220)-BUV805 (BD Bioscience) (1:200), GL7-Alexa Fluor 488 (Biolegend) (1:200) and PE-Klickmer-coupled RBD. The staining buffer also included BD Horizon Brilliant Stain Buffer (BD Bioscience) (1: 50). 2 pL PE-Klickmer (Immudex) was used per sample, and it was coupled prior to use in a 1: 15 molar ratio to biotinylated 6xHis- and Avi-tagged Wuhan RBD 69 according to the manufacturer's protocol, yielding approximately 3.5 pL total volume per sample. The PE-Klickmer-coupled RBD was added to the cells first along with 5 pL FACS buffer and was allowed to incubate for 10 minutes before addition of 45 pL of the premixed surface antibody staining solution, followed by incubation for 30 minutes. Following washing, the cells were fixated with 100 pL 4% paraformaldehyde (Sigma) for 10 minutes, washed, and finally resuspended in 350 pL FACS buffer before analysis on a FACSymphony A5 (BD Bioscience) running FACSDiva (BD Bioscience).
In vivo lymph node distribution
The Albumus (hFcRn+/+, hAlb+/+ mice) were injected with Alexa 680-conjugated albumin variants wild type (WT) and high-binder (HB) (both variants manufactured by Albumedix, no ref #). The albumin variants diluted in PBS (#cat: 7778-77-0) were injected subcoutaneously at the tail base, at a dose of 3 nmol/mouse and max volume of 100 uL/20 g mouse. Blood samples of 20 uL were drawn from clipped tail tip 24 h after the injection. After 24 h mice were euthanized by cervical dislocation under isoflurane (#cat: 26675-46-7) anesthesia and organs were be harvested from the mice for subsequent analysis with an Amersham Typhoon 5 (Cytiva).
Example 2 - ODN-albumin constructs
Aim of study
Design, produce and purify different albumin sequences for tuneable FcRn engagement, to which a single CpG-ODN is conjugated on albumin cysteine 34. This compound has a longer half-life in circulation and an enhanced effect on dendritic cells, macrophages and B-cells. The CpG-ODN used in the design belongs to class C, and it can stimulate both murine and human cells.
The endosomal co-localisation of TLR9 and FcRn is a crucial aspect of this project. The use of albumin variants with high FcRn affinity not only enables the extension of the in vivo circulatory half-life and accumulation in tumours, but also enhances the effect of on immune cells.
Generation of CpG-rHA
The CpG was conjugated to rHA through an SM(PEG)8 crosslinker, followed by purification with IEX HPLC. The purified construct CpG-rHA WT was run on a Native-PAGE, with unconjugated rHA as a control, to evaluate the purity of the constructs (Figure 2). In the native-PAGE nucleic acids were stained with SYBR Gold, and after SYBR staining the gels were stained with Coomassie Blue to detect proteins. The native-PAGE stained with SYBR Gold shows two bands of CpG (lane 1). The appearance of two CpG bands (lane 1) is most likely due to the presence of a 3' palindromic sequence, that could result in dimerisation or formation of a 3' hairpin. As shown in figure 2, the band shift between lane 1 and 3 clearly shows successful conjugation of CpG to albumin.
CpG-rHA induced TNFo secretion
The secretion of TNFo was evaluated in murine RAW 264.7 and human PBMC cells (Figure 3). Both cell lines were incubated for 24 h with the samples before collection of the supernatant. The presence of secreted TNFo, either murine or human, in the supernatant was then detected with an ELISA kit. The amount of TNFa secretion from RAW 264.7 cells (Figure 3a) in the untreated (UT) control (vehicle control) is equal to that of ODN and rHA WT, while CpG induces a low, but detectable signal. The amount of TNFa secretion stimulated by the three CpG-rHA variants (around 2000 pg/mL) is ~9-10 times higher than the amount induced by free CpG. Showing that the constructs works as intended. The equal stimulation achieved by the three CpG-rHA variants could indicate that a 24 h incubation is too short to detect differences between variants. LPS was added as a positive control.
The same trend can be seen in the TNFa secretion from human PBMCs (Figure 3b). As before, UT control, ODN and rHA WT have equal amount of secreted TNFa. The secretion induced by CpG is higher of that induced by the ODN control, however, the PBMCs show a lower secretion compared to that detected in RAW 264.7 cells. The three CpG-rHA variants induce different amounts of TNFa (CpG- rHA NB, WT and HB induce 700 pg/mL, 1100 pg/mL and 800 pg/mL, respectively), but all higher than the free CpG. The higher TNFa secretion induced by CpG-rHA variants compared to free CpG in both human and murine cells could be the result of the rHA endosomal trafficking facilitating the accumulation of CpG in the endosomes, where TLR9 is localised. ODN-rHA WT also promotes secretion of TNFa, an amount 3.6 times lower compared to CpG-rHA WT. The experiment showed a strong effect of CpG-rHA on both murine RAW 264.7 and human PBMC TNFa secretion.
Overall, these experiments show that CpG induces a TNFa response in both human and murine cells, which is increased when CpG is conjugated to albumin, and thus confirms that the constructs works as intended.
TLR stimulation
The measurement of the TNFa was performed in collaboration with Professor Soren Riis Paludan (Department of Biomedicine, Aarhus University), with the qPCR experiment being performed by Marie Beck Iversen.
Among the pattern recognition receptors (PRR) expressed by immune cells, TLRs and STING can recognise DNA in the endosomes and the cytoplasm, respectively. The ability of CpG-rHA to specifically stimulate TLRs was tested by incubation of the conjugate with three cell lines: THP-1, THP-1 STING KO and THP-1 MyD88 KO. THP-1 is a human monocytic cell line; the THP-1 STING KO line does not express the cytosolic DNA sensor STING, while knockout of MyD88 inhibits TLR signaling in THP-1 MyD88 KO. The cells were differentiated in macrophages with PMA for 24 h, then allowed to stabilise in PMA-free medium for 24 h. CpG-rHA variants and controls were incubated with the cells for 6 h, and the amount of TNFo and IFNP mRNA in the cells, compared to 0-actin, was quantified through qPCR (Figure 4). The stimulation of wild-type THP-1 (Figure 4a) with the three variants of CpG-rHA resulted in a strong response, with ~30-36 times more TNFo expression compared to the UT cells. The type of rHA variant conjugated to CpG appears to have only a small effect on TNFo expression after 6 h of incubation. The control ODN-rHA WT also stimulated TNFo expression, but only 10 times more than the UT cells. The TNFo expression of rHA WT was equal to that of the UT control, indicating that rHA has no immunostimulatory effect. The lack of TNFo expression in the rHA WT control support the hypothesis that the high response seen with CpG-rHA is caused by CpG-mediated TLR signalling. As stated previously, rHA could improve CpG internalisation in the endosomal compartment, where TLR9 is localised, thus improving CpG stimulation. The coadministration of CpG and rHA WT did not influence TNFo expression, demonstrating that the improvement in CpG efficacy is dependent on the conjugation to rHA. The free CpG control also did not have an effect on TNFo expression. This is probably due to the low concentration, or the short 6 hours incubation times, used in the experiment.
The IFNP expression was affected only by the presence of cGAMP, which was added only in wild-type THP-1 cells (Figure 4a) as a positive control. The rest of the samples had no effect on IFNP expression in either of the cells lines used in the experiment (Figure 4a, b and c). This could be explained by IFN[3 being mainly a marker of STING activation, suggesting CpG-rHA is not entering the cells cytoplasm; for detection of TLR9 activation, IFNo expression has been canonically used.
The TNFo expression level of the UT control, and other controls, in THP-1 STING KO is higher than that in the wild-type THP-1 cells (Figure 4b). It has been previously shown that mouse STING-deficient macrophages were hyperresponsive to TLR ligands and produced "abnormally high levels of proinflammatory cytokines". The knockout of STING could therefore cause the increased TNFo expression in THP-1 STING KO cells. The high levels of cytokines could also be related to the differentiation of THP-1 cells with PMA; a longer "rest period" in PMA free medium could help to restore the expression of TNFo to the levels detected with the wild-type THP-1 cells (Figure 4a).
TNFo expression by the CpG conjugated are similar to the wild-type THP-1 cells (figure 4b). The TNFo induction in cells not expressing STING supports the hypothesis that CpG-rHA is not activating the cytosolic STING pathway, but that the response is caused by CpG-mediated activation of TLRs in the endosomes. The hypothesis is reinforced by the complete absence of TNFo induction, compared to the UT control, in the THP-1 MyD88 KO cells (Figure 4c), in which TLR signaling is inhibited. Additionally, the absence of TNFo induction in the MyD88 KO cells suggest that the release of TNFo in the experiment with THP-1 wild-type and STING KO (Figure 4 a and b) is not the result of a cytotoxic effect of the conjugate, but instead of the activation of the TLR receptors. To specifically determine if TLR9 is activated by CpG-rHA, the experiment could be repeated on THP-1 cells in which TLR9 has been edited out with CRISPR. These results complement the TNFo secretion data of (Figure 3), supporting the conclusion that the strong TNFo induction, by CpG-rHA variants, seen in RAW 264.7 and PBMCs is mediated by TLR activation.
Conclusion
Overall, these experiments show that CpG induces a TNFo response mediated by TLR activation. When CpG is conjugated to albumin the response is increased, and, thus, confirms that the constructs works as intended.
Example 3 - ODN-Albumin-anti-PD-Ll constructs
Aim of study
The design of a partner molecule, comprising an anti-PD-Ll nanobody (patent WO 2019/166622A1) fused to albumin variants, to which a single CpG-ODN is conjugated on albumin cysteine 34, synergistically combines the effect of checkpoint inhibition with TLR9 stimulation.
The use of albumin as a scaffold for the combination of checkpoint inhibitor and CpG ODN in one single construct would ensure spatiotemporal control on the effect of the drug, synergistic effect of the two drugs to boost both the innate and adaptive immune systems, and long lasting effects. Furthermore, the combination would reduce the number of injections required per cycle of treatment, improving patient compliance.
Generation of rHA-anti-PD-Ll and CpG-rHA-anti-PD-Ll
The proteins were designed using the software CLC Main Workbench (version 21.0.4). The secreted version, without propeptide, of the rHA sequence (UniProtKB - Q56G89), modified to obtain NB and HB (QMP) variants, were fused at the N-terminal to the anti-PD-Ll nanobody (patent WO 2019/166622A1) C- terminal with a flexible GGGGS (SEQ ID NO: 15) peptide linker and inserted into pcDNA3.1 plasmids. The plasmids encoding the three rHA-anti-PD-Ll variants (NB, WT and HB) were amplified in E. coli, isolated with the NucleoBond®Xtra Maxi kit, and mixed with PEI MAX for the transient transfection of HEK293E cells. After 7 days, the conditioned medium was harvested, and the cells removed by centrifugation. The secreted rHA-anti-PD-Ll was purified from the supernatant through affinity chromatography with an anti-HSA CaptureSelect® column. The albumin-containing fractions were pooled, concentrated and the buffer exchanged to PBS with 30 MWCO spin filters.
The purified proteins were run on an SDS-PAGE and stained with Coomassie brilliant blue (Figure 5) Coomassie stained SDS-PAGE showing purified rHA-anti- PD-Ll NB, WT and HB variants. rHA included as control. The thick bands of the three rHA-anti-PD-Ll variants are aligned with the protein ladder 80 kDa band (predicted MW of rHA-anti-PD-Ll of 80.11 kDa). This result confirms the integrity and purity of the proteins.
The CpG was conjugated to rHA-anti-PD-Ll through an SM(PEG)8 crosslinker, followed by purification with IEX HPLC. The purified construct CpG-rHA-anti-PD-Ll was run on a Native-PAGE, with unconjugated rHA, rHA-anti-PD-Ll and CpG-rHA WT as controls, to evaluate the purity of the constructs (Figure 2). In the native- PAGE nucleic acids were stained with SYBR Gold, and after SYBR staining the gels were stained with Coomassie Blue to detect proteins. The bands of CpG-rHA-anti- PD-L1 in the SYBR Gold stain show high degree of CpG conjugation and purity. The presence of multiple bands indicates the formation of dimers. Affinity studies towards FcRn
The binding affinity of rHA-anti-PD-Ll variants for FcRn at pH 5.5 was measured with BLI. Biotinylated FcRn was immobilised on streptavidin biosensors, which were exposed to the recombinant proteins to obtain the binding curves and KD (Figure 6). The observed KD of rHA WT (2800 nM) (Figure 6a) concords with the value found in literature ( 1000 nM). The affinity of rHA-anti-PD-Ll WT (3600 nM) (Figure 6b) for FcRn was comparable to that of rHA WT, although slightly lower. The HB variants of rHA (Figure 6c) and rHA-anti-PD-Ll (Figure 6d) showed an affinity of 30 nM and 43 nM respectively, ~93 and ~83 times higher than the WT counterparts. The steric hindrance of fused moieties at the N-terminal of albumin can lower the affinity of rHA for FcRn, however, the difference in the KD between rHA and corresponding rHA-anti-PD-Ll variants detected with BLI are not likely to produce appreciable differences physiologically.
FcRn-mediated Recycling
The cellular FcRn-mediated rescue of albumin-based designs from lysosomal degradation was measured with a recycling assay. The ability of rHA-anti-PD-Ll and CpG-rHA-anti-PD-Ll to be internalised by cells, interact with FcRn in the endosome and be directed and released in the supernatant was compared to the rHA variants (Figure 7). While the rHA NB (non-binder) was not rescued, the rHA WT and HB variants show recycling, with 2.7-fold more of the rHA HB released back into the medium compared with the WT. For rHA-anti-PD-Ll and CpG-rHA- anti-PD-Ll the recycled amount was respectively 2.4-fold and 11.6-fold higher than the rHA WT. The high recycling detected for CpG-rHA-anti-PD-Ll is likely the result of the conjugation of the fusion protein with the phosphorothioate backbone oligo, that interact with other receptor on the cell surface and allow internalisation.
Blockade of PD-L1-PD1 interaction
Once the albumin functionality was confirmed, it was important to test whether the fused anti-PD-Ll nanobody conserved the ability to bind its ligand. The constructs mediated inhibition of the PD-1/PD-L1 axis was measured with a PD- 1/PD-L1 Blockade Bioassay (Promega©). The assay includes two engineered cells lines: Jurkat T cells expressing PD-1 and, upon activation of TCR signaling, a luciferase reporter; and CHO-K1 cells expressing human PD-L1 and an engineered surface protein that activates TCR in an antigen-independent manner. When the cells are co-cultured, the PD-1/PD-L1 interaction inhibits the TCR induced production of luciferase in Jurkat cells. Disruption of the checkpoint molecules interaction results in the production of luciferase, that can in turn be used to generate a measurable signal upon the addition of luciferin.
The PD-1/PD-L1 inhibition as a function of concentration, after 6 h incubation, was tested for an anti-PD-Ll antibody, rHA-anti-PD-Ll, CpG-rHA-anti-PD-Ll and the anti-PD-Ll nanobody (Figure 8). The half maximal effective concentration (EC50) values determined for anti-PD-Ll antibody (5.17 nM), rHA-anti-PD-Ll (64.07 nM), CpG-rHA-anti-PD-Ll (15.46 nM) anti-PD-Ll nanobody (15.53 nM) indicate that all the constructs retain their ability to block the PD-1/PD-L1 axis and similar affinity for PD-L1. The EC50 of the anti-PD-Ll antibody positive is an order of magnitude lower of those of the other constructs, possibly due to antibodies possessing two antigen-binding sites, while the rHA-anti-PD-Ll has only one.
CpG-rHA-anti-PD-Ll effect on RAW 264.7 cells
The secretion of TNFo induced by CpG-rHA-anti-PD-Ll was evaluated in murine RAW 264.7 (Figure 9). The cells were incubated for 24 h with the samples before collection of the supernatant. The presence of secreted TNFo in the supernatant was then detected with an ELISA kit. The same trend as in Figure 3) is seen, with the low TNFo secretion in the vehicle control, ODN and ODN-rHA WT. CpG induces TNFo secretion ~4 times lower than CpG-rHA WT. To much of a surprise, the combination of CpG and anti-PD-Ll on CpG-rHA-anti-PD-Ll induces a TNFo secretion of 5010 pg/mL, which is more than what is secreted by CpG-rHA (1428 pg/mL) and rHA-anti-PD-Ll (824 pg/mL) combined and clearly shows that the combination induces an unexpected synergistic effect.
CpG-rHA-anti-PD-Ll effect on human PBMCs
The human PBMCs are exposed to CpG-rHA-anti-PD-Ll, following the protocol used in disclosed above (Secretion of TNFo). Conclusion
The experiments show that CpG-rHA-anti-PD-Ll are produced efficiently, and the functions are retained. The constructs are recycled via FcRn and more efficiently than albumin on its own, the checkpoint inhibitors can be blocked by the nanobody and most importantly CpG-rHA-anti-PD-Ll induces an unexpected synergistic secretion of TNFo.
Example 4 - ODN-Albumin-bispecific antibody constructs
Aim of study
The design follows the same rationale as CpG-rHA-anti-PD-Ll, using albumin as a scaffold to combine LiTE (see below) and CpG ODN in order to boost the innate and adaptive immune systems, and long lasting effects.
Albu-LiTE comprises a bispecific "light" T-cell engager (LiTE) antibody, formed by an anti-EGFR nanobody fused with an anti-CD3 scFv, fused to albumin variants. A single CpG-ODN is conjugated on albumin cysteine 34, to combine the T-cell redirection with TLR9 stimulation.
CpG-Albu-LiTE effect on RAW 264.7 cells
The secretion of TNFo induced by CpG-Albu-LiTE was also evaluated in murine RAW 264.7 (Figure 10), following the same protocol as for CpG-rHA-anti-PD-Ll. Albu-LiTE induces a TNFo secretion of 3579 pg/mL, 2.39 times higher compared to CpG-rHA WT (1428 pg/mL). The conjugation of CpG and Albu-LiTE on CpG- Albu-LiTE, however, increases the amount of secreted TNFo to 11670 pg/mL, showing the same synergistic effect measured with CpG-rHA-anti-PD-Ll.
Conclusion
The experiments show that CpG-Albu-LiTE are produced efficiently, and the functions are retained, most importantly CpG-Albu-LiTE also induces an unexpected synergistic secretion of TNFo, as seen in example 3.
Example 5 - ODN-Albumin-tolerance constructs
Aim of study
Design, conjugation and purification of constructs containing CpG ODN (Class B) conjugated to albumin fused to Fel dl cat allergen, to identify whether a vaccine construct, capable of inducing tolerance, could be inserted into the compound. Results
CpG-AlbuFel dl induced TNFa secretion
The secretion of TNFa induced by CpG-AlbuFel dl (AlbuFel dl conjugated to CpG 2006) was evaluated in human PBMC cells (Figure 12). The cell lines were incubated for 24 h with the samples before collection of the supernatant. The results show a very low secretion when PBMCs are stimulated with free CpG ODN (13 pg/mL). The stimulation with AlbuFel dl alone, or mixed with CpG ODN, resulted in similar secretion of 402 pg/mL and 353 pg/mL, respectively. The conjugate CpG-AlbuFel dl elicited a surprisingly high level of TNFa secretion, with 977 pg/mL. The RPMI control showed no TNFa secretion (below detection of the ELISA kit). ODN-rHA WT also promotes secretion of TNFa, an amount 3.6 times lower compared to CpG-rHA WT. The experiment showed a strong effect of CpG- AlbuFel dl on human PBMC TNFa secretion.
Conclusion
This work presents the successful production of a tolerance-inducing molecule, of a vaccine.
The tolerance-inducing CpG-AlbuFel dl exploits albumin for the co-delivery of a CpG ODN class B (2006, human specific) and the cat allergen Fel dl. The conjugation of CpG to the protein fusion yielded higher TNFa secretion from PBMCs in vitro, an unexpected synergistic secretion of TNFa, as seen in the previous examples.
Example 6 - ODN-Albumin-vaccine constructs
Aim of study
Design, conjugation and purification of constructs containing CpG ODN (Class B) conjugated to albumin fused to the antigen SARS-CoV-2 receptor binding domain (RBD), to identify whether an immunogenic vaccine construct could be inserted into the compound.
The study further tests, whether engagement to FcRn and ACE2 is retained within the construct, and retained availability of epitopes on the RBD antigen. Lastly, the study further shows improved intranasal vaccination in vivo, measurement of antigen-positive B-cells in lymph nodes and antibody titer. Results
Direct conjugation of the adjuvant CpG 1826 to RBD-QMP was performed as described above. After successful conjugation and isolation (Figure 13-a), the purified proteins were run on a Native-PAGE and stained with SYBR Gold and Coomassie brilliant blue (Figure 13-b). The lack of CpG ODN bands at the bottom of the gel for CpG-RBD-QMP confirms that the conjugation and purification were successful.
Next, the binding of CpG-RBD-QMP to ACE2 (Figure 14-a), human FcRn (Figure 14-b), as well as monoclonal SARS-CoV-2 antibodies (Figure 14 c), was confirmed and shown to be similar to that of RBD-QMP. This confirms that the constructs, and the RBD domains are correctly folded, and important epitopes are exposed to the immune system.
Following intranasal vaccination of Tg32-hFc mice, an increased population of RBD-specific B-cells was measured in the mediastinal lymph nodes for CpG-RBD- QMP, when compared with RBD-QMP with CpG in solution (RBD-QMP+CpG nonconjugated) (Figure 15 a). The generated antibody responses showed an increase in the antibody amount when the conjugate CpG-RBD-QMP is used Figure 15 b). No antigen-specific immune responses were detected when an albumin-fusion was intranasally administrated in the absence of CpG, and this was independent of the vaccine subunit (Figure 15).
It was further tested whether the injection of human albumin only into mice resulted in a production of anti-albumin antibodies, and a minimal induction of anti-albumin antibodies was seen (data not shown).
Conclusion
The vaccine CpG-RBD-QMP joins a CpG class B (1826, mouse specific) and an antigen, on an albumin scaffold with high-FcRn affinity (QMP). The CpG-RBD-QMP conjugate retained the affinity for FcRn, ACE2 and the accessibility to the epitopes on RBD. Furthermore, after intranasal mouse vaccination, CpG-RBD-QMP yielded higher amount of antigen-specific B-cells in LN and antibody titer in the nose mucosa compared to RBD-QMP mixed with CpG non-covalently. Example 7 wt and HB albumin accumulation in lymph nodes
Aim of study
To investigate the accumulation of albumin (WT) or variant with higher FcRn affinity (HB) in lymph nodes and serum.
Results
The Alexa680-albumin variants WT and HB were injected subcutaneously at the tail base, and after 24 h a blood sample and the inguinal, axillary and brachial lymph nodes were collected (Figure 16 a). The background signal from PBS control was subtracted from the lymph nodes (LN) signal and the resulting signal normalised to WT albumin. The average signal from all the LN of each mouse shows a ~2 fold increase of HB accumulation (Figure 16 b). When considered separately, the inguinal LN show a ~2 fold increase from WT to HB (Figure 16 ci), the axillary a ~3 fold increase (Figure 16 cii) and the brachial a ~2 fold increase (Figure 16 ciii). The similar concentration of WT and HB variants in the inguinal LN could be due to the proximity to the injection site, while more distant LN show a different trend, with higher HB accumulation. The concentration of albumin WT and HB in the serum were significantly different at 24 h, with a 1.5 fold increase for the HB variant (Figure 16 d).
Conclusion
Albumin variants accumulate in the lymph nodes, verifying the rationale for conjugating to albumin, to increase the immune system presentation.
Further, the difference in FcRn affinity between albumin WT and HB resulted in different pharmacokinetic profiles of the two variants. Albumin HB concentration in the serum and LN, 24 h after the injection, was higher than the WT variant. This result shows that in some instances, albumin variants, with high FcRn affinity, may preferably be used, for example to further increase lymph node accumulation and increase immune system presentation.
Combined conclusions
This work demonstrates the successful use of rHA as a scaffold on which a CpG ODN immunostimulatory effect can be combined with a fused checkpoint inhibitor or bispecific T-cell engager. rHA-anti-PD-Ll fusions retained the affinity of rHA variants for FcRn and of the nanobody for PD-L1, as demonstrated by Bio-Layer Interferometry, cellular recycling and PD-1/PD-L1 Blockade Bioassay, respectively. CpG-rHA induced higher TNFo secretion, compared to free CpG, in murine RAW 264.7 and human PBMC cells. Additionally, it was shown that CpG-rHA induces TNFo expression through activation of TLR, and not STING, in THP-1 cells. CpG-rHA-anti-PD-Ll showed a high degree of endosomal-recycling and retained function of CpG, anti-PD-Ll nanobody and albumin. Both CpG-rHA-anti-PD-Ll and CpG-Albu-LiTE exhibited a surprisingly high degree of synergistic effect, compared to the previous constructs, as shown in the stimulation of TNFo secretion from RAW 264.7 murine cells.
Further, this work presents the successful production of vaccines, namely a tolerance-inducing molecule (CpG-AlbuFel dl) and an immunogenic molecule CpG-RBD-QMP.
The tolerance-inducing CpG-AlbuFel dl exploits albumin for the co-delivery of a CpG ODN class B (2006, human specific) and the cat allergen Fel dl. The conjugation of CpG to the protein fusion yielded higher TNFo secretion from PBMCs in vitro.
The vaccine CpG-RBD-QMP joins a CpG class B (1826, mouse specific) and an antigen, on an albumin scaffold with high-FcRn affinity (QMP). The CpG-RBD-QMP conjugate retained the affinity for FcRn, ACE2 and the accessibility to the epitopes on RBD. Furthermore, after intranasal mouse vaccination, CpG-RBD-QMP yielded higher amount of antigen-specific B-cells in LN and antibody titer in the nasal mucosa compared to RBD-QMP mixed with CpG non-covalently.
Albumin variants accumulate in the lymph nodes, verifying the rationale for conjugating to albumin, to increase the immune system presentation.
Further, the difference in FcRn affinity between albumin WT and HB resulted in different pharmacokinetic profiles of the two variants. Albumin HB concentration in the serum and LN, 24 h after the injection, was higher than the WT variant. This result shows that in some instances, albumin variants, with high FcRn affinity, may preferably be used, for example to further increase lymph node accumulation and increase immune system presentation.
Lastly, these experiments show that the construct functions with different albumin variants both wildtype (Albu-LITE and AlbuFel dl), non-binders (rHA-anti-PD-Ll) and high-binders (rHA-anti-PD-Ll and RBD-QMP). These results form a promising foundation for the future use of CpG-conjugated albumin-based therapeutics as a platform with a broad range of applications.
References
Otagiri et al, 2009, Biol. Pharm. Bull. 32(4), 527-534.
Zhu et al Albumin/vaccine nanocomplexes that assemble in vivo for combination cancer immunotherapy. Nature Communications volume 8, Article number: 1954 (2017).
Liu et al. Structure-based programming of lymph node targeting in molecular vaccines. 2014 | Vol 507 | NATURE | 519.
J.D. Thompson et al. improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 PMID: 7984417.
Dinesen A et al. Albumin Biomolecular Drug Designs Stabilized through Improved Thiol Conjugation and a Modular Locked Nucleic Acid Functionalized Assembly. Bioconjug Chem. 2022 Feb 16;33(2) : 333-342. PMID: 35129956.
Sequence listing
CDR sequences of nanobodies are underlined.
Cys34 is marked in bold.
SEQ ID NO: 1 - HA wildtype - signal peptide (aal-18) and propeptide (aa!9-24)
MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFED HVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERN ECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYK
AAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRF PKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEK SHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLR LAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRY TKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKH KPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL SEQ ID NO: 2 - rHA WT (secreted protein sequence)
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENC
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM
CTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRD
EGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADI-AKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVE
SKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRI-AKTYETTLEKCCAAADPHECYAKVF
DEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKC CKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVP KEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA DDKETCFAEEGKKLVAASQAALGL
SEQ ID NO: 3 - rHA NB - K500A
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENC
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM
CTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRD
EGKASSAKQGLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFV
GSKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGS KCCKHPEAKRMPCAEDCLSVFLNQLCVLHEKTPVSDRVTKCCTESLVNGRPCFSALEVDETY
VPAEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK
ADDKETCFAEEGKKLVAASQAALGL
SEQ ID NO: 4 - rHA HBI - K573P
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENC
DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM
CTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRD
EGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG
DLLECADDRADI-AKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVE
SKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRI-AKTYETTLEKCCAAADPHECYAKVF
DEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKC CKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVP KEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA DDKETCFAEEGPKLVAASQAALGL
SEQ ID NO: 5 - rHA HBII - E492G/K573P/K574H/Q580K DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENC DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM CTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRD EGKASSAKQGLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG DLLECADDRADI_AKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSI_AADFV GSKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAK
VFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGS KCCKHPEAKRMPCAEDCLSVFLNQLCVLHEKTPVSDRVTKCCTESLVNGRPCFSALGVDETY VPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK ADDKETCFAEEGPHLVAASKAALGL
SEQ ID NO: 6 - rHA HB (QMP)
DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENC DKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVM CTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRD EGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG DLLECADDRADI-AKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVE SKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRI-AKTYETTLEKCCAAADPHECYAKVF
DEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKC CKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVP KEFNAQTFTFHADICTLSEKERQIKKQMALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKA DDKETCFAEEGPKLVAASQAALGL
SEQ ID NO: 7 - PD-L1 NB-rHA
QVQLQESGGGLVQPGGSLRLSCAASGFTFSSFAMSWVRQAPGKGLEWVSDIDTTGRTTDY ADSVKGRFTISRDNAENTLYLQMNDLKPEDTAVYYCANVPKELVLSFGSWGPGTQVTVSST GGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVR PEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKV
HTECCHGDLLECADDRADI-AKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLP SI_AADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRI_AKTYETTLEKCCAAADP HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRN LGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSAL EVDETYVPAEFNAETFTFQADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAF VEKCCKADDKETCFAEEGKKLVAASQAALGL
SEQ ID NO: 8 - PD-L1 HB-rHA OVOLOESGGGLVOPGGSLRLSCAASGFTFSSFAMSWVROAPGKGLEWVSDIDTTGRTTDY ADSVKGRFTISRDNAENTLYLOMNDLKPEDTAVYYCANVPKELVLSFGSWGPGTOVTVSST GGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVAD ESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVR PEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKV HTECCHGDLLECADDRADI-AKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLP
SI_AADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRI_AKTYETTLEKCCAAADP HECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRN LGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSAL EVDETYVPKEFNAQTFTFHADICTLSEKERQIKKQMALVELVKHKPKATKEQLKAVMDDFAA FVEKCCKADDKETCFAEEGPKLVAASQAALGL
SEQ ID NO: 9 - PD-L1 HB-rHA
CAGGTCCAGTTACAGGAGTCGGGTGGAGGTCTCGTTCAGCCAGGCGGGTCTCTGAGGC TGTCTTGCGCCGCCTCTGGTTTCACTTTCTCCTCGTTTGCGATGTCTTGGGTTAGACAGG CGCCTGGGAAAGGACTGGAGTGGGTGTCCGATATCGACACGACAGGACGGACCACCGA CTACGCAGATAGTGTTAAAGGGCGATTCACAATATCCCGAGACAACGCCGAGAACACGC TATACCTCCAGATGAATGACCTCAAGCCCGAAGATACCGCCGTTTACTACTGTGCAAACG TCCCTAAGGAATTAGTTTTATCATTCGGGTCCTGGGGGCCTGGCACCCAAGTAACGGTCA GCAGTACCGGTGGCGGGGGAAGCGATGCACACAAGAGTGAGGTTGCTCATCGGTTTAA
AGATTTGGGAGAAGAAAATTTCAAAGCTTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAG CAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACAT GTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGGAGACA AATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTGTGCAA AACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAACCTCC CCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGA CATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGGA
ACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTGCTGAT AAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCTTCGTC TGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTCAAAGC ATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAA GTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAATG TGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGATCTCCAG TAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGCCGAAGT
GGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGAAAGTAA GGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGA ATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACATA TGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGCCAAAGT GTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAATTGTGAG CTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTACACCAAG AAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTG GGGTCCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATCTA
TCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGACAGAGTC ACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCTGGAAGTC GATGAAACATACGTTCCCAAAGAGTTTAATGCTCAAACATTCACCTTCCATGCAGATATAT GCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAATGGCACTTGTTGAGCTCGTGA AACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAGCTT TTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGGAGGGTCCC AAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTA
SEQ ID NO: 10 - Albu-LiTE WT
MAQVQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAIRWSGGY TYYTDSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDY WGQGTQVTVSSAAAGGGGSRQVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVK QRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARY YDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIDLTQSPAIMSASPGEKVTMTCS ASSSVSYMNWYQQKSGTSPKRWIYDTSKI-ASGVPAHFRGSGSGTSYSLTISGMEAEDAAT YYCQQWSSNPFTFGSGTKLEINRATGAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFE
DHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPER NECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRY KAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQR
FPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLE KSHCIAEVENDEMPADLPSI-AADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLL RLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVR YTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDR VTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKH KPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL
SEQ ID NO: 11 - Albu-LiTE WT
ATGGCCCAGGTGCAGCTGCAGGAGAGCGGCGGCGGCCTGGTGCAGCCCGGCGGCAGC CTGAGGCTGAGCTGCGCCGCCAGCGGCAGGACCTTCAGCAGCTACGCCATGGGCTGGT TCAGGCAGGCCCCCGGCAAGGAGAGGGAGTTCGTGGCCGCCATCAGGTGGAGCGGCG GCTACACCTACTACACCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCC
AAGACCACCGTGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTA
CTGCGCCGCCACCTACCTGAGCAGCGACTACAGCAGGTACGCCCTGCCCCAGAGGCCCC
TGGACTACGACTACTGGGGCCAGGGCACCCAGGTGACCGTGAGCAGCGCCGCCGCCGG
CGGCGGCGGCAGCAGGCAGGTGCAGCTGCAGCAGAGCGGCGCCGAGCTGGCCAGGCC
CGGCGCCAGCGTGAAGATGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGGTACACCA
TGCACTGGGTGAAGCAGAGGCCCGGCCAGGGCCTGGAGTGGATCGGCTACATCAACCC
CAGCAGGGGCTACACCAACTACAACCAGAAGTTCAAGGACAAGGCCACCCTGACCACCG
ACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACCAGCGAGGACAGCGC
CGTGTACTACTGCGCCAGGTACTACGACGACCACTACTGCCTGGACTACTGGGGCCAGG
GCACCACCCTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGCG
GCGGCGGCAGCGACATCGACCTGACCCAGAGCCCCGCCATCATGAGCGCCAGCCCCGG
CGAGAAGGTGACCATGACCTGCAGCGCCAGCAGCAGCGTGAGCTACATGAACTGGTATC
AGCAGAAGAGCGGCACCAGCCCCAAGAGGTGGATCTACGACACCAGCAAGCTGGCCAG
CGGCGTGCCCGCCCACTTCAGGGGCAGCGGCAGCGGCACCAGCTACAGCCTGACCATC
AGCGGCATGGAGGCCGAGGACGCCGCCACCTACTACTGCCAGCAGTGGAGCAGCAACC
CCTTCACCTTCGGCAGCGGCACCAAGCTGGAGATCAACAGGGCCACCGGTGCACACAAG
AGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCTTTGGTGTTG
ATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATG
AAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAAT
CACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGG
TGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACA
CAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCA
CTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAG
ACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTA
CAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTC
GGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAA
TTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAA
AGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATG
CTGCCATGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCT
GTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGG
AAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCAT
TAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATG
TCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCT
GCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAG ATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTC
AGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGA
ATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAG
AGGTCTCAAGAAACCTAGGAAAAGTGGGGTCCAAATGTTGTAAACATCCTGAAGCAAAAA
GAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATG
AGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGG
CGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCT
GAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAG
AAACAAACTGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACT
GAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAA
GGAGACCTGCTTTGCCGAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAG
GCTTA
SEQ ID NO: 12 - CpG 2006 - (5 ' ) NH2-C6, * = phosphoroth ioate backbones
T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T
SEQ ID NO: 13 - CpG 2395 (PS) - (5 ) NH2-C6, * = phosphoroth ioate backbones G*G*T*C*A*T*C*C*A*T*G*A*C*A*A*C*T*T*T*T*T
SEQ ID NO: 14
GGTGGCGGGGGAAGC
SEQ ID NO: 15
GGGGS
SEQ ID NO: 16 - CpG 1826 (5 ' ) NH2-C6, * = phosphoroth ioate backbones
T*C*C*A*T*G*A*C*G*T*T*C*C*T*G*A*C*G*T*T
SEQ ID NO: 17 - RBD-QMP
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYG
VSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS
KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY
QPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFDAHKSEVAHRFKDLGEENFKALVLIAF
AQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMA
DCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFY
APELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFK
AWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADI-AKYICENQDSISSK
LKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYAR
RHPDYSVVLLLRI-AKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLG
EYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLC VLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAQTFTFHADICTLSEKERQIK
KQMALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLVAASQAALGL
SEQ ID NO: 18 - RBD-QMP
AGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTG
GTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCA
GCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGT
TATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATT
TGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTG
ATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAAcTCTAACAAT
CTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATC
TCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAA
TGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATG
GTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGC
AACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTCGAT
GCACACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCC
TTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATTTGAAGATCATGTAAAAT
TAGTGAATGAAGTAACTGAATTTGCAAAAACATGTGTTGCTGATGAGTCAGCTGAAAATT
GTGACAAATCACTTCATACCCTTTTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGA
AACCTATGGTGAAATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTT
CTTGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAGGTTGATGT
GATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAAAAAATACTTATATGAAATT
GCCAGAAGACATCCTTACTTTTATGCCCCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAG
CTGCTTTTACAGAATGTTGCCAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCG
ATGAACTTCGGGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAGTGTGCCAGT
CTCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTAGCTCGCCTGAGCCAGAG
ATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGACAGATCTTACCAAAGTCCAC
ACGGAATGCTGCCAcGGAGATCTGCTTGAATGTGCTGATGACAGGGCGGACCTTGCCAA
GTATATCTGTGAAAATCAAGATTCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACC
TCTGTTGGAAAAATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTT
GCCTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTATGCTGAGGC
AAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAGAAGGCATCCTGATTACTCT
GTCGTGCTGCTGCTGAGACTTGCCAAGACATATGAAACCACTCTAGAGAAGTGCTGTGCC
GCTGCAGATCCTCATGAATGCTATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAA
GAGCCTCAGAATTTAATCAAACAAAATTGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAA
TTCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCAACTCCAACT CTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGatcCAAATGTTGTAAACATCCTGAA GCAAAAAGAATGCCCTGTGCAGAAGACTATCTATCCGTGGTCCTGAACCAGTTATGTGTG TTGCATGAGAAAACGCCAGTAAGTGACAGAGTCACCAAATGCTGCACAGAATCCTTGGT
GAACAGGCGACCATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTT TAATGCTcAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAGGAGAGACAA ATCAAGAAACAAAtgGCACTTGTTGAGCTCGTGAAACACAAGCCCAAGGCAACAAAAGAG CAACTGAAAGCTGTTATGGATGATTTCGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGAC GATAAGGAGACCTGCTTTGCCGAGGAGGGTcccAAACTTGTTGCTGCAAGTCAAGCTGCC TTAGGCTTATAA
SEQ ID NO: 19 - RBD
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYG VSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDS KVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY QPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF
SEQ ID NO: 20 - RBD
AGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTG GTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCA
GCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGT TATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATT TGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTG
ATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAAcTCTAACAAT CTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATC TCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAA TGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATG GTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGC AACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTC
SEQ ID NO: 21 - AlbuFel dl
EICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSLLDK IYTSPLCVKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTAMKKIQDCYVENGLISR VLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGRTGGGGSDAHKSEVAHRFKDLGEENF KALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRE TYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKF GERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICEN QDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSI-AADFVESKDVCKNYAEAKDVFLGM FLYEYARRHPDYSVVLLLRI_AKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCE
LFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSV
VLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSE
KERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAAS
QAALGL
SEQ ID NO: 22 - AlbuFel dl
GAAATCTGTCCGGCGGTGAAGAGGGACGTAGACCTGTTTCTGACTGGGACTCCTGACGA
GTACGTGGAGCAGGTCGCCCAGTACAAGGCTCTGCCCGTGGTCCTCGAGAATGCCCGGA
TCCTGAAAAACTGTGTCGACGCCAAAATGACTGAGGAAGACAAAGAGAACGCACTGTCT
CTTCTAGACAAAATCTACACGTCCCCACTGTGCGTGAAAATGGCCGAGACATGCCCAATC
TTCTACGATGTGTTCTTTGCCGTGGCCAATGGCAATGAACTGCTGCTGGACTTGTCACTG
ACCAAGGTGAACGCCACCGAGCCTGAGAGGACCGCCATGAAGAAAATCCAGGATTGTTA
CGTTGAGAATGGTCTGATTTCAAGGGTGCTTGACGGACTCGTGATGACCACCATCTCAAG
CAGCAAGGATTGTATGGGAGAAGCGGTCCAGAACACCGTCGAAGATCTGAAGTTGAATA
CCCTCGGCCGGACCGGTGGCGGGGGAAGCGATGCACACAAGAGTGAGGTTGCTCATCG
GTTTAAAGATTTGGGAGAAGAAAATTTCAAAGCTTTGGTGTTGATTGCCTTTGCTCAGTAT
CTTCAGCAGTGTCCATTTGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAA
AAACATGTGTTGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTTTTTGG
AGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGAAATGGCTGACTGCTG
TGCAAAACAAGAACCTGAGAGAAATGAATGCTTCTTGCAACACAAAGATGACAACCCAAA
CCTCCCCCGATTGGTGAGACCAGAGGTTGATGTGATGTGCACTGCTTTTCATGACAATGA
AGAGACATTTTTGAAAAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCC
CCGGAACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGCCAAGCTG
CTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCGGGATGAAGGGAAGGCT
TCGTCTGCCAAACAGAGACTCAAGTGTGCCAGTCTCCAAAAATTTGGAGAAAGAGCTTTC
AAAGCATGGGCAGTAGCTCGCCTGAGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGT
TTCCAAGTTAGTGACAGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCT
TGAATGTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAAGATTCGAT
CTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAAATCCCACTGCATTGC
CGAAGTGGAAAATGATGAGATGCCTGCTGACTTGCCTTCATTAGCTGCTGATTTTGTTGA
AAGTAAGGATGTTTGCAAAAACTATGCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTT
GTATGAATATGCAAGAAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAA
GACATATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGCTATGC
CAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAATTTAATCAAACAAAAT
TGTGAGCTTTTTGAGCAGCTTGGAGAGTACAAATTCCAGAATGCGCTATTAGTTCGTTAC ACCAAGAAAGTACCCCAAGTGTCAACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGA
AAAGTGGGGTCCAAATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGA
CTATCTATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTAAGTGA
CAGAGTCACCAAATGCTGCACAGAATCCTTGGTGAACAGGCGACCATGCTTTTCAGCTCT
GGAAGTCGATGAAACATACGTTCCCAAAGAGTTTAATGCTGAAACATTCACCTTCCATGC
AGATATATGCACACTTTCTGAGAAGGAGAGACAAATCAAGAAACAAACTGCACTTGTTGA
GCTCGTGAAACACAAGCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTT
CGCAGCTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCCGAGG
AGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTA
SEQ ID NO: 23 - Fel dl
EICPAVKRDVDLFLTGTPDEYVEQVAQYKALPVVLENARILKNCVDAKMTEEDKENALSLLDK
IYTSPLCVKMAETCPIFYDVFFAVANGNELLLDLSLTKVNATEPERTAMKKIQDCYVENGLISR
VLDGLVMTTISSSKDCMGEAVQNTVEDLKLNTLGR
SEQ ID NO: 24 - Fel dl
GAAATCTGTCCGGCGGTGAAGAGGGACGTAGACCTGTTTCTGACTGGGACTCCTGACGA
GTACGTGGAGCAGGTCGCCCAGTACAAGGCTCTGCCCGTGGTCCTCGAGAATGCCCGGA
TCCTGAAAAACTGTGTCGACGCCAAAATGACTGAGGAAGACAAAGAGAACGCACTGTCT
CTTCTAGACAAAATCTACACGTCCCCACTGTGCGTGAAAATGGCCGAGACATGCCCAATC
TTCTACGATGTGTTCTTTGCCGTGGCCAATGGCAATGAACTGCTGCTGGACTTGTCACTG
ACCAAGGTGAACGCCACCGAGCCTGAGAGGACCGCCATGAAGAAAATCCAGGATTGTTA
CGTTGAGAATGGTCTGATTTCAAGGGTGCTTGACGGACTCGTGATGACCACCATCTCAAG
CAGCAAGGATTGTATGGGAGAAGCGGTCCAGAACACCGTCGAAGATCTGAAGTTGAATA
CCCTCGGCCGG
Items
1. A compound comprising: a. a protein conjugate comprising an albumin, preferably a highbinder albumin, and one or more proteinaceous parts; b. one or more CpG oligonucleotides (ODN) covalently conjugated to the albumin, preferably via thiols such as Cys34 of albumin; and c. optionally, a linker connecting the albumin and the one or more proteinaceous parts; wherein the one or more proteinaceous parts are selected from the group consisting of an antigen-targeting moiety or fragment thereof, an immunogenic protein or a fragment thereof, and a tolerogenic protein or fragment thereof.
2. The compound according to item 1, wherein the ODN is conjugated to the albumin via a thioether bond, such as to Cys34 of albumin.
3. The compound according to any of the preceding items, wherein the ODN is conjugated to the albumin at the 5'end or the 3'end of the ODN, preferably the 5' end.
4. The compound according to any of the preceding items, wherein the ODN comprises a class A ODN, class B ODN, such as CpG 2006 according to SEQ ID NO: 12, or a class C ODN, preferably class C ODN, such as CpG 2395 according to SEQ ID NO: 13.
5. The compound according to any of the preceding items, wherein the ODN is selected from the group consisting of CpG 2395 according to SEQ ID NO: 13, and CpG 2006 according to SEQ ID NO: 12.
6. The compound according to any of the preceding items, wherein the ODN comprises modified nucleotides, such as comprising one or more artificial nucleotides, such as one or more locked nucleic acids (LNAs), such as comprising or consisting of one or more phosphorothioate bonds between individual nucleotides in the backbone of the ODN. 7. The compound according to any of the preceding items, wherein the ODN activates an immune response, such as via activation of TLR preferably via the TLR9 receptor.
8. The compound according to any of the preceding items, wherein the ODN activates one or more immune cells, such as dendritic cells, B cells, macrophages and/or NK cells, preferably antigen-presenting cells.
9. The compound according to any of the preceding items, wherein the binding of the ODN to the TLR receptor, such as the TLR9 receptor, stimulates secretion of one or more cytokines, such as TNFo, TNF[3, IL- 12, IL-6, IL-10, IFN-y, and/or IFN-o.
10. The compound according to any of the preceding items, wherein the one or more proteinaceous parts is an antigen-targeting moiety or a fragment thereof such as an antibody, single domain antibody, diabody, a singlechain variable fragment, affibody or a DARPin.
11. The compound according to item 10, wherein the antigen-targeting moiety is a multispecific antigen-targeting moiety, such as a bispecific antigen-targeting moiety.
12. The compound according to any of items 10-11, wherein the antigentargeting moiety is an immune checkpoint inhibitor.
13. The compound according to item 12, wherein the immune checkpoint inhibitor inhibits an immune checkpoint selected from the group consisting of PD-1, PD-L1, CTLA-4, VISTA, TIM-3, LAG-3, IDO, KIR2D, A2AR, B7-1, B7-H3, TIGIT and BTLA.
14. The compound according to any of items 12-13, wherein the immune checkpoint inhibitor is selected from the group consisting of PD-1 antagonists, PD-L1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-1 antagonists, B7- H3 antagonists, B7-H4 antagonists, and BTLA antagonists. The compound according to any of items 12-14, wherein the immune checkpoint inhibitor is capable of blocking the signal of the immune checkpoint. The compound according to any of items 10-11 and item 15, wherein the antigen-targeting moiety is an immune checkpoint costimulatory agonist, such as 4-1BB, CD28 or CD16A. The compound according to any of items 12-15, wherein the immune checkpoint inhibitor is a PD-L1 antagonist, such as the PD-L1 antagonist according to amino acids 1-121 of SEQ ID NO: 8. The compound according to any of items 11-15, wherein the multispecific antigen-targeting moiety targets at least one antigen located on a cancer cell or an immune cell. The compound according to item 18, wherein the at least one antigen located on a cancer cell is selected from the group consisting of EGFR, PSMA, folate receptor, RGD peptide, transferrin, FcRn, and HEU2. The compound according to item 18, wherein the at least one antigen located on an immune cell is selected from the group consisting of CD3, CD16, and CD16A. The compound according to any of items 18-20, wherein the multispecific antigen-targeting moiety is a bispecific antigen-targeting moiety targeting EGFR and CD3, such as the bispecific antibody according to SEQ ID NO: 10. The compound according to any of items 1-9, wherein the one or more proteinaceous parts is an immunogenic- or tolerogenic protein or a fragment thereof. The compound according to item 22, wherein the immunogenic- or tolerogenic protein or a fragment thereof is capable of binding to a B-cell or an MHC-I or MHC-II molecule on an antigen-presenting cell. The compound according to any of the preceding items, wherein the albumin has at least 80% sequence identity to the sequence according to SEQ ID NO: 2. The compound according to any of the preceding items, wherein the albumin is capable of binding to the FcRn receptor. The compound according to any of the preceding items, wherein the albumin comprises one or more additional artificial cysteines, such as the cysteine introducing mutations K93C, or E294C of albumin. The compound according to any of the preceding items, wherein the albumin is a highbinder albumin, such as having one or more improved pharmacokinetic properties when compared with wild type albumin such as providing a long lasting effect, having an increased binding affinity towards the FcRn receptor, having a longer half-life, having a higher binding affinity to FcRn, and/or endosomal-mediated cellular recycling, preferably wherein the highbinder is selected from the group consisting of: a. the albumin according to any of the preceding items having one or more of the mutations E505Q, T527M, and/or K573P, such as the QMB highbinder according to SEQ ID NO: 6; b. highbinder I (HBI) according to SEQ ID NO: 4; and c. highbinder II (HBII) according to SEQ ID NO: 5. The compound according to any of the preceding items, wherein the albumin and the one or more proteinaceous parts is connected via a linker, such as a flexible- or a rigid linker, preferably a peptide linker, such as a GS linker. The compound according to any of the preceding items, wherein the albumin and the one or more proteinaceous parts is connected via the ODN. 30. The compound according to any of the preceding items, wherein at least one of the one or more proteinaceous parts is connected via a peptide linker and at least one of the one or more proteinaceous parts is connected via the ODN.
31. The compound according to any of items 29-30, wherein the connection via the ODN is through a double stranded ODN, wherein the albumin is conjugated to a first ODN and the one or more proteinaceous parts is conjugated to a second ODN, the first- and second ODN being complementary to each other thereby capable of forming the double stranded ODN upon contact.
32. The compound according to any of the preceding items, wherein the albumin and the one or more proteinaceous parts are translated from a single transcript, such as the albumin and the one or more proteinaceous parts is consisting of one continuous polypeptide chain.
33. The compound according to any of the preceding items, wherein the albumin is positioned at or near the N-terminal- or the C-terminal end of the protein conjugate, in relation to the one or more proteinaceous parts, such as at most 50 additional amino acids between albumin and the N- terminal- or the C-terminal end of the protein conjugate.
34. A composition, comprising the compound according to any of the preceding items.
35. The composition according to item 34, wherein the composition comprises a pharmaceutically acceptable carrier.
36. The compound according to any of items 1-33 or the composition according to any of items 34-35, for use as a medicament.
37. The compound according to any of items 1-33, or the composition according to any of items 34-35, for use as a vaccine.
38. The compound according to any of items 1-33, or the composition according to any of items 34-35 for use in the treatment of cancer, such as a solid cancer, such as a solid cancer selected from the group consisting of breast cancer, colorectal cancer, lung cancers, pancreatic cancers, liver cancers, intestinal cancers, prostate cancers, bladder cancers, kidney cancers, such as renal clear cell carcinoma, ovarian cancers, cervical cancers, adenocarcinomas, squamous cell carcinomas, head and neck cancer, ear or nose or throat cancers, skin cancers, such as basal cell carcinomas, melanomas, non-melanoma skin cancers, squamous cell carcinomas of the skin, such as a liquid cancer such as leukemia, such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML). A method of treatment, comprising administering a therapeutic amount of the compound according to any of items 1-33, or the composition according to any of items 34-35 to a subject in need thereof. A method for producing the compound or the composition according to any of items 1-35, the method comprising: a. providing one or more nucleic acid(s) encoding the protein conjugate or its individual parts according to any of items 1-33, or one or more expression vector(s) comprising said nucleic acid(s); b. introducing the nucleic acid(s) or the vector(s) into one or more host cell(s), such as host cells selected from the group consisting of: bacteria and eukaryotes; c. growing the host cell(s) under conditions that allow for expression of the protein conjugate from the nucleic acid(s) or the vector(s); d. purifying the protein conjugate, or its individual parts; e. providing CpG oligonucleotides; and f. conjugating the CpG oligonucleotides to the purified protein conjugate or its individual parts, thereby obtaining the compound or the composition. The method according to item 40, wherein the CpG oligonucleotides are Maleimide modified oligonucleotides, such as monobromide maleimide (MBM), lysine-modified oligonucleotides, preferably Maleimide modified oligonucleotides. The method according to item 40 or item 41, wherein the compound obtained in step f is purified. The method according to any of items 40-42, wherein the protein conjugate's individual parts of step f are brought in contact with each other to allow them anneal. The method according to any of items 40-43, wherein Maleimide modified oligonucleotides are prepared by combining a CpG-NH2 oligonucleotide with Maleimide, such as Maleimide-(PEG)n-NHS-ester, wherein n is a number between 1-10, succinimidyl 4-(N-maleimidomethyl)cyclohexane- 1-carboxylate (SMCC), and optionally purifying the Maleimide modified oligonucleotides. The method according to any of items 40-44, wherein the nucleic acids encoding the protein conjugate is operably linked to a promotor and optionally, additional regulatory sequences that regulate expression of said nucleic acid. A kit comprising: a. the protein conjugate or its individual parts according to any of the items items 1-33; b. a CpG oligonucleotide, such as a CpG-NH2, such as a Maleimide modified oligonucleotide; c. reagents for conjugating the CpG oligonucleotide to the protein conjugate. A vaccine comprising the compound or the composition as defined in any of items 1-9 and 22-33. Embodiments
1. A compound comprising: a. a protein conjugate comprising an albumin, preferably a highbinder albumin, and one or more proteinaceous parts; b. one or more CpG oligonucleotides (ODN) covalently conjugated to the albumin, preferably via thiols such as Cys34 of albumin; and c. optionally, a linker connecting the albumin and the one or more proteinaceous parts; wherein the one or more proteinaceous parts are selected from the group consisting of an antigen-targeting moiety or fragment thereof, an immunogenic protein or a fragment thereof, and a tolerogenic protein or fragment thereof.
2. The compound according to embodiment 1, wherein the ODN activates one or more immune cells via TLR9, such as dendritic cells, B cells, macrophages and/or NK cells, preferably antigen-presenting cells.
3. The compound according to any of the preceding embodiments, wherein the one or more proteinaceous parts is an antigen-targeting moiety or a fragment thereof such as a multispecific antigen-targeting moiety, such as an antibody, single domain antibody, diabody, a single-chain variable fragment, affibody or a DARPin.
4. The compound according to any of the preceding embodiments, wherein the antigen-targeting moiety is an immune checkpoint inhibitor selected from the group consisting of PD-L1 antagonists, such as SEQ ID NO: 8, PD-1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-1 antagonists, B7-H3 antagonists, B7-H4 antagonists, and BTLA antagonists. The compound according to embodiment 4, wherein the multispecific antigen-targeting moiety targets at least one antigen located on a cancer cell or an immune cell. The compound according to embodiment 5, wherein the at least one antigen located on a cancer cell is selected from the group consisting of EGFR, PSMA, folate receptor, RGD peptide, transferrin, FcRn, and HEU2 or wherein the at least one antigen located on an immune cell is selected from the group consisting of CD3, CD16, and CD16A. The compound according to any of embodiments 5-6, wherein the multispecific antigen-targeting moiety is a bispecific antigen-targeting moiety targeting EGFR and CD3, such as the bispecific antibody according to SEQ ID NO: 10. The compound according to any of embodiments 1-2, wherein the one or more proteinaceous parts is an immunogenic protein or a fragment thereof or a tolerogenic protein or a fragment thereof, capable of binding to a B-cell or an MHC-I or MHC-II molecule on an antigen-presenting cell. The compound according to any of the preceding embodiments, wherein the albumin is a highbinder albumin, such as having one or more improved pharmacokinetic properties when compared with wild type albumin such as providing a long lasting effect, having an increased binding affinity towards the FcRn receptor, having a longer half-life, having a higher binding affinity to FcRn, and/or endosomal-mediated cellular recycling, preferably wherein the highbinder is selected from the group consisting of: a. the albumin according to any of the preceding embodiments having one or more of the mutations E505Q, T527M, and/or K573P, such as the QMB highbinder according to SEQ ID NO: 6; b. highbinder I (HBI) according to SEQ ID NO: 4; and c. highbinder II (HBII) according to SEQ ID NO: 5. The compound according to any of the preceding embodiments, wherein the albumin and the one or more proteinaceous parts are connected via the ODN. A composition, comprising the compound according to any of the preceding embodiments. The compound according to any of embodiments 1-10 or the composition according to embodiment 11, for use as a medicament. The compound according to any of embodiment 1-10 or the composition according to embodiment 11 for use in the treatment of cancer, such as a cancer selected from the group consisting of: a. a solid cancer, such as a solid cancer selected from the group consisting of breast cancer, colorectal cancer, lung cancers, pancreatic cancers, liver cancers, intestinal cancers, prostate cancers, bladder cancers, kidney cancers, such as renal clear cell carcinoma, ovarian cancers, cervical cancers, adenocarcinomas, squamous cell carcinomas, head and neck cancer, ear or nose or throat cancers, skin cancers, such as basal cell carcinomas, melanomas, non-melanoma skin cancers, squamous cell carcinomas of the skin, and b. a liquid cancer such as leukemia, such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML). A method for producing the compound according to any of embodiments 1-10 or the composition according to embodiment 11, the method comprising: a. providing one or more nucleic acid(s) encoding the protein conjugate or its individual parts according to any of embodiments 1-10, or one or more expression vector(s) comprising said nucleic acid(s); b. introducing the nucleic acid(s) or the vector(s) into one or more host cell(s), such as host cells selected from the group consisting of: bacteria and eukaryotes; c. growing the host cell(s) under conditions that allow for expression of the protein conjugate from the nucleic acid(s) or the vector(s); d. purifying the protein conjugate, or its individual parts; e. providing CpG oligonucleotides, such as Maleimide modified oligonucleotides; and f. conjugating the CpG oligonucleotides to the purified protein conjugate or its individual parts, thereby obtaining the compound or the composition. vaccine comprising the compound as defined in any of embodiments 1- or 8-10.

Claims

Claims
1. A compound comprising: a. a protein conjugate comprising an albumin, preferably a highbinder albumin, and one or more proteinaceous parts; b. one or more CpG oligonucleotides (ODN) covalently conjugated to the albumin, preferably via thiols such as Cys34 of albumin; and c. optionally, a linker connecting the albumin and the one or more proteinaceous parts; wherein the one or more proteinaceous parts are selected from the group consisting of an antigen-targeting moiety or antigen binding fragment thereof, an immunogenic protein or an immunogenic fragment thereof, and a tolerogenic protein or a tolerogenic fragment thereof.
2. The compound according to claim 1, wherein the ODN activates one or more immune cells via TLR9, such as dendritic cells, B cells, macrophages and/or NK cells, preferably antigen-presenting cells.
3. The compound according to any of the preceding claims, wherein the one or more proteinaceous parts is an antigen-targeting moiety or an antigen binding fragment thereof such as a multispecific antigen-targeting moiety, such as an antibody, single domain antibody, diabody, a singlechain variable fragment, affibody or a DARPin.
4. The compound according to any of the preceding claims, wherein the antigen-targeting moiety is an immune checkpoint inhibitor selected from the group consisting of PD-L1 antagonists, such as SEQ ID NO: 8, PD-1 antagonists, PD-L2 antagonists, CTLA-4 antagonists, VISTA antagonists, TIM-3 antagonists, LAG-3 antagonists, IDO antagonists, KIR2D antagonists, A2AR antagonists, B7-1 antagonists, B7-H3 antagonists, B7- H4 antagonists, and BTLA antagonists.
5. The compound according to claim 3, wherein the multispecific antigentargeting moiety targets at least one antigen located on a cancer cell or an immune cell, preferably an antigen-presenting cell. The compound according to claim 5, wherein the at least one antigen located on a cancer cell is selected from the group consisting of BTLA, 0X40, LAG3, NRP1, VEGF, HER2, CEA, CD19, CD20, Amyloid beta, HER3, IGF-1R, MUC1, EpCAM, CD22, VEGFR-2, PSMA, GM-CSF, CXCR4, CD30, CD70, FGFR2, BCMA, CD44, ICAM-1, Notchl, MHC, CD28, IL-1R1, TCR, Notch3, FGFR3, TGF-g, TGFBR1, TGFBR2, CD109, GITR, CD47, Alpha- synuclein, CD26, LRP1, CD52, IL-4Ro, VAP-1, EPO Receptor, Integrin ov, TIM-3, Grp78, LIGHT, TLR2, TLR3, PAR-2, NRP2, GLP-1 receptor, Hedgehog, and Syndecan 1 or wherein the at least one antigen located on an immune cell is selected from the group consisting of CD3, CD16, and CD16A. The compound according to any of claims 5-6, wherein the multispecific antigen-targeting moiety is a bispecific antigen-targeting moiety targeting EGFR and CD3, such as the bispecific antibody according to SEQ ID NO: 10. The compound according to any of claims 1-2, wherein the one or more proteinaceous parts is an immunogenic protein or an immunogenic fragment thereof or a tolerogenic protein or a tolerogenic fragment thereof, capable of binding to a B-cell and/or an MHC-I or MHC-II molecule on an antigen-presenting cell. The compound according to claim 8, wherein the immunogenic protein is selected from an infectious agent known to cause disease in subjects, or an immunogenic fragment thereof, or wherein the tolerogenic protein peptide is selected from an allergy antigen known to cause allergies in subjects or a tolerogenic fragment thereof. The compound according to claim 8 or claim 9, wherein the immunogenic protein is the RBD protein according to SEQ ID NO: 19 or an immunogenic fragment thereof or wherein the tolerogenic protein peptide is Fel dl according to SEQ ID NO: 23 or a tolerogenic fragment thereof. The compound according to any of the preceding claims, wherein the albumin is a highbinder albumin, such as having one or more improved pharmacokinetic properties when compared with wild type albumin such as providing a long lasting effect, having an increased binding affinity towards the FcRn receptor, having a longer half-life, having a higher binding affinity to FcRn, and/or endosomal-mediated cellular recycling, preferably wherein the highbinder is selected from the group consisting of: a. the albumin according to any of the preceding claims having one or more of the mutations E505Q, T527M, and/or K573P, such as the QMP highbinder according to SEQ ID NO: 6; b. highbinder I (HBI) according to SEQ ID NO: 4; and c. highbinder II (HBII) according to SEQ ID NO: 5. The compound according to any of the preceding claims, wherein the albumin and the one or more proteinaceous parts are connected via the ODN. The compound according to any of the preceding claims, wherein the antigen-targeting moiety is a moiety targeting a target, such as a protein, present on the surface of a cell. The compound according to any of the preceding claims, wherein the antigen-targeting moiety targets a cell in need of immunestimulation. The compound according to any of the preceding claims, wherein the antigen-targeting moiety targets a tissue in need of immunestimulation. A composition, comprising the compound according to any of the preceding claims. The compound according to any of claims 1-15 or the composition according to claim 16, for use as a medicament. The compound according to any of claim 1-15 or the composition according to claim 16 for use in the treatment of an infectious disease, an allergy, or a cancer, such as a cancer selected from the group consisting of: a. a solid cancer, such as a solid cancer selected from the group consisting of breast cancer, colorectal cancer, lung cancers, pancreatic cancers, liver cancers, intestinal cancers, prostate cancers, bladder cancers, kidney cancers, such as renal clear cell carcinoma, ovarian cancers, cervical cancers, adenocarcinomas, squamous cell carcinomas, head and neck cancer, ear or nose or throat cancers, skin cancers, such as basal cell carcinomas, melanomas, non-melanoma skin cancers, squamous cell carcinomas of the skin, and b. a liquid cancer such as leukemia, such as acute lymphocytic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML).
19. A method for producing the compound according to any of claims 1-15 or the composition according to claim 16, the method comprising: a. providing one or more nucleic acid(s) encoding the protein conjugate or its individual parts according to any of claims 1-15, or one or more expression vector(s) comprising said nucleic acid(s); b. introducing the nucleic acid(s) or the vector(s) into one or more host cell(s), such as host cells selected from the group consisting of: bacteria and eukaryotes; c. growing the host cell(s) under conditions that allow for expression of the protein conjugate from the nucleic acid(s) or the vector(s); d. purifying the protein conjugate, or its individual parts; e. providing CpG oligonucleotides, such as Maleimide modified oligonucleotides; and f. conjugating the CpG oligonucleotides to the purified protein conjugate or its individual parts, thereby obtaining the compound or the composition. 0. A vaccine comprising the compound as defined in any of claims 1-2 or 8- 15.
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